Molecular Biological Identification of Endogenous Smooth Muscle Cell Elastase in Pulmonary Artery.

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Vascular elastase is reported to activate the growth factors such as basic fibroblast growth factor and transforming growth factor β stored in the extracellular matrix, and leads to hypertrophy and proliferation of vascular smooth muscle cells (SMC), resulting in more increased production of extracellular matrix. The vascular SMC phenotype change induced by these growth factors is playing the central role in the development of vascular remodeling. We tried to extract total RNA from porcine pulmonary artery SMCs by guanidinium method to identify pulmonary artery SMC elastase DNA sequences. We made PCR primers (primer 1 named pEL up (neutro) : GCCGCGCACTGCGTG, primer 2 named pEL down : CAAGGGGCCGCCTGAGTCCCC, primer 3 named pEL up (pancreas) : CACACGTGTGGCGGT) from conservative sequences of both human leukocyte elastase and pancreas elastase DNA sequences, then followed by reverse transcription polymerase chain reaction (RT-PCR). We obtained 284bp RT-PCR product following primer 2-3 RT-PCR, however, the DNA sequence analysis demonstrated nothing homologous to any proteinase. Contamination troubles in the SMC cultures caused schedule delay of this project. We finally obtained another RT-PCR product with 59% homology to a serine proteinase derived from T lymphocyte of mouse. We have started the screening step of this prroject using this cDNA primers. We will also confirm whether this primers are appropriate for the probe of the screening or not, using Northern blotting. We hope this project will be one of the promissing steps clarifying the mechanism of the pulmonary hypertension.