27-nt miRNA对血管内皮细胞eNOS表达/活性的调节及其代谢产物的影响

目的:前期工作表明,27-nt miRNA对eNOS的转录和表达有负反馈调节作用。本实验进一步探讨27-nt miRNA对血管内皮细胞eNOS的基因表达、活性调节及其代谢产物的影响。方法:构建27-nt miRNA高表达质粒,并将其转染至HUVECs。MTT法检测细胞的增殖情况,划痕实验检测细胞的迁移能力,Western Blot检测27-nt miRNA及细胞eNOS蛋白的表达情况,ELISA法检测eNOS活性,硝酸还原法测定细胞培养上清液中NO的含量。结果:27-nt miRNA对HUVECs的增殖有强烈的抑制作用(0.674±0.093 vs 0.315±0.013,0.743±0.076 vs 0.315±0.013,P0.05);27-nt miRNA对HUVECs的迁移有显著的抑制作用(0.483±0.009vs 0.806±0.017,0.465±0.047 vs 0.806±0.017,P0.05);27-nt miRNA显著降低eNOS蛋白的表达(0.410±0.004 vs 0.645±0.007,0.483±0.009 vs 0.645±0.007,P0.05)及明显抑制eNOS的活性(1.093±0.357 vs 5.034±0.509,1.707±0.652 vs 5.034±0.509,P0.05);27-nt miRNA明显抑制NO的合成与释放(70.687±4.432 vs 136.803±6.913,75.264±4.481 vs 136.803±6.913,P0.05)。结论:27-nt miRNA高表达明显抑制eNOS基因的表达及活性;27-nt miRNA明显抑制NO的合成和释放,可能成为血管性疾病治疗的分子靶点。     

内含子源性microRNA对内皮型一氧化氮合酶表达及血管内皮细胞增殖的作用

前期工作表明,内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)第4内含子中的27碱基 (nucleotide,nt)重复序列是27-nt microRNA的来源,并对eNOS具有重要的调节作用．为进一步探讨该内含子源性27-nt microRNA参与调节eNOS表达的分子机制及其在内皮细胞增殖中的可能作用,通过构建27-nt microRNA高表达质粒,用脂质体将该质粒转染人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC),Western blot和RT-PCR检测该细胞系中eNOS蛋白和mRNA表达情况以及胞核转录因子的表达改变,并观察HUVEC增殖的变化情况．结果发现：27-nt microRNA 高表达能降低eNOSmRNA的水平和蛋白质表达;同时对转录因子Sp1、Ap1的蛋白质表达也产生了不同程度的抑制作用;转染后细胞的生长速度比未转染的细胞明显减慢,尤其转染了27-nt microRNA的双倍长度突变体(pEGP-mut-54nt-mi)质粒的HUVEC,其生长倍增时间比正常对照组明显延长达49.4%．结果表明,27-nt microRNA明显抑制eNOS蛋白及其mRNA表达,同时 HUVEC增殖受到明显抑制,转录因子Sp1 和Ap1 在27-nt microRNA对eNOS的表达调节中起重要作用．实验提示,内含子源性microRNA与转录因子共同参与对内皮细胞增殖及其相关性基因的表达调节,可能是众多真核细胞中某些疾病相关性基因表达自我调节的重要机制之一．     

Biogenesis of short intronic repeat 27-nucleotide small RNA from endothelial nitric-oxide synthase gene

Endothelial nitric-oxide synthase (eNOS) is a constitutively expressed gene in endothelium that produces NO and is critical for vascular integrity. Previously, we reported that the 27-nucleotide (nt) repeat polymorphism in eNOS intron 4, a source of 27-nt small RNA, which inhibits eNOS expression, were associated with cardiovascular risk and expression of the eNOS gene. In the current study, we investigated the biogenesis of the intron 4-derived 27-nt small RNA. Using Northern blot, we showed that the eNOS-derived 27-nt short intronic repeat RNA (sir-RNA) expressed only in the eNOS expressing endothelial cells. Cells containing 10 x 27- or 5 x 27-nt repeats produced higher levels of 27nt sir-RNA and lower levels of eNOS mRNA than the cells with 4 x 27-nt repeats. The 27nt sir-RNA was mostly present within the endothelial nuclei. When the splicing junctions of the 27-nt repeat containing intron 4 in the full-length eNOS cDNA vector were mutated, 27nt sir-RNA biogenesis was abolished. Suppression of Drosha or Dicer diminished the biogenesis of the 27nt sir-RNA. Our study suggests that the 27nt sir-RNA derived through eNOS pre-mRNA splicing may represent a new class of small RNA. The more eNOS is transcribed or higher number of the 27-nt repeats, the more 27nt sir-RNA is produced, which functions as a negative feedback self-regulator by specifically inhibiting the host gene eNOS expression. This novel molecular model may be responsible for quantitative differences between individuals carrying different numbers of the polymorphic repeats hence the cardiovascular risk.     

Interleukin-6-induced JAK2/STAT3 signaling pathway in endothelial cells is suppressed by hemodynamic flow

Endothelial cells (ECs) are constantly exposed to shear stress, the action of which triggers signaling pathways and cellular responses. During inflammation, cytokines such as IL-6 increase in plasma. In this study, we examined the effects of steady flow on IL-6-induced endothelial responses. ECs exposed to IL-6 exhibited STAT3 activation via phosphorylation of Tyr705. However, when ECs were subjected to shear stress, shear force-dependent suppression of IL-6-induced STAT3 phosphorylation was observed. IL-6 treatment increased the phosphorylation of JAK2, an upstream activator of STAT3. Consistently, shear stress significantly reduced IL-6-induced JAK2 activation. Pretreatment of ECs with an inhibitor of MEK1 did not alter this suppression by shear stress, indicating that extracellular signal-regulated kinase (ERK1/2) was not involved. However, pretreatment of ECs with an endothelial nitric oxide synthase inhibitor (nitro-L-arginine methyl ester) attenuated this inhibitory effect of shear stress on STAT3 phosphorylation. Shear stress-treated ECs displayed decreased nuclear transmigration of STAT3 and reduced STAT3 binding to DNA. Intriguingly, ECs exposed to IL-6 entered the cell cycle, as evidenced by increasing G₂/M phase, and shear stress to these ECs significantly reduced IL-6-induced cell cycle progression. STAT3-mediated IL-6-induced cell cycle was confirmed by the inhibition of the cell cycle in ECs infected with adenovirus carrying the inactive mutant of STAT3. Our study clearly shows that shear stress exerts its inhibitory regulation by suppressing the IL-6-induced JAK2/STAT3 signaling pathway and thus inhibits IL-6-induced EC proliferation. This shear force-dependent inhibition of IL-6-induced JAK2/STAT3 activation provides new insights into the vasoprotective effects of steady flow on ECs against cytokine-induced responses. shear stress; nitric oxide; cell cycle     

Crosstalk between hydrogen sulfide and nitric oxide in endothelial cells

Hydrogen sulfide (H₂S) and nitric oxide (NO) are major gasotransmitters produced in endothelial cells (ECs), contributing to the regulation of vascular contractility and structural integrity. Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H₂S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs‐926) with NaHS (a H₂S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H₂S had little effect on eNOS protein expression in ECs. L‐cysteine, a precursor of H₂S, stimulated NO production whereas blockage of the activity of H₂S‐generating enzyme, cystathionine gamma‐lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3‐K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H₂S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS‐specific siRNA or nitro‐L‐arginine methyl ester (L‐NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H₂S on ECs. Our results suggest that H₂S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt‐dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H₂S‐induced angiogenesis.     

Inhibition of fibroblast-induced angiogenic phenotype of cultured endothelial cells by the overexpression of tissue inhibitor of metalloproteinase (TIMP)-3

In this study, we examined the effect of overexpression of tissue inhibitor of metalloproteinase (TIMP)-3 on the angiogenic phenotype expressed by vascular endothelial cells (ECs). ECs were infected with a recombinant adenovirus carrying the TIMP-3 gene at various multiplicities of infection, and TIMP-3 expression by transfected cells was confirmed by Western blotting and reverse zymography. At transfection doses of 6.25, 12.5, 25, 50 and 100 multiplicity of infection, EC migration was reduced to 66, 45, 25, 17 and 5%, respectively, of that of the control. At the multiplicity of infection of 20, capillary tube length was reduced by 80% compared to that of the control. Thus, expression of TIMP-3 by ECs effectively inhibited EC migration and tube formation. Overexpression of TIMP-3 by ECs may be considered a gene therapy strategy for the treatment of pathological angiogenesis such as cancer and diabetic retinopathy.     

A role for endothelial cells in promoting the maturation of astrocytes through the apelin/APJ system in mice

Interactions between astrocytes and endothelial cells (ECs) are crucial for retinal vascular formation. Astrocytes induce migration and proliferation of ECs via their production of vascular endothelial growth factor (VEGF) and, conversely, ECs induce maturation of astrocytes possibly by the secretion of leukemia inhibitory factor (LIF). Together with the maturation of astrocytes, this finalizes angiogenesis. Thus far, the mechanisms triggering LIF production in ECs are unclear. Here we show that apelin, a ligand for the endothelial receptor APJ, induces maturation of astrocytes mediated by the production of LIF from ECs. APJ (Aplnr)- and Apln-deficient mice show delayed angiogenesis; however, aberrant overgrowth of endothelial networks with immature astrocyte overgrowth was induced. When ECs were stimulated with apelin, LIF expression was upregulated and intraocular injection of LIF into APJ-deficient mice suppressed EC and astrocyte overgrowth. These data suggest an involvement of apelin/APJ in the maturation process of retinal angiogenesis.     

Glycogen synthase kinase-3β is involved in C-reactive protein-induced endothelial cell activation

C-reactive protein (CRP) is a significant contributor to atherosclerosis and a powerful predictor of cardiovascular risk. The role of CRP in endothelial cell (EC) activation has been extensively investigated, but the underlying mechanisms have not been fully elucidated. The effect of glycogen synthase kinase-3β (GSK-3β) on CRP-induced EC activation was evaluated in this study. We observed that CRP decreased endothelial nitric oxide synthase (eNOS) activity during EC activation. CRP also activated GSK-3β by dephosphorylating its Ser9 level and reducing β-catenin protein expression in a time-dependent manner. We also found that the GSK-3β inhibitors TDZD-8 and SB415286 partially restored eNOS activity and suppressed the release of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 from ECs. These data provide new evidence for the involvement of GSK-3β in EC activation.     

MARCH5 restores endothelial cell function against ischaemic/hypoxia injury via Akt/eNOS pathway

MARCH5 is a critical regulator of mitochondrial dynamics, apoptosis and mitophagy. However, its role in cardiovascular system remains poorly understood. This study aimed to investigate the role of MARCH5 in endothelial cell (ECs) injury and the involvement of the Akt/eNOS signalling pathway in this process. Rat models of myocardial infarction (MI) and human cardiac microvascular endothelial cells (HCMECs) exposed to hypoxia (1% O₂) were used in this study. MARCH5 expression was significantly reduced in ECs of MI hearts and ECs exposed to hypoxia. Hypoxia inhibited the proliferation, migration and tube formation of ECs, and these effects were aggravated by knockdown of MARCH5 but antagonized by overexpressed MARCH5. Overexpression of MARCH5 increased nitric oxide (NO) content, p-eNOS and p-Akt, while MARCH5 knockdown exerted the opposite effects. The protective effects mediated by MARCH5 overexpression on ECs could be inhibited by eNOS inhibitor L-NAME and Akt inhibitor LY294002. In conclusion, these results indicated that MARCH5 acts as a protective factor in ischaemia/hypoxia-induced ECs injury partially through Akt/eNOS pathway.