Mineralocorticoid Receptors in Vascular Smooth Muscle: Blood Pressure and Beyond

Abstract

After half a century of evidence suggesting the existence of mineralocorticoid receptors (MR) in the vasculature, the advent of technology to specifically knockout (KO) the MR from smooth muscle cells (SMCs) in mice has elucidated contributions of SMC-MR to cardiovascular function and disease, independent of the kidney. This review summarizes the latest understanding of the molecular mechanisms by which SMC-MR contributes to: 1) regulation of vasomotor function and blood pressure to contribute to systemic and pulmonary hypertension; 2) vascular remodeling in response to hypertension, vascular injury, obesity and aging, and the impact on vascular calcification; and 3) cardiovascular pathologies including aortic aneurysm, heart valve dysfunction, and heart failure. Data are reviewed from in vitro studies using SMCs and in vivo findings from SMC-specific MR KO mice that implicate target genes and signaling pathways downstream of SMC-MR. By regulating expression of the L-type calcium channel (LTCC) subunit Cav1.2 and angiotensin II type-1 receptor, SMC-MR contributes to myogenic tone and vasoconstriction, thereby contributing to systemic blood pressure. MR activation also promotes SMC proliferation, migration, production and degradation of extracellular matrix, and osteogenic differentiation by regulating target genes including connective tissue growth factor, osteopontin, bone morphogenetic protein-2, galectin-3, and matrix metallopeptidase-2. By these mechanisms SMC-MR promotes disease progression in models of aging-associated vascular stiffness, vascular calcification, mitral and aortic valve disease, pulmonary hypertension, and heart failure. While rarely tested, when sexes were compared, the mechanisms of SMC-MR-mediated disease were sexually dimorphic. These advances support targeting SMC-MR-mediated mechanisms to prevent and treat diverse cardiovascular disorders.

1. MR in Vascular Smooth Muscle: Historical Background

The mineralocorticoid receptor (MR) is a hormone-activated transcription factor that regulates renal sodium absorption to control blood pressure (BP). The MR coordinates cellular functions in response to the steroid hormone aldosterone, by functioning in the nucleus as a transcription factor to regulate gene expression and in the cytoplasm to induce rapid signaling pathways.¹ Even before the MR gene was identified, animal studies in the 1950s associated mineralocorticoid-induced hypertension with cation flux in the rat vasculature, suggesting the possibility of aldosterone receptors in vessels.² Publications in the 1980s provided evidence of receptors in rabbit arteries and rat aortic smooth muscle cells (SMCs) that bind with high affinity to radiolabeled aldosterone.^3–5 The MR was cloned in 1987 by homology to the glucocorticoid receptor.⁶ In addition to activation by mineralocorticoids, aldosterone in humans and deoxycorticosterone (DOCA) in mice, the MR binds glucocorticoids with high affinity. Aldosterone specificity is maintained in the kidney by co-expression of the cortisol-inactivating enzyme 11-β hydroxysteroid dehydrogenase-2 (11βHSD2). Studies in the 1990s demonstrated co-localization in SMCs of aldosterone-binding capacity with 11βHSD2 activity⁷, suggesting that aldosterone is a ligand for SMC-MR. Over time, individual SMC-MR target genes were identified including the Na-K-ATPase,⁸ Na-K-2Cl cotransporter,⁹ and plasminogen activator inhibitor-1.¹⁰ With the advent of high throughput transcriptomics in the 2000s, the aldosterone-regulated transcriptome was determined in cultured human coronary SMCs¹¹ and in whole mouse vessels.¹² The vascular MR-regulated transcriptome is enriched with genes involved in calcification, extracellular matrix, metalloendopeptidase activity, and angiogenesis.¹² Further in vitro studies showed that aldosterone enhances SMC osteogenic differentiation and calcification,^13–16 fibrosis gene expression¹⁴ and proliferation.^15,16

Ample in vivo data reveal substantial impact of pharmacologic MR activation or inhibition on vascular function (reviewed in ^17,18); however, a direct role for SMC-MR cannot be confirmed from systemic drug studies. It was not until Cre-lox technology made cell type-specific gene deletion possible in vivo, that the direct contribution of SMC-MR to vascular pathophysiology could be formally interrogated. The first manuscript examining the impact of SMC-specific MR knockout (KO) in vivo was published in 2012.¹⁹ This review summarizes advances in our understanding of the role of SMC-MR in vascular function since that first study over 20 years ago, with greater emphasis on more recent findings. The review is organized into 3 sections: 1) SMC-MR regulation of vasomotor function and blood pressure to contribute to systemic and pulmonary hypertension; 2) SMC-MR in vascular remodeling, in response to; a) hypertension, b) vascular injury c) risk factors including obesity and aging, and d) leading to vascular calcification; and 3) Recent studies implicating SMC-MR in other cardiovascular pathologies including; a) aortic aneurysm, b) heart valve dysfunction and c) heart failure. For each area, we summarize relevant in vitro data in SMC, potential SMC-MR target genes and signaling pathways that may mediate the effects, and available data in vivo implicating SMC-MR using cell specific KO mice.

2. The Role of Smooth Muscle Cell Mineralocorticoid Receptors in Blood Pressure Regulation

A. Evidence Supporting Extra-Renal MR Contribution to Systemic Blood Pressure

The critical role for renal MR in BP regulation is established. The results of the PATHWAY-2 clinical trial demonstrated that spironolactone was the most effective add-on drug for the treatment of multi-drug-resistant hypertension compared to doxazosin, bisoprolol, and placebo, suggesting that the pathophysiological cause of treatment-resistant hypertension may involve the MR.²⁰ MR activation by aldosterone in the distal nephron induces sodium reabsorption in exchange for potassium wasting to maintain stable serum potassium levels, blood volume and BP.²¹ As such, total body KO of the MR in mice caused neonatal lethality from dehydration, unless rescued by salt supplementation.²² Hence, for some time is was concluded that renal MR alone mediates its impact on systemic BP. However, renal tubule-specific MR deficiency in mice did not fully reproduce the neonatal lethality phenotype of total body MR KO. Rather salt-wasting and death occurred only upon low-salt challenge²³ suggesting the existence of extra-renal MRs that also contribute to systemic BP. Further support for this notion comes from clinical studies of hypertensive patients treated with the MR antagonist eplerenone, showing that the degree of rise in serum potassium, a marker of MR inhibition in the kidney, does not correlate with reduction in BP.²⁴ MR expression has subsequently been discovered in other cells in which it may contribute to BP regulation including the colonic epithelium, leukocytes, vascular endothelial cells (ECs), and vascular smooth muscle cells (SMCs). Substantial data support that EC MR also contributes substantially to vascular function and cardiovascular disease however, this review specifically focuses on the role of MR in SMC, as the role of EC MR has recently been reviewed in detail.^25–27

B. In Vivo Evidence for SMC-MR Contribution to Systemic Blood Pressure Regulation

The first SMC-MR-KO model conditionally deleted MR in mice using the tamoxifen-inducible Cre-recombinase driven by the smooth muscle actin promoter.¹⁹ When SMC-MR deletion was induced in adulthood (2 months), by 7 months of age, SMC-MR-KO mice had significantly lower basal BP compared to MR-intact littermates.¹⁹ A second model with constitutive SMC-MR-KO confirmed lower BP in SMC-MR-KO mice as early as 5 months of age, consistent with SMC-MR deletion from conception (i.e. 2 months earlier).²⁸ Importantly, in both models SMC-MR-KO mice showed no alterations in acute or chronic renal sodium handling or basal vascular structural defects compared to MR-intact littermates.^19,28 Rather, as they aged, inducible SMC-MR-KO mice were found to have lower resistance vessel myogenic tone and attenuated agonist-mediated vasoconstriction,¹⁹ vascular mechanisms that contribute to peripheral vascular resistance and hence to BP. In addition to a role in basal BP control, SMC-MR also contributes to the rise in BP in hypertension models induced by high salt,¹⁹ AngII infusion,¹⁹ and uninephrectomy with high salt.²⁸ However, when aldosterone and high salt were given together,¹⁹ the rise in BP was the same in MR-intact and SMC-MR-KO mice, supporting the capacity for renal MR to overcome the lack of SMC-MR with sufficiently robust renal hypertensive stimuli. Overall, a growing body of evidence supports a direct contribution of SMC-MR to physiologic and pathophysiological systemic BP regulation.

C. Mechanisms of SMC-MR Control of Systemic Blood Pressure

1). SMC-MR Contributes to Vasoconstriction and Tone:

Resistance vessels constrict in response to contractile agonists or increased intravascular pressure, thereby generating myogenic tone which contributes to peripheral vascular resistance and hence to BP. Aged SMC-MR-KO mice generate less resistance vessel myogenic tone.¹⁹ SMC-MR also contributes to vasoconstriction in response to calcium channel agonists and extracellular potassium (stimuli that open voltage-gated calcium channels), AngII, and thromboxane activators, with no impact on vasoconstriction to phenylephrine.^19,29 Whether deficiency of SMC-MR contributes to endothelium-dependent or –independent vasodilation is more controversial with differences between studies potentially depending on the specific dilators tested^19,29 or vascular bed studied (mesenteric¹⁹, aorta²⁹, coronary³⁰). As coronary arteries and the aorta do not contribute to vascular resistance, mesenteric vessels are the only vessel examined in which MR contributes to systemic blood pressure control.

2). Mechanisms by which SMC-MR Contributes to Vascular Constriction:

SMC constriction is initiated by opening of voltage gated L-type calcium channels (LTCC), allowing calcium influx which promotes the release of intracellular calcium stores via sarcoplasmic reticulum calcium channels (SERCA). Increased intracellular calcium leads to phosphorylation of myosin light chain by myosin light chain kinase that ultimately results in SMC contraction. Large-conductance calcium-activated potassium channels (BKCa) act as a brake on vasoconstriction by releasing potassium to repolarize the membrane and close the LTCC, thereby reducing myogenic tone and blood pressure.³¹ DuPont et al demonstrated that SMC-MR induces expression of Cav1.2, the pore-forming subunit of the LTCC, in the aging vasculature by inhibiting transcription of microRNA (miR)-155, which targets Cav1.2 mRNA for degradation.³² More recent data reveal that MR also directly induces Cav1.2 exon1a transcription via its promoter in rat cardiomyocytes.³³ In vivo, aldosterone increased coronary, aortic, and mesenteric artery Cav1.2 exon1a expression and increased coronary vasoconstriction to potassium chloride or LTCC activation.³³ Subsequent research in rat mesenteric arterioles showed that aldosterone may also induce protein expression of SERCA via MR which contributes to sarcoplasmic reticulum calcium reuptake.³⁴ Ultimately, decreased LTCC activity in SMC-MR-KO mice results in decreased phosphorylation of myosin light-chain kinase, myosin regulatory light chain 2, and myosin phosphatase-targeting protein subunit-1 in resistance vessels, which ultimately decreased SMC contraction (Figure 1).²⁹

Figure 1. Roles of Smooth Muscle Cell Mineralocorticoid Receptor in Vasomotor Function and Blood Pressure Regulation.

Upon aldosterone activation of mineralocorticoid receptors in smooth muscle cells (SMCs), MR induces expression of the angiotensin II type 1 receptor (AT1R) and L-type calcium channel (LTCC) subunit Cav1.2. MR induces these genes by direct binding to their promoter or by inhibiting expression of microRNA (miR)-155, which inhibits degradation of AT1R and Cav1.2 mRNA. Increased AT1R enhances angiotensin II (AngII)-mediated vasoconstriction and leads to increased reactive oxygen species (ROS) which inhibit soluble guanylate cyclase (sGC), thereby preventing vasodilation. Aldosterone-mediated activation of SMC-MR also decreases large-conductance calcium-activated potassium (BK_Ca) channel expression which promotes membrane depolarization and hence LTCC activity. Increased Cav1.2 expression and LTCC activity leads to increased Ca²⁺ influx into SMC, resulting in myosin light chain (MLC) phosphorylation by myosin light chain kinase (MLCK) and SMC constriction. Vascular aging leads to an increase in MR expression to contribute to vascular calcium channel activity and AngII-induced vasoconstriction. In obesity, SMC-MR exacerbates western diet-induced coronary dysfunction and AngII-related vasoconstriction while impairing acetylcholine-induced vasodilation in females. SMC-MR also mediates calcineurin inhibitor-induced renal vasoconstriction and oxidative stress to contribute to drug-induced kidney damage. Following a pre-eclampic pregnancy, SMC-MR mediates the exaggerated vasoconstrictive response to hypertensive stimuli including high salt and AngII. All these mechanisms are inhibited by mineralocorticoid receptor antagonists (MRA) or by knock-out of the MR gene specifically in SMC (SMC-MR-KO) mice.

The role of SMC-MR in regulating the BKCa appears to be more context dependent. In aging-associated hypertension, McCurley et al. found no impact of SMC-MR deletion on aortic mRNA expression of BKCa subunits nor BKCa channel function in mesenteric SMCs via patch clamp electrophysiology studies.¹⁹ On the contrary, in mice with cardiomyocyte-specific aldosterone synthase overexpression, BKCa subunit expression was decreased in the coronary arteries and acetylcholine-mediated vasodilation was impaired in a BKCa-dependent manner.³⁵ This suggests that MR activation locally by cardiomyocyte-derived aldosterone decreased coronary vessel BKCa channel expression to enhance vasoconstriction. Whether this was mediated directly by SMC-MR was not tested in that study. Finally, one study demonstrated that MR contributes to vasoconstrictive responses to phenylephrine and KCl in female rats using an MR antagonist. The study was performed in endothelium-denuded aortic rings, thereby implicating SMC-MR in vasoconstriction in females, and using a BKCa antagonist to implicate BKCa channels in the mechanism. Provencer et al. further compared pregnant to non-pregnant rats and showed that the pregnancy associated decrease in vasoconstriction, was dependent on BKCa channels and this was prevented with MR inhibition.³⁶ Thus, it appears that regulation of BKCa by MR is dependent on physiologic state (pregnancy) and the vascular bed in question (coronary versus mesenteric). Whether any of these effects of SMC-MR on ion channels differs by sex has not been studied.

In addition to regulating vascular calcium channels, multiple cross talk mechanisms between SMC-MR and AT1R signaling also contribute to vasoconstriction and BP (reviewed in ³⁷). Studies have shown that AT1R signaling contributes to classical aldosterone-induced activation of SMC-MR via ERK1/2, JNK, and NFkB pathways.³⁸ SMC-MR also induces AT1R expression in aging vascular SMCs via downregulation of miR-155, a miR that targets the AT1R mRNA for degradation.³² Ligand-independent activation of MR by AngII has also been demonstrated in human SMCs via AT1R signaling to protein kinase C; however, the potency for MR activation by AngII is substantially lower (about 50%) than the classical ligand.³⁹ Furthermore, stimulation of VSMCs with aldosterone or AngII individually had no effect on c-Src activation, whereas simultaneous stimulation with both agents activated c-Src, illustrating another dimension of this AngII-MR signaling cross talk.³⁷ Finally, AngII induces oxidative stress to contribute to vasoconstriction and this was prevented in vessels from SMC-MR-KO mice consistent with a decreased BP response to AngII infusion.¹⁹ Together, these data suggest a complex interaction between AngII and aldosterone signaling via MR in SMC to contribute to myogenic tone and BP control.

The SMC-MR-mediated increase in vascular oxidative stress may further exacerbate hypertension in feedforward fashion. One study showed that oxidative stress induced by aldosterone in SMC causes impaired guanylyl cyclase activity, thereby reducing SMC dilation capacity.⁴⁰ Oxidative stress can further induce expression of SMC-MR itself via activation of the transcription factor HIF1α, which was recently found to transcriptionally regulate the MR gene.⁴¹ SMC-MR-mediated oxidative stress and vasoconstriction also contribute directly to hypertension and renal damage induced by calcineurin inhibitors used for immune suppression in transplant recipients.^42,43 SMC-MR-KO ameliorated oxidative stress, vascular fibrosis, hypertension, and renal injury due to high-dose tacrolimus treatment in mice⁴³ and attenuated renal microvascular contraction and impaired renal perfusion in response to cyclosporine.⁴² Furthermore, during renal ischemia, activated SMC-MR in the renal vasculature was found to induce Rac1 thereby increasing oxidative stress in SMCs. Induction of ROS production by SMC-MR was found to promote post-translational sulfenic acid modification of endothelin B receptor in ECs, impairing eNOS activation and diminishing NO production in ECs.⁴⁴ In this way, SMC-MR-induced oxidative stress contributes to endothelial dysfunction in the setting of renal ischemia, which leads to sustained vasoconstriction and reduced kidney perfusion. Similar post-translational modifications of endothelin B receptors have also been demonstrated in pulmonary arterial endothelial cells when treated with aldosterone.⁴⁵

3). Advances in Understanding SMC-MR Contribution to Vasoconstriction and Blood Pressure in Females:

At the time of the last review of SMC-MR in vasomotor function in 2019⁴⁶, only one study had considered vascular function in female SMC-MR-KO mice.⁴² More information has since become available regarding the role of SMC-MR in regulating vasomotor function in females in response to risk factors including aging, obesity, and exposure to pregnancy complications. A recent study explored sex-differences in the role of SMC-MR in mesenteric resistance vessel aging by comparing male and female, young (3 month), middle aged (12 month), and old (18 month) MR-intact to SMC-MR-KO mice.⁴⁷ MR expression and AngII-induced vasoconstriction both increased with age in mesenteric arteries from both sexes. In males, SMC-MR-KO mice had decreased mesenteric AngII constriction by 12 months of age, with lower expression of the AT1R. These aging-associated changes occurred at an older age in females (18 months), when most mice have ceased estrous cycling. Female SMC-MR-KO were also protected from rising AngII vasoconstriction with aging, but the expression of AT1R was unchanged.⁴⁷ Thus, SMC-MR contributes to enhanced AngII vasoconstriction with aging in both sexes by sexually dimorphic mechanisms. As pre-menopausal protection from cardiovascular disease in women is lost after menopause, estrogen receptors were examined and found to inhibit the transcriptional impact of MR in ECs,⁴⁸ and estrogen abrogated aldosterone-induced oxidative stress in SMC by increasing glucose-6-phosphate dehydrogenase enzyme activity and cellular NADPH.⁴⁹

While a role for MR in obesity-induced vascular dysfunction has been extensively studied, most studies have focused on the role of MR in ECs, with much less known about the role of SMC-MR. New studies in female mice reveal a specific role of SMC-MR in coronary artery function in response to obesity. Western diet-induced obesity in female mice significantly impaired coronary artery dilation to acetylcholine and enhanced constriction to the thromboxane agonist U46619 in MR-intact but not in SMC-MR-KO mice.³⁰

Finally SMC-MR is also implicated in the vascular impact of pregnancy complications.^50,51 Preeclampsia is a common hypertensive disorder of pregnancy which predisposes women to a high risk of developing early hypertension and cardiovascular disease post-partum. Biwer et al examined the response of SMC-MR-KO mice to hypertensive stimuli after exposure to an experimental model of preeclampsia during pregnancy.⁵⁰ Despite no difference in systemic BP between MR-intact and SMC-MR-KO mice during pre-eclamptic pregnancy, SMC-MR-KO mice were protected from the enhanced BP response to high salt or AngII several months post-partum.⁵⁰ After exposure to preeclampsia and post-partum hypertensive challenges, mesenteric arteries from previously preeclamptic mice had enhanced AngII-mediated constriction, myogenic tone, and AT1R expression, all of which were prevented in SMC-MR-KO mice. In vitro studies show that after exposure to anti-angiogenic pre-eclamptic proteins, SMC-MR is more transcriptionally active. Similarly, resistance vessel vasoconstriction was recently shown to be increased post-partum in both the mothers and offspring exposed to obesity during pregnancy, in a MR-dependent manner.⁵¹ Zheng et al demonstrated that after exposure to diet-induced obesity in utero, mesenteric SMCs from adult male offspring had increased calcium currents.⁵¹ Aldosterone increased calcium current more in offspring from an obese pregnancy, and this was ameliorated by the MR antagonist eplerenone, however, this was not tested in SMC-MR-KO mice.⁵¹

Overall, ample data now support that SMC-MR contributes to vasoconstriction, myogenic tone and systemic BP in males and females by sexually dimorphic mechanisms involving AT1R expression, oxidative stress, and regulation of L-type calcium channels. By these mechanisms, SMC-MR contributes to vasoconstriction in response to a variety of physiologic or pathologic stimuli (Figure 1) including; 1) aging¹⁹, 2) hypertensive stimuli including, renin-angiotensin aldosterone activation, high salt, and preeclampsia^19,50, 3) after myocardial infarction⁵² and 4) during acute kidney injury.⁴²

B. SMC-MR and its Role in Pulmonary Arterial Hypertension (PAH)

Pulmonary arterial hypertension (PAH) is characterized by a progressive rise in pulmonary artery pressure resulting in death from right heart failure. Serum aldosterone levels are elevated and correlate with disease severity in human PAH^53,54 and MR expression is increased in pulmonary vessels from PAH patients and in experimental rodent models.^55–57 Multiple in vitro studies show that MR is increased, and 11βHSD2 is decreased, in SMC from PAH patients compared to controls, supporting a potential role for SMC-MR in PAH progression.^46,54,55,58 In idiopathic PAH, the elevated pulmonary artery pressure occurs due to a combination of pulmonary vasoconstriction and adverse pulmonary vessel remodeling, processes to which SMC-MR contributes in other vascular beds.

Multiple groups have demonstrated that MR activation promotes human pulmonary artery SMC proliferation and provide in vitro data exploring mechanisms.^46,54,55,58 Yamanaka et al. showed that bone morphogenetic protein-2 (BMP2) signaling, which is increased in PAH, induces SMC-MR expression in human pulmonary artery (PA)SMCs.⁵⁵ MR, in turn, has been shown to regulate BMP2 expression^11,13 in SMC, resulting in a feed-forward loop.⁵⁴ Amplified aldosterone-induced ERK signaling promotes proliferation of PASMCs, which was abrogated by the MR antagonist eplerenone.⁵⁵ In PASMCs from the monocrotaline-induced rat model, MR activation induced aquaporin-1 expression and SMC proliferation.⁵⁹ Finally, aldosterone induced AKT signaling via mTOR and Raptor thereby activating p70S6K which increases proliferation and viability of PASMCs.⁵⁸ How these many proliferation mechanisms are connected remains to be clarified, but a role of MR in PASMC proliferation in vitro is well supported.

Multiple in vivo studies using diverse rodent PAH models reveal a role for MR in vascular remodeling and PAH progression.^{45,56,57,60–64} Spironolactone, eplerenone, and finerenone (the recently approved non-steroidal MR antagonist) have all been shown to prevent PAH and/or to treat established disease in rodent models induced by chronic hypoxia, sugen/hypoxia or monocrotaline. Only one study examined the specific role of SMC-MR in PH in vivo. In the mouse sugen/hypoxia model, Menon et. al. showed that while spironolactone decreased pulmonary vessel remodeling, perivascular inflammation and right ventricular pressure, SMC-MR-KO reduced only pulmonary vascular inflammation without attenuating right ventricular systolic pressure or SMC remodeling,⁶¹ consistent with other literature showing a complex role for MR in multiple cell types in PAH pathogenesis (Reviewed in ⁶⁵). Indeed, in vitro studies suggest that cross talk between MR in ECs and SMCs contributes to pathologic gene expression in PAH. Hypoxia-exposed human pulmonary artery ECs promoted aldosterone-dependent transcription of the profibrotic regulator connective tissue growth factor (CTGF) in PASMCs.⁶⁶ Similarly, aldosterone-induced NEDD9 activation in pulmonary ECs caused exosome-mediated NEDD9 activation of PASMCs and increased COL3A1 expression via SMC-MR.⁶⁶ Together, ample data from human tissues and cultured cells and from multiple preclinical PAH models support a role for MR in PAH with SMC-MR likely contributing to SMC proliferation, inflammation and fibrosis.^56,57,59,61 The possibility that SMC-MR or its downstream targets could serve as therapeutic targets for PAH warrants additional exploration as most current therapies only target pulmonary vasodilation.

3. Smooths Muscle Cell Mineralocorticoid Receptors in Vascular Remodeling

A. SMC MR in Vascular Remodeling in Response to Hypertension

When exposed to chronic high pressure, arterial wall thickening occurs due to SMC proliferation and extracellular matrix remodeling. Over time, vascular remodeling contributes to rising vascular stiffness, which further exacerbates hypertension and contributes to microvascular damage resulting in end-organ dysfunction including heart and kidney failure.⁶⁷ Thus, understanding the mechanisms by which elevated BP drives vascular remodeling and stiffness is critical to mitigating the adverse effects of hypertension on cardiovascular disease.

Early in vitro studies using cultured aortic SMC showed that AngII and aldosterone synergistically induce SMC proliferation by mechanisms involving ERK and epidermal growth factor signaling.¹⁶ Aldosterone also induced SMC migration via MR signaling to Rho-kinase.⁶⁸ A role for these mechanisms in the vascular response to hypertension was confirmed in SMC from hypertensive rats in which MR activation by aldosterone increased ERK and MAPK signaling, NADPH oxidase activity, SMC proliferation and collagen synthesis significantly more than in cells from non-hypertensive rats.⁶⁹

Studies in rodent hypertension models have similarly supported a role for MR in regulating fibrotic remodeling induced by hypertension (reviewed in ⁷⁰). In a rat model, aldosterone-induced hypertension resulted in increased aortic SMC proliferation, vascular fibrosis, and expression of transforming growth factor (TGF)-beta and collagen.⁷¹ Hypertension-induced vascular remodeling was prevented the MR antagonist eplerenone but not with the same degree of BP lowering with hydralazine, suggesting a BP-independent role of MR in vascular remodeling. This was ultimately confirmed using constitutive SMC-MR-KO mice in which aldosterone/salt-induced hypertension increased vascular stiffness in MR-intact mice but not in SMC-MR-KO littermates, despite the same degree of BP elevation.²⁸

Mechanistically, several MR-regulated genes have been implicated in vascular remodeling in the setting of hypertension (Figure 2). CTGF is a SMC-MR target gene that induces collagen expression.¹² Osteopontin, a protein induced in the setting of hypertension that contributes to arterial remodeling⁷², was induced by aldosterone in rat SMCs via a classical MR binding site in the gene promoter.⁷³ MR activation in rat SMC also induces galectin-3 (Gal-3) thereby driving type I collagen expression in vitro and vascular hypertrophy, inflammation and fibrosis in the aldosterone/salt-induced hypertension model were prevented by MR inhibition or Gal-3 knockout.⁷⁴ Finally, in SMC-MR-intact mice, hypertension-induced vascular stiffness and increased collagen, fibronectin, and integrin-alpha5 expression were lost in SMC-MR-KO mice.²⁸

Figure 2. Smooth Muscle Cell Mineralocorticoid Receptor-Mediated Mechanisms of Vascular Remodeling and Impact on Cardiovascular Diseases.

Activation of mineralocorticoid receptor (MR) by aldosterone (A) in smooth muscle cells (SMC) leads to enhanced SMC proliferation by extracellular signal-regulated kinase (ERK) and epidermal growth factor (EGF) signaling and migration via Rho signaling (Rho). SMC-MR increases vascular fibrosis by induction of osteopontin (OPN), galectin-3 (Gal-3), and connective tissue growth factor (CTGF); and contributes to calcification by inducing SMC dedifferentiation to an osteoblastic phenotype by regulation of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), and microRNA (miRNA)-34b/c. Vascular aging induces vascular stiffness by epigenetic histone modifications that are regulated by SMC-MR. In mineralocorticoid-induced aneurysm, brain and muscle Arnt-like protein-1 (BMAL1) inhibits tissue inhibitor metalloproteinase 4 and cellular communication network factor 2 (CCN2) activates metalloproteinase (MMP) 2 and 9, leading to elastin degradation, inflammatory cell infiltration and SMC degeneration. In heart valvular interstitial cells (VICs), activation of MR by aldosterone induces calcification, proteoglycan production and fibrosis. In a model of pressure-overload-induced heart failure (by transverse aortic constriction (TAC)), SMC-MR contributes to cardiac perivascular fibrosis, oxidative stress and ventricular dysfunction by regulating coronary flow reserve and angiogenesis. All these mechanisms are inhibited by mineralocorticoid receptor antagonists (MRA) or by knock-out of the MR gene specifically in SMC (SMC-MR-KO) mice.

B. SMC-MR Contributes to Injury-Associated Vascular Fibrosis Aging-Associated Vascular Stiffness

Another situation in which vascular SMC proliferation and fibrosis contribute to adverse outcomes is after vessel injury. In response to mechanical injury, vascular SMCs dedifferentiate from a contractile state into a phenotype that can proliferate, migrate, and produce extracellular matrix. This injury response contributes to vessel healing but is detrimental when excessive, as in transplant allograft vasculopathy, in-stent restenosis, vein graft failure, or arteriovenous fistula dysfunction (Reviewed in ^46,75). A role for MR in adverse vessel remodeling after injury has been demonstrated (Reviewed in ⁷⁶). For arterovenous fistula failure, a role for MR has been postulated, but it has never been directly explored in preclimical models or in humans.⁷⁷ MR is expressed in venous SMC and is induced when veins are grafted into the arterial circulation in humans.⁷⁸ In a mouse model of vein grafting, MR inhibition attenuated vein graft SMC hypertrophy, fibrosis and inflammation, but a specific role for SMC-MR has not been tested in vivo.⁷⁹ Aldosterone enhances, and MR antagonism inhibits, SMC proliferation and vessel fibrosis in multiple preclinical models of arterial injury.⁷⁰ In the mouse wire-induced carotid injury model, aldosterone induced a significant increase in the proliferative and fibrotic responses to injury and the degree of vessel fibrosis after injury was attenuated in mice lacking SMC-MR.⁸⁰ Aldosterone-dependent induction of both proliferation and fibrosis was also completely dependent on SMC-MR.⁸⁰ One mechanism involved SMC-MR regulation of placental growth factor and of the VEGF type-1 receptor specifically in injured SMCs.⁸⁰ Overall, ample data support that SMC-MR directly contributes to exuberant vascular responses to injury that limit the benefits of many surgical treatments for vascular disease.

Vascular fibrosis and stiffness also increase with aging in humans. This has significant clinical implications as aortic stiffness is an independent predictor of MI, stroke and cardiovascular death in men and women.⁸¹ In mice, vascular stiffness similarly increases with age in males and females.⁴⁷ The progression of vascular stiffness was prevented when middle aged mice (12 months) were treated with the MR antagonist, spironolactone.⁸² Aging-associated vascular stiffness is prevented in male and female mice with SMC-MR-KO but again, the mechanism appears to differ by sex.^47,82,83 In males, SMC-MR-KO prevented aortic, carotid, and renal arteriolar fibrosis by downregulating expression of a pro-fibrotic gene program including CTGF, BMPs, and collagen genes, while these gene expression changes did not occur in female mice.^41,47,82 Additional mechanistic studies only performed in male mice revealed that shifts in aging-induced vascular gene expression programs associate with global changes in epigenetic histone modifications. Specifically, aging associates with induction of acetylation of histone H3 at lysine 27 (H3K27) in the aorta that is driven by SMC-MR suppression of the H3K27 methyltransferase, EZH2.⁸⁴ H3K27 acetylation is associated with open chromatin in aging human aortic SMCs,^85,86 with enrichment of MR binding to the promoter of CTGF and integrin-a5 resulting in increased expression of those fibrosis genes.⁸⁴ Atomic force microscopy studies showed that MR inhibition decreased intrinsic stiffness of human SMCs and SMC adhesion to fibronectin via integrin.⁸⁴ Although MR expression and vascular stiffness both rise with age in human SMCs and mice of both sexes, the mechanism in females does not appear to involve vessel fibrosis.⁴⁷ The female-specific mechanism of vascular stiffness in aging deserves further study. Preeclampsia is thought to lead to a situation of early vascular aging and recent data reveal enhanced vascular stiffness in a model of post-preeclampsia hypertension, and this was also prevented in SMC-MR-KO mice.⁵⁰ In vitro, transient exposure of SMC to anti-angiogenic proteins that are induced in preeclampsia exacerbated aldosterone induction of the profibrotic gene, CTGF.⁵⁰ These studies support an important role for SMC-MR in driving vascular aging pathophysiology.⁸³

C. Contribution of the SMC-MR to Vascular Calcification

Vascular calcification occurs when calcium mineral deposits in the intimal and medial layers of the vessel wall.⁸⁷ Diabetes, aging, and chronic kidney disease (CKD) are associated with vascular calcification and the burden of calcification is associated with greater risk of adverse cardiovascular outcomes.⁸⁷ Vascular calcification is an actively regulated process in which vascular SMCs dedifferentiate into an osteoblast-like phenotype via processes typically involved in bone mineralization, but in the vessel wall.⁸⁷ An early study of the aldosterone-regulated human coronary SMC transcriptome revealed overrepresentation of calcification genes, including BMP2 and alkaline phosphatase.¹¹ A follow up study demonstrated that MR activation by aldosterone in aortic SMCs promoted an osteoblastic phenotype with enhanced alkaline phosphatase expression and mineral deposition, although BMP2 signaling was not required.¹³ More recent studies have explored potential mechanisms; One study showed that aldosterone-induced vascular calcification was mediated by MR-mediated downregulation of miR-34b/c resulting in upregulation of special AT-rich sequence-binding protein 2 (SATB2), to induce Runx2, a master calcification regulator;⁸⁸ Another study implicated inhibition of autophagy and activation of AMPK pathway.⁸⁹ A common feature of all of these studies is that basal, aldosterone-induced, and glucocorticoid-induced SMC calcification are all prevented by MR antagonists^13,88–90 supporting a direct role of MR in SMC calcification in vitro.

In vivo studies confirm a role for MR in rodent vascular calcification models. Chronic kidney disease (CKD) is the most profound risk factor for vascular calcification, which is induced by high serum calcium and phosphate levels (Review in ^91,92). In a mouse model of high phosphate diet-induced vascular calcification without CKD, mineralocorticoid co-administration exacerbated vascular calcification. In the hyperphosphatemic Klotho vascular calcification model, spironolactone blocked vascular calcification, further implicating the MR. This was true even after adrenalectomy, so Alesutan et al suggested that elevated phosphate levels may induce aldosterone production by SMC via induction of CYP11B2, the gene encoding aldosterone synthase.⁹³ Whether SMC can generate aldosterone remains controversial, as others have not demonstrated aldosterone production by human SMC using multiple methods¹¹ (although others have not examined the impact of high phosphate). Even in the Alesutan et al study, aldosterone was not detectable in the SMC media.⁹³ Overall, ample data support a role for MR in vascular calcification, but the source of MR activation is not totally clear. Finally, while MR inhibition to prevent vascular calcification has never been tested in clinical trials, one study showed that serum from CKD patients randomized to MR inhibition (MiREnDa trial) induced less calcification versus placebo, in human aortic SMCs in vitro, and in the nephrectomy/high calcium vascular calcification. mouse model in vivo.⁹⁴ While ample data support that MR contributes to vascular calcification, studies are needed using SMC-MR-KO mice to confirm the direct role of SMC-MR in calcification in vivo.

4. Emerging Roles for SMC-MR in Aneurysm, Valve Disease and Heart Failure

A. Involvement of Aldosterone in Aortic Aneurysm

Aortic aneurysm is a localized dilation of the aorta caused by disruption of vascular structure characterized by vessel inflammation, medial smooth muscle cell death and elastin degradation. Advanced age, male sex, smoking, and hypertension are risk factors for aortic aneurysm development.⁹⁵ Aneurysm expansion can lead to rupture which is often lethal. While the incidence of aneurysm is four times greater in males, the rate of expansion and rupture is higher in females.⁹⁶ Since SMC-MR is induced in the aging vasculature and contributes to vessel remodeling in response to aging and hypertension by sexually dimorphic mechanisms, SMC-MR might be expected to contribute to aortic aneurysm pathogenesis.

Activation of the MR in mice by DOCA or aldosterone infusion with high salt diet induces thoracic and abdominal aortic aneurysm formation and rupture.⁹⁷ Aneurysm progression was significantly more rapid when older mice (10 months), in which SMC-MR expression is known to be elevated,⁸⁴ were exposed to DOCA/salt versus younger mice (2 months).⁹⁷ Pathologically, DOCA infusion induced elastin degradation, inflammatory cell infiltration and SMCs degeneration and this was prevented by MR inhibition with spironolactone or eplerenone⁹⁷, confirming a role for MR in this aneurysm model. Mechanistically, DOCA induced vascular expression of MMP2 and MMP9⁹⁷, enzymes previously implicated in matrix degradation in aneurysm.⁹⁸ Substantial data supports a role for SMC-MR in regulating MMP expression, particularly in the aging vasculature. The original aldosterone-regulated vascular transcriptome was significantly overrepresented for the molecular function “metalloendopeptidase activity”.¹² Gene expression profiling in aging mouse vessels with MR-intact or SMC-MR-KO mice revealed that specifically in SMC-MR-KO mice, vascular MMP2 declines with age.⁸² Finally, when an aging phenotype was induced in primary human aortic SMC by exposure to oxidative stress, MR expression increased along with enrichment of MR at the MMP2 promoter and MMP2 expression.⁸⁴ While no study has specifically examined the role of SMC-MR in aneurysm formation using cell-specific KO mice, other SMC genes have been implicated in mineralocorticoid-induced aneurysm. Brain and muscle Arnt-like protein-1 (BMAL1) is a circadian regulator that was recently shown to be induced by mineralocorticoid/salt infusion in mice, and SMC-BMAL1-KO protected from aortic aneurysm in this model.⁹⁹ Mechanistically, BMAL1 was found to bind to the promoter of tissue inhibitor of metalloproteinase-4 (TIMP4) and suppressed TIMP4 transcription. As such, BMAL1-KO induced TIMP4 which inhibited MMP and elastin degradation.⁹⁹ Another study examined the role of cellular communication network factor 2 (CCN2) in aneurysm, showing that CCN2-KO mice have increased AngII-induced aneurysm formation and mortality in association with induction of MMP2 and MMP9 activity, all of which was significantly attenuated by MR inhibition.¹⁰⁰ Future studies should directly explore the role of SMC-MR in aortic aneurysm and consider clinical testing of MR inhibition to prevent aneurysm progression to rupture.

B. Contribution of MR in Heart Valve Diseases.

While we focus this review on vascular SMCs, analogous cell types contribute to the normal and pathologic function in heart valves. Heart valves, like vessels, are composed of a surface layer of valve ECs overlying multiple layers of valve interstitial cells (VICs) that share many functions of vascular SMCs and contribute to the pathophysiology of heart valve disease (reviewed in ¹⁰¹). Heart valves separate the chambers of the heart, allowing for regulated and efficient flow of blood between chambers. The prevalence of valve disorders is rising, in part due to the aging population, with the most common being stenosis of the aortic valve (that allows blood to pass from the left ventricle into the aorta) and leakage of the mitral valve (which normally prevents blood from flowing from the left ventricle back into the left atrium during ventricular systole).⁷⁶ MR was recently shown to be expressed in VICs from male and female patients with mitral valve prolapse or aortic stenosis and to correlate with VIC activation markers, suggesting that MR could contribute to valve disease.^102,103 In cells from MV prolapse patients, aldosterone increased VIC proliferation and proteoglycan expression in a MR-dependent manner.¹⁰² In a small nonrandomized observational study, MV prolapse patients on an MR antagonist had less proteoglycan and MMP2 expression in valve tissue at the time of surgery.¹⁰² In vivo, in a mouse model of nordexfenfluramine-induced mitral valve prolapse, MR inhibition decreased mitral leaflet thickening and proteoglycan deposition.¹⁰² Using cell specific MR-KO mice in this model of mitral valve prolapse, Ibarrola et al. showed that EC-MR-KO mice are protected from the deleterious effects of NDF, reducing mitral valve area and thickness as well as proteoglycan deposition while SMC-MR-KO (using the alpha-smooth muscle actin-Cre), no protective effects were observed.¹⁰² Together, the data support a predominant role for MR in valve endothelial cells in mitral valve prolapse. Activated VICs express smooth muscle actin so we might expect that MR was deleted; however, as the authors did not present evidence confirming whether the SMA Cre induces MR deletion from VICs in this model, it is possible that MR in activated VICs may still contribute to mitral valve prolapse and further studies are needed to clarify this.

In VICs from aortic stenosis patients, while MR expression correlated with activation markers in both sexes, it correlated more tightly with calcification markers in males.⁹⁵ MR activation induced calcification genes in male-derived VICs, including BMP2, BMP4, osteopontin, and periostin. There are important sex differences in the histopathology (males more calcified, females more fibrotic) and outcomes of aortic stenosis (males greater prevalence, females worse surgical outcomes).¹⁰⁴ While surgical valve replacement is the only solution for heart valve diseases,¹⁰⁵ these studies open the possibility of considering pharmacologic approaches to prevent the progression of valve disease, with MR inhibition as a testable target.

C. A Direct Role for SMC-MR in Heart Failure Development

A role for the MR in heart failure has long been appreciated as multiple large randomized clinical trials show that MR antagonists decrease mortality in heart failure patients (reviewed in ¹⁰⁶). This has long been attributed to MR in cardiomyocytes (reviewed in ¹⁰⁷). SMC are, of course, present in the coronary vessels in the heart, where they contribute to the critical regulation of cardiac blood supply. Here we summarize the available studies examining a potential role for SMC-MR in the response to stresses that lead to heart failure including MI, aging, and pressure overload. In a mouse model of MI, a common cause of heart failure with reduced ejection fraction, one study directly compared the impact of MR inhibition with finerenone, to SMC-MR-KO. Global MR inhibition improved ventricular function and reduced cardiac fibrosis after MI.⁵² SMC-MR-KO alone reproduced the improvement in cardiac remodeling seen with finerenone, and some parameters of LV function, including elasticity and compliance.⁵² Furthermore, SMC-MR-KO improved coronary flow reserve post-MI by improving NO bioavailability and reducing oxidative stress-mediated coronary endothelial dysfunction. This suggests that SMC-MR contributes significantly to post-MI vascular ROS production that impairs EC function and hence coronary vascular function after MI.⁵² These data support a role for SMC-MR in progression of heart failure post MI, by impairing coronary blood flow and contributing to adverse cardiac remodeling and the decline LV function, although the more complete protection by global MR inhibition highlights additive roles of MR in other cell types in post-MI heart failure. A role for SMC-MR was also tested in aging-associated cardiac dysfunction.⁴⁷ The heart dilates and cardiac systolic function declines with age in male and female mice. SMC-MR-KO mice were protected from cardiac dilation and systolic dysfunction with aging in association with decreased cardiac perivascular fibrosis, but the mechanisms again appear to differ by sex.⁴⁷ Finally, in a model of heart failure induced by pressure overload due to transverse aortic constriction, SMC-MR-KO mice had reduced cardiomyocyte hypertrophy, cardiac interstitial and perivascular fibrosis, improved systolic function, and developed less pulmonary edema and exercise intolerance.¹⁰⁸ Mechanistically, deficiency of SMC-MR enhanced capillary density and improved coronary flow reserve in response to pressure overload.¹⁰⁸ Together, these findings confirm that SMC-MR not only contributes to vascular disease but also to the development of heart failure in response to the common etiologic factors including MI, pressure overload and aging.

5. Conclusions

Following up on studies suggested a role for vascular MR since the 1950s, 20 years of data using tissue-specific knockout mice have now demonstrated that the MR in vascular SMCs is an important driver of diverse cardiovascular diseases. SMC-MR directly contributes to systemic blood pressure control and hypertension by regulating calcium channels, angiotensin receptors, and oxidative stress to modulate myogenic tone and vasoconstriction (Figure 1). SMC-MR is also a critical driver of vascular remodeling in response to hypertension, vascular injury, and cardiovascular risk factors including obesity and aging thereby contributing to vascular, cardiac, and renal hypertrophy, fibrosis, and stiffness (Figure 2). As such, SMC-MR has been found to induce disease progression in diverse models of human disease including pulmonary hypertension, chronic renal failure, vascular calcification, aging-associated vascular stiffness, and vascular injury. Emerging evidence also links SMC-MR to aortic aneurysm formation, cardiac valve dysfunction, and even heart failure. SMC-MR-mediated mechanisms are sexually dimorphic whenever tested, highlighting the need for careful study and direct comparison of both sexes. Future research should directly examine the role of SMC-MR in aortic aneurysm and vascular calcification using cell-specific knockout models, and additional clinical trials of MR antagonists are warranted, leveraging this recent knowledge. The continued elucidation of SMC-MR’s diverse roles offers promising avenues for innovative and targeted treatments to advance care of diverse cardiovascular pathologies.

Abbreviations

11βHSD2

11-Beta Hydroxysteroid Dehydrogenase-2

AngII

Angiotensin II

AT1R

Angiotensin II Type-1 Receptor

BMP2

Bone Morphogenetic Protein 2

BP

Blood Pressure

CKD

Chronic Kidney Disease

CTGF

Connective Tissue Growth Factor

DOCA

Deoxycorticosterone

EC

Endothelial Cell

ERK

Extracellular Signal-Regulated Kinase

KO

Knockout

MI

Myocardial Infarction

MR

Mineralocorticoid Receptor

PAH

Pulmonary Arterial Hypertension

PASMC

Pulmonary Artery Smooth Muscle Cell

SERCA

Sarcoplasmic/endoplasmic Reticulum Calcium-ATPase

SMC

Smooth Muscle Cell