Intraluminal Cell Transplantation Prevents Growth and Rupture in a Model of Rupture-Prone Saccular Aneurysms
Background and Purpose—Aneurysm occlusion by intraluminal thrombus formation is the desired effect of all endovascular treatments. Intraluminal thrombus may, however, recanalize and be absorbed, unless it is infiltrated by cells that turn it into fibrous tissue (neointima). Because ruptured aneurysm walls are characterized by loss of smooth muscle cells, we assessed the impact of mural cell loss on wall remodeling of thrombosed aneurysms and investigated whether neointima formation could be enhanced by direct transplantation of cells into the thrombus.
Methods—Sidewall aneurysms were microsurgically created in rats (n=81). Certain aneurysms were decellularized. Thrombosis was induced using direct injection of a fibrin polymer into the aneurysm. CM-Dil–labeled smooth muscle cells were injected into 25 of 46 fibrin embolized aneurysms. Recanalization and aneurysm growth were monitored with magnetic resonance angiography. Endoscopy, optical projection tomography, histology, and immunohistochemistry were used to study the fate of transplanted cells, thrombus organization, and neointima formation.
Results—Decellularized embolized aneurysms demonstrated higher angiographic recurrence compared with decellularized embolized aneurysms with transplanted cells (P=0.037). Local cell replacement at the time of thrombosis resulted in better histological neointima formation than both nondecellularized embolized aneurysms (P<0.001) and decellularized embolized aneurysms (P=0.002). Aneurysm growth and rupture were observed exclusively in decellularized embolized aneurysms.
Conclusions—Lack of smooth muscle cells in the aneurysm wall promotes wall degradation, aneurysm growth and rupture, even if the aneurysm is occluded by luminal thrombus. Transplantation of smooth muscle cells into the luminal thrombus can reduce this degenerative remodeling.
- cell transplantation
- intracranial aneurysm
- smooth muscle cells
Inducing flow stasis-mediated luminal thrombosis of saccular intracranial aneurysms (IAs) is the goal of all current neuroendovascular treatments. Ideally, the intraluminal thrombus results in immediate exclusion of the aneurysm from circulation, promotes re-endothelialization, and serves as scaffold for recruitment of cells that synthesize extracellular matrix and turn the clot into fibrotic scar tissue (neointima). This cell-mediated remodeling process eventually leads to permanent occlusion of the aneurysm and reconstitution of the luminal surface of the arterial segment harboring the aneurysm.
Thrombolytic cascades are activated at the same time as the coagulation cascade. The luminal thrombus in an aneurysm may therefore eventually be recanalized and reabsorbed if neointima formation and re-endothelialization of the aneurysm ostium do not occur. Growing evidence indicates that the balance between healing and reverse remodeling is mainly determined by the condition of the aneurysm wall.1–4 Loss of cells from the aneurysm wall is characteristic to ruptured human IAs.5–7 Previously it has been shown in models of fusiform aortic aneurysms that restoration of the cell content of the aneurysm with local cell transplantation can reduce the progression of the aneurysm.8,9
We investigated the hypothesis that loss of mural cells will promote degenerative remodeling of the saccular aneurysm wall in thrombosed aneurysms, and that this degenerative remodeling can be reduced or compensated by direct transplantation of smooth muscle cells (SMCs) into the luminal thrombus.
Materials and Methods
Animals, Study Design, and Anesthesia
A total of 81 rats were used for this study. Male Wistar rats (Harlan, Horst, The Netherlands) weighing 290 to 400 g served either as experimental pilots (n=10, fibrin polymer induced thrombosis; n=8, optical projection tomography tissue processing and staining protocols) or were randomly allocated to 1 of the 3 groups: thrombus induction in healthy nondecellularized aneurysms (n=17), sick decellularized aneurysms (n=21), and sick decellularized SMC-transplanted aneurysms (n=25). We did not study 1 single group of rats with repeated follow-up contrast-enhanced (CE) MRI, but rather 3 different subgroups representing different time points (groups at days 3, 7, and 21) within each experimental group. After interim analysis, a replication of experiments was performed for time point day 7 (n=4, for all groups). Animals were excluded from final histological analysis when autopsy could not be performed within 12 hours. Study design, drop outs, follow-up modalities, and schedule are given in Figure 1. Surgical characteristics are given in Table I in the online-only Data Supplement.
Aneurysm Model, Graft Decellularization, and Thrombus Induction
Sidewall aneurysms were created under operating microscope by microsurgical end-to-side anastomosis of an arterial saccular graft from the descending thoracic aorta of syngeneic donor rat to the infrarenal abdominal aorta of the recipient animal, as described in detail in the Methods in the online-only Data Supplement.
The untreated donor grafts were immediately reimplanted in thrombus-induced nondecellularized aneurysms rats. The saccular aneurysms to be decellularized before use in thrombus-induced decellularized aneurysms and thrombus-induced decellularized smooth muscle cell–transplanted aneurysms groups were processed as described in detail previously.10,11 Briefly, donor grafts were harvested and frozen in PBS at −4°C. The next day the grafts were thawed, incubated for 10 hours at 37°C in 0.1% sodium dodecyl sulfate (SDS), and finally refrozen until use. Thrombus induction was performed using fibrin polymer according to the manufacturer’s protocol (Tisseel, Baxter). All saccular aneurysms were maximally filled with fibrin clots with or without SMC. To standardize thrombus induction, fibrin clot was carefully aligned with the orifice of the graft leaving no neck remnant. To minimize group differences in hemodynamics, rate of thrombosis and potential recanalization rate, we standardized surgical procedures including aneurysm dimensions and created anastomosis perpendicular to the parent artery (see Methods in the online-only Data Supplement).
Cell Culture, Labeling, and Immunofluorescence
Primary cell cultures were obtained using freshly isolated cells from abdominal aorta segments from the same breeder as the recipient Wistar rats. Although not strictly syngeneic, the histocompatibility is sufficient to avoid graft rejection immune response. Cells were grown, maintained, and underwent 6 to 10 passages before transplantation. Further details of SMC culture are given in the Methods in the online-only Data Supplement. Cells were labeled using a carbocyanine lipid cell membrane tracer (CellTraker CM-Dil, life Technologies; Invitrogen), homogenously suspended in thrombin solution in a concentration of 1×106 cells/mL and mixed with the fibrinogen component to form a clot. The switch from the contractile to the synthetic SMC phenotype (myofibroblast cell phenotype) was confirmed by immunostaining of cytoplasmic smooth muscle actin and vimentin (Figures I–III and Methods in the online-only Data Supplement).
MRI and Angiographic Evaluation
Contrast-enhanced (CE) magnetic resonance angiography (MRA) studies were performed with a 4.7 T scanner (PharmaScan, Bruker BioSpin, Ettlingen, Germany). Existing protocols for high-resolution time-of-flight MRA12 and CE-MRA11 were performed. CE-MRA were analyzed and scored for the rate of thrombosis as described previously, with slight modification (Figure IV in the online-only Data Supplement)11,13: 0=no recurrence (no filling); 1=partial recurrence (<50% filling); and 2=complete recurrence (>50% filling). Growing aneurysms were further analyzed using 3-dimensional active contour segmentation software itk-SNAP (V2.4, Pennsylvania, PA).14
Tissue Preparation and Combined Macroscopic and Endoscopic Scoring
After final follow-up MRA, the animals underwent laparotomy and dissection of the aneurysm (Movie I in the online-only Data Supplement). The tissues were perfusion fixed with 4% paraformaldehyde in PBS and measured in all dimensions. The posterior wall of the aorta was opened and evaluated by macroscopic and endoscopic intraluminal aneurysm surface inspection (Leica Stereomicroscope, Wetzlar, and Karl Storz Endoscopy, Tuttlingen, Germany). Neointima formation was graded as described previously, with slight modification (Figure V in the online-only Data Supplement)15: 0=thick neointima, no recanalization, completely sealed; 1=thick neointima, minimal recanalization; 2=thin neointima covering completely; 3=deficient neointima or recurrent aneurysm; and 4=no neointima or complete recanalization. Each case underwent observer-blinded analysis of neointima formation based on ≥1 macroscopic and endoscopic video screen-shot by S.M. and P.H. Scores were dichotomized as complete or near-complete occlusion (scores, 0–1) and incomplete occlusion (scores, ≥2), and compared using the Fisher exact test.
Histology and Histological Analysis
Aneurysms were stained for hematoxylin–eosin, Elastica van Gieson, and Masson–Goldner trichrome. Fluorescent images were examined to determine the fate of transplanted CM-Dil–labeled SMC. All histological slides underwent qualitative analysis by 2 observers (J.F. and S.M.) as described in detail in the Methods in the online-only Data Supplement. Histological scoring was performed blinded for treatment allocation. Slides were visualized under light microscope (Carl Zeiss) and fluorescence microscope (Axiovision, Carl Zeiss). Postprocessing was performed using Adobe Photoshop CS 6 (V13.0, Adobe Systems) and Image J 1.47e (National Institutes of Health, Bethesda, MD) software.16
Optical Projection Tomography
Spatial migration of CM-Dil–labeled SMCs was followed up for 1 week after transplantation using optical projection tomography (Bioptonics OPT 3001).17 Aneurysm and parent artery were stained using in vivo lectin perfusion and visualized in Bioptonics viewer (v2.01, Bioptonics). Three-dimensional animations were created using Imaris 7 (Bitplane Scientific Software; Figure VI and Movie II in the online-only Data Supplement).
Data Analysis and Statistics
Two-tailed Fisher exact test was used for comparison of dichotomized histological, macroscopic and endoscopic grades, and aneurysm growth and recurrence rate between thrombus-induced nondecellularized aneurysms and thrombus-induced decellularized aneurysms, thrombus-induced nondecellularized aneurysms and thrombus-induced decellularized smooth muscle cell–transplanted aneurysms, and thrombus-induced decellularized aneurysms and thrombus-induced decellularized smooth muscle cell–transplanted aneurysms, respectively. Student t test was performed to assess differences in surgical characteristics. Data were analyzed and visualized using Graph Pad Prism statistical software V6.04 for Windows (GraphPad Software, San Diego, CA). Values are expressed as mean±SD and 95% confidence interval. A probability value of <0.05 was considered statistically significant.
Luminal thrombus was successfully induced by fibrin polymer injection in all aneurysms with the exception of 1 decellularized aneurysm and 2 cases of decellularized and cell-transplanted aneurysm. In these 3 cases, the aneurysm healed completely (complete fibrin clot reorganization). There was endothelial cell loss in all groups. There was a complete decellularization of all SDS-treated aneurysm grafts without microscopic changes in the extracellular matrix. Case-by-case analysis of macroscopic and endoscopic findings including baseline and follow-up CE-MRA of all animals in all groups including replicates and drop outs are summarized in Figure VII in the online-only Data Supplement.
Recanalization, Growth, and Wall Structure in Embolized Aneurysms With a Healthy Wall
All but 1 aneurysm (1/4) showed partial or complete recurrence after 3 weeks. With the exception of 1 case, all aneurysms showed incomplete neointima formation (Figure 2). In the replication series, half of the aneurysms (2/4) recurred and remained stable. Almost half (5/12) of healthy nondecellularized aneurysm walls showed complete loss (n=3) or only a few remaining viable cells (n=2). Aneurysms with an initially healthy wall that suffered mural cell loss showed significant more wall inflammation and a trend to more neutrophil accumulation in the thrombus when compared with aneurysms without loss of mural cells (P=0.01 and P=0.072; Table II in the online-only Data Supplement). Two of 3 growing aneurysms demonstrated complete mural cell loss.
Recanalization, Growth, and Wall Structure in Embolized Aneurysms With a Sick Wall
First recurrence was seen 7 days after thrombus induction. After 3 weeks, all decellularized aneurysms were partially or completely recanalized. Three aneurysms had grown including 1 aneurysm that grew into a giant partially thrombosed multilobulated aneurysm (Figure VIII in the online-only Data Supplement). One of the growing aneurysms ruptured 10 days after creation. With the exception of 2 cases (2/8), neointima formation was incomplete in all decellularized aneurysms. In the replication series, half of the aneurysm (2/4) demonstrated recurrence and growth. All aneurysms that had experienced growth demonstrated (1) enhanced endoluminal and intrathrombus neutrophil accumulation, (2) inflammatory and hemorrhagic transformation of the wall, and (3) enhanced periadventitial fibrosis (Figure IX in the online-only Data Supplement).
Recanalization, Growth, and Wall Structure After Cell Transplantation in Embolized Aneurysms With a Sick Wall
All cell-transplanted aneurysms with a sick wall remained occluded with complete or near-complete neointima formation in all but 1 aneurysm (1/12). The single case of incomplete neointima formation demonstrated increase in aneurysm size at 3-week follow-up. There was no recurrence or growth in the replicate experiments. Intraluminal cell replacement promoted rapid thrombus organization, neointima formation, and aneurysm healing (Figure 3A). After transplantation, cells were equally distributed within the intraluminal thrombus, became confluent within the fibrin clot, and demonstrated no trend of migration toward the aneurysm wall or the aneurysm ostium (Figure 3B). Spindle-shaped CM-Dil–labeled cells embedded in collagen bundles were found to organize the fibrin clot and neointima along the neck (Figure 4).
Comparison of Thrombus-Induced and Cell-Transplanted Aneurysms With Thrombus-Induced Aneurysms Without Cell Transplantation
Overall recurrence rate of thrombus-induced sick decellularized aneurysms was significantly higher when compared with thrombus-induced sick decellularized aneurysms with concomitant cell replacement (P=0.037). Decellularized aneurysm treated with local cell replacement at the time of thrombosis demonstrated significantly superior histological neointima formation than thrombus-induced healthy nondecellularized aneurysms (P<0.001) and thrombosed sick decellularized aneurysms (P=0.002).
There was significantly more neutrophil accumulation in the thrombus in decellularized aneurysms (P=0.03) and a trend (P=0.15) toward increased neutrophils in the thrombus of nondecellularized aneurysms as compared with decellularized aneurysms with local cell transplantation (Figure X in the online-only Data Supplement). Healed aneurysm had significantly less neutrophils in the thrombus as compared with aneurysms with missing neointima formation (P=0.017; Table III in the online-only Data Supplement).
Our results demonstrate that loss of SMC in the aneurysm wall impairs thrombus organization and neointima formation in thrombosed aneurysms and promotes recurrence, growth, and eventual rupture of embolized aneurysms. Furthermore, our results suggest that thrombus induction in healthy aneurysms can provoke mural cell loss and increase intramural and intrathrombus inflammation. Local cell transplantation to the thrombus enhances thrombus organization, compensates for the loss of mural cells, attenuates inflammatory reactions, promotes aneurysm healing, and reduces recurrence, growth, and rupture rate of thrombosed aneurysms.
The sidewall aneurysm model only partially reflects the actual pathogenesis of human IA. The model allows to study basic biological concepts although one needs to be aware of differences in hemodynamic characteristics and vascular biology between the aorta and cerebral arteries. None of the available aneurysm models that can be embolized ideally represent a human saccular cerebral artery aneurysm or reproduce the exact pathobiology behind aneurysm formation or rupture.
The models developed by Hashimoto et al18 are the most physiological IA models to date in terms of representing human morphology, histology, and IA hemodynamics. Because we assessed the impact of mural cell loss on wall remodeling of thrombosed aneurysms, we performed the experiments in the presented saccular sidewall model, which allowed us to induce thrombosis and perform cell transplantation in arterial grafts with different wall conditions (cell-rich healthy and decellularized sick walls).
Aneurysm Wall Condition Predicts Natural Course and Fate of Intraluminal Thrombosis
MRA, macroscopic and microscopic evaluation, and histology of aneurysms with loss of mural cells revealed their inability to organize induced thrombosis. Initially healthy aneurysms that suffered mural cell loss because of rapid intraluminal thrombus induction showed an evolution similar to sick decellularized aneurysms in terms of recurrence, growth, and increase in wall inflammation and intrathrombus neutrophil accumulation. Taken together, the findings corroborate previous experimental research suggesting that neointimal SMCs originate from the wall of the aneurysm1 and that aneurysms missing mural cells are subject to increased inflammatory reaction, severe wall degeneration, aneurysm growth, and eventual rupture.11
If the experimental findings holds true for the human IA wall it would mean that decreased number of SMCs would be a potential surrogate marker for not only prediction of rupture risk but also estimation of healing response after embolization. Macrophage imaging within human cerebral aneurysm walls using ferumoxytol-enhanced MRI demonstrated that knowledge of the histological correlation of aneurysm risk can be valuable for clinical decision making. In human histopathologic series loss of mural SMCs was found to be even more characteristic for rupture-prone aneurysm wall than inflammation.6,7
Local Cell Therapy Heals Aneurysms With Degenerated/Decellularized Walls
The findings demonstrate that transplanted viable SMCs compensate the loss of mural cells and improve early thrombus reorganization and neointima formation. Neointima formation prevents recurrence and additional thrombus formation. Consequently, intraluminal amount of new red blood cells, platelets, and macromolecular plasma components such as lipids, complement, and immunoglobulins are reduced, which in turn attenuates wall inflammation. This could explain the finding that healed aneurysm had significantly fewer neutrophils in the thrombus as compared with aneurysms with missing neointima formation.
The compensation of mural SMC loss by local cell therapy may have great therapeutic potential. It has been demonstrated that bone marrow mesenchymal stem cell and SMC transplantation can stabilize growing abdominal aortic aneurysms in rats8,9 and promote healing of saccular venous pouch aneurysms in dogs.19 In a rabbit model of elastase-induced saccular aneurysms, endovascular seeding of bone marrow mesenchymal stem cells promoted thrombus organization and caused the aneurysm to shrink in size.20 For a more detailed discussion of potential cell-mediated methods to promote aneurysm wall healing and repair please see our prior review.21
In previous experiments, spontaneous stepwise thrombosis did not result in increased inflammation, mural cell loss in healthy aneurysms, and did not promote aneurysm growth or rupture.11 A potential explanation for these (contradictory) findings might be that acute thrombosis induces inflammation on such a large scale that it over-runs the aneurysm wall defense mechanisms or that fibrin biopolymer leads to a thicker clot than normal coagulation. Acute thrombus induction has been linked to mural destabilization not only in experimental aneurysms22,23 but also in the clinical setting after application of flow diverters to treat intracranial aneurysms.24,25 These studies consistently found histopathologic characteristics comparable with those found in our study, with large amounts of inflamed cells and mural cells loss in destabilized aneurysm wall segments after rapid thrombosis.22,23,25
However, the cell loss observed in healthy aneurysms could also be attributed to ischemic or inflammatory reactions induced by the fibrin polymer thrombus. It might also be possible that luminal fibrin polymer lead to a thicker clot than normal coagulation and impaired diffusion of nutrients to the healthy media and promoted inflammation as a secondary reaction. In future experiments it would be of much interest to study the course of clinically more relevant coil-induced thrombosis in decellularized and healthy (cell-rich) aneurysms.
Hemodynamic characteristics, rate of spontaneous thrombosis and recanalization, and healing/remodeling processes after intraluminal thrombosis are significantly influenced by the experimental aneurysm angioarchitecture.13,26,27 Despite the efforts made to standardize aneurysm dimensions, we cannot exclude completely the possibility of group differences in hemodynamics and hence rate of thrombosis and recanalization.
The present study provides additional evidence that the condition of the aneurysm wall is of utmost importance for the net effect that a thrombus has on aneurysm healing. Methods that can be used in the clinical setting to evaluate the number and function of mural SMCs may significantly help in the diagnostics of rupture-prone IA and in prediction of accuracy of healing capacity after embolization. Novel therapies that increase the number of functioning SMC could compensate for mural cell loss and reduce the recurrence, growth, and rupture rate of IA and may also improve the healing capacity after intraluminal thrombus induction.
We thank N. Lim and O. Mattila for their excellent technical assistance. We thank J. Kenkkila for support in optical projection tomography (OPT) imaging acquisition, image processing, and in 3-dimensional contrast-enhanced magnetic resonance angiography volumetry. We thank G.V.D. Amico Lago for sharing her expertise in OPT tissue processing. We thank T. Holopainen for sharing her expertise for in vivo fluorescein isothiocyanate lectin staining. We are grateful to A. Reijula and M. Berthold for tissue processing and staining. The Biomedicum Molecular Imaging Unit is acknowledged for microscopy services. We are grateful for the support of the Kari Alitalo laboratory for cell culture staining. Dr Marbacher contributed to the study concept, performed the experiments, analyzed the data, prepared the figures, obtained personal funding, and wrote the article. Dr Frösén developed the study concept, helped to perform initial surgical experiments, provided expertise in cell culture and pathology, analyzed the histopathology, and helped to write the article. Dr Marjamaa performed surgical experiments and provided expertise in MRI. Dr Anisimov provided expertise in cell culture and cellular labeling. Dr Honkanen analyzed macroscopic and endoscopic video screenshots. Dr von Gunten provided expertise in pathology, tissue processing, and staining. Dr Abo-Ramadan performed MR scans and provided expertise in and designed MRI sequences. Drs Hernesniemi and Niemelä directed the studies, provided funding, and helped to write the article.
Sources of Funding
This work was supported by the research funds of the Helsinki University Central Hospital, Helsinki, Finland, and by grants from The Sigrid Juselius Foundation (Helsinki, Finland). Dr Marbacher was supported by a grant from the Swiss National Science Foundation (PBSKP3-123454).
We are solely responsible for the design and conduct of the presented study and report no conflict of interest concerning the materials and methods used in this study or the findings specified in the article. They confirm the adherence of ethical standards. Dr Frösén has received research grants or research support from Cook Medical and speaker honorariums from the European Society for Minimally Invasive Neurological Therapy. The other authors report no conflicts.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.114.006600/-/DC1.
- Received July 19, 2014.
- Revision received September 20, 2014.
- Accepted September 24, 2014.
- © 2014 American Heart Association, Inc.
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