RNA-sequencing reveals significant differences in the inflammatory response of iPSC-derived endothelial cells from ACS patients and healthy controls

Abstract

Abstract Background Coronary artery disease (CAD) and cardiovascular diseases (CVD) in general still represent the leading cause of death worldwide. As a multifactorial disease entity, genetic predisposition and lifestyle strongly impact disease development and progression. Here, induced pluripotent stem cells (iPSC) are an ideal tool to differentiate (epi-)genetic from lifestyle disease risk modulation and identify the underlying molecular mechanisms. Aim This study aims to determine differences in iPSC-derived endothelial cell (iPSC-EC) characteristics and functionality between healthy donors and high-risk CAD patients. Methods IPSC from participants with similar lifestyle but different disease status (healthy >65 y/o, n=5, acute coronary syndrome (ACS) <65y/o, n=6) were differentiated into iPSC-EC. Cells were exposed to inflammatory stimulation with tumour necrosis factor alpha (TNF-a) followed by bulk RNA-seq. Monocyte (THP-1) recruitment was assessed under protective and atherogenic flow conditions. Mitotic age was calculated based on DNA-methylation via Hannum estimate (1) for each donor at the iPSC and iPSC-EC (passage 4) stage. Results ACS-iPSC-EC had significantly higher upregulation of E-selectin upon TNF-a stimulation on mRNA level (ACS: 532.1-fold±48.7 vs healthy: 322.3-fold±55.7 with p=0.02). Similarly, ICAM1 protein upregulation was higher in ACS-iPSC-EC compared to healthy iPSC-EC as determined by flow cytometry (ACS: 1.4-fold±0.01 vs healthy: 1.1-fold±0.0007; p<0.001). ACS-iPSC-EC recruited significantly more THP-1 per view field under protective (high shear stress, laminar) flow conditions (ACS: 78.4±20.7 vs healthy: 33.25±12.3 with p=0.04) compared to healthy iPSC-EC. While both groups recruited significantly more THP-1 under atherogenic than protective conditions (ACS: 302±38.9 with p=0.0055 vs healthy: 340±70.4 with p=0.0005), there was no difference between the donor groups (p=0.43). Analysis of bulk RNA-seq data showed significant differences in TNF-a-induced transcriptome changes. Gene set enrichment analysis identified differences in signalling pathways related to leukocyte and lymphocyte-mediated immunity, adaptive immune response, cytokine activity, and many more as shown in Figure 1 (p.adj ≤1e-10). IPSC of both groups had a negative calculated mitotic age (in years) which significantly increased after endothelial differentiation and four population doublings (ACS-iPSC: −14.1±0.7 vs ACS-EC: −0.1±1.1, p<0.0001 and healthy-iPSC: −13.7±0.8 vs healthy-EC: 2.5±1.5, p=0.0001). Conclusion The higher rate of basal THP-1 recruitment and upregulation of adhesion molecules on mRNA and protein level and the distinct transcriptome changes suggest intrinsic differences between patient and healthy iPSC-EC both in the basal state and in inflammatory response. Furthermore, this demonstrates iPSC as a viable in vitro model also for polygenetic diseases such as CAD. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): German Cardiac Society, Research Scholarship