Biochemistry and Molecular Biology Inhibition of Elastin Peptide-Mediated Angiogenic Signaling Mechanism(s) in Choroidal Endothelial Cells by the a6(IV)NC1 Collagen Fragment Venugopal Gunda,1,2 Raj Kumar Verma,1,3 and Yakkanti Akul Sudhakar1,4 1Cell Signaling, Retinal & Tumor Angiogenesis Laboratory, Boys Town National Research Hospital, Omaha, Nebraska 2The Eppley Institute for Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 3Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, Texas 4Center for Cancer & Metabolism, Cell Signaling Laboratory, Bioscience Division, Stanford Research Institute (SRI) International, Menlo Park, California Correspondence:YakkantiAkulSud- PURPOSE. The inhibitory effects and mechanism(s) of type IV collagen a-6 chain–derived hakar, Center for Cancer & Metabo- noncollagenous domain (a6[IV]NC1 or hexastatin) on elastin-derived peptide (EDP)–activated lism, Cell Signaling Laboratory, choroidal endothelial cell migration, kinase signaling, and membrane type 1 metalloprotei- Bioscience Division, SRI Interna- nase (MT1-MMP) activation are explored. tional, 333 Ravenswood Avenue, Menlo Park, CA 94025-3493; METHODS. Mouse choroidal endothelial cells (MCECs) were incubated in media with soluble [email protected]. EDPs (kappa elastin, mouse elastin, and Val-Gly-Val-Ala-Pro-Gly [VGVAPG] hexapeptide) for Submitted: August 29, 2012 different time intervals with or without a6(IV)NC1. The MCECs proliferation, migration, tube Accepted: May 22, 2013 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 Citation: Gunda V, Verma RK, Sudha- kar YA. Inhibition of elastin peptide- inhibitors for evaluation of focal adhesion kinase (FAK) and protein kinase B (Akt) mediated angiogenic signaling mecha- phosphorylation. nism(s) in choroidal endothelial cells RESULTS. Kappa elastin, mouse elastin, and VGVAPG enhanced the migration, without affecting by the a6(IV)NC1 collagen fragment. the proliferation of MCECs. The a6(IV)NC1 inhibited survival and EDP-activated migration of Invest Ophthalmol Vis Sci. MCECs. The EDP-activated MCEC tube formation on matrigel also was inhibited by 2013;54:7828–7835. DOI:10.1167/ iovs.12-10870 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 athologic progression of choroidal neovascularization CNV further established the role of elastolysis in CNV of AMD.2 P (CNV) in the wet form of age-related macular degeneration Such increased EDPs in CNV were generated through the (AMD) includes the activation of angiogenic behavior in elastolysis in skin, Bruch’s membrane, and choriocapillaries. choroidal endothelial cells. This is promoted by different Thus, elastolysis in the extraocular tissues also affected pathologic factors, including extracellular matrix (ECM) deriv- CNV.2,8,9 The role of EDPs in promoting CNV has been atives.1,2 Elastins and type IV collagen constitute major demonstrated at cellular level through the elucidation of components of ECM in different tissues, such as skin, lungs, angiogenic effects activated by the EDPs on choroidal vascular basement membrane (VBM) of blood vessels, and endothelial cells. These studies used soluble elastins generated ocular tissues.3,4 Degradation of elastin was identified as a through the elastolysis of bovine ligament elastins (kappa- pathologic factor leading to the progression of CNV, through elastin [jE]) and the synthetic elastin-derived bioactive peptide, substantial histologic, genetic, and clinical studies, with Val-Gly-Val-Ala-Pro-Gly hexapeptide (VGVAPG [BP]).10 significant focus on elastin degradation products (EDPs).1,2 In contrast with the generation of angiogenic promoting Histologic studies revealed conspicuous fragmentation and EDPs through elastin metabolism, type IV collagen metabolism decrease in the thickness of Bruch’s membrane–elastin layer in leads to the release of noncollagenous domains (NC1) with patients with AMD. Thus, damage in elastic Bruch’s membrane antiangiogenic properties on endothelial cells (ECs).11–18 Type was identified to promote intrusion of pathologic choriocapil- IV collagen a-2 chain–derived noncollagenous domain laries in CNV.5–7 Elevation of serum EDP levels in patients with (a2[IV]NC1) induced apoptotic activity in ECs and suppressed Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783 7828 Downloaded from iovs.arvojournals.org on 09/29/2021 Elastin Peptide-Mediated Angiogenic Signaling IOVS j December 2013 j Vol. 54 j No. 13 j 7829 FIGURE 1. Inhibition of MCEC survival by a6(IV)NC1. (A) Upper graph showing survival of MCECs without polymyxin-B treatments. (B) Lower graph showing MCECs treated with polymyxin-B. Status bars indicate average cell survival of MCECs (mean 6 SD of 3 replicates) after 48 hours of treatment. Presence (þ) and absence (À) of factor in respective treatments are indicated. CNV.19 The NC1 domain of type IV collagen a-1 sub-chain in levels in the purified protein were estimated using the suppressed angiogenic activation in retinal endothelial cells.20 Limulus amoebocyte lysate assay kit (Lonza), following Thus, contrasting roles of elastin and type IV collagen manufacturer’s instructions. derivatives have been established in regulation of CNV. However, these earlier studies characterized the individual Cell Viability or Proliferation Assay roles of either EDPs or type IV collagen NC1 domains on angiogenesis. Our study investigated the combined effects of The MCECs were cultured in endothelial basal medium EGM- different EDPs and a6(IV)NC1 in regulating angiogenic 2 (Lonza) medium and approximately 80% confluence cells signaling in mouse choroidal endothelial cells (MCECs). were subjected to serum starvation for 8 hours. Serum- Recombinant a6(IV)NC1 exhibited antiangiogenic properties starved cells were trypsinized, resuspended in media with in vitro and in vivo, though to our knowledge its angioinhi- respective treatments, and distributed into 96-well plates at bitory signaling mechanism is not known yet.21–23 In our study, density of 5 3 104 cells per well. The endothelial cell basal the effects of a6(IV)NC1 on EDP-mediated angiogenic activities medium (EBM)-2 without serum was used as negative control and signaling were evaluated systematically using MCECs. We and EMB-2 with 2% fetal calf serum (FCS) was used as positive showed that a6(IV)NC1 inhibits MCECs proliferation, and EDP control. Treated wells contained cells resuspended in EBM-2 mediated angiogenic signaling by negatively regulating focal medium containing 2% FCS, added with one of the EDPs adhesion kinase (FAK)/phosphoinositide-3-kinase (PI-3K)/ (bovine neck ligament soluble elastins/kappa-elastin [jE-2; mammalian target of rapamycin (mToR)/protein kinase B lg/mL] or mouse lung elastin peptides/m-elastins [ME-2; lg/ (Akt) phosphorylation. This resulted in inhibition of membrane mL], or Bio active peptide-VGVAPG [BP-200; ng/mL]) from type 1 metalloproteinase (MT1-MMP) expression, reducing Elastin Products Company, Inc. (Owensville, MO) with or migratory potential. without 0.5 and 1.0 lM a6(IV)NC1. Cells treated with 0.5 and 1.0 lM a6(IV)NC1 alone also were maintained to evaluate the individual effect of MATERIALS AND METHODS a6(IV)NC1 on MCECs. Three replicates were maintained for each treatment and experiments were repeated twice. Mouse Choroidal Endothelial Cell Culture Similar replicates of control and treated wells also were maintained by adding polymyxin-B (5 mg/mL) to the above- The MCECs used in our study were a gift from Sheibani Nader mentioned treatments, to evaluate the effects of endotoxin (University of Wisconsin, Madison, WI). The MCECs were neutralization on cell proliferation. Cells were incubated for cultured in endothelial cell growth Medium (EGM-2; Lonza, 48 hours in a 5% CO2 incubator at 378Candproliferation Walkersville, MD) on 1% gelatin-coated plates and used for all was analyzed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5- 24 in vitro studies. diphenyl tetrazolium bromide assay (MTT) reagent meth- od.20 Recombinant Expression and Purification of a6(IV)NC1 Migration Assay Recombinant human a6(IV)NC1 was cloned, expressed, and Assessment of MCEC migration in the presence of EDPs, purified from bacterial inclusion bodies by column chroma- a6(IV)NC1, and their combinations was done using a 48-well tography using the L-arginine renaturation method.22 Endotox- Boyden chamber. The MCECs (1 3 104/30 lL) were Downloaded from iovs.arvojournals.org on 09/29/2021 Elastin Peptide-Mediated Angiogenic Signaling IOVS j December 2013 j Vol. 54 j No. 13 j 7830 matrigel was thawed overnight at 48C, and 250 lL were added to each well and allowed to polymerize at 378C, for approximately 30 minutes. The MCECs were suspended at a density of 5 3 104 cells/mL in EGM-2 (without antibiotics) containing different combinations of EDPs and a6(IV)NC1, and released onto the polymerized matrigel. Wells contain- ing culture medium were used as controls, medium with only EDPs (2 lg/mL jE/ME or 200 ng/mL VGVAPG) were used as positive controls, and EDPs along with 0.5 or 1.0 lM purified a6(IV)NC1 were considered as treated wells. Tube formation was recorded after 48 hours of incubation at 378C, using a Juli light microscope (Bulldog Bio, Inc., Portsmouth, NH).