Dataset Information

Role of alternative splicing of VEGF-A in the development of atherosclerosis.

ABSTRACT: Vascular endothelial cell growth factor A (VEGF-A) signaling promotes the endothelial cell proliferation, macrophage infiltration and foam cell formation, which play pivotal roles in the pathogenesis of atherosclerosis (AS). However, the role of alternative splicing of VEGF here is not known. Here, ApoE (-/-) mice supplied high-fat diet (HFD mice) were used to generate AS, while ApoE (-/-) mice supplied with normal diet (NOR mice) were used as a control. Aortic endothelial cells (AECs) and infiltrated macrophages were purified and quantified by flow cytometry. Alternative splicing of VEGF and the regulator of VEGF splicing, SRPK1, were assessed by RT-qPCR and immunoblotting in both AECs and aortic macrophages. We found that HFD mice developed AS in 12 weeks, while the NOR did not. Compared to NOR mice, HFD mice possessed significantly more AECs and AEC proliferation, and had significantly more aortic infiltrated macrophages and more apoptosis of them. Significant shift of VEGF-A splicing to pro-angiogenic VEGF₁₆₅ was detected in both AECs and macrophages from HFD mice, seemingly through upregulation of SRPK1. In vitro, SRPK1 overexpression significantly increased EC proliferation and macrophage apoptosis. Thus, our data suggest that alternative splicing of VEGF-A to pro-angiogenic VEGF₁₆₅ may contribute to the development of AS.

SUBMITTER: Zhao N

PROVIDER: S-EPMC6224261 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Role of alternative splicing of VEGF-A in the development of atherosclerosis.

Zhao Naishi N Zhang Jianfeng J

Aging 20181001 10

Vascular endothelial cell growth factor A (VEGF-A) signaling promotes the endothelial cell proliferation, macrophage infiltration and foam cell formation, which play pivotal roles in the pathogenesis of atherosclerosis (AS). However, the role of alternative splicing of VEGF here is not known. Here, ApoE (-/-) mice supplied high-fat diet (HFD mice) were used to generate AS, while ApoE (-/-) mice supplied with normal diet (NOR mice) were used as a control. Aortic endothelial cells (AECs) and infil ...[more]

PMID: 30317225

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Project description:RNA-binding proteins (RBPs) are involved in the regulation of RNA splicing, stability, and localization. How RBPs control the development of atherosclerosis, is not fully understood. To explore the relevant RNA-binding proteins (RBPs) and alternative splicing events (ASEs) in atherosclerosis. We made a comprehensive work to integrate analyses of differentially expressed genes, including differential RBPs, and variable splicing characteristics related to different stages of atherosclerosis in dataset GSE104140. A total of 3712 differentially expressed genes (DEGs) were identified, including 2921 upregulated genes and 791 downregulated genes. Further analysis screened out 54 RBP genes, and 434 AS genes overlapped DEGs. We selected high expression ten RBP genes (SAMHD1, DDX60 L, TLR7, RBM47, MYEF2, RNASE6, PARP12, APOBEC3G, SMAD9, and RNASE1) for co-expression analysis. Meanwhile, we found seven regulated alternative splicing genes (RASGs) (ABI1, FXR1, CHID1, PLEC, PRKACB, BNIP2, PPP3CB) that could be regulated by RBPs. The co-expression network was used to further elucidate the regulatory and interaction relationship between RBPs and AS genes. Apoptotic process and innate immune response, revealed by the functional enrichment analysis of RASGs regulated by RBPs were closely related to atherosclerosis. In addition, 26 of the 344 alternative splicing genes regulated by the above 10 RBPs were transcription factors (TFs), We selected high expression nine TFs (TFDP1, RBBP7, STAT2, CREB5, ERG, ELF1, HMGN3, BCLAF1, and ZEB2) for co-expression analysis. The target genes of these TFs were mainly enriched in inflammatory and immune response pathways that were associated with atherosclerosis. indicating that AS abnormalities of these TFs may have a function in atherosclerosis. Furthermore, the expression of differentially expressed RBPs and the alternative splicing events of AS genes was validated by qRT-PCR in umbilical vein endothelial cells (HUVEC). The results showed that RBM47 were remarkedly difference in HUVEC treated with ox-LDL and the splicing ratio of AS in BCLAF1which is regulated by RBM47 significantly changed. In conclusion, the differentially expressed RBPs identified in our analysis may play important roles in the development of atherosclerosis by regulating the AS of these TF genes.

Project description:Alternative splicing generates protein diversity and allows for post-transcriptional gene regulation. Estimates suggest that 10% of the genes in Caenorhabditis elegans undergo alternative splicing. We constructed a splicing-sensitive microarray to detect alternative splicing for 352 cassette exons and tested for changes in alternative splicing of these genes during development. We found that the microarray data predicted that 62/352 (approximately 18%) of the alternative splicing events studied show a strong change in the relative levels of the spliced isoforms (>4-fold) during development. Confirmation of the microarray data by RT-PCR was obtained for 70% of randomly selected genes tested. Among the genes with the most developmentally regulated alternatively splicing was the hnRNP F/H splicing factor homolog, W02D3.11 - now named hrpf-1. For the cassette exon of hrpf-1, the inclusion isoform comprises 65% of hrpf-1 steady state messages in embryos but only 0.1% in the first larval stage. This dramatic change in the alternative splicing of an alternative splicing factor suggests a complex cascade of splicing regulation during development. We analyzed splicing in embryos from a strain with a mutation in the splicing factor sym-2, another hnRNP F/H homolog. We found that approximately half of the genes with large alternative splicing changes between the embryo and L1 stages are regulated by sym-2 in embryos. An analysis of the role of nonsense-mediated decay in regulating steady-state alternative mRNA isoforms was performed. We found that 8% of the 352 events studied have alternative isoforms whose relative steady-state levels in embryos change more than 4-fold in a nonsense-mediated decay mutant, including hrpf-1. Strikingly, 53% of these alternative splicing events that are affected by NMD in our experiment are not obvious substrates for NMD based on the presence of premature termination codons. This suggests that the targeting of splicing factors by NMD may have downstream effects on alternative splicing regulation.

Dataset Information

Role of alternative splicing of VEGF-A in the development of atherosclerosis.

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Role of alternative splicing of VEGF-A in the development of atherosclerosis.

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