Vascular expression of the alpha vbeta 3-integrin in lung and other organs

Baljit Singh, Chenzhong Fu, and Jahar Bhattacharya

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The expression of the alpha vbeta 3-integrin in nonproliferating vascular beds remains unclear. To determine possible organ-specific differences, we compared alpha vbeta 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 alpha vbeta 3-integrin. Immunogold electron microscopy was used to localize alpha vbeta 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 alpha v or beta 3 mRNA. To confirm specific amplification, PCR products were further hybridized in situ with an alpha v or beta 3 cDNA probe. In the lung, the alpha vbeta 3-integrin protein as well as alpha v and beta 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 alpha v and beta 3 mRNAs and the alpha vbeta 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 alpha vbeta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE alpha vbeta 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 alpha vbeta 3-integrin, for which a functional role is implicated in the proliferating vascular phenotype. Supportive findings are that the alpha vbeta 3-integrin is well expressed in proliferating vessels but not in normal nonproliferating vessels (4-6, 16). Moreover, alpha vbeta 3 antagonists downregulate alpha vbeta 3 expression and inhibit endothelial, and hence vascular, proliferation (5, 11).

Although its relative absence in systemic vessels accords little biological significance to the alpha vbeta 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 alpha vbeta 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 alpha vbeta 3-integrin requires consideration because of the potential importance of the integrin in vascular pathology. The alpha vbeta 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 alpha vbeta 3-integrin (1, 7, 19).

Because of these potential interactions, ligation of the alpha vbeta 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 alpha vbeta 3 binding in the lung microvessel. Using intravital microscopy in isolated lungs, we determined that ligation of the alpha vbeta 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 alpha vbeta 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 alpha vbeta 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 alpha vbeta 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 alpha vbeta 3 heterodimer in the lung microvascular endothelium. Our findings indicate that under nonstressed conditions, the alpha vbeta 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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha v (RAV 611) and beta 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 alpha v and beta 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 alpha v 3'primer alone, Shinar et al. used a redundant oligomer. Therefore, we designed the alpha v 3'primer according to the published human alpha v cDNA sequence (2). The 5'primer for alpha 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 alpha v cDNA (GenBank accession no. M14648). For beta 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 beta 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 alpha v and beta 3 cDNAs flanked by the primer sequences were used to prepare hybridization probes.

Probes. To prepare probes labeled with digoxigenin, cDNAs for alpha v, beta 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-alpha vbeta 3-integrin polyclonal antibody (AB1903, Chemicon), an anti-alpha vbeta 3-integrin monoclonal antibody LM609 (MAB1976, Chemicon), an anti-CD31 monoclonal antibody (JC/70A, DAKO), and an anti-alpha -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 alpha v or beta 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 alpha v, beta 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 alpha v or beta 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 alpha v or beta 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Light microscopy of blood vessels. We determined alpha vbeta 3-integrin distributions using both a polyclonal (AB1903) and a monoclonal (LM609) antibody against the alpha vbeta 3-integrin. Although the polyclonal antibody may recognize several integrins containing the alpha v-subunit, LM609 is specific for the alpha vbeta 3-integrin dimer because Tsukada et al. (22) previously determined that the antibody immunoprecipitates bands corresponding only to the alpha v- and beta 3-subunits from homogenates of rat lung.

Rat lung sections incubated with either the monoclonal (LM609) or the polyclonal (AB1903) anti-alpha vbeta 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-alpha vbeta 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 alpha vbeta 3-integrin were coincident in that both the green and the purple stains were most pronounced on the microvascular intima.


View larger version (143K):
[in this window]
[in a new window]
 
Fig. 1.   Immunohistochemistry of rat lung with monoclonal antibody LM609. a: absence of staining to an isotype-matched nonspecific primary antibody. Original magnification, ×100. b: extensive and uniform staining in parenchymal microvessels (arrows). Original magnification, ×100. c: high-magnification view of the 3 microvessels in b (arrows) demonstrating reaction with LM609 along margins. Original magnification, ×500. d: alpha vbeta 3-integrin staining is seen in luminal margin (arrows) of a large vessel and in vascular mononuclear cells (arrowhead). Original magnification, ×100. The results were replicated 3 times.



View larger version (120K):
[in this window]
[in a new window]
 
Fig. 2.   Immunohistochemistry of rat lung with polyclonal antibody AB1903. a-d: serial sections taken at ~50-µm intervals. a: differential immunostaining with antibodies CD31 and RT-1 distinguishes, respectively, microvascular endothelial layer in green (arrow) from surrounding alveolar type 1 epithelial cell layer in brown (arrowhead). Original magnification, ×400. b: purple staining for anti-alpha vbeta 3-integrin (arrow) occurs predominantly in endothelial layer, with poor staining in septum (arrowhead). Original magnification, ×400. c: no significant staining is evident with nonimmune IgG. Original magnification. ×400. d: lack of staining for anti-alpha -actin (arrow) indicates that alveolar microvessels lacked smooth muscle. Original magnification, ×400. e: Anti-alpha -actin staining was well evident in large-vessel media (arrow). f: Presence of alpha vbeta 3-integrin staining on bronchial epithelium (arrow) and in media of a muscular artery (star ). Original magnification, ×100. These results were replicated 3 times.

Because the vascular alpha vbeta 3-integrin may exist on both endothelium and smooth muscle, we stained the microvascular tissue sections for smooth muscle alpha -actin. Absence of alpha -actin staining (Fig. 2d) indicated that the microvessels were smooth muscle free, thereby confirming expression of the alpha vbeta 3-integrin specifically on the microvascular endothelium. Figures 1 and 2 indicate that vascular smooth muscle when detected by positive alpha -actin staining or by the presence of thick vascular media always stained positive for alpha vbeta 3-integrin.

Electron microscopy of blood vessels. To determine the distribution of the alpha vbeta 3-integrin in lung capillaries, we prepared lung sections for immunogold cytochemistry using LM609. By electron microscopy, gold particles that localized alpha vbeta 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 alpha vbeta 3-integrin (Fig. 3a). No tissue labeling was evident when LM609 was replaced by a control, isotype-matched, nonspecific antibody.


View larger version (102K):
[in this window]
[in a new window]
 
Fig. 3.   Immunogold staining of rat lung with anti-alpha vbeta 3-integrin antibody (LM609). Electron micrographs show high (a)- and low (b)-power views of capillary endothelium (E) labeled with gold particles at multiple sites on both luminal (single thin arrows) and abluminal aspects (double arrows). Gold particles are also present in an intraendothelial vesicle (thick arrow) and a luminal platelet (P). Note absence of labeling on alveolar epithelium (arrowheads). AS: alveolar space. Original magnification: ×50,000 in a; ×30,000 in b. These results were replicated 3 times.

The airway. To determine distribution of the alpha vbeta 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-alpha vbeta 3-integrin antibody (1:100), through the catheter. The lungs were then prepared for immunohistochemistry (see METHODS). Figure 4 shows that although staining for alpha vbeta 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 alpha vbeta 3-integrin in the lining of conducting airways. These light-microscopic data indicated that alpha vbeta 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 alpha vbeta 3-integrin was detectable on the apical surface of alveolar epithelium (Fig. 3, a and b).


View larger version (81K):
[in this window]
[in a new window]
 
Fig. 4.   Immunostaining with anti-alpha vbeta 3-integrin (R838) and anti-alveolar type I cell (RT-1) antibodies instilled through airway. Staining for alpha vbeta 3-integrin is strong on epithelium of a large bronchus (LB; a), less pronounced on epithelium of a small bronchus (SB; b), and not present on epithelia of an alveolar duct (AE) and adjoining alveoli (b). Positive staining for anti-alveolar type I cell antibody RT-1 (arrowheads) confirms that injected liquid reached the alveolar lumen (c). Original magnification, ×400. Note that no alpha vbeta 3-integrin staining was evident in bronchial vein (V; a). d: alpha vbeta 3-integrin staining (single arrows) in rat bronchiolar epithelium with anti-alpha vbeta 3-integrin monoclonal antibody LM609. These results were replicated 3 times.

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 alpha v or beta 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 alpha v and beta 3 cDNA, respectively.


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 5.   Southern blots on reverse-transcribed RNAs from lung (Lu), kidney (K), heart (H), liver (Li), and brain (B) with cDNA probes for alpha v (A), beta 3 (B), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; C).

alpha v and beta 3 mRNA expression in lung vessels. To localize alpha v and beta 3 mRNA expression, we applied in situ RT-PCR followed by in situ hybridization on paraffin-embedded tissue sections. To determine the expression of alpha v and beta 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 alpha vbeta 3 protein expression, namely, for both mRNAs, staining was strong in the endothelial but not in the alveolar epithelial lining.


View larger version (116K):
[in this window]
[in a new window]
 
Fig. 6.   In situ RT-PCR and hybridization of lung microvessels. a and b: distribution of alpha v and beta 3 mRNAs, respectively. c: background staining of lung tissue in absence of PCR probes. Original magnification, ×400. d and e: staining for distribution of anti-alveolar type I cell antibody after sections in a and b, respectively, were destained. Arrows, microvascular endothelium; arrowheads, alveolar epithelial lining. Original magnification, ×400. f: alpha v mRNA expression in smooth muscle of large vessel (star ) and bronchial epithelium (arrow). Original magnification, ×100. These results were replicated 3 times.

alpha vbeta 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 alpha vbeta 3-integrin expression was largely not evident in these nonpulmonary beds. Some exceptions were the hepatic portal venous system that stained weakly for alpha vbeta 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).


View larger version (168K):
[in this window]
[in a new window]
 
Fig. 7.   Immunohistochemistry of nonpulmonary organs with LM609. a: liver. Bulk of parenchyma fails to stain, although weak staining for alpha vbeta 3-integrin is evident in a branch of portal vein (arrow). b: brain. No staining is evident in blood vessels (double arrows) or parenchyma. Staining was positive in nonidentified isolated cells (single arrows). c: skeletal muscle. Staining was evident in connective tissue (single arrows) but not in blood vessels (double arrows). d: skin. Staining was absent over blood vessels (double arrows) but present on epithelium (single arrows) and connective tissue (*). Original magnification, ×150. These results were replicated 3 times.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We show here that under resting conditions, alpha vbeta 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 alpha vbeta 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 alpha vbeta 3-integrin.

Second, we demonstrate the presence of not only alpha vbeta 3 protein but also of alpha v and beta 3 mRNAs in lung microvessels. This mRNA evidence indicates for the first time that gene transcription for the alpha v- and beta 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 alpha v and beta 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 alpha vbeta 3-integrin subunits is unique in lung blood vessels.

Third, our ultrastructural data are the first direct evidence for alpha vbeta 3-integrin expression on both the luminal and abluminal aspects of lung microvascular endothelium. Previously, bipolar expression of the endothelial alpha vbeta 3-integrin has been reported only in cultured endothelial cells (22). In our present data, occasionally alpha vbeta 3-integrin-localizing gold particles appeared aggregated (Fig. 3a). Because the alpha vbeta 3-integrin aggregates when ligated (3), the presence of aggregated gold particles suggests that in some regions the alpha vbeta 3-integrin may have undergone ligation. Although we carried out no procedures to ligate the integrin, luminal platelets may release alpha vbeta 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 alpha vbeta 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-alpha vbeta 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 alpha vbeta 3-integrin labeling on the alveolar epithelium. We conclude from these findings that although the alpha vbeta 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 alpha vbeta 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 alpha vbeta 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 alpha v-subunit but the absence of the beta 3-subunit in human bronchial epithelium using subunit-specific antibodies. These and other workers (13) also reported little or no staining for the alpha vbeta 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-alpha vbeta 3-integrin antibodies, namely monoclonal antibody LM609 and polyclonal antibody AB1903. Although AB1903, being polyclonal, may recognize other alpha v-containing integrins, LM609 is widely recognized to be specific for the alpha vbeta 3-integrin (5, 11, 16) because it immunoprecipitates only the alpha v- and the beta 3-subunits from homogenates of rat lung (22). However, both antibodies gave highly similar immunostaining patterns. Not only was the alpha vbeta 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 alpha vbeta 3-integrin.

We localized mRNA expression in lung histological sections using in situ RT-PCR (Fig. 6). alpha v and beta 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 alpha v and 723 bp for beta 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 alpha v- and beta 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 alpha vbeta 3-integrin expression under unstressed conditions. Nevertheless, the extensive presence of the alpha vbeta 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 alpha vbeta 3-integrin in lung blood vessels and airway require further investigation.


    ACKNOWLEDGEMENTS

B. Singh and C. Fu contributed equally to this work.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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[Abstract/Free Full Text].

3.   Bhattacharya, S., C. Fu, J. Bhattacharya, and S. Greenberg. Soluble ligands of the alpha vbeta 3 integrin mediate enhanced tyrosine phosphorylation of multiple proteins in adherent bovine pulmonary artery endothelial cells. J. Biol. Chem. 270: 16781-16787, 1995[Abstract/Free Full Text].

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[Abstract/Free Full Text].

12.   Gavrilovskaya, I. N., M. Shepley, R. Shaw, M. H. Ginsberg, and E. R. Mackow. beta 3 Integrins mediate the cellular entry of hantaviruses that cause respiratory failure. Proc. Natl. Acad. Sci. USA 95: 7074-7079, 1998[Abstract/Free Full Text].

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 alpha 5beta 1 is required for internalization of vitronectin by integrin alpha vbeta 3. J. Biol. Chem. 272: 2736-2743, 1997[Abstract/Free Full Text].

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 alpha v and beta 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 alpha vbeta 3 integrin increases capillary hydraulic conductivity of rat lung. Circ. Res. 77: 651-659, 1995[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 278(1):L217-L226
0002-9513/00 $5.00 Copyright © 2000 the American Physiological Society