Endothelial Cell Dysfunction in Pulmonary Hypertension

Introduction: Primary pulmonary hypertension, now known as idiopathic pulmonary arterial hypertension (IPAH), a subgroup of pulmonary arterial hypertension (PAH), is a rare disorder characterized by severe morbidity and high mortality rates. There are no routine screening tests or validated markers of disease activity in IPAH, or the broader group of PAH. Therefore, patients usually present at advanced stages of disease. The pathogenesis of IPAH and other forms of PAH remain unclear. Prior theories stressed a "one-hit" hypothesis. Current thinking focuses on a "two-hit" hypothesis: 1) genetic susceptibility, and 2) a triggering stimulus that initiates pulmonary vascular injury, resulting in endothelial cell (EC) dysfunction and the mobilization of endothelial progenitor cells (EPC). Loss of function mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene, has been implicated in the pathogenesis of IPAH. EC dysfunction in IPAH has been associated with decreases in both endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production. Peripheral blood mononuclear cells (PBMCs) interact with an altered endothelial cell surface, which may also be important in the disease process.

Objectives: We plan to define a subset of differentially regulated biomarkers in IPAH and PAH that may lead to earlier diagnosis and better methods for measuring responses to therapy. Specifically, we hope to identify biomarkers of IPAH and other forms of PAH that is suggestive of NO therapeutic response and which may be useful in titrating NO therapy. We also hope to identify novel targets for the development of new therapeutic strategies.

Methods: This study will consist of a pilot study and a primary study. The pilot study will enroll up to 30 patients and 30 controls in order to obtain completed studies on 10 normal subjects and 10 patients with PAH. The goal of the pilot study is to determine the best technique for circulating endothelial cell (CEC) and PBMC identification, quantification, and isolation and EPC identification and quantification. The subjects in the pilot phase undergo right heart catheterization to obtain hemodynamics and pulmonary artery blood. Pulmonary artery and peripheral blood will be used for EPC quantification and CEC and PBMC isolation. CECs and PBMCs will be studied in depth using high density oligonucleotide microarrays. In addition, plasma obtained from PAH patients and healthy volunteers will be applied in vitro to various cell populations suspected to be central to disease pathogenesis including but not limited to ECs, circulating mononuclear cells, cardiac myocytes and/or vascular smooth muscle cells. Phenotypic alterations induced by plasma exposure will be assessed using in vitro assays.

The primary study will recruit the following subject groups: 1) patients with IPAH and other forms of PAH (vascular injury-induced pulmonary hypertension) who currently are on no therapy, less than or equal to 6 months of IV therapy, or less than or equal to one year of oral therapy, 2) patients with pulmonary hypertension (PH) ascribed to a nonvascular injury process and 3) normal individuals. The following baseline studies will be performed in all groups: 1) noninvasive assessment of right ventricular (RV) function by echocardiogram and magnetic resonance imaging (MRI), 2) determination of exercise capacity by cardiopulmonary stress test and six minute walk, 3) measurement of hemodynamic parameters by right heart catheterization and 4) characterization of disease phenotype by cell surface markers, oligonucleotide microarrays, and proteomics using peripheral and pulmonary arterial blood. EPCs will be quantitated and CECs and PBMCs will be isolated and analyzed by flow cytometry for expression of cell surface markers involved in coagulation, adhesion, and angiogenesis, as these are important processes in IPAH and PAH. Furthermore, ECs (identified by positive and negative selection and isolated by cell sorting) and PBMCs will be studied in depth using high density oligonucleotide microarrays to more fully characterize their transcriptome.

A major impediment to the widespread use of chronic home based inhaled NO are related to its delivery system and duration of effects. In PAH patients we plan to study a novel NO delivery system (INO pulse delivery device). Patients with PAH will be given inhaled NO (20 and 40 ppm) and then placed on inhaled NO using the INO pulse delivery device for 24 hours. Hemodynamics will be obtained serially with each dose and upon completion of 24 hours of therapy, pulmonary artery and peripheral blood will be drawn and reexamined by flow cytometry, microarrays, and proteomics.

We also plan to follow response to standard therapy (as determined by the referring physician). After the initial day 0 studies, we will restudy the same parameters (excluding NO studies) in patients with PAH at approximately 4 months, and yearly for 5 years after therapeutic intervention.