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Anabolic resistance to a mechanical stimulus may contribute to the loss of skeletal muscle mass observed with age. In this study, young and aged mice were injected with saline or human LM-111 (1 mg/kg). One week later, the myotendinous junction of the gastrocnemius muscle was removed via myotenectomy (MTE), thus placing a chronic mechanical stimulus on the remaining plantaris muscle for 2 weeks. LM-111 increased α7B integrin protein expression and clustering of the α7B integrin near DAPI+ nuclei in aged muscle in response to MTE. LM-111 reduced CD11b+ immune cells, enhanced repair, and improved the growth response to loading in aged plantaris muscle. These results suggest that LM-111 may represent a novel therapeutic approach to prevent and/or treat sarcopenia.

Extended periods of bed rest and limb immobilization are required for healing post-injury or disease, yet disuse can result in significant muscle atrophy and decreased quality of life in older adults. Physical rehabilitation is commonly prescribed to recover these deficits, yet accumulation of reactive oxygen species and sustained rates of protein degradation persist during the rehabilitation period that can significantly delay or prevent recovery. Pericytes, considered the primary mesenchymal and vascular stromal cell in skeletal muscle, secrete beneficial factors that maintain baseline muscle mass, yet minimal information exists regarding the pericyte response to disuse and recovery. In the current study, single-cell RNA sequencing and functional assays were performed to demonstrate that pericytes in mouse skeletal muscle lose the capacity to synthesize antioxidants during disuse and recovery. This information was used to guide the design of a strategy in which healthy donor pericytes were stimulated with hydrogen peroxide (H

Unaccustomed resistance exercise can initiate skeletal muscle remodeling and adaptive mechanisms that can confer protection from damage and enhanced strength with subsequent stimulation. The myofiber may provide the primary origin for adaptation, yet multiple mononuclear cell types within the surrounding connective tissue may also contribute. The purpose of this study was to evaluate the acute response of muscle-resident interstitial cells to contraction initiated by electrical stimulation (e-stim) and subsequently determine the contribution of pericytes to remodeling as a result of training. Mice were subjected to bilateral e-stim or sham treatment. Following a single session of e-stim, NG2+CD45−CD31− (NG2+Lin−) pericyte, CD146+Lin− pericyte, and PDGFRα+ fibroadipogenic progenitor cell quantity and function were evaluated via multiplex flow cytometry and targeted quantitative PCR. Relative quantity was not significantly altered 24 h postcontraction, yet unique gene signatures were observed for each cell population at 3 h postcontraction. CD146+Lin− pericytes appeared to be most responsive to contraction, and upregulation of genes related to immunomodulation and extracellular matrix remodeling was observed via RNA sequencing. Intramuscular injection of CD146+Lin− pericytes did not significantly increase myofiber size yet enhanced ECM remodeling and angiogenesis in response to repeated bouts of e-stim for 4 wk. The results from this study provide the first evidence that CD146+Lin− pericytes are responsive to skeletal muscle contraction and may contribute to the beneficial outcomes associated with exercise.

Cholesterol has been implicated in the clinical progression of breast cancer, a disease that continues to be the most commonly diagnosed cancer in women. Previous work has identified the cholesterol metabolite 27-hydroxycholesterol (27HC) as a major mediator of the effects of cholesterol on breast tumor growth and progression. 27HC can act as an estrogen receptor (ER) modulator to promote the growth of ERα+ tumors, and as a liver X receptor (LXR) ligand in myeloid immune cells to establish an immune-suppressive program. In fact, the metastatic properties of 27HC require the presence of myeloid cells with neutrophils (polymorphonuclear neutrophils; PMNs) being essential for the increase in lung metastasis in murine models. In an effort to further elucidate the mechanisms by which 27HC alters breast cancer progression, we made the striking finding that 27HC promoted the secretion of extracellular vesicles (EVs), a diverse assortment of membrane bound particles that includes exosomes. The resulting EVs had a size distribution that was skewed slightly larger than EVs generated by treating cells with vehicle. The increase in EV secretion and size was consistent across 3 different subtypes: primary murine PMNs, RAW264.7 monocytic cells, and 4T1 murine mammary cancer cells. Label-free analysis of 27HC-EVs indicated that they had a different metabolite composition to those from vehicle-treated cells. Importantly, 27HC-EVs from primary PMNs promoted tumor growth and metastasis in 2 different syngeneic models, demonstrating the potential role of 27HC-induced EVs in the progression of breast cancer. EVs from PMNs were taken up by cancer cells, macrophages, and PMNs, but not T cells. Since EVs did not alter proliferation of cancer cells, it is likely that their protumor effects are mediated through interactions with myeloid cells. Interestingly, RNA-seq analysis of tumors from 27HC-EV-treated mice do not display significantly altered transcriptomes, suggesting that the effects of 27HC-EVs occur early on in tumor establishment and growth. Future work will be required to elucidate the mechanisms by which 27HC increases EV secretion, and how these EVs promote breast cancer progression. Collectively, however, our data indicate that EV secretion and content can be regulated by a cholesterol metabolite, which may have detrimental effects in terms of disease progression, important findings given the prevalence of both breast cancer and hypercholesterolemia.

Skeletal muscle structure and function are dependent on the presence or absence of mechanical cues in the tissue microenvironment. High intensity or sustained contractions can build muscle mass with exercise training, whereas bedrest or limb immobilization result in loss of muscle mass. Molecular signaling pathways, such as mTORC1 and induction of activating transcription factor 4 (ATF4), regulate intrinsic myofiber growth and atrophy respectively, yet the extent to which mononuclear cells outside the fiber contribute to muscle structure remodeling remains unclear. The skeletal muscle microenvironment is enriched with mononuclear cells, including progenitor and stem cells, fibroblasts, immune cells, and vascular stromal cells. All possess the potential to sense mechanical cues and contribute to structural remodeling, but the intricate positioning of vascular stromal cells around vessels make these cells particularly receptive to changes in mechanical stimuli due to alterations in both blood perfusion and contraction. Recent studies suggest that pericytes, a population of vascular stromal cells supporting capillaries and venules, are highly responsive to conditions of use and disuse. This chapter discusses the characteristics and behavior of skeletal muscle-derived pericytes as well as the specific responses to physiological stimuli with which muscle can be subjected. Determining how pericytes behave in situations of muscle activation and immobilization is important for the development of cell-based therapies for the treatment of skeletal muscle disorders.

The α7β1integrin is concentrated at the costameres of skeletal muscle and provides a critical link between the actin cytoskeleton and laminin in the basement membrane. We previously demonstrated that expression of the α7BX2 integrin subunit (MCK:α7BX2) preserves muscle integrity and enhances myofiber cross-sectional area following eccentric exercise. The purpose of this study was to utilize gene expression profiling to reveal potential mechanisms by which the α7BX2-integrin contributes to improvements in muscle growth after exercise. A microarray analysis was performed using RNA extracted from skeletal muscle of wild-type or transgenic mice under sedentary conditions and 3 h following an acute bout of downhill running. Genes with false discovery rate probability values below the cutoff of P < 0.05 ( n = 73) were found to be regulated by either exercise or transgene expression. KEGG pathway analysis detected upregulation of genes involved in endoplasmic reticulum protein processing with integrin overexpression. Targeted analyses verified increased transcription of Rpl13a, Nosip, Ang, Scl7a5, Gys1, Ndrg2, Hspa5, and Hsp40 as a result of integrin overexpression alone or in combination with exercise ( P < 0.05). A significant increase in HSPA5 protein and a decrease in CAAT-enhancer-binding protein homologous protein (CHOP) were detected in transgenic muscle ( P < 0.05). In vitro knockdown experiments verified integrin-mediated regulation of Scl7a5. The results from this study suggest that the α7β1integrin initiates transcription of genes that allow for protection from stress, including activation of a beneficial unfolded protein response and modulation of protein synthesis, both which may contribute to positive adaptations in skeletal muscle as a result of engagement in eccentric exercise.

β-Hydroxy β-methylbutyrate (HMB) is a metabolite of the essential amino acid leucine. Recent studies demonstrate a decline in plasma HMB concentrations in humans across the lifespan, and HMB supplementation may be able to preserve muscle mass and strength in older adults. However, the impact of HMB supplementation on hippocampal neurogenesis and cognition remains largely unexplored. The purpose of this study was to simultaneously evaluate the impact of HMB on muscle strength, neurogenesis and cognition in young and aged mice. In addition, we evaluated the influence of HMB on muscle-resident mesenchymal stem/stromal cell (Sca-1+CD45-; mMSC) function to address these cells potential to regulate physiological outcomes. Three month-old (n=20) and 24 month-old (n=18) female C57BL/6 mice were provided with either Ca-HMB or Ca-Lactate in a sucrose solution twice per day for 5.5weeks at a dose of 450mg/kg body weight. Significant decreases in relative peak and mean force, balance, and neurogenesis were observed in aged mice compared to young (age main effects, p≤0.05). Short-term HMB supplementation did not alter activity, balance, neurogenesis, or cognitive function in young or aged mice, yet HMB preserved relative peak force in aged mice. mMSC gene expression was significantly reduced with age, but HMB supplementation was able to recover expression of select growth factors known to stimulate muscle repair (HGF, LIF). Overall, our findings demonstrate that while short-term HMB supplementation does not appear to affect neurogenesis or cognitive function in young or aged mice, HMB may maintain muscle strength in aged mice in a manner dependent on mMSC function.

Peripheral arterial disease (PAD) is a debilitating complication of diabetes mellitus (DM) that leads to thousands of injuries, amputations, and deaths each year. The use of mesenchymal stem cells (MSCs) as a regenerative therapy holds the promise of regrowing injured vasculature, helping DM patients live healthier and longer lives. We report the use of muscle-derived MSCs to treat surgically-induced hindlimb ischemia in a mouse model of type 1 diabetes (DM-1). We serially evaluate several facets of the recovery process, including αVβ3 -integrin expression (a marker of angiogenesis), blood perfusion, and muscle function. We also perform microarray transcriptomics experiments to characterize the gene expression states of the MSC-treated is- chemic tissues, and compare the results with those of non-ischemic tissues, as well as ischemic tissues from a saline-treated control group. The results show a multifaceted impact of mMSCs on hindlimb ischemia. We determined that the angiogenic activity one week after mMSC treatment was enhanced by approximately 80% relative to the saline group, which resulted in relative increases in blood perfusion and muscle strength of approximately 42% and 1.7-fold, respectively. At the transcriptomics level, we found that several classes of genes were affected by mMSC treatment. The mMSCs appeared to enhance both pro-angiogenic and metabolic genes, while suppressing anti-angiogenic genes and certain genes involved in the inflammatory response. All told, mMSC treatment appears to exert far-reaching effects on the microenvironment of ischemic tissue, enabling faster and more complete recovery from vascular occlusion.

During a traditional set configuration of resistance exercise (TRD), characterized by a continuous completion of repetitions, a decrease in power output tends to occur throughout a set of repetitions. Inclusion of intraset rest, otherwise known as a cluster set configuration (CLU), counteracts this power decline. However, the effect of a CLU configuration on postexercise myofibrillar protein synthesis rates (MPS) and anabolic signaling has not been investigated. PURPOSE We aimed to determine if any mechanistic differences exist between TRD and CLU signaling events associated with muscle anabolism. METHODS In randomized crossover trials, eight resistance-trained participants (23 ± 1 yr, 81 ± 4.7 kg, body fat: 18% ± 1.9%; 1 repetition maximum [1RM], 150 ± 9.1 kg) performed an acute bout of CLU (4 sets × (2 × 5) repetitions, 30-s intraset rest, 90-s interset rest) and TRD (4 sets × 10 repetitions, 120-s interset rest) barbell back squats at approximately 70% 1RM with total volume load equated during primed continuous L-[ring-C6]phenylalanine infusions. Blood and muscle biopsy samples were collected at rest and after exercise at 0, 2, and 5 h. RESULTS There was no difference in postexercise MPS between the CLU and TRD condition (P > 0.05) and no changes in phosphorylation of mTORC1 downstream targets (p70S6K and 4EBP1). Total and phosphorylated yes-associated protein on Ser127 transiently increased (P < 0.01) immediately after exercise (t = 0) in CLU (~2.1-fold) and TRD condition (~2.2-fold). CONCLUSIONS Our results show that CLU is a viable anabolic option by preserving power output with similar MPS stimulation when compared with the TRD condition in trained young adults.

The α7β1-integrin is a transmembrane adhesion protein that connects laminin in the extracellular matrix (ECM) with actin in skeletal muscle fibers. The α7β1-integrin is highly expressed in skeletal muscle and is concentrated at costameres and myotendious junctions, providing the opportunity to transmit longitudinal and lateral forces across the membrane. Studies have demonstrated that α7-integrin subunit mRNA and protein are upregulated following eccentric contractions as a mechanism to reinforce load-bearing structures and resist injury with repeated bouts of exercise. It has been hypothesized for many years that the integrin can also promote protein turnover in a manner that can promote beneficial adaptations with resistance exercise training, including hypertrophy. This review provides basic information about integrin structure and activation and then explores its potential to serve as a critical mechanosensor and activator of muscle protein synthesis and growth. Overall, the hypothesis is proposed that the α7β1-integrin can contribute to mechanical-load induced skeletal muscle growth via an mammalian target of rapamycin complex 1-independent mechanism.

β-hydroxy β-methylbutyrate (HMB) is a nutritional supplement purported to enhance skeletal muscle mass and strength, as well as cognitive function in older adults. The purpose of this study was to determine the potential for long-term HMB supplementation to preserve muscle function and cognition in aged mice, as well as provide evidence of a link between vessel-associated pericyte function and outcomes. Four- (Adult/Ad) and 17 month-old (Aged/Ag) C57BL/6J mice consumed chow containing 600 mg/kg BW/day of either Ca-HMB (Ad, n=16; Ag, n=17) or Ca-Lactate (Ad, n=16; Ag, n=17) for 6 months. HMB did not prevent age-related reductions in muscle mass, strength and coordination (Age main effect, P

Freeze-dried hydrogels are increasingly used to create 3D interconnected micropores that facilitate biomolecular and cellular transports. However, freeze-drying is often plagued by variance in micropore architecture based on polymer choice. We hypothesized that water-polymer binding affinity plays a significant role in sizes and numbers of micropores formed through freeze-drying, influencing cell-derived tissue quality. Poly(ethylene glycol)diacrylate (PEGDA) hydrogels with alginate methacrylate (AM) were used due to AM's higher binding affinity for water than PEGDA. PEGDA-AM hydrogels with larger AM concentrations resulted in larger sizes and numbers of micropores than pure PEGDA hydrogels, attributed to the increased mass of water binding to the PEGDA-AM gel. Skeletal myoblasts loaded in microporous PEGDA-AM hydrogels were active to produce 3D muscle-like tissue, while those loaded in pure PEGDA gels were localized on the gel surface. We propose that this study will be broadly useful in designing and improving the performance of various microporous gels.

AB Purpose: The [alpha]7[beta]1 integrin is a transmembrane protein expressed in the skeletal muscle that can link the actin cytoskeleton to the surrounding basal lamina. We have previously demonstrated that transgenic mice overexpressing the [alpha]7B integrin in the skeletal muscle (MCK:[alpha]7B; [alpha]7Tg) mount an enhanced satellite cell and growth response to single or multiple bouts of eccentric exercise. In addition, interstitial stem cells characterized as mesenchymal stem cells (MSCs) accumulate in [alpha]7Tg muscle (mMSCs) in the sedentary state and after exercise. The results from these studies prompted us to determine the extent to which mMSC underlie the beneficial adaptive responses observed in [alpha]7Tg skeletal muscle after exercise. Methods: mMSCs (Sca-1+CD45-) were isolated from [alpha]7Tg mice, dye-labeled, and intramuscularly injected into adult wild type recipient mice. After injection of mMSCs or saline, mice remained sedentary (SED) or were subjected to eccentric exercise training (TR) (downhill running) on a treadmill (three times per week) for 2 or 4 wk. Gastrocnemius-soleus complexes were collected 24 h after the last bout of exercise. Results: mMSCs did not directly fuse with existing fibers; however, mMSCs injection enhanced Pax7+ satellite cell number and myonuclear content compared with all other groups at 2 wk after exercise. Mean CSA, percentage of larger caliber fibers (>3000 [mu]m2), and grip strength were increased in mMSCs/TR compared with saline/SED and mMSCs/SED at 4 wk. mMSC transplantation did not enhance repair or growth in the absence of exercise. Conclusions: The results from this study demonstrate that mMSCs contribute to beneficial changes in satellite cell expansion and growth in [alpha]7Tg muscle after eccentric exercise. Thus, MSCs that naturally accumulate in the muscle after eccentric contractions may enhance the adaptive response to exercise.

For the past few decades, efforts have been extensively made to reproduce tissue of interests for various uses including fundamental bioscience studies, clinical treatments, and even soft robotic systems. In these studies, cells are often cultured in micropores introduced in a provisional matrix despite that bulk rigidity may negatively affect cellular differentiation involved in tissue formation. To this end, we hypothesized that suspending cells within a soft fibrous matrix that is encapsulated within the microchannels of a provisional matrix would allow us to mediate effects of the matrix rigidity on cells and, in turn, to increase the cell differentiation level. We examined this hypothesis by filling microchannels interpenetrating alginate matrices with collagen gels of controlled elastic moduli (i.e., 125 to 1 Pa). Myoblasts used as a model predifferentiated cell were suspended within the collagen gels. The elastic modulus of the collagen gels was decreased through the addition of poly(ethylene glycol) during the gel preparation. Myoblasts loaded in the collagen gel exhibited a higher myogenic differentiation level than those adhered to the collagen-coated microchannel wall. Furthermore, the collagen gel softened by poly(ethylene glycol) further increased the volume of the multinucleated myofibers. The role of collagen gel softness on cell differentiation became more significant when the bulk elastic modulus of the alginate matrix was tuned to be close to that of muscle tissue (i.e., 11 kPa). We believe that the results of this study would be useful to understanding phenotypic activities of a wide array of cells involved in tissue development and regeneration.

Laminin-111 (α1, β1, γ1; LM-111) is an important component of the extracellular matrix that is required for formation of skeletal muscle during embryonic development. Recent studies suggest that LM-111 supplementation can enhance satellite cell proliferation and muscle function in mouse models of muscular dystrophy. The purpose of this study was to determine the extent to which LM-111 can alter satellite and nonsatellite stem cell quantity following eccentric exercise-induced damage in young adult, healthy mice. One week following injection of LM-111 or saline, mice either remained sedentary or were subjected to a single bout of downhill running (EX). While one muscle was preserved for evaluation of satellite cell number, the other muscle was processed for isolation of mesenchymal stem cells (MSCs; Sca-1+CD45−) via FACS at 24 hours postexercise. Satellite cell number was approximately twofold higher in LM-111/EX compared with all other groups (p < .05), and the number of satellite cells expressing the proliferation marker Ki67 was 50% to threefold higher in LM-111/EX compared with all other groups (p < .05). LM-111 also increased the quantity of embryonic myosin heavy chain-positive (eMHC+) fibers in young mice after eccentric exercise (p < .05). Although MSC percentage and number were not altered, MSC proinflammatory gene expression was decreased, and hepatocyte growth factor gene expression was increased in the presence of LM-111 (p < .05). Together, these data suggest that LM-111 supplementation provides a viable solution for increasing skeletal muscle stem cell number and/or function, ultimately allowing for improvements in the regenerative response to eccentric exercise.

In article number 1801521, Hyunjoon Kong and co‐workers demonstrate that substrate topography regulates neural innervation by enhancing maturity of the myotubes. Neural stem cells are differentiated on the myotubes, formed on grooved substrates. The motor neurons innervate more actively to the mature myotubes formed on the 1600 nm groove width. Accordingly, those myotubes respond more rapidly to neurotransmitter and neuromuscular antagonists. The results of this study will be useful for improving the quality of engineered muscle used in applications such as drug screening, regeneration therapies, and biological machinery assembly.

Recreation of a muscle that can be controlled by the nervous system would provide a major breakthrough for treatments of injury and diseases. However, the underlying basis of how neuron–muscle interfaces are formed is still not understood sufficiently. Here, it is hypothesized that substrate topography regulates neural innervation and synaptic transmission by mediating the cross‐talk between neurons and muscles. This hypothesis is examined by differentiating neural stem cells on the myotubes, formed on the substrate with controlled groove width. The substrate with the groove width of 1600 nm, a similar size to the myofibril diameter, serves to produce larger and aligned myotubes than the flat substrate. The myotubes formed on the grooved substrate display increases in the acetylcholine receptor expression. Reciprocally, motor neuron progenitor cells differentiated from neural stem cells innervate the larger and aligned myotubes more actively than randomly oriented myotubes. As a consequence, mature and aligned myotubes respond to glutamate (i.e., an excitatory neurotransmitter) and curare (i.e., a neuromuscular antagonist) more rapidly and homogeneously than randomly oriented myotubes. The results of this study will be broadly useful for improving the quality of engineered muscle used in a series of applications including drug screening, regeneration therapies, and biological machinery assembly.

A major challenge for translating cell-based therapies is understanding the dynamics of cells and cell populations in complex in vivo environments. Intravital microscopy has shown great promise for directly visualizing cell behavior in vivo. However, current methods are limited to relatively short imaging times (hours), by ways to track cell and cell population dynamics over extended time-lapse periods (days to weeks to months), and by relatively few imaging contrast mechanisms that persist over extended investigations. We present technology to visualize and quantify complex, multifaceted dynamic changes in natural deformable skin over long time periods using novel multimodal imaging and a non-rigid image registration method. These are demonstrated in green fluorescent protein (GFP) bone marrow (BM) transplanted mice to study dynamic skin regeneration. This technology provides a novel perspective for studying dynamic biological processes and will enable future studies of stem, immune, and tumor cell biology in vivo.

Bone-marrow (BM)-derived cells have been shown to be capable of aiding skin regeneration in vivo by differentiating into keratinocytes. However, the conditions under which this occurs are not fully understood. Characterizing innate mechanisms of skin regeneration by stem cells in vivo is important for the area of stem cell biology. In this study, we investigate the use of novel in vivo imaging technology for characterizing the contribution of BM-derived cells to regeneration of the epidermis in mouse skin in vivo. In vivo imaging provides the ability to non-invasively observe the spatial positions and morphology of the BM-derived cells. Using a GFP BM-transplanted mouse model and in vivo multimodal microscopy, BM-derived cells can be observed in the skin. Our in vivo imaging method was used to search for the presence and identify the 3D spatial distribution of BM-derived cells in the epidermis of the skin under normal conditions, following wound healing, and after syngeneic skin grafting. We did not observe any evidence of BM-derived keratinocytes under these conditions, but we did observe BM-derived dendritic cells in the skin grafts. In vivo multimodal imaging has great potential for characterizing the conditions under which BM-derived cells contribute to skin regeneration.

The α7β1-integrin is an adhesion molecule highly expressed in skeletal muscle that can enhance regeneration in response to eccentric exercise. We have demonstrated that mesenchymal stem cells (MSCs), predominantly pericytes, accumulate in muscle (mMSCs) overexpressing the α7B-integrin (MCK:α7B; α7Tg) and contribute to new fiber formation following exercise. Since vascularization is a common event that supports tissue remodeling, we hypothesized that the α7-integrin and/or mMSCs may stimulate vessel growth following eccentric exercise. Wild-type (WT) and α7Tg mice were subjected to single or multiple (3 times/wk, 4 wk) bouts of downhill running exercise. Additionally, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) -labeled mMSCs were intramuscularly injected into WT recipients. A subset of recipient mice were run downhill before injection to recapitulate the exercised microenvironment. While total number of CD31+ vessels declined in both WT and α7Tg muscle following a single bout of exercise, the number of larger CD31+ vessels with a visible lumen was preferentially increased in α7Tg mice following eccentric exercise training ( P < 0.05). mMSC transplantation similarly increased vessel diameter and the total number of neuron-glial antigen-2 (NG2+) arterioles postexercise. Secretion of arteriogenic factors from mMSCs in response to mechanical strain, including epidermal growth factor and granulocyte macrophage-colony stimulating factor, may account for vessel remodeling. In conclusion, this study demonstrates that the α7-integrin and mMSCs contribute to increased vessel diameter size and arteriolar density in muscle in response to eccentric exercise. The information in this study has implications for the therapeutic treatment of injured muscle and disorders that result in vessel occlusion, including peripheral artery disease.

Impaired skin wound healing is a significant comorbid condition of diabetes, which often results in nonhealing diabetic ulcers due to poor peripheral microcirculation, among other factors. The effectiveness of the regeneration of adipose-derived stem cells (ADSCs) and muscle-derived stem cells (MDSCs) was assessed using an integrated multimodal microscopy system equipped with two-photon fluorescence and second-harmonic generation imaging. These imaging modalities, integrated in a single platform for spatial and temporal coregistration, allowed us to monitor in vivo changes in the collagen network and cell dynamics in a skin wound. Fluorescently labeled ADSCs and MDSCs were applied topically to the wound bed of wild-type and diabetic (db/db) mice following punch biopsy. Longitudinal imaging demonstrated that ADSCs and MDSCs provided remarkable capacity for improved diabetic wound healing, and integrated microscopy revealed a more organized collagen remodeling in the wound bed of treated mice. The results from this study verify the regenerative capacity of stem cells toward healing and, with multimodal microscopy, provide insight regarding their impact on the skin microenvironment. The optical method outlined in this study, which has the potential for in vivo human use, may optimize the care and treatment of diabetic nonhealing wounds.

Mechanical stimuli increase skeletal muscle growth in a mammalian target of rapamycin (mTOR)- and p70S6K-dependent manner. It has been proposed that costameric proteins at Z bands may sense and transfer tension to these initiators of protein translation, but few candidates have been identified. The purpose of this study was to determine whether a role exists for the α7-integrin in the activation of hypertrophic signaling and growth following eccentric exercise training. Five-week-old, wild-type (WT) and α7BX2-integrin transgenic (α7Tg) mice were randomly assigned to one of two groups: 1) sedentary (SED), or 2) exercise training (EX). Exercise training consisted of downhill running 3 sessions/wk for 4 wk (−20°, 17 m/min, 30 min). Downhill running was used to induce physiological mechanical strain. Twenty-four hours following the final training session, maximal isometric hindlimb plantar flexor force was measured. Gastrocnemius-soleus complexes were collected for further analysis of signaling changes, which included AKT, mTOR and p70S6K, and muscle growth. Despite increased p70S6Kactivity in WT/EX, no significant changes in cross-sectional area or force were observed in WT/EX compared with WT/SED. AKT, mTOR, and p70S6Kactivation was higher, and whole muscle hypertrophy, relative muscle weight, myofibrillar protein, and force were significantly elevated in α7Tg/EX compared with α7Tg/SED. A marked increase in average myofiber cross-sectional area was observed in α7Tg/EX compared with all groups. Our findings demonstrate that the α7β1-integrin sensitizes skeletal muscle to mechanical strain and subsequent growth. Thus the α7β1-integrin may represent a novel molecular therapy for the treatment of disuse muscle atrophy.

Fourier transform light scattering (FTLS) has been recently developed as a novel, ultrasensitive method for studying light scattering from inhomogeneous and dynamic structures. FTLS relies on quantifying the optical phase and amplitude associated with a coherent image field and propagating it numerically to the scattering plane. In this paper, we review the principle and applications of FTLS to static and dynamic light scattering from biological tissues and live cells. Compared with other existing light scattering techniques, FTLS has significant benefits of high sensitivity, speed, and angular resolution. We anticipate that FTLS will set the basis for disease diagnosis based on intrinsic tissue optical properties and provide an efficient tool for quantifying cell structures and dynamics.

We review the principle and application of Fourier transform light scattering (FTLS), a new technique developed in our laboratory to study static and dynamic light scattering (DLS) from the biological tissues and live cells. The results demonstrate that FTLS has significant benefits over existing light scattering techniques in terms of sensitivity and resolution. We anticipate that FTLS will set the basis for disease diagnosis based on the intrinsic tissue optical properties and provide an efficient tool for quantifying cell structures and dynamics.

Stem cells are traditionally studied in the context of embryonic development, yet studies confirm that a fraction remains in the adult organism for the purpose of daily remodeling and rejuvenation of multiple tissues following injury. Adult stem cells (ASCs) are found in close proximity to vessels and respond to tissue-specific cues in the microenvironment that dictate their fate and function. Exercise can dramatically alter strain sensing, extracellular matrix composition, and inflammation, and such changes in the niche likely alter ASC quantity and function postexercise. The field of stem cell biology is still in its infancy and identification and terminology of ASCs continues to evolve; thus, current information regarding exercise and stem cells is lacking. This chapter summarizes the literature that reports on the ASC response to acute exercise and exercise training, with particular emphasis on hematopoietic stem cells, endothelial progenitor cells, and mesenchymal stem cells.

α7β1-Integrin links laminin in the extracellular matrix with the cell cytoskeleton and therein mediates transduction of mechanical forces into chemical signals. Muscle contraction and stretching ex vivo result in activation of intracellular signaling molecules that are integral to postexercise injury responses. Because α7β1-integrin stabilizes muscle and provides communication between the matrix and cytoskeleton, the role of this integrin in exercise-induced cell signaling and skeletal muscle damage was assessed in wild-type and transgenic mice overexpressing the α7BX2 chain. We report here that increasing α7β1-integrin inhibits phosphorylation of molecules associated with muscle damage, including the mitogen-activated protein kinases (JNK, p38, and ERK), following downhill running. Likewise, activation of molecules associated with hypertrophy (AKT, mTOR, and p70S6k) was diminished in mice overexpressing integrin. While exercise resulted in Evans blue dye-positive fibers, an index of muscle damage, increased integrin protected mice from injury. Moreover, exercise leads to an increase in α7β1protein. These experiments provide the first evidence that α7β1-integrin is a negative regulator of mechanotransduction in vivo and provides resistance to exercise-induced muscle damage.

Obesity is a primary cause of muscle insulin resistance and is also associated with morphological and functional changes in the skeletal muscle including fibrosis. Studies suggest that macrophages in obese skeletal muscle may be primed to secrete transforming growth factor β1 (TGFβ1), a factor that can stimulate type I collagen gene expression via Smad3 activation but the extent to which exercise could modulate high fat (HF) diet-induced inflammation and fibrosis in skeletal muscle remains to be determined. The purpose of this study was to determine the extent to which moderate intensity exercise training can attenuate pro-inflammatory cytokine gene expression and markers of fibrosis in skeletal muscle in response to concomitant HF diet. Male C57BL/6J mice (6 wk old) were randomly assigned to one of four treatment groups: (1) Control diet-No Exercise (CON-No Ex), (2) CON-Ex, (3) HF-No Ex, or (4) HF-Ex. Mice were exercised on a motorized treadmill 40min/day at 12m/min, 5% grade, 5days/wk, for 12weeks. Macrophage (F4/80, CD11c, CD206), inflammatory cytokine (TNFα, IL-6, IL-10), TGFβ1, and collagen (Col1α) gene expression were evaluated in skeletal muscle by qPCR. Frozen muscle sections were stained to assess collagen content and fiber cross sectional area (CSA). F4/80, CD206 and IL-6 gene expression were increased by HF diet, and exercise only attenuated the increase in F4/80 and IL-6 (p

Objective: To investigate the role of mesenchymal stem cells (MSCs) in adipose tissue (AT) inflammation and fibrosis during high fat diet and exercise training. Methods: C57BL/6 mice were fed high fat (HF, 60%) or low fat (LF, 10%) diets for 8 weeks. Half of the mice were then exercised for 16 weeks. Sca1+CD45- MSCs were extracted from epididymal fat, isolated by FACS and analyzed by Q-PCR. AT inflammation and fibrosis were evaluated using Q-PCR and histology. Results: HF diet (HFD) increased body weight and exercise attenuated weight gain. AT F4/80, TNFα, and IL-10 gene expression increased with HF (p

This study demonstrates that a new method to align microparticles releasing bioactive molecules in microchannels of a hydrogel allows the guiding of growth direction and spacing of vascular networks.

Physical exercise and contraction increase c-Jun NH2-terminal kinase (JNK) activity in rat and human skeletal muscle, and eccentric contractions activate JNK to a greater extent than concentric contractions in human skeletal muscle. Because eccentric contractions include a lengthening or stretch component, we compared the effects of isometric contraction and static stretch on JNK and p38, the stress-activated protein kinases. Soleus and extensor digitorum longus (EDL) muscles dissected from 50- to 90-g male Sprague-Dawley rats were subjected to 10 min of electrical stimulation that produced contractions and/or to 10 min of stretch (0.24 N tension, 20–25% increase in length) in vitro. In the soleus muscle, contraction resulted in a small, but significant, increase in JNK activity (1.8-fold above basal) and p38 phosphorylation (4-fold). Static stretch had a much more profound effect on the stress-activated protein kinases, increasing JNK activity 19-fold and p38 phosphorylation 21-fold. Increases in JNK activation and p38 phosphorylation in response to static stretch were fiber-type dependent, with greater increases occurring in the soleus than in the EDL. Immunohistochemistry performed with a phosphospecific antibody revealed that activation of JNK occurred within the muscle fibers. These studies suggest that the stretch component of a muscle contraction may be a major contributor to the increases in JNK activity and p38 phosphorylation observed after exercise in vivo.

Eccentric contractions require the lengthening of skeletal muscle during force production and result in acute and prolonged muscle injury. Because a variety of stressors, including physical exercise and injury, can result in the activation of the c-Jun NH2-terminal kinase (JNK) intracellular signaling cascade in skeletal muscle, we investigated the effects of eccentric exercise on the activation of this stress-activated protein kinase in human skeletal muscle. Twelve healthy subjects (7 men, 5 women) completed maximal concentric or eccentric knee extensions on a KinCom isokinetic dynamometer (10 sets, 10 repetitions). Percutaneous needle biopsies were obtained from the vastus lateralis muscle 24 h before exercise (basal), immediately postexercise, and 6 h postexercise. Whereas both forms of exercise increased JNK activity immediately postexercise, eccentric contractions resulted in a much higher activation (15.4 ± 4.5 vs. 3.5 ± 1.4-fold increase above basal, eccentric vs. concentric). By 6 h after exercise, JNK activity decreased back to baseline values. In contrast to the greater activation of JNK with eccentric exercise, the mitogen-activated protein kinase kinase 4, the immediate upstream regulator of JNK, was similarly activated by concentric and eccentric exercise. Because the activation of JNK promotes the phosphorylation of a variety of transcription factors, including c-Jun, the results from this study suggest that JNK may be involved in the molecular and cellular adaptations that occur in response to injury-producing exercise in human skeletal muscle.

Bone marrow (BM)-derived stem and immune cells play critical roles in maintaining the health, regeneration, and repair of many tissues. Given their important functions in tissue regeneration and therapy, tracking the dynamic behaviors of BM-derived cells has been a long-standing research goal of both biologists and engineers. Because of the complex cellular-level processes involved, real-time imaging technologies that have sufficient spatial and temporal resolution to visualize them are needed. In addition, in order to track cellular dynamics, special attention is needed to account for changes in the microenvironment where the cells reside, for example, tissue contraction, stretching, development, etc. In this chapter, we introduce methods for real-time imaging and longitudinal tracking of BM-derived immune and stem cells in in vivo three-dimensional (3-D) tissue environments with an integrated optical microscope. The integrated microscope combines multiple imaging functions derived from optical coherence tomography (OCT) and multiphoton microscopy (MPM), including optical coherence microscopy (OCM), micro-vasculature imaging, two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) microscopy. Short- and long-term tracking of the dynamic behavior of BM-derived cells involved in cutaneous wound healing and skin grafting in green fluorescent protein (GFP) BM-transplanted mice is demonstrated. Methods and algorithms for nonrigid registration of time-lapse images are introduced, which allows for long-term tracking of cell dynamics over several months.

Skeletal muscle repair is essential for effective remodeling, tissue maintenance, and initiation of beneficial adaptations post-eccentric exercise. A series of well characterized events, such as recruitment of immune cells and activation of satellite cells, constitute the basis for muscle regeneration. However, details regarding the fine-tuned regulation of this process in response to different types of injury are open for investigation. Muscle-resident non-myogenic, non-satellite stem cells expressing conventional mesenchymal stem cell (MSC) markers, have the potential to significantly contribute to regeneration given the role for bone marrow-derived MSCs in whole body tissue repair in response to injury and disease. The purpose of this mini-review is to highlight a regulatory role for non-satellite stem cells in the process of skeletal muscle healing post-eccentric exercise. The non-myogenic, non-satellite stem cell fraction will be defined, its role in tissue repair will be briefly reviewed, and recent studies demonstrating a contribution to eccentric exercise-induced regeneration will be presented.

Mechanical stimuli increase skeletal muscle growth in a mammalian target of rapamycin (mTOR)- and p70(S6K)-dependent manner. It has been proposed that costameric proteins at Z bands may sense and transfer tension to these initiators of protein translation, but few candidates have been identified. The purpose of this study was to determine whether a role exists for the α(7)-integrin in the activation of hypertrophic signaling and growth following eccentric exercise training. Five-week-old, wild-type (WT) and α(7)BX2-integrin transgenic (α(7)Tg) mice were randomly assigned to one of two groups: 1) sedentary (SED), or 2) exercise training (EX). Exercise training consisted of downhill running 3 sessions/wk for 4 wk (-20°, 17 m/min, 30 min). Downhill running was used to induce physiological mechanical strain. Twenty-four hours following the final training session, maximal isometric hindlimb plantar flexor force was measured. Gastrocnemius-soleus complexes were collected for further analysis of signaling changes, which included AKT, mTOR and p70(S6K), and muscle growth. Despite increased p70(S6K) activity in WT/EX, no significant changes in cross-sectional area or force were observed in WT/EX compared with WT/SED. AKT, mTOR, and p70(S6K) activation was higher, and whole muscle hypertrophy, relative muscle weight, myofibrillar protein, and force were significantly elevated in α(7)Tg/EX compared with α(7)Tg/SED. A marked increase in average myofiber cross-sectional area was observed in α(7)Tg/EX compared with all groups. Our findings demonstrate that the α(7)β(1)-integrin sensitizes skeletal muscle to mechanical strain and subsequent growth. Thus the α(7)β(1)-integrin may represent a novel molecular therapy for the treatment of disuse muscle atrophy.

The α7β1-integrin is a heterodimeric transmembrane protein that adheres to laminin in the extracellular matrix, representing a critical link that maintains structure in skeletal muscle. In addition to preventing exercise-induced skeletal muscle injury, the α7-integrin has been proposed to act as an intrinsic mechanosensor, initiating cellular growth in response to mechanical strain. The purpose of this study was to determine the extent to which the α7-integrin regulates muscle hypertrophy following eccentric exercise. Wild-type (WT) and α7-integrin transgenic (α7Tg) mice completed a single bout of downhill running exercise (−20°, 17 m/min, 60 min), and gastrocnemius-soleus complexes were collected 1, 2, 4, and 7 days (D) postexercise (PE). Maximal isometric force was maintained and macrophage accumulation was suppressed in α7Tg muscle 1D PE. Mean fiber cross-sectional area was unaltered in WT mice but increased 40% in α7Tg mice 7D PE. In addition, a rapid and striking fivefold increase in embryonic myosin heavy chain-positive fibers appeared in α7Tg mice 2D PE. Although Pax7-positive satellite cells were increased in α7Tg muscle 1D PE, the number of nuclei per myofiber was not altered 7D PE. Phosphorylation of mammalian target of rapamycin (mTOR) was significantly elevated in α7Tg 1D PE. This study provides the first demonstration that the presence of the α7β1-integrin in skeletal muscle increases fiber hypertrophy and new fiber synthesis in the early time course following a single bout of eccentric exercise. Further studies are necessary to elucidate the precise mechanism by which the α7-integrin can enhance muscle hypertrophy following exercise.

High speed intravital microscopy has emerged as an essential tool for studying cellular dynamics in live tissue. A limitation of this technique, however, is that the timescale that a sample can be continuously imaged is limited by practical considerations to several hours. Long term observation of live tissue is of great interest for a variety of research areas. We present methods for observing long term cellular dynamics in live tissue based on three-dimensional registration of time-lapse intravital microscopy images. For these experiments we utilized a custom multimodal microscope that allows simultaneous and co-registered acquisition of optical coherence (OCM) and multiphoton (MPM) microscopy images. OCM allows the structure of a sample to be visualized based on backscattered light while MPM excited fluorescence allows individual cells and cell function to be visualized. The OCM images of tissue structure are used to register data sets taken at different time points. The transformations of the OCM images are applied to MPM images to determine the migration of cell populations. This method of image registration is applied to in vivo tracking of bone-marrow derived GFP-labeled stem cells in mouse skin following bone marrow transplants from GFP donors into species-matched wildtype hosts. The use of three-dimensional image registration of time-lapse microscopy images enables tracking these cells after local cutaneous injury, and for investigating the role of skin stem cells in wound healing.

Rapamycin-sensitive signaling is required for skeletal muscle differentiation and remodeling. In cultured myoblasts, the mammalian target of rapamycin (mTOR) has been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of mTOR kinase activity. However, the kinase-independent function of mTOR remains controversial, and no in vivo studies have examined those mTOR myogenic mechanisms previously identified in vitro. In this study, we find that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of mTOR function, we have generated and characterized transgenic mice expressing two mutants of mTOR under the control of human skeletal actin (HSA) promoter: rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI). Our results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in wild-type muscles but partially resistant to rapamycin in both RR- and RR/KI-mTOR muscles, consistent with mTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of mTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, mTOR regulates muscle regeneration through kinase-independent and kinase-dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively.