Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA.
Toxicol Lett. 2013 Jul 4;220(2):126-34. doi: 10.1016/j.toxlet.2013.04.011. Epub 2013 Apr 22.
Cigarette smoke is the major cause of chronic obstructive pulmonary disease (COPD), yet pathogenic mechanisms are not fully understood. Vascular endothelial growth factor (VEGF) is one of the major regulators of endothelial cell survival and is believed to play a role in the pathogenesis of COPD. Fibroblasts are a significant source of VEGF in the lungs; however the effect of cigarette smoke exposure on VEGF release by fibroblasts is not fully understood. We hypothesized that cigarette smoke-induced disturbed VEGF release by human lung fibroblasts is a potential pathogenic mechanism that could contribute to COPD. Cigarette smoke extract (CSE) was prepared by modification of the methods of Carp and Janoff (American Review of Respiratory Disease, 1978). Human fetal lung fibroblasts (HFL-1) were exposed to different concentrations of CSE and for different durations. VEGF release into the media was measured using ELISA. TGF-β1 receptor (TβR1)/Smad3 as a potential pathway for CSE modulated VEGF release was also investigated using biochemical analyses and siRNA inhibition of Smad3 and siRNA and pharmacologic inhibition of TβR1. CSE induced VEGF release by HFL-1 in concentration and time dependent manner. This was confirmed in two additional types of primary human fetal lung fibroblasts. CSE induced Smad3 phosphorylation and nuclear translocation in HFL-1 cells. Silencing of Smad3 by siRNA not only eliminated the stimulatory effect of CSE on VEGF release but also inhibited baseline VEGF production. Suppression of TβR1 by the pharmacological inhibitor (SB431542) markedly reduced VEGF release by HFL-1 in response to CSE and this effect was confirmed by TβR1 siRNA. In contrast, nicotine inhibited VEGF release by HFL-1 in a dose and time dependent manner. Our findings indicate that CSE stimulates Smad3-mediated VEGF release by lung fibroblasts. Nicotine does not account for the CSE stimulation of VEGF in HFL-1. The ability of lung fibroblasts to produce VEGF may play a role in pathogenesis of cigarette smoke induced lung disease.
香烟烟雾是慢性阻塞性肺疾病（COPD）的主要原因，但发病机制尚不完全清楚。血管内皮生长因子（VEGF）是内皮细胞存活的主要调节因子之一，被认为在 COPD 的发病机制中起作用。成纤维细胞是肺部 VEGF 的重要来源；然而，香烟烟雾暴露对成纤维细胞 VEGF 释放的影响尚不完全清楚。我们假设香烟烟雾诱导的人肺成纤维细胞 VEGF 释放紊乱是一种潜在的致病机制，可能导致 COPD。香烟烟雾提取物（CSE）通过修改 Carp 和 Janoff 的方法（美国呼吸疾病评论，1978）制备。将人胎肺成纤维细胞（HFL-1）暴露于不同浓度的 CSE 和不同时间。使用 ELISA 测量培养基中 VEGF 的释放。还通过生化分析和 Smad3 的 siRNA 抑制以及 TβR1 的 siRNA 和药理学抑制研究了 CSE 调节的 VEGF 释放的 TGF-β1 受体（TβR1）/Smad3 作为潜在途径。CSE 以浓度和时间依赖性方式诱导 HFL-1 中 VEGF 的释放。这在另外两种类型的原代人胎肺成纤维细胞中得到了证实。CSE 诱导 HFL-1 细胞中 Smad3 的磷酸化和核转位。Smad3 的 siRNA 不仅消除了 CSE 对 VEGF 释放的刺激作用，而且还抑制了基础 VEGF 的产生。药理学抑制剂（SB431542）抑制 TβR1 显著降低了 CSE 对 HFL-1 中 VEGF 释放的作用，并且该作用通过 TβR1 siRNA 得到了证实。相反，尼古丁以剂量和时间依赖性方式抑制 HFL-1 中 VEGF 的释放。我们的发现表明，CSE 刺激肺成纤维细胞中 Smad3 介导的 VEGF 释放。尼古丁不能解释 CSE 刺激 HFL-1 中 VEGF 的作用。肺成纤维细胞产生 VEGF 的能力可能在香烟烟雾诱导的肺部疾病的发病机制中起作用。
