Human Cerebral Arteriovenous Malformations Research Articles

DNA methylation signatures on vascular differentiation genes are aberrant in vessels of human cerebral arteriovenous malformation nidus

Arteriovenous malformation (AVM) is a tangle of arteries and veins, rupture of which can result in catastrophic hemorrhage in vulnerable sites such as the brain. Cerebral AVM is associated with a high mortality rate in humans. The causative factor or the stimulus at the artery-venous junction and the molecular basis of the development and progression of cerebral AVM remain unknown. While it is known that aberrant hemodynamic forces in the artery-vein junction contribute to the development of AVMs, the mechanistic pathways are unclear. Given that various environmental stimuli modulate epigenetic modifications on the chromatin of cells, we speculated that misregulated DNA methylome could lead to cerebral AVM development. To identify the aberrant epigenetic signatures, we used AVM nidus tissues and analyzed the global DNA methylome using the Infinium DNA methylome array. We observed significant alterations of DNA methylation in the genes associated with the vascular developmental pathway. Further, we validated the DNA hypermethylation by DNA bisulfite sequencing analysis of selected genes from human cerebral AVM nidus. Taken together, we provide the first experimental evidence for aberrant epigenetic signatures on the genes of vascular development pathway, in human cerebral AVM nidus.

Procedure for the Isolation of Endothelial Cells from Human Cerebral Arteriovenous Malformation (cAVM) Tissues.

In this study, we successfully established a stable method for the isolation of endothelial cells (ECs) from human cerebral arteriovenous malformation (cAVM) tissues. Despite human cAVM tissues having a minor population of ECs, they play an important role in the manifestation and development of cAVM as well as in hemorrhagic stroke and thrombogenesis. To characterize and understand the biology of ECs in human cAVM (cAVM-ECs), methods for the isolation and purification of these cells are necessary. We have developed this method to reliably obtain pure populations of ECs from cAVMs. To obtain pure cell populations, cAVM tissues were mechanically and enzymatically digested and the resulting single cAVM-ECs suspensions were then labeled with antibodies of specific cell antigens and selected by flow cytometry. Purified ECs were detected using specific makers of ECs by immunostaining and used to study different cellular mechanisms. Compared to the different methods of isolating ECs from tissues, we could isolate ECs from cAVMs confidently, and the numbers of cAVM-ECs harvested were almost similar to the amounts present in vessel components. In addition to optimizing the protocol for isolation of ECs from human cAVM tissues, the protocol could also be applied to isolate ECs from other human neurovascular-diseased tissues. Depending on the tissues, the whole procedure could be completed in about 20 days.

In vitro characteristics of endothelial cells prepared from human cerebral arteriovenous malformation lesions using a novel method

Background and purposeThe cerebral arteriovenous malformation (cAVM) is a usual and continually unaware reason of heamorrhage and seizure. It contains of feeder arteries, drain veins and abnormal vessel nets. However, pathologic mechanisms of the development of cAVM are unknown. The purpose of this study was to explore a novel protocol to isolate, culture and passage endothelial cells (ECs) from human cAVM lesions. MethodsWe developed a protocol for isolating and growing ECs from eight patients with cAVM. The tissues were microsurgically removed from cAVM lesion and were digested by 0.25% Trypsin-EDTA, and cultured in ECM medium. ECs were selected by FACS and confirmed their EC origin by immunocytochemistry of the basic expression patterns of CD31 and CD34. LDL-uptake and capillary tube formation were used to determine their functional features. ResultsThe isolated cAVM-ECs exhibited contact inhibition of growth and appearance of rounded cobblestone. cAVM-ECs were CD31- and CD34-positive. In functional assays, cAVM-ECs were able to uptake LDL and form capillary tubes. cAVM-ECs from younger patients proliferated faster than that from elders, and cAVM-ECs were less stable than normal artery ECs. In addition, cAVM-ECs appeared to more easily transform into mesenchymal cells than normal artery ECs. ConclusionUsing the protocol, isolated cAVM-ECs are stably established, and retain their endothelial phenotypes. These cAVM-ECs may provide a biological tool to examine molecular phenotypes and mechanisms responsible for human cAVM.

An efficient and non-enzymatic method for isolation and culture of endothelial cells from the nidus of human cerebral arteriovenous malformations

In this report, we describe an efficient and non-enzymatic method for isolating and culturing endothelial cells (ECs) from the nidus of surgically resected arteriovenous malformation (AVM) specimens. These cultured cells possessed typical phenotypic markers (i.e. von Willebrand factor and CD34), as well as morphological and ultrastructural characteristics of ECs. However, they had activated Notch-1 signaling, which plays a critical role in the development of AVM. The present study suggests that hypoxic endothelial cells from the nidus of human cerebral arteriovenous malformation (CAVMECs) have angiogenic potentials, as our data showed that VEGF gene expression and cell proliferation were more evident with prolonged hypoxia. In our study, we successfully used the vascular tissue explants adherent method to isolate and culture CAVMECs with high purity. This may prove to be a useful tool for studying the molecular mechanisms that mediate abnormal vessel development and maintenance in AVM.

Gene Expression Signatures in the Peripheral Blood After Radiosurgery of Human Cerebral Arteriovenous Malformations

To unravel biological mechanisms potentially resulting in the obliteration process after radiosurgery (RS) of human cerebral arteriovenous malformations (AVMs) by investigating molecular signatures on the transcriptomic level in peripheral blood of patients. Venous blood samples were obtained at definite points of time before and after RS. The samples were tested for radiation-induced changes regarding biological markers (mRNA) using cDNA and oligo-microarray technology. The corresponding expression profiles were correlated with clinical data and obliteration signs in radiologic imaging. The proof of principle that RS outcome can be successfully correlated with transcriptomics of cellular blood components as disease parameter was demonstrated. The authors identified 76 differentially regulated genes (p < 0.001) after RS. Interestingly, in particular genes with known roles in anti-angiogenic and pro-coagulative pathways were identified as potentially relevant. In particularly, the authors found a significant downregulation of neuropilin-2, protein C inhibitor and cyclin-dependent kinase 6. They also found that low pretreatment blood mRNA levels of TLR4 (toll-like receptor 4) and STAT3 (signal transducer and activator of transcription 3) correlated with fast obliteration of AVMs. The authors report on a novel technique for molecular biological analysis of blood from patients with cerebral AVM treated with RS. Differential regulation of genes in peripheral blood was successfully correlated with RS and time to obliteration of AVMs. The identified genes indicate a potential new methodology to monitor RS, which may result in an individualized therapy and optimized follow-up.

Neuronal expression of Fas-associated death domain protein and caspase-8 in the perinidal parenchyma of cerebral arteriovenous malformations

The expression and localization of phosphorylated Fas-associated death domain protein (pFADD) and cleaved caspase-8 was examined in human cerebral arteriovenous malformations (AVMs). The authors focused on the perinidal parenchyma to clarify the effect of AVMs on perinidal brain tissue. Seventeen cerebral AVMs were analyzed using immunohistochemical methods. Specimens were removed from patients during surgical procedures. The characteristics of the areas that stained positively for pFADD or cleaved caspase-8 were also assessed using an image analysis system. Eleven (65%) of the 17 lesions demonstrated anti-pFADD immunoreactivity and 12 (71%) showed anti-cleaved caspase-8 immunoreactivity. The immunoreactive cells in the perinidal parenchyma demonstrated obvious neuronal morphological characteristics. The characteristics of pFADD-positive and cleaved caspase-8-positive areas were assessed using the image analysis system. The mean distance from the nidus adjacent to either area was not affected by preoperative hemorrhage. The neuronal densities of pFADD-positive and cleaved caspase-8-positive areas were analyzed using the same system. The density of the control area (samples that were pFADD-negative and cleaved caspase-8 negative) was significantly higher when compared with that of pFADD-positive and cleaved caspase-8-positive areas (p < 0.05). The expressions of cleaved caspase-9, cleaved poly(adenosine diphosphate-ribose) polymerase, and apoptotic cells were analyzed using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method. Neuronal areas that stained positively for pFADD and cleaved caspase-8 existed around the nidus of AVMs. In these areas, the neuronal density was lower than that in the other parenchyma around the AVM. Neuronal loss around the nidus may be the origin of brain dysfunction around AVMs.

Proliferative Activity through Extracellular Signal-regulated Kinase of Smooth Muscle Cells in Vascular Walls of Cerebral Arteriovenous Malformations

We examined the expression and localization of phosphorylated extracellular signal-regulated kinase (pERK) and proliferation-related antigens in human cerebral arteriovenous malformations (AVMs) to clarify the role of vascular remodeling via this pathway in the development of the lesions. Thirteen cerebral AVMs and five control specimens were analyzed using immunohistochemical methods. Specimens were obtained from the patients during the surgical procedures. Control middle cerebral artery samples were obtained during autopsies. We performed immunohistochemical analysis of AVMs by using an antibody specifically recognizing pERK. pERK immunoreactivity was detected in all specimens. Among the control specimens, only weak pERK immunoreactivity was detected, mainly in the intima. pERK immunoreactivity was located in nuclei of cells in the endothelial layer and media. Semiquantitative analysis for pERK immunoreactivity showed that the immunoreactivity score in the media was significantly higher for the AVM than for the control specimens. The results of double staining for pERK and proliferating cell nuclear antigen indicated that these immunoreactivities were colocalized in the same cells. Moreover, those cells in the media were immunoreactive for alpha-actin, indicating that they were smooth muscle cells. pERK was detected in smooth muscle cells of the vascular walls of AVMs. It may function in the proliferative activity of smooth muscle cells. Vascular remodeling through pERK may play an important role in the growth and maintenance of cerebral vascular malformations.

Hypoxia inducible factor-1α and expression of vascular endothelial growth factor and its receptors in cerebral arteriovenous malformations

Vascular endothelial growth factor (VEGF) and its tyrosine kinase family of receptors (VEGFR) (Flt-1, Flk-1, Flt-4) have been implicated in vascular angiogenesis and remodelling in cerebral arteriovenous malformations (CAVM). In this study, we investigate the role of hypoxia inducible factor-1 (HIF-1alpha) in CAVM and its relationship to VEGF and VEGFR. Surgical specimens from 26 patients undergoing CAVM resection were studied for HIF-1alpha , VEGF, Flt-1, Flk-1 and Flt-4. The mean age was 34.08 +/- 14.18 years. Twenty-one patients presented with intracerebral haemorrhage. VEGF, Flt-1 and Flt-4 were expressed in all specimens. Flk-1 was expressed in 15 of 26 patients. HIF-1alpha was expressed in 15 of 26 patients. HIF-1alpha expression was significantly associated with VEGF, Flt-1 and Flk-1 expression (p < 0.05) HIF-1alpha is expressed in human CAVM. The expression of HIF-1alpha is significantly related to VEGF and VEGFR expression, suggesting a possible role for its induction and role in maintaining angiogenesis and vascular remodelling.

Effect of a single high-dose gamma irradiation on cultured cells in human cerebral arteriovenous malformation

Object. The purpose of this study was to analyze the effect of single high-dose gamma irradiation at a cellular biological level on tissue cultures obtained in patients who underwent surgery for cerebral arteriovenous malformation (AVM). Methods. The cell proliferation indices and changes in activation of p53, p21Waf-1, and mdm-2 were determined. Additionally, immunohistochemical investigations for vimentin, desmin, α—smooth muscle actin (α-SMA), glial fibrillary acidic protein, Factor VIII—related antigen (F-VIII), cytokeratin, S100, and transforming growth factor—β (TGFβ) were performed on cultured AVM cells after a single high-dose irradiation. Normal human brain microvessel endothelial (HBE) cells and aortic smooth muscle cells served as controls. The proliferation index decreased on the 5th day after irradiation and remained depressed over the observation period in the irradiated AVM cultures. The p53, p21Waf-1, and mdm-2 messenger RNA measurements showed considerable elevation both in AVM cultures and HBE cells after 15-Gy irradiation, which indicated apoptosis. Immunohistochemistry revealed strong vimentin positivity in the nonirradiated cultures, which gradually decreased in the irradiated cultures. Transforming growth factor—β positivity was demonstrated in the irradiated specimens, indicating transformation of fibroblastic cells into activated myofibroblastic elements. This transformation was confirmed by demonstrating elevated SMA expression as well in the radiation-treated fibroblasts. Conclusions. The presence of TGFβ and α-SMA activity in the irradiated AVM cells suggests that along with the genetically confirmed apoptotic activity, fibroblast transformation into myofibroblasts might be one of the mechanisms leading to shrinkage and obliteration of AVMs after single high-dose gamma irradiation.

A new rat model of chronic cerebral hypoperfusion associated with arteriovenous malformations.

A new experimental model of chronic cerebral hypoperfusion was developed to study the effects of systemic arterial shunting and obstruction of the primary vessel that drains intracranial venous blood on cerebral perfusion pressure (CPP), as well as cerebral pathological changes during restoration of normal perfusion pressure. Twenty-four Sprague-Dawley rats were randomly assigned to either a sham-operated group, an arteriovenous fistula (AVF) group, or a model group (eight rats each). The animal model was readied by creating a fistula through an end-to-side anastomosis between the right distal external jugular vein (EJV) and the ipsilateral common carotid artery (CCA), followed by ligation of the left vein draining the transverse sinus and bilateral external carotid arteries. Systemic mean arterial pressure (MAP), draining vein pressure (DVP), and CPP were monitored and compared among the three groups preoperatively, immediately postoperatively, and again 90 days later. Following occlusion of the fistula after a 90-day interval, blood-brain barrier (BBB) disruption and water content in the right cortical tissues of the middle cerebral artery territory were confirmed and also quantified with transmission electron microscopy. Formation of a fistula resulted in significant decreases in MAP and CPP, and a significant increase in DVP in the AVF and model groups. Ninety days later, there were still significant increases in DVP and decreases in CPP in the model group compared with the other groups (p < 0.05). Damage to the BBB and brain edema were noted in animals in the model group during restoration of normal perfusion pressure by occlusion of the fistula. Electron microscopy studies revealed cerebral vasogenic edema and/or hemorrhage in various amounts, which correlated with absent astrocytic foot processes surrounding some cerebral capillaries. The results demonstrated that an end-to-side anastomosis between the distal EJV and CCA can induce a decrease in CPP, whereas a further chronic state of cerebral hypoperfusion may be caused by venous outflow restriction, which is associated with perfusion pressure breakthrough. This animal model conforms to the basic hemodynamic characteristics of human cerebral arteriovenous malformations.

Abnormal Pattern of Tie-2 and Vascular Endothelial Growth Factor Receptor Expression in Human Cerebral Arteriovenous Malformations

Human cerebral arteriovenous malformations (AVMs) are speculated to result from abnormal angiogenesis. Vascular endothelial growth factor receptors (VEGF-Rs) and Tie-2 play critical roles in vasculogenesis and angiogenesis. We hypothesized that the abnormal vascular phenotype of AVMs may be associated with abnormal expression of VEGF-Rs and Tie-2. We measured the expression of Tie-2, VEGF-R1, and VEGF-R2 in AVMs and normal brain tissue, using immunoblotting. To assess active vascular remodeling, we also measured endothelial nitric oxide synthase expression. CD31 expression was used to control for endothelial cell mass for Tie-2, VEGF-Rs, and endothelial nitric oxide synthase. Immunoblotting data were presented as relative expression, using normal brain tissue values as 100%. CD31 was expressed to similar degrees in AVMs and normal brain tissue (99+/-29% versus 100+/-20%, mean +/- standard error, P = 0.98). Tie-2 expression was markedly decreased in all AVMs, compared with normal brain tissue (16+/-9% versus 100+/-37%, P = 0.04). VEGF-R1 expression was decreased in four of five AVMs, but the difference between the mean values was not significant (35+/-8% versus 100+/-42%, P = 0.14). VEGF-R2 expression was decreased in all AVMs, compared with normal brain tissue (28+/-6% versus 100+/-29%, P = 0.03). There was no difference in endothelial nitric oxide synthase expression between AVMs and normal brain tissue (106+/-42% versus 100+/-25%, P = 0.91). AVM vessels exhibited abnormal expression of Tie-2 and VEGF-Rs, both of which may contribute to the pathogenesis of AVMs.

Room C, 10/17/2000 9: 00 AM - 11: 00 AM (PS) Abnormal Expression of Angiopoietin-2 and Tie-2 in Human Cerebral Arteriovenous Malformations

<b>Room C, 10/17/2000 9: 00 AM - 11: 00 AM (PS) Abnormal Expression of Angiopoietin-2 and Tie-2 in Human Cerebral Arteriovenous Malformations</b>

Room C, 10/17/2000 9: 00 AM - 11: 00 AM (PS) Increased Endothelial Cell Turnover in Human Cerebral Arteriovenous Malformations

<b>Room C, 10/17/2000 9: 00 AM - 11: 00 AM (PS) Increased Endothelial Cell Turnover in Human Cerebral Arteriovenous Malformations</b>

Normal perfusion pressure breakthrough: the role of capillaries.

Excision of human cerebral arteriovenous malformations (AVMs) can be complicated by postoperative edema and hemorrhage in adjacent brain tissue, despite the complete excision of the malformation. Various theories have purported to explain the hemodynamic basis for this predisposition, including disordered autoregulation causing "normal perfusion pressure breakthrough" and obstruction of venous drainage leading to "occlusive hyperemia." This study did not evaluate the arterial or venous circulations in this scenario, but rather examined the capillaries in adjacent brain parenchyma for any structural deficiencies that would predispose the brain to the postoperative formation of edema and hemorrhage. Arteriovenous fistulas (AVFs) were created surgically in the necks of 10 male Sprague-Dawley rats, which caused chronic cerebral hypoperfusion with a reduction in cerebral blood flow of between 25% and 50%. Ten age-matched animals were used as controls. Twenty-six weeks after AVF formation the animals were killed and perfusion fixed. Their brain tissue was prepared for light microscopic studies by staining for glial fibrillary acidic protein or for transmission electron microscopy. In the CA1 pyramidal cell region of the hippocampus, it was found that in the animals with AVFs there was increased capillary density and absent astrocytic foot processes in some of these vessels. It was concluded that these vessels had developed as a result of neovascularization in response to chronic cerebral ischemia and that their anatomical configuration made them prone to mechanical weakness and instability following the increase in perfusion pressure that occurs in adjacent brain parenchyma after AVM excision. The authors believe that this study pinpoints a structural accompaniment to the hemodynamic changes that occur in brain tissue in the vicinity of cerebral AVMs that predispose these areas to the formation of edema and hemorrhage after AVM excision.