Gβγ 鸟苷酸结合蛋白在调节鼠股动脉重塑过程中蛋白酶的作用。
Role for Gβγ G-proteins in protease regulation during remodeling of the murine femoral artery.
机构信息
Vascular Biology and Therapeutics Program, The Methodist Hospital Research Institute, and Department of Cardiovascular Surgery, Methodist DeBakey Heart & Vascular Center, The Methodist Hospital, Houston, Texas 77030, USA.
出版信息
J Surg Res. 2012 Nov;178(1):40-7. doi: 10.1016/j.jss.2011.08.006. Epub 2011 Sep 5.
BACKGROUND
Intimal hyperplasia remains the principal lesion in the development of restenosis after vessel wall injury. G-protein coupled receptors are involved in smooth muscle cell proliferation but the role of Gβγ in arterial intimal hyperplasia has not been well defined. The aim of this study is to characterize the expression of Gβγ G-proteins in the developing intimal hyperplasia in a murine model and the impact of disruption of Gβγ signaling on intimal hyperplasia development.
METHODS
The murine femoral wire injury model was employed. Specimens were perfusion-fixed and sections were stained with H&E and Movat's stains such that morphometry could be performed using an Image-Pro system. Additional specimens of femoral artery were also harvested and snap frozen for Western blotting for the Gβγ expression and for Western blotting and zymography to allow for the study of gelatinase and plasminogen activator expression and activation. Contralateral vessels were used as controls. Additional vessels were immersed in pluronic gel containing an adenovirus with the Gβγ inhibitor βARK(CT).
RESULTS
The injured femoral arteries developed intimal hyperplasia, while sham vessels did not produce such a response. Cell proliferation peaked at 3-5 d and cell migration at 7 d after injury. There was a marked time-dependent increase in Gβγ over the 28 d following injury. Inhibition of Gβγ with βARK(CT) inhibited cell proliferation, cell migration and the development of intimal hyperplasia. Inhibition of Gβγ decreased peak uPA activity and expression without increasing early PAI-1 activity and expression. Inhibition of Gβγ reduced peak MMP-2 activity at d 1 but not at d 7 and also reduced peak MMP-9 activity at d 3. Protein expression for both MMP-2 and MMP-9 was also transiently decreased. There were no changes in TIMP-1 and TIMP-2 expression and activity.
CONCLUSIONS
These data demonstrate a time-dependent increase in Gβγ G-protein expression following wire injury in the mouse. Inhibition of Gβγ alters cell proliferation and migration with associated changes in MMP-2, MMP-9, and uPA expression and activity.
背景
血管壁损伤后再狭窄的主要病变仍然是内膜增生。G 蛋白偶联受体参与平滑肌细胞增殖，但 Gβγ 在动脉内膜增生中的作用尚未得到很好的定义。本研究的目的是描述 Gβγ G 蛋白在小鼠模型中发展中的内膜增生中的表达，以及破坏 Gβγ 信号对内膜增生发展的影响。
方法
采用小鼠股动脉钢丝损伤模型。标本经灌注固定，用 H&E 和 Movat 染色进行染色，以便使用 Image-Pro 系统进行形态计量学分析。还采集了股动脉的其他标本，用于 Western blot 分析 Gβγ 表达，用于 Western blot 和明胶酶谱分析，以研究明胶酶和纤溶酶原激活物的表达和激活。对对照侧血管进行了相应的处理。将其他血管浸入含有 Gβγ 抑制剂 βARK(CT)的腺病毒的 pluronic 凝胶中。
结果
损伤的股动脉发生了内膜增生，而假手术血管则没有产生这种反应。细胞增殖在损伤后 3-5 天达到高峰，细胞迁移在 7 天达到高峰。损伤后 28 天内，Gβγ 的表达呈明显的时间依赖性增加。用 βARK(CT)抑制 Gβγ 抑制了细胞增殖、细胞迁移和内膜增生的发展。抑制 Gβγ 减少了 uPA 的活性和表达峰值，而没有增加早期 PAI-1 的活性和表达。抑制 Gβγ 减少了 MMP-2 的活性峰值，但没有减少 MMP-7 的活性峰值，也减少了 MMP-9 的活性峰值。MMP-2 和 MMP-9 的蛋白表达也短暂减少。TIMP-1 和 TIMP-2 的表达和活性没有变化。
结论
这些数据表明，在小鼠的钢丝损伤后，Gβγ G 蛋白表达呈时间依赖性增加。抑制 Gβγ 改变了细胞增殖和迁移，与 MMP-2、MMP-9 和 uPA 的表达和活性变化有关。
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