Oxidative Protein Cross-linking Reactions Involving L-Tyrosine in Transforming Growth Factor-beta 1-stimulated Fibroblasts*

Jose M. Larios, Rohit Budhiraja, Barry L. Fanburg, and Victor J. ThannickalDagger

From the Pulmonary and Critical Care Division, Department of Medicine, New England Medical Center/Tupper Research Institute, Tufts University School of Medicine, Boston, Massachusetts 02111

Received for publication, January 17, 2001, and in revised form, January 31, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mechanisms by which ligand-stimulated generation of reactive oxygen species in nonphagocytic cells mediate biologic effects are largely unknown. The profibrotic cytokine, transforming growth factor-beta 1 (TGF-beta 1), generates extracellular hydrogen peroxide (H2O2) in contrast to intracellular reactive oxygen species production by certain mitogenic growth factors in human lung fibroblasts. To determine whether tyrosine residues in fibroblast-derived extracellular matrix (ECM) proteins may be targets of H2O2-mediated dityrosine-dependent cross-linking reactions in response to TGF-beta 1, we utilized fluorophore-labeled tyramide, a structurally related phenolic compound that forms dimers with tyrosine, as a probe to detect such reactions under dynamic cell culture conditions. With this approach, a distinct pattern of fluorescent labeling that seems to target ECM proteins preferentially was observed in TGF-beta 1-treated cells but not in control cells. This reaction required the presence of a heme peroxidase and was inhibited by catalase or diphenyliodonium (a flavoenzyme inhibitor), similar to the effect on TGF-beta 1-induced dityrosine formation. Exogenous addition of H2O2 to control cells that do not release extracellular H2O2 produced a similar fluorescent labeling reaction. These results support the concept that, in the presence of heme peroxidases in vivo, TGF-beta 1-induced H2O2 production by fibroblasts may mediate oxidative dityrosine-dependent cross-linking of ECM protein(s). This effect may be important in the pathogenesis of human fibrotic diseases characterized by overexpression/activation of TGF-beta 1.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tissue repair responses to injurious agents are complex processes involving resident/structural (e.g. fibroblasts) and circulating/immune (e.g. macrophages) cells. A "normal" repair response most often restores tissue architecture and maintains organ function. However, in some cases "dysregulated" repair leads to tissue fibrosis and organ failure. Cell-cell communication in this context is mediated by a network of growth factors, cytokines, and other soluble mediators (1-3). Transforming growth factor-beta 1 (TGF-beta 1)1 is a multifunctional cytokine that plays a central role in tissue injury/repair. Persistent or exaggerated expression and/or activation of TGF-beta 1 in areas of active injury/repair is linked to a number of human fibrotic diseases involving diverse organ systems (4, 5).

Multiple actions of TGF-beta 1 contribute to its profibrotic effects including its ability to transform fibroblasts both in vitro and in vivo into activated contractile phenotypes known as "myofibroblasts" (6-8). Myofibroblasts are key effector cells in injury/repair processes and fibrosis because of their high synthetic capacity for extracellular matrix (ECM) proteins (9, 10), integrins (11), growth factors (12), growth factor receptors (13), and oxidants (14). Recent studies suggest that TGF-beta 1-induced myofibroblast differentiation depends on the compliance/deformability of the matrix on which cells are grown (15, 16). This observation is consistent with the growing recognition that biomechanical properties of the ECM regulate cell function (17-19).

We have reported previously on the ability of TGF-beta 1 to induce extracellular release of H2O2 in association with cell surface-associated NADH:flavin:O2 oxidoreductase (NADH oxidase) activity in human lung fibroblasts (14). The enzymatic source and regulation of this activity is unrelated to the p21ras-dependent generation of intracellular reactive oxygen species (ROS) by certain mitogenic growth factors (20). Intracellular ROS have been reported to function as signaling molecules in growth factor-induced mitogenesis (21-23). Recent work by Barrett et al. (24) suggests that this effect may be mediated by intracellular O&cjs1138;2-dependent reversible oxidation of critical cysteine residues within the catalytic site of protein-tyrosine phosphatases.

The precise function of TGF-beta 1-induced extracellular H2O2 production is yet to be determined. Our previous data indicate that although tyrosine phosphorylation regulates NADH oxidase activity/H2O2 production, H2O2 does not itself seem to mediate protein tyrosine phosphorylation in cultured lung fibroblasts (25). Moreover, TGF-beta 1-induced extracellular H2O2 is not associated with cell proliferation in these cells (20). H2O2 is a relatively mild oxidant in most biological systems, but it functions as a potent oxidant (by inducing dimerization) of phenolic compounds in the presence of heme peroxidases (26). This property of H2O2 is utilized for useful purposes in the formation of a protective coat, composed of highly cross-linked ECM proteins, around freshly fertilized oocytes in sea urchins and in the plant hypersensitivity response (27, 28). Heinecke et al. (29) have demonstrated that phagocyte-derived myeloperoxidases, which normally convert H2O2 to hypochlorous acid, are capable of catalyzing similar reactions in human atherosclerotic plaques.

Based on the availability of heme peroxidases (such as phagocyte-derived myeloperoxidases) in the extracellular milieu of "activated" fibroblasts, we postulated that extracellular H2O2 production by these cells may mediate similar reactions involving the phenolic amino acid L-tyrosine. In this study, we examined the ability of TGF-beta 1 to induce dimerization of exogenous L-tyrosine by H2O2/heme peroxidase-dependent oxidation. Moreover, the possibility that this reaction may target endogenous protein targets under dynamic conditions in which cultured fibroblasts are actively generating extracellular H2O2 was assessed utilizing a novel fluorescent labeling approach that identifies susceptible tyrosine residues.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Reagents-- All cell culture experiments were performed on normal human fetal lung fibroblasts (IMR-90; Institute for Medical Research, Camden, NJ). Cells were plated on 35-mm Petri dishes at a density of 105 cells/dish and incubated in 5% CO2, 95% air. For fluoroscopy-based assays, cells were plated on microscope glass slides sterilely placed in Petri dishes. Cells were maintained in medium consisting of RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Sigma), 100 units/ml penicillin/streptomycin (Sigma), and fungizone (Life Technologies, Inc.). Medium was changed every 3 days. Cells were grown to 80-100% confluency and serum-deprived for 48 h prior to assays. Porcine platelet-derived TGF-beta 1 was obtained from R&D Systems (Minneapolis, MN). All other reagents including L-tyrosine were from Sigma.

Measurement of Dityrosine Formation-- Dityrosine formation was measured by a fluorometric assay described by Heinecke et al. (30) with minor modifications. The feasibility and reliability of this method in our cell culture system was determined first. Known concentrations of H2O2 (0-3 µM) were added to a reaction mixture containing 1 mM L-tyrosine, 5 units/ml horseradish peroxidase (HRP), and 1 mM HEPES, pH 7.4, in Hanks' balanced salt solution (HBSS). After a 5-min incubation, the reaction was terminated by the addition of 0.1 M glycine-NaOH buffer, pH 12.0, and the final pH was adjusted to 10.0. Fluorescence of the samples was monitored at excitation and emission wavelengths of 325 and 410, respectively. Values were recorded as relative fluorescence with the sample buffer containing no H2O2 as reference ("0" value, Fig. 2, inset).

For dityrosine measurements in cell culture, cells were washed first with Dulbecco's phosphate-buffered saline and incubated with the same HBSS-based reaction mixture containing L-tyrosine and HRP for 1 h at 37 °C. The overlying medium was then removed, the pH was adjusted to 10.0 with 0.1 M glycine-NaOH buffer, and fluorescence was measured as described above. Samples with the reaction mixture alone (without cells) that were processed similarly served as control and the reference against which the relative fluorescence for each of the experimental samples was recorded. "Spontaneous" dityrosine formation was not observed in these control samples.

Labeling of Endogenous Proteins Targeted by H2O2/HRP-mediated Oxidative Tyrosine Cross-linking-- To detect tyrosine cross-linking on endogenous protein substrates, we utilized a commercially available laboratory reagent designed for signal amplification protocols. This reagent, tyramide, labeled with fluorescein (tyramide-FITC, TSATM Fluorescence Systems, PerkinElmer Life Sciences), is distributed as "TSA" kits for amplification of HRP-generated signals in immunoblotting, immunohistochemistry, and in situ hybridization assays (31). Specifically, HRP, in the presence of exogenously added H2O2 (found in the "amplification diluent" provided with the TSA kit), catalyzes the "cross-linking" of tyramide-FITC with tyrosine residues of proteins in close proximity to the site of enzyme (HRP) catalysis. This reportedly results in the deposition of the fluorophore at or near the site of the bound antibody-HRP conjugate resulting in "amplification" of the signal (31). An important feature of this assay is the absolute requirement for exogenous H2O2 to mediate these HRP-catalyzed cross-linking reactions. By eliminating exogenous H2O2 from the reaction, we have exploited this property of tyramide-FITC to localize "susceptible" tyrosine residues on cellular or extracellular proteins to mediate similar cross-linking reactions in response to endogenous cytokine-generated H2O2 in cultured lung fibroblasts.

Cultured cells grown on Petri dishes were washed first with HBSS and incubated with 1 ml of HBSS containing 1 mM HEPES buffer (pH 7.4), 5 units/ml HRP, and 1:1000 dilution of freshly reconstituted tyramide-FITC. Tyramide-FITC was supplied by the manufacturer as lyophilized powder (weight not indicated). Initial reconstitution was with Me2SO using the 1:10 volume recommended by the manufacturer. The final concentration of tyramide-FITC used in our assays amounted to one-half that recommended for use in signal amplification protocols (kit insert, PerkinElmer Life Sciences). The amplification diluent (which contains H2O2) provided with the TSA kit was discarded and is not required for this application. Cells were allowed to incubate for 1 h at 37 °C in the buffer solution containing tyramide-FITC and HRP. Cells then were washed two times with HBSS and visualized (and photographed) with a fluorescent microscope (Zeiss IM 35 inverted microscope, Oberkochen, Germany).

Statistical Analysis-- Data from the various groups were expressed as means ± S.D. Statistical comparisons were made using the Student's t test for unpaired samples. For studies involving more than two groups, two-way analysis of variance was determined using the Scheffe's test (GB-STAT, Dynamic Microsystems, Silver Spring, MD). Statistical significance in all cases was defined as p < 0.05.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TGF-beta 1 Induces Dimerization of Exogenous L-Tyrosine by an H2O2- and Heme Peroxidase-dependent Mechanism-- The ability of H2O2 in the presence of heme peroxidases such as HRP to induce oxidative dimerization of phenolic compounds is well established (Fig. 1a; see Ref. 26 for details). We hypothesized that TGF-beta 1, by stimulating H2O2 production in lung fibroblasts, may mediate a similar effect on the phenolic amino acid, L-tyrosine. First, the feasibility of measuring dityrosine formation in our cell culture system using the method described by Heinecke et al. (30) was verified by adding increasing concentrations of H2O2 (0-3 µM) to assay medium containing L-tyrosine (1 mM) and HRP (5 units/ml) for 5 min followed by an assessment of dityrosine formation (see "Experimental Procedures" for details). A dose-dependent linear relationship between H2O2 concentration and dityrosine formation was observed (Fig. 2, inset). When HRP or L-tyrosine was excluded from the reaction, dityrosine formation was not detectable (results not shown).


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Fig. 1.   a, dimerization of substituted phenolic compounds by heme peroxidase-catalyzed oxidation by H2O2. The ability of H2O2 to induce heme peroxidase-catalyzed dimerization of substituted phenolic compounds is well recognized. This effect is mediated by a series of reactions that begins with the reversible oxidization of HRP by H2O2 to an intermediate compound capable of reacting with phenolic substrates to form radicals that combine to form dimers (see Ref. 26 for details). R', alkyl groups. b, structural comparison of the p-substituted phenolic compounds, tyrosine and tyramide. Both compounds are capable of undergoing homodimerization and heterodimerization reactions by the mechanism described in a. R', fluorescein moiety in tyramide-FITC.


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Fig. 2.   Dityrosine formation by TGF-beta 1-induced H2O2 production in the presence of a heme peroxidase, HRP, in cultured human lung fibroblasts. Inset, increasing H2O2 concentrations were added to assay medium containing HRP (5 units/ml) and L-tyrosine (1 mM) for 5 min, and dityrosine formation was measured as described under "Experimental Procedures." Using this assay system, the effect of endogenous H2O2 production on dityrosine formation was assessed in control and TGF-beta 1-stimulated (2 ng/ml × 16 h) fibroblasts in the presence/absence of HRP (5 units/ml), catalase (1000 units/ml), or diphenyliodonium (DPI, 10 µM, a flavoprotein inhibitor added 15 min prior to assay). The starting concentration of L-tyrosine in all cases was 1 mM. Values of dityrosine formation are expressed as mean ± SD, n = 4.

To determine then whether physiological concentrations of H2O2 generated by cytokine-stimulated fibroblasts are capable of inducing L-tyrosine dimerization, the same assay medium (with and without HRP) was added to TGF-beta 1-treated fibroblasts at the peak time point when these cells have been shown to actively release H2O2 into the overlying media (14). In the presence of HRP, dityrosine formation was stimulated markedly in TGF-beta 1-treated cells, whereas it was undetectable in control cells (Fig. 2). The elimination of HRP from the reaction mixture resulted in undetectable dityrosine formation, indicating the requirement for peroxidative chemistry to mediate this reaction and supporting the general reaction mechanism shown in Fig. 1a. In further support of this, the substitution of catalase in place of HRP failed to generate any dityrosine (Fig. 2). Additionally, cotreatment of catalase (1000 units/ml) with HRP significantly inhibited TGF-beta 1-induced dityrosine formation (Fig. 2). Relatively high concentrations of catalase were required for inhibition, most likely related to the fact that catalase has to "compete" with HRP for H2O2, and the rate constants for these reactions are several orders of magnitude higher for HRP than for catalase (32). Almost complete inhibition of TGF-beta 1-induced dityrosine formation was observed when cells were preincubated with diphenyliodonium (DPI, 10 µM, added 15 min prior to measurement), a flavoprotein inhibitor that blocks TGF-beta 1-induced H2O2 production (14) (Fig. 2). Taken together, these results indicate the ability of H2O2 generated by TGF-beta 1-stimulated fibroblasts to mediate HRP-catalyzed dimerization reactions involving the phenolic amino acid L-tyrosine in the extracellular milieu of cultured fibroblasts.

Detection and Localization of Endogenous Protein Cross-linking Reactions Using a Fluorophore-labeled Phenolic Compound (Tyramide)-- Given the finding that the TGF-beta 1-induced H2O2 is capable of inducing dimerization of exogenous L-tyrosine, we hypothesized that such reactions might occur on tyrosine residues of endogenous cellular or extracellular proteins. We utilized a novel approach to test this hypothesis by making use of a fluorophore-labeled phenolic compound (tyramide-FITC) that undergoes similar reactions to those described for p-substituted phenolic compounds (Fig. 1a; see Fig. 1b for structural comparisons of tyrosine and tyramide). In fact, this property of tyramide is the basis for its utilization in signal amplification (31). This reagent was added to the same HBSS-based assay medium used in the L-tyrosine experiments described above. The amplification diluent provided with the assay kits for signal amplification was not used (because it contains H2O2; see "Experimental Procedures" for details). Fig. 3 demonstrates the characteristic fluorescent pattern observed when TGF-beta 1-treated cells are incubated with tyramide-FITC and HRP. Similar to TGF-beta 1-induced dityrosine formation, the elimination of HRP from the reaction resulted in a loss of the fluorescent signal (Fig. 3). Both catalase and DPI significantly inhibited the fluorescent signal generated by the tyramide-FITC/HRP system (Fig. 3), suggesting a requirement for H2O2 to mediate this effect. These results suggest that TGF-beta 1-stimulated H2O2, in the presence of a heme peroxidase, is capable of mediating cross-linking reactions between tyramide-FITC and tyrosine residues of fibroblast or fibroblast-derived proteins. Based on the localization of this fluorescent labeling reaction and pattern of distribution, the protein(s) that are "targeted" by this reaction seem to reside primarily in the ECM.


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Fig. 3.   Localization of endogenous proteins targeted by H2O2/HRP-dependent tyrosine cross-linking in cultured human lung fibroblasts. Control and TGF-beta 1-treated (2 ng/ml × 16 h) cells were incubated in assay medium containing fluorescein-labeled tyramide in the absence/presence of HRP (5 units/ml) for 1 h followed by washing and fluorescent microscopy (see "Experimental Procedures" and text for details). The effect of catalase (1000 units/ml, coincubated during the assay period) and the flavoprotein inhibitor, diphenyliodonium (DPI, 10 µM added 15 min prior to assay) on the TGF-beta 1-induced labeling (cross-linking) reaction is shown.

Effect of Exogenous H2O2 on the Pattern of Protein Cross-linking in Non-H2O2-producing Cells-- The effect of adding H2O2 exogenously to control cells (not treated with TGF-beta 1) that do not generate extracellular H2O2 on tyrosine cross-linking utilizing the same tyramide-FITC assay system was examined. Cells were exposed to varying concentrations of H2O2 in the assay medium containing HRP and tyramide-FITC for 5 min (see "Experimental Procedures" for details). Fig. 4 demonstrates a dose-dependent effect of H2O2 on fluorescent labeling intensity. The pattern of fluorescent labeling was similar to that observed with endogenous H2O2 production in TGF-beta 1-treated cells, suggesting that the same ECM proteins may be targeted by these cross-linking reactions.


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Fig. 4.   Effect of exogenous H2O2 on fluorophore-labeled tyramide/HRP-dependent fluorescent intensity and pattern in cultured lung fibroblasts. Cultured cells were incubated with assay medium containing tyramide and HRP (5 units/ml) for 5 min. Cells then were washed and viewed under a fluorescence microscope (see "Experimental Procedures" for details). The effect of increasing H2O2 concentrations (0, 1, 5, and 10 µM) on the intensity and pattern of fluorescent labeling is shown.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Growth factors and cytokines are now well recognized to induce the generation of ROS in a variety of nonphagocytic cells for the purposes of cell signaling and in regulation of normal physiological processes (reviewed in Ref. 33). The biological effects of ROS are likely to depend on several factors including the specific reactive species (e.g. O&cjs1138;2 versus H2O2) and its concentration, kinetics, and site of production. The latter factor may be relevant particularly to ROS actions because of the inherently high reactivity of these molecules in biological systems. Thus, the chemistry of specific ROS and the microenvironment in which they are produced may be key determinants of their biological targets and effects.

In this study, we show that "physiological" concentrations of H2O2 generated by TGF-beta 1-stimulated fibroblasts are capable of mediating dimerization of L-tyrosine by a peroxidative mechanism. Moreover, this reaction seems to target tyrosine residues on endogenous protein substrates, suggesting that this mechanism might be important for mediating oxidative cross-linking of proteins under certain physiologic/pathologic conditions. This is the first report, to our knowledge, that demonstrates oxidative cross-linking reactions in response to cytokine-generated ROS. Interestingly, the ability of H2O2 to mediate such reactions on tyrosine residues of ECM proteins has been well demonstrated in the so-called oxidative burst of fertilization to protect the freshly fertilized sea urchin oocyte (27) and to prevent pathogen spread in the "plant hypersensitivity response" (34). Such examples of a physiologic "defense mechanism" in humans are lacking. The best, and likely only, known example of a "purposeful" role for H2O2 in humans of heme peroxidase-catalyzed dimerization reactions involving tyrosine is in the biosynthesis of thyroxine, which is catalyzed by thyroid peroxidase (35, 36). Under pathological conditions, cross-linking of proteins by phagocyte-derived myeloperoxidase has been proposed as a marker of oxidative stress in human atherosclerotic lesions (30).

Similar to arteriosclerosis, pulmonary fibrosis also has been characterized as a disease of oxidative stress (37), although the mechanisms by which oxidants might contribute to disease pathogenesis are not known (38). The data presented here suggest one possible, and even likely, mechanism. The strong association of TGF-beta 1 and myofibroblasts in lesion formation and the involvement of phagocytic cells in this process provide all of the necessary components for this reaction to occur in vivo. The recent data suggesting that the biomechanical properties of the ECM may regulate myofibroblast differentiation are intriguing (15, 16). Oxidative cross-linking of ECM would be expected to reduce tissue compliance and further promote myofibroblast differentiation by this mechanism, suggesting a potential positive feedback loop. Alternatively, cross-linking of ECM proteins may render them less susceptible to digestion by matrix metalloproteinases (39). If such effects can be confirmed in vivo, it would present multiple potential targets for therapy of fibrotic diseases including carefully designed antioxidant strategies.

The specific ECM protein(s) targeted by H2O2/HRP-dependent cross-linking reactions induced by TGF-beta 1 in our study are currently unknown. It is quite clear, however, that these proteins are localized primarily in the ECM based not only on the observed pattern of fluorescent labeling but also on the finding that the extracellular enzymes, HRP and catalase, which do not freely cross the plasma membrane, regulate/alter these reactions when added extracellularly. Candidate molecules for this reaction include collagen and elastin, both of which have been shown to undergo dityrosine-dependent cross-linking when H2O2 and HRP are introduced exogenously (40, 41). Moreover, both of these ECM proteins are well recognized to be up-regulated by TGF-beta 1 (42, 43). The possibility that such reactions also may involve plasma membrane proteins at the cell surface requires further study.

Whether oxidative cross-linking reactions of the type discussed may occur under physiologic rather than pathologic conditions is unclear. Physiologic cross-linking of collagen and elastin is mediated by lysyl oxidase, a copper amine oxidase that modifies the epsilon -amino group of lysine side chains in these ECM proteins to form inter- and intrachain cross-links (44). Interestingly, a "by-product" of this reaction is H2O2. However, no role for lysyl oxidase-generated H2O2 in protein cross-linking has been demonstrated. We have excluded the possibility that lysyl oxidase activity may be responsible for TGF-beta 1-induced H2O2 production. The lysyl oxidase inhibitors, beta -aminopropionitrile and ethylenediamine, had no effect on TGF-beta 1-induced NADH oxidase activity/H2O2 production (20). Moreover, we have demonstrated that this enzyme activity involves a flavoprotein (14), which is not required for lysyl oxidase activity (45). Recent data suggest that the enzyme responsible for this effect of TGF-beta 1 in fibroblasts is related closely to the phagocytic NAD(P)H oxidase family of plasma membrane oxidases.2 Our ability to measure extracellular H2O2 without detectable O&cjs1138;2 in these adherent cells (14) bears some resemblance to the oxidative burst activity of neutrophils adherent to biological surfaces (46, 47), raising the possibility of O&cjs1138;2 generation with subsequent rapid dismutation to H2O2. Homologues of the NAD(P)H oxidase family have been described in various mammalian tissues (36, 48, 49). The findings in this study add to the growing complexity of these enzymes in nonphagocytic cells and potential novel roles for their activity under physiologic/pathologic conditions.

    ACKNOWLEDGEMENT

We thank Dr. Jay W. Heinecke (Washington University School of Medicine, St. Louis) for helpful discussions and suggestions with regard to the measurement of dityrosine.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants K08 HL-03552 and HL-42376.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.

Dagger To whom correspondence should be addressed: Pulmonary and Critical Care Division, New England Medical Center, 750 Washington St., NEMC 257, Boston, MA 02111. Tel.: 617-636-7608; Fax: 617-636-5953; E-mail: vthannickal@lifespan.org.

Published, JBC Papers in Press, February 6, 2001, DOI 10.1074/jbc.M100426200

2 J. M. Larios and V. J. Thannickal, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: TGF-beta 1, transforming growth factor-beta 1; ECM, extracellular matrix; ROS, reactive oxygen species; HRP, horseradish peroxidase; HBSS, Hanks' balanced salt solution; FITC, fluorescein isothiocyanate.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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