Vascular Biology Center, Georgia Regents University, Augusta, Georgia, United States of America.
PLoS One. 2013 Apr 16;8(4):e59733. doi: 10.1371/journal.pone.0059733. Print 2013.
In a neonatal model of hypoxic pulmonary hypertension, a dramatic pulmonary artery adventitial thickening, accumulation of inflammatory cells in the adventitial compartment, and angiogenic expansion of the vasa vasorum microcirculatory network are observed. These pathophysiological responses suggest that rapidly proliferating vasa vasorum endothelial cells (VVEC) may exhibit increased permeability for circulating blood cells and macromolecules. However, the molecular mechanisms underlying these observations remain unexplored. Some reports implicated extracellular adenosine in the regulation of vascular permeability under hypoxic and inflammatory conditions. Thus, we aimed to determine the role of adenosine in barrier regulation of VVEC isolated from the pulmonary arteries of normoxic (VVEC-Co) or chronically hypoxic (VVEC-Hyp) neonatal calves.
We demonstrate via a transendothelial electrical resistance measurement that exogenous adenosine significantly enhanced the barrier function in VVEC-Co and, to a lesser extent, in VVEC-Hyp. Our data from a quantitative reverse transcription polymerase chain reaction show that both VVEC-Co and VVEC-Hyp express all four adenosine receptors (A1, A2A, A2B, and A3), with the highest expression level of A1 receptors (A1Rs). However, A1R expression was significantly lower in VVEC-Hyp compared to VVEC-Co. By using an A1R-specific agonist/antagonist and siRNA, we demonstrate that A1Rs are mostly responsible for adenosine-induced enhancement in barrier function. Adenosine-induced barrier integrity enhancement was attenuated by pretreatment of VVEC with pertussis toxin and GSK690693 or LY294002, suggesting the involvement of Gi proteins and the PI3K-Akt pathway. Moreover, we reveal a critical role of actin cytoskeleton in VVEC barrier regulation by using specific inhibitors of actin and microtubule polymerization. Further, we show that adenosine pretreatment blocked the tumor necrosis factor alpha (TNF-α)-induced permeability in VVEC-Co, validating its anti-inflammatory effects.
We demonstrate for the first time that stimulation of A1Rs enhances the barrier function in VVEC by activation of the Gi/PI3K/Akt pathway and remodeling of actin microfilament.
在缺氧性肺动脉高血压的新生儿模型中，观察到肺动脉外膜显著增厚，外膜腔中炎症细胞积聚，以及血管腔网络的血管生成扩张。这些病理生理反应表明，快速增殖的血管外膜血管（Vasa Vasorum）内皮细胞（VVEC）可能表现出对循环血细胞和大分子的通透性增加。然而，这些观察结果的分子机制仍未被探索。一些报道表明，细胞外腺苷在缺氧和炎症条件下调节血管通透性。因此，我们旨在确定腺苷在来自正常氧合（VVEC-Co）或慢性低氧（VVEC-Hyp）新生牛肺血管的 VVEC 中对屏障的调节作用。
我们通过跨内皮电阻测量证明，外源性腺苷显著增强了 VVEC-Co 的屏障功能，并在一定程度上增强了 VVEC-Hyp 的屏障功能。我们的定量逆转录聚合酶链反应数据显示，VVEC-Co 和 VVEC-Hyp 均表达所有四种腺苷受体（A1、A2A、A2B 和 A3），其中 A1 受体（A1Rs）的表达水平最高。然而，与 VVEC-Co 相比，VVEC-Hyp 中的 A1R 表达明显较低。通过使用 A1R 特异性激动剂/拮抗剂和 siRNA，我们证明 A1Rs 主要负责腺苷诱导的屏障功能增强。用百日咳毒素和 GSK690693 或 LY294002 预处理 VVEC 可减弱腺苷诱导的屏障完整性增强，表明 Gi 蛋白和 PI3K-Akt 途径的参与。此外，我们通过使用肌动蛋白和微管聚合的特异性抑制剂，揭示了肌动蛋白细胞骨架在 VVEC 屏障调节中的关键作用。此外，我们还表明，腺苷预处理可阻断 VVEC-Co 中肿瘤坏死因子-α（TNF-α）诱导的通透性，验证了其抗炎作用。
我们首次证明，通过激活 Gi/PI3K/Akt 途径和重塑肌动蛋白微丝，刺激 A1R 可增强 VVEC 的屏障功能。
