The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

Citation: Gunda V, Verma RK, Sudhakar YA. Inhibition of elastin peptidemediated angiogenic signaling mechanism(s) in choroidal endothelial cells by the a6(IV)NC1 collagen fragment. Invest Ophthalmol Vis Sci. 2013;54:7828-7835. DOI:10.1167/ iovs.12-10870 PURPOSE. The inhibitory effects and mechanism(s) of type IV collagen a-6 chain-derived noncollagenous domain (a6[IV]NC1 or hexastatin) on elastin-derived peptide (EDP)-activated choroidal endothelial cell migration, kinase signaling, and membrane type 1 metalloproteinase (MT1-MMP) activation are explored. METHODS.Mouse choroidal endothelial cells (MCECs) were incubated in media with soluble EDPs (kappa elastin, mouse elastin, and Val-Gly-Val-Ala-Pro-Gly [VGVAPG] hexapeptide) for different time intervals with or without a6(IV)NC1. The MCECs proliferation, migration, tube formation, MT1-MMP expression, and angiogenic signaling were analyzed in cells subjected to EDP and a6(IV)NC1 treatments. The MCECs also were subjected to EDPs, and specific inhibitors for evaluation of focal adhesion kinase (FAK) and protein kinase B (Akt) phosphorylation.RESULTS. Kappa elastin, mouse elastin, and VGVAPG enhanced the migration, without affecting the proliferation of MCECs. The a6(IV)NC1 inhibited survival and EDP-activated migration of MCECs. The EDP-activated MCEC tube formation on matrigel also was inhibited by a6(IV)NC1. Further, EDP-activated MT1-MMP expression and FAK/phosphoinositide-3-kinase (PI-3K)/mammalian target of rapamycin (mToR)/Akt phosphorylation in MCECs, were reduced by a6(IV)NC1. The EDP-induced FAK and Akt phosphorylation was blocked by FAK-and Akt-specific inhibitors.CONCLUSIONS. The EDPs and a6(IV)NC1 are identified to exhibit opposing effects on MCEC angiogenic behavior and signaling. The a6(IV)NC1 inhibited cell survival, EDP-mediated migration, MT1-MMP expression and, FAK/PI-3K/mToR/Akt phosphorylation in MCECs. This work demonstrates a6(IV)NC1 as a prospective endogenous molecule for the treatment of diseases involving choroidal neovascularization in the eye.Keywords: elastin-derived peptides, noncollagenous domains of type IV collagen, angiogenesis P athologic progression of choroidal neovascularization (CNV) in the wet form of age-related macular degeneration (AMD) includes the activation of angiogenic behavior in choroidal endothelial cells. This is promoted by different pathologic factors, including extracellular matrix (ECM) derivatives. 1,2 Elastins and type IV collagen constitute major components of ECM in different tissues, such as skin, lungs, vascular basement membrane (VBM) of blood vessels, and ocular tissues.3,4 Degradation of elastin was identified as a pathologic factor leading to the progression of CNV, through substantial histologic, genetic, and clinical studies, with significant focus on elastin degradation products (EDPs). 1,2Histologic studies revealed conspicuous fragmentation and decrease in the thickness of Bruch's membrane-elastin layer in patients with AMD. Thus, damage in elastic Bruch's membrane was identified to promote ...