Functional improvement of collagen-based bioscaffold to enhance periodontal-defect healing via combination with dietary antioxidant and COMP-angiopoietin 1.

Scaffolds combined with bioactive agents can enhance bone regeneration at therapeutic sites. We explore whether combined supplementation with coumaric acid and recombinant human-cartilage oligomeric matrix protein-angiopoietin 1 (rhCOMP-Ang1) is an ideal approach for bone tissue engineering. We developed coumaric acid-conjugated absorbable collagen scaffold (CA-ACS) and investigated whether implanting CA-ACS in combination with rhCOMP-Ang1 facilitates ACS- or CA-ACS-mediated bone formation using a rat model of critically sized mandible defects. We examined the mechanisms by which coumaric acid and rhCOMP-Ang1 regulate behaviors of human periodontal ligament fibroblasts (hPLFs). The CA-ACS exhibits greater anti-degradation and mechanical strength properties than does ACS alone. Implanting CA-ACS loaded with rhCOMP-Ang1 greatly enhances bone regeneration at the defect via the activation of angiogenic, osteogenic, and anti-osteoclastic responses compared with other rat groups implanted with an ACS alone or CA-ACS. Treatment with both rhCOMP-Ang1 and coumaric acid increases proliferation, mineralization, and migration of cultured hPLFs via activation of the Ang1/Tie2 signaling axis at a greater rate than treatment with either of them alone. Collectively, this study demonstrates that CA-ACS impregnated with rhCOMP-Ang1 enhances bone regeneration at therapeutic sites, and this enhancement is associated with a synergistic interaction between rhCOMP-Ang1-mediated angiogenesis and coumaric acid-related antioxidant responses.

Angiopoietin-1 protects 3T3-L1 preadipocytes from saturated fatty acid-induced cell death.

Cross talk between endothelial cells and adipocytes is vital to adipocyte functions, but little is known about the mechanisms or factors controlling the process. Angiogenesis is a critical component linking the endothelium to healthy adipogenesis, yet it is not known if or how it is involved in adipocyte physiology. Therefore, the purpose of this study was to determine the effect of angiopoietin-1 (Ang-1) and -2 (Ang-2) as well as their receptor, Tie-2, on adipocyte physiology. 3T3-L1 pre- and mature adipocytes were found to express Ang-1, Ang-2, and Tie-2, which decrease upon polyunsaturated fatty acid treatment. Furthermore, 3T3-L1 cells treated with recombinant Ang-1 or Ang-2 increased expression of the antiapoptotic gene Bcl-x and decreased expression of the proapoptotic gene Casp-8. Next, preadipocytes were treated with saturated fatty acids (SFAs) to induce cell stress. SFA-mediated splicing of X-box-binding protein-1 was reduced by co-treatment with Ang-1, and cell viability was improved in the presence of SFAs + Ang-1. Taken together, these results indicate that Ang-1 may protect preadipocytes from SFA-induced apoptosis and endoplasmic reticulum stress.

Tie2 in tumor endothelial signaling and survival: implications for antiangiogenic therapy.

Signaling through the Tie2 receptor on endothelial cells has been shown to play an important role in normal and pathologic vascular development. We generated K1735 murine melanoma tumor cells that inducibly express soluble Tie2 receptor (Tie2Ex) to study the effects of inhibiting Tie2 signaling on tumor vasculature. Tie2Ex induction rapidly decreased AKT activation but not extracellular signal-regulated kinase (ERK) activation in tumor endothelial cells as detected by immunostaining. This was accompanied by an increase in endothelial cell TUNEL staining but no change in Ki-67 expression. Together with a decrease in the percentage of perfused vessels, this suggested that tumor vessel regression and impaired vascular function rather than angiogenesis inhibition was responsible for the delay in tumor growth following Tie2Ex treatment. However, Tie2Ex failed to inhibit the growth of larger, more established K1735 tumors. These tumors were additionally treated with sorafenib, a multikinase inhibitor that inhibits tumor endothelial cell ERK activation but not AKT activation. Combining Tie2Ex and sorafenib decreased both endothelial cell AKT and ERK activation, decreased endothelial cell survival and proliferation, and significantly inhibited growth of the more established tumors. These studies indicate that activity of specific signaling pathways and prosurvival effects are brought about by Tie2 activation in tumor endothelial cells, and knowledge of the effects of Tie2 inhibition can lead to development of more effective therapeutic regimens for inhibiting tumor neovascularization.

Effects of regulatory factors on engineered cardiac tissue in vitro.

We tested the hypothesis that supplemental regulatory factors can improve the contractile properties and viability of cardiac tissue constructs cultured in vitro. Neonatal rat heart cells were cultured on porous collagen sponges for up to 8 days in basal medium or medium supplemented with insulin-like growth factor-I (IGF), insulin-transferrin-selenium (ITS), platelet-derived growth factor-BB (PDGF), or angiopoietin-1 (ANG). IGF and ITS enhanced contractile properties of the 8-day constructs significantly more than with unsupplemented controls according to contractile amplitude and excitation threshold, and IGF also significantly increased the amount of cardiac troponin-I and enhanced cell viability according to different assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and terminal deoxynucleotidyl transferase biotin-2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL)). PDGF significantly increased the contractile amplitude of 4-day constructs and enhanced cell viability according to MTT, LDH, and TUNEL; ANG enhanced cell viability according to the LDH assay. Our results demonstrate that supplemental regulatory molecules can differentially enhance properties of cardiac tissue constructs and imply that these constructs can provide a platform for systematic in vitro studies of the effects of complex stimuli that occur in vivo to improve our basic understanding of cardiogenesis and identify underlying mechanisms that can potentially be exploited to enhance myocardial regeneration.