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The aim of the study was to characterize the role of Rac1 GTPase for the mineralocorticoid receptor (MR)-mediated pro-fibrotic remodeling. Transgenic mice with cardiac overexpression of constitutively active Rac1 (RacET) develop an age-dependent phenotype with atrial dilatation, fibrosis, and atrial fibrillation. Expression of MR was similar in RacET and WT mice. The expression of 11β hydroxysteroid dehydrogenase type 2 (11β-HSD2) was age-dependently up-regulated in the atria and the left ventricles of RacET mice on mRNA and protein levels. Statin treatment inhibiting Rac1 geranylgeranylation reduced 11β-HSD2 up-regulation. Samples of human left atrial myocardium showed a positive correlation between Rac1 activity and 11β-HSD2 expression ( r = 0.7169). Immunoprecipitation showed enhanced Rac1-bound 11β-HSD2 relative to Rac1 expression in RacET mice that was diminished with statin treatment. Both basal and phorbol 12-myristate 13-acetate (PMA)-induced NADPH oxidase activity were increased in RacET and correlated positively with 11β-HSD2 expression ( r = 0.788 and r = 0.843, respectively). In cultured H9c2 cardiomyocytes, Rac1 activation with l-buthionine sulfoximine increased; Rac1 inhibition with NSC23766 decreased 11β-HSD2 mRNA and protein expression. Connective tissue growth factor (CTGF) up-regulation induced by aldosterone was prevented with NSC23766. Cardiomyocyte transfection with 11β-HSD2 siRNA abolished the aldosterone-induced CTGF up-regulation. Aldosterone-stimulated MR nuclear translocation was blocked by the 11β-HSD2 inhibitor carbenoxolone. In cardiac fibroblasts, nuclear MR translocation induced by aldosterone was inhibited with NSC23766 and spironolactone. NSC23766 prevented the aldosterone-induced proliferation and migration of cardiac fibroblasts and the up-regulation of CTGF and fibronectin. In conclusion, Rac1 GTPase regulates 11β-HSD2 expression, MR activation, and MR-mediated pro-fibrotic signaling., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

We studied the role of the mineralocorticoid receptor (MR) in the signaling that promotes atrial fibrosis. Left atrial myocardium of patients with atrial fibrillation (AF) exhibited 4-fold increased hydroxyproline content compared with patients in sinus rhythm. Expression of MR was similar, as was 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which also increased. 11β-HSD2 converts cortisol to receptor-inactive metabolites allowing aldosterone occupancy of MR. 11β-HSD2 was up-regulated by arrhythmic pacing in cultured cardiomyocytes and in a mouse model of spontaneous AF (RacET). In cardiomyocytes, aldosterone induced connective tissue growth factor (CTGF) in the absence but not in the presence of cortisol. Hydroxyproline expression was increased in cardiac fibroblasts exposed to conditioned medium from aldosterone-treated cardiomyocytes but not from cardiomyocytes treated with both cortisol and aldosterone. Aldosterone increased connective tissue growth factor and hydroxyproline expression in cardiac fibroblasts, which were prevented by BR-4628, a dihydropyridine-derived selective MR antagonist, and by spironolactone. Aldosterone activated RhoA GTPase. Rho kinase inhibition by Y-27632 prevented CTGF and hydroxyproline, whereas the RhoA activator CN03 increased CTGF expression. Aldosterone and CTGF increased lysyl oxidase, and aldosterone enhanced miR-21 expression. MR antagonists reduced the aldosterone but not the CTGF effect. In conclusion, MR signaling promoted fibrotic remodeling. Increased expression of 11β-HSD2 during AF leads to up-regulation of collagen and pro-fibrotic mediators by aldosterone, specifically RhoA activity as well as CTGF, lysyl oxidase, and microRNA-21 expression. The MR antagonists BR-4628 and spironolactone prevent these alterations. MR inhibition may, therefore, represent a potential pharmacologic target for the prevention of fibrotic remodeling of the atrial myocardium.

Increased interferon (IFN)-β signaling in patients with insufficient coronary collateralization and an inhibitory effect of IFNβ on collateral artery growth in mice have been reported. The mechanisms of IFNβ-induced inhibition of arteriogenesis are unknown. In stimulated monocytes from patients with chronic total coronary artery occlusion and decreased arteriogenic response, whole genome expression analysis showed increased expression of IFNβ-regulated genes. Immunohistochemically, the IFNβ receptor was localized in the vascular media of murine collateral arteries. Treatment of vascular smooth muscle cells (VSMC) with IFNβ resulted in an attenuated proliferation, cell-cycle arrest, and increased expression of cyclin-dependent kinase inhibitor-1A (p21). The growth inhibitory effect of IFNβ was attenuated by inhibition of p21 by RNA interference. IFNβ-treated THP1 monocytes showed enhanced apoptosis. Subsequently, we tested if collateral artery growth can be stimulated by inhibition of IFNβ-signaling. RNA interference of the IFNβ receptor-1 (IFNAR1) increased VSMC proliferation, cell cycle progression, and reduced p21 gene expression. IFNβ signaling and FAS and TRAIL expression were attenuated in monocytes from IFNAR1(-/-) mice, indicating reduced monocyte apoptosis. Hindlimb perfusion restoration 1 week after femoral artery ligation was improved in IFNAR1(-/-) mice compared with wild-type mice as assessed by infusion of fluorescent microspheres. These results demonstrate that IFNβ inhibits collateral artery growth and VSMC proliferation through p21-dependent cell cycle arrest and induction of monocyte apoptosis. Inhibition of IFNβ stimulates VSMC proliferation and collateral artery growth.

Angiotensin II induces cardiomyocyte hypertrophy, but its consequences on cardiomyocyte metabolism and energy supply are not completely understood. Here we investigate the effect of angiotensin II on glucose and fatty acid utilization and the modifying role of AMP-activated protein kinase (AMPK), a key regulator of metabolism and proliferation. Treatment of H9C2 cardiomyocytes with angiotensin II (Ang II, 1 microm, 4 h) increased [(3)H]leucine incorporation, up-regulated the mRNA expression of the hypertrophy marker genes MLC, ANF, BNP, and beta-MHC, and decreased the phosphorylation of the negative mTOR-regulator tuberin (TSC-2). Rat neonatal cardiomyocytes showed similar results. Western blot analysis revealed a time- and concentration-dependent down-regulation of AMPK-phosphorylation in the presence of angiotensin II, whereas the protein expression of the catalytic alpha-subunit remained unchanged. This was paralleled by membrane translocation of glucose-transporter type 4 (GLUT4), increased uptake of [(3)H]glucose and transient down-regulation of phosphorylation of acetyl-CoA carboxylase (ACC), whereas fatty acid uptake remained unchanged. Similarly, short-term transaortic constriction in mice resulted in down-regulation of P-AMPK and P-ACC but up-regulation of GLUT4 membrane translocation in the heart. Preincubation of cardiomyocytes with the AMPK stimulator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mM, 4 h) completely prevented the angiotensin II-induced cardiomyocytes hypertrophy. In addition, AICAR reversed the metabolic effects of angiotensin II: GLUT4 translocation was reduced, but ACC phosphorylation and TSC phosphorylation were elevated. In summary, angiotensin II-induced hypertrophy of cardiomyocytes is accompanied by decreased activation of AMPK, increased glucose uptake, and decreased mTOR inhibition. Stimulation with the AMPK activator AICAR reverses these metabolic changes, increases fatty acid utilization, and inhibits cardiomyocyte hypertrophy.

Rac1 GTPase is essential for the activation of the NAD(P)H oxidase complex and, thereby, regulates the release of reactive oxygen species (ROS) in the vessel wall. 17 beta-estradiol (E2) inhibits vascular ROS production. To elucidate the underlying molecular mechanisms we investigated the potential regulation of Rac1 by E2 in vascular smooth muscle cells. Treatment of vascular smooth muscle cells with angiotensin II as well as overexpression of the constitutively active mutant RacL61 increased ROS release as assessed by dichlorofluorescein fluorescence, whereas inhibition of Rac1 by Clostridium sordellii lethal toxin or overexpression of dominant-negative RacN17 inhibited ROS production. Treatment with E2 (100 nm) completely prevented angiotensin II-induced NAD(P)H oxidase activity and ROS production. E2 time and concentration dependently decreased angiotensin II-induced and basal Rac1 mRNA and protein expression as well as Rac1 activity. Down-regulation of Rac1 expression by E2 was mediated by inhibition of gene transcription (nuclear run-on assays), but E2 had no effect on Rac1 mRNA stability. Regulation of Rac1 was mediated by estrogen receptors since co-incubation with ICI 182.780 prevented down-regulation of Rac1. To test these observations in vivo, ovariectomized spontaneously hypertensive rats were treated with E2 or vehicle. Real-time PCR and Western blotting showed reduction of aortic Rac1 mRNA and protein by 32 and 58%, respectively. Furthermore, down-regulation of Rac1 by E2 was observed in human mononuclear cells of women with elevated E2 levels after controlled ovarian hyperstimulation. Rac1 GTPase gene-transcription and activity is regulated by 17 beta-estradiol, which may be an important molecular mechanism contributing to the cardiovascular effects of estrogens.