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High levels of testosterone (Testo) are associated with cardiovascular risk by increasing reactive oxygen species (ROS) formation. NADPH oxidases (NOX) are the major source of ROS in the vasculature of cardiovascular diseases. NOX4 is a unique isotype, which produces hydrogen peroxide (H2O2), and its participation in cardiovascular biology is controversial. So far, it is unclear whether NOX4 protects from Testo-induced endothelial injury. Thus, we hypothesized that supraphysiological levels of Testo induce endothelial NOX4 expression to attenuate endothelial injury. Human mesenteric vascular endothelial cells (HMECs) and human umbilical vein endothelial cells (HUVEC) were treated with Testo (10-7 M) with or without a NOX4 inhibitor [GLX351322 (10-4 M)] or NOX4 siRNA. In vivo, 10-wk-old C57Bl/6J male mice were treated with Testo (10 mg/kg) for 30 days to study endothelial function. Testo increased mRNA and protein levels of NOX4 in HMECs and HUVECs. Testo increased superoxide anion (O2-) and H2O2 production, which were abolished by NOX1 and NOX4 inhibition, respectively. Testo also attenuated bradykinin-induced NO production, which was further impaired by NOX4 inhibition. In vivo, Testo decreased H2O2 production in aortic segments and triggered endothelial dysfunction [decreased relaxation to acetylcholine (ACh)], which was further impaired by GLX351322 and by a superoxide dismutase and catalase mimetic (EUK134). Finally, Testo led to a dysregulated endothelial cell migration, which was exacerbated by GLX351322. These data indicate that supraphysiological levels of Testo increase the endothelial expression and activity of NOX4 to counterbalance the deleterious effects caused by Testo in endothelial function.NEW & NOTEWORTHY By inducing ROS formation, high levels of testosterone play a major role in the pathogenesis of cardiovascular disease. NOXs are the major sources of ROS in the vasculature of cardiovascular diseases. Herein, we describe a novel compensatory mechanism by showing that NOX4 is a protective oxidant enzyme and counterbalances the deleterious effects of testosterone in endothelial cells by modulating hydrogen peroxide formation.

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BACKGROUND: Aldosterone has been described to initiate cardiovascular diseases by triggering exacerbated sterile vascular inflammation. The functions of CCL5 (C-C motif chemokine ligand 5) and its receptor CCR5 (C-C motif chemokine receptor 5) are well known in infectious diseases, their contributions to aldosterone-induced vascular injury and hypertension remain unknown. METHODS: We analyzed the vascular profile, blood pressure, and renal damage in wild-type (CCR5+/+) and CCR5 knockout (CCR5-/-) mice treated with aldosterone (600 µg/kg per day for 14 days) while receiving 1% saline to drink. Vascular function was analyzed in aorta and mesenteric arteries, blood pressure was measured by telemetry and renal injury and inflammation were analyzed via histology and flow cytometry. Endothelial cells were used to study the molecular signaling whereby CCL5 induces endothelial dysfunction. RESULTS: Aldosterone treatment resulted in exaggerated CCL5 circulating levels and vascular CCR5 expression in CCR5+/+ mice accompanied by endothelial dysfunction, hypertension, and renal inflammation and damage. CCR5-/- mice were protected from these aldosterone-induced effects. Mechanistically, we demonstrated that CCL5 increased NOX1 (NADPH oxidase 1) expression, reactive oxygen species formation, NFκB (nuclear factor kappa B) activation, and inflammation and reduced NO production in isolated endothelial cells. These effects were abolished by antagonizing CCR5 with Maraviroc. Finally, aorta incubated with CCL5 displayed severe endothelial dysfunction, which is prevented by blocking NOX1, NFκB, or CCR5. CONCLUSIONS: Our data demonstrate that CCL5/CCR5, through activation of NFκB and NOX1, is critically involved in aldosterone-induced vascular and renal damage and hypertension placing CCL5 and CCR5 as potential therapeutic targets for conditions characterized by aldosterone excess.

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Objective: Cardiovascular disease (CVD) is a global health crisis and a leading cause of mortality. The intricate interplay between vascular contractility and mitochondrial function is central to CVD pathogenesis. The progranulin gene (GRN) encodes glycoprotein progranulin (PGRN), a ubiquitous molecule with known anti-inflammatory property. However, the role of PGRN in CVD remains enigmatic. In this study, we sought to dissect the significance of PGRN in the regulation vascular contractility and investigate the interface between PGRN and mitochondrial quality. Method: Our investigation utilized aortae from male and female C57BL6/J wild-type (PGRN+/+) and B6(Cg)-Grntm1.1Aidi/J (PGRN-/-) mice, encompassing wire myograph assays to assess vascular contractility and primary aortic vascular smooth muscle cells (VSMCs) for mechanistic insights. Results: Our results showed suppression of contractile activity in PGRN-/- VSMCs and aorta, followed by reduced α-smooth muscle actin expression. Mechanistically, PGRN deficiency impaired mitochondrial oxygen consumption rate (OCR), complex I activity, mitochondrial turnover, and mitochondrial redox signaling, while restoration of PGRN levels in aortae from PGRN-/- mice via lentivirus delivery ameliorated contractility and boosted OCR. In addition, VSMC overexpressing PGRN displayed higher mitochondrial respiration and complex I activity accompanied by cellular hypercontractility. Furthermore, increased PGRN triggered lysosome biogenesis by regulating transcription factor EB and accelerated mitophagy flux in VSMC, while treatment with spermidine, an autophagy inducer, improved mitochondrial phenotype and enhanced vascular contractility. Finally, angiotensin II failed to induce vascular contractility in PGRN-/- suggesting a key role of PGRN to maintain the vascular tone. Conclusion: Our findings suggest that PGRN preserves the vascular contractility via regulating mitophagy flux, mitochondrial complex I activity, and redox signaling. Therefore, loss of PGRN function appears as a pivotal risk factor in CVD development.

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BACKGROUND: Renin-angiotensin (Ang II)-aldosterone system (RAAS) is crucial for the cardiovascular risk associated with excessive ethanol consumption. Disturbs in mitochondria have been implicated in multiple cardiovascular diseases. However, if mitochondria dysfunction contributes to ethanol-induced vascular dysfunction is still unknown. We investigated whether ethanol leads to vascular dysfunction via RAAS activation, mitochondria dysfunction, and mitochondrial reactive oxygen species (mtROS). METHODS: Male C57/BL6J or mt-keima mice (6-8-weeks old) were treated with ethanol (20% vol./vol.) for 12 weeks with or without Losartan (10 mg/kg/day). RESULTS: Ethanol induced aortic hypercontractility in an endothelium-dependent manner. PGC1α (a marker of biogenesis), Mfn2, (an essential protein for mitochondria fusion), as well as Pink-1 and Parkin (markers of mitophagy), were reduced in aortas from ethanol-treated mice. Disturb in mitophagy flux was further confirmed in arteries from mt-keima mice. Additionally, ethanol increased mtROS and reduced SOD2 expression. Strikingly, losartan prevented vascular hypercontractility, mitochondrial dysfunction, mtROS, and restored SOD2 expression. Both MnTMPyP (SOD2 mimetic) and CCCP (a mitochondrial uncoupler) reverted ethanol-induced vascular dysfunction. Moreover, L-NAME (NOS inhibitor) and EUK 134 (superoxide dismutase/catalase mimetic) did not affect vascular response in ethanol group, suggesting that ethanol reduces aortic nitric oxide (NO) and H2O2 bioavailability. These responses were prevented by losartan. CONCLUSION: AT1 receptor modulates ethanol-induced vascular hypercontractility by promoting mitochondrial dysfunction, mtROS, and reduction of NO and H2O2 bioavailability. Our findings shed a new light in our understanding of ethanol-induced vascular toxicity and open perspectives of new therapeutic approaches for patients with disorder associated with abusive ethanol drinking.

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Background: Aldosterone, a mineralocorticoid steroid hormone, has been described to initiate cardiovascular diseases by triggering exacerbated sterile vascular inflammation. The functions of C-C Motif Chemokine Ligand 5 (CCL5) and its receptor, C-C Motif Chemokine Receptor 5 (CCR5), are well known in infectious diseases, but their roles in the genesis of aldosterone-induced vascular injury and hypertension are unknown. Methods: We analyzed the vascular profile, blood pressure, and renal damage in wild-type (CCR5+/+) and CCR5 knockout (CCR5-/-) mice treated with aldosterone (600 µg/kg/day for 14 days) while receiving 1% saline to drink. Results: Here, we show that CCR5 plays a central role in aldosterone-induced vascular injury, hypertension, and renal damage. Long-term infusion of aldosterone in CCR5+/+ mice resulted in exaggerated CCL5 circulating levels and vascular CCR5 expression. Aldosterone treatment also triggered vascular injury, characterized by endothelial dysfunction and inflammation, hypertension, and renal damage. Mice lacking CCR5 were protected from aldosterone-induced vascular damage, hypertension, and renal injury. Mechanistically, we demonstrated that CCL5 increased NADPH oxidase 1 (Nox1) expression, reactive oxygen species (ROS) formation, NFκB activation, and inflammation and reduced nitric oxide production in isolated endothelial cells. These effects were abolished by antagonizing CCR5 with Maraviroc. Finally, aortae incubated with CCL5 displayed severe endothelial dysfunction, which is prevented by blocking Nox1, NFκB, or with Maraviroc treatment. Conclusions: Our data demonstrate that CCL5/CCR5, through activation of NFkB and Nox1, is critically involved in aldosterone-induced vascular and renal damage and hypertension. Our data place CCL5 and CCR5 as potential targets for therapeutic interventions in conditions with aldosterone excess.

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Background The mechanisms determining vascular tone are still not completely understood, even though it is a significant factor in blood pressure management. Many circulating proteins have a significant impact on controlling vascular tone. Progranulin displays anti-inflammatory effects and has been extensively studied in neurodegenerative illnesses. We investigated whether progranulin sustains the vascular tone that helps regulate blood pressure. Methods and Results We used male and female C57BL6/J wild type (progranulin+/+) and B6(Cg)-Grntm1.1Aidi/J (progranulin-/-) to understand the impact of progranulin on vascular contractility and blood pressure. We found that progranulin-/- mice display elevated blood pressure followed by hypercontractility to noradrenaline in mesenteric arteries, which is restored by supplementing the mice with recombinant progranulin. In ex vivo experiments, recombinant progranulin attenuated the vascular contractility to noradrenaline in male and female progranulin+/+ arteries, which was blunted by blocking EphrinA2 or Sortilin1. To understand the mechanisms whereby progranulin evokes anticontractile effects, we inhibited endothelial factors. N(gamma)-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor) prevented the progranulin effects, whereas indomethacin (cyclooxygenase inhibitor) affected only the contractility in arteries incubated with vehicle, indicating that progranulin increases nitric oxide and decreases contractile prostanoids. Finally, recombinant progranulin induced endothelial nitric oxide synthase phosphorylation and nitric oxide production in isolated mesenteric endothelial cells. Conclusions Circulating progranulin regulates vascular tone and blood pressure via EphrinA2 and Sortilin1 receptors and endothelial nitric oxide synthase activation. Collectively, our data suggest that deficiency in progranulin is a cardiovascular risk factor and that progranulin might be a new therapeutic avenue to treat high blood pressure.

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BACKGROUND: Obesity is the number one cardiovascular risk factor for both men and women and is a complex condition. Although a sex dimorphism on vascular function has already been noted, the underlying processes remain unclear. The Rho-kinase pathway has a unique role in controlling vascular tone, and in obese male mice, hyperactivation of this system results in worsened vascular constriction. We investigated whether female mice exhibit decreased Rho-kinase activation as a protective mechanism in obesity. METHODS: We exposed male and female mice to a high-fat diet (HFD) for 14 weeks. At the end, energy expenditure, glucose tolerance, adipose tissue inflammation, and vascular function were investigated. RESULTS: Male mice were more sensitive to HFD-induced body weight gain, glucose tolerance, and inflammation than female mice. After establishing obesity, female mice demonstrated increase in energy expenditure, characterized by an increase in heat, whereas male mice did not. Interestingly, obese female mice, but not male, displayed attenuated vascular contractility to different agonists, such difference was blunted by inhibition of Rho-kinase, which was accompanied by a suppressed Rho-kinase activation, measured by Western blot. Finally, aortae from obese male mice displayed an exacerbated inflammation, whereas obese female demonstrated a mild vascular inflammation. CONCLUSION: In obesity, female mice demonstrate a vascular protective mechanism-suppression of vascular Rho-kinase-to minimize the cardiovascular risk associated with obesity, whereas male mice do not generate any adaptive response. Future investigations can help to understand how Rho-kinase becomes suppressed in female during obesity.

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ABSTRACT: Kawasaki disease (KD) is a systemic vasculitis of childhood characterized by vascular damage in the acute stage, which can persist into the late stage. The vascular mechanisms in the cardiovascular risk of KD are not fully studied. We investigated the vascular function and blood pressure in a murine model of KD. We used the Candida albicans water-soluble (CAWS) fraction model. Mice were injected with 4 mg CAWS for 5 consecutive days and separated into three groups. Control, CAWS 7 days (C7), and CAWS 28 days (C28). Hearts and arteries were harvested for vascular characterization. Rat aortic smooth muscle cells were used to studies in vitro. C7 presented elevated inflammatory markers in the coronary area and abdominal aortas, whereas C28 showed severe vasculitis. No difference was found in blood pressure parameters. Vascular dysfunction characterized by higher contractility to norepinephrine in C7 and C28 in aortic rings was abolished by blocking nitric oxide (NO), reactive oxygen species, and cyclooxygenase (COX)-derived products. The CAWS complex increased COX2 expression in rat aortic smooth muscle cells, which was prevented by Toll-like receptor 4 antagonist. Our data indicate that the murine model of KD is associated with vascular dysfunction likely dependent on COX-derived products, oxidant properties, and NO bioavailability. Furthermore, vascular smooth muscle cell may present an important role in the genesis of vascular dysfunction and vasculitis via the Toll-like receptor 4 pathway. Finally, the CAWS model seems not to be appropriate to study KD-associated shock. More studies are necessary to understand whether vascular dysfunction and COXs are triggers for vasculitis.

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Obesity and hypertension are intimately linked due to the various ways that the important cell types such as vascular smooth muscle cells (VSMC), endothelial cells (EC), immune cells, and adipocytes, communicate with one another to contribute to these two pathologies. Adipose tissue is a very dynamic organ comprised primarily of adipocytes, which are well known for their role in energy storage. More recently adipose tissue has been recognized as the largest endocrine organ because of its ability to produce a vast number of signaling molecules called adipokines. These signaling molecules stimulate specific types of cells or tissues with many adipokines acting as indicators of adipocyte healthy function, such as adiponectin, omentin, and FGF21, which show anti-inflammatory or cardioprotective effects, acting as regulators of healthy physiological function. Others, like visfatin, chemerin, resistin, and leptin are often altered during pathophysiological circumstances like obesity and lipodystrophy, demonstrating negative cardiovascular outcomes when produced in excess. This review aims to explore the role of adipocytes and their derived products as well as the impacts of these adipokines on blood pressure regulation and cardiovascular homeostasis.

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BACKGROUND: Chemokine (C- Cmotif) ligand 5 (CCL5) and its receptor C-C motif chemokine receptor 5 (CCR5), have been broadly studied in conjunction with infectious pathogens, however, their involvement in cardiovascular disease is not completely understood. NADPH oxidases (Noxs) are the major source of reactive oxygen species (ROS) in the vasculature. Whether the activation of Noxs is CCL5/CCR5 sensitive and whether such interaction initiates vascular injury is unknown. We investigated whether CCL5/CCR5 leads to vascular damage by activating Noxs. MATERIAL AND METHODS: We used rat aortic smooth muscle cells (RASMC) to investigate the molecular mechanisms by which CCL5 leads to vascular damage and carotid ligation (CL) to analyze the effects of blocking CCR5 on vascular injury. RESULTS: CCL5 induced Nox1 expression in concentration and time-dependent manners, with no changes in Nox2 or Nox4. Maraviroc pre-treatment (CCR5 antagonist, 40uM) blunted CCL5-induced Nox1 expression. Furthermore, CCL5 incubation led to ROS production and activation of Erk1/2 and NFkB, followed by increased vascular cell migration, proliferation, and inflammatory markers. Notably, Nox1 inhibition (GKT771, 10uM) blocked CCL5-dependent effects. In vivo, CL induced pathological vascular remodeling and inflammatory genes and increased Nox1 and CCR5 expression. Maraviroc treatment (25 mg/Kg/day) reduced pathological vascular growth and Nox1 expression. CONCLUSIONS: Our findings suggest that CCL5 activates Nox1 in the vasculature, leading to vascular injury likely via NFkB and Erk1/2. Herein, we place CCR5 antagonists and/or Nox1 inhibitors might be preeminent antiproliferative compounds to reduce the cardiovascular risk associated with medical procedures (e.g. angioplasty) and vascular diseases associated with vascular hyperproliferation.

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