Ashrafpour Homa, Huang Ning, Neligan Peter C, Forrest Christopher R, Addison Patrick D, Moses Michael A, Levine Ronald H, Pang Cho Y
The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8.
Am J Physiol Heart Circ Physiol. 2004 Mar;286(3):H946-54. doi: 10.1152/ajpheart.00901.2003. Epub 2003 Nov 26.
Various laboratories have reported that local subcutaneous or subdermal injection of VEGF(165) at the time of surgery effectively attenuated ischemic necrosis in rat skin flaps, but the mechanism was not studied and enhanced angiogenesis was implicated. In the present study, we used the clinically relevant isolated perfused 6 x 16-cm pig buttock skin flap model to 1) test our hypothesis that VEGF(165) is a potent vasodilator and acute VEGF(165) treatment increases skin perfusion; and 2) investigate the mechanism of VEGF(165)-induced skin vasorelaxation. We observed that VEGF(165) (5 x 10(-16)-5 x 10(-11) M) elicited a concentration-dependent decrease in perfusion pressure (i.e., vasorelaxation) in skin flaps preconstricted with a submaximal concentration of norepinephrine (NE), endothelin-1, or U-46619. The VEGF(165)-induced skin vasorelaxation was confirmed using a dermofluorometry technique for assessment of skin perfusion. The vasorelaxation potency of VEGF(165) in NE-preconstricted skin flaps (pD(2) = 13.57 +/- 0.31) was higher (P < 0.05) than that of acetylcholine (pD(2) = 7.08 +/- 0.24). Human placental factor, a specific VEGF receptor-1 agonist, did not elicit any vasorelaxation effect. However, a specific antibody to VEGF receptor-2 (1 microg/ml) or a specific VEGF receptor-2 inhibitor (5 x 10(-6) M SU-1498) blocked the vasorelaxation effect of VEGF(165) in NE-preconstricted skin flaps. These observations indicate that the potent vasorelaxation effect of VEGF(165) in the skin vasculature is initiated by the activation of VEGF receptor-2. Furthermore, using pharmacological probes, we observed that the postreceptor signaling pathways of VEGF(165)-induced skin vasorelaxation involved activation of phospholipase C and protein kinase C, an increase in inositol 1,4,5-trisphosphate activity, release of the intra-cellular Ca(2+) store, and synthesis/release of endothelial nitric oxide, which predominantly triggered the effector mechanism of VEGF(165)-induced vasorelaxation. This information provides, for the first time, an important insight into the mechanism of VEGF(165) protein or gene therapy in the prevention/treatment of ischemia in skin flap surgery and skin ischemic diseases.
多个实验室报告称，手术时在大鼠皮瓣局部皮下或皮内注射VEGF(165)可有效减轻缺血性坏死，但未对其机制进行研究，推测与血管生成增强有关。在本研究中，我们使用临床相关的离体灌注6×16厘米猪臀部皮瓣模型来：1) 验证我们的假设，即VEGF(165)是一种强效血管舒张剂，急性给予VEGF(165)可增加皮肤灌注；2) 研究VEGF(165)诱导皮肤血管舒张的机制。我们观察到，在预先用次最大浓度去甲肾上腺素(NE)、内皮素-1或U-46619预收缩的皮瓣中，VEGF(165)(5×10⁻¹⁶ - 5×10⁻¹¹ M)引起灌注压力呈浓度依赖性降低（即血管舒张）。使用皮肤荧光测定技术评估皮肤灌注，证实了VEGF(165)诱导的皮肤血管舒张。VEGF(165)在NE预收缩皮瓣中的血管舒张效能（pD₂ = 13.57 ± 0.31）高于（P < 0.05）乙酰胆碱（pD₂ = 7.08 ± 0.24）。人胎盘因子，一种特异性VEGF受体-1激动剂，未引起任何血管舒张效应。然而，VEGF受体-2特异性抗体（1微克/毫升）或特异性VEGF受体-2抑制剂（5×10⁻⁶ M SU-1498）可阻断VEGF(165)在NE预收缩皮瓣中的血管舒张效应。这些观察结果表明，VEGF(165)在皮肤血管系统中的强效血管舒张作用是由VEGF受体-2的激活引发的。此外，使用药理学探针，我们观察到VEGF(165)诱导皮肤血管舒张的受体后信号通路涉及磷脂酶C和蛋白激酶C的激活、肌醇1,4,5-三磷酸活性增加、细胞内钙储存释放以及内皮一氧化氮的合成/释放，这主要触发了VEGF(165)诱导血管舒张的效应机制。这一信息首次为VEGF(165)蛋白或基因疗法在预防/治疗皮瓣手术缺血和皮肤缺血性疾病中的机制提供了重要见解。
