Transcriptional profiles of pulmonary artery endothelial cells in pulmonary hypertension

Abstract

Pulmonary arterial hypertension (PAH) is characterized by endothelial cell (EC) dysfunction. There are no data from living patients to inform whether differential gene expression of pulmonary artery ECs (PAECs) can discern disease subtypes, progression and pathogenesis. We aimed to further validate our previously described method to propagate ECs from right heart catheter (RHC) balloon tips and to perform additional PAEC phenotyping. We performed bulk RNA sequencing of PAECs from RHC balloons. Using unsupervised dimensionality reduction and clustering we compared transcriptional signatures from PAH to controls and other forms of pulmonary hypertension. Select PAEC samples underwent single cell and population growth characterization and anoikis quantification. Fifty-four specimens were analyzed from 49 subjects. The transcriptome appeared stable over limited passages. Six genes involved in sex steroid signaling, metabolism, and oncogenesis were significantly upregulated in PAH subjects as compared to controls. Genes regulating BMP and Wnt signaling, oxidative stress and cellular metabolism were differentially expressed in PAH subjects. Changes in gene expression tracked with clinical events in PAH subjects with serial samples over time. Functional assays demonstrated enhanced replication competency and anoikis resistance. Our findings recapitulate fundamental biological processes of PAH and provide new evidence of a cancer-like phenotype in ECs from the central vasculature of PAH patients. This "cell biopsy" method may provide insight into patient and lung EC heterogeneity to advance precision medicine approaches in PAH.

Figure 1

Differential gene expression in Group 1 pulmonary arterial hypertension vs. controls without pulmonary hypertension. (A) Volcano plot of fold-change in differential gene expression: Group 1 pulmonary arterial hypertension vs. controls.* (B) Heatmap of top differentially expressed genes. (C) Biologic pathways mapped from differentially downregulated genes. *p-values adjusted for multiple comparisons based on a false discovery rate (FDR) < 0.05. A p_FDR < 0.05 was considered significant. PAH pulmonary arterial hypertension. CTD connective tissue disease. HIV human immunodeficiency virus. PoPH portopulmonary hypertension. CHD congenital heart disease. APAH associated pulmonary arterial hypertension.

Figure 2

Differential gene expression in subjects with heritable pulmonary arterial hypertension (HPAH) versus controls. (A) Volcano-plot of unsupervised fold-change analysis of differentially expressed genes between HPAH1 vs controls*. (B) Heatmap of genes related to anoikis (GO: 0043276) demonstrated significant between-subject heterogeneity with increased expression in HPAH1, who has classical heritable PAH, and equivocal expression in HPAH2, who has hereditary hemorrhagic telangiectasia. (C) In an unsupervised analysis, one gene was differently expressed greater in PAECs from HPAH1 as compared to controls (PLAC8) with a splice-variant (PLAC8-203) identified as responsible for positive regulation of cell proliferation and negative regulation of apoptosis. *p-values adjusted for multiple comparisons based on a false discovery rate (FDR) < 0.05. A p_FDR < 0.05 was considered significant.

Figure 3

PAECs isolated from patients with pulmonary hypertension exhibit anoikis resistance as compared to commercial controls. (A) Some PAECs survive in suspension over 7 days and proliferate when reseeded on gelatin-coated plastic culture dishes, characteristic of anoikis resistance. Phase-contrast images of PAECs from a patient with hereditary pulmonary arterial hypertension (HPAH1) are shown, including cells at confluence (left-hand panel), 7 days following suspension in ultra-low attachment 6-well plates (middle panel), and 8 days after re-seeding the cells in suspension on a culture dish (right-hand panel). (B) The growth of anoikis-resistant cells is distinctive among patients with pulmonary hypertension. ~ 10–30% of HPAECs survived in suspension for 24 h, whereas ~ 5% of cells survived for 3- and 7-days (left-hand panel). The right-hand panel demonstrates percent confluency on day 4 (y-axis) of cells that have been in suspension for 1, 3 and 7 days (x-axis). Anoikis-resistant cells from one HPAH patient (HPAH1) exhibited rapid proliferation, whereas the growth capacity of anoikis-resistant cells from patients pulmonary veno-occlussive disease (PVOD), idiopathic PAH (IPAH1), IPAH2, and a second HPAH patient (HPAH2) was limited. (C) Representative phase-contrast images taken on the 14th day of incubation of the single-cell cloning wells from two different subjects are shown. The insert on the top demonstrates that the colony size in each well occupied less than 25% of the well. HPAH1 cells (left-hand panel), anoikis-resistant PAECs collected after 7 days in suspension from HPAH1 (HPAH1-ARD7, middle panel), and IPAH1 cells are shown. IPAH1 cells did not survive after 7 days in suspension. (D) Low density and single-cell proliferation are variable among PAECs isolated from patients with pulmonary hypertension. Confluency of serially diluted cells from six different cell types (Control, HPAH1, HPAH1-ARD7, PVOD, IPAH1, and IPAH2) is plotted after 10 days of culture, with densities ranging from 125K/well to 62 cells (left-hand panel). Anoikis-resistant cells are more replication-competent in single-cell cloning assays, 2 weeks after single-cell cloning (right-hand). (E) Lectin-induced agglutination discriminates endothelial cell phenotypes in vitro. Helix pomatia agglutinates PAECs, but not pulmonary microvascular endothelial cells. Helix pomatia agglutinated HPAECs from HPAH1, whereas Griffonia simplicifolia did not. Griffonia simplicifolia agglutinates pulmonary microvascular endothelial cells and endothelial progenitor cells. Here, Griffonia simplicifolia agglutinated anoikis resistant, highly proliferative cells from HPAH1-ARD7. Arrows indicate agglutinated cells. Data in panels (B) and (C) represent technical replicates for PH patients and n = 3 for commercial control. All phase-contrast images are taken with a 10 × objective, and the scale bar represents 100 μm. HPAH heritable pulmonary arterial hypertension. PVOD pulmonary vent-occlusive disease. IPAH idiopathic pulmonary arterial hypertension.

Figure 4

Biological replicates in a subject with portopulmonary hypertension. (A) Hemodynamics before and after liver transplant. Near resolution of pulmonary hypertension was seen after liver transplantation off all PAH therapy. (B) Heatmap of differential gene expression in genes related to portopulmonary hypertension (gene list assembled from Roberts, K., Kawut, S., et al. AJRCCM. 2009). A “normalization” of gene expression toward control levels was seen after liver transplantation (C) DAVID was used to mine GO biologic pathways of downregulated genes of pre-transplant endothelial cells as compared to post-transplant cells. Pathways related to the immune response were downregulated pre-transplant as compared to post-transplant. RAP right atrial pressure, mPAP mean pulmonary artery pressure, PCWP pulmonary capillary wedge pressure, CO cardiac output, PVR pulmonary vascular resistance, TID three times daily. PoPH portopulmonary hypertension.

Figure 5

Biological replicates in a subject with systemic sclerosis-associated pulmonary arterial hypertension. (A) Hemodynamics at baseline and clinical worsening. The subject’s clinical course was marked by profound hypoxemia at the time of the second right heart catheterization; the development of pulmonary veno-occlusive disease (PVOD) was suspected clinically. (B) Heatmap of differential expression of genes related to PVOD and hypoxia showed increased expression of genes including HIF-3α, TWIST1 and EIK2AK4 along with decreased expression of STOX1 and CD34 (gene list assembled from Hypoxia (GO: 0071456) and EIF2AK4). (C) Histology of explanted lung demonstrating venule involvement.  RAP right atrial pressure. mPAP mean pulmonary artery pressure. PCWP pulmonary capillary wedge pressure. CO cardiac output. PVR pulmonary vascular resistance.

Figure 6

Gene expression is similar across all subjects with pre-capillary pulmonary hypertension. (A) Heatmap and (B) principal component analysis (PCA) plot of differential gene expression between subjects with Group 1 pulmonary arterial hypertension (PAH) vs Group 2–5 precapillary pulmonary hypertension (PH), defined as mPAP > 20 mmHg, a PCWP ≤ 15 mmHg, and a PVR ≥ 3 Wood units. Despite within group heterogeneity, there appears to be similarities in gene expression between Group 1 PAH and precapillary Group 2–5 PH and there was no evidence of differential gene expression across precapillary PH from Group 1 versus Group 2–5. mPAP mean pulmonary artery pressure, PCWP pulmonary capillary wedge pressure, PVR pulmonary vascular resistance, CpcPH combined pre-post capillary pulmonary hypertension.