Chronic Kidney Disease: Runx2-NLRP3 Axis Regulates Matrix Stiffness-Induced Vascular Smooth Muscle Cell Inflammation

Academic Background

Chronic Kidney Disease (CKD) is a common condition worldwide, leading to high morbidity and mortality rates among patients and significantly increasing the risk of cardiovascular events. Arterial stiffening, characterized by increased arterial stiffness and reduced vascular elasticity, is one of the hallmarks of cardiovascular complications in CKD patients. Studies have shown that arterial stiffening is closely related to low-grade vascular inflammation, but the specific role of matrix stiffness in the onset of inflammation remains unclear. Therefore, exploring the causal relationship between arterial stiffening and vascular inflammation, especially how matrix stiffness regulates the inflammatory phenotype of vascular smooth muscle cells (VSMCs), has become a focal point in current research.

Source of the Paper

This paper was co-authored by researchers Zhiqing Li, Hao Wu, Fang Yao, and others, with team members from multiple institutions including Peking University and Fuwai Hospital Chinese Academy of Medical Sciences. The study was first published on January 7, 2025, in the journal American Journal of Physiology-Cell Physiology, titled “Runx2-NLRP3 axis orchestrates matrix stiffness-evoked vascular smooth muscle cell inflammation.”

Research Process and Results

1. Research Process

a) Animal Model Establishment and Arterial Stiffening Assessment

The study initially used an adenine-induced CKD mouse model to evaluate the occurrence of arterial stiffening and its relationship with vascular inflammation. Mice were divided into control and CKD groups, with pulse-wave velocity (PWV) measurements taken at 2, 4, and 8 weeks to assess arterial stiffness. Additionally, Masson’s staining and immunofluorescence analysis were conducted to evaluate collagen accumulation and the expression of inflammatory markers in the aorta.

b) Effect of Matrix Stiffness on VSMC Inflammatory Phenotype

To investigate the impact of matrix stiffness on the inflammatory phenotype of VSMCs, the research team cultured primary rat VSMCs on collagen-coated polyacrylamide hydrogels of varying stiffness. F-actin staining was used to assess changes in cell morphology, while real-time quantitative PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were employed to detect the expression and secretion of inflammatory factors such as MCP-1, IL-6, IL-1β, and IL-18.

c) RNA Sequencing and Bioinformatics Analysis

To further explore the molecular mechanisms by which matrix stiffness regulates the inflammatory phenotype of VSMCs, the research team performed RNA sequencing (RNA-seq) on VSMCs cultured on soft and stiff matrices and conducted bioinformatics analysis of differentially expressed genes (DEGs). Through the Chip-Atlas database, potential regulatory transcription factors were screened.

d) Validation of the Regulatory Relationship Between Runx2 and NLRP3

The research team verified the transcriptional regulation of NLRP3 by Runx2 through chromatin immunoprecipitation sequencing (ChIP-seq) and luciferase reporter gene experiments. Additionally, siRNA knockdown of Runx2 or NLRP3 was used to evaluate their effects on matrix stiffness-induced inflammatory phenotypes in VSMCs.

2. Main Results

a) Arterial Stiffening Precedes Vascular Inflammation

In the CKD mouse model, PWV significantly increased early in the disease progression (at 2 weeks), while the expression of vascular inflammatory markers (such as MCP-1, IL-6, IL-1β, and IL-18) was upregulated only at 8 weeks. This indicates that arterial stiffening precedes vascular inflammation, suggesting that arterial stiffening may be a trigger for vascular inflammation.

b) Matrix Stiffness Induces Inflammatory Phenotype in VSMCs

Increased matrix stiffness significantly altered the morphology of VSMCs, transitioning them from a contractile to a synthetic phenotype. Simultaneously, matrix stiffness upregulated the expression and secretion of inflammatory factors in VSMCs, indicating that matrix stiffness promotes vascular inflammation by regulating the inflammatory phenotype of VSMCs.

c) Runx2 Is a Key Transcription Factor in Matrix Stiffness Regulation of VSMC Inflammation

RNA sequencing and bioinformatics analysis revealed that Runx2 is a key transcription factor in matrix stiffness regulation of the inflammatory phenotype in VSMCs. Matrix stiffness significantly upregulated the expression of Runx2 and promoted its nuclear translocation. Knockdown of Runx2 significantly inhibited matrix stiffness-induced inflammatory responses in VSMCs.

d) NLRP3 Is a Downstream Target Gene of Runx2

ChIP-seq and luciferase reporter gene experiments confirmed that Runx2 directly binds to the promoter region of NLRP3 and regulates its transcription. Knockdown of NLRP3 or treatment with its specific inhibitor MCC950 significantly suppressed Runx2-induced inflammatory responses in VSMCs.

Conclusions and Significance

This study found that in a CKD mouse model, arterial stiffening precedes vascular inflammation, and matrix stiffness induces the transition of VSMCs to an inflammatory phenotype through the activation of the Runx2-NLRP3 axis, thereby promoting vascular inflammation. This discovery not only reveals the causal relationship between arterial stiffening and vascular inflammation but also provides new potential targets for the treatment of CKD-related cardiovascular complications.

Highlights of the Study

1. First Demonstration That Arterial Stiffening Precedes Vascular Inflammation: The study clarified the temporal relationship between arterial stiffening and vascular inflammation through time-series analysis, providing important clues for subsequent mechanistic studies.
2. Discovery of the Runx2-NLRP3 Axis: The study first discovered that Runx2 mediates matrix stiffness-induced inflammatory phenotypes in VSMCs by directly regulating NLRP3, offering a new perspective for understanding the molecular mechanisms of vascular inflammation.
3. Mechanism of Matrix Stiffness Regulation of VSMC Inflammatory Phenotype: Using techniques such as RNA sequencing and ChIP-seq, the study systematically elucidated how matrix stiffness regulates the inflammatory phenotype of VSMCs through the Runx2-NLRP3 axis, providing new theoretical support for the treatment of CKD-related cardiovascular diseases.

Other Valuable Information

The research team also proposed potential future therapeutic strategies, including targeting Runx2 or NLRP3 with small-molecule inhibitors or RNA-based therapies to suppress vascular inflammation in CKD patients. Additionally, the research team plans to further validate these findings in human samples and explore their clinical application value.