Stiffness of Extracellular Matrix Components Modulates the Phenotype of Human Smooth Muscle Cells in Vitro and Allows for the Control of Properties of Engineered Tissues

Abstract

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.

Collagen content and extracellular matrix cause cytoskeletal remodelling in pancreatic fibroblasts

“…AFM nanoscale characterization demonstrated that gels consisted of collagen fibres exhibiting the characteristic D-band periodicity and had random orientation ( figure 6). As the collagen concentration increased, the gel density, stiffness and collagen fibre diameter were significantly increased, confirming previous research demonstrating that alterations in collagen concentration are translated into changes in ECM stiffness [28,40]. Concerning fibre diameters, we found a range of 50-300 nm, similar to previous studies performed in high concentrated collagen gels [25] and within the range of fibre diameter of native tissues [28].…”

“…Furthermore, we developed 3D collagen models to study cell invasion in a more physiologically relevant approach [40]. In our 3D experiments, we formed tumour cell spheroids that were embedded in collagen gels of different concentration.…”

“…Given our results from the 2D experiments, we sought to find out how the metastatic potential of FBs and CAFs was affected by ECM stiffness in a 3D set-up that better mimics real ECM and tumour setting [40] as it is evident that the tissue microenvironment regulates cancer cell motility and invasion [41]. Our experimental approach involved the formation of cell spheroids embedded in 3D gels made by purified type I collagen, with tunable stiffness.…”

Vascular smooth muscle cell durotaxis depends on extracellular matrix composition

“…A loss of laminin and an increase in fibronectin surrounding smooth muscle cells has been observed during the progression of neointima formation in vascular disease, suggesting these proteins may play an important role in regulation of cell phenotype in disease (39). Additionally, increasing stiffness has been shown to drive smooth muscle cells toward the synthetic, migratory phenotype observed in atherosclerosis (40,41), This evidence that VSMC phenotype is modulated by both matrix stiffness and composition, coupled with our previous observations of matrix typedependent responses to stiffness by VSMCs, led us to hypothesize that the durotactic migratory behavior of VSMC s on substrates with mechanical gradients may also be differentially modulated by fibronectin and laminin.…”

Vascular smooth muscle cell contractile function and mechanotransduction

“…In addition to altered actomyosin activity, matrix stiffness also potentially influences VSMC differentiation. VSMC phenotype is regulated by numerous environmental cues, including the ECM [92] . Atherosclerotic plaques display reduced levels of elastin and collagen-I accumulation [93] .…”