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 i n neointima formation in mechanically injured vessels and in restenosi s after angioplasty. To further understand the significance of TGF-bet a s in neointima formation, we examined the temporal expression of thr ee TGF-beta isoforms (-beta(1), -beta(2), and -beta(3)), their recepto rs (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 cathet er-injured rat carotid arteries and their dependency on tyrosine kinas e activity. Using a standardized reverse transcriptase-polymerase chai n reaction assay optimized to estimate mRNA levels, we observed distin ct 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 ti me while TGF-beta(3) mRNA also increased almost 2-fold. There were als o 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 hi gher; mRNA encoding the type II receptor increased approximately 3-fol d by 24 hours. There were also associated increases in TGF-beta(1), TG F-beta(3), ALK-5, and T beta RII immunoreactive peptide levels. Peak i ncreases in mRNAs for integrins alpha(v) and beta(3) averaged approxim ately 2-fold and 2.5-fold, respectively. Perivascular administration o f the tyrosine kinase inhibitor genistein at the time of vessel injury markedly (>85%) inhibited elevations in mRNAs encoding TGF-beta(1), T GF-beta(3), T beta RII, and the two integrins alpha(v) and beta(3), wh ile application of its inactive chemically similar homologue daidzein did not prevent the injury-induced elevations in mRNA levels. Since th e increases in integrins alpha(v) and beta(3) mRNA. could be theoretic ally attributed to TGF-beta actions despite being dependent on tyrosin e kinase activity, we examined whether the observed elevations in inte grins alpha(v) and beta(3) were due to TGF-beta(1) secretion, using cu ltured rat carotid artery smooth muscle cells. TGF-beta(1) neutralizin g antibodies specifically inhibited elevations in integrins alpha(v) a nd beta(3) mRNAs due to platelet derived growth factor-BB and fibrobla st growth factor-2. We conclude that multiple components of the TGF-be ta system in vessels are activated following injury and influence expr ession of integrin receptors important for smooth muscle cell migratio n. Activation of the TGF-beta system appears to be highly dependent on tyrosine kinases.