IPSC-derived vascular smooth muscle cell model recapitulates Loeys-Dietz-Syndrome type V aortic phenotype

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Abstract Introduction Thoracic aortic aneurysm and dissection (TAAD) is a hallmark of Loeys-Dietz syndrome (LDS) and a prominent cause of morbidity and sudden death. Current drug therapies are only able to slow down but not stop or reverse aneurysm formation. TAAD pathomechanisms remain largely elusive, complicating the development of more effective treatments. Purpose The creation of a disease-relevant in vitro model, that recapitulates the disease phenotype, is incredibly valuable to gain better insights in the pathomechanisms underlying TAAD and to develop new treatments. Methods Validated induced pluripotent stem cells (iPSCs) of two male TAAD patients carrying the same pathogenic p.Asp263His variant in TGFB3 (LDS type V) and one male healthy individual were used in this study. One patient underwent a Bentall procedure at age 66 years because of insufficiency of a bicuspid aortic valve and TAA (50 mm, z-score= 5.2). The second patient presented with a type A dissection at age 31 years, also requiring Bentall surgery. The patients are distantly related, having a common ancestor allele, estimated to be arisen >400 years ago. We created and characterized iPSC-derived neural crest-vascular smooth muscle cell (NC-VSMC) lines from both LDS type V patients and the healthy control. Addition of a chemical thromboxane A2 receptor agonist initiated NC-VSMC contraction which was quantified by the change in impedance using the xCELLigence system and RTCA software. Additionally, the proliferation rate was quantified with the CyQuant Direct Cell Proliferation Assay Kit and inverted DMi8 fluorescence microscope (Leica). With an univariate ANOVA test, we assessed similarity of the variance between groups, after which the corresponding two-way t-test was performed. Results Within 30 minutes after thromboxane A2 receptor agonist addition, a maximal contracted state was reached, which was significantly lower for the first patient (2 clones) as compared to the control (2 cloness) (p=0.03). The time to get to this maximal contracted state was comparable between patient and control, while the surface area coverage over a duration of 1h stimulation was reduced for patient NC-VSMCs (p=0.04). Contraction of the NC-VSMC line (1 clone) from the second LDS patient did not significantly differ from the contraction profile of the control line. After one hour, U46619 was removed resulting in relaxation and full recovery for all NC-VSMCs. Proliferation rate of NC-VSMCs from all patient and control lines was around 1.5, with no significant difference between the groups (p=0.59). Conclusion We created an NC-VSMC model that recapitulates the LDS aortic phenotype with regards to reduced VSMC contractility. Although NC-VSMC lines created from the same individual showed extremely little variability, there is important variability between the lines of different patients which might be due to immaturity of the cells, environmental factors and the presence of genetic modifiers.