CD13 regulates anchorage and differentiation of the skeletal muscle satellite stem cell population in ischemic injury

Abstract

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, CD13(KO) 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 CD13(KO) muscles and adhesion of isolated CD13(KO) 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 CD13(KO) 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.

Keywords: Angiogenesis; CD13; Hind limb ischemia; Muscle stem cells.

Fig 1. CD13 plays a protective role in skeletal muscle regeneration after ischemic injury

A) Representative color-coded images of WT and CD13^KO 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 CD13^KO mice (n=8 each) are shown graphically as ratios of blood flow in ischemic limb (I) to that in the non-ischemic 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/non-ischemic) on day 21. (n=8 in each group and values are shown as mean±SEM *P<0.05; assessment criteria in Methods). F) Hematoxylin and eosin (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<0.05) (n=8 per group; 20× objective; Bar=100µm).

Fig 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=50µm). 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=200µm) and 20× (Bar=100µm). E,F) αSMA positive vessels were measured per cross section in gastrocnemius and tibialis muscles harvested at 21 days after FAL. Capillaries and vessels were quantified by Image J software (NIH). Data represent the mean±SEM (n=8 mice per group) (*P<0.05). G,H) Endothelial progenitor cells (EPC) and mature endothelial cells (EC) were analyzed by flow cytometry at day 3 and day 7 compared to non-ischemic (NI) muscle (n=5 per group).

Fig 3. Inflammatory cells, cytokines and growth factor profiles are altered in the muscles of CD13^KO mice in response to hindlimb ischemia

A) Profile of total hematopoietic cells (CD45+) at d3 and d7 compared to non-ischemic muscle, B) Flow cytometric analysis of macrophages and C) DCs in the peripheral blood (PB) and muscle of WT and CD13^KO 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 bone marrow (BM), Peripheral Blood (PB) and muscles isolated from WT and its knockout counterpart. Error bars represent mean±SEM for WT (n=5) and CD13^KO (n=5) mice; *P<0.05. Gating strategy is shown in Supplemental Fig S3. F–I) Western blot analysis of protein expression levels of the indicated angiogenic and inflammatory factors in injured muscles of WT and CD13^KO mice (n=4 per group) post-ischemic d3. Detected band sizes are: MCP1 22.5kDa, PDGF 19kDa, IL6 25kDa, TGFβ 25kDa and TNFα 22kDa. Band intensities were quantified with NIH Image J and are expressed relative to tubulin loading control (*P<0.05). J. VEGF levels were quantitated by ELISA assay.

Fig 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 CD13^KO muscle cell suspensions at d3 post-ischemia. 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 (58kDa) protein levels. Muscles were lysed at d3 after ligation. Band intensities were quantified with NIH Image J. (n=6) (**P<0.01).

Fig 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 d3 post-ischemic muscles of WT and CD13 null mice were plated on Matrigel coated Transwell filters and cells migrating through the filter visualized with DAPI (Supplemental Fig S5). Data represents the mean±S.E.M n=4/group of two independent experiments (*P<0.05). D) Colorimetric quantification of adhesion of primary satellite cells on different ECM substrates. Data represents the mean±S.E.M n=6/group of three independent experiments (*P<0.05). E) Proliferation kinetics of primary cultured satellite cells measured by MTT assay. Each bar is presented as mean ±S.E.M. (n=6 from two independent experiments, *P<0.05, **P<0.01, ***P<0.001). F) Pooled cultures of isolated stem cells were differentiated and stained for the early differentiation marker MyoD at d1 post-induction. CD13^KO cultures contain more differentiated cells (20× objective; Bar=100µm). G) Cultures of isolated CD13^KO stem cells display more fusion events as indicated by eMHC-positive myotubes containing two or more DAPI-stained nuclei at 3d post-induction (20× objective; Bar=100µm). H–K) Primary satellite cells (20 cells/ml) 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 S.E.M (*P<0.05, **P<0.01).

Fig 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 non-ischemic 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 hindlimb among three groups at 20d post-ischemia. C) Gait dynamics of paw length, paw width and paw area. DigiGait-treadmill speed= 15 cm/s. Significance was determined by t test and indicated between groups by *P<0.05, **P<0.01, ***P<0.001 (WT-R WT-D vs. KO-R WT-D), ^$P<0.05, ^$$P<0.01 (WT-R WT-D vs. WT-R KO-D) and ^#P<0.05 (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.

Fig 7. Satellite cell defects in CD13^KO mice are cell intrinsic

A) phalloidin-stained primary satellite cells isolated from injured muscles of CD13^KO mice showed remarkable cytoskeletal disruption compared to cells from WT mice; Objective 20× (Bar=100µm) and 63× (Bar=30µm). B and C) Protein lysates of d3 post-ischemic injured muscles were probed for phospho-FAK (125kDa) and phospho-ERK (44/42kDa) with tubulin as the loading control. The band intensities were quantified with NIH Image J. Data represents mean±SEM (n=7/group, *P<0.05). D) Co-immunostaining 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=100µm). E) Primary satellite cell lysates were probed for CD13 and Pax7 expression. CD13^KO 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=30µm) 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 represents the mean±S.E.M. n=8 from three independent experiments (***P<0.001), or I) human satellite cells treated with the CD13 activating mAb 452. Data represents the mean±S.E.M. n=9 from two independent experiments (**P<0.01).