Samuel Herberg

The adipokine leptin increases skeletal muscle mass and significantly alters skeletal muscle miRNA expression profile in aged mice.

Age-associated loss of muscle mass, or sarcopenia, contributes directly to frailty and an increased risk of falls and fractures among the elderly. Aged mice and elderly adults both show decreased muscle mass as well as relatively low levels of the fat-derived hormone leptin. Here we demonstrate that loss of muscle mass and myofiber size with aging in mice is associated with significant changes in the expression of specific miRNAs. Aging altered the expression of 57 miRNAs in mouse skeletal muscle, and many of these miRNAs are now reported to be associated specifically with age-related muscle atrophy. These include miR-221, previously identified in studies of myogenesis and muscle development as playing a role in the proliferation and terminal differentiation of myogenic precursors. We also treated aged mice with recombinant leptin, to determine whether leptin therapy could improve muscle mass and alter the miRNA expression profile of aging skeletal muscle. Leptin treatment significantly increased hindlimb muscle mass and extensor digitorum longus fiber size in aged mice. Furthermore, the expression of 37 miRNAs was altered in muscles of leptin-treated mice. In particular, leptin treatment increased the expression of miR-31 and miR-223, miRNAs known to be elevated during muscle regeneration and repair. These findings suggest that aging in skeletal muscle is associated with marked changes in the expression of specific miRNAs, and that nutrient-related hormones such as leptin may be able to reverse muscle atrophy and alter the expression of atrophy-related miRNAs in aging skeletal muscle.

Evaluation of an injectable rhGDF-5/PLGA construct for minimally invasive periodontal regenerative procedures: a histological study in the dog.

AIM To evaluate the injectability, biocompatibility, safety, and periodontal wound healing/regeneration following application of a novel bioresorbable recombinant human growth/differentiation factor-5 (rhGDF-5)/poly(lactic-co-glycolic acid) (PLGA) construct. MATERIAL AND METHODS Periodontal pockets (3 x 6 mm, width x depth) were surgically created over the buccal roots of the second and fourth mandibular pre-molars in eight adult Hound Labrador mongrel dogs. Surgeries including injection of the rhGDF-5/PLGA construct into the pockets were sequenced that four animals provided 2-/4-week and four animals 6-/8-week observations of sites receiving rhGDF-5/PLGA or serving as sham-surgery control. RESULTS The rhGDF-5/PLGA construct was easy to prepare and apply. Approximately 0.2 ml (93 microg rhGDF-5)/tooth was used. Clinical and radiographic healing was exemplary without adverse events. Healing was characterized by a non-specific connective tissue attachment, acellular/cellular cementum, periodontal ligament (PDL), bone regeneration, and a junctional epithelium. PLGA fragments were observed in 4/7, 2/8, and 1/8 sites at 2, 4, and 6 weeks, respectively. Associated inflammatory reactions exhibited no limiting effect on periodontal wound healing/regeneration. Root resorption/ankylosis was not observed. Bone formation showed apparent increased maturity (lamellar bone) at 6 weeks in sites receiving rhGDF-5/PLGA compared with the control. Both protocols exhibited significant increases in PDL, cementum, and bone regeneration over time, without significant differences between treatments. In time, PDL and cementum regeneration was twofold greater for the control at 4 weeks (p=0.04) while increased bone formation was observed at sites receiving rhGDF-5/PLGA (p<0.01). CONCLUSIONS In conclusion, the rhGDF-5/PLGA construct appears to be a safe technology for injectable, ease-of-use application of rhGDF-5-stimulated periodontal wound healing/regeneration. Additional work to optimize the polymer carrier and rhGDF-5 release kinetics/dose might be required before evaluating the efficacy of this technology in clinical settings using minimally invasive approaches.

Effects of the activin A-myostatin-follistatin system on aging bone and muscle progenitor cells.

The activin A-myostatin-follistatin system is thought to play an important role in the regulation of muscle and bone mass throughout growth, development, and aging; however, the effects of these ligands on progenitor cell proliferation and differentiation in muscle and bone are not well understood. In addition, age-associated changes in the relative expression of these factors in musculoskeletal tissues have not been described. We therefore examined changes in protein levels of activin A, follistatin, and myostatin (GDF-8) in both muscle and bone with age in C57BL6 mice using ELISA. We then investigated the effects of activin A, myostatin and follistatin on the proliferation and differentiation of primary myoblasts and mouse bone marrow stromal cells (BMSCs) in vitro. Myostatin levels and the myostatin:follistatin ratio increased with age in the primarily slow-twitch mouse soleus muscle, whereas the pattern was reversed with age in the fast-twitch extensor digitorum longus muscle. Myostatin levels and the myostatin:follistatin ratio increased significantly (+75%) in mouse bone marrow with age, as did activin A levels (+17%). Follistatin increased the proliferation of primary myoblasts from both young and aged mice, whereas myostatin increased proliferation of younger myoblasts but decreased proliferation of older myoblasts. Myostatin reduced proliferation of both young and aged BMSCs in a dose-dependent fashion, and activin A increased mineralization in both young and aged BMSCs. Together these data suggest that aging in mice is accompanied by changes in the expression of activin A and myostatin, as well as changes in the response of bone and muscle progenitor cells to these factors. Myostatin appears to play a particularly important role in the impaired proliferative capacity of muscle and bone progenitor cells from aged mice.

Stromal cell-derived factor-1β mediates cell survival through enhancing autophagy in bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) hold great potential for cell-based therapy, yet the therapeutic efficacy remains uncertain. Transplanted BMSCs often fail to engraft within the bone marrow (BM), in part due to the poor survival of donor cells in response to inflammatory reactions, hypoxia, oxidative stress, or nutrient starvation. Two basic cell processes, apoptosis and autophagy, could potentially be responsible for the impaired survival of transplanted BMSCs. However, the functional relationship between apoptosis and autophagy in BMSC homeostasis is complex and not well understood. The stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signaling axis appears to be critical in maintaining proliferation and survival of BM stem cell populations through improving cell proliferation and survival in response to stress; however, the exact mechanisms remain unclear. We recently described novel genetically engineered Tet-Off-SDF-1β BMSCs, which over-express SDF-1β under tight doxycycline-control, thus providing an ideal model system to investigate the isolated effects of SDF-1β. In this study we tested the hypothesis that SDF-1β can mediate cell survival of BMSCs in vitro through increasing autophagy. We found that SDF-1β had no effect on BMSC proliferation; however, SDF-1β significantly protected genetically engineered BMSCs from H2O2-induced cell death through increasing autophagy and decreasing caspase-3-dependent apoptosis. Taken together, we provide novel evidence that the SDF-1/CXCR4 axis, specifically activated by the SDF-1β isoform, plays a critical role in regulating BMSC survival under oxidative stress through increasing autophagy.

Development of an injectable composite as a carrier for growth factor-enhanced periodontal regeneration.

AIM Biomaterials are often applied in periodontal therapy; however, not always well adapted for tissue regeneration. The objective of this study was to evaluate the physico-chemical properties and biocompatibility of an injectable, in situ setting composite for growth factor-enhanced periodontal regeneration. MATERIAL AND METHODS The composite constitutes bioresorbable poly(lactic-co-glycolic acid) (PLGA) and additives forming in situ a matrix designed as a carrier for recombinant human growth/differentiation factor-5 (rhGDF-5). In vitro characterization included the porosity, biointeraction, biodegradation, injectability, and biological activity of released rhGDF-5. Biocompatibility was compared with granular beta-tricalcium phosphate and an absorbable collagen sponge using a canine periodontal defect model. RESULTS The PLGA composite showed a highly porous (500-1000 mum) space-providing structure. It effectively induced coagulation exhibiting an intimate interaction with the fibrin clot. The biphasic biodegradation was complete within 4 weeks. The composite was conveniently injectable (90.4+/-3.6 N) for ease of use. It exhibited a sustained rhGDF-5 release over 4 weeks (40.8%) after initial burst (3.4%) detected by ALP activity. Sites receiving the composite showed limited, if any, residuals and had no appreciable negative effect on periodontal wound healing. There were no noteworthy inflammatory lesions in sites receiving the PLGA composite. CONCLUSION Characteristics of the PLGA composite makes it an attractive matrix to support native wound healing and rhGDF-5-enhanced periodontal regeneration.

Role of Myostatin (GDF-8) Signaling in the Human Anterior Cruciate Ligament

Myostatin, also referred to as growth and differentiation factor‐8 (GDF‐8), is expressed in muscle tissue where it functions to suppress myoblast proliferation and myofiber hypertrophy. Recently, myostatin and its receptor, the type IIB activin receptor (ActRIIB), were detected in the leg tendons of mice, and recombinant myostatin was shown to increase cellular proliferation and the expression of type 1 collagen in primary fibroblasts from mouse tendons. We sought to determine whether myostatin and its receptor were present in human anterior cruciate ligament (ACL) tissue, and if myostatin treatment had any effect on primary ACL fibroblasts. ACL tissue samples were obtained from material discarded during ACL reconstruction surgery. Real‐time PCR and immunohistochemistry demonstrate that both myostatin and its receptor are abundant in the human ACL. Primary fibroblasts isolated from human ACL specimens were treated with recombinant myostatin, and myostatin treatment increased fibroblast proliferation as well as the expression of tenascin C (TNC), type 1 collagen, and transforming growth factor‐β1. Real‐time PCR analysis of TNC and type 1 collagen expression in ACL specimens from normal mice and mice lacking myostatin supported these findings by showing that both TNC and type 1 collagen were downregulated in ACL tissue from myostatin‐deficient mice. Together, these data suggest that myostatin is a pro‐fibrogenic factor that enhances cellular proliferation and extracellular matrix synthesis by ACL fibroblasts. Recombinant myostatin may therefore have therapeutic applications in the area of tendon and ligament engineering and regeneration.

Inkjet-based biopatterning of SDF-1β augments BMP-2-induced repair of critical size calvarial bone defects in mice

BACKGROUND A major problem in craniofacial surgery is non-healing bone defects. Autologous reconstruction remains the standard of care for these cases. Bone morphogenetic protein-2 (BMP-2) therapy has proven its clinical utility, although non-targeted adverse events occur due to the high milligram-level doses used. Ongoing efforts explore the use of different growth factors, cytokines, or chemokines, as well as co-therapy to augment healing. METHODS Here we utilize inkjet-based biopatterning to load acellular DermaMatrix delivery matrices with nanogram-level doses of BMP-2, stromal cell-derived factor-1β (SDF-1β), transforming growth factor-β1 (TGF-β1), or co-therapies thereof. We tested the hypothesis that bioprinted SDF-1β co-delivery enhances BMP-2 and TGF-β1-driven osteogenesis both in-vitro and in-vivo using a mouse calvarial critical size defect (CSD) model. RESULTS Our data showed that BMP-2 bioprinted in low-doses induced significant new bone formation by four weeks post-operation. TGF-β1 was less effective compared to BMP-2, and SDF-1β therapy did not enhance osteogenesis above control levels. However, co-delivery of BMP-2+SDF-1β was shown to augment BMP-2-induced bone formation compared to BMP-2 alone. In contrast, co-delivery of TGF-β1+SDF-1β decreased bone healing compared to TGF-β1 alone. This was further confirmed in vitro by osteogenic differentiation studies using MC3T3-E1 pre-osteoblasts. CONCLUSIONS Our data indicates that sustained release delivery of a low-dose growth factor therapy using biopatterning technology can aid in healing CSD injuries. SDF-1β augments the ability for BMP-2 to drive healing, a result confirmed in vivo and in vitro; however, because SDF-1β is detrimental to TGF-β1-driven osteogenesis, its effect on osteogenesis is not universal.

Abstract 4846: IGF1 attenuates autophagic catabolism and apoptotic cell death in hormonally treated breast cancer cells via a MEK1 pathway

In recent studies, we and others showed that autophagy is one of the prosurvival mechanisms during anti-estrogen therapy that plays a significant role in the development of resistance. In this study, we sought to determine if autophagy plays a role in the protective effects of insulin like growth factor 1 (IGF-1)/MEK1 signaling on breast cancer cells treated with hormonal therapy. In initial studies, we established that IGF1 protects estrogen receptor-positive (ER+) breast cancer cells from cell death induced by the anti-estrogen 4-hydroxytamoxifen (4-OHT) and the anti-progestin mifepristone (MIF) and that this protection was mediated by MEK1 signaling. To determine if the mechanism of MEK1-mediated protection involved modulation of autophagy, ER+ breast cancer cells alone or transfected with MEK1-GFP or MEK1 dominant negative (MEK1-DN) expression vectors were treated with 4-OHT and/or MIF, in the presence or absence of IGF1. Effects on autophagy were monitored using immunoblotting with antibodies to key autophagy proteins, electron microscopy, GFP-LC3 reporter-based expression analysis, quantitative PCR, and autophagic proteolysis (turnover of long-lived proteins). Apoptosis was monitored with the JC-1 assay (mitochondrial membrane permeability determination), trypan blue exclusion, cytokeratin 18 cleavage assay, and determination of the levels of cleaved PARP and lamin A. The results showed that IGF1 significantly increased the levels of several autophagy genes, including Atg8, Atg6, and Atg5. However, even with the increased levels of these autophagy proteins, IGF-1 was shown to clearly suppress hormonally-induced long-lived protein turnover or macroautophagy. Thus, the levels of key autophagy proteins were increased, while the catabolic function of the mature autolysosomes was reduced. MEK1 overexpression also significantly protected breast cancer cells from death, while MEK1DN expression significantly enhanced 4-OHT and/or MIF induced apoptotic cell death. Our interpretation of the concomitant attenuation of macroautophagy and cell death by IGF1/MEK signaling is not that autophagy is a prerequisite to cell death, but that autophagic catabolism is a readout of the significant stress experienced by MEK1 inhibition in breast cancer cells. Based on our results, we are now addressing three key questions: (1) will blockade of autophagy enhance the cytotoxicity of MEK1 inhibitors; (2) is the elevated expression of autophagy genes in IGF-1-treated breast cancer cells mediated by MEK1 signaling; and (3) is mitophagy enhanced in breast cancer cells overexpressing MEK1. This knowledge will be used to support the proposed use of MEK1 blockade to enhance the cytotoxic action of hormonal therapy and to circumvent the occurrence of anti-estrogen resistance of breast cancer, particularly in patients with high circulating levels of IGF-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4846.