Background: The exact pathways of collagen remodeling in tendon tissue are not well understood. Therefore, we have established a 3D collagen gel system and studied the remodeling capacity of two different TSPC lines: young, Y-TSPC and aged/degenerative, A-TSPC. We specifically investigated the involvement of integrin receptors in the remodeling process. Methods: Y- and A-TSPC were derived from human Achilles tendon. RT-PCR was used to assess the expression of collagen-binding integrins. Integrins a1 and a11 were silenced by lentiviral delivery of shRNA in the Y-TSPC. Control-shRNA, a1-shRNA and a11-shRNA virus was given for 24h and then cells were selected with zeocin for 10 days. The integrin knockdown (KD) efficiency was assessed by quantitative PCR and western blotting. Last, time-lapse recording of gel contraction of Y-TSPC+con, Y-TSPC+a1KD, Y-TSPC+a11KD, and A-TSPC were performed. Results: Integrin a1 and a11 were significantly downregulated in A-TSPC. Therefore, to mimic the A-TSPC we carried out a1 and a11 KD in Y-TSPC. PCR and western blot validated very efficient KD. Analyses of collagen contraction revealed that Y-TSPC+a11KD had significant reduction in collagen contractibility comparable to A-TSPC phenotype. Regarding integrin a1, we found that this receptor had no effect on the contraction rate of TSPC. Thus, to our knowledge we have now identified for the first time a novel role of a11 integrin in tendon matrix remodeling, and a follow up analyses of the exact downstream cascade are on the way.
The exact pathways of collagen remodeling in tendon tissue are not well understood. Therefore, we have established an ex vivo 3D collagen gel-based system and we studied the remodeling capacity of two different TSPC lines from young, Y-TSPC and aged/degenerative, A-TSPC donors. Here, we specifically focused on investigating the involvement of integrin receptors in the remodeling process. Integrins are transmembrane receptors consisting of alpha (a) and beta (b) subunits, which form cell-to-matrix bonds, activate various pathways and thereby control cell proliferation, differentiation and survival. Y- and A-TSPC were derived from human Achilles tendons and are fully described in Kohler et al. 2013. RT-PCR was used to assess the expression of collagen-binding integrins in the TSPC cultivated in collagen gels. Next, a1 and a11 integrins were silenced by stable lentiviral delivery of target-specific shRNA in the Y-TSPC. Control (con-shRNA), integrin (a1-shRNA) and integrin a11 (a11-shRNA) virus-containing supernatant was given for 24h and then cells were selected with 50 microg./ml zeocin for 10 days. The integrin knockdown (KD) efficiency was assessed by quantitative PCR and western blotting. Last, functional tests were carried out by time-lapse recording gel contraction of four cell groups (Y-TSPC+con, Y-TSPC+a1KD, Y-TSPC+a11KD, and A-TSPC). Among the screened integrins we found that integrin a1 and a11 were significantly downregulated in A-TSPC with 3.8 and 5.6 folds, correspondingly. Therefore, to mimic the A-TSPC we carried out a gene KD of a1 and a11 in Y-TSPC. PCR and western blot clearly validated the efficient KD. Analyses of collagen contraction, revealed that Y-TSPC+a11KD significantly reduced collagen contractability comparable to A-TSPC. This indicated the indispensable role of this integrin in the signaling pathway of collagen matrix remodeling. In respect to integrin a1, we found that this receptor did not affect the contraction rate of Y-TSPC, which was similar to Y-TSPC+con. To our knowledge we have now identified for the first time the critical role of a11 integrin receptor in tendon collagen remodeling, and a follow up analysis of its exact downstream cascade is on the way. Future efforts in deciphering how tendon matrix makeover is regulated can lead to innovation in preventive strategies for tendon degeneration.
