Molecular Mechanisms of Arterial-Venous Differentiation

动静脉分化的分子机制

• 批准号: 8449174
• 负责人: Saulius Sumanas
• 金额: $ 36.41万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8449174
• 关键词: Adult; Affect; Angioblast; Arteries; Biological Models; Blood Circulation; Blood Vessels; CD44 Antigens; Cells; Data; Defect; Development; Diabetic Retinopathy; Disease; Embryo; Endothelial Cells; Exhibits; Genes; Hemorrhage; Hyaluronan; Hyaluronic Acid; Image; Knowledge; Lead; Location; Maps; Modeling; Molecular; Notch Signaling Pathway; Pathway interactions; Pattern; Phosphorylation; Play; Process; Role; Signal Transduction; Signaling Molecule; Staging; Stem cells; Stroke; System; Testing; Time; Transgenic Organisms; Translating; Vascular Diseases; Vascular Endothelial Growth Factors; Veins; Venous; Venous Malformation; Vertebrates; Wound Healing; Zebrafish; angiogenesis; base; cell motility; experience; hyaluronan synthase 1; loss of function; malformation; migration; morphogens; notch protein; novel; overexpression; prevent; progenitor; public health relevance; receptor; tissue repair; transcription factor; tumor; tumorigenesis; vasculogenesis

DESCRIPTION (provided by applicant): Arterial and venous (A-V) patterning is critical for the establishment of functional embryonic and adult vasculature. Proper A-V differentiation is critical for the formation of functional vessels during tissue repair processes such as wound healing and is associated with a number of pathological conditions including A-V malformations and tumor induced angiogenesis. Therefore understanding the mechanisms of A-V differentiation will lead to new treatments in tumorigenesis, wound repair and multiple vascular disorders. While it is difficult to study A-V patterning in mammalian systems, transparent zebrafish embryos are easily accessible for observation and experimental manipulations. During formation of the major vessels, vascular endothelial progenitor cells (EPCs) assume arterial or venous identity prior to the initiation of circulation. However, it is not understood how EPCs choose among the arterial or venous fates if they are all exposed to the same signaling molecules such as Vegf and Hh. We have found that the expression timing of an evolutionarily conserved master regulator of vasculogenesis Etsrp / Etv2 is one of the critical factors in A-V differentiation. Furthermore, arterial and venous progenitors may originate at different spatial locations, the inner and the outer lines and thus experience different Vegf concentrations. We hypothesize that the arterial- venous fate of EPCs is determined by the combination of etsrp-dependent timing of Vegf receptor flk1 and hyaluronan (HA) receptor stabilin 2 (stab2) expresion and their spatial location within Vegf gradient. To demonstrate that the timing of etsrp expression affects A-V differentiation, photoactivatable morpholinos will be used to inhibit etsrp function at different developmental stages in zebrafish embryos. Fate mapping and time- lapse imaging will be performed to characterize the cell movements and to determine the arterial-venous fates of the inner and outer lines of EPCs. To determine if Vegf and Hh gradients play a role in the activation of Notch signaling and arterial differentiation within the inner line angioblasts, overexpression and loss of function approaches will be used to inhibit Vegf, Hh and Notch signaling combined with lineage tracing to determine the A-V cell fates. To determine if Etsrp downstream target Stab2 functions as a receptor for HA and induces activation of Notch signaling during A-V differentiation, A-V defects in Stab2 and HA synthase Has2 knockdown embryos will be investigated. It will be analyzed if HA-Stab2 signaling leads to Tyr-phosphorylation of Stab2 and ERK phosphorylation, resulting in Notch pathway activation and arterial marker expression. Upon completion of this study, we will have identified the molecular mechanism how the timing of etsrp expression and Vegf gradient lead to the differential expression of arterial and venous genes. The acquired knowledge will be critical in our understanding of molecular mechanisms of A-V differentiation and will have an impact on developing treatments for multiple diseases and pathological conditions related to vasculature formation which include A-V malformations, diabetic retinopathy, wound healing and tumorigenesis.!

描述（由申请人提供）：动脉和静脉（A-V）模式对于建立功能性胚胎和成人脉管系统至关重要。正确的 A-V 分化对于伤口愈合等组织修复过程中功能性血管的形成至关重要，并且与许多病理状况相关，包括 A-V 畸形和肿瘤诱导的血管生成。因此，了解 A-V 分化机制将带来肿瘤发生、伤口修复和多血管疾病的新治疗方法。 虽然研究哺乳动物系统中的 A-V 模式很困难，但透明的斑马鱼胚胎很容易进行观察和实验操作。在大血管形成过程中，血管内皮祖细胞（EPC）在循环开始之前呈现出动脉或静脉特征。然而，如果 EPC 都暴露于相同的信号分子（例如 Vegf 和 Hh），则尚不清楚它们如何在动脉或静脉命运中进行选择。我们发现，进化上保守的血管生成主调节因子 Etsrp / Etv2 的表达时间是 A-V 分化的关键因素之一。此外，动脉和静脉祖细胞可能起源于不同的空间位置、内线和外线，因此经历不同的 Vegf 浓度。我们假设 EPC 的动静脉命运是由 Vegf 受体 flk1 和透明质酸 (HA) 受体稳定蛋白 2 (stab2) 表达的 etsrp 依赖性时序及其在 Vegf 梯度内的空间位置的组合决定的。为了证明 etsrp 表达的时间影响 A-V 分化，将使用光激活吗啉来抑制斑马鱼胚胎不同发育阶段的 etsrp 功能。将进行命运图谱和延时成像来表征细胞运动并确定 EPC 内线和外线的动静脉命运。为了确定 Vegf 和 Hh 梯度是否在内线成血管细胞内的 Notch 信号传导和动脉分化中发挥作用，将使用过度表达和功能丧失方法来抑制 Vegf、Hh 和 Notch 信号传导，并结合谱系追踪来确定 A-V 细胞的命运。为了确定 Etsrp 下游目标 Stab2 是否作为 HA 受体发挥作用并在 A-V 分化过程中诱导 Notch 信号激活，将研究 Stab2 和 HA 合酶 Has2 敲低胚胎中的 A-V 缺陷。将分析 HA-Stab2 信号传导是否导致 Stab2 的 Tyr 磷酸化和 ERK 磷酸化，从而导致 Notch 通路激活和动脉标志物表达。 完成这项研究后，我们将确定 etsrp 表达时间和 Vegf 梯度如何导致动脉和静脉基因差异表达的分子机制。所获得的知识对于我们理解 A-V 分化的分子机制至关重要，并将对开发与脉管系统形成相关的多种疾病和病理状况的治疗方法产生影响，其中包括 A-V 畸形、糖尿病视网膜病变、伤口愈合和肿瘤发生。