Regulation of granulosa cell proliferation and EGF-like ligands during the periovulatory interval in monkeys

BACKGROUND: This study seeks to clarify cell cycle dynamics of granulosa cells following hCG and elucidate the expression of epidermal growth factor (EGF)-like ligands during luteinization. METHODS: Granulosa cells were obtained from rhesus macaques undergoing controlled ovarian stimulation protocols before or after an ovulatory hCG bolus. Cell cycle characteristics were determined by flow cytometry and levels of EGF receptor (EGFR), amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC) mRNAs were measured by real-time RT-PCR. RESULTS: The proportion of cells in S-phase was 7.5% prior to hCG and did not decline until 24 h after hCG (3.1%). EGFR protein and BTC mRNA did not change following hCG, whereas AREG and EREG mRNA increased starting at 3 and 12 h post-hCG, respectively, and remained elevated thereafter. CONCLUSIONS: Cell cycle transit of macaque granulosa cells does not change until 24 h after an ovulatory stimulus, whereas the EGF-like ligands EREG and AREG are increased rapidly. This suggests that luteinizing granulosa cells are refractory to mitogenic stimulation by EGFR ligands.     

Canonical WNT signaling pathway and human AREG

AREG (Amphiregulin), BTC (beta-cellulin), EGF, EPGN (Epigen), EREG (Epiregulin), HBEGF, NRG1, NRG2, NRG3, NRG4 and TGFA (TGFalpha) constitute EGF family ligands for ERBB family receptors. Cetuximab (Erbitux), Pertuzumab (Omnitarg) and Trastuzumab (Herceptin) are anti-cancer drugs targeted to EGF family ligands, while Gefitinib (Iressa), Erlotinib (Tarceva) and Lapatinib (GW572016) are anti-cancer drugs targeted to ERBB family receptors. AREG and TGFA are biomarkers for Gefitinib non-responders. The TCF/LEF binding sites within the promoter region of human EGF family members were searched for by using bioinformatics and human intelligence (Humint). Because three TCF/LEF-binding sites were identified within the 5'-promoter region of human AREG gene, comparative genomics analyses on AREG orthologs were further performed. The EPGN-EREG-AREG-BTC cluster at human chromosome 4q13.3 was linked to the PPBP-CXCL segmental duplicons. AREG was the paralog of HBEGF at human chromosome 5q31.2. Chimpanzee AREG gene, consisting of six exons, was located within NW_105918.1 genome sequence. Chimpanzee AREG was a type I transmembrane protein showing 98.0% and 71.4% total amino-acid identity with human AREG and mouse Areg, respectively. Three TCF/LEF-binding sites within human AREG promoter were conserved in chimpanzee AREG promoter, but not in rodent Areg promoters. Primate AREG promoters were significantly divergent from rodent Areg promoters. AREG mRNA was expressed in a variety of human tumors, such as colorectal cancer, liver cancer, gastric cancer, breast cancer, prostate cancer, esophageal cancer and myeloma. Because human AREG was characterized as potent target gene of WNT/beta-catenin signaling pathway, WNT signaling activation could lead to Gefitinib resistance through AREG upregulation. AREG is a target of systems medicine in the field of oncology.     

REM sleep deprivation impairs muscle regeneration in rats

Introduction: The aim was observe the influence of sleep deprivation (SD) and sleep recovery on muscle regeneration process in rats submitted to cryolesion.

Methods: Thirty-two Wistar rats were randomly allocated in four groups: control (CTL), SD for 96?h (SD96), control plus sleep recovery period (CTL?+?R) and SD96h plus 96?h of sleep recovery (SD96?+?R). The animals were submitted to muscle injury by cryolesioning, after to SD and sleep recovery.

Results: The major outcomes of this study were the reduction of muscular IGF-1 in both legs (injured and uninjured) and a delay in muscle regeneration process of animals submitted to SD compared to animals that slept, with increase connective tissue, inflammatory infiltrate and minor muscle fibers.

Conclusions: SD impairs muscle regeneration in rats, moreover reduces muscular IGF-1 and sleep recovery was able to restore it to basal levels, but it was not enough to normalize the muscle regeneration.     

Human Betacellulin,a Member of the EGF Family Dominantly Expressed in Pancreas and Small Intestine,is Fully Active in a Monomeric Form

Abstract

Betacellulin (BTC) was found to be expressed mainly in human pancreas and small intestine. This finding suggests that BTC possesses some specific function distinguished from the other members of epidermal growth factor (EGF) family. To clarify this function, the released form of human BTC has been expressed in Edi, purified, and characterized. The recombinant human BTC was produced as an inclusion body. This material was dissolved in guanidine-HC1 under reducing conditions, refolded, and purified through sequential liquid chromatography. Purified BTC was electrophoresed under reducing conditions and a molecular size of 18 kDa was determined, which is the supposed size of a dimer of the peptide. However, chemical analysis failed to show a covalently linked dimer. The molecular mass of BTC analyzed by mass spectrometry revealed it to be 9 kDa, which is consistent with theoretical value for a monomer. Recombinant BTC showed growth promoting activity for mouse fibroblasts and rat aortic smooth muscle cells which was equivalent to EGF. On the other hand, BTC was found to exhibit a growth inhibitory effect on the cells overexpressing EGF receptor.     

Announcement

Binding of iodinated IGF-I to tissue sections from regenerating muscle was studied by autoradiography in normal and in hypophysectomized rats. Binding of IGF-I was low in control muscle in both groups of animals, but increased transiently about 10-fold during regeneration after injury. Maximal binding occurred later in hypophysectomized rats than in control rats, and there was also a slower regeneration process in these animals. IGF-I, as demonstrated by immunohistochemistry, and IGF-I mRNA, as demonstrated by in situ hybridization, were expressed by the regenerating muscle cells in both groups of animals. It is concluded that locally produced IGF-I is the most likely ligand for IGF-I receptors during muscle regeneration.     

Long‐Term Therapy With Omega‐3 Ameliorates Myonecrosis and Benefits Skeletal Muscle Regeneration in Mdx Mice

In Duchenne muscle dystrophy (DMD) and in the mdx mouse model of DMD, a lack of dystrophin leads to myonecrosis and cardiorespiratory failure. Several lines of evidence suggest a detrimental role of the inflammatory process in the dystrophic process. Previously, we demonstrated that short‐term therapy with eicosapentaenoic acid (EPA), at early stages of disease, ameliorated dystrophy progression in the mdx mouse. In the present study, we evaluated the effects of a long‐term therapy with omega‐3 later in dystrophy progression. Three‐month‐old mdx mice received omega‐3 (300 mg/kg) or vehicle by gavage for 5 months. The quadriceps and diaphragm muscles were removed and processed for histopathology and Western blot. Long‐term therapy with omega‐3 increased the regulatory protein MyoD and muscle regeneration and reduced markers of inflammation (TNF‐α and NF‐kB) in both muscles studied. The present study supports the long‐term use of omega‐3 at later stages of dystrophy as a promising option to be investigated in DMD clinical trials. Anat Rec, 298:1589–1596, 2015. © 2015 Wiley Periodicals, Inc.     

Synergistic effects of dimethyloxalylglycine and butyrate incorporated into α-calcium sulfate on bone regeneration

Osteogenesis is closely related to angiogenesis, and the combined delivery of angiogenic and osteogenic factors has been suggested to enhance bone regeneration. Small molecules have been explored as alternatives to growth factors for tissue regeneration applications. In this study, we examined the effects of the combined application of angiogenic and osteogenic small molecules on bone regeneration using a prolyl hydroxylase, dimethyloxalylglycine (DMOG), and a histone deacetylase inhibitor, butyrate. In a critical size bone defect model in rats, DMOG and butyrate, which were incorporated into α calcium sulfate (αCS), resulted in synergistic enhancements in bone and blood vessel formation, eventually leading to bone healing, as confirmed by micro-CT and histological analyses. In MC4 pre-osteoblast cultures, DMOG and butyrate enhanced the pro-angiogenic responses and osteoblast differentiation, respectively, which were evaluated based on the levels of hypoxia inducible factor (HIF)-1α protein and the expression of pro-angiogenic molecules (VEGF, home oxidase-1, glucose transporter-1) and by alkaline phosphatase (ALP) activity and the expression of osteoblast phenotype marker molecules (ALP, α1(I)col, osteocalcin, and bone sialoprotein). DMOG combined with butyrate synergistically improved osteoblast differentiation and pro-angiogenic responses, the levels of which were drastically increased in the cultures on αCS disks. Furthermore, it was demonstrated that αCS increased the level of HIF-1α and as a consequence VEGF expression, and supported osteoblast differentiation through the release of calcium ions from the αCS. Altogether, the results of this study provide evidence that a combination treatment with the small molecules DMOG and butyrate can expedite the process of bone regeneration and that αCS can be an efficient delivery vehicle for the small molecules for bone regeneration.     

Expression of oestrogen receptor α and β is higher in skeletal muscle of highly endurance‐trained than of moderately active men

Aim: Two known oestrogen receptors (ERs), ERα and the recently cloned ERβ, are expressed in the human skeletal muscle of both males and females. The effects of oestrogen and the role of ERs in skeletal muscle tissue are not well known. Oestrogen receptors and some of their target genes are involved in angiogenic processes. It was hypothesized that ERs are expressed at a higher level in a group with higher oxidative capacity, and that such an enhanced expression would parallel expression of the angiogenic factor – vascular endothelial growth factor (VEGF). Method: Muscle biopsies were taken from vastus lateralis in 10 highly endurance‐trained males and 10 moderately active males and analysed for the expression of ERs and VEGF. Results: The major findings in the present study were the higher mRNA levels of ERα, ERβ and VEGF in the highly endurance‐trained than in the moderately active group. Conclusion: These data suggest that the greater mRNA expression of ERα and ERβ and the oestrogen‐associated angiogenic factor VEGF support the hypothesis of an involvement of ERs in the adaptation of skeletal muscle to endurance training.     

Promotion of muscle regeneration in the toad (Bufo viridis) gastrocnemius muscle by low-energy laser irradiation

The effect of low-energy laser (He-Ne) irradiation on the process of skeletal muscle regeneration after cold injury to the gastrocnemius muscle of the toad (Bufo viridis) was studied using quantitative histological and morphometric methods. The injured zones in the experimental toads were subjected to five direct He-Ne laser (632.8 nm wavelength) irradiations (6.0 mW for 2.3 min) every alternate day starting on the fourth day postinjury. Muscles that were injured as above, and subjected to redlight irradiation, served as a control group. Morphometric analysis was performed on histological sections of injured areas at 9, 14, and 30 days postinjury. At 9 days postinjury, mononucleated cells populated 69.3% ± 16.8% of the total area of injury. Thereafter, their volume fraction (percent of total injured zone) decreased gradually but more rapidly in the laserirradiated muscle than in the control. The volume fraction of the myotubes in the laser-irradiated muscles at 9 days of muscle regeneration was significantly higher (7.0% ± 2.2%) than in the control muscle (1.2% ± 0.4%). Young myofibers in the laser-irradiated muscles populated 15.5% ± 7.9% and 65.0% ± 9.5% of the injured area at 9 and 14 days of muscle regeneration, respectively, while in control muscles these structures were not evident at 9 days and made up only 5.3% ± 2.9% of the traumatized area at 14 days postinjury. The volume fraction of the young myofibers further increased by 30 days of muscle regeneration making up 75.7% ± 13.2% of the traumatized area, while in the laser-irradiated muscles most of the injured zone was filled with mature muscle fibers. It is concluded that He-Ne laser irradiation during the regeneration process markedly promotes muscle maturation in the injured zone following cold injury to the toad gastrocnemius muscle. © 1993 Wiley-Liss, Inc.     

Transgenic Analysis of Signaling Pathways Required for Xenopus Tadpole Spinal Cord and Muscle Regeneration

The Xenopus tadpole has the capacity fully to regenerate its tail after amputation. Previously, we have established that this regeneration process requires the operation of several signaling pathways including the bone morphogenic protein, Wnt, and Fgf pathways. Here, we have addressed the signaling requirements for spinal cord and muscle regeneration in a tissue‐specific manner. Two methods were used namely grafts of transgenic spinal cord to a wild type host, and the use of the Tet‐on conditional transgenic system to express inhibitors in the individual tissues. For the grafting experiments, the tail was amputated through the graft, which contained a temperature inducible inhibitor of the Wnt‐β‐catenin pathway. For the Tet‐on experiments, treatment with doxycycline was used to induce cell autonomous inhibitors of the Wnt‐β‐catenin or the Fgf pathway in either spinal cord or muscle. The results show that both spinal cord and muscle regeneration depend on both the Wnt‐β‐catenin and the Fgf pathways. This experimental design also enables us to observe the effect of inhibition of regeneration of one tissue on the regeneration of the others. Regardless of the method of inhibition, we find that reduction of spinal cord regeneration reduces regeneration of other parts of the tail, including the myotomal muscles. In contrast, reduction of muscle regeneration has no effect on the regeneration of the spinal cord. In common with other regeneration systems, this indicates that soluble factors from the spinal cord are needed to promote the regeneration of the other tissues in the tail. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Nrf2 augments skeletal muscle regeneration after ischaemia–reperfusion injury

Skeletal muscles harbour a resident population of stem cells, termed satellite cells (SCs). After trauma, SCs leave their quiescent state to enter the cell cycle and undergo multiple rounds of proliferation, a process regulated by MyoD. To initiate differentiation, fusion and maturation to new skeletal muscle fibres, SCs up‐regulate myogenin. However, the regulation of these myogenic factors is not fully understood. In this study we demonstrate that Nrf2, a major regulator of oxidative stress defence, plays a role in the expression of these myogenic factors. In both promoter studies with myoblasts and a mouse model of muscle injury in Nrf2‐deficient mice, we show that Nrf2 prolongs SC proliferation by up‐regulating MyoD and suppresses SC differentiation by down‐regulating myogenin. Moreover, we show that IL‐6 and HGF, both factors that facilitate SC activation, induce Nrf2 activity in myoblasts. Thus, Nrf2 activity promotes muscle regeneration by modulating SC proliferation and differentiation and thereby provides implications for tissue regeneration.Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Toll‐like receptor signalling in regenerative myogenesis: friend and foe

Skeletal muscle regeneration in normal and diseased muscle is regulated by multiple factors and cells present in the injured muscle micro‐environment. In addition to muscle progenitor cells, several immunocytes participate in the regenerative response. Among them, macrophages are one of the most important components of the immune response that governs the step‐wise progression of muscle regeneration. The initial role of macrophages is to phagocytose muscle cell debris and later, through their transition to an anti‐inflammatory phenotype, they promote regeneration. However, in several genetic muscle disorders, continuous muscle injury disrupts the balance between pro‐inflammatory and anti‐inflammatory macrophages, leading to an overall inflammatory milieu and inhibition of muscle regeneration. Accumulating evidence suggests that Toll‐like receptor (TLR)‐mediated signalling plays an important role in the regulation of macrophage phenotypes during regenerative myogenesis in response to both acute and chronic muscle injury. Here, we discuss the role of TLR signalling in regulating macrophage phenotypes and skeletal muscle regeneration in healthy and diseased muscle. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Surgical manipulation,but not moderate exercise,is associated with increased cytokine mRNA expression in the rat soleus muscle

Interleukin (IL)‐6 production in contracting skeletal muscle and IL‐6 concentration in plasma are increased after prolonged and strenuous exercise. However, as tissue stress or damage are unspecific triggers of increased cytokine levels, we examined whether moderate muscle activity is an independent stimulus for cytokine expression, and to which extent invasive procedures might affectthe results. Soleus muscles were isolated from sedentary rats or from rats that had been running on a treadmill at moderate intensity (70% of maximal oxygen uptake) for 1 h. In another group the soleus muscle was prepared in situ and stimulated intermittently at 5 Hz for 1 h, so that maximal developed force declined by 30%. In situ prepared soleus muscles not subjected to electrical stimulation were used as controls. Messenger RNA (mRNA) expression of 11 cytokines was analysed in the soleus muscles using multiprobe RNAse protection assay, and IL‐6 plasma concentration was measured by enzyme‐linked immunosorbent assay. Treadmill exercise did not affect the mRNA expression of any of the measured cytokines in the soleus muscle. Irrespective of electrical stimulation, mRNA expression of IL‐6 and IL‐1β were significantly increased in the surgically manipulated soleus muscles. Interleukin‐6 plasma concentration was not affected by treadmill running or electrical stimulation. Conclusion, gentle surgical manipulation is a strong stimulus for IL‐6 and IL‐1β mRNA synthesis in skeletal muscle, whereas exercise or electrical muscle stimulation at moderate intensity does not independently affect cytokine mRNA levels in the contracting soleus.     

Prostaglandins in muscle regeneration

Muscle regeneration is a tightly orchestrated process where activated satellite cells (myoblasts) respond to external stimuli in order to proliferate, differentiate and fuse to damaged myofibers. Simultaneously, the injured tissue undergoes an inflammatory response and communication between leukocytes and the spectrum of differentiated and undifferentiated muscle cells is essential for proper healing. This communication is mediated by cytokines, growth factors and prostaglandins and dissecting the role of these signaling molecules might be the key to positively manipulate muscle regeneration in the future. This review will focus on the roles of prostaglandins and will consider the potential cost of using non-steroidal inflammatory drugs as popular treatment of muscle injury.     

IFATS collection: Fibroblast growth factor-2-induced hepatocyte growth factor secretion by adipose-derived stromal cells inhibits postinjury fibrogenesis through a c-Jun N-terminal kinase-dependent mechanism

Adipose-derived stem/stromal cells (ASCs) not only function as tissue-specific progenitor cells but also are multipotent and secrete angiogenic growth factors, such as hepatocyte growth factor (HGF), under certain circumstances. However, the biological role and regulatory mechanism of this secretion have not been well studied. We focused on the role of ASCs in the process of adipose tissue injury and repair and found that among injury-associated growth factors, fibroblast growth factor-2 (FGF-2) strongly promoted ASC proliferation and HGF secretion through a c-Jun N-terminal kinase (JNK) signaling pathway. In a mouse model of ischemia-reperfusion injury of adipose tissue, regenerative changes following necrotic and apoptotic changes were seen for 2 weeks. Acute release of FGF-2 by injured adipose tissue was followed by upregulation of HGF. During the adipose tissue remodeling process, adipose-derived 5-bromo-2-deoxyuridine-positive cells were shown to be ASCs (CD31-CD34+). Inhibition of JNK signaling inhibited the activation of ASCs and delayed the remodeling process. In addition, inhibition of FGF-2 or JNK signaling prevented postinjury upregulation of HGF and led to increased fibrogenesis in the injured adipose tissue. Increased fibrogenesis also followed the administration of a neutralizing antibody against HGF. FGF-2 released from injured tissue acts through a JNK signaling pathway to stimulate ASCs to proliferate and secrete HGF, contributing to the regeneration of adipose tissue and suppression of fibrogenesis after injury. This study revealed a functional role for ASCs in the response to injury and provides new insight into the therapeutic potential of ASCs.     

Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1α mediated BMSCs

The successful clinical outcome of the implanted tissue-engineered bone is dependent on the establishment of a functional vascular network. A gene-enhanced tissue engineering represents a promising approach for vascularization. Our previous study indicated that hypoxia-inducible factor-1α (HIF-1α) can up-regulate the expression of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF-1) in bone mesenchymal stem cells (BMSCs). The angiogenesis is a co-ordinated process that requires the participation of multiple angiogenic factors. To further explore the angiogenic effect of HIF-1α mediated stem cells, in this study, we systematically evaluated the function of HIF-1α in enhancing BMSCs angiogenesis in vitro and in vivo. A constitutively active form of HIF-1α (CA5) was inserted into a lentivirus vector and transduced into BMSCs, and its effect on vascularization and vascular remodeling was further evaluated in a rat critical-sized calvarial defects model with a gelatin sponge (GS) scaffold. The expression of the key angiogenic factors including VEGF, SDF-1, basic fibroblast growth factor (bFGF), placental growth factor (PLGF), angiopoietin 1 (ANGPT1), and stem cell factor (SCF) at both mRNAs and proteins levels in BMSCs were significantly enhanced by HIF-1α overexpression compared to the in vitro control group. In addition, HIF-1α-over expressing BMSCs showed dramatically improved blood vessel formation in the tissue-engineered bone as analyzed by photography of specimen, micro-CT, and histology. These data confirm the important role of HIF-1α in angiogenesis in tissue-engineered bone. Improved understanding of the mechanisms of angiogenesis may offer exciting therapeutic opportunities for vascularization, vascular remodeling, and bone defect repair using tissue engineering strategies in the future.