BackgroundTo determine whether and how exosomes from human umbilical vein endothelial cells (HUVEC-Exos) regulates vascular smooth muscle cells (VSMCs) calcification/senescence in high glucose condition.MethodsHUVEC-Exos were isolated from normal glucose (NG) and high glucose (HG) stimulated HUVECs (NG/HG-HUVEC-Exos) by super speed centrifugation. HUVEC-Exos were identified by transmission electron microscopy and Western blot of CD63. Protein profile in HUVEC-Exos was examined to screen the candidate molecules that mediate HUVEC-Exos function. VSMCs were incubated with HUVEC-Exos. A series of functional assays in vitro were performed to assess the effects of HUVEC-Exos on the calcification/senescence of VSMCs. The role of the candidate protein in HUVEC-Exos-induced VSMCs dysfunction was assessed.ResultsExosomes isolated from HG-HUVEC-Exos induced calcification/senescence in VSMCs as assessed by Alizarin Red Staining, senescence-associated β-galactosidase (SA-β-gal) staining, and the expression of ALP and p21. HG-HUVEC-Exos significantly increased LDH activity, as well as the product of lipid peroxidation (MDA content), and decreased oxidative stress marker activity, as compared with NG-HUVEC-Exos. Moreover, mechanism studies showed that mitochondrial membrane potential and the expression levels of mitochondrial function related protein HADHA and Cox-4 were significantly decreased in HG-HUVEC-Exos compared to controls. Proteomic analysis showed that HG-HUVEC-Exos consisted of higher level of versican (VCAN), as compared with NG-HUVEC-Exos. Observation under laser confocal microscopy revealed that most green fluorescence of VCAN could overlap with the red fluorescence came from mitochondria, indicating VCAN is mainly localized to the mitochondria of VSMCs. Knockdown of VCAN with siRNA in HUVECs, inhibited HG-HUVEC-Exos-induced mitochondrial dysfunction and calcification/senescence of VSMCs.ConclusionsOur data indicate an intracellular role for VCAN in VSMCs. VCAN participates in hyperglycemia-induced calcification/senescence via modulation of mitochondrial function in VSMCs.Electronic supplementary materialThe online version of this article (10.1186/s13578-018-0263-x) contains supplementary material, which is available to authorized users.

In the elderly with atherosclerosis, hypertension and diabetes, vascular calcification and ageing are ubiquitous. Melatonin (MT) has been demonstrated to impact the cardiovascular system. In this study, we have shown that MT alleviates vascular calcification and ageing, and the underlying mechanism involved. We found that both osteogenic differentiation and senescence of vascular smooth muscle cells (VSMCs) were attenuated by MT in a MT membrane receptor‐dependent manner. Moreover, exosomes isolated from VSMCs or calcifying vascular smooth muscle cells (CVSMCs) treated with MT could be uptaken by VSMCs and attenuated the osteogenic differentiation and senescence of VSMCs or CVSMCs, respectively. Moreover, we used conditional medium from MT‐treated VSMCs and Transwell assay to confirm exosomes secreted by MT‐treated VSMCs attenuated the osteogenic differentiation and senescence of VSMCs through paracrine mechanism. We also found exosomal miR‐204/miR‐211 mediated the paracrine effect of exosomes secreted by VSMCs. A potential target of these two miRs was revealed to be BMP2. Furthermore, treatment of MT alleviated vascular calcification and ageing in 5/6‐nephrectomy plus high‐phosphate diet‐treated (5/6 NTP) mice, while these effects were partially reversed by GW4869. Exosomes derived from MT‐treated VSMCs were internalised into mouse artery detected by in vivo fluorescence image, and these exosomes reduced vascular calcification and ageing of 5/6 NTP mice, but both effects were largely abolished by inhibition of exosomal miR‐204 or miR‐211. In summary, our present study revealed that exosomes from MT‐treated VSMCs could attenuate vascular calcification and ageing in a paracrine manner through an exosomal miR‐204/miR‐211.

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