Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis

Abstract

Background: The family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb.

Methodology/principal findings: Ectopic expression of VEGF-B in the insulin-producing β-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors.

Conclusions/significance: Taken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic.

Figure 1. Characterization of angiogenesis in pancreatic islets from RIP1-VEGFB mice.

A) Pancreatic sections of control C57BL/6 (left) and of RIP1-VEGFB mice (right) were stained for human VEGF-B (red) to detect transgene expression (upper panel), for CD31 (red) to examine intra-insular blood vessel distribution (middle panel) and were perfusion stained with FITC-coupled tomato lectin to evaluate intra-insular blood vessel functionality (lower panel). To visualize islets of Langerhans, pancreatic sections were co-stained with insulin. Nuclei were visualized by DAPI stain. Scale bar: 100 µm. B) Quantification of islet microvessel area and density of C57BL/6 (N =  5, n =  37) and RIP1-VEGFB (N =  4, n =  36) mice. Analysis was performed by determination of the CD31 stained area (left panel) or CD31 counts (right panel) in relation to the islet area using computer-assisted image analysis. * P =  0.0112. N =  number of analyzed mice, n =  number of islets. C) Islets isolated from RIP1-VEGF-A (n = 23, N = 2), RIP1-VEGFB167 (n = 60, N = 10) and C57BL/6 (n = 38, N = 9), mice were co-cultured with HUVEC in a collagen gel matrix and their ability to induce an angiogenic response was determined. The data points represent the average from two independent experiments using C57Bl/6 and RIP1-VEGFB167 mice, while all islets from RIP1-VEGFA mice were analyzed in a single experiment. n =  number of islets, N =  number of mice.

Figure 2. Characterization of the phenotype of tumors from RIP1-Tag2; RIP1-VEGFB mice.

A) RT-PCR analysis of VEGF-R1 expression in GLP1R+ β-tumor-cells and CD31+ tumor-derived blood-endothelial cells (BEC) isolated from 12 weeks old RIP1-Tag2 mice. B) Pancreatic tumor sections of control RIP1-Tag2 (left) and RIP1-Tag2; RIP1-VEGFB (right) mice were stained for human VEGF-B (red) to detect transgene expression. Nuclei were counterstained with DAPI. T =  Tumor, E =  Exocrine pancreas. Scale bar: 100 µm. C) Tumor incidence (left) and volumes (right) of RIP1-Tag2 (N =  36) and RIP1-Tag2; RIP1-VEGFB (N =  38) mice were determined at the age of 12 weeks. Single points represent the total tumor volume (or tumor number) per mouse as indicated. * P  =  0.0149 (Student's t-test). D) Tumor cell proliferation (left) and apoptosis (right) in RIP1-Tag2 and RIP1-Tag2; RIP1-VEGFB mice was determined by counting the number of BrdU and TUNEL positive tumor cells in a total of 7 to 10 microscopic fields (magnification 400×) per mouse.

Figure 3. Characterization of the vascular and angiogenic profile of tumors derived from RIP1-Tag2; RIP1-VEGFB mice.

A) Representative immunofluorescence microphotographs of pancreatic tumor sections of RIP1-Tag2 (left) and RIP1-Tag2; RIP1-VEGFB (right) mice stained for CD31. Scale bar: 100 µm. B) Quantification of intratumoral vessel density in RIP1-Tag2 (N =  5, n =  20) and RIP1-Tag2; RIP1-VEGFB (N =  5, n =  20) was performed using computer-assisted image analysis. Results are displayed as relation of CD31 stained area or CD31 positive cell counts to tumor area. N =  number of analyzed mice, n =  number of islets. C) Representative immunofluorescence microphotographs of pancreatic tumor sections of RIP1-Tag2 (left) and of RIP1-Tag2; RIP1-VEGFB (right) mice double-stained with CD31 and the pericyte marker NG2 (upper panel) or with FITC-coupled tomato lectin (lower panel). Scale bar: 100 µm. D) Evaluation of mVEGFR-1, mVEGFR-2, mVEGF-A, mPlGF, mPDGF-BB, mFGF2 and mAng2 mRNA expression by quantitative RT-PCR in total tumors of RIP1-Tag2 (n = 5) and RIP1-Tag2; RIP1-VEGFB (n = 5) mice. The mRNA expression profiles of the indicated genes are normalized to the expression of the internal control gene ribosomal protein 19 (mRPL19). E) Upper panel: Immunoblot for mVEGFR-1, mVEGFR-2 and vinculin (loading control) of total tumor lysate prepared from pancreatic tumors of a 12 weeks-old RIP1-Tag2 and RIP1-Tag2; RIP1-VEGFB. Lower panel: Quantitation. F) Determination of the percentage of dysplastic islets isolated from RIP1-Tag2 (white bar, n =  24) and RIP1-Tag2; RIP1-VEGFB mice (grey bar, n =  25) which are able to induce an angiogenic response when they were co-cultured with HUVECs in a collagen gel matrix.

Figure 4. Analysis of inflammatory cell infiltration in tumors derived from RIP1-Tag2; RIP1-VEGFB mice.

A) Representative immunofluorescence microphotographs of pancreatic tumor sections of RIP1-Tag2 (left) and RIP1-Tag2; RIP1-VEGFB (right) mice stained for CD45. Scale bar: 100 µm. B) Quantification of tumor-infiltrating immune cells in RIP1-Tag2 (white bar, N =  5, n =  26-46) and RIP1-Tag2; RIP1-VEGFB (grey bar, N =  5, n = 27–45). The number of tumor-infiltrating CD45+ and F4/80+ cells as well as 7/4+ neutrophils was determined by quantification of the area stained for the selected marker in relation to the total tumor area using computer-assisted image analysis. N =  number of analyzed mice, n =  number of tumors.

Figure 5. Characterization of the tumor and angiogenic phenotype of tumors derived from Vegfb-deficient RIP1-Tag2 mice.

A) Quantification of tumor number (left) and total tumor burden (right) of 12 weeks-old RIP1-Tag2; Vegfb ^+/− (n = 30) and RIP1-Tag2; Vegfb ^−/− (n = 18) mice. B) Quantification of tumor cell apoptosis in lesions (n = 40 for each genotype) from RIP1-Tag2; Vegfb ^+/− and RIP1-Tag2; Vegfb ^−/− mice, as assessed by TUNEL assay. C,D) Vascular density (C) and morphology (D) in lesions from RIP1-Tag2; Vegfb ^+/− (n = 27) and RIP1-Tag2; Vegfb ^−/− (n = 23) mice. Scale bar, 50 µm. E) Pericyte coverage (n = 24 for each genotype), as visualized by immunostaining for the pericyte marker NG2 (green) in relation to the endothelial cell marker CD31 (red). Cell nuclei were visualized using DAPI (blue).