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Gene profile for differentiation of vascular adventitial myofibroblasts

Guo Shujie, Wu Lingyun, Shen Weili, Chen Wendong, Wei Jian, Gao Pingjin, Zhu Dingliang

Shanghai Key Laboratory of Vascular Biology, Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine.Shanghai 200025；China

Abstract

Our previous study demonstrated that TGF-#beta#1 could induce the differentiation of vascular adventitial fibroblasts (AFs) to myofibroblasts (MFs). The aim of this study was to identify the genes which might be responsible for the cell phenotypic change using genechips. Cultured rat AFs were treated with TGF-#beta#1 (10 ng/ml) for 0 min, 5 min, 15 min, 2 h, 12 h and 24 h, respectively. Then the cells were gathered to prepare total RNA. We examined TGF-#beta#1-induced gene expression profiling using Affymetrix oligonucleotide microarrays and analyzed data by GCOS 1.2 software. Moreover, expressional similarity was measured by hierarchical clustering. Some of genechip results were confirmed by real-time quantitative RT-PCR. Microarray analysis identified 2 121 genes with a 2-fold change or above after TGF-#beta#1 stimulation. 1 318 genes showed a greater than 2-fold increase and 761 genes were reduced 2 folds or more at mRNA levels, whereas a small portion of the total regulated genes (42 genes) displayed dynamically up- and down-regulated pattern.Genes were further segregated for early (peak at 5 min, 15 min and/or 2 h), late (peak at 12 h and/or 24 h), and sustained (2-fold change or above at five time points) temporal response groups according to the time of their peak expression level. Among 1 318 up-regulated genes, 333 genes (25.3%) responded rapidly to TGF-#beta#1 and 159 genes (12.1%) responded in a sustained manner. Most genes (826, 62.6%) were regulated at 12 h or later. For the 761 down-regulated genes, numbers of early and late responsive genes were 335 (44%) and 267 (36.1%),respectively. There were also 159 genes, 19.9% of total down-regulated genes, decreased at five time points treated by TGF-#beta#1. Theresults suggested that the gene expressions of secreted phosphoprotein 1 (APP1) and Rho-associated coiled-coil forming kinase 2(ROCK2) had the same trends as #alpha#-smooth muscle-actin, a marker of MF differentiation. In addition, the gene expression of potassiumvoltage-gated channel, Shal-related family and member 2 (KCND2) was up-regulated. Furthermore, it was found that endothelin 1(EDN1), some complement components, NADPH oxidase 4 (NOX4) and NAD(P)H dehydrogenase, quinone 1 (NQO1) might beinvolved in MF differentiation. Using microarrary technique, we confirmed some genes that have been identified by other techniqueswere implicated in MF differentiation and observed new genes involved in this process. Our results suggest that gene expressionprofiling study is helpful in identifying genes and pathways potentially involved in cell differentiation.

Key words: oligonucleotide arrays;adventitia;Fibroblast;differentiation

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Citing This Article：

Guo Shujie, Wu Lingyun, Shen Weili, Chen Wendong, Wei Jian, Gao Pingjin, Zhu Dingliang. Gene profile for differentiation of vascular adventitial myofibroblasts. Acta Physiol Sin 2006; 58 (4): (in Chinese with English abstract).