iPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia

Abstract

Defects in the low-density lipoprotein receptor (LDLR) are associated with familial hypercholesterolemia (FH), manifested by atherosclerosis and cardiovascular disease. LDLR deficiency in hepatocytes leads to elevated blood cholesterol levels, which damage vascular cells, especially endothelial cells, through oxidative stress and inflammation. However, the distinctions between endothelial cells from individuals with normal and defective LDLR are not yet fully understood. In this study, we obtained and examined endothelial derivatives of induced pluripotent stem cells (iPSCs) generated previously from conditionally healthy donors and compound heterozygous FH patients carrying pathogenic LDLR alleles. In normal iPSC-derived endothelial cells (iPSC-ECs), we detected the LDLR protein predominantly in its mature form, whereas iPSC-ECs from FH patients have reduced levels of mature LDLR and show abolished low-density lipoprotein uptake. RNA-seq of mutant LDLR iPSC-ECs revealed a unique transcriptome profile with downregulated genes related to monocarboxylic acid transport, exocytosis, and cell adhesion, whereas upregulated signaling pathways were involved in cell secretion and leukocyte activation. Overall, these findings suggest that LDLR defects increase the susceptibility of endothelial cells to inflammation and oxidative stress. In combination with elevated extrinsic cholesterol levels, this may result in accelerated endothelial dysfunction, contributing to early progression of atherosclerosis and other cardiovascular pathologies associated with FH.

Keywords: LDLR; directed differentiation; endothelial cells; familial hypercholesterolemia; patient-specific iPSCs.

Figure 1

Characteristics of endothelial derivatives obtained by differentiating iPSCs from patients with FH and a healthy donor. (a) VE-cadherin quantification of iPSC-derived ECs from a healthy donor (K7-4) and patients with FH (FH 1.3.1S and FH 3.2.8T). The experiment was performed in three biological and three technical replicates. (b) iPSC-ECs from a healthy donor and patients with FH are positively stained with antibodies against CD31 (red) and von Willebrand factor (green). Nuclei are stained with DAPI. The scale bar is 100 microns. Magnification 200×. (c) Capillary-like structures formed by endothelial derivatives of iPSCs from a healthy donor (EC K7-4) and patients with FH (FH EC 3.1S and FH EC 3.2.8T). The scale bar is 100 microns. Magnification 100×. (d–f) Quantification of angiogenic potential based on (d) total and (e) average capillary length, and (f) total number of branching points calculated in 10 random fields of view for triplicate.

Figure 2

iPSC-derived endothelium from patients with FH shows reduced LDL uptake capacity. (a) Representative images showing differences in the fluorescently labeled LDL uptake capacity (red signal) between endothelial derivatives obtained from iPSCs with normal (EC K7-4) and pathological (EC FH 1.3.1 and EC FH 3.2.8) LDLR allelic variants. Nuclei are stained with DAPI. The scale bar is 100 microns. Magnification 200×. The experiment was performed in triplicate. (b) Quantification of LDL uptake capacity for control and patient-specific IPSC-derived endothelial cells in 10 fields of view for each sample.

Figure 3

LDLR protein expression in iPSCs and their endothelial derivatives from patients with FH and a healthy donor. (a) Immunoblotting revealed a decreased level of mature LDLR in iPSCs and iPSC-ECs from patients with FH (FH 1.3.1 and FH 3.2.8) and an increased level of immature LDLR in iPSCs and iPSC-ECs from FH 1.3.1 compared to a healthy donor (K7-4). (b) Relative densitometric quantification of total LDL (mature and immature forms together) in iPSCs and iPSC-ES from patients with FH (FH 1.3.1 and FH 3.2.8) and a healthy donor (K7-4). The experiment was performed in triplicate. (c) Relative densitometric quantification of mature and immature LDL in iPSCs and iPSC-ES from patients with FH (FH 1.3.1 and FH 3.2.8) and a healthy donor (K7-4). The experiment was performed in triplicate.

Figure 4

Transcriptional profiling of the control (CTRL) and LDLR mutant (FH) iPSC-ECs. (a) PCA plot for two control (CTRL) and two LDLR mutant (FH) iPSC-EC cultures. (b) Volcano plot of 39 differentially expressed genes (DEGs) between CTRL and FH. FDR, false discovery rate; FC, fold change. The top five DEGs are shown. (c) Heat map of 39 DEGs. The color of each dot represents the gene expression in the sample. The brighter the red, the higher the expression; the brighter the blue, the lower the expression. (d) Histograms showing gene ontology of biological processes and reactome gene sets with upregulated (logFC  ≥  1) and downregulated (logFC  ≤ −1) expression in FH compared to CTRL. (e) Bioinformatic analysis of DEGs using STRING to identify functional interactions between deregulated proteins. Each node represents a protein, and each edge represents an interaction.