Endothelial cells derived extracellular vesicles promote diabetic arterial calcification via circ_0008362/miR-1251-5p/Runx2 axial

Abstract

Introduction: Arterial calcification, an independent predictor of cardiovascular events, increases morbidity and mortality in patients with diabetes mellitus (DM), but its mechanisms remain unclear. Extracellular vesicles (EVs) play an important role in intercellular communication. The study investigates the role and potential mechanisms of EVs derived from endothelial cells (ECs) in regulating vascular smooth muscle cell (VSMC) calcification under high glucose (HG) condition, with a goal of developing effective prevention and treatment strategies for diabetic arterial calcification.

Results: The results showed that EVs derived from HG induced ECs (EC^HG-EVs) exhibited a bilayer structure morphology with a mean diameter of 74.08 ± 31.78 nm, expressing EVs markers including CD9, CD63 and TSG101, but not express calnexin. EC^HG-EVs was internalized by VSMCs and induced VSMC calcification by increasing Runx2 expression and mineralized nodule formation. The circ_0008362 was enriched in EC^HG-EVs, and it can be transmitted to VSMCs to promote VSMC calcification both in vitro and in vivo. Mechanistically, miR-1251-5p might be one of the targets of circ_0008362 and they were co-localization in the cytoplasm of VSMCs. Runx2 was identified as the downstream target of miR-1251-5p, and circ_0008362 acted as a sponge, enhancing Runx2 expression and then promoted VSMC calcification. Besides, circ_0008362 could directly interact with Runx2 to aggravate VSMC calcification. Notably, DiR-labelled EC^HG-EVs was detected in the vessels of mice. Meanwhile, the level of circ_0008362 and Runx2 were increased significantly, while the expression of miR-1251-5p was decreased significantly in calcified artery tissues of mice. However, inhibiting the release of EVs by GW4869 attenuated arterial calcification in diabetic mice. Finally, the level of circulation of plasma EVs circ_0008362 was significantly higher in patients with DM compared with normal controls. Elevated levels of plasma EVs circ_0008362 were associated with more severe coronary and aorta artery calcification in patients with DM.

Conclusions: Our findings suggested that circ_0008362 was enriched in EVs derived from ECs and promoted VSMC calcification under HG conditions, both by sponging miR-1251-5p to upregulate Runx2 expression and through direct interaction with Runx2. Furthermore, elevated levels of plasma EVs circ_0008362 were associated with more severe coronary and aorta artery calcification in patients with DM. These results may serve as a potential prevention and therapeutic target for diabetic arterial calcification.

Keywords: Arterial calcification; Circ_0008362; Diabetes mellitus; Endothelial cells; Extracellular vesicles; Vascular smooth muscle cells.

Fig. 1

EVs derived from HG-stimulated ECs exacerbated VSMC calcification. A Representative morphological analysis of EC-EVs by TEM (Scale bar = 100 nm). B Diameter distribution of EC-EVs by NTA; C the expression of CD9, CD63, TSG101 and Calnexin were detected by WB. D–E Laser scanning confocal microscope and Z-stack analysis showed that PKH26-labeled EC-EVs would be taken up by VSMCs and mainly located at the cytoplasm of VSMCs (PKH26 in red, DAPI in blue, and a-SMA in green) (scale bar = 20 μm). F ARS staining and quantification of the calcium content in VSMCs treated with EC^NG-EVs, EC^HG-Sup, EC^HG-EVs, EC^HG-Sup^−EVs, respectively. The blue arrows indicate mineralized nodules in VSMCs (Scale bar = 100 μm). G The expression of Runx2 protein was determined by WB in VSMCs incubated with EC^NG-EVs, EC^HG-Sup, EC^HG-EVs, EC^HG-Sup^−EVs, respectively. H ECs were pre-treated with HG and co-cultured with VSMCs in the trans-well units, either with or without the addition of GW4869; I the expression of Runx2 protein was measured by using WB. One-way ANOVA with Tukey’s multiple comparisons test (F, G, I) was used. Three independent experiments were performed, and the representative data were shown. Data were shown as mean ± SD. ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05. EVs, extracellular vesicles; TEM, transmission electron micrographs; NTA, nanoparticle tracking analysis; WB, western blot; EC^NG-EVs, EVs derived from normal-glucose induced ECs; EC^HG-EVs, EVs derived from high-glucose induced ECs; EC^HG-Sup, supernatant derived from high-glucose induced ECs; EC^HG-Sup^−EVs, supernatant derived from EC^HG with deprivation of EVs; ARS: alizarin red s

Fig. 2

hsa_circ_0008362 promoted VSMC calcification both in vitro and in vivo. A the location of hsa_circ_0008362 and mFBXW4 in EC^HG was demonstrated by qRT-PCR analysis. B qRT-PCR detected the different expression of circ_0008362 and mFBXW4 between EC^NG-EVs and EC^HG-EVs; C AGE detected the size of the PCR amplification product of hsa_circ_0008362 with double parallel experiment in EC^NG-EVs and EC^HG-EVs. D ECs was treated with RNase R and the expression of circ_0008362 and mFBXW4 were measured by qRT-PCR; E ECs was treated with 5 μg/ml actinomycin D (0, 2, 4, 8, 12 h) and the expression of circ_0008362 and mFBXW4 was detected by qRT-PCR; F the expression of Runx2 was measured by WB in VSMCs treated with EVs^Circ_Control or EVs^{Circ_0008362−WT}, respectively. G ARS staining and quantification of the calcium content in VSMCs treated with EVs^Circ_Control or EVs^{Circ_0008362−WT}. The blue arrows indicate mineralized nodules in VSMCs (scale bar = 100 μm). H qRT-PCR detected the expression of circ_0008362 in the aorta tissues of CircCtrl and Circ_0008362 OE mice. I ARS staining showed the arterial calcification in different mice (n = 5/group). The arrows indicate calcified arteries (scale bar = 200 μm). J Immunohistochemistry staining analysis of Runx2 expression in different mice. The arrows indicate the positive expression of Runx2 in the mice aorta (scale bar = 200 μm). One-way ANOVA with Tukey’s multiple comparisons test (B, D, F, G ) and Student’s t-test (E, H, I, J) were used. Three independent experiments were performed, and representative data were shown. Data were shown as mean ± SD. ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05. ns, no significance. WB, western blot; EC^NG-EVs, EVs derived from normal-glucose induced ECs; EC^HG-EVs, EVs derived from high-glucose induced ECs; EVs^Circ_Control, EVs transfected with circular RNA negative control; EVs^{Circ_0008362−WT}, EVs transfected with circ_0008362-wild type plasmid; CircCtrl, adeno-associated virus 9 negative control; Circ_0008362 OE, adeno-associated virus 9 circ_0008362 overexpression; AGE, agarose gel electrophoresis; ARS: alizarin red s

Fig. 3

hsa_circ_0008362/miR-1251-5p/Runx2 axial regulated VSMC calcification.A Schematic representation of putative target sites between miR-1251-5p and circ_0008362 as well as in Runx2 3′-UTR. B FISH showed the presence of circ_0008362 and miR-1251-5p in VSMCs. Scale bar = 50 μm. C the expression of miR-1251-5p in VSMCs transfected the vector of Circ_Control or Circ_0008362-WT. D the expression of hsa_circ_0008362 was detected by qPCR in VSMCs transfected by miR-1251-5p mimics or inhibitor. E The expression of circ_0008362 was detected by qRT‐PCR after biotin‐labeled miR‐1251‐5p pull‐down assay. F RIP assay showed the binding efficiency of miR‐1251‐5p and circ_0008362 to Ago2 protein in VSMCs. G the expression of Runx2 was measured by WB in VSMCs treated with EC^NG-EVs or EC^HG-EVs. H Luciferase reporter assays showed the luciferase activities of Runx2 in VSMCs transfected with miR‐1251‐5p mimics or control oligos. I VSMCs were transfected with circ_0008362 plasmid and the level of Runx2 protein was measured by WB. J The level of Runx2 protein was measured by WB in VSMCs with different treatment. K ARS staining showed the mineralized nodules in VSMCs with different treatment, and the calcium content was quantified by spectrophotometry. One-way ANOVA with Tukey’s multiple comparisons test (C, D, E, F, H, I, J, K) and the Student's test (G) were used. Three independent experiments were performed, and representative data were shown. Data were shown as mean ± SD. ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05. ns: no significance. EC^NG-EVs, EVs derived from normal-glucose induced ECs; EC^HG-EVs, EVs derived from high-glucose induced ECs; Circ_Control, circular RNA negative control; circ_0008362-WT, circ_0008362-wild type plasmid; ARS, alizarin red s; RISC: RNA-induced silencing complex

Fig. 4

hsa_circ_0008362 interacted directly with Runx2 to promote VSMC calcification. A hsa_circ_0008362 had strong binding region with Runx2 protein. B The expression of Runx2 was measured by WB after biotin‐labelled circ_0008362 pull‐down assay. C, D The expression of hsa_circ_0008362 and mFBXW4 detected by qRT-PCR in VSMCs via anti-Runx2 RIP experiments, respectively. E The half-life of Runx2 protein was measured by WB in VSMCs treated with EC^NG-EVs or EC^HG-EVs, accompanied with CHX (5ug/ml) treatment. F The degradation of Runx2 protein was measured by WB in VSMCs treated with hsa_circ_0008362 plasmid, accompanied with CHX (5 ug/ml) treatment. G The level of Runx2 protein was measured by WB in VSMCs with different treatment. H ARS staining showed the mineralized nodules in VSMCs with different treatment, and the calcium content was quantified by spectrophotometry. One-way ANOVA with Tukey’s multiple comparisons test (B, C, D, H, G) was used. one-way ANOVA and Student’s t-test (E, F) were used. Three independent experiments were performed, and representative data were shown. Data were shown as mean ± SD. ****p < 0.0001, ***p < 0.001, **p < 0.005. ns, no significance; WB, western blot; EC^NG-EVs, EVs derived from normal-glucose induced ECs; EC^HG-EVs, EVs derived from high-glucose induced ECs; circ_0008362-WT, circ_0008362-wild type plasmid; ARS, alizarin red s; CHX, cycloheximide

Fig. 5

EC^HG-EVs mediated arterial calcification in diabetic mice. A–C Fluorescence signals were detected in the living mice and organs (aorta, heart, lung, kidney, liver and spleen), respectively, after 12 h post-injection of DiR-labeled EC^HG-EVs in mice. D ARS staining analysis showed the arterial calcification in different mice (n = 5/group). The arrows indicate calcified arteries (scale bar = 200 μm). E–F The expression of circ_0008362 and miR-1251-5p in different mice arterial tissues (n = 5/group). G Immunohistochemistry staining analysis of Runx2 expression in different mice arterial tissues. The arrows indicate the positive expression of Runx2 in the mice aorta (scale bar = 50 μm). One-way ANOVA with Tukey’s multiple comparisons test (C–G) was used. Three independent experiments were performed, and data were shown as mean ± SD. ****p < 0.0001, ***p < 0.001, **p < 0.005. EC^HG-EVs: EVs derived from high-glucose induced ECs; DM: diabetes mellitus; DMSO: Dimethyl sulfoxide; ARS: Alizarin red s

Fig. 6

Circulating plasma EVs hsa_circ_0008362 was correlated with arterial calcification in patients with DM. A–C CT images showed the coronary artery calcification as well as aorta artery calcification and quantified as CACs and AACs in normal control and DM group (n = 20). D The relative expression of hsa_circ_0008362 was detected by qRT-PCR in plasma EVs of normal control and patients with DM (n = 20). E–H Correlations between circ_0008362 of plasma EVs and CACs as well as AACs were analyzed using a Spearman’s correlation analysis in normal control and patients with DM (n = 20). The normality of data distribution was assessed by Shapiro–Wilk test and Non-parametric Mann–Whitney U test for C and D. ****p < 0.0001, ***p < 0.001. ns, no significance; DM, diabetes mellitus; CACs, coronary artery calcification scores; AACs, aorta artery calcification scores

Fig. 7

Mechanism of hsa_circ_0008362 derived from EC-EVs regulating diabetic arterial calcification. Under hyperglycemia, ECs are stimulated by HG to release EVs and carry circ_0008362 to VSMCs. Circ_0008362, on the one hand, sponge with miR-1251-5p to promote VSMC calcification by upregulating the target gene-Runx2. On the other hand, circ_0008362 would also directly interact with Runx2 protein to increase the expression of Runx2, and then further enhance VSMC calcification. Finally, the accumulation of VSMC calcification resulted in arterial calcification in patients with DM. HG: high glucose; DM: diabetes mellitus. The figure created with BioRender.com