Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor

Allen D. Everett,1,2 Jill V. Narron,1 Tamara Stoops,1 Hideji Nakamura,3 and Amy Tucker2,4

1Department of Pediatrics, Division of Pediatric Cardiology, 4Department of Internal Medicine, Cardiovascular Division, 2Cardiovascular Research Center, University of Virginia Health System, Charlottesville, Virginia 22908; and 3Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan

Submitted 3 December 2003 ; accepted in final form 27 January 2004


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Hepatoma-derived growth factor (HDGF) was previously identified as a developmentally regulated cardiovascular and renal gene that is mitogenic for vascular smooth muscle and aortic endothelial cells. As reciprocal interactions of smooth muscle and endothelial cells are necessary for vascular formation, we examined whether HDGF plays a role in angiogenesis. According to immunohistochemistry, HDGF was highly expressed in endothelial cells of nonmuscularized, forming blood vessels of the fetal lung. HDGF was also expressed in endothelial cells of small (20 µm) mature arteries and veins. By Western immunoblotting, HDGF was highly expressed by human pulmonary microvascular endothelial cells in vitro. Adenoviral overexpression of HDGF was mitogenic for human pulmonary microvascular endothelial cells in serum-free medium, stimulating a 1.75-fold increase in bromodeoxyuridine (BrdU) uptake and a twofold increase in cell migration. With the chick chorioallantoic membrane (CAM), a biologic assay for angiogenesis, exogenous recombinant HDGF significantly stimulated blood vessel formation and a dose-dependent reorganization of cells within the CAM into a more compact, linear alignment reminiscent of tube formation. According to double immunostaining for endothelial cells with a transforming growth factor-{beta}II receptor antibody and BrdU as a marker of cell proliferation, exogenous HDGF selectively stimulated endothelial cell BrdU uptake. HDGF also activated specific ERK1/2 signaling and did not overlap with VEGF SAPK/JNK, Akt-mediated pathways. We conclude that HDGF is a highly expressed vascular endothelial cell protein in vivo and is a potent endothelial mitogen and regulator of endothelial cell migration by mechanisms distinct from VEGF.

lung; angiogenesis; growth factors; cell migration


EARLY VASCULAR DEVELOPMENT is a complex process involving the differentiation of pluripotential hemangioblasts into endothelial cells to line-muscularized tubes of specialized smooth muscle (reviewed in Ref. 4). The development of the pulmonary vasculature has largely been descriptive. It was observed, that unlike other organs, vascularization of lung likely includes both the de novo development of intrinsic blood vessels (vasculogenesis) and the ingrowth of existing vessels (angiogenesis) (5). During lung development the microvasculature transforms from a loose mesenchymal capillary network to an extensive subepithelial bed of capillaries. The development of the microvasculature continues in concert with the airway during the canalicular phase, where the lung is canalized by the proliferation of capillaries. During this stage, as the airway septa begin to thin, the capillaries become closely aligned with the distal epithelium (2, 5). This is associated with a flattening of the differentiating type I and type II cells so that a thin air-blood interface is formed (5).

Pulmonary microvascular growth and development require endothelial cell differentiation, migration, and recruitment of smooth muscle cells. However, the diverse group of molecules involved in regulating pulmonary endothelial and vessel formation, including extracellular matrix, growth factors, and their receptors, is still poorly defined (2, 13, 17). Essential to this process is vascular endothelial growth factor (VEGF). VEGF, acting through its cognate receptors, is mitogenic for endothelial cells and induces migration and organization into tubes (reviewed in Ref. 4). VEGF is of critical importance in all vascular formation as loss of a single VEGF allele is lethal in the embryo (3). VEGF regulates endothelial function via well-established signaling pathways involving activation of ERK1/2, SAPK/JNK, and Akt (10, 16). However, it is not known whether other growth factors may play a complementary role to VEGF in endothelial growth.

We recently identified hepatoma-derived growth factor (HDGF) as a highly expressed endothelial protein (6) in the fetal cardiovascular system. Although HDGF lacks a secretory leader sequence, it was originally purified from the conditioned media of the human hepatoma cell line HuH-7 (14) and later from the rat metanephrogenic mesenchymal cell line 7.1.1 (15). Consistent with the evidence that HDGF is a released protein, in vitro studies have shown HDGF to be an exogenous mitogen for HuH-7 (14), Swiss 3T3 fibroblasts (14), and aortic endothelial cells (15). HDGF may play an important role in vascular growth, as we identified HDGF as a potent mitogen for vascular smooth muscle cells in vitro with re-expression in response to vascular injury in pathologic animal models and in human atherosclerotic vascular disease (7).

We report here that HDGF is highly expressed in vascular endothelial cells of the fetal and adult lung. According to adenovirus-targeted expression, HDGF stimulated migration and proliferation of human peripheral pulmonary endothelial cells. Furthermore, we demonstrate that HDGF is an angiogenic factor in vivo using the chick chorioallantoic membrane (CAM) assay. These observations strongly implicate HDGF as an important factor in the regulation of pulmonary vascular growth.


    METHODS
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Endothelial cell culture. Human primary pulmonary microvascular endothelial cells (HMVEC) and culture media and supplements were obtained from Biowhittaker/Clonetics (East Rutherford, NJ). Cells were cultured on fibronectin-coated tissue culture plates in endothelial cell basal medium containing 10 µg/ml human recombinant epidermal growth factor, 1.0 mg/ml hydrocortisone, 50 mg/ml gentamicin, 50 µg/ml amphotericin, and 3 mg/ml bovine brain extract with 2% FBS (complete growth media) or without serum [serum-free media (SF)] for 6–12 passages. For cell signaling studies, HMVEC in six-well plates were incubated serum-free overnight and stimulated with recombinant HDGF (14). At the end of the treatment period cells were washed with saline and lysed with Laemmli loading buffer.

CAM assay for angiogenesis. One-day-old fertilized White Leghorn chicken eggs were incubated in a rotating incubator at 37°C for 7 days before windowing. On day 7 a 1 x 1-cm window was cut into the eggshell and covered with cellophane tape, and the eggs were placed in a tissue culture incubator at 37°C with 2% CO2. Recombinant HDGF (190 ng) (7) or PBS vehicle was applied daily on days 11–14 to a filter paper disc placed on the CAM through the window. The windows were covered with cellophane tape after each application. On day 15 the CAMs were harvested, and the vessels intersecting the filter paper disc at an angle >=45° were counted under a dissecting microscope. The paper discs were then removed, and the membranes were photographed.

We performed single growth factor applications by drying 100–1,000 ng of HDGF in PBS with 0.4% BSA, 500 ng VEGF (Sigma) in PBS with 0.4% BSA, or vehicle on a sterile 5 x 5-mm Thermonox coverslip and then inverting them onto 9-day CAMs through a 1 x 1-cm window. Windows were covered with cellophane tape, and the eggs were incubated at 37°C until the CAMs were harvested on day 14. The number of vessels intersecting the margins of the coverslips was counted.

Changes in blood vessel number between groups were compared by paired t-test with significance set at P <= 0.05.

For histological evaluation of HDGF effects on the CAM, CAMs were fixed in 10% buffered formalin for 1 h at 4°C, cryoprotected in 20% sucrose overnight, embedded in optimal cutting temperature mounting medium, cryosectioned, and stained with propidium iodide. For bromodeoxyuridine (BrdU) studies, CAMs had 10 µM BrdU (Sigma) applied overnight on day 14. CAMs were double immunostained for BrdU (monoclonal antibody 1:100, Roche Biochemicals) and transforming growth factor {beta}II receptor (1; TGF{beta}IIR, polyclonal antibody, 1:100; Exalpha Biologicals, Boston, MA) with the use of an Oregon green-conjugated anti-rabbit (1:1,000, Molecular Probes) for TGF{beta}IIR and an AlexaFluor red-conjugated anti-mouse secondary antibody (1:1,000, Molecular Probes) for BrdU. All CAM slides were examined and photographed under an Olympus BX5OWI Fluoview confocal microscope.

HDGF-expressing adenovirus. Replication-deficient adenovirus type 5 was constructed to express a green fluorescent protein (GFP)-HDGF fusion or GFP alone as a control after the method of Hardy et al. (11). A rat GFP-HDGF cDNA was subcloned as a HindIII/EcoRI restriction fragment into the Ad5 shuttle plasmid pAdlox (11). 293 CRE8 cells, a cell line that stably expresses Cre recombinase (11), were transfected with 3 µg of GFP-HDGF-pAdlox or GFP-pAdlox and 3 µg of {psi}5 viral DNA using CaPO4. {psi}5 is an E1- and E3-deleted version of Ad5 containing loxP sites flanking the packaging site (11). Transfected cells were GFP positive by fluorescent microscopy after 24 h. CRE8 cells containing recombined Ad5 expressing GFP-HDGF were lysed by repeat freeze-thaw. Ad5-GFP-HDGF was plaque purified, and high-titer virus was recovered over a CsCl gradient. We determined the multiplicity of infection (MOI) for HMVEC by infecting HMVEC in complete growth media for 4 h with increasing MOI, and the expression of HDGF was measured by immunodot blotting for GFP.

Cell migration. BioCoat Control Cell Culture Inserts (24-well, 8.00-µm pore size; Becton Dickinson, Bedford, MA) were incubated with 0.02 mg/ml fibronectin (Sigma) in PBS for 1 h at 37°C and removed. Fifty thousand HMVEC were plated per upper chamber in complete growth media and allowed to attach for 12–24 h. To express HDGF, HMVEC were infected with Ad5-GFP-HDGF or Ad5-GFP at an MOI of 100 in complete growth media for 4 h. To allow recovery of the cells after infection, we replaced virus and media from the upper chambers with complete growth media and incubated them for 14–16 h. Medium in both the upper and lower chambers was then replaced with SF and incubated for 24 h to allow migration. After 24 h, cells were washed once with PBS and fixed with 2% paraformaldehyde in PBS for 20 min at room temperature. Cells adherent to the upper side of the chamber membrane (nonmigrating) were removed by gentle scraping with a cotton swab. The membrane was removed from the insert and coverslipped bottom side up with Vectashield Mounting medium (Vector Laboratories, Burlingame, CA) containing propidium iodide. Migration assays were done in triplicate for each condition, and the experiment was repeated twice. Five random high-power fields/membrane were photographed under a Nikon Microphot-SA equipped with a Hamamatsu C4742 charge coupled device camera driven by OpenLab 2.0.6 (Improvision, Lexington, MA). The number of migrating cells per field was counted, averaged, and compared by t-test.

Cell proliferation assay. To assess HMVEC cell proliferation, we used the Colorimetric Cell Proliferation ELISA (Roche) with the following modifications. A clear 96-well, tissue culture-treated flat-bottom culture plate (Corning) was coated with 0.02 mg/ml fibronectin (Sigma). In each well, 3,200 HMVEC in complete growth medium were plated, allowed to attach, and infected with Ad5-GFP-HDGF or Ad5-GFP at a MOI of 100 for 4 h. The virus was removed, and the cells were incubated in complete growth medium for 14–16 h to recover. Stabilized cells were then transferred to SF for 8–12 h and then incubated overnight with 10 µM BrdU (Roche). Each condition was performed in pentuplicate per assay. BrdU uptake was determined with a µQuant plate reader (Bio-Tek Instruments, Highland Park, VT). Blanks include culture media + fibronectin, BrdU (Roche) + fibronectin, and anti-BrdU-POD (Roche) + fibronectin. Controls include cells + fibronectin + culture media and anti-BrdU-peroxidase + cells in culture media + fibronectin. Results for each condition were averaged and compared by one-way ANOVA and t-test between individual groups.

Immunocytochemistry. Immunohistochemical analysis was performed as previously described (7). Whole rat embryos or adult lungs were fixed in 4% buffered paraformaldehyde for 30 min at room temperature, paraffin embedded, and sectioned. Separate tissue sections were incubated with the HDGF antibody (6, 7) (1:2,000) or in combination with platelet endothelial cell adhesion molecule (PECAM, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) described above. Controls slides were incubated with nonspecific rabbit IgG (Vector Laboratories) at the same concentration as the primary antibody. Images were acquired on a Nikon Eclipse 400 microscope equipped with a MicroPublisher digital camera (Qimaging, Burnaby, Canada).

Western blotting. Western blotting was performed as previously described (7). Briefly, for HDGF analysis, HMVEC were grown between 50 and 70% confluent in complete growth medium under standard cell culture conditions. Soluble homogenates (10 µg) were separated under denaturing conditions in a 10% SDS-PAGE gel, followed by blotting of the proteins to nitrocellulose (Bio-Rad, Burlingame, CA). Blots were blocked at room temperature for 1 h in 50 mM Tris·HCl pH 7.4, 0.15 M NaCl, 2% BSA, and 0.1% Tween 20. Subsequently blots were incubated with a rabbit anti-HDGF polyclonal antibody (dilution 1:5,000) for 1 h at room temperature. For cell signaling studies membranes of cell lysates (50 µg of protein/lane) were immunodetected in parallel with phosphospecific antibodies against ERK1/2, Akt, and SAPK/JNK (Cell Signaling Technology, Beverly, MA). For Ad5 GFP-HDGF studies membranes were incubated with GFP or VEGF antisera (Santa Cruz) at 1:1,000 dilution. Subsequently, membranes were washed at room temperature and incubated with an anti-rabbit IgG-conjugated to horseradish peroxidase (Sigma) for 1 h at room temperature. Immunoreactive proteins were detected with enhanced chemiluminescence (ECL; Amersham Pharmacia, Piscataway, NJ) and exposure to film (Kodak AR).


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Immunohistochemical expression of HDGF in rat pulmonary vascular endothelium. Using a specific anti-HDGF polyclonal antibody (6, 7), we determined the pulmonary endothelial expression of HDGF. Fetal lung vascular endothelial cells [embryonic day 16 (E16)] express HDGF at high levels as shown by dual immunostaining with antibodies for HDGF and the vascular endothelial cell marker PECAM (Fig. 1A). In the early E13 lung, HDGF was expressed in the nuclei of endothelial cells forming loose capillary networks (Fig. 1B) in mesenchyme not spatially associated with epithelium. In the E18 lung now in the canalicular stage before birth, HDGF continues to be expressed in the nuclei of capillary endothelial cells that lie within the thinning septa. In the fully developed adult lung, HDGF expression persists in endothelial cells of small capillaries (Fig. 1C) and small pulmonary arteries and veins (Fig. 1, F and G). HDGF was also expressed in the nuclei of large and small pulmonary arteries and venous smooth muscle cells (Fig. 1, D–F). HDGF is also expressed in vitro in HMVEC (Fig. 2); as expected, minor 48- and major 43-kDa bands with specific HDGF antisera were observed by Western blotting (6, 7).



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Fig. 1. Immunohistochemical expression of hepatoma-derived growth factor (HDGF) in the developing rat pulmonary vasculature. A: specific embryonic day 16 pulmonary vascular endothelial cell expression of HDGF by dual immunostaining for platelet endothelial cell adhesion molecule (PECAM, arrows) and HDGF (red, nuclei marked with *). The merged image demonstrates HDGF expression in PECAM-positive cells. B: HDGF was nuclearly expressed in pulmonary capillary endothelial cells (arrows) from embryonic day 13 (A), day 18 (B), and in the adult rat (C). In the adult lung, HDGF was also expressed in a nuclear pattern in vascular endothelium (arrows) in large pulmonary arteries (D), pulmonary veins (E) and small arteries (F), and veins (G). Magnification: A, B, D–G, x40; C, x100.

 


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Fig. 2. Western blotting for endogenous HDGF in human microvascular endothelial cells (HMVEC). HMVEC cell lysate (10 g) was separated by SDS-PAGE and immunoblotted for HDGF. Endogenous HDGF expression is shown as specific major 43- and minor 48-kDa bands.

 
HDGF is a mitogen for human pulmonary endothelial cells. Proliferation of endothelial cells is necessary for the formation of the vasculature. HMVEC were used as an in vitro model to study the mitogenic effects of HDGF. Adenovirus to express a GFP-HDGF fusion (Ad5-GFP-HDGF) or GFP (Ad5-GFP) was developed to test the mitogenic effects of increased expression of HDGF. The appropriate MOI for HMVEC was determined after a 4-h infection, and the expression of GFP was examined by immunodot blotting for GFP. As shown in Fig. 3, A and B, an MOI of 100 induced maximal expression of GFP with minimal cytotoxic effects. To examine whether HDGF is an HMVEC mitogen, we infected HMVEC with Ad5-GFP-HDGF to express GFP-HDGF or Ad-GFP to express GFP as a control, both at an MOI of 100 for 4 h. As a positive control for proliferation a group of cells were treated with 2% FBS without viral transfection. The HMVEC were then changed to SF and loaded with BrdU overnight. As shown in Fig. 3C, HMVEC in SF expressing GFP-HDGF demonstrated a significant (P <= 0.009) 1.75-fold increase in BrdU uptake compared with the GFP controls. FBS had a slightly greater effect with a 2.4-fold increase in BrdU uptake compared with HMVEC in SF (Fig. 3C).



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Fig. 3. Adenoviral green fluorescent protein (GFP)-HDGF expression in HMVEC induces cell proliferation. A: multiplicity of infection (MOI) determination of Ad5-GFP-HDGF in HMVEC. HMVEC were infected with increasing MOI and lysed 24 h later and immunodot blotted for GFP. B: HMVEC infected with Ad5-GFP-HDGF or Ad5-GFP for 4 h at a MOI of 100 demonstrate high levels of GFP expression with normal cell spreading and attachment to the plate. As expected, GFP-HDGF is expressed in the nucleus of HMVEC compared with the GFP control. C: bromodeoxyuridine (BrdU) ELISA assay of HMVEC proliferation. HMVEC were grown in serum-free complete media (SF) and infected with Ad5 to express GFP or GFP-HDGF at a MOI of 100 for 4 h. As a positive control for proliferation, noninfected cells were grown in complete growth media containing FBS (2% FBS). Cells were BrdU loaded overnight and assayed 24 h after infection. Each condition was performed in pentuplicate and expressed as means ± SE. *P <= 0.009 GFP-HDGF vs. GFP; **P <= 0.005 SF vs. 2% FBS.

 
HDGF stimulates HMVEC migration. Endothelial cell migration is necessary for sprouting and formation of the neovasculature. To examine whether HDGF affects HMVEC migration, we used a modified Boyden chamber assay. HMVEC seeded onto the fibronectin-coated migration support were infected with adenovirus to express GFP-HDGF or GFP as a control. The cells were changed to SF, and the number of GFP-positive cells migrating after 24 h to the underside of the support was counted. As shown in Fig. 4, expression of HDGF in HMVEC in SF resulted in increased cell migration. When migrating cells were counted, HDGF expression stimulated a significant (P <= 0.005) twofold increase in HMVEC migration.



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Fig. 4. HDGF expression induces HMVEC migration. A: HMVEC seeded onto fibronectin-coated cell culture inserts with 8-m pores were infected with Ad5-GFP-HDGF (HDGF) or Ad5-GFP (GFP). Cell migration in SF through the membrane was observed 24 h later (magnification x20). B: when the number of migrating cells was counted (3 membranes, 5 high-power fields/membrane), we saw that HDGF expression stimulated increased cell migration (*P <= 0.001).

 
Functional role of HDGF in angiogenesis. As shown above, HDGF is highly expressed by pulmonary endothelial cells in the lung and by HMVEC in culture. Furthermore, HDGF is a mitogen for HMVEC. To directly measure HDGF's angiogenic effect, 190 ng of HDGF were applied daily to CAMs for 4 days. Compared with control (Fig. 5, A and B), treatment with HDGF significantly increased the formation of small vessels in the area where the HGDF-containing disc was placed. New vessels could be seen to branch toward the disc (Fig. 5A). According to our counts, HDGF treatment resulted in significant new vessel formation (P < 0.05) compared with the vehicle control (Fig. 5B) in two separate experiments. A single application of 500 ng of HDGF to CAMs via an inverted coverslip elicited a less robust response, increasing the number of new vessels intersecting the margin of the coverslip by 21% (n = 7, P < 0.05) compared with control (n = 10). This was similar to the response obtained using a 500-ng application of VEGF (24% increase, n = 5).



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Fig. 5. Exogenous HDGF increases blood vessels in the chorioallantoic membrane (CAM) assay. A: representative CAMs are shown after 5 days of 190 ng/day treatment with HDGF or diluent control (control, PBS) with the filter disc removed, demonstrating the increase in blood vessel formation. B: when the number of blood vessels entering the area of the filter disc from multiple CAMs was counted, we saw that HDGF treatment increased blood vessel numbers (*P <= 0.05, HDGF vs. PBS). The results of 2 separate experiments are shown.

 
To examine the cell-specific effect of HDGF on CAMs, we performed a single application of recombinant HDGF exogenously to CAMs, and the CAMs were sectioned for microscopic analysis. Striking reorganization of the cellular architecture was observed with HDGF treatment. As shown in Fig. 6, there was a dose-dependent increase in cellular reorganization of the CAM in response to HDGF, which peaked at 100–500 ng. A gradient of effect could be seen in the CAMs (Fig. 6, top low-power panels) with the greatest increase in blood vessels and cellularity with proximity to the HDGF-containing patch. With increasing concentrations of HDGF, the cellular density of the CAMs increased, with the cells becoming linearly oriented, reminiscent of tubes. There was also an obvious increase in the number of blood vessels (Fig. 6, arrows) present in the CAMs with increasing HDGF treatment.



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Fig. 6. Exogenous HDGF induces CAM cellular reorganization and increases blood vessel formation. Representative CAMs were treated for 5 days with a single exogenous application of 100–1,000 ng of HDGF, fixed, sectioned, and stained with propidium iodide for DNA. There was a maximal dose-dependent increase in cell density and blood vessel formation (arrows) at 100–500 ng.

 
To examine whether exogenous HDGF stimulated endothelial cell proliferation in the CAMs, a requirement for vascular formation and growth, we treated CAMs with exogenous HDGF and loaded them with BrdU. Double immunohistochemistry for BrdU and the chicken endothelial cell marker TGF{beta}IIR (1) was performed to determine whether proliferating cells were vascular endothelium. As shown in Fig. 7, there is little BrdU uptake in cells of a CAM treated with the PBS control. However, in the HDGF-treated CAMs, there were many TGF{beta}IIR-positive endothelial cells organized into blood vessels per high-power field (Fig. 7). Importantly, HDGF stimulated endothelial cell proliferation as indicated by uptake of the thymidine analog BrdU in these TGF{beta}IIR-positive cells (Fig. 7). Strikingly, with HDGF treatment, only endothelial cells were BrdU positive in the CAM, indicating a selective sensitivity of endothelial cells to respond to HDGF. Also, BrdU-positive cells were seen at vascular branch points, consistent with the stimulation of new vascular sprouts (Fig. 7).



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Fig. 7. HDGF selectively increases endothelial cell proliferation in the CAM assay. CAMs were treated with a single application of 500 ng of HDGF or PBS control for 5 days with BrdU loading overnight. CAMs were fixed, sectioned, and double immunostained for the endothelial cell marker transforming growth factor-{beta}II receptor (TGF{beta}IIR) and BrdU as a marker of cell proliferation. In the first panel there is very little uptake of BrdU in the PBS-treated CAM. In a representative CAM treated with HDGF, a large number of TGF{beta}IIR blood vessels (arrows, green) are present with endothelial cell nuclei positive for BrdU (red) and in the merged image.

 
HDGF treatment stimulates a MAPK ERK1/2 signaling that is distinct from VEGF. The mechanism of HDGF-stimulated HMVEC proliferation is unknown. VEGF stimulation, however, activates the distinct signaling intermediates MAPK ERK1/2, Akt, and SAPK/JNK (10, 16). To explore the mechanism for HDGF-stimulated endothelial cell proliferation of HMVEC and overlap with VEGF signaling pathways, we treated HMVEC with recombinant HDGF and assayed them for ERK1/2-, SAPK/JNK-, and Akt-activated signaling using phosphospecific (activated) antisera. As shown in Fig. 8A, HDGF stimulates a rapid ERK1/2 signal at 5 min but does not activate SAPK/JNK or Akt (Fig. 8B) signaling. We confirmed equivalent protein loading by blotting for total MAPK, SAPK/JNK, and Akt. VEGF is a required endothelial survival factor and mitogen. To determine whether HDGF stimulates HMVEC proliferation by regulating VEGF expression, we induced HMVEC to overexpress a GFP-HDGF fusion using recombinant adenovirus. As shown in Fig. 8C, HDGF expression for up to 72 h had no effect on VEGF protein levels. Similar levels of HDGF expression at all three time points were demonstrated by equivalent levels of GFP expression. Growth factors can amplify their effects by stimulating the downstream expression of other growth factors. To determine whether VEGF regulates the expression of HDGF as part of its regulation of HMVEC growth, we treated cells with exogenous VEGF and Western blotted cell lysates for HDGF. VEGF had a potent negative effect on HDGF expression with a single application of VEGF, resulting in a 50% decrease in HDGF protein levels by 48 h.



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Fig. 8. HDGF treatment initiates a specific MAPK signal that is distinct from VEGF. A: HMVEC were treated with 50 ng of recombinant HDGF for 0–30 min, and cell lysates were blotted for MAPKs ERK1/2 and SAPK/JNK. B: HMVEC were treated with exogenous HDGF (0–120 min) and Western blotted for the VEGF signaling pathway intermediate phospho-Akt (P-Akt) or total Akt (T-Akt) as a loading control. C: HMVEC were infected with adenovirus to express GFP or a GFP-HDGF fusion (24–72 h), with cell lysates Western blotted for VEGF or GFP as a control for expression. As shown, HDGF expression did not alter VEGF protein levels. D: HMVEC were treated (6–48 h) with a single application of VEGF (concentration) or diluent and Western blotted for HDGF.

 

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The processes necessary for pulmonary vascular development are endothelial differentiation, proliferation, migration, and formation of cell-cell junctions and attraction of smooth muscle cells. In the present study we explored the role of HDGF in pulmonary angiogenesis using developmental expression of HDGF in pulmonary endothelial cells, mitogenic and cell migration assays for human peripheral pulmonary endothelial cells, angiogenesis assays using the CAM model of fetal angiogenesis, and assays to determine the mechanism of HDGF effects. We found that HDGF is highly expressed in early fetal and mature lung endothelial cells in situ. HDGF was also a potent mitogen and stimulant of endothelial cell migration according to a modified Boyden chamber assay. The involvement of HDGF in vascular development was confirmed by a biologic model of fetal neovascularization, the CAM assay. HDGF was determined to elicit a distinct ERK1/2 signaling event and did not regulate VEGF or known VEGF signaling pathways.

Evidence for HDGF involvement in vascular growth has come from the previous demonstration that HDGF purified from the conditioned media of a fetal kidney metanephric mesenchymal cell line stimulated bovine aortic endothelial cell proliferation (15). Previously we showed that HDGF is a developmentally regulated cardiovascular gene and protein (6) and a mitogen for vascular smooth muscle cells (7, 8). Consistent with a role in vascular growth, in the present study, we demonstrate that HDGF is a human pulmonary endothelial cell mitogen and is endogenously expressed in rat early (E13) and adult pulmonary endothelial cells and by HMVEC in culture. These findings are consistent with the previous report by Oliver and Al-Awqati (15) that HDGF was a mitogen for bovine and rat aortic endothelial cells. However, unlike Oliver and Al-Awqati (15), we demonstrate that endogenous HDGF and HDGF overexpressed as a GFP-HDGF fusion exist only as an endothelial nuclear protein. This is consistent with our previous work in several cell types including vascular smooth cells (7, 8, 12). It is clear from recent HDGF mutation and deletion studies that HDGF nuclear targeting is necessary for its mitogenic effect (8, 12), possibly by regulating transcription of target genes as has been demonstrated for the HDGF-related protein p52/75 (9).

HDGF is a true vascular growth factor as HDGF treatment resulted in increased blood vessel formation equivalent to VEGF. HDGF also induced a dose-dependent cell reorganization in the CAM assay in which cells became linearly oriented, resembling tube formation. This was complemented by an increase in blood vessel formation. The cellular reorganization of the CAM suggests HDGF plays a role in the phenotypic modulation of cells to stimulate differentiation into endothelial cells or their activation. This is consistent with the observation that HDGF selectively stimulated endothelial cell proliferation in the CAM assay and not the surrounding fibroblasts, suggesting that endothelial cells are particularly sensitive to HDGF. The observation that HDGF stimulated proliferation of endothelial cells at branch points could be quite important for the process of angiogenic sprouting. For angiogenic sprouting to occur, activated and proliferating endothelial cells must migrate and form lumens (4). In the CAM assay, HDGF stimulated proliferation of endothelial cells at branch points and, in the in vitro cell assays, stimulated a significant over twofold increase in cell migration in vitro. HDGF has been implicated in the activation of cell migration in an independent screen to identify migration associated genes in wound repair (18) of retinal epithelial cells.

HDGF was originally identified as a exogenous mitogen from the conditioned media of Huh-7, a human hepatoma cell line, and later from 7.1.1 fetal kidney epithelial cells. HDGF has previously been shown to be an exogenous mitogen from several research groups and for many cell types. However, the mechanism of HDGF stimulation of cell proliferation was unknown. We present evidence that exogenous HDGF stimulates a rapid and specific activation of the MAPK ERK1/2 growth factor signaling pathway. ERK1/2 activation is a common downstream signaling intermediate for many vascular growth factors including VEGF, PDGF, and FGF. Interestingly, HDGF-stimulated cell signaling is distinct from the well-documented VEGF pathway, which includes activation of both Akt (10) and SAPK/JNK (16) signals. In fact, HDGF had no effect on activation of Akt or SAPK/JNK, suggesting that HDGF cell surface signaling is distinct from VEGF. It is possible that VEGF could act downstream of HDGF, with HDGF treatment increasing the expression of VEGF. However, high-level overexpression of HDGF had no effect on VEGF protein levels. Therefore, the HDGF effect on HMVEC proliferation and migration is unlikely mediated through downstream VEGF signaling. Interestingly, VEGF had a potent effect on HDGF, decreasing its expression by 50% after 48 h. The significance of HDGF regulation by VEGF is unclear but could be important in VEGF-stimulated differentiation and survival of endothelial cells. It is unclear at this time whether HDGF signals at the cell surface via a receptor but is the focus of future work.

In summary, these data provide evidence for an HDGF role in vascular growth and formation by regulating endothelial cell proliferation and migration. The expression and mitogenic function of HDGF in human pulmonary endothelial cells implicate an important new molecule in pulmonary vascular development and possibly angiogenesis in general.


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This research was supported by National Heart, Lung, and Blood Institute Grant R01HL-69938, The University of Virginia Children's Medical Center (A. D. Everett), and the Cardiovascular Division, University of Virginia (A. Tucker).


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. D. Everett, Johns Hopkins Hospital, Pediatric Cardiology, Brady 5, 600 N. Wolfe St., Baltimore, MD 21287 (E-mail: aeveret3{at}jhmi.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.


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