Stromal cell–derived factor-1 promoted angiogenesis and inflammatory cell infiltration in aneurysm walls

Laboratory investigation

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Object

A small percentage of cerebral aneurysms rupture, but when they do, the effects are devastating. Current management of unruptured aneurysms consists 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. The authors studied the role of stromal cell–derived factor-1 (SDF-1) in inflammation-mediated wall remodeling in cerebral aneurysms.

Methods

Human aneurysms, murine carotid artery aneurysms, and murine intracranial aneurysms were studied using immunohistochemistry. Flow cytometry analysis was performed on blood from mice developing carotid 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 with 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 expressed SDF-1, and mice with developing carotid or intracranial aneurysms had increased progenitor cells expressing CXCR4, the receptor for SDF-1 (p < 0.01 and p < 0.001, respectively). Human aneurysms and murine carotid aneurysms had endothelial cells, macrophages, and capillaries in the walls of the aneurysms, and the presence of capillaries in the walls of human aneurysms was associated with the presence of macrophages (p = 0.01). Stromal cell–derived factor-1 promoted endothelial cell and macrophage migration (p < 0.01 for each), and promoted capillary tube formation (p < 0.001). When mice were given anti–SDF-1 blocking antibody, there was 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 developed significantly fewer intracranial aneurysms (33% vs 89% in mice given control immunoglobulin G, respectively; p < 0.05).

Conclusions

These data suggest SDF-1 is 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.

Abbreviations used in this paper:FBS = fetal bovine serum; HUVEC = human umbilical vein endothelial cell; IgG = immunoglobulin G; SCA-1 = stem cell antigen-1; SDF-1 = stromal cell–derived factor-1; STA = superficial temporal artery; VEGF = vascular endothelial growth factor.

Object

A small percentage of cerebral aneurysms rupture, but when they do, the effects are devastating. Current management of unruptured aneurysms consists 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. The authors studied the role of stromal cell–derived factor-1 (SDF-1) in inflammation-mediated wall remodeling in cerebral aneurysms.

Methods

Human aneurysms, murine carotid artery aneurysms, and murine intracranial aneurysms were studied using immunohistochemistry. Flow cytometry analysis was performed on blood from mice developing carotid 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 with 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 expressed SDF-1, and mice with developing carotid or intracranial aneurysms had increased progenitor cells expressing CXCR4, the receptor for SDF-1 (p < 0.01 and p < 0.001, respectively). Human aneurysms and murine carotid aneurysms had endothelial cells, macrophages, and capillaries in the walls of the aneurysms, and the presence of capillaries in the walls of human aneurysms was associated with the presence of macrophages (p = 0.01). Stromal cell–derived factor-1 promoted endothelial cell and macrophage migration (p < 0.01 for each), and promoted capillary tube formation (p < 0.001). When mice were given anti–SDF-1 blocking antibody, there was 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 developed significantly fewer intracranial aneurysms (33% vs 89% in mice given control immunoglobulin G, respectively; p < 0.05).

Conclusions

These data suggest SDF-1 is 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.

Cerebral aneurysms are believed to occur in up to 5% of the population,53 but the incidence of subarachnoid hemorrhage is only approximately 14.5 per 100,000 in the US.44 This implies that most unruptured aneurysms remain unruptured, but for the small percentage that do rupture, the effects are devastating: a mortality rate as high as 50%, and as many as 30% of patients with subarachnoid hemorrhage ending up permanently disabled.52

Current treatments for cerebral aneurysms are directed at the surgical isolation of the aneurysm from the circulation (clipping), or intrasaccular packing to halt blood flow into the aneurysm (coiling). However, an alternative approach, that has yet to be identified, would be the development of potential drug therapies that could stabilize an unruptured cerebral aneurysm and prevent it from developing into one that is prone to rupture.

Ruptured cerebral aneurysms are characterized by inflammation-mediated wall remodeling.9–11,22,23,50 It is believed that aneurysm wall remodeling consists of matrix degeneration by macrophages and other inflammatory cells, and regeneration by smooth muscle cells, but that a failure of regeneration leads to wall thinning resulting in aneurysm rupture.9–11,22,23,50 Angiogenesis within the aneurysm wall is believed to regulate aneurysm wall remodeling and is suspected to have a critical role in aneurysm formation and rupture.23,25,45

Stromal cell–derived factor-1 (SDF-1, also known as CXCL12) is a chemokine with a robust role in angiogenesis27,28,39,46 and in activation of the inflammatory cascade.26,40 SDF-1 has been shown to be involved in the recruitment of lymphocytes within the wall of abdominal aortic aneurysms.34 Our hypothesis is that SDF-1 has a key role in angiogenesis and inflammatory cell infiltration in the walls of aneurysms. We studied this hypothesis in human cerebral aneurysm specimens, and in 2 murine models: an elastase carotid aneurysm model and a hypertensive elastase circle-of-Willis intracranial aneurysm model. In this study, the following hypotheses were proved by the experiments: 1) angiogenesis occurs in the walls of aneurysms; 2) angiogenesis is associated with inflammatory cell infiltration in the walls of aneurysms; 3) SDF-1 is expressed in aneurysms and aneurysm formation activates circulation of progenitor cells expressing CXCR4, the receptor for SDF-1; 4) SDF-1 promotes angiogenesis (by promoting endothelial cell migration and tube formation); 5) SDF-1 promotes inflammatory cell migration; 6) blocking SDF-1 inhibits angiogenesis in the walls of aneurysms; and 7) blocking SDF-1 inhibits cell proliferation in the walls of aneurysms.

Methods

Human Aneurysm Specimens

All collection and studies of human aneurysm specimens and control arteries (superficial temporal arteries [STAs]) were performed according to a research protocol approved by the Institutional Review Board at the University of Florida. Patients signed an informed consent form before aneurysm and control STA specimens were harvested at the time of craniotomy and aneurysm clipping surgery. Tissue was collected from aneurysm domes and immediately fixed by 4% paraformaldehyde.

Animals

All animal experimentation was performed in accordance with a protocol approved by the University of Florida's Institutional Animal Care and Use Committee, and all studies were performed using C57BL6 female mice, 6–8 weeks old, weighing 20–25 grams (Charles River Laboratories).

Carotid and Intracranial Aneurysm Models

Carotid aneurysms were induced in C57BL6 mice by an elastase method we have previously described.16,17 Murine circle-of-Willis intracranial aneurysms were created in C57BL6 mice using a modified version of a method previously described.33 Briefly, in our modified version, the left common carotid artery and the right renal artery are ligated in mice fed a hypertensive diet (8% NaCl diet with 0.12% β-aminopropionitril; Harlan Laboratories). One week later, 20 μl of 10 units/ml porcine pancreatic elastase solution (Worthington Biochemical Corp.) diluted in phosphate-buffered saline (Invitrogen) is stereotactically injected into the right basal cistern at 1.2 mm rostral of bregma, 1.2 mm lateral of midline, and 5.3 mm deep to the surface of the brain. Angiotensin II (Bachem) is continually infused via a subcutaneously placed osmotic pump (Alzet) at a dose of 1000 ng/kg/min in phosphate-buffered saline. Three weeks later, intracranial aneurysms are observed at the circle of Willis.

SDF-1 Blockade

Stromal cell–derived factor-1 blockade in mice was performed using anti–SDF-1 blocking antibody (monoclonal anti–human/mouse CXCL12/SDF-1 antibody; R&D Systems) intravenously 24 hours before elastase injection (100 μg/animal) and every 48 hours (50 μg/animal) for 3 weeks after elastase injection, for both the murine carotid aneurysm model and the murine intracranial aneurysm model. This antibody, human/mouse CXCL12/SDF-1 antibody, detects human and mouse CXCL12/SDF-1α and human CXCL12/SDF-1β. The source of the antibody is monoclonal mouse immunoglobulin G (IgG)1 clone 79014. Regarding the neutralization, according to the R&D data sheet, 111 μg/ml of this antibody will neutralize more than 50% of the chemotactic effect due to 2 ng/ml recombinant human CXCL12/SDF-1α in vitro. Control was performed using isotype-matched IgG control (mouse IgG1 isotype control; R&D Systems) using the same dose and schedule.

Mice were randomly selected from the cage and received either SDF-1 blockade or isotype-matched IgG control by computer-generated random selection of syringes that were blindly labeled with numbers. The assigned agent for each numbered syringe was kept in a master data sheet to which the mouse surgeon and data collectors were blinded.

Immunohistochemical Analysis

Immunohistochemical analysis was performed on human and mouse aneurysm specimens. Prior to harvesting of murine aneurysms, the mice underwent cardiac perfusion with 4% paraformaldehyde. After 24 hours of paraformaldehyde fixation, specimens were either frozenprepared or paraffin-embedded. The tissues frozen-prepared were transferred into 18% sucrose for 24 hours at 4°C, then embedded in a cassette with optimal cutting temperature compound (Sakura/Tissue-Tek Company) and frozen with dry ice and 2-methylbutane. Tissues were stored at −80°C before they were sectioned by a cryostat into 5-μm sections. Tissues paraffin-embedded were transferred into 70% ethyl alcohol followed by paraffin embedding using a rapid microwave histoprocessing unit (Milestone). The specimens were sectioned by a microtome into 5-μm sections.

Immunohistochemistry was performed with the following antibodies: anti–human/mouse SDF-1 (Santa Cruz Biotechnology), anti–human CD31 (DAKO Cytomation), anti–human CD45 (DAKO Cytomation), anti–human CD68 (Abcam), anti–MECA32 (BD Pharmingen), anti–mouse CXCR4 (Novus Biologicals), anti–mouse CD45 (Abcam), and anti–Ki 67 (Leica). Antigen retrieval with sodium citrate buffer (pH 6.0) was required for optimal staining with CXCR4. Anti–human CD31, anti–CD45, MECA-32, SDF-1, and Ki 67 were heat-retrieved in Target Retrieval Solution (DAKO Cytomation) following the manufacturer's instructions. Trilogy (Cell Marque) heat retrieval was used with CD68 staining. All slides were detected using 1:500 dilutions of species-appropriate Alexa Fluor antibodies created in donkeys. Sections were mounted in VectaShield with DAPI (Vector Laboratories) prior to imaging. Positive control tissues and concentration-matched IgG controls were included with each immunoassay (data not shown).

Stereological counting rules were used for the cell and capillary counts. A total of 5 sections from each sample were created every 200 μm. The sections were then stained for MECA-32 and imaged at high resolution. MECA-32–positive cells and capillaries were counted by 2 blinded observers (D.P.D. and K.W.N.). The area of the aneurysmal wall was measured by the blinded observers using Image-Pro software (Media Cybernetics).

Human and murine aneurysms were imaged using Optronics Magnafire digital microscopy (Meyer Instruments) and an Olympus IX71 inverted fluorescent microscope (Olympus Inc.). Cell and capillary counting was performed by 2 independent blinded observers.

Human aneurysm data collectors were blinded to the clinical and aneurysm details of the human aneurysm specimens they were given. Murine aneurysm data collectors were blinded to any treatment mice were given.

Flow Cytometry

Peripheral blood samples were collected from mice 3 days after elastase administration. Mononuclear cells from peripheral blood were isolated using Ficoll-Paque (GE Healthcare Biosciences) and incubated with anti–mouse CXCR4 antibody conjugated with fluorescein isothiocyanate (BD Pharmingen) and anti–mouse stem cell antigen-1 (SCA-1) antibody conjugated with phycoerythrin (BD Pharmingen).

Cell Culture

Mouse J774 macrophage (ATCC) was grown in Dulbecco modified Eagle medium (Mediatech) supplemented with 10% fetal bovine serum (FBS), 2 μM GlutaMAX (Invitrogen), and 100 units/ml penicillin/streptomycin (Invitrogen). Human umbilical vein endothelial cells (HUVEC; Invitrogen) were grown in Vasculife vascular endothelial growth factor (VEGF) medium (Lifeline Cell Technology) prepared as per the manufacturer's instructions.

Cell Migration Assay

Cell migration assays were performed with endothelial cells and macrophages using 6.5-mm, 8-μm Transwell Cell Inserts (Corning Life Sciences). Cells were grown to 80% confluence, serum-starved overnight, and harvested. Cells were then resuspended in serum-free medium and seeded into the upper chamber of cell inserts at a concentration of 30,000 cells/well for endothelial cells, and 200,000 cells/well for macrophages. The bottom chamber was filled with 0.6 ml of medium with or without SDF-1 (100 ng/ml) and with or without anti–SDF-1 blocking antibody (1600 ng/ml). Endothelial cells and macrophages were allowed to migrate for 24 hours at 37°C in 5% CO2. At the end of the migration period the inserts were washed and fixed in 100% methanol and stained with hematoxylin. The top portion of the membrane was wiped twice with a cotton swab and migrated cells were counted by a blinded observer.

Bottom chambers of the transwell assays were randomly assigned to SDF-1, no SDF-1, anti–SDF-1 blocking antibody, or no anti–SDF-1 blocking antibody, with syringes blindly labeled with numbers. The assigned agent for each numbered syringe was kept in a master data sheet to which the person performing the cell migration assay and data collectors were blinded.

For all wells in each group (n = 5) from each assay, 3 microscopic images per well were obtained using a ×20 objective lens with Volocity 3D Image Analysis Software, and all images were tested. All images were not overlapped with other fields. Because the cotton swabs were not able to wipe off the cells around the edges of membranes, the image of those areas was not taken. All fields were imaged at high resolution by a blinded observer, and all hematoxylin-positive cells were counted by a blinded observer.

Endothelial Cell Proliferation Assay

Human umbilical vein endothelial cells were grown to 80% confluence, harvested by 0.05% trypsin-EDTA, and resuspended in Vasculife VEGF basal medium with 2% FBS, 50 μg/ml ascorbic acid, 1.0 μg/ml hydrocortisone sulfate, and 10 μM L-glutamine. Approximately 7500 cells were seeded into wells in a tissue culture–treated 96-well plate (Techno Plastic Products) with or without SDF-1 at 100 ng/ml concentration and incubated for 48 hours. To confirm the effect of SDF-1 on endothelial cell proliferation, the activity of SDF-1 was blocked in some wells using anti–SDF-1 antibody in 1:1 molar concentration. Pictures were taken of each well with a ×5 objective on an inverted phase-contrast microscope shortly after, 24 hours after, and 48 hours after seeding. No medium was replaced or added during the experimental period.

For all wells in each group (n = 5) from each assay, 3 microscopic images per well were obtained using a ×5 objective lens with Volocity 3D Image Analysis Software, and all images were tested. All images were not overlapped with other fields. All fields were imaged with high resolution by a blinded observer, and the numbers of cells were quantified by a blinded observer.

Tube Formation Assay

Briefly, HUVECs were grown to 80% confluence, harvested, and resuspended in Vasculife VEGF basal medium with 2% FBS, 50 μg/ml ascorbic acid, 1.0 μg/ml hydrocortisone sulfate, and 10 μM L-glutamine. Approximately 10,000 cells were seeded into wells in a tissue culture–treated 96-well plate (Techno Plastic Products) with or without SDF-1 (100 ng/ml) and incubated for 24 hrs. To confirm the effect of SDF-1 on endothelial cell tube formation, the activity of SDF-1 was blocked in some wells using anti–SDF-1 antibody in 1:1 molar concentration. The number of complete loop networks formed by endothelial cells was quantified by a blinded observer. The number of cells was counted in each well to confirm that the effect of SDF-1 could not be attributed to an effect on cell proliferation.

Tissue culture wells were randomly assigned to SDF-1, no SDF-1, anti–SDF-1 blocking antibody, or no anti–SDF-1 blocking antibody with syringes blindly labeled with numbers. The assigned agent for each numbered syringe was kept in a master data sheet to which the person performing the tube formation assay and data collectors were blinded. For all wells in each group (n = 5) from each assay, 3 microscopic images per well were obtained using a ×5 objective lens with Volocity 3D Image Analysis Software, and all images were tested. All images were not overlapped with other fields. All fields were imaged with high resolution by a blinded observer, and the number of complete loops networks formed by endothelial cells was quantified by a blinded observer.

Statistical Analysis

Data are given as means and 95% CIs. The Fisher's exact test was performed to test for an association between the presence of capillaries and monocytes, macrophages, and hematopoietic-derived inflammatory cells in the walls of aneurysms. Data are summarized with means and SDs as well as medians and ranges. Because sample sizes for the 2-group comparisons were small and possibly from nonnormal distributions, making t-tests inappropriate, 2-sided permutation tests (R software, version 2.13.1) were used to determine whether group differences existed. For multiple-group comparisons, an ANOVA (SAS GLM procedure, version 9.3) to evaluate overall group differences was used, and Tukey's method was applied to maintain the Type I error rate at 0.05 when making post hoc pairwise comparisons. The ANOVA assumptions were verified by checking the normality of the residuals visually with a histogram and a Q-Q plot, and by plotting the residuals versus predicted values to check for homogeneity of variance.

Power Calculations

For the comparison of cells expressing CXCR4 (the receptor for SDF-1) by flow cytometry analysis between mice given anti–SDF-1 blocking antibody, mice given IgG control, and sham-operated mice for the carotid aneurysm model and for the hypertensive circle-of-Willis intracranial aneurysm model, we powered the experiment to have an 80% probability of detecting a true difference in 3 percentage 3 points between groups. We anticipated an overall mean response of approximately 5% and a standard deviation of approximately 2 percentage points in both groups. Although we later chose nonparametric tests for the final analysis because our data were not normally distributed, we initially assumed we would be able to use t-tests to determine whether observed differences were significant. These assumptions yielded a required sample size of 8 per group. To ensure adequate power in the event that some mice died, we increased the sample size to 10 per group.

For the comparison of endothelial cells, capillaries, and cell proliferation in the aneurysm walls between mice given anti–SDF-1 blocking antibody and mice given IgG control, we arrived at a sample size of 10 per group after making similar assumptions and performing similar calculations for each test. At assumed standard deviations of 100 for endothelial cells and cell proliferation and 10 for capillaries, this sample size gave us 80% power to detect true differences by t-test of 130 (true difference in cells/mm2) and 13 (true difference in capillaries/mm2), respectively.

For the comparison of endothelial cell migration, capillary tube formation, and inflammatory cell migration between SDF-1, SDF-1 with anti–SDF-1 blocking antibody, or none, we powered the experiment to have an 80% probability of detecting a true difference in cells/field of 30% between any 2 groups. We assumed we would observe an overall mean of approximately 225 cells/field with a standard deviation of approximately 50 in both groups on all tests. Although we later chose nonparametric tests for the final analysis because our data were not normally distributed, we initially assumed we would be able to use t-tests to determine whether observed differences were significant. These assumptions led us to set our sample size at 10 per group.

Animal Exclusion

An initial total sample size of 100 mice was used. A total sample size of 80 mice was used for data analysis. The animal experiments conducted in the flow cytometry analysis and the anti–SDF-1 blocking antibody testing were performed with an initial sample size of 10 per group. Only mice surviving to the full time point were included in the analysis. The number of excluded animals due to death were as follows: 1) flow cytometry analysis, circulation of progenitor cells in mice developing carotid aneurysms: C57BL6 (n = 1), sham (n = 2), anti–SDF-1 (n = 3) and IgG control (n = 3); 2) flow cytometry analysis, circulation of progenitor cells in mice developing intracranial aneurysms: elastase (n = 4) and sham (n = 1); and 3) blocking SDF-1 in angiogenesis and cell proliferation in carotid aneurysm wall: anti–SDF-1 (n = 3) and IgG control (n = 3).

Results

Angiogenesis in the Walls of Aneurysms

Human aneurysm specimens were harvested at the time of aneurysm clipping surgery, and immunohistochemical staining was performed demonstrating endothelial cells (CD31+) and angiogenesis (capillary formation) within the media of the aneurysm walls, whereas control STAs did not (Table 1; Fig. 1A). Elastase-induced carotid aneurysms from C57BL6 mice were harvested 3 weeks after elastase adminstration, and immunohistochemical staining was performed demonstrating endothelial cells (MECA-32+) and angiogenesis within the media of the aneurysm walls, but not in normal control murine carotid arteries (Table 2; Fig. 1B).

Fig. 1.
Fig. 1.

Photomicrographs of angiogenesis in the walls of aneurysms. A: Human aneurysms contain endothelial cells (CD31+; red) and angiogenesis (capillary formation) within the media of the aneurysm walls. Blue = DAPI. Original magnification ×20. Bar = 200 μm. B: Elastase-induced murine carotid aneurysms contain endothelial cells (MECA-32+; green) and angiogenesis (capillary formation) within the media of the aneurysm walls, but not in normal control murine carotid arteries. Blue = DAPI. Original magnification ×20. Bar = 200 μm.

TABLE 1:

Immunostaining of human aneurysms and control arteries*

LocationSize (mm)RupturedCD31+CapillariesCD68+CD45+SDF-1+
PCoA4noyesyesyesyesno
MCA4noyesnoyesyesyes
ICA terminus5noyesnonoyesyes
ACoA5yesnonoyesyesyes
ACoA6noyesyesyesyesyes
ACoA6nononoyesyesyes
MCA7nononononono
MCA7noyesnononoyes
ACoA7nononononono
MCA7nononononoyes
ACoA7noyesyesnonoyes
ACoA7noyesnoyesyesyes
ICA terminus7nononononoyes
ACA7noyesyesyesyesno
ACoA8noyesyesyesyesyes
ICA terminus8noyesyesyesyesyes
ACoA8noyesyesnonoyes
ACoA8nononononoyes
A2–A3 of ACA8noyesyesnoyesno
MCA8noyesnoyesyesyes
MCA9noyesnononoyes
MCA9noyesnoyesyesno
MCA9noyesyesyesyesyes
PCoA10nononoyesyesyes
ACoA10nononononoyes
ACoA11noyesyesyesyesyes
ACoA11noyesnononoyes
MCA11noyesnoyesyesno
MCA11noyesyesyesyesno
MCA11noyesyesnoyesyes
ICA terminus12nononononono
ACoA13noyesyesyesyesyes
MCA13noyesnononoyes
MCA14nononononono
MCA14yesyesyesyesyesyes
MCA15yesyesnoyesyesno
control STANAnononoyesyesno
control STANAnononoyesyesno
control STANAnononononono
control STANAnononononono
control STANAyesyesnononono
control STANAnononononono
control STANAnoyesyesnoyesno
control STANAyesnonoyesyesyes
control STANAnononononono

ACA = anterior cerebral artery; ACoA = anterior communicating artery; ICA = internal carotid artery; MCA = middle cerebral artery; NA = not applicable; PCoA = posterior communicating artery.

TABLE 2:

Immunostaining of murine aneurysms

ModelTime StainedTreatmentNo. of AnimalsFindings*
carotid3 hrsnone6SDF-1+, CXCR4+
intracranial3 daysnone5SDF-1+
carotid1 wknone7CD45+, CD11b+, Ki 67+
carotid3 wksIgG control7CD45++, Cd11b++, MECA-32++, capillaries++, Ki 67+
carotid3 wksanti–SDF-1 blocking antibody7CD45+/−, CD11b+/−, MECA-32−, capillaries−, Ki 67+

+ = positive expression; ++ = robust positive expression; +/− = minimal positive expression; − = no expression.

Angiogenesis and Inflammatory Cell Invasion in the Walls of Aneurysms

Human aneurysm specimens contained abundant monocytes and macrophages (CD68+) and hematopoietic-derived inflammatory cells (CD45+) within the media of the walls of the aneurysms (Table 1; Fig. 2). The presence of capillaries in the walls of aneurysms was associated with the presence of monocytes and macrophages (p = 0.01, Fisher's exact test) and hematopoietic-derived inflammatory cells in the walls of the aneurysm specimens (p = 0.01, Fisher's exact test).

Fig. 2.
Fig. 2.

Photomicrographs showing angiogenesis is associated with inflammatory cell invasion in the walls of aneurysms. Human aneurysms contain abundant monocytes and macrophages (CD68+), and hematopoietic-derived inflammatory cells (CD45+), within the media of the walls of the aneurysms. Red = CD68+ (left), CD45+ (right); blue = DAPI. Original magnification ×40. Bar = 100 μm.

SDF-1 Expression, Aneurysm Formation, and Circulation of Progenitor Cells

Human aneurysm specimens demonstrated positive SDF-1 expression by immunohistochemical staining, whereas control STAs did not (Table 1; Fig. 3A). Elastase-induced murine carotid aneurysms harvested 3 hours after elastase expressed SDF-1 and its receptor, CXCR4, but normal control murine carotid arteries did not (Table 2; Fig. 3B).

Fig. 3.
Fig. 3.

Photomicrographs showing SDF-1 is expressed in aneurysms. A: Human aneurysms express SDF-1. Red = SDF-1+, blue = DAPI. Original magnification ×60. Bar = 50 μm. B: Elastase-induced murine carotid aneurysms express SDF-1 and its receptor, CXCR4, but normal control murine carotid arteries do not. Green = SDF-1+ (left column), CXCR4+ (right column); blue = DAPI. Original magnification ×60 (left column), ×20 (right column). Bar = 50 μm (left column), 200 μm (right column). C: Murine intracranial aneurysms express SDF-1, but normal murine circle-of-Willis (COW) aneurysms do not. Red = SDF-1+, blue = DAPI. Original magnification ×60 (left), ×10 (right). Bar = 50 μm (left), 400 μm (right).

C57BL6 mice underwent right renal and left carotid ligation, and infusion of angiotensin II (by subcutaneous osmotic pump), were fed a hypertensive diet, and underwent stereotactic injection of elastase into the right basal cistern. The circle of Willis was harvested at 3 days after elastase injection. Immunohistochemical staining demonstrated positive SDF-1 expression, but not in sham-operated mice (Table 2; Fig. 3C).

Flow cytometric analysis of blood collected 3 days after elastase injection from 9 mice induced to develop carotid aneurysms demonstrated increased circulation of progenitor cells (SCA-1+) expressing CXCR4, the receptor for SDF-1, compared with 8 sham-operated mice (p < 0.01; Fig. 4A). When anti–SDF-1 blocking antibody was given to 7 mice, there were significantly fewer progenitor cells (SCA-1+) expressing CXCR4 than 7 mice given IgG control (p < 0.01; Fig. 4A).

Fig. 4.
Fig. 4.

Graphs (A and B) and photomicrograph (C) showing aneurysm formation activates circulation of progenitor cells expressing CXCR4, the receptor for SDF-1. A: Mice developing carotid aneurysms had increased circulation of progenitor cells (SCA-1+) expressing CXCR4, the receptor for SDF-1, compared with sham-operated mice. When anti–SDF-1 blocking antibody was given to mice, there were significantly fewer progenitor cells (SCA-1+) expressing CXCR4 than mice given IgG control. B: Mice developing intracranial aneurysms had increased circulation of progenitor cells (SCA-1+) expressing CXCR4 compared with sham-operated mice. C: Murine intracranial aneurysms demonstrated invasion of hematopoietic-derived inflammatory cells (CD45+, red), whereas none were found in normal circle of Willis (COW). Blue = DAPI. Original magnification ×40 (left), ×10 (right). Bar = 100 μm (left), 400 μm (right).

Flow cytometric analysis of blood collected 3 days after elastase injection from 6 mice induced to develop intracranial circle-of-Willis aneurysms demonstrated increased circulation of progenitor cells (SCA-1+) expressing CXCR4, compared with 9 sham-operated mice (p < 0.001; Fig. 4B). Additionally, immunohistochemical staining of the circle of Willis of mice that developed aneurysms demonstrated invasion of hematopoietic-derived inflammatory cells (CD45+), whereas none were found in normal circle of Willis (Fig. 4C).

SDF-1 Promoted Angiogenesis

The effect of SDF-1 on endothelial cell migration was demonstrated by in vitro cell migration assays with HUVECs cultured in the upper chamber and the bottom chamber containing either serum-free medium with SDF-1 (100 ng/ml), SDF-1 (100 ng/ml) with anti–SDF-1 blocking antibody (1600 ng/ml), or none. After 24 hours, there was significant migration of endothelial cells in the chambers containing SDF-1 compared with the chambers containing SDF-1 and anti–SDF-1 blocking antibody, or none (p < 0.01; Fig. 5A).

Fig. 5.
Fig. 5.

Graphs showing SDF-1 promotes angiogenesis (by promoting endothelial cell migration, endothelial cell proliferation, and tube formation) and inflammatory cell migration. A: In transwell cell migration assays, there was significant migration of endothelial cells in chambers containing SDF-1 compared with chambers containing SDF-1 and anti–SDF-1 blocking antibody, or neither. B: In endothelial cell proliferation assays, there was no significant difference between cells exposed to 100 ng/ml SDF-1 compared with the control and anti–SDF-1 blocked groups at 24 hours (p = 0.18) by ANOVA. However, at 48 hours, cells exposed to 100 ng/ml SDF-1 showed 31% higher proliferation than both the control (p = 0.0012) and anti–SDF-1 blocked groups (p = 0.0012) by ANOVA with Tukey's pairwise comparison. The control and anti–SDF-1 blocked groups were not found to be significantly different at 48 hours by the same analysis. C: In capillary tube formation assays, there were significantly greater complete loop networks formed by endothelial cells cultured with SDF-1 compared with SDF-1 and anti–SDF-1 blocking antibody, or neither. D: There was significantly greater macrophage migration in chambers containing SDF-1 compared with SDF-1 and anti–SDF-1 blocking antibody, or neither.

The effect of SDF-1 on endothelial cell proliferation was demonstrated in proliferation assays. The proliferation trajectory of cells exposed to 100 ng/ml SDF-1 was found to be significantly increased compared with the control (p = 0.0004) and anti–SDF-1 blocked (p = 0.0003) groups. No difference in trajectories was found between the control and anti–SDF-1 blocked groups (p = 0.919). Next, endothelial cell growth at 24 and 48 hours was compared. No significant difference among the groups was found at 24 hours (p = 0.18) by ANOVA. However, at 48 hours, cells exposed to 100 ng/ml SDF-1 showed 31% higher proliferation than both the control (p = 0.0012) and the anti–SDF-1 blocked group (p = 0.0012) by ANOVA using Tukey's pairwise comparison (Fig. 5B). The control and the anti–SDF-1 blocked group were not found to be significantly different at 48 hours by the same analysis.

The effect of SDF-1 on angiogenesis was demonstrated by in vitro endothelial cell tube formation assay, in which HUVECs were cultured with SDF-1 (100 ng/ml), SDF-1 (100 ng/ml) with anti–SDF-1 blocking antibody (1600 ng/ml), or none. After 24 hours, there were significantly greater complete loop networks formed by endothelial cells cultured with SDF-1 compared with SDF-1 and anti–SDF-1 blocking antibody, or none (p < 0.05; Fig. 5C). There was no significant difference in cell numbers between each group after the assay, which indicates that the increased tube formation was not due to an effect of SDF-1 on increased cell proliferation.

SDF-1 Promoted Inflammatory Cell Migration

The effect of SDF-1 on inflammatory cell invasion was demonstrated by in vitro cell migration assays in which macrophages were cultured in the upper chamber of Transwell Cell Inserts (Corning Life Sciences) and the bottom chamber containing serum-free medium with SDF-1 (100 ng/ml), SDF-1 (100 ng/ml) with anti–SDF-1 blocking antibody (1600 ng/ml), or none. After 24 hours, there was significantly greater macrophage migration in the chambers containing SDF-1 compared with SDF-1 and anti–SDF-1 blocking antibody, or none (p < 0.001; Fig. 5D).

Blocking SDF-1 and Its Effects

Carotid aneurysms harvested 3 weeks after elastase injection from 7 C57BL6 mice given anti–SDF-1 blocking antibody had significantly fewer endothelial cells (p < 0.05) and number of formed capillaries (p < 0.05) in the walls of the aneurysms compared with 7 mice given IgG control (Table 2; Fig. 6A and B).

Fig. 6.
Fig. 6.

Bar graphs and photomicrographs demonstrating that blocking SDF-1 inhibits angiogenesis and cell proliferation in the walls of aneurysms. A: Carotid aneurysms from mice given anti–SDF-1 blocking antibody (Ab) have significantly fewer endothelial cells in the walls of the aneurysms compared with mice given IgG control. Red = MECA-32+, blue = DAPI. Original magnification ×10 (left), ×60 (right). Bar = 400 μm (left), 50 μm (right). B: Carotid aneurysms from mice given anti–SDF-1 blocking antibody have significantly fewer formed capillaries in the walls of the aneurysms compared with mice given IgG control. C: Carotid aneurysms from mice given anti–SDF-1 blocking antibody have significantly less cell proliferation compared with mice given IgG control. Red = Ki 67+, blue = DAPI. Original magnification ×10 (left), ×60 (right). Bar = 400 μm (left), 50 μm (right).

Ki 67 antibody staining performed on murine carotid aneurysms 3 weeks after elastase injection from 7 C57BL6 mice given anti-SDF-1 blocking antibody demonstrated significantly less cell proliferation compared with 7 mice given IgG control (p < 0.05; Table 2, Fig. 6C).

The 10 C57BL6 mice given anti–SDF-1 blocking antibody developed significantly fewer intracranial aneurysms than 10 C57BL6 mice given IgG control (33% vs 89%, respectively; p < 0.05; Fig. 7A–C). One mouse from each group died perioperatively due to anesthetic issues. Mice given anti–SDF-1 blocking antibody also had fewer ruptured intracranial aneurysms (1 of 9) than mice given IgG control (3 of 9), but this difference was not statistically significant (Fig. 7D).

Fig. 7.
Fig. 7.

Graph (A) and photographs (B–D) showing that blocking SDF-1 is associated with reduced intracranial aneurysm formation. A: The 10 C57BL6 mice given anti–SDF-1 blocking antibody developed significantly fewer intracranial aneurysms than the 10 C57BL6 mice given IgG control (33% vs 89%, respectively; p < 0.05). One mouse from each group died perioperatively due to anesthetic issues. B–D: Representative examples of an intracranial aneurysm in a mouse given IgG control (B), no intracranial aneurysm in a mouse given anti–SDF-1 blocking antibody (C), and a ruptured intracranial aneurysm in a mouse given IgG control (D). Bar = 5 mm.

Discussion

The outcomes of cerebral aneurysm rupture can be devastating, with 30%–50% mortality and 50% significant morbidity.20,29,36,49 Only a small percentage of unruptured cerebral aneurysms go on to rupture, however, and their management consists of either watchful waiting, surgical clipping, or endovascular coiling. If the pathophysiology of how unruptured aneurysms go on to rupture were better understood, then a drug could potentially be developed that could stabilize and protect an unruptured aneurysm from becoming prone to rupture.

The same or similar type of drug could also stabilize aneurysms that have undergone coil embolization. Aneurysms with coil embolization are at risk for regrowth, which can occur in as many as 25% of coiled aneurysms.3 Aneurysm regrowth after coiling is believed to represent growth of the aneurysm sac.14 A drug that prevents aneurysm growth could be given to patients who have undergone coil embolization to prevent aneurysm regrowth after this procedure.

The pathophysiology of aneurysm growth is not well elucidated. Operative findings55 and histopathological analysis1,2,19,32,45 of human cerebral aneurysm specimens have identified angiogenesis or vasa vasorum in the walls of aneurysms. Additionally, angiogenic growth factors have been demonstrated in the walls of aneurysms,9,23,45 and specifically in ruptured aneurysms compared with unruptured aneurysms.9,23

Histopathological analysis of human ruptured compared with unruptured cerebral aneurysms has demonstrated a significant association between the degree of inflammatory cell invasion and the fragility of the aneurysm wall.22 Inflammatory cell, particularly macrophage, infiltration of the walls of aneurysms is associated with extracellular matrix degradation and loss of structural integrity of the wall, and these are associated with aneurysm rupture.10,22 Activation of the complement system has also been found in the walls of human cerebral aneurysm specimens, with a greater degree of complement activation in the walls of ruptured aneurysms compared with unruptured aneurysms. The presence of complement activation in the aneurysm wall further supports the hypothesis that inflammation is involved in aneurysm wall degeneration and rupture.51

Neovascularization of the vasa vasorum is believed to be the conduit by which inflammatory cells such as macrophages gain entry to the walls of diseased arteries.31 Aneurysm wall remodeling consists of matrix degeneration by macrophages and other inflammatory cells, and wall regeneration by smooth muscle cells. It is believed that a failure of regeneration is what leads to aneurysm wall thinning and eventual rupture. It has been suggested by other authors that a mechanism for cerebral aneurysm growth and rupture is: 1) the angiogenesis of vasa vasorum in the walls of aneurysms; 2) inflammatory cell infiltration of the aneurysm wall via the vasa vasorum; and 3) degeneration of the extracellular matrix of the aneurysm wall by inflammatory cell secretion of proteases resulting in fragility and loss of structural integrity.19,25,32,41,42,56

In this study we show that the presence of inflammatory cells in the media of human cerebral aneurysm walls is directly associated with the presence of capillaries (angiogenesis). We also show that SDF-1 is expressed in human cerebral aneurysms, murine carotid aneurysms, and murine circle-of-Willis intracranial aneurysms, and that aneurysm formation triggers the circulation of progenitor cells expressing CXCR4, the receptor for SDF-1. We demonstrate in vitro that SDF-1 promotes endothelial cell and macrophage migration and promotes endothelial cell tube formation (angiogenesis). We show that blocking SDF-1 inhibits murine aneurysm wall angiogenesis and cell proliferation, and inhibits the formation of murine intracranial aneurysms.

The effect of SDF-1 in promoting angiogenesis has been demonstrated in animal models of myocardial infarction,8 intracranial tumors,24 and Ewing's sarcoma.38 Additionally, blockade of SDF-1 has been shown to attenuate tumor growth by inhibiting angiogenesis.13 One theory is that SDF-1–mediated angiogenesis is a response to ischemia or arterial injury. Ischemia increases SDF-1 levels, which then promote endothelial progenitor cell formation of new blood vessels in ischemic tissue.6 In arterial injury and repair models, SDF-1/CXCR4 is a key mediator of vascular proliferation in response to injury.35 It has been shown that SDF-1 promotes proliferation, recruitment, and incorporation of endothelial-type progenitor cells into newly formed blood vessels.7,21,43,54 In vitro studies have demonstrated that SDF-1/CXCR4 signaling modifies the capillary-like organization of human embryonic stem cell–derived endothelium.5 Additionally, SDF-1 induces endothelial cell migration and capillary tube formation.30 The mechanism for endothelial cell migration is believed to be SDF-1–stimulated JNK3 activity via endothelial nitric oxide synthase–dependent nitrosylation of MKP7 to enhance endothelial migration.37 Furthermore, in gene transfer studies of SDF-1, ischemic angiogenesis and angiogenesis is enhanced by SDF-1 expression via a VEGF/endothelial nitric oxide synthase–related pathway.15

SDF-1 promotes inflammatory cell migration both directly and by promoting angiogenesis, which provides the capillary network by which inflammatory cells can infiltrate ischemic or injured tissue. Again, response to ischemia or injury appears to be critical. The recruitment of monocytes/macrophages is mediated by SDF-1, which is upregulated at the site of tissue injury. SDF-1 is key in the recruitment of monocytes/macrophages and their perivascular retention around new blood vessels that arise from neovascularlization.12 SDF-1 mediates proliferation, adhesion, migration, and homing of circulating progenitor cells that express the SDF-1 receptor CXCR4 and monocytes, thereby promoting tissue regeneration.4,18,48 Further, SDF-1 causes proliferation of CD34+ cells and differentiation of these cells into macrophages and foam cells.47

Conclusions

The data from this study combined with those discussed from previous studies leads us to believe that arterial injury or ischemia at the site of an aneurysm causes upregulation of SDF-1. SDF-1 is associated with angiogenesis by promoting proliferation, recruitment, migration, and incorporation of endothelial-type progenitor cells into newly formed blood vessels. SDF-1 is also associated with inflammatory cell infiltration of the aneurysm wall by promoting proliferation, recruitment, and migration of monocytes, macrophages, and progenitor cells. It has been suggested by other authors that newly formed capillaries in the vessel wall are the conduit by which inflammatory cells can infiltrate the aneurysm wall. Future studies are needed to investigate the role of infiltrating inflammatory cells in creating conditions contributing to aneurysm growth and possible rupture.

Disclosure

Funding for this study was provided by NIH, the Brain Aneurysm Foundation, the Thomas H. Maren Foundation, and the AANS Neurosurgery Research and Education Foundation.

Author contributions to the study and manuscript preparation include the following. Conception and design: Hoh, Hosaka, Scott. Acquisition of data: Hoh, Hosaka, Downes, Nowicki, Wilmer, Velat. Analysis and interpretation of data: Hoh, Hosaka, Scott. Drafting the article: Hoh. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Hoh. Administrative/technical/material support: Hoh, Scott. Study supervision: Hoh.

This article contains some figures that are displayed in color online but in black-and-white in the print edition.

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Article Information

Address correspondence to: Brian L. Hoh, M.D., University of Florida Department of Neurosurgery, McKnight Brain Institute, 1149 Newell Dr., Rm. L2-100, Gainesville, FL 32611. email: brian.hoh@neurosurgery.ufl.edu.

Please include this information when citing this paper: published online October 25, 2013; DOI: 10.3171/2013.9.JNS122074.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Photomicrographs of angiogenesis in the walls of aneurysms. A: Human aneurysms contain endothelial cells (CD31+; red) and angiogenesis (capillary formation) within the media of the aneurysm walls. Blue = DAPI. Original magnification ×20. Bar = 200 μm. B: Elastase-induced murine carotid aneurysms contain endothelial cells (MECA-32+; green) and angiogenesis (capillary formation) within the media of the aneurysm walls, but not in normal control murine carotid arteries. Blue = DAPI. Original magnification ×20. Bar = 200 μm.

  • View in gallery

    Photomicrographs showing angiogenesis is associated with inflammatory cell invasion in the walls of aneurysms. Human aneurysms contain abundant monocytes and macrophages (CD68+), and hematopoietic-derived inflammatory cells (CD45+), within the media of the walls of the aneurysms. Red = CD68+ (left), CD45+ (right); blue = DAPI. Original magnification ×40. Bar = 100 μm.

  • View in gallery

    Photomicrographs showing SDF-1 is expressed in aneurysms. A: Human aneurysms express SDF-1. Red = SDF-1+, blue = DAPI. Original magnification ×60. Bar = 50 μm. B: Elastase-induced murine carotid aneurysms express SDF-1 and its receptor, CXCR4, but normal control murine carotid arteries do not. Green = SDF-1+ (left column), CXCR4+ (right column); blue = DAPI. Original magnification ×60 (left column), ×20 (right column). Bar = 50 μm (left column), 200 μm (right column). C: Murine intracranial aneurysms express SDF-1, but normal murine circle-of-Willis (COW) aneurysms do not. Red = SDF-1+, blue = DAPI. Original magnification ×60 (left), ×10 (right). Bar = 50 μm (left), 400 μm (right).

  • View in gallery

    Graphs (A and B) and photomicrograph (C) showing aneurysm formation activates circulation of progenitor cells expressing CXCR4, the receptor for SDF-1. A: Mice developing carotid aneurysms had increased circulation of progenitor cells (SCA-1+) expressing CXCR4, the receptor for SDF-1, compared with sham-operated mice. When anti–SDF-1 blocking antibody was given to mice, there were significantly fewer progenitor cells (SCA-1+) expressing CXCR4 than mice given IgG control. B: Mice developing intracranial aneurysms had increased circulation of progenitor cells (SCA-1+) expressing CXCR4 compared with sham-operated mice. C: Murine intracranial aneurysms demonstrated invasion of hematopoietic-derived inflammatory cells (CD45+, red), whereas none were found in normal circle of Willis (COW). Blue = DAPI. Original magnification ×40 (left), ×10 (right). Bar = 100 μm (left), 400 μm (right).

  • View in gallery

    Graphs showing SDF-1 promotes angiogenesis (by promoting endothelial cell migration, endothelial cell proliferation, and tube formation) and inflammatory cell migration. A: In transwell cell migration assays, there was significant migration of endothelial cells in chambers containing SDF-1 compared with chambers containing SDF-1 and anti–SDF-1 blocking antibody, or neither. B: In endothelial cell proliferation assays, there was no significant difference between cells exposed to 100 ng/ml SDF-1 compared with the control and anti–SDF-1 blocked groups at 24 hours (p = 0.18) by ANOVA. However, at 48 hours, cells exposed to 100 ng/ml SDF-1 showed 31% higher proliferation than both the control (p = 0.0012) and anti–SDF-1 blocked groups (p = 0.0012) by ANOVA with Tukey's pairwise comparison. The control and anti–SDF-1 blocked groups were not found to be significantly different at 48 hours by the same analysis. C: In capillary tube formation assays, there were significantly greater complete loop networks formed by endothelial cells cultured with SDF-1 compared with SDF-1 and anti–SDF-1 blocking antibody, or neither. D: There was significantly greater macrophage migration in chambers containing SDF-1 compared with SDF-1 and anti–SDF-1 blocking antibody, or neither.

  • View in gallery

    Bar graphs and photomicrographs demonstrating that blocking SDF-1 inhibits angiogenesis and cell proliferation in the walls of aneurysms. A: Carotid aneurysms from mice given anti–SDF-1 blocking antibody (Ab) have significantly fewer endothelial cells in the walls of the aneurysms compared with mice given IgG control. Red = MECA-32+, blue = DAPI. Original magnification ×10 (left), ×60 (right). Bar = 400 μm (left), 50 μm (right). B: Carotid aneurysms from mice given anti–SDF-1 blocking antibody have significantly fewer formed capillaries in the walls of the aneurysms compared with mice given IgG control. C: Carotid aneurysms from mice given anti–SDF-1 blocking antibody have significantly less cell proliferation compared with mice given IgG control. Red = Ki 67+, blue = DAPI. Original magnification ×10 (left), ×60 (right). Bar = 400 μm (left), 50 μm (right).

  • View in gallery

    Graph (A) and photographs (B–D) showing that blocking SDF-1 is associated with reduced intracranial aneurysm formation. A: The 10 C57BL6 mice given anti–SDF-1 blocking antibody developed significantly fewer intracranial aneurysms than the 10 C57BL6 mice given IgG control (33% vs 89%, respectively; p < 0.05). One mouse from each group died perioperatively due to anesthetic issues. B–D: Representative examples of an intracranial aneurysm in a mouse given IgG control (B), no intracranial aneurysm in a mouse given anti–SDF-1 blocking antibody (C), and a ruptured intracranial aneurysm in a mouse given IgG control (D). Bar = 5 mm.

References

  • 1

    Atkinson JLOkazaki HSundt TM JrNichols DARufenacht DA: Intracranial cerebrovascular vasa vasorum associated with atherosclerosis and large thick-walled aneurysms. Surg Neurol 36:3653691991

    • Search Google Scholar
    • Export Citation
  • 2

    Bavinzski GTalazoglu VKiller MRichling BGruber AGross CE: Gross and microscopic histopathological findings in aneurysms of the human brain treated with Guglielmi detachable coils. J Neurosurg 91:2842931999

    • Search Google Scholar
    • Export Citation
  • 3

    Campi ARamzi NMolyneux AJSummers PEKerr RSSneade M: Retreatment of ruptured cerebral aneurysms in patients randomized by coiling or clipping in the International Subarachnoid Aneurysm Trial (ISAT). Stroke 38:153815442007

    • Search Google Scholar
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