Identification of stretch-responsive genes in pulmonary artery smooth muscle cells by a two arbitrary primer-based mRNA differential display approach

Physical forces induce profound changes in cell phenotype, shape and behavi or. These changes can occur in vascular structures as a result of pressure overload and their effects can be seen in atherosclerotic vessels in which smooth muscle cells have undergone hyperplastic and hypertrophic changes. A t the molecular level, mechanical stimuli are converted into chemical ones and lead to modulation of gene expression and/or the activation of a new re pertoire of genes whose encoded proteins help the cells to adapt to their m icroenvironment. In this study, we have used a two primer-based mRNA differ ential display technique to identify candidate mechano-responsive genes in pulmonary artery smooth muscle cells. As compared to the original method de scribed by Liang and Pardee, this technique uses two arbitrary primers inst ead of an anchored oligo(dt) plus an arbitrary primer in the polymerase cha in reaction. The chief advantages of these modifications are an increase in the efficiency of the amplification and in the identification of different ially expressed clones. Using this approach, we compared the pattern of exp ressed genes in cells cultured under static conditions with those in cells that were mechanically stretched (1 Hz) for 24 h in a well-defined in vitro mechanical system. Three candidate genes that showed reproducible differen ces were chosen for further characterization and cloning. One clone was und er expressed in stretched cells and had a DNA sequence with 90% homology to the human fibronectin gene. Two other clones were highly expressed in stre tched cells and had a 92% and a 83% sequence homology with human platelet-a ctivating factor (PAF) receptor and rat insulin-like growth factor-I (IGF-I ) genes respectively. Northern blot analysis confirmed low levels of fibron ectin mRNA transcripts in stretched cells. In contrast, accumulation of PAF receptor mRNA occurred 30 min after mechanical stretch was initiated where as IGF-I mRNA levels peaked at 8 h. Both mRNA levels were sustained for up to 24 h of mechanical stretching. These results demonstrate the usefulness of the two primer-based mRNA differential display that enabled us to identi fy and characterize alterations at the level of gene expression among matri x proteins, G-protein coupled receptors and growth factors, each of whose r esponse to mechanical strain is different. A more complete understanding of these responses will provide further insight into the pathologic processes associated with hypertension and atherosclerosis.