Fat Accumulation with Altered Inflammation and Regeneration in Skeletal

1

Fat Accumulation with Altered Inflammation and Regeneration in Skeletal

Muscle of CCR2 -/- Mice Following Ischemic Injury

Running head: Fat Accumulation During Regeneration in CCR2-/- Mice

Verónica Contreras-Shannon1, Oscar Ochoa1, Sara M. Reyes-Reyna1, Dongxu Sun1, Joel E.

Michalek2, William A. Kuziel7, Linda M. McManus3,4,6 and Paula K. Shireman1,5,6,8

Departments of Surgery1, Epidemiology and Biostatistics2, Pathology3, Periodontics4 and

Medicine5

Sam and Ann Barshop Institute for Longevity and Aging Studies6, at the University of Texas

Health Science Center, San Antonio, TX,

Protein Design Labs7, Fremont, CA, and

The South Texas Veterans Health Care System8, San Antonio, TX

Corresponding author:

Paula K. Shireman, MD

7703 Floyd Curl Drive

MC 7741

San Antonio, TX 78229-3900

Office: 210-567-5715

Fax: 210-567-1762

Email: [email protected]

of 50Articles in PresS. Am J Physiol Cell Physiol (October 4, 2006). doi:10.1152/ajpcell.00154.2006

Copyright © 2006 by the American Physiological Society.

mailto:[email protected]

2

Abstract

Chemokines recruit inflammatory cells to sites of injury, but the role of the CC chemokine

receptor 2 (CCR2) during regenerative processes following ischemia is poorly understood. We

studied injury, inflammation, perfusion, capillary formation, monocyte chemotactic protein-1

(MCP-1) levels, muscle regeneration, fat accumulation and transcription factor activation in hind

limb muscles of CCR2-/- and wild type (WT) mice following femoral artery excision (FAE). In

both groups, muscle injury and restoration of vascular perfusion were similar. Nevertheless,

edema and neutrophil accumulation were significantly elevated in CCR2 -/- compared to WT

mice at day 1 post-FAE and fewer macrophages were present at day 3. MCP-1 levels in post-

ischemic calf muscle of CCR2-/- animals were significantly elevated over baseline through 14

days post-FAE and were higher than WT mice at days 1, 7, and 14. Additionally, CCR2-/- mice

exhibited impaired muscle regeneration, decreased muscle fiber size, and increased

intermuscular adipocytes with similar capillaries/mm2 post-injury. Finally, the transcription

factors, MyoD and signal transducers of and activators of transcription-3 (STAT3), were

significantly increased above baseline but did not differ significantly between groups at any time

point post-FAE. These findings suggest that increases in MCP-1, and possibly, MyoD and

STAT3, may modulate molecular signaling in CCR2-/- mice during inflammatory and

regenerative events. Further, alterations in neutrophil and macrophage recruitment in CCR2-/-

mice may critically alter the normal progression of downstream regenerative events in injured

skeletal muscle, and may direct myogenic precursor cells in the regenerating milieu towards an

adipogenic phenotype.

Keywords: adipocyte, macrophage, MyoD, myogenic progenitor cell, neutrophil, regeneration,

STAT3

of 50

3

Introduction

The CC chemokine receptor 2 (CCR2) and its ligand, monocyte chemotactic protein-1

(MCP-1, also known as CCL2), are crucial for the recruitment of macrophages to the sites of

injury (7), however, the contribution of the MCP-1/CCR2 axis during post-ischemic renewal

processes such as muscle regeneration and restoration of perfusion remains poorly understood.

In addition, while previous work suggests that the MCP-1/CCR2 axis is important in both

angiogenesis (50) and arteriogenesis (16), the mechanisms of these interactions are not well

established. For example, previous studies have provided conflicting results regarding the role

of CCR2 in the restoration of perfusion following hind limb ischemia (17, 60). Nevertheless,

angiogenic and arteriogenic events are of importance since the restoration of adequate blood

flow is crucial to myogenesis and the regeneration of ischemic skeletal muscle (51). Injury

triggers inflammation and stimulates a series of highly coordinated cellular activities leading to

restoration of perfusion, the removal of necrotic tissue, and the regeneration of damaged muscle

(63). Macrophages may contribute both to skeletal muscle regeneration by facilitating myofiber

repair via the production of growth factors and cytokines (42), and the restoration of perfusion by

promoting collateral artery formation, also known as arteriogenesis (16), as well as angiogenesis

(50). Thus, diminished monocyte/macrophage recruitment in the absence of MCP-1 or CCR2

could have profound effects upon the reparative process.

Of growing interest is the parallel and possibly direct contribution of this chemokine

system to the progression of cellular events in skeletal muscle regeneration following ischemic

injury. For instance, MCP-1 and CCR2 are expressed in injured skeletal muscle and the

recovery of muscle strength is delayed in CCR2-/- mice following injury (64, 65). MCP-1 has

of 50

4

also been immunolocalized to endothelial cells and macrophages in the ischemic muscles of

C57Bl/6J mice (53). Further, in unrelated in vitro studies, isolated macrophages enhanced

proliferation, while delaying differentiation of myogenic progenitor cells (MPC) (38). MPC,

also known as satellite cells, are normally quiescent, but upon injury become activated and

proliferate (21). Activated satellite cells are referred to as myoblasts. Independently, we have

observed impaired perfusion and altered muscle regeneration in MCP-1 -/- mice following

femoral artery excision (FAE, unpublished observations). In combination, these findings suggest

that the recruitment and activation of macrophages via MCP-1/CCR2 forms the foundation for

normal muscle regeneration. We hypothesize that recruitment and activation of macrophages via

MCP-1/CCR2 during early inflammatory events can impact muscle regeneration at the level of

the MPC responsible for replacing damaged muscle fibers. Because MPC are activated

immediately following muscle injury (21), the relationship between CCR2, macrophages, and

regeneration is an intriguing one. However, whether CCR2-mediated signaling acts directly or

indirectly to regulate MPC during regeneration remains speculative.

Given the diversity of cells that are involved in responses following muscle injury, a

complex repertoire of signaling mechanisms is required to regulate dynamic reparative

processes. For example, in regenerating muscle, the expression of the myogenic regulatory

transcription factor, MyoD, promotes proliferation and myogenic differentiation of MPC (49).

While in vitro MPC are multipotent and are capable of transdifferentiation into other cell types,

including adipocytes (62), osteoblasts (57) and endothelial cells (55), the ability of MPC to

transdifferentiate in vivo is not well established. These alternative cellular outcomes are the

result of modified transcriptional steps critical to directing terminal differentiation. In addition to

MyoD, other molecular signaling factors are involved in MPC regulation. One such factor,

of 50

5

signal transducers of and activators of transcription-3 (STAT3) was associated with MPC

proliferation in regenerating rat skeletal muscle (25, 26) and cultured C2C12 myoblasts (19).

STAT3 is also involved in inflammatory cell activation and function (32). Thus, complex

signaling cascades are capable of modulating multiple types of cellular responses in the healing

microenvironment following ischemic injury.

The present study examined the role of CCR2 during inflammation, restoration of

perfusion, and skeletal muscle regeneration following FAE. Despite similar levels of ischemic

muscle injury and restoration of perfusion compared to WT mice, CCR2-/- mice had increased

neutrophil infiltration, decreased macrophage accumulation, impairments in muscle regeneration,

and increased intermuscular adipocyte deposition in areas of regeneration. In parallel, MCP-1

levels were increased in the ischemic muscle of CCR2-/- mice and persisted for an extended time

period. Though no significant differences in the MyoD and STAT3 activities were observed at

any individual time point post-FAE between WT and CCR2-/- animals, overall transcription

factor activity was increased in CCR2-/- mice. These findings suggest that alterations in

inflammatory cell recruitment modulate signaling events via the MCP-1 and CCR2 axis, and

may impinge on MPC fate and skeletal muscle regeneration.

of 50

6

Materials and Methods

Experimental Animals

CCR2-/- mice on a C57Bl/6J background were derived as previously described (29) and

backcrossed to C57Bl/6J mice from Jackson Labs (Bar Harbor, Maine) for 6 generations. CCR2

-/- mice on the C57Bl/6J background were bred at the Audie Murphy Veterans Hospital and

C57Bl/6J wild type (WT), control mice were purchased from Jackson Labs. Male mice 10-20

weeks old were used in this study. All procedures complied with the NIH Animal Use and Care

Guidelines and were approved by the Institutional Animal Care and Use Committee of the

University of Texas Health Science Center at San Antonio and at the South Texas Veterans

Health Care System.

Mouse FAE model and laser doppler imaging (LDI)

Mice were anesthetized and the femoral artery excised as previously described (54).

Briefly, the right femoral artery was removed from the inguinal ligament to just proximal to the

bifurcation of the popliteal and saphena arteries; the femoral vein and nerve were preserved. A

sham surgery was performed on the left leg by making and closing a skin incision. For vascular

perfusion studies, laser doppler imaging (LDI) (Moor LDI, Moor Instruments, Wilmington,

Delaware) was sequentially performed on each animal at all time points to determine perfusion

to both hind limbs as previously described (54).

Tissue weights and lysate preparation

of 50

7

Mice were sacrificed at various time points following FAE and sham surgery (days 1, 3,

7 and 14; n=5/time point) and all of the muscles of the hind limb were removed, weighed and

immediately utilized to prepare tissue lysates as described previously in detail (53). For western

blots, lysates were prepared as previously described(53) using a modified buffer (10 mM Tris,

pH 7.4 containing 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 5.5

mM EDTA, 1 mM EGTA, 20 mM sodium pyrophosphate, 1 mm 4-(2-aminoethyl)

benzenesulfonyl fluoride hydrochloride, 65 µm bestatin hydrochloride, 7 µm trans-

epoxysuccinyl-l-leucylamido-(4-guanidino) butane, 11 µm leupeptin, 0.15 µm aprotinin, and 1

mm phenylmethylsulfonylfluoride). “Calf” was designated as all muscles from the knee to the

ankle and “thigh” was designated as all muscles above the knee to the inguinal ligament. Similar

samples were collected from animals not subjected to surgery and served as negative controls

(baseline).

Measurement of Protein and MCP-1 Levels and Activity of Myeloperoxidase (MPO) and Lactate

Dehydrogenase (LDH)

Protein in tissue lysates was determined by the Pierce BCA™ protein assay (Pierce

Biotechnology, Inc., Rockford, IL) (53). MPO and LDH activity in tissue lysates was measured

and adjusted for the amount of protein in the tissue lysates (53). MCP-1 levels were assessed by

an enzyme-linked immunosorbent assay (ELISA) from Biosource (Camarillo, CA) with a slight

modification of manufacturer’s specifications as previously described (53). Data for weight,

protein/weight, MPO, LDH and MCP-1 for WT animals has been previously published (53) and

is included here for comparison to the CCR2-/- mice.

of 50

8

Determination of MyoD and STAT3 activity and expression

MyoD and STAT3 activity assays were performed in tissue lysates using TransAM

kits(10, 36) from Active Motif (Carlsbad, CA). This ELISA-like assay measures activated

transcription factor binding to an oligonucleotide consensus binding sequence (5’–CACCTG-3’

or 5’-TTCCCGGAA-3’ for MyoD and STAT3, respectively) on a microtiter plate; the active

form of the transcription factors specifically binds to the consensus sequence bound to the

microtiter well is then identified via an enzyme-linked antibody detection system with

colorimetric readout(45). For this assay, tissue lysates were thawed on ice and centrifuged at

16,100 g for 10 minutes at 4ºC. All samples were initially diluted in tissue lysate buffer to a

protein concentration of 2.0 mg/ml followed by further serial dilutions. For MyoD

determinations, 5 and 10 µg of protein from each lysate were assayed. For STAT3 assays, 5 µg

of protein from each lysate were assayed. Nuclear extracts (Active Motif) of C2C12 or HepG2

cells were diluted in lysate buffer and used in every assay as a positive control for MyoD or

STAT3 activity, respectively. Lysate buffer alone was used to determine background absorbance

at 450 nm; the OD450 values of all unknown samples were corrected for background absorbance

and values reported as OD450 after adjustment for mg of protein in the assay.

MyoD and STAT3 expression were evaluated in representative samples for each mouse

strain and post-FAE time point by western blot analyses. For western blot analyses,

polyacrylamide/sodium dodecyl sulfate (SDS) gel electrophoresis was conducted as described by

Laemmli and Favre (30) using 12.5% polyacrylamide/SDS resolving gels (Bio-Rad, Hercules,

CA). Proteins were transferred to Immobilon (Millipore, Billerica, MA) polyvinylidene fluoride

(PVDF) membrane. Immunoblot analyses were conducted using rabbit polyclonal STAT3 and

rabbit monoclonal phospho-STAT3 (Tyr705) antibodies (Cell Signaling, Danvers, MA) and

of 50

9

rabbit polyclonal MyoD (C-18) (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive

proteins were detected with AlexaFluor 680 goat anti-rabbit secondary antibody (Invitrogen,

Carlsbad, CA) using the LiCor Odyssey Infrared Imaging System (LiCor, Lincoln, Nebraska).

Measurement of tissue macrophages and neutrophils by fluorescence-activated cell sorting

(FACS)

Cells from FAE and sham surgery calf muscles were isolated as previously described by

Rando and Blau (43). Briefly, all the muscles between the knee and ankle were removed from

WT and CCR2 -/- mice 3 days post-FAE. Fat was carefully removed from the tissues. Cells

were enzymatically dissociated by incubation in a solution containing 1% collagenase type II and

2.4 units/ml dispase (both from Invitrogen Corporation, Carlsbad, CA) in phosphate-buffered

saline (PBS) supplemented with calcium chloride to a final concentration of 2.5 mM at 37 °C for

90 min. Muscle tissues were triturated with fire polished pasture pipettes every 15 min, and then

passed through 40 µm cell strainer (BD Biosciences, San Jose, CA). The filtrate was centrifuged

at 350 g to harvest the dissociated cells which were further quantitatively analyzed for

macrophages and neutrophils by FACS.

Macrophages and neutrophils were analyzed by a modified procedure for muscle-

associated cells as previously described by Lagasse and Weissman (31). Neutrophils were

defined as CD11b+/Gr-1+ and macrophages were defined as CD11b+/Gr-1-. Single cell

suspensions were treated with mAb 2.4G2 (1 µg/106 cells, BD Biosciences) for 20 min to block

Fcγ II/III receptors and incubated with conjugated antibodies at optimal concentration at 4°C for

25 min. The conjugated monoclonal antibodies were as follows (all from BD Biosciences):

APC-anti-mouse Gr-1 (Clone RB6-8C5) at 0.2 µg/106 cells, PE-anti-mouse CD11b at 0.1 µg/106

of 50

10

cells (Clone, M1/70), as well as the corresponding isotype control antibodies. Cell analysis was

performed on a FACSAria (BD Biosciences) with data analysis accomplished with FACSDiva

software. (BD Biosciences).

Histology, Immunohistochemistry, Oil Red O, Capillary Counts and Histomorphometry

Mice were sacrificed at 1, 3, 7, 14, 21 or 28 days (4-8 mice/time point) following FAE

and sham surgery (right and left leg, respectively), and hind limb tissues collected for histology

and immunohistochemistry with and without perfusion fixation as previously described (53).

Routine, indirect immunohistochemical procedures were used for the localization of

monocytes/macrophages (F4/80 and mac3) or neutrophils (Ly-6) in deparaffinized sections.

Non-specific binding of antibodies was blocked by treatment of sections (30 min) with 1%

human serum albumin (HSA) in PBS. Primary rat monoclonal antibodies were directed against

mouse F4/80 (Serotec, Inc, Raleigh, NC), mouse mac3 (BD Biosciences) and mouse Ly-6 (BD

Biosciences). Antibodies were diluted 1:500, 1:200 and 1:10, respectively, in 1% HSA in PBS.

A biotinylated secondary antibody (mouse absorbed rabbit anti-rat IgG used at a dilution of

1:200) and streptavidin-HRP were obtained from Vector Laboratories (Burlingame, CA). After

enzymatic development in diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide,

sections were counterstained with hematoxylin. MyoD and F4/80 immunolocalization on frozen

sections was performed as previously described (53) using a primary antibody dilution of 1:80

for MyoD. Oil Red O staining was performed on 4 µm cross-sections of frozen muscle using a

kit from Poly Scientific (Bay Shore, NY) per manufacturer protocol. The biotinylated lectin,

Griffonia (Bandeiraea) Simplicifolia lectin I (Vector Laboratories) at 1:50 dilution was used to

identify endothelial cells on deparaffinized cross-sections. Endogenous peroxidase activity was

of 50

11

blocked by incubation in 3% H2O2 and nonspecific binding was blocked by treatment of sections

with 1.67% horse serum in PBS. After incubation with lectin, sections were sequentially

incubated with HRP-Streptavidin followed by DAB, and counterstained with hematoxylin and

eosin (H&E).

Histology and immunohistochemistry images were captured with a Nikon E600 Eclipse

microscope (Nikon, Melville, NY) equipped with a high-resolution digital camera (Nikon DXM

1200) interfaced to a personal computer equipped with Act-1 (Nikon) software for image capture

and archiving. Capillary counts and morphometric analyses were performed on images captured

using a Nikon Eclipse TE2000-U microscope equipped with a high resolution digital camera

(Nikon DXM 1200F) interfaced with a personal computer equipped with Metamorph (Nikon)

software.

For morphometric analyses, anterior compartment specimen were removed en bloc at 21

days post-FAE (n=5 mice/strain), 2-3 µm cross-sections were obtained through the mid portion

of the muscle and stained with H&E or Masson’s trichrome. Additional sections were processed

for capillary density. Within each section, eight non-overlapping areas of the tibialis anterior

(TA) muscle were randomly selected and digitally captured (20x magnification); areas

containing large blood vessels or fibrous tissue bands between muscle bundles were excluded.

The percentage of the total cross sectional area of the TA covered by the 8 digital images was

35-50%. Preliminary studies demonstrated no significant difference in fiber cross-sectional area

between H&E and trichrome stained sections (data not shown). For fiber size and percent of

intermuscular fat, two different microscopic sections separated by at least 100 µm were used for

morphometric analyses for both the FAE and sham surgery muscle; data were similar at both

levels. Results from replicate sections were averaged for a given animal.

of 50

12

In each of the digitally captured images, intermuscular adipocytes (i.e., between and

among individual muscle fibers in a given muscle bundle) were manually outlined and used to

calculate the total area of fat that was then divided by the total area of the image (0.278 µm2) to

calculate the percent fat. The average percent fat area of 8 images for each TA section was then

determined. The average percent fat among replicate TA sections for each animal was calculated

for subsequent use in comparisons of results for each group of animals.

The average cross-sectional area (µm2) of individual muscle fibers for a given animal was

determined after manually outlining individual muscle fibers in each of the digitally captured

images for a given TA; fibers that were only partially included within the images were excluded.

In the TA of FAE limbs, only regenerated fibers (identified by the presence of a centrally-located

nucleus (6)) were measured whereas only fibers with peripherally-located nuclei (i.e. mature,

non-regenerated fibers) were measured in the TA of the sham surgery limbs for both WT and

CCR2 -/-mice.

Capillary counts were performed on both the TA of FAE and sham surgery specimen

using the 8 fields described above. Images were digitally captured using phase contrast

microscopy at 20X to enhance the identification of cell borders and lectin staining. The number

of capillaries and muscle fibers in each image were counted using NIS-Elements Software

(Nikon). Only capillaries associated with muscle fibers were counted and expressed as

capillaries/muscle fiber. In addition, areas of fat were manually outlined and subtracted from the

total area and capillary density was also expressed as capillaries/mm2.

Data Analysis

of 50

13

SAS software (SAS, Cary, North Carolina) was used for all statistical analyses. Results

from corresponding time points of each group were averaged and used to calculate descriptive

statistics (mean + SEM).

Sequentially derived LDI ratios were analyzed by two different methods. First, to

determine if LDI ratios within a given group had returned to pre-operative (baseline) levels, a

Bonferroni corrected multiple comparisons procedure was used. Data collected pre-operatively

was subtracted from each of the other time points to produce a dataset of comparable LDI ratio

differences. Secondly, to determine if there were differences between CCR2-/- and WT mice at

each time point, an analysis of variance incorporating repeated measures across time was used

with a Bonferroni correction for multiple comparisons. Within animals, time correlations and

heterogeneity of the variances across time, were modeled using the ANTE(1) covariance

structure. Animal was considered a random effect.

Protein/weight, weight, enzyme activity, transcription factors and MCP-1 data were

analyzed by a Dunnett-corrected multiple comparisons procedure utilizing a 2-way analysis of

variance of least square means to determine whether significant differences existed at different

time points (1, 3, 7 and 14 days post-FAE) as compared to baseline values. For transcription

factors, MCP-1 and MPO, results were log transformed to adjust for unequal variances. For

lysates samples with MCP-1 below the level of detection in the ELISA (<78 pg/ml), a value of

78/ 2 pg/ml was assigned to these samples (20) and this value was corrected for the protein in

each specimen.

FACS data for neutrophil and macrophage quantitation was analyzed by paired (within

strain) and unpaired (between strains) t tests.

of 50

14

Histomorphometry data for percent fat and fiber cross-sectional area were analyzed as

follows. First, a repeated measures linear model was used to determine that there were no

significant interactions between the 2 different sections of the TA muscle for a given animal.

Replicate data for all images and levels were then averaged to generate a single number for each

limb for each mouse, i.e., fiber cross-sectional area was determined for both FAE and the

contralateral limb whereas percent fat was only calculated for the FAE limb. Paired t-tests were

used to assess the significance of mean differences in fiber cross-sectional area between FAE vs

sham limbs within each strain. One-way analyses of variance were used to assess the

significance of mean differences between the limbs of CCR2-/- and WT mice for FAE (fiber

cross-sectional area and percent fat) or sham (fiber cross-sectional area). Analysis of

capillaries/fiber and capillaries/mm2 were performed in a similar manner, except only 1 level of

the TA was used to generate a single number for each limb. All statistical testing was two-sided

with a significance level of 5%.

of 50

15

Results

Injury, regeneration and perfusion after FAE

LDH activity in calf muscles was used as a marker of muscle injury (indicated by

decreased activity) and regeneration (indicated by restored activity) (11, 53). LDH activity in

tissue lysates were measured, normalized to protein concentration, and compared to baseline

levels from tissue obtained from mice with no surgery. LDH activities in calf muscles obtained

following sham surgery were not significantly different from baseline values in either strain of

mice (data not shown). Muscle injury in WT and CCR2-/- mice was similar following FAE (Fig.

1). In WT mice, maximal muscle injury occurred at day 3 where LDH activity was

approximately 50% of that in the normal, uninjured muscle (p<0.001). LDH activity in post-

ischemic calf muscle of WT mice remained significantly decreased at day 7 (p=0.002) but WT

LDH activity were similar to baseline by day 14 post-FAE. In contrast, LDH activity in CCR2-/-

mice was significantly decreased at day 3 (p=0.003) and remained significantly decreased as

compared to baseline at both days 7 and 14 (p<0.001). Furthermore, LDH activity was

significantly decreased in CCR2-/- as compared to WT mice at day 14 (p=0.004). This

enzymatic measure of tissue injury and repair paralleled the histological evaluation and extent of

muscle regeneration described below.

Following FAE, the restoration of perfusion was sequentially measured in each animal by

LDI. Prior to surgery, the perfusion ratio was approximately 1 in both mouse strains (Fig. 2),

i.e., perfusion was comparable in the right and left legs. Immediately following FAE, perfusion

dropped to ~15% of the perfusion levels observed in the contralateral, sham surgery leg.

Thereafter, restoration of perfusion was similar in both mouse strains, increasing gradually

of 50

16

throughout days 3-28 following FAE. In both WT and CCR2-/- mice, perfusion was not fully

restored and remained at ~75% of pre-operative perfusion levels at day 28 (p<0.001 compared to

baseline). There were no significant differences in the perfusion ratios between WT and CCR2-

/- mice at any time point.

Histological analyses of inflammation and skeletal muscle regeneration following FAE

The pattern of neutrophil and macrophage infiltration as well as the regeneration of

muscle was followed histologically in specimen obtained at various time points following FAE

(Figs. 3 and 4). In the thighs of both WT and CCR2-/- mice, an inflammatory infiltrate was

primarily associated with the skin incision; and, occasionally, focal areas of skeletal muscle

necrosis were identified adjacent to the skin incision as well as distally, near the knee.

In WT mice at 1 day post-FAE, ischemic injury of calf skeletal muscle was widespread

and an inflammatory infiltrate consisting mainly of neutrophils was present; injured fibers were

readily identified by the intense eosin staining of swollen cells and the absence of myocyte

nuclei in association with abundant edema. By day 3 post-FAE, a prominent mononuclear cell

infiltrate was associated with muscle injury in WT mice (Fig. 3A); F4/80+ cells (macrophages)

predominated (Fig. 3C). At day 7 in WT mice, the inflammatory infiltrate was diminished and

largely replaced by regenerated muscle which was identified as myofibers containing multiple,

centrally-located nuclei (Fig. 3E).

The calf muscle of CCR2-/- mice had a similar histological appearance to WT mice at

day 1 post-FAE, except neutrophils appeared to be more numerous. However, the injured

muscle of CCR2-/- mice differed from that of WT mice at all subsequent times. CCR2-/- mice

had a minimal mononuclear cell infiltrate at day 3 (Fig. 3B) and very few F4/80+ cells were

of 50

17

identified in areas of necrotic muscle fibers (Fig. 3D), indicating that CCR2-/- mice have fewer

intramuscular macrophages compared to WT mice. At day 7 post-FAE in CCR2-/- mice, an

interstitial inflammatory infiltrate was prevalent in association with extensive residual necrotic

myofibers (Fig. 3F). Necrotic myofibers persisted and were widespread at day 14 in CCR2-/-

animals (Figs. 4C, D) whereas muscle regeneration was essentially complete in WT mice at this

time (Fig. 4A). Necrosis was still present in CCR2 -/- mice at 21 days post-FAE (Fig 4E). Thus,

CCR2 -/- mice had decreased and delayed macrophage accumulation into the injured muscle in

association with residual tissue necrosis. In addition, by day 14 post-FAE in CCR2-/- mice,

there was a considerable accumulation of adipocytes between regenerated fibers (Figs. 4C, D)

while WT mice exhibited only small, scattered foci of intermuscular adipocytes (Fig. 4B). The

lipid content within the cytoplasmic vacuoles of these cells was confirmed by Oil Red O staining

(Fig. 5A, B). Interestingly, at 7 days post-FAE, large, vacuolated cells were prevalent among

necrotic myofibers of CCR2-/- mice (Fig. 3G); these cells may represent the early precursor cells

of adipocytes that prevailed at 14 and 21 days post-FAE (24). Note that the histological

progression of muscle regeneration in both groups closely paralleled LDH measurements of

tissue injury and regeneration (Fig. 1).

Intermuscular fat accumulation, cross-sectional muscle fiber size and capillary density on post-

operative day 21

To quantify increased fat accumulation and impaired muscle regeneration in CCR2-/-

mice, the area of adipocytes present in regenerated muscle as well as the average cross-sectional

fiber area of regenerated TA muscle were morphometrically assessed. The 21 day time point

was chosen as only small foci of necrotic muscle were present in the TA post-FAE in both

of 50

18

strains of mice. The largest muscle in the anterior compartment, the TA, was chosen for analysis

as the entire muscle consistently underwent necrosis and regeneration.

Intermuscular fat was not identified in the sham surgery TA muscles of either mouse

strain on post-operative day 21. However, in the TA of FAE limbs, intermuscular adipocytes

were present in areas of muscle regeneration (Figs. 3H, 4B and 4E). In comparison to WT mice,

CCR2-/- mice had significantly increased intermuscular fat within the TA (18.2+3.6% vs

3.3+0.2% for CCR2-/- and WT mice, respectively, p=0.003) (Fig. 6A).

There was no significant difference in the muscle mature fiber cross-sectional area of

CCR2-/- and WT mice in the contralateral, sham surgery TA (Fig. 6B). However, at day 21

post-FAE, the cross-sectional area of regenerated muscle fibers in the TA was significantly

(p=0.001) decreased in CCR2-/- mice (672+29 µm2) as compared to that of WT mice (1,080+58

µm2) (Fig. 6B). Moreover, in WT mice, the cross-sectional area of regenerated muscle fibers was

not significantly different than that of uninjured muscle fibers in the contralateral leg at 21 days

post-FAE (Fig. 6B). In contrast, the regenerated muscle fibers in CCR2-/- mice were

significantly (p<0.001) smaller than myofibers of the corresponding contralateral limb.

Finally, capillary density was measured in the FAE and sham 21 day TA muscles of

CCR2 -/- and WT mice (Fig 5C, D) and expressed as either capillaries/muscle fiber or

capillaries/mm2. In the FAE TA muscles, only areas of regeneration, identified by centrally-

located nuclei in the muscle fibers were used. For these measurements, only capillaries

associated with muscle fibers were counted and areas of adipocyte accumulation or necrosis

were excluded so that the significant increased adipocyte accumulation in the CCR2 -/-

regenerating muscle would not influence capillary density measurements. There were no

significant differences in capillaries/fiber (Fig. 6C) or capillaries/mm2 (Fig. 6D) in the

of 50

19

contralateral, sham TA of CCR2 -/- and WT mice. However, in the FAE TA, CCR2 -/- mice

exhibited significantly (p<0.001) decreased capillaries/fiber (0.97+0.03) compared to WT mice

(1.66+0.04, Fig. 6C). Furthermore, as compared to the sham TA, CCR2 -/- mice had a

significant decrease in capillaries/fiber in the FAE TA (p=0.001) while the FAE TA of WT mice

had similar capillaries/fiber.

Interestingly, the significant differences in capillary density between CCR2 -/- and WT

mice were not exhibited when capillary density was expressed in relation to the area of the

regenerated muscle. Thus, between strains, similar numbers of capillaries/mm2 were present in

regenerated and normal muscle (Fig. 6D). Therefore, the significant decrease in capillaries/fiber

in the FAE TA of CCR2 -/- animals reflected the decreased size of the regenerated muscle fibers

(Fig. 6B) rather than differences in the number of capillaries at the 21 day time point.

FAE-induced changes in tissue weight, protein, MPO activity and macrophage recruitment

Tissue weight was measured at various time points following FAE as an indication of

edema. To adjust for small differences in animal size, thigh and calf FAE specimen weights

were normalized to the contralateral, sham surgery leg. For thigh muscles, there were no

significant changes in the relationship between right and left weight ratios as compared to

baseline at any time point following FAE for either strain of mice (data not shown). In contrast,

the relative weight of the ischemic calf of WT mice was significantly increased at day 3

(p<0.001) and returned to baseline at day 7 (Fig. 7A). In comparison, the calf weight ratio of

CCR2-/- was significantly elevated at day 1 and remained elevated through day 7 before

returning to baseline at day 14. CCR2-/- calf weight ratios were significantly elevated over WT

at days 1 and 7 (p<0.01). The protein/weight ratios in calf tissue lysates had a similar but inverse

of 50

20

pattern to tissue weight except for day 14 post-CT in the CCR2 -/- mice, where the

protein/weight ratio remained decreased from baseline secondary to decreased protein levels in

the lysates (Fig. 7B). Thus, the weight and protein/weight ratios during post-CT days 1-7 were

consistent with edema in the CCR2 -/- mice while the decreased protein in the 14 day post-CT

lysates in the CCR2 -/- mice suggested impaired regeneration; both of these observations were

consistent with the histological features of the post-ischemic muscle tissues.

MPO activity was measured to quantify neutrophils associated with inflammation in

calves following ischemia. MPO activity in calf muscle following sham surgery did not differ

from baseline for either strain (data not shown). However, following ischemic injury, WT mice

had significantly elevated MPO activity over baseline at 3 and 7 days (p<0.002) post FAE (Fig.

7C). In comparison, MPO activity in the calf muscle of CCR2-/- mice was elevated over

baseline in an accelerated pattern, i.e., at day 1 post-FAE. At this time, MPO activity in the calf

muscle of CCR2-/- mice was significantly elevated compared to WT mice (p<0.001), indicating

an increased presence of neutrophils.

Further quantitation of tissue neutrophils and macrophages (cells x106/gram of tissue)

was accomplished by FACS analysis at day 3 post-FAE (Fig. 8). Neutrophils (p<0.04) and

macrophages (p<0.03) in both WT and CCR2 -/- mice were elevated in the FAE vs the sham calf

tissues. In the sham surgery calves, WT and CCR2 -/- mice had similar numbers of neutrophils

(0.15+0.06 vs 0.21+0.19 cells x106/gram of tissue, WT and CCR2 -/-, respectively). In contrast,

the CCR2 -/- sham calves had significantly less macrophage recruitment than WT mice

(0.07+0.02 vs 0.01+0.00 cells x106/gram of tissue for WT and CCR2 -/- mice, respectively,

p=0.03). In the ischemic, FAE calves, neutrophil recruitment was similar at day 3 post-FAE

(Fig. 8C) and this was consistent with the MPO data at day 3 post-FAE (Fig. 7C), described

of 50

21

above. On the other hand, there was a severe impairment in macrophage recruitment in CCR2 -/-

compared to WT mice (Fig. 8C).

FAE- and inflammation-induced changes in MCP-1 levels and transcription factor activities

Levels of the CCR2 ligand, MCP-1, were measured in the post-FAE calf (where muscle

regenerative processes occur) and thigh muscle (where collateral artery development takes

place). At baseline, MCP-1 was not detectable in the calf muscle of WT mice and only modest

levels of MCP-1 were present in CCR2-/- mice (Fig. 9A). MCP-1 levels in calf muscles

increased in both strains following FAE, were maximal at day 3 and decreased at days 7 and 14.

Despite this similar pattern of changes, MCP-1 was significantly elevated in the calf muscle of

CCR2-/- mice at days 1, 7 and 14 compared to WT (p<0.002) which had returned to baseline

levels at day 7.

In thigh muscles, MCP-1 levels were considerably lower than in calf muscles post-FAE

(Fig. 9B). MCP-1 in thigh muscle at baseline was minimal in WT mice and not detectable in

CCR2-/- mice. Following FAE, MCP-1 was not increased over baseline at any time point in

thigh muscles of WT mice. In contrast, MCP-1 levels in the thigh muscle of CCR2-/- mice were

increased above baseline levels at days 1, 3, and 7 (p<0.01) and significantly elevated at days 3

and 7 compared to WT mice (p<0.004) (Fig. 9B).

To examine the molecular regulation of muscle regeneration and other cellular responses,

MyoD and STAT3 transcription factor activities were measured in calf muscle tissue lysates

following FAE. For both mouse strains, MyoD activity progressively increased after ischemic

injury and decreased as the muscle regenerated (Fig. 10A). In WT mice, MyoD activity was

significantly higher than baseline at days 3, 7 and 14 (p<0.02) with maximal expression at days 3

and 7. In CCR2-/- mice, MyoD activity was significantly elevated over baseline at all time

of 50

22

points post-FAE (p<0.001). STAT3 activity in the post-ischemic calf muscle of WT mice was

only increased above baseline at day 3 (p=0.05, Fig. 10B) whereas in CCR2-/- mice, STAT3

activity was significantly elevated over baseline at days 7 and 14 post-FAE (p<0.05). Although

there was a trend towards increased MyoD and STAT3 activities in CCR2 -/- mice, there were

no significant differences in the activities of these transcription factors between WT and CCR2-/-

mice at any individual time point post-FAE. Nevertheless, when results from all time points

post-FAE were taken altogether, there were significant differences between WT and CCR2 –/-

mice for both myoD and STAT3 (p=0.02 and p=0.05, respectively). Western blot analyses for

MyoD, STAT3 and phosphorylated STAT3 showed a pattern of expression consistent with those

observed by transcription factor activity assay (Fig. 10C).

MyoD immunolocalization in CCR2 -/- mice exhibited a nuclear staining pattern in

mononuclear cells associated with injured muscle fibers (Fig 5E, G); this pattern of MyoD

localization in CCR2 -/- mice post-FAE was similar to that previously published for WT animals

(53). Interestingly and in contrast to WT animals, only a few F4/80 positive mononuclear cells

were present within injured skeletal muscle in association with MyoD positive cells in CCR2 -/-

mice at day 3 post-FAE (Fig 5F). While F4/80 positive cells were increased in CCR2 -/- mice at

day 7 post-FAE (Fig. 5H), the cellular distribution of MyoD and F4/80 remained distinctly

different (Fig 5G, H).

of 50

23

Discussion

In the present study, quantification of skeletal muscle injury, restoration of perfusion,

capillary density, inflammation, muscle regeneration and signaling events were used to establish

the involvement of CCR2 during post-ischemic regenerative processes. We hypothesized that

alterations in the recruitment and activation of macrophages during early inflammatory events,

due to the absence of CCR2, impacted muscle regeneration, possibly at the level of the MPC

responsible for replacing damaged muscle fibers. Most notably, these studies demonstrate that

CCR2-/- mice had smaller regenerated fibers and increased intermuscular adipocytes following

FAE despite similar levels of injury between WT and knockout mice. However, the transcription

factors MyoD and STAT3 only showed an overall increase in transcription factor activity in

CCR2-/- mice but did not differ between WT and CCR2-/- mice at any individual time point. In

addition, early inflammatory events were altered in CCR2-/- mice; these animals exhibited

decreased macrophages and an increased neutrophil population compared to WT mice.

Interestingly, MCP-1 chemokine levels were elevated in CCR2 -/- mice compared to WT mice.

In combination, these findings suggest that imbalanced recruitment of neutrophils and

macrophages into ischemic tissues of CCR2-/- mice profoundly affects muscle regeneration and

may modulate MCP-1 mediated signaling events within the healing microenvironment.

Recently, Reichel, et. al. (44), reported that mice deficient in certain CC-chemokine

receptors, including CCR2, exhibited decreased neutrophil infiltration following ischemia. In

contrast, we observed increased neutrophil infiltration into the ischemic calf muscle of CCR2-/-

mice following FAE. The differences between these studies may arise from the assessment,

theirs being made in an ischemia-reperfusion model immediately following reperfusion. In

of 50

24

addition to using the FAE model, the current study used myeloperoxidase measurements to

quantify neutrophils present in ischemic muscle. Other investigators have described increased

neutrophil accumulation in CCR2-/- mice using various injury models (23, 29). Moreover, the

antagonism of MCP-1 has resulted in delayed neutrophil clearance due to decreased numbers of

macrophages and the reduced ability of macrophages to phagocytize apoptotic neutrophils (1,

35). Thus, inadequate macrophage recruitment and activation in the present study may underlie

the increased accumulation of neutrophils.

Previously, Tang et. al. (60) reported reduced macrophage recruitment following hind-

limb ischemia in CCR2-/- mice. In that study, macrophages in ischemic calf muscle were

decreased, but in the thigh, an area corresponding to the site of active arteriogenesis, equivalent

amounts of macrophages and increased levels of MCP-1 mRNA were observed. Tang et. al. also

described similar restoration of perfusion between CCR2-/- and WT mice following ischemia

(60). In contrast, another study described decreased restoration of perfusion in CCR2-/- mice

(17). The earlier findings by Tang et. al. taken in conjunction with the findings described here,

pose a confounding contradiction: that despite an apparent normal restoration of perfusion,

muscle was unable to regenerate properly in CCR2-/- mice. This indicates that events other than

perfusion (i.e., inflammation, satellite cell activation, etc) account for the delayed and impaired

muscle regeneration observed in CCR2-/- mice. With regard to MCP-1-related arteriogenesis,

the present study demonstrated that MCP-1 protein levels in thigh were negligible. Rather,

elevated levels of MCP-1 protein observed in the calf suggest a more crucial function for MCP-1

and CCR2 at the site of ischemic tissue injury and regeneration.

Skeletal muscle regeneration is primarily dependent upon perfusion, inflammation, and

satellite cells, the multipotent progenitor cells that reside in skeletal muscle (14). Although there

of 50

25

appeared to be no major differences in restoration of perfusion in WT and CCR2-/- mice herein,

it is still possible that the impairment in skeletal muscle regeneration was secondary to a more

severe or prolonged ischemic insult. Alternatively, the impairment of skeletal muscle

regeneration could be a primary defect, i.e., satellite cells may require CCR2 for efficient

proliferation and differentiation. It is also possible that modifications in the nature or magnitude

of the inflammatory infiltrate, especially macrophages, including the presence or absence of

mediators derived following cell activation, lead to impairments in skeletal muscle regeneration.

Previous studies of ischemic muscle damage have demonstrated that inflammation is

important for the removal of necrotic tissue and influences many aspects of regenerative

processes (33, 63). Consistent with these observations, decreased macrophage recruitment in

CCR2-/- mice in the present study was associated with impaired muscle regeneration and

increased intermuscular fat deposition. Similar impairments in muscle regeneration were

provided in a recent study where macrophage influx was reduced by liposomal clodronate

injections (56). It is conceivable that with reduced macrophage recruitment in both of these

studies, inadequate macrophage-dependent growth factors in the regenerating environment lead

to impaired and altered muscle regeneration. Indeed, macrophages secrete factors that modulate

both physiological responses and differentiation of nearby cells (58). Thus, alterations in

inflammation may influence not only the removal of damaged skeletal muscle, but also MPC

proliferation and/or differentiation.

MCP-1 is just one of many factors secreted by macrophages (23). This chemokine is also

generated by muscle (46). We observed significantly elevated levels of MCP-1 protein in CCR2

-/- mice, however, the physiological consequence of increased MCP-1 remains unknown. At

concentrations exceeding normal physiologic levels, MCP-1 may interact with alternative

of 50

26

chemokine receptors that are not typically favored in the presence of CCR2. In this regard,

higher concentrations of MCP-1 are required to activate CCR3 while subnanomolar

concentrations are sufficient for CCR2 activation (41). Conversely, CCR2 can bind other

chemokines, including MCP-2, MCP-3, MCP-4, MCP-5 (27, 47) and eotaxin-1 (41). Normal

cellular responses to these other chemokines could also be inhibited in the absence of CCR2.

Likewise, potential interactions between excessive amounts of MCP-1 and other chemokine

receptors may have functions redundant to the normal MCP-1/CCR2 axis, but may also direct an

array of unforeseen events.

The mechanism of MCP-1 signal transduction via the CCR2 receptor is not well

established. It has been suggested that MCP-1 may trigger activation of the Janus kinase 2

(JAK2)/STAT3 pathway and tyrosine phosphorylation of the CCR2 receptor in macrophages (4).

However, based on the current study, it appears that MCP-1 does not directly activate JAK2 via

the CCR2 receptor. Further studies are needed to clarify the role of the MCP-1/CCR2 axis in

STAT3 signaling.

The effect of STAT3 signaling during regeneration, particularly in multipotent MPC, is

unknown. However, STAT3 activation normally regulates several biological functions in

myoblasts including proliferation, differentiation, and survival (25, 26). While STAT3

activation in CCR2-/- mice was not significantly different than WT at specific time points,

STAT3 activity in CCR2-/- mice tended to be elevated overall compared to WT. Interestingly,

in human adipogenesis, STATs, including STAT3, are upregulated (15). Further evaluation of

STAT3 activation in CCR2 -/- mice will be important in determining the contribution of this

factor to the increased adipocyte accumulation. In addition, the kinetics of STAT3 activation

paralleled macrophage recruitment in both WT and CCR2 -/- mice. This is of note since

of 50

27

macrophage activation is also associated with the activation of STAT3 (13, 32). Additional

studies are required to clarify the role of CCR2 and STAT3 signaling in regenerating muscle

following ischemic injury.

In addition to the above, STAT3 induces the expression of the proangiogenic cytokine,

vascular endothelial growth factor (VEGF) (19) and macrophages can also produce VEGF (58).

Since capillary density was similar in CCR2 -/- and WT mice in both uninjured and ischemic

muscle at day 21 post-FAE and STAT3 activities leading to this point were similar between

mice, it seems unlikely that STAT3-dependent angiogenic events were modified in CCR2 -/-

mice. While capillaries/muscle fiber was decreased in regenerated muscle of CCR2 -/- mice, this

reflected the smaller size of regenerated fibers. Thus, although it remains to be determined if

earlier angiogenic events are also intact in CCR2 -/- mice, the current results do not support the

idea that revascularization underlies the observed alterations in skeletal muscle regeneration.

Rather, our findings support the hypothesis that the primary defect in skeletal muscle

regeneration in CCR2 -/- mice involves impairment of myoblast regeneration and/or

differentiation.

The results of the present study demonstrate that myoblast regeneration, as indicated by

MyoD activity, is independent of CCR2-mediated signaling as the amount and duration of MyoD

activity increased similar to WT in the absence of CCR2. Earlier studies reported no difference

in MyoD mRNA levels following macrophage depletion (56) or between WT and CCR2-/- mice

following freeze injury (64). Current findings of MyoD activity and protein expression are

consistent with this previous study. However, we observed an overall trend toward elevated

MyoD activity in CCR2-/- mice. Clearly, further studies are required to establish the relative

activities of transcription factors that are known to regulate muscle regeneration in CCR2-/- mice

of 50

28

compared to WT and thus define the basis for CCR2-dependent impairments in muscle

regeneration.

An important question that was not addressed in the current study is what cell types are

expressing MyoD and STAT3. Previous studies have documented that MyoD localization is

limited to satellite cells, myoblasts and some muscle fibers (9) in regenerating skeletal muscle;

the result herein are consistent with this observation. In contrast to the cell specific localization

of MyoD to muscle, STAT3 is expressed in a variety of cells, including muscle,(25)

inflammatory cells (34) and vascular endothelial cells (18). A detailed study of STAT3

expression in regenerating rat muscle demonstrated that phosphorylated STAT3 was first

detected in the nuclei of activated satellite cells and remained activated in proliferating, but not

differentiating, myoblasts. STAT3 nuclear staining was also identified in cells outside of the

myofiber and presumably represented inflammatory cells (25). Additional studies are required to

identify the cellular sources of STAT3 signaling in regenerating muscle.

The most significant observation in this study was adipocyte formation in regenerating

skeletal muscle of CCR2-/- mice. This suggests that locally impaired CCR2-mediated signaling,

in association with reduced myogenic and/or regenerative capacity of skeletal muscle progenitor

cells following injury, might favor differentiation of mesenchymal progenitor cells toward an

adipocytic phenotype (28). This possibility is supported by results from in vitro studies

documenting that satellite cells can be redirected, by altered molecular signaling, to accumulate

intracellular fat and express adipocyte markers (61, 62). Alternatively, adipocytes from the

surrounding areas may migrate into the injured muscle (2) or the adipocytes may be the result of

differentiation/transdifferentiation of other progenitor populations, such as bone marrow-derived

progenitor cells (22, 52). Regardless of the origin of the adipocytes, our results are consistent

of 50

29

with those of a recent report demonstrating adipocyte formation in regenerating muscles of

CCR2-/- mice following freeze-induced skeletal muscle injury (64). Together, these findings

suggest that the propensity to develop adipocytes in mice with impairments in CCR2 is a

generalized reaction to muscle injury, regardless of the basis for muscle tissue damage.

Additional evidence for a relationship between altered macrophage recruitment/function and

increased intermuscular fat has been observed when macrophages are partially depleted in WT

mice by clodronate-containing liposomes (56).

Interestingly, in humans, skeletal muscle regeneration is also impaired with aging (8, 14)

and there is an increase in fat within and between the muscle fibers (40, 48). Furthermore, aged

mice (12, 39, 59), similar to elderly humans (3, 37), have alterations in macrophage function.

Thus, CCR2-/- mice may represent a unique animal model for studies designed to elucidate the

inflammation-based mechanisms involved in impaired skeletal muscle regeneration that may

occur in normal human aging. This is particularly important because in addition to the

physiological consequences of impaired muscle regeneration, such as reductions in force-

producing muscle fibers, impairments in contraction, and metabolic dysfunction (28), adipose

tissue is recognized as having a paracrine/endocrine function with secretion of many factors that

influence diverse cell types as well as attract macrophages (5). Therefore, the accumulation of

adipocytes could serve as a compensatory mechanism to recruit macrophages for the purpose of

enhancing impaired healing and regeneration in this model. Interestingly, the majority of

adipose tissue-derived cytokines may emanate from tissue-resident macrophages (5). The

possible paracrine effects of intermuscular adipocytes on the surrounding muscle tissue are an

active area of ongoing investigation in our lab.

of 50

30

In conclusion, the current study supports a role for CCR2-mediated events in skeletal

muscle regeneration, but not restoration of perfusion. Increases in MCP-1 indicate potentially

profound changes in cellular signaling events in CCR2-/- mice in association with enhanced

neutrophil and impaired macrophage recruitment. In these animals, there was defective muscle

regeneration and increased fat accumulation, despite apparently similar activities of MyoD and

STAT3. These events suggest that the functional outcome of the reparative process, including

signaling events, is impaired in the absence of CCR2. Importantly, these alterations in

neutrophil and macrophage recruitment, and the reparative processes, direct regenerating muscle

towards an adipogenic lineage by mechanisms which remain to be established.

of 50

31

Acknowledgements

The authors would like to acknowledge the expert technical assistance of Lourdes Ruiz,

Didier Nuno, Masahiko Kobayashi and Jefferey Jimenez in the completion of these studies.

Statistical support was provided by Gary Chisholm and John Cornell.

Verónica Contreras-Shannon, Ph.D. is currently affiliated with the Department of

Cellular & Structural Biology and Sara M. Reyes-Reyna, Ph.D. is currently affiliated with the

Department of Medicine, both at the University of Texas Health Science Center, San Antonio,

TX.

Grants

Supported, in part, by the San Antonio Area Foundation, Veterans Administration Merit

Review, The American Heart Association, Texas Affiliate (0365123Y) and the NIH (HL070158,

HL074236, HL04776 and AR052610).

Disclosures

William Kuziel is employed by Protein Design Labs, otherwise, the authors have no

conflicts of interest to disclose.

of 50

32

References

1. Amano H, Morimoto K, Senba M, Wang H, Ishida Y, Kumatori A, Yoshimine H, Oishi K, Mukaida N, and Nagatake T. Essential contribution of monocyte chemoattractant protein-1/C-C chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia. J Immunol 172: 398-409, 2004.

2. Anghelina M, Krishnan P, Moldovan L, and Moldovan NI. Monocytes/macrophages cooperate with progenitor cells during neovascularization and tissue repair: conversion of cell columns into fibrovascular bundles. Am J Pathol 168: 529-541, 2006.

3. Antonaci S, Jirillo E, Ventura MT, Garofalo AR, and Bonomo L. Non-specific immunity in aging: deficiency of monocyte and polymorphonuclear cell-mediated functions. Mech Ageing Dev 24: 367-375, 1984.

4. Biswas SK and Sodhi A. Tyrosine phosphorylation-mediated signal transduction in MCP-1-induced macrophage activation: role for receptor dimerization, focal adhesion protein complex and JAK/STAT pathway. Int Immunopharmacol 2: 1095-1107, 2002.

5. Bouloumie A, Curat CA, Sengenes C, Lolmede K, Miranville A, and Busse R. Role of macrophage tissue infiltration in metabolic diseases. Curr Opin Clin Nutr Metab Care8: 347-354, 2005.

6. Charge SB and Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev 84: 209-238, 2004.

7. Charo IF and Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res 95: 858-866, 2004.

8. Conboy IM, Conboy MJ, Smythe GM, and Rando TA. Notch-mediated restoration of regenerative potential to aged muscle. Science 302: 1575-1577, 2003.

9. Cooper RN, Tajbakhsh S, Mouly V, Cossu G, Buckingham M, and Butler-Browne GS. In vivo satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle. J Cell Sci 112 (Pt 17): 2895-2901, 1999.

10. Fenton JI, Hursting SD, Perkins SN, and Hord NG. Interleukin-6 production induced by leptin treatment promotes cell proliferation in an Apc (Min/+) colon epithelial cell line. Carcinogenesis 27: 1507-1515, 2006.

11. Fink E, Fortin D, Serrurier B, Ventura-Clapier R, and Bigard AX. Recovery of contractile and metabolic phenotypes in regenerating slow muscle after notexin-induced or crush injury. J Muscle Res Cell Motil 24: 421-429, 2003.

12. Forner MA, Collazos ME, Barriga C, De la Fuente M, Rodriguez AB, and Ortega E.Effect of age on adherence and chemotaxis capacities of peritoneal macrophages. Influence of physical activity stress. Mech Ageing Dev 75: 179-189, 1994.

13. Gao H, Guo RF, Speyer CL, Reuben J, Neff TA, Hoesel LM, Riedemann NC, McClintock SD, Sarma JV, Van Rooijen N, Zetoune FS, and Ward PA. Stat3 activation in acute lung injury. J Immunol 172: 7703-7712, 2004.

14. Grounds MD. Age-associated changes in the response of skeletal muscle cells to exercise and regeneration. Ann N Y Acad Sci 854: 78-91, 1998.

15. Harp JB, Franklin D, Vanderpuije AA, and Gimble JM. Differential expression of signal transducers and activators of transcription during human adipogenesis. Biochem Biophys Res Commun 281: 907-912, 2001.

of 50

33

16. Heil M and Schaper W. Cellular mechanisms of arteriogenesis. Exs: 181-191, 2005.17. Heil M, Ziegelhoeffer T, Wagner S, Fernandez B, Helisch A, Martin S, Tribulova S,

Kuziel WA, Bachmann G, and Schaper W. Collateral Artery Growth (Arteriogenesis) After Experimental Arterial Occlusion Is Impaired in Mice Lacking CC-Chemokine Receptor-2. Circ Res 94: 671-677, 2004.

18. Hilfiker-Kleiner D, Hilfiker A, and Drexler H. Many good reasons to have STAT3 in the heart. Pharmacol Ther 107: 131-137, 2005.

19. Hilfiker-Kleiner D, Limbourg A, and Drexler H. STAT3-mediated activation of myocardial capillary growth. Trends Cardiovasc Med 15: 152-157, 2005.

20. Hornung RW and Reed DR. Estimation of average concentration in the presence of nondetectable values. Applied Occupational and Environmental Hygiene 5: 46-51, 1990.

21. Hurme T and Kalimo H. Activation of myogenic precursor cells after muscle injury. Med Sci Sports Exerc 24: 197-205, 1992.

22. Jin-Xiang F, Xiaofeng S, Jun-Chuan Q, Yan G, and Xue-Guang Z. Homing efficiency and hematopoietic reconstitution of bone marrow-derived stroma cells expanded by recombinant human macrophage-colony stimulating factor in vitro. Exp Hematol 32: 1204-1211, 2004.

23. Jinnouchi K, Terasaki Y, Fujiyama S, Tomita K, Kuziel WA, Maeda N, Takahashi K, and Takeya M. Impaired hepatic granuloma formation in mice deficient in C-C chemokine receptor 2. J Pathol 200: 406-416, 2003.

24. Junqueira LC and Carneiro J. Adipose tissue. In: Basic Histology Text & Atlas (11th ed.), edited by Malley J, Lebowitz H and Boyle PJ. New York: McGraw-Hill, 2005, p. 123-127.

25. Kami K and Senba E. In vivo activation of STAT3 signaling in satellite cells and myofibers in regenerating rat skeletal muscles. J Histochem Cytochem 50: 1579-1589, 2002.

26. Kataoka Y, Matsumura I, Ezoe S, Nakata S, Takigawa E, Sato Y, Kawasaki A, Yokota T, Nakajima K, Felsani A, and Kanakura Y. Reciprocal inhibition between MyoD and STAT3 in the regulation of growth and differentiation of myoblasts. J Biol Chem 278: 44178-44187, 2003.

27. Kim CH and Broxmeyer HE. Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J Leukoc Biol 65: 6-15, 1999.

28. Kirkland JL, Tchkonia T, Pirtskhalava T, Han J, and Karagiannides I.Adipogenesis and aging: does aging make fat go MAD? Exp Gerontol 37: 757-767, 2002.

29. Kuziel WA, Morgan SJ, Dawson TC, Griffin S, Smithies O, Ley K, and Maeda N.Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2. Proc Natl Acad Sci U S A 94: 12053-12058, 1997.

30. Laemmli UK and Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol 80: 575-599, 1973.

31. Lagasse E and Weissman IL. Flow cytometric identification of murine neutrophils and monocytes. J Immunol Methods 197: 139-150, 1996.

32. Lang R. Tuning of macrophage responses by Stat3-inducing cytokines: molecular mechanisms and consequences in infection. Immunobiology 210: 63-76, 2005.

33. Lescaudron L, Peltekian E, Fontaine-Perus J, Paulin D, Zampieri M, Garcia L, and Parrish E. Blood borne macrophages are essential for the triggering of muscle regeneration following muscle transplant. Neuromuscul Disord 9: 72-80, 1999.

of 50

34

34. Levy DE and Lee CK. What does Stat3 do? J Clin Invest 109: 1143-1148, 2002.35. Li P, Garcia GE, Xia Y, Wu W, Gersch C, Park PW, Truong L, Wilson CB,

Johnson R, and Feng L. Blocking of monocyte chemoattractant protein-1 during tubulointerstitial nephritis resulted in delayed neutrophil clearance. Am J Pathol 167: 637-649, 2005.

36. Liby K, Voong N, Williams CR, Risingsong R, Royce DB, Honda T, Gribble GW, Sporn MB, and Letterio JJ. The synthetic triterpenoid CDDO-Imidazolide suppresses STAT phosphorylation and induces apoptosis in myeloma and lung cancer cells. Clin Cancer Res 12: 4288-4293, 2006.

37. McLachlan JA, Serkin CD, Morrey-Clark KM, and Bakouche O. Immunological functions of aged human monocytes. Pathobiology 63: 148-159, 1995.

38. Merly F, Lescaudron L, Rouaud T, Crossin F, and Gardahaut MF. Macrophages enhance muscle satellite cell proliferation and delay their differentiation. Muscle Nerve22: 724-732, 1999.

39. Ortega E, Forner MA, Barriga C, and De la Fuente M. Effect of age and of swimming-induced stress on the phagocytic capacity of peritoneal macrophages from mice. Mech Ageing Dev 70: 53-63, 1993.

40. Pahor M and Kritchevsky S. Research hypotheses on muscle wasting, aging, loss of function and disability. J Nutr Health Aging 2: 97-100, 1998.

41. Parody TR and Stone MJ. High level expression, activation, and antagonism of CC chemokine receptors CCR2 and CCR3 in Chinese hamster ovary cells. Cytokine 27: 38-46, 2004.

42. Pimorady-Esfahani A, Grounds MD, and McMenamin PG. Macrophages and dendritic cells in normal and regenerating murine skeletal muscle. Muscle Nerve 20: 158-166, 1997.

43. Rando TA and Blau HM. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol 125: 1275-1287, 1994.

44. Reichel CA, Khandoga A, Anders HJ, Schlondorff D, Luckow B, and Krombach F.Chemokine receptors Ccr1, Ccr2, and Ccr5 mediate neutrophil migration to postischemic tissue. J Leukoc Biol 79: 114-122, 2006.

45. Renard P, Ernest I, Houbion A, Art M, Le Calvez H, Raes M, and Remacle J.Development of a sensitive multi-well colorimetric assay for active NFkappaB. Nucleic Acids Res 29: E21, 2001.

46. Reyes-Reyna SM and Krolick KA. Chemokine production by rat myocytes exposed to interferon-gamma. Clin Immunol 94: 105-113, 2000.

47. Rollins BJ. Chemokines. Blood 90: 909-928, 1997.48. Roubenoff R. Sarcopenia and its implications for the elderly. Eur J Clin Nutr 54 Suppl

3: S40-47, 2000.49. Sabourin LA and Rudnicki MA. The molecular regulation of myogenesis. Clin Genet

57: 16-25, 2000.50. Salcedo R, Ponce ML, Young HA, Wasserman K, Ward JM, Kleinman HK,

Oppenheim JJ, and Murphy WJ. Human endothelial cells express CCR2 and respond to MCP-1: direct role of MCP-1 in angiogenesis and tumor progression. Blood 96: 34-40, 2000.

of 50

35

51. Scholz D, Thomas S, Sass S, and Podzuweit T. Angiogenesis and myogenesis as two facets of inflammatory post-ischemic tissue regeneration. Mol Cell Biochem 246: 57-67, 2003.

52. Sekiya I, Larson BL, Smith JR, Pochampally R, Cui JG, and Prockop DJ. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells 20: 530-541, 2002.

53. Shireman PK, Contreras-Shannon V, Reyes-Reyna SM, Robinson SC, and McManus LM. MCP-1 parallels inflammatory and regenerative responses in ischemic muscle. J Surg Res 134: 145-157, 2006.

54. Shireman PK, Quinones MP. Differential necrosis despite similar perfusion in mouse strains after ischemia. J Surg Res 129: 242-250, 2005.

55. Steffel J, Wernig M, Knauf U, Kumar S, Wiestler OD, Wernig A, and Brustle O.Migration and differentiation of myogenic precursors following transplantation into the developing rat brain. Stem Cells 21: 181-189, 2003.

56. Summan M, Warren GL, Mercer RR, Chapman R, Hulderman T, Van Rooijen N, and Simeonova PP. Macrophages and skeletal muscle regeneration: a clodronate-containing liposome depletion study. Am J Physiol Regul Integr Comp Physiol 290: R1488-1495, 2006.

57. Sun JS, Wu SY, and Lin FH. The role of muscle-derived stem cells in bone tissue engineering. Biomaterials 26: 3953-3960, 2005.

58. Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, and Sorg C. Macrophages and angiogenesis. J Leukoc Biol 55: 410-422, 1994.

59. Swift ME, Burns AL, Gray KL, and DiPietro LA. Age-related alterations in the inflammatory response to dermal injury. J Invest Dermatol 117: 1027-1035, 2001.

60. Tang G, Charo DN, Wang R, Charo IF, and Messina L. CCR2-/- knockout mice revascularize normally in response to severe hindlimb ischemia. J Vasc Surg 40: 786-795, 2004.

61. Taylor-Jones JM, McGehee RE, Rando TA, Lecka-Czernik B, Lipschitz DA, and Peterson CA. Activation of an adipogenic program in adult myoblasts with age. Mech Ageing Dev 123: 649-661, 2002.

62. Teboul L, Gaillard D, Staccini L, Inadera H, Amri EZ, and Grimaldi PA.Thiazolidinediones and fatty acids convert myogenic cells into adipose-like cells. J Biol Chem 270: 28183-28187, 1995.

63. Tidball JG. Inflammatory processes in muscle injury and repair. Am J Physiol Regul Integr Comp Physiol 288: R345-353, 2005.

64. Warren GL, Hulderman T, Mishra D, Gao X, Millecchia L, O'Farrell L, Kuziel WA, and Simeonova PP. Chemokine receptor CCR2 involvement in skeletal muscle regeneration. FASEB J 19: 413-415, 2005.

65. Warren GL, O'Farrell L, Summan M, Hulderman T, Mishra D, Luster MI, Kuziel WA, and Simeonova PP. Role of CC chemokines in skeletal muscle functional restoration after injury. Am J Physiol Cell Physiol 286: C1031-1036, 2004.

of 50

36

Figure Legends

Figure 1. Hind limb skeletal muscle injury and regeneration in CCR2-/- and WT mice

following FAE. LDH activity was measured in calf muscle lysates prepared from CCR2-/-

and WT mice and used as a measure of injury and regeneration following FAE. LDH activity

was expressed in U/mg of protein. Baseline results represent LDH activity from samples

collected in the absence of surgery. Results represent the mean+SEM; n=5 mice/time

point/strain. *significant difference (p<0.003) as compared to baseline activity; # significant

difference (p=0.004) between CCR2-/- and WT groups.

Figure 2. Restoration of perfusion in CCR2-/- and WT mice after FAE. Using LDI,

perfusion ratios (FAE/sham) for CCR2-/- and WT mice were sequentially measured for each

mouse at every time point. Perfusion in both groups was significantly different at all time

points compared to pre-excision LDI measurements (p<0.001) and there were no significant

differences between CCR2-/- and WT groups at any time point. Results represent the mean

+SEM; n=10 mice/strain.

Figure 3. Morphological alterations in ischemic skeletal muscle of CCR2-/- and WT mice.

At 3 days post-FAE, skeletal muscle necrosis and inflammation were extensive in ischemic calf

muscles in WT mice (A) and this included an abundant mononuclear cell infiltrate whereas in

CCR2-/- mice (B) this reflected persistent neutrophils (arrow), necrotic myocytes (asterisks) and

a scarce mononuclear cell infiltrate. Also at the day 3 time point, F4/80 immunolocalization

demonstrated abundant macrophages in WT mice (C) and minimal macrophages (arrows) in

CCR2-/- animals (D). By 7 days post-FAE in WT mice (E), skeletal muscle regeneration was

of 50

37

widespread as evidenced by the presence of multiple, centrally-located myocyte nuclei whereas

in CCR2-/- animals (F, G), necrotic tissue remained and muscle regeneration was substantially

reduced; regenerated fibers often encircled residual necrotic myofibers (asterisks). Large

vacuolated cells (arrows) were prevalent throughout the injured tissue in a CCR2-/- mice at 7

days post-FAE (G). By 21 days, intermuscular adipocytes accumulated in areas of regenerated

muscle in CCR2 -/ mice (H). *indicates necrotic muscle fiber. H&E stain except for C and D

that were counterstained with hematoxylin.

Figure 4. Necrosis and intermuscular adipocyte accumulation in CCR2-/- mice 14-21 days

post-FAE. Muscle regeneration was essentially complete at day 14 in WT mice (A);

longitudinal regenerated muscle fibers contained multiple, centrally located nuclei. Minimal

intermuscular fat was present between regenerated fibers in WT mice at 21 days post-FAE (B).

In CCR2-/- mice, residual myofiber necrosis, small regenerated muscle fibers, and extensive

intermuscular fat were prevalent at 14 days post-FAE (C, D). At 21 days post-FAE, widespread

intermuscular fat and persistent necrotic myofibers were evident in CCR2-/- mice (E); an

extensive area of injured muscle transitions from regenerated myofibers with interspersed

adipocytes to a region of necrotic muscle fibers (upper right). s=regenerated soleus muscle;

n=normal gastrocnemius muscle, H&E stain, *indicates necrotic muscle fibers.

Figure 5. Intermuscular adipocytes, capillaries and MyoD immunolocalization in CCR2 -/-

mice post-FAE. Oil red O staining of lipid between regenerated myofibers (arrows) at day 21

post-FAE in WT (A) and CCR2 -/- (B) animals. Lectin localization of capillaries (arrowheads)

in the regenerated TA of WT (C) and CCR2 -/- (D) mice at 21 days post-FAE (H&E

of 50

38

counterstain). Immunolocalization of MyoD at post-FAE day 3 (E) and day 7 (G) and F4/80

localization at post-FAE day 3 (F) and day 7 (H) in CCR2 -/- mice (hematoxylin counterstain).

In (E), MyoD positive nuclei were either within myofibers in rounded nuclei (arrows) or in peri-

fiber flat nuclei (arrowheads). F4/80 immunolocalization in an adjacent frozen section (F)

indicated that these nuclei did not correspond to those of macrophages. In (G) and (H), arrows

indicate MyoD positive nuclei and F4/80 positive cells, respectively. n=nerve; 1, 2, 3 indicate

corresponding myofibers in adjacent frozen sections.

Figure 6. Intermuscular fat, muscle fiber cross-sectional area and capillary density in the

TA muscle at day 21 post-FAE. Fat area (%) in regenerated muscle (A), cross-sectional area

(µm2) of muscle fibers (B), capillaries/muscle fiber (C) and capillaries/mm2 (D) in the TA

muscle at 21 days post-FAE. For FAE specimen, results were derived from regions comprised

exclusively of regenerated myocytes (i.e., all myofibers contained centrally located nuclei); for

sham surgery specimen, results were derived only from areas with non-regenerated, mature

fibers (i.e., fibers with peripherally-displaced nuclei). Data presented as the mean+SEM; n=5

mice/group. *significant difference (p<0.01) as compared to the contralateral, sham surgery leg;

#significant difference (p<0.003) between CCR2-/- and WT groups.

Figure 7. FAE-induced changes in calf tissue weight, protein/weight ratios and MPO

activity. At various times following FAE in CCR2-/- and WT mice, calf muscles were collected

and weighed. Weights of calf muscles were reported as the ratio of the FAE/sham calf muscle

weights (A). Changes in protein/weight ratios of calf muscle lysates following FAE were

inversely related to the muscle weight (B). MPO activity in calf muscle lysates prepared after

of 50

39

FAE was used to assess neutrophil accumulation (C). Baseline results were derived from

samples collected in the absence of surgery. Results represent the mean+SEM; n=5 mice/time

point/strain. *significant difference (p<0.04) as compared to baseline; #significant difference

(p<0.01) between CCR2-/- and WT groups.

Figure 8. Decreased tissue macrophages with comparable neutrophils in ischemic hind

limb muscles of CCR2 -/- and WT mice at day 3 post-FAE. Macrophages and neutrophils in

(A) WT and (B) CCR2 -/- calf tissues were identified by FACS where macrophages were

defined as CD11b+/Gr-1- (upper left quadrant) and neutrophils were defined as CD11b+/Gr-1+

(upper right quadrant). (C) Absolute numbers of tissue neutrophils and macrophages, as

measured by FACS analysis, in ischemic calf muscle. Results represent the mean+SEM; n=4

mice/strain. #significant difference (p=0.009) between CCR2-/- and WT groups.

Figure 9. MCP-1 tissue levels following FAE. MCP-1 levels were measured in the (A) calf

and (B) thigh of CCR2-/- and WT mice following FAE. Baseline results were derived from

samples collected in the absence of surgery. Among specimen from 5 mice for each time point,

MCP-1 protein was not detectable (ND) in some specimens including WT calf muscles at

baseline (n=5), days 7 (n=2) and 14 (n=5), WT thigh muscles at baseline (n=3), days 1 (n=2), 3

(n=1), 7 (n=5) and 14 (n=5), and CCR2-/- calf muscles at baseline (n=3) and CCR2-/- thigh

muscles at baseline (n=5), days 7 (n=2) and 14 (n=3). Results represent the mean+SEM of only

those samples with detectable levels of MCP-1. *significant difference (p<0.01) as compared to

baseline levels; #significant difference (p<0.004) between CCR2-/- and WT groups.

of 50

40

Figure 10. Transcription factor activity in the regenerating milieu of CCR2-/- and WT mice

following FAE. MyoD (A) and STAT3 (B) activities were assayed in calf muscle lysates of

CCR2-/- and WT mice. DNA binding activity of transcription factors was expressed in OD/mg

of protein. Baseline results were derived from samples collected in the absence of surgery.

MyoD (p=0.02) and STAT3 (p=0.05) activities were significantly elevated in CCR2-/- vs WT

mice, combined data for all post-FAE time points. Results represent the mean+SEM; n=5

mice/time point/strain. *significant difference (p<0.05) as compared to baseline. (C)

Immunoblot analyses of MyoD, phosphorylated STAT3 (Tyr705) and STAT3 protein expression

in 100 µg of right calf muscle protein lysate from representative CCR2-/- and WT mouse

samples for all post-FAE time points. MyoD, which migrates as a doublet, is delineated in

selected lanes by a right brace, }.

of 50

Figure 1

of 50

Figure 2

of 50

Figure 3

of 50

Figure 4

of 50

Figure 5

of 50

Figure 6

of 50

Figure 7

of 50

Figure 8

of 50

Figure 9

of 50

Figure 10

of 50

Fat Accumulation with Altered Inflammation and Regeneration in Skeletal

Documents

Transcript of Fat Accumulation with Altered Inflammation and Regeneration in Skeletal