Division of Neonatology, Department of Pediatrics, Strong Children's Research Center, University of Rochester School of Medicine, Rochester, New York 14642
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ABSTRACT |
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Cellular fibronectin (cFN) expression is characteristic of injured tissues. Unlike plasma FN, cFN mRNA often contains the EIIIA or EIIIB domains. We examined the lung cell-specific expression of total cFN mRNA and the EIIIA and EIIIB splice variants in rabbits after acute oxygen injury. By in situ hybridization, control lung had low cFN mRNA. After exposure to >95% oxygen, mRNAs for total cFN and EIIIA were noted primarily in alveolar macrophages and large-vessel endothelial cells. By 3-5 days recovery, cFN and EIIIA mRNA abundance was increased in alveolar septal cells (i.e., alveolar epithelial, interstitial, or endothelial cells) and in some large-vessel endothelial cells but was low in bronchial epithelial cells. During recovery, EIIIB mRNA was low in alveolar septal cells but was noted mainly in chondrocytes. Immunostaining for EIIIA increased during recovery, paralleling the in situ hybridizations. Because FN may modulate alveolar type II cell phenotype, we investigated type II cell cFN mRNA expression in vivo. During recovery, neither isolated type II cells nor cells with surfactant protein C mRNA in vivo contained FN mRNA. In summary, these data suggest that cFN with the EIIIA domain has a role in alveolar cell recovery from oxygen injury and that type II cells do not express cFN during recovery.
alveolar type II cells; endothelial cells; surfactant protein C
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INTRODUCTION |
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FIBRONECTIN (FN) is an adhesive extracellular matrix glycoprotein with many putative functions, including regulation of cell proliferation, differentiation, and migration (16). A large, multidomain heterodimer, FN has multiple sites for binding to other extracellular matrix constituents and to signal-transducing cellular integrins. Two major isoforms of FN, plasma FN (pFN) and cellular FN (cFN), result from alternative splicing of the primary mRNA transcript, which is the product of a single gene (28). pFN is synthesized by hepatocytes, is soluble in blood plasma, and lacks sequences coded by two alternatively spliced exons, EIIIA and EIIIB. cFN, which is less soluble, frequently contains either or both of the EIIIA and EIIIB domains. Many cell types can express cFN mRNA; however, its expression is often greatest in developing and injured tissues (4, 17, 24, 31). The biological functions of cFN and the various splice variants are incompletely understood, but the EIIIA and EIIIB domains have been implicated in formation of pericellular matrices and modulation of cell migration and differentiation (10, 11).
In normal adult lung, transcripts for FN can be found in alveolar interstitial cells, endothelial cells, chondrocytes, and vascular smooth muscle cells (31). Normal air space epithelial cells are devoid of FN message. cFN mRNA and FN protein are more abundant in developing lung than in mature lung (31, 32). Normal adult lung expresses cFN mRNA primarily with the EIIIA domain and with little EIIIB (22). In fibrotic lung, several studies have documented increased FN mRNA or protein, especially in fibrotic areas (3, 18, 25). Although cFN probably has roles in repair of injured tissues, limited data are available on cFN expression during repair of acute lung injury (6, 8). In acute, severe oxygen injury, we documented that whole lung cFN mRNA abundance increased during a 5-day recovery in air (22). A role for FN in lung repair is also suggested by its in vitro effects on cell phenotype, including alveolar epithelial type II cells (26, 29, 33, 34), which probably migrate, proliferate, and differentiate into type I cells after severe oxidant injury (1). In vitro studies show that cFN is expressed by migrating airway epithelial cells and that exogenous FN accelerates type II cell migration and closure of an artificial epithelial wound (13, 14, 19). These data suggest that cFN has a role in repair of oxidant injury, but in vivo data are lacking.
In vitro, many lung cell types can express FN, including alveolar epithelial type II cells (7, 21, 27), bronchial epithelial cells (20), endothelial cells (17), and other mesenchymal cells (6). However, type II cell FN mRNA expression in vitro, including the EIIIA and EIIIB variants, is associated with decreased surfactant protein C (SP-C) expression (21, 22). Because SP-C is specific to differentiated type II cells, this observation suggests that type II cells might not express cFN in vivo. In support of this hypothesis, we found that type II cells isolated from normal lung have low cFN mRNA abundance (21). Alveolar macrophages express FN during recovery from oxidant lung injury (30), but the cell-specific expression of cFN by resident lung cells, including type II cells, during recovery has not been investigated. cFN expression by recovering alveolar cells would support the speculation that cFN has a role in alveolar repair. The objective of the current study was to determine the cell-specific expression of total cFN (without regard to splicing) and the EIIIA and EIIIB splice variants during acute oxygen injury and recovery. Our findings show that total cFN mRNA and the EIIIA variant increased in alveolar septal cells, alveolar macrophages, and large-vessel endothelial cells during recovery. cFN transcripts were rare in bronchial epithelial cells, SP-C-expressing cells, or freshly isolated type II cells during recovery. Immunostaining for EIIIA followed temporal and spatial patterns similar to mRNA. Together, these data support the speculation that expression of cFN and the EIIIA splice variant by alveolar and endothelial cells contributes to repair after severe oxygen injury.
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MATERIAL AND METHODS |
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Animals, hyperoxia exposures, and recovery. Hyperoxic exposures were conducted as previously described (23). Adult male rabbits were placed in Plexiglas chambers with constant oxygen flow (7 l/min) for 64 h. Oxygen concentration was checked twice daily and was always >95%. This treatment causes severe alveolar injury as determined by increased alveolar proteins (15). Some animals were allowed to recover in air. At 0, 1, 3, 5, and 7 days of recovery, animals were killed by intravenous injection of pentobarbital sodium. Control animals were maintained in standard vivarium conditions. Both groups were fed standard rabbit chow and were supplied with water. Two separate hyperoxia experiments were performed, which resulted in a total of two control animals and two animals at each time point. Use of animals for these experiments was approved by the University Committee on Animal Resources.
Tissue preparation. To preserve tissue architecture, phosphate-buffered 10% Formalin was instilled in situ into the trachea at a pressure of 20 cmH2O for 20-25 min. The trachea and lungs were then removed together and immersed in Formalin. Tracheal instillation of fixative continued for 2 h at a pressure of 20 cmH2O. The lungs were cut into cubes and embedded in paraffin for later sectioning.
Alveolar type II cell isolation. Alveolar type II epithelial cells were isolated from rabbits recovering in air for 3 days using methods previously described (23). Briefly, the lungs were perfused in situ with saline, removed, and lavaged with protease solution (trypsin, DNase, and elastase). The lungs were minced and filtered, and type II cells were purified on a discontinuous Percoll gradient. The cells were sedimented on slides and prepared for in situ hybridization.
Riboprobe preparation. Rat cDNAs for total cFN (SR270) and the EIIIA and EIIIB exons were gifts from R. O. Hynes (11). The SR270 probe hybridizes to sequences in the 10th and 11th type I repeats, which are not alternatively spliced. It hybridizes to all known cFN mRNA variants. The EIIIA and EIIIB probes are exon specific. The rat cDNAs for total cFN, EIIIA, and EIIIB are 270, 213, and 255 bp, respectively. For double in situ hybridization for cFN and SP-C, we used a rabbit cDNA (2.5 kb) for total FN (30) and a rabbit cDNA (0.85 kb) for SP-C (a gift from F. Possmayer, Univ. of Western Ontario). Sense and antisense complementary RNA (cRNA) probes for rat cFN, the EIIIA and EIIIB variants, and rabbit SP-C were synthesized and labeled with 35S to a specific activity of ~2 × 109 dpm/µg. The rabbit FN probe was synthesized using digoxigenin 11-UTP. The rabbit probes were hydrolyzed to a length of ~200 bp by alkaline hydrolysis.
In situ hybridization.
In situ hybridization was performed as described previously (23).
Briefly, lung tissue sections of 4-µm thickness were
cut, placed on
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic
acid (TES)-treated slides, and dried, and the paraffin was
removed. Cytospun type II cells on TES-treated slides were fixed in
buffered Formalin and rinsed in SSC (1× SSC is 0.15 M NaCl and
0.015 M sodium citrate, pH 7.0). They were then dehydrated and dried
for storage at 70°C. Before hybridization, the tissue
sections were treated with proteinase K. All slides were then
equilibrated with 100 mM triethanolamine-HCl (pH 8.0) and treated with
0.25% acetic anhydride. The slides were then washed in SSC,
dehydrated, and dried. Prehybridization was at 55°C for 2 h. The
slides were then rinsed in SSC and dehydrated. Hybridization was
performed for 16 h at 53°C using a hybridization solution
containing 30 ng · kb
1 · ml
probe
1. After
hybridization, slides were rinsed, digested with RNases A and T1, and
rinsed in RNase buffer. The slides were then rinsed in 0.2× SSC
at 60°C and in 0.1× SSC at room temperature. The slides were
dehydrated, dipped in a 1:1 dilution of NTB-2 emulsion
(Eastman Kodak, Rochester, NY), exposed at 4°C for 3 wk, developed,
fixed, and stained with hematoxylin and eosin.
Double in situ hybridization.
Double in situ hybridization was performed as detailed previously (23).
Hybridization with the radiolabeled SP-C probe was conducted as in
In situ hybridization
until the RNase digestion step. The slides were not
digested with RNases but were washed in 0.5× SSC for 1 h.
The slides were then rinsed in 2× SSC and 0.1× SSC,
followed by dehydration, dipping in emulsion, exposure, development,
and fixation as above. Slides for double in situ hybridization were not
counterstained but were dehydrated, and the hybridization was repeated
as above using hybridization solution containing 150 ng · kb1 · ml
digoxigenin-labeled probe
1
for rabbit FN. Washes were as above, including RNase digestion. After
the washes, the double in situ hybridization slides were treated with
anti-digoxigenin antibody-alkaline phosphatase conjugate and then
incubated for the chromogen reaction as previously described (23). These slides were not counterstained but were rinsed
in water and coverslipped using Aqua Polymount (Polysciences,
Warrington, PA).
Immunohistochemistry for total FN and EIIIA. Goat polyclonal anti-rabbit FN antibody was used to detect total FN (Cappel, Durham, NC). This antibody detects both pFN and cFN. After deparaffinization and rehydration, slides were placed in 0.3% H2O2 in methanol for 30 min and rinsed in Tris-buffered saline (TBS) and in TBS containing 0.05% Triton X-100. Slides were then blocked with 3% BSA in TBS for 45 min and incubated overnight at 4°C with the primary antibody diluted 1:2,000 with TBS and 0.1% BSA. (Purified goat IgG diluted to the same concentration was used as a negative control.) The slides were rinsed in TBS for 15 min, and then biotinylated rabbit anti-goat IgG antibody diluted 1:200 was applied for 1 h. After a rinsing in TBS, avidin-biotinylated horseradish peroxidase complex (ABC Elite) reagent (Vector Laboratories, Burlingame, CA) was applied for 30 min. Slides were rinsed in TBS, and 3,3'-diaminobenzidine substrate was applied for 10 min. The slides were rinsed and lightly counterstained with 0.01% methylene blue.
Mouse monoclonal anti-human cFN (DH1) was used to detect cFN containing the EIIIA domain (ICN Biochemicals, Costa Mesa, CA). This antibody does not cross-react with pFN. Antigen retrieval was necessary to unmask the EIIIA epitope on Formalin-fixed, paraffin-embedded tissues. After deparaffinization and rehydration, the slides were boiled by microwave in 100 mM citric acid, pH 2.0, for 10 min. The immunostaining was performed as for total FN, except slides were blocked with 3% horse serum, and the primary antibody was diluted 1:150. (Mouse monoclonal IgG1 antibody against Aspergillus niger glucose oxidase diluted 1:150 was used as a negative control.) The secondary antibody was biotinylated horse anti-mouse IgG diluted 1:200. ![]() |
RESULTS |
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Expression of total cFN mRNA increases in alveolar and endothelial cells during recovery from acute oxygen injury. The rat probes for total cFN and the splice variants used in the current investigations detect appro-priately sized transcripts in rabbit tissues (22). Lung tissue sections from control and oxygen-injured rabbits were hybridized with the rat cRNA probe for total cFN, which detects all FN splice variants. The results from two separate experiments were nearly identical. In control lung, little or no total cFN mRNA was detected in alveolar septal cells (i.e., alveolar epithelial, interstitial, or endothelial cells), bronchial epithelial cells, large-vessel endothelial cells, or loose adventitial fibroblasts (Fig. 1, A and B). Weak hybridization was noted in vascular smooth muscle cells (not shown). Hybridization with sense-strand probe gave very low background (Fig. 1C). After 64 h of hyperoxia with no recovery, the alveolar septa were thickened, and inflammatory cells were abundant. Overall, cFN mRNA signal intensity appeared increased, due mostly to cells located in the alveolar spaces (Fig. 1D). Higher magnification indicated that these cells were alveolar macrophages (Fig. 1E). Polymorphonuclear cells had no cFN mRNA, and little cFN message was noted in alveolar septal cells. Chondrocytes and endothelial cells in muscularized arteries had increased cFN mRNA after injury (Fig. 1F), but little or no signal was noted in bronchial epithelial cells.
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FN EIIIA and EIIIB mRNA expression. In situ hybridization using the exon-specific rat probe for the EIIIA splice variant gave results that were similar to total cFN. Relatively low levels of EIIIA mRNA were detected in control lung (Fig. 2A). Alveolar macrophages and endothelial cells of muscularized vessels in the 64-h hyperoxic lung had abundant EIIIA mRNA (Fig. 2B). Unlike total cFN, EIIIA transcripts were not seen in chondrocytes. Sense-strand hybridization gave low signal (Fig. 2C). At 1 day of recovery, hybridization was somewhat increased compared with controls, and signal intensity increased further in alveolar septal cells at 3 and 5 days of recovery (Fig. 2, D-I). Similar to total cFN, EIIIA message was also detected in large-vessel endothelial cells and adventitial cells in the recovering animals but was not abundant in bronchial epithelial cells. In situ hybridization using the EIIIB probe gave a pattern that differed from EIIIA. EIIIB mRNA was very low in all cells of the control lung; after 64 h of hyperoxia without recovery, some signal was detected in alveolar macrophages but not in endothelial cells (not shown). Unlike EIIIA, EIIIB mRNA was abundant in chondrocytes of oxygen-injured animals (Fig. 3). During recovery, EIIIB mRNA remained low in alveolar septal cells compared with EIIIA but remained abundant in chondrocytes (not shown).
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cFN mRNA is not expressed in isolated type II cells or SP-C-expressing cells in recovering lung. The identity of the recovering alveolar septal cells with total cFN or EIIIA messages could not be completely discerned by light microscopy of the tissue sections. Alveolar type II cells identified by morphological criteria in fibrotic lung have been reported to have FN mRNA in vivo (3), and type II cells can express FN in vitro (7, 21, 22). To determine whether type II cells express cFN during recovery, we performed in situ hybridization for total cFN on cytospin preparations of freshly isolated type II cells from 3-day recovering animals. These cells rarely had significant cFN mRNA signal (Fig. 4A). As a positive control, we analyzed cytospin preparations of alveolar macrophages isolated from recovering lung. These cells had abundant FN mRNA (Fig. 4B).
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Immunolocalization of total FN and the EIIIA isoform. The antibody used to immunolocalize total FN recognizes all forms of FN including pFN. Control lung had immunostaining noted primarily around blood vessels. Little or no staining was detected in airway epithelial cells or in the alveolar septa (Fig. 6A). After 64 h of hyperoxia without recovery, staining intensity increased around blood vessels, and increased staining was noted in alveolar septa (Fig. 6B). At 1 day of recovery, substantial immunostaining was present in the alveolar septa (Fig. 6C) and was also noted in chondrocytes (Fig. 6D). Debris in the alveolar spaces was also positively stained. No staining was noted in airway epithelial cells. By 3 and 5 days of recovery, alveolar septal staining was reduced compared with 1-day recovery but was still noted around blood vessels (Fig. 6, E and F). By 7 days of recovery (Fig. 6G), immunostaining was at relatively low levels in the alveolar septa compared with 1-day recovery. Nonimmune control antibody had no staining (Fig. 6H).
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DISCUSSION |
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Acute hyperoxic lung injury is characterized by destruction of alveolar cells, particularly endothelial and alveolar type I cells, infiltration of inflammatory cells, and increased capillary permeability, which results in accumulation of serum proteins such as pFN. Repair of tissue injuries, including hyperoxic alveolar damage, is associated with cell migration, proliferation, differentiation, and production of provisional extracellular matrix. Each of these cellular functions can be regulated by FN (16). A role for cFN in repair of various tissue injuries is suggested by several in vitro and in vivo studies (4, 14, 17, 24). The major findings of the current investigation are 1) cFN mRNA and immunostaining for EIIIA increased in alveolar septal cells during recovery from acute, severe oxygen injury, consistent with a role for cFN in alveolar repair; 2) the patterns of EIIIA and EIIIB mRNA expression differ, suggesting lung cell-specific cFN alternative splicing and different functions of the EIIIA and EIIIB variants; and 3) neither isolated alveolar type II cells nor SP-C-expressing cells in vivo had cFN mRNA during recovery.
Total FN protein, detected by immunostaining, increases in various
experimental and human lung injuries, including the current study (3,
8, 18). Much of the FN protein in acute lung injury is probably pFN,
resulting from capillary leakage. Increased cFN mRNA and protein during
recovery of injured lung, as documented in this study, imply a role for
cFN that may be separate from pFN. The molecular differences between
pFN and cFN are known (16, 28), but the functional differences between
these isoforms or among the splice variants of cFN are just emerging.
In general, cFN is expressed at low levels in adult tissues but is
increased in injury, coincident with cell migration and proliferation
(9, 11). For example, in an in vitro model of airway epithelial injury,
cFN and the
5
1-integrin
were expressed by migrating cells (14). Some injured tissues reexpress
fetal variants of cFN that contain EIIIA or EIIIB domains (4, 11, 24).
cFN with these domains is often located in injured tissues where cells are migrating or proliferating (9).
The present study found a restricted pattern of cFN expression during recovery from oxygen injury. Confirming previous work (30), we noted that alveolar macrophages had abundant FN mRNA, but polymorphonuclear cells and lymphoid cells did not. Similar to injured skin and kidney (2, 4), cFN expression by infiltrating macrophages preceded cFN expression by resident cells. Limited data in fibrotic human lung suggested that resident pulmonary parenchymal cells have cFN mRNA and protein (3, 18). In the present study of recovery from severe hyperoxic injury, cFN mRNA in resident lung cells was restricted mainly to alveolar septal cells, endothelial cells of muscularized arteries, and chondrocytes. The limited resolution of light microscopy precluded exact identification of the alveolar septal cells with cFN mRNA. Little cFN message and protein were detected in bronchial epithelial cells. Increased endothelial cell cFN expression has been noted during repair of endothelium (17). Both alveolar septal cells and endothelial cells are injured in oxygen toxicity (12). The finding of increased cFN in these cells during recovery suggests a role for cFN in repair of hyperoxic injury.
The cell-specific patterns of EIIIA and EIIIB mRNA expression differed in recovering lung: EIIIB mRNA was limited largely to chondrocytes, whereas EIIIA mRNA was detected in alveolar septal cells, alveolar macrophages, and endothelial cells but not in chondrocytes. A predominance of EIIIA mRNA in infiltrating macrophages has also been noted in injured kidney and peritoneal macrophages, whereas dermal macrophages had both EIIIA and EIIIB mRNA (2, 4). Lack of EIIIA immunostaining in chondrocytes confirmed the mRNA findings. Preferential expression of EIIIB by chondrocytes and EIIIA by endothelial cells has been noted in other organs (9). These data suggest that FN splicing is lung cell-type specific.
The cell-specific roles of different cFN splice variants are not yet known. cFN with the EIIIA domain promotes cellular adherence and stress fiber formation in the presence of pFN (35). EIIIA may be vectorially secreted and incorporate efficiently into pericellular matrices. Thus EIIIA expression by cells such as alveolar macrophages and resident lung cells may modify the composition and adhesive qualities of the provisional matrix after acute lung injury. EIIIB-containing cFN has been implicated in regulating cell proliferation (9). The EIIIB domain may alter the three-dimensional conformation of FN, which would modulate interactions with other matrix components and integrins (9). For example, inclusion of the EIIIB domain exposed a previously cryptic FN epitope, suggesting that the tertiary structure of FN was altered (9). Expression of EIIIB by damaged cartilage underscores its role in chondrocyte response to injury (5), but its unique functions have not been elucidated.
In previous work using Northern hybridizations, we found that fetal rabbit lung had both EIIIA and EIIIB mRNA (22). The finding of increased EIIIA and EIIIB mRNA in recovering lung in the current studies supports the general concept that a fetal pattern of FN expression is reestablished during lung repair. However, several factors limit the conclusion that recovering lung recapitulates fetal patterns of cFN mRNA. First, the cell-specific expression of FN splice variants in fetal lung is not known; therefore, it is not clear that repairing lung cells reexpress variants characteristic of that cell type in development. Second, we found EIIIA mRNA by Northern hybridization in normal adult lung in previous studies (22). Thus the increase in EIIIA mRNA found in the current study may represent increased cFN mRNA abundance rather than a change in FN splicing to reflect fetal patterns.
The identity of cFN-expressing alveolar septal cells is particularly interesting because FN has substantial effects on type II cell phenotype in vitro. Culture of type II cells on an FN-rich matrix promotes loss of type II cell characteristics and acquisition of a spread, flattened type I cell-like phenotype (26, 29, 34) similar to the changes in type II cell phenotype during repair of the alveolar epithelium. FN also stimulates type II cell motility in vitro (19), which may be important in alveolar repair. The finding that isolated type II cells from recovering lung did not have cFN mRNA is similar to findings in cells isolated from normal lung (21). Type II cells in vitro can express cFN mRNA, but this occurred when SP-C mRNA was decreased (21, 22). Similarly, the current study found that SP-C-positive cells rarely had FN mRNA in vivo. These findings suggest that differentiated type II cells do not express FN mRNA in vivo in either normal or recovering lung. A lack of markers for type I cells or for type II cells in transition to type I cells precludes identification of either of these cells as putative FN-expressing cells. Nevertheless, it is unlikely that differentiated type II cells contribute cFN to provisional matrix during alveolar repair of acute oxygen injury.
The temporal and spatial patterns of EIIIA immunostaining generally paralleled the EIIIA message detected by in situ hybridization. EIIIA protein was detected in alveolar septal regions and blood vessels but not in bronchial epithelial cells or chondrocytes. EIIIA immunostaining was greatest during recovery. In contrast, immunostaining for total FN, probably mainly pFN, increased during injury and declined during 7 days of recovery. It is likely that staining for total FN detected abundant pFN in extravasated serum proteins during injury and early recovery. The finding of differing patterns of pFN and EIIIA immunostaining implies differing functions of these molecules in lung injury. Some differences between EIIIA immunostaining and in situ hybridizations were noted. For example, EIIIA mRNA returned to control levels by 7 days of recovery, whereas immunostaining for the protein remained elevated at this time, possibly reflecting protein accumulation during recovery.
These findings have certain limitations. In previous work (22), we found that EIIIB mRNA increased on Northern hybridization during recovery. Although this may be due to increased EIIIB in chondrocytes, it is possible that other cell types had EIIIB mRNA that was below the sensitivity of in situ hybridization. We noted occasional SP-C-positive cells in vivo that also had FN mRNA. This finding is likely due to overlapping cells in the tissue section. We have documented previously that type II cells may have variable SP-C abundance after hyperoxic injury (23). Thus our double in situ hybridization data do not exclude the possibility that low SP-C-expressing type II cells may also express cFN. However, the finding that isolated type II cells had minimal cFN mRNA supports our speculation that these cells do not express cFN in vivo. The methods we used to immunodetect total FN and EIIIA were optimized for the individual antibodies, including the tissue preparation and the concentration of antibody. This allows comparing the patterns of antibody staining over time, but it precludes comparing the abundance of total FN and EIIIA at any particular time point.
In summary, cFN and EIIIA mRNA and immunostaining increase in alveolar septal cells and some large-artery endothelial cells during recovery from acute, severe hyperoxic injury. This pattern is consistent with a role for cFN in repair of alveolar septal and endothelial cells, both of which are damaged by hyperoxia. The paucity of cFN mRNA in type II cells during recovery from injury suggests that putative effects of cFN on type II cell phenotype are mediated by other cell types. The divergent patterns of EIIIA and EIIIB mRNA expression imply lung cell-type specific regulation of cFN alternative splicing.
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ACKNOWLEDGEMENTS |
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We thank R. O. Hynes and F. Possmayer for providing cDNA probes.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-54632 and Specialized Center of Research Grant HL-365443.
Present address of S. Horowitz: CardioPulmonary Research Institute, Winthrop Univ. Hospital, State Univ. of New York at Stony Brook, Mineola, NY 11501.
Address for reprint requests: W. M. Maniscalco, Box 651, Dept. of Pediatrics, Univ. of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642.
Received 17 March 1997; accepted in final form 5 January 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adamson, I. Y. R.,
and
D. H. Bowden.
The type II cell as progenitor of alveolar epithelial regeneration.
Lab. Invest.
30:
35-42,
1974[Medline].
2.
Barnes, J. L.,
E. S. Torres,
R. J. Mitchell,
and
J. H. Peters.
Expression of alternatively spliced fibronectin variants during remodeling in proliferative glomerulonephritis.
Am. J. Pathol.
147:
1361-1371,
1995[Abstract].
3.
Broekelmann, T. J.,
A. H. Limper,
T. V. Colby,
and
J. A. McDonald.
Transforming growth factor beta 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis.
Proc. Natl. Acad. Sci. USA
88:
6642-6646,
1991[Abstract].
4.
Brown, L. F.,
D. Dubin,
L. Lavigne,
B. Logan,
H. F. Dvorak,
and
L. Vandewater.
Macrophages and fibroblasts express embryonic fibronectins during cutaneous wound healing.
Am. J. Pathol.
142:
793-801,
1993[Abstract].
5.
Chevalier, X.,
N. Groult,
and
W. Hornbeck.
Increased expression of the Ed-B-containing fibronectin (an embryonic isoform of fibronectin) in human osteoarthritic cartilage.
Br. J. Rheumatol.
35:
407-415,
1996[Medline].
6.
Dubayo, B. A.,
G. J. Rubeiz,
and
S. E. G. Fligiel.
Dynamic changes in the functional characteristics of the interstitial fibroblast during lung repair.
Exp. Lung Res.
18:
461-477,
1992[Medline].
7.
Dunsmore, S. E.,
C. Martinez-Williams,
R. A. Goodman,
and
D. E. Rannels.
Composition of extracellular matrix of type II pulmonary epithelial cells in primary culture.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L754-L765,
1995
8.
Durr, R. A.,
B. A. Dubayo,
and
L. A. Thet.
Repair of chronic hyperoxic lung injury: changes in lung ultrastructure and matrix.
Exp. Mol. Pathol.
47:
219-240,
1987[Medline].
9.
Ffrench-Constant, C.
Alternative splicing of fibronectinmany different proteins but few different functions.
Exp. Cell Res.
221:
261-271,
1995[Medline].
10.
Ffrench-Constant, C.,
and
R. O. Hynes.
Patterns of fibronectin gene expression and splicing during cell migration in chicken embryos.
Development
104:
369-382,
1988[Abstract].
11.
Ffrench-Constant, C.,
L. Van de Water,
H. F. Dvorak,
and
R. O. Hynes.
Reappearance of an embryonic pattern of fibronectin splicing during wound healing in the adult rat.
J. Cell Biol.
109:
903-914,
1989[Abstract].
12.
Fracica, P. J.,
S. P. Caminiti,
C. A. Piantadosi,
F. G. Duhaylongsod,
J. D. Crapo,
and
S. L. Young.
Natural surfactant and hyperoxic lung injury in primates. II. Morphometric analysis.
J. Appl. Physiol.
76:
1002-1010,
1994
13.
Garat, C.,
F. Kheradmand,
K. H. Albertine,
H. G. Folkesson,
and
M. A. Matthay.
Soluble and insoluble fibronectin increases alveolar epithelial wound healing in vitro.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L844-L853,
1996
14.
Herard, A.,
D. Pierrot,
J. Hinnrasky,
H. Kaplan,
D. Sheppard,
E. Puchelle,
and
J. Zahm.
Fibronectin and its 5
1-integrin receptor are involved in the wound repair process of airway epithelium.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L726-L733,
1996
15.
Holm, B. A.,
S. Matalon,
J. N. Finkelstein,
and
R. H. Notter.
Type II pneumocyte changes during hyperoxic lung injury and recovery.
J. Appl. Physiol.
65:
2672-2678,
1988
16.
Hynes, R. O.
Fibronectins. New York: Springer-Verlag, 1990.
17.
Ishiwata, T.,
T. Aida,
M. Yokoyama,
and
G. Asano.
Fibronectin biosynthesis in endothelial regeneration after intimal injury.
Exp. Mol. Pathol.
60:
1-11,
1994[Medline].
18.
Kuhn, C.,
J. Boldt,
T. E. King,
E. Crouch,
T. Vartio,
and
J. A. McDonald.
An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis.
Am. Rev. Respir. Dis.
140:
1693-1703,
1989[Medline].
19.
Lesur, O.,
K. Arsalane,
and
D. Lane.
Lung alveolar epithelial cell migration in vitro: modulators and regulation process.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L311-L319,
1996
20.
Linnala, A.,
V. Kinnula,
L. A. Laitinen,
V. P. Lehto,
and
I. Virtanen.
Transforming growth factor- regulates the expression of fibronectin and tenascin in beas 2b human bronchial epithelial cells.
Am. J. Respir. Cell Mol. Biol.
13:
578-585,
1995[Abstract].
21.
Maniscalco, W. M.,
R. A. Sinkin,
R. H. Watkins,
and
M. H. Campbell.
Transforming growth factor-1 modulates type II cell fibronectin and surfactant protein C expression.
Am. J. Physiol.
267 (Lung Cell. Mol. Physiol. 11):
L569-L577,
1994
22.
Maniscalco, W. M.,
R. H. Watkins,
and
M. H. Campbell.
Expression of fibronectin mRNA splice variants by rabbit lung in vivo and by type II cells in vitro.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L972-L980,
1996
23.
Maniscalco, W. M.,
R. H. Watkins,
J. N. Finkelstein,
and
M. H. Campbell.
Vascular endothelial growth factor mRNA increases in alveolar epithelial cells during recovery from oxygen injury.
Am. J. Respir. Cell Mol. Biol.
13:
377-386,
1995[Abstract].
24.
Mathews, G. A.,
and
C. Ffrench-Constant.
Embryonic fibronectins are up-regulated following peripheral nerve injury in rats.
J. Neurobiol.
26:
1171-1188,
1995.
25.
Raghow, R.,
S. Lurie,
J. M. Seyer,
and
A. H. Kang.
Profiles of steady-state levels of messenger RNAs coding for type I procollagen, elastin, and fibronectin in hamster lungs undergoing bleomycin-induced interstitial pulmonary fibrosis.
J. Clin. Invest.
76:
1733-1739,
1985[Medline].
26.
Rannels, S. R.,
C. S. Fisher,
L. J. Heuser,
and
D. E. Rannels.
Culture of type II pneumocytes on a type II cell-derived fibronectin-rich matrix.
Am. J. Physiol.
253 (Cell Physiol. 22):
C759-C765,
1987
27.
Sage, H.,
F. M. Farin,
G. E. Striker,
and
A. B. Fisher.
Granular pneumocytes in primary culture secrete several major components of the extracellular matrix.
Biochemistry
22:
2148-2155,
1983[Medline].
28.
Schwarzbauer, J. E.
Alternative splicing of fibronectin: three variants, three functions.
Bioessays
13:
527-533,
1991[Medline].
29.
Shannon, J. M.,
P. A. Emrie,
J. H. Fisher,
Y. Kuroki,
S. D. Jennings,
and
R. J. Mason.
Effect of a reconstituted basement membrane on expression of surfactant apoproteins in cultured adult rat alveolar type II cells.
Am. J. Respir. Cell Mol. Biol.
2:
183-192,
1990[Medline].
30.
Sinkin, R. A.,
M. B. LoMonaco,
J. N. Finkelstein,
R. H. Watkins,
C. Cox,
and
S. Horowitz.
Increased fibronectin mRNA in alveolar macrophages following in vivo hyperoxia.
Am. J. Respir. Cell Mol. Biol.
7:
548-555,
1992[Medline].
31.
Sinkin, R. A.,
R. S. Sanders,
S. Horowitz,
J. N. Finkelstein,
and
M. B. LoMonaco.
Cell-specific expression of fibronectin in adult and developing rabbit lung.
Pediatr. Res.
37:
189-195,
1995[Abstract].
32.
Snyder, J. M.,
J. A. O'Brien,
and
H. F. Rodgers.
Localization and accumulation of fibronectin in rabbit fetal lung tissue.
Differentiation
34:
32-39,
1987[Medline].
33.
Sugahara, K.,
T. Kiyota,
R. A. Clark,
and
R. J. Mason.
The effect of fibronectin on cytoskeleton structure and transepithelial resistance of alveolar type II cells in primary culture.
Virchows Arch. B Cell Pathol. Incl. Mol. Pathol.
64:
115-122,
1993[Medline].
34.
Woodcock-Mitchell, J.,
S. R. Rannels,
J. Mitchell,
D. E. Rannels,
and
R. B. Low.
Modulation of keratin expression in type II pneumocytes by the extracellular matrix.
Am. Rev. Respir. Dis.
139:
343-351,
1989[Medline].
35.
Xia, P.,
and
L. A. Culp.
Adhesion activity in fibronectin's alternatively spliced domain EDa (EIIIA): complementarity to plasma fibronectin functions.
Exp. Cell Res.
217:
517-527,
1995[Medline].
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