Tao Y, Bazan H E, Bazan N G
LSU Eye Center, Louisiana State University Medical Center, New Orleans, LA 70112.
Invest Ophthalmol Vis Sci. 1995 Feb;36(2):345-54.
The inflammatory mediator platelet-activating factor (PAF) induces the expression of interstitial collagenase (metalloproteinase-1) messenger RNA in rabbit corneal epithelium. In this study, the authors investigated the effect of PAF on gene expression and protein activity of other matrix metalloproteinases (MMPs) in the cornea.
Rabbit corneas were incubated in an organ culture with 100 nM of cPAF (a nonhydrolyzable PAF analog), PAF, or lyso-PAF, an inactive metabolite of PAF. In some experiments, the corneas were preincubated for 1 hour with 10 microM BN50730, a PAF antagonist, before cPAF was added to the medium. Corneal epithelial cells and/or conditioned medium were collected at different times for analysis. Also, in vivo experiments were done by injecting 2 micrograms of cPAF intrastromally into rabbit eyes and collecting the epithelium 8 hours later for study. Northern blot analysis and zymography were performed to determine the mRNA abundance and/or enzyme activity of 92 kd gelatinase (MMP-9), 72 kd gelatinase (MMP-2), and stromelysin (MMP-3). The activity of MMP-1 was tested by collagenase assays.
cPAF induced the expression of MMP-9 mRNA, but not MMP-3 mRNA. The message was induced at 4 hours and remained elevated at 48 hours, with a peak at 36 hours. In corneas preincubated with BN50730, MMP-9 mRNA activation by cPAF was inhibited. In vivo injection of cPAF also induced the expression of MMP-9. Furthermore, cPAF increased MMP-9 activity in the epithelial cells and in the conditioned media. The effect was blocked by BM50730. cPAF did not affect MMP-2 activity. Finally, cPAF also increased MMP-1 collagenolytic activity of the corneal epithelium, which was blocked by the PAF antagonist.
These results suggest a novel mechanism by which PAF activates MMPs. The lipid mediator selectively enhances the expression of MMP-1 and MMP-9 in rabbit corneal epithelium. This activation by PAF may be involved in the remodeling mechanisms of the cornea after injury and, when overexpressed, may lead to the formation of corneal ulcers. Specific PAF antagonists could therapeutically deter corneal ulcer formation and facilitate corneal wound healing.
炎症介质血小板活化因子（PAF）可诱导兔角膜上皮细胞中间质胶原酶（金属蛋白酶-1）信使核糖核酸的表达。在本研究中，作者调查了PAF对角膜中其他基质金属蛋白酶（MMPs）基因表达和蛋白活性的影响。
将兔角膜置于器官培养中，分别用100 nM的cPAF（一种不可水解的PAF类似物）、PAF或溶血PAF（PAF的无活性代谢产物）进行孵育。在一些实验中，在向培养基中添加cPAF之前，将角膜先用10 microM的BN50730（一种PAF拮抗剂）预孵育1小时。在不同时间收集角膜上皮细胞和/或条件培养基进行分析。此外，通过向兔眼基质内注射2微克cPAF并在8小时后收集上皮组织进行体内实验。进行Northern印迹分析和酶谱分析以确定92 kD明胶酶（MMP-9）、72 kD明胶酶（MMP-2）和基质溶解素（MMP-3）的信使核糖核酸丰度和/或酶活性。通过胶原酶测定法检测MMP-1的活性。
cPAF诱导MMP-9信使核糖核酸的表达，但不诱导MMP-3信使核糖核酸的表达。该信使在4小时时被诱导，并在48小时时保持升高，在36小时时达到峰值。在用BN50730预孵育的角膜中，cPAF对MMP-9信使核糖核酸的激活受到抑制。体内注射cPAF也诱导MMP-9的表达。此外，cPAF增加了上皮细胞和条件培养基中MMP-9的活性。该作用被BM50730阻断。cPAF不影响MMP-2的活性。最后，cPAF还增加了角膜上皮的MMP-1胶原分解活性，这被PAF拮抗剂阻断。
这些结果提示了一种PAF激活MMPs的新机制。这种脂质介质选择性地增强兔角膜上皮中MMP-1和MMP-9的表达。PAF的这种激活可能参与角膜损伤后的重塑机制，并且当过度表达时，可能导致角膜溃疡的形成。特异性PAF拮抗剂在治疗上可阻止角膜溃疡的形成并促进角膜伤口愈合。
