Carmine Savoia

Inflammation in hypertension

Purpose of reviewIn this review we summarize the recent evidence that highlights the involvement of low-grade inflammation in the development and pathophysiology of hypertension. Recent findingsEssential hypertension is characterized by increased peripheral vascular resistance to blood flow, due in large part to vascular remodeling. Vascular changes in hypertension are associated with mechanical and humoral factors that modulate signaling events, resulting in abnormal function, media growth, extracellular matrix deposition and inflammation. Recent evidence suggests that inflammation is present in the vasculature in animal models of hypertension. Inflammatory markers, such as C-reactive protein, are associated with vascular lesions in humans, and are predictive of cardiovascular outcome. In animal and human studies, pro-inflammatory components of the renin–angiotensin–aldosterone system have been demonstrated in large conduit and small arteries in the kidney and heart. Peroxisome proliferator-activated receptor activators are drugs with metabolic properties that have been demonstrated to exert anti-inflammatory effects on the vasculature, and there is now evidence that these actions may be protective for blood vessels. SummaryInflammatory processes are important participants in the pathophysiology of hypertension and cardiovascular disease. The identification of the mechanisms leading to the activation of inflammation should contribute to the development of specific therapeutic approaches to apply in hypertension and its complications.

Deletion of p66shc Gene Protects Against Age-Related Endothelial Dysfunction

Background—Enhanced production of reactive oxygen species (ROS) has been recognized as the major determinant of age-related endothelial dysfunction. The p66shc protein controls cellular responses to oxidative stress. Mice lacking p66shc (p66shc−/−) have increased resistance to ROS and a 30% prolonged life span. The present study investigates age-dependent changes of endothelial function in this model. Methods and Results—Aortic rings from young and old p66shc−/− or wild-type (WT) mice were suspended for isometric tension recording. Nitric oxide (NO) release was measured by a porphyrinic microsensor. Expression of endothelial NO synthase (eNOS), inducible NOS (iNOS), superoxide dismutase, and nitrotyrosine-containing proteins was assessed by Western blotting. Nitrotyrosine residues were also identified by immunohistochemistry. Superoxide (O2−) production was determined by coelenterazine-enhanced chemiluminescence. Endothelium-dependent relaxation in response to acetylcholine was age-dependently impaired in WT mice but not in p66shc−/− mice. Accordingly, an age-related decline of NO release was found in WT but not in p66shc−/− mice. The expression of eNOS and manganese superoxide dismutase was not affected by aging either in WT or in p66shc−/− mice, whereas iNOS was upregulated only in old WT mice. It is interesting that old WT mice displayed a significant increase of O2− production as well as of nitrotyrosine expression compared with young animals. Such age-dependent changes were not found in p66shc−/− mice. Conclusions—We report that inactivation of the p66shc gene protects against age-dependent, ROS-mediated endothelial dysfunction. These findings suggest that the p66shc is part of a signal transduction pathway also relevant to endothelial integrity and may represent a novel target to prevent vascular aging.

Transient Receptor Potential Melastatin 7 Ion Channels Regulate Magnesium Homeostasis in Vascular Smooth Muscle Cells: Role of Angiotensin II

Magnesium modulates vascular smooth muscle cell (VSMC) function. However, molecular mechanisms regulating VSMC Mg2+ remain unknown. Using biochemical, pharmacological, and genetic tools, the role of transient receptor potential membrane melastatin 7 (TRPM7) cation channel in VSMC Mg2+ homeostasis was evaluated. Rat, mouse, and human VSMCs were studied. Reverse transcriptase polymerase chain reaction and immunoblotting demonstrated TRPM7 presence in VSMCs (membrane and cytosol). Angiotensin II (Ang II) and aldosterone increased TRPM7 expression. Gene silencing using small interfering RNA (siRNA) against TRPM7, downregulated TRPM7 (mRNA and protein). Basal [Mg2+]i, measured by mag fura-2AM, was reduced in siRNA-transfected cells (0.39±0.01 mmol/L) versus controls (0.54±0.01 mmol/L; P<0.01). Extracellular Mg2+ dose-dependently increased [Mg2+]i in control cells (Emax 0.70±0.02 mmol/L) and nonsilencing siRNA-transfected cells (Emax 0.71±0.04 mmol/L), but not in siRNA-transfected cells (Emax 0.5±0.01 mmol/L). The functional significance of TRPM7 was evaluated by assessing [Mg2+]i and growth responses to Ang II in TRPM7 knockdown cells. Acute Ang II stimulation decreased [Mg2+]i in control and TRPM7-deficient cells in a Na+-dependent manner. Chronic stimulation increased [Mg2+]i in control, but not in siRNA-transfected VSMCs. Ang II–induced DNA and protein synthesis, measured by 3[H]-thymidine and 3[H]-leucine incorporation, respectively, were increased in control and nonsilencing cells, but not in TRPM7 knockdown VSMCs. Our data indicate that VSMCs possess membrane-associated, Ang II–, and aldosterone-regulated TRPM7 channels, which play a role in regulating basal [Mg2+]i, transmembrane Mg2+ transport and DNA and protein synthesis. These novel findings identify TRPM7 as a functionally important regulator of Mg2+ homeostasis and growth in VSMCs.

Selective Mineralocorticoid Receptor Blocker Eplerenone Reduces Resistance Artery Stiffness in Hypertensive Patients

Some antihypertensive agents may improve resistance artery remodeling in hypertensive patients whereas other agents may not, for similar blood pressure reduction. We questioned whether the selective mineralocorticoid receptor blocker eplerenone improves resistance artery remodeling in hypertensive patients versus the β-blocker atenolol. Sixteen hypertensive patients were randomly assigned to double-blind daily treatment with eplerenone or atenolol. Resistance arteries from gluteal subcutaneous tissue were assessed on a pressurized myograph. After 1 year of treatment, systolic and diastolic blood pressures were similarly well controlled in both groups. Endothelial function did not change with treatment in either group. Media/lumen ratio and cross-sectional area were unchanged in either the atenolol or the eplerenone group. In atenolol-treated patients, the arterial wall became stiffer, whereas in the eplerenone-treated patients, it became less stiff and similar to that of a normotensive control group. The media collagen/elastin ratio was reduced only after eplerenone treatment. Circulating concentrations of osteopontin, monocyte chemoattractant protein-1, basic fibroblast growth factor, interleukin-8, and interleukin-10 were significantly reduced only by eplerenone. However, plasma interleukin-1 receptor a concentration was significantly reduced by both drugs. In conclusion, in hypertensive patients, blood pressure control for 1 year with atenolol was associated with increased wall stiffness of resistance arteries, whereas eplerenone treatment was associated with reduced stiffness, decreased collagen/elastin ratio, and a reduction in circulating inflammatory mediators. These data raise the possibility that eplerenone treatment of hypertensive patients when normalizing blood pressure could potentially be associated with better vascular protection and outcomes than the β-blocker atenolol, which remains to be demonstrated.

Differential calcium regulation by hydrogen peroxide and superoxide in vascular smooth muscle cells from spontaneously hypertensive rats.

We investigated the role of reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2) and superoxide anion (•O2−) in the regulation of vascular smooth muscle cell (VSMC) Ca2+ concentration ([Ca2+]i) and vascular contraction and assessed whether redox-dependent Ca2+ signaling and contraction are altered in hypertension. VSMCs and mesenteric arteries from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were studied. Cells were stimulated with H2O2 (10−4 mol/l) or LY83583 (•O2− generator, 10−5 mol/l). [Ca2+]i and cytosolic •O2 − were measured by fura-2AM and tempo-9-AC fluorescence respectively. L-type and T-type Ca2+ channels were assessed using verapamil/diltiazem and mibefradil respectively and mRNA and protein expression of these channels was assessed by real-time PCR and immunoblotting respectively. H2O2 time-dependently increased [Ca2+]i and contraction with significantly greater effects in SHR versus WKY (P < 0.001). LY83583 increased [Ca2+]i in both strains, but responses were blunted in SHR. Removal of extracellular Ca2+ abrogated [Ca2+]i responses to H2O2 and •O2−. Verapamil and diltiazem, but not mibefradil, significantly decreased H2O2 -induced [Ca2+]i responses with greater effects in SHR (P < 0.01). L-type and T-type Ca2+ channel inhibition reduced LY83583-mediated [Ca2+]i increase only in WKY cells. Both types of Ca2+ channels were expressed (mRNA and protein) in VSMCs from WKY and SHR, with greater abundance in SHR than WKY (2- to 3-fold). These results demonstrate that ROS increase vascular [Ca2+]i and contraction, primarily via extracellular Ca2+ influx. Whereas responses to H2O2 are enhanced, •O2− -mediated actions are blunted in SHR. These effects may relate to differential activation of Ca2+ channels by H2O2 and •O2 −. Enhanced activation of L-type Ca2+ channels and increased Ca2+ influx by H2O2 may contribute to increased Ca2+ signaling in VSMCs from SHR.

Angiotensin Type 2 Receptor in Resistance Arteries of Type 2 Diabetic Hypertensive Patients

The role of angiotensin type 2 receptor (AT2R) on vascular responses to angiotensin II in humans remains unclear. In this study we explored whether AT2R is expressed and functionally active on peripheral resistance arteries of hypertensive diabetic patients treated for 1 year with either the angiotensin receptor blocker valsartan or the &bgr;-blocker atenolol. Twenty-six hypertensive type 2 diabetic patients treated with oral hypoglycemic and antihypertensive agents (not receiving angiotensin receptor blockers or &bgr;-blockers) were randomly assigned to double-blind treatment for 1 year with valsartan or atenolol once daily added to their previous therapy in a clinical trial that we reported recently and compared with 10 normal subjects. Resistance arteries dissected from gluteal subcutaneous tissues were assessed on a pressurized myograph. Vasomotor response curves to angiotensin II (1 nmol/L to 1 &mgr;mol/L) were performed on norepinephrine precontracted vessels in the presence of valsartan (10 &mgr;mol/L) with or without the AT2R inhibitor PD123319 (1 &mgr;mol/L). AT2R expression was evaluated by confocal microscopy. After 1 year of treatment, systolic and diastolic blood pressure was controlled and comparable in the valsartan and atenolol groups. Angiotensin II evoked a significant vasodilatory response only on resistance arteries from patients treated with valsartan, effect blocked by PD123319. AT2R expression was 4-fold higher in small arteries of valsartan-treated patients. In conclusion, AT2Rs are upregulated and contribute to angiotensin II–induced vasodilation in resistance arteries of hypertensive diabetic patients treated with angiotensin type 1 receptor blockers and may mediate, in part, vascular actions of these drugs in high cardiovascular risk patients.

Angiotensin II AT2 receptor subtype: an uprising frontier in cardiovascular disease?

The renin–angiotensin system (RAS) plays a pivotal role in the regulation of fluid, electrolyte balance and blood pressure, and is a modulator of cellular growth and proliferation. Biological actions of RAS are linked to the binding of the effector molecule, angiotensin II (AngII), to specific membrane receptors, mostly the AT1 subtype and, to a lesser extent, other subtypes. Following the identification and characterization of the AT2 subtype receptor, it has been proposed that a complex interaction between AngII and its receptors may play an important role in the effects of RAS. In this paper current information on AngII subtype receptors – their structure, regulation and intracellular signalling – are reviewed, with a particular emphasis on the potential relevance for cardiovascular pathophysiology. In addition, we discuss modulation of expression of the AT2 receptor and its interaction with the AT1 receptor subtype, as well as the potential effects of this receptor on blood pressure regulation. A better understanding of the integrated effects of the AngII subtype receptors may help to elucidate the function of the RAS, as well as their participation in the mechanisms of cardiovascular disease and attendant therapeutic implications.

Eplerenone Prevents Salt-Induced Vascular Remodeling and Cardiac Fibrosis in Stroke-Prone Spontaneously Hypertensive Rats

We examined the effect of different levels of salt intake on the role of aldosterone on cardiac and vascular changes in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). Eleven-week-old SHRSP were fed high-salt (4.2% NaCl), normal-salt (0.28%), or low-salt (0.03%) diets with or without eplerenone (100 mg/kg per day, in food) for 5 weeks. A group of high-salt SHRSP was also treated with hydralazine (25 mg/kg per day). Blood pressure increased more in high-salt rats than in other groups (P < 0.001). Eplerenone prevented further blood pressure rise in salt-loaded rats, with little effect on control and low-salt SHRSP. Increased media-to-lumen ratio of mesenteric resistance arteries induced by salt (P < 0.01) was prevented by eplerenone (P < 0.01). Maximal acetylcholine-induced vasodilation was impaired under salt loading (P < 0.01), but improved under eplerenone (P < 0.01). Eplerenone prevented (P < 0.01) increased heart weight and left and right ventricular collagen deposition induced by high salt. Blood pressure lowering by hydralazine in high-salt SHRSP did not influence endothelial function or left ventricular collagen. Our study demonstrates salt-dependency of aldosterone effects on severity of hypertension, endothelial dysfunction, and cardiac and vascular remodeling in SHRSP. These effects were attenuated by eplerenone, particularly in the salt-loaded state, underlining the pathophysiological role of aldosterone in salt-sensitive hypertension.

Persistent Remodeling of Resistance Arteries in Type 2 Diabetic Patients on Antihypertensive Treatment

Abstract—We hypothesized that resistance arteries from diabetic patients with controlled hypertension have less remodeling than vessels from untreated hypertensive subjects. Eight normotensive subjects (aged 44±3 years, 3 men; values are mean±SEM), 19 untreated hypertensive subjects (46±2 years, 9 men), and 23 hypertensive subjects with type 2 diabetes mellitus under antihypertensive treatment (58±1 years, 15 men) were studied. Resistance arteries dissected from gluteal subcutaneous tissue were assessed on a pressurized myograph. Most diabetic patients (70%) were being treated with angiotensin-converting enzyme inhibitors. Although systolic blood pressure was still above the normotensive range in these patients (144±2 versus 150±3 mm Hg in hypertensive and 114±4 mm Hg in normotensive subjects), diastolic blood pressure was well controlled (83±2 mm Hg) and significantly lower compared with that in untreated hypertensives (100±1 mm Hg; P <0.001) but higher than in normotensives (76±3 mm Hg; P <0.05). Thus, pulse pressure was higher in diabetic patients (P <0.05). The media-to-lumen ratio of resistance arteries was greater in hypertensives (0.083±0.002) compared with normotensive controls (0.059±0.003; P <0.05) and was even higher in diabetic hypertensive subjects (0.105±0.004; P <0.001 versus normotensive controls). The medial cross-sectional area was greater in diabetic and hypertensive patients compared with normotensive controls (P <0.001). Acetylcholine-induced relaxation was impaired in vessels from hypertensive patients and from patients with both diabetes mellitus and hypertension (P <0.05 versus normotensive controls), whereas endothelium-independent vasorelaxation was similar in all groups. Despite effective antihypertensive treatment, resistance arteries from hypertensive diabetic patients showed marked remodeling, greater than that of vessels from untreated, nondiabetic, hypertensive subjects, in agreement with the high cardiovascular risk of subjects suffering from both diabetes and hypertension.

Negative regulation of RhoA/Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats.

Objective To test whether angiotensin II (Ang II) through the Ang II type 2 receptor (AT2R), downregulates RhoA/Rho kinase, which plays a role in AT1 receptor (AT1R)-mediated function. Methods In vitro studies were performed in A10 vascular smooth muscle cells (VSMC) and in vivo studies in mesenteric arteries from Wistar–Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) rats. VSMC were stimulated with Ang II (10−7 mol/l), CGP42112A (10−7 mol/l, a selective AT2R agonist) ± valsartan (10−5 mol/l, an AT1R antagonist), or the Rho kinase inhibitor fasudil (10−5 mol/l). AT1R and AT2R expression and myosin light chain (MLC) phosphorylation were determined by immunoblotting. RhoA activity was assessed by measuring membrane translocation. Functional significance between AT2R, RhoA/Rho kinase and vasodilation was assessed in arteries from valsartan-treated (30 mg/kg per day, 14 days) WKY and SHRSP rats. Vasodilatory responses to Ang II (10−9–10−6 mol/l) were performed in norepinephrine pre-contracted vessels ± valsartan(10−6 mol/l), PD123319 (10−6 mol/l, an AT2R antagonist) or fasudil (10−6 mol/l). Results A10 VSMC expressed AT1R and AT2R. In valsartan-treated cells, Ang II-induced RhoA translocation was reduced versus controls (42 ± 6%, P < 0.05). Similar responses were obtained with CGP42112A (45 ± 6%, P < 0.05). This was associated with decreased MLC activation. Fasudil abrogated Ang II- and CGP42112A-mediated effects. Ang II evoked a significant vasodilatory response only in valsartan-treated SHRSP (max dilation 40 ± 7%). PD123319 blocked these effects. Fasudil increased AngII-induced relaxation in SHRSP vessels. AT2R expression was increased by valsartan (two- to three-fold) in SHRSP arteries. RhoA translocation was increased two-fold in untreated SHRSP (P < 0.05) and was reduced by valsartan (P < 0.05). These changes were associated with decreased MLC phosphorylation. Conclusions Ang II/AT2R negatively regulates vascular RhoA/Rho kinase/MLC phosphorylation. These processes may play a role in Ang II-mediated vasodilation in conditions associated with vascular AT2R upregulation, such as in SHRSP chronically treated with AT1R blockers, which may contribute to blood pressure lowering by these antihypertensive agents.