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.

Substrate Stiffness Affects Endothelial Cell Junctions and MAPK Signaling Pathway

Vascular homeostasis is critical for maintaining normal vascular structure and function. Aging is an irreversible trigger of vascular sclerosis, which causes structural and functional damage to blood vessels, leading to severe atherosclerosis. Endothelial cells (ECs) can respond to mechanical stimuli from the extracellular matrix, causing disruption of endothelial barrier function and activating signaling pathways to regulate cellular behavior under pathological conditions. In this paper, we investigated the effect of substrate stiffness on endothelial cell junctions, and the activation of mitogen-activated protein kinase (MAPK) signaling pathways. An in vitro stiffness model was established using polyacrylamide hydrogels of 1 kPa, 20 kPa and 100 kPa. By transcriptome analysis, we found that the cell-cell junction, cadherin binding, cytoskeleton and classical signaling pathways such as MAPK and Rho GTPase of endothelial cells were regulated by substrate stiffness. The expression of cell junction-related molecules TJP1, TJP2, JAM3 and JCAD was also found to be reduced at higher stiffness. The MAPK signaling pathway-related molecules MAP2K3, MAP2K7, MAP3K3, MAP3K6, MAPK3, MAPK7 were upregulated with increased stiffness. qRT-PCR analysis showed that the gene expression of JCAD was reduced with increased stiffness. Immunofluorescence staining of VE-cadherin indicated that the total fluorescence level of VE-cadherin decreased significantly with increased stiffness, and stiffness impaired the cell-cell junction with increased punctuation and discontinuity. Western blotting analysis confirmed that the protein expression ratio of pp38MAPK/p38MAPK increased with stiffness. Our research suggested that substrate stiffness played an important role in regulating endothelial cell integrity and MAPK signaling pathway.

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.

Substrate stiffness modulates bone marrow-derived macrophage polarization through NF-κB signaling pathway

The stiffness of the extracellular matrix(ECM)plays an important role in regulating the cellular programming.However,the mechanical characteristics of ECM affecting cell differentiation are still under investigated.Herein,we aimed to study the effect of ECM substrate stiffness on macrophage polarization.We prepared polyacrylamide hydrogels with different substrate stiffness,respectively.After the hydrogels were confirmed to have a good biocompatibility,the bone marrow-derived macrophages(BMMs)from mice were incubated on the hydrogels.With simulated by the low substrate stiffness,BMMs displayed an enhanced expression of CD86 on the cell surface and production of reactive oxygen species(ROS)in cells,and secreted more IL-1βand TNF-αin the supernatant.On the contrary,stressed by the medium stiffness,BMMs expressed more CD206,produced less ROS,and secreted more IL-4 and TGF-β.In vivo study by delivered the hydrogels subcutaneously in mice,more CD68+CD86+cells around the hydrogels with the low substrate stiffness were observed while more CD68+CD206+cells near by the middle stiffness hydrogels.In addition,the expressions of NIK,phosphorylated p65(pi-p65)and phosphorylated IκB(pi-IκB)were significantly increased after stimulation with low stiffness in BMMs.Taken together,these findings demonstrated that substrate stiffness could affect macrophages polarization.Low substrate stiffness promoted BMMs to shift to classically activated macrophages(M1)and the middle one to alternatively activated macrophages(M2),through modulating ROS-initiated NF-κB pathway.Therefore,we anticipated ECM-based substrate stiffness with immune modulation would be under consideration in the clinical applications if necessary.

Substrate stiffness differentially impacts autophagy of endothelial cells and smooth muscle cells

Stiffening of blood vessels is one of the most important characteristics in the process of many cardiovascular pathologies such as atherosclerosis,angiosteosis,and vascular aging.Increased stiffness of the vascular extracellular matrix drives artery pathology and alters phenotypes of vascular cell.Understanding how substrate stiffness impacts vascular cell behaviors is of great importance to the biomaterial design in tissue engineering,regenerative medicine,and medical devices.Here we report that changing substrate stiffness has a significant impact on the autophagy of vascular endothelial cells(VECs)and smooth muscle cells(VSMCs).Interestingly,our findings demonstrate that,with the increase of substrate stiffness,the autophagy level of VECs and VSMCs showed differential changes:endothelial autophagy levels reduced,leading to the reductions in a range of gene expression associated with endothelial function;while,autophagy levels of VSMCs increased,showing a transition from contractile to the synthetic phenotype.We further demonstrate that,by inhibiting cell autophagy,the expressions of endothelial functional gene were further reduced and the expression of VSMC calponin increased,suggesting an important role of autophagy in response of the cells to the challenge of microenvironment stiffness changing.Although the underlying mechanism requires further study,this work highlights the relationship of substrate stiffness,autophagy,and vascular cell behaviors,and enlightening the design principles of surface stiffness of biomaterials in cardiovascular practical applications.

An overview of substrate stiffness guided cellular response and its applications in tissue regeneration

Cell-matrix interactions play a critical role in tissue repair and regeneration.With gradual uncovering of substrate mechanical characteristics that can affect cell-matrix interactions,much progress has been made to unravel substrate stiffness-mediated cellular response as well as its underlying mechanisms.Yet,as a part of cell-matrix interaction biology,this field remains in its infancy,and the detailed molecular mechanisms are still elusive regarding scaffold-modulated tissue regeneration.This review provides an overview of recent progress in the area of the substrate stiffness-mediated cellular responses,including 1)the physical determination of substrate stiffness on cell fate and tissue development;2)the current exploited approaches to manipulate the stiffness of scaffolds;3)the progress of recent researches to reveal the role of substrate stiffness in cellular responses in some representative tissue-engineered regeneration varying from stiff tissue to soft tissue.This article aims to provide an up-to-date overview of cell mechanobiology research in substrate stiffness mediated cellular response and tissue regeneration with insightful information to facilitate interdisciplinary knowledge transfer and enable the establishment of prognostic markers for the design of suitable biomaterials.

Consistent apparent Young’s modulus of human embryonic stem cells and derived cell types stabilized by substrate stiffness regulation promotes lineage specificity maintenance

Background:Apparent Young’s modulus(AYM),which reflects the fundamental mechanical property of live cells measured by atomic force microscopy and is determined by substrate stiffness regulated cytoskeletal organization,has been investigated as potential indicators of cell fate in specific cell types.However,applying biophysical cues,such as modulating the substrate stiffness,to regulate AYM and thereby reflect and/or control stem cell lineage specificity for downstream applications,remains a primary challenge during in vitro stem cell expansion.Moreover,substrate stiffness could modulate cell heterogeneity in the single-cell stage and contribute to cell fate regulation,yet the indicative link between AYM and cell fate determination during in vitro dynamic cell expansion(from single-cell stage to multi-cell stage)has not been established.Results:Here,we show that the AYM of cells changed dynamically during passaging and proliferation on substrates with different stiffness.Moreover,the same change in substrate stiffness caused different patterns of AYM change in epithelial and mesenchymal cell types.Embryonic stem cells and their derived progenitor cells exhibited distinguishing AYM changes in response to different substrate stiffness that had significant effects on their maintenance of pluripotency and/or lineage-specific characteristics.On substrates that were too rigid or too soft,fluctuations in AYM occurred during cell passaging and proliferation that led to a loss in lineage specificity.On a substrate with‘optimal’stiffness(i.e.,3.5 kPa),the AYM was maintained at a constant level that was consistent with the parental cells during passaging and proliferation and led to preservation of lineage specificity.The effects of substrate stiffness on AYM and downstream cell fate were correlated with intracellular cytoskeletal organization and nuclear/cytoplasmic localization of YAP.Conclusions:In summary,this study suggests that optimal substrate stiffness regulated consistent AYM during passaging and proliferation reflects and contributes to hESCs and their derived progenitor cells lineage specificity maintenance,through the underlying mechanistic pathways of stiffness-induced cytoskeletal organization and the downstream YAP signaling.These findings highlighted the potential of AYM as an indicator to select suitable substrate stiffness for stem cell specificity maintenance during in vitro expansion for regenerative applications.

Effect of mechanical stretching and substrate stiffness on the morphology,cytoskeleton and nuclear shape of corneal endothelial cells

Due to the limited capacity of corneal endothelial cells(CECs)division,corneal endothelial diseases have become a great challenge.The cornea is subjected to various mechanical stimuli in vivo,which may have a positive or negative influence.Thus,it is significant to gain an insight into the mechanism of mechanobiology of CECs for seeking more possible treatment.The purpose of this study was to determine the impacts of mechanical stretch and substrate stiffness on the morphology and fundamental cell behavior of CECs.Rabbit corneal endothelial cells(RCECs)were subjected to a 5%mechanical stretch or cultured on substrates of different stiffness.The impacts of mechanical stimulus on cell area,aspect ratio,circularity,cell density,nuclear shape,cytoskeleton,and cell viability were investigated.The expressions of the corneal endothelium-related markers ZO-1 and Na^(+)/K^(+) ATPase were also evaluated by confocal immunofluorescence microscopy in the stiffness group.Our results suggested that mechanical stretch promoted the rearrangement of the cytoskeleton while decreasing the cell circularity,nuclear area,and cell density as well as cell viability.RCECs cultured on 10 kPa substrates,which was close to the physiological stiffness of rabbit Descemet's membrane(DM),showed better cell morphology,more stable actin cytoskeleton assembly,and more robust expression of the functional marker compared with other softer or stiffer substrates.In summary,mechanical stretch and substrate stiffness have profound influences on the morphology and function of CECs,which may have implications for the understanding and possible treatment of corneal endothelial diseases.

Soft Substrate Induces Endothelial Cell Inflammation and Disrupts Endothelium Integrity

Atherosclerosis (AS) is the main cause of death and disability all over the world. A lot of efforts have been devoted to treat AS, among which tissue engineering blood vessel materials, including artificial blood vessels, stents and vascular patches, have brought hope to ameliorate the symptoms in AS patients. However, there remains a large percentage of implantation failure due to the incompatibility of the material with the body. AS is a multi-factor related disease, and chronic inflammation is a major event that involves with its pathogenesis and development. Since previous studies suggested that the stiffness of the blood vessel might affect the inflammatory conditions, in this paper, we investigate the mechanism of how substrate stiffness could affect the inflammation response of the endothelial cells (ECs). Polyacrylamide (PA) based hydrogels at different concentrations were used as the culture substrate for ECs. The mRNA expression level of VCAM-1 and ICAM-1 was determined by qRT-PCR. EC chemotactic effect was evaluated by the number of THP-1 adhered to EC monolayer. The protein levels of IκBα and NF-κB were determined by western blotting analysis. The expression and localization of the major adherens junctions (AJs) proteins, VE-cadherin and β-catenin, were evaluated by western blotting and immunofluorescence staining. Our results showed that ECs cultured on soft substrate (1 kPa) demonstrated more chemotactic effect and the amount of the monocytes adhered to them was higher than that on harder substrate (20 kPa, p < 0.05). Moreover, NF-κB signaling pathway in ECs on 1 kPa substrate was more activated compared to those on 20 kPa substrate, with the IκBα protein expression level in the cytoplasm decreasing and NF-κB translocating more into the nuclear. In addition, the AJs of the endothelial monolayer changed with the substrate stiffness. Compared with ECs on normal substrate (20 kPa), the protein expression level of β-catenin decreased (p < 0.05), and immunofluorescence staining of VE-cadherin and β-catenin showed the AJs between the ECs on soft substrate (1 kPa) were punctuated. Taken together, our results suggested the stiffness of the substrate was important in regulating inflammation of the ECs and the integrity of the cell-cell junction. Therefore, the stiffness of the tissue engineering blood vessel material should be considered as an important criterium to avoid EC inflammation.

Dynamically softened substrate regulates malignancy of breast tumor cells

It has long been hypothesized that an increase in the extracellular matrix(ECM)stiffness mechanoactivates malignant phenotypes of breast tumor cells by regulating an array of processes underlying cancer biology.Although the contribution of substrate stiffening to drive malignant phenotype traits and other biological functions of a tumor is increasingly understood,the functional role of substrate softening on breast cancer cellular responses has rarely been investigated.Herein,we employed matrix metalloproteinase(MMP)-sensitive film to perform assays to explore the consequences of lowering stiffness on the biological behaviors of breast cancer cell MDA-MB-231.We demonstrated that cells underwent dramatic changes in migration,cellular conjunction,and expression of malignance-associated proteins and genes when the substrate stiffness decreased.Based on RNA sequencing and analysis,we found that hub genes including TP53,CCND1,MYC,CTNNB1,and YAP were validated to play central parts in regulating stiffness-dependent cellular manner change.Moreover,through visualization of differentially expressed genes(DEGs),cells on dynamically softened substrate appeared less influenced by transfer to tissue culture polystyrene(TCPS).These results suggest substrates with decreasing stiffness could normalize breast tumor malignant phenotype and help cells store the mechanical memory of the consequential weaker malignance.

Trans-scale surface wrinkling model and scaling relationship analysis of stiff film-compliant substrate structures

The self-assembly of surface-order structures based on the surface wrinkling of stiff film-compliant substrate structures(SFCS)is potentially useful in the fabrication of functional devices,the manufacture of superhydrophobic or self-cleaning surfaces,and so on.Due to the influence of the intrinsic characteristic length(g),the surface wrinkling behavior of SFCS at the micro scale is different from that at the macro scale.In this work,based on the strain gradient theory,a trans-scale surface wrinkling model for SFCS is established.First,the effectiveness of this model is verified by previous experiments.Then,based on the model and dimensional analysis,the effect of g on the surface wrinkling behavior is investigated,and the scaling relationship of surface wrinkling of SFCS at different scales is analyzed.The results show that the influence of g cannot be neglected when the film thickness decreases to the one comparable to g.At the micro scale,g will lead to the increase of the critical wrinkling wavelength and load.In addition,the scaling relationship of surface wrinkling at the micro scale will not follow the traditional one.Our study explains the underlying mechanism of the dissimilarity of surface wrinkling behaviors of SFCS at different scales and lays a theoretical foundation for the precise control of surface-order structures.