ABSTRACT
An important objective of vascularized tissue regeneration is to develop agents for osteonecrosis. We aimed to identify the pro-angiogenic and osteogenic efficacy of adipose tissue-derived (AD) pericytes combined with Nel-like protein-1 (NELL-1) to investigate the therapeutic effects on osteonecrosis. Tube formation and cell migration were assessed to determine the pro-angiogenic efficacy. Vessel formation was evaluated in vivo using the chorioallantoic membrane assay. A mouse model with a 2.5 mm necrotic bone fragment in the femoral shaft was used as a substitute for osteonecrosis in humans. Bone formation was assessed radiographically (plain radiographs, three-dimensional images, and quantitative analyses), and histomorphometric analyses were performed. To identify factors related to the effects of NELL-1, analysis using microarrays, qRT-PCR, and Western blotting was performed. The results for pro-angiogenic efficacy evaluation identified synergistic effects of pericytes and NELL-1 on tube formation, cell migration, and vessel formation. For osteogenic efficacy analysis, the mouse model for osteonecrosis was treated in combination with pericytes and NELL-1, and the results showed maximum bone formation using radiographic images and quantitative analyses, compared with other treatment groups and showed robust bone and vessel formation using histomorphometric analysis. We identified an association between FGF2 and the effects of NELL-1 using array-based analysis. Thus, combinatorial therapy using AD pericytes and NELL-1 may have potential as a novel treatment for osteonecrosis.
Subject(s)
Adipose Tissue/cytology , Calcium-Binding Proteins/metabolism , Neovascularization, Physiologic , Osteogenesis , Osteonecrosis/therapy , Pericytes/cytology , Adipose Tissue/metabolism , Aged , Aged, 80 and over , Animals , Apoptosis , Calcium-Binding Proteins/genetics , Cell Movement , Cell Proliferation , Cells, Cultured , Embryo, Mammalian/blood supply , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Female , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Osteonecrosis/etiology , Osteonecrosis/metabolism , Osteonecrosis/pathology , Pericytes/metabolismABSTRACT
Inadequate vessel maintenance or growth causes ischemia in diseases such as myocardial infarction, stroke, and neurodegenerative disorders. Therefore, developing an effective strategy to salvage ischemic tissues using a novel compound is urgent. Drug repurposing has become a widely used method that can make drug discovery more efficient and less expensive. Additionally, computational virtual screening tools make drug discovery faster and more accurate. This study found a novel drug candidate for pro-angiogenesis by in silico virtual screening. Using Gene Expression Omnibus (GEO) microarray datasets related to angiogenesis studies, differentially expressed genes were identified and characteristic direction signatures extracted from GEO2EnrichR were used as input data on L1000CDS2 to screen pro-angiogenic molecules. After a thorough review of the candidates, a list of compounds structurally similar to TWS-119 was generated using ChemMine Tools and its clustering toolbox. ChemMine Tools and ChemminR structural similarity search tools for small-molecule analysis and clustering were used for second screening. A molecular docking simulation was conducted using AutoDock v.4 to evaluate the physicochemical effect of secondary-screened chemicals. A cell viability or toxicity test was performed to determine the proper dose of the final candidate, ellipticine. As a result, we found ellipticine, which has pro-angiogenic effects, using virtual computational methods. The noncytotoxic concentration of ellipticine was 156.25 nM. The phosphorylation of glycogen synthase kinase-3ß was decreased, whereas the ß-catenin expression was increased in human endothelial cells treated with ellipticine. We concluded that ellipticine at sublethal dosage could be successfully repositioned as a pro-angiogenic substance by in silico virtual screening.
Subject(s)
Ellipticines/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Neovascularization, Pathologic/drug therapy , Cell Survival/drug effects , Cells, Cultured , Drug Discovery/methods , Drug Repositioning/methods , Gene Expression/drug effects , Glycogen Synthase Kinase 3 beta/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Molecular Docking Simulation/methods , Molecular Dynamics Simulation , Neovascularization, Pathologic/metabolism , Protein Binding/drug effects , beta Catenin/metabolismABSTRACT
BACKGROUND: In this study, we investigated whether substance P (SP) or calcitonin gene-related peptide (CGRP) expression is associated with tendon degeneration in patients with lateral epicondylitis. METHODS: Twenty-nine patients who underwent surgical treatment for lateral epicondylitis were enrolled in the final analyses. Extensor carpi radialis brevis tendon origins were harvested for histological analysis. RESULTS: SP and CGRP immunostaining were negative in healthy tendons but positive in degenerative tendons; moreover, their immunoreactivity increased with degeneration severity. Univariate analysis indicated that variables such as the preoperative visual analog scale (VAS) score or SP or CGRP expression levels were significantly associated with the Movin score. However, multivariate analysis revealed that only higher SP and/or CGRP signals were associated with higher Movin scores. Elevations in SP or CGRP expression were also linked with significantly severe preoperative VAS scores. CONCLUSION: We demonstrated that tendon degeneration severity is associated with increased SP and CGRP expression in the biopsy samples of lateral epicondylitis.
