Pulmonary Vascular Biology Research Laboratory, Providence Veterans Affairs Medical Center, and Department of Medicine, Brown Medical School, Providence, Rhode Island 02908
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ABSTRACT |
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Treatment of cultured bovine pulmonary endothelial cells (BPAEC) with adenosine (Ado) alone or in combination with homocysteine (Hc) leads to disruption of focal adhesion complexes, caspase-dependent degradation of components of focal adhesion complexes, and subsequent apoptosis. Endothelial cells transiently overexpressing paxillin or p130Cas cDNAs underwent Ado-Hc-induced apoptosis to an extent similar to that of cells transfected with vector alone. However, overexpression of focal adhesion kinase (FAK) cDNA blunted Ado-Hc-induced apoptosis. FAK constructs lacking the central catalytic domain or containing a point mutation, rendering the catalytic domain enzymatically inactive, did not provide protection from apoptosis. Constructs containing a mutation in the major autophosphorylation site (tyrosine-397) similarly did not prevent cell death. A FAK mutant in amino acid 395, deficient in phosphatidylinositol 3-kinase (PI 3-kinase) binding, was not able to blunt apoptosis. Finally, overexpression of FAK did not provide protection from apoptosis in the presence of LY-294002, a PI 3-kinase inhibitor. Taken together, these data suggest that the survival signals mediated by overexpression of FAK in response to Ado-Hc-induced apoptosis require a PI 3-kinase-dependent pathway.
cell survival; paxillin; p130Cas; endothelium; focal adhesion complexes
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INTRODUCTION |
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APOPTOSIS (programmed cell death) plays a role in normal morphogenesis and homeostasis of tissues (43). The apoptotic process can be initiated by a variety of factors, including various cytokines, loss of adhesion to substratum, or oxidative stress (2, 17, 43). Once initiated, apoptosis programs involve multiple biochemical pathways, including activation of kinases, phosphatases, and proteases, and also altered mitochondrial function (43). The end results of these processes include DNA condensation, blebbing of the plasma membrane, and cytoplasmic shrinkage that ultimately leads to the formation of apoptotic bodies destined for destruction by neighboring cells. Ultimately, whether a cell traverses this pathway to completion is a function of a balance between conflicting pro- and antiapoptotic signals conveyed via a large number of apoptosis-related gene products, including the caspase and Bcl-2 families.
Endothelial cell apoptosis is associated with vascular injury in atherosclerosis (6), hyperoxia-induced lung injury (2), acute respiratory distress syndrome (31), primary pulmonary hypertension (27), and allograft rejection of heart transplant (38). Moreover, the antiangiogenic properties of angiostatin and endostatin are due to their ability to induce endothelial cell apoptosis (12, 15). Thus apoptosis is an important aspect of endothelial cell biology.
We previously showed that extracellular ATP or adenosine (Ado) at 100 µM induces endothelial apoptosis (14, 34). Such a concentration is likely achieved in the local microenvironment, at least transiently, during processes such as degranulation of platelets, cellular necrosis (as might occur during sepsis, tissue injury, ischemia, or rhabdomyolysis), after sympathetic nerve stimulation, and after membrane transporter-mediated release (20). The mechanism of apoptosis induction includes extracellular ATP hydrolysis and cellular uptake of Ado by cells (14). The ability of the methionine metabolite homocysteine (Hc) to potentiate Ado apoptosis, taken together with the similar induction of apoptosis by S-adenosylhomocysteine hydrolase inhibitors, suggested that the mechanism of ATP- or Ado-induced apoptosis involved S-adenosylhomocysteine hydrolase inhibition (34).
We previously demonstrated that Ado-Hc-induced apoptosis is accompanied by disruption of focal adhesion complexes (FAC), followed by caspase-dependent degradation of their component proteins, including focal adhesion kinase (FAK), paxillin, and p130Cas (22). FAC consist of proteins that link intracellular actin cytoskeleton to extracellular matrix (ECM) components via cell surface integrins (13, 21). Among the proteins in the complexes are nonreceptor tyrosine kinases such as c-Src and FAK. FAK is a 125-kDa nonreceptor kinase important in the assembly of FAC and organization of the cytoskeleton (21). On cell adhesion, FAK undergoes autophosphorylation at tyrosine-397 (21). Once phosphorylated, this tyrosine residue serves as a binding site for the SH2 domains of c-Src or phosphatidylinositol 3-kinase (PI 3-kinase) (36). Autophosphorylation of FAK leads to further FAC formation and recruitment of adaptor proteins such as paxillin or p130Cas.
In light of the fact that Ado-Hc treatment causes FAK, paxillin, and p130Cas degradation, we sought to determine whether 1) expression of FAC components would provide endothelial cell survival signals and 2) the downstream signaling pathways were involved in endothelial cell survival. We employed a strategy of analyzing apoptosis in single cells transiently transfected with expression vectors for these proteins. We found that overexpression of FAK, but not paxillin or p130Cas, partially rescues endothelial cells from Ado-Hc-induced apoptosis and that this rescue requires PI 3-kinase activity.
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MATERIALS AND METHODS |
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Cell culture. Bovine pulmonary artery endothelial cells (BPAEC) were obtained via a enzyme-free scraping technique as previously described (14) and maintained in MEM containing 10% fetal bovine serum, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B (Fungizone). Cells were used at passages 4-13.
Reagents. Ado, DL-homocysteine, and Hoechst-33342 were obtained from Sigma Chemical (St. Louis, MO), LY-294002 from Calbiochem (San Diego, CA), antibodies directed against green fluorescent protein (GFP) from Molecular Probes (Eugene, OR), antibodies against hemagglutinin (HA) from BabCO (Richmond, CA), antibodies against CD2 from BD Pharmingen (San Diego, CA), TdT from Promega (Madison, WI), Texas red-conjugated streptavidin from Jackson Immunochemicals (Bar Harbor, ME), and biotin-dUTP from Roche Molecular Biochemicals (Mannheim, Germany).
Expression plasmids.
The vectors expressing GFP and the GFP-FAK fusion proteins
(pEGFP-FAK, pEGFP-FAT, and pEGFP-FRNK) were kindly provided by C. Damsky (25). The vectors pCD2, pCD2FAK, and pCD2FAK454
(lysine-phenylalanine) were kindly provided by A. Aruffo
(10). The vectors expressing the HA-tagged FAK proteins
pHAFAK, pHAFAK395 (aspartic acid-alanine), and pHAFAK397
(tyrosine-phenylalanine) were kindly provided by H. C. Chen
(9). The vector expressing paxillin (pCEFL-HA-paxillin) was kindly provided by J. S. Gutkind (24). The vector
expressing p130Cas (pSSRCas-FLAG) was kindly provided by
K. Yamada and H. Hirai (39).
Transfections. BPAEC were transfected by a calcium phosphate procedure. Briefly, 24 h after the cells were plated, culture medium was replaced with fresh medium and cells were incubated for 1 h. DNA in 250 mM CaCl2 was added to an equal volume of 2× HBS (270 mM NaCl, 10 mM KCl, 1.4 mM NaH2PO4, and 42 mM HEPES, pH 7.08) and incubated at room temperature for 30 min. In cases where a GFP cDNA was cotransfected, the ratio of effector DNA to GFP cDNA was 3:1 (wt/wt). The DNA-calcium phosphate mixture was then added to the cultures, and the cells were incubated for 5 h. The medium was removed, and the cultures were incubated with 15% glycerol-HBS for 2 min. The glycerol solution was removed and replaced with fresh medium, and the cells were cultured for an additional 24-48 h.
Apoptosis assay.
Cells grown on coverslips in duplicate were washed three times in
phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min, and incubated for 10 min at room temperature with the DNA-specific
stain Hoechst-33342 at 0.1 µg/ml. The coverslips were then mounted in
an antifade solution (5% n-propyl gallate, 0.25% DABCO,
and 0.0025% p-phenylenediamine in glycerol). The cells were
then viewed by fluorescence microscopy (×200) using a fluorescence
microscope (model E6400, Nikon). Successfully transfected cells,
identified by GFP fluorescence or HA immunofluorescence, were
classified as apoptotic or normal on the basis of nuclear morphology. Apoptotic cells had condensed, brightly staining
chromatin, while normal cells had larger, faintly staining nuclei.
Assays were conducted 16 h after treatment with Ado or Ado-Hc. For
each coverslip, enough random fields were evaluated until the total number of GFP- or HA-positive cells analyzed was 100 at the end of a
complete count of the last field. Results are expressed as percentage
of transfected cells with apoptotic nuclei.
Immunoblot analysis.
Immunoblot analysis was performed as previously described
(22). Briefly, BPAEC were scraped and lysed in a buffer
containing 50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet
P-40, 10% glycerol (vol/vol), 5 mM EDTA, 50 mM NaF, 500 µM
Na3VO4, 10 mM -glycerophosphate, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 20 µg/ml
aprotinin. Protein was fractionated by SDS-PAGE, transferred to
polyvinylidene difluoride, and analyzed by immunoblot essentially as
described previously (22). Horseradish peroxidase-conjugated secondary antibodies were obtained from Bio-Rad
(Hercules, CA). Detection was achieved by chemiluminescence (ECL kit,
Amersham Pharmacia Biotech, Piscataway, NJ) according to
manufacturer's instructions.
Immunofluorescence. Cells were analyzed for immunofluorescence as previously described (22). Briefly, cells grown on coverslips were washed once with PBS, fixed for 10 min with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate, pH 7.2. After initial blocking, cells were incubated with primary antibodies in PBS/serum for 1 h at 37°C, washed, and incubated with fluoresceinated secondary antibody in PBS/serum. After final washing, cells were washed and mounted in antifade solution. Photographic images were obtained using a fluorescence microscope (model E6400, Nikon).
Statistical analysis. Values are means ± SD. Differences among the means were analyzed for significance by ANOVA plus Fisher's least significant difference test.
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RESULTS |
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Overexpression of FAK, but not paxillin or p130Cas,
blunts Ado-Hc-induced apoptosis.
We previously demonstrated that Ado-Hc-induced endothelial cell
apoptosis was accompanied by disruption of focal adhesion contacts and degradation of FAC proteins, including FAK, paxillin, and
p130Cas (22). We therefore sought to determine
whether ectopic expression of these proteins would blunt Ado-Hc-induced
apoptosis. We employed an experimental approach of combining
transient transfection of cDNA vectors expressing the proteins of
interest with fluorescence analysis of transfected cells for
apoptosis. We transfected exponentially growing BPAEC with
expression vectors for FAK, paxillin, or p130Cas. As
controls, the vectors lacking cDNA inserts were transfected. To confirm
successful transfection, cells were analyzed by immunoblot and/or
immunofluorescence. Figure 1 demonstrates
that BPAEC transfected with a GFP-FAK construct had a punctate pattern
of fluorescence, consistent with localization of GFP-FAK in FAC (Fig.
1, A and B). Immunoblotting with antibody to GFP
revealed expression of GFP-FAK with the expected molecular mass (Fig.
1C). Similar analyses were performed for all constructs used
in these studies, and confirmation of expression and localization to
FAC was obtained for all constructs (data not shown).
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Central catalytic domain is required for protection from
apoptosis.
To determine which protein domains of FAK were necessary for
protection, we compared the ability of various mutants to blunt apoptosis. FAT is a construct expressing only the focal
adhesion-targeting domain; FRNK contains all the FAT sequences plus an
additional 150 amino acids NH2 terminus to FAT (Fig.
4) (25). Both of these deletion mutants lack the central catalytic domain but bind to integrins (8). Thus these mutants lack tyrosine kinase
activity (25). We also transfected a full-length FAK cDNA
fused to CD2 sequences or a mutant in lysine-454, which abrogates
catalytic activity (10). The CD2 moiety targets the
protein to membranes, yielding a constitutively active wild-type FAK.
To identify successfully transfected cells for these constructs, which
were not GFP fusions, we cotransfected a GFP expression vector. At
24 h after transfection, cells were treated with Ado or Ado-Hc,
and after 16 h cells were assessed for apoptosis. Neither
FAT nor FRNK elicited protection from Ado- or Ado-Hc-induced
apoptosis (Fig. 5A).
Overexpression of FAT induced some apoptosis in the absence of
treatment, in agreement with other reports (25, 26). In
addition, FAT increased Ado-induced apoptosis significantly
compared with Ado-treated, GFP-expressing cells alone. FRNK did not
induce apoptosis in the absence of treatment, in contrast to
the effects seen with FAT. However, FRNK significantly increased the
amount of apoptosis in response to Ado treatment. FAK with
mutation at K454 did not protect, while CD2-FAK did blunt Ado- and
Ado-Hc-induced apoptosis (Fig. 5B). These data
support the notion that the catalytic activity of FAK is necessary for
protection from Ado- and Ado-Hc-induced apoptosis.
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FAK mutants incapable of signaling through PI 3-kinase are unable to prevent cell death. FAK undergoes autophosphorylation on tyrosine-397 and serves as a docking site for the SH2 domains of PI 3-kinase and Src (8). Survival signaling of FAK can proceed through either of these pathways depending on the cellular signal. To assess the roles of PI 3-kinase and Src in FAK-mediated protection from apoptosis, we compared two mutants with respect to their ability to protect against apoptosis. Chan and colleagues (9) recently described a series of HA-tagged FAK constructs, a Y397F mutant and a mutant in D395A (Fig. 4, position denoted by an asterisk). Similar to the Y397F mutant, the D395A mutant is unable to bind PI 3-kinase. However, unlike the Y397F mutant, the D395A binds c-Src and can therefore signal through the c-Src. Thus the D395A mutant is more selective than the Y397F mutant in assessing the role of PI 3-kinase in FAK-mediated signaling.
We transfected cells with wild-type FAK, FAKY397F, or FAKD395A. As expected, overexpression of HA-FAK protected cells from apoptosis (Fig. 6A). The Y397F mutant failed to protect, suggesting that Src or PI 3-kinase binding may be necessary for protection. However, the D395A mutant, which retains Src binding and signaling, also failed to protect, suggesting that expression of a mutant capable of signaling through c-Src could not rescue endothelial cells from Ado-Hc-induced apoptosis. These results suggest that FAK-mediated protection against Ado-Hc-induced apoptosis may require signaling through PI 3-kinase.
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PI 3-kinase inhibition blocks the ability of FAK to protect.
We next sought to determine whether the ability of FAK to mediate
protection required PI 3-kinase activity. We repeated our CD2-FAK
rescue experiments in the presence of 25 µM LY-294002, an inhibitor
of PI 3-kinase. In these experiments, we lowered the doses of Ado-Hc to
500 µM Ado or 50 µM Ado-Hc to better assess the effects of
LY-294002. Ado and Ado-Hc enhanced apoptosis in cultures
transfected with CD2 alone (Fig. 7). As
in Fig. 5B, overexpression of CD2-FAK blunted Ado- and
Ado-Hc-induced apoptosis. However, coincubation with LY-294002
increased apoptosis and blocked the ability of wild-type FAK to
protect from apoptosis induced by Ado or Ado-Hc. These results
suggest that PI 3-kinase activity is necessary for FAK-mediated
protection from Ado-Hc-induced endothelial cell apoptosis.
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DISCUSSION |
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The data presented here indicate that FAK overexpression can protect endothelial cells from apoptosis in response to Ado and Ado-Hc. This result was observed with three distinct FAK constructs. In contrast, paxillin or p130Cas overexpression was ineffective. The inability of FAT, FRNK (both of which lack the catalytic domain), or K454 mutants (which via point mutation lacks catalytic activity) to protect against apoptosis suggests that a functional catalytic domain for FAK is required for this protection. The inability of Y397F mutants (a point mutation in the major autophosphorylation site) to protect suggests that tyrosine-397 phosphorylation is crucial for protection, an observation that is consistent with our previous observations that Ado-Hc-induced apoptosis is protein tyrosine phosphatase (PTPase) dependent (22). The inability of the Y397F (lacking Src and PI 3-kinase binding) or D395A (lacking PI 3-kinase binding but maintaining Src binding) mutants to protect, along with the ability of a PI 3-kinase inhibitor to 1) potentiate Ado-Hc apoptosis and 2) prevent FAK rescue, suggests that PI 3-kinase binding and activity are required for protection.
Ado-Hc-induced endothelial apoptosis is accompanied by FAC disruption and caspase-dependent proteolysis of FAK, paxillin, and p130Cas (22). Normal cultured adherent cells receive survival signals from growth factors and ECM (5). ECM-initiated signals are transmitted through integrins and associated kinases such as FAK (8). Our data suggest that Ado-Hc-mediated degradation of FAC prevents normal survival signaling from proceeding through FAK and PI 3-kinase and extends our knowledge with respect to the mechanisms of Ado-Hc-induced apoptosis.
FAK has previously been demonstrated to provide survival signals in canine kidney epithelial cells (9, 17), HL-60 cells (37), and fibroblasts (25). Apoptosis was induced by microinjection of Madin-Darby canine kidney cells with anti-FAK antibodies or peptides corresponding to a region of the integrin molecule thought to be required for FAK interaction (17). A membrane-tethered, and thereby constitutively activated, FAK rescues COS cells from apoptosis induced by loss of cell surface contact (10), and wild-type FAK overexpression protects serum-deprived primary fibroblasts from apoptosis (25). The catalytic activity of FAK enzyme and tyrosine-397 are necessary for FAK-mediated protection of fibroblasts from apoptosis, a finding consistent with our data (10, 25). However, the signaling mechanism by which FAK protects depends on cell type and culture conditions. Ilic and co-workers (26) demonstrated that binding of the SH3 domain of p130Cas to proline rich-1 region of FAK is required for rat synovial fibroblast survival when cultured on fibronectin (in the absence of serum). This signaling activates c-Jun NH2-terminal kinase via a Ras-Rac-Pak1-MKK4 pathway. Under these conditions, the PI 3-kinase pathway is not activated. However, they observed that if these cells are deprived of attachment in the presence of serum, signaling proceeds via a PI 3-kinase-Akt pathway. Thus it appears that extracellular survival signals through FAK are dependent on cell type and soluble factors.
The reason for protection from apoptosis by FAK overexpression is not clear. FAK can be cleaved in vitro by caspases, and Ado-Hc-induced FAK degradation is caspase dependent (22). It is possible that the presence of excess FAK acts as a competitive inhibitor of caspases and stabilizes focal adhesions by preventing caspase-dependent proteolysis, thus preserving normal PI 3-kinase signaling. Alternatively, FAK overexpression may act as a competitor of some other enzyme, such as a PTPase, thus increasing FAK phosphorylation and maintaining PI 3-kinase signaling. Our previous data implicate a PTPase in Ado-Hc-induced apoptosis and would be consistent with this alternative (21, 22).
The inability of paxillin or p130Cas overexpression to protect suggests that, under these conditions, these FAK-associated proteins do not mediate survival signals, although evidence suggests that they are involved in signaling. Paxillin was first identified as a cytoskeletal protein with increased tyrosine phosphorylation in Src-transformed fibroblasts (4, 19). Paxillin becomes phosphorylated on tyrosine residues in response to a variety of stimuli, including bombesin, platelet-derived growth factor, and cell adhesion (32, 42, 44). It is believed to act as a scaffolding protein in FAC by mediating interactions with other signaling or cytoskeletal proteins (41), suggesting that paxillin may be involved in diverse signaling pathways. Indeed, phosphorylation of paxillin on tyrosines-31 and -118 regulates rat bladder carcinoma cell migration (30).
Recent data also implicate p130Cas in mediating intracellular signaling. As a member of a recently identified family of proteins that serve in mediating cytoskeletal signaling pathways, p130Cas undergoes increased tyrosine phosphorylation in Src-transformed fibroblasts and serves as an adapter protein with one SH3 domain and multiple tyrosines in SH2 consensus sites (29, 35). p130Cas binds to and is phosphorylated by FAK. p130Cas has been implicated in G protein-coupled receptor signaling, in growth factor receptor stimulation, and in antigen receptor stimulation (29). Ilic and co-workers (26) observed that binding of the SH3 domain of p130Cas to proline rich-1 region of FAK is required for fibroblast survival on fibronectin in the absence of serum.
Although paxillin and p130Cas can participate in intracellular signaling, we observe no evidence that they participate in survival signaling required in Ado-Hc-treated endothelial cells. This may be due to the fact that they are structural proteins, in contrast to FAK, which has enzymatic activity. Alternatively, degradation of endogenous FAK may prevent the association of overexpressed paxillin and p130Cas into complexes necessary for providing survival signals.
The downstream effector of FAK survival signals in Ado-Hc-treated cells
is PI 3-kinase, an enzyme that is required for multiple cellular
processes, including cell proliferation, differentiation, and
apoptosis (40). PI 3-kinase comprises a
family of agonist-stimulated lipid-signaling enzymes
that initiate signaling cascades by generating three distinct
membrane phospholipids, the phosphoinositides phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and
phosphatidylinositol 3,4,5-trisphosphate. Many enzymes, including
protein kinases, phospholipases, and G proteins are effector molecules
of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol
3,4,5-trisphosphate, and their activities are affected by lipid-protein
interaction (7). The regulatory unit, p85, of PI 3-kinase
contains an NH2-terminal SH3 domain, a breakpoint cluster
homology (BH) domain, and two SH2 domains. These domains allow p85 to
simultaneously interact with multiple intracellular signaling
molecules. Thus PI 3-kinase can recruit a variety of signaling
molecules. There are several phosphoinositide-binding proteins that are
potential downstream effectors of PI 3-kinase. One potential mechanism
is activation of Akt (also known as protein kinase B), a
serine/threonine kinase that is recruited to the plasma membrane
(7). At the plasma membrane, the PI 3-kinase effector
phosphatidylinositol-dependent kinase-1 (PDK1) phosphorylates Akt at
threonine-308 and serine-473 (1, 16). Activated Akt can
provide survival signals through various downstream pathways, including
nuclear factor-B activation, phosphorylation and inactivation of the
proapoptotic Bcl-2 family member BAD, forkhead transcription
factors (FKHR, FKHRL1, and AFX), glycogen synthase kinase-3
(GSK-3
), CREB, and caspase 9 (40). Thus there are
multiple potential pathways relating FAK and PI 3-kinase to cell survival.
In summary, overexpression of FAK protects endothelial cells from apoptosis induced by Ado-Hc, and this protection is dependent on PI 3-kinase activity and binding. Thus FAK signaling through PI 3-kinase may be important in regulation of endothelial cell survival. Better understanding of factors regulating endothelial cell survival may result in improved treatments of acute lung injury.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Department of Veterans Affairs (VA) Merit Review (S. Rounds), VA Merit Review Type II (E. O. Harrington), and VA/Department of Defense Collaborative Research Project (S. Rounds), National Heart, Lung, and Blood Institute Grant RO1 HL-64936 (S. Rounds) and HL-67795 (E. O. Harrington), and American Heart Association Beginning Grant-in-Aid NG60157T (R. E. Bellas).
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FOOTNOTES |
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Some of the reported studies have been published in abstract form (Am J Respir Crit Care 161: A755, 2000).
Address for reprint requests and other correspondence: S. Rounds, Providence VA Medical Center, Pulmonary/Critical Care Medicine Section, 830 Chalkstone Ave., Providence, RI 02908 (E-mail: Sharon_Rounds{at}brown.edu).
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.
First published January 4, 2002;10.1152/ajplung.00174.2001
Received 23 May 2001; accepted in final form 14 December 2001.
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