A critical role for macrophages in neovessel ... - The FASEB Journal

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A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts Narutoshi Hibino, Tai Yi, Daniel R. Duncan, Animesh Rathore, Ethan Dean, Yuji Naito, Alan Dardik, Themis Kyriakides, Joseph Madri, Jordan S. Pober, Toshiharu Shinoka, and Christopher K. Breuer1 Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine, New Haven, Connecticut, USA The primary graft-related complication during the first clinical trial evaluating the use of tissue-engineered vascular grafts (TEVGs) was stenosis. We investigated the role of macrophages in the formation of TEVG stenosis in a murine model. We analyzed the natural history of TEVG macrophage infiltration at critical time points and evaluated the role of cell seeding on neovessel formation. To assess the function of infiltrating macrophages, we implanted TEVGs into mice that had been macrophage depleted using clodronate liposomes. To confirm this, we used a CD11bdiphtheria toxin-receptor (DTR) transgenic mouse model. Monocytes infiltrated the scaffold within the first few days and initially transformed into M1 macrophages. As the scaffold degraded, the macrophage infiltrate disappeared. Cell seeding decreased the incidence of stenosis (32% seeded, 64% unseeded, P!0.024) and the degree of macrophage infiltration at 2 wk. Unseeded TEVGs demonstrated conversion from M1 to M2 phenotype, whereas seeded grafts did not. Clodronate and DTR inhibited macrophage infiltration and decreased stenosis but blocked formation of vascular neotissue, evidenced by the absence of endothelial and smooth muscle cells and collagen. These findings suggest that macrophage infiltration is critical for neovessel formation and provides a strategy for predicting, detecting, and inhibiting stenosis in TEVGs.— Hibino, N., Yi, T., Duncan, D. R., Rathore, A., Dean, E., Naito, Y., Dardik, A., Kyriakides, T., Madri, J., Pober, J. S., Shinoka, T., Breuer, C. K. A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts. FASEB J. 25, 4253– 4263 (2011). www.fasebj.org

ABSTRACT

Key Words: monocytes ! clodronate liposomes Tissue-engineered vascular grafts (TEVGs) hold great promise for advancing the field of congenital heart surgery, where their growth capacity can be used to its fullest potential (1–3). We designed and developed a TEVG for use in congenital heart surgery by seeding a biodegradable scaffold with autologous bone 0892-6638/11/0025-4253 © FASEB

marrow-derived mononuclear cells (BM-MNCs); with this TEVG, we performed the first clinical trial evaluating the use of TEVGs in humans (1, 4 –7). These TEVGs were used as vascular interposition grafts in a high-flow low-pressure system connecting the inferior vena cava (IVC) to the pulmonary artery in pediatric patients with single ventricle cardiac anomalies undergoing modified Fontan surgery (8 –9). The late-term results of this pilot study were promising, demonstrating no graftrelated mortality and no graft failures in 25 patients in a mean follow-up of 5.8 yr (7). In addition, results of postoperative serial imaging confirmed the results of our preclinical studies demonstrating the growth capacity of the TEVG, making it the first human-made vascular conduit with growth potential (6, 10). However, results of this study also demonstrated that stenosis was the primary graft-related complication, affecting 4 of the 25 patients (7) between 1 and 5 yr after implantation. While all 4 patients were successfully managed with angioplasty or angioplasty and stenting, the development of an improved TEVG rationally designed to inhibit the formation of stenosis would be an important advance in the translation of this promising technology. The rational design of improved, second-generation TEVGs will be predicated on our understanding of the cellular and molecular mechanisms underlying the formation of TEVG stenosis. We recently reported our results using a murine model to investigate neovessel formation, the process by which a biodegradable tubular scaffold seeded with BM-MNCs transforms into a living vascular conduit with the ability to grow, repair, and remodel (11). We have demonstrated that neovessel formation is a regenerative process in which the neovessel forms from host-derived cells rather than from the autologous seeded cells as a result of migration of endothelial and smooth muscle cells from adjacent vessel segments (11, 12). Because of the early 1 Correspondence: 10 Amistad St., Amistad Bldg. Rm. 301 C, New Haven CT 06510, USA. E-mail: christopher.breuer@ yale.edu doi: 10.1096/fj.11-186585

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abundance of host-derived inflammatory cells, we hypothesized that neovessel formation is an immune mediated phenomenon similar to the proposed role for monocytes-macrophages in other human vascular biological processes, such as vein graft adaptation (13, 14). We suggested that the seeded cells participate in neovessel formation via a paracrine mechanism by initiating an immunomodulatory cytokine cascade necessary for vascular neotissue formation, including endothelial cell and smooth muscle cell proliferation and migration in addition to extracellular matrix production and tissue remodeling (11). Herein, we test this hypothesis and evaluate the role of host-derived macrophages on vascular neotissue formation and the development of TEVG stenosis.

Histology Explanted grafts were pressure fixed in 10% formalin overnight and then embedded in paraffin or glycolmethacrylate using previously published methods (11). Sections were stained with hematoxylin and eosin (H&E). Morphometry of TEVG Graft luminal diameter was measured on histological specimens using ImageJ software (Image Processing and Analysis in Java; National Institutes of Health, Bethesda, MD, USA). Stenosis was defined as greater than 50% decrease in luminal diameter compared to graft at time of implantation. Critical stenosis was defined as 75% narrowing of the luminal diameter. Graft occlusion was defined as 100% narrowing of the luminal diameter. Immunohistochemistry

MATERIALS AND METHODS Scaffold fabrication Scaffolds were constructed from a nonwoven polyglycolic acid (PGA) mesh (Concordia Fibers, Coventry, RI, USA) and a copolymer sealant solution of poly-l-lactide and -ε-caprolactone [P(CL/LA)] using previously described methods (15). Each scaffold was !3 mm in length and 0.9 mm in diameter. BM-MNC isolation and TEVG assembly Bone marrow was collected from femurs of syngeneic CB57BL/6 mice (Jackson Laboratories, Bar Harbor, ME, USA). Following purification of the mononuclear cell component using Histopaque-1086 (Sigma, St. Louis, MO, USA) centrifugation, 106 mononuclear cells were manually seeded by directly pipetting the 10-"l cell suspension in DMEM (Life Technologies, North Andover, MA, USA) into the lumen through both ends. Each seeded scaffold was then allowed to sit for 10 min to allow cell adhesion. A 21-gauge needle was then gently threaded through the lumen of the graft to prevent occlusion, and each graft was incubated overnight in 1 ml of medium prior to implantation. TEVG implantation TEVG implantations were performed using microsurgical technique. The scaffolds were inserted into the infrarenal IVC of 3- to 4 mo-old female mice (Jackson Laboratories), as described previously (11, 15). A total of 160 animals were implanted with TEVG. All animal experiments were done in accordance with the Yale University institutional guidelines for the use and care of animals, and the institutional review board approved the experimental procedures described. Ultrasound Serial ultrasonography (Vevo Visualsonics 770; Visualsonics, Toronto, ON, Canada) was utilized for surveillance of the TEVG. Prior to ultrasonography, mice were anesthetized with 1.5% inhaled isoflurane. Graft luminal diameter was determined sonographically at the indicated time points postimplantation. 4254

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Primary antibodies included rat-anti-mouse Mac-3 (BD Bioscience, Franklin Lakes, NJ, USA), F4/80 (AbD Serotec, Raleigh, NC, USA), mouse-anti-human calponin (Dako, Glostrup, Denmark), rabbit-anti-human vWF (Dako), and collagen type III (16). Antibody binding was detected using appropriate biotinylated secondary antibodies, followed by binding of streptavidin-HRP and color development with 3,3-diaminobenzidine (Vector Laboratories, Burlingame, CA, USA). Nuclei were then counterstained with hematoxylin. For immunofluorescence detection, a goat-anti-rabbit IgGAlexa Fluor 568 (Invitrogen, Carlsbad, CA, USA) or a goatanti-mouse IgG-Alexa Fluor 488 (Invitrogen) was used with subsequent 4#,6-diamidino-2-phenylindole nuclear counterstaining. Computer-assisted image analysis (quantitative immunohistochemistry) Macrophages, identified by positive F4/80 expression, were quantified for each explanted scaffold. Two separate sections of each explant were counterstained with hematoxalin and imaged at $400. The nuclei were counted in 5 regions of each section and averaged for a total of 9 seeded and 9 unseeded samples. RNA isolation and qRT-PCR to characterize macrophage phenotype Explanted tissue grafts were frozen in optimal cutting temperature (OCT) compound (Tissue-Tek; Sakura Finetek, Torrance, CA, USA), and each was sectioned into forty 10-"m sections using a Cryocut 1800 (Leica Microsystems, Wetzlar, Germany). Excess OCT compound was removed by centrifugation in water. RNA was extracted and purified using the RNeasy kit (Qiagen, Venlo, The Netherlands), as described previously (11). qRT-PCR was performed using predeveloped assay reagents from Applied Biosystems (Carlsbad, CA, USA), as described previously (11). Primers for the following genes were used: MCP-1 (Mm00441242_m1), CCR2 (Mm00438270_m1), CCL3 (Mm00441258_m1), fizz1 (Mm00445110_g1), and ym1 (Mm00657889_mH). Values were normalized to HPRT expression (Mm00441258_m1). DNA quantification Explanted tissue grafts were incubated for 10 min in 180 "l of lysis buffer (Qiagen) and proteinase K (12 mAU/reaction) at 56°C. Following tissue digestion, DNA was isolated from

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samples using DNeasy Blood and Tissue Kit (Qiagen) following manufacturer’s instructions. DNA concentrations were determined using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). Macrophage depletion with clodronate liposomes Depletion of macrophages in vivo was achieved with dichloromethylene diphosphonate-liposomes (CL2MDP-lip). Clodronate (Roche Diagnostics, Mannheim, Germany) was encapsulated in these liposomes, as described previously (17). Systemic macrophage depletion was achieved by 600 "l of CL2MDP-lip (200 "l%1 mg) via intraperitoneal administration at 3 d and 24 h before graft implantation and then every 2 d after graft implantation for 2 wk. Control groups received administration of PBS liposomes. Flow cytometric analysis was performed to evaluate the efficacy of systemic macrophage depletion. Briefly, white blood cells were separated from EDTA-anticoagulated whole blood using lysis buffer (BD Bioscience). White blood cells were labeled with rat antimouse CD115 antigen-PE conjugate (eBioscience, San Diego, CA, USA). Local depletion of macrophages on the graft was evaluated by counting F4/80 cells at $400 view. CD11b-diphtheria toxin receptor (DTR) transgenic mice (DT mice) DT mice were obtained from Jackson Laboratories. Systemic macrophage depletion was achieved by 25 ng/g body weight of diphtheria toxin injection (Santa Cruz Biotechnology, Santa Cruz, CA, USA) via intraperitoneal administration at 3 d and 24 h before graft implantation and then every 2 d after graft implantation for 2 wk. Statistical analysis Statistical differences were measured using Student’s t test, &2, or ANOVA. Values of P ' 0.05 were considered statistically significant.

RESULTS The natural history of neovessel formation in C57BL/6 murine hosts The first objective of this study was to determine whether the C57 BL/6 mouse is a relevant animal

model to study human TEVG stenosis. Specifically, we determined whether similar results could be obtained in the C57BL/6 model as compared to the human clinical trial, i.e., whether there is similar vascular neotissue formation and the development of stenosis in some TEVG. Both BM-MNC-seeded (n%10) and unseeded (n%10) scaffolds were implanted as IVC interposition grafts in C57BL/6 mice. The TEVGs were serially monitored using ultrasound over a 10-wk course, after which all TEVGs were explanted and examined. Vascular neotissue, consisting of mural vascular smooth muscle cells and an endothelial cell lining, formed in both seeded and unseeded TEVGs, within the lumen of the scaffold. Early stenosis (defined as (50% reduction in luminal diameter on ultrasound examination) occurred in 80% of unseeded TEVGs, but only 20% of seeded TEVGs, and all stenoses were detectable within 2 wk of implantation (Fig. 1). Critical stenosis (defined as (75% reduction in luminal diameter on final histology) only developed in grafts that developed early stenosis detectable on ultrasound. In addition, no grafts demonstrated evidence of reversal of stenosis at later time points on serial ultrasound interrogation. Finally no grafts failed due to aneurysmal dilation and rupture or thrombosis, in the absence of critical stenosis. These results show that the C57BL/6 mouse model develops vascular neotissue that may be complicated by stenoses similar to the human TEVG clinical results, and therefore is an appropriate model for the investigation of neovessel formation. Characterization of macrophage infiltration during neovessel formation To assess the role of macrophage infiltration during TEVG maturation, we implanted TEVGs (n%19) as IVC interposition grafts and characterized macrophage infiltration of the TEVGs over a 6-mo course. TEVGs were pressure fixed and harvested at 3 d (n%4), 1 wk (n%4), 2 wk (n%4), and 6 mo (n%7)

Figure 1. Natural history of neovessel formation. A) Graph tracking changes in diameter of TEVGs by ultrasound over 10-wk course (seeded: n%10, unseeded: n%10). Note that the critical time point for developing TEVG stenosis in the C57BL/6 mouse model is 2 wk after TEVG implantation. B) Time course demonstrating neovessel formation over 6 mo. H&E staining demonstrates neotissue formation and scaffold degradation. Note that peak cellularity was developed over 14 d, while scaffold degradation required 180 d. Also, note similarity in histological appearance of TEVG 180 d after implantation and the native IVC. 3 d: n % 4, 7 d: n % 4, 14 d: n % 4, 180 d: n % 7. MACROPHAGES IN TISSUE ENGINEERED VASCULAR GRAFTS

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after implantation. Early time points were selected based on the findings above that all significant TEVG stenosis in this model occurred within 2 wk (Fig. 1). Histological and immunohistochemical characterization of the TEVG demonstrated that vascular neotissue formation occurred in an organized, and timedependent manner (Fig. 2). Early (3 d) neotissue formation was characterized by fibrin and platelet deposition on the luminal surface of the TEVG and monocyte migration into the TEVG (Fig. 2). The next phase (1 wk) of neotissue formation was characterized by macrophage infiltration into the scaffold wall (Fig. 2). This was followed by peak macrophage infiltration in the scaffold wall at 2 wk (Fig. 2). In addition, there was near-complete formation of concentric layers of smooth muscle cells lined with endothelium at the 2-wk time point (Fig. 2). By 6 mo after implantation, the scaffolding was nearly completely degraded, with only scattered macrophage infiltration (Fig. 2). The resulting neovessel resembled the native IVC, including a widely patent lumen and a thin laminated wall with an intima, media, and adventitia (Fig. 1). Finally, we characterized the natural history of the development of TEVG stenosis at both early (2 wk) and late (6 mo) time points. We observed that at the 2-wk time point, the outer diameter of the TEVG remained constant, while the luminal diameter narrowed in stenosed TEVG, suggesting that stenosis occurred via thickening of the graft wall (Fig. 3A). At the 6-mo time point, we observed that both the outer diameter and

luminal diameter of the TEVG narrowed, suggesting the stenosis occurred via inward remodeling (Fig. 3B). Investigation of the effect of cell seeding on macrophage infiltration and development of TEVG stenosis Since our results suggest that cell seeding of the TEVGs correlates with development of stenosis in TEVGs, we evaluated the role of cell seeding on macrophage infiltration, vascular neotissue formation, and TEVG patency (Fig. 4). Both seeded (n%25) and unseeded (n%25) TEVGs were implanted as IVC interposition grafts and evaluated for the development of stenosis using both ultrasound and quantitative histological morphometry. Both ultrasound and histological morphometric analysis demonstrated that cell seeding decreased the formation of TEVG stenosis and increased luminal diameter (Fig. 4A, B). Interestingly, whereas syngeneic BM-MNC cell seeding of the TEVG correlated with diminished macrophage infiltration (Fig. 4C), macrophage infiltration directly correlated with the degree of TEVG stenosis (Fig. 4D). To determine whether cell seeding altered the macrophage phenotype, we implanted both seeded (n%6) and unseeded (n%6) TEVGs and harvested the grafts at either 1 or 2 wk after implantation to assess the TEVGs for markers of macrophage phenotype. In unseeded TEVGs, there was high expression of M1-associated markers and low expression of M2-associated mark-

Figure 2. Time course of neotissue development in TEVGs. A) F4/80-positive macrophage infiltration started at 3 d and reached a peak at 2 wk after implantation. At 24 wk after implantation, the scaffold was degraded, and the degree of macrophage infiltration was comparable to native IVC. B) Development of CD31-positive endothelial cell layer. Endothelialization was completed by 2 wk after implantation. C) Development of calponin-positive smooth muscle cell layer. Smooth muscle layer creation was completed by 2 wk after implantation. 3 d: n % 4, 1 wk: n % 4, 2 wk: n % 4, 24 wk: n % 7. 4256

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Figure 3. Characterization of TEVG stenosis over time. A) Representative images of patent and stenosed TEVGs 2 wk after implantation with histological morphometric analyses of the inner and outer diameters (patent: n%17, stenosed: n%8). Note how the outer diameter remains constant in the stenosed grafts, suggesting wall thickening. B) Representative images of patent and stenosed (occluded) TEVGs 6 mo after implantation with histological morphometric analysis of the inner and outer diameter (patent: n%5, stenosed: n%2). Note how the outer diameter decreases in size in the stenosed graft, suggesting inward remodeling.

ers at 1 wk; however, this pattern was reversed at 2 wk, suggesting a phenotypic switch (Fig. 5). Cell seeding decreased the overall expression of the M1 phenotypic markers, similar to levels of unseeded TEVG, and expression levels decreased further between 1

and 2 wk (Fig. 5). In contrast, M2 markers did not increase in the cell-seeded grafts. These results are consistent with TEVG cell seeding correlating with macrophage infiltration, phenotype, and development of TEVG stenosis.

Figure 4. Characterization of seeded or unseeded TEVGs. A) Patency rate was significantly higher in seeded grafts compared with unseeded grafts. B) Luminal diameter was significantly higher in seeded grafts compared with unseeded grafts (seeded: n%25, unseeded: n%25). C) Number of F4/80-positive macrophages in the graft was significantly higher in unseeded grafts than in seeded grafts (seeded: n%9, unseeded: n%9). D) Stenotic grafts included significantly higher numbers of F4/80-positive macrophages compared with patent grafts.

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Figure 5. Phenotype of macrophages in the scaffold over time. Unseeded grafts expressed higher relative mRNA level of M1 markers (MCP1, CCR2, CCL3) at 1 wk after implantation and M2 markers (Fizz1, ym1) at 2 wk after implantation compared with seeded grafts (n%3/group at each time point).

Determination of the role of macrophage infiltration on neovessel formation and the development of TEVG stenosis In order to determine whether the relationship between macrophage infiltration and TEVG stenosis was correlative or causative, we implanted TEVG into mice that were macrophage-depleted with low-dose clodronate liposomes (200 "l; n%10), high-dose clodronate liposomes (600 "l; n%10), or control mice treated with PBS liposomes (n%9). Clodronate liposomes are a well-described method for macrophage depletion: following endocytosis by macrophages, they cause cell apoptosis induced by clodronate (17). As a control experiment to confirm macrophage depletion, we took blood samples and measured the percentage of CD115-positive cells at 1 and 3 wk after

implantation. We confirmed a dose-dependent response for the clodronate liposomes (Fig. 6). We then harvested and examined the TEVGs at 2 wk. Treatment with clodronate liposomes decreased the degree of macrophage infiltration and decreased the incidence of TEVG stenosis, as demonstrated by increased luminal diameters in the clodronate liposome-treated group (Fig. 7). Furthermore, treatment with clodronate liposomes dramatically reduced the cellularity of the TEVGs (Fig. 7A), suggesting that inhibition of macrophage infiltration led to inhibition of vascular neotissue formation, as well as the concentration of DNA per scaffold (Fig. 8B). Furthermore, there was complete absence of staining for vWF and SMA on the luminal surface of the TEVGs, as well as diminished collagen staining (Fig. 8A), additionally suggesting inhibition of endothelial or

Figure 6. A) Dose-response study for clodronate liposome treatment. Treatment with either 200 "l (low dose) or 600 "l (high dose) clodronate liposomes significantly reduced the number of CD 115-positive monocytes in the blood at both 1 and 3 wk after starting injections. B) Clodronate liposome administration increased the luminal diameter of the TEVGs at 2 wk after implantation in a dose-dependent manner; however, only TEVGs treated with high-dose clondronate liposomes demonstrated a statistically significant increase in luminal diameter. C) Treatment with either low-dose or high-dose clodronate liposomes significantly decreased the degree of macrophage infiltration in the TEVGs (PBS: n%9, low dose: n%10, high dose: n%10). 4258

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Figure 7. Effect of macrophage depletion on morphology of graft. A) H&E staining of graft implanted in mouse treated with PBS or clodronate liposomes. Clodronate-treated group demonstrated inhibition of cellular infiltration into the scaffold. B) Ultrasound demonstrated significantly higher luminal diameter in both the clodronate-treated group and the CD11b-DTR transgenic mice treated with diphtheria toxin (DTR). C) Quantitative histological morphometry also demonstrated significantly higher luminal diameter in both macrophage-depleted groups. D) Both the clodronate treated group and the CD11b-DTR transgenic mice treated with diphtheria toxin showed a significantly decreased number of macrophages infiltrating the scaffold. *P ' 0.01.

smooth muscle cell migration onto the luminal surface of the TEVGs. Taken together, these findings are consistent with the interpretation that macrophages are critical for vascular neotissue formation, such that blockade of macrophage infiltration inhibits neovessel formation. Because clodronate liposome treatment could have other effects, we confirmed these results using an alternative approach to deplete monocytes/macrophages. We implanted TEVGs as IVC interposition grafts into DT mice treated with diphtheria toxin (n%10) or control mice (DT mice treated with carrier solution; n%10) and harvested the TEVGs at 2 wk. Again, we noted no evidence of stenosis, diminished F4/80 staining, and the absence of vWF, SMA, and collagen staining in the TEVGs treated with DT compared to control mice, in which the TEVGs matured (Fig. 8A). These findings suggest that inhibition of macrophage infiltration, rather than nonspecific effects of clodronate liposomes, leads to inhibition of vascular neotissue formation in TEVG.

DISCUSSION The results of this study highlight the critical role of host-derived macrophages in both vascular neotissue formation and the formation of TEVG stenosis. These findings have several important implications for the future development and continued translation of this promising technology. The direct correlation between the degree of macrophage infiltration and the formation of stenosis at critical time points suggests that measurement of the degree of macrophage infiltration at specific time points during neovessel formation could provide a biomarker for detecting or even predicting TEVG stenosis. The development of magnetic MACROPHAGES IN TISSUE ENGINEERED VASCULAR GRAFTS

particles for selectively labeling macrophages has already been used to detect the accumulation of macrophages in blood vessel walls in vivo using magnetic resonance imaging (MRI), thus demonstrating the feasibility of developing a noninvasive method for serially monitoring macrophage infiltration (18 –21). Whether a similar method could be developed with adequate sensitivity to detect the differences in macrophage infiltration needed to identify TEVG at risk for the formation of TEVG stenosis is an area of active investigation in our laboratory. Alternatively, measurement of the number of circulating monocytes may provide a surrogate measure for the number of infiltrating macrophages, analogous to its ability to predict the potential for postnatal arteriogenesis (collateral formation), providing another potential method for identifying TEVGs at risk for stenosis (22, 23). There is an abundance of literature supporting the critical role of circulating monocytes and infiltrating macrophages in other types of vascular repair or remodeling processes (23–25). Macrophages have been shown to play critical roles in postnatal neovascularization in addition to the formation of intimal hyperplasia (23–25). For example, inhibition of macrophage infiltration into the area of vascular injury by depletion of macrophages using clodronate liposomes reduced neointimal formation after balloon injury in rats and rabbits (25). Macrophages have also been found to play a role in the response of veins to implantation into the arterial circulation. Stark et al. (26) first proposed a critical role for macrophages in the development of vein graft intimal hyperplasia, with up-regulation of MCP-1 in the vein graft, resulting in monocyte recruitment into the graft wall. These investigators went on to show, using liposomal clodronate, that macrophages are critical to the formation of vein graft neointimal hyperplasia, as well as expression of both MCP-1 and 4259

Figure 8. Macrophage depletion inhibits vascular neotissue formation. A) Grafts implanted in mice treated with clodronate liposomes (n%10) demonstrated no vWF-positive endothelial cell layer, no SMA-positive smooth muscle cell layer, and inhibition of deposition of collagen in graft. Grafts implanted into CD11b-DTR transgenic mice treated with diphtheria toxin (DTR; n%10) replicated this result. Scale bars % 100 "m. B) DNA content in clodronate-treated group was significantly lower compared with control at 14 d (P%0.001). DNA content was also significantly higher at 14 d compared to 3 d for each group. (P'0.01). DNA content was equivalent in each group at 3 d (P%0.444). *P ' 0.01.

TGF-) protein (27). Schepers et al. (28) showed similar results in a mouse vein graft model in hypercholesterolemic ApoE3Leiden mice using CCR-2 inhibition, and Tatewaki et al. (29) showed similar results inhibiting MCP-1 in dogs. This literature suggests that vein graft adaptation to the arterial circulation is a finely regulated system that balances the need to thicken the wall to the stress of the arterial environment without excessive thickening and creation of vein graft stenosis. We believe that similar processes are occurring in our mouse model of TEVGs, i.e., that excessive macrophage infiltration promotes scarring, resulting in the formation of a stenotic or occluded neovessel, while nearcomplete inhibition of macrophage infiltration can block neotissue formation and inhibit vascular repair. In this way, achieving the proper balance of macrophage infiltration into the TEVG, at critical time points, will determine whether remodeling will be physiological, resulting in a healthy neovessel, or pathophysiological, resulting in stenosis. Recent studies have suggested that macrophage phenotype could be influenced by acellular and cell-containing scaffolds and plays an important role in remodeling (30, 31). Specifically, it was shown that seeded scaffolds induced a predominant M1 type macrophage, 4260

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leading to excessive formation of dense tissue and scarring. In contrast, our observations suggest that cells seeded into TEVGs reduce the overall influx of macrophages and the magnitude of M1 activation. Moreover, macrophages that infiltrate cell-seeded grafts do not undergo M1 to M2 transition, as was observed with macrophages in unseeded grafts. It should be noted that the concept of M1 and M2 activation in macrophages was recently challenged by the suggestion that macrophages can assume a wide spectrum of activation phenotypes (32). Consistent with this suggestion, a recent study described wound-healing macrophages with features of both M1 and M2 activation (33). Thus, we conclude that macrophages that populate cellseeded grafts assume a unique, yet to be defined, activation state that promotes remodeling. If we can find specific markers of macrophage subtypes that affect graft patency, these would be useful for predicting graft stenosis in the clinical setting. There are several limitations to this study. We investigated the role of macrophages in vascular neotissue formation and the development of TEVG stenosis using a murine vascular interposition graft model. We have previously used this model to investigate neovessel formation (34 –36). In this study, we demonstrate that

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the C57 BL/6 model faithfully recapitulates the results of our human clinical trial with respect to development of stenosis. As in our clinical trial, stenosis was the primary graft-related complication and the primary mode of graft failure. In addition, as in the clinical trial, there was no evidence of alternative modes of graft failure, such as aneurysmal dilation or graft occlusion due to thrombosis in the absence of significant stenosis. However as in all animal models species-specific differences can limit the validity and applicability of findings to the clinical experience. Of note, in our prior work, we used SCID-beige mice, which readily tolerate xenotransplantation due to lack of a functional adaptive immune system in order to enable implantation of human BM-MNC; however, in this study we used syngeneic murine BM-MNC. On the basis of results of our prior work, which suggested a critical role for inflammation in neovessel formation, we believe it was essential to study this phenomenon in an immunocompetent animal model (11). Interestingly, we did note several differences in the results of this study. Unlike our prior studies using SCID-beige mice, with implantation of TEVGs into C57 BL/6 mice, we were able to demonstrate a statistically significant effect of cell seeding on decreasing the incidence of TEVG stenosis (Fig. 3). This finding is consistent with our prior work using immunocompetent large-animal models, including both lambs and dogs (10, 37– 40). Similar to results from previous studies using SCIDbeige mice, cell seeding in the C57BL/6 mice altered the kinetics of macrophage infiltration and neotissue formation but on a more accelerated time course, as the TEVG stenosis occurred within 2 wk of implantation in the C57 BL6 mice and 4 wk in the SCID-beige mice (11, 12). This highlights the importance of adequately characterizing the natural history of stenosis prior to selecting specific time points for evaluating stenosis and macrophage infiltration. While every effort was made to create a TEVG for use in the mouse model that closely resembles the TEVG used in our clinical trial, there are some differences. The TEVG used in this study is composed of the same materials as the TEVG used in the clinical trial and has the same porosity and degradation profile; however because of differences in the fabrication method, the scaffold wall is relatively thicker. In addition, we had to increase the number of cells seeded onto the scaffold and duration of incubation of the seeded scaffold in the mouse model in order to attach similar numbers of cells compared to the TEVG used in the clinical trial. We have also noted a significant difference in the time course for the development of stenosis between mice and humans, with stenosis developing over the course of weeks in mice and years in humans. The ultimate effect of these differences on the relevance of our finding to the clinical experience will require validation in large-animal studies prior to any attempt at clinical implementation. Additional limitations of this investigation include our selection of methods to deplete macrophages. We MACROPHAGES IN TISSUE ENGINEERED VASCULAR GRAFTS

opted to use clodronate liposomes to decrease the number of macrophages infiltrating the TEVGs. Use of clodronate liposomes is a well-established method for evaluating the role of macrophages on various physiological and pathophysiological processes, including restenosis and is widely used to deplete macrophages for experimental purposes (25, 27). However, despite the fact that clodronate liposomes are relatively macrophage specific they also decrease the number of neutrophils and may have other nonspecific side effects that result in systemic toxicity (17). In addition, clodronate liposomes not only decrease the number of macrophages but also alter the function of the remaining macrophages (17). To compensate for these confounding variables, we elected to validate the findings using the conditional knockout mouse model (CD11b-DTR transgenic mouse model). However, similar to the clodronate liposome model, the surviving macrophages from the conditional knockout are also dysfunctional (41). As would be expected, the operative mortality rates were !35% higher in our macrophage-depleted mice, highlighting the toxicity of these agents in addition to the critical role of macrophages in wound healing after major surgery. We acknowledge that this is a potential confounding variable for this study. Our initial attempts to apply the concept of macrophage depletion to this technology as a strategy of inhibiting the formation of TEVG stenosis involved titrating the dose of clodronate liposomes to inhibit but not block macrophage infiltration into the TEVGs. Results of this pilot experiment showed that we were able to decrease the degree of macrophage infiltration and decrease the incidence of stenosis using low-dose clodronate liposomes; however, the decrease in the incidence of stenosis did not achieve statistical significance (Fig. 6). Whether the failure to achieve statistical significance is because of small sample size, inadequate dosing, or macrophage dysfunction due to the clodronate liposomes is an area of active investigation in our laboratory, where we are currently evaluating the use of local, controlled release of clodronate liposomes in an attempt to decrease the systemic toxicity and minimize the side effects of the clodronate. Perhaps one of the most important aspects of this study is its implication for the rational design of an improved TEVG. The identification of manipulation of macrophage infiltration as a logical target for inhibiting the development of TEVG stenosis could be very important. The rational design of the next-generation TEVG should be based on developing methods for optimizing macrophage infiltration of the TEVG. We are currently investigating the mechanism by which autologous cell seeding decreases macrophage infiltration of the TEVG. Results of this study suggest that autologous cell seeding of BM-MNC attenuates the foreign body response to the scaffold in such a way as to promote tissue regeneration and inhibit TEVG stenosis. Seeded cells, therefore, seem to be essential for guiding the degree and nature of the macrophage infiltrate and preventing the initiation of mechanisms that can lead 4261

to stenosis. From our previous work, we know that the seeded cells rapidly disappear after implantation, and, therefore, presumably exert this effect via a paracrine mechanism (11). We have identified several cytokines critical to this process and have developed a cytokineeluting scaffold that can mimic the effect of cell seeding (11). We are currently developing methods for optimizing macrophage infiltration through controlled release of immunomodulatory cytokines from the TEVG scaffold. We contend that this cytokine-eluting scaffold is the prototype for the next-generation TEVG that will offer improved patency and better off-the-shelf availability and thus broader utility than its current counterpart. Research support was provided through the following grants: U.S. National Institutes of Health (NIH) K08HL083980, NIH R01HL098228, NIH UL1RR024139, NIH P30DK072442, and NIH P01HL070295; Howard Hughes Medical Institute; and the Doris Duke Charitable Foundation Clinical Scientist Development Award. Histological processing was done by the Yale Core Center for Musculoskeletal Disorders (NIH AR46032). Gunze Ltd. has provided research support for the clinical trial evaluating the use of tissueengineered vascular grafts. None of the funding for the work done in this manuscript was provided by Gunze Ltd.

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