Zuckerbraun Brian S, McCloskey Carol A, Mahidhara Raja S, Kim Peter K M, Taylor Bradley S, Tzeng Edith
Department of Surgery, University of Pittsburgh School of Medicine, 3459 Fifth Avenue, Pittsburgh, PA 15213, USA.
J Vasc Surg. 2003 Oct;38(4):812-9. doi: 10.1016/s0741-5214(03)00427-0.
Vascular injury and inflammation are associated with elaboration of a number of cytokines that signal through multiple pathways to act as smooth muscle cell (SMC) mitogens. Activation of the nuclear factor-kappa B (NF-kappaB) transcription factor is essential for SMC proliferation in vitro and is activated by vascular injury in vivo. Activation of NF-kappaB is controlled by several upstream regulators, including the inhibitors of kappa B (IkappaB). These proteins bind to and keep NF-kappaB inactivated. The purpose of this study was to determine whether adenoviral gene transfer of a mutated IkappaBalpha super-repressor (AdIkappaBalphaSR) could inhibit development of intimal hyperplasia in vivo and to investigate how over-expression of this construct influences in vitro SMC proliferation and cell cycle regulatory proteins.
A rat carotid injury model was used to study prevention of intimal hyperplasia. Arteries were assayed 14 days after injury and infection with AdIkappaBalphaSR or adenoviral beta-galactosidase (AdLacZ). Untreated SMC or SMC infected with AdLacZ or AdIkappaBalphaSR were stimulated with 10% fetal bovine serum, interleukin-1beta, or tumor necrosis factor-alpha. Electrophoretic mobility shift assays were used to assay for NF-kappaB activation. Protein levels of IkappaBalpha and cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27(Kip1) were determined with Western blot analysis. Proliferation was measured with (3)H-thymidine incorporation assays.
AdIkappaBalphaSR inhibited the development of intimal hyperplasia by 49% (P <.05). Infection with AdIkappaBalphaSR significantly suppressed in vitro SMC proliferation when stimulated with serum, interleukin 1, or tumor necrosis factor alpha, and did not result in cell death. Inhibition of proliferation was associated with increased p21(Cip1/Waf1) and p27(Kip1) protein levels.
Gene transfer of IkappaBalpha super-repressor inhibited development of intimal hyperplasia in vivo and SMC proliferation in vitro. The antiproliferative activity may be related to cell cycle arrest through upregulation of the cyclin-dependent kinase inhibitors p21 and p27. Overexpression of IkappaBalpha may be a future therapeutic option in treatment of vascular diseases.
血管损伤和炎症与多种细胞因子的产生有关，这些细胞因子通过多种途径发挥信号作用，充当平滑肌细胞（SMC）的促有丝分裂原。核因子-κB（NF-κB）转录因子的激活对于体外SMC增殖至关重要，并且在体内可被血管损伤激活。NF-κB的激活受多种上游调节因子控制，包括κB抑制因子（IkappaB）。这些蛋白质与NF-κB结合并使其保持失活状态。本研究的目的是确定突变的IkappaBalpha超级抑制因子（AdIkappaBalphaSR）的腺病毒基因转移是否能在体内抑制内膜增生的发展，并研究该构建体的过表达如何影响体外SMC增殖和细胞周期调节蛋白。
使用大鼠颈动脉损伤模型研究内膜增生的预防。在损伤和用AdIkappaBalphaSR或腺病毒β-半乳糖苷酶（AdLacZ）感染后14天对动脉进行检测。用10%胎牛血清、白细胞介素-1β或肿瘤坏死因子-α刺激未处理的SMC或感染AdLacZ或AdIkappaBalphaSR的SMC。采用电泳迁移率变动分析来检测NF-κB的激活。用蛋白质印迹分析测定IkappaBalpha以及细胞周期蛋白依赖性激酶抑制剂p21（Cip1/Waf1）和p27（Kip1）的蛋白水平。用（3）H-胸腺嘧啶核苷掺入试验测量增殖情况。
AdIkappaBalphaSR使内膜增生的发展受到49%的抑制（P<.05）。用血清、白细胞介素1或肿瘤坏死因子α刺激时，AdIkappaBalphaSR感染显著抑制体外SMC增殖，且未导致细胞死亡。增殖的抑制与p21（Cip1/Waf1）和p27（Kip1）蛋白水平升高有关。
IkappaBalpha超级抑制因子的基因转移在体内抑制了内膜增生的发展，并在体外抑制了SMC增殖。抗增殖活性可能与通过上调细胞周期蛋白依赖性激酶抑制剂p21和p27导致细胞周期停滞有关。IkappaBalpha的过表达可能是未来治疗血管疾病的一种选择。
