Smad-3 as a mediator of the fibrotic response

Introduction Smad‐3, a key cytoplasmic mediator of transforming growth factor‐β (TGF‐β) signalling, mediates many of its inflammatory and fibrotic effects in vivo ( Roberts et al. 2001 ). Smad‐3 null mice are protected against cutaneous injury induced by ionizing irradiation ( Flanders et al. 2002 ). Here, we report on our continuing studies on radioprotection as well as protection against tubulointerstitial fibrosis following unilateral ureteral obstruction (UUO) in Smad‐3 null mice. Methods For radioprotection studies, the flank skin of Smad‐3^+/+ wild‐type (WT) and Smad‐3^–/– knockout (KO) mice was exposed to 30 Gy of localized γ‐irradiation and analysed for histology and gene expression at various times post irradiation. In the UUO model, the right proximal ureter of WT and KO mice was ligated, and 1–2 weeks later kidneys were analysed for inflammation, fibrosis and gene expression. Results Six weeks after exposure to irradiation, skin from KO mice shows less epidermal acanthosis and influx of mast cells, macrophages and neutrophils than skin of WT mice. Paradoxically, at 6–8 h post irradiation, KO skin shows a significantly greater number of neutrophils. Irradiated KO skin also exhibits less immunoreactive TGF‐β, fewer myofibroblasts and less scarring than does WT. Smad‐3 null dermal fibroblasts do not respond to the chemotactic effects of TGF‐β and show less induction of fibrogenic cytokines when treated with irradiation plus TGF‐β compared to WT cells. Following UUO, normal kidney architecture is preserved in KO mice, while kidneys from WT mice are enlarged with an influx of mononuclear cells and increased expression of collagen and TGF‐β1. Additionally, renal tubules in obstructed kidneys of KO mice remain positive for E‐cadherin without expression of α‐smooth muscle actin, while the opposite expression pattern is seen in obstructed kidneys of WT mice. TGF‐β treatment of primary cultures of WT renal tubular epithelial cells results in a phenotypic change from a cobblestone pattern to a spindle‐shaped fibroblastic appearance, while KO cells treated with TGF‐β maintain their original appearance. Conclusion Smad‐3 plays an important role in mediating pathogenic inflammation and fibrosis in several model systems and is also essential for TGF‐β1‐induced epithelial–mesenchymal transition in renal tubular epithelial cells. Inhibitors of the Smad‐3 pathway may have clinical applications in the treatment of a number of fibrotic conditions.     

Interference with transforming growth factor-beta/ Smad3 signaling results in accelerated healing of wounds in previously irradiated skin

Transforming growth factor (TGF)-beta regulates many aspects of wound repair including inflammation, chemotaxis, and deposition of extracellular matrix. We previously showed that epithelialization of incisional wounds is accelerated in mice null for Smad3, a key cytoplasmic mediator of TGF-beta signaling. Here, we investigated the effects of loss of Smad3 on healing of wounds in skin previously exposed to ionizing radiation, in which scarring fibrosis complicates healing. Cutaneous wounds made in Smad3-null mice 6 weeks after irradiation showed decreased wound widths, enhanced epithelialization, and reduced numbers of neutrophils and myofibroblasts compared to wounds in irradiated wild-type littermates. Differences in breaking strength of wild-type and Smad3-null wounds were not significant. As shown previously for neutrophils, chemotaxis of primary dermal fibroblasts to TGF-beta required Smad3, but differentiation of fibroblasts to myofibroblasts by TGF-beta was independent of Smad3. Previous irradiation-enhanced induction of connective tissue growth factor mRNA in wild-type, but not Smad3-null fibroblasts, suggested that this may contribute to the heightened scarring in irradiated wild-type skin as demonstrated by Picrosirius red staining. Overall, the data suggest that attenuation of Smad3 signaling might improve the healing of wounds in previously irradiated skin commensurate with an inhibition of fibrosis.     

Chronic ultraviolet B irradiation causes loss of hyaluronic acid from mouse dermis because of down-regulation of hyaluronic acid synthases

Remodeling of the dermal extracellular matrix occurs during photoaging. Here, the effect of repetitive UVB irradiation on dermal hyaluronic acid (HA) was examined. C57/BL6 mice were chronically (182 days) irradiated with UVB, and consecutive skin biopsies were collected during the irradiation period and afterward (300 and 400 days of age). UVB caused marked loss of HA from the papillary dermis and down-regulation of HA synthase 1 (HAS1), HAS2, and HAS3 mRNA expression. In contrast, hyaluronidases (HYAL) 1, HYAL2, and HA receptor CD44 were unchanged. Furthermore, transforming growth factor beta-1 (TGF-beta1) and TGF-beta1-receptor II expression were decreased in UVB-irradiated biopsies, and TGF-beta1 strongly induced HAS1 and HAS2 expression in cultured dermal fibroblasts. Therefore, TGF-beta1 might be one factor involved in UVB-induced down-regulation of HAS enzymes. In addition, total cell number and the percentage of proliferating fibroblasts in the papillary dermis of UVB-irradiated mice were decreased. Down-regulation of HAS2 by lentiviral overexpression of short hairpin RNA in vitro caused inhibition of HA synthesis, DNA synthesis, and migration of dermal fibroblasts. In conclusion, chronic UVB irradiation induces loss of HA from the dermis, thereby contributing to the quiescent phenotype of dermal fibroblasts.     

Transforming growth factor-beta-dependent and -independent pathways of induction of tubulointerstitial fibrosis in beta6(-/-) mice

Transforming growth factor-beta1 (TGF-beta1) and the renin-angiotensin-aldosterone system are key mediators in kidney fibrosis. Integrin alphavbeta6, a heterodimeric matrix receptor expressed in epithelia, binds and activates latent TGF-beta1. We used beta6 integrin-null mice (beta6(-/-)) to determine the role of local TGF-beta1 activation in renal fibrosis in the unilateral ureteral obstruction (UUO) model. Obstructed kidneys from beta6(-/-) mice showed less injury than obstructed kidneys from wild-type (WT) mice, associated with lower collagen I, collagen III, plasminogen activator inhibitor (PAI-1), and TGF-beta1 mRNA levels and lower collagen content. Infusion with either angiotensin II (Ang II) or aldosterone (Aldo) or combination in beta6(-/-) UUO mice significantly increased collagen contents to levels comparable to those in identically treated WT. Active TGF-beta protein expression in beta6(-/-) mice was less in UUO kidneys with or without Ang II infusion compared to matched WT mice. Activated Smad 2 levels in beta6(-/-) obstructed kidneys were lower than in WT UUO mice, and did not increase when fibrosis was induced in beta6(-/-) UUO mice by Ang II infusion. Anti-TGF-beta antibody only partially decreased this Ang II-stimulated fibrosis in beta6(-/-) UUO kidneys. In situ hybridization and immunostaining showed low expression of PAI-1 mRNA and protein in tubular epithelium in beta6(-/-) UUO kidneys, with increased PAI-1 expression in response to Ang II, Aldo, or both. Our results indicate that interruption of alphavbeta6-mediated activation of TGF-beta1 can protect against tubulointerstitial fibrosis. Further, the robust induction of tubulointerstitial fibrosis without increase in activated Smad 2 levels in obstructed beta6(-/-) mice by Ang II suggests the existence of a TGF-beta1-independent pathway of induction of fibrosis through angiotensin.     

Irradiation of the skin and systemic graft-versus-host disease synergize to produce cutaneous lesions.

In this report, the relationship between irradiation and graft-versus-host disease (GVHD)-induced cutaneous injury was investigated. Unirradiated F1 hybrid mice were grafted with irradiated skin and then injected with parental strain lymphoid cells to induce GVHD. Although low grade dermal lymphoid infiltrates were observed in unirradiated skin grafts of some GVH-reactive mice, and irradiated grafts of normal animals showed occasional fibrosis, only the irradiated grafts of GVH-reactive mice developed lesions consisting of vacuolar degeneration of the epidermal-dermal junction and necrotic keratinocytes accompanied by pronounced epidermal infiltrates, characteristic of clinical cutaneous GVHD. The results suggest that cutaneous irradiation exerts a permissive effect on lesion formation in the skin of mice undergoing GVHD. Furthermore, systemic irradiation, known to exacerbate the severity of GVHD, is not required. Cutaneous lesions may be triggered by radiation injury of keratinocytes, up-regulation of adhesion molecules on irradiated endothelium, destruction of protective radiosensitive intraepithelial lymphocytes, and radiation-induced priming of intradermal macrophages.     

Loss of Smad3 gives rise to poor soft callus formation and accelerates early fracture healing

Smad3 is an intracellular signaling molecule in the transforming growth factor β (TGF-β) pathway that serves as a regulator of chondrogenesis and osteogenesis. To investigate the role of the TGF-β/Smad3 signaling in the process of fracture healing, an open fracture was introduced in mouse tibiae, and the histology of the healing process was compared between wild-type (WT) and Smad3-null (KO) mice. In KO mice, the bone union formed more rapidly with less formation of cartilage in the callus and eventually the fracture was repaired more rapidly than in WT mice. Alkaline phosphatase staining showed that osteoblastic differentiation in the fracture callus was promoted in KO mice. Additionally, TRAP staining and the TUNEL assay revealed that the induction of osteoclasts and apoptotic cells was significantly promoted in the healing callus of KO mice. Sox9 expression clearly decreased at both mRNA and protein levels in the early stage of fracture in KO mice. In contrast, the expression of genes for osteogenesis and osteoclast formation increased from day 5 until day 14 post-fracture in KO mice compared to WT mice. From these results, we concluded that the loss of TGF-β/Smad3 signaling promoted callus formation by promoting osteogenesis and suppressing chondrogenesis, which resulted in faster fracture healing.     

Influence of endothelial cells on vascular smooth muscle cells phenotype after irradiation: implication in radiation-induced vascular damages

Damage to vessels is one of the most common effects of therapeutic irradiation on normal tissues. We undertook a study in patients treated with preoperative radiotherapy and demonstrated in vivo the importance of proliferation, migration, and fibrogenic phenotype of vascular smooth muscle cells (VSMCs) in radiation-induced vascular damage. These lesions may result from imbalance in the cross talk between endothelial cells (ECs) and VSMCs. Using co-culture models, we examined whether ECs influence proliferation, migration, and fibrogenic phenotype of VSMCs. In the presence of irradiated ECs, proliferation and migration of VSMCs were increased. Moreover, expressions of alpha-smooth muscle actin, connective tissue growth factor, plasminogen activator inhibitor type 1, heat shock protein 27, and collagen type III, alpha 1 were up-regulated in VSMCs exposed to irradiated ECs. Secretion of transforming growth factor (TGF)-beta1 was increased after irradiation of ECs, and irradiated ECs activated the Smad pathway in VSMCs by inducing Smad3/4 nuclear translocation and Smad-dependent promoter activation. Using small interferring RNA targeting Smad3 and a TGFbeta-RII neutralizing antibody, we demonstrate that a TGF-beta1/TGF-beta-RII/Smad3 pathway is involved in the fibrogenic phenotype of VSMCs induced by irradiated ECs. In conclusion, we show the importance of proliferation, migration, and fibrogenic phenotype of VSMCs in patients. Moreover, we demonstrate in vitro that ECs influence these fundamental mechanisms involved in radiation-induced vascular damages.     

Imbalance of receptor-regulated and inhibitory Smads in lung fibroblasts from bleomycin-exposed rats

Transforming growth factor (TGF)-beta plays a central role in lung fibrosis, stimulating extracellular matrix deposition. Intracellular signaling of TGF-beta is mediated by Smad proteins. We questioned whether the expression and activation of Smads would be altered in lung fibroblasts from rats exposed to bleomycin, an agent used to provoke an experimental model of lung fibrosis. Fibroblasts were isolated from rat lungs 14 d after intratracheal instillation of bleomycin (BLF) or saline (NLF), and cell cultures established. Whole cell lysates were obtained at baseline, and after stimulation with TGF-beta1 (10 ng/ml). Western blot analysis was performed to measure levels of phosphorylated Smad3 (p-Smad3) and Smad7. Real-time PCR was used to determine changes in Smad7 mRNA after TGF-beta stimulation. We found increased baseline levels of p-Smad3 in BLF versus NLF (P < 0.05). In contrast, baseline levels of Smad7 were comparable. The ratio of stimulatory to inhibitory Smads was increased in BLF compared with NLF (P < 0.05). After stimulation with TGF-beta, levels of p-Smad3 were increased in both groups, with maximal responses at 30 min (P < 0.01). While Smad7 mRNA levels were significantly upregulated (at 1 h) after TGF-beta in both groups, the increase in Smad7 protein was significant in NLF only. We conclude there is sustained activation of Smad signaling in lung fibroblasts isolated from bleomycin-exposed rats, with an imbalance between the levels of p-Smad3 and Smad7. Insufficient levels of the inhibitory Smad7 at baseline, and inadequate response to TGF-beta, may contribute to the fibrotic phenotype characteristic of BLF.     

Fibrin-induced skin fibrosis in mice deficient in tissue plasminogen activator

The deposition of fibrin is an integral part of the tissue repair process, but its persistence is also associated with a number of fibrotic conditions. This study addressed the hypothesis that reduced fibrinolysis and fibrin persistence are associated with an enhanced accumulation of collagen and the development of skin fibrosis. Decreased fibrinolysis was confirmed in fibrin gel cultures that contained human dermal fibroblasts plus the specific plasmin inhibitor alpha(2)-antiplasmin or dermal fibroblasts isolated from plasminogen activator (PA)-deficient mice. Collagen accumulation was significantly increased in the presence of inhibitor and in tPA-deficient, but not uPA-deficient, fibroblasts compared with controls. These findings were also confirmed using a skin fibrosis model in which multiple injections of fibrin were given subcutaneously to PA-deficient mice. Injection sites from tPA-deficient mice displayed significantly increased collagen levels compared with uPA-deficient mice and wild-type controls. Up-regulation of fibroblast procollagen gene expression and reduced activation of pro-MMP-1 appeared to mediate the increase in collagen by human dermal fibroblasts in the presence of alpha2-antiplasmin. These findings suggest that persistent fibrin is associated with enhanced collagen accumulation that may result in the development of fibrotic skin disorders in which reduced fibrinolysis is a feature.     

Heparin fragments modulate the collagen phenotype of fibroblasts from radiation-induced subcutaneous fibrosis

Acute local gamma irradiation of porcine skin induces, as in human skin, an extensive and mutilating sclerosis characterized by continuous expansion of the fibrosis invading the adjacent muscle and by accumulation of the macromolecular components of the extracellular matrix. Collagen synthesis, content, and types were studied in the presence of heparin fragments (100 micrograms/10(6) cells) in the culture medium, by measuring the incorporation of the radiolabeled precursor [3H]proline into confluent primary cultures of porcine fibroblasts obtained from normal and irradiated fibrotic dermis. Enhancement in collagen biosynthesis and deposition and preferential increase in collagen type III synthesis were observed in fibrotic fibroblast cultures when compared to those in normal dermis fibroblasts. The total collagen synthesis and the rate of collagen hydroxylation appear unmodified by heparin fragments both in normal and in fibrotic fibroblast cultures. But heparin fragments induce a 10- and 2-fold decrease, respectively, in collagen type III and type V syntheses by fibrosis fibroblasts. As only minor effects upon collagen type III and V are observed in cultures of normal dermis fibroblasts, these results highly suggest that heparin fragments are capable of specifically modulating the collagen phenotype of fibroblasts derived from radiation-induced dermis fibrosis and thus are able to regulate the fibrotic process.     

Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice

To clarify interleukin (IL)-6 roles in wound healing, we prepared skin excisions in wild-type (WT) and IL-6-deficient BALB/c [knockout (KO)] mice. In WT mice, the wound area was reduced to 50% of original size at 6 days after injury. Microscopically, leukocyte infiltration was evident at wound sites. Furthermore, the re-epithelialization rate was approximately 80% at 6 days after injury with increases in angiogenesis and hydroxyproline contents. The gene expression of IL-1, chemokines, adhesion molecules, transforming growth factor-beta1, and vascular endothelial growth factor was enhanced at the wound sites. In contrast, the enhanced expression of these genes was significantly reduced in KO mice. Moreover, in KO mice, the reduction of wound area was delayed with attenuated leukocyte infiltration, re-epithelialization, angiogenesis, and collagen accumulation. Finally, the administration of a neutralizing anti-IL-6 monoclonal antibody significantly delayed wound closure in WT mice. These observations suggest that IL-6 has crucial roles in wound healing, probably by regulating leukocyte infiltration, angiogenesis, and collagen accumulation.     

Animal model of sclerotic skin. V: Increased expression of alpha-smooth muscle actin in fibroblastic cells in bleomycin-induced scleroderma.

Scleroderma is a connective tissue disorder with unknown etiology. Myofibroblasts appear during fibrotic processes such as scleroderma, hypertrophic scarring, and wound healing. We previously established a mouse model for scleroderma by local injections of bleomycin. To determine the phenotype of the fibroblasts in sclerotic skin after bleomycin treatment, we examined the expression of alpha-smooth muscle actin (alpha-SMA), a marker for myofibroblasts, in lesional skin as well as in fibrous lung in this model. Dermal sclerosis was induced by daily local injections of bleomycin (100 microg/ml) for 3 weeks in C3H mice. Immunohistochemical examination showed that alpha-SMA-reactive cells were detectable on fibroblastic cells in bleomycin-injected skin at 1 week. There was a significant increase in the immunoreactive fibroblastic cells for alpha-SMA in lesional skin in parallel with the induction of dermal sclerosis. After 3 weeks' treatment with bleomycin, the number of alpha-SMA-reactive fibroblasts showed an 11-fold increase compared with that in control PBS-treated mice. alpha-SMA-positive cells were also detected in lung parenchyma after bleomycin treatment. Following concomitant treatment with anti-transforming growth factor-beta (TGF-beta) antibody with bleomycin, the number of alpha-SMA-positive fibroblastic cells was significantly reduced up to 50%, along with the reduction of dermal sclerosis. To confirm the protein level of alpha-SMA, immunoblotting was carried out. Results showed an increase of alpha-SMA expression in lesional skin at 3 weeks of bleomycin treatment, which was reduced following anti-TGF-beta antibody treatment. These data suggest that fibroblastic cells are phenotypically altered into myofibroblasts during the fibrotic process in the experimental model of bleomycin-induced scleroderma, which was considered mediated, for the most part, by TGF-beta. Blockade of TGF-beta may be a therapeutic intervention for scleroderma.     

Lack of ataxia telangiectasia mutated kinase induces structural and functional changes in the heart: role in β-adrenergic receptor-stimulated apoptosis

Ataxia telangiectasia mutated kinase (ATM) is involved in cell cycle checkpoints, DNA repair and apoptosis. β-Adrenergic receptor (β-AR) stimulation induces cardiac myocyte apoptosis. Here we analysed basal myocardial structure and function in ATM knockout (KO) mice and tested the hypothesis that ATM modulates β-AR-stimulated myocyte apoptosis. Left ventricular (LV) structure and function, myocyte apoptosis, fibrosis and expression of fibrosis-, hypertrophy- and apoptosis-related proteins were examined in wild-type (WT) and KO mice with or without l-isoprenaline treatment for 24 h. Body and heart weights were lower in KO mice. M-Mode echocardiography showed reduced septal wall thicknesses and LV diameters in KO mice. Doppler echocardiography showed an increased ratio of early peak velocity (E wave) to that of the late LV filling (A wave) in KO mice. Basal fibrosis and myocyte cross-sectional area were greater in KO hearts. Expression of fibrosis-related genes (connective tissue growth factor and plasminogen activator inhibitor-1) and hypertrophy-related gene (atrial natriuretic peptide) was higher in KO hearts. β-Adrenergic receptor stimulation increased myocyte apoptosis to a similar extent in both groups. Activation of c-Jun N-terminal kinases and expression and phosphorylation of p53 in response to β-AR stimulation were only observed in the WT group. Akt phosphorylation was lower in KO sham-treated animals and remained lower following β-AR stimulation in the KO group. β-Adrenergic receptor stimulation activated glycogen synthase kinase-3β to a similar extent in both groups. Thus, lack of ATM induces structural and functional changes in the heart, with enhanced myocardial fibrosis and myocyte hypertrophy. β-Adrenergic receptor-stimulated apoptosis in WT hearts is associated with a p53- and JNKs-dependent mechanism, while decreased Akt activity may play a role in increased myocyte apoptosis in the absence of ATM.