Michael R. Ward

Inhibition of Protein Tyrosine Kinases Attenuates Increases in Expression of Transforming Growth Factor-β 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 vessels 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.

Tranilast Prevents Activation of Transforming Growth Factor-β System, Leukocyte Accumulation, and Neointimal Growth in Porcine Coronary Arteries After Stenting

N (3,4-Dimethoxycinnamoyl) anthranilic acid (tranilast) prevents the synchronous upregulation of isoforms and receptors of the transforming growth factor (TGF)-&bgr; system after arterial injury and reduces restenosis after human coronary angioplasty. However, the effects of tranilast and the importance of the TGF-&bgr; system in stent restenosis, in which inward remodeling is unimportant but inflammatory cell stimulation of neointima formation is exaggerated, are uncertain. Boston minipigs, treated with tranilast or vehicle, were subjected to endoluminal stenting, and the expression of TGF-&bgr;1 and TGF-&bgr;3, the expression of their signaling receptors ALK-5 and T&bgr;R-II, leukocyte numbers around the stent struts, and neointima development were assessed over 28 days. Stenting greatly increased early (5-day) mRNA expression of the 2 TGF-&bgr; isoforms and their receptors. Immunohistochemical localization later showed that their concentrations were greatest in regions adjacent to stent struts, where leukocytes and collagen deposition were prevalent. Tranilast suppressed these elevations in TGF-&bgr; mRNAs and reduced their immunoreactive peptides detectable around stent struts. The accumulation of leukocytes and deposition of collagen in these regions was also greatly inhibited by tranilast. These effects were associated with a 48% reduction in maximal neointimal cross-sectional area and 43% reduction in mean neointimal cross-sectional area at 28 days (P <0.05). We conclude that tranilast suppresses neointima development after stenting, effects that can be at least partly attributed to its ability to attenuate the induction of the TGF-&bgr; system and leukocyte accumulation around stent struts.

Vascular types I and II transforming growth factor-beta receptor expression: differential dependency on tyrosine kinases during induction by TGF-β

Recent evidence indicates that the type II transforming growth factor‐β (TGF‐β) receptor (TβRII) is a serine‐threonine‐tyrosine kinase. However, the significance of its tyrosine kinase is unclear. We investigated in vascular smooth muscle cells the effects of tyrosine kinase inhibition on the expression of TGF‐β receptor types I (ALK‐5) and II (TβRII) mRNA, induced by TGF‐β 1. TGF‐β 1 elevated ALK‐5 mRNA levels 5‐fold; essentially similar TGF‐β 1‐dependent elevations were observed with growth factors, PDGF‐BB and FGF‐2. The tyrosine kinase inhibitor genistein abolished these TGF‐β 1 and growth factor responses. TGF‐β 1 also elevated TβRII mRNA levels which were not inhibited by genistein. We conclude that tyrosine kinases participate in defining how cells respond to TGF‐β.

The pathobiology of restenosis after percutaneous revascularisation: Current concepts and therapeutic objectives

Abstract Restenosis after angioplasty, which causes recurrent angina in 15–20% of patients treated, remains the Achilles heel of percutaneous intervention, and results from a combination of elastic recoil, intimal hyperplasia and remodelling. Recent intravascular ultrasound-based studies have emphasised the importance of vascular remodelling, and the factors controlling it, which include abnormal flow-mediated remodelling, late recoil and adventitial fibrotic constriction. Antioxidants probably reduce restenosis by positively affecting remodelling. Other current preventive treatments for restenosis include: (i) stenting (which prevents recoil and remodelling); (ii) intracoronary irradiation (which affects smooth muscle cell proliferation); (iii) growth factor antagonists such as trapidil and tranilast; and (iv) abciximab (the monoclonal antibody to β 3 integrins). Future therapies are likely to include coated drugeluting stents and gene therapy. This review examines the pathobiology of restenosis, how different devices affect different components of the process, and current and future therapies.