Abstract-Proliferation of pulmonary arterial smooth muscle cells, endothelial dysfunction, oxidative stress, and inflammation promotes the development of pulmonary hypertension. Resveratrol is a polyphenolic compound that exerts antioxidant and anti-inflammatory protective effects in the systemic circulation, but its effects on pulmonary arteries remain poorly defined. The present study was undertaken to investigate the efficacy of resveratrol to prevent pulmonary hypertension. Rats injected with monocrotaline progressively developed pulmonary hypertension. Resveratrol treatment (25 mg/kg per day, PO, from day 1 postmonocrotaline) attenuated right ventricular systolic pressure and pulmonary arterial remodeling, decreased expression of inflammatory cytokines (tumor necrosis factor-␣, interleukin 1␤, interleukin 6, and platelet-derived growth factor-␣/␤), and limited leukocyte infiltration in the lung. Resveratrol also inhibited proliferation of pulmonary arterial smooth muscle cells. Treatment of rats with resveratrol increased expression of endothelial NO synthase, decreased oxidative stress, and improved endothelial function in small pulmonary arteries. Pulmonary hypertension was associated with an upregulation of NAD(P)H oxidase in small pulmonary arteries, which was significantly attenuated by resveratrol treatment. Our studies show that resveratrol exerts anti-inflammatory, antioxidant, and antiproliferative effects in the pulmonary arteries, which may contribute to the prevention of pulmonary hypertension. Key Words: pulmonary hypertension Ⅲ resveratrol Ⅲ oxidative stress Ⅲ endothelial dysfunction Ⅲ inflammation P ulmonary hypertension is a syndrome that encompasses several diseases, all of which have in common increased pulmonary artery pressures. Idiopathic ("primary") pulmonary hypertension is a rare disease caused by genetic defects in the bone morphogenetic protein signaling pathways. Common causes of "secondary" forms of pulmonary hypertension include the following: (1) pulmonary hypertension associated with chronic obstructive pulmonary disease; (2) pulmonary embolism; and (3) pressure/volume overload-related pulmonary hypertension. In addition, pulmonary hypertension often develops in patients with autoimmune diseases or as a severe adverse effect of anorectic drug treatment. Despite the diverse etiologic differences, many similarities in the pathological alterations in pulmonary arteries occur among the various forms of pulmonary hypertension, ie, vascular remodeling, including cellular proliferation in both the intima and media; endothelial dysfunction/increased vasoconstriction; and activation of inflammatory processes (eg, inflammatory cytokine expression and monocyte infiltration).Current therapies for chronic pulmonary hypertension are designed to reduce pulmonary arterial resistance by inducing vasodilation (eg, NO inhalation, stimulation of cGMP production by phosphodiesterase inhibitors, endothelin receptor antagonists, and prostacyclin analogs). These therapeutic approaches mainly provide ...

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its vasculoprotective effects are not completely understood. Because oxidative stress and endothelial cell injury play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits oxidative stress-induced endothelial apoptosis. We found that oxidized LDL and TNF-␣ elicited significant increases in caspase-3/7 activity in endothelial cells and cultured rat aortas, which were prevented by resveratrol pretreatment (10 Ϫ6 -10 Ϫ4 mol/l). The protective effect of resveratrol was attenuated by inhibition of glutathione peroxidase and heme oxygenase-1, suggesting a role for antioxidant systems in the antiapoptotic action of resveratrol. Indeed, resveratrol treatment protected cultured aortic segments and/or endothelial cells against increases in intracellular H 2O2 levels and H2O2-mediated apoptotic cell death induced by oxidative stressors (exogenous H 2O2, paraquat, and UV light). Resveratrol treatment also attenuated UV-induced DNA damage (comet assay). Resveratrol treatment upregulated the expression of glutathione peroxidase, catalase, and heme oxygenase-1 in cultured arteries, whereas it had no significant effect on the expression of SOD isoforms. Resveratrol also effectively scavenged H2O2 in vitro. Thus resveratrol seems to increase vascular oxidative stress resistance by scavenging H2O2 and preventing oxidative stress-induced endothelial cell death. We propose that the antioxidant and antiapoptotic effects of resveratrol, together with its previously described anti-inflammatory actions, are responsible, at least in part, for its cardioprotective effects. endothelial cell; comet assay; caloric restriction mimetics; apoptosis; polyphenol; heme oxygenase antioxidant EPIDEMIOLOGICAL STUDIES have shown that, in southern France and other Mediterranean territories, the morbidity and mortality of coronary artery disease is low, despite a diet rich in saturated fats and smoking habits (17,30). This unexpected epidemiological finding was termed the "French paradox." It has been proposed that resveratrol, an important constituent of Mediterranean diets, is involved in vasculoprotection. Resveratrol has been identified in more than 70 species of plants, including grapevines (Vitis vinifera), mulberries (Morus rubra), Vaccinum species, and peanuts (Arachis hypogea), and it is thought to have diverse antiatherogenic activities (42, 55-57, 60, 61), such as the inhibition of LDL oxidation (22) and platelet aggregation (46) and regulation of vascular smooth muscle proliferation (24 -26, 54). Recently, studies from this and other laboratories have shown that resveratrol inhibits endothelial activation and monocyte adhesion (12, 21, 39) and attenuates proinflammatory gene expression by inhibition of NF-B activation in coronary arterial endothelial cells (12).There is overwhelming evidence that oxidative stres...

Protein kinase C (PKC) stimulation of NAD(P)H oxidases (Nox) is an important component of multiple vascular disease processes; however, the relationship between oxidase activation and the regulation of vascular smooth muscle contraction by PKC remains poorly understood. Therefore, we examined the signaling cascade of PKC-elicited Nox activation and the role of superoxide and hydrogen peroxide in mediating PKC-induced vascular contraction. Endothelium-denuded bovine coronary arteries showed a PKC-dependent basal production of lucigenin (5 muM)-detected Nox oxidase-derived superoxide, which was stimulated fourfold by PKC activation with 10 muM phorbol 12,13-dibutyrate (PDBu). PDBu appeared to increase superoxide generation by Nox2 through both p47(phox) and peroxide-dependent Src activation mechanisms based on the actions of inhibitors, properties of Src phosphorylation, and the loss of responses in aorta from mice deficient in Nox2 and p47(phox). The actions of inhibitors of contractile regulating mechanisms, scavengers of superoxide and peroxide, and responses in knockout mouse aortas suggest that a major component of the contraction elicited by PDBu appeared to be mediated through peroxide derived from Nox2 activation stimulating force generation through Rho kinase and calmodulin kinase-II mechanisms. Superoxide generated by PDBu also attenuated relaxation to nitroglycerin. Peroxide-derived from Nox2 activation by PKC appeared to be a major contributor to the thromboxane A(2) receptor agonist U46619 (100 nM)-elicited contraction of coronary arteries. Thus a p47(phox) and Src kinase activation of peroxide production by Nox2 appears to be an important contributor to vascular contractile mechanisms mediated through activation of PKC.

Reduced bone morphogenetic protein (BMP) receptor (BMPR) expression and BMP signaling have been implicated in vascular cell proliferation and remodeling associated with pulmonary arterial hypertension (PAH). The low penetrance of the BMPR II disease gene in familial PAH suggests that additional genetic or environmental factors are involved in clinical manifestation of PAH. Smurf1 ubiquitin ligase, together with inhibitory SMAD 6/7, forms a negative feedback loop for the attenuation of BMP signals by downregulating BMPR and signaling molecules and, in addition, functions in the integration of MAPK/Ras mitogenic pathways. The present study found that Smurf1 was significantly elevated in pulmonary arteries of monocrotaline and hypoxia-induced PAH rats. In the pulmonary artery of hypoxia-exposed mice, elevation of Smurf1 and SMAD7 was correlated with reduced expression of BMPR II protein. Over-expression of Smurf1 in cultured cells induced ubiquitination and degradation of BMPR I and II whereas ligase-inactive Smurf1 reduced ubiquitination and elevated their protein levels, thus serving a dominant-negative function. Smurf1-induced receptor degradation was inhibited by both proteasomal and lysosomal inhibitors. Thus, Smurf1 reduces steady-state levels of BMPRs by ubiquitination and subsequent degradation involving proteasomes and lysosomes. Therefore, these results show that Smurf1 induction could be a key event for triggering downregulation of BMP signaling and causing vascular cell proliferation and remodeling in PAH and that abrogating Smurf1 function could be a strategy for PAH therapeutics.

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We previously reported that all-trans retinoic acid (RA) and fenretinide (4HPR) suppress HL-60 leukemia cell growth and cause partial cell arrest in the G 1 -to-S phase. Moreover, 4HPR but not RA induces apoptosis in HL-60 cells. To investigate further the observed biological effects, cyclin D1 and cdk4 expression and the level of phosphorylation of the retinoblastoma protein Rb were assessed. Cyclin D1 and cdk4 expression and Rb phosphorylation were significantly reduced, by 40-75%, after 24 hr of treatment with RA or 4HPR; these decreases were either transient, e.g., only at 24 hr for cdk4, or sustained for 72 hr. In general, more pronounced decreases were seen in the 4HPR-treated cells. Evidence for 4HPR-induced apoptosis comes from (1) cleavage of the enzyme poly(ADP-ribose) polymerase (PARP) to an 89-kDa truncated product, (2) appearance of DNA ladders on agarose gel electrophoresis, and (3) higher incorporation in situ of digoxigenin nucleotides into the free 3'-ends of DNA. Overnight pretreatment with 0.5-5.0 µM of the CPP32 inhibitor DEVD, but not the ICE inhibitor YVAD, significantly reduced the specific processing of PARP, suggesting that CPP32 is involved in the mechanism of action of 4HPR. Analysis of 2 lipid-derived second messengers, ceramide and diacylglycerol (DAG), as a function of time of treatment with RA or 4HPR, showed ceramide but not DAG to be significantly albeit transiently increased 2-fold at 3 hr, by 4HPR. To test further whether ceramide may be involved in the signaling cascade that culminates in the induction of apoptosis in 4HPR-treated HL-60 cells, the effects of fumonisin B 1 , an inhibitor of ceramide synthase, were studied. Simultaneous treatment of cells with 4HPR and 25-100 µM fumonisin B 1 resulted in a dose-dependent reduction in the elevation in ceramide, the extent of PARP cleavage, and induction of apoptosis. Pretreatment with DEVD or YVAD, on the other hand, had no effect on the 4HPR-induced increase in ceramide. Int.

/ ajplung.00312.2003.-We previously reported that angiotensin II stimulates an increase in nitric oxide production in pulmonary artery endothelial cells. The aims of this study were to determine which receptor subtype mediates the angiotensin II-dependent increase in nitric oxide production and to investigate the roles of the angiotensin type 1 and type 2 receptors in modulating angiotensin II-dependent vasoconstriction in pulmonary arteries. Pulmonary artery endothelial cells express both angiotensin II type 1 and type 2 receptors as assessed by RT-PCR, Western blot analysis, and flow cytometry. Treatment of the endothelial cells with PD-123319, a type 2 receptor antagonist, prevented the angiotensin II-dependent increase in nitric oxide synthase mRNA, protein levels, and nitric oxide production. In contrast, the type 1 receptor antagonist losartan enhanced nitric oxide synthase mRNA levels, protein expression, and nitric oxide production. Pretreatment of the endothelial cells with either PD-123319 or an anti-angiotensin II antibody prevented this losartan enhancement of nitric oxide production. Angiotensin II-dependent enhanced hypoxic contractions in pulmonary arteries were blocked by the type 1 receptor antagonist candesartan; however, PD-123319 enhanced hypoxic contractions in angiotensin II-treated endothelium-intact vessels. These data demonstrate that angiotensin II stimulates an increase in nitric oxide synthase mRNA, protein expression, and nitric oxide production via the type 2 receptor, whereas signaling via the type 1 receptor negatively regulates nitric oxide production in the pulmonary endothelium. This endothelial, type 2 receptor-dependent increase in nitric oxide may serve to counterbalance the angiotensin II-dependent vasoconstriction in smooth muscle cells, ultimately regulating pulmonary vascular tone. nitric oxide synthase; angiotensin type 2 receptor; pulmonary endothelium THE RENIN-ANGIOTENSIN SYSTEM (RAS) plays a major role in the control of cardiovascular, renal, and adrenal functions (15). The main effector peptide molecule of the RAS, angiotensin II (ANG II), and its metabolites elicit cellular responses through at least three receptor subtypes (15,33,45): type 1 (AT 1 ), type 2 (AT 2 ), and type 4 (AT 4 ). Signaling pathways mediated via the AT 1 receptor include stimulation of phospholipases, protein kinases, and gene transcription; calcium mobilization; and inhibition of adenylate cyclase (15). Although the AT 2 receptor has been linked to inhibition of cell growth, neuronal differentiation, apoptosis, and regulation of blood pressure, there are conflicting data regarding the specific signaling pathways linked to this receptor (15, 45). For example, it has been reported that activation of the AT 2 receptor can lead to both an increase (5) and decrease (27) in protein phosphatase activity. Moreover, depending on experimental conditions, the p42/p44 MAPK pathway can be either activated (19) or inhibited (24) via the AT 2 receptor. Whereas cloning of the AT 1 and AT 2 receptors reveal...

Although angiotensin II (ANG II) is a known pulmonary vasoconstrictor, the purpose of this study was to examine the effect of ANG II on pulmonary artery endothelial cell nitric oxide synthase (ecNOS) mRNA and protein expression. Cultured bovine pulmonary artery endothelial (BPAE; passages 5-8) cells were incubated for 0-12 h with 10(-6) M ANG II. Total RNA was extracted, and ecNOS expression was assessed by Northern blot analysis. In BPAE cells, ecNOS mRNA was significantly increased 2.4 +/- 0.3-fold (P < 0.05 vs. basal; n = 5) 6 h after the addition of ANG II over basal levels. In & similar time course, it was found that ecNOS protein concentrations are increased 247 +/- 62% (P < 0.05 vs. basal; n = 8) over basal levels 4 h after ANG II addition. There is a second protein peak 8 h after ANG II addition in which ecNOS was increased 333 +/- 145% over basal (P < 0.05, n = 3). These data suggest that ANG II stimulates ecNOS mRNA expression and are followed by increased levels of ecNOS protein in cultured BPAE cells, consistent with an observed increase in nitrite production. Both the increase in ecNOS protein and mRNA expression could be inhibited with the ANG II receptor antagonist saralasin. Additionally, actinomycin D, an inhibitor of transcription, prevented the rise in mRNA at 6 h while cycloheximide inhibited the initial protein peak. The effects of ANG II on ecNOS were specific for the pulmonary artery endothelium. Addition of ANG II did not increase ecNOS protein or mRNA expression in parallel studies in bovine coronary artery endothelium. The stimulation of ecNOS by ANG II may act to protect the lung and maintain low pulmonary artery pressures in the renin-angiotensin model of systemic hypertension.