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.
The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box‐counting is used to infer a fractal dimension (D f) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin‐treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to D f, having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in D f and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. © 2016 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.
The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box-counting is used to infer a fractal dimension (D f ) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin-treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to D f , having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in D f and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. V C 2016The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.
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