PAI-1 Regulates the Cytoskeleton and Intrinsic Stiffness of Vascular Smooth Muscle Cells.

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.

Sitting leg vasculopathy: potential adaptations beyond the endothelium.

Increased sitting time, the most common form of sedentary behavior, is an independent risk factor for all-cause and cardiovascular disease mortality; however, the mechanisms linking sitting to cardiovascular risk remain largely elusive. Studies over the last decade have led to the concept that excessive time spent in the sitting position and the ensuing reduction in leg blood flow-induced shear stress cause endothelial dysfunction. This conclusion has been mainly supported by studies using flow-mediated dilation in the lower extremities as the measured outcome. In this review, we summarize evidence from classic studies and more recent ones that collectively support the notion that prolonged sitting-induced leg vascular dysfunction is likely also attributable to changes occurring in vascular smooth muscle cells (VSMCs). Indeed, we provide evidence that prolonged constriction of resistance arteries can lead to modifications in the structural characteristics of the vascular wall, including polymerization of actin filaments in VSMCs and inward remodeling, and that these changes manifest in a time frame that is consistent with the vascular changes observed with prolonged sitting. We expect this review will stimulate future studies with a focus on VSMC cytoskeletal remodeling as a potential target to prevent the detrimental vascular ramifications of too much sitting.

Integrating molecular and cellular components of endothelial shear stress mechanotransduction.

The lining of blood vessels is constantly exposed to mechanical forces exerted by blood flow against the endothelium. Endothelial cells detect these tangential forces (i.e., shear stress), initiating a host of intracellular signaling cascades that regulate vascular physiology. Thus, vascular health is tethered to the endothelial cells' capacity to transduce shear stress. Indeed, the mechanotransduction of shear stress underlies a variety of cardiovascular benefits, including some of those associated with increased physical activity. However, endothelial mechanotransduction is impaired in aging and disease states such as obesity and type 2 diabetes, precipitating the development of vascular disease. Understanding endothelial mechanotransduction of shear stress, and the molecular and cellular mechanisms by which this process becomes defective, is critical for the identification and development of novel therapeutic targets against cardiovascular disease. In this review, we detail the primary mechanosensitive structures that have been implicated in detecting shear stress, including junctional proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1), the extracellular glycocalyx and its components, and ion channels such as piezo1. We delineate which molecules are truly mechanosensitive and which may simply be indispensable for the downstream transmission of force. Furthermore, we discuss how these mechanosensors interact with other cellular structures, such as the cytoskeleton and membrane lipid rafts, which are implicated in translating shear forces to biochemical signals. Based on findings to date, we also seek to integrate these cellular and molecular mechanisms with a view of deciphering endothelial mechanotransduction of shear stress, a tenet of vascular physiology.

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.

Multi-omic analysis of the cardiac cellulome defines a vascular contribution to cardiac diastolic dysfunction in obese female mice.

Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Clinical data further support that CMD associates with development of heart failure with preserved ejection fraction and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the impact of smooth muscle cell (SMC)-specific MR deletion on obesity-associated coronary and cardiac diastolic dysfunction in female mice. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Global MR blockade with spironolactone prevented coronary and cardiac dysfunction in obese females and specific deletion of SMC-MR was sufficient to prevent obesity-associated coronary and cardiac diastolic dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in non-myocyte populations including B/T cells, macrophages, and endothelium as well as increased coronary VCAM-1 protein expression, independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction, thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.

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.

New insights into mechanisms of endothelial insulin resistance in type 2 diabetes.

Insulin resistance in the vasculature is a hallmark of type 2 diabetes (T2D), and blunting of insulin-induced vasodilation is its primary consequence. Individuals with T2D exhibit a marked impairment in insulin-induced dilation in resistance arteries across vascular beds. Importantly, reduced insulin-stimulated vasodilation and blood flow to skeletal muscle limits glucose uptake and contributes to impaired glucose control in T2D. The study of mechanisms responsible for the suppressed vasodilatory effects of insulin has been a growing topic of interest for not only its association with glucose control and extension to T2D but also its relationship with cardiovascular disease development and progression. In this mini-review, we integrate findings from recent studies by our group with the existing literature focused on the mechanisms underlying endothelial insulin resistance in T2D.

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.

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.

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 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.

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.

Guidelines for the measurement of vascular function and structure in isolated arteries and veins.

The measurement of vascular function in isolated vessels has revealed important insights into the structural, functional, and biomechanical features of the normal and diseased cardiovascular system and has provided a molecular understanding of the cells that constitutes arteries and veins and their interaction. Further, this approach has allowed the discovery of vital pharmacological treatments for cardiovascular diseases. However, the expansion of the vascular physiology field has also brought new concerns over scientific rigor and reproducibility. Therefore, it is appropriate to set guidelines for the best practices of evaluating vascular function in isolated vessels. These guidelines are a comprehensive document detailing the best practices and pitfalls for the assessment of function in large and small arteries and veins. Herein, we bring together experts in the field of vascular physiology with the purpose of developing guidelines for evaluating ex vivo vascular function. By using this document, vascular physiologists will have consistency among methodological approaches, producing more reliable and reproducible results.

Structure and excited-state dynamics of dimeric copper(i) photosensitizers investigated by time-resolved X-ray and optical transient absorption spectroscopy.

Time-resolved X-ray (tr-XAS) and optical transient absorption (OTA) spectroscopy in the picosecond time scale coupled with Density Functional theory (DFT) and X-ray absorption near-edge structure (XANES) calculations are applied to study three homoleptic Cu(i) dimeric chromophores with ethyl and longer propyl spacers, denoted as [Cu2(mphenet)2]Cl2 (C1), [Cu2(mphenet)2](ClO4)2 (C2) and [Cu2(mphenpr)2](ClO4)2 (C3) (where mphenet = 1,2-bis(9-methyl-1,10-phenanthrolin-2-yl)ethane and mphenpr = 1,3-bis(9-methyl-1,10-phenanthrolin-2-yl)propane). Tr-XAS analysis after light illumination at ∼ 100 ps illustrate the formation of a flattened triplet excited state in all 3 complexes. Optical transient absorption (OTA) analysis for C1 monitored in water and C2 and C3 measured in acetonitrile reveals distinct excited-state lifetimes of 169 ps, 670 ps and 1600 ps respectively. These differences are associated to changes in the solvent (comparing C1 and C2) and the flexibility of the ligand to adapt after Cu flattening upon excitation (C2 and C3). Our results are important for the improved structural dynamics of these types of Cu-based dimeric compounds, and can guide the integration of these chromophores into more complex solar energy conversion schemes.

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.

Obesity and cardiovascular disease in women.

As the prevalence of obesity continues to grow worldwide, the health and financial burden of obesity-related comorbidities grows too. Cardiovascular disease (CVD) is clearly associated with increased adiposity. Importantly, women are at higher risk of CVD when obese and insulin resistant, in particular at higher risk of developing heart failure with preserved ejection fraction and ischemic heart disease. Increased aldosterone and mineralocorticoid receptor activation, aberrant estrogenic signaling and elevated levels of androgens are among some of the proposed mechanisms explaining the heightened CVD risk. In addition to traditional cardiovascular risk factors, understanding nontraditional risk factors specific to women, like excess weight gain during pregnancy, preeclampsia, gestational diabetes, and menopause are central to designing personalized interventions aimed to curb the epidemic of CVD. In the present review, we examine the available evidence supporting a differential cardiovascular impact of increased adiposity in women compared with men and the proposed pathophysiological mechanisms behind these differences. We also discuss women-specific cardiovascular risk factors associated with obesity and insulin resistance.

Mechanisms of Connexin-Related Lymphedema.

RATIONALE: Mutations in GJC2 and GJA1, encoding Cxs (connexins) 47 and 43, respectively, are linked to lymphedema, but the underlying mechanisms are unknown. Because efficient lymph transport relies on the coordinated contractions of lymphatic muscle cells (LMCs) and their electrical coupling through Cxs, Cx-related lymphedema is proposed to result from dyssynchronous contractions of lymphatic vessels. OBJECTIVE: To determine which Cx isoforms in LMCs and lymphatic endothelial cells are required for the entrainment of lymphatic contraction waves and efficient lymph transport. METHODS AND RESULTS: We developed novel methods to quantify the spatiotemporal entrainment of lymphatic contraction waves and used optogenetic techniques to analyze calcium signaling within and between the LMC and the lymphatic endothelial cell layers. Genetic deletion of the major lymphatic endothelial cell Cxs (Cx43, Cx47, or Cx37) revealed that none were necessary for the synchronization of the global calcium events that triggered propagating contraction waves. We identified Cx45 in human and mouse LMCs as the critical Cx mediating the conduction of pacemaking signals and entrained contractions. Smooth muscle-specific Cx45 deficiency resulted in 10- to 18-fold reduction in conduction speed, partial-to-severe loss of contractile coordination, and impaired lymph pump function ex vivo and in vivo. Cx45 deficiency resulted in profound inhibition of lymph transport in vivo, but only under an imposed gravitational load. CONCLUSIONS: Our results (1) identify Cx45 as the Cx isoform mediating the entrainment of the contraction waves in LMCs; (2) show that major endothelial Cxs are dispensable for the entrainment of contractions; (3) reveal a lack of coupling between lymphatic endothelial cells and LMCs, in contrast to arterioles; (4) point to lymphatic valve defects, rather than contraction dyssynchrony, as the mechanism underlying GJC2- or GJA1-related lymphedema; and (5) show that a gravitational load exacerbates lymphatic contractile defects in the intact mouse hindlimb, which is likely critical for the development of lymphedema in the adult mouse.

Hypomethylation of the aquatic invasive plant, Ludwigia grandiflora subsp. hexapetala mimics the adaptive transition into the terrestrial morphotype.

Ongoing global changes affect ecosystems and open up new opportunities for biological invasion. The ability of invasive species to rapidly adapt to new environments represents a relevant model for studying short-term adaptation mechanisms. The aquatic invasive plant, Ludwigia grandiflora subsp. hexapetala, is classified as harmful in European rivers. In French wet meadows, this species has shown a rapid transition from aquatic to terrestrial environments with emergence of two distinct morphotypes in 5 years. To understand the heritable mechanisms involved in adjustment to such a new environment, we investigate both genetic and epigenetic as possible sources of flexibility involved in this fast terrestrial transition. We found a low overall genetic differentiation between the two morphotypes arguing against the possibility that terrestrial morphotype emerged from a new adaptive genetic capacity. Artificial hypomethylation was induced on both morphotypes to assess the epigenetic hypothesis. We analyzed global DNA methylation, morphological changes, phytohormones and metabolite profiles of both morphotype responses in both aquatic and terrestrial conditions in shoot and root tissues. Hypomethylation significantly affected morphological variables, phytohormone levels and the amount of some metabolites. The effects of hypomethylation depended on morphotypes, conditions and plant tissues, which highlighted differences among the morphotypes and their plasticity. Using a correlative integrative approach, we showed that hypomethylation of the aquatic morphotype mimicked the characteristics of the terrestrial morphotype. Our data suggest that DNA methylation rather than a new adaptive genetic capacity is playing a key role in L. grandiflora subsp. hexapetala plasticity during its rapid aquatic to terrestrial transition.

Reactivity of CuIN4 Flattened Complexes: Interplay between Coordination Geometry and Ligand Flexibility.

The relation between redox activity and coordination geometry in CuIN4 complexes indicates that more flattened structures tend to be more reactive. Such a preorganization of the ligand confers to the complex geometries closer to a transition state, which has been termed the "entatic" state in metalloproteins, more recently extending this concept for copper complexes. However, many aspects of the redox chemistry of CuI complexes cannot be explained only by flattening. For instance, the role of ligand flexibility in this context is an open debate nowadays. To analyze this point, we studied oxidation properties of a series of five monometallic CuI Schiff-base complexes, [CuI(Ln)]+, which span a range of geometries from a distorted square planar (n = 3) to a distorted tetrahedron (n = 6, 7). This stepped control of the structure around the CuI atom allows us to explore the effect of the flattening distortion on both the electronic and redox properties through the series. Experimental studies were complemented by a theoretical analysis based on density functional theory calculations. As expected, oxidation was favored in the flattened structures, spanning a broad potential window of 370 mV for the complete series. This orderly behavior was tested in the reductive dehalogenation reaction of tetrachloroethane (TCE). Kinetic studies show that CuI oxidation by TCE is faster as the flattening distortion is higher and the oxidation potentials of the metal are lower. However, the most reactive complex was not the more planar, contradicting the trend expected from oxidation potentials. The origin of this irregularity is related to ligand flexibility and its connection with the atom/electron transfer reaction path, highlighting the need to consider effects beyond flattening distortion to better understand the reactivity of this important class of complexes.