Xiaowu Wu

The promotion of a functional fibrosis in skeletal muscle with volumetric muscle loss injury following the transplantation of muscle-ECM

Tissue engineering strategies that primarily use biological extracellular matrices (ECMs) with or without the inclusion of a stem or progenitor cell source are under development for the treatment of trauma resulting in the loss of a large volume of skeletal muscle (i.e., volumetric muscle loss; VML). The explicit goal is to restore functional capacity to the injured tissue by promoting generation of muscle fibers. In the current study, a syngeneic muscle-derived ECM (mECM) was transplanted in a rat tibialis anterior (TA) muscle VML model. Instead of muscle fiber generation a large fibrotic mass was produced by mECM transplantation out to six months post-injury. Surprisingly, recovery of one-third of the original functional deficit was still achieved by two months post-injury following mECM transplantation. These counterintuitive findings may be due, at least in part, to the ability of mECM to attenuate muscle damage in the remaining muscle as compared to non-repaired muscle. These findings point to a novel role of biological ECMs for the treatment of VML, wherein the remaining muscle mass is protected from prolonged overload injury.

A novel significance score for gene selection and ranking.

MOTIVATION When identifying differentially expressed (DE) genes from high-throughput gene expression measurements, we would like to take both statistical significance (such as P-value) and biological relevance (such as fold change) into consideration. In gene set enrichment analysis (GSEA), a score that can combine fold change and P-value together is needed for better gene ranking. RESULTS We defined a gene significance score π-value by combining expression fold change and statistical significance (P-value), and explored its statistical properties. When compared to various existing methods, π-value based approach is more robust in selecting DE genes, with the largest area under curve in its receiver operating characteristic curve. We applied π-value to GSEA and found it comparable to P-value and t-statistic based methods, with added protection against false discovery in certain situations. Finally, in a gene functional study of breast cancer profiles, we showed that using π-value helps elucidating otherwise overlooked important biological functions. AVAILABILITY http://gccri.uthscsa.edu/Pi_Value_Supplementary.asp CONTACT xy@ieee.org, cheny8@uthscsa.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

A Standardized Rat Model of Volumetric Muscle Loss Injury for the Development of Tissue Engineering Therapies

Abstract Soft tissue injuries involving volumetric muscle loss (VML) are defined as the traumatic or surgical loss of skeletal muscle with resultant functional impairment and represent a challenging clinical problem for both military and civilian medicine. In response, a variety of tissue engineering and regenerative medicine treatments are under preclinical development. A wide variety of animal models are being used, all with critical limitations. The objective of this study was to develop a model of VML that was reproducible and technically uncomplicated to provide a standardized platform for the development of tissue engineering and regenerative medicine solutions to VML repair. A rat model of VML involving excision of ∼20% of the muscles mass from the superficial portion of the middle third of the tibialis anterior (TA) muscle was developed and was functionally characterized. The contralateral TA muscle served as the uninjured control. Additionally, uninjured age-matched control rats were also tested to determine the effect of VML on the contralateral limb. TA muscles were assessed at 2 and 4 months postinjury. VML muscles weighed 22.7% and 19.5% less than contralateral muscles at 2 and 4 months postinjury, respectively. These differences were accompanied by a reduction in peak isometric tetanic force (Po) of 28.4% and 32.5% at 2 and 4 months. Importantly, Po corrected for differences in body weight and muscle wet weights were similar between contralateral and age-matched control muscles, indicating that VML did not have a significant impact on the contralateral limb. Lastly, repair of the injury with a biological scaffold resulted in rapid vascularization and integration with the wound. The technical simplicity, reliability, and clinical relevance of the VML model developed in this study make it ideal as a standard model for the development of tissue engineering solutions for VML.

Growth hormone down-regulation of Interleukin-1beta and Interleukin-6 induced acute phase protein gene expression is associated with increased gene expression of suppressor of cytokine signal-3.

Severe burn induces the hepatic acute phase response. We previously showed that recombinant human growth hormone (GH) treatment after burn down-regulated acute phase protein (APP) production and gene expression in vivo. In this study, we hypothesized that the inhibitory effect of GH on the hepatic acute phase response was due to increased suppressor of cytokine signaling (SOCS) gene expression. HepG2 cells were treated with Interleukin-1&bgr; (IL-1&bgr;; 2 ng/mL) and interleukin 6 (IL-6; 20 ng/mL) alone or combined with GH (2 &mgr;g/mL) for 15 and 30 min, and 1, 2, and 4 h. The levels of gene expression for &agr;1-acid glycoprotein (AGP), &agr;1-antitrypsin (ATT), and SOCS (CIS, SOCS-1, 2, and 3) were measured by reverse transcript-polymerase chain reaction (RT-PCR). APP levels in the supernatant were determined by enzyme-linked immunosorbent sandwich assay (ELISA). The gene expression of AGP and ATT were also measured in HepG2 cells transfected with pEF-Flag-I/mSOCS-3 plasmid after IL-1&bgr; or IL-6 treatment. Data are expressed as means ± SEM, and statistical analysis was performed by one- or two-way analysis of variance. IL-1&bgr; and IL-6 induced AGP and ATT gene expression and protein production, respectively, which was down-regulated by GH treatment. SOCS-3 but not CIS, SOCS-1, or SOCS-2 gene expression was significantly increased by GH treatment. APP gene expression was significantly decreased in cells transfected with plasmid over expressing SOCS-3 after IL-6 and IL-1&bgr; treatment. GH attenuates IL-1&bgr; or IL-6 induced APP gene expression, which is associated with increased expression of SOCS-3. This study suggests that SOCS-3 plays an important role in the suppression of cytokine signaling by GH in down-regulating the acute phase response after injury.

Skeletal muscle satellite cell activation following cutaneous burn in rats

BACKGROUND Cutaneous burn distant from skeletal muscles induces atrophy; however, its effect on muscle stem cells resident in skeletal muscle (satellite cells) distal to burn is not known. METHODS Satellite cell activation was measured in predominantly fast-twitch [tibialis anterior, extensor digitorum longus (EDL), plantaris, and gastrocnemius] and slow-twitch (soleus) muscles of rats that received either 40% total body surface area full-thickness scald burn or sham burn to the trunk area by determining bromodeoxyuridine incorporation, MyoD, and Pax7 immunohistochemistry in vivo ≤48 h after burn. To determine the effects of circulating factors on satellite cell activation, satellite cell cultures were treated with serum from sham or burn rats. RESULTS In vivo activation of satellite cells was increased in fast muscles isolated from burn as compared to sham animals, whereas a significant response was not seen in slow muscles. Serum taken from animals in the burn group increased the activation of satellite cells isolated from both sham and burn animals in vitro, suggesting that circulating factors have the potential to increase satellite cell activation following burn. CONCLUSIONS Increases in satellite cell activation in muscles distal to burn are fiber-type-dependent, and circulating factors may play a role in the activation of satellite cells following burn. A better understanding of the impact of burn on satellite cell functionality will allow us to identify the cellular mechanisms of long-term muscle atrophy.

The impact of muscle disuse on muscle atrophy in severely burned rats.

BACKGROUND Severe burn induces a sustained hypermetabolic response, which causes long-term loss of muscle mass and decrease in muscle strength. In this study, we sought to determine whether muscle disuse has additional impact on muscle atrophy after severe burn using a rat model combining severe cutaneous burn and hindlimb unloading. METHODS Forty Sprague-Dawley rats (≈ 300 g) were randomly assigned to sham ambulatory (S/A), sham hindlimb unloading (S/HLU), burn ambulatory (B/A), or burn hindlimb unloading (B/HLU) groups. Rats received a 40% total body surface (TBSA) full thickness scald burn, and rats with hindlimb unloading were placed in a tail traction system. At d 14, lean body mass (LBM) was determined using DEXA scan, followed by measurement of the isometric mechanical properties in the predominantly fast-twitch plantaris muscle (PL) and the predominantly slow-twitch soleus muscle (SL). Muscle weight (wt), protein wt, and wet/dry wt were determined. RESULTS At d 14, body weight had decreased significantly in all treatment groups; B/HLU resulted in significantly greater loss compared with the B/A, S/HLU, and S/A. The losses could be attributed to loss of LBM. PL muscle wt and Po were lowest in the B/HLU group (<0.05 versus S/A, S/HLU, or B/A). SL muscle wt and Po were significantly less in both S/HLU and B/HLU compared with that of S/A and B/A; no significant difference was found between S/HLU and B/HLU. CONCLUSIONS Cutaneous burn and hindlimb unloading have an additive effect on muscle atrophy, characterized by loss of muscle mass and decrease in muscle strength in both fast (PL) and slow (SL) twitch muscles. Of the two, disuse appeared to be the dominant factor for continuous muscle wasting after acute burn in this model.

Muscle contractile properties in severely burned rats.

Burn induces a sustained catabolic response which causes massive loss of muscle mass after injury. A better understanding of the dynamics of muscle wasting and its impact on muscle function is necessary for the development of effective treatments. Male Sprague-Dawley rats underwent either a 40% total body surface area (TBSA) scald burn or sham burn, and were further assigned to subgroups at four time points after injury (days 3, 7, 14 and 21). In situ isometric contractile properties were measured including twitch tension (Pt), tetanic tension (Po) and fatigue properties. Body weight decreased in burn and sham groups through day 3, however, body weight in the sham groups recovered and increased over time compared to burned groups, which progressively decreased until day 21 after injury. Significant differences in muscle wet weight and protein weight were found between sham and burn. Significant differences in muscle contractile properties were found at day 14 with lower absolute Po as well as specific Po in burned rats compared to sham. After burn, the muscle twitch tension was significantly higher than the sham at day 21. No significant difference in fatigue properties was found between the groups. This study demonstrates dynamics of muscle atrophy and muscle contractile properties after severe burn; this understanding will aid in the development of approaches designed to reduce the rate and extent of burn induced muscle loss and function.

Growth hormone effects on hypertrophic scar formation: A randomized controlled trial of 62 burned children

The hypercatabolism after massive pediatric burns has been effectively treated with recombinant human growth hormone, an anabolic agent that stimulates protein synthesis and abrogates growth arrest. While experimental studies have shown increased potential for fibrosis induced by growth hormone therapy, adverse effects on human scars have not been investigated. Our aim was to evaluate hypertrophic scar formation in 62 patients randomized to receive injections of 0.05 mg/kg/day of recombinant human growth hormone or placebo, from discharge until 1 year after burn. Scar scales were used to evaluate scar‐severity at discharge, 6, 9, 12, and 18–24 months after burn, by three observers blinded to treatment. Computer‐assisted planimetry allowed quantification of percentage of hypertrophic scar formation. Types I and III collagens were localized and quantified in scars and normal skin of patients from both groups, using immunohistochemistry with confocal laser microscopy analysis. Insulin‐like growth factor‐1 blood levels helped assess compliance. Statistical analysis showed that scar hypertrophy significantly increased from 6 to 12 months after injury in both groups, while decreasing at 18–24 months postburn. Types I and III collagens were statistically increased in the reticular layer of scars from both groups when compared to paired normal skin. Insulin‐like growth factor‐1 was significantly increased in the recombinant human growth factor‐treated group. No differences were seen when recombinant human growth factor and control groups were compared using the scar scales, planimetry, or immunohistochemistry. We concluded that recombinant human growth hormone therapy did not adversely affect scar formation and should not contraindicate the administration of recombinant human growth hormone as a therapeutic approach to severely burned children.

Insulin effects on glucose tolerance, hypermetabolic response, and circadian-metabolic protein expression in a rat burn and disuse model

Insulin controls hyperglycemia after severe burns, and its use opposes the hypermetabolic response. The underlying molecular mechanisms are poorly understood, and previous research in this area has been limited because of the inadequacy of animal models to mimic the physiological effects seen in humans with burns. Using a recently published rat model that combines both burn and disuse components, we compare the effects of insulin treatment vs. vehicle on glucose tolerance, hypermetabolic response, muscle loss, and circadian-metabolic protein expression after burns. Male Sprague-Dawley rats were assigned to three groups: cage controls (n = 6); vehicle-treated burn and hindlimb unloading (VBH; n = 11), and insulin-treated burn and hindlimb unloading (IBH; n = 9). With the exception of cage controls, rats underwent a 40% total body surface area burn with hindlimb unloading, then IBH rats received 12 days of subcutaneous insulin injections (5 units·kg(-1)·day(-1)), and VBH rats received an equivalent dose of vehicle. Glucose tolerance testing was performed on day 14, after which blood and tissues were collected for analysis. Body mass loss was attenuated by insulin treatment (VBH = 265 ± 17 g vs. IBH = 283 ± 14 g, P = 0.016), and glucose clearance capacity was increased. Soleus and gastrocnemius muscle loss was decreased in the IBH group. Insulin receptor substrate-1, AKT, FOXO-1, caspase-3, and PER1 phosphorylation was altered by injury and disuse, with levels restored by insulin treatment in almost all cases. Insulin treatment after burn and during disuse attenuated the hypermetabolic response, increased glucose clearance, and normalized circadian-metabolic protein expression patterns. Therapies aimed at targeting downstream effectors may provide the beneficial effects of insulin without hypoglycemic risk.

193: SEVERE BURN AND DISUSE EFFECTS ON MUSCLE AND BONE STRENGTH IN RATS OVER TIME

observed between males and females in the %EMGmax measured for the scalenus muscle (p < 0.05). However, when PImax was used as the standard, there is no significant difference observed. For the sternocleidomastoid muscle (STERNO), changing the evaluation standard had no influence on measurements observed. For the trapezius muscle, there was a significant difference (p < 0.05) observed at comparatively high points of inspiratory resistance load. When PImax was the standard, the value of STERNO showed high correlation, rs = 0.77 for males and rs = 0.85 for females (p < 0.001). Conclusions: For evaluating accessory muscles during labored inspiration, STERNO is least influenced by changes in evaluation standards. This study demonstrated that evaluation of labored inspiration during mechanical ventilation is possible using PImax as an evaluation standard.