Promotion Of Corneal Neovascularization By IL-1β-treated Senescent Corneal Fibroblasts In Vitro And In Vivo

PurposeWe do this research to examine changes of cells and cell products of normal andsenescent fibroblasts after IL-1β treatment,evaluate the the differences of theirproliferation, migration and tube formation of HUVECs and in vivo observe of their effectson corneal neovascularization.Methods1.Cell culture and senescence inductionPrimary culture of HCF(human corneal fibroblasts) was from accidental death eye donors,using digestion method and cultured4~5generation before test.2.The HCFs were classified into four groups: normal fibroblasts, senescent fibroblasts,normal fibroblasts induced by IL-1β and senescent fibroblasts induced by IL-1β. We used300μM H2O2to induce senescence and concentration of IL-1β was10ng/ml. We usedSA-β-gel to identify cells’ senescence before other tests.3. The production of MMPs，VEGF，PEDF，tPA，uPA were assessed by real-time PCR．In the four groups the proliferation was estimated by MTT, migration capacity wasexamined by migration assay, and tube formation of HUVECs were estimated by tube formation assay. The results of all factors were analyzed.4. Senescent fibroblasts promoted corneal neovascularization in vivo.Equal number ofthese four cells were injected into the corneal stroma of normal C57BL/6mice. All themice eyes were photographed under slitlamp for the evaluation of cornealneovascularization.Results1.Under the light microscope we observed that the primary cells grew spindle-shaped afteradherence and came to fusion after1week.2weeks after passage, cell growth can be flaky,network connection.2.After the treatments of IL-1β and H2O2four groups of cells grew with differentshapes.And we examined SA-β-gel+to identify cells’ senescence.3. The cell proliferation, the tubes and the number of migration cells were significantlyhigher in the last group than that in the normal group (P<0.05)，and the expression ofMMPs，VEGF，PEDF，tPA and uPA was also increased in the last group (P<0．05)．Thecell proliferation, the tubes and the number of migration cells were significantly increasedin the senescent cells added IL-1β than that in the cells added IL-1and senescent cells．Theproduction of MMPs， VEGF， PEDF， tPA and uPA was significantly increased in thesenescent cells added IL-1β than that in the cells added IL-1and senescent cells．4. Compared with normal corneal fibroblasts, the other three groups caused early onset ofneovascularization. The intrastromal injection of IL-1β senescent fibroblasts caused morenumber of new vessels than IL-1β and senescent groups.Conclusions1.Senescent corneal fibroblasts secreted higher levels of MMPs，VEGF，PEDF，tPA anduPA and promoted the proliferation, tube-formation,and migration of HUVECs moresignificantly than did normal fibroblasts.2.Senescent corneal fibroblasts added IL-1β secreted higher levels of MMPs， VEGF， PEDF， tPA and uPA and promoted the proliferation, tube-formation,and migration ofHUVECs more significantly than did normal and senescent fibroblasts.3.After IL-1β treatment on normal and senescent fibroblasts, their effects on cornealneovascularization enhanced as well as their ability of the proliferation, migration,andtube-formation of HUVECs.