Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5222, USA.
J Virol. 2011 Mar;85(5):2296-303. doi: 10.1128/JVI.02319-10. Epub 2010 Dec 22.
Hantaviruses predominantly infect human endothelial cells and, in the absence of cell lysis, cause two diseases resulting from increased vascular permeability. Andes virus (ANDV) causes a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). ANDV infection enhances the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF) by increasing signaling responses directed by the VEGFR2-Src-VE-cadherin pathway, which directs adherens junction (AJ) disassembly. Here we demonstrate that inhibiting pathway-specific VEGFR2 and Src family kinases (SFKs) blocks ANDV-induced endothelial cell permeability. Small interfering RNA (siRNA) knockdown of Src within ANDV-infected endothelial cells resulted in an ∼70% decrease in endothelial cell permeability compared to that for siRNA controls. This finding suggested that existing FDA-approved small-molecule kinase inhibitors might similarly block ANDV-induced permeability. The VEGFR2 kinase inhibitor pazopanib as well as SFK inhibitors dasatinib, PP1, bosutinib, and Src inhibitor 1 dramatically inhibited ANDV-induced endothelial cell permeability. Consistent with their kinase-inhibitory concentrations, dasatinib, PP1, and pazopanib inhibited ANDV-induced permeability at 1, 10, and 100 nanomolar 50% inhibitory concentrations (IC(50)s), respectively. We further demonstrated that dasatinib and pazopanib blocked VE-cadherin dissociation from the AJs of ANDV-infected endothelial cells by >90%. These findings indicate that VEGFR2 and Src kinases are potential targets for therapeutically reducing ANDV-induced endothelial cell permeability and, as a result, capillary permeability during HPS. Since the functions of VEGFR2 and SFK inhibitors are already well defined and FDA approved for clinical use, these findings rationalize their therapeutic evaluation for efficacy in reducing HPS disease. Endothelial cell barrier functions are disrupted by a number of viruses that cause hemorrhagic, edematous, or neurologic disease, and as a result, our findings suggest that VEGFR2 and SFK inhibitors should be considered for regulating endothelial cell barrier functions altered by additional viral pathogens.
汉坦病毒主要感染人类内皮细胞，在不发生细胞裂解的情况下，引起两种因血管通透性增加而导致的疾病。安第斯病毒（ANDV）引起一种高度致命的急性肺水肿，称为汉坦病毒肺综合征（HPS）。ANDV 感染通过增加 VEGFR2-Src-VE-cadherin 途径的信号反应来增强内皮细胞对血管内皮生长因子（VEGF）的通透性，该途径指导着黏着连接（AJ）的解体。在这里，我们证明抑制特定于途径的 VEGFR2 和Src 家族激酶（SFKs）可阻断 ANDV 诱导的内皮细胞通透性。在 ANDV 感染的内皮细胞中用小干扰 RNA（siRNA）敲低 Src，与 siRNA 对照相比，内皮细胞通透性降低约 70%。这一发现表明，现有的美国食品和药物管理局批准的小分子激酶抑制剂可能同样阻断 ANDV 诱导的通透性。VEGFR2 激酶抑制剂帕唑帕尼以及 SFK 抑制剂达沙替尼、PP1、博舒替尼和Src 抑制剂 1 可显著抑制 ANDV 诱导的内皮细胞通透性。与它们的激酶抑制浓度一致，达沙替尼、PP1 和帕唑帕尼分别在 1、10 和 100 纳摩尔 50%抑制浓度（IC（50））下抑制 ANDV 诱导的通透性。我们进一步证明，达沙替尼和帕唑帕尼阻断了 ANDV 感染的内皮细胞中 VE-cadherin 从 AJ 上的解离，超过 90%。这些发现表明，VEGFR2 和 Src 激酶是治疗性降低 ANDV 诱导的内皮细胞通透性的潜在靶点，因此，在 HPS 期间降低毛细血管通透性。由于 VEGFR2 和 SFK 抑制剂的功能已经得到很好的定义，并被美国食品和药物管理局批准用于临床使用，因此，这些发现为评估其在降低 HPS 疾病中的疗效提供了合理性。许多病毒会破坏内皮细胞的屏障功能，导致出血、水肿或神经病，因此，我们的发现表明，VEGFR2 和 SFK 抑制剂应被考虑用于调节由其他病毒病原体改变的内皮细胞屏障功能。
