Mechanical Regulation of Apoptosis in the Cardiovascular System.

Apoptosis is a highly conserved physiological process of programmed cell death which is critical for proper organism development, tissue maintenance, and overall organism homeostasis. Proper regulation of cell removal is crucial, as both excessive and reduced apoptotic rates can lead to the onset of a variety of diseases. Apoptosis can be induced in cells in response to biochemical, electrical, and mechanical stimuli. Here, we review literature on specific mechanical stimuli that regulate apoptosis and the current understanding of how mechanotransduction plays a role in apoptotic signaling. We focus on how insufficient or excessive mechanical forces may induce apoptosis in the cardiovascular system and thus contribute to cardiovascular disease. Although studies have demonstrated that a broad range of mechanical stimuli initiate and/or potentiate apoptosis, they are predominantly correlative, and no mechanisms have been established. In this review, we attempt to establish a unifying mechanism for how various mechanical stimuli initiate a single cellular response, i.e. apoptosis. We hypothesize that the cytoskeleton plays a central role in this process as it does in determining myriad cell behaviors in response to mechanical inputs. We also describe potential approaches of using mechanomedicines to treat various diseases by altering apoptotic rates in specific cells. The goal of this review is to summarize the current state of the mechanobiology field and suggest potential avenues where future research can explore.

Heterogeneity Profoundly Alters Emergent Stress Fields in Constrained Multicellular Systems.

Stress fields emerging from the transfer of forces between cells within multicellular systems are increasingly being recognized as major determinants of cell fate. Current analytical and numerical models used for the calculation of stresses within cell monolayers assume homogeneous contractile and mechanical cellular properties; however, cell behavior varies by region within constrained tissues. Here, we show the impact of heterogeneous cell properties on resulting stress fields that guide cell phenotype and apoptosis. Using circular micropatterns, we measured biophysical metrics associated with cell mechanical stresses. We then computed cell-layer stress distributions using finite element contraction models and monolayer stress microscopy. In agreement with previous studies, cell spread area, alignment, and traction forces increase, whereas apoptotic activity decreases, from the center of cell layers to the edge. The distribution of these metrics clearly indicates low cell stress in central regions and high cell stress at the periphery of the patterns. However, the opposite trend is predicted by computational models when homogeneous contractile and mechanical properties are assumed. In our model, utilizing heterogeneous cell-layer contractility and elastic moduli values based on experimentally measured biophysical parameters, we calculate low cell stress in central areas and high anisotropic stresses in peripheral regions, consistent with the biometrics. These results clearly demonstrate that common assumptions of uniformity in cell contractility and stiffness break down in postconfluence confined multicellular systems. This work highlights the importance of incorporating regional variations in cell mechanical properties when estimating emergent stress fields from collective cell behavior.

Reproducible in vitro model for dystrophic calcification of cardiac valvular interstitial cells: insights into the mechanisms of calcific aortic valvular disease.

Calcific aortic valvular disease (CAVD) is the most prevalent valvular pathology in the United States. Development of a pharmacologic agent to slow, halt, or reverse calcification has proven to be unsuccessful as still much remains unknown about the mechanisms of disease initiation. Although in vitro models of some features of CAVD exist, their utility is limited by the inconsistency of the size and time course of the calcified cell aggregates. In this study, we introduce and verify a highly reproducible in vitro method for studying dystrophic calcification of cardiac valvular interstitial cells, considered to be a key mechanism of clinical CAVD. By utilizing micro-contact printing, we were able to consistently reproduce cell aggregation, myofibroblastic markers, programmed cell death, and calcium accumulation within aggregates of 50-400 µm in diameter on substrates with moduli from 9.6 to 76.8 kPa. This method is highly repeatable, with 70% of aggregates staining positive for Alizarin Red S after one week in culture. Dense mineralized calcium-positive nanoparticles were found within the valvular interstitial cell aggregates as shown by scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS). The area of micro-contact printed aggregates staining positive for caspase 3/7 activity increased from 5.9 ± 0.9% to 12.6 ± 4.5% over one week in culture. Z-VAD-FMK reduced aggregates staining positive for Alizarin Red S by 60%. The state of cell stress is hypothesized to play a role in the disease progression; traction force microscopy indicates high substrate stresses along the aggregate periphery which can be modulated by altering the size of the aggregates and the modulus of the substrate. Micro-contact printing is advantageous over the currently used in vitro model as it allows the independent study of how cytokines, substrate modulus, and pharmacologic agents affect calcification. This controlled method for aggregate creation has the potential to be used as an in vitro assay for the screening of promising therapeutics to mitigate CAVD.

Mechanoregulation of aortic valvular interstitial cell life and death.

Valvular interstitial cells (VICs) are the major cell type within aortic valve leaflets. VICs are able to exhibit a spectrum of phenotype characteristics including those of fibroblasts, smooth muscle cells, and myofibroblasts. VICs are responsible for valve maintenance and repair, yet excessive persistence of the myofibroblast phenotype is implicated in a number of valve diseases, including calcific aortic valve disease and fibrosis. Despite the prevalence of these diseases, the stimuli regulating the transition to the activated myofibroblast state and reversal to quiescent fibroblast and/or induction of apoptosis are not fully understood. The purpose of this article is to review in vitro studies that have contributed to the current understanding of mechanical regulation of VIC phenotype and fate. In particular, we have focused on studies utilizing advanced in vitro systems that allow modulation and measurement of cell tension and cell-generated forces in two-dimensional and three-dimensional cultures. In addition, we discuss the importance of cell tension in phenotype modulation and how cytoskeletal tension may contribute to aggregation and calcification. Future directions of pharmaceutical development aimed at reducing VIC cytoskeletal tension are also highlighted.

Nonlinear strain stiffening is not sufficient to explain how far cells can feel on fibrous protein gels.

Recent observations suggest that cells on fibrous extracellular matrix materials sense mechanical signals over much larger distances than they do on linearly elastic synthetic materials. In this work, we systematically investigate the distance fibroblasts can sense a rigid boundary through fibrous gels by quantifying the spread areas of human lung fibroblasts and 3T3 fibroblasts cultured on sloped collagen and fibrin gels. The cell areas gradually decrease as gel thickness increases from 0 to 150 µm, with characteristic sensing distances of >65 µm below fibrin and collagen gels, and spreading affected on gels as thick as 150 µm. These results demonstrate that fibroblasts sense deeper into collagen and fibrin gels than they do into polyacrylamide gels, with the latter exhibiting characteristic sensing distances of <5 µm. We apply finite-element analysis to explore the role of strain stiffening, a characteristic mechanical property of collagen and fibrin that is not observed in polyacrylamide, in facilitating mechanosensing over long distances. Our analysis shows that the effective stiffness of both linear and nonlinear materials sharply increases once the thickness is reduced below 5 µm, with only a slight enhancement in sensitivity to depth for the nonlinear material at very low thickness and high applied traction. Multiscale simulations with a simplified geometry predict changes in fiber alignment deep into the gel and a large increase in effective stiffness with a decrease in substrate thickness that is not predicted by nonlinear elasticity. These results suggest that the observed cell-spreading response to gel thickness is not explained by the nonlinear strain-stiffening behavior of the material alone and is likely due to the fibrous nature of the proteins.

Eccentric rheometry for viscoelastic characterization of small, soft, anisotropic, and irregularly shaped biopolymer gels and tissue biopsies.

Quantification of the physical properties of tissue biopsies and cell-remodeled hydrogels is critical for understanding tissue development and pathophysiological tissue remodeling. However, due to the low modulus, small size, irregular shape, and anisotropy of samples from these materials, accurate viscoelastic characterization using standard rheometric methods is problematic. The goal of this work is to utilize image analysis to extend rotational rheometry to these samples. In this method, the sample is offset to increase the torque generated; a custom clear glass geometry, right angle prism, and camera are used to determine the exact shape and location of the sample relative to the axis of rotation for calculation of the sample shear modulus, G'. Values of G' for standard polydimethylsiloxane gels tested in centered and eccentric configurations were not statistically different (respectively 137 ± 37 kPa and 126 ± 8 kPa, p = 0.58), indicating accuracy of the method. Additionally, G' values from circular and irregularly shaped collagen gels yielded equivalent results (31 ± 1.8 Pa and 31 ± 5.1 Pa, p = 0.29). A blood clot and a lipid plaque sample recovered from human patients (G' ~ 4 kPa) were successfully tested with this method demonstrating applicability to clinical diagnostics.