Hermansky-Pudlak syndrome

The natural anticoagulant-activated protein C may inhibit inflammation and fibrosis in the lung. Platelet-derived growth factor is involved in the pathogenesis of lung fibrosis. This study assessed the effect of activated protein C on platelet-derived growth factor expression in human cell lines and in an in vivo model of lung fibrosis. Activated protein C significantly inhibited the secretion and expression of platelet-derived growth factor in human lung cell lines, primary bronchial epithelial cells, and macrophages. In vitro studies also showed that the endothelial activated protein C receptor is expressed by lung epithelial cells and macrophages, and that this receptor and the proteolytic activity of activated protein are implicated in the inhibition of platelet-derived growth factor expression. In the in vivo model of lung fibrosis, intratracheal administration of activated protein C decreased the expression of platelet-derived growth factor and suppressed the development of lung fibrosis. Concomitant intratracheal administration of activated protein C and anti-endothelial activated protein C receptor or antiplatelet-derived growth factor suppressed the inhibitory activity of activated protein C in vivo. In brief, this study describes a novel biological function of activated protein C that may further explain its inhibitory activity on lung inflammation and fibrosis.

Keywords: activated protein C receptor; coagulation; epithelial cells; growth factor; lung fibrosis

Activated protein C (APC) is the active enzyme of the anticoagulant protein C (PC) pathway formed after activation of PC zymogen by the thrombin-thrombomodulin (TM) complex on the phospholipid-rich surface of endothelial cells, platelets, and monocytes (1). APC also indirectly promotes fibrinolysis by directly inactivating plasminogen activation inhibitor-1. In addition to its regulatory function in coagulation and fibrinolysis, APC also plays important roles in inflammatory processes. The systemic administration of APC prevents the lethal effects of Escherichia coli-associated sepsis in experimental animal models, reduces endotoxin-induced pulmonary vascular injury in rats, and is effective for the treatment of patients with meningo-coccemia or acquired PC deficiency (2). Further, a double-blind randomized trial has demonstrated that treatment with APC improves the clinical outcome of patients with sepsis (3). The antiinflammatory activity of APC appears to depend on its ability to suppress the secretion of tumor necrosis factor-[alpha] (TNF-[alpha]) and interleukin-1[beta] (IL-1[beta]) from inflammatory cells, and the activation and extravasation of leukocytes at sites of tissue injury (2-4). The identification of the endothelial PC/ APC receptor (EPCR) reinforces the importance of APC in the inflammatory response (5). EPCR enhances the thrombinthrombomodulin complex-mediated activation of PC and plays an adjuvant role in the host defense against E. coli (6).

Platelet-derived growth factor (PDGF) is a 30-kD dimeric, cationic glycoprotein containing two polypeptide chains, A and B, linked by disulfide bonds; the A and B chains may combine to form three possible PDGF dimers, termed AA, BB, and AB (7). PDGF is a potent mitogen and chemoattractant for mesenchymal cells and induces gene expression of cell matrix-related molecules such as collagen, fibronectin, and glycosaminoglycans (7). Correlation between high expression of PDGF and lung fibrosis in human disease and animal models suggests the role of PDGF in fibroproliferative processes of the lung. Alveolar macrophages from patients with idiopathic pulmonary fibrosis showed increased transcription rates of PDGF-B gene and exhibited an exaggerated production of PDGF-B protein (8). Elevated levels of PDGF-B mRNA have also been demonstrated in alveolar macrophages by in situ hybridization and in bronchoalveolar lavage fluid obtained from rats with bleomycin-induced lung fibrosis (9). High levels of PDGF have also been detected in lavage fluid from patients with Hermansky-Pudlak syndrome, which is a disorder characterized by severe pulmonary fibrosis (10). Induction of lung fibrosis by intratracheal instillation of PDGF or inhibition of lung fibrosis by blockers of PDGF receptor phosphorylation in experimental animals also illustrates the relevance of this growth factor in the pathogenesis of lung fibrosis (11, 12).

We have previously demonstrated that activated protein C generation is decreased in the lungs of patients with interstitial lung disease and that this decreased APC generation is associated with enhanced collagen formation in the lungs of these patients (13, 14). In addition, we have previously shown that intratracheal administration of APC inhibits the development of lung fibrosis in a mouse model of lung injury, further supporting the role of APC in the pathogenesis of interstitial lung disease (15). The mechanism of the inhibitory activity of APC on lung fibrosis is not completely clear. In the present study, we evaluated whether APC inhibits the expression of PDGF in human lung cells and in an experimental animal model of lung fibrosis.

METHODS

(For more details see the online supplement.)

Cell Culture

Human alveolar epithelial (A549) cells, human bronchial epithelial (BEAS-2B) cells, and THP-1 cells were obtained from the American Type Culture Collection (Manassas, VA), and primary human umbilical vein endothelial cells (HUVECs), human pulmonary artery endothelial cells (HPAECs), and primary normal human bronchial epithelial (NHBE) cells were obtained from Clonetics (Walkersville, MD). A549 cells and THP-1 cells were cultured in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO), primary NHBE cells in CCMD161 (Clonetics), and HUVECs and HPAECs in MCDB131 medium (Clonetics), all of them containing 10% heat-inactivated fetal bovine serum and supplements. BEAS-2B cells were cultured in serum-free Ham's F-12 medium.

Preparation of Conditioned Medium

After incubating the cells for 24 hours in basal medium without supplements, all cells except THP-1 cells were washed and incubated for 48 hours in the presence of 125 nM thrombin and various concentrations of APC. THP-1 cells were incubated in the presence of phorbol ester myristate to induce macrophage differentiation and then stimulated with LPS in the presence of various concentrations of APC. In separate experiments, before treating the cells with 125 nM APC, BEAS-2B cells were pretreated with various concentrations of monoclonal anti-human EPCR antibody, RCR-252, in the presence of 125 nM thrombin. Furthermore, BEAS-2B cells were stimulated with 125 nM thrombin and 125 nM APC in the presence of monoclonal anti-APC antibody or normal mouse IgG for 48 hours or with 25 nM thrombin and 125 nM diisopropyl fluorophosphate (DIPF)-APC for the same period of time. The conditioned medium of each cell culture was then harvested, centrifuged at 1,200 x g for 15 minutes, and stored at -80[degrees]C until use. To evaluate the time course of APC-mediated inhibition of PDGF secretion, BEAS-2B cells were culture in the presence of both 125 nM thrombin and 250 nM APC and then the conditioned medium was harvested 3, 6, 12, 24, and 48 hours after starting cell stimulation.

Enzyme Immunoassay

Sandwich enzyme immunoassay of PDGF was performed as previously described (16). The bronchoalveolar lavage fluid (BALF) concentrations of interleukin-6 (IL-6) and tumor necrosis factor-[alpha] (TNF-[alpha]) were measured with mouse-specific enzyme immunoassay kits purchased from Biosource International (Camarillo, CA).

Animal Model of Lung Fibrosis

Pathogen-free, 8- to 10-week-old, female C57BL/6 mice, weighing 18-22 g, were purchased from Nihon SLC (Hamamatsu, Japan) and maintained in a specific pathogen-free environment in the animal house of Mie University (Tsu City, Mie, Japan). The animal study was approved by the Mie University Review Board. The induction of lung fibrosis with bleomycin and treatment with APC by intratracheal administration were performed essentially as described previously (15). One group of animals was treated with sterile saline by minipump (SAL group), and another group with BLM by minipump (BLM group). On Day 7 of saline or BLM minipump treatment, some mice from the SAL group were treated with intratracheal vehicle (SAL/Veh group; n = 5) and some were treated with APC (SAL/APC group; n = 5), and some animals from the BLM group were treated by intratracheal instillation of vehicle (BLM/Veh group; n = 5), APC (BLM/APC group; n = 5), or DIPF-APC (BLM/ DIPF-APC group, n = 5). To evaluate the role of EPCR in APC-mediated inhibition of lung fibrosis in the mouse, another set of in vivo experiments including a group of animals treated with BLM by subcutaneous minipump infusion on Day 0 and intratracheal APC in combination with anti-mouse EPCR (1 [mu]g/kg of mouse body weight) in addition to SAL/Veh, BLM/Veh, and BLM/APC groups of animals was performed. In another separate experiment, to demonstrate that the APC effect is indeed due to its modulation of endogenous PDGF, exogenous PDGF (1 [mu]g/kg of mouse body weight) was also administered intratracheally in combination with APC on Day 7 after BLM subcutaneous administration.

Pulmonary Function Tests, Bronchoalveolar Lavage Fluid, and Lung Tissue Sampling

Functional respiratory variables in the animal models were measured with a double-chamber plethysmograph as described (17). BALF samples were taken on Day 14 and lung tissues on Day 21 after BLM or saline subcutaneous infusion as described. The tissue sections were embedded in paraffin and then prepared for hematoxylin-eosin or Azan-Mallory staining.

Immunohistochemistry of EPCR in Lung Specimens and Primary NHBECs

After appropriate preparations, lung histologic samples were treated with monoclonal anti-mouse EPCR (mRCR-16; 1 [mu]g/ml) or with IgG from a nonimmunized rat as first antibody for 60 minutes and then with biotinlabeled rabbit anti-rat IgG and peroxidase-labeled streptavidin, using a Vectastain kit (Vector Laboratories, Burlingame, CA), and then developed by treatment with peroxidase substrate, using a diaminobenzidine (DAB) kit (Funakoshi, Tokyo, Japan) in accordance with the manufacturer's instructions. The sections were counterstained with methyl green (Vector Laboratories).

Immunofluorescence Staining

A confocal laser scanning microscope (Axiovert 100; Zeiss, Thornwood, NY) was used to evaluate the expression of EPCR. After treating cells with or without APC, they were incubated in the presence of anti-human EPCR IgG, washed, and then treated with fluorescein isothiocyanate (FITC)-labeled anti-rat IgG rabbit antibody. Flow cytometry analysis was performed with a FACSCalibur HG flow cytometer (Becton Dickinson, Tokyo, Japan). Cells (1 x 10^sup 5^) were stained with RCR-49 (10 [mu]g/ml), an anti-EPCR mAb, or with rat IgG as control, followed by FITC-conjugated anti-rat IgG.

Semiquantitative Reverse Transcriptase-Polymerase Chain Reaction for Gene Expression

Total RNA was extracted from cultured cells and tissues by the guanidine isothiocyanate procedure, using TRIzol reagent (GIBCO Life Technologies, Grand Island, NY). Two micrograms of total RNA was reverse transcribed with oligo(dT) primers and then the cDNA was amplified by polymerase chain reaction (PCR), using a Superscript preamplification system kit (GIBCO Life Technologies) according to the manufacturer's instructions and a thermal cycle program (ASTEC, Fukuoka, Japan). The sequences of the primers used in the experiments are described in Table 1. All PCR studies were performed under preplateau conditions for each amplification. PCR products were run on a 2% agarose gel and the bands were visualized by ethidium bromide staining and ultraviolet transillumination. The intensity of the bands was quantified by densilometric analysis, using the public domain NIH Image program (W. Rasband, Research Service Branch, National Institutes of Health, Bethesda, MD) on a Macintosh computer.

EPCR cDNA Cloning, Southern and Northern Hybridizations

The PCR product of EPCR was cloned by the TA cloning method, using a TOPO TA cloning kit (Invitrogen, San Diego, CA) in accordance with the manufacturer's protocol. The sequence of the double-stranded stXI cDNA fragment was determined with a dye-terminator cycle sequencing FS Ready Reaction kit and an ABI 373A DNA sequencer (PE Biosystems, Foster City, CA). The EPCR clone was then used for Southern hybridization after labeling with [[alpha]-^sup 32^P]dCTP. For Northern hybridization of PDGF-A, PDGF-B, and glyccraldehyde-3-phosphate dehydrogenase commercially available cDNA probes were used after labeling with [[alpha]-^sup 32^P]dCTP.

Generation of Human Protein C Inhibitor Gene Transgenic Mice

Transgenic mice were generated by microinjection of the complete (1.5-kb) human protein C inhibitor (PCI) gene (18) into fertilized oocytes of B6C3F1 strain mice (T. Hayashi and colleagues, unpublished data). Transgenic mice were identified by PCR analysis with a pair of primers described in Table 1 for amplification of the 5'-flanking region of the PCI gene, and by an enzyme immunoassay that specifically detects human PCI. Founders and their offspring were bred with B6C3F1 strain mice. Transgenic mice were then housed under a constant 12-hour light and 12-hour dark cycle, and were allowed free access to standard food and water.

Statistical Analysis

All data are expressed as means + or - standard error (SE) unless otherwise specified. The difference between three or more variables was calculated by analysis of variance. Statistical analyses were performed with the Slat View 4.1 package software for the Macintosh (Abacus Concepts, Berkeley, CA).

RESULTS

Expression of Endothelial Protein C Receptor by Lung Epithelial Cell Lines, Primary Epithelial Cells, and Macrophage-differentiated THP-1 Cells

Reverse transcriptase (RT)-PCR showed that, in addition to being expressed on HUVECs, EPCR is also expressed on A549 and BEAS-2B epithelial cell lines, primary NHBE bronchial epithelial cells, and macrophage-differentiated monocytic THP-1 cells. Hybridization of PCR products with cDNA specific for the EPCR gene corroborated these findings (Figure 1A). Immunoperoxidase staining of histologic specimens from mice also revealed the expression of EPCR antigens in airway epithelial cells (Figure 1B). Flow cytometry analysis also showed the expression of EPCR on cell lines and primary bronchial epithelial cells (Figure 1C). Semiquantitative analysis by RT-PCR showed that APC enhances the expression of EPCR in epithelial cells (Figure 1D). Immunofluorescence of EPCR visualized by confocal microscopy also revealed the presence of this receptor on the cell membrane of lung cells (Figure 1E) and macrophages (data not shown), and that EPCR is internalized in A549 and BEAS-2B epithelial cells after treatment with APC (Figure 1E).

APC Inhibits PDGF Expression in Lung Epithelial Cells and Macrophage-differentiated THP-1 Cells

Thrombin induced a marked increase in PDGF concentration in the conditioned media of epithelial cells (Figures 2A-2C) and endothelial cells (Figure 2D); LPS also induced significant expression of PDGF in macrophage-differentiated macrophages (Figure 2E). The expression of PDGF induced by thrombin in A549 and BEAS-2B epithelial cell lines (Figures 2A, 2B) and that induced by LPS in macrophage-differentiated THP-1 cell lines (Figure 2E) were significantly inhibited by APC in a dose-dependent manner. Inhibition assays were also performed in primary epithelial (NHBE) cells to evaluate the potential relevance of the effect of APC in vivo. APC significantly suppressed the secretion of PDGF from primary NHBE cells in a dose-dependent fashion (Figure 2C). The common physiopathological process of all interstitial lung diseases is the occurrence of epithelial/endothelial injury, and thus we also assessed the effect of APC on PDGF secretion from primary endothelial cells (HPAECs) (19, 20). APC also markedly inhibited the secretion of PDGF from primary HPAECs in a concentration-dependent manner (Figure 2D). Viability of cells, as evaluated with a cell counting kit [2-(4-isophenyl)-3-(4-nitrophenyl) -5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1)], was not affected when they were treated for 72 hours with 125 nM thrombin alone or with 125 nM thrombin plus various concentrations of APC in medium without fetal bovine serum (data not shown). The polarity of PDGF secretion in differentiated primary airway epithelial cells was also assessed by an air-liquid interface culture method as described previously (21). Thrombin increased the concentration of PDGF in the culture media of both apical and basolateral regions of epithelial cells; the addition of APC in this assay system decreased the thrombin-induced PDGF secretion from both apical and basolateral regions of epithelial cells (data not shown). After demonstrating that APC exerts the same effects in alveolar (A549) and bronchial (BEAS-2B) epithelial cell lines, as well as in primary epithelial (NHBE) cells and primary endothelial cells (HPAECs), subsequent experiments were performed with only BEAS-2B bronchial epithelial cells. The time course of the APC effect on PDGF secretion was also evaluated. APC-mediated inhibition of PDGF secretion from BEAS-2B cells was statistically significant 24 hours (85.7 + or - 38.3 versus 60.9 + or - 20.9) and 48 hours (783.1 + or - 105.4 versus 439.5 + or - 102.5) after stimulation of cells with thrombin and APC. The effect of APC on PDGF gene expression was also evaluated. Northern blot analysis showed that APC inhibits significantly the thrombin-induced mRNA expression of both PDGF-A, -B in BEAS-2B cells (Figure 2F).

APC-induced Inhibition of PDGF Expression Is Mediated by Endothelial Protein C Receptor

Increasing concentrations of the anti-EPCR antibody blocked in a dose-dependent manner the effect of APC on PDGF secretion from BEAS-2B cells treated with thrombin, suggesting that binding of APC to EPCR is a fundamental step in the inhibitory process induced by APC (Figure 3A). The proteolytic activity of APC was also found lo be necessary for Ihis APC action because the monoclonal anti-APC antibody, which is known to inhibit the activity of APC, significantly suppressed the effect of APC on thrombin-induced secretion of PDGF from BEAS-2B cells (Figure 3B); in addition, diisopropyl fluorophosphate (DIPF)-APC was unable to inhibit PDGF secretion (Figure 3B). Interestingly, the baseline level of PDGF secretion from epithelial cells was also inhibited by APC alone (Figure 3B).

Bleomycin-induced Lung Fibrosis Model

The relevance of this new in vitro biological effect of the natural anticoagulant APC prompted us to prove whether APC also inhibits in vivo PDGF in a mouse model of BLM-induced lung fibrosis. Under profound anesthesia, all animals were killed 3 weeks after BLM administration and lung tissue samples were drawn for histologic staining and mRNA measurements. Before APC treatment, hematoxylin-eosin staining of the lungs showed only mild inflammatory changes on Day 3 (Figure 4B) but more severe inflammatory reaction and early fibrotic changes on Day 7 after BLM administration (BLM group; Figure 4C) compared with animals receiving only saline through a minipump (SAL group; Figure 4A). On Day 21, compared with mice of the SAL/Veh group (Figure 4D), animals of the BLM/Veh group (Figure 4F) showed severe fibrotic changes in the lungs, expanding to the central regions of the lung parenchyma, involving the perivascular and peribronchiolar areas, and with more uniform areas of consolidation in subpleural regions of the lung. By contrast, histologic findings at 21 days in mice of the BLM/APC group showed fewer fibrotic lesions in the subpleural areas, with central areas of lung parenchyma appearing almost normal (Figure 4G). Fibrotic changes were not observed in the SAL/APC group (Figure 4E). BLM/DIFP-APC mice showed severe fibrotic and functional changes in the lungs, suggesting the specificity of APC for the inhibition of BLM-induced pulmonary fibrosis (Figure 4H). Evaluation of lung functional parameters showed a significant decrease in respiratory frequency (Figure 5A) and increased tidal volume (Figure 5B) in mice of the BLM/APC group compared with animals of the BLM/Veh group. Total lung compliance also markedly improved in mice of the BLM/APC group compared with the BLM/Veh group (Figure 5C). There was no significant difference in functional parameters between BLM/Veh and BLM/DIPF-APC groups.

To evaluate whether APC also inhibits PDGF expression in vivo, the levels of PDGF were measured in the lungs of mice with lung fibrosis treated with APC on Day 7. The BALF concentrations of PDGF were significantly decreased in animals of the BLM/APC group compared with those of the BLM/Veh group (Figure 6A). RT-PCR analysis also showed that the relative abundance of mRNA of each PDGF isoform (Figure 6B) is significantly lower in the lung tissue of mice of the BLM/APC group than in animals of the BLM/Veh group. Another important mediator of inflammation, tissue remodeling, and repair in the lung is TGF-[beta]1 (22). Intratracheal instillation of APC was also associated with relatively decreased TGF-[beta] mRNA in the lungs of animals of the BLM/APC group compared with the BLM/Veh group (Figure 6C).

APC Failed to Inhibit PDGF Expression in Lung Fibrosis Induced in a Protein C Inhibitor Transgenic Mouse

PCI is the most potent inhibitor of APC. An increased concentration of human PCI was detected in BALF of our PCI-transgenic (TG) mice compared with non-TG mice. Lung fibrosis was induced in PCI-TG mice secreting increased levels of human PCI in the lung. Induction of lung fibrosis with BLM, and intratracheal administration of APC (TG BLM/APC group) or vehicle (TG BLM/SAL group) on Day 7, were performed according to the same protocol described above. On Day 21 of BLM treatment, compared with the TG SAL group (Figure 7A), both TG BLM/ APC and TG BLM/SAL groups showed severe fibrotic changes in the subpleural and central areas of both lungs (Figures 7B and 7C). The pulmonary function tests showed restrictive patterns in both TG BLM/APC and TG BLM/SAL groups (Figure 7D). There was no significant difference in the BALF concentrations of PDGF between TG BLM/APC and TG BLM/SAL groups (Figure 7E).

Anti-endothelial Protein C Receptor Antibody Inhibits APC-mediated Suppression of PDGF Secretion in the Lung

To investigate whether the inhibitory activity of APC in our animal model is mediated by its receptor, the effect of intratracheal administration of a mixture of APC and monoclonal anti-mouse EPCR antibody on bleomycin (BLM)-induced lung fibrosis was evaluated. Mice of the BLM/SAL group showed increased collagen deposition in the lung compared with the SAL/Veh group (Figures 8A and 8B). Significantly more severe interstitial deposition of collagen was observed in the lungs of mice of the BLM/ APC + anti-EPCR group (Figure 8D) compared with the BLM/ APC group (Figure 8C) on Day 21 of BLM treatment. BALF analysis disclosed a significantly decreased count of total leukocytes, and significantly decreased levels of IL-6, TNF-[alpha], and PDGF in the BLM/APC group compared with the BLM/SAL group; however, total leukocyte count and the BALF levels of IL-6, TNF-[alpha] and PDGF were significantly increased in the BLM/APC + anti-EPCR group compared with the BLM/APC group (Figures 8E-8H).

Exogenous PDCF Suppresses the Inhibitory Activity of APC on Lung Fibrosis

To demonstrate that the APC effect is indeed due to its modulation of endogenous PDGF, exogenous PDGF was also administered intratracheally to mice in combination with APC. The degree of lung fibrosis was significantly less in the BLM/APC group compared with BLM/SAL mice, but it was markedly rescued in the BLM/APC/PDGF group (Figures 9A-9D).

DISCUSSION

A novel finding in the present study is the observation that EPCR is expressed not only by vascular endothelial cells but also by lung epithelial cells and macrophage-differentiated THP-1 cells. EPCR is a Type 1 transmembrane glycoprotein composed of an N-terminal signal sequence, [alpha]^sub 1^ and [alpha]^sub 2^ domains, a transmembrane domain, and a short cytoplasmic tail; it shows high homology with the CD1/major histocompatibility complex Class I family of molecules (23). Previous studies have suggested that part of the antiinflammatory activity of APC is mediated by EPCR. For example, suppression of APC binding to its receptor by a monoclonal antibody was associated with a dramatic enhancement of inflammation in response to sublethal levels of E. coli infusion (6). In the present study, we showed that EPCR mediates the inhibitory activity of APC on the expression and secretion of PDGF, another important mediator of inflammation, from lung epithelial and endothelial cells. The cellular events that ensue after APC binding to EPCR and the intracellular mechanisms leading to inhibition of PDGF expression by APC remain unclear. Some evidence suggests that, after APC binds to its receptor, the APC-EPCR complex is translocated to the cytoplasm (24). Our present results also suggest that internalization of EPCR occurs in bronchial and alveolar epithelial cells after APC treatment. However, further studies should be performed to clarify the link between this intracellular translocation and inhibition of PDGF secretion by APC.

The antiinflammatory activity of APC has been ascribed to its ability to suppress the expression of inflammatory cytokines and the activation and infiltration of neutrophils (2). In accord with this, in our present animal model, BALF analysis showed that treatment with intratracheal APC is associated with significant reduction in leukocyte count and in the level of inflammatory cytokines. In addition, suppression of PDGF expression as seen in our present study may also constitute a pathway through which APC may indirectly decrease the inflammatory response because PDGF itself may also induce inflammation by multiple mechanisms. PDGF may promote inflammation by acting as a chemotactic factor for eosinophils, by inducing the secretion of chemokines such as the chemoattractant macrophage chemotactic protein-1 in mesenchymal cells, or by favoring thrombin formation through the induction of tissue thromboplastin (tissue factor, TF), the initiator of coagulation system activation (25-27). The occurrence of severe inflammatory reaction in the lungs of transgenic mice overexpressing PDGF also supports the role of this growth factor as a triggering factor of inflammation (28). Thus, by inhibiting PDGF expression APC may also be an important modulator of inflammation in the lungs.

Our present investigation showed that inhibition of lung fibrosis by APC in an experimental animal model is associated with relatively lower abundance of PDGF mRNA and with decreased protein concentration of PDGF in the lungs compared with control animals. Although we cannot draw a definite conclusion regarding the cause-effect relationship of this association, it is likely that suppression of lung fibrosis by APC resulted, at least in part, from a decreased lung expression of PDGF. Previous data demonstrating the participation of PDGF in the process of lung fibrosis support this assumption. For example, macrophages and epithelial cells from patients with idiopathic pulmonary fibrosis expressed increased levels of PDGF mRNA and secreted increased amounts of PDGF compared with cells from normal lung (8, 9, 29). In an animal model of pulmonary fibrosis, asbestos inhalation induced upregulation of PDGF-A and -B genes in lung epithelial cells at sites of asbestos fiber deposition (30). In addition, induction of lung fibrosis in animal models by overexpressing PDGF-B from an adenovirus vector administered intratracheally, by intratracheal instillation of recombinant human PDGF-BB, or by developing transgenic mice secreting increased lung concentration of PDGF illustrates the importance of PDGF in the pathogenesis of lung fibrosis (11, 28, 31). Furthermore, the demonstration in the present study that APC is unable to inhibit lung fibrosis when the animals are simultaneously treated with intratracheal APC and PGDF also suggests that APC suppresses the development of lung fibrosis by inhibiting the expression of endogenous PDGF.

A question that needs to be clarified concerns the specificity of the inhibitory effect of APC on lung fibrosis and on PDGF levels in lung injury. To investigate this point, we induced lung fibrosis in TG mice secreting high levels of PCI in the lung and treated them with APC. PCI is the main physiological inhibitor of APC (32). Theoretically, the suppressive effect of APC would not work in the presence of high levels of human PCI. The results of these experiments showed that APC is unable to suppress fibrosis and PDGF upregulation in the lungs of human PCI-TG mice. Although these findings suggest specificity of APC action, they should be interpreted with cautious because PCI, as a multifunctional serine protease inhibitor, inhibits not only APC but also several other proteases such as thrombin, factor Xa, kallikrein, plasminogen activator inhibitor, trypsin, and plasmin (32). To further support the specificity of APC action, the effect of APC inactivated with the protease inhibitor DIPF on lung fibrosis and BALF PDGF levels was also assessed. DIPF-treated APC suppressed neither BLM-induced fibrosis nor PDGF upregulation in the lungs of mice, suggesting the specificity of APC inhibitory activity in our animal model.

Another important finding in this study is that EPCR mediates the suppressive effect of APC on PDGF secretion in vivo. In this experiment, a group of animals was treated simultaneously with APC and anti-mouse EPCR antibody by intratracheal instillation and the degree of fibrosis and cytokine levels were evaluated in comparison with a control group. Anti-mouse EPCR antibody significantly inhibited the suppressive activity of APC on BLM-induced lung fibrosis and on the secretion of PDGF, TNF-[alpha], and IL-6 in the lung. These findings suggest that the therapeutic benefit of APC in our in vivo model may also be partially explained by the EPCR-mediated inhibitory activity of APC on the secretion of PDGF and other inflammatory cytokines. It is worth noting that functional and structural conservation exists in human and murine EPCR. Both human and murine EPCR can bind human APC and, compared with human EPCR, the murine EPCR sequence was found to be 69% identical (33). These findings may explain the efficacy of human APC in our murine in vivo model of lung fibrosis.

Current therapies for interstitial lung diseases, which commonly end up causing lung fibrosis, aim only at suppressing the inflammatory process. The classic therapy for the interstitial lung diseases continues being the chronic administration of corticosteroids despite the lack of blinded therapeutic trial to support the efficacy of this therapy (7). One of the limitations of corticosteroid therapy is its inability to block the overexpression of growth factors, the secretion of which has been found to be upregulatecl by this therapy (34). Workshops have reviewed the current therapies for pulmonary fibrosis and concluded that all of them are ineffective and recommended the development of new compounds that may exert both antiinflammatory and antifibrotic effects (35-37). The results of our present study demonstrating the ability of APC to inhibit PDGF expression and secretion from lung cells extends the spectrum activity of APC and further supports its potential usefulness for the treatment of lung fibrogenic diseases.

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Shino Shimizu, Esteban C. Gabazza, Osamu Taguchi, Hiroki Yasui, Yukiko Taguchi, Tatsuya Hayashi, Masaru Ido, Takeshi Shimizu, Tomohiro Nakagaki, Hiroshi Kobayashi, Kenji Fukudome, Naoko Tsuneyoshi, Corina N. D'Alessandro-Gabazza, Masahiko Izumizaki, Michiko Iwase, Ikuo Homma, Yukihiko Adachi, and Koji Suzuki

Department of Molecular Pathobiology, Third Department of Internal Medicine, Department of Otorhinolaryngology, Mie University School of Medicine, Tsu City, Mie; Chemo-Sero-Therapeutic Research Institute, Kumamoto; Department of Gynecology and Obstetrics, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka; Department of Immunology, Saga Medical School, Saga; and Second Department of Physiology, Showa University School of Medicine, Tokyo, Japan

(Received in original form June 5, 2002; accepted in final form December 3, 2002)

Supported by Grants-in-Aid (nos. 12357017, 13670597,14370055, and 14021044) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Correspondence and requests for reprints should be addressed to Dr. Esteban Cesar Gabazza, Ph.D., M.D., Third Department of Internal Medicine, Mie University School of Medicine, Edobashi 2-174, Tsu City, Mie 514-8507, Japan. E-mail: gabazza@clin.medic.mie-u.ac.jp

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Am J Respir Crit Care Med Vol 167. pp 1416-1426, 2003

DOI: 10.1164/rccm.200206-51SOC

Internet address: www.atsjournals.org

Copyright American Thoracic Society May 15, 2003
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