acetyl-prolyl-histidyl-seryl-cysteinyl-asparaginamide and Disease-Models--Animal

Topics: Animals; Brain; Cytokines; Disease Models, Animal; Inflammation; Integrin alpha5beta1; Ischemic Stroke; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Neutrophil Infiltration; Oligopeptides

BACKGROUND ATN-161 (Ac-PHSCN-NH2), an antagonist of integrin α5β1, has shown an important influence in inhibiting tumor angiogenesis and metastasis of other tumor types. However, the mechanism of action of ATN-161 and whether it can inhibit ocular neovascularization (NV) are unclear. This study investigated the role of ATN-161 in regulating ocular angiogenesis in mouse models and explored the underlying signaling pathway. MATERIAL AND METHODS An oxygen-induced retinopathy (OIR) mouse model and a laser-induced choroidal neovascularization (CNV) mouse model were used to test integrin a5b1 expression and the effect of ATN-161 on ocular NV by immunofluorescence staining, Western blot analysis, and flat-mount analysis. The activation of nuclear factor-κB (NF-κB), matrix metalloproteinase-2/9 (MMP-2/9), and cell apoptosis were detected by immunofluorescence staining, Western blot, real-time RT-PCR, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). The cell proliferation was detected by BrdU labeling. RESULTS In OIR and CNV mice, the protein expression level of integrin α5β1 increased compared with that in age-matched controls. The mice given ATN-161 had significantly reduced retinal neovascularization (RNV) and CNV. Blocking integrin a5b1 by ATN-161 strongly inhibited nuclear factor-κB (NF-κB) activation and matrix metalloproteinase-2/9 (MMP-2/9) expression and promoted cell apoptosis, but the effect of ATN-161 on proliferation in CNV mice was indirect and required the inhibition of neovascularization. Inhibiting NF-κB activation by ammonium pyrrolidinedithiocarbamate (PDTC) reduced RNV and promoted cell apoptosis in ocular NV. CONCLUSIONS Blocking integrin α5β1 by ATN-161 reduced ocular NV by inhibiting MMP-2/MMP-9 expression and promoting the cell apoptosis of ocular NV.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Cell Movement; Cell Proliferation; Choroidal Neovascularization; Disease Models, Animal; Endothelial Cells; Eye; Female; Integrin alpha5beta1; Intravitreal Injections; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; NF-kappa B; Oligopeptides; Retinal Neovascularization

Integrin α5β1 is involved in the development of choroidal neovascularization (CNV). Thus, the inhibition of integrin α5β1 may provide an alternative to the current standard of CNV therapy, which involves inhibition of vascular endothelial growth factor (VEGF) and is not effective in all patients. This study evaluated the antiangiogenic effects of ATN-161, a small peptide inhibitor of integrin α5β1, in human choroidal endothelial cells (hCECs) and in laser-induced CNV in rats. Furthermore, the utility of spectral-domain optical coherence tomography (SD-OCT) for dynamic observation of the development of CNV in animal models was assessed.. The antiangiogenic potential of ATN-161 was evaluated in VEFG-stimulated hCECs by MTS proliferation assays, migration assays, and synthetic matrix capillary tube formation assays. To evaluate the antiangiogenic effects of ATN-161 in vivo, CNV was induced in rats by laser photocoagulation. ATN-161, scrambled peptide, or AF564 anti-VEGF antibody, were injected intravitreally immediately after photocoagulation. Eyes were examined by SD-OCT and fluorescein angiography on days 1, 7, and 14 after injection, and the areas of CNV were measured by analysis of choroidal flatmounts at day 14.. ATN-161 inhibited VEGF-induced migration and capillary tube formation in hCECs, but did not inhibit proliferation. In vivo, injection of ATN-161 after laser photocoagulation inhibited CNV leakage and neovascularization to an extent similar to AF564. Furthermore, SD-OCT and histologic examinations indicated that ATN-161 significantly decreased the size of laser-induced lesions.. Integrin α5β1 inhibition by ATN-161 may be a promising alternative therapy for CNV-related angiogenesis. In addition, SD-OCT technology allows excellent visualization of experimentally induced CNV in vivo.

Topics: Angiogenesis Inhibitors; Animals; Blotting, Western; Capillaries; Cell Movement; Cell Proliferation; Cells, Cultured; Choroid; Choroidal Neovascularization; Disease Models, Animal; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Fluorescein Angiography; Humans; Integrin alpha5beta1; Laser Coagulation; Oligopeptides; Rats; Rats, Inbred BN; Tomography, Optical Coherence; Vascular Endothelial Growth Factor A

Neoangiogenesis is a critical component of chronic inflammatory disorders. Inhibition of angiogenesis is an effective treatment in animal models of inflammation, but has not been tested in experimental colitis.. To investigate the effect of ATN-161, an anti-angiogenic compound, on the course of experimental murine colitis.. Interleukin 10-deficient (IL10(-/-)) mice and wild-type mice were kept in ultra-barrier facilities (UBF) or conventional housing, and used for experimental conditions. Dextran sodium sulphate (DSS)-treated mice were used as a model of acute colitis. Mice were treated with ATN-161 or its scrambled peptide ATN-163. Mucosal neoangiogenesis and mean vascular density (MVD) were assessed by CD31 staining. A Disease Activity Index (DAI) was determined, and the severity of colitis was determined by a histological score. Colonic cytokine production was measured by ELISA, and lamina propria mononuclear cell proliferation by thymidine incorporation.. MVD increased in parallel with disease progression in IL10(-/-) mice kept in conventional housing, but not in IL10(-/-) mice kept in UBF. Angiogenesis also occurred in DSS-treated animals. IL10(-/-) mice with established disease treated with ATN-161, but not with ATN-163, showed a significant and progressive decrease in DAI. The histological colitis score was significantly lower in ATN-161-treated mice than in scrambled peptide-treated mice. Inhibition of angiogenesis was confirmed by a significant decrease of MVD in ATN-161-treated mice than in ATN-163-treated mice. No therapeutic effects were observed in the DSS model of colitis. ATN-161 showed no direct immunomodulatory activity in vitro.. Active angiogenesis occurs in the gut of IL10(-/-) and DSS-treated colitic mice and parallels disease progression. ATN-161 effectively decreases angiogenesis as well as clinical severity and histological inflammation in IL10(-/-) mice but not in the DDS model of inflammatory bowel disease (IBD). The results provide the rational basis for considering anti-angiogenic strategies in the treatment of IBD in humans.

Topics: Acute Disease; Angiogenesis Inhibitors; Animals; Cell Proliferation; Cells, Cultured; Colitis; Colon; Cytokines; Dextran Sulfate; Disease Models, Animal; Disease Progression; Interleukin-10; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Oligopeptides