The primary objective of this R21 is to use a novel strategy to purify murine endogenous vascular elastase (EVE) from lung tissue and to identify its human homolog. This is important because we have linked EVE to the progression of experimental and clinical pulmonary vascular changes and pulmonary hypertension (PAH). We have also shown that inhibition of EVE is sufficient to reverse experimentally induced PAH and we now present preliminary data indicating that inhibition of EVE can regress advanced neointimal lesions in cultured explants of lung obtained from PAH patients undergoing lung transplant. Thus, determining the protein structure of EVE is essential in defining its role in PAH and in developing selective inhibitors. We plan to purify EVE from the lungs of transgenic mice that overexpress S100A4 one week after these mice have been infected with a murine form of the Kaposi Sarcoma virus (MHV-68). Elevation of serine elastase activity in lung following infection, and after re-activation of the virus three months later, is associated with severe neointimal lesions similar to those seen in patients with clinical PAH. Besides mimicking the pulmonary vascular pathology of PAH, the S100A4 mouse infected with MHV-68 is relevant to clinical PAH in other ways. Viral antigens homologous to MHV-68 have been linked to human PAH. There is heightened S100A4 expression in pulmonary arteries from patients with PAH and advanced neointimal lesions, and S100A4 stimulates proliferation and migration of cultured human pulmonary artery smooth muscle cells. Our proposal has three specific aims.
Aim 1 seeks to purify EVE from the S100A4 mouse lung using FLAG-tagged recombinant elafin, the inhibitor that binds EVE with high affinity. We will use peptide sequence information from mass spectrometry (MS/MS MALDI-TOF) analyses to identify the full-length cDNA encoding EVE. The peptide and cDNA sequences will be used to make antibodies and riboprobes to localize the enzyme in the lung.
Aim 2 applies sequence information from murine EVE to search databases to identify or clone the human homolog, and to localize EVE in lung tissue from PAH patients. The secondary objective of this R21 pursues the observation that there might be a link between reduced levels of microRNA (miR)-155 in S100A4 compared to C57BL6 lungs, and heightened EVE in response to MHV-68. This notion derives from the fact that PU.1, a transcription factor for serine elastase, is a putative target of miR-155.
Aim 3 seeks to determine whether the miR- 155 knockout mouse exhibits heightened EVE activity following MHV-68 inoculation, and if this in turn leads to pulmonary vascular disease. Taken together, these studies will establish the protein identify of EVE, and provide new insight into how a genetic factor that confers host susceptibility (S100A4 gene) interacts with an environmental factor (MHV-68 virus) to cause pulmonary vascular disease.

Public Health Relevance

Our group has shown that an enzyme called endogenous vascular elastase (EVE) plays a pivotal role in the pathogenesis of pulmonary arterial hypertension (PAH) and that inhibition of EVE may be a strategy to reverse the disease. We therefore plan to establish the structure of both the murine form of EVE and of the human enzyme, so that in the future highly targeted therapies can be developed to inhibit its activity. We also propose to determine whether EVE may be regulated by a microRNA and whether the level of this microRNA determines host susceptibility to virus-induced pulmonary vascular disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Exploratory/Developmental Grants (R21)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-CVS-Q (90))
Program Officer
Moore, Timothy M
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Stanford University
Schools of Medicine
United States
Zip Code
Kim, Yu-Mee; Haghighat, Leila; Spiekerkoetter, Edda et al. (2011) Neutrophil elastase is produced by pulmonary artery smooth muscle cells and is linked to neointimal lesions. Am J Pathol 179:1560-72