Cholesterol Metabolite 27-Hydroxycholesterol Enhances the Secretion of Cancer Promoting Extracellular Vesicles by a Mitochondrial ROS-Induced Impairment of Lysosomal Function.

Extracellular vesicles (EVs) serve as crucial mediators of cell-to-cell communication in normal physiology as well as in diseased states, and have been largely studied in regard to their role in cancer progression. However, the mechanisms by which their biogenesis and secretion are regulated by metabolic or endocrine factors remain unknown. Here, we delineate a mechanism by which EV secretion is regulated by a cholesterol metabolite, 27-Hydroxycholesterol (27HC), where treatment of myeloid immune cells (RAW 264.7 and J774A.1) with 27HC impairs lysosomal homeostasis, leading to shunting of multivesicular bodies (MVBs) away from lysosomal degradation, towards secretion as EVs. This impairment of lysosomal function is caused by mitochondrial dysfunction and subsequent increase in reactive oxygen species (ROS). Interestingly, cotreatment with a mitochondria-targeted antioxidant rescued the lysosomal impairment and attenuated the 27HC-mediated increase in EV secretion. Overall, our findings establish how a cholesterol metabolite regulates EV secretion and paves the way for the development of strategies to regulate cancer progression by controlling EV secretion.

Mechanisms of mechanical overload-induced skeletal muscle hypertrophy: current understanding and future directions.

Mechanisms underlying mechanical overload-induced skeletal muscle hypertrophy have been extensively researched since the landmark report by Morpurgo (1897) of "work-induced hypertrophy" in dogs that were treadmill trained. Much of the preclinical rodent and human resistance training research to date supports that involved mechanisms include enhanced mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, an expansion in translational capacity through ribosome biogenesis, increased satellite cell abundance and myonuclear accretion, and postexercise elevations in muscle protein synthesis rates. However, several lines of past and emerging evidence suggest that additional mechanisms that feed into or are independent of these processes are also involved. This review first provides a historical account of how mechanistic research into skeletal muscle hypertrophy has progressed. A comprehensive list of mechanisms associated with skeletal muscle hypertrophy is then outlined, and areas of disagreement involving these mechanisms are presented. Finally, future research directions involving many of the discussed mechanisms are proposed.

Underpinning the Food Matrix Regulation of Postexercise Myofibrillar Protein Synthesis by Comparing Salmon Ingestion With the Sum of Its Isolated Nutrients in Healthy Young Adults.

BACKGROUND: Protein is most commonly consumed as whole foods as opposed to single nutrients. However, the food matrix regulation of the postprandial muscle protein synthetic response has received little attention. OBJECTIVES: The purpose of this study was to assess the effects of eating salmon (SAL) and of ingesting the same nutrients as an isolated mixture of crystalline amino acids and fish oil (ISO) on the stimulation of postexercise myofibrillar protein synthesis (MPS) and whole-body leucine oxidation rates in healthy young adults. METHODS: Ten recreationally active adults (24 ± 4 y; 5 men, 5 women) performed an acute bout of resistance exercise, followed by the ingestion of SAL or ISO in a crossover fashion. Blood, breath, and muscle biopsies were collected at rest and after exercise during primed continuous infusions of L-[ring-2H5]phenylalanine and L-[1-13C]leucine. All data are presented as means ± SD and/or mean differences (95% CIs). RESULTS: Postprandial essential amino acid (EAA) concentrations peaked earlier (P = 0.024) in the ISO group than those in the SAL group. Postprandial leucine oxidation rates increased over time (P < 0.001) and peaked earlier in the ISO group (1.239 ± 0.321 nmol/kg/min; 63 ± 25 min) than those in the SAL group (1.230 ± 0.561 nmol/kg/min; 105 ± 20 min; P = 0.003). MPS rates for SAL (0.056 ± 0.022 %/h; P = 0.001) and ISO (0.046 ± 0.025 %/h; P = 0.025) were greater than the basal rates (0.020 ± 0.011 %/h) during the 0- to 5-h recovery period, with no differences between conditions (P = 0.308). CONCLUSION: We showed that the postexercise ingestion of SAL or ISO stimulate postexercise MPS rates with no differences between the conditions. Thus, our results indicate that ingesting protein from SAL as a whole-food matrix is similarly anabolic to ISO in healthy young adults. This trial was registered at www. CLINICALTRIALS: gov as NCT03870165.

Development of a cell-free strategy to recover aged skeletal muscle after disuse.

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 (H2 O2 ) to produce small extracellular vesicles (sEVs) that effectively restored myofibre size in adult and aged muscle after disuse. Proteomic assessment detected 11 differentially regulated proteins in primed sEVs that may account for recovery of muscle, including proteins associated with extracellular matrix composition and anti-inflammatory and antioxidant processes. This study demonstrates that healthy H2 O2 -primed pericyte-derived sEVs effectively improve skeletal muscle recovery after immobilization, presenting a novel acellular approach to rebuild muscle mass in older adults after a period of disuse. KEY POINTS: Previous studies suggest that prolonged oxidative stress is a barrier to skeletal muscle recovery after a period of immobilization. In this study we demonstrate that muscle-resident perivascular stromal cells (pericytes) become dysfunctional and lack the capacity to mount an antioxidant defence after disuse in mice. Hydrogen peroxide treatment of healthy pericytes in vitro simulates the release of small extracellular vesicles (sEVs) that effectively recover skeletal muscle fibre size and extracellular matrix remodelling in young adult and aged mice after disuse. Pericyte-derived sEVs present a novel acellular strategy to recover skeletal muscle after disuse.

Optimization of a pericyte therapy to improve muscle recovery after limb immobilization.

Extended bed rest or limb immobilization can significantly reduce skeletal muscle mass and function. Recovery may be incomplete, particularly in older adults. Our laboratory recently reported that vascular mural cell (pericyte) quantity is compromised after immobilization and appropriate replacement immediately before remobilization can effectively recover myofiber size in mice. Identification of a single cell surface marker for isolation of the most therapeutic pericyte would streamline efforts to optimize muscle recovery. The purpose of this study was to compare the capacity for neural/glial antigen 2 (Cspg4/NG2+) and melanoma cell adhesion molecule (Mcam/CD146+) positive pericytes to uniquely recover skeletal muscle post-disuse. A single hindlimb from adult C57BL/6J mice was immobilized in full dorsiflexion via a surgical staple inserted through the center of the foot and body of the gastrocnemius. Fourteen days after immobilization, the staple was removed and pericytes, either NG2+CD45-CD31-[Lin-], CD146+NG2-Lin-, or CD146+Lin- pericytes, were injected into the atrophied tibialis anterior (TA) muscle. TA muscles were excised 14 days after transplantation and remobilization. Pericyte transplantation did not significantly improve muscle mass or myofiber cross-sectional area (CSA) after 14 days of remobilization. However, injection of CD146+ pericytes significantly increased Type IIa quantity, capillarization, and collagen remodeling compared with NG2+ pericytes (P < 0.05). Our results suggest that selection of pericytes based on CD146 rather than NG2 results in the isolation of therapeutic mural cells with high capacity to positively remodel skeletal muscle after a period of immobilization.NEW & NOTEWORTHY In this study, pericytes were isolated from mouse skeletal muscle based on cell surface marker expression of neural/glial antigen 2 (NG2) or melanoma cell adhesion molecule (Mcam/CD146) and then compared for the capacity to recover skeletal muscle after a period of immobilization in recipient mice. We report that CD146+Lin- pericytes exhibit higher capacity than NG2+Lin- pericytes to recover Type IIa fiber quantity, capillary content, and collagen turnover after disuse.

Increases in Integrin-ILK-RICTOR-Akt Proteins, Muscle Mass, and Strength after Eccentric Cycling Training.

PURPOSE: Recently, it has been suggested that a cellular pathway composed of integrin, integrin-linked kinase (ILK), rapamycin-insensitive companion of mTOR (RICTOR), and Akt may facilitate long-term structural and functional adaptations associated with exercise, independent of the mTORC1 pathway. Therefore, we examined changes in integrin-ILK-RICTOR-Akt protein in vastus lateralis (VL) before and after 8 wk of eccentric cycling training (ECC), which was expected to increase muscle function and VL cross-sectional area (CSA). METHODS: Eleven men (23 ± 4 yr) completed 24 sessions of ECC with progressive increases in intensity and duration, resulting in a twofold increase in work from the first three (75.4 ± 14.1 kJ) to the last three sessions (150.7 ± 28.4 kJ). Outcome measures included lower limb lean mass, VL CSA, static strength, and peak and average cycling power output. These measures and VL samples were taken before and 4-5 d after the last training session. RESULTS: Significant (P < 0.05) increases in integrin-ß1 (1.64-fold) and RICTOR (2.99-fold) protein as well as the phosphorylated-to-total ILK ratio (1.70-fold) were found, but integrin-α7 and Akt did not change. Increases in lower limb, thigh, and trunk lean mass (2.8%-5.3%, P < 0.05) and CSA (13.3% ± 9.0%, P < 0.001) were observed. Static strength (18.1% ± 10.8%) and both peak (8.6% ± 10.5%) and average power output (7.4% ± 8.3%) also increased (P < 0.05). However, no significant correlations were found between the magnitude of increases in protein and the magnitude of increases in CSA, static strength, or power output. CONCLUSIONS: In addition to increased muscle mass, strength, and power, we demonstrate that ECC increases integrin-ß1 and RICTOR total protein and p-ILK/t-ILK, which may play a role in protection against muscle damage as well as anabolic signaling to induce muscle adaptations.

The Cholesterol Metabolite 27HC Increases Secretion of Extracellular Vesicles Which Promote Breast Cancer Progression.

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.

Laminin-111 Improves the Anabolic Response to Mechanical Load in Aged Skeletal Muscle.

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.

Electrically stimulated hind limb muscle contractions increase adult hippocampal astrogliogenesis but not neurogenesis or behavioral performance in male C57BL/6J mice.

Regular exercise is crucial for maintaining cognitive health throughout life. Recent evidence suggests muscle contractions during exercise release factors into the blood which cross into the brain and stimulate adult hippocampal neurogenesis. However, no study has tested whether muscle contractions alone are sufficient to increase adult hippocampal neurogenesis and improve behavioral performance. Adult male, C57BL/6J mice were anesthetized and exposed to bilateral hind limb muscle contractions (both concentric and eccentric) via electrical stimulation (e-stim) of the sciatic nerve twice a week for 8 weeks. Each session lasted approximately 20 min and consisted of a total of 40 muscle contractions. The control group was treated similarly except without e-stim (sham). Acute neuronal activation of the dentate gyrus (DG) using cFos immunohistochemistry was measured as a negative control to confirm that the muscle contractions did not activate the hippocampus, and in agreement, no DG activation was observed. Relative to sham, e-stim training increased DG volume by approximately 10% and astrogliogenesis by 75%, but no difference in neurogenesis was detected and no improvement in behavioral performance was observed. E-stim also increased astrogliogenesis in CA1/CA2 hippocampal subfields but not in the cortex. Results demonstrate that muscle contractions alone, in absence of DG activation, are sufficient to increase adult hippocampal astrogliogenesis, but not neurogenesis or behavioral performance in mice.

Cognitive function is preserved in aged mice following long-term ß-hydroxy ß-methylbutyrate supplementation.

ß-hydroxy ß-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<0.05). The rate of muscle protein synthesis decreased within the mitochondrial fraction (age main effect, P<0.05), and this decline was not prevented with HMB. Despite no change in muscle mass or function, an age-dependent reduction in active avoidance learning was attenuated with HMB (Age and HMB main effects, P<0.05). Age detrimentally impacted muscle-resident pericyte gene expression with no recovery observed with HMB, whereas no changes in brain-resident pericyte quantity or function were observed with age or HMB. The findings from this study suggest that prolonged HMB supplementation starting in adulthood may preserve cognition with age.

Resistance Exercise-induced Regulation of Muscle Protein Synthesis to Intraset Rest.

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 impact of skeletal muscle contraction on CD146+Lin- pericytes.

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.

Integrin signaling: linking mechanical stimulation to skeletal muscle hypertrophy.

The α7ß1-integrin is a transmembrane adhesion protein that connects laminin in the extracellular matrix (ECM) with actin in skeletal muscle fibers. The α7ß1-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 α7ß1-integrin can contribute to mechanical-load induced skeletal muscle growth via an mammalian target of rapamycin complex 1-independent mechanism.

Matrix Topography Regulates Synaptic Transmission at the Neuromuscular Junction.

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.

Pericyte transplantation improves skeletal muscle recovery following hindlimb immobilization.

Conditions of extended bed rest and limb immobilization can initiate rapid and significant loss of skeletal muscle mass and function. Physical rehabilitation is standard practice following a period of disuse, yet mobility may be severely compromised, and recovery is commonly delayed or incomplete in special populations. Thus, a novel approach toward recovery of muscle mass is highly desired. Pericytes [neuron-glial antigen 2 (NG2)+CD31-CD45- (Lineage- [Lin-]) and CD146+Lin-] demonstrate capacity to facilitate muscle repair, yet the ability to enhance myofiber growth following disuse is unknown. In the current study, 3-4-mo-old mice were unilaterally immobilized for 14 d (IM) or immobilized for 14 d followed by 14 d of remobilization (RE). Flow cytometry and targeted gene expression analyses were completed to assess pericyte quantity and function following IM and RE. In addition, a transplantation study was conducted to assess the impact of pericytes on recovery. Results from targeted analyses suggest minimal impact of disuse on pericyte gene expression, yet NG2+Lin- pericyte quantity is reduced following IM (P < 0.05). Remarkably, pericyte transplantation recovered losses in myofiber cross-sectional area and the capillary-to-fiber ratio following RE, whereas deficits remained with vehicle alone (P = 0.01). These findings provide the first evidence that pericytes effectively rehabilitate skeletal muscle mass following disuse atrophy.-Munroe, M., Dvoretskiy, S., Lopez, A., Leong, J., Dyle, M. C., Kong, H., Adams, C. M., Boppart, M. D. Pericyte transplantation improves skeletal muscle recovery following hindlimb immobilization.

The impact of mechanically stimulated muscle-derived stromal cells on aged skeletal muscle.

Perivascular stromal cells, including mesenchymal stem/stromal cells (MSCs), secrete paracrine factor in response to exercise training that can facilitate improvements in muscle remodeling. This study was designed to test the capacity for muscle-resident MSCs (mMSCs) isolated from young mice to release regenerative proteins in response to mechanical strain in vitro, and subsequently determine the extent to which strain-stimulated mMSCs can enhance skeletal muscle and cognitive performance in a mouse model of uncomplicated aging. Protein arrays confirmed a robust increase in protein release at 24h following an acute bout of mechanical strain in vitro (10%, 1Hz, 5h) compared to non-strain controls. Aged (24month old), C57BL/6 mice were provided bilateral intramuscular injection of saline, non-strain control mMSCs, or mMSCs subjected to a single bout of mechanical strain in vitro (4×104). No significant changes were observed in muscle weight, myofiber size, maximal force, or satellite cell quantity at 1 or 4wks between groups. Peripheral perfusion was significantly increased in muscle at 4wks post-mMSC injection (p<0.05), yet no difference was noted between control and preconditioned mMSCs. Intramuscular injection of preconditioned mMSCs increased the number of new neurons and astrocytes in the dentate gyrus of the hippocampus compared to both control groups (p<0.05), with a trend toward an increase in water maze performance noted (p=0.07). Results from this study demonstrate that acute injection of exogenously stimulated muscle-resident stromal cells do not robustly impact aged muscle structure and function, yet increase the survival of new neurons in the hippocampus.

Multimodal Assessment of Mesenchymal Stem Cell Therapy for Diabetic Vascular Complications.

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 αVß3 -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.

In Vivo Assessment of Engineered Skin Cell Delivery with Multimodal Optical Microscopy.

The healing process is often significantly impaired under conditions of chronic or large area wounds, which are often treated clinically using autologous split-thickness skin grafts. However, in many cases, harvesting of donor tissue presents a serious problem such as in the case of very large area burns. In response to this, engineered biomaterials have emerged that attempt to mimic the natural skin environment or deliver a suitable therapy to assist in the healing process. In this study, a custom-built multimodal optical microscope capable of noninvasive structural and functional imaging is used to investigate both the engineered tissue microenvironment and the in vivo wound healing process. Investigation of various engineered scaffolds show the strong relationship among the microenvironment of the scaffold, the organization of the cells within the scaffold, and the delivery pattern of these cells onto the healing wound. Through noninvasive tracking of these processes and parameters, multimodal optical microscopy provides an important tool in the assessment of engineered scaffolds both in vitro and in vivo.

α7ß1 Integrin regulation of gene transcription in skeletal muscle following an acute bout of eccentric exercise.

The α7ß1 integrin 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 α7ß1 integrin 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.

Impact of ß-hydroxy ß-methylbutyrate (HMB) on age-related functional deficits in mice.

ß-Hydroxy ß-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.