c-Src inhibition improves cardiovascular function but not remodeling or fibrosis in Ang II-induced hypertension

c-Src plays an important role in angiotensin II (Ang II) signaling. Whether this member of the Src family kinases is involved in the development of Ang II–induced hypertension and associated cardiovascular damage in vivo remains unknown. Here, we studied Ang II–infused (400 ng/kg/min) mice in which c-Src was partially deleted (c-Src+/−) and in wild-type (WT, c-Src+/+) mice treated with a c-Src inhibitor (CGP077675; 25 mg/kg/d). Ang II increased blood pressure and induced endothelial dysfunction in WT mice, responses that were ameliorated in c-Src+/− and CGP077675-treated mice. Vascular wall thickness and cross-sectional area were similarly increased by Ang II in WT and c-Src+/− mice. CGP077675 further increased cross-sectional area in hypertensive mice. Cardiac dysfunction (ejection fraction and fractional shortening) in Ang II–infused WT mice was normalized in c-Src+/− mice. Increased oxidative stress (plasma thiobarbituric acid–reactive substances, hydrogen peroxide, and vascular superoxide generation) in Ang II–infused WT mice was attenuated in c-Src–deficient and CGP077675-treated mice. Hyperactivation of vascular c-Src, ERK1/2 (extracellular signal–regulated kinase 1/2), and JNK (c-Jun N-terminal kinase) in hypertensive mice was normalized in CGP077675-treated and c-Src+/− mice. Vascular fibronectin was increased by Ang II in all groups and further augmented by CGP077675. Cardiac fibrosis and inflammation induced by Ang II were amplified in c-Src+/− and CGP-treated mice. Our data indicate that although c-Src downregulation attenuates development of hypertension, improves endothelial and cardiac function, reduces oxidative stress, and normalizes vascular signaling, it has little beneficial effect on fibrosis. These findings suggest a divergent role for c-Src in Ang II–dependent hypertension, where c-Src may be more important in regulating redox-sensitive cardiac and vascular function than fibrosis and remodeling

Transient receptor potential melastatin 7 cation channel kinase new player in angiotensin II–induced hypertension

Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg2+)/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase–deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg2+ was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II–infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule–1 expression was increased in Ang II–infused TRPM7 kinase–deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II–treated groups. In TRPM7 kinase–deficient mice, Ang II–induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II–induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II–induced hypertension and associated vascular and target organ damage

Atorvastatin inhibits pro-inflammatory actions of aldosterone in vascular smooth muscle cells by reducing oxidative stress

Vascular inflammatory responses play an important role in several cardiovascular diseases. Of the many pro-inflammatory vasoactive factors implicated in this process, is aldosterone, an important mediator of vascular oxidative stress. Statins, such as atorvastatin, are cholesterol-lowering drugs that have pleiotropic actions, including anti-oxidant properties independently of their cholesterol-lowering effect. This study investigated whether atorvastatin prevents aldosterone-induced VSMC inflammation by reducing reactive oxygen species (ROS) production. Vascular smooth muscle cells (VSMC) from WKY rats were treated with 1 μM atorvastatin for 60 min or for 72 h prior to aldosterone (100 nM) stimulation. Atorvastatin inhibited Rac1/2 and p47phox translocation from the cytosol to the membrane, as well as reduced aldosterone-induced ROS production. Atorvastatin also attenuated aldosterone-induced vascular inflammation and macrophage adhesion to VSMC. Similarly EHT1864, a Rac1/2 inhibitor, and tiron, ROS scavenger, reduced macrophage adhesion. Through its inhibitory effects on Rac1/2 activation and ROS production, atorvastatin reduces vascular ROS generation and inhibits VSMC inflammation. Our data suggest that in conditions associated with aldosterone-induced vascular damage, statins may have vasoprotective effects by inhibiting oxidative stress and inflammation

Aldosterone signaling through transient receptor potential melastatin 7 cation channel (TRPM7) and its α-kinase domain

We demonstrated a role for the Mg2 + transporter TRPM7, a bifunctional protein with channel and α-kinase domains, in aldosterone signaling. Molecular mechanisms underlying this are elusive. Here we investigated the function of TRPM7 and its α-kinase domain on Mg2 + and pro-inflammatory signaling by aldosterone. Kidney cells (HEK-293) expressing wild-type human TRPM7 (WThTRPM7) or constructs in which the α-kinase domain was deleted (ΔKinase) or rendered inactive with a point mutation in the ATP binding site of the α-kinase domain (K1648R) were studied. Aldosterone rapidly increased [Mg2 +]i and stimulated NADPH oxidase-derived generation of reactive oxygen species (ROS) in WT hTRPM7 and TRPM7 kinase dead mutant cells. Translocation of annexin-1 and calpain-II and spectrin cleavage (calpain target) were increased by aldosterone in WT hTRPM7 cells but not in α-kinase-deficient cells. Aldosterone stimulated phosphorylation of MAP kinases and increased expression of pro-inflammatory mediators ICAM-1, Cox-2 and PAI-1 in Δkinase and K1648R cells, effects that were inhibited by eplerenone (mineralocorticoid receptor (MR) blocker). 2-APB, a TRPM7 channel inhibitor, abrogated aldosterone-induced Mg2 + responses in WT hTRPM7 and mutant cells. In 2-APB-treated ΔKinase and K1648R cells, aldosterone-stimulated inflammatory responses were unchanged. These data indicate that aldosterone stimulates Mg2 + influx and ROS production in a TRPM7-sensitive, kinase-insensitive manner, whereas activation of annexin-1 requires the TRPM7 kinase domain. Moreover TRPM7 α-kinase modulates inflammatory signaling by aldosterone in a TRPM7 channel/Mg2 +-independent manner. Our findings identify novel mechanisms for non-genomic actions of aldosterone involving differential signaling through MR-activated TRPM7 channel and α-kinase

Renoprotective effects of atorvastatin in diabetic mice: downregulation of RhoA and upregulation of Akt/GSK3

Potential benefits of statins in the treatment of chronic kidney disease beyond lipid-lowering effects have been described. However, molecular mechanisms involved in renoprotective actions of statins have not been fully elucidated. We questioned whether statins influence development of diabetic nephropathy through reactive oxygen species, RhoA and Akt/GSK3 pathway, known to be important in renal pathology. Diabetic mice (db/db) and their control counterparts (db/+) were treated with atorvastatin (10 mg/Kg/day, p.o., for 2 weeks). Diabetes-associated renal injury was characterized by albuminuria (albumin:creatinine ratio, db/+: 3.2 ± 0.6 vs. db/db: 12.5 ± 3.1*; *P<0.05), increased glomerular/mesangial surface area, and kidney hypertrophy. Renal injury was attenuated in atorvastatin-treated db/db mice. Increased ROS generation in the renal cortex of db/db mice was also inhibited by atorvastatin. ERK1/2 phosphorylation was increased in the renal cortex of db/db mice. Increased renal expression of Nox4 and proliferating cell nuclear antigen, observed in db/db mice, were abrogated by statin treatment. Atorvastatin also upregulated Akt/GSK3β phosphorylation in the renal cortex of db/db mice. Our findings suggest that atorvastatin attenuates diabetes-associated renal injury by reducing ROS generation, RhoA activity and normalizing Akt/GSK3β signaling pathways. The present study provides some new insights into molecular mechanisms whereby statins may protect against renal injury in diabetes

Downregulation of C-terminal Src kinase (csk) and Csk-binding protein (cbp) is associated with increased aldosterone-induced C-src phosphorylation in Shr vascular smooth muscle cells

c-Src phosphorylation is controlled by the recruitment of enzyme regulators, such as C-terminal Src kinase (CSK) which inhibits Src activity, and interactions with transmembrane adaptors. These complex regulatory mechanisms coordinate activity of c-Src at multiple levels. We previously showed that in aldosterone-stimulated SHR vascular smooth muscle cells (VSMCs), c-Src phosphorylation and its downstream signaling are upregulated. Here we hypothesized that mechanisms underlying vascular c-Src hyperactivation in SHR are related to dysregulated CSK and altered autophosphorylation at Tyr416 and Tyr527 in aldosterone-stimulated SHR cells. Studies were performed in cultured VSMCs from WKY and SHR. C-terminal Src kinase (Csk) cytosol/membrane translocation, Csk-binding protein (CBP), and c-Src phosphorylation were evaluated by western blot. Cholesterol-enriched fractions were obtained by sucrose-gradient centrifugation. Aldosterone (100 nM) induced Tyr527 c-Src phosphorylation (153.5 ± 13.6 %) which locks the kinase in an inactive conformation. This was blunted in SHR cells. Csk is a cytosolic kinase that catalyzes c-Src Tyr527 phosphorylation. ASN (10 uM), a Csk inhibitor, prevented the kinase translocation to the membrane and inhibited Tyr527 c-Src phosphorylation induced by aldosterone in WKY cells. Inhibition of Csk induced an increase in Tyr416 c-Src (180 ± 21%) under basal conditions. In SHR cells, Csk translocation to the membrane was reduced by aldosterone compared with WKY cells. Aldosterone induced an increase in expression (180.3 ± 39 %) and phosphorylation (169 ± 23 %) of the adaptor protein CBP in WKY, but not in SHR cells. Aldosterone stimulation increased Csk trafficking into lipid rafts/caveolae in WKY cells, without affecting the kinase content in the cholesterol-enriched fractions from SHR. Our findings demonstrate that 1) key regulators of c-Src activation by aldosterone, specifically Csk and CBP, are altered in SHR VSMCs; 2) c-Src regulation by aldosterone involves lipid rafts/caveolae. These novel findings suggest that modulation of Csk could be an important strategy to blunt c-Src-dependent aldosterone vascular effects

Vitamin C prevents the endothelial dysfunction induced by acute ethanol intake

Aims: Investigate the effect of ascorbic acid (vitamin C) on the endothelial dysfunction induced by acute ethanol intake. Main methods: Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. Key findings: Ethanol intake decreased the endothelium-dependent relaxation induced by acetylcholine in the rat aorta and treatment with vitamin C (250 mg/kg; p.o. gavage, 5 days) prevented this response. Ethanol increased superoxide anion (O2−) generation and decreased aortic nitrate/nitrite levels and these responses were not prevented by vitamin C. Superoxide dismutase (SOD) and catalase (CAT) activities as well as hydrogen peroxide (H2O2) and reduced glutathione (GSH) levels were not affected by ethanol. RhoA translocation as well as the phosphorylation levels of protein kinase B (Akt), eNOS (Ser1177 or Thr495 residues), p38MAPK, SAPK/JNK and ERK1/2 was not affected by ethanol intake. Vitamin C increased SOD activity and phosphorylation of Akt, eNOS (Ser1177 residue) and p38MAPK in aortas from both control and ethanol-treated rats. Incubation of aortas with tempol prevented ethanol-induced decrease in the relaxation induced by acetylcholine. Ethanol (50 mM/1 min) increased O2− generation in cultured aortic vascular smooth muscle cells (VSMC) and vitamin C did not prevent this response. In endothelial cells, vitamin C prevented the increase on ROS generation and the decrease in the cytosolic NO content induced by ethanol. Significance: Our study provides novel evidence that vitamin C prevents the endothelial dysfunction induced by acute ethanol intake by a mechanism that involves reduced ROS generation and increased NO availability in endothelial cells

Upregulation of the mineralocorticoid system in obesity-associated diabetes leads to increased adipocyte Rho kinase activation and dysregulation of adipocytokines

Increased activation of the renin-angiotensin-aldosterone (RAAS) system is often associated with obesity and metabolic syndrome. We previously reported that aldosterone (aldo) produced by adipocytes regulate vascular function. Despite the fact that aldo production by adipocytes is increased in obesity, the role of aldo and its receptor, the mineralocorticoid receptor (MR), in the regulation of adipocytes biology and their downstream signaling remain elusive. Since Rho kinase (Rock) signaling has recently been implicated in the development of obesity, we tested the hypothesis that MR upregulation leads to dysregulation of adipokines through Rock-dependent mechanisms in obesity. Here we used obese db/db and lean db/+ mice, treated for 4 weeks with K canrenoate (MR antagonist, 30 mg/kg/day) and fasudil (Rock inhibitor, 30 mg/kg/day). Aldo production and Rock activation were measured by ELISA. mRNA and protein levels of adipokines and Rock signaling were assayed by real time PCR and immunoblotting. Plasma and adipocyte-derived aldo levels were increased in db/db mice (plasma: pg/mL, db/+ 310±33, db/db 567±43, p<0.05; adipocytes: pg/mL/μgRNA, db/+ 329±130, db/db 3125±494, p<0.002), an effect partially prevented by MR blockade (pg/mL/μgRNA: db/db 1278±176, p<0.01) and not by fasudil. Aldosterone synthase mRNA levels were increased (2.3 fold) as well as Nr3c2 (MR) (1.8 fold) and markers of MR activation (Sgk1 and Ngal- 2.3 and 2.9 fold) in mature adipocytes from db/db mice. In mature adipocytes from db/db, adiponectin mRNA levels were decreased (2.6 fold; p<0.01), whereas leptin and IL-6 mRNA levels were increased (2 and 4.8 fold; p<0.01). All changes were blocked by K canrenoate. Rock activity and downstream effectors, such as activation of MYPT1 and ERM, were increased in perivascular adipose tissue from db/db mice, an effect prevented by MR blockade. In conclusion, our data demonstrate that in db/db mice adipocyte MR-dependent activation of Rock is associated with a pro-inflammatory adipose phenotype that is normalized by MR blockade. Our results implicate a potential role of adipocyte aldo/MR through RhoA/Rock in adipocyte dysfunction in obesity/diabetes, important co-morbidities often associated with metabolic syndrome and hypertension

Atorvastatin Inhibits Src/ROS-mediated Redox Sensitive And Pro-Inflammatory Actions of Aldosterone in Vascular Smooth Muscle Cells: Focus on Statins Pleiotropic Effects

Statins, described as cholesterol-lowering drugs, are now recognized to induce effects throught pleiotropic actions, including oxidative stress reduction and protein geranylgeranylation inhibition. Mechanisms whereby aldosterone associates with c-Src and related redox signaling molecules involve lipid rafts. This study aims to identify whether c-Src/NADPH oxidases (Nox) pathway is a statin-sensitive target independent of membrane cholesterol depletion mechanisms. In order to discriminate statin pleiotropic effects from its classic cholesterol synthesis inhibition, VSMCs from WKY were treated with 1 uM atorvastatin for 60 min or 72h prior 100 nM aldosterone stimulations respectively. Aldosterone-induced c-Src phosphorylation (% vehicle, 267 ± 35) was inhibited by long (150 ± 23 %) and short (114 ± 33 %) term atorvastatin treatment. To restore cholesterol synthesis, cells were incubated with its intermediate, mevalonate (100 μM). Mevalonate reload restored c-Src phosphorylation (287 ± 27 %) induced by aldosterone in atorvastatin long term-treated VSMCs. Geranylgeranyl-pyrophosphate (GGPP) was used to prevent protein geranylgeranylation inhibition. GGPP reposition recovered the effects of aldosterone on c-Src phosphorylation in both short (268 ± 22 %) and long (203 ± 35 %) term VSMCs treated with atorvastatin. Aldosterone-induced increase of Nox1, 2 and 4 expression and the associated ROS-generation (215 ± 38 %) were inhibited by long and short term atorvastatin incubation. Aldosterone-induced RAC1/2 and p47phox translocation (cytosol to membrane) was prevented by atorvastatin treatments. Aldosterone stimulation increased Nox1 and p47phox content in cholesterol-enriched fractions, an effect inhibited by atorvastatin short term treatment. Atorvastatin also prevented the increase of redox-signaling phosphorylation (ERK1/2, p38 and JNK) and pro-inflammatory markers (VCAM-1 expression, NFkB p65 phosphorylation) by aldosterone. We demonstrate that atorvastatin influences c-Src/Nox-mediated effects of aldosterone involving lipid rafts through classic and pleiotropic actions independent of cholesterol depletion. This study identifies a novel mechanism for statins in aldosterone-associated vascular injury