Isogenic pairs of induced-pluripotent stem-derived endothelial cells identify DYRK1A/PPARG/EGR1 pathway is responsible for Down syndrome-associated pulmonary hypertension

Figures

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  Figure 1. Cellular physiology and mitochondrial respiratory function of T21-ECs and cDi21-ECs. (A) Proliferation assay evaluating the percentages of EdU-positive cells. There is no significant difference among cDi21-, T21-, and T21’-ECs. (B) Relative migration distance 12 h after the wound scratch of the cells. There is no significant difference among cDi21-, T21-, and T21’-ECs. (C) Cell adhesion assay by counting the number of attached cells 2 h after seeding. There is no significant difference among cDi21-, T21-, and T21’-ECs. (D) Representative images of apoptosis assay by flow cytometry for annexin V and propidium iodide (PI). (E) The quantitative analyses of annexin V-positive and PI-negative cell populations show that apoptotic cells are significantly increased in T21- and T21’-ECs as compared with cDi21-ECs. (F) Representative images of phase contrast microscopy in tube formation assay. Note that lots of cellular tubes are formed in cDi21-ECs. Scale bar: 500 μm. (G) The quantitative assessments tube formation show that ability of angiogenesis is impaired in T21- and T21’-ECs as compared with cDi21-ECs. (H) Representative fluorescent images of MitoSOX red staining for mitochondrial…

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  Figure 2. The RNA-sequencing analyses of T21-, cDi21-, and healthy control-ECs. RNAs were extracted from two independent iPSC-derived EC lines of patients with Down syndrome (T21 and T21’), two independent iPSC-derived EC lines of healthy controls (HC_1 and HC_2), and an isogenic iPSC-derived EC line of corrected to disomy 21 (cDi21). (A) T-distributed stochastic neighbor embedding (tSNE) plots of RNA-sequencing. The comprehensive expression patterns of T21- and T21’-ECs are separated from cDi21-ECs and HC-ECs. (B) The pathway analysis of RNA-sequencing reveals that the mitochondria related genes are dysregulated in T21- and T21’-ECs as compared to HC-ECs and cDi21-ECs. (C) The RNA-sequencing reveals that the endothelial cell related genes are dysregulated in T21- and T21’-ECs. (D) Differential gene expression (DEG) analysis reveals significantly upregulated and downregulated genes in T21-ECs. (E) Quantitative PCR analysis of EGR1 in T21-, T21’-, cDi21-, and HC-ECs. Data are presented as mean ± SEM. Data were analyzed using the Tukey–Kramer multiple comparison test. **P < 0.01.

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  Figure 3. Knockdown of EGR1 expression in T21-EC restores angiogenesis and mitochondrial respiratory function. (A) Representative phase contrast images of tube formation assay in scrambled siRNA-treated or siEGR1-treated ECs. Scale bar: 500 μm. (B) The quantitative analyses of tube formation assays reveal that angiogenesis is significantly recovered in siEGR1 treated T21- and T21’-ECs as same level as that of cDi21-ECs. (C) OCR of each cell line and condition at baseline and after sequential supplementation of oligomycin (Oligo), FCCP, and Rotenone/Antimycin A (R/AA). (D) Quantitative analyses of basal respiration and maximal respiration show significant improvement of OCR in siEGR1 treated T21- and T21’-ECs. (E) Quantitative analyses of annexin V-positive and PI-negative cell populations show that apoptotic cells are significantly decreased in siEGR1-treated T21- and T21’-ECs, similar to cDi21-ECs. (F) Quantitative measurements of MitoSOX fluorescence intensity show that the level of mitochondrial superoxide production is significantly recovered in siEGR1-treated T21- and T21’-ECs, which is similar to the level in cDi21-ECs.Three to four independent experiments were conducted in each.…

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  Figure 4. Pioglitazone suppresses EGR1 mRNA expression in DS-ECs and can restore angiogenesis and mitochondrial respiratory function. (A and B) Relative mRNA expression of PPARG and EGR1 in T21-ECs treated with DMSO or pioglitazone (PIO). PIO treatment significantly increases PPARG expression and suppresses EGR1 expression. (C) Representative phase contrast images of tube formation assays in each cell line and condition. Scale bar: 500 μm. (D) The quantitative analyses of tube formation assays demonstrate that angiogenesis is significantly recovered in PIO-treated T21- and T21’-ECs as same level as that of cDi21-ECs. (E) OCR of each cell line and condition at baseline and after sequential treatment of oligomycin (Oligo), FCCP, and Rotenone/Antimycin A (R/AA). (F) Quantitative analyses of basal respiration and maximal respiration show significant improvement of OCR in PIO-treated T21- and T21’-ECs. (G) Quantitative analyses of annexin V-positive and PI-negative cell populations show that apoptotic cells are significantly decreased in PIO-treated T21- and T21’-ECs, similar to cDi21-ECs. (H) Quantitative measurements of MitoSOX fluorescence intensity show that the level of mitochondrial…

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  Figure 5. DYRK1A inhibitor, harmine reduces EGR1 mRNA expression in T21-EC via PPARG pathway and restores angiogenesis and mitochondrial respiratory function. (A) Relative mRNA expressions of DYRK1A in T21- and T21’-ECs, evaluated by quantitative PCR. Approximately 1.5-fold higher expression is observed both in T21- and T21’-ECs as compared with cDi21-ECs. (B) Relative mRNA expression of PPARG in cDi21-ECs, T21-, and T21’-ECs treated with DMSO or harmine; the latter significantly increases PPARG expression in T21- and T21’-ECs, but not in cDi21-ECs. (C) Relative mRNA expression of EGR1 treated with DMSO or harmine; the latter significantly reduces EGR1 expression in T21- and T21’-ECs. (D) Representative phase contrast images of tube formation assays in each cell line and condition. Scale bar: 500 μm. (E) Quantitative analyses of tube formation assays reveal that angiogenesis is significantly recovered in harmine-treated T21- and T21’-ECs as same level as that of cDi21-ECs. (F) OCR of each cell line and condition at baseline and after sequential treatment of oligomycin (Oligo), FCCP, and Rotenone/Antimycin A (R/AA). (G) Quantitative analyses of basal respiration and maximal respiration show…

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  Figure 6. DYRK1A overexpression impairs angiogenesis and mitochondrial respiratory function in cDi21- and HC-ECs. (A) Transfection of the DYRK1A-expression vector induced significant higher mRNA level of DYRK1A in cDi21-ECs as compared with that of empty vector (EV). Overexpression of DYRK1A results in a significant decrease of PPARG and an increase of EGR1 levels in cDi21-ECs compared with EV-treated-ECs. (B) Representative images of the tube formation assays in cDi21- and two HC-EC lines treated with EV and DYRK1A expression vector, visualized by phase contrast microscopy. Scale bar: 500 μm. (C) Quantitative analyses of the tube formation assays show that angiogenesis is significantly impaired in DYRK1A-overexpressed cDi21- and HC-ECs. (D) OCR at baseline and after sequential treatment of oligomycin (Oligo), FCCP, and Rotenone/Antimycin A (R/AA) in each line treated with EV and DRYK1A expression vector. (E) Quantitative analyses of basal respiration and maximal respiration show significant impairment of OCR in cells treated with DYRK1A-expression vector. (F) Quantitative analyses of annexin V-positive and PI-negative cell populations demonstrate that the proportion of apoptotic cells…

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  Figure 7. Immunohistochemistry of EGR1 in pulmonary arteries of patients with Down syndrome and PAH. (A) Representative images of immunohistochemistry of EGR1 in the lung tissues of healthy control (upper panels) and patients with DS and PAH (lower panels). Scale bar: 200 μm. (B) Representative images of quantitative evaluation for EGR1 positive pulmonary ECs. The parentages of 3,3′-Diaminobenzidine (DAB) positive nuclei of pulmonary arterial ECs were counted. The black arrowheads indicate EGR1-positive nuclei and the white arrowhead indicates EGR1-negative nucleus. Scale bar: 50 μm. (C) EGR1-positive cell ratio of pulmonary ECs in DS is significantly higher than that of healthy control. Seven arteries and nine arteries were analyzed in healthy control and in DS, respectively. Data are presented as mean ± SEM. Data were analyzed by an unpaired Student’s t test. ***P < 0.001.