doi: 10.1161/01.CIRCULATIONAHA.104.525071
(Circulation. 2005;112:I-111 – I-116.)
© 2005 American Heart Association, Inc.
Cell Transplantation and Tissue Engineering |
Method of Cell Transplantation Promoting the Organization of Intraarterial Thrombus
From the Departments of Thoracic and Cardiovascular Surgery (K.H., T.S., K.F., M.K., Y.M., H.S.), Orthopaedics (H.H., A.U.), Hematology (M.N.), and Pathology (K.I.-Y., T.Y.), Mie University School of Medicine, Tsu; the Department of Chemistry for Materials (K.M.), Faculty of Engineering, Mie University, Tsu; and Graduate School of Health Science (I.Y.), Suzuka University of Medical Science, Suzuka, Japan.
Correspondence to Takatsugu Shimono, MD, Department of Thoracic and Cardiovascular Surgery, Mie University School of Medicine, 2-172, Edobashi, Tsu, Mie 514-8507, Japan. E-mail simono-t{at}clin.medic.mie-u.ac.jp
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Abstract |
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Background— Endovascular aortic repairs have been developed as less invasive treatments for aortic aneurysms. Some aneurismal cavities, however, remain without organization, causing a re-expansion of the aneurysms. We studied cell transplantation into the aneurismal sac to promote the organization of thrombus for the complete healing of aneurysms.
Methods and Results— Skin fibroblasts and skeletal myoblasts were isolated from rats for cell transplantation. An intraarterial thrombus model was made by ligation of the carotid artery. Culture medium (medium group, n=11), collagen gel (gel group, n=11), fibroblasts with collagen gel (F group, n=15), myoblasts with collagen gel (M group, n=12), or mixture of fibroblasts and myoblasts with collagen gel (F+M group, n=14) were injected into the thrombus. After 28 days, histologically, the arterial lumens of the F and M groups were partly filled with fibrous tissues, whereas in the F+M group organization was almost completed and luminal sizes diminished. Immunohistochemical staining demonstrated that 
-smooth muscle actin-positive cells were more abundantly contained in the organized area of the F+M group than in the other groups. We also analyzed cellular function in vitro with immunofluorescence; coculture of fibroblasts and myoblasts showed that the fraction of -smooth muscle actin-positive fibroblasts increased. This phenomenon accounts for the rapid organization of thrombus in the F+M group in vivo.
Conclusions— Cell transplantation accelerated thrombus organization. Especially, myoblasts enhanced differentiation of fibroblasts into myofibroblasts, contributing to rapid thrombus organization. Cell transplantation into unorganized spaces seems applicable to endovascular treatment of aneurysms.
Key Words: aorta • aneurysm • thrombus • cells • transplantation
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Introduction |
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Recently, endovascular aortic repair, as a less invasive surgical procedure, has been successfully applied in patients with aortic aneurysms.1–3 We have reported the efficacy of an endovascular stent-graft treatment for aortic dissections, especially for acute onset dissections.4,5 In successful cases, thrombi of excluded aneurismal sacs are gradually organized, followed by contraction of the aneurismal wall. In some cases, however, persistent endoleak prevents the organization of thrombus in excluded spaces, raising endotension and causing the re-expansion of an aneurysm or false lumen.6,7
Various attempts, including transcatheter embolization with coils and agents such as thrombin, lipiodol, and gelfoam powder, have been performed to block persistent endoleaks. Thrombin is the most commonly used agent, but insufficient effects on clot formation in the sac and peripheral emboli have been reported.8 Liquid adhesive, n-butyl 2-cyanoacrylate (n-BCA), and liquid embolic agent, ethylene-vinyl-alcohol copolymer (Onyx), are also applied to occlude the sac cavity.9,10 These agents appear to provide favorable results with their immediate effects; however, long-term outcomes are still of concern because these artificial agents are not physiological and may block the normal healing mechanism.
During the natural process of organization of a thrombus, a crucial step can be the recruitment of fibroblasts into a blood clot. Fibroblasts transform into myofibroblasts, which produce collagen fibers and generate contraction forces, resulting in stable organized tissues and the contraction of the aneurysm.11,12 In this study, we examined whether cell transplantation into the thrombus can promote its organization. A matrix of collagen gel was used to retain cells in the space, as it is known that collagen triggers the blood clotting system of platelets and coagulation factors. Thus, the effects of fibroblasts on promoting the organization of fresh thrombus were examined in an intraarterial thrombus model of a dead-ended rat lumen with a ligated carotid artery. We also used myoblasts, muscle-derived stem cells that are commonly used in cell transplantation therapy into infarcted myocardium,13–15 as the transplanted cells and tested their combined effect with fibroblasts.
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Methods |
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Animals
All procedures used in the study, as well as animal care, were in accordance with the guidelines approved by the Mie University Animal Experiment and Care Committee. Isogenic Lewis rats (SLC Japan Co, Shizuoka, Japan) were used as donors and recipients to simulate clinical autologous transplants.
Isolation and Culture of Skeletal Myoblasts and Skin Fibroblasts
After an overdose with pentobarbital, skeletal muscles, the musculus gluteus maximus, or the musculus quadriceps femoris of adult rats were excised. Muscles were then minced with scissors and muscle fragments were incubated in culture medium consisting of Dulbecco’s modified Eagle medium (Invitrogen), plus 10% heat-inactivated fetal bovine serum (FBS, ICN Biomedicals) and 100 µg/mL kanamycin (Invitrogen) supplemented with 0.2% type XI collagenase (Sigma) for 120 minutes at 37°C in a shaken water bath. The remaining muscle mass was then spun at 1000 rpm for 5 minutes. Sedimented muscle fragments were then incubated in 50 U/mL dispase (BD Biosciences) for 60 minutes at 37°C to dissociate myogenic cells. The supernatant containing the cells was then collected and washed twice with the culture medium. By taking advantage of their differential adherence to a dish, myoblasts were separated from contaminating cells such as fibroblasts; the cells obtained were initially plated on fibronectin-coated culture dishes (BD Biosciences) in culture medium and incubated at 37°C for 30 minutes. Nonadherent cells, myoblasts, were then collected and plated on laminin-coated culture dishes (BD Biosciences). Cells were grown at 37°C in 5% CO2/95% air and the culture medium was changed 2 days later. When the cells reached confluence, they were harvested by trypsinization, washed, and preserved with a cell preservation solution (Cellbanker, Mitsubishi Kagaku Iatron) at –80°C in a freezer until they were to be transplanted.
To isolate skin fibroblasts, neonatal rats were anesthetized with an inhalation of ethanol. Their back skins were harvested and washed 3 times with phosphate buffered saline. The skins were then incubated in a culture medium consisting of Dulbecco’s modified Eagle medium, plus 10% FBS and 100 µg/mL kanamycin supplemented with 0.2% type XI collagenase for 48 hours at 4°C. After removing the epidermis, the remaining corium fibroblasts were then suspended in culture medium, plated on culture dishes, and incubated at 37°C in 5% CO2/95% air. Passage was done when the cells reached confluence. Fibroblasts at 3 passages were used in this study.
Cell Transplantation
On the day of transplantation, cells (myoblasts and/or fibroblasts) were defrosted and washed by centrifugation. The pelleted cells were labeled with chloromethylbenzamido derivatives of DiI (1 µg/mL in Hanks’ balanced salt solution, Molecular Probes), resuspended in type-I collagen gel (3.0 mg/mL, Cellmatrix Type 1-A, Nitta Gelatin Inc), and kept on ice.
Rats (weight, 280 to 330g) were anesthetized by intraperitoneal administration of sodium pentobarbital (50 mg/kg). A midline incision was made over the neck, the left carotid sheath was exposed and carefully incised, and the common carotid artery was then isolated. The artery was ligated near the carotid bifurcation, and a 24-gauge catheter was inserted into the artery from a site just proximal of the ligation point in a retrograde fashion. A cell suspension of 100 µL gel was injected into the artery through the catheter, which was then removed and the insertion point closed to prevent the leakage of any injected gel. The wound was closed with a running suture, and the animal returned to the animal care facility.
Experimental Groups
Rats (n=63) were divided into 5 groups. The medium group (n=11) was injected with 100 µL culture medium only, the gel group (n=11) with the 100 µL type I collagen gel, the F group (n=15) with the 100 µL gel containing 2x106 skin fibroblasts, the M group (n=12) with 100 µL gel containing 2x106 skeletal myoblasts, and the F+M group (n=14) with 100 µL gel containing 1x106 skin fibroblasts and 1x106 skeletal myoblasts.
Histological Analyses
Animals were anesthetized with pentobarbital 14 or 28 days after injection, and transcardiac perfusion was performed with 4% paraformaldehyde. The left common carotid arteries along with the surrounding soft tissue were then carefully harvested and fixed in 4% paraformaldehyde at 4°C overnight. Specimens were divided into 6 parts; 3 parts were embedded in paraffin, and the other 3 were snap-frozen in OCT Compound (Tissue Tek Inc). Frozen sections were made using a cryostat and examined by fluorescence microscopy to evaluate implanted cells labeled with DiI. Nuclear staining was performed with Hoechst 33342 (Sigma) for identification of cell viability. Expression of -smooth muscle actin (SMA) in transplanted cells was examined using an antibody to anti-SMA (1A4, Sigma) and fluorescein isothiocyanate-conjugated (FITC) goat anti-mouse immunoglobulin G secondary antibody (MBL) for identification of cell characteristics. Paraffin sections from 28 days after injection in each group (medium group, n=6; gel group, n=6; F group, n=7; M group, n=6; F+M group, n=9) were stained with hematoxylin and eosin and modified Masson’s trichrome. Cross-sections of proximal sites 6 mm from the permanent ligature were photographed using a digital camera and processed with the analysis software, NIH image (version 1.61, Macintosh). The inside area encircled by the internal elastic lamina of the arterial media was measured as a vascular lumen, and the cellular and fibrotic compartments in the lumen were defined as organized areas. Averages of the luminal sizes and the ratio of the organized areas were calculated for each group. To characterize cells forming organized tissues at an earlier stage, we histologically examined intraarterial thrombus using paraffin sections from 14 days after injection (medium group, n=5; gel group, n=5; F group, n=8; M group, n=6; F+M group, n=5). Immunohistochemistry was performed using an antibody to anti-SMA and anti-sarcomeric myosin (MF20, Developmental Studies Hybridoma Bank). Immunoreactions were detected with 3,3'diaminobenzidine. The ratio of SMA positive cells to total cells in the organized area was measured. All histological assessments in this study were performed by 2 pathologists blinded to treatment.
Coculture of Fibroblasts With Myoblasts
We used a coculture system composed of myoblasts and fibroblasts to investigate the effect of skeletal myoblasts on the differentiation of skin fibroblasts into myofibroblasts. We labeled 1x105 skin fibroblasts with DiI (1 µg/mL in Hanks’ balanced salt solution) and plated on cover glasses inserted in 35-mm dishes. Cover glasses were incubated in a culture medium plus 0.1% bovine serum albumin instead of 10% fetal bovine serum at 37°C in 5% CO2/95% air followed by additional plating of 1x105 myoblasts 2 days later to make a coculture. After incubating for 3 days, the culture medium was removed, and the cover glasses were washed in phosphate buffered saline. Cells were then fixed in 4% paraformaldehyde with 0.25% Triton-X and incubated in 10% normal goat serum for 30 minutes. Next, they were incubated with an antibody against SMA at 1:50 dilution for 1 hour at room temperature, followed by FITC secondary antibody at 1:100 dilution for 1 hour at room temperature. SMA-positive and DiI-positive fibroblasts were counted under a fluorescent microscope and the percentage of SMA -positive fibroblasts calculated. SMA-positive and DiI-negative myoblasts were also counted.
Statistical Analysis
Values are expressed as the mean±standard deviation (SD). Statistical analysis between groups was performed by ANOVA. When a statistically significant overall effect was detected, individual data were compared using Mann-Whitney test or Bonferroni correction. Differences between groups were considered significant at P<0.05.
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Results |
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Organization of Intraarterial Thrombus After Cell Transplantation in a Rat Model
Twenty-eight days after injection, the organization of thrombi of the dead-ended artery was almost completed in the F+M group (Figure 1A-e). In the F and M groups (Figure 1A-c and 1A-d, respectively), lumens were partly organized, whereas only small amounts of organization were observed in the medium and gel groups (Figure 1A-a and 1A-b, respectively). Percentages of organized areas in the luminal cavities were 4.0±7.4% in the medium group, 14.0±21.2 in the gel group, 50.3±30.4 in the F group, 40.2±27.3 in the M group, and 85.7±30.4 in the F+M group (Figure 1B). The organizing ratio in the F+M group was significantly higher than those in the other groups. In addition, the luminal size of the F+M group was smaller than those in the other groups. Luminal sizes were 0.23±0.07 mm2 in the medium, 0.26±0.14 in the gel, 0.48±0.22 in F, 0.51±0.21 in M, and 0.16±0.21 in the F+M groups (Figure 1C). The size of the lumen in the F+M group was significantly smaller than those in the F and M groups. Thus, after injection of the mixed cells, the luminal cavities were almost fully organized and apparently minimized. On the other hand, the sizes in the medium and gel groups were smaller than those in F and M group. Because the majority of luminal spaces in the medium and gel groups contained only a small amount of clot, the vascular wall shrunk at paraformaldehyde fixation, and the luminal sizes measured smaller than before. In the cell transplantation groups (F, M, and F+M groups), however, vascular lumens were filled with thrombus, cells, and extracellular matrix. Therefore, they were hardly influenced by fixation and kept their luminal size as before. All the vascular lumen in the F+M group was completely filled with cellular component; therefore, it is apparent that the arterial sacs contracted because of thrombus organization in the F+M group.
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Transplanted Cells in Organized Areas of Lumens and the Characteristics of These Cells
Transplanted cells labeled with DiI were detected in the F, M, and F+M groups at 14 days (Figure 2a, 2e, and 2i) and 28 days after the injections; those cells were also stained with Hoechst 33342 (Figure 2b, 2f, and 2j). Some DiI-labeled cells were also positive for SMA in the F, M, and F+M groups at 14 days after the injection (Figure 2c, 2g, and 2k); such cells were especially prevalent in the F+M group (Figure 2l).
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Immunohistological Studies of Cells in Organized Areas of the Lumens
To clarify the mechanism of the acceleration of intraluminal thrombus, we characterized cell components in the organized areas. It is well known that myofibroblasts expressing SMA generate contraction forces and minimize organized tissues.11,12 In immunohistological findings of SMA, positive cells were predominantly detected in the areas of the F+M group (Figure 3A-c). In the F group, positive cells were less than in the F+M, whereas a small number of cells were seen in the M group (Figure 3A-a and 3A-b). The ratio of SMA positive cells to total cells in the organized area in the F+M group reached 54.8±13.1%, showing significant differences in comparison with other groups except for the F group. In the F and M groups, the ratios were 22.4±20.0% and 10.8±16.7%, respectively. Those in the medium and gel groups were less than 6% (Figure 3B). Cells expressing SMA constituted a major cellular component in the organized areas of the F+M group. Sarcomeric myosin, on the contrary, was not expressed in the organized areas of the M and F+M groups (data not shown).
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SMA Expression by Fibroblasts Cocultured With Myoblasts In Vitro
The fractions of SMA-positive fibroblasts incubated with or without skeletal myoblasts for 3 days were 55.2±3.5 and 74.92±5.0, respectively (Figure 4). Coculture with myoblasts significantly increased the ratio of SMA-positive fibroblasts. In contrast, coculture with fibroblasts did not affect the expression of SMA by myoblasts. The ratios of SMA positive myoblasts cocultured with or without fibroblasts were 18.4±2.2 and 18.7±5.7, respectively, showing no significant difference.
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Discussion |
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Endovascular aortic repair is widely accepted as being a less invasive treatment for aortic aneurysms or dissections; however, a problem with this treatment is that in some cases, aneurysms or false lumens do not contract and heal. Recently, Allaire’s group reported a cellular therapy for aortic aneurysms; they directly seeded vascular smooth muscle cells on aneurismal walls in animal models and promoted the stabilization of aneurismal walls and prevented further expansion.16–18 We have also studied cellular therapies for aortic aneurysms; however, the purpose of our study is the development of an ideal method for a cell therapy as a combination treatment with endovascular aortic repair.
Endografts inserted into aortic aneurysms are surrounded with fresh blood clots, which in a normal healing process after an endovascular stent graft placement will be organized and change into connective tissue. This process leads to the contraction and complete healing of the aneurysm sac. However, if an endoleak exists, the blood clot is always renewed and so there can be no contraction and healing.
In this study, we demonstrated that cell transplantation can promote thrombus organization in a rat model. During a favorable healing process for an aneurysm, fibroblasts migrate into the blood clot, transform into myofibroblasts, form granulation tissue, and finally replace it with solid connective tissue. In line with our intent to accelerate this natural response, we tested 2 types of cell sources, skin fibroblasts and skeletal myoblasts, both of which can be noninvasively harvested with minor surgery from the patient’s own tissue. As we anticipated, transplanted skin fibroblasts along with collagen matrices retained in the dead-ended arterial lumen functioned as seeds for clot organization. Another cell type, skeletal myoblasts, are recognized to contain multipotential stem cells19,20 that have the ability to differentiate into fibroblasts/myofibroblasts21 or hematopoietic, chondrogenic, adipogenic, myogenic, and osteogenic cells.22,23 In the present study, transplantation of skeletal myoblasts alone caused thrombus organization, at least to some extend, but the effect was weaker than that of fibroblasts alone. However, cotransplantation of myoblasts with fibroblasts not only dramatically accelerated thrombus organization but also significantly reduced the size of the arterial lumen. Immunohistochemical characterization of the cells demonstrated that numerous SMA-positive cells were present in the organizing tissue of the cotransplanted cases, more so than those transplanted with either fibroblasts or myoblasts alone. As sarcomeric myosin was negative, the transplanted myoblasts did not differentiate toward skeletal myocytes in that environment. It seems likely that the interaction between skeletal myoblasts and fibroblasts increases the number of myofibroblasts and strengthens their contraction forces. Myofibroblasts share characteristics with both fibroblasts and smooth muscle cells, which have a well-developed contractile apparatus and express smooth muscle-specific proteins such as SMA,24,25 and appear during wound repair of various tissues, synthesize and organize collagen fibrils, and exert mechanical forces on matrix that minimizes the wound area.26
Our in vitro study demonstrated that cocultured skeletal myoblasts upregulated the expression of SMA of skin fibroblasts, whereas myoblasts themselves were not affected by fibroblasts. These results suggest that in our rat model, myoblasts enhance the phenotypic change of skin fibroblasts to myofibroblasts, causing the generation of contraction forces that could be responsible for the acceleration of thrombus organization and a reduction of the arterial lumen. Although it cannot be assured that the transplanted skeletal myoblasts have a potent activity to enhance the phenotypic change of the skin fibroblasts, various growth factors such as transforming growth factor-ß1, cytokines, newly synthesized extracellular matrix protein, or other factors could stimulate phenotypic changes from fibroblasts to myofibroblasts.27,28 Myoblasts might synthesize these factors so that they can promote the differentiation of fibroblasts.
This study has some limitations; it is represents just our preliminary study for a new cell therapy for aneurismal repair combined with endovascular therapy. A wider study using a model of an excluded aneurismal sac is also needed. Further, the exact mechanisms of the acceleration of the organization of thrombi, especially the interactions between transplanted myoblasts and fibroblasts, need to be elucidated.
In conclusion, the organization of thrombi in the arterial lumen was dramatically accelerated by transplantation of a mixture of myoblasts and fibroblasts contained in collagen gel. Cell transplantation into unorganized spaces in an excluded aneurismal sac and false lumen of an aortic dissection might be applicable as an additional procedure for endovascular treatments for aortic aneurysms and dissections.
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Acknowledgments |
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This study was supported by a Grant-in-Aid 2002 from the Mie Medical Research Foundation and a Grant-in-Aid for General Scientific Research (B) (2) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (No. 15390409).
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