Published online before print January 17, 2008, doi: 10.1161/ATVBAHA.107.158030
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
SM16, an Orally Active TGF-β Type I Receptor Inhibitor Prevents Myofibroblast Induction and Vascular Fibrosis in the Rat Carotid Injury Model
From the Departments of Pharmacology (K.F., D.C., F.L., A.G.), Molecular and Cellular Biology (M.J.C., X.Z., J.N.M., H.-K.C., L.E.L.), Medicinal Chemistry (L.S., F.S., M.C., M.C., M.B.C., W.-C.L.), Assay Development and Compound Profiling (J.E.F., J.L.P., S.B., D.S., M.N.N., S.J.), Research Pathology (H.S.), Structural Biochemistry (P.A.B.-S.), Target Biochemistry (R.M.A.), and Structural Informatics (S.B., C.E.C., X.Z., J.S.) Biogen Idec, Cambridge, Mass.
Correspondence to Leona E. Ling, Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142. E-mail leona.ling{at}biogenidec.com
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Objective— TGF-β plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis.
Methods and Results— The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF-β and activin-induced Smad2/3 phosphorylation and TGF-β-induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle 
-actin–positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells.
Conclusion— These results are the first to demonstrate the efficacy of an orally active, small-molecule ALK5/ALK4 inhibitor in a vascular fibrosis model and suggest the potential therapeutic application of these inhibitors in vascular fibrosis.
Key Words: TGF beta • restenosis • fibrosis • neointimal formation • activin
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
TGF-β promotes key cellular events in vascular fibrosis including the induction of adventitial myofibroblasts and activated smooth muscle cells as well as their proliferation, survival, contraction, and increased collagen deposition.1 TGF-β is overexpressed in animal models of vascular fibrosis as well as human diseases such as restenosis, cardiac hypertrophy, primary pulmonary hypertension, and organ transplant vasculopathy.1–7 Activin, another TGF-β superfamily member, is also upregulated during vascular remodeling after balloon injury of the rat carotid artery.8,9 The association of overexpression of these 2 cytokines with inflammation and fibrotic responses to injury suggest that inhibition of either or both of these TGF-β superfamily pathways may prevent vascular fibrosis.1–10
TGF-β and activin signal through binding to their respective type II receptors, TGF-βRII and ActRII. The subsequent interaction of this complex with their respective type I receptors, ALK5 and ALK4, allows the interaction with and phosphorylation of type I receptor-associated signaling proteins, Smad2 and Smad3.11,12 Blockade of TGF-β signaling with either the soluble TGF-β type II receptor extracellular domain-Fc fusion protein (sTGF-βRII:Fc),5,7 antibodies to TGF-β1,13 decorin,14 TGF-β antisense oligonucleotides,15–17 or adenovirus Smad718 was shown previously to inhibit vascular fibrosis in animal models. Here, we identified SM16, a potent, selective and orally active ALK5/ALK4 kinase inhibitor which potently inhibits TGF-β-induced phosphorylation of Smad2 and Smad3 in cells and neointimal thickening and vascular remodeling in the rat carotid balloon injury model. This orally active, small molecule TGF-β/activin signaling inhibitor prevents neointimal thickening and lumenal loss primarily through inhibition of myofibroblast induction and collagen production and provides a novel therapeutic option for treating vascular fibrotic disease.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Reagents
SM16 was synthesized at Biogen Idec and can be obtained by contacting the corresponding author. His-tagged human ALK5 kinase domain was expressed and purified as described previously.19,20 His-tagged ALK1 (residues 155 to 502), ALK4 (residues 165 to 505), and ALK6 (residue 165 to 501) kinase domains were generated according to the same protocols. Purified, active p38
/SAPK2a kinase was from Upstate (#14-251).
Competitive Displacement Assays
Competitive displacement of 3H-HTS446284, 4-(3-pyridin-2-yl-1H-pyrazol-4-yl)-quinoline,20 was assayed by incubation of varying concentrations of SM16 with 50 nmol/L 3H- HTS446284, nmol/L ALK5 kinase, 50 mmol/L HEPES, pH 7.5, 60 mmol/L NaCl, 1 mmol/L MgCl2, 5 mmol/L MnCl2, 2 mmol/L dithiothreitol (DTT) and 2% Brij-35 (1 h, RT) in a nickel-chelate Flashplate Plus (PerkinElmer #SMP107) and was measured using a TopCount scintillation counter (Perkin Elmer). EC50 values for SM16 were determined by nonlinear regression with the bottom of the curve set to 0 and the Ki was calculated from the Cheng-Prusoff equation.
Biacore Analysis
Analysis of binding kinetics was carried out on a Biacore S51 instrument (http://www.biacore.com/lifesciences). ALK5 was immobilized on a Biacore CM5 chip by anti-His antibody capture, and various concentrations (0.75 to 50 nmol/L) of SM16 were flowed over the chip. The rate constants ka and kd were determined by globally fitting 6 concentration curves to the equation for a single-site binding model. The affinity constant was calculated as KD=kd/ka.
Cellular PAI-Luc Assay
HepG2 cells carrying a TGF-β–inducible PAI-1 promoter-driven luciferase reporter were grown in 96-well Wallac Isoplates. Cells were stimulated overnight with 2.5 ng/mL human TGF-β1 (R&D Systems) in DMEM/0.5% FBS containing varying concentrations of SM16 (final DMSO concentration 1%). The LucLite Reporter Gene Assay (Perkin-Elmer) and a Packard Top Count NXT were used to measure luminescence.
Smad Phosphorylation
Confluent HepG2 cells were incubated in DMEM/0.5%FBS containing SM16 and stimulated with 10 ng/mL TGF-β1 (1.5 h, 37°C) or 100 ng/mL Activin A (45 min, 37°C). Lysates were analyzed by 10% SDS-PAGE and Western blotting. Block: 5% milk/PBS/0.1% Tween-20. Primary antibodies: anti-Smad2/3 antibody (Cell Signaling #3102); anti–Phospho-Smad2/3 (Cell Signaling #3101), both 1:2000 in 5% milk (O/N, 4°). Secondary: HRP-linked goat antirabbit (Bio-Rad), 1:4000 (1 h, RT).
Kinase Selectivity Assays
Kinase selectivity assays were performed using Upstate KinaseProfiler at 10 µmol/L SM16 and with ATP concentrations at the Km for each kinase. ALK1 and ALK6 kinase assays were performed in Flashplates by incubating 40 nmol/L ALK1 or ALK6 in 50 µL of 50 mmol/L HEPES, pH 7.5, 60 mmol/L NaCl, 1 mmol/L MgCl2, 5 mmol/L MnCl2, 2 mmol/L DTT, 2.2% glycerol, 0.015% Brij-35, 1 µmol/L ATP, containing 0.5 µCi 33P-ATP, varying concentrations of SM16, and 1% DMSO (30 min, RT). ALK4 conditions were as above, but with 20 nmol/L his-tagged ALK4-K for 10 min. A p38/SAPK2a kinase inhibition assay used a DELFIA format in myelin basic protein (MBP) coated microplates (Upstate #30-011).
Rat Carotid Balloon Injury Model
Male Sprague-Dawley rats (300 to 400 g, Charles River Labs, Wilmington, MA) were anesthetized by i.p. injection with 7 to 10 mg/kg xylazine (AnaSed, Lloyd laboratories) and 70 to 90 mg/kg ketamine (Ketalar, Parke-Davis). The common carotid artery was denuded with a balloon catheter as previously described.5 Buprenorphine, 0.01 to 0.05 mg/kg, was given subcutaneously for postoperative analgesia. SM16 in 10% Captisol (CyDex Inc) was administered by oral gavage 30 min before surgery and daily thereafter until sacrifice at 14 days. Tissues were perfusion-fixed at physiological pressure with 4% paraformaldehyde in PBS, the carotid arteries excised fixed in neutral-buffered formalin. All animal study protocols were approved by the Biogen Idec animal use committee and Institutional Animal Care and Use Committee guidelines were observed.
Tissue Staining, Immunohistochemistry, and Morphometric Analysis
Morphometric analysis for intimal thickening was performed using Image-Pro, version 4.0 software (Media Cybernetics) on carotid artery crossections after modified Verhoeff staining of the elastic lamina.21 Ratios of areas were calculated, and ANOVA with a subsequent Dunnett Multiple Comparison test was used to determine significance (P<0.05). Masson trichrome staining for collagen and extracellular matrix. Alpha-SMA staining used a biotinylated antihuman –SMA (clone 1A4, DAKO #M0851), diluted 1:50 in Biocare DaVinci diluent, followed by streptavidin-HRP and diaminobenzidene (DAB). Ki67 was immunostained using a polyclonal rabbit antihuman Ki-67 (Novocastra Laboratories), (1:1000, 60 min), followed by biotinylated goat antirabbit IgG (1:200, for 30 min), avidin/biotin-HRP, and DAB substrate (all Vector Laboratories) and counterstained with Mayer Hematoxylin (Sigma). Staining was quantitated with MetaMorph v6.0 software using a brown (–SMA, Ki67) or blue (collagen) color threshold with density of –SMA, Ki67 or collagen measured as the area above the threshold versus total area for each region. Error bars represent standard error of the mean where n=3 or 4 (Vehicle: Day 2 to 7 and 14; SM16: Day 2 to 7 for neointimal collagen and day 2 and 4 for all others) or the range of values for n<3 (Vehicle: Day 10; SM16: day 10 and 14 for neointimal collagen and day 7 to 14 for all others).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
SM16 Binds in ALK5 in the ATP Site and Is a Potent Inhibitor of Cellular TGF-β Signaling
SM16 (4-(5-(benzo[d][1,3]dioxol-5-yl)-4-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)bicyclo[2.2.2]octane-1-carboxamide),a 430.5 MW ALK5/ALK4 kinase inhibitor (Figure 1A), was optimized for potency, selectivity and in vivo activity.22 SM16 occupies the ATP binding site of ALK5 kinase and competitively displaced a previously identified potent, ATP-competitive ALK5 inhibitor HTS46628420 with a Ki of 44 nmol/L (range=38 to 49 nmol/L) (supplemental Figure I, available online at http://atvb.ahajournals.org). Biacore analysis confirmed binding of SM16 to the ALK5 kinase domain and showed association (ka=9.2x105±3.8mol/L–1s–1) and dissociation constants (kd=5.8x10–3±1.7 s–1) which indicate a rapid, but reversible association of SM16 with ALK5 kinase. The affinity constant (KD=kd/ka was 6.6 nmol/L±1.6 nmol/L) was in good agreement with the Ki determined by displacement of HTS466284 (supplemental Figure II). Kinetic curves derived from these experiments are consistent with a single binding site for SM16 and also with the X-ray cocrystal structure of SM16 bound to the ATP binding site of the ALK5 kinase domain (data not shown).
|
In addition to potent binding to ALK5 kinase, SM16 inhibited TGF-β signaling in cells. SM16 inhibited TGF-β1-induced Smad2/3 phosphorylation in a dose-dependent manner (IC50 between 160 and 620 nmol/L) with complete inhibition between 620 nmol/L and 2.5 µmol/L. (Figure 1B). Also, inhibition of TGF-β-induced PAI-luciferase reporter activity was dose-dependent (IC50=64 nmol/L) (supplemental Figure III).
SM16 Is Selective Against Other Kinases
Because considerable homology exists among the ATP-binding sites of various kinases, SM16 was tested against a panel of 60 kinases at 10 µmol/L. Supplemental Table I shows the selectivity of SM16 against 25 kinases, including those for which SM16 showed the greatest crossreactivity. SM16 showed weak inhibitory activity against p38/SAPK2a and Raf with IC50 of 0.8 µmol/L and 1 µmol/L, respectively (Table). However, all ALK5 kinase inhibitors to date have shown no selectivity against ALK4, a highly homologous type I receptor family member.23 Similarly, SM16 exhibited potent ALK4 and ALK5 activity either by competitive displacement or autophosphorylation assays and inhibited activin-induced Smad2/3 phosphorylation in cells in a dose-dependent manner (Figure 1C and supplemental Table I). However, SM16 demonstrated selectivity against other members of the TGF-β type I receptor superfamily such as ALK1 and ALK6 (supplemental Table I).
|
SM16 Prevents Intimal Thickening and Vascular Remodeling in the Rat Carotid Balloon Injury Model
The significant potency and selectivity of SM16 for ALK4 and ALK5 suggested that this small molecule kinase inhibitor might be capable of inhibiting the fibrotic hyperplastic vascular response in the rat carotid balloon injury model. Rats that underwent unilateral balloon injury of the right carotid artery were administered a single daily oral dose of SM16 (0, 15, or 30 mg/kg) beginning on the day of injury and continuing for 14 days. Arterial cross-sections from vehicle- and SM16-treated injured rats on day 14 show that SM16 treatment caused a statistically significant inhibition of neointimal formation and lumenal narrowing with both 15 and 30 mg/kg SM16 treatments. There was a 44% reduction of the intimal-to-medial ratio (I/M ratio) in the 15 mg/kg SM16-treated group compared to the vehicle-treated group (Figure 2A and 2B). The I/M ratio was reduced by 78% in the 30 mg/kg SM16-treated group compared to the vehicle-treated group. In addition, the cross-sectional area of the artery lumen was increased by 37% and 49% at the 15 mg/kg and 30 mg/kg doses, respectively (Figure 2A and 2C). The rats tolerated SM16 treatment well with no changes in weight gain at either the 15 and 30 mg/kg doses (data not shown). These results show that SM16 is a potent inhibitor of vascular fibrosis in response to balloon catheter injury.
|
SM16 Inhibits Early Adventitial Myofibroblast Formation That Precedes Intimal Thickening
To determine the mechanism by which SM16 inhibits intimal thickening and vascular remodeling resulting in decreased lumenal narrowing, the events occurring after vascular injury were examined over the time course of 14 days at 2, 4, 7, 10, and 14 days postinjury. The vehicle-treated group showed a progressive increase in intimal thickening whereas once daily oral dosing with 25 mg/kg SM16 significantly suppressed this increase in intimal area throughout the 14-day period (Figures 3 and 4
A).
|
|
indicates vehicle-treated; X, SM16-treated.
Activation of myofibroblasts in the adventitia is one of the early responses to balloon injury and can contribute to the growth of the neointima.7,24 Immunohistochemical staining for -smooth muscle actin, a marker of myofibroblasts showed a distinct layer of -SMA-positive cells at the periphery of the adventitia in the vehicle-treated rat carotid sections which was apparent by day 2 postinjury, peaked at day 4, and returned to nearly baseline by day 14 (Figure 3A, arrows). Treatment with 25 mg/kg SM16 completely abolished these -SMA–positive adventitial myofibroblasts throughout the 14-day period (Figures 3A and 4
B). Collagen production was also evident in the neointima and adventitia after injury in vehicle-treated rats7 (Figure 3B). Treatment with SM16 appears to reduce the density of collagen staining in the neointima at 10 and 14 days (Figures 3C and 4C). The ring of adventitial collagen induced after injury contributes to vessel contraction and luminal loss at 14 days7 (Figure 3B, arrows).
Proliferation of myofibroblasts and vascular smooth muscle cells also contributes to neointimal thickening.2 There was a notable increase in the total number of neointimal proliferating cells which peaked at day 7 in the intima (Figure 4D), but not the media or adventitia (data not shown). However, the number of proliferating cells per neointimal area was unaffected by SM16-treatment (Figure 4D). Also, no difference was seen in the number of apoptotic cells per area (caspase-3 staining) in any of the vascular tissue layers between SM16- or vehicle-treated injured rat carotid arteries (data not shown). Together these results suggest that SM16 prevents neointimal thickening and vascular remodeling by inhibiting adventitial myofibroblast activation and collagen production without affecting proliferation.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
These results are the first to demonstrate inhibition of vascular fibrosis by an orally available ALK5/ALK4 kinase inhibitor. The ability of SM16 to prevent adventitial myofibroblast induction, collagen production, and neointimal thickening without significant effect on proliferation is similar to that seen with the soluble TGF-βRII:Fc.5,7 These results and those obtained with anti-TGF-β1 ribozymes15,16 and anti-TGF-β1 antibodies13 highlight the key role played by TGF-β in vascular fibrosis.
SM16 treatment has a significant effect on the early adventitial myofibroblast induction response to balloon catheter injury. This effect is consistent with the likelihood that early upregulation of adventitial TGF-β seen previously in this model7 drives myofibroblast induction. Additionally, the collagen staining present in control-treated rats was ameliorated by SM16 treatment. There was no effect of SM16 on the number of neointimal cells per tissue area undergoing proliferation, although the overall number of neointimal cells and area are significantly reduced, similar to the findings with soluble TGF-βRII:Fc.5 Thus, the efficacy of SM16 may be attributable to its early effects on inhibition of myofibroblast induction which may significantly decrease the overall number of neointimal cells available and decrease overall collagen production. Alternatively, direct inhibition of collagen production by the few myofibroblasts or activated smooth muscle cells present in SM16 treated rat carotid arteries may also contribute to decreased collagen content in the drug-treated rat carotid artery.
The specific effect of SM16 on the early induction of adventitial myofibroblasts may be attributable either to decreased induction of existing stromal adventitial cells or decreased trafficking of nearby or circulating myofibroblast precursors to the site of injury. Indeed, recent data suggests that TGF-β promotes circulating mesenchymal progenitor cell migration to sites of tissue injury as well as their differentiation to myofibroblasts contributing to scar formation.25,26
Although the effects of SM16 are consistent with the effect of other TGF-β antagonists such as soluble TGF-βRII:Fc,5,7 the decrease in inward remodeling shown previous with the soluble TGF-βRII:Fc was not evident in our model of rat carotid injury. We found no statistically significant difference in the circumference of the IEL or EEL of the injured vessel either between vehicle and SM16-treated groups or between vehicle and (soluble mouse) TGF-βRII:Fc–treated groups (data not shown). It is unclear whether these differences with previous findings are attributable to differences in the model or because the TGF-βRII:Fc used in these studies consisted of the mouse TGF-βRII extracellular region whereas the TGF-βRII:Fc used in the previous studies contained the rabbit receptor extracellular region.5,7
The similar effects of SM16 treatment, soluble mouse TGF-βRII:Fc, and other TGF-β antagonists on responses to balloon carotid injury suggest these effects may be accounted for by the potent inhibition by SM16 of ALK5. However, the ability of SM16 to inhibit activin signaling through ALK4 could also contribute to its efficacy in this model. Activin was shown to be elevated in balloon injured carotid arteries and demonstrated to have profibrotic activities which are similar to and synergistic with those of TGF-β in other models of tissue fibrosis.9–11,27–33 SM16 may at high concentrations also inhibit p38 and Raf, 2 signaling pathways also implicated in neointimal formation in the rat carotid injury model.1,34,35 However, the primary effect of SM16 is likely TGF-β inhibition because the Ras-Raf-extracellular signal regulated kinase (ERK) pathway is associated with proliferation and no significant inhibition of proliferation was noted with either SM16 treatment or with soluble TGF-βRII:Fc.5 The likelihood that ALK5/ALK4 inhibition rather than p38 inhibition is the primary mechanism for blockade of myofibroblast induction and fibrosis by ALK5/ALK4 inhibitors was also demonstrated in the bleomycin-induced lung injury model.36 Interestingly, partial TGF-β pathway inhibition in Smad3-null mice enhanced SMC proliferation and intimal thickening in a mouse femoral artery injury model.37 Although the biological response in the mouse femoral model differs somewhat from that of the rat in carotid injury, it may be that inhibition of the entire TGF-β signaling pathway via TGF-β ligand or receptor activity is preferred for preventing vascular fibrosis.
The efficacy of ALK5/ALK4 inhibitors in decreasing fibrosis and myofibroblast induction was recently demonstrated in animal models of lung,38–40 renal,41–43 liver,44 and skin45 fibrosis. The data presented here suggest that ALK5/ALK4 kinase inhibitors like SM16 may provide therapeutic utility also for vascular fibrosis. Although potential adverse effects of significant chronic TGF-β blockade have been demonstrated in animal models of atherosclerosis and suggested in human atherosclerosis,46 acute or subacute inhibition of TGF-β to prevent vascular fibrosis after angioplasty, stent placement, or after transplant rejection in the presence of immunosuppresants, may provide a favorable risk/benefit outcome. If so, small molecule therapeutics are likely to provide more flexible, controlled, and convenient routes of administration as well as more cost-effective treatment options than protein, viral, or antisense-based TGF-β antagonists.
|
Acknowledgments |
|---|
We thank Adrian Whitty for helpful discussions in enzyme kinetics and Thomas Crowell and Linda Biagini for helpful discussions in and management of histology.
Sources of Funding
This work was funded entirely by Biogen Idec.
Disclosures
Biogen Idec is not currently developing SM16 for clinical use. All authors are or were employees of Biogen Idec. Some authors own shares of Biogen Idec stock.
|
Footnotes |
|---|
Original received November 5, 2007; final version accepted January 7, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Ruiz-Ortega M, Rodriguez-Vita J, Sanchez-Lopez E, Carvajal G, Egido J. TGF-beta signaling in vascular fibrosis. Cardiovasc Res. 2007; 74: 196–206.
2. Khan A, Agrotis A, Bobik A. Understanding the role of transforming growth factor-beta1 in intimal thickening after vascular injury. Cardiovasc Res. 2007; 74 (2): 223–34.
3. Ozdemir BH, Ozdemir FN, Demirhan B, Haberal M. TGF-beta1 expression in renal allograft rejection and cyclosporine A toxicity. Transplantation. 2005; 80: 1681–1652.[CrossRef][Medline] [Order article via Infotrieve]
4. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor beta 1 during repair of arterial injury. J Clin Invest. 1991; 88: 904–910.[Medline] [Order article via Infotrieve]
5. Smith JD, Bryant SR, Couper LL, Vary CP, Gotwals PJ, Koteliansky VE, Lindner V. Soluble transforming growth factor-beta type II receptor inhibits negative remodeling, fibroblast transdifferentiation, and intimal lesion formation but not endothelial growth. Circ Res. 1999; 84: 1212–1222.
6. Chamberlain J, Gunn J, Francis SE, Holt CM, Arnold ND, Cumberland DC, Ferguson MW, Crossman DC. TGFbeta is active, and correlates with activators of TGFbeta, following porcine coronary angioplasty. Cardiovasc Res. 2001; 50: 125–136.
7. Ryan ST, Koteliansky VE, Gotwals PJ, Lindner V. Transforming growth factor-beta-dependent events in vascular remodeling following arterial injury. J Vasc Res. 2003; 40: 37–46.[CrossRef][Medline] [Order article via Infotrieve]
8. Pawlowski JE, Taylor DS, Valentine M, Hail ME, Ferrer P, Kowala MC, Molloy CJ. Stimulation of activin A expression in rat aortic smooth muscle cells by thrombin and angiotensin II correlates with neointimal formation in vivo. J Clin Invest. 1997; 100: 639–648.[Medline] [Order article via Infotrieve]
9. Molloy CJ, Taylor DS, Pawlowski JE. Novel cardiovascular actions of the activins. J Endocrinol. 1999; 161: 179–185.[Abstract]
10. Werner S, Alzheimer C. Roles of activin in tissue repair, fibrosis, and inflammatory disease. Cytokine Growth Factor Rev. 2006; 17: 157–171.[CrossRef][Medline] [Order article via Infotrieve]
11. Feng XH, Derynck R. Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol. 2005; 21: 659–693.[CrossRef][Medline] [Order article via Infotrieve]
12. Abe Y, Minegishi T, Leung PC. Activin receptor signaling. Growth Factors. 2004; 22: 105–110.[CrossRef][Medline] [Order article via Infotrieve]
13. Wolf YG, Rasmussen LM, Ruoslahti E. Antibodies against transforming growth factor-beta 1 suppress intimal hyperplasia in a rat model. J Clin Invest. 1994; 93: 1172–1178.[Medline] [Order article via Infotrieve]
14. Fischer JW, Kinsella MG, Clowes MM, Lara S, Clowes AW, Wight TN. Local expression of bovine decorin by cell-mediated gene transfer reduces neointimal formation after balloon injury in rats. Circ Res. 2000; 86: 676–683.
15. Merrilees M, Beaumont B, Scott L, Hermanutz V, Fennessy P. Effect of TGF-beta(1) antisense S-oligonucleotide on synthesis and accumulation of matrix proteoglycans in balloon catheter-injured neointima of rabbit carotid arteries. J Vasc Res. 2000; 37: 50–60.[CrossRef][Medline] [Order article via Infotrieve]
16. Wolff RA, Ryomoto M, Stark VE, Malinowski R, Tomas JJ, Stinauer MA, Hullett DA, Hoch JR. Antisense to transforming growth factor-beta1 messenger RNA reduces vein graft intimal hyperplasia and monocyte chemotactic protein 1. J Vasc Surg. 2005; 41: 498–508.[CrossRef][Medline] [Order article via Infotrieve]
17. Ando H, Fukuda N, Kotani M, Yokoyama S, Kunimoto S, Matsumoto K, Saito S, Kanmatsuse K, Mugishima H. Chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor-beta 1 mRNA inhibits neointima formation in rat carotid artery after balloon injury. Eur J Pharmacol. 2004; 483: 207–214.[CrossRef][Medline] [Order article via Infotrieve]
18. Mallawaarachchi CM, Weissberg PL, Siow RC. Smad7 gene transfer attenuates adventitial cell migration and vascular remodeling after balloon injury. Arterioscler Thromb Vasc Biol. 2005; 25: 1383–1387.
19. Huse M, Chen YG, Massague J, Kuriyan J. Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12. Cell. 1999; 96: 425–436.[CrossRef][Medline] [Order article via Infotrieve]
20. Singh J, Chuaqui CE, Boriack-Sjodin PA, Lee WC, Pontz T, Corbley MJ, Cheung HK, Arduini RM, Mead JN, Newman MN, Papadatos JL, Bowes S, Josiah S, Ling LE. Successful shape-based virtual screening: the discovery of a potent inhibitor of the type I TGFbeta receptor kinase (TbetaRI). Bioorg Med Chem Lett. 2003; 13: 4355–4359.[CrossRef][Medline] [Order article via Infotrieve]
21. Verhoeff FH. Some new staining methods of wide applicability. Including a rapid differential stain for elastic tissue. JAMA. 1908; 50: 876–877.
22. Ling LE, Chuaqui C, Boriack-Sjodin PA, Lepage D, Silverio E, Corbley MJ, Sun L, Papadatos J, Shan F, Pontz T, Cheung H-K, Zhang X, Arduini R, Mead J, Newman M, Bowes S, Josiah S, Lee WC, Singh J. "The Use of Virtual Screening in ALK5 Kinase Inhibitor Discovery and Validation of Orally Active ALK5 Kinase Inhibitors in Oncology". Transforming Growth Factor-Beta in Cancer Therapy, S.Jakowlew, ed. 2006. The Humana Press. Vol. 2: "Cancer Treatment and Therapy", Chapter 41.
23. Carcamo J, Weis FM, Ventura F, Wieser R, Wrana JL, Attisano L, Massague J. Type I receptors specify growth-inhibitory and transcriptional responses to transforming growth factor beta and activin. Mol Cell Biol. 1994; 14: 3810–3821.
24. Sartore S, Chiavegato A, Faggin E, Franch R, Puato M, Ausoni S, Pauletto P. Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant. Circ Res. 2001; 89: 1111–1121.
25. Iwata H, Sata M. Potential contribution of bone marrow-derived precursors to vascular repair and lesion formation: lessons from animal models of vascular disease. Frontiers in Bioscience. 2007; 12: 4157–4167.[CrossRef][Medline] [Order article via Infotrieve]
26. Wu GD, Bowdish ME, Jin YS, Zhu H, Mitsuhashi N, Barsky LW, Barr ML. Contribution of mesenchymal progenitor cells to tissue repair in rat cardiac allografts undergoing chronic rejection. J Heart Lung Transplant. 2005; 24: 2160–2169.[CrossRef][Medline] [Order article via Infotrieve]
27. You L, Kruse FE. Differential effect of activin A and BMP-7 on myofibroblast differentiation and the role of the Smad signaling pathway. Invest Ophthalmol Vis Sci. 2002; 43: 72–81.
28. Wada W, Kuwano H, Hasegawa Y, Kojima I. The dependence of transforming growth factor-beta-induced collagen production on autocrine factor activin A in hepatic stellate cells. Endocrinology. 2004; 145: 2753–2759.
29. Gaedeke J, Boehler T, Budde K, Neumayer HH, Peters H. Glomerular activin A overexpression is linked to fibrosis in anti-Thy1 glomerulonephritis. Nephrol Dial Transplant. 2005; 20: 319–328.
30. Aoki F, Kurabayashi M, Hasegawa Y, Kojima I. Attenuation of Bleomycin-induced pulmonary fibrosis by follistatin. Am J Respir Crit Care Med.. 2005; 172: 713–720.
31. Sugiyama M, Ichida T, Sato T, Ishikawa T, Matsuda Y, Asakura H. Expression of activin A is increased in cirrhotic and fibrotic rat livers. Gastroenterology. 1998; 114: 550–558.[CrossRef][Medline] [Order article via Infotrieve]
32. Yamashita S, Maeshima A, Kojima I, Nojima Y. Activin A is a potent activator of renal interstitial fibroblasts. J Am Soc Nephrol. 2004; 15: 91–101.
33. Matsuse T, Ikegami A, Ohga E, Hosoi T, Oka T, Kida K, Fukayama M, Inoue S, Nagase T, Ouchi Y, Fukuchi Y. Expression of immunoreactive activin A protein in remodeling lesions associated with interstitial pulmonary fibrosis. Am J Pathol. 1996; 148: 707–713.[Abstract]
34. Ohashi N, Matsumori A, Furukawa Y, Ono K, Okada M, Iwasaki A, Miyamoto T, Nakano A, Sasayama S. Role of p38 mitogen-activated protein kinase in neointimal hyperplasia after vascular injury. Arterioscler Thromb Vasc Biol. 2000; 20: 2521–2526.
35. Ju H, Nerurkar S, Sauermelch CF, Olzinski AR, Mirabile R, Zimmerman D, Lee JC, Adams J, Sisko J, Berova M, Willette RN. Sustained activation of p38 mitogen-activated protein kinase contributes to the vascular response to injury. J Pharmacol Exp Ther. 2002; 301: 15–20.
36. Kapoun AM, Gaspar NJ, Wang Y, Damm D, Liu YW, O’Young G, Quon D, Lam A, Munson K, Tran TT, Ma JY, Murphy A, Dugar S, Chakravarty S, Protter AA, Wen FQ, Liu X, Rennard SI, Higgins LS. TGF-βR1 kinase activity, but not p38 activation is required for TGF-βR1-induced myofibroblast differentiation and pro-fibrotic gene expression. Mol Pharmacol. 2006; 70: 518–31.
37. Kobayashi K, Yokote K, Fujimoto M, Yamashita K, Sakamoto A, Kitahara M, Kawamura H, Maezawa Y, Asaumi S, Tokuhisa T, Mori S, Saito Y. Targeted disruption of TGF-beta-Smad3 signaling leads to enhanced neointimal hyperplasia with diminished matrix deposition in response to vascular injury. Circ Res. 2005; 96: 904–912.
38. Bonniaud P, Margetts PJ, Kolb M, Schroeder JA, Kapoun AM, Damm D, Murphy A, Chakravarty S, Dugar S, Higgins L, Protter AA, Gauldie J. Progressive transforming growth factor beta1-induced lung fibrosis is blocked by an orally active ALK5 kinase inhibitor. Am J Respir Crit Care Med. 2005; 171: 889–898.
39. Leung SY, Niimi A, Noble A, Oates T, Williams AS, Medicherla S, Protter AA, Chung KF. Effect of transforming growth factor-beta receptor I kinase inhibitor 2,4-disubstituted pteridine (SD-208) in chronic allergic airway inflammation and remodeling. J Pharmacol Exp Ther. 2006; 319: 586–594.
40. Higashiyama H, Yoshimoto D, Kaise T, Matsubara S, Fujiwara M, Kikkawa H, Asano S, Kinoshita M. Inhibition of activin receptor-like kinase 5 attenuates Bleomycin-induced pulmonary fibrosis. Exp Mol Pathol. 2007; 83: 39–46.[CrossRef][Medline] [Order article via Infotrieve]
41. Grygielko ET, Martin WM, Tweed C, Thornton P, Harling J, Brooks DP, Laping NJ. Inhibition of gene markers of fibrosis with a novel inhibitor of transforming growth factor-beta type I receptor kinase in puromycin-induced nephritis. J Pharmacol Exp Ther. 2005; 313: 943–951.
42. Gellibert F, de Gouville AC, Woolven J, Mathews N, Nguyen VL, Bertho-Ruault C, Patikis A, Grygielko ET, Laping NJ, Huet S. Discovery of 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro- 2H- pyran-4-yl)benzamide (GW788388): a potent, selective, and orally active transforming growth factor-beta type I receptor inhibitor. J Med Chem. 2006; 49: 2210–2221.[CrossRef][Medline] [Order article via Infotrieve]
43. Moon JA, Kim HT, Cho IS, Sheen YY, Kim DK. IN-1130, a novel transforming growth factor-beta type I receptor kinase (ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy. Kidney Int. 2006; 70: 1234–1243.[CrossRef][Medline] [Order article via Infotrieve]
44. de Gouville AC, Boullay V, Krysa G, Pilot J, Brusq JM, Loriolle F, Gauthier JM, Papworth SA, Laroze A, Gellibert F, Huet S. Inhibition of TGF-beta signaling by an ALK5 inhibitor protects rats from dimethylnitrosamine-induced liver fibrosis. Br J Pharmacol. 2005; 145: 166–177.[CrossRef][Medline] [Order article via Infotrieve]
45. Ishida W, Mori Y, Lakos G, Sun L, Shan F, Bowes S, Josiah S, Lee WC, Singh J, Ling LE, Varga J. Intracellular TGF-beta receptor blockade abrogates Smad-dependent fibroblast activation in vitro and in vivo. J Invest Dermatol. 2006; 126: 1733–1744.[CrossRef][Medline] [Order article via Infotrieve]
46. Bobik A. Transforming Growth Factor-b’s and Vascular Disorders. Arterioscler Thromb Vasc Biol. 2006; 26: 1712–1720.
This article has been cited by other articles:
 |
|
 |
|
  C. Gerhardinger, Z. Dagher, P. Sebastiani, Y. S. Park, and M. Lorenzi The Transforming Growth Factor-{beta} Pathway Is a Common Target of Drugs That Prevent Experimental Diabetic Retinopathy Diabetes, July 1, 2009; 58(7): 1659 - 1667. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Hirschberg, T. Radovits, S. Loganathan, L. Entz, C. J. Beller, M.-L. Gross, P. Sandner, M. Karck, and G. Szabo Selective phosphodiesterase-5 inhibition reduces neointimal hyperplasia in rat carotid arteries after surgical endarterectomy. J. Thorac. Cardiovasc. Surg., June 1, 2009; 137(6): 1508 - 1514. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. RAUSCH, T. HAHN, L. RAMANATHAPURAM, D. BRADLEY-DUNLOP, D. MAHADEVAN, M. E. MERCADO-PIMENTEL, R. B. RUNYAN, D. G. BESSELSEN, X. ZHANG, H.-K. CHEUNG, et al. An Orally Active Small Molecule TGF-{beta} Receptor I Antagonist Inhibits the Growth of Metastatic Murine Breast Cancer Anticancer Res, June 1, 2009; 29(6): 2099 - 2109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Thomas, C. Docx, A. M. Holmes, S. Beach, N. Duggan, K. England, C. Leblanc, C. Lebret, F. Schindler, F. Raza, et al. Activin-Like Kinase 5 (ALK5) Mediates Abnormal Proliferation of Vascular Smooth Muscle Cells from Patients with Familial Pulmonary Arterial Hypertension and Is Involved in the Progression of Experimental Pulmonary Arterial Hypertension Induced by Monocrotaline Am. J. Pathol., February 1, 2009; 174(2): 380 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kim, G. Buchlis, Z. G. Fridlender, J. Sun, V. Kapoor, G. Cheng, A. Haas, H. K. Cheung, X. Zhang, M. Corbley, et al. Systemic Blockade of Transforming Growth Factor-{beta} Signaling Augments the Efficacy of Immunogene Therapy Cancer Res., December 15, 2008; 68(24): 10247 - 10256. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||