Study On Angiogenesis Effect Of β₂-glycoprotein On Human Umbilical Vein Endothelial Cells

Aims:Angiogenesis, the formation of new blood vessels from a pre-existing vascular network, is an essential process in a variety of physiological and pathological conditions, including wound healing, embryonic development, menstrual cycle, chronic inflammation, rheumatoid arthritis, diabetic retinopathy, cancer and metastasis. Angiogenesis is also a complex and tightly regulated process, and is stimulated by a large number of growth factors, notably including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Complex sequential steps are involved in angiogenesis, such as basement membrane degradation by proteases, endothelial cell proliferation and migration, formation of capillary tubes and survival of newly formed blood vessels.VEGF is a potent mitogen for endothelial cells. In particular, a number of studies have shown that VEGF is the most important angiogenic factor closely associated with neovascularization in human tumors. VEGF acts on endothelial cells through two high-affinity tyrosine kinase receptors, namely Flt-1 (or VEGF-R1) and KDR/Flk-1 (or VEGF-R2). KDR/Flk-1 mainly mediates the VEGF-dependent mitogenic and chemotactic effects. VEGF induces proliferation through activation of the extracellular signal-regulated kinase (ERK) 1/2 pathway and endothelial cell survival through activation of phosphatidylinositol 3-kinase (PI3K) and its downstream proteins. bFGF, mainly released by damaged cells at wound edges, stimulates survival, proliferation, migration and differentiation of primary and stable endothelial cells. Both VEGF and bFGF activate the ERK1/2 cascade via MAPK pathway.Proliferative diabetic retinopathy is characterized by the formation of neovasculars within retina, which is frangible to bleed. Moreover, fibrosis tissue will lead to the aberrant of retina and contributes to the blindness. Meanwhile, angiogenesis also plays an important role in the development of athersclerosis, which will devastate the stability of plaque and induce the outbreak of unstable angina. Thus, the inhibition of abnormal angiogenesis is a crucial step to management the development of diabetic vascular complications.Methods:To determine the effects ofβ2GPI on VEGF and bFGF-induced HUVEC migration, an in vitro wound-healing assay was performed. Cells were seeded into 96-well microtitre plates, cell titer 96(?) AQueous One Solution (MTS reagent) was added to each well. The absorbance at 490 nm is directly proportional to the number of live cells. We performed a tube formation assay to investigate the effect of nβ2GPI on the capillary-like structure formation of HUVECs. Following stimulation of HUVECs on the Matrigel with VEGF or bFGF. We evaluated whether nP2GPI decreased KDR/Flk-1 expression, the receptor that is involved in endothelial cell proliferation, migration and angiogenesis, by real-time PCR. To further evaluate the effects of nβ2GPI on downstream signalling molecules of KDR/Flk-1 triggered by VEGF and bFGF, we examined the activation of ERK1/2, Akt, and GSK3βwith Western blot. And also gene chip was used to find out the genes that affected by P2GPI on HUVECs after stimulating by VEGF. To evaluate the relationship between of plasmaβ2GPI, blood lipid and vascular diseases, we measured the concentration ofβ2GPI and other indexes, such as blood pressure, plasma glucose, insulin level, lipids,24h urine microalbumin et al, in 231 patients with diabetes and 119 control. The correlations among them were analyzed by software SPSS.Results:Results from cell culture experiments are as follows:1. Nativeβ2GPI, clippedβ2GPI as well as DI-IV dose-dependently inhibit VEGF-and bFGF-induced HUVECs proliferation(P<0.05, P<0.01). There is no significant effect of DⅡ-Ⅴon the VEGF-or bFGF-induced proliferation of HUVECs(P>0.05).2. nβ2GPI, cβ2GPI and mutantβ2GPI (DⅠ-Ⅳ) significantly inhibited VEGF and bFGF-induced migration of HUVECs in a dose-dependent manner(P<0.05, P<0.01). There was no significant difference between the effects of native, clippedβ2GPI and mutant p2GPI (DⅠ-Ⅳ) on endothelial migration (P>0.05). However, mutantβ2GPI (DⅡ-Ⅴ) did not inhibit rhVEGF-or rhbFGF-induced HUVEC migration (P>0.05).3. nβ2GPI, cβ2GPI and DⅠ-Ⅳinhibit VEGF-induced tube formaion of HUVECs on Matrigel in a dose-dependent manner(P<0.05, P<0.01). np2GPI, cp2GPI and DⅠ-Ⅳalso dose-dependently inhibit bFGF-induced tube formaion of HUVECs on Matrigel(P<0.05, P<0.01). However, mutantβ2GPI (DⅡ-Ⅴ) did not inhibit rhVEGF-or rhbFGF-induced tube formation on Matrigel (P>0.05).4. nβ2GPI in the presence of VEGF or bFGF (40ng/ml) significantly reduced the mRNA level of KDR/Flk-1 in HUVECs(P<0.05, P<0.01). In contrast, np2GPI had no significant effects on the mRNA level of Flt-1 in HUVECs treated with rhVEGF or rhbFGF (P>0.05).5. Relative density quantitative analyses (compared withβ-actin) demonstrated that nβ2GPI inhibits VEGF-and bFGF-stimulated phosphorylation of Akt (Ser 473), GSK-3p (Ser 9) and ERK1/2 in a dose-dependent manner (P<0.05, P<0.01).6.50 genes were screened by gene chip, which might down-regulated VEGF expression. Among them, IL-8, Epithelial membrane proteinl and Matrix gla protein regulated cell angiogenesis, cell motility, calcium-mediated signaling, cell cycle arrest, cell-cell signaling, chemokine activity, chemotaxis, extracelluar region, extracelluar space, G-protein coupled receptor protein signaling pathway, immune response, inflammatory response, intracellular signaling cascade, signal transduction and cell adhesion. On the other hand,β2GPI could decrease the expression of transcription factors and MMPs family, which will damage the base membrane of endothelial, inhibits the formation of vascular.Results from clinic analysis are as follows:1. Plasmaβ2GPI level of diabetics was significant increased compared to normal control (P<0.05). There are strong correlations betweenβ2GPI and hypertriglyceride and cholesteremia (P<0.01).2. There is a significant correlation betweenβ2GPI and systolic pressure (P<0.01). And also there is a relationship betweenβ2GPI and glomerulus diseases.Conclusions:Our experimental results show that native and clippedβ2GPI as well as domain deletion mutantβ2GPI (DⅠ-Ⅳ) inhibit VEGF-and bFGF-induced endothelial cell proliferation, migration and tube formation in vitro. These effects were mainly regulated through KDR/Flk-1 and its downstream signaling pathway, including MAPK/ERK and PI3K/Akt/GSK3βpathways.On the other hand, the concentration ofβ2GPI will affect the disturbance of glucose and lipids, and also is one of monitors of diabetes and diabetic nephropathy.β2GPI might both positively and negatively regulate the development of vascular diseases. Slight increase ofβ2GPI aggregated the vascular disease by disturbing the balance of blood lipids. And a distinct rise ofβ2GPI might slow down the process of vascular diseases by inhibiting the angiogenesis and correcting lipids disturbance. DomainⅠ-Ⅳor clippedβ2GPI could be used to treat the diseases involving angiogenesis. However, DⅡ-Ⅴcould not inhibit formation of abnormal angiogenesis. So domain I ofβ2GPI might contribute to the regulation of angiogenesis. It will benefit the treatment of diabetic vascular complications characterized by over-angiogenesis, such as diabetic retinopathy, nephropathy and atherosclerosis.