Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, 5501 Hopkins Bayview Circle, JHAAC 4A.52, Baltimore, MD 21224, USA.
Am J Physiol Lung Cell Mol Physiol. 2012 Aug 15;303(4):L343-53. doi: 10.1152/ajplung.00130.2012. Epub 2012 Jun 8.
Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration (Ca(2+)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, Ca(2+), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC Ca(2+), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in Ca(2+) that results in increased AQP1 protein levels and cell migration.
肺血管平滑肌细胞 (PASMC) 迁移是低氧性肺动脉高压发展过程中血管重构的关键组成部分，尽管其调控机制仍知之甚少。水通道蛋白-1（AQP1）是一种完整的膜水通道蛋白，最近已被证明有助于内皮细胞迁移。由于 AQP1 在某些类型的血管平滑肌中表达，我们假设 AQP1 将在 PASMC 中表达，并在缺氧时迁移是必需的。通过 PCR 和免疫印迹技术，我们确定了肺血管平滑肌中 AQP 的表达以及缺氧对 AQP 水平的影响，并通过 Transwell 滤器测定法检查了 AQP1 在缺氧诱导的大鼠 PASMC 迁移中的作用。此外，由于 AQP1 的细胞质尾部含有一个假定的钙结合位点，并且细胞内钙浓度增加 (Ca(2+)) 是 PASMC 缺氧暴露的标志，我们还确定了这些反应是否依赖于 Ca(2+)。结果与主动脉平滑肌细胞 (AoSMCs) 中的结果进行了比较。我们发现，尽管 AQP1 在 PASMC 和 AoSMCs 中都很丰富，但缺氧选择性地增加了 PASMC 中的 AQP1 蛋白水平、Ca(2+) 和迁移。通过电压依赖性 Ca(2+) 或非选择性阳离子通道阻断 Ca(2+) 内流可防止缺氧引起的 PASMC Ca(2+)、AQP1 水平和迁移增加。通过 siRNA 沉默 AQP1 也可防止缺氧诱导的 PASMC 迁移。我们的结果表明，缺氧诱导 PASMC 中 Ca(2+) 的特异性增加，导致 AQP1 蛋白水平和细胞迁移增加。
