Role of vascular endothelial growth factors in experimental colitis

人类和实验性炎症性肠病(IBD)都以淋巴管密度显著增加和肠道淋巴系统重新分布为特征。虽然已经证实淋巴管生成是对炎症条件(介导免疫反应、限制组织水肿和清除免疫细胞)的重要生理反应，但淋巴管生成如何影响炎症过程还没有很好的定义。此前，已发现多种信号分子参与炎症诱导的淋巴管生成。其中，血管内皮生长因子家族的成员(VEGF-C/D)及其受体(VEGFR-2/3)被认为是中枢调节因子。然而，这些VEGFs和VEGFRs之间复杂且经常重叠的相互作用影响淋巴管的形成和功能，以及它们对血管和淋巴管生成同步的影响仍不清楚。初步研究表明，干预淋巴管生成的血管内皮生长因子信号通路能够影响小鼠结肠炎的严重程度。这些研究表明，与血管生成加剧肠道炎症不同，增强的淋巴管生成在IBD中是一种保护性事件，可能成为新的治疗靶点。因此，我们推测：在实验性结肠炎模型中，淋巴管生成因子在肠道炎症过程中释放出来，通过血管内皮生长因子-C/D和血管内皮生长因子受体-3信号通路激活淋巴管的增殖，抑制实验性结肠炎的肠水肿、炎症和组织损伤的发展。为了验证这一假设，我们将使用葡聚糖硫酸钠诱导的急性结肠炎(AIM1)小鼠体内模型，确定基线和炎症诱导的结肠和全身的变化：1)VEGF-C/D水平；2)它们相关的VEGFR-2/3的表达；3)它们的可溶性形式(sVR2、sVR3)。接下来，我们将使用突变小鼠模型(Chy-3，FOXC2+/-，Pax6+/-)和腺病毒介导的淋巴管生成调节剂基因转移(Ad-VEGFR3-Ig，Ad-VEGF-C156S，Ad-VEGF-D)的特定组合来阐明与炎症诱导的淋巴管生成(AIM 2)相关的通路。最后，我们将检测VEGF-C/D和Ad-VEGFR-3-Ig对体外淋巴管内皮细胞屏障功能和淋巴管平滑肌收缩功能(AIM3)的分子和细胞机制作用。这三个目标的成功完成将识别和区分血管生成和淋巴管生成通路在炎症诱导的实验性结肠炎血管变化中的作用(目标1)，并揭示限制肠道水肿和炎性细胞聚集的可能机制途径(目标3)。特别是，AIM 2将为腺病毒介导的调节血管内皮生长因子信号的可能的治疗应用提供新的和潜在的结论性见解，使其从已建立的炎症诱导的血管生成转变为更具淋巴管生成的方向。

Human and experimental inflammatory bowel disease (IBD) are both characterized by significant increased lymphatic vessel density and redistributions in the intestinal lymphatic system. While it has been established that lymphangiogenesis is an important physiological response to inflammatory conditions (mediating immune response, limiting tissue edema and clearing immune cells), it is not well defined how lymphangiogenesis affects the course of inflammation. Previously, a variety of signaling molecules have been found to be involved in inflammation induced lymphangiogenesis. Among these, members of the vascular endothelial growth factor family (VEGF-C/D) and their receptors (VEGFR- 2/3) have been revealed as central regulators. However, the complex and often over-lapping interactions between these VEGFs and VEGFRs that affect lymphatic formation and functioning, as well as their influences on synchronization of angio- and lymphangiogenesis, remain unclear. Preliminary studies have shown that interventions in lymphangiogenic VEGF signaling pathways were able to influence the severity of murine colitis. These studies indicate that unlike angiogenesis, which exacerbates gut inflammation, enhanced lymphangiogenesis is a protective event in IBD, and may be a novel therapeutic target. Thus, we hypothesize that: Lymphangiogenic factors, liberated during intestinal inflammation, activate lymphatic proliferation via VEGF-C/D and VEGFR-3 signaling and suppress the development of intestinal edema, inflammation and tissue injury in experimental colitis. To test this hypothesis we will: determine baseline and inflammation induced changes in colonic as well as systemic: 1) VEGF-C/D levels 2) Expression of their associated VEGFR-2/3 and 3) their soluble forms (sVR2, sVR3), using the in vivo murine model of acute dextran sodium sulfate induced colitis (AIM1). Next we will use specific combinations of mutant mouse models (Chy-3, FOXC2+/-, Pax6+/-) with adenovirus-mediated gene transfers of lymphangiogenic modulators (Ad-VEGFR3-Ig, Ad-VEGF-C156S, Ad-VEGF-D) to clarify pathways related to inflammation-induced lymphangiogenesis (AIM 2). Lastly we will test the molecular and cellular mechanistic roles of VEGF-C/D and Ad-VEGFR-3-Ig on in vitro lymphatic endothelial barrier function and lymphatic smooth muscle contractility (AIM 3). The successful completion of these 3 aims will identify and discriminate the role of angiogenic and lymphangiogenic pathways in inflammation induced vascular changes in experimental colitis (Aim 1), and reveal possible mechanistic pathways involved in limiting intestinal edema and inflammatory cell accumulation (Aim 3). In particular, Aim 2 will provide novel and potentially conclusive insights into possible therapeutic applications of adenovirus-mediated modulations in VEGF signaling to shift from the established inflammation-induced angiogenesis into a more lymphangiogenic direction.