Endothelial cells are important role of the new vessel formation, respectively physiological conditions as well as pathophysiological conditions, such as wound healing, atherosclerosis, rheumatoid arthritis, menstruation, hypertension, and ischemia/re ...
Endothelial cells are important role of the new vessel formation, respectively physiological conditions as well as pathophysiological conditions, such as wound healing, atherosclerosis, rheumatoid arthritis, menstruation, hypertension, and ischemia/reperfusion damage. In these conditions, endothelial cells modulate angiogenesis and cell death and regulated by various growth factors, cytokines, and metabolite. However, these molecular mechanisms are not elucidated. I investigated angiogenesis by fractalkine and apoptosis by homocysteine and their underlying signaling mechanism.
Part I. Fractalkine stimulates angiogenesis by activating the Raf1/MEK/ERK- and PI3K/Akt/ eNOS-dependent signal pathways
I here investigated the molecular mechanism by which FKN regulates angiogenesis. I here found that recombinant FKN increased in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs), as well as stimulated in vivo angiogenesis. FKN-induced angiogenesis was accompanied by phosphorylation of ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as an increase in NO production. These biochemical events and angiogenesis were completely inhibited by the G protein-coupled receptor inhibitor pertussis toxin. Inhibitors of Raf-1, MEK, PI3K, and eNOS or transfection with dominant negative ERK and Akt significantly suppressed the angiogenic activity of FKN. However, inhibitors of Raf-1 and MEK blocked FKN-induced ERK phosphorylation, but not Akt and eNOS phosphorylation. The PI3K inhibitor suppressed Akt and eNOS phosphorylation and NO production. My results demonstrated that FKN stimulated angiogenesis by activating both the Raf-1/MEK/ERK and PI3K/Akt/eNOS/NO signal pathways via the G protein-coupled receptor CX3CR1, indicating that two pathways are required for full angiogenic activity of FKN. This study suggests that FKN may play an important role in pathophysiological process of inflammatory angiogenesis.
Part II. Nitric oxide inhibition of homocysteine-induced human endothelial cell apoptosis by down-regulation of p53-dependent Noxa expression through the formation of S-nitrosohomocysteine
I examined the molecular mechanism by which homocysteine (HCy) causes endothelial cell apoptosis and by which nitric oxide (NO) affects HCy-induced apoptosis. My data demonstrated that HCy caused caspase-dependent apoptosis in cultured HUVECs, as determined by cell viability, nuclear condensation, and caspase-3 activation and activity. These apoptotic characteristics were correlated with reactive oxygen species (ROS) production, lipid peroxidation, p53 and Noxa expression, and mitochondrial cytochrome c release following HCy treatment. HCy also induced p53 and Noxa expression and apoptosis in endothelial cells from wild type mice, but not in the p53-deficient cells. The NO donor S-nitroso-N-acetlylpenicillamine, adenoviral iNOS vector (AdiNOS) transfection, and antioxidants (α-tocopherol and superoxide dismutase plus catalase), but not oxidized SNAP, 8-Br-cGMP, nitrite, and nitrate, suppressed ROS production, p53-dependent Noxa expression, and apoptosis induced by HCy. The cytotoxic effect of HCy was decreased by siRNA-mediated suppression of Noxa expression, indicating that Noxa upregulation plays an important role in HCy-induced endothelial cell apoptosis. AdiNOS transfection increased the formation of S-nitrosoHCy (S-NOHCy), which was inhibited by the NOS inhibitor N-monomethyl-L-arginine. Moreover, S-NOHCy did not increase ROS generation, p53-dependent Noxa expression, and apoptosis. These results suggest that up-regulation of p53-dependent Noxa expression may play an important role in the pathogenesis of atherosclerosis induced by HCy and that an increase in vascular NO production may prevent HCy-induced endothelial dysfunction by S-nitrosylation.