Impaired vascular smooth muscle cell force-generating capacity and phenotypic deregulation in Marfan Syndrome mice.

Mechanisms whereby fibrillin-1 mutations determine thoracic aorta aneurysms/dissections (TAAD) in Marfan Syndrome (MFS) are unclear. Most aortic aneurysms evolve from mechanosignaling deregulation, converging to impaired vascular smooth muscle cell (VSMC) force-generating capacity accompanied by synthetic phenotype switch. However, little is known on VSMC mechanoresponses in MFS pathophysiology. Here, we investigated traction force-generating capacity in aortic VSMC cultured from 3-month old mg∆lpn MFS mice, together with morpho-functional and proteomic data. Cultured MFS-VSMC depicted marked phenotype changes vs. wild-type (WT) VSMC, with overexpressed cell proliferation markers but either lower (calponin-1) or higher (SM alpha-actin and SM22) differentiation marker expression. In parallel, the increased cell area and its complex non-fusiform shape suggested possible transition towards a mesenchymal-like phenotype, confirmed through several markers (e.g. N-cadherin, Slug). MFS-VSMC proteomic profile diverged from that of WT-VSMC particularly regarding lower expression of actin cytoskeleton-regulatory proteins. Accordingly, MFS-VSMC displayed lower traction force-generating capacity and impaired contractile moment at physiological substrate stiffness, and markedly attenuated traction force responses to enhanced substrate rigidity. Such impaired mechanoresponses correlated with decreased number, altered morphology and delocalization of focal adhesions, as well as disorganized actin stress fiber network vs. WT-VSMC. In VSMC cultured from 6-month-old mice, phenotype changes were attenuated and both WT-VSMC and MFS-VSMC generated less traction force, presumably involving VSMC aging, but without evident senescence. In summary, MFS-VSMC display impaired force-generating capacity accompanying a mesenchymal-like phenotype switch connected to impaired cytoskeleton/focal adhesion organization. Thus, MFS-associated TAAD involves mechanoresponse impairment common to other TAAD types, but through distinct mechanisms.

Effect of M1-M2 Polarization on the Motility and Traction Stresses of Primary Human Macrophages.

Macrophages become polarized by cues in their environment and this polarization causes a functional change in their behavior. Two main subsets of polarized macrophages have been described. M1, or "classically activated" macrophages, are pro-inflammatory and M2, or "alternatively activated" macrophages, are anti-inflammatory. In this study, we investigated the motility and force generation of primary human macrophages polarized down the M1 and M2 pathways using chemokinesis assays and traction force microscopy on polyacrylamide gels. We found that M1 macrophages are significantly less motile and M2 macrophages are significantly more motile than unactivated M0 macrophages. We also showed that M1 macrophages generate significantly less force than M0 or M2 macrophages. We further found that M0 and M2, but not M1, macrophage force generation is dependent on ROCK signaling, as identified using the chemical inhibitor Y27632. Finally, using the chemical inhibitor blebbistatin, we found that myosin contraction is required for force generation by M0, M1, and M2 macrophages. This study represents the first investigation of the changes in the mechanical motility mechanisms used by macrophages after polarization.

Numeric reconstruction of 2D cellular actomyosin network from substrate displacement

Introduction: One of the fundamental structural elements of the cell is the cytoskeleton. Along with myosin, actin microfilaments are responsible for cellular contractions, and their organization may be related to pathological changes in myocardial tissue. Due to the complexity of factors involved, numerical modeling of the cytoskeleton has the potential to contribute to a better understanding of mechanical cues in cellular activities. In this work, a systematic method was developed for the reconstruction of an actomyosin topology based on the displacement exerted by the cell on a flexible substrate. It is an inverse problem which could be considered a phenomenological approach to traction force microscopy (TFM). Methods An actomyosin distribution was found with a topology optimization method (TOM), varying the material density and angle of contraction of each element of the actomyosin domain. The routine was implemented with a linear material model for the bidimensional actomyosin elements and tridimensional substrate. The topology generated minimizes the nodal displacement squared differences between the generated topology and experimental displacement fields obtained by TFM. The structure resulting from TOM was compared to the actin structures observed experimentally with a GFP-attached actin marker. Results The optimized topology reproduced the main features of the experimental actin and its squared displacement differences were 11.24 µm2, 27.5% of the sum of experimental squared nodal displacements (40.87 µm2). Conclusion This approach extends the literature with a model for the actomyosin structure capable of distributing anisotropic material freely, allowing heterogeneous contraction over the cell extension.