Matrix Stiffness-Mediated DNA Methylation in Endothelial Cells

Oncology Medicine Life Sciences Bioengineering Cell Biology
DNA methylation endothelial cells matrix stiffness DNMT1 tumor microenvironment

In pathological conditions, alterations in tissue mechanical properties are one of the prominent features of many diseases, such as cancer. The tumor vascular system plays a critical role in tumor growth, but its structure and function often become abnormal, manifesting as disorganized, tortuous, and leaky blood vessels. Research has shown that the stiffness of the extracellular matrix (ECM) plays an important role in regulating endothelial cell behavior. Tumor tissues are typically stiffer than normal tissues, and this increase in stiffness is partly due to excessive matrix deposition or cross-linking. Previous studies have demonstrated that reducing matrix stiffness can improve certain pathological features of the tumor vascular system, such as decreasing angiogenesis and reducing vascular permeability. Therefore, understanding how matrix stiffness affects epigenetic changes in endothelial cells, particularly DNA methylation, is crucial for revealing the pathological mechanisms of the tumor vascular system.

DNA methylation is one of the key mechanisms of epigenetics, regulating gene expression by covalently attaching a methyl group to the cytosine base in DNA. In endothelial cells, abnormal DNA methylation is closely related to the development and progression of various diseases, such as atherosclerosis. In recent years, the intersection of mechanobiology and epigenetics has garnered increasing attention, especially as the mechanisms by which matrix stiffness affects DNA methylation remain unclear. Thus, this study aims to explore the impact of matrix stiffness on DNA methylation in endothelial cells and uncover the underlying molecular mechanisms.

Source of the Paper

This paper was co-authored by Paul V. Taufalele, Hannah K. Kirkham, and Cynthia A. Reinhart-King from the Department of Biomedical Engineering at Vanderbilt University and Rice University. The paper was published online on January 17, 2025, in the journal Cellular and Molecular Bioengineering, with the DOI 10.1007/s12195-024-00836-9.

Research Process and Results

Research Process

  1. Cell Culture and Matrix Preparation
    The study used Human Umbilical Vein Endothelial Cells (HUVECs) as the research subject. HUVECs were seeded on collagen-coated polyacrylamide (PA) gels with stiffnesses of 2.5 kPa and 20 kPa to simulate the range of stiffness observed in the tumor microenvironment. The cells were cultured on the gels for 5 days to form monolayers.

  2. DNA Methylation Detection
    DNA methylation levels were assessed via immunofluorescent staining of 5-methylcytosine and ELISA (enzyme-linked immunosorbent assay). Immunofluorescent staining was used to detect 5-methylcytosine signals in the nucleus, while ELISA was used to quantify 5-methylcytosine levels in genomic DNA.

  3. Gene Expression Analysis
    Quantitative PCR (qPCR) was performed to measure the expression levels of genes encoding enzymes related to DNA methylation, including DNMT1, DNMT3A, DNMT3B, TET1, and TET2.

  4. Temporal Dynamics Analysis
    To investigate the dynamic effects of matrix stiffness on DNA methylation, the study measured DNA methylation levels in HUVECs at different time points (24, 48, 72, 96, and 120 hours). Additionally, the impact of passaging on DNA methylation levels was evaluated.

Key Results

  1. Effect of Matrix Stiffness on DNA Methylation
    The study found that HUVECs cultured on stiffer substrates (20 kPa) exhibited significantly lower global DNA methylation levels compared to those cultured on softer substrates (2.5 kPa). Both immunofluorescent staining and ELISA results confirmed this finding, indicating that increased matrix stiffness leads to reduced DNA methylation.

  2. Downregulation of DNMT1 Expression
    qPCR results showed that the expression of the DNMT1 gene was significantly downregulated on stiffer substrates, while the expression levels of other DNA methylation-related enzymes (DNMT3A, TET1, and TET2) remained unchanged. DNMT1 is a key enzyme responsible for maintaining DNA methylation patterns, and its downregulation may be one of the reasons for the reduced DNA methylation levels.

  3. Temporal Dynamics of DNA Methylation
    Temporal dynamics analysis revealed that DNA methylation levels were significantly lower on stiffer substrates after just 24 hours of culture. Over time, DNA methylation levels decreased in both stiffness conditions, but the difference between them remained significant. Additionally, the passaging process significantly increased DNA methylation levels, suggesting that cells may re-establish methylation states through certain mechanisms after detaching from the matrix.

Conclusions and Significance

This study highlights the significant impact of matrix stiffness on DNA methylation in endothelial cells, particularly the reduction in global DNA methylation levels on stiffer substrates. This finding provides new insights into the epigenetic changes in vascular endothelial cells within the tumor microenvironment. Furthermore, the study suggests that downregulation of DNMT1 expression may be a key factor contributing to the reduced DNA methylation levels. These results emphasize the importance of considering matrix mechanical properties in cell culture to ensure that in vitro experiments accurately recapitulate in vivo conditions.

The scientific value of this study lies in revealing the direct link between matrix stiffness and DNA methylation, offering a new explanation for the pathological mechanisms of the tumor vascular system. Additionally, the findings provide a theoretical basis for developing therapeutic strategies targeting tumor matrix stiffness, with potential applications in clinical practice.

Research Highlights

  1. Key Findings
    The study is the first to demonstrate the direct impact of matrix stiffness on DNA methylation in endothelial cells, particularly the significant reduction in global DNA methylation levels on stiffer substrates.

  2. Innovative Research Methods
    The study employed collagen-coated polyacrylamide gels as cell culture substrates, successfully simulating the range of stiffness observed in the tumor microenvironment. Additionally, the combination of immunofluorescent staining and ELISA enabled precise detection of DNA methylation levels.

  3. Specialized Research Focus
    The study focused on endothelial cells, which play a critical role in the formation and function of the tumor vascular system, providing a new perspective for understanding the pathological mechanisms of tumor vasculature.

Other Valuable Information

The study also explored the impact of passaging on DNA methylation levels, finding that the passaging process significantly increased DNA methylation levels. This discovery provides new clues for understanding epigenetic changes in cells after detachment from the matrix, which may have important implications for cell culture and tumor metastasis research.