Nitric oxide down-regulates MKP-3 mRNA levels: involvement in endothelial cell protection from apoptosis

MAP kinase-dependent phosphorylation processes have been shown to interfere with the degradation of the antiapoptotic protein Bcl-2. The cytosolic MAP kinase phosphatase MAP kinase phosphatase-3 (MKP-3) induces apoptosis of endothelial cells in response to tumor necrosis factor alpha (TNFalpha) via dephosphorylation of the MAP kinase ERK1/2, leading to Bcl-2 proteolysis. Here we report that the endothelial cell survival factor nitric oxide (NO) down-regulated MKP-3 by destabilization of MKP-3 mRNA. This effect of NO was paralleled by a decrease in MKP-3 protein levels. Moreover, ERK1/2 was found to be protected against TNFalpha-induced dephosphorylation by coincubation of endothelial cells with the NO donor. Subsequently, both the decrease in Bcl-2 protein levels and the mitochondrial release of cytochrome c in response to TNFalpha were largely prevented by exogenous NO. In cells overexpressing MKP-3, no differences in phosphatase activity in the presence or absence of NO were found, excluding potential posttranslational modifications of MKP-3 protein by NO. These data demonstrate that upstream of the S-nitrosylation of caspase-3, NO exerts additional antiapoptotic effects in endothelial cells, which rely on the down-regulation of MKP-3 mRNA.     

Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration

Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration. The protein kinase Akt activates the endothelial NO synthase (eNOS) by phosphorylation of Ser-1177. Therefore, we investigated the contribution of Akt-mediated eNOS phosphorylation to VEGF-induced EC migration. Inhibition of NO synthase or overexpression of a dominant negative Akt abrogated VEGF-induced cell migration. In contrast, overexpression of constitutively active Akt was sufficient to induce cell migration. Moreover, transfection of an Akt site phospho-mimetic eNOS (S1177D) potently stimulated EC migration, whereas a non-phosphorylatable mutant (S1177A) inhibited VEGF-induced EC migration. Our data indicate that eNOS activation via phosphorylation of Ser-1177 by Akt is necessary and sufficient for VEGF-mediated EC migration.     

Reactive oxygen species and vascular cell apoptosis in response to angiotensin II and pro-atherosclerotic factors

Reactive oxygen species (ROS) are known to induce apoptotic cell death in various cell types. In the vessel wall, ROS can be formed by macrophages within the atherosclerotic plaque or can act on the endothelium after adhesion of monocytes or leucocytes. Moreover, ROS are endogenously synthesized by endothelial and vascular smooth muscle cells by NAD(P)H oxidase. Enhanced ROS production is a very early hallmark in the atherogenic process, suggesting a link between ROS and apoptosis. In endothelial cells, the endogenous generation of ROS is induced by different pro-inflammatory and pro-atherosclerotic factors such as Ang II, oxLDL or TNFalpha, which all promote the execution of programmed cell death. ROS synthesis is thereby causally involved in apoptosis induction, because antioxidants prevent endothelial cell death. The pro-apoptotic effects of endogenous ROS in endothelial cells mechanistically seems to involve the disturbance of mitochondrial membrane permeability followed by cytochrome c release, which finally activates the executioner caspases. In contrast to the pro-apoptotic capacity of ROS in endothelial cells, in vascular smooth muscle cells emerging evidence suggests that endogenous ROS synthesis promotes cell proliferation and hypertrophy and does not affect cell survival. However, high concentrations of exogenous ROS can also stimulate smooth muscle cell apoptosis as shown for other cell types probably via activation of p53. Taken together, the double-edged effects of endogenously derived ROS in endothelial cells versus VSMC may provide a mechanistic clue to the anti-atherosclerotic effects of antioxidants shown in experimental studies, given that the promotion of endothelial survival in combination with inhibition of VSMC proliferation blocks two very important steps in the pathogenesis of atherosclerosis.     

尿毒症皮肤瘙痒患者采取腹膜透析与血液透析治疗的效果探究

目的：探究尿毒症皮肤瘙瘁患者采取腹膜透析与血液透析治疗的临床效果,并为该病的临床治疗提供经验积累。方法：选取我院肾内科于2005年1月-2012年12月收治的52例尿毒症皮肤瘙痒患者,利用随机数字表法进行分组,分别设为研究组和对照组。其中对照组实施血液透析治疗,而研究组开展腹膜透析治疗。记录两组在治疗前和治疗后第8周末血生化客观指标及皮肤瘙痒程度,并做好对比。结果：两组在治疗前的皮肤瘙痒评分、β2-MG、PTH、p3,BUN、SCr值差异无统计学意义（P〉0．05）;治疗后,研究组皮肤瘙瘁评分为（3．4±0．8）分,对照组为（5．8±0．9）分,差异有统计学意义（P〈0．05）。治疗后,研究组β2-MG、PTH及Ps．均低于对照组,差异有统计学意义（P〈0．05）。结论：尿毒症皮肤瘙痒患者的主要致敏因子为大中分子,采取腹膜透析能够有效清除大分子物质,进而达到瘙痒程度的有效改善。     

丝素蛋白/羟基磷灰石复合物对磷酸钙骨水泥性能的影响

目的：磷酸钙骨水泥（Calcium phosphate cement,CPC）以其诸多优点正得到了越来越多的应用,但其较差的力学性能表现也限制了它的使用范围。本研究目的在于改善磷酸钙骨水泥的力学性能,同时评估改性后的磷酸钙骨水泥的其他性能。方法：通过丝素蛋白（Silk fibroin,SF）的矿化自组装方法制备丝素蛋白／羟基磷灰石复合物（silk fibroin/hydroxyapitite composite, SF/HA）。按照1％、2％、3％、4％的质量分数加入磷酸钙骨水泥中,与磷酸钙骨水泥组对比。比较内容包括力学强度、抗渍散性能及细胞毒性。结果：以丝素蛋白溶液为液相组的磷酸钙骨水泥强度大约为35MPa。随后随着添加丝素蛋白／羟基磷灰石复合物的质量分数从1％增至3％,磷酸钙骨水泥的强度逐渐增加（P〈0．05）,最高约至45MPa。而当丝素蛋白／羟基磷灰石的质量分数达到4％时,磷酸钙骨水泥的强度较质量分数3％组小幅度下降至43MPa（P〈0．05）。以丝素蛋白溶液作为液相时,磷酸钙骨水泥的抗溃散能力也得到了加强。在MTT法测定细胞活力的对照实验中,无论是加入丝素蛋白溶液或丝素蛋白／羟基磷灰石复合物,都未观察到细胞毒性。结论：在磷酸钙骨水泥中加入3％质量分数的丝素蛋白／羟基磷灰石复合物,能显著提高磷酸钙骨水泥的抗压强度。而丝素蛋白溶液作为液相可改善磷酸钙骨水泥的抗溃散能力。同时,丝素蛋白和丝素蛋白／羟基磷灰石复合物都不表现出细胞毒性。更理想的力学强度和更强的抗溃散能力,大大扩展了磷酸钙骨水泥的应用范围。     

重组人MASP2蛋白在大肠杆菌中的表达和纯化

目的:获得酶原形式的重组人甘露聚糖结合凝集素相关丝氨酸蛋白酶2(MASP2)。方法:在大肠杆菌中诱导表达重组人MASP2全长蛋白,包涵体裂解后,经复性、透析、浓缩、考马斯亮蓝染色、SDS-PAGE及Western印迹,鉴定纯化结果及酶活性。结果:复性后的MASP2蛋白经考马斯亮蓝染色未见杂带。自激活实验表明,当MASP2浓度在1μmool/L以下时,无论在4℃还是37℃,都能较稳定地保持酶原形式;蛋白浓度为3.5μmool/L时只能在4℃保持稳定,37℃发生自激活;蛋白浓度达到12μmool/L后,在4℃时已不能稳定存在。结论:获得了较纯的重组人MASP2蛋白,且具有自激活活性。     

变性高效液相色谱技术对创伤弧菌检测的研究

应用PCR结合变性高效液相色谱技术对创伤弧菌进行检测,建立创伤弧菌快速准确的检测新方法。经过DHPLC分析条件优化,在DHPLC非变性温度下分析创伤弧菌特异性PCR扩增产物。同时进行方法特异性、灵敏度、重复性实验。实验结果表明所建立的创伤弧菌PCR-DHPLC检测方法特异性强、灵敏度高、重现性好、结果稳定可靠、检测时间短,检测低限可达到124 CFU/mL,是创伤弧菌快速检测的新技术。     

胸内正压对正常人左室功能影响及其力学原理

目的：探讨胸内正压对正常人左室射血及充盈的影响及其力学原理。方法：超声心动图观测30例正常人初始时与标准乏氏动作张力期10s时左室舒张末容积（LVEDV）、左室收缩末容积（LVESV）、每搏量（SV）、射血分值（EF）、流入道血流速度（E峰、A峰）、E／A值、二尖瓣环舒张早期运动速度（e）及舒张早期充盈压（E/e）的变化。结果：与初始时比较,标准乏氏动作张力期LVEDV、LVESV及SV减低而心率（陬）增快（P均〈0．001）,EF值增加,但无统计学意义（P〉0．05）;E峰与E／A值减低（P均〈O．05）;e没有变化（P〉0．05）．E／e值减低（P〈O．05）。结论：胸内正压对左室游离壁的力学作用促进了左室收缩运动而阻碍了左室舒张运动,会引起EF值增加,E峰及E／A值减低;2,胸内正压降低了肺静脉系统与心脏的跨壁压力,增加了血流阻力也是导致肺静脉系统与左室血液回流减少．E峰减低．E／e值减低的一个原因。     

Class IIb HDAC6 regulates endothelial cell migration and angiogenesis by deacetylation of cortactin

Histone deacetylases (HDACs) deacetylate histones and non-histone proteins, thereby affecting protein activity and gene expression. The regulation and function of the cytoplasmic class IIb HDAC6 in endothelial cells (ECs) is largely unexplored. Here, we demonstrate that HDAC6 is upregulated by hypoxia and is essential for angiogenesis. Silencing of HDAC6 in ECs decreases sprouting and migration in vitro and formation of functional vascular networks in matrigel plugs in vivo. HDAC6 regulates zebrafish vessel formation, and HDAC6-deficient mice showed a reduced formation of perfused vessels in matrigel plugs. Consistently, overexpression of wild-type HDAC6 increases sprouting from spheroids. HDAC6 function requires the catalytic activity but is independent of ubiquitin binding and deacetylation of α-tubulin. Instead, we found that HDAC6 interacts with and deacetylates the actin-remodelling protein cortactin in ECs, which is essential for zebrafish vessel formation and which mediates the angiogenic effect of HDAC6. In summary, we show that HDAC6 is necessary for angiogenesis in vivo and in vitro, involving the interaction and deacetylation of cortactin that regulates EC migration and sprouting.