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Keywords:

  • CD13;
  • Angiogenesis;
  • Muscle stem cells;
  • Hind limb ischemia

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

CD13 is a multifunctional cell surface molecule that regulates inflammatory and angiogenic mechanisms in vitro, but its contribution to these processes in vivo or potential roles in stem cell biology remains unexplored. We investigated the impact of loss of CD13 on a model of ischemic skeletal muscle injury that involves angiogenesis, inflammation, and stem cell mobilization. Consistent with its role as an inflammatory adhesion molecule, lack of CD13 altered myeloid trafficking in the injured muscle, resulting in cytokine profiles skewed toward a prohealing environment. Despite this healing-favorable context, CD13KO animals showed significantly impaired limb perfusion with increased necrosis, fibrosis, and lipid accumulation. Capillary density was correspondingly decreased, implicating CD13 in skeletal muscle angiogenesis. The number of CD45−/Sca1−/α7-integrin+/β1-integrin+ satellite cells was markedly diminished in injured CD13KO muscles and adhesion of isolated CD13KO satellite cells was impaired while their differentiation was accelerated. Bone marrow transplantation studies showed contributions from both host and donor cells to wound healing. Importantly, CD13 was coexpressed with Pax7 on isolated muscle-resident satellite cells. Finally, phosphorylated-focal adhesion kinase and ERK levels were reduced in injured CD13KO muscles, consistent with CD13 regulating satellite cell adhesion, potentially contributing to the maintenance and renewal of the satellite stem cell pool and facilitating skeletal muscle regeneration. Stem Cells 2014;32:1564–1577


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Healing in response to ischemic injury universally involves the processes of inflammation and angiogenesis [1-3]. During inflammation, monocytes use adhesion molecules as addresses to traffic to and populate the injured muscle. Once at the site of injury they differentiate to macrophages and participate in the healing process by clearing the necrotic tissue [4-6], facilitating angiogenesis [5], and promoting muscle regeneration [7]. The critical role of myeloid cells in postischemic healing is illustrated by studies in which systemic depletion of these cells showed markedly impaired wound healing and perfusion recovery [8, 9]. Similarly, new vessel formation or angiogenesis is driven by tissue hypoxia and cytokines elicited by infiltrating inflammatory cells where nascent vessels increase capillary density, perfuse the hypoxic tissue, and restore oxygen and nutrient supply routes [10]. We have previously demonstrated that the myeloid cell marker CD13 is an angiogenic regulator as well as an inflammatory adhesion molecule that forms a homotypic complex containing both monocytic and endothelial CD13 in vitro, and thus could contribute to wound healing in vivo on many levels.

While ischemic injury triggers similar responses, different organs also rely on tissue-specific mechanisms for optimal repair, many involving populations of resident regenerative/stem cells [11-13]. Pertinent to this study, healing of skeletal muscle injury is highly dependent on a well-characterized population of quiescent resident stem cells, the satellite cells. In response to trauma, these become activated, proliferate, and form new multinucleated myofibers or fuse to damaged myofibers to contribute substantially to muscle regeneration [14]. A second critical property of satellite cells is their ability to self-renew and thus maintain a pool of quiescent regenerative cells. Interestingly, in addition to its role as a myeloid marker, CD13 has been identified as a marker of human adult stem cells isolated from many tissues [15-20]. However, potential functional roles for CD13 in these cells have not been investigated. We designed this study to determine the contribution of CD13 in the wound healing response to severe peripheral ischemia in vivo, a model of occlusive peripheral artery disease which is highly dependent upon inflammatory cell trafficking, neovessel formation, and stem cell function [21].

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Additional methods and details are included in Supporting Information Methods Online.

Surgery

Surgical grade anesthesia was induced by intraperitoneal injection of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). The right femoral artery was ligated proximal to the deep femoral artery and distal to saphenous artery. The deep femoral artery, superficial branches, and bifurcation of the popliteal artery were cauterized, and the femoral artery was completely removed between the two ligatures avoiding injury of the femoral vein and nerve to preclude influence of inflammation and edema on arteriogenesis and angiogenesis. Postoperative analgesia was provided with buprenorphine (0.05 mg/kg).

Laser-Doppler Perfusion Imaging

Noninvasive measurements of superficial hind limb perfusion were obtained before and 0, 3, 7, 14, and 21 days after ligation using a Laser Doppler perfusion imager (model LDI2-IR, Moor Instruments, Wilmington, DE, http://www.moor.co.uk) that was modified for high resolution and depth of penetration (2 mm) with and 830 nm wavelength infrared 2.5 mW laser diode, 100 µm beam diameter, and 15 kHz bandwidth. At each time point, an average of four measurements per animal was made on anesthetized (1.5% isoflurane on an isothermal heating pad). To avoid the influence of light and temperature, the results were expressed as a ratio of perfusion in the right (ischemic) versus left (nonischemic) limb [21].

In Vivo Assessment of Limb Function and Ischemic Damage

Semiquantitative assessment of impaired use of the ischemic limb (ambulation score) was performed using the following criterion: 3 = most severe, unable to use the foot, dragging foot; 2 = no dragging, but no plantar flexion (ability to flex the ankle); 1 = positive plantar flexion; and 0 = able to flex toes to grasp cage in response to gentle traction on the tail [22]. Semiquantitative measurement of the ischemic damage (necrosis score) was also assessed (1–5 = one to five fingernails damaged, 6–10 = one to five fingers fully damaged, 11 = total paw damage).

Muscle Satellite Cell Isolation and Culture

Single muscle fibers were isolated from postischemic mice at day 3. Gastrocnemius and tibialis muscles were digested in 2 mg/ml collagenase II (Worthington, Lakewood, NJ), 1% penicillin/streptomycin no fetal bovine serum in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, www.lifetechnologies.com) for 2 hours at 37°C with mild agitation on orbital shaker (50–60 rpm). After aspirating collagenase solution, triturate with a wide-bored pipette in growth medium (Ham's F-10, 20% fetal bovine serum, 1% penicillin/streptomycin, and 5 ng/ml basic fibroblast growth factor) to release single fibers. For isolation of primary satellite cells, freshly isolated fibers were stripped of their basal lamina using 19-gauge needle and syringe, filtered, and plated for 2–4 hours until attached [23, 24]. Debris was aspirated from the plate and media gently replaced. All cultures were incubated at 37°C, with 5% CO2 and atmospheric O2 concentrations in growth medium on Matrigel (Franklln Lakes, NJ, www.bdbiosciences.com, 1:400 dilutions) coated tissue culture plate. All primary cell experiments used cells at passage 2–3 to avoid effects of long-term culture.

Satellite Cell Migration from Parent Myofibers

To compare satellite cell migration from parent myofibers and colony formation ability [24], postischemic WT and CD13KO myofiber samples of equal numbers (200–220) were plated in 60 mm tissue culture dish. After 1 week, cells were fixed and stained with 0.5% crystal violet for colony visualization. Image was taken with Nikon SLR D40 camera.

Clonal Analysis

Single-cell colony assay: isolated mouse satellite cells were diluted (20 cells per milliliter) and plated 100 µl per well in 96-well plates, such that approximately each well received one cell so resultant colonies originated from a single cell. Cultures were grown in growth medium for 8 days or with a switch to differentiation medium (DMEM, penicillin/streptomycin, 2% horse serum) for a further 4 days on Matrigel (www.bdbiosciences.com, 1:300 dilution in minimal volume for coverage) [23]. At the appropriate time points, cells were fixed in 4% paraformaldehyde for 15 minutes, washed twice, stained with 0.04% trypan blue for visualization, and washed three times. Images were acquired at ×20 magnification (×2 objective) using a Nikon T-BPA camera attached to the Nikon Eclipse TE2000-U. The software used was SPOT version 4.1. Cells were seeded in 48 wells for each of WT and CD13KO and quantitated as indicated.

Western Blotting

At day 3 postsurgery, gastrocnemius muscles or isolated primary muscle satellite cells were lysed in ice-cold buffer (1% Nonidet P40 lysis buffer with protease and phosphatase inhibitors). Equal amount of protein from each group was separated by SDS-PAGE and transferred on to polyvinylidene difluoride membrane and incubated with respective primary antibodies; CD13 SL-13 for mouse CD13 (ProMab Biotechnologies, Richmond, CA, www.promab.com); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); TGFβ (Minneapolis, MN, www.rndsystems.com); TNFα, IL-6, MCP-1, Pax7, and Platelet-derived growth factor (Cambridge, MA, www.abcam.com); pFAK 397 and pERK (www.cellsignal.com); tubulin and GAPDH (St. Louis, MO, www.sigma-aldrich.com) followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected with the use of a chemiluminescence reagent kit (Rockford, IL, www.thermoscientific.com). The band intensities were quantified with the NIH Image J program.

Murine Bone Marrow Transplantation Model

Recipient mice were treated with a suspension of 800 mg/l sulfamethoxazole and 160 mg/l trimethoprim (SMZ) for 1 week prior to irradiation and 2 weeks after bone marrow transplantation [25]. Tibias and femurs of 7-week WT and CD13KO donor mice were flushed with phosphate buffered saline to obtain bone marrow cells, triturated to form single-cell suspensions. Mononuclear cells were isolated by density centrifugation over Histopaque-1083 (St. Louis, MO, www.sigma-aldrich.com), yielding an average 2 × 107 cells per animal. Six-week-old WT and CD13KO recipient mice were lethally irradiated and intravenously infused with approximately 5 × 106 donor bone marrow cells in 200 ml per animal in three groups; WT recipient-WT Donor n = 7, WT recipient-CD13KO Donor n = 9, CD13KO recipient-WT Donor n = 8. Six weeks post-transplantation, mice were checked by flow cytometry for reconstitution and underwent femoral artery ligation (FAL) surgery. Hind limb perfusion was measured at 0, 3, 7, 14, and 21 days after ligation using Doppler. Transplanted mice were also assessed using the DigiGait apparatus (Mouse Specifics Inc., Boston, MA) at day 20 postligation, which provides numerous spatial and temporal indices of gait dynamics. Detailed methods for Digigait are described in Supporting Information.

Statistical Analysis

The data were represented as mean ± SEM of the indicated number of measurements. Statistical significance was calculated by two-tailed unpaired t test for two datasets. Two-way ANOVA was used to compare values between groups over time. Differences were considered significant at p < .05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

CD13 Expression Is Increased in Response to Peripheral Artery Occlusion and Contributes to Perfusion and Functional Recovery in Occluded Limbs

While we have previously shown that CD13 is an inflammatory adhesion molecule in vitro [26, 27] and a regulator of angiogenesis [28-30], its role in the healing muscle in vivo has not been examined. To address this issue, we chose a modification of the model of occlusive peripheral artery disease, permanent FAL, where the artery is clamped, blocking blood flow but retaining the collateral arteries. Conventional FAL induces two distinct vascular processes, angiogenesis (formation of new vessels), and arteriogenesis (strengthening and remodeling of existing collateral arteries) [21]. To focus this study on the processes of inflammatory infiltration and the angiogenic vascular response, we surgically removed the femoral artery and its collateral branches, thus precluding arteriogenesis [10]. We initially determined that CD13 expression in the wounded area was temporally upregulated following surgery of wild-type animals, peaking between 3 days and 7 days postinjury and decreasing thereafter in a pattern consistent with its expression on infiltrating inflammatory cells and angiogenic vasculature (Supporting Information Fig. S1A). Quantitative analysis of the gastrocnemius muscles of the murine hind limb shows that CD13 protein levels are upregulated by over threefold (Supporting Information Fig. S1B). Analysis of perfusion in ischemic limbs and in particular, the paw and digits, by Dopplar imaging showed a significant and prolonged delay in recovery of blood flow over 21 days postinjury in the CD13KO as compared to wild-type animals (Fig. 1A, 1B). In agreement with this result, we found a higher degree of paw necrosis and reduced ambulatory capacity (impaired limb function) in the CD13KO animals (Fig. 1C, 1D, criteria as outlined in Materials and Methods). Finally, recovery of muscle mass in the gastrocnemius and tibialis muscles was also impaired in CD13KO animals (Fig. 1E).

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Figure 1. CD13 plays a protective role in skeletal muscle regeneration after ischemic injury. (A): Representative color-coded images of WT and CD13KO mice on day 0, 3, 7, 14, and 21 after surgery assessed by laser Doppler imaging. Red is highest velocity, green intermediate, and blue, lowest velocity. (B): Cumulative results for WT and CD13KO mice (n = 8 each) are shown graphically as ratios of blood flow in ischemic limb (I) to that in the nonischemic limb (NI) at each time point. (C–E): Functional assessments of ischemic muscles. Cumulative results are shown graphically as (C) the ambulatory impairment score; (D) ischemic tissue damage score; and (E) injured muscle weight ratio (ischemic/nonischemic) on day 21 (n = 8 in each group and values are shown as mean ± SEM, *, p < .05; assessment criteria in Materials and Methods). (F): H&E staining of gastrocnemius muscle regeneration was confirmed by the presence of multiple, centrally located myocyte nuclei. A significant reduction in muscle regeneration (average) was observed in CD13 null mice when compared with WT at day 21. (G): Masson's trichrome stain was used to measure the area of fibrosis. CD13 null mice showed significant increase of interstitial fibrosis (blue) in the ischemic limb. (H): Lipid adjacent to regenerated myofibers was detected with oil red O staining shows significantly more fat accumulation in KO mice. All data were quantified by ImagePro Plus. Values are shown as mean ± SEM (*, p < .05) (n = 8 per group; ×20 objective; bar = 100 µm). Abbreviations: H&E, hematoxylin and eosin; NI, nonischemic; WT, wild type.

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Regenerating muscle was clearly evident upon histologic analysis of wild-type animals at 21 days postsurgery as illustrated by numerous myofibers with centrally located nuclei (gastrocnemius—Fig. 1F and tibialis—Supporting Information Fig. S2A). In contrast, CD13KO muscles displayed marked metaplasia with loss of myofibers, significant increases in collagen deposition (Fig. 1F, 1G and Supporting Information Fig. S2A, S2B), an increase in oil red O-positive lipid accumulation (Fig. 1H and Supporting Information Fig. S2C), and decreased muscle regeneration characteristic of impaired muscle recovery [21], suggesting that CD13 promotes wound healing in this model of ischemic injury.

CD13 Is Required for Angiogenesis During Healing

Since we and others have shown that CD13 is an angiogenic regulator [29-32], we began our investigation by analyzing the vascular response to peripheral ischemia in our wild-type or CD13KO mice at 21 days postinjury. Immunofluorescent analysis of CD31+ endothelial cell-lined luminal structures indicated a significant decrease in capillary density in the gastrocnemius and tibialis muscles of the CD13KO animals (Fig. 2A–2C). In addition, in the CD13KO animals these structures appeared more immature with fewer characteristic branches, confirming our earlier in vitro observations that CD13 is required for angiogenesis. Similarly, the number and diameter of more mature vessels covered with α-smooth muscle actin-positive mural cells were also diminished (Fig. 2D–2F). Flow cytometric analysis of cells from day 3-postinjury collagenase-digested muscle showed that the progressive accumulation of Sca1+/CD31+ endothelial progenitor or total CD31+ endothelial cells in the wound was not significantly different between genotypes (Fig. 2G, 2H), supporting our previous findings that CD13 regulates angiogenesis by controlling endothelial invasion but not proliferation [32]. Together, these data are consistent with an angiogenic basis for impaired healing in ischemic muscles.

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Figure 2. Skeletal muscle vessel formation is impaired in CD13 null mice following ischemic injury. (A): Capillaries were visualized by immunofluorescent staining with CD31 (red) and nuclei with DAPI (blue); Objective ×40 (bar = 50 µm). (B, C): Capillary density per fiber ratio was measured in gastrocnemius and tibialis muscles. (D): Vessels detected by double staining of CD31 (red) and αSMA (green); Objective ×10 (bar = 200 µm) and ×20 (bar = 100 µm). (E, F): αSMA-positive vessels were measured per cross section in gastrocnemius and tibialis muscles harvested at 21 days after femoral artery ligation. Capillaries and vessels were quantified by Image J software (NIH). Data represent the mean ± SEM (n = 8 mice per group) (*, p < .05). (G, H): EPC and mature EC were analyzed by flow cytometry at day 3 and day 7 compared to NI muscle (n = 5 per group). Abbreviations: αSMA, α-smooth muscle actin; EC, endothelial cells; EPC, endothelial progenitor cell; H&E, hematoxylin and eosin; NI, nonischemic; WT, wild type.

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CD13 Regulates the Profile of Infiltrating Inflammatory Cells and Cytokine Levels

Tissue injury induces a strong inflammatory response that involves temporally regulated phases of extravasation of functionally distinct myeloid cell subsets into the site of injury to orchestrate the removal of dead cells, attract additional cells and promote an environment optimal for wound healing [33]. CD13 can also function in vitro as an adhesion molecule to mediate the monocyte/endothelial interactions critical for inflammation and healing of injured tissue, an effect mediated by CD13 expressed on both the monocytes as well as endothelial cells [26, 34]. Flow cytometric analysis of cell suspensions isolated from wild-type or CD13KO injured muscles (day 3) showed equivalent numbers of infiltrating hematopoietic cells in ischemic peripheral tissues (Fig. 3A, gating strategy—Supporting Information Fig. S3). Similarly, analysis of myeloid cell subsets in the peripheral blood and muscle tissue indicated that while percentages of tissue-resident and circulating macrophages are not significantly different (Fig. 3B), the profiles of other populations were highly skewed in the CD13KO animals. Dendritic cells, which contribute to tissue damage by producing proinflammatory cytokines, chemokines, and other soluble inflammatory mediators (Fig. 3C) from both peripheral blood and muscle, were significantly decreased. Importantly, although the profiles of the inflammatory (Gr-1high, proinflammatory, Fig. 3D upper) or resident (Gr-1low, prohealing, Fig. 3D lower) monocytes were equivalent in bone marrow and peripheral blood from both genotypes, the normally high ratio of inflammatory/reparative monocytes at day 3 was decreased in muscles of CD13KO animals (WT = 4.6/1, KO = 2.4/1). Therefore, this data indicate that the lack of CD13 alters the patterns of inflammatory cell trafficking in response to injury in a manner which would be expected to promote healing.

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Figure 3. Inflammatory cells, cytokines, and growth factor profiles are altered in the muscles of CD13KO mice in response to hind limb ischemia. (A): Profile of total hematopoietic cells (CD45+) at day 3 and day 7 compared to nonischemic muscle. (B) Flow cytometric analysis of macrophages and (C) DCs in the PB and muscle of WT and CD13KO mice. (D): Infiltrating inflammatory monocytes− CD11b+ Gr-1hi, and (E) reparative monocytes− CD11b+ Gr-1lo in the live CD45+ cell population were analyzed in the BM, PB, and muscles isolated from WT and its knockout counterpart. Error bars represent mean ± SEM for WT (n = 5) and CD13KO (n = 5) mice; *, p < .05. Gating strategy is shown in Supporting Information Figure S3. (F–I): Western blot analysis of protein expression levels of the indicated angiogenic and inflammatory factors in injured muscles of WT and CD13KO mice (n = 4 per group) postischemic day 3. Detected band sizes are: MCP1 22.5 kDa, PDGF 19 kDa, IL6 25 kDa, TGFβ 25 kDa, and TNFα 22 kDa. Band intensities were quantified with NIH Image J and are expressed relative to tubulin loading control (*, p < .05). (J): VEGF levels were quantitated by ELISA assay. Abbreviations: BM, bone marrow; PB, peripheral blood; WT, wild type.

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Once in the wound, the infiltrating inflammatory cell subsets elicit specific patterns of cytokines required to orchestrate the subsequent wound healing process [35]. Logically, these altered cell profiles in the CD13KO animals would also result in changes in the relative levels of cytokines in the wound. Indeed, characterization of protein (Fig. 3F–3J) or mRNA expression (Supporting Information Fig. S4) of a panel of cytokines that are produced by these subsets showed that cytokine profiles are also distorted in a manner that corresponds to the profiles of infiltrating cells, with decreases in expression of the proinflammatory cytokines produced by inflammatory monocytes (TGFβ, MCP-1, TNFα, and IL-6) and an increase in IL-10, a product of the reparative monocytic subpopulation. Decreases in the proangiogenic cytokines vascular endothelial growth factor, platelet-derived growth factor, and Ang1 may contribute to impaired angiogenesis as well (Fig. 2). Remarkably, this phenotype (an increase in prohealing and proangiogenic and decreased proinflammatory myeloid cells and cytokines) would be predicted to result in an environment beneficial for healing. This stark contrast to the compromised repair observed in the CD13 null animals prompted further investigation into potential underlying mechanisms in addition to impaired angiogenesis.

Muscle Satellite Cell Numbers Are Decreased in CD13 Null Animals

Skeletal muscle contains a well-characterized population of self-renewing regenerative cells known as satellite stem cells [36, 37] that supply a significant proportion of the cells that form the new myofibers critical to healing damaged peripheral muscles. A second critically important function of these satellite cells is to replenish this regenerative pool through a process of self-renewal via asymmetric division, resulting in a balance between differentiated myofibers and multipotent satellite cells [38, 39]. CD13 has been reported to be a marker of adult mesenchymal stem cells in numerous tissues and although its function on these cells is currently unknown, it is possible that the impaired muscle regeneration may involve stem cell CD13. Interestingly, flow cytometric analysis of cells isolated from collagenase-disrupted injured muscles at day 3 showed that a lower percentage of the cells isolated from CD13KO muscles displayed the CD45−/Sca1−/α7-integrin+/β1-integrin+ muscle satellite cell phenotype as compared to wild-type (Fig. 4A, 4B). Accordingly, lower levels of Pax7 protein are found in CD13KO muscle lysates (Fig. 4C).

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Figure 4. The satellite pool is decreased in CD13 null skeletal muscles. (A): Pseudo-colored plots of flow cytometric analysis for the CD45−/Sca1− population (left), CD45−/Sca1−/α7integrin+/β1integrin+ satellite cells (center), and isotype controls (right) in wild-type and CD13KO muscle cell suspensions at day 3 postischemia. The satellite cell pool is significantly reduced in CD13 null mice compared to WT (right, n = 3 per group). (B): Western blot of ischemic muscles for Pax7 (58 kDa) protein levels. Muscles were lysed at day 3 after ligation. Band intensities were quantified with NIH Image J. (n = 6) (**, p < .01). Abbreviation: WT, wild type.

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Adhesion of CD13KO Satellite Cells Is Impaired

Proper adhesion to the niche is critically important for maintaining satellite cell pluripotency and impaired adhesion results in increased differentiation and compromised self-renewal, leading to depletion of the pool of renewable satellite cells [40-42]. Our previous studies demonstrating that CD13 mediates cell-cell adhesion during inflammation lead us to hypothesize that it may also play a role in adhesion of satellite cells to the extracellular matrix (ECM), which we investigated from various perspectives using a number of approaches (Fig. 5A). Indeed, in vitro migration assays demonstrated that fewer satellite cells migrated away from CD13KO parent myofibers to form colonies (Fig. 5B) in agreement with our flow cytometric data. Furthermore, isolated satellite cells showed reduced migration in Transwell assays (Fig. 5C), adhered at significantly lower levels to both Matrigel and fibronectin matrices (Fig. 5D) and proliferated more slowly (Fig. 5E) in the absence of CD13. Similarly, in vitro cultures of CD13KO cells showed significantly more early-differentiating, MyoD+ cells at day 1 postinduction of differentiation (Fig. 5F) and correspondingly more fusion events (two or more nuclei per myotube) at day 3 as visualized by DAPI-stained nuclei in embryonic myosin heavy chain positive (eMHC+) fibers (Fig. 5G). Consistent with this data, limiting dilution analysis of primary isolated satellite cells indicates that wild-type tissues contained nearly twice as many cells capable of expansion (Fig. 5H) and these colonies produced significantly more progeny in culture (Fig. 5I). However, although fewer fusion events were seen in each CD13KO well (Fig. 5J), a higher percentage of the CD13KO-derived cells had undergone fusion events when compared with wild-type clones (Fig. 5K). These data are consistent with CD13 functioning to regulate muscle satellite cell adhesion and its loss leads to impaired adhesion and increased differentiation which could potentially contribute to lower rates of self-renewal and a diminished satellite stem cell pool, in agreement with our in vivo results (Fig. 4).

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Figure 5. Lack of CD13 affects satellite cell anchorage and differentiation. (A): Outline of procedures for in vitro functional assessment. (B): Primary satellite cell colonies migrating from equal numbers of myofibers after 2 weeks of culture visualized with 0.5% crystal violet. (C): Transwell migration assay—equal numbers of primary satellite cells derived from day 3 postischemic muscles of WT and CD13 null mice were plated on Matrigel coated Transwell filters and cells migrating through the filter visualized with DAPI (Supporting Information Fig. S5). Data represent the mean ± SEM, n = 4/group of two independent experiments (*, p < .05). (D): Colorimetric quantification of adhesion of primary satellite cells on different ECM substrates. Data represent the mean ± SEM, n = 6/group of three independent experiments (*, p < .05). (E): Proliferation kinetics of primary cultured satellite cells measured by MTT assay. Each bar is presented as mean ±SEM (n = 6 from two independent experiments, *, p < .05; **, p < .01; ***, p < .001). (F): Pooled cultures of isolated stem cells were differentiated and stained for the early differentiation marker MyoD at day 1 postinduction. CD13KO cultures contain more differentiated cells (×20 objective; bar = 100 µm). (G): Cultures of isolated CD13KO stem cells display more fusion events as indicated by eMHC-positive myotubes containing two or more DAPI-stained nuclei at 3 days postinduction (×20 objective; bar = 100 µm). (H–K): Primary satellite cells (20 cells per milliliter) were plated in 96-well plates, such that each well received approximately one cell. Manual counts were made of: (H) % wells containing cells, (I) total number of cells per well, (J) number of fusion events (myotubes containing two or more nuclei, black arrow bar), and (K) calculated % differentiation/well (# of fusion events/# of total cells). n = 48 for each group and each bar is presented as mean SEM (*, p < .05; **, p < .01). Abbreviation: WT, wild type.

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Impaired Healing in the Absence of CD13 Is Due to Defects in Both the Infiltrating and Resident Cells

While we have concentrated on the effects of a lack of CD13 in satellite cell renewal, we also observed distorted inflammatory cell profiles in the CD13KO mice which could also contribute to the impaired healing. To assess the contribution of each of these populations in our model, we transplanted WT or CD13KO bone marrow into wild-type recipients or wild-type bone marrow into CD13KO recipients and performed surgery following reconstitution. Comparison of the rates of perfusion by Doppler (Fig. 6A) and functional recovery using the Digigait apparatus (described in Materials and Methods; Fig. 6B, 6C) indicated that CD13 expression on the circulating cells is necessary for optimal recovery. CD13KO bone marrow is unable to recapitulate the degree of perfusion achieved by an entirely wild-type system, suggesting additional CD13-dependent effects due to impaired trafficking. However, healing in CD13KO recipient animals reconstituted with wild-type marrow is clearly diminished when compared with wild-type recipients, illustrating a critical, cell-intrinsic basis for the CD13KO defect that cannot be overcome by the presence of wild-type inflammatory cells.

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Figure 6. Lack of CD13 in both the infiltrating and resident cells impairs healing. (A): Cumulative results of laser Doppler imaging analysis for mice transplanted with the bone marrow of the indicated genotype are shown as the ratio of blood flow in ischemic (I) limb to that in the nonischemic limb (NI) over time. (B): Gait dynamics of transplanted/injured mice. The horizontal axis represents the dependent variables acquired from the DigiGait Imaging System for the indicated groups. The graphic shows differences in swing, brake, propel, stance, and stride duration of ischemic hind limb among three groups at 20 days postischemia. (C): Gait dynamics of paw length, paw width, and paw area. DigiGait-treadmill speed = 15 cm/second. Significance was determined by t test and indicated between groups by *, p < .05; **, p < .01; ***, p < .001 (WT-R WT-D vs. KO-R WT-D), $, p < .05; $$, p < .01 (WT-R WT-D vs. WT-R KO-D), and #, p < .05 (WT-R KO-D vs. KO-R WT-D). Groups WT-R WT-D, n = 7; WT-R KO-D, n = 9; and KO-R WT-D, n = 8.

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Signaling Pathways Downstream of Adhesion Are Disrupted in the Absence of CD13

To determine the mechanism underlying CD13's role as a regulator of satellite cell adhesion, we visualized actin filaments in satellite cell-derived primary myoblasts isolated from injured wild-type and CD13KO muscles plated on Matrigel (Fig. 7A). Analysis of phalloidin-stained cells showed remarkable alterations in overall cell and cytoskeletal morphology in CD13KO cells, where cells exhibited long thin protrusions with largely cortical or patches of actin instead of assembled stress fibers, suggesting disrupted adhesion. Cell adhesion to the ECM leads to phosphorylation of focal adhesion kinase (FAK), and inhibition or blocking of FAK phosphorylation has been shown to inhibit the development of focal adhesions and stress fibers [43]. To determine the status of FAK activation in the absence of CD13, protein lysates of day 3-injured muscles were analyzed and demonstrated reduced levels of phospho-FAK (Fig. 7B). Moreover, adhesion-dependent activation of FAK stimulates the MAP kinase pathway to further promote proper adhesion [44, 45]. A dramatic reduction in the levels of phosphorylated ERK in CD13KO muscles is consistent with CD13 participating in adhesion mechanisms in the injured muscle (Fig. 7C). Coimmunostaining clearly demonstrated that CD13 is consistently expressed on Pax7+ isolated wild-type murine muscle satellite cells (Fig. 7D) and that satellite cells isolated from CD13KO muscles expressed lower levels of Pax7 protein (Fig. 7E), consistent with enhanced differentiation. CD13 is also expressed in isolated human Pax7-positive satellite cells indicating a potential human relevance for our studies (Fig. 7F, 7G). Finally, we have previously characterized two species-specific anti-CD13 mAbs as either blocking or enhancing CD13-dependent adhesion [26, 27, 34, 46]. In agreement with our genetic data, treatment of isolated wild-type murine satellite cells with the CD13-blocking mAb SL13 significantly inhibits adhesion (Fig. 7H), while treatment of human satellite cells with the adhesion-activating mAb 452 increases their adhesion to Matrigel (Fig. 7I), further supporting a role for CD13 as a regulator of muscle satellite cell adhesion. Taken together, these data are consistent with a fundamental contribution of CD13 to muscle satellite cell function, leading to compromised healing in CD13KO muscles. Therefore, in addition to mediating cell-cell adhesion, CD13 plays a novel role in cell-ECM interactions that has important implications for the maintenance of satellite cell pluripotency and self-renewal.

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Figure 7. Satellite cell defects in CD13KO mice are cell intrinsic. (A): Phalloidin-stained primary satellite cells isolated from injured muscles of CD13KO mice showed remarkable cytoskeletal disruption compared to cells from WT mice; objective ×20 (bar = 100 µm) and ×63 (bar = 30 µm). (B, C): Protein lysates of day 3 postischemic injured muscles were probed for phospho-FAK (125 kDa) and phospho-ERK (44/42 kDa) with tubulin as the loading control. The band intensities were quantified with NIH Image J. Data represent mean ± SEM (n = 7/group, *, p < .05). (D): Coimmunostaining of CD13 (green) and Pax7 (red) clearly confirmed that isolated primary satellite cells from wild-type mice expressed both CD13 and Pax7; objective ×20 (bar = 100 µm). (E): Primary satellite cell lysates were probed for CD13 and Pax7 expression. CD13KO satellite cells expressed lower levels of Pax7 protein. GAPDH is shown as a loading control. (F): Human satellite cells also express CD13 (green); objective ×63 (bar = 30 µm) and (G) Pax7 by immunoblot of human cell lysates. Mouse satellite cell lysate was used as a positive control. (H): Colorimetric quantification of adhesion to matrigel of WT mouse satellite cells treated with the CD13 blocking mAb, SL13. Data represent the mean ± SEM. n = 8 from three independent experiments (***, p < .001), or (I) human satellite cells treated with the CD13-activating mAb 452. Data represent the mean ± SEM. n = 9 from two independent experiments (**, p < .01). Abbreviation: WT, wild type.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

CD13 is a multifunctional cell surface peptidase expressed in a number of tissues where it acts in both enzyme-dependent and -independent manners to regulate disparate processes such as tumor angiogenesis [30-32, 47], endothelial filopodia formation [47], and dendritic cell antigen uptake and presentation [46] among others [48]. We have recently identified CD13 as a homotypic adhesion molecule that mediates in vitro monocyte adhesion to anti-CD13 activated, but not to classic, TNF-activated endothelial cells [26]. These studies suggest additional complexity in inflammatory trafficking processes and raise questions regarding the specific circumstances under which CD13 may participate in monocyte trafficking. These questions have prompted us to investigate whether CD13 also regulates inflammation in vivo in a model of ischemic injury following peripheral femoral artery dissection of the hind limb. We find that CD13KO mice are impaired in their ability to repair the ischemic wound, resulting in significantly reduced perfusion and ambulation and increased paw necrosis. Cellular and molecular analysis of injured muscles showed deleterious effects of the lack of CD13 on angiogenesis with reductions in numbers of both neovessels and more mature vessels. In addition, inflammatory cell trafficking was indeed altered and resulted in skewed inflammatory profiles in CD13KO wounds, producing a prohealing environment in contrast to the defective repair. However, we found that a lack of CD13 also apparently perturbs adhesion of the well-characterized satellite cell population to the niche, thus promoting differentiation, perhaps at the expense of renewal and potentially leading to depletion of the regenerative pool, thus adding cell-ECM adhesion to the list of pleiotropic functional effects of CD13 and implicating it in satellite stem cell function. A potential role for CD13 in satellite cell self-renewal is currently under investigation.

In contrast to general concepts of ischemic wound healing, skeletal muscle repair and regeneration have been shown to be particularly dependent on the population of normally quiescent cells that upon injury, become activated, differentiate, and fuse to repair damaged myofibers and importantly, self-renew by asymmetric division (reviewed in [39, 49]). In addition to angiogenic defects, we see a striking decrease in the satellite cell population in CD13KO muscles postinjury, which may contribute substantially to impaired repair. Whether satellite cell number is limiting for repair is not clear; however, a relationship between satellite number and function seems logical. If stem cells are abundant, the functional demand on each cell is modest and dispersed in response to injury. However, if this population is scarce, demand on these cells will be increased and they may soon be overwhelmed [38]. This notion is supported by studies showing that depletion or compromise of satellite cells by various methods universally impairs regeneration (reviewed in [39]). Pertinent to this study, perturbation of a number of components of the Notch signaling pathway leads to increased satellite cell differentiation, decreased self-renewal, and exhaustion of the pool leading to reduced muscle repair in response to injury [50, 51]. While potential interplay between Notch and CD13 is currently unknown, the Notch studies support the concept that a reduction in satellite stem cell numbers could be an underlying mechanism of altered repair in CD13KO mice.

Many factors have been shown to influence maintenance of the satellite cell population [14, 38, 39]. The composition of the microenvironment is essential to maintain an adequate supply of quiescent cells in the niche in case of injury, termed “niche addiction” [52]. For example, the majority of progenitor cells in resting muscles reside within a few microns of capillaries and capillary density correlates with satellite cell numbers, linking endothelial cells and myogenesis [53]. Thus, angiogenic defects observed in the CD13KO muscles may also contribute to a reduction in the satellite cell pool. Alternatively, trafficking defects presumably would not be a factor since satellite cells are tissue resident and unable to migrate through the endothelium [49]. Similarly, inflammatory monocyte/macrophage subsets have been postulated to differentially control maintenance of the niche and support wound healing through production of cytokine programs, which recruit additional immune cells to clear damaged tissue, promote neovascularization, stimulate precursor cell proliferation and renewal, and promote myocyte fusion and repair [7, 54]. We find that although the percentages of muscle-resident CD11b+ myeloid cells are comparable in wild-type and CD13KO animals, the profiles of the pro- and anti-inflammatory monocytic subsets are clearly skewed in the null animals, which would accordingly result in alterations in the levels of cytokines produced. Indeed, cytokine protein and/or mRNA profiles in CD13KO injured muscles show reduced expression of a number of proinflammatory molecules such as IL-6, TNFa, and MCP-1, consistent with fewer proinflammatory Gr1hi monocytes. Similarly, impaired trafficking could underlie the decrease in levels of VEGF, PDGF, and Ang-1 and thus contribute to diminished angiogenesis. Finally, CD13KO muscle repair is impaired despite a decline in expression levels of the potent inhibitor of skeletal muscle cell differentiation and regeneration, TGFβ [14], although our studies in aging muscles have demonstrated that TGFβ and TNF levels that fall below physiological levels can also impair regeneration, and thus may contribute to the CD13 phenotype [55]. Overall, these findings support the concept that proper healing requires a balance and favorable conditions in one aspect may not be sufficient to overcome deleterious defects in others.

Alternatively, stem cell adhesion to the niche is critical to the maintenance of pluripotency in the embryonic brain, as was recently demonstrated to involve Id1 transcriptional control of adhesion of neural stem cells, where proliferation and self-renewal of Id1 null stem cells were reduced and differentiation enhanced [42]. This study illustrated that cell intrinsic differentiation mechanisms are kept in check by specific signals derived from the niche but that once released, cells proceed along default differentiation pathways. Similarly, we find that following injury, CD13KO muscle satellite cells proliferate and adhere at significantly lower rates than wild-type cells, CD13KO injured muscles show clear decreases in the activation levels of FAK and ERK kinases critical to adhesion, and there are nearly 30% fewer CD45−/Sca1−/α7-integrin+/β1-integrin+ cells in the injured muscles lacking CD13, consistent with impaired adhesion and enhanced differentiation; perhaps leading to decreased self-renewal and depletion of the compartment, resulting in impaired regeneration. Interestingly, one of us has previously reported that pERK levels decline in aging muscles which contributes to the decreased activation of Notch and the failure to activate aged satellite cells in response to muscle attrition [56, 57], perhaps suggesting a role for CD13 in aging muscles as well. Alternatively, animals lacking the endothelial-expressed adhesion molecule E-selectin in the bone marrow vascular niche demonstrated increased hematopoietic cell quiescence and enhanced self-renewal [58], suggesting the niche microenvironment both positively and negatively regulates stem cell fate. Whether niche mechanisms and molecules are conserved between tissues or if they differ in developmental, homeostatic versus injury responses is a fascinating area of future investigation.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Finally, taken together, this study may begin to speak to the diversity and potential hierarchical ranking of interdependent processes with regard to the complex response to injury. When addressed individually or in vitro, particular angiogenic responses, inflammatory cell profiles, cytokine “signatures” or vascular addresses have been determined to either support or undermine the healing process. For example, high numbers of Ly6Clow monocytes accompanied by low levels of TNFa, IL-6 and TGFβ would be considered “prohealing” and predict improved repair. Interestingly, in the CD13KO animal, healing and muscle regeneration are unambiguously impaired despite the decidedly prohealing cytokine environment provided by distorted ratios of regulatory monocytes, illustrating that a healthy progenitor cell pool may be more fundamental to repair and thus be a more effective therapeutic target for skeletal muscle injury. Dissection of the relative contribution of these interconnected healing processes may provide valuable insights leading to more focused and successful treatment programs.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

We would like to thank Dr. Kotaro Takeda for technical support in hind limb ischemia, Dr. Kevin Claffey for use of his microscope, Dr. Anu Maharjan for helping with culturing human myoblast cells, and Charan Devarakonda for confocal microscopy. In addition, we thank the staff of the UCHC Gene Targeting and Transgenic Facility (GTTF) and the Histology Core Facility. This work was supported by Public Health Service Grants CA-106345 from the National Cancer Institute and HL-70694 from the National Heart, Lung and Blood Institute and the State of Connecticut Stem Cell Research Program Grant #09-SCA-UCHC-009.

Author Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

M.M.R.: developed study concept, designed experiments, performed experiments, interpreted data, wrote manuscript; L.H.S.: developed study concept, designed experiments, interpreted data, wrote manuscript; G.-H.F.: developed study concept; M.E.C.: developed study concept, designed experiments, interpreted data; M.G. and J.S.: performed experiments and interpreted data.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References
  12. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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