Ma Xiang, Ottino Paulo, Bazan Haydee E P, Bazan Nicolas G
Department of Ophthalmology, Neuroscience Center of Excellence, Louisiana State University Health Sciences Center School of Medicine in New Orleans, New Orleans, Louisiana 70112, USA.
Invest Ophthalmol Vis Sci. 2004 Sep;45(9):2915-21. doi: 10.1167/iovs.04-0128.
Platelet-activating factor (PAF) is a potent proinflammatory mediator that accumulates in the cornea after injury and induces the expression of genes related to inflammation and wound healing. The current study was conducted to investigate the direct effect of PAF on corneal neovascularization and on the expression of angiogenic growth factors in vascular endothelial cells.
Pellets containing carbamyl-PAF (cPAF) were implanted in corneas of wild-type or PAF-receptor (PAF-R)-knockout mice, and the progression of angiogenesis was monitored by microscope. In some experiments, mice were treated with a daily intraperitoneal injection of the PAF-R antagonist LAU8080. Migration assays of human umbilical cord vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMVECs) were performed in a Boyden chamber after addition of various concentrations of cPAF or bovine fibroblast growth factor (FGF-2). Cell proliferation was assessed by fluorescence-binding assay in the presence of cPAF or FGF-2 for 8 days. Vascular endothelial growth factor (VEGF) and FGF-2 expression was studied by RT-PCR and Northern- and Western-blot analyses in cells stimulated with cPAF at different concentrations and for different times.
Six days after cPAF pellet implantation, there were new vessels growing from the limbus to the center of the cornea. The PAF-induced neovascularization was significantly reduced in PAF-R-knockout mice and in mice treated with the PAF antagonist. cPAF added to the lower well of the Boyden chamber produced a dose-dependent migration of HUVECs and HMVECs that was inhibited in cells preincubated with LAU8080 or with a VEGF-blocking antibody. In contrast, cPAF did not stimulate proliferation of endothelial cells. cPAF induced VEGF mRNA and protein expression but not FGF-2 expression in HUVECs and HMVECs.
PAF stimulates corneal neovascularization by a receptor-mediated mechanism. Induction of VEGF expression and stimulation of vascular endothelial cell migration are initial events in PAF-promoted corneal angiogenesis.
血小板活化因子（PAF）是一种强效促炎介质，在损伤后会在角膜中蓄积，并诱导与炎症和伤口愈合相关的基因表达。本研究旨在探讨PAF对角膜新生血管形成以及血管内皮细胞中血管生成生长因子表达的直接影响。
将含有氨甲酰-PAF（cPAF）的微丸植入野生型或PAF受体（PAF-R）基因敲除小鼠的角膜中，通过显微镜监测血管生成的进程。在一些实验中，小鼠每日腹腔注射PAF-R拮抗剂LAU8080进行治疗。在添加不同浓度的cPAF或牛成纤维细胞生长因子（FGF-2）后，在博伊登小室中进行人脐静脉内皮细胞（HUVECs）和人真皮微血管内皮细胞（HMVECs）的迁移试验。在存在cPAF或FGF-2的情况下培养8天，通过荧光结合试验评估细胞增殖。采用逆转录-聚合酶链反应（RT-PCR）、Northern印迹和Western印迹分析，研究不同浓度和不同时间的cPAF刺激细胞后血管内皮生长因子（VEGF）和FGF-2的表达情况。
植入cPAF微丸6天后，有新血管从角膜缘向角膜中心生长。在PAF-R基因敲除小鼠和用PAF拮抗剂治疗的小鼠中，PAF诱导的新生血管形成明显减少。添加到博伊登小室下层孔中的cPAF可使HUVECs和HMVECs产生剂量依赖性迁移，在用LAU8080或VEGF阻断抗体预孵育的细胞中这种迁移受到抑制。相比之下，cPAF不刺激内皮细胞增殖。cPAF可诱导HUVECs和HMVECs中VEGF mRNA和蛋白表达，但不诱导FGF-2表达。
PAF通过受体介导的机制刺激角膜新生血管形成。诱导VEGF表达和刺激血管内皮细胞迁移是PAF促进角膜血管生成的初始事件。
