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Am J Physiol Heart Circ Physiol 291: H1351-H1359, 2006. First published February 17, 2006; doi:10.1152/ajpheart.01042.2005
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LRP and {alpha}vbeta3 mediate tPA activation of smooth muscle cells

Sa'ed Akkawi,1 Taher Nassar,1 Mark Tarshis,2 Douglas B. Cines,3 and Abd Al-Roof Higazi1,3

1Department of Clinical Biochemistry and 2Interdepartmental Unit, Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem, Israel; and 3Departments of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Submitted 3 October 2005 ; accepted in final form 17 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue-type plasminogen activator (tPA) regulates vascular contractility through the low-density lipoprotein-related receptor (LRP), and this effect is inhibited by plasminogen activator inhibitor type 1 (PAI-1). We now report that tPA-mediated vasocontraction also requires the integrin {alpha}vbeta3. tPA-induced contraction of rat aortic rings is inhibited by the Arg-Gly-Asp (RGD) peptide and by monoclonal anti-{alpha}vbeta3 antibody. tPA induces the formation of a complex between LRP and {alpha}vbeta3 in vascular smooth muscle cells. The three proteins are internalized within 10 min, causing the cells to become refractory to the readdition of tPA. LRP and {alpha}vbeta3 return to the cell surface by 90 min, restoring cell responsiveness to tPA. PAI-1 and the PAI-1-derived hexapeptide EEIIMD abolish the vasocontractile activity of tPA and inhibit the tPA-mediated interaction between LRP and {alpha}vbeta3. tPA induces calcium mobilization from intracellular stores in vascular smooth muscle cells, and this effect is inhibited by PAI-1, RGD, and antibodies to both LRP and {alpha}vbeta3. These data indicate that tPA-mediated vasocontraction involves the coordinated interaction of LRP with {alpha}vbeta3. Delineating the mechanism underlying these interactions and the nature of the signals transduced may provide new tools to regulate vascular tone and other consequences of tPA-mediated signaling.

lipoprotein-related receptor; tissue-type plasminogen activator; integrins; vasoactivity

TISSUE-TYPE PLASMINOGEN ACTIVATOR (tPA) is one of the principal plasminogen activators in mammals (3). However, several other activities have also been attributed to tPA, for example, within the central nervous system. In an experimental model of stroke, the size of the infarcted area was smaller in tPA knockout mice than in wild-type animals (27) and larger in plasminogen activator inhibitor type-1 (PAI-1) knockout mice (16). Administration of exogenous tPA increased infarct size as well (4). However, no change in infarct size was observed in urokinase plasminogen activator knockout mice in a similar model (16). These data suggest that the deleterious role of tPA in this setting may not be due entirely to its plasminogen activator activity.

Recent studies, including some from our laboratory, have shown that the binding of tPA to the low-density lipoprotein-related protein (LRP) initiates intracellular signal transduction and leads to multifaceted changes in vascular contractility by inducing vasodilation at low concentrations (1 nM) and vasoconstriction at therapeutic concentrations (≥20 nM) (19) and by increasing blood brain barrier permeability (28). We have also observed that under pathological conditions, such as hypoxia/ischemia, tPA induces constriction in the cerebral vasculature in pigs (2). PAI-1 neutralizes the vasoactive effects of tPA in isolated aortic rings (19) and on cerebral vasculature in vivo (2) through a catalytic site-independent mechanism. Taken together, these observations may offer an explanation for the poor outcome of PAI-1 knockout mice after stroke (16) and suggest a role for LRP in the process.

In addition to LRP, several integrins, including {alpha}vbeta3 (14), {alpha}5beta1 (13), and {alpha}4beta1 (26), have been implicated in regulating vascular contractility. The behavior of {alpha}vbeta3 and {alpha}vbeta5 is modified by LRP and by at least one of its ligands, urokinase plasminogen activator (5), which shares enzymatic, signal-transducing (19, 20), and some structural properties with tPA. Therefore, we pursued the hypothesis that tPA-induced vasocontraction may be mediated, in part, through an interaction between one or more integrins with LRP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. All experiments in rats were performed in accordance with protocols approved by the Institutional Review Board for the Care of Animals of Hadassah Hospital/Hebrew University of Jerusalem and the International Animal Care and Use Committee of Univeristy of Pennsylvania School of Medicine. Recombinant tPA was obtained from Genentech (South San Francisco, CA). Phenylephrine (PE), Arg-Gly-Asp (RGD), and Arg-Gly-Glu (RGE) were purchased from Sigma (St. Louis, MO). Anti-LRP antibodies were kindly provided by D. Strickland (American Red Cross, Rockville, MD) and by American Diagnostica (Greenwich, CT); anti-tPA antibodies were generously provided by American Diagnostica (cat. no. 385-R). Mouse monoclonal anti-human {alpha}vbeta3 (cat. no. 1976) and mouse anti-human LRP (cat. no. 1932) antibodies were purchased from Biotest (Kfar Saba Israel). Alexa 488-labeled goat IgG anti-mouse was purchased from Jackson Immuno Research. Fluo-4 was purchased from Molecular Probes (Invitrogen, Carlsbad, CA). The peptide Ac-EEIIMD-amide was synthesized as previously described (29). Human umbilical vein vascular smooth muscle cells (SMC) and endothelial cells were prepared as previously described (8).

Contractile response of isolated aortic rings. Experiments were performed as previously described (9, 18, 20). Briefly, rats were killed by exsanguination. Thoracic aortas were removed with care to avoid damage to the endothelium, dissected free of fat and connective tissue, and cut into transverse rings 5 mm in length (21). To record isometric tension, the rings were mounted in a 10-ml bath containing an oxygenated (95% O2-5% CO2) solution of Krebs-Henseleit (KH) buffer (144 mM NaCl, 5.9 mM KCl, 1.6 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11.1 mM D-glucose). The rings were equilibrated for 1.5 h at 37°C and maintained under a resting tension of 2 g throughout the experiment. Each ring was then contracted by adding PE in stepwise increments from 10–10 to 10–5 M. The maximal contraction of the rings (100%) was determined by adding 20–40 mM of KCl. Where indicated, tPA was added 10 min before the addition of PE. In other experiments, anti-{alpha}vbeta3 antibody, RGD, or RGE (100 µM) was added alone or 5 min before tPA. To examine the capacity of tPA to induce vasocontraction directly, aortic rings were preincubated with PE at a concentration that induces ~15% of its maximal contractile capacity. Ten minutes later, tPA (20 nM) was added. In each experiment, rings exposed to KH buffer alone were analyzed in parallel. Isometric tension was measured with a force displacement transducer and recorded online with a computerized system (ExperimentiaÆ). The EC50 was calculated by measuring the response of the rings to increasing concentration of PE as described previously (18, 20).

Confocal microscopy. Confocal microscopy was performed as described previously (18). Briefly, SMC grown on coverslips were incubated in DMEM media containing 10% FCS supplemented with tPA (50 nM) for the indicated periods of time at 37°C. The cells were washed three times with PBS, fixed for 10 min with 4% formaldehyde in PBS, and permeabilized or not, as indicated in each figure legend, with 0.2% Triton X-100 in PBS-BSA buffer for 3 min. The coverslips were overlaid for 20 min with 2% normal horse serum and then incubated for 40 min with anti-tPA or anti-{alpha}vbeta3 (where indicated) or with irrelevant antibodies diluted 1:500 in PBS. After four washes with PBS, the cells were stained for 40 min with Alexa 488-labeled goat IgG anti-mouse or anti-rabbit IgG (Molecular Probes, Eugene, OR), washed four times with PBS, and mounted in 80% glycerol-20% PBS supplemented with 3% DABCO (1,4-diazabicyclo-[2,2]-octane) as an antibleaching agent. No staining was seen when either the primary or the secondary antibody was omitted or when irrelevant IgG was used instead of the primary antibody.

Confocal microscopy was performed with a Zeiss LSM 410 confocal laser scanning system attached to a Zeiss axiovert 135M inverted microscope using a x40/1.3 plain oil immersion lens. The system was equipped with a 25 mW argon laser (488 nm excitation line with 515 nm low-pass barrier filter) to excite Alexa 488 green fluorescence. The differential interference contrast Nomarski images were collected simultaneously by a transmitted light detector. Autofluorescence was set to background. To reduce the visual noise, all optical sections were studied in the fast line-scan acquisition mode (512 pixels per line) by averaging eight images before the final image was produced on the monitor. In each experiment, the background level, exciting light intensity, photomultiplier, imaging filters, aperture (pinhole) contrast, and electronic zoom size were monitored at the same level. Confocal images were converted to a Tag Image File (TIF) format and transferred to a Zeiss imaging workstation to provide pseudocolor representation.

Uptake of radiolabeled calcium. The uptake of 45Ca2+ was measured as previously described (10, 18). Briefly, SMC were grown in 60-mm dishes and overlaid with KH buffer containing 25 µM 45Ca2+ at 37°C. PE (0.1 µM) was then added. In some experiments, tPA (20 or 50 nM), alone or together with equimolar concentrations of PAI-1 or the PAI-1-derived peptide EEIIMD (2 µM), was added. In other experiments, 100 nM anti-LRP, anti-{alpha}vbeta3, or control (anti-LDL receptor) antibody or 100 µM RGD or RGE was added 5 min before PE and/or tPA. Ten minutes after the addition of PE alone or in the presence of inhibitors or controls, the cells were washed to remove unincorporated 45Ca2+. The cells were then solubilized, and the 45Ca2+ content was measured. No differences in total protein concentration were detected between cell incubation with PE and/or tPA compared with control cells, excluding the possible loss of cells during the incubation.

Measurement of intracellular calcium by fluorescence. The change in concentration of intracellular Ca2+ over time in response to tPA in SMC was measured with the use of a fluorescent dye as previously described (18). Briefly, the cells were washed with PBS, and the washed cells were then incubated with fluo-4 (5 µM; Molecular Probes) mixed with the nonionic detergent pluronic acid F-127 in DMSO (Molecular Probes) for 20 min at 37°C in a 5% CO2 atmosphere. The cells were washed again with PBS to remove any dye nonspecifically associated with the cell membrane and then incubated for another 30 min to complete deesterification of intracellular AM esters. Ca2+ mobilization was determined in Krebs-Ringer bicarbonate solution in the absence or presence of 2 µM Ca2+. Where indicated, tPA (20 nM) or PE (0.1 µM) was added after the basal cytoplasmic concentration of Ca2+ was determined. The Fluo-4 fluorescence was measured with a x20/0.6 plan Neofluor lens (Zeiss) in a Zeiss LSM 410 confocal system with an Axiovert 135 inverted microscope. Fluorescence excitation was induced by an argon ion laser with a 515-nm emission filter and was measured every 10 s for the indicated time. Several random fields for each experiment were scored. Confocal TIF images were transferred to a Zeiss imaging workstation to analyze fluorescence intensity. The results were expressed numerically in arbitrary fluorescence units (0–250), where white is the most intense fluorescence above background.

Coimmunoprecipitation. Cultured SMC were incubated in media alone or in media containing either 50 nM tPA, 20 nM tPA and an equimolar concentration of PAI-1, or 20 nM tPA plus 1 µM EEIIMD for the indicated time, and washed twice with PBS. The cells were lysed, and the lysates were pelleted. To study aortic extracts, isolated aortic rings were incubated in an oxygenated (95% O2-5% CO2) Krebs-Ringer bicarbonate solution. Where indicated, tPA (20 nM) alone or in the presence of an equimolar concentration of PAI-1 or 1 µM EEIIMD was added for the indicated time. The rings were homogenized in 5 vol of cold Krebs-Ringer bicarbonate solution and lysate; the lysates were centrifuged. The supernatant fractions of the lysates prepared from SMC and from aortic rings were precleared with protein A-agarose beads preblocked with 1% BSA. The precleared supernatants were then incubated for 2 h with beads containing rabbit anti-LRP or irrelevant IgG. The beads were washed five times with PBS, the proteins were eluted three times with 0.1 glycine buffer for 5 min each and centrifuged, and the supernatant was analyzed by dot blotting and Western blotting. Samples were applied to nitrocellulose membranes. The membranes were blocked with horse serum, incubated initially with anti-{alpha}vbeta3 or anti-tPA antibodies, and then incubated with a species-specific secondary antibody conjugated to horseradish peroxidase (HRP). All experiments were performed in triplicate and were repeated a minimum of three times. Irrelevant IgG was added instead of the specific primary antibodies as a control. Western blotting to detect {alpha}vbeta3 was performed as previously described (25). Briefly, the immunoprecipitates were electrophoresed on a 8–10% SDS glycine polyacrylamide gel. Separated proteins were electroblotted onto nitrocellulose membranes. The membranes were blocked with low-fat dry milk in 10 mM Tris·HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20, and the integrin subunits were detected with the same antibodies used for the dot blots. The membranes were incubated with secondary antibodies conjugated with peroxidase and developed with the appropriate colometric substrates.

Cell surface biotinylation. SMC monolayers were washed twice in ice-cold PBS, surface proteins were biotinylated by adding 400 µM Biotin-XX (Molecular Probes) on ice for 35 min, and the reaction was quenched with 20 mM glycine in PBS, pH 7.4, for 15 min. The biotinylated cells were incubated alone or with tPA (50 nM) at 4°C or at 18°C for 20 min. Incubation at 18°C permits endocytosis but impedes further migration of the protein from early to late endosomal/lysosomal compartments. Subcellular fractionation was then carried out as previously described (6). Briefly, postnuclear supernatants were loaded onto isosmolar 20%-Percoll (Amersham Pharmacia Biotech, Alameda, CA), and gradients were established by centrifugation at 20,000 g for 52 min at 4°C. Fractions were collected from the top of the gradient, and the distribution of biotinylated protein was determined by immunoprecipitation with anti-{alpha}vbeta3 followed by blotting with HRP-coupled avidin.

Statistical analysis. Data are presented as means (SD), except in Fig. 6B, where means ± SE are shown. When indicated, differences were analyzed by t-test. Statistical significance was set at P < 0.05.


Figure 6
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  		Fig. 6. A: effect of tPA on {alpha}vbeta3/LRP complex formation is time dependent. SMC were incubated with tPA (50 nM) for indicated periods of time. Cell supernatants were prepared as described in MATERIALS AND METHODS and in Fig. 5. Proteins were precipitated with anti-LRP and detected with anti-{alpha}vbeta3 using dot blotting (A, top) and Western blotting (A, bottom). Experiments were performed in triplicate and repeated a minimum of three times. A representative experiment is shown. B: density of each dot was analyzed using public domain NIH Image program (developed at National Institutes of Health and available at http://rsb.info.nih.gov/nih-image) and expressed as %control (time 0 = 100%). Each experiment was performed in triplicate. Means ± SE of three independent experiments are shown. *P < 0.05 compared with time 0.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Vasocontractile effect of tPA is integrin dependent. We have previously observed that tPA regulates the contraction of isolated aortic rings induced by PE in a dose-dependent manner (19); vasoconstriction induced by PE is increased by tPA at concentrations (10–50 nM) that are well below those used therapeutically (~0.5 µM). The vasocontractile effect of tPA is also observed in vivo (2). The capacity of tPA to increase vasoconstriction is blocked by inhibitors of LRP (19). Several integrins have also been reported to regulate blood vessel contractility, including {alpha}vbeta3 (14). Therefore, we pursued the hypothesis that tPA-induced vasoconstriction may be mediated, in part, through an interaction between LRP and {alpha}vbeta3.

The stimulatory effect of tPA (20 nM) on vasoconstriction induced by PE was inhibited by RGD but not by RGE, supporting a requirement for integrin(s) in the contractile process (Fig. 1A). To determine the involvement of {alpha}vbeta3 in tPA vasoactivity, we examined the effect of anti-{alpha}vbeta3 antibodies on the tPA-mediated contraction. A monoclonal ligand-blocking anti-{alpha}vbeta3 antibody inhibited the effect of tPA on the contraction of isolated aortic rings, whereas an irrelevant antibody did not (Fig. 1A). Furthermore, the addition of 20 nM tPA to aortic rings that had been minimally contracted by PE (14% of maximum) increased the contraction more than sixfold (to 87% of maximum) (Fig. 1B). As in Fig. 1A, the contractile effect of tPA (20 nM) was inhibited by RGD and the monoclonal ligand-blocking anti-{alpha}vbeta3 antibody but not by RGE (Fig. 1B) or an irrelevant antibody (Fig. 1B).


Figure 1
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  		Fig. 1. A: procontractile effect of tissue-type plasminogen activator (tPA) is blocked by antagonists of integrins and low-density lipoprotein-related receptor (LRP). Contraction of isolated aorta rings was induced by increasing concentrations of phenylephrine (PE) alone ({blacktriangleup}) or PE with 20 nM tPA ({blacksquare}) in the presence of 100 µM Arg-Gly-Asp (RGD) ({triangleup}) or its control peptide Arg-Gly-Glu (RGE) ({lozenge}), anti-{alpha}vbeta3 ({square}) or irrelevant IgG (x). B: tPA induces integrin- and LRP-dependent vasoconstriction. Rat aortic rings were minimally contracted by PE (Cont., control) before addition of 20 nM tPA either alone (tPA) or together with 100 µM RGD or RGE, anti-{alpha}vbeta3, anti-LRP, or irrelevant IgG immediately before tension was measured. Means (SD) of three experiments each performed in triplicates are shown.

 
tPA regulates {alpha}vbeta3 expression in a time-dependent fashion. The data presented in Fig. 1 indicate that the {alpha}vbeta3 integrin is involved in the tPA-induced vasoconstriction. We next asked whether tPA interacts with {alpha}vbeta3. {alpha}vbeta3 was identified on the surface of cultured vascular SMC using confocal microscopy, as expected (Fig. 2A, top left). The addition of tPA (20 nM) caused the apparent disappearance of {alpha}vbeta3 from the cell surface as recognized by the monoclonal antibody MAb 1976 (Fig. 2A, top right). This tPA-induced loss of {alpha}vbeta3 recognition occurred within 10 min. The effect was transient, because expression of {alpha}vbeta3 on the cell surface was completely restored by 90 min (Fig. 2A, bottom left).


Figure 2
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  		Fig. 2. tPA reduces expression of {alpha}vbeta3 on the cell surface. A: vascular smooth muscle cells (SMC) grown on coverslips were incubated in medium containing 50 nM tPA (tPA), 50 nM tPA + 1 µM EEIIMD (tPA + EEIIMD) or medium alone (Cont.) for 10 or 90 min at 37°C as indicated. Cells were stained with a monoclonal anti-{alpha}vbeta3 antibody and Cy5–5-labeled goat anti-mouse IgG as described in MATERIALS AND METHODS. No staining was seen when either anti-{alpha}vbeta3 or secondary antibody was omitted or with an isotype control (data not shown). Staining was analyzed with confocal microscopy. Experiments were performed in triplicate and were performed a minimum of three times. A representative experiment is shown. B: experiment was performed as described in A except that cells were preincubated with anti-LRP or irrelevant IgG for 15 min before addition of tPA for 10 min. Experiments were performed in triplicate and were performed a minimum of three times. A representative experiment is shown.

 
PAI-1 and anti-LRP inhibit redistribution of {alpha}vbeta3 caused by tPA. We have previously shown that the induction of vascular contraction by tPA is inhibited by its physiological inhibitor PAI-1 and by the peptide EEIIMD (19), which corresponds to amino acids 350–355 of PAI-1 (1, 11, 12). In accordance with this observation, EEIIMD inhibited the capacity of tPA to downregulate expression of {alpha}vbeta3 (Fig. 2A, bottom right). The same effect was seen when PAI-1 was added (data not shown). tPA had no effect on {alpha}vbeta3 expression when anti-LRP antibodies (Fig. 2B) or receptor-associated protein (RAP) was also present (not shown). In view of our previous finding that the tPA-induced contraction of aortic rings is mediated through LRP (19), this result suggests that, in the presence of tPA, LRP and {alpha}vbeta3 interact either directly or indirectly.

tPA induces internalization of {alpha}vbeta3. The seeming disappearance of {alpha}vbeta3 from the cell surface after addition of tPA could be due to a physical translocation of the integrin or to competition between tPA and the ligand-blocking monoclonal anti-{alpha}vbeta3 that we used. To distinguish between these possibilities, the experiments were repeated by using permeabilized cells. {alpha}vbeta3 was found on the surface of nonpermeabilized SMC (Fig. 3A). Ten minutes after the addition of tPA, {alpha}vbeta3 could no longer be detected on nonpermeabilized cells (Fig. 3B) but was readily detected in permeabilized cells (Fig. 3C), indicating translocation of the integrin.


Figure 3
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  		Fig. 3. tPA induces internalization of {alpha}vbeta3: confocal microscopy. SMC were incubated with (B and C) or without (A) 50 nM tPA for 10 min as in Fig. 2. Cells were permeabilized (C) or not (A and B), and anti-{alpha}vbeta3 antibody was added. Cells were then studied using confocal microscopy. Experiments were performed in triplicate and were performed a minimum of three times. A representative experiment is shown.

 
To analyze the effect of tPA on the subcellular distribution of {alpha}vbeta3, cell surface proteins were biotinylated and then incubated with PBS or PBS containing tPA (50 nM). The cells were incubated at 18°C for 20 min to permit endocytosis but prevent transfer of the ligand from early to late endosomes/lysosomes (5). As a control, biotinylated cells were incubated at 4°C for 20 min in the presence or absence of 50 nM tPA. The cells were lysed, and postnuclear supernatants were prepared and separated on Percoll gradients. Fractions containing the plasma membrane and early endosomes were analyzed for the presence of biotinylated {alpha}vbeta3 by immunoprecipitation with anti-{alpha}vbeta3, followed by blotting with HRP-coupled avidin (5). When cells were incubated with PBS alone, {alpha}vbeta3 was only found in the plasma membrane fraction (Fig. 4, lane 1). The addition of tPA induced a significant shift in the distribution of {alpha}vbeta3, such that the majority of biotinylated integrin was now found in the endosomal fraction (Fig. 4, lane 2), indicating that tPA induced internalization of the integrin. When the incubation was performed at 4°C, the presence of tPA did not change the distribution of {alpha}vbeta3 (not shown). Internalization of {alpha}vbeta3 induced by tPA was blocked by EEIIMD (Fig. 4, lane 3).


Figure 4
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  		Fig. 4. tPA induces internalization of {alpha}vbeta3. SMC surface proteins were biotinylated. Cells were incubated in the absence (lane 1) or presence (lane 2) of 50 nM tPA or tPA ± EEIIMD (lane 3) for 20 min at 18°C. Postnuclear supernatants were prepared and separated by centrifugation. Presence of biotinylated {alpha}vbeta3 integrin in membrane (M) or endosomal (E) fractions was determined by immunoprecipitation with anti-{alpha}vbeta3, followed by blotting with horseradish peroxidase (HRP)-coupled avidin.No protein was detected when irrelevant IgG was added instead of anti-{alpha}vbeta3 (lanes 4 and 5). Experiments were performed in triplicate and were repeated a minimum of three times. A representative experiment is shown.

 
tPA promotes formation of LRP/{alpha}vbeta3 complex. To further elucidate the relationship between these observations and tPA-mediated vasoconstriction, we took into account several observations: 1) tPA signaling is both integrin- and LRP-dependent; 2) both receptors are located primarily on the plasma membrane; and 3) tPA causes the transient disappearance of {alpha}vbeta3 from the cell surface, a process that is inhibited by anti-LRP antibodies (Fig. 2B). On the basis of these findings, we asked whether LRP interacts with {alpha}vbeta3, whether the interaction is regulated by tPA, and whether this interaction affects the internalization of the integrin.

To approach these issues, we first performed coimmunoprecipitation experiments using lysates prepared from SMC and extracts of rat aorta incubated with anti-LRP to precipitate and anti-{alpha}vbeta3 antibodies to detect complexes that might form between the two proteins. Complexes between LRP and {alpha}vbeta3 were detected in extracts from both resting SMC and aortic rings (Fig. 5, A and B). Addition of tPA (20 nM) augmented complex formation in both. The induction of LRP/{alpha}vbeta3 complexes by tPA was inhibited by PAI-1 (Fig. 5, A and B) and by EEIIMD (not shown), whereas PAI-1 and EEIIMD had no effect on the amount of complex detected in cells not exposed to tPA (not shown). In addition to {alpha}vbeta3, tPA and LRP were also detected in the immunoprecipitate obtained from SMC (Fig. 5, C and D, respectively). These results suggest that tPA induced the formation of a ternary complex. The effect of tPA on cellular {alpha}vbeta3/LRP complex formation was time dependent (Fig. 6), and the time course of complex formation mirrored the kinetics of the disappearance of {alpha}vbeta3 from the cell surface (Fig. 2). In extracts from SMC, stimulation of {alpha}vbeta3/LRP complex formation by tPA was evident by 10 min. Over the next 60 min, there was a progressive decrease in the amount of complex detected. However, by 90 min, the amount of LRP/{alpha}vbeta3 complexes was again comparable to that seen at baseline. Essentially identical results were seen when extracts from aorta were studied (not shown).


Figure 5
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  		Fig. 5. tPA induces formation of LRP/{alpha}vbeta3 complexes. A: SMC were incubated for 10 min in medium alone or medium containing 50 nM tPA or tPA and an equimolar concentration of plasminogen activator inhibitor type 1 (PAI-1). Cells were managed as described in in MATERIALS AND METHODS; immunodetection was performed with the use of monoclonal anti-{alpha}vbeta3 and then with a secondary antibody conjugated to HRP. No protein was detected by anti-{alpha}vbeta3 when irrelevant IgG was added instead of anti-LRP (B). B, left: Western blotting of {alpha}vbeta3 immunoprecipitated by anti-LRP antibodies from lysates of SMC. Immunoprecipitates were electrophoresed, managed as described in MATERIALS AND METHODS, and integrins were detected with anti-{alpha}vbeta3 (lanes 1-3) or irrelevant IgG (lane 4). Blots were then incubated with secondary antibodies conjugated with peroxidase. B, right: same experiment described in left panel of B was performed with homogenates of rat aorta. Precipitated proteins were detected with anti-{alpha}vbeta3 antibody (lanes 1-3) or irrelevant IgG (lane 4). MW, molecular weight. C: experiment was performed as in A and B except that proteins precipitated by anti-LRP antibodies were detected with anti-tPA (lanes 1 and 2) or irrelevant IgG (lane 3). D: after immunoprecipitates were separated by electrophoresis and transferred onto nitrocellulose membranes, anti-LRP monoclonal antibodies (lane 1) or irrelevant IgG (lane 2) was added, followed by secondary antibodies conjugated with peroxidase. All experiments were performed in triplicate and were repeated a minimum of three times. A representative experiment is shown.

 
LRP and {alpha}vbeta3 mediate tPA-induced Ca2+ mobilization. We next asked whether tPA-induced vasocontraction is accompanied by an increase in intracellular Ca2+ (a universal trigger of muscle contraction) and whether LRP and integrins are involved in this cellular response to tPA. The addition of tPA (50 nM) to SMC increased intracellular Ca2+ and augmented the effect of PE on Ca2+ internalization (Fig. 7), as expected. A similar effect was obtained at 20 nM tPA (data not shown) As would be predicted from the results shown in Fig. 1B, the effect of tPA on Ca2+ mobilization in the presence of PE was inhibited by antagonists of LRP, by RGD, by anti-{alpha}vbeta3 as well as by PAI-1 and the PAI-1-derived peptide EEIIMD (Fig. 7). As a second approach, we examined tPA-induced Ca2+ mobilization using an indicator dye. tPA increases cytoplasmic Ca2+ in the presence or absence of extracellular Ca2+ (Fig. 8). This result indicates that tPA induces release of intracellular Ca2+, likely from the endoplasmic reticulum.


Figure 7
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  		Fig. 7. LRP and integrin are required for signal transduction induced by tPA. SMC were incubated with 45Ca2+ in the absence (Cont.) or presence of 50 nM tPA, phenylephrine (PE), tPA + PE, tPA + PE + PAI-1, tPA + PE + EEIIMD, tPA + PE + anti-LRP antibodies or anti-{alpha}vbeta3 antibodies (AvB3), tPA + PE + RGD, RGE or irrelevant control antibodies (IR Ab). Means (SD) of three independent experiments, each performed in triplicate, are shown. cpm, counts/min.

 

Figure 8
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  		Fig. 8. tPA induces Ca2+ mobilization from intracellular stores. SMC were incubated in media containing tPA (20 nM) in Ca2+-containing (A) or Ca2+-free (B) medium. C and D are negative and positive controls with media containing either no additives (C) or 100 nM PE (D), respectively. Fluorescence excitation was induced by an argon ion laser equipped with a 515-nm emission filter. Signal was measured every 10 s. Experiments were performed in triplicate and were repeated three times. A representative experiment is shown.

 
We then asked whether the entry of labeled calcium into the cells in response to tPA correlates with the various stages of LRP/{alpha}vbeta3 complex formation and dissociation. Sixty minutes after the addition of tPA to SMC, the cells were refractory to readdition of tPA (Fig. 9). This parallels the time course with which LRP/{alpha}vbeta3 complexes induced by tPA were seen to dissociate (Figs. 6 and 10). Ca2+ mobilization in response to tPA was restored by 90 min (Fig. 8), a time at which the expression of {alpha}vbeta3/LRP complexes returned to baseline (Figs. 6 and 10), indicating that the refractory period had passed.


Figure 9
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  		Fig. 9. Response of SMC cells to tPA is time limited and time dependent. SMC were incubated with 45Ca2+ in the absence (Cont.) or presence of 20 nM tPA for 10 min. In some cells, internalized Ca2+ was measured 10 min later (tPA 10 min). In others, supernatant was changed to one that did not contain tPA. Sixty minutes later, Ca2+ was measured in some cells (tPA 60 min), whereas in other cells, tPA was readded. Ca2+ was measured either 10 min after second addition of tPA (tPAx2 60 min) or tPA was readded 90 min later and Ca2+ was measured 10 min after the second addition (tPAx2 90 min). Means (SD) of three independent experiments, each performed in triplicate, are shown.

 

Figure 10
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  		Fig. 10. Effect of tPA on LRP and {alpha}vbeta3: hypothetical schema. In resting SMC, free and complexed LRP and {alpha}vbeta3 are in equilibrium. Presence of tPA enhances binding of LRP to integrin and triggers an increase in intracellular calcium. Ternary complex (LRP, tPA, and {alpha}vbeta3) is internalized and dissociates within the cell. tPA is degraded, whereas LRP and {alpha}vbeta3 recycle to the cell membrane. In the presence of PAI-1, tPA binds to LRP, and resultant complex is internalized without integrin. LRP recycles to the membrane, where tPA-PAI-1 complex is degraded.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although tPA promotes fibrinolysis, it has been less effective in the treatment of thrombotic stroke than had been anticipated based on experience in treating acute coronary arterial thrombosis. Indeed, tPA has been shown to be neurotoxic (7, 17, 23), to open the blood-brain barrier (28), and to increase the infarct area after stroke (16, 27) or head trauma (15) in several experimental models. The mechanism(s) underlying these deleterious effects is only partially understood. We previously reported that tPA exerts a dose-dependent effect on vascular contractility (19), including causing cerebral vasoconstriction under pathological conditions, such as hypoxia/ischemia (2). Others have observed that tPA enhances blood-brain barrier permeability through an LRP-mediated pathway (28). These unintended effects on vascular function may impede the beneficial effects of tPA-mediated fibrinolysis.

In the present study, we found that the procontractile properties of tPA depend on {alpha}vbeta3 as well as LRP. Binding of tPA to LRP causes a rapid time-dependent loss of {alpha}vbeta3 expression from the surface of vascular SMC. The decrease in integrin expression is due to its internalization, which is induced by tPA. We also found that LRP and {alpha}vbeta3 form a complex in resting SMC and extracts of blood vessels. The amount of LRP/{alpha}vbeta3 complex formed is regulated by tPA in a time-dependent manner that parallels the rate of disappearance of {alpha}vbeta3 from the cell. These observations support the interrelationship among the three processes.

The observation that anti-LRP antibodies inhibited the effect of tPA on the internalization of {alpha}vbeta3 makes it less likely that endocytosis was caused by a direct interaction between tPA and {alpha}vbeta3. This suggests that tPA interacts with {alpha}vbeta3 primarily through LRP, although a direct interaction between tPA and the integrin cannot be excluded. On the basis of these findings, we propose a model in which {alpha}vbeta3 and LRP can form heterodimeric complexes. Binding of tPA to LRP shifts the equilibrium between the free proteins toward the formation of these complexes (Fig. 5). Formation of the complex generates an intracellular signal that releases calcium from intracellular stores, likely the endoplasmic reticulum, which promotes vasoconstriction.

tPA-mediated signal transduction is interrupted by its cognate inhibitor PAI-1 and a PAI-1-related peptide EEIIMD, which, as we have shown, does not bind to the catalytic site or impede the catalytic activity of tPA (19). In support of the proposed model, PAI-1 inhibits tPA-mediated loss of {alpha}vbeta3 expression on the cell surface and the formation of LRP/{alpha}vbeta3 complexes. At the same time, PAI-1 accelerates the transfer of tPA to lysosomes and its subsequent degradation (19).

We postulate that tPA binds to LRP, inducing its interaction with {alpha}vbeta3 (Fig. 10). The interaction between LRP and {alpha}vbeta3 leads to initiation of signal transduction before the ligand continues to the lysosomes, where it is degraded. Once tPA has been released, LRP and {alpha}vbeta3 dissociate and return to the cell membrane as free proteins, as suggested by the loss of immunoprecipitable complexes 60 min after tPA addition (Figs. 6 and 7). Once LRP and {alpha}vbeta3 have recycled to the membrane, the equilibrium between free and complexed proteins is restored. PAI-1 inhibits the procontractile activity of tPA by impeding the formation of LRP/{alpha}vbeta3 complexes (Fig. 10). tPA/PAI-1 complexes are rapidly transported to the lysosomes via LRP. This model suggests that the LRP/integrin complex participates in the process of tPA signaling and the formation of the complex is inhibited by PAI-1 (Fig. 10). These data add to the diversity of the possible interactions between LRP and integrins that have been described in various cell types and in response to other ligands (22). A similar or opposing noncatalytic process may underlie the tPA- and LRP-dependent increase in blood-brain barrier permeability (28).

Additional studies are necessary to clarify the details of how LRP/{alpha}vbeta3 complexes are formed, the signal transduction system that is initiated, and the role of this process in physiological and pathophysiological responses of the vasculature. Detailed elucidation of the interaction between LRP and {alpha}vbeta3 may suggest novel means to regulate vascular contractility and tPA-mediated damage to the central nervous system and other organs.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-06831, HL-076406, and HL-67381 and Grant 930/04 from the Israel Science Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Al-Roof Higazi, Dept. of Pathology and Laboratory Medicine, Univ. of Pennsylvania, 513A Stellar-Chance, 422 Curie Boulevard, Philadelphia, PA 19104 (e-mail: higazi{at}mail.med.upenn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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