Disease Model of Atherosclerosis Using Werner Syndrome Patient-Derived Induced Pluripotent Stem Cells Reveals Excessive Inflammatory Response Against Ox-LDL

Atherosclerosis is a major cause of various fatal diseases, such as myocardial infarction, stroke, and ischemic heart failure worldwide. The risk factors of atherosclerosis are well known, including hypertension, diabetes mellitus, dyslipidemia, smoking and obesity. Growing evidence also suggests that aging is the strongest determinant of stroke, which is less common before 40 years old (Soler and Ruiz 2010). Werner syndrome (WS) is a rare human inherited disorder characterized by the appearance of premature aging followed by early onset of aging-associated diseases including atherosclerosis. Myocardial infarction and cancer are being the most common causes of death in patients with WS (Oshima et al. 1993). However, despite being one of the most common causes of death, the etiology of atherosclerosis in WS patients have not been well documented yet. Therefore, the aim of the study was to assess in vitro model of atherosclerosis using WS patient-derived induced pluripotent stem cells (iPSC) to better understand the etiology and pathogenesis of atherosclerosis in WS patients.

Atherosclerosis is a chronic inflammatory disease in which vascular endothelial cells (VEC), vascular smooth muscle cells (VSMC) and macrophages (Mφ) play interactive roles in the progression of the disease. To obtain these cells in vitro, we induced VEC, VSMC and Mφ from the progenitor cell populations, KDR⁺/CD34⁺/TRA-1-60^-, KDR⁺/CD34^-/TRA-1-60^- and CD43⁺/CD34⁺ cells respectively, derived from iPSCs by the iPS-sac method we developed previously (Takayama et al. 2008, 2010). We found by co-culture with C3H10T1/2 mouse mesenchymal stromal cell line, KDR⁺/CD34⁺ and KDR⁺/CD34^- cell populations were able to efficiently induce VEC and VSMC respectively on day 17. Especially, VEC induction efficiency was ~ 4 and ~ 21 fold as compared with the previously established method with collagen IV and laminin-411 respectively (Ohta et al. 2016; Sone et al. 2007; Taura et al. 2009). Furthermore, to induce Mφ, iPS-sac-like structures in culture were maintained for two weeks to get CD43⁺ hematopoietic cells. CD43⁺ cells were then cultured for an additional 7 days with a cytokine cocktail on C3H10T1/2 feeder layer. After 3 weeks of initiating culture, ~ 54% of CD11b⁺/CD33⁺ Mφ-like cells were obtained.

To examine the responses of the induced VEC- and Mφ-like cells in atherosclerotic conditions, we applied oxidized low-density lipoprotein (Ox-LDL) at the concentration of 10, 50 and 100 μg/mL, and performed qRT-PCR analysis. It is well known that inflammatory response is triggered with the uptake of LDL both in VEC and Mφ, thereby contribute to local inflammation, cell necrosis, apoptosis, VSMC proliferation and fibrosis. Accordingly, in Ox-LDL treated iPSC-derived VEC and Mφ, mRNA expression of IL-1β, IL-6 and TNF-α were increased in a dose dependent manner.

A critical event in the early stages of atherosclerosis is the internalization of lipid particles by VEC, and then, these lipids particles are oxidized to form Ox-LDL in the endothelium, leading to an increase in inflammatory cell adhesion molecules (ICAM-1 and VCAM-1) on the endothelium. Subsequently, monocytes are then recruited and differentiated into Mφ and start to uptake Ox-LDL particles ultimately forming foam-cells (Wu et al. 2017). Next, we were enthusiastic to compare the effect of Ox-LDL for inflammatory response in WS iPSC-derived VEC and Mφ with healthy iPSC-derived VEC and Mφ. Although no differences were observed in the induction efficiency, cell proliferation and mean fluorescent intensity of Ox-LDL uptake between WS- and healthy-iPSC-derived VEC and Mφ, qRT-PCR analysis revealed that, VEC derived from WS-iPSC shows much higher mRNA expression of cell adhesion molecule gene (ICAM-1; 2.5 fold and VCAM-1; 3.6 fold), inflammatory cytokine gene (IL-6; 12.5 fold and TGF-β; 4.3 fold) and endothelial dysfunction related gene (ET-1; 5.9 fold and PAI-1; 25.2 fold) compared with healthy-iPSC-derived VEC.

Consequently, our results suggest that the higher risk of atherosclerosis in WS patients might be due to excessive inflammation response against Ox-LDL in WS-iPSC-derived cells, which is independent of other risk factors frequently observed in WS, such as diabetes and hyperlipidemia, and might be implicated to understand the pathogenesis and therapeutic strategy against WS and general atherosclerosis.