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Changes in vascular permeability and expression of different angiogenic factors following anti-angiogenic treatment in rat glioma.

Ali MM, Janic B, Babajani-Feremi A, Varma NR, Iskander AS, Anagli J, Arbab AS - PLoS ONE (2010)

Bottom Line: The findings were validated with histochemical and western blot studies.Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased K(trans) at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors.These findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital, Detroit, Michigan, United States of America.

ABSTRACT

Background: Anti-angiogenic treatments of malignant tumors targeting vascular endothelial growth factor receptors (VEGFR) tyrosine kinase are being used in different early stages of clinical trials. Very recently, VEGFR tyrosine kinase inhibitor (Vetanalib, PTK787) was used in glioma patient in conjunction with chemotherapy and radiotherapy. However, changes in the tumor size, tumor vascular permeability, vascular density, expression of VEGFR2 and other angiogenic factors in response to PTK787 are not well documented. This study was to determine the changes in tumor size, vascular permeability, fractional plasma volume and expression of VEGFR2 in PTK787 treated U-251 glioma rat model by in vivo magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). The findings were validated with histochemical and western blot studies.

Methodologies and principal findings: Seven days after implantation of U251 glioma cells, animals were treated with either PTK787 or vehicle-only for two weeks, and then tumor size, tumor vascular permeability transfer constant (K(trans)), fractional plasma volume (fPV) and expression of VEGFR2 and other relevant angiogenic factors were assessed by in vivo MRI and SPECT (Tc-99-HYNIC-VEGF), and by immunohistochemistry and western blot analysis. Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased K(trans) at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors. Size of the tumors was also increased in the treated group. Expression of VEGFR2 detected by Tc-99m-HYNIC-VEGF SPECT also showed significantly increased activity in the treated tumors. In PTK787-treated tumors, histological staining revealed increase in microvessel density in the close proximity to the tumor border. Western blot analysis indicated increased expression of VEGF, SDF-1, HIF-1alpha, VEGFR2, VEGFR3 and EGFR at the peripheral part of the treated tumors compared to that of central part of the treated tumors. Similar expression patters were not observed in vehicle treated tumors.

Conclusion: These findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot.

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Related in: MedlinePlus

Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGF, HIF-1α, SDF-1 and vessel morphology.Expression of vascular endothelial growth factor (VEGF) (dark brown colored) at different parts of the PTK787 treated and vehicle treated tumors. There were no differences observed in the expression of VEGF on immunohistochemistry at different parts of the tumors treated with either PTK787 or vehicle. However, delineation of vessels using FITC tagged tomato lectin indicated higher number of dilated vessels at the tumor periphery in rats that received PTK787 treatment. These dilated vessels may be indicative of increased permeability, fPV and signal intensity changes in PTK787 treated tumors (see Figures 1 and 2). Increased permeability may also be due to increased VEGF expression. HIF-1α expression was mostly seen in the central part of the vehicle treated tumors (arrows), however, SDF-1 expressions were observed both in PTK787 and vehicle treated tumors (arrows).
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pone-0008727-g004: Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGF, HIF-1α, SDF-1 and vessel morphology.Expression of vascular endothelial growth factor (VEGF) (dark brown colored) at different parts of the PTK787 treated and vehicle treated tumors. There were no differences observed in the expression of VEGF on immunohistochemistry at different parts of the tumors treated with either PTK787 or vehicle. However, delineation of vessels using FITC tagged tomato lectin indicated higher number of dilated vessels at the tumor periphery in rats that received PTK787 treatment. These dilated vessels may be indicative of increased permeability, fPV and signal intensity changes in PTK787 treated tumors (see Figures 1 and 2). Increased permeability may also be due to increased VEGF expression. HIF-1α expression was mostly seen in the central part of the vehicle treated tumors (arrows), however, SDF-1 expressions were observed both in PTK787 and vehicle treated tumors (arrows).

Mentions: There were no signs of necrosis within the tumor in both, PTK787 and vehicle treated tumors. Figure 4 shows the expression of HIF-1α, SDF-1, VEGF and vascular morphology and density as detected by FITC-labeled tomato lectin. Numerous HIF-1α positive cells were observed in the central part of the vehicle treated tumors, whereas none to very few positive cells were observed in the central part of the PTK-787 treated tumors. Despite the differences in HIF-1α expression, both PTK787 and vehicle treated tumors showed the expression of SDF-1, especially at the peripheral parts. Both groups of tumors exhibited strong expression of VEGF in the central tumor mass as well as at the satellite tumor foci. FITC-labeled tomato lectin staining showed multiple dilated vessels at the peripheral part of the PTK787 treated tumors. Both, the numbers and the morphology of the vessels seen in PTK787 treated tumors were different from the vessels that were observed in vehicle treated tumors. PTK787 treated tumors showed high level of expression of VRGFR2 and VEGFR3 receptors both, in the lining of dilated vessels and in the tumor cells (Figure 5). Multiple cells in the brain (possibly microglia) near the tumors also exhibited high expression of VEGFR2 and VEGFR3. Although both receptors were also expressed in the vehicle treated tumors, the number of cells expressing the receptors in the surrounding brain was less compared to that of PTK787 treated tumors. Both vehicle and PTK787 treated tumors showed EGFR positive cells in the tumors.


Changes in vascular permeability and expression of different angiogenic factors following anti-angiogenic treatment in rat glioma.

Ali MM, Janic B, Babajani-Feremi A, Varma NR, Iskander AS, Anagli J, Arbab AS - PLoS ONE (2010)

Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGF, HIF-1α, SDF-1 and vessel morphology.Expression of vascular endothelial growth factor (VEGF) (dark brown colored) at different parts of the PTK787 treated and vehicle treated tumors. There were no differences observed in the expression of VEGF on immunohistochemistry at different parts of the tumors treated with either PTK787 or vehicle. However, delineation of vessels using FITC tagged tomato lectin indicated higher number of dilated vessels at the tumor periphery in rats that received PTK787 treatment. These dilated vessels may be indicative of increased permeability, fPV and signal intensity changes in PTK787 treated tumors (see Figures 1 and 2). Increased permeability may also be due to increased VEGF expression. HIF-1α expression was mostly seen in the central part of the vehicle treated tumors (arrows), however, SDF-1 expressions were observed both in PTK787 and vehicle treated tumors (arrows).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806917&req=5

pone-0008727-g004: Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGF, HIF-1α, SDF-1 and vessel morphology.Expression of vascular endothelial growth factor (VEGF) (dark brown colored) at different parts of the PTK787 treated and vehicle treated tumors. There were no differences observed in the expression of VEGF on immunohistochemistry at different parts of the tumors treated with either PTK787 or vehicle. However, delineation of vessels using FITC tagged tomato lectin indicated higher number of dilated vessels at the tumor periphery in rats that received PTK787 treatment. These dilated vessels may be indicative of increased permeability, fPV and signal intensity changes in PTK787 treated tumors (see Figures 1 and 2). Increased permeability may also be due to increased VEGF expression. HIF-1α expression was mostly seen in the central part of the vehicle treated tumors (arrows), however, SDF-1 expressions were observed both in PTK787 and vehicle treated tumors (arrows).
Mentions: There were no signs of necrosis within the tumor in both, PTK787 and vehicle treated tumors. Figure 4 shows the expression of HIF-1α, SDF-1, VEGF and vascular morphology and density as detected by FITC-labeled tomato lectin. Numerous HIF-1α positive cells were observed in the central part of the vehicle treated tumors, whereas none to very few positive cells were observed in the central part of the PTK-787 treated tumors. Despite the differences in HIF-1α expression, both PTK787 and vehicle treated tumors showed the expression of SDF-1, especially at the peripheral parts. Both groups of tumors exhibited strong expression of VEGF in the central tumor mass as well as at the satellite tumor foci. FITC-labeled tomato lectin staining showed multiple dilated vessels at the peripheral part of the PTK787 treated tumors. Both, the numbers and the morphology of the vessels seen in PTK787 treated tumors were different from the vessels that were observed in vehicle treated tumors. PTK787 treated tumors showed high level of expression of VRGFR2 and VEGFR3 receptors both, in the lining of dilated vessels and in the tumor cells (Figure 5). Multiple cells in the brain (possibly microglia) near the tumors also exhibited high expression of VEGFR2 and VEGFR3. Although both receptors were also expressed in the vehicle treated tumors, the number of cells expressing the receptors in the surrounding brain was less compared to that of PTK787 treated tumors. Both vehicle and PTK787 treated tumors showed EGFR positive cells in the tumors.

Bottom Line: The findings were validated with histochemical and western blot studies.Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased K(trans) at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors.These findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital, Detroit, Michigan, United States of America.

ABSTRACT

Background: Anti-angiogenic treatments of malignant tumors targeting vascular endothelial growth factor receptors (VEGFR) tyrosine kinase are being used in different early stages of clinical trials. Very recently, VEGFR tyrosine kinase inhibitor (Vetanalib, PTK787) was used in glioma patient in conjunction with chemotherapy and radiotherapy. However, changes in the tumor size, tumor vascular permeability, vascular density, expression of VEGFR2 and other angiogenic factors in response to PTK787 are not well documented. This study was to determine the changes in tumor size, vascular permeability, fractional plasma volume and expression of VEGFR2 in PTK787 treated U-251 glioma rat model by in vivo magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). The findings were validated with histochemical and western blot studies.

Methodologies and principal findings: Seven days after implantation of U251 glioma cells, animals were treated with either PTK787 or vehicle-only for two weeks, and then tumor size, tumor vascular permeability transfer constant (K(trans)), fractional plasma volume (fPV) and expression of VEGFR2 and other relevant angiogenic factors were assessed by in vivo MRI and SPECT (Tc-99-HYNIC-VEGF), and by immunohistochemistry and western blot analysis. Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased K(trans) at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors. Size of the tumors was also increased in the treated group. Expression of VEGFR2 detected by Tc-99m-HYNIC-VEGF SPECT also showed significantly increased activity in the treated tumors. In PTK787-treated tumors, histological staining revealed increase in microvessel density in the close proximity to the tumor border. Western blot analysis indicated increased expression of VEGF, SDF-1, HIF-1alpha, VEGFR2, VEGFR3 and EGFR at the peripheral part of the treated tumors compared to that of central part of the treated tumors. Similar expression patters were not observed in vehicle treated tumors.

Conclusion: These findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot.

Show MeSH
Related in: MedlinePlus