The Mineralocorticoid Receptor in the Vasculature: Friend or Foe?

Abstract

Originally described as the renal aldosterone receptor that regulates sodium homeostasis, it is now clear that mineralocorticoid receptors (MRs) are widely expressed, including in vascular endothelial and smooth muscle cells. Ample data demonstrate that endothelial and smooth muscle cell MRs contribute to cardiovascular disease in response to risk factors (aging, obesity, hypertension, atherosclerosis) by inducing vasoconstriction, vascular remodeling, inflammation and oxidative stress. Extrapolating from its role in disease, evidence supports beneficial roles of vascular MRs in the context of hypotension by promoting inflammation, wound healing and vasoconstriction to enhance survival from bleeding or sepsis. Advances in understanding how vascular MRs become activated are also reviewed, describing transcriptional, ligand-dependent, and ligand-independent mechanisms. By synthesizing evidence describing how vascular MRs convert cardiovascular risk factors into disease (the vascular MR as a “Foe”), we postulate that the teleological role of the MR is to coordinate responses to hypotension (the MR as a “Friend”).

1. INTRODUCTION

The mineralocorticoid receptor (MR) was cloned in 1987 as the steroid receptor that responds to the adrenal hormone, aldosterone (1). Its name comes from its high homology to the glucocorticoid receptor, but with the capacity to regulate sodium and potassium (“mineral”) levels. The ability to produce aldosterone evolved when lung fish emerged from the salt replete sea to inhabit the land, where sodium is scarce (2; 3). Thus, the initial function attributed to the MR was regulation of renal sodium retention and fluid homeostasis to maintain blood pressure in the face of sodium scarcity (4–6). When low blood pressure is sensed by the kidney, the renin-angiotensin-aldosterone system (RAAS) is engaged by the release of renin from the juxtaglomerular cells of the kidney. Renin initiates a proteolytic cascade, cleaving liver-derived angiotensinogen, that culminates in production of the peptide hormone angiotensin II (AngII). AngII has multiple functions that are mediated by binding to AngII type-1 and -2 receptors (AT1R and AT2R). One critical function of AngII is to induce production and release of aldosterone from the adrenal gland, situated just superior to the kidney (7–9). Aldosterone then circulates from the adrenal throughout the body, ultimately arriving back at the kidney to bind to the MR. Aldosterone binding activates the MR, a hormone-activated transcription factor, to induce transcription of genes in the kidney that promote sodium retention thereby increasing blood pressure (10). The capacity to retain sodium is crucial to terrestrial survival and as such, MR activation in the renal tubules is critical to maintenance of electrolyte homeostasis, blood volume and blood pressure (2).

While originally thought to function exclusively in the kidney, the MR has since been found to be expressed in diverse non-renal tissues including in the heart, vessels, adipose, brain and leukocytes. This review focuses on the MR in the vasculature as ample data over several decades has confirmed that MR is expressed in all cells of the vessel where it contributes to cardiovascular disease. Blood vessels are organized into three layers termed the intima, media, and adventitia. The intimal layer is composed of a monolayer of endothelial cells (ECs) that lines the lumen and contacts the blood circulation. The healthy endothelium is antithrombotic and anti-inflammatory but when injured, ECs are critical for initiating inflammation, thrombosis, and wound healing. ECs also produce vasodilators that regulate vessel diameter by acting on adjacent smooth muscle cells (SMCs). The media layer is composed of vascular SMCs which constrict and relax to regulate lumen diameter. When the vessel is injured, SMC migration, proliferation, and production of extracellular matrix (ECM) promotes wound healing. The adventitia contains fibroblasts embedded in an ECM rich in collagen and elastin fibers, which confers structure and elasticity to larger vessels (11). The MR is expressed in vascular ECs, SMCs, and fibroblasts where it contributes to vascular constriction, inflammation, fibrosis, stiffness and calcification (12; 13). As such, enhanced MR activation is associated with increased risk of cardiovascular diseases (CVD) including hypertension, heart attack, stroke and heart failure (14–16). However, in scenarios of hypotension, such as vascular injury with bleeding or infection with sepsis, these same actions of the MR in ECs and SMCs to promote vasoconstriction, inflammation and fibrosis, are likely beneficial in healing the damaged vessel, clearing the infection, and promoting overall survival. This review summarizes the substantial recent advances in our understanding of how vascular MR activation is induced by cardiovascular disease risk factors to promote vascular disease development and the less well studied actions of vascular MR when activated in response to hypotension in providing survival benefits. By synthesizing substantial recent evidence describing mechanisms by which vascular MRs convert cardiovascular risk factors into disease (the vascular MR as a “Foe”), we postulate that the teleological role of vascular MRs is as part of a coordinated response to mitigate hypotension (the vascular MR as a “Friend”, Figure 1).

Figure 1. The Vascular Mineralocorticoid Receptor: A Friend in Hypotension Turned into a Foe by Cardiovascular Disease Risk Factors.

Injury with bleeding, low salt intake, or sepsis each induce hypotension along with acute oxidative stress, inflammation and appropriate aldosterone production that activate the vascular mineralocorticoid receptor (MR) to induce localized inflammation, wound healing and restoration of blood pressure. Obesity, aging, atherosclerosis or hypertension induce long-term and diffuse vascular oxidative stress and inflammation with unregulated aldosterone production that chronically activates the vascular MR to promote vasoconstriction, inflammatory atherosclerosis, adverse vascular remodeling, and endothelial dysfunction, thereby promoting cardiovascular disease.

2. THE MINERALOCORTICOID RECEPTOR AS A FOE: INAPPROPRIATE VASCULAR MINERALOCORTICOID RECEPTOR ACTIVATION IN THE SETTING OF RISK FACTORS PROMOTES CARDIOVASCULAR DISEASE

2.1. Mineralocorticoid Receptor Activation Associates with Increased Cardiovascular Disease Risk in Humans

Clinical studies associating activation of the MR with cardiovascular disease risk have driven an explosion of research into the role of vascular MRs in preclinical cardiovascular disease models. Specifically, multiple clinical trials have demonstrated that the MR antagonists spironolactone and eplerenone, decrease mortality and hospitalization of patients with heart failure (17–19). MR antagonists are also particularly effective at treating resistant hypertension (20), when other antihypertensives are inadequate. In addition to heart failure and hypertension, aldosterone levels correlate with a higher risk of cardiovascular ischemic events caused by atherosclerosis, including myocardial infarction, stroke and cardiovascular death (14; 15). Such clinical observations of increased cardiovascular risk with enhanced MR activation, and cardiovascular benefits of MR inhibition, generally demonstrate a blood pressure-independent impact of the MR, leading to the search for non-renal mechanisms. For example, patients with primary hyperaldosteronism have a 4-6 fold increased risk of heart attack and stroke even when compared to blood pressure-matched control patients (14). In general, clinical data demonstrate that MR inhibition has cardiovascular disease benefits when the MR is inappropriately activated, as occurs in the setting of traditional risk factors such as hypertension, obesity, metabolic syndrome, diabetes, advanced age and hyperlipidemia. This section summarizes recent advances in our mechanistic understanding of how activation vascular MRs links cardiovascular risk factors to vascular dysfunction and hence contributes to CVD.

2.2. The Mineralocorticoid Receptor Contributes to Vascular Remodeling and Stiffness in Response to Aging, Vessel Injury or High Blood Pressure

Aging is a universal and very potent risk factor for CVD and the population is rapidly aging. Vascular aging is characterized by progressive stiffening of large vessels. Ample clinical data show that the degree of vascular stiffness predicts the risk of coronary heart disease, stroke, and cardiovascular death, independent of blood pressure (21). Multiple studies also demonstrate that expression of the MR in SMCs increases with age in male and female humans and rodents (22–26). Studies in mice with the MR specifically knocked out (KO) from SMCs compared to MR-intact littermates confirm the rise in vessel stiffness with aging, and reveal that KO of the SMC MR prevents aging-induced vascular stiffness in both sexes (23; 24). These results support a causative role for the SMC MR in aging-associated vascular stiffness with further mechanistic studies revealing sexually dimorphic mechanisms. The MR contributes to vascular stiffness in males by inducing vascular fibrosis, but not in females (24; 27). Recent data show that in males, vascular fibrosis and stiffness are mediated by an epigenetic mechanism whereby up-regulation of the MR in human SMCs suppresses expression of the histone methyltransferase EZH2, thereby decreasing histone H3 lysine 27 (H3K27) methylation and promoting recruitment of MRs and H3K27 acetylation at fibrosis gene promoters (26). This leads to global alterations in vascular gene expression, including induction of connective tissue growth factor (CTGF), a known target gene of the MR and a driver of collagen gene expression and vascular fibrosis (23; 26). In females, vascular fibrosis increases with age, but KO of the SMC MR does not attenuate the fibrosis (24), supporting alternative mechanisms driving vascular fibrosis and stiffness in females that remain to be determined.

Interestingly, the MR in SMC also contributes to changes in cardiac function with aging. When mice are aged to 18 months, cardiac dilation with systolic dysfunction develops in both sexes, which is significantly attenuated by genetic deletion of the SMC MR in females (24). When heart failure is induced by pressure overload in males, the SMC MR was also found to contribute substantially to heart failure pathophysiology (28). KO of the MR in SMCs attenuated pressure-induced cardiac hypertrophy, systolic and diastolic dysfunction, and the rise in lung weight and associated exercise intolerance in this heart failure model. Specifically, KO of the SMC MRs is associated with reduced cardiac interstitial and perivascular fibrosis with improved capillary density and coronary flow reserve (28), none of which were impacted by KO of the EC MR (29). Thus, the SMC MR contributes to adverse vascular remodeling in the aging vasculature, contributing substantially to the vascular and cardiac dysfunction that occur with advancing age and pressure overload.

The SMC MR has also been shown to contribute to vascular remodeling and fibrosis in non-aging models. Differentiated SMC do not proliferate but rather express proteins involved in vasoconstriction. However, when injured, SMCs de-differentiate and regain the capacity to migrate and proliferate. This contributes to neointima formation in the setting of percutaneous vascular procedures (30), vein grafting (31), atherosclerosis (32), and pulmonary hypertension (33). In animal models of all of those disorders of increased SMC proliferation, activation of the MR promotes SMC proliferation and MR antagonism prevents neointima formation (mechanisms reviewed in (34)). For example, in the carotid wire injury model, the SMC MR directly contributed to vascular proliferation and fibrosis (30). Importantly, in the absence of vascular injury or aging, deletion of the MR from SMCs did not impact SMC proliferation nor vascular fibrosis suggesting that the SMC MR has less impact in healthy vessels, but rather is poised to respond to vascular damage by contributing to vessel remodeling.

In addition to aging and mechanical injury, hypertension itself induces vascular remodeling and stiffness. In multiple rodent models of hypertension, the MR contributes to vascular stiffness and fibrosis in response to elevated blood pressure (35). In a study in which hypertension is induced by aldosterone and salt, carotid stiffness increases along with expression of integrin alpha-5, a component of the fibronectin receptor that mediates attachment of SMCs to the ECM (36). In that study, genetic deletion of the SMC MR prevents hypertension-induced vascular stiffness as well as the rise in integrin expression. Finally, in addition to SMCs, endothelial cells (ECs) also contribute to vascular stiffness. In the hypertension model induced by deoxycorticosterone with high salt, EC MRs contribute to vascular stiffness by activation of the endothelial sodium channel (EnNaC) (37), a traditional MR target gene in the kidney that is also expressed in vascular ECs, leading to sodium influx and stiffening of the endothelium (Reviewed in (38)).

Overall, substantial data support the concept that the MR in the vasculature is poised to respond to vessel injury, which may be induced by direct mechanical damage or exposure to risk factors like aging or hypertension. Once activated, vascular MRs contribute to vessel remodeling by inducing SMC proliferation and migration, ECM production, adhesion of SMCs to the ECM, and stiffening of the endothelium. By these mechanisms, vascular MRs contribute to vascular fibrosis and stiffness and the associated risk of CVD.

2.3. Inappropriate Mineralocorticoid Receptor Activation in Obesity Drives the Vascular Complications

Like aging, obesity, and its complications (metabolic syndrome and diabetes), are cardiovascular risk factors with rapidly growing prevalence. Aldosterone levels correlate positively with body mass index, and hence the MR is more activated, in obesity, (39). Healthy adipose tissue secretes adipokines that support healthy metabolism and normal vascular function however, with obesity, the profile of adipokines changes to contribute to metabolic dysfunction and vascular disease. The adipokine leptin is increased in obesity and has recently been shown to induce production of aldosterone by cells of the adrenal gland, thereby contributing to hyperaldosteronism and inappropriate activation of the MR (40). The higher aldosterone level in obesity likely also contributes to the development of obesity-associated hypertension (40–42) and vascular EC dysfunction, an early step in the progression to CVD.

The MR contributes to obesity-induced vascular dysfunction in multiple cell types in the vessel wall and by varied mechanisms. A potential role for the MR in adipose and SMC dysfunction is implicated in the db/db mouse model of obesity. As obesity develops in the db/db model, the anti-contractile effects of perivascular adipose tissue are lost, leading to vasoconstriction (43; 44). The mechanism involves obesity-induced mitochondrial dysfunction with increased reactive oxygen species (ROS) production by adipocytes. These ROS act in a paracrine manner to activate SMC Rho signaling, inducing myosin phosphorylation and vascular constriction. MR antagonism in obese mice prevents all of these signaling events, implicating the MR in obesity-induced vascular dysfunction, although the specific cell type is not clear in these studies (43; 44).

Obesity classically impairs endothelial function to which the EC MR contributes by multiple mechanisms. Endothelial function is often measured by quantifying EC-dependent vasodilation, which is mediated by EC-derived vasodilators including nitric oxide (NO), prostacyclin, and potassium ions. Activation of the MR in cultured ECs induces oxidative stress by upregulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases leading to production of ROS. ROS inactivate NO, making it less bioavailable for vasodilation. The EC MR also decreases phosphorylation of endothelial NO synthase (eNOS), resulting in reduced NO production which, together with increased ROS, impairs overall NO bioavailability (45). In mice, obesity or aldosterone infusion impairs aortic endothelial function, increases aortic EC NADPH oxidase gene expression and decreases anti-oxidant genes, all of which are prevented by MR antagonism or by deletion of the EC MR (45). Belin de Chantemèle’s group further demonstrated that leptin induces endothelial dysfunction in obese female mice and that this is prevented by deletion of the EC MR, supporting that leptin-induced aldosterone production may play a role in obesity-induced vascular dysfunction via direct effects on the EC MR (46).

Studies in resistance mesenteric vessels from obese and lean mice with KO of the EC MR compared to MR-intact littermates further confirms a role for the EC MR in obesity-induced endothelial dysfunction by sexually dimorphic mechanisms (47). Males are less sensitive to obesity-induced endothelial dysfunction and the impact of obesity in males is predominantly a decrease in NO production that is independent of the EC MR. However, mesenteric vessels from females are more sensitive to obesity-induce endothelial dysfunction and KO of the EC MR is protective via increasing NO availability (47). Recent studies using double KO mice with the estrogen receptor alpha (ERα) deleted from ECs (in addition to the MR) reveals potential mechanisms for the sex difference. In obese females, the EC MR suppresses expression of the ERα and also decreases ERα recruitment to the membrane by the scaffolding protein striatin thereby preventing estrogen induction of eNOS activity and lowering the NO synthase contribution to vasodilation (48). Epidemiologic data shows that premenopausal women are protected from CVD relative to men however this protection is lost in young obese women. The inappropriate activation of the EC MR in the setting of obesity may contribute to the loss of cardiovascular protection of obese women by decreasing both the expression of the EC ERα and by preventing ERα from inducing NO production in ECs.

In addition to resistance microvessel endothelial dysfunction, obesity induces large vessel stiffness. The Sowers’ group showed that obesity-associated vascular stiffness is also prevented by KO of the EC MR (49). In an elegant series of studies, the group showed that the mechanism involves enhanced EnNaC channel activity leading to cortical actin stiffening (Reviewed in (38)). EnNaC activation further decreases NO production which also leads to SMC proliferation, infiltration of inflammatory cells into the vessel, and fibrosis, further contributing to vascular stiffness and disease (50). Taken together, ample data support that obesity induces inappropriate activation of the MR in the vasculature, particularly in ECs, where the MR contributes to vascular dysfunction by reducing NO, inducing oxidative stress, enhancing inflammation, and activating EnNaC to promote vascular stiffness (51–53).

2.4. The Mineralocorticoid Receptor Contributes to Atherosclerosis

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of cholesterol in the arterial wall that leads to plaque formation. The plaques are composed of inflammatory cells that are recruited by ECs that have been damaged by oxidized lipids and other risk factors (54). As the process of atherogenesis progresses, leukocytes in the vessel engulf lipids, and these lipid laden macrophages form the plaque core, surrounded by migrating and proliferating SMCs that secrete collagen to form a fibrous cap over the thrombogenic plaque. Damaged ECs induce the expression of adhesion molecules and along with macrophage-derived cytokines, recruit additional leukocytes thereby increasing plaque inflammation. Inflammatory SMCs and leukocytes in the plaque secrete matrix metalloproteinases (MMPs) that degrade the fibrous cap, predisposing the plaque to rupture (54). Plaque rupture is the predominant cause of acute heart attack and ischemic stroke, and these leading causes of death are both associated with increased activation of the MR in clinical studies (14; 15).

These clinical findings have prompted multiple studies in animal atherosclerosis models that directly implicate the MR in atherogenesis (Reviewed in (55)). In the apolipoprotein E (ApoE)-KO mouse model of atherosclerosis, activation of the MR by low salt diet, AngII or aldosterone infusion all accelerate atherosclerosis with increased plaque size, inflammation and lipid content in the aorta (32; 56; 57). Conversely, treatment of ApoE-KO mice with an antagonists of the MR attenuates early atherosclerotic plaque development (57; 58).

Tissue specific KO mice have been used to explore the molecular mechanism. Although the MR in SMCs was shown to release factors that promote leukocyte chemotaxis in vitro (32) and SMC proliferation in vivo (55), SMC-specific deletion of the MR did not impact atherosclerosis phenotypes in male ApoE-KO mice (59). Rather, the MR in ECs was found to contribute directly to vascular inflammation in atherosclerosis. Activation of the MR induces the expression of EC adhesion molecules including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecular-1 (VCAM-1) and E selectin in human coronary ECs and in animal models of atherosclerosis (55; 60; 61). Further studies show that ICAM-1 is necessary for aldosterone-induced atherosclerosis (62). These adhesion molecules facilitate leukocyte rolling, adhesion and infiltration into the vasculature and the plaque. EC-specific KO of the MR reduces leukocyte-endothelial interactions by intravital microscopy, plaque inflammation by flow cytometry, and expression of E-selectin and ICAM-1 in the PCSK9-driven atherosclerosis model in male mice (61). Interestingly, MR-intact female mice have less inflamed plaques than males. In vitro studies reveal that the ER, when bound by estrogen, can block the transcriptional activity of the MR on the ICAM1 promoter (63). As such, deletion of the EC MR in young female mice does not impact plaque inflammation, potentially because the ER is already blocking the pro-inflammatory actions of the EC MR in ECs from females.

The MR is also expressed in inflammatory cells including macrophages and monocytes (Reviewed in (64)), further contributing to atherogenesis. Deletion of the MR in myeloid cells attenuates AngII-induce atherosclerosis in ApoE-KO mice (65). In that model, KO of the myeloid MR blocks macrophage foam cell formation, upregulates cholesterol efflux genes, and increases the phagocytic capacity of macrophages (65). The myeloid MR also contributes to atherosclerotic plaque size and inflammation in the ApoE-KO (without AngII) by contributing to leukocyte trafficking via transcriptional regulation of the selectin ligand, P-selectin glycoprotein ligand-1 (PSGL1) (66). Thus, MRs in ECs and myeloid cells act in a coordinated fashion to recruit leukocytes to the damaged endothelium. This contributes to vascular inflammation and atherosclerotic plaque progression (12; 53; 55; 67; 68), potentially explaining the increased risk of atherosclerosis complications in settings of chronic MR activation such as obesity or hyperaldosteronism.

2.5. The Vascular Mineralocorticoid Receptor Contributes to Hypertension

In addition to mediating the adverse impact of hypertension on vascular remodeling as described in section 2.2, the vascular MR also directly contributes to blood pressure regulation. Of course, the MR functions in the kidney to regulate blood volume and blood pressure hence, excessive sodium intake with dysregulated RAAS activation are implicated in hypertension (69). As such, MR antagonists are effective antihypertensive agents, particularly in patients with resistant hypertension (19; 70). However, clinical studies in those taking MR antagonists also indicate that the degree of potassium rise, a marker of renal MR inhibition, does not directly correlate with the blood pressure-lowering effects, supporting a role for the MR outside the kidney in blood pressure control (71).

ECs and SMCs contribute to blood pressure by impacting resistance vessel tone and constriction with a role for the vascular MR recently demonstrated using tissue-specific KO and transgenic mice. Two different mouse lines with the EC MR deleted show no impact on basal blood pressure (72; 73). However, when the human MR is overexpressed specifically in ECs, blood pressure increases independent of renal sodium homeostasis and vessel remodeling (74). Mice with increased expression of the MR in ECs have an enhanced blood pressure response to AngII and endothelin infusion. Similarly, vasoconstriction of ex vivo vessels from these mice is exacerbated in response to these and other contractile agonists. While little is known about regulation of the EC MR at the level of gene expression, one study showed that expression of EC MRs is increased specifically in obese compared to lean female mice, potentially driven by progesterone (46). Thus, while the EC MR does not appear to contribute to basal blood pressure, under conditions in which the MR is upregulated in ECs (like females with obesity), the EC MR may drive hypertension.

Multiple studies demonstrate a direct contribution of the MR in SMCs to blood pressure regulation. Genetic deletion of the SMC MR in mice results in decreased vascular myogenic tone and lower blood pressure, particularly with aging (75; 76). Myogenic tone is regulated by the action of L-type calcium channels in resistance vessel SMCs. The SMC MR has been shown to regulate L-type calcium channel expression and to enhance channel function thereby modulating resistance vessel tone (75) and renal vasoconstriction (77). Mechanistically, the SMC MR regulates expression of the L-type calcium channel subunit Cav1.2 and the AT1R by downregulating expression of their target microRNA, miR-155, and as such contributes to myogenic tone and hypertension induced by AngII infusion (78–80). Together, these data reveal that while the vascular MR does not substantially contribute to basal blood pressure homeostasis, in the setting of risk factors such as aging and obesity, upregulation and activation of the MR in ECs and SMCs contributes to vasoconstriction and elevates blood pressure.

2.6. What Have We Learned About the Vascular Mineralocorticoid Receptor as a Foe?

Decades of active investigation have substantially enhanced our understanding of how chronic, diffuse, and inappropriate vascular MR activation induces cardiovascular disease in the large proportion of the population exposed to common risk factors including obesity, hyperlipidemia, hypertension or advanced age. MR in SMC contributes to cell proliferation and ECM deposition to promote neointima formation and vascular stiffness, specifically in response to vessel injury either mechanically, during aging, or with pulmonary or systemic hypertension. MR in endothelial cells and leukocytes coordinately regulate leukocyte recruitment and trafficking into the vessel wall in response to hyperlipidemia or inflammatory stimuli and EC MR contributes to endothelial dysfunction in obesity. Finally, vascular MR contributes directly to blood pressure control by regulating vasoconstriction, with MR in SMC promoting vasoconstriction by regulating L-type calcium channels and AT1R expression and EC MR inducing oxidative stress and impairing nitric oxide production when overexpressed or overactivate, as in obesity.

3. THE MINERALOCORTICOID RECEPTOR AS A FRIEND: APPROPRIATE VASCULAR MINERALOCORTICOID RECEPTOR ACTIVATION

3.1. Aldosterone as an Endocrine Coordinator of the Response to Hypotension

With the greater understanding of the detrimental roles of the vascular MR in CVD development, the larger question arises as to what is the teleological role for the existence of the MR in the vasculature? The primary signal triggering release of the MR ligand aldosterone from the adrenal gland is low blood pressure. Hypotension is a symptom of multiple potentially life-threatening conditions, including dehydration, bleeding, and sepsis. Low blood pressure is sensed by the decrease in kidney perfusion which triggers renal renin to activate the RAAS. The RAAS culminates with the release of adrenal aldosterone which circulates to all tissues of the body. For 75 years, the principal impact of aldosterone was known to be induction of sodium reabsorption and fluid homeostasis in the kidney to restore blood pressure. However, if the only role of aldosterone in mitigating hypotension is to function in the renal collecting system, a paracrine mechanism linking decreased kidney perfusion to renal sodium reabsorption would be more rapid and efficient. Indeed, the pressure-natriuresis mechanism intrinsic to the kidney serves this purpose. Aldosterone is released from the adrenal gland, which one might argue is conveniently situated just superior to the kidney where aldosterone is known to act. However, rather than act locally, endocrine hormones circulate to coordinate the function of multiple organs to meet physiological needs (i.e. cortisol to respond to stress, female sex hormones to coordinate pregnancy). Hypotension is one of those situations in which coordinated response of multiple organs has benefits. Thus, we put forth the concept that aldosterone is released to coordinate the activity of the MR in multiple tissues to respond to situations of hypotension. This next section summarizes recent advances in our understanding of how the vascular MR may have beneficial roles when appropriately activated to mitigate hypotension. Much less investigation has been performed to understand these physiological roles of the vascular MR. Thus, much of the information in this next section is extrapolated from the extensive studies summarized in section 2 exploring the pathological functions of the vascular MR and applying those concepts to how they might be beneficial in hypotensive situations.

3.2. Role of the Vascular Mineralocorticoid Receptor in the Response to Low Blood Pressure from Salt Scarcity or Dehydration

The capacity to make mineralocorticoids (hormones distinct from glucocorticoids that specifically regulate sodium avidity) evolved when lung fish emerged from the salt rich sea onto the land (2; 81). Salt scarcity is a critical threat to survival of terrestrial animals and hence requires potent mechanisms to maintain sodium balance (82). This includes brain regions where the MR is expressed to promote salt appetite (83) as well as the renal RAAS cascade involving activation of MR in the distal nephron to upregulate expression of the ENaC to promote sodium reabsorption thereby increasing blood volume and pressure (82; 84; 85). The critical role of the MR in retaining sodium to maintain blood volume was confirmed by the finding that mice with total body KO of the MR die of dehydration in the neonatal period unless rescued with sodium supplementation (86; 87). However, mice with renal cell-specific KO of the MR survive the neonatal period and become hypotensive only when exposed to a low salt diet (85). These findings indicate that while the renal MR plays a critical role in basal blood pressure control, there are additional non-renal mechanisms of MR action that contribute to blood pressure control under stress, including salt scarcity. Here we posit that MR in vascular cells acts in concert with the kidney to contribute to maintenance of blood pressure in the setting of hypotensive stress.

Extrapolating from section 2.2 summarizing the role of the vascular MR in hypertension, it is known that when the MR is deleted specifically from ECs (73) or from SMCs (75), there is no immediate impact on basal blood pressure. However, when the MR is overexpressed specifically in ECs in mice, blood pressure increases significantly (74). Similarly, when expression of the MR in SMCs rises with aging, SMC MR contributes directly to rising blood pressure in those aging mice (75). These studies support a role for the vascular MR in raising blood pressure under situations in which the EC or SMC MR is upregulated. In ECs, the MR regulates the balance between oxidative stress and NO and enhances vasoconstriction responses to stimuli including phenylephrine (adrenergic), endothelin and AngII (73; 74). In SMCs, the MR regulates expression of the L-type calcium channel and the AT1R to modulate vascular tone (75; 79). Whether the vascular MR is upregulated under low salt conditions has not been carefully studied.

Overall, the available data support the concept that the vascular MR does not contribute substantially to basal blood pressure regulation but is poised in ECs and SMCs to respond in settings of hypotension. A decline in blood volume due to dehydration or bleeding would activate the RAAS resulting in a rise in AngII, which acts through the SMC MR to induce vasoconstriction. RAAS activation also induces aldosterone release, which acts via the EC MR to decreases NO production to prevent vasodilation. Together, these functions of vascular MRs would coordinate with the renal MR-mediated sodium retention to restore blood pressure more rapidly.

3.3. Role of the Vascular Mineralocorticoid Receptor in Wound Healing in Hypotension from Bleeding

Injury with blood loss is a potential life threatening cause of hypotension. In addition to retention of sodium by the renal MR and induction of vasoconstriction by the vascular MR, the ability to rapidly heal the wound and restore hemostasis would improve survival from hemorrhage. While not the focus of this review, MR is also expressed in cells of the skin (Reviewed in (88; 89)) where the MR may also contribute to wound healing (90–93). A role for the MR in thrombosis, a critical early step in maintenance of hemostasis, remains controversial. Some data supports that the MR activation may be pro-thrombotic, either via MRs in ECs or platelets, which would fit with the model of MR as a coordinator of cell types needed to respond to acute hemorrhage (94–99).

When a wound occurs, the associated injury to the vessels is the primary cause of bleeding. In response to mechanical injury to the vasculature, the normally differentiated, contractile and quiescent SMCs dedifferentiate into cells that can migrate, proliferate, and produce extracellular matrix (ECM) to contribute to restoration of vessel integrity (reviewed in (100)). Simultaneously, the re-endothelization process is induced, involving EC migration and proliferation to reestablish the barrier function of the vessel (101). When the EC layer is completely healed, further SMC proliferation is supressed. Multiple studies show that MR antagonists prevent excessive vascular remodeling in response to injury (reviewed in (102)). Conversely, aldosterone enhances the pro-fibrotic and the SMC proliferative response in a mouse model of wire-induced carotid injury, without changes in blood pressure (103). This effect of aldosterone to enhance vessel remodeling after injury is dependent on the MR in SMC. As such, deletion of the SMC MR prevented aldosterone from enhancing SMC proliferation and fibrosis without impacting the rate of re-endothelialization of the injured vessel (30). While the role of the SMC MR has not been directly tested in a model of hemorrhage, we extrapolate this notion from experiments in which carotid vessel injury was induced in combination with low-dose aldosterone infusion in MR-intact compared to SMC-MR-KO mice, thereby providing insight into the role of the SMC MR in vessel healing when aldosterone might be induced by the hypotensive response to blood loss (30). Mechanistically, SMC-MR regulates expression of the vascular endothelial growth factor (VEGF) family member, placental growth factor (PlGF), in an oxidative stress-dependent manner (98). PlGF is necessary for aldosterone-induced vascular remodeling (103) and the SMC MR further contributes to upregulation of the VEGF type-1 receptor on injured SMC which mediates the impact of PlGF (30). Of note, KO of the SMC MR had no impact on the structure of the contralateral uninjured vessel, further supporting the idea that the MR in SMC is poised to respond to a vascular injury (mechanisms reviewed in (13; 34; 102)). Thus, the SMC MR promotes vasoconstriction and enhances wound healing when vessels are mechanically injured which, along with MR activation in the kidney, skin, and potentially platelets, could coordinately mitigate the potential lethal impact of hypotension due to hemorrhage.

3.4. Role of the Vascular Mineralocorticoid Receptor in Response to Hypotension due to Infection and Sepsis

Sepsis is characterized by an excessive host immune system response to infection resulting in a systemic inflammatory response, vasodilatation, and hypotension which often progresses to multi-organ failure and death (104; 105). Several clinical and basic research studies support a beneficial role for MR activation in sepsis. Epidemiological data comparing patients with primary hyperaldosteronism to matched individuals with essential hypertension, revealed an 80% relative risk reduction of sepsis and sepsis-related mortality in those with hyperaldosteronism (106). While clinical trials testing glucocorticoids alone in septic shock have had mixed results (reviewed in (107)), a clinical trial randomizing septic patients to glucocorticoid plus mineralocorticoid treatment significantly decreased the need for pressors, the degree of organ failure and mortality (108). Similarly in a canine model of staphylococcal sepsis, mineralocorticoid (but not glucocorticoid) treatment improved blood pressure, cardiac function, and survival (109). In a mouse model of sepsis, aldosterone treatment also improved blood pressure and survival (110). Mechanistic studies in rodent sepsis models show that mineralocorticoids increase vascular reactivity to catecholamines with increased vascular alpha1-adrenergic receptor expression (110–112), indirectly implicating the vasculature. In addition, aldosterone reduces the levels of histamine, serotonin and bradykinin, circulating factors that contribute to hypotension in septic physiology, as well as interleukin-6 (IL-6), tumor necrosis factor (TNF) and other inflammatory mediators (107).

These findings support the concept that appropriate activation of the MR in the setting of sepsis improves blood pressure and reduces sepsis mortality. Although the specific role of vascular MRs has not been tested in sepsis models, ample data reveal that the EC MR regulates transcription of adhesion molecules in ECs (60–62). This may contribute to the inflammatory response as mice with the MR deleted from ECs have significantly decreased TNF-induced leukocyte rolling and adhesion by intrivital microscopy and reduced TNF-induced expression of ICAM1 and E-selectin in the vasculature. MR is also expressed in myeloid cells (monocytes, macrophages, dendritic cells and neutrophils) and T cells. Activation of the MR in leukocytes contributes to a pro-inflammatory myeloid cell phenotype, induces the NLRP3 inflammasome, and increases inflammatory cytokine production (reviewed in (64; 107)). Our lab recently showed that the myeloid MR also plays a role in leukocyte trafficking into the peritoneum in a peritonitis model and through the vasculature in response to TNFα. The mechanism was shown to involve transcriptional regulation by the myeloid MR of PSGL1 resulting in increased expression on the surface of monocytes (66). In the context of atherosclerosis, increased leukocyte trafficking contributes adversely to the pathophysiology. However, in the context of infection, activation of the MR in ECs and in leukocytes would be expected to be beneficial. Recruiting inflammatory cells to the site of infection would more rapidly clear external pathogens and in this way, may contribute to the benefits of mineralocorticoids in sepsis. Once again, this fits with the concept that MR coordinates the response to hypotension by simultaneously promoting pro-inflammatory effects of the MR in ECs and leukocytes, vasoconstrictive actions of the MR in ECs and SMCs, and volume retention by the renal MR. Together, these effects of the MR would clear the infection while raising blood pressure, thereby promoting survival in sepsis as is seen in hyperaldosteronism patients (106).

4. MECHANISMS OF MINERALOCORTICOID RECEPTOR ACTIVATION

As we previously described, the MR is activated in multiple physiologic and pathologic settings, being beneficial in some situations (injury, bleeding, dehydration, sepsis) and harmful in others (aging, obesity, hypertension, atherosclerosis). Activation of vascular MRs leads to increase inflammation, oxidative stress, vasoconstriction and vascular healing/remodeling. Substantial advances have also recently been made in our understanding of the multiple mechanisms by which MR activity may be induced. Understanding the mechanisms of appropriate or inappropriate MR activation may allow us to better manipulate the RAAS to enhance the benefits while mitigating the harm (Figure 2).

Figure 2. Mechanisms of inappropriate activation of the mineralocorticoid receptor in response to cardiovascular risk factors.

Cardiovascular risk factors lead to chronic mineralocorticoid receptor (MR) activity by inducing inappropriate activation of the renin-angiotensin-aldosterone system (RAAS) and diffuse vascular oxidative stress and inflammation. RAAS activation increases aldosterone (A) and angiotensin II (ANG). oxidative stress activates Rac1 signaling and hypoxia inducible factor-1α (HIF1α), and inflammation induces the nuclear factor kappa-B (NFκB) transcription factor. Aldosterone mediates ligand-dependent MR activation, ANG and Rac1 signaling pathways induce post translational modifications (PM) of the MR to mediate ligand-independent MR activation. ANG does this by activating protein kinase C delta (PKCδ in smooth muscle cells. Finally, HIF1α and NFκB induce transcription of the MR gene.

4.1. Hormone-dependent Mineralocorticoid Receptor Activation

MR has two hormone ligands, aldosterone and cortisol, both of which bind with similar affinity to MR and thus compete for binding to the C-terminal ligand binding domain (1). Cortisol circulates at much higher concentrations than aldosterone, but specificity for aldosterone is maintained by several mechanisms (reviewed in (113)). First, much of the cortisol circulates bound to proteins in plasma while aldosterone circulates in a free form. Also, once bound to the receptor, aldosterone dissociates more slowly from the MR and this more stable binding perpetuates the transcriptional response of the MR when bound to aldosterone. Finally, aldosterone-responsive tissues express the enzyme 11-beta hydroxysteroid dehydrogenase-2 (11βHSD2) that locally inactivates cortisol by converting it into cortisone, thereby conferring tissue-specific aldosterone responsiveness (13; 68; 114; 115). Multiple authors have demonstrated that vascular cells, including SMCs and ECs, express 11βHSD2, supporting the concept of the vasculature as an aldosterone-responsive tissue (78; 116–119). In leukocytes and cardiomyocytes, 11βHSD2 expression has not been confirmed, hence glucocorticoids likely act as ligands for the MR in those cell types.

In situations of appropriate MR activation due to hypotension, aldosterone is released from the adrenal gland and acts as a classical activator of the MR, restoring blood pressure in dehydration, bleeding or infection. As such, when adrenal insufficiency occurs, outcomes are worse in these hypotensive conditions. Some conditions, most notably heart or liver failure, produce hypotension in the absence of low sodium or decreased blood volume. In such cases, the resulting rise in aldosterone contributes to volume overload and associates with worse outcomes. As such, MR antagonists have proven clinical benefit in heart or liver failure (17; 18; 120). In a growing segment of the population with obesity, hypertension or advanced age, the MR may also be inappropriately activated due to dysregulated aldosterone production in the absence of hypotension. As we described in the section on obesity, adipose tissue from obese individuals releases adipokines, including leptin, that raise serum aldosterone levels by inducing adrenal aldosterone release (40; 121). In people with hypertension, recent data reveals that inappropriately elevated aldosterone is much more common than previously described, occurring in 15%, 21%, and 22% of patients with stage 1, stage 2, and resistant hypertension respectively (122). In aging individuals, aldosterone production by the normal adrenal gland decreases while, autonomously regulated aldosterone-producing cell clusters increase in the adrenals (123). Thus, the vascular MR can be activated by the traditional ligand aldosterone appropriately in the setting of hypotension with beneficial effects, and also inappropriately, as in obesity, hypertension and aging, where inappropriate and dysregulated activation of the vascular MR contributes to development of vascular disease (Figure 2).

4.2. Ligand-independent Mineralocorticoid Receptor Activation by Angiotensin II

Other steroid hormone receptors, including the estrogen, progesterone and androgen receptors, can be activated in a hormone-independent manner by growth factor signaling that induces receptor phosphorylation (reviewed in (124)). When the RAAS is activated, the peptide hormone AngII is produced. In addition to acting on the adrenal to induce aldosterone release, AngII acts directly on the blood vessel causing rapid and potent vasoconstriction that contributes to a rapid increase in blood pressure. AngII binds to two G protein-coupled receptors, AT1R or AT2R. Activation of the AT1R mediates many of the traditional AngII responses including vasoconstriction while AT2R binding on ECs has the opposite effect, inducing modest vasodilation (125). The AT1R is expressed in many cells, including SMCs, ECs, and cardiomyocytes. AngII activation of AT1R activates signaling cascades that stimulate ROS production and induces SMC hypertrophy, proliferation, and migration (125; 126). Ample data support substantial crosstalk between AngII and aldosterone signaling in the vasculature (127; 128). Independent of aldosterone production, studies show that AngII directly activates the MR in primary human vascular SMCs (78). More recently, the mechanism by which AngII activates the SMC MR was shown to require protein kinase C-delta (PKCδ). AngII binding to SMC AT1R induces formation of a MR-PKCδ complex, phosphorylation and nuclear translocation of the MR, transcription of SMC MR target genes, and SMC proliferation (129). In addition, multiple studies show that the SMC MR upregulates vascular expression of the AT1R, thereby enhancing the vasoconstrictive response to AngII. Specifically in aging resistance vessels, expression of the SMC MR increases with age and suppresses miR-155 transcription leading to an increase in the expression of the miR-155 target, the AT1R (80). Thus, in addition to ligand-dependent activation by aldosterone, the MR in vascular SMC can be directly activated by AngII via AT1R to contribute to vascular SMC proliferation, oxidative stress, and fibrosis and the MR activity further enhances the vascular actions of AngII by upregulating AT1R (12; 78; 129). In the setting of hypotension, AngII and aldosterone would thus act synergistically to coordinate the response and restore blood pressure. In the setting of CVD, MR antagonists often have benefits that are independent of aldosterone levels, including in those with hypertension, atherosclerosis, and heart failure. In those cases, ligand-independent MR activation may be contributing to adverse cardiovascular outcomes.

4.3. Ligand-Independent Mineralocorticoid Receptor Activation via Rac1 Signaling

In addition to AngII, ample recent data has implicated Rac1 signaling as an additional mechanism of ligand-independent activation of the MR with substantial contributions to disease development (reviewed in (130)). Rac1 is a Rho-family small GTPase that has been implicated in renal and cardiovascular disease when activated by oxidative stress. Nagese et al identified Rac1 as the mediator that links enhanced oxidative stress to activation of the MR. In cultured cardiomyocytes, induction of oxidative stress increases active Rac1 and enhances transcriptional reporter activity of the MR. The link from oxidative stress to enhanced MR activity is Rac1-dependent yet independent of the MR ligand-binding domain, confirming Rac1 as a ligand-independent MR activator (131). This mechanism has been found to contribute to cardio-renal disease in multiple models using cardiomyocyte-specific Rac1 KO mice and Rac1 and MR antagonists. In a mouse model of pressure overload-induced heart failure, pressure overload activates Rac1 to induce nuclear localization of the MR and expression of MR target genes resulting in cardiac hypertrophy and failure (132). In a rat model of high salt diet-induced hypertension, Rac1 is activated in the kidney, leading to activation of the renal MR in the setting of low aldosterone and contributing to hypertension and kidney injury (133). Similarly, in a rat model of obesity with type 2 diabetes, high glucose induces Rac1 activity to activate MR in kidney mesangial cells and contributes to diabetes-induced kidney damage (134). More recently, Rac1 activation of MR signaling in SMCs has been demonstrated to contribute to acute kidney injury (135). This raises the possibility that Rac1-mediated ligand-independent MR activation in the vasculature may contribute to the adverse roles of vascular MR in the setting of hypertension, aging or obesity, conditions in which Rac1 is activated.

4.4. Regulation of the Mineralocorticoid Receptor at the Level of Transcription by Oxidative Stress and Inflammation

The MR gene structure consists of 10 exons, with two alternatively spliced first exons (1α and 1β) that are not translated, and exons 2-9 that encode the single MR protein sequence (Reviewed in (136)). In 1995, Zennaro and colleagues characterized the human MR gene, demonstrating that the 1α and 1β isoforms are regulated by two alternative promoters, termed P1 and P2, corresponding to the 5’-flanking regions of the untranslated exons 1α and 1β (137). Isoform-specific in situ hybridization (138) and promoter-reporter mouse studies (139) revealed that the two promoters can drive tissue-specific expression and as such, the 1α and 1β MR isoforms can be differentially regulated while ultimately translating into the same MR protein.

Ample data has demonstrated a role for the MR in CVD that is often driven by disease-induced upregulation (or experimental overexpression) of the MR in specific cells (ie the EC MR increases in obesity, the SMC MR increases in aging). However, specific transcriptional regulatory mechanisms driving expression of the MR have only recently begun to be elucidated. We recently demonstrated that both of the MR isoforms are expressed in human SMCs and increase with age (25). Two transcription factors were identified, hypoxia-inducible factor-1 alpha (HIF1α and nuclear factor kappa-B (NFκB), which regulate the MR promoter. HIF1α is induced by hypoxia or oxidative stress and NFκB is activated by inflammatory stimuli, both of which increase in aging mouse and human vessels. Both transcription factors induce the expression of the MR protein in aged human SMCs and correlate with MR expression in human aortic tissue. HIF1α specifically regulates the MR-1α isoform and NFKB regulates both the MR-1α and MR-1β isoforms (25). These recent findings provide a potential mechanistic underpinning for increases in the vascular MR expression in conditions of enhanced oxidative stress and inflammation including aging, obesity, atherosclerosis, or hypertension, in which the vascular MR drives disease. Further studies are needed to confirm if these transcriptional mechanisms contribute to the role of the vascular MR in the setting of obesity, atherosclerosis, hypertension, sepsis, or bleeding. The combination of HIF1α-driven MR overexpression with Rac1-induced MR activation may help explain the substantial role of the vascular MR in driving CVD, independent of the hormone aldosterone, when oxidative stress is high. This same mechanism could mediate physiological benefits of the MR by inducing receptor expression in the setting of active inflammation and oxidative stress as occurs in infection and sepsis.

4.5. Post-translational Modifications of the Mineralocorticoid Receptor

In addition to activation by ligand, the MR protein can be modified by post-translational modifications including phosphorylation, ubiquitination, acetylation, sumoylation or oxidation (Reviewed in (140; 141)). These modifications can impact the function of the MR by altering the MR protein stability, modulating activation by ligand binding, changing the MR cellular localization or directly activating the MR to regulate target genes. Mass spectrometry studies reveal that the MR is a highly phosphorylated protein with multiple phosphorylation sites in the large and unique N-terminal domain, most of which have unknown function. An inhibitory phosphosite was identified in the C-terminal ligand-binding domain that is phosphorylated by ULK1 and contributes to function of the MR in the kidney (142). As described above, AngII promotes phosphorylation of the SMC MR via PKCδ resulting in induction of MR-target gene transcription and SMC proliferation (129). MR acetylation has been shown to reduce the MR transcriptional activity (143). Finally, as with other steroid hormone receptors, chronic hormone binding induces ubiquitination of the MR resulting in degradation and decreased protein levels, acting as a feedback break in the setting of chronic stimulation. It is possible that this negative feedback mechanism may be overcome in settings of disease in which the MR expression is driven at the level of transcription by oxidative stress or inflammation. Overall, diverse protein modifications can alter the MR function however, much remains to be learned, particularly as it relates to the physiologic and pathologic roles of the MR in the vasculature (reviewed in (140; 141)).

5. Conclusion: The Vascular Mineralocorticoid Receptor: A Friend Turned into a Foe by Cardiovascular Disease Risk Factors

This review summarized the different scenarios in which activation of the MR act as a “friend”, helping to combat hypotension by retaining salt, inducing vasoconstriction, promoting wound healing and driving vascular inflammation to improve outcomes from dehydration, bleeding or sepsis. Even more data reveals multiple cardiovascular diseases in which induced expression or function of the MR acts as a “foe” to promote vascular dysfunction and cardiovascular disease development (Figure 1). The MR can be activated by three pathways, RAAS activation (either via aldosterone or directly via AngII in SMCs), oxidative stress (via Rac1 signaling or HIF1α induction of MR gene expression) and inflammation (via transcriptional upregulation by NFkB (Figure 2)). These three regulatory pathways are commonly activated in hypotension and infection. These same pathways are active in the setting of cardiovascular risk factors, in which aldosterone is increased, as in obesity and hypertension, oxidative stress is induced, as in aging and obesity, and chronic inflammation occurs, as in obesity, aging and atherosclerosis. In conditions of hypotension, MR is activated appropriately by these three signaling pathways, acutely, for a short time, and in some cases (ie vascular injury or focal infection), in a localized area. This short-term, localized, appropriate activation of the vascular MR is beneficial to restore blood pressure, heal vascular damage, and clear infection. Obesity, vascular aging, and hypertension are all associated with chronically enhanced oxidative stress, inflammation, and RAAS system activity and hence induce the MR via HIF1α, NFκB, aldosterone, AngII, and Rac1. This chronic, diffuse, and unregulated vascular MR activation, acts synergistically with the risk factors themselves, to accelerate cardiovascular disease. This paradigm expands the potential conditions for which MR antagonists may be considered beneficial from current use in hypertension, heart failure, and renal protection, to consideration of testing MR inhibitors for atheroprotection, after stenting or vein grafting, or to slow progression of cardiovascular disease in obesity and aging. Greater understanding of the appropriate and inappropriate mechanisms driving expression and activation of the MR will be essential to reap the benefits of MR activation to protect from hypotension while exploiting MR antagonism as a more broad cardiovascular disease prevention strategy.

Terms/Definitions List

MR

Mineralocorticoid receptor

RAAS

Renin angiotensin aldosterone system

AngII

Angiotensin II

AT1R

Angiotensin 1 receptor

AT2R

Angiotensin 2 receptor

EC

Endothelial cell

SMC

Smooth muscle cell

CVD

Cardiovascular disease

KO

Knock out

CTGF

Connective tissue growth factor

ENaC

Epithelial sodium channels

ROS

Reactive oxygen species

NO

Nitric Oxide

MMP

Matrix metalloproteinase

ApoE

Apolipoprotein E

PSGL1

P-selectin glycoprotein ligand-1

SGK1

Serine/threonine-protein kinase 1

VEGF

Vascular endothelial growth factor

HIF1α

Hypoxia Inducible Factor-alpha

NFκB

Nuclear Factor Kappa B