Nitric oxide: Is it the culprit for the continued expansion of keloids?

Abstract

Keloids are characterized by excessive proliferation of fibroblasts and invasion of surrounding healthy skin. High levels of Nitric Oxide (NO) are thought to be the crucial factor within the micro-environment in promoting keloid formation. However, the effects and mechanisms of NO on the proliferation of Keloid Fibroblasts (KDFs) remain unclear. In this study, we investigated the effect of NO on KDFs proliferation by Sodium Nitroprusside (SNP), an NO donor. Our results show that SNP significantly enhanced KDFs proliferation. Moreover, with prolonged treatment with SNP after cell confluence, the growth of KDFs escape contact inhibition and experience significant pile up growth. Furthermore, PTIO, an NO scavenger, attenuated SNP-enhanced cell proliferation effectively. The mechanism involved in SNP-induced KDFs proliferation was soluble Guanylyl Cyclase (sGC) and cGMP independent. ODQ, a specific sGC inhibitor, failed to suppress SNP-enhanced KDFs proliferation. 8-Bromo-c GMP, a cell-permeable cGMP analogue, could not stimulate KDFs proliferation. Erk and Akt provide important signaling for cell growth. U0126 and LY294002, inhibitors of Erk and Akt respectively, block SNP-enhanced KDFs proliferation effectively. As expected, a Western blot showed that SNP up-regulated the phosphorylation levels of Erk and Akt. Moreover, it decreased the expression of p²⁷, a cell cycle inhibitor. Our results reveal that SNP induced KDFs proliferation and loss contact inhibition led to pile up growth via activation of the Erk and Akt pathways, as well as a decreased expression of p²⁷. Thus, we speculate that the pathological feature of continuous expansion in keloids is caused by NO-induced KDFs sustained growth.

Keywords: 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one; 2-phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxide; 3′-5′-cyclicguanosine monophosphate; 8-br-cGMP; 8-bromoadenosine 3′,5′-cyclic monophosphate; Akt; BH4; Cell proliferation; Erk; HSFs; KDFs; Keloid fibroblasts; NO; NSFs; Nitric oxide; ODQ; PTIO; S-Nitroso-N-acetylpenicillamine; SNAP; SNP; Sodium nitroprusside; TGF-β1; cGMP; extracellular-signal-regulated kinase; hypertrophic scar fibroblasts; keloid fibroblasts; nitric oxide; normal skin fibroblasts; protein kinase B; sGC; sodium nitroprusside; soluble guanylyl cyclase; tetrahydrobiopterin; transforming growth factor-β 1; α-SMA; α-smooth muscle actin.