Ward M R, Agrotis A, Kanellakis P, Dilley R, Jennings G, Bobik A
Cell Biology Laboratory, Baker Medical Research Institute, Prahran, Australia.
Arterioscler Thromb Vasc Biol. 1997 Nov;17(11):2461-70. doi: 10.1161/01.atv.17.11.2461.
Transforming growth factor-beta 1 (TGF-beta 1) has been implicated in neointima formation in mechanically injured vessels and in restenosis after angioplasty. To further understand the significance of TGF-beta s in neointima formation, we examined the temporal expression of three TGF-beta isoforms (-beta 1, -beta 2, and -beta 3), their receptors (ALK-2, ALK-5, and T beta RII), and two putative TGF-beta responses (elevations in alpha v and beta 3 integrin mRNAs) in balloon catheter-injured rat carotid arteries and their dependency on tyrosine kinase activity. Using a standardized reverse transcriptase-polymerase chain reaction assay optimized to estimate mRNA levels, we observed distinct patterns of mRNA regulation for TGF-beta 1, -beta 2, and -beta 3 during the 48 hours immediately after injury, which were localized to the vessel's media. TGF-beta 1 mRNA increased 10-fold during this time while TGF-beta 3 mRNA also increased almost 2-fold. There were also increases in mRNAs encoding the TGF-beta type I receptors ALK-5 and ALK-2, as well as the type II receptor (T beta RII). Eight hours after the injury, mRNA levels for ALK-2 and ALK-5 were on average 2-fold higher; mRNA encoding the type II receptor increased approximately 3-fold by 24 hours. There were also associated increases in TGF-beta 1, TGF-beta 3, ALK-5, and T beta RII immunoreactive peptide levels. Peak increases in mRNAs for integrins alpha v and beta 3 averaged approximately 2-fold and 2.5-fold, respectively. Perivascular administration of the tyrosine kinase inhibitor genistein at the time of vessel injury markedly (> 85%) inhibited elevations in mRNAs encoding TGF-beta 1, TGF-beta 3, T beta RII, and the two integrins alpha v and beta 3, while application of its inactive chemically similar homologue daidzein did not prevent the injury-induced elevations in mRNA levels. Since the increases in integrins alpha v and beta 3 mRNA could be theoretically attributed to TGF-beta actions despite being dependent on tyrosine kinase activity, we examined whether the observed elevations in integrins alpha v and beta 3 were due to TGF-beta 1 secretion, using cultured rat carotid artery smooth muscle cells. TGF-beta 1 neutralizing antibodies specifically inhibited elevations in integrins alpha v and beta 3 mRNAs due to platelet-derived growth factor-BB and fibroblast growth factor-2. We conclude that multiple components of the TGF-beta system in vessels are activated following injury and influence expression of integrin receptors important for smooth muscle cell migration. Activation of the TGF-beta system appears to be highly dependent on tyrosine kinases.
转化生长因子-β1（TGF-β1）与机械损伤血管的新生内膜形成以及血管成形术后的再狭窄有关。为了进一步了解TGF-βs在新生内膜形成中的意义，我们检测了三种TGF-β亚型（-β1、-β2和-β3）、它们的受体（ALK-2、ALK-5和TβRII）以及两种假定的TGF-β反应（αv和β3整合素mRNA升高）在球囊导管损伤的大鼠颈动脉中的时间表达情况，以及它们对酪氨酸激酶活性的依赖性。使用优化的标准化逆转录聚合酶链反应检测来估计mRNA水平，我们观察到在损伤后立即的48小时内，TGF-β1、-β2和-β3的mRNA调节模式不同，这些调节定位于血管中膜。在此期间，TGF-β1 mRNA增加了10倍，而TGF-β3 mRNA也增加了近2倍。编码TGF-β I型受体ALK-5和ALK-2以及II型受体（TβRII）的mRNA也有所增加。损伤后8小时，ALK-2和ALK-5的mRNA水平平均高出2倍；编码II型受体的mRNA在24小时时增加了约3倍。TGF-β1、TGF-β3、ALK-5和TβRII免疫反应性肽水平也相应增加。整合素αv和β3的mRNA峰值增加分别平均约为2倍和2.5倍。在血管损伤时血管周围给予酪氨酸激酶抑制剂金雀异黄素可显著（>85%）抑制编码TGF-β1、TGF-β3、TβRII以及两种整合素αv和β3的mRNA升高，而应用其无活性的化学类似物大豆苷元并不能阻止损伤诱导的mRNA水平升高。由于整合素αv和β3 mRNA的增加理论上尽管依赖于酪氨酸激酶活性但可能归因于TGF-β的作用，我们使用培养的大鼠颈动脉平滑肌细胞检测了观察到的整合素αv和β3升高是否由于TGF-β1分泌所致。TGF-β1中和抗体特异性抑制了血小板衍生生长因子-BB和成纤维细胞生长因子-2导致的整合素αv和β3 mRNA升高。我们得出结论，血管中TGF-β系统的多个成分在损伤后被激活，并影响对平滑肌细胞迁移重要的整合素受体的表达。TGF-β系统的激活似乎高度依赖于酪氨酸激酶。
