Bone Marrow-derived Cells Contribute To Chorioidal Neovascularization By MMPs Expression Regulated By MiRNA

Background:Choroidal neovascularization(CNV) is up to 40 kinds of eye diseases common pathological process which can affect central vision. It is one of intraocular neovascularization important form of expression, including angiogenesis and vascularization of two forms, which can cause the retina and choroid relatively hypoxic ischemic tissue and neovascularization formation. Angiogenesis is defined as intrinsic vessel cells participates in and forms into new blood vessels through a series of stu hyperplasia, migration. And vascularization refers to the formation of new blood vessels by the mesodermal cells differentiate into vascular endothelial cells(VEC) and the bone marrow-derived cells(BMCs).CNV is a prerequisite for growth: the basement membrane and extracellular matrix(ECM) decomposition, where the VEC to proliferate to form a bud, and then developed to generate CNV. I, III, IV, V collagen is the main composition of the extracelluar matrix in CNV. IV collagen belongs to the main composition of the basement membrane in CNV. A variety of factors inflammation, different hypoxia and a lot of stress injury also can affect the whole process of CNV and accompanied through the development of CNV.Our preliminary studies have shown that bone marrow-derived cells(BMCs) can not only proliferation and differentiation of constituent VEC, vascular smooth muscle cells(VSMC) of the vessel wall and secrete angiogenic factors involved in the generation process of CNV. After induced injury, BMCs the CNV area rapid and specific chemokines in the initiation of the CNV lesion to be chemotactic rather than stay in the other normal tissues. BMCs role in angiogenesis in pathological angiogenesis therapy provides a new way of thinking, on the one hand can be formed by behavioral interventions BMCs control vessels, on the other hand can also be BMCs as a carrier, the drug, the drug activating substances brought to the site in need of treatment of neovascularization play a role in targeting therapy.Matrix metalloproteinase(MMPs) plays an essential part in CNV formation, it can be widely degrade vascular basement membrane and ECM, vascular cell migration budding, BMCs chemokines involved in angiogenesis prerequisite. Matrix metalloproteinases-2/-13(MMP-2/-13) takes a major role in CNV generation process, MMP-13 can main degrade I, III collagen, MMP-2 can main degrade IV, type V collagen, while its mail origin is not ocular cells in situ. BMCs which participate in the production of CNV, is another source of cell composition of CNV. Probably, it is an important source of MMPs in CNV. And according to our earlier research, it is suggested that MMP-2/MMP-13 might be the regulating target genes of mi R188-5p. Objective:To prove whether BMCs, which are involved in the development of CNV, are the main source of MMPs, and whether the MMP-2/MMP-13 expression has a relationship with mi R188-5p, and whether they affect the development of CNV. Methods:Bone marrow cells were transplanted from green fluorescent protein(GFP) transgenic mice to wild-type C57BL/6J mice in order to establish chimeras. Those whose chimeric degrees are more than 85% were included into the follow-up experiments. CNV was induced by laser on chimeric mice(treatment group) and wild-type mice(control group). MMP-2/MMP-13 levels in laser induced eyes were quantified by ELISA, Western Blot and Zymography at time points of day1, 3, 5, 7, 10, 14, and 28. Mi R188-5p expression in laser induced eyes was quantified by q RT-PCR at the same time points. Immunofluorescence staining was used to identify MMP-2/MMP-13 and mi R188-5p expressed by GFP-positive BMCs in CNV, and the expression level was quantified by images analysis. Results:Results of tissue ELISA showed similar trends of MMP-2/MMP-13 expression between the treatment group and control group(ANOVA MMP-2:F=0.060,p=0.810;MMP-13:F=0.012,p=0.915). ELISA, Western Blot, Zymography and immunofluorescence staining all showed that MMP-2 expression and active began with a rapid increase in the early stage of CNV and reached its climax on the third day. Moreover, at that time, the corresponding expression of BMCs reached the peak as well, and occupied 64.21% of the total expression, then began to decline. MMP-13 expression and active showed slowly increases at the early stage of CNV and reached its climax on the day 7, meanwhile the expression of BMCs also reached the highest and accounts for 79.61% of the total expression, then fell. On the other hand, firstly, the predict result of target genes showed that 3′UTR of MMP-2 or MMP-13 has a complementary association point of mi R188-5p. Secondly, both the results of q RT-PCR and immunofluorescent staining demonstrated that mi R188-5p fell rapidly at the early stage of CNV, the lowest expression level observed on the day 7, then increased. MMP-2 / MMP-13 protein expression was negatively correlated with mi R188-5p expression. Immunofluorescence localization semi-quantitative analysis of the expression of MMP-13 in CNV by BMCs showed a negative correlation with the expression of mi R188-5p in CNV by BMCs(r=-0.868,p<0.05).The statistical analysis of the expression of MMP-2 by BMCs showed a negative correlation with the expression of mi R188-5p by BMCs in the first 5 days after CNV induction(r=-0.997,p<0.05). Conclusions:MMP-2/MMP-13 and mi R188-5p expression change as CNV development in mice model. They mainly or partially expressed by BMCs. The expression of MMP-2/MMP-13 in CNV by BMCs showed a negative correlation with the expression of mi R188-5p in CNV by BMCs. Those results demonstrated that BMCs play the main role of regulating MMPs expression in CNV. The expression of MMP-2/MMP-13 from BMCs might be regulated negatively by mi R188-5p, which further inhibits the development of CNV.