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【摘要】
Objectives— S1P acts via the S1PR family of G protein–coupled receptors to regulate a variety of physiological responses. Whereas S1P1R activates G i - and PI-3-kinase–dependent signals to inhibit vascular permeability, the related S1P2R inhibits the PI-3-kinase pathway by coupling to the Rho-dependent activation of the PTEN phosphatase. However, cellular consequences of S1P2R signaling in the vascular cells are not well understood.
Methods and Results— Selective signaling of the S1P2R was achieved by adenoviral-mediated expression in endothelial cells. Secondly, endogenously expressed S1P2R was blocked by the specific pharmacological antagonist JTE013. Activation of S1P2R in endothelial cells resulted in Rho-ROCK– and PTEN-dependent disruption of adherens junctions, stimulation of stress fibers, and increased paracellular permeability. JTE013 treatment of naive endothelial cells potentiated the S1P1R-dependent effects such as formation of cortical actin, blockade of stress fibers, stimulation of adherens junction assembly, and improved barrier integrity. This observation was extended to the in vivo model of vascular permeability in the rat lung: the S1P2R antagonist JTE013 significantly inhibited H 2 O 2 -induced permeability in the rat lung perfused model.
Conclusions— S1P2R activation in endothelial cells increases vascular permeability. The balance of S1P1 and S1P2 receptors in the endothelium may determine the regulation of vascular permeability by S1P.
We tested the hypothesis that S1P2R signaling via Rho–ROCK–PTEN pathway is a critical modulator of vascular permeability. Our results show that activation of S1P2R in endothelial cells results in Rho-, p160-Rho–associated kinase (ROCK)- and PTEN-dependent disruption of endothelial cell–cell junctions, profound modulation of actin cytoskeletal dynamics, and increased paracellular permeability.
【关键词】 sphingosinephosphate Rho ROCK PTEN permeability
Introduction
Vascular permeability is a tightly regulated process in various organ beds and an essential component of angiogenesis, tumor metastasis, and inflammation. Several in vitro and in vivo studies indicate the regulation of endothelial cell permeability by the bioactive lipid sphingosine-1-phosphate (S1P). 1–4 S1P, a sphingolipid enriched in plasma, mediates multiple cellular responses in many different cell types by activating the endothelial differentiation gene family of G protein–coupled receptors, renamed as S1P1–5R. 5,6
Various endothelial cells express different combinations of S1P1R, S1P2R, and S1P3R receptors. 2,7 Therefore, under physiological conditions, S1P in the plasma is available to bind to its receptors and activate different signal transduction pathways in the endothelium. S1P1R couples exclusively to G i, whereas S1P2R and S1P3R couple to G i, G q, and G 12/13. 8 In vitro studies indicate a role for S1P on endothelial cell survival, migration, adherens junction assembly, morphogenesis, 2,9 and barrier integrity. 1,4 These effects are inhibited by pertussis toxin, indicating the importance of G i signaling for these cellular actions. Downstream effectors such as the small GTPase Rac, protein kinase B/Akt, and the phosphoinositide-3-kinase (PI3K) pathways are critical for the function of S1P1R. 1,9,10 In vivo, S1P and the S1P receptor agonist FTY720-phosphate (FTY720-P), which is a high affinity ligand for all S1P receptors except S1P2R 11,12 potently inhibited pulmonary edema 3 and vascular endothelial cell growth factor (VEGF)-induced vascular permeability. 4 Therefore, S1P1R is a potent regulator of endothelial cell function in vitro and blood vessel integrity in vivo.
The regulation of endothelial cell function by S1P2R is less understood. S1P2R and S1P1R mediate opposite effects on endothelial cell migration attributable to the coupling of S1P2R to the G 12/13 /Rho pathway, 13 which results in activation of PTEN (phosphatase and tensin homolog deleted on chromosome 10), 14 a phosphatase with enzymatic activities toward 3'-phosphoinositides. In vivo, S1P2R cooperates with S1P1R during embryonic vascular development. 15 In a recent study, S1p2r –/– mice were shown to be deaf because of a defective regulation of vascular tone of the spiral modiolar artery that supplies the stria vascularis of the inner ear. Lack of S1P2R resulted in degenerative changes in inner ear epithelium, vasculature, and hair cells. 16,17 Therefore, the regulation of endothelial cell functions by S1P2R needs to be further examined, especially with respect to the signal transduction pathways. In this report, we investigated the regulation of adherens junction assembly and endothelial cell permeability by S1P2R. In sharp contrast to S1P1R, the S1P2R receptor induced adherens junction disruption, and increased endothelial cell permeability in vitro. These effects were mediated by the S1P2R downstream effectors Rho, ROCK, and PTEN, which negatively regulate the small GTPase Rac. In addition, blockade of S1P2R significantly inhibited H 2 O 2 -induced vascular permeability in the rat lung perfused model. Therefore, the balance of S1P1R and S1P2R receptors may be critical in determining the effects of the blood-borne S1P on the regulation of endothelial cell permeability and the maintenance of vascular homeostasis.
Materials and Methods
Materials, cell culture and adenoviral transduction, immunofluorescence analysis, paracellular permeability, affinity precipitation of Rho-GTP and Rac-GTP, immunoprecipitation, Western Blot Analysis, perfused rat lung model and statistical analysis are described in detail in the supplemental materials, available online at http://atvb.ahajournals.org.
Results
S1P2R Disrupts Adherens Junctions and Induces Paracellular Permeability
To study the regulation of adherens junction assembly and endothelial cell permeability by S1P2R we used human umbilical vein endothelial cells (HUVEC), which express high levels of S1P1R and S1P3R and lower levels of S1P2R (supplemental Figure I, available online at http://atvb. ahajournals.org). Physiological levels of S1P2R receptor were expressed in HUVEC by adenoviral transduction (20 MOI) as we have previously described. 14 At this MOI, S1P2R expression was comparable to endogenous S1P1R levels. The effects of S1P on actin cytoskeleton in control adenovirus (β-Gal) and S1P2R transduced HUVEC was assessed by Rhodamine-Phalloidin staining. In addition, adherens junction assembly was assessed by VE–cadherin localization using immunofluorescence confocal microscopy. S1P stimulation induced cortical actin structures in the membrane ruffles and stress fibers in control HUVEC ( Figure 1 A). This effect was accompanied by VE–cadherin translocation to the adherens junctions, as it can be assessed by continuous VE–cadherin staining at the cell–cell contact sites ( Figure 1 A). S1P2R expression in HUVEC completely abolished the ability of S1P to induce actin polymerization in membrane ruffles. In contrast, a cortical ring of stress fibers was observed in S1P2R-expressing cells. In addition, VE–cadherin translocation to the adherens junctions was profoundly inhibited by S1P2R.
Figure 1. Effects of S1P2R expression on actin cytoskeleton, adherens junctions, and paracellular permeability. A, Control (β-Gal) or S1P2R (S1P 2 )-transduced HUVEC were stimulated with vehicle (–S1P) or 100 nmol/L S1P (+S1P) for 1 hour. F-actin and VE–cadherin staining is shown (n=6). Scale bar: 20 µm. B, Confluent HUVEC grown on transwell filters were transduced with control (β-Gal) or S1P2R (S1P 2 ) adenoviruses, and basal (C) paracellular permeability of FITC-labeled dextran over the time was quantified. Where indicated (+S1P), 100 nmol/L S1P was added to the medium. Results are mean±SE. of triplicates. n=3. * P <0.05 S1P-treated vs nontreated; # P <0.05 S1P2R vs β-Gal–transduced HUVEC.
To study how the changes in VE–cadherin localization affected the endothelial cell barrier function, we assessed the permeability to fluorescein isothiocyanate (FITC)-dextran (average Mr=2000 kDa) through the endothelial cell monolayer. S1P enhanced the barrier properties of control virus transduced HUVEC by decreasing basal permeability ( Figure 1 B). However, S1P2R expression increased basal permeability. In addition, the ability of S1P to enhance barrier integrity was completely abolished by the expression of S1P2R. Similar results were obtained when 50 kDa FITC-dextran was used in this assay. Altogether these results indicate that expression of S1P2R to similar levels of S1P1R in HUVEC counteracted the effects of S1P1R by modulation of actin cytoskeletal architecture and adherens junction disassembly, which resulted in decreased barrier function of the endothelial cell monolayer.
Regulation of Rho and Rac GTPases by the S1P2R
Because Rho GTPases are key regulators of the actin cytoskeleton dynamics and paracellular permeability, we next studied the regulation of Rho and Rac GTPases by S1P receptors in endothelial cells. S1P stimulation activated Rho both in control and S1P2R-transduced HUVEC ( Figure 2 A). However, basal levels of active Rho were significantly higher in S1P2R HUVEC compared with β-Gal, most likely because of autocrine signaling of S1P2R by the endothelial-derived S1P ligand, 18 and they were further increased by S1P.
Figure 2. Regulation of Rho and Rac activation by the S1P2R. Control (β-Gal) or S1P2R (S1P 2 )-transduced HUVEC were stimulated with 100 nmol/L S1P for the indicated times. A, GTP-Rho levels normalized to total Rho. B, GTP-Rac levels normalized to total Rac levels. Data are mean±SE. n=3.
S1P stimulation of adenovirus control-transduced HUVECs led to a modest increase in the levels of Rac-GTP (from 0.8±0.04 to 1.35±0.09 and 1.28±0.08, after 5 and 15 minutes, respectively; P <0.05), as assessed by Rac pull down assays ( Figure 2 B). Concomitantly, S1P stimulation triggered translocation of Rac to the plasma membrane. As shown in supplemental Figure II, in nonstimulated cells, Rac was predominantly localized in the cytoplasmic and perinuclear areas. On S1P stimulation, Rac was redistributed to the cell periphery and cell–cell contact sites. When S1P2R was expressed in HUVECs, the ability of S1P to activate Rac was abrogated, and S1P stimulation led to decreased levels of active Rac after 15 minutes (from 0.87±0.05 to 0.6±0.074 in S1P treated cells, Figure 2 B). Accordingly, Rac translocation to the plasma membrane by S1P stimulation was blocked in S1P2R-expressing HUVEC (supplemental Figure II). These results indicate that S1P2R potently activates the GTPase Rho and inhibits S1P-induced Rac activation in HUVEC.
S1P2R–Rho–ROCK Pathway Induces Phosphorylation of VE–Cadherin Complexes
Because disruption of VE–cadherin–mediated cell–cell junctions and increased Tyr phosphorylation of VE–cadherin have been reported to be important for VEGF-induced disruption of endothelial cell barrier function, 19 we studied the regulation of Tyr phosphorylation of VE–cadherin by S1P. S1P stimulation did not trigger significant changes in Tyr phosphorylated VE–cadherin in control adenovirus-transduced HUVEC ( Figure 3 ). Similar results were obtained with the S1P receptor agonist FTY720-P, which is a high affinity ligand for all S1P receptors but S1P2R. 11,12 On the contrary, when S1P2R was expressed in HUVEC, S1P but not FTY720-P, brought about a significant increase in the levels of Tyr phosphorylated VE–cadherin (2.4±0.3-fold induction). No changes in levels of phospho-Ser/Thr VE–cadherin were detected by immunoprecipitation of VE–cadherin and blotting with a phospho Ser/Thr antibody (data not shown).
Figure 3. Activation of S1P2R regulates VE–cadherin Tyr phosphorylation. Control (β-Gal) and S1P2R-transduced HUVEC (S1P 2 ) were stimulated with S1P or FTY720-P (Fp) for 1 hour. Cell extracts were immunoprecipitated with VE–cadherin antibody, followed by immunoblotting with anti–phospho-Tyr (pY) or VE–cadherin antibodies. n=5.
Next, we set out to determine whether the ability of S1P to induce Tyr phosphorylation of VE–cadherin was dependent on Rho/ROCK signaling. As shown in supplemental Figure III, treatment of HUVECs with Y-27632, an inhibitor the Rho effector ROCK, 20 resulted in lower levels of Tyr phosphorylated VE–cadherin after S1P stimulation (1.5±0.2-fold induction). These results point to the pivotal role of S1P2R dependent activation of Rho in VE–cadherin Tyr-phosphorylation and adherens junction disruption.
S1P2R Induces Rho-, ROCK-, and PTEN-Dependent Disruption of Adherens Junctions and Increased Paracellular Permeability
The Rho GTPase has been shown to be critical for increased permeability induced by various edemagenic agents, such as thrombin 21 or VEGF. 22 Activation of Rho leads to increased myosin light chain phosphorylation 20 through activation of its effector ROCK, which results in actomyosin assembly and cell contraction, ultimately leading to increased permeability. Because S1P2R is a strong activator of the G 12/13 –Rho–PTEN pathway, we examined the role of Rho and its downstream signaling partners, ROCK and PTEN, in the regulation of actin cytoskeleton dynamics, adherens junction assembly and paracellular permeability. Inhibition of Rho by the dominant negative (dn) N19Rho mutant abrogated the ability of S1P2R to induce cell contraction and intercellular gap formation. Indeed, S1P stimulation in S1P2R- and dnRho-transduced cells, resulted in membrane ruffle formation in a similar extent compared with control virus transduced (β-Gal) HUVEC ( Figure 4 A). In addition, inhibition of Rho rescued the ability of S1P to promote VE–cadherin translocation to cell–cell contact sites, giving rise to a continuous pattern of the VE–cadherin staining instead of the zigzag pattern observed in S1P2R-expressing HUVEC. Similar results were obtained when the downstream effectors of Rho, ROCK, and PTEN were inhibited, by incubation with the ROCK inhibitor Y-27632 (10 µmol/L) for 2 hours or adenoviral transduction with the enzymatically inactive mutant C124S PTEN (dnPTEN), 23 respectively. Interestingly, inhibition of Rac by adenoviral transduction with N17 dnRac, dnAkt 9 or wild-type PTEN in β-Gal HUVECs, abrogated the ability of S1P to induce membrane ruffles and VE–cadherin translocation to adherens junctions (supplemental Figure IV), pointing to the pivotal role of Rac on the regulation of adherens junction assembly by S1P.
Figure 4. S1P2R-induced disruption of adherens junctions and increased permeability are dependent on the Rho–ROCK–PTEN pathway. A, S1P2R (S1P 2 ) or control (β-Gal) HUVEC were transduced with dn Rho or dn PTEN adenoviruses (100 M.O.I.). Cells were pretreated with 10 µmol/L Y-27632 (+Y) for 2 hours before stimulation with 100nmol/L S1P (+S1P). F-actin and VE–cadherin staining is shown. n=3. Scale bar: 20 µm. B, HUVEC grown on transwell filters were transduced with control (β-Gal) or S1P2R (S1P 2 ), and dn PTEN or dn Rho adenoviruses and transcellular permeability over the time was quantified. Where indicated (+Y), overnight 10 µmol/L Y-27632 pretreatment was conducted. Results are mean±SE of triplicates. n=3. * P <0.05 S1P 2 vs β-Gal.
Next, we investigated the role of the Rho–ROCK–PTEN pathway in the S1P2R-induced barrier dysfunction in endothelial cells. Inhibition of the Rho pathway by dnRho, the ROCK inhibitor, Y-27632, or dnPTEN blocked the ability of S1P2R to disrupt barrier function ( Figure 4 B). These results indicate that the effects of S1P2R on barrier disruption are mediated by the Rho–ROCK–PTEN pathway.
Effects of Endogenous S1P2R Blockade on Adherens Junction Assembly and Vascular Permeability
Because HUVEC express high levels of S1P1R and lower levels of S1P2R, 24 we examined the role of endogenous S1P2R on the regulation of adherens junction assembly by using the S1P2R-selective antagonist JTE013. 24 JTE013 specifically blocked S1P2R and not S1P1R signaling in a heterologous expression system (supplemental Figure Va). In HUVECs, blockade of S1P2R with JTE013 resulted in higher Akt phosphorylation levels after S1P stimulation (supplemental Figure Vb), in agreement with the activation of PTEN by S1P via S1P2R. 14 As shown in Figure 5 A, JTE013 enhanced S1P-induced VE–cadherin translocation to adherens junction sites compared with cells preincubated with vehicle and treated with S1P. In addition, S1P2R blockade inhibited S1P-induced stress fibers and potentiated the ability of S1P to induce cortical actin assembly. These data suggest that activation of endogenous S1P2R in endothelial cells counteracts S1P1R and S1P3R-mediated effects on adherens junction assembly and actin cytoskeleton dynamics.
Figure 5. Effects of endogenous S1P2R blockade on adherens junction assembly, actin cytoskeleton, paracellular and vascular permeability. A, HUVEC monolayers were pretreated with vehicle (C) or JTE013 (0.2 µmol/L) for 30 minutes before stimulation with 100nmol/L S1P (+S1P) for 1 hour. VE–cadherin and F-actin staining of a representative experiment of 3 is shown. Scale bar: 20 µm. B, HUVEC grown on transwell filters were pretreated with vehicle (C) or JTE013 (0.2 µmol/L) (JTE) for 30 minutes. Paracellular permeability after 1 hour was quantified. Where indicated (+S1P), 100nmol/L S1P was added. Results are mean±SE of triplicates. n=3. * P <0.05 S1P-treated vs nontreated. # P <0.05 JTE pretreated vs vehicle pretreated. C, Lungs were perfused with either vehicle (Control) or 0.5 µmol/L JTE013 (JTE013) during 15 minutes and K f measurements were taken (BL). Then, 50 µmol/L H 2 O 2 was added to the reservoir, allowed to circulate for 15 minutes, and Kf was measured (H 2 O 2 ). Results are mean±SE, n=4. * P <0.01 before vs after H 2 O 2 -treatment, # P <0.01 JTE013-treated vs vehicle-treated.
Then, we determined the ability of S1P to enhance barrier properties of HUVEC monolayers after S1P2R blockade with JTE013. HUVEC monolayers were incubated with vehicle or with the S1P2R antagonist for 30 minutes. Paracellular permeability in the presence or absence of S1P was measured after 60 minutes. The S1P2R antagonist JTE013 significantly inhibited basal permeability and potentiated the effects of S1P on barrier integrity ( Figure 5 B). Altogether these data indicate that endogenous S1P2R in HUVEC induced stress fiber formation, disassembly of adherens junctions and increased paracellular permeability.
We further studied the effects of the blockade of S1P2R on vascular permeability in the ex vivo model of perfused rat lungs. Lungs were perfused with either vehicle or 0.5 µmol/L JTE013 during 15 minutes. No significant changes in the capillary filtration coefficient (K f ) were induced by these treatments relative to untreated lungs (data not shown). Then, lung edema was induced by addition of 50 µmol/L H 2 O 2 to the perfusate. We observed an increase up to 0.647±0.064 in the rate of lung wet weight gain after 15 minutes of H 2 O 2 treatment in the vehicle-treated lungs ( Figure 5 C). Interestingly, in lungs treated with the S1P2R antagonist, H 2 O 2 -induced lung edema was markedly inhibited (K f =0.374±0.013). These data indicate that the inhibition of S1P2R signaling results in decreased vascular permeability and H 2 O 2 -induced lung edema.
Discussion
Plasma-derived S1P activates multiple S1P receptor subtypes expressed on vascular endothelial cells. S1P1R is required for vascular maturation, 25 and inhibits vascular permeability 3,4 and the inflammatory response of the endothelium. 7 However, the regulation of endothelial cell function by S1P2R is less understood. S1P2R is implicated in the regulation of vascular development, in cooperation with S1P1R. 15 Because S1P1R and S1P2R couple to opposing signaling pathways, 14 we hypothesized that S1P2R signaling via Rho–ROCK–PTEN pathway is a critical modulator of vascular permeability by counterbalancing the actions of S1P through the S1P1R-Gi-PI3K pathway. Herein, we describe the novel regulation of endothelial cell permeability by the S1P2R-Rho–ROCK–PTEN axis.
Our data show that expression of S1P2R in endothelial cells increases paracellular permeability. In HUVEC, which primarily express the S1P1R, 2 ligand activation stimulates barrier integrity and inhibits paracellular permeability. S1P2R expression induced endothelial cell barrier dysfunction, even in cells not stimulated with the ligand. This could be attributable to autocrine stimulation by endogenous S1P produced by HUVEC. 18,26 Indeed, blockade of S1P2R by incubation with JTE013 in S1P2R-HUVEC completely abrogated the effects of S1P2R expression on the cytoskeleton and adherens junctions (supplemental Figure Vc), indicating that S1P2R activation by endogenous S1P produced by endothelial cells accounts for the effects observed in the absence of exogenous ligand. In addition, blockade of endogenous S1P2R in HUVEC decreased paracellular permeability without addition of exogenous ligand, effect that was further enhanced after addition of S1P. Our data suggest that spatial and temporal regulation of expression of S1P1R and S1P2R may determine the permeability response of various vascular beds.
The actin cytoskeleton dynamics is profoundly regulated by S1P in endothelial cells. In HUVEC, S1P stimulation induces cortical actin assembly at lamellapodia, cell spreading, and stress fiber formation. 2,27 In contrast, when S1P2R is expressed, cortical actin and lamellapodia are completely inhibited. In addition, a strong peripheral ring of stress fibers is induced. Interestingly, inhibition of endogenous S1P2R by the S1P2R antagonist JTE013 resulted in increased cortical actin assembly after S1P stimulation. This is consistent with the previous findings in Chinese Hamster Ovary cells that S1P2R induces myosin light chain phosphorylation 28 and inhibits IGF-induced membrane ruffling. 29
VE–cadherin, a major adherens junction protein in endothelial cells, is incorporated into adherens junctions after S1P treatment, an effect that requires the signal transduction of the S1P1R-Rac pathway. 2 Because adherens junctions are coupled to the cytoskeleton via the catenin proteins, changes in actin dynamics may contribute to this effect. However, when S1P2R is expressed, VE–cadherin translocation into adherens junction is inhibited. Furthermore, S1P2R strongly induces Tyr phosphorylaton of VE–cadherin. This is in agreement with the fact that other inducers of vascular permeability, such as VEGF, also induce the Tyr phosphorylation of VE–cadherin, 19 which may be involved in junction disruption. Inhibition of adherens junction structures by S1P2R may be involved in the increased paracellular permeability induced by this receptor.
Cytoskeleton dynamics and adherens junctions are regulated by the Rho family of GTPases. 21 In endothelial cells, S1P binding to S1P1R and S1P3R activates both Rac and Rho GTPases. 27 Our data indicate that S1P2R in HUVECs strongly activates Rho and inhibits Rac activity and membrane translocation, in contrast with the actions of S1P1R, which is a potent inducer of the GTPases Rac and Rho. 27,29 Our previous work showed that S1P2R activates the phosphatase PTEN, which antagonizes the PI3K–Akt pathway. PTEN downregulates the activity of critical signaling proteins such as Akt and Rac. Because PI3K/Akt is important for optimal Rac activation 9 and this GTPase plays a pivotal role on the regulation of barrier integrity by S1P1R, 10 the inhibitory action of S1P2R on the Rac GTPase may be important for the vascular effects this receptor. Moreover, strong activation of Rho may have independent effects on paracellular permeability, as other edemagenic agents such as thrombin and histamine also induce this GTPase. 21,22
A major finding of this study is that S1P2R-Rho–ROCK–PTEN signaling is required for the disruption of adherens junctions and the induction of paracellular permeability. The phosphatase PTEN has been shown to regulate embryonic vascular development and tumor angiogenesis. 30 Interestingly, genetic disruption of Akt1 results in increased vascular permeability, inhibition of vascular maturation, and enhanced tumor angiogenesis. 31 Our results indicate that inhibition of Rho, ROCK, and PTEN abrogated the effects of S1P2R on the adherens junction disruption and actin cytoskeleton. In addition, ROCK, which activates PTEN, 32 is needed for Tyr phosphorylation of VE–cadherin. These data describe a novel pathway for the regulation of adherens junctions in the endothelium. Further work is needed for molecular details of regulation of adherens junctional complexes by S1P2R, such as the identification of specific kinases and description of protein-protein interactions.
In conclusion, we show that S1P2R regulates Rho–ROCK–PTEN dependent pathways to induce permeability in the endothelium. This contrasts sharply with the signaling of the S1P1R, which inhibits vascular permeability. The opposite effects of S1P1R and S1P2R can be explained by the antagonistic activities of its downstream effectors, PI3K and PTEN, respectively. A combined therapy of a S1P1R agonist and S1P2R antagonist could eventually be used for the treatment of pulmonary edema in sepsis or other vascular permeability disorders. Further studies are needed to understand how the expression, trafficking, and signaling of S1P1R and S1P2R in a particular vascular bed regulate vascular permeability, angiogenesis, and the inflammatory response of the endothelium.
Acknowledgments
We thank Drs Shuh Narumiya, Martin Schwartz, William Sessa, Volker Brinkmann, and Christopher Kontos for their gift of reagents used in this work.
Sources of Funding
This work is supported by AHA SDG grant 0630384N (to T.S.), NIH grants HL70694 and HL67330 (to T.H.), and VA Merit Review and NIH HL67795 grants (to E.H.).
Disclosures
None.
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作者单位：Center for Vascular Biology (T.S., A.S., M.T.W., T.H.), Department of Cell Biology, University of Connecticut Health Center, Farmington, Conn; and the Vascular Research Laboratory (B.C., E.O.H.), Providence Veterans Affairs Medical Center, Department of Medicine, Brown Medical School, Providence, RI