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Keywords:

  • cell cultures;
  • EPCR polymorphism;
  • shedding

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Competing interests statement
  8. References

Summary.  The endothelial cell protein C receptor (EPCR) plays an important role in regulating blood coagulation and in activated protein C-mediated anti-inflammatory and antiapoptotic processes. Recent studies reported that there are polymorphisms in the human EPCR gene. One of the polymorphisms (haplotype A3) results in substitution of the Ser at residue 219 with Gly in the transmembrane domain. This haplotype is associated with increased plasma levels of soluble EPCR and is a candidate risk factor for thrombosis. We established stable cell lines expressing either the EPCR A1 (Ser at residue 219) or A3 (Gly at residue 219) haplotype. Both constitutive and PMA-stimulated shedding are five- to sevenfold higher in the A3 cell line than the A1 cell line. We also isolated human umbilical vein endothelial cells (HUVEC) from A1/A1 or A1/A3 origins. PMA-stimulated shedding is fourfold higher in HUVEC derived from A1/A3 origin than from A1/A1 origin. After PMA treatment, the rate of human protein C activation decreased 36% in HUVEC derived from A1/A3 origin, while it only decreased 18% in HUVEC derived from A1/A1 origin. These results indicate that the A3 haplotype does promote cellular shedding in either 293 or endothelial cells and therefore is likely directly contributory to the higher soluble EPCR levels seen in patients carrying this haplotype.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Competing interests statement
  8. References

The protein C anticoagulant pathway is critical to the negative regulation of the blood clotting cascade [1]. The endothelial protein C receptor (EPCR) plays an important role in this pathway [2]. EPCR binds protein C and increases the rate of protein C activation by thrombin/thrombomodulin (TM) complex [3,4]. EPCR is a type I transmembrane protein. It shares homology with the major histocompatibility class 1/CD1 family of proteins involved in the immune response [5,6]. Although a transmembrane protein, soluble EPCR (sEPCR) is present in normal plasma [7]. sEPCR is generated by ectodomain shedding from the endothelium mediated by an unidentified metalloproteinase [8].

Recently, two groups have reported that plasma sEPCR levels in healthy subjects display a bimodal distribution [9,10]. Higher levels of sEPCR are associated with one of the three EPCR haplotypes, A3, and may be a candidate risk factor for thrombosis [9]. There are several base differences between each of the haplotypes but the only difference in coding sequences is between A3 and the others. The A3 haplotype sequence has an adenosine in the place of a guanosine at EPCR genomic sequence position 6936, resulting in the substitution of serine with glycine (Gly) at EPCR protein residue 219 in the transmembrane domain.

When our laboratory reported the cloned human EPCR cDNA sequence for the first time [5], the cDNA library was derived from a single umbilical cord. Our human EPCR cDNA sequence contains guanosine at position 679, resulting in Gly at residue 219 of the protein sequence, corresponding to the less common A3 haplotype.

To determine whether this amino acid change is sufficient to explain the higher level of sEPCR found with the A3 haplotype, a cell line expressing the EPCR A1 haplotype was established. We find that both constitutive and PMA-stimulated EPCR shedding is five to sevenfold higher in the A3 than the A1 EPCR cell line. We also isolated human umbilical vein endothelial cells (HUVEC) derived from A1/A1 origin and A1/A3 origin, and PMA-stimulated EPCR shedding and protein C activation were studied on these cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Competing interests statement
  8. References

Materials

Phorbol-12-myristate 13-acetate (PMA) and bovine serum albumin (BSA) were from Sigma (St Louis, MO, USA). Recombinant human interleukin-1β (IL-1β) was purchased from Roche Applied Science (Indianapolis, IN, USA). Human protein C, bovine thrombin, FITC-labeled activated protein C (fl-APC) and monoclonal antibodies (mAbs) against human EPCR (JRK1494, JRK1496, JRK1513) were prepared as described in Ref. [3]. FITC-conjugated goat antimouse IgG was purchased from Becton Dickson (San Jose, CA, USA). pZeoSV vector, LipofectAMINE transfection reagent, Zeocin, Micro-FastTrack 2.0TM mRNA Isolation kit and SuperScriptTM First-Strand Synthesis System for RT-PCR were from Invitrogen (Carlsbad, CA, USA). SYBR Green PCR Core Reagents were from PE Biosystems (Warrington, UK). DNAzol genomic DNA isolation reagent was purchased from Molecular Research Center, Inc (Cincinnati, OH, USA). Immobilon-P membranes were purchased from Millipore (Bedford, MA, USA). The sheep antimouse IgG conjugated with horseradish peroxidase and the enhanced chemiluminescence system were purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA).

Construction of hEPCR G679A cDNA

cDNA corresponding to the A3 haplotype (EPCR-G219) was inserted into pZeoSV vector via XhoI and NotI restriction sites. The QuickChange site-directed mutagenesis method was used to generate G679A substitution. The sense primer was: 5′-GGCGTCCTGGTGGGCAGTTTCATCATTGCTGGT-3′, and the antisense primer was: 5′-ACCAGCAATGATGAAACTGCCCACCAGGACGCC-3′. The sequence was confirmed by the automatic sequencing.

Establishing stable cell lines

HEK293 cells were maintained in DMEM containing 10% heat-inactivated fetal bovine serum. For transfection, cells were seeded into 6-well plates. At 50% confluence, the cells were transfected with 1 μg of the human EPCRG219 cDNA (pZeoSV-EPCRG219) plasmid or with 1 μg of the human EPCRS219 cDNA (pZeoSV-EPCRS219) using 6 μL of LipofectAMINE according to the manufacturer's instructions. In 72 h post-transfection, the cells were selected for transfectants with medium containing 400 μg mL−1 of Zeocin. After 2 weeks, colonies were isolated, expanded, and screened for EPCR expression by fluorescent-activated cell sorting (FACS) analysis. We designated positive cell lines as EPCR-G219 and EPCR-S219.

FACS analysis

Cells were incubated with 5 μg mL−1 of anti-EPCR mAb JRK1494 in Hank's balanced salt solution (HBSS), 1% BSA, 25 mm HEPES, pH 7.5 for 30 min at 4 °C, washed twice with the same buffer, and then incubated with FITC-conjugated goat antimouse IgG (1:100 dilution) in the same buffer for 30 min at 4 °C. The cells were washed again with the same buffer and subjected for FACS analysis using a FACSCalibur (Becton Dickson). Fluorescein-labeled human APC (Fl-APC) was prepared as described in Ref. [5]. For fl-APC binding, the cells were incubated with 0, 10, 30, 60, 120 and 180 nm of fl-APC in HBSS, 1% BSA, 25 mm HEPES, pH 7.5, 3 mm CaCl2, 0.6 mm MgCl2 for 30 min at 4 °C, washed twice with the same buffer and subjected to FACS analysis.

mRNA isolation and real-time quantitative PCR analysis

mRNAs were isolated from EPCR-G219 and EPCR-S219 cells (3 × 106 cells) using the Micro-FastTrack 2.0TM mRNA Isolation kit. First strand cDNA was synthesized using the SuperScriptTM First-strand Synthesis System for RT-PCR, then subjected to real-time quantitative PCR using SYBR Green PCR reagents. The primers for EPCR were: forward primer: 5′-CGCCTCAGATGGCCTCC-3′, reverse primer: 5′-CGCGTTGCCCTGGTACC-3′. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. The primers for GAPDH were: forward primer: 5′-GAAGGTGAAGGTCGGAGTC-3′, reverse primer: 5′-GAAGATGGTGATGGGATTTC-3′. The quantitative real-time PCR was carried out using an ABI PRISM 7000 system (PE Biosystems).

Isolating and genotyping of HUVECS

Human umbilical vein endothelial cells were isolated from individual umbilical cords as previously described in Ref. [5]. HUVECs were maintained in M199 containing 10% heat-inactivated fetal bovine serum, 2% bovine brain extract, 50 μg mL−1 of heparin, penicillin and streptomycin. The cells were used within two passages. Genomic DNAs were isolated from HUVECs using DNAzol genomic DNA isolation reagent following the manufacturer's instructions. The EPCR genomic DNA sequences of exon four were amplified using the polymerase chain reaction (PCR) method. The PCR products were then sequenced using a sense primer and an antisense primer to determine the sequence at position 679. Umbilical cords were collected under an Institutional Review Board approved protocol.

Constitutive shedding, PMA treatment, ELISA and Western blot detection of EPCR

To study the constitutive shedding, EPCR-G219 and EPCR-S219 cells were seeded into 6-well plates. At approximately 90% confluence, the cells were incubated with fresh DMEM for 16 h at 37 °C. To study the PMA-stimulated EPCR shedding, the cells were seeded into 6-well plates and grown to confluence. The cells were then incubated for 1 h at 37 °C with fresh DMEM medium in the presence or absence of 0.1 μm PMA. The conditioned media were collected, passed through a 0.2 μm filter, and Triton X-100 was added to a final concentration of 1%. The cells were washed with PBS once and lyzed with 1% Triton X-100 in PBS at room temperature. EPCR in the conditioned media and cell lysates was measured by ELISA using mAb JRK1494 as the coating antibody and biotinylated mAb JRK1500 as the second antibody. TM in the cell lysates was measured by ELISA using mAb CTM1029 as the coating antibody and biotinylated mAb CTM 1009 as the second antibody. For Western blot analysis, the equivalent amount of protein from conditioned media or cell lysates was subjected to SDS–PAGE on 4–20% gradient gels under the reducing conditions, and then transferred to Immobilon-P membranes in a semi-dry apparatus (Bio-Rad, Hercules, CA, USA). Membranes were blocked with non-fat milk, incubated with anti-EPCR mAb JRK1513, washed and incubated with sheep antimouse IgG conjugated with horseradish peroxidase. The membranes were developed with the enhanced chemiluminescent detection reagent, and scanned with the GS-670 imaging densitometer for quantification (Bio-Rad).

Iodination of monoclonal antibodies

Anti-EPCR mAb JRK1494 was labeled with Na125I using the IODO-GEN reagent (Pierce, Rockford, IL, USA) as described previously in Ref. [8].

Internalization assay

EPCR-G219 and EPCR-S219 cells were seeded into 24-well plates. At confluence, the cells were placed on ice and washed three times with cold HBSS and 1% BSA. The cells were incubated with 30 nm125I-JRK1494 in 200 μL of DMEM/1% FBS on ice for 30 min followed by three washes with cold DMEM/1% FBS. For one plate, the cells were lyzed immediately with 200 μL of 1 m NaOH. The radioactive counts from these cell lysates were considered to reflect total bound antibody. For another plate, the cells were incubated with 200 μL of DMEM/1% FBS at 37 °C for 2, 5, 10 and 15 min. At each time interval, the medium was collected and counted to determine released radioactivity. After removing the cell medium at each time point, 200 μL of 20 mm Gly–HCl in 0.15 m NaCl, pH 2.5 was added to each well and incubated for 1 min at 37 °C. The radioactive counts in the acid wash were considered the cell surface bound antibody. After removing the acid buffer, the cells were lyzed with 200 μL of 1 m NaOH. The radioactive counts from these cell lysates were considered internalized antibody. 125I was counted using a γ-counter (Iso-Data, Rolling Meadows, IL, USA). Duplicate wells were used for each time point and two separate experiments were performed.

Protein C Activation on HUVEC

Confluent HUVEC in 24-well plates were incubated with fresh DMEM medium in the presence of 0, 0.1, 0.5 and 1 μm PMA for 1 h at 37 °C. The cells were then washed two times with HBSS, 1% BSA, 25 mm HEPES, pH 7.5, 3 mm CaCl2, 0.6 mm MgCl2. The human protein C activation was initiated by the addition of bovine thrombin (5 nm final concentration) to 200 nm protein C in a total volume of 0.2 mL. Where indicated, 50 μg mL−1 of anti-EPCR mAb JRK1494 was added to the cells 15 min prior to the addition of protein C. This antibody blocks the ability of EPCR to enhance protein C activation by TM. After 30 min at 37 °C, the reactions were stopped by addition of 20 μL of antithrombin (1.66 mg mL−1). Aliquots of the supernatants were transferred into the 96-well microplate and amidolytic activities of APC were determined using 0.2 mm Spectrozyme PCa as the substrate in 0.15 m NaCl, 20 mm Tris–HCl, pH 7.5. The rates of substrate cleavage were measured with a Vmax microplate reader (Molecular Devices, Sunnyvale, CA, USA). The concentration of APC in the reaction mixtures was determined by the comparison to a standard curve of amidolytic activity vs. APC concentration constructed with freshly prepared, fully activated protein C as described in Ref. [3]. Under the conditions employed in this study, <10% of the protein C was activated during the assay and all assays were linear as a function of time between the initiation and termination of the assay. All determinations were performed in duplicate.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Competing interests statement
  8. References

Comparison of EPCR-S219 and EPCR-G219 cell lines

To study the effect of EPCR polymorphisms, we established two cell lines stably expressing EPCR-S219 or EPCR-G219. As seen in Table 1, these two cell lines expressed similar amounts of EPCR mRNA and protein. EPCR-S219 and EPCR-G219 cells also bound fl-APC with similar affinity (Fig. 1).

Table 1.  Comparison of EPCR-S219 and EPCR-G219 cell
 EPCR/ GAPDH mRNA ratio EPCR protein (ng/5 μg total protein)mAb binding to cell surface EPCR (MCF)
  1. mRNAs were isolated from EPCR-S219 and EPCR-G219 cells and subjected to RT-PCR followed by quantitative real-time PCR. The relative level of EPCR mRNA compared with GAPDH mRNA is presented. The amount of EPCR protein in 5 μ total protein of each cell lysate was detected by ELISA. The results represent two separate experiments performed in duplicate ± SD. EPCR on the cell surface was detected using mAb against EPCR by FACS analysis. The mean channel fluorescent counts represent the average of two determinations ± SD.

EPCR-S2191289 ± 20.88442.5 ± 39.14
EPCR-G2191.04300 ± 29.87417.4 ± 76.39
image

Figure 1. The plasma membrane expression of EPCR on EPCR-S219 and EPCR-G219 cells. (A) FACS analysis of fl-APC (60 nm) binding to HEK293, EPCR-S219 and EPCR G219 cells. (B) Concentration dependence of fl-APC binding to EPCR-S219 (open circles) and EPCR-G219 (solid squares) cells. Fl-APC binding to HEK293, EPCR-S219 and EPCR-G219 cells was analyzed by FACS. The MCF counts of fl-APC binding to HEK293 cells were subtracted from the MCF counts of fl-APC binding to EPCR-S219 and EPCR-G219 cells. The MCF counts plotted represent two separate experiments performed in duplicate ± SD.

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125I-labeled monoclonal antibody against EPCR was used to study the rate of EPCR internalization. Both EPCR-S219 and EPCR-G219 have similar internalization rates. Approximately, 35% of surface EPCR can be internalized over 20 min (Fig. 2). These results indicate that both EPCR-S219 and EPCR-G219 cells express EPCR at similar levels and EPCRs are internalized at similar rates.

image

Figure 2. Measurement of internalization of EPCR in EPCR-G219 and EPCR-S219 cells. EPCR-G219 and EPCR-S219 cells were seeded into 24-well plates. At confluence, the cells were analyzed for rate of internalization as described in the text. (A) Internalization of EPCR in EPCR-G219 cells. (B) Internalization of EPCR in EPCR-S219 cells. Solid circles (•), cell surface bound mAb; solid squares (bsl00001), released mAb; solid triangles (bsl00066), internalized mAb.

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EPCR-G219 cells release more sEPCR than EPCR-S219 cells

For the constitutive shedding, both cell lines were incubated with DMEM for 48 h. EPCR was more than fivefold higher in conditioned medium (CM) from EPCR-G219 cells than from EPCR-S219 cells (Fig. 3B). After PMA stimulation, EPCR in the CM was about sevenfold higher from EPCR-G219 than from EPCR-S219 cells (Fig. 3A and C). These results indicate that the Ser to Gly substitution is probably responsible for the increased plasma EPCR levels seen in people with the A3 haplotype. As the concentration of PMA was increased, EPCR shedding from EPCR-G219 cells appeared optimal by 0.5 μm PMA, whereas shedding from the EPCR-S219 cells continued to increase (Fig. 3D).

image

Figure 3. The constitutive and PMA-stimulated shedding of EPCR is higher in EPCR-G219 cells. (A) EPCR-G219 and EPCR-S219 cells were treated with 0.1 μm PMA at 37 °C for 1 h. The CM and cell lysate were analyzed by Western blot to detect EPCR. (B) EPCR-G219 and EPCR-S219 cells were incubated with DMEM only for 48 h. The CM and cell lysates were collected and analyzed by ELISA to detect EPCR. The percentage of EPCR in CM compared with total (CM + lysate) is presented. The results represent two separate experiments performed in duplicate ± SD. (C) EPCR-G219 (solid bar) and EPCR-S219 (open bar) cells were treated with or without 0.1 μm PMA at 37 °C for 1 h. The CM and cell lysate were analyzed by ELISA to detect EPCR. The percentage of EPCR in CM compared with total (CM + lysate) is presented. The results represent two separate experiments performed in duplicate ± SD. (D) EPCR-S219 (solid squares) and EPCR-G219 (solid circles) cells were incubated with 0, 0.1, 0.5 and 1 μm PMA at 37 °C for 1 h. The CM and cell lysate were analyzed by ELISA to detect EPCR. The percentage of EPCR in CM compared with total (CM + lysate) is presented. The results represent two separate experiments performed in duplicate ± SD.

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sEPCR level is higher in HUVEC A1/A3 compared with HUVEC A1/A1

We have screened HUVEC derived from 35 individual umbilical cords. Four cultures were A1/A3 (S219/G219) heterozygous, 31 were A1/A1 (S219/S219) homozygous, and no A3/A3 (G219/G219) was found. To determine constitutive shedding, both HUVEC A1/A1 and A1/A3 were incubated with fresh complete growth medium for 24 h at confluence. EPCR was 50% higher in CM from HUVEC A1/A3 than from HUVEC A1/A1 (Fig. 4A). After PMA stimulation, EPCR was approximately four- to sixfold higher in the CM from HUVEC A1/A3 than from HUVEC A1/A1 (Fig. 4B). EPCR shedding from both cell types appeared optimal by 0.1 μm PMA treatment. This result suggests that EPCR on endothelial cells is more sensitive to PMA-stimulated shedding than EPCR on transfected 293 cells. As a more physiological relevant reagent, recombinant IL-1β was used to stimulate these cells (Fig. 4C). EPCR was approximately twofold higher in the CM from HUVEC A1/A3 than from HUVEC A1/A1. EPCR shedding from both cell types appeared optimal by 100 U mL−1 IL-1β treatment.

image

Figure 4. Constitutive and stimulated shedding of EPCR is higher in HUVEC A1/A3 than in HUVEC A1/A1. (A) HUVEC A1/A3 and HUVEC A1/A1 were incubated with fresh growth medium for 24 h. The CM and cell lysates were collected and analyzed by ELISA to detect EPCR. (B) HUVEC A1/A1 (squares) and HUVEC A1/A3 (circles) were incubated with 0, 0.01, 0.05, 0.1, 0.5 and 1 μm PMA at 37 °C for 1 h. The CM and cell lysate were analyzed by ELISA to detect EPCR. (C) HUVEC A1/A1 (squares) and HUVEC A1/A3 (circles) were incubated with 0, 1, 5, 50, 100 and 200 U mL−1 IL-1β at 37 °C for 1 h. The CM and cell lysate were analyzed by ELISA to detect EPCR. The percentage of EPCR in CM compared with total (CM + lysate) is presented. The results represent two separate experiments performed in duplicate +SD.

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Protein C activation rate is higher in HUVEC A1/A1 compared with HUVEC A1/A3

Protein C activation on both cells was compared (Fig. 5). The rate of protein C activation on HUVEC A1/A3 is approximately 73% of the rate on HUVEC A1/A1 in the absence of PMA. This is most likely because of the decrease of EPCR on HUVEC A1/A3 caused by the constitutive shedding. After 1 μm PMA stimulation, the rate of protein C activation on HUVEC A1/A3 decreased approximately 36%, but decreased only about 18% on HUVEC A1/A1 cells. This suggests that the A3 haplotype is not only associated with an increased sEPCR level, but is also associated with the decreased protein C activation. A decrease in protein C activation can be because of a loss of EPCR and/or TM upon PMA treatment [11]. To test how much of the observed changes were because of EPCR vs. TM loss, cells were incubated with mAb JRK1494 to block the EPCR contribution to protein C activation [3]. Blocking protein C binding to EPCR dramatically decreased protein C activation to an equivalent level (Fig. 5B) (∼1.4 nm/105 cells in the presence of blocking antibody, vs. ∼6 or 9 nm/105 cells in the absence of blocking antibody). Total TM level was measured by ELISA. There was approximately 23.4 ng mL−1 of TM from HUVEC A1/A1 cell lysates, and 22.1 ng mL−1 after 1 μm PMA treatment. There was approximately 24.6 ng mL−1 of TM from HUVEC A1/A3 cell lysates, and 24.6 ng mL−1 after 1 μm PMA treatment. These results indicate that both the total and surface-expressed TM (as determined functionally) were not affected by the EPCR genotype. PMA treatment caused an approximately 11% decrease of protein C activation on HUVEC A1/A3 cells and approximately 15% decrease on HUVEC A1/A1 cells (Fig. 5B; note that the y-axis in A and B are on different scales). In a report from Dittman et al. [11], protein C activation decreased 40% after 0.32 μm PMA treatment for 30 min on mouse hemangioma cells. Part of this decrease may have been because of the shedding of mouse EPCR from these cells.

image

Figure 5. The rate of protein C activation is lower in HUVEC A1/A3 than in HUVEC A1/A1. HUVEC A1/A3 (solid bar) and HUVEC A1/A1 (open bar) were incubated with 0, 0.1, 0.5 and 1 μm PMA at 37 °C for 1 h. Protein C activation was carried out at 37 °C for half-an-hour as described in the text. The amount of nm APC/105 cells generated is presented. The results represent two separate experiments performed in duplicate ± SD. (A) Protein C activation in the absence of anti-EPCR mAb, JRK1494. (B) Protein C activation in the presence of anti-EPCR mAb, JRK1494.

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We have demonstrated that, at the tissue culture level, EPCR shedding is increased in cells bearing the G219 polymorphism compared with S219. This suggests that the S to G substitution is primarily responsible for the higher plasma EPCR concentration seen in the A3 haplotype individuals. Amino acid residue 218 of the EPCR transmembrane domain is Gly, with another Gly at residue 219 for the A3 haplotype. Gly–Gly in a transmembrane domain would likely disrupt the transmembrane helix [12]. This may favor rapid sorting into plasma membranes or rapid partition into different micro-domains on plasma membranes, resulting in increased exposure to the metalloproteinase. In one study, the A3 haplotype was over-represented in patients with venous thromboses, suggesting that carrying the A3 haplotype contributed to an increased risk of thrombosis [9]. Studies of protein C activation on HUVEC A1/A3 and HUVEC A1/A1 suggest that the increased shedding of cellular EPCR may affect some important physiological functions, such as augmenting protein C activation. It has been reported that patients with severe sepsis vary markedly in their ability to generate APC [13]. This may be due in part to the different levels of EPCR expressed on endothelial cells.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Competing interests statement
  8. References

We thank Gary L. Ferrell for preparing all the antibodies used, Virginia Conley for helping with the manuscript. This work was supported in part by the National Institute of Health (NIH) Grant P50 HL54502. C.T.E. is an investigator of the Howard Hughes Medical Institute.

Competing interests statement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Competing interests statement
  8. References

The authors declare that they have no competing financial interests.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Competing interests statement
  8. References
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