Fig 1: Monocyte chemoattractant protein-1 induces fibrogenic activation of hepatic stellate cells. (A) Research hypothesis of the study. Following the identification of extracellular MCP-1 level as a novel biomarker of liver fibrosis associated with TMD in DS,11 we hypothesized that prolonged hSC activation by paracrine and/or autocrine MCP-1 mediation plays a fundamental role during the progression of liver fibrosis associated with TMD. (B) MCP-1 expression in hSCs. Left, representative image of an affected liver specimen showing characteristic MCP-1 expression in the hSCs. Original magnification ×200. Right, co-expression of CCR2 and MCP-1 in LX-2 cells. Scale bar, 100 µm. (C) LX-2 cells were treated with an increasing dose of MCP-1 (1 ng/mL to 100 ng/mL). Cell growth was assessed 3 days later. Upper panel shows their significant growth rate compared to unstimulated control cells (# P < 0.01). Bottom panel shows a dose-dependent increase of phosphorylated p38 and p42/44. ACTB expression was used as a protein loading control. Representative images of three independent experiments are shown. (D) qRT-PCR analysis of profibrogenic genes. Graphs show a 1.89-fold increase of type IV collagen and a 1.78-fold increase of a-SMA expression compared to unstimulated cells (# P < 0.01). (E) Ex vivo evaluation of the patient's serum activity. LX-2 cells were stimulated with the patient's serum obtained during the active stage (postnatal day 90) and the remission stage (postnatal day 144). Cell growth ratio and type IV collagen protein expression levels were examined after serum stimulation. Note the strong fibrogenic activity of the patient serum obtained at the active phase and prominent inhibition of serum activity in the presence of MCP-1 blocking antibody (# P < 0.01). Data in C-E represent mean ± SD. Abbreviations: a-SMA, a-smooth muscle actin; ACTB, beta actin; CCR2, C-C chemokine receptor type 2; mRNA, messenger RNA; qRT-PCR, quantitative reverse-transcription polymerase chain reaction.