Advances on studies of miR-21 in vascular endothelial function and angiogenesis

MicroRNAs (miRNAs)are a class of non-coding, endogenous, single-stranded small RNA molecules composed of 19~25 nucleotides. miRNAs are widely involved in the process of human life activities. Recent studies have shown that part of miRNAs regulate the vascular endothelial function and angiogenesis. High expression of miRNA-21 is found to play important roles in the cell proliferation, cell apoptosis, cell growth and death of vascular endothelial cells. This review will focus on the recent progress related to miRNAs in vascular endothelial function and angiogenesis, providing a new insight in cardiovascular disease prevention, clinical diagnosis, prognosis and target therapeutics.     

Mechanism of endothelial microparticle generation in hypertension and its pathophysiological roles

Hypertension is closely related to many target organ damage. Endothelial microparticles (EMPs), derived from endothelial cells in response to endothelial cell activation or apoptosis, are complex vesicular structures with a membrane skeleton and express various antigens specific to parental endothelial cells. EMPs circulate in human plasma and show elevated levels in many vascular damage diseases, such as hypertension, atherosclerotic vascular diseases, sepsis and diabetes. Recent studies have shown that EMPs could be a comprehensive index for endothelial homeostasis monitoring, such as vasomotor activity, anti-inflammatory status and so on. Especially, more and more evidence suggests that EMPs play an important role in hypertension. Patients with hypertension show higher circulating levels of EMPs compared with healthy controls. Furthermore, increasing evidence demonstrates that EMPs can induce endothelial dysfunction in vitro and in vivo, and then further promote the development of hypertension and its complications. This review will summarize the progress in the definition and formation mechanisms of EMPs, levels of EMPs and their phenotypes in patients with hypertension, and the pathophysiological roles of EMPs in hypertension.     

Advances in function of circular RNA and regulation of atherosclerosis development

Atherosclerosis is one of the cardiovascular diseases that seriously endanger human health. The pathogenesis of atherosclerosis is very complicated and related to a variety of risk factors. In recent years, many studies have found that circular RNAs play an important regulatory role in the occurrence and development of atherosclerosis by inducing proliferation, differentiation, migration and apoptosis of vascular smooth muscle cells, endothelial cells and macrophages. This article reviews the basic functions of circular RNAs and its recent research on the regulation of atherosclerosis.     

Progress in function of HDL-eNOS signaling pathway in cardiovascular system and it's influencing factors

High-density lipoprotein (HDL) is negatively related to the risk of cardiovascular diseases such as atherosclerosis. Recent studies have shown that HDL activates a variety of target cells, such as vascular endothelial cells and macrophages, and activates the related cell signaling pathway to exert an anti-atherosclerosis role. HDL is a complex substance which composes of multiple particles. The changes of many factors affect the characteristics and functions of HDL, and then affect the activation of endothelial nitric oxide synthase (eNOS).This paper summarizes the recent correlation studies, and expounds the related factors that affect the HDL-eNOS signaling pathway.     

Role of endothelial cell glucose metabolism in vascular development

As an important organ in the body, blood vessels transport oxygen and nutrients to the tissues of the body. Endothelial cells are layers of cells lined with blood vessels whose metabolism is involved in regulating multiple links of vascular development and vascular diseases. Glucose metabolism provides the main energy for endothelial cells, and changes in glucose metabolism regulation affect vascular development and even lead to various diseases. Recently, strategies have been proposed to target endothelial cell metabolism for the treatment of vascular diseases. This article reviews the role of endothelial cell glucose metabolism in vascular development and vascular diseases in recent years.     

Progress in role of lncRNA in cardiovascular diseases

Cardiovascular diseases are closely related to proliferation, injury and apoptosis of the cells in the cardiovascular system. For instance, endothelial cells play an important role in the pathogenic process of hypertension and atherosclerosis, and smooth muscle cells and monocytes/macrophages involve in the formation of atherosclerotic plaque. Recently, it has been confirmed that long noncoding RNA (lncRNA) regulates proliferation, apoptosis, injury, autophagy and differentiation of the cells by a series of regulatory mechanisms, thus participating in the development and progression of cardiovascular diseases. This article is to review the recent research progress on the function of lncRNAs and their regulatory roles in the cardiovascular diseases at cellular and molecular levels.     

Recent advances in apolipoprotein M and atherosclerosis

Atherosclerosis is a complex pathological process, which has a close relationship with inflammatory response and disorder of lipid metabolism. The cardioprotective role of high-density lipoprotein (HDL) is related to its characters of protecting the vascular endothelial cells and the properties of anti-inflammation and anti-oxidation. Low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) are thought to have the adverse effects on human atherosclerosis. Apolipoprotein M (apoM, found in 1999) is an apolipoprotein mainly associated with HDLs. ApoM has a remarkable property of anti-atherosclerosis in animal experiment. However, recent studies have not yet been able to establish that apoM is a defining risk factor for human coronary heart disease, and the biological functions of apoM, including its potential role in human atherogenesis, need to be established.     

ECV304 cells can be used as a general model,tool or target in the research of biomedicine and pharmacology

ECV304 was reported first in 1990 as a spontaneously-transformed and immortalized cell line derived from a Japanese HUVEC. Subsequently, many studies validated that the ECV304 is a permanent endothelial cell line. It has been used widely as an endothelial cell model and an useful research tool in biomedicine and pharmacology. However, several distinct differences exist between ECV304 and HUVEC. Some studies even pointed out that ECV304 is not of HUVEC origin. According to the research data including ours, this reportedly endothelial-derived permanent human cell line ECV304 may be dedifferentiated towards an epithelial phenotype. It is therefore not an appropriate cell line to study endothelial cell biology. But cultured ECV304 cells can still be used as a model, tool or target in the pathophysiological and pharmacological studies, depending on whether or not their functional expression or markers are suitable for the research work.     

Protective effect of heat-stress preconditioning on anoxic endothelial cells and its mechanism

AIM:To observe the protective effect of heat stress preconditioning on endothelial cells under anoxia and explore its mechanism. METHODS:The endothelial cells were divided into 4 groups: (1) anoxia; (2) heat stress; (3) heat stress preconditioning + hypoxia; (4) control. LDH activity was measrued by using Automatic Biochemistry Analysis-Meter. Cell death rate was determined by trypan blue, NO production was tested by measuring NO₂^-/NO₃^- content in cellular culture medium by using Griess assay. RESULTS:LDH release and cell death rate of the anoxia endothelial cells significantly increased compared with control; 39℃ heat stress preconditioning reduced those increment by 29.47%, 33.67% respectively. 41℃ heat stress preconditioning has no protection against the anoxia-induced injury in endothelial cells. The NO production in anoxic endothelial cells decreased markedly. 39℃ heat stress preconditioning induced the increase in NO production in endothelial cells, but 41℃ heat stress preconditioning made the NOS activity decrease. The NO production was correlated negatively with LDH release and cell death rate in anoxic endothelial cells. CONCLUSION:The heat stress preconditioning within the limits can protect the endothelial cells from anoxia injury. The increase in NO in endothelial cells may play an important role in the mechanism of the protective effect.     

超敏蛋白对低温胁迫下茶树生理特性的影响

 超敏蛋白是一种诱导植物抗逆性的新型生物制剂。为了解超敏蛋白在低温胁迫过程中对茶树耐寒性的影响，以茶树品种‘迎霜’为试材，分别在常温和4 ℃下喷施超敏蛋白，研究了在低温胁迫下超敏蛋白对茶树叶片生理特性的影响。结果表明：常温下，超敏蛋白喷施的茶树叶片，在6 d内略微提高了渗透调节物质含量和抗氧化酶活性。低温胁迫下，茶树叶片中可溶性糖含量、可溶性蛋白含量、游离脯氨酸含量、丙二醛（MDA）含量、超氧化物歧化酶活性（SOD）、过氧化物酶活性（CAT）、过氧化氢酶（POD）活性均增加，叶绿素含量降低；喷施24 μg ? L^-1超敏蛋白后，低温下可在6 d内提高茶树叶片叶绿素、可溶性蛋白、可溶性糖、游离脯氨酸的含量，提高SOD、CAT、POD活性，从而缓解低温胁迫对茶树的伤害，增强茶树耐寒性。试验表明，喷施一定浓度的超敏蛋白可以有效提高茶树的耐寒性。     

Microparticles derived from bone marrow mesenchymal stem cells promote angiogenesis in rat myocardial infarction model

AIM: To observe the effects of microparticles derived from bone marrow mesenchymal stem cells (MSC-MPs) on angiogenesis and cardiac function in a rat myocardial infarction model. METHODS: MSCs were obtained from Sprague-Dawley rats. MSCs were treated under serum-free condition in hypoxia for 72 h, and the microparticles were isolated from the supernatants. The phenotypic profile of MSC-MPs was determined by bead-based flow cytometry and the morphology was observed under a transmission electron microscope. The rat myocardial infarction model was established. The cardiac function was evaluated by echocardiography after the intramyocardial injection of MSC-MPs. The myocardial infarct size was observed by Masson staining. The blood vessel density in the peri-infarcted area was measured using immunohistochemical staining for von Willebrand factor and α-smooth muscle actin. The expression of vascular endothelial growth factor (VEGF) was analyzed by real-time PCR. RESULTS: Apoptotic MSCs released a large quantity of microparticles which were phenotypically similar to the parent MSCs and 100~1 000 nm in diameter. The cardiac functions of myocardial infarction rat model were improved at 7 d and 28 d after intramyocardial injection of MSC-MPs compared with control group. The myocardial infarct size was reduced and angiogenesis was promoted significantly in the infarcted heart injected with MSC-MPs 28 d after treatment. MSC-MPs treatment also increased the expression level of VEGF within 7 d.CONCLUSION: MSC-MPs protect cardiac tissue from ischemic injury and improve cardiac function by promoting angiogenesis after myocardial infarction.     

The plasticity of hematopoietic stem cells and clinical application

Traditional concept has been that hematopoietic stem cells (HSC) are tissue-specific stem cells, which are restricted to generate the cell types of the blood and immune system. However, recent studies have shown that there is a higher plasticity of the HSC than previous expected, it can be induced to transdifferentiated into mature cells of other nonhematopoietic organs after transplantation in vivo. In this article, the recent advances in the plasticity of HSC and its potential clinical application are reviewed.     

ceRNA mechanism of circRNA-miRNA-mRNA in atherosclerosis

Atherosclerosis (AS) is a common cardiovascular disease and a major cause of coronary heart di-sease, cerebral infarction and peripheral vascular disease. Circular RNA (circRNA) is a class of circularly closed non-co-ding RNAs that play an important role in the formation of AS. Physiological and pathological significances of the processes such as lipid metabolism, endothelial cell damage, vascular smooth muscle cell proliferation and migration, monocyte pha-gocytosis and foam-like cell formation, and inflammatory responses are involved in the development of AS. Most circRNA adjust mRNA expression by regulating microRNAs (miRNAs). circRNA provides new ideas and targets for the diagnosis and treatment of AS.     

Protective effect of onychin on the human umbilical vein endothelial cells injured by menadione

AIM: To investigate the protective effect of onychin on the endothelial cells injured by oxidative stress. METHODS: The injured model was established by endothelial cells treated with menadione. The growth inhibitory rate of endothelial cell was determined by MTT assay; NO₂^-／NO₃^- concentration in the medium was determined by nitrate reductase assay; eNOS and caveolin-1 protein levels were determined by Western blot. RESULTS: Onychin significantly decreased the growth inhibitory rate of endothelial cells injured by menadione, increased NO₂^-／NO₃^- concentration in the medium and eNOS activity and up-regulated caveolin-1 expression. CONCLUSION: Onychin possesses a protective effect against endothelial cell injury induced by menadione via caveolin-1/eNOS pathway.     

2,3,5,4’-Tetrahydroxystilbene-2-O-β-D-glucoside protects against LPC-induced endothelial cell injury

AIM: To observe the protective effect of 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) on lysophosphatidylcholine (LPC)-induced vascular endothelial cell injury. METHODS: The 3rd and 4th generations of human umbilical vein endothelial cells (HUVECs) were cultured in vitro and propagated. The cells were randomly divided into 3 groups: control group, model group (LPC) and experimental group (TSG+LPC). The cells in control group were not treated with any reagent. To establish endothelial cell injury model, LPC was administered to HUVECs at concentration of 10 mg/L and incubated with the cells for 24 h. In TSG+LPC group, TSG was administered to HUVECs at concentrations of 10.0, 1.0 and 0.1 μmol/L 1 h before administration of LPC, and then the cells were incubated for 24 h. The cell viability, the content of asymmetric dimethyl arginine (ADMA) and NO, and apoptotic rate were detected. RESULTS: Compared with control group, ADMA content in the cell culture supernatants and apoptotic rate of the HUVECs in LPC group were significantly increased, while the NO content and cell viability were notably decreased. Compared with LPC group, ADMA content and apoptotic rate in TSG+LPC group was significantly decreased, while the NO content and cell viability were notably increased. CONCLUSION: TSG may protect LPC-injured vascular endothelial cells by attenuating the expression of ADMA and enhancing the production of NO, thus inhibiting apoptosis and increasing cell survival rate.     

Effects of atovastatin calcium on vascular endothelial cells in hypertensive rats

AIM: To investigate the effects of atorvastatin calcium on vascular endothelial cells and circulating endothelial progenitor cells (CEPCs) in hypertensive rats. METHODS: Renal hypertension rat model was constructed with two-kidney-one-clip technique. Twenty-four male SD rats were randomized into hypertension group, statins group and sham-operated group (n=8 each). On the 4th week, the animals in statins group were injected with 20 mg·kg-1·d-1 atorvastatin calcium intra-abdominally for 8 weeks. Blood pressure and blood lipid were measured at 4th and 12th week post-operation. On the 12th week, scanning electron microscopy was used to observe the damage of aortic endothelial cells. The circulatory endothelial cell count, CEPCs count, CEPCs proliferation ability, CEPCs adhesion ability and CEPCs apoptosis were also measured. RESULTS: The injury of endothelial cells in statins group was observed, milder than that in hypertension group, but worsen than that in sham-operated group. The numbers of circulatory endothelial cells in hypertension group, statin treatment group and sham-operated group were 5.9×106, 3.9×106 and 2.0×106 respectively, and the CEPCs apoptosis rate was 22.1%±2.1%, 13.4%±1.6 %and 7.4%±1.3%, respectively. Meanwhile, the numbers of CEPCs were 21.63±2.33, 40.38±6.00 and 65.38±2.97, respectively, the proliferative abilities of CEPCs were 0.13±0.01, 0.17±0.01 and 0.29±0.03, respectively, and the adhesion of CEPCs was 12.25±2.49, 21.50±2.20 and 28.88±2.85, respectively. CONCLUSION: (1) The severity of vascular endothelial cell injury is related to hypertension states. (2) Atorvastatin calcium has direct protective effects on endothelial cells, possibly through increasing CEPCs count, reducing CEPCs apoptosis and markedly enhancing reparation ability of CEPCs to endothelial cells.     

Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.     

MicroRNA expression in hepatocytes with hydrogen peroxide-induced oxidative stress-injury and alleviating effect of mesenchymal stem cell-conditioned medium

AIM: To evaluate the changes of microRNA (miRNA) in hepatocytes during hydrogen peroxide-induced oxidative stress injury, and to observe the alleviating effect of mesenchymal stem cell-conditioned medium (MSC-CM) in this progress. METHODS: The hepatocyte oxidative stress injury model was established using hydrogen peroxide and human normal liver cell line L02. MSC-CM was prepared using centrifugation and filter. The effects of MSC-CM on hepatocyte injury were evaluated by apoptosis analysis, cell viability detection, cell cycle, and mitochondrial membrane potential (MMP). Twenty-one differentially expressed miRNAs were selected by gene chip hybridization, in which miR-143, miR-145, miR-301a and let-7a were confirmed by RT-qPCR. Bioinformatics software was utilized to predict target proteins of these miRNAs, and then the proteins were verified by Western blot.RESULTS: MSC-CM markedly attenuated hydrogen peroxide-induced oxidative stress injury by reducing apoptosis, promoting cell viability and regulating cell cycle. The expression of miR-143, miR-145, miR-301a and let-7a, indentified by RT-qPCR, increased under the condition of oxidative stress injury, while decreased after MSC-CM treatment. The expression of miR-143 predicted target proteins, HK2 and ADRB1, decreased under the hydrogen peroxide-exposure, while increased after MSC-CM treatment, which is consistent with the regulatory trend of miR-143. CONCLUSION: MSC-CM might attenuate hydrogen peroxide induced oxidative stress injury via inhibiting apoptosis and regulating some miRNA expression.