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

Distinct Effects of Unfractionated Heparin versus Bivalirudin on Circulating Angiogenic Peptides

Background: Human studies of therapeutic angiogenesis, stem-cell, and progenitor-cell therapy have failed to demonstrate consistent clinical benefit. Recent studies have shown that heparin increases circulating levels of anti-angiogenic peptides. Given the widely prevalent use of heparin in percutaneous and surgical procedures including those performed as part of studies examining the benefit of therapeutic angiogenesis and cell-based therapy, we compared the effects of unfractionated heparin (UFH) on angiogenic peptides with those of bivalirudin, a relatively newer anticoagulant whose effects on angiogenic peptides have not been studied. Methodology/Principal Findings: We measured soluble fms-like tyrosine kinase-1 (sFLT1), placental growth factor (PlGF), vascular endothelial growth factor (VEGF), and soluble Endoglin (sEng) serum levels by enzyme linked immunosorbent assays (ELISA) in 16 patients undergoing elective percutaneous coronary intervention. Compared to baseline values, sFLT1 and PlGF levels increased by 26296313 % and 253654%, respectively, within 30 minutes of UFH therapy (p,0.01 for both; n = 8). VEGF levels decreased by 93.265 % in patients treated with UFH (p,0.01 versus baseline). No change in sEng levels were observed after UFH therapy. No changes in sFLT1, PlGF, VEGF, or sEng levels were observed in any patients receiving bivalirudin (n = 8). To further explore the direct effect of anticoagulation on circulating angiogenic peptides, adult, male wild-type mice received venous injections of clinically dosed UFH or bivalirudin. Compared to saline controls, sFLT1 an

Direct contribution of vascular mineralocorticoid receptors to blood pressure regulation

Photograph of a scene on Lake Murray, in the Lake Murray State Park

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 humans160921012114FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2014/26192-6This work was supported by National Institutes of Health Grants HL095590 and HL119290 (to I.Z.J.), American Heart Association Grants EIA18290005 (to I.Z.J.) and 11POST5390010 (to A.P.M.), and by São Paulo Research Foundation (FAPESP) Grant 2014/26192-6 to (A.P.D.

Mineralocorticoid receptor antagonism inhibits vein graft remodeling in mice

ObjectiveVein 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.MethodsReverse 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.ResultsMessenger RNAs encoding the MR, 11-β-hydroxysteroid dehydrogenase 2, angiotensin type 1 receptor, and the angiotensin-converting enzyme are expressed in whole HSVSMC. MR and 11-β-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.ConclusionsMR 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