血管生成素样蛋白3表达调控及生物学功能

血管生成素样蛋白3（angiopoietin-like protein3,ANGPTL3）是血管生成素和血管生成素样蛋白家族成员,主要表达于肝脏。其具有调节脂质代谢和促进血管生成的作用,其中尤以调节脂质代谢的作用显著。探讨ANGPTL3表达调控及功能的分子机制,对于了解代谢性疾病发病机制及寻找新的治疗途径有积极意义。     

血管生成素样蛋白3与疾病

血管生成素样蛋白3（angiopoietin-like protein3,ANGPTL3）是一种分泌蛋白,因其具有血管生成素家族的特征性结构而得名。该蛋白质主要在人和鼠的肝脏表达,可能与脂代谢紊乱、冠心病与动脉粥样硬化、糖尿病、代谢综合征、肾病以及肝癌等疾病有关。因此深入研究该蛋白质与疾病的关系,可望为诊断、治疗、预防相关疾病提供新的途径。     

血管生成素样蛋白家族表达调控及其在动物分子育种领域的现状分析

血管生成素样蛋白(Angiopoietin-like proteins,Angptls)是与脂类、葡萄糖、能量代谢和血管生成密切相关的蛋白质家族,现已发现8个成员,因其在代谢调控和动物辅助选育方面发挥有重要作用而备受研究人员的关注。以下综述了该蛋白家族的结构、介导的信号通路、上游调控基因及代谢网络等方面,并结合课题组研究工作对其应用于动物育种领域的现状和问题进行了分析,对前景作了展望。     

下调血管生成素样蛋白7表达对血管紧张素Ⅱ介导的血管平滑肌细胞炎症反应的影响

目的探讨RNA干扰血管生成素样蛋白7 （Angptl7）基因对血管紧张素Ⅱ（AngⅡ）诱导的血管平滑肌细胞（VSMC）炎症因子的影响及其作用机制。 方法体外培养人VSMC,分为常规F12K培养基培养（对照）和1 μg/mL AngII培养24 h。VSMC用AngⅡ（1 μg/mL）处理24 h后,采用siRNA-Angptl7和阴性对照siRNA-NC在Lipofectamine 2000介导下转染VSMC。RT-qPCR检测mRNA表达水平;Griess反应测定一氧化氮（NO）含量;蛋白免疫印记法检测相关蛋白的改变;酶联免疫吸附法检测VSMC中炎症因子肿瘤坏死因子α（TNF-α）、白细胞介素-1β （IL-1β）和IL-6水平。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验,两组间比较采用独立样本t检验。 结果与对照比较,1 μg/mL AngⅡ处理可促进VSMC中Angptl7 mRNA （0.97±0.06比3.05±0.21）和蛋白表达（1.01±0.12比1.61±0.14）,亦可促进VSMC中IL-1β[（45.21±8.10）比（126.17±11.77） pg/mL]、IL-6[（50.50±7.51）比（108.50±9.51）pg/mL]和TNF-α的表达[（60.77±9.58）比（185.67±17.35）pg/mL],差异有统计学意义（P均< 0.01）。与对照和转染siRNA-NC相比,转染siRNA-Angptl7下调Angptl7蛋白表达（0.99±0.12,0.98±0.12比0.44±0.14,P < 0.01）。与AngⅡ干预组相比,siRNA-Angptl7降低AngⅡ介导的VSMC炎症反应相关蛋白TNF-α、IL-6和IL-1β的表达,核因子κB （NF-κB）/诱导型一氧化氮合酶（iNOS）/环氧化酶2 （COX-2）信号通路相关蛋白NF-κB、iNOS和COX-2表达及NO含量亦降低,差异有统计学意义（P均< 0.01）。与siRNA-NC相比,siRNA-Angptl7组AngⅡ诱导的VSMC炎症反应相关蛋白TNF-α （0.99±0.13比0.51±0.12）、IL-6 （1.00±0.12比0.38±0.05）和IL-1β的表达（0.99±0.14比0.48±0.11）,NF-κB （1.00±0.10比0.42±0.08）、iNOS （1.02±0.12比0.42±0.10）和COX-2表达（1.00±0.11比0.52±0.12）均降低,NO含量[（54.78±2.76）比（18.08±3.61）μmol/L]亦降低,差异有统计学意义（P均< 0.01）。 结论AngⅡ可通过Angptl7促进VSMC炎症反应,下调Angptl7蛋白表达可以抑制VSMC的炎症反应,其作用机制可能与抑制NF-κB/iNOS-COX-2信号通路有关。     

血管生成系及其相关蛋白的研究进展

对血管生成素及其相关蛋白的研究进展和分子生物学特性进行了综述,迄今,已经发现了4种血管生成素及某些血管生成素的同工蛋白或变异体。这些蛋白质具有共同的结构,即氨基端与分泌相关的信号肽,介导同源寡聚体形成的螺旋样结构域,和羧基末端介导配体活性的纤维蛋白原样结构域。血管生成素均可与Tie-2受体结合,但只有血管生成素1和4可激活Tie-2受体。血管生成素1和4为效应剂而血管生成素2和3为拮抗剂。对于血管生成素是否可与Tie-1结合尚不清楚。血管生成素相关蛋白具有公认的血管生成素的特征性结构,对其功能及其作用途径还不十分了解,有待于进一步研究。Tie-1和Tie-2的其他配体以及血管生成素相关蛋白类似物的特异受体有可能在今后得到阐明。     

基于血管生成素的药物研发

血管生成素（angiogenin,ANG）在伤口愈合、月经周期、妊娠、胚胎发育、先天性免疫、细胞应激保护和维持机体稳态等生理病理过程,特别是肿瘤的生存与进展、神经细胞的存活和生长中扮演着重要角色,是药物研发的重要靶点.本文综述了ANG在功能上的特殊性及其药物研发潜力.在肿瘤中,ANG扮演促进肿瘤细胞增生和促进血管生成的双重角色,且是其它血管生成因子如血管内皮生长因子（vascular endothelial growth factor, VEGF）、酸性成纤维生长因子（acidic fibroblast growth factor, aFGF）、碱性成纤维生长因子（basic fibroblast growth factor, bFGF）和表皮生长因子（epidermal growth factor, EGF）发挥作用的必需准许因子. ANG的抗肿瘤治疗较之目前常用的针对单一血管生成因子的抑制剂更有效,具有良好的药物研发和临床运用前景.由于ANG通过核转位促进rRNA转录是发挥促进肿瘤细胞增生和血管生成活性所必须的,因此,它的核转位抑制剂如新霉胺,将有望首先获得抗肿瘤临床应用.另外,业已证明重组ANG能促进体内外运动神经元的存活,且可明显改善肌萎缩侧索硬化症（amyotrophic lateral sclerosi, ALS）模型小鼠的行为,其在神经退行性疾病治疗方面也将有良好的研发前景.     

血管生成素与炎症性疾病

血管生成素（angiogenin,ANG）是首个被发现来源于肿瘤的具有血管生成能力的蛋白质,但其在炎症中的作用机制尚未完全阐明. 研究表明, ANG在炎症性疾病的发生发展中起重要作用,并与炎症的调控密切相关,而慢性炎症正是导致肿瘤形成、生长和转移的因素之一. 本文以ANG与炎症的关联为基础,结合我们的工作阐述ANG在炎症性疾病特别是肿瘤中的作用和调控机制,明确ANG与蛋白质的相互作用、对信号通路的调控及核内作用是其发挥功能的重要机制,也可能是调控肿瘤炎症的重要机制. 因而,深入研究ANG与炎症的关系不仅可加深我们对ANG兼具抗炎、抗新生血管双重功能的认识,更可为炎症性疾病的治疗提供潜在的作用靶点和新的思路和方法.     

人血管生成素研究进展

人血管生成素对正常机体的组织发育和再生具有较重要的生理功能,同时还可能参与肿瘤组织的新生血管生成,在肿瘤转移中发挥病理作用。本文对其分类及生理学作用,基因结构,及在微生物中的过量表达和医学领域的应用作一综述。     

血管生成素样蛋白2在糖尿病肾病小鼠肾脏中的表达研究

利用半定量RT-PCR和免疫组化的方法同时从mRNA水平和蛋白质水平对血管生成素样蛋白2在不同病理阶段的2型糖尿病肾病模型小鼠--db/db小鼠肾脏中的表达情况进行了较为系统的分析.结果发现:a.在糖尿病前的db/db小鼠(4周龄的db/db小鼠),血管生成素样蛋白2与作为正常对照的db/m小鼠相比,差异不是很大,随着肥胖的加剧,高血糖、蛋白尿的出现,血管生成素样蛋白2在db/db小鼠肾脏中的表达无论从mRNA水平还是从蛋白质水平均显著升高.b.从免疫组化的分析结果来看,血管生成素样蛋白2主要分布于小鼠肾脏的肾小球部分,主要是沿毛细血管袢呈线性分布,其位置与足细胞的位置重叠,足细胞是小鼠肾脏中血管生成素样蛋白2的主要分泌细胞.c.小鼠肾脏血管生成素样蛋白2的表达水平似乎还与鼠龄相关:虽然变化幅度不是很大,但在周龄较大的小鼠(如20周龄以上),其表达水平相对较高.上述工作不仅印证了先前对2型糖尿病肾病患者肾小球基因表达谱的分析结果,更加明确了血管生成素样蛋白2与糖尿病肾病的相关性,同时揭示了血管生成素样蛋白2在正常小鼠和糖尿病肾病小鼠肾脏中的表达、分布和变化规律,有利于进一步揭示血管生成素样蛋白2的功能及其在糖尿病肾病发生、发展过程中的可能作用,探讨糖尿病肾病的分子机制.     

血管生成素相互作用蛋白的发现及其在哺乳动物细胞中的验证

通过RT-PCR从人外周血白细胞中钓取血管生成素(angiogenin,Ang)cDNA,构建诱饵蛋白载体pAS-2-1-Ang,对其自身转录激活活性进行鉴定后,通过酵母双杂交系统筛选人肝细胞cDNA文库,获得两个双阳性克隆.序列分析和同源检索表明所获候选蛋白分别为人上皮细胞素和λ晶体蛋白.构建Ang及候选蛋白标签融合表达载体并共转染COS-7细胞,利用免疫共沉淀和蛋白质印迹方法在哺乳动物细胞中验证了Ang与候选蛋白间的相互作用.为阐明Ａng促血管新生的分子机制创造了条件.     

Leptin and insulin down-regulate angiopoietin-like protein 3, a plasma triglyceride-increasing factor

We reported previously that angiopoietin-like protein3 (ANGPTL3), a liver-specific secretory factor, increased plasma triglyceride (TG) via inhibition of lipoprotein lipase and free fatty acid (FFA) by activating adipose-lipolysis. The current study examined the regulation of Angptl3 by leptin and insulin, both of which are key players in the metabolic syndrome. Angptl3 expression and plasma ANGPTL3 levels were increased in leptin-resistant C57BL/6J(db/db) and -deficient C57BL/6J(ob/ob) mice, relative to the control. Leptin supplements decreased Angptl3 gene expression and plasma ANGPTL3 in C57BL/6J(ob/ob) mice. The changes of Angptl3 were associated with alterations of plasma TG and FFA levels. Leptin treatment directly suppressed Angptl3 gene expression in hepatocytes. Angptl3 gene expression and plasma protein levels were also increased in insulin-deficient streptozotocin-treated mice. Insulin treatment of hepatocytes decreased Angptl3 gene expression and protein secretion. Our results suggest that elevated ANGPTL3 by leptin- or insulin-resistance is attributed to increased plasma TG and FFA concentrations in obesity.     

The role of angiopoietin-like protein 2 in pathogenesis of dermatomyositis

Dermatomyositis (DM) is an autoimmune disease marked by chronic inflammation of skin and muscle tissues and characterized clinically by proximal muscle weakness and skin eruption, including heliotrope rash, and Gottron's sign. Treatment with a non-specific immunosuppressive agent or anti-inflammatory corticosteroids is beneficial, although some patients are resistant to these therapies. Proinflammatory cytokines derived from infiltrating inflammatory cells and activated resident cells within skin and muscle tissues likely promote chronic inflammation in DM pathogenesis; however, molecular mechanisms underlying the disease are not completely defined. Here we show that mRNA and protein levels of angiopoietin-like protein 2 (Angptl2), a recently identified chronic inflammation mediator, are abundant in keratinocytes from DM patients' skin eruptions. To examine whether skin cell-derived Angptl2 promotes DM manifestations, we analyzed transgenic (Tg) mice expressing Angptl2 driven by the keratinocyte specific promoter K14 (K14-Angptl2) and therefore constitutively expressing Angptl2 in skin tissue. We found that K14-Angptl2 Tg mice exhibited skin phenotypes similar to those observed in DM patients. In addition, treatment of keratinocytes with exogenous Angptl2 activated the NF-κB inflammatory cascade, resulting in increased expression of the proinflammatory cytokines IL-1β and IL-6. We propose that keratinocyte-derived Angptl2 functions in DM pathogenesis by inducing chronic inflammation in skin tissue.     

Oligomerization state-dependent hyperlipidemic effect of angiopoietin-like protein 4

Angiopoietin-like protein 4 (Angptl4) is the second member of the angiopoietin-like family of proteins previously shown to increase plasma triglyceride (TG) levels in vivo. We recently reported that Angptl4 is a variable-sized oligomer formed by intermolecular disulfide bonds and undergoes regulated proteolytic processing upon secretion. We now show that adenoviral overexpression of Angptl4 potently increases plasma TG levels by a mechanism independent of food intake or hepatic VLDL secretion. We determined that cysteine residues at positions 76 and 80 of Angptl4, conserved among mouse, rat, and human, are required to form higher order structures. By generating adenoviral expression vectors of Angptl4 containing different epitope tags at both N and C termini, we show that loss of oligomerization results in decreased stability of the N-terminal coiled-coil domain of Angptl4 as well as decreased ability to increase plasma TG levels, suggesting that intermolecular disulfide bond formation plays important roles in determining the magnitude of the hyperlipidemic effect of Angptl4. Because Angptl4 is more potent than Angptl3 in increasing plasma TG levels in mice, inappropriate oligomerization of Angptl4 could be associated with disorders of lipid metabolism in vivo.     

GPIHBP1 stabilizes lipoprotein lipase and prevents its inhibition by angiopoietin-like 3 and angiopoietin-like 4

Glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) binds both LPL and chylomicrons, suggesting that GPIHBP1 is a platform for LPL-dependent processing of triglyceride (TG)-rich lipoproteins. Here, we investigated whether GPIHBP1 affects LPL activity in the absence and presence of LPL inhibitors angiopoietin-like (ANGPTL)3 and ANGPTL4. Like heparin, GPIHBP1 stabilized but did not activate LPL. ANGPTL4 potently inhibited nonstabilized LPL as well as heparin-stabilized LPL but not GPIHBP1-stabilized LPL. Like ANGPTL4, ANGPTL3 inhibited nonstabilized LPL but not GPIHBP1-stabilized LPL. ANGPTL3 also inhibited heparin-stabilized LPL but with less potency than nonstabilized LPL. Consistent with these in vitro findings, fasting serum TGs of Angptl4^−/−/Gpihbp1^−/− mice were lower than those of Gpihbp1^−/− mice and approached those of wild-type littermates. In contrast, serum TGs of Angptl3^−/−/Gpihbp1^−/− mice were only slightly lower than those of Gpihbp1^−/− mice. Treating Gpihbp1^−/− mice with ANGPTL4- or ANGPTL3-neutralizing antibodies recapitulated the double knockout phenotypes. These data suggest that GPIHBP1 functions as an LPL stabilizer. Moreover, therapeutic agents that prevent LPL inhibition by ANGPTL4 or, to a lesser extent, ANGPTL3, may benefit individuals with hyperlipidemia caused by gene mutations associated with decreased LPL stability.