RNA Sequencing Analysis Detection of a Novel Pathway of Endothelial Dysfunction in Pulmonary Arterial Hypertension

Abstract

Rationale: Pulmonary arterial hypertension is characterized by endothelial dysregulation, but global changes in gene expression have not been related to perturbations in function.

Objectives: RNA sequencing was used to discriminate changes in transcriptomes of endothelial cells cultured from lungs of patients with idiopathic pulmonary arterial hypertension versus control subjects and to assess the functional significance of major differentially expressed transcripts.

Methods: The endothelial transcriptomes from the lungs of seven control subjects and six patients with idiopathic pulmonary arterial hypertension were analyzed. Differentially expressed genes were related to bone morphogenetic protein type 2 receptor (BMPR2) signaling. Those down-regulated were assessed for function in cultured cells and in a transgenic mouse.

Measurements and main results: Fold differences in 10 genes were significant (P < 0.05), four increased and six decreased in patients versus control subjects. No patient was mutant for BMPR2. However, knockdown of BMPR2 by siRNA in control pulmonary arterial endothelial cells recapitulated 6 of 10 patient-related gene changes, including decreased collagen IV (COL4A1, COL4A2) and ephrinA1 (EFNA1). Reduction of BMPR2-regulated transcripts was related to decreased β-catenin. Reducing COL4A1, COL4A2, and EFNA1 by siRNA inhibited pulmonary endothelial adhesion, migration, and tube formation. In mice null for the EFNA1 receptor, EphA2, versus control animals, vascular endothelial growth factor receptor blockade and hypoxia caused more severe pulmonary hypertension, judged by elevated right ventricular systolic pressure, right ventricular hypertrophy, and loss of small arteries.

Conclusions: The novel relationship between BMPR2 dysfunction and reduced expression of endothelial COL4 and EFNA1 may underlie vulnerability to injury in pulmonary arterial hypertension.

Keywords: collagen IV; ephrin; pulmonary hypertension; transcriptome; vascular endothelium.

Figure 1.

RNAseq analysis of pulmonary arterial endothelial cells (PAECs) from patients with idiopathic pulmonary arterial hypertension (IPAH) and unused donor controls (Con) reveals differences in gene expression. (A) Volcano plots illustrate fold differences in individual gene expression in IPAH versus control subjects, and associated P values (negative log10), in two RNAseq experiments. (B) Mean ± SEM fold differences (e.g., +1 = 100% higher, +0.5 = 50% higher) in gene candidates as measured by quantitative polymerase chain reaction (qPCR) and RNAseq. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control value, by t test. (C) Comparison of RNAseq and qPCR measurements in individual IPAH and control cells for COL4A1 and COL4A2. Data are arbitrary units normalized to the mean value in each experiment. (D) Representative immunoblot for COL4 protein in PAEC lysates, with β-actin as a loading control, and densitometry analysis below. Bars represent mean ± SEM of n = 3 with ****P < 0.0001. COL4A = collagen IV-A.

Figure 2.

Reduced bone morphogenetic protein (BMP) signaling recapitulates many of the changes in transcripts identified by RNAseq analysis in idiopathic pulmonary arterial hypertension (IPAH) versus control pulmonary arterial endothelial cells (PAECs). (A) Fold change in gene expression of siBMPR2-transfected PAECs versus scrambled small interfering RNA (siRNA)-transfected cells calculated as in Figure 1B. Bars represent mean ± SEM of n = 3, **P < 0.01 and ***P < 0.001 by t test. (B) Immunoblot analysis of collagen IV (COL4) and ephrinA1 (EFNA1) protein in media from PAECs transfected with scrambled (Con) or BMPR2 targeting siRNA. Ponceau indicates loading. Bars represent mean ± SEM. ****P < 0.0001 by t test versus control siRNA. (C) Quantitative polymerase chain reaction of transcripts altered by BMPR2 siRNA or β-catenin (β-CAT) siRNA versus nontargeting control siRNA. Bars represent mean ± SEM of n = 3 different PAEC harvests. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way analysis of variance and post hoc Bonferroni tests. BMPR2 = bone morphogenetic protein type 2 receptor; eNOS = endothelial nitric oxide synthase; HMGA1 = high-mobility group AT-hook 1; mRNA = messenger RNA.

Figure 3.

The functional consequences of loss of collagen IV (COL4) and ephrinA1 (EFNA1) expression in pulmonary arterial endothelial cells (PAECs). Representative fields are shown above and quantitative data below. (A) PAECs transfected with EFNA1 and/or COL4 small interfering RNA (siRNA), normalized to scrambled siRNA-transfected cells (Con) were adherent to gelatin or collagen-IV-coated wells after 40 minutes, assessed by semiautomated counting of 4′6-diamidino-2-phenylindole–positive nuclei. (B) PAECs transfected with EFNA1 and/or COL4 siRNA versus control siRNA and migration assessed at 8 hours by modified Boyden Chamber assay. (C) Matrigel assays in cells transfected with EFNA1, COL4 siRNA, or both versus scrambled (Con) siRNA, over 19 hours, in response to 0.2% FBS + 25 ng/ml vascular endothelial growth factor. Scale bars = 100 μm. Bars represent mean ± SEM of n = 3. ***P < 0.001, **P < 0.01, and *P < 0.05 versus Con siRNA, ^##P < 0.01 and ^###P < 0.001 versus gelatin coated condition by one-way analysis of variance or two-way analysis of variance when comparing collagen with gelatin and siRNA condition, and post hoc Bonferroni test. FBS = fetal bovine serum.

Figure 4.

Mice with deletion of EphA2 develop exaggerated pulmonary hypertension (PH). EphA2^−/− (KO) and wild-type (WT) mice were exposed to room air (normoxia), 10% O₂ (hypoxia), or subcutaneous injection of the vascular endothelial growth factor receptor blocker Sugen 5416 (Su) in addition to hypoxia for 4 weeks and development of PH assessed. n = 5 (3 males, 2 females) for each group. Bars indicate mean ± SEM. (A) Right ventricular systolic pressure (RVSP). (B) Right ventricular hypertrophy (RVH) given by the Fulton index (weight of right ventricle [RV]/left ventricle [LV] and septum [S]). **P < 0.01, ***P < 0.001 versus WT by two-way analysis of variance with Bonferroni post test. (C) Representative histology of distal pulmonary arteries (DPA) from mice exposed to hypoxia following Sugen injection; scale bars = 50 μm. The photomicrographs show similar muscularization of DPAs in the two genotypes but a greater reduction in the number of DPAs in the EphA2^−/− versus WT. Left images show high magnification of α-smooth muscle actin–immunostained vessels, and right images lower-magnification hematoxylin and eosin–stained tissue sections. (D) Muscularization of distal pulmonary arteries. FM = fully muscularized; NM = nonmuscularized; PM = partially muscularized. (E) Number of vessels (DPA) per 100 alveoli. (F) Alveoli per square millimeter. Bars represent mean ± SEM, ****P < 0.0001 versus normoxia, ^####P < 0.0001 versus WT, by two-way analysis of variance and post hoc Bonferroni test.