Background
Vascular calcification (VC) is a pathological deposition of hydroxyapatite crystals in vascular wall, which is common in chronic kidney disease (CKD) [
1]. As an independent predictor of cardiovascular events, VC is also significantly associated with mortality in CKD [
2,
3]. There are multifactor (including metabolic disorders of serum phosphate and calcium, inflammatory cytokines, oxidative stress, and uremic toxins) accelerating VC in CKD patients, among which hyperphosphatemia (HP) is most strongly associated with VC [
2]. It has been reported that endothelial extracellular vesicles (EC-EVs) can be released under HP circumstances [
4]. However, there is rare information about the relationship between HP-EC-EVs and vascular smooth muscle cell (VSMC) calcification.
EC-EVs are formed from the outward budding of endothelial cells (EC) plasma membranes, and secreted to the extracellular chamber during stimuli such as HP [
5,
6]. More and more researchers focus on the function of microRNAs (miRs) in EC-EVs. Several studies implicated that EC-EVs play an important role in inflammation, angiogenesis, and thrombosis by delivering miRs [
7,
8]. There is a report showing that miR-126 and miR-26a in EC-EVs are significantly reduced in diabetic patients compared to non-diabetic patients [
9]. Moreover, it has been demonstrated that miR-145-5p and miR-320a in EC-EVs could contribute to the progression of vasculitis [
10]. Additionally, it has been reported that miR-29b, miR-133b, and miR-211 regulated VSMC calcification induced by HP [
11]. Nevertheless, the different expression of miRs in HP-EC-EVs has not been determined.
In the current study, we firstly isolated HP-EC-EVs from endothelial cells treated with HP. We then observed the process of HP-EC-EVs being up-taken by VSMCs. The VSMC calcium deposition mediated by HP-EC-EVs was characterized by Alizarin Red S, quantified by colourimetric analysis and ALP activity. To better understand the mechanism of HP-EC-EVs-induced VSMC calcification in vitro, we detected the expression of miRs in HP-EC-EVs via high-throughput sequencing. Differentially expressed miRs (DEMs) from HP-EC-EVs, target genes of DEMs, and their related signaling pathway were performed by using bioinformatics analysis. The content of HP-EC-EVs may drive activation or down regulation of pathways related to vascular calcification.
Discussion
There is increasing evidence that miRs play an important role in vascular calcification, and several researches have investigated the association among hyperphosphatemia, miRs in VSMC or its vesicles, and vascular calcification [
11,
15]. However, there is rare information about the miR expression in HP-EC-EVs.
There are report that endothelial dysfunction can cause vascular calcification through BMP activation [
16]. Hyperphosphatemia is a well-known factor that induces vascular calcification in CKD [
17]. In the current study, our results indicated that HP-EC-EVs from injured endothelial cells might have the ability to induce VSMC calcification, and we observed the capture of HP-EC-EVs by VSMCs. Those captured HP-EC-EVs packaging miRs, might lead VSMCs calcification. Our results implicated that HP-EC-EVs and its cargos such as miRs potentially provide a link between the communication of activated endothelial cells and VSMCs to induce vascular calcification under hyperphosphatemia circumstances. Thus, we decided to explore the miR expression of HP-EC-EVs to better understand the CKD calcification.
RNA-seq and bioinformatics analysis were carried out to understand the different expression of miRs between HP-EC-EVs group and PBS-EC-EVs group. Those miRs (including hsa-miR-143-3p, hsa-miR-30c-2-3p, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-486-5p, and hsa-miR-193b-5p) were associated with vascular calcification, and statistically down-regulated in HP-EC-EVs group. miR-143 is a potential biomarker of vascular calcification and cardiovascular disease associated with CKD, and it was down-regulated in VSMCs during the time course of Pi-induced vascular calcification [
18‐
20]. Our current results showed that miR-143-3p was remarkably down-regulated in HP-EC-EVs. In consistent with our results, miR-143 expression in extracellular vesicles (EV) derived from urea and indoxyl sulphate-stimulated EC (EV
UR) were also down-regulated, and mimicking of miR-143 in EV
UR blocked the pro-calcifying effects of EV
UR [
21]. Among those miRs, the miR-30 family has been reported to play an important role in osteogenesis [
22]. Our analysis revealed low level of miR-30c-2-3p in HP-EC-EVs. The published research that BMP-2 down-regulation miR-30c to increase Runx2 expression in human coronary artery SMCs and promoting mineralization may partly explain our result [
23]. Additionally, the expression of miR-30a was significantly higher in VSMCs during vascular calcification [
24‐
26]. However, miR-30a-3p and miR-30a-5p was significantly reduced in the HP-EC-EVs compared to controls in this study. This discrepancy may be due to miR different expression in VSMCs and HP-EC-EVs. Three novel miR (hsa-miR-novel-Chr11_14558, hsa-miR-novel-Chr11_14340, hsa-miR-novel-Chr1_1551) were also significantly down-regulated in HP-EC-EVs, but its structure and function remain elusive. Therefore, HP-EMP-induced VSMC calcification may be associated with the down-regulation of the abovementioned miRs.
In contrast, hsa-miR-3182 was markedly up-regulated in HP-EC-EVs. A recent research showed that hsa-miR-3182 and its host coding genes are genetically associated with cardiovascular disease [
27], which supported our findings. Our analysis demonstrated that those target mRNAs for hsa-miR-3182 included NOD2, ZCCHC14, DMTF1, STAM2, SLC22A25, AOAH, KIAA1109, HECTD4, SOGA3, RBM47, ADGRL3, PTPRT, KIAA0408, SLC16A7, PSTPIP1. In consistent with our results, several lines of evidence demonstrated that NOD2 deficiency enhanced pulmonary VSMC proliferation, and exacerbated plaque necrosis in advanced atherosclerotic lesions [
28,
29]. Furthermore, Sanneke et al. observed that SLC22A25 gene had strong signals associated with plaque morphology [
30]. Additionally, RBM47 was identified to be associated with blood pressure or hypertension [
31]. Taken together, target genes of hsa-miR-3182 may be associated with vascular calcification through regulation of blood pressure, VSMC proliferation, plaque morphology and necrosis. Surprisingly, only one up-regulated known miR was found while others were all belong to novel miRs. This remains us that we lack the understanding for those miRs, and further work about those differentially expressed miRs, especially those novel miRs, in HP-EC-EVs is still needed in the future.
In order to explore the regulatory mechanisms of differentially expressed miRs between HP-EC-EVs and PBS-EC-EVs, we annotated biological functions of miRs, predicted their targets and constructed regulation networks. This is the first study to identify a number of putative miR-mRNA interactions for DEMs in HP-EC-EVs (Fig.
4). GO enrichment analysis of DEMs in HP-EC-EVs is useful to describe BP, CC, and MF in relation to predicted target gene candidates.
In the current study, our top-20 KEGG pathway analysis revealed that calcium signaling pathway, cAMP signaling pathway, and ABC transporters were closely related to vascular calcification [
32]. Early studies have suggested that roles for cellular calcium signaling involved in the regulation of calcification [
33]. Integrative genomic study confirmed calcium signaling pathway genes RUNX2 and CACNA1C are associated with calcific disease [
34]. Previous study has demonstrated that cAMP pathway promotes in vitro vascular calcification by enhancing osteoblast-like differentiation of calcifying vascular cells [
35]. Later, Prosdocimo et al. reported that increased cAMP signaling and elevated extracellular inorganic phosphate (Pi) act synergistically to induce calcification of VSMC [
36]. Thus, it is reasonable to understand that increased calcium signaling pathway or cAMP signaling pathway could be involved in the HP-EMP-induced VSMC calcification in vitro. ABC transporters represent a large family of ATP-driven transmembrane transporters involved in uni- or bidirectional transfer of substrates [
37]. ABCC6, a unidirectional exporter protein, has been reported that its deficiency could alter ABC transporter gene expression and cause the ectopic mineralization disorder, characterized by calcification [
37,
38]. Taken together, up-regulation of calcium signaling pathway or cAMP signaling pathway, and down-regulation of ABC transporters may be important in HP-EMP-induced VSMC calcification.
Surprisingly, except the vascular calcification-related pathways mentioned above, signaling pathways associated with cancer (such as Choline metabolism in cancer, MicroRNAs in cancer, and proteoglycans in cancer), endocrine system (such as Cushing syndrome, thyroid hormone signaling pathway, GnRH signaling pathway and endocrine resistance), nervous system (such as Rap1 signaling pathway, Axon guidance, signaling pathway regulating pluripotency of stem cells, and neurotrophin signaling pathway), and cardiomyopathy (such as dilated cardiomyopathy, arrhythmogenic right ventriculart cardiomyopathy, and hypertrophic cardiomyopathy) were also found in the HP-EC-EVs. Actually, EC-EVs have been reported to be a potentially useful biomarker of endothelial dysfunction in heart failure risk stratification [
39]. However, there is rare information about the role of HP-EC-EVs in CKD or uremia patients. Our current study revealed that HP-EC-EVs may not only play important role in vascular calcification, but also in cardiomyopathy, nervous disease such as uremic encephalopathy, cancer, and endocrine disease in ESRD patients. This shed new light on the possible mechanism of several diseases in uremia patients, and further study about the potential role of HP-EC-EVs in those diseases especially for the cardio cerebrovascular disease leading high morbidity in CKD, should be explored in the future.
There are some limitations in this study. Our current study only focused on EVs induced by HP-ECs in vitro, which are not an arterial model. Although HP is important in VC, it is not the only factor and Pi binders is disappointed in the management of vascular calcification in CKD [
40]. Thus, it is not clear whether these findings would apply to human disease. Considering the limitation of this study, further researches are absolutely needed to evaluate these findings, especially in vivo.
Conclusion
This is the first study to perform miR-seq analysis in HP-EC-EVs. We identified 12 known down-regulated miRs (hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-143-3p, hsa-miR-193b-5p, hsa-miR-30c-2-3p, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-365a-5p, hsa-miR-486-5p, hsa-miR-7706, hsa-miR-941 and hsa-miR-99b-5p) and 1 (hsa-miR-3182) known up-regulated miR. We also constructed miR-mRNA network, and performed GO term and related pathway analysis of the targets to predict the biological function of the altered miRs. We find that calcium signaling pathway, cAMP signaling pathway, and ABC transporters may have their special role in regulating vascular calcification. Our findings indicated that HP-EC-EVs might induce VSMC calcification and they do activate several pathways in the VSMCs. The differentially expressed miRs packaged in HP-EC-EVs might shed light on the mechanism of vascular calcification.
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