Xu Zhenhua, Yu Yilin, Duh Elia J
Department of Ophthalmology, The Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
Invest Ophthalmol Vis Sci. 2006 Sep;47(9):4059-66. doi: 10.1167/iovs.05-1528.
ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs) has been demonstrated to inhibit angiogenesis in vivo and to suppress endothelial cell proliferation in vitro. The purpose of this study was to investigate the expression of ADAMTS1 in endothelial cells and in a mouse model of ischemia-induced retinal neovascularization and to study the regulation of ADAMTS1 expression in endothelial cells by vascular endothelial growth factor (VEGF). In addition, the potential function of endothelial cell-derived ADAMTS1 on cell proliferation was investigated.
Expression of ADAMTS1 in human retinal endothelial cells (HRECs), human umbilical vein endothelial cells (HUVECs), and the mouse model of ischemia-induced retinal neovascularization was assayed by real-time PCR and Western blot analysis. The effect of ADAMTS1 on endothelial cell proliferation was evaluated using siRNA knockdown and [3H] thymidine incorporation.
ADAMTS1 mRNA and protein levels were increased in a mouse model of ischemia-induced retinal neovascularization, and VEGF induced time- and dose-dependent increases in ADAMTS1 mRNA and protein expression in endothelial cells. This upregulation was inhibited by the VEGF receptor (VEGFR)2 inhibitor SU1498, anti-VEGFR2 neutralizing antibody, and the phospholipase C (PLC)-gamma inhibitor U73122. VEGF upregulation of ADAMTS1 expression was completely abolished by the inhibition of protein kinase C by calphostin C and largely blocked by the specific inhibition of PKCbeta. Knockdown of endogenous ADAMTS1 resulted in increased proliferation of endothelial cells.
These results indicate that VEGF significantly induces ADAMTS1 expression in endothelial cells in a PKC-dependent fashion. ADAMTS1 expression is also increased, along with VEGF expression, in vivo in ischemia-induced retinal neovascularization. In addition, ADAMTS1 appears to be an endogenous regulator of endothelial cell proliferation. Therefore, VEGF upregulation of ADAMTS1, a potent angiogenesis inhibitor, may represent a mechanism for feedback inhibition of angiogenesis and retinal neovascularization.
含血小板反应蛋白基序的解聚素和金属蛋白酶1（ADAMTS1）已被证明在体内可抑制血管生成，在体外可抑制内皮细胞增殖。本研究的目的是调查ADAMTS1在内皮细胞以及缺血诱导的视网膜新生血管小鼠模型中的表达情况，并研究血管内皮生长因子（VEGF）对内皮细胞中ADAMTS1表达的调控。此外，还研究了内皮细胞源性ADAMTS1对细胞增殖的潜在作用。
通过实时聚合酶链反应（PCR）和蛋白质免疫印迹分析检测ADAMTS1在人视网膜内皮细胞（HREC）、人脐静脉内皮细胞（HUVEC）以及缺血诱导的视网膜新生血管小鼠模型中的表达。使用小干扰RNA（siRNA）敲低技术和[3H]胸苷掺入法评估ADAMTS1对内皮细胞增殖的影响。
在缺血诱导的视网膜新生血管小鼠模型中，ADAMTS1的信使核糖核酸（mRNA）和蛋白水平升高，VEGF在内皮细胞中诱导ADAMTS1的mRNA和蛋白表达呈时间和剂量依赖性增加。VEGF受体（VEGFR）2抑制剂SU1498、抗VEGFR2中和抗体以及磷脂酶C（PLC）-γ抑制剂U73122可抑制这种上调。钙泊三醇C抑制蛋白激酶C可完全消除VEGF对ADAMTS1表达的上调作用，PKCβ的特异性抑制可很大程度上阻断这种上调作用。敲低内源性ADAMTS1会导致内皮细胞增殖增加。
这些结果表明，VEGF以蛋白激酶C依赖性方式显著诱导内皮细胞中ADAMTS1的表达。在缺血诱导的视网膜新生血管形成的体内过程中，ADAMTS1的表达也随着VEGF表达的增加而升高。此外，ADAMTS1似乎是内皮细胞增殖的内源性调节因子。因此，VEGF上调强效血管生成抑制剂ADAMTS1可能代表一种对血管生成和视网膜新生血管形成进行反馈抑制的机制。
