Neuraminidase-induced externalization of phosphatidylserine activates ADAM17 and impairs insulin signaling in endothelial cells.

Endothelial insulin resistance represents a causal factor in the pathogenesis of type 2 diabetes (T2D) and vascular disease, thus the need to identify molecular mechanisms underlying defects in endothelial insulin signaling. We previously have shown that a disintegrin and metalloproteinase-17 (ADAM17) is increased while insulin receptor α-subunit (IRα) is decreased in the vasculature of patients with T2D, leading to impaired insulin-induced vasodilation. We have also demonstrated that ADAM17 sheddase activity targets IRα; however, the mechanisms driving endothelial ADAM17 activity in T2D are largely unknown. Herein, we report that externalization of phosphatidylserine (PS) to the outer leaflet of the plasma membrane causes ADAM17-mediated shedding of IRα and blunting of insulin signaling in endothelial cells. Furthermore, we demonstrate that endothelial PS externalization is mediated by the phospholipid scramblase anoctamin-6 (ANO6) and that this process can be stimulated by neuraminidase, a soluble enzyme that cleaves sialic acid residues. Of note, we demonstrate that men and women with T2D display increased levels of neuraminidase activity in plasma, relative to age-matched healthy individuals, and this occurs in conjunction with increased ADAM17 activity and impaired leg blood flow responses to endogenous insulin. Collectively, this work reveals the neuraminidase-ANO6-ADAM17 axis as a novel potential target for restoring endothelial insulin sensitivity in T2D.NEW & NOTEWORTHY This work provides the first evidence that neuraminidase, an enzyme increased in the circulation of men and women with type 2 diabetes (T2D), promotes anoctamin-6 (ANO6)-dependent externalization of phosphatidylserine in endothelial cells, which in turn leads to activation of a disintegrin and metalloproteinase-17 (ADAM17) and consequent shedding of the insulin receptor-α from the cell surface. Hence, this work supports that consideration should be given to the neuraminidase-ANO6-ADAM17 axis as a novel potential target for restoring endothelial insulin sensitivity in T2D.

Neuraminidase inhibition improves endothelial function in diabetic mice.

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.

Impact of sex and diet-induced weight loss on vascular insulin sensitivity in type 2 diabetes.

Vascular insulin resistance, a major characteristic of obesity and type 2 diabetes (T2D), manifests with blunting of insulin-induced vasodilation. Although there is evidence that females are more whole body insulin sensitive than males in the healthy state, whether sex differences exist in vascular insulin sensitivity is unclear. Also uncertain is whether weight loss can reestablish vascular insulin sensitivity in T2D. The purpose of this investigation was to 1) establish if sex differences in vasodilatory responses to insulin exist in absence of disease, 2) determine whether female sex affords protection against the development of vascular insulin resistance with long-term overnutrition and obesity, and 3) examine if diet-induced weight loss can restore vascular insulin sensitivity in men and women with T2D. First, we show in healthy mice and humans that sex does not influence insulin-induced femoral artery dilation and insulin-stimulated leg blood flow, respectively. Second, we provide evidence that female mice are protected against impairments in insulin-induced dilation caused by overnutrition-induced obesity. Third, we show that men and women exhibit comparable levels of vascular insulin resistance when T2D develops but that diet-induced weight loss is effective at improving insulin-stimulated leg blood flow, particularly in women. Finally, we provide indirect evidence that these beneficial effects of weight loss may be mediated by a reduction in endothelin-1. In aggregate, the present data indicate that female sex confers protection against obesity-induced vascular insulin resistance and provide supportive evidence that, in women with T2D, vascular insulin resistance can be remediated with diet-induced weight loss.

Role of adropin in arterial stiffening associated with obesity and type 2 diabetes.

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.

Endothelial Glycocalyx.

The glycocalyx is a polysaccharide structure that protrudes from the body of a cell. It is primarily conformed of glycoproteins and proteoglycans, which provide communication, electrostatic charge, ionic buffering, permeability, and mechanosensation-mechanotransduction capabilities to cells. In blood vessels, the endothelial glycocalyx that projects into the vascular lumen separates the vascular wall from the circulating blood. Such a physical location allows a number of its components, including sialic acid, glypican-1, heparan sulfate, and hyaluronan, to participate in the mechanosensation-mechanotransduction of blood flow-dependent shear stress, which results in the synthesis of nitric oxide and flow-mediated vasodilation. The endothelial glycocalyx also participates in the regulation of vascular permeability and the modulation of inflammatory responses, including the processes of leukocyte rolling and extravasation. Its structural architecture and negative charge work to prevent macromolecules greater than approximately 70 kDa and cationic molecules from binding and flowing out of the vasculature. This also prevents the extravasation of pathogens such as bacteria and virus, as well as that of tumor cells. Due to its constant exposure to shear and circulating enzymes such as neuraminidase, heparanase, hyaluronidase, and matrix metalloproteinases, the endothelial glycocalyx is in a continuous process of degradation and renovation. A balance favoring degradation is associated with a variety of pathologies including atherosclerosis, hypertension, vascular aging, metastatic cancer, and diabetic vasculopathies. Consequently, ongoing research efforts are focused on deciphering the mechanisms that promote glycocalyx degradation or limit its syntheses, as well as on therapeutic approaches to improve glycocalyx integrity with the goal of reducing vascular disease. © 2022 American Physiological Society. Compr Physiol 12: 1-31, 2022.

ADAM17 cleaves the insulin receptor ectodomain on endothelial cells and causes vascular insulin resistance.

Inflammation and vascular insulin resistance are hallmarks of type 2 diabetes (T2D). However, several potential mechanisms causing abnormal endothelial insulin signaling in T2D need further investigation. Evidence indicates that the activity of ADAM17 (a disintegrin and metalloproteinase-17) and the presence of insulin receptor (IR) in plasma are increased in subjects with T2D. Accordingly, we hypothesized that in T2D, increased ADAM17 activity sheds the IR ectodomain from endothelial cells and impairs insulin-induced vasodilation. We used small visceral arteries isolated from a cross-sectional study of subjects with and without T2D undergoing bariatric surgery, human cultured endothelial cells, and recombinant proteins to test our hypothesis. Here, we demonstrate that arteries from subjects with T2D had increased ADAM17 expression, reduced presence of tissue inhibitor of metalloproteinase-3 (TIMP3), decreased extracellular IRα, and impaired insulin-induced vasodilation versus those from subjects without T2D. In vitro, active ADAM17 cleaved the ectodomain of the IRß subunit. Endothelial cells with ADAM17 overexpression or exposed to the protein kinase-C activator, PMA, had increased ADAM17 activity, decreased IRα presence on the cell surface, and increased IR shedding. Moreover, pharmacological inhibition of ADAM17 with TAPI-0 rescued PMA-induced IR shedding and insulin-signaling impairments in endothelial cells and insulin-stimulated vasodilation in human arteries. In aggregate, our findings suggest that ADAM17-mediated shedding of IR from the endothelial surface impairs insulin-mediated vasodilation. Thus, we propose that inhibition of ADAM17 sheddase activity should be considered a strategy to restore vascular insulin sensitivity in T2D.NEW & NOTEWORTHY To our knowledge, this is the first study to investigate the involvement of ADAM17 in causing impaired insulin-induced vasodilation in T2D. We provide evidence that ADAM17 activity is increased in the vasculature of patients with T2D and support the notion that ADAM17-mediated shedding of endothelial IRα ectodomains is a novel mechanism causing vascular insulin resistance. Our results highlight that targeting ADAM17 activity may be a potential therapeutic strategy to correct vascular insulin resistance in T2D.

Endothelial HSP72 is not reduced in type 2 diabetes nor is it a key determinant of endothelial insulin sensitivity.

Impaired endothelial insulin signaling and consequent blunting of insulin-induced vasodilation is a feature of type 2 diabetes (T2D) that contributes to vascular disease and glycemic dysregulation. However, the molecular mechanisms underlying endothelial insulin resistance remain poorly known. Herein, we tested the hypothesis that endothelial insulin resistance in T2D is attributed to reduced expression of heat shock protein 72 (HSP72). HSP72 is a cytoprotective chaperone protein that can be upregulated with heating and is reported to promote insulin sensitivity in metabolically active tissues, in part via inhibition of JNK activity. Accordingly, we further hypothesized that, in individuals with T2D, 7 days of passive heat treatment via hot water immersion to waist level would improve leg blood flow responses to an oral glucose load (i.e., endogenous insulin stimulation) via induction of endothelial HSP72. In contrast, we found that: 1) endothelial insulin resistance in T2D mice and humans was not associated with reduced HSP72 in aortas and venous endothelial cells, respectively; 2) after passive heat treatment, improved leg blood flow responses to an oral glucose load did not parallel with increased endothelial HSP72; and 3) downregulation of HSP72 (via small-interfering RNA) or upregulation of HSP72 (via heating) in cultured endothelial cells did not impair or enhance insulin signaling, respectively, nor was JNK activity altered. Collectively, these findings do not support the hypothesis that reduced HSP72 is a key driver of endothelial insulin resistance in T2D but provide novel evidence that lower-body heating may be an effective strategy for improving leg blood flow responses to glucose ingestion-induced hyperinsulinemia.

SGLT2 inhibition attenuates arterial dysfunction and decreases vascular F-actin content and expression of proteins associated with oxidative stress in aged mice.

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.

Cystamine reduces vascular stiffness in Western diet-fed female mice.

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.

Mutation of the 5'-untranslated region stem-loop mRNA structure reduces type I collagen deposition and arterial stiffness in male obese mice.

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.

TRAF3IP2 (TRAF3 Interacting Protein 2) Mediates Obesity-Associated Vascular Insulin Resistance and Dysfunction in Male Mice.

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.

LIMK (LIM Kinase) Inhibition Prevents Vasoconstriction- and Hypertension-Induced Arterial Stiffening and Remodeling.

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.

Western diet induces renal artery endothelial stiffening that is dependent on the epithelial Na+ channel.

Consumption of a Western diet (WD) induces central aortic stiffening that contributes to the transmittance of pulsatile blood flow to end organs, including the kidney. Our recent work supports that endothelial epithelial Na+ channel (EnNaC) expression and activation enhances aortic endothelial cell stiffening through reductions in endothelial nitric oxide (NO) synthase (eNOS) and bioavailable NO that result in inflammatory and oxidant responses and perivascular fibrosis. However, the role that EnNaC activation has on endothelial responses in the renal circulation remains unknown. We hypothesized that cell-specific deletion of the α-subunit of EnNaC would prevent WD-induced central aortic stiffness and protect the kidney from endothelial dysfunction and vascular stiffening. Twenty-eight-week-old female αEnNaC knockout and wild-type mice were fed either mouse chow or WD containing excess fat (46%), sucrose, and fructose (17.5% each). WD feeding increased fat mass, indexes of vascular stiffening in the aorta and renal artery (in vivo pulse wave velocity and ultrasound), and renal endothelial cell stiffening (ex vivo atomic force microscopy). WD further impaired aortic endothelium-dependent relaxation and renal artery compliance (pressure myography) without changes in blood pressure. WD-induced renal arterial stiffening occurred in parallel to attenuated eNOS activation, increased oxidative stress, and aortic and renal perivascular fibrosis. αEnNaC deletion prevented these abnormalities and support a novel mechanism by which WD contributes to renal arterial stiffening that is endothelium and Na+ channel dependent. These results demonstrate that cell-specific EnNaC is important in propagating pulsatility into the renal circulation, generating oxidant stress, reduced bioavailable NO, and renal vessel wall fibrosis and stiffening.

Chronic Elevation of Endothelin-1 Alone May Not Be Sufficient to Impair Endothelium-Dependent Relaxation.

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.

Sexual Dimorphism in Obesity-Associated Endothelial ENaC Activity and Stiffening in Mice.

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.

Diet-Induced Obesity Promotes Kidney Endothelial Stiffening and Fibrosis Dependent on the Endothelial Mineralocorticoid Receptor.

Obesity is characterized by enhanced MR (mineralocorticoid receptor) activation, vascular stiffness, and associated cardiovascular and kidney disease. Consumption of a Western-style diet (WD), high in saturated fat and refined carbohydrates, by female mice, leads to obesity and vascular stiffening. Use of ECMR (endothelial cell-specific MR) knockout mice supports that ECMR activation is critical for development of vascular and cardiac fibrosis and stiffening. However, the role of ECMR activation in kidney inflammation and fibrosis remains unknown. We hypothesized that cell-specific deletion of ECMR would prevent WD-induced central aortic stiffness and protect the kidney from endothelial dysfunction and vascular stiffening. Four-week-old female ECMR KO and wild-type mice were fed either mouse chow or WD for 16 weeks. WD feeding increased body weight and fat mass, proteinuria, as well as vascular stiffness indices (pulse wave velocity and kidney artery stiffening) and impaired endothelial-dependent vasodilatation without blood pressure changes. The WD-induced kidney arterial stiffening was associated with attenuated eNOS (endothelial NO synthase) activation, increased oxidative stress, proinflammatory immune responses, alterations in extracellular matrix degradation pathways, and fibrosis. ECMR deletion prevented these abnormalities by improving eNOS activation and reducing macrophage proinflammatory M1 polarization, expression of TG2 (transglutaminase 2), and MMP (matrix metalloproteinase)-9. Our data support the concept that ECMR activation contributes to endothelial dysfunction, increased kidney artery fibrosis/stiffening, and impaired NOS (NO synthase) activation, processes associated with macrophage infiltration and polarization, inflammation, and oxidative stress, collectively resulting in tubulointerstitial fibrosis in females consuming a WD.

IGF-1 Deficiency Promotes Pathological Remodeling of Cerebral Arteries: A Potential Mechanism Contributing to the Pathogenesis of Intracerebral Hemorrhages in Aging.

Clinical and experimental studies show that age-related decline in circulating insulin-like growth factor-1 (IGF-1) levels promotes the pathogenesis of intracerebral hemorrhages, which critically contribute to the development of vascular cognitive impairment and disability in older adults. Yet, the mechanisms by which IGF-1 deficiency compromises structural integrity of the cerebral vasculature are not completely understood. To determine the role of IGF-1 deficiency in pathological remodeling of middle cerebral arteries (MCAs), we compared alterations in vascular mechanics, morphology, and remodeling-related gene expression profile in mice with liver-specific knockdown of IGF-1 (Igf1f/f + TBG-Cre-AAV8) and control mice with or without hypertension induced by angiotensin-II treatment. We found that IGF-1 deficiency resulted in thinning of the media and decreased wall-to-lumen ratio in MCAs. MCAs of control mice exhibited structural adaptation to hypertension, manifested as a significant increase in wall thickness, vascular smooth muscle cell (VSMC) hypertrophy, decreased internal diameter and up-regulation of extracellular matrix (ECM)-related genes. IGF-1 deficiency impaired hypertension-induced adaptive media hypertrophy and dysregulated ECM remodeling, decreasing elastin content and attenuating adaptive changes in ECM-related gene expression. Thus, circulating IGF-1 plays a critical role in maintenance of the structural integrity of cerebral arteries. Alterations of VSMC phenotype and pathological remodeling of the arterial wall associated with age-related IGF-1 deficiency have important translational relevance for the pathogenesis of intracerebral hemorrhages and vascular cognitive impairment in elderly hypertensive patients.

Glycemic control by the SGLT2 inhibitor empagliflozin decreases aortic stiffness, renal resistivity index and kidney injury.

BACKGROUND: Arterial stiffness is emerging as an independent risk factor for the development of chronic kidney disease. The sodium glucose co-transporter 2 (SGLT2) inhibitors, which lower serum glucose by inhibiting SGLT2-mediated glucose reabsorption in renal proximal tubules, have shown promise in reducing arterial stiffness and the risk of cardiovascular and kidney disease in individuals with type 2 diabetes mellitus. Since hyperglycemia contributes to arterial stiffness, we hypothesized that the SGLT2 inhibitor empagliflozin (EMPA) would improve endothelial function, reduce aortic stiffness, and attenuate kidney disease by lowering hyperglycemia in type 2 diabetic female mice (db/db). MATERIALS/METHODS: Ten-week-old female wild-type control (C57BLKS/J) and db/db (BKS.Cg-Dock7m+/+Leprdb/J) mice were divided into three groups: lean untreated controls (CkC, n = 17), untreated db/db (DbC, n = 19) and EMPA-treated db/db mice (DbE, n = 19). EMPA was mixed with normal mouse chow at a concentration to deliver 10 mg kg-1 day-1, and fed for 5 weeks, initiated at 11 weeks of age. RESULTS: Compared to CkC, DbC showed increased glucose levels, blood pressure, aortic and endothelial cell stiffness, and impaired endothelium-dependent vasorelaxation. Furthermore, DbC exhibited impaired activation of endothelial nitric oxide synthase, increased renal resistivity and pulsatility indexes, enhanced renal expression of advanced glycation end products, and periarterial and tubulointerstitial fibrosis. EMPA promoted glycosuria and blunted these vascular and renal impairments, without affecting increases in blood pressure. In addition, expression of "reversion inducing cysteine rich protein with Kazal motifs" (RECK), an anti-fibrotic mediator, was significantly suppressed in DbC kidneys and partially restored by EMPA. Confirming the in vivo data, EMPA reversed high glucose-induced RECK suppression in human proximal tubule cells. CONCLUSIONS: Empagliflozin ameliorates kidney injury in type 2 diabetic female mice by promoting glycosuria, and possibly by reducing systemic and renal artery stiffness, and reversing RECK suppression.

Uric acid promotes vascular stiffness, maladaptive inflammatory responses and proteinuria in western diet fed mice.

OBJECTIVE: Aortic vascular stiffness has been implicated in the development of cardiovascular disease (CVD) and chronic kidney disease (CKD) in obese individuals. However, the mechanism promoting these adverse effects are unclear. In this context, promotion of obesity through consumption of a western diet (WD) high in fat and fructose leads to excess circulating uric acid. There is accumulating data implicating elevated uric acid in the promotion of CVD and CKD. Accordingly, we hypothesized that xanthine oxidase(XO) inhibition with allopurinol would prevent a rise in vascular stiffness and proteinuria in a translationally relevant model of WD-induced obesity. MATERIALS/METHODS: Four-week-old C57BL6/J male mice were fed a WD with excess fat (46%) and fructose (17.5%) with or without allopurinol (125mg/L in drinking water) for 16weeks. Aortic endothelial and extracellular matrix/vascular smooth muscle stiffness was evaluated by atomic force microscopy. Aortic XO activity, 3-nitrotyrosine (3-NT) and aortic endothelial sodium channel (EnNaC) expression were evaluated along with aortic expression of inflammatory markers. In the kidney, expression of toll like receptor 4 (TLR4) and fibronectin were assessed along with evaluation of proteinuria. RESULTS: XO inhibition significantly attenuated WD-induced increases in plasma uric acid, vascular XO activity and oxidative stress, in concert with reductions in proteinuria. Further, XO inhibition prevented WD-induced increases in aortic EnNaC expression and associated endothelial and subendothelial stiffness. XO inhibition also reduced vascular pro-inflammatory and maladaptive immune responses induced by consumption of a WD. XO inhibition also decreased WD-induced increases in renal TLR4 and fibronectin that associated proteinuria. CONCLUSIONS: Consumption of a WD leads to elevations in plasma uric acid, increased vascular XO activity, oxidative stress, vascular stiffness, and proteinuria all of which are attenuated with allopurinol administration.

Amiloride Improves Endothelial Function and Reduces Vascular Stiffness in Female Mice Fed a Western Diet.

Obese premenopausal women lose their sex related cardiovascular disease protection and develop greater arterial stiffening than age matched men. In female mice, we have shown that consumption of a Western diet (WD), high in fat and refined sugars, is associated with endothelial dysfunction and vascular stiffening, which occur via activation of mineralocorticoid receptors and associated increases in epithelial Na+ channel (ENaC) activity on endothelial cells (EnNaC). Herein our aim was to determine the effect that reducing EnNaC activity with a very-low-dose of amiloride would have on decreasing endothelial and arterial stiffness in young female mice consuming a WD. To this end, we fed female mice either a WD or control diet and treated them with or without a very-low-dose of the ENaC-inhibitor amiloride (1 mg/kg/day) in the drinking water for 20 weeks beginning at 4 weeks of age. Mice consuming a WD were heavier and had greater percent body fat, proteinuria, and aortic stiffness as assessed by pulse-wave velocity than those fed control diet. Treatment with amiloride did not affect body weight, body composition, blood pressure, urinary sodium excretion, or insulin sensitivity, but significantly reduced the development of endothelial and aortic stiffness, aortic fibrosis, aortic oxidative stress, and mesenteric resistance artery EnNaC abundance and proteinuria in WD-fed mice. Amiloride also improved endothelial-dependent vasodilatory responses in the resistance arteries of WD-fed mice. These results indicate that a very-low-dose of amiloride, not affecting blood pressure, is sufficient to improve endothelial function and reduce aortic stiffness in female mice fed a WD, and suggest that EnNaC-inhibition may be sufficient to ameliorate the pathological vascular stiffening effects of WD-induced obesity in females.