The mechanism involved in the inhibitory action of tranilast on collagen biosynthesis of keloid fibroblasts.

Tranilast, an anti-allergic drug inhibiting the release of substances such as histamine and prostaglandins from mast cells, was previously reported to suppress collagen synthesis of fibroblasts derived from keloid tissues. However, the inhibitory mechanism on collagen synthesis is unknown. We studied its inhibitory mechanism on collagen synthesis by culturing fibroblasts from keloid and hypertrophic scar tissues of humans. Collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue is greater than that from healthy human skin. Tranilast (3-100 microM) did not inhibit prolyl hydroxylase (the rate-limiting enzyme in collagen synthesis) activity. Tranilast (3-300 microM) suppressed the collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue but not healthy skin fibroblasts. Tranilast (30-300 microM) inhibited the release of transforming growth factor (TGF)-beta 1 from keloid fibroblasts, which enhances the collagen synthesis of keloid fibroblasts. Anti-TGF-beta 1 antibody (50 microliter/ml) inhibited the collagen synthesis, although diphenhydramine (10 microM) and indomethacin (10 microM) did not show any inhibition. These results suggest that tranilast inhibits collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue through suppressing the release of TGF-beta 1 from the fibroblasts themselves.     

Effect of tranilast, an anti-allergic drug, on the human keloid tissues]

We studied the inhibitory effects of tranilast, an anti-allergic drug, on the human keloid tissues implanted into the dorsal skin of athymic nude mice and on the growth of keloid fibroblast in vitro. In the keloid tissue-implanted model, tranilast (50-200 mg/kg, p.o.) decreased the weight of the keloid tissue as triamcinolone (25 mg/kg, p.o.) did. Tranilast (200 mg/kg, p.o.) reduced the hydroxyproline content of implanted tissues. Tranilast (3-300 microM) also inhibited the collagen synthesis by keloid fibroblast in vitro. Only a high concentration of tranilast (300 microM) suppressed the glycosaminoglycan synthesis and cell proliferation of keloid fibroblasts. Moreover, tranilast scarcely affected the fibronectin production. Triamcinolone (10 microM) also inhibited glycosaminoglycan synthesis and cell proliferation. These results suggest that the inhibitory effect of tranilast on the keloid tissues is related to its inhibition of the collagen synthesis of fibroblasts. Tranilast would be useful as a therapeutic drug for the treatment of keloids.     

Effect of tranilast, an anti-allergic drug, on carrageenin-induced granulation and capillary permeability in rats]

We studied the effect of tranilast on the growth of carrageenin-induced granulation and the increase in capillary permeability induced by inflammatory agents in rats. In the carrageenin-induced granulation model, tranilast (50 or 100-200 mg/kg, p.o.) decreased significantly and dose-dependently the weight and the hydroxyproline content of the granulation tissue. Tranilast, however, showed no effect on the healing day of locally wounded dorsal skin of rats. Triamcinolone (10 mg/kg, p.o.) also showed an inhibitory effect on the carrageenin-induced granulation model. Tranilast (50-400 mg/kg, p.o.) dose-dependently inhibited the enhancement of capillary permeability induced by the Ca ionophore A23187, bradykinin and xanthine oxidase. Moreover, tranilast (30 and 300 microM) suppressed superoxide production induced by FMLP in human neutrophils, but did not act as a superoxide scavenger. Considering that hypertrophic scar and keloid are conditions characterized by abnormal cell proliferation and excessive collagen accumulation accompanied with itch and pain, these results suggest that tranilast is useful as a therapeutic drug for hypertrophic scars and keloids.     

Inhibition by tranilast of nifedipine-induced proliferation of cultured human gingival fibroblasts

The appropriate method of etiologic therapy for gingival overgrowth is yet unknown. In this study drug-induced proliferation of Gin-1 cells, a normal human gingival fibroblast cell line, was examined by using the reagent water-soluble tetrazolium-1. Tranilast (100 microM) inhibited the nifedipine (10 microM)-induced proliferation of gingival fibroblasts. The level of basic fibroblast growth factor (bFGF) was determined by using an enzyme-linked immunosorbent assay kit. Tranilast inhibited the release of bFGF from the cells. In conclusion, tranilast depresses the nifedipine-induced proliferation of gingival fibroblasts by inhibiting the release of bFGF. Administration of tranilast may thus be clinically effective for the treatment of gingival overgrowth.     

Uncontrollable prurigo nodularis effectively treated by roxithromycin and tranilast

Treatment of prurigo nodularis (PN) is often very difficult even with strong corticosteroid dressing and other available means. Macrolide roxithromycin (RXM) is used in consideration of its immunosuppressive effects in treating several skin disorders. Tranilast (N-(3,4-dimethoxycinnamoyl) is useful for treating atopic disorders and hypertrophic scars as well, suggesting its capacity to inhibit fibroblast proliferation. More adequate and effective therapy for this disorder has been requested. We report 3 cases of uncontrollable PN treated with 300 mg/day roxithromycin and 200 mg/day tranilast. Complete and/or remarkable regression of PN was observed on treatment with roxithromycin and tranilast in combination within 4 to 6 months. The 2 agents in combination can be used effectively for the treatment of uncontrollable PN.     

p53基因在病理性瘢痕成纤维细胞中的表达

目的　探讨 p5 3基因在病理性瘢痕成纤维细胞中的表达情况及其意义。方法　取手术切除的正常皮肤、增生性瘢痕和瘢痕疙瘩组织各 8例为标本 ,通过细胞培养 6～ 8代后 ,应用反转录 -聚合酶链反应 (RT-PCR)的方法检测 p5 3m RNA的表达。结果　 p5 3m RNA在正常皮肤高表达 ,而在增生性瘢痕和瘢痕疙瘩组明显减弱 ,差异具有显著性 (P<0 .0 5 )。结论　 p5 3基因介导了瘢痕疙瘩成纤维细胞的异常增生 ,是病理性瘢痕发生的分子机制之一。     

The kallikrein-kininogen-kinin system in chronic inflammation

We examined bradykinin's effects on macrophages and fibroblasts, two cell types important in the pathogenesis of chronic inflammation. Bradykinin stimulated release of proteins of 18 kDa from macrophages. These proteins caused increased thymocyte proliferation (interleukin 1, IL-1) and completely inhibited lipoprotein lipase (tumor necrosis factor, TNF). When fibroblasts were incubated with bradykinin, PGE2 synthesis was stimulated. Pretreatment with IL-1 or TNF dramatically amplified bradykinin-stimulated PGE2 synthesis. Thus, bradykinin is involved in a positive feedback loop in which bradykinin activates macrophages to release potent inflammatory cytokines; these in turn amplify responsiveness of bradykinin target tissues.     

Tranilast inhibits transplant-associated coronary arteriosclerosis in a murine model of cardiac transplantation.

Accelerated coronary arteriosclerosis remains a major problem for the long-term survival of cardiac transplant recipients. However, the pathogenesis of graft vasculopathy is poorly understood and there is no effective therapy. Tranilast is a promising drug that may prevent post-angioplasty restenosis. Here, we investigated whether orally administered tranilast inhibits the development of intima hyperplasia in a mouse model of cardiac transplantation. Cardiac allografts from BALB/c mice were transplanted heterotopically into C3H/He mice. Mice were administered either vehicle or tranilast everyday by gavage. Morphometrical analysis of the cardiac allografts harvested at 2 months revealed that the administration of tranilast significantly reduced the development of coronary atherosclerosis. In the mice treated with tranilast, up-regulation of the cyclin-dependent kinase inhibitor p21 was observed in the allografts, accompanied by a reduced number of proliferating cells. Tranilast also suppressed transforming growth factor-beta (TGF-beta) expression. Tranilast may be effective in preventing transplant-associated arteriosclerosis through its anti-inflammatory and anti-proliferative effects.     

Effect of enoxaparin and onion extract on human skin fibroblast cell line – Therapeutic implications for the treatment of keloids

Context: Keloids and hypertrophic scars are hyperproliferative skin disorders resulting in abnormal wound healing. In the prevention and treatment of keloids and hypertrophic scars, ointments containing heparin and onion extract are very popular. Their therapeutic effects, however, are still controversial and the mechanism of action is not fully understood.

Objective: The aim of this study was to assess the effect of enoxaparin and dry onion extract on proliferation, apoptosis and β1 integrin expression in human fibroblasts.

Materials and methods: Fibroblast human cell lines (46 BR.1?N) were treated for 48?h with various concentrations of enoxaparin sodium (20, 100, 500?µg/mL) and/or onion [Allium cepa L. (Alliaceae)] extract (50, 250, 1000?µg/mL). The cell proliferation was evaluated by [³H]-thymidine incorporation assay. Furthermore, the expression of β1 integrin and apoptosis was determined by flow cytometry.

Results and discussion: The results demonstrate that enoxaparin and onion extract inhibited the proliferation of human fibroblasts. Almost complete inhibition of cell proliferation was achieved by enoxaparin in 500?µg/mL concentration (91.5% reduction). The onion extract at a concentration of 250?µg/mL also strongly inhibited the proliferation of cells (50.8% reduction). Depending on concentration, enoxaparin and onion extract induced apoptosis (500 and 1000?µg/mL, respectively) and, depending on concentration, downregulated the expression of β1 integrin on human fibroblasts.

Conclusion: This work points at possible mechanism of action of enoxaparin and onion extract, when administered in the treatment of patients with keloids and hypertrophic scars.     

Differential regulation of bradykinin receptor density, intracellular Ca2+, and prostanoid release in skin and foreskin fibroblasts. Effects of cell density and interleukin-1alpha.

1. Bradykinin (BK) receptors, cytosolic Ca2+, and prostanoids were studied in human skin and foreskin fibroblasts. 2. Bmax values of BK receptors were higher in foreskin than in skin fibroblasts, increasing with cell densities in both cell types. IL-1alpha-dependent receptor induction was blocked by cycloheximide. 3. BK-stimulated cytosolic Ca2+ elevation was higher in confluent than in non-confluent cultures and larger in foreskin than in skin fibroblasts. Responses were not enhanced after IL-1-alpha-induced up-regulation of BK receptors. 4. Intrinsic prostanoid production was higher in foreskin than in skin fibroblasts at comparable cell densities. In foreskin, but not in skin fibroblasts, BK stimulation increased the release of PGE2 10 fold and that of 6-oxo-PGF1alpha 6-7 fold. 5. Preincubation with IL-1alpha had a marked effect on prostanoid release in foreskin fibroblasts only. Subsequent BK stimulation increased the release of PGE2 and 6-oxo-PGF1alpha 7-10 fold in skin fibroblasts while this increase was only 30% in foreskin fibroblasts. Release of TXA2 reached values up to one third of the other prostanoids. The IL-1alpha induced rise in BK-stimulated PGE2 synthesis was fully abolished by specific inhibition of cyclo-oxygenase 2. 6. IL-1alpha sensitized BK-stimulated prostanoid synthesis and modulated prostanoid patterns differently in fibroblasts from skin and foreskin. The IL-1alpha effects on prostanoid release were not related to BK receptor numbers nor to the BK-stimulated Ca2+ signal but appear to be due to induction of prostanoid synthesizing enzymes. Foreskin fibroblasts seem to be unique and significantly different from fibroblasts of other skin locations in respect to their response to inflammation-associated kinins and cytokines.     

Effect of tranilast oily gel on carrageenin-induced granulation in rats

Tranilast (TL) oily gels consisting of hydrogenated soybean phospholipid and fatty-acid ester were prepared, and the inhibitory effect of the gels on the growth of granulation tissue were evaluated in a carrageenin-induced rat granulation model. By the application of 0.1 and 0.2% TL oily gel, the weight of granulation tissue was significantly reduced to 64 and 55%, respectively, of control value. Furthermore, these gels reduced their respective hydroxyproline content to 64 and 51% of the control. On the other hand, the inhibitory effect of 10% TL ointments, which are clinically used for the treatment of keloids and hypertrophic scars as hospital preparations, was much lower than that of the oily gels. In addition, the application of 0.1 and 0.2% oily gel led to high concentration (0.1% gel, 168+/-18 microg/g; 0.2% gel, 221+/-16 microg/g) of TL in the dermis as compared with the 10% TL ointments. These results suggest that TL oily gels may be a useful topical formulation for the treatment of keloids and hypertrophic scars.     

Botulinum Toxin A for the Treatment of Keloids

Introduction: Keloids are the result of excessive scar tissue formation. Besides their poor aesthetic appearance, keloids can be associated with severe clinical symptoms such as pain, itching, and rigidity. Unfortunately, most therapeutic approaches remain clinically unsatisfactory. Recently, injections with botulinum toxin A (BTA) were proposed for the treatment of established keloids in a clinical trial. In this study, we aimed to verify the effects of intralesional BTA for the treatment of therapy-resistant keloids using objective measurements. In addition, the underlying molecular mechanisms were investigated using cultured keloid-derived fibroblasts. Materials and Methods: Four patients received BTA (doses varying from 70 to 140 Speywood units per session) injected directly into their keloids every 2 months for up to 6 months. Differences in height and volume were evaluated clinically and measured with a 3-D optical profiling system. Keloid-derived fibroblasts were treated with different concentrations of BTA, and expression of collagen (COL)1A1, COL1A2, COL3A1, TGF-β1, TGF-β2, TGF-β3, fibronectin-1, laminin-β2, and α-SMA was determined by real-time quantitative PCR. MTT and BrdU assays were used to analyze the effects of BTA on fibroblast proliferation and metabolism. Results: Intralesional administration of BTA did not result in regression of keloid tissue. No differences in expression of ECM markers, collagen synthesis, or TGF-β could be observed after BTA treatment of keloid fibroblasts. In addition, cell proliferation and metabolism of keloid fibroblasts was not affected by BTA treatment. Conclusion: The suggested clinical efficiency of intralesional BTA for the therapy of existent keloids could not be confirmed in this study. Based on our data, the potential mechanisms of action of BTA on keloid-derived fibroblasts remain unclear.     

Tranilast inhibits protein kinase C-dependent signalling pathway linked to angiogenic activities and gene expression of retinal microcapillary endothelial cells.

1. Tranilast, first developed as an anti-allergic drug, has been reported to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis and vasopermeability. To further clarify the inhibitory mechanism, we investigated the effects of tranilast on VEGF binding and subsequent intracellular signalling pathway linked to angiogenic activities and gene expression of bovine retinal microcapillary endothelial cells. 2. Tranilast significantly (P<0.01) inhibited VEGF, basic fibroblast growth factor (bFGF), and hypoxia conditioned media-induced BREC proliferation in a dose dependent manner with IC50's of 22, 82 and 10 microM, respectively. 3. VEGF-induced migration was also inhibited by tranilast in a dose dependent manner, with IC50 of 18 microM, and complete inhibition was observed at 300 microM (P<0.01). Tranilast suppressed VEGF-induced tube formation in a dose dependent manner with maximum (46%) inhibition observed at 300 microM (P<0.05). 4. Tranilast inhibited phorbol myristate acetate (PMA)-dependent stimulation of [3H]-thymidine incorporation and VEGF- and PMA-induced gene expression of integrin alpha v and c-fos in BREC. 5. Tranilast suppressed VEGF- and PMA-stimulated PKC activity in BREC. 6. Tranilast did not affect VEGF binding or VEGF-induced phosphorylation of tyrosine residues of VEGF receptor- and phospholipase Cgamma and their associated proteins. 7. These data suggest that tranilast might prove an effective inhibitor to prevent retinal neovascularization in ischaemic retinal diseases, and that its inhibitory effect might be through suppression of PKC-dependent signal transduction in BREC.     

Study of the mechanism of inhibitory action of tranilast on chemical mediator release

We investigated the mechanism of inhibitory action of tranilast on chemical mediator release by antigen-antibody reactions. Tranilast (10(-5)-10(-3) M) inhibited antigen (DNP-Ascaris)-induced histamine release from sensitized purified rat mast cells (PMC), but did not show an obvious influence on intracellular cyclic AMP. 45Ca uptake into PMC induced by antigen (300 micrograms/ml) was obviously suppressed by tranilast (10(-6)-10(-3) M). Tranilast (10(-4) M) inhibited antigen-induced histamine release from and 45Ca uptake into PMC independently of the presence or absence of glucose in the medium. On the other hand, 2-deoxyglucose (10(-2) M) markedly inhibited both responses in the absence but not in the presence of glucose. Tranilast slightly inhibited Ca-induced contraction of guinea pig taenia coli, but had no influence on aggregation of rabbit platelets. Verapamil (10(-6)-10(-4) M) had no effect on antigen-induced histamine release, but it markedly suppressed Ca-induced contraction and platelet aggregation. From these results, we suggest that the mechanism of inhibitory action of tranilast on the release of antigen-induced chemical mediator from mast cells involves the suppression of Ca uptake, but that its mode of action is apparently different from those of 2-deoxyglucose and verapamil.     

Suppressive effect of tranilast on interleukin-5 prolonged eosinophils survival via apoptosis.

Tranilast has long been used clinically to treat allergic diseases such as bronchial asthma. To further clarify the antiinflammatory machanism, we examined the ability of tranilast to counteract the prolongation of eosinophil survival induced by interleukin (IL)-5. Tranilast reduced the IL-5 prolonged survival of eosinophils at the concentration range of 30 microg/ml to 100 microg/ml. The DNA extracted from eosinophils cultured with tranilast showed signs of fragmentation that was comparable with apoptosis. Electron-microscopic analysis of activated eosinophils cultured with 100 microg/ml of tranilast also revealed morphologic features of apoptosis. These data suggest that tranilast may act in vivo on activated eosinophils to reduce inflammation in allergic diseases.