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 (10-7 mol/L) 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.

Vascular dysfunction in obese diabetic db/db mice involves the interplay between aldosterone/mineralocorticoid receptor and Rho kinase signaling.

Activation of aldosterone/mineralocorticoid receptors (MR) has been implicated in vascular dysfunction of diabetes. Underlying mechanisms are elusive. Therefore, we investigated the role of Rho kinase (ROCK) in aldosterone/MR signaling and vascular dysfunction in a model of diabetes. Diabetic obese mice (db/db) and control counterparts (db/+) were treated with MR antagonist (MRA, potassium canrenoate, 30 mg/kg/day, 4 weeks) or ROCK inhibitor, fasudil (30 mg/kg/day, 3 weeks). Plasma aldosterone was increased in db/db versus db/+. This was associated with enhanced vascular MR signaling. Norepinephrine (NE)-induced contraction was increased in arteries from db/db mice. These responses were attenuated in mice treated with canrenoate or fasudil. Db/db mice displayed hypertrophic remodeling and increased arterial stiffness, improved by MR blockade. Vascular calcium sensitivity was similar between depolarized arteries from db/+ and db/db. Vascular hypercontractility in db/db mice was associated with increased myosin light chain phosphorylation and reduced expression of PKG-1α. Vascular RhoA/ROCK signaling and expression of pro-inflammatory and pro-fibrotic markers were exaggerated in db/db mice, effects that were attenuated by MRA. Fasudil, but not MRA, improved vascular insulin sensitivity in db/db mice, evidenced by normalization of Irs1 phosphorylation. Our data identify novel pathways involving MR-RhoA/ROCK-PKG-1 that underlie vascular dysfunction and injury in diabetic mice.

c-Src Inhibition Improves Cardiovascular Function but not Remodeling or Fibrosis in Angiotensin 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.

Differential renal effects of candesartan at high and ultra-high doses in diabetic mice-potential role of the ACE2/AT2R/Mas axis.

High doses of Ang II receptor (AT1R) blockers (ARBs) are renoprotective in diabetes. Underlying mechanisms remain unclear. We evaluated whether high/ultra-high doses of candesartan (ARB) up-regulate angiotensin-converting enzyme 2 (ACE2)/Ang II type 2 receptor (AT2R)/Mas receptor [protective axis of the of the renin-angiotensin system (RAS)] in diabetic mice. Systolic blood pressure (SBP), albuminuria and expression/activity of RAS components were assessed in diabetic db/db and control db/+ mice treated with increasing candesartan doses (intermediate, 1 mg/kg/d; high, 5 mg/kg/d; ultra-high, 25 and 75 mg/kg/d; 4 weeks). Lower doses candesartan did not influence SBP, but ultra-high doses reduced SBP in both groups. Plasma glucose and albuminuria were increased in db/db compared with db/+ mice. In diabetic mice treated with intermediate dose candesartan, renal tubular damage and albuminuria were ameliorated and expression of ACE2, AT2R and Mas and activity of ACE2 were increased, effects associated with reduced ERK1/2 phosphorylation, decreased fibrosis and renal protection. Ultra-high doses did not influence the ACE2/AT2R/Mas axis and promoted renal injury with increased renal ERK1/2 activation and exaggerated fibronectin expression in db/db mice. Our study demonstrates dose-related effects of candesartan in diabetic nephropathy: intermediate-high dose candesartan is renoprotective, whereas ultra-high dose candesartan induces renal damage. Molecular processes associated with these effects involve differential modulation of the ACE2/AT2R/Mas axis: intermediate-high dose candesartan up-regulating RAS protective components and attenuating pro-fibrotic processes, and ultra-high doses having opposite effects. These findings suggest novel mechanisms through the protective RAS axis, whereby candesartan may ameliorate diabetic nephropathy. Our findings also highlight potential injurious renal effects of ultra-high dose candesartan in diabetes.

Adipocyte-Specific Mineralocorticoid Receptor Overexpression in Mice Is Associated With Metabolic Syndrome and Vascular Dysfunction: Role of Redox-Sensitive PKG-1 and Rho Kinase.

Mineralocorticoid receptor (MR) expression is increased in adipose tissue from obese individuals and animals. We previously demonstrated that adipocyte-MR overexpression (Adipo-MROE) in mice is associated with metabolic changes. Whether adipocyte MR directly influences vascular function in these mice is unknown. We tested this hypothesis in resistant mesenteric arteries from Adipo-MROE mice using myography and in cultured adipocytes. Molecular mechanisms were probed in vessels/vascular smooth muscle cells and adipose tissue/adipocytes and focused on redox-sensitive pathways, Rho kinase activity, and protein kinase G type-1 (PKG-1) signaling. Adipo-MROE versus control-MR mice exhibited reduced vascular contractility, associated with increased generation of adipocyte-derived hydrogen peroxide, activation of vascular redox-sensitive PKG-1, and downregulation of Rho kinase activity. Associated with these vascular changes was increased elastin content in Adipo-MROE. Inhibition of PKG-1 with Rp-8-Br-PET-cGMPS normalized vascular contractility in Adipo-MROE. In the presence of adipocyte-conditioned culture medium, anticontractile effects of the adipose tissue were lost in Adipo-MROE mice but not in control-MR mice. In conclusion, adipocyte-MR upregulation leads to impaired contractility with preserved endothelial function and normal blood pressure. Increased elasticity may contribute to hypocontractility. We also identify functional cross talk between adipocyte MR and arteries and describe novel mechanisms involving redox-sensitive PKG-1 and Rho kinase. Our results suggest that adipose tissue from Adipo-MROE secrete vasoactive factors that preferentially influence vascular smooth muscle cells rather than endothelial cells. Our findings may be important in obesity/adiposity where adipocyte-MR expression/signaling is amplified and vascular risk increased.

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 (Mg(2+))/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 Mg(2+) 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.

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 (1g/kg; p.o. gavage) effects were assessed within 30min 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 (250mg/kg; p.o. gavage, 5days) 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 (Ser(1177) or Thr(495) residues), p38MAPK, SAPK/JNK and ERK1/2 was not affected by ethanol intake. Vitamin C increased SOD activity and phosphorylation of Akt, eNOS (Ser(1177) 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 (50mM/1min) 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.

Vascular injury in diabetic db/db mice is ameliorated by atorvastatin: role of Rac1/2-sensitive Nox-dependent pathways.

Oxidative stress [increased bioavailability of reactive oxygen species (ROS)] plays a role in the endothelial dysfunction and vascular inflammation, which underlie vascular damage in diabetes. Statins are cholesterol-lowering drugs that are vasoprotective in diabetes through unknown mechanisms. We tested the hypothesis that atorvastatin decreases NADPH oxidase (Nox)-derived ROS generation and associated vascular injury in diabetes. Lepr(db)/Lepr(db) (db/db) mice, a model of Type 2 diabetes and control Lepr(db)/Lepr(+) (db/+) mice were administered atorvastatin (10 mg/kg per day, 2 weeks). Atorvastatin improved glucose tolerance in db/db mice. Systemic and vascular oxidative stress in db/db mice, characterized by increased plasma TBARS (thiobarbituric acid-reactive substances) levels and exaggerated vascular Nox-derived ROS generation respectively, were inhibited by atorvastatin. Cytosol-to-membrane translocation of the Nox regulatory subunit p47(phox) and the small GTPase Rac1/2 was increased in vessels from db/db mice compared with db/+ mice, an effect blunted by atorvastatin. The increase in vascular Nox1/2/4 expression and increased phosphorylation of redox-sensitive mitogen-activated protein kinases (MAPKs) was abrogated by atorvastatin in db/db mice. Pro-inflammatory signalling (decreased IκB-α and increased NF-κB p50 expression, increased NF-κB p65 phosphorylation) and associated vascular inflammation [vascular cell adhesion molecule-1 (VCAM-1) expression and vascular monocyte adhesion], which were increased in aortas of db/db mice, were blunted by atorvastatin. Impaired acetylcholine (Ach)- and insulin (INS)-induced vasorelaxation in db/db mice was normalized by atorvastatin. Our results demonstrate that, in diabetic mice, atorvastatin decreases vascular oxidative stress and inflammation and ameliorates vascular injury through processes involving decreased activation of Rac1/2 and Nox. These findings elucidate redox-sensitive and Rac1/2-dependent mechanisms whereby statins protect against vascular injury in diabetes.

Acute ethanol intake induces mitogen-activated protein kinase activation, platelet-derived growth factor receptor phosphorylation, and oxidative stress in resistance arteries

In the present study, we investigated the role of angiotensin type I (AT1) receptor in reactive oxygen species (ROS) generation and mitogen-activated protein kinases (MAPK) activation induced by acute ethanol intake in resistance arteries. We also evaluated the effect of ethanol on platelet-derived growth factor receptors (PDGF-R) phosphorylation and the role of this receptor on ROS generation by ethanol. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. Acute ethanol intake did not alter angiotensin I or angiotensin II levels in the rat mesenteric arterial bed (MAB). Ethanol induced vascular oxidative stress, and this response was not prevented by losartan (10 mg/kg; p.o. gavage), a selective AT1 receptor antagonist. MAB from ethanol-treated rats displayed increased SAPK/JNK and PDGF-R phosphorylation, responses that were not prevented by losartan. The phosphorylation levels of protein kinase B (Akt) and eNOS were not affected by acute ethanol intake. MAB nitrate levels and the reactivity of this tissue to acetylcholine, phenylephrine, and sodium nitroprusside were not affected by ethanol intake. Ethanol did not alter plasma antioxidant capacity, the levels of reduced glutathione, or the activities of superoxide dismutase and catalase in the rat MAB. Short-term effects of ethanol (50 mmol/l) were evaluated in vascular smooth muscle cells (VSMC) isolated from rat MAB. Ethanol increased ROS generation, and this response was not affected by AG1296, a PDGF-R inhibitor, or losartan. Finally, ethanol did not alter MAPK or PDGF-R phosphorylation in cultured VSMC. Our study provides novel evidence that acute ethanol intake induces ROS generation, PDGF-R phosphorylation, and MAPK activation through AT(1)-independent mechanisms in resistance arteries in vivo. MAPK and PDGF-R play a role in vascular signaling and cardiovascular diseases and may contribute to the vascular pathobiology of ethanol

Acute ethanol intake induces mitogen-activated protein kinase activation, platelet-derived growth factor receptor phosphorylation, and oxidative stress in resistance arteries.

In the present study, we investigated the role of angiotensin type I (AT1) receptor in reactive oxygen species (ROS) generation and mitogen-activated protein kinases (MAPK) activation induced by acute ethanol intake in resistance arteries. We also evaluated the effect of ethanol on platelet-derived growth factor receptors (PDGF-R) phosphorylation and the role of this receptor on ROS generation by ethanol. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. Acute ethanol intake did not alter angiotensin I or angiotensin II levels in the rat mesenteric arterial bed (MAB). Ethanol induced vascular oxidative stress, and this response was not prevented by losartan (10 mg/kg; p.o. gavage), a selective AT1 receptor antagonist. MAB from ethanol-treated rats displayed increased SAPK/JNK and PDGF-R phosphorylation, responses that were not prevented by losartan. The phosphorylation levels of protein kinase B (Akt) and eNOS were not affected by acute ethanol intake. MAB nitrate levels and the reactivity of this tissue to acetylcholine, phenylephrine, and sodium nitroprusside were not affected by ethanol intake. Ethanol did not alter plasma antioxidant capacity, the levels of reduced glutathione, or the activities of superoxide dismutase and catalase in the rat MAB. Short-term effects of ethanol (50 mmol/l) were evaluated in vascular smooth muscle cells (VSMC) isolated from rat MAB. Ethanol increased ROS generation, and this response was not affected by AG1296, a PDGF-R inhibitor, or losartan. Finally, ethanol did not alter MAPK or PDGF-R phosphorylation in cultured VSMC. Our study provides novel evidence that acute ethanol intake induces ROS generation, PDGF-R phosphorylation, and MAPK activation through AT(1)-independent mechanisms in resistance arteries in vivo. MAPK and PDGF-R play a role in vascular signaling and cardiovascular diseases and may contribute to the vascular pathobiology of ethanol.

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

We demonstrated a role for the Mg(2+) 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 Mg(2+) 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 [Mg(2+)]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 Mg(2+) 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 Mg(2+) 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/Mg(2+)-independent manner. Our findings identify novel mechanisms for non-genomic actions of aldosterone involving differential signaling through MR-activated TRPM7 channel and α-kinase.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: a role for angiotensin type 1 receptor.

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT1 receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT1-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage.

Testosterone induces vascular smooth muscle cell migration by NADPH oxidase and c-Src-dependent pathways.

Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10(-7) mol/L, 0-120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase-derived ROS and c-Src-dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs.

Adipocytes produce aldosterone through calcineurin-dependent signaling pathways: implications in diabetes mellitus-associated obesity and vascular dysfunction.

We reported aldosterone as a novel adipocyte-derived factor that regulates vascular function. We aimed to investigate molecular mechanisms, signaling pathways, and functional significance of adipocyte-derived aldosterone and to test whether adipocyte-derived aldosterone is increased in diabetes mellitus-associated obesity, which contributes to vascular dysfunction. Studies were performed in the 3T3-L1 adipocyte cell line and mature adipocytes isolated from human and mouse (C57BL/6J) adipose tissue. Mesenteric arteries with and without perivascular fat and mature adipocytes were obtained from obese diabetic db/db and control db/+ mice. Aldosterone synthase (CYP11B2; mRNA and protein) was detected in 3T3-L1 and mature adipocytes, which secrete aldosterone basally and in response to angiotensin II (Ang II). In 3T3-L1 adipocytes, Ang II stimulation increased aldosterone secretion and CYP11B2 expression. Ang II effects were blunted by an Ang II type 1 receptor antagonist (candesartan) and inhibitors of calcineurin (cyclosporine A and FK506) and nuclear factor of activated T-cells (VIVIT). FAD286 (aldosterone synthase inhibitor) blunted adipocyte differentiation. In candesartan-treated db/db mice (1 mg/kg per day, 4 weeks) increased plasma aldosterone, CYP11B2 expression, and aldosterone secretion were reduced. Acetylcholine-induced relaxation in db/db mesenteric arteries containing perivascular fat was improved by eplerenone (mineralocorticoid receptor antagonist) without effect in db/+ mice. Adipocytes possess aldosterone synthase and produce aldosterone in an Ang II/Ang II type 1 receptor/calcineurin/nuclear factor of activated T-cells-dependent manner. Functionally adipocyte-derived aldosterone regulates adipocyte differentiation and vascular function in an autocrine and paracrine manner, respectively. These novel findings identify adipocytes as a putative link between aldosterone and vascular dysfunction in diabetes mellitus-associated obesity.

Ethanol induces vascular relaxation via redox-sensitive and nitric oxide-dependent pathways.

We investigated the role of reactive oxygen species (ROS) and nitric oxide (NO) in ethanol-induced relaxation. Vascular reactivity experiments showed that ethanol (0.03-200 mmol/L) induced relaxation in endothelium-intact and denuded rat aortic rings isolated from male Wistar rats. Pre-incubation of intact or denuded rings with l-NAME (non selective NOS inhibitor, 100 µmol/L), 7-nitroindazole (selective nNOS inhibitor, 100 µmol/L), ODQ (selective inhibitor of guanylyl cyclase enzyme, 1 µmol/L), glibenclamide (selective blocker of ATP-sensitive K(+) channels, 3 µmol/L) and 4-aminopyridine (selective blocker of voltage-dependent K(+) channels, 4-AP, 1 mmol/L) reduced ethanol-induced relaxation. Similarly, tiron (superoxide anion (O(2)(-)) scavenger, 1 mmol/L) and catalase (hydrogen peroxide (H(2)O(2)) scavenger, 300 U/mL) reduced ethanol-induced relaxation to a similar extent in both endothelium-intact and denuded rings. Finally, prodifen (non-selective cytochrome P450 enzymes inhibitor, 10 µmol/L) and 4-methylpyrazole (selective alcohol dehydrogenase inhibitor, 10 µmol/L) reduced ethanol-induced relaxation. In cultured aortic vascular smooth muscle cells (VSMCs), ethanol stimulated generation of NO, which was significantly inhibited by l-NAME. In endothelial cells, flow cytometry studies showed that ethanol increased cytosolic Ca(2+) concentration ([Ca(2+)]c), O(2)(-) and cytosolic NO concentration ([NO]c). Tiron inhibited ethanol-induced increase in [Ca(2+)]c and [NO]c. The major new finding of this work is that ethanol induces relaxation via redox-sensitive and NO-cGMP-dependent pathways through direct effects on ROS production and NO signaling. These findings identify putative molecular mechanisms whereby ethanol, at pharmacological concentrations, influences vascular reactivity.

Adipocyte-derived factors regulate vascular smooth muscle cells through mineralocorticoid and glucocorticoid receptors.

Adipose tissue influences vascular function through adipocyte-derived factors, including components of the renin-angiotensin-aldosterone system. Molecular mechanisms underlying these phenomena are elusive. We investigated the role of adipocyte-derived factors on mitogen-activated protein kinases (MAPKs), proinflammatory status, apoptosis, and mitogenic signaling in vascular smooth muscle cells (VSMCs) and questioned whether these effects involve mineralocorticoid receptor (MR), glucocorticoid receptor (GR), and angiotensin II type 1 receptor (AT(1)R). Cultured mouse VSMCs were exposed to adipocyte-conditioned medium (ACM) from differentiated 3T3-L1 adipocytes. ACM induced phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase, p38MAPK, and extracellular signal-regulated kinase 1/2 and increased expression of proinflammatory and proliferative markers in VSMCs. Eplerenone (MR antagonist), mifepristone (GR antagonist), and candesartan (AT(1)R antagonist) inhibited ACM-induced effects on extracellular signal-regulated kinase 1/2, p38MAPK, and proliferating cell nuclear antigen, without influencing apoptosis (Bax, Bcl, and caspase 3). Stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation was inhibited by mifepristone and candesartan but not by eplerenone. ACM-induced increase of fibronectin, vascular cell adhesion molecule 1, and plasminogen activator inhibitor 1 expression was blocked by MR and AT(1)R antagonism but not by GR inhibition. ACM has no effect on GR, MR, and AT(1)R expression. Our data show that adipocyte-derived factors influence MAPK signaling, leading to VSMC proinflammatory and profibrotic responses through distinct pathways. Although ACM stimulates p38MAPK and extracellular signal-regulated kinase 1/2 phosphorylation through MR, GR, and AT(1)R, activation of stress-activated protein kinase/c-Jun N-terminal kinase involves GR and AT(1)R. These findings suggest that adipocyte-derived factors regulate VSMC function through specific MAPKs linked to MR, GR, and AT(1)R, a posttranslational phenomenon, because ACM did not influence receptor expression. Such cross-talk between adipocytes and VSMCs may provide a potential molecular mechanism linking renin-angiotensin-aldosterone system, adipocytes, and vascular function.

Dysregulation of renal transient receptor potential melastatin 6/7 but not paracellin-1 in aldosterone-induced hypertension and kidney damage in a model of hereditary hypomagnesemia.

RATIONALE: Hyperaldosteronism, important in hypertension, is associated with electrolyte alterations, including hypomagnesemia, through unknown mechanisms. OBJECTIVE: To test whether aldosterone influences renal Mg(2+) transporters, (transient receptor potential melastatin (TRPM) 6, TRPM7, paracellin-1) leading to hypomagnesemia, hypertension and target organ damage and whether in a background of magnesium deficiency, this is exaggerated. METHODS AND RESULTS: Aldosterone effects in mice selectively bred for high-normal (MgH) or low (MgL) intracellular Mg(2+) were studied. Male MgH and MgL mice received aldosterone (350 µg/kg per day, 3 weeks). SBP was elevated in MgL. Aldosterone increased blood pressure and albuminuria and increased urinary Mg(2+) concentration in MgH and MgL, with greater effects in MgL. Activity of renal TRPM6 and TRPM7 was lower in vehicle-treated MgL than MgH. Aldosterone increased activity of TRPM6 in MgH and inhibited activity in MgL. TRPM7 and paracellin-1 were unaffected by aldosterone. Aldosterone-induced albuminuria in MgL was associated with increased renal fibrosis, increased oxidative stress, activation of mitogen-activated protein kinases and nuclear factor-NF-κB and podocyte injury. Mg(2+) supplementation (0.75% Mg(2+)) in aldosterone-treated MgL normalized plasma Mg(2+), increased TRPM6 activity and ameliorated hypertension and renal injury. Hence, in a model of inherited hypomagnesemia, TRPM6 and TRPM7, but not paracellin-1, are downregulated. Aldosterone further decreased TRPM6 activity in hypomagnesemic mice, a phenomenon associated with hypertension and kidney damage. Such effects were prevented by Mg(2+) supplementation. CONCLUSION: Amplified target organ damage in aldosterone-induced hypertension in hypomagnesemic conditions is associated with dysfunctional Mg(2+)-sensitive renal TRPM6 channels. Novel mechanisms for renal effects of aldosterone and insights into putative beneficial actions of Mg(2+), particularly in hyperaldosteronism, are identified.

Vascular proinflammatory responses by aldosterone are mediated via c-Src trafficking to cholesterol-rich microdomains: role of PDGFR.

AIMS: We demonstrated c-Src activation as a novel non-genomic signalling pathway for aldosterone in vascular smooth muscle cells (VSMCs). Here, we investigated molecular mechanisms and biological responses of this phenomenon, focusing on the role of lipid rafts/caveolae and platelet-derived growth factor receptor (PDGFR) in c-Src-regulated proinflammatory responses by aldosterone. METHODS AND RESULTS: Studies were performed in cultured VSMCs from Wistar-Kyoto (WKY) rats and caveolin-1 knockout (Cav 1(-/-)) and wild-type mice. Aldosterone stimulation increased c-Src phosphorylation and trafficking to lipid rafts/caveolae. Cholesterol depletion with methyl-ß-cyclodextrin abrogated aldosterone-induced phosphorylation of c-Src and its target, Pyk2. Aldosterone effects were recovered by cholesterol reload. Aldosterone-induced c-Src and cortactin phosphorylation was reduced in caveolin-1-silenced and Cav 1(-/-) VSMCs. PDGFR is phosphorylated by aldosterone within cholesterol-rich fractions of VSMCs. AG1296, a PDGFR inhibitor, prevented c-Src phosphorylation and translocation to cholesterol-rich fractions. Aldosterone induced an increase in adhesion molecule protein content and promoted monocyte adhesion to VSMCs, responses that were inhibited an by cholesterol depletion, caveolin-1 deficiency, AG1296 and PP2, a c-Src inhibitor. Mineralocorticoid receptor (MR) content in flotillin-2-rich fractions and co-immunoprecipitation with c-Src and PDGFR increased upon aldosterone stimulation, indicating MR-lipid raft/signalling association. CONCLUSION: We demonstrate that aldosterone-mediated c-Src trafficking/activation and proinflammatory signalling involve lipid rafts/caveolae via PDGFR.

Sphingosine-1-phosphate-induced inflammation involves receptor tyrosine kinase transactivation in vascular cells: upregulation in hypertension.

Sphingosine-1-phosphate (S1P), a multifunctional phospholipid, regulates vascular cell function. Whether S1P influences vascular inflammatory responses, particularly in hypertension, is unclear. We tested the hypothesis that S1P is a proinflammatory mediator signaling through receptor tyrosine kinase transactivation and that responses are amplified in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats (SHRSPs), a model in which we demonstrated Edg1 (S1P1 receptor) to be a candidate gene for salt-sensitive hypertension. Vascular smooth muscle cell from Wistar-Kyoto rats and SHRSPs were studied. S1P receptor subtypes, S1P1 and S1P2, were similarly expressed in Wistar-Kyoto rats and SHRSPs. S1P induced phosphorylation of epidermal growth factor receptor and platelet-derived growth factor and activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, with amplified effects in SHRSPs versus Wistar-Kyoto rats. Inhibition of epidermal growth factor receptor and platelet-derived growth factor (with AG1478 and AG1296, respectively) abolished S1P-induced phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase in Wistar-Kyoto rats with variable effects in SHRSPs. Vascular smooth muscle cell inflammation was evaluated by expression of adhesion molecules and functional responses assessed by monocyte adhesion. S1P stimulated expression of intercellular adhesion molecule 1 and vascular cell adhesion protein 1 and promoted monocyte adhesion, particularly in SHRSP cells. S1P-mediated inflammation was blunted by AG1478 and AG1296 in SHRSP cells. VPC23019, a S1P1 receptor antagonist, inhibited S1P-induced mitogen-activated protein kinase phosphorylation, intercellular adhesion molecule 1 and vascular cell adhesion protein 1 expression, and monocyte adhesion. Our data indicate that molecular processes underlying vascular inflammation and cell adhesion in SHRSPs involve S1P/S1P1 receptors and phosphorylation of receptor tyrosine kinases. We identify a novel pathway linking S1P/S1P1 receptors to specific proinflammatory signaling pathways through epidermal growth factor receptor and platelet-derived growth factor transactivation, a process that is upregulated in SHRSPs. Such molecular events may contribute to vascular inflammation in hypertension.

Differential regulation of Nox1, Nox2 and Nox4 in vascular smooth muscle cells from WKY and SHR.

The functional significance and regulation of NAD(P)H oxidase (Nox) isoforms by angiotensin II (Ang II) and endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs) from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) was studied. Expression of Nox1, Nox2, and Nox4 (gene and protein) and NAD(P)H oxidase activity were increased in SHR. Basal NAD(P)H oxidase activity was blocked by GKT136901 (Nox1/4 inhibitor) and by Nox1 siRNA in WKY cells and by siNOX1 and siNOX2 in SHR. Whereas Ang II increased expression of all Noxes in WKY, only Nox1 was influenced in SHR. Ang II-induced NAD(P)H activity was inhibited by siNOX1 in WKY and by siNOX1 and siNOX2 in SHR. ET-1 upregulated Nox expression only in WKY and increased NAD(P)H oxidase activity, an effect inhibited by siNOX1 and siNOX2. Nox1 co-localized with Nox2 but not with Nox4, implicating association between Nox1 and Nox2 but not between Nox1 and Nox4. These data highlight the complexity of Nox biology in VSMCs, emphasising that more than one Nox member, alone or in association, may be involved in NAD(P)H oxidase-mediated •O(2)(-) production. Nox1 regulation by Ang II, but not by ET-1, may be important in •O(2)(-) formation in VSMCs from SHR.