ABSTRACT
Editor's Note: this Article has been retracted; the Retraction Note is available at https://www.nature.com/articles/s41598-020-76500-9.
ABSTRACT
α1(IV)NC1 inhibits angiogenesis by regulating MAPK activation, this biological function was partly attributed α1(IV)NC1 binding to α1ß1-integrin. However, its potent antiangiogenic activity and the molecular targets of α1(IV)NC1 has not been investigated. In the present study, the regulation of MMP-2 activation by α1(IV)NC1 was evaluated. α1ß1-integrin which is required for inhibition of angiogenesis is not playing a role in cellular invasion and inhibition of MMP-2 activation by α1(IV)NC1. We found that α1(IV)NC1 binds the CBD of MMP-2 and forming a stable complex that prevents activation of MMP-2. The antiangiogenic activity of α1(IV)NC1 is mediated, in part, by this binding activity. In addition, up-regulation of TIMP-2 by α1(IV)NC1 led to saturation of MT1-MMP binding sites, which in turn led to inhibition of MMP-2 activation. In-vivo studies using α1-integrin null-mice treated with higher doses of α1(IV)NC1 showed integrin independent inhibition of tumor growth and active-MMP-2, without affecting MMP-9, MMP-7 and angiostatin.
Subject(s)
Collagen Type IV/metabolism , Matrix Metalloproteinase 2/metabolism , Protein Interaction Domains and Motifs , Angiostatins/metabolism , Animals , Collagen Type IV/chemistry , Collagen Type IV/genetics , Enzyme Activation/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Integrin alpha1/genetics , Matrix Metalloproteinase 14/metabolism , Matrix Metalloproteinase 7/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Knockout , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Neovascularization, Physiologic/drug effects , Phenylmercuric Acetate/analogs & derivatives , Phenylmercuric Acetate/pharmacology , Protein Interaction Domains and Motifs/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
OBJECT: Antiangiogenic treatments are beginning to give promising outcomes in many vascular diseases including tumor angiogenesis. In this current study the antiangiogenic and pro-apoptotic actions of α1(IV)NC1 and its N- and C- peptides α1S1(IV)NC1, α1S2(IV)NC1 were investigated in-vitro and in-vivo. STUDY METHOD: Endothelial cells (ECs) were treated with α1(IV)NC1, α1S1(IV)NC1, α1S2(IV)NC1 and in-vitro proliferation, migration, tube formation and apoptotic assays were executed. FasL, Fas, Caspase-8, -3 and PARP activations were studied using immunoblotting analysis using specific antibodies. Also the in-vivo antiangiogenic and pro-apoptotic effects were tested using α1(IV)NC1 in a mice model. RESULTS: Like α1(IV)NC1, its N- and C- terminal α1S2(IV)NC1 and α1S1(IV)NC1 domains posses anti-proliferative, pro-apoptotic activity and inhibit ECs migration and tube formation in-vitro. Both α1S1(IV)NC1 and α1S2(IV)NC1 domains promote apoptosis by activating FasL and down stream apoptotic events including activation of caspase-8, -3 and PARP cleavage in a dose dependent manner in-vitro in ECs. Tumors in mice showed apoptotic TUNEL positive microvasculature upon α1(IV)NC1 treatment, indicating inhibition of tumor angiogenesis and tumor growth. Further, the antitumor activity of α1(IV)NC1 was abrogated when caspase-3 inhibitor was used. These results conform additional properties of α1(IV)NC1 as an endogenous angioinhibitor that induces apoptosis in-vitro and in-vivo by activating FasL mediated caspase-3. SIGNIFICANCE: α1(IV)NC1 and its N- and C- terminal α1S1(IV)NC1 and α1S2(IV)NC1 domains also posses pro-apoptotic and angioinhibitory activity in-vitro and in-vivo. α1(IV)NC1 regulates tumor angiogenesis by activating FasL mediated apoptosis in-vitro and in-vivo. These results demonstrate that α1(IV)NC1 and its peptides inhibit neo-vascular diseases.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Collagen Type IV/pharmacology , Fas Ligand Protein/genetics , Gene Expression Regulation, Neoplastic , Skin Neoplasms/blood supply , Teratocarcinoma/blood supply , Animals , Apoptosis/drug effects , Caspase 3/genetics , Caspase 3/metabolism , Caspase 8/genetics , Caspase 8/metabolism , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Fas Ligand Protein/agonists , Fas Ligand Protein/metabolism , Humans , Mice , Neovascularization, Pathologic , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Protein Isoforms/pharmacology , Signal Transduction , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Teratocarcinoma/drug therapy , Teratocarcinoma/genetics , Teratocarcinoma/pathology , fas Receptor/genetics , fas Receptor/metabolismABSTRACT
PURPOSE: The inhibitory effects and mechanism(s) of type IV collagen α-6 chain-derived noncollagenous domain (α6[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 α6(IV)NC1. The MCECs proliferation, migration, tube formation, MT1-MMP expression, and angiogenic signaling were analyzed in cells subjected to EDP and α6(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 α6(IV)NC1 inhibited survival and EDP-activated migration of MCECs. The EDP-activated MCEC tube formation on matrigel also was inhibited by α6(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 α6(IV)NC1. The EDP-induced FAK and Akt phosphorylation was blocked by FAK- and Akt-specific inhibitors. CONCLUSIONS: The EDPs and α6(IV)NC1 are identified to exhibit opposing effects on MCEC angiogenic behavior and signaling. The α6(IV)NC1 inhibited cell survival, EDP-mediated migration, MT1-MMP expression and, FAK/PI-3K/mToR/Akt phosphorylation in MCECs. This work demonstrates α6(IV)NC1 as a prospective endogenous molecule for the treatment of diseases involving choroidal neovascularization in the eye.
Subject(s)
Choroid/metabolism , Choroidal Neovascularization/drug therapy , Collagen Type IV/pharmacology , Elastin/metabolism , Endothelial Cells/metabolism , Animals , Blotting, Western , Cell Movement/drug effects , Cell Survival/drug effects , Cells, Cultured , Choroid/drug effects , Choroid/pathology , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Endothelial Cells/drug effects , Endothelial Cells/pathology , Mice , Prospective Studies , Signal Transduction/drug effectsABSTRACT
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.