Stiffness model was used to establish an in vitro cell culture system to mimic the different stiffness condition. Acrylamide and bis-acrylamide (Sangon Biotech, China) was mixed at different ratio described in previous studies [17] [18] [19] to manufacture polyacrylamide hydrogels of different stiffnesses. The crosslinking reagents were 1M HEPES (Sigma, USA), 10% APS (Sangon Biotech, China) and TEMED (Klamar, China). Sulfo-SANPAH (ProteoChem, USA) was used for UV cross-linking. The surface of the polyacrylamide hydrogel was coated with 0.1 mg/ml of rat collagen I (Corning, USA) solution at 4˚C overnight to ensure proper cell adhesion. According to previous studies, 20 kPa was selected as normal stiffness control. One kPa and 100 kPa were selected as pathological stiffness condition [15] [18].

Human umbilical vein endothelial cells (HUVECs) (Sciencell, USA) were used as a representative EC in this study. The cells were maintained in Endothelial Cell Medium (ECM) provided by ScienCell (USA) in a 37˚C, 5% CO₂ incubator.

Before cell seeding, the hydrogels were irradiated with UV for 30 min and treated with 75% alcohol for 10 s. After washing with DPBS, the ECs were diluted to a concentration of 10 × 102 cells/mm² and uniformly added dropwise to the surface of the hydrogels and cultured in an incubator at 37˚C with 5% CO₂.

In this experiment, total RNA was extracted from ECs grown on softer (1 kPa) and stiffer (100 kPa) hydrogels and then subjected to transcriptome analysis. The genes expressed on different stiffnesses were explored by differentially expressed gene analysis and prediction analysis of transcription factors and their target genes. The transcriptome analysis was provided by the services of Oebiotech, China.

The total RNA extraction, reverse transcription and qRT-PCR were performed using kits from Tiangen, China, followed by the instructions of the manufacturer. The primers used in the experiment were purchased from BioTNT, China. The primers are as follows, human JCAD: (forward) 5'-CGG AGG CGG GGT TTT GTA AT-3', (reverse) 5'-GGG GTC ATT GAT GGC AAC A-3', human GAPDH: (forward) 5'-GGG AAG GTG AAG GTC GGA GT-3', (reverse) 5'-CAC CCT GTT GCT GTA GCC AAA TTC-3'.

RIPA (Beyotime, China) was used as the lysis solution and protein concentration was determined using BCA Protein Assay Kit (Thermo Scientific, USA). The protein samples were denatured with loading buffer at 95˚C for 5 minutes. The membrane was blocked using 10% BSA at room temperature for 2 hours and incubated with primary antibodies overnight at 4˚C. The primary antibodies were p-p38MAPK, p38MAPK (both from Cell Signaling Technology, USA), and GAPDH (Proteintech, USA), respectively. Then the membrane was incubated with HRP-conjugated secondary antibody for 1.5 hours at room temperature. Protein chemiluminescence was performed using Chemiluminescent HRP Substrate (MILLIPORE, USA). The images were acquired by Biorad imaging system (USA).

Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.5% TritonX-100 at room temperature. After blocking with 5% BSA, VE-cadherin antibody (Cell Signaling Technology, USA, 1:500) was incubated overnight at 4˚C. The fluorescence labeled secondary antibodies were incubated at room temperature for 1.5 hours. Cell nucleus were stained with DAPI (Beyotime, China, 1:5000). The images were acquired by a laser confocal microscope (Leica, TCS SP5 II, Germany) and the intensity of the signal was quantified by ImageJ V1.8.0.

Unless otherwise stated, results were shown as the mean of at least three independent experiments, and error bars represent the standard error of the mean (SEM). Pairwise comparisons were performed using the Student t-test. P values less than 0.05 were considered statistically significant in this experiment.

Vascular Sclerosis is manifested by increased mitochondrial oxidative stress, increased levels of inflammatory factors, and increased vascular permeability. When vascular function is impaired, the composition and structure of the extracellular matrix is altered, leading to damage in endothelial cell-cell junctions. At the same time, signaling pathways are activated in response to external mechanical stimuli. We performed transcriptome analysis in ECs cultured on softer (1 kPa) and stiffer (100 kPa) substrates, as shown by the transcriptome enrichment analysis (Figure 1). The results indicated that cell-cell junction assembly was the major biological process affected. In addition, cytoskeleton organization and focal adhesion were significantly involved as well. In view of cellular components, cell-cell junction proteins, including adherens and gap junction proteins, were all affected. As to molecular functions, cadherin binding and cell-cell junction assembly were significantly affected by substrate stiffness. Mitogen-activated protein kinase (MAPK), Rho GTPase and other classical signal transduction pathways were also regulated by substrate stiffness. In summary, the above results suggested that substrate stiffness as an important external mechanical signal has profound impact on the integrity of cell-cell junctions and the classical signaling pathway MAPK.

MAPK is a protein kinase that plays a role in cell growth, proliferation, differentiation and apoptosis. MAPK was activated by phosphorylation of its kinase MAPKK, which in turn can be activated by phosphorylation of its kinase MAPKKK. The process may involve small GTPases or other proteases, among which small GTPases are Rac, Rho, etc. Activation of the MAPK/ERK pathway in ECs at wound healing can regulate the angiogenic response of microvascular ECs. p38MAPK is activated by environmental stress or inflammatory cytokines, which in turn regulate cell behavior and function. Transcriptome analysis of ECs

cultured on softer (1 kPa) and stiffer (100 kPa) substrates revealed that the expression of MAPK signaling pathway-related molecules MAP2K3, MAP2K7, MAP3K3, MAP3K6, MAPK3, MAPK7, as well as RHO family interacting cell polarization regulator 1 (RIPOR1), ras homolog family member (RHOF), ras homolog family member related to GTPase activity T2 (RHOT2), TGF-β activated kinase 1 (MAP3K7) binding protein 1 (TAB1) and MAPK activated protein kinase 2 (MAPKAPK2), which are molecules related to MAPK activation, were also upregulated with increased stiffness (Figure 2(a), Figure 2(b)). The ratio of protein expression p-p38MAPK/p38MAPK was found to increase with stiffness by culturing ECs in vitro at three stiffnesses of 1 kPa, 20 kPa and 100 kPa (Figure 2(c), Figure 2(d)).

Cell junctions assume key roles in pathological processes, which confer resilience and stiffness to tissues, mediate signal transmission, control the transport of ions and molecules, and act as channels to mediate the transport of ions and molecules. Tight junctions (TJs), anchoring junctions, and gap junctions play different roles in pathological processes. Zona occludens1 (ZO-1), encoded by the tight junction protein 1 (TJP1) gene, is a regulator of endothelial cell permeability [20]. TJP2, linked to the actin cytoskeleton is an important component of tight junction proteins, which is also associated with signaling pathways [21]. Junctional adhesion molecules (JAMs) are involved in different cellular functions, such as regulating the formation of cell junctions, cell migration or mitotic spindle orientation. Thus, all the above proteins play an important role in endothelial barrier formation and angiogenesis [22]. Transcriptome analysis of ECs cultured on softer (1 kPa) and stiffer (100 kPa) substrates revealed that the expression of cell junction-related molecules TJP1, TJP2, JAM3 and JCAD were reduced on stiffer substrates (Figure 3(a)). Junctional Cadherin 5 Associated (JCAD) is an endothelial cell ligand protein that regulates pathological angiogenesis, EC proliferation and migration, and the CAD/MI risk gene JCAD identified by Genome-wide association study (GWAS) promotes endothelial dysfunction and atherosclerosis in mice [23]. We found that the gene expression level of JCAD showed a negative correlation to stiffness (Figure 3(b)).

VE-cadherin is an important component of EC adherent junctions and has a key role in maintaining vascular integrity by controlling vascular permeability and inhibiting unrestricted vascular growth. It was found that its changes may involve endothelial cytoskeleton remodeling and regulation of gene transcription, etc. Therefore, we performed immunofluorescence staining for VE-cadherin (Figure 4). Immunostaining results showed the punctuation and discontinuity of cell-cell junction (as indicated by red arrows) tended to be increased (Figure 4(a)). The total fluorescence intensity of VE-cadherin in ECs differed significantly between softer and stiffer substrates. The quantitative analysis of fluorescence intensity demonstrated that the expression level of VE-cadherin decreased significantly with increased substrate stiffness (Figure 4(b)). The above results suggested that high substrate stiffness impaired endothelial cell-cell junctions and the integrity of endothelial cell.