ABSTRACT
INTRODUCTION: Vascular neointimal hyperplasia, a pathological process observed in cardiovascular diseases such as atherosclerosis and pulmonary hypertension, involves the abundant presence of vascular smooth muscle cells (VSMCs). The proliferation, migration, and autophagy of VSMCs are associated with the development of neointimal lesions. Circular RNAs (circRNAs) play critical roles in regulating VSMC proliferation and migration, thereby participating in neointimal hyperplasia. However, the regulatory roles of circRNAs in VSMC autophagy remain unclear. OBJECTIVES: We aimed to identify circRNAs that are involved in VSMC autophagy-mediated neointimal hyperplasia, as well as elucidate the underlying mechanisms. METHODS: Dual-luciferase reporter gene assay was performed to validate two competing endogenous RNA axes, hsa_circ_0001402/miR-183-5p/FKBP prolyl isomerase like (FKBPL) and hsa_circ_0001402/miR-183-5p/beclin 1 (BECN1). Cell proliferation and migration analyses were employed to investigate the effects of hsa_circ_0001402, miR-183-5p, or FKBPL on VSMC proliferation and migration. Cell autophagy analysis was conducted to reveal the role of hsa_circ_0001402 or miR-183-5p on VSMC autophagy. The role of hsa_circ_0001402 or miR-183-5p on neointimal hyperplasia was evaluated using a mouse model of common carotid artery ligation. RESULTS: Hsa_circ_0001402 acted as a sponge for miR-183-5p, leading to the suppression of miR-183-5p expression. Through direct interaction with the coding sequence (CDS) of FKBPL, miR-183-5p promoted VSMC proliferation and migration by decreasing FKBPL levels. Besides, miR-183-5p reduced BECN1 levels by targeting the 3'-untranslated region (UTR) of BECN1, thus inhibiting VSMC autophagy. By acting as a miR-183-5p sponge, overexpression of hsa_circ_0001402 increased FKBPL levels to inhibit VSMC proliferation and migration, while simultaneously elevating BECN1 levels to activate VSMC autophagy, thereby alleviating neointimal hyperplasia. CONCLUSION: Hsa_circ_0001402, acting as a miR-183-5p sponge, increases FKBPL levels to inhibit VSMC proliferation and migration, while enhancing BECN1 levels to activate VSMC autophagy, thus alleviating neointimal hyperplasia. The hsa_circ_0001402/miR-183-5p/FKBPL axis and hsa_circ_0001402/miR-183-5p/BECN1 axis may offer potential therapeutic targets for neointimal hyperplasia.
ABSTRACT
Background: The crotonylation of histones is discovered of late as one of the post-translational modifications (PTMs) that can regulate gene expression. However, the function of crotonylation on nonhistone proteins in vascular smooth muscle cells (VSMCs) is unclear. Here, we aim to find the cellular characteristics of crotonylated nonhistone proteins and the cross talk with ubiquitinated proteins in VSMC phenotypic remodeling using the modified omics and proteomic analysis. Methods: We performed the modified omics and proteomic analysis of VSMCs before and after the stimulation with platelet-derived growth factor-BB (PDGF-BB). The crotonylated and ubiquitinated pan-antibody was used to enrich proteins and then subjected to a high-throughput mass spectrometry analysis. The enrichment analysis was performed within differentially modified proteins in regard to GO terms, KEGG, and protein domains. Results: As a result, there were 2,138 crotonylation sites in 534 proteins and 1,359 ubiquitination sites corresponding to 657 proteins. These crotonylated proteins detected after PDGF-BB stimulation might be involved in various vital cellular pathways and carry out important functions in VSMCs. Some of them closely took part in significant physiological processes of VSMC phenotypic remodeling, including glycolysis/gluconeogenesis, vascular smooth muscle contraction, and the PI3K-Akt signaling pathway. Furthermore, the KEGG pathway enrichment analysis showed the involvement of ubiquitinated proteins in the physiological processes of VSMC phenotypic remodeling, including glycolysis/gluconeogenesis, vascular smooth muscle contraction, RAS signaling pathway, or the PI3K-Akt signaling pathway. A cross talk analysis showed that there were 199 sites within the 177 proteins modified by crotonylation and ubiquitination simultaneously. Protein-protein interaction (PPI) network analysis indicated that crotonylated and ubiquitinated proteins play an important role in cellular bioprocess commonly and possibly have a synergistic effect. Conclusion: In summary, our bioinformatics analysis shows that the crotonylation and ubiquitination of nonhistone proteins play an essential role in VSMC phenotypic transformation induced by PDGF-BB stimulation. The cross talk between crotonylation and ubiquitination in glycolysis is possibly a novel mechanism during VSMC phenotypic remodeling.
