敲除miR-223促进血管炎症和动脉粥样硬化的发生

目的研究miR-223对血管炎症和动脉粥样硬化的影响,为临床动脉硬化性疾病提供新的诊疗方向。方法miR-223敲除鼠与ApoE敲除鼠(ApoE KO)繁殖制备miR-223/ApoE双敲鼠(miR-223/ApoE DKO);检测小鼠血浆脂质水平;处死取材后检测主动脉根部及血管全长的斑块含量;通过免疫组化检测斑块的炎症细胞浸润;转录组学测序分析血管中炎症相关基因的表达;结合microRNA靶基因数据库寻找并验证其可能的靶基因。结果miR-223/ApoE双敲鼠主动脉根部及血管全长斑块量显著增加(P<0.05)。免疫组化染色显示,主动脉根部炎症细胞浸润增加;血管转录组学测序发现炎症相关基因血管细胞黏附分子1(VCAM-1)、白细胞介素1α(IL-1α)等在双敲鼠中显著上调。通过靶基因数据库筛选,发现白细胞介素6(IL-6)是miR-223的靶基因并且在双敲鼠的血管中表达显著上调;使用miR-223模拟物刺激成纤维细胞,显著抑制了IL-6的表达。结论miR-223抑制靶基因IL-6的表达降低炎症反应,敲除miR-223显著升高血管炎症水平促进动脉粥样硬化的进展。     

Apolipoprotein E deficiency and a mouse model of accelerated liver aging

The liver is the central metabolic organ which regulates several key aspects of lipid metabolism. The liver changes with age leading to an impaired ability to respond to hepatic insults and an increased incidence of liver disease in the elderly. Apolipoprotein E (ApoE) null mice have proved to be a very popular model to study spontaneous atherosclerosis, but recently it has been demonstrated that in ApoE?/? mice liver there are enzymatic and structural alterations, normally linked to the age. The purpose of this study was to consider ApoE?/? mice as a model for oxidative stress induced hepatic disease and to clarify how ApoE inactivation accelerates the aging process and causes liver disease.We used ApoE null mice and control mice at different ages (6 weeks and 15 months).Liver morphological damage as well as proteins involved in oxidative stress and liver ageing were all analyzed.Our study showed that ApoE null mice develop important age-related changes including oxidative stress, pseudocapillarization, increased polyploidy, decreased hepatocyte number and increased nuclear size. Our findings provide evidence that hypercholesterolemic ApoE?/? mice are more likely to develop severe liver injury, suggesting that in addition to vascular disease, increased cholesterol products and oxidative stress may also play a role in accelerating the progression of aging in the liver.     

LncRNA PVT1 knockdown alleviated ox-LDL-induced vascular endothelial cell injury and atherosclerosis by miR-153-3p/GRB2 axis via ERK/p38 pathway

Background and aimsLncRNA plasmacytoma variant translocation 1 (PVT1) plays a regulatory role in some cardiovascular diseases, but its role in atherosclerosis (AS) remains barely explored. The study aimed to investigate the effects of PVT1 on high fat diet-induced AS and its potential mechanisms.Methods and resultsApoE −/− mice were fed with high fat diet for 8 weeks to establish an AS model. Lentiviral vectors containing PVT1 short hairpin RNA (PVT1-shRNA) or NC-shRNA were administered by tail vein injection. Cell viability, apoptosis, inflammatory factor secretion, and cellular oxidative stress were measured to evaluate oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cell (HUVEC) injury. Dual-luciferase reporter gene and RNA immunoprecipitation assays were used to confirm the interaction between miR-153-3p and PVT1 or growth factor receptor binding protein 2 (GRB2). Atherosclerotic lesions, lipid deposition, and cell apoptosis in aorta were analyzed by H&E, Oil Red O, and TUNEL straining. PVT1 knockdown alleviated ox-LDL-induced inflammation, apoptosis and oxidative stress in HUVECs. PVT1 acted as a sponge of miR-153-3p, and GRB2 was confirmed as a target of miR-153-3p. MiR-153-3p overexpression attenuated the enhanced effects of PVT1 on ox-LDL-induced cell damage. GRB2 overexpression reversed the mitigating effects of miR-153-3p on ox-LDL-caused injury. Inhibiting PVT1 restrained the activation of ERK1/2 and p38 pathway via miR-153-3p/GRB2 axis. Additionally, silencing PVT1 in vivo reduced atherosclerotic plaques, lipid deposition, inflammation, oxidative stress, and apoptosis in AS mice.ConclusionPVT1 knockdown alleviated ox-LDL-induced vascular endothelial cell injury and atherosclerosis through miR-153-3p/GRB2 axis via ERK1/2 and p38 pathway.     

Effect of miR-467b on atherosclerosis of rats

Objective:To observe the effect of miR-467 b on the atherosclerosis(AS) of rats with apolipoprotein E(ApoE) gene knockout(ApoE~(-/-)).Methods:ApoE~(-/-) rats were fed with high fat and high cholesterol diet and were randomly divided into group A,group B and group C,with10 rats in each group.Group A:rats were injected with ApoE agonist through the caudal vein;Group B:rats were injected with ApoE antagonist through the caudal vein;Group C:as negative control group.Enzyme oxidation method was used to detect the blood lipid levels of rats.Western blotting method was used to detect the aortic lipoprotein lipase(LPL) cxprcssion IcvcLs of rats.HE staining and oil rod o staining were performed to observe the AS lesions and lipid accumulation state.Results:Compared with Group C,blood lipid level,aortic intima and aortic sinus lipid accumulation area ratio,aortic sinus lesion area and LPL expression level in Group A significantly reduced:while blood lipid level,aortic intima and aortic sinus lipid accumulation area ratio,aortic sinus lesion area,and LPL expression level in Group B significantly increased,with the statistical difference(P0.05).Conclusions:miR-467 b can alleviate the AS lesions of ApoE~(-/-) rats,and its inhibiting effect on AS may be related to LPL expression.     

Atheroprotection via cannabinoid receptor-2 is mediated by circulating and vascular cells in vivo

Low-dose oral tetrahydrocannabinol (THC) reduces progression of atherosclerosis in mice. THC activates central cannabinoid-1 receptors (CB1) with subsequent psychoactive effects as well as peripheral cannabinoid-2 receptors (CB2). In order to dissect the underlying mechanisms, we performed experiments under selective CB2 stimulation as well as after genetic disruption of the CB2 receptor. Atherosclerosis prone apolipoprotein E-deficient mice were crossed with cannabinoid receptor-2 deficient mice to obtain ApoE -/- CB2 -/- double knockout mice. After 8weeks of a high-cholesterol diet, immunohistochemical stainings of the aortic root revealed that vascular leukocyte infiltration in atherosclerotic plaques was accelerated in ApoE -/- CB2 -/- mice compared with ApoE -/- mice. This was accompanied by increased release of reactive oxygen species as measured using L012-enhanced chemiluminescence, and by decreased endothelial function as assessed in isolated aortic rings in organ chamber experiments. ApoE -/- mice treated with the selective CB2 agonist JWH 133 during a high-cholesterol diet showed decreased atherosclerotic lesion formation, improved endothelial function and reduced levels of reactive oxygen species. To assess whether CB2 expression in circulating cells influences atherosclerosis, irradiated ApoE -/- mice were repopulated with bone marrow-derived cells from ApoE -/- and ApoE -/- CB2 -/- mice and were fed a high-cholesterol diet for 8weeks. CB2 deficiency in bone marrow-derived cells increased leukocyte infiltration into the vessel wall, but had no impact on plaque formation. Cell culture experiments revealed that CB2 activation diminishes ROS generation in vascular cells. Selective CB2 receptor stimulation modulates atherogenesis via impact on both circulating proinflammatory and vascular cells.     

Melatonin prevents endothelial cell pyroptosis via regulation of long noncoding RNA MEG3/miR‐223/NLRP3 axis

Atherosclerosis (AS ) is an inflammatory disease linked to endothelial dysfunction. Melatonin is reported to possess substantial anti‐inflammatory properties, which has proven to be effective in AS . Emerging literature suggests that pyroptosis plays a critical role during AS progression. However, whether pyroptosis contributes to endothelial dysfunction and the underlying molecular mechanisms remained unexploited. This study was designed to investigate the antipyroptotic effects of melatonin in atherosclerotic endothelium and to elucidate the potential mechanisms. In this study, high‐fat diet (HFD )‐treated ApoE^?/? mice were used as an atherosclerotic animal model. We found intragastric administration of melatonin for 12 weeks markedly reduced the atherosclerotic plaque in aorta. Meanwhile, melatonin also attenuated the expression of pyroptosis‐related genes, including NLRP 3, ASC , cleaved caspase1, NF ‐κB/GSDMD , GSDMD N‐termini, IL ‐1β, and IL ‐18 in aortic endothelium of melatonin‐treated animals. Consistent antipyroptotic effects were also observed in ox‐LDL ‐treated human aortic endothelial cells (HAEC s). We found that lncRNA MEG 3 enhanced pyroptosis in HAEC s. Moreover, MEG 3 acted as an endogenous sponge by sequence complementarity to suppress the function of miR‐223 and to increase NLRP 3 expression and enhance endothelial cell pyroptosis. Furthermore, knockdown of miR‐223 blocked the antipyroptotic actions of melatonin in ox‐LDL ‐treated HAEC s. Together, our results suggest that melatonin prevents endothelial cell pyroptosis via MEG 3/miR‐223/NLRP 3 axis in atherosclerosis, and therefore, melatonin replacement might be considered a new strategy for protecting endothelium against pyroptosis, thereby for the treatment of atherosclerosis associated with pyroptosis.     

Atherosclerosis-associated endothelial dysfunction is promoted by miR-199a-5p/SIRT1 axis regulated by circHIF1ɑ

Background and aimsAtherosclerosis (AS) is a chronic inflammatory disease that damages the arterial wall as a result of hyperlipidemia and causes endothelial cell dysfunction, which increases the risk of atherothrombotic events. Multiple pathological conditions have shown ectopic miR-199a-5p levels to cause endothelial injury, but its role in the AS competitive endogenous RNA (CeRNA) network is still unknown.Methods and resultsThe high-fat diet (HFD) apoE−/− mouse model was constructed in vivo, and ECs were cultured under ox-LDL treatment to induce EC injury in vitro. Immunohistochemistry and immunofluorescence staining were used to assess the effect of miR-199a-5p on the macrophage, SMC, collagen content, and endothelial coverage in the artery wall of mouse model. miR-199a-5p level was validated to be overexpression in the aorta tissue of HFD apoE−/− mice and in the ox-LDL-treated ECs, and even in the plasma EVs of the patients with cerebral AS. Silencing of miR-199a-5p significantly attenuated atherosclerotic progress in HFD apoE−/− mice, and the gain/loss-of-function assay indicated that miR-199a-5p overexpression aggravated ox-LDL-induced disabilities of endothelial proliferation, motility, and neovascularization based on cell counting kit-8 assay, transwell assay and matrigel assay. Mechanistically, miR-199a-5p prevented EC activation by activating the FOXO signaling pathway by targeting SIRT1. Additionally, circular RNA (circRNA) circHIF1ɑ was identified as having a low expression in the ox-LDL-treated EC and mediated SIRT1 expression via sponging miR-199a-5p to rescue ox-LDL-induced EC injury.ConclusionsOur study demonstrated the vital role of miR-199a-5p/SIRT1 axis regulated by circHIF1ɑ in AS pathogenesis and provided novel effective targets for AS treatment.     

Atheroprotection via cannabinoid receptor-2 is mediated by circulating and vascular cells in vivo

Low-dose oral tetrahydrocannabinol (THC) reduces progression of atherosclerosis in mice. THC activates central cannabinoid-1 receptors (CB1) with subsequent psychoactive effects as well as peripheral cannabinoid-2 receptors (CB2). In order to dissect the underlying mechanisms, we performed experiments under selective CB2 stimulation as well as after genetic disruption of the CB2 receptor. Atherosclerosis prone apolipoprotein E-deficient mice were crossed with cannabinoid receptor-2 deficient mice to obtain ApoE −/− CB2 −/− double knockout mice. After 8 weeks of a high-cholesterol diet, immunohistochemical stainings of the aortic root revealed that vascular leukocyte infiltration in atherosclerotic plaques was accelerated in ApoE −/− CB2 −/− mice compared with ApoE −/− mice. This was accompanied by increased release of reactive oxygen species as measured using L012-enhanced chemiluminescence, and by decreased endothelial function as assessed in isolated aortic rings in organ chamber experiments. ApoE −/− mice treated with the selective CB2 agonist JWH 133 during a high-cholesterol diet showed decreased atherosclerotic lesion formation, improved endothelial function and reduced levels of reactive oxygen species. To assess whether CB2 expression in circulating cells influences atherosclerosis, irradiated ApoE −/− mice were repopulated with bone marrow-derived cells from ApoE −/− and ApoE −/− CB2 −/− mice and were fed a high-cholesterol diet for 8 weeks. CB2 deficiency in bone marrow-derived cells increased leukocyte infiltration into the vessel wall, but had no impact on plaque formation. Cell culture experiments revealed that CB2 activation diminishes ROS generation in vascular cells. Selective CB2 receptor stimulation modulates atherogenesis via impact on both circulating proinflammatory and vascular cells.     

Disruption of the 12/15-lipoxygenase gene (Alox15) protects hyperlipidemic mice from nonalcoholic fatty liver disease

We have shown that Alox15, the gene encoding for 12/15-lipoxygenase (12/15-LO), is markedly up-regulated in livers from apolipoprotein E-deficient (ApoE(-/-)) mice, which spontaneously develop nonalcoholic fatty liver disease secondary to hyperlipidemia. In the current study, we used ApoE(-/-) mice with a targeted disruption of the Alox15 gene to assess the role of 12/15-LO in the development and progression of hepatic steatosis and inflammation. Compared with ApoE(-/-) mice, which exhibited extensive hepatic lipid accumulation and exacerbated inflammatory injury, ApoE/12/15-LO double-knockout (ApoE(-/-)/12/15-LO(-/-)) mice showed reduced serum alanine aminotransferase levels; decreased hepatic steatosis, inflammation, and macrophage infiltration; and decreased fatty acid synthase, tumor necrosis factor α (TNFα), monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-18, and IL-6 expression. Remarkably, disruption of Alox15 attenuated glucose intolerance and high-fat diet-induced insulin resistance, up-regulated insulin receptor substrate-2, and exerted opposite effects on hepatic c-Jun amino-terminal kinase and adenosine monophosphate-activated protein kinase phosphorylation, known negative and positive regulators of insulin signaling, respectively. In adipose tissue, the absence of Alox15 induced significant reductions in the expression of the proinflammatory and insulin-resistant adipokines MCP-1, TNFα, and resistin while increasing the expression of glucose transporter-4. Interestingly, compared with ApoE(-/-) mice, which exhibited increased hepatic caspase-3 staining, ApoE(-/-)/12/15-LO(-/-) mice showed attenuated hepatocellular injury. Consistent with this finding, hepatocytes isolated from ApoE(-/-) mice were more vulnerable to TNFα-induced programmed cell death, an effect that was not observed in hepatocytes carrying a targeted disruption of the Alox15 gene. Conclusion: Collectively, our data suggest a potentially relevant mechanism linking 12/15-LO to the promotion of hepatic steatosis, insulin resistance, and inflammation in experimental liver disease of metabolic origin.     

Endothelial arginase II: a novel target for the treatment of atherosclerosis

Oxidized low-density lipoproteins increase arginase activity and reciprocally decrease endothelial NO in human aortic endothelial cells. Here, we demonstrate that vascular endothelial arginase activity is increased in atherogenic-prone apolipoprotein E-null (ApoE(-/-)) and wild-type mice fed a high cholesterol diet. In ApoE(-/-) mice, selective arginase II inhibition or deletion of the arginase II gene (Arg II(-/-) mice) prevents high-cholesterol diet-dependent decreases in vascular NO production, decreases endothelial reactive oxygen species production, restores endothelial function, and prevents oxidized low-density lipoprotein-dependent increases in vascular stiffness. Furthermore, arginase inhibition significantly decreases plaque burden. These data indicate that arginase II plays a critical role in the pathophysiology of cholesterol-mediated endothelial dysfunction and represents a novel target for therapy in atherosclerosis.     

The potential role of resistin in atherogenesis

Resistin, an adipocyte-derived cytokine linked to insulin resistance and obesity, has recently been shown to activate endothelial cells (ECs). Using microarrays, we found that along with numerous other pro-atherosclerotic genes, resistin expression levels are elevated in the aortas of C57BL/6J apoE-/- mice; these findings led us to further explore the relation between resistin and atherosclerosis. Using TaqMan PCR and immunohistochemistry, we found that ApoE-/- mice had significantly higher resistin mRNA and protein levels in their aortas, and elevated serum resistin levels, compared to C57BL/6J wild-type mice. Incubation of murine aortic ECs with recombinant resistin increased monocyte chemoattractant protein (MCP)-1 and soluble vascular cell adhesion molecule (sVCAM)-1 protein levels in the conditioned medium. Furthermore, human carotid endarterectomy samples stained positive for resistin protein, while internal mammary artery did not show strong staining. Patients diagnosed with premature coronary artery disease (PCAD) were found to have higher serum levels of resistin than normal controls. In summary, resistin protein is present in both murine and human atherosclerotic lesions, and mRNA levels progressively increase in the aortas of mice developing atherosclerosis. Resistin induces increases in MCP-1 and sVCAM-1 expression in murine vascular endothelial cells, suggesting a possible mechanism by which resistin might contribute to atherogenesis. Finally, PCAD patients exhibited increased serum levels of resistin when compared to controls. These findings suggest a possible role of resistin in cardiovascular disease.     

Decorin overexpression reduces atherosclerosis development in apolipoprotein E-deficient mice

Atherosclerosis results from accumulation of macrophages and extracellular matrix in the arterial wall. Decorin, a small matrix proteoglycan, is able to regulate cell proliferation, migration and growth factors' activity. We investigated the effect of decorin overexpression on atherosclerosis progression in apolipoprotein E-deficient (ApoE(-/-)) mice. Female ApoE(-/-) mice, 10 weeks old (early treatment, n = 20) and 20 weeks old (delayed treatment, n = 20) were administered intravenously with either an adenovirus (2.5 x 10(9) plaque-forming units/mouse) containing human decorin gene (Ad-Dcn) or beta-galactosidase (LacZ), or PBS. Transgenic decorin was mainly expressed in the liver, and was secreted in the plasma up to 4 weeks. Six weeks after treatment, no significant difference in aortic root lesion size was observed between LacZ- and PBS-control groups. In contrast, Ad-Dcn-treated mice showed significantly reduced atherosclerotic lesions as compared to controls in both early and delayed treatment groups (2.9 +/- 1.1% versus 5.5 +/- 0.4%; p = 0.004 and 13.4 +/- 1.3% versus 19.9 +/- 1.41%; p = 0.009, respectively). In parallel, macrophage, gelatinase activity and collagen plaque content were also reduced. Interestingly, plasma triglycerides were reduced and decorin formed complexes with transforming growth factor-beta1 (TGF-beta1) that resulted in reduced circulating free-TGF-beta1. In conclusion, systemic overexpression of decorin reduces inflammation, triglycerides and fibrosis in atherosclerotic plaques of ApoE(-/-) mice resulting in slowing down of disease progression.     

Heart rate reduction by ivabradine improves aortic compliance in apolipoprotein e-deficient mice

Background: Impaired vascular compliance is associated with cardiovascular mortality. The effects of heart rate on vascular compliance are unclear. Therefore, we characterized effects of heart rate reduction (HRR) by I(f) current inhibition on aortic compliance and underlying molecular mechanisms in apolipoprotein E-deficient (ApoE(-)/(-)) mice. Methods: ApoE(-)/(-) mice fed a high-cholesterol diet and wild-type (WT) mice were treated with ivabradine (20 mg/kg/d) or vehicle for 6 weeks. Compliance of the ascending aorta was evaluated by MRI. Results: Ivabradine reduced heart rate by 113 ± 31 bpm (～19%) in WT mice and by 133 ± 6 bpm (～23%) in ApoE(-)/(-) mice. Compared to WT controls, ApoE(-)/(-) mice exhibited reduced distensibility and circumferential strain. HRR by ivabradine increased distensibility and circumferential strain in ApoE(-)/(-) mice but did not affect both parameters in WT mice. Ivabradine reduced aortic protein and mRNA expression of the angiotensin II type 1 (AT1) receptor and reduced rac1-GTPase activity in ApoE(-)/(-) mice. Moreover, membrane translocation of p47(phox) was inhibited. In ApoE(-)/(-) mice, HRR induced anti-inflammatory effects by reduction of aortic mRNA expression of IL-6, TNF-alpha and TGF-beta. Conclusion: HRR by ivabradine improves vascular compliance in ApoE(-)/(-) mice. Contributing mechanisms include downregulation of the AT1 receptor, attenuation of oxidative stress and modulation of inflammatory cytokine expression.     

软脉降脂胶囊对载脂蛋白E基因敲除小鼠动脉粥样硬化影响的实验研究

目的观察软脉降脂胶囊对载脂蛋白E基因敲除载脂蛋白E基因敲除小鼠(ApoE)高脂血症及动脉粥样硬化(atherosclerosis,AS)的影响.方法6周龄ApoE小鼠,随机分为高脂血症组与软脉降脂胶囊大、中、小剂量组,相同遗传背景的同龄正常C57BL/6J小鼠作为正常对照组.软脉降脂胶囊组灌服其生药,高脂血症组、正常对照组均灌服生理盐水.连续灌胃24周后,下腔静脉取血测血脂;取主动脉做形态学观察及图像分析.结果高脂血症组血清总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL-C)明显高于正常对照组(P＜0.05),软脉降脂胶囊组明显低于高脂血症组(P＜0.05);图像分析测量结果显示高脂血症组AS病变显著,与正常对照组比较斑块总面积明显增加(P＜0.01);软脉降脂胶囊组AS病变较高脂血症纽明显减轻,斑块总面积明显减小(P＜0.01).结论软脉降脂胶囊可降低ApoE小鼠血清TC、TG、LDL-C含量,有对抗动脉粥样硬化斑块形成的作用.     

Junctional adhesion molecule-A deficiency increases hepatic ischemia-reperfusion injury despite reduction of neutrophil transendothelial migration

The endothelial receptors that control leukocyte transmigration in the postischemic liver are not identified. We investigated the role of junctional adhesion molecule-A (JAM-A), a receptor expressed in endothelial tight junctions, leukocytes, and platelets, for leukocyte transmigration during hepatic ischemia-reperfusion (I/R) in vivo. We show that JAM-A is up-regulated in hepatic venular endothelium during reperfusion. I/R-induced neutrophil transmigration was attenuated in both JAM-A-/- and endothelial JAM-A-/- mice as well as in mice treated with an anti-JAM-A antibody, whereas transmigration of T cells was JAM-A independent. Postischemic leukocyte rolling remained unaffected in JAM-A-/- and endothelial JAM-A-/- mice, whereas intravascular leukocyte adherence was increased. The extent of interactions of JAM-A-/- platelets with the postischemic endothelium was comparable with that of JAM-A+/+ platelets. The I/R-induced increase in the activity of alanine aminotransferase (ALT)/aspartate aminotransferase (AST) and sinusoidal perfusion failure was not reduced in JAM-A-/- mice, while the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive hepatocytes was significantly higher. Thus, we show for the first time that JAM-A is up-regulated in hepatic venules and serves as an endothelial receptor of neutrophil transmigration, but it does not mediate leukocyte rolling, adhesion, or platelet-endothelial cell interactions. JAM-A deficiency does not reduce I/R-induced microvascular and hepatocellular necrotic injury, but increases hepatocyte apoptosis, despite attenuation of neutrophil infiltration.     

Inhibition of miR-92a increases endothelial proliferation and migration in vitro as well as reduces neointimal proliferation in vivo after vascular injury

The role of miR-92a on vascular remodelling after injury is currently unknown. Thus, the aim of the present study was to evaluate the role of miR-92a on rat endothelial and vascular smooth muscle cells proliferation and migration in vitro as well as after balloon injury or arterial stenting in vivo. MiR-92a was highly expressed in RAO-ECs and vascular endothelium, but not in RAO-SMCs or medial smooth muscle as assessed by real-time RT-PCR. Importantly, BrdU incorporation and wound healing assay provide evidence that functional inhibition of miR-92a resulted in an increased RAO-ECs proliferation and migration, but had no effect on RAO-SMCs proliferation or migration in vitro. Immunoblotting analysis revealed an increased phosphorylation of ERK1/2, JNK/SAPK as well as eNOS and phospho-eNOS increased expression level in RAO-ECs as a consequence of miR-92a inhibition. Using gain and loss of function experiments, we showed that miR-92a modulates regulation of KLF4 and MKK4 expression level in endothelial cells. Finally, in vivo administration of antagomiR-92a significantly enhanced re-endothelialization in injured carotid arteries and reduced neointimal formation after balloon injury or arterial stenting. These data provide the first evidence that inhibition of miR-92a may represent a novel strategy to improve endothelial regeneration and reduce restenosis after vascular injury.     

普伐他汀和辛伐他汀对载脂蛋白E缺陷小鼠主动脉粥样硬化形成及主动脉壁血管细胞粘附分子-1表达的影响

为观察普伐他汀和辛伐他汀对载脂蛋白E缺陷小鼠主动脉粥样斑块形成的影响及主动脉壁血管细胞粘附分子 1表达的影响 ,将载脂蛋白E缺陷小鼠分为普伐他汀组 (每天 10mg/kg)、辛伐他汀组 (每天 5mg/kg)和阳性对照组 (等量生理盐水 ) ,从主动脉血管根部连续切片 ,常规HE染色 ,计算机图像扫描 ,分析主动脉粥样硬化斑块的面积和斑块占管腔面积等 ;采用免疫组织化学及Western杂交方法测定主动脉壁血管细胞粘附分子 1表达。结果发现 ,除降胆固醇作用外 ,普伐他汀和辛伐他汀皆延缓斑块形成 ,与对照载脂蛋白E缺陷小鼠比 ,用药组小鼠的主动脉粥样斑块明显缩小 ;普伐他汀和辛伐他汀还明显抑制载脂蛋白E缺陷小鼠主动脉壁血管细胞粘附分子 1的表达 ;其中 2药对 14和 2 4周龄小鼠主动脉壁血管细胞粘附分子 1表达的抑制作用强于 34周龄小鼠。结果提示 ,普伐他汀和辛伐他汀可延缓或缩小载脂蛋白E缺陷小鼠主动脉粥样斑块的形成 ,抑制或下调主动脉壁血管细胞粘附分子 1表达 ,其效果与降胆固醇作用不成比例 ,可能是独立于调脂作用以外的抗动脉粥样硬化机制     

Deletion of the alternatively spliced fibronectin EIIIA domain in mice reduces atherosclerosis

The alternatively spliced and highly conserved EIIIA domain of fibronectin (FN) is included in most FN of the extracellular matrix in embryos. In adults, both extracellular matrix and plasma FN essentially lack EIIIA. In diverse inflammatory situations however, EIIIA is specifically included by regulated RNA splicing. In atherosclerotic lesions, FN, including the EIIIA domain (EIIIA-FN), is abundant, whereas FN in the flanking vessel wall lacks EIIIA. Lesional EIIIA-FN is localized with endothelial cells and macrophage foam cells. To directly test the function of EIIIA-FN, we generated EIIIA-null (EIIIA(-/-)) mice that lack the EIIIA exon and crossed them with apolipoprotein E (ApoE)-null (ApoE(-/-)) mice that develop arterial wall lesions. Compared with ApoE(-/-) controls, EIIIA(-/-)ApoE(-/-) mice had significantly smaller lesions throughout the aortic tree. EIIIA-FN was increased in ApoE(-/-) plasma, and total plasma cholesterol was reduced in EIIIA(-/-)ApoE(-/-) mice, specifically in large lipoprotein particles, suggesting a functional role for plasma EIIIA-FN. To assess a role for macrophage EIIIA-FN in the vessel wall, we conducted in vitro foam cell assays. EIIIA(-/-)ApoE(-/-) macrophages accumulated significantly less intracellular lipid than control ApoE(-/-) cells. These results provide genetic evidence that suggests roles for EIIIA-FN in plasma lipoprotein metabolism and in foam cell formation.     

Ischemia and reperfusion: the endothelial perspective. A radical view

Ischemia and reperfusion (IR) injury causes a variety of changes in tissue homeostasis that lead to necrosis and/or programmed cell death. Due to its strategic location at the luminal surface of vessels, the vascular endothelium is particularly sensitive to IR. In particular, endothelial biosynthetic activities (and their protective effects) appear to be impaired by the oxidative burst induced by a sudden increase in oxygen free radical species upon reperfusion. Importantly, this endothelial damage can be easily assessed in vivo in humans by measuring endothelium-dependent vasorelaxation. Paradoxically, recent studies have emphasized the central role of free radicals (including oxygen free radicals and nitric oxide) also in a protective process, denominated ischemic preconditioning, i.e. a condition whereby a given stimulus can increase the tolerance of a tissue to IR damage. We discuss the role of the endothelium in determining the mechanism of IR injury, and on the other side, the effect of IR injury on endothelial function. In particular, we focus on the role of reactive free radicals in endothelial IR injury and in the development of ischemic preconditioning.     

Targeted vascular delivery of antisense molecules using intravenous microbubbles.

OBJECTIVE: Perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles bind the antisense to the c-myc protooncogene (anti-c-myc) which prevents neointimal hyperplasia following vascular endothelial injury. The microbubbles also adhere to sites of damaged vascular endothelium and thus may be a method of systemically targeting delivery of anti-c-myc. METHODS: Laser scanning microscopy was performed on the aorta of 10 mice (five which were complement depleted) that received intravenous FITC-PESDA following aortic endothelial injury. C-myc expression was quantified following selective intracoronary injury in nine pigs that received intravenous (IV) anti-c-myc bound to PESDA. Finally, neointimal formation was measured following intracoronary stent deployment in 30 pigs that received either IV anti-c-myc alone or the same dose bound to PESDA. RESULTS: Fluorescent microscopy confirmed selective PESDA microbubble adherence to aortic endothelium in all mice with aortic injury. This binding was nearly abolished when serum complement was depleted prior to injury. C-myc expression at the site of coronary endothelial injury was significantly lower in pigs treated with systemic anti-c-myc bound to PESDA. There was a 33% reduction in % stenosis and a 28% reduction in intimal area at 45 days post-stent deployment in pigs that received IV antisense plus PESDA. The stent margins also had reduced neointimal formation. CONCLUSION: Systemic administration of anti-c-myc bound to PESDA microbubbles may be a good method for preventing coronary neointimal formation within and around implanted stents.