ABSTRACT: The senescence of vascular smooth muscle cells (VSMCs), characterized by the acquisition of senescence-associated secretory phenotype (SASP), is relevant for VSMCs osteoblastic differentiation and vascular calcification (VC). MicroRNA-34a (miR-34a) is a driver of such phenomena and could play a role in vascular inflammaging. Herein, we analyzed the relationship between miR-34a and the prototypical SASP component IL6 in in vitro and in vivo models. miR-34a and IL6 levels increased and positively correlated in aortas of 21 months-old male C57BL/6J mice and in human aortic smooth muscle cells (HASMCs) isolated from donors of different age and undergone senescence. Lentiviral overexpression of miR-34a in HASMCs enhanced IL6 secretion. HASMCs senescence and calcification accelerated after exposure to conditioned medium of miR-34a-overexpressing cells. Analysis of miR-34a-induced secretome revealed enhancement of several pro-inflammatory cytokines and chemokines, including IL6, pro-senescent growth factors and matrix-degrading molecules. Moreover, induction of aortas medial calcification and concomitant IL6 expression, with an overdose of vitamin D, was reduced in male C57BL/6J Mir34a-/- mice. Finally, a positive correlation was observed between circulating miR-34a and IL6 in healthy subjects of 20-90 years. Hence, the vascular age-associated miR-34a promotes VSMCs SASP activation and contributes to arterial inflammation and dysfunctions such as VC.
Project description:Vascular calcification/aging is common in diabetes and is associated with increased morbidity and mortality of patients. MiR-34c-5p, not miR-34c-3p, was suppressed significantly in calcification/senescence of human aorta vascular smooth muscle cells (HA-VSMCs) induced by high glucose, which was proven by the formation of mineralized nodules and staining of senescence associated-β-galactosidase staining (SA β-gal) positive cells. Overexpression of miR-34c-5p alleviated calcification/senescence of HA-VSMCs, whereas inhibition of miR-34c-5p received the opposite results. Bcl-2 modifying factor (BMF) was a functional target of miR-34c-5p and it was involved in the process of calcification/senescence of HA-VSMCs. Besides, lncRNA-ES3 acted as a competing endogenous RNAs (ceRNA) of miR-34c-5p to enhance BMF expression. Further, lncRNA-ES3 inhibited miR-34c-5p expression by direct interaction and its knockdown suppressed the calcification/senescence of HA-VSMCs. Our results showed for the first time that the calcification/senescence of VSMCs was regulated by lncRNA-ES3 /miR-34c-5p/BMF axis.
Project description:Background: The osteogenic differentiation of vascular smooth muscle cell (VSMCs) is important for the development of vascular calcification (VC), particularly in diabetes. Exosomes derived from Mesenchymal Stromal Cells (MSCs) are effective against cardiovascular diseases, yet their role in VC remains unclear. Advanced glycation end products (AGEs) inhibit bone marrow stromal cell osteogenesis by targeting osteogenesis-associated genes. Thus, we investigated the role of exosomes derived from MSCs pretreated with AGEs-BSA in VC and its potential mechanisms. Methods: Primary VSMCs and MSCs were isolated from the aorta and bone marrow of Sprague-Dawley rats, respectively. VSMCs were cultured with AGEs-BSA to induce osteogenic differentiation. Exosomes were harvested from MSCs by ultracentrifugation. MSCs and VSMCs were cocultured in Transwells, and exosomes were added to VSMC culture medium to assess their effects on osteogenic differentiation. Double luciferase reporter assay was applied to confirm that miR-146a directly targets the 3' UTR of the thioredoxin-interacting protein (TXNIP) gene. Results: Pretreatment of VSMCs with AGEs-BSA increased the expression of thioredoxin-interacting protein (TXNIP) by inhibiting that of miR-146a, resulting in enhanced ROS production and VSMC calcification. By contrast, the expression of miR-146a in MSCs was increased by AGEs-BSA treatment. Thus, miR-146a was transferred from AGEs-BSA-pretreated or miR-146a-transfected MSCs to VSMCs via exosomes. After coculture with miR-146a-containing exosomes, the AGEs-BSA-mediated increase in VSMC calcification was diminished, accompanied by decreased TXNIP expression and ROS production. Furthermore, TXNIP overexpression counteracted the anti-calcification effects of MSC-derived miR-146a-containing exosomes. In addition, TXNIP was identified as a target gene of miR-146a, and the results of double luciferase reporter assay confirmed that TXNIP was the direct target gene of miR-146a. Conclusions: Exosomes secreted by MSCs pretreated with AGEs-BSA contained a high level of miR-146a, which was transferred to VSMCs and inhibited AGEs-BSA-induced calcification in a TXNIP-dependent manner. Thus, miR-146a-containing exosomes may be a potential therapeutic target for VC.
Project description: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.
Project description:Unraveling the complex regulatory pathways that mediate the effects of phosphate on vascular smooth muscle cells (VSMCs) may provide novel targets and therapies to limit the destructive effects of vascular calcification (VC) in patients with chronic kidney disease (CKD). Our previous studies have highlighted several signaling networks associated with VSMC autophagy, but the underlying mechanisms remain poorly understood. Thereafter, the current study was performed to characterize the functional relevance of O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) in high phosphate-induced VC in CKD settings. We generated VC models in 5/6 nephrectomized rats in vivo and VSMC calcification models in vitro. Artificial modulation of OGT (knockdown and overexpression) was performed to explore the role of OGT in VSMC autophagy and VC in thoracic aorta, and in vivo experiments were used to substantiate in vitro findings. Mechanistically, co-immunoprecipitation (Co-IP) assay was performed to examine interaction between OGT and kelch like ECH associated protein 1 (KEAP1), and in vivo ubiquitination assay was performed to examine ubiquitination extent of nuclear factor erythroid 2-related factor 2 (NRF2). OGT was highly expressed in high phosphate-induced 5/6 nephrectomized rats and VSMCs. OGT silencing was shown to suppress high phosphate-induced calcification of VSMCs. OGT enhances KEAP1 glycosylation and thereby results in degradation and ubiquitination of NRF2, concurrently inhibiting VSMC autophagy to promote VSMC calcification in 5/6 nephrectomized rats. OGT inhibits VSMC autophagy through the KEAP1/NRF2 axis and thus accelerates high phosphate-induced VC in CKD.
Project description:Aims:Vascular calcification (VC) is a primary risk factor for cardiovascular mortality in chronic renal failure (CRF) patients; thus, effective therapeutic targets are urgently needed to be explored. Here, we identified the role of intestinal bacterial translocation in CRF-related VC. Methods and Results:Antibiotic supplementation by oral gavage significantly suppressed intestinal bacterial translocation, CRF-related VC, and aortic osteogenic gene and Toll-like receptor (TLR) gene expression in CRF rats. Furthermore, TLR4 and TLR9 activation in vascular smooth muscle cells (VSMCs) aggravated inorganic phosphate- (Pi-) induced calcification. TLR9 inhibition, but not TLR4 inhibition, by both a pharmacological inhibitor and genetic methods could significantly reduce CRF rats' serum or CRF-induced VC. Interestingly, bone morphogenic protein-2 (BMP-2) levels were increased in the aorta and sera from CRF rats. Increased BMP-2 levels were also observed in VSMCs treated with TLR9 agonist, which was blocked by NF-?B inhibition. Both siRNA knockdown of BMP-2 and NF-?B inhibitor significantly blocked TLR9 agonist-induced VSMC calcification. Conclusions:Gut bacterial translocation inhibited by oral antibiotic significantly reduces CRF-related VC through inhibition of TLR9/NF-?B/BMP-2 signaling.
Project description:Children on dialysis have a cardiovascular mortality risk equivalent to older adults in the general population, and rapidly develop medial vascular calcification, an age-associated pathology. We hypothesized that premature vascular ageing contributes to calcification in children with advanced chronic kidney disease (CKD). Vessels from children with Stage 5 CKD with and without dialysis had evidence of increased oxidative DNA damage. The senescence markers p16 and p21 were also increased in vessels from children on dialysis. Treatment of vessel rings ex vivo with calcifying media increased oxidative DNA damage in vessels from children with Stage 5 CKD, but not in those from healthy controls. Vascular smooth muscle cells cultured from children on dialysis exhibited persistent DNA damage, impaired DNA damage repair, and accelerated senescence. Under calcifying conditions vascular smooth muscle cells from children on dialysis showed increased osteogenic differentiation and calcification. These changes correlated with activation of the senescence-associated secretory phenotype (SASP), an inflammatory phenotype characterized by the secretion of proinflammatory cytokines and growth factors. Blockade of ataxia-telangiectasia mutated (ATM)-mediated DNA damage signaling reduced both inflammation and calcification. Clinically, children on dialysis had elevated circulating levels of osteogenic SASP factors that correlated with increased vascular stiffness and coronary artery calcification. These data imply that dysregulated mineral metabolism drives vascular "inflammaging" by promoting oxidative DNA damage, premature senescence, and activation of a pro-inflammatory SASP. Drugs that target DNA damage signaling or eliminate senescent cells may have the potential to prevent vascular calcification in patients with advanced CKD.
Project description:Vascular calcification is one of the most important factors for cardiovascular and all-cause mortality in patients with end-stage renal diseases (ESRD). The current study was aimed to investigate the function and mechanisms of miR-34b on the calcification of vascular smooth muscle cells (VSMCs) both in vitro and in vivo. We found that the expression of miR-34b was significantly suppressed in VSMCs with high inorganic phosphate (Pi) treatment, as well as mouse arteries derived from 5/6 nephrectomy with a high-phosphate diet (0.9% Pi, 5/6 NTP) and human renal arteries from uraemia patients. Overexpression of miR-34b alleviated calcification of VSMCs, while VSMCs calcification was enhanced by inhibiting the expression of miR-34b. Bisulphite sequencing PCR (BSP) uncovered that CpG sites upstream of miR-34b DNA were hypermethylated in calcified VSMCs and calcified arteries due to 5/6 NTP, as well as calcified renal arterial tissues from uraemia patients. Meantime, increased DNA methyltransferase 3a (DNMT3a) resulted in the hypermethylation of miR-34b in VSMCs, while 5-aza-2'-deoxycytidine (5-aza) reduced the methylation rate of miR-34b and restored the expression of miR-34b in VSMCs. When DNMT3a was knocked down using DNMT3a siRNA, the effect of 3.5 mM of Pi on calcification of VSMCs was abrogated. In addition, Notch1 was validated as the functional target of miR-34b and involved in the process of calcification of VSMCs. Taken together, our data showed a specific role for miR-34b in regulating calcification of VSMCs both in vitro and in vivo, which was regulated by upstream DNA methylation of miR-34b and modulated by the downstream target gene expression, Notch1. These results suggested that modulation of miR-34b may offer new insight into a novel therapeutic approach for vascular calcification.
Project description:Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP-/- mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, ?-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs.
Project description:Vascular calcification (VC) is characterized by calcium deposition inside arteries and is closely associated with the morbidity and mortality of atherosclerosis, chronic kidney disease, diabetes, and other cardiovascular diseases (CVDs). VC is now widely known to be an active process occurring in vascular smooth muscle cells (VSMCs) involving multiple mechanisms and factors. These mechanisms share features with the process of bone formation, since the phenotype switching from the contractile to the osteochondrogenic phenotype also occurs in VSMCs during VC. In addition, VC can be regulated by epigenetic factors, including DNA methylation, histone modification, and noncoding RNAs. Although VC is commonly observed in patients with chronic kidney disease and CVD, specific drugs for VC have not been developed. Thus, discovering novel therapeutic targets may be necessary. In this review, we summarize the current experimental evidence regarding the role of epigenetic regulators including histone deacetylases and propose the therapeutic implication of these regulators in the treatment of VC.
Project description:Vascular calcification is a common phenomenon in older adults. Intermedin (IMD) is a cardiovascular bioactive peptide inhibiting vascular calcification. In this study, we aimed to investigate whether IMD1-53 attenuates aging-associated vascular calcification. Vascular calcification was induced by vitamin D3 plus nicotine (VDN) in young and old rats. The calcification in aortas was more severe in old rats treated with VDN than young control rats, and IMD expression was lower. Exogenous administration of IMD1-53 significantly inhibited the calcium deposition in aortas and the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) in VDN-treated old rats. Moreover, levels of aging-related p16, p21 and ?-galactosidase were all greatly decreased by IMD1-53. These results were further confirmed in rat and human VSMCs in vitro. In addition, IMD-deficient mouse VSMCs showed senescence features coinciding with osteogenic transition as compared with wild-type mouse VSMCs. Mechanistically, IMD1-53 significantly increased the expression of the anti-aging factor sirtuin 1 (sirt1); the inhibitory effects of IMD1-53 on calcification and senescence were blocked by sirt1 knockdown. Furthermore, preincubation with inhibitors of PI3K, AMPK or PKA efficiently blunted the upregulatory effect of IMD1-53 on sirt1. Consequently, IMD1-53 could attenuate aging-associated vascular calcification by upregulating sirt1 via activating PI3K/Akt, AMPK and cAMP/PKA signaling.