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CARDIOVASCULAR
Departments of Comparative Medicine (C.N.M.), Pathology (C.N.M., C.J.L.), and Medicine (K.M., C.J.L.), The Johns Hopkins University School of Medicine, Baltimore, Maryland
Received December 20, 2004; accepted March 16, 2005.
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Abstract |
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). P-selectin-null mice exhibit an absence of leukocyte rolling on an activated endothelium and a delayed recruitment of neutrophils to the peritoneal cavity following experimentally induced inflammation (Mayadas et al., 1993). Following activation of endothelial exocytosis, further inflammatory stimuli can trigger leukocyte activation, which leads to expression of intracellular adhesion molecules mediating tight adherence to the vessel wall and diapedesis across the endothelium and into the tissue. However, endothelial exocytosis is the initial step in leukocyte trafficking.
Several sets of proteins mediate exocytosis, including N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment receptors (SNAREs), Sec/Munc proteins, and members of the Rab superfamily (Rothman and Wieland, 1996; Jahn and Sudhof, 1999; Mellman and Warren, 2000; Wickner and Haas, 2000; Jahn et al., 2003). SNAREs are transmembrane proteins associated with vesicle membranes and target membranes. Members of the SNARE superfamily include syntaxins, SNAP-25, and VAMP (or synaptobrevins) (Flaumenhaft et al., 1999). The SNAREs expressed in endothelial cells include syntaxin-2, syntaxin-4, SNAP-23, and VAMP-3 (Matsushita et al., 2003). Three members of the SNARE family can associate via coiled-coil SNARE domains, forming a stable ternary complex and bringing vesicle and target membranes into apposition. The original SNARE hypothesis of Rothman proposed that unique combinations of SNARE molecules specified membrane fusion partners.
Vesicle trafficking also requires NSF, a member of the ATPase-associated activity superfamily. NSF is comprised of three domains (Fig. 1A) (Wilson et al., 1992; Tagaya et al., 1993). The N-terminal domain is responsible for NSF binding to the SNARE complex through the accessory protein 
-soluble NSF attachment protein (-SNAP). The D1 domain contains an ATPase site; hydrolysis of ATP provides the energy for NSF disassembly activity (Whiteheart et al., 1994; Matveeva et al., 1997). The D2 domain binds to ATP (without hydrolysis) and mediates hexamerization of NSF.
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We hypothesized that inhibitors of NSF would decrease endothelial exocytosis, leukocyte trafficking, and tissue inflammation. We therefore developed a novel compound that can cross cell membranes and inhibit NSF. This NSF inhibitory compound is a fusion polypeptide consisting of two domains: an amino-terminal domain derived from HIV transactivator of transcription (TAT) protein fused to a carboxy-terminal domain derived from NSF. The region of TAT that mediates cell penetration is an 11-amino acid residue sequence (YGRKKRRQRRR) (Frankel and Pabo, 1988; Green and Loewenstein, 1988; Green et al., 1989). Mammalian cells internalize TAT-fusion polypeptides by a lipid raft-dependent, macropinocytosis-mediated process (Wadia et al., 2004). Polypeptides consisting of this TAT domain fused to proteins can cross membranes of cells ex vivo and in vivo (Schwarze et al., 1999; Schwarze and Dowdy, 2000; Snyder and Dowdy, 2001; Vocero-Akbani et al., 2001). The carboxy-terminal portion of the NSF inhibitor is derived from amino acid residues 700 to 722 of NSF. This region within the D2 domain of NSF mediates NSF homohexamerization. We refer to this fusion polypeptide as TAT-NSF700.
We used this novel fusion polypeptide to test the hypothesis that inhibition of endothelial cell exocytosis decreases leukocyte trafficking and inflammation. We now show, using a murine model of leukocyte rolling and peritonitis, that TAT-NSF700 decreases endothelial exocytosis in vitro, leukocyte adherence ex vivo, leukocyte rolling along venules in vivo, and leukocyte trafficking into the peritoneal cavity following experimentally induced inflammation. These data suggest that inhibitors of exocytosis are potentially valuable in the treatment of inflammatory disorders.
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Materials and Methods |
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NSF ATPase Assay. The ATPase activity of NSF was measured by a coupled assay, in which ATP utilization is linked to the pyruvate kinase reaction, which generates pyruvate, which in turn is measured continuously with lactate dehydrogenase (Huang and Hackney, 1994). Recombinant NSF (0.2 µg/µl) was pretreated with TAT-NSF700 or TAT-NSF700scr for 10 min at 22°C. ATPase reaction buffer (100 mM HEPES buffer, pH 7.0, 100 mM KCl, 10 mM MgCl2, 5 mM CaCl2, 10 mM ATP, 5 mM phosphoenol pyruvate, 50 U lactate dehydrogenase, and 50 U pyruvate kinase) was added to the mixture, followed by 10 µl of NADH (2 mg/ml in 1% sodium bicarbonate). The mixture was incubated for 10 min at 22°C, and the absorbance was measured at 340 nm.
Disassembly Assay. The effect of TAT-NSF700 on SNARE complex disassembly activity of NSF was studied in vitro as described previously (Matsushita et al., 2003). Recombinant (His)6-NSF, (His)6--SNAP, GST-syntaxin-4, GST-VAMP3, and GST-SNAP23 were expressed in bacteria and purified. Recombinant NSF (0.5 µg) was incubated with control, TAT-NSF700, or TAT-NSF700scr for 10 min and then added to equal amounts of -SNAP, GST-syntaxin-4, GST-VAMP3, and GST-SNAP23. The incubation buffer was 4 mM HEPES, 0.1M NaCl, 1 mM EDTA, 3.5 mM CaCl2, and 0.5% NP40. Either 10 mM ATP or ATP-
S with 20 mM MgCl2 was added along with 50 µl of binding buffer and 20 µl of 50% glutathione-Sepharose beads. The mixture was incubated for 1 h at 4°C, washed in binding buffer, and boiled for 3 min with SDS sample buffer. Samples were fractionated on 4 to 15% precast gels (Bio-Rad, Hercules, CA) and immunoblotted.
Cell Exocytosis Studies. HAEC (Cambrex Bio Science Walkersville, Walkersville, MD) were incubated with TAT-NSF700 or control for 15 min and activated with 1 U/ml thrombin. vWF release was measured by an ELISA. In some experiments examining the persistence of the effects of TAT-NSF700, HAEC were washed after the 15-min incubation with TAT-NSF700 and then activated with thrombin 0 to 8 h after TAT-NSF700 treatment. Human platelets were acquired from normal healthy blood donors. Subjects were excluded if they had used aspirin or a nonsteroidal anti-inflammatory agent 10 days prior to the blood draw. Blood was collected by venipuncture into sodium citrate anticoagulant tubes. Whole blood was centrifuged at 180g for 15 min to isolate the top layer of platelet rich plasma. Platelets were incubated for 15 min with TAT-NSF700 or control and activated with TRAP (thrombin receptor-activating peptide). Surface P-selectin translocation was measured by FACS.
TAT-NSF700 Inhibition of Leukocyte Binding to Endothelial Cells. HAEC cells were grown in six-well plates. Cells were incubated with 1 µM of TAT-NSF700, TAT-NSF700Scr, or control for 20 min. HAEC cells were activated with 1 U/ml of thrombin (Sigma-Aldrich, St. Louis, MO) or control for 20 min. Whole blood from human donors was collected into EDTA-containing tubes by venipuncture and fluorescently labeled with 50 µl of 0.05% rhodamine 6G (Molecular Probes, Eugene, OR) in 3 ml of blood. Blood (25 µl) was added to the cells for 20 min with gentle rocking. Cells were washed three times. Separate fields were imaged using a fluorescent microscope (Nikon, Melville, NY) and a digital imaging camera (Retiga, Burnaby, BC, Canada) for each experiment. The mean fluorescence was determined in each field. As a negative control, cells were also activated or not as above, and cells were incubated with anti-P-selectin antibody (BD Biosciences PharMingen, San Diego, CA) prior to the addition of leukocytes.
Leukocyte Rolling in Vivo. Intravital microscopy was performed as previously described (Cambien et al., 2003). Leukocytes were fluorescently labeled with rhodamine 6G (50 µl of 0.05%) intravenous. Mice were anesthetized with ketamine (80 mg/kg) and xylazine (13 mg/kg) and then injected intravenously with control, 0.5 mg/kg TAT-NSF700, or TAT-NSF700Scr. The mesentery was exteriorized, venules (120–150 µM diameter) were selected, and the mouse mesentery was prepared on an inverted fluorescent microscope (Nikon) with a 37°C stage warmer. Endothelial degranulation was induced by the superfusion of 30 µl of 1 µM histamine, and images of leukocyte rolling were captured each minute (Retiga). Leukocyte rolling on the activated endothelium was quantified at each time point.
Wall shear rate was measured by intravital microscopy according to the methods of others (Kubes et al., 1991; Scalia et al., 1996; Sperandio et al., 2003). Erythrocytes were isolated from mice, labeled with calcein-acetoxymethyl ester, and injected into wild-type mice, which were then treated or not with TAT-NSF700. Erythrocyte velocity was measured by determining the distance traveled in a 50-ms period. Venule diameter was measured by direct observation using image analysis software. Wall shear rate was calculated as follows: g = 8(erythrocyte velocity/venule diameter).
Peritonitis Model. C57BL6/J mice were injected intraperitoneally with 1 ml of 2% glycogen and intravenously with 0.5 mg/kg TAT-NSF700 peptide. After 2 or 4 h, the mice underwent a peritoneal lavage with 5 ml of a solution containing 0.1% albumin and 0.5 mM EDTA. One milliliter of lavage solution was withdrawn from the peritoneal cavity, centrifuged at 3000 rpm for 5 min, and resuspended in 250 µl of solution. Cell counts were performed using a Coulter counter.
Statistical Analysis. Results are expressed as mean ± standard deviation (S.D.). Comparison between groups was performed by Student's paired two-tailed t test. Two-way analysis of variance was used to describe differences between groups, with post hoc analysis performed by the methods of Student-Newman-Keuls. A value of P < 0.05 was considered significant.
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Results |
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We first explored the effect of the fusion polypeptide TAT-NSF700 upon the ATPase activity of NSF. We synthesized recombinant (His)6-NSF and (His)6--SNAP in bacteria and purified them using metal chelate chromatography. We then added TAT-NSF700 or its control, TAT-NSF700scr, to recombinant NSF and -SNAP and measured ATPase activity using a coupled assay. TAT-NSF700 inhibits the ATPase activity of NSF in a dose-dependent manner as compared with its control peptide TAT-NSF700scr (Fig. 1B). The IC50 of TAT-NSF700 is between 0.1 to 1.0 µM.
We next investigated TAT fusion peptide inhibition of NSF disassembly activity. TAT-NSF700 or TAT-NSF700scr was added to a mixture of recombinant NSF, -SNAP, GST-syntaxin-4, GST-VAMP-3, and GST-SNAP-23. Glutathione beads were added to precipitate GST-tagged peptides, and the precipitate was immunoblotted against NSF. As expected, NSF cannot disassemble the SNARE complex in the presence of ATP-S (Fig. 1C, left panel). Furthermore, NSF can disassemble the SNARE complex in the presence of ATP (Fig. 1C, left panel). However, TAT-NSF700 dose-dependently inhibits the disassembly activity of NSF (Fig. 1C, left panel). The TAT-NSF700scr control peptide does not inhibit the disassembly activity of NSF (Fig. 1C, right panel). These data demonstrate that TAT-NSF700 inhibits the ATPase and disassembly activities of NSF.
TAT-NSF700 Inhibits Exocytosis from HAEC. We next explored the effect of TAT-NSF700 upon endothelial cell exocytosis. HAEC were incubated with TAT-NSF700, TAT-NSF700scr, or media alone for 20 min and then activated with thrombin. Exocytosis was measured using an ELISA for vWF. Thrombin triggers HAEC release of vWF (Fig. 2A). TAT-NSF700 inhibits vWF release (Fig. 2A). In contrast, the control peptide TAT-NSF700scr has no effect.
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We also explored the effect of TAT-NSF700 upon platelet exocytosis. Platelets were incubated with control or TAT-NSF700 for 15 min and then activated with TRAP. Platelet granule exocytosis was measured by surface P-selectin translocation using FACS. TAT-NSF700 does not inhibit granule exocytosis from platelets (Fig. 2C).
TAT-NSF700 Decreases Leukocyte Adherence to Endothelial Cells in Vitro. Since endothelial exocytosis externalizes P-selectin, which in turn contributes to leukocyte adherence, we predicted that inhibitors of endothelial exocytosis would decrease leukocyte adherence to endothelial cells. Accordingly, we next examined the effect of TAT-NSF700 upon leukocyte binding to endothelial cells in vitro. HAEC were incubated with TAT-NSF700, TAT-NSF700scr, or control. Endothelial cells were then activated with thrombin or not, and fluorescently labeled leukocytes were added. The cells were washed to remove nonadherent leukocytes, images of fluorescent adherent cells were taken, and the mean fluorescence of each condition was determined. Thrombin increases leukocyte binding to endothelial cells (Fig. 3, A and B). TAT-NSF700 inhibits leukocyte binding to stimulated endothelial cells (Fig. 3, A and B). However, TAT-NSF700scr does not inhibit leukocyte binding to activated endothelial cells. These data demonstrate that the NSF inhibitor TAT-NSF700 decreases leukocyte binding to stimulated endothelial cells in culture.
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P-selectin, a mediator of leukocyte-endothelial cell interactions, is delivered to the surface of endothelial cells by WPB exocytosis. To demonstrate that in our model system leukocyte binding to HAEC is mediated in part by P-selectin surface translocation, HAECs were prepared as above and activated or not with thrombin, and antibody to P-selectin was added following thrombin activation. Fluorescent leukocytes were added to HAEC cells and washed, and the number of adherent leukocytes was counted. Thrombin increases leukocyte binding to endothelial cells (Fig. 3C). In contrast, antibody to P-selectin inhibits leukocyte binding to activated endothelial cells (Fig. 3C). These data show that leukocyte binding to HAEC is at least partially dependent on P-selectin translocation.
TAT-NSF700 Inhibits Leukocyte Rolling in Vivo. We next investigated the in vivo effect of the TAT-NSF700 peptide using a model of leukocyte rolling in mice. Mice were injected with rhodamine 6G to fluorescently label leukocytes in vivo. Vehicle, TAT-NSF700, or TAT-NSF700scr (0.5 mg/kg) were injected intravenously, and the mesentery was exteriorized on the stage of an inverted microscope. Endothelial cell Weibel-Palade body degranulation was induced by super-fusion of 30 µl of 1 µM histamine. Images of rolling leukocytes on venules were captured using a digital camera. Histamine increases leukocyte rolling within 3 min of treatment (Fig. 4, A and B). TAT-NSF700 inhibits leukocyte rolling. The TAT-NSF700scr control peptide has no effect upon histamine-activated leukocyte rolling (Fig. 4, A and B). TAT-NSF700 has no effect on the vessel hemodynamics as assessed by measuring vessel diameter and erythrocyte velocity before and after histamine treatment (Table 1). These data demonstrate that TAT-NSF700 decreases leukocyte rolling in vivo.
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We next explored the effect of TAT-NSF700 in a murine model of acute peritonitis. Following injection of TAT-NSF700, TAT-NSF700scr peptide, or control intravenously, mice were then injected intraperitoneally with 1 ml of 2% glycogen or control to induce peritonitis. The peritoneum was lavaged 2 and 4 h after glycogen treatment, and the number of leukocytes was counted. TAT-NSF700 decreases the number of leukocytes in the peritoneum following glycogen injury compared with control (Fig. 4C). In particular, TAT-NSF700 decreases peritonitis within 2 h after glycogen treatment; however, the effect of TAT-NSF700 wears off, and it has no effect 4 h after glycogen treatment (Fig. 4C). TAT-NSF700scr does not reduce leukocyte infiltrates into the peritoneum. The percentage of each cell type stays constant between treatment groups, with only the total number of leukocytes differing (data not shown). These data show that TAT-NSF700 decreases leukocyte translocation into the peritoneum following experimental peritoneal inflammation.
We then administered the TAT-NSF700 peptide after glycogen treatment to see if this peptide could alter the course of peritonitis after inflammatory insults. Mice were injected i.p. with glycogen at time 0 and treated with TAT-NSF700 peptide 1 or 2 h after glycogen. Mice were then lavaged 4 h after glycogen treatment, and the number of leukocytes was counted. TAT-NSF700 administered 2 h after glycogen decreases inflammation (Fig. 4D). However, TAT-NSF700 has no effect on inflammation when administered 1 h after glycogen. These data suggest that TAT-NSF700 administered as a single i.v. bolus can suppress exocytosis over a 2-h period. Together, these data demonstrate that the novel peptide inhibitor of NSF TAT-NSF700 reduces endothelial Weibel-Palade body exocytosis, leukocyte rolling, and adherence in vitro and in vivo.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Our data suggest that endothelial exocytosis is critical for leukocyte trafficking. Various knockout mice lacking WPB components demonstrate the importance of endothelial exocytosis to cell trafficking. Knockout mice lacking vWF display severe defects in hemostasis and thrombosis, whereas P-selectin-deficient mice exhibit compromised leukocyte rolling and extravasation (Mayadas et al., 1993; Denis et al., 1998). TAT-NSF700-treated mice have a functional phenotype very similar to P-selectin knockout mice (Fig. 4). Studies utilizing anti-adhesion molecule therapies also demonstrate the importance of WPB components in regulating leukocyte and endothelial cell interactions. Monoclonal antibodies to P-selectin or its ligand or other adhesion molecules decrease vascular inflammation and tissue injury, particularly in models of inflammatory bowel disease, multiple sclerosis, and psoriasis (Harlan and Winn, 2002). TAT-NSF700 similarly decreases P-selectin-mediated leukocyte adherence to endothelial cells (Fig. 3); however, clinical trials of anti-adhesion molecule therapy aimed at ischemia-reperfusion have been less fruitful (Harlan and Winn, 2002; Yonekawa and Harlan, 2005). A possible reason for the lack of benefit is that many of these therapies target events following endothelial exocytosis, such as expression of individual intercellular adhesion molecules or isolated integrins on leukocytes and endothelial cells. Our TAT-NSF700 peptide may provide a means to target acute inflammatory events and suppress endothelial cell inflammatory activation.
We found that TAT fusion polypeptides will enter endothelial cells but not platelets. No previous studies have identified a cell type into which TAT polypeptides cannot enter. TAT PTD-mediated uptake was initially thought to occur by direct penetration across the cell membrane. However, a recent study by Wadia indicates that cellular uptake of TAT-fusion protein is mediated by rapid TAT internalization through lipid raft-dependent macropinocytosis (Wadia et al., 2004). Platelets also take up proteins from their surroundings by endocytic mechanisms. Platelet endocytosis is part of granule maturation. Platelet granules acquire much of their protein content by endocytosis and pinocytosis in both megakaryocytes and platelets (Harrison and Cramer, 1993). Our finding that platelets cannot take up TAT-NSF700 suggests that platelets lack lipid raft-dependent macropinocytosis. Alternatively, our data may indicate that TAT PTD uptake is mediated by a receptor present in the lipid raft-rich membrane domain of some cells, such as endothelial cells, but absent from other cells, such as platelets.
We designed TAT-NSF700 to interact with the hexamerization domain of endogenous NSF. We hypothesize that our peptide is a dominant negative inhibitor of NSF, interfering with NSF assembly into homohexamers. The reversibility of TAT-NSF700 may be related to dynamic NSF hexamerization or synthesis of new NSF molecules.
The effects of a single dose of TAT-NSF700 are brief: TAT-NSF700 inhibits exocytosis of cultured cells for 0 to 2 h after treatment (Fig. 2B). The peptide may be degraded by cell proteolytic pathways. TAT-NSF700 also inhibits inflammation for a brief period in vivo: the peptide inhibits inflammation within 2 h of treatment but has no effect 4 h after treatment (Fig. 4C). The catabolic pathways of TAT-tagged peptides are unknown.
Our data and the work of others suggest that NSF is a critical regulator of endothelial exocytosis. Since endothelial exocytosis regulates leukocyte trafficking, novel inhibitors of NSF may prove useful in the treatment of inflammatory diseases.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: WPB, Weibel-Palade bodies; vWF, von Willebrand's factor; NSF, N-ethylmaleimide-sensitive factor; SNARE, soluble NSF attachment receptor; SNAP, soluble NSF attachment protein; VAMP, vesicle-associated membrane protein; -SNAP, -soluble NSF attachment protein; TAT, transactivator of transcription; GST, glutathione S-transferase; ATP-S, adenosine-5'-O-(3-thio)triphosphate; HAEC, human aortic endothelial cells; ELISA, enzyme-linked immunosorbent assay; TRAP, thrombin receptor-activating peptide; FACS, fluorescence-activated cell sorting; HIV, human immunodeficiency virus; PTD, protein transduction domain.
Address correspondence to: Charles J. Lowenstein, 950 Ross Building, 720 Rutland Avenue, Baltimore, MD 21205. E-mail: clowenst{at}jhmi.edu
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