Fabrication and Characterization of Self-Assembled Tissue Rings using Patient Cells with Variants in Aneurysm Genes

Abstract

Aortic Abdominal Aneurysm (AAA) is a chronic degenerative disease of the arterial wall. The aortic vessel wall abnormally dilates due to multiple possible causes and may eventually rupture. It is characterized by several factors leading to the extreme dilation due to the degeneration of the vessel wall extracellular matrix (ECM). It is often asymptomatic making it difficult to diagnose before rupture. The etiology of AAA is complex and not yet fully understood. Hallmarks of the disease are; loss of elastin and vascular smooth muscle cells, influx of inflammatory cells which produce proteases, increased stiffness due to higher collagen content, disturbed ECM network organization and an eventual destruction of the ECM leading to the vessel rupture. These changes may be related to environmental factors in combination with a genetic susceptibility, or by major defects in genes involved in the ECM homeostasis. Family history and genetic conditions play an important role in aneurysms. In approximately 20 % of aneurysm patients there is a familial disease or a family history of aneurysms, and in these families a major genetic defect is to be expected. Aneurysm patients show pathogenic variants in less than 5 % of the screened patients however, variants of unknown clinical significance in aneurysm genes occur at a much higher frequency. To determine the effect of these variants in aneurysm genes, functional assays which display a change in the function of the gene product needs to be established. Tissue engineering, in recent years, have become an alternative approach to animal models in developing tissue repair/replacement grafts or disease models. For this study, a cell-derived self-assembly tissue ring culture method was chosen and optimized for the use of patient cells with different pathogenic variants in aneurysm genes. After optimizing the culture conditions, the ring formation and structural ECM characteristics were compared for patient and control cases. The kinetics of ring formation showed differences in behavior and speed for patient and control cell lines. Patient lines took longer to settle into the ring shape and contract to their final thickness. The incidence of ring failure was higher in patient cases. The final patient rings were significantly thinner and non-uniform, additionally they showed a significantly lower area fraction of collagen compared to the controls. The tissue rings could not be mechanically tested due to their fragility. However, it was possible to identify the structural differences between the patient and control cases which were expected due to the pathogenic variants in the aneurysm genes of these patients. With further improvements, this method could be a potential functional assay for revealing the effects of variants of unknown significance in aneurysm genes, waiting to be identified.