Woodward Heather N, Anwar Adil, Riddle Suzette, Taraseviciene-Stewart Laimute, Fragoso Miguel, Stenmark Kurt R, Gerasimovskaya Evgenia V
Department of Pediatrics, 12700 E. 19th Ave., University of Colorado Denver, Research Complex 2, Box B131, Aurora, CO 80045, USA.
Am J Physiol Lung Cell Mol Physiol. 2009 Nov;297(5):L954-64. doi: 10.1152/ajplung.00038.2009. Epub 2009 Aug 14.
We recently reported that vasa vasorum expansion occurs in the pulmonary artery (PA) adventitia of chronically hypoxic animals and that extracellular ATP is a pro-angiogenic factor for isolated vasa vasorum endothelial cells (VVEC). However, the sources of extracellular ATP in the PA vascular wall, as well as the molecular mechanisms underlying its release, remain elusive. Studies were undertaken to explore whether VVEC release ATP in response to hypoxia and to determine signaling pathways involved in this process. We found that hypoxia (1-3% O2) resulted in time- and O2-dependent ATP release from VVEC. Preincubation with the inhibitors of vesicular transport (monensin, brefeldin A, and N-ethylmaleimide) significantly decreased ATP accumulation in the VVEC conditioned media, suggesting that hypoxia-induced ATP release occurs through vesicular exocytosis. Additionally, both hypoxia and exogenously added ATP resulted in the activation of PI3K and accumulation of GTP-bound RhoA in a time-dependent manner. Pharmacological inhibition of PI3K and ROCK or knockout of RhoA by small interfering RNA significantly abolished hypoxia-induced ATP release from VVEC. Moreover, RhoA and ROCK play a critical role in ATP-induced increases in VVEC DNA synthesis, migration, and tube formation, indicating a functional contribution of PI3K, Rho, and ROCK to both the autocrine mechanism of ATP release and ATP-mediated angiogenic activation of VVEC. Taken together, our findings provide novel evidence for the signaling mechanisms that link hypoxia-induced increases in extracellular ATP and vasa vasorum expansion.
我们最近报道，慢性低氧动物的肺动脉（PA）外膜中发生了血管滋养管扩张，并且细胞外ATP是分离的血管滋养管内皮细胞（VVEC）的促血管生成因子。然而，PA血管壁中细胞外ATP的来源及其释放的分子机制仍不清楚。本研究旨在探讨VVEC是否会对低氧作出反应而释放ATP，并确定该过程中涉及的信号通路。我们发现低氧（1-3% O₂）导致VVEC随时间和氧浓度依赖性地释放ATP。用囊泡运输抑制剂（莫能菌素、布雷菲德菌素A和N-乙基马来酰亚胺）预孵育可显著降低VVEC条件培养基中的ATP积累，这表明低氧诱导的ATP释放是通过囊泡胞吐作用发生的。此外，低氧和外源性添加的ATP均导致PI3K激活和GTP结合的RhoA随时间依赖性积累。PI3K和ROCK的药理学抑制或小干扰RNA敲除RhoA可显著消除低氧诱导的VVEC ATP释放。此外，RhoA和ROCK在ATP诱导的VVEC DNA合成、迁移和管形成增加中起关键作用，表明PI3K、Rho和ROCK对ATP释放的自分泌机制以及ATP介导地VVEC血管生成激活均有功能贡献。综上所述，我们的研究结果为连接低氧诱导的细胞外ATP增加和血管滋养管扩张的信号机制提供了新证据。
