**4.2. Role of MR in vascular remodeling**

Vessel injury induces a pathological response termed vascular remodeling which contributes to human ischemic vascular disease. Adverse vascular remodeling limits vessel lumen diameter and increases vascular stiffness associated with fibrosis, thereby contributing to organ ischemia and hypertension. MR activation contributes to vascular remodeling by acting synergistically with endothelial damage, angiotensin II (Ang II), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) [52–55]. These processes involve both genomic (upregulation of genes involved in cell migration, proliferation, and matrix modulation) and non-genomic mechanisms (via MAPK and the c-Src/Rho) [56]. In the pulmonary artery, MR activation induced the proliferation of VSMC, an effect prevented by spironolactone [57]. Moreover, in a VSMC-MR knockout mouse model, carotid injury-induced and aldosterone-enhanced vascular fibroses were attenuated; thus, VSMC-MR is necessary for aldosterone-induced vascular remodeling [58]. In aged VSMC-MR-deficient mice (18-monthold), a decrease in aortic collagen content was found [42], suggesting that VSMC-MR play a role in vascular fibrosis. Unlike in an aldosterone/salt hypertension model, the specific VSMC-MR inactivation also leads to the attenuation of arterial stiffening preventing the cellmatrix attachment proteins but without significant modification in vascular collagen/elastin ratio [22]. Other studies support that, after injury, aldosterone-infused animal developed vascular remodeling and MR antagonist reversed those effects [59–61]. Pharmacological inhibition of MR has also demonstrated beneficial effects such as increased lumen and outer diameters of the middle cerebral artery of spontaneously hypertensive stroke-prone rats [62]. Moreover, in a clinical study, treatment with the MR antagonist eplerenone improves the degree of arterial stiffness in hypertensive patients [63]. In conclusion, these studies support that VSMC-MR plays a direct role in vascular remodeling.

**4.4. Vascular MR and hypertension**

Hypertension represents an aging-associated cardiovascular risk factor. It is known that renal MR regulates the BP and the MR has been considered an antihypertensive target for decades. The association between high levels of Aldo and hypertension was proposed when some forms of hypertension were found associated to primary hyperaldosteronism; also, the positive correlation of high levels of Aldo with high MR expression and hypertension has pointed out to a key role of MR in the establishment of the hypertensive phenotype. Moreover, about 50 years ago, the MR antagonist spironolactone decreased BP in hypertensive patients [74] and in patients with other types of hypertension [75]. The antihypertensive effects of MRA have been analyzed in clinical trials demonstrating a BP reduction in hypertensive patients

Mineralocorticoid Receptor in Calcium Handling of Vascular Smooth Muscle Cells

of renal MR activation [44, 79]. The meta-analysis by Dahal et al. showed that spironolactone reduced systolic BP and this effect was not associated with an increased risk of hyperkalemia compared to placebo [80]. Antihypertensive effects of MRA were analyzed also in resistant hypertension that affects at least 10–15% of all patients. Several studies support that low-dose spironolactone provides significant additive BP reduction in subjects with resistant hypertension [75, 81–85]. PATHWAY-2 was the first randomized and controlled trial to compare spironolactone with other BP-lowering drugs in a well-characterized population of patients with resistant hypertension. In this study it was demonstrated that MRA reduced systolic BP with no hyperkalemia risk [86]. In addition, a role of MR in pulmonary hypertension has been recently identified. MRA treatment initiated at the time of the pulmonary arterial hypertension stimulus prevents the pulmonary vascular hyperplasia and reduces systemic BP [57, 87]. Thus, MR activation may be equally important in patients with and without an established diagnosis of primary aldosteronism. The pathogenesis of MR-associated hypertension in the presence of physiological levels of Aldo in plasma might be mediated by MR activation by other pathways,

for instance, MR overexpression, sensitivity, and/or overstimulation by other factors.

results in neonatal lethality from dehydration by renal Na<sup>+</sup>

been reported yet.

The effect of MR blockade in the development of hypertension has been also assessed in experimental models. In the Dahl salt-sensitive model, MRA attenuated the progressive rise in systolic BP in rats fed with a high-salt diet [88, 89]. Whole body disruption of MR in mice

mouse models allowing cell-specific targeting of MR expression have been used to understand the role of MR in vascular tissues and its potential implication in BP regulation. The participation of vascular MR in BP regulation has been analyzed by using two different mouse models: a tamoxifen-inducible VSMC-specific MR inactivation model (via the smooth muscle actin promoter [42]) and a constitutive model of VSMC-specific MR inactivation (via the SM22 promoter; [22]). No transgenic models with targeted MR overexpression in the VSMCs have

The genetic inactivation of MR in adult (2 months of age) mice prevented the increase in BP induced by aging. SMC-MR-deficient mice developed reduced spontaneous myogenic tone. However, the vascular structure and stiffness of resistance arteries from aged SMC-MR-deficient were similar to those from control mice, supporting the notion that SMC-MR contributes to vascular tone and BP regulation independently of structural changes in the

concentration, a marker

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73

and water loss; thus, transgenic

with primary aldosteronism [76–78] with no changes in plasma K<sup>+</sup>

#### **4.3. Participation of MR in vascular inflammation**

MR plays a key role in the pathogenesis of vascular disease including atherosclerosis and hypertensive vasculopathy, where the role of inflammation has been studied in the last years. In patients with atherosclerosis, high levels of aldosterone in serum predict a substantial increase in subsequent myocardial infarction or death. In vitro studies with human VSMC and ECs have shown that MR activation directly promotes the expression of inflammatory genes [64]. Interestingly, in an in vivo model, spironolactone reduced the number of inflammatory cells in the grafted vein without changing total SMC content, suggesting that MR signaling may contribute to graft remodeling through inflammatory processes rather than SMC hypertrophy [65]. Moreover, in experimental models of atherosclerosis, it was confirmed that the plaque progression was enhanced by Aldo and prevented by MR antagonists [66–68]. The pro-atherogenic genes (*CTGF*, *MT1*, and *PGF*) are also vascular MR-regulated genes [41]. MR signaling also contributes to vascular inflammation in animal models of hypertension. In experimental models of hypertension, MR inhibition reduced the vascular inflammation even without changes in BP, supporting that MR activation participates in vascular inflammation and damage through a BP-independent process [69]. Vascular calcification is a late stage found in atherosclerosis, particularly in the elderly and in patients with renal failure [70]. In VSMCs from human coronary artery, MR activation by Aldo upregulated the expression of genes implicated in vascular calcification, including bone morphogenetic protein 2 (*BMP-2*), alkaline phosphatase (*ALP*), and osteoprotegerin [16]. Also, in an in vitro model, MR activation by Aldo or cortisol stimulated vascular *ALP* [71]. MR is also involved in vascular calcification by regulating the expression of the phosphate transporter *Pit1*, which has an osteogenic function in the smooth muscle ameliorated by spironolactone [72]. However, in a different in vitro study using VSMC isolated from the aorta, it was showed that pro-calcification effects of corticosterone and 11-DHC are mediated directly by MR, but the expression of *Osterix*, *BMP-2*, and *Pit-1* was unaltered [73]. These in vitro studies support that MR is involved in the late stage of atherosclerosis: vascular calcification. All these studies support that vascular MR activation participates in the inflammatory response and contributes to the complications associated with atherosclerotic vascular disease.

#### **4.4. Vascular MR and hypertension**

aldosterone-enhanced vascular fibroses were attenuated; thus, VSMC-MR is necessary for aldosterone-induced vascular remodeling [58]. In aged VSMC-MR-deficient mice (18-monthold), a decrease in aortic collagen content was found [42], suggesting that VSMC-MR play a role in vascular fibrosis. Unlike in an aldosterone/salt hypertension model, the specific VSMC-MR inactivation also leads to the attenuation of arterial stiffening preventing the cellmatrix attachment proteins but without significant modification in vascular collagen/elastin ratio [22]. Other studies support that, after injury, aldosterone-infused animal developed vascular remodeling and MR antagonist reversed those effects [59–61]. Pharmacological inhibition of MR has also demonstrated beneficial effects such as increased lumen and outer diameters of the middle cerebral artery of spontaneously hypertensive stroke-prone rats [62]. Moreover, in a clinical study, treatment with the MR antagonist eplerenone improves the degree of arterial stiffness in hypertensive patients [63]. In conclusion, these studies support

MR plays a key role in the pathogenesis of vascular disease including atherosclerosis and hypertensive vasculopathy, where the role of inflammation has been studied in the last years. In patients with atherosclerosis, high levels of aldosterone in serum predict a substantial increase in subsequent myocardial infarction or death. In vitro studies with human VSMC and ECs have shown that MR activation directly promotes the expression of inflammatory genes [64]. Interestingly, in an in vivo model, spironolactone reduced the number of inflammatory cells in the grafted vein without changing total SMC content, suggesting that MR signaling may contribute to graft remodeling through inflammatory processes rather than SMC hypertrophy [65]. Moreover, in experimental models of atherosclerosis, it was confirmed that the plaque progression was enhanced by Aldo and prevented by MR antagonists [66–68]. The pro-atherogenic genes (*CTGF*, *MT1*, and *PGF*) are also vascular MR-regulated genes [41]. MR signaling also contributes to vascular inflammation in animal models of hypertension. In experimental models of hypertension, MR inhibition reduced the vascular inflammation even without changes in BP, supporting that MR activation participates in vascular inflammation and damage through a BP-independent process [69]. Vascular calcification is a late stage found in atherosclerosis, particularly in the elderly and in patients with renal failure [70]. In VSMCs from human coronary artery, MR activation by Aldo upregulated the expression of genes implicated in vascular calcification, including bone morphogenetic protein 2 (*BMP-2*), alkaline phosphatase (*ALP*), and osteoprotegerin [16]. Also, in an in vitro model, MR activation by Aldo or cortisol stimulated vascular *ALP* [71]. MR is also involved in vascular calcification by regulating the expression of the phosphate transporter *Pit1*, which has an osteogenic function in the smooth muscle ameliorated by spironolactone [72]. However, in a different in vitro study using VSMC isolated from the aorta, it was showed that pro-calcification effects of corticosterone and 11-DHC are mediated directly by MR, but the expression of *Osterix*, *BMP-2*, and *Pit-1* was unaltered [73]. These in vitro studies support that MR is involved in the late stage of atherosclerosis: vascular calcification. All these studies support that vascular MR activation participates in the inflammatory response and contributes to the complications

that VSMC-MR plays a direct role in vascular remodeling.

**4.3. Participation of MR in vascular inflammation**

72 Calcium and Signal Transduction

associated with atherosclerotic vascular disease.

Hypertension represents an aging-associated cardiovascular risk factor. It is known that renal MR regulates the BP and the MR has been considered an antihypertensive target for decades. The association between high levels of Aldo and hypertension was proposed when some forms of hypertension were found associated to primary hyperaldosteronism; also, the positive correlation of high levels of Aldo with high MR expression and hypertension has pointed out to a key role of MR in the establishment of the hypertensive phenotype. Moreover, about 50 years ago, the MR antagonist spironolactone decreased BP in hypertensive patients [74] and in patients with other types of hypertension [75]. The antihypertensive effects of MRA have been analyzed in clinical trials demonstrating a BP reduction in hypertensive patients with primary aldosteronism [76–78] with no changes in plasma K<sup>+</sup> concentration, a marker of renal MR activation [44, 79]. The meta-analysis by Dahal et al. showed that spironolactone reduced systolic BP and this effect was not associated with an increased risk of hyperkalemia compared to placebo [80]. Antihypertensive effects of MRA were analyzed also in resistant hypertension that affects at least 10–15% of all patients. Several studies support that low-dose spironolactone provides significant additive BP reduction in subjects with resistant hypertension [75, 81–85]. PATHWAY-2 was the first randomized and controlled trial to compare spironolactone with other BP-lowering drugs in a well-characterized population of patients with resistant hypertension. In this study it was demonstrated that MRA reduced systolic BP with no hyperkalemia risk [86]. In addition, a role of MR in pulmonary hypertension has been recently identified. MRA treatment initiated at the time of the pulmonary arterial hypertension stimulus prevents the pulmonary vascular hyperplasia and reduces systemic BP [57, 87]. Thus, MR activation may be equally important in patients with and without an established diagnosis of primary aldosteronism. The pathogenesis of MR-associated hypertension in the presence of physiological levels of Aldo in plasma might be mediated by MR activation by other pathways, for instance, MR overexpression, sensitivity, and/or overstimulation by other factors.

The effect of MR blockade in the development of hypertension has been also assessed in experimental models. In the Dahl salt-sensitive model, MRA attenuated the progressive rise in systolic BP in rats fed with a high-salt diet [88, 89]. Whole body disruption of MR in mice results in neonatal lethality from dehydration by renal Na<sup>+</sup> and water loss; thus, transgenic mouse models allowing cell-specific targeting of MR expression have been used to understand the role of MR in vascular tissues and its potential implication in BP regulation. The participation of vascular MR in BP regulation has been analyzed by using two different mouse models: a tamoxifen-inducible VSMC-specific MR inactivation model (via the smooth muscle actin promoter [42]) and a constitutive model of VSMC-specific MR inactivation (via the SM22 promoter; [22]). No transgenic models with targeted MR overexpression in the VSMCs have been reported yet.

The genetic inactivation of MR in adult (2 months of age) mice prevented the increase in BP induced by aging. SMC-MR-deficient mice developed reduced spontaneous myogenic tone. However, the vascular structure and stiffness of resistance arteries from aged SMC-MR-deficient were similar to those from control mice, supporting the notion that SMC-MR contributes to vascular tone and BP regulation independently of structural changes in the vasculature [42]. The constitutive model of VSMC-specific MR inactivation reported a similar basal BP decrease in 5-month-old MR-KO mice [22]. The BP phenotype in both inactivated VSMC-MR model mice is independent of Na<sup>+</sup> intake and renal MR function supporting a role for VSMC-MR in BP regulation. Interestingly, tamoxifen-inducible VSMC-MR inactivation prevented the in vivo increase in BP induced by Ang II infusion but not by aldosteronesalt challenge [22, 42]. Inactivation of VSMC-MR was also shown to decrease the contractile response to KCl and extracellular Ca2+ [62]. The role of the vascular MR could also depend on the vascular bed that is considered. In the future, the use of transgenic models will allow us to decipher the contribution of endothelial MR and VSMC-MR in the different vascular beds and the possible implication in BP regulation [90].

VSMC-MR-KO mice [102]. Furthermore, during the aging process, MR expression increases in resistance vessels along with a decline in the microRNA (miR)-155 abundance, suggesting

Adding more pieces to the puzzle, we recently showed in cardiomyocytes that aldosterone

P1-promoter [19]. Importantly, we deciphered that aldosterone, through MR-dependent mechanism, dramatically activates the "cardiac"-specific *Cacna1c* P1-promoter, even in blood

channel blocker actions, a mechanism that might participate to treatment-resistant hypertension, as recently proposed [86]. These findings were further validated using a hypertensive rat aldosterone-salt model, as previously described [104]. Although our data showed that aldosterone/MR impairs 1,4-dihydropyridine sensitivity in VSMC through alternative splic-

Ca2+-activated potassium channels (KCa), mainly the large conductance KCa channels (BKCa), have been recognized as another important target of MR in blood vessels [105]. BKCa plays a critical role in limiting arterial contraction by producing VSMC hyperpolarization

tion [106]. However, three subtypes of KCa have been identified in blood vessels and categorized according to their conductance: small (SKCa), intermediate, and BKCa. Small- and intermediate-conductance channels are mainly expressed in the ECs, while BKCa channels

Previous studies have shown that increased plasma aldosterone concentration enhances vascular KCa function [105]. Oppositely, it was demonstrated that mice lacking the poreforming BKCaα subunit led to an elevation of BP resulting from hyperaldosteronism, which

arteries [107]. Accordingly, impaired acetylcholine-mediated relaxation in isolated coronary arteries has been shown in mice model with cardiac-specific overexpression of aldosterone synthase (MAS mice) [30]. These findings correlate with decreased mRNA and protein expression of BKCa α and β1 subunits in the heart and coronary artery of MAS mice. Moreover, in vitro treatment of rat aortic VSMCs with increasing concentrations of aldosterone led to a reduced BKCa subunit expression in a concentration-dependent manner. Thus, these findings suggest that augmented local aldosterone production likely acts in a paracrine fashion way suppressing BKCa expression in the surrounding coronary VSMC, thereby contributing to the impaired endothelium-dependent VSMC relaxation. Intriguingly, despite aged VSMC-MR-KO mice displaying lower BP than age-matched WT mice, no significant changes were observed in aortic mRNA expression and function of BKCa in mesenteric VSMC [42]. Furthermore, aldosterone-treated aorta for 24 h with 10−8 M of aldosterone did not modify mRNA expression of BKCa α and β1 subunits [19]; thus, further studies are needed to clarify

the effect of MR activation in the expression and activity of BKCa channels.

1.2-LNT by recruiting MR onto targeted genomic regions in "cardiac" *Cacna1c*

Mineralocorticoid Receptor in Calcium Handling of Vascular Smooth Muscle Cells

1.2α1C, further studies are needed to validate whether this mechanism participates

current in response to increased intracellular Ca2+ concentra-

levels, as well as increased vascular tone in small

1.2α1C in this tissue that minimizes Ca2+

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75

1.2 is a downstream target of miR-155 regulation [103].

vessels, conferring a new molecular signature to Cav

that Cav

regulates Cav

ing of Cav

in the resistant hypertension.

through transient outward K<sup>+</sup>

**5.2. Ca2+-activated potassium channels**

are predominately expressed in VSMCs.

was accompanied by decreased serum K<sup>+</sup>
