Induced Pluripotent Stem Cell Model of Pulmonary Arterial Hypertension Reveals Novel Gene Expression and Patient Specificity

Abstract

Rationale: Idiopathic or heritable pulmonary arterial hypertension is characterized by loss and obliteration of lung vasculature. Endothelial cell dysfunction is pivotal to the pathophysiology, but different causal mechanisms may reflect a need for patient-tailored therapies.

Objectives: Endothelial cells differentiated from induced pluripotent stem cells were compared with pulmonary arterial endothelial cells from the same patients with idiopathic or heritable pulmonary arterial hypertension, to determine whether they shared functional abnormalities and altered gene expression patterns that differed from those in unused donor cells. We then investigated whether endothelial cells differentiated from pluripotent cells could serve as surrogates to test emerging therapies.

Methods: Functional changes assessed included adhesion, migration, tube formation, and propensity to apoptosis. Expression of bone morphogenetic protein receptor type 2 (BMPR2) and its target, collagen IV, signaling of the phosphorylated form of the mothers against decapentaplegic proteins (pSMAD1/5), and transcriptomic profiles were also analyzed.

Measurements and main results: Native pulmonary arterial and induced pluripotent stem cell-derived endothelial cells from patients with idiopathic and heritable pulmonary arterial hypertension compared with control subjects showed a similar reduction in adhesion, migration, survival, and tube formation, and decreased BMPR2 and downstream signaling and collagen IV expression. Transcriptomic profiling revealed high kisspeptin 1 (KISS1) related to reduced migration and low carboxylesterase 1 (CES1), to impaired survival in patient cells. A beneficial angiogenic response to potential therapies, FK506 and Elafin, was related to reduced slit guidance ligand 3 (SLIT3), an antimigratory factor.

Conclusions: Despite the site of disease in the lung, our study indicates that induced pluripotent stem cell-derived endothelial cells are useful surrogates to uncover novel features related to disease mechanisms and to better match patients to therapies.

Keywords: CES1; KISS1; SLIT3; endothelial cells; precision medicine.

Figure 1.

Idiopathic and heritable pulmonary arterial hypertension (IPAH/HPAH) pulmonary arterial endothelial cells (PAEC) and endothelial cells differentiated from induced pluripotent stem cells (iPSC-EC) show similar functional abnormalities. (A) PAEC isolated and cultured from explanted donor lungs and iPSC-EC differentiated from skin fibroblast-derived iPSC have the similar cobblestone morphology, incorporate acetylated low-density lipoprotein (Ac-LDL, green), and express the endothelial marker cluster of differentiation 144 (CD144) (green; blue = 4′,6-diamidino-2-phenylindole, dihydrochloride [DAPI]). (B) Adherent cells per ×10 field were counted 30 minutes after seeding on 10 μg/cm² laminin (n = 7). (C) A scratch was applied to cell monolayers, and migration of the cells toward the wound was recorded by photomicrographs at 6 hours (n = 5 Control, and 6 IPAH/HPAH; 4 IPAH and 2 HPAH). Representative images of wound closure on top. Related quantitative data are below. (D) Apoptosis was evaluated by the caspase-3/7 assay 12 hours after serum withdrawal in Control and IPAH/HPAH PAEC and iPSC-EC (n = 6 Control, and 6 IPAH/HPAH; 4 IPAH and 2 HPAH). (E) Angiogenesis assays: representative images of tube formation (top), and quantitative analysis (bottom) indicates the number of tubes formed and average tube length 4 hours after seeding cells in Matrigel (n = 6 control and 7 IPAH/HPAH; 5 IPAH and 2 HPAH). Bars represent mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005, unpaired Student’s t test IPAH/HPAH versus control. High and low values for patients and control subjects are designated by identifiers in the tables (see Figures E1–E5). RLU = relative light unit.

Figure 1.

Idiopathic and heritable pulmonary arterial hypertension (IPAH/HPAH) pulmonary arterial endothelial cells (PAEC) and endothelial cells differentiated from induced pluripotent stem cells (iPSC-EC) show similar functional abnormalities. (A) PAEC isolated and cultured from explanted donor lungs and iPSC-EC differentiated from skin fibroblast-derived iPSC have the similar cobblestone morphology, incorporate acetylated low-density lipoprotein (Ac-LDL, green), and express the endothelial marker cluster of differentiation 144 (CD144) (green; blue = 4′,6-diamidino-2-phenylindole, dihydrochloride [DAPI]). (B) Adherent cells per ×10 field were counted 30 minutes after seeding on 10 μg/cm² laminin (n = 7). (C) A scratch was applied to cell monolayers, and migration of the cells toward the wound was recorded by photomicrographs at 6 hours (n = 5 Control, and 6 IPAH/HPAH; 4 IPAH and 2 HPAH). Representative images of wound closure on top. Related quantitative data are below. (D) Apoptosis was evaluated by the caspase-3/7 assay 12 hours after serum withdrawal in Control and IPAH/HPAH PAEC and iPSC-EC (n = 6 Control, and 6 IPAH/HPAH; 4 IPAH and 2 HPAH). (E) Angiogenesis assays: representative images of tube formation (top), and quantitative analysis (bottom) indicates the number of tubes formed and average tube length 4 hours after seeding cells in Matrigel (n = 6 control and 7 IPAH/HPAH; 5 IPAH and 2 HPAH). Bars represent mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005, unpaired Student’s t test IPAH/HPAH versus control. High and low values for patients and control subjects are designated by identifiers in the tables (see Figures E1–E5). RLU = relative light unit.

Figure 2.

Reduced expression of bone morphogenetic protein receptor type 2 (BMPR2) and collagen type IV and alpha 2 chain (COL4A2); and reduced BMPR2 signaling in idiopathic and heritable pulmonary arterial hypertension (IPAH/HPAH) pulmonary arterial endothelial cells (PAEC) and endothelial cells differentiated from induced pluripotent stem cells (iPSC-EC). (A) BMPR2 and (B) COL4A2 mRNA were measured by quantitative reverse-transcriptase polymerase chain reaction. n = 6 control and n = 7 IPAH/HPAH (5 IPAH and 2 HPAH). *P < 0.05 versus control, unpaired Student’s t test. (C) Representative Western immunoblot of BMPR2, pSMAD1/5, ID1, and glyceraldehyde phosphate dehydrogenase (top) with BMP9 (10 ng/ml) or vehicle (phosphate-buffered saline) treatment in PAH-4, with densitometry on the bottom (n = 3; two IPAH and one HPAH). Bars represent mean ± SEM. *P < 0.05 versus untreated ^#P < 0.05 versus genotype, two-way analysis of variance with Bonferroni test. High and low values for patients and control subjects are designated by identifiers in the tables. BMP9 = bone morphogenetic protein 9; GAPDH = glyceraldehyde phosphate dehydrogenase; ID1 = inhibitor of DNA binding 1; pSMAD1/5 = phosphorylated form of the mothers against decapentaplegic proteins; Veh = vehicle.

Figure 3.

Proangiogenic responders to Elafin and FK506 in idiopathic and heritable pulmonary arterial hypertension (IPAH/HPAH) pulmonary arterial endothelial cells (PAEC) and endothelial cells differentiated from induced pluripotent stem cells (iPSC-EC). (A) Representative images of angiogenesis assay in patient IPAH-5 PAEC and iPSC-EC following treatment for 4 hours with vehicle (0.9% sodium chloride for Elafin; dimethyl sulfoxide for FK506), Elafin (1 μg/ml), or FK506 (15 μg/ml) show improved angiogenesis with both agents. (B) Elafin and FK506 versus vehicle, assessed by the number of tubes or tube length (pixels) in eight matched IPAH/HPAH PAEC and iPSC-EC. Patients IPAH, 8, 9, 1, 2, 4, 5, and HPAH 6, 7 (Table 1) are represented on the figure. Bars represent mean ± SEM. **P < 0.005, ***P < 0.0005 versus vehicle by one-way analysis of variance, followed by Bonferroni test. (C) Data from wound closure assay show that migration may be predictive of angiogenic response to Elafin and FK506.

Figure 4.

RNA-seq analysis of pulmonary arterial endothelial cells (PAEC) and endothelial cells differentiated from induced pluripotent stem cells (iPSC-EC) from control versus idiopathic and heritable pulmonary arterial hypertension (IPAH/HPAH), and drug responders versus nonresponders, reveals differences in gene expression. (A) Principal component analysis plot showing that the first and second principal components separate PAEC and iPSC-EC from undifferentiated iPSC and fibroblasts (control n = 6, CON-3, 4, 6, 8, 9, and 10; IPAH/HPAH n = 6, PAH-1, 2, 4, 5, 6, and 7). (B) Venn diagram shows differentially expressed genes (DEG) with a maximum false discovery rate of 0.1. On the top are genes up-regulated in control cells, and on the bottom are genes up-regulated in IPAH/HPAH cells, with the overlap indicating common genes. (C) Heatmap displaying the 16 overlapping DEG in IPAH/HPAH PAEC and iPSC-EC versus control PAEC and iPSC-EC. Hierarchical clustering was performed using the average gene expression from each of the indicated groups. (D) KISS1, an up-regulated gene in I/HPAH, was reduced by small interfering RNA (siRNA) and wound closure assessed for improvement in both PAEC and iPSC-EC of PAH patient 1, 4, and 7. (E) CES1, a down-regulated gene in IPAH/HPAH, was reduced by siRNA in CON 4, 9, and 10 PAEC and iPSC-EC and vulnerability to apoptosis assessed by active caspase after serum withdrawal at 96 hours. (F) Heatmap displaying 11 overlapping DEG between Elafin and FK506 responders versus nonresponders, in both PAEC and iPSC-EC (n = 3; two IPAH and one HPAH). Hierarchical clustering used the average gene expression from each of the indicated conditions. DEG revealed three up-regulated and eight down-regulated genes in nonresponders versus responders. (G) SLIT3, a gene up-regulated in the nonresponders, was reduced by siRNA in PAEC and iPSC-EC of IPAH nonresponders PAH-1, 2, and 7, and wound closure assessed 96 hours after transfection. Scr = scrambled siRNA. Bars represent mean ± SEM. *P < 0.05, unpaired Student’s t test versus siScr, n = 3 (see Figure E6). ADRB1 = adrenoceptor β1; AFP = α fetoprotein; C17orf53 = chromosome 17 open reading frame 53; CES1 = carboxylesterase 1; CLIC3 = chloride intracellular channel 3; CRHBP = corticotropin releasing hormone binding protein; CTNNA3 = catenin α3; ESX1 = ESX homeobox 1; GDA = guanine deaminase; GLIS3 = GLIS family zinc finger 3; GPR183 = G protein–coupled receptor 183; KCNS3 = potassium voltage–gated channel modifier subfamily S member 3; KISS1 = kisspeptin 1; LINC00960 = long intergenic non-protein coding RNA 960; OLFML2A = olfactomedin like 2A; OPCML = opioid binding protein/cell adhesion molecule like; PLA1A = phospholipase A1 member A; PTPN20A = protein tyrosine phosphatase, non-receptor type 20A; PTPN20B = protein tyrosine phosphatase, non-receptor type 20B; RERG = RAS-like estrogen regulated growth inhibitor; RLU = relative light unit; SATB1 = SATB homeobox 1; SLC2A14 = solute carrier family 2 member 14; SLIT3 = slit guidance ligand 3; TGFA = transforming growth factor α; UTS2 = urotensin 2.