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

Albrecht I, Kopfstein L, Strittmatter K, Schomber T, Falkevall A, Hagberg CE, Lorentz P, Jeltsch M, Alitalo K, Eriksson U, Christofori G, Pietras K - PLoS ONE (2010)

Bottom Line: 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.No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, Institute of Biochemistry and Genetics, University of Basel, Basel, Switzerland.

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.

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Related in: MedlinePlus

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.
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pone-0014109-g001: 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.

Mentions: To investigate the role of VEGF-B in normal and pathological angiogenesis, we generated transgenic mice expressing the human VEGF-B167 isoform under the control of the rat insulin promoter (RIP1-VEGFB mice), thus directing expression of VEGF-B to the β-cells of the pancreatic islets of Langerhans. Human VEGF-B167 activates VEGFR-1 downstream target genes FATP3 and FATP4 to the same extent as mouse VEGF-B167 and VEGF-B186 isoforms in the mouse pancreatic islet endothelial cell line MS1, indicating that human VEGF-B readily binds mouse VEGFR-1 (Figure S1). Expression of the transgene in vivo was confirmed by immunostaining of tissue sections from the pancreas of RIP1-VEGFB mice for human VEGF-B (Figure 1a). No changes were found in the pancreatic islets of transgenic mice in terms of islet architecture, number, or size (Figure S2a-c). Moreover, β-cell density and functionality, as measured by glucose tolerance tests, were normal in RIP1-VEGFB mice (Figure S2d-e). Next, we analyzed the effects of the transgenic expression of VEGF-B on the vascular tree by immunostaining for the endothelial cell marker CD31 and by perfusion with fluorescein-labeled tomato lectin. Whereas there was no difference in the number of islet blood vessels (vascular density; Figure 1a-b), pancreatic islets of RIP1-VEGFB mice exhibited a 20% increase in the fraction of the islet area covered by vessels, as compared to wildtype mice (Figure 1a–b; 13.2±0.6% vs 11.0±0.6%, p<0.05). The increase in vessel area was consequent to an apparent increase in the diameter of pancreatic islet microvessels from 8.0±0.25 µm in non-transgenic mice to 9.7±0.50 µm in RIP1-VEGFB mice (Table 1; p<0.01), while vessel length was unchanged (Table 1). No overt differences in perfusion of the islet capillaries were noted (Figure 1a). Finally, to investigate whether islets of Langerhans from RIP1-VEGFB mice exhibited an increased angiogenic potential, we made use of an ex vivo collagen gel sprouting assay. Pancreatic islets were purified by limited collagenase digestion of the pancreas, and subsequently seeded into collagen gels together with human umbilical vein endothelial cells (HUVEC). Factors produced by the islet will diffuse into the gel and affect the phenotype of the co-cultured endothelial cells. Islets from RIP1-VEGFA mice were used to demonstrate migration and sprouting of HUVEC towards the islet upon the release of an angiogenic factor (Figure 1c). Whereas 30% of islets from RIP1-VEGFA mice exhibited angiogenic properties, only 13.6% of islets from RIP1-VEGFB mice were able to attract the co-cultured endothelial cells (Figure 1c). No islets from wildtype mice were overtly angiogenic in this assay (Figure 1c).


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

Albrecht I, Kopfstein L, Strittmatter K, Schomber T, Falkevall A, Hagberg CE, Lorentz P, Jeltsch M, Alitalo K, Eriksson U, Christofori G, Pietras K - PLoS ONE (2010)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991338&req=5

pone-0014109-g001: 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.
Mentions: To investigate the role of VEGF-B in normal and pathological angiogenesis, we generated transgenic mice expressing the human VEGF-B167 isoform under the control of the rat insulin promoter (RIP1-VEGFB mice), thus directing expression of VEGF-B to the β-cells of the pancreatic islets of Langerhans. Human VEGF-B167 activates VEGFR-1 downstream target genes FATP3 and FATP4 to the same extent as mouse VEGF-B167 and VEGF-B186 isoforms in the mouse pancreatic islet endothelial cell line MS1, indicating that human VEGF-B readily binds mouse VEGFR-1 (Figure S1). Expression of the transgene in vivo was confirmed by immunostaining of tissue sections from the pancreas of RIP1-VEGFB mice for human VEGF-B (Figure 1a). No changes were found in the pancreatic islets of transgenic mice in terms of islet architecture, number, or size (Figure S2a-c). Moreover, β-cell density and functionality, as measured by glucose tolerance tests, were normal in RIP1-VEGFB mice (Figure S2d-e). Next, we analyzed the effects of the transgenic expression of VEGF-B on the vascular tree by immunostaining for the endothelial cell marker CD31 and by perfusion with fluorescein-labeled tomato lectin. Whereas there was no difference in the number of islet blood vessels (vascular density; Figure 1a-b), pancreatic islets of RIP1-VEGFB mice exhibited a 20% increase in the fraction of the islet area covered by vessels, as compared to wildtype mice (Figure 1a–b; 13.2±0.6% vs 11.0±0.6%, p<0.05). The increase in vessel area was consequent to an apparent increase in the diameter of pancreatic islet microvessels from 8.0±0.25 µm in non-transgenic mice to 9.7±0.50 µm in RIP1-VEGFB mice (Table 1; p<0.01), while vessel length was unchanged (Table 1). No overt differences in perfusion of the islet capillaries were noted (Figure 1a). Finally, to investigate whether islets of Langerhans from RIP1-VEGFB mice exhibited an increased angiogenic potential, we made use of an ex vivo collagen gel sprouting assay. Pancreatic islets were purified by limited collagenase digestion of the pancreas, and subsequently seeded into collagen gels together with human umbilical vein endothelial cells (HUVEC). Factors produced by the islet will diffuse into the gel and affect the phenotype of the co-cultured endothelial cells. Islets from RIP1-VEGFA mice were used to demonstrate migration and sprouting of HUVEC towards the islet upon the release of an angiogenic factor (Figure 1c). Whereas 30% of islets from RIP1-VEGFA mice exhibited angiogenic properties, only 13.6% of islets from RIP1-VEGFB mice were able to attract the co-cultured endothelial cells (Figure 1c). No islets from wildtype mice were overtly angiogenic in this assay (Figure 1c).

Bottom Line: 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.No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, Institute of Biochemistry and Genetics, University of Basel, Basel, Switzerland.

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.

Show MeSH
Related in: MedlinePlus