Sherzad Rashid

Redox-Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in Response to EPA:DHA 6:1

Aims Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have vasoprotective effects, in part, by stimulating the endothelial formation of nitric oxide (NO). This study determined the role of the EPA:DHA ratio and amount, and characterized the mechanism leading to endothelial NO synthase (eNOS) activation. Methods and Results EPA:DHA 6∶1 and 9∶1 caused significantly greater endothelium-dependent relaxations in porcine coronary artery rings than EPA:DHA 3∶1, 1∶1, 1∶3, 1∶6, 1∶9, EPA and DHA alone, and EPA:DHA 6∶1 with a reduced EPA + DHA amount, which were inhibited by an eNOS inhibitor. Relaxations to EPA:DHA 6∶1 were insensitive to cyclooxygenase inhibition, and reduced by inhibitors of either oxidative stress, Src kinase, PI3-kinase, p38 MAPK, MEK, or JNK. EPA:DHA 6∶1 induced phosphorylation of Src, Akt, p38 MAPK, ERK, JNK and eNOS; these effects were inhibited by MnTMPyP. EPA:DHA 6∶1 induced the endothelial formation of ROS in coronary artery sections as assessed by dihydroethidium, and of superoxide anions and hydrogen peroxide in cultured endothelial cells as assessed by electron spin resonance with the spin probe CMH, and the Amplex Red based assay, respectively. Conclusion Omega-3 fatty acids cause endothelium-dependent NO-mediated relaxations in coronary artery rings, which are dependent on the EPA:DHA ratio and amount, and involve an intracellular activation of the redox-sensitive PI3-kinase/Akt and MAPKs pathways to activate eNOS.

Bilberry extract (Antho 50) selectively induces redox-sensitive caspase 3-related apoptosis in chronic lymphocytic leukemia cells by targeting the Bcl-2/Bad pathway

Defect in apoptosis has been implicated as a major cause of resistance to chemotherapy observed in B cell chronic lymphocytic leukaemia (B CLL). This study evaluated the pro-apoptotic effect of an anthocyanin-rich dietary bilberry extract (Antho 50) on B CLL cells from 30 patients and on peripheral blood mononuclear cells (PBMCs) from healthy subjects, and determined the underlying mechanism. Antho 50 induced concentration- and time-dependent pro-apoptotic effects in B CLL cells but little or no effect in PBMCs. Among the main phenolic compounds of the bilberry extract, delphinidin-3-O-glucoside and delphinidin-3-O-rutinoside induced a pro-apoptotic effect. Antho 50-induced apoptosis is associated with activation of caspase 3, down-regulation of UHRF1, a rapid dephosphorylation of Akt and Bad, and down-regulation of Bcl-2. Antho 50 significantly induced PEG-catalase-sensitive formation of reactive oxygen species in B CLL cells. PEG-catalase prevented the Antho 50-induced induction of apoptosis and related signaling. The present findings indicate that Antho 50 exhibits strong pro-apoptotic activity through redox-sensitive caspase 3 activation-related mechanism in B CLL cells involving dysregulation of the Bad/Bcl-2 pathway. This activity of Antho 50 involves the glucoside and rutinoside derivatives of delphinidin. They further suggest that Antho 50 has chemotherapeutic potential by targeting selectively B CLL cells.

Probiotics (VSL#3) Prevent Endothelial Dysfunction in Rats with Portal Hypertension: Role of the Angiotensin System

Aims Portal hypertension characterized by generalized vasodilatation with endothelial dysfunction affecting nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) has been suggested to involve bacterial translocation and/or the angiotensin system. The possibility that ingestion of probiotics prevents endothelial dysfunction in rats following common bile duct ligation (CBDL) was evaluated. Methods Rats received either control drinking water or the probiotic VSL#3 solution (50 billion bacteria.kg body wt−1.day−1) for 7 weeks. After 3 weeks, rats underwent surgery with either resection of the common bile duct or sham surgery. The reactivity of mesenteric artery rings was assessed in organ chambers, expression of proteins by immunofluorescence and Western blot analysis, oxidative stress using dihydroethidium, and plasma pro-inflammatory cytokine levels by flow cytometry. Results Both NO- and EDH-mediated relaxations to acetylcholine were reduced in the CBDL group compared to the sham group, and associated with a reduced expression of Cx37, Cx40, Cx43, IKCa and SKCa and an increased expression of endothelial NO synthase (eNOS). In aortic sections, increased expression of NADPH oxidase subunits, angiotensin converting enzyme, AT1 receptors and angiotensin II, and formation of ROS and peroxynitrite were observed. VSL#3 prevented the deleterious effect of CBDL on EDH-mediated relaxations, vascular expression of connexins, IKCa, SKCa and eNOS, oxidative stress, and the angiotensin system. VSL#3 prevented the CBDL-induced increased plasma TNF-α, IL-1α and MCP-1 levels. Conclusions These findings indicate that VSL#3 ingestion prevents endothelial dysfunction in the mesenteric artery of CBDL rats, and this effect is associated with an improved vascular oxidative stress most likely by reducing bacterial translocation and the local angiotensin system.

Redox-sensitive up-regulation of eNOS by purple grape juice in endothelial cells: role of PI3-kinase/Akt, p38 MAPK, JNK, FoxO1 and FoxO3a.

The vascular protective effect of grape-derived polyphenols has been attributable, in part, to their direct action on blood vessels by stimulating the endothelial formation of nitric oxide (NO). The aim of the present study was to determine whether Concord grape juice (CGJ), which contains high levels of polyphenols, stimulates the expression of endothelial NO synthase (eNOS) in porcine coronary artery endothelial cells and, if so, to determine the signaling pathway involved. CGJ dose- and time-dependently increased eNOS mRNA and protein levels and this effect is associated with an increased formation of NO in endothelial cells. The stimulatory effect of CGJ on eNOS mRNA is not associated with an increased eNOS mRNA stability and inhibited by antioxidants such as MnTMPyP, PEG-catalase, and catalase, and by wortmannin (an inhibitor of PI3-kinase), SB 203580 (an inhibitor of p38 MAPK), and SP 600125 (an inhibitor of JNK). Moreover, CGJ induced the formation of reactive oxygen species (ROS) in endothelial cells and this effect is inhibited by MnTMPyP, PEG-catalase, and catalase. The CGJ-induced the phosphorylation of p38 MAPK and JNK kinases is abolished by MnTMPyP. CGJ induced phosphorylation of transcription factors FoxO1 and FoxO3a, which regulate negatively eNOS expression, and this effect is prevented by MnTMPyP, PEG-catalase, wortmannin, SB203580 and SP600125. Moreover, chromatin immunoprecipitation assay indicated that the FoxO3a protein is associated with the eNOS promoter in control cells and that CGJ induced its dissociation. Thus, the present study indicates that CGJ up-regulates the expression of eNOS mRNA and protein leading to an increased formation of NO in endothelial cells. The stimulatory effect of CGJ is a redox-sensitive event involving PI3-kinase/Akt, p38 MAPK and JNK pathways, and the inactivation of the FoxO transcription factors, FoxO1 and FoxO3a, thereby preventing their repression of the eNOS gene.

Effect of the oestrogen receptor antagonist fulvestrant on the cirrhotic rat lung

It has been postulated that cirrhosis‐related lung vasodilatation and the subsequent hepatopulmonary syndrome are partly explained by an increased estradiol level through an enhanced endothelial formation of nitric oxide (NO). In this study, we assessed whether the oestrogen receptor antagonist fulvestrant (F) improves cirrhosis‐related lung abnormalities. Cirrhosis was induced in rats by chronic bile duct ligation (CBDL). Four groups were studied: CBDL, CBDL+F, sham, and sham+F. Histological, immunohistochemical, and Western blot analyses were performed on lung samples. In the lung, the endothelial NO synthase and the nitrotyrosine protein expressions were increased in CBDL as compared to sham rats. Both parameters were significantly reduced by fulvestrant in the CBDL rats. Surprisingly, the level of pVASP (an indirect marker of NO formation and action) was decreased in CBDL rats, and fulvestrant had no effect on this parameter. The level of the vascular endothelial growth factor, the diameter of small lung vessels, and the number of macrophages were increased in CBDL lungs in comparison with sham lungs, and these parameters were unaffected by fulvestrant treatment. In conclusion, fulvestrant may not be relevant to improve lung abnormalities in cirrhosis because NO may not be biologically active and because key events contributing to the lung abnormalities are not affected by fulvestrant.

0040: The EPA: DHA 6:1-evoked endothelium-dependent NO-mediated relaxation in the coronary artery involves a copper-dependent pro-oxidant response triggering the PI3-kinase/Akt-mediated activation of eNOS

Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to reduce the risk of cardiovascular disease, in part, by stimulating the endothelial formation of nitric oxide (NO), a potent vasoprotective factor. This study determined the mechanism leading to endothelial NO synthase (eNOS) activation in response to the highly active EPA:DHA 6:1 product. Vascular reactivity was assessed using porcine coronary artery rings suspended in organ chambers, the level of oxidative stress in coronary artery sections using the redox-sensitive probe, dihydroethidine, and the phosphorylation level of target proteins in cultured coronary artery endothelial cells by Western blot analysis. EPA:DHA 6:1 caused pronounced endothelium-dependent relaxations in porcine coronary artery rings. Relaxations to EPA:DHA 6:1 were slightly but significantly reduced by an eNOS inhibitor, not affected by inhibition of endotheliumdependent hyperpolarization and abolished by both treatments. Relaxations to EPA:DHA 6:1 were reduced by inhibitors of oxidative stress (MnTMPyP, PEG-catalase), an inhibitor of either Src kinase (PP2) or PI3-kinase (wortmannin), and intracellular copper chelating agents (neocuproine, tetrathiomolybdate) and were insensitive to cyclooxygenase inhibition (indomethacin), chelating agents for iron (desferroxamine), zinc (histidine), extracellular copper (bathocuproine). EPA:DHA 6:1 induced phosphorylation of Src, Akt and eNOS at Ser 1177; these effects were inhibited by MnTMPyP and PEGcatalase. EPA:DHA 6:1 induced the endothelial formation of ROS in coronary artery sections, this effect was inhibited by MnTMPyP, PEG-catalase, and intracellular copper chelating agents. EPA:DHA 6:1 causes endotheliumdependent NO-mediated relaxations in coronary artery rings, and this effect involves an intracellular copper-dependent event triggering the redox-sensitive PI3-kinase/Akt pathway to activate eNOS by phosphorylation at Ser 1177.

0051: Lipid emulsions used in parenteral nutrition induce endothelial dysfunction in porcine coronary artery rings: role of oxidative stress and cyclooxygenase-derived vasoconstrictors

Lipid emulsions are used to provide a source of calories and essential fatty acids for patients requiring parenteral nutrition. They have been associated with hypertriglyceridemia, hypercholesterolemia and metabolic stress, which may promote the development of endothelial dysfunction. The aim of the present study was to determine the possibility that five different commercial lipid emulsions affect the endothelial function of coronary arteries, and, if so, to investigate the underlying mechanism. Porcine coronary arteries were incubated with lipid emulsions for 30 minutes before the determination of vascular reactivity in organ chambers and the level of oxidative stress using the redox-sensitive fluorescent dye dihydroethidium (DHE). Incubation of coronary artery rings with either Lipidem®, Medialipid® or Smoflipid® (medium-chain triglycerides), but not with Intralipid ® or Clinoleic® (long-chain triglycerides), significantly reduced the bradykinin- induced endothelium-dependent relaxations mediated by both nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH). In contrast, Lipidem® did not affect endothelium-independent relaxations to sodium nitroprusside. The endothelial dysfunction induced by Lipidem® was significantly improved by indomethacin, a cyclooxygenase (COX) inhibitor, and by inhibitors of oxidative stress (N-acetylcysteine, superoxide dismutase, catalase) and transition metal chelating agents (neocuproine, L-histidine, desferoxamine). Lipidem® markedly increased the vascular oxidative stress as indicated by increased DHE signal throughout the arterial wall. The present findings indicate that several but not all lipid emulsions induce an endothelial dysfunction in coronary artery rings, involving both blunted NO- and EDH-mediated relaxations. The Lipidem®-induced endothelial dysfunction is associated with increased vascular oxidative stress and the formation of COX-derived vasoconstrictor prostanoids.