Department of Physiology and Cellular Biophysics, Department of Medicine, and St. Luke's-Roosevelt Institute of Health Sciences, College of Physicians and Surgeons, Columbia University, New York, New York 10019
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ABSTRACT |
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The expression of
the v
3-integrin in nonproliferating
vascular beds remains unclear. To determine possible organ-specific differences, we compared
v
3-integrin
expression in the lung and other organs. Paraffin-embedded tissue
sections of lung, liver, brain, muscle and skin obtained from rats were
processed for immunohistochemistry with a monoclonal (LM609) and a
polyclonal antibody (AB1903) against the
v
3-integrin. Immunogold electron
microscopy was used to localize
v
3-integrin in rat lung microvasculature.
With the use of custom-designed primers, lung sections were subjected
to in situ PCR in a thermal cycler to amplify
v
or
3 mRNA. To confirm specific amplification, PCR
products were further hybridized in situ with an
v or
3 cDNA probe. In the lung, the
v
3-integrin protein as well as
v and
3 mRNAs was extensively evident in the endothelium of extra-alveolar and alveolar microvessels, in vascular smooth muscle, and in large bronchial epithelium but not in
the epithelium of alveolar ducts or alveoli. Ultrastructural immunogold
labeling showed the presence of the integrin on the luminal and
abluminal faces of the lung microvascular endothelium but not on the
apical surface of the alveolar epithelium. Staining for the integrin
was generally negative in blood vessels of several systemic organs,
although weak staining was evident in branches of the hepatic portal
vein. The constitutive presence of the
v and
3 mRNAs and the
v
3-integrin in the lung microvascular bed suggests that gene transcription for the integrin is ongoing in lung
vessels. Because it binds vitronectin, the lung vascular
v
3-integrin may play a role in ligation
of bloodborne, vitronectin-containing macromolecular complexes formed
in inflammation.
lung endothelium; in situ polymerase chain reaction; immunohistochemistry
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INTRODUCTION |
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THE
v
3-INTEGRIN
BELONGS to the family of cytoadhesive integrins that are
functionally important for establishing cell-matrix interactions. These
interactions promote cell stability and support cell proliferation and
motility in conditions such as wound healing and development. In blood
vessels, increasing attention has focused on the
v
3-integrin, for which a functional role
is implicated in the proliferating vascular phenotype. Supportive
findings are that the
v
3-integrin is well
expressed in proliferating vessels but not in normal nonproliferating
vessels (4-6, 16). Moreover,
v
3
antagonists downregulate
v
3 expression
and inhibit endothelial, and hence vascular, proliferation (5, 11).
Although its relative absence in systemic vessels accords little
biological significance to the
v
3-integrin in the resting vasculature,
lung findings suggest the opposite. Immunocytochemical evidence from
our laboratory (22) as well as from others (9, 13, 14) indicates that
the
v
3 antigen exists in unstressed lung
vessels. However, the finding is not universal because some (21) have
failed to confirm such an expression. The possibility of preferential
lung vascular expression of the
v
3-integrin requires consideration
because of the potential importance of the integrin in vascular
pathology. The
v
3-integrin binds
vitronectin (18), which is itself capable of binding bacteria (7).
Vitronectin also forms part of the assembly of macromolecular complexes
such as SC5b-9 that is formed in complement activation and the
thrombin-antithrombin complex that is formed during thrombin activation
(18). Moreover, several bacteria, the tat protein that promotes human
immunodeficiency virus infection and some tumor cells such as the
melanoma cell ligate the
v
3-integrin (1,
7, 19).
Because of these potential interactions, ligation of the
v
3-integrin either directly or indirectly
through vitronectin may constitute a mechanism by which some
pathological bloodborne substances are localized to the lung. Although
the consequences of such a ligation remain poorly understood, our
recent data indicate a possible pathophysiological role for
v
3 binding in the lung microvessel. Using
intravital microscopy in isolated lungs, we determined that ligation of
the
v
3-integrin with multivalent vitronectin or with the vitronectin-containing complement complex SC5b-9 increases lung capillary permeability as quantified in terms of capillary hydraulic conductivity (22). These findings implicate the
v
3-integrin in lung
inflammatory responses in that ligation of the integrin in
complement-activated states may lead to the capillary barrier
deterioration that leads to pulmonary edema.
In previous studies in the lung, the
v
3-integrin was localized to extraseptal
vessels, namely, large vessels (9, 13, 14) and venular capillaries
(22). This extraseptal distribution may be of little consequence if
expression is nonexistent in the septal capillaries that comprise the
major lung vascular segment in terms of both vascular surface area and
pathophysiological significance. Hence a critical question is whether
septal capillaries express the
v
3-integrin under resting conditions.
Here we addressed this issue through a direct comparison of the lung
versus major systemic organs of rats. Because mRNA detection confirms
the existence of constitutive protein synthesis, we applied a reverse
transcriptase-polymerase chain reaction to lung histological sections
(in situ RT-PCR) in conjunction with immunohistochemistry to determine
the relative profiles of mRNA and heterodimer expression. Furthermore,
we used immunogold electron microscopy to determine the luminal versus abluminal expression of the
v
3
heterodimer in the lung microvascular endothelium. Our findings
indicate that under nonstressed conditions, the
v
3 heterodimer is extensively expressed
in lung vessels including lung capillaries, although it is expressed
weakly or not at all in vascular beds of several systemic organs. In
addition, we report an unexpected, region-specific expression of the
integrin in the airway.
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METHODS |
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Reagents. We purchased the following: RNase-free DNase I and RNase H (GIBCO BRL, Life Technologies, Gaithersburg, MD), RNase inhibitor (RNasin, Perkin-Elmer, Norwalk, CT), an RT-PCR kit (EZ RT-PCR, Perkin-Elmer), digoxigenin-11-dUTP and anti-digoxigenin antibody conjugated with alkaline phosphatase (AP; Boehringer Mannheim, Indianapolis, IN), ultrapure PCR water (Research Genetics), an RNA isolation kit (RNAzol B, Tel-Test), a hybridization kit (Dig Easy Hyb, Boehringer Mannheim), and pepsin (Sigma).
cDNA. Rat v (RAV 611) and
3 (RIB3
494/3) cDNA were kindly provided by Dr. D. Shinar (Merck Sharpe & Dohme
Research Laboratories, West Point, PA) (20).
Primer designing. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and v and
3 primers were
purchased from GIBCO BRL. For the primer design, we used the sequences
in Shinar et al. (20) because the sequences successfully amplified rat
cDNA. For the
v 3'primer alone, Shinar et al. used
a redundant oligomer. Therefore, we designed the
v
3'primer according to the published human
v cDNA
sequence (2). The 5'primer for
v was
5'-GACTGTGTGGAAGACAATGTCTGT- AAACCC, and the 3'primer was
5'-CCAGCTAAGAGTTGAG- TTCCAGCC. These primers
extended, respectively, from positions 1914 to 1943 and positions 2196 to 2219 of human
v cDNA (GenBank accession no. M14648).
For
3, the 5'primer was 5'-TTCGACGAGATCATGCA
and the 3'primer was 5'-AAGGTCCCGTTCCCGTTGTTGCA. These
primers extended, respectively, from positions 762 to 778 and positions
1462 to 1485 of human
3 cDNA (GenBank accession no.
L28832). The GADPH 5'and 3' primers were
5'-TGAAGGTCGGTGTGAACGGATTTGG-3' and
5'-ACGACATACTCAGCACCAGCATCAC-3', respectively. To check the
specificity of the PCR product amplified by these primers, rat
v and
3 cDNAs flanked by the primer
sequences were used to prepare hybridization probes.
Probes. To prepare probes labeled with digoxigenin, cDNAs for
v,
3, and GAPDH were amplified by PCR in
a thermal cycler (Perkin-Elmer) for 30 cycles with digoxigenin-11-dUTP.
The labeled products were recovered by 0.8% agarose gel electrophoresis.
Antibodies. We purchased an
anti-v
3-integrin polyclonal antibody
(AB1903, Chemicon), an anti-
v
3-integrin
monoclonal antibody LM609 (MAB1976, Chemicon), an anti-CD31 monoclonal
antibody (JC/70A, DAKO), and an anti-
-actin monoclonal antibody
(CG7, ENZO). A monoclonal antibody against alveolar type I cells (RT1)
was kindly provided by Dr. Leland Dobbs (University of
California, San Francisco) (10). The secondary antibodies and
substrates used were goat anti-rabbit or anti-mouse IgG conjugated with
horseradish peroxidase (HRP) and HRP substrates
[3,3'-diaminobenzidine (DAB), DAKO;
3,3',5,5'-tetramethylbenzidine (TMB), Vector
Laboratories], goat anti-rabbit or anti-mouse IgG antibodies conjugated with AP and AP substrates
[5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium
(BCIP/NBT), Bio-Rad; Vector red, Vector Laboratories], and goat
anti-mouse IgG conjugated to 10-nm gold particles (Sigma).
Tissue preparation. The lungs, kidneys, heart, liver, intestines, brain, muscle, and skin were excised from each of seven anesthetized (pentobarbital sodium 35 mg/kg ip) rats. With the use of methods previously described (22), the lungs were cannulated at the pulmonary artery, left atrium, and trachea. The blood-filled vasculature was held at pulmonary artery = left atrial pressure of 8 cmH2O. The tracheal cannula was connected to an air supply and inflated at an airway pressure of 5 cmH2O. A section of the lung was clamped, and 0.2 g of tissue was excised and then homogenized in RNAzol solution. Total RNA was isolated with standard methods with an RNA isolation kit. The remainder of the lung as well as the other organs were fixed in 4% formaldehyde by immersion (16 h). The tissues were embedded and sectioned (8 µm) for immunostaining and in situ PCR.
Southern blots. With the use of standard methods, the isolated
RNA from the lung and other organs was subjected to reverse transcription and then to PCR as follows: 40 µl of the reagent mixture provided with the RT-PCR kit [1× EZ buffer, 200 µM deoxynucleotide triphosphate, 2.5 mM manganese acetate solution,
2.5 U of rTth DNA polymerase, 60 U of RNasin, and 0.45 µM
each v or
3 5'and 3'primers] were added to 10 µl of each sample of isolated
RNA. After incubation for 25 min at 62°C for reverse transcription, the samples were inserted in a thermal cycler (Ampligen PCR 1000, Perkin-Elmer), and 15 PCR cycles were instituted under the following conditions per cycle: 94°C for 60 s, 65°C for 60 s, and
72°C for 120 s.
The PCR products were separated on 1% agarose gel, then transferred to
nylon membrane (Schleicher & Schuell, Keene, NH). After transfer, the
membrane was denatured (80°C for 1 h), treated with prehybridization buffer (30 min at 68°C; DIG Easy Hyb, Boehringer Mannheim), and incubated with denatured, digoxigenin-labeled
v,
3, or GAPDH probe in prehybridization
buffer (overnight at 68°C). Then, after stringency washes with
saline-sodium citrate (SSC) with 0.1% SDS buffer and maleic acid
buffer, the membranes were treated for 30 min with blocking solution
provided with the hybridization kit. Finally, anti-digoxigenin-AP
antibody in blocking solution was applied to the membranes for 30 min.
To detect chemiluminescence, the membranes were incubated with AP
substrate and exposed to X-ray film.
Immunohistochemistry. Serial histological sections were processed for immunohistochemistry as previously described (22). Briefly, paraffin-embedded tissue sections were dewaxed by immersion in xylene. Then the sections were rehydrated in decreasing concentrations of ethanol, inhibited for endogenous peroxidase by immersion in 0.5% hydrogen peroxide in absolute methanol (20 min), washed in running water (10 min), and equilibrated in phosphate-buffered saline (PBS; 0.01 M, pH 7.4).
An important part of the immunohistology protocol was to treat tissue sections with pepsin at an optimal concentration and for a sufficient period to adequately expose the antigenic sites. The protocol used was developed through trial experiments in which incubation of tissue sections in 2 mg/ml of pepsin in 0.1 M HCl for 75 min resulted in successful staining. After digestion, pepsin was inactivated by washes in water. Subsequently, the sections were blocked with 0.2% BSA in PBS to prevent nonspecific binding. Different antibodies were applied to the sections overnight at 4°C in the following concentrations: AB1903, 1:100; LM609, 1:80; RT1, 1:20; CG7, 1:20; and anti-CD31, 1:20. After washes with PBS, the sections were exposed to the appropriate secondary antibody for 1 h at room temperature. Then after further washes with PBS, the sections were incubated for 10 min at room temperature with either DAB or TMB solution containing 0.01% hydrogen peroxide, BCIP/NBT, or Vector red solution depending on whether the secondary antibody was, respectively, HRP or AP linked. The sections were finally washed in running water, counterstained with hematoxylin for DAB or nuclear fast red for BCIP/NBT, dehydrated in a reversed alcohol-xylene series, and prepared for microscopy.
Immunogold electron microscopy. Lung pieces collected from three rats were immersion fixed in 0.2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer for 2 h. To prevent damage to antigenic sites, we avoided the usual postfixation in osmium tetroxide. Tissues were embedded in Unicryl (50°C for 48 h; Electron Microscopy Sciences). Thin sections (~100 nm) were prepared from tissue blocks and placed on uncoated nickel grids. The sections were etched with 4% sodium metaperiodate, blocked with 1% BSA for 30 min, and then incubated with LM609 (1:100) overnight at 4°C. After several washes with PBS, the sections were incubated at room temperature (30 min) with anti-mouse IgG antibody conjugated to 10-nm gold particles (1:45). After postfixation in a mixture of 2% glutaraldehyde and 2.5% paraformaldehyde for 10 min, the sections were stained with uranyl acetate, then finally examined in a Zeiss transmission electron microscope at 80 kV.
In situ RT-PCR. Tissue sections were processed according to the
modified Nuovo (15) method with a RT-PCR kit. After being dewaxed and
air-dried, all sections were digested in 2 mg/ml of pepsin in 0.01 N
HCl (75 min at room temperature). The pepsin was inactivated by 1-min
washes in ultrapure water and absolute ethanol, and then the sections
were air-dried (for a positive control, directly to RT-PCR cycles).
This was followed by RNase-free DNase I digestion (400 U/ml in
digestive buffer) overnight at 37°C (for detected sections) and
RNase digestion (50 U/ml in digestive buffer) for 2 h at room
temperature (for negative control). For RT and in situ PCR processes,
50 µl of the reagent mixture [1× EZ buffer, 200 µM
deoxynucleotide triphosphate, 2.5 mM manganese acetate solution, 0.45 µM each v or
3 5'and
3'primers, 2.5 U/50 µl of rTth DNA polymerase, and 60 U
of RNasin] were added to each section and sealed by an in situ
PCR assembly tool (Perkin-Elmer). After incubation for 25 min at
62°C for reverse transcription, the PCR cycles were instituted in
an in situ PCR cycler (Ampligen PCR 1000, Perkin-Elmer) for 10 cycles.
In situ hybridization. The steps were briefly as follows. After
fixation in 3.7% paraformaldehyde in PBS (pH 7.4) and incubation with
0.02 M HCl and 0.01% Triton X-100-PBS, the slides were digested with
proteinase K (100 µg/ml) in Tris-EDTA buffer and endogenous AP was
removed in cold 20% acetic acid. After washes with PBS, the sections
were prehybridized in prewarmed DIG Easy Hyb prehybridization buffer
for 30 min and incubated with denatured digoxigenin-labeled v or
3 probe in DIG Easy Hyb overnight at
68°C. After stringency washes with SSC with 0.1% SDS buffer and
maleic acid buffer, the sections were blocked in block solution for 30 min and incubated with anti-digoxigenin-AP in block solution for 30 min. Then the slides were further blocked with 0.2% BSA in 0.1×
SSC solution at 45°C for 10 min and incubated in goat
anti-digoxigenin antibody-conjugated AP for 30 min at room temperature.
After three washes with Tris buffer (0.1 M, pH 7.5), the sections were
treated for 10 min at room temperature with NBT (2.5 µl/ml)/BCIP (2.5 µl/ml) in Tris-buffered CaCl2 solution (pH 9.5), and
development was stopped by washing in running tap water. The sections
were counterstained with either nuclear fast red or
immunohistochemistry with anti-alveolar type I antibody and rabbit
anti-mouse IgG conjugated to HRP after Vector red staining. The slides
were then dehydrated and mounted for viewing.
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RESULTS |
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Light microscopy of blood vessels. We determined
v
3-integrin distributions using both a
polyclonal (AB1903) and a monoclonal (LM609) antibody against the
v
3-integrin. Although the polyclonal antibody may recognize several integrins containing the
v-subunit, LM609 is specific for the
v
3-integrin dimer because Tsukada et al.
(22) previously determined that the antibody
immunoprecipitates bands corresponding only to the
v-
and
3-subunits from homogenates of rat lung.
Rat lung sections incubated with either the monoclonal (LM609) or the
polyclonal (AB1903) anti-v
3 antibody
showed extensive and uniform staining of alveolar septal capillaries,
corner microvessels, and medium- to large-size parenchymal vessels
(Figs. 1 and
2). Differential lung immunohistochemistry
with multicolor staining allowed discrimination between endothelial and
epithelial cells. Figure 2a shows a section in which green and
brown discolorations, respectively, were attributable to counterstains
on the anti-CD31 antibody that targeted endothelial cells and the
antibody RT-1 that targeted alveolar type 1 cells. In Fig. 2b,
the purple discoloration is attributable to counterstain on the
anti-
v
3 antibody. A comparison of Fig. 2,
a and b, which were taken ~50 µm apart from the
same microvessel, indicates that the distributions of the endothelial marker and the
v
3-integrin were
coincident in that both the green and the purple stains were most
pronounced on the microvascular intima.
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Because the vascular v
3-integrin may
exist on both endothelium and smooth muscle, we stained the
microvascular tissue sections for smooth muscle
-actin. Absence of
-actin staining (Fig. 2d) indicated that the microvessels
were smooth muscle free, thereby confirming expression of the
v
3-integrin specifically on the microvascular endothelium. Figures 1 and 2 indicate that vascular smooth muscle when detected by positive
-actin staining or by the
presence of thick vascular media always stained positive for
v
3-integrin.
Electron microscopy of blood vessels. To determine the
distribution of the v
3-integrin in lung
capillaries, we prepared lung sections for immunogold cytochemistry
using LM609. By electron microscopy, gold particles that localized
v
3-integrin were evident on both the
luminal and abluminal endothelial surfaces. This result is exemplified
by the capillary shown in Fig. 3,
a and b, although all vessels displayed this bipolar
distribution. The labeling was evident mostly as single electron-dense
particles on the endothelial plasma membrane, but occasionally
aggregated particles were also observed in endothelial cells (Fig.
3a). Figure 3b is a low-magnification micrograph that
captures a larger surface area of endothelium showing labeling with
gold particles at multiple locations. Vascular cells such as platelets
also stained positive for the presence of
v
3-integrin (Fig. 3a). No tissue
labeling was evident when LM609 was replaced by a control,
isotype-matched, nonspecific antibody.
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The airway. To determine distribution of the
v
3-integrin on the airway luminal
surface, in three lungs, we introduced a polythene catheter (PE-10)
through the trachea until it was wedged in the peripheral airway. Then
at pulmonary artery = left atrial pressure of 8 cmH2O and
constant airway pressure of 5 cmH2O, we injected 1 ml of
PBS containing RT-1, the anti-alveolar type 1 cell antibody (1:100),
and AB1903, the anti-
v
3-integrin antibody (1:100), through the catheter. The lungs were then prepared for immunohistochemistry (see METHODS). Figure
4 shows that although staining for
v
3 was well detected on the epithelial
linings of large (diameter >1 mm) and small (diameter 100-200
µm) bronchi, no staining was evident on the alveolar or alveolar
ductal epithelia. This lack of staining was not attributable to a
failed delivery of the infused liquid to peripheral lung regions
because the alveolar surface stained positively for the coinfused RT-1
antibody. Figure 4d shows that LM609 also recognized airway
epithelium, which further affirms the presence of the
v
3-integrin in the lining of conducting airways. These light-microscopic data indicated that
v
3-integrin was expressed luminally in
the epithelial lining of conducting airways but not of alveoli. In
support, by ultrastructural immunogold cytochemistry, no expression of
v
3-integrin was detectable on the apical
surface of alveolar epithelium (Fig. 3, a and b).
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RT-PCR and Southern blot. Total mRNA extracted from rat lung,
kidney, heart, liver, and brain was amplified by RT-PCR and tested for
the presence of v or
3 mRNA by Southern
blot. Parallel Southern blots were also conducted with a GAPDH cDNA
probe as a control. As shown for one experiment in Fig.
5, the presence of bands corresponding to
308 (a) and 723 (b) bp indicated successful amplification of
v and
3 cDNA,
respectively.
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v and
3 mRNA expression in lung
vessels. To localize
v and
3 mRNA expression, we applied in situ
RT-PCR followed by in situ hybridization on
paraffin-embedded tissue sections. To determine the expression of
v and
3 mRNAs in microvessel endothelium
as distinct from the closely adjacent type I alveolar epithelium, a
series of sections was subjected to both RT-PCR and in situ
hybridization as well as to immunostaining with anti-type I cell
antibody. As shown for a single experiment in Fig.
6, a-e, the
PCR-hybridization procedures resulted in staining patterns that were
similar to those of
v
3 protein
expression, namely, for both mRNAs, staining was strong in the
endothelial but not in the alveolar epithelial lining.
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v
3-Integrin
expression in nonpulmonary vessels. Immunohistochemistry of
sections from liver, brain, muscle, and skin were obtained with LM609
and AB1903. Because the staining patterns were identical, we only show
the LM609 data (Fig. 7). The findings in
these organs were in striking contrast to those of the lung. Vascular
v
3-integrin expression was largely not evident in these nonpulmonary beds. Some exceptions were the hepatic portal venous system that stained weakly for
v
3-integrin in both the central vein and
its branches (Fig. 7a). However, neither hepatic arterial
vessels nor interlobular ducts stained for the integrin. No staining
for the integrin was detectable in blood vessels of brain, skeletal
muscle, and skin (Fig. 7, b-d).
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DISCUSSION |
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We show here that under resting conditions,
v
3-integrin expression is abundant at all
levels of the normal lung vascular bed of rat. Our new findings are as
follows. First, this is the first evidence for extensive
v
3-integrin expression in septal capillaries. Because septal capillaries comprise >95% of the lung vascular surface area, expression of the integrin in this vascular segment indicates that ligational responses to the integrin, such as
capillary barrier deterioration (22), may be extensive if induced in
the lung. Because we compared expression within the same animal, we may
conclude that this well-developed expression in the lung occurs despite
poor or absent expression in the vascular beds of systemic organs.
These considerations lead to the important conclusion that lung
vascular proliferation is not a requirement for lung vascular
expression of the
v
3-integrin.
Second, we demonstrate the presence of not only
v
3 protein but also of
v
and
3 mRNAs in lung microvessels. This mRNA evidence indicates for the first time that gene transcription for the
v- and
3-subunits was constitutive and
ongoing in lung vessels rather than being driven by episodic events.
Interestingly, the positive PCR findings in whole organ liquid extracts
(Fig. 5) indicate that systemic organs do express
v and
3 mRNA, although in view of the negative vascular
immunohistochemistry (Fig. 7), we conclude that this expression must
occur in nonvascular cells (e.g., macrophages). These nonpulmonary
findings reaffirm our view that constitutive gene transcription for
v
3-integrin subunits is unique in lung blood vessels.
Third, our ultrastructural data are the first direct evidence for
v
3-integrin expression on both the
luminal and abluminal aspects of lung microvascular endothelium.
Previously, bipolar expression of the endothelial
v
3-integrin has been reported only in
cultured endothelial cells (22). In our present data, occasionally
v
3-integrin-localizing gold particles
appeared aggregated (Fig. 3a). Because the
v
3-integrin aggregates when ligated (3),
the presence of aggregated gold particles suggests that in some regions
the
v
3-integrin may have undergone
ligation. Although we carried out no procedures to ligate the integrin, luminal platelets may release
v
3 ligands
(e.g., von Willebrand factor). Thus in one instance, we observed
aggregated particles in the region of a platelet-endothelial encounter
(Fig. 3b).
Fourth, for the first time, we report differential
v
3-integrin distribution in the airway.
When we instilled antibodies into the airway, the anti-epithelial
antibody recognized alveolar epithelial cells, indicating that the
instillation reached the alveolar surface. However, the coinstilled
anti-
v
3-integrin antibody stained
epithelial cells only of large airways but not of alveolar ducts or
alveoli (Fig. 4). Furthermore, immunogold microscopy also failed to
reveal
v
3-integrin labeling on the alveolar epithelium. We conclude from these findings that although the
v
3-integrin is well expressed on the
luminal aspect of epithelium in the large airways, expression is absent
in the distal airway and alveolar epithelium. Hence, a decreasing
proximal-distal gradient in the luminal distribution of the
v
3-integrin exists in lung airway
epithelium. This gradient may result from exposure of the proximal
airway epithelium to airborne particles because the large airways of
fetal or newborn rats lack such expression (B. Singh, S. Bhattacharya
and J. Bhattacharya, unpublished observations). The functional
significance of the airway distribution of the
v
3-integrin remains unclear, although it
may be significant for clearance of inhaled particles.
Previously, Damjanovich et al. (9) and Mette et al. (14)
reported expression of the v-subunit but the absence of
the
3-subunit in human bronchial epithelium using
subunit-specific antibodies. These and other workers (13) also reported
little or no staining for the
v
3-integrin
in lung microvessels. We cannot explain these differences from our data
except to suggest that our immunocytochemical methods were different.
As opposed to their frozen tissue sections, we used paraffin-embedded
sections that permitted digestion with pepsin to achieve optimal
unmasking of antigen sites. For immunostaining, we used two
anti-
v
3-integrin antibodies, namely
monoclonal antibody LM609 and polyclonal antibody AB1903. Although
AB1903, being polyclonal, may recognize other
v-containing integrins, LM609 is widely recognized to be
specific for the
v
3-integrin (5, 11, 16)
because it immunoprecipitates only the
v- and the
3-subunits from homogenates of rat lung (22). However,
both antibodies gave highly similar immunostaining patterns. Not only
was the
v
3-integrin well expressed in the endothelium and smooth muscle of large- and medium-size vessels, but
the expression was well evident in the endothelium of smooth muscle-free microvessels. The similar staining patterns for the two
different antibodies together strengthen our conclusion that lung
vessels normally express the
v
3-integrin.
We localized mRNA expression in lung histological sections using in
situ RT-PCR (Fig. 6). v and
3
mRNAs were well expressed in all lung vessels as indicated by these PCR
data. To rule out nonspecific cDNA amplification due to mispriming, we
conducted in situ hybridization of the PCR-amplified product in
the same tissue section using a probe of sufficient sequence length to exclude nonspecific ligation. For our probes consisting of 308 bp for
v and 723 bp for
3, hybridization
confirms specific recognition of selected mRNA and virtually eliminates
the probability of nonspecific ligation. Because mRNA species typically
have a short half-life, the in situ and liquid RT-PCR data provide the
first direct evidence for ongoing gene transcription of the
v- and
3-subunits in lung microvessels.
Finally, our data must be interpreted with the caution that the present
conclusions are applicable only to the rat. It remains unclear as to
whether other species also exhibit a lung-specific vascular
v
3-integrin expression under unstressed
conditions. Nevertheless, the extensive presence of the
v
3-integrin in lung vessels suggests a
potential role for the integrin in lung microvascular function.
Although the full scope remains inadequately understood, possible roles
may be based on the ability of the integrin to regulate microvascular
permeability (22) and affect endocytosis of ligands (17) such as
vitronectin, which associates with complement proteins and the
thrombin-antithrombin complex (18), and entry of viruses such as
hantavirus into endothelial cells (12). These and other potential
functions of the
v
3-integrin in lung
blood vessels and airway require further investigation.
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ACKNOWLEDGEMENTS |
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B. Singh and C. Fu contributed equally to this work.
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FOOTNOTES |
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Rashmi Patel helped with tissue preparation and Jason Reidy with electron microscopy. Dr. David Sims (University of Prince Edward Island, Charlottetown, Canada) provided use of an image-analysis system.
This work was supported by National Heart, Lung, and Blood Grants HL-36024 and HL-53625 (to J. Bhattacharya).
Present address of B. Singh: Department of Veterinary Anatomy, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B4.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Bhattacharya, St. Luke's-Roosevelt Hospital Center, Columbia College of Physicians & Surgeons, 1000 10th Ave., New York, NY 10019 (E-mail: jb39{at}columbia.edu).
Received 12 February 1999; accepted in final form 7 September 1999.
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REFERENCES |
---|
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---|
1.
Albini, A.,
G. Barillari,
R. Benelli,
R. C. Gallo,
and
B. Ensoli.
Angiogenic properties of human immunodeficiency virus type 1 Tat protein.
Proc. Natl. Acad. Sci. USA
92:
4838-4842,
1995[Abstract].
2.
Aznavoorian, S.,
M. L. Stracke,
J. Parsons,
J. McClanahan,
and
L. A. Liotta.
Integrin avb3 mediates chemotactic and haptotactic motility in human melanoma cells through different signaling pathways.
J. Biol. Chem.
271:
3247-3254,
1996
3.
Bhattacharya, S.,
C. Fu,
J. Bhattacharya,
and
S. Greenberg.
Soluble ligands of the v
3 integrin mediate enhanced tyrosine phosphorylation of multiple proteins in adherent bovine pulmonary artery endothelial cells.
J. Biol. Chem.
270:
16781-16787,
1995
4.
Brooks, P. C.,
R. A. Clark,
and
D. A. Cheresh.
Requirement of vascular integrin alpha v beta 3 for angiogenesis.
Science
264:
569-571,
1994[ISI][Medline].
5.
Brooks, P. C.,
S. Stromblad,
R. Klemke,
D. Visscher,
F. H. Sarkar,
and
D. A. Cheresh.
Anti-integrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin.
J. Clin. Invest.
96:
1815-1822,
1995[ISI][Medline].
6.
Casaroli Marano, R. P.,
K. T. Preissner,
and
S. Vilaro.
Fibronectin, laminin, vitronectin and their receptors at newly-formed capillaries in proliferative diabetic retinopathy.
Exp. Eye Res.
60:
5-17,
1995[ISI][Medline].
7.
Chatwal, G. S.,
K. T. Preissner,
G. Muller-Berghaus,
and
H. Blobel.
Specific binding of the human S protein (vitronectin) to streptococci, Staphylococcus aureus, and Escherichia coli.
Infect. Immun.
55:
1878-1883,
1987[ISI][Medline].
8.
Conforti, G.,
C. Dominguez-Jimenez,
A. Zanetti,
M. A. Gimbrone,
O. Cremona,
P. C. Marchisio,
and
E. Dejana.
Human endothelial cells express integrin receptors on the luminal aspect of their membrane.
Blood
80:
437-446,
1992[Abstract].
9.
Damjanovich, L.,
S. M. Albelda,
S. Mette,
and
C. Buck.
Distribution of integrin cell adhesion receptors in normal and malignant lung tissue.
Am. J. Respir. Cell Mol. Biol.
6:
197-206,
1992[ISI][Medline].
10.
Dobbs, L. G.,
M. C. Williams,
and
R. Gonzalez.
Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells.
Biochim. Biophys. Acta
970:
146-156,
1988[ISI][Medline].
11.
Drake, C. J.,
D. A. Cheresh,
and
C. D. Little.
An antagonist of integrin alpha v beta 3 prevents maturation of blood vessels during embryonic neovascularization.
J. Cell Sci.
108:
2655-1661,
1995
12.
Gavrilovskaya, I. N.,
M. Shepley,
R. Shaw,
M. H. Ginsberg,
and
E. R. Mackow.
3 Integrins mediate the cellular entry of hantaviruses that cause respiratory failure.
Proc. Natl. Acad. Sci. USA
95:
7074-7079,
1998
13.
Koukoulis, G. D.,
W. H. Warren,
I. Virtanen,
and
V. E. Gould.
Immunolocalization of integrins in the normal lung and in pulmonary carcinomas.
Hum. Pathol.
28:
1018-1025,
1997[ISI][Medline].
14.
Mette, S. A.,
J. Pilewski,
C. A. Buck,
and
S. M. Albelda.
Distribution of integrin cell adhesion receptors on normal bronchial epithelial cells and lung cancer cells in vitro and in vivo.
Am. J. Respir. Cell Mol. Biol.
8:
562-572,
1993[ISI][Medline].
15.
Nuovo, G. J.
PCR In Situ Hybridization Protocols and Applications (2nd ed.). New York: Raven, 1994, p. 1-416.
16.
Okada, Y.,
B. R. Copeland,
G. F. Hamann,
J. A. Koziol,
D. A. Cheresh,
and
G. J. del Zoppo.
Integrin alphavbeta3 is expressed in selected microvessels after focal cerebral ischemia.
Am. J. Pathol.
149:
37-44,
1996[Abstract].
17.
Pijuan-Thompson, V.,
and
C. L. Gladson.
Ligation of integrin 5
1 is required for internalization of vitronectin by integrin
v
3.
J. Biol. Chem.
272:
2736-2743,
1997
18.
Preissner, K.
Structure and biological role of vitronectin.
Annu. Rev. Cell Biol.
7:
275-310,
1991[ISI].
19.
Rao, S. P.,
K. Ogata,
and
A. Catanzaro.
Mycobacterium avium-M. intracellulare binds to the integrin receptor alpha v beta 3 on human monocytes and monocyte-derived macrophages.
Infect. Immun.
61:
663-670,
1993[Abstract].
20.
Shinar, D. M.,
A. Schmidt,
D. Halperin,
G. A. Rodan,
and
M. Weinred.
Expression of v and
3 integrin subunits in rat osteoclasts in situ.
J. Bone Miner. Res.
8:
403-414,
1993[ISI][Medline].
21.
Suzuki, S.,
T. Takahashi,
S. Nakamura,
K. Koike,
Y. Ariyoshi,
T. Takahashi,
and
R. Ueda.
Alterations of integrin expression in human lung cancer.
Jpn. J. Cancer Res.
84:
168-174,
1993[Medline].
22.
Tsukada, H.,
X. Ying,
C. Fu,
S. Ishikawa,
P. McKeown-Long,
S. Albelda,
S. Bhattacharya,
B. Anderson,
and
J. Bhattacharya.
Ligation of endothelial v
3 integrin increases capillary hydraulic conductivity of rat lung.
Circ. Res.
77:
651-659,
1995