Mario Fritsch Neves

Spironolactone Improves Angiotensin-Induced Vascular Changes and Oxidative Stress

Abstract—Angiotensin II plays an important role in vascular remodeling. We investigated the role of aldosterone, which is stimulated by angiotensin II, as a mediator of angiotensin II–induced vascular structural and functional alterations. Sprague-Dawley rats (n=8 to 12/group) received angiotensin II (120 ng/kg per minute, subcutaneously) for 14 days ± spironolactone or hydralazine (25 mg/kg per day). An additional group received aldosterone (750 ng/h, subcutaneously) ± spironolactone. Systolic blood pressure was increased by angiotensin II (P <0.001) and reduced by spironolactone and hydralazine (P <0.001). Aldosterone-induced increase of blood pressure was reduced by spironolactone (P <0.05). In mesenteric small arteries studied on a pressurized myograph, media/lumen ratio was increased (P <0.001) and acetylcholine-mediated relaxation was impaired in angiotensin II–infused rats (P <0.001); both were partially improved by spironolactone (P <0.05) but not by hydralazine. Aldosterone-induced increase of media/lumen ratio (P <0.001) and impaired response to acetylcholine (P <0.001) were normalized by spironolactone. Response to sodium nitroprusside was similar in all groups. Aortic NADPH oxidase activity was increased (P <0.01) by angiotensin II and reduced by spironolactone and hydralazine. Aldosterone also increased (P <0.05) activation of NADPH oxidase, an effect abolished by spironolactone. Plasma thiobarbituric acid–reactive substances (a marker of oxidative stress), higher in angiotensin II and aldosterone rats (P <0.001), were normalized by spironolactone. In conclusion, spironolactone, which inhibited aldosterone actions, partially corrected structural and functional angiotensin II–induced abnormalities. These effects were associated with reduced vascular NADPH oxidase activity and decreased plasma markers of oxidative stress. Our findings suggest that aldosterone may mediate some of angiotensin II–induced vascular effects in hypertension, in part via increased oxidative stress.

Structure, Endothelial Function, Cell Growth, and Inflammation in Blood Vessels of Angiotensin II–Infused Rats Role of Peroxisome Proliferator–Activated Receptor-γ

Background—Pioglitazone and rosiglitazone, thiazolidinedione peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) activators, reduce blood pressure (BP) in some hypertensive models by unclear mechanisms. We tested the hypothesis that pioglitazone or rosiglitazone would prevent BP elevation and vascular dysfunction in angiotensin (Ang) II–infused rats by direct vascular effects. Methods and Results—Sprague-Dawley rats received Ang II (120 ng · kg−1 · min−1 SC) with or without pioglitazone (10 mg · kg−1 · d−1) or rosiglitazone (5 mg · kg−1 · d−1) for 7 days. Systolic BP, elevated in Ang II–infused rats (176±5 mm Hg) versus controls (109±2 mm Hg, P <0.01), was reduced by pioglitazone (134±2 mm Hg) or rosiglitazone (123±2 mm Hg). In mesenteric small arteries studied in a pressurized myograph, media/lumen ratio was increased (P <0.05) and acetylcholine-induced relaxation impaired in Ang II–infused rats (P <0.05); both were normalized by the thiazolidinediones. In Ang II–infused rats, vascular DNA synthesis (by 3H-thymidine incorporation); expression of cell cycle proteins cyclin D1 and cdk4, angiotensin II type 1 receptors, vascular cell adhesion molecule-1, and platelet and endothelial cell adhesion molecule; and nuclear factor-&kgr;B activity were increased. These changes were abrogated by pioglitazone or rosiglitazone. Conclusions—Thiazolidinedione PPAR-&ggr; activators attenuated the development of hypertension, corrected structural abnormalities, normalized cell growth, and improved endothelial dysfunction induced by Ang II and prevented upregulation of angiotensin II type 1 receptors, cell cycle proteins, and proinflammatory mediators. Thiazolidinediones may be useful in the prevention and/or treatment of hypertension, particularly when it is associated with insulin resistance or diabetes mellitus.

Endothelium-Restricted Overexpression of Human Endothelin-1 Causes Vascular Remodeling and Endothelial Dysfunction

Background—Endothelin (ET)-1 is a potent vasoconstrictor that contributes to vascular remodeling in hypertension and other cardiovascular diseases. Endogenous ET-1 is produced predominantly by vascular endothelial cells. To directly test the role of endothelium-derived ET-1 in cardiovascular pathophysiology, we specifically targeted expression of the human preproET-1 gene to the endothelium by using the Tie-2 promoter in C57BL/6 mice. Methods and Results—Ten-week-old male C57BL/6 transgenic (TG) and nontransgenic (wild type; WT) littermates were studied. TG mice exhibited 3-fold higher vascular tissue ET-1 mRNA and 7-fold higher ET-1 plasma levels than did WT mice but no significant elevation in blood pressure. Despite the absence of significant blood pressure elevation, TG mice exhibited marked hypertrophic remodeling and oxidant excess-dependent endothelial dysfunction of resistance vessels, altered ET-1 and ET-3 vascular responses, and significant increases in ETB expression compared with WT littermates. Moreover, TG mice generated significantly higher oxidative stress, possibly through increased activity and expression of vascular NAD(P)H oxidase than did their WT counterparts. Conclusions—In this new murine model of endothelium-restricted human preproET-1 overexpression, ET-1 caused structural remodeling and endothelial dysfunction of resistance vessels, consistent with a direct nonhemodynamic effect of ET-1 on the vasculature, at least in part through the activation of vascular NAD(P)H oxidase.

T Regulatory Lymphocytes Prevent Angiotensin II–Induced Hypertension and Vascular Injury

Angiotensin (Ang) II induces hypertension by mechanisms mediated in part by adaptive immunity and T effector lymphocytes. T regulatory lymphocytes (Tregs) suppress T effector lymphocytes. We questioned whether Treg adoptive transfer would blunt Ang II–induced hypertension and vascular injury. Ten- to 12-week–old male C57BL/6 mice were injected IV with 3×105 Treg (CD4+CD25+) or T effector (CD4+CD25−) cells, 3 times at 2-week intervals, and then infused or not with Ang II (1 &mgr;g/kg per minute, SC) for 14 days. Ang II increased systolic blood pressure by 43 mm Hg (P<0.05), NADPH oxidase activity 1.5-fold in aorta and 1.8-fold in the heart (P<0.05), impaired acetylcholine vasodilatory responses by 70% compared with control (P<0.05), and increased vascular stiffness (P<0.001), mesenteric artery vascular cell adhesion molecule expression (2-fold; P<0.05), and aortic macrophage and T-cell infiltration (P<0.001). All of the above were prevented by Treg but not T effector adoptive transfer. Ang II caused a 43% decrease in Foxp3+ cells in the renal cortex, whereas Treg adoptive transfer increased Foxp3+ cells 2-fold compared with control. Thus, Tregs suppress Ang II–mediated vascular injury in part through anti-inflammatory actions. Immune mechanisms modulate Ang II–induced blood pressure elevation, vascular oxidative stress, inflammation, and endothelial dysfunction.

PPARα Activator Effects on Ang II–Induced Vascular Oxidative Stress and Inflammation

Docosahexaenoic acid (DHA), a peroxisome proliferator–activated receptor-α (PPARα) activator, reduces blood pressure (BP) in some hypertensive models by unclear mechanisms. We tested the hypothesis that DHA would prevent BP elevation and improve vascular dysfunction in angiotensin (Ang) II–infused rats by modulating of NADPH oxidase activity and inflammation in vascular wall. Sprague-Dawley rats received Ang II (120 ng/kg per minute SC) with or without DHA (2.5 mL of oil containing 40% DHA/d PO) for 7 days. Systolic BP (mm Hg), elevated in Ang II–infused rats (172±3) versus controls (108±2, P P P

Endothelin Antagonism on Aldosterone-Induced Oxidative Stress and Vascular Remodeling

Abstract—Endothelin A (ETA) receptor blockade has prevented vascular remodeling in aldosterone and salt-induced hypertension. To evaluate effects of the ETA receptor antagonist, BMS 182874, compared with the aldosterone antagonist, spironolactone, on vascular remodeling in aldosterone-infused rats not exposed to a high salt diet, Sprague-Dawley rats were infused subcutaneously with aldosterone (0.75 &mgr;g/h) and treated with BMS 182874 (40 mg · kg−1 · d−1), spironolactone, or hydralazine (both 25 mg · kg−1 · d−1) while receiving a normal salt diet for 6 weeks. Aldosterone increased systolic BP (P <0.01), plasma endothelin (3.33±0.32 versus 1.85±0.40 pmol/L in control, P <0.05), systemic oxidative stress as shown by plasma thiobarbituric acid–reacting substances and vascular nicotinamide adenine dinucleotide phosphate (NADPH) activity. Aldosterone increased small artery media thickness (17.7±0.9 versus 13.6±0.8 &mgr;m in control, P <0.05) and media/lumen ratio (7.6±0.4 versus 5.5±0.4% in control, P <0.05), with growth index of 21% indicating hypertrophic remodeling. Laser confocal microscopy showed increased collagen and fibronectin deposition and intercellular adhesion molecule-1 (ICAM-1) content in the vessel wall of aldosterone-infused rats. The 3 treatments lowered BP, although hydralazine was slightly less effective. BMS 182874 and spironolactone decreased oxidative stress, normalized the hypertrophic remodeling, decreased collagen and fibronectin deposition, and reduced ICAM-1 abundance in the vascular wall of aldosterone-infused rats, whereas hydralazine only reduced NADPH activity in aorta but did not affect the remaining parameters. Vascular remodeling of small arteries occurs in aldosterone-infused rats exposed to a normal salt diet and may be mediated in part by ET-1 via stimulation of ETA receptors. Endothelin blockade may exert beneficial effects on vascular remodeling, fibrosis, oxidative stress, and adhesion molecule expression in aldosterone-induced hypertension.

Role of NAD(P)H oxidase on vascular alterations in angiotensin II-infused mice.

Objectives Angiotensin (Ang) II stimulates vascular reactive oxygen species generation via NAD(P)H oxidase activation. We investigated whether vascular NAD(P)H oxidase influences structure and function of resistance arteries from Ang II-infused mice. Methods Mice received Ang II alone (400 ng/kg per min, subcutaneously), Ang II + apocynin (NAD(P)H oxidase inhibitor, 2.5 mg/day, in the food), apocynin alone or Ang II + hydralazine (50mg/kg per day) for 14 days. Systolic blood pressure (SBP) was measured by tail-cuff methodology and function and structure of small mesenteric arteries were studied in pressurized vessels. Vascular collagen type I/III content was evaluated by confocal immunofluorescence microscopy and by immunoblotting. Results The rise in SBP induced by Ang II (P < 0.001) was prevented by apocynin and hydralazine. Media/lumen ratio increase in Ang II-infused mice (P < 0.01) was prevented by apocynin. Acetylcholine-mediated relaxation, which was impaired in Ang II-infused mice (P < 0.001), was improved by apocynin. Confocal microscopy and immunoblotting demonstrated increased collagen type I/III content in mesenteric arteries from Ang II-infused mice. Apocynin, but not hydralazine, prevented the increase in collagen abundance in Ang II-infused mice. The increase in vascular NAD(P)H oxidase activity by Ang II (P < 0.001) was prevented by apocynin. Conclusions The NAD(P)H oxidase inhibitor apocynin reduced blood pressure elevation and prevented structural alterations, endothelial dysfunction, and collagen deposition in the media of small arteries in Ang II-infused mice. Although hydralazine also decreased blood pressure, it had no effects on vascular collagen content. Our findings suggest that NAD(P)H oxidase activity plays an important role in vascular functional and structural changes and in the composition of the vascular wall in Ang II-dependent hypertension.

T Regulatory Lymphocytes Prevent Aldosterone-Induced Vascular Injury

Aldosterone mediates actions of the renin-angiotensin-aldosterone system inducing hypertension, oxidative stress, and vascular inflammation. Recently, we showed that angiotensin II–induced hypertension and vascular damage are mediated at least in part by macrophages and T-helper effector lymphocytes. Adoptive transfer of suppressor T-regulatory lymphocytes (Tregs) prevented angiotensin II action. We hypothesized that Treg adoptive transfer would blunt aldosterone-induced hypertension and vascular damage. Thirteen to 15-week–old male C57BL/6 mice were injected intravenously at 1-week intervals with 3×105 CD4+CD25+ cells (representing Treg) or control CD4+CD25− cells and then infused or not for 14 days with aldosterone (600 &mgr;g/kg per day, SC) while receiving 1% saline to drink. Aldosterone induced a small but sustained increase in blood pressure (P<0.001), decreased vasodilatory responses to acetylcholine by 66% (P<0.001), increased both media:lumen ratio (P<0.001) and media cross-sectional area of resistance arteries by 60% (P<0.05), and increased NADPH oxidase activity 2-fold in aorta (P<0.001), kidney and heart (P<0.05), and aortic superoxide production. As well, aldosterone enhanced aortic and renal cortex macrophage infiltration and aortic T-cell infiltration (all P<0.05), and tended to decrease Treg in the renal cortex. Treg adoptive transfer prevented all of the vascular and renal effects induced by aldosterone. Adoptive transfer of CD4+CD25− cells exacerbated aldosterone effects except endothelial dysfunction and increases in media:lumen ratio of resistance arteries. Thus, Tregs suppress aldosterone-mediated vascular injury, in part through effects on innate and adaptive immunity, suggesting that aldosterone-induced vascular damage could be prevented by an immunomodulatory approach.

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.

Effect of Hyperhomocystinemia and Hypertension on Endothelial Function in Methylenetetrahydrofolate Reductase–Deficient Mice

Objective—We evaluated the effect of hyperhomocystinemia and angiotensin (Ang) II on vascular function and structure in methylenetetrahydrofolate reductase knockout mice (Mthfr+/−). Methods and Results—Mthfr+/− and controls (Mthfr+/+) received Ang II (400 ng/kg per min SC) or saline (14 days). Blood pressure, similar in Mthfr+/− and Mthfr+/+, was increased by Ang II. Acetylcholine- and bradykinin-induced relaxations were impaired in mesenteric resistance arteries (pressurized myograph) in Mthfr+/− and in Ang II–infused Mthfr+/+ mice and additionally blunted in Ang II–infused Mthfr+/− mice. The inhibition by L-NAME on acetylcholine was reduced in Mthfr+/− and in Ang II–Mthfr+/+ and absent in Ang II–Mthfr+/− mice. In these groups, vitamin C improved the response to acetylcholine and restored the inhibition by L-NAME. The media to lumen ratio of small arteries, similar in Mthfr+/− and Mthfr+/+, was increased by Ang II. Vascular NADPH oxidase activity, similar in Mthfr+/− and Mthfr+/+, increased after Ang II infusion. Vascular xanthine oxidase activity was also similar in Mthfr+/− and Mthfr+/+. Superoxide production in the aorta was reduced by sepiapterin and by L-NAME, suggesting that reduced bioavailability of tetrahydrobiopterin and uncoupling of nitric oxide synthase were the origin of increased reactive oxygen species in this model. Conclusions—Mthfr+/− mice show endothelial dysfunction of mesenteric vessels probably attributable to a reduced nitric oxide bioavailability caused by oxidative excess due to uncoupling of nitric oxide synthase without vascular structural alterations. Concurrent Ang II–induced hypertension additionally reduced nitric oxide, increased NADPH oxidase activity, and induced structural alterations. Our findings suggest additive adverse effect of Ang II–dependent hypertension and hyperhomocystinemia on endothelial function.