Development of a Rat Model of Fasciotomy Treatment for Compartment Syndrome.

Skeletal muscle injuries are a major cause of disability for military and civilian populations. Compartment syndrome (CS) in skeletal muscle results from an edema-induced increase in intracompartmental pressure (ICP) after primary injury. Untreated ICP will occlude the tissue vasculature, tissue necrosis, and potential loss of limb. The current standard of care for CS is surgical fasciotomy, an incision through the muscle fascia to relieve ICP. Early fasciotomy will preserve the limb, but often leaves patients with long-term scarring and reduced muscle function. Our group previously developed and characterized a rat model of CS to explore the pathophysiology of CS and test new therapies. We present an expansion of this CS model, including the fasciotomy, to better simulate clinical treatment. CS was induced on the hind limb of adult male Lewis rats and fasciotomy was performed 24 h later. Less than 20% of the rats that underwent fasciotomy showed detectable force 4 days after injury, compared with the 75% of rats that underwent CS induction without fasciotomy. Muscles undergoing fasciotomy showed a significant increase in fibrosis and an increased number of macrophages, Pax7+ satellite cells, and α-smooth muscle actin+ myofibroblasts at 7 days postinjury. These data indicate that the use of fasciotomy in a rat model of CS resulted in injury sequelae that reflect the severity of human clinical disease presentation along with current standard of care. Impact Statement Current animal models of skeletal muscle injury struggle to accurately reflect the injury sequelae seen in humans, particularly in rats and mice. These animals also recover faster than humans do. More accurate recapitulation of the injury is needed to better study the injury progression, as well as screen for novel therapies. This research combines an existing model of compartment syndrome with its clinical standard of care (fasciotomy), creating a more accurate rat model of injury, and providing for a better treatment screening tool. These results show how our model leads to a sustained skeletal muscle deficit with increased inflammation.

The Use of Pulsed Electromagnetic Field to Modulate Inflammation and Improve Tissue Regeneration: A Review.

Pulsed electromagnetic field (PEMF) is emerging as innovative treatment for regulation of inflammation, which could have significant effects on tissue regeneration. PEMF modulates inflammatory processes through the regulation of pro- and anti-inflammatory cytokine secretion during different stages of inflammatory response. Consistent outcomes in studies involving animal and human tissue have shown promise for the use of PEMF as an alternative or complementary treatment to pharmaceutical therapies. Thus, PEMF treatment could provide a novel nonpharmaceutical means of modulating inflammation in injured tissues resulting in enhanced functional recovery. This review examines the effect of PEMF on immunomodulatory cells (e.g., mesenchymal stem/stromal cells [MSCs] and macrophages [MΦ]) to better understand the potential for PEMF therapy to modulate inflammatory signaling pathways and improve tissue regeneration. This review cites published data that support the use of PEMF to improve tissue regeneration. Our studies included herein confirm anti-inflammatory effects of PEMF on MSCs and MΦ.

A real-time monitoring platform of myogenesis regulators using double fluorescent labeling.

Real-time, quantitative measurement of muscle progenitor cell (myoblast) differentiation is an important tool for skeletal muscle research and identification of drugs that support skeletal muscle regeneration. While most quantitative tools rely on sacrificial approach, we developed a double fluorescent tagging approach, which allows for dynamic monitoring of myoblast differentiation through assessment of fusion index and nuclei count. Fluorescent tagging of both the cell cytoplasm and nucleus enables monitoring of cell fusion and the formation of new myotube fibers, similar to immunostaining results. This labeling approach allowed monitoring the effects of Myf5 overexpression, TNFα, and Wnt agonist on myoblast differentiation. It also enabled testing the effects of surface coating on the fusion levels of scaffold-seeded myoblasts. The double fluorescent labeling of myoblasts is a promising technique to visualize even minor changes in myogenesis of myoblasts in order to support applications such as tissue engineering and drug screening.

The role of endothelial cells in myofiber differentiation and the vascularization and innervation of bioengineered muscle tissue in vivo.

Musculoskeletal disorders are a major cause of disability and effective treatments are currently lacking. Tissue engineering affords the possibility of new therapies utilizing cells and biomaterials for the recovery of muscle volume and function. A major consideration in skeletal muscle engineering is the integration of a functional vasculature within the regenerating tissue. In this study we employed fluorescent cell labels to track the location and differentiation of co-cultured cells in vivo and in vitro. We first utilized a co-culture of fluorescently labeled endothelial cells (ECs) and muscle progenitor cells (MPCs) to investigate the ability of ECs to enhance muscle tissue formation and vascularization in an in vivo model of bioengineered muscle. Scaffolds that had been seeded with both MPCs and ECs showed significantly greater vascularization, tissue formation and enhanced innervation as compared to scaffolds seeded with MPCs alone. Subsequently, we performed in vitro experiments using a 3-cell type system (ECs, MPCs, and pericytes (PCs)) to demonstrate the utility of fluorescent cell labeling for monitoring cell growth and differentiation. The growth and differentiation of individual cell types was determined using live cell fluorescent microscopy demonstrating the utility of fluorescent labels to monitor tissue organization in real time.

Compression-induced muscle injury in rats that mimics compartment syndrome in humans.

Compartment syndrome (CS) is a serious complication arising from a variety of extremity injuries and resultant swelling within the fascicles of the muscle tissue. The current standard of care for CS is fasciotomy, which relieves the intracompartmental pressure of CS but inflicts further tissue damage. The development of new techniques to treat CS include angiogenic therapy, antifibrosis treatments, and stem cell therapy, all which aim to enhance tissue regeneration and functional recovery. Current rodent models of skeletal muscle injury do not accurately mimic the complex physiological tissue damage found in CS in human patients, and large-animal models of CS cannot be used as an experimental model of human cell therapy because of the lack of immunocompromised animals. We developed a rat model of CS that mimics the sequelae of the human condition. Compression of the hindlimb of rats using neonatal blood pressure cuffs maintaining 120 to 140 mmHg for 3 hours resulted in considerable muscular, vascular, and neural damage. Histological and functional analyses documented the initial degeneration and subsequent regeneration of the muscle tissue over time. The complex muscular, vascular, and neural injury observed in this model provides an ideal platform for testing cellular, biological, and pharmacological agents for the restoration of muscle volume and function.

Design of isoform-selective phospholipase D inhibitors that modulate cancer cell invasiveness.

Phospholipase D (PLD) is an essential enzyme responsible for the production of the lipid second messenger phosphatidic acid. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase signal transduction networks. The lack of potent and isoform-selective inhibitors has limited progress in defining the cellular roles of PLD. We used a diversity-oriented synthetic approach and developed a library of PLD inhibitors with considerable pharmacological characterization. Here we report the rigorous evaluation of that library, which contains highly potent inhibitors, including the first isoform-selective PLD inhibitors. Specific members of this series inhibit isoforms with >100-fold selectivity both in vitro and in cells. A subset of inhibitors was shown to block invasiveness in metastatic breast cancer models. These findings demonstrate the power of diversity-oriented synthesis combined with biochemical assays and mass spectrometric lipid profiling of cellular responses to develop the first isoform-selective PLD inhibitors--a new class of antimetastatic agents.

Knockdown of the transforming growth factor-beta type III receptor impairs motility and invasion of metastatic cancer cells.

The transforming growth factor-beta (TGF-beta) signaling pathway plays dual roles in epithelial cell tumorigenesis. TGF-beta is initially growth inhibitory, but as tumorigenesis progresses, TGF-beta becomes prometastatic. Although the role of the types I and II TGF-beta receptors is fairly well established, the role of the ubiquitously expressed TGF-beta type III receptor (TbetaRIII) in tumorigenesis is less defined. To examine the role of TbetaRIII in breast cancer cells, we stably expressed short hairpin RNAs specific to TbetaRIII in MDA-231 human breast cancer cells and mouse mammary carcinoma cells expressing the polyomavirus middle T oncogene (PMTLuc). MDA-231 and PMTLuc cells with down-regulated TbetaRIII expression (231-kd; PMTLuc-kd) exhibited decreased growth rate, motility, and invasion into Matrigel, as well as an increase in apoptosis, compared with control cells. MDA-231 xenografts established in nude mice metastasized, whereas tumors made by 231-kd cells did not. Nuclear factor-kappaB (NF-kappaB) activity, which is known to regulate cell growth and motility, was lower in the MDA-231 and PMTLuc knockdown cells compared with control cells. Transfection of an expression vector encoding constitutively active IKK2 into the 231-kd cells restored the ability of TbetaRIII-deficient cells to invade Matrigel and decreased their basal level of apoptosis. These data indicate that TbetaRIII differentially regulates cell growth, motility, and invasion in tumorigenic MDA-231 and PMTLuc cells and that these growth changes occur through the modulation of NF-kappaB activity.

Modulation of NFkappaB activity and E-cadherin by the type III transforming growth factor beta receptor regulates cell growth and motility.

Transforming growth factor beta is growth-inhibitory in non-transformed epithelial cells but becomes growth-promoting during tumorigenesis. The role of the type I and II receptors in tumorigenesis has been extensively studied, but the role of the ubiquitously expressed type III receptor (TbetaRIII) remains elusive. We developed short hairpin RNAs directed against TbetaRIII to investigate the role of this receptor in breast cancer tumorigenesis. Nontumorigenic NMuMG mouse cells stably expressing short hairpin RNA specific to mouse TbetaRIII (NM-kd) demonstrated increased cell growth, motility, and invasion as compared with control cells expressing shRNA to human TbetaRIII (NM-con). Reconstitution of TbetaRIII expression with rat TbetaRIII abrogated the increased growth and motility seen in the NM-kd cells. In addition, the NM-kd cells exhibited marked reduction in the expression of the adherens junction protein, E-cadherin. This loss of E-cadherin was due to increased NFkappaB activity that, in turn, resulted in increased expression of the transcriptional repressors of E-cadherin such as Snail, Slug, Twist, and Sip1. Finally, NMuMG cells in which TbetaRIII had been knocked down formed invasive tumors in athymic nude mice, whereas the control cells did not. These data indicate that TbetaRIII acts as a tumor suppressor in nontumorigenic mammary epithelial cells at least in part by inhibiting NFkappaB-mediated repression of E-cadherin.

Mornings with Art, lessons learned: feedback regulation, restriction threshold biology, and redundancy govern molecular stress responses.

Work from the laboratory of Dr. Arthur B. Pardee has highlighted basic principles that govern cellular and molecular biological processes in living cells. Among the most important governing principles in cellular and molecular responses are: (i) threshold "restriction" responses, wherein a level of response is reached and a "point of no return" is achieved; (ii) feedback regulation; and (iii) redundancy. Lessons learned from the molecular biology of cellular stress responses in mammalian cancer versus normal cells after ionizing radiation (IR) or chemotherapeutic agent exposures reveal similar instances of these guiding principles in mammalian cells. Among these are the: (i) induction of cell death responses by beta-lapachone (beta-lap), a naphthoquinone anti-tumor agent that kills cancer cells via an NQO1 (i.e., X-ray-inducible protein-3, xip3)-dependent mechanism; (ii) induction of secretory clusterin (sCLU) in response to TGF-beta1 exposure, and the ability of induced sCLU protein to down-regulate TGF-beta1 signaling; and (iii) induction of DNA mismatch repair-dependent G(2) cell cycle checkpoint responses after exposure to alkylating agents. We have learned these lessons and now adopted strategies to exploit them for improved therapy. These examples will be discussed and compared to the pioneering findings of researchers in the Pardee laboratory over the years.