Wick Marilee J, Loomis Zoe L, Harral Julie W, Le Mysan, Wehling Carol A, Miller York E, Dempsey Edward C
Cardiovascular Pulmonary Research Laboratory, University of Colorado Denver, RC-2, Box B-133, 12700 E. 19th Ave., Aurora, CO, 80045, USA.
Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Aurora, CO, 80045, USA.
Transgenic Res. 2016 Dec;25(6):773-784. doi: 10.1007/s11248-016-9969-x. Epub 2016 Jul 1.
Neprilysin (NEP) is a cell surface metallopeptidase found in many tissues. Based mostly on pharmacological manipulations, NEP has been thought to protect blood vessels from plasma extravasation. We have suggested that NEP may protect against pulmonary vascular injury. However, these prior studies did not utilize mice which overexpress NEP. The aims of the present investigation were to develop and characterize doubly transgenic (DT) mice that overexpress NEP universally and conditionally, and to investigate the protective effect that overexpressed NEP may have against plasma extravasation in the vasculature. The duodenum, which is often used to assess vascular permeability, and in which the NEP protein was overexpressed in our DT mice two-fold, was selected as our experimental preparation. We found that substance P-induced plasma extravasation was decreased substantially (3.5-fold) in the duodenums of our doxycycline-treated DT mice, giving independent evidence of NEP's protective effects against plasma extravasation. Transgenic lung NEP protein was not stably expressed in the DT mice, so we were not able to test the effect of NEP overexpression in the lung. Although initially overexpressed nearly nine-fold at that site, pulmonary NEP protein overexpression eventually dissipated. Surprisingly, at a time when there was no lung transgenic NEP protein overexpression, lung NEP mRNA expression was still increased 23-fold, indicating that the expression defect probably is not transcriptional. These studies help to characterize our complex transgenic model of NEP overexpression and further demonstrate NEP's protective effects against plasma extravasation.
中性肽链内切酶（NEP）是一种在许多组织中发现的细胞表面金属肽酶。主要基于药理学操作，人们认为NEP可保护血管免受血浆外渗的影响。我们曾提出NEP可能预防肺血管损伤。然而，这些先前的研究并未使用过表达NEP的小鼠。本研究的目的是构建并鉴定普遍且条件性过表达NEP的双转基因（DT）小鼠，并研究过表达的NEP对脉管系统中血浆外渗可能具有的保护作用。十二指肠常用于评估血管通透性，且在我们的DT小鼠中NEP蛋白过表达两倍，因此被选作我们的实验标本。我们发现，在经强力霉素处理的DT小鼠的十二指肠中，P物质诱导的血浆外渗显著降低（3.5倍），这为NEP对血浆外渗的保护作用提供了独立证据。转基因肺NEP蛋白在DT小鼠中未稳定表达，因此我们无法测试NEP过表达在肺中的作用。尽管最初在该部位过表达近9倍，但肺NEP蛋白的过表达最终消失。令人惊讶的是，在没有肺转基因NEP蛋白过表达的时候，肺NEP mRNA表达仍增加了23倍，这表明表达缺陷可能不是转录性的。这些研究有助于描述我们复杂的NEP过表达转基因模型，并进一步证明NEP对血浆外渗的保护作用。
