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* Department of Histology and Medical Embryology, CE-BEMM and Interuniversity Institute of Myology, University of Rome "La Sapienza," Rome, Italy;
Neuroimmunology Unit, Santa Lucia Foundation Scientific Institute, Rome, Italy;
IRCCS San Raffaele, Rome, Italy;
EMBL Mouse Biology Program, Monterotondo, Italy;
|| Harefield Heart Science Centre, National Heart and Lung Institute, Imperial College London, London, UK; and
¶ Edith Cowan University, Perth, Western Australia, Australia
2Correspondence: Department of Histology and Medical Embryology, University of Rome "La Sapienza," Via A. Scarpa, 14 Rome 00161, Italy. E-mail: antonio.musaro{at}uniroma1.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B. The rapid restoration of injured mIGF-1 transgenic muscle was also associated with connective tissue remodeling and a rapid recovery of functional properties. By modulating the inflammatory response and reducing fibrosis, supplemental mIGF-1 creates a qualitatively different environment for sustaining more efficient muscle regeneration and repair.—Pelosi, L., Giacinti, C., Nardis, C., Borsellino, G., Rizzuto, E., Nicoletti, C., Wannenes, F., Battistini, L., Rosenthal, N., Molinaro, M., Musarò, A. Local expression of IGF-1 accelerates muscle regeneration by rapidly modulating inflammatory cytokines and chemokines.
Key Words: fibrosis • inflammatory response • injury
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Injury results in the rapid necrosis of myofibers and the activation of inflammation, which contributes to the removal of the necrotic material and also to the secretion of several cytokines and growth factors stimulating satellite cell activation (4
5
6)
.
Inflammation is clearly a critical component of muscle physiology and is an important phase in the regenerative process. Nevertheless, the inflammatory response must be resolved to allow muscle repair. In fact, functional impairment is associated with perturbed spatial distribution of inflammatory cells, altered identity of the inflammatory infiltrate (cell type and magnitude of influx) and disrupted temporal sequence, resulting in a persistent rather than resolved inflammatory phase (7)
. Thus, fine tuning of the regenerative response is a critical prerequisite for maintaining functional skeletal musculature.
Insulin-like growth factor-1 (IGF-1) has been implicated as a central regulator of muscle regeneration (8)
. We have previously reported that transgenic expression of a locally acting IGF-1 isoform (mIGF-1) safely enhanced and preserved muscle fibers’ integrity, even at advanced ages (8
, 9)
, suggesting that mIGF-1 acts as a survival factor by prolonging the regenerative potential of skeletal muscle through increases in satellite cell activity. High levels of MLC/mIGF-1 transgene expression in the mdx mouse model of muscular dystrophy also preserved muscle function in the absence of dystrophin, reducing fibrosis and myonecrosis, and elevating signaling pathways associated with muscle survival and regeneration (10)
. Local mIGF-1 expression also counteracted muscle decline in ALS mouse model, activating satellite cells and improving the survival of motor neurons (11)
. In addition, mIGF-1-mediated improvement in muscle regeneration reflected increased recruitment and incorporation of circulating stem cells at sites of muscle injury (12)
.
In the present study, we investigated whether accelerating the course of muscle repair through mIGF-1 expression involves modulation of the inflammatory response. To determine which processes dominating the different phases of muscle regeneration are modulated by mIGF-1 expression, we induced muscle injury by cardiotoxin (CTX) injection (8
, 12)
and examined the regenerative process at mechanical, cellular and molecular levels. Gene expression and proteomic analysis revealed that persistent expression of the mIGF-1 transgene rapidly modulated specific inflammatory factors, accelerated the timing of regeneration, and restored muscle function and architecture soon after injury. Specifically, mIGF-1 suppresses the expression and activity of macrophage migration inhibitory factor (MIF), high mobility group protein 1 (HMGB1) and transcription factor NF-B, all of which are involved in the persistence of the inflammatory response (13
14
15
16)
, often associated with severe and progressive fibrosis (17)
. Together, these results show how mIGF-1 accelerates the healing process, establishing a balance between inflammation and connective remodeling, and promoting a qualitative improvement in the tissue environment to ensure a rapid functional recovery.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C, and data not shown). The muscles were harvested at different time points following injury. Contralateral TA was used as a control. Seven micrometers of cryosections were fixed in 4% paraformaldehyde and processed for histological and immunofluorescence analysis, as described (8)
. Antibody against alpha-smooth actin (
-SMA) (Sigma) was used for immunofluorescence analysis. Stained cells were observed under an inverted microscope (model Axioskop 2 plus; Carl Zeiss Microimaging, Inc., Thornwood, NY), and images were processed using Axiovision 3.1. Twelve fields within the injured areas (four mice/strain) were analyzed and the absolute area of -SMA positive cells (pixel/µm2) were measured for each field using Scion Image 4.0.3.2. software. To determine the extent of infiltrating area, muscle sections from both wild-type and MLC/mIGF-1 injured muscle were stained with hematoxylin and eosin and analyzed at different time points (2, 5, and 10 days after CTX injection). Scion Image 4.0.3.2. software was used to measure the absolute area of infiltration (pixel/µm2). To determine the degree and the stage of muscle regeneration, cross sections were stained with X-gal (8
, 21)
. Muscle regeneration was assessed by counting the number of fibers with blue-positive nuclei. Twelve fields within the injured areas (four mice/strain) were analyzed, and the fibers with blue-positive nuclei were counted for each field using Scion Image 4.0.3.2. software and expressed as a percentage of the total number of fibers. All animal studies have been approved by the authors’ Institutional Review Board.
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Evans blue staining
Evans blue was injected into the tail vein of 3-mo-old wild-type and MLC/mIGF-1 mice 24 h or 5 days after muscle injury with CTX injection. Animals were killed 6 h after Evans blue injection, and muscles were removed and rapidly frozen in melting isopentane. Ten-micrometer frozen sections were fixed in cold acetone, rinsed in PBS, and covered with aqueous mounting media containing 4',6'-diamidino-2-phenylidole nuclear stain (Vectashield; Vector Laboratories). Fluorescent fibers were viewed under an inverted microscope (model Axioskop 2 plus; Carl Zeiss Microimaging, Inc), and images were processed using Axiovision 3.1. Twelve fields within the damaged areas (four mice/strain) were analyzed and the Evans blue positive fibers were counted for each field using Scion Image 4.0.3.2. software.
RNA preparation and RT-MultiplexPCR
RNA were prepared as described (18)
; 10 µg of RNA was incubated with DNase for 15 min at 37°C and reverse-transcribed for 1 h at 37°C using Oligo-(dT)20 mers and the RTeasy Reverse Transcription Kit (Maxim Biotech, Rockville, MD). RT-MultiplexPCR (RT-MPCR) provides an accurate method to detect multiple gene expression by amplifying all of the selected genes under the same conditions (19)
. Maxim’s Mouse Chemokines MPCR kits (Maxim Biotech) have been designed to accurately detect the expression pattern of inflammatory cytokines. We performed six independent M-polymerase chain reaction (PCR) experiments, using 6 samples/strain (wild-type, MLC/mIGF-1) for each time point. For MCP-1 (233 bp), MCP-2 (275 bp), RANTES (176 bp), and GAPDH (532 bp) (kit components) the two steps PCR thermocycle reaction profile consisted of an initial denaturation at 96°C for 1 min, 63°C for 4 min (2 cycles), followed by 32 cycles at 94°C for 1 min, 63°C for 2m, with a final elongation step at 70°C for 10 min. For MIP-1 (211 bp), MIP-1ß (187 bp), ENA-78 (295 bp), and GAPDH (532 bp) (kit primers), the reaction profile for amplification consisted of an initial 96°C for 1 min, 64°C for 4 min (2 cycles), followed by 32 cycles at 94°C for 1 min, 64°C for 2 min, with a final elongation step at 70°C for 10 min. EIIIA, collagen I, and collagen III expression was analyzed by real-time PCR. Real-time PCR was conducted using 1 µl of RT reaction and Platinum SYBR Green qPCR SuperMix UDG with ROX (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. The following primers have been designed to detect EIIIA, procollagen I, procollagen III, and GAPDH expression:
EIIIA sense 5'-CTAGACCAGGTGATGAGTGT-3' and antisense 5'-GAACAGAAGACTTTTAAATAATAACTA-3'
Procollagen I sense: 5'-GGGCGAGTGCTGTGCTTTCTG-3' and antisense: 5'-CCTCGGTGTCCCTTCATTCCA-3'
Procollagen III sense: 5'-AGCCACCTTGGTCAGTCCTA-3'- and antisense: 5'-TTCCTCCCACTCCAGACTTG-3'
GAPDH sense 5'-GGAGCAAAAGGGTCATCATCTC-3' and antisense 5'-AAGGTGGAAGAGTGGGAGTTG CT-3'.
We performed four independent real time-PCR experiments, using four samples/strain (wild-type, MLC/mIGF-1) for each time point, with the data averaged per mouse at each time point.
Protein array analysis
Proteomic analysis was performed using RayBio® Mouse Cytokine Antibody Array 1.1 kit (RayBiotech, Inc., Norcross, GA) (20)
. Muscle proteins were extracted with Array Lysis Buffer (kit component) and incubated at final concentration of 1 µg/µl. Each membrane was blocked for 30 min at RT with Array Blocking Buffer 1X. Membranes were incubated with protein extracts at 4°C overnight. Membranes were washed for 30 min at RT, incubated with biotin-conjugated antibody for 2 h at RT. Membranes were washed for 30 min and incubated with HRP-conjugated streptavidin for 2 h at RT, followed by 30 min of wash. Then, each membrane was placed into the Detection Solution and incubated for 1 min at RT and exposed to X-ray film. The intensities of signals were quantified by densitometric analysis and the positive control (biotin-conjugated IgG) was used to normalize the results from each membrane. We performed 3 separate proteomic analyses, using 3 different pooled samples from 2 animals/strain (wild-type, MLC/mIGF-1).
Western blot analysis
TA muscles, from both wild-type and MLC/mIGF-1 transgenic mice, injected with CTX and contralateral control TA were homogenized in modified lysis buffer (10 mM Tris-HCl, pH 7.4/150 mM NaCl/1% Nonidet P-40/1% sodium deoxycholate/0.1% SDS/10% glycerol). Equal amounts of protein were separated by 12% SDS-PAGE, and standard blotting procedures were used. Membranes were blotted with the following antibodies: antiphosho-IkB- (Ser-32 (Cell Signaling Technology, Danvers, MA); anti-MIF (AbCam); anti-HMGB1 (BD Pharmingen, Franklin Lakes, NJ); anti--tubulin (Sigma, St. Louis, MO).
Densitometric analysis
Densitometric analysis of RT-MPCR, Western blotting and proteomic array results were performed using Imaging Fluor S instrument (Bio-Rad, Hercules, CA). The optic density (OD) of each signal was normalized with GAPDH signal (for RT-MPCR), with -tubulin signal (for Western blot) and with biotin-conjugated IgG signal (for Proteomic Array).
Flow cytometry
Muscle-derived cells were purified from injured and control muscle of both wt and MLC/mIGF-1 transgenic mice. Same amount of muscle-derived cells, isolated from both wt and MLC/mIGF-1 mice, were overlaid on a Percoll gradient (70–40% in PBS) (12)
and analyzed for CD11b (Becton Dickinson Biosciences) expression. Cell analyses were performed on a MoFlo triple-laser flow cytometer (Dako, Glostrup, Denmark), and data were analyzed using the software FlowJo (Treestar). A 488-nm argon laser was used for PE-Cy5 excitation.
Mechanical measurements
Mice, age 2–3 mo, were killed by cervical dislocation under anesthesia, and EDL muscles were removed for isolated muscle force measurements as described (9)
. The tendons were attached to a rigid post and to an isometric force transducer in a bath of Ringer solution gas-equilibrated with 95% O2 and 5% CO2. Optimum length of the muscle was determined by twitch force from supramaximal stimulation. To evaluate time to peak, 
relaxation time and twitch contractile force, the muscle was stimulated with three single pulses of 500 ms. Tetanic force was measured with three stimuli at 120 Hz and two stimuli at 180 Hz using a 100-ms pulses delivered through two parallel platinum electrodes. Maximal tetanic force was determined at 180-Hz stimulation frequency.
Statistics
Student’s t test was used for comparison of histological, Western blots, RT-MPCR, and proteomic analysis between age-matched wild-type and MLC/mIGF-1 mice. Statistical significance was accepted for comparisons where P < 0.05. Values presented are means ± SEM. Each time point (9 h, 24 h, 2, 5, and 10 days postinjury) of either wild-type or MLC/mIGF-1 transgenic mice was compared with the correspondent baseline level (time=0). Statistical comparison between wild-type and MLC/mIGF-1 is also indicated in the figures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Notably, at 5 days’ post CTX injection mononucleated cells still actively infiltrated the injured area of wild-type muscle (Fig. 1A
), whereas a conspicuous percentage of newly regenerating myofibers, with the characteristic central nuclei, appeared in injured MLC/mIGF-1 transgenic muscle (Fig. 1A
). By 10 days post CTX injection, the injured muscle of MLC/mIGF-1 mice was largely restored, whereas the wild-type muscle had only just activated the reparative phase, as indicated by the presence of regenerating fibers with central nuclei (Fig. 1A
).
To further document stimulation of rapid repair in MLC/mIGF-1 muscle, we monitored induction of a coinherited nls-lacZ transgene driven by the desmin promoter as a marker of satellite cell mobilization (21)
. Histological analysis of desmin/nls-lacZ (Wt) and bigenic desmin/nls-lacZxMLC/mIGF-1 (Tg) muscles at 10 and 15 days post CTX injection revealed elevated numbers of centrally located nuclei in Wt muscle fibers, with the desmin transgene activated in regenerating fibers (Fig. 1B
, fibers with blue nuclei). In contrast, at the same stage, Tg mice presented few fibers with central nuclei, mostly located at the cell periphery (Fig. 1B
, fibers with blue nuclei; Fig. 1B
, inset).
To exclude that mIGF-1 expression elicits protective effects against CTX-induced injury, we evaluated, by Evans Blue Dye labeling, the extent of fiber damage at early (24 h) and late (5 days) stages postinjury. Fluorescent microscopy performed at 24 h after CTX injection (Fig. 1C
) did not reveal significant difference in the percentage of necrotic fibers between wild-type and MLC/mIGF-1 transgenic mice. In contrast, Evans blue staining performed at 5 days postinjury (Fig. 1C
), revealed the elevated presence of necrotic fibers in wild-type muscle compared with MLC/mIGF-1 mice littermates (Fig. 1C
). This suggests that mIGF-1 does not protect skeletal muscle from initial CTX-induced damage but acts subsequently to modulate the muscle milieu, improving muscle regeneration.
Inflammatory cytokine production is modulated in regenerating mIGF-1 skeletal muscle
Muscle injury and healing processes are intimately related to inflammatory cell recruitment. To verify whether the enhanced regenerative response of transgenic muscle is associated with a modulation of the inflammatory response, we examined accumulation of CD11b+ cells in the postinjury extracellular matrix (ECM) (22)
. Cytofluorimetric analysis of mononucleate cells in skeletal muscle 5 days after CTX injection documented greater numbers of CD11b+ cells in wild-type muscle compared to MLC/mIGF-1 muscle (Fig. 2
A).
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To examine more precisely how mIGF-1 expression accelerates the timing of inflammation and contributes to muscle repair, we analyzed expression patterns of specific cytokines and chemokines associated with the infiltration of inflammatory cells into damaged muscle. Semiquantitative RT-MPCR analysis revealed that the expression of TNF- and IL-1ß, two important mediators of the early inflammatory response (1
, 23)
, was rapidly up-regulated within 9 h after CTX injection, with a peak of expression at 24 h (Fig. 2B
). Importantly, TNF- and IL-1ß expression decreased significantly at 5 days postinjury in MLC/mIGF-1 muscle, while high expression levels persisted in wild-type damaged muscles (Fig. 2B
).
A biphasic expression pattern of ENA-78, a potent chemoattractant and activator of neutrophil function (24)
, was observed in injured wild-type muscle (Fig. 2B
): ENA-78 mRNA increased at 9 h after CTX injection and remained at high levels until 24 h. At 2 days postinjury, expression levels dropped, with a further significant increase at 5 days postinjury, returning to basal levels by 10 days post injury (Fig. 2B
). In contrast, expression of ENA-78 in MLC/mIGF-1 injured muscle followed a simpler course, increasing within 9 h after CTX injection and progressively decreasing, showing a significant reduction at 5 days compared to wild-type injured muscle (Fig. 2B
). By 5 days postinjury the MLC/mIGF-1 injured muscle displayed basal levels of ENA-78 expression (Fig. 2B
), further confirming a rapid resolution of the inflammatory response.
At later stages, the inflammatory response to muscle injury is associated with elevated expression of CC chemokines involved in the recruitment of monocytes/macrophages (25
, 26)
. Significant increases in monocyte chemoattractant protein-1, -2 (MCP-1, MCP-2), macrophage inflammatory protein-1, -1ß (MIP-1, MIP-1ß), and RANTES expression were detected within 9 h of CTX injection in both wild-type and MLC/mIGF-1 transgenic muscle (Fig. 2C
). Notably, the mRNA transcripts of MCP-1, MCP-2, MIP-1, MIP-1ß, and RANTES returned to control levels by day 5 after CTX injection in MLC/mIGF-1 muscle, whereas the expression of these CC chemokines was maintained at higher levels in wild-type injured muscle for an extended period of time (Fig. 2C
).
Chemotactic signals modulated by mIGF-1
To relate RNA expression levels to their corresponding proteins, we used a reliable and very sensitive approach that detects multiple proteins in an antibody-based protein microarray (RayBiotech) (20)
. We analyzed 40 different cyto-chemokines in both wild-type and MLC/mIGF-1 transgenic injured muscle. Figure 3
A shows the expression profile of cytokines that are modulated at 5 days postinjury in both wild-type and MLC/mIGF-1 transgenic mice. Densitometric analysis validated the results obtained by multiplex RT-polymerase chain reaction (RT-PCR) analysis, revealing a significant reduction in TNF-, IL-1ß, MCP-1, and MIP-1 protein expression in the MLC/mIGF-1 injured muscle compared to the wild-type counterpart (Fig. 3A
). In addition, the expression of macrophage colony stimulating factor (M-CSF), a cytokine that regulates the survival, proliferation, and chemotaxis of macrophages (27)
, was significantly reduced in MLC/mIGF-1 injured muscle at 5 days post-CTX injection (Fig. 3A
).
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mIGF-1 modulates molecular mechanisms involved in the inflammatory response
Persistence of inflammation occurs when cell recruitment or proliferation is ongoing and emigration and death are inhibited (7)
. One cytokine gaining recognition for its importance in inflammation is macrophage migration inhibitory factor (MIF) (13)
. Western blots analysis (Fig. 3B
) revealed that MIF accumulated in wild-type muscle at 2 days postinjury and persisted until 5 days post CTX injection, whereas its expression remained at baseline levels in MLC/mIGF-1 transgenic injured muscle during the time course of regeneration (Fig. 3B
). In addition, immunoblot analysis (Fig. 3B
) revealed that high-mobility group protein 1 (HMGB1) expression was increased within 2 days of CTX injection in the MLC/mIGF-1 muscle and returned to basal levels within 10 days postinjury (Fig. 3B
), confirming the histological evidences that mIGF-1 accelerates the repair process. In contrast, in the wild-type damaged muscle HMGB1 expression significantly rose at 2 days postinjury and persisted over 10 days of CTX injection (Fig. 3B
).
NF-B (NFB) is one of the central players of the inflammatory system (14
15
16
, 28)
. Inactive NF-B is present within the cytoplasm bound to its inhibitory protein Ba (IB). Proinflammatory cytokines induce the phosphorylation of IB with subsequent degradation, resulting in the activation of NF-B (28)
. Western blot analysis (Fig. 3B
) did not reveal significant differences in the phospho-IB expression between wild-type and MLC/mIGF-1 injured muscle at 2 days post-CTX injection; however phospho-IB was dramatically down-modulated in MLC/mIGF-1 injured muscle at 5 days post-CTX injection, whereas its phosphorylation persisted for an extended period of time in wild-type muscle (Fig. 3B
), suggesting a constant activation of NF-kB in the extended inflammatory response.
Remodeling of connective tissue in regenerating mIGF-1 transgenic muscle
The full reconstruction of muscle tissue undergoing regeneration is frequently hindered by the formation of scar tissue and by activation of myofibroblasts, leading to ECM generation (2
, 17)
. Significant differences in the expression of -SMA, a molecular marker of activated myofibroblasts, were observed between wild-type and MLC/mIGF-1 injured muscle at 10 days post-CTX injection (Fig. 4
A). Analysis of the -SMA-positive area revealed that the transgenic injured muscle was largely restored compared to wild-type muscle, with a significant reduction (60%) in the accumulation of activated myofibroblasts (Fig. 4A
, right). Myofibroblasts are responsible for fibrous tissue formation, expressed principally as type I (Col1a1) and III (Col3a1) fibrillar collagens. Real-time PCR revealed that collagen I and III expression was up-regulated at 2 days post-CTX injection and returned to basal levels within 10 days postinjury in MLC/mIGF-1 transgenic muscle (Fig. 4B
). In contrast, in wild-type injured muscle, collagen I and III expression remained prominently up-regulated over 10 days post CTX injection (Fig. 4B
).
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Myofibroblast activation is regulated by transforming growth factor beta1 (TGF-ß1) and an ECM component, specifically the ED-A(EIIIA) domain of fibronectin, which is necessary for TGF-ß to trigger -SMA expression (29)
. Real-time PCR analysis revealed that EIIIA expression was up-regulated in MLC/mIGF-1 injured muscle at 2 days post-CTX injection, with a peak of expression within 5 days, and returned to basal levels within 10 days postinjury (Fig. 4C
). In contrast, wild-type injured muscle up-regulated EIIIA expression at 2 days post-CTX injection (Fig. 4C
); its expression constantly increased during the time course of regeneration, remaining at high levels over 10 days postinjury (Fig. 4C
and data not shown).
Transgenic mIGF-1 expression improves the recovery of muscle strength after injury
The full recovery of muscle strength after injury is also hampered by the persistence of inflammatory response and the development of fibrosis (30)
. To evaluate the restoration of performance in damaged muscle conferred by mIGF-1, we analyzed functional parameters of 2-mo-old wild-type and MLC/mIGF-1 mice 15 days after direct cardiotoxin (CTX) injection. No significant differences were observed in twitch force, time to peak, and relaxation time between wild-type and transgenic mice in both uninjured and injured muscle (Table 1
). In contrast, tetanic force measurements of EDL muscle (Fig. 5
A) demonstrated that at 15 days postinjury, mIGF-1 transgenic muscle recovered the 85% of its maximum force, compared to wild-type muscle, which covered only 65% of its maximum force. Notably, specific force decreased in both wild-type and MLC/mIGF-1 transgenic EDL muscle after CTX injection (Fig. 5B
). The increase in muscle strength in injured MLC/mIGF-1 transgenic muscle suggests that in addition to the improvement of muscle regeneration and the modulation of inflammatory cytokines, mIGF-1 expression also modulates connective remodeling and accelerates the functional recovery of injured muscle.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A balance must therefore be struck between excessive and insufficient inflammatory action. From a therapeutic standpoint, while it is often difficult to intervene in primary pathogenetic events, restriction of subsequent inflammation may be the next best option (14)
.
The results of the present study suggest new therapeutic avenues by identifying a potential temporal window to modulate the inflammatory response in skeletal muscle subject to trauma or injury. The interaction between mIGF-1 and the molecular players of the inflammatory response is important in determining the course of muscle injury, repair, and remodeling. Early phases of the inflammatory response are not affected by mIGF-1 expression, indicating that mIGF-1 does not alter or block the activation of inflammatory response on injury, a critical component of muscle physiology that represents an important phase in the regenerative process. Rather, enhanced regenerative capacity of MLC/mIGF-1 mice correlates with a dampening of the later stages of inflammation, such that necrosis and prolonged infiltration by monocytes/macrophages typical of wild-type injured muscle is more rapidly resolved in the presence of mIGF-1. In this regard, local expression of mIGF-1 selectively down-regulated the proinflammatory cytokines, such as IL1, TNF-, ENA-78, and the CC chemokines at 5 days postinjury, when the persistence of cytokine and chemokine expression is associated with sustained mononuclear cell influx and therefore in the switch from acute to chronic inflammatory process (7)
.
Acceleration of the inflammatory choreography by mIGF-1 is well illustrated by its regulation of MIF expression, a cytokine involved in the production of proinflammatory molecules, including TNF-, IL-1ß, and MIP-2, and that plays a critical role in the emigration/retention of macrophages in injured tissue (13)
. In wild-type injured muscle, MIF remained expressed at higher levels for an extended period of time. In contrast, the significant down-regulation of MIF at 5 days post-CTX injection in MLC/mIGF-1 injured muscle may facilitate the emigration of infiltrating cell pools, leading to a rapid resolution of the inflammatory response. This was also supported by the repression of other molecular players involved in the activation and maintenance of the inflammatory response, such as NF-B and HMGB1, a potent proinflammatory cytokine that acts as a late modulator of inflammatory response and can also serve as a signal of tissue damage (14
15
16)
.
The persistence of the inflammatory response is often associated with severe and progressive fibrosis (17)
, with a direct role of CC chemokines in the activation of myofibroblasts, which express -SMA as a hallmark of tissue scarring and fibrosis (33)
. The significant reduction in -SMA expression and type I and III collagen at 10 days after damage of MLC/mIGF-1 muscle may be related to a rapid down-regulation of the fibronectin ED-A domain, which is necessary for TGF-ß to trigger -SMA expression by myofibroblasts (29)
. The positive effects of mIGF-1 on muscle regeneration and connective remodeling are also supported by a recent report in which expression of IGF-1 reduces fibrogenesis and enhances regeneration after liver injury (34)
. In the setting of heart failure, involvement of elevated myocardial IGF-1 may similarly act to limit cardiac atrophy and apoptosis during reverse remodeling and to promote repair and regeneration (35)
.
Maintaining a balance between inflammation and subsequent connective remodeling is also of particular relevance to the treatment of several muscle diseases. The accelerated resolution of the inflammatory response in mIGF-1 transgenic mice may underlie the dramatic maintenance of muscle homeostasis during exercise and aging (8)
and the resistance to muscle decline in models of ALS (11)
and muscular dystrophy (7
, 32)
in which virtually all skeletal muscles succumb to fibrosis (10)
. Our studies are also relevant to stem cell applications, in which scar tissue inevitably forms a barrier to repopulation by implanted cells. Modulating the host tissue’s fibrotic response to cell implantation is therefore a critical component of future success in cell therapies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication November 6, 2006. Accepted for publication December 7, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B inhibition for the treatment of human disease. Cell. Cycle 5,1160-1163[Medline]This article has been cited by other articles:
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relaxation time in wild-type and transgenic mice in both uninjured and injured muscle