Stefany B.A. Cau

Differential Modulation of Nitric Oxide Synthases in Aging: Therapeutic Opportunities

Vascular aging is the term that describes the structural and functional disturbances of the vasculature with advancing aging. The molecular mechanisms of aging-associated endothelial dysfunction are complex, but reduced nitric oxide (NO) bioavailability and altered vascular expression and activity of NO synthase (NOS) enzymes have been implicated as major players. Impaired vascular relaxation in aging has been attributed to reduced endothelial NOS (eNOS)-derived NO, while increased inducible NOS (iNOS) expression seems to account for nitrosative stress and disrupted vascular homeostasis. Although eNOS is considered the main source of NO in the vascular endothelium, neuronal NOS (nNOS) also contributes to endothelial cells-derived NO, a mechanism that is reduced in aging. Pharmacological modulation of NO generation and expression/activity of NOS isoforms may represent a therapeutic alternative to prevent the progression of cardiovascular diseases. Accordingly, this review will focus on drugs that modulate NO bioavailability, such as nitrite anions and NO-releasing non-steroidal anti-inflammatory drugs, hormones (dehydroepiandrosterone and estrogen), statins, resveratrol, and folic acid, since they may be useful to treat/to prevent aging-associated vascular dysfunction. The impact of these therapies on life quality in elderly and longevity will be discussed.

Time course involvement of matrix metalloproteinases in the vascular alterations of renovascular hypertension

Increased vascular matrix metalloproteinases (MMPs) levels play a role in late phases of hypertensive vascular remodeling. However, no previous study has examined the time course of MMPs in the various phases of two-kidney, one-clip hypertension (2K1C). We examined structural vascular changes, collagen and elastin content, vascular oxidative stress, and MMPs levels/activities during the development of 2K1C hypertension. Plasma angiotensin converting enzyme (ACE) activity was measured to assess renin-angiotensin system activation. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NADPH activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. 2K1C hypertension was associated with increased ACE activity, which decreased to normal after 10 weeks. We found increased aortic collagen and elastin content in the early phase of hypertension, which were associated with vascular hypertrophy, increased vascular MMP-2 and MMP-14 (but not TIMP-2) levels, and increased gelatinolytic activity, possibly as a result of increased vascular NADPH oxidase activity and oxidative stress. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with early increases in MMPs activities, enhanced matrix deposition and oxidative stress. Using antioxidants or MMPs inhibitors in the early phase of hypertension may prevent the vascular alterations of hypertension.

Vitamin D Induces Increased Systolic Arterial Pressure via Vascular Reactivity and Mechanical Properties

Background/Aims The aim of this study was to evaluate whether supplementation of high doses of cholecalciferol for two months in normotensive rats results in increased systolic arterial pressure and which are the mechanisms involved. Specifically, this study assesses the potential effect on cardiac output as well as the changes in aortic structure and functional properties. Methods Male Wistar rats were divided into three groups: 1) Control group (C, n = 20), with no supplementation of vitamin D, 2) VD3 (n = 19), supplemented with 3,000 IU vitamin D/kg of chow; 3) VD10 (n = 21), supplemented with 10,000 IU vitamin D/kg of chow. After two months, echocardiographic analyses, measurements of systolic arterial pressure (SAP), vascular reactivity, reactive oxygen species (ROS) generation, mechanical properties, histological analysis and metalloproteinase-2 and -9 activity were performed. Results SAP was higher in VD3 and VD10 than in C rats (p = 0.001). Echocardiographic variables were not different among groups. Responses to phenylephrine in endothelium-denuded aortas was higher in VD3 compared to the C group (p = 0.041). Vascular relaxation induced by acetylcholine (p = 0.023) and sodium nitroprusside (p = 0.005) was impaired in both supplemented groups compared to the C group and apocynin treatment reversed impaired vasodilation. Collagen volume fraction (<0.001) and MMP-2 activity (p = 0.025) was higher in VD10 group compared to the VD3 group. Elastin volume fraction was lower in VD10 than in C and yield point was lower in VD3 than in C. Conclusion Our findings support the view that vitamin D supplementation increases arterial pressure in normotensive rats and this is associated with structural and functional vascular changes, modulated by NADPH oxidase, nitric oxide, and extracellular matrix components.

Nitrite or sildenafil, but not BAY 41-2272, blunt acute pulmonary embolism-induced increases in circulating matrix metalloproteinase-9 and oxidative stress

INTRODUCTION Inhibition of matrix metalloproteinases (MMPs) improves the hemodynamics during acute pulmonary embolism (APE) and oxidative stress upregulates MMPs. We compared the effects of different NO-cGMP pathway activators on APE-induced increases in MMPs. MATERIALS AND METHODS Hemodynamic and biochemical evaluations were performed in non-embolized dogs treated with saline (N=5), and in microspheres embolized dogs receiving saline (n=9), or nitrite (6.75 micromol/kg i.v. over 15 min followed by 0.28 micromol/kg/min; n=5), or sildenafil (0.25 mg/kg; n=5), or BAY 41-2272 (0.03, 0.1, 0.3, and 1 mg/kg/h; n=5). Plasma thiobarbituric acid reactive substances (TBARS) concentrations were determined. Zymograms of plasma samples were performed, and in vitro antioxidant effects or inhibition of MMPs by these drugs were examined. RESULTS APE increased mean pulmonary artery pressure by ~25 mmHg. Nitrite, BAY 41-2272, or sildenafil reversed this increase by ~40% (P<0.05). Similar effects were seen on the pulmonary vascular resistance. While both nitrite and sildenafil produced no systemic effects, the highest dose of BAY 41-2272 produced systemic hypotension (P<0.05). While nitrite and sildenafil blunted the increases in plasma pro-MMP-9 levels and TBARS (all P<0.05), BAY 41-2272 produced no such effects. Nitrite and sildenafil produced in vitro antioxidant effects and inhibited MMPs only at high concentrations. BAY 41-2272 produced no such effects. CONCLUSIONS Activation of the NO-cGMP pathway with nitrite or sildenafil, but not with BAY 41-2272, attenuates APE-induced oxidative stress and increased MMP-9 levels. These findings are consistent with the idea that NO-cGMP pathway activators with antioxidant effects prevent the release of MMP-9 during APE.

Evidence for the involvement of matrix metalloproteinases in the cardiovascular effects produced by nicotine.

Nicotine plays a role in smoking-associated cardiovascular diseases, and may upregulate matrix metalloproteinase (MMP)-2 and MMP-9. We examined whether nicotine induces the release of MMP-2 and MMP-9 by rat smooth muscle cells (SMC), and whether doxycycline (non-selective MMP inhibitor) inhibits the vascular effects produced by nicotine. SMC were incubated with nicotine 0, 50, and 150 nM for 48 h. MMP-2 and MMP-9 levels in the cell supernatants were determined by gelatin zymography. The acute changes in mean arterial pressure caused by nicotine 2 micromol/kg (or saline) were assessed in rats pretreated with doxycycline (or saline). We also examined whether doxcycline (30 mg/Kg, i.p., daily) modifies the effects of nicotine (10mg/kg/day; 4 weeks) on the endothelium-dependent relaxations of rat aortic rings. Aortic MMP-2 levels were assessed by gelatin zymography. Aortic gelatinolytic activity was assessed using a gelatinolytic activity kit. MMP-2 and MMP-9 levels increased in the supernatant of SMC cells incubated with nicotine 150 nM (P<0.05) but not with 50 nM. Nicotine (2 micromol/kg) produced lower increases in the mean arterial pressure in rats pretreated with doxycycline than those found in rats pretreated with saline (26+/-4 vs. 37+/-4 mm Hg, respectively; P<0.05). Nicotine impaired of the endothelium-dependent responses to acetylcholine, and treatment with doxycycline increased the potency (pD2) by approximately 25% (P<0.05). While we found no significant differences in aortic MMP-2 levels, nicotine significantly increased gelatinolytic activity (P<0.05). These findings suggest that nicotine produces cardiovascular effects involving MMPs. It is possible that MMPs inhibition may counteract the effects produced by nicotine.

Papel do óxido nítrico na regulação da circulação pulmonar: implicações fisiológicas, fisiopatológicas e terapêuticas

Nitric oxide (NO) is an endogenous vasoactive compound that contributes to pulmonary vascular homeostasis and is produced by three nitric oxide synthase (NOS) isoforms-neuronal NOS (nNOS); inducible NOS (iNOS); and endothelial NOS (eNOS)-all three of which are present in the lung. Studies using pharmacological inhibitors or knockout mice have shown that eNOS-derived NO plays an important role in modulating pulmonary vascular tone and attenuating pulmonary hypertension. However, studies focusing on the role of iNOS have shown that this isoform contributes to the pathophysiology of acute lung injury and acute respiratory distress syndrome. This review aimed at outlining the role played by NO in the control of pulmonary circulation, both under physiological and pathophysiological conditions. In addition, we review the evidence that the L-arginine-NO-cyclic guanosine monophosphate pathway is a major pharmacological target in the treatment of pulmonary vascular diseases.

The Nuclear Factor kappaB Inhibitor Pyrrolidine Dithiocarbamate Prevents Cardiac Remodelling and Matrix Metalloproteinase‐2 Up‐Regulation in Renovascular Hypertension

Imbalanced matrix metalloproteinase (MMP) activity is involved in hypertensive cardiac hypertrophy. Pharmacological inhibition of nuclear factor kappaB (NF‐кB) with pyrrolidine dithiocarbamate (PDTC) can prevent MMP up‐regulation. We suggested that treatment with PDTC could prevent 2‐kidney, 1‐clip (2K1C) hypertension‐induced left ventricular remodelling. Sham‐operated controls or 2K1C rats with hypertension received either vehicle or PDTC (100 mg/kg/day) by gavage for 8 weeks. Systolic blood pressure was monitored every week. Histological assessment of left ventricles was carried out with haematoxylin/eosin sections, and fibrosis was quantified in picrosirius red‐stained sections. Oxidative stress was evaluated in heart samples with the dihydroethidium probe. Cardiac MMP activity was determined by in situ zymography, and cardiac MMP‐2 was assessed by immunofluorescence. 2K1C surgery significantly increased systolic blood pressure in the 2K1C vehicle. PDTC exerted antihypertensive effects after 2 weeks of treatment. Histology revealed increased left ventricular and septum wall thickness associated with augmented myocyte diameter in hypertensive rats, which were reversed by treatment with PDTC. Hypertensive rats developed pronounced cardiac fibrosis with increased interstitial collagen area, increased cardiac reactive oxygen species levels, gelatinase activity and MMP‐2 expression. PDTC treatment decreased these alterations. These findings show that PDTC modulates myocardial MMP‐2 expression and ameliorates cardiac remodelling in renovascular hypertension. These results suggest that interfering with MMP expression at transcriptional level may be an interesting strategy in the therapy of organ damage associated with hypertension.

Spironolactone treatment attenuates vascular dysfunction in type 2 diabetic mice by decreasing oxidative stress and restoring NO/GC signaling.

Type 2 diabetes (DM2) increases the risk of cardiovascular disease. Aldosterone, which has pro-oxidative and pro-inflammatory effects in the cardiovascular system, is positively regulated in DM2. We assessed whether blockade of mineralocorticoid receptors (MR) with spironolactone decreases reactive oxygen species (ROS)-associated vascular dysfunction and improves vascular nitric oxide (NO) signaling in diabetes. Leptin receptor knockout [LepRdb/LepRdb (db/db)] mice, a model of DM2, and their counterpart controls [LepRdb/LepR+, (db/+) mice] received spironolactone (50 mg/kg body weight/day) or vehicle (ethanol 1%) via oral per gavage for 6 weeks. Spironolactone treatment abolished endothelial dysfunction and increased endothelial nitric oxide synthase (eNOS) phosphorylation (Ser1177) in arteries from db/db mice, determined by acetylcholine-induced relaxation and Western Blot analysis, respectively. MR antagonist therapy also abrogated augmented ROS-generation in aorta from diabetic mice, determined by lucigenin luminescence assay. Spironolactone treatment increased superoxide dismutase-1 and catalase expression, improved sodium nitroprusside and BAY 41-2272-induced relaxation, and increased soluble guanylyl cyclase (sGC) β subunit expression in arteries from db/db mice. Our results demonstrate that spironolactone decreases diabetes-associated vascular oxidative stress and prevents vascular dysfunction through processes involving increased expression of antioxidant enzymes and sGC. These findings further elucidate redox-sensitive mechanisms whereby spironolactone protects against vascular injury in diabetes.

mTOR Inhibition: A Promise for a Young Heart.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that senses nutritional and cellular energy status and regulates cell growth, proliferation, and survival. Rapamycin (sirolimus), a naturally occurring antifungal macrolide isolated from the bacterium Streptomyces hygroscopicus in a soil sample from Easter Island of the Pacific Ocean (or Rapa Nui in the native language; Vezina et al., 1975), inhibits mTOR interaction with other molecular components (Brown et al., 1994). Rapamycin was shown to extend life span in mice, even when administered late in life (Harrison et al., 2009), suggesting that inhibition of the mTOR pathway may prolong human life span. In this review, Ming et al. (2012) address the role of mTOR complex 1 and its downstream effector S6K1 (mTORC1–S6K1) signaling pathway in aging and age-associated diseases. The authors discuss the important new concept that augmented mTORC1–S6K1 signaling is not only critical in aging-related processes, but also provides a link between aging and cardiovascular disturbances, such as vascular and cardiac remodeling seen, e.g., in diabetes, arterial hypertension, atherosclerosis, and heart failure. It has been hypothesized that some dietary regimes, like caloric restriction and methionine restriction, extends lifespan by decreasing mTOR activity (Kaeberlein et al., 2005). Accordingly, a comparison between the beneficial effects of pharmacological intervention with rapamycin and life style modification (caloric restriction) is also provided by the authors. One should keep in mind that the effects produced by caloric restriction and inhibition mTOR signaling are not straightforward correlated. Unlike caloric restriction, rapamycin treatment does not reduce animal size (Harrison et al., 2009) and caloric restriction fails to extend life span when initiated late in life (Masoro, 2005). Mammalian target of rapamycin–S6K1 signaling and its specific inhibition emerge as a promising “treatment for aging,” mainly through the prevention or reversion of cardiovascular aging. However, only intense research will clarify whether potential adverse side effects of mTOR inhibitors, such as suppression of the immune system (Weir et al., 2010), impairment of glucose tolerance (Houde et al., 2010), can be overcome by the beneficial effects in the treatment of age-related diseases (especially if they are to be used as a prophylactic treatment). Future research targeting mTOR downstream proteins, which would exhibit more specific actions, will also clarify the relevance of mTOR inhibition for the prophylactic treatment of aging or age-related diseases.

Inducible Nitric Oxide Synthase Inhibition as a Target for the Treatment of Vascular Dysfunction in Hypertension

To the Editor: Since its discovery, NO has been recognized as a protective molecule for vascular function via its vasodilatory, antithrombotic, and antiapoptotic effects. The role of NO synthase (NOS) in the physiological control of vascular tone was clarified by experiments with the systemic administration of the pharmacological competitor NOS inhibitor, NG -nitro-l-arginine, which increases blood pressure in experimental animals. The spontaneous development of hypertension in mice with the endothelial isoform of NOS deleted demonstrated that endothelial cell–derived NO promotes vasodilation and opposes the development of hypertension. On the other hand, Smith et al1 demonstrated an upregulation of inducible NOS (iNOS) in microvessels of hypertensive subjects and an impressive restoration of …