Mineralocorticoid receptors in vascular disease: connecting molecular pathways to clinical implications.

The mineralocorticoid receptor (MR), a steroid-hormone-activated transcription factor, plays a substantial role in cardiovascular diseases. MR antagonists (MRAs) have long been appreciated as effective treatments for heart failure and hypertension; however, recent research suggests that additional patient populations may also benefit from MRA therapy. Experimental evidence demonstrates that in addition to its classic role in the regulating sodium handling in the kidney, functional MR is expressed in the blood vessels and contributes to hypertension, vascular inflammation and remodeling, and atherogenesis. MR activation drives pathological phenotypes in smooth muscle cells, endothelial cells, and inflammatory cells, whereas MRAs inhibit these effects. Collectively, these studies demonstrate a new role for extrarenal MR in cardiovascular disease. This review summarizes these new lines of evidence and how they contribute to the mechanisms of atherosclerosis, pulmonary and systemic hypertension, and vein graft failure, and describes new patient populations that may benefit from MRA therapy.

Aldosterone regulates vascular gene transcription via oxidative stress-dependent and -independent pathways.

OBJECTIVE: Aldosterone (Aldo) antagonism prevents cardiovascular mortality by unclear mechanisms. Aldo binds to the mineralocorticoid receptor (MR), a ligand-activated transcription factor, which is expressed in human vascular cells. Here we define the early Aldo-regulated vascular transcriptome and investigate the mechanisms of gene regulation by Aldo in the vasculature that may contribute to vascular disease. METHODS AND RESULTS: Gene expression profiling of Aldo-treated mouse aortas identified 72 genes regulated by Aldo. These genes are overrepresented in Gene Ontology categories involved in vascular function and disease. Quantitative reverse transcription-polymerase chain reaction was used to confirm and further explore mechanisms of vascular gene regulation by Aldo. Aldo-regulated vascular gene expression was inhibited by actinomycin D and MR antagonists supporting a transcriptional MR-dependent mechanism. Aldo regulation of a subset of genes was enhanced in the setting of vascular endothelial denudation and blocked by the free radical scavenger Tempol, supporting synergy between Aldo and vascular injury that is oxidative stress dependent. In the aortic arch, a region predisposed to atherosclerosis, the injury-enhanced genes also demonstrated enhanced expression compared with the descending aorta, both at baseline and after Aldo exposure. Furthermore, the clinically beneficial MR antagonist spironolactone inhibited expression of the identified genes in aortic tissue from humans with atherosclerosis. CONCLUSIONS: This study defines the Aldo-regulated vascular transcriptome and characterizes a subset of proatherogenic genes with enhanced Aldo-stimulated, oxidative stress-dependent expression in the setting of vascular injury and in areas predisposed to atherosclerosis. Inhibition of MR regulation of these genes may play a role in the protective effects of Aldo antagonists in patients with vascular disease, and these pathways may provide novel drug targets to prevent atherosclerosis in humans.

Molecular basis of TRAP-5'SL RNA interaction in the Bacillus subtilis trp operon transcription attenuation mechanism.

Expression of the Bacillus subtilis trpEDCFBA operon is regulated by the interaction of tryptophan-activated TRAP with 11 (G/U)AG trinucleotide repeats that lie in the leader region of the nascent trp transcript. Bound TRAP prevents folding of an antiterminator structure and favors formation of an overlapping intrinsic terminator hairpin upstream of the trp operon structural genes. A 5'-stem-loop (5'SL) structure that forms just upstream of the triplet repeat region increases the affinity of TRAP-trp RNA interaction, thereby increasing the efficiency of transcription termination. Single-stranded nucleotides in the internal loop and in the hairpin loop of the 5'SL are important for TRAP binding. We show here that altering the distance between these two loops suggests that G7, A8, and A9 from the internal loop and A19 and G20 from the hairpin loop constitute two structurally discrete TRAP-binding regions. Photochemical cross-linking experiments also show that the hairpin loop of the 5'SL is in close proximity to the flexible loop region of TRAP during TRAP-5'SL interaction. The dimensions of B. subtilis TRAP and of a three-dimensional model of the 5'SL generated using the MC-Sym and MC-Fold pipeline imply that the 5'SL binds the protein in an orientation where the helical axis of the 5'SL is perpendicular to the plane of TRAP. This interaction not only increases the affinity of TRAP-trp leader RNA interaction, but also orients the downstream triplet repeats for interaction with the 11 KKR motifs that lie on TRAP's perimeter, increasing the likelihood that TRAP will bind in time to promote termination.

Woody Plant Encroachment has a Larger Impact than Climate Change on Dryland Water Budgets.

Woody plant encroachment (WPE) into grasslands is a global phenomenon that is associated with land degradation via xerification, which replaces grasses with shrubs and bare soil patches. It remains uncertain how the global processes of WPE and climate change may combine to impact water availability for ecosystems. Using a process-based model constrained by watershed observations, our results suggest that both xerification and climate change augment groundwater recharge by increasing channel transmission losses at the expense of plant available water. Conversion from grasslands to shrublands without creating additional bare soil, however, reduces transmission losses. Model simulations considering both WPE and climate change are used to assess their relative roles in a late 21st century condition. Results indicate that changes in focused channel recharge are determined primarily by the WPE pathway. As a result, WPE should be given consideration when assessing the vulnerability of groundwater aquifers to climate change.

PKCδ Mediates Mineralocorticoid Receptor Activation by Angiotensin II to Modulate Smooth Muscle Cell Function.

Angiotensin II (AngII) and the mineralocorticoid receptor (MR) ligand aldosterone both contribute to cardiovascular disorders, including hypertension and adverse vascular remodeling. We previously demonstrated that AngII activates MR-mediated gene transcription in human vascular smooth muscle cells (SMCs), yet the mechanism and the impact on SMC function are unknown. Using an MR-responsive element-driven transcriptional reporter assay, we confirm that AngII induces MR transcriptional activity in vascular SMCs and endothelial cells, but not in Cos1 or human embryonic kidney-293 cells. AngII activation of MR was blocked by the MR antagonist spironolactone or eplerenone and the protein kinase C-δ (PKCδ) inhibitor rottlerin, implicating both in the mechanism. Similarly, small interfering RNA knockdown of PKCδ in SMCs prevented AngII-mediated MR activation, whereas knocking down of MR blocked both aldosterone- and AngII-induced MR function. Coimmunoprecipitation studies reveal that endogenous MR and PKCδ form a complex in SMCs that is enhanced by AngII treatment in association with increased serine phosphorylation of the MR N terminus. AngII increased mRNA expression of the SMC-MR target gene, FKBP51, via an MR-responsive element in intron 5 of the FKBP51 gene. The impact of AngII on FKBP51 reporter activity and gene expression in SMCs was inhibited by spironolactone and rottlerin. Finally, the AngII-induced increase in SMC number was also blocked by the MR antagonist spironolactone and the PKCδ inhibitor rottlerin. These data demonstrate that AngII activates MR transcriptional regulatory activity, target gene regulation, and SMC proliferation in a PKCδ-dependent manner. This new mechanism may contribute to synergy between MR and AngII in driving SMC dysfunction and to the cardiovascular benefits of MR and AngII receptor blockade in humans.

No Significant Role for Smooth Muscle Cell Mineralocorticoid Receptors in Atherosclerosis in the Apolipoprotein-E Knockout Mouse Model.

Objective: Elevated levels of the hormone aldosterone are associated with increased risk of myocardial infarction and stroke in humans and increased progression and inflammation of atherosclerotic plaques in animal models. Aldosterone acts through the mineralocorticoid receptor (MR) which is expressed in vascular smooth muscle cells (SMCs) where it promotes SMC calcification and chemokine secretion in vitro. The objective of this study is to explore the role of the MR specifically in SMCs in the progression of atherosclerosis and the associated vascular inflammation in vivo in the apolipoprotein E knockout (ApoE-/-) mouse model. Methods and Results: Male ApoE-/- mice were bred with mice in which MR could be deleted specifically from SMCs by tamoxifen injection. The resulting atheroprone SMC-MR-KO mice were compared to their MR-Intact littermates after high fat diet (HFD) feeding for 8 or 16 weeks or normal diet for 12 months. Body weight, tail cuff blood pressure, heart and spleen weight, and serum levels of glucose, cholesterol, and aldosterone were measured for all mice at the end of the treatment period. Serial histologic sections of the aortic root were stained with Oil Red O to assess plaque size, lipid content, and necrotic core area; with PicroSirius Red for quantification of collagen content; by immunofluorescent staining with anti-Mac2/Galectin-3 and anti-smooth muscle α-actin antibodies to assess inflammation and SMC marker expression; and with Von Kossa stain to detect plaque calcification. In the 16-week HFD study, these analyses were also performed in sections from the brachiocephalic artery. Flow cytometry of cell suspensions derived from the aortic arch was also performed to quantify vascular inflammation after 8 and 16 weeks of HFD. Deletion of the MR specifically from SMCs did not significantly change plaque size, lipid content, necrotic core, collagen content, inflammatory staining, actin staining, or calcification, nor were there differences in the extent of vascular inflammation between MR-Intact and SMC-MR-KO mice in the three experiments. Conclusion: SMC-MR does not directly contribute to the formation, progression, or inflammation of atherosclerotic plaques in the ApoE-/- mouse model of atherosclerosis. This indicates that the MR in non-SMCs mediates the pro-atherogenic effects of MR activation.

Effect of Spironolactone on Myocardial Fibrosis and Other Clinical Variables in Patients with Hypertrophic Cardiomyopathy.

BACKGROUND: Myocardial fibrosis has proved to be an important marker and determinant in the pathogenesis of hypertrophic cardiomyopathy. In particular, scar formation, if substantial, can promote ventricular tachyarrhythmias or progressive heart failure in the absence of left ventricular outflow obstruction. Therefore, an intervention to mitigate myocardial fibrosis would be potentially advantageous to hypertrophic cardiomyopathy patients. METHODS: Eligible hypertrophic cardiomyopathy patients were randomized 1:1 in a prospective double-blind fashion to spironolactone 50 mg or placebo to be taken over a 12-month period. The primary endpoint was the effect of mineralocorticoid receptor blockade on serum markers of collagen synthesis and degradation. A number of other functional and morphologic variables and biomarkers comprised secondary exploratory measures. RESULTS: Fifty-three hypertrophic cardiomyopathypatients (41 ± 13 years old; 72% men) were randomized; demographic and clinical variable were well matched at baseline. Absolute change between baseline and 12 months did not differ between hypertrophic cardiomyopathy patients treated with spironolactone and those receiving placebo with respect to serum markers of collagen synthesis or degradation, fibrosis by late gadolinium enhancement on cardiac magnetic resonance imaging, or other clinical variables, including objective measure of functional capacity (peak VO2), New York Heart Association functional class, left ventricular wall thickness, mass and volume, and left atrial size, as well as assessment of diastolic function (P = .4-1.0). CONCLUSIONS: These findings do not support the use of spironolactone in hypertrophic cardiomyopathy to improve left ventricular remodeling by mitigating myocardial fibrosis or altering clinical course.

Mineralocorticoid receptor-mediated vascular insulin resistance: an early contributor to diabetes-related vascular disease?

Two-thirds of adults in the U.S. are overweight or obese, and another 26 million have type 2 diabetes (T2D). Patients with diabetes and/or the metabolic syndrome have a significantly increased risk of heart attack and stroke compared with people with normal insulin sensitivity. Decreased insulin sensitivity in cardiovascular tissues as well as in traditional targets of insulin metabolic signaling, such as skeletal muscle, is an underlying abnormality in obesity, hypertension, and T2D. In the vasculature, insulin signaling plays a critical role in normal vascular function via endothelial cell nitric oxide production and modulation of Ca(2+) handling and sensitivity in vascular smooth muscle cells. Available evidence suggests that impaired vascular insulin sensitivity may be an early, perhaps principal, defect of vascular function and contributor to the pathogenesis of vascular disease in persons with obesity, hypertension, and T2D. In the overweight and obese individual, as well as in persons with hypertension, systemic and vascular insulin resistance often occur in concert with elevations in plasma aldosterone. Indeed, basic and clinical studies have demonstrated that elevated plasma aldosterone levels predict the development of insulin resistance and that aldosterone directly interferes with insulin signaling in vascular tissues. Furthermore, elevated plasma aldosterone levels are associated with increased heart attack and stroke risk. Conversely, renin-angiotensin-aldosterone system and mineralocorticoid receptor (MR) antagonism reduces cardiovascular risk in these patient populations. Recent and accumulating evidence in this area has implicated excessive Ser phosphorylation and proteosomal degradation of the docking protein, insulin receptor substrate, and enhanced signaling through hybrid insulin/IGF-1 receptor as important mechanisms underlying aldosterone-mediated interruption of downstream vascular insulin signaling. Prevention or restoration of these changes via blockade of aldosterone action in the vascular wall with MR antagonists (i.e., spironolactone, eplerenone) may therefore account for the clinical benefit of these compounds in obese and diabetic patients with cardiovascular disease. This review will highlight recent evidence supporting the hypothesis that aldosterone and MR signaling represent an ideal candidate pathway linking early promoters of diabetes, especially overnutrition and obesity, to vascular insulin resistance, dysfunction, and disease.

Mineralocorticoid receptor antagonism inhibits vein graft remodeling in mice.

OBJECTIVE: Vein graft failure rates resulting from adverse graft remodeling remain high with no effective therapy. The mineralocorticoid receptor (MR) plays a role in pathologic arterial remodeling. We demonstrated recently that the MR is upregulated in venous tissues after grafting and hypothesized that MR inhibition would reduce vein graft remodeling. METHODS: Reverse transcription polymerase chain reaction and immunoblotting were used to examine the expression of the MR and other components of the renin-angiotensin-aldosterone system in human vein and primary human saphenous vein smooth muscle cells (HSVSMC). Adenoviral reporter gene assays were used to explore MR transcriptional activity in HSVSMC. The effect of MR inhibition on vein graft remodeling in vivo was characterized in a mouse vein graft model. RESULTS: Messenger RNAs encoding the MR, 11-ß-hydroxysteroid dehydrogenase 2, angiotensin type 1 receptor, and the angiotensin-converting enzyme are expressed in whole HSVSMC. MR and 11-ß-hydroxysteroid dehydrogenase 2 protein expression is confirmed, and MR-dependent transcriptional regulation is demonstrated at physiologic aldosterone concentrations in HSVSMC. Treatment of mice with the MR antagonist spironolactone, at doses that do not lower blood pressure (20 mg/kg per day), reduces maximal vein graft intima-media thickness by 68%, with an associated reduction in graft inflammatory cell infiltration and fibrosis. CONCLUSIONS: MR is expressed in human venous tissue and cells and modulates gene expression in HSVSMC in response to physiologic aldosterone concentrations. In vivo, MR inhibition reduces vein graft thickening and inflammation. These preclinical data support the potential to use MR antagonists as novel treatments to preserve vein graft patency.

Aldosterone increases early atherosclerosis and promotes plaque inflammation through a placental growth factor-dependent mechanism.

BACKGROUND: Aldosterone levels correlate with the incidence of myocardial infarction and mortality in cardiovascular patients. Aldosterone promotes atherosclerosis in animal models, but the mechanisms are poorly understood. METHODS AND RESULTS: Aldosterone was infused to achieve pathologically relevant levels that did not increase blood pressure in the atherosclerosis-prone apolipoprotein E-knockout mouse (ApoE-/-). Aldosterone increased atherosclerosis in the aortic root 1.8±0.1-fold after 4 weeks and in the aortic arch 3.7±0.2-fold after 8 weeks, without significantly affecting plaque size in the abdominal aorta or traditional cardiac risk factors. Aldosterone treatment increased lipid content of plaques (2.1±0.2-fold) and inflammatory cell content (2.2±0.3-fold), induced early T-cell (2.9±0.3-fold) and monocyte (2.3±0.3-fold) infiltration into atherosclerosis-prone vascular regions, and enhanced systemic inflammation with increased spleen weight (1.52±0.06-fold) and the circulating cytokine RANTES (regulated and normal T cell secreted; 1.6±0.1-fold). To explore the mechanism, 7 genes were examined for aldosterone regulation in the ApoE-/- aorta. Further studies focused on the proinflammatory placental growth factor (PlGF), which was released from aldosterone-treated ApoE-/- vessels. Activation of the mineralocorticoid receptor by aldosterone in human coronary artery smooth muscle cells (SMCs) caused the release of factors that promote monocyte chemotaxis, which was inhibited by blocking monocyte PlGF receptors. Furthermore, PlGF-deficient ApoE-/- mice were resistant to early aldosterone-induced increases in plaque burden and inflammation. CONCLUSIONS: Aldosterone increases early atherosclerosis in regions of turbulent blood flow and promotes an inflammatory plaque phenotype that is associated with rupture in humans. The mechanism may involve SMC release of soluble factors that recruit activated leukocytes to the vessel wall via PlGF signaling. These findings identify a novel mechanism and potential treatment target for aldosterone-induced ischemia in humans.

Placental growth factor mediates aldosterone-dependent vascular injury in mice.

In clinical trials, aldosterone antagonists reduce cardiovascular ischemia and mortality by unknown mechanisms. Aldosterone is a steroid hormone that signals through renal mineralocorticoid receptors (MRs) to regulate blood pressure. MRs are expressed and regulate gene transcription in human vascular cells, suggesting that aldosterone might have direct vascular effects. Using gene expression profiling, we identify the pro-proliferative VEGF family member placental growth factor (PGF) as an aldosterone-regulated vascular MR target gene in mice and humans. Aldosterone-activated vascular MR stimulated Pgf gene transcription and increased PGF protein expression and secretion in the mouse vasculature. In mouse vessels with endothelial damage and human vessels from patients with atherosclerosis, aldosterone enhanced expression of PGF and its receptor, FMS-like tyrosine kinase 1 (Flt1). In atherosclerotic human vessels, MR antagonists inhibited PGF expression. In vivo, aldosterone infusion augmented vascular remodeling in mouse carotids following wire injury, an effect that was lost in Pgf-/- mice. In summary, we have identified PGF as what we believe to be a novel downstream target of vascular MR that mediates aldosterone augmentation of vascular injury. These findings suggest a non-renal mechanism for the vascular protective effects of aldosterone antagonists in humans and support targeting the vascular aldosterone/MR/PGF/Flt1 pathway as a therapeutic strategy for ischemic cardiovascular disease.

TRAP-5' stem loop interaction increases the efficiency of transcription termination in the Bacillus subtilis trpEDCFBA operon leader region.

TRAP regulates expression of the Bacillus subtilis trpEDCFBA operon by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the nascent trp leader transcript. Bound TRAP blocks formation of an antiterminator structure and allows formation of an overlapping intrinsic terminator upstream of the trp operon structural genes. A 5' stem-loop (5'SL) structure located upstream of the triplet repeat region also interacts with TRAP. TRAP-5'SL RNA interaction participates in the transcription attenuation mechanism by preferentially increasing the affinity of TRAP for the nascent trp leader transcript during the early stages of transcription, when only a few triplet repeats have been synthesized. Footprinting assays indicated that the 5'SL contacts TRAP through two discrete groups of single-stranded nucleotides that lie in the hairpin loop and in an internal loop. Filter binding and in vivo expression assays of 5'SL mutants established that G7, A8, and A9 from the internal loop, and A19 and G20 from the hairpin loop are critical for proper 5'SL function. These nucleotides are conserved among certain other 5'SL-containing organisms. Single-round transcription results indicated that the 5'SL increases the termination efficiency when transcription is fast; however, the influence of the 5'SL was lost when transcription was slowed by reducing the ribonucleoside triphosphate concentration. Since there is a limited amount of time for TRAP to bind to the nascent transcript and promote termination, our data suggest that the contribution of TRAP-5'SL interaction increases the rate of TRAP binding, which, in turn, increases the efficiency of transcription termination.