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Am J Physiol Lung Cell Mol Physiol 294: L17-L23, 2008. First published October 12, 2007; doi:10.1152/ajplung.00178.2007
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EDITORIAL FOCUS

Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxia-induced cell death

Dong Xu, Ricardo E. Perez, Ikechukwu I. Ekekezie, Angels Navarro, and William E. Truog

Neonatology Research Laboratory, Neonatology Section, Department of Pediatrics, Children's Mercy Hospital, School of Medicine, University of Missouri-Kansas City, Kansas City, Missouri

Submitted 2 May 2007 ; accepted in final form 11 October 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hyperoxia is one of the major contributors to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants. Emerging evidence suggests that the arrested lung development of BPD is associated with pulmonary endothelial cell death and vascular dysfunction resulting from hyperoxia-induced lung injury. A better understanding of the mechanism of hyperoxia-induced endothelial cell death will provide critical information for the pathogenesis and therapeutic development of BPD. Epidermal growth factor-like domain 7 (EGFL7) is a protein secreted from endothelial cells. It plays an important role in vascular tubulogenesis. In the present study, we found that Egfl7 gene expression was significantly decreased in the neonatal rat lungs after hyperoxic exposure. The Egfl7 expression was returned to near normal level 2 wk after discounting oxygen exposure during recovery period. In cultured human endothelial cells, hyperoxia also significantly reduced Egfl7 expression. These observations suggest that diminished levels of Egfl7 expression might be associated with hyperoxia-induced endothelial cell death and lung injury. When we overexpressed human Egfl7 (hEgfl7) in EA.hy926 human endothelial cell line, we found that hEgfl7 overexpression could partially block cytochrome c release from mitochondria and decrease caspase-3 activation. Further Western blotting analyses showed that hEgfl7 overexpression could reduce expression of a proapoptotic protein, Bax, and increase expression of an antiapoptotic protein, Bcl-xL. Theses findings indicate that hEGFL7 may protect endothelial cell from hyperoxia-induced apoptosis by inhibition of mitochondria-dependent apoptosis pathway.

apoptosis; necrosis; lung injury; chronic lung disease

SUPPLEMENTAL OXYGEN (hyperoxia) therapy is a common life-saving practice for critically ill patients such as premature infants with very low birth weight (6). Unfortunately, the prolonged hyperoxia can generate reactive oxygen species and result in oxidative stress, which may lead to respiratory failure and death (1a). Although the endothelial cell has been identified as a vulnerable target and hyperoxia-induced endothelial cell death occurs before onset of respiratory failure, the pathogenesis of hyperoxia-induced endothelial cell death is not completely understood. Emerging evidence suggests that the arrested lung development of bronchopulmonary dysplasia (BPD) is associated with pulmonary vascular dysfunction resulting from hyperoxia-induced lung injury (3, 17, 20). It is crucial to understand how hyperoxia regulates angiogenic factors, induces endothelial cell death, and causes pulmonary vascular dysfunction in the developing lung. A better understanding of cellular and molecular mechanisms of hyperoxia-induced endothelial cell death will provide critical information in the development of effective therapy for BPD.

Maturation of pulmonary vasculature is a complex process that involves endothelial cell proliferation, differentiation, migration, tube formation, and stabilization. Pulmonary vascular formation is tightly regulated by stimulators and inhibitors in both physiological and pathological conditions (8, 27). It appears that there is a relationship between capillary invasion and alveolar septation. Blockage of VEGF receptor disrupts pulmonary vasculature development and attenuates lung alveolarization, suggesting that pulmonary vasculogenesis and angiogenesis are necessary for alveolarization during normal lung development (17, 21, 33). Hyperoxia is known to modulate the expression of angiogenic factors such as VEGF. An imbalance of angiogenic growth factors after hyperoxic exposure can disrupt normal pulmonary vascularization and alveolarization, which appear to be critical in the pathogenesis of hyperoxia-induced lung injury including BPD and acute lung injury (24, 26, 32). However, the mechanisms of pulmonary vasculogenesis and angiogenesis regulation are not completely understood during normal lung development. The pathophysiological causes that lead to disrupted alignment of the vasculature and alveolar formation in hyperoxia-induced lung injury remain to be investigated.

Epidermal growth factor-like domain 7 (EGFL7) is a recently identified protein secreted from vascular endothelial cells and is expressed at a high level in the lung, heart, and kidney (30). Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis, indicating that EGFL7 plays a specific role in the crucial steps of vasculogenesis and angiogenesis (25, 29). EGFL7 also inhibits vascular smooth muscle cell migration induced by PDGF (30). The expression of EGFL7 is upregulated in regenerating endothelium after injury (5). It appears that Egfl7 expression is upregulated in response to hypoxic preconditioning in the premature brain (16), although the hypoxia-responsive elements have not been identified in the Egfl7 upstream promoter region. However, the roles of EGFL7 in hyperoxia-induced endothelial cell death and lung injury are unknown.

Using a hyperoxia-induced lung injury model of neonatal rat, which mimics some aspects of the pathology of BPD, we have demonstrated that Egfl7 expression is significantly decreased during the neonatal rat lung injury caused by hyperoxia. These observations suggest that a reduced level of EGFL7 is associated with endothelial cell death and hyperoxia-induced lung injury. Overexpression of human Egfl7 (hEgfl7) could prevent hyperoxia-induced endothelial cell death. Further elucidation of EGFL7 cytoprotective effects on endothelial cell death will enable us to understand the cellular and molecular pathogenesis of BPD and hyperoxia-induced lung injury.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oxygen exposure. The animal use was approved by the Institutional Animal Care and Use Committee, University of Missouri-Kansas City. The newborn rats at 4 days of age were randomly divided into two groups, room air (normoxia) and oxygen (hyperoxia) exposure groups according to our previous published procedure (34). The animals were housed in regular rat cages that were placed into Lucite chambers. The newborn rats in the chambers breathed either room air or humidified 95% oxygen. Oxygen concentration was monitored continuously with an oxygen analyzer. Dams were given food and water ad libitum, kept on a 12:12-h on-off light cycle, and fostered by rotating in and out of the chamber every 24 h to avoid oxygen toxicity. At the designated exposure time points, the animals from both treatment groups were killed by exsanguination after receiving intraperitoneal pentobarbital for anesthesia. Lung tissues from each group were collected, minced, and stored in liquid nitrogen for total RNA extraction.

Plasmid construction and transfection. For hEgfl7 plasmid construction, full-length hEgfl7 cDNA without stop codon was amplified from human umbilical vein endothelial cell (HUVEC) cDNA library by RT-PCR using the following primers: sense, 5'-CACAGGCCATGAGGGGCTCTCAGG-3'; antisense, 5'-CGAGTCTTTCTTGCAGGAGCAGG-3'. The resulting hEgfl7 cDNA was subcloned into plasmid vector pcDNA3.1 (Invitrogen, Carlsbad, CA). The hEgfl7 DNA sequence was confirmed by direct nucleotide sequencing. hEgfl7-pcDNA3 and empty Neo-pcDNA3 plasmids were transfected into EA.hy926 cells using Lipofectamine (Invitrogen). The transfected cells were then selected by G418 sulfate at 500 µg/ml for 10 days.

Cell culture and cell treatment. The human endothelial cell line EA.hy926 (a gift from Dr. C. J. Edgell, University of North Carolina at Chapel Hill; Ref. 10) was grown in DMEM containing 10% fetal bovine serum, 50 µg/ml penicillin, and 50 µg/ml streptomycin in 5% CO2 at 37°C. Normoxic exposure of the cells was conducted under room air and 5% CO2 in a humidified cell culture incubator at 37°C. Hyperoxic exposure of the cells was conducted in a humidified chamber (Billups-Rothenberg, Del Mar, CA), and the chamber was flushed with 95% O2-5% CO2 at a flow rate of 10 l/min for 15 min before incubation at 37°C.

Total RNA isolation, cDNA synthesis, and real-time PCR. Treated and nontreated endothelial cells were directly lysed in culture plates with TRIzol reagent from Invitrogen for total RNA isolation according to manufacturer's protocol. One microgram of total RNA was used for the first strand cDNA synthesis using oligo(dT) primers according to the first strand cDNA synthesis kit protocol from Invitrogen. Before cDNA synthesis, total RNA will be treated with RNase-free DNase I to eliminate any DNA contamination; non-reverse-transcription (without adding reverse-transcriptase in cDNA synthesis) samples were also included as negative controls. The resulting cDNAs were then used for the assessment of hEgfl7 gene expression using real-time PCR (iCycler, Bio-Rad). Specific primers used for hEgfl7 real-time PCR are: sense, 5'-TACACTCTGTGTGCCCAAGG-3'; antisense, 5'-CAGCCTCTGCACTTCTTCCT-3'. Rat Egfl7 (rEgfl7) primers are: sense, 5'-GAATGAAGGGAGTTGCATCC-3'; antisense, 5'-GACACCTGGCCTCTCCTGTA-3'. The real-time PCR was run with SYBR Green using two-step PCR protocol (95°C for 3 min or 95°C for 10 s and 55°C, 45 s) with melting curve. The threshold cycle was used to quantify the sample mRNA levels with housekeeping gene β-actin normalization.

LDH cytotoxicity assay. Lactate dehydrogenase (LDH) assay kit was from BioVision (Mountain View, CA), and LDH activity was measured per manufacturer's instruction. Briefly, cells were incubated in an incubator (5% CO2, 37°C) for the appropriate time of treatment. The cultured media were collected and saved. Adherent cells were washed with PBS and lysed with 1% Triton X-100 in 50 mM Tris·HCl, pH 7.5. Both cell-cultured media and cell lysates (100 µl per well) were carefully transferred into the corresponding wells of a 96-well plate. Reaction mixture (100 µl) was then added to each well and incubated for 30 min at room temperature. The absorbance of all samples at 490 nm was measured using a microplate reader. The cytotoxicity was determined by the percentage of LDH activity in cultured medium over combined LDH activities of the cultured medium and cell lysate.

Measurement of apoptotic cell death. Apoptosis detection kit was from R&D Systems (Minneapolis, MN). Treated cells were trypsinized and collected by centrifugation at 500 g for 5 min. Cells were washed with cold PBS once and resuspended in 100-µl binding buffer containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2. Cells were stained with annexin V-FITC (0.025 µg per sample) for 15 min according to manufacturer's instruction. The stained cells were subjected to flow cytometry analysis.

Western blotting analysis. Antibodies were purchased from Cell Signaling Technology (Beverly, MA) and used according to manufacturer's instructions. Cultured cells after treatment were washed with cold PBS three times, and then 300 µl of sample lysis buffer (62.5 mM Tris·HCl, pH 6.8, 2% wt/vol SDS, 10% glycerol, 200 mM dithiothreitol, and protease cocktails) was added to each plate. Cell lysates were centrifuged at 12,000 g for 10 min. The supernatants were saved for analysis. To prepare samples for cytochrome c Western blotting, the treated cells were lysed with 100 µl of 10 mM Tris·HCl buffer containing 250 mM sucrose and the protease cocktails for 20 min at 4°C. The supernatants were used for the analysis. Protein concentration was determined by BCA protein assay kit (Sigma, St. Louis, MO). Samples in loading sample buffer were boiled for 5 min and loaded on Tris-glycine SDS-PAGE gels. Gels were run at 120 V for ~2 h and transferred overnight at 20 V to nitrocellulose membranes. Membranes were incubated with the blocking buffer containing 5% nonfat milk in PBST (0.1% Tween-20 in PBS) for 1 h, washed with PBST, and incubated overnight with the primary antibody. The membranes were washed in PBST, and proteins were visualized using horseradish peroxidase-conjugated secondary anti-IgG and the enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). The membranes were stripped using a standard stripping solution (62.5 mM Tris·HCl, pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol) at 50°C and reprobed with β-actin antibody. Protein band intensities on autoradiogram were analyzed with AlphaImager (Innotech, San Leandro, CA) and normalized by β-actin in the same sample.

Measurement of cytochrome c release. Cytochrome c release was measured by Western blotting (28). Treated cells (~0.6–1.0 x 107 cells) were collected and washed twice in ice-cold PBS, resuspended, and lysed in buffer containing 10 mM Tris·HCl, pH 7.5, 10 mM KCl, 1.9 mM MgCl2, 1 mM EGTA, 1 mM EDTA, and protease cocktail inhibitors for 20 min at 4°C. The cell lysates were spun at 20,000 g for 30 min, and the supernatants were saved for cytochrome c Western blotting analysis. Cytochrome c antibody was purchased from Cell Signaling Technology (Beverly, MA).

Statistical analysis. The results are expressed as the means ± SE of data obtained from two or more experiments or, where appropriate, as means ± SD. Statistical analysis was performed using Student's t-test for paired comparisons and ANOVA for multiple comparisons. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hyperoxia reduces Egfl7 gene expression in the neonatal rat lungs and cultured HUVEC endothelial cells. We measured rEgfl7 mRNA expression by real-time PCR in the neonatal rat lungs during normoxia (room air) and hyperoxia (95% O2). rEgfl7 mRNA expression in the neonatal rat lungs was significantly decreased when exposed to hyperoxia for 3–10 days. The rEgfl7 reduction in the hyperoxic group was ~60% compared with the normoxic group (Fig. 1A; P < 0.001; n = 9). The expression of rEgfl7 mRNA in the hyperoxic group returned to near normal levels 2 wk after discontinuing hyperoxic exposure. In cultured HUVEC endothelial cells, when we measured hEgfl7 mRNA expression by real-time PCR in cells exposed to normoxia or hyperoxia for 48 h, we found that prolonged hyperoxia for 48 h significantly decreased hEgfl7 mRNA expression by fivefold (Fig. 1B; P < 0.001; n = 5).


Figure 1
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  		Fig. 1. A and B: effect of hyperoxia on epidermal growth factor-like domain 7 Egfl7 gene mRNA expression in the neonatal rat lungs and human umbilical vein endothelial cells (HUVEC). A: neonatal rats at 4 days of age were treated with room air (normoxia) or 95% oxygen (hyperoxia) for 3–10 days (n = 9). After 10-day room air or 95% oxygen exposure, neonatal rats were kept in room air to recover for 2–8 wk (n = 8). Total RNA was isolated from the neonatal rat lung at each time point. Rat Egfl7 (rEgfl7) mRNA expression was measured by real-time PCR. Results are expressed as means ± SE. *P < 0.001 compared with time-matched normoxic group. B: HUVEC cells were treated with room air (normoxia) or 95% oxygen (hyperoxia) for 48 h. Human Egfl7 (hEgfl7) mRNA expression was measured by real-time PCR. Results are expressed as means ± SE. *P < 0.001 compared with time-matched normoxic group (n = 5).

 
Overexpression of hEgfl7 gene in EA.hy926 endothelial cell line.

We cloned hEgfl7 cDNA from the HUVEC cDNA library, and hEgfl7 cDNA was then subcloned into pcDNA3 expression vector with a V5 tag. The resulting hEgfl7-pcDNA3 and Neo-pcDNA plasmids were transfected into EA.hy926 endothelial cells. Neo-EA.hy926 and hEgfl7-EA.hy926 stable cell lines were established after G418 selection. The level of hEgfl7 mRNA in hEgfl7-EA.hy926 cells was nearly twice as much as that in Neo-EA.hy926 cells (Fig. 2A). The overexpressed hEGFL7-V5 fusion protein could be detected by an anti-V5 antibody in hEgfl7-EA.hy926 cell lysates (Fig. 2B). We also measured the secreted hEGFL7-V5 fusion protein in the media of cultured Neo-EA.hy926 and hEgfl7-EA.hy926 cells by Western blotting analysis with an anti-V5 antibody. hEGFL7-V5 fusion protein secreted into cell culture medium was detectable in hEgfl7-EA.hy926 cells but not in Neo-EA.hy926 cells (Fig. 2C). There were no significant differences of cell morphology between Neo-EA.hy926 cell line and hEgfl7-EA.hy926 cell line when observed under light microscopy (Fig. 2D).


Figure 2
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  		Fig. 2. A–D: hEgfl7 overexpression in EA.hy926 endothelial cells. A: hEgfl7 mRNA expression in Neo-EA.hy926 (control) and hEgfl7-EA.hy926 cells was measured by real-time PCR. B: cell lysates from Neo-EA.hy926 (control) and hEgfl7-EA.hy926 cells were analyzed by Western blotting with an anti-V5 antibody. C: cell culture media (900 µl) from Neo-EA.hy926 (control) or hEgfl7-EA.hy926 cells were precipitated with 10% TCA, and the precipitated proteins were analyzed by Western blotting with an anti-V5 antibody. D: the morphologies of Neo-EA.hy926 and hEgfl7-EA.hy926 cells; contrast phase, 100x.

 
Overexpression of hEgfl7 attenuates hyperoxia-induced endothelial cell death. We treated Neo-EA.hy926 and hEgfl7-EA.hy926 cells with normoxia (room air) or hyperoxia (95% O2) for 48 h. The treated cells were measured for necrotic cell death by LDH release assay and apoptotic cell death by annexin V staining. Hyperoxia significantly increased necrotic cell death in both Neo-EA.hy926 and hEgfl7-EA.hy926 cells. The necrotic cell death was increased from 7.4% to 34.0% in Neo-EA.hy926 cells (Fig. 3A; n = 6; P < 0.001) and from 6.2% to 23.9% in hEgfl7-EA.hy926 cells (Fig. 3A; n = 6; P < 0.001) after 48-h hyperoxic exposure. Compared with Neo-EA.hy926 cells, the necrotic cell death was significantly reduced in hEgfl7-EA.hy926 cells after 48-h hyperoxic exposure (Fig. 3A; n = 6; P < 0.001). Hyperoxia also significantly induced apoptotic cell death in both Neo-EA.hy926 and hEgfl7-EA.hy926 cells (Fig. 3B; n = 6; P < 0.001). Apoptotic cell death was increased from 0.9% in normoxia-treated Neo-EA.hy926 cells to 10.6% in hyperoxia-treated Neo-EA.hy926 cells and from 0.9% in normoxia-treated hEgfl7-EA.hy926 cells to 6.7% in hyperoxia-treated hEgfl7-EA.hy926 cells. Compared with hyperoxia-treated Neo-EA.hy926 cells, the apoptotic cell death was significantly reduced in hyperoxia-treated hEgfl7-EA.hy926 cells (Fig. 3B; n = 6; P < 0.001).


Figure 3
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  		Fig. 3. hEgfl7 overexpression reduces hyperoxia-induced necrosis and apoptosis. A: cultured Neo-EA.hy926 and hEgfl7-EA.hy926 cells in 6-well plates were treated with room air (normoxia) or 95% oxygen (hyperoxia) for 48 h. The cytotoxicity was determined by lactate dehydrogenase release (n = 6). B: cultured Neo-EA.hy926 and hEgfl7-EA.hy926 cells in 6-well plates were treated with room air (normoxia) or 95% oxygen (hyperoxia) for 48 h. Treated cells were stained with annexin V-FITC, and fluorescence was measured by flow cytometry (n = 6).

 
Overexpression of hEgfl7 partially blocks cytochrome c release from mitochondria and attenuates caspase-3 activation. We treated Neo-EA.hy926 and hEgfl7-EA.hy926 cells with normoxia or hyperoxia for 24 and 48 h and then measured cytochrome c release and caspase-3 activation. Western blotting analysis showed that cytochrome c release was increased by 1.4-fold in Neo-EA.hy926 after 48-h hyperoxic exposure compared with Neo-EA.hy926 cells after 48-h normoxic exposure (Fig. 4, A and B). But the cytochrome c release in hEgfl7-EA.hy926 cells after 48-h normoxic or hyperoxic exposure remained unchanged or even lower compared with Neo-EA.hy926 cells under the same conditions (Fig. 4, A and B). Caspase-3 activation/cleavage was observed in both Neo-EA.hy926 and hEgfl7-EA.hy926 cells after 48-h hyperoxic exposure (Fig. 5A). It was increased by 2.1-fold in Neo-EA.hy926 cells and 1.3-fold in hEgfl7-EA.hy926 cells after 48-h hyperoxic exposure. The caspase-3 activation in hEgfl7-EA.hy926 cells was much lower than that in Neo-EA.hy926 cells (Fig. 5B).


Figure 4
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  		Fig. 4. hEgfl7 overexpression suppresses hyperoxia-induced cytochrome c (Cyt c) release. A: a representative Western blot analysis for cytosolic cytochrome c from 2 independent experiments after 48-h normoxic or hyperoxic exposure in Neo-EA.hy926 and hEgfl7-EA.hy926 cells. N, normoxia; H, hyperoxia. B: the levels of cytosolic cytochrome c from 2 independent experiments after 48-h hypoxic exposure in Neo-EA.hy926 and hEgfl7-EA.hy926 cells were quantified by densitometry and normalized by β-actin. Data are expressed as means ± SD.

 

Figure 5
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  		Fig. 5. hEgfl7 overexpression reduces hyperoxia-induced caspase-3 activation. A: a representative Western blot analysis for cleaved caspase-3 from 2 independent experiments. B: the levels of cleaved caspase-3 from 2 independent experiments after 48-h normoxic or hyperoxic exposure in Neo-EA.hy926 and hEgfl7-EA.hy926 cells were quantified by densitometry and normalized by total caspase-3 and β-actin. Data are expressed as means ± SD.

 
Overexpression of hEgfl7 decreases Bax expression but increases Bcl-xL expression. The level of Bax protein was increased by ~1.7-fold in Neo-EA.hy926 cells compared with hEgfl7-EA.hy926 cells after 48-h hyperoxic exposure. The level of Bax protein in hEgfl7-EA.hy926 cells after 24- and 48-h hyperoxic exposure was not significantly altered (Fig. 6, A and B). However, the level of Bcl-xL protein was elevated in hEgfl7-EA.hy926 cells after either normoxic or hyperoxic exposure for 48 h compared with Neo-EA.hy926 cells under the same conditions (Fig. 7, A and B). Bcl-xL level was increased by 1.56-fold in hEgfl7-EA.hy926 cells compared with Neo-EA.hy926 cells after 48-h normoxic exposure. Hyperoxia exposure increased Bcl-xL levels in both Neo-EA.hy926 and hEgfl7-EA.hy926 cells, but the level of Bcl-xL in hEgfl7-EA.hy926 cells was higher than that in Neo-EA.hy926 cells (Fig. 7, A and B).


Figure 6
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  		Fig. 6. hEgfl7 overexpression reduces proapoptotic protein, Bax. A: a representative Western blotting analysis for Bax from 2 independent experiments. B: the levels of Bax from 2 independent experiments after 48-h normoxic or hyperoxic exposure in Neo-EA.hy926 and hEgfl7-EA.hy926 cells were quantified by densitometry and normalized by β-actin. Data are expressed as means ± SD.

 

Figure 7
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  		Fig. 7. hEgfl7 overexpression elevates antiapoptotic protein, Bcl-xL. A: a representative Western blot analysis for the antiapoptotic protein, Bcl-xL, from 3 independent experiments. B: the levels of Bcl-xL from 2 independent experiments after 48-h normoxic or hyperoxic exposure in Neo-EA.hy926 and hEgfl7-EA.hy926 cells were quantified by densitometry and normalized by β-actin. Data are expressed as means ± SD.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we found that hyperoxia downregulated Egfl7 expression in the neonatal rat lungs and the cultured endothelial cells. Overexpression of hEgfl7 protected the endothelial cells from hyperoxia-induced cell death. hEgfl7 upregulated an antiapoptotic protein, Bcl-xL, and downregulated a proapoptotic protein, Bax. Overexpression of hEgfl7 partially blocked cytochrome c release from mitochondria and suppressed activation of caspase-3, suggesting the cytoprotective effects of EGFL7 may be due to inhibition of mitochondria-dependent, intrinsic apoptotic pathway.

EGFL7 may play a specific role in the crucial steps of vasculogenesis and angiogenesis (5, 25). It has been shown that loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis (25, 29). In the current study, we observed that Egfl7 expression was decreased significantly in the neonatal rat lungs and cultured endothelial cells after hyperoxic treatment, suggesting decreased Egfl7 expression is associated with hyperoxia-induced lung injury. EGFL7 is a secreted protein from endothelial cells (12, 20). Expression of the Egfl7 gene can be detected at the early stage of vasculogenesis, which implies that EGFL7 could be an early endothelial marker and might be essential for the establishment of the vasculature (5, 30). Recombinant EGFL7 purified from the conditioned medium inhibits PDGF-induced aortic smooth muscle cell migration, but it has no effect on smooth muscle cell proliferation (30). EGFL7 does not affect endothelial cell proliferation (25). EGFL7 appears to induce endothelial cell migration (5). Immunofluorescence staining has shown that EGFL7 protein is secreted from endothelial cells and remained in the vicinity of the endothelial cells, suggesting that EGFL7 protein is an autocrine/paracine factor and associated with the cell membrane receptor or extracellular matrix (25, 29). We overexpressed hEgfl7 in EA.hy926 endothelial cell line. The overexpressed hEgfl7 gene and hEGFL7 protein were detectable in hEgfl7-EA.hy926 endothelial cell line by real-time PCR and Western blotting analysis, respectively. Western blotting analysis also showed that the secreted hEGFL7 protein was present in the cultured hEgfl7-EA.hy926 cell medium. It reported that EGFL7 recombinant protein was difficult to be purified with biological activity (4). The hEgfl7-EA.hy926 cell line we created provided us an ideal cell model to study EGFL7 functions in the endothelial cells.

Exposure to high levels of inspired oxygen (hyperoxia) could lead to respiratory failure and death (1a, 11). Endothelial cell death occurs early after hyperoxic injury and before the onset of respiratory failure. Endothelial cell death and repair play important roles in the pathogenesis of the oxidative stress-related diseases such as BPD and acute respiratory distress syndrome (ARDS). In our cell culture model, we found that overexpression of hEgfl7 in EA.hy926 cell line protected the endothelial cells from hyperoxia-induced cell death and increased cell viability. hEgfl7 reduced the level of a proapoptotic protein, Bax, but increased the level of an antiapoptotic protein, Bcl-xL in the endothelial cells. The decreased Bax and elevated Bcl-xL probably lead to maintain mitochondrial permeability potential, to decrease cytochrome c release, to inhibit caspase-3 activation, and eventually to attenuate apoptotic cell death in the endothelial cells. There are two apoptosis pathways, extrinsic and intrinsic, in endothelial cells (9, 17). Both pathways require a cascade of caspase activation for executions of apoptosis. We did not observe the overexpression of hEgfl7-altered caspase-8 activation that is an indication of activating extrinsic apoptosis pathway. The cytochrome c release and caspase-3 activation were significantly inhibited by overexpression of hEgfl7 in the EA.hy926 endothelial cells after hyperoxic exposure. Taken together, the cytoprotective effect of hEGFL7 might suppress mitochondria-dependent, intrinsic apoptotic pathway and protect endothelial cells against hyperoxia-induced apoptosis. It has been reported that endothelial cell apoptosis induced by hyperoxia is associated with decreased angiogenic growth factor such as VEGF (8, 20, 35). The decreased VEGF level after hyperoxic exposure is associated with the activation of caspase-3 and increase of endothelial cell apoptosis. VEGF overexpression or treatment suppresses hyperoxia-induced apoptosis in the endothelial cells (21, 33), which may be through the activation of phosphatidylinositol 3-kinase-Akt and ERK-MAPK signaling pathways and the inhibition of p38 MAPK signaling pathway (13–15, 22). Western blotting analysis of VEGF protein expression in Neo-EA.hy926 and hEgfl7-EA.hy926 cells under either normoxic or hyperoxic conditions showed that hyperoxia decreased VEGF expression that is consistent with previously reported data (8, 20, 35). However, hEgfl7 overexpression did not influence the level of VEGF under either normoxic or hyperoxic conditions (unpublished data). It is unclear whether overexpression of VEGF would affect the levels of EGFL7 in vivo and in vitro. It is possible that EGFL7 is regulated by VEGF because the expression of VEGF is downregulated under hyperoxic condition as well.

Endothelial cells are heterogeneous among different vascular beds in addition to the common features such as capillary formation and platelet endothelial cell adhesion molecule expression on cell surface. For example, pulmonary endothelial cell is very distinct compared with the endothelial cells from other vascular beds because of its unique function in gas exchange. EA.hy926 permanent endothelial cell line is a hybrid cell line from HUVEC and human lung adenocarcinoma cell line (A549; Ref. 10). EA.hy926 endothelial cell line expresses factor VIII-related antigen (von Willebrand factor) with the same morphological distribution as in primary HUVEC. In the present study, we found that hyperoxia decreased Egfl7 expression in the neonatal rat lungs, which is probably due to the decreased EGFL7 expression in pulmonary endothelial cells because EGFL7 is only expressed and secreted from endothelial cells (25, 29, 30). We also found that hyperoxia downregulated hEgfl7 expression in primary HUVEC. Therefore, we utilized EA.hy926 cells to overexpress hEgfl7 for our studies. EA.hy926 cell line has been previously used for a variety of vascular endothelial cell studies, including investigations in endothelial cell death (2, 18, 23, 31).

In summary, EGFL7 is a secreted protein from vascular endothelial cells. Hyperoxia reduces Egfl7 expression in vitro and in vivo. Overexpression of hEgfl7 protects the endothelial cells from hyperoxia-induced cell death. The cytoprotective effect of hEGFL7 is mediated by suppressing the mitochondria-dependent, intrinsic apoptosis pathway.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by Clinical Scholar Award and Katherine B. Richardson Grants from Children's Mercy Hospital (DX) and, in part, by National Heart, Lung, and Blood Institute Grant R01-HL-70560 (W. E. Truog).


   ACKNOWLEDGMENTS
 
We thank Dr. D. Zwick for expertise in apoptotic cell death assay (the clinical and research flow-cytometry core facility at Children's Mercy Hospital). We thank Dr. T. Finkel (the cardiology branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD) for comments and Dr. C. J. Edgell (Pathology, University of North Carolina at Chapel Hill) for providing EA.hy926 cell line. We thank R. Morgan, M. Rezaiekhaligh, and S. Mabry for their technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Xu, Neonatology Research Laboratory, Children's Mercy Hospital, Pediatric Research Center, 4th Floor, 2401 Gillham Rd., Kansas City, MO 64108 (e-mail: xud{at}umkc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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