Background and Aims: Atherosclerosis, a major contributor to cardiovascular and cerebrovascular diseases, remains a leading cause of global mortality and morbidity.
More...Background and Aims: Atherosclerosis, a major contributor to cardiovascular and cerebrovascular diseases, remains a leading cause of global mortality and morbidity. This study focuses on the role of chemokine-like receptor 1 (CMKLR1) in VSMCs and its potential impact on atherosclerotic plaque formation and remodelling. Methods: Cmklr1-/-Apoe-/- mice, generated using CRISPR/Cas9 technology, were fed a high-fat western diet for 16 weeks to model atherosclerosis. Primary VSMCs were isolated from Cmklr1-/- and Cmklr1+/+ mice for in vitro experiments. Cell viability, senescence-associated β-galactosidase staining, and RNA interference were performed to assess the effect of CMKLR1 deficiency on VSMCs. Various assays, including flow cytometry, immunocytochemistry, and RNA-seq analysis, were employed to investigate the molecular mechanisms and downstream signals associated with CMKLR1 deficiency. Results: CMKLR1 deficiency was found to disrupt the cell cycle, leading to VSMC senescence both in vitro and in vivo. Cmklr1-/-Apoe-/- mice exhibited increased atherosclerotic plaque burden, emphasizing the role of CMKLR1 in plaque stability. Transcriptome analysis revealed significant changes in cellular components and biological processes, with a particular impact on the extracellular matrix and cellular response to stimulus. The TGFβ signalling pathway emerged as a potential downstream target of CMKLR1, affecting VSMC differentiation, vessel development, and extracellular matrix synthesis. The deficiency of CMKLR1 led to a reduction in TGFβ1 expression and downstream molecule SM22α, influencing plaque composition and stability. Conclusion: This study demonstrates that CMKLR1 deficiency promotes VSMC senescence and exacerbates atherosclerosis. The disrupted TGFβ signalling pathway, identified as a potential molecular mechanism, provides insights into the complex regulatory network associated with CMKLR1 in atherosclerotic plaque formation and remodelling. These findings highlight the importance of CMKLR1 in atherosclerosis and its potential as a therapeutic target for plaque stability.
Overall design: Total RNA was extracted from the VSMCs of Cmklr1-/- and Cmklr1+/+ mice following standard protocols of RNA isolation. Quantification and quality of RNA was assessed. The library preparation and sequencing were performed by Wenzheng Biotechnology Co., Ltd. on an Illumina NovaSeq platform following the manufacturer’s instructions, and the paired-end reads were generated. The raw data were stored as FASTQ files. The clean data was obtained by removing adapters and low-quality reads. All downstream analyses were based on clean, high-quality data. The RPKM (reads per kilobase per million reads) of each gene was calculated based on the read counts mapped to this gene and the length of the gene. Differential expression analysis between the two groups was performed using the DESeq R package (1.16.1). Genes with the P <0.05 and abs (log2 (fold change)) >2 were defined as differentially expressed. Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.kegg.jp) and Gene Ontology (GO) (http://www.geneontology.org) analyses of differentially expressed genes were performed using the OmicShare tools, a free online platform for data analysis (https://www.omicshare.com/tools). Statistical enrichment in gene functions and biological pathways was obtained. The transcription factor enrichment analysis was conducted with ChEA3 (ChIP-X Enrichment Analysis Version 3, https://maayanlab.cloud/chea3/). The STRING database (http://string-db.org) was used for assessing the interactions of protein-protein, including direct (physical) and indirect (functional) associations.
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