Effect Of Substrate Stiffness Research Articles

Substrate stiffness promotes vascular smooth muscle cell calcification by reducing the levels of nuclear actin monomers

BackgroundVascular calcification (VC) is a prevalent independent risk factor for adverse cardiovascular events and is associated with diabetes, hypertension, chronic kidney disease, and atherosclerosis. However, the mechanisms regulating the osteogenic differentiation of vascular smooth muscle cells (VSMC) are not fully understood. MethodsUsing hydrogels of tuneable stiffness and lysyl oxidase-mediated stiffening of human saphenous vein ex vivo, we investigated the role of substrate stiffness in the regulation of VSMC calcification. ResultsWe demonstrate that increased substrate stiffness enhances VSMC osteogenic differentiation and VSMC calcification. We show that the effects of substrate stiffness are mediated via a reduction in the level of actin monomer within the nucleus. We show that in cells interacting with soft substrate, elevated levels of nuclear actin monomer repress osteogenic differentiation and calcification by repressing YAP-mediated activation of both TEA Domain transcription factor (TEAD) and RUNX Family Transcription factor 2 (RUNX2). ConclusionThis work highlights for the first time the role of nuclear actin in mediating substrate stiffness-dependent VSMC calcification and the dual role of YAP-TEAD and YAP-RUNX2 transcriptional complexes.

The Effect of Substrate Stiffness on Elastic Force Transmission in the Epithelial Monolayers over Short Timescales

PurposeThe importance of mechanical forces and microenvironment in guiding cellular behavior has been widely accepted. Together with the extracellular matrix (ECM), epithelial cells form a highly connected mechanical system subjected to various mechanical cues from their environment, such as ECM stiffness, and tensile and compressive forces. ECM stiffness has been linked to many pathologies, including tumor formation. However, our understanding of the effect of ECM stiffness and its heterogeneities on rapid force transduction in multicellular systems has not been fully addressed.MethodsWe used experimental and computational methods. Epithelial cells were cultured on elastic hydrogels with fluorescent nanoparticles. Single cells were moved by a micromanipulator, and epithelium and substrate deformation were recorded. We developed a computational model to replicate our experiments and quantify the force distribution in the epithelium. Our model further enabled simulations with local stiffness gradients.ResultsWe found that substrate stiffness affects the force transduction and the cellular deformation following an external force. Also, our results indicate that the heterogeneities, e.g., gradients, in the stiffness can substantially influence the strain redistribution in the cell monolayers. Furthermore, we found that the cells’ apico-basal elasticity provides a level of mechanical isolation between the apical cell–cell junctions and the basal focal adhesions.ConclusionsOur simulation results show that increased ECM stiffness, e.g., due to a tumor, can mechanically isolate cells and modulate rapid mechanical signaling between cells over distances. Furthermore, the developed model has the potential to facilitate future studies on the interactions between epithelial monolayers and elastic substrates.

Stiffness sensing by smooth muscle cells: Continuum mechanics modeling of the acto-myosin role

Aortic smooth muscle cells (SMCs) play a vital role in maintaining homeostasis in the aorta by sensing and responding to mechanical stimuli. However, the mechanisms that underlie the ability of SMCs to sense and respond to stiffness change in their environment are still partially unclear. In this study, we focus on the role of acto-myosin contractility in stiffness sensing and introduce a novel continuum mechanics approach based on the principles of thermal strains. Each stress fiber satisfies a universal stress-strain relationship driven by a Young's modulus, a contraction coefficient scaling the fictitious thermal strain, a maximum contraction stress and a softening parameter describing the sliding effects between actin and myosin filaments. To account for the inherent variability of cellular responses, large populations of SMCs are modeled with the finite-element method, each cell having a random number and a random arrangement of stress fibers. Moreover, the level of myosin activation in each stress fiber satisfies a Weibull probability density function. Model predictions are compared to traction force measurements on different SMC lineages. It is demonstrated that the model not only predicts well the effects of substrate stiffness on cellular traction, but it can also successfully approximate the statistical variations of cellular tractions induced by intercellular variability. Finally, stresses in the nuclear envelope and in the nucleus are computed with the model, showing that the variations of cytoskeletal forces induced by substrate stiffness directly induce deformations of the nucleus which can potentially alter gene expression. The predictability of the model combined to its relative simplicity are promising assets for further investigation of stiffness sensing in 3D environments. Eventually, this could contribute to decipher the effects of mechanosensitivity impairment, which are known to be at the root of aortic aneurysms.

Stiffness-Tunable Hydrogel-Sandwich Culture Modulates the YAP-Mediated Mechanoresponse in Induced-Pluripotent Stem Cell Embryoid Bodies and Augments Cardiomyocyte Differentiation.

Microenvironmental factors, including substrate stiffness, regulate stem cell behavior and differentiation. However, the effects of substrate stiffness on the behavior of induced pluripotent stem cell- (iPSC) derived embryoid bodies (EB) remain unclear. To investigate the effects of mechanical cues on iPSC-EB differentiation, we developed a three-dimensional hydrogel-sandwich culture (HGSC) system that controls the microenvironment surrounding iPSC-EBs using a stiffness-tunable polyacrylamide hydrogel assembly. Mouse iPSC-EBs were seeded between upper and lower polyacrylamide hydrogels of differing stiffness (Young's modulus [E'] = 54.3 ± 7.1kPa [hard], 28.1 ± 2.3kPa [moderate], and 5.1 ± 0.1kPa [soft]) and cultured for 2 days. HGSC induced stiffness-dependent activation of the yes-associated protein (YAP) mechanotransducer and actin cytoskeleton rearrangement in the iPSC-EBs. Moreover, moderate-stiffness HGSC specifically upregulated the mRNA and protein expression of ectoderm and mesoderm lineage differentiation markers in iPSC-EBs via YAP-mediated mechanotransduction. Pretreatment of mouse iPSC-EBs with moderate-stiffness HGSC promoted cardiomyocyte differentiation and structural maturation of myofibrils. Our proposed HGSC system provides a viable platform for investigating the role of mechanical cues on the pluripotency and differentiation of iPSCs that could be beneficial for research into tissue regeneration and engineering. This article is protected by copyright. All rights reserved.

Substrate stiffness in nerve cells

Recently, substrate stiffness has been involved in the physiology and pathology of the nervous system. However, the role and function of substrate stiffness remain unclear. Here, we review known effects of substrate stiffness on nerve cell morphology and function in the central and peripheral nervous systems and their involvement in pathology. We hope this review will clarify the research status of substrate stiffness in nerve cells and neurological disorder.

A Case for Material Stiffness as a Design Parameter in Encapsulated Islet Transplantation.

Diabetes is a disease that plagues over 463 million people globally. Approximately 40 million of these patients have type 1 diabetes mellitus (T1DM), and the global incidence is increasing by up to 5% per year. T1DM is where the body's immune system attacks the pancreas, specifically the pancreatic beta cells, with antibodies to prevent insulin production. Although current treatments such as exogenous insulin injections have been successful, exorbitant insulin costs and meticulous administration present the need for alternative long-term solutions to glucose dysregulation caused by diabetes. Encapsulated islet transplantation (EIT) is a tissue-engineered solution to diabetes. Donor islets are encapsulated in a semipermeable hydrogel, allowing the diffusion of oxygen, glucose, and insulin but preventing leukocyte infiltration and antibody access to the transplanted cells. Although successful in small animal models, EIT is still far from commercial use owing to necessary long-term systemic immunosuppressants and consistent immune rejection. Most published research has focused on tailoring the characteristics of the capsule material to promote clinical viability. However, most studies have been limited in scope to biochemical changes. Current mechanobiology studies on the effect of substrate stiffness on the function of leukocytes, especially macrophages-primary foreign body response (FBR) orchestrators, show promise in tailoring a favorable response to tissue-engineered therapies such as EIT. In this review, we explore strategies to improve the clinical viability of EIT. A brief overview of the immune system, the FBR, and current biochemical approaches will be elucidated throughout this exploration. Furthermore, an argument for using substrate stiffness as a capsule design parameter to increase EIT efficacy and clinical viability will be posed.

Discreteness of cell–surface contacts affects spatio-temporal dynamics, adhesion, and proliferation of mouse embryonic stem cells

The self-renewal and lineage-specific differentiation of stem cells are regulated by interactions with their microenvironments, called stem cell niche. Stem cells receive both biochemical and biophysical cues from their niche, which leads to the activation of signaling pathways, resulting in the modulation of gene expressions to guide their fate. Most of previous studies are focused on the effect of substrate stiffness using hydrogels with different Young’s moduli, and information is lacking on the effect of the discreteness of cell–substrate contacts on stem cells. Using mouse pluripotent, embryonic stem cells (mESCs) as the model system for early development, we quantitatively investigated the migration, dynamic deformation, and adhesion of mESCs on sparse and dense gelatin nanofibers deposited on glass surfaces, with a continuous layer of gelatin coated on glass substrates as the control. After confirming the maintenance of pluripotency on all the surfaces throughout the experiments, the centroid trajectories were monitored using timelapse imaging. The mean square displacement analysis indicated that both the diffusion coefficient and exponent were largest on sparse nanofibers, while the diffusion coefficient of mESCs on dense nanofibers was comparable to that on the control. Moreover, power spectral analysis of the shape deformation in the Fourier mode indicated that mESCs predominantly underwent elliptic deformation (mode 2), with the largest energy dissipation on sparse nanofibers. These data suggest that mESCs can deform and move on sparse nanofibers owing to the discrete cell–surface contact points. Intriguingly, using a self-developed technique based on laser-induced shock waves, a distinctly larger critical pressure was required to detach cells from nanofibers than from continuous gelatin. This finding suggests that the continuous but weak cell-substrate contacts suppress the deformation-driven mESC migration. As one of the key biological functions of stem cells, the proliferation rate of mESCs on these surfaces was determined. Although the observed difference was not statistically significant, the highest proliferation rate was observed on nanofibers, suggesting that the discreteness of cell–surface contacts can be used to regulate not only spatio-temporal dynamics but also the biological function of pluripotent stem cells.

Soft substrates promote direct chemical reprogramming of fibroblasts into neurons

Fibroblasts can be directly reprogrammed via a combination of small molecules to generate induced neurons (iNs), bypassing intermediate stages. This method holds great promise for regenerative medicine; however, it remains inefficient. Recently, studies have suggested that physical cues may improve the direct reprogramming of fibroblasts into neurons, but the underlying mechanisms remain to be further explored, and the physical factors reported to date do not exhibit the full properties of the extracellular matrix (ECM). Previous in vitro studies mainly used rigid polystyrene dishes, while one of the characteristics of the native in-vivo environment of neurons is the soft nature of brain ECM. The reported stiffness of brain tissue is very soft ranging between 100 Pa and 3 kPa, and the effect of substrate stiffness on direct neuronal reprogramming has not been explored. Here, we show for the first time that soft substrates substantially improved the production efficiency and quality of iNs, without needing to co-culture with glial cells during reprogramming, producing more glutamatergic neurons with electrophysiological functions in a shorter time. Transcriptome sequencing indicated that soft substrates might promote glutamatergic neuron reprogramming through integrins, actin cytoskeleton, Hippo signalling pathway, and regulation of mesenchymal-to-epithelial transition, and competing endogenous RNA network analysis provided new targets for neuronal reprogramming. We demonstrated that soft substrates may promote neuronal reprogramming by inhibiting microRNA-615-3p-targeting integrin subunit beta 4. Our findings can aid the development of regenerative therapies and help improve our understanding of neuronal reprogramming. Statement of significanceFirst, we have shown that low stiffness promotes direct reprogramming on the basis of small molecule combinations. To the best of our knowledge, this is the first report on this type of method, which may greatly promote the progress of neural reprogramming.Second, we found that microRNA (miR)-615-3p may interact with integrin subunit beta 4 (ITGB4), and the soft substrates may promote neural reprogramming by inhibiting miR-615-3p targeting ITGB4. We are the first to report on this mechanism.Our findings will provide more functional neurons for subsequent basic and clinical research in neurological regenerative medicine, and will help to improve the overall understanding of neural reprogramming. This work also provides new ideas for the design of medical biomaterials for nerve regeneration.

Effects of substrate stiffness on the viscoelasticity and migration of prostate cancer cells examined by atomic force microscopy.

The stiffness of the extracellular matrix of tumour cells plays a key role in tumour cell metastasis. However, it is unclear how mechanical properties regulate the cellular response to the environmental matrix. In this study, atomic force microscopy (AFM) and laser confocal imaging were used to qualitatively evaluate the relationship between substrate stiffness and migration of prostate cancer (PCa) cells. Cells cultured on stiff substrates (35 kPa) undergone several interesting phenomena compared to those on soft substrates (3 kPa). Here, the stimulation generated by the stiff substrates triggered the F-actin skeleton to bundle its filaments, increasing the polarity index of the external contour of PCa cells. Analysis of AFM force–distance curves indicated that the elasticity of the cells cultured on 35 kPa substrates increased while the viscosity decreased. Wound-healing experiments showed that PCa cells cultured on 35 kPa substrates have higher migration potential. These phenomena suggested that the mechanical properties may be correlated with the migration of PCa cells. After actin depolymerisation, the elasticity of the PCa cells decreased while the viscosity increased, and the migration ability was correspondingly decreased. In conclusion, this study clearly demonstrated the relationship between substrate stiffness and the mechanical properties of cells in prostate tumour metastasis, providing a basis for understanding the changes in the biomechanical properties at a single-cell level.

Effect of substrate stiffness on the mechanical properties of cervical cancer cells

BackgroundCervical cancer microenvironment is involved in the regulation of the behavior, morphology, and mechanical properties of the cervical cancer cells. Integrins expressed on the cell membrane combine with the factors of the microenvironment to determine cervical cancer cells’ properties. The mechanical properties of integrin-extracellular matrix (ECM) interactions are important for the mechanotransduction of cervical cancer cells. However, the quantified study on the adhesion force and binding probabilities between collagen and integrins on cervical cancer cells grown on different stiffness substrates have not been reported. MethodsPolyacrylamide (PA) gel was used as substrate to mimic the mechanical microenvironment of cancer cells. ImageJ software was used to measure the perimeter and area of the cells. SiHa cells were stained with FITC phalloidine to observe the cytoskeleton. Atomic force microscopy (AFM) was used to measure the cell mechanical properties. ResultThe perimeters of SiHa cells grown on different stiffness substrates were 63.4 ± 1.3, 102.8 ± 4.0, and 152.6 ± 4.1 μm, for soft, intermediate, and stiff substrates, respectively. These areas were 277.2 ± 13.3, 428.9 ± 26.0, and 1166.0 ± 63.2 μm2, for soft, intermediate, and stiff substrates, respectively. The Young's modulus of SiHa cells grown on stiff substrates (3.0 ± 0.02 kPa) was higher compared with those on soft substrates (0.6 ± 0.01 kPa) or intermediate substrates (bimodal distribution, 1.36 and 1.67 kPa). The adhesion force between the functionalized probe and SiHa cells grown on glass (55.65 ± 0.78 pN) was significantly greater than that on stiff (47.03 ± 0.85 pN), intermediate (34.07 ± 0.58 pN) and soft (27.94 ± 0.47 pN) substrates. The binding probabilities of the collagen-integrin of the four rigid substrates were significantly differed. ConclusionThe changes in substrate stiffness can obviously regulate SiHa cells' mechanical properties, such as the Young's modulus. The adhesion force and binding probabilities of SiHa cells both increased with increasing substrate strength.

Effect of substrate stiffness on friction in collective cell migration

In collective cell migration, the motion results from forces produced by each cell and transmitted to the neighboring cells and to the substrate. Because inertia is negligible and the migration occurs over long time scales, the cell layer exhibits viscous behavior, where force and motion are connected by an apparent friction that results from the breaking and forming of adhesive bonds at the cell–cell and cell–substrate interfaces. Most theoretical models for collective migration include an apparent friction to connect force and motion, with many models making predictions that depend on the ratio of cell–cell and cell–substrate friction. However, little is known about factors that affect friction, leaving predictions of many theoretical models untested. Here, we considered how substrate stiffness and the number of adhesions affected friction at the cell–substrate interface. The experimental data were interpreted through prior theoretical models, which led to the same conclusion, that increased substrate stiffness increased the number of cell–substrate adhesions and caused increased cell–substrate friction. In turn, the friction affected the collective migration by altering the curvature at the edge of the cell layer. By revealing underlying factors affecting friction and demonstrating how friction perturbs the collective migration, this work provides experimental evidence supporting prior theoretical models and motivates the study of other ways to alter the collective migration by changing friction.

Effects of substrate stiffness on astrocyte migration: Piezo1 regulation in neuroinflammatory condition

Neuroinflammation is critical to the pathology of acute and chronic diseases and cause detrimental effects on the central nervous system. Astrocytes play prominent roles in neuroinflammation and interestingly, they respond to changes in their external biomechanical environment. It is well known that mechanosensitive ion channels respond to changes to their extracellular environment, especially to changes in stiffness. Our previous studies have found that mechanosensitive ion channel Piezo1was significantly upregulated in cultured astrocytes (C8-S cell) when treated with lipopolysaccharide (LPS), a treatment to induce inflammation.

Substrate stiffness-dependent regulatory volume decrease and calcium signaling in chondrocytes.

The pericellular matrix stiffness is strongly associated with its biochemical and structural changes during the aging and osteoarthritis progress of articular cartilage. However, how substrate stiffness modulates the chondrocyte regulatory volume decrease (RVD) and calcium signaling in chondrocytes remains unknown. This study aims to investigate the effects of substrate stiffness on the chondrocyte RVD and calcium signaling by recapitulating the physiologically relevant substrate stiffness. Our results showed that substrate stiffness induces completely different dynamical deformations between the cell swelling and recovering progresses. Chondrocytes swell faster on the soft substrate but recovers slower than the stiff substrate during the RVD response induced by the hypo-osmotic challenge. We found that stiff substrate enhances the cytosolic Ca oscillation of chondrocytes in the iso-osmotic medium. Furthermore, chondrocytes exhibit a distinctive cytosolic Ca oscillation during the RVD response. Soft substrate significantly improves the Ca oscillation in the cell swelling process whereas stiff substrate enhances the cytosolic Ca oscillation in the cell recovering process. Our work also suggests that the TRPV4 channel is involved in the chondrocyte sensing substrate stiffness by mediating Ca signaling in a stiffness-dependent manner. This helps to understand a previously unidentified relationship between substrate stiffness and RVD response under the hypo-osmotic challenge. A better understanding of substrate stiffness regulating chondrocyte volume and calcium signaling will aid the development of novel cell-instructive biomaterial to restore cellular functions.

Effects of substrate stiffness on mast cell migration.

Mast cells (MCs) play important roles in multiple pathologies, including fibrosis; however, their behaviors in different extracellular matrix (ECM) environments have not been fully elucidated. Accordingly, in this study, the migration of MCs on substrates with different stiffnesses was investigated using time-lapse video microscopy. Our results showed that MCs could appear in round, spindle, and star-like shapes; spindle-shaped cells accounted for 80-90 % of the total observed cells. The migration speed of round cells was significantly lower than that of cells with other shapes. Interestingly, spindle-shaped MCs migrated in a jiggling and wiggling motion between protrusions. The persistence index of MC migration was slightly higher on stiffer substrates. Moreover, we found that there was an intermediate optimal stiffness at which the migration efficiency was the highest. These findings may help to improve our understanding of MC-induced pathologies and the roles of MC migration in the immune system.

Effect of substrate stiffness on human intestinal enteroids’ infectivity by enteroaggregative Escherichia coli

Human intestinal enteroids (HIE) models have contributed significantly to our understanding of diarrheal diseases and other intestinal infections, but their routine culture conditions fail to mimic the mechanical environment of the native intestinal wall. Because the mechanical characteristics of the intestine significantly alter how pathogens interact with the intestinal epithelium, we used different concentrations of polyethylene glycol (PEG) to generate soft (~2 kPa), medium (~10 kPa), and stiff (~100 kPa) hydrogel biomaterial scaffolds. The height of HIEs cultured in monolayers atop these hydrogels was 18 µm whereas HIEs grown on rigid tissue culture surfaces (with stiffness in the GPa range) were 10 µm. Substrate stiffness also influenced the amount of enteroaggregative E. coli (EAEC strain 042) adhered to the HIEs. We quantified a striking difference in adherence pattern; on the medium and soft gels, the bacteria formed clusters of > 100 and even > 1000 on both duodenal and jejunal HIEs (such as would be found in biofilms), but did not on glass slides and stiff hydrogels. All hydrogel cultured HIEs showed significant enrichment for gene and signaling pathways related to epithelial differentiation, cell junctions and adhesions, extracellular matrix, mucins, and cell signaling compared to the HIEs cultured on rigid tissue culture surfaces. Collectively, these results indicate that the HIE monolayers cultured on the hydrogels are primed for a robust engagement with their mechanical environment, and that the soft hydrogels promote the formation of larger EAEC aggregates, likely through an indirect differential effect on mucus. Statement of significanceEnteroids are a form of in vitro experimental mini-guts created from intestinal stem cells. Enteroids are usually cultured in 3D within Matrigel atop rigid glass or plastic substrates, which fail to mimic the native intestinal mechanical environment. Because intestinal mechanics significantly alter how pathogens interact with the intestinal epithelium, we grew human intestinal enteroids in 2D atop polyethylene glycol (PEG) hydrogel scaffolds that were soft, medium, or stiff. Compared with enteroids grown in 2D atop glass or plastic, the enteroids grown on hydrogels were taller and more enriched in mechanobiology-related gene signaling pathways. Additionally, enteroids on the softest hydrogels supported adhesion of large aggregates of enteroaggregative E. coli. Thus, this platform offers a more biomimetic model for studying enteric diseases.

Stiffness Regulates the Morphology, Adhesion, Proliferation, and Osteogenic Differentiation of Maxillary Schneiderian Sinus Membrane-Derived Stem Cells.

Recent studies, which aim to optimize maxillary sinus augmentation, have paid significant attention exploring osteogenic potential of maxillary Schneiderian sinus membrane-derived cells (MSSM-derived cells). However, it remains unclear that how MSSM-derived cells could respond to niche's biomechanical properties. Herein, this study investigated the possible effects of substrate stiffness on rMSSM-derived stem cell fate. Initially, rMSSM-derived stem cells with multiple differentiation potential were successfully obtained. We then fabricated polyacrylamide substrates with varied stiffness ranging from 13 to 68 kPa to modulate the mechanical environment of rMSSM-derived stem cells. A larger cell spreading area and increased proliferation of rMSSM-derived stem cells were found on the stiffer substrates. Similarly, cells became more adhesive as their stiffness increased. Furthermore, the higher stiffness facilitated osteogenic differentiation of rMSSM-derived stem cells. Overall, our results indicated that increase in stiffness could mediate behaviors of rMSSM-derived stem cells, which may serve as a guide in future research to design novel biomaterials for maxillary sinus augmentation.

Decreased Substrate Stiffness Promotes a Hypofibrotic Phenotype in Cardiac Fibroblasts.

A hypofibrotic phenotype has been observed in cardiac fibroblasts (CFs) isolated from a volume overload heart failure model, aortocaval fistula (ACF). This paradoxical phenotype results in decreased ECM synthesis despite increased TGF-β presence. Since ACF results in decreased tissue stiffness relative to control (sham) hearts, this study investigates whether the effects of substrate stiffness could account for the observed hypofibrotic phenotype in CFs isolated from ACF. CFs isolated from ACF and sham hearts were plated on polyacrylamide gels of a range of stiffness (2 kPa to 50 kPa). Markers related to cytoskeletal and fibrotic proteins were measured. Aspects of the hypofibrotic phenotype observed in ACF CFs were recapitulated by sham CFs on soft substrates. For instance, sham CFs on the softest gels compared to ACF CFs on the stiffest gels results in similar CTGF (0.80 vs. 0.76) and transgelin (0.44 vs. 0.57) mRNA expression. The changes due to stiffness may be explained by the observed decreased nuclear translocation of transcriptional regulators, MRTF-A and YAP. ACF CFs appear to have a mechanical memory of a softer environment, supported by a hypofibrotic phenotype overall compared to sham with less YAP detected in the nucleus, and less CTGF and transgelin on all stiffnesses.

Substrate Stiffness Regulates Cholesterol Efflux in Smooth Muscle Cells.

The infiltration and deposition of cholesterol in the arterial wall play an important role in the initiation and development of atherosclerosis. Smooth muscle cells (SMCs) are the major cell type in the intima. Upon exposure to cholesterol, SMCs may undergo a phenotype switching into foam cells. Meanwhile, the pathological processes of the blood vessel such as cholesterol deposition and calcification induce the changes in the substrate stiffness around SMCs. However, whether substrate stiffness affects the cholesterol accumulation in SMCs and the formation of foam cells is not well-understood. In this study, SMCs were cultured on the substrates with different stiffnesses ranging from 1 to 100 kPa and treated with cholesterol. We found that cholesterol accumulation in SMCs was higher on 1 and 100 kPa substrates than that on intermediate stiffness at 40 kPa; consistently, total cholesterol (TC) content on 1 and 100 kPa substrates was also higher. As a result, the accumulation of cholesterol increased the expression of macrophage marker CD68 and downregulated SMC contractile marker smooth muscle α-actin (ACTA2). Furthermore, the mRNA and protein expression level of cholesterol efflux gene ATP-binding cassette transporter A1 (ABCA1) was much higher on 40 kPa substrate. With the treatment of a liver X receptor (LXR) agonist GW3965, the expression of ABCA1 increased and cholesterol loading decreased, showing an additive effect with substrate stiffness. In contrast, inhibition of LXR decreased ABCA1 gene expression and increased cholesterol accumulation in SMCs. Consistently, when ABCA1 gene was knockdown, the cholesterol accumulation was increased in SMCs on all substrates with different stiffness. These results revealed that substrate stiffness played an important role on SMCs cholesterol accumulation by regulating the ABCA1 expression. Our findings on the effects of substrate stiffness on cholesterol efflux unravel a new mechanism of biophysical regulation of cholesterol metabolism and SMC phenotype, and provide a rational basis for the development of novel therapies.

Substrate Stiffness Affected the Inflammatory Response of SMCs

The pathogenesis of atherosclerosis is accompanied by chronic inflammation with changes in the stiffness of the coronary artery wall. Being the main component of the vascular media, the smooth muscle cells (SMCs) are crucial to maintain blood vessel function. SMCs are mechano-sensitive, which can rapidly adapt to the fluctuations in the microenvironment of the blood vessel, including the subtle changes of the vascular stiffness. However, how substrate stiffness influences the phenotype and inflammatory response of SMCs is not well understood. In this study, we investigated the effects of substrate stiffness on SMCs phenotype, inflammatory gene expression and the nuclear factorkappa B (NFκB) signaling pathway of vascular SMCs. From 1 kPa to 100 kPa, the SMCs cytoskeleton became more and more organized with the increase of the substrate stiffness, representing by the uniformed distribution of the stress fibers. SMCs cultured on both soft (1 kPa) and hard (100 kPa) substrate increased the expression of macrophage marker CD68 molecule (CD68) and Galectin 3 (LGALS3) and the inflammatory gene Interleukin-6 (IL-6) and Interleukin-1β (IL-1β) than those on 40 kPa substrate. Moreover, the protein expression level of phosphorylated nuclear factor kappa B inhibitor (p-IκB) was higher on either soft (1 kPa) or hard (100 kPa) substrate. In consistent, the dephosphorylated IκB showed a higher expression level on the substrate stiffness of 40 kPa. These results suggested that substrate stiffness played an important role in SMCs cell morphology, phenotype and inflammatory response by affecting NFκB signaling pathway.

Probing coordinated co-culture cancer related motility through differential micro-compartmentalized elastic substrates

Cell development and behavior are driven by internal genetic programming, but the external microenvironment is increasingly recognized as a significant factor in cell differentiation, migration, and in the case of cancer, metastatic progression. Yet it remains unclear how the microenvironment influences cell processes, especially when examining cell motility. One factor that affects cell motility is cell mechanics, which is known to be related to substrate stiffness. Examining how cells interact with each other in response to mechanically differential substrates would allow an increased understanding of their coordinated cell motility. In order to probe the effect of substrate stiffness on tumor related cells in greater detail, we created hard–soft–hard (HSH) polydimethylsiloxane (PDMS) substrates with alternating regions of different stiffness (200 and 800 kPa). We then cultured WI-38 fibroblasts and A549 epithelial cells to probe their motile response to the substrates. We found that when the 2 cell types were exposed simultaneously to the same substrate, fibroblasts moved at an increased speed over epithelial cells. Furthermore, the HSH substrate allowed us to physically guide and separate the different cell types based on their relative motile speed. We believe that this method and results will be important in a diversity of areas including mechanical microenvironment, cell motility, and cancer biology.