Intrinsic Mechanics of Human Stem Cell Derived Aortic Smooth Muscle Cells Support a Developmental Basis for Aneurysm Localization in Marfan Syndrome

Marfan Syndrome (MFS), a connective tissue disorder caused by a mutation in the fibrillin-1 gene, occurs in approximately 1 in 5,000 people worldwide. As an important constituent of the extracellular matrix, mutated fibrillin-1 in Marfan Syndrome leads to aortic medial degeneration, aneurysm, and dissection. TGF{beta} in the matrix, which is controlled by fibrillin-1, is known to cause pathological effects in smooth muscle cells (SMCs) within the aortic wall during MFS. TGF{beta} as well as other cytokines have been shown to impact neural crest derived SMCs differently than mesodermal derived SMCs. Furthermore, outcomes of variable cytokine responsiveness of neural crest SMCs are compounded by genetically imposed changes to neural crest SMC integrin distributions in MFS. Thus, it has been hypothesized that neural crest derived SMCs, which give rise to ascending aortic SMCs, are intrinsically mechanically susceptible to aneurysm formation in MFS. This hypothesis has been linked to the clinical observation of aneurysm formation preferentially occurring in the ascending versus descending aorta in MFS. We aim to test the hypothesis that aortic smooth muscle cells (ASMCs) have intrinsic mechanobiological properties which cause cell weakening in Marfan Syndrome. Human induced pluripotent stem cells (hiPSC) from Marfan patients and healthy volunteers were differentiated into either ascending- or descending-ASMCs via their respective developmental lineages, and cultured to either an early (6 days) or late (30 days) stage of post-differentiation maturation. Mass spectrometry-based proteomics of early-stage iPSC-ASMCs revealed an array of depleted proteins unique to MFS ascending-SMCs that were associated with cell mechanics and aortic aneurysm. Targeted examination of the proteomics dataset revealed intracellular proteins (ACTA2, CNN1, TAGLN) were significantly depleted in MFS ascending-ASMCs. The intrinsic, matrix-independent, hiPSC-ASMC stiffness quantified by atomic force microscopy (AFM) revealed that MFS ascending-ASMCs, but not descending-ASMCs, were significantly less stiff than healthy, at the late cell-maturation stage (p<0.0005). Late-stage ascending- and descending-ASMCs also showed clear functional impairments via calcium flux in MFS. AFM revealed a similar mechanical phenotype in early-stage ASMCs, with MFS ascending-ASMCs, but not descending-ASMCs, being significantly less stiff than healthy (p<0.005). In summary, this study supports an emerging hypothesis of ontogenetic predisposition for aneurysm susceptibility in Marfan Syndrome based on locally altered mechanobiology of developmental origin-specific ASMC subtypes. This may lead to new cell-targeted approaches for treating aortic aneurysm in patients with MFS.