RESUMO
BACKGROUND: Plasma concentration of PAI-1 (plasminogen activator inhibitor-1) correlates with arterial stiffness. Vascular smooth muscle cells (SMCs) express PAI-1, and the intrinsic stiffness of SMCs is a major determinant of total arterial stiffness. We hypothesized that PAI-1 promotes SMC stiffness by regulating the cytoskeleton and that pharmacological inhibition of PAI-1 decreases SMC and aortic stiffness. METHODS: PAI-039, a specific inhibitor of PAI-1, and small interfering RNA were used to inhibit PAI-1 expression in cultured human SMCs. Effects of PAI-1 inhibition on SMC stiffness, F-actin (filamentous actin) content, and cytoskeleton-modulating enzymes were assessed. WT (wild-type) and PAI-1-deficient murine SMCs were used to determine PAI-039 specificity. RNA sequencing was performed to determine the effects of PAI-039 on SMC gene expression. In vivo effects of PAI-039 were assessed by aortic pulse wave velocity. RESULTS: PAI-039 significantly reduced intrinsic stiffness of human SMCs, which was accompanied by a significant decrease in cytoplasmic F-actin content. PAI-1 gene knockdown also decreased cytoplasmic F-actin. PAI-1 inhibition significantly increased the activity of cofilin, an F-actin depolymerase, in WT murine SMCs, but not in PAI-1-deficient SMCs. RNA-sequencing analysis suggested that PAI-039 upregulates AMPK (AMP-activated protein kinase) signaling in SMCs, which was confirmed by Western blotting. Inhibition of AMPK prevented activation of cofilin by PAI-039. In mice, PAI-039 significantly decreased aortic stiffness and tunica media F-actin content without altering the elastin or collagen content. CONCLUSIONS: PAI-039 decreases intrinsic SMC stiffness and cytoplasmic stress fiber content. These effects are mediated by AMPK-dependent activation of cofilin. PAI-039 also decreases aortic stiffness in vivo. These findings suggest that PAI-1 is an important regulator of the SMC cytoskeleton and that pharmacological inhibition of PAI-1 has the potential to prevent and treat cardiovascular diseases involving arterial stiffening.
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Neuraminidases cleave sialic acids from glycocalyx structures and plasma neuraminidase activity is elevated in type 2 diabetes (T2D). Therefore, we hypothesize circulating neuraminidase degrades the endothelial glycocalyx and diminishes flow-mediated dilation (FMD), whereas its inhibition restores shear mechanosensation and endothelial function in T2D settings. We found that compared with controls, subjects with T2D have higher plasma neuraminidase activity, reduced plasma nitrite concentrations, and diminished FMD. Ex vivo and in vivo neuraminidase exposure diminished FMD and reduced endothelial glycocalyx presence in mouse arteries. In cultured endothelial cells, neuraminidase reduced glycocalyx coverage. Inhalation of the neuraminidase inhibitor, zanamivir, reduced plasma neuraminidase activity, enhanced endothelial glycocalyx length, and improved FMD in diabetic mice. In humans, a single-arm trial (NCT04867707) of zanamivir inhalation did not reduce plasma neuraminidase activity, improved glycocalyx length, or enhanced FMD. Although zanamivir plasma concentrations in mice reached 225.8 ± 22.0 ng/mL, in humans were only 40.0 ± 7.2 ng/mL. These results highlight the potential of neuraminidase inhibition for ameliorating endothelial dysfunction in T2D and suggest the current Food and Drug Administration-approved inhaled dosage of zanamivir is insufficient to achieve desired outcomes in humans.NEW & NOTEWORTHY This work identifies neuraminidase as a key mediator of endothelial dysfunction in type 2 diabetes that may serve as a biomarker for impaired endothelial function and predictive of development and progression of cardiovascular pathologies associated with type 2 diabetes (T2D). Data show that intervention with the neuraminidase inhibitor zanamivir at effective plasma concentrations may represent a novel pharmacological strategy for restoring the glycocalyx and ameliorating endothelial dysfunction.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Doenças Vasculares , Camundongos , Humanos , Animais , Zanamivir/farmacologia , Neuraminidase/química , Neuraminidase/farmacologia , Células Endoteliais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Antivirais/farmacologia , Inibidores Enzimáticos/farmacologiaRESUMO
Consumption of diets high in fat, sugar, and salt (Western diet, WD) is associated with accelerated arterial stiffening, a major independent risk factor for cardiovascular disease (CVD). Women with obesity are more prone to develop arterial stiffening leading to more frequent and severe CVD compared with men. As tissue transglutaminase (TG2) has been implicated in vascular stiffening, our goal herein was to determine the efficacy of cystamine, a nonspecific TG2 inhibitor, at reducing vascular stiffness in female mice chronically fed a WD. Three experimental groups of female mice were created. One was fed regular chow diet (CD) for 43 wk starting at 4 wk of age. The second was fed a WD for the same 43 wk, whereas a third cohort was fed WD, but also received cystamine (216 mg/kg/day) in the drinking water during the last 8 wk on the diet (WD + C). All vascular stiffness parameters assessed, including aortic pulse wave velocity and the incremental modulus of elasticity of isolated femoral and mesenteric arteries, were significantly increased in WD- versus CD-fed mice, and reduced in WD + C versus WD-fed mice. These changes coincided with respectively augmented and diminished vascular wall collagen and F-actin content, with no associated effect in blood pressure. In cultured human vascular smooth muscle cells, cystamine reduced TG2 activity, F-actin:G-actin ratio, collagen compaction capacity, and cellular stiffness. We conclude that cystamine treatment represents an effective approach to reduce vascular stiffness in female mice in the setting of WD consumption, likely because of its TG2 inhibitory capacity.NEW & NOTEWORTHY This study evaluates the novel role of transglutaminase 2 (TG2) inhibition to directly treat vascular stiffness. Our data demonstrate that cystamine, a nonspecific TG2 inhibitor, improves vascular stiffness induced by a diet rich in fat, fructose, and salt. This research suggests that TG2 inhibition might bear therapeutic potential to reduce the disproportionate burden of cardiovascular disease in females in conditions of chronic overnutrition.
Assuntos
Cistamina/farmacologia , Dieta Ocidental/efeitos adversos , Inibidores Enzimáticos/farmacologia , Proteína 2 Glutamina gama-Glutamiltransferase/antagonistas & inibidores , Rigidez Vascular/efeitos dos fármacos , Actinas/metabolismo , Animais , Aorta/metabolismo , Aorta/fisiologia , Células Cultivadas , Colágeno/metabolismo , Elasticidade , Feminino , Humanos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Análise de Onda de PulsoRESUMO
Adropin is a peptide largely secreted by the liver and known to regulate energy homeostasis; however, it also exerts cardiovascular effects. Herein, we tested the hypothesis that low circulating levels of adropin in obesity and type 2 diabetes (T2D) contribute to arterial stiffening. In support of this hypothesis, we report that obesity and T2D are associated with reduced levels of adropin (in liver and plasma) and increased arterial stiffness in mice and humans. Establishing causation, we show that mesenteric arteries from adropin knockout mice are also stiffer, relative to arteries from wild-type counterparts, thus recapitulating the stiffening phenotype observed in T2D db/db mice. Given the above, we performed a set of follow-up experiments, in which we found that 1) exposure of endothelial cells or isolated mesenteric arteries from db/db mice to adropin reduces filamentous actin (F-actin) stress fibers and stiffness, 2) adropin-induced reduction of F-actin and stiffness in endothelial cells and db/db mesenteric arteries is abrogated by inhibition of nitric oxide (NO) synthase, and 3) stimulation of smooth muscle cells or db/db mesenteric arteries with a NO mimetic reduces stiffness. Lastly, we demonstrated that in vivo treatment of db/db mice with adropin for 4 wk reduces stiffness in mesenteric arteries. Collectively, these findings indicate that adropin can regulate arterial stiffness, likely via endothelium-derived NO, and thus support the notion that "hypoadropinemia" should be considered as a putative target for the prevention and treatment of arterial stiffening in obesity and T2D.NEW & NOTEWORTHY Arterial stiffening, a characteristic feature of obesity and type 2 diabetes (T2D), contributes to the development and progression of cardiovascular diseases. Herein we establish that adropin is decreased in obese and T2D models and furthermore provide evidence that reduced adropin may directly contribute to arterial stiffening. Collectively, findings from this work support the notion that "hypoadropinemia" should be considered as a putative target for the prevention and treatment of arterial stiffening in obesity and T2D.
Assuntos
Diabetes Mellitus Tipo 2 , Rigidez Vascular , Actinas , Animais , Células Endoteliais , Humanos , Artérias Mesentéricas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico , Óxido Nítrico Sintase , Obesidade/complicações , Peptídeos/farmacologia , Rigidez Vascular/fisiologiaRESUMO
Arterial stiffening, a characteristic feature of obesity and type 2 diabetes, contributes to the development and progression of cardiovascular diseases (CVD). Currently, no effective prophylaxis or therapeutics is available to prevent or treat arterial stiffening. A better understanding of the molecular mechanisms underlying arterial stiffening is vital to identify newer targets and strategies to reduce CVD burden. A major contributor to arterial stiffening is increased collagen deposition. In the 5'-untranslated regions of mRNAs encoding for type I collagen, an evolutionally conserved stem-loop (SL) structure plays an essential role in its stability and post-transcriptional regulation. Here, we show that feeding a high-fat/high-sucrose (HFHS) diet for 28 wk increases adiposity, insulin resistance, and blood pressure in male wild-type littermates. Moreover, arterial stiffness, assessed in vivo via aortic pulse wave velocity, and ex vivo using atomic force microscopy in aortic explants or pressure myography in isolated femoral and mesenteric arteries, was also increased in those mice. Notably, all these indices of arterial stiffness, along with collagen type I levels in the vasculature, were reduced in HFHS-fed mice harboring a mutation in the 5'SL structure, relative to wild-type littermates. This protective vascular phenotype in 5'SL-mutant mice did not associate with a reduction in insulin resistance or blood pressure. These findings implicate the 5'SL structure as a putative therapeutic target to prevent or reverse arterial stiffening and CVD associated with obesity and type 2 diabetes.NEW & NOTEWORTHY In the 5'-untranslated (UTR) regions of mRNAs encoding for type I collagen, an evolutionally conserved SL structure plays an essential role in its stability and posttranscriptional regulation. We demonstrate that a mutation of the SL mRNA structure in the 5'-UTR decreases collagen type I deposition and arterial stiffness in obese mice. Targeting this evolutionarily conserved SL structure may hold promise in the management of arterial stiffening and CVD associated with obesity and type 2 diabetes.
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Aorta/fisiopatologia , Doenças Cardiovasculares/genética , Colágeno Tipo I/genética , Sequências Repetidas Invertidas/genética , Obesidade/fisiopatologia , RNA Mensageiro/genética , Rigidez Vascular/genética , Regiões 5' não Traduzidas/genética , Adiposidade , Animais , Doenças Cardiovasculares/fisiopatologia , Cadeia alfa 1 do Colágeno Tipo I , Dieta Hiperlipídica , Sacarose Alimentar , Artéria Femoral/fisiopatologia , Resistência à Insulina , Masculino , Artérias Mesentéricas/fisiopatologia , Camundongos , Microscopia de Força Atômica , Mutação , Análise de Onda de PulsoRESUMO
Obstructive sleep apnea (OSA), characterized by episodes of intermittent hypoxia (IH), is highly prevalent in patients with abdominal aortic aneurysm (AAA). However, whether IH serves as an independent risk factor for AAA development remains to be investigated. Here, we determined the effects of chronic (6 months) IH on angiotensin (Ang II)-induced AAA development in C57BL/6J male mice, and IH underlying mechanisms in cultured vascular smooth muscle cells (SMCs). IH increased abdominal aortic diameter and the incidence of AAA in mice infused with Ang II as assessed by transabdominal ultrasound imaging. Importantly, IH with Ang II augmented aortic elastin degradation and expression of matrix metalloproteinase (MMP)s, mainly MMP8, MMP12 and a disintegrin and metalloproteinase-17 (ADAM17) as measured by histology and immunohistochemistry. Mechanistically, IH increased the activities of MMP2, MMP8, MMP9, MMP12, and ADAM17, while reducing the expression of the MMP regulator, reversion inducing cysteine rich protein with kazal motifs (RECK) in cultured SMCs. Aortic samples from human AAA were associated with decreased RECK and increased expression of ADAM17 and MMPs. These data suggest that IH promotes the development of AAA in association with an increased expression of MMPs and ADAM17, while decreased expression of RECK may be responsible for the increased protease activity. These findings support a potential causal link between OSA and AAA and provide a better understanding of the molecular mechanisms underlying the pathogenesis of AAA.
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Misfolded protein aggregation causes disease and aging; autophagy counteracts this by eliminating damaged components, enabling cells to survive starvation. The cytoplasm-to-vacuole targeting pathway in yeast encompasses the aggregation of the premature form of aminopeptidase 1 (prApe1) in cytosol and its sequestration by autophagic proteins into a vesicle for vacuolar transport. We show that the propeptide of Ape1 is important for aggregation and vesicle formation and that it is sufficient for binding to prApe1 and Atg19. Defective aggregation disrupts vacuolar transport, suggesting that aggregate shape is important in vesicle formation, whereas Atg19 binding is not sufficient for vacuolar transport. Aggregation involves hydrophobicity, whereas Atg19 binding requires additional electrostatic interactions. Ape1 dodecamerization may cluster propeptides into trimeric structures, with sufficient affinity to form propeptide hexamers by binding to other dodecamers, causing aggregation. We show that Ape1 aggregates bind Atg19 and Atg8 in vitro; this could be used as a scaffold for an in vitro assay of autophagosome formation to elucidate the mechanisms of autophagy.
Assuntos
Aminopeptidases/metabolismo , Citoplasma/enzimologia , Precursores Enzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Vesículas Secretórias/enzimologia , Vacúolos/enzimologia , Aminopeptidases/genética , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Citoplasma/genética , Precursores Enzimáticos/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Multimerização Proteica/fisiologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vesículas Secretórias/genética , Vacúolos/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Aging of the vasculature is characterized by endothelial dysfunction and arterial stiffening, two key events in the pathogenesis of cardiovascular disease (CVD). Treatment with sodium glucose transporter 2 (SGLT2) inhibitors is now known to decrease cardiovascular morbidity and mortality in type 2 diabetes. However, whether SGLT2 inhibition attenuates vascular aging is unknown. We first confirmed in a cohort of adult subjects that aging is associated with impaired endothelial function and increased arterial stiffness and that these two variables are inversely correlated. Next, we investigated whether SGLT2 inhibition with empagliflozin (Empa) ameliorates endothelial dysfunction and reduces arterial stiffness in aged mice with confirmed vascular dysfunction. Specifically, we assessed mesenteric artery endothelial function and stiffness (via flow-mediated dilation and pressure myography mechanical responses, respectively) and aortic stiffness (in vivo via pulse wave velocity and ex vivo via atomic force microscopy) in Empa-treated (14 mg/kg/day for 6 weeks) and control 80-week-old C57BL/6 J male mice. We report that Empa-treated mice exhibited improved mesenteric endothelial function compared with control, in parallel with reduced mesenteric artery and aortic stiffness. Additionally, Empa-treated mice had greater vascular endothelial nitric oxide synthase activation, lower phosphorylated cofilin, and filamentous actin content, with downregulation of pathways involved in production of reactive oxygen species. Our findings demonstrate that Empa improves endothelial function and reduces arterial stiffness in a preclinical model of aging, making SGLT2 inhibition a potential therapeutic alternative to reduce the progression of CVD in older individuals.
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Diabetes Mellitus Tipo 2 , Inibidores do Transportador 2 de Sódio-Glicose , Doenças Vasculares , Actinas/metabolismo , Idoso , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Análise de Onda de Pulso , Transportador 2 de Glucose-Sódio/metabolismo , Transportador 2 de Glucose-Sódio/uso terapêutico , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêuticoRESUMO
Insulin resistance in the vasculature is a characteristic feature of obesity and contributes to the pathogenesis of vascular dysfunction and disease. However, the molecular mechanisms underlying obesity-associated vascular insulin resistance and dysfunction remain poorly understood. We hypothesized that TRAF3IP2 (TRAF3 interacting protein 2), a proinflammatory adaptor molecule known to activate pathological stress pathways and implicated in cardiovascular diseases, plays a causal role in obesity-associated vascular insulin resistance and dysfunction. We tested this hypothesis by employing genetic-manipulation in endothelial cells in vitro, in isolated arteries ex vivo, and diet-induced obesity in a mouse model of TRAF3IP2 ablation in vivo. We show that ectopic expression of TRAF3IP2 blunts insulin signaling in endothelial cells and diminishes endothelium-dependent vasorelaxation in isolated aortic rings. Further, 16 weeks of high fat/high sucrose feeding impaired glucose tolerance, aortic insulin-induced vasorelaxation, and hindlimb postocclusive reactive hyperemia, while increasing blood pressure and arterial stiffness in wild-type male mice. Notably, TRAF3IP2 ablation protected mice from such high fat/high sucrose feeding-induced metabolic and vascular defects. Interestingly, wild-type female mice expressed markedly reduced levels of TRAF3IP2 mRNA independent of diet and were protected against high fat/high sucrose diet-induced vascular dysfunction. These data indicate that TRAF3IP2 plays a causal role in vascular insulin resistance and dysfunction. Specifically, the present findings highlight a sexual dimorphic role of TRAF3IP2 in vascular control and identify it as a promising therapeutic target in vasculometabolic derangements associated with obesity, particularly in males.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endotélio Vascular/metabolismo , Resistência à Insulina/genética , Insulina/metabolismo , Obesidade/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Aorta/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Humanos , Masculino , Camundongos , Obesidade/genética , Transdução de Sinais/fisiologia , Vasodilatação/fisiologiaRESUMO
Increased arterial stiffness and vascular remodeling precede and are consequences of hypertension. They also contribute to the development and progression of life-threatening cardiovascular diseases. Yet, there are currently no agents specifically aimed at preventing or treating arterial stiffening and remodeling. Previous research indicates that vascular smooth muscle actin polymerization participates in the initial stages of arterial stiffening and remodeling and that LIMK (LIM kinase) promotes F-actin formation and stabilization via cofilin phosphorylation and consequent inactivation. Herein, we hypothesize that LIMK inhibition is able to prevent vasoconstriction- and hypertension-associated arterial stiffening and inward remodeling. We found that small visceral arteries isolated from hypertensive subjects are stiffer and have greater cofilin phosphorylation than those from nonhypertensives. We also show that LIMK inhibition prevents arterial stiffening and inward remodeling in isolated human small visceral arteries exposed to prolonged vasoconstriction. Using cultured vascular smooth muscle cells, we determined that LIMK inhibition prevents vasoconstrictor agonists from increasing cofilin phosphorylation, F-actin volume, and cell cortex stiffness. We further show that localized LIMK inhibition prevents arteriolar inward remodeling in hypertensive mice. This indicates that hypertension is associated with increased vascular smooth muscle cofilin phosphorylation, cytoskeletal stress fiber formation, and heightened arterial stiffness. Our data further suggest that pharmacological inhibition of LIMK prevents vasoconstriction-induced arterial stiffening, in part, via reductions in vascular smooth muscle F-actin content and cellular stiffness. Accordingly, LIMK inhibition should represent a promising therapeutic means to stop the progression of arterial stiffening and remodeling in hypertension.
Assuntos
Artérias/efeitos dos fármacos , Quinases Lim/antagonistas & inibidores , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Rigidez Vascular/fisiologia , Vasoconstrição/efeitos dos fármacos , Adulto , Animais , Artérias/fisiologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiologia , Feminino , Humanos , Hipertensão/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/fisiologia , Vasoconstrição/fisiologiaRESUMO
Obesity and insulin resistance stiffen the vasculature, with females appearing to be more adversely affected. As augmented arterial stiffness is an independent predictor of cardiovascular disease (CVD), the increased predisposition of women with obesity and insulin resistance to arterial stiffening may explain their heightened risk for CVD. However, the cellular mechanisms by which females are more vulnerable to arterial stiffening associated with obesity and insulin resistance remain largely unknown. In this study, we provide evidence that female mice are more susceptible to Western diet-induced endothelial cell stiffening compared with age-matched males. Mechanistically, we show that the increased stiffening of the vascular intima in Western diet-fed female mice is accompanied by enhanced epithelial sodium channel (ENaC) activity in endothelial cells (EnNaC). Our data further indicate that: (i) estrogen signaling through estrogen receptor α (ERα) increases EnNaC activity to a larger extent in females compared with males, (ii) estrogen-induced activation of EnNaC is mediated by the serum/glucocorticoid inducible kinase 1 (SGK-1), and (iii) estrogen signaling stiffens endothelial cells when nitric oxide is lacking and this stiffening effect can be reduced with amiloride, an ENaC inhibitor. In aggregate, we demonstrate a sexual dimorphism in obesity-associated endothelial stiffening, whereby females are more vulnerable than males. In females, endothelial stiffening with obesity may be attributed to estrogen signaling through the ERα-SGK-1-EnNaC axis, thus establishing a putative therapeutic target for female obesity-related vascular stiffening.
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Endotélio Vascular/fisiopatologia , Canais Epiteliais de Sódio/metabolismo , Obesidade/fisiopatologia , Caracteres Sexuais , Rigidez Vascular , Animais , Células Cultivadas , Endotélio Vascular/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Obesidade/metabolismoRESUMO
Endothelin-1 (ET-1) is a powerful vasoconstrictor peptide considered to be causally implicated in hypertension and the development of cardiovascular disease. Increased ET-1 is commonly associated with reduced NO bioavailability and impaired vascular function; however, whether chronic elevation of ET-1 directly impairs endothelium-dependent relaxation (EDR) remains elusive. Herein, we report that (1) prolonged ET-1 exposure (ie, 48 hours) of naive mouse aortas or cultured endothelial cells did not impair EDR or reduce eNOS (endothelial NO synthase) activity, respectively (P>0.05); (2) mice with endothelial cell-specific ET-1 overexpression did not exhibit impaired EDR or reduced eNOS activity (P>0.05); (3) chronic (8 weeks) pharmacological blockade of ET-1 receptors in obese/hyperlipidemic mice did not improve aortic EDR or increase eNOS activity (P>0.05); and (4) vascular and plasma ET-1 did not inversely correlate with EDR in resistance arteries isolated from human subjects with a wide range of ET-1 levels (r=0.0037 and r=-0.1258, respectively). Furthermore, we report that prolonged ET-1 exposure downregulated vascular UCP-1 (uncoupling protein-1; P<0.05), which may contribute to the preservation of EDR in conditions characterized by hyperendothelinemia. Collectively, our findings demonstrate that chronic elevation of ET-1 alone may not be sufficient to impair EDR.