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.

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.

Rationale: Despite increasing understanding of the prognostic importance of vascular stiffening linked to perivascular fibrosis in hypertension, the molecular and cellular regulation of this process is poorly understood. Objectives: To study the functional role of microRNA-214 (miR-214) in the induction of perivascular fibrosis and endothelial dysfunction driving vascular stiffening. Methods and Results: Out of 381 miRs screened in the perivascular tissues in response to Ang II (angiotensin II)-mediated hypertension, miR-214 showed the highest induction (8-fold, P =0.0001). MiR-214 induction was pronounced in perivascular and circulating T cells, but not in perivascular adipose tissue adipocytes. Global deletion of miR-214 − /− prevented Ang II-induced periaortic fibrosis, Col1a1 , Col3a1, Col5a1 , and Tgfb1 expression, hydroxyproline accumulation, and vascular stiffening, without difference in blood pressure. Mechanistic studies revealed that miR-214 −/− mice were protected against endothelial dysfunction, oxidative stress, and increased Nox2, all of which were induced by Ang II in WT mice. Ang II-induced recruitment of T cells into perivascular adipose tissue was abolished in miR-214 −/− mice. Adoptive transfer of miR-214 −/− T cells into RAG1 −/− mice resulted in reduced perivascular fibrosis compared with the effect of WT T cells. Ang II induced hypertension caused significant change in the expression of 1380 T cell genes in WT, but only 51 in miR-214 −/− . T cell activation, proliferation and chemotaxis pathways were differentially affected. MiR-214 −/− prevented Ang II-induction of profibrotic T cell cytokines ( IL-17 , TNF-α, IL-9 , and IFN-γ ) and chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR6, and CXCR3). This manifested in reduced in vitro and in vivo T cell chemotaxis resulting in attenuation of profibrotic perivascular inflammation. Translationally, we show that miR-214 is increased in plasma of patients with hypertension and is directly correlated to pulse wave velocity as a measure of vascular stiffness. Conclusions: T-cell–derived miR-214 controls pathological perivascular fibrosis in hypertension mediated by T cell recruitment and local profibrotic cytokine release.

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.

Obesity is a major risk factor that contributes 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 WT 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.

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.

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.

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.

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.

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 EC 50 to noradrenaline without affecting maximum contraction. BAT increased EC 50 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 β 3 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.

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.

Objective: Hypertension (HT) is associated with perivascular inflammation and increased vascular fibrosis. MicroRNAs (miR) are a novel gene expression regulation mechanism and play a pivotal role in a range of pathological processes. The role and mechanism of miR214 in vascular fibrosis is unknown. Methods: 3-month-old C57BL/6, miR214KO and wild-type littermates were treated with angiotensin II (AngII, 490ng/kg/min; n=6-10) or control buffer for 14 days. PVATs from C57BL/6 animals were analysed using TaqMan_Rodent_microRNA_Arrays. Histological studies, wire myography, lucigenin-enhanced luminometry and cytometrical analysis was conducted, followed by statistical analysis with ANOVA or t-test. Data are expressed as a mean±SEM. Results: Out of 381 miRs, 16 were significantly overexpressed in C57BL/6 AngII animals, with only miR214 showing 8-fold induction (p<0.01) after Bonferroni correction. Also, 3-fold elevation of pri-miR-214 was observed. Interestingly, hydralazine treatment prevented both these changes (p<0.01). AngII infusion in miR214 KO animals did not alter blood pressure when compared to WT mice. Mir214 KOs exhibited diminished peri-aortic fibrosis (44779±2491 vs 78805±8696μm, p<0.01), upon AngII hypertension. This was associated with a significantly reduced induction of COL1A1, COL3A1 and TGFβ1 mRNA expression in PVAT and aortas (p<0.05). Vascular studies revealed improved endothelial function (69±10 vs. 22±4%, p<0.01), protection against oxidative stress (66±7 vs 118±19 RLU/sec/mg, p<0.001) and NOX2 mRNA expression (1.9±0.2 vs1.1±0.1, p<0.05) in AngII miR-214-KO aortas, while these parameters were not altered in mesenteric arteries. Recruitment of T cells into aortic PVAT was abolished in KO HT animals in comparison to control group (192±65 vs. 603±164 cell/mg; p<0.05). AngII HT was associated with 4-fold increase of miR-214 expression in the circulating peripheral blood T cells and 2-fold in the spleen. Moreover, AngII infusion increased TNFα mRNA expression in WT T cells (1±0.1 vs 1.6±0, p<0.01) whereas this effect was not seen in miR214 KO T cells (0.9±0.3 vs 0.9±0.1). Conclusions: MiR-214 plays a major role in modulation of aortic fibrosis, vascular function, oxidative stress and perivascular inflammation.

The study of adipose tissue and more specifically of adipocytes is considered pivotal for dissecting molecular mechanisms responsible for alterations in several organs and systems, including adipose tissue, not only in obesity but also in other diseases (hypertension, heart failure). Adipose tissue is a complex tissue composed of adipocytes and the stromal vascular fraction which includes a heterogeneous population of preadipocytes, blood cells, endothelial cells, and macrophages. In the present chapter, methods are detailed to generate purified mature adipocytes from white adipose tissue by using enzymatic digestion. Such methods should help laboratories to study the specific roles of adipocytes in different pathologies and are easily adaptable to different animal models. Moreover, as gene activity is controlled at both transcriptional and posttranscriptional levels, it is very important to determine the levels of messenger ribonucleic acid (mRNA) of genes of interest. This process involves the isolation of total RNA and subsequent analysis of the mRNA of interest by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Given the unique nature of adipose tissue and adipocytes (i.e., containing high amounts of lipid), we have set up a special RNA isolation technique in both white adipose tissue and isolated mature adipocytes from mice. In summary, isolation and culture of adipocytes in vivo and gene expression studies will help to understand the mechanisms that control adipocyte function in physiological and pathological states and may lead to design interventions that might affect the adipocyte birth-death balance or phenotype.

Objective: Visceral adipose tissue (VAT) influences vascular function through aldosterone and mineralocorticoid receptors (MR). We demonstrated that adipocyte MR over-activation in mice leads to metabolic changes. Whether this is associated with vascular dysfunction remains unclear. Design and method: To address this, we generated mice with conditional MR overexpression targeted to adipocytes (Adipo-MROE) and studied the effect of fat conditioned medium (Fcm, collected from visceral adipose tissue) on vascular reactivity of resistant mesenteric arteries and associated molecular mechanisms. Endothelial-dependent relaxation to acetylcholine (ACh), in the presence or absence of Fcm, was assessed by wire myography. Levels of reactive oxygen species (ROS) were determined in mitochondria isolated from adipose tissues (mito-AT) by electron paramagnetic spin resonance and Amplex Red kit. Inhibitors of NADPH oxidases and mitochondrial ROS were used in our experiments. Results: We previously showed that 1) ROS were increased in adipose tissues from Adipo-MROE vs control (CTR) mice; b) Fcm from Adipo-MROE (Fcm-MR) induced endothelial dysfunction in control arterie, through redox-sensitive mechanisms. Here, we identified the source of ROS in adipocytes, with the hypothesis that NADPH oxidases and mitochondria may play an important role. Levels of superoxide were 3 fold-increased in mito-AT from Adipo-MROE compared to CTR mice (n = 7 mice per group, p < 0.01). We pre-incubated Fcm-MR with either the inhibitor of NADPH oxidase isoforms 1 and 2 (ML171, 10–6 M), either a specific inhibitor of mitochondrial-derived ROS (mito-TEMPO, 10–7 M), and assessed ACh-induced relaxation (10–6 M) in CTR arteries. Normal relaxation was restored by mito-TEMPO (% relaxation: CTR+Fcm-MR, without vs with mito-TEMPO: 47.5 ± 4.4 vs 87.1 ± 2.2, n = 6 mice per group, p < 0.05), whereas Nox1/2 inhibitor-ML171 did not improve endothelial function (% relaxation: 47.7 ± 4.2, ns). Conclusions: Our data demonstrate that adipocyte MR over-activation leads to increased mitochondrial-derived ROS generation in visceral adipose tissue, which contributes to endothelial dysfunction. We describe a novel mechanism that directly links adipose tissue and vascular function. Our study identifies novel mechanisms linking vascular/adipose tissue biology and aldosterone/MR activation, which may be particularly important in vascular dysfunction associated with metabolic syndrome.

Hypertension (HT) and CADASIL are clinical conditions of small vessel disease. Vascular dementia is a major feature in CADASIL, and a serious consequence of HT. CADASIL is a monogenic condition due to mutations in NOTCH3 , which is expressed almost exclusively in VSMCs. We hypothesised that altered NOTCH3 signalling in CADASIL and HT are associated with small vessel disease. Small arteries from gluteal biopsies from CADASIL patients (n=14), HT patients (n=3) and healthy controls (n=10) were investigated. Vascular function was assessed by myography. Cultured VSMCs were used to assess signaling through NOTCH3, NO, ER stress (gene array) and Rho kinase (ELISA). CADASIL and HT patients exhibited endothelial dysfunction (Max response: CADASIL 41.7±3%, HT 54.1±2% vs Control 98.2±4%). Pre-incubation with N-acetyl-cysteine ameliorated vasorelaxation. Only CADASIL displayed impaired endothelium-independent relaxation (Max response: CADASIL 53±1.9% vs Control 93±8.9%) and contraction (Max response: CADASIL 78±1.3% vs control 102±5%) (p<0.05). AngII-induced contraction was elevated in HT (98%), yet reduced in CADASIL (28%) (vs control 64% max contraction: p<0.05), despite VSMCs from both conditions displaying increased AT 1 R mRNA expression (HT: 5.1; CADASIL: 3.8; fold vs control; p<0.05). VSMCs from CADASIL and HT have decreased expression of CAMK1, SIRT2 and VEGFA; important in NO signalling (0.5 fold; p<0.05 vs control). VSMC levels of NOTCH3 and NOTCH ligand, JAG1, were increased in CADASIL (3.5, 2.5 fold) and HT (3.0, 2.6 fold, p<0.05). Downstream targets, HEY1 and HEYL, were elevated in CADASIL (3.8, 4.2 fold) and HT (1.9, 2.6 fold) (p<0.05). CADASIL but not HT VSMCs exhibited increased expression of ER stress markers. Rho kinase activity was increased in VSMCs from CADASIL (2.5 fold) and HT (2 fold) vs control (p<0.05). These data demonstrate that in CADASIL and HT, vascular dysfunction, is associated with aberrant NOTCH3 and Rho kinase signalling. In CADASIL, but not HT, endothelium-independent relaxation and ER stress were increased. Our results demonstrate a putative role for NOTCH3 -Rho kinase in vascular dysfunction in conditions of small vessel disease and suggest that ER stress and oxidative stress may be important in vascular injury in CADASIL.

High doses of Ang II receptor (AT1R) blockers (ARBs) are renoprotective in diabetes. Underlying mechanisms remain unclear. We evaluated whether high/ultra-high doses of candesartan (ARB) up-regulate angiotensin-converting enzyme 2 (ACE2)/Ang II type 2 receptor (AT2R)/Mas receptor [protective axis of the of the renin–angiotensin system (RAS)] in diabetic mice. Systolic blood pressure (SBP), albuminuria and expression/activity of RAS components were assessed in diabetic db/db and control db/+ mice treated with increasing candesartan doses (intermediate, 1 mg/kg/d; high, 5 mg/kg/d; ultra-high, 25 and 75 mg/kg/d; 4 weeks). Lower doses candesartan did not influence SBP, but ultra-high doses reduced SBP in both groups. Plasma glucose and albuminuria were increased in db/db compared with db/+ mice. In diabetic mice treated with intermediate dose candesartan, renal tubular damage and albuminuria were ameliorated and expression of ACE2, AT2R and Mas and activity of ACE2 were increased, effects associated with reduced ERK1/2 phosphorylation, decreased fibrosis and renal protection. Ultra-high doses did not influence the ACE2/AT2R/Mas axis and promoted renal injury with increased renal ERK1/2 activation and exaggerated fibronectin expression in db/db mice. Our study demonstrates dose-related effects of candesartan in diabetic nephropathy: intermediate–high dose candesartan is renoprotective, whereas ultra-high dose candesartan induces renal damage. Molecular processes associated with these effects involve differential modulation of the ACE2/AT2R/Mas axis: intermediate–high dose candesartan up-regulating RAS protective components and attenuating pro-fibrotic processes, and ultra-high doses having opposite effects. These findings suggest novel mechanisms through the protective RAS axis, whereby candesartan may ameliorate diabetic nephropathy. Our findings also highlight potential injurious renal effects of ultra-high dose candesartan in diabetes.

Understanding the mechanisms linking obesity with hypertension is important in the current obesity epidemic as it may improve therapeutic interventions. Plasma aldosterone levels are positively correlated with body mass index and weight loss in obese patients is reported to be accompanied by decreased aldosterone levels. This suggests a relationship between adipose tissue and the production/secretion of aldosterone. Aldosterone is synthesized principally by the adrenal glands, but its production may be regulated by many factors, including factors secreted by adipocytes. In addition, studies have reported local synthesis of aldosterone in extra-adrenal tissues, including adipose tissue. Experimental studies have highlighted a role for adipocyte-secreted aldosterone in the pathogenesis of obesity-related cardiovascular complications via the mineralocorticoid receptor. This review focuses on how aldosterone secretion may be influenced by adipose tissue and the importance of these mechanisms in the context of obesity-related hypertension.

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.