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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 β-adrenergic receptor (βAR) 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 βAR 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 β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR 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 βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs 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 βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

β2-adrenergic receptor (β2AR) agonists (β2-agonist) are the most commonly used therapy for acute relief in asthma, but chronic use of these bronchodilators paradoxically exacerbates airway hyper-responsiveness. Activation of βARs by β-agonist leads to desensitization (inactivation) by phosphorylation through G-protein coupled receptor kinases (GRKs) which mediate β-arrestin binding and βAR internalization. Resensitization occurs by dephosphorylation of the endosomal βARs which recycle back to the plasma membrane as agonist-ready receptors. To determine whether the loss in β-agonist response in asthma is due to altered βAR desensitization and/or resensitization, we used primary human airway smooth muscle cells (HASMCs) isolated from the lungs of non-asthmatic and fatal-asthmatic subjects. Asthmatic HASMCs have diminished adenylyl cyclase activity and cAMP response to β-agonist as compared to non-asthmatic HASMCs. Confocal microscopy showed significant accumulation of phosphorylated β2ARs in asthmatic HASMCs. Systematic analysis of desensitization components including GRKs and β-arrestin showed no appreciable differences between asthmatic and non-asthmatic HASMCs. However, asthmatic HASMC showed significant increase in PI3Kγ activity and was associated with reduction in PP2A activity. Since reduction in PP2A activity could alter receptor resensitization, endosomal fractions were isolated to assess the agonist ready β2ARs as a measure of resensitization. Despite significant accumulation of β2ARs in the endosomes of asthmatic HASMCs, endosomal β2ARs cannot robustly activate adenylyl cyclase. Furthermore, endosomes from asthmatic HASMCs are associated with significant increase in PI3Kγ and reduced PP2A activity that inhibits β2AR resensitization. Our study shows that resensitization, a process considered to be a homeostasis maintaining passive process is inhibited in asthmatic HASMCs contributing to β2AR dysfunction which may underlie asthma pathophysiology and loss in asthma control.