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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5137fjev1 20/9/1495    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, B. Articles by Young, P. P. Search for Related Content PubMed PubMed Citation Articles by Li, B. Articles by Young, P. P.

VEGF and PlGF promote adult vasculogenesis by enhancing EPC recruitment and vessel formation at site of tumor neovascularization

Bin Li^*, Emerson E. Sharpe^*, Amanda B. Maupin^*, Amylynn A. Teleron^*, Amy L. Pyle^*, Peter Carmeliet and Pampee P. Young^*,,1

Departments of

^* Pathology and

Internal Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA; and

Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven, Leuven, Belgium

¹ Correspondence: Vanderbilt University School of Medicine, Department of Pathology, 1161 21st Ave. South, C2217 MCN, Nashville, TN 37232, USA. E-mail: pampee.young{at}vanderbilt.edu

SPECIFIC AIMS

Vascular endothelial growth factor (VEGF) expression is dramatically up-regulated by hypoxia and results in enhanced neovascularization. A related family member, placental growth factor (PlGF), is also expressed during tumor growth. Although their role in angiogenesis has been characterized, their role in adult vasculogenesis remains poorly understood. The primary aim of this study was to examine the role of VEGF and PlGF in promoting tumor vasculogenesis. VEGF’s enhancement of tumor vasculogenesis was further studied to determine if this effect was mediated downstream of its effect to enhance endothelial progenitor cell (EPC) mobilization and/or intravascular survival. The role of VEGF receptors flt1 (VEGFR1) and flk1 (VEGFR2) was studied in culture-expanded (CE)-EPCs to determine their contribution to EPC recruitment and vessel formation at the site of tumor growth.

PRINCIPAL FINDINGS

1. VEGF and PlGF promote tumor vasculogenesis in a melanoma mouse model
We used transgenic mice that constitutively expressed ßbeta;-galactosidase (ßbeta;-gal; lacZ) transcriptionally regulated by an endothelial cell (EC)-specific promoter, VEGFR2 (flk1) as bone marrow (BM) donors to congenic, wild-type mice to facilitate identification of BM-derived (lacZ-positive) vessels within tumors of engrafted animals. The transplanted mice were administered subcutaneously with multiple, stable B16F10 melanoma cell clones exhibiting an approximately twofold overexpression of either VEGF or PlGF into the left flank. The right flank was implanted with equal numbers of B16F10 cells that had been stably transfected with a control plasmid (Fig. 1 A depicts the experimental design). The numbers of BM-derived (lacZ positive) vessels were compared between VEGF (or PlGF)-overexpressing tumor and the contralateral control tumor within the same animal. A greater number of lacZ-positive cells were identified in most sections of tumors obtained either from VEGF- and PlGF-overexpressing tumors, as compared with their respective contralateral control tumors (Fig. 1B-E only VEGF data shown). To quantify tumor vasculogenesis, we measured ßbeta;-gal enzyme activity in tumor homogenates obtained from half of each resected tumor mass. The average ßbeta;-gal activity in control tumors vs. VEGF-overexpressing tumors was 0.65 ± 0.33 and 1.67 ± 0.77 optical density (OD)/mg total protein, respectively (Fig. 1F ). The average ßbeta;-gal activity in control tumors vs. PlGF-overexpressing tumors was 0.33 ± 0.14 and 0.64 ± 0.09 OD/mg total protein, respectively (Fig. 1G ). Hence, tumor cells that over-expressed VEGF or PlGF exhibited an 2 and 2.5-fold increase, respectively, in vasculogenesis over control.

View larger version (39K): [in this window] [in a new window]   Figure 1. Enhanced tumor VEGF or PlGF expression promotes tumor vasculogenesis in flk/LacZ/BMT model. A) Schematic demonstrates flk/lacZ/BMT model. After engraftment, animals were implanted subcutaneously with tumor cells stably transfected with vector in right flank and tumor cells stably transfected with vector encoding VEGF or PlGF in left flank. Tumors were analyzed at day 14 after implantation. B) Tumors implanted into flk/lacZ mice (positive controls) were analyzed by LacZ immunohistochemistry (arrows point to immunoreactive ECs of both large and small vessels). Parallel analysis of negative controls in inset. C) Representative histology from Bl6F10 vector tumor obtained from flk/lacZ/BMT demonstrates a longitudinal section through a LacZ positive small vessel containing red blood cells (arrow). D) Representative histology from Bl6F10VEGF tumor obtained from flk/lacZ/BMT demonstrates numerous LacZ-positive vessels (arrows). E) Fold change in ßbeta;-gal enzyme activity, as a marker for donor (BM-derived) vessels, was measured in VEGF-overexpressing (F) or PlGF-overexpressing (G) tumor homogenates as compared withcontralateral control tumor residing within same mouse to quantify effect of VEGF and PlGF on vasculogenesis.

2. An independent mouse tumor model confirmed the effect of VEGF to promote tumor vasculogenesis
We confirmed our finding that VEGF promoted tumor vasculogenesis using a second model: the ßbeta;-glucuronidase (GUSB)-deficient mouse engrafted with GUSB-positive (wild-type) BM, resulting in GUSB/BMT. GUSB is a lysosomal enzyme that is expressed in virtually all cell types, including BM-derived cells, and on transplantation into a GUSB-deficient host, cells from normal mouse or human donors can be identified by virtue of their GUSB expression with a number of sensitive biochemical and histochemical assay. A spontaneously occurring, high-grade sarcoma was isolated from a GUSB-deficient mouse. Both polymerase chain reaction and histochemical stain for GUSB demonstrated absence of GUSB enzyme activity. Each transplanted recipient received subcutaneous implantation of 2 x 10⁶ GUSB^–/– tumor cells with and without admixed VEGF in opposing flanks. By both immunofluorescent and histochemical analysis, GUSB-positive cells were detected in tumor sections that resembled vascular structures and colocalized with BS1 lectin-positive cells. We used flow cytometric analysis of collagenase-treated tumor suspensions to quantify the numbers of GUSB-positive (BM-derived) cells that coexpressed VE-cadherin, a marker specific for ECs. VEGF-containing tumors isolated after 5 days of implantation contained a significantly greater percentage of BM-derived ECs (GUSB/VE-cadherin dual positive), 30.8 ± 8.2 vs. 10 ± 4.8%, P < 0.05. This effect of VEGF was not statistically different from control in tumors obtained 25 days after implantation. The lack of effect of VEGF at day 25 was likely due to the short bioavailability of rVEGF. The analysis of tumor homogenates after 5 days of implantation supported our findings from the flk1/lacZ/BMT-melanoma tumor model that tumor-associated-increased VEGF levels promoted increased vasculogenesis at the site of tumor growth.

3. VEGFR1 mediates EPC recruitment to site of tumorigenesis
The role of VEGFRs in EPC recruitment in vivo was investigated by preincubation of human EPCs with receptor-specific antibodies that blocked both ligand binding and activation. GUSB^–/– mice were inoculated subcutaneously with 1 x 10⁶ GUSB^–/– sarcoma cells. After 10 days of tumor growth (tumor vol. 0.5 cm³), mice were injected intravenously with 5 x 10⁵ human peripheral blood-derived CE-EPCs preincubated with either anti-VEGFR1- or -VEGFR2-blocking antibodies or control IgG. The mice were killed 12 h after administration, and their tumors were homogenized and assayed for GUSB enzyme activity as a quantitative marker for administered CE-EPCs. GUSB specific activity (1 U=nmol/mg protein/h) in lung (as an example of normal tissue) was very low and similar between treated and control animals, 140 ± 20 vs. 110 ± 29 U, respectively. By contrast, GUSB specific activity in tumor tissue was 10-fold (1320±80 U) higher than that in the lung of control animals. Preincubation with VEGFR1 antibody resulted in fivefold decrease in total GUSB activity in tumors as compared with tumors from mice receiving CE-EPCs preincubated with control IgG. Preincubation of EPCs with anti-VEGFR2 antibody diminished CE-EPC recruitment, but this was not statistically significant. Tumors from mice receiving PBS injections served as negative control and demonstrated no GUSB activity.

4. VEGFR-blocking antibodies prevent CE-EPC mediated vessel formation in tumors
To distinguish CE-EPC survival and vessel formation from recruitment effects of receptor-blocking antibodies, we preincubated 5 x 10⁵ CE-EPCs in the presence of anti-VEGFR2 or -VEGFR1 antibodies or IgG control before subcutaneous administration with 2 x 10⁶ tumor cells. Each mouse was implanted with all three conditions, thereby generating three foci of solid tumors. Tumors were removed after 8 days for analysis of functional vessel formation by GUSB-positive CE-EPCs. Control tumors contained numerous GUSB-positive cells, many that had assembled into vascular-like structures. CE-EPC-derived functional vessels were identified by colocalizing CE-EPC-derived vessels using human-specific anti-von Willebrand factor (vWF) antibody with BS1-lectin positive structures, which marked functional tumor vessels. Abundant GUSB-positive cells were also evident in tumors in which CE-EPCs preincubated with VEGFR1 antibodies had been added; however, no donor-derived, vWF-positive vessels in tumors were found. Although clusters of cells were evident, no cord-like structures were evident in experiments in which cells were preincubated with anti-VEGFR1 or VEGFR2. There were far fewer intact GUSB-positive cells derived from CE-EPCs preincubated with anti-VEGFR2 than control IgG. Unlike VEGFR1-blocking antibodies, the presence of VEGFR2-blocking antibodies significantly diminished (3-fold) the numbers of intact CE-EPCs in tumors as determined by counting intact numbers of GUSB-positive cells in representative fields from three different tumors. Thus, either VEGFR1- or VEGFR2-blocking antibodies prevented CE-EPC assembly into vesselswithin the tumor mass; VEGFR2 inhibition diminished CE-EPC viability.

5. VEGF/VEGFR1 interactions play a unique role in VEGF-mediated CE-EPC migration in vitro
By using a modified Boyden chamber migration assay, we showed that CE-EPCs migrated in a dose-dependent manner to both VEGF and the VEGFR1-specific agonist PlGF. To examine receptor specificity, CE-EPCs were preincubated in receptor-blocking anti-VEGFR1 or -VEGFR2 for 60 min before migration assay. Migration of CE-EPCs was equally responsive to PlGF, a VEGFR1-specific agonist, as VEGF. Concentration of inhibition by VEGFR1-blocking antibody was commensurate with blocking anti-VEGFR2. By contrast, in vitro migration in response to VEGF of differentiated ECs was mediated primarily by VEGFR2, consistent with published studies. Hence, unlike ECs, CE-EPC migration in vitro was mediated by both receptors.

CONCLUSIONS AND SIGNIFICANCE
Published data have shown that VEGF enhances EPC mobilization and survival in circulation (Fig. 2 ). In this study, we showed that VEGF promotes tumor vasculogenesis by mechanisms that include events downstream of these effects (Fig. 2) . We also found that tumor-expressed PlGF, a VEGFR1-specific agonist, also promotes tumor vasculogenesis (Fig. 2) . Consistent with this, we found that VEGF/VEGFR1 interactions modulate CE-EPC recruitment to tumors. Pretreatment of CE-EPCs with blocking antibodies to either VEGFRs blocked assembly into functional vessels. In addition, blocking VEGFR2, but not VEGFR1, resulted in reduced viability of CE-EPCs resident in tumors. This suggests that VEGF/VEGFR2 signaling may provide necessary survival cues. These data also support the hypothesis that the local microenvironment (i.e., local VEGF and/or PlGF levels) plays an important role in vasculogenesis.

View larger version (51K): [in this window] [in a new window]   Figure 2. Schematic representation of effects of VEGF and PlGF on tumor vasculogenesis. VEGF has been shown to increase the numbers of circulating EPCs in several models. In this study, we show that enhanced tumor-associated VEGF and PlGF can enhance vasculogenesis. VEGFRs modulate EPC recruitment and vessel formation at tumor site.

FOOTNOTES

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5137fjev1 20/9/1495    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, B. Articles by Young, P. P. Search for Related Content PubMed PubMed Citation Articles by Li, B. Articles by Young, P. P.