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

Abstract 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

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

<div><p>Objective</p><p>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.</p><p>Methodology</p><p>We performed immunohistochemistry and western blot analysis of vascular differentiation (<i>HEY2</i>, <i>DLL4</i>, <i>EFNB2</i>, and <i>COUP-TFII</i>), vascular maturation (<i>ENG</i> and <i>KLF2</i>) and brain capillary specific genes (<i>GGTP</i> and <i>GLUT1</i>) on ten surgically excised human brain AVMs and ten normal human brain tissues.</p><p>Results</p><p>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).</p><p>Summary</p><p>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.</p></div

Gene expression analysis of nidus of cerebral arteriovenous malformations reveals vascular structures with deficient differentiation and maturation - Fig 8

<p><b>(A) Photomicrograph of tissues stained with the antibody against KLF2 in AVM and control tissues.</b> KLF2 is not expressed in AVM nidus structures. In control vessels, KLF2 is expressed (green) in the endothelial cell lining (arrowhead) and smooth muscle cells (arrow). Magnified images are shown in a and b. Hoechst 33342 (blue) is used to counter stain nuclei. 60X magnification. <b>(B) Graphical representation of fluorescence intensity of KLF2 in AVM and control tissues.</b> KLF2 expression is significantly low in AVM nidus structures (n = 10) compared to control tissues (n = 10) (*P < 0.001).</p

(A) Hematoxylin and Eosin staining of control tissues and (B, C, D and E) AVM nidus.

<p>(A) H & E staining of control brain reveals normal histological pattern of brain parenchyma (arrowhead) and blood vessels (arrow). (B) H & E staining of AVM nidus reveals that nidus consists of blood vessels of different size and morphology. There are both thin walled (arrowheads) and thick walled vessels (arrow). (C) Partial denudation of endothelial cell layer is seen (arrowhead). Large sized veins (arrows) with uneven thickness of tunica media layer are seen. Thickened tunica media layer (asterisk) is seen in AVM vessels. Magnification 10X. (D) Higher magnification image of AVM nidus clearly demonstrates partial denudation of endothelial cell layer (arrowhead) and large sized vein (arrows) with an uneven thickness of tunica media layer. (E) A blood vessel with thickened smooth muscle cell layer (asterisk) is seen. Magnification 20X. <b>(F and G) EVG staining of control vessels and (H and I) AVM nidus structures.</b> (F) A control artery with a thick internal elastic lamina layer (arrow) is depicted. (G) Staining for elastic fibres (arrow) in the wall of a normal vein is evident. (H) Dark brown colour (arrow) indicates the internal elastic lamina layer of a large artery in AVM nidus. (I) A dysplastic vein in AVM nidus with a thin internal elastic layer (arrow) is shown. Magnification 10X.</p

Clinical data of patients with brain arteriovenous malformation.

<p>Clinical data of patients with brain arteriovenous malformation.</p

Shear Stress Alterations Activate BMP4/pSMAD5 Signaling and Induce Endothelial Mesenchymal Transition in Varicose Veins

Chronic venous diseases, including varicose veins, are characterized by hemodynamic disturbances due to valve defects, venous insufficiency, and orthostatism. Veins are physiologically low shear stress systems, and how altered hemodynamics drives focal endothelial dysfunction and causes venous remodeling is unknown. Here we demonstrate the occurrence of endothelial to mesenchymal transition (EndMT) in human varicose veins. Moreover, the BMP4-pSMAD5 pathway was robustly upregulated in varicose veins. In vitro flow-based assays using human vein, endothelial cells cultured in microfluidic chambers show that even minimal disturbances in shear stress as may occur in early stages of venous insufficiency induce BMP4-pSMAD5-based phenotype switching. Furthermore, low shear stress at uniform laminar pattern does not induce EndMT in venous endothelial cells. Targeting the BMP4-pSMAD5 pathway with small molecule inhibitor LDN193189 reduced SNAI1/2 expression in venous endothelial cells exposed to disturbed flow. TGF&beta; inhibitor SB505124 was less efficient in inhibiting EndMT in venous endothelial cells exposed to disturbed flow. We conclude that disturbed shear stress, even in the absence of any oscillatory flow, induces EndMT in varicose veins via activation of BMP4/pSMAD5-SNAI1/2 signaling. The present findings serve as a rationale for the possible use of small molecular mechanotherapeutics in the management of varicose veins

Representative photomicrographs of immunohistochemical analysis of arterial markers (A) Hey2, (B) Dll4 and (C) EphrinB2 in AVM and control tissues obtained from temporal lobe epilepsy patients.

<p>(A) There is intense staining of Hey2 in AVM nidus structures compared to control vessels. Hey2 is expressed in the endothelial cell lining (arrowhead) and smooth muscle cells (thin arrow) of AVM nidus structures. In control vessels (arrowhead), the Hey2 expression is much less. (B) In AVM nidus, Dll4 is expressed with high staining intensity in the endothelial cell lining (arrowhead), smooth muscle cells (thin arrow) and tunica adventitia layer (thick arrow). There is much less expression of Dll4 in blood vessels in control brain (arrowhead). In control tissue, Dll4 positive cells are found in adjacent brain parenchyma cells (thin arrow). (C) Nidus of AVM has high expression of EphrinB2 in tunica intima (arrowhead), tunica media (thin arrow) and tunica adventitia (thick arrow) layer of blood vessels. In control tissue, brain parenchyma expresses EphrinB2 positive cells (arrow) and blood vessels have much less expression of EphrinB2 (arrowhead). (D) Photomicrograph of tissue stained with secondary antibody alone. 10X magnification.</p

Immunohistochemical staining of α-SMA in (A) AVM nidus structures and (C) control tissues.

<p>(A) There is intense staining of α-SMA in the endothelial cell lining (arrow) and in the peri-endothelial cell layer (arrowhead) of AVM vessels (AVM 1). AVM nidus structures consist of both circular (arrow) and longitudinal bundles (arrowhead) of smooth muscle cells (AVM 2). (C) α-SMA is expressed in the smooth muscle cells of control vein (control 1) and control arteries (control 2). (B and D) Secondary isotype controls for AVM and control tissues respectively. Magnification 20X.</p