The microRNA miR-134-5p induces calcium deposition by inhibiting histone deacetylase 5 in vascular smooth muscle cells.

Calcium deposition in vascular smooth muscle cells (VSMCs) is a form of ectopic ossification in blood vessels. It can result in rigidity of the vasculature and an increase in cardiac events. Here, we report that the microRNA miR-134-5p potentiates inorganic phosphate (Pi)-induced calcium deposition in VSMCs by inhibiting histone deacetylase 5 (HDAC5). Using miRNA microarray analysis of Pi-treated rat VSMCs, we first selected miR-134-5p for further evaluation. Quantitative RT-PCR confirmed that miR-134-5p was increased in Pi-treated A10 cells, a rat VSMC line. Transfection of miR-134-5p mimic potentiated the Pi-induced increase in calcium contents. miR-134-5p increased the amounts of bone runt-related transcription factor 2 (RUNX2) protein and bone morphogenic protein 2 (BMP2) mRNA in the presence of Pi but decreased the expression of osteoprotegerin (OPG). Bioinformatic analysis showed that the HDAC5 3'untranslated region (3'UTR) was one of the targets of miR-134-5p. The luciferase construct containing the 3'UTR of HDAC5 was down-regulated by miR-134-5p mimic in a dose-dependent manner in VSMCs. Overexpression of HDAC5 mitigated the calcium deposition induced by miR-134-5p. Our results suggest that a Pi-induced increase of miR-134-5p may cause vascular calcification through repression of HDAC5.

Regulation of acetylation of histone deacetylase 2 by p300/CBP-associated factor/histone deacetylase 5 in the development of cardiac hypertrophy.

RATIONALE: Histone deacetylases (HDACs) are closely involved in cardiac reprogramming. Although the functional roles of class I and class IIa HDACs are well established, the significance of interclass crosstalk in the development of cardiac hypertrophy remains unclear. OBJECTIVE: Recently, we suggested that casein kinase 2α1-dependent phosphorylation of HDAC2 leads to enzymatic activation, which in turn induces cardiac hypertrophy. Here we report an alternative post-translational activation mechanism of HDAC2 that involves acetylation of HDAC2 mediated by p300/CBP-associated factor/HDAC5. METHODS AND RESULTS: Hdac2 was acetylated in response to hypertrophic stresses in both cardiomyocytes and a mouse model. Acetylation was reduced by a histone acetyltransferase inhibitor but was increased by a nonspecific HDAC inhibitor. The enzymatic activity of Hdac2 was positively correlated with its acetylation status. p300/CBP-associated factor bound to Hdac2 and induced acetylation. The HDAC2 K75 residue was responsible for hypertrophic stress-induced acetylation. The acetylation-resistant Hdac2 K75R showed a significant decrease in phosphorylation on S394, which led to the loss of intrinsic activity. Hdac5, one of class IIa HDACs, directly deacetylated Hdac2. Acetylation of Hdac2 was increased in Hdac5-null mice. When an acetylation-mimicking mutant of Hdac2 was infected into cardiomyocytes, the antihypertrophic effect of either nuclear tethering of Hdac5 with leptomycin B or Hdac5 overexpression was reduced. CONCLUSIONS: Taken together, our results suggest a novel mechanism by which the balance of HDAC2 acetylation is regulated by p300/CBP-associated factor and HDAC5 in the development of cardiac hypertrophy.

Small heterodimer partner blocks cardiac hypertrophy by interfering with GATA6 signaling.

RATIONALE: Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA-binding domain. Through interactions with other transcription factors, SHP regulates diverse biological events, including glucose metabolism in liver. However, the role of SHP in adult heart diseases has not yet been demonstrated. OBJECTIVE: We aimed to investigate the role of SHP in adult heart in association with cardiac hypertrophy. METHODS AND RESULTS: The roles of SHP in cardiac hypertrophy were tested in primary cultured cardiomyocytes and in animal models. SHP-null mice showed a hypertrophic phenotype. Hypertrophic stresses repressed the expression of SHP, whereas forced expression of SHP blocked the development of hypertrophy in cardiomyocytes. SHP reduced the protein amount of Gata6 and, by direct physical interaction with Gata6, interfered with the binding of Gata6 to GATA-binding elements in the promoter regions of natriuretic peptide precursor type A. Metformin, an antidiabetic agent, induced SHP and suppressed cardiac hypertrophy. The metformin-induced antihypertrophic effect was attenuated either by SHP small interfering RNA in cardiomyocytes or in SHP-null mice. CONCLUSIONS: These results establish SHP as a novel antihypertrophic regulator that acts by interfering with GATA6 signaling. SHP may participate in the metformin-induced antihypertrophic response.

Circular RNA circSMAD4 regulates cardiac fibrosis by targeting miR-671-5p and FGFR2 in cardiac fibroblasts.

Heart failure is a leading cause of death and is often accompanied by activation of quiescent cardiac myofibroblasts, which results in cardiac fibrosis. In this study, we aimed to identify novel circular RNAs that regulate cardiac fibrosis. We applied transverse aortic constriction (TAC) for 1, 4, and 8 weeks in mice. RNA sequencing datasets were obtained from cardiac fibroblasts isolated by use of a Langendorff apparatus and then further processed by use of selection criteria such as differential expression and conservation in species. CircSMAD4 was upregulated by TAC in mice or by transforming growth factor (TGF)-ß1 in primarily cultured human cardiac fibroblasts. Delivery of si-circSMAD4 attenuated myofibroblast activation and cardiac fibrosis in mice treated with isoproterenol (ISP). si-circSmad4 significantly reduced cardiac fibrosis and remodeling at 8 weeks. Mechanistically, circSMAD4 acted as a sponge against the microRNA miR-671-5p in a sequence-specific manner. miR-671-5p was downregulated during myofibroblast activation and its mimic form attenuated cardiac fibrosis. miR-671-5p mimic destabilized fibroblast growth factor receptor 2 (FGFR2) mRNA in a sequence-specific manner and interfered with the fibrotic action of FGFR2. The circSMAD4-miR-671-5p-FGFR2 pathway is involved in the differentiation of cardiac myofibroblasts and thereby the development of cardiac fibrosis.

Histone methyltransferase SETD3 regulates muscle differentiation.

Histone lysine methylation, as one of the most important factors in transcriptional regulation, is associated with a various physiological conditions. Using a bioinformatics search, we identified and subsequently cloned mouse SET domain containing 3 (SETD3) with SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) and Rubis-subs-bind domains. SETD3 is a novel histone H3K4 and H3K36 methyltransferase with transcriptional activation activity. SETD3 is expressed abundantly in muscular tissues and, when overexpressed, activates transcription of muscle-related genes, myogenin, muscle creatine kinase (MCK), and myogenic factor 6 (Myf6), thereby inducing muscle cell differentiation. Conversely, knockdown of SETD3 by shRNA significantly retards muscle cell differentiation. In this study, SETD3 was recruited to the myogenin gene promoter along with MyoD where it activated transcription. Together, these data indicate that SETD3 is a H3K4/K36 methyltransferase and plays an important role in the transcriptional regulation of muscle cell differentiation.

Casein kinase-2α1 induces hypertrophic response by phosphorylation of histone deacetylase 2 S394 and its activation in the heart.

BACKGROUND: Cardiac hypertrophy is characterized by transcriptional reprogramming of fetal gene expression, and histone deacetylases (HDACs) are tightly linked to the regulation of those genes. We previously demonstrated that activation of HDAC2, 1 of the class I HDACs, mediates hypertrophy. Here, we show that casein kinase-2α1 (CK2α1)-dependent phosphorylation of HDAC2 S394 is required for the development of cardiac hypertrophy. METHODS AND RESULTS: Hypertrophic stimuli phosphorylated HDAC2 S394, which was necessary for its enzymatic activation, and therefore the development of hypertrophic phenotypes in rat neonatal cardiomyocytes or in isoproterenol-administered mice hearts. Transgenic mice overexpressing HDAC2 wild type exhibited cardiac hypertrophy, whereas those expressing phosphorylation-resistant HDAC2 S394A did not. Compared with that in age-matched normal human hearts, phosphorylation of HDAC2 S394 was dramatically increased in patients with hypertrophic cardiomyopathy. Hypertrophy-induced phosphorylation of HDAC2 S394 and its enzymatic activity were completely blocked either by CK2 blockers or by CK2α1 short interfering RNA. Hypertrophic stimuli led CK2α1 to be activated, and its chemical inhibitors blocked hypertrophy in both phenylephrine-treated cardiomyocytes and isoproterenol-administered mice. CK2α1-transgenic mice developed hypertrophy, which was attenuated by administration of trichostatin A, an HDAC inhibitor. Overexpression of CK2α1 caused hypertrophy in cardiomyocytes, whereas chemical inhibitors of both CK2 and HDAC as well as HDAC2 S394A blunted it. Hypertrophy in CK2α1-transgenic mice was exaggerated by crossing these mice with wild-type-HDAC2-overexpressing mice. By contrast, however, it was blocked when CK2α1-transgenic mice were crossed with HDAC2 S394A-transgenic mice. CONCLUSIONS: We have demonstrated a novel mechanism in the development of cardiac hypertrophy by which CK2 activates HDAC2 via phosphorylating HDAC2 S394.

Regulation of mouse steroidogenesis by WHISTLE and JMJD1C through histone methylation balance.

The dynamic exchange of histone lysine methylation status by histone methyltransferases and demethylases has been previously implicated as an important factor in chromatin structure and transcriptional regulation. Using immunoaffinity TAP analysis, we purified the WHISTLE-interacting protein complexes, which include the heat shock protein HSP90α and the jumonji C-domain harboring the histone demethylase JMJD1C. In this study, we demonstrate that JMJD1C specifically demethylates histone H3K9 mono- and di-methylation, and mediates transcriptional activation. We also provide evidence suggesting that both WHISTLE and JMJD1C performs functions in the development of mouse testes by regulating the expression of the steroidogenesis marker, p450c17, via SF-1-mediated transcription. Furthermore, we demonstrate that WHISTLE is recruited to the p450c17 promoter via SF-1 and represses the transcription of prepubertal stages of steroidogenesis, after which JMJD1C replaces WHISTLE and activates the expression of target genes via SF-1-mediated interactions. Our results demonstrate that the histone methylation balance mediated by HMTase WHISTLE and demethylase JMJD1C perform a transcriptional regulatory function in mouse testis development.

Circular RNA circSmoc1-2 regulates vascular calcification by acting as a miR-874-3p sponge in vascular smooth muscle cells.

Vascular calcification (VC), or calcium deposition inside the blood vessels, is common in patients with atherosclerosis, cardiovascular disease, and chronic kidney disease. Although several treatments are available to reduce calcification, the incidence of VC continues to rise. Recently, there have been several reports describing the regulation of circular RNAs (circRNAs) in various diseases. However, the role of circRNAs in VC has not yet been fully explored. Here, we investigated the function of circSmoc1-2, one of the circRNAs generated from the Smoc1 gene, which is downregulated in response to VC. CircSmoc1-2 is localized primarily to the cytoplasm and is resistant to exonuclease digestion. Inhibition of circSmoc1-2 worsens VC, while overexpression of circSmoc1-2 reduces VC, suggesting that circSmoc1-2 can prevent calcification. We went on to investigate the mechanism of circSmoc1-2 as a microRNA sponge and noted that miR-874-3p, the predicted target of circSmoc1-2, promotes VC, while overexpression of circSmoc1-2 reduces VC by suppressing miR-874-3p. Additionally, we identified the potential mRNA target of miR-874-3p as Adam19. In conclusion, we revealed that the circSmoc1-2/miR-874-3p/Adam19 axis regulates VC, suggesting that circSmoc1-2 may be a novel therapeutic target in the treatment of VC.

Regulation of MDM2 E3 ligase-dependent vascular calcification by MSX1/2.

Vascular calcification increases morbidity and mortality in patients with cardiovascular and renal diseases. Previously, we reported that histone deacetylase 1 prevents vascular calcification, whereas its E3 ligase, mouse double minute 2 homolog (MDM2), induces vascular calcification. In the present study, we identified the upstream regulator of MDM2. By utilizing cellular models and transgenic mice, we confirmed that E3 ligase activity is required for vascular calcification. By promoter analysis, we found that both msh homeobox 1 (Msx1) and msh homeobox 2 (Msx2) bound to the MDM2 promoter region, which resulted in transcriptional activation of MDM2. The expression levels of both Msx1 and Msx2 were increased in mouse models of vascular calcification and in calcified human coronary arteries. Msx1 and Msx2 potentiated vascular calcification in cellular and mouse models in an MDM2-dependent manner. Our results establish a novel role for MSX1/MSX2 in the transcriptional activation of MDM2 and the resultant increase in MDM2 E3 ligase activity during vascular calcification.

Activation of histone deacetylase 2 by inducible heat shock protein 70 in cardiac hypertrophy.

Diverse cardiac diseases induce cardiac hypertrophy, which leads to dilatation and heart failure. We previously reported that hypertrophy can be blocked by class I histone deacetylase (HDAC) inhibitor, which prompted us to investigate the regulatory mechanism of class I HDACs. Cardiac hypertrophy was introduced by aortic banding, by infusion of isoproterenol or angiotensin II, or by swimming. Hypertrophic stimuli transiently elevated the activity of histone deacetylase-2 (Hdac2), a class I HDAC. In cardiomyocytes, forced expression of Hdac2 simulated hypertrophy in an Akt-dependent manner, whereas enzymatically inert Hdac2 H141A failed to do so. Hypertrophic stimuli induced the expression of heat shock protein (Hsp)70. The induced Hsp70 physically associated with and activated Hdac2. Hsp70 overexpression produced a hypertrophic phenotype, which was blocked either by siHdac2 or by a dominant negative Hsp70DeltaABD. In Hsp70.1(-/-) mice, cardiac hypertrophy and Hdac2 activation were significantly blunted. Heat shock either to cardiomyocytes or to mice activated Hdac2 and induced hypertrophy. However, heat shock-induced Hdac2 activation was blunted in the cardiomyocytes isolated from Hsp70.1(-/-) mice. These results suggest that the induction of Hsp70 in response to diverse hypertrophic stresses and the ensuing activation of HDAC2 trigger cardiac hypertrophy, emphasizing HSP70/HDAC2 as a novel mechanism regulating hypertrophy.

Characterization of Circular RNAs in Vascular Smooth Muscle Cells with Vascular Calcification.

Circular RNAs (circRNAs) are generally formed by back splicing and are expressed in various cells. Vascular calcification (VC), a common complication of chronic kidney disease (CKD), is often associated with cardiovascular disease. The relationship between circRNAs and VC has not yet been studied. Inorganic phosphate (Pi) was used to treat rat vascular smooth muscle cells to induce VC. circRNAs were identified by analyzing RNA sequencing (RNA-seq) data, and their expression change during VC was validated. The selected circRNAs, including circSamd4a, circSmoc1-1, circMettl9, and circUxs1, were resistant to RNase R digestion and mostly localized in the cytoplasm. While silencing circSamd4a promoted VC, overexpressing it reduced VC in calcium assay and Alizarin red S (ARS) staining. In addition, microRNA (miRNA) microarray, luciferase reporter assay, and calcium assay suggested that circSamd4a could act as a miRNA suppressor. Our data show that circSamd4a has an anti-calcification role by functioning as a miRNA sponge. Moreover, mRNAs that can interact with miRNAs were predicted from RNA-seq and bioinformatics analysis, and the circSamd4a-miRNA-mRNA axis involved in VC was verified by luciferase reporter assay and calcium assay. Since circSamd4a is conserved in humans, it can serve as a novel therapeutic target in resolving VC.

miR-27a-3p Targets ATF3 to Reduce Calcium Deposition in Vascular Smooth Muscle Cells.

Vascular calcification, the ectopic deposition of calcium in blood vessels, develops in association with various metabolic diseases and atherosclerosis and is an independent predictor of morbidity and mortality associated with these diseases. Herein, we report that reduction of microRNA-27a-3p (miR-27a-3p) causes an increase in activating transcription factor 3 (ATF3), a novel osteogenic transcription factor, in vascular smooth muscle cells. Both microRNA (miRNA) and mRNA microarrays were performed with rat vascular smooth muscle cells, and reciprocally regulated pairs of miRNA and mRNA were selected after bioinformatics analysis. Inorganic phosphate significantly reduced the expression of miR-27a-3p in A10 cells. The transcript level was also reduced in vitamin D3-administered mouse aortas. miR-27a-3p mimic reduced calcium deposition, whereas miR-27a-3p inhibitor increased it. The Atf3 mRNA level was upregulated in a cellular vascular calcification model, and miR-27a-3p reduced the Atf3 mRNA and protein levels. Transfection with Atf3 could recover the miR-27a-3p-induced reduction of calcium deposition. Our results suggest that reduction of miR-27a-3p may contribute to the development of vascular calcification by de-repression of ATF3.

Histone methyltransferase PRDM8 regulates mouse testis steroidogenesis.

A family of PRDM proteins are similar to histone methyltransferases (HMTases) with SET domain in that they modulate different cellular processes, including transcriptional regulation, through chromatin modifying activities. By applying a bioinformatic approach, we searched for proteins containing the SET domain and identified a double zinc-finger domain containing PRDM8 with HMTase activity. In vitro HMTase assay and immunoblot analysis revealed that PRDM8 specifically methylates H3K9 of histones which indicates transcriptional repression activity of PRDM8. Direct recruitment of PRDM8 to the promoter mediated transcriptional repression and indicated no involvement of HDAC. Tissue blot analyses identified PRDM8 transcripts from brain and testis in adult mouse. Consistent with these observations, we demonstrate that PRDM8 repressed the expression of steroidogenic markers, p450c17c and LHR, which indicates its regulatory role in mouse testis development.

Long noncoding RNAs in vascular smooth muscle cells regulate vascular calcification.

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.

Sumoylation of histone deacetylase 1 regulates MyoD signaling during myogenesis.

Sumoylation, the conjugation of a small ubiquitin-like modifier (SUMO) protein to a target, has diverse cellular effects. However, the functional roles of the SUMO modification during myogenesis have not been fully elucidated. Here, we report that basal sumoylation of histone deacetylase 1 (HDAC1) enhances the deacetylation of MyoD in undifferentiated myoblasts, whereas further sumoylation of HDAC1 contributes to switching its binding partners from MyoD to Rb to induce myocyte differentiation. Differentiation in C2C12 skeletal myoblasts induced new immunoblot bands above HDAC1 that were gradually enhanced during differentiation. Using SUMO inhibitors and sumoylation assays, we showed that the upper band was caused by sumoylation of HDAC1 during differentiation. Basal deacetylase activity was not altered in the SUMO modification-resistant mutant HDAC1 K444/476R (HDAC1 2R). Either differentiation or transfection of SUMO1 increased HDAC1 activity that was attenuated in HDAC1 2R. Furthermore, HDAC1 2R failed to deacetylate MyoD. Binding of HDAC1 to MyoD was attenuated by K444/476R. Binding of HDAC1 to MyoD was gradually reduced after 2 days of differentiation. Transfection of SUMO1 induced dissociation of HDAC1 from MyoD but potentiated its binding to Rb. SUMO1 transfection further attenuated HDAC1-induced inhibition of muscle creatine kinase luciferase activity that was reversed in HDAC1 2R. HDAC1 2R failed to inhibit myogenesis and muscle gene expression. In conclusion, HDAC1 sumoylation plays a dual role in MyoD signaling: enhancement of HDAC1 deacetylation of MyoD in the basally sumoylated state of undifferentiated myoblasts and dissociation of HDAC1 from MyoD during myogenesis.

The microRNA miR-124 inhibits vascular smooth muscle cell proliferation by targeting S100 calcium-binding protein A4 (S100A4).

S100 calcium-binding protein A4 (S100A4) induces proliferation and migration of vascular smooth muscle cells (VSMCs). We aimed to find the microRNA regulating S100A4 expression. S100A4 transcripts are abruptly increased in the acute phase of carotid arterial injury 1 day later (at day 1) but gradually decreases at days 7 and 14. Bioinformatics analysis reveals that miR-124 targets S100A4. VSMC survival is attenuated by miR-124 mimic but increased by miR-124 inhibitor. miR-124 decreases immediately after carotid arterial injury but dramatically increases at days 7 and 14. miR-124 inhibitor-induced cell proliferation is blocked by S100A4 siRNA, whereas miR-124-induced cell death is recovered by S100A4. Our findings suggest that miR-124 is a novel regulator of VSMC proliferation and may play a role in the development of neointimal proliferation.

MDM2 E3 ligase-mediated ubiquitination and degradation of HDAC1 in vascular calcification.

Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC.

The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor.

The fine balance between proliferation and differentiation of vascular smooth muscle cells (VSMCs) is indispensable for the maintenance of healthy blood vessels, whereas an increase in proliferation participates in pathologic cardiovascular events such as atherosclerosis and restenosis. Here we report that microRNA-34c (miR-34c) targets stem cell factor (SCF) to inhibit VSMC proliferation and neointimal hyperplasia. In an animal model, miR-34c was significantly increased in the rat carotid artery after catheter injury. Transient transfection of miR-34c to either VSMCs or A10 cells inhibited cell survival by inducing apoptosis, which was accompanied by an increase in expression of p21, p27, and Bax. Transfection of miR-34c also attenuated VSMC migration. Bioinformatics showed that SCF is a target candidate of miR-34c. miR-34c down-regulated luciferase activity driven by a vector containing the 3'-untranslated region of SCF in a sequence-specific manner. Forced expression of SCF in A10 cells induced proliferation and migration, whereas knocking-down of SCF reduced cell survival and migration. miR-34c antagomir-induced VSMC proliferation was blocked by SCF siRNA. Delivery of miR-34c to rat carotid artery attenuated the expression of SCF and blocked neointimal hyperplasia. These results suggest that miR-34c is a new modulator of VSMC proliferation and that it inhibits neointima formation by regulating SCF.

Involvement of miR-34c in high glucose-insulted mesenchymal stem cells leads to inefficient therapeutic effect on myocardial infarction.

High glucose-insulted bone marrow-derived mesenchymal stem cells (BMCs) showed impaired angiogenesis along with downregulation of stem cell factor (SCF). This study was designed to determine the involvement of microRNAs (miR), which are actively involved in the physiological function of stem cells. We observed that miR-34c was significantly induced by high glucose treatment and blunted tube formation of BMCs. Stem cell factor (SCF) was confirmed as a target of miR-34c by 3'-UTR promoter analysis and Western blot. SCF knockdown by siRNA induced Krüppel-like factor 4 (KLF4) and resulted in the blockade of angiogenesis of BMCs. Sequentially, KLF4 overexpression completely blocked tube formation through inducing PAI-1 (plasminogen activator inhibitor-1). To study the action of miR-34c in terms of the therapeutic potential of BMCs, myocardial infarction (MI) was induced by ligation of the coronary artery in nude mice, BMCs transfected with miR-control or miR-34c were injected into the infarcted myocardium 7 days later, and histological studies were performed 2 weeks later. Cardiac fibrosis was 18.24±4.7% in the miR-34c-BMC group and 10.01±0.2% in the miR-control-BMC group (p<0.05). Cardiac function and vessel density were decreased in the miR-34c-BMC group compared with the miR-con-BMC group. Particularly, miR-34c-BMCs failed to incorporate into vessels. Our results show that the angiogenic activity of BMCs is finely regulated by the miR-34c-SCF-KLF4 axis, which is a potent translational target for optimizing the therapeutic activity of autologous BMCs for cardiac repair.

Angiopoietin-like 4 is involved in the poor angiogenic potential of high glucose-insulted bone marrow stem cells.

BACKGROUND AND OBJECTIVES: Diabetes is reported to reduce the function or number of progenitor cells. We compared the gene expression patterns of bone marrow-derived mesenchymal stem cells from diabetic (DM-BMCs) and healthy (non-DM-BMCs) rats and suggested Angiopoietin-like 4 (Angptl4) could be a responsible factor for impaired angiogenesis of DM-BMCs. SUBJECTS AND METHODS: BMCs were isolated from DM or non-DM rat, and in vitro angiogenesis activity was compared by tube formation assay on Matrigel and complementary deoxyribonucleic acid expression was analyzed by microarray with or without oxytocin treatment. Human BMCs (hBMCs) were treated with high glucose, and were performed polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Angptl4 plasmid DNA and micro ribonucleic acid-132 (miR-132) were transfected to immortalized hBMCs. RESULTS: In vitro angiogenesis assay showed the impaired tube formation in DM-BMCs, and slightly recovery by oxytocin treatment. Angptl4, an adipokine, was upregulated in DM-BMCs compared to non-DM-BMCs. Oxytocin treatment reduced Angptl4 in DM-BMCs. In hBMCs, overexpression of Angptl4 attenuated the tube formation. In addition to Angptl4, miR-132 was increased by high glucose treatment. Collectively, high glucose resulted in impaired tube formation through miR-132 induction and Angptl4 upregulation in BMCs. CONCLUSION: Our results show that the angiogenic activity of BMCs is impaired by high glucose stress, which would be mediated by Angptl4 and miR-132.