Inhibition of protein tyrosine kinases attenuates increases in expression of transforming growth factor-beta isoforms and their receptors following arterial injury

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.