R. William Caldwell

Pravastatin sodium activates endothelial nitric oxide synthase independent of its cholesterol-lowering actions.

OBJECTIVES We tested the hypothesis that pravastatin (PRA) activates endothelial nitric oxide synthase (eNOS). BACKGROUND Pravastatin has been found to have clinical benefits beyond those predicted by its actions in reducing plasma low density lipoprotein cholesterol (LDL). Both PRA and simvastatin (SIM) are equally effective in reducing LDL, but only PRA reduces platelet aggregation and is an effective vasodilator. Nitric oxide (NO) also inhibits platelet aggregation and vasodilates. METHODS We determined PRA and SIM effects on vasorelaxation in aortic rings and NO production by cultured bovine aortic endothelial cells. Nitric oxide was measured by using a NO electrode and by an assay for conversion of hemoglobin to methemoglobin. Specificity of NOS activation was tested by using the NOS inhibitor nitro-L-arginine methyl ester (L-NAME, 1 mmol/liter) in the presence or absence of excess L-arginine (L-ARG, 1 mmol/liter). RESULTS Endothelium-dependent vasorelaxation was maximal with acetylocholine (ACH, 100%), followed by PRA (62.8%) and then SIM (37.1%). Direct measurement of NO confirmed that vasorelaxation is due to NO release and showed that PRA and ACH had similar dose-dependent effects on NO production, while SIM was only 25% to 30% as effective. Methemoglobin assay confirmed these results and demonstrated their specificity for NOS activity. The L-NAME blunted the responses to 45% of initial values. Excess L-ARG reversed this effect and potentiated NO production to 133% of initial levels. CONCLUSIONS Both PRA and SIM activate eNOS, but SIM is much less effective. Clinical benefits with PRA not explained by LDL reductions may be the result of an independent action of PRA on eNOS activation.

Vascular endothelial growth factor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives.

Retinal neovascularization and macular edema are central features of diabetic retinopathy, the major cause of blindness in the developed world. Current treatments are limited in their efficacy and are associated with significant adverse effects. Characterization of the molecular and cellular processes involved in vascular growth and permeability has led to the recognition that the angiogenic growth factor and vascular permeability factor vascular endothelial growth factor (VEGF) plays a pivotal role in the retinal microvascular complications of diabetes. Therefore, VEGF represents an exciting target for therapeutic intervention in diabetic retinopathy. This review highlights the current understanding of the mechanisms that regulate VEGF gene expression and mediate its biological effects and how these processes may become altered during diabetes. The cellular and molecular alterations that characterize experimental models of diabetes are considered in relation to the influence of high glucose‐mediated oxidative stress on VEGF expression and on the mechanisms of VEGFs actions under hyperglycemic induction. Finally, potential therapeutic strategies for preventing VEGF overexpression or blocking its pathological effects in the diabetic retina are considered. Copyright

Role of NADPH Oxidase and Stat3 in Statin-Mediated Protection against Diabetic Retinopathy

PURPOSE Inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (statins) reduce signs of diabetic retinopathy in diabetic patients and animals. Indirect clinical evidence supports the actions of statins in improving cardiovascular function, but the mechanisms of their protective actions in the retina are not understood. Prior studies have implicated oxidative stress and NADPH oxidase-mediated activation of signal transducer and activator of transcription 3 (STAT3) in diabetes-induced increases in expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1 and breakdown of the blood-retinal barrier (BRB). Because statins are known to be potent antioxidants, the hypothesis for the current study was that the protective effects of statins in preventing diabetic retinopathy involve blockade of diabetes-induced activation of NADPH oxidase and STAT3. METHODS The hypothesis was tested by experiments in which rats with streptozotocin (STZ)-induced diabetes and retinal endothelial cells maintained in high-glucose medium were treated with simvastatin. Blood-retinal barrier (BRB) function was assayed by determining extravasation of albumin. Oxidative stress was assayed by measuring lipid peroxidation, protein nitration of tyrosine, dihydroethidine oxidation, and chemiluminescence. Immunoprobe techniques were used to determine the levels of NADPH oxidase subunit expression and STAT3 activation. RESULTS These studies showed that simvastatin blocks diabetes or high-glucose-induced increases in VEGF and ICAM-1 and preserves the BRB by a process involving blockade of diabetes/high-glucose-induced activation of STAT3 and NADPH oxidase. Statin treatment also prevents diabetes-induced increases in expression of the NADPH oxidase catalytic and subunit NOX2. CONCLUSIONS These results suggest that simvastatin protects against the early signs of diabetic retinopathy by preventing NADPH oxidase-mediated activation of STAT3.

Angiotensin II-induced vascular endothelial dysfunction through RhoA/Rho kinase/p38 mitogen-activated protein kinase/arginase pathway

Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 μM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 μM). Blockade of ANG II AT(1) receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 μM) or Rho kinase (ROCK) (Y-27632, 10 μM; H1152, 0.5 μM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 μM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 μg·kg(-1)·day(-1)) or arginase (ABH, 8 mg·kg(-1)·day(-1)) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT(1) receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.

Downregulation of nitrovasodilator‐induced cyclic GMP accumulation in cells exposed to endotoxin or interleukin‐1β

1 Induction of nitric oxide synthase (iNOS) results in overproduction of nitric oxide (NO), which may be a principal cause of the massive vasodilatation and hypotension observed in septic shock. Since NO‐induced vasorelaxation is mediated via the soluble isoform of guanylate cyclase (sGC), the regulation of sGC activity during shock is of obvious importance, but yet poorly understood. The aim of the present study was to investigate the activation of sGC by sodium nitroprusside (SNP) before and after exposure of rat aortic smooth muscle cells to endotoxin (LPS) or interleukin‐1β (IL‐1β). 2 Exposure of rat aortic smooth muscle cells to SNP (10 μm) elicited up to 200 fold increases in cyclic GMP. This effect was attenuated by 30–70% in IL‐1β‐ or LPS‐pretreated cells, in a pretreatment time‐and IL‐1β‐ or LPS‐concentration‐dependent manner. When, however, cells were exposed to IL‐1β or LPS and then stimulated with the particulate guanylate cyclase activator, atriopeptin II, no reduction in cyclic GMP accumulation was observed. 3 Pretreatment of rats with LPS (5 mg kg−1, i.v.) for 6 h led to a decrease in aortic ring SNP‐induced cyclic GMP accumulation. 4 The IL‐1β‐induced reduction in SNP‐stimulated cyclic GMP accumulation in cultured cells was dependent on NO production, as arginine depletion abolished the downregulation of cyclic GMP accumulation in response to SNP. 5 Reverse‐transcriptase‐polymerase chain reaction analysis revealed that the ratio of steady state mRNA for the α1 subunit of sGC to glyceraldehyde phosphate dehydrogenase was decreased in LPS‐ or IL‐1β‐treated cells, as compared to vehicle‐treated cells. 6 Protein levels of the α1 sGC subunit remained unaltered upon exposure to LPS or IL‐1β, suggesting that the early decreased cyclic GMP accumulation in IL‐1β‐ or LPS‐pretreated cells was probably due to reduced sGC activation. Thus, the observed decreased responsiveness of sGC to NO stimulation following cytokine or LPS challenge may represent an important homeostatic mechanism to offset the extensive vasodilatation seen in sepsis.

Arginase: an old enzyme with new tricks

Arginase has roots in early life-forms. It converts L-arginine to urea and ornithine. The former provides protection against NH3; the latter serves to stimulate cell growth and other physiological functions. Excessive arginase activity in mammals has been associated with cardiovascular and nervous system dysfunction and disease. Two relevant aspects of this elevated activity may be involved in these disease states. First, excessive arginase activity reduces the supply of L-arginine needed by nitric oxide (NO) synthase to produce NO. Second, excessive production of ornithine leads to vascular structural problems and neural toxicity. Recent research has identified inflammatory agents and reactive oxygen species (ROS) as drivers of this pathologic elevation of arginase activity and expression. We review the involvement of arginase in cardiovascular and nervous system dysfunction, and discuss potential therapeutic interventions targeting excess arginase.

Simvastatin Improves Diabetes-Induced Coronary Endothelial Dysfunction

3-Hydroxy-3-methylglutaryl CoA reductase inhibitors decrease cardiovascular morbidity in diabetic patients, but the mechanism is unclear. We studied the actions of simvastatin (SIM) in enhancing NO bioavailability and reducing oxidative stress in coronary vessels from diabetic rats and in rat coronary artery endothelial cells (RCAEC) exposed to high glucose. Coronary arteries isolated from diabetic rats showed decreases in acetylcholine (ACh)-mediated maximal relaxation from 81.0 ± 4.5% in controls to 43.5 ± 7.6% at 4 weeks and 22.3 ± 0.6% at 10 weeks of diabetes. This effect was associated with oxidative stress in coronary vessels as shown by dichlorofluorescein (DCF) imaging and nitrotyrosine labeling. Diabetes also reduced trans-coronary uptake of [3H]l-arginine. Supplemental l-arginine (50 mg/kg/day p.o.) did not improve coronary vasorelaxation to ACh. However, SIM treatment (5 mg/kg/day subcutaneously) improved maximal ACh relaxation to 65.8 ± 5.1% at 4 weeks and 47.1 ± 3.9% at 10 weeks. Coronary arteries from rats treated with both SIM and l-arginine demonstrated the same maximal relaxation to ACh (66.1 ± 3%) as SIM alone. Mevalonate and l-NAME (Nω-nitro-l-arginine methyl ester hydrochloride) inhibited the response to ACh in SIM-treated diabetic rats. Coronary arteries from all groups relaxed similarly to sodium nitroprusside. SIM increased endothelial NO synthase protein levels and blocked diabetes-induced increases in DCF and nitrotyrosine labeling in diabetic coronary vessels. SIM treatment restored normal NO levels in media from high-glucose-treated RCAEC and plasma of diabetic rat. Treatment with SIM or the NADPH oxidase inhibitor apocynin also blocked high-glucose-induced increases in reactive oxygen species and superoxide formation in RCAEC. Taken together, these data suggest that SIM improves diabetes-induced coronary dysfunction by reducing oxidative stress and increasing NO bioavailability.

Calpain mediates pulmonary vascular remodeling in rodent models of pulmonary hypertension, and its inhibition attenuates pathologic features of disease

Pulmonary hypertension is a severe and progressive disease, a key feature of which is pulmonary vascular remodeling. Several growth factors, including EGF, PDGF, and TGF-β1, are involved in pulmonary vascular remodeling during pulmonary hypertension. However, increased knowledge of the downstream signaling cascades is needed if effective clinical interventions are to be developed. In this context, calpain provides an interesting candidate therapeutic target, since it is activated by EGF and PDGF and has been reported to activate TGF-β1. Thus, in this study, we examined the role of calpain in pulmonary vascular remodeling in two rodent models of pulmonary hypertension. These data showed that attenuated calpain activity in calpain-knockout mice or rats treated with a calpain inhibitor resulted in prevention of increased right ventricular systolic pressure, right ventricular hypertrophy, as well as collagen deposition and thickening of pulmonary arterioles in models of hypoxia- and monocrotaline-induced pulmonary hypertension. Additionally, inhibition of calpain in vitro blocked intracellular activation of TGF-β1, which led to attenuated Smad2/3 phosphorylation and collagen synthesis. Finally, smooth muscle cells of pulmonary arterioles from patients with pulmonary arterial hypertension showed higher levels of calpain activation and intracellular active TGF-β. Our data provide evidence that calpain mediates EGF- and PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells via an intracrine TGF-β1 pathway in pulmonary hypertension.

Peroxynitrite mediates diabetes-induced endothelial dysfunction: possible role of Rho kinase activation.

Endothelial dysfunction is characterized by reduced bioavailability of NO due to its inactivation to form peroxynitrite or reduced expression of eNOS. Here, we examine the causal role of peroxynitrite in mediating diabetes-induced endothelial dysfunction. Diabetes was induced by STZ-injection, and rats received the peroxynitrite decomposition catalyst (FeTTPs, 15 mg/Kg/day) for 4 weeks. Vasorelaxation to acetylcholine, oxidative-stress markers, RhoA activity, and eNOS expression were determined. Diabetic coronary arteries showed significant reduction in ACh-mediated maximal relaxation compared to controls. Diabetic vessels showed also significant increases in lipid-peroxides, nitrotyrosine, and active RhoA and 50% reduction in eNOS mRNA expression. Treatment of diabetic animals with FeTTPS blocked these effects. Studies in aortic endothelial cells show that high glucose or peroxynitrite increases the active RhoA kinase levels and decreases eNOS expression and NO levels, which were reversed with blocking peroxynitrite or Rho kinase. Together, peroxynitrite can suppress eNOS expression via activation of RhoA and hence cause vascular dysfunction.

Prevention of diabetes-induced arginase activation and vascular dysfunction by Rho kinase (ROCK) knockout

AIMS We determined the role of the Rho kinase (ROCK) isoforms in diabetes-induced vascular endothelial dysfunction and enhancement of arginase activity and expression. METHODS AND RESULTS Studies were performed in aortic tissues from haplo-insufficient (H-I) ROCK1 and ROCK2 mice and wild-type (WT) mice rendered diabetic with streptozotocin and in bovine aortic endothelial cells (BAECs) treated with high glucose (HG, 25 mM). Protein expression of both ROCK isoforms was substantially elevated in aortas of WT mice after 8 weeks of diabetes and in BAECs after 48 h in HG. Impairment of endothelium-dependent vasorelaxation of aortas was observed in diabetic WT mice. However, there was no impairment in aortas of diabetic ROCK1 H-I mice and less impairment in aortas of diabetic ROCK2 H-I mice, compared with non-diabetic mice. These vascular effects were associated with the prevention of diabetes-induced decrease in nitric oxide (NO) production and a rise in arginase activity/expression. Acute treatment with the arginase inhibitor, BEC, improved endothelium-dependent vasorelaxation of aortas of both diabetic WT and ROCK2, but not of ROCK1 mice. CONCLUSION Partial deletion of either ROCK isoform, but to a greater extent ROCK1, attenuates diabetes-induced vascular endothelial dysfunction by preventing increased arginase activity and expression and reduction in NO production in type 1 diabetes. Limiting ROCK and arginase activity improves vascular function in diabetes.