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1-induced connective tissue growth factor expression in airway smooth muscle cells
1Thoracic Medicine, National Heart and Lung Institute, Imperial College and 2Pathology Department, Royal Brompton Hospital, London, United Kingdom
Submitted 30 April 2004 ; accepted in final form 12 September 2004
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ABSTRACT |
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may play an important role in airway remodeling, and the fibrogenic effect of TGF- may be mediated through connective tissue growth factor (CTGF) release. We investigated the role of MAPKs and phosphatidylinositol 3-kinase (PI3K) and the effects of inflammatory cytokines on TGF--induced CTGF expression in human airway smooth muscle cells (ASMC). We examined whether Smad signal was involved in the regulatory mechanisms. TGF-1 induced a time- and concentration-dependent expression of CTGF gene and protein as analyzed by real-time RT-PCR and Western blot. Inhibition of ERK and c-jun NH2-terminal kinase (JNK), but not of p38 MAPK and PI3K, blocked the effect of TGF-1 on CTGF mRNA and protein expression and on Smad2/3 phosphorylation. T helper lymphocyte 2-derived cytokines, IL-4 and IL-13, attenuated TGF-1-stimulated mRNA and protein expression of CTGF and inhibited TGF-1-stimulated ERK1/2 and Smad2/3 activation in ASMC. The proinflammatory cytokines tumor necrosis factor-
and IL-1 reduced TGF-1-stimulated mRNA expression of CTGF but did not inhibit TGF--induced Smad2/3 phosphorylation. TGF-1-stimulated CTGF expression is mediated by mechanisms involving ERK and JNK pathways and is downregulated by IL-4 and IL-13 through modulation of Smad and ERK signals. mitogen-activated protein kinases; signal transduction; Smad; cytokines; asthma; transforming growth factor
is a polypeptide commonly associated with various disorders of inflammation and repair, such as asthma and chronic obstructive airway disease. TGF- is expressed in human ASMC, and increased expression of the isoform TGF-1 has been demonstrated in ASMC of patients with asthma compared with nonasthmatic airways (3, 12, 16). A variety of cells, including macrophages, platelets, epithelial cells, and ASMC, can produce TGF-, and increased levels of TGF- have been detected in bronchial biopsy specimens (36, 43) and bronchoalveolar lavage fluid samples (39) from subjects with asthma. TGF- induces proliferation of ASMC (7, 15), enhances the production and release of ECM proteins from airway smooth muscle, and may induce collagen I synthesis by an autocrine effect (16). Therefore, TGF- may be an important mediator of airway remodeling.
Connective tissue growth factor (CTGF), originally identified as a growth factor secreted by endothelial cells (8), promotes fibroblast proliferation, migration, adhesion, and ECM production (23). In addition, CTGF exhibits other biological actions in vitro such as vascular smooth muscle migration, angiogenesis, and apoptosis (21, 27). CTGF upregulates collagen I and fibronectin in human lung fibroblasts and vascular smooth muscle cells (21, 22). Overproduction of CTGF may therefore be an important pathway leading to fibrosis. Synergy between CTGF and TGF-1 could also occur through the direct binding of TGF-1 to CTGF or through enhancement of TGF- binding to its receptor by CTGF or by the potentiation of Smad2/3 phosphorylation induced by TGF-1 (2). Of particular interest to the current work, CTGF is transcriptionally activated by TGF- and has been shown as a downstream mediator of TGF- mediating its profibrotic activity (19, 23). A recent study showed that ASMC also express CTGF, and increased expression of CTGF stimulated by TGF- has been demonstrated in ASMC cultured from asthmatic subjects (10). Therefore, CTGF induction by TGF- may be an important mechanism of airway remodeling in asthma.
The mechanism mediating increased CTGF expression by TGF- in ASMC remains unknown. MAPKs are a family of protein kinases that phosphorylate specific serines and threonines of target protein substrates to regulate cellular activities (32). Three subfamilies of MAPKs that differ in their substrate specificity have been well characterized, including ERK, c-jun NH2-terminal kinase (JNK), and p38 MAPKs. ERK plays an important role in cell proliferation and differentiation as well as in survival mediated by many growth factors, whereas JNK and p38 MAPK are activated by various inflammatory cytokines and environmental stimuli and play an important role in cytokine production and apoptosis. A central general role of Smads in TGF--induced expression of target genes has been identified; Smads act as transcriptional modulators that recruit transcription factors to the promoter (47). A number of cell-specific interactions have been described between the MAPK cascade and TGF--driven Smad signaling, such as the interaction of JNK and Smad (20). Regulation of CTGF by MAPKs and phosphatidylinositol 3-kinase (PI3K) in human mesangial cells and in fibroblasts has been reported (17). The T helper lymphocyte 2 (Th2) cytokines IL-4 and IL-13 and the proinflammatory cytokines tumor necrosis factor (TNF)- and IL-1 play an active role in stimulating mediator production from ASMC and can promote allergic airway inflammation (14). These cytokines may interact with TGF- and influence CTGF expression.
To understand the intracellular mechanisms of CTGF activation by TGF-, we investigated the role of ERK, JNK, p38 MAPKs, and PI3K in ASMC. We also examined whether cytokines involved in allergen inflammation, IL-4 and IL-13, and proinflammation cytokines IL-1 and TNF- can interfere with CTGF production induced by TGF- and whether these cytokines could modulate TGF--induced Smad signaling.
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MATERIALS AND METHODS |
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1, IL-4, IL-13, IL-1, and TNF- were purchased from R&D Systems (Abingdon, UK). Rabbit polyclonal CTGF antibody (CTGF-ab6992) was from Abcam (Cambridge, UK). Primers for CTGF and GAPDH were obtained from Sigma Genosys (Pampisford, Cambridgeshire, UK). PD-98059, SB-203580, and wortmannin were purchased from Calbiochem (Nottingham, UK). SP-600125 was a kind gift from Celgene (San Diego, CA). Antibodies for phosphospecific ERK1/2 (p-ERK1/2), ERK1/2, phospho-Smad2/3 (p-Smad2/3; Ser465/467), and anti-rabbit horseradish peroxidase (HRP)-conjugated IgG were obtained from Cell Signaling Technology (Hertfordshire, UK). Anti-Smad2/3 antibody was from Upstate (Milton Keynes, UK). Enhanced chemiluminescence (ECL) reagent was from Amersham International (Bucks, UK). Tissue culture media and reagents were purchased from Sigma or GIBCO-BRL (Paisley, Strathclyde, UK).
Cell culture and treatment.
ASMC were isolated from fresh lobar or main bronchus, obtained from lung resection donors, by treatment with collagenase and cultured in DMEM supplemented with 10% heat-inactivated FCS as described previously (31). ASMC characteristics were identified by light microscopy with typical "hill and valley" appearance and by positive immunostaining of smooth muscle (SM) -actin, SM myosin heavy chain, calponin, and SM-22. The cells were maintained in T175 or T75 culture flasks at 37°C in a humidified atmosphere of 5% CO2. For these experiments, ASMC were studied from passages 3–6.
Cells were trypsinized and subcultured in six-well plates for total protein and RNA extractions. After reaching confluence in 10% FCS DMEM, cells were incubated for 2–3 days in serum-free medium containing 0.5% BSA before treatment. Cells were treated with TGF- or the appropriate test reagents in serum-free medium containing 0.5% BSA. Control cultures were incubated in the medium-containing vehicle alone.
Western blot. Total cell protein was extracted on ice with lysis buffer (1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS in PBS, pH 7.4) in the presence of freshly added protease inhibitors including 1 mM PMSF, 5 µg/ml aprotinin, 1 mM Na3VO4, and 5 µg/ml leupeptin. Protein concentration was determined using the Bradford method with a Bio-Rad protein assay reagent. Protein extract (30 µg/lane) was fractionated by SDS-PAGE on a 10% Tris-glycine precast gel (Invitrogen) and then transferred to a nitrocellulose membrane (Amersham). The membrane was incubated overnight at 4°C with an antibody for CTGF (0.5 µg/ml) or p-ERK1/2 (1:1,000), ERK1/2 (1:1,000), p-Smad2/3 (1:1,000), or Smad2/3 (2 µg/ml). The next day, the membrane was incubated for 1 h with an HRP-conjugated secondary antibody raised against rabbit IgG (1:2,000) at room temperature. Antibody-bound proteins were visualized by ECL. The membranes were stripped and then reprobed with a mouse anti-GAPDH monoclonal antibody (1:5,000; Biogenesis, Poole, UK) to control for the loading. Relevant band intensities were quantified by scanning densitometric analysis using software from Ultra-Violet Products (Cambridge, UK). Densitometric data were normalized for GAPDH values.
Real-time RT-PCR. Total RNA was isolated from ASMC by using the RNeasy Mini Kit (Qiagen, West Sussex, UK) or TRIzol (Invitrogen) according to the manufacturer’s instructions. cDNA was generated by RT using random hexamers and an avian myeloblastosis virus reverse transcriptase (Promega). The cDNA (42 ng/reaction) was used as a template in the subsequent PCR analyses. Transcript levels were determined by real-time PCR (Rotor Gene 3000) using the SYBR Green PCR Master Mix Reagent Kit (Qiagen). The human CTGF forward and reverse primers were 5'-GAGGAAAACATTAAGAAGGGCAAA-3' and 5'-CGGCACAGGTCTTGATGA-3'. Primer was used at a concentration of 0.5 µM in each reaction. Cycling conditions were as follows: step 1, 15 min at 95°C; step 2, 20 s at 94°C; step 3, 20 s at 60°C; step 4, 20 s at 72°C, with repeat from step 2 to step 4 for 35 times. Data from the reaction were collected and analyzed by the complementary computer software (Corbett Research) using a standard curve. The standard curves are used to determine copy number for GAPDH by running GAPDH standard over 3 x 103 to 3 x 107 copies/reaction with analyzed samples, or relative expression for CTGF by diluting a sample, for example, to 1:1, 1:10, and 1:100 and giving the relative concentration to 1, 0.1, and 0.01 for each detection. Relative quantitations of gene expression were normalized to GAPDH.
Immunocytochemistry and immunofluorescence.
Immunocytochemistry was performed to detect the presence of CTGF in ASMC grown on chamber slides in the presence or absence of TGF-. The cells were fixed in 3.7% formaldehyde in PBS for 10 min followed by cold methanol for 4 min and cold acetone for 1 min. Immunostaining was carried out in accordance with the directions supplied by the Vectastain Kit (Vector Laboratories). CTGF was detected using the rabbit polyclonal CTGF antibody (5 µg/ml, 1 h). Sections were incubated with the avidin/biotinylated alkaline phosphatase complex and then with Sigma Fast Red chromogenic solution. Sections were counterstained with 2% hematoxylin.
Immunofluorescence was carried out to detect translocation of p-Smad2/3 and Smad4 following stimulation of TGF-. The cells were fixed with 4% paraformaldehyde for 15 min at 4°C. Smad signal was detected using the rabbit antibody for p-Smad2/3 (1:100) or Smad4 (4 µg/ml, Santa Cruz Biotechnology) by incubating cells at 4°C overnight. Sections were incubated with a rhodamine-conjugated donkey anti-rabbit IgG (1:50) for 45 min and photographed with a laser scanning confocal microscope.
Data analysis. Data were analyzed by ANOVA using the software program Statview (Abacus Concepts, Berkeley, CA). Results are expressed as means ± SE and are representative of at least three separate experiments. P < 0.05 was used to determine the statistical significance of the data.
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RESULTS |
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1 induces CTGF mRNA expression in ASMC.
ASMC were treated with TGF-1 (0.3–10 ng/ml) in serum-free medium at different time points (1–24 h). Real-time RT-PCR showed that unstimulated cells expressed baseline levels of CTGF mRNA and that TGF-1 induced a time-dependent increase in CTGF mRNA expression (Fig. 1A). The expression of CTGF mRNA was almost doubled following 1-h treatment with 10 ng/ml of TGF-1 and increased sevenfold at 3 h, and the maximal effect was seen at 24 h. TGF-1-stimulated expression of CTGF mRNA was sustained after a 72-h treatment (data not shown). The effect of TGF-1 on CTGF mRNA was dose dependent over the range of 0.3–10 ng/ml after 24-h treatments (Fig. 1B). TGF-1 at 1 ng/ml increased CTGF transcript by 6-fold, and maximal stimulation was seen at 10 ng/ml by 14-fold compared with unstimulated controls. This concentration was chosen for subsequent experiments.
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1 induces CTGF protein expression of ASMC.
ASMC were incubated in six-well plates in the presence or absence of TGF-1 (0.3–10 ng/ml) for 3–48 h, and protein expression of CTGF was analyzed by Western blot. There was a time-dependent increase in the expression of CTGF protein in ASMC treated with TGF-1 (10 ng/ml) for 3–48 h (Fig. 2, A and C). An approximate twofold increase in CTGF level was observed following a 6-h treatment with TGF-1, with an eightfold increase after 24 h, reaching maximal levels after 48 h (
12-fold). Similar to the effects on CTGF mRNA, TGF-1 also stimulated a dose-dependent increase in the protein expression of CTGF in ASMC treated for 48 h (Fig. 2, B and D). Immunostaining showed increased expression of CTGF in ASMC following a 24-h treatment with 10 ng/ml of TGF-1 and was sustained up to 7 days of treatment (data not shown).
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1.
Cells were preincubated with specific blockers (PD-98059 for ERK, SP-600125 for JNK, SB-203580 for p38 MAPK, and wortmannin for PI3K) for 30 min and then cotreated with 10 ng/ml of TGF-1 for 24 h for analysis of mRNA expression or for 48 h for protein expression. Treatment of ASMC with PD-98059 (10–50 µM) inhibited TGF-1-induced expression of CTGF protein (Fig. 3, A and B) and mRNA (Fig. 3C) in a dose-dependent manner. SP-600125 induced a similar inhibition in CTGF protein production (Fig. 4, A and B) and mRNA expression (Fig. 4C) by TGF-1. SB-203580 (1 µM) and wortmannin (100 nM) had no effect on TGF-1-stimulated protein expression of CTGF (Fig. 4D). The inhibitors PD-98059, SP-600125, SB-203580, or wortmannin alone at the used concentrations had no effect on CTGF expression and also did not affect cell viability (data not shown).
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-induced Smad signaling.
TGF-1 induced a time- and concentration-dependent activation of Smad2/3 in ASMC (Fig. 5). Phosphorylation of Smad2/3 by TGF- was observed following a 10-min treatment (data not shown) and was sustained for up to 48 h (Fig. 5, A and B). The maximal activity was seen at 10 ng/ml of TGF- (Fig. 5, C and D). Immunofluorescence showed the translocation of p-Smad2/3 and Smad4 from cytosol to nucleus after TGF- stimulation, and there was increasing signal in the nucleus and corresponding decreases in the cytosol for Smad4 (data not shown). Treatment with PD-98059 (10–50 µM), a selective blocker for ERK, led to inhibition of the TGF--induced p-Smad2/3 in a concentration-dependent manner (Fig. 6, A and B). A similar inhibition was achieved when cells were treated with the JNK blocker SP-600125 (Fig. 6B). In contrast, SB-203580 (1 µM) and wortmannin (100 nM) did not affect Smad2/3 phosphorylation (data not shown).
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1-induced CTGF expression and ERK and Smad activation.
After 48 h of stimulation with 10 ng/ml of IL-4 or IL-13, basal CTGF protein levels were not changed as assessed by Western blot (Fig. 7, A and B). However, IL-4 and IL-13 attenuated TGF-1-stimulated protein expression of CTGF by 50%. Similar results were obtained using real-time RT-PCR to determine CTGF mRNA after a 24-h treatment (Fig. 7C). To investigate whether the downregulation of TGF--induced CTGF expression may be mediated by modulation of ERK or Smad activity, ASMC were treated for 48 h with TGF- (10 ng/ml) alone or in combination with IL-4 or IL-13. TGF-1 induced marked phosphorylation of ERK in ASMC, whereas IL-4 inhibited the stimulated phosphorylation by TGF- (Fig. 8A), and IL-13 had a similar effect (data not shown). The basal and TGF-1-induced ERK phosphorylation was blocked when cells were treated with the ERK blocker PD-98059 at 50 µM concentration (Fig. 8A). IL-4 and IL-13 also inhibited TGF--induced activation of Smad2/3 in a concentration-dependent manner (Fig. 8, B and C).
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1 with TNF- and IL-1 on CTGF expression and Smad activation.
To investigate the interaction between TGF-1 and the proinflammatory cytokines TNF- and IL-1 on CTGF expression, cells were incubated with TGF-1 (10 ng/ml) in the presence of TNF- (20 ng/ml) or IL-1 (10 ng/ml). Significant suppression of basal and TGF-1-induced expression of CTGF mRNA was observed when cells were incubated with TNF- (Fig. 9A). IL-1 modestly decreased the basal and TGF-1-induced mRNA expression of CTGF. TNF- slightly reduced CTGF protein production by TGF-, but IL-1 had no prominent effect as determined by Western blot (Fig. 9B). There was no effect on CTGF protein production when cells were treated with either TNF- alone or IL-1 alone (data not shown). Both TGF- and IL-1 did not inhibit TGF--induced phosphorylation of Smad2/3 in ASMC (Fig. 9C).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1 stimulates CTGF mRNA expression and protein production in ASMC, mediated by mechanisms involving the ERK and JNK pathways, but not the p38 MAPK and PI3K pathways. TGF-1-stimulated activation of Smad 2/3 was inhibited by ERK and JNK inhibitors, implying a role for these pathways in TGF-1-induced Smad signaling. We also observed that the Th2 cytokines IL-4 and IL-13 inhibited TGF-1-stimulated mRNA and protein expression of CTGF and TGF--induced ERK and Smad2/3 activation. Of the proinflammatory cytokines, TNF- and IL-1 reduced the TGF-1-induced mRNA expression of CTGF but had less effect on TGF--stimulated protein production, with no effect on Smad 2/3 phosphorylation.
The stimulation of CTGF by TGF-1 is consistent with a recent report that ASMC from bronchial biopsies or cultures express detectable levels of CTGF, which is increased by TGF-1 (10). The sustained expression of CTGF up to 48 h may be secondary to the prolonged activation of ERK pathway and of Smad2/3 phosphorylation. In addition, CTGF and TGF- may interact, such as CTGF potentiating TGF-1 binding to its receptor and signaling (2). Many actions of TGF- on connective tissue cells are mediated through the induction of CTGF, for example, blocking the effect of CTGF by transfection with antisense CTGF or with anti-CTGF antibody blocking TGF--stimulated fibronectin and collagen (19, 45). In addition, as with TGF--induced proliferation or collagen synthesis, TGF- induces the myofibroblast phenotype through CTGF (25). Therefore, TGF- actions on fibrogenesis are mediated to a large extent by CTGF; whether this also relates to TGF- fibrogenic actions on ASMC is not known. There are also interactions between CTGF and TGF- that are important to consider. In vivo experiments show that CTGF enhances the profibrotic response to TGF-; for example, application of TGF- or CTGF alone causes a transient fibrotic response (4, 37), but both in combination cause a sustained fibrotic response (37). TGF- also mediates fibrogenesis in CTGF-independent pathways (29), although further investigation is expected so that we can have a better understanding of the CTGF-independent effects of TGF-.
The mechanisms that mediate the TGF--induced CTGF in ASMC are unknown. We investigated the role of ERK, JNK, p38 MAPK, and PI3K pathways as previously observed in fibroblasts and mesangial cells (11, 42). TGF--induced CTGF expression of mRNA and protein was dose dependently inhibited by the ERK blocker and the JNK blocker, but not by the p38 MAPK and PI3K blockers as analyzed by real-time PCR and Western blot. The inhibition of ERK and JNK pathway activities abrogated the TGF--induced CTGF, suggesting that activation of ERK and JNK pathways forms the basis of an early intracellular signaling response to TGF--induced CTGF.
TGF- signal transduction is first initiated by binding to two cell membrane serine-threonine kinase receptors, termed type I (TRI) and type II (TRII), followed by their phosphorylation (41). Two TRII receptor subunits phosphorylate and activate two TRI receptors. In turn, the Smad family of proteins are the primary substrates of the phosphorylated TGF- receptors. Phosphorylation of Smad2 and Smad3 leads to the formation of heteromeric complexes with Smad4. These complexes then translocate to the nucleus and regulate gene transcription by binding DNA directly or in association with other transcriptional factors. Transcriptional activation may be further regulated by coactivators or corepressors binding to the Smad complex (47). We showed that TGF--induced phosphorylation of Smad 2/3 was downregulated by inhibitors of ERK and JNK in ASMC, suggesting that TGF--induced CTGF expression involved the Smad signaling pathway (11). A functional Smad binding site on the promoter of the CTGF gene has also been described (28). TGF--induced CTGF may also be mediated through a Smad-independent pathway as TGF- response element on CTGF gene promoter has been reported in fibroblasts (24).
MAPK pathways have also been implicated in both positive and negative regulation of TGF- signaling, either dependently or independently of the Smad signaling pathway (20, 38, 44). TGF- also induces the direct activation of several MAPK pathways through the upstream mediators RhoA, Ras, and possibly via TGF--activated kinase (5, 20, 33, 46). In the human ASMC, TGF- signaling transcriptional activation of CTGF was positively regulated by ERK and JNK pathways because addition of selective blockers for these pathways blocked TGF--induced CTGF transcription. However, we found no involvement of the p38 MAPK pathway and the PI3K pathway. This is somewhat different from the recent finding in fibroblasts where CTGF mRNA expression by TGF- is dependent on the JNK and PI3K pathways but not on the p38 MAPK and ERK pathways (42), illustrating the differential involvement of the different MAPK pathways in different cell types. TGF- activated ERK or JNK to cause phosphorylation of Smad2/3 with potentiation of Smad signaling as seen with EGF receptor activation (9, 18).
Because IL-4 and IL-13 are important cytokines that underlie the allergic inflammation of asthma (40), we investigated their effects on TGF--induced CTGF. IL-4 has been shown to inhibit TGF--induced CTGF mRNA expression in fibroblasts by reducing the rate of transcription (40). Although basal CTGF mRNA levels were unchanged by IL-4, this cytokine attenuated the TGF--stimulated expression of CTGF mRNA and protein by 50%. We determined whether regulation of TGF--induced CTGF expression by IL-4 was mediated via the ERK pathway. Indeed, TGF--stimulated activation of ERK (p42/44 MAPK) was inhibited by IL-4 and was blocked by PD-98059, a specific ERK inhibitor that also reduced the basal activity of ERK. Inhibitory effects of both IL-4 and IL-13 have been observed in ASMC such as the inhibition of release of RANTES and IL-8 and mitogen-induced proliferation (26, 30, 31). IL-13 had similar inhibitory effects on TGF--stimulated CTGF expression in ASMC, which may be mediated through inhibiting TGF--induced activation of Smad 2/3.
We also found that TNF- downregulated basal and TGF--stimulated CTGF mRNA, but only slightly reduced the TGF--induced protein level of CTGF, whereas IL-1 did not significantly affect the CTGF expression by TGF-. The discrepancy between CTGF mRNA and the protein levels after TNF- stimulation may be due to the effect of TNF- in increasing CTGF protein stability by reducing the rate of proteasomal degradation, thereby maintaining CTGF protein expression despite reduced mRNA expression. Because changes in CTGF protein level are more biologically relevant, we consider that TNF- and IL-1 are not important in CTGF regulation compared with IL-4 and IL-13. Neither IL-1 nor TNF- alters TGF--induced Smad2/3 phosphorylation. In previous studies, TNF- suppressed basal CTGF mRNA expression in bovine aortic endothelial cells (34) and inhibited TGF--induced CTGF expression in human dermal fibroblasts through activation of NF-
B (1). There is evidence that inhibition of TGF- signaling by TNF- is through the induction of the repressive transcription factor Smad-7 by NF-B that is in turn activated by TNF- (6).
Our findings demonstrate that TGF-1-stimulated CTGF expression in ASMC is mediated by mechanisms involving ERK and JNK pathways and is downregulated by IL-4 and IL-13 through modulation of Smad and ERK signals.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 P. R. A. Johnson, J. K. Burgess, Q. Ge, M. Poniris, S. Boustany, S. M. Twigg, and J. L. Black Connective Tissue Growth Factor Induces Extracellular Matrix in Asthmatic Airway Smooth Muscle Am. J. Respir. Crit. Care Med., January 1, 2006; 173(1): 32 - 41. [Abstract] [Full Text] [PDF]
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J. K. Burgess, Q. Ge, M. H. Poniris, S. Boustany, S. M. Twigg, J. L. Black, and P. R. A. Johnson Connective tissue growth factor and vascular endothelial growth factor from airway smooth muscle interact with the extracellular matrix Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L153 - L161. [Abstract] [Full Text] [PDF]
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 C. S. Samuel Relaxin: Antifibrotic Properties and Effects in Models of Disease Clin. Med. Res., November 1, 2005; 3(4): 241 - 249. [Abstract] [Full Text] [PDF]
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C. Bronner Is the upregulation of bradykinin B2 receptors by TGF-{beta}1 one of the missing pieces in the "airway hyperresponsiveness" puzzle? Am J Physiol Lung Cell Mol Physiol, October 1, 2005; 289(4): L509 - L510. [Full Text] [PDF]
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