RNA-sequencing reveals significant differences in the inflammatory response of iPSC-derived endothelial cells from ACS patients and healthy controls
Authors: Straessler E T , Kraenkel N , Landmesser U 
Published: October 2022

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 &gt;65 y/o, n=5, acute coronary syndrome (ACS) &lt;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&lt;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&lt;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