THE ENDOGENOUS VASCULAR ELASTASE THAT GOVERNS DEVELOPMENT AND PROGRESSION OF MONOCROTALINE-INDUCED PULMONARY-HYPERTENSION IN RATS IS A NOVEL ENZYME RELATED TO THE SERINE PROTEINASE ADIPSIN

We showed previously a cause and effect relationship between increased activity of an endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in rats, We now report the isolation an d characterization of EVE. Degenerate oligonucleotides synthesized to homologous sequences in serine elastases were used in a PCR with rat p ulmonary artery (PA) cDNA. The PCR product hybridized to a 1.2-kb mRNA and the intensity of hybridization was threefold increased in RNA fro m rat hypertensive PA at a timepoint when EVE activity was increased. The PCR product was used to screen a cDNA library and sequences obtain ed encoded rat adipsin. We then used immunoaffinity to purify EVE. An antibody to the elastin-binding protein was used to remove this compet itor of elastase from the PA extract and the elastolytic activity incr eased 100-fold. The enzyme was purified using an antibody that recogni zes NH2-terminal sequences of serine proteinases and the eluate was fu rther purified using an antibody raised against recombinant adipsin. A single band at 20 kD immunoreactive with the adipsin antibody was res olved as an active enzyme on an elastin substrate gel. Immunogold labe ling with an antibody to an adipsin peptide sequence localized EVE to PA smooth muscle cells. This is the first isolation of EVE; it appears to be a novel enzyme related to the serine proteinase adipsin origina lly found in adipose tissue.