Novel Role of Lactosylceramide in Vascular Endothelial Growth FactoreCMediated Angiogenesis in Human Endothelial Cells
http://www.100md.com
循环研究杂志 2005年第10期
the Johns Hopkins Singapore and National Heart Center, Vascular Biology Program (M.R., S.C.)
Department of Pediatrics, Lipid Research Unit, Johns Hopkins University (A.K., S.C.), Baltimore, Md
the Department of Biochemistry, National University of Singapore (S.C). M.R. is currently at the Department of Biological Sciences, National University of Singapore.
Abstract
Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis associated with coronary heart disease, vascular complications in diabetes, inflammatory vascular diseases, and tumor metastasis. The mechanism of VEGF-driven angiogenesis involving glycosphingolipids such as lactosylceramide (LacCer), however, is not known. To demonstrate the involvement of LacCer in VEGF-induced angiogenesis, we used small interfering RNA (siRNA)-mediated silencing of LacCer synthase expression (GalT-V) in human umbilical vein endothelial cells. This gene silencing markedly inhibited VEGF-induced platelet endothelial cell adhesion molecule-1 (PECAM-1) expression and angiogenesis. Second, we used D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of LacCer synthase and glucosylceramide synthase, that significantly mitigated VEGF-induced PECAM-1 expression and angiogenesis. Interestingly, these phenotypic changes were reversed by LacCer but not by structurally related compounds such as glucosylceramide, digalactosylceramide, and ceramide. In a human mesothelioma cell line (REN) that lacks the endogenous expression of PECAM-1, VEGF/LacCer failed to stimulate PECAM-1 expression and tube formation/angiogenesis. In REN cells expressing human PECAM-1 gene/protein, however, both VEGF and LacCer-induced PECAM-1 protein expression and tube formation /angiogenesis. In fact, VEGF-induced but not LacCer-induced angiogenesis was mitigated by SU-1498, a VEGF receptor tyrosine kinase inhibitor. Also, VEGF/LacCer-induced PECAM-1 expression and angiogenesis was mitigated by protein kinase C and phospholipase A2 inhibitors. These results indicate that LacCer generated in VEGF-treated endothelial cells may serve as an important signaling molecule for PECAM-1 expression and in angiogenesis. This finding and the reagents developed in our report may be useful as anti-angiogenic drugs for further studies in vitro and in vivo.
Key Words: vascular endothelial growth factor lactosylceramide platelet endothelial cell adhesion molecule-1 angiogenesis
Introduction
Vascular endothelial growth factor (VEGF) has been implicated in the process of vasculogenesis and angiogenesis.1,2 Aberrant expression of VEGF has been reported in several vascular pathologies such as inflammation, complications of diabetes mellitus, cardiovascular diseases, and tumor metastasis.3 VEGF binds to its receptors KDR/Flk-1 to mediate its effect on angiogenesis in physiological conditions and in human atherosclerosis.4eC6
Platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31 is a constitutively expressed integral protein in endothelial cells.7 In addition, PECAM-1 is expressed in platelets, monocytes, neutrophils, and a certain subset of T cells.8 Recent studies implicate PECAM-1 in angiogenesis and in vitro endothelial cell migration.9,10 For example, human mesothelioma cell line (REN) that is deficient in PECAM-110 and cells from PECAM-1 deficient mice11 did not form tubes in vitro (angiogenesis). In contrast, REN cells overexpressing PECAM-1 did form tubes in vitro. Furthermore, the use of monoclonal PECAM-1 antibody inhibited tumor angiogenesis in mice.12 More recently, the pivotal role of PECAM-1 in angiogenesis was unraveled, wherein transfection of human full-length PECAM-1 cDNA carrying mutation in immunotyrosine-based inhibitory motifs (ITIM) in REN cells inhibited migration of these cells in response to VEGF and failed to form tubes in the in vitro angiogenesis assays.13
Lactosylceramide (LacCer) is a member of the glycosphingolipid (GSL) family. LacCer is ubiquitously present in mammalian tissues and plays a pivotal role as a precursor for the synthesis of complex GSLs.14 Moreover, LacCer has been implicated in critical phenotypic changes such as proliferation and adhesion in mammalian cells.15eC21 Recently, in a promonocytic cell line (U-937), we have shown that LacCer stimulates the transcriptional expression and protein expression of PECAM-1 by recruiting protein kinase C (PKC) and and phospholipase A2 (PLA2).22 An increased level of LacCer has been reported in plasma of patients with familial hypercholesterolemia23 and in calcified and un-calcified human atherosclerotic plaques.24 Similarly,an increased plasma level of soluble PECAM-1 has been reported in patients with cardiovascular disease25,26 and in animal models of atherosclerosis such as the apolipoprotein E knockout mice.27
Because PECAM-1 expression may be a prerequisite for VEGF-induced vasculogenesis and also angiogenesis, and because LacCer can upregulate PECAM-1 expression in U937 cells, we rationalized that LacCer may well play a second messenger role in VEGF-induced PECAM-1 expression and angiogenesis in human endothelial cells. In the present article, we describe how LacCer is critical in mediating VEGF- induced PECAM-1 expression and angiogenesis in human umbilical vein endothelial cells (HUVECs).
Materials and Methods
Expanded Materials and Methods can be found in online data supplement available at http://circres.ahajournals.org.
Cell Culture
HUVECs and endothelial cell growth medium were purchased from Cambrex (Walkersville, Md) and were cultured with 10% fetal bovine serum (FBS). Human mesothelioma cell line (REN-wild type; WT) that lacks endogenous PECAM-1 expression and REN (mt-rhPECAM-1) expressing human PECAM-1 were kindly provided by Dr Steven Albelda, University of Pennsylvania Medical Center, Philadelphia. REN-WT was grown in RPMI 1640 supplemented medium with 10% FBS. REN (mt-rhPECAM-1) was cultured in the same medium with G418 (0.5g/L Gibco).
Determination of LacCer Synthesis
Determination of LacCer by high performance thin-layer chromatography was performed as described previously.19
Determination of LacCer Synthase Activity
The activity of LacCer synthase in HUVECs was determined as described earlier.19 (In this article, we have used the term GalT-V to specifically designate the HUVEC enzyme. Where we are not sure whether the enzyme is GalT-V or GalT-VI, we have referred to it as LacCer synthase.)
LacCer Synthase (GalT-V) siRNA Synthesis and Transfection
The siRNA sequence for human GalT-V cDNA (Gene Bank Accession No. AF038663) according to the (N19) TT rule was 5'-CGG AGU GAG UGG CUU AAC A dTdT-3' (sense), 5'-UGU UAA GCC ACU CAC UCC G dTdT- 3' (antisense) respectively. Scrambled (negative control) siRNA used were 5'- AUG GUG AUU AGA CUG UAC C dTdT-3' (sense), 5'- AAG CGU ACU AGG AUC AGU A dTdT-3'(antisense), respectively. HUVECs were transfected with siRNA duplexes using Oligofectamine reagent (Invitrogen) following protocol supplied by the manufacturer.
Real-Time Reverse Transcriptase Polymerase Chain Reaction
Further information on real-time reverse transcriptase polymerase chain reaction (RT-PCR) is available in the online data supplement.
Western Immunoblot Analysis
Analysis information is available in the online data supplement.
In Vitro Angiogenesis and Tube Formation Assay
In vitro angiogenesis assays were performed using a commercially available kit from Chemicon Inc.
Statistical Analysis
All assays were performed in duplicate or triplicate. Values were expressed as mean±SD. Student’s t test was used to evaluate the statistical significance of data. P<0.05 was considered significant.
Results
VEGF Induces PECAM-1 mRNA and Protein Expression
There was a dose-dependent increase in the expression of PECAM-1 mRNA. Maximal mRNA expression of PECAM-1 was observed when HUVECs were treated with VEGF (30 ng/mL for 4 hours) as determined by real-time RT-PCR (Figure 1A). Similarly, when HUVECs were treated with 25 ng/mL VEGF for different time points, maximal PECAM-1 mRNA expression was observed at 4 to 5 hours and decreased thereafter, as demonstrated by real-time RT-PCR (Figure 1C). Further, PECAM-1 protein expression was maximal after incubation with VEGF (30 ng/mL) for 4 hours (Figure 1B). When HUVECs were incubated with VEGF (25 ng/mL) for various time intervals, PECAM-1 protein expression was maximal at 4 hours (Figure 1D).
VEGF Stimulates LacCer Synthesis and This Is Abrogated by D-PDMP
As shown in Figure 2A, treatment of HUVECs with VEGF (25 ng/mL) significantly stimulated the de novo biosynthesis of LacCer (Figure 2A, panel A, open squares) in a time-dependent fashion, which occurred early at 10 minutes of incubation and continued to be higher as compared with control. In sharp contrast, HUVECs pretreated with 20 eol/L D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) (an inhibitor of glucosylceramide synthase that blocks the synthesis of glucosylceramide [GlcCer] from ceramide) and LacCer synthase mitigated VEGF-induced LacCer biosynthesis (Figure 2A, panel A, solid squares). VEGF also stimulated the biosynthesis of GlcCer (Figure 2A, panel B, open squares) and D-PDMP pretreatment inhibited VEGF-induced GlcCer synthesis as early as at 10 minutes of incubation (Figure 2A, panel B, solid squares).
VEGF-Induced PECAM-1 Expression Is Abrogated by D-PDMP and Reversed by LacCer
Preincubation of HUVECs with D-PDMP (10 to 30 eol/L) exerted a concentration-dependent inhibition of VEGF-induced PECAM-1 expression (Figure 2B). Preincubation of HUVECs with D-PDMP (20 eol/L) for 90 minutes followed by incubation with VEGF (25 ng/mL) also abrogated PECAM-1 mRNA and protein expression, and this was bypassed by LacCer (Figure 2C).
LacCer Specifically Reversed the Inhibitory Effect of D-PDMP on PECAM-1 Expression and Tube Formation/Angiogenesis
When HUVECs were incubated with GlcCer, DGDG or C2 ceramide (2.5 eol/L each) for 4 hours did not induce PECAM-1 expression (Figure 3A). Moreover, treatment of HUVECs with D-PDMP followed by incubation with GlcCer, DGDG, or C2 ceramide failed to bypass the inhibitory effect of D-PDMP on VEGF-induced PECAM-1 expression (Figure 3B) and angiogenesis (Figure 3C, panels e and f, and Figure 3D). In contrast, LacCer significantly induced PECAM-1 expression and angiogenesis independent of the presence/absence of D-PDMP and VEGF (Figure 3B, 3C panel b, and Figure 3D). These observations suggest that VEGF-induced PECAM-1 expression and angiogenesis are closely associated and regulated by LacCer.
PPMP Inhibits VEGF-Induced PECAM-1 Expression and Tube Formation/Angiogenesis and Is Bypassed by LacCer
We found that pretreatment of HUVECs with 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP; 20 eol/L), a specific inhibitor for glucosylceramide synthase, resulted in mitigation of VEGF-induced PECAM-1 expression and angiogenesis (Figure 4A and 4B). LacCer reversed the inhibitory effect of PPMP on VEGF-induced PECAM-1 expression and angiogenesis (Figure 4A and 4B). GlcCer also reversed the inhibitory effect of PPMP with regard to PECAM-1 expression and angiogenesis (Figure 4A and 4B) but to a lesser extent than LacCer. Thus, these findings suggest that VEGF targeting of LacCer synthase is critical in PECAM-1 expression and angiogenesis in HUVECs.
LacCer Synthase (GalT-V) Gene Ablation Mitigates PECAM-1 Expression and Angiogenesis
To investigate whether LacCer is specifically required to mediate VEGF-induced PECAM-1 expression and angiogenesis, we silenced GalT-V gene expression using siRNA duplex directed for human GalT-V. Western immunoblot assay using cell lysates prepared from 2 separate preparations of HUVECs and a mutant CHO cell line Pro5Lec20 expressing GalT-V but missing GalT-1 and GalT-VI genes (gift from Dr Pamela Stanley, Albert Einstein University, New York, NY) revealed that the rabbit polyclonal GalT-V antibody (IgG) specifically reacted with GalT-V with an apparent molecular weight of 55kDa (Figure 5A). Moreover, transfection of GalT-VeCspecific siRNA duplex (100 nmol/L) markedly decreased (70% GalT-V) protein expression in HUVECs (Figure 5B). In addition, the activity of GalT-V enzyme in these cells also decreased 62% when compared with scrambled siRNA treated cells (Figure 5C). Further, we were interested in understanding the effects of VEGF in GalT-VeCsilenced HUVECs on PECAM-1 expression and angiogenesis. We observed no change in PECAM-1 expression (Figure 6A) and blunted angiogenesis (Figure 6 B panel D and Figure 6C) in LacCer synthase (GalT-V) silenced cells treated with VEGF when compared with scrambled siRNA transfected cells (Figure 6B panel B). These results provide convincing evidence that LacCer is critical in mediating VEGF-induced PECAM-1 expression and angiogenesis in HUVECs.
PECAM-1 Is Required for VEGF/LacCer-Induced Tube Formation/Angiogenesis
To investigate whether PECAM-1 is absolutely required for VEGF/LacCer-induced angiogenesis, we pretreated HUVECs with PECAM-1 monoclonal antibody, followed by incubation with VEGF or LacCer. In PECAM-1 monoclonal antibody but not mouse IgG pretreated cells, VEGF/LacCer after treatment did not significantly induce angiogenesis (Figure 7A, panels e and f). Further, to understand whether PECAM-1 is pivotal for VEGF/LacCer angiogenesis, we performed experiments in REN (WT) cells, which phenotypically resemble endothelial cells but lack endogenous PECAM-1 expression. On the other hand, 2.5 kb human PECAM-1 cDNA was cloned in to mammalian expression vector pcDNA3 (Invitrogen) under cytomegalovirus promoter for constitutive expression of PECAM-1 and was transfected in REN WT and established REN (mt-rhPECAM-1) as previously described.10 In REN (WT) cells, VEGF/LacCer did not form tube-like structures in the in vitro angiogenesis assays (Figure 8B and 8C) when compared with 2% FBS treated cells (Figure 8A). On the other hand, in REN cells transfected with human PECAM-1 gene, VEGF/LacCer induced tube formation in the in vitro angiogenesis assays (Figure 8E and 8F) when compared with control (Figure 8D). Therefore, the results from these experiments reveal that PECAM-1 expression is necessary for VEGF/LacCer-induced angiogenesis. In addition, we observed that pretreatment of HUVECs with VEGF receptor (KDR/Flk-1) antagonist SU 1498 followed by incubation with VEGF but not LacCer failed to induce angiogenesis (Figure 7A, panels c, d, and h). These observations suggest that LacCer is downstream of the KDR/Flk-1 in VEGF-induced signaling pathway, leading to PECAM-1 expression and angiogenesis in HUVECs.
PKC and PLA2 Inhibitors Mitigate VEGF/LacCer-Induced PECAM-1 and Angiogenesis
PKC inhibitors CC (5.0 eol/L), G 6850, and 6976 (50 nmol/L) and PLA2 inhibitors BPB (10 eol/L) and MAFP (3.0 eol/L) abrogated VEGF/LacCer-induced PECAM-1 expression (supplemental Figure IA) and tube formation (supplemental Figure IB) when compared with cells that were treated with vehicle alone. Thus, LacCer induces PECAM-1 expression by recruiting PKC and PLA2 and these are downstream signaling events, which LacCer can recruit to induce PECAM-1expression and angiogenesis.
L-PDMP Stimulates PECAM-1 Expression and Tube Formation/Angiogenesis
Because L-PDMP is a potent activator of LacCer synthase,16,28 we examined whether this compound may alter PECAM-1 expression and angiogenesis. L-PDMP significantly induced PECAM-1 expression (supplemental Figure IIA) and angiogenesis (supplemental Figure IIB). These observations stress the importance of PDMP stereoisomers in the upregulation and downregulation of LacCer synthase, PECAM-1 expression, and angiogenesis.
Discussion
Angiogenesis involves a series of steps wherein endothelial cells degrade their basement membrane locally. Next, the endothelial cells migrate into the connective tissue stroma, proliferate, and finally differentiate into capillary loops. VEGF is a mediator of angiogenesis and is of considerable interest, as it is known to augment collateral blood flow in experimental animals and in patients with limb and myocardial ischemia.29
Although most studies have focused on the role of VEGF in angiogenesis, little is known in regard to mechanisms underlying this critical phenotypic change. Lactosylceramide is a member of the neutral glycosphingolipid family and plays a pivotal role by virtue of serving as a precursor for the biosynthesis of gangliosides such as monosialoganglioside GM3 and disialoganglioside GD3, as well as globotriosylceramide and LacCer sulfate. Although these glycosphingolipids have been shown to impart diverse biological functions, LacCer by its own right has been implicated in cell proliferation, cell adhesion, and cell migration, events that are collectively required for angiogenesis. Most importantly, LacCer was found to induce PECAM-1 gene/protein expression,22 a pre-requisite to initiate angiogenesis.10,11
Consequently, the focus of the current study was to determine whether LacCer is implicated in VEGF-induced angiogenesis. The initial step in VEGF-induced angiogenesis in endothelial cells requires its binding to a receptor having intrinsic tyrosine kinase domain designated as KDR/Flk-1.3 SU1498, a small lipophilic molecule, has been shown to specifically inhibit the tyrosine kinase activity of VEGF and abrogate angiogenesis/tube formation in endothelial cells.30 In our present study, SU1498 abrogated VEGF- but not LacCer-induced angiogenesis. This observation suggests that LacCer synthase/LacCer are downstream components of the VEGF-induced angiogenesis-signaling pathway.
To determine the mechanism by which VEGF may recruit LacCer in inducing angiogenesis, we have used both pharmacological and molecular approaches to manipulate enzymes responsible for LacCer biosynthesis. Because VEGF induced LacCer synthase activity, we first used D-PDMP, initially shown to be an inhibitor of GlcCer synthase31 but later proven to be an inhibitor of purified LacCer synthase.28,32 Our studies provided evidence that VEGF-induced LacCer/GlcCer synthesis, PECAM-1 gene/protein expression, and angiogenesis was inhibited by D-PDMP in a dose-dependent fashion. Moreover, this inhibitory effect was by passed by LacCer but not GlcCer, suggesting that VEGF targets the LacCer synthase to induce angiogenesis. Recently, Pannu et al33 demonstrated that interferon- or lipopolysaccharide treatment in neuronal cells also recruited LacCer to induce inducible nitric oxide synthase and accelerated spinal cord injury in mice and that tumor necrosis factor- induced proliferation of astrocytes and astrogliosis in spinal cord injury in rats. These events were abrogated by D-PDMP and antisense-mediated silencing of LacCer synthase.34
Previously, D-PDMP has also been shown to mitigate neurite outgrowth and ameliorate osteoclast formation35,36 and aortic smooth muscle cell proliferation.15 Although D-PDMP can also induce apoptosis by raising the cellular level of ceramide, in studies above35,36 and in the present study, D-PDMP (20 eol/L) up to 4 to 6 hours did not induce apoptosis in HUVEC (data not shown). Collectively, D-PDMP has been widely used to elaborate the role of LacCer synthase/LacCer in multiple phenotypic changes in vivo and in vitro. In contrast, a stereoisomer L-PDMP that stimulates the activity of LacCer synthase28 stimulated PECAM-1 expression and angiogenesis in our present study. Thus, stereoisomers of PDMP, by virtue of targeting LacCer synthase, altered phenotypic changes such as cell proliferation in previous studies15eC22 and angiogenesis/tube formation in the present study.
To further determine that the target for VEGF action was LacCer synthase and not GlcCer synthase, we used PPMP, a specific inhibitor of GlcCer synthase. Again, PPMP, like D-PDMP, also mitigated VEGF-induced PECAM-1 expression and angiogenesis, and this was bypassed by LacCer. A more direct approach to ascertain the role of LacCer synthase/LacCer in VEGF-induced PECAM-1 expression and angiogenesis in the present study was to use siRNA-mediated gene ablation. Herein, we silenced the LacCer synthase/GalT-V expression in HUVECs and then compared its effect on VEGF-induced PECAM-1 protein expression and angiogenesis. These studies showed that LacCer synthase (GalT-V) siRNA silencing in HUVECs contributed to a 70% decrease in the GalT-V gene/protein ablation and significantly mitigated VEGF-induced PECAM-1 gene expression and angiogenesis. On the basis of Northern blot assays, however, GalT-V constitutes 90% of the total LacCer synthase in HUVECs (data not shown), whereas GalT-VI represents the rest of LacCer synthase. These observations and the 70% ablation of the GalT-V gene could explain why complete inhibition of VEGF-induced angiogenesis and inhibition of LacCer synthase activity (Figure 6B panel D and Figure 6D) in HUVECs was not achieved in our study.
Previous studies have shown a vital role for PECAM-1 in angiogenesis.10,11 In our present study, REN cells, which are devoid of PECAM-1, were unresponsive to VEGF/LacCer-induced angiogenesis. In contrast, VEGF/LacCer treatment in REN cells expressing full-length cDNA for PECAM-1 responded strongly with regard to PECAM-1 expression and formation of tube-like structures in the in vitro assay of angiogenesis (Figure 8). Thus, both pharmacological and/or genetic manipulations of LacCer synthase adversely affected PECAM-1 gene/protein expression and angiogenesis. Therefore, our studies accede to the tenet that indeed PECAM-1 gene/protein expression and angiogenesis are closely associated.
Several studies have shown that VEGF recruits PKC/PLA to induce angiogenesis in HUVECs.37,38 Using specific inhibitors of PKC/PLA2, in the present study we found that VEGF induced de novo synthesis of LacCer, which in turn recruits PKC/PLA2 to induce angiogenesis/tube formation in HUVECs. In addition, we have also documented that in a promonocytic cell line (U-937), LacCer specifically stimulated the migration of cytosolic PKC / to the cell membrane considered to be due to the activation of these proteins.22
To date, several in vitro angiogenesis assays have been developed. Often, these assays involve the study of one particular step in the angiogenic cascade, such as proliferation, migration, or differentiation of endothelial cells.39 Tube formation assay using extracellular matrix derived from Engelbreth-Holm-Swarm sarcoma has been extensively used for studying the angiogenic/anti-angiogeic potential of a molecule over a decade.40 In vitro angiogenesis, such as the tube formation assay that we have used in our study, offers the opportunity to investigate angiogenic mechanisms with a greater speed and simplicity than with in vivo assays. It is important to note, however, that tube formation in vitro does not mimic the whole process of in vivo angiogenesis. Nonetheless, tube formation assays were widely used because of the ability to study 2 key steps in the angiogenesis process, the migration and differentiation of endothelial cells.40 Because there could also be variation in the tube formation assays, therefore one has to repeat the assays several times to arrive at the convincing conclusions. Because of these limitations in the in vitro angiogenesis/tube formation assays, one has to verify the in vitro angiogenesis data with in vivo angiogenesis assays. Presently, our laboratory is engaged in selecting and optimizing the assays to investigate the in vivo angiogenic potential of LacCer.
Recently, the requirement for sphingosine-1-phosphate receptor-1 in tumor angiogenesis was demonstrated using in vivo RNA interference.41 The present study suggests that because angiogenesis is a critical multifaceted event, cells may recruit various sphingolipids to meet the demands of organ repair, growth, and development. Our present study indicates that the use of antibodies against PECAM-1, LacCer synthase inhibitors, and/or LacCer synthase siRNA can mitigate VEGF-induced angiogenesis and well may serve as invaluable pharmacological reagents in anti-angiogenic therapy.
Acknowledgments
This work was supported by grants from the Johns Hopkins Singapore Pte. Ltd and the National Medical Research Council of Singapore (NMRC#10618.1200) and by a CAM-NIH grant. We thank Dr Steven Albelda (University of Pennsylvania Medical Center, Philadelphia) for the gift of REN cells.
The intellectual property right developed in our laboratory has been licensed out by Johns Hopkins University to Merlion Pharmaceuticals. This license deals with use of two compounds D-PDMP and L-PDMP for use in mitigating cell proliferation/promoting cell proliferation, respectively.
This manuscript was sent to Donald D. Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
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Xia P, Aiello LP, Ishii H, Jiang ZY, Park DJ, Robinson GS, Takagi H, Newsome WP, Jirousek MR, King GL. Characterization of vascular endothelial growth factor’s effect on the activation of protein kinase C, its isoforms, and endothelial cell growth. J Clin Invest. 1996; 98: 2018eC2026.
Jain RK, Schlenger K, Hockel M, Yuan F. Quantitative angiogenesis assays: progress and problems. Nat Med. 1997; 3: 1203eC1208.
Auerbach A, Lewis R, Shinners B, Kubai L, Akhtar N. Angiogenesis assays: a critical overview. Clin Chem. 2003; 49: 32eC40.
Chae S, Paike JH, Furneaux H, Hla T. Requirement of sphingosine 1-phosphate receptor-1 in tumor angiogenesis demonstrated by in vivo RNA interference. J Clin Invest. 2004; 114: 1082eC1089.(Mohanraj Rajesh, Antonina)
Department of Pediatrics, Lipid Research Unit, Johns Hopkins University (A.K., S.C.), Baltimore, Md
the Department of Biochemistry, National University of Singapore (S.C). M.R. is currently at the Department of Biological Sciences, National University of Singapore.
Abstract
Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis associated with coronary heart disease, vascular complications in diabetes, inflammatory vascular diseases, and tumor metastasis. The mechanism of VEGF-driven angiogenesis involving glycosphingolipids such as lactosylceramide (LacCer), however, is not known. To demonstrate the involvement of LacCer in VEGF-induced angiogenesis, we used small interfering RNA (siRNA)-mediated silencing of LacCer synthase expression (GalT-V) in human umbilical vein endothelial cells. This gene silencing markedly inhibited VEGF-induced platelet endothelial cell adhesion molecule-1 (PECAM-1) expression and angiogenesis. Second, we used D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of LacCer synthase and glucosylceramide synthase, that significantly mitigated VEGF-induced PECAM-1 expression and angiogenesis. Interestingly, these phenotypic changes were reversed by LacCer but not by structurally related compounds such as glucosylceramide, digalactosylceramide, and ceramide. In a human mesothelioma cell line (REN) that lacks the endogenous expression of PECAM-1, VEGF/LacCer failed to stimulate PECAM-1 expression and tube formation/angiogenesis. In REN cells expressing human PECAM-1 gene/protein, however, both VEGF and LacCer-induced PECAM-1 protein expression and tube formation /angiogenesis. In fact, VEGF-induced but not LacCer-induced angiogenesis was mitigated by SU-1498, a VEGF receptor tyrosine kinase inhibitor. Also, VEGF/LacCer-induced PECAM-1 expression and angiogenesis was mitigated by protein kinase C and phospholipase A2 inhibitors. These results indicate that LacCer generated in VEGF-treated endothelial cells may serve as an important signaling molecule for PECAM-1 expression and in angiogenesis. This finding and the reagents developed in our report may be useful as anti-angiogenic drugs for further studies in vitro and in vivo.
Key Words: vascular endothelial growth factor lactosylceramide platelet endothelial cell adhesion molecule-1 angiogenesis
Introduction
Vascular endothelial growth factor (VEGF) has been implicated in the process of vasculogenesis and angiogenesis.1,2 Aberrant expression of VEGF has been reported in several vascular pathologies such as inflammation, complications of diabetes mellitus, cardiovascular diseases, and tumor metastasis.3 VEGF binds to its receptors KDR/Flk-1 to mediate its effect on angiogenesis in physiological conditions and in human atherosclerosis.4eC6
Platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31 is a constitutively expressed integral protein in endothelial cells.7 In addition, PECAM-1 is expressed in platelets, monocytes, neutrophils, and a certain subset of T cells.8 Recent studies implicate PECAM-1 in angiogenesis and in vitro endothelial cell migration.9,10 For example, human mesothelioma cell line (REN) that is deficient in PECAM-110 and cells from PECAM-1 deficient mice11 did not form tubes in vitro (angiogenesis). In contrast, REN cells overexpressing PECAM-1 did form tubes in vitro. Furthermore, the use of monoclonal PECAM-1 antibody inhibited tumor angiogenesis in mice.12 More recently, the pivotal role of PECAM-1 in angiogenesis was unraveled, wherein transfection of human full-length PECAM-1 cDNA carrying mutation in immunotyrosine-based inhibitory motifs (ITIM) in REN cells inhibited migration of these cells in response to VEGF and failed to form tubes in the in vitro angiogenesis assays.13
Lactosylceramide (LacCer) is a member of the glycosphingolipid (GSL) family. LacCer is ubiquitously present in mammalian tissues and plays a pivotal role as a precursor for the synthesis of complex GSLs.14 Moreover, LacCer has been implicated in critical phenotypic changes such as proliferation and adhesion in mammalian cells.15eC21 Recently, in a promonocytic cell line (U-937), we have shown that LacCer stimulates the transcriptional expression and protein expression of PECAM-1 by recruiting protein kinase C (PKC) and and phospholipase A2 (PLA2).22 An increased level of LacCer has been reported in plasma of patients with familial hypercholesterolemia23 and in calcified and un-calcified human atherosclerotic plaques.24 Similarly,an increased plasma level of soluble PECAM-1 has been reported in patients with cardiovascular disease25,26 and in animal models of atherosclerosis such as the apolipoprotein E knockout mice.27
Because PECAM-1 expression may be a prerequisite for VEGF-induced vasculogenesis and also angiogenesis, and because LacCer can upregulate PECAM-1 expression in U937 cells, we rationalized that LacCer may well play a second messenger role in VEGF-induced PECAM-1 expression and angiogenesis in human endothelial cells. In the present article, we describe how LacCer is critical in mediating VEGF- induced PECAM-1 expression and angiogenesis in human umbilical vein endothelial cells (HUVECs).
Materials and Methods
Expanded Materials and Methods can be found in online data supplement available at http://circres.ahajournals.org.
Cell Culture
HUVECs and endothelial cell growth medium were purchased from Cambrex (Walkersville, Md) and were cultured with 10% fetal bovine serum (FBS). Human mesothelioma cell line (REN-wild type; WT) that lacks endogenous PECAM-1 expression and REN (mt-rhPECAM-1) expressing human PECAM-1 were kindly provided by Dr Steven Albelda, University of Pennsylvania Medical Center, Philadelphia. REN-WT was grown in RPMI 1640 supplemented medium with 10% FBS. REN (mt-rhPECAM-1) was cultured in the same medium with G418 (0.5g/L Gibco).
Determination of LacCer Synthesis
Determination of LacCer by high performance thin-layer chromatography was performed as described previously.19
Determination of LacCer Synthase Activity
The activity of LacCer synthase in HUVECs was determined as described earlier.19 (In this article, we have used the term GalT-V to specifically designate the HUVEC enzyme. Where we are not sure whether the enzyme is GalT-V or GalT-VI, we have referred to it as LacCer synthase.)
LacCer Synthase (GalT-V) siRNA Synthesis and Transfection
The siRNA sequence for human GalT-V cDNA (Gene Bank Accession No. AF038663) according to the (N19) TT rule was 5'-CGG AGU GAG UGG CUU AAC A dTdT-3' (sense), 5'-UGU UAA GCC ACU CAC UCC G dTdT- 3' (antisense) respectively. Scrambled (negative control) siRNA used were 5'- AUG GUG AUU AGA CUG UAC C dTdT-3' (sense), 5'- AAG CGU ACU AGG AUC AGU A dTdT-3'(antisense), respectively. HUVECs were transfected with siRNA duplexes using Oligofectamine reagent (Invitrogen) following protocol supplied by the manufacturer.
Real-Time Reverse Transcriptase Polymerase Chain Reaction
Further information on real-time reverse transcriptase polymerase chain reaction (RT-PCR) is available in the online data supplement.
Western Immunoblot Analysis
Analysis information is available in the online data supplement.
In Vitro Angiogenesis and Tube Formation Assay
In vitro angiogenesis assays were performed using a commercially available kit from Chemicon Inc.
Statistical Analysis
All assays were performed in duplicate or triplicate. Values were expressed as mean±SD. Student’s t test was used to evaluate the statistical significance of data. P<0.05 was considered significant.
Results
VEGF Induces PECAM-1 mRNA and Protein Expression
There was a dose-dependent increase in the expression of PECAM-1 mRNA. Maximal mRNA expression of PECAM-1 was observed when HUVECs were treated with VEGF (30 ng/mL for 4 hours) as determined by real-time RT-PCR (Figure 1A). Similarly, when HUVECs were treated with 25 ng/mL VEGF for different time points, maximal PECAM-1 mRNA expression was observed at 4 to 5 hours and decreased thereafter, as demonstrated by real-time RT-PCR (Figure 1C). Further, PECAM-1 protein expression was maximal after incubation with VEGF (30 ng/mL) for 4 hours (Figure 1B). When HUVECs were incubated with VEGF (25 ng/mL) for various time intervals, PECAM-1 protein expression was maximal at 4 hours (Figure 1D).
VEGF Stimulates LacCer Synthesis and This Is Abrogated by D-PDMP
As shown in Figure 2A, treatment of HUVECs with VEGF (25 ng/mL) significantly stimulated the de novo biosynthesis of LacCer (Figure 2A, panel A, open squares) in a time-dependent fashion, which occurred early at 10 minutes of incubation and continued to be higher as compared with control. In sharp contrast, HUVECs pretreated with 20 eol/L D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) (an inhibitor of glucosylceramide synthase that blocks the synthesis of glucosylceramide [GlcCer] from ceramide) and LacCer synthase mitigated VEGF-induced LacCer biosynthesis (Figure 2A, panel A, solid squares). VEGF also stimulated the biosynthesis of GlcCer (Figure 2A, panel B, open squares) and D-PDMP pretreatment inhibited VEGF-induced GlcCer synthesis as early as at 10 minutes of incubation (Figure 2A, panel B, solid squares).
VEGF-Induced PECAM-1 Expression Is Abrogated by D-PDMP and Reversed by LacCer
Preincubation of HUVECs with D-PDMP (10 to 30 eol/L) exerted a concentration-dependent inhibition of VEGF-induced PECAM-1 expression (Figure 2B). Preincubation of HUVECs with D-PDMP (20 eol/L) for 90 minutes followed by incubation with VEGF (25 ng/mL) also abrogated PECAM-1 mRNA and protein expression, and this was bypassed by LacCer (Figure 2C).
LacCer Specifically Reversed the Inhibitory Effect of D-PDMP on PECAM-1 Expression and Tube Formation/Angiogenesis
When HUVECs were incubated with GlcCer, DGDG or C2 ceramide (2.5 eol/L each) for 4 hours did not induce PECAM-1 expression (Figure 3A). Moreover, treatment of HUVECs with D-PDMP followed by incubation with GlcCer, DGDG, or C2 ceramide failed to bypass the inhibitory effect of D-PDMP on VEGF-induced PECAM-1 expression (Figure 3B) and angiogenesis (Figure 3C, panels e and f, and Figure 3D). In contrast, LacCer significantly induced PECAM-1 expression and angiogenesis independent of the presence/absence of D-PDMP and VEGF (Figure 3B, 3C panel b, and Figure 3D). These observations suggest that VEGF-induced PECAM-1 expression and angiogenesis are closely associated and regulated by LacCer.
PPMP Inhibits VEGF-Induced PECAM-1 Expression and Tube Formation/Angiogenesis and Is Bypassed by LacCer
We found that pretreatment of HUVECs with 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP; 20 eol/L), a specific inhibitor for glucosylceramide synthase, resulted in mitigation of VEGF-induced PECAM-1 expression and angiogenesis (Figure 4A and 4B). LacCer reversed the inhibitory effect of PPMP on VEGF-induced PECAM-1 expression and angiogenesis (Figure 4A and 4B). GlcCer also reversed the inhibitory effect of PPMP with regard to PECAM-1 expression and angiogenesis (Figure 4A and 4B) but to a lesser extent than LacCer. Thus, these findings suggest that VEGF targeting of LacCer synthase is critical in PECAM-1 expression and angiogenesis in HUVECs.
LacCer Synthase (GalT-V) Gene Ablation Mitigates PECAM-1 Expression and Angiogenesis
To investigate whether LacCer is specifically required to mediate VEGF-induced PECAM-1 expression and angiogenesis, we silenced GalT-V gene expression using siRNA duplex directed for human GalT-V. Western immunoblot assay using cell lysates prepared from 2 separate preparations of HUVECs and a mutant CHO cell line Pro5Lec20 expressing GalT-V but missing GalT-1 and GalT-VI genes (gift from Dr Pamela Stanley, Albert Einstein University, New York, NY) revealed that the rabbit polyclonal GalT-V antibody (IgG) specifically reacted with GalT-V with an apparent molecular weight of 55kDa (Figure 5A). Moreover, transfection of GalT-VeCspecific siRNA duplex (100 nmol/L) markedly decreased (70% GalT-V) protein expression in HUVECs (Figure 5B). In addition, the activity of GalT-V enzyme in these cells also decreased 62% when compared with scrambled siRNA treated cells (Figure 5C). Further, we were interested in understanding the effects of VEGF in GalT-VeCsilenced HUVECs on PECAM-1 expression and angiogenesis. We observed no change in PECAM-1 expression (Figure 6A) and blunted angiogenesis (Figure 6 B panel D and Figure 6C) in LacCer synthase (GalT-V) silenced cells treated with VEGF when compared with scrambled siRNA transfected cells (Figure 6B panel B). These results provide convincing evidence that LacCer is critical in mediating VEGF-induced PECAM-1 expression and angiogenesis in HUVECs.
PECAM-1 Is Required for VEGF/LacCer-Induced Tube Formation/Angiogenesis
To investigate whether PECAM-1 is absolutely required for VEGF/LacCer-induced angiogenesis, we pretreated HUVECs with PECAM-1 monoclonal antibody, followed by incubation with VEGF or LacCer. In PECAM-1 monoclonal antibody but not mouse IgG pretreated cells, VEGF/LacCer after treatment did not significantly induce angiogenesis (Figure 7A, panels e and f). Further, to understand whether PECAM-1 is pivotal for VEGF/LacCer angiogenesis, we performed experiments in REN (WT) cells, which phenotypically resemble endothelial cells but lack endogenous PECAM-1 expression. On the other hand, 2.5 kb human PECAM-1 cDNA was cloned in to mammalian expression vector pcDNA3 (Invitrogen) under cytomegalovirus promoter for constitutive expression of PECAM-1 and was transfected in REN WT and established REN (mt-rhPECAM-1) as previously described.10 In REN (WT) cells, VEGF/LacCer did not form tube-like structures in the in vitro angiogenesis assays (Figure 8B and 8C) when compared with 2% FBS treated cells (Figure 8A). On the other hand, in REN cells transfected with human PECAM-1 gene, VEGF/LacCer induced tube formation in the in vitro angiogenesis assays (Figure 8E and 8F) when compared with control (Figure 8D). Therefore, the results from these experiments reveal that PECAM-1 expression is necessary for VEGF/LacCer-induced angiogenesis. In addition, we observed that pretreatment of HUVECs with VEGF receptor (KDR/Flk-1) antagonist SU 1498 followed by incubation with VEGF but not LacCer failed to induce angiogenesis (Figure 7A, panels c, d, and h). These observations suggest that LacCer is downstream of the KDR/Flk-1 in VEGF-induced signaling pathway, leading to PECAM-1 expression and angiogenesis in HUVECs.
PKC and PLA2 Inhibitors Mitigate VEGF/LacCer-Induced PECAM-1 and Angiogenesis
PKC inhibitors CC (5.0 eol/L), G 6850, and 6976 (50 nmol/L) and PLA2 inhibitors BPB (10 eol/L) and MAFP (3.0 eol/L) abrogated VEGF/LacCer-induced PECAM-1 expression (supplemental Figure IA) and tube formation (supplemental Figure IB) when compared with cells that were treated with vehicle alone. Thus, LacCer induces PECAM-1 expression by recruiting PKC and PLA2 and these are downstream signaling events, which LacCer can recruit to induce PECAM-1expression and angiogenesis.
L-PDMP Stimulates PECAM-1 Expression and Tube Formation/Angiogenesis
Because L-PDMP is a potent activator of LacCer synthase,16,28 we examined whether this compound may alter PECAM-1 expression and angiogenesis. L-PDMP significantly induced PECAM-1 expression (supplemental Figure IIA) and angiogenesis (supplemental Figure IIB). These observations stress the importance of PDMP stereoisomers in the upregulation and downregulation of LacCer synthase, PECAM-1 expression, and angiogenesis.
Discussion
Angiogenesis involves a series of steps wherein endothelial cells degrade their basement membrane locally. Next, the endothelial cells migrate into the connective tissue stroma, proliferate, and finally differentiate into capillary loops. VEGF is a mediator of angiogenesis and is of considerable interest, as it is known to augment collateral blood flow in experimental animals and in patients with limb and myocardial ischemia.29
Although most studies have focused on the role of VEGF in angiogenesis, little is known in regard to mechanisms underlying this critical phenotypic change. Lactosylceramide is a member of the neutral glycosphingolipid family and plays a pivotal role by virtue of serving as a precursor for the biosynthesis of gangliosides such as monosialoganglioside GM3 and disialoganglioside GD3, as well as globotriosylceramide and LacCer sulfate. Although these glycosphingolipids have been shown to impart diverse biological functions, LacCer by its own right has been implicated in cell proliferation, cell adhesion, and cell migration, events that are collectively required for angiogenesis. Most importantly, LacCer was found to induce PECAM-1 gene/protein expression,22 a pre-requisite to initiate angiogenesis.10,11
Consequently, the focus of the current study was to determine whether LacCer is implicated in VEGF-induced angiogenesis. The initial step in VEGF-induced angiogenesis in endothelial cells requires its binding to a receptor having intrinsic tyrosine kinase domain designated as KDR/Flk-1.3 SU1498, a small lipophilic molecule, has been shown to specifically inhibit the tyrosine kinase activity of VEGF and abrogate angiogenesis/tube formation in endothelial cells.30 In our present study, SU1498 abrogated VEGF- but not LacCer-induced angiogenesis. This observation suggests that LacCer synthase/LacCer are downstream components of the VEGF-induced angiogenesis-signaling pathway.
To determine the mechanism by which VEGF may recruit LacCer in inducing angiogenesis, we have used both pharmacological and molecular approaches to manipulate enzymes responsible for LacCer biosynthesis. Because VEGF induced LacCer synthase activity, we first used D-PDMP, initially shown to be an inhibitor of GlcCer synthase31 but later proven to be an inhibitor of purified LacCer synthase.28,32 Our studies provided evidence that VEGF-induced LacCer/GlcCer synthesis, PECAM-1 gene/protein expression, and angiogenesis was inhibited by D-PDMP in a dose-dependent fashion. Moreover, this inhibitory effect was by passed by LacCer but not GlcCer, suggesting that VEGF targets the LacCer synthase to induce angiogenesis. Recently, Pannu et al33 demonstrated that interferon- or lipopolysaccharide treatment in neuronal cells also recruited LacCer to induce inducible nitric oxide synthase and accelerated spinal cord injury in mice and that tumor necrosis factor- induced proliferation of astrocytes and astrogliosis in spinal cord injury in rats. These events were abrogated by D-PDMP and antisense-mediated silencing of LacCer synthase.34
Previously, D-PDMP has also been shown to mitigate neurite outgrowth and ameliorate osteoclast formation35,36 and aortic smooth muscle cell proliferation.15 Although D-PDMP can also induce apoptosis by raising the cellular level of ceramide, in studies above35,36 and in the present study, D-PDMP (20 eol/L) up to 4 to 6 hours did not induce apoptosis in HUVEC (data not shown). Collectively, D-PDMP has been widely used to elaborate the role of LacCer synthase/LacCer in multiple phenotypic changes in vivo and in vitro. In contrast, a stereoisomer L-PDMP that stimulates the activity of LacCer synthase28 stimulated PECAM-1 expression and angiogenesis in our present study. Thus, stereoisomers of PDMP, by virtue of targeting LacCer synthase, altered phenotypic changes such as cell proliferation in previous studies15eC22 and angiogenesis/tube formation in the present study.
To further determine that the target for VEGF action was LacCer synthase and not GlcCer synthase, we used PPMP, a specific inhibitor of GlcCer synthase. Again, PPMP, like D-PDMP, also mitigated VEGF-induced PECAM-1 expression and angiogenesis, and this was bypassed by LacCer. A more direct approach to ascertain the role of LacCer synthase/LacCer in VEGF-induced PECAM-1 expression and angiogenesis in the present study was to use siRNA-mediated gene ablation. Herein, we silenced the LacCer synthase/GalT-V expression in HUVECs and then compared its effect on VEGF-induced PECAM-1 protein expression and angiogenesis. These studies showed that LacCer synthase (GalT-V) siRNA silencing in HUVECs contributed to a 70% decrease in the GalT-V gene/protein ablation and significantly mitigated VEGF-induced PECAM-1 gene expression and angiogenesis. On the basis of Northern blot assays, however, GalT-V constitutes 90% of the total LacCer synthase in HUVECs (data not shown), whereas GalT-VI represents the rest of LacCer synthase. These observations and the 70% ablation of the GalT-V gene could explain why complete inhibition of VEGF-induced angiogenesis and inhibition of LacCer synthase activity (Figure 6B panel D and Figure 6D) in HUVECs was not achieved in our study.
Previous studies have shown a vital role for PECAM-1 in angiogenesis.10,11 In our present study, REN cells, which are devoid of PECAM-1, were unresponsive to VEGF/LacCer-induced angiogenesis. In contrast, VEGF/LacCer treatment in REN cells expressing full-length cDNA for PECAM-1 responded strongly with regard to PECAM-1 expression and formation of tube-like structures in the in vitro assay of angiogenesis (Figure 8). Thus, both pharmacological and/or genetic manipulations of LacCer synthase adversely affected PECAM-1 gene/protein expression and angiogenesis. Therefore, our studies accede to the tenet that indeed PECAM-1 gene/protein expression and angiogenesis are closely associated.
Several studies have shown that VEGF recruits PKC/PLA to induce angiogenesis in HUVECs.37,38 Using specific inhibitors of PKC/PLA2, in the present study we found that VEGF induced de novo synthesis of LacCer, which in turn recruits PKC/PLA2 to induce angiogenesis/tube formation in HUVECs. In addition, we have also documented that in a promonocytic cell line (U-937), LacCer specifically stimulated the migration of cytosolic PKC / to the cell membrane considered to be due to the activation of these proteins.22
To date, several in vitro angiogenesis assays have been developed. Often, these assays involve the study of one particular step in the angiogenic cascade, such as proliferation, migration, or differentiation of endothelial cells.39 Tube formation assay using extracellular matrix derived from Engelbreth-Holm-Swarm sarcoma has been extensively used for studying the angiogenic/anti-angiogeic potential of a molecule over a decade.40 In vitro angiogenesis, such as the tube formation assay that we have used in our study, offers the opportunity to investigate angiogenic mechanisms with a greater speed and simplicity than with in vivo assays. It is important to note, however, that tube formation in vitro does not mimic the whole process of in vivo angiogenesis. Nonetheless, tube formation assays were widely used because of the ability to study 2 key steps in the angiogenesis process, the migration and differentiation of endothelial cells.40 Because there could also be variation in the tube formation assays, therefore one has to repeat the assays several times to arrive at the convincing conclusions. Because of these limitations in the in vitro angiogenesis/tube formation assays, one has to verify the in vitro angiogenesis data with in vivo angiogenesis assays. Presently, our laboratory is engaged in selecting and optimizing the assays to investigate the in vivo angiogenic potential of LacCer.
Recently, the requirement for sphingosine-1-phosphate receptor-1 in tumor angiogenesis was demonstrated using in vivo RNA interference.41 The present study suggests that because angiogenesis is a critical multifaceted event, cells may recruit various sphingolipids to meet the demands of organ repair, growth, and development. Our present study indicates that the use of antibodies against PECAM-1, LacCer synthase inhibitors, and/or LacCer synthase siRNA can mitigate VEGF-induced angiogenesis and well may serve as invaluable pharmacological reagents in anti-angiogenic therapy.
Acknowledgments
This work was supported by grants from the Johns Hopkins Singapore Pte. Ltd and the National Medical Research Council of Singapore (NMRC#10618.1200) and by a CAM-NIH grant. We thank Dr Steven Albelda (University of Pennsylvania Medical Center, Philadelphia) for the gift of REN cells.
The intellectual property right developed in our laboratory has been licensed out by Johns Hopkins University to Merlion Pharmaceuticals. This license deals with use of two compounds D-PDMP and L-PDMP for use in mitigating cell proliferation/promoting cell proliferation, respectively.
This manuscript was sent to Donald D. Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
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