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

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

(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

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

Immunohistochemical staining of PECAM-1 antibody in AVM nidus structures and control tissues.

<p>(A) PECAM-1 is expressed in the endothelial cell lining of (arrow) AVM and control vessels. (B) Photomicrograph of tissues stained with secondary antibody alone Magnification 20X.</p