Inhibition of Elastin Peptide-Mediated Angiogenic Signaling Mechanism(s) in Choroidal Endothelial Cells by the α6(IV)NC1 Collagen Fragment

Abstract

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 ...

Collagen‐mediated hemostasis

“…Arresten, canstatin, tumstatin, tetrastatin, pentastatin and hexastatin are all proteins derived from the NC1 domains of collagen type IV α1, α2, α3, α4, α5 and α6 chains, respectively. All of them inhibit angiogenesis by blocking endothelial cell proliferation and migration, tube formation, and proapoptotic function . For instance, canstatin inhibits angiopoietin‐1, a vascular growth factor that is necessary for angiogenesis .…”

NC1‐peptide regulates spermatogenesis through changes in cytoskeletal organization mediated by EB1

“…9 This NC1-peptide has also been shown to be a biologically active peptide, capable of modulating a number of cellular functions including MMP activation, cell adhesion, angiogenesis, tumorigenesis, and others based on studies in multiple endothelia and/or epithelia. [10][11][12][13] Studies have shown that NC1-peptide was also generated in the testis, capable of modulating Sertoli cell tight junction (TJ)-permeability barrier function, 14 and its cloning into the mammalian expression vector pCI-neo to be used for its overexpression in Sertoli cells in vitro as well as in the testis in vivo was shown to perturb the Sertoli cell BTB function both in vitro and in vivo. 15 These studies thus illustrate that the NC1-peptide exerts its modulating effects to support spermatogenesis are mediated through changes in the cytoskeletal organization of both actin-and MT-cytoskeletons, in particular the organization of actin filament bundles and MTs across the Sertoli cell at the ultrastructures known as the basal ectoplasmic specialization (ES) or apical ES at the Sertoli cell-cell or Sertoli-spermatid interface, respectively.…”

Developments in purification methods for obtaining and evaluation of collagen derived endogenous angioinhibitors

“…Simultaneous renaturation and purification methods provide another alternative strategy for obtaining refolded protein during purification process [45]. In a recent study, L-arginine mediated on-column renaturation coupled with size exclusion chromatography was applied for obtaining higher yields of soluble hexastatin from bacterial inclusion bodies [46, 47]. In this study recombinant human hexastatin was expressed in transformed E. coli as inclusion bodies.…”