Cytoskeletal and Integrin Dysregulation in Stem Cell-derived Neural Crest Smooth Muscle Cells Supports a Mechanobiological Basis for Marfan Syndrome Phenotype

Introduction: : Marfan Syndrome (MFS), an autosomal dominant disorder that affects more than 1 in 5,000 individuals, is associated with aortic medial degeneration and aneurysm, preferentially of the ascending aorta. While this putatively reflects regionally elevated hemodynamics stress, aortic smooth muscle cells (SMCs) also have regionally specific developmental origins, including the neural crest (NC) and paraxial mesoderm (PM), that can give rise to distinct phenotypes in ascending vs. descending aorta, respectively [Granata+, Nat Genet, 2017]. In this study, we used healthy and MFS-patient human induced pluripotent stem cell (hiPSC)-derived SMCs, Atomic Force Microscopy (AFM), and mass spectrometry-based proteomics to test the hypothesis that Neural Crest (NC)-SMCs in MFS have intrinsically abnormal mechanobiological properties that may underlie local susceptibility to degeneration in the ascending aorta.

Materials and

Methods: : Human iPSCs reprogrammed from dermal fibroblasts of Healthy and MFS donors (with heterozygous missense mutation in Fbn1 gene, and history of ascending aortic aneurysm/replacement) were differentiated to SMCs through NC or PM lineages [Cheung+, Nat Protocol, 2014]. Briefly, NC induction involved inhibition of TGFβ/Activin/NODAL signaling via ALK inhibition and FGF stimulation; PM induction involved a combination of PI3K inhibition, FGF and BMP activation. Smooth muscle cell terminal differentiation was then completed with TGFβ and PDGF. Terminally differentiated SMCs were matured in serum-containing media for 6 days (early-stage, S6) or 30 days (late-stage, S30). For AFM, a 0.1 N/m silicon nitride probe with pyramidal tip was used to perform a 4x4 array of indentations targeting a 5x5 µm area of the cell body, and analyzed using Hertz theory to obtain a median value of elastic modulus for each cell [Zhou+, Cardiovasc Res, 2017]. Calcium signaling was measured by Fluo-4AM fluorescent reporter. Western blot measured levels of contractile proteins (αSMA, SM22α) and osteopontin (OPN) after 48 hrs of TGF-β1 treatment. For proteomics, early-stage SMCs (n=4 x four conditions) underwent lysis, enzymatic digestion, and 16-plex Tandem-Mass-Tagged labeling before fractionation into 13 peptide groups. Orbitrap Fusion Lumos mass spectrometry and statistical analysis (R, LIMMA) identified differentially abundant proteins with a log2 fold-change cutoff of ±1 and adjusted p-value < 10⁻⁴. Statistical analysis was conducted in MATLAB using an unbalanced multiway-ANOVA with two levels (genotype and origin, grouped by maturity), to test for interaction effect between levels and pairwise comparisons, with significance at p< 0.05.

Results, Conclusions, and Discussions:: AFM measurements of late-stage MFS NC-SMCs revealed significantly lower cell stiffness than Healthy NC-SMCs (p < 0.0001) and PM-SMCs (p=0.0010), suggesting a mechanically weakened diseased state. Early-stage MFS NC-SMCs also showed reduced stiffness compared to Healthy, confirming an early onset of the mechanical phenotype (p=0.0020). In response to carbachol treatment (2.5 mM), late-stage MFS NC-SMCs exhibited faster-decaying and less oscillating calcium flux vs. Healthy NC-SMCs, indicating impaired calcium signaling. Western blot revealed a TGF-β1 dose-dependent (0, 2, 20 ng/mL) increase in key proteins (αSMA, SM22α, OPN) in MFS NC-SMCs, in contrast to a decreasing TGF-β1 dose-dependence in Healthy NC-SMCs. Proteomic analysis of early-stage MFS NC-SMCs revealed significant depletion of key mechanical proteins, including αSMA, CNN1, and MYH11, supporting the observed mechanical deficiencies. Further proteomic analysis identified 88 significantly depleted and 148 abundant proteins in MFS vs. Healthy NC-SMCs, with depleted proteins linked to gene ontologies for focal adhesion, cytoskeletal regulation, extracellular matrix organization, and aortic disease. In conclusion, our results suggest MFS hiPSC-derived NC-SMCs are less stiff and have impaired calcium signaling compared to Healthy. The dependence of SMC cytoskeletal proteins (αSMA, SM22α, OPN) on TGF-β1 treatment in MFS NC-SMCs was opposite to that in healthy NC-SMCs. Moreover, MFS NC-SMCs show significant depletion of proteins related to cytoskeletal stiffness and contraction, and these depleted proteins significantly associate with adhesion, mechanosensing, matrix organization, and aortic aneurysm disease. Our findings align with previous results showing medial tissue softening in MFS model mice [Lee+, Ann Biomed Eng, 2016] and impaired function of MFS NC-SMCs [Granata+, Nat Genet, 2017]. This study supports a hypothesized mechanosignaling pathway in which intrinsically depleted integrin mechanosensing in MFS NC-ASMC, leads to a reduction in actinin-mediated actin crosslinking, yielding a SMC disease phenotype with impaired mechanics and function that could predispose the ascending aorta to aneurysm in Marfan Syndrome.

Acknowledgements and/or References (Optional):: Funding from NIH F31 HL149271, NIH T32 HD075735, NIH U54 HG008098, NIH R01 HL115195, NIH S10 RR027609, NIH S10 OD025047, T32 HL007824-25, and support from Mount Sinai AFM/Confocal Core Facility, Azeloglu Lab (ISMMS), Rutgers University Proteomic Facility