Patient-specific iPSC-derived endothelial cells reveal aberrant p38 MAPK signaling in atypical hemolytic uremic syndrome

Abstract

Atypical hemolytic uremic syndrome (aHUS) is a rare disease associated with high morbidity and mortality. Existing evidence suggests that the central pathogenesis to aHUS might be endothelial cell damage. Nevertheless, the role of endothelial cell alterations in aHUS has not been well characterized and the underlying mechanisms remain unclear. Utilizing an induced pluripotent stem cell-derived endothelial cell (iPSC-EC) model, we showed that anti-complement factor H autoantibody-associated aHUS patient-specific iPSC-ECs exhibited an intrinsic defect in endothelial functions. Stimulation using aHUS serums exacerbated endothelial dysfunctions, leading to cell apoptosis in iPSC-ECs. Importantly, we identified p38 as a novel signaling pathway contributing to endothelial dysfunctions in aHUS. These results illustrate that iPSC-ECs can be a reliable model to recapitulate EC pathological features, thus providing a unique platform for gaining mechanistic insights into EC injury in aHUS. Our findings highlight that the p38 MAPK signaling pathway can be a therapeutic target for treatment of aHUS.

Keywords: aHUS; anti-CFH autoantibodies; endothelial dysfunction; iPSC-ECs; p38 MAPK.

Graphical abstract

Figure 1

Generation and characterization of patient-specific aHUS iPSCs (A) Typical morphology of skin fibroblasts derived from three pediatric patients with anti-CFH autoantibody-associated aHUS and two healthy control subjects (shown as CON). Scale bars, 100 μm. (B) Typical morphology of iPSC colony derived from control and aHUS skin fibroblasts. Scale bars, 100 μm. (C) Karyotype analysis of control and aHUS iPSCs. (D) Alkaline phosphatase staining of control and aHUS iPSCs. Scale bars, 50 μm. (E and F) Pluripotent staining of control and aHUS iPSCs using OCT4 (green), SOX2 (red), NANOG (green), and SSEA4 (red). DAPI indicates nuclear staining (blue). Scale bars, 100 μm.

Figure 2

Generation and characterization of endothelial cells derived from patient-specific aHUS iPSCs (A) Schematic demonstration of iPSC-derived endothelial cell (EC) differentiation. CHIR, CHIR-99021; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; EPCs, endothelial progenitor cells. (B) Typical morphology of control and aHUS iPSC-ECs. Scale bars, 100 μm. (C) CD144 (green) staining of control and aHUS iPSC-ECs. DAPI indicates nuclear staining (blue). Scale bars, 50 μm. (D) Dil-LDL (red) staining of control and aHUS iPSC-ECs. DAPI indicates nuclear staining (blue). Scale bars, 100 μm.

Figure 3

Endothelial dysfunction phenotype in aHUS iPSC-ECs (A) Representative images of wound closure in control and aHUS iPSC-ECs assessed at 12 h. Scale bars, 100 μm. (B) Scatterplot to compare the percentage of wound closure between control and aHUS iPSC-ECs. ^∗∗∗∗p < 0.0001; n = 8 independent experiments. (C) Representative images of tube formation on Matrigel in control and aHUS iPSC-ECs assessed at 6 h. Scale bars, 100 μm. (D and E) Scatterplots to compare the number of tube-like structures (D) or tube length (E) between control and aHUS iPSC-ECs. ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; n = 8 independent experiments. (F) Scatterplot to compare the cell proliferation between control and aHUS iPSC-ECs using absorbance reads at 450 nm. ^∗∗∗p < 0.001; n = 8 independent experiments.

Figure 4

aHUS serum stimulation exacerbates EC function and induces EC loss (A) Representative images of wound closure in control and aHUS iPSC-ECs under different conditions, including basal, normal or aHUS-diseased serum alone (NS or DS), and normal or aHUS-diseased serum plus compstatin (NS Comp or DS Comp). Scale bars, 100 μm. (B) Scatterplot to compare the percentage of wound closure between different groups. ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001, compared with basal; ^#p < 0.05, ^###p < 0.001, and ^####p < 0.0001, compared with serum alone. n = 4–8 independent experiments. (C) Representative images of tube formation on Matrigel in control and aHUS iPSC-ECs under basal, normal serum, and aHUS serum stimulation conditions. Scale bars, 100 μm. (D–F) Scatterplots to compare the number of tube-like structures (D), tube length (E), or cell viability (F) between different groups. ^∗∗∗∗p < 0.0001, compared with basal; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, and ^####p < 0.0001, compared with serum alone. n = 4–8 independent experiments.

Figure 5

RNA-seq analysis of aHUS iPSC-ECs (A) Heatmap demonstrating the differential gene expression between control and aHUS iPSC-ECs. (B and C) Top 20 genes showing the greatest differences in expression between control and aHUS iPSC-ECs, respectively. (D and E) Gene ontology (GO) analysis using upregulated (D) and downregulated (E) genes. (F) Bar graphs to compare the FPKM values of PECAM1, BMP4, HOXA3, and CDH5 between control and aHUS iPSC-ECs. (G) Bar graphs to compare the mRNA expression of PECAM1, BMP4, HOXA3, and CDH5 between control and aHUS iPSC-ECs by qPCR. ^∗∗∗∗p < 0.0001.

Figure 6

Impaired p38 MAPK signaling in aHUS iPSC-ECs (A–C) Upper panels: western blot analysis of p-ERK (A), p-JNK (B), or p-p38 (C) expression in control and aHUS iPSC-ECs. Lower panel: scatterplot to compare the p-ERK (A), p-JNK (B), or p-p38 (C) expression between control and aHUS iPSC-ECs. ^∗∗∗∗p < 0.0001; n = 3 independent EC differentiation experiments. (D–F) Upper panels: western blot analysis of p-p38 expression in aHUS iPSC-ECs derived from patient #1 (D), patient #2 (E), and patient #3 (F) upon basal or anisomycin (ANS) treatment, respectively. Lower panels: scatterplots to compare the p-p38 expression between basal and ANS-treated aHUS iPSC-ECs. ^∗∗p < 0.01 and ^∗∗∗∗p < 0.0001; n = 3 independent experiments.

Figure 7

Activation of p38 by anisomycin or MKK6 overexpression rescues endothelial phenotype in aHUS iPSC-ECs (A) Representative images of wound closure in basal control iPSC-ECs, basal or anisomycin (ANS)-treated aHUS iPSC-ECs. Scale bars, 100 μm. (B) Scatterplot to compare the percentage of wound closure between different groups. ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001, compared with basal control iPSC-ECs; ^####p < 0.0001, compared with basal aHUS iPSC-ECs. n = 4 independent experiments. (C) Representative images of tube formation on Matrigel in basal control iPSC-ECs, basal, or ANS-treated aHUS iPSC-ECs. Scale bars, 100 μm. (D and E) Scatterplots to compare the number of tube-like structures (D) or tube length (E) between different groups. ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001, compared with basal control iPSC-ECs; ^####p < 0.0001, compared with basal aHUS iPSC-ECs. n = 4 independent experiments. (F) Representative images of wound closure in aHUS iPSC-ECs overexpressing vector only (Vector OE) or MKK6 (MKK6 OE). Scale bars, 100 μm. (G) Scatterplot to compare the percentage of wound closure between different groups. ^∗p < 0.05; n = 4 independent experiments. (H) Representative images of tube formation on Matrigel in aHUS iPSC-ECs overexpressing vector only or MKK6. Scale bars, 100 μm. (I and J) Scatterplots to compare the number of tube-like structures (I) or tube length (J) between different groups. ^∗p < 0.05; n = 4 independent experiments.