Effect Of HGF In Guinea Pig With Lens Induced Myopia

Myopia is commonly and frequently encountered disease in ophthalmology, which greatly affects people s life. The incidence of myopia is increasing year by year. Therefore the pathogenesis and control of myopia has become very important.With the development of molecular biology, it is found that abnormal vision modulates the development of local sclera by changing the level of various neurotransmitters and growth factors in the eye. These neurotransmitters and growth factors work by means of cooperate parallel and across, and lead to the ultrastructural and biomechanical alteration of sclera through a series of signal transudations. Sclera is remodeled and eye axial is lengthened and induced to axis myopia. Many studies had indicated that the expression of MMP-2 was significantly higher in the posterior sclera in the myopia. Matrix metalloproteinase-2 (MMP-2) play a pivotal role in the process of extracellular matrix (ECM) remodeling in the posterior sclera. But the reason of elevated levels of MMP-2 is not clear.Hepatocyte growth factor (HGF) is a multi-function growth factor. The c-Met tyrosine kinase receptor is the only known receptor for HGF. Downstream Met signaling is essential for multiple biological actions involving proliferation, morphogenesis, apoptosis, and breakdown of extracellular matrix (ECM) in target cells. It was reported that the hepatic growth factor gene was closely related to normal variation in eye size in any mammal and HGF can elevate MMP-2 expression and attenuates fibrogenic responses which was mediated by MMP-2. So, we hypothesized that HGF may be involved in the development of myopia.The c-Jun N-terminal kinase (JNK) is a member of the MAPkinase family. It is one such family of multifunctional-signaling molecules, activated in response to wide range of cellular stresses as well as in response to cytokine such as HGF, NGF and so on. A large body of evidence indicates that JNK pathway regulates various processes by up-regulating MMP-2 expression in multiple cell types.So for, there are no reports about whether HGF and c-Met are dispersed in sclera and sclera fibroblasts, whether HGF can up-regulate MMP-2 expression in myopia and whether HGF up-regulates MMP-2 expression through JNK pathway.In this study, we established the guinea pigs model of lens induced myopia and cultured guinea pig scleral fibroblasts, detected the expressions of HGF and MMP-2 in the sclera and scleral fibroblasts of induced and control eyes, sanalyzed the MMP-2 expression in scleral fibroblasts of control eyes treated with HGF and of induced eye after suppressed the expression of HGF by RNA interference, investigated whether JNK pathway is activated by HGF and involved in the HGF-induced MMP-2 expressions in scleral fibroblasts, and investigated whether SP600125 by intravitreal injection method, could inhibit the development of lens induced myopia. The finding will provide theory bases for the opinion that HGF-JNK-MMP-2 signaling pathway could be considered potential therapeutic target for myopia.Part I:Expression of HGF in guinea pig with lens induced myopiaObjectiveTo study the relationship between HGF and myopia, we established the guinea pigs model of lens induced myopia and detected the expressions of HGF, c-Met and MMP-2 mRNA and protein in the sclera of induced and control eyes by immunohistochemistry, Western blot and RT-PCR. Methods1. Animal models and groups:Ninty flower color guinea pigs (one-week-old) were randomly divided into control and induced groups. The both eyes of thirty guinea pigs were untreated as control group. The right eyes of sixty guinea pigs were monocularly defocused by-10.OD lens. After 6 weeks of defocus, twenty eyes of each group were used for immunohistochemistry, another twenty eyes for RT-PCR, the last twenty eyes for Western blot.2. Measure of the refractive state and axial length:Before and after defocused for 6 weeks, the refractive state and axial length were measured by radioscopy and A-scan ultrasonography respectively.3. Hematoxylin-eosin staining and morphology:After the refractive state and axial length were measured, the eyes were extracted, routine hematoxylin-eosin (HE) staining were done, and morphological changes were observed under light microscope.4. The expressions of HGF, c-Met and MMP-2 protein in sclera of induced and control eye were detected by immunohistochemistry.5. The expressions of HGF, c-Met and MMP-2 mRNA in sclera of induced and control eye were detected by RT-PCR.6. The expressions of HGF, c-Met, p-c-Met and MMP-2 protein in sclera of induced and control eye were detected by Western blot.7. Statistical analysis:All the data were analyzed by SPSS 13.0 statistical package. Measurement data were expressed by standard deviation, the mean of two groups used the pair sample t-test. The relation of two variables was analyzed by the Pearson s correlation analysis. A less than 0.05 was considered statistical significance.Results1. Comparison of refraction state and axial length between the induced and control eyes:The refraction state and axial length showed significant difference (P<0.05) between the induced eyes and control eyes. 2. Morphology change:Hematoxylin-eosin (HE) staining showed the posterior retina and sclera of the induced eyes were thinner than that of the control eyes.3. Immunohistochemical analysis:The expressions of HGF, c-Met and MMP-2 protein were low in sclera of the control eyes. The expressions of HGF and MMP-2 protein were significantly higher in sclera of the induced eyes than that of the control eyes (all P<0.05). The expressions of c-Met protein were not significantly different between the induced and control eyes(P>0.05).4. RT-PCR analysis:The expressions of HGF, c-Met and MMP-2 mRNA were low in sclera of the control eyes. The expressions of HGF and MMP-2 mRNA were significantly higher in sclera of the induced eyes than that of the control eyes (all P<0.05). The expression of c-Met mRNA was not significantly different between the induced and control eyes (P>0.05).5. Western blot analysis:The expressions of HGF, c-Met and MMP-2 protein were low in sclera of the control eyes. The expressions of HGF, p-c-Met and MMP-2 protein were significantly higher in sclera of the induced eyes than that of the control eyes (all P< 0.05).6. The expressions of HGF protein was positively correlated with the expression levels of p-c-Met protein (r= 0.902, P<0.05), p-c-Met protein was positively correlated with MMP-2 protein (r=0.885, P<0.05), and HGF protein was positively correlated with MMP-2 protein (r=0.900, P<0.05) in sclera of the induced eyes.Conclusions1. We established the guinea pigs model of lens induced myopia.2. This study reveals first time that the expressions of HGF and c-Met mRNA and protein were higher in sclera of the induced eyes than that of the control eyes. The results suggestted that HGF maybe involved in the development of myopia.3. The expressions of HGF protein was positively correlated with the expression levels of p-c-Met protein and MMP-2 protein in sclera of the induced eyes. It is possible that abnonnal visual signals might induce HGF protein synthesis in sclera of the induced eyes, then increase HGF bind to c-met and induce tyrosine phosphorylation of c-met and then phosphorylated c-met activate the downstream signal transduction pathway, thereby upregulate exprssion of MMP-2, and ultimately result in breakdown of extracellular matrix (ECM) and myopia.PartⅡ:Regulation of HGF on MMP-2 in guinea pig scleral fibroblasts cultured in vitroObjectiveWe found that HGF maybe involved in the development of lens induced myopia by upregulating the exprssion of MMP-2. For further study, we established the guinea pigs model of lens induced myopia and cultured scleral fibroblasts of guinea pigs, detected the expressions of HGF, c-Met and MMP-2 protein in the scleral fibroblasts of induced and control eyes, analyzed the MMP-2 expression in scleral fibroblasts of control eyes treated with HGF and of induced eyes after suppressed the expression of HGF by RNA interference, investigated whether JNK pathway is activated by HGF and involved in the HGF- induced MMP-2 expression in scleral fibroblasts.Methods1. Animal models and groups:Thirty flower color guinea pigs (one-week-old) were divided randomly into control and induced groups. The both eyes of ten guinea pigs were untreated as control group. The right eyes of twenty guinea pigs were monocularly defocused by-10D lens. Four weeks later, the induced and control guinea pig scleral fibroblasts were isolated, cultured and identificated.2. The expressions of HGF, c-Met and MMP-2 portein in two types of scleral fibroblasts were detected by Immunocytochemistry and Western blot.3. To study whether MMP-2 is a downstream mediator of HGF in guinea pig scleral fibroblasts:3.1 MTT assayed the effect of HGF with different doses on proliferation of scleral fibroblast of control eye after 72h. 3.2 We stimulated the scleral fibroblasts of control eyes with HGF(10 ng/ml) with different doses, the expression of p-c-Met protein was detected by Western blot and the expressions of MMP-2 mRNA and protein were detected by RT-PCR and Western blot. We chosed the HGF (10ng/ml) for the latter study because of its effective and cheap.3.3 We chosed the scleral fibroblasts of induced eyes, in which the expression of HGF was higer, to culture and transfect with siRNA. The expression of HGF protein was detected by Western blot. The expressions of MMP-2 mRNA and protein were detected by RT-PCR and Western blot.4. To study whether HGF-induced MMP-2 expression is mediated by JNK pathway in guinea pig scleral fibroblasts:4.1 We stimulated scleral fibroblasts of control eyes with HGF (10 ng/ml), the expression of p-JNK and MMP-2 protein was detected by Western blot.4.2 MTT assayed the effect of SP600125 with different doses on proliferation of scleral fibroblast of control eye after 72h. We chosed the SP600125 (10μmol/L) for the latter study because of its effective and cheap.4.3 The expressions of p-JNK and MMP-2 protein stimulated with HGF(1 Ong/ml) in the presence or absence of SP600125 (10μmol/L) were detected by Western blot.5. Statistical analysis:All the data were analyzed by SPSS 13.0 statistical package. Measurement data were expressed by standard deviation, the mean of two groups used the pair sample t-test. The mean among more groups used the ANVOA. A less than 0.05 was considered statistical significance.Results1. The cells shape showed typical spindle morphology, and there was no difference between the two groups of cells. Cells showed a vimentin-positive and cytokeratin-negative phenotype, and thus were concluded to be fibroblasts.2. The expressions of HGF, c-Met and MMP-2 portein in two types of scleral fibroblasts: 2.1 Immunohistochemical analysis:Specific brown-colored staining for HGF, c-Met and MMP-2 was recognized in the cytoplasm of two types of scleral fibroblasts, and the immunohistochemical signals of HGF and MMP-2 in scleral fibroblasts of induced eyes was stronger than that in control eyes (P<.0.05). The immunohistochemical signals of c-Met was not significantly different between two types of scleral fibroblasts(P>0.05).2.2 Western blot analysis:HGF, c-Met and MMP-2 protein expression could be detected in two types of scleral fibroblasts. The expressions of HGF and MMP-2 protein in scleral fibroblasts of induced eyes were significantly higher as compared to that in control eyes (P<0.05). There was a marked increase in the tyrosine phosphorylation of c-Met in scleral fibroblasts of induced eyes(P< 0.05), total c-Met levels was unchanged(P>0.05).3. In scleral fibroblasts of guinea pigs:3.1 HGF with different doses have no signifieant influence on proliferation of scleral fibroblast of control eye after 72h(P>0.05).3.2 The expressions of p-c-Met and MMP-2 protein were increased in HGF-treated scleral fibroblasts of control eyes in a dose dependent manner. The expressions of MMP-2 mRNA and protein was obviously reduced by silencing HGF with siRNA in scleral fibroblasts of induced eyes in a time dependent manner.4. In scleral fibroblasts of guinea pigs:4.1 HGF elicited the phosphorylation of JNK and then the expression of MMP-2 protein was markedly increased (P<0.05).4.2 SP600125 with different doses have no signifieant influence on proliferation of scleral fibroblasts of control eye after 72h(P>0.05).4.3 The expression of HGF (10ng/ml)-induced MMP-2 protein was markedly inhibited by treatment with SP600125 (10μmol/L) in scleral fibroblasts of control eye(P<0.05). Conclusions1. This study reveals first time that the expressions of HGF and MMP-2 protein were significantly higher in scleral fibroblasts of the induced eyes than that of the control eyes.2. This study reveals first time that MMP-2 is a downstream mediator of HGF in scleral fibroblasts of guinea pigs.3. This study reveals first time that HGF-induced MMP-2 expression is mediated by JNK pathway in scleral fibroblasts of guinea pigs. The findings will provide theory bases for the opinion that HGF-JNK-MMP-2 signaling pathway could be considered potential therapeutic target for myopia.PartⅢ:Blocking activation of JNK signaling pathway in sclera of guinea pigs with lens induced myopia by SP600125ObjectiveIn this study, we established the guinea pigs model of lens induced myopia and investigated whether JNK inhibitor SP600125 by intravitreal injection method, could inhibit the development of lens induced myopia.Methods1. Animal models and groups:Seventy flower color guinea pigs (one-week-old) were randomly divided into 4 groups:groupⅠ(6 weeks’induced), group II (6 weeks’induced plus PBS), groupⅢ(6 weeks’induced plus SP600125 (10μmol/L)), groupⅣ(control group). Each group had 20 guinea pigs and the right eyes in each group were defocused by-10.0D lens as experimental eyes except for group IV in which both eyes of ten guinea pigs were untreated as control eyes.2. Measure of the refractive state and axial length:referred to partⅠ3. Hematoxylin-eosin staining and morphology:referred to partⅠ 4. The expressions of p-JNK and MMP-2 portein in sclera of each group were detected by Western blot.5. Statistical analysis referred to partⅠ.Results1. Comparison of refraction state and axial length between each groups:The refraction state and axial length of groupⅠ, groupⅡand groupⅢwere sihnificantly deeper and longer than that of groupIV(P<0.05). The refraction state and axial length showed significant difference (P<0.05) between the groupⅢand groupⅠor groupⅡ.2. Morphology change:The posterior sclera of eyes in groupⅠor groupⅡwas obviously thinner than that of groupⅣ(P<0.05). The posterior sclera of eyes in groupⅢwas thicker than that of groupⅠor groupⅡ(P<0.05), but thinner than that of groupIV(P<0.05).3. The expressions of p-JNK and MMP-2 portein induced by abnormal visual signals were partly inhibited by SP600125:The expressions of p-JNK and MMP-2 portein in sclera of groupⅠor groupⅡwas obviously higher than that of groupⅣ(P<0.05). Tthe expressions of p-JNK and MMP-2 portein in sclera of groupⅢwere lower than that of groupⅠor groupⅡ(P<0.05), but higher than that of grouplV(P<0.05).Conclusions1. This study reveals first time that during the development of lens induced myopia, SP600125 could partly inhibit the activation of JNK signal pathway and then down-regulate the expression of MMP-2 in vivo.2. The results indicate that SP600125 could partly inhibit the development of lens induced myopia.