Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration.

The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.

Endothelium-derived nitric oxide (NO) activates the NO-epidermal growth factor receptor-mediated signaling pathway in bradykinin-stimulated angiogenesis.

Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel®-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.

Hyperbaric oxygen therapy protects the liver from apoptosis caused by ischemia-reperfusion injury in rats.

PURPOSE: : The present paper aimed to investigate the role of hyperbaric oxygen treatment (HBO) and the apoptosis in rat liver ischemia-reperfusion injury (IRI). METHODS: : Thirty-seven male Wistar rats were subjected to 30 minutes of hepatic ischemia and 30 minutes of reperfusion and randomly distributed into six groups: G-I/R (n = 8), control without HBO; G-HBO/I (n = 8), HBO only during the ischemia period; G-HBO/R (n = 8), HBO only during the reperfusion period; G-HBO-I/R (n = 8), HBO during both the ischemia and reperfusion periods; G-Sh (n = 3), HBO without ischemia or reperfusion as sham group; G-C (n = 2) for control of current apoptosis expression on the normal liver tissue. HBO was carried out using a transparent, cylindrical acrylic chamber with a pressure of 2.0 ATA. Hepatic samples were stained for caspase-3 cleavage. RESULTS: : Apoptotic cells were identified in all groups. In the hepatic specimens of animals HBO-treated during ischemia (GHBO-I), there was a significant decrease (P < 0.001) in the number of cells undergoing apoptosis (1.62 +/- 0.91). The apoptotic index showed no significant difference in the animals HBO-treated during ischemia/reperfusion (5.75 +/- 1.28) compared with the G-I/R (3.5 +/- 0.75), which had no HBO treatment. The apoptosis index (11.25 +/- 1.90) was significantly higher (P < 0.01) in HBO-treated animals during the reperfusion period when compared with any of the other groups. CONCLUSION: : A favorable effect was obtained when hyperbaric oxygen was administered early during ischemia. The hyperbaric oxygen in later periods of reperfusion was associated with a more severe apoptosis index. (c) 2009 Wiley-Liss, Inc. Microsurgery 2009.