VEGF regulates remodeling during permanent anatomic closure of the ductus arteriosus

¹ Cardiovascular Research Institute and ² Department of Pediatrics, University of California, San Francisco, California 94143; ³ Department of Pediatrics, University of Texas Health Science Center and the Southwest Foundation for Biomedical Research, San Antonio, Texas 78284; ⁴ Department of Radiation Oncology, University of Pennsylvania, Philadelphia, Pennsylvania 19104; ⁵ Genentech, South San Francisco, California 94080; ⁶ SRI International, Menlo Park, California 78284; and ⁷ Department of Pediatrics, University of New Mexico, Albuquerque, New Mexico 87131

	ABSTRACT

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Anatomic remodeling and permanent closure of the newborn ductus arteriosus appears to require the development of intense hypoxia within the constricted vessel wall. Hypoxic ductus smooth muscle cells express vascular endothelial cell growth factor (VEGF). We studied premature baboons and sheep to determine the effects of VEGF inhibition (in baboons) and VEGF stimulation (in sheep) on ductus remodeling in vivo. For study of VEGF inhibition, 13 premature newborn baboons (68% gestation) were treated with inhibitors of both prostaglandin and nitric oxide production to constrict the ductus and induce ductus wall hypoxia. Six received a neutralizing monoclonal antibody against VEGF (A.4.6.1, mAbVEGF), while seven did not. Both groups developed the same degree of ductus constriction, tissue hypoxia, and VEGF expression. The mAbVEGF treatment produced a significant (P < 0.05) reduction in ductus vasa vasorum ingrowth and neointima formation (due to both a decrease in luminal endothelial cell proliferation and a decrease in smooth muscle cell migration into the neointima). For study of VEGF stimulation, nine sheep fetuses (70% gestation) had their ductus wall injected with either VEGF (n = 6) or vehicle (n = 4) in vivo. VEGF administration produced a significant (P < 0.05) increase in vasa vasorum ingrowth and neointima formation. We conclude that VEGF plays an important role in the formation of neointimal mounds and vasa vasorum ingrowth during permanent ductus closure.

nitric oxide; neointima; hypoxia; vasa vasorum

	INTRODUCTION

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IN THE FULL-TERM INFANT, closure of the ductus arteriosus (DA) occurs in two phases: first, smooth muscle cell constriction produces a functional closure of the DA lumen; this is followed by permanent anatomic occlusion of the lumen due to extensive neointimal thickening and loss of smooth muscle cells from the inner muscle media (6). The initial functional constriction appears to be responsible for the ultimate anatomic closure of the DA. Loss of luminal blood flow produces a zone of hypoxia in the DA's muscle media that, depending on its severity, induces the following anatomic changes: angiogenesis, neointima formation, and cell death (6). Hypoxia of the vessel wall appears to be the required stimulus for anatomic remodeling. Failure to develop hypoxia of the muscle media leads to failure of DA remodeling and subsequent DA reopening despite the initial constriction (6, 30). The preterm newborn is also capable of remodeling its DA, just like the full-term newborn, if it can develop the same degree of hypoxia as found at term (19, 32).

The mechanism(s) by which hypoxia induces the changes that occur during anatomic remodeling is currently unknown. Hypoxic vascular tissue can express increased amounts of fibronectin (11) and vascular endothelial cell growth factor (VEGF; Ref. 4). Inhibition of fibronectin production has recently been shown to inhibit ductus remodeling (29).

A consistent finding during the development of ductus wall hypoxia is the early appearance of VEGF in the muscle media of the full-term and preterm ductus (6, 19, 32). VEGF is a hypoxia-induced growth factor (4, 26) that can stimulate endothelial cell proliferation and migration as well as fluid and protein extravasation from blood vessels (20, 25). In the following experiments we studied premature sheep and baboons to identify the effects of both VEGF stimulation and VEGF inhibition on DA remodeling in vivo. We hypothesized that VEGF may play an important role in producing the neointimal expansion and vasa vasorum ingrowth that occur after the development of hypoxia in the ductus wall.

	METHODS

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In vivo study of preterm baboons: VEGF inhibition. All studies were approved by the Committees on Animal Research at the University of California San Francisco, the University of Texas Health Science Center, and the Southwest Foundation for Biomedical Research.

We used preterm newborn baboons (Papio sp.) to examine the effects of VEGF inhibition on ductus remodeling. Animal care, surgery, and necropsy were performed as previously described (6, 32). Preterm newborn baboons (n = 29) were delivered by cesarean section at 125 days gestation (68% of full term) and were euthanized on the 6th day after delivery.

Preterm newborn baboons fail to constrict their DA unless they are treated with inhibitors of both prostaglandin and nitric oxide production (32). Therefore, preterm newborn baboons were randomly assigned to one of three treatment protocols: group 1) indomethacin (Indo) plus N^G-nitro-L-arginine (L-NNA), a nitric oxide synthase (NOS) inhibitor (Indo + L-NNA, n = 7); group 2) Indo plus L-NNA plus a neutralizing monoclonal antibody (mAb) made against human VEGF (mAbVEGF) (A.4.6.1; Refs. 3 and 21) (Indo + L-NNA + mAb, n = 6); group 3) no treatment (control, n = 16). Indo (Indocin, 0.1 mg · kg¹ · dose¹) was given intravenously at 24, 48, 72, 84, 96, 108, 120, and 132 h after delivery to animals in group 1 (Indo + L-NNA) and group 2 (Indo + L-NNA + mAb). A continuous infusion of L-NNA (Sigma) (6 mg · kg¹ · h¹) was given to the same two groups starting at 50 h after delivery. A.4.6.1 was administered as a single intravenous dose (10 mg/kg) 24 h after delivery. A.4.6.1 has a plasma half-life of 2 wk (3, 21). The animals in groups 1 and 3 have been described previously (32).

At necropsy, the DA was dissected in 4°C phosphate-buffered saline solution (D-PBS). The minimal luminal diameter was measured, and the DA was embedded in Tissuetek (Miles) and frozen in liquid nitrogen or was embedded in paraffin (after fixation with 4% paraformaldehyde).

Most of the findings for animals in the control and Indo + L-NNA groups have been reported previously (32). We are including these data in the current report so that the changes that occurred in the Indo + L-NNA + mAb group can be more easily compared.

Detection of hypoxia with EF5. To detect regions of hypoxia within the DA, we used the 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) detection system that we have described previously (6, 18, 19, 32). Premature baboons were given EF5 (10⁴ mol/kg iv) 36 h before necropsy. Blood samples were collected and analyzed for EF5 as previously described (6). We used mAb (ELK 3-51), which is highly specific for EF5 and its tissue adducts, to detect the presence of bound EF5 in the tissue (6, 23). EF5 binding was corrected for the in vivo EF5 drug exposure and was expressed as a percentage of the calculated maximal expected binding (23, 32).

Vasa vasorum perfusion: intravenous Hoechst dye technique. The DNA-binding dye, Hoechst (no. 33342)-bisbenzimide (Sigma) (20 mg/kg iv), was administered to the baboons 15 min before necropsy (32). This fluorescent dye readily intercalates into cellular DNA and can be used to demonstrate regions of the DA that were perfused and had access to the dye (as measured by Hoechst-bisbenzimide uptake) just before necropsy (32).

In vivo study of preterm fetal lambs: VEGF addition. We used preterm fetal lambs (mixed Western breed: 105 ± 2 days gestation, 70% of term) to examine the effects of VEGF addition on DA remodeling. A midline laparotomy was performed on the ewe, and the DA was visualized through a fetal thoracotomy. A 10-µl aliquot of control solution [sheep albumin, 1 mg/ml D-PBS, plus Accuprime ink (Beckman, Schiller Park, IL)] or the same solution containing 100 nM recombinant human VEGF₁₆₅ (R&D Systems, Minneapolis, MN; Sf21 insect cell expression system) was injected into four contiguous sites along the exposed/ipsilateral wall of the DA. The fetal thoracotomy was closed and the fetus was returned to the uterus. The fetal DA was removed 72 h later for immunohistochemical analysis.

Immunohistochemistry. Protocols for the immunohistochemistry of endothelial cell NOS (eNOS), CD-31, proliferating cell nuclear antigen (PCNA), VEGF, -smooth muscle actin, and EF5 were similar to those reported previously (7, 32). Endothelial cells were detected with either anti-eNOS (clone 3, Transduction Lab, Lexington, KY) or anti-CD-31 (R&D Systems). We present only the eNOS staining results because sequential sections stained for CD-31 had identical findings (data not shown).

Scoring for VEGF immunostaining (Santa Cruz Biotechnology, Santa Cruz, CA) was as follows: 0, none; 1+, mild; 2+, moderate. All sections were stained in the same assay as previously reported (32).

Histological measurements in the newborn baboon DA were made at the level of minimal luminal area, which was determined from 6-µm serial sections. Tissue dimensions were determined by averaging measurements made from eight predetermined regions of the section, using an overlay template and NIH Image software. The neointimal zone was defined as the region between the luminal endothelial cells and the internal elastic lamina (identified by phase-contrast microscopy).

In the fetal baboon (125 days gestation), the thickness of the DA wall (distance from the luminal endothelium to the adventitia) is only 470 ± 16 µm (6), and vasa vasorum are present only in the adventitia (6, 19). After birth, vasa vasorum invade the muscle media (6, 19). To determine the depth of vasa vasorum ingrowth in the preterm baboon, we partitioned the DA wall into concentric regions on the basis of the percentage of wall thickness between the luminal endothelium and adventitia. Vessel ingrowth was based on the innermost region of the wall that contained at least one vasa vasorum (8).

In contrast to the fetal baboon, the thickness of the fetal sheep DA (100 days gestation) is 630 ± 78 µm, and vasa vasorum are normally present in both the adventitia and outer half of the muscle media. To determine the effect of VEGF injections on vasa vasorum proliferation within the fetal sheep DA, we measured the vasa vasorum area within predetermined regions of the outer half of the DA muscle media. Endothelial cells lining the individual vasa were outlined, and the area contained within each vasa was measured. The total vasa vasorum area contained in three contiguous ×40 fields of outer muscle media was recorded for each side of the DA wall. The total vasa vasorum area of the injected side of the DA wall was compared with the noninjected, contralateral side of the DA as a ratio. The vasa vasorum ratio (injected/contralateral) for each DA was the average ratio determined from three sections obtained at intervals of 36 µm. We compared the ratios calculated from the VEGF-injected DA with those obtained from control-injected DA.

Cell death. We used the TdT-mediated dUTP nick end labeling (TUNEL) technique to detect cells in the early stages of DNA fragmentation and cell death as we have described previously (6). The number of TUNEL-positive nuclei per 100 nuclei was scored in the region of EF5 staining.

In vitro study of DA endothelial and smooth muscle cells: hypoxia vs. VEGF. We used endothelial cells and smooth muscle cells, isolated from 100-day-gestation fetal lamb DA, to determine the effects of different degrees of hypoxia on VEGF production. The isolated cells were grown in monolayer culture and passaged as previously described (18). The dishes containing the cells were incubated with different O₂ concentrations for 4 h. After RNA isolation, Northern blotting was performed with a ³²P-labeled cDNA probe specific for sheep VEGF (22, 34). To determine the level of hypoxia in the endothelial and smooth muscle cells, we measured the binding of EF5 to the isolated cells using an immunoblot assay as previously described (18, 19).

Statistics. Results are presented as means ± SD, percentages, and correlation coefficients. Intergroup differences were evaluated with either a chi-square analysis or unpaired t-test. When more than one comparison was made, Bonferroni's correction was used.

	RESULTS

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In vitro study of DA endothelial and smooth muscle cells: hypoxia and VEGF mRNA. VEGF mRNA expression in DA endothelial and smooth muscle cells was increased within 2 h of exposure to hypoxia; however, maximal expression was not achieved until 4 h of hypoxia (data not shown). DA smooth muscle cells had a substantial increase in VEGF mRNA after even mild degrees of hypoxia (3% O₂). In contrast, VEGF mRNA was only mildly increased in endothelial cells during hypoxia (Fig. 1).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effects of hypoxia on vascular endothelial cell growth factor (VEGF) mRNA expression and 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) binding in ductus arteriosis (DA) endothelial and smooth muscle cells. For VEGF mRNA studies (bottom), cells were incubated with different O2 concentrations for 4 h before RNA isolation and Northern blotting. The intensities of the hybridized bands were normalized to the amount of 28S RNA. Similar results were obtained in 2 additional experiments. For EF5 binding studies (top), cells were incubated with 104 M EF5 for 3 h at the indicated O2 concentrations before immunoblot assay. EF5 values were expressed as a percentage of maximal binding at the lowest O2 concentration, 0.01%.

Premature newborn baboons. Combined treatment of preterm baboons with Indo and L-NNA produced a much greater degree of DA constriction than baboons receiving no treatment. A neutralizing anti-VEGF antibody was given to some baboons to test the hypothesis that VEGF plays an important role during the neointimal expansion and vasa vasorum ingrowth that occur after ductus constriction and hypoxia. As anticipated, administration of the neutralizing mAbVEGF did not alter the rate of DA constriction [as measured by daily pulsed-Doppler exams (data not shown)] or the final degree of DA constriction at necropsy (Fig. 2A), nor did it alter the development of hypoxia and cell death in the DA wall (see below).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of DA treatment on DA diameter at necropsy (A) and VEGF expression (B). Units of VEGF (0, 1+, 2+) refer to intensity of immunostaining. Numbers in parentheses are number of animals. * P < 0.05 vs. control group. Data for control and the indomethacin (Indo) + NG-nitro-L-arginine (L-NNA) groups have been taken from Ref. 32. mAb, monoclonal antibody against VEGF.

We have shown previously that DA wall hypoxia (measured by EF5 binding) depends on the degree of luminal constriction [r = 0.98, log (EF5 binding expressed as a percentage of maximal EF5 binding) vs. log (luminal area)]. Similarly, cell death in the DA wall (measured by the number of TUNEL-positive cells) depends on the degree of luminal constriction and EF5 binding (r = 0.99) (32). DA from the control animals had negligible EF5 binding (median = 1%; range = 1-2% of maximal binding) (Fig. 3) and no TUNEL-positive nuclei (0 TUNEL-positive nuclei/100 cells, n = 3). In contrast, DA from newborns treated with Indo + L-NNA (either with or without mAbVEGF) had intense EF5 binding (median = 33%; range = 13-115% of maximal binding) (Fig. 3) and a moderate number of TUNEL-positive nuclei (median = 11 positive nuclei/100 cells; range = 3-69). The use of mAbVEGF did not alter the relationship between the rate of EF5 binding and the degree of luminal constriction, nor did it alter the relationship between the number of TUNEL-positive cells and the intensity of EF5 binding.

View larger version (109K): [in this window] [in a new window]   Fig. 3.   Hypoxia, neointimal formation, and vasa vasorum ingrowth in DA from 3 different treatment groups. EF5 (top): 10-µm sections were stained with Cy3-conjugated ELK 3-51 and photographed at the same magnification. Dashed white line represents outer edge of DA muscle media. Bar, 500 µm. Lumen (middle): endothelial nitric oxide synthase (eNOS) = brown stain. Cell nuclei counterstained with hematoxylin. Dashed black line represents internal elastic lamina (identified by phase-contrast microscopy). Bars, 50 and 100 µm. Vasa vasorum (bottom): eNOS stains vasa vasorum. Bar, 100 µm.

DA from baboons treated with Indo + L-NNA expressed moderate levels of VEGF in the region of muscle media that had intense EF5 binding (Fig. 2B). Administration of the neutralizing mAbVEGF did not affect the detection of VEGF in the newborns treated with Indo + L-NNA + mAb.

DA from the premature baboons treated with Indo + L-NNA developed the same anatomic changes that have been described previously, after ductus constriction and hypoxia (6). DA from the premature baboons treated with Indo + L-NNA developed a significantly thicker neointima than DA from the control baboons (Figs. 3 and 4C). The neointima was composed of proliferating mounds of endothelial cells (Figs. 3 and 4, B and C) and an expanding layer of nonendothelial cells (Figs. 3 and 4C) [that expressed -smooth muscle actin (data not shown)]. The mAbVEGF inhibited both the endothelial and nonendothelial increase in neointimal thickness. We examined the rate of luminal endothelial proliferation by monitoring the number of PCNA-positive cells that lined the DA lumen (Fig. 4). DA from the baboons treated with Indo + L-NNA had an increased number of PCNA-positive cells compared with DA from the control animals; mAbVEGF reduced the number of PCNA-positive cells (Fig. 4A) and partially reduced the thickness of the luminal endothelial mounds (Fig. 4, B and C). The mAbVEGF also completely blocked the influx of nonendothelial cells into the neointima (Fig. 4C).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of DA treatment on measurements of DA endothelial proliferation (A), luminal endothelial accumulation (B), neointimal thickness (C), and vasa vasorum ingrowth (D). A: proliferating cell nuclear antigen (PCNA), expressed as positive cells per 100 cells lining the DA lumen. B: luminal endothelium study. DA were categorized as having either a single or double layer of eNOS-positive luminal cells or a lumen filled with multiple layers (plug). C: neointimal thickness (mean ± SD). Total thickness is distance between lumen and internal elastic lamina; endothelial thickness is width of cell layer that stains for eNOS; nonendothelial thickness is difference between total and endothelial thicknesses. D: vasa vasorum ingrowth. Four separate, random sections from the middle of the DA were stained for eNOS to measure vessel ingrowth (see METHODS). * P < 0.05 vs. control group; # P < 0.05, Indo + L-NNA group vs. Indo + L-NNA + mAbVEGF group. Data in A-D belonging to control and Indo + L-NNA groups are taken from Ref. 32 with minor modifications.

After birth, vasa vasorum invade the muscle media of the preterm baboon DA (Figs. 3 and 4D). The baboons treated with Indo + L-NNA had a much greater ingrowth of vasa vasorum than those in the control group. The mAbVEGF decreased vasa vasorum invasion to control values (Fig. 4D). Similarly, mAbVEGF decreased vasa vasorum perfusion of the outer muscle media (as measured by Hoechst-bisbenzimide uptake) (Fig. 5).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Nuclear staining of cells in the outer third of DA muscle media after intravenous administration of Hoechst-bisbenzimide dye. Horizontal bar in A, 500 µm. The DA wall was partitioned into 3 concentric regions based on the percentage of wall thickness between the luminal endothelium and adventitia. More than 200 nuclei in the outer third of the muscle media were counted in each of 2 sections from a single DA. The number of bisbenzimide stained nuclei/100 nuclei counted was expressed as a percentage (B). * P < 0.05 vs. Indo + L-NNA group.

Premature fetal lambs. Fetal sheep DA were injected with either control or VEGF-containing solution to examine the direct effects of VEGF on DA remodeling. In three preliminary experiments, the wall of the fetal sheep DA was injected with control solution containing Hoechst-bisbenzimide to determine the distribution of the injectate. We found that the Hoechst-bisbenzimide (which is a smaller molecule than VEGF) stained cells only on the ipsilateral side of the injected DA (Fig. 6B).

View larger version (93K): [in this window] [in a new window]   Fig. 6.   A: DA from 4 100-day fetal lambs injected with either control (1 and 3) or VEGF (2 and 4) solutions. eNOS (endothelial cells) = brown stain. Smooth muscle cell nuclei counterstained with hematoxylin. Dashed back line = internal elastic lamina. Intima 1-4: endothelial cells at lumen of the 4 fetal DA. Vasa vasorum 1-4: endothelial cells lining vasa vasorum in the outer half of the muscle media of the 4 fetal DA. Bar, 100 µm. B: Hoechst-bisbenzimide (white) injected into wall of DA. Bar, 1,000 µm.

Three days after injecting the control solution, there was no difference in the histology between the injected and the noninjected/contralateral wall of the DA (Fig. 7).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   DA from VEGF-injected fetal lambs have greater neointimal thickness and vasa vasorum ingrowth than control-injected DA. Within each experimental injection group (control, VEGF), we compared the histology of the injected/ipsilateral wall (inject) with the histology of the noninjected/contralateral wall (contra) of the DA. A: neointimal thickness (µm): mean ± SD; * P < 0.05, ipsilateral (inject) vs. contralateral (contra). B: luminal endothelial invasion: % of injected DA demonstrating luminal endothelial invasion (see Fig. 6A, intima 4, for an example). C: vasa vasorum area.  P < 0.05, total vasa vasorum area ratio of VEGF-injected DA vs. control DA (see METHODS).

When VEGF was injected into the DA wall, marked VEGF immunostaining was apparent in the ipsilateral wall when the fetus was necropsied 1 h after the injection (n = 2, data not shown). However, by 3 days after the injection, VEGF was no longer detectable by immunohistochemistry (n = 6).

Three days after the VEGF injection, there was an increase in the vasa vasorum in the muscle media (Figs. 6 and 7C) and expansion of the neointima with cells containing -smooth muscle actin (data not shown). There were only occasional sites where the luminal endothelium appeared to be piling up; however, in two of six of the VEGF-injected DA, luminal endothelial cells were observed to be invading the neointima (Fig. 7B; see Fig. 6A, intima 4). None of these changes was observed on the noninjected/contralateral wall of the VEGF-injected DA.

	DISCUSSION

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Inhibitors of prostaglandin and nitric oxide production produce constriction of the preterm baboon DA, hypoxia of the DA wall, and VEGF expression (Fig. 2) (32). Although factors other than hypoxia could account for the appearance of VEGF in the wall of the constricted DA (10, 12, 16, 27, 28, 31, 33), VEGF's appearance and geographic distribution correspond exactly with the distribution and intensity of hypoxia in the DA wall as measured by EF5 (6, 19, 32). The range of EF5 binding observed in the baboons treated with Indo + L-NNA corresponds to tissue O₂ concentrations between 0.2 and 0.01% (median = 0.06%) (see Fig. 1) (18, 19). This degree of in vivo hypoxia is capable of producing an intense increase in VEGF mRNA in cultured DA smooth muscle cells (Fig. 1).

The baboons treated with Indo + L-NNA that received mAbVEGF had the same degree of DA constriction, tissue hypoxia, and VEGF induction as the baboons treated with Indo + L-NNA that did not receive the mAbVEGF (Figs. 2 and 3). The mAbVEGF treatment produced a marked reduction in vasa vasorum ingrowth and perfusion of the outer muscle media (Figs. 3, 4D, and 5). The mAbVEGF treatment inhibited the rate of luminal endothelial cell proliferation and endothelial cell accumulation in the expanding neointima (Fig. 4, A-C). The mAbVEGF treatment also inhibited smooth muscle cell migration into the neointima (Figs. 3 and 4C). Despite these changes, the mAbVEGF-treated newborns still had a significantly thicker luminal endothelium than the control animals (Fig. 4, B and C). These findings suggest that VEGF may play an essential role in vasa vasorum ingrowth and smooth muscle expansion of the neointima but that other factors, in addition to VEGF, may be responsible for the accumulation of luminal endothelial cells after DA closure.

Our studies administering VEGF directly into the wall of the preterm fetal DA support the conclusions from the mAbVEGF experiments. Direct administration of VEGF into the DA wall increased the number of vasa vasorum in the muscle media and expanded the thickness of the neointima with smooth muscle; luminal endothelial cell accumulation was less consistently observed (Figs. 6 and 7). This may be due to the short exposure time to exogenous VEGF (secondary to removal by intramural vasa vasorum) or to the need for additional angiogenic factors during neointima formation.

Our observations differ from an earlier study that found that VEGF inhibited neointimal hyperplasia after balloon-induced arterial injury (1). In that study, VEGF appeared to have an indirect effect on neointimal thickness by accelerating reendothelialization of the denuded intimal surface (1). On the other hand, our observations are consistent with observations made in several other models of vascular disease, where VEGF was found to promote neointimal thickening after vascular injury (5, 17, 24, 35).

There are several ways in which VEGF could play a role in neointima formation. VEGF stimulates endothelial cell proliferation and migration (20, 25). The _v5-integrin, which mediates VEGF-induced cell migration (13), is upregulated during DA closure (7). VEGF has a direct chemotactic effect on vascular smooth muscle cells themselves (14), in addition to potentiating the promigratory response of other growth factors (9). VEGF alters the normal barrier function of the endothelium (20, 25), thereby exposing smooth muscle cells to promigratory serum factors. VEGF also is a direct chemoattractant for monocytes (2), which play a role in smooth muscle cell migration. We are presently trying to establish which of these mechanisms might play a role during DA closure.

Perspectives

Our experiments emphasize the importance of VEGF as a mediator of the anatomic remodeling that follows DA constriction and hypoxia. Our findings are consistent with other reports that suggest that VEGF is capable of inducing neointimal hyperplasia and angiogenesis (5, 15, 17, 24, 35). The present findings have important implications for other types of vascular pathology (e.g., neointima formation in vascular grafts) where tissue hypoxia is known to occur.

	ACKNOWLEDGEMENTS

We thank W. Cox for performing the Doppler studies, Dr. B. Yoder for expertise in managing the preterm baboons, Dr. J. Coalson for managing the Bronchopulmonary Dysplasia Resource Center, P. Oprysko for EF5 analysis, D. Fleming for editorial assistance, V. Winter and L. Buchanan for distributing the tissues, and all the technicians in the Neonatal Intensive Care Unit at the Southwest Foundation, San Antonio, TX, for help in caring for the preterm baboons.

	FOOTNOTES

This research was supported in part by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-46691 and HL-56061, National Cancer Institute Grant CA-74071, and a gift from the Perinatal Associates Research Foundation. The BPD Resource Center is funded by NHLBI Grant HL-52636.

Address for correspondence: R. I. Clyman, Box 0544, HSE 1492, Univ. of California, San Francisco, CA 94143-0544.

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.

10.1152/ajpregu.00298.2001

Received 29 May 2001; accepted in final form 18 September 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

1.   Asahara, T, Bauters C, Pastore C, Kearney M, Rossow S, Bunting S, Ferrara N, Symes JF, and Isner JM. Local delivery of vascular endothelial growth factor accelerates reendothelialization and attenuates intimal hyperplasia in balloon-injured rat carotid artery. Circulation 91: 2793-2801, 1995[Abstract/Free Full Text].

2.   Barleon, B, Sozzani S, Zhou D, Weich HA, Mantovani A, and Marme D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 87: 3336-3343, 1996[Abstract/Free Full Text].

3.   Borgstrèom, P, Bourdon MA, Hillan KJ, Sriramarao P, and Ferrara N. Neutralizing anti-vascular endothelial growth factor antibody completely inhibits angiogenesis and growth of human prostate carcinoma microtumors in vivo. Prostate 35: 1-10, 1998[ISI][Medline].

4.   Brogi, E, Wu T, Namiki A, and Isner JM. Indirect angiogenic cytokines upregulate VEGF and bFGF gene expression in vascular smooth muscle cells, whereas hypoxia upregulates VEGF expression only. Circulation 90: 649-652, 1994[Abstract/Free Full Text].

5.   Celletti, FL, Waugh JM, Amabile PG, Brendolan A, Hilfiker PR, and Dake MD. Vascular endothelial growth factor enhances atherosclerotic plaque progression. Nat Med 7: 425-429, 2001[ISI][Medline].

6.   Clyman, RI, Chan CY, Mauray F, Chen YQ, Cox W, Seidner SR, Lord EM, Weiss H, Wale N, Evan SM, and Koch CJ. Permanent anatomic closure of the ductus arteriosus in newborn baboons: the roles of postnatal constriction, hypoxia, and gestation. Pediatr Res 45: 19-29, 1999[ISI][Medline].

7.   Clyman, RI, Goetzman BW, Chen YQ, Mauray F, Kramer RH, Pytela R, and Schnapp LM. Changes in endothelial cell and smooth muscle cell integrin expression during closure of the ductus arteriosus: an immunohistochemical comparison of the fetal, preterm newborn, and full-term newborn rhesus monkey ductus. Pediatr Res 40: 198-208, 1996[ISI][Medline].

8.   Clyman, RI, Waleh N, Black SM, Riemer RK, Mauray F, and Chen YQ. Regulation of ductus arteriosus patency by nitric oxide in fetal lambs. The role of gestation, oxygen tension and vasa vasorum. Pediatr Res 43: 633-644, 1998[ISI][Medline].

9.   Couper, L, Bryant S, Eldrup-Jorgensen J, Bredenberg C, and Linder V. Vascular endothelial growth factor increases the mitogenic response to fibroblast growth factor-2 in vascular smooth muscle cells in vivo via expression of fms-like tyrosine kinase-1. Circ Res 81: 932-939, 1997[Abstract/Free Full Text].

10.   Cullinan-Bove, K, and Koos RD. Vascular endothelial growth factor/vascular permeability factor expression in the rat uterus: rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth. Endocrinology 133: 829-837, 1993[Abstract].

11.   Durmowicz, AG, Parks WC, Hyde DM, Mecham RP, and Stenmark KR. Persistence, re-expression, and induction of pulmonary arterial fibronectin, tropoelastin, and type I procollagen mRNA expression in neonatal hypoxic pulmonary hypertension. Am J Pathol 145: 1411-1420, 1994[Abstract].

12.   Finkenzeller, G, Marme D, Weich HA, and Hug H. Platelet-derived growth factor-induced transcription of the vascular endothelial growth factor gene is mediated by protein kinase C. Cancer Res 52: 4821-4823, 1992[Abstract/Free Full Text].

13.   Friedlander, M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA, and Cheresh DA. Definition of two angiogenic pathways by distinct alpha v integrins. Science 270: 1500-1502, 1995[Abstract/Free Full Text].

14.   Grosskreutz, CL, Anand-Apte B, Dupláa C, Quinn TP, Terman BI, Zetter B, and D'Amore PA. Vascular endothelial growth factor-induced migration of vascular smooth muscle cells in vitro. Microvasc Res 58: 128-136, 1999[ISI][Medline].

15.   Hamamichi, Y, Ichida F, Yu X, Hirono KI, Uese KI, Hashimoto I, Tsubata S, Yoshida T, Futatani T, Kanegane H, and Miyawaki T. Neutrophils and mononuclear cells express vascular endothelial growth factor in acute kawasaki disease: its possible role in progression of coronary artery lesions. Pediatr Res 49: 74-80, 2001[ISI][Medline].

16.   Harada, S, Nagy JA, Sullivan KA, Thomas KA, Endo N, Rodan GA, and Rodan SB. Induction of vascular endothelial growth factor expression by prostaglandin E₂ and E₁ in osteoblasts. J Clin Invest 93: 2490-2496, 1994.

17.   Inoue, M, Itoh H, Ueda M, Naruko T, Kojima A, Komatsu R, Doi K, Ogawa Y, Tamura N, Takaya K, Igaki T, Yamashita J, Chun TH, Masatsugu K, Becker AE, and Nakao K. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions: possible pathophysiological significance of VEGF in progression of atherosclerosis. Circulation 98: 2108-2116, 1998[Abstract/Free Full Text].

18.   Kajino, H, Chen YQ, Chemtob S, Waleh N, Koch CJ, and Clyman RI. Tissue hypoxia inhibits prostaglandin and nitric oxide production and prevents ductus arteriosus reopening. Am J Physiol Regulatory Integrative Comp Physiol 279: R278-R286, 2000[Abstract/Free Full Text].

19.   Kajino, H, Chen YQ, Seidner SR, Waleh N, Mauray F, Roman C, Chemtob S, Koch CJ, and Clyman RI. Factors that increase the contractile tone of the ductus arteriosus also regulate its anatomic remodeling. Am J Physiol Regulatory Integrative Comp Physiol 281: R291-R301, 2001[Abstract/Free Full Text].

20.   Keck, PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, and Connolly DT. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 246: 1309-1312, 1989[Abstract/Free Full Text].

21.   Kim, KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, and Ferrara N. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 362: 841-844, 1993[Medline].

22.   Klekamp, JG, Jarzecka K, Hoover RL, Summar ML, Redmond N, and Perkett EA. Vascular endothelial growth factor is expressed in ovine pulmonary vascular smooth muscle cells in vitro and regulated by hypoxia and dexamethasone. Pediatr Res 42: 744-749, 1997[ISI][Medline].

23.   Koch, CJ, Evans SM, and Lord EM. Oxygen dependence of cellular uptake of EF5 (2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide): analysis of drug adducts by fluorescent antibodies vs. bound radioactivity. Br J Cancer 72: 869-874, 1995[ISI][Medline].

24.   Lazarous, DF, Shou M, Scheinowitz M, Hodge E, Thirumurti V, Kitsiou AN, Stiber JA, Lobo AD, Hunsberger S, Guetta E, Epstein SE, and Unger EF. Comparative effects of basic fibroblast growth factor and vascular endothelial growth factor on coronary collateral development and the arterial response to injury. Circulation 94: 1074-1082, 1996[Abstract/Free Full Text].

25.   Leung, DW, Cachianes G, Kuang WJ, Goeddel DV, and Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246: 1306-1309, 1989[Abstract/Free Full Text].

26.   Levy, AP, Levy NS, Loscalzo J, Calderone A, Takahashi N, Yeo KT, Koren G, Colucci WS, and Goldberg MA. Regulation of vascular endothelial growth factor in cardiac myocytes. Circ Res 76: 758-766, 1995[Abstract/Free Full Text].

27.   Li, J, Perrella MA, Tsai JC, Yet SF, Hsieh CM, Yoshizumi M, Patterson C, Endege WO, Zhou F, and Lee ME. Induction of vascular endothelial growth factor gene expression by interleukin-1 in rat aortic smooth muscle cells. J Biol Chem 270: 308-312, 1995[Abstract/Free Full Text].

28.   Liu, Y, Christou H, Morita T, Laughner E, Semenza GL, and Kourembanas S. Carbon monoxide and nitric oxide suppress the hypoxic induction of vascular endothelial growth factor gene via the 5' enhancer. J Biol Chem 273: 15257-15262, 1998[Abstract/Free Full Text].

29.   Mason, CA, Bigras JL, O'Blenes SB, Zhou B, McIntyre B, Nakamura N, Kaneda Y, and Rabinovitch M. Gene transfer in utero biologically engineers a patent ductus arteriosus in lambs by arresting fibronectin-dependent neointimal formation. Nat Med 5: 176-182, 1999[ISI][Medline].

30.   Narayanan, M, Cooper B, Weiss H, and Clyman RI. Prophylactic indomethacin: factors determining permanent ductus arteriosus closure. J Pediatr 136: 330-337, 2000[ISI][Medline].

31.   Pertovaara, L, Kaipainen A, Mustonen T, Orpana A, Ferrara N, Saksela O, and Alitalo K. Vascular endothelial growth factor is induced in response to transforming growth factor- in fibroblastic and epithelial cells. J Biol Chem 269: 6271-6274, 1994[Abstract/Free Full Text].

32.   Seidner, SR, Chen YQ, Oprysko PR, Mauray F, Tse MM, Lin E, Koch C, and Clyman RI. Combined prostaglandin and nitric oxide inhibition produces anatomic remodeling and closure of the ductus arteriosus in the premature newborn baboon. Pediatr Res 50: 365-373, 2001[ISI][Medline].

33.   Shweiki, D, Neeman M, Itin A, and Keshet E. Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implications for tumor angiogenesis. Proc Natl Acad Sci USA 92: 768-772, 1995[Abstract/Free Full Text].

34.   Tannenbaum, JE, Waleh NS, Mauray F, Gold L, Perkett EA, and Clyman RI. Transforming growth factor- protein and mRNA expression is increased in the closing ductus arteriosus. Pediatr Res 39: 427-434, 1996[ISI][Medline].

35.   Yonemitsu, Y, Kaneda Y, Morishita R, Nakagawa K, Nakashima Y, and Sueishi K. Characterization of in vivo gene transfer into the arterial wall mediated by the Sendai virus (hemagglutinating virus of Japan) liposomes: an effective tool for the in vivo study of arterial diseases. Lab Invest 75: 313-323, 1996[ISI][Medline].