Plexogenic human pulmonary arterial hypertension (hPAH) is a severe disease with no cure and nearly always lethal. While it is evident that plexiform lesions (PLs) contain predominantly phenotypically altered, proliferative and dysfunctional endothelial cells (ECs), the cellular and molecular factors that contribute to abnormal proliferation of ECs are poorly understood. The objective for this application is to understand how cleavage of intersectin-1s (ITSN) by granzyme B (GrB) during pulmonary inflammation associated with PAH contributes to EC proliferation and formation of PLs. ITSN, a major pro-survival protein of lung ECs, is a recently identified GrB substrate. GrB cleaves ITSN and generates two biologically active protein fragments with the N-terminal fragment (GrB-EHITSN) possessing EC proliferative potential, mediated via phosphorylation of p38MAPK and Elk- 1 transcription factor, and abolished by chemical inhibition of p38 kinase. Moreover, lung tissue of PAH animal models and hPAH lungs with PLs, as well as pulmonary artery ECs of PAH subjects, contain the GrB- EHITSN and express lower levels of full-length ITSN compared to controls. GrB immunoreactivity is associated with PLs in hPAH lungs. The C-terminal fragment (GrB-SH3A-EITSN) has dominant negative effects on Erk1/2 signaling and the concurrent expression of the two GrB/ITSN fragments results in a high p38 to Erk1/2 activity ratio, critical for the EC proliferative phenotype associated with hPAH. Furthermore, mice transduced with the GrB-EHITSN show a PAH phenotype (i.e. increased lung EC proliferation, elevated right ventricular systolic pressure, right heart hypertrophy, pulmonary arteriopathy) which is ameliorated by treatment with a GrB-EHITSN inhibitory peptide. Thus, we hypothesize that decrease of full-length ITSN expression due to GrB cleavage during pulmonary inflammation associated with hPAH and the high p38 to Erk1/2 activity ratio caused by the two GrB/ITSN cleavage products lead to EC proliferation and selection of a proliferative/plexiform EC phenotype.
Three specific aims (SA) will test this hypothesis: SA1 will demonstrate that decrease of full-length ITSN expression due to GrB cleavage and the presence of GrB-EHITSN account for the EC proliferative phenotype in hPAH lungs. SA2 will demonstrate that GrB-EHITSN-induced p38 activation favors transcriptional activity of Elk-1 and preferential transcription of c-fos immediate early response gene leading to EC proliferation and overgrowth, in vivo. SA3 will demonstrate that GrB-EHITSN elicits a mouse lung EC proliferative response and vascular remodeling that can be ameliorated via a GrB-EHITSN inhibitory peptide. Successful completion of the aims will identify the GrB-EHITSN as the trigger in the emergence of the proliferative/plexiform EC phenotype and as a critical factor of a patho-physiological mechanism for PAH initiation and progression. The findings are central for understanding the mechanisms responsible for abnormal EC proliferation in severe hPAH and they may provide therapeutic targets for a new generation of small molecules that, by inhibiting the proliferative effect of GrB EHITSN can effectively ameliorate EC proliferation and halt PAH progression, making survival a realistic expectation for PAH patients.

Public Health Relevance

Angioproliferative pulmonary arterial hypertension (PAH) is a severe disease, with high incidence, no cure and is nearly always lethal. The molecular mechanism that promotes angioproliferation is unknown. Our recent studies indicated that intersectin-1s, a protein highly expressed in lung tissue, is cleaved during inflammatory reactions. The cleavage generates two fragments, one of them possessing endothelial cell proliferative potential and perhaps, responsible for the emergence of the angioproliferative endothelial cells in PAH. This is significant because, preventing the cleavage of intersectin-1s or reducing the proliferative effect of the fragment, may provide new therapeutic options that can be used to effectively treat patients with PAH.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Respiratory Integrative Biology and Translational Research Study Section (RIBT)
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Xiao, Lei
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Rush University Medical Center
Schools of Medicine
United States
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