Investigation of the role of Eph/Ephrin signaling in vasculogenesis and angiogenesis

尽管调控血管生成的细胞和分子机制才刚刚开始被了解，但通过内皮限制性受体，特别是受体酪氨酸激酶的信号传递已经被证明在这些事件中发挥了关键作用。最近的报道表明，EphB受体酪氨酸激酶及其跨膜型ePhin-B2配体在胚胎血管系统中起着重要的作用。Eph受体酪氨酸激酶及其与细胞表面结合的配体，介导细胞迁移和黏附。它们有助于发育中的血管组装和身体模式，包括指导轴突引导和在发育中的大脑中建立。这表明双向EphB/ePhin-B2信号的细胞间排斥效应可能对血管发育至关重要，类似于所描述的神经元发育的机制。为了验证这一假说，我们通过产生转基因(CAGP-Ephin…)来扰乱EphB/Ephin-B2在体内的精确表达模式More-B2Tg)小鼠，在无处不在的结构性启动子CAg的控制下表达ePhin-B2。这些小鼠的躯体间血管节段性排列异常，而在仅在血管内皮细胞(ECs)过表达的Tie-2p-eaffin-B2TG小鼠中没有观察到这种异常。这一发现表明，不表达Ephin-B2的内皮细胞改变了表达EphB受体的ECs向通常不表达Ephin-B2的躯体间区的迁移。CAGP-EBARIN-B2TG小鼠在新生阶段因升主动脉血管平滑肌细胞缺陷募集而猝死于主动脉夹层动脉瘤。接下来，我们研究了间充质细胞上的ewitinB2在血管发育中的作用。为此，我们分析了腹主动脉旁内脏胸膜中胚层(P-Sp)外植体的血管床和网络的形成，其中P-Sp外植体富含来自小鼠胚胎(E9.5)的内皮前体细胞，使用共培养系统与OP9基质细胞共培养，其中OP9基质细胞转染载体或ePhin B2。有趣的是，OP9/ewitinB2间质细胞促进ewitinB2阳性内皮细胞和平滑肌肌动蛋白阳性周细胞的增殖和出芽/重塑。这些发现表明，内皮细胞和周围间充质细胞之间的EphB/eaffin-B2信号通路在血管生成、血管生成和血管成熟过程中起着至关重要的作用。由于血管投射在许多病理条件下都是初始事件，它可能为(抗)血管生成治疗提供新的策略。较少

Although the cellular and molecular mechanisms governing angiogenesis are only beginning to be understood, signaling through endothelial-restricted receptors, particularly receptor tyrosine kinases, have been shown to play a pivotal role in these events. Recent reports show that EphB receptor tyrosine kinases and their transmembrane-type ephrin-B2 ligands play essential roles in the embryonic vasculature. Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins mediate cell migration and adhesion. They contribute to developmental vascular assembly and body patterning, including directing axon guidance and building in the developing brain. These suggest that cell-to-cell repellent effects due to bi directional EphB/ephrin-B2 signaling may be crucial for vascular development similar to the mechanism described for neuronal development. To test this hypothesis, we disrupted the precise expression pattern of EphB/ephrin-B2 in vivo by generating transgenic (CAGp-ephrin … More -B2 Tg) mice that express ephrin-B2 under the control of a ubiquitous and constitutive promoter, CAG. These mice displayed an abnormal segmental arrangement of intersomitic vessels, while such anomalies were not observed in Tie-2p-ephrin-B2 Tg mice in which ephrin-B2 was overexpressed in only vascular endothelial cells (ECs). This finding suggests that non ECs expressing ephrin-B2 alter the migration of ECs expressing EphB receptors into the intersomitic region where ephrin-B2 expression is normally absent. CAGp-ephrin-B2 Tg mice show sudden death at neonatal stages from aortic dissecting aneurysms due to defective recruitment of vascular smooth muscle cells to the ascending aorta. Next, we investigate the role of ephrinB2 on mesenchymal cells in vascular development. To do so, we analyzed the vascular bed and network formation by para-aortic splanchnopleural mesoderm (P-Sp) explants which are rich of endothelial precursor cells from mouse embryos (E9.5) using a co-culture system with OP9 stromal cells transfected with vector or ephrin B2. Interestingly, OP9/ephrinB2 stromal cells promote both proliferation and sprouting/remodeling of ephrinB2-positive endothelial cells and smooth muscle actin-positive pericytes. These findings demonstrate that EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells plays an essential role in vasculogenesis, angiogenesis and vessel maturation. As vessel projection is initial event in many pathological condition, it may provide new strategies in (anti)-angiogenic treatment. Less