Patient-specific and gene-corrected induced pluripotent stem cell-derived endothelial cells elucidate single-cell phenotype of pulmonary veno-occlusive disease

Figure 1. Generation and characterization of PVOD patients-specific induced pluripotent stem cells (iPSCs) and gene-corrected PVOD-iPSCs (GC-PVOD-iPSCs) (A) The high-resolution computed tomography (CT) of the chest from three PVOD patients showed significant centrilobular ground-glass opacification (The red arrows indicated) and interlobular septal thickening. (B) The EIF2AK4 mutation sites information in three PVOD patients and their pathogenicity classification. VUS means uncertain significance, and LP means likely pathogenic. (C) Strategy of correcting EIF2AK4 c.2965 C>T (PVOD#1) variant, c.3460A>T variant (PVOD#2), and c.4832_4833insAAAG variant (PVOD#3). The homologous sequences are shown between the red lines. (D) DNA sequencing demonstrates variant sites on EIF2AK4 (PVOD#1: c.2965C>T and c.4724T>C; PVOD#2: c.4736T>C and c.3460A>T; PVOD#3: c.1942A>T and c.4832_4833insAAAG) of the three iPSC lines.

Figure 2. Excessive angiogenesis and proliferation of PVOD patients induced pluripotent stem cell-derived endothelial cells (PVOD-iPSC-ECs) (A) Endothelial cells (ECs) differentiated from control, gene-corrected (GC), and PVOD-iPSCs exhibit typical cobblestone morphology, incorporate 3,3′-dioctadecylindocarbocyanine labeled low-density lipoprotein (Dil-LDL, red), and are positive for the EC surface marker CD144 (green). Blue: DAPI. Scale bar represents 100 μm (top), 200 μm (middle), and 200 μm (bottom). (B) EIF2AK4 gene expression quantified by western blot and densitometric quantification of GCN2 protein and real-time qPCR in iPSC-ECs from controls, PVODs, and GCs. ∗p < 0.05 by one-way ANOVA followed by the Bonferroni test. (C) Representative images of Ki67 immunostaining and quantitative data to compare the proliferation capacity of iPSC-ECs from controls, PVODs, and GCs. Scale bar, 50 μm. ∗p < 0.05 by one-way ANOVA followed by the Bonferroni test. (D) MTT assay was conducted to evaluate the proliferation capacity of iPSC-ECs from control, PVODs, and GCs during a 4-day time period. ∗p < 0.05 by two-way ANOVA followed by Bonferroni post hoc test. (E) Representative images of tube…

Figure 3. Disrupted proliferation and angiogenesis phenotype in HUVECs with EIF2AK4 siRNA knockdown treatment (A and B) Western blot and real-time qPCR show EIF2AK4 expression in HUVECs with EIF2AK4 siRNA knockdown. (C) MTT assay was conducted to evaluate the proliferation capacity of HUVECs between scramble siRNA and EIF2AK4 siRNA groups during a 4-day time period. ∗p < 0.05 by two-way ANOVA followed by Bonferroni post hoc test. (D) Representative images of Ki67 immunostaining and quantitative data to compare the proliferation capacity of HUVECs between scramble siRNA and EIF2AK4 siRNA groups. Scale bar, 50 μm. (E) Representative images of tube formation of HUVECs from scramble siRNA and EIF2AK4 siRNA groups with quantitative analysis. Scale bar, 100 μm. Three independent experiments were performed in duplicate.

Figure 4. Differential mRNA-level expression of proliferation and angiogenesis in PVOD induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) (A) Volcano plots of genes in RNA sequencing between PVOD-iPSC-ECs and GC-PVOD-iPSC-ECs. Blue plots indicate the downregulated genes, and red plots indicate the upregulated genes. (B) KEGG pathways analysis of genes regulated by EIF2AK4 compound heterozygous variants. (C) Heatmap comparative analysis of genes related to proliferation and angiogenesis biological processes between PVOD-iPSC-ECs and GC-PVOD-iPSC-ECs. (D) Phospho-AKT/total-AKT expression quantified by western blot and densitometric quantification in PVOD-iPSC-ECs and GC-PVOD-iPSC-ECs. ∗p < 0.05 by one-way ANOVA followed by the Bonferroni test. Three independent experiments were performed in duplicate. (E and F) Real-time qPCR of representative genes related to proliferation and angiogenesis biological processes between PVOD-iPSC-ECs and GC-PVOD-iPSC-ECs. Including PCNA, MCM7, MCM4, PDGFD, VEGFA, TNC, and ANGPTL1. Data are presented as the mean ± SEM. ∗p < 0.05 vs. GC-PVOD-iPSC-ECs by Student’s t test. Three independent experiments were performed in duplicate.

Figure 5. AZD5363 and MK2206 are beneficial to PVOD-iPSC-ECs disease phenotype with EIF2AK4 compound heterozygous variants (A and B) Representative images of Ki67 immunostaining of PVOD-iPSC-ECs with DMSO, 8 nM AZD5363, or 10 μM MK2206 treatment and quantitative analysis. Red: Ki67; blue: DAPI. Scale bar, 100 μm. Data are presented as the mean ± SEM. ∗p < 0.05 vs. DMSO group by Student’s t test. (C) MTT assay to assess the inhibitory effect of AZD5363 on PVOD-iPSC-ECs at dose-dependent concentration during a 4-day time period. ∗p < 0.05 vs. DMSO group. Two-way ANOVA followed by the Bonferroni post hoc test. (D) MTT assay to assess the inhibitory effect of MK2206 on PVOD-iPSC-ECs at dose-dependent concentration during a 4-day time period. ∗p < 0.05 vs. DMSO group. Two-way ANOVA followed by the Bonferroni post hoc test. (E) Representative images of tube formation of PVOD-iPSC-ECs with DMSO, 8 nM AZD5363, or 10 μM MK2206 treatment and quantitative analysis. Scale bar, 100 μm.

Figure 6. Relative PAH-drugs testing on PVOD-iPSC-ECs (A and B) Representative images of Ki67 immunostaining of PVOD-iPSC-ECs with DMSO, 10 μM imatinib, 2 mM amifostine, 10 μM bosentan, and 10 μM slidenafil treatment and quantitative analysis. Scale bar, 100 μm. (C–F) MTT assay to assess the inhibitory effect of imatinib, amifostine, bosentan, and slidenafil on PVOD-iPSC-ECs at dose-dependent concentration during a 4-day time period. (G and H) Representative images of tube formation of PVOD-iPSC-ECs with DMSO, amifostine, bosentan, and slidenafil treatment and quantitative analysis. Scale bar, 100 μm. Data are presented as the mean ± SEM. ∗p < 0.05 vs. DMSO group by Student’s t test. Three independent experiments were performed in duplicate.

Figure 7. Mechanistic overview of the study Normal iPSCs and patient-specific iPSCs are generated from two healthy subjects and three PVOD patients. CRISPR-Cas9 genome editing is used to correct the EIF2AK4 variants. The normal iPSCs, PVOD-iPSCs, and GC-PVOD-iPSCs are differentiated into ECs. PVOD-iPSC-ECs exhibited disease phenotype featured by excessive proliferation and increased tube formation capacity when compared with those in control-iPSC-ECs and GC-PVOD-iPSC-ECs. Drug testing reveals that AZD5363, MK2206, imatinib, and amifostine, but not bosentan and sildenafil, normalize the disease phenotype of PVOD-iPSC-EC.