Abdulkadir Ozkan

Endovascular treatment increases but gamma knife radiosurgery decreases angiogenic activity of arteriovenous malformations: an in vivo experimental study using a rat cornea model.

OBJECTIVETo compare the angiogenic potentials of embolized, gamma knife–treated or untreated cerebral arteriovenous malformations (AVMs), using a rat cornea angiogenesis model. METHODSTissue samples from cerebral AVM patients who were either untreated or had previously been treated with embolization or gamma knife radiosurgery and who had undergone operations for hemorrhage at the Neurosurgery Department or the Neurological Sciences Institute of Marmara University were used. For the macroscopic evaluation of angiogenesis, tissue samples were inoculated in a micropocket created on the rat eye, and the level of angiogenic activity was graded macroscopically for 15 days, with glioblastoma multiforme and normal brain artery tissues serving as positive and negative controls, respectively. For the other part of the experiment, eyes of another set of rats were inoculated with the study samples only using the same cornea angiogenesis model, in which microvessel count and vascular endothelial growth factor assessment was done at days 3, 7, 11, and 15. RESULTSBased on our macroscopic findings in the cornea angiogenesis model, embolized AVMs exhibited the highest angiogenic activity, followed by untreated AVMs and gamma knife–treated AVMs. Evaluations of vascular endothelial growth factor expression and microvessel counts showed a similar relation among the 3 tissue groups with regard to the level of angiogenic activity, supporting the results of macroscopic examinations. CONCLUSIONThis study, for the first time, provides experimental semiquantitative data to compare the angiogenic potentials of embolized and gamma knife–treated AVM tissues. Embolization may increase angiogenic activity, and gamma knife radiosurgery may decrease it when compared with activity in previously untreated AVMs. These data can be useful to understand why recurrence of AVMs after angiographically demonstrated endovascular occlusion is common but after gamma knife occlusion is rare.

Expressions of angiogenesis associated matrix metalloproteinases and extracellular matrix proteins in cerebral vascular malformations

This study aimed to compare cerebral arteriovenous malformations (cAVM) and cerebral cavernous malformations (CCM) with regard to the immunohistochemical expressions of matrix metalloproteinases (MMP) and selected extracellular matrix (ECM) proteins, which have a role in the regulation of angiogenesis. Fresh-frozen surgical specimens from patients with cAVM (n=14) and CCM (n=15) were immunohistochemically stained with antibodies for MMP-2, MMP-9, laminin, fibronectin and tenascin. To compare cAVM and CCM, expression of each protein was graded using a four-point scoring system for each histological layer of the lesion. MMP-2 and MMP-9 were more strongly expressed in the vascular walls of CCMs compared to cAVMs for all comparable layers: endothelium, subendothelium and the perivascular space. The stronger expression of MMP and other EMP associated with early angiogenesis in CCMs compared to AVMs may support the hypothesis that CCMs occur at earlier embryogenic stages than AVMs.

Expression of angiogenic factors in craniopharyngiomas: implications for tumor recurrence.

BACKGROUNDThe primary treatment for craniopharyngiomas is total excision, but recurrence is common. However, current knowledge on the mechanisms of recurrence is limited. OBJECTIVEWe hypothesized that recurrence is linked to the angiogenesis of the tumor. Recurrent and nonrecurrent tumor samples were compared with regard to expression of angiogenesis-related factors and angiogenic capacity in a corneal angiogenesis model. METHODSSpecimens of 4 recurrent and 6 nonrecurrent tumors were selected from 57 patients with adamantinomatous craniopharyngiomas. Sections were immunohistochemically stained with antibodies for vascular endothelial growth factor (VEGF), fibronectin, fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-A, PDGF-B, platelet-derived growth factor receptor (PDGFR)-α, and PDGFR-β. Expression levels were graded using a 4-point scoring system and were compared. For corneal angiogenesis assay, tissue samples were inoculated in a micropocket created on the rat eye, and microvessels were counted on days 3, 5, 7, and 9 to evaluate angiogenic potential. RESULTSExpression of PDGFR-α and FGF-2 were significantly higher for recurrent tumors (P = .02 and P = .01). However, recurrent and nonrecurrent tumors did not differ in the expressions of other ligands and receptors (PDGF-A, PDGF-B, and PDGFR-β). Recurrent tumors displayed a higher angiogenic potential starting from the fifth day of corneal angiogenesis assay. CONCLUSIONThese findings suggest a relationship between recurrence of craniopharyngiomas and angiogenesis. New treatment modalities with selective PDGFR-α blockers may represent a novel and effective therapeutic option for the treatment of craniopharyngiomas.

Identification of novel neutralizing single-chain antibodies against vascular endothelial growth factor receptor 2.

Human vascular endothelial growth factor (VEGF) and its receptor (VEGFR‐2/kinase domain receptor [KDR]) play a crucial role in angiogenesis, which makes the VEGFR‐2 signaling pathway a major target for therapeutic applications. In this study, a single‐chain antibody phage display library was constructed from spleen cells of mice immunized with recombinant human soluble extracellular VEGFR‐2/KDR consisting of all seven extracellular domains (sKDR D1–7) to obtain antibodies that block VEGF binding to VEGFR‐2. Two specific single‐chain antibodies (KDR1.3 and KDR2.6) that recognized human VEGFR‐2 were selected; diversity analysis of the clones was performed by BstNI fingerprinting and nucleotide sequencing. The single‐chain variable fragments (scFvs) were expressed in soluble form and specificity of interactions between affinity purified scFvs and VEGFR‐2 was confirmed by ELISA. Binding of the recombinant antibodies for VEGFR‐2 receptors was investigated by surface plasmon resonance spectroscopy. In vitro cell culture assays showed that KDR1.3 and KDR2.6 scFvs significantly suppressed the mitogenic response of human umbilical vein endothelial cells to recombinant human VEGF165 in a dose‐dependent manner, and reduced VEGF‐dependent cell proliferation by 60% and 40%, respectively. In vivo analysis of these recombinant antibodies in a rat cornea angiogenesis model revealed that both antibodies suppressed the development of new corneal vessels (p < 0.05). Overall, in vitro and in vivo results disclose strong interactions of KDR1.3 and KDR2.6 scFvs with VEGFR‐2. These findings indicate that KDR1.3 and KDR2.6 scFvs are promising antiangiogenic therapeutic agents.

Relationship of angiogenic potential with clinical features in cranial meningiomas: a corneal angiogenesis study.

BACKGROUND: Intracranial meningiomas constitute approximately one fourth of all primary intracranial tumors. The invention of cranial angiographic techniques has led to the recognition of the angiogenic potential of meningiomas, which has been the subject of extensive research. OBJECTIVE: To test the relationship between the angiogenetic potential of intracranial meningiomas and clinical/prognostic features such as World Health Organization (WHO) grade, peritumoral edema, tumor border shape, and recurrence using rat corneal angiogenesis assay. METHODS: Fifteen WHO grade I (typical), 10 WHO grade II (atypical), and 5 WHO grade III (malignant) meningioma samples were implanted in the micropockets formed on rat corneas, and the number of developed vessels were counted on days 5, 10, 15, and 20. Normal brain and glioblastoma multiforme tissues served as negative and positive controls, respectively. Patients were evaluated by magnetic resonance imaging preoperatively and every 6 months thereafter. RESULTS: The angiogenic potential of WHO grade II tumors was significantly lower than that of grade III tumors and higher than that of grade I tumors throughout the experiment. Tumors with a smooth border shape and nonrecurrent tumors exhibited significantly lower angiogenic activity compared with the tumors with irregular border shape and recurrent tumors, respectively. No association was found between angiogenic activity and peritumoral edema. However, multivariate analysis identified WHO grade, recurrence, and peritumoral edema as significant predictors of a high angiogenic potential. CONCLUSION: Our findings, based on a dynamic in vivo model to examine angiogenesis, demonstrate that the angiogenic potential of meningiomas is correlated with WHO grade, recurrence, and possibly with tumor border shape and peritumoral edema. Angiogenesis seems to be an important factor in the natural course of meningiomas, suggesting that inhibition of angiogenesis may be an option, particularly in the treatment of meningiomas with an aggressive course.

Expression of platelet-derived growth factor ligand and receptor in cerebral arteriovenous and cavernous malformations.

The aim of this study was to investigate the expression of platelet-derived growth factor (PDGF) ligands A and B and receptors α and β in cerebral arteriovenous and cavernous malformations. Fifteen arteriovenous malformation (AVM) and 15 cerebral cavernous malformation (CCM) tissue samples were immunostained for PDGF ligands A and B, PDGF receptors (PDGFR) α and β, and vascular endothelial growth factor. Tissues were compared in terms of expression levels within various vascular layers, and the results were confirmed using western blotting. AVM had higher levels of PDGF-A expression than CCM (p = 0.004, 0.009, 0.001, and 0.027, for endothelium, media, adventitia, and perilesional tissue, respectively) and western blotting showed that there was higher expression of PDGFR-α in AVM tissues. In contrast, CCM endothelium, media, and adventitia had higher PDGF-B expression compared with AVM (p = 0.007, 0.001, and 0.039, respectively). PDGFR-β expression was also significantly higher in the endothelium of CCM tissue (p = 0.007). Overexpression of PDGF ligands and receptors in AVM and CCM may mean that therapeutic strategies targeting the PDGF pathway could be useful in the treatment of these two malformations.

Temporal expression analysis of angiogenesis-related genes in brain development

BackgroundThe current knowledge on molecular pathogenesis of cerebral vascular malformations (CVM), which are believed to arise during development, is very limited. To unravel the molecular mechanisms involved in CVMs, a detailed understanding of the brain vascular development at molecular level is crucial. In this study, we aimed to explore the temporal and comparative expression profile of angiogenesis-related genes in the establishment of brain vasculature.MethodsExpression of a total of 113 angiogenesis-related genes during murine brain development has been analyzed using low-density array systems designed for angiogenesis-related genes. Bai1 (brain specific angiogenesis inhibitor-1), a recently identified novel anti-angiogenic gene, has been selected for further characterization.ResultsWe found that 62 out of 113 analyzed genes have expression in brain development at varying levels. Nineteen of these were differentially expressed between embryonic and postnatal stages (>1.5 fold). Bai1 is strongly expressed on growing blood vessels of cerebral cortex and hippocampus, partially expressed in the lateral regions of striatum, but mostly absent on the thalamus.ConclusionBy showing the comparative expression analysis of angiogenesis-related genes throughout brain development, the data presented here will be a crucial addition to further functional studies on cerebrovascular research.

Temporal Expression of Angiogenesis-Related Genes in Developing Neonatal Rodent Retina: A Novel in Vivo Model to Study Cerebral Vascular Development

BACKGROUNDExperimental models to study cerebrovascular malformations are limited therefore we used the neonatal rodent retina as a model to study cerebral angiogenesis. OBJECTIVEWe performed a gene expression analysis to define temporal changes in the expression of 96 angiogenesis-related genes during retinal vascularization. METHODSA total of 72 retinas from 36 newborn C57BL/6 mice were used. Sets of neonatal mouse retinas were surgically isolated by 2-day intervals starting from postnatal day 0 to day 20 and at the 32nd day (representing adult retinas). For each of these 12 time points in the postnatal developmental period of mouse retinas, separate sets of 6 retinas from 3 mice were pooled, and their RNA was hybridized to an angiogenesis-specific gene array. Temporal expression patterns of each of the 96 angiogenesis-related genes were analyzed. For confirmation, vascular endothelial growth factor protein expression was also studied by immunohistochemistry. RESULTSTwenty-two of the 96 genes analyzed displayed a significantly different temporal expression profile, and the rest exhibited a static expression, as compared to the human glyceraldehyde-3-phosphate dehydrogenase gene. Among these genes, the temporal pattern of expression was variable, but peaks were seen mostly on days 8, 10, 12, and 16. This timing corresponds well to morphologic changes that occur in the retina during different stages of angiogenesis. CONCLUSIONThe neonatal rodent retina, which has a cellular architecture similar to that of the brain, has active and quantifiable angiogenic activity during the neonatal period and can be used as a simple and convenient model to study cerebral angiogenesis.

High angiogenic potential in an in vivo rat corneal model is associated with shorter disease-free survival in low-grade oligodendrogliomas

This study aimed to examine the association between time to tumor recurrence, angiogenic potential and tumor contrast-enhancement. Tumor samples were taken from 20 patients with low-grade oligodendroglioma and examined for their angiogenic potential using an in vivo rat corneal model of angiogenesis. Patients were evaluated for tumor contrast enhancement prior to surgical excision using MRI and they were followed for tumor recurrence. Patients who had tumors without contrast enhancement had longer disease-free survival (median time to tumor recurrence, 72 months) compared to those who had tumors with contrast enhancement (median, 42 months; p=0.0068). Based on corneal angiogenesis assay results, a high angiogenic potential was associated with a significantly shorter disease-free survival. Our findings suggest that radiological contrast enhancement and a high angiogenic potential based on an in vivo corneal angiogenesis assay were related to a shorter disease-free survival. This might have important prognostic implications in patients with low-grade oligodendrogliomas.