Vascular Endothelial Growth Factor Receptor-2 Couples Cyclo-Oxygenase-2 with Pro-Angiogenic Actions of Leptin on Human Endothelial Cells

BackgroundThe adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs.
Methodology/Principal FindingsImmunoblotting studies showed that leptin increased cyclo-oxygenase-2 (COX-2) expression (but not COX-1) in cultured human umbilical vein ECs (HUVEC) through pathways that depend upon activation of both p38 mitogen-activated protein kinase (p38MAPK) and Akt, and stimulated rapid phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) on Tyr1175. Phosphorylation of VEGFR2, p38MAPK and Akt, and COX-2 induction in cells challenged with leptin were blocked by a specific leptin peptide receptor antagonist. Pharmacological inhibitors of COX-2, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and p38MAPK abrogated leptin-induced EC proliferation (assessed by quantifying 5-bromo-2′-deoxyuridine incorporation, calcein fluorescence and propidium iodide staining), slowed the increased migration rate of leptin-stimulated cells (in vitro wound healing assay) and inhibited leptin-induced capillary-like tube formation by HUVEC on Matrigel. Inhibition of VEGFR2 tyrosine kinase activity reduced leptin-stimulated p38MAPK and Akt activation, COX-2 induction, and pro-angiogenic EC responses, and blockade of VEGFR2 or COX-2 activities abolished leptin-driven neo-angiogenesis in a chick chorioallantoic membrane vascularisation assay in vivo.
Conclusions/SignificanceWe conclude that a functional endothelial p38MAPK/Akt/COX-2 signalling axis is required for leptin's pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin's regulation of the vasculature in both non-obese and obese individuals.

背景 脂肪细胞衍生激素瘦素（leptin）可调控多种细胞类型的行为，如今已被证实是一种促血管生成与促炎因子。在血管系统中，这类效应部分通过其直接结合瘦素受体（ObRb）对内皮细胞（ECs）产生的作用介导，但上述活性的具体分子机制尚未阐明。本研究旨在更全面地解析瘦素刺激后人内皮细胞的炎症与血管生成应答之间的分子关联。

研究方法与主要结果 免疫印迹实验显示，瘦素可通过依赖p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase, p38MAPK）与Akt共同激活的通路，在培养的人脐静脉内皮细胞（human umbilical vein ECs, HUVEC）中上调环氧合酶-2（cyclo-oxygenase-2, COX-2）的表达（但不影响环氧合酶-1（COX-1）的水平），并快速磷酸化血管内皮生长因子受体2（vascular endothelial growth factor receptor 2, VEGFR2）的Tyr1175位点。瘦素处理后细胞中VEGFR2、p38MAPK与Akt的磷酸化水平，以及COX-2的诱导表达，均可被特异性瘦素肽类受体拮抗剂阻断。环氧合酶-2、磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase, PI3K）/Akt通路以及p38MAPK的药理学抑制剂，均可抑制瘦素诱导的内皮细胞增殖（通过定量5-溴-2′-脱氧尿苷掺入、钙黄绿素荧光染色与碘化丙啶染色评估），减缓瘦素刺激后细胞迁移速率的升高（体外划痕愈合实验），并抑制瘦素诱导的人脐静脉内皮细胞在Matrigel上形成毛细血管样管腔结构。抑制VEGFR2酪氨酸激酶活性可降低瘦素刺激下p38MAPK与Akt的激活、COX-2的诱导表达以及促血管生成的内皮细胞应答；阻断VEGFR2或COX-2的活性，则可在体内鸡胚尿囊膜血管生成实验中完全取消瘦素介导的新生血管形成。

结论与意义 本研究证实，功能性内皮细胞p38MAPK/Akt/COX-2信号轴是瘦素发挥促血管生成作用的必需条件，且该信号轴上游受ObRb依赖的VEGFR2激活调控。本研究揭示了VEGFR2作为瘦素刺激下COX-2表达与血管生成介质的新功能，为理解瘦素对非肥胖与肥胖个体血管系统的调控机制提供了新的参考依据。