David W. Hammers

Controlled release of IGF‐I from a biodegradable matrix improves functional recovery of skeletal muscle from ischemia/reperfusion

Ischemia/reperfusion (I/R) injury is a considerable insult to skeletal muscle, often resulting in prolonged functional deficits. The purpose of the current study was to evaluate the controlled release of the pro‐regenerative growth factor, insulin‐like growth factor‐I (IGF‐I), from a biodegradable polyethylene glycol (PEG)ylated fibrin gel matrix and the subsequent recovery of skeletal muscle from I/R. To accomplish this, the hind limbs of male Sprague–Dawley rats were subjected to 2‐h tourniquet‐induced I/R then treated with saline, bolus IGF‐I (bIGF), PEGylated fibrin gel (PEG‐Fib), or IGF‐I conjugated PEGylated fibrin gel (PEG‐Fib‐IGF). Functional and histological evaluations were performed following 14 days of reperfusion, and muscles from 4‐day reperfusion animals were analyzed by Western blotting and histological assessments. There was no difference in functional recovery between saline, bIGF, or PEG‐Fib groups. However, PEG‐Fib‐IGF treatment resulted in significant improvement of muscle function and structure, as observed histologically. Activation of the PI3K/Akt pathway was significantly elevated in PEG‐Fib‐IGF muscles, compared to PEG‐Fib treatment, at 4 days of reperfusion, suggesting involvement of the pathway PI3K/Akt as a mediator of the improved function. Surprisingly, myoblast activity was not evident as a result of PEG‐Fib‐IGF treatment. Taken together, these data give evidence for a protective role for the delivered IGF. These results indicate that PEG‐Fib‐IGF is a viable therapeutic technique in the treatment of skeletal muscle I/R injury. Biotechnol. Bioeng. 2012; 109:1051–1059.

Functional deficits and insulin-like growth factor-I gene expression following tourniquet-induced injury of skeletal muscle in young and old rats

This study investigated the effect of age on recovery of skeletal muscle from an ischemia-reperfusion (I/R)-induced injury. Young (6 mo old) and old (24-27 mo old) Sprague-Dawley rats underwent a 2-h bout of hindlimb ischemia induced by a pneumatic tourniquet (TK). The TK was released to allow reperfusion of the affected limb, and animals were divided into 7- and 14-day recovery groups. Maximum plantar flexor force production was assessed in both 7- and 14-day recovery groups of both ages, followed by histological evaluation. Subsequent analysis of IGF-I gene expression and intracellular signaling in 7-day recovery muscles was performed by RT-PCR and Western blotting, respectively. Old rats had significantly greater deficits in force production and exhibited more evidence of histological pathology than young at both 7 and 14 days postinjury. In addition, old rats demonstrated an attenuated upregulation of IGF-I mRNA and induction of proanabolic signaling compared with young in response to injury. We conclude that aged skeletal muscle exhibits more damage and/or defective regeneration following I/R and identify an age-associated decrease in local IGF-I responsiveness as a potential mechanism for this phenomenon.

Impairment of IGF-I expression and anabolic signaling following ischemia/reperfusion in skeletal muscle of old mice.

With the advancement of age, skeletal muscle undergoes a progressive decline in mass, function, and regenerative capacity. Previously, our laboratory has reported an age-reduction in recovery and local induction of IGF-I gene expression with age following tourniquet (TK)-induced skeletal muscle ischemia/reperfusion (I/R). In this study, young (6 mo) and old (24-28 mo) mice were subjected to 2h of TK-induced ischemia of the hindlimb followed by 1, 3, 5, or 7 days of reperfusion. Real time-PCR analysis revealed clear age-related reductions and temporal alterations in the expression of IGF-I and individual IGF-I Ea and Eb splice variants. ELISA verified a reduction of IGF-I peptide with age following 7 day recovery from TK. Western blotting showed that the phosphorylation of Akt, mTOR, and FoxO3, all indicators of anabolic activity, were reduced in the muscles of old mice. These data indicate that an age-related impairment of IGF-I expression and intracellular signaling does exist following injury, and potentially has a role in the impaired recovery of skeletal muscle with age.

Anti-inflammatory macrophages improve skeletal muscle recovery from ischemia-reperfusion

The presence of macrophages (MPs) is essential for skeletal muscle to properly regenerate following injury. The aim of this study was the evaluation of MP profiles and their importance in skeletal muscle recovering from tourniquet-induced ischemia-reperfusion (I/R). Using flow cytometry, we identified two distinct CD11b(+) MP populations that differ in expression of the surface markers Ly-6C and F4/80. These populations are prominent at 3 and 5 days of reperfusion and molecularly correspond to inflammatory and anti-inflammatory MP phenotypes. Sorted MP populations demonstrated high levels of IGF-I expression, and whole muscle post-I/R IGF-I expression strongly correlates with F4/80 expression. This suggests MPs largely influence postinjury IGF-I upregulation. We additionally demonstrate that direct intramuscular injection of FACS-isolated CD11b(+)Ly-6C(lo)F4/80(hi) MPs improves the functional and histological recovery of I/R-affected muscle. Taken together, these data further support the substantial influence of the innate immune system on muscle regeneration and suggest MP-focused therapeutic approaches may greatly facilitate skeletal muscle recovery from substantial injury.

Controlled delivery of SDF-1α and IGF-1: CXCR4+ cell recruitment and functional skeletal muscle recovery

Therapeutic delivery of regeneration-promoting biological factors directly to the site of injury has demonstrated its efficacy in various injury models. Several reports describe improved tissue regeneration following local injection of tissue specific growth factors, cytokines and chemokines. Evidence exists that combined cytokine/growth factor treatment is superior for optimizing tissue repair by targeting different aspects of the regeneration response. The purpose of this study was to evaluate the therapeutic potential of the controlled delivery of stromal cell-derived factor-1alpha (SDF-1α) alone or in combination with insulin-like growth factor-I (SDF-1α/IGF-I) for the treatment of tourniquet-induced ischemia/reperfusion injury (TK-I/R) of skeletal muscle. We hypothesized that SDF-1α will promote sustained stem cell recruitment to the site of muscle injury, while IGF-I will induce progenitor cell differentiation to effectively restore muscle contractile function after TK-I/R injury while concurrently reducing apoptosis. Utilizing a novel poly-ethylene glycol PEGylated fibrin gel matrix (PEG-Fib), we incorporated SDF-1α alone (PEG-Fib/SDF-1α) or in combination with IGF-I (PEG-Fib/SDF-1α/IGF-I) for controlled release at the site of acute muscle injury. Despite enhanced cell recruitment and revascularization of the regenerating muscle after SDF-1α treatment, functional analysis showed no benefit from PEG-Fib/SDF-1α therapy, while dual delivery of PEG-Fib/SDF-1α/IGF-I resulted in IGF-I-mediated improvement of maximal force recovery and SDF-1α-driven in vivo neovasculogenesis. Histological data supported functional data, as well as highlighted the important differences in the regeneration process among treatment groups. This study provides evidence that while revascularization may be necessary for maximizing muscle force recovery, without modulation of other effects of inflammation it is insufficient.