Kiemer Alexandra K, Weber Nina C, Fürst Robert, Bildner Nicole, Kulhanek-Heinze Stefanie, Vollmar Angelika M
Department of Pharmacy, Center of Drug Research, University of Munich, Munich, Germany. Alexandra.Kiemer@ cup.uni-muenchen.de
Circ Res. 2002 May 3;90(8):874-81. doi: 10.1161/01.res.0000017068.58856.f3.
The atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory potential due to its inhibitory action on the production of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). The aim of this study was to determine whether ANP is able to attenuate inflammatory effects of TNF-alpha on target cells. Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha in the presence or absence of ANP. Changes in permeability, cytoskeletal alterations, phosphorylation of p38 MAPK and HSP27, and expression of MKP-1 were determined by macromolecule permeability assay, fluorescence labeling, RT-PCR, and immunoblotting. Antisense studies were done by transfecting cells with MKP-1 antisense oligonucleotides. Activation of HUVECs with TNF-alpha lead to a significant increase of macromolecule permeability and formation of stress fibers. Treatment of cells with ANP (10(-8) to 10(-6) mol/L) significantly reduced the formation of stress fibers and elevated permeability. Both TNF-alpha-induced effects were shown to be mediated via the activation of p38 using SB203580, a specific inhibitor of p38. ANP significantly reduced the TNF-alpha-induced activation of p38 and attenuated the phosphorylation of HSP27, a central target downstream of p38. ANP showed no effect on p38 upstream kinases MKK3/6. However, a significant induction of the MAPK phosphatase MKP-1 mRNA and protein could be observed in ANP-treated cells. Antisense experiments proved a causal role for MKP-1 induction in the ANP-mediated inhibition of p38. These data show the inhibitory action of ANP on TNF-alpha-induced changes in endothelial cytoskeleton and macromolecule permeability involving an MKP-1-induced inactivation of p38 MAPK. These effects point to an antiinflammatory and antiatherogenic potential of this cardiovascular hormone.
心房利钠肽（ANP）是一种心血管激素，因其对炎症介质如肿瘤坏死因子-α（TNF-α）的产生具有抑制作用而具有抗炎潜力。本研究的目的是确定ANP是否能够减弱TNF-α对靶细胞的炎症作用。在有或没有ANP存在的情况下，用人肿瘤坏死因子-α（TNF-α）处理人脐静脉内皮细胞（HUVECs）。通过大分子通透性测定、荧光标记、逆转录-聚合酶链反应（RT-PCR）和免疫印迹法测定通透性变化、细胞骨架改变、p38丝裂原活化蛋白激酶（MAPK）和热休克蛋白27（HSP27）的磷酸化以及丝裂原活化蛋白激酶磷酸酶-1（MKP-1）的表达。通过用MKP-1反义寡核苷酸转染细胞进行反义研究。用TNF-α激活HUVECs导致大分子通透性显著增加和应力纤维形成。用ANP（10^(-8)至10^(-6)mol/L）处理细胞可显著减少应力纤维的形成并降低通透性。使用p38的特异性抑制剂SB203580表明，TNF-α诱导的两种效应均通过p38的激活介导。ANP显著降低TNF-α诱导的p38激活，并减弱HSP27的磷酸化，HSP27是p38下游的主要靶点。ANP对p38上游激酶MKK3/6没有影响。然而，在ANP处理的细胞中可观察到MAPK磷酸酶MKP-1 mRNA和蛋白的显著诱导。反义实验证明了MKP-1诱导在ANP介导的p38抑制中的因果作用。这些数据表明ANP对TNF-α诱导的内皮细胞骨架和大分子通透性变化具有抑制作用，涉及MKP-1诱导的p38 MAPK失活。这些效应表明这种心血管激素具有抗炎和抗动脉粥样硬化潜力。
