Pulmonary arterial hypertension (PAH) is currently an incurable disease diagnosed at an advanced state and when there is often compromise of cardiac function. The major drawback in developing therapies to reverse this disease has been the lack of lung tissue and cells that could be studied from patients. The PHBI Network has therefore developed a resource for the investigative community by harvesting and archiving tissue and vascular cells from patients with idiopathic PAH (IPAH) and from PAH associated with other medical conditions (APAH), as well as from unused donor lungs, as controls. Our group contributes as a PHBI lung procurement site. We have also harvested and cultured CD31 positive(+) (endothelial) cells from patients with IPAH and from control lungs, and we study gene expression in these cells using 'next generation'tools including RNA-Seq. Having identified transcripts that are significantly up- or downregulated in CD31+ cells from IPAH vs. control lungs, our first aim in this proposal is to localize expression of these transcripts to the PAECs in the lung tissue. The CD31+ PAECs will be isolated by laser-capture microdissection from intra-acinar arteries where lesions are found in the IPAH patients vs. controls, and will be analyzed by RNA extraction and qRT-PCR. These studies will allow us to verify that the changes are differentially induced by culture but are expressed in vessels where the vascular lesions of PAH are present. We will then determine whether some of the changes in gene expression observed in IPAH CD31+ cells in tissue are also present in CD31+ cells from lungs in APAH patients. This will position us to focus on the functional significance of these abnormalities as they relate to the advanced pathology of IPAH. In other studies, we analyzed changes in gene expression in freshly isolated CD31+ cells using microfluidics assays, in which single cells are sorted and subjected to 48 PCR reactions. We identified expanded subpopulations of vascular cells in IPAH vs control lungs that are double positive for CD31+ and GM-CSFR?+, or CD31+ and PDGFR?+. Those CD31+GM-CSFR?+ cells are equally subdivided into cells that express primarily EC markers, and those that express monocyte/macrophage markers. The expanded CD31+PDGFR?+ cells from IPAH patient lungs express primarily monocyte/macrophage markers.
Our second aim i s therefore to localize these subpopulations in the lung vasculature by confocal microscopy, and to determine whether they are associated with a vessel of a particular size, or with a specific lesion. We can then further assess, by microfluidics and by immunofluorescence, which of the identified subpopulations are actively proliferating, and whether they selectively express the abnormal transcripts identified in CD31+ cells in Specific Aim I. By identifying the abnormally expanded sub-populations of cells in IPAH lesions, we can further interrogate these cells ex-vivo to determine whether they are in a state of transformation or de-differentiation, and whether they are giving rise to the cels in the obliterative and plexiform lesions, and could be good targets for emerging therapies.
Recent studies from our group identified aberrant gene expression in endothelial cells cultured from pulmonary arteries from lungs of patients with idiopathic pulmonary arterial hypertension, compared to those from healthy (unused donor) lungs. We now have the opportunity, through the PHBI, to analyze endothelial cells from frozen tissue sections to determine whether the cells in the diseased tissue show the altered expression of these genes in vessels with lesions and do not reflect a change related to the condition of culture. We have used microfluidic technology to identify expanded sub-populations of endothelial cells in IPAH vs. control lungs, and will now use confocal microscopy to determine the location of these cells, and which of the sub- populations might be the source of the cells that occlude the vessel lumen, and might therefore be important to target using emerging therapies.