Oscillatory shear stress modulates Notch-mediated endothelial mesenchymal plasticity in cerebral arteriovenous malformations.

BACKGROUND: Cerebral arteriovenous malformations (cAVM) are a significant cause of intracranial hemorrhagic stroke and brain damage. The arteriovenous junctions in AVM nidus are known to have hemodynamic disturbances such as altered shear stress, which could lead to endothelial dysfunction. The molecular mechanisms coupling shear stress and endothelial dysfunction in cAVMs are poorly understood. We speculated that disturbed blood flow in artery-vein junctions activates Notch receptors and promotes endothelial mesenchymal plasticity during cAVM formation. METHODS: We investigated the expression profile of endothelial mesenchymal transition (EndMT) and cell adhesion markers, as well as activated Notch receptors, in 18 human cAVM samples and 15 control brain tissues, by quantitative real-time PCR (qRT-PCR) and immunohistochemical evaluation. Employing a combination of a microfluidic system, qRT-PCR, immunofluorescence, as well as invasion and inhibitor assays, the effects of various shear stress conditions on Notch-induced EndMT and invasive potential of human cerebral microvascular endothelial cells (hCMEC/d3) were analyzed. RESULTS: We found evidence for EndMT and enhanced expression of activated Notch intracellular domain (NICD3 and NICD4) in human AVM nidus samples. The expression of transmembrane adhesion receptor integrin α9/ß1 is significantly reduced in cAVM nidal vessels. Cell-cell adhesion proteins such as VE-cadherin and N-cadherin were differentially expressed in AVM nidus compared with control brain tissues. Using well-characterized hCMECs, we show that altered fluid shear stress steers Notch3 nuclear translocation and promotes SNAI1/2 expression and nuclear localization. Oscillatory flow downregulates integrin α9/ß1 and VE-cadherin expression, while N-cadherin expression and endothelial cell invasiveness are augmented. Gamma-secretase inhibitor RO4929097, and to a lesser level DAPT, prevent the mesenchymal transition and invasiveness of cerebral microvascular endothelial cells exposed to oscillatory fluid flow. CONCLUSIONS: Our study provides, for the first time, evidence for the role of oscillatory shear stress in mediating the EndMT process and dysregulated expression of cell adhesion molecules, especially multifunctional integrin α9/ß1 in human cAVM nidus. Concomitantly, our findings indicate the potential use of small-molecular inhibitors such as RO4929097 in the less-invasive therapeutic management of cAVMs.

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.

Aberrant regulation of retinoic acid signaling genes in cerebral arterio venous malformation nidus and neighboring astrocytes.

BACKGROUND: Cerebral arterio venous malformations (AVM) are a major causal factor for intracranial hemorrhage, which result in permanent disability or death. The molecular mechanisms of AVM are complex, and their pathogenesis remains an enigma. Current research on cerebral AVM is focused on characterizing the molecular features of AVM nidus to elucidate the aberrant signaling pathways. The initial stimuli that lead to the development of AVM nidus structures between a dilated artery and a vein are however not known. METHODS: In order to understand the molecular basis of development of cerebral AVM, we used in-depth RNA sequencing with the total RNA isolated from cerebral AVM nidus. Immunoblot and qRT-PCR assays were used to study the differential gene expression in AVM nidus, and immunofluorescence staining was used to study the expression pattern of aberrant proteins in AVM nidus and control tissues. Immunohistochemistry was used to study the expression pattern of aberrant proteins in AVM nidus and control tissues. RESULTS: The transcriptome study has identified 38 differentially expressed genes in cerebral AVM nidus, of which 35 genes were upregulated and 3 genes were downregulated. A final modular analysis identified an upregulation of ALDH1A2, a key rate-limiting enzyme of retinoic acid signaling pathway. Further analysis revealed that CYR61, a regulator of angiogenesis, and the target gene for retinoic acid signaling is upregulated in AVM nidus. We observed that astrocytes associated with AVM nidus are abnormal with increased expression of GFAP and Vimentin. Triple immunofluorescence staining of the AVM nidus revealed that CYR61 was also overexpressed in the abnormal astrocytes associated with AVM tissue. CONCLUSION: Using high-throughput RNA sequencing analysis and immunostaining, we report deregulated expression of retinoic acid signaling genes in AVM nidus and its associated astrocytes and speculate that this might trigger the abnormal angiogenesis and the development of cerebral AVM in humans.

Correction to: Genetic and epigenetic mechanisms in the development of arteriovenous malformations in the brain.

Upon publication of the original article [1] the authors noticed that the affiliation Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, India was missing.

Gene expression analysis of nidus of cerebral arteriovenous malformations reveals vascular structures with deficient differentiation and maturation.

OBJECTIVE: Arteriovenous malformations (AVMs) are characterised by tangles of dysplastic blood vessels which shunt blood from arteries to veins with no intervening capillary bed. It is not known at what stage of development and differentiation, AVM vessels became aberrant. To address this, we have analysed the expression of vascular differentiation, vascular maturation and brain capillary specific genes in AVM nidus. METHODOLOGY: We performed immunohistochemistry and western blot analysis of vascular differentiation (HEY2, DLL4, EFNB2, and COUP-TFII), vascular maturation (ENG and KLF2) and brain capillary specific genes (GGTP and GLUT1) on ten surgically excised human brain AVMs and ten normal human brain tissues. RESULTS: Immunohistochemical analysis revealed that AVM vessels co-express both artery and vein differentiation genes. H-score analysis revealed that there is statistically significant (P < 0.0001) increase in expression of these proteins in AVM vessels compared to control vessels. These findings were further confirmed by western blot analysis and found to be statistically significant (P < 0.0001 and P < 0.001) for all proteins except Hey2. Both immunostaining and western blot analysis revealed that AVM vessels express GGTP and GLUT1, markers specific to brain capillaries. Immunofluorescent staining demonstrated that expression of KLF2, a vascular maturation marker is significantly (P <0.001) decreased in AVM vessels and was further confirmed by western blot analysis (P < 0.001). Immunohistochemical and western blot analysis demonstrated that another vascular maturation protein Endoglin had high expression in AVM vessels compared to control vessels. The results were found to be statistically significant (P < 0.0001). SUMMARY: Our findings suggest that vascular structures of AVMs co-express markers specific for arteries, veins and capillaries. We conclude that AVM nidus constitutes of aberrant vessels which are not terminally differentiated and inadequately matured.

Genetic and epigenetic mechanisms in the development of arteriovenous malformations in the brain.

Vascular malformations are developmental congenital abnormalities of the vascular system which may involve any segment of the vascular tree such as capillaries, veins, arteries, or lymphatics. Arteriovenous malformations (AVMs) are congenital vascular lesions, initially described as "erectile tumors," characterized by atypical aggregation of dilated arteries and veins. They may occur in any part of the body, including the brain, heart, liver, and skin. Severe clinical manifestations occur only in the brain. There is absence of normal vascular structure at the subarteriolar level and dearth of capillary bed resulting in aberrant arteriovenous shunting. The causative factor and pathogenic mechanisms of AVMs are unknown. Importantly, no marker proteins have been identified for AVM. AVM is a high flow vascular malformation and is considered to develop because of variability in the hemodynamic forces of blood flow. Altered local hemodynamics in the blood vessels can affect cellular metabolism and may trigger epigenetic factors of the endothelial cell. The genes that are recognized to be associated with AVM might be modulated by various epigenetic factors. We propose that AVMs result from a series of changes in the DNA methylation and histone modifications in the genes connected to vascular development. Aberrant epigenetic modifications in the genome of endothelial cells may drive the artery or vein to an aberrant phenotype. This review focuses on the molecular pathways of arterial and venous development and discusses the role of hemodynamic forces in the development of AVM and possible link between hemodynamic forces and epigenetic mechanisms in the pathogenesis of AVM.

Leucas aspera inhibits the Dalton's ascitic lymphoma in Swiss albino mice: A preliminary study exploring possible mechanism of action.

BACKGROUND: North East India is a rich source of medicinal plants and a number of plant extracts are used by tribal peoples living in this area for various disorders. L.aspera is such a plant, traditionally used as an antitumor agent. AIM: In the present study, aerial parts of L.aspera were investigated for antitumor activity in Dalton's lymphoma (DAL) bearing mice. The ability of plant extract in free radical scavenging, neoangiogenesis inhibition and macrophage stimulation were also checked. MATERIALS AND METHODS: Based on the preliminary in vitro cytotoxicity studies ethyl acetate fraction of L.aspera (EALA) was selected for the detailed study. DAL ascites tumor model was performed to check the antitumor activity of EALA (200 and 400mg/kg of body weight). Hematological and histopathological parameters were estimated. Antioxidant levels, neoangiogenesis and peritoneal macrophage count were also determined. RESULTS: In vitro MTT and Trypan blue assay results showed the cytotoxic effect of EALA in DAL cells lines. EALA treatment resulted in significant decrease in ascites tumor volume and viable cell count. Hematological and liver antioxidant parameters were normalised by EALA treatment. It was also found that EALA treatment inhibits neovascularisation and produce macrophage stimulation in treated mice. CONCLUSION: The results showed that EALA is a promising anticancer agent and its activity is comparable to the standard drug 5-Flouro uracil (5-FU).