Inhibition of RIPK1 alleviating vascular smooth muscle cells osteogenic transdifferentiation via Runx2
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ABSTRACT:
SUBMITTER: Li Y
PROVIDER: S-EPMC10839642 | biostudies-literature | 2023 Dec
REPOSITORIES: biostudies-literature
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Project description:Vascular calcification (VC) is regarded as an important pathological change lacking effective treatment and associated with high mortality. Sirtuin 6 (SIRT6) is a member of the Sirtuin family, a class III histone deacetylase and a key epigenetic regulator. SIRT6 has a protective role in patients with chronic kidney disease (CKD). However, the exact role and molecular mechanism of SIRT6 in VC in patients with CKD remain unclear. Here, we demonstrated that SIRT6 was markedly downregulated in peripheral blood mononuclear cells (PBMCs) and in the radial artery tissue of patients with CKD with VC. SIRT6-transgenic (SIRT6-Tg) mice showed alleviated VC, while vascular smooth muscle cell-specific (VSMC-specific) SIRT6 knocked-down mice showed severe VC in CKD. SIRT6 suppressed the osteogenic transdifferentiation of VSMCs via regulation of runt-related transcription factor 2 (Runx2). Coimmunoprecipitation (co-IP) and immunoprecipitation (IP) assays confirmed that SIRT6 bound to Runx2. Moreover, Runx2 was deacetylated by SIRT6 and further promoted nuclear export via exportin 1 (XPO1), which in turn caused degradation of Runx2 through the ubiquitin-proteasome system. These results demonstrated that SIRT6 prevented VC by suppressing the osteogenic transdifferentiation of VSMCs, and as such targeting SIRT6 may be an appealing therapeutic target for VC in CKD.
| S-EPMC8718147 | biostudies-literature
Project description:Vascular calcification is associated with significant morbidity and mortality within diabetes, involving activation of osteogenic regulators and transcription factors. Recent evidence demonstrates the beneficial role of Sirtuin 1 (SIRT1), an NAD+ dependant deacetylase, in improved insulin sensitivity and glucose homeostasis, linking hyperglycaemia and SIRT1 downregulation. This study aimed to determine the role of SIRT1 in vascular smooth muscle cell (vSMC) calcification within the diabetic environment. An 80% reduction in SIRT1 levels was observed in patients with diabetes, both in serum and the arterial smooth muscle layer, whilst both RUNX2 and Osteocalcin levels were elevated. Human vSMCs exposed to hyperglycaemic conditions in vitro demonstrated enhanced calcification, which was positively associated with the induction of cellular senescence, verified by senescence-associated β-galactosidase activity and cell cycle markers p16 and p21. Activation of SIRT1 by SRT1720 reduced Alizarin red staining by a third, via inhibition of the RUNX2 pathway and prevention of senescence. Conversely, inhibition of SIRT1 via Sirtinol and siRNA increased RUNX2 by over 50%. These findings demonstrate the key role that SIRT1 plays in preventing calcification in a diabetic environment, through the inhibition of RUNX2 and senescence pathways, suggesting a downregulation of SIRT1 may be responsible for perpetuating vascular calcification in diabetes.
| S-EPMC6351547 | biostudies-literature
Project description:Extracellular vesicles (EV) function as messengers between endothelial cells (EC) and vascular smooth muscle cells (VSMC). Since chronic kidney disease (CKD) increases the risk for vascular calcifications, we investigated whether EV derived from uraemic milieu-stimulated EC and derived from uraemic rats impact the osteogenic transdifferentiation/calcification of VSMC. For that purpose, human EC were treated with urea and indoxyl sulphate or left untreated. Experimental uraemia in rats was induced by adenine feeding. 'Uraemic' and control EV (EVUR ; EVCTRL ) were isolated from supernatants and plasma by using an exosome isolation reagent. Rat VSMC were treated with a pro-calcifying medium (CM) with or without EV supplementation. Gene expressions, miRNA contents and protein expressions were determined by qPCR and Western blots, respectively. Calcifications were determined by colorimetric assays. Delivery of miRNA inhibitors/mimics to EV and siRNA to VSMC was achieved via transfection. EVCTRL and EVUR differed in size and miRNA contents. Contrary to EVCTRL , EC- and plasma-derived EVUR significantly increased the pro-calcifying effects of CM, including altered gene expressions of osterix, runx2, osteocalcin and SM22α. Further, EVUR enhanced the protein expression of the phosphate transporter PiT-1 in VSMC and induced a phosphorylation of AKT and ERK. Knock down of PiT-1 and individual inhibition of AKT and ERK signalling in VSMC blocked the pro-calcifying effects of EVUR . Similar effects were achieved by inhibition of miR-221/-222 and mimicking of miR-143/-145 in EVUR . In conclusion, EVUR might represent an additional puzzle piece of the complex pathophysiology of vascular calcifications in CKD.
| S-EPMC8184672 | biostudies-literature
Project description:Vascular calcification is a complication that is frequently encountered in patients affected by atherosclerosis, diabetes, and chronic kidney disease (CKD), and that is characterized by the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs). At present, there remains a pressing lack of any effective therapies that can treat this condition. The sodium-glucose transporter 2 (SGLT2) inhibitor dapagliflozin (DAPA) has shown beneficial effects in cardiovascular disease. The role of this inhibitor in the context of vascular calcification, however, remains largely uncharacterized. Our findings revealed that DAPA treatment was sufficient to alleviate in vitro and in vivo osteogenic transdifferentiation and vascular calcification. Interestingly, our study demonstrated that DAPA exerts its anti-calcification effects on VSMCs by directly targeting SGLT2, with the overexpression of SGLT2 being sufficient to attenuate these beneficial effects. DAPA was also able to limit the glucose levels and NAD+/NADH ratio in calcified VSMCs, upregulating sirtuin 1 (SIRT1) in a caloric restriction (CR)-dependent manner. The SIRT1-specific siRNA and the SIRT1 inhibitor EX527 attenuated the anti-calcification effects of DAPA treatment. DAPA was also to drive SIRT1-mediated deacetylation and consequent degradation of hypoxia-inducible factor-1α (HIF-1α). The use of cobalt chloride and proteasome inhibitor MG132 to preserve HIF-1α stability mitigated the anti-calcification activity of DAPA. These analyses revealed that the DAPA/SGLT2/SIRT1 axis may therefore represent a viable novel approach to treating vascular calcification, offering new insights into how SGLT2 inhibitors may help prevent and treat vascular calcification.
| S-EPMC11550308 | biostudies-literature
Project description:Vascular calcification occurs in arterial aging, atherosclerosis, diabetes mellitus, and chronic kidney disease. Transforming growth factor-β1 (TGF-β1) is a key modulator driving the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs), leading to vascular calcification. We hypothesize that milk fat globule-epidermal growth factor 8 (MFG-E8), a glycoprotein expressed in VSMCs, promotes the osteogenic transdifferentiation of VSMCs through the activation of TGF-β1-mediated signaling. We observe that the genetic deletion of MFG-E8 prevents calcium chloride-induced vascular calcification in common carotid arteries (CCAs). The exogenous application of MFG-E8 to aged CCAs promotes arterial wall calcification. MFG-E8-deficient cultured VSMCs exhibit decreased biomineralization and phenotypic transformation to osteoblast-like cells in response to osteogenic medium. MFG-E8 promotes β1 integrin-dependent MMP2 expression, causing TGF-β1 activation and subsequent VSMC osteogenic transdifferentiation and biomineralization. Thus, the established molecular link between MFG-E8 and vascular calcification suggests that MFG-E8 can be therapeutically targeted to mitigate vascular calcification.
| S-EPMC9018696 | biostudies-literature
Project description:Osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) is a risk factor associated with vascular diseases. Wnt signaling is one of the major mechanisms implicated in the osteogenic conversion of VSMCs. Since Cdon has a negative effect on Wnt signaling in distinct cellular processes, we sought to investigate the role of Cdon in vascular calcification. The expression of Cdon was significantly downregulated in VSMCs of the aortas of patients with atherosclerosis and aortic stenosis. Consistently, calcification models, including vitamin D3 (VD3)-injected mice and VSMCs cultured with calcifying media, exhibited reduced Cdon expression. Cdon ablation mice (cKO) exhibited exacerbated aortic stiffness and calcification in response to VD3 compared to the controls. Cdon depletion induced the osteogenic conversion of VSMCs accompanied by cellular senescence. The Cdon-deficient aortas showed a significant alteration in gene expression related to cell proliferation and differentiation together with Wnt signaling regulators. Consistently, Cdon depletion or overexpression in VSMCs elevated or attenuated Wnt-reporter activities, respectively. The deletion mutant of the second immunoglobulin domain (Ig2) in the Cdon ectodomain failed to suppress Wnt signaling and osteogenic conversion of VSMCs. Furthermore, treatment with purified recombinant proteins of the entire ectodomain or Ig2 domain of Cdon displayed suppressive effects on Wnt signaling and VSMC calcification. Our results demonstrate a protective role of Cdon in VSMC calcification by suppressing Wnt signaling. The Ig2 domain of Cdon has the potential as a therapeutic tool to prevent vascular calcification.
| S-EPMC9898282 | biostudies-literature
Project description:Vascular calcification is a hallmark of atherosclerosis, a major cause of morbidity and mortality in the United States. We have previously reported that the osteogenic transcription factor Runx2 is an essential and sufficient regulator of calcification of vascular smooth muscle cells (VSMC) in vitro.To determine the contribution of osteogenic differentiation of VSMC to the pathogenesis of vascular calcification and the function of VSMC-derived Runx2 in regulating calcification in vivo.SMC-specific Runx2-deficient mice, generated by breeding SM22?-Cre mice with the Runx2 exon 8 floxed mice, exhibited normal aortic gross anatomy and expression levels of SMC-specific marker genes. Runx2 deficiency did not affect basal SMC markers, but inhibited oxidative stress-reduced expression of SMC markers. High-fat-diet-induced vascular calcification in vivo was markedly inhibited in the Runx2-deficient mice in comparison with their control littermates. Runx2 deficiency inhibited the expression of receptor activator of nuclear factor ?B ligand, which was accompanied by decreased macrophage infiltration and formation of osteoclast-like cells in the calcified lesions. Coculture of VSMC with bone marrow-derived macrophages demonstrated that the Runx2-deficient VSMC failed to promote differentiation of macrophages into osteoclast-like cells.These data have determined the importance of osteogenic differentiation of VSMC in the pathogenesis of vascular calcification in mice and defined the functional role of SMC-derived Runx2 in regulating vascular calcification and promoting infiltration of macrophages into the calcified lesion to form osteoclast-like cells. Our studies suggest that the development of vascular calcification is coupled with the formation of osteoclast-like cells, paralleling the bone remodeling process.
| S-EPMC3678289 | biostudies-literature
Project description:BACKGROUND:In vitro cell culture is a widely used technique for investigating a range of processes such as stem cell behavior, regenerative medicine, tissue engineering, and drug discovery. Conventional cell culture is performed in Petri dishes or flasks where cells typically attach to a flat glass or plastic surface as a cell monolayer. However, 2D cell monolayers do not provide a satisfactory representation of in vivo conditions. A 3D culture could be a much better system for representing the conditions that prevail in vivo. METHODS AND RESULTS:To simulate 3D conditions, vascular smooth muscle cells (VSMCs) were loaded with gold-polyvmer-iron oxide hydrogel, enabling levitation of the cells by using spatially varying magnetic fields. These magnetically levitated 3D cultures appeared as freely suspended, clustered cells which proliferated 3-4 times faster than cells in conventional 2D cultures. When the levitated cells were treated with 10nM lysophosphatidylcholine (LPC), for 3days, cell clusters exhibited translucent extensions/rods 60-80?m wide and 200-250?m long. When 0.5?g/?l Schnurri-3 was added to the culture containing LPC, these extensions were smaller or absent. When excited with 590-650nm light, these extensions emitted intrinsic fluorescence at >667nm. When the 3D cultures were treated with a fluorescent probe specific for calcium hydroxyapatite (FITC-HABP-19), the cell extensions/rods emitted intensely at 518nm, the ?max for FITC emission. Pellets of cells treated with LPC were more enriched in calcium, phosphate, and glycosaminoglycans than cells treated with LPC and Schnurri-3. CONCLUSIONS:In 3D cultures, VSMCs grow more rapidly and form larger calcification clusters than cells in 2D cultures. Transdifferentiation of VSMC into calcifying vascular cells is enhanced by LPC and attenuated by Schnurri-3. GENERAL SIGNIFICANCE:The formation of calcified structures in 3D VSMC cultures suggests that similar structures may be formed in vivo.
| S-EPMC4383529 | biostudies-literature
Project description:Vascular calcification is prominent in patients with chronic kidney disease (CKD) and is a strong predictor of cardiovascular mortality in the CKD population. However, the mechanism underlying CKD-associated vascular calcification remains unclear. To identify potential therapeutic targets, a 5/6 nephrectomy rat model was established by feeding of a high-phosphorous diet as the CKD group and compared with sham group rats at 4 and 16 weeks. Sequencing analyses of the rat aorta revealed 643 upregulated and 1023 downregulated genes at 4 weeks, as well as 899 upregulated and 1185 downregulated genes at 16 weeks in the CKD group compared to the sham group. Bioinformatics analyses suggested that SOST (which encodes sclerostin) and Wnt signaling are involved in CKD-associated vascular calcification. Furthermore, protein-protein interactions analysis revealed interactions between SOST, WNT5A, and WNT5B, that involved runt-related transcription factor 2 (RUNX2) and transgelin (TAGLN). SOST was increased in CKD-associated vascular calcification following reduction of the Wnt signaling, including WNT5A and WNT5B, both in vivo and in vitro. TargetScan was used to predict the microRNAs (miRNAs) targeting WNT5A and WNT5B. The expression levels of miR-542-3p, miR-298-3p, miR-376b-5p, and miR-3568 were significantly reduced, whereas that of miR-742-3p was significantly increased in calcified rat aortic vascular smooth muscle cells (VSMCs). In CKD rat aortas, the expression of miR-542-3p, miR-298-3p, miR-376b-5p, miR-3568, miR-742-3p, and miR-22-5p were significantly reduced at both 4 and 16 weeks. Altogether, owing to several assessments, potentially diagnostic and prognostic biomarkers for improving common CKD diagnostic tools were identified in this study. Abbreviations: BUN: blood urea nitrogen; CKD: chronic kidney disease; CKD-MBD: chronic kidney disease-mineral bone disorder; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GO: the Gene Ontology; HE: hematoxylin-eosin; HRP: horseradish peroxidase; KEGG: Kyoto Encyclopedia of Genes and Genomes; MiRNAs: microRNAs; PAS: periodic acid-Schiff; RUNX2: runt-related transcription factor 2; SCr: serum creatinine; STRING: the Search Tool for the Retrieval of Interacting Genes/Proteins; TAGLN: transgelin; VSMC: vascular smooth muscle cell.
| S-EPMC9423850 | biostudies-literature
Project description:Moyamoya disease (MMD) is an occlusive, chronic cerebrovascular disease affected by genetic mutation and the immune response. Furthermore, vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) participate in the neointima of MMD, but the etiology and pathophysiological changes in MMD vessels remain largely unknown. Therefore, we established the circZXDC (ZXD family zinc finger C)–miR-125a-3p–ABCC6 (ATP-binding cassette subfamily C member 6) axis from public datasets and online tools based on “sponge-like” interaction mechanisms to investigate its possible role in VSMCs. The results from a series of in vitro experiments, such as dual luciferase reporter assays, cell transfection, CCK-8 assays, Transwell assays, and Western blotting, indicate a higher level of circZXDC in the MMD plasma, especially in those MMD patients with the RNF213 mutation. Moreover, circZXDC overexpression results in a VSMC phenotype switching toward a synthetic status, with increased proliferation and migration activity. CircZXDC sponges miR-125a-3p to increase ABCC6 expression, which induces ERS (endoplasmic reticulum stress), and subsequently regulates VSMC transdifferentiation from the contractive phenotype to the synthetic phenotype, contributing to the intima thickness of MMD vessels. Our findings provide insight into the pathophysiological mechanisms of MMD and indicate that the circZXDC–miR-125a-3p–ABCC6 axis plays a pivotal role in the progression of MMD. Furthermore, circZXDC might be a diagnostic biomarker and an ABCC6-specific inhibitor and has the potential to become a promising therapeutic option for MMD.
| S-EPMC9741004 | biostudies-literature