Nitric Oxide Synthase Activity and Angiogenesis Measured by Expression of CD34 in Burns Treated With Chitosan Films
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8 Many cell like keratinocytes, melanocytes, fibroblasts, endothelial cells, and adipose cells are able to synthesize different enzyme forms and release NO.27 The authors suggest that this free radical may be interacting with chitosan or could be influencing cell migration or infiltration during stages of skin repair. In wound healing, the majority of NO synthesis is due to the inflammatory cells present during the early phase of healing, especially macrophages.28 NO has a peak at 4–6 days post wounding and remains at a high level for 3 weeks or longer. However, fibroblasts, keratinocytes, and endothelial cells contribute to ongoing NO synthesis, although to a lesser degree. Therefore, the overall time course of iNOS activity and NO generation during wound healing has to be viewed as a decreasing curve over time.23 Carls-Grieson et al29 revealed that NO in low concentration (0.01 mM–0.25 mM) stimulates cell division, while high concentrations (> 0.5 mM) have cytostatic effects. The use of iNOS inhibitor caused a decrease of NO in the fluids of wounds and cell cultures adjacent to wounds showed a high correlation with a decrease in collagen deposition and failure of wound closure, which is in accordance with the present results that demonstrate an association with higher NO release and collagen deposit with a higher cell infiltration, especially in chitosan-treated burns.10 The activity of the NOS enzyme detected in the authors’ experiments was highest in the treated burns. NOS activity was concentrated after day 7 and extended to day 14. On these days significant differences were observed and such findings can be linked to the type of infiltrate described in the histopathological analysis, where a notable number of PMNs and macrophages were found during the previously mentioned period of time. Consequently, the increase in the NOS enzyme activity would not be caused by a direct effect of chitosan, but an indirect effect promoted by the higher cell infiltration in wounds, which could lead to a higher release of NO. The present results demonstrated that the highest NOS activity was during the inflammatory and proliferative stages.8 The lowest activity of NOS was found on day 21. This presumably associated to iNOS activity can be downregulated by the resolution of the inflammatory response or by cytokine signaling.30 In the present study NOS activity was not differentiated between eNOS or iNOS, but it can be assumed that most or nearly the entire released NO was caused by the iNOS activity associated to cellular infiltrate. The previous data are supported by experiments carried out by Carter et al31 who found a significant iNOS increase on day 21 in rat burns. The beneficial effects of NO on wound repair may be attributed to its functional influences on angiogenesis and inflammation.32
A higher number of capillaries per field were observed in treated burns compared to controls. According to statistical analysis, there were significant differences by direct chitosan treatment influence. This agrees with the results obtained by Fukumura et al33 who associated neovascularization with proportional presence of NO. Also, the present study confirmed their results that VEGF increased NO production at the gene expression level and that the angiogenic effects of VEGF appear to be dependent on NO.
NO plays a meaningful and important role in angiogenesis, suggesting a mechanism that causes upregulation of αvβ3 integrins, which facilitate cell adhesion to the base of the matrix and stimulate cell migration,34 which would explain the relationship between the increase of NOS activity in chitosan-treated lesions and the increased number of counted capillaries.