Riikka Tulamo

Saccular intracranial aneurysm: pathology and mechanisms

Saccular intracranial aneurysms (sIA) are pouch-like pathological dilatations of intracranial arteries that develop when the cerebral artery wall becomes too weak to resist hemodynamic pressure and distends. Some sIAs remain stable over time, but in others mural cells die, the matrix degenerates, and eventually the wall ruptures, causing life-threatening hemorrhage. The wall of unruptured sIAs is characterized by myointimal hyperplasia and organizing thrombus, whereas that of ruptured sIAs is characterized by a decellularized, degenerated matrix and a poorly organized luminal thrombus. Cell-mediated and humoral inflammatory reaction is seen in both, but inflammation is clearly associated with degenerated and ruptured walls. Inflammation, however, seems to be a reaction to the ongoing degenerative processes, rather than the cause. Current data suggest that the loss of mural cells and wall degeneration are related to impaired endothelial function and high oxidative stress, caused in part by luminal thrombosis. The aberrant flow conditions caused by sIA geometry are the likely cause of the endothelial dysfunction, which results in accumulation of cytotoxic and pro-inflammatory substances into the sIA wall, as well as thrombus formation. This may start the processes that eventually can lead to the decellularized and degenerated sIA wall that is prone to rupture.

COMPLEMENT ACTIVATION ASSOCIATES WITH SACCULARCEREBRAL ARTERY ANEURYSM WALL DEGENERATION AND RUPTURE

OBJECTIVE Saccular cerebral artery aneurysm (SCAA) wall degeneration and inflammatory cell infiltrations associate with aneurysm rupture and subarachnoid hemorrhage, resulting in a devastating form of stroke. The complement system is the key mediator of inflammation and household processing of injured tissue. We studied how complement activation associates with SCAA wall degeneration and rupture to better understand the pathobiology of SCAA wall rupture. METHODS Unruptured (n = 26) and ruptured (n = 32) SCAA fundi resected after microsurgical clipping were studied by immunostaining for complement activation (membrane attack complex [MAC]) and by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction for related cell death. Complement activation was correlated with clinical and other histological parameters. Electromicroscopy and immunoelectron microscopy were used for locating MAC depositions at the ultrastructural level. RESULTS MAC localized consistently in a decellularized layer in the outer SCAA wall, and was found in all SCAA samples. The percentage of MAC-positive area relative to the total SCAA wall surface area (range, 5-77%) was greater in ruptured (n = 25; median, 39%) than in unruptured SCAAs (n = 18; median, 20%; P = 0.005). It also associated significantly with SCAA wall degeneration (P < 0.001), de-endothelialization(P < 0.001), and CD163+ macrophage (P = 0.023) and T-lymphocyte (P = 0.030) infiltrations. Apoptotic terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive nuclei and MAC were located at the same wall areas in four out of 14 double-stained samples, but no double-positive cells were found. Electromicroscopy and immunoelectron microscopy of an unruptured SCAA showed cell death in the MAC-positive layers in the outer SCAA wall. CONCLUSION These data suggests that complement activation and MAC formation are involved in SCAA wall degeneration and rupture.

Upregulated signaling pathways in ruptured human saccular intracranial aneurysm wall: an emerging regulative role of Toll-like receptor signaling and nuclear factor-κB, hypoxia-inducible factor-1A, and ETS transcription factors.

BACKGROUND:Aneurysmal subarachnoid hemorrhage, almost always from saccular intracranial aneurysm (sIA), is a devastating form of stroke that affects the working-age population. Cellular and molecular mechanisms predisposing to the rupture of the sIA wall are largely unknown. This knowledge would facilitate the design of novel diagnostic tools and therapies for the sIA disease. OBJECTIVE:To investigate gene expression patterns distinguishing ruptured and unruptured sIA. METHODS:We compared the whole-genome expression profile of 11 ruptured sIA wall samples with that of 8 unruptured ones using oligonucleotide microarrays. Signaling pathways enriched in the ruptured sIA walls were identified with bioinformatic analyses. Their transcriptional control was predicted in silico by seeking the enrichment of conserved transcription factor binding sites in the promoter regions of differentially expressed genes. RESULTS:Overall, 686 genes were significantly upregulated and 740 were downregulated in the ruptured sIA walls. Significantly upregulated biological processes included response to turbulent blood flow, chemotaxis, leukocyte migration, oxidative stress, vascular remodeling; and extracellular matrix degradation. Toll-like receptor signaling and nuclear factor-κB, hypoxia-inducible factor-1A, and ETS transcription factor binding sites were significantly enriched among the upregulated genes. CONCLUSION:We identified pathways and candidate genes associated with the rupture of human sIA wall. Our results may provide clues to the molecular mechanism in sIA wall rupture and insight for novel therapeutic strategies to prevent rupture.

Complement system becomes activated by the classical pathway in intracranial aneurysm walls.

Inflammation and activation of the complement system in the intracranial aneurysm (IA) wall predispose to IA rupture. We have previously shown that increased C5b-9 accumulation correlates with IA rupture and wall degeneration. To elucidate the underlying mechanisms, we investigated initiators and the pathway of complement activation in unruptured and ruptured IAs. Unruptured and ruptured IA wall samples were studied in parallel sections by immunohistochemical and immunofluorescence stainings for the location and relations of classical and alternative pathway complement components (C1q, C3b/iC3b, C3d, C4b/iC4b; n=35 and properdin, n=10), putative complement activators IgG (n=90), IgM, CRP and OxLDL (n=10), and complement activation endproduct C5b-9. Classical pathway components were seen in all IAs, and they were located mostly in the extracellular matrix. The early pathway complement components colocalized with each other, but were present in larger areas than C5b-9. The areas positive for complement component accumulation were significantly broader in ruptured than in unruptured IAs. The potential complement activators IgG, IgM, CRP and OxLDL were found mostly in the extracellular matrix and in partial overlap with C5b-9. Lipids were seen in Oil-Red-O staining in colocalization with C5b-9. Complement becomes activated by the classical pathway in the IA wall. The activation appears to be induced by multiple factors, which, in addition to the traditional activators (immunoglobulins, CRP, OxLDL), could involve vascular pressure-induced tissue damage. Despite wide early pathway activation, the terminal pathway is focused on a distinct lipid-rich layer. The profile of the complement components and the association of C5b-9 with lipids in the extracellular matrix indicate a long-term chronic inflammatory process rather than an acute targeted inflammatory reaction. The observed pattern of complement activation may be the consequence of local stress-induced insufficiency of complement regulation in IA walls.

Contribution of Mural and Bone Marrow-derived Neointimal Cells to Thrombus Organization and Wall Remodeling in a Microsurgical Murine Saccular Aneurysm Model

OBJECTIVE:Endovascular occlusive therapy of human saccular cerebral artery aneurysms may fail because of thrombus recanalization and incomplete neointima formation. Bone marrow-derived progenitor cells may contribute to these processes, but their role in human saccular cerebral artery aneurysms and experimental aneurysm models remains unclear. METHODS:Experimental saccular aneurysms were constructed from syngeneic thoracic aortas transplanted end-to-side to the abdominal aorta of Wistar rats (n = 14), C57/B6 mice (n = 13), ApoE mice (n = 7), reporter gene expressing ROSA mice (n = 7), and mice with labeled bone marrow (ROSA [n = 12] or green fluorescent protein [n = 3]). Magnetic resonance imaging or angiography was used to monitor patency of the experimental aneurysms. Histology and immunohistochemistry were used to study thrombus organization and neointima formation and X-gal staining and confocal microscopy to study the origin of neointimal cells. RESULTS:Experimental aneurysms developed luminal pads of neointimal hyperplasia or organizing thrombosis that became thicker and occluded partly the lumen at later time points during the first week. Reporter gene mice (ROSA) revealed that 42 to 81% (median, 58%) of neointimal hyperplasia/organizing thrombosis was derived from the experimental aneurysm wall. Bone marrow-derived neointimal cells were found in only 5 of 15 mice (range, 11–73 per section; a median of 22 cells among a total of 2000–6000 wall cells). CONCLUSION:Thrombus organizing or neointimal cells were mostly derived from the experimental aneurysm wall, with only a minor contribution from the bone marrow. In human saccular cerebral artery aneurysms, the contribution of bone marrow-derived neointimal cells might be more important and should be compared with that found in other experimental models used to develop endovascular therapies.

Lipid accumulation, lipid oxidation, and low plasma levels of acquired antibodies against oxidized lipids associate with degeneration and rupture of the intracranial aneurysm wall

BackgroundRupture of a saccular intracranial aneurysm (sIA) causes an often fatal subarachnoid hemorrhage (SAH). Why some sIAs rupture remains unknown. Since sIA walls bear some histological similarities with early atherosclerotic lesions, we hypothesized that accumulation and oxidation of lipids might occur in the sIA wall and might associate with sIA wall degeneration. Tissue samples from sIA fundi (n = 54) were studied with histochemistry and a panel of previously characterized antibodies for epitopes of oxidized LDL (OxLDL). Plasma samples from sIA carriers (n = 125) were studied with ELISA and EIA for IgG and IgM -antibodies against a panel of OxLDL epitopes.ResultsLipid accumulation, foam cells, and oxidized lipids were found both in unruptured and ruptured sIA walls. Lipid accumulation associated with wall degeneration (P < 0.001), as did the expression of adipophilin, a marker of lipid ingestion by cells. Lipid accumulation associated also with loss of mural cells (P < 0.001), as did the accumulation of OxLDL (P < 0.001). Plasma IgG antibody titers against OxLDL or malondialdehyde modified LDL were higher in patients with unruptured sIAs than in patients with aneurysmal SAH (P ≤ 0.001). A trend but not statistically significant differences were found in plasma IgM antibodies against oxidized lipids.ConclusionsAccumulation of lipids and their oxidation in the sIA wall associates with the degeneration of the sIA wall. Acquired immunity against oxidized lipid epitopes may be protective of lipid associated sIA wall degeneration, but warrants further studies.

Lack of Complement Inhibitors in the Outer Intracranial Artery Aneurysm Wall Associates with Complement Terminal Pathway Activation

Inflammation and activation of the complement system predispose to intracranial artery aneurysm (IA) rupture. Because disturbances in complement regulation may lead to increased susceptibility to complement activation and inflammation, we looked for evidence for dysregulation of the complement system in 26 unruptured and 26 ruptured IAs resected intraoperatively. Immunohistochemical and immunofluorescence results of parallel IA sections showed that deposition of the complement activation end-product C5b-9 was lacking from the luminal part of the IA wall that contained complement inhibitors factor H, C4b binding protein, and protectin as well as glycosaminoglycans. In contrast, the outer, less cellular part of the IA wall lacked protectin and had enabled full complement activation and C5b-9 formation. Decay accelerating factor and membrane cofactor protein had less evident roles in complement regulation. The Factor H Y402H variant, studied in 97 IA patients, was seen as often in aneurysm patients with or without aneurysm rupture as in the control population. The regulatory capacity of the complement system thus appears disturbed in the outer part of the IA wall, allowing full proinflammatory complement activation to occur before aneurysm rupture. Insufficient complement control might be due to matrix remodeling and cell loss by mechanical hemodynamics and/or inflammatory stress. Apparently, disturbed complement regulation leads to an increased susceptibility to complement activation, inflammation, and tissue damage in the IA wall.

Oxidative stress is associated with cell death, wall degradation, and increased risk of rupture of the intracranial aneurysm wall.

BACKGROUND The cause of rupture of intracranial aneurysms (IA) is not well understood. We previously demonstrated that loss of cells from the IA wall is associated with wall degeneration and rupture. OBJECTIVE To investigate the mechanisms mediating cell death in the IA wall. METHODS Snap-frozen tissue samples from aneurysm fundi were studied with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and immunostaining (14 unruptured and 20 ruptured), as well as with Western blot (12 unruptured and 12 ruptured). RESULTS : Ruptured IA walls had more TUNEL-positive cells than unruptured walls (P < .001). Few cells positive for cleaved caspase-3 were detected. Cleaved caspase-9 (intrinsic activation of apoptosis) was significantly increased in ruptured IA walls, whereas cleaved caspase-8 (extrinsic activation of apoptosis) was not detected. Increased expression of hemeoxygenase-1, a marker for oxidative stress, was associated with IA wall degeneration and rupture. CONCLUSION Our results show that programmed cell death is activated in the IA wall via the intrinsic pathway. High oxidative stress in the IA wall is probably a significant cause of the intrinsic activation of cell death.

Mast Cells, Neovascularization, and Microhemorrhages are Associated With Saccular Intracranial Artery Aneurysm Wall Remodeling

Abstract Chronic inflammation contributes to remodeling, degeneration, and rupture of saccular intracranial artery aneurysms. Mast cells are important proinflammatory and proangiogenic cells in chronic inflammatory vascular diseases. Here we studied mast cells and neovascularization in 36 intraoperatively resected aneurysms using histology and immunohistochemistry and analyzed the clinical characteristics of the aneurysms according to bleeding status (unruptured vs ruptured). Among the 36 aneurysms, 9 contained mast cells (tryptase-positive cells) and 15 contained neovessels (CD34- and CD31-positive capillarylike structures). The density of neovessels was significantly higher in aneurysm walls containing mast cells than in walls not containing them. In particular, wall areas with abundant mast cells and neovessels also contained iron deposits, indicating damage of newly formed endothelium with ensuing microhemorrhages. Walls with the highest neovessel density and the greatest iron deposition also showed evidence of degeneration. Finally, none of the mast cell–containing aneurysms showed an intact luminal endothelium. Thus, mast cells may adversely affect both neovascular and luminal endothelia. The novel association of mast cells with neovessels and injurious microhemorrhages, as well as with luminal endothelial erosion, suggests that mast cells contribute to remodeling and degeneration of saccular intracranial artery aneurysms.

Intracellular signaling pathways and size, shape, and rupture history of human intracranial aneurysms.

BACKGROUND Size and morphological features are associated with intracranial aneurysm (IA) rupture. The cellular mechanisms of IA development and rupture are poorly known. OBJECTIVE We studied the expression and phosphorylation of different intracellular signaling molecules in the IA wall compared with IA morphological features to understand better the cellular pathways involved in IA development and wall degeneration. METHODS Nine ruptured and 17 unruptured human IA samples were collected intraoperatively. The expression levels and phosphorylation state of 3 mitogen-activated protein kinases (c-Jun N-terminal kinase [JNK], p38, extracellular signal-regulated kinase [ERK]), Bcl-2 antagonist of cell death (Bad), mammalian target of rapamycin (mTOR), cyclic AMP response element binding protein (CREB), and Akt were determined by Western blotting. The localization of signaling proteins was determined by immunofluorescence. From 3-dimensional segmentation of computed tomography angiographic data, size and shape indexes were calculated. RESULTS We found a 5-fold difference in phospho-Bad levels between ruptured and unruptured IAs. Phospho-mTOR was downregulated 2.5-fold in ruptured IAs. Phospho-p54 JNK, phospho-p38, and phospho-Akt levels correlated positively with IA size. Phospho-CREB levels were significantly associated with nonsphericity and ellipticity indexes. Phospho-Akt and phospho-p38 correlated negatively with undulation index. CONCLUSION The signaling pathway profile (apoptosis, cell proliferation, stress signaling) differs between ruptured and unruptured IAs and is associated with IA geometry. Our results increase the knowledge of IA development and wall degeneration.