Arnott Clare, Punnia-Moorthy Gaya, Tan Joanne, Sadeghipour Sara, Bursill Christina, Patel Sanjay
Department of Cardiology, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia.
Sydney Medical School, The University of Sydney, New South Wales, Australia.
PLoS One. 2016 Mar 9;11(3):e0150688. doi: 10.1371/journal.pone.0150688. eCollection 2016.
Recent studies have suggested that the VEGF inhibitors, Ranibizumab and Aflibercept may be associated with an excess of cardiovascular events, potentially driven by increasing atheroma instability, leading to plaque rupture and clinical events. Inflammation plays a key role in the progression of atherosclerotic plaque and particularly conversion to an unstable phenotype. Here, we sought to assess the in vitro effects of these drugs on the expression of key inflammatory mediators on endothelial cells.
Human coronary artery endothelial cells were co-incubated for 16h with Ranibizumab (0.11nM) or Aflibercept (0.45nM), as determined by each drug's peak serum concentration (Cmax). Expression at protein (ELISA) and gene (RT-PCR) level of inflammatory chemokines CCL2, CCL5 and CXC3L1 as well as gene expression for the cell adhesion molecules VCAM-1, ICAM-1 and the key NF-κb protein p65 was assessed. VEGF-A protein levels were also determined.
Both drugs significantly increased chemokine, cell adhesion molecule (CAM) and p65 expression, while decreasing VEGF-A protein secretion. At equivalent Cmax concentrations, Aflibercept was significantly more pro-inflammatory than Ranibizumab. Reduction of secreted VEGF-A levels significantly attenuated inflammatory effects of both drugs, whereas blockade of the VEGF-A receptor or silencing of VEGF-A gene synthesis alone had no effect, suggesting that binding of drug to secreted VEGF-A is crucial in promoting inflammation. Finally, blockade of Toll-like receptor 4 significantly reduced inflammatory effects of both drugs.
We demonstrated here, for the first time, that both drugs have potent pro-inflammatory effects, mediated via activation of Toll-like receptor 4 on the endothelial cell surface by drug bound to VEGF-A. Further studies are required to investigate whether these effects are also seen in vivo.
最近的研究表明，血管内皮生长因子（VEGF）抑制剂雷珠单抗和阿柏西普可能与心血管事件增多有关，这可能是由于动脉粥样硬化斑块不稳定性增加，导致斑块破裂和临床事件。炎症在动脉粥样硬化斑块进展中起关键作用，尤其是在转变为不稳定表型方面。在此，我们试图评估这些药物对内皮细胞关键炎症介质表达的体外影响。
将人冠状动脉内皮细胞与雷珠单抗（0.11nM）或阿柏西普（0.45nM）共同孵育16小时，这是根据每种药物的血清峰值浓度（Cmax）确定的。评估炎症趋化因子CCL2、CCL5和CXC3L1在蛋白（酶联免疫吸附测定法）和基因（逆转录聚合酶链反应）水平的表达，以及细胞黏附分子血管细胞黏附分子-1（VCAM-1）、细胞间黏附分子-1（ICAM-1）和关键的核因子κB蛋白p65的基因表达。还测定了VEGF-A蛋白水平。
两种药物均显著增加趋化因子、细胞黏附分子（CAM）和p65的表达，同时降低VEGF-A蛋白分泌。在等效Cmax浓度下，阿柏西普的促炎作用明显强于雷珠单抗。分泌的VEGF-A水平降低显著减弱了两种药物的炎症作用，而单独阻断VEGF-A受体或沉默VEGF-A基因合成则没有效果，这表明药物与分泌的VEGF-A结合在促进炎症方面至关重要。最后，阻断Toll样受体4显著降低了两种药物的炎症作用。
我们首次在此证明，两种药物均具有强大的促炎作用，是通过与VEGF-A结合的药物激活内皮细胞表面的Toll样受体4介导的。需要进一步研究来调查这些作用在体内是否也会出现。
