Smooth muscle cells (SMCs) play a significant role in the pathogenesis of atherosclerosis. 2D cultures elucidated valuable information about the interaction between SMCs and extracellular matrix (ECM) components. However, 3D constructs better represent the native vascular environment. Furthermore, a limited number of studies addressed the effect of ECM stiffness on SMCs phenotype. We investigated the effect of stiffness of different ECM substrates by modulating their concentrations, including the effect on morphology, proliferation, expression of the contractile protein α-smooth muscle actin (α-SMA) and deposition of collagen type I (Col I) and collagen type III (Col III) proteins. At low concentrations of Col I gels and Col I gels supplemented with 10% fibronectin (Fn), SMCs exhibited non-elongated, 'hill-and-valley' shape and large mean cellular area, indicating a hypertrophic morphology, characteristic of the synthetic phenotype. However, with increasing concentration, mean cellular area and proliferation relative to cells cultured in 2D dropped. Whole protein secretion into the culture media and deposition of Col I and Col III generally decreased with increasing stiffness. Moreover, percentage of α-SMA+ SMCs decreased with increasing gel concentration, pointing to a shift towards the synthetic phenotype. Supplementing Col I with 10% Laminin (Ln) maintained higher cellular area and aspect ratio at all gel concentrations and did not change α-SMA expression significantly, compared to Col I alone or Col I + Fn. Overall, these results demonstrate that ECM components and stiffness could provide the tools to modulate the phenotype and function of SMCs in vitro, which allows for the control of properties of engineered tissues.
As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 5′ and 3′-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5′ or 3′ extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.