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(Circulation Research. 1995;77:651-659.)
© 1995 American Heart Association, Inc.

Articles

Ligation of Endothelial _vß₃ Integrin Increases Capillary Hydraulic Conductivity of Rat Lung

Hiroshi Tsukada, Xiaoyou Ying, Chenzhong Fu, Shigemi Ishikawa, Paula McKeown-Longo, Steven Albelda, Sunita Bhattacharya, Bonnie Anderson Bray, Jahar Bhattacharya

From the Departments of Medicine (H.T., X.Y., C.F., S.I., B.A.B., J.B.), Physiology & Cellular Biophysics (J.B.), and Pediatrics (S.B.), College of Physicians & Surgeons, Columbia University, St. Luke's-Roosevelt Hospital Center, New York, NY; the Department of Physiology (P.M.-L.), Albany (NY) Medical College; and the Department of Medicine (S.A.), University of Pennsylvania School of Medicine, Philadelphia.

Correspondence to Dr. J. Bhattacharya, Columbia University, St. Luke's-Roosevelt Hospital Center, 1000 10th Ave, New York, NY 10019.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Complement-mediated pulmonary edema results from increases in lung capillary hydraulic conductivity (Lp), possibly by receptor-mediated mechanisms. We considered the Lp effects of vitronectin and the vitronectin-containing complement complex SC5b-9, which ligate the integrin _vß₃. Vitronectin, SC5b-9, and SC5b-9–enriched zymosan-activated serum all rapidly increased Lp, as determined by the split-drop technique in single lung capillaries of rat lung. The Lp increases were inhibited by a monospecific (LM609) and a polyclonal (R838) antibody against the _vß₃ integrin but not by an irrelevant monoclonal antibody isotype matched with LM609, by a monoclonal antibody against the _vß₅ integrin, or by preimmune rabbit serum. Vitronectin monomers failed to increase Lp. The tyrosine kinase blockers genistein and methyl 2,5-dihydroxycinnamate caused significant concentration-dependent inhibitions of Lp increases due to vitronectin and zymosan-activated serum. By contrast, the protein kinase C blocker calphostin C had no major effect. We conclude that (1) multivalent ligation of the luminally located _vß₃ integrin of lung capillary endothelium increases transcapillary liquid flux, and (2) the dominant signal transduction pathway for this effect occurs through tyrosine kinase activation.

Key Words: endothelium • permeability • immunology • vitronectin • integrin

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Complement activation is a potential cause of the lung microvascular injury that underlies pulmonary edema characteristic of conditions such as ARDS. Although complement-mediated microvascular injury has been attributed to several products formed in the complement pathway,¹ specific receptor mechanisms that may lead to complement-induced pulmonary edema are not well understood. In general, the increase of microvascular permeability in complement-mediated pulmonary edema may result from the ligation of endothelial receptors that affect microvascular barrier properties. In lung microvessels, the quantification of the barrier by the single capillary method^{2 3 4} achieves accurate determinations of the capillary Lp. This provides a sensitive index for the identification of receptor-mediated barrier regulatory mechanisms.

In the present study, we consider a new class of endothelial receptors that may play a role in the pathophysiology of complement-mediated pulmonary edema, namely, the integrins of the cytoadhesin family. Of particular interest is the integrin _vß₃, a cytoadhesin that stabilizes endothelial monolayers in vitro⁵ and may, by extension, stabilize the endothelial barrier in microvessels. However, relevant microvascular data are lacking. Cytoadhesins, including the _vß₃ integrin, tend to be prominent at focal adhesions at which the extracellular domain of an integrin heterodimer binds ECM components and the cytoplasmic tail binds cytoskeletal elements. Because of these linkages, the functional importance of cytoadhesins is attributed mainly to the establishment of cell anchorage on the ECM.⁶ Recently, integrin _vß₃ has been shown to be essential for angiogenesis, and mAb LM609, which blocks the binding of ligands to _vß₃, promoted tumor regression of angiogenic blood vessels in chick embryos.^{7 8}

In the cultured endothelial cell, the _vß₃ integrin is located on both the abluminal and luminal surfaces.⁹ Since a similar distribution of the integrin probably occurs in endothelial cells of intact microvessels, a function for the lumen-facing integrin needs to be defined. Although the ECM-facing _vß₃ integrin may subserve cell anchorage, the function of a luminal _vß₃ is likely to be different. One possibility is that ligation of the luminal _vß₃ integrin by blood-borne factors induces endothelial signaling that ultimately results in increases of microvascular permeability to water and solutes. In other cell types, integrins have been shown to be capable of inducing cell signaling, as indicated in the integrin-mediated increases of cellular Ca²⁺,¹⁰ tyrosine phosphorylation of cell proteins,¹¹ induction of gene expression,¹² and regulation of ion transporters.¹³ However, specific integrin-mediated signaling events that affect endothelial barrier properties remain poorly understood.

The link between complement activation and the endothelial _vß₃ integrin may be attributable to the formation of the complement complex SC5b-9 in the terminal part of the complement pathway. SC5b-9 is known to contain several molecules of the prototypic _vß₃ ligand vitronectin, which recognizes the integrin by means of its RGD (Arg-Gly-Asp) tripeptide sequence.¹⁴ In previous experiments, we showed that complement-activated serum increases lung microvascular Lp and that the critical factor responsible is SC5b-9.³ However, no direct data attest to the importance of the endothelial luminal _vß₃ integrin in the regulation of microvascular barrier properties. In the present study, we tested endothelial barrier effects of _vß₃ ligation through determinations of lung capillary Lp by using three products, namely, vitronectin, SC5b-9, and SC5b-9–enriched ZAS, as ligands. We report below that these products caused an _vß₃-mediated Lp increase through a signaling pathway involving tyrosine kinase activation. These data are the first direct evidence that link the endothelial luminal _vß₃ integrin of lung endothelium to barrier regulation in lung microvessels.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Experimental Materials
Antibodies
mAbs LM609 and P1F6, directed against the human _vß₃ and _vß₅ integrins, respectively, were purchased from Chemicon. One batch of mAb LM609 was the generous gift of Dr David A. Cheresh, Scripps Institute. Both LM609 and P1F6 are isotype matched (IgG₁) and grown in mouse ascites fluid. Anti-laminin mAb LAM 89, raised in mouse ascites fluid (Chemicon), was used as an irrelevant isotype-matched (IgG₁) control for LM609. Purified polyclonal antibody R838 was raised against purified _vß₃ integrin isolated from human endothelial cells as previously described.¹⁵ The ability of this antibody to cross-react with _vß₃ integrin from bovine and rat cells is well characterized.^{16 17}

Vitronectin
Plasma vitronectin was purified by heparin affinity chromatography by the method of Yatohgo et al,¹⁸ as previously described.^{19 20} We used human plasma depleted of vitamin K–dependent clotting factors (barium sulfate precipitation), fibrinogen (glycine precipitation), and fibronectin (gelatin affinity). In the purified sample, we confirmed the presence of vitronectin by Western blotting with anti-vitronectin antibody (not shown). The purified vitronectin was largely in the form of high-molecular-weight disulfide-bonded multimers (Fig 1, top).

View larger version (40K): [in this window] [in a new window]   Figure 1. SDS-PAGE of purified products. Top, Vitronectin. In lane A, vitronectin was run under reducing conditions in the presence of mercaptoethanol to show the typical two-chain form (75 and 65 kD).21 In the absence of mercaptoethanol, a significant proportion of disulfide-bonded high-molecular-weight material remained at the top of the gel (lane C, arrowheads). Purified vitronectin was reduced and alkylated as described in "Materials and Methods." When this product was run in the absence of mercaptoethanol, the disulfide-bonded multimeric material was greatly reduced (lane D, arrowheads). When the reduced and alkylated vitronectin was run with mercaptoethanol, bands similar to lane A were seen (lane B). Bands were visualized by staining with Coomassie blue. Bottom, SC5b-9. Samples of the extract from the SC5b-9 purification procedure were run on SDS-PAGE under reducing conditions. Bands (left lane) show the expected complement proteins,22 which were absent in the sample depleted of SC5b-9 by treatment with beads coated with anti–SC5b-9 mAb (50 µg/mL). Molecular mass markers on the left are given in kilodaltons.

Alkylated Vitronectin
To cleave disulfide bonds, the purified vitronectin was treated with 10 mmol/L dithiothreitol for 3 hours and then alkylated with 60 mmol/L iodoacetamide for 1 hour at room temperature. The precipitate was dialyzed against PBS overnight, with three changes of PBS. This procedure yielded monomeric vitronectin (Fig 1, top, lane D). This reduced and alkylated vitronectin was able to support adhesion and spreading of cells when used to coat tissue culture wells (not shown), suggesting that this form of vitronectin was still capable of recognizing cell surface integrin receptors.

Fibronectin
Human plasma fibronectin was purified from a fibronectin and fibrinogen-rich byproduct of factor VIII production by previously described methods.²³ The plasma fraction was dissolved in 0.05 mol/L Tris and 0.02 mol/L sodium citrate at pH 7.5. Fibrinogen was precipitated by heating at 56°C for 3 minutes. The solution was clarified by centrifugation and chromatographed on DEAE-cellulose (not shown). The fibronectin peak was pooled, and the protein was precipitated with ammonium sulfate, dialyzed against Tris-buffered saline, and frozen at -70°C until use. For Lp measurements, the fibronectin was used at 660 µg/mL in 40 mg/mL albumin.

ZAS
We have shown previously that ZAS derived from rat or human serum gives qualitatively similar Lp responses³ ; hence, we used rat serum in the present study. As before,³ we activated serum complement by using zymosan (Sigma Chemical Co) according to the method of Gawryl et al.²² Briefly, we first boiled and cooled zymosan, and then we added it to cell-free serum (10 mg/mL) prepared from freshly collected anticoagulant-free blood under cold conditions (4°C). The serum was incubated (1 hour at 37°C) and then cleared of zymosan particles by centrifugation (20 000g for 10 minutes) and filtration (0.8-µm filter, Millipore Co). Control nonactivated serum was prepared in parallel without the addition of zymosan.

Aliquots of nonactivated sera and ZAS were microscopically examined to confirm the absence of cells and then analyzed for protein oncotic pressure, protein concentration, osmolality, and electrolyte (K⁺ and Ca²⁺) concentrations, as previously described.³ These variables, which could potentially affect the interpretation of Lp, were identical for ZAS and nonactivated serum. All sera aliquots were stored in plastic containers at -70°C. Samples were used within 4 weeks of preparation; each aliquot was thawed once.

SC5b-9
SC5b-9 was purified by the method of Gawryl et al,²² with some modifications as described. SC5b-9 was precipitated from ZAS (9 mL) by the addition of ammonium sulfate to a 37.5% concentration while the mixture was stirred in an ice bath. The precipitate was recovered by centrifugation, dissolved in PBS (pH 7.4, 2 mL) containing 1% Triton X-100, and immediately chromatographed on a column of Sepharose CL-6B equilibrated with PBS. Fractions were electrophoresed on cellulose acetate and stained (with amido black) to identify proteins (not shown). Some samples were Western blotted with anti-vitronectin antibody (Chemicon) to confirm the presence of vitronectin in SC5b-9. The product migrated to the ₁ region on cellulose acetate electrophoresis and contained the appropriate peptide subunits,²² as evident on SDS-PAGE (Fig 1, bottom). As before,³ SC5b-9 levels in the fractions were quantified by enzyme-linked immunosorbent assay using anti–SC5b-9 mAb (Quidel). Lp data were obtained by using the fraction containing SC5b-9 at 119 µg/mL. An aliquot of this sample was immunodepleted, as described before,³ by using beads coated with the anti–SC5b-9 antibody (Quidel) (Fig 1, bottom).

Other Proteins
Human -thrombin (a generous gift from Dr John W. Fenton II, New York State Department of Health, Albany) was dissolved in 0.75% NaCl, dialyzed against Ringer's lactate, and diluted in 40 mg/mL albumin to obtain required concentrations. As a vehicle for purified substances and chemicals, we used delipidated BSA (fraction V, Sigma) in Ringer's lactate containing (mmol/L) Na⁺ 144, K⁺ 4, Ca²⁺ 1.5, and lactate 28, at pH 7.4 and 300 mOsm (micro-osmometer, model 3M0, Advanced Instruments, Inc). At 40 mg/mL, albumin provided the requisite split-solution oncotic pressure (21 cm H₂O) for Lp measurements.⁴

Chemicals
The peptides GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) and GRGESP (Gly-Arg-Gly-Glu-Ser-Pro) (Peninsula Laboratories, Inc) were used at 1 mg/mL in 40 mg/mL albumin. In some experiments, the RGD tripeptide (Sigma) was used instead of GRGDSP. Stock solutions of the tyrosine kinase inhibitors genistein (4',5,7-trihydroxyisofravone, Sigma) and MDC (GIBCO BRL), a stable analogue of erbstatin,²⁴ and of the PKC inhibitor calphostin C (Kamiya Biomedical Co) were prepared in dimethyl sulfoxide (Sigma). For experiments, aliquots from the stock solutions were diluted in 40 mg/mL albumin, as required.

Experimental Procedures
Immunoprecipitation
Cross reactivity of mAbs LM609 and P1F6 with rat lung tissue was determined by immunoprecipitation. Through a pulmonary artery cannula, the vasculature of a rat lung was filled with a membrane-impermeant biotinylating reagent, sulfosuccinimidobiotin (Pierce), at 4°C in PBS, pH 7.4. The lung was stored at 4°C overnight, and then the vasculature was washed out with PBS to remove unbound reagent. The lung was homogenized in lysis buffer and centrifuged (14 000g for 5 minutes). The supernatant was incubated (22°C for 1 hour) with beads (protein A/G–agarose) coated with anti-integrin antibodies or IgG₁ (control) and centrifuged (14 000g for 5 minutes). The pellet containing the beads was washed three times with PBS, then boiled (3 minutes) with reducing buffer, and recentrifuged. The supernatant was run on SDS-PAGE using polyacrylamide gels under reducing conditions, transferred to a nitrocellulose membrane, and treated with horseradish peroxidase–streptavidin (Jackson Immuno Research Lab, Inc). Blots were developed by enhanced chemiluminescence (Dupont NEN).

Lp Measurement
Lung Preparation
The lung preparation and split-drop methods have been reported several times^{3 4} and are described here briefly. Lungs removed from anesthetized (2% halothane inhalation followed by 40 mg/kg IP sodium pentobarbital) rats (500 to 600 g, Harlan Sprague-Dawley Inc, Indianapolis, Ind) were pump-perfused with autologous rat blood through cannulas inserted in the pulmonary artery and left atrium. A tracheal cannula was used for lung inflation with gas (30% O₂/6% CO₂/64% N₂) that maintained blood PO₂, PCO₂, and pH at, respectively, 140 mm Hg, 35 mm Hg, and pH 7.3 (178 pH/blood gas analyzer, Corning). A heat exchanger (model 44TD, Yellow Springs) maintained perfusate temperature at 37°C. Recordings of lung vascular and airway pressures (model P23 ID pressure transducer, Gould Statham), referred to the micropuncture level, were displayed on a multichannel recorder (Gould RS 3400). Lung vascular pressures were varied by adjusting the height of the venous outflow. Airway pressure was held constant at 5 cm H₂O during micropuncture but was cyclically varied to induce ventilation during nonmicropuncture intervals lasting 10 to 15 minutes.

The Split-Drop Procedure
Lungs were positioned on a vibration-free air table (Micro-G, Technical Manufacturing Corp) for microscopy and videorecording (x350; microscope, Olympus model SZH; camera, Panasonic WV-CD51; monitor, Panasonic WV-5410; and videorecorder, Panasonic AG-6050). To prevent drying, the experimental lung surface was layered with a drip of silicone oil (Dow Corning 200) warmed to 37°C (Sensortek Thermalert TH-5). Micropipettes with beveled tips (diameter, 5 µm) prepared as previously described³ were filled with either castor oil colored with carbon black B or the experimental solution. The split-drop procedure was carried out in venular capillaries (diameter, 20 to 25 µm), which were identified by their convergent flow patterns.

The micropuncture steps of our split-drop procedure have been previously described in detail.^{3 4} In brief, blood flow is stopped at a defined pulmonary artery pressure, which therefore equals Pc because of stop-flow conditions. By use of successive micropunctures, the experimental capillary is filled with an oil drop, which is then split by an injection of the experimental solution (split solution) to separate the split margins by 60 µm (split length). Further oil injections are instituted to seal the micropuncture sites. Finally, pipettes are removed, and split-length changes are videorecorded for 1 to 1.5 minutes. The oil-endothelial contact does not deteriorate the endothelial barrier within the <2-minute duration of the split-drop procedure,⁴ after which blood flow is reinstated.

Split length decreases with liquid exit from the split drop (filtration) but increases with liquid entry (absorption). Microvessel diameter remains constant. Jv at time zero is estimated according to our previously reported analysis.^{2 4} Briefly, split length and capillary diameter measurements determined by microcaliper (Mitsutoyo Corp) from a frame-by-frame video replay (x400; resolution, 1 µm) are applied to the cylinder formula to calculate split-drop volume and surface area. Jv is calculated by differentiating, at time zero, the slope of the exponential regression of split-drop volume versus time in the first minute. Lp is determined as the slope of the linear Jv-Pc relation, calculated by linear regression of three or more Jv measurements at different Pc values. Lp remains unchanged for up to 4 hours,⁴ within which all determinations are completed.

Imaging
Capillary fluorescence of fluorescein isothiocyanate was determined by mercury lamp (Ushio) excitation through a 510-nm barrier filter. Fluorescence collected through a x40 objective (Nikon Fluor LWD; Olympus microscope, Vanox), an image intensifier (Videoscope KS 1381), and a high-gain video camera (Dage CCD-72) was recorded by imaging software (Imaging Research MCID-M4) on a microcomputer platform.

Statistics and Units
Data are mean±SD; n indicates the number of experiments. Paired differences were tested by the paired t test, and differences among more than two groups were determined by ANOVA (Newman-Keuls test). Significance was accepted at P<.05. NS denotes nonsignificant differences from controls that are not shown.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Integrin Distribution in the Lung Capillary
Although mAbs LM609 and P1F6 are well characterized with regard to their ability to recognize several different types of tissue, their specific cross-reactivity with rat lung is not reported. The results of immunoprecipitation shown in Fig 2 confirmed that both mAbs cross-react with rat lung (Fig 2A). The experiment shown in Fig 2B confirmed the antigenic specificity of mAb LM609 for the _vß₃ integrin, in that LM609 failed to recognize proteins in rat lung homogenates previously immunodepleted with the _vß₃-recognizing polyclonal antibody R838. To determine the distribution of the endothelial luminal _vß₃ integrin, we intravitally imaged _vß₃ distribution in the lung venular capillary (Fig 3A). Sequential capillary infusions at 4°C of the anti-_vß₃ mAb LM609 and fluorescent anti-mouse IgG resulted in significant fluorescence of the capillary wall (Fig 3A, left), whereas no capillary fluorescence was evident when fluorescent IgG alone was infused (Fig 3A, right). These findings, together with the immunohistochemical data shown in Fig 3B, confirm the presence of the _vß₃ and _vß₅ integrins on the capillary luminal surface of rat lung.

View larger version (28K): [in this window] [in a new window]   Figure 2. Immunoprecipitation of integrins. Rat lung blood vessels were biotin-labeled by perfusion and homogenized; then homogenate supernatants were incubated with separate batches of protein A/G–Sepharose beads coated with anti-integrin antibodies. Horseradish peroxidase–streptavidin treatment of supernatants recovered from the beads gave appropriate bands25 in SDS-PAGE under reducing conditions corresponding to integrin subunits (see "Materials and Methods" for details). A, Homogenates were incubated with the anti-vß3 mAb LM609 (lane 1), the vß5 mAb P1F6 (lane 2), or mouse IgG (MlgG) (lane 3). The 9% gel shows bands corresponding to the v and ß3 subunits in lane 1 and the v and ß5 subunits in lane 2. B, The homogenates were incubated with LM609 alone (lane 1), rabbit serum (RS) followed by MlgG (lane 2), or R838, an anti-vß3 polyclonal antibody, followed by LM609 (lane 3). The 6% gel shows, in lane 3, that after antigen depletion by R838, no proteins were recognized by LM609.

View larger version (0K): [in this window] [in a new window]   Figure 3. Integrin distribution in lung capillary. A, Intravital fluorescent images of lung venular capillaries. Single capillary microinfusions were carried out at 4°C to block endocytosis. Blood flow was stopped during the microinfusions but was reinstated after infusion to wash out fluorescence from the vessel lumen. A 5-minute microinfusion of the anti-vß3 monoclonal antibody LM609 followed by microinfusion of a secondary anti-mouse FITC-IgG (IgG Fc-labeled with the fluorophore fluorescein isothiocyanate) resulted in diffuse, sustained fluorescent staining in each of four venular capillaries in three lungs (left). Infusion of FITC-IgG alone resulted in black images with no residual fluorescence (right). B, Histological sections of capillaries from rat lung (magnification x200). The sections were reacted first with anti-vß3 mAb LM609 (top left) or anti-vß5 mAb P1F6 (top right) and then with secondary antibody labeled with horseradish peroxidase. Capillary wall staining (arrows) indicates the presence of the vß3 and vß5 integrins. Control sections exposed first to mouse IgG1 and then to the horseradish peroxidase–conjugated secondary antibody stained negatively (bottom). One of three experiments is shown.

Histology of the Oil-Filled Capillary Segment
To determine the possible presence of blood cells in the split drop, we microinjected oil drops into the venular capillaries of three lungs, fixed the lungs in formaldehyde, and then examined the histological sections of the capillaries by light microscopy (Fig 4). Although the oil drop was always lost during sectioning, the oil-filled segment appeared as an empty space, forming well-defined margins with the adjoining plasma-containing segments. No blood cells were detected by toluidine or hematoxylin-eosin staining in 11 such segments.

View larger version (100K): [in this window] [in a new window]   Figure 4. Histological section through a subpleural venular capillary. A 100-µm-length oil drop was microinjected into a subpleural venular capillary (diameter, 15 µm) of an isolated, blood-perfused rat lung. The lung was fixed by immersion in 4% buffered formaldehyde; then 1-µm tissue sections were stained with toluidine blue and examined under light microscopy (magnification x400). The arrows demarcate the oil-filled space. During tissue fixation, the oil drop moved from the immediate subpleural location. Note the absence of blood cells in the oil-injected space. PL indicates pleura; Alv, alveolus; and Cap, venular capillary.

Vitronectin
The single experiment shown in Fig 5 (top) exemplifies our method for quantifying Lp as the regression slope of the linear Jv-Pc relation. In this example, vitronectin at 100 µg/mL increased Lp from a control of 5.1x10^-7 to 12.2x10^-7 mL/(cm² · s · cm H₂O). The group data in Fig 5 (middle) show that the Lp increase to vitronectin was concentration dependent and that the effect was inhibited completely in the presence of a pentapeptide containing the RGD sequence. In agreement with our previous findings,³ RGD itself had no effect on control Lp (n=3). In three experiments, different concentrations of the GRGES pentapeptide, which lacks the RGD sequence, failed to block the increase of Lp (Fig 5, open squares in middle panel). To rule out possible platelet effects, in three experiments we gave 100 µg/mL vitronectin in platelet-rich plasma, obtained by centrifuging blood and collecting the supernatant above the buffy coat. As we previously reported, this species of rat has a high blood platelet count.²⁶ The Lp determined with vitronectin in platelet-rich serum [11±0.1x10^-7 mL/(cm² · s · cm H₂O)] was not different from the control response to an identical vitronectin concentration [12.5±0.7x10^-7 mL/(cm² · s · cm H₂O), n=3], indicating that the presence of platelets did not enhance vitronectin's Lp effect. We conclude that these Lp increases reflect vitronectin-induced increases of transendothelial liquid flux, attributable to an endothelial integrin that recognizes the RGD sequence of vitronectin.

View larger version (16K): [in this window] [in a new window]   Figure 5. Vitronectin-induced transendothelial flux in lung capillary. Top, Plots of Jv vs Pc from a single experiment. The split solutions were 100 µg/mL vitronectin in 40 mg/mL albumin () and albumin alone (). Lines drawn were by linear regression (P<.05). Negative Jv values at low pressures signify lumen-directed liquid absorption; positive Jv values signify filtration. Middle, Effect of vitronectin (VN) on Lp. Lp values were determined by using vitronectin alone () and vitronectin with 1 mg/mL GRGDSP (). In the experiments shown as open squares, vitronectin was given with GRGESP at 5 (top square), 1 (middle square), and 10 (bottom square) mg/mL. Lines of best fit were by linear regression (P<.5). Bottom, Lumen-directed flux in lung capillaries. Volume increase signifies liquid entry into split drop in single capillary. indicates 40 mg/mL albumin (n=3); , 200 µg/mL vitronectin (n=4). Points are mean±SD. *P<.05 vs control.

To determine the onset of the vitronectin-induced flux increase, lumen-directed fluxes were generated at low capillary pressure (5 cm H₂O).⁴ Within 5 seconds of injecting vitronectin, the lumen-directed flux was significantly higher than the control value (Fig 5, bottom). This rapid response attributes the vitronectin-induced flux increase to ligation of a luminal rather than an abluminal vitronectin receptor, because transendothelial ligand transport in this time scale is highly unlikely under the present conditions. Transendothelial transport processes are convective, diffusive, or transcellular. The large size of the vitronectin multimer (>200 kD) and the presence of lumen-directed convection preclude the possibility that ligand was transported by either convection or diffusion in a time scale of seconds. Transcellular transport is also unlikely, because for a much smaller molecule, albumin, transcellular transport occurs in minutes²⁷ ; hence, for these larger ligands, transcellular effects are expected with a delay of minutes rather than seconds.

The Lp increase to vitronectin at 200 µg/mL was inhibited 88% by coinjection of the anti-_vß₃ mAb LM609 and 91% by that of the polyclonal anti-_vß₃ antibody R838 (Table). No inhibitory effects were evident when we coinjected the anti-_vß₅ mAb P1F6 (20 µg/mL; n=3; Lp, 12.6±1.3x10^-7 mL/(cm² · s · cm H₂O). Since both mAbs are isotype matched and raised in mouse ascites fluid, P1F6 serves as a control for LM609, and the negative response with P1F6 indicates that the inhibition by LM609 was not due to contaminants or nonspecific factors. As a second control for the LM609 effect, we coinjected the anti-laminin mAb LAM89 (30 µg/mL) with vitronectin (n=3). This mAb, which is also isotype matched with LM609 though irrelevant for the _vß₃ integrin (see "Materials and Methods"), also failed to block the Lp increases. As control for the Lp-inhibiting effect of R838, in four experiments coinjection of preimmune rabbit serum with vitronectin failed to inhibit the Lp increase. An antibody-induced Lp effect was ruled out because none of the antibodies given alone in the present concentrations increased Lp. We conclude that the antibodies LM609 and R838 competitively inhibited the ligation of vitronectin to the _vß₃ integrin and thereby blocked the vitronectin-induced Lp increase.

View this table: [in this window] [in a new window]   Table 1. Effects of Anti-vß3 Antibodies on Capillary Lp of Rat Lung

Alkylated Vitronectin
To determine whether vitronectin needed to be multimeric to elicit changes in Lp, vitronectin was reduced and alkylated as previously described ("Materials and Methods"). Alkylation of vitronectin resulted in a marked decrease in the proportion of high-molecular-weight species in the preparation (Fig 1, top). An aliquot of this preparation (300 µg/mL) gave an Lp of 5.5±0.5x10^-7 mL/(cm² · s · cm H₂O) (n=3), which was not significantly different from baseline. The adhesive glycoprotein fibronectin, which ligates the ₅ß₁ integrin,⁶ also failed to increase Lp above baseline at a concentration considerably greater (660 µg/mL) than the effective vitronectin concentrations (n=3).

ZAS and SC5b-9
The Table also shows that the Lp increases to ZAS and SC5b-9 (P<.01) were inhibited 60% and 93%, respectively, by LM609 and 75% and 94% by R838. Again, no inhibitory effects were evident when we coinjected ZAS and SC5b-9 with the anti-laminin mAb LAM89 (30 µg/mL) as a control for mAb LM609 (n=1 each) or with preimmune serum as a control for R838 (n=3 each). Coinfusion of the RGD tripeptide with SC5b-9 gave an Lp of 4.3±0.5x10^-7 mL/(cm² · s · cm H₂O) (n=5), which was not significantly different from baseline. This confirms that similar to our previous finding with ZAS,³ the SC5b-9–induced Lp increase is inhibited by RGD. To verify that the Lp increases to the purified products were not due to contaminants, we confirmed that aliquots of vitronectin and SC5b-9, when immunodepleted by exposure to anti-vitronectin or anti–SC5b-9 mAb, failed to increase Lp (n=3 each).

Tyrosine Kinase
To determine evidence for signaling mechanisms, 20-minute preinfusions of capillaries with genistein or MDC were each followed by determinations of Lp responses to a fixed concentration of ZAS or vitronectin. Control Lp values for the ZAS and vitronectin groups were obtained with control serum [3.3±0.2x10^-7 mL/(cm² · s · cm H₂O), n=3] and 40 mg/mL albumin [5.4±0.2x10^-7 mL/(cm² · s · cm H₂O), n=3], respectively. ZAS and vitronectin (100 µg/mL) increased Lp to 8.4±1.2x10^-7 and 14.5±1.4x10^-7 mL/(cm² · s · cm H₂O) (n=8), respectively. Both genistein and MDC inhibited these Lp increases in a concentration-dependent manner (Fig 6, top and middle). Using exponential analysis for the genistein inhibition plot in Fig 6, we estimated an IC₅₀ (concentration at 50% inhibition) of 17 µmol/L for vitronectin and 24 µmol/L for ZAS. A linear analysis for the MDC inhibition plot (Fig 6) gave an IC₅₀ of 25 µmol/L for both ligands. These effects of the tyrosine kinase inhibitors suggest that the present Lp increases were attributable to tyrosine kinase activation.

View larger version (13K): [in this window] [in a new window]   Figure 6. Inhibition of Lp increases by kinase inhibitors. Top and middle, Lp increases attributable to ZAS () or 100 µg/mL vitronectin () given alone were normalized to 100%. Significantly smaller Lp increases occurred when the same ZAS or vitronectin concentrations were given together with the tyrosine kinase inhibitors genistein and MDC. Bottom, Lp increases obtained with 200 µg/mL vitronectin () or 10-7 mol/L -thrombin () were normalized to 100%. Note that in the presence of calphostin C, the -thrombin–induced Lp increases were significantly reduced, although the vitronectin-induced Lp increases were not significantly different from the 100% response. Values are mean±SD (n=3 for each point). *P<.05 vs 100% response.

PKC
Since tyrosine phosphorylation of phospholipase C- may induce a diacylglycerol-mediated PKC activation, we determined the effects of PKC inhibition by calphostin C on the vitronectin-induced Lp increase. For comparison we used -thrombin, a known PKC activator.²⁸ Baseline Lp obtained with the vehicle, 40 mg/mL albumin, averaged 5.2±0.2x10^-7 mL/(cm² · s · cm H₂O) (n=4). After 15-minute preinfusions of vehicle in paired vessels, -thrombin at 10^-7 mol/L and vitronectin at 200 µg/mL increased Lp to 15.6±1.5x10^-7 and 14.5±1.4x10^-7 mL/(cm² · s · cm H₂O) (n=8), respectively. In a parallel protocol, preinfusions of calphostin C markedly inhibited the Lp increase to -thrombin but not to vitronectin (Fig 6, bottom). Thus, at 1 µmol/L calphostin C inhibited the -thrombin response by >50% but the vitronectin response by <5% (P<.01). The inhibition disparity was also evident at 20 µmol/L, at which calphostin C inhibited the -thrombin response completely but the vitronectin response by only 20%. We conclude that PKC activation did not play a major role in the present Lp increases.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We show for the first time that ligation of the _vß₃ integrin on the endothelial luminal surface increases transcapillary liquid conductance in the lung. Vitronectin, a classic _vß₃ ligand,¹⁴ SC5b-9, which also binds the _vß₃ integrin,²⁹ and ZAS, which is essentially SC5b-9–enriched serum,³ each markedly increased lung capillary Lp. We demonstrated the presence of the _vß₃ integrin in the lung capillary by histochemistry. Moreover, we confirmed that the integrin exists on the luminal surface of the lung capillary endothelium by imaging capillary fluorescence attributable to the distribution of anti-_vß₃ mAb LM609 (Fig 3). The inhibitions of the Lp increases by two different anti-_vß₃ antibodies, namely, mAb LM609 and the polyclonal antibody R838, confirm that the Lp effects of vitronectin, ZAS, and SC5b-9 resulted from _vß₃ ligation. Involvement of the vitronectin-recognizing _vß₅ integrin,²⁵ the existence of which we demonstrate here in lung capillary endothelium (Fig 3), may be ruled out because LM609, which does not recognize _vß₅,²⁵ effectively blocked the entire Lp responses to vitronectin and SC5b-9. The inability of anti-_vß₅ mAb P1F6 to inhibit the vitronectin-induced Lp increase is also consistent with the noninvolvement of the _vß₅ integrin. However, the _vß₅-blocking effect of P1F6, which we used essentially as a control for LM609, remains unconfirmed in rat tissues.

The Lp increases, which always occurred within 1 minute of ligand injection, indicate that ligation of the endothelial _vß₃ integrin caused rapid increases of transcapillary liquid flux. In fact, when we timed the vitronectin response more accurately by inducing lumen-directed fluxes, we recorded flux increases within seconds (Fig 5, bottom). For the reasons given in "Results," we interpret that these rapid flux increases follow _vß₃ ligation on the endothelial luminal surface and that together with the Lp data, they signify a decrease of endothelial barrier function. By contrast, the _vß₃ integrin appears to be barrier protective when ligated on the abluminal side. Inhibition of integrin-ECM binding by RGD-containing peptides or by integrin-specific antisera causes monolayer disruption, as is evident in cultured endothelial monolayers by the appearance of holes and increased permeability to tracers such as horseradish peroxidase.⁵ Tang et al³⁰ concluded that maintenance of _vß₃-containing focal adhesions protects against endothelial retraction in cultured monolayers. It is likely that as part of the focal adhesion, the abluminal _vß₃ integrin affects endothelial permeability to the extent that it serves a cell-stabilizing function. In the luminal location, the _vß₃ integrin evidently behaves more as a receptor that mediates opening of the interendothelial junction and, in this role, may be important in endothelial inflammatory responses.

Our histological evidence indicates that the oil injection in the split-drop technique effectively clears the lumen of blood cells that could potentially complicate the interpretation of Lp responses. Nevertheless, we considered the possibility that the split drop contained resident endothelium-attached platelets that, if activated, could indirectly affect Lp. We believe that this is unlikely for the following reasons: (1) We have shown previously that the split-drop technique does not injure the endothelium⁴ ; hence, it is unlikely that platelets adhered to the normally nonthrombogenic endothelial surface. (2) Vitronectin in platelet-rich serum did not augment its platelet-free effect on Lp; hence, platelets did not contribute to the vitronectin effect. (3) We have not detected endothelial attachment of platelets or other blood cells in the oil-filled capillary segment by conventional light microscopy.

The fact that the Lp increases following _vß₃ ligation were inhibited by tyrosine kinase inhibitors provides the first evidence that signaling pathways involving tyrosine kinase activation are important in microvascular barrier regulation. The tyrosine kinase inhibitors genistein and MDC caused concentration-dependent inhibitions of the Lp increases to ZAS and vitronectin. These inhibitors have different modes of action: genistein inhibits ATP binding to tyrosine kinase,³¹ whereas MDC, a synthetic erbstatin analogue, inhibits by competing for the kinase substrate.²⁴ The specificity of genistein and MDC as tyrosine kinase inhibitors has been obtained from assays of kinase activity and of kinase-dependent effects. These assays indicate that genistein inhibits tyrosine kinase at an IC₅₀ of <50 µmol/L^{32 33 34 35} but serine/threonine kinases such as PKC at an IC₅₀ of >185 µmol/L.³¹ The IC₅₀ of MDC for tyrosine kinase, determined from the inhibition of tyrosine kinase–dependent effects in different cell types, is <70 µmol/L.^{36 37 38} Our Lp inhibition data for ZAS and vitronectin gave IC₅₀ values for genistein and MDC that are consistent with reported data and lie in the range of tyrosine kinase but not PKC inhibition. Hence, the present Lp inhibitions by these agents were attributable to tyrosine kinase inhibition. PKC activation, if present, played a minor role in these Lp increases, as is evident in the weak inhibition of the vitronectin effect by calphostin C (Fig 5). These results support tyrosine phosphorylation events as the principal postligational effect that led to the Lp increases and are consistent with our recent findings that vitronectin and SC5b-9–induced _vß₃ ligation causes protein tyrosine phosphorylation in endothelial cells.³⁹

Since integrins are not known to possess intrinsic enzymatic activity, tyrosine phosphorylation of critical proteins is the important intermediary step that initiates signaling.⁶ Ligation of several integrins of the ß₁ family (eg, ₃ß₁ in human carcinoma cells and ₅ß₁ in fibroblasts) phosphorylates cytoplasmic proteins on tyrosine.^{11 40} In platelets, -thrombin–induced activation of the _IIbß₃, an integrin similar to _vß₃, causes tyrosine phosphorylation of several proteins.⁴¹ _vß₃ ligation in fibroblasts mediates phosphorylation of paxillin and the kinase pp125 FAK.¹¹ The integrin-cytoskeleton link also suggests that integrin ligation may cause tyrosine phosphorylation–induced cytoskeletal reorganization and cell-shape change,⁴² which in endothelium could result in the Lp increases seen here.

An important result was obtained when we used a predominantly monomeric form of vitronectin obtained by the reduction and alkylation of vitronectin multimers (see "Materials and Methods"). Although the postalkylation product was capable of recognizing integrins to the extent that it supported cell spreading and adhesion, it did not increase Lp above the control value. This result indicates that in its monomeric form, vitronectin has no detectable effect on the lung endothelial barrier. Vitronectin is monomeric in the native state, but several vitronectin molecules are incorporated into SC5b-9 following complement activation.⁴³ The juxtaposition of multiple vitronectin molecules, as in SC5b-9 or a vitronectin multimer, is likely to increase the density of RGD sites, which may lead to ligation of multiple _vß₃ receptors, receptor aggregation, and the induction of signal transduction mechanisms.

Although we did not obtain direct evidence for receptor clustering in the lung capillary, integrins are known to cluster when ligated.^{40 44} In the present experiments, aggregation of the _vß₃ integrin caused by the vitronectin multimer may have induced the critical signaling that finally led to endothelial retraction⁴⁵ and increased Lp. The binding studies of Zanetti et al⁴⁶ and our recent findings on protein tyrosine phosphorylation,³⁹ taken together, indicate that multivalent ligation of the _vß₃ receptor, presumably followed by receptor aggregation,⁴⁴ is the prerequisite for _vß₃-induced intracellular signaling. We showed that the binding of the receptor by vitronectin multimers but not by small molecules such as the RGD peptide or by monomeric vitronectin enhanced tyrosine phosphorylation of endothelial protein substrates.³⁹ These findings are consistent with the present negative Lp effect of monomers obtained by alkylating the vitronectin multimer or of the RGD peptide given alone.³ However, the RGD inhibition of the Lp response to _vß₃ ligation proves that RGD binding to the receptor occurred. This binding, though ineffective for Lp responses, was nevertheless sufficient to inhibit the effects of multivalent ligands, presumably because the ligands could no longer bind and aggregate the receptor. Interestingly, fibronectin, which also binds the _vß₃ integrin by an RGD mechanism, did not increase Lp, presumably because fibronectin does not form multimers and probably does not cluster the receptor. The biological potency of the multivalent form of vitronectin is further indicated in that although both the multimer and the native monomer of vitronectin bind fibroblast monolayers, only the multimer is internalized by _vß₅-induced endocytosis.²⁰ These considerations indicate that because of its ability to form polymers, vitronectin's effectiveness as a ligand may be conformer sensitive. Although <5% of total plasma vitronectin normally occurs in the multivalent form,¹⁴ during inflammatory conditions the increased formation of multivalent vitronectin, as in SC5b-9 or perhaps as resulting from the interaction of vitronectin with bacteria,¹⁴ may prove to be a significant factor in the increase of lung microvascular permeability.

In conclusion, our findings provide the first evidence (1) that ligation of luminal _vß₃ of the lung capillary by multimeric vitronectin and by a vitronectin-containing complement complex, SC5b-9, increases lung endothelial liquid conductance and (2) that tyrosine kinase activation by this integrin plays a role in the signaling cascade that leads to increases of Lp. In the present study, we demonstrate that in its luminal location, the endothelial _vß₃ integrin is capable of exerting a significant effect on the lung endothelial barrier; hence, the barrier regulatory function of this luminal integrin needs to be addressed separate from its ECM-linked biological functions such as blood vessel growth.^{7 8} Since blood levels of vitronectin-containing complement complexes increase in ARDS,⁴⁷ the ligation of the endothelial luminal _vß₃ integrin by these complexes presents a new mechanism that may decrease lung microvascular barrier function and promote pulmonary edema.

	Selected Abbreviations and Acronyms

 

ARDS = adult respiratory distress syndrome ECM = extracellular matrix Jv = transendothelial flux (liquid flux per unit surface area) Lp = hydraulic conductivity mAb(s) = monoclonal antibody(ies) MDC = methyl 2,5-dihydroxycinnamate Pc = capillary pressure PKC = protein kinase C ZAS = zymosan-activated serum

	Acknowledgments

 
This study was supported by grant HL-36024 from the National Institutes of Health, Bethesda, Md (Dr Bhattacharya), and by grants CA-58626 from NIH and AHA 93013270 from the American Heart Association (Dr McKeown-Longo). We thank Rashmi Patel for technical assistance.

Received May 4, 1995; accepted July 13, 1995.

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