Background Up to 5% of the population may have a brain aneurysm, and if the brain aneurysm ruptures, there is greater than 50% mortality, and more than onethird of survivors are dependent. Brain aneurysms detected before rupture can be treated to prevent rupture, or ruptured aneurysms can be treated to prevent re-rupture. Endovascular coiling of brain aneurysms is the treatment of choice for some aneurysms; however, up to one quarter of aneurysms may recur. The coiled aneurysms that do not recur are characterized by inflammatory intra-aneurysmal tissue healing; therefore, we studied the biology of this process, specifically, the role of monocyte chemotactic protein-1 (MCP-1), a cytokine known for tissue healing. Methods and Results We created coils with a PLGA coating that released MCP-1 at 3 different doses (100 μg/mL; 1 mg/mL; and 10 mg/mL) and performed a dose-response study for effect on intra-aneurysmal tissue healing in a murine carotid aneurysm model. We then demonstrated MCP-1(100 μg/mL)-releasing coils promote significantly greater aneurysm tissue ingrowth than bare platinum or PLGA-only coils. We show that MCP-1 recruits the migration of fibroblasts, macrophages, smooth muscle cells, and endothelial cells in vitro, in cell migration assays; and in vivo, in murine carotid aneurysms. Using gfp bone marrow transplant chimeric mice, we demonstrate that the MCP-1-recruited fibroblasts and macrophages are derived from the bone marrow. We demonstrate that this MCP-1-mediated vascular inflammatory repair occurs via amacrophage inflammatory protein-1α (MIP-1α) and macrophage inflammatory protein-2 (MIP-2)-dependent pathway. MCP-1 released from coiled murine aneurysms causes significant upregulation of MIP-1α and MIP-2 expression by cytokine array assay. Blocking MIP-1α and MIP-2 with antagonist antibody causes significant decrease in MCP-1-mediated intra-aneurysmal tissue healing. Conclusions Our findings suggest that MCP-1 has a critical role in promoting inflammatory intra-aneurysmal tissue healing in a MIP-1α and MIP-2-dependent pathway.

IntroductionCerebral aneurysms occur in up to 5% of the population. There are several murine models of aneurysms; however, all have limitations and none reproducibly model aneurysm rupture. To fulfill this need, we modified two current rodent aneurysm models to create a murine model which reproducibly produces intracranial aneurysms and rupture.MethodsThe left common carotid arteries and the right renal arteries were ligated in C57BL/6 female mice with a hypertensive diet. One week later, small burr holes were created with a stereotactic frame using the following stereotactic measurements: 1.2 mm rostral and 0.7 mm lateral to the right of the bregma. A 26 G needle was gradually advanced via the burr hole until contact with the skull base, upon which the needle was pulled back 0.3 mm. Five, 10 and 20 μL of 10 U/mL elastase solution and 10 μL of 1 U/mL elastase solution were stereotactically injected into the basal cisterns. Angiotensin II was then continually infused at a dose of 1000 ng/kg/min via an osmotic pump placed subcutaneously. In the control mice, 20 μL bromophenol blue solution was injected. Three weeks later, or earlier if mice expired prior to 3 weeks, the circle of Willis was inspected by microscopy for aneurysm formation and/or signs of rupture. Histological analyses were then performed to evaluate elastic lamina destruction, inflammatory cell and macrophage infiltration, absence of intimal endothelial cells and thickening of the smooth muscle layer within the aneurysm wall. To compare with human aneurysms, human aneurysm specimens (n=35; 34 unruptured and 1 ruptured) and normal control superficial temporal arteries (STAs) (n=9) were examined.ResultsAll mice given 5, 10 and 20 μL of 10 U/mL elastase solution developed intracranial aneurysms within the circle of Willis; 40%, 60% and 50% of mice had ruptured aneurysms, respectively. In mice given 10 μL of 1.0 U/mL elastase solution, 90% developed intracranial aneurysms and 20% had ruptured aneurysms. Aneurysms were confirmed by examining the destruction of the elastic lamina. Aneurysms consistently demonstrated CD45 positive inflammatory cell and F4/80 positive macrophage infiltration within the aneurysm wall which was not present in the circle of Willis of normal sham-operated mice. These results were similar to those in human aneurysms and STA control arteries.ConclusionsWe modified two current rodent aneurysm models to create a murine model that produces consistent aneurysms and rupture and can be used for studying cerebral aneurysm formation, rupture and treatment.

Object A small percentage of cerebral aneurysms rupture, but when they do, the effects are devastating. Current management of unruptured aneurysms consist of surgery, endovascular treatment, or watchful waiting. If the biology of how aneurysms grow and rupture were better known, a novel drug could be developed to prevent unruptured aneurysms from rupturing. Ruptured cerebral aneurysms are characterized by inflammation-mediated wall remodeling. We studied the role of stromal cell-derived factor-1 (SDF-1) in inflammation-mediated wall remodeling in cerebral aneurysms. Methods Human aneurysms; murine carotid aneurysms; and murine intracranial aneurysms were studied by immunohistochemistry. Flow cytometry analysis was performed on blood from mice developing carotid aneurysms or intracranial aneurysms. The effect of SDF-1 on endothelial cells and macrophages was studied by chemotaxis cell migration assay and capillary tube formation assay. Anti-SDF-1 blocking antibody was given to mice and compared to control (vehicle)-administered mice for its effects on the walls of carotid aneurysms and the development of intracranial aneurysms. Results Human aneurysms, murine carotid aneurysms, and murine intracranial aneurysms, all express SDF-1; and mice with developing carotid aneurysms or intracranial aneurysms have increased progenitor cells expressing CXCR4, the receptor for SDF-1 (P<0.01 and P<0.001, respectively). Human aneurysms and murine carotid aneurysms have endothelial cells, macrophages, and capillaries in the walls of the aneurysms; and the presence of capillaries in the walls of human aneurysms is associated with presence of macrophages (P=0.01). SDF-1 promotes endothelial cell and macrophage migration (P<0.01 for each), and promotes capillary tube formation (P<0.001). When mice are given anti-SDF-1 blocking antibody, there is a significant reduction in endothelial cells (P<0.05), capillaries (P<0.05), and cell proliferation (P<0.05) in the aneurysm wall. Mice given anti-SDF-1 blocking antibody develop significantly fewer intracranial aneurysms (33% versus 89% in mice given control IgG)(P<0.05). Conclusions These data suggest SDF-1 associated with angiogenesis and inflammatory cell migration and proliferation in the walls of aneurysms, and may have a role in the development of intracranial aneurysms.

BackgroundEstrogen deficiency is associated with the development of cerebral aneurysms; however, the mechanism remains unknown. We explored the pathway of cerebral aneurysm development by investigating the potential link between estrogen deficiency and inflammatory factors.Methods and ResultsFirst, we established the role of interleukin‐17 (IL‐17)A. We performed a cytokine screen demonstrating that IL‐17A is significantly expressed in mouse and human aneurysms (P=0.03). Likewise, IL‐17A inhibition was shown to prevent aneurysm formation by 42% (P=0.02) and rupture by 34% (P<0.05). Second, we found that estrogen deficiency upregulates T helper 17 cells and IL‐17A and promotes aneurysm rupture. Estrogen‐deficient mice had more ruptures than control mice (47% versus 7%; P=0.04). Estradiol supplementation or IL‐17A inhibition decreased the number of ruptures in estrogen‐deficient mice (estradiol 6% versus 37%; P=0.04; IL‐17A inhibition 18% versus 47%; P=0.018). Third, we found that IL‐17A‐blockade protects against aneurysm formation and rupture by increased E‐cadherin expression. IL‐17‐inhibited mice had increased E‐cadherin expression (P=0.003). E‐cadherin inhibition reversed the protective effect of IL‐17A inhibition and increased the rate of aneurysm formation (65% versus 28%; P=0.04) and rupture (12% versus 0%; P=0.22). However, E‐cadherin inhibition alone does not significantly increase aneurysm formation in normal mice or in estrogen‐deficient mice. In cell migration assays, E‐cadherin inhibition promoted macrophage infiltration across endothelial cells (P<0.05), which may be the mechanism for the estrogen deficiency/IL‐17/E‐cadherin aneurysm pathway.ConclusionsOur data suggest that estrogen deficiency promotes cerebral aneurysm rupture by upregulating IL‐17A, which downregulates E‐cadherin, encouraging macrophage infiltration in the aneurysm vessel wall.

Cerebral aneurysms are thought to develop at locations of hemodynamic shear stress, via an inflammatory process. The molecular mechanism that links shear stress to inflammation, however, is not completely understood. Progress in studying this disease is limited by a lack of a suitable in vitro model. To address this, we designed novel in vitro parallel-plate flow chamber models of a straight artery, a bifurcation, and a bifurcation aneurysm. We compared endothelial cell phenotypes across the three different models, and among microenvironments within each flow model, by cytokine array, ELISA and relative immunofluorescence. Human aneurysms express IL-8 and CXCL1, whereas normal arteries do not. The bifurcation aneurysm model showed significantly higher IL-8 and CXCL1 levels than both the straight artery and bifurcation models. Within the bifurcation and bifurcation aneurysm models, endothelial cells near the bifurcation or within the aneurysm sac microenvironments have significantly higher expression of CXCL1, and IL-8 and CXCL1, respectively, than at the straight proximal segment or the limbs of the bifurcation. Murine aneurysms express CXCL1, and it is the primary ELR+ CXC chemokine expressed, whereas normal arteries do not. CXCL1 antibody blockade results in significantly fewer murine aneurysms (13.3 vs 66.7%, p=0.0078), decreased neutrophil infiltration and VCAM-1 expression than an IgG control. We successfully designed and validated a novel hemodynamic model of cerebral aneurysms in vitro. We also show that shear stress-induced CXCL1 plays a critical role in cerebral aneurysm formation.