Supplementary Material for: Protective Actions of Globular and Full-Length Adiponectin on Human Endothelial Cells: Novel Insights into Adiponectin-Induced Angiogenesis

<b><i>Background/Aims:</i></b> Adiponectin levels are decreased in diabetes and atherosclerosis. Coexisting hyperglycaemia and systemic inflammation predisposes to dysregulated angiogenesis and vascular disease. We investigated the effect of globular adiponectin (gAd) and full-length adiponectin (fAd) on angiogenesis and pro-angiogenic molecules, i.e. matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF), in human microvascular endothelial cells (HMEC-1). <b><i>Methods:</i></b> Angiogenesis was assessed by studying capillary tube formation in HMEC-1 on growth factor-reduced Matrigel. Endothelial cell migration assay was performed in a modified Boyden chamber. <b><i>Results:</i></b> Endothelial cell proliferation, in vitro migration and angiogenesis were significantly increased by gAd (mediated by AdipoR1, AMPK-Akt pathways), and gAd significantly increased MMP-2, MMP-9 and VEGF expression levels. The effect of gAd on VEGF appears to be mediated by AdipoR1, whilst the effect of gAd on MMP-2 and MMP-9 appears to be mediated by AdipoR1 and AdipoR2. Only endothelial cell proliferation was significantly increased by fAd in human microvascular endothelial cells and appears to be mediated by AdipoR2. No significant effects on MMP-2, MMP-9 and VEGF were observed. Importantly, gAd decreased glucose and C-reactive protein-induced angiogenesis with a concomitant reduction in MMP-2, MMP-9 and VEGF in HMEC-1 cells. <b><i>Conclusion:</i></b> We report novel insights into the mechanisms of adiponectin on angiogenesis.

背景与目的：糖尿病与动脉粥样硬化患者的脂联素（Adiponectin）水平显著降低。合并存在的高血糖与全身性炎症易诱发血管生成失调及血管疾病。本研究探讨了球状脂联素（globular adiponectin，gAd）与全长脂联素（full-length adiponectin，fAd）对人微血管内皮细胞（human microvascular endothelial cells，HMEC-1）中血管生成及促血管生成分子——基质金属蛋白酶（matrix metalloproteinase，MMP）-2、基质金属蛋白酶-9（MMP-9）与血管内皮生长因子（vascular endothelial growth factor，VEGF）的调控效应。
方法：通过观察HMEC-1细胞在生长因子减毒基质胶（Matrigel）上的毛细血管管腔形成能力，评估血管生成水平；采用改良Boyden小室（Boyden chamber）开展内皮细胞迁移实验。
结果：gAd可显著增强内皮细胞增殖、体外迁移及血管生成能力，该作用由脂联素受体1（AdipoR1）、AMPK-Akt信号通路介导；同时gAd可显著上调MMP-2、MMP-9及VEGF的表达水平。gAd对VEGF的调控作用仅由AdipoR1介导，而其对MMP-2与MMP-9的调控作用则由AdipoR1及脂联素受体2（AdipoR2）共同介导。在HMEC-1细胞中，仅fAd可显著促进内皮细胞增殖，且该作用由AdipoR2介导，未对MMP-2、MMP-9及VEGF的表达产生显著影响。值得注意的是，gAd可抑制高血糖与C反应蛋白（C-reactive protein）诱导的血管生成，同时伴随HMEC-1细胞中MMP-2、MMP-9及VEGF表达水平的下调。
结论：本研究揭示了脂联素调控血管生成的全新机制。