Role of vascular endothelial growth factors in experimental colitis

人类和实验性炎症性肠病（IBD）的特征都是淋巴管密度显著增加和肠淋巴系统中的再分布。虽然已经确定淋巴管生成是对炎性病症的重要生理反应（介导免疫反应、限制组织水肿和清除免疫细胞），但淋巴管生成如何影响炎症过程还没有很好地定义。以前，已经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（VEGF-C/D）及其受体（VEGFR-2/3）的成员已被揭示为中央调节器。然而，这些VEGF和VEGF受体之间复杂且经常重叠的相互作用影响淋巴形成和功能，以及它们对血管和淋巴管生成同步化的影响仍然不清楚。初步研究表明，淋巴管生成VEGF信号通路的干预能够影响小鼠结肠炎的严重程度。这些研究表明，与加剧肠道炎症的血管生成不同，增强的淋巴管生成是IBD中的保护性事件，并且可能是新的治疗靶点。因此，我们假设：在实验性结肠炎中，在肠道炎症期间释放的淋巴管生成因子通过VEGF-C/D和VEGFR-3信号传导激活淋巴增殖，并抑制肠水肿、炎症和组织损伤的发展。为了检验这一假设，我们将：使用急性葡聚糖硫酸钠诱导的结肠炎（AIM 1）的体内鼠模型，确定结肠以及全身的基线和炎症诱导的变化：1）VEGF-C/D水平，2）其相关VEGFR-2/3的表达和3）其可溶形式（sVR 2、sVR 3）。接下来，我们将使用突变小鼠模型（Chy-3、FOXC 2 +/-、Pax6 +/-）与腺病毒介导的淋巴管生成调节剂（Ad-VEGF 3-IG、Ad-VEGF-C156 S、Ad-VEGF-D）的基因转移的特定组合来阐明与炎症诱导的淋巴管生成（AIM 2）相关的途径。最后，我们将测试VEGF-C/D和Ad-VEGFR-3-IG对体外淋巴管内皮屏障功能和淋巴管平滑肌收缩性（AIM 3）的分子和细胞机制作用。这3个目标的成功完成将确定和区分血管生成和淋巴管生成途径在实验性结肠炎中炎症诱导的血管变化中的作用（目标1），并揭示限制肠水肿和炎性细胞积聚的可能机制途径（目标3）。特别是，目标2将提供新的和潜在的结论性的见解，可能的治疗应用的腺病毒介导的调节VEGF信号从建立炎症诱导的血管生成转移到一个更淋巴管生成的方向。

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.