Centre de Recherche Cardio-Thoracique de Bordeaux, Univ Bordeaux, 33076, Bordeaux, France.
Pflugers Arch. 2012 Sep;464(3):261-72. doi: 10.1007/s00424-012-1136-5. Epub 2012 Jul 22.
Pulmonary hypertension, the main disease of the pulmonary circulation, is characterized by an increase in pulmonary vascular resistance, involving proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). However, cellular and molecular mechanisms underlying these phenomena remain to be identified. In the present study, we thus investigated in rat intrapulmonary arteries (1) the expression and the functional activity of TRPV1 and TRPV4, (2) the PASMC migration triggered by these TRPV channels, and (3) the associated reorganization of the cytoskeleton. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated expression of TRPV1 and TRPV4 mRNA in rat intrapulmonary arteries. These results were confirmed at the protein level by western blot. Using microspectrofluorimetry (indo-1), we show that capsaicin and 4α-phorbol-12,13-didecanoate (4α-PDD), selective agonists of TRPV1 and TRPV4, respectively, increased the intracellular calcium concentration of PASMC. Furthermore, stimulation of TRPV1 and TRPV4 induced PASMC migratory responses, as assessed by two different methods (a modified Boyden chamber assay and a wound-healing migration assay). This response cannot seem to be attributed to a proliferative effect as assessed by BrdU and Wst-1 colorimetric methods. Capsaicin- and 4α-PDD-induced calcium and migratory responses were inhibited by the selective TRPV1 and TRPV4 blockers, capsazepine and HC067047, respectively. Finally, as assessed by immunostaining, these TRPV-induced migratory responses were associated with reorganization of the F-actin cytoskeleton and the tubulin and intermediate filament networks. In conclusion, these data point out, for the first time, the implication of TRPV1 and TRPV4 in rat PASMC migration, suggesting the implication of these TRPV channels in the physiopathology of pulmonary hypertension.
肺动脉高压是肺循环的主要疾病，其特征为肺血管阻力增加，涉及到肺动脉平滑肌细胞（PASMC）的增殖和迁移。然而，这些现象的细胞和分子机制仍有待确定。在本研究中，我们因此研究了大鼠肺内动脉：（1）TRPV1 和 TRPV4 的表达和功能活性；（2）这些 TRPV 通道触发的 PASMC 迁移；（3）相关的细胞骨架重排。逆转录聚合酶链反应（RT-PCR）分析表明 TRPV1 和 TRPV4 mRNA 在大鼠肺内动脉中的表达。这些结果在蛋白质水平上通过 Western blot 得到了证实。使用微光谱荧光法（indo-1），我们表明辣椒素和 4α-佛波醇-12,13-二癸酸酯（4α-PDD），分别为 TRPV1 和 TRPV4 的选择性激动剂，增加了 PASMC 的细胞内钙离子浓度。此外，刺激 TRPV1 和 TRPV4 诱导了 PASMC 的迁移反应，如通过两种不同的方法（改良 Boyden 室测定和划痕愈合迁移测定）评估的。该反应似乎不能归因于通过 BrdU 和 Wst-1 比色法评估的增殖作用。辣椒素和 4α-PDD 诱导的钙和迁移反应分别被选择性 TRPV1 和 TRPV4 阻断剂，辣椒素和 HC067047 抑制。最后，如通过免疫染色评估的，这些 TRPV 诱导的迁移反应与 F-肌动蛋白细胞骨架和微管和中间丝网络的重排有关。总之，这些数据首次指出 TRPV1 和 TRPV4 参与大鼠 PASMC 迁移，提示这些 TRPV 通道参与肺动脉高压的病理生理学。
