Heat Shock Protein 70 (HSP70) has emerged as a modulator of vascular behavior, and its physiological role is sex- and age-dependent. In diabetic conditions, pharmacological blockade of HSP70 improves the vasculature in males. Yet, in diabetic females, the HSP70 profile and its role in vascular hypercontractility, a hallmark of type 2 diabetes (T2D), remain unknown. We showed that inhibiting aortic HSP70 in female T2D ameliorates vascular dysfunction, while the eHSP70-to-iHSP70 ratio is preserved, suggesting sex-specific differences persist in T2D.
","acknowledgements":"We would like to acknowledge Dr. Samuel Mozen for his help with handling the animals and Ahmed Araara for technical support.
","authors":[{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","formalAnalysis","methodology","dataCuration","writing_originalDraft","investigation"],"email":"vochoamendoz2019@my.fit.edu","firstName":"Valentina ","lastName":"Ochoa Mendoza","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0003-1125-8499"},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","formalAnalysis","writing_reviewEditing"],"email":"srastogi2022@my.fit.edu","firstName":"Swasti","lastName":"Rastogi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["methodology","writing_reviewEditing"],"email":"cbiby2024@my.fit.edu","firstName":"Chloe","lastName":"Biby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","methodology","writing_reviewEditing"],"email":"jliaw2025@my.fit.edu","firstName":"Jessica ","lastName":"Liaw","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","fundingAcquisition","project","supervision","writing_reviewEditing"],"email":"knunes@fit.edu","firstName":"Kenia Pedrosa","lastName":" Nunes","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0002-4838-7617"}],"awards":[{"awardId":"1R15DK131511-01A1","funderName":"National Institute of Diabetes and Digestive and Kidney Diseases (United States)","awardRecipient":"Kenia Pedrosa Nunes"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This research was funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), grant number 1R15DK131511-01A1, PI: Nunes, Kenia Pedrosa
","image":{"url":"https://portal.micropublication.org/uploads/f7844a4c18b2e4d3765e6dc1361fe773.png"},"imageCaption":"(A) VER155008 (VER) reduces PE-induced vasoconstriction in female diabetic (db/db) and non-diabetic (db/+) aorta. Cumulative concentration-response curves to PE; db/+-Vehicle (black filled circles), db/+-VER (red filled circles), db/db-Vehicle (black empty circles), and db/db-VER (red empty circles). (B) Area under the curve (AUC) quantification. (C) Maximal force of contraction (Emax). (D) Representative time vs. force curves in response to a single dose of PE; db/+ (continuous lines) and db/db (dashed lines) tissues incubated with vehicle (black) or VER (red). (E) Emax of the phasic part of the curve and (F) tonic part of the curve in db/+ (solid symbols) and db/db (open symbols) mice. A-F, data are shown as means ± SEM and were analyzed by two-way ANOVA with Holm–Sidak’s post hoc test; *** p ≤ 0.001, and **** p ≤ 0.0001; n = 6–7. (G) Representative immunoblots of intracellular HSP70 (iHSP70) in aorta (normalized to β-actin). (H) Systemic extracellular HSP70 (eHSP70) in the serum is shown for db/+ and db/db mice. (I) Quantification of iHSP70/β-actin expression and (J) eHSP70 levels (arbitrary units, a.u.) in db/+ and db/db. (K) eHSP70-to-iHSP70 ratio and (L) H index calculated from the same animals. G-L, data are presented as mean ± SEM; n = 4–5 mice per group. *p ≤ 0.05, ***p ≤ 0.001 vs. db/+, unpaired Student’s t-test or Mann–Whitney U-test as appropriate.
","imageTitle":"Female db/db aorta shows hypercontractility to Phenylephrine (PE) reversed by HSP70 inhibition, while the HSP70 ratio is not disrupted.
","methods":"Ex vivo assessment of thoracic aortic function by wire myograph
Thoracic aortic rings (2 mm in length) from db/db and db/+ mice were mounted in a DMT620M multi-wire myograph system (Danish Myo Technology, Copenhagen, Denmark) under isometric conditions and set to an optimal resting tension of approximately 5 mN in PSS, continuously aerated with carbogen (95% O2, 5% CO2) at 37 °C. The protocol for the equilibration period and verification of functional integrity of each ring was assessed as previously described (Ochoa Mendoza et al., 2025).
Cumulative concentration-response curves
After equilibration and viability confirmation, the aortic rings were incubated for 30 min with vehicle (DMSO) or VER155008, a small-molecule HSP70 inhibitor (10 μmol/L in DMSO; Sigma Aldrich, St. Louis, MO, USA; catalog SML0271). The cumulative concentration-response curves for phenylephrine (PE; 1 nmol/L-100 μmol/L) were then generated, with contractile responses recorded at each concentration. Area under the curve (AUC) was calculated using the trapezoidal rule.
Time-force curves
For time-force analysis, aortic segments were incubated for 30 min with vehicle (DMSO) or VER155008 (10 µmol/L), followed by exposure to a single dose of PE (10⁻6 mol/L). Isometric force was recorded continuously for 15 min (1 s sampling rate). Time-force curves were fit to a bi-exponential function using least-squares regression:
The phasic component was quantified as the peak force at the fast-to-slow transition (approximated as Ffast), and the tonic component as the sustained force at 15 min (approximating Fslow), both expressed relative to baseline (time 0).
Western blot analysis of HSP70
Thoracic aortas were homogenized in Tissue Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA, Cat. 78510) supplemented with Protease Inhibitor Cocktail (Sigma Aldrich, St. Louis, MO, USA; Cat. P8340). Total protein concentration in aortic lysates was determined using the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA, Cat. 23225). For electrophoresis, 10 µg of aortic protein or 10 µL of serum were loaded onto 10% SDS‑PAGE gels, followed by transfer to nitrocellulose membranes. Membranes were briefly stained with Ponceau S to confirm transfer efficiency, imaged, and then washed in Tris-buffered saline containing 1% Tween-20 (TBS-T). Non-specific binding was blocked by incubating membranes for 1 h at room temperature in 5% non-fat dry milk in TBS-T. Membranes were then probed overnight at 4 °C with a primary anti-HSP70 antibody (Cell Signaling Technology, Cat. 4872; 1:2000 in TBS-T containing 0.5 g BSA). After washing, membranes were incubated for 1 h at room temperature with an HRP-conjugated anti-rabbit secondary antibody (Cell Signaling Technology; Cat.7074; 1:10,000 in TBS-T) under gentle agitation. A chemiluminescent signal was developed using SuperSignal West Femto substrate (Thermo Fisher Scientific, Rockford, IL, USA, Cat. 34096), and bands were detected with a Bio-Rad ChemiDoc MP imaging system (Hercules, CA, USA). Band intensities were quantified using ImageJ and normalized to β-actin (Abcam, Waltham, MA, USA; ab8227) for aortic samples. For serum samples, normalization was performed against total protein load rather than a housekeeping protein, given the high protein complexity and variability of β-actin in serum.
Statistical analysis
All data analysis and graph generation were performed using GraphPad Prism 10.3.1 (GraphPad Software, San Diego, CA, USA). Results are expressed as means ± SEM. Data distribution was evaluated using the D’Agostino–Pearson test for normality, and potential outliers were identified and removed using Grubb’s test where appropriate. Depending on data distribution and experimental design, comparisons between two groups were made using unpaired Student’s t-tests or Mann–Whitney U-tests, while multiple comparisons were assessed using two-way ANOVA followed by Sidak’s post hoc test. A p-value ≤ 0.05 was considered statistically significant. The reported sample size (n) refers to the number of mice per group.
","reagents":"Male db/db mice (B6.BKS(D)-Leprdb/J) and age-matched heterozygous db/+ controls were obtained from The Jackson Laboratory (Bar Harbor, ME, USA; total n = 30). At 16 weeks of age, body weight was recorded, and non-fasting blood glucose was measured using a Metene TD-4116 Blood Glucose Monitor (TaiDoc Technology Corporation, New Taipei City, Taiwan). Mice were then anesthetized with 4% isoflurane in 100% O2, and blood was collected from the abdominal aorta into BD Vacutainer serum tubes. Serum was separated and snap-frozen in liquid nitrogen for subsequent extracellular HSP70 (eHSP70) quantification by Western blotting. Animals were euthanized by exsanguination via cardiac puncture for harvesting thoracic aortas, which were snap-frozen or prepared for vascular reactivity evaluation in the myograph. All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Florida Institute of Technology (IACUC; protocol #2022-04) and conducted under veterinary supervision in accordance with the ARRIVE guidelines.
","patternDescription":"The onset and progression of vascular dysfunction in T2D are complex and influenced by multiple factors (Dunn & Nelson, 2010; Fernández-Velasco et al., 2014). HSP70 plays critical roles across different tissues (de Oliveira et al., 2022). In skeletal and cardiac muscle, deletion of inducible HSP70 genes disrupts calcium (Ca2+) handling and impairs contractile function (Kim et al., 2006; Senf et al., 2013), while in vascular smooth muscle, HSP70 is required for receptor-mediated contraction (de Oliveira & Nunes, 2020a). Importantly, hyperglycemia has been associated with elevated circulating HSP70 levels (Nakhjavani et al., 2010), and it is now suggested that HSP70 is not merely a bystander but a potential mechanistic player in this setting. HSP70 is ubiquitously expressed, residing intracellularly (iHSP70) or released extracellularly (eHSP70), and its effects in diabetes depend critically on both location and relative abundance (de Oliveira et al., 2022). An imbalance between eHSP70 and iHSP70 has been proposed as an inflammatory biomarker, and a disruption of this ratio is associated with vascular dysfunction in both type 1 (de Oliveira et al., 2022) and T2D (Ochoa Mendoza et al., 2025). In the aorta of male rodents, HSP70 contributes to receptor-mediated vasoconstriction by regulating Ca2+-handling mechanisms in the vasculature, supporting an important role for HSP70 in maintaining physiological vascular tone (de Oliveira, et al., 2021).
Recent evidence also indicates that vascular HSP70 expression and function are age (de Oliveira, et al., 2022) and sex-dependent (de Oliveira et al., 2021). Healthy female rats have lower aortic HSP70 levels than males, despite reports of higher HSP70 expression in some female tissues such as the heart (Voss et al., 2003). Under non-pathological conditions, pharmacological blockade of HSP70 with VER155008 has a greater impact on phenylephrine-induced contraction in female aortic rings than in male rings, affecting both phasic and tonic components of the response (de Oliveira, et al., 2021). These findings suggest that impaired vascular HSP70 expression in females augments their dependence on HSP70-mediated Ca²⁺ handling for the maintenance of contractile tone, providing a potential molecular axis for sex-specific differences in vascular reactivity.
Given that females are generally protected from cardiovascular disease for much of their lives but may lose this advantage in T2D, and HSP70 might be a possible therapeutic target (de Oliveira et al., 2019), we examined the contribution of HSP70 to vascular dysfunction in a T2D diabetic female (db/db) model. Our findings show that (1) PE-induced vasoconstriction is enhanced in the diabetic female aorta, consistent with a hypercontractile phenotype, and that HSP70 inhibitor VER155008 markedly attenuates this response, (2) HSP70 impacts both phases of the biphasic contraction curve, and (3) the eHSP70-to-iHSP70 ratio is preserved in T2D females.
In the aorta of female db/db mice, pharmacological inhibition of HSP70 showed a more pronounced effect when compared to control db/+ (Figure 1A-C), indicating that HSP70 contributes to the exaggerated vasoconstriction observed in T2D. Considering that HSP70 blockade reduces Ca²⁺ influx–mediated contraction and lowers free cytosolic Ca²⁺ levels in aortic tissues from T2D males (Ochoa Mendoza et al., 2025), we evaluated the biphasic contraction curve in response to a single dose of PE in the aorta of diabetic females. This biphasic behavior reflects a two‑phase rise in cytosolic Ca²⁺. The phasic phase is driven predominantly by IP₃ receptor (IP₃R) ‑mediated Ca²⁺ release from the SR, whereas the tonic phase depends on continued Ca²⁺ influx through voltage‑dependent and receptor‑operated channels, together with Ca²⁺‑sensitizing mechanisms that maintain force at relatively sustained Ca²⁺ levels (Webb, 2003). HSP70 inhibition reduced both phases of the biphasic curve (Figure 1D): the phasic (Figure 1E) and the tonic (Figure 1F) in db/+ and db/db vessels, with a more pronounced difference in the tonic phase, in which diabetic aortas displayed greater contraction than controls. Combined, the results evaluating the vascular reactivity of the diabetic female aorta corroborate the hypercontractile profile triggered by diabetes, which is decreased by HSP70 inhibition, thereby improving overall vascular function.
Interestingly, elevated levels of iHSP70 are associated with an anti-inflammatory state, whereas higher levels of circulating eHSP70 are associated with inflammation [32]. Mechanistically, eHSP70 acts as a damage-associated molecular pattern (DAMP) and activates the innate immune receptor Toll-like receptor 4 (TLR4) in the cardiovascular system (de Oliveira et al., 2019). Thus, eHSP70 is suggested as a therapeutic target for diabetic vasculopathies (de Oliveira & Nunes, 2020). T1D and T2D disrupt the balance between eHSP70 and iHSP70, leading to vascular dysfunction, and this phenotype can be prevented by targeting HSP70 (Ochoa Mendoza et al., 2025). These studies were conducted only in a male diabetic model. To fill this gap, we evaluated aortic and systemic levels of HSP70. Our data showed that both iHSP70 and eHSP70 were reduced when comparing non-diabetic db/+ versus diabetic db/db (Figure 1 G-J). However, the eHSP70-to-iHSP70 ratio (Krause et al., 2015) and the H index (Costa-Beber et al., 2020) for this protein, both used as indirect biomarkers for low-grade inflammation, were not disrupted (Figure 1K and L, respectively), contrasting reports in male diabetic mice (Ochoa Mendoza et al., 2025).
An important mediator of sex differences in cardiovascular function across species is the nitric oxide (NO)/NO synthase (NOS) pathway. Although the molecular mechanisms underlying these sex differences remain incompletely defined, the NO/NOS pathway has been strongly implicated in both humans (Forte et al., 1998; Sader & Celermajer, 2002) and rats (Kauser & Rubanyi, 1994). In humans, premenopausal women display superior NO-dependent endothelial vasodilation compared with men, attributable to estrogen's ability to upregulate eNOS expression, enhance its phosphorylation, and boost NO bioavailability while mitigating its oxidative inactivation (Steinberg et al., 2000; White, 2002). Rodent models corroborate this pattern, with female rats exhibiting elevated basal NO production and greater NO-mediated vascular relaxation than males (Kauser & Rubanyi, 1994).
These estrogen-driven effects not only promote endothelium-dependent relaxation but also likely influence chaperone homeostasis by curbing hyperglycemia-induced inflammation and oxidative stress, which can otherwise disrupt the eHSP70-to-iHSP70 balance according to the chaperone balance hypothesis (Krause et al., 2015). The preservation of the eHSP70:iHSP70 ratio and H index in our diabetic females, despite downregulation of both iHSP70 and eHSP70, may reflect a more stable, coordinated adaptation to diabetic stress. In T2D, where metabolic stressors typically compromise NO signaling, one could say that the preserved HSP70 equilibrium in female db/db mice suggests a lingering protective NO/NOS axis. This may explain the exaggerated attenuation of tonic hypercontractility by VER155008 in diabetic females (Figure A and D, red lines), as HSP70 inhibition could synergize with residual NO-mediated vasodilation to more effectively counteract sustained vascular tone. Interestingly, under physiological conditions, inhibition of HSP70 does not affect NO in the vasculature of males (de Oliveira & Nunes, 2020a), which does not exclude the possibility that this landscape changes in females. Overall, the data presented corroborate HSP70’s involvement in vascular function, expand our understanding of HSP70's sex-specific role in pathological conditions, and highlight a worthwhile therapeutic window for targeting HSP70 to restore vascular function in women with T2D.
","references":[{"reference":"Costa Beber LC, Hirsch GE, Heck TG, Ludwig MS. 2020. Chaperone duality: the role of extracellular and intracellular HSP70 as a biomarker of endothelial dysfunction in the development of atherosclerosis. Archives of Physiology and Biochemistry: 1.","pubmedId":"","doi":"10.1080/13813455.2020.1745850"},{"reference":"De Oliveira AA, Faustino J, De Lima ME, Menezes R, Nunes KP. 2019. Unveiling the Interplay between the TLR4/MD2 Complex and HSP70 in the Human Cardiovascular System: A Computational Approach. International Journal of Molecular Sciences. 20","pubmedId":"","doi":"10.3390/ijms20133121"},{"reference":"De Oliveira AA, Mendoza VO, Priviero F, Webb RC, Nunes KP. 2022. Age-Related Decline in Vascular Responses to Phenylephrine Is Associated with Reduced Levels of HSP70. Biomolecules. 12","pubmedId":"","doi":"10.3390/biom12081125"},{"reference":"De Oliveira AA, Nunes KP. 2020. An additional physiological role for HSP70: Assistance of vascular reactivity. Life Sciences. 256: 117986.","pubmedId":"","doi":"10.1016/j.lfs.2020.117986"},{"reference":"De Oliveira AA, Priviero F, Tostes RC, Webb RC, Nunes KP. 2021. Dissecting the interaction between HSP70 and vascular contraction: role of Ca^2+ handling mechanisms. Scientific Reports. 11: 1420.","pubmedId":"","doi":"10.1038/s41598-021-80966-6"},{"reference":"De Oliveira AA, Priviero F, Webb RC, Nunes KP. 2021. Impaired HSP70 Expression in the Aorta of Female Rats: A Novel Insight Into Sex-Specific Differences in Vascular Function. Frontiers in Physiology. 12","pubmedId":"","doi":""},{"reference":"Dunn KM, Nelson MT. 2010. Calcium and diabetic vascular dysfunction. Focus on “Elevated Ca2+ sparklet activity during acute hyperglycemia and diabetes in cerebral arterial smooth muscle cells”. American Journal of Physiology-Cell Physiology. 298: C203.","pubmedId":"","doi":"10.1152/ajpcell.00499.2009"},{"reference":"Fernandez Velasco M, Ruiz Hurtado G, Gomez AM, Rueda A. 2014. Ca2+ handling alterations and vascular dysfunction in diabetes. Calcium in diabetes. 56: 397.","pubmedId":"","doi":"10.1016/j.ceca.2014.08.007"},{"reference":"Forte P, Kneale BJ, Milne E, Chowienczyk PJ, Johnston A, Benjamin N, Ritter JM. 1998. Evidence for a difference in nitric oxide biosynthesis between healthy women and men. Hypertension. 32: 730.","pubmedId":"","doi":""},{"reference":"Kauser K, Rubanyi GM. 1994. Gender difference in bioassayable endothelium-derived nitric oxide from isolated rat aortae. American Journal of Physiology-Heart and Circulatory Physiology. 267: H2311.","pubmedId":"","doi":""},{"reference":"Kim YK, Suarez J, Hu Y, Mc Donough PM, Boer C, Dix DJ, Dillmann WH. 2006. Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy. Circulation. 113: 2589.","pubmedId":"","doi":"10.1161/CIRCULATIONAHA.105.598409"},{"reference":"Krause M, Heck TG, Bittencourt A, Scomazzon SP, Newsholme P, Curi R, Homem De Bittencourt PI. 2015. The chaperone balance hypothesis: the importance of the extracellular to intracellular HSP70 ratio to inflammation-driven type 2 diabetes, the effect of exercise, and the implications for clinical management. Mediators of inflammation. 2015: 249205.","pubmedId":"25814786","doi":"10.1155/2015/249205"},{"reference":"Nakhjavani M, Morteza A, Khajeali L, Esteghamati A, Khalilzadeh O, Asgarani F, Outeiro TF. 2010. Increased serum HSP70 levels are associated with the duration of diabetes. Cell stress & chaperones. 15: 959.","pubmedId":"20496051","doi":"10.1007/s12192-010-0204-z"},{"reference":"Ochoa Mendoza V, De Oliveira AA, Nunes KP. 2025. Blockade of HSP70 Improves Vascular Function in a Mouse Model of Type 2 Diabetes. Cells. 14: 424.","pubmedId":"","doi":"10.3390/cells14060424"},{"reference":"Oliveira AA, Mendoza VO, Rastogi S, Nunes KP. 2022. New insights into the role and therapeutic potential of HSP70 in diabetes. Pharmacological Research. 178: 106173.","pubmedId":"","doi":"https://doi.org/10.1016/j.phrs.2022.106173"},{"reference":"Oliveira AA, Priviero F, Webb RC, Nunes KP. 2022. Increased eHSP70-to-iHSP70 ratio disrupts vascular responses to calcium and activates the TLR4-MD2 complex in type 1 diabetes. Life Sciences. 310: 121079.","pubmedId":"","doi":"https://doi.org/10.1016/j.lfs.2022.121079"},{"reference":"Sader MA, Celermajer DS. 2002. Endothelial function, vascular reactivity and gender differences in the cardiovascular system. Cardiovascular research. 53: 597.","pubmedId":"","doi":""},{"reference":"Senf SM, Howard TM, Ahn B, Ferreira LF, Judge AR. 2013. Loss of the inducible Hsp70 delays the inflammatory response to skeletal muscle injury and severely impairs muscle regeneration. PloS one. 8: e62687.","pubmedId":"23626847","doi":"10.1371/journal.pone.0062687"},{"reference":"Steinberg HO, Paradisi G, Cronin J, Crowde K, Hempfling A, Hook G, Baron AD. 2000. Type II Diabetes Abrogates Sex Differences in Endothelial Function in Premenopausal Women. Circulation. 101: 2040.","pubmedId":"","doi":"10.1161/01.CIR.101.17.2040"},{"reference":"Voss MR, Stallone JN, Li M, Cornelussen RNM, Knuefermann P, Knowlton AA. 2003. Gender differences in the expression of heat shock proteins: the effect of estrogen. American Journal of Physiology-Heart and Circulatory Physiology. 285: H687.","pubmedId":"","doi":"10.1152/ajpheart.01000.2002"},{"reference":"Webb RC. 2003. SMOOTH MUSCLE CONTRACTION AND RELAXATION. Advances in Physiology Education. 27: 201.","pubmedId":"","doi":"10.1152/advan.00025.2003"},{"reference":"White RE. 2002. Estrogen and vascular function. Vascular Pharmacology. 38: 73.","pubmedId":"","doi":"10.1016/S0306-3623(02)00129-5"}],"title":"
Heat Shock Protein 70 (HSP70) has emerged as a modulator of vascular behavior, and its physiological role is sex- and age-dependent. In diabetic conditions, pharmacological blockade of HSP70 improves the vasculature in males. Yet, in diabetic females, the HSP70 profile and its role in vascular hypercontractility, a hallmark of type 2 diabetes (T2D), remain unknown. We showed that inhibiting aortic HSP70 in female T2D ameliorates vascular dysfunction, while the eHSP70-to-iHSP70 ratio is preserved, suggesting sex-specific differences persist in T2D.
","acknowledgements":"We would like to acknowledge Dr. Samuel Mozen for his help with handling the animals and Ahmed Araara for technical support.
","authors":[{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","formalAnalysis","methodology","dataCuration","writing_originalDraft","investigation"],"email":"vochoamendoz2019@my.fit.edu","firstName":"Valentina ","lastName":"Ochoa Mendoza","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0003-1125-8499"},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","formalAnalysis","writing_reviewEditing"],"email":"srastogi2022@my.fit.edu","firstName":"Swasti","lastName":"Rastogi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["methodology","writing_reviewEditing"],"email":"cbiby2024@my.fit.edu","firstName":"Chloe","lastName":"Biby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","methodology","writing_reviewEditing"],"email":"jliaw2025@my.fit.edu","firstName":"Jessica ","lastName":"Liaw","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","fundingAcquisition","project","supervision","writing_reviewEditing"],"email":"knunes@fit.edu","firstName":"Kenia Pedrosa","lastName":" Nunes","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0002-4838-7617"}],"awards":[{"awardId":"1R15DK131511-01A1","funderName":"National Institute of Diabetes and Digestive and Kidney Diseases (United States)","awardRecipient":"Kenia Pedrosa Nunes"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This research was funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), grant number 1R15DK131511-01A1, PI: Nunes, Kenia Pedrosa
","image":{"url":"https://portal.micropublication.org/uploads/f7844a4c18b2e4d3765e6dc1361fe773.png"},"imageCaption":"(A) VER155008 (VER) reduces PE-induced vasoconstriction in female diabetic (db/db) and non-diabetic (db/+) aorta. Cumulative concentration-response curves to PE; db/+-Vehicle (black filled circles), db/+-VER (red filled circles), db/db-Vehicle (black empty circles), and db/db-VER (red empty circles). (B) Area under the curve (AUC) quantification. (C) Maximal force of contraction (Emax). (D) Representative time vs. force curves in response to a single dose of PE; db/+ (continuous lines) and db/db (dashed lines) tissues incubated with vehicle (black) or VER (red). (E) Emax of the phasic part of the curve and (F) tonic part of the curve in db/+ (solid symbols) and db/db (open symbols) mice. A-F, data are shown as means ± SEM and were analyzed by two-way ANOVA with Holm–Sidak’s post hoc test; *** p ≤ 0.001, and **** p ≤ 0.0001; n = 6–7. (G) Representative immunoblots of intracellular HSP70 (iHSP70) in aorta (normalized to β-actin). (H) Systemic extracellular HSP70 (eHSP70) in the serum is shown for db/+ and db/db mice. (I) Quantification of iHSP70/β-actin expression and (J) eHSP70 levels (arbitrary units, a.u.) in db/+ and db/db. (K) eHSP70-to-iHSP70 ratio and (L) H index calculated from the same animals. G-L, data are presented as mean ± SEM; n = 4–5 mice per group. *p ≤ 0.05, ***p ≤ 0.001 vs. db/+, unpaired Student’s t-test or Mann–Whitney U-test as appropriate.
","imageTitle":"Female db/db aorta shows hypercontractility to Phenylephrine (PE) reversed by HSP70 inhibition, while the HSP70 ratio is not disrupted.
","methods":"Ex vivo assessment of thoracic aortic function by wire myograph
Thoracic aortic rings (2 mm in length) from db/db and db/+ mice were mounted in a DMT620M multi-wire myograph system (Danish Myo Technology, Copenhagen, Denmark) under isometric conditions and set to an optimal resting tension of approximately 5 mN in PSS, continuously aerated with carbogen (95% O2, 5% CO2) at 37 °C. The protocol for the equilibration period and verification of functional integrity of each ring was assessed as previously described (Ochoa Mendoza et al., 2025).
Cumulative concentration-response curves
After equilibration and viability confirmation, the aortic rings were incubated for 30 min with vehicle (DMSO) or VER155008, a small-molecule HSP70 inhibitor (10 μmol/L in DMSO; Sigma Aldrich, St. Louis, MO, USA; catalog SML0271). The cumulative concentration-response curves for phenylephrine (PE; 1 nmol/L-100 μmol/L) were then generated, with contractile responses recorded at each concentration. Area under the curve (AUC) was calculated using the trapezoidal rule.
Time-force curves
For time-force analysis, aortic segments were incubated for 30 min with vehicle (DMSO) or VER155008 (10 µmol/L), followed by exposure to a single dose of PE (10⁻6 mol/L). Isometric force was recorded continuously for 15 min (1 s sampling rate). Time-force curves were fit to a bi-exponential function using least-squares regression:
The phasic component was quantified as the peak force at the fast-to-slow transition (approximated as Ffast), and the tonic component as the sustained force at 15 min (approximating Fslow), both expressed relative to baseline (time 0).
Western blot analysis of HSP70
Thoracic aortas were homogenized in Tissue Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA, Cat. 78510) supplemented with Protease Inhibitor Cocktail (Sigma Aldrich, St. Louis, MO, USA; Cat. P8340). Total protein concentration in aortic lysates was determined using the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA, Cat. 23225). For electrophoresis, 10 µg of aortic protein or 10 µL of serum were loaded onto 10% SDS‑PAGE gels, followed by transfer to nitrocellulose membranes. Membranes were briefly stained with Ponceau S to confirm transfer efficiency, imaged, and then washed in Tris-buffered saline containing 1% Tween-20 (TBS-T). Non-specific binding was blocked by incubating membranes for 1 h at room temperature in 5% non-fat dry milk in TBS-T. Membranes were then probed overnight at 4 °C with a primary anti-HSP70 antibody (Cell Signaling Technology, Cat. 4872; 1:2000 in TBS-T containing 0.5 g BSA). After washing, membranes were incubated for 1 h at room temperature with an HRP-conjugated anti-rabbit secondary antibody (Cell Signaling Technology; Cat.7074; 1:10,000 in TBS-T) under gentle agitation. A chemiluminescent signal was developed using SuperSignal West Femto substrate (Thermo Fisher Scientific, Rockford, IL, USA, Cat. 34096), and bands were detected with a Bio-Rad ChemiDoc MP imaging system (Hercules, CA, USA). Band intensities were quantified using ImageJ and normalized to β-actin (Abcam, Waltham, MA, USA; ab8227) for aortic samples. For serum samples, normalization was performed against total protein load rather than a housekeeping protein, given the high protein complexity and variability of β-actin in serum.
Statistical analysis
All data analysis and graph generation were performed using GraphPad Prism 10.3.1 (GraphPad Software, San Diego, CA, USA). Results are expressed as means ± SEM. Data distribution was evaluated using the D’Agostino–Pearson test for normality, and potential outliers were identified and removed using Grubb’s test where appropriate. Depending on data distribution and experimental design, comparisons between two groups were made using unpaired Student’s t-tests or Mann–Whitney U-tests, while multiple comparisons were assessed using two-way ANOVA followed by Sidak’s post hoc test. A p-value ≤ 0.05 was considered statistically significant. The reported sample size (n) refers to the number of mice per group.
","reagents":"Male db/db mice (B6.BKS(D)-Leprdb/J) and age-matched heterozygous db/+ controls were obtained from The Jackson Laboratory (Bar Harbor, ME, USA; total n = 30). At 16 weeks of age, body weight was recorded, and non-fasting blood glucose was measured using a Metene TD-4116 Blood Glucose Monitor (TaiDoc Technology Corporation, New Taipei City, Taiwan). Mice were then anesthetized with 4% isoflurane in 100% O2, and blood was collected from the abdominal aorta into BD Vacutainer serum tubes. Serum was separated and snap-frozen in liquid nitrogen for subsequent extracellular HSP70 (eHSP70) quantification by Western blotting. Animals were euthanized by exsanguination via cardiac puncture for harvesting thoracic aortas, which were snap-frozen or prepared for vascular reactivity evaluation in the myograph. All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Florida Institute of Technology (IACUC; protocol #2022-04) and conducted under veterinary supervision in accordance with the ARRIVE guidelines.
","patternDescription":"The onset and progression of vascular dysfunction in T2D are complex and influenced by multiple factors (Dunn & Nelson, 2010; Fernández-Velasco et al., 2014). HSP70 plays critical roles across different tissues (de Oliveira et al., 2022). In skeletal and cardiac muscle, deletion of inducible HSP70 genes disrupts calcium (Ca2+) handling and impairs contractile function (Kim et al., 2006; Senf et al., 2013), while in vascular smooth muscle, HSP70 is required for receptor-mediated contraction (de Oliveira & Nunes, 2020a). Importantly, hyperglycemia has been associated with elevated circulating HSP70 levels (Nakhjavani et al., 2010), and it is now suggested that HSP70 is not merely a bystander but a potential mechanistic player in this setting. HSP70 is ubiquitously expressed, residing intracellularly (iHSP70) or released extracellularly (eHSP70), and its effects in diabetes depend critically on both location and relative abundance (de Oliveira et al., 2022). An imbalance between eHSP70 and iHSP70 has been proposed as an inflammatory biomarker, and a disruption of this ratio is associated with vascular dysfunction in both type 1 (de Oliveira et al., 2022) and T2D (Ochoa Mendoza et al., 2025). In the aorta of male rodents, HSP70 contributes to receptor-mediated vasoconstriction by regulating Ca2+-handling mechanisms in the vasculature, supporting an important role for HSP70 in maintaining physiological vascular tone (de Oliveira, et al., 2021).
Recent evidence also indicates that vascular HSP70 expression and function are age (de Oliveira, et al., 2022) and sex-dependent (de Oliveira et al., 2021). Healthy female rats have lower aortic HSP70 levels than males, despite reports of higher HSP70 expression in some female tissues such as the heart (Voss et al., 2003). Under non-pathological conditions, pharmacological blockade of HSP70 with VER155008 has a greater impact on phenylephrine-induced contraction in female aortic rings than in male rings, affecting both phasic and tonic components of the response (de Oliveira, et al., 2021). These findings suggest that impaired vascular HSP70 expression in females augments their dependence on HSP70-mediated Ca²⁺ handling for the maintenance of contractile tone, providing a potential molecular axis for sex-specific differences in vascular reactivity.
Given that females are generally protected from cardiovascular disease for much of their lives but may lose this advantage in T2D, and HSP70 might be a possible therapeutic target (de Oliveira et al., 2019), we examined the contribution of HSP70 to vascular dysfunction in a T2D diabetic female (db/db) model. Our findings show that (1) PE-induced vasoconstriction is enhanced in the diabetic female aorta, consistent with a hypercontractile phenotype, and that HSP70 inhibitor VER155008 markedly attenuates this response, (2) HSP70 impacts both phases of the biphasic contraction curve, and (3) the eHSP70-to-iHSP70 ratio is preserved in T2D females.
In the aorta of female db/db mice, pharmacological inhibition of HSP70 showed a more pronounced effect when compared to control db/+ (Figure 1A-C), indicating that HSP70 contributes to the exaggerated vasoconstriction observed in T2D. Considering that HSP70 blockade reduces Ca²⁺ influx–mediated contraction and lowers free cytosolic Ca²⁺ levels in aortic tissues from T2D males (Ochoa Mendoza et al., 2025), we evaluated the biphasic contraction curve in response to a single dose of PE in the aorta of diabetic females. This biphasic behavior reflects a two‑phase rise in cytosolic Ca²⁺. The phasic phase is driven predominantly by IP₃ receptor (IP₃R) ‑mediated Ca²⁺ release from the SR, whereas the tonic phase depends on continued Ca²⁺ influx through voltage‑dependent and receptor‑operated channels, together with Ca²⁺‑sensitizing mechanisms that maintain force at relatively sustained Ca²⁺ levels (Webb, 2003). HSP70 inhibition reduced both phases of the biphasic curve (Figure 1D): the phasic (Figure 1E) and the tonic (Figure 1F) in db/+ and db/db vessels, with a more pronounced difference in the tonic phase, in which diabetic aortas displayed greater contraction than controls. Combined, the results evaluating the vascular reactivity of the diabetic female aorta corroborate the hypercontractile profile triggered by diabetes, which is decreased by HSP70 inhibition, thereby improving overall vascular function.
Interestingly, elevated levels of iHSP70 are associated with an anti-inflammatory state, whereas higher levels of circulating eHSP70 are associated with inflammation [32]. Mechanistically, eHSP70 acts as a damage-associated molecular pattern (DAMP) and activates the innate immune receptor Toll-like receptor 4 (TLR4) in the cardiovascular system (de Oliveira et al., 2019). Thus, eHSP70 is suggested as a therapeutic target for diabetic vasculopathies (de Oliveira & Nunes, 2020). T1D and T2D disrupt the balance between eHSP70 and iHSP70, leading to vascular dysfunction, and this phenotype can be prevented by targeting HSP70 (Ochoa Mendoza et al., 2025). These studies were conducted only in a male diabetic model. To fill this gap, we evaluated aortic and systemic levels of HSP70. Our data showed that both iHSP70 and eHSP70 were reduced when comparing non-diabetic db/+ versus diabetic db/db (Figure 1 G-J). However, the eHSP70-to-iHSP70 ratio (Krause et al., 2015) and the H index (Costa-Beber et al., 2020) for this protein, both used as indirect biomarkers for low-grade inflammation, were not disrupted (Figure 1K and L, respectively), contrasting reports in male diabetic mice (Ochoa Mendoza et al., 2025).
An important mediator of sex differences in cardiovascular function across species is the nitric oxide (NO)/NO synthase (NOS) pathway. Although the molecular mechanisms underlying these sex differences remain incompletely defined, the NO/NOS pathway has been strongly implicated in both humans (Forte et al., 1998; Sader & Celermajer, 2002) and rats (Kauser & Rubanyi, 1994). In humans, premenopausal women display superior NO-dependent endothelial vasodilation compared with men, attributable to estrogen's ability to upregulate eNOS expression, enhance its phosphorylation, and boost NO bioavailability while mitigating its oxidative inactivation (Steinberg et al., 2000; White, 2002). Rodent models corroborate this pattern, with female rats exhibiting elevated basal NO production and greater NO-mediated vascular relaxation than males (Kauser & Rubanyi, 1994).
These estrogen-driven effects not only promote endothelium-dependent relaxation but also likely influence chaperone homeostasis by curbing hyperglycemia-induced inflammation and oxidative stress, which can otherwise disrupt the eHSP70-to-iHSP70 balance according to the chaperone balance hypothesis (Krause et al., 2015). The preservation of the eHSP70:iHSP70 ratio and H index in our diabetic females, despite downregulation of both iHSP70 and eHSP70, may reflect a more stable, coordinated adaptation to diabetic stress. In T2D, where metabolic stressors typically compromise NO signaling, one could say that the preserved HSP70 equilibrium in female db/db mice suggests a lingering protective NO/NOS axis. This may explain the exaggerated attenuation of tonic hypercontractility by VER155008 in diabetic females (Figure A and D, red lines), as HSP70 inhibition could synergize with residual NO-mediated vasodilation to more effectively counteract sustained vascular tone. Interestingly, under physiological conditions, inhibition of HSP70 does not affect NO in the vasculature of males (de Oliveira & Nunes, 2020a), which does not exclude the possibility that this landscape changes in females. Overall, the data presented corroborate HSP70’s involvement in vascular function, expand our understanding of HSP70's sex-specific role in pathological conditions, and highlight a worthwhile therapeutic window for targeting HSP70 to restore vascular function in women with T2D.
","references":[{"reference":"Costa Beber LC, Hirsch GE, Heck TG, Ludwig MS. 2020. Chaperone duality: the role of extracellular and intracellular HSP70 as a biomarker of endothelial dysfunction in the development of atherosclerosis. Archives of Physiology and Biochemistry: 1.","pubmedId":"","doi":"10.1080/13813455.2020.1745850"},{"reference":"De Oliveira AA, Faustino J, De Lima ME, Menezes R, Nunes KP. 2019. Unveiling the Interplay between the TLR4/MD2 Complex and HSP70 in the Human Cardiovascular System: A Computational Approach. International Journal of Molecular Sciences. 20","pubmedId":"","doi":"10.3390/ijms20133121"},{"reference":"De Oliveira AA, Mendoza VO, Priviero F, Webb RC, Nunes KP. 2022. Age-Related Decline in Vascular Responses to Phenylephrine Is Associated with Reduced Levels of HSP70. Biomolecules. 12","pubmedId":"","doi":"10.3390/biom12081125"},{"reference":"De Oliveira AA, Nunes KP. 2020. An additional physiological role for HSP70: Assistance of vascular reactivity. Life Sciences. 256: 117986.","pubmedId":"","doi":"10.1016/j.lfs.2020.117986"},{"reference":"De Oliveira AA, Priviero F, Tostes RC, Webb RC, Nunes KP. 2021. Dissecting the interaction between HSP70 and vascular contraction: role of Ca^2+ handling mechanisms. Scientific Reports. 11: 1420.","pubmedId":"","doi":"10.1038/s41598-021-80966-6"},{"reference":"De Oliveira AA, Priviero F, Webb RC, Nunes KP. 2021. Impaired HSP70 Expression in the Aorta of Female Rats: A Novel Insight Into Sex-Specific Differences in Vascular Function. Frontiers in Physiology. 12","pubmedId":"","doi":""},{"reference":"Dunn KM, Nelson MT. 2010. Calcium and diabetic vascular dysfunction. Focus on “Elevated Ca2+ sparklet activity during acute hyperglycemia and diabetes in cerebral arterial smooth muscle cells”. American Journal of Physiology-Cell Physiology. 298: C203.","pubmedId":"","doi":"10.1152/ajpcell.00499.2009"},{"reference":"Fernandez Velasco M, Ruiz Hurtado G, Gomez AM, Rueda A. 2014. Ca2+ handling alterations and vascular dysfunction in diabetes. Calcium in diabetes. 56: 397.","pubmedId":"","doi":"10.1016/j.ceca.2014.08.007"},{"reference":"Forte P, Kneale BJ, Milne E, Chowienczyk PJ, Johnston A, Benjamin N, Ritter JM. 1998. Evidence for a difference in nitric oxide biosynthesis between healthy women and men. Hypertension. 32: 730.","pubmedId":"","doi":""},{"reference":"Kauser K, Rubanyi GM. 1994. Gender difference in bioassayable endothelium-derived nitric oxide from isolated rat aortae. American Journal of Physiology-Heart and Circulatory Physiology. 267: H2311.","pubmedId":"","doi":""},{"reference":"Kim YK, Suarez J, Hu Y, Mc Donough PM, Boer C, Dix DJ, Dillmann WH. 2006. Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy. Circulation. 113: 2589.","pubmedId":"","doi":"10.1161/CIRCULATIONAHA.105.598409"},{"reference":"Krause M, Heck TG, Bittencourt A, Scomazzon SP, Newsholme P, Curi R, Homem De Bittencourt PI. 2015. The chaperone balance hypothesis: the importance of the extracellular to intracellular HSP70 ratio to inflammation-driven type 2 diabetes, the effect of exercise, and the implications for clinical management. Mediators of inflammation. 2015: 249205.","pubmedId":"25814786","doi":"10.1155/2015/249205"},{"reference":"Nakhjavani M, Morteza A, Khajeali L, Esteghamati A, Khalilzadeh O, Asgarani F, Outeiro TF. 2010. Increased serum HSP70 levels are associated with the duration of diabetes. Cell stress & chaperones. 15: 959.","pubmedId":"20496051","doi":"10.1007/s12192-010-0204-z"},{"reference":"Ochoa Mendoza V, De Oliveira AA, Nunes KP. 2025. Blockade of HSP70 Improves Vascular Function in a Mouse Model of Type 2 Diabetes. Cells. 14: 424.","pubmedId":"","doi":"10.3390/cells14060424"},{"reference":"Oliveira AA, Mendoza VO, Rastogi S, Nunes KP. 2022. New insights into the role and therapeutic potential of HSP70 in diabetes. Pharmacological Research. 178: 106173.","pubmedId":"","doi":"https://doi.org/10.1016/j.phrs.2022.106173"},{"reference":"Oliveira AA, Priviero F, Webb RC, Nunes KP. 2022. Increased eHSP70-to-iHSP70 ratio disrupts vascular responses to calcium and activates the TLR4-MD2 complex in type 1 diabetes. Life Sciences. 310: 121079.","pubmedId":"","doi":"https://doi.org/10.1016/j.lfs.2022.121079"},{"reference":"Sader MA, Celermajer DS. 2002. Endothelial function, vascular reactivity and gender differences in the cardiovascular system. Cardiovascular research. 53: 597.","pubmedId":"","doi":""},{"reference":"Senf SM, Howard TM, Ahn B, Ferreira LF, Judge AR. 2013. Loss of the inducible Hsp70 delays the inflammatory response to skeletal muscle injury and severely impairs muscle regeneration. PloS one. 8: e62687.","pubmedId":"23626847","doi":"10.1371/journal.pone.0062687"},{"reference":"Steinberg HO, Paradisi G, Cronin J, Crowde K, Hempfling A, Hook G, Baron AD. 2000. Type II Diabetes Abrogates Sex Differences in Endothelial Function in Premenopausal Women. Circulation. 101: 2040.","pubmedId":"","doi":"10.1161/01.CIR.101.17.2040"},{"reference":"Voss MR, Stallone JN, Li M, Cornelussen RNM, Knuefermann P, Knowlton AA. 2003. Gender differences in the expression of heat shock proteins: the effect of estrogen. American Journal of Physiology-Heart and Circulatory Physiology. 285: H687.","pubmedId":"","doi":"10.1152/ajpheart.01000.2002"},{"reference":"Webb RC. 2003. SMOOTH MUSCLE CONTRACTION AND RELAXATION. Advances in Physiology Education. 27: 201.","pubmedId":"","doi":"10.1152/advan.00025.2003"},{"reference":"White RE. 2002. Estrogen and vascular function. Vascular Pharmacology. 38: 73.","pubmedId":"","doi":"10.1016/S0306-3623(02)00129-5"}],"title":"
Heat Shock Protein 70 (HSP70) has emerged as a modulator of vascular behavior, and its physiological role is sex- and age-dependent. In diabetic conditions, pharmacological blockade of HSP70 improves the vasculature in males. Yet, in diabetic females, the HSP70 profile and its role in vascular hypercontractility, a hallmark of type 2 diabetes (T2D), remain unknown. We showed that inhibiting aortic HSP70 in T2D females ameliorates vascular dysfunction, while the eHSP70-to-iHSP70 ratio is preserved, suggesting sex-specific differences during T2D.
","acknowledgements":"We would like to acknowledge Dr. Samuel Mozen for his help with handling the animals and Ahmed Araara for technical support.
","authors":[{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","formalAnalysis","methodology","dataCuration","writing_originalDraft","investigation"],"email":"vochoamendoz2019@my.fit.edu","firstName":"Valentina ","lastName":"Ochoa Mendoza","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0003-1125-8499"},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","formalAnalysis","writing_reviewEditing"],"email":"srastogi2022@my.fit.edu","firstName":"Swasti","lastName":"Rastogi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["methodology","writing_reviewEditing"],"email":"cbiby2024@my.fit.edu","firstName":"Chloe","lastName":"Biby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","methodology","writing_reviewEditing"],"email":"jliaw2025@my.fit.edu","firstName":"Jessica ","lastName":"Liaw","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","fundingAcquisition","project","supervision","writing_reviewEditing"],"email":"knunes@fit.edu","firstName":"Kenia Pedrosa","lastName":" Nunes","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0002-4838-7617"}],"awards":[{"awardId":"1R15DK131511-01A1","funderName":"National Institute of Diabetes and Digestive and Kidney Diseases (United States)","awardRecipient":"Kenia Pedrosa Nunes"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Embed equation in Methods section>Time forces curves>to a bi-exponential function using least-squares regression
","doi":null,"resourceType":"Text","name":"Equation.mathml.docx","url":"https://portal.micropublication.org/uploads/daa047aee154c3bbd364f27ff76b3377.docx"}],"funding":"This research was funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), grant number 1R15DK131511-01A1, PI: Nunes, Kenia Pedrosa
","image":{"url":"https://portal.micropublication.org/uploads/a3d54d788ee6439ed7301b3d5cf8ba29.png"},"imageCaption":"(A) Cumulative PE concentration-response curves in db/+ vehicle (grey filled circles), db/+ VER (lilac filled circles), db/db vehicle (grey unfilled circles), and db/db VER (lilac unfilled circles) aortas. (B) AUC quantification. (C) Maximal contractile response (Emax). (D) Representative time–force traces in response to a single PE challenge in db/+ (continuous lines) and db/db (dashed lines) vessels treated with vehicle (grey) or VER (lilac). (E) Emax of the phasic phase and (F) tonic phase in db/+ (filled symbols) and db/db (unfilled symbols) mice. Data in A–F are mean ± SEM; two-way ANOVA with Holm–Sidak’s post hoc test; *p ≤ 0.001, **p ≤ 0.0001; n = 6–7. (G) Representative immunoblots of aortic iHSP70 normalized to β-actin. (H) Serum eHSP70 levels in db/+ (filled circles) and db/db (unfilled circles) mice. (I) iHSP70/β-actin and (J) eHSP70 quantification (a.u.). (K) eHSP70-to-iHSP70 ratio and (L) H index. Data in G–L are mean ± SEM; n = 4–5 mice per group. *p ≤ 0.05, **p ≤ 0.001 vs. db/+, unpaired Student’s t-test or Mann–Whitney U-test as appropriate.
","imageTitle":"Female db/db aorta shows hypercontractility to Phenylephrine (PE) reversed by HSP70 inhibition, which impacts the tonic phase of contraction mediated by Ca2+ influx, while the HSP70 ratio is preserved
","methods":"Ex vivo assessment of thoracic aortic function by wire myograph
Thoracic aortic rings (2 mm in length) from db/db and db/+ female mice were mounted in a DMT620M multi-wire myograph system (Danish Myo Technology, Copenhagen, Denmark) under isometric conditions and set to an optimal resting tension of approximately 5 mN in PSS, continuously aerated with carbogen (95% O2, 5% CO2) at 37 °C. The protocol for the equilibration period and verification of functional integrity of each ring was assessed as previously described (Ochoa Mendoza et al., 2025).
Cumulative concentration-response curves
After equilibration and viability confirmation, the aortic rings were incubated for 30 min with vehicle (DMSO) or VER155008, a small-molecule HSP70 inhibitor (10 μmol/L in DMSO; Sigma Aldrich, St. Louis, MO, USA; catalog SML0271). The cumulative concentration-response curves for phenylephrine (PE; 1 nmol/L-100 μmol/L) were then generated, with contractile responses recorded at each concentration. Area under the curve (AUC) was calculated using the trapezoidal rule.
Time-force curves
For time-force analysis, aortic segments were incubated for 30 min with vehicle (DMSO) or VER155008 (10 µmol/L), followed by exposure to a single dose of PE (10⁻6 mol/L). Isometric force was recorded continuously for 15 min (1 s sampling rate). Time-force curves were fit to a bi-exponential function using least-squares regression:
**Embed equation here**
The phasic component was quantified as the peak force at the fast-to-slow transition (approximated as Ffast), and the tonic component as the sustained force at 15 min (approximating Fslow), both expressed relative to baseline (time 0).
Western blot analysis of HSP70
Thoracic aortas were homogenized in Tissue Protein Extraction Reagent supplemented with protease inhibitors, and protein concentration was determined by BCA assay. Equal amounts of aortic protein (10 µg) or serum (10 µL) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were briefly stained with Ponceau S to confirm transfer, washed in TBS-T, and blocked for 1 h at room temperature in 5% non-fat dry milk. They were then incubated overnight at 4 °C with an anti-HSP70 primary antibody, followed by 1 h at room temperature with an HRP-conjugated anti-rabbit secondary antibody. Signal was developed using a chemiluminescent substrate and detected with a ChemiDoc MP imaging system. Band intensity was quantified in ImageJ and normalized to β-actin for aortic samples. Serum samples were normalized to total protein load, since housekeeping proteins are not suitable for this matrix.
Statistical analysis
Statistical analyses and graphing were performed with GraphPad Prism 10.3.1 (GraphPad Software, San Diego, CA, USA). Data are presented as means ± SEM. Normality was assessed using the D’Agostino–Pearson test, and outliers were identified and excluded with Grubb’s test when appropriate. Two-group comparisons were performed using unpaired Student’s t-tests or Mann–Whitney U-tests, depending on data distribution, whereas multiple-group analyses were conducted by two-way ANOVA followed by Sidak’s post hoc test. Statistical significance was set at *p ≤ 0.05. Sample size (n) indicates the number of mice per group.
","reagents":"Female db/db mice (B6.BKS(D)-Leprdb/J) and age-matched heterozygous db/+ controls were purchased from The Jackson Laboratory (Bar Harbor, ME, USA; n = 20 total). Body weight was recorded, and non-fasting blood glucose were measured biweekly with a Metene TD-4116 glucose monitor (TaiDoc Technology Corporation, New Taipei City, Taiwan). Diabetic profile was confirmed by hyperglycemia (350 mg/dl or higher). Mice were anesthetized with 4% isoflurane in 100% O2, and blood was collected from the abdominal aorta into BD Vacutainer serum tubes. Serum was isolated, snap-frozen in liquid nitrogen, and later used for eHSP70 analysis by Western blotting. Animals were then euthanized at 16 weeks by exsanguination via cardiac puncture, and thoracic aortas were harvested for snap-freezing or vascular reactivity studies in the myograph. All procedures were approved by the Institutional Animal Care and Use Committee of the Florida Institute of Technology (protocol #2022-04) and performed under veterinary supervision in accordance with ARRIVE guidelines.
","patternDescription":"T2D-associated vascular dysfunction arises from a network of interacting mechanisms rather than a single cause, making its progression difficult to explain by a single pathway alone (Dunn & Nelson, 2010; Fernández-Velasco et al., 2014).HSP70 plays critical roles across different tissues (de Oliveira et al., 2022). It is increasingly recognized as a multifunctional protein with tissue-specific roles, including important effects in muscle biology. In skeletal and cardiac muscle, loss of inducible HSP70 disrupts Ca2+ homeostasis and weakens contractile performance (Kim et al., 2006; Senf et al., 2013), while in vascular smooth muscle, HSP70 supports receptor-driven contraction (de Oliveira & Nunes, 2020). Importantly, hyperglycemia has been associated with elevated circulating HSP70 levels (Nakhjavani et al., 2010), and it is now suggested that HSP70 is not merely a bystander but a potential mechanistic player in this setting. HSP70 is ubiquitously expressed, residing intracellularly (iHSP70) or released extracellularly in the circulation (eHSP70), and its effects in diabetes depend critically on both location and surplus (de Oliveira et al., 2022). An imbalance between eHSP70 and iHSP70 has been proposed as an inflammatory biomarker, and a disruption of this ratio is associated with vascular dysfunction in both type 1 (de Oliveira et al., 2022) and T2D (Ochoa Mendoza et al., 2025). In the aorta of male rodents, HSP70 contributes to receptor-mediated vasoconstriction by regulating Ca2+-handling mechanisms, supporting an important role for HSP70 in maintaining physiological vascular tone (de Oliveira, et al., 2021).
Recent evidence indicates that vascular HSP70 expression and function are age- (de Oliveira, et al., 2022) and sex-dependent (de Oliveira et al., 2021). Healthy female rats have lower aortic HSP70 levels than males, despite reports of higher HSP70 expression in some female tissues such as the heart (Voss et al., 2003). Under non-pathological conditions, pharmacological blockade of HSP70 with VER155008 has a greater impact on phenylephrine-induced contraction in female aortic rings than in male rings, affecting both phasic and tonic components of the response (de Oliveira, et al., 2021). These findings suggest that impaired vascular HSP70 expression in females augments their dependence on HSP70-mediated Ca²⁺ handling for the maintenance of contractile tone, providing a potential molecular axis for sex-specific differences in vascular reactivity.
Given that females are generally protected from cardiovascular disease for much of their lives but may lose this advantage in T2D, and HSP70 might be a possible therapeutic target (de Oliveira et al., 2019), we examined the contribution of HSP70 to vascular dysfunction in a T2D diabetic female (db/db) model. Our findings show that (1) the hypercontractile phenotype in the aorta of T2D females is prevented by HSP70 inhibitor VER155008, similar to results in males, (2) but it only impacts the tonic phase of contraction when comparing control (db/+) versus diabetic (db/db), and (3) the eHSP70-to-iHSP70 ratio is preserved in T2D females, contrasting data in males (Ochoa Mendoza et al., 2025).
In the aorta of female db/db mice, pharmacological inhibition of HSP70 showed a more pronounced effect when compared to control db/+ (Figure 1A-C), indicating that HSP70 contributes to the exaggerated vasoconstriction observed in T2D. Considering that HSP70 blockade reduces Ca²⁺ influx–mediated contraction and lowers free cytosolic Ca²⁺ levels in aortic tissues from T2D males (Ochoa Mendoza et al., 2025), we evaluated the biphasic contraction curve in response to a single dose of PE in the aorta of diabetic females. This biphasic behavior reflects a two‑phase rise in cytosolic Ca²⁺. The phasic phase is driven predominantly by IP₃ receptor (IP₃R) ‑mediated Ca²⁺ release from the SR, whereas the tonic phase depends on continued Ca²⁺ influx through voltage‑dependent and receptor‑operated channels, together with Ca²⁺‑sensitizing mechanisms that maintain force at relatively sustained Ca²⁺ levels (Webb, 2003). HSP70 inhibition reduced contraction in both phases of the biphasic curve (Figure 1D, 1E, and 1F) in db/+ and db/db vessels. However, a significant difference between the db/+ and db/db groups was only observed during the tonic phase (Figure 1F), suggesting Ca²⁺ influx impairment. The aorta's vascular reactivity corroborates the hypercontractile profile triggered by diabetes, which is reduced by HSP70 inhibition, thereby improving overall vascular function in T2D females.
Circulating eHSP70 is suggested as a target for the treatment of diabetic vasculopathies as it can act like a damage-associated molecular pattern (DAMP) and lead to an innate immune response via TLR4 (de Oliveira, et al., 2022), thus it is associated with low-grade inflammation, whereas increased iHSP70 is regarded as protective and anti-inflammatory (de Oliveira, Mendoza, Rastogi, et al., 2022). A disruption in the balance between eHSP70 and iHSP70 can lead to vascular dysfunction in diabetic males (Ochoa Mendoza et al., 2025). Still, the HSP70 ratio was unknown in adult females. Thus, we evaluated aortic and systemic HSP70 levels. Our data showed that both iHSP70 and eHSP70 were reduced in control db/+ versus diabetic db/db (Figure 1 G-J), which led to an undisrupted eHSP70-to-iHSP70 ratio (Krause et al., 2015) and H index (Costa-Beber et al., 2020), both used as indirect biomarkers for low-grade inflammation (Figure 1K and L, respectively), differing from male diabetic mice (Ochoa Mendoza et al., 2025).
An important mediator of sex differences in cardiovascular function across species is the nitric oxide (NO)/NO synthase (NOS) pathway. Although the molecular mechanisms underlying these sex differences remain incompletely defined, the NO/NOS pathway has been strongly implicated in both humans (Forte et al., 1998; Sader & Celermajer, 2002) and rats (Kauser & Rubanyi, 1994). In humans, premenopausal women display superior NO-dependent endothelial vasodilation compared to men, attributable to estrogen's ability to upregulate eNOS expression, enhance its phosphorylation, and boost NO bioavailability while mitigating its oxidative inactivation (Steinberg et al., 2000; White, 2002). Rodent models corroborate this pattern, with female rats exhibiting elevated basal NO production and greater NO-mediated vascular relaxation than males (Kauser & Rubanyi, 1994).
These estrogen-driven effects not only promote endothelium-dependent relaxation but also likely influence chaperone homeostasis by curbing hyperglycemia-induced inflammation and oxidative stress, which can otherwise disrupt the eHSP70-to-iHSP70 balance according to the chaperone balance hypothesis (Krause et al., 2015). The preservation of the eHSP70:iHSP70 ratio and H index in our diabetic females, may reflect a more stable, coordinated adaptation to diabetic stress. Noteworthy, a disrupted ratio has only been reported in postmenopausal T2D women, corroborating that at the adult stage, our females are still protected (Seibert et al., 2022). In T2D, where metabolic stressors typically compromise NO signaling, one could say that the preserved HSP70 equilibrium in the female db/db mice suggests a lingering protective NO/NOS axis. This may explain the exaggerated attenuation of hypercontractility by VER155008 in diabetic females (Figure A and D, lilac lines), as HSP70 inhibition could synergize with residual NO-mediated vasodilation to more effectively counteract sustained vascular tone. Interestingly, under physiological conditions, inhibition of HSP70 does not affect NO in the vasculature of males (de Oliveira & Nunes, 2020), which does not exclude the possibility that this landscape changes in females. Overall, the data presented corroborate HSP70’s involvement in vascular function, expand our understanding of HSP70's sex-specific role in pathological conditions, and highlight a worthwhile therapeutic window for targeting HSP70 to restore vascular function in women with T2D.
","references":[{"reference":"Costa Beber LC, Hirsch GE, Heck TG, Ludwig MS. 2020. Chaperone duality: the role of extracellular and intracellular HSP70 as a biomarker of endothelial dysfunction in the development of atherosclerosis. Archives of Physiology and Biochemistry: 1.","pubmedId":"","doi":"10.1080/13813455.2020.1745850"},{"reference":"De Oliveira AA, Faustino J, De Lima ME, Menezes R, Nunes KP. 2019. Unveiling the Interplay between the TLR4/MD2 Complex and HSP70 in the Human Cardiovascular System: A Computational Approach. International Journal of Molecular Sciences. 20","pubmedId":"","doi":"10.3390/ijms20133121"},{"reference":"De Oliveira AA, Mendoza VO, Priviero F, Webb RC, Nunes KP. 2022. Age-Related Decline in Vascular Responses to Phenylephrine Is Associated with Reduced Levels of HSP70. Biomolecules. 12","pubmedId":"","doi":"10.3390/biom12081125"},{"reference":"De Oliveira AA, Nunes KP. 2020. An additional physiological role for HSP70: Assistance of vascular reactivity. Life Sciences. 256: 117986.","pubmedId":"","doi":"10.1016/j.lfs.2020.117986"},{"reference":"De Oliveira AA, Priviero F, Tostes RC, Webb RC, Nunes KP. 2021. Dissecting the interaction between HSP70 and vascular contraction: role of Ca^2+ handling mechanisms. Scientific Reports. 11: 1420.","pubmedId":"","doi":"10.1038/s41598-021-80966-6"},{"reference":"De Oliveira AA, Priviero F, Webb RC, Nunes KP. 2021. Impaired HSP70 Expression in the Aorta of Female Rats: A Novel Insight Into Sex-Specific Differences in Vascular Function. Frontiers in Physiology. 12","pubmedId":"","doi":""},{"reference":"Dunn KM, Nelson MT. 2010. Calcium and diabetic vascular dysfunction. Focus on “Elevated Ca2+ sparklet activity during acute hyperglycemia and diabetes in cerebral arterial smooth muscle cells”. American Journal of Physiology-Cell Physiology. 298: C203.","pubmedId":"","doi":"10.1152/ajpcell.00499.2009"},{"reference":"Fernandez Velasco M, Ruiz Hurtado G, Gomez AM, Rueda A. 2014. Ca2+ handling alterations and vascular dysfunction in diabetes. Calcium in diabetes. 56: 397.","pubmedId":"","doi":"10.1016/j.ceca.2014.08.007"},{"reference":"Forte P, Kneale BJ, Milne E, Chowienczyk PJ, Johnston A, Benjamin N, Ritter JM. 1998. Evidence for a difference in nitric oxide biosynthesis between healthy women and men. Hypertension. 32: 730.","pubmedId":"","doi":""},{"reference":"Kauser K, Rubanyi GM. 1994. Gender difference in bioassayable endothelium-derived nitric oxide from isolated rat aortae. American Journal of Physiology-Heart and Circulatory Physiology. 267: H2311.","pubmedId":"","doi":""},{"reference":"Kim YK, Suarez J, Hu Y, Mc Donough PM, Boer C, Dix DJ, Dillmann WH. 2006. Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy. Circulation. 113: 2589.","pubmedId":"","doi":"10.1161/CIRCULATIONAHA.105.598409"},{"reference":"Krause M, Heck TG, Bittencourt A, Scomazzon SP, Newsholme P, Curi R, Homem De Bittencourt PI. 2015. The chaperone balance hypothesis: the importance of the extracellular to intracellular HSP70 ratio to inflammation-driven type 2 diabetes, the effect of exercise, and the implications for clinical management. Mediators of inflammation. 2015: 249205.","pubmedId":"25814786","doi":"10.1155/2015/249205"},{"reference":"Nakhjavani M, Morteza A, Khajeali L, Esteghamati A, Khalilzadeh O, Asgarani F, Outeiro TF. 2010. Increased serum HSP70 levels are associated with the duration of diabetes. Cell stress & chaperones. 15: 959.","pubmedId":"20496051","doi":"10.1007/s12192-010-0204-z"},{"reference":"Ochoa Mendoza V, De Oliveira AA, Nunes KP. 2025. Blockade of HSP70 Improves Vascular Function in a Mouse Model of Type 2 Diabetes. Cells. 14: 424.","pubmedId":"","doi":"10.3390/cells14060424"},{"reference":"Oliveira AA, Mendoza VO, Rastogi S, Nunes KP. 2022. New insights into the role and therapeutic potential of HSP70 in diabetes. Pharmacological Research. 178: 106173.","pubmedId":"","doi":"https://doi.org/10.1016/j.phrs.2022.106173"},{"reference":"Oliveira AA, Priviero F, Webb RC, Nunes KP. 2022. Increased eHSP70-to-iHSP70 ratio disrupts vascular responses to calcium and activates the TLR4-MD2 complex in type 1 diabetes. Life Sciences. 310: 121079.","pubmedId":"","doi":"https://doi.org/10.1016/j.lfs.2022.121079"},{"reference":"Sader MA, Celermajer DS. 2002. Endothelial function, vascular reactivity and gender differences in the cardiovascular system. Cardiovascular research. 53: 597.","pubmedId":"","doi":""},{"reference":"Seibert P, Anklam CFV, Costa-Beber LlCa, Sulzbacher LM, Sulzbacher MM, Sangiovo AMB, et al., Ludwig. 2022. Increased eHSP70-to-iHSP70 ratio in prediabetic and diabetic postmenopausal women: a biomarker of cardiometabolic risk. Cell Stress and Chaperones 27: 523-534.
","pubmedId":"35767179","doi":"10.1007/s12192-022-01288-8"},{"reference":"Senf SM, Howard TM, Ahn B, Ferreira LF, Judge AR. 2013. Loss of the inducible Hsp70 delays the inflammatory response to skeletal muscle injury and severely impairs muscle regeneration. PloS one. 8: e62687.","pubmedId":"23626847","doi":"10.1371/journal.pone.0062687"},{"reference":"Steinberg HO, Paradisi G, Cronin J, Crowde K, Hempfling A, Hook G, Baron AD. 2000. Type II Diabetes Abrogates Sex Differences in Endothelial Function in Premenopausal Women. Circulation. 101: 2040.","pubmedId":"","doi":"10.1161/01.CIR.101.17.2040"},{"reference":"Voss MR, Stallone JN, Li M, Cornelussen RNM, Knuefermann P, Knowlton AA. 2003. Gender differences in the expression of heat shock proteins: the effect of estrogen. American Journal of Physiology-Heart and Circulatory Physiology. 285: H687.","pubmedId":"","doi":"10.1152/ajpheart.01000.2002"},{"reference":"Webb RC. 2003. SMOOTH MUSCLE CONTRACTION AND RELAXATION. Advances in Physiology Education. 27: 201.","pubmedId":"","doi":"10.1152/advan.00025.2003"},{"reference":"White RE. 2002. Estrogen and vascular function. Vascular Pharmacology. 38: 73.","pubmedId":"","doi":"10.1016/S0306-3623(02)00129-5"}],"title":"Heat Shock Protein 70 (HSP70) has emerged as a modulator of vascular behavior, and its physiological role is sex- and age-dependent. In diabetic conditions, pharmacological blockade of HSP70 improves the vasculature in males. Yet, in diabetic females, the HSP70 profile and its role in vascular hypercontractility, a hallmark of type 2 diabetes (T2D), remain unknown. We showed that inhibiting aortic HSP70 in T2D females ameliorates vascular dysfunction, while the eHSP70-to-iHSP70 ratio is preserved, suggesting sex-specific differences during T2D.
","acknowledgements":"We would like to acknowledge Dr. Samuel Mozen for his help with handling the animals and Ahmed Araara for technical support.
","authors":[{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","formalAnalysis","methodology","dataCuration","writing_originalDraft","investigation"],"email":"vochoamendoz2019@my.fit.edu","firstName":"Valentina ","lastName":"Ochoa Mendoza","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0003-1125-8499"},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","formalAnalysis","writing_reviewEditing"],"email":"srastogi2022@my.fit.edu","firstName":"Swasti","lastName":"Rastogi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["methodology","writing_reviewEditing"],"email":"cbiby2024@my.fit.edu","firstName":"Chloe","lastName":"Biby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","methodology","writing_reviewEditing"],"email":"jliaw2025@my.fit.edu","firstName":"Jessica ","lastName":"Liaw","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","fundingAcquisition","project","supervision","writing_reviewEditing"],"email":"knunes@fit.edu","firstName":"Kenia Pedrosa","lastName":" Nunes","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0002-4838-7617"}],"awards":[{"awardId":"1R15DK131511-01A1","funderName":"National Institute of Diabetes and Digestive and Kidney Diseases (United States)","awardRecipient":"Kenia Pedrosa Nunes"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Embed equation in Methods section>Time forces curves>to a bi-exponential function using least-squares regression
","doi":null,"resourceType":"Text","name":"Equation.mathml.docx","url":"https://portal.micropublication.org/uploads/daa047aee154c3bbd364f27ff76b3377.docx"}],"funding":"This research was funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), grant number 1R15DK131511-01A1, PI: Nunes, Kenia Pedrosa
","image":{"url":"https://portal.micropublication.org/uploads/a3d54d788ee6439ed7301b3d5cf8ba29.png"},"imageCaption":"(A) Cumulative PE concentration-response curves in db/+ vehicle (grey filled circles), db/+ VER (lilac filled circles), db/db vehicle (grey unfilled circles), and db/db VER (lilac unfilled circles) aortas. (B) AUC quantification. (C) Maximal contractile response (Emax). (D) Representative time–force traces in response to a single PE challenge in db/+ (continuous lines) and db/db (dashed lines) vessels treated with vehicle (grey) or VER (lilac). (E) Emax of the phasic phase and (F) tonic phase in db/+ (filled symbols) and db/db (unfilled symbols) mice. Data in A–F are mean ± SEM; two-way ANOVA with Holm–Sidak’s post hoc test; *p ≤ 0.001, **p ≤ 0.0001; n = 6–7. (G) Representative immunoblots of aortic iHSP70 normalized to β-actin. (H) Serum eHSP70 levels in db/+ (filled circles) and db/db (unfilled circles) mice. (I) iHSP70/β-actin and (J) eHSP70 quantification (a.u.). (K) eHSP70-to-iHSP70 ratio and (L) H index. Data in G–L are mean ± SEM; n = 4–5 mice per group. *p ≤ 0.05, **p ≤ 0.001 vs. db/+, unpaired Student’s t-test or Mann–Whitney U-test as appropriate.
","imageTitle":"Female db/db aorta shows hypercontractility to Phenylephrine (PE) reversed by HSP70 inhibition, which impacts the tonic phase of contraction mediated by Ca2+ influx, while the HSP70 ratio is preserved
","methods":"Ex vivo assessment of thoracic aortic function by wire myograph
Thoracic aortic rings (2 mm in length) from db/db and db/+ female mice were mounted in a DMT620M multi-wire myograph system (Danish Myo Technology, Copenhagen, Denmark) under isometric conditions and set to an optimal resting tension of approximately 5 mN in PSS, continuously aerated with carbogen (95% O2, 5% CO2) at 37 °C. The protocol for the equilibration period and verification of functional integrity of each ring was assessed as previously described (Ochoa Mendoza et al., 2025).
Cumulative concentration-response curves
After equilibration and viability confirmation, the aortic rings were incubated for 30 min with vehicle (DMSO) or VER155008, a small-molecule HSP70 inhibitor (10 μmol/L in DMSO; Sigma Aldrich, St. Louis, MO, USA; catalog SML0271). The cumulative concentration-response curves for phenylephrine (PE; 1 nmol/L-100 μmol/L) were then generated, with contractile responses recorded at each concentration. Area under the curve (AUC) was calculated using the trapezoidal rule.
Time-force curves
For time-force analysis, aortic segments were incubated for 30 min with vehicle (DMSO) or VER155008 (10 µmol/L), followed by exposure to a single dose of PE (10⁻6 mol/L). Isometric force was recorded continuously for 15 min (1 s sampling rate). Time-force curves were fit to a bi-exponential function using least-squares regression:
**Embed equation here**
The phasic component was quantified as the peak force at the fast-to-slow transition (approximated as Ffast), and the tonic component as the sustained force at 15 min (approximating Fslow), both expressed relative to baseline (time 0).
Western blot analysis of HSP70
Thoracic aortas were homogenized in Tissue Protein Extraction Reagent supplemented with protease inhibitors, and protein concentration was determined by BCA assay. Equal amounts of aortic protein (10 µg) or serum (10 µL) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were briefly stained with Ponceau S to confirm transfer, washed in TBS-T, and blocked for 1 h at room temperature in 5% non-fat dry milk. They were then incubated overnight at 4 °C with an anti-HSP70 primary antibody, followed by 1 h at room temperature with an HRP-conjugated anti-rabbit secondary antibody. Signal was developed using a chemiluminescent substrate and detected with a ChemiDoc MP imaging system. Band intensity was quantified in ImageJ and normalized to β-actin for aortic samples. Serum samples were normalized to total protein load, since housekeeping proteins are not suitable for this matrix.
Statistical analysis
Statistical analyses and graphing were performed with GraphPad Prism 10.3.1 (GraphPad Software, San Diego, CA, USA). Data are presented as means ± SEM. Normality was assessed using the D’Agostino–Pearson test, and outliers were identified and excluded with Grubb’s test when appropriate. Two-group comparisons were performed using unpaired Student’s t-tests or Mann–Whitney U-tests, depending on data distribution, whereas multiple-group analyses were conducted by two-way ANOVA followed by Sidak’s post hoc test. Statistical significance was set at *p ≤ 0.05. Sample size (n) indicates the number of mice per group.
","reagents":"Female db/db mice (B6.BKS(D)-Leprdb/J) and age-matched heterozygous db/+ controls were purchased from The Jackson Laboratory (Bar Harbor, ME, USA; n = 20 total). Body weight was recorded, and non-fasting blood glucose were measured biweekly with a Metene TD-4116 glucose monitor (TaiDoc Technology Corporation, New Taipei City, Taiwan). Diabetic profile was confirmed by hyperglycemia (350 mg/dl or higher). Mice were anesthetized with 4% isoflurane in 100% O2, and blood was collected from the abdominal aorta into BD Vacutainer serum tubes. Serum was isolated, snap-frozen in liquid nitrogen, and later used for eHSP70 analysis by Western blotting. Animals were then euthanized at 16 weeks by exsanguination via cardiac puncture, and thoracic aortas were harvested for snap-freezing or vascular reactivity studies in the myograph. All procedures were approved by the Institutional Animal Care and Use Committee of the Florida Institute of Technology (protocol #2022-04) and performed under veterinary supervision in accordance with ARRIVE guidelines.
","patternDescription":"T2D-associated vascular dysfunction arises from a network of interacting mechanisms rather than a single cause, making its progression difficult to explain by a single pathway alone (Dunn & Nelson, 2010; Fernández-Velasco et al., 2014).HSP70 plays critical roles across different tissues (de Oliveira et al., 2022). It is increasingly recognized as a multifunctional protein with tissue-specific roles, including important effects in muscle biology. In skeletal and cardiac muscle, loss of inducible HSP70 disrupts Ca2+ homeostasis and weakens contractile performance (Kim et al., 2006; Senf et al., 2013), while in vascular smooth muscle, HSP70 supports receptor-driven contraction (de Oliveira & Nunes, 2020). Importantly, hyperglycemia has been associated with elevated circulating HSP70 levels (Nakhjavani et al., 2010), and it is now suggested that HSP70 is not merely a bystander but a potential mechanistic player in this setting. HSP70 is ubiquitously expressed, residing intracellularly (iHSP70) or released extracellularly in the circulation (eHSP70), and its effects in diabetes depend critically on both location and surplus (de Oliveira et al., 2022). An imbalance between eHSP70 and iHSP70 has been proposed as an inflammatory biomarker, and a disruption of this ratio is associated with vascular dysfunction in both type 1 (de Oliveira et al., 2022) and T2D (Ochoa Mendoza et al., 2025). In the aorta of male rodents, HSP70 contributes to receptor-mediated vasoconstriction by regulating Ca2+-handling mechanisms, supporting an important role for HSP70 in maintaining physiological vascular tone (de Oliveira, et al., 2021).
Recent evidence indicates that vascular HSP70 expression and function are age- (de Oliveira, et al., 2022) and sex-dependent (de Oliveira et al., 2021). Healthy female rats have lower aortic HSP70 levels than males, despite reports of higher HSP70 expression in some female tissues such as the heart (Voss et al., 2003). Under non-pathological conditions, pharmacological blockade of HSP70 with VER155008 has a greater impact on phenylephrine-induced contraction in female aortic rings than in male rings, affecting both phasic and tonic components of the response (de Oliveira, et al., 2021). These findings suggest that impaired vascular HSP70 expression in females augments their dependence on HSP70-mediated Ca²⁺ handling for the maintenance of contractile tone, providing a potential molecular axis for sex-specific differences in vascular reactivity.
Given that females are generally protected from cardiovascular disease for much of their lives but may lose this advantage in T2D, and HSP70 might be a possible therapeutic target (de Oliveira et al., 2019), we examined the contribution of HSP70 to vascular dysfunction in a T2D diabetic female (db/db) model. Our findings show that (1) the hypercontractile phenotype in the aorta of T2D females is prevented by HSP70 inhibitor VER155008, similar to results in males, (2) but it only impacts the tonic phase of contraction when comparing control (db/+) versus diabetic (db/db), and (3) the eHSP70-to-iHSP70 ratio is preserved in T2D females, contrasting data in males (Ochoa Mendoza et al., 2025).
In the aorta of female db/db mice, pharmacological inhibition of HSP70 showed a more pronounced effect when compared to control db/+ (Figure 1A-C), indicating that HSP70 contributes to the exaggerated vasoconstriction observed in T2D. Considering that HSP70 blockade reduces Ca²⁺ influx–mediated contraction and lowers free cytosolic Ca²⁺ levels in aortic tissues from T2D males (Ochoa Mendoza et al., 2025), we evaluated the biphasic contraction curve in response to a single dose of PE in the aorta of diabetic females. This biphasic behavior reflects a two‑phase rise in cytosolic Ca²⁺. The phasic phase is driven predominantly by IP₃ receptor (IP₃R) ‑mediated Ca²⁺ release from the SR, whereas the tonic phase depends on continued Ca²⁺ influx through voltage‑dependent and receptor‑operated channels, together with Ca²⁺‑sensitizing mechanisms that maintain force at relatively sustained Ca²⁺ levels (Webb, 2003). HSP70 inhibition reduced contraction in both phases of the biphasic curve (Figure 1D, 1E, and 1F) in db/+ and db/db vessels. However, a significant difference between the db/+ and db/db groups was only observed during the tonic phase (Figure 1F), suggesting Ca²⁺ influx impairment. The aorta's vascular reactivity corroborates the hypercontractile profile triggered by diabetes, which is reduced by HSP70 inhibition, thereby improving overall vascular function in T2D females.
Circulating eHSP70 is suggested as a target for the treatment of diabetic vasculopathies as it can act like a damage-associated molecular pattern (DAMP) and lead to an innate immune response via TLR4 (de Oliveira, et al., 2022), thus it is associated with low-grade inflammation, whereas increased iHSP70 is regarded as protective and anti-inflammatory (de Oliveira, Mendoza, Rastogi, et al., 2022). A disruption in the balance between eHSP70 and iHSP70 can lead to vascular dysfunction in diabetic males (Ochoa Mendoza et al., 2025). Still, the HSP70 ratio was unknown in adult females. Thus, we evaluated aortic and systemic HSP70 levels. Our data showed that both iHSP70 and eHSP70 were reduced in control db/+ versus diabetic db/db (Figure 1 G-J), which led to an undisrupted eHSP70-to-iHSP70 ratio (Krause et al., 2015) and H index (Costa-Beber et al., 2020), both used as indirect biomarkers for low-grade inflammation (Figure 1K and L, respectively), differing from male diabetic mice (Ochoa Mendoza et al., 2025).
An important mediator of sex differences in cardiovascular function across species is the nitric oxide (NO)/NO synthase (NOS) pathway. Although the molecular mechanisms underlying these sex differences remain incompletely defined, the NO/NOS pathway has been strongly implicated in both humans (Forte et al., 1998; Sader & Celermajer, 2002) and rats (Kauser & Rubanyi, 1994). In humans, premenopausal women display superior NO-dependent endothelial vasodilation compared to men, attributable to estrogen's ability to upregulate eNOS expression, enhance its phosphorylation, and boost NO bioavailability while mitigating its oxidative inactivation (Steinberg et al., 2000; White, 2002). Rodent models corroborate this pattern, with female rats exhibiting elevated basal NO production and greater NO-mediated vascular relaxation than males (Kauser & Rubanyi, 1994).
These estrogen-driven effects not only promote endothelium-dependent relaxation but also likely influence chaperone homeostasis by curbing hyperglycemia-induced inflammation and oxidative stress, which can otherwise disrupt the eHSP70-to-iHSP70 balance according to the chaperone balance hypothesis (Krause et al., 2015). The preservation of the eHSP70:iHSP70 ratio and H index in our diabetic females, may reflect a more stable, coordinated adaptation to diabetic stress. Noteworthy, a disrupted ratio has only been reported in postmenopausal T2D women, corroborating that at the adult stage, our females are still protected (Seibert et al., 2022). In T2D, where metabolic stressors typically compromise NO signaling, one could say that the preserved HSP70 equilibrium in the female db/db mice suggests a lingering protective NO/NOS axis. This may explain the exaggerated attenuation of hypercontractility by VER155008 in diabetic females (Figure A and D, lilac lines), as HSP70 inhibition could synergize with residual NO-mediated vasodilation to more effectively counteract sustained vascular tone. Interestingly, under physiological conditions, inhibition of HSP70 does not affect NO in the vasculature of males (de Oliveira & Nunes, 2020), which does not exclude the possibility that this landscape changes in females. Overall, the data presented corroborate HSP70’s involvement in vascular function, expand our understanding of HSP70's sex-specific role in pathological conditions, and highlight a worthwhile therapeutic window for targeting HSP70 to restore vascular function in women with T2D.
","references":[{"reference":"Costa-Beber LlCa, Hirsch GE, Heck TG, Ludwig MS. 2020. Chaperone duality: the role of extracellular and intracellular HSP70 as a biomarker of endothelial dysfunction in the development of atherosclerosis. Archives of Physiology and Biochemistry 128: 1016-1023.
","pubmedId":"","doi":"10.1080/13813455.2020.1745850"},{"reference":"de Oliveira AA, Faustino J, De Lima ME, Menezes R, Nunes KP. 2019. Unveiling the Interplay between the TLR4/MD2 Complex and HSP70 in the Human Cardiovascular System: A Computational Approach. International Journal of Molecular Sciences. 20
","pubmedId":"","doi":"10.3390/ijms20133121"},{"reference":"de Oliveira AA, Mendoza VO, Priviero F, Webb RC, Nunes KP. 2022. Age-Related Decline in Vascular Responses to Phenylephrine Is Associated with Reduced Levels of HSP70. Biomolecules. 12
","pubmedId":"","doi":"10.3390/biom12081125"},{"reference":"de Oliveira AA, Mendoza VO, Rastogi S, Nunes KP. 2022. New insights into the role and therapeutic potential of HSP70 in diabetes. Pharmacological Research 178: 106173.
","pubmedId":"","doi":"10.1016/j.phrs.2022.106173"},{"reference":"de Oliveira AA, Nunes KP. 2020. An additional physiological role for HSP70: Assistance of vascular reactivity. Life Sciences. 256: 117986.
","pubmedId":"","doi":"10.1016/j.lfs.2020.117986"},{"reference":"de Oliveira AA, Priviero F, Tostes RC, Webb RC, Nunes KP. 2021. Dissecting the interaction between HSP70 and vascular contraction: role of Ca^2+ handling mechanisms. Scientific Reports. 11: 1420.
","pubmedId":"","doi":"10.1038/s41598-021-80966-6"},{"reference":"de Oliveira AA, Priviero F, Webb RC, Nunes KP. 2021. Impaired HSP70 Expression in the Aorta of Female Rats: A Novel Insight Into Sex-Specific Differences in Vascular Function. Front Physiol 12: 666696.
","pubmedId":"33967836","doi":""},{"reference":"de Oliveira AA, Priviero F, Webb RC, Nunes KP. 2022. Increased eHSP70-to-iHSP70 ratio disrupts vascular responses to calcium and activates the TLR4-MD2 complex in type 1 diabetes. Life Sciences 310: 121079.
","pubmedId":"","doi":"10.1016/j.lfs.2022.121079"},{"reference":"Forte P, Kneale BJ, Milne E, Chowienczyk PJ, Johnston A, Benjamin N, Ritter JM. 1998. Evidence for a difference in nitric oxide biosynthesis between healthy women and men. Hypertension 32(4): 730-4.
","pubmedId":"9774371","doi":""},{"reference":"Sader MA, Celermajer DS. 2002. Endothelial function, vascular reactivity and gender differences in the cardiovascular system. Cardiovasc Res 53(3): 597-604.
","pubmedId":"11861030","doi":""},{"reference":"Seibert P, Anklam CFV, Costa-Beber LC, Sulzbacher LM, Sulzbacher MM, Sangiovo AMB, et al., Ludwig MS. 2022. Increased eHSP70-to-iHSP70 ratio in prediabetic and diabetic postmenopausal women: a biomarker of cardiometabolic risk. Cell Stress Chaperones 27(5): 523-534.
","pubmedId":"35767179","doi":"10.1007/s12192-022-01288-8"},{"reference":"Dunn KM, Nelson MT. 2010. Calcium and diabetic vascular dysfunction. Focus on “Elevated Ca2+ sparklet activity during acute hyperglycemia and diabetes in cerebral arterial smooth muscle cells”. American Journal of Physiology-Cell Physiology. 298: C203.","pubmedId":"","doi":"10.1152/ajpcell.00499.2009"},{"reference":"Fernandez Velasco M, Ruiz Hurtado G, Gomez AM, Rueda A. 2014. Ca2+ handling alterations and vascular dysfunction in diabetes. Calcium in diabetes. 56: 397.","pubmedId":"","doi":"10.1016/j.ceca.2014.08.007"},{"reference":"Kauser K, Rubanyi GM. 1994. Gender difference in bioassayable endothelium-derived nitric oxide from isolated rat aortae. American Journal of Physiology-Heart and Circulatory Physiology. 267: H2311.","pubmedId":"","doi":""},{"reference":"Kim YK, Suarez J, Hu Y, Mc Donough PM, Boer C, Dix DJ, Dillmann WH. 2006. Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy. Circulation. 113: 2589.","pubmedId":"","doi":"10.1161/CIRCULATIONAHA.105.598409"},{"reference":"Krause M, Heck TG, Bittencourt A, Scomazzon SP, Newsholme P, Curi R, Homem De Bittencourt PI. 2015. The chaperone balance hypothesis: the importance of the extracellular to intracellular HSP70 ratio to inflammation-driven type 2 diabetes, the effect of exercise, and the implications for clinical management. Mediators of inflammation. 2015: 249205.","pubmedId":"25814786","doi":"10.1155/2015/249205"},{"reference":"Nakhjavani M, Morteza A, Khajeali L, Esteghamati A, Khalilzadeh O, Asgarani F, Outeiro TF. 2010. Increased serum HSP70 levels are associated with the duration of diabetes. Cell stress & chaperones. 15: 959.","pubmedId":"20496051","doi":"10.1007/s12192-010-0204-z"},{"reference":"Ochoa Mendoza V, De Oliveira AA, Nunes KP. 2025. Blockade of HSP70 Improves Vascular Function in a Mouse Model of Type 2 Diabetes. Cells. 14: 424.","pubmedId":"","doi":"10.3390/cells14060424"},{"reference":"Senf SM, Howard TM, Ahn B, Ferreira LF, Judge AR. 2013. Loss of the inducible Hsp70 delays the inflammatory response to skeletal muscle injury and severely impairs muscle regeneration. PloS one. 8: e62687.","pubmedId":"23626847","doi":"10.1371/journal.pone.0062687"},{"reference":"Steinberg HO, Paradisi G, Cronin J, Crowde K, Hempfling A, Hook G, Baron AD. 2000. Type II Diabetes Abrogates Sex Differences in Endothelial Function in Premenopausal Women. Circulation. 101: 2040.","pubmedId":"","doi":"10.1161/01.CIR.101.17.2040"},{"reference":"Voss MR, Stallone JN, Li M, Cornelussen RNM, Knuefermann P, Knowlton AA. 2003. Gender differences in the expression of heat shock proteins: the effect of estrogen. American Journal of Physiology-Heart and Circulatory Physiology. 285: H687.","pubmedId":"","doi":"10.1152/ajpheart.01000.2002"},{"reference":"Webb RC. 2003. SMOOTH MUSCLE CONTRACTION AND RELAXATION. Advances in Physiology Education. 27: 201.","pubmedId":"","doi":"10.1152/advan.00025.2003"},{"reference":"White RE. 2002. Estrogen and vascular function. Vascular Pharmacology. 38: 73.","pubmedId":"","doi":"10.1016/S0306-3623(02)00129-5"}],"title":"Preserved eHSP70-to-iHSP70 Ratio Masks a Functional Role of HSP70 in Vascular Hypercontractility in Female Diabetic Mice","reviews":[{"reviewer":{"displayName":"Thiago Heck"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"277392c6-2096-4bea-9490-d09c0b680e93","decision":"accept","abstract":"Heat Shock Protein 70 (HSP70) has emerged as a modulator of vascular behavior, and its physiological role is sex- and age-dependent. In diabetic conditions, pharmacological blockade of HSP70 improves the vasculature in males. Yet, in diabetic females, the HSP70 profile and its role in vascular hypercontractility, a hallmark of type 2 diabetes (T2D), remain unknown. We showed that inhibiting aortic HSP70 in T2D females ameliorates vascular dysfunction, while the eHSP70-to-iHSP70 ratio is preserved, suggesting sex-specific differences during T2D.
","acknowledgements":"We would like to acknowledge Dr. Samuel Mozen for his help with handling the animals and Ahmed Araara for technical support.
","authors":[{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","formalAnalysis","methodology","dataCuration","writing_originalDraft","investigation"],"email":"vochoamendoz2019@my.fit.edu","firstName":"Valentina ","lastName":"Ochoa Mendoza","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0003-1125-8499"},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","formalAnalysis","writing_reviewEditing"],"email":"srastogi2022@my.fit.edu","firstName":"Swasti","lastName":"Rastogi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["methodology","writing_reviewEditing"],"email":"cbiby2024@my.fit.edu","firstName":"Chloe","lastName":"Biby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","methodology","writing_reviewEditing"],"email":"jliaw2025@my.fit.edu","firstName":"Jessica ","lastName":"Liaw","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","fundingAcquisition","project","supervision","writing_reviewEditing"],"email":"knunes@fit.edu","firstName":"Kenia Pedrosa","lastName":" Nunes","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0002-4838-7617"}],"awards":[{"awardId":"1R15DK131511-01A1","funderName":"National Institute of Diabetes and Digestive and Kidney Diseases (United States)","awardRecipient":"Kenia Pedrosa Nunes"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Embed equation in Methods section>Time forces curves>to a bi-exponential function using least-squares regression
","doi":null,"resourceType":"Text","name":"Equation.mathml.docx","url":"https://portal.micropublication.org/uploads/daa047aee154c3bbd364f27ff76b3377.docx"}],"funding":"This research was funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), grant number 1R15DK131511-01A1, PI: Nunes, Kenia Pedrosa
","image":{"url":"https://portal.micropublication.org/uploads/a3d54d788ee6439ed7301b3d5cf8ba29.png"},"imageCaption":"(A) Cumulative PE concentration-response curves in db/+ vehicle (grey filled circles), db/+ VER (lilac filled circles), db/db vehicle (grey unfilled circles), and db/db VER (lilac unfilled circles) aortas. (B) AUC quantification. (C) Maximal contractile response (Emax). (D) Representative time–force traces in response to a single PE challenge in db/+ (continuous lines) and db/db (dashed lines) vessels treated with vehicle (grey) or VER (lilac). (E) Emax of the phasic phase and (F) tonic phase in db/+ (filled symbols) and db/db (unfilled symbols) mice. Data in A–F are mean ± SEM; two-way ANOVA with Holm–Sidak’s post hoc test; *p ≤ 0.05, ****p ≤ 0.0001; n = 6–7. Representative immunoblot of (G) aortic iHSP70 normalized to β-actin and (H) serum eHSP70 levels normalized to total protein. (I) iHSP70/β-actin (a.u.) and (J) eHSP70/Total protein (a.u.). (K) eHSP70-to-iHSP70 ratio and (L) H index. Data in I–L are mean ± SEM; n = 4–5 mice per group in db/+ (filled circles) and db/db (unfilled circles). *p ≤ 0.05, ***p ≤ 0.001 vs. db/+, unpaired Student’s t-test or Mann–Whitney U-test as appropriate.
","imageTitle":"Female db/db aorta shows hypercontractility to Phenylephrine (PE) reversed by HSP70 inhibition, which impacts the tonic phase of contraction mediated by Ca2+ influx, while the HSP70 ratio is preserved
","methods":"Ex vivo assessment of thoracic aortic function by wire myograph
Thoracic aortic rings (2 mm in length) from db/db and db/+ female mice were mounted in a DMT620M multi-wire myograph system (Danish Myo Technology, Copenhagen, Denmark) under isometric conditions and set to an optimal resting tension of approximately 5 mN in PSS, continuously aerated with carbogen (95% O2, 5% CO2) at 37 °C. The protocol for the equilibration period and verification of functional integrity of each ring was assessed as previously described (Ochoa Mendoza et al., 2025).
Cumulative concentration-response curves
After equilibration and viability confirmation, the aortic rings were incubated for 30 min with vehicle (DMSO) or VER155008, a small-molecule HSP70 inhibitor (10 μmol/L in DMSO; Sigma Aldrich, St. Louis, MO, USA; catalog SML0271). The cumulative concentration-response curves for phenylephrine (PE; 1 nmol/L-100 μmol/L) were then generated, with contractile responses recorded at each concentration. Area under the curve (AUC) was calculated using the trapezoidal rule.
Time-force curves
For time-force analysis, aortic segments were incubated for 30 min with vehicle (DMSO) or VER155008 (10 µmol/L), followed by exposure to a single dose of PE (10⁻6 mol/L). Isometric force was recorded continuously for 15 min (1 s sampling rate). Time-force curves were fit to a bi-exponential function using least-squares regression:
F (t) = FFast ( 1 - e -kFast x t ) + FSlow ( 1 - e -kSlow x t )
The phasic component was quantified as the peak force at the fast-to-slow transition (approximated as Ffast), and the tonic component as the sustained force at 15 min (approximated as Fslow), both expressed relative to baseline (time 0).
Western blot analysis of HSP70
Thoracic aortas were homogenized in Tissue Protein Extraction Reagent supplemented with protease inhibitors, and protein concentration was determined by BCA assay. Equal amounts of aortic protein (10 µg) or serum (10 µL) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were briefly stained with Ponceau S to confirm transfer, washed in TBS-T, and blocked for 1 h at room temperature in 5% non-fat dry milk. They were then incubated overnight at 4 °C with a HSP70 primary antibody (Cell Signaling Technology, Danvers, MA, USA, #4872), followed by 1 h at room temperature with an HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA, #7074). Signal was developed using a chemiluminescent substrate and detected with a ChemiDoc MP imaging system. Band intensity was quantified in ImageJ and normalized to β-actin for aortic samples. Serum samples were normalized to total protein load, since housekeeping proteins are not suitable for this matrix.
Statistical analysis
Statistical analyses and graphing were performed with GraphPad Prism 10.3.1 (GraphPad Software, San Diego, CA, USA). Data are presented as means ± SEM. Normality was assessed using the D’Agostino–Pearson test, and outliers were identified and excluded with Grubb’s test when appropriate. Two-group comparisons were performed using unpaired Student’s t-tests or Mann–Whitney U-tests, depending on data distribution, whereas multiple-group analyses were conducted by two-way ANOVA followed by Sidak’s post hoc test. Statistical significance was set at *p ≤ 0.05. Sample size (n) indicates the number of mice per group.
","reagents":"Female db/db mice (B6.BKS(D)-Leprdb/J) and age-matched heterozygous db/+ controls were purchased from The Jackson Laboratory (Bar Harbor, ME, USA; n = 20 total). Body weight was recorded, and non-fasting blood glucose were measured biweekly with a Metene TD-4116 glucose monitor (TaiDoc Technology Corporation, New Taipei City, Taiwan). Diabetic profile was confirmed by hyperglycemia (350 mg/dl or higher). Mice were anesthetized with 4% isoflurane in 100% O2, and blood was collected from the abdominal aorta into BD Vacutainer serum tubes. Serum was isolated, snap-frozen in liquid nitrogen, and later used for eHSP70 analysis by Western blotting. Animals were then euthanized at 16 weeks by exsanguination via cardiac puncture, and thoracic aortas were harvested for snap-freezing or vascular reactivity studies in the myograph. All procedures were approved by the Institutional Animal Care and Use Committee of the Florida Institute of Technology (protocol #2022-04) and performed under veterinary supervision in accordance with ARRIVE guidelines.
","patternDescription":"T2D-associated vascular dysfunction arises from a network of interacting mechanisms rather than a single cause, making it difficult to explain its progression through a single pathway (Dunn and Nelson, 2010; Fernández-Velasco et al., 2014). HSP70 plays critical roles across different tissues (de Oliveira et al., 2022). It is increasingly recognized as a multifunctional protein with tissue-specific roles, including important effects in muscle biology. In skeletal and cardiac muscle, loss of inducible HSP70 disrupts Ca2+ homeostasis and weakens contractile performance (Kim et al., 2006; Senf et al., 2013), while in vascular smooth muscle, HSP70 supports receptor-driven contraction (de Oliveira and Nunes, 2020). Importantly, hyperglycemia has been associated with elevated circulating HSP70 levels (Nakhjavani et al., 2010), and HSP70 is now suggested to be not merely a bystander but a potential mechanistic player in this setting. HSP70 is ubiquitously expressed, residing intracellularly (iHSP70) or released extracellularly in the circulation (eHSP70), and its effects in diabetes depend critically on both location and ratio (de Oliveira et al., 2022). In this manuscript, HSP70 refers to the entire 13-isoform family, as our experimental design targets total HSP70 levels without distinguishing among variants. An imbalance between eHSP70 and iHSP70 has been proposed as an inflammatory biomarker, and a disruption of this ratio is associated with vascular dysfunction in both type 1 (de Oliveira et al., 2022) and T2D (Ochoa Mendoza et al., 2025). In the aorta of male rodents, HSP70 contributes to receptor-mediated vasoconstriction by regulating Ca2+-handling mechanisms, supporting an important role for HSP70 in maintaining physiological vascular tone (de Oliveira et al., 2021).
Recent evidence indicates that vascular HSP70 expression and function are age- (de Oliveira et al., 2022) and sex-dependent (de Oliveira et al., 2021). Healthy female rats have lower aortic HSP70 levels than males, despite reports of higher HSP70 expression in some female tissues such as the heart (Voss et al., 2003). Under non-pathological conditions, pharmacological blockade of HSP70 with VER155008 has a greater impact on phenylephrine-induced contraction in female aortic rings than in male rings, affecting both phasic and tonic components of the response (de Oliveira et al., 2021). VER155008 is a competitive HSP70 inhibitor that binds to the ATPase pocket of HSP70, preventing ATP hydrolysis (Williamson et al., 2009), without compromising vessel viability (De Oliveira and Nunes, 2020). These findings suggest that impaired vascular HSP70 expression in females increases their dependence on HSP70-mediated Ca²⁺ handling to maintain contractile tone, providing a potential molecular axis for sex-specific differences in vascular reactivity.
Given that females are generally protected from cardiovascular disease for much of their lives but may lose this advantage in T2D, and HSP70 might be a possible therapeutic target (de Oliveira et al., 2019), we examined the contribution of HSP70 to vascular dysfunction in a T2D diabetic female (db/db) model. Our findings show that (1) the hypercontractile phenotype in the aorta of T2D females is prevented by HSP70 inhibitor VER155008, similar to results in males, (2) but it only impacts the tonic phase of contraction when comparing control (db/+) versus diabetic (db/db), and (3) the eHSP70-to-iHSP70 ratio is preserved in T2D females, contrasting with data in males (Ochoa Mendoza et al., 2025).
In the aorta of female db/db mice, pharmacological inhibition of HSP70 showed a more pronounced effect than in control db/+ mice (Figure 1A-C), indicating that HSP70 contributes to the exaggerated vasoconstriction observed in T2D. Considering that HSP70 blockade reduces Ca²⁺ influx–mediated contraction and lowers free cytosolic Ca²⁺ levels in aortic tissues from T2D males (Ochoa Mendoza et al., 2025), we evaluated the biphasic contraction curve in response to a single dose of PE in the aorta of diabetic females. This biphasic behavior reflects a two‑phase rise in cytosolic Ca²⁺. The phasic phase is driven predominantly by IP₃ receptor (IP₃R) ‑mediated Ca²⁺ release from the SR, whereas the tonic phase depends on continued Ca²⁺ influx through voltage‑dependent and receptor‑operated channels, together with Ca²⁺‑sensitizing mechanisms that maintain force at relatively sustained Ca²⁺ levels (Webb, 2003). HSP70 inhibition reduced contraction in both phases of the biphasic curve (Figure 1D, 1E, and 1F) in db/+ and db/db vessels. However, a significant difference between the db/+ and db/db groups was observed only during the tonic phase (Figure 1F), suggesting impaired Ca²⁺ influx. The aorta's vascular reactivity corroborates the hypercontractile profile triggered by diabetes, which HSP70 inhibition reduces, thereby improving overall vascular function in T2D females.
Circulating eHSP70 is suggested as a target for the treatment of diabetic vasculopathies, as it can act like a damage-associated molecular pattern (DAMP) and lead to an innate immune response via TLR4 (de Oliveira et al., 2022), thus it is associated with low-grade inflammation, whereas increased iHSP70 is regarded as protective and anti-inflammatory (de Oliveira, et al., 2022). A disruption in the balance between eHSP70 and iHSP70 can lead to vascular dysfunction in diabetic males (Ochoa Mendoza et al., 2025). Still, the HSP70 ratio was unknown in adult females. Therefore, we evaluated aortic and systemic HSP70 levels. Importantly, a limitation here is that HSP70’s tissue levels include both cytosolic and membrane-bound HSP70. Our data showed that both iHSP70 and eHSP70 were reduced in control db/+ versus diabetic db/db (Figure 1 G-J), which led to an undisrupted eHSP70-to-iHSP70 ratio (Krause et al., 2015) and H index (Costa-Beber et al., 2020), both used as indirect biomarkers for low-grade inflammation (Figure 1K and L, respectively), differing from male diabetic mice previously reported (Ochoa Mendoza et al., 2025).
An important mediator of sex differences in cardiovascular function across species is the nitric oxide (NO)/NO synthase (NOS) pathway. Although the molecular mechanisms underlying these sex differences remain incompletely defined, the NO/NOS pathway has been strongly implicated in both humans (Forte et al., 1998; Sader and Celermajer, 2002) and rats (Kauser and Rubanyi, 1994). In humans, premenopausal women display superior NO-dependent endothelial vasodilation compared to men, attributable to estrogen's ability to upregulate eNOS expression, enhance its phosphorylation, and boost NO bioavailability while mitigating its oxidative inactivation (Steinberg et al., 2000; White, 2002). Rodent models corroborate this pattern, with female rats exhibiting elevated basal NO production and greater NO-mediated vascular relaxation than males (Kauser and Rubanyi, 1994).
These estrogen-driven effects not only promote endothelium-dependent relaxation but also likely influence chaperone homeostasis by curbing hyperglycemia-induced inflammation and oxidative stress, which can otherwise disrupt the eHSP70-to-iHSP70 balance according to the chaperone balance hypothesis (Krause et al., 2015). The preserved eHSP70:iHSP70 ratio and H index in our diabetic females may reflect a more stable, coordinated adaptation to diabetic stress. Notably, a disrupted ratio has been reported only in postmenopausal T2D women, corroborating that at the adult stage, our females are still protected (Seibert et al., 2022). In T2D, where metabolic stressors typically compromise NO signaling, one could say that the preserved HSP70 equilibrium in the female db/db mice suggests a lingering protective NO/NOS axis. This may explain the exaggerated attenuation of hypercontractility by VER155008 in diabetic females (Figure A and D, lilac lines), as HSP70 inhibition could synergize with residual NO-mediated vasodilation to more effectively counteract sustained vascular tone. Interestingly, under physiological conditions, HSP70 inhibition does not affect NO in the vasculature of males (de Oliveira and Nunes, 2020), which does not exclude the possibility that this landscape changes in females. Overall, the data presented corroborate HSP70’s involvement in vascular function, expand our understanding of HSP70's sex-specific role in pathological conditions, and highlight a worthwhile therapeutic window for targeting HSP70 to restore vascular function in women with T2D.
","references":[{"reference":"Costa-Beber LlCa, Hirsch GE, Heck TG, Ludwig MS. 2020. Chaperone duality: the role of extracellular and intracellular HSP70 as a biomarker of endothelial dysfunction in the development of atherosclerosis. Archives of Physiology and Biochemistry 128: 1016-1023.
","pubmedId":"","doi":"10.1080/13813455.2020.1745850"},{"reference":"de Oliveira AA, Faustino J, De Lima ME, Menezes R, Nunes KP. 2019. Unveiling the Interplay between the TLR4/MD2 Complex and HSP70 in the Human Cardiovascular System: A Computational Approach. International Journal of Molecular Sciences. 20
","pubmedId":"","doi":"10.3390/ijms20133121"},{"reference":"de Oliveira AA, Mendoza VO, Priviero F, Webb RC, Nunes KP. 2022. Age-Related Decline in Vascular Responses to Phenylephrine Is Associated with Reduced Levels of HSP70. Biomolecules. 12
","pubmedId":"","doi":"10.3390/biom12081125"},{"reference":"de Oliveira AA, Mendoza VO, Rastogi S, Nunes KP. 2022. New insights into the role and therapeutic potential of HSP70 in diabetes. Pharmacological Research 178: 106173.
","pubmedId":"","doi":"10.1016/j.phrs.2022.106173"},{"reference":"de Oliveira AA, Nunes KP. 2020. An additional physiological role for HSP70: Assistance of vascular reactivity. Life Sciences. 256: 117986.
","pubmedId":"","doi":"10.1016/j.lfs.2020.117986"},{"reference":"de Oliveira AA, Priviero F, Tostes RC, Webb RC, Nunes KP. 2021. Dissecting the interaction between HSP70 and vascular contraction: role of Ca^2+ handling mechanisms. Scientific Reports. 11: 1420.
","pubmedId":"","doi":"10.1038/s41598-021-80966-6"},{"reference":"de Oliveira AA, Priviero F, Webb RC, Nunes KP. 2021. Impaired HSP70 Expression in the Aorta of Female Rats: A Novel Insight Into Sex-Specific Differences in Vascular Function. Front Physiol 12: 666696.
","pubmedId":"33967836","doi":""},{"reference":"de Oliveira AA, Priviero F, Webb RC, Nunes KP. 2022. Increased eHSP70-to-iHSP70 ratio disrupts vascular responses to calcium and activates the TLR4-MD2 complex in type 1 diabetes. Life Sciences 310: 121079.
","pubmedId":"","doi":"10.1016/j.lfs.2022.121079"},{"reference":"Forte P, Kneale BJ, Milne E, Chowienczyk PJ, Johnston A, Benjamin N, Ritter JM. 1998. Evidence for a difference in nitric oxide biosynthesis between healthy women and men. Hypertension 32(4): 730-4.
","pubmedId":"9774371","doi":""},{"reference":"Sader MA, Celermajer DS. 2002. Endothelial function, vascular reactivity and gender differences in the cardiovascular system. Cardiovasc Res 53(3): 597-604.
","pubmedId":"11861030","doi":""},{"reference":"Seibert P, Anklam CFV, Costa-Beber LC, Sulzbacher LM, Sulzbacher MM, Sangiovo AMB, et al., Ludwig MS. 2022. Increased eHSP70-to-iHSP70 ratio in prediabetic and diabetic postmenopausal women: a biomarker of cardiometabolic risk. Cell Stress Chaperones 27(5): 523-534.
","pubmedId":"35767179","doi":"10.1007/s12192-022-01288-8"},{"reference":"Dunn KM, Nelson MT. 2010. Calcium and diabetic vascular dysfunction. Focus on “Elevated Ca2+ sparklet activity during acute hyperglycemia and diabetes in cerebral arterial smooth muscle cells”. American Journal of Physiology-Cell Physiology. 298: C203.","pubmedId":"","doi":"10.1152/ajpcell.00499.2009"},{"reference":"Fernandez Velasco M, Ruiz Hurtado G, Gomez AM, Rueda A. 2014. Ca2+ handling alterations and vascular dysfunction in diabetes. Calcium in diabetes. 56: 397.","pubmedId":"","doi":"10.1016/j.ceca.2014.08.007"},{"reference":"Kauser K, Rubanyi GM. 1994. Gender difference in bioassayable endothelium-derived nitric oxide from isolated rat aortae. American Journal of Physiology-Heart and Circulatory Physiology. 267: H2311.","pubmedId":"","doi":""},{"reference":"Kim YK, Suarez J, Hu Y, Mc Donough PM, Boer C, Dix DJ, Dillmann WH. 2006. Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy. Circulation. 113: 2589.","pubmedId":"","doi":"10.1161/CIRCULATIONAHA.105.598409"},{"reference":"Krause M, Heck TG, Bittencourt A, Scomazzon SP, Newsholme P, Curi R, Homem De Bittencourt PI. 2015. The chaperone balance hypothesis: the importance of the extracellular to intracellular HSP70 ratio to inflammation-driven type 2 diabetes, the effect of exercise, and the implications for clinical management. Mediators of inflammation. 2015: 249205.","pubmedId":"25814786","doi":"10.1155/2015/249205"},{"reference":"Nakhjavani M, Morteza A, Khajeali L, Esteghamati A, Khalilzadeh O, Asgarani F, Outeiro TF. 2010. Increased serum HSP70 levels are associated with the duration of diabetes. Cell stress & chaperones. 15: 959.","pubmedId":"20496051","doi":"10.1007/s12192-010-0204-z"},{"reference":"Ochoa Mendoza V, De Oliveira AA, Nunes KP. 2025. Blockade of HSP70 Improves Vascular Function in a Mouse Model of Type 2 Diabetes. Cells. 14: 424.","pubmedId":"","doi":"10.3390/cells14060424"},{"reference":"Senf SM, Howard TM, Ahn B, Ferreira LF, Judge AR. 2013. Loss of the inducible Hsp70 delays the inflammatory response to skeletal muscle injury and severely impairs muscle regeneration. PloS one. 8: e62687.","pubmedId":"23626847","doi":"10.1371/journal.pone.0062687"},{"reference":"Steinberg HO, Paradisi G, Cronin J, Crowde K, Hempfling A, Hook G, Baron AD. 2000. Type II Diabetes Abrogates Sex Differences in Endothelial Function in Premenopausal Women. Circulation. 101: 2040.","pubmedId":"","doi":"10.1161/01.CIR.101.17.2040"},{"reference":"Voss MR, Stallone JN, Li M, Cornelussen RNM, Knuefermann P, Knowlton AA. 2003. Gender differences in the expression of heat shock proteins: the effect of estrogen. American Journal of Physiology-Heart and Circulatory Physiology. 285: H687.","pubmedId":"","doi":"10.1152/ajpheart.01000.2002"},{"reference":"Webb RC. 2003. SMOOTH MUSCLE CONTRACTION AND RELAXATION. Advances in Physiology Education. 27: 201.","pubmedId":"","doi":"10.1152/advan.00025.2003"},{"reference":"White RE. 2002. Estrogen and vascular function. Vascular Pharmacology. 38: 73.","pubmedId":"","doi":"10.1016/S0306-3623(02)00129-5"},{"reference":"Williamson DS, Borgognoni J, Clay A, Daniels Z, Dokurno P, Drysdale MJ, et al., Massey. 2009. Novel Adenosine-Derived Inhibitors of 70 kDa Heat Shock Protein, Discovered Through Structure-Based Design. Journal of Medicinal Chemistry 52: 1510-1513.
","pubmedId":"19256508","doi":"10.1021/jm801627a"}],"title":"Preserved eHSP70-to-iHSP70 Ratio Masks a Functional Role of HSP70 in Vascular Hypercontractility in Female Diabetic Mice","reviews":[],"curatorReviews":[]},{"id":"61bebd06-30df-496a-b510-baf367ecd959","decision":"publish","abstract":"Heat Shock Protein 70 (HSP70) has emerged as a modulator of vascular behavior, and its physiological role is sex- and age-dependent. In diabetic conditions, pharmacological blockade of HSP70 improves the vasculature in males. Yet, in diabetic females, the HSP70 profile and its role in vascular hypercontractility, a hallmark of type 2 diabetes (T2D), remain unknown. We showed that inhibiting aortic HSP70 in T2D females ameliorates vascular dysfunction, while the eHSP70-to-iHSP70 ratio is preserved, suggesting sex-specific differences during T2D.
","acknowledgements":"We would like to acknowledge Dr. Samuel Mozen for his help with handling the animals and Ahmed Araara for technical support.
","authors":[{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","formalAnalysis","methodology","dataCuration","writing_originalDraft","investigation"],"email":"vochoamendoz2019@my.fit.edu","firstName":"Valentina ","lastName":"Ochoa Mendoza","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0003-1125-8499"},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","formalAnalysis","writing_reviewEditing"],"email":"srastogi2022@my.fit.edu","firstName":"Swasti","lastName":"Rastogi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["methodology","writing_reviewEditing"],"email":"cbiby2024@my.fit.edu","firstName":"Chloe","lastName":"Biby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","methodology","writing_reviewEditing"],"email":"jliaw2025@my.fit.edu","firstName":"Jessica ","lastName":"Liaw","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","fundingAcquisition","project","supervision","writing_reviewEditing"],"email":"knunes@fit.edu","firstName":"Kenia Pedrosa","lastName":" Nunes","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"https://orcid.org/0000-0002-4838-7617"}],"awards":[{"awardId":"1R15DK131511-01A1","funderName":"National Institute of Diabetes and Digestive and Kidney Diseases (United States)","awardRecipient":"Kenia Pedrosa Nunes"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Embed equation in Methods section>Time forces curves>to a bi-exponential function using least-squares regression
","doi":null,"resourceType":"Text","name":"Equation.mathml.docx","url":"https://portal.micropublication.org/uploads/daa047aee154c3bbd364f27ff76b3377.docx"}],"funding":"This research was funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), grant number 1R15DK131511-01A1, PI: Nunes, Kenia Pedrosa
","image":{"url":"https://portal.micropublication.org/uploads/a3d54d788ee6439ed7301b3d5cf8ba29.png"},"imageCaption":"(A) Cumulative PE concentration-response curves in db/+ vehicle (grey filled circles), db/+ VER (lilac filled circles), db/db vehicle (grey unfilled circles), and db/db VER (lilac unfilled circles) aortas. (B) AUC quantification. (C) Maximal contractile response (Emax). (D) Representative time–force traces in response to a single PE challenge in db/+ (continuous lines) and db/db (dashed lines) vessels treated with vehicle (grey) or VER (lilac). (E) Emax of the phasic phase and (F) tonic phase in db/+ (filled symbols) and db/db (unfilled symbols) mice. Data in A–F are mean ± SEM; two-way ANOVA with Holm–Sidak’s post hoc test; *p ≤ 0.05, ****p ≤ 0.0001; n = 6–7. Representative immunoblot of (G) aortic iHSP70 normalized to β-actin and (H) serum eHSP70 levels normalized to total protein. (I) iHSP70/β-actin (a.u.) and (J) eHSP70/Total protein (a.u.). (K) eHSP70-to-iHSP70 ratio and (L) H index. Data in I–L are mean ± SEM; n = 4–5 mice per group in db/+ (filled circles) and db/db (unfilled circles). *p ≤ 0.05, ***p ≤ 0.001 vs. db/+, unpaired Student’s t-test or Mann–Whitney U-test as appropriate.
","imageTitle":"Female db/db aorta shows hypercontractility to Phenylephrine (PE) reversed by HSP70 inhibition, which impacts the tonic phase of contraction mediated by Ca2+ influx, while the HSP70 ratio is preserved
","methods":"Ex vivo assessment of thoracic aortic function by wire myograph
Thoracic aortic rings (2 mm in length) from db/db and db/+ female mice were mounted in a DMT620M multi-wire myograph system (Danish Myo Technology, Copenhagen, Denmark) under isometric conditions and set to an optimal resting tension of approximately 5 mN in PSS, continuously aerated with carbogen (95% O2, 5% CO2) at 37 °C. The protocol for the equilibration period and verification of functional integrity of each ring was assessed as previously described (Ochoa Mendoza et al., 2025).
Cumulative concentration-response curves
After equilibration and viability confirmation, the aortic rings were incubated for 30 min with vehicle (DMSO) or VER155008, a small-molecule HSP70 inhibitor (10 μmol/L in DMSO; Sigma Aldrich, St. Louis, MO, USA; catalog SML0271). The cumulative concentration-response curves for phenylephrine (PE; 1 nmol/L-100 μmol/L) were then generated, with contractile responses recorded at each concentration. Area under the curve (AUC) was calculated using the trapezoidal rule.
Time-force curves
For time-force analysis, aortic segments were incubated for 30 min with vehicle (DMSO) or VER155008 (10 µmol/L), followed by exposure to a single dose of PE (10⁻6 mol/L). Isometric force was recorded continuously for 15 min (1 s sampling rate). Time-force curves were fit to a bi-exponential function using least-squares regression:
F (t) = FFast ( 1 - e -kFast x t ) + FSlow ( 1 - e -kSlow x t )
The phasic component was quantified as the peak force at the fast-to-slow transition (approximated as Ffast), and the tonic component as the sustained force at 15 min (approximated as Fslow), both expressed relative to baseline (time 0).
Western blot analysis of HSP70
Thoracic aortas were homogenized in Tissue Protein Extraction Reagent supplemented with protease inhibitors, and protein concentration was determined by BCA assay. Equal amounts of aortic protein (10 µg) or serum (10 µL) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were briefly stained with Ponceau S to confirm transfer, washed in TBS-T, and blocked for 1 h at room temperature in 5% non-fat dry milk. They were then incubated overnight at 4 °C with a HSP70 primary antibody (Cell Signaling Technology, Danvers, MA, USA, #4872), followed by 1 h at room temperature with an HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA, #7074). Signal was developed using a chemiluminescent substrate and detected with a ChemiDoc MP imaging system. Band intensity was quantified in ImageJ and normalized to β-actin for aortic samples. Serum samples were normalized to total protein load, since housekeeping proteins are not suitable for this matrix.
Statistical analysis
Statistical analyses and graphing were performed with GraphPad Prism 10.3.1 (GraphPad Software, San Diego, CA, USA). Data are presented as means ± SEM. Normality was assessed using the D’Agostino–Pearson test, and outliers were identified and excluded with Grubb’s test when appropriate. Two-group comparisons were performed using unpaired Student’s t-tests or Mann–Whitney U-tests, depending on data distribution, whereas multiple-group analyses were conducted by two-way ANOVA followed by Sidak’s post hoc test. Statistical significance was set at *p ≤ 0.05. Sample size (n) indicates the number of mice per group.
","reagents":"Female db/db mice (B6.BKS(D)-Leprdb/J) and age-matched heterozygous db/+ controls were purchased from The Jackson Laboratory (Bar Harbor, ME, USA; n = 20 total). Body weight was recorded, and non-fasting blood glucose were measured biweekly with a Metene TD-4116 glucose monitor (TaiDoc Technology Corporation, New Taipei City, Taiwan). Diabetic profile was confirmed by hyperglycemia (350 mg/dl or higher). Mice were anesthetized with 4% isoflurane in 100% O2, and blood was collected from the abdominal aorta into BD Vacutainer serum tubes. Serum was isolated, snap-frozen in liquid nitrogen, and later used for eHSP70 analysis by Western blotting. Animals were then euthanized at 16 weeks by exsanguination via cardiac puncture, and thoracic aortas were harvested for snap-freezing or vascular reactivity studies in the myograph. All procedures were approved by the Institutional Animal Care and Use Committee of the Florida Institute of Technology (protocol #2022-04) and performed under veterinary supervision in accordance with ARRIVE guidelines.
","patternDescription":"T2D-associated vascular dysfunction arises from a network of interacting mechanisms rather than a single cause, making it difficult to explain its progression through a single pathway (Dunn and Nelson, 2010; Fernández-Velasco et al., 2014). HSP70 plays critical roles across different tissues (de Oliveira et al., 2022). It is increasingly recognized as a multifunctional protein with tissue-specific roles, including important effects in muscle biology. In skeletal and cardiac muscle, loss of inducible HSP70 disrupts Ca2+ homeostasis and weakens contractile performance (Kim et al., 2006; Senf et al., 2013), while in vascular smooth muscle, HSP70 supports receptor-driven contraction (de Oliveira and Nunes, 2020). Importantly, hyperglycemia has been associated with elevated circulating HSP70 levels (Nakhjavani et al., 2010), and HSP70 is now suggested to be not merely a bystander but a potential mechanistic player in this setting. HSP70 is ubiquitously expressed, residing intracellularly (iHSP70) or released extracellularly in the circulation (eHSP70), and its effects in diabetes depend critically on both location and ratio (de Oliveira et al., 2022). In this manuscript, HSP70 refers to the entire 13-isoform family, as our experimental design targets total HSP70 levels without distinguishing among variants. An imbalance between eHSP70 and iHSP70 has been proposed as an inflammatory biomarker, and a disruption of this ratio is associated with vascular dysfunction in both type 1 (de Oliveira et al., 2022) and T2D (Ochoa Mendoza et al., 2025). In the aorta of male rodents, HSP70 contributes to receptor-mediated vasoconstriction by regulating Ca2+-handling mechanisms, supporting an important role for HSP70 in maintaining physiological vascular tone (de Oliveira et al., 2021).
Recent evidence indicates that vascular HSP70 expression and function are age- (de Oliveira et al., 2022) and sex-dependent (de Oliveira et al., 2021). Healthy female rats have lower aortic HSP70 levels than males, despite reports of higher HSP70 expression in some female tissues such as the heart (Voss et al., 2003). Under non-pathological conditions, pharmacological blockade of HSP70 with VER155008 has a greater impact on phenylephrine-induced contraction in female aortic rings than in male rings, affecting both phasic and tonic components of the response (de Oliveira et al., 2021). VER155008 is a competitive HSP70 inhibitor that binds to the ATPase pocket of HSP70, preventing ATP hydrolysis (Williamson et al., 2009), without compromising vessel viability (De Oliveira and Nunes, 2020). These findings suggest that impaired vascular HSP70 expression in females increases their dependence on HSP70-mediated Ca²⁺ handling to maintain contractile tone, providing a potential molecular axis for sex-specific differences in vascular reactivity.
Given that females are generally protected from cardiovascular disease for much of their lives but may lose this advantage in T2D, and HSP70 might be a possible therapeutic target (de Oliveira et al., 2019), we examined the contribution of HSP70 to vascular dysfunction in a T2D diabetic female (db/db) model. Our findings show that (1) the hypercontractile phenotype in the aorta of T2D females is prevented by HSP70 inhibitor VER155008, similar to results in males, (2) but it only impacts the tonic phase of contraction when comparing control (db/+) versus diabetic (db/db), and (3) the eHSP70-to-iHSP70 ratio is preserved in T2D females, contrasting with data in males (Ochoa Mendoza et al., 2025).
In the aorta of female db/db mice, pharmacological inhibition of HSP70 showed a more pronounced effect than in control db/+ mice (Figure 1A-C), indicating that HSP70 contributes to the exaggerated vasoconstriction observed in T2D. Considering that HSP70 blockade reduces Ca²⁺ influx–mediated contraction and lowers free cytosolic Ca²⁺ levels in aortic tissues from T2D males (Ochoa Mendoza et al., 2025), we evaluated the biphasic contraction curve in response to a single dose of PE in the aorta of diabetic females. This biphasic behavior reflects a two‑phase rise in cytosolic Ca²⁺. The phasic phase is driven predominantly by IP₃ receptor (IP₃R) ‑mediated Ca²⁺ release from the SR, whereas the tonic phase depends on continued Ca²⁺ influx through voltage‑dependent and receptor‑operated channels, together with Ca²⁺‑sensitizing mechanisms that maintain force at relatively sustained Ca²⁺ levels (Webb, 2003). HSP70 inhibition reduced contraction in both phases of the biphasic curve (Figure 1D, 1E, and 1F) in db/+ and db/db vessels. However, a significant difference between the db/+ and db/db groups was observed only during the tonic phase (Figure 1F), suggesting impaired Ca²⁺ influx. The aorta's vascular reactivity corroborates the hypercontractile profile triggered by diabetes, which HSP70 inhibition reduces, thereby improving overall vascular function in T2D females.
Circulating eHSP70 is suggested as a target for the treatment of diabetic vasculopathies, as it can act like a damage-associated molecular pattern (DAMP) and lead to an innate immune response via TLR4 (de Oliveira et al., 2022), thus it is associated with low-grade inflammation, whereas increased iHSP70 is regarded as protective and anti-inflammatory (de Oliveira, et al., 2022). A disruption in the balance between eHSP70 and iHSP70 can lead to vascular dysfunction in diabetic males (Ochoa Mendoza et al., 2025). Still, the HSP70 ratio was unknown in adult females. Therefore, we evaluated aortic and systemic HSP70 levels. Importantly, a limitation here is that HSP70’s tissue levels include both cytosolic and membrane-bound HSP70. Our data showed that both iHSP70 and eHSP70 were reduced in control db/+ versus diabetic db/db (Figure 1 G-J), which led to an undisrupted eHSP70-to-iHSP70 ratio (Krause et al., 2015) and H index (Costa-Beber et al., 2020), both used as indirect biomarkers for low-grade inflammation (Figure 1K and L, respectively), differing from male diabetic mice previously reported (Ochoa Mendoza et al., 2025).
An important mediator of sex differences in cardiovascular function across species is the nitric oxide (NO)/NO synthase (NOS) pathway. Although the molecular mechanisms underlying these sex differences remain incompletely defined, the NO/NOS pathway has been strongly implicated in both humans (Forte et al., 1998; Sader and Celermajer, 2002) and rats (Kauser and Rubanyi, 1994). In humans, premenopausal women display superior NO-dependent endothelial vasodilation compared to men, attributable to estrogen's ability to upregulate eNOS expression, enhance its phosphorylation, and boost NO bioavailability while mitigating its oxidative inactivation (Steinberg et al., 2000; White, 2002). Rodent models corroborate this pattern, with female rats exhibiting elevated basal NO production and greater NO-mediated vascular relaxation than males (Kauser and Rubanyi, 1994).
These estrogen-driven effects not only promote endothelium-dependent relaxation but also likely influence chaperone homeostasis by curbing hyperglycemia-induced inflammation and oxidative stress, which can otherwise disrupt the eHSP70-to-iHSP70 balance according to the chaperone balance hypothesis (Krause et al., 2015). The preserved eHSP70:iHSP70 ratio and H index in our diabetic females may reflect a more stable, coordinated adaptation to diabetic stress. Notably, a disrupted ratio has been reported only in postmenopausal T2D women, corroborating that at the adult stage, our females are still protected (Seibert et al., 2022). In T2D, where metabolic stressors typically compromise NO signaling, one could say that the preserved HSP70 equilibrium in the female db/db mice suggests a lingering protective NO/NOS axis. This may explain the exaggerated attenuation of hypercontractility by VER155008 in diabetic females (Figure A and D, lilac lines), as HSP70 inhibition could synergize with residual NO-mediated vasodilation to more effectively counteract sustained vascular tone. Interestingly, under physiological conditions, HSP70 inhibition does not affect NO in the vasculature of males (de Oliveira and Nunes, 2020), which does not exclude the possibility that this landscape changes in females. Overall, the data presented corroborate HSP70’s involvement in vascular function, expand our understanding of HSP70's sex-specific role in pathological conditions, and highlight a worthwhile therapeutic window for targeting HSP70 to restore vascular function in women with T2D.
","references":[{"reference":"Costa-Beber LlCa, Hirsch GE, Heck TG, Ludwig MS. 2020. Chaperone duality: the role of extracellular and intracellular HSP70 as a biomarker of endothelial dysfunction in the development of atherosclerosis. Archives of Physiology and Biochemistry 128: 1016-1023.
","pubmedId":"","doi":"10.1080/13813455.2020.1745850"},{"reference":"de Oliveira AA, Faustino J, De Lima ME, Menezes R, Nunes KP. 2019. Unveiling the Interplay between the TLR4/MD2 Complex and HSP70 in the Human Cardiovascular System: A Computational Approach. International Journal of Molecular Sciences. 20
","pubmedId":"","doi":"10.3390/ijms20133121"},{"reference":"de Oliveira AA, Mendoza VO, Priviero F, Webb RC, Nunes KP. 2022. Age-Related Decline in Vascular Responses to Phenylephrine Is Associated with Reduced Levels of HSP70. Biomolecules. 12
","pubmedId":"","doi":"10.3390/biom12081125"},{"reference":"de Oliveira AA, Mendoza VO, Rastogi S, Nunes KP. 2022. New insights into the role and therapeutic potential of HSP70 in diabetes. Pharmacological Research 178: 106173.
","pubmedId":"","doi":"10.1016/j.phrs.2022.106173"},{"reference":"de Oliveira AA, Nunes KP. 2020. An additional physiological role for HSP70: Assistance of vascular reactivity. Life Sciences. 256: 117986.
","pubmedId":"","doi":"10.1016/j.lfs.2020.117986"},{"reference":"de Oliveira AA, Priviero F, Tostes RC, Webb RC, Nunes KP. 2021. Dissecting the interaction between HSP70 and vascular contraction: role of Ca^2+ handling mechanisms. Scientific Reports. 11: 1420.
","pubmedId":"","doi":"10.1038/s41598-021-80966-6"},{"reference":"de Oliveira AA, Priviero F, Webb RC, Nunes KP. 2021. Impaired HSP70 Expression in the Aorta of Female Rats: A Novel Insight Into Sex-Specific Differences in Vascular Function. Front Physiol 12: 666696.
","pubmedId":"33967836","doi":""},{"reference":"de Oliveira AA, Priviero F, Webb RC, Nunes KP. 2022. Increased eHSP70-to-iHSP70 ratio disrupts vascular responses to calcium and activates the TLR4-MD2 complex in type 1 diabetes. Life Sciences 310: 121079.
","pubmedId":"","doi":"10.1016/j.lfs.2022.121079"},{"reference":"Forte P, Kneale BJ, Milne E, Chowienczyk PJ, Johnston A, Benjamin N, Ritter JM. 1998. Evidence for a difference in nitric oxide biosynthesis between healthy women and men. Hypertension 32(4): 730-4.
","pubmedId":"9774371","doi":""},{"reference":"Sader MA, Celermajer DS. 2002. Endothelial function, vascular reactivity and gender differences in the cardiovascular system. Cardiovasc Res 53(3): 597-604.
","pubmedId":"11861030","doi":""},{"reference":"Seibert P, Anklam CFV, Costa-Beber LC, Sulzbacher LM, Sulzbacher MM, Sangiovo AMB, et al., Ludwig MS. 2022. Increased eHSP70-to-iHSP70 ratio in prediabetic and diabetic postmenopausal women: a biomarker of cardiometabolic risk. Cell Stress Chaperones 27(5): 523-534.
","pubmedId":"35767179","doi":"10.1007/s12192-022-01288-8"},{"reference":"Dunn KM, Nelson MT. 2010. Calcium and diabetic vascular dysfunction. Focus on “Elevated Ca2+ sparklet activity during acute hyperglycemia and diabetes in cerebral arterial smooth muscle cells”. American Journal of Physiology-Cell Physiology. 298: C203.","pubmedId":"","doi":"10.1152/ajpcell.00499.2009"},{"reference":"Fernandez Velasco M, Ruiz Hurtado G, Gomez AM, Rueda A. 2014. Ca2+ handling alterations and vascular dysfunction in diabetes. Calcium in diabetes. 56: 397.","pubmedId":"","doi":"10.1016/j.ceca.2014.08.007"},{"reference":"Kauser K, Rubanyi GM. 1994. Gender difference in bioassayable endothelium-derived nitric oxide from isolated rat aortae. American Journal of Physiology-Heart and Circulatory Physiology. 267: H2311.","pubmedId":"","doi":""},{"reference":"Kim YK, Suarez J, Hu Y, Mc Donough PM, Boer C, Dix DJ, Dillmann WH. 2006. Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy. Circulation. 113: 2589.","pubmedId":"","doi":"10.1161/CIRCULATIONAHA.105.598409"},{"reference":"Krause M, Heck TG, Bittencourt A, Scomazzon SP, Newsholme P, Curi R, Homem De Bittencourt PI. 2015. The chaperone balance hypothesis: the importance of the extracellular to intracellular HSP70 ratio to inflammation-driven type 2 diabetes, the effect of exercise, and the implications for clinical management. Mediators of inflammation. 2015: 249205.","pubmedId":"25814786","doi":"10.1155/2015/249205"},{"reference":"Nakhjavani M, Morteza A, Khajeali L, Esteghamati A, Khalilzadeh O, Asgarani F, Outeiro TF. 2010. Increased serum HSP70 levels are associated with the duration of diabetes. Cell stress & chaperones. 15: 959.","pubmedId":"20496051","doi":"10.1007/s12192-010-0204-z"},{"reference":"Ochoa Mendoza V, De Oliveira AA, Nunes KP. 2025. Blockade of HSP70 Improves Vascular Function in a Mouse Model of Type 2 Diabetes. Cells. 14: 424.","pubmedId":"","doi":"10.3390/cells14060424"},{"reference":"Senf SM, Howard TM, Ahn B, Ferreira LF, Judge AR. 2013. Loss of the inducible Hsp70 delays the inflammatory response to skeletal muscle injury and severely impairs muscle regeneration. PloS one. 8: e62687.","pubmedId":"23626847","doi":"10.1371/journal.pone.0062687"},{"reference":"Steinberg HO, Paradisi G, Cronin J, Crowde K, Hempfling A, Hook G, Baron AD. 2000. Type II Diabetes Abrogates Sex Differences in Endothelial Function in Premenopausal Women. Circulation. 101: 2040.","pubmedId":"","doi":"10.1161/01.CIR.101.17.2040"},{"reference":"Voss MR, Stallone JN, Li M, Cornelussen RNM, Knuefermann P, Knowlton AA. 2003. Gender differences in the expression of heat shock proteins: the effect of estrogen. American Journal of Physiology-Heart and Circulatory Physiology. 285: H687.","pubmedId":"","doi":"10.1152/ajpheart.01000.2002"},{"reference":"Webb RC. 2003. SMOOTH MUSCLE CONTRACTION AND RELAXATION. Advances in Physiology Education. 27: 201.","pubmedId":"","doi":"10.1152/advan.00025.2003"},{"reference":"White RE. 2002. Estrogen and vascular function. Vascular Pharmacology. 38: 73.","pubmedId":"","doi":"10.1016/S0306-3623(02)00129-5"},{"reference":"Williamson DS, Borgognoni J, Clay A, Daniels Z, Dokurno P, Drysdale MJ, et al., Massey. 2009. Novel Adenosine-Derived Inhibitors of 70 kDa Heat Shock Protein, Discovered Through Structure-Based Design. Journal of Medicinal Chemistry 52: 1510-1513.
","pubmedId":"19256508","doi":"10.1021/jm801627a"}],"title":"Preserved eHSP70-to-iHSP70 Ratio Masks a Functional Role of HSP70 in Vascular Hypercontractility in Female Diabetic Mice","reviews":[],"curatorReviews":[]},{"id":"903c1ccf-b428-43b6-a708-db164fd2e72a","decision":"publish","abstract":"Heat Shock Protein 70 (HSP70) has emerged as a modulator of vascular behavior, and its physiological role is sex- and age-dependent. In diabetic conditions, pharmacological blockade of HSP70 improves the vasculature in males. Yet, in diabetic females, the HSP70 profile and its role in vascular hypercontractility, a hallmark of type 2 diabetes (T2D), remain unknown. We showed that inhibiting aortic HSP70 in T2D females ameliorates vascular dysfunction, while the eHSP70-to-iHSP70 ratio is preserved, suggesting sex-specific differences during T2D.
","acknowledgements":"We would like to acknowledge Dr. Samuel Mozen for his help with handling the animals and Ahmed Araara for technical support.
","authors":[{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","formalAnalysis","methodology","dataCuration","writing_originalDraft","investigation"],"email":"vochoamendoz2019@my.fit.edu","firstName":"Valentina ","lastName":"Ochoa Mendoza","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0003-1125-8499"},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","formalAnalysis","writing_reviewEditing"],"email":"srastogi2022@my.fit.edu","firstName":"Swasti","lastName":"Rastogi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["methodology","writing_reviewEditing"],"email":"cbiby2024@my.fit.edu","firstName":"Chloe","lastName":"Biby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["dataCuration","methodology","writing_reviewEditing"],"email":"jliaw2025@my.fit.edu","firstName":"Jessica ","lastName":"Liaw","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Florida Institute of Technology, Melbourne, FL, US"],"departments":["Biomedical Engineering and Science, Laboratory of Vascular Biology"],"credit":["conceptualization","fundingAcquisition","project","supervision","writing_reviewEditing"],"email":"knunes@fit.edu","firstName":"Kenia Pedrosa","lastName":" Nunes","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4838-7617"}],"awards":[{"awardId":"1R15DK131511-01A1","funderName":"National Institute of Diabetes and Digestive and Kidney Diseases (United States)","awardRecipient":"Kenia Pedrosa Nunes"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Embed equation in Methods section>Time forces curves>to a bi-exponential function using least-squares regression
","doi":null,"resourceType":"Text","name":"Equation.mathml.docx","url":"https://portal.micropublication.org/uploads/daa047aee154c3bbd364f27ff76b3377.docx"}],"funding":"This research was funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), grant number 1R15DK131511-01A1, PI: Nunes, Kenia Pedrosa
","image":{"url":"https://portal.micropublication.org/uploads/a3d54d788ee6439ed7301b3d5cf8ba29.png"},"imageCaption":"(A) Cumulative PE concentration-response curves in db/+ vehicle (grey filled circles), db/+ VER (lilac filled circles), db/db vehicle (grey unfilled circles), and db/db VER (lilac unfilled circles) aortas. (B) AUC quantification. (C) Maximal contractile response (Emax). (D) Representative time–force traces in response to a single PE challenge in db/+ (continuous lines) and db/db (dashed lines) vessels treated with vehicle (grey) or VER (lilac). (E) Emax of the phasic phase and (F) tonic phase in db/+ (filled symbols) and db/db (unfilled symbols) mice. Data in A–F are mean ± SEM; two-way ANOVA with Holm–Sidak’s post hoc test; *p ≤ 0.05, ****p ≤ 0.0001; n = 6–7. Representative immunoblot of (G) aortic iHSP70 normalized to β-actin and (H) serum eHSP70 levels normalized to total protein. (I) iHSP70/β-actin (a.u.) and (J) eHSP70/Total protein (a.u.). (K) eHSP70-to-iHSP70 ratio and (L) H index. Data in I–L are mean ± SEM; n = 4–5 mice per group in db/+ (filled circles) and db/db (unfilled circles). *p ≤ 0.05, ***p ≤ 0.001 vs. db/+, unpaired Student’s t-test or Mann–Whitney U-test as appropriate.
","imageTitle":"Female db/db aorta shows hypercontractility to Phenylephrine (PE) reversed by HSP70 inhibition, which impacts the tonic phase of contraction mediated by Ca2+ influx, while the HSP70 ratio is preserved
","methods":"Ex vivo assessment of thoracic aortic function by wire myograph
Thoracic aortic rings (2 mm in length) from db/db and db/+ female mice were mounted in a DMT620M multi-wire myograph system (Danish Myo Technology, Copenhagen, Denmark) under isometric conditions and set to an optimal resting tension of approximately 5 mN in PSS, continuously aerated with carbogen (95% O2, 5% CO2) at 37 °C. The protocol for the equilibration period and verification of functional integrity of each ring was assessed as previously described (Ochoa Mendoza et al., 2025).
Cumulative concentration-response curves
After equilibration and viability confirmation, the aortic rings were incubated for 30 min with vehicle (DMSO) or VER155008, a small-molecule HSP70 inhibitor (10 μmol/L in DMSO; Sigma Aldrich, St. Louis, MO, USA; catalog SML0271). The cumulative concentration-response curves for phenylephrine (PE; 1 nmol/L-100 μmol/L) were then generated, with contractile responses recorded at each concentration. Area under the curve (AUC) was calculated using the trapezoidal rule.
Time-force curves
For time-force analysis, aortic segments were incubated for 30 min with vehicle (DMSO) or VER155008 (10 µmol/L), followed by exposure to a single dose of PE (10⁻6 mol/L). Isometric force was recorded continuously for 15 min (1 s sampling rate). Time-force curves were fit to a bi-exponential function using least-squares regression:
F (t) = FFast ( 1 - e -kFast x t ) + FSlow ( 1 - e -kSlow x t )
The phasic component was quantified as the peak force at the fast-to-slow transition (approximated as Ffast), and the tonic component as the sustained force at 15 min (approximated as Fslow), both expressed relative to baseline (time 0).
Western blot analysis of HSP70
Thoracic aortas were homogenized in Tissue Protein Extraction Reagent supplemented with protease inhibitors, and protein concentration was determined by BCA assay. Equal amounts of aortic protein (10 µg) or serum (10 µL) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were briefly stained with Ponceau S to confirm transfer, washed in TBS-T, and blocked for 1 h at room temperature in 5% non-fat dry milk. They were then incubated overnight at 4 °C with a HSP70 primary antibody (Cell Signaling Technology, Danvers, MA, USA, #4872), followed by 1 h at room temperature with an HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA, #7074). Signal was developed using a chemiluminescent substrate and detected with a ChemiDoc MP imaging system. Band intensity was quantified in ImageJ and normalized to β-actin for aortic samples. Serum samples were normalized to total protein load, since housekeeping proteins are not suitable for this matrix.
Statistical analysis
Statistical analyses and graphing were performed with GraphPad Prism 10.3.1 (GraphPad Software, San Diego, CA, USA). Data are presented as means ± SEM. Normality was assessed using the D’Agostino–Pearson test, and outliers were identified and excluded with Grubb’s test when appropriate. Two-group comparisons were performed using unpaired Student’s t-tests or Mann–Whitney U-tests, depending on data distribution, whereas multiple-group analyses were conducted by two-way ANOVA followed by Sidak’s post hoc test. Statistical significance was set at *p ≤ 0.05. Sample size (n) indicates the number of mice per group.
","reagents":"Female db/db mice (B6.BKS(D)-Leprdb/J) and age-matched heterozygous db/+ controls were purchased from The Jackson Laboratory (Bar Harbor, ME, USA; n = 20 total). Body weight was recorded, and non-fasting blood glucose were measured biweekly with a Metene TD-4116 glucose monitor (TaiDoc Technology Corporation, New Taipei City, Taiwan). Diabetic profile was confirmed by hyperglycemia (350 mg/dl or higher). Mice were anesthetized with 4% isoflurane in 100% O2, and blood was collected from the abdominal aorta into BD Vacutainer serum tubes. Serum was isolated, snap-frozen in liquid nitrogen, and later used for eHSP70 analysis by Western blotting. Animals were then euthanized at 16 weeks by exsanguination via cardiac puncture, and thoracic aortas were harvested for snap-freezing or vascular reactivity studies in the myograph. All procedures were approved by the Institutional Animal Care and Use Committee of the Florida Institute of Technology (protocol #2022-04) and performed under veterinary supervision in accordance with ARRIVE guidelines.
","patternDescription":"T2D-associated vascular dysfunction arises from a network of interacting mechanisms rather than a single cause, making it difficult to explain its progression through a single pathway (Dunn and Nelson, 2010; Fernández-Velasco et al., 2014). HSP70 plays critical roles across different tissues (de Oliveira et al., 2022). It is increasingly recognized as a multifunctional protein with tissue-specific roles, including important effects in muscle biology. In skeletal and cardiac muscle, loss of inducible HSP70 disrupts Ca2+ homeostasis and weakens contractile performance (Kim et al., 2006; Senf et al., 2013), while in vascular smooth muscle, HSP70 supports receptor-driven contraction (de Oliveira and Nunes, 2020). Importantly, hyperglycemia has been associated with elevated circulating HSP70 levels (Nakhjavani et al., 2010), and HSP70 is now suggested to be not merely a bystander but a potential mechanistic player in this setting. HSP70 is ubiquitously expressed, residing intracellularly (iHSP70) or released extracellularly in the circulation (eHSP70), and its effects in diabetes depend critically on both location and ratio (de Oliveira et al., 2022). In this manuscript, HSP70 refers to the entire 13-isoform family, as our experimental design targets total HSP70 levels without distinguishing among variants. An imbalance between eHSP70 and iHSP70 has been proposed as an inflammatory biomarker, and a disruption of this ratio is associated with vascular dysfunction in both type 1 (de Oliveira et al., 2022) and T2D (Ochoa Mendoza et al., 2025). In the aorta of male rodents, HSP70 contributes to receptor-mediated vasoconstriction by regulating Ca2+-handling mechanisms, supporting an important role for HSP70 in maintaining physiological vascular tone (de Oliveira et al., 2021).
Recent evidence indicates that vascular HSP70 expression and function are age- (de Oliveira et al., 2022) and sex-dependent (de Oliveira et al., 2021). Healthy female rats have lower aortic HSP70 levels than males, despite reports of higher HSP70 expression in some female tissues such as the heart (Voss et al., 2003). Under non-pathological conditions, pharmacological blockade of HSP70 with VER155008 has a greater impact on phenylephrine-induced contraction in female aortic rings than in male rings, affecting both phasic and tonic components of the response (de Oliveira et al., 2021). VER155008 is a competitive HSP70 inhibitor that binds to the ATPase pocket of HSP70, preventing ATP hydrolysis (Williamson et al., 2009), without compromising vessel viability (De Oliveira and Nunes, 2020). These findings suggest that impaired vascular HSP70 expression in females increases their dependence on HSP70-mediated Ca²⁺ handling to maintain contractile tone, providing a potential molecular axis for sex-specific differences in vascular reactivity.
Given that females are generally protected from cardiovascular disease for much of their lives but may lose this advantage in T2D, and HSP70 might be a possible therapeutic target (de Oliveira et al., 2019), we examined the contribution of HSP70 to vascular dysfunction in a T2D diabetic female (db/db) model. Our findings show that (1) the hypercontractile phenotype in the aorta of T2D females is prevented by HSP70 inhibitor VER155008, similar to results in males, (2) but it only impacts the tonic phase of contraction when comparing control (db/+) versus diabetic (db/db), and (3) the eHSP70-to-iHSP70 ratio is preserved in T2D females, contrasting with data in males (Ochoa Mendoza et al., 2025).
In the aorta of female db/db mice, pharmacological inhibition of HSP70 showed a more pronounced effect than in control db/+ mice (Figure 1A-C), indicating that HSP70 contributes to the exaggerated vasoconstriction observed in T2D. Considering that HSP70 blockade reduces Ca²⁺ influx–mediated contraction and lowers free cytosolic Ca²⁺ levels in aortic tissues from T2D males (Ochoa Mendoza et al., 2025), we evaluated the biphasic contraction curve in response to a single dose of PE in the aorta of diabetic females. This biphasic behavior reflects a two‑phase rise in cytosolic Ca²⁺. The phasic phase is driven predominantly by IP₃ receptor (IP₃R) ‑mediated Ca²⁺ release from the SR, whereas the tonic phase depends on continued Ca²⁺ influx through voltage‑dependent and receptor‑operated channels, together with Ca²⁺‑sensitizing mechanisms that maintain force at relatively sustained Ca²⁺ levels (Webb, 2003). HSP70 inhibition reduced contraction in both phases of the biphasic curve (Figure 1D, 1E, and 1F) in db/+ and db/db vessels. However, a significant difference between the db/+ and db/db groups was observed only during the tonic phase (Figure 1F), suggesting impaired Ca²⁺ influx. The aorta's vascular reactivity corroborates the hypercontractile profile triggered by diabetes, which HSP70 inhibition reduces, thereby improving overall vascular function in T2D females.
Circulating eHSP70 is suggested as a target for the treatment of diabetic vasculopathies, as it can act like a damage-associated molecular pattern (DAMP) and lead to an innate immune response via TLR4 (de Oliveira et al., 2022), thus it is associated with low-grade inflammation, whereas increased iHSP70 is regarded as protective and anti-inflammatory (de Oliveira, et al., 2022). A disruption in the balance between eHSP70 and iHSP70 can lead to vascular dysfunction in diabetic males (Ochoa Mendoza et al., 2025). Still, the HSP70 ratio was unknown in adult females. Therefore, we evaluated aortic and systemic HSP70 levels. Importantly, a limitation here is that HSP70’s tissue levels include both cytosolic and membrane-bound HSP70. Our data showed that both iHSP70 and eHSP70 were reduced in control db/+ versus diabetic db/db (Figure 1 G-J), which led to an undisrupted eHSP70-to-iHSP70 ratio (Krause et al., 2015) and H index (Costa-Beber et al., 2020), both used as indirect biomarkers for low-grade inflammation (Figure 1K and L, respectively), differing from male diabetic mice previously reported (Ochoa Mendoza et al., 2025).
An important mediator of sex differences in cardiovascular function across species is the nitric oxide (NO)/NO synthase (NOS) pathway. Although the molecular mechanisms underlying these sex differences remain incompletely defined, the NO/NOS pathway has been strongly implicated in both humans (Forte et al., 1998; Sader and Celermajer, 2002) and rats (Kauser and Rubanyi, 1994). In humans, premenopausal women display superior NO-dependent endothelial vasodilation compared to men, attributable to estrogen's ability to upregulate eNOS expression, enhance its phosphorylation, and boost NO bioavailability while mitigating its oxidative inactivation (Steinberg et al., 2000; White, 2002). Rodent models corroborate this pattern, with female rats exhibiting elevated basal NO production and greater NO-mediated vascular relaxation than males (Kauser and Rubanyi, 1994).
These estrogen-driven effects not only promote endothelium-dependent relaxation but also likely influence chaperone homeostasis by curbing hyperglycemia-induced inflammation and oxidative stress, which can otherwise disrupt the eHSP70-to-iHSP70 balance according to the chaperone balance hypothesis (Krause et al., 2015). The preserved eHSP70:iHSP70 ratio and H index in our diabetic females may reflect a more stable, coordinated adaptation to diabetic stress. Notably, a disrupted ratio has been reported only in postmenopausal T2D women, corroborating that at the adult stage, our females are still protected (Seibert et al., 2022). In T2D, where metabolic stressors typically compromise NO signaling, one could say that the preserved HSP70 equilibrium in the female db/db mice suggests a lingering protective NO/NOS axis. This may explain the exaggerated attenuation of hypercontractility by VER155008 in diabetic females (Figure A and D, lilac lines), as HSP70 inhibition could synergize with residual NO-mediated vasodilation to more effectively counteract sustained vascular tone. Interestingly, under physiological conditions, HSP70 inhibition does not affect NO in the vasculature of males (de Oliveira and Nunes, 2020), which does not exclude the possibility that this landscape changes in females. Overall, the data presented corroborate HSP70’s involvement in vascular function, expand our understanding of HSP70's sex-specific role in pathological conditions, and highlight a worthwhile therapeutic window for targeting HSP70 to restore vascular function in women with T2D.
","references":[{"reference":"Costa-Beber LlCa, Hirsch GE, Heck TG, Ludwig MS. 2020. Chaperone duality: the role of extracellular and intracellular HSP70 as a biomarker of endothelial dysfunction in the development of atherosclerosis. Archives of Physiology and Biochemistry 128: 1016-1023.
","pubmedId":"","doi":"10.1080/13813455.2020.1745850"},{"reference":"de Oliveira AA, Faustino J, De Lima ME, Menezes R, Nunes KP. 2019. Unveiling the Interplay between the TLR4/MD2 Complex and HSP70 in the Human Cardiovascular System: A Computational Approach. International Journal of Molecular Sciences. 20
","pubmedId":"","doi":"10.3390/ijms20133121"},{"reference":"de Oliveira AA, Mendoza VO, Priviero F, Webb RC, Nunes KP. 2022. Age-Related Decline in Vascular Responses to Phenylephrine Is Associated with Reduced Levels of HSP70. Biomolecules. 12
","pubmedId":"","doi":"10.3390/biom12081125"},{"reference":"de Oliveira AA, Mendoza VO, Rastogi S, Nunes KP. 2022. New insights into the role and therapeutic potential of HSP70 in diabetes. Pharmacological Research 178: 106173.
","pubmedId":"","doi":"10.1016/j.phrs.2022.106173"},{"reference":"de Oliveira AA, Nunes KP. 2020. An additional physiological role for HSP70: Assistance of vascular reactivity. Life Sciences. 256: 117986.
","pubmedId":"","doi":"10.1016/j.lfs.2020.117986"},{"reference":"de Oliveira AA, Priviero F, Tostes RC, Webb RC, Nunes KP. 2021. Dissecting the interaction between HSP70 and vascular contraction: role of Ca^2+ handling mechanisms. Scientific Reports. 11: 1420.
","pubmedId":"","doi":"10.1038/s41598-021-80966-6"},{"reference":"de Oliveira AA, Priviero F, Webb RC, Nunes KP. 2021. Impaired HSP70 Expression in the Aorta of Female Rats: A Novel Insight Into Sex-Specific Differences in Vascular Function. Front Physiol 12: 666696.
","pubmedId":"33967836","doi":""},{"reference":"de Oliveira AA, Priviero F, Webb RC, Nunes KP. 2022. Increased eHSP70-to-iHSP70 ratio disrupts vascular responses to calcium and activates the TLR4-MD2 complex in type 1 diabetes. Life Sciences 310: 121079.
","pubmedId":"","doi":"10.1016/j.lfs.2022.121079"},{"reference":"Forte P, Kneale BJ, Milne E, Chowienczyk PJ, Johnston A, Benjamin N, Ritter JM. 1998. Evidence for a difference in nitric oxide biosynthesis between healthy women and men. Hypertension 32(4): 730-4.
","pubmedId":"9774371","doi":""},{"reference":"Sader MA, Celermajer DS. 2002. Endothelial function, vascular reactivity and gender differences in the cardiovascular system. Cardiovasc Res 53(3): 597-604.
","pubmedId":"11861030","doi":""},{"reference":"Seibert P, Anklam CFV, Costa-Beber LC, Sulzbacher LM, Sulzbacher MM, Sangiovo AMB, et al., Ludwig MS. 2022. Increased eHSP70-to-iHSP70 ratio in prediabetic and diabetic postmenopausal women: a biomarker of cardiometabolic risk. Cell Stress Chaperones 27(5): 523-534.
","pubmedId":"35767179","doi":"10.1007/s12192-022-01288-8"},{"reference":"Dunn KM, Nelson MT. 2010. Calcium and diabetic vascular dysfunction. Focus on “Elevated Ca2+ sparklet activity during acute hyperglycemia and diabetes in cerebral arterial smooth muscle cells”. American Journal of Physiology-Cell Physiology. 298: C203.","pubmedId":"","doi":"10.1152/ajpcell.00499.2009"},{"reference":"Fernandez Velasco M, Ruiz Hurtado G, Gomez AM, Rueda A. 2014. Ca2+ handling alterations and vascular dysfunction in diabetes. Calcium in diabetes. 56: 397.","pubmedId":"","doi":"10.1016/j.ceca.2014.08.007"},{"reference":"Kauser K, Rubanyi GM. 1994. Gender difference in bioassayable endothelium-derived nitric oxide from isolated rat aortae. American Journal of Physiology-Heart and Circulatory Physiology. 267: H2311.","pubmedId":"","doi":""},{"reference":"Kim YK, Suarez J, Hu Y, Mc Donough PM, Boer C, Dix DJ, Dillmann WH. 2006. Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy. Circulation. 113: 2589.","pubmedId":"","doi":"10.1161/CIRCULATIONAHA.105.598409"},{"reference":"Krause M, Heck TG, Bittencourt A, Scomazzon SP, Newsholme P, Curi R, Homem De Bittencourt PI. 2015. The chaperone balance hypothesis: the importance of the extracellular to intracellular HSP70 ratio to inflammation-driven type 2 diabetes, the effect of exercise, and the implications for clinical management. Mediators of inflammation. 2015: 249205.","pubmedId":"25814786","doi":"10.1155/2015/249205"},{"reference":"Nakhjavani M, Morteza A, Khajeali L, Esteghamati A, Khalilzadeh O, Asgarani F, Outeiro TF. 2010. Increased serum HSP70 levels are associated with the duration of diabetes. Cell stress & chaperones. 15: 959.","pubmedId":"20496051","doi":"10.1007/s12192-010-0204-z"},{"reference":"Ochoa Mendoza V, De Oliveira AA, Nunes KP. 2025. Blockade of HSP70 Improves Vascular Function in a Mouse Model of Type 2 Diabetes. Cells. 14: 424.","pubmedId":"","doi":"10.3390/cells14060424"},{"reference":"Senf SM, Howard TM, Ahn B, Ferreira LF, Judge AR. 2013. Loss of the inducible Hsp70 delays the inflammatory response to skeletal muscle injury and severely impairs muscle regeneration. PloS one. 8: e62687.","pubmedId":"23626847","doi":"10.1371/journal.pone.0062687"},{"reference":"Steinberg HO, Paradisi G, Cronin J, Crowde K, Hempfling A, Hook G, Baron AD. 2000. Type II Diabetes Abrogates Sex Differences in Endothelial Function in Premenopausal Women. Circulation. 101: 2040.","pubmedId":"","doi":"10.1161/01.CIR.101.17.2040"},{"reference":"Voss MR, Stallone JN, Li M, Cornelussen RNM, Knuefermann P, Knowlton AA. 2003. Gender differences in the expression of heat shock proteins: the effect of estrogen. American Journal of Physiology-Heart and Circulatory Physiology. 285: H687.","pubmedId":"","doi":"10.1152/ajpheart.01000.2002"},{"reference":"Webb RC. 2003. SMOOTH MUSCLE CONTRACTION AND RELAXATION. Advances in Physiology Education. 27: 201.","pubmedId":"","doi":"10.1152/advan.00025.2003"},{"reference":"White RE. 2002. Estrogen and vascular function. Vascular Pharmacology. 38: 73.","pubmedId":"","doi":"10.1016/S0306-3623(02)00129-5"},{"reference":"Williamson DS, Borgognoni J, Clay A, Daniels Z, Dokurno P, Drysdale MJ, et al., Massey. 2009. Novel Adenosine-Derived Inhibitors of 70 kDa Heat Shock Protein, Discovered Through Structure-Based Design. Journal of Medicinal Chemistry 52: 1510-1513.
","pubmedId":"19256508","doi":"10.1021/jm801627a"}],"title":"Preserved eHSP70-to-iHSP70 Ratio Masks a Functional Role of HSP70 in Vascular Hypercontractility in Female Diabetic Mice","reviews":[],"curatorReviews":[]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon 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