2019, Vol. 46
血管新生相关的疾病(如眼疾、癌症等)严重威胁着人类健康。年龄相关性黄斑变性(age-related macular degeneration, AMD)、增生性糖尿病视网膜病变(proliferative diabetic retinopathy, PDR)等眼部视网膜血管新生引起的视力下降甚至丧失, 是发达国家中青年劳动力致盲的主要原因[1]。目前临床上已经将血管内皮生长因子(vascular endothelial growth factor, VEGF)途径的靶向药物(如贝伐单抗、雷珠单抗、康柏西普等)用于抗血管新生治疗, 但是长期眼部抑制VEGF或会引起神经元毒性和一些眼部并发症[2-3], 为进一步了解血管新生的生理学和病理学机制, 并寻找更有效的治疗靶点, 本文对最近发现的糖酵解过程中生理性和病理性血管新生作用进行总结, 首先介绍糖酵解的重要调节剂6-磷酸果糖-2-激酶/果糖-2, 6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3, PFKFB3)在不同情况下的表达水平的改变及其调节机制, 然后介绍PFKFB3调节的糖酵解过程对内皮细胞功能的影响, 并由此讨论其对血管新生过程的影响。
PFKFB3在缺氧条件下的表达 缺氧是多种疾病共有的病理生理特点, 尤其是病理性血管新生性疾病[4-5]。在缺氧期间细胞代谢转变为主要靠糖酵解代谢来以满足其能量需求。这种代谢途径的转变是否在血管新生过程中具有某种作用, 尚不清楚。PFKFB3也是糖酵解通量的重要控制因素。PFKFB3基因位于染色体10p15-p14[6], 基因中至少含有19个外显子, 并且由于COOH-末端的可变区可以进行可变剪接, 所以目前在人中至少发现了6种分别具有不同组织选择性的结构同种型——UBI2K1~6[7]。该基因编码的蛋白质PFKFB3, 属于双功能酶家族(PFKFB1-4), 该家族蛋白在氨基末端含有激酶结构域6-磷酸果糖-2-激酶(6-phosphofructo-2-kinase, PFK-2)以及在羧基末端含有双磷酸酶结构域果糖-2, 6-二磷酸酶(fructose-2, 6-bisphosphatase 2, FBPase-2), 并通过PFK-2催化2, 6-二磷酸果糖的合成; 通过FBPase-2催化其分解, 当两者平衡调节时, 2, 6-二磷酸果糖(fructose 2, 6-diphosphate, F2, 6BP)在体内的浓度达到稳态[8]。在体内F2, 6BP不仅是糖酵解关键酶PFK-1的变构激活剂, 也是果糖1, 6-二磷酸酶(FBPase-1)的抑制剂[9-10](图 1)。由于PFKFB3缺乏像PFKFB1的Ser32磷酸化位点[11], 无法通过该位点的磷酸化下调激酶活性, 所以PFKFB3的激酶/磷酸酶活性的比例比其他家族成员高[12]。已知在PFKFB3基因的增强子区域中含有2个拷贝的缺氧诱导因子-1(HIF-1)结合基序(5’-ACGTG-3’)[13], 在缺氧条件下通过HIF-1α介导PFKFB3表达上调[14]。
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| There are two different domains at the amino terminus and the carboxy terminus, which is the structural feature of the bifunctional enzyme.The amino-terminal kinase domain PFK-2 is capable to catalyze the synthesis of F2, 6BP, while the carboxy-terminal diphosphatase domain FBPase-2 is responsible for catalyzing the decomposition of F2, 6BP, which is the functional feature of the bifunctional enzyme.F2, 6BP:Fructose 2, 6-diphosphate. 图 1 双功能酶的结构和功能 Fig 1 Structure and functions of the bifunctional enzyme |
PFKFB3的其他调节机制 研究发现, 肿瘤组织中的血管内皮细胞具有高糖酵解代谢, 并且当肿瘤组织的血管内皮细胞中PFKFB3等位基因中的一个拷贝失活, 或者在阻断PFKFB3的作用时, 可以通过使肿瘤组织的血管正常化来减少癌细胞的侵袭, 血管内渗和转移[15]。PFKFB3在人类多种肿瘤组织和细胞中的表达水平都较正常组织和细胞要高, 并且与HIF-1α水平有关[16-17]。PFKFB3分子中含有多个丝氨酸磷酸化位点, 包括s461、s467和s478等。当细胞处于缺氧、高渗透压等应激状态时, 可以通过激活p38/MK2、MAPK、ERK/RSK等激酶的作用, 对PFKFB3进行磷酸化, 从而增强PFKFB3激酶活性, 提高糖酵解通量以适应细胞增殖的能量需求[18-20]。在其mRNA的3’非编码区中, 存在多个拷贝的AUUUA序列, 使得mRNA不稳定, 可以被多种调节因素改变蛋白的表达水平[21]。miR-206和miR-26a、miR-26b、miR-449c等微小核苷酸还可以通过直接与该3’-UTR相互作用抑制PFKFB3的转录活性, 从而抑制肿瘤细胞的增殖和迁移[22-24]。转化生长因子1β(transforming growth factor beta 1, TGF-1β)、雌二醇和胰岛素等通过受体介导的信号通路促进PFKFB3的转录活性, 提高蛋白表达水平[19, 25-26](图 2)。
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| In the nucleus, estradiol regulates PFKFB3 gene transcription via ER, high osmotic pressure through the P38/MK2/SRF pathway and hypoxia through the MAPK/HIF-1 pathway.MiR-26a, miR-26b, miR-206, miR-449c affect the translation by direct interaction with PFKFB3 mRNA.Kinases MK2 and MAPK can also play a role in affecting the phosphorylation of PFKFB3 protein; APC/C-Cdk-1 exerts its function by directly degrading PFKFB3.When these factors affect the synthesis or function of PFKFB3 protein, it will have an effect on the glycolysis process. 图 2 PFKFB3的调节机制和功能 Fig 2 Function and mechanism of PFKFB3 |
PFKFB3的生物学功能 PFKFB3表达增高促进糖酵解通量增加, 乳酸增多, 高水平乳酸可刺激血管新生。PFKFB3还可定位到核内, 通过细胞周期蛋白依赖性激酶, 如细胞周期蛋白依赖激酶1(cyclin dependent kinase 1, Cdk-1), 促进细胞周期进程, 从而促进细胞增殖[27]。泛素蛋白酶体途径是目前已知的、所有真核生物体内具有的高度选择性的、重要的蛋白质降解途径, 泛素连接酶APC/C-Cdk-1可以通过促进PFKFB3泛素化降解, 降低细胞的糖酵解, 减少进入S期的细胞, 抑制细胞增殖[28]。此外, APC/C-Cdk-1通过降解PFKFB3抑制糖酵解途径, 促进磷酸戊糖途径, 细胞中还原型谷胱甘肽合成增多, 细胞抗氧化能力增强[28](图 2)。
内皮细胞的糖酵解与血管新生
糖酵解参与调节血管新生 最近有研究证明人脐静脉内皮细胞中约80%的ATP是通过糖酵解途径产生的, 即使在正常氧分压下, 血管内皮细胞也主要利用葡萄糖进行糖酵解产能[29-30]。虽然血管内皮细胞与循环系统中的氧气有密切的接触, 但是由于细胞内线粒体数量少、体积小, 并且为了能将更多的氧气供给远处的组织、细胞, 所以内皮细胞的代谢主要是糖酵解。在成年人中血管是相对静止的, 很少形成新的分支; 但是血管内皮细胞却保持很高的可塑性, 可以敏锐感知微环境中的血管生成信号, 如VEGF、血小板衍生生长因子(platelet derived growth factor, PDGR)等信号分子, 并对其做出反应。这种改变不仅仅包括分子方面的改变, 细胞对能量的需求也在短时间内发生了很大变化[31]。人脐静脉内皮细胞在缺氧时, 己糖激酶活性和膜葡萄糖转运蛋白1表达被提高, 促进了18F-氟脱氧葡萄糖(18F-FDG)摄取和乳酸的产生[32]。这表明内皮细胞的糖酵解代谢过程可能在血管新生中发挥重要作用。
PFKFB3通过影响发芽过程影响血管新生 近期有报道发现细胞糖酵解过程及其调节剂PFKFB3在血管新生过程尤其是发芽过程中具有重要作用[29]。原代人脐静脉内皮细胞的体外实验和小鼠的体内实验发现, PFKFB3在体外可以刺激内皮细胞增殖, PFKFB3基因缺陷小鼠体内的血管存在缺陷; 进一步研究发现, PFKFB3主要调节血管发芽过程, 即主要对Notch-DLL4信号通路介导的尖端细胞和茎细胞的行为进行调控。内皮细胞中的PFKFB3基因沉默时, 细胞形成较小且不规则的片状伪足, 丝状伪足更少、更短, 尖端细胞运动能力受限, 无法引导血管发芽。该研究者还发现, 发芽诱导信号VEGF, 成纤维细胞生长因子2(fibroblast growth factor 2, FGF2)等增加PFKFB3表达和糖酵解, 而发芽限制信号DLL4(激活Notch)引起相反的效应; 但是, PFKFB3蛋白表达改变时却并没有调节内皮细胞中这些分子的表达[29](图 3)。这可能是因为PFKFB3引起的糖酵解增加主要是为尖端细胞和茎细胞迁移时肌动蛋白的活动提供ATP[33]。因此, 内皮细胞的代谢调节可以作为独立于血管新生分子调节机制之外的另一个抗血管新生治疗的靶点[34]。
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| The VEGF pathway and the FGF2 pathway can promote the expression of PFKFB3 gene, while the DLL4 signal can inhibit the expression of PFKFB3 gene, thereby affecting the level of glycolysis in endothelial cells. 图 3 血管新生相关通路对PFKFB3的调节 Fig 3 Regulation of PFKFB3 by angiogenesis-related pathways |
PFKFB3抑制剂对生理性和病理性血管新生具有抑制作用 最近研究表明, 次优剂量的PFKFB3拮抗剂, 即小分子3-(3-吡啶基)-1-(4-吡啶基)-2-丙烯-1-酮(3PO)和VEGFR抑制剂SU5416单独作用于斑马鱼胚胎时, 对椎血管发育的损伤很小, 但是二者联合使用时对胚胎中椎血管的损伤加重, 高剂量SU5416联合3PO时可以废除几乎所有胚胎中椎血管的发育[35]。在激光诱导的脉络膜新生血管(choroidal neovascularization, CNV)的小鼠模型中, 3PO可以剂量依赖性地降低CNV损伤体积, 并且增加VEGFR2单克隆抗体DC101的抗血管生成活性, 当使用次优剂量的DC101时, CNV面积减少38%, 而DC101与3PO的组合导致CNV面积减少67%[35]。与CNV一样, 早产儿视网膜病变的氧诱导小鼠模型主要病变也是病理性血管新生。在出生后12天(post-natal 12 day, P12)至17天(post-natal 17 day, P17)的血管增殖期间用3PO处理幼崽, P17血管簇形成减少[35]。已知内源性雌激素17β-雌二醇(E2)也是促进血管生成的关键因子[36-37]。雌激素通过选择性G蛋白偶联雌激素受体介导人脐静脉内皮细胞的迁移, 该过程必需有PFKFB3参与, 这为雌激素受体诱导的病理性血管新生的治疗提供了更多的思路[38]。
结语 细胞代谢产生ATP以及其他中间产物是维持细胞生存和活动的重要过程。现已发现内皮糖酵解过程在病理性血管新生中有重要作用, 主要是通过调节尖端细胞伪足形成和迁移能力促进血管发芽。通过调节内皮异常糖酵解过程抑制异常血管新生, 可能是治疗眼部或者其他部位病理性血管新生的新方法。
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