Jiaxin ChenHongjiao YuXiao TanSimon Wing Fai MokYuchen XieYueheng WangXueyan JiangVicky E. MacRaeLan LanXiaodong FuDongxing Zhu
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A link between increased glycolysis and vascular calcification has recently been reported, but it remains unclear how increased glycolysis contributes to vascular calcification. We therefore investigated the role of PFKFB3, a critical enzyme of glycolysis, in vascular calcification. We found that PFKFB3 expression was upregulated in calcified mouse VSMCs and arteries. We showed that expression of miR-26a-5p and miR-26b-5p in calcified mouse arteries was significantly decreased, and a negative correlation between Pfkfb3 mRNA expression and miR-26a-5p or miR-26b-5p was seen in these samples. Overexpression of miR-26a/b-5p significantly inhibited PFKFB3 expression in VSMCs. Intriguingly, pharmacological inhibition of PFKFB3 using PFK15 or knockdown of PFKFB3 ameliorated vascular calcification in vD3 -overloaded mice in vivo or attenuated high phosphate (Pi)-induced VSMC calcification in vitro. Consistently, knockdown of PFKFB3 significantly reduced glycolysis and osteogenic transdifferentiation of VSMCs, whereas overexpression of PFKFB3 in VSMCs induced the opposite effects. RNA-seq analysis and subsequent experiments revealed that silencing of PFKFB3 inhibited FoxO3 expression in VSMCs. Silencing of FoxO3 phenocopied the effects of PFKFB3 depletion on Ocn and Opg expression but not Alpl in VSMCs. Pyruvate or lactate supplementation, the product of glycolysis, reversed the PFKFB3 depletion-mediated effects on ALP activity and OPG protein expression in VSMCs. Our results reveal that blockade of PFKFB3-mediated glycolysis inhibits vascular calcification in vitro and in vivo. Mechanistically, we show that FoxO3 and lactate production are involved in PFKFB3-driven osteogenic transdifferentiation of VSMCs. PFKFB3 may be a promising therapeutic target for the treatment of vascular calcification.【Objective】 To determine whether autophagy is involved in the process of vascular smooth muscle cell calcification induced by Ox-LDL. 【Methods】 The in vitro model of vascular calcification was used in this study. Vascular smooth muscle cells were randomly divided into controls,an Ox-LDL-treated group,and a 3MA plus Ox-LDL-treated group. Cells were grown in DMEM supplemented with 10 mM BGP. Calcification was assessed by alizarin red staining. The mRNA and protein expression of cbfa1 and Beclin1 were analyzed by Q-PCR and Western blotting respectively. 【Results】 Ox-LDL promotes vascular smooth muscle cell calcification,up-regulates the mRNA and protein expression of cbfa1 and Beclin1. The specific autophagy inhibitor attenuates vascular calcification,and inhibits the up-regulation of cbfa1 and Beclin1 expression induced by Ox-LDL. 【Conclusion】 The pathogenesis of vascular smooth muscle cell calcification induced by Ox-LDL involves autophagy.
Pathogenesis
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RUNX2
Ex vivo
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Small interfering RNA (siRNA) molecules achieve sequence- specific gene silencing through a process known as RNA interference (RNAi). Compared to other nucleic acid-based therapeutics aimed at post-transcriptional gene silencing, such as antisense oligodeoxynucleotides, siRNA molecules achieve greater magnitude and duration of gene silencing at significantly lower doses. While the duration of gene knockdown by siRNA typically lasts around 1 week in rapidly dividing cells, recent reports of knockdown lasting for several weeks in nondividing cells indicate that dilution due to cell division may be a limiting factor in rapidly dividing cells. To determine if cell division directly impacts the duration of gene knockdown by siRNA, we chose to investigate the kinetics of siRNA-mediated gene silencing in luciferase-expressing cell lines with different observed doubling times using noninvasive bioluminescent imaging and a mathematical model of siRNA delivery and function. In vitro and in vivo, the duration of gene knockdown is inversely proportional to the rate of cell division. Consistent with previous reports, luciferase protein levels recover to pre-treatment values within less than 1 week in rapidly dividing cell lines, but take longer than 3 weeks to return to steady-state levels in nondividing fibroblasts. Similar results are observed in vivo, with knockdown lasting around 1 week in subcutaneous tumors in A/J mice and 3-4 weeks in the nondividing hepatocytes of BALB/c mice. These data indicate that dilution due to cell division, and not intracellular siRNA half-life, governs the duration of gene silencing under these conditions. Here, we will present our latest results describing the effects of cell doubling time, siRNA stability, and dosing schedule on siRNA- mediated gene silencing. Specifically, we will investigate whether the duration of knockdown using chemically modified siRNA molecules exhibits a similar dependence on cell doubling time. The implications of these findings will be highlighted using model calculations to determine the dosing schedule required to maintain persistent silencing of target proteins and to predict when maximum mRNA or protein knockdown will occur, an especially important factor when trying to observe a therapeutic effect resulting from protein knockdown. The approach of bioluminescent imaging combined with mathematical modeling provides insights into siRNA function that will hopefully be of practical use for both researchers and clinicians alike.
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Zinc is considered to be involved in maintaining healthy vascular condition. Atherosclerotic calcification of vascular smooth muscle cells (VSMCs) occurs via the mechanism of cell death; therefore, cell viability is a critical factor for preventing VSMC calcification. In this study, we tested whether zinc affected VSMC viability under both normal physiological non-calcifying (0 mM P) and atherosclerotic calcifying conditions (3 and 5 mM P), since VSMC physiological characters change during the VSMC calcification process. The study results showed that an optimal zinc level (15 μM) restored the decreased VSMC viability which was induced under low zinc levels (0 and 1 μM) and calcifying conditions (3 and 5 mM P) at 9 and 15 days culture. This zinc-protecting effect for VSMC viability is more prominent under atherosclerotic calcifying condition (3 and 5 mM P) than normal condition (0 mM P). Also, the increased VSMC viability was consistent with the decreased Ca and P accumulation in VSMC cell layers. The results suggested that zinc could be an effective biomineral for preventing VSMC calcification under atherosclerotic calcifying conditions.
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Objective To explore the effects of epigallocatechingallate(EGCG) on vascular calcification.Methods Calcific vascular smooth muscular cells(VSMCs) were induced by β-glycerophosphate in rats.Rats were invided into four groups:the control,the calcification,EGCG and EGCG intervention groups(10-7,10-6,10-5 and 10-4 mol·L-1 subgroups).Von Kossa staining,the Ca2+ content and the alkaline phosphatase(ALP) activity in VSMCs were measured.Results Lots of gathered black granules were found among the smooth muscle cells and its matrix by Von Kossa staining in VSMCs calcifacation group;more Calcium contents and ALP activities were observed in calcification group than those in control group;after treated with EGCG 10-7~10-4mol·L-1,the calcification in VSMCs was also attenuated and the total Ca2+ content and ALP activity in VSMCs showed decrease and a concentration-dependent manner.Conclusion The results indicated that EGCG can significantly attenuate the VSMCs calcification.
Von Kossa stain
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Objective To study the effects of salvianolate on the calcification in rat vascular smooth muscle cells(VSMCs).Methods Calcification of cultured rat VSMCs was prepared by incubation with β-glycerophosphate.Calcification was confirmed by measurement of calcium content and alkaline phosphatase(ALP) activity in VSMCs.The mRNA and protein levels of BMP-2 were detected by using real-time PCR and Western blotting,respectively.Results Compared with control group,3.92-fold calcium content and 10.5-fold increase in ALP activity were observed in calcified VSMCs group,respectively.Levels of mRNA and protein of BMP-2 were significantly increased in calcified group.Salvianolate decreased the calcium and ALP activity in a dose-dependent manner,these data were decreased by 32.3 %,42.2%,60.9%(P 0.05);36.4%,48.8% and 74.7%(P 0.01),respectively.And salvianolate decreased the mRNA and protein levels of BMP-2.Conclusion Salvianolate may have cardiovascular protecting effect by antagonizing vascular calcification,maybe by regulating BMP-2 level.
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Abstract Vascular calcification is characterized by the accumulation of hydroxyapatite crystals, which is a result of aberrant mineral metabolism. Although many clinical studies have reported its adverse effects on cardiovascular morbidity, the molecular mechanism of vascular calcification, especially the involvement of long noncoding RNAs (lncRNAs), is not yet reported. From the transcriptomic analysis, we discovered hundreds of lncRNAs differentially expressed in rat vascular smooth muscle cells (VSMCs) treated with inorganic phosphate, which mimics vascular calcification. We focused on Lrrc75a-as1 and elucidated its transcript structure and confirmed its cytoplasmic localization. Our results showed that calcium deposition was elevated after knockdown of Lrrc75a-as1, while its overexpression inhibited calcium accumulation in A10 cells. In addition, Lrrc75a-as1 attenuated VSMCs calcification by decreasing the expression of osteoblast-related factors. These findings suggest that Lrrc75a-as1 acts as a negative regulator of vascular calcification, and may serve as a possible therapeutic target in vascular calcification.
Vascular tissue
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Objective— CCN1 (Cyr61) is an extracellular matrix-associated protein involved in cell proliferation and survival. CCN1 is bound to vascular smooth muscle cells (VSMCs) via integrins and is expressed in VSMCs in atherosclerotic lesions, suggesting involvement in the regulation of vascular smooth muscle cell (VSMC) proliferation and atherosclerosis. We hypothesized that knockdown of CCN1 may inhibit VSMC proliferation and suppress neointimal hyperplasia. Methods and Results— We examined the effect of the knockdown of CCN1 using rat cultured VSMCs and a rat balloon injury model. CCN1 stimulated adhesion and migration of VSMCs in a dose-dependent manner, and this was blocked by an antibody for integrin α6β1. Moreover, knockdown of endogenous CCN1 by lentiviral delivery of siRNA significantly inhibited proliferation of VSMCs and the uptake of 5-bromo-2′-deoxyuridine (BrdU). Replenishment with recombinant CCN1 reversed the effect of siRNA knockdown. Interestingly, knockdown of CCN1 significantly suppressed neo...
CYR61
Neointimal hyperplasia
Neointima
Intimal hyperplasia
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Objective To develope gene knockdown cell model with artificial microRNA in setting up gene knockdown cell model.Methods We constructed vectors,and prepared siRNA fragments targeting on DJ-1.Then we transciently transfected the artificial miRNA and siRNA into MN9D cells by lipofectamine2000 reagent,the mRNA and protein expression level of DJ-1 gene were detected by RT-PCR and Western blot.Results Compared with control group,DJ-1 expression level was significantly decreased in both artificial miRNA and siRNA groups.DJ-1 was knockdowned and DJ-1 was decreased 90%(P0.05)at mRNA expression level,and decreased 70%~85%(P0.05) at protein level in MN9D cells transfected with the artificial miRNA.While DJ-1 was decreased by 50%~70%(P0.05)at mRNA level,and decreased by 20%~50%(P0.05)at protein level in MN9D cells transfected with siRNA.Comparing with siRNA,miRNA was more effective in silencing DJ-1.Conclusion The artificial miRNA and siRNA are both effective in silencing gene.miRNA has more significant function in knockingdown DJ-1 than siRNA.
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We recently developed a piggyback knockdown method that was used to knockdown genes in adult zebrafish. In this method, a vivo morpholino (VMO) piggybacks an antisense deoxyoligonucleotide (dO) into the somatic cells and reduces the cognate mRNA levels. In this paper, we tested whether we can piggyback more than one dO with one VMO. We designed various hybrids that had more than one dO that could be piggybacked with one VMO. We chose f7, f8, and αIIb genes and tested their knockdown by the appropriate assays. The knockdown with piggybacking either two or three dOs by one VMO yielded > 85% knockdown efficiency. We also performed knockdown of argonautes and rnaseh separately along with f7. We found the knockdown of f7 occurs when knockdown of argonautes happens and not when rnaseh knockdown was performed, suggesting that RNaseH is involved in mRNA degradation. In conclusion, we developed a method where we could knockdown three genes at one time, and by increasing the concentration of VMO by twofold, we could knockdown six genes simultaneously. These multiple gene knockdowns will not only increase the efficiency of the method in whole genome-wide knockdowns but will also be useful to study multifactorial disorders.
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