Smooth Muscle Cell Mineralocorticoid Receptors Research Articles

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HomeCirculation ResearchVol. 132, No. 6In this Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn this Issue Originally published16 Mar 2023https://doi.org/10.1161/RES.0000000000000602Circulation Research. 2023;132:671sFlt1-Induced Preeclampsia Causes Persistent Post-partum Sensitivity to Hypertension Via Smooth Muscle Mineralocorticoid Receptor (p 674)Download figureDownload PowerPointThe mineralocorticoid receptor is central to post-preeclampsia hypertension, say Biwer et al.Preeclampsia (PE) is a serious, sometimes fatal, condition affecting around five percent of pregnancies and is characterized by high blood pressure and kidney dysfunction that starts after the twentieth gestational week. While delivery of the baby usually resolves PE, survivors have an increased risk of cardiovascular issues including increased sensitivity to hypertensive stimuli. Because the smooth muscle cell mineralocorticoid receptor (SMC-MR) is a key factor in the body’s response to these stimuli, Biwer and colleagues investigated its role in post-PE hypertension sensitivity. First, they created a mouse model of PE, wherein the growth factor receptor sFlt1 was overexpressed in late gestation. This factor is found at high levels in patients with PE and its overproduction in pregnant mice results in PE-like physiology. Compared with control animals, the PE mice had increased post-partum sensitivity to hypertensive stimuli (angiotensin II and salt) as well as aortic stiffness and vascular constriction. If the PE-model mice lacked SMC-MR, however, they were protected from these post-partum effects. Together with supporting cell culture experiments, these results highlight SMC-MR’s role in post-PE hypertension and suggest blocking the receptor may lower the risk.Hypoxia Sensing of β-Adrenergic Receptor Is Regulated by Endosomal PI3Kγ (p 690)Download figureDownload PowerPointSun et al unravel the molecular consequences of acute hypoxia in the heart.Reduced oxygen availability in the myocardium resulting from ischemic heart disease is known to impair functioning of the cardiomyocyte beta-adrenergic receptors (βARs)—critical controllers of the heart’s rhythm and output. A full understanding of this molecular pathology is lacking, however. Sun and colleagues now show that hypoxia-induced βAR dysfunction is mediated in large part by the membrane-associated lipid kinase PI3Kγ. After several hours of hypoxia exposure, βAR phosphorylation (a mark of dysfunction) was significantly increased in human and mouse cells, particularly within the endosomes (a site of βAR recycling). Endosomal activity of PI3Kγ was increased too, the team showed, and this resulted in the repression of endosomal PP2A—a protein phosphatase that normally dephosphorylates, and thus reactivates, βAR. Confirming the role of PI3Kγ, the team showed that mice lacking the kinase were protected from the heart damaging effects of acute hypoxia—including reduced ejection fraction and cardiac remodeling—that were seen in wild type animals. The authors thus conclude that preserving myocardial βAR function, via the inhibition of PI3Kγ, may be a therapeutic approach for safeguarding the heart in situations where the tissue is acutely starved of oxygen.Dectin-1 Acts as a Non-Classical Receptor of Ang II to Induce Cardiac Remodeling (p 707)Download figureDownload PowerPointDectin-1 binds angiotensin II and mediates heart failure pathology, say Ye et al.Angiotensin II (AngII) is a vasoconstricting hormone central to blood pressure regulation and which contributes to heart failure progression through its promotion of cardiomyocyte hypertrophy, arrythmia and fibrosis. The latter involves activation of inflammatory macrophages, but the precise molecular details of AngII’s effects on these cells is unknown. Ye and colleagues now show that the macrophage receptor Dectin-1, which normally functions as part of the innate immune response, mediates AngII’s inflammatory effects. Previous work had shown Dectin-1 is increased in the heart following ischemia-reperfusion injury, and the new work shows levels of the receptor are also elevated following chronic AngII infusion. Wild type mice receiving such an infusion developed signs of heart failure including reduced ejection fraction, enlarged left ventricle diameter, cardiac hypertrophy, inflammation and fibrosis, but these signs were notably reduced in animals genetically engineered to lack Dectin-1. The team went on to show AngII directly interacts with Dectin-1 and identified the particular residues involved. The importance of this AngII-Dectin-1 interaction in heart pathology, suggests that preventing it could potentially slow the progression to heart failure, say the team. Previous Back to top Next FiguresReferencesRelatedDetails March 17, 2023Vol 132, Issue 6 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000602 Originally publishedMarch 16, 2023 PDF download Advertisement

Smooth Muscle Mineralocorticoid Receptor Promotes Hypertension After Preeclampsia.

Preeclampsia is a syndrome of high blood pressure (BP) with end organ damage in late pregnancy that is associated with high circulating soluble VEGF receptor (sFlt1 [soluble Fms-like tyrosine kinase 1]). Women exposed to preeclampsia have a substantially increased risk of hypertension after pregnancy, but the mechanism remains unknown, leaving a missed interventional opportunity. After preeclampsia, women have enhanced sensitivity to hypertensive stress. Since smooth muscle cell mineralocorticoid receptors (SMC-MR) are activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure in pregnancy induces a postpartum state of enhanced SMC-MR responsiveness. Postpartum BP response to high salt intake was studied in women with prior preeclampsia. MR transcriptional activity was assessed in vitro in sFlt1-treated SMC by reporter assays and PCR. Preeclampsia was modeled by transient sFlt1 expression in pregnant mice. Two months post-partum, mice were exposed to high salt and then to AngII (angiotensin II) and BP and vasoconstriction were measured. Women exposed to preeclampsia had significantly enhanced salt sensitivity of BP verses those with a normotensive pregnancy. sFlt1 overexpression during pregnancy in mice induced elevated BP and glomerular endotheliosis, which resolved post-partum. The sFlt1 exposed post-partum mice had significantly increased BP response to 4% salt diet and to AngII infusion. In vitro, SMC-MR transcriptional activity in response to aldosterone or AngII was significantly increased after transient exposure to sFlt1 as was aldosterone-induced expression of AngII type 1 receptor. Post-partum, SMC-MR-KO mice were protected from the enhanced response to hypertensive stimuli after preeclampsia. Mechanistically, preeclampsia mice exposed to postpartum hypertensive stimuli develop enhanced aortic stiffness, microvascular myogenic tone, AngII constriction, and AngII type 1 receptor expression, all of which were prevented in SMC-MR-KO littermates. These data support that sFlt1-induced vascular injury during preeclampsia produces a persistent state of enhanced sensitivity of SMC-MR to activation. This contributes to postpartum hypertension in response to common stresses and supports testing of MR antagonism to mitigate the increased cardiovascular risk in women after PE.

SFlt1‐Induced Preeclampsia Enhances Cardiovascular Response to Post Partum Hypertensive Stimuli via Smooth Muscle Mineralocorticoid Receptor

BackgroundPreeclampsia (PE), a syndrome of high blood pressure (BP) and renal damage in late pregnancy, is associated with increased soluble VEGF receptor (sFlt1) and survivors have increased risk of future hypertension and cardiovascular disease.HypothesisSince smooth muscle cell mineralocorticoid receptor (SMC‐MR) can be activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure during pregnancy may induce a post‐partum state of enhanced vascular sensitivity via SMC‐MR activation.Methods/ResultsA PE model was induced by transient viral expression of sFlt1 in pregnant C57Bl6 mice. Elevated serum sFlt1, BP and glomerular endotheliosis was confirmed which all resolve post‐partum. In a small cohort of women with prior PE, we confirm salt sensitivity of BP. Thus, postpartum mice were implanted with telemetric BP monitors and exposed to repeated hypertensive stimuli (high salt and subsequent AngII). Mice with prior PE had a significantly increased BP response to hypertensive stimuli. Microvascular arteries from mice after PE and hypertensive stimuli had enhanced ex vivomyogenic tone and AngII vasoconstriction. To test the direct impact of sFlt1 on SMC‐MR function, cultured SMC (Pac1) were transiently exposed to sFlt1 (24 hours on then off) to mimic the post‐PE state. SMC‐MR transcriptional activity in response to aldosterone and AngII were significantly increased after sFlt1 exposure as measured by luciferase reporter assay. Target gene expression was also enhanced after sFlt1 exposure. Finally, the role of SMC‐MR in the post‐PE phenotype was tested in vivo by PE induction in SMC‐MR‐KO vs MR‐intact littermates. Post partum, SMC‐MR‐KO mice were protected from the PE‐induced increase in aortic stiffness, microvascular myogenic tone and AngII vasoconstriction.ConclusionsFlt1‐induced PE produces a state of enhanced sensitivity to stimuli that may be mediated by increased activation of SMC‐MR. These data support the use of MR antagonists to mitigate the increased risk of cardiovascular disease in women exposed to PE.

Smooth muscle mineralocorticoid receptor as an epigenetic regulator of vascular ageing.

Vascular stiffness increases with age and independently predicts cardiovascular disease risk. Epigenetic changes, including histone modifications, accumulate with age but the global pattern has not been elucidated nor are the regulators known. Smooth muscle cell-mineralocorticoid receptor (SMC-MR) contributes to vascular stiffness in ageing mice. Thus, we investigated the regulatory role of SMC-MR in vascular epigenetics and stiffness. Mass spectrometry-based proteomic profiling of all histone modifications completely distinguished 3 from 12-month-old mouse aortas. Histone-H3 lysine-27 (H3K27) methylation (me) significantly decreased in ageing vessels and this was attenuated in SMC-MR-KO littermates. Immunoblotting revealed less H3K27-specific methyltransferase EZH2 with age in MR-intact but not SMC-MR-KO vessels. These ageing changes were examined in primary human aortic (HA)SMC from adult vs. aged donors. MR, H3K27 acetylation (ac), and stiffness gene (connective tissue growth factor, integrin-α5) expression significantly increased, while H3K27me and EZH2 decreased, with age. MR inhibition reversed these ageing changes in HASMC and the decline in stiffness genes was prevented by EZH2 blockade. Atomic force microscopy revealed that MR antagonism decreased intrinsic stiffness and the probability of fibronectin adhesion of aged HASMC. Conversely, ageing induction in young HASMC with H2O2; increased MR, decreased EZH2, enriched H3K27ac and MR at stiffness gene promoters by chromatin immunoprecipitation, and increased stiffness gene expression. In 12-month-old mice, MR antagonism increased aortic EZH2 and H3K27 methylation, increased EZH2 recruitment and decreased H3K27ac at stiffness genes promoters, and prevented ageing-induced vascular stiffness and fibrosis. Finally, in human aortic tissue, age positively correlated with MR and stiffness gene expression and negatively correlated with H3K27me3 while MR and EZH2 are negatively correlated. These data support a novel vascular ageing model with rising MR in human SMC suppressing EZH2 expression thereby decreasing H3K27me, promoting MR recruitment and H3K27ac at stiffness gene promoters to induce vascular stiffness and suggests new targets for ameliorating ageing-associated vascular disease.

Abstract 14155: Exposure to sFlt1-Induced Preeclampsia Enhances the Future Vascular Response to Hypertensive Stimuli in Mice in a Smooth Muscle Mineralocorticoid Receptor Dependent Manner

Preeclampsia (PE), a syndrome of high blood pressure (BP) and renal damage in late pregnancy, is associated with increased soluble VEGF receptor (sFlt1) and survivors have increased risk of future hypertension with increased angiotensin II (AngII) and salt sensitivity. Since smooth muscle cell mineralocorticoid receptor (SMC-MR) can be activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure during pregnancy may induce a post-partum state of enhanced vascular sensitivity via SMC-MR activation. A PE model was induced by transient viral expression of sFlt1 in pregnant C57Bl6 mice. Elevated serum sFlt1, BP and glomerular endotheliosis was confirmed which all resolve post-partum. Two months later, resistance arteries from post-PE mice show no change in vasoconstriction to AngII but with increased mRNA ratio of the pro-constrictive AngII receptor-1(ATR1) to the vasodilatory ATR2. In a small cohort of women with prior PE, we confirm salt sensitivity of BP thus, postpartum mice were implanted with telemetric BP monitors and exposed to high salt or AngII (600mg/kg/day) infusion. Mice with prior PE had a significantly increased BP response to hypertensive stimuli. Microvessels from mice after PE and hypertensive stimuli had enhanced ex vivo myogenic tone and AngII vasoconstriction. To test the direct impact of sFlt1 on SMC-MR function, cultured SMC (Pac1) were transiently exposed to sFlt1 (24 hours on then off) to mimic the post-PE state. SMC-MR transcriptional activity in response to aldosterone and AngII were significantly increased after sFlt1 exposure as measured by luciferase reporter assay. SMC-MR activation and target gene expression was also enhanced after sFlt1 exposure (including AT1R/AT2R ratio). Finally, the role of SMC-MR in the post-PE phenotype was tested in vivo by PE induction in SMC-MR-KO vs MR-intact littermates. Post partum, SMC-MR-KO mice were protected from the PE-induced increase in aortic stiffness, microvascular myogenic tone and AngII vasoconstriction. sFlt1-induced PE produces a state of enhanced sensitivity to AngII that may be mediated by increased activation of SMC-MR. These data support the use of MR antagonists to mitigate the increased risk of cardiovascular disease in women exposed to PE.

Vascular cell-specific roles of mineralocorticoid receptors in pulmonary hypertension.

Abnormalities that characterize pulmonary arterial hypertension include impairment in the structure and function of pulmonary vascular endothelial and smooth muscle cells. Aldosterone levels are elevated in human pulmonary arterial hypertension and in experimental pulmonary hypertension, while inhibition of the aldosterone-binding mineralocorticoid receptor attenuates pulmonary hypertension in multiple animal models. We explored the role of mineralocorticoid receptor in endothelial and smooth muscle cells in using cell-specific mineralocorticoid receptor knockout mice exposed to sugen/hypoxia-induced pulmonary hypertension. Treatment with the mineralocorticoid receptor inhibitor spironolactone significantly reduced right ventricular systolic pressure. However, this is not reproduced by selective mineralocorticoid receptor deletion in smooth muscle cells or endothelial cells. Similarly, spironolactone attenuated the increase in right ventricular cardiomyocyte area independent of vascular mineralocorticoid receptor with no effect on right ventricular weight or interstitial fibrosis. Right ventricular perivascular fibrosis was significantly decreased by spironolactone and this was reproduced by specific deletion of mineralocorticoid receptor from endothelial cells. Endothelial cell-mineralocorticoid receptor deletion attenuated the sugen/hypoxia-induced increase in the leukocyte-adhesion molecule, E-selectin, and collagen IIIA1 in the right ventricle. Spironolactone also significantly reduced pulmonary arteriolar muscularization, independent of endothelial cell-mineralocorticoid receptor or smooth muscle cell-mineralocorticoid receptor. Finally, the degree of pulmonary perivascular inflammation was attenuated by mineralocorticoid receptor antagonism and was fully reproduced by smooth muscle cell-specific mineralocorticoid receptor deletion. These studies demonstrate that in the sugen/hypoxia pulmonary hypertension model, systemic-mineralocorticoid receptor blockade significantly attenuates the disease and that mineralocorticoid receptor has cell-specific effects, with endothelial cell-mineralocorticoid receptor contributing to right ventricular perivascular fibrosis and smooth muscle cell-mineralocorticoid receptor participating in pulmonary vascular inflammation. As mineralocorticoid receptor antagonists are being investigated to treat pulmonary arterial hypertension, these findings support novel mechanisms and potential mineralocorticoid receptor targets that mediate therapeutic benefits in patients.

Experimental preeclampsia results in post‐partum sensitivity to hypertensive stimuli

Preeclampsia (PE) is a syndrome of new onset high blood pressure (BP) with renal damage during late pregnancy that is associated with increased circulating anti‐angiogenic proteins, including soluble VEGF receptor (sFlt1). Women exposed to PE have increased risk of future cardiovascular disease, including hypertension, with evidence of a “hyper‐responsive” renin‐angiotensin‐aldosterone system (RAAS) persisting post‐partum. Aldosterone (Aldo) and angiotensin II (AngII) can activate vascular smooth muscle cell mineralocorticoid receptor (SMC‐MR), causing changes in gene transcription that contribute to increased BP. Thus, we hypothesized that exposure to increased sFlt1 during pregnancy may induce a state of persistent sensitivity to hypertensive stress by enhanced SMC‐MR activity post‐partum .We established a model that reproduces the hallmarks of PE by viral overexpression of sFlt1 compared to control virus in pregnant C57Bl6 mice. Using telemetry to measure blood pressure, we demonstrate that sFlt1 injection results in increased serum sFlt1, elevated BP and glomerular endotheliosis, the hallmark of PE‐induced renal damage, in late pregnancy that all resolve post‐partum. Two months post‐partum, wire myography studies performed on 3rd order mesenteric resistance vessels ex vivo reveal that sFlt1‐injected females (“prior‐PE”) have a vasoconstrictor sensitivity to AngII (1e‐9 dose: 0.16mN vs. 1.42mN, p=0.059) . To test whether prior PE results in increased sensitivity to hypertensive stimuli, post‐partum mice were exposed to various high salt diet conditions or to a moderate pressor dose of AngII (600mg/kg/day infusion) while BP was monitored via telemetry. Mice with prior PE had a trend toward an increased peak BP in response to 2% sodium (11.71mmHg vs. 3.62mmHg, p=0.09) and a significantly increased BP response to 4% sodium (8.88mmHg vs 5.34mmHg, p<0.05). Prior PE mice also had a significantly enhanced BP response to AngII versus those with normotensive pregnancies (22.83 mmHg vs 4.46 mmHg, p=0.0005). Plasma Aldo measured via radioimmunoassay tended to be higher in mice with prior PE (4.65nM vs 2.09nM, P=0.08). Since SMC‐MR can be activated by AngII, we investigated the extent of AngII‐induced SMC‐MR activation after PE using qPCR to measure known SMC‐MR target genes. In microvessels from mice with prior PE, mRNA expression showed increased AT1R (relative expression 3.69 vs 1.30, p<0.05), Cav1.2 (2.29 vs 1.11, p=0.06) and PlGF (1.92 vs 1.20, p=0.11). Studies are underway to determine whether SMC‐specific deletion of MR or post‐partum pharmacologic MR inhibition can protect against enhanced BP sensitivity to AngII and high salt diet after sFlt1‐induced PE.sFlt1‐induced PE produces a state of enhanced sensitivity to hypertensive stimuli that may be mediated by increased activation of SMC‐MR. If confirmed, these data support the use of spironolactone as a therapy to mitigate the increased risk of cardiovascular disease in women exposed to PE.Support or Funding InformationNIH T32HL007609 (LAB), NIH R25GM066567 (BVC)

Prevention of Obesity‐Associated Coronary and Cardiac Diastolic Dysfunction by Deletion of Smooth Muscle Cell Mineralocorticoid Receptor in Females

Coronary microvascular dysfunction is independently predictive of cardiac diastolic dysfunction and both conditions are common in obesity and associated metabolic syndrome. Recent work by us and others indicates that mineralocorticoid receptor (MR) antagonism treats obesity‐associated coronary and cardiac diastolic dysfunction. Vascular cell‐specific MR involvement in these obesity‐related defects remains unclear. Thus, we evaluated the hypothesis that deletion of smooth muscle cell (SMC) MR would prevent obesity‐associated coronary and cardiac diastolic dysfunction in females. Female wild‐type (WT) and SMC MR knockout (KO) mice were fed a standard chow or western diet (WD) to induce obesity for 16 weeks. WD feeding induced obesity, hyperinsulinemia, hypercholesterolemia, and visceral adipose inflammation in both WT and SMC MR KO mice. Assessment of coronary vascular function via wire myography revealed WD‐induced impairment of endothelium‐dependent vasodilation and enhanced vasoconstriction to the thromboxane A2 analog U46619 in WT but not SMC MR KO mice. Vasodilation to the nitric‐oxide donor sodium nitroprusside was unchanged by WD feeding. Global quantitative proteomics of aortas from each group revealed a number of differentially expressed proteins following WD feeding including upregulation of the oxidative stress‐related small GTPase Rac‐1 in WT but not SMC MR KO aortas. Accordingly, pretreatment of coronary arteries with the superoxide dismutase mimetic Tempol restored endothelium‐dependent vasodilation in WD‐fed WT mice with no effect in SMC MR KO vessels. Furthermore, WD feeding induced cardiac diastolic dysfunction assessed by echocardiography (i.e., increased E/E′, reduced E′/A′, increased diastolic stiffness). Coincident with prevention of WD‐induced coronary dysfunction, SMC MR deletion prevented WD‐induced cardiac diastolic dysfunction. Importantly, this cardioprotective effect of SMC MR deletion occurred independent of changes in left ventricular hypertrophy, blood pressure, aortic stiffening, and proteinuria. Taken together, our data demonstrate a central role of SMC MR signaling underlying WD‐induced coronary and cardiac diastolic dysfunction in females. Importantly, this study adds to an emerging body of evidence suggesting a vascular origin of cardiac dysfunction in obesity.Support or Funding InformationSupported by NIH (HL136386 to SBB, HL119290 to IZJ) and the Department of Veterans Affairs BLR&D (CDA‐2 IK2 BX002030). BC is supported by Department of Veterans Affairs (IK6 BX004016 and I01 BX002255).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sex differences in the role of the smooth muscle cell mineralocorticoid receptor in cardiovascular aging

Vascular aging is a process that involves the development of vasomotor dysfunction and vessel stiffening, which leads to cardiovascular disease (CVD). Clinical data supports that differential mechanisms may be involved in the progression of vascular aging in males compared to females. We have previously demonstrated that male mice with smooth muscle cell (SMC)‐specific deletion of the mineralocorticoid receptor (MR‐KO), a steroid hormone receptor known to regulate renal sodium handling, have lower blood pressure, vascular tone, and vasoconstriction as they age. The purpose of this study was to determine whether there are sex differences in the role of SMC‐MR in vascular aging. We demonstrated that vascular MR expression increases and miR‐155 decreases from 3 to 12 months of age in male mice. Additional studies revealed that SMC‐MR transcriptionally represses vascular miR‐155, which then promotes an increase in expression of miR‐155 target genes, Cav1.2, the pore‐forming subunit of the L‐type calcium channel (LTCC) and the angiotensin II type 1 receptor (AT1R) at 12 months of age in male mice. When compared directly to males, aging female mice do not display an increase in vascular MR expression until 18 months of age, accompanied by a decrease in miR‐155. Similarly, 12 month old female mice do not have altered vascular expression of Cav1.2 or AT1R compared to young females. Further, LTCC function is increased at 12 months of age in males and decreased at 12 months of age in females and then ultimately rose in females by 18 months of age. Additionally, Ang II‐mediated vasoconstriction increases at 12 months of age in males and is attenuated by SMC‐MR‐KO, however, in females, Ang II constriction is unaltered at 12 months of age and then surpasses the male Ang II constriction response at 18 months of age. We also observed an increase in vascular stiffness, measured by aortic pulse wave velocity, in male mice at 12 months of age that persists at 18 months of age and is prevented by SMC‐MR‐KO. In contrast, female mice do not exhibit increases in stiffness until 18 months of age, which is also attenuated by SMC‐MR‐KO. To explore potential mechanisms of vascular stiffness, we performed atomic force microscopy studies in 16–19 month old male and female mice. Females exhibited significantly more SMC‐stiffness than males, and SMC‐MR‐KO partially restored the aging‐associated stiffness. We also examined cardiac function, measured by echocardiography, and found that systolic and diastolic cardiac function declines with age in both males and females, but is modulated by SMC‐MR‐KO in females only. Further, we found that exercise capacity, measured by a graded exercise test, declines in both males and females with age, but is modulated by SMC‐MR‐KO in males only. Together, these results suggest that the mechanisms and time course of cardiovascular aging are distinct between males and females, and that SMC‐MR appears to play a differential role in males versus females. Furthermore, these studies suggest that sex‐specific therapies may be essential to improve CVD outcomes in the aging population.Support or Funding InformationSupported by HL095590‐05, HL119290, and AHA15POST21300000.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Smooth Muscle Cell-Mineralocorticoid Receptor as a Mediator of Cardiovascular Stiffness With Aging.

Stiffening of the vasculature with aging is a strong predictor of adverse cardiovascular events, independent of all other risk factors including blood pressure, yet no therapies target this process. MRs (mineralocorticoid receptors) in smooth muscle cells (SMCs) have been implicated in the regulation of vascular fibrosis but have not been explored in vascular aging. Comparing SMC-MR-deleted male mice to MR-intact littermates at 3, 12, and 18 months of age, we demonstrated that aging-associated vascular stiffening and fibrosis are mitigated by MR deletion in SMCs. Progression of cardiac stiffness and fibrosis and the decline in exercise capacity with aging were also mitigated by MR deletion in SMC. Vascular gene expression profiling analysis revealed that MR deletion in SMC is associated with recruitment of a distinct antifibrotic vascular gene expression program with aging. Moreover, long-term pharmacological inhibition of MR in aged mice prevented the progression of vascular fibrosis and stiffness and induced a similar antifibrotic vascular gene program. Finally, in a small trial in elderly male humans, short-term MR antagonism produced an antifibrotic signature of circulating biomarkers similar to that observed in the vasculature of SMC-MR-deleted mice. These findings suggest that SMC-MR contributes to vascular stiffening with aging and is a potential therapeutic target to prevent the progression of aging-associated vascular fibrosis and stiffness.

Smooth Muscle Cell Mineralocorticoid Receptors Are Necessary for Cardiovascular Fibrosis with Aging

Vascular aging involves fibrosis leading to stiffening of the vascular wall, an independent predictor of adverse cardiovascular events. Mineralocorticoid receptors (MR) are expressed in vascular smooth muscle cells (SMC) and MR signaling has been implicated in the regulation of cardiovascular fibrosis but has not been explored in cardiovascular aging. We previously demonstrated that aged mice lacking MR in SMCs (SMC‐MR‐KO) have lower blood pressure and reduced responsiveness to angiotensin II. The aim of this study was to investigate the role of SMC‐MR in cardiovascular structural and functional changes with aging and to explore mechanisms by which SMC‐MR contributes to vascular aging by modulating global vascular gene expression in mice. Vascular and cardiac fibrosis, aortic pulse wave velocity (PWV), cardiac echocardiography, intra‐cardiac pressure‐volume loops, and endurance exercise capacity were assessed in 3, 12 and 18 month‐old male MR‐intact and SMC‐MR‐KO mice. Vascular fibrosis in aortas, carotid arteries, and renal arterioles and aortic stiffness increased with aging in MR‐intact mice. Importantly, these aging‐induced increases were attenuated in SMC‐MR‐KO mice. Cardiac hypertrophy, wall thickness, fibrosis and stiffness increased and cardiac function and endurance exercise capacity decreased with aging in MR‐intact mice. Aging‐induced changes in cardiac hypertrophy, wall thickness and function were unaffected by the absence of SMC‐MR, however, SMC‐MR‐KO mice were protected from the aging‐induced increases in cardiac fibrosis and stiffness and the decrease in endurance exercise capacity. There was a significant correlation between aortic PWV and end‐systolic pressure volume relationship, suggesting that the changes in cardiac systolic stiffness are likely secondary to changes in aortic stiffness with aging. Renal fibrosis increased with aging but this increase was independent of the presence of SMC‐MR. To explore the mechanism by which SMC‐MR contributes to vascular fibrosis and stiffness with aging, we characterized global gene expression profiles in aortas from 3 and 12 month‐old MR‐intact and SMC‐MR‐KO mice. 552 and 477 genes were differentially expressed with aging in aortas from MR‐intact and SMC‐MR‐KO, respectively. Only 79 genes were differentially expressed with aging in both MR‐intact and SMC‐MO‐KO mice. Pathway analysis revealed that changes in cardiovascular development pathways with aging are predicted to act in opposing directions in SMC‐MR‐KO mice compared to MR intact mice. We also identified a network of fibrosis‐related genes that are down‐regulated with aging in SMC‐MR‐KO mice, providing a possible mechanism for the reduced vascular fibrosis with aging in SMC‐MR‐KO mice. Our findings provide evidence that SMC‐MR plays a role in aging‐induced increase in vascular fibrosis that contributes to cardiovascular tissue stiffening and dysfunction with aging. Further studies are underway to treat aged mice with MR antagonist to attempt to reverse the aging phenotype.Support or Funding InformationNIH/NHLBI R01 HL119290 and AHA EIA18290005

Mechanism of Aging‐associated Hypertension: Role of Smooth Muscle Cell Mineralocorticoid Receptor Regulation of Vascular L‐type Calcium Channels

Hypertension (HTN), the most prevalent cardiovascular disease risk factor, increases with age. We found that mice with SMC-specific deletion of the mineralocorticoid receptor (MR KO) lack the aging-associated rise in blood pressure (BP). We hypothesize that SMC MR contributes to BP regulation with aging by regulating vascular L-type calcium channels (LTCC). Patch clamp studies on mesenteric resistance vessel (MRV) SMC from young (3-4 mo.) and aged (9-12 mo.) MR intact and MR KO reveal reduced LTCC current density in aged MR KO (p<0.05 vs. MR intact). Fura-2 photometry studies reveal decreased MRV Ca flux in aged MR KO (p<0.05 vs. MR intact) in response to BayK (LTCC agonist). Contraction to BayK is blunted in aged MR KO MRV (p<0.05 vs. MR intact). RNA expression of Cav1.2, the pore-forming subunit of LTCC, is reduced by 60% in aged MR KO vs. MR intact MRV (p<0.05). Micro-RNA expression profiling identified miR-155 as being modulated by SMC MR with aging. MRV miR-155 expression is increased in aged MR KO (p<0.05 vs. MR intact). Ingenuity pathway analysis identified Cav1.2 as a potential target of miR-155. Overexpression of miR-155 in mouse MRV SMC reduced Cav1.2 expression by 45% (p<0.05 vs. ctrl). These data suggest that SMC MR contributes to HTN through regulating Cav1.2 expression/function, potentially via miR-155. These results enhance our basic understanding of BP control with aging, and provide support for innovative therapeutic targets to treat aging-associated HTN. Supported by HL095590-05.

Role of smooth muscle cell mineralocorticoid receptor in vascular tone.

Identification of the mineralocorticoid receptor (MR) in the vasculature (i.e., endothelial and smooth muscle cells) raised the question of its role in vascular function and blood pressure control. Using a mouse model with conditional inactivation of MR in vascular smooth muscle cell (VSMC) (MR(SMKO)), we have recently shown that the VSMC MR is crucial for aldosterone-salt-induced carotid stiffening. In the present study, we have investigated the specific contribution of the VSMC MR in the regulation of vascular tone in large vessels. In MR(SMKO) mice, contractions induced by potassium chloride and calcium (Ca(2+)) are decreased in the aorta, whereas contraction is normal in response to phenylephrine and caffeine. The difference in response to Ca(2+) suggests that the VSMC-specific deficiency of the MR modifies VSM Ca(2+) signaling but without altering the intracellular Ca(2+) store handling. The relaxation induced by acetylcholine is not affected by the absence of MR. However, the relaxation induced by Ach in the presence of indomethacin and the relaxation induced by sodium nitroprussiate are significantly reduced in MR(SMKO) mice compared to controls. Since endothelial nitric oxide synthase (eNOS) activity is increased in mutant mice, their altered relaxation reflects impairment of the nitric oxide (NO) signaling pathway. In addition to altered NO and Ca(2+) signaling, the activity of myosin light chain and its regulators, myosin light chain kinase (MLCK) and myosin phosphatase (MLCP), is reduced. In conclusion, MR expressed in VSMC is required for NO and Ca(2+) signaling pathways and contractile protein activity leading to an altered contraction/relaxation coupling.

Direct Role for Smooth Muscle Cell Mineralocorticoid Receptors in Vascular Remodeling: Novel Mechanisms and Clinical Implications

The mineralocorticoid receptor (MR) is a key regulator of blood pressure. MR antagonist drugs are used to treat hypertension and heart failure, resulting in decreased mortality by mechanisms that are not completely understood. In addition to the kidney, MR is also expressed in the smooth muscle cells (SMCs) of the vasculature, where it is activated by the hormone aldosterone and affects the expression of genes involved in vascular function at the cellular and systemic levels. Following vascular injury due to mechanical or physiological stresses, vessels undergo remodeling resulting in SMC hypertrophy, migration, and proliferation, as well as vessel fibrosis. Exuberant vascular remodeling is associated with poor outcomes in cardiovascular patients. This review compiles recent findings on the specific role of SMC-MR in the vascular remodeling process. The development and characterization of a SMC-specific MR-knockout mouse has demonstrated a direct role for SMC-MR in vascular remodeling. Additionally, several novel mechanisms contributing to SMC-MR-mediated vascular remodeling have been identified and are reviewed here, including Rho-kinase signaling, placental growth factor signaling through vascular endothelial growth factor type 1 receptor, and galectin signaling.

Aldosterone Promotes Vascular Remodeling by Direct Effects on Smooth Muscle Cell Mineralocorticoid Receptors

Vascular remodeling occurs after endothelial injury, resulting in smooth muscle cell (SMC) proliferation and vascular fibrosis. We previously demonstrated that the blood pressure-regulating hormone aldosterone enhances vascular remodeling in mice at sites of endothelial injury in a placental growth factor-dependent manner. We now test the hypothesis that SMC mineralocorticoid receptors (MRs) directly mediate the remodeling effects of aldosterone and further explore the mechanism. A wire-induced carotid injury model was performed in wild-type mice and mice with inducible SMC-specific deletion of the MR. Aldosterone did not affect re-endothelialization after injury in wild-type mice. Deletion of SMC-MR prevented the 79% increase in SMC proliferation induced by aldosterone after injury in MR-Intact littermates. Moreover, both injury-induced and aldosterone-enhanced vascular fibrosis were attenuated in SMC-specific MR knockout mice. Further exploration of the mechanism revealed that aldosterone-induced vascular remodeling is prevented by in vivo blockade of the placental growth factor-specific receptor, type 1 vascular endothelial growth factor receptor (VEGFR1), the receptor for placental growth factor. Immunohistochemistry of carotid vessels shows that the induction of VEGFR1 expression in SMC after vascular injury is attenuated by 72% in SMC-specific MR knockout mice. Moreover, aldosterone induction of vascular placental growth factor mRNA expression and protein release are also prevented in vessels lacking SMC-MR. These studies reveal that SMC-MR is necessary for aldosterone-induced vascular remodeling independent of renal effects on blood pressure. SMC-MR contributes to induction of SMC VEGFR1 in the area of vascular injury and to aldosterone-enhanced vascular placental growth factor expression and hence the detrimental effects of aldosterone are prevented by VEGFR1 blockade. This study supports exploring MR antagonists and VEGFR1 blockade to prevent pathological vascular remodeling induced by aldosterone.

Smooth Muscle Cell Mineralocorticoid Receptors Are Mandatory for Aldosterone–Salt to Induce Vascular Stiffness

Arterial stiffness is recognized as a risk factor for many cardiovascular diseases. Aldosterone via its binding to and activation of the mineralocorticoid receptors (MRs) is a main regulator of blood pressure by controlling renal sodium reabsorption. Although both clinical and experimental data indicate that MR activation by aldosterone is involved in arterial stiffening, the molecular mechanism is not known. In addition to the kidney, MR is expressed in both endothelial and vascular smooth muscle cells (VSMCs), but the specific contribution of the VSMC MR to aldosterone-induced vascular stiffness remains to be explored. To address this question, we generated a mouse model with conditional inactivation of the MR in VSMC (MR(SMKO)). MR(SMKO) mice show no alteration in renal sodium handling or vascular structure, but they have decreased blood pressure when compared with control littermate mice. In vivo at baseline, large vessels of mutant mice presented with normal elastic properties, whereas carotids displayed a smaller diameter when compared with those of the control group. As expected after aldosterone/salt challenge, the arterial stiffness increased in control mice; however, it remained unchanged in MR(SMKO) mice, without significant modification in vascular collagen/elastin ratio. Instead, we found that the fibronectin/α5-subunit integrin ratio is profoundly altered in MR(SMKO) mice because the induction of α5 expression by aldosterone/salt challenge is prevented in mice lacking VSMC MR. Altogether, our data reveal in the aldosterone/salt hypertension model that MR activation specifically in VSMC leads to the arterial stiffening by modulation of cell-matrix attachment proteins independent of major vascular structural changes.

Smooth muscle cell mineralocorticoid receptors: role in vascular function and contribution to cardiovascular disease

The mineralocorticoid receptor (MR), a member of the steroid receptor family, regulates blood pressure by mediating the effects of the hormone aldosterone on renal sodium handling. In recent years, it has become clear that MR is expressed in vascular smooth muscle cells (SMCs), and interest has grown in understanding the direct role of SMC MR in regulating vascular function. This interest stems from multiple clinical studies where MR inhibitor treatment reduced the incidence of cardiovascular events and mortality. This review summarizes the most recent advances in our understanding of SMC MR in regulating normal vascular function and in promoting vascular disease. Many new studies suggest a role for SMC MR activation in stimulating vascular contraction and contributing to vessel inflammation, fibrosis, and remodeling. These detrimental vascular effects of MR activation appear to be independent of changes in blood pressure and are synergistic with the presence of endothelial dysfunction or damage. Thus, in humans with underlying cardiovascular disease or cardiovascular risk factors, SMC MR activation may promote hypertension, atherosclerosis, and vascular aging. Further exploration of the molecular mechanisms for the effects of SMC MR activation has the potential to identify novel therapeutic targets to prevent or treat common cardiovascular disorders.

Abstract 302: Smooth Muscle Cell Mineralocorticoid Receptors Directly Mediate Aldosterone-induced Remodeling After Vascular Injury Via Type 1 Vegf Receptors

Background and Significance Endothelial damage initiates vascular remodeling with smooth muscle cell (SMC) proliferation and vascular fibrosis. Excessive remodeling, as in restenosis after vascular procedures or atherosclerosis, limits blood flow to distal tissues. In animals, aldosterone (aldo) infusion enhances remodeling which is reversed by aldo antagonists, implicating the mineralocorticoid receptor (MR). We previously showed that MR is expressed in SMC and that aldo induces the type 1 VEGF receptor (VEGFR1) in injured vessels. Hypothesis and Results We hypothesize that aldo promotes pathological vascular remodeling by directly activating MR in SMC and promoting SMC proliferation via VEGFR1 signaling. We show in vivo that aldo does not affect re-endothelialization after injury supporting our focus on SMC. To test this hypothesis we measured aldo-induced remodeling after wire carotid injury in mice with inducible SMC-specific deletion of MR (SMC-MR-KO). The 80% increase in SMC proliferation with aldo in control littermates is completely prevented in SMC-MR-KO mice (p&lt;0.005, n=11). To examine the mechanism, mouse carotid SMC were treated with aldo in vitro producing a dose dependent proliferative response (1,5,10 and100nM aldo, 9%±2, 17%±3, 21%±6, and 25%±7 vs control, respectively, p&lt;0.05, n=8). The increase in proliferation with 10 nM aldo+IgG was completely inhibited by treatment with specific VEGFR1-blocking antibody (Ab, p=0.04, n=8). Immunohistochemistry of carotid vessels reveals that VEGFR1 is induced in SMC after injury and further enhanced by aldo (55%±0.5 increase with injury, 74%± 0.5 with aldo+injury vs uninjured control, p&lt;0.05, n=8).We examined the role of VEGFR1 in vivo and VEGFR1 blockade significantly reduces aldo-induced SMC proliferation after injury (46%±4 decrease vs aldo+IgG, p&lt;0.05, n=9). Tail cuff measurements reveal no difference in blood pressure with any of the treatments. Conclusions These data support that SMC MR directly mediates aldo-induced vascular remodeling independent blood pressure. The mechanism involves VEGFR1 that is induced on SMC in the area of injury. This study supports exploring VEGFR1-blocking drugs to prevent pathological vascular remodeling induced by aldo in humans.

Smooth muscle cell mineralocorticoid receptors—role in blood pressure control and vascular ageing

Smooth muscle cell mineralocorticoid receptors—role in blood pressure control and vascular ageing

Direct regulation of blood pressure by smooth muscle cell mineralocorticoid receptors

Hypertension is a cardiovascular risk factor present in over two-thirds of people over age 60 in North America; elevated blood pressure correlates with increased risk of heart attack, stroke and progression to heart and kidney failure. Current therapies are insufficient to control blood pressure in almost half of these patients. The mineralocorticoid receptor (MR), acting in the kidney, is known to regulate blood pressure through aldosterone binding and stimulation of sodium retention. However, recent studies support the concept that the MR also has extrarenal actions and that defects in sodium handling alone do not fully explain the development of hypertension and associated cardiovascular mortality. We and others have identified functional MR in human vascular smooth muscle cells (SMCs), suggesting that vascular MR might directly regulate blood pressure. Here we show that mice with SMC-specific deficiency of the MR have decreased blood pressure as they age without defects in renal sodium handling or vascular structure. Aged mice lacking MR in SMCs (SMC-MR) have reduced vascular myogenic tone, agonist-dependent contraction and expression and activity of L-type calcium channels. Moreover, SMC-MR contributes to angiotensin II–induced vascular oxidative stress, vascular contraction and hypertension. This study identifies a new role for vascular MR in blood pressure control and in vascular aging and supports the emerging hypothesis that vascular tone contributes directly to systemic blood pressure.