Retinal diseases as proliferative diabetic retinopathy and retinopathy of pematurity are characterized by the formation of anormal new blood vessels which breaks through the inner limiting membrane into the vitreous.
These proliferative retinopathies are postulated to result from hypoxic insults to the retina that stimulate growth factors, integrins and extracellular proteinases, which result in endothelial cell migration and proliferation. Normally, there is a tight balance between neovascular factors and neovascular inhibitors that help to keep the neovascular process under control. It has been demonstrated that a2 Macroglobulin an important proteinase inhibitor, can also activates cellular proliferation in cells expressing LRP-1.
In this sense, the main focus of our laboratory has been the role of the a2 macroglobulin receptor, LRP-
These antecedents suggest that LRP-1/a2M system has a major role in modulating uncontrolled cell proliferation as in retinal neovascularization.
In order to test this assumption the objective of this work is to demonstrate how the LRP-1/alpha 2 Macroglobulin system is involved in the retinal neovascularization and the molecular mechanism of this participation
First we investigated the expression of LRP-1 and receptor-associated ligands in a rat model of oxygen-induced retinal neovascularization. This model has been extensively characterized and resembles that seen with retinopathy of prematurity.
In this model by WB, we showed that LRP-1 was expressed, along with a2M, at P20 in neovascular retinas .
Then we further examined the LRP-1 retinal localization by immunofluorescence and we identified the cells as Muller cells. We performed simultaneous demonstration of LRP-1 and CRALBP immunoreactivity. Finally by zymographic analysis demonstrated increased activity of MMP-2 and MMP-9 under neovascular conditions.
With these results then we investigated the LRP-1 expression and the a2-M effect in an in vitro model using glial cells
The Muller cells were isolated from C57BL6 retinas and then characterized for expression and localization of specific cell markers by indirect inmunofluorescence staining. We obtained a highly pure muller cell preparation and approximately the 95-100% of cells expressed CRALPB (a Muller marker) and 5% GFAP (an astrocyte marker). In conclusion, we obtained a highly pure glial cell preparation from mouse retinas. In these cells we were able to demonstrated the expression of LRP-1 by western blot and IF using a mouse monoclonal antibody against the 85-kDa b chain of LRP-1.
In an in vivo model we observed a relationship between LRP-1/a2M system and MMPs activity. Using this MC culture our interest was to study if a2 M treatment induces MMPs via LRP-1. With this idea in mind we analyzed the a-2M kinetics in the supernatants of MC. A significantly increased in the amount of MMP-2 activity was observed at 24 hs by gelatine zymography. As positive controls we used IGF-
Finally, we investigated the presence of LRP and receptor-associated ligands in patients with neovascular eye diseases. Human vitreous samples of patients with proliferative diabetic retinopathy (PDR), neovascular glaucoma and other vitreoretinal diseases were extracted. Immunoreactive band of a2-Macroglobulin (a2-M) was detected both in extracts of neovascular rat retinas and human vitreous samples from neovascular glaucoma and PDR. These results correlated with activity level of MMP-2 and MMP-9.
In summary the present study demonstrates that a2-M/LRP-1 system is involved in the regulation of extracellular proteinase production during retinal neovascularization in an in vivo and in vitro model. However future experiments are necessary in order to understand the molecular mechanism involved in this regulation.
