Functional Characterization of the 28 kDa Endostatin-spanning Fragment of Collagen XVIII in Mouse Cornea

Abstract

Abstract: : Purpose: To determine the effects of the C-terminal 28 kDa endostatin-spanning proteolytic fragment generated from matrix metalloproteinase matrilysin (MMP-7) cleavage of type XVIII collagen on corneal neovascularization. Methods: We have previously reported the action of MMP-7 on collagen XVIII to generate a 28 kDa endostatin-spanning fragment. We generated extensive quantities of various Histidine-tagged portions 28 kDa endostatin-spanning fragment and endostatin from bacterial cultures (BL21DE3) through IPTG induction and affinity-purified by using Ni-beads. His-28 kDa endostatin-spanning fragment and His-endostatin were eluted with (40 mM and 150 mM) imidazole and then dialyzed with PBS. Calf pulmonary artery endothelial cell (CPAE) proliferation and migration assays were performed in the presence of 2.5-10 ug/ml of His-endostatin-spanning fragment and His-endostatin. After the 48 hour proliferation period, cell density was determined using a Formazin kit. Corneal lysates from immortalized cells were then subjected to immunodepletion with anti-endostatin and anti-hinge antibodies. Corneal lysates with/without antibody depletion were used for CPAE proliferation assays. In addition, recombinant NC1 fragment (1 ug) of collagen XVIII, was subjected to enzymatic cleavage with various MMPs (MMP-7 and -14). Results: Recombinant 28 kDa endostatin-spanning fragment and recombinant endostatin inhibited CPAE proliferation, OD₄₉₅ = 0.378 + 0.04 and OD₄₉₅ = 0.375 + 0.015, respectively, when compared to the control (10% FCS) OD₄₉₅ = 0.46 + 0.02. Corneal epithelial cellular lysates inhibit CPAE proliferation (OD₄₉₅ = 0.61 + 0.02) while corneal lysates depleted with anti-endostatin enhanced CPAE proliferation (OD₄₉₅ = 0.69 + 0.05) when compared to control (FCS only OD₄₉₅ = 0.63 + 0.037). MMP-14 cleaves NC1 fragment of collagen XVIII to generate fragments (20-24 kDa) differ that from that of MMP-7 (28 kDa). Conclusions: Our results suggest that the 28 kDa endostatin-spanning fragment generated by the enzymatic cleavage of type XVIII collagen by matrilysin, has an anti-angiogenic effect similar to that of 20 kDa endostatin.

Keywords: cornea: basic science • molecular biology • wound healing