title: Inhibition of protein tyrosine kinases attenuates increases in expression of transforming growth factor-beta isoforms and their receptors following arterial injury.
abstract: 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.
authors.affiliation: Cell Biology Laboratory, Baker Medical Research Institute, Prahran, Australia. mward@Baker.edu.au
authors.forename: M R
authors.initials: MR
authors.lastname: Ward
authors.forename: A
authors.initials: A
authors.lastname: Agrotis
authors.forename: P
authors.initials: P
authors.lastname: Kanellakis
authors.forename: R
authors.initials: R
authors.lastname: Dilley
authors.forename: G
authors.initials: G
authors.lastname: Jennings
authors.forename: A
authors.initials: A
authors.lastname: Bobik
mesh_terms: D030201:Activin Receptors, Type I; D000800:Angioplasty, Balloon / Q000009:adverse effects; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics; D002339:Carotid Arteries / Q000378:metabolism*; D020212:Carotid Artery Injuries; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D019833:Genistein / Q000494:pharmacology*; D039564:Integrin alphaV; D039661:Integrin beta3; D007529:Isoflavones / Q000494:pharmacology*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D017978:Receptors, Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics; D017539:Tunica Intima / Q000293:injuries / Q000473:pathology*; D014945:Wound Healing / Q000502:physiology*
chemical_list: D015703:Antigens, CD; D004791:Enzyme Inhibitors; D039564:Integrin alphaV; D039661:Integrin beta3; D007529:Isoflavones; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger; D017978:Receptors, Growth Factor; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; C004742:daidzein; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; C000625843:Tgfbr1 protein, rat