Deletion Of Mineralocorticoid Receptor Research Articles

Eplerenone Reduces Blood Pressure and Improves Vascular Function in a Mouse Model of Leptin-induced Preeclampsia

Preeclampsia is a hypertensive pregnancy disease that increases the risks for adverse outcomes of pregnancy for both mother and baby, notably by reducing fetal growth in utero. We have shown that mid-late gestation leptin infusion induces preeclampsia characteristics in pregnant mice in inducing hypertension, endothelial dysfunction, and fetal growth restriction, which is ablated in mice with endothelial mineralocorticoid receptor deletion (ECMR). However, there is a lack of preclinical data and rationale for MR antagonism for use in preeclampsia in the existing literature. Therefore, we hypothesized that leptin increases ECMR expression and that MR antagonism decreases hypertension, endothelial dysfunction, and fetal growth restriction in leptin-induced preeclamptic mice. We infused timed-pregnant Balb/C mice with saline (sham) or leptin (LEP, 0.9 mg/kg/day) via s.c. osmotic minipump and administered vehicle or eplerenone (EPL, 200mg/kg/day) in drinking water from gestation day (GD)11-18 (n=7-12). We measured mean arterial blood pressure (BP) via radiotelemetry, vascular function in 2nd order mesenteric arteries by wire myography, and pup/placental weights on GD18. We isolated endothelial cells from placentas of our mice via magnetic cell sorting. Pairwise comparisons were by student’s t test and myography curves analyzed by 2-W ANOVA w/RM. Leptin increased ECMR mRNA expression (3.1±0.32 WT LEP vs 1.1±0.48 WT sham, fold change, *p<0.05) compared to WT sham mice in placenta indicating a direct role for leptin to upregulate ECMR in pregnancy. EPL ablated leptin-induced increases in BP (98±4 LEP/EPL vs 113±3 lep/veh mmHg, *p<0.05), and when treated with EPL leptin did not induce endothelial dysfunction, as assessed as a lack of decrease in vascular relaxation to acetylcholine (p>0.05 LEP/EPL vs sham/EPL). Interestingly, EPL treatment did not prevent leptin from decreasing placental effciency in pregnant mice (7.42±0.17 LEP/veh vs 6.71±0.15 LEP/EPL, pup/placenta weight ratio, p>0.05). Collectively, these data show that leptin infusion from GD11-18 in pregnant mice increases ECMR activation by increasing expression. Furthermore, MR antagonism by eplerenone ablates the maternal hypertension and endothelial dysfunction in leptin-induced preeclampsia mice, however, fetal growth is not restored by eplerenone. Therefore, eplerenone may be a promising therapeutic for the maternal cardiovascular syndrome of preeclampsia patients, however, MR antagonism therapies may need additional agents to improve fetal outcomes. 1R01HL169576, 4R00HL146948. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Atlas of cardiac endothelial cell enhancer elements linking the mineralocorticoid receptor to pathological gene expression.

Endothelial cells play crucial roles in physiology and are increasingly recognized as therapeutic targets in cardiovascular disease. Here, we analyzed the regulatory landscape of cardiac endothelial cells by assessing chromatin accessibility, histone modifications, and 3D chromatin organization and confirmed the functional relevance of enhancer-promoter interactions by CRISPRi-mediated enhancer silencing. We used this dataset to explore mechanisms of transcriptional regulation in cardiovascular disease and compared six different experimental models of heart failure, hypertension, or diabetes. Enhancers that regulate gene expression in diseased endothelial cells were enriched with binding sites for a distinct set of transcription factors, including the mineralocorticoid receptor (MR), a known drug target in heart failure and hypertension. For proof of concept, we applied endothelial cell-specific MR deletion in mice to confirm MR-dependent gene expression and predicted direct MR target genes. Overall, we have compiled here a comprehensive atlas of cardiac endothelial cell enhancer elements that provides insight into the role of transcription factors in cardiovascular disease.

Multi-omic analysis of the cardiac cellulome defines a vascular contribution to cardiac diastolic dysfunction in obesity

Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Emerging evidence suggests development of heart failure with preserved ejection fraction in females with CMD and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the hypothesis that smooth muscle cell (SMC)-specific MR deletion prevents obesity-associated coronary and cardiac diastolic dysfunction in females. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Initial studies revealed that global MR blockade with spironolactone prevented impaired coronary vasodilation and diastolic dysfunction in obese females. Importantly, specific deletion of SMC-MR similarly prevented obesity-associated coronary and cardiac dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in multiple non-myocyte populations (B/T cells, macrophages, and endothelium), independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling broadly impacting the cardiac cellulome and underlying obesity-associated coronary and cardiac dysfunction thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity. Supported by NIH R01 HL136386 (Bender), NIH R01 HL119290 (Jaffe), the University of Missouri Research Core Facilities Grant Program (Bender), NHMRC Ideas Grant GNT1188503 (Pinto), VA Merit I01 BX004220 and RCS IK2 6004016 (Chandrasekar). The work was also supported by resources and the use of facilities at the Harry S. Truman Memorial Veterans Hospital in Columbia, MO. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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

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

Midgestation Leptin Infusion Induces Characteristics of Clinical Preeclampsia in Mice, Which Is Ablated by Endothelial Mineralocorticoid Receptor Deletion.

Patients with preeclampsia demonstrate increases in placental leptin production in midgestation, and an associated increase in late gestation plasma leptin levels. The consequences of mid-late gestation increases in leptin production in pregnancy is unknown. Our previous work indicates that leptin infusion induces endothelial dysfunction in nonpregnant female mice via leptin-mediated aldosterone production and endothelial mineralocorticoid receptor (ECMR) activation, which is ablated by ECMR deletion. Therefore, we hypothesized that leptin infusion in mid-gestation of pregnancy induces endothelial dysfunction and hypertension, hallmarks of clinical preeclampsia, which are prevented by ECMR deletion. Leptin was infused via miniosmotic pump (0.9 mg/kg per day) into timed-pregnant ECMR-intact (WT) and littermate-mice with ECMR deletion (KO) on gestation day (GD)11-18. Leptin infusion decreased fetal weight and placental efficiency in WT mice compared with WT+vehicle. Radiotelemetry recording demonstrated that blood pressure increased in leptin-infused WT mice during infusion. Leptin infusion reduced endothelial-dependent relaxation responses to acetylcholine (ACh) in both resistance (second-order mesenteric) and conduit (aorta) vessels in WT pregnant mice. Leptin infusion increased placental ET-1 (endothelin-1) production evidenced by increased PPET-1 (preproendothelin-1) and ECE-1 (endothelin-converting enzyme-1) expressions in WT mice. Adrenal aldosterone synthase (CYP11B2) and angiotensin II type 1 receptor b (AT1Rb) expression increased with leptin infusion in pregnant WT mice. KO pregnant mice demonstrated protection from leptin-induced reductions in pup weight, placental efficiency, increased BP, and endothelial dysfunction. Collectively, these data indicate that leptin infusion in midgestation induces endothelial dysfunction, hypertension, and fetal growth restriction in pregnant mice, which is ablated by ECMR deletion.

Abstract 8929: Mineralocorticoid Receptor as an Epigenetic Regulator of Vascular Aging

Introduction: Vascular stiffness increases with age and independently predicts cardiovascular events but vascular fibrosis mechanisms are unclear. Epigenetic changes accumulate with age and contribute to aging pathology. We showed that aging-associated increase in vascular stiffness/fibrosis are mitigated by treating old mice with mineralocorticoid receptor (MR) antagonist (MRA) or by genetic deletion of MR in smooth muscle cells (SMC-MR-KO). Hypothesis: We hypothesized that SMC-MR regulates vascular fibrosis genes by modulating epigenetic histone modifications in the aging vasculature. Methods and Results: Global histone proteomic profiling revealed increased acetylation (ac) and decreased methylation (me) of histone H3 lysine 27 (H3K27) in old vs young mouse aorta that is attenuated by SMC-MR-KO. Similarly, RNA profiling shows downregulation of fibrotic genes (CTGF, MMP2 and Integrin-α5) with aging in mouse aortas from SMC-MR-KO mice. Mechanism was explored in primary low passage human aortic SMC (HASMC). Comparison of protein levels in HASMCs from old vs young people shows increased MR, decreased H3K27-specific methyltransferase EZH2, increased H3K27ac and increased fibrosis gene expression in aged HASMC. Treatment of young HASMC with H 2 O 2 (aging inductor) increased MR and decreased EZH2 in association with decreased H3K27me. These epigenetic changes were associated with increased H3K27ac and MR enrichment at the promoter of fibrosis genes by ChIP along with increased fibrosis gene expression. Conversely, treatment of aged HASMC with MRA increased EZH2 and H3K27me with decreased fibrosis gene expression. Finally, treatment of 12 month old mice with MR antagonist for 4 months or deletion of MR from SMC prevented aging induction of vascular stiffness by pulse wave velocity in association with decreased H3K27ac at promoters and decreased expression of fibrosis genes in aging aortas. Conclusion: These data support a model in which rising SMC-MR with aging contributes to vascular fibrosis via EZH2 reduction leading to decreased H3K27me and increased H3K27ac and MR recruitment to promote transcription of fibrotic genes. These findings suggest the therapeutic potential for MRAs for aging-associated vascular stiffening/fibrosis.

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.

Endothelial Mineralocorticoid Receptor Deletion Ablates Leptin-Induced Preeclampsia Characteristics in Pregnancy

Endothelial Mineralocorticoid Receptor Deletion Ablates Leptin-Induced Preeclampsia Characteristics in Pregnancy

Eplerenone Improves Pulmonary Vascular Remodeling and Hypertension by Inhibition of the Mineralocorticoid Receptor in Endothelial Cells.

[Figure: see text].

Mineralocorticoid and Estrogen Receptors in Endothelial Cells Coordinately Regulate Microvascular Function in Obese Female Mice.

[Figure: see text].

Endothelial mineralocorticoid receptor deletion ablates leptin‐induced cardiovascular and fetal growth preeclampsia characteristics in pregnant mice

Obesity increases the risk for preeclampsia (PE), a hypertensive syndrome of pregnancy characterized by placental ischemia. Obese PE patients demonstrate increases in plasma leptin levels in midgestation due to increased placental leptin production. We have shown that pregnancy increases endothelial mineralocorticoid receptor (ECMR) expression and that ECMR mediates leptin-induced endothelial dysfunction and hypertension in nonpregnant females. We therefore hypothesized that ECMR deletion protects from the development endothelial dysfunction, hypertension and fetal growth restriction in pregnant leptin-infused mice. Pregnant ECMR+/+ (WT) and ECMR-/- (KO) littermate mice were infused with vehicle or leptin (N=4-6/group) by osmotic minipump (0.9mg/kg/day, s.c.) from GD11-18 and thoracic aortas assessed for vascular function by wire myography. Separate groups (N=1-2/group) were implanted with radiotelemeters for systolic blood pressure (BP) measurement. Leptin decreased pup weight (0.86±0.04g WT vs 0.52±0.11g WT+lep, *P<0.05) and placental efficiency (9.7±0.7 WT vs 7.9±0.6 WT+lep, pup/placenta weight ratio, *P<0.05) indicating fetal growth restriction. ECMRdeletion prevented leptin-mediated decreases in pup weight (0.91±0.06g KO vs 1.0±0.07g KO+lep) and placental efficiency (9.6±0.5 KO vs 9.4±1.2 KO+lep). Preliminary data shows that prior to leptin infusion (GD8-11) all groups demonstrated similar BP and circadian rhythm of BP. Leptin infusion increased BP and ablated circadian rhythm (GD11-18, 114 mmHg WT vs 137 mmHg WT+lep), however, ECMR deletion blunted leptin-induced BP increases (112 KO mmHg vs 120 mmHg KO+lep) and preserved circadian rhythm. Leptin infusion reduced endothelial function (acetylcholine-mediated relaxation) in aortas of WT, but not KO, pregnant mice (2-way ANOVA, repeated measures, *P<0.05). Relaxation to acetylcholine with LNAME showed restoration of endothelial function in KO+lep mice was NO-dependent. In addition, ECMR deletion protected pregnant mice from leptin-induced increases in vascular α1 receptor-mediated constriction (phenylephrine) (*P<0.05). Endothelial-independent relaxation to sodium nitroprusside was similar across all groups. Moreover, ECMR deletion protected from leptin-mediated increases in placental mRNA expression of: sFlt-1 (WT+leptin: 1.2±0.03-fold from WT, *P<0.05, KO+leptin: 1.1±0.1-fold change from KO), HIF1α (WT+leptin: 1.6±0.1-fold, P=0.09, KO+leptin: 1.5±0.2-fold) and galectin 3 (WT+leptin: 3.2±0.5-fold, *P<0.05, KO+leptin: 0.8±0.1-fold). Collectively, these data indicate that ECMR deletion decreases BP, improves vascular function and placental function in association with increased vascular NO bioavailability and fetal growth in an obesity-associated, leptin-infusion model of PE.

Mineralocorticoid Receptor in Smooth Muscle Contributes to Pressure Overload-Induced Heart Failure.

Mineralocorticoid receptor (MR) antagonists decrease heart failure (HF) hospitalization and mortality, but the mechanisms are unknown. Preclinical studies reveal that the benefits on cardiac remodeling and dysfunction are not completely explained by inhibition of MR in cardiomyocytes, fibroblasts, or endothelial cells. The role of MR in smooth muscle cells (SMCs) in HF has never been explored. Male mice with inducible deletion of MR from SMCs (SMC-MR-knockout) and their MR-intact littermates were exposed to HF induced by 27-gauge transverse aortic constriction versus sham surgery. HF phenotypes and mechanisms were measured 4 weeks later using cardiac ultrasound, intracardiac pressure measurements, exercise testing, histology, cardiac gene expression, and leukocyte flow cytometry. Deletion of MR from SMC attenuated transverse aortic constriction-induced HF with statistically significant improvements in ejection fraction, cardiac stiffness, chamber dimensions, intracardiac pressure, pulmonary edema, and exercise capacity. Mechanistically, SMC-MR-knockout protected from adverse cardiac remodeling as evidenced by decreased cardiomyocyte hypertrophy and fetal gene expression, interstitial and perivascular fibrosis, and inflammatory and fibrotic gene expression. Exposure to pressure overload resulted in a statistically significant decline in cardiac capillary density and coronary flow reserve in MR-intact mice. These vascular parameters were improved in SMC-MR-knockout mice compared with MR-intact littermates exposed to transverse aortic constriction. These results provide a novel paradigm by which MR inhibition may be beneficial in HF by blocking MR in SMC, thereby improving cardiac blood supply in the setting of pressure overload-induced hypertrophy, which in turn mitigates the adverse cardiac remodeling that contributes to HF progression and symptoms.

A New Role for the Aldosterone/Mineralocorticoid Receptor Pathway in the Development of Mitral Valve Prolapse.

Mitral valve prolapse (MVP) is one of the most common valvular disorders. However, the molecular and cellular mechanisms involved in fibromyxomatous changes in the mitral leaflet tissue have not been elucidated. Aldosterone (Aldo) promotes fibrosis in myocardium, and MR (mineralocorticoid receptor) antagonists (MRAs) improve cardiac function by decreasing cardiac fibrosis. We investigated the role of the Aldo/MR in the fibromyxomatous modifications associated with MVP. Aldo enhanced valvular interstitial cell activation markers and induced endothelial-mesenchymal transition in valvular endothelial cells, resulting in increased proteoglycan secretion. MRA blocked all the above effects. Cytokine arrays showed CT-1 (cardiotrophin-1) to be a mediator of Aldo-induced valvular interstitial cell activation and proteoglycan secretion and CD (cluster of differentiation) 14 to be a mediator of Aldo-induced endothelial-mesenchymal transition and proteoglycan secretion in valvular endothelial cells. In an experimental mouse model of MVP generated by nordexfenfluramine administration, MRA treatment reduced mitral valve thickness and proteoglycan content. Endothelial-specific MR deletion prevented fibromyxomatous changes induced by nordexfenfluramine administration. Moreover, proteoglycan expression was slightly lower in the mitral valves of MVP patients treated with MRA. These findings demonstrate, for the first time, that the Aldo/MR pathway regulates the phenotypic, molecular, and histological changes of valvular interstitial cells and valvular endothelial cells associated with MVP development. MRA treatment appears to be a promising option to reduce fibromyxomatous alterations in MVP.

Endothelial sodium channel (EnNaC) activation contributes to mineralocorticoid receptormediated increases in coronary artery and cardiac fibrosis/stiffness leading to diastolic dysfunction in obesity

BackgroundWhile obesity, induced by an industrialized or Western diet (WD), has been shown to impact structural and functional properties of both coronary arteries and myocardium, the exact mechanisms by which these interact to cause diastolic dysfunction (DD) are uncertain. We hypothesized that the mineralocorticoid receptor (MR) activation in coronary vascular endothelial cells (ECs) impairs endothelial function via EC Na+ channel, EnNaC, activation. Further, we posited that such coronary dysfunction initiates increased myocyte stiffness and fibrosis ultimately leading to DD. The objective was to demonstrate that WD‐induced DD is dependent on EnNaC activation.MethodsTo cause MR activation, aldosterone was administered to female or male mice (C57BL6) by osmotic minipump for 3 wks. Further, female wild‐type and EC MR deficient mice (2–3 mths of age) or low aldosterone implanted as subcutaneous pellet were fed a WD, high in saturated fat and fructose (16 wks). In an additional experiment, female wild‐type and EnNaC α subunit deficient mice (3–4 mths of age) were fed the WD (12–16 wks). Vascular function was assessed in isolated coronary arteries by pressure myography, cardiac and perivascular fibrosis was evaluated by picosirius red (PSR) staining and diastolic function was assessed by echocardiography. Using in vitro cellular approaches, intrinsic cardiomyocyte stiffness was measured by atomic force microscopy (AFM) and EnNaC activity (Na+ current) determined by whole cell patch clamp.ResultsAldosterone infusion caused impaired endothelium‐dependent relaxation and coronary vascular fibrosis, which were prevented by the MR antagonist, spironolactone (1 mg/kg/day). WD feeding was associated with DD, but not systolic dysfunction, that was prevented by either spironolactone or EC‐specific MR deletion. Furthermore, DD was accompanied by increased vascular EnNaC currents and increased stiffness of both ECs and cardiomyocytes, which were attenuated by EC MR deletion. Finally, WD‐induced increases in activation of coronary EnNaC, EC stiffness and cardiomyocyte stiffness, together with DD, were suppressed in EnNaC α deficient mice.ConclusionsCollectively, these results demonstrate that MR activation (aldosterone infusion and diet‐induced) elicits coronary vascular dysfunction associated with tissue and cellular cardiac changes and DD. Further, activation of EnNaC, a known target of MR signaling, promotes coronary EC‐dependent cardiomyocyte stiffness, cardiac fibrosis and impairs DD. Therapeutic targeting to prevent EnNaC activation may be a viable approach for the management of obesity‐associated diastolic dysfunction.

Endothelial Mineralocorticoid Receptor Deletion Abrogates Leptin‐Induced Endothelial Dysfunction in Pregnant Mice

Obesity is a risk factor for endothelial dysfunction and hypertension in pregnancy, both of which are hallmarks of preeclampsia. Our lab has demonstrated that obesity‐associated endothelial dysfunction and hypertension in female mouse models is mediated via leptin‐induced aldosterone production and subsequent mineralocorticoid receptor (MR) activation. We also showed that progesterone increases endothelial MR (ECMR) expression and that high progesterone levels in pregnant mice are associated with highly elevated ECMR expression, potentially rendering pregnant female mice susceptible to leptin‐induced endothelial dysfunction in obese pregnancy. Therefore, we hypothesized that ECMR deletion would prevent leptin‐induced endothelial dysfunction in pregnant mice in late gestation. Endothelial function was experimentally measured by wire myography concentration response curves to acetylcholine in mouse aorta. Chronic leptin infusion (0.9mg/kg/day, s.c. osmotic minipump) throughout late‐gestation (gestational days 11–18) induced endothelial impairment in wild‐type pregnant mice that was impaired both compared to sham‐treated pregnant (2‐way ANOVA with repeated measures, *P&lt;0.05) and non‐pregnant leptin‐infused females (*P&lt;0.05). However, leptin infusion in pregnant ECMR−/− mice did not induce similar endothelial dysfunction (*P&lt;0.05). Relaxation responses to acetylcholine in the presence of LNAME indicated that endothelial dysfunction in pregnant leptin‐infused mice was attributable to reduction in nitric oxide activity. In addition, no changes in endothelial‐independent sodium nitroprusside responses were observed among groups. Placental dysfunction is a characteristic of preeclampsia that precludes the development of endothelial dysfunction and hypertension. In homogenized placentas, mRNA expression of hypoxia‐inducible factor 1α (HIF1α), a marker of ischemia, was increased by leptin infusion in wild‐type (1.9±0.2‐fold change from sham wild‐type, P=0.08), but not in ECMR−/− (1.2±0.2‐fold change from sham ECMR−/−) pregnant mice. These changes in placental function were associated with adverse fetal effects in leptin‐infused wild‐type mice as indicated by decreased pup weight (0.86±0.04g sham wild‐type vs 0.52±0.11g wild‐type +leptin, *P&lt;0.05). However, ECMR deletion rescued pup weight in leptin‐infused pregnant mice (0.79±0.08g, sham ECMR−/− vs 0.86±0.11g, ECMR−/− +leptin). Collectively, these data indicate that high leptin levels in obese pregnancies may predispose to placental dysfunction and endothelial dysfunction, which are characteristics of preeclampsia.Support or Funding Information1R01HL130301‐01, 19EIA34760167, Adrenal Center pilot grant, 1K99HL146948‐01A1

Attenuation of Mineralocorticoid Receptor Signaling Promotes Vascular Recovery from Hind Limb Ischemia in a Sex Specific Manner

Peripheral arterial disease (PAD) is a growing concern affecting 8.5 million Americans annually and contributing to both fatal and non‐fatal cardiovascular ischemic events. Importantly, available evidence indicates that women experience higher rates of occlusion, reintervention, and mortality than males, however, mechanisms underlying these sex differences remain unclear. Recent evidence has revealed sex‐specific deleterious effects of mineralocorticoid receptor (MR) signaling in a variety of cardiovascular conditions including vascular inflammation and endothelial dysfunction, which have been implicated in PAD. Whether MR signaling differentially impairs microvascular adaptations in males and females following an ischemic insult, however, has not been examined. Accordingly, we evaluated the hypothesis that attenuation of MR signaling promotes microvascular compensation and perfusion recovery following hind limb ischemia (HLI) to a greater extent in females than in males involving enhanced capillary neovascularization and augmented outward remodeling of existing collaterals. Male and female C57BL/6J mice (10–12 weeks old) underwent unilateral femoral artery ligation and excision to induce HLI after implantation of pellets containing either placebo or the MR antagonist spironolactone (20mg/kg/day, sc). The contralateral leg underwent sham surgery with no ligation or excision. Hind limb perfusion was serially assessed for 28 days with laser Doppler imaging. At 28 days post occlusion, the hind limb vasculature was perfusion fixed following maximal dilation and perfused with bismuth for assessment of collateral morphology by micro‐CT imaging. These studies were complemented with immunohistochemistry and angiogenic assay experiments. Additional preliminary HLI experiments were conducted in male mice with endothelial cell‐specific MR (EC MR) deletion. Systemic inhibition of MR signaling enhanced recovery of hind limb perfusion in female, not male, mice. This effect of MR blockade was associated with greater outward collateral remodeling in females compared to males. Interestingly, spironolactone similarly increased skeletal muscle capillary density in both hind limbs of males and females consistent with impairment of VEGF‐stimulated capillary sprouting by the MR agonist aldosterone in an aortic angiogenesis assay. Finally, preliminary studies revealed enhanced recovery of hind limb perfusion in male mice with EC MR deletion and that EC MR deletion enhanced aortic capillary sprouting in vitro. Taken together, these data suggest that MR signaling limits the microvascular response to ischemia and that enhanced perfusion recovery following MR blockade in females involves greater collateral expansion compared to males. Significant vascular cell‐specificity of MR signaling underlying these responses may contribute to these sex differences and warrant further investigation.Support or Funding InformationFunded by HL136386, HL095590, and HL088105.

Two Mineralocorticoid Receptor–Mediated Mechanisms of Pendrin Activation in Distal Nephrons

Regulation of sodium chloride transport in the aldosterone-sensitive distal nephron is essential for fluid homeostasis and BP control. The chloride-bicarbonate exchanger pendrin in β-intercalated cells, along with sodium chloride cotransporter (NCC) in distal convoluted tubules, complementarily regulate sodium chloride handling, which is controlled by the renin-angiotensin-aldosterone system. Using mice with mineralocorticoid receptor deletion in intercalated cells, we examined the mechanism and roles of pendrin upregulation via mineralocorticoid receptor in two different models of renin-angiotensin-aldosterone system activation. We also used aldosterone-treated NCC knockout mice to examine the role of pendrin regulation in salt-sensitive hypertension. Deletion of mineralocorticoid receptor in intercalated cells suppressed the increase in renal pendrin expression induced by either exogenous angiotensin II infusion or endogenous angiotensin II upregulation via salt restriction. When fed a low-salt diet, intercalated cell-specific mineralocorticoid receptor knockout mice with suppression of pendrin upregulation showed BP reduction that was attenuated by compensatory activation of NCC. In contrast, upregulation of pendrin induced by aldosterone excess combined with a high-salt diet was scarcely affected by deletion of mineralocorticoid receptor in intercalated cells, but depended instead on hypokalemic alkalosis through the activated mineralocorticoid receptor-epithelial sodium channel cascade in principal cells. In aldosterone-treated NCC knockout mice showing upregulation of pendrin, potassium supplementation corrected alkalosis and inhibited the pendrin upregulation, thereby lowering BP. In conjunction with NCC, the two pathways of pendrin upregulation, induced by angiotensin II through mineralocorticoid receptor activation in intercalated cells and by alkalosis through mineralocorticoid receptor activation in principal cells, play important roles in fluid homeostasis during salt depletion and salt-sensitive hypertension mediated by aldosterone excess.

Role of Mineralocorticoid Receptor in Adipogenesis and Obesity in Male Mice.

Increased visceral adiposity and hyperglycemia, 2 characteristics of metabolic syndrome, are also present in conditions of excess glucocorticoids (GCs). GCs are hormones thought to act primarily via the glucocorticoid receptor (GR). GCs are commonly prescribed for inflammatory disorders, yet their use is limited due to many adverse metabolic side effects. In addition to GR, GCs also bind the mineralocorticoid receptor (MR), but there are many conflicting studies about the exact role of MR in metabolic disease. Using MR knockout mice (MRKO), we find that both white and brown adipose depots form normally when compared with wild-type mice at P5. We created mice with adipocyte-specific deletion of MR (FMRKO) to better understand the role of MR in metabolic dysfunction. Treatment of mice with excess GCs for 4 weeks, via corticosterone in drinking water, induced increased fat mass and glucose intolerance to similar levels in FMRKO and floxed control mice. Separately, when fed a high-fat diet for 16 weeks, FMRKO mice had reduced body weight, fat mass, and hepatic steatosis, relative to floxed control mice. Decreased adiposity likely resulted from increased energy expenditure since food intake was not different. RNA sequencing analysis revealed decreased enrichment of genes associated with adipogenesis in inguinal white adipose of FMRKO mice. Differentiation of mouse embryonic fibroblasts (MEFs) showed modestly impaired adipogenesis in MRKO MEFs compared with wild type, but this was rescued upon the addition of peroxisome proliferator-activated receptor gamma (PPARγ) agonist or PPARγ overexpression. Collectively, these studies provide further evidence supporting the potential value of MR as a therapeutic target for conditions associated with metabolic syndrome.

Mineralocorticoid Receptor and Endothelial Dysfunction in Hypertension.

To review the latest reports of the contributions of the endothelial mineralocorticoid receptor to endothelial dysfunction and hypertension to begin to determine the clinical potential for this pathway for hypertension treatment. Endothelial mineralocorticoid receptor expression is sex-specifically increased in female mice and humans compared with males. Moreover, the expression of endothelial mineralocorticoid receptors is increased by endothelial progesterone receptor activation and naturally occurring fluctuations in progesterone levels (estrous, pregnancy) predict endothelial mineralocorticoid receptor expression levels in female mice. These data follow many previous reports that have indicated that endothelial mineralocorticoid receptor deletion is protective in the development of obesity- and diabetes-associated endothelial dysfunction in female mouse models. These studies have more recently been followed up by reports indicating that both intact endothelial mineralocorticoid receptor and progesterone receptor expression are required for obesity-associated, leptin-mediated endothelial dysfunction in female mice. In addition, the intra-endothelial signaling pathway for endothelial mineralocorticoid receptors to induce dysfunction requires the intact expression of α-epithelial sodium channels (αENaC) in endothelial cells in females. Endothelial mineralocorticoid receptors are sex-specifically upregulated in the vasculature of females, a sex difference which is driven by endothelial progesterone receptor activation, and increased activity of these endothelial mineralocorticoid receptors is a crucial mediator of endothelial dysfunction, and potentially hypertension, in obese female experimental models.

Endothelial Mineralocorticoid Receptors Contribute to Vascular Inflammation in Atherosclerosis in a Sex-Specific Manner.

MR (mineralocorticoid receptor) activation is associated with cardiovascular ischemia in humans. This study explores the role of the MR in atherosclerotic mice of both sexes and identifies a sex-specific role for endothelial cell (EC)-MR in vascular inflammation. Approach and Results: In the AAV-PCSK9 (adeno-associated virus-proprotein convertase subtilisin/kexin type 9) mouse atherosclerosis model, MR inhibition attenuated vascular inflammation in males but not females. Further studies comparing male and female littermates with intact MR or EC-MR deletion revealed that although EC-MR deletion did not affect plaque size in either sex, it reduced aortic arch inflammation specifically in male mice as measured by flow cytometry. Moreover, MR-intact females had larger plaques but were protected from vascular inflammation compared with males. Intravital microscopy of the mesenteric vasculature demonstrated that EC-MR deletion attenuated TNFα (tumor necrosis factor α)-induced leukocyte slow rolling and adhesion in males, while females exhibited fewer leukocyte-endothelial interactions with no additional effect of EC-MR deletion. These effects corresponded with decreased TNFα-induced expression of the endothelial adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and E-selectin in males with EC-MR deletion compared with MR-intact males and females of both genotypes. These observations were also consistent with MR and estrogen regulation of ICAM-1 transcription and E-selectin expression in primary cultured mouse ECs and human umbilical vein ECs. In male mice, EC-MR deletion attenuates leukocyte-endothelial interactions, plaque inflammation, and expression of E-selectin and ICAM-1, providing a potential mechanism by which the MR promotes vascular inflammation. In females, plaque inflammation and leukocyte-endothelial interactions are decreased relative to males and EC-MR deletion is not protective.