Culture of pulmonary artery endothelial cells from pulmonary artery catheter balloon tips: considerations for use in pulmonary vascular disease

Abstract

Endothelial dysfunction is a hallmark of pulmonary arterial hypertension (PAH) but there are no established methods to study pulmonary artery endothelial cells (PAECs) from living patients. We sought to culture PAECs from pulmonary artery catheter (PAC) balloons used during right-heart catheterisation (RHC) to characterise successful culture attempts and to describe PAEC behaviour.PAECs were grown in primary culture to confluence and endothelial cell phenotype was confirmed. Standard assays for apoptosis, migration and tube formation were performed between passages three to eight. We collected 49 PAC tips from 45 subjects with successful PAEC culture from 19 balloons (39%).There were no differences in subject demographic details or RHC procedural details in successful versus unsuccessful attempts. However, for subjects who met haemodynamic criteria for PAH, there was a higher but nonsignificant (p=0.10) proportion amongst successful attempts (10 out of 19, 53%) versus unsuccessful attempts (nine out of 30, 30%). A successful culture was more likely in subjects with a lower cardiac index (p=0.03) and higher pulmonary vascular resistance (p=0.04). PAECs from a subject with idiopathic PAH were apoptosis resistant compared to commercial PAECs (p=0.04) and had reduced migration compared to PAECs from a subject with portopulmonary hypertension with high cardiac output (p=0.01). PAECs from a subject with HIV-associated PAH formed fewer (p=0.01) and shorter (p=0.02) vessel networks compared to commercial PAECs.Sustained culture and characterisation of PAECs from RHC balloons is feasible, especially in PAH with high haemodynamic burden. This technique may provide insight into endothelial dysfunction during PAH pathogenesis.

Figure 1.

Representative images confirming endothelial cell phenotype. Top left) commercial human pulmonary artery endothelial cells; passage 6. Top middle) No PAH = subject 14; passage 4. Top right) idiopathic PAH = subject 18; passage 3. Bottom left) HIV APAH = subject 13; passage 4. Bottom right) PoPH, high CO = subject 7; passage All images: VE-cadherin staining (red), acetylated low density lipoprotein uptake (green), images at 40X magnification. Scale bars = 50 μm. PAH = pulmonary arterial hypertension; HIV = human immunodeficiency virus; APAH = associated pulmonary arterial hypertension; PoPH = portopulmonary hypertension; CO = cardiac output.

Figure 2.

Apoptosis assessed by TUNEL assay following treatment with vehicle and TNF-α. Panel A) Representative image of PAECs exposed to tert-butyl hydroperoxide for 6 hours TUNEL stained for apoptosis and counterstained with 4’,6-diamidino-2-phenylindole (DAPI). TUNEL positive cells are indicated by yellow arrow. Image at 20X magnification. Scale bar = 50 μm. Panel B) PAECs were exposed to vehicle or TNF-α for six hours. Data are presented as mean ± SD. n = 2 – 5; each n is derived from a different passage of cells from a single balloon tipped catheter from one right heart catheterization procedure for a single subject. *p < 0.05. X axis: No PAH = subject 14; idiopathic PAH = subject 18; HIV APAH = subject 13; PoPH, high CO = subject 7. PAEC = pulmonary artery endothelial cell; PAH = pulmonary arterial hypertension; PoPH = portopulmonary hypertension; CO = cardiac output.

Figure 3.

Migration assessed by scratch assay under low, enriched, and VEGF in low serum conditions. Representative phase image of PAECs at 0 (Panel A) and 6 hours (Panel B) after scratch. Images at 4X magnification. Scale bar = 100 μm. Panel C) Data are presented as mean ± SD. n = 3 – 8; each n is derived from a different passage of cells from a single balloon tipped catheter from one right heart catheterization procedure for a single subject. *p < 0.05. X axis: No PAH = subject 14; idiopathic PAH = subject 18; PoPH, high CO = subject 7. VEGF = vascular endothelial growth factor; PAEC = pulmonary artery endothelial cells; PAH = pulmonary arterial hypertension; PoPH=portopulmonary hypertension; CO=cardiac output.

Figure 4.

Tube formation following Matrigel treatment as quantified by AngioTool. Panel A) Representative phase images of tube formation at 2 and 6 hours respectively following seeding of PAECs on Matrigel. Images at 4X magnification. Scale bar = 100 μm. Panel B) number of segments, Panel C) number of branches, Panel D) mesh size (pixels²), Panel E) segment length (pixels²), and Panel F) branch length (pixels²). Data are presented as mean ± SD. n = 4 – 8; each n is derived from a different passage of cells from a single balloon tipped catheter from one right heart catheterization procedure for a single subject. *p < 0.05. X axis: No PAH = subject 14; idiopathic PAH = subject 18; HIV APAH = subject 13; PoPH, high CO = subject 7. PAH = pulmonary arterial hypertension; HIV = human immunodeficiency virus; APAH = associated pulmonary arterial hypertension; PoPH = portopulmonary hypertension; CO = cardiac output.