Cyclo-oxygenase 2, a putative mediator of vessel remodeling, is expressed in the brain AVM vessels and associates with inflammation

Abstract

Background: Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions.

Methods: Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records.

Results: COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels' lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage.

Conclusion: COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies.

Keywords: Brain arteriovenous malformation; Cyclo-oxygenase-2; Inflammation; Intracranial hemorrhage; Non-steroidal anti-inflammatory drugs; Vessel remodeling.

Fig. 1

COX2 is expressed in the endothelial cells of bAVMs. The figure shows the expression of COX2 (in brown) in the endothelial cells of multiple small bAVM vessels (marked by black arrows) in a lower magnification view (A, scale bar 250 µm) and in a higher magnification close-up (B, scale bar 25 µm. Negative control from the same region is shown in the insert at the right upper corner of B). Another example of a lower magnification view with multiple COX2 expressing small bAVM vessels (marked by black arrows) is given in (C) (scale bar 100 µm), in which also an adjacent larger bAVM vessel (marked by an asterisk) shows COX2 expression (in brown) on the luminal and adventitial surface. Immunofluorescence double staining (D–F) show that COX2 is expressed in luminal and adventitial cells positive for CD31, supporting the endothelial (luminal double-positive cells) and inflammatory cell (adventitial double-positive cells) phenotype. Double-positive cells are visible as yellow in the overlay image (D), whereas COX2 is shown in red in the corresponding single channel image (E) and CD31 in green (F). The negative control section imaged with the same excitation and image acquisition settings as for (D–F) is shown in the upper corner of (F). The markings A and L in the microphotographs stand for adventitia and lumen, respectively

Fig. 2

COX2 is expressed in the smooth muscle cells of bAVMs. The figure demonstrates COX2 expression (in brown) in the smooth muscle cell layer of large- and middle-sized bAVM vessels (A–C, scale bar 50 µm). Immunofluorescence double stainings (D–G) show in a lower magnification view (D, E) and in a higher magnification view (F, G) the wall of a small bAVM vessel with alfa smooth muscle actin-positive cells (in red in (D)) that show COX2 expression (in green in (E, F)). Double-positive cells are visible as yellow in the overlay image (G). An asterisk (*) shows red blood cells, which autofluorescate in all channels, also in the negative control. A double asterisk (**) marks vessel lumen. The negative control section imaged with the same excitation and image acquisition settings as for (D) and (G) is shown in the upper corner of (G). The markings A and L in the microphotographs stand for adventitia and lumen, respectively

Fig. 3

EP2, receptor for prostaglandin E2 is expressed in bAVMs. The figure shows EP2 expression (in brown) in smooth muscle cells of bAVM vessel in a lower magnification view (A) and in a higher magnification view (B). Immunofluorescence double staining (alfa smooth muscle actin in green and EP2 in red) in a lower magnification view (C) and in a higher magnification view (D) demonstrate EP2 expression in luminal cells of a large bAVM vessel. Based on their morphology these cells are endothelial cells (C, D). EP2 expression was also observed in the endothelium of smaller bAVM vessels (in brown in (E), and same area in negative control from adjacent section is presented in the upper corner of (E)). Immunofluorescence double staining in a lower magnification view (F) and in a higher magnification view (G) also demonstrates EP2 expression (in red) in the endothelium of small bAVM vessels (F). In addition, EP2 expression was observed in inflammatory cells (adventitial cells negative for alfa smooth muscle actin in green and with a mononuclear morphology) (G). The negative control section (H) imaged with the same excitation and image acquisition settings as for (F) and (G) is shown in the upper corner of (G). The markings A and L in the microphotographs stand for adventitia and lumen, respectively

Fig. 4

EP2 is expressed in bAVM vessels undergoing remodeling. The figure shows EP2 expression (in brown) in lower magnification views (A, D) and in higher magnification views (B, C and E, F) in adaptive (upper pictures, site of intimal hyperplasia in a large bAVM vessel) and destructive (lower pictures, site of vessel wall inflammation with infiltration of mononuclear and polymorphonuclear inflammatory cells and subsequent destruction of the vessel wall) bAVM vessel remodeling. The markings A and L in the microphotographs stand for adventitia and lumen, respectively

Fig. 5

MMP9 is expressed in bAVMs. The figure shows MMP9 expression (in brown) in endothelial cells (marked by black arrows) of a bAVM vessel (A, scale bar 25 um). MMP9 expression was also observed in mural cells of the smooth muscle cell layer (lower magnification in (B) (scale bar 250 µm) and higher magnification in (C) (scale bar 50 µm)). Example of a large bAVM vessel with inflamed wall and MMP9 expression is presented in lower magnification in (D) (scale bar 2.5 mm), with a higher magnification view showing MMP9 expression (in brown) in (E) (scale bar 25 µm) and in inflammatory cells (CD45 in brown) in adjacent section in (F)

Fig. 6

Inflammatory cells are present in bAVMs and express COX2. Figure presents in a lower (A) and in a higher (B) magnification view CD45 (panleukocyte marker, in brown) positive cells in the luminal surface, smooth muscle layer, and in the adventitia of a bAVM vessel. COX2 staining from an adjacent section (C) shows COX2-positive cells (in brown) at the same region as CD45 positive inflammatory cells. In C, the perivascular inflammatory cells are marked by black arrows and an asterisk (*) marks vessel lumen. Another example of co-localization of perivascular inflammatory cells (in brown, D) and COX2 expression (in brown, E) in adjacent sections is given in (D) and (E). The correlation between inflammation (x-axis) and COX2 expression (y-axis) is shown in (F). The following scale was used to score CD45 and COX2 immunostainings: 0 = no positive signal, 1 = 2–15 positive cells, 2 = more than 15 positive cells but < 1/3 of the surface area showing positivity, 3 = over 1/3 of the sample surface area showing positive signal

Fig. 7

COX2 is expressed in parenchymal cells of bAVMs. The figure presents COX2 expression (in brown) in bAVM’s perinidal neural cells in a lower magnification (A) and in a higher magnification (B). The CD45 staining from adjacent section shows only few inflammatory cells (in brown) around vessels (C), demonstrating that the COX2-positive parenchymal cells (A) and (B) are not inflammatory cells. Morphologically, these COX2 expressing cells correspond to neurons and astrocytes COX2-positivity co-localized in adjacent sections with both GFAP-positive and GFAP-negative parenchymal cells (Supplemental data, Fig. S3)