Division of Pulmonary and Critical Care Medicine and Center for Translational Respiratory Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224-6801
Submitted 13 September 2002 ; accepted in final form 30 January 2003
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ABSTRACT |
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endothelium; lung; vasculature; injury; G protein; differentiation; cell differentiating gene
Sphingosine-1-phosphate (S1P) is a recently described serum-borne sphingolipid that is released by activated platelets. Through its interaction with the endothelial differention gene (EDG) family of receptors, S1P mediates numerous biological effects including EC migration (8, 9, 22, 28, 3436, 49), adherens junction assembly (26), cell proliferation (3, 22, 41), wound healing (25), and inhibition of apoptosis (20, 24, 41). Platelets have a highly active form of sphingosine kinase that rapidly converts sphingosine into S1P and a relative deficiency of sphingosine lyase, which is the enzyme responsible for S1P catabolism and breakdown (52). As a result, platelets store abundant amounts of S1P and are the cellular component that is primarily responsible for the concentration of S1P found in plasma (51). We recently described (11) the ability of S1P to potently and rapidly enhance transendothelial electrical resistance (TER) and reorganize filamentous actin into a prominently thickened cortical actin band.
The ability of platelets to secrete S1P into the bloodstream, in the
context of the potent biological effects that S1P has on ECs, warrants a
closer examination of S1P as a potential mediator of platelet-induced barrier
enhancement. We analyzed the effects of isolated washed human platelets on the
transmonolayer electrical resistance of confluent human pulmonary artery ECs
(HPAECs), which is a measure of endothelial integrity, as well as on actin
cytoskeletal arrangement in these cells. Our data show that platelets and S1P
produce similar concentration- and dose-dependent increases in EC barrier
resistance and comparable reorganization of the actin cytoskeleton into
thickened cortical bands. In addition, pharmacological inhibition of
Gi-coupled protein signal transduction, EDG-1 receptor
antisense oligonucleotide strategies, and incubation of the stimulus with
activated charcoal diminish the effects of both exogenously added platelets
and S1P on HPAECs. This correlation supports our hypothesis that S1P
represents the major bioactive platelet-derived factor that affects the
enhancement of the EC permeability barrier.
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MATERIALS AND METHODS |
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Cell culture. HPAECs were obtained from Clonetics (Walkersville, MD) and were cultured in endothelial basal medium (EBM)-2 growth media supplemented with 0.2 ml of hydrocortisone, 2 ml of human FGF-B, 0.5 ml of VEGF, 0.5 ml of long-arm insulin-like growth factor-1 (R3-IGF-1), 0.5 ml of ascorbic acid, 0.5 ml of human epidermal growth factor (EGF), 0.5 ml of GA-1000 (Clonetics), and 0.5 ml of heparin with 10% FBS. The cultures were grown in gelatin-coated tissue culture-treated flasks and maintained in a humidified atmosphere of 5% CO2-95% air until confluency was reached with contact-inhibited monolayers. Cells were then seeded onto gelatinized glass coverslips (for immunofluoresence studies) or gelatinized gold-coated microelectrodes (for electrical resistance measurements) and again grown to 100% confluent monolayers before use. All experiments were performed in DMEM serum-free media.
Measurement of cell monolayer TER. Electrical resistance across EC
monolayers was measured by using an electrical cell-substate sensor (ECIS)
system (Applied Biophysics, Troy, NY) as previously described
(13). Cells grown on gold
microelectrodes (10-3 cm2) in polycarbonate wells act as
insulating particles, and the resistance across the monolayers (TER) is
measured in real time. TER increases as cells adhere on the microelectrode and
intercellular cell contacts are formed or in response to agents that increase
cell-to-cell adhesive interactions
(11). In contrast, cell
retraction, rounding, or loss of adhesion is reflected by decreases in TER
(13). These measurements
provide a highly sensitive biophysical assay that indicates the state of cell
shape, focal adhesion, and endothelial barrier function
(16,
47). Briefly, current was
applied across the electrodes by a 4,000-Hz AC voltage source with an
amplitude of 1 V in series with a 1 M resistance to approximate a
constant current source (
1 µA). The small gold electrode and the larger
counterelectrode (1 cm2) were connected to a phase-sensitive
lock-in amplifier (model 5301A, EG&G Instruments, Princeton, NJ) with a
built-in differential preamplifier (model 5316A, EG&G Instruments). The
in-phase and out-of-phase voltages between the electrodes were monitored in
real time with the lock-in amplifier and converted to scalar measurements of
transendothelial impedance of which resistance was the primary focus. TER was
monitored for 30 min to establish a baseline resistance. Wells with baselines
that exceeded 2 standard deviations from the pooled mean were rejected from
analysis. All electrical resistance data are presented as values normalized by
the basal resistance for each well.
Platelet isolation. Whole blood was collected from healthy donors into 10-ml vacutainers (Becton-Dickinson) that contained 15% K3-EDTA. Blood was transferred to round-bottom polypropylene tubes (Falcon) and centrifuged at 500 g (Allegra 6 centrifuge, Beckman) for 10 min to obtain PRP. The PRP was removed and spun at 1,200 g for an additional 10 min to separate platelets from plasma. The platelet button was resuspended and washed four times in a modified Tyrode's buffer (2.37 mM KCl, 124 mM NaCl, 11 mM NaCHO3, 0.4 mM Na2HPO4, 0.3% BSA, 4.6 mM dextrose, and 10 mM EDTA). After the final washing, platelet counts were adjusted to 1.52.5 x 108/ml (Beckman Coulter Z1) in Tyrode's buffer that contained 100 µM EDTA. Platelet supernatants were prepared by centrifuging platelet suspensions in a microcentrifuge for 45 s.
Platelet function analysis. Platelets suspended in Tyrode's buffer
that contained 100 µM EDTA were stimulated with 100 nM human
-thrombin either in the presence or absence of 1 mM CaCl2.
Light transmission, a reflection of platelet aggregation, was measured by a
Chrono-log aggregometer and recorded on a Chrono-log chart-strip recorder.
Washed human platelets remain functional after the isolation and washing procedure. After carefully isolating and washing human platelets, we tested their responsiveness, viability, and function by measuring platelet aggregation in response to the known platelet-aggregating agent thrombin. Tyrode's buffer is able to prevent platelet activation and aggregation in response to thrombin. Replenishing the amount of usable calcium (which is required for platelet aggregation) in the Tyrode's buffer with 1 mM CaCl2 allowed us to rapidly and effectively induce platelet aggregation after stimulation with thrombin. This data assures us that the isolation procedure does not affect platelet function or cause premature platelet activation.
Immunofluorescence. After treatment, HPAECs were washed with PBS and fixed with 3.7% formaldehyde for 15 min. Cells were then rinsed twice with PBS, permeablilized with 0.25% Triton X-100 for 5 min at room temperature, and rinsed twice with Tris-buffered saline that contained 0.1% Tween 20 (TBST). Coverslips were incubated in Texas red phallodin for 60 min at room temperature and rinsed four times with TBST before being mounted on glass slides. A Nikon Eclipse TE3000 microscope and Sony DKC-5000 digital camera connected to a personal computer were used to visualize and analyze filamentous F-actin.
Charcoal-stripped supernatants derived from washed human platelets. Supernatant derived from 2.5 x 106 platelets/ml was incubated with washed activated charcoal overnight at 4°C to remove S1P and lipid growth factors (50). After incubation, suspensions were filtered through a 0.22-µm filter to remove charcoal and added to electrical cell impedance-sensor (ECIS) chambers immediately after filtration.
Construction of antisense EDG-1 adenovirus. The recombinant Adv-AS-EDG1 was constructed by using the method of He et al. (19). Briefly, with the use of pcDNA3.1/EDG1 (kindly provided by Dr. Timothy Hla, University of Connecticut Health Center, Farmington, CT) as a template, PCR primers that contained the indicated restriction enzymes were synthesized to amplify the EDG-1 sequence from -8 (relative to ATG) to 577 bp (primers: 5' asEDG1-Xba, 5'-TGCTCTAGATTGGCACCATGGGGCCCACCAGCGTCCCG-3'; 3' asEDG1-Kpn, 5'-CGGGGTACCGGAGCAGCTGGACAGCGCACTG-3'). The 585-bp fragment was cleaned (Qiagen), cut with the two enzymes, and gel purified. This 585-bp fragment was cloned in reverse orientation into the pAdTrack-CMV shuttle vector cut with KpnI and XbaI. After this plasmid was confirmed and purified, it was cut with PmeI and transformed into BJ5183 containing pAdeasy1. Recombinant viruses were isolated on kanamycin plates and verified by restriction analysis. A recombinant clone was then cut with PacI and transfected with Lipofectamine (according to manufacturer's protocol) into human embryonic kidney (HEK)-293 cells. After 10 days, cell lysate was prepared and used to infect more HEK-293 cells. The presence of infectious virus was confirmed by green fluorescent protein (GFP) fluorescence.
Inhibition of EDG-1 receptor expression with overexpression of antisense EDG-1. HPAECs were seeded into ECIS chambers at 100,000 cells/well. Cells were infected with GFP control or antisense EDG-1 adenoviruses (multiplicity of infection of 25) 3 h after seeding. After 24 h of infection, the media was replaced. At 48 h postinfection, the cells were placed in serum-free media for 1.5 h and allowed to equilibrate. TER was monitored over time in response to 1 µM S1P or activated platelet supernatant obtained from 6 x 106 platelets.
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RESULTS |
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EC barrier enhancement induced by platelets is dependent on
Gi-coupled receptor. Pertussis toxin
(PTX) is commonly used as a tool to inhibit G protein-coupled cell signaling
by Gi
-coupled proteins. To evaluate whether the
barrier-enhancing factor present in platelet supernatants exerted its effect
via ligation of a G protein-coupled receptor, we examined the effects of PTX
on platelet- and platelet supernatant-induced increases in endothelial TER
(Fig. 2). We previously
reported that PTX (1 µg/ml) resulted in the ADP ribosylation of a range of
proteins, induced stress-fiber formation, and increased transmonlayer albumin
flux in bovine endothelium
(14,
38). Consistent with our prior
report, PTX pretreatment resulted in decreased TER but also abolished the
barrier-enhancing effect of both freshly isolated intact platelets and
platelet supernatant (Fig. 2).
These data indicate that platelet-mediated effects on endothelial barrier
properties are mediated by a G protein-coupled receptor.
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Removal of lipid growth factors from platelet supernatants attenuates
increase in TER. Activated platelets release a wide variety of bioactive
molecules including fibrinogen, fibronectin, thrombospondin, transforming
growth factor-, platelet-derived growth factor, calcium, ATP, ADP, and
platelet factor-4. Previous studies have established that the permeability
barrier-enhancing activity in serum derived from platelets is resistant to
heat denaturing, which suggests that the platelet-derived barrier-enhancing
factor is not a peptide. We confirmed this observation by heating platelet
supernatants to 100°C for 15 min and challenging confluent endothelial
monolayers with the boiled material. Heat treatment resulted in only a minimal
loss (<10%) of the maximal increase in TER (data not shown). We therefore
hypothesized that the platelet-associated endothelial barrier-enhancing factor
was a platelet-derived lipid, and that the barrier-promoting activity of
platelet supernatants would be lost by delipidation. To examine this
hypothesis, we used activated charcoal to remove lipids from platelet
supernatants. This strategy has been previously validated by Lee et al.
(27), who demonstrated that,
after coincubation in serum, materials eluted from activated charcoal
activated cells that had been transfected with the EDG-1 receptor, whereas the
charcoal-treated serum did not, thereby confirming the ability of charcoal to
remove the EDG-1 ligand from serum. We therefore adsorbed lipid components
from platelet supernatants onto activated charcoal as previously described
(9,
27,
50). Delipidation of platelet
supernatants with this strategy resulted in marked attenuation in the maximal
increase in TER after addition of the charcoal-treated platelet supernatants
to the endothelial monolayers (Fig.
3). This data supports the conclusion that the platelet-derived
barrier-promoting factor is a lipid product.
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S1P shares similar physiological effects and signal transduction pathways with human platelets. Our previous work indicated that S1P is the primary endothelial chemotatic factor in serum (9) and that it exerts prominent effects on EC barrier function. As our data indicated a platelet-derived barrier-promoting lipid factor, we next explored whether S1P was the platelet-derived factor. When human pulmonary endothelium was cultured on gold microelectrodes to confluence and challenged with S1P (1 µM), TER was seen to increase in a similar time scope and to a similar magnitude when compared with the TER increases induced by either intact platelets or platelet supernatants (Fig. 3). To validate our usage of activated charcoal as a means to remove lipids (primarily S1P) in this model, we also incubated aliquots of S1P with the activated charcoal and tested the effects on TER. As illustrated in Fig. 3, the biological activity of S1P was abrogated by activated charcoal treatment, which is consistent with our prior report (9) that demonstrates a loss of chemoattractive activity of both platelet-rich plasma and fetal bovine serum after delipidation.
Both platelets and S1P reverse thrombin-induced endothelial paracellular gap formation and barrier dysfunction. Thrombin induces endothelial paracellular gap formation in in vitro and in vivo models, is found at sites of vascular injury and wounding, and is a known platelet activator. To mimic a physiologically relevant setting in which platelets and their released factors can interact with thrombin in the presence of compromised endothelial monolayers, we tested the effects of platelets after treatment of monolayers with thrombin. As expected, thrombin produced a robust decrease in TER that was reversed by the addition of S1P (Fig. 4A), platelets (Fig. 4B), and platelet supernatants (data not shown). These data provide convincing evidence that platelets and S1P are important cellular mediators of vascular barrier function.
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Both platelets and S1P induce actin cytoskeletal rearrangement in HPAECs. The actin cytoskeleton plays a crucial role in many signaling pathways and in barrier regulation (12, 43). To demonstrate the critical involvement of the cytoskeleton in the S1P- and platelet-mediated increase in electrical resistance, we pretreated cells with the microfilament-disrupting agent cytochalasin B and found that both the platelet supernatant- and S1P-induced increases in electrical resistance were completely abolished (Fig. 5A). This suggested that the enhancement of endothelial barrier function induced by both S1P and platelets was essentially dependent on the integrity or organization of F-actin. To study the effect of platelet-derived vasoactive soluble factors on EC cytoskeletal organization, platelet supernatant- or S1P-challenged endothelium was stained with Texas red phalloidin to localize F-actin. Both S1P and platelets induced a dramatic change in actin staining found throughout the cell (Fig. 5B). In control endothelium, actin is found in a reticular pattern dispersed throughout the cell (Fig. 5B, left), whereas the addition of either S1P (middle) or platelets (right) caused actin to rearrange into a thickened cortical band especially enriched at the periphery. The manner in which actin was reorganized in endothelium by challenge with platelet supernatants was qualitatively highly similar to that induced by challenge with S1P.
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EDG-1 receptor inhibition significantly blocks effects of platelets and S1P on HPAEC electrical resistance. The previous results are highly consistent with a role for S1P as the mediator of platelet-induced barrier protection. S1P binds a number of EDG receptors including EDG-1, -3, -5, -6, and -8 and increases TER in a PTX-sensitive manner (11). Our studies thus far have demonstrated a correlation between the events evoked by treatment of ECs with isolated washed human platelets, platelet-derived supernatants, and commercially available S1P. To confirm a central role for EDG1-receptor ligation by S1P in platelet supernatant-mediated enhancement of endothelial barrier function, we constructed an adenoviral vector that expresses an antisense oligonucleotide directed against EDG-1. We have previously shown that this antisense oligonucleotide strategy successfully reduces the biological effects of S1P in ECs as measured by both EC migration (28) and TER (11). In addition, we have shown that the levels of EDG-5 and -8 expression are extremely low or undetectable compared with the levels of EDG-1 and -3 expression in our cells. Consistent with a role for S1P in the platelet-mediated barrier enhancement, infection with adenoviral EDG-1 antisense oligonucleotide significantly attenuated the HPAEC response to platelets and to S1P (Fig. 6A). We observed an incomplete attenuation of the S1P- and platelet-induced enhancement of barrier function by the adenoviral vector. We therefore examined the effect of the vector on EDG-1 protein level and observed that transduction of the EDG-1 antisense oligonucleotide resulted in a 40% reduction in the expression of EDG-1 protein (Fig. 6B). Therefore, the subtotal reduction in EDG-1 expression correlates with the incomplete attenuation of enhanced barrier function.
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DISCUSSION |
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We and others have reported on vasoactive platelet-derived lipids that activate endothelium, including our first report that S1P activated EC phospholipase D (7, 10, 23, 33, 48). Subsequent studies have since demonstrated that platelet-derived phospholipids modulate the barrier function of the vascular endothelium via both endothelial barrier-perturbing lipid products such as phosphatidate (6) and barrier-stabilizing/enhancing lipid products such as lysophosphatidic acid (LPA) (8, 32) and S1P (11). Although these platelet-derived factors are implicated in the vascular endothelial barrier-enhancing effect of platelets, the specific platelet-derived factors remain incompletely characterized. Our data clearly demonstrate that both platelet supernatants and exogenously added S1P enhance endothelial barrier function (see Fig. 1) in a Gi-dependent manner (see Fig. 2), which is in agreement with earlier findings (11). We also found that both platelet supernatants and exogenously added S1P reversed agonist-mediated barrier dysfunction (see Fig. 4), and each produced a highly similar reorganization of cellular F-actin (see Fig. 5). The implicated role of S1P in mediating the barrier-enhancing effect of platelet supernatants was confirmed by demonstrating that diminished expression of the specific S1P receptor EDG-1 attenuated the barrier-enhancing effects of platelet supernatants and exogenously added S1P (see Fig. 6). Taken together, these observations support a significant role for S1P in mediating the vessel-stabilizing effect of circulating platelets.
Our data agree with published reports that platelet-released products decrease endothelial permeability (18) and increase TER (32). Although our data strongly support the concept that S1P is a platelet-derived factor that significantly underlies this effect, our data do not exclude the possibility that other platelet-derived products may also participate in the barrier-protective effect of platelets. We and others have demonstrated that LPA, released from thrombin-stimulated platelets, possesses endothelial barrier-stabilizing activity (1, 8, 32). Although in our experiments LPA-induced endothelial barrier enhancement was weak in vitro (8), LPA released from platelets may form an active complex with serum albumin that further enhances its barrier-enhancing activity (32), which may in part explain the previously observed trypsin sensitivity of the barrier-enhancing platelet-derived factor (18). Although we did not examine trypsin sensitivity or possible cooperation between S1P and other serum proteins in these studies, we have previously established that S1P, unlike LPA, induces significant enhancement of endothelial barrier function when added exogenously even in the absence of albumin or serum (11).
The mechanisms by which platelets and platelet phospholipids such as S1P enhance the vascular barrier are unknown. Although mechanistic approaches designed to understand EC paracellular gap and barrier regulation have revealed the extreme complexity of these processes, several valuable paradigms have been developed. One useful model describes paracellular gap formation as regulated by the balance of competing contractile and adhesive forces that together regulate cell shape changes (5, 12). In this paradigm, cell-cell and cell-matrix tethering forces exist in equilibrium with contractile mechanisms that generate centripetal tension via an actin and myosin motor. Both competing forces in this model are intimately linked to the actin-based endothelial cytoskeleton by a variety of actin-binding proteins that are critical to tensile force generation as well as the linkage of the actin cytoskeleton to adhesive membrane components. We have shown that platelets and S1P produce rapid and dramatic enhancement of polymerized F-actin and myosin staining that was spatially confined to the cortical cytoskeletal ring. To link this observation to human EC monolayer barrier enhancement, we used the actin-depolymerizing agent cytochalasin, which produced a prompt decline in barrier function (see Fig. 5). Unlike the dramatic protective response to S1P challenge after established thrombin-induced barrier dysfunction, neither platelets nor S1P increased barrier function when added after cytochalasin challenge, which indicates a critical requirement for dynamic cytoskeletal rearrangement and an intact actin cytoskeleton for S1P-mediated barrier protection. We speculate that platelets, via elaboration of S1P, may enhance barrier function by promoting a rearrangement of actin structures that either reduce or redistribute tensile forces. Because Rho-family small GTPases are key molecular switches for regulating actin assembly and dynamics in cells, they are likely to be essential effectors in the enhancement of endothelial barrier function by platelets and platelet-released products. We have previously demonstrated that the Rho-family GTPases Rac and Rho play a central role in S1P-mediated endothelial cytoskeletal rearrangement (11).
S1P is a biologically active lipid generated by hydrolysis of glycerophospholipids and sphingomyelin in the membranes of activated cells that is formed by the phosphorylation of sphingosine by sphingosine kinase. Whereas in most cell types the levels of S1P are tightly controlled by the rapid metabolism of S1P via sphingosine lyase and cellular lipid phosphohydrolase activities (52), platelets are unique in lacking in these catabolic activities, which thereby enables the accumulation of S1P in cells and allows for its release by activated platelets (51, 52). S1P is a normal constituent in plasma with increased (micromolar) levels in serum that are consistent with S1P release during whole blood coagulation (51). S1P exerts diverse biological effects on cells and is now recognized as an important courier of cellular information (7, 10, 23, 33, 48). S1P stimulates proliferation, calcium mobilization, adhesion molecule expression, and suppression of caspase-dependent apoptosis (7, 20, 40, 46) via ligation of G protein-coupled receptors of the EDG family (26). EDG-1 was cloned from RNA expressed in ECs stimulated to undergo angiogenic responses in vitro (21). Whereas S1P binds to EDG-1, -3, -5, and -6, LPA preferentially binds EDG-2 and -4 and perhaps other unidentified members of this family of receptors (2, 46). In cultured human artery endothelium, the expressed EDG-family receptors are EDG-1 and -3 (26, 49) with EDG-1 being significantly more abundant than EDG-3 in our system (8).
We have shown that platelet supernatants and S1P each invoke similar physiological and histochemical responses in pulmonary artery endothelium, and that the endothelial barrier-enhancing activity of platelets is EDG-1 dependent, which implicates an important role for S1P as a key platelet-derived endothelial barrier-enhancing factor. Thus the vascular endothelium is well positioned both molecularly and anatomically to respond to the barrier-modulating effects of platelets. We speculate that this intimate relationship between platelet-derived S1P and the vascular endothelium reflects a critical linkage between coagulation, inflammation, and angiogenesis that works in a concerted fashion with other factors to initiate neovascularization and potentiate the function of nascent vessels by optimizing endothelial barrier integrity.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-50533, HL-03666, and HL-69340.
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FOOTNOTES |
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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.
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REFERENCES |
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