Ming Sheng Zhou

Atorvastatin Prevents End-Organ Injury in Salt-Sensitive Hypertension Role of eNOS and Oxidant Stress

Statins, inhibitors of cholesterol biosynthesis, are endowed with pleiotropic effects that may contribute to their favorable clinical results. Hypertensive Dahl salt-sensitive (DS) rats have endothelial dysfunction and cardiorenal injury associated with decreased NO bioavailability and increased superoxide (O2−) production linked to a functional upregulation of angiotensin II. We investigated whether atorvastatin (30 mg/kg per day; by gavage) would prevent endothelial nitric oxide (eNOS) downregulation and the increase in O2− in DS rats, thereby reducing end-organ injury. DS rats given a high-salt diet (4% NaCl) for 10 weeks developed hypertension (systolic blood pressure [SBP] 200±8 versus 150±2 mm Hg in DS rats fed 0.5% NaCl diet [NS]; P <0.05), impaired endothelium-dependent relaxation, functional upregulation of endothelin-1, left ventricular hypertrophy (LVH; 30%), and proteinuria (167%), accompanied by downregulation of aortic eNOS activity (0.7±0.2 versus 1.8±0.3 nmol/min per gram protein in NS; P <0.05) and increased aortic O2−(2632±316 versus 1176±112 counts/min per milligram in NS; P <0.05) and plasma 8-F2&agr; isoprostanes. Atorvastatin prevented the decrease in eNOS activity (1.5±0.3 nmol/min per gram protein) as well as the increase in O2−(1192±243 counts/min per milligram) and plasma 8-F2&agr; isoprostanes, reduced LVH and proteinuria, and normalized endothelial function and vascular response to endothelin-1, although reduction in SBP was modest (174±8 mm Hg). Atorvastatin combined with removal of high salt normalized aortic eNOS activity, SBP, LVH, and proteinuria. These findings strongly suggest that concomitant prevention of vascular eNOS downregulation and inhibition of oxidative stress may contribute to the protection against end-organ injury afforded by this statin in salt-sensitive hypertension.

Reduced NAD(P)H Oxidase in Low Renin Hypertension: Link Among Angiotensin II, Atherogenesis, and Blood Pressure

Endothelial dysfunction (ED) complicates hypertension and is a precursor of atherosclerosis. Reduced NO bioactivity, because of increased reduced NAD(P)H oxidase–derived reactive oxygen species (ROS), plays a critical role in ED. gp91phox, predominantly expressed in the endothelium and adventitia, is a subunit of NAD(P)H oxidase important for its activation in response to angiotensin (Ang) II. Human atherosclerotic plaques are heavy laden with gp91phox. We have shown that in Dahl salt-sensitive (DS) rats, a paradigm of low renin salt-sensitive (SS) hypertension in humans, Ang II receptor blockade normalizes ROS production and endothelium-dependent relaxation (EDR) without significantly affecting systolic blood pressure (SBP). To additionally elucidate the mechanisms involved in the functional association of Ang II in SS hypertension, we administered a cell-permeable inhibitor of the assembly of p47phox with gp91phox in NAD(P)H oxidase, gp91ds-tat (10 mg/kg body weight, 3 weeks by minipump), to DS rats fed a 4% salt diet. Control rats received either vehicle or an inactive scramb-tat peptide. Vehicle-treated DS developed hypertension (SBP 168±5 mm Hg), left ventricular hypertrophy (LVH), proteinuria, impaired EDR, and increased aortic ROS production (superoxide 115% and peroxynitrite 157%) and expression of the proatherogenic molecules LOX-1 (130%) and MCP-1 (166%). gp91ds-tat, but not scramb-tat, normalized ROS and EDR, as well as LOX-1 and MCP-1, despite nonsignificant effects on SBP (159±5 mm Hg; P>0.05), left ventricular hypertrophy, and proteinuria. Our findings support the notion that in SS hypertension, activation of NAD(P)H oxidase promotes ED and atherogenesis via decreased nitric oxide bioactivity and increased LOX-1 and MCP-1, independent of blood pressure.

Renoprotection by statins is linked to a decrease in renal oxidative stress, TGF-β, and fibronectin with concomitant increase in nitric oxide bioavailability

Clinical and experimental studies have provided evidence suggesting that statins exert renoprotective effects. To investigate the mechanisms by which statins may exert renoprotection, we utilized the hypertensive Dahl salt-sensitive (DS) rat model, which manifests cardiovascular and renal injury linked to increased angiotensin II-dependent activation of NADPH oxidase and decreased nitric oxide (NO) bioavailability. DS rats given high salt diet (4% NaCl) for 10 wk exhibited hypertension [systolic blood pressure (SBP) 200 +/- 8 vs. 150 +/- 2 mmHg in normal salt diet (0.5% NaCl), P < 0.05], glomerulosclerosis, and proteinuria (158%). This was associated with increased renal oxidative stress demonstrated by urinary 8-F(2alpha)-isoprostane excretion and NADPH oxidase activity, increased protein expression of transforming growth factor (TGF)-beta (63%) and fibronectin (181%), increased mRNA expression of the proinflammatory molecules monocyte chemoattractant protein-1 (MCP-1) and lectin-like oxidized LDL receptor-1 (LOX-1), as well as downregulation of endothelial NO synthase (eNOS) activity (-44%) and protein expression. Return to normal salt had no effect on SBP or any of the measured parameters. Atorvastatin (30 mg.kg(-1).day(-1)) significantly attenuated proteinuria and glomerulosclerosis and normalized renal oxidative stress, TGF-beta1, fibronectin, MCP-1 and LOX-1 expression, and eNOS activity and expression. Atorvastatin-treated rats showed a modest reduction in SBP that remained in the hypertensive range (174 +/- 8 mmHg). Atorvastatin combined with removal of high salt normalized SBP and proteinuria. These findings suggest that statins mitigate hypertensive renal injury by restoring the balance among NO, TGF-beta1, and oxidative stress and explain the added renoprotective effects observed in clinical studies using statins in addition to inhibitors of the renin-angiotensin system.

Interaction between nitric oxide and angiotensin II in the endothelium: role in atherosclerosis and hypertension.

Background Although there is overwhelming evidence that hypertension promotes atherosclerosis, the relative contribution and/or interaction of vasoactive and hemodynamic factors remain undefined. Endothelial dysfunction complicates hypertension and is a precursor of atherosclerosis. It is characterized by a reduction in the bioavailability of vasodilators, particularly nitric oxide, and an increase in the activity of vasoconstrictors, including angiotensin (Ang) II and reactive oxygen species (ROS). Nitric oxide antagonizes the vasoconstrictive and pro-atherosclerotic effects of Ang II, whereas Ang II decreases nitric oxide bioavailability by promoting oxidative stress. Objectives The present review will focus on the interaction among nitric oxide, Ang II, and ROS in the endothelium and will examine their role in vascular tone and atherogenesis. In this context, studies from our laboratory will be reviewed demonstrating that salt-sensitive hypertension is a vascular diathesis characterized by a local activation of Ang II and NAD(P)H oxidase-derived ROS in the setting of insufficient nitric oxide. In hypertensive Dahl salt-sensitive rats, a paradigm of human salt-sensitive hypertension, inhibition of Ang II type 1 receptor or NAD(P)H oxidase-derived ROS prevented the development of endothelial dysfunction, upregulation of pro-atherogenic molecules, and vascular ROS generation, independently of blood pressure. Conclusions Salt sensitivity, an independent risk factor for increased cardiovascular morbidity and mortality, affects approximately 50% of hypertensives. Our studies suggest that, in salt-sensitive hypertension, atherogenesis is more closely linked to oxidative stress than to the hemodynamic stress of hypertension. To prevent or arrest atherosclerosis, antihypertensive therapy should aim at restoring the homeostatic balance between vasoactive factors in the vascular wall.

Vascular inflammation, insulin resistance, and endothelial dysfunction in salt-sensitive hypertension: role of nuclear factor kappa B activation

Objectives Activation of the nuclear factor kappa B (NFκB) inflammatory pathway by angiotensin II and reactive oxygen species may play an important role in the development of insulin resistance and cardiovascular injury in hypertensive and metabolic diseases. We have shown that in hypertensive Dahl salt-sensitive rats, upregulation of angiotensin II and reactive oxygen species contributed to increased vascular inflammatory gene expression, endothelial dysfunction, and insulin resistance. Herein, we investigated whether activation of NFκB contributes to the development of endothelial dysfunction, vascular injury, and vascular and peripheral insulin resistance in salt-sensitive hypertension. Methods Dahl salt-sensitive rats were fed a normal (0.5% NaCl) or high-salt diet (4% NaCl), or high-salt diet and pyrrolidine dithiocarbamat (150 mg/kg in drinking water), an inhibitor of NFκB activation, for 6 weeks. Results Hypertensive Dahl salt-sensitive rats manifested impaired endothelium-dependent relaxation to acetylcholine, aortic hypertrophy (35%), increased plasma C-reactive protein (25%), vascular superoxide (O2−) production (148%), and expression of monocyte chemoattractant protein-1, tumor necrosis factor alpha, phospho-IκBα, and phospho-(Ser536)-p65NFκB. Pyrrolidine dithiocarbamat significantly improved endothelium-dependent relaxation, reduced vascular O2−, and normalized aortic hypertrophy and systemic and local inflammation, despite only mildly reducing blood pressure. Hypertensive Dahl salt-sensitive rats also manifested impaired insulin-mediated vasorelaxation and Akt/endothelial nitric oxide synthase phosphorylation and decreased insulin sensitivity by hyperinsulinemic–euglycemic clamp (glucose infusion rate, −32%). Pyrrolidine dithiocarbamat significantly improved insulin-mediated vascular relaxation and Akt/endothelial nitric oxide synthase phosphorylation as well as insulin sensitivity. Conclusion The current findings strongly suggest that activation of the NFκB inflammatory pathway by angiotensin II-induced reactive oxygen species generation may importantly contribute to vascular injury, systemic inflammation, as well as vascular and peripheral insulin resistance in salt-sensitive hypertension.

Role of angiotensin II and oxidative stress in vascular insulin resistance linked to hypertension

Insulin activation of the phosphatidylinositol 3-kinase (PI3K) pathway stimulates glucose uptake in peripheral tissues and synthesis of nitric oxide (NO) in the endothelium. Insulin resistance (IR) and hypertension frequently coexist, particularly among individuals with salt-sensitive hypertension. The mechanisms underlying this association are poorly understood. We investigated these mechanisms in a model of salt-sensitive hypertension in which we have previously shown that endothelial dysfunction is mediated by superoxide anion (O(2)(-)) linked to local ANG II. Dahl salt-sensitive rats were fed, for 6 wk, a normal salt diet (NS; 0.5% NaCl), high-salt diet (HS; 4% NaCl), HS plus the ANG II type 1 receptor (AT(1)R) blocker (ARB) candesartan (10 mg.kg(-1).day(-1)), or HS plus the antioxidant tempol (172 mg/l in drinking water). Hypertensive (mean arterial pressure: 145 +/- 4 vs. 102 +/- 5 mmHg in NS, P < 0.05) rats manifested increased aortic AT(1)R mRNA (210%) and protein (101%) expression and O(2)(-) production (104%) and impaired endothelium-dependent relaxation (EDR) to acetylcholine [maximal response (E(max)): 68 +/- 9 vs. 91 +/- 8% in NS, P < 0.05]. ARB or tempol normalized O(2)(-) and EDR despite that they did not normalize mean arterial pressure, which was reduced only 25%. Hypertensive rats manifested metabolic IR (36% reduction in the glucose infusion rate by insulin clamp), impaired NO-mediated insulin-induced EDR (E(max): 12 +/- 5 vs. 32 +/- 4% in NS, P < 0.05), and impaired insulin activation of PI3K/endothelial NO synthase. ARB or tempol improved insulin-mediated EDR, PI3K, Akt/ endothelial NO synthase phosphorylation, and metabolic IR (all P < 0.05). This study provides insight into the mechanisms that underlie the association between metabolic and hypertensive cardiovascular diseases and support the notion that O(2)(-) overproduction linked to tissue ANG II interferes with shared insulin signaling pathways in metabolic and cardiovascular tissues.

Thiazide diuretics, endothelial function, and vascular oxidative stress.

Objectives Increased endothelial production of reactive oxygen species and decreased nitric oxide bioactivity, associated with the upregulation of monocyte chemoattractant protein (MCP)-1 and lectin-like oxidized low-density lipoprotein receptor (LOX)-1, link hypertension with atherogenesis. We investigated whether the beneficial effects of thiazide diuretics are exclusively related to a reduction in the biomechanical stress of hypertension or are also endowed with pleiotropic vasculoprotective effects that are independent of their effect upon blood pressure. Methods Dahl salt-sensitive (DSS) rats, a paradigm of human salt-sensitive hypertension, were given a diet with normal salt (0.5% NaCl), high salt (4% NaCl), or a high salt diet plus either hydrochlorothiazide 75 mg/l, chlorthalidone 37 or 75 mg/l in their drinking water for 6 weeks. We determined systolic blood pressure (SBP), left ventricular hypertrophy (LVH), proteinuria, aortic superoxide anion (O2−) production, endothelium-dependent relaxation (EDR) to acetylcholine, and aortic angiotensin II type 1 (AT1) receptor, LOX-1, and MCP-1 messenger RNA expression (by real-time polymerase chain reaction). Results DSS rats on a high salt diet developed hypertension, LVH, proteinuria, increased production of aortic O2− (106%), impaired EDR, and aortic upregulation of AT1 receptor (198%), LOX-1 (135%), and MCP-1 (145%). Hydrochlorothiazide as well as the high and low dose of chlorthalidone reduced SBP, LVH, and proteinuria, but did not reduce O2− production, AT1 receptor, LOX-1, or MCP-1 expression, or improved EDR. Conclusions This study demonstrates that thiazide diuretics do not reduce oxidative stress, improve endothelial function, or prevent the expression of pro-atherogenic molecules. We conclude that thiazide diuretics may not fully provide long-term global cardiovascular protection beyond lowering blood pressure.

Link between the renin-angiotensin system and insulin resistance: Implications for cardiovascular disease

The incidence of metabolic syndrome is rapidly increasing in the United States and worldwide. The metabolic syndrome is a complex metabolic and vascular disorder that is associated with inappropriate activation of the renin–angiotensin–aldosterone system (RAAS) in the cardiovascular (CV) system and increased CV morbidity and mortality. Insulin activation of the phosphatidylinositol-3-kinase (PI3K) pathway promotes nitric oxide (NO) production in the endothelium and glucose uptake in insulin-sensitive tissues. Angiotensin (Ang) II inhibits insulin-mediated PI3K pathway activation, thereby impairing endothelial NO production and Glut-4 translocation in insulin-sensitive tissues, which results in vascular and systemic insulin resistance, respectively. On the other hand, Ang II enhances insulin-mediated activation of the mitogen-activated protein kinase (MAPK) pathway, which leads to vasoconstriction and pathologic vascular cellular growth. Therefore, the interaction of Ang II with insulin signaling is fully operative not only in insulin-sensitive tissues but also in CV tissues, thereby linking insulin resistance and CV disease. This notion is further supported by an increasing number of experimental and clinical studies indicating that pharmacological blockade of RAAS improves insulin sensitivity and endothelial function, as well as reduces the incidence of new-onset diabetes in high-risk patients with CV disease. This article reviews experimental and clinical data elucidating the physiological and pathophysiological role of the interaction between insulin and RAAS in the development of insulin resistance as well as CV disease.

Vascular but not cardiac remodeling is associated with superoxide production in angiotensin II hypertension.

Objective Angiotensin (Ang) II increases reactive oxygen species (ROS), decreases nitric oxide (NO) bioavailability and promotes cardiovascular remodeling. ROS have been identified as critical second messengers of the trophic responses by Ang II. In rats with Ang II-induced hypertension, we investigated the role of ROS in cardiac hypertrophy as well as the remodeling of aortas and mesenteric (resistance) arteries. Methods Sprague–Dawley rats received Ang II (0.7 mg/kg per day by mini-pump, n = 7) or vehicle (n = 7) for 5 days. Endothelium-dependent relaxation to acetylcholine (EDR) in aortas was determined in organ baths and in mesenteric resistance vessels in a pressurized myograph. Superoxide (O2−) production was measured by lucigenin chemiluminescence, laser-confocal fluorescence microscopy (LCM) and NADPH oxidase assay. Results Ang II-treated rats developed hypertension (183 ± 3 versus 138 ± 4 mmHg, P < 0.05), increased aortic O2− (50%), aortic hypertrophy (12%) and impaired EDR. Mesenteric arteries manifested impaired EDR, increased NADPH oxidase activity (356%) and eutrophic inward remodeling (decreased lumen diameter and increased wall/lumen ratio). However, although Ang II-treated rats developed cardiac hypertrophy (13%), this was not accompanied by an increase in cardiac O2−, as measured by lucigenin, LCM or NADPH oxidase assay. On the other hand, cardiac calcineurin, a molecule that promotes cardiac hypertrophy linked to Ang II, was increased by 40% (52 ± 8 versus 33 ± 5 pmol/min per mg protein, P < 0.05). Conclusion These studies demonstrate that the role of ROS in Ang II-induced vascular remodeling differ across vascular territories. Although in conduit and resistance vessels, vascular hypertrophy and endothelial dysfunction are linked to increased ROS production, cardiac hypertrophy is not. Instead, cardiac hypertrophy is associated, at least in part, with an increase in calcineurin. These studies unveil novel mechanisms that may play an important role in the pathogenesis of cardiac and vascular injury in hypertension.

Altered Renal Expression of Angiotensin II Receptors, Renin Receptor, and ACE-2 Precede the Development of Renal Fibrosis in Aging Rats

Background: The susceptibility to fibrosis and progression of renal disease is mitigated by inhibition of the renin-angiotensin system (RAS). We hypothesized that activation of the intrarenal RAS predisposes to renal fibrosis in aging. Methods: Intrarenal expression of angiotensin II type 1 (AT1R), type 2 (AT2R), and (pro)renin receptors, ACE and ACE-2, as well as pro- and antioxidant enzymes were measured in 3-month-old (young), 14-month-old (middle-aged), and 24-month-old (old) male Sprague-Dawley rats. Results: Old rats manifested glomerulosclerosis and severe tubulointerstitial fibrosis with increased fibronectin and TGF-β expression (7-fold). AT1R /AT2R ratios were increased in middle-aged (cortical 1.6-fold, medullary 5-fold) and old rats (cortical 2-fold, medullary 4-fold). Similarly, (pro)renin receptor expression was increased in middle-aged (cortical 2-fold, medullary 3-fold) and old (cortical 5-fold, medullary 3-fold) rats. Cortical ACE was increased (+35%) in old rats, whereas ACE-2 was decreased (–50%) in middle-aged and old rats. NADPH oxidase activity was increased (2-fold), whereas antioxidant capacity and expression of the mitochondrial enzyme manganese superoxide dismutase (cortical –40%, medullary –53%) and medullary endothelial nitric oxide synthase (–48%) were decreased in old rats. Conclusion: Age-related intrarenal activation of the RAS preceded the development of severe renal fibrosis, suggesting that it contributes to the increased susceptibility to renal injury observed in the elderly.