INDUCTION OF ENDOSTATIN EXPRESSION IN MENSICAL FIBROCHONDROCYTE BY COCULTUR WITH ENDOTHELIAL CELLS

Abstract

Introduction: The highest goal after meniscus damage is the preservation of the meniscus, which is often not possible due to the bad healing of meniscus lesions in the avascular zone. Therefore, the goal of our investigations was the analysis of expression of different angiogenic factors, growth hormones and cytokines in human meniscus cells (fibrochondrocytes). The mutual influence of the fibrochondrocytes by endothelial cell cocultures was analyzed, in order to examine the molecular bases of the healing of meniscus tears in vascularized zones more exactly. For this purpose, commercially available HUVEC [human umbilical vein endothelial cells] were used as well established and stable endothelial cell model.

Material and Methods: Meniscal fibrochondrocytes were expanded in DMEM medium enriched with antibiotics and 10 % FCS. Cocultures of mensical cells and HUVEC were incubated in transwells over four and twelve days, separated by a semipermeable membrane. The expression of Angiopoietin-1, Angiopoietin-2, End-ostatin, VEGF, SMAD-4, Thrombospondin-1, Aggrecan, Biglycan, Fibronectin, Vimentin, Connexin-43, IL-1β, iNOS, MMP-1, MMP-3, MMP-13, collagen-I, -II, -III, -VI, X, and -XVIII were examined by RT-PCR and immunhistochemistry in fibrochondrocytes in the comparison to cultures without endothelial coculture. A proliferation assay was used to investigate the mitotic activity in the coculture compared to the control culture after 4 and 12 days.

Results: In presence of HUVEC, meniscal fibrochon-drocytes expressed the following factors at rates comparable to cells w/o HUVECS: Angiopoietin-1, Angiopoietin-2, VEGF, SMAD-4, Aggrecan, Biglycan, Fibronectin, Vimentin, Connexin-43, iNOS, MMP-1, MMP-3, MMP-13, Thrombostatin-1, collagen-I, -II, -III, -VI, X, and -XVIII. In contrast, expression of end-ostatin (5.1-fold ± 1.2, p< 0.01) and IL-1β (10.3-fold ± 2.3, p< 0.003) were expressed significantly higher in the coculture when compared to the individual cell cultures. The proliferation rate of HUVEC was significantly decreased in coculture when compared to controls: 22 % after 7 days and 35 % after 14 days (p< 0.001).

Discussion/ Conclusion: We were able to cultivate and characterize human fibrochondrocytes from menisci of the knee joint. We could show that coculture of meniscus cells with endothelial cells revealed an increased expression of the anti-angiogenetic factor endostatin and the pro-inflammatory IL-1β. This suggests that meniscus cells are trying to inhibit proliferation of endothelial cells in their neigbourhood, which implicates huge problems in the research field of neoangiogenisis and tissue engineering in meniscus tissue for new healing methods after meniscus trauma.