Research Fellow University of Michigan Ann Arbor, Michigan, United States
Introduction:: Introduction: Thoracic aortic aneurysm (TAA) is a life-threatening vascular disease frequently associated with an underlying genetic cause. Despite significant efforts, there have been no effective therapeutic advances to prevent TAA formation or reverse its progression due to the inadequate understanding of human aneurysm pathogenesis. End-stage aneurysmal tissue with compensatory changes accumulating over many years is not ideal to investigate the early molecular causes of TAA formation indicating the need to develop better human aortic aneurysm models. To fill the current gap in knowledge about the molecular events leading to human aneurysm formation, we develope a novel human TAA model in an animal host by combining human induced pluripotent stem cells (hiPSCs) and bioengineered vascular grafts (BVGs). This platform enables us to capture features of LDS root aneurysm formation associated with a pathogenic TGBFR1 variant. We present evidence that structural collagen defects likely caused by reduced collagen modifying enzyme activity contribute to the weaker mechanical performance of TGBFR1A230T BVGs in vivo leading to BVG dilation.
Materials and Methods:: Materials and Methods: To model Loeys-Dietz Syndrome (LDS)-associated root aneurysms using functional three-dimensional constructs, we compared PatientA230T/+ hiPSC derived cardiovascular progenitor lineage smooth muscle cells (CPC-SMCs) with patient corrected cells (iWT+/+). CPC-SMCs from different conditions were seeded on biodegradable polyglycolic acid meshes and cultured for 8 weeks in vitro to generate arteriole-scale BVGs (Fig. A). Mechanical studies including burst pressure and suture retention were performed on the BVGs prior to implantation. An in vivo human aneurysm model was established by implanting control and experimental BVGs into the common carotid arteries of the same nude rat. Each matched BVG pair was closely monitored using ultrasonography. The BVGs were harvested at week 8 post-operation and spatial transcriptomics profiling was performed to investigate the molecular and cellular defects. The findings were validated by quantitative and qualitative collagen assays in BVGs and patient aortic root samples.
Results, Conclusions, and Discussions:: Results and Discussion: We generated BVGs from PatientA230T/+ and iWT+/+ CPC-SMCs, both formed opaque BVGs similar to native vessels (Fig. B). In vitro mechanical tests revealed that the iWT+/+ BVGs displayed a significantly higher burst pressure (1,414±122 mmHg) compared with PatientA230T/+ BVGs (758±86 mmHg) with a nearly 80% increase (Fig. C). To assess their in vivo performance, PatientA230T/+ and iWT+/+ BVGs were implanted into the common carotid arteries of the same Foxn1RNU nude rats. Strikingly, harvested PatientA230T/+ BVGs showed a uniform dilation pattern along the vessel at week 8 (Fig. D). Comparison of ultrasound cross-section images also confirmed this clear dilation by week 8 post-operation (Fig. E). The quantification of inner dimeter change overtime displayed a gradual increase in PatientA230T/+ BVGs (a nearly 35% increase in inner diameter) (Fig. F). Meanwhile, the iWT+/+ BVGs displayed a small increase in inner diameter by week 8. Molecular analysis using spatial transcriptomics profiling revealed that PatientA230T/+ BVGs were enriched in injury- and repair-related gene sets including Interferon signaling and DNA repair (Fig. G). ECM organization, collagen formation and modifying enzymes, and elastic fiber formation gene sets showed enrichment in iWT+/+ BVGs consistent with their stronger mechanical performance in vivo. Histological analysis and protein assays indicated quantitative and qualitative collagen defects in PatientA230T/+ BVGs including decreased collagen hydroxylation, essential for collagen fiber stability (Fig. H-J). Collagen fibers were disorganized in patient root samples with lower amounts of thick collagen compared with the control aortic roots (Fig. K-L) confirming our model’s ability to capture key features of root aneurysm samples.
Conclusions: We established a functional human aneurysm model using BVGs to mimic human TAA formation, which represents the first human aortic aneurysm model in an animal host. Our findings highlight reduced collagen modifying enzyme activity as a contributor to the dilation phenotype in PatientA230T/+ BVGs informing the aortic root aneurysm pathogenesis in LDS patients. Our study offers an avenue to develop novel therapeutic approaches for the prevention and treatment of aortic aneurysm and dissection as an alternative to surgical repair.
Acknowledgements (Optional): : Acknowledgement: This study was supported by the American Heart Association Postdoctoral Fellowship (830938) and National Institutes of Health (R01-HL151776).