(343a) Substrate Mechanics And Rhoa Regulate Smooth Muscle Cell Phenotype In A 3-D Extracellular Matrix Analog

In order to engineer functional tissue replacements, it is increasingly clear that a variety of inputs must be provided to cells with precise spatial and temporal control to direct tissue development. These inputs include not only soluble (e.g., growth factors, mitogens, etc.) and insoluble (e.g., extracellular matrix (ECM)) biochemical cues, but also mechanical cues to dictate cell fate. This is particularly true for vascular smooth muscle, a tissue that is normally subjected to cyclic mechanical strain in vivo and that can experience significant changes in passive mechanical properties as a result of numerous pathologies. However, while the effects of substrate mechanics on cells in 2-D cultures have been well characterized, the impact of ECM mechanics on cell function in 3-D remains unclear.

Addressing this question in 3-D requires a material in which substrate mechanical properties can be tuned independently from adhesion ligand density. These parameters cannot be decoupled using native biopolymers (e.g., collagen, fibrin, Matrigel). In this study, we utilized a novel biosynthetic hybrid hydrogel based on a copolymer of poly(ethylene glycol) (PEG) and fibrinogen to examine how the static, intrinsic mechanical properties of this synthetic ECM analog affect the phenotype of smooth muscle cells (SMCs) in 3-D. The hybrid features of this copolymer system permit the formation of PEG-based synthetic cross-links via UV photopolymerization while retaining the biologic functionality of fibrinogen, including its ability to support cell adhesion and be remodeled by cell-secreted proteases. For this study, we added increasing amounts of acrylated PEG while maintaining the fibrinogen concentration constant at 6.3 mg/ml, thereby tuning the compressive mechanical properties of our 3-D substrates across a range from 448 to 5408 Pa. Scanning electron microscopy revealed that the microstructure of these hydrogels changed simultaneously with changes in crosslink density.

Exploiting the mechanical fidelity of this system, we found via confocal microscopy that the presence of F-actin bundles increased with matrix stiffness in 3-D, consistent with our previous findings in 2-D. However, there was a notable absence of the classic focal adhesions characteristic of SMCs on rigid 2-D substrates. Interestingly, when SMCs were virally transduced with an active RhoA mutant, F-actin bundling markedly increased in the softest matrices, and vinculin began to associate into sites resembling focal adhesions irrespective of ECM mechanics. Phenotypically, normal SMCs proliferated only marginally in 3-D, with seemingly no dependence on the surrounding matrix compliance. However, when induced to overexpress active RhoA, SMC proliferation in 3-D surprisingly decreased in all stiffness conditions, most significantly so in the stiffest matrix tested. Our interpretation of these novel data, along with the results of ongoing studies to dissect the relationship between ECM mechanical properties, RhoA activity, and the expression of SMC differentiation markers in 3-D, will be presented.