Nitric Oxide Synthase Expression and Nitric Oxide Production Are Reduced in Hypertrophic Scar Tissue and Fibroblasts

Abstract

We have recently demonstrated that human dermal fibroblasts express both constitutive and inducible nitric oxide synthases (NOS) and produce nitric oxide (NO). In this study, NOS expression and NO production were compared in human fibroblasts derived from hypertrophic scar (HSc) and site-matched normal dermis. NOS expression and NO production in HSc fibroblasts are significantly reduced when compared with normal fibroblasts. Without stimulation, the HSc fibroblasts produced 7.37 +/- 1.17 nmol and the normal fibroblasts produced 11.15 +/- 0.79 nmol/10(6) cells/96 h (paired t test, p < 0.01, n = 6) NO as determined by the Griess reaction. After stimulation with 200 units interferon-gamma per ml and 40 microg lipopolysaccharide per ml, both HSc and normal fibroblasts produced significantly higher NO, indicating that the HSc fibroblasts retain the capacity to express inducible NOS (iNOS). The Ca2+-dependent NOS (cNOS) activity in the cytosol of HSc fibroblasts (1.43 pmol/min/g of protein) was significantly lower than that in normal fibroblasts (2.60 pmol/min/g of protein), as determined by citrulline assay (p < 0.01, n = 4). The mRNAs for endothelial cNOS and iNOS in both HSc and normal fibroblasts were detectable by RT-PCR. Flow cytometry confirmed that the untreated HSc fibroblasts expressed less endothelial cNOS protein than untreated normal fibroblasts. Because NO markedly inhibits cell proliferation, our results suggest that after thermal injury, fibroblasts in the scar tissue may undergo phenotypic alteration leading to reduced endothelial cNOS expression. The low levels of endogenous NO in HSc fibroblasts might be responsible for the cellularity of post-burn HSc, a characteristic feature of this fibrotic condition.