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Timothy Jee Kam Koh Department of Renal Medicine, Tan Tock Seng Hospital, 308433, Singapore Abstract: Conventional hemodialysis is associated with high morbidity and mortality rates, as well as a reduced quality of life. There is a growing interest in the provision of more intensive hemodialysis, due to associated benefits in terms of reduced cardiovascular morbidity, better regulation of mineral metabolism, as well as its impact on quality of life measures, fertility, and sleep. Nocturnal hemodialysis, both in center and at home, allows the delivery of more intensive hemodialysis. This review discusses the benefits of nocturnal hemodialysis and evaluates the evidence based on available literature. Keywords: nocturnal hemodialysis, quality of life, benefits, outcomes, survival, cardiac

Research articles about the effects of rumen-protected choline (RPC) supplementation on metabolic response, and reproductive and productive performances in transitional dairy cows were reviewed and presented. Analysis was conducted on 32 research papers that were published. The papers examined treatments without RPC supplementation and RPC supplementation levels varying from 10 to 100 gm/day. The feeding duration of RPC started from 40 to 140 days prepartum and continued until 20 to 140 days postpartum in multiparous dairy cows. Studies indicated that adding herbal choline (Cho) to the diet of transition dairy cows resulted in increased milk production and improved milk quality, leading to enhanced energy balance and reduced oxidative stress. The concentration and yield of IgG in colostrum provide passive immunity to dairy newborns and can be enhanced by dietary Cho supplementation. The inconsistent effects of RPC supplementation on reproduction may be due to several factors such as heat stress, genetics, and management. RPC supplementation improved the transition dairy cows' milk yield and quality, but dosage response was not observed as in the prior publication. Remarkably, the length of RPC supplementation had a positive correlation with an increase in milk yield. Based on this review, 45-50 gm/day dietary RPC supplementation between 3 weeks pre-calving to 8 weeks post-calving is suggested to increase at least 10% milk yield in dairy cows., Competing Interests: The authors declare that they have no conflicts of interest., (© The authors.)

Background: (1)Sarcopenia, or low skeletal mass index (SMI), contributes to higher lung cancer mortality. The SMI at third lumbar vertebrae (L3) is the reference standard for body composition analysis. However, there is a need to explore the validity of alternative landmarks in this population. We compared the agreement of sarcopenia identification at the first lumbar (L1) and second lumbar (L2) to L3 in non-Hispanic Black (NHB) and White (NHW) individuals with lung cancer., Methods: (2)This retrospective, cross-sectional study included 214 NHB and NHW adults with lung cancer. CT scans were analyzed to calculate the SMI at L1, L2, and L3. T-tests, chi-square, Pearson's correlation, Cohen's kappa, sensitivity, and specificity analysis were used., Results: (3)Subjects presented with a mean age of 68.4 ± 9.9 years and BMI of 26.3 ± 6.0 kg/m 2 . Sarcopenia prevalence varied from 19.6% at L1 to 39.7% at L3. Cohen's kappa coefficient was 0.46 for L1 and 0.64 for L2, indicating weak and moderate agreement for the identification of sarcopenia compared to L3., Conclusions: (4)Sarcopenia prevalence varied greatly depending on the vertebral landmark used for assessment. Using L2 or L1 alone resulted in a 16.8% and 23.8% misclassification of sarcopenia in this cohort of individuals with lung cancer., Competing Interests: Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Obesity is associated with low-grade chronic inflammation and impaired glucose metabolism, both of which are detrimental to wound healing. C-C motif chemokine receptor 2 (CCR2) plays an important role in cell recruitment during healing, and our recent studies revealed the significance of CCR2-CCL2 signaling in promoting the proliferation of pro-inflammatory monocytes/macrophages in wounds. Therefore, we sought to determine whether diet-induced obesity increases monocyte/macrophage proliferation and their accumulation in skin wounds. We first confirmed that wound closure was delayed in obese CCR2 RFP/+ mice fed with a high-fat diet (HFD) compared to mice fed with a normal diet (ND). Using in vivo imaging and flow cytometry analysis, we found that HFD mice had significantly increased accumulation of CCR2+ monocytes/macrophages, particularly pro-inflammatory CCR2+Ly6C+ cells in wounds compared to their ND counterparts. Importantly, HFD mice exhibited an increased proliferation of wound CCR2+Ly6C+ compared to ND mice. Together, our data suggest that obesity leads to an increased proliferation and accumulation of pro-inflammatory CCR2+Ly6C+ monocytes/macrophages in skin wounds, which may contribute to delayed healing.

Many factors regulate scar formation, which yields a modified extracellular matrix (ECM). Among ECM components, microfibril-associated proteins have been minimally explored in the context of skin wound repair. Microfibril-associated protein 5 (MFAP5), a small 25 kD serine and threonine rich microfibril-associated protein, influences microfibril function and modulates major extracellular signaling pathways. Though known to be associated with fibrosis and angiogenesis in certain pathologies, MFAP5's role in wound healing is unknown. Using a murine model of skin wound repair, we found that MFAP5 is significantly expressed during the proliferative and remodeling phases of healing. Analysis of existing single-cell RNA-sequencing data from mouse skin wounds identified two fibroblast subpopulations as the main expressors of MFAP5 during wound healing. Furthermore, neutralization of MFAP5 in healing mouse wounds decreased collagen deposition and refined angiogenesis without altering wound closure. In vitro, recombinant MFAP5 significantly enhanced dermal fibroblast migration, collagen contractility, and expression of pro-fibrotic genes. Additionally, TGF-ß1 increased MFAP5 expression and production in dermal fibroblasts. Our findings suggest that MFAP5 regulates fibroblast function and influences scar formation in healing wounds. Our work demonstrates a previously undescribed role for MFAP5 and suggests that microfibril-associated proteins may be significant modulators of wound healing outcomes and scarring., (© 2023. The Author(s).)

Introduction: Chronic, non-healing skin wounds such as diabetic foot ulcers (DFUs) are common in patients with type 2 diabetes mellitus (T2DM) and often result in limb amputation and even death. However, mechanisms by which T2DM and inflammation negatively impact skin wound healing remains poorly understood. Here we investigate a mechanism by which an excessive level of chemokine CCL28, through its receptor CCR10, impairs wound healing in patients and mice with T2DM. Methods & Results: Firstly, a higher level of CCL28 was observed in skin and plasma in both patients with T2DM, and in obesity-induced type 2 diabetic db/db mice. Compared with WT mice, adipose tissue from db/db mice released 50% more CCL28, as well as 2- to 3-fold more IL-1β, IL-6, and TNF-α, and less VEGF, as determined by ELISA measurements. Secondly, overexpression of CCL28 with adenovirus (Adv-CCL28) caused elevation of proinflammatory cytokines as well as CCR10 expression and also reduced eNOS expression in the dorsal skin of WT mice as compared with control Adv. Thirdly, topical application of neutralizing anti-CCL28 Ab dose-dependently accelerated wound closure and eNOS expression, and decreased IL-6 level, with an optimal dose of 1 μg/wound. In addition, mRNA levels of eNOS and anti-inflammatory cytokine IL-4 were increased as shown by real-time RT-PCR. The interaction between eNOS and CCR10 was significantly reduced in diabetic mouse wounds following application of the optimal dose of anti-CCL28 Ab, and eNOS expression increased. Finally, enhanced VEGF production and increased subdermal vessel density as indicated by CD31 immunostaining were also observed with anti-CCL28 Ab. Discussion: Taken together, topical application of neutralizing anti-CCL28 Ab improved dorsal skin wound healing by reducing CCR10 activation and inflammation in part by preventing eNOS downregulation, increasing VEGF production, and restoring angiogenesis. These results indicate anti-CCL28 Ab has significant potential as a therapeutic strategy for treatment of chronic non-healing diabetic skin wounds such as DFUs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Haus, DiPietro, Koh and Minshall.)

Background: Lung cancer incidence and mortality rates are higher in Non-Hispanic Black (NHB) compared to Non-Hispanic White (NHW) individuals in the Chicago metropolitan area, which may be related to exposure to chronic stress which may increase inflammation., Specific Aim: We investigated disparities in inflammation as measured by neutrophil to lymphocyte ratio (NLR) in individuals with lung cancer by race and by neighborhood concentrated disadvantage index (CDI)., Methods: This retrospective, cross-sectional study included 263 NHB and NHW adults with lung cancer. We analyzed NLR as a continuous and categorical variable to determine degree and prevalence of inflammation. We used Mann Whitney U, t-tests, Chi square tests, linear and logistic regression models as appropriate., Results: More than 60% of subjects had inflammation (NLR ≥ 3) at lung cancer diagnosis. The degree of inflammation was significantly lower in NHB (NLR 5.50 +/- 7.45) compared to NHW individuals (NLR 6.53 +/- 6.53; p=0.01) but did not differ by neighborhood CDI. The prevalence of inflammation (NLR ≥ 3) was significantly lower in NHB (55.07%) compared to NHW individuals (71.20%; p<0.01) and in those from the most disadvantaged (54.07%) compared to the least disadvantaged (71.88%; p<0.01) neighborhoods., Conclusion: At lung cancer diagnosis, there is a lower degree and prevalence of inflammation in NHB compared to NHW individuals, and lower prevalence in those residing in the most disadvantaged neighborhoods. Further research is needed to determine mechanisms of inflammation that may be contributing to lung cancer disparities as well as whether NLR is an appropriate biomarker when examining racial differences in inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Byrne, Gomez, Kim, Oddo, Koh and Fantuzzi.)

People with spinal cord injury (SCI) are at high risk of developing a pressure injury. It is unclear why some people with SCI develop pressure injury while others with similar predisposing risk factors do not during acute hospitalisation. This may hinder healthcare utilisation to prevent pressure injuries. The purpose of the study was to examine the proof-of-concept objective bedside skin blood flow measurements before a pressure injury develops in spinal cord injured patients during acute hospitalisation. This was an observational study. All participants had acute traumatic SCI and were pressure injury-free upon enrollment. Skin blood flow patterns were collected at both heels under two circumstances: localised pressure for reactive hyperemia, and localised heating for heat hyperemia. Our results showed that reactive and heat hyperemia were successfully induced in all eleven participants. Two participants developed pressure injury and nine did not have pressure injury at discharge. Heat hyperemia was smaller in participants with pressure injury. No difference was observed in reactive hyperemia between the groups. In conclusion, skin blood flow measurements could be obtained at bedside during acute hospitalisation of SCI for the purpose of research. Further examination of a larger group is warranted to determine clinical use of heat hyperemia pattern as predictor for pressure injury development., (© 2022 The Authors. International Wound Journal published by Medicalhelplines.com Inc (3M) and John Wiley & Sons Ltd.)

Chronic, nonhealing skin wounds, such as diabetic foot ulcers (DFUs), are common in patients with type 2 diabetes. Here, we investigated the role of chemokine (C-C motif) ligand 28 (CCL28) and its receptor C-C chemokine receptor type 10 (CCR10) in downregulation of endothelial nitric (NO) oxide synthase (eNOS) in association with delayed skin wound healing in the db/db mouse model of type 2 diabetes. We observed reduced eNOS expression and elevated CCL28/CCR10 levels in dorsal skin of db/db mice and subdermal leg biopsy specimens from human subjects with type 2 diabetes. Further interrogation revealed that overexpression of CCR10 reduced eNOS expression, NO bioavailability, and tube formation of human dermal microvascular endothelial cells (HDMVECs) in vitro, which was recapitulated in mouse dorsal skin. In addition, incubation of HDMVECs with CCL28 led to internalization of the CCR10/eNOS complex and colocalization with lysosome-associated membrane protein 1. Finally, topical application of myristoylated CCR10 binding domain 7 amino acid (Myr-CBD7) peptide prevented CCR10-eNOS interaction and subsequent eNOS downregulation, enhanced eNOS/NO levels, eNOS/VEGF-R2+ microvessel density, and blood perfusion, reduced inflammatory cytokine levels, and importantly, decreased wound healing time in db/db mice. Thus, endothelial cell CCR10 activation in genetically obese mice with type 2 diabetes promotes eNOS depletion and endothelial dysfunction, and targeted disruption of CCR10/eNOS interaction improves wound healing., (© 2022 by the American Diabetes Association.)

Monocytes and macrophages (Mo/MΦ) rapidly accumulate in skin wounds after injury, then disappear as healing progresses. However, the mechanisms underlying their ultimate fate in wounds remain to be elucidated. Here, we show that apoptosis of Mo/MΦ parallels their reduction as wound healing progresses in non-diabetic mice. scRNAseq analysis confirmed enriched apoptosis GO pathways on day 6 post-injury in wound Mo/MΦ from non-diabetic mice. In contrast, there was significantly less Mo/MΦ apoptosis in wounds from diabetic mice, particularly in the pro-inflammatory Ly6C+ population, which may contribute to persistent Mo/MΦ accumulation and chronic inflammation. scRNAseq analysis implicated TNF, MAPK, Jak-STAT, and FoxO signaling pathways in promoting wound Mo/MΦ apoptosis in non-diabetic mice while cell proliferation related pathways appeared to be activated in diabetic mice. These novel findings indicate that reduced apoptosis is a contributor to persistent Mo/MΦ accumulation in diabetic wounds. These findings also highlight pathways that may regulate Mo/MΦ apoptosis during wound healing, which could be targeted to help resolve inflammation and improve healing., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Chronic wounds in diabetic patients represent an escalating health problem, leading to significant morbidity and mortality. Our group previously reported that whole body low-intensity vibration (LIV) can improve angiogenesis and wound healing in diabetic mice. The purpose of the current study was to determine whether effects of LIV on wound healing are frequency and/or amplitude dependent. Wound healing was assessed in diabetic (db/db) mice exposed to one of four LIV protocols with different combinations of two acceleration magnitudes (0.3 and 0.6 g) and two frequencies (45 and 90 Hz) or in non-vibration sham controls. The low acceleration, low frequency protocol (0.3 g and 45 Hz) was the only one that improved wound healing, increasing angiogenesis and granulation tissue formation, leading to accelerated re-epithelialization and wound closure. Other protocols had little to no impact on healing with some evidence that 0.6 g accelerations negatively affected wound closure. The 0.3 g, 45 Hz protocol also increased levels of insulin-like growth factor-1 and tended to increase levels of vascular endothelial growth factor in wounds, but had no effect on levels of basic fibroblast growth factor or platelet derived growth factor-bb, indicating that this LIV protocol induces specific growth factors during wound healing. Our findings demonstrate parameter-dependent effects of LIV for improving wound healing that can be exploited for future mechanistic and therapeutic studies., Competing Interests: TK has a patent pending on the application of vibration for therapeutic treatment for tissue repair “Method and system for physical stimulation of tissue” (#2013/0165,824). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Roberts, Bilgen, Kineman and Koh.)

Macrophages are prominent cells in normally healing adult skin wounds, yet their exact functions and functional significance to healing outcomes remain enigmatic. Many functional attributes are ascribed to wound macrophages, including host defense and support of the proliferation of new tissue to replace that lost by injury. Indeed, the depletion of macrophages is unmistakably detrimental to normal skin healing in adult mammals. Yet in certain systems, dermal wounds seem to heal well with limited or even no functional macrophages, creating an apparent paradox regarding the function of this cell in wounds. Recent advances in our understanding of wound macrophage phenotypes, along with new information about cellular plasticity in wounds, may provide some explanation for the apparently contradictory findings and suggest new paradigms regarding macrophage function in wounds. Continued study of this remarkable cell is needed to develop effective therapeutic options to improve healing outcomes.

Background: After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. The sequence of M1 and M2 macrophage accumulation and the duration of each subtype in the injured area may help to direct the relative extent of fibrogenesis and myogenesis during healing. We hypothesized that increasing the number of M1 macrophages early after traumatic muscle injury would produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing., Methods: To test this hypothesis, we transfected skeletal muscle with a plasmid vector to transiently express GM-CSF shortly after injury to drive the polarization of macrophages towards the M1 subset. C57BL/6 mouse tibialis anterior (TA) muscles were injured by contusion and electroporated with uP-mGM, which is a plasmid vector that transiently expresses GM-CSF. Myogenesis, angiogenesis, and fibrosis were evaluated by histology, immunohistochemistry, and RT-qPCR; subpopulations of macrophages by flow cytometry; and muscle functioning by the maximum running speed on the treadmill and the recovery of muscle mass., Results: Muscle injury increased the number of local M1-like macrophages and decreased the number of M2-like macrophages on day 4, and uP-mGM treatment enhanced this variation. uP-mGM treatment decreased TGF-β1 protein expression on day 4, and the Sirius Red-positive area decreased from 35.93 ± 15.45% (no treatment) to 2.9% ± 6.5% (p < 0.01) on day 30. uP-mGM electroporation also increased Hgf, Hif1α, and Mtor gene expression; arteriole density; and muscle fiber number during regeneration. The improvement in the quality of the muscle tissue after treatment with uP-mGM affected the increase in the TA muscle mass and the maximum running speed on a treadmill., Conclusion: Collectively, our data show that increasing the number of M1-like macrophages immediately after traumatic muscle injury promotes muscle recovery with less fibrosis, and this can be achieved by the transient expression of GM-CSF.

Monocytes (Mo) and macrophages (Mϕ) play important roles in normal skin wound healing, and dysregulation of wound Mo/Mϕ leads to impaired wound healing in diabetes. Although skin wound Mϕ originate both from tissue resident Mϕ and infiltrating bone marrow-derived Mo, the latter play dominant roles during the inflammatory phase of wound repair. Increased production of bone marrow Mo caused by alterations of hematopoietic stem and progenitor cell (HSPC) niche and epigenetic modifications of HSPCs likely contributes to the enhanced number of wound Mϕ in diabetes. In addition, an impaired transition of diabetic wound Mϕ from "pro-inflammatory" to "pro-healing" phenotypes driven by the local wound environment as well as intrinsic changes in bone marrow Mo is also thought to be partly responsible for impaired diabetic wound healing. The current brief review describes the origin, heterogeneity and function of wound Mϕ during normal skin wound healing followed by discussion of how dysregulated wound Mϕ numbers and phenotype are associated with impaired diabetic wound healing. The review also highlights the possible links between altered bone marrow myelopoiesis and increased Mo production as well as extrinsic and intrinsic factors that drive wound macrophage dysregulation leading to impaired wound healing in diabetes., (Copyright © 2020 Barman and Koh.)

Monocytes and macrophages (Mo/MΦ) play critical roles in all phases of skin wound healing. The majority of these cells are thought to be recruited from blood Mo; however, the role local proliferation of Mo/MΦ in the wound has not been defined. Therefore, we tested the hypothesis that local proliferation of Mo and/or MΦ contributes to their accumulation during wound healing. Male C57Bl/6 mice (N = 4-9/group) were subjected to excisional skin wounding. Proliferating Mo/MΦ (F4/80+Ki67+) were observed in wound cryosections, peaking on day 5 post-wounding. Cell cycle analysis on cells isolated from skin tissue revealed that wounding increased both the number and percentage of inflammatory Ly6C+F4/80lo/- Mo/MΦ in the S/G2/M phases, peaking on day 6 post-wounding. In contrast, more mature Ly6C-F4/80+ cells were found predominantly in the G0 phase with less than 1% cells in S/G2/M phase following injury. In peripheral blood, Mo were very rarely found in the S/G2/M phase, suggesting that the wound environment triggered the Ly6C+F4/80lo/- Mo proliferative response. Furthermore, injury induced several potential regulators of proliferation in wounds, including IL-1β and IL-6, and wound Mo/MΦ expressed surface receptors for these cytokines. However, wound Mo/MΦ proliferation was not altered in IL-1R1 knockout (KO) or IL-6 KO mice. In summary, our findings indicate that proliferation contributes to Mo/MΦ accumulation in wounds and, contrary to findings in other pathophysiologic conditions, Ly6C+/F4/80lo/- Mo/MΦ proliferate during skin wound healing whereas mature Ly6C-F4/80+ MΦ do not., (©2019 Society for Leukocyte Biology.)

Recovery from traumatic muscle injuries is typically prolonged and incomplete. Our previous study demonstrated that whole-body low-intensity vibration (LIV) enhances healing in a mouse laceration model. We sought to determine whether locally applied LIV (a) improves muscle repair following injury in mice and (b) is directly transduced by cultured muscle cells, via increased IGF-1 activity. C57BL/6J mice were subjected to laceration of the gastrocnemius muscle and were treated with LIV applied directly to the lower leg for 30 min/day or non-LIV sham treatment (controls) for 7 or 14 days. LIV was also applied to differentiating myotubes in culture for 30 min/day for 3 or 6 days. Compared with control mice, LIV increased myofiber cross-sectional area, diameter, and percent area of peripherally nucleated fibers, and decreased percent damaged area after 14 days of treatment. In cultured myotubes, LIV increased fusion and diameter compared with controls after 6 days of treatment. These LIV-induced effects were associated with increased total Akt on day 7 in injured muscle and on day 3 in myotubes, whereas phosphorylated-to-total Akt ratio increased on day 14 in injured muscle and on day 6 in myotubes but were not associated with increased IGF-1 levels at any time point. These changes were also associated with LIV-induced suppression of FOXO1 and Atrogin-1 gene expression at day 7 in injured muscle. These findings demonstrate that muscle cells can directly transduce LIV signals into increased growth and differentiation, and this effect is associated with increased Akt signaling., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Background: Cachexia occurs in individuals affected by chronic diseases in which systemic inflammation leads to fatigue, debilitation, decreased physical activity and sarcopenia. The pathogenesis of cachexia-associated sarcopenia is not fully understood., Objectives: The aim of this systematic review is to summarize the current evidence on genes expressed in the skeletal muscles of humans with chronic disease-associated cachexia and/or sarcopenia (cases) compared to controls and to assess the strength of such evidence., Methods: We searched PubMed, EMBASE and CINAHL using three concepts: cachexia/sarcopenia and associated symptoms, gene expression, and skeletal muscle., Results: Eighteen genes were studied in at least three research articles, for a total of 27 articles analyzed in this review. Participants were approximately 60 years of age and majority male; sample size was highly variable. Use of comparison groups, matching criteria, muscle biopsy location, and definitions of cachexia and sarcopenia were not homogenous. None of the studies fulfilled all four criteria used to assess the quality of molecular analysis, with only one study powered on the outcome of gene expression. FOXO1 was the only gene significantly increased in cases versus healthy controls. No study found a significant decrease in expression of genes involved in autophagy, apoptosis or inflammation in cases versus controls. Inconsistent or non-significant findings were reported for genes involved in protein degradation, muscle differentiation/growth, insulin/insulin growth factor-1 or mitochondrial transcription., Conclusion: Currently available evidence on gene expression in the skeletal muscles of humans with chronic disease-associated cachexia and/or sarcopenia is not powered appropriately and is not homogenous; therefore, it is difficult to compare results across studies and diseases., Competing Interests: The authors have declared that no competing interests exist.

Aim: Investigate the immediate effect of low intensity vibration on skin blood flow and its underlying control mechanisms in healthy human participants., Materials and Methods: One-group pre-post design in a university laboratory setting. Nine adults underwent two bouts of 10-minute vibration (30Hz, peak acceleration 0.4g). Outcome measures include skin blood flow, and skin temperature on the right foot. To examine the control mechanisms underlying the vibration-induced blood flow response, SHORT-TIME Fourier analyses were computed to obtain the spectral densities for three frequency bands: metabolic (0.0095-0.02Hz), neurogenic (0.02-0.06Hz), and myogenic (0.06-0.15Hz). Non-parametric Friedman's tests were computed to compare changes of the outcome measures and control mechanisms over the course of vibration., Results: Vibration increased skin blood flow during both bouts of vibration, however the effect did not last after vibration was terminated. Myogenic spectral density increased during both bouts of vibration, whereas the metabolic and neurogenic spectral densities increased only during the 2nd bout of vibration. Interestingly, only the metabolic spectral density remained elevated after vibration ended., Conclusion: Low intensity vibration produced acute increases in skin blood flow mediated in part by vascular control mechanisms of myogenic origin. Further investigation is warranted to determine whether low intensity vibration induces similar increases in skin blood flow in populations prone to developing chronic non-healing wounds, such as spinal cord injury and diabetes., Competing Interests: The author of this manuscript (Koh) has the following competing interest: patent pending on the application of vibration for therapeutic treatment. The name for the pending patent is, “Method and system for physical stimulation of tissue” (#2013/0165,824). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Recovery from traumatic muscle injuries is typically prolonged and incomplete, leading to impaired muscle and joint function. We sought to determine whether mechanical stimulation via whole-body low-intensity vibration (LIV) could (1) improve muscle regeneration and (2) reduce muscle fibrosis following traumatic injury. C57BL/6J mice were subjected to a laceration of the gastrocnemius muscle and were treated with LIV (0.2 g at 90 Hz or 0.4 g at 45 Hz for 30 min/day) or non-LIV sham treatment (controls) for seven or 14 days. Muscle regeneration and fibrosis were assessed in hematoxylin and eosin or Masson's trichrome stained muscle cryosections, respectively. Compared to non-LIV control mice, the myofiber cross-sectional area was larger in mice treated with each LIV protocol after 14 days of treatment. Minimum fiber diameter was also larger in mice treated with LIV of 90 Hz/0.2 g after 14 days of treatment. There was also a trend toward a reduction in collagen deposition after 14 days of treatment with 45 Hz/0.4 g ( p = 0.059). These findings suggest that LIV may improve muscle healing by enhancing myofiber growth and reducing fibrosis. The LIV-induced improvements in muscle healing suggest that LIV may represent a novel therapeutic approach for improving the healing of traumatic muscle injuries., Competing Interests: Conflicts of Interest: Timothy J. Koh and Stefan Judex have a patent pending regarding the application of vibrations for therapeutic treatment. Eileen M. Weinheimer-Haus and Thomas F. Corbiere certify that he or she, or a member of his or her immediate family, has no funding or commercial associations (e.g., consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article.

The purpose of this study was to determine whether thrombospondin (TSP)-1 promotes macrophage activity and disease progression in dysferlinopathy. First, we found that levels of TSP-1 are elevated in blood of non-ambulant dysferlinopathy patients compared with ambulant patients and healthy controls, supporting the idea that TSP-1 levels are correlated with disease progression. We then crossed dysferlinopathic BlaJ mice with TSP-1 knockout mice and assessed disease progression longitudinally with magnetic resonance imaging (MRI). In these mice, deletion of TSP-1 ameliorated loss in volume and mass of the moderately affected gluteal muscle but not of the severely affected psoas muscle. T2 MRI parameters revealed that loss of TSP-1 modestly inhibited inflammation only in gluteal muscle of male mice. Histological assessment indicated that deletion of TSP-1 reduced inflammatory cell infiltration of muscle fibers, but only early in disease progression. In addition, flow cytometry analysis revealed that, in males, TSP-1 knockout reduced macrophage infiltration and phagocytic activity, which is consistent with TSP-1-enhanced phagocytosis and pro-inflammatory cytokine induction in cultured macrophages. In summary, TSP-1 appears to play an accessory role in modulating Mp activity in BlaJ mice in a gender, age and muscle-dependent manner, but is unlikely a primary driver of disease progression of dysferlinopathy., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Chronic inflammation plays a key role in the pathogenesis of myriad complications associated with diabetes and thus anti-inflammatory therapies may ameliorate these complications. Quercus infectoria (Qi) extract has been shown to downregulate inflammatory processes; however, the molecular mechanisms of this anti-inflammatory activity remain unclear. The hypothesis of our study was that Qi extract exerts its anti-inflammatory effect by downregulating the Set7/NF-κB pathway. Bone marrow-derived macrophages (BMM) were treated with high glucose plus palmitate medium (HG/Pa) to simulate the diabetic environment. Compared with control conditions, HG/Pa elevated expression Set7, expression and activity of NF-κB along with expression of several inflammatory cytokines. These changes were associated with increased levels of intracellular reactive oxygen species (ROS). Moreover, similar alterations were demonstrated in BMM derived from mice fed a high fat diet (HFD) compared to those from lean mice, suggesting that HFD-induced changes in BM progenitors persist throughout differentiation and culture. Importantly, Qi extract dose-dependently reduced Set7, p65 and inflammatory cytokine expression relative to vehicle controls in both HG/Pa-and HFD-treated BMM. Finally, macrophages/monocytes isolated from wounds of diabetic mice that were treated with Qi solution exhibited lower expression of the inflammatory cytokines, IL-1β and TNF-α, compared with vehicle treated wounds, demonstrating translation to the in vivo diabetic environment. Taken together, data from this study suggests that Qi downregulates diabetes-induced activity of the Set7/NF-kB pathway., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system. Current treatment modalities do not fully exploit the genetic basis between the different molecular subtypes and little is known about the targets discovered in recent mutational and genetic studies. Neuroblastomas with poor prognosis are often characterized by 1p36 deletion, containing the kinesin gene KIF1B. Its beta isoform, KIF1Bβ, is required for NGF withdrawal-dependent apoptosis, mediated by the induction of XIAP-associated Factor 1 (XAF1). Here, we showed that XAF1 low expression correlates with poor survival and disease status. KIF1Bβ deletion results in loss of XAF1 expression, suggesting that XAF1 is indeed a downstream target of KIF1Bβ. XAF1 silencing protects from NGF withdrawal and from KIF1Bβ-mediated apoptosis. Overexpression of XAF1 impairs tumor progression whereas knockdown of XAF1 promotes tumor growth, suggesting that XAF1 may be a candidate tumor suppressor in neuroblastoma and its associated pathway may be important for developing future interventions., Competing Interests: FINANCIAL SUPPORT AND CONFLICTS OF INTEREST Zhang'e Choo and the project is supported by the National Medical Research Council, Singapore New Investigator Grant awarded to Zhi Xiong Chen. Susanne Schlisio is supported by grants from the Swedish Children Cancer Foundation and the Swedish Research Council. In addition, all authors declare that there is no conflict of interest.

Background: Uremic neuropathy is a common complication in patients with end stage kidney disease. Its pathogenesis has been attributed to accumulation of uremic toxins. Kidney transplantation has been the best therapeutic option., Case Presentation: We describe a case of severe uremic peripheral neuropathy, which improved after conversion from a conventional renal replacement therapy to nocturnal hemodialysis., Conclusion: Enhanced uremic control by intensive hemodialysis may contribute at least in part to clinical and neurophysiological improvements of uremic neuropathy.

The Nod-like receptor protein (NLRP)-3 inflammasome/IL-1β pathway is involved in the pathogenesis of various inflammatory skin diseases, but its biological role in wound healing remains to be elucidated. Since inflammation is typically thought to impede healing, we hypothesized that loss of NLRP-3 activity would result in a downregulated inflammatory response and accelerated wound healing. NLRP-3 null mice, caspase-1 null mice and C57Bl/6 wild type control mice (WT) received four 8 mm excisional cutaneous wounds; inflammation and healing were assessed during the early stage of wound healing. Consistent with our hypothesis, wounds from NLRP-3 null and caspase-1 null mice contained lower levels of the pro-inflammatory cytokines IL-1β and TNF-α compared to WT mice and had reduced neutrophil and macrophage accumulation. Contrary to our hypothesis, re-epithelialization, granulation tissue formation, and angiogenesis were delayed in NLRP-3 null mice and caspase-1 null mice compared to WT mice, indicating that NLRP-3 signaling is important for early events in wound healing. Topical treatment of excisional wounds with recombinant IL-1β partially restored granulation tissue formation in wounds of NLRP-3 null mice, confirming the importance of NLRP-3-dependent IL-1β production during early wound healing. Despite the improvement in healing, angiogenesis and levels of the pro-angiogenic growth factor VEGF were further reduced in IL-1β treated wounds, suggesting that IL-1β has a negative effect on angiogenesis and that NLRP-3 promotes angiogenesis in an IL-1β-independent manner. These findings indicate that the NLRP-3 inflammasome contributes to the early inflammatory phase following skin wounding and is important for efficient healing.

The objective of this study was to determine the relative contributions of different cell subsets to the production of cytokines and growth factors during normal and impaired wound healing. Cells were isolated from wounds of non-diabetic and diabetic mice and separated by magnetic sorting into neutrophils/T cells/B cells (NTB cell subset), monocytes/macrophages (Mo/Mp subset) and non-leukocytic cells including keratinocyte/fibroblast/endothelial cells (KFE subset). On both per cell and total contribution bases, the Mo/Mp subset was the dominant producer of pro-inflammatory cytokines interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 in both non-diabetic and diabetic mice and was a significant producer of vascular endothelial cell growth factor (VEGF)-A, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-β1. The NTB subset was also a significant producer of TNF-α and IL-10 whereas the KFE subset contributed significant amounts of VEGF, IGF-1 and TGF-β1. Sustained production of pro-inflammatory cytokines and impaired production of healing-associated factors were evident in each subset in diabetic mice. These data will be useful for further experimental and modeling studies on the role of cell subsets in wound healing as well as for designing therapeutic strategies for improving healing., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Chronic wounds represent a significant health problem, especially in diabetic patients. In the current study, we investigated a novel therapeutic approach to wound healing--whole body low-intensity vibration (LIV). LIV is anabolic for bone, by stimulating the release of growth factors, and modulating stem cell proliferation and differentiation. We hypothesized that LIV improves the delayed wound healing in diabetic mice by promoting a pro-healing wound environment. Diabetic db/db mice received excisional cutaneous wounds and were subjected to LIV (0.4 g at 45 Hz) for 30 min/d or a non-vibrated sham treatment (controls). Wound tissue was collected at 7 and 15 d post-wounding and wound healing, angiogenesis, growth factor levels and wound cell phenotypes were assessed. LIV increased angiogenesis and granulation tissue formation at day 7, and accelerated wound closure and re-epithelialization over days 7 and 15. LIV also reduced neutrophil accumulation and increased macrophage accumulation. In addition, LIV increased expression of pro-healing growth factors and chemokines (insulin-like growth factor-1, vascular endothelial growth factor and monocyte chemotactic protein-1) in wounds. Despite no evidence of a change in the phenotype of CD11b+ macrophages in wounds, LIV resulted in trends towards a less inflammatory phenotype in the CD11b- cells. Our findings indicate that LIV may exert beneficial effects on wound healing by enhancing angiogenesis and granulation tissue formation, and these changes are associated with increases in pro-angiogenic growth factors.

The hypothesis of this study was that sustained activity of the Nod-like receptor protein (NLRP)-3 inflammasome in wounds of diabetic humans and mice contributes to the persistent inflammatory response and impaired healing characteristic of these wounds. Macrophages (Mp) isolated from wounds on diabetic humans and db/db mice exhibited sustained inflammasome activity associated with low level of expression of endogenous inflammasome inhibitors. Soluble factors in the biochemical milieu of these wounds are sufficient to activate the inflammasome, as wound-conditioned medium activates caspase-1 and induces release of interleukin (IL)-1β and IL-18 in cultured Mp via a reactive oxygen species-mediated pathway. Importantly, inhibiting inflammasome activity in wounds of db/db mice using topical application of pharmacological inhibitors improved healing of these wounds, induced a switch from proinflammatory to healing-associated Mp phenotypes, and increased levels of prohealing growth factors. Furthermore, data generated from bone marrow-transfer experiments from NLRP-3 or caspase-1 knockout to db/db mice indicated that blocking inflammasome activity in bone marrow cells is sufficient to improve healing. Our findings indicate that sustained inflammasome activity in wound Mp contributes to impaired early healing responses of diabetic wounds and that the inflammasome may represent a new therapeutic target for improving healing in diabetic individuals.

Macrophages are essential for the efficient healing of numerous tissues, and they contribute to impaired healing and fibrosis. Tissue repair proceeds through overlapping phases of inflammation, proliferation, and remodeling, and macrophages are present throughout this progression. Macrophages exhibit transitions in phenotype and function as tissue repair progresses, although the precise factors regulating these transitions remain poorly defined. In efficiently healing injuries, macrophages present during a given stage of repair appear to orchestrate transition into the next phase and, in turn, can promote debridement of the injury site, cell proliferation and angiogenesis, collagen deposition, and matrix remodeling. However, dysregulated macrophage function can contribute to failure to heal or fibrosis in several pathological situations. This review will address current knowledge of the origins and functions of macrophages during the progression of tissue repair, with emphasis on skin and skeletal muscle. Dysregulation of macrophages in disease states and therapies targeting macrophage activation to promote tissue repair are also discussed., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Diabetes is associated with persistent inflammation and defective tissue repair responses. The hypothesis of this study was that interleukin (IL)-1β is part of a proinflammatory positive feedback loop that sustains a persistent proinflammatory wound macrophage phenotype that contributes to impaired healing in diabetes. Macrophages isolated from wounds in diabetic humans and mice exhibited a proinflammatory phenotype, including expression and secretion of IL-1β. The diabetic wound environment appears to be sufficient to induce these inflammatory phenomena because in vitro studies demonstrated that conditioned medium of both mouse and human wounds upregulates expression of proinflammatory genes and downregulates expression of prohealing factors in cultured macrophages. Furthermore, inhibiting the IL-1β pathway using a neutralizing antibody and macrophages from IL-1 receptor knockout mice blocked the conditioned medium-induced upregulation of proinflammatory genes and downregulation of prohealing factors. Importantly, inhibiting the IL-1β pathway in wounds of diabetic mice using a neutralizing antibody induced a switch from proinflammatory to healing-associated macrophage phenotypes, increased levels of wound growth factors, and improved healing of these wounds. Our findings indicate that targeting the IL-1β pathway represents a new therapeutic approach for improving the healing of diabetic wounds.

Mp are crucial for tissue repair and regeneration but can also contribute to tissue damage and fibrosis. Mp can adopt a variety of functional phenotypes in response to different stimuli; two of the best-characterized in vitro phenotypes are a proinflammatory "M1" phenotype, produced by exposure to IFN-γ and TNF-α, and an anti-inflammatory "M2a" phenotype, produced by IL-4 or IL-13. M2a Mp are frequently termed "wound healing" Mp, as they express factors that are important for tissue repair. This review will summarize current knowledge of Mp phenotypes during tissue repair and will argue that these in vivo Mp populations are heterogeneous and temporally regulated and do not conform to existing, in vitro-defined M1 or M2 phenotypes. Mp during the early stages of tissue repair exhibit a more proinflammatory phenotype than their later counterparts, which in turn may exhibit some M2a-associated characteristics. However, phenotypic markers that appear to be coregulated in cultured Mp can be expressed independently of each other in vivo. Additionally, M1- and M2-associated markers may be expressed simultaneously by actual tissue-repair Mp. Improved understanding of Mp phenotypes and their regulation may assist in generation of novel therapies based on manipulating Mp function to improve healing.

The hypothesis of this study was that cells of the monocyte/macrophage lineage (Mo/Mp) exhibit an impaired transition from pro-inflammatory to pro-healing phenotypes in wounds of diabetic mice, which contributes to deficient healing. Mo/Mp isolated from excisional wounds in non-diabetic db/+ mice exhibited a pro-inflammatory phenotype on day 5 post-injury, with high level expression of the pro-inflammatory molecules interleukin-1β, matrix metalloprotease-9 and inducible nitric oxide synthase. Wound Mo/Mp exhibited a less inflammatory phenotype on day 10 post-injury, with decreased expression of the pro-inflammatory molecules and increased expression of the alternative activation markers CD206 and CD36. In contrast, in db/db mice, the pro-inflammatory phenotype persisted through day 10 post-injury and was associated with reduced expression of insulin-like growth factor-1, transforming growth factor-β1 and vascular endothelial growth factor. Reduced levels of these growth factors in wounds of db/db mice may have contributed to impaired wound closure, reduced granulation tissue formation, angiogenesis and collagen deposition. The persistent pro-inflammatory wound Mo/Mp phenotype in db/db mice may have resulted from elevated levels of pro-inflammatory interleukin-1β and interferon-γ and reduced levels of anti-inflammatory interleukin-10 in the wound environment. Our findings are consistent with the hypothesis that dysregulation of Mo/Mp phenotypes contributes to impaired healing of diabetic wounds., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Plasminogen activator inhibitor-1 (PAI-1) is increased in the lungs of patients with pulmonary fibrosis, and animal studies have shown that experimental manipulations of PAI-1 levels directly influence the extent of scarring that follows lung injury. PAI-1 has 2 known properties that could potentiate fibrosis, namely an antiprotease activity that inhibits the generation of plasmin, and a vitronectin-binding function that interferes with cell adhesion to this extracellular matrix protein. To determine the relative importance of each PAI-1 function in lung fibrogenesis, we administered mutant PAI-1 proteins that possessed either intact antiprotease or vitronectin-binding activity to bleomycin-injured mice genetically deficient in PAI-1. We found that the vitronectin-binding capacity of PAI-1 was the primary determinant required for its ability to exacerbate lung scarring induced by intratracheal bleomycin administration. The critical role of the vitronectin-binding function of PAI-1 in fibrosis was confirmed in the bleomycin model using mice genetically modified to express the mutant PAI-1 proteins. We conclude that the vitronectin-binding function of PAI-1 is necessary and sufficient in its ability to exacerbate fibrotic processes in the lung.

In obesity and type 2 diabetes, efficient skeletal muscle repair following injury may be required, not only for restoring muscle structure and function, but also for maintaining exercise capacity and insulin sensitivity. The hypothesis of this study was that muscle regeneration would be impaired in ob/ob and db/db mice, which are common mouse models of obesity and type 2 diabetes. Muscle injury was produced by cardiotoxin injection, and regeneration was assessed by morphological and immunostaining techniques. Muscle regeneration was delayed in ob/ob and db/db mice, but not in a less severe model of insulin resistance - feeding a high-fat diet to wild-type mice. Angiogenesis, cell proliferation, and myoblast accumulation were also impaired in ob/ob and db/db mice, but not the high-fat diet mice. The impairments in muscle regeneration were associated with impaired macrophage accumulation; macrophages have been shown previously to be required for efficient muscle regeneration. Impaired regeneration in ob/ob and db/db mice could be due partly to the lack of leptin signaling, since leptin is expressed both in damaged muscle and in cultured muscle cells. In summary, impaired muscle regeneration in ob/ob and db/db mice was associated with reduced macrophage accumulation, angiogenesis, and myoblast activity, and could have implications for insulin sensitivity in the skeletal muscle of obese and type 2 diabetic patients.

Selenium-binding protein (SBP) 1 is present in reduced levels in several cancer types as compared with normal tissues, and lower levels are associated with poor clinical prognosis. Another selenium-containing protein, glutathione peroxidase 1 (GPX1), has been associated with cancer risk and development. The interaction between these representatives of different classes of selenoproteins was investigated. Increasing SBP1 levels in either human colorectal or breast cancer cells by transfection of an expression construct resulted in the reduction of GPX1 enzyme activity. Increased expression of GPX1 in the same cell types resulted in the transcriptional and translational repression of SBP1, as evidenced by the reduction of SBP1 messenger RNA and protein and the inhibition of transcription measured using an SBP1 reporter construct. The opposing effects of SBP1 and GPX1 on each other were also observed when GPX1 was increased by supplementing the media of these tissue culture cells with selenium, and the effect of selenium on SBP1 was shown to be GPX1 dependent. Decreasing or increasing GPX1 levels in colonic epithelial cells of mice fed a selenium-deficient, -adequate or -supplemented diet resulted in the opposing effect on SBP1 levels. These data are explained in part by the demonstration that SBP1 and GPX1 form a physical association, as determined by coimmunoprecipitation and fluorescence resonance energy transfer assay. The results presented establish an interaction between two distinct selenium-containing proteins that may enhance the understanding of the mechanisms by which selenium and selenoproteins affect carcinogenesis in humans.

The plasminogen system plays a crucial role in the repair of a variety of tissues, including skeletal muscle. We hypothesized that urokinase-type plasminogen activator (uPA) promotes muscle regeneration by activating hepatocyte growth factor (HGF), which, in turn, stimulates proliferation of myoblasts required for regeneration. In our studies, levels of active HGF and phosphorylation of the HGF receptor c-met were increased after muscle injury in wild-type mice. Compared with wild-type animals, mice deficient in uPA (uPA(-/-)) had markedly reduced HGF levels and c-met activation after muscle damage. This reduced HGF activity in uPA(-/-) animals was associated with decreased cell proliferation, myoblast accumulation, and new muscle fiber formation. On the other hand, HGF activity was enhanced at early time points in PAI-1(-/-) mice compared with wild-type mice and the PAI-1(-/-) animals exhibited accelerated muscle fiber regeneration. Furthermore, administration of exogenous uPA rescued HGF levels and muscle regeneration in uPA(-/-) mice, and an HGF-blocking antibody reduced HGF activity and muscle regeneration in wild-type mice. We also found that uPA promotes myoblast proliferation in vitro through its proteolytic activity, and this process was inhibited by an HGF-blocking antibody. Together, our findings demonstrate that uPA promotes muscle regeneration through HGF activation and subsequent myoblast proliferation.

Macrophages are thought to play important roles during wound healing, but definition of these roles has been hampered by our technical inability to specifically eliminate macrophages during wound repair. The purpose of this study was to test the hypothesis that specific depletion of macrophages after excisional skin wounding would detrimentally affect healing by reducing the production of growth factors important in the repair process. We used transgenic mice that express the human diphtheria toxin (DT) receptor under the control of the CD11b promoter (DTR mice) to specifically ablate macrophages during wound healing. Mice without the transgene are relatively insensitive to DT, and administration of DT to wild-type mice does not alter macrophage or other inflammatory cell accumulation after injury and does not influence wound healing. In contrast, treatment of DTR mice with DT prevented macrophage accumulation in healing wounds but did not affect the accumulation of neutrophils or monocytes. Such macrophage depletion resulted in delayed re-epithelialization, reduced collagen deposition, impaired angiogenesis, and decreased cell proliferation in the healing wounds. These adverse changes were associated with increased levels of tumor necrosis factor-alpha and reduced levels of transforming growth factor-beta1 and vascular endothelial growth factor in the wound. In summary, macrophages seem to promote both wound closure and dermal healing, in part by regulating the cytokine environment of the healing wound.

Anti-inflammatory strategies are often used to reduce muscle pain and soreness that can result from high-intensity muscular activity. However, studies indicate that components of the acute inflammatory response may be required for muscle repair and growth. The hypothesis of this study was that cyclooxygenase (COX)-2 activity is required for compensatory hypertrophy of skeletal muscle. We used the synergist ablation model of skeletal muscle hypertrophy, along with the specific COX-2 inhibitor NS-398, to investigate the role of COX-2 in overload-induced muscle growth in mice. COX-2 was expressed in plantaris muscles during compensatory hypertrophy and was localized mainly in or near muscle cell nuclei. Treatment with NS-398 blunted the increases in mass and protein content in overloaded muscles compared with vehicle-treated controls. Additionally, the COX-2 inhibitor decreased activity of the urokinase type plasminogen activator, macrophage accumulation, and cell proliferation, all of which are required for hypertrophy after synergist ablation. Expression of insulin-like growth factor-1 and phosphorylation of Akt, mammalian target of rapamycin, and p70S6K were increased following synergist ablation, but were not affected by NS-398. Additionally, expression of atrogin-1 was reduced during hypertrophy, but was also not affected by NS-398. These results demonstrate that COX-2 activity is required for skeletal muscle hypertrophy, possibly through facilitation of extracellular protease activity, macrophage accumulation, and cell proliferation.

The inflammatory response is thought to play important roles in tissue healing. The hypothesis of this study was that the inflammatory cytokine interferon (IFN)-gamma is produced endogenously following skeletal muscle injury and promotes efficient healing. We show that IFN-gamma is expressed at both mRNA and protein levels in skeletal muscle following injury, and that the time course of IFN-gamma expression correlated with the accumulation of macrophages, T-cells, and natural killer cells, as well as myoblasts, in damaged muscle. Cells of each type were isolated from injured muscle, and IFN-gamma expression was detected in each cell type. We also demonstrate that administration of an IFN-gamma receptor blocking antibody to wild-type mice impaired induction of interferon response factor-1, reduced cell proliferation, and decreased formation of regenerating fibers. IFN-gamma null mice showed similarly impaired muscle healing associated with impaired macrophage function and development of fibrosis. In vitro studies demonstrated that IFN-gamma and its receptor are expressed in the C2C12 muscle cell line, and that the IFN-gamma receptor blocking antibody reduced proliferation and fusion of these muscle cells. In summary, our results indicate that IFN-gamma promotes muscle healing, in part, by stimulating formation of new muscle fibers.

Adult skeletal muscle possesses remarkable potential for growth in response to mechanical loading; however, many of the cellular and molecular mechanisms involved remain undefined. The hypothesis of this study was that the extracellular serine protease, urokinase-type plasminogen activator (uPA), is required for muscle hypertrophy, in part by promoting macrophage accumulation in muscle subjected to increased mechanical loading. Compensatory muscle hypertrophy was induced in mouse plantaris (PLT) muscles by surgical ablation of synergist muscles. Following synergist ablation, PLT muscles in wild-type mice demonstrated edema and infiltration of neutrophils and macrophages but an absence of overt muscle fiber damage. Sham procedures resulted in no edema or accumulation of inflammatory cells. In addition, synergist ablation was associated with a large increase in activity of uPA in the PLT muscle. uPA-null mice demonstrated complete abrogation of compensatory hypertrophy associated with reduced macrophage accumulation, indicating that uPA is required for hypertrophy. Macrophages isolated from wild-type PLT muscle during compensatory hypertrophy expressed uPA and IGF-I, both of which may contribute to hypertrophy. To determine whether macrophages are required for muscle hypertrophy, clodronate liposomes were administered to deplete macrophages in wild-type mice; this resulted in reduced muscle hypertrophy. Decreased macrophage accumulation was associated with reduced cell proliferation but did not alter signaling through the mammalian target of rapamycin pathway. These data indicate that uPA and macrophages are required for muscle hypertrophy following synergist ablation.

The hypothesis of this study was the urokinase-type plasminogen activator receptor (uPAR) is required for accumulation of inflammatory cells in injured skeletal muscle and for efficient muscle regeneration. Expression of uPAR was elevated at 1 and 3 days after cardiotoxin-induced muscle injury in wild-type mice before returning to baseline levels. Neutrophil accumulation peaked 1 day postinjury in muscle from both wild-type (WT) and uPAR null mice, while macrophage accumulation peaked between 3 and 5 days postinjury, with no differences between strains. Histological analyses confirmed efficient muscle regeneration in both wild-type and uPAR null mice, with no difference between strains in the formation or growth of regenerating fibers, or recovery of normal morphology. Furthermore, in vitro experiments demonstrated that chemotaxis is not different between WT and uPAR null macrophages. Finally, fusion of cultured satellite cells into multinucleated myotubes was not different between cells isolated from WT and uPAR null mice. These results demonstrate that uPAR is not required for the accumulation of inflammatory cells or the regeneration of skeletal muscle following injury, suggesting uPA can act independently of uPAR to regulate events critical for muscle regeneration.

Hypergastrinemia in INS-GAS mice leads to accelerated carcinogenesis of the stomach, but the mechanisms have not been well defined. We investigated the possible role of gastrin-induced gastric cell apoptosis in the development of gastric cancer. We examined apoptosis and the expression of Bcl-2 family proteins in INS-GAS mice of different ages, as well as in gastrin-deficient (GAS-KO) mice after gastrin-17 (G-17) infusion. In addition, we studied the effects of the gastrin/cholecystokinin-2 (CCK-2) receptor antagonist YF476 and/or histamine H2 (H-2) receptor antagonist loxtidine on apoptosis and atrophy in INS-GAS mice with or without Helicobacter felis (H. felis) infection. INS-GAS mice had age-associated increases in Bax protein expression and decreases in Bcl-2 protein expression, along with increased glandular and epithelial cell apoptosis. At 8-week gastrin infusions in GAS-KO mice resulted in a similar pattern of altered Bax and Bcl-2 expression, followed by gastric cell apoptosis. H. felis infection of INS-GAS mice led to increased apoptosis and the development of atrophy, whereas treatment with either YF476 and/or loxtidine strongly inhibited both apoptosis and atrophy. In vitro studies with Fas-expressing RGM1 cells showed that gastrin stimulation alone directly induced apoptosis via gastrin/CCK-2 receptor and synergized with FasL stimulation. These results indicate that gastrin can induce apoptosis in gastric epithelial cells and contribute to the development of gastric carcinogenesis.

Skeletal muscle possesses a remarkable capacity for regeneration. Although the regulation of this process at the molecular level remains largely undefined, the plasminogen system appears to play a critical role. Specifically, mice deficient in either urokinase-type plasminogen activator (uPA-/- mice) or plasminogen demonstrate markedly impaired muscle regeneration after injury. In the present study, we tested the hypothesis that loss of the primary inhibitor of uPA, plasminogen activator inhibitor-1 (PAI-1), would improve muscle regeneration. Repair of the extensor digitorum longus muscle was assessed after cardiotoxin injury in wild-type, uPA-/-, and PAI-1-deficient (PAI-1-/-) mice. As expected, there was no uPA activity in the injured muscles of uPA-/- mice, and muscles from these transgenic animals demonstrated impaired regeneration. On the other hand, uPA activity was increased in injured muscle from PAI-1-/- mice to a greater extent than in wild-type controls. Furthermore, PAI-1-/- mice demonstrated increased expression of MyoD and developmental myosin after injury as well as accelerated recovery of muscle morphology, protein levels, and muscle force compared with wild-type animals. The injured muscles of PAI-1-null mice also demonstrated increased macrophage accumulation, contrasting with impaired macrophage accumulation in uPA-deficient mice. The extent of macrophage accumulation correlated with both the clearance of protein after injury and the efficiency of regeneration. Taken together, these results indicate that PAI-1 deficiency promotes muscle regeneration, and this protease inhibitor represents a therapeutic target for enhancing muscle regeneration.

We tested the hypotheses that: (1) neutrophil accumulation after contraction-induced muscle injury is dependent on the beta(2) integrin CD18, (2) neutrophils contribute to muscle injury and oxidative damage after contraction-induced muscle injury, and (3) neutrophils aid the resolution of contraction-induced muscle injury. These hypotheses were tested by exposing extensor digitorum longus (EDL) muscles of mice deficient in CD18 (CD18(-/-); Itgb2(tm1Bay)) and of wild type mice (C57BL/6) to in situ lengthening contractions and by quantifying markers of muscle inflammation, injury, oxidative damage and regeneration/repair. Neutrophil concentrations were significantly elevated in wild type mice at 6 h and 3 days post-lengthening contractions; however, neutrophils remained at control levels at these time points in CD18-/- mice. These data indicate that CD18 is required for neutrophil accumulation after contraction-induced muscle injury. Histological and functional (isometric force deficit) signs of muscle injury and total carbonyl content, a marker of oxidative damage, were significantly higher in wild type relative to CD18-/- mice 3 days after lengthening contractions. These data show that neutrophils exacerbate contraction-induced muscle injury. After statistically controlling for differences in the force deficit at 3 days, wild type mice also demonstrated a higher force deficit at 7 days, a lower percentage of myofibres expressing embryonic myosin heavy chain at 3 and 7 days, and a smaller cross sectional area of central nucleated myofibres at 14 days relative to CD18-/- mice. These observations suggest that neutrophils impair the restoration of muscle structure and function after injury. In conclusion, neutrophil accumulation after contraction-induced muscle injury is dependent on CD18. Furthermore, neutrophils appear to contribute to muscle injury and impair some of the events associated with the resolution of contraction-induced muscle injury.

Several lines of evidence suggest that muscle cells can distinguish between specific mechanical stimuli. To test this concept, we subjected C(2)C(12) myotubes to cyclic uniaxial or multiaxial stretch. Both types of stretch induced an increase in extracellular signal-regulated kinase (ERK) and protein kinase B (PKB/Akt) phosphorylation, but only multiaxial stretch induced ribosomal S6 kinase (p70(S6k)) phosphorylation. Further results demonstrated that the signaling events specific to multiaxial stretch (p70(S6k) phosphorylation) were elicited by forces delivered through the elastic culture membrane and were not due to greater surface area deformations or localized regions of large tensile strain. Experiments performed using medium that was conditioned by multiaxial stretched myotubes indicated that a release of paracrine factors was not sufficient for the induction of signaling to p70(S6k). Furthermore, incubation with gadolinium(III) chloride (500 microM), genistein (250 microM), PD-98059 (250 microM), bisindolylmaleimide I (20 microM), or LY-294002 (100 microM ) did not block the multiaxial stretch-induced signaling to p70(S6k). However, disrupting the actin cytoskeleton with cytochalasin D did block the multiaxial signaling to p70(S6k), with no effect on signaling to PKB/Akt. These results demonstrate that specific types of mechanical stretch activate distinct signaling pathways, and we propose that this occurs through direct mechanosensory-mechanotransduction mechanisms and not through previously defined growth factor/receptor binding pathways.

Recently we have reported synergistic effects between glycine-extended gastrin (G-gly) and amidated gastrin-17 on acid secretion in short-term infusion studies. In the present study, we examined the long-term effect of G-gly on the atrophy-promoting effects of amidated gastrin in the mouse stomach with or without Helicobacter infection. Transgenic mice overexpressing amidated gastrin (INS-GAS mice), G-gly (MTI/G-gly mice), and both peptides (INS-GAS/G-gly mice) were used for assessment of acid secretion and ulcer susceptibility and histologic examination and scoring of preneoplastic lesions in response to the 3 and 6 months Helicobacter felis (H. felis) infection. We found that MTI/G-gly mice had normal gastric histology and acid secretion. Double transgenic (INS-GAS/G-gly) mice showed 2-fold increases in acid secretion compared with INS-GAS mice. Acute peptic ulcers after pyloric ligation were noted in 50% of the INS-GAS/G-gly mice but in none of the INS-GAS mice at 6 months of age. Whereas male INS-GAS mice had a >50% decrease in the numbers of parietal cell and enterochromaffin-like cell at 6 months of age, the male double transgenic mice had no such decrease. Overexpression of G-gly reduced the scores of preneoplasia in the stomach; however, it did not prevent the development of amidated gastrin-dependent gastric cancer in both H. felis-infected mice and uninfected mice. We conclude that G-gly synergizes with amidated gastrin to stimulate acid secretion and inhibits parietal cell loss in INS-GAS/G-gly mice. The overexpression of G-gly seems to increase the susceptibility to peptic ulcer disease and delay the development of Helicobacter-mediated gastric preneoplasia in this model.

Gastrin is a known growth/differentiation factor for the gastric mucosa. Its effects are likely mediated by the induction of heparin-binding epidermal-like growth factor (HB-EGF), a member of the EGF family of growth factors that is expressed by gastric parietal cells. In this study, we investigated the regulation of the HB-EGF promoter by gastrin in a human gastric cancer cell line. Serial human HB-EGF promoter-luciferase reporter deletion constructs and heterologous promoter constructs were transfected into AGS-E cells and stimulated with gastrin (10(-7) M) with or without various signal transduction inhibitors. EMSA were also performed. Gastrin stimulation resulted in a fivefold increase in HB-EGF-luciferase activity. The cis-acting element mediating gastrin responsiveness was mapped to the -69 to -58 region of the HB-EGF promoter. Gastrin stimulation was PKC dependent and at least partially mediated by activation of the EGF receptor.

The purposes of this study were to determine whether, immediately after lengthening contractions, 1) levels of specific force-transmitting cytoskeletal elements are reduced in skeletal muscle cells and 2) cytosolic small heat shock proteins (HSPs) translocate to structures prone to disruption. Western blot analysis demonstrated decreased concentrations of z-disk proteins alpha-actinin and plectin and membrane scaffolding proteins dystrophin and beta-spectrin in muscle exposed to lengthening contractions compared with contralateral control muscle. Lengthening contractions also resulted in immediate translocation of constitutively expressed HSP25 and alphaB-crystallin from the soluble to the insoluble fraction of muscle homogenates, and cryosections showed translocation from a diffuse, cytosolic localization to striations that corresponded to z-disks. Lengthening contraction-induced translocation of HSP25 and alphaB-crystallin was associated with phosphorylation of these small HSPs, which may trigger their protective activity. In summary, these findings demonstrate loss of z-disk and membrane scaffolding proteins immediately after lengthening contractions, and concomitant translocation of HSP25 and alphaB-crystallin to the z-disk, which may help to stabilize or repair cytoskeletal elements at this site.

The less processed forms of gastrin have recently been shown to act as trophic factors for both normal and malignant colonic cells. Although incompletely processed forms of gastrin such as glycine-extended gastrin and progastrin are also expressed in human lung cancers, the clinical significance of this expression has not been addressed. Consequently, we investigated the effects of overexpression of glycine-extended gastrin in a mouse strain that is prone to developing lung cancer and also examined the expression of incompletely processed gastrins in primary human lung cancers. We found that transgenic overexpression of glycine-extended gastrin in FVB/N mice resulted in a significant increase in the prevalence and growth of bronchoalveolar carcinoma. In addition, a substantial subset of human lung cancers was found to express progastrin and/or glycine-extended gastrin. Overexpression of glycine-extended gastrin by human lung cancers was associated with a significantly decreased survival. Taken together, these results suggest that glycine-extended gastrin may play a role in the growth and progression of some human lung cancers.