Die Rolle von Stickstoffmonoxid im Tissue Engineering von Fettgewebe

Insufficient differentiation of precursor cells and inadequate vascularization in central parts of three-dimensional constructs are major problems in adipose tissue engineering. This study aimed at finding a solution to these problems by analyzing the effects of nitric oxide (NO) on preadipocytes and endothelial cells in a setting as seen after transplantation of a three-dimensional biohybrid composed of viable endothelial cells and adipose precursor cells. NO is an important signalling molecule regulating physiological and pathophysiological functions throughout the body, including endothelial and adipose tissue activities. NO is generated by the enzyme family of NO synthases that metabolize arginine and molecular oxygen to citrulline and NO. Preadipocytes were isolated from human subcutaneous adipose tissue samples, cultured in DMEM/F12 with 10% fetal calf serum (FCS), and differentiated after 7–14 days of expansion by insulin, isobutylmethylxanthine, pioglitazone, dexamethasone, and transferrin in the absence of FCS. To evaluate the influence of NO on proliferation and differentiation, the NO donor molecule DETA/NO was added at various concentrations. Proliferation was evaluated by microscopy and the extent of differentiation was assayed after 15 days by cell counting and analysis of the enzyme glycerophosphate dehydrogenase highly expressed in mature adipocytes. In parallel, primary rat aorta endothelial cells (EC) were isolated, cultured in RPMI 1640/20% FCS and treated with DETA/NO to analyze the effect of NO on the stress response genes Bcl-2, vascular endothelial growth factor (VEGF), and heme oxygenase (HO)-1. We find that NO in combination with the conventionally used differentiation-inducing factors significantly enhances maturation of precursor cells to adipocytes whereas proliferation, in contrast, is inhibited in the presence of NO. Treatment of EC with DETA/NO causes the upregulation of the antiapoptotic protein Bcl-2, the pro-angiogenic VEGF, and the antioxidative HO-1. Our results emphasize the pivotal role of iNOS activity in regulating gene expression. Further, they are encouraging for applying NO-donors during transplantation of preadipocytes and EC in a three-dimensional setting since it helps optimizing differentiation of adipogenic precursor cells and stimulating angiogenesis by inducing proangiogenic VEGF.