VEGF Regulation of Hepatic Erythropoietin Synthesis

VEGF 对肝促红细胞生成素合成的调节

• 批准号: 6873237
• 负责人: CALVIN J KUO
• 金额: $ 38.51万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6873237
• 关键词: DNA footprinting; RNA; cell cell interaction; chromatin immunoprecipitation; erythropoietin; gel mobility shift assay; gene deletion mutation; gene expression; genetic enhancer element; genetic promoter element; genetic transcription; genetically modified animals; hypoxia; intermolecular interaction; laboratory mouse; liver cells; liver metabolism; mixed tissue /cell culture; organ culture; protein structure function; transcription factor; vascular endothelial growth factors; vascular endothelium

DESCRIPTION (provided by applicant): Erythropolesis, the production of red blood cells (RBCs), is regulated by an intricate network of signals involving the hormone erythropoietin (Epo), and sensing of hypoxia through inhibition of both HIF-1alpha proline hydroxylation and its subsequent interaction with the Von Hippel-Lindau (VHL) protein. Either insufficient or excess erythropoiesis produces disease in humans, with too few RBCs resulting in anemia, and too many RBCs resulting in polycythemia vera. The treatment of anemia alone consumes significant economic resources, with the necessity for either blood transfusions or Epo treatment. We have developed adenoviruses which express soluble ectodomains of the Flkl and Fltl receptors for Vascular Endothelial Growth Factor (VEGF). Single injections in mice of these adenoviruses produce high levels of circulating soluble Flk1 and Flt1 ectodomains which persist for >3-4 weeks, potently suppressing tumor growth and angiogenesis, and producing stringent conditional inactivation of-VEGF in adult animals. Surprisingly, adenoviral expression of Flk1 or Flt1 ectodomains (soluble VEGF receptors, or sVEGFRs) in normal mice strongly stimulates erythropoiesis, with increase in hematocrit increasing from baseline 45% to new levels of 55-75 %. Circulating erythropoietin levels are elevated in parallel; however, the Epo originates from the liver as in embryonic development, not the usual site of synthesis in the kidney. This data implicates VEGF as an unsuspected repressor of hepatic Epo synthesis, and this grant application is focused on determining the underlying moIecular mechanisms. In Aim 1, experiments will expand upon preliminary data implicating perturbation of hepatocyte--endothelial cross-talk in the sVEGFR induction of hepatic Epo. Adenoviral and transgenic expression of Cre recombinase will be used to test the effects of specific inhibition of hepatocyte-produced VEGF on hepatocyte Epo expression. Conversely, co-culture experiments manipulating VEGF will be used to test the effects of endothelial cells on hepatocyte Epo production. Aim 2 will define the specific factors mediating the transcriptional regulation of Epo by VEGF and VEGF blockade. Candidate factors (HIF proteins, HNF4, RXRalpha, GATA factors) will be directly tested by chromatin immunoprecipitation assays and functionally by adenoviral and transgenic expression of Cre to delete floxed alleles of relevant candidates. Additionally, novel factors will be sought by unbiased approaches of DNAse1 footprinting and hypersensitivity assays. The therapeutic implications of the ability to reactivate hepatic Epo synthesis are substantial. These studies should also yield significant insight into the action of VEGF as an unsuspected upstream regulator of RBC homeostasis, into the regulation of hepatic Epo production which is strongly repressed post-natally, and indicate the potential utility of alterations in erythropoiesis as surrogate markers for or a desirable consequence of stringent VEGF blockade.

描述（由申请人提供）：红细胞的产生（RBC）受涉及激素促红细胞生成素（EPO）的信号网络的调节，以及通过抑制HIF-1Alpha脯氨酸羟基化和随后与Von hippell-lindauu的相互作用（Vhlindaue）（EPO）（EPO）的感应（EPO）（EPO）（EPO）。不足或过量的红细胞生成会在人类中产生疾病，RBC太少会导致贫血，而RBC太多导致了多余的Vera。仅贫血的治疗就消耗了大量的经济资源，因此需要输血或EPO治疗。我们开发了腺病毒，这些腺病毒表达FLKL和FLTL受体的可溶性外生域，用于血管内皮生长因子（VEGF）。这些腺病毒的小鼠的单次注射会产生高水平的可溶性FLK1和FLT1胞外域，持续持续> 3-4周，从而有效抑制了肿瘤的生长和血管生成，并在成年动物中产生严格的条件灭活-VEGF。令人惊讶的是，正常小鼠中FLK1或FLT1胞菌（可溶性VEGF受体或SVEGFRS）的腺病毒表达强烈刺激红细胞生成，而血压计的增加增加了基线的增加，从基线45％增加到55-75％的新水平。循环红细胞生成素水平平行升高；但是，EPO源自肝脏中的肝脏发育，而不是肾脏合成的通常位点。该数据暗示VEGF是肝EPO合成的未经使用的阻遏物，该赠款的应用集中在确定基本的摩尔机制上。在AIM 1中，实验将扩展基于初步数据，暗示肝细胞 - 内皮跨词的扰动肝EPO诱导。 CRE重组酶的腺病毒和转基因表达将用于测试肝细胞生产的VEGF对肝细胞EPO表达的特异性抑制作用。相反，操纵VEGF的共培养实验将用于测试内皮细胞对肝细胞EPO产生的影响。 AIM 2将定义介导VEGF和VEGF封锁对EPO转录调控的特定因素。候选因子（HIF蛋白，HNF4，RXRALPHA，GATA因子）将通过染色质免疫沉淀测定直接测试，并在功能上通过腺病毒和CRE的转基因表达来删除相关候选者的floxed flox erox earles。另外，新的因素将通过DNASE1足迹和超敏反应测定的无偏方法来寻求新因素。重新激活肝EPO合成能力的治疗意义是重要的。这些研究还应对VEGF作为RBC稳态的一种未使用的上游调节剂的作用进行重大洞察力，并在肝EPO生产的调节中受到了强烈的压制，并表明了红细胞动物改变的潜在效用，作为对或希望强烈的vegf blokeent blokent blokeent blokent blokeent blokeent blognent vegf blockade blockade blockade blockade blockade blockade的替代标志。