Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although beta-adrenergic receptor (beta AR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of beta AR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated beta-blocker alprenolol was synthesized and validated as a beta AR specific probe that was combined with immunophenotyping to quantify beta AR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that beta AR density was decreased in most WBC subsets in PAH samples compared to healthy controls. EcMPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell beta AR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.