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Identification of a Novel Angiogenesis Signalling circSCRG1/miR-1268b/NR4A1 Pathway in Atherosclerosis and the Regulatory Effects of TMP-PF In Vitro.

ABSTRACT: Angiogenesis contributes to plaque instability in atherosclerosis and further increases cardio-cerebrovascular risk. Circular RNAs (circRNAs) are promising biomarkers and potential therapeutic targets for atherosclerosis. Previous studies have demonstrated that tetramethylpyrazine (TMP) and paeoniflorin (PF) combination treatment (TMP-PF) inhibited oxidized low-density lipoprotein (ox-LDL)-induced angiogenesis in vitro. However, whether circRNAs regulate angiogenesis in atherosclerosis and whether TMP-PF can regulate angiogenesis-related target circRNAs in atherosclerosis are unknown. In this study, human RNA sequencing (RNA-seq) data were analysed to identify differentially expressed (DE) circRNAs in atherosclerosis and to obtain angiogenesis-associated circRNA-microRNA (miRNA)-messenger RNA (mRNA) networks. Target circRNA-related mechanisms in angiogenesis in atherosclerosis and the regulatory effects of TMP-PF on target circRNA signalling were studied in ox-LDL-induced human umbilical vein endothelial cells (HUVECs) by cell proliferation, migration, tube formation, and luciferase reporter assays, real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. A novel circRNA (circular stimulator of chondrogenesis 1, circSCRG1) was initially identified associated with angiogenesis in atherosclerosis, and circSCRG1 silencing up-regulated miR-1268b expression, increased nuclear receptor subfamily 4 group A member 1 (NR4A1) expression and then promoted ox-LDL-induced angiogenesis. TMP-PF (1 μmol/L TMP combined with 10 μmol/L PF) up-regulated circSCRG1 expression, mediated miR-1268b to suppress NR4A1 expression and then inhibited ox-LDL-induced angiogenesis. However, circSCRG1 silencing abolished the inhibitory effects of TMP-PF on ox-LDL-induced angiogenesis, which were rescued by the miR-1268b inhibitor. In conclusion, circSCRG1 might serve as a new target regulating angiogenesis in atherosclerosis via the circSCRG1/miR-1268b/NR4A1 axis and TMP-PF could regulate the circSCRG1/miR-1268b/NR4A1 axis to inhibit angiogenesis in atherosclerosis in vitro, indicating a novel angiogenesis signalling circSCRG1/miR-1268b/NR4A1 pathway in atherosclerosis and the regulatory effects of TMP-PF, which might provide a new pharmaceutical strategy to combat atherosclerotic plaque instability.

SUBMITTER: Yuan R

PROVIDER: S-EPMC9919304 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Identification of a Novel Angiogenesis Signalling circSCRG1/miR-1268b/NR4A1 Pathway in Atherosclerosis and the Regulatory Effects of TMP-PF In Vitro.

Yuan Rong R Xin Qiqi Q Ma Xiaochang X Yu Meng M Miao Yu Y Chen Keji K Cong Weihong W

Molecules (Basel, Switzerland) 20230128 3

Angiogenesis contributes to plaque instability in atherosclerosis and further increases cardio-cerebrovascular risk. Circular RNAs (circRNAs) are promising biomarkers and potential therapeutic targets for atherosclerosis. Previous studies have demonstrated that tetramethylpyrazine (TMP) and paeoniflorin (PF) combination treatment (TMP-PF) inhibited oxidized low-density lipoprotein (ox-LDL)-induced angiogenesis in vitro. However, whether circRNAs regulate angiogenesis in atherosclerosis and wheth ...[more]

PMID: 36770940

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Project description:Aims of the studyTo investigate the regulatory mechanism of Caesalpinia sappan L. ethyl acetate extract (CSEAE) on angiogenesis in atherosclerosis (AS) based on the miR-126/VEGF signalling pathway.Materials and methodsOur study first screened for differentially expressed microRNAs (miRNAs) associated with AS using the Gene Expression Omnibus (GEO) public database at the National Center for Biotechnology Information (NCBI) and R language software. Subsequently, our study verified the target-regulatory relationship between miR-126 and vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs) by using the "TargetScan" website and dual-luciferase reporter assay. In cellular experiments, Our study used cell proliferation assays and flow cytometry to assess the effects of CSEAE-Mediated serum on the proliferation and apoptosis of HUVECs. In animal experiments, our study used HE staining, Oil Red O staining and immunohistochemistry (IHC) staining to detect plaque area/lumen area (%), lipid area/plaque area (%) and microvessel density (MVD) in mouse aortas. In addition, our study performed RT‒PCR, ELISA and Western blot assays in ex vivo and in vivo experiments.ResultsA total of 39 differentially expressed miRNAs of AS were identified, among which the miR-126 expression level was significantly downregulated. Dual luciferase reporter gene assay results showed that miR-126 and VEGF have a targeting relationship, and the miR-126 mimic could inhibit the luciferase activity of the wild-type VEGF reporter gene vector (p value < 0.01). In cellular experiments, cell proliferation assays and flow cytometry results showed that CSEAE-Mediated serum significantly increased the proliferative activity after 24-72 h of treatment (p-value <0.01) and decreased the apoptotic level of HUVECs (p value < 0.01), and RT‒PCR results showed that CSEAE-Mediated serum significantly upregulated the expression of miR-126 (p value < 0.01) and downregulated the expression of VEGF mRNA in HUVECs (p value < 0.01). In vivo experiments, HE staining and IHC staining showed that CSEAE significantly reduced the MVD in the aorta and plaques of mice (p value < 0.01) and significantly reduced the aortic plaque area/lumen area (%) (p value < 0.01). Moreover, RT‒PCR assay and Western blot analysis results showed that CSEAE significantly upregulated the expression of miR-126 (p value < 0.01), downregulated the expression of VEGF mRNA (p value < 0.01), and decreased the protein expression levels of VEGF (p value < 0.01), phosphatidyl-inositol-3-kinase (PI3K) (p value < 0.01), and Ser/Thr-protein kinase (AKT1) (p value < 0.01) in mouse aortas, while ELISA showed that CSEAE significantly reduced the serum levels of vascular endothelial growth factor receptor (VEGFR2) (p value < 0.01) and hypoxia-inducible factor-1 (HIF-1) (p value < 0.01) in mice.ConclusionThis study emphasises CSEAE as a natural medicinal extract for the treatment of AS that can improve the migratory viability and reduce the apoptosis of HUVECs to maintain the health of the arterial endothelial microenvironment, while CSEAE also inhibits angiogenesis and delays plaque formation in ApoE-/- mice, suggesting that the therapeutic effect of CSEAE for AS may be related to its inhibition of neovascularisation and that its molecular mechanism may be related to the miR-126/VEGF signalling pathway.

Project description:BackgroundNontraumatic osteonecrosis of the femoral head (NONFH) is a common, progressive, and refractory orthopaedic disease. Decreased osteogenesis and angiogenesis are considered the main factors in the pathogenesis of NONFH. We aimed to figure out whether exosomes and exosomal miRNA from necrotic bone tissues of patients with NONFH are involved in the pathogenesis of NONFH and reveal the underlying mechanisms.MethodsRT-PCR and western blotting (WB) were used to detect the expression of osteogenic, adipogenic, and angiogenic markers. ALP staining and Alizarin Red S (ARS) staining were used to evaluate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Oil Red O staining was performed to assess the adipocyte deposition. A tube formation assay was used to study angiogenesis of human umbilical vascular endothelial cells (HUVECs). H&E staining and immunohistochemistry (IHC) staining were used to detect the effect of the NONFH exosomes in vivo. MicroRNA sequencing was conducted to identify potential regulators in the NONFH exosomes. The target relationship between miR-100-5p and BMPR2 was predicted and confirmed by a dual luciferase reporter assay and WB.ResultsThe NONFH exosomes reduced the osteogenic differentiation of hBMSCs and angiogenesis of HUVECs. In addition, the injection of the NONFH exosomes caused thinning and disruption of bone trabeculae in the femoral heads of rats. MiR-100-5p expression was upregulated in the NONFH exosomes and inhibited the osteogenesis of hBMSCs and angiogenesis of HUVECs by targeting BMPR2 and suppressing the BMPR2/SMAD1/5/9 signalling pathway. Silencing miR-100-5p expression rescued the reduction in osteogenesis and angiogenesis caused by the NONFH exosomes by activating the BMPR2/SMAD1/5/9 signalling pathway.ConclusionThe NONFH exosomal miR-100-5p can lead to NONFH-like damage by targeting BMPR2 and suppressing the BMPR2/SMAD1/5/9 signalling pathway, which may be involved in the pathophysiological mechanisms of nontraumatic osteonecrosis of the femoral head (NONFH).

Project description:Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis in vitro leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis.

Dataset Information

Identification of a Novel Angiogenesis Signalling circSCRG1/miR-1268b/NR4A1 Pathway in Atherosclerosis and the Regulatory Effects of TMP-PF In Vitro.

Publications

Identification of a Novel Angiogenesis Signalling circSCRG1/miR-1268b/NR4A1 Pathway in Atherosclerosis and the Regulatory Effects of TMP-PF In Vitro.

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