Important Role Of Increased Matrix Metalloproteinase-2 Activity Regulated By Transforming Growth Factor Beta1 In The Active Scleral Remodeling Process During The Development Of Form-Deprivation Myopia In Chicks

Purpose: To investigate the key role of matrix metalloproteinase-2 (MMP-2) in the active scleral remodeling process of extracellular matrix(ECM) and the effect of transforming growth factor-β1(TGF-β1) on the expression of mRNA, pro-enzyme and enzymic activity of MMP-2 during the development of form-deprivation myopia (FDM) of chicks. Methods: The current study was divided into three parts. The first part was that: one-day-old chicks were randomly chosen to establish the animal models of FDM and recovery of form-deprivation myopia (RFDM), and one step reverse transcriptiontase-polymerase chain reaction (RT-PCR) and histochemical method were performed respectively to measure the expression of mRNA, and pro-or active protein of MMP-2 and its specific tissue inhibitor (TIMP-2). and meanwhile gelatin enzymography was also used to determined the level of activity of MMP-2 in the posterior sclera of these experimental eyes. To determine the important role of MMP-2 in the development of FDM different dose of a synthetic inhibitor of matrix metalloproteinase GM6001were daily administered into the deprived eyes to inhibit the activity of MMP-2. After four or fourteen days'treatment the effect of GM6001 on the active remodeling of ECM in the posterior sclera and the development of FDM was determined by changes of axial length and diopters of experimental chick eyes. The second part was that: on the basis of what had been attained in the first part the posterior sclera required from normal and deprived eyes were cultured, and at the same time five interference factors, that is tumor necrosic factor-α(TNF-α) (2.5ng/ml), basic fibroblast growth factor(bFGF) (10ng/ml), Ovotransferrin(500ng/ml), fibronectin (0.2mg/ml) and TGF-β1 (10ng/ml) were added into the culture medium. After 24 hours' incubation the level of MMP-2 activity was measured by gelatin enzymography, and the levels of MMP-2 and TIMP-2 mRNA were evaluated by one step RT-PCR to chosen the postive modulation factors for MMP-2 and TIMP-2 gene expression. The last part was that: Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) selected were administered to determined the effect of TGF-β 1 activation on the modulation of MMP-2 activity and its mRNA expression, and on TIMP-2 gene expression, and on changes of the axial length and diopters in normal and in FDM eyes. Results: The results attained from first part were as follows: ⑴compared with normal and self-controlled groups, the expression levels of MMP-2 activity, mRNA and its pro-enzyme in deprived eyes were much higher(P<0.001), and while the levels of TIMP-2 mRNA and its protein were lower(P<0.001). With increase of the time of monocular deprivation these changes were more significant and reached the highest or lowest point on 4-14th day with an intergroup statistical difference(P<0.01).⑵After removed the diffuser for 4d, changes in gene expression of MMP-2 and TIMP-2 metioned above could be recovered ultimately or partly in chicks deprived vision for 7d or 21d respectively. ⑶GM6001 could completely inhibit the development of FDM in chicks deprived vision for 4d, and while in chicks deprived for 14d its effects lessened obviously, and there was no effect on that of nondeprived chicks. What had been got from the second part was that: compared with groups treated with TNF-α, bFGF and Ovotransferrin both in the normal and MD eyes the expression levels of MMP-2 activity and pro-enzyme and its mRNA in the groups treated with TGF-β1 were lower (P<0.001). As for TIMP-2 mRNA expression levels the thing was different. In MD group treated with TGF-β1 the level of TIMP-2 mRNA increased slightly(P<0.05), and no changes were found in normal group(P>0.05). The results found in the last part were as follows: ⑴ In the non-deprived eyes, the axial length and diopter degree were reduced after uPA treatment, whereasPAI-1 increased them. In the deprived eyes, uPA inhibited axial length elongation and the development of FDM, but PAI-1 had no effect on it.⑵The contents of TGF-β1 mRNA and protein in the posterior sclera were reduced significantly in FDM eyes compared with the control specimen(P<0.001). In the non-deprived and deprived eyes, the levels of TGF-β1 mRNA and protein were increased after uPA treatment, whereas the effects of PAI-1 on them in non-deprived group was just contrary, and there was no effect was found in FDM group. ⑶In the non-deprived and deprived eyes, the levels of MMP-2 mRNA and enzymic activity and pro-enzyme were reduced after uPA treatment, whereas the effects of PAI-1 on them in non-deprived group was increased , and there was no effect was found in FDM group.⑷uPA could only slightly increase the TIMP-2 gene expression levels in deprived group, and PAI-1 could only reduced it in non-deprived group. Conclusion:⑴That MMP-2 activity expressed significant high in the posterior sclera would be the directed key factor to trigger sclera ECM remodeling process therefore to develop FDM, in which inhibition effect of TIMP-2 and transcriptional modulation of MMP-2 gene expression would be important in modulation of MMP-2 activity.⑵Probably by influencing its transcriptional modulation of gene expression TGF-β1 could significantly inhibit the expression of MMP-2 gene in the posterior sclera in FDM; ⑶Specific high level of MMP-2 activity would be resulted from elimation of the inhibition effect of TGF-β 1 whose expressional level were reduced significantly during the development of FDM.