Kenia Pedrosa Nunes

The cornea exhibits significant swelling behavior during mechanical characterization in a liquid environment. This technical brief quantified the impact of swelling on nanomechanical characterization during an atomic force microscopy (AFM) test. Corneal sections from the African green monkey were tested in this study. Results showed that lamellar stiffness in swollen corneal section was slightly lower than that in controls, particularly in the anterior and posterior regions (172.4 ± 107.8 kPa and 107.6 ± 69.3 kPa versus 221.7 ± 62.5 kPa and 124.3 ± 42.2 kPa, respectively), while the middle region remained nearly unchanged (105.9 ± 30.1 kPa versus 116.2 ± 44.3 kPa); however, these differences were not statistically significant (p > 0.05). These results indicate that the increased interlamellar spacing leads to a larger thickness, which likely contributes to reduced bulk stiffness measured by uniaxial testing. During AFM testing in liquid, adhesion between the tissue section and the glass slide mechanically constrained free swelling. In addition, 14 days of frozen storage at −20 °C significantly increased lamellar stiffness in both control and swollen corneal sections. After storage, control corneal section exhibited stiffness values of 307.7 ± 103.4 kPa, 161.4 ± 30.6 kPa, and 171.9 ± 52.0 kPa at the anterior, middle, and posterior regions, respectively, and 268.8 ± 154.0 kPa, 163.1 ± 101.5 kPa, and 163.2 ± 72.0 kPa in the swollen corneal section. These results indicate that immediate testing following cryosectioning is necessary to better preserve the nanomechanical integrity of the tissue.

Diabetes and hypertension are closely interlinked and among the most prevalent diseases that significantly increase cardiovascular risks. They can be caused by a wide range of factors, which often affect overlapping molecular pathways, resulting in vascular dysfunction. Hyperglycemia in diabetes and increased angiotensin II levels in hypertension act similarly to promote vascular dysfunction by triggering pathways leading to oxidative stress, low-grade inflammation, and vessel remodeling. In this context, Heat Shock Protein 70 (HSP70), a ubiquitous molecular chaperone that exists locally (iHSP70) and systemically (eHSP70) and has opposing biological effects, is emerging as a new component associated with vascular dysfunction in chronic conditions. While elevated levels of eHSP70 in diabetes and hypertension have been associated with low-grade inflammation, iHSP70 appears to regulate intracellular calcium handling—a hallmark mechanism disrupted in both pathologies. This review focuses on the role of HSP70 as an evolving component of the intricate mechanisms underlying vascular dysfunction in these two pervasive diseases, which are very likely to coexist and represent a growing burden worldwide, while addressing whether HSP70 actively contributes to the vascular dysfunction or reflects an adaptive response to disease-associated stress across tissues and experimental models. Finally, we discuss emerging evidence on the pharmacological modulation of HSP70 and the challenges remaining in defining its role as a biomarker and therapeutic target.

Type 2 diabetes (T2D) is a chronic metabolic disorder leading to increased cardiovascular risk and vascular dysfunction. Hyperglycemia, a hallmark of T2D, drives hypercontractility, thereby compromising vascular function. Heat shock protein 70 (HSP70) has emerged as an important player in vascular reactivity under physiological conditions via its interaction with calcium mobilization, and in T2D, blocking this protein prevents hypercontractility. Circulating extracellular HSP70 (eHSP70) has also been proposed as a biomarker in chronic diseases, as it can function as a damage-associated molecular pattern (DAMP) to activate the innate immune system and promote low-grade inflammation. Berberine (BBR), a natural alkaloid with anti-inflammatory properties, has been shown to attenuate vascular contraction by modulating intracellular calcium handling. Yet the link between HSP70 and BBR in modulating vascular contraction in T2D remains unknown. Therefore, we investigated whether acute and/or chronic BBR treatment modulates HSP70 to prevent vascular hypercontractility in the T2D mouse model. For acute ex vivo treatment, db/+ and db/db aortic rings were incubated for 30 min with or without the HSP70 inhibitor VER155008, in the presence or absence of BBR or vehicle. For chronic in vivo treatment, db/+ and db/db mice received intraperitoneal BBR injections (10 mg/kg, 3 times per week) and BBR in their drinking water (0.5 mg/mL) for 28 days. Following chronic (4 weeks, in vivo) or acute ex vivo (30 min) BBR treatment, vascular function was assessed in aortic rings isolated from male T2D (db/db) and age-matched non-diabetic (db/+) mice using wire myography. Rings were incubated with or without the HSP70 inhibitor VER155008, in the presence or absence of BBR or vehicle. Overt hyperglycemia and hypercontractility were observed in diabetic animals compared with non-diabetic controls. While acute BBR treatment attenuated vasoconstriction in both diabetic and nondiabetic groups, the combination of BBR and VER155008 produced a stronger inhibitory effect only in the diabetic group. Chronic BBR treatment prevented aortic hypercontractility in diabetic mice; however, the synergistic effect with VER155008 was no longer observed. Additionally, BBR reduced systemic HSP70 levels. Collectively, these findings indicate that BBR improves vascular smooth muscle cells’ function in T2D, at least in part, through HSP70-dependent mechanisms during chronic treatment.

Background: Individuals with type 1 diabetes (T1D) exhibit elevated arterial stiffness and are at a higher risk of developing cardiovascular disease, both of which are sex dependent. However, whether sex differentially affects aortic structure at macro-, micro-, and nano-levels, which represent evaluations of the aorta at multiple scales, remains poorly understood. Therefore, we investigated sex-based differences in arterial stiffness by assessing pulse wave velocity (PWV), extracellular matrix remodeling, and atomic force microscopy (AFM) based medial-layer biomechanics in the aorta of a rat model of T1D. Methods: Male and female Sprague Dawley rats were injected intraperitoneally with streptozotocin (65 mg/kg) to induce T1D. After 4 weeks, arterial stiffness and vascular alterations were evaluated in aortas of both sexes across multiple levels. At the macro-level, in vivo arterial stiffness was measured using ultrasound-based PWV. At the micro-level, structural remodeling was evaluated by quantifying collagen and elastin content in the aortic extracellular matrix. At the nano-level, biomechanical properties were assessed using AFM to determine Young’s modulus in the aortic tissue. Results: T1D increased in vivo PWV in both sexes, with significantly higher PWV in diabetic males compared to diabetic females. Structural analysis revealed that diabetic males exhibited higher collagen deposition than diabetic females, whereas the Young’s modulus increased with diabetes but showed no differences associated with sex. Discussion: These findings suggest that sex differences in T1D-related arterial stiffness at the macro level are primarily associated with extracellular matrix remodeling rather than nanoscale vascular smooth muscle cell stiffness at this disease stage.

Vascular Smooth Muscle Cells (VSMC) dysfunction is a major contributor to Type 1 diabetes (T1D)-associated vascular complications. Ca²⁺ is a key messenger responsible for maintaining VSMC tone and function, and alterations in its cytosolic levels are central to diabetes-related vasculopathy. Heat Shock Protein 70 (HSP70), a multifaceted chaperone present intracellularly (iHSP70), regulates vascular reactivity by supporting Ca²⁺ handling, and extracellularly (eHSP70) activates immune signaling. Disruption of eHSP70/iHSP70 balance has been implicated in T1D-associated VSMC dysfunction. Curcumin, a phytochemical found in turmeric, is an emerging therapeutic adjuvant for treating a wide range of pathologies, including diabetes. However, whether curcumin modulates Ca²⁺ dynamics and HSP70 expression, thereby improving VSMC function, in diabetic aorta remains unclear. To investigate this, Streptozotocin-induced diabetic rats (i.p. 65 mg/kg) were treated with curcumin (300 mg/kg) for 28 days. Vascular function was evaluated using wire myography to assess changes in biphasic contraction curve and Ca²⁺ dynamics, while HSP70 was quantified using Western blotting and ELISA. Structural alterations were analyzed by assessing collagen and elastin using Picrosirius staining and fluorescence microscopy. Chronic curcumin treatment improved vascular function by normalizing Ca²⁺ mishandling, restoring the eHSP70/iHSP70 ratio, reducing hypercontractility, and mitigating arterial structural alterations. These findings indicate that curcumin could potentially ameliorate diabetes-related VSMC dysfunction by restoring Ca²⁺ homeostasis and modulating HSP70.

Hypertension, a leading factor for cardiovascular diseases (CVD), is a particularly heavy burden in women during middle age, when cardioprotective hormones begin to decline. The abnormal handling of calcium (Ca²⁺) in vascular smooth muscle cells (VSMCs) leads to increased vasoconstriction, remodeling, and altered arterial compliance during hypertension. The Spontaneously Hypertensive Rats (SHR) is a model of essential hypertension, and middle-aged females with hypertension represent a stage of disease where vascular dysfunction is prominent but understudied. Losartan, a widely prescribed angiotensin II (AngII) receptor (AT1R) blocker, exerts antihypertensive effects by affecting Ang II/Ca²⁺ signaling. However, whether it corrects the Ca²⁺ mishandling in the aorta of middle-aged female SHR has not been established. In this study, the thoracic aorta from 36-week-old female SHRs treated with losartan was assessed for Ca²⁺ mishandling using myography and biochemical assays. Meanwhile, biomechanical properties and stiffness were evaluated using Pulse Wave Velocity (PWV), Atomic Force Microscopy (AFM), and assessments of collagen and elastin contents. Compared with normotensive controls, SHR demonstrated disrupted Ca²⁺ handling, increased stiffness, and Extracellular Matrix (ECM) remodeling in middle-aged females. Treatment with losartan abrogated Ca²⁺ mishandling influx and efflux in the VSMC, decreased stiffness, and restored the aortic structural changes. These findings demonstrate that losartan abolishes Ca²⁺ mishandling and highlight a mechanistic role of AT1R blockade in restoring vascular function in the aorta of middle-aged females during hypertension.

Type 2 diabetes (T2D) is a chronic disease that damages blood vessels and increases the risk of cardiovascular disease (CVD). Heat-shock protein 70 (HSP70), a family of chaperone proteins, has been recently reported as a key player in vascular reactivity that affects large blood vessels like the aorta. Hyperglycemia, a hallmark of diabetes, correlates with the severity of vascular damage and circulating HSP70 levels. In diabetes, blood vessels often show impaired contractility, contributing to vascular dysfunction. However, HSP70’s specific role in T2D-related vascular contraction remains unclear. We hypothesized that blocking HSP70 would improve vascular function in a widely used diabetic mouse model (db/db). To test this, we measured both vascular intracellular and serum circulating HSP70 levels in control and diabetic male mice using immunofluorescence and Western blotting. We also examined the aorta’s contractile response using a wire myograph system, which measured the force produced in response to phenylephrine (PE), both with and without VER155008, a pharmacological inhibitor that targets the ATPase domain of HSP70, and after removing extracellular calcium. Our findings show that intracellular HSP70 (iHSP70) levels were similar in control and diabetic groups, while circulating HSP70 (eHSP70) levels were higher in the serum of diabetic mice, altering the iHSP70/eHSP70 ratio. Even though VER155008 attenuated both phases of the contractile curve in the diabetic and control groups, enhanced vasoconstriction to PE was only observed in the tonic phase of the curve in the db/db group, which was prevented by iHSP70 inhibition. This effect involved calcium mobilization, as both the maximal and total contraction forces to PE were restored in groups treated with VER155008. Additionally, internal calcium levels in aortic rings treated with VER155008 decreased, as observed in force generation upon calcium reintroduction, which was further corroborated using a biochemical calcium assay. In conclusion, our study demonstrates that blocking HSP70 improves vascular reactivity in the hyperglycemic state of T2D by restoring proper vascular contraction.