Abstract
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.
Highlights
Pulmonary arterial hypertension (PAH) is a heterogeneous group of debilitating diseases characterized by hyperproliferative apoptosis-resistant pulmonary artery endothelial cells contributing to vascular remodeling of the small pulmonary arteries, which results in elevated pulmonary artery blood pressure and right ventricular failure [1,2,3]. β-adrenergic receptors are transmembrane G-protein coupled receptors that are essential to many aspects of human physiology and are best known for control of cardiac chronotropy, inotropy, and vascular tone [4,5]
We report the development of new cytometric methods for assessing β-adrenergic receptors (βAR) density in peripheral blood cells with an alprenolol probe and enumeration of endothelial cell-derived microparticles (Ec-MPs) in urine samples
Specificity of the alprenolol-probe for βAR was confirmed by demonstrating dose-dependent saturable binding that was reversed by a βAR ligand but not by a scrambled competitor losartan consistent with, classical radio-ligand studies [12]
Summary
Pulmonary arterial hypertension (PAH) is a heterogeneous group of debilitating diseases characterized by hyperproliferative apoptosis-resistant pulmonary artery endothelial cells contributing to vascular remodeling of the small pulmonary arteries, which results in elevated pulmonary artery blood pressure and right ventricular failure [1,2,3]. β-adrenergic receptors (βAR) are transmembrane G-protein coupled receptors that are essential to many aspects of human physiology and are best known for control of cardiac chronotropy, inotropy, and vascular tone [4,5]. Current methods for quantifying βAR density are laborious and time consuming that require radioactive detection of cell membrane extracts [9,10,12] which are not suitable for easy routine high throughput testing of patients. Microparticles such as those derived from endothelial cells (Ec-MP) are vesicles generated by exocytic budding [13] and display surface antigens from their cell of origin [14].
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