Milkiewicz Malgorzata, Mohammadzadeh Forough, Ispanovic Eric, Gee Eric, Haas Tara L
Department of Laboratory Diagnostics and Molecular Medicine, Pomeranian Medical University, Szczecin, Poland.
J Cell Biochem. 2007 Feb 15;100(3):750-61. doi: 10.1002/jcb.21055.
VEGF and MMP protein production are both required for exercise-induced capillary growth in skeletal muscle. The underlying process by which muscle activity initiates an angiogenic response is not established, but it is known that mechanical forces such as muscle stretch are involved. We hypothesized that stretch of skeletal muscle microvascular endothelial cells induces production of MMP-2 and VEGF through a common signal pathway. Endothelial cells were grown on Bioflex plates and exposed to 10% static stretch for up to 24 h. MMP-2 protein level was measured by gelatin zymography and VEGF, MMP-2, and MT1-MMP mRNA levels were quantified by real-time quantitative PCR. ERK1/2 and JNK phosphorylation and VEGF protein levels were assessed by Western blotting. Effects of mitogen-activated protein kinases (MAPKs) (ERK1/2, JNK) and reactive oxygen species (ROS) on stretch-induced expression of MMP-2 and VEGF were tested using pharmacological inhibitors. Stretching of endothelial cells for 24 h caused significant increases in MMP-2 protein and mRNA level, but no change in MT1-MMP mRNA. While MMP-2 protein production was enhanced by H(2)O(2) in unstretched cells, ROS inhibition during stretch did not diminish MMP-2 mRNA or protein production. Inhibition of JNK suppressed stretch-induced MMP-2 protein and mRNA, but inhibition of ERK had no effect. In contrast, inhibition of ERK but not JNK attenuated the stretch-induced increase in VEGF mRNA. Our results demonstrate that differential regulation of MMP-2 and VEGF by MAPK signal pathways contribute to stretch-induced activation of microvascular endothelial cells.
血管内皮生长因子（VEGF）和基质金属蛋白酶（MMP）的蛋白生成都是运动诱导骨骼肌毛细血管生长所必需的。肌肉活动引发血管生成反应的潜在过程尚未明确，但已知诸如肌肉拉伸等机械力参与其中。我们推测骨骼肌微血管内皮细胞的拉伸通过共同信号通路诱导MMP-2和VEGF的生成。将内皮细胞培养在Bioflex板上，并暴露于10%的静态拉伸下长达24小时。通过明胶酶谱法测量MMP-2蛋白水平，通过实时定量PCR对VEGF、MMP-2和MT1-MMP mRNA水平进行定量。通过蛋白质印迹法评估细胞外信号调节激酶1/2（ERK1/2）和应激活化蛋白激酶（JNK）的磷酸化以及VEGF蛋白水平。使用药理学抑制剂测试丝裂原活化蛋白激酶（MAPKs）（ERK1/2、JNK）和活性氧（ROS）对拉伸诱导的MMP-2和VEGF表达的影响。内皮细胞拉伸24小时导致MMP-2蛋白和mRNA水平显著增加，但MT1-MMP mRNA无变化。虽然在未拉伸的细胞中过氧化氢（H₂O₂）增强了MMP-2蛋白生成，但拉伸过程中抑制ROS并未减少MMP-2 mRNA或蛋白生成。抑制JNK可抑制拉伸诱导的MMP-2蛋白和mRNA，但抑制ERK则无作用。相反，抑制ERK而非JNK可减弱拉伸诱导的VEGF mRNA增加。我们的结果表明，MAPK信号通路对MMP-2和VEGF的差异调节有助于拉伸诱导的微血管内皮细胞活化。
