Melatonin alleviates vascular calcification and ageing through exosomal miR‐204/miR‐211 cluster in a paracrine manner

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  Figure 1. Melatonin can antagonise VSMC osteogenic differentiation and senescence via an MT membrane receptor‐dependent manner. A‐C, Effects of melatonin (MT) on RUNX2 expression, ALP activity and senescence level. VSMCs were treated with 10 mM β‐glycerophosphate (β‐GP) and different concentrations of MT (10 nM, 100 nM, 1 μM, 10 μM and 100 μM) or vehicle for the control. A, The expression of RUNX2 was determined by Western blotting. B, ALP activity was measured using an ALP kit, normalised to the cellular protein content. C, SA‐β‐gal staining was performed to detect cell senescence level. Semi‐quantitative analysis of SA‐β‐gal–positive cells (left panel) and representative microphotographs (right panel) are shown. D, The protein levels of RUNX2 and p21 were determined by Western blotting in vehicle, luzindole, 10 μM MT plus luzindole or 10 μM MT‐treated VSMCs. E, ALP activity assays were measured using an ALP kit for vehicle, luzindole, 10 μM MT plus luzindole or 10 μM MT‐treated VSMCs. F, β‐GP–induced VSMCs were treated with vehicle, luzindole, 10 μM MT plus luzindole or 10 μM MT for 14 days and then subjected to Alizarin Red S staining. Calcium deposition was extracted with…

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  Figure 2. VSMC‐derived exosomes induced by MT inhibit VSMC calcification and senescence. A, Transmission electron microscopic analysis of exosomes is shown. Scale bar represents 100 nm. B, Nanoparticle tracking analysis of exosomes derived from VSMCs showed that the size of most of VSMC‐exos was approximately 80‐140 nm. C, Western blotting showed exosome‐specific proteins CD81, CD63, Synt‐1 and ALIX were abundant in exosomes compared with cell lysate. D, VSMCs incubated with PKH26 fluorescently labelled exosomes for 12 h. Confocal microscopy analyses used to identify the uptake of exosomes by VSMCs are shown (PKH26 in red, DAPI in blue). Magnification, 100×. E, The expression of RUNX2 and p21 was determined by Western blotting in VSMCs incubated with exosomes from MT‐treated VSMCs or exosomes from vehicle‐treated VSMCs. F, ALP activity was determined by an ALP kit in VSMCs incubated with exosomes from MT‐treated VSMCs or exosomes from vehicle‐treated VSMCs. G, Alizarin Red S staining and calcium content were measured in VSMCs incubated with exosomes from MT‐treated VSMCs or exosomes from vehicle‐treated VSMCs. Representative microscopic views are shown. Scale bar represents 200 µm. H,…

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  Figure 3. MT attenuated the calcification and senescence of VSMCs by affecting exosomes in a paracrine manner. VSMCs were treated with fresh conditional medium (control), whole MT‐treated VSMCs conditional medium (MT‐VSMCs‐CM), MT‐treated VSMCs exosome‐free conditional medium (MT‐VSMCs‐exo‐free‐CM) or MT‐treated VSMCs exosome with GW4869 conditional medium (MT‐VSMCs‐GW4869‐CM), respectively. All groups were incubated with β‐GP at the same time. A, The protein levels of RUNX2 and p21 were determined by Western blotting. B, ALP activity assays were determined with an ALP measurement kit. C, CCK8 assay was performed to detect cell proliferation. D, Schematic representation of coculture Transwell system to analyse the exosomal paracrine effects of VSMCs. VSMCs in lower chamber incubated with MT and treated with or without GW4869 were cocultured with VSMCs without treatment in the upper chamber in six‐well Transwell units. And the upper chamber VSMCs were used to determine the expression of RUXN2, p21, ALP activities and cell viability. E, Western blotting showed that RUNX2 and p21 increased in the VSMCs from the upper chamber in MT plus GW4869 group compared with MT plus vehicle. F, ALP…

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  Figure 4. MT induced exosomal miR‐204 and miR‐211 antagonise VSMC osteogenic differentiation and senescence. A, qRT‐PCR analysis of eight microRNAs in exosomes derived from MT‐treated VSMCs or vehicle‐treated VSMCs. B, qRT‐PCR analysis of miR‐204 and miR‐211 expression in VSMCs treated with MT or vehicle. C, qRT‐PCR analysis of miR‐204 and miR‐211 in VSMCs after miR‐204/211 mimics or mimics control transfection. D, Confocal microscopy analysis was used to verify exosomal miR‐204 could be uptaken by VSMCs. VSMCs were cultured with PKH26‐labelled exosomes derived from VSMCs transfected FAM‐miR‐204‐mimics. The FAM‐miR‐204 signals were detected in the cytoplasm of VSMCs (Green), and FAM‐miR‐204 signals were co‐localised with PKH26 in VSMCs (PKH26 in red, DAPI in blue). Magnification, 100×. E, The expression of RUNX2 and p21 was determined with Western blotting in VSMCs treated with exosomes from miR‐204/211 overexpression VSMCs or exosomes from mimic control transfected VSMCs. F, ALP activity was determined using an ALP kit in VSMCs treated with exosomes from miR‐204/211 overexpression VSMCs or exosomes from mimic control transfected VSMCs. G, VSMCs treated with exosomes from miR‐204/211…

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  Figure 5. BMP2 is the direct target gene of miR‐204 and miR‐211. A, Schematic representation of miR‐204/211 putative target sites in BMP2 3′‐UTR and alignment of miR‐204/211 with WT and MUT BMP2 3′‐UTR showing pairing. B, Luciferase reporter assays were performed using luciferase constructs carrying a WT or mutant BMP2 3′‐UTR cotransfected into VSMCs with miR‐204/211 mimics compared with an empty vector control. Firefly luciferase activity was normalised to Renilla luciferase activity. C, miR‐204/211 mimics or miR‐204/211 inhibitor regulated BMP2, RUNX2 and p21 protein expression as indicated by Western blotting. D, Knock‐down of BMP2 by siRNA decreased the protein level of BMP2, RUNX2 and p21 as indicated by Western blotting. BMP2 siRNA attenuated the effect of exosomes from MT‐treated VSMCs (MT exo) on RUNX2 and p21 expression detected by Western blotting. E, ALP activity was measured with an ALP measurement kit in the VSMCs with BMP2 siRNA group, BMP2 siRNA plus exosomes from MT‐treated VSMC group (MT exo) and the siRNA group. F, CCK8 assay was measured in the VSMCs with BMP2 siRNA group, BMP2 siRNA plus exosomes from MT‐treated VSMCs (MT exo) and the siRNA group. G and H, VSMCs treat…

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  Figure 6. MT alleviates vascular calcification and ageing in the 5/6 NTP‐induced mouse model. A, Schematic flow diagram representing the in vivo treatment of MT with or without GW4869 in the 5/6 NTP mouse model. Sham operation mice with high‐phosphate diet were used as control (n = 5 per group). B, Calcium content of the thoracic aorta of mice was measured by o‐cresolphthalein complexone method. C, The ALP activity of thoracic aortic tissues was measured by the specific kit and normalised to total proteins contents. D, Representative microscopic pictures of Von Kossa‐stained and Alizarin Red S‐stained sections from the thoracic aorta (left panel) and quantitation of positive staining area (right panel) are shown. Scare bar 200 µm (Black). E, The expression of RUNX2 in thoracic aorta was examined by immunohistochemistry (IHC) (left panel), and quantitation of positive staining area (right panel) is shown. Scale bar 50 µm (Blue) and 500 µm (Red). F, Representative vascular SA‐β‐gal staining pictures are presented (left panel), and quantitation of positive staining area (right panel) is shown. Scale bar 50 µm (Blue). Green staining area indicates ageing tissues. G, The expression of p21 in…

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  Figure 7. Exosomal miR‐204/miR‐211 contributes to the inhibition effects of VSMCsMT‐exos on vascular calcification and ageing in a 5/6 NTP mouse model. A, The 5/6 NTP‐induced mice were subjected to vehicle (sterile PBS), DiR or DiR‐labelled VSMC‐derived exosomes (VSMCs‐exos) intravenously (100 μg/mice, n = 5 per group). Representative in vivo images of mice (A) and ex vivo images of thoracic aorta (B, upper panel), as well as quantification of fluorescence intensities in thoracic aorta (B, lower panel), are shown after treatment for 24 h. (C and D) Exosomes from MT‐treated VSMCs (VSMCsMT‐exos, 100 μg/mice) or vehicle‐treated VSMCs (VSMCsVeh‐exos, 100 μg/mice) were injected intravenously every 3 d for 4 wk in 5/6 NTP mice (n = 5 per group). C, Representative confocal fluorescence images of exosome marker TSG101 (red fluorescence) and smooth muscle marker αSMA (green fluorescence) in thoracic aorta sections are shown. Scare bar represents 50 µm (yellow). D, Relative expression of miR‐204 and miR‐211 in the artery was detected by qRT‐PCR. E, Schematic flow diagram of injections of exosomes with or without miR‐204 and miR‐211 in 5/6 NTP‐induced mice (n = 5 per group). F, Calcium content of…

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  Figure 8. The mechanism diagram about exosomes from MT‐treated VSMCs could attenuate vascular calcification and ageing in a paracrine manner through an exosomal miR‐204/miR‐211. MT can antagonise VSMC osteogenic differentiation and senescence through exosome paracrine mechanism. In addition, MT‐treated VSMCs can secrete exosomal miR‐204/miR‐211 to antagonise VSMC osteogenic differentiation and senescence as well as vascular calcification and ageing. BMP2 was revealed to be a potential target of miR‐204 and miR‐211 and increased VSMC osteogenic differentiation and senescence