Date Published: January 15, 2010
Publisher: Public Library of Science
Author(s): Meser M. Ali, Branislava Janic, Abbas Babajani-Feremi, Nadimpalli R. S. Varma, A. S. M. Iskander, John Anagli, Ali S. Arbab, Andrew Boswell. http://doi.org/10.1371/journal.pone.0008727
Abstract: 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.
Partial Text: Malignant gliomas are among the most devastating tumors, with survival of only one to three years after diagnosis, even with the best of treatments combining surgery, radiation and chemotherapy , . Because of the hypervascular nature of glioblastoma and the associated active angiogenesis, investigators have added anti-angiogenic treatment as an adjuvant to normalize blood vessels and control abnormal angiogenesis , , , . Angiogenesis is essential for glioma tumor growth and metastasis. Malignant glioma cells release vascular endothelial growth factor (VEGF), an important regulator and promoter of angiogenesis . Animal studies have indicated that angiogenesis and increased vascular permeability are essential for the proliferation and survival of glioma cells . Vascular endothelial growth factor, also termed vascular permeability factor (VPF), is well-studied multifunctional cytokine considered to play a pivotal role in the induction of tumor angiogenesis. In vitro and in vivo data suggest that VEGF/VPF is an endothelial-cell specific mitogen . In addition to having a mitogenic activity, VEGF/VPF is a potent vascular permeability enhancer . VEGF/VPF has been shown to increase the permeability of micro vessels to plasma macromolecules with a potency approximating 50,000 times that of histamine . Expression of VEGF and its receptors correlates to the degree of tumor vascularization and has been proposed as a prognostic factor for assessing patient survival . High-affinity cognate VEGF endothelial receptors are VEGFR-1/Flt-1 and VEGFR-2/Flk-1/KDR and both receptors have been shown to be important regulatory factors for vasculogenesis and physiological angiogenesis . However, the interaction of VEGF/VPF with Flk-1/VEGFR2 is thought to be more important for tumor angiogenesis because it is essential for induction of the full spectrum of VEGF/VPF functions . In fact, many compounds and molecules developed to block VEGF/VPF activities mediated by Flk-1/VEGFR2 have been shown to have antiangiogenic activity in animal models , . One such molecule is PTK787 that inhibits the phosphorylation of Flk-1/VEGFR2 and Flt-1 receptors by binding to and inhibiting the protein kinase domain of these receptors . By directly inhibiting the phosphorylation of the VEGF/VPF receptor tyrosine kinases, PTK787 suppresses angiogenesis induced by VEGF/VPF. At slightly higher doses, it also inhibits PDGF receptor tyrosine kinase . PTK787 demonstrated efficacy in preclinical and Phase I/II trials where it significantly reduced tumor vessel density and in some cases induced tumor regression . PTK787 significantly inhibited growth of breast tumors in vivo and disrupted tumor microvasculature after five days of treatment . However, it has also been noted that continued anti-angiogenic therapy targeting only the VEGF-VEGFR system might activate pro-angiogenic factors other than VEGF, such as basic fibroblast growth factor (bFGF), stromal derived factor 1 (SDF-1) and Tie2 , and may mobilize circulating endothelial cells and bone marrow derived precursor cells that are known to promote angiogenesis , , . Thus, the inhibitory therapy targeting VEGF and/or VEGFRs may paradoxically end up enhancing angiogenic and pro-growth responses. Moreover, abundant VEGF over expression has been demonstrated in human malignant glioma animal model . However, there has been no report on the changes in the expression pattern of different angiogenic factors in glioma following treatment with antiangiogenic agents.
Results from these studies showed that orally administered PTK787 drug increased the tumor vascular permeability and tumor growth in rat model of human glioma. Surprisingly, even post contrast T1WI showed distinct differences between PTK787- and vehicle-treated tumors. The MR images showed bright contrast enhancement at the tumor rim and less contrast in the tumor center after therapy. T2WI also showed larger high signal intensity areas beyond the margin of post-contrast enhancement on T1WI. Analysis of the signal intensity ratio (tumor rims versus contra lateral brains) showed significantly higher values as early as 3 minutes after the administration of contrast agents. Our results demonstrated the unexpected, paradoxical effects of antiangiogenic treatment with VEGFRs inhibitors. Malignant gliomas are hypervascular tumors characterized by release of vascular endothelial growth factor (VEGF), an important regulator and promoter of angiogenesis . Animal studies indicated that angiogenesis and increased vascular permeability are essential for the proliferation and survival of glioma cells . Therefore, it was thought that anti-angiogenic therapy targeting VEGF or VEGF receptors (VEGFRs) would become an effective tool for controlling malignant glioma. However, our results are contradictory to this hypothesis and contradict the recent studies that reported decrease in tumor size of implanted glioma (and other tumors) as well as the decrease in permeability after treatment with VEGFRs inhibitor , . However, in these reported studies, the investigators started the treatment soon after implantation and therefore might have not allowed for the sufficient time for the clinically relevant tumor angiogenesis to be generated. In our study, we started PTK787 treatment 7 days after tumor implantation, to mimic the clinical scenario of a developed malignant glioma with extensive angiogenesis. Recently, tyrosine kinase inhibitors (such as vetanalib) that target different VEGFRs have also been used in clinical trials ,  with a limited success. However, it has been noted that continued anti-angiogenic therapy targeting only the VEGF-VEGFR system might activate pro-angiogenic factors other than VEGF, such as basic fibroblast growth factor (bFGF), stromal derived factor 1 (SDF-1) and Tie2 , and may mobilize circulating endothelial cells and bone marrow derived precursor cells that are known to promote angiogenesis , , . Our results also showed increased expression of VEGF, HIF-1α, SDF-1 following the treatment with PTK787, indicating activation of pro-angiogenic factors, which may cause more angiogenesis and increase in vascular permeability. This effect is in part due to the accumulation of endothelial progenitor cells, since HIF-1α induced increase in SDF-1expression is a strong chemo-attractant for endothelial progenitor cells .