Proteomic analysis of homocysteine inhibition of microvascular endothelial cell angiogenesis.
Although homocysteine (Hcy) inhibits angiogenesis in vivo and in vitro, the mechanism(s) underlying this phenomenon are largely unclear. The hypothesis of the present work is that Hcy, while inducing the expression of antiangiogenic factors, inhibits the production of angiogenic factors. Mouse brain microvascular endothelial cells (MVEC) were cultured in the presence and absence of 20 microM Hcy for 24 hr in serum-free medium. Cell homogenates were incubated with Trans-Signal Angiogenesis Antibody Array containing antibodies to angiogenic activators (ANG, HGF, leptin, VEGF, IL-6, IL-8, PIGF, FGF-alpha/beta, TNF-alpha and TGF-alpha) and inhibitors (IFN-gamma, IL-12, IP-10, TIMP-1 and -2). The array membranes were scanned and normalized with positive controls. Angiogenesis and formation of capillaries were measured by culturing the MVEC in Matrigels. The capillary-like structures were identified by transmission microscopy. Hcy decreased the expression of leptin, IL-6, -8, PIGF, FGF-alpha and VEGF, while the levels of anti-angiogenic IL-12, IP-10 (chemokine) and TIMP-1 were increased by Hcy. The vascular tube-like structures by MVEC were decreased by increased Hcy. However, the addition of VEGF to Hcy-treated MVEC ameliorated the decreased Hcy-mediated capillary formation. The results suggest that Hcy inhibits angiogenesis, in part, by decreasing VEGF and increasing TIMP-1.
Shastry
S
S
Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.
Tyagi
N
N
Hayden
M R
MR
Tyagi
S C
SC
D000818:Animals; D001921:Brain / Q000098:blood supply / Q000378:metabolism; D002196:Capillaries; D049109:Cell Proliferation; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D018925:Chemokines; D003094:Collagen / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D004338:Drug Combinations; D004730:Endothelium, Vascular / Q000378:metabolism; D006710:Homocysteine / Q000737:chemistry* / Q000378:metabolism*; D007797:Laminin / Q000494:pharmacology; D020738:Leptin / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008833:Microcirculation; D046529:Microscopy, Electron, Transmission; D009389:Neovascularization, Pathologic; D020411:Oligonucleotide Array Sequence Analysis; D011509:Proteoglycans / Q000494:pharmacology; D040901:Proteomics / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D018925:Chemokines; D016895:Culture Media, Serum-Free; D004338:Drug Combinations; D007797:Laminin; D020738:Leptin; D011509:Proteoglycans; D019715:Tissue Inhibitor of Metalloproteinase-1; D042461:Vascular Endothelial Growth Factor A; D006710:Homocysteine; C059485:matrigel; D003094:Collagen