Adipocyte-Mineralocorticoid Receptor Alters Mitochondrial Quality Control Leading to Mitochondrial Dysfunction and Senescence of Visceral Adipose Tissue.

Mineralocorticoid receptor (MR) expression is increased in the adipose tissue (AT) of obese patients and animals. We previously demonstrated that adipocyte-MR overexpression in mice (Adipo-MROE mice) is associated with metabolic alterations. Moreover, we showed that MR regulates mitochondrial dysfunction and cellular senescence in the visceral AT of obese db/db mice. Our hypothesis is that adipocyte-MR overactivation triggers mitochondrial dysfunction and cellular senescence, through increased mitochondrial oxidative stress (OS). Using the Adipo-MROE mice with conditional adipocyte-MR expression, we evaluated the specific effects of adipocyte-MR on global and mitochondrial OS, as well as on OS-induced damage. Mitochondrial function was assessed by high throughput respirometry. Molecular mechanisms were probed in AT focusing on mitochondrial quality control and senescence markers. Adipo-MROE mice exhibited increased mitochondrial OS and altered mitochondrial respiration, associated with reduced biogenesis and increased fission. This was associated with OS-induced DNA-damage and AT premature senescence. In conclusion, targeted adipocyte-MR overexpression leads to an imbalance in mitochondrial dynamics and regeneration, to mitochondrial dysfunction and to ageing in visceral AT. These data bring new insights into the MR-dependent AT dysfunction in obesity.

ER stress and Rho kinase activation underlie the vasculopathy of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) leads to premature stroke and vascular dementia. Mechanism-specific therapies for this aggressive cerebral small vessel disease are lacking. CADASIL is caused by NOTCH3 mutations that influence vascular smooth muscle cell (VSMC) function through unknown processes. We investigated molecular mechanisms underlying the vasculopathy in CADASIL focusing on endoplasmic reticulum (ER) stress and RhoA/Rho kinase (ROCK). Peripheral small arteries and VSMCs were isolated from gluteal biopsies of CADASIL patients and mesentery of TgNotch3R169C mice (CADASIL model). CADASIL vessels exhibited impaired vasorelaxation, blunted vasoconstriction, and hypertrophic remodeling. Expression of NOTCH3 and ER stress target genes was amplified and ER stress response, Rho kinase activity, superoxide production, and cytoskeleton-associated protein phosphorylation were increased in CADASIL, processes associated with Nox5 upregulation. Aberrant vascular responses and signaling in CADASIL were ameliorated by inhibitors of Notch3 (γ-secretase inhibitor), Nox5 (mellitin), ER stress (4-phenylbutyric acid), and ROCK (fasudil). Observations in human CADASIL were recapitulated in TgNotch3R169C mice. These findings indicate that vascular dysfunction in CADASIL involves ER stress/ROCK interplay driven by Notch3-induced Nox5 activation and that NOTCH3 mutation-associated vascular pathology, typical in cerebral vessels, also manifests peripherally. We define Notch3-Nox5/ER stress/ROCK signaling as a putative mechanism-specific target and suggest that peripheral artery responses may be an accessible biomarker in CADASIL.

MR (Mineralocorticoid Receptor) Induces Adipose Tissue Senescence and Mitochondrial Dysfunction Leading to Vascular Dysfunction in Obesity.

Adipose tissue (AT) senescence and mitochondrial dysfunction are associated with obesity. Studies in obese patients and animals demonstrate that the MR (mineralocorticoid receptor) contributes to obesity-associated cardiovascular complications through its specific role in AT. However, underlying mechanisms remain unclear. This study aims to elucidate whether MR regulates mitochondrial function in obesity, resulting in AT premature aging and vascular dysfunction. Obese (db/db) and lean (db/+) mice were treated with an MR antagonist or a specific mitochondria-targeted antioxidant. Mitochondrial and vascular functions were determined by respirometry and myography, respectively. Molecular mechanisms were probed by Western immunoblotting and real-time polymerase chain reaction in visceral AT and arteries and focused on senescence markers and redox-sensitive pathways. db/db mice displayed AT senescence with activation of the p53-p21 pathway and decreased SIRT (sirtuin) levels, as well as mitochondrial dysfunction. Furthermore, the beneficial anticontractile effects of perivascular AT were lost in db/db via ROCK (Rho kinase) activation. MR blockade prevented these effects. Thus, MR activation in obesity induces mitochondrial dysfunction and AT senescence and dysfunction, which consequently increases vascular contractility. In conclusion, our study identifies novel mechanistic insights involving MR, adipose mitochondria, and vascular function that may be of importance to develop new therapeutic strategies to limit obesity-associated cardiovascular complications.

Chemerin receptor blockade improves vascular function in diabetic obese mice via redox-sensitive and Akt-dependent pathways.

Chemerin and its G protein-coupled receptor [chemerin receptor 23 (ChemR23)] have been associated with endothelial dysfunction, inflammation, and insulin resistance. However, the role of chemerin on insulin signaling in the vasculature is still unknown. We aimed to determine whether chemerin reduces vascular insulin signaling and whether there is interplay between chemerin/ChemR23, insulin resistance, and vascular complications associated with type 2 diabetes (T2D). Molecular and vascular mechanisms were probed in mesenteric arteries and cultured vascular smooth muscle cells (VSMCs) from C57BL/6J, nondiabetic lean db/m, and diabetic obese db/db mice as well as in human microvascular endothelial cells (HMECs). Chemerin decreased insulin-induced vasodilatation in C57BL/6J mice, an effect prevented by CCX832 (ChemR23 antagonist) treatment. In VSMCs, chemerin, via oxidative stress- and ChemR23-dependent mechanisms, decreased insulin-induced Akt phosphorylation, glucose transporter 4 translocation to the membrane, and glucose uptake. In HMECs, chemerin decreased insulin-activated nitric oxide signaling. AMP-activated protein kinase phosphorylation was reduced by chemerin in both HMECs and VSMCs. CCX832 treatment of db/db mice decreased body weight, insulin, and glucose levels as well as vascular oxidative stress. CCX832 also partially restored vascular insulin responses in db/db and high-fat diet-fed mice. Our novel in vivo findings highlight chemerin/ChemR23 as a promising therapeutic target to limit insulin resistance and vascular complications associated with obesity-related diabetes. NEW & NOTEWORTHY Our novel findings show that the chemerin/chemerin receptor 23 axis plays a critical role in diabetes-associated vascular oxidative stress and altered insulin signaling. Targeting chemerin/chemerin receptor 23 may be an attractive strategy to improve insulin signaling and vascular function in obesity-associated diabetes.

Minor role of mature adipocyte mineralocorticoid receptor in high fat induced obesity.

Obesity is a major risk factor that contribute to the development of cardiovascular disease and type 2 diabetes. Mineralocorticoid receptor (MR) expression is increased in the adipose tissue of obese patients and several studies provide evidence that MR pharmacological antagonism improves glucose metabolism in genetic and diet-induced mouse models of obesity. In order to investigate whether the lack of adipocyte MR is sufficient to explain these beneficial metabolic effects, we generated a mouse model with inducible adipocyte-specific deletion of Nr3c2 gene encoding MR (adipo-MRKO). We observed a significant, yet not complete, reduction of Nr3c2 transcript and MR protein expression in subcutaneous and visceral adipose depots of adipo-MRKO mice. Notably, only mature adipocyte fraction lacks MR, whereas the stromal vascular fraction maintains normal MR expression in our mouse model. Adipo-MRKO mice fed a 45% high fat diet for 14 weeks did not show any significant difference in body weight and fat mass compared to control littermates. Glucose and insulin tolerance tests revealed that mature adipocyte MR deficiency did not improve insulin sensitivity in response to a metabolic homeostatic challenge. Accordingly, no significant changes were observed in gene expression profile of adipogenic and inflammatory markers in adipose tissue of adipo-MRKO mice. Moreover, pharmacological MR antagonism in mature primary murine adipocytes, which differentiated ex vivo from wild-type mice, did not display any effect on adipokine expression. Taken together, these data demonstrate that the depletion of MR in mature adipocytes displays a minor role in diet-induced obesity and metabolic dysfunctions.

Mitochondrial oxidative stress in obesity: role of the mineralocorticoid receptor.

Obesity is a multifaceted, chronic, low-grade inflammation disease characterized by excess accumulation of dysfunctional adipose tissue. It is often associated with the development of cardiovascular (CV) disorders, insulin resistance and diabetes. Under pathological conditions like in obesity, adipose tissue secretes bioactive molecules called 'adipokines', including cytokines, hormones and reactive oxygen species (ROS). There is evidence suggesting that oxidative stress, in particular, the ROS imbalance in adipose tissue, may be the mechanistic link between obesity and its associated CV and metabolic complications. Mitochondria in adipose tissue are an important source of ROS and their dysfunction contributes to the pathogenesis of obesity-related type 2 diabetes. Mitochondrial function is regulated by several factors in order to preserve mitochondria integrity and dynamics. Moreover, the renin-angiotensin-aldosterone system is over-activated in obesity. In this review, we focus on the pathophysiological role of the mineralocorticoid receptor in the adipose tissue and its contribution to obesity-associated metabolic and CV complications. More specifically, we discuss whether dysregulation of the mineralocorticoid system within the adipose tissue may be the upstream mechanism and one of the early events in the development of obesity, via induction of oxidative stress and mitochondrial dysfunction, thus impacting on systemic metabolism and the CV system.

Vascular dysfunction in obese diabetic db/db mice involves the interplay between aldosterone/mineralocorticoid receptor and Rho kinase signaling.

Activation of aldosterone/mineralocorticoid receptors (MR) has been implicated in vascular dysfunction of diabetes. Underlying mechanisms are elusive. Therefore, we investigated the role of Rho kinase (ROCK) in aldosterone/MR signaling and vascular dysfunction in a model of diabetes. Diabetic obese mice (db/db) and control counterparts (db/+) were treated with MR antagonist (MRA, potassium canrenoate, 30 mg/kg/day, 4 weeks) or ROCK inhibitor, fasudil (30 mg/kg/day, 3 weeks). Plasma aldosterone was increased in db/db versus db/+. This was associated with enhanced vascular MR signaling. Norepinephrine (NE)-induced contraction was increased in arteries from db/db mice. These responses were attenuated in mice treated with canrenoate or fasudil. Db/db mice displayed hypertrophic remodeling and increased arterial stiffness, improved by MR blockade. Vascular calcium sensitivity was similar between depolarized arteries from db/+ and db/db. Vascular hypercontractility in db/db mice was associated with increased myosin light chain phosphorylation and reduced expression of PKG-1α. Vascular RhoA/ROCK signaling and expression of pro-inflammatory and pro-fibrotic markers were exaggerated in db/db mice, effects that were attenuated by MRA. Fasudil, but not MRA, improved vascular insulin sensitivity in db/db mice, evidenced by normalization of Irs1 phosphorylation. Our data identify novel pathways involving MR-RhoA/ROCK-PKG-1 that underlie vascular dysfunction and injury in diabetic mice.

Benefit of Mineralocorticoid Receptor Antagonism in AKI: Role of Vascular Smooth Muscle Rac1.

AKI is a frequent complication in hospitalized patients. Unfortunately, there is no effective pharmacologic approach for treating or preventing AKI. In rodents, mineralocorticoid receptor (MR) antagonism prevents AKI induced by ischemia-reperfusion (IR). We investigated the specific role of vascular MR in mediating AKI induced by IR. We also assessed the protective effect of MR antagonism in IR-induced AKI in the Large White pig, a model of human AKI. In mice, MR deficiency in smooth muscle cells (SMCs) protected against kidney IR injury. MR blockade by the novel nonsteroidal MR antagonist, finerenone, or genetic deletion of MR in SMCs associated with weaker oxidative stress production. Moreover, ischemic kidneys had higher levels of Rac1-GTP, required for NADPH oxidase activation, than sham control kidneys, and genetic deletion of Rac1 in SMCs protected against AKI. Furthermore, genetic deletion of MR in SMCs blunted the production of Rac1-GTP after IR. Pharmacologic inhibition of MR also prevented AKI induced by IR in the Large White pig. Altogether, we show that MR antagonism, or deletion of the MR gene in SMCs, limited the renal injury induced by IR through effects on Rac1-mediated MR signaling. The benefits of MR antagonism in the pig provide a rational basis for future clinical trials assessing the benefits of this approach in patients with IR-mediated AKI.

Brown Adipose Tissue Regulates Small Artery Function Through NADPH Oxidase 4-Derived Hydrogen Peroxide and Redox-Sensitive Protein Kinase G-1α.

OBJECTIVE: Biomedical interest in brown adipose tissue (BAT) has increased since the discovery of functionally active BAT in adult humans. Although white adipose tissue (WAT) influences vascular function, vascular effects of BAT are elusive. Thus, we investigated the regulatory role and putative vasoprotective effects of BAT, focusing on hydrogen peroxide, nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4), and redox-sensitive signaling. APPROACH AND RESULTS: Vascular reactivity was assessed in wild-type and Nox4-knockout mice (Nox4-/-) by wire myography in the absence and presence of perivascular adipose tissue of different phenotypes from various adipose depots: (1) mixed WAT/BAT (inguinal adipose tissue) and (2) WAT (epididymal visceral fat) and BAT (intrascapular fat). In wild-type mice, epididymal visceral fat and perivascular adipose tissue increased EC50 to noradrenaline without affecting maximum contraction. BAT increased EC50 and significantly decreased maximum contraction, which were prevented by a hydrogen peroxide scavenger (polyethylene glycated catalase) and a specific cyclic GMP-dependent protein kinase G type-1α inhibitor (DT-3), but not by inhibition of endothelial nitric oxide synthase or guanylate cyclase. BAT induced dimerization of cyclic GMP-dependent protein kinase G type-1α and reduced phosphorylation of myosin light chain phosphatase subunit 1 and myosin light chain 20. BAT from Nox4-knockout mice displayed reduced hydrogen peroxide levels and no anticontractile effects. Perivascular adipose tissue from ß3 agonist-treated mice displayed browned perivascular adipose tissue and an increased anticontractile effect. CONCLUSIONS: We identify a novel vasoprotective action of BAT through an anticontractile effect that is mechanistically different to WAT. Specifically, BAT, via Nox4-derived hydrogen peroxide, induces cyclic GMP-dependent protein kinase G type-1α activation, resulting in reduced vascular contractility. BAT may constitute an interesting therapeutic target to restore vascular function and prevent vascular complications in cardiovascular diseases.

Adipocyte-Specific Mineralocorticoid Receptor Overexpression in Mice Is Associated With Metabolic Syndrome and Vascular Dysfunction: Role of Redox-Sensitive PKG-1 and Rho Kinase.

Mineralocorticoid receptor (MR) expression is increased in adipose tissue from obese individuals and animals. We previously demonstrated that adipocyte-MR overexpression (Adipo-MROE) in mice is associated with metabolic changes. Whether adipocyte MR directly influences vascular function in these mice is unknown. We tested this hypothesis in resistant mesenteric arteries from Adipo-MROE mice using myography and in cultured adipocytes. Molecular mechanisms were probed in vessels/vascular smooth muscle cells and adipose tissue/adipocytes and focused on redox-sensitive pathways, Rho kinase activity, and protein kinase G type-1 (PKG-1) signaling. Adipo-MROE versus control-MR mice exhibited reduced vascular contractility, associated with increased generation of adipocyte-derived hydrogen peroxide, activation of vascular redox-sensitive PKG-1, and downregulation of Rho kinase activity. Associated with these vascular changes was increased elastin content in Adipo-MROE. Inhibition of PKG-1 with Rp-8-Br-PET-cGMPS normalized vascular contractility in Adipo-MROE. In the presence of adipocyte-conditioned culture medium, anticontractile effects of the adipose tissue were lost in Adipo-MROE mice but not in control-MR mice. In conclusion, adipocyte-MR upregulation leads to impaired contractility with preserved endothelial function and normal blood pressure. Increased elasticity may contribute to hypocontractility. We also identify functional cross talk between adipocyte MR and arteries and describe novel mechanisms involving redox-sensitive PKG-1 and Rho kinase. Our results suggest that adipose tissue from Adipo-MROE secrete vasoactive factors that preferentially influence vascular smooth muscle cells rather than endothelial cells. Our findings may be important in obesity/adiposity where adipocyte-MR expression/signaling is amplified and vascular risk increased.

Chemerin Regulates Crosstalk Between Adipocytes and Vascular Cells Through Nox.

Adipocytes produce adipokines, including chemerin, a chemoattractant that mediates effects through its ChemR23 receptor. Chemerin has been linked to endothelial dysfunction and vascular injury in pathological conditions, such as obesity, diabetes mellitus, and hypertension. Molecular mechanisms underlying this are elusive. Here we assessed whether chemerin through redox-sensitive signaling influences molecular processes associated with vascular growth, apoptosis, and inflammation. Human microvascular endothelial cells and vascular smooth muscle cells were stimulated with chemerin (50 ng/mL). Chemerin increased generation of reactive oxygen species and phosphorylation of mitogen-activated protein kinases, effects that were inhibited by ML171, GKT137831 (Nox inhibitors), and N-acetylcysteine (reactive oxygen species scavenger). Chemerin increased mRNA expression of proinflammatory mediators in vascular cells and increased monocyte-to-endothelial cell attachment. In human vascular smooth muscle cells, chemerin induced phosphorylation of mitogen-activated protein kinases and stimulated proliferation (increased proliferating cell nuclear antigen expression [proliferation marker] and BrdU incorporation [proliferation assay]). Chemerin decreased phosphatidylinositol 3-kinase/protein kinase B activation and increased TUNEL-positive human vascular smooth muscle cells. In human microvascular endothelial cells, chemerin reduced endothelial nitric oxide synthase activity and nitric oxide production. Adipocyte-conditioned medium from obese/diabetic mice (db/db), which have elevated chemerin levels, increased reactive oxygen species generation in vascular smooth muscle cells, whereas adipocyte-conditioned medium from control mice had no effect. Chemerin actions were blocked by CCX 832, a ChemR23 inhibitor. Our data demonstrate that chemerin, through Nox activation and redox-sensitive mitogen-activated protein kinases signaling, exerts proapoptotic, proinflammatory, and proliferative effects in human vascular cells. These findings elucidate some molecular mechanisms through chemerin, which is increased in obesity, whereby adipocytes may influence vascular function. We identify chemerin as a novel vasoactive adipokine, which may be important in obesity-related vascular injury.

Adipocyte Mineralocorticoid Receptor Activation Leads to Metabolic Syndrome and Induction of Prostaglandin D2 Synthase.

Metabolic syndrome is a major risk factor for the development of diabetes mellitus and cardiovascular diseases. Pharmacological antagonism of the mineralocorticoid receptor (MR), a ligand-activated transcription factor, limits metabolic syndrome in preclinical models, but mechanistic studies are lacking to delineate the role of MR activation in adipose tissue. In this study, we report that MR expression is increased in visceral adipose tissue in a preclinical mouse model of metabolic syndrome and in obese patients. In vivo conditional upregulation of MR in mouse adipocytes led to increased weight and fat mass, insulin resistance, and metabolic syndrome features without affecting blood pressure. We identified prostaglandin D2 synthase as a novel MR target gene in adipocytes and AT56, a specific inhibitor of prostaglandin D2 synthase enzymatic activity, blunted adipogenic aldosterone effects. Moreover, translational studies showed that expression of MR and prostaglandin D2 synthase is strongly correlated in adipose tissues from obese patients.

Mineralocorticoid receptor blockade prevents vascular remodelling in a rodent model of type 2 diabetes mellitus.

Mineralocorticoid receptors (MRs), which are activated by mineralocorticoids and glucocorticoids, actively participate in mechanisms that affect the structure and function of blood vessels. Although experimental and clinical evidence shows that vascular damage in diabetes is associated with structural alterations in large and small arteries, the role of MR in this process needs further studies. Thus, we tested the hypothesis that MR, through redox-sensitive mechanisms, plays a role in diabetes-associated vascular remodelling. Male, 12-14-weeks-old db/db mice, a model of type 2 diabetes and their non-diabetic counterpart controls (db/+) were treated with spironolactone (MR antagonist, 50 mg/kg/day) or vehicle for 6 weeks. Spironolactone treatment did not affect blood pressure, fasting glucose levels or weight gain, but increased serum potassium and total cholesterol in both, diabetic and control mice. In addition, spironolactone significantly reduced serum insulin levels, but not aldosterone levels in diabetic mice. Insulin sensitivity, evaluated by the HOMA (homoeostatic model assessment)-index, was improved in spironolactone-treated diabetic mice. Mesenteric resistance arteries from vehicle-treated db/db mice exhibited inward hypertrophic remodelling, increased number of smooth muscle cells and increased vascular stiffness. These structural changes, determined by morphometric analysis and with a myography for pressurized arteries, were prevented by spironolactone treatment. Arteries from vehicle-treated db/db mice also exhibited augmented collagen content, determined by Picrosirius Red staining and Western blotting, increased reactive oxygen species (ROS) generation, determined by dihydroethidium (DHE) fluorescence, as well as increased expression of NAD(P)H oxidases 1 and 4 and increased activity of mitogen-activated protein kinases (MAPKs). Spironolactone treatment prevented all these changes, indicating that MR importantly contributes to diabetes-associated vascular dysfunction by inducing oxidative stress and by increasing the activity of redox-sensitive proteins.

Cholesteryl ester-transfer protein inhibitors stimulate aldosterone biosynthesis in adipocytes through Nox-dependent processes.

Hyperaldosteronism and hypertension were unexpected side effects observed in trials of torcetrapib, a cholesteryl ester-transfer protein (CETP) inhibitor that increases high-density lipoprotein. Given that CETP inhibitors are lipid soluble, accumulate in adipose tissue, and have binding sites for proteins involved in adipogenesis, and that adipocytes are a source of aldosterone, we questioned whether CETP inhibitors (torcetrapib, dalcetrapib, and anacetrapib) influence aldosterone production by adipocytes. Studies were performed using human adipocytes (SW872), which express CETP, and mouse adipocytes (3T3-L1), which lack the CETP gene. Torcetrapib, dalcetrapib, and anacetrapib increased expression of CYP11B2, CYP11B1, and steroidogenic acute regulatory protein, enzymes involved in mineralocorticoid and glucocorticoid generation. These effects were associated with increased reactive oxygen species formation. Torcetrapib, dalcetrapib, and anacetrapib upregulated signal transducer and activator of transcription 3 (STAT3) and peroxisome proliferation-activated receptor-γ, important in adipogenesis, but only torcetrapib stimulated production of chemerin, a proinflammatory adipokine. To determine mechanisms whereby CETP inhibitors mediate effects, cells were pretreated with inhibitors of Nox1/Nox4 [GKT137831; 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione], Nox1 (ML171 [2-acetylphenothiazine]), mitochondria (rotenone), and STAT3 (S3I-201 [2-hydroxy-4-(((4-methylphenyl)sulfonyloxy)acetyl)amino)-benzoic acid]). In torcetrapib-stimulated cells, Nox inhibitors, rotenone, and S3I-201 downregulated CYP11B2 and steroidogenic acute regulatory protein and reduced aldosterone. Dalcetrapib and anacetrapib effects on aldosterone were variably blocked by GKT137831, ML171, rotenone, and S3I-201. In adipocytes, torcetrapib, dalcetrapib, and anacetrapib inhibit enzymatic pathways responsible for aldosterone production through Nox1/Nox4- and mitochondrial-generated reactive oxygen species and STAT3. CETP inhibitors also influence adipokine production. These processes may be CETP independent. Our findings identify novel adipocyte-related mechanisms whereby CETP inhibitors increase aldosterone production. Such phenomena may contribute to hyperaldosteronism observed in CETP inhibitor clinical trials.

Vascular injury in diabetic db/db mice is ameliorated by atorvastatin: role of Rac1/2-sensitive Nox-dependent pathways.

Oxidative stress [increased bioavailability of reactive oxygen species (ROS)] plays a role in the endothelial dysfunction and vascular inflammation, which underlie vascular damage in diabetes. Statins are cholesterol-lowering drugs that are vasoprotective in diabetes through unknown mechanisms. We tested the hypothesis that atorvastatin decreases NADPH oxidase (Nox)-derived ROS generation and associated vascular injury in diabetes. Lepr(db)/Lepr(db) (db/db) mice, a model of Type 2 diabetes and control Lepr(db)/Lepr(+) (db/+) mice were administered atorvastatin (10 mg/kg per day, 2 weeks). Atorvastatin improved glucose tolerance in db/db mice. Systemic and vascular oxidative stress in db/db mice, characterized by increased plasma TBARS (thiobarbituric acid-reactive substances) levels and exaggerated vascular Nox-derived ROS generation respectively, were inhibited by atorvastatin. Cytosol-to-membrane translocation of the Nox regulatory subunit p47(phox) and the small GTPase Rac1/2 was increased in vessels from db/db mice compared with db/+ mice, an effect blunted by atorvastatin. The increase in vascular Nox1/2/4 expression and increased phosphorylation of redox-sensitive mitogen-activated protein kinases (MAPKs) was abrogated by atorvastatin in db/db mice. Pro-inflammatory signalling (decreased IκB-α and increased NF-κB p50 expression, increased NF-κB p65 phosphorylation) and associated vascular inflammation [vascular cell adhesion molecule-1 (VCAM-1) expression and vascular monocyte adhesion], which were increased in aortas of db/db mice, were blunted by atorvastatin. Impaired acetylcholine (Ach)- and insulin (INS)-induced vasorelaxation in db/db mice was normalized by atorvastatin. Our results demonstrate that, in diabetic mice, atorvastatin decreases vascular oxidative stress and inflammation and ameliorates vascular injury through processes involving decreased activation of Rac1/2 and Nox. These findings elucidate redox-sensitive and Rac1/2-dependent mechanisms whereby statins protect against vascular injury in diabetes.

Crosstalk between adipose tissue and blood vessels in cardiometabolic syndrome: implication of steroid hormone receptors (MR/GR).

Crosstalk between adipose tissue and blood vessels is vital to vascular homeostasis and is disturbed in cardiovascular and metabolic diseases such as hypertension, diabetes and obesity. Cardiometabolic syndrome (CMS) refers to the clustering of obesity-related metabolic disorders such as insulin resistance, glucose and lipid profile alterations, hypertension and cardiovascular diseases. Mechanisms underlying these associations remain unclear. Adipose tissue associated with the vasculature [known as perivascular adipose tissue (PVAT)] has been shown to produce myriads of adipose tissue-derived substances called adipokines, including hormones, cytokines and reactive oxygen species (ROS), which actively participate in the regulation of vascular function and local inflammation by endocrine and/or paracrine mechanisms. As a result, the signaling from PVAT to the vasculature is emerging as a potential therapeutic target for obesity and diabetes-related vascular dysfunction. Accumulating evidence supports a shift in our understanding of the crucial role of elevated plasma levels of aldosterone in obesity, promoting insulin resistance and hypertension. In obesity, aldosterone/mineralocorticoid receptor (MR) signaling induces an abnormal secretion of adipokines, ROS production and systemic inflammation, which in turn contribute to impaired insulin signaling, reduced endothelial-mediated vasorelaxation, and associated cardiovascular abnormalities. Thus, aldosterone excess exerts detrimental metabolic and vascular effects that participate to the development of the CMS and its associated cardiovascular abnormalities. In this review, we focus on the physiopathological roles of corticosteroid receptors in the interplay between PVAT and the vasculature, which underlies their potential as key regulators of vascular function.

Angiotensin II and vascular injury.

Vascular injury, characterized by endothelial dysfunction, structural remodelling, inflammation and fibrosis, plays an important role in cardiovascular diseases. Cellular processes underlying this include altered vascular smooth muscle cell (VSMC) growth/apoptosis, fibrosis, increased contractility and vascular calcification. Associated with these events is VSMC differentiation and phenotypic switching from a contractile to a proliferative/secretory phenotype. Inflammation, associated with macrophage infiltration and increased expression of redox-sensitive pro-inflammatory genes, also contributes to vascular remodelling. Among the many factors involved in vascular injury is Ang II. Ang II, previously thought to be the sole biologically active downstream peptide of the renin-angiotensin system (RAS), is converted to smaller peptides, [Ang III, Ang IV, Ang-(1-7)], that are functional and that modulate vascular tone and structure. The actions of Ang II are mediated via signalling pathways activated upon binding to AT1R and AT2R. AT1R activation induces effects through PLC-IP3-DAG, MAP kinases, tyrosine kinases, tyrosine phosphatases and RhoA/Rho kinase. Ang II elicits many of its (patho)physiological actions by stimulating reactive oxygen species (ROS) generation through activation of vascular NAD(P)H oxidase (Nox). ROS in turn influence redox-sensitive signalling molecules. Here we discuss the role of Ang II in vascular injury, focusing on molecular mechanisms and cellular processes. Implications in vascular remodelling, inflammation, calcification and atherosclerosis are highlighted.

Endothelial mineralocorticoid receptor activation enhances endothelial protein C receptor and decreases vascular thrombosis in mice.

Previous studies have shown that aldosterone, which activates the mineralocorticoid receptor (MR), promotes thrombosis in animal models. Our objective was to determine whether MR activation/expression in the vascular endothelium could modify thrombotic risk in vivo and to examine thrombin generation at the surface of aortic endothelial cells (HAECs). MR was conditionally overexpressed in vivo in vascular endothelial cells in mice (MR-EC mice) or stimulated with aldosterone in HAECs. Thrombosis after ferric chloride injury was delayed in MR-EC mice compared with controls as well as in wild-type FVB/NRj mice treated with aldosterone (60 µg/kg/d for 21 d). Thrombin generation in platelet-poor plasma did not differ between MR-EC mice and controls. In MR-EC mice, aortic endothelial cell protein C receptor (EPCR) expression was increased. Aldosterone (10(-8) M) attenuated thrombin generation at the surface of cultured HAECs, and this effect was associated with up-regulation of expression of EPCR, which promotes formation of activated protein C. Aldosterone increases EPCR expression via a transcriptional mechanism involving interaction of MR with the specificity protein 1 site. These findings demonstrate that MR activation acts on endothelial cells to protect against thrombosis in physiological conditions and that MR-mediated EPCR overexpression drives this antithrombotic property through enhancing protein C activation.

Renoprotective effects of a novel Nox1/4 inhibitor in a mouse model of Type 2 diabetes.

Nox (NADPH oxidase)-derived ROS (reactive oxygen species) have been implicated in the development of diabetic nephropathy. Of the Nox isoforms in the kidney, Nox4 is important because of its renal abundance. In the present study, we tested the hypothesis that GKT136901, a Nox1/4 inhibitor, prevents the development of nephropathy in db/db (diabetic) mice. Six groups of male mice (8-week-old) were studied: (i) untreated control db/m, (ii) low-dose GKT136901-treated db/m (30 mg/kg of body weight per day), (iii) high-dose GKT136901-treated db/m (90 mg/kg of body weight per day), (iv) untreated db/db; (v) low dose GKT136901-treated db/db; and (vi) high-dose GKT136901-treated db/db. GKT136901, in chow, was administered for 16 weeks. db/db mice developed diabetes and nephropathy as evidenced by hyperglycaemia, albuminuria and renal injury (mesangial expansion, tubular dystrophy and glomerulosclerosis). GKT136901 treatment had no effect on plasma glucose or BP (blood pressure) in any of the groups. Plasma and urine TBARSs (thiobarbituric acid-reacting substances) levels, markers of systemic and renal oxidative stress, respectively, were increased in diabetic mice. Renal mRNA expression of Nox4, but not of Nox2, increased, Nox1 was barely detectable in db/db. Expression of the antioxidant enzyme SOD-1 (superoxide dismutase 1) decreased in db/db mice. Renal content of fibronectin, pro-collagen, TGFß (transforming growth factor ß) and VCAM-1 (vascular cell adhesion molecule 1) and phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2) were augmented in db/db kidneys, with no change in p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase). Treatment reduced albuminuria, TBARS and renal ERK1/2 phosphorylation and preserved renal structure in diabetic mice. Our findings suggest a renoprotective effect of the Nox1/4 inhibitor, possibly through reduced oxidative damage and decreased ERK1/2 activation. These phenomena occur independently of improved glucose control, suggesting GKT136901-sensitive targets are involved in complications of diabetes rather than in the disease process.

Angiotensin II, NADPH oxidase, and redox signaling in the vasculature.

SIGNIFICANCE: Angiotensin II (Ang II) influences the function of many cell types and regulates many organ systems, in large part through redox-sensitive processes. In the vascular system, Ang II is a potent vasoconstrictor and also promotes inflammation, hypertrophy, and fibrosis, which are important in vascular damage and remodeling in cardiovascular diseases. The diverse actions of Ang II are mediated via Ang II type 1 and Ang II type 2 receptors, which couple to various signaling molecules, including NADPH oxidase (Nox), which generates reactive oxygen species (ROS). ROS are now recognized as signaling molecules, critically placed in pathways activated by Ang II. Mechanisms linking Nox and Ang II are complex and not fully understood. RECENT ADVANCES: Ang II regulates vascular cell production of ROS through various recently characterized Noxs, including Nox1, Nox2, Nox4, and Nox5. Activation of these Noxs leads to ROS generation, which in turn influences many downstream signaling targets of Ang II, including MAP kinases, RhoA/Rho kinase, transcription factors, protein tyrosine phosphatases, and tyrosine kinases. Activation of these redox-sensitive pathways regulates vascular cell growth, inflammation, contraction, and senescence. CRITICAL ISSUES: Although there is much evidence indicating a role for Nox/ROS in Ang II function, there is still a paucity of information on how Ang II exerts cell-specific effects through ROS and how Nox isoforms are differentially regulated by Ang II. Moreover, exact mechanisms whereby ROS induce oxidative modifications of signaling molecules mediating Ang II actions remain elusive. FUTURE DIRECTIONS: Future research should elucidate these issues to better understand the significance of Ang II and ROS in vascular (patho) biology.