Alvaro Yogi

Aldosterone Activates Vascular p38MAP Kinase and NADPH Oxidase Via c-Src

Increasing evidence indicates that aldosterone elicits vascular effects through nongenomic signaling pathways. We tested the hypothesis that aldosterone induces activation of vascular mitogen-activated protein (MAP) kinases and NADPH oxidase via c-Src–dependent mechanisms in vascular smooth muscle cells (VSMCs). Aldosterone effects on activation of c-Src, p38MAP kinase, and NADPH oxidase, and incorporation of [3H]proline, an index of collagen synthesis, were assessed in cultured rat VSMCs. Studies were performed in the absence and presence of eplerenone, a selective mineralocorticoid receptor blocker, PP2, a selective Src inhibitor, and SB212190, a selective p38MAPK inhibitor. Phosphorylation of c-Src was dose-dependently increased by aldosterone, with maximal responses obtained at 10−7 mol/L. Aldosterone increased p38MAP kinase phosphorylation, NAD(P)H oxidase activation, and [3H]proline incorporation. These responses were abrogated by eplerenone and almost abolished by PP2. Aldosterone-stimulated incorporation of [3H]proline was significantly reduced by SB212190, indicating that p38MAP kinase plays a role in profibrotic actions of aldosterone. To unambiguously demonstrate the importance of aldosterone in c-Src signaling, VSMCs from c-Src+/+ and c-Src+/− mice were also studied. Aldosterone increased phosphorylation of c-Src, p38MAP kinase, and cortactin, a Src-specific substrate, in c-Src+/+ VSMCs, but not in c-Src-deficient cells. Taken together, our findings demonstrate that nongenomic signaling by aldosterone occurs through c-Src–dependent pathways. These processes may play an important role in profibrotic actions of aldosterone.

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.

Angiotensin(1-7) Blunts Hypertensive Cardiac Remodeling by a Direct Effect on the Heart

Angiotensin-converting enzyme 2 (ACE2) converts the vasopressor angiotensin II (Ang II) into angiotensin (1-7) [Ang(1-7)], a peptide reported to have vasodilatory and cardioprotective properties. Inactivation of the ACE2 gene in mice has been reported by one group to result in an accumulation of Ang II in the heart and an age-related defect in cardiac contractility. A second study confirmed the role of ACE2 as an Ang II clearance enzyme but failed to reproduce the contractility defects previously reported in ACE2-deficient mice. The reasons for these differences are unclear but could include differences in the accumulation of Ang II or the deficiencies in Ang(1-7) in the mouse models used. As a result, the roles of ACE2, Ang II, and Ang(1-7) in the heart remain controversial. Using a novel strategy, we targeted the chronic overproduction of either Ang II or Ang(1-7) in the heart of transgenic mice and tested their effect on age-related contractility and on cardiac remodeling in response to a hypertensive challenge. We demonstrate that a chronic accumulation of Ang II in the heart does not result in cardiac contractility defects, even in older (8-month-old) mice. Likewise, transgenic animals with an 8-fold increase in Ang(1-7) peptide in the heart exhibited no differences in resting blood pressure or cardiac contractility as compared to age-matched controls, but they had significantly less ventricular hypertrophy and fibrosis than their nontransgenic littermates in response to a hypertensive challenge. Analysis of downstream signaling cascades demonstrates that cardiac Ang(1-7) selectively modulates some of the downstream signaling effectors of cardiac remodeling. These results suggest that Ang(1-7) can reduce hypertension-induced cardiac remodeling through a direct effect on the heart and raise the possibility that pathologies associated with ACE2 inactivation are mediated in part by a decrease in production of Ang(1-7).

Aldosterone and Angiotensin II Synergistically Stimulate Migration in Vascular Smooth Muscle Cells Through c-Src-Regulated Redox-Sensitive RhoA Pathways

Objective—Synergistic interactions between aldosterone (Aldo) and angiotensin II (Ang II) have been implicated in vascular inflammation, fibrosis, and remodeling. Molecular mechanisms underlying this are unclear. We tested the hypothesis that c-Src activation, through receptor tyrosine kinase transactivation, is critically involved in synergistic interactions between Aldo and Ang II and that it is upstream of promigratory signaling pathways in vascular smooth muscle cells (VSMCs). Methods and Results—VSMCs from WKY rats were studied. At low concentrations (10−10 mol/L) Aldo and Ang II alone did not influence c-Src activation, whereas in combination they rapidly increased phosphorylation (P<0.01), an effect blocked by eplerenone (Aldo receptor antagonist) and irbesartan (AT1R blocker). This synergism was attenuated by AG1478 and AG1296 (inhibitors of EGFR and PDGFR, respectively), but not by AG1024 (IGFR inhibitor). Aldo and Ang II costimulation induced c-Src–dependent activation of NAD(P)H oxidase and c-Src–independent activation of ERK1/2 (P<0.05), without effect on ERK5, p38MAPK, or JNK. Aldo/Ang II synergistically activated RhoA/Rho kinase and VSMC migration, effects blocked by PP2, apocynin, and fasudil, inhibitors of c-Src, NADPH oxidase, and Rho kinase, respectively. Conclusions—Aldo/Ang II synergistically activate c-Src, an immediate signaling response, through EGFR and PDGFR, but not IGFR transactivation. This is associated with activation of redox-regulated RhoA/Rho kinase, which controls VSMC migration. Although Aldo and Ang II interact to stimulate ERK1/2, such effects are c-Src–independent. These findings indicate differential signaling in Aldo-Ang II crosstalk and highlight the importance of c-Src in redox-sensitive RhoA, but not ERK1/2 signaling. Blockade of Aldo/Ang II may be therapeutically useful in vascular remodeling associated with abnormal VSMC migration.

Angiotensin II-Dependent Chronic Hypertension and Cardiac Hypertrophy Are Unaffected by gp91phox-Containing NADPH Oxidase

The gp91phox-containing NADPH oxidase is the major source of reactive oxygen species (ROS) in the cardiovascular system and inactivation of gp91phox has been reported to blunt hypertension and cardiac hypertrophy seen in angiotensin (Ang) II-infused animals. In the current study, we sought to determine the role of gp91phox-derived ROS on cardiovascular outcomes of chronic exposure to Ang II. The gp91phox-deficient mice were crossed with transgenic mice expressing active human renin in the liver (TTRhRen). TTRhRen mice exhibit chronic Ang II–dependent hypertension and frank cardiac hypertrophy by age 10 to 12 weeks. Four genotypes of mice were generated: control, TTRhRen trangenics (TTRhRen), gp91phox-deficient (gp91−), and TTRhRen transgenic gp91phox-deficient (TTRhRen/gp91−). Eight to 10 mice/group were studied. ROS levels were significantly reduced (P <0.05) in the heart and aorta of TTRhRen/gp91− and gp91−mice compared with control counterparts, and this was associated with reduced cardiac, aortic, and renal NADPH oxidase activity (P <0.05). Systolic blood pressure (SBP), cardiac mass, and cardiac fibrosis were increased in TTRhRen versus controls. In contrast to its action on ROS generation, gp91phox inactivation had no effect on development of hypertension or cardiac hypertrophy in TTRhRen mice, although interstitial fibrosis was reduced. Cardiac and renal expression of gp91phox homologues, Nox1 and Nox4, was not different between groups. Thus, although eliminating gp91phox-associated ROS production may be important in cardiovascular consequences in acute insult models, it does not prevent the development of hypertension and cardiac hypertrophy in a model in which the endogenous renin-angiotensin system is chronically upregulated.

Endothelin-1-induced oxidative stress in DOCA-salt hypertension involves NADPH-oxidase-independent mechanisms

We have demonstrated recently [Callera, Touyz, Teixeira, Muscara, Carvalho, Fortes, Schiffrin and Tostes (2003) Hypertension 42, 811-817] that increased vascular oxidative stress in DOCA (deoxycorticosterone acetate)-salt rats is associated with activation of the ET (endothelin) system via ETA receptors. The exact source of ET-1-mediated oxidative stress remains unclear. The aim of the present study was to investigate whether ET-1 increases generation of ROS (reactive oxygen species) in DOCA-salt hypertension through NADPH-oxidase-dependent mechanisms. Xanthine oxidase, eNOS (endothelial nitric oxide synthase) and COX-2 (cyclo-oxygenase-2) were also examined as potential ET-1 sources of ROS as well as mitochondrial respiration. DOCA-salt and control UniNX (uninephrectomized) rats were treated with the ETA antagonist BMS182874 (40 mg.day(-1).kg(-1) of body weight) or vehicle. Plasma TBARS (thiobarbituric acid-reacting substances) were increased in DOCA-salt compared with UniNX rats. Activity of NADPH and xanthine oxidases in aorta, mesenteric arteries and heart was increased in DOCA-salt rats. BMS182874 decreased plasma TBARS levels without influencing NADPH and xanthine oxidase activities in DOCA-salt rats. Increased p22(phox) protein expression and increased p47(phox) membrane translocation in arteries from DOCA-salt by rats were not affected by BMS182874 treatment. Increased eNOS and COX-2 expression, also observed in aortas from DOCA-salt rats, was unaltered by BMS182874. Increased mitochondrial generation of ROS in DOCA-salt rats was normalized by BMS182874. ETA antagonism also increased the expression of mitochondrial MnSOD (manganese superoxide dismutase) in DOCA-salt rats. In conclusion, activation of NADPH oxidase does not seem to be the major source of oxidative stress induced by ET-1/ETA in DOCA-salt hypertension, which also appears to be independent of increased activation of xanthine oxidase or eNOS/COX-2 overexpression. Mitochondria may play a role in ET-1-driven oxidative stress, as evidenced by increased mitochondrial-derived ROS in this model of hypertension.

c-Src–Dependent Nongenomic Signaling Responses to Aldosterone Are Increased in Vascular Myocytes From Spontaneously Hypertensive Rats

Aldosterone plays an important role in the pathogenesis of hypertension. We previously demonstrated that nongenomic signaling by aldosterone in vascular smooth muscle cells occurs through c-Src–dependent pathways. Here we tested the hypothesis that upregulation of c-Src by aldosterone plays a role in increased mitogen-activated protein (MAP) kinase activation, [3H]-proline incorporation, and NADPH-driven generation of reactive oxygen species, thereby inducing cell growth, collagen production, and inflammation, respectively, in vascular smooth muscle cells from spontaneously hypertensive rats. The time course of c-Src phosphorylation by aldosterone was shifted to the left in vascular myocytes from hypertensive animals. Aldosterone rapidly increased phosphorylation of p38 MAP kinase and extracellular signal–regulated kinase with significantly greater effects in cells from spontaneously hypertensive rats versus control cells (P<0.05). Aldosterone increased NADPH oxidase activity with significantly greater responses in vascular smooth muscle cells from hypertensive animals (P<0.05). These events were associated with enhanced [3H]proline incorporation (index of collagen synthesis) in cells from spontaneously hypertensive rats (P<0.05). The NADPH oxidase activity increase, collagen synthesis, c-Src, and MAP kinase phosphorylation induced by aldosterone were significantly reduced by eplerenone (selective mineralocorticoid receptor blocker) and PP2 (selective c-Src inhibitor). In conclusion, nongenomic signaling by exogenous aldosterone, mediated through c-Src, is increased in vascular smooth muscle cells from spontaneously hypertensive rats. Upregulation of c-Src signaling may be important in the profibrotic and proinflammatory actions of aldosterone in this genetic model of hypertension.

Renal Redox-Sensitive Signaling, but Not Blood Pressure, Is Attenuated by Nox1 Knockout in Angiotensin II–Dependent Chronic Hypertension

We demonstrated previously that, in mice with chronic angiotensin II–dependent hypertension, gp91phox-containing NADPH oxidase is not involved in the development of high blood pressure, despite being important in redox signaling. Here we sought to determine whether a gp91phox homologue, Nox1, may be important in blood pressure elevation and activation of redox-sensitive pathways in a model in which the renin-angiotensin system is chronically upregulated. Nox1-deficient mice and transgenic mice expressing human renin (TTRhRen) were crossed, and 4 genotypes were generated: control, TTRhRen, Nox1-deficient, and TTRhRen Nox1-deficient. Blood pressure and oxidative stress (systemic and renal) were increased in TTRhRen mice (P<0.05). This was associated with increased NADPH oxidase activation. Nox1 deficiency had no effect on the development of hypertension in TTRhRen mice. Phosphorylation of c-Src, mitogen-activated protein kinases, and focal adhesion kinase was significantly increased 2- to 3-fold in kidneys from TTRhRen mice. Activation of c-Src, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and focal adhesion kinase but not of extracellular signal regulated kinase 1/2 or extracellular signal regulated kinase 5, was reduced in TTRhRen/Nox1-deficient mice (P<0.05). Expression of procollagen III was increased in TTRhRen and TTRhRen/Nox1-deficient mice versus control mice, whereas vascular cell adhesion molecule-1 was only increased in TTRhRen mice. Our findings demonstrate that, in Nox1-deficient TTRhRen mice, blood pressure is elevated despite reduced NADPH oxidase activation, decreased oxidative stress, and attenuated redox signaling. Our results suggest that Nox1-containing NADPH oxidase plays a key role in the modulation of systemic and renal oxidative stress and redox-dependent signaling but not in the elevation of blood pressure in a model of chronic angiotensin II–dependent 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.

Functional characterization and expression of endothelin receptors in rat carotid artery: involvement of nitric oxide, a vasodilator prostanoid and the opening of K+ channels in ETB-induced relaxation

We aimed to functionally characterize endothelin (ET) receptors in the rat carotid artery. mRNA and protein expressions of both ETA and ETB receptors, evaluated by reverse transcription–polymerase chain reaction (RT–PCR) and Western immunoblotting, were detected in carotid segments. Immunohistochemical assays showed that ETB receptors are expressed in the endothelium and smooth muscle cells, while ETA receptors are expressed only in the smooth muscle cells. In endothelium‐denuded vessels, levels of ETB receptor mRNA were reduced. Vascular reactivity experiments, using standard muscle bath procedures, showed that ET‐1 induces contraction in endothelium‐intact and ‐denuded carotid rings in a concentration‐dependent manner. Endothelial removal enhanced ET‐1‐induced contraction. BQ123 and BQ788, selective antagonists for ETA and ETB receptors, respectively, produced concentration‐dependent rightward displacements of the ET‐1 concentration–response curves. IRL1620, a selective agonist for ETB receptors, induced a slight vasoconstriction that was abolished by BQ788, but not affected by BQ123. IRL1620‐induced contraction was augmented after endothelium removal. ET‐1 concentration dependently relaxed phenylephrine‐precontracted rings with intact endothelium. The relaxation was augmented in the presence of BQ123, reduced in the presence of BQ788 and completely abolished after endothelium removal. IRL1620 induced vasorelaxation that was abolished by BQ788 and endothelium removal, but not affected by BQ123. Preincubation of intact rings with NG‐nitro‐L‐arginine methyl ester (L‐NAME), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), indomethacin or tetraethylammonium (TEA) reduced IRL1620‐induced relaxation. The combination of L‐NAME, indomethacin and TEA completely abolished IRL1620‐induced relaxation while sulfaphenazole did not affect this response. 4‐aminopyridine (4‐AP), but not apamin, glibenclamide or charybdotoxin, reduced IRL1620‐induced relaxation. The major finding of this work is that it firstly demonstrated functionally the existence of both ETA and ETB vasoconstrictor receptors located on the smooth muscle of rat carotid arteries and endothelial ETB receptors that mediated vasorelaxation via NO–cGMP pathway, vasodilator cyclooxygenase product(s) and the activation of voltage‐dependent K+ channels.