Caiyu Chen

LincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis and Atherosclerosis by Enhancing p53 Activity

Background— Long noncoding RNAs (lncRNAs) have recently been implicated in many biological processes and diseases. Atherosclerosis is a major risk factor for cardiovascular disease. However, the functional role of lncRNAs in atherosclerosis is largely unknown. Methods and Results— We identified lincRNA-p21 as a key regulator of cell proliferation and apoptosis during atherosclerosis. The expression of lincRNA-p21 was dramatically downregulated in atherosclerotic plaques of ApoE−/− mice, an animal model for atherosclerosis. Through loss- and gain-of-function approaches, we showed that lincRNA-p21 represses cell proliferation and induces apoptosis in vascular smooth muscle cells and mouse mononuclear macrophage cells in vitro. Moreover, we found that inhibition of lincRNA-p21 results in neointimal hyperplasia in vivo in a carotid artery injury model. Genome-wide analysis revealed that lincRNA-p21 inhibition dysregulated many p53 targets. Furthermore, lincRNA-p21, a transcriptional target of p53, feeds back to enhance p53 transcriptional activity, at least in part, via binding to mouse double minute 2 (MDM2), an E3 ubiquitin-protein ligase. The association of lincRNA-p21 and MDM2 releases MDM2 repression of p53, enabling p53 to interact with p300 and to bind to the promoters/enhancers of its target genes. Finally, we show that lincRNA-p21 expression is decreased in patients with coronary artery disease. Conclusions— Our studies identify lincRNA-p21 as a novel regulator of cell proliferation and apoptosis and suggest that this lncRNA could serve as a therapeutic target to treat atherosclerosis and related cardiovascular disorders.

Extracellular vesicle-mediated transfer of donor genomic DNA to recipient cells is a novel mechanism for genetic influence between cells

Extracellular vesicles (EVs) carry signals within or at their limiting membranes, providing a mechanism by which cells can exchange more complex information than what was previously thought. In addition to mRNAs and microRNAs, there are DNA fragments in EVs. Solexa sequencing indicated the presence of at least 16434 genomic DNA (gDNA) fragments in the EVs from human plasma. Immunofluorescence study showed direct evidence that acridine orange-stained EV DNAs could be transferred into the cells and localize to and inside the nuclear membrane. However, whether the transferred EV DNAs are functional or not is not clear. We found that EV gDNAs could be homologously or heterologously transferred from donor cells to recipient cells, and increase gDNA-coding mRNA, protein expression, and function (e.g. AT1 receptor). An endogenous promoter of the AT1 receptor, NF-κB, could be recruited to the transferred DNAs in the nucleus, and increase the transcription of AT1 receptor in the recipient cells. Moreover, the transferred EV gDNAs have pathophysiological significance. BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia, could be transferred from K562 EVs to HEK293 cells or neutrophils. Our present study shows that the gDNAs transferred from EVs to cells have physiological significance, not only to increase the gDNA-coding mRNA and protein levels, but also to influence function in recipient cells.

Prolyl Hydroxylase Domain Protein 2 Silencing Enhances the Survival and Paracrine Function of Transplanted Adipose-Derived Stem Cells in Infarcted Myocardium

Rationale: Transplantation of stem cells into damaged hearts has had modest success as a treatment for ischemic heart disease. One of the limitations is the poor stem cell survival in the diseased microenvironment. Prolyl hydroxylase domain protein 2 (PHD2) is a cellular oxygen sensor that regulates 2 key transcription factors involved in cell survival and inflammation: hypoxia-inducible factor and nuclear factor-&kgr;B. Objective: We studied whether and how PHD2 silencing in human adipose-derived stem cells (ADSCs) enhances their cardioprotective effects after transplantation into infarcted hearts. Methods and Results: ADSCs were transduced with lentiviral short hairpin RNA against prolyl hydroxylase domain protein 2 (shPHD2) to silence PHD2. ADSCs, with or without shPHD2, were transplanted after myocardial infarction in mice. ADSCs reduced cardiomyocyte apoptosis, fibrosis, and infarct size and improved cardiac function. shPHD2-ADSCs exerted significantly more protection. PHD2 silencing induced greater ADSC survival, which was abolished by short hairpin RNA against hypoxia-inducible factor-1&agr;. Conditioned medium from shPHD2-ADSCs decreased cardiomyocyte apoptosis. Insulin-like growth factor-1 (IGF-1) levels were significantly higher in the conditioned medium of shPHD2-ADSCs versus ADSCs, and depletion of IGF-1 attenuated the cardioprotective effects of shPHD2-ADSC–conditioned medium. Nuclear factor-&kgr;B activation was induced by shPHD2 to induce IGF-1 secretion via binding to IGF-1 gene promoter. Conclusions: PHD2 silencing promotes ADSCs survival in infarcted hearts and enhances their paracrine function to protect cardiomyocytes. The prosurvival effect of shPHD2 on ADSCs is hypoxia-inducible factor-1&agr; dependent, and the enhanced paracrine function of shPHD2-ADSCs is associated with nuclear factor-&kgr;B–mediated IGF-1 upregulation. PHD2 silencing in stem cells may be a novel strategy for enhancing the effectiveness of stem cell therapy after myocardial infarction.

Angiotensin II AT2 receptor decreases AT1 receptor expression and function via nitric oxide/cGMP/Sp1 in renal proximal tubule cells from Wistar–Kyoto rats

Background: The renin–angiotensin (Ang) system controls blood pressure, in part, by regulating renal tubular sodium transport. In the kidney, activation of the angiotensin II type 1 (AT1) receptor increases renal sodium reabsorption, whereas the angiotensin II type 2 (AT2) receptor produces the opposite effect. We hypothesized that the AT2 receptor regulates AT1 receptor expression and function in the kidney. Methods and results: In immortalized renal proximal tubule (RPT) cells from Wistar–Kyoto rats, CGP42112, an AT2 receptor agonist, decreased AT1 receptor mRNA and protein expression (P < 0.05), as assessed by reverse transcriptase-polymerase chain reaction and immunoblotting. The inhibitory effect of the AT2 receptor on AT1 receptor expression was blocked by the AT2 receptor antagonist, PD123319 (10−6 mol/l), the nitric oxide synthase inhibitor Nw-nitro-L-arginine methyl ester (10−4 mol/l), or the nitric oxide-dependent soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10−5 mol/l), indicating that both nitric oxide and cyclic guanosine monophosphate (cGMP) were involved in the signaling pathway. Furthermore, CGP42112 decreased Sp1 serine phosphorylation and reduced the binding of Sp1 to AT1 receptor DNA. Stimulation with Ang II (10−11 mol/l per 30 min) enhanced Na+-K+-ATPase activity in RPT cells, which was prevented by pretreatment with CGP42112 (10−7 mol/l per 24 h) (P < 0.05). The above-mentioned results were confirmed in RPT cells from AT2 receptor knockout mice; AT1 receptor expression and Ang II-stimulated Na+-K+-ATPase activity were greater in these cells than in RPT cells from wild-type mice (P < 0.05). AT1/AT2 receptors co-localized and co-immunoprecipitated in RPT cells; short-term CGP42112 (10−7 mol/l per 30 min) treatment increased AT1/AT2 receptor co-immunoprecipitation (P < 0.05). Conclusions: These results indicate that the renal AT2 receptor, via nitric oxide/cGMP/Sp1 pathway, regulates AT1 receptor expression and function, which may be important in the regulation of sodium excretion and blood pressure.

Transferred BCR/ABL DNA from K562 Extracellular Vesicles Causes Chronic Myeloid Leukemia in Immunodeficient Mice

Our previous study showed that besides mRNAs and microRNAs, there are DNA fragments within extracellular vesicles (EVs). The BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia (CML), could be transferred from K562 EVs to neutrophils and decrease their phagocytic activity in vitro. Our present study provides evidence that BCR/ABL DNAs transferred from EVs have pathophysiological significance in vivo. Two months after injection of K562 EVs into the tail vein of Sprague-Dawley (SD) rats, they showed some characteristics of CML, e.g., feeble, febrile, and thin, with splenomegaly and neutrophilia but with reduced neutrophil phagocytic activity. These findings were also observed in immunodeficient NOD/SCID mice treated with K562 EVs; BCR/ABL mRNA and protein were found in their neutrophils. The administration of actinomycin D, an inhibitor of de novo mRNA synthesis, prevented the abnormalities caused by K562 EVs in NOD/SCID mice related to CML, including neutrophilia and bone marrow hyperplasia. As a specific inhibitor of tyrosine kinases, imatinib blocked the activity of tyrosine kinases and the expression of phospho-Crkl, induced by the de novo BCR/ABL protein caused by K562 EVs bearing BCR/ABL DNA. Our current study shows the pathophysiological significance of transferred tumor gene from EVs in vivo, which may represent an important mechanism for tumorigenesis, tumor progression, and metastasis.

EGCG attenuates high glucose-induced endothelial cell inflammation by suppression of PKC and NF-κB signaling in human umbilical vein endothelial cells.

AIMS Vascular inflammation is a key factor in the pathogenesis of diabetes-related vascular complications. Our previous study showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits high glucose-induced vascular smooth muscle cell proliferation, thus it may have beneficial effects in diabetes and its complications. However, the effect of EGCG on inflammation in diabetes is not known. In the present study, we investigated whether EGCG suppresses the vascular inflammation induced by high glucose in human umbilical vein endothelial cells (HUVECs). MAIN METHODS The inhibitory effect of EGCG on high glucose-induced up-regulation of the expression of vascular cell adhesion molecule 1 (VCAM-1) was measured using enzyme-linked immunosorbent, RT-PCR, immunoblotting and cell adhesion assays. The effect of EGCG on high glucose-induced nuclear factor-kappa B (NF-κB) activation was investigated by immunoblotting, immunofluorescence and electrophoretic mobility shift assays. KEY FINDINGS High glucose increased VCAM-1 expression and enhanced the adhesion of monocytes to HUVECs. Pretreatment with EGCG in a concentration-dependent manner (1.0-50 μM) significantly attenuated these effects. High glucose (25 mM)-mediated vascular inflammation was blocked by PKC pseudosubstrate (PKC inhibitor 19-31) or the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Stimulation with high glucose increased the NF-κB translocation from the cytoplasm to the nucleus, and increased IκB-α phosphorylation, decreased its expression, and in the presence of EGCG, the effect of high glucose on NF-κB and IκB-α were blocked. SIGNIFICANCE EGCG suppresses high glucose-induced vascular inflammatory process via the inhibition of PKC and NF-κB activation in HUVECs, suggesting that EGCG may be a potential candidate for the treatment and prevention of diabetic vascular complications.

(-)-Epigallocatechin gallate suppresses proliferation of vascular smooth muscle cells induced by high glucose by inhibition of PKC and ERK1/2 signalings.

Proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development and progression of diabetes-related vascular complications. (-)-Epigallocatechin gallate (EGCG), the major catechin derived from green tea, is able to exert antidiabetes effects in animal models. However, it is not known whether or not EGCG inhibits VSMC proliferation induced by high glucose. This study tested the hypothesis that EGCG might have an inhibitory effect on VSMC proliferation induced by high glucose. VSMC proliferation was determined by [(3)H]-thymidine incorporation and uptake of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT). Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was determined by immunoblotting, and ERK 1/2 activity was detected by measuring the ability to phosphorylate its substrate Elk-1. Glucose increased VSMC proliferation in a concentration-dependent manner, which was reduced in the presence of EGCG. VSMC proliferation mediated by high glucose (30 mM) was involved in protein kinase C (PKC) and ERK1/2 signalings, because its effect was blocked by PKC inhibitor (PKC inhibitor 19-31) and ERK1/2 inhibitor (PD98059). Pretreatment of VSMCs with EGCG significantly inhibited the stimulatory effect of high glucose on PKC and ERK1/2 activation, followed by attenuation of its downstream transcription factor Elk-1 phosphorylation. Taken together, these results suggest that EGCG could suppress VSMC proliferation induced by high glucose by inhibition of PKC and ERK1/2 signalings in VSMCs, which indicates that EGCG might be a possible medicine to reduce vascular complications in diabetes.

Role of GRK4 in the Regulation of Arterial AT1 Receptor in Hypertension

G-protein–coupled receptor kinase 4 (GRK4) gene variants, via impairment of renal dopamine receptor and enhancement of renin–angiotensin system functions, cause sodium retention and increase blood pressure. Whether GRK4 and the angiotensin type 1 receptor (AT1R) interact in the aorta is not known. We report that GRK4 is expressed in vascular smooth muscle cells of the aorta. Heterologous expression of the GRK4&ggr; variant 142V in A10 cells increased AT1R protein expression and AT1R-mediated increase in intracellular calcium concentration. The increase in AT1R expression was related to an increase in AT1R mRNA expression via the NF-&kgr;B pathway. As compared with control, cells expressing GRK4&ggr; 142V had greater NF-&kgr;B activity with more NF-&kgr;B bound to the AT1R promoter. The increased AT1R expression in cells expressing GRK4&ggr; 142V was also associated with decreased AT1R degradation, which may be ascribed to lower AT1R phosphorylation. There was a direct interaction between GRK4&ggr; and AT1R that was decreased by GRK4&ggr; 142V. The regulation of AT1R expression by GRK4&ggr; 142V in A10 cells was confirmed in GRK4&ggr; 142V transgenic mice; AT1R expression was higher in the aorta of GRK4&ggr; 142V transgenic mice than control GRK4&ggr; wild-type mice. Angiotensin II–mediated vasoconstriction of the aorta was also higher in GRK4&ggr; 142V than in wild-type transgenic mice. This study provides a mechanism by which GRK4, via regulation of arterial AT1R expression and function, participates in the pathogenesis of conduit vessel abnormalities in hypertension.

Cholesteryl Ester Transfer Protein Inhibitors in the Treatment of Dyslipidemia: A Systematic Review and Meta-Analysis

Cholesteryl ester transfer protein (CETP) inhibitors are gaining substantial research interest for raising high density lipoprotein cholesterol levels. The aim of the research was to estimate the efficacy and safety of cholesteryl ester transfer protein inhibitors as novel lipid modifying drugs. Systematic searches of English literature for randomized controlled trials (RCT) were collected from MEDLINE, EBASE, CENTRAL and references listed in eligible studies. Two independent authors assessed the search results and only included the double-blind RCTs by using cholesteryl ester transfer protein inhibitors as exclusively or co-administrated with statin therapy irrespective of gender in enrolled adult subjects. Two independent authors extracted the data by using predefined data fields. Of 503 studies identified, 14 studies met the inclusion criteria, and 12 studies were included into the final meta-analysis. Our meta-analysis revealed that CETP inhibitors increased the HDL-c levels (n = 2826, p<0.00001, mean difference (MD)  = 20.47, 95% CI [19.80 to 21.15]) and total cholesterol (n = 3423, p = 0.0002, MD = 3.57, 95%CI [1.69 to 5.44] to some extent combined with a reduction in triglyceride (n = 3739, p<0.00001, MD = −10.47, 95% CI [−11.91 to −9.03]) and LDL-c (n = 3159, p<0.00001, MD = −17.12, 95% CI [−18.87 to −15.36]) irrespective of mono-therapy or co-administration with statins. Subgroup analysis suggested that the lipid modifying effects varied according to the four currently available CETP inhibitors. CETP inhibitor therapy did not increase the adverse events when compared with control. However, we observed a slight increase in blood pressure (SBP, n = 2384, p<0.00001, MD = 2.73, 95% CI [2.14 to 3.31], DBP, n = 2384, p<0.00001, MD = 1.16, 95% CI [0.73 to 1.60]) after CETP inhibitor treatment, which were mainly ascribed to the torcetrapib treatment subgroup. CETP inhibitors therapy is associated with significant increase in HDL-c and decrease in triglyceride and LDL-c with satisfactory safety and tolerability in patients with dyslipidemia. However, the side-effect on blood pressure deserves more consideration in future studies.

Mesenchymal stem cells-derived extracellular vesicles, via miR-210, improve infarcted cardiac function by promotion of angiogenesis

Mesenchymal stem cells (MSCs) exert therapeutic effect on treating acute myocardial infarction. Recent evidence showed that paracrine function rather than direct differentiation predominately contributes to the beneficial effects of MSCs, but how the paracrine factors function are not fully elucidated. In the present study, we tested if extracellular vesicles (EVs) secreted by MSC promotes angiogenesis in infracted heart via microRNAs. Immunostaining of CD31 and matrigel plug assay were performed to detect angiogenesis in a mouse myocardial infarction (MI) model. The cardiac function and structure was examined with echocardiographic analysis. Capillary-like tube formation, migration and proliferation of human umbilical vein endothelial cells (HUVECs) were determined. As a result, MSC-EVs significantly improved angiogenesis and cardiac function in post-MI heart. MSC-EVs increased the proliferation, migration and tube formation capacity of HUVECs. MicroRNA (miR)-210 was found to be enriched in MSC-EVs. The EVs collected from MSCs with miR-210 silence largely lost the pro-angiogenic effect both in-vitro and in-vivo. The miR-210 target gene Efna3, which plays a role in angiogenesis, was down-regulated by MSC-EVs treatment in HUVECs. In conclusion, MSC-EVs are sufficient to improve angiogenesis and exert therapeutic effect on MI, its pro- angiogenesis effect might be associated with a miR-210-Efna3 dependent mechanism. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure - edited by Jun Ren & Megan Yingmei Zhang.