Myostatin Inhibitors: Panacea or Predicament for Musculoskeletal Disorders?

Myostatin, also known as growth differentiation factor 8 (GDF8), is a transforming growth factor-β (TGF-β) family member that functions to limit skeletal muscle growth. Accordingly, loss-of-function mutations in myostatin result in a dramatic increase in muscle mass in humans and various animals, while its overexpression leads to severe muscle atrophy. Myostatin also exerts a significant effect on bone metabolism, as demonstrated by enhanced bone mineral density and bone regeneration in myostatin null mice. The identification of myostatin as a negative regulator of muscle and bone mass has sparked an enormous interest in developing myostatin inhibitors as therapeutic agents for treating a variety of clinical conditions associated with musculoskeletal disorders. As a result, various myostatin-targeting strategies involving antibodies, myostatin propeptides, soluble receptors, and endogenous antagonists have been generated, and many of them have progressed to clinical trials. Importantly, most myostatin inhibitors also repress the activities of other closely related TGF-β family members including GDF11, activins, and bone morphogenetic proteins (BMPs), increasing the potential for unwanted side effects, such as vascular side effects through inhibition of BMP 9/10 and bone weakness induced by follistatin through antagonizing several TGF-β family members. Therefore, a careful distinction between targets that may enhance the efficacy of an agent and those that may cause adverse effects is required with the improvement of the target specificity. In this review, we discuss the current understanding of the endogenous function of myostatin, and provide an overview of clinical trial outcomes from different myostatin inhibitors.

Development of Educational Content for Nurses to Prevent Safety Accidents of Hospitalized Children: Delphi Approach

PURPOSE: The purpose of this study was to develop content for safety education to prevent hospital safety accidents among hospitalized children and to investigate the status of safety education performed by nurses. METHODS: First, the Delphi method was used, with 18 experts, to develop educational contents for preventing safety accidents. Second, an exploratory survey was performed of the actual status of safety education for preventing safety accidents among hospitalized children using a questionnaire developed based on the Delphi method. The participants of this study were 159 nurses with at least 6 months of work experience. RESULTS: The educational content developed through the Delphi method for preventing safety accidents among hospitalized children contained seven domains (falls, injury, electric shocks/burns, suffocation/aspiration, poisoning/abuse, kidnapping, medical devices) with 44 topics. The item mean of nurses'perceptions of the importance of child safety education was 4.18, and the actual performance score was 3.72, which was a statistically significant difference (t=11.58, p < .001). CONCLUSION: These seven comprehensive domains of accident prevention education for hospitalized children are expected to be useful for interventions to support the safety of hospitalized children.

Effects on G2/M Phase Cell Cycle Distribution and Aneuploidy Formation of Exposure to a 60 Hz Electromagnetic Field in Combination with Ionizing Radiation or Hydrogen Peroxide in L132 Nontumorigenic Human Lung Epithelial Cells

The aim of the present study was to assess whether exposure to the combination of an extremely low frequency magnetic field (ELF-MF; 60 Hz, 1 mT or 2 mT) with a stress factor, such as ionizing radiation (IR) or H2O2, results in genomic instability in non-tumorigenic human lung epithelial L132 cells. To this end, the percentages of G2/M-arrested cells and aneuploid cells were examined. Exposure to 0.5 Gy IR or 0.05 mM H2O2 for 9 h resulted in the highest levels of aneuploidy; however, no cells were observed in the subG1 phase, which indicated the absence of apoptotic cell death. Exposure to an ELF-MF alone (1 mT or 2 mT) did not affect the percentages of G2/M-arrested cells, aneuploid cells, or the populations of cells in the subG1 phase. Moreover, when cells were exposed to a 1 mT or 2 mT ELF-MF in combination with IR (0.5 Gy) or H2O2 (0.05 mM), the ELF-MF did not further increase the percentages of G2/M-arrested cells or aneuploid cells. These results suggest that ELF-MFs alone do not induce either G2/M arrest or aneuploidy, even when administered in combination with different stressors.

Release of heat shock protein 70 (Hsp70) and the effects of extracellular Hsp70 on matric metalloproteinase-9 expression in human monocytic U937 cells

Heat shock protein 70 (Hsp70) release and its effects on pro-inflammatory cytokine production have been controversial. In this study, we investigated whether Hsp70 could be released from monocytes and activates matrix metalloproteinase-9 (MMP-9) gene expression. Hsp70 overexpression in human monocytic cell line U937 was found to increase PMA- induced MMP-9 expression and enhance cell motility. Hsp70 cDNA transfectants released Hsp70 protein into culture supernatants, and a part of released Hsp70 subsequently was bound to the surface of U937 cells. Addition of culture medium containing the extracelluar Hsp70 led to an increase not only in proMMP-9 secretion, but also the invasiveness of U937 cells through Matrigel or human umbilical vascular endothelial cells (HUVEC) in vitro. Immunodepletion of Hsp70 abolished its effect on MMP-9 expression. The released Hsp70 activated nuclear factor kappa B (NF-kappa B) and activating protein-1 (AP-1), which led to the activation of MMP-9 transcription. Taken together, these results suggest that extracellular Hsp70 induces the expression of MMP-9 gene through activation of NF-kappa B and AP-1.

Ionizing radiation induces blockade of c-Jun N-terminal kinasedependent cell death pathway in amanner correlated with p21Cip/WAF1 induction in primary cultured normal human fibroblasts

During radiotherapy of cancer, neighboring normal cells may receive sub-lethal doses of radiation. To investigate whether such low levels of radiation modulate normal cell responses to death stimuli, primary cultured human fibroblasts were exposed to various doses of gamma-rays. Analysis of cell viability using an exclusion dye propidium iodide revealed that the irradiation up to 10 Gy killed the fibroblasts only to a minimal extent. In contrast, the cells efficiently lost their viability when exposed to 0.5-0.65 mM H2O2. This type of cell death was accompanied by JNK activation, and was reversed by the use of a JNK-specific inhibitor SP600125. Interestingly, H2O2 failed to kill the fibroblasts when these cells were pre-irradiated, 24 h before H2O2 treatment, with 0.25-0.5 Gy of gamma-rays. These cytoprotective doses of gamma-rays did not enhance cellular capacity to degrade H2O2, but elevated cellular levels of p21Cip/WAF1, a p53 target that can suppress H2O2-induced cell death by blocking JNK activation. Consistently, H2O2-induced JNK activation was dramatically suppressed in the pre-irradiated cells. The overall data suggests that ionizing radiation can impart normal fibroblasts with a survival advantage against oxidative stress by blocking the process leading to JNK activation.

PKC alpha induces differentiation through ERK1/2 phosphorylation in mouse keratinocytes

Epidermal keratinocyte differentiation is a tightly regulated stepwise process that requires protein kinase C (PKC) activation. Studies on cultured mouse keraitnocytes induced to differentiate with Ca2+ have indirectly implicated the involvement of PKC alpha isoform. When PKC alpha was overexpressed in undifferentiated keratinocytes using adenoviral system, expressions of differentiation markers such as loricrin, filaggrin, keratin 1 (MK1) and keratin 10 (MK10) were increased, and ERK1/2 phosphorylation was concurrently induced without change of other MAPK such as p38 MAPK and JNK1/2. Similarly, transfection of PKC alphakinase active mutant (PKC alpha- CAT) in the undifferentiated keratinocyte, but not PKC beta-CAT, also increased differentiation marker expressions. On the other hand, PKC alphadominant negative mutant (PKC beta-KR) reduced Ca2+ -mediated differentiation marker expressions, while PKC beta-KR did not, suggesting that PKC alphais responsible for keratinocyte differentiation. When downstream pathway of PKC alphain Ca2+ - mediated differentiation was examined, ERK1/2, p38 MAPK and JNK1/2 phosphorylations were increased by Ca2+ shift. Treatment of keratinocytes with PD98059, MEK inhibitor, and SB20358, p38 MAPK inhibitor, before Ca2+ shift induced morphological changes and reduced expressions of differentiation markers, but treatment with SP60012, JNK1/2 inhibitor, did not change at all. Dominant negative mutants of ERK1/2 and p38 MAPK also inhibited the expressions of differentiation marker expressions in Ca2+ shifted cells. The above results indicate that both ERK1/2 and p38 MAPK may be involved in Ca2+- mediated differentiation, and that only ERK1/2 pathway is specific for PKCa-mediated differentiation in mouse keratinocytes.

Dose-Incidence Relationships on the Prenatal Effects of Gamma-Radiation in Mice

The objective of this investigation was to evaluate dose-incidence relationships on the prenatal effects of gamma-radiation. Pregnant ICR mice were exposed on day 11.5 after conception, coincident with the most sensitive stage for the induction of major congenital malformations, with 0.5-4.0 Gy of gamma-radiations. The animals were sacrificed on day 18 of gestation and the fetuses were examined for mortality, growth retardation, change in head size and any other morphological abnormalities. With increasing radiation dose, incidence of small head, growth retarded fetuses, cleft palate, dilatation of cerebral ventricle and abnormalities of the extremities in live fetuses rose. The threshold doses of radiation that induced cleft palate and dilatation of cerebral ventricle, and abnormal extremities were between 1.0 and 2.0 Gy, and between 0.5 and 1.0 Gy, respectively.

Influence of gestational age at exposure on the prenatal effects of gamma-radiation

The objective of this investigation was to evaluate the influence of gestational age at exposure on the prenatal effects of gamma-radiation. Pregnant ICR mice were exposed to a single dose of 2.0 Gy gamma-radiation at a gestational 2.5 to 15.5 days post-coitus (p.c.). The animals were sacrificed on day 18 of gestation and the fetuses were examined for mortality, growth retardation, change in head size and any other morphological abnormalities. The only demonstrable effect of irradiation during the preimplantation period was an increase in prenatal mortality. Resorptions were maximal on post-exposure day 2.5 after conception. The pre-implantation irradiated embryos which survived did not show any major fetal abnormalities. Small head, growth retardation, cleft palate, dilatation of the cerebral ventricle, dilatation of the renal pelvis and abnormalities of the extremities and tail were prominent after exposure during the organogenesis period, especially on day 11.5 of gestation. Our results indicate that the late period of organogenesis in the mouse is a particularly sensitive phase in terms of the development of the brain, skull and extremities.

Anticarcinogenic Effect of Panax ginseng C.A. Meyer and Identification of Active Compounds

The failure to improve the five-year survival rate of cancer patients, from one in three in the 1960s to one in two in the 1970s, stimulated awareness of the importance of primary prevention of cancer. Korean investigators carried out extensive long-term anticarcinogenicity experiments with 2000 newborn mice to investigate whether Panax ginseng C.A. Meyer inhibited carcinogenesis induced by several chemical carcinogens in 1978. There was a 22% decrease (p<0.05) in the incidence of urethane induced lung adenoma by the combined use of red ginseng extract. In the group sacrificed at 56 weeks after the treatment with aflatoxin B1, the incidence of hepatoma significantly decreased to 75% by the addition of red ginseng extract (p<0.05). The result showed that natural products can provide hope for human cancer prevention. By the newly established '9 week medium-term anticarcinogenicity test model of lung tumors in mice' (Yun's model), we confirmed significant anticarcinogenic effects of powders and extracts of the 6- yr-old dried fresh ginseng, 5- and 6-yr old white ginsengs, and 4-, 5-, and 6-yr old red ginseng. We also demonstrated that the anticarcinogencity of ginseng was more prominent in aged or heat treated extracts of ginseng and red ginseng made by steaming. To investigate the active components for cancer prevention, several fractions of 6-yr old fresh ginseng and red ginseng, four semi-synthetic ginsenoside Rh1, Rh2, Rg3 and Rg5, major saponin components in red ginseng, were prepared. Among the ginsenosides, Rg3 and Rg5 showed statistically significant reduction of lung tumor incidence and Rh2 had a tendency of decreasing the incidence. Ginsenoside Rg3, Rg5 and Rh2 were found to be active anticarcinogenic compounds. Rg3, Rg5 and Rh2 are active components in red ginseng, and they prevent cancer either singularly or synergistically.

Correlation of changes in natural killer cell activity and glutathione S-transferase placental form positive hepatocytes in diethylnitrosamine-induced rat hepatocarcinogenesis

To evaluate the induction of preneoplastic hepatic foci in relation to natural killer cell (NK) activity, we sequentially analyzed glutathione S-transferase placental form positive (GST-P+) hepatocytes and NK activity during diethylnitrosamine (DEN) and phenobarbital (PB)-induced hepatocarcinogenesis in Sprague-Dawley rats. Previous studies have shown that NK activity can modulate the carcinogenic process induced by chemical carcinogens. Newborn females were initially given a single intraperitoneal injection of 15 mg DEN/kg and three weeks later, they were treated with 500 ppm phenobarbital (PB). From week 3, PB was administered in drinking water for 9 weeks. Interim and terminal sacrifices were performed at weeks 12, 15 and 30. GST-P+ hepatocytes increased with age in DEN-treated rats, especially in the population of more than two GST-P+ hepatocytes. The NK activity of DEN-treated rats did not significantly differ from that of control rats until week 12, but it progressively decreased from week 15 to 30. These results indicate that changes of NK activity inversely correlated with the induction of preneoplastic hepatic foci. This strong correlation of decreased NK activity with enhanced induction of GST-P+ foci suggests that NK activity is important in the early progression of hepatocarcinogenesis in rats.

Ito Cell Activity and Hepatocyte Proliferation Activity According to Collagen Content in Liver Cirrhosis / 대한간학회지

BACKGROUND/AIMS: Liver cirrhosis is an end-stage liver disease. Ito cell is known to have central role in fibrogenes is of liver cirrhosis. But collagen content and Ito cell activity in liver cirr hosis have received little attention. So Ito cell activity and hepatocyte proliferation activity according to collagen content was investigated. WAF-1 and c- met were studied to evaluate the effect of cell cycle. METHODS: We analyzed 56 cases of liver cirrhosis ( viral:41, biliary:11, alcoholic:2, Wilson' s disease:2). Collagen content was measured by spectrophot ometry. Ito cell activity and prolifer ation index was measured by-SMA and Ki- 67 immunohistochemistry. RESULTS: In viral cirrhosis, high collagen group showed increased Ito cell activity compared to low collagen group. There was no difference in hepatocyte prolifer ation activity bet ween high and low collagen group in viral cirrhosis. In biliary cirrhos is, high collagen group showed increased Ito cell activity in septal zones compared to low collagen group. WAF- 1and c- met were negative in most of cases. CONCLUSION: Collagen content of liver cirrhosis is closely related to increment of activated Ito cells . Ito cell activity was prominent in septal zones than in parenchymal areas of viral cirrhosis and that was only significant in septal zones of biliary cirrhosis. There is no correlation bet ween collagen content and hepatocyte proliferation activity.

The Relevance of Degree of Liver Fibrosis, Ito cell, and PKC Activity in Hepatic Fibrogenesis / 대한간학회지

BACKGROUND/AIMS: Hepatic fibrosis in rat induced by thioacet amide shares similar morphological and biochemical characteristics with human liver cirrhosis. Thioacetamide (T AA) initially induces accumulation of collagen in Disse space and eventually leads to macro- and micronodular cirrhos is. Ito cell was believed to play a main role in hepatic fibrosis. And it s activity was known to be regulated by the expression of various genes. But little has been discovered about the upstream signal trans duction pathway of these genes in hepatic fibrosis. The expression of genesrelated to Ito cell activity was regulated by many transcription factors , the activity of which was regulated by protein kinase C( PKC) is oforms. So it is s upposed that PKC could be as s ociated with fibrosis in liver. METHODS: We investigated the correlation of PKC is oforms and It ocell activity in the course of hepatic fibrosis using TAA induced rat liver cirrhosis model. We used six week- old male rats , and administered 0.03% TAA in drinking water. The animals were sacrificed at 9, 20, and 30 weeks after TAA administration. The degree of hepatic fibrosis was evaluated by measuring the total amount of collagen.-SMA immunohist ochemical st aining of liver tissue was done to determine the Ito cell activity. The expression pattern of PKC isoforms was investigated by West ern blotting. RESULTS: In TAA- treated group, collagen cont ent and Ito cell activity did not increase until 30 weeks and 20 weeks of treatment , respectively, while in control group collagen cont ent and Ito cell activity were not detected. Collagen content showed linear correlation with Ito cell activity. This implied that the proliferation of activated Ito cells was prior to the increase of collagen content. In view of expression pattern of PKC is oforms, PKC alpha showed no difference in TAA- treated group and control group. In TAA-treated group, PKCbeta1 exhibited increased level of expression in both particulate and cytosolic forms at 9 weeks, while PKCdelta and PKC epsilon showed striking shift to particulated form. After 20 weeks, all of the PKC beta1, delta, and epsilon degenerated and showed remarkably decreased level of expression. This suggested PKC alpha had no relation to hepatic fibrosis,while PKC beta1, delta, and epsilon, showing activity at 9 weeks, were related to fibrosis og liver. In response to fibrogenic factors, molecules engaged in intracellular signal transduction pathway like PKC beta1, delta, and epsilon, began to change prior to the increase of Ito cell activity, morphologic changes and alterations of collagen content. CONCLUSION: Our results strongly suggest that the activity of PKC isoforms play an important role in early step of hepatic fibrosis, while accompanying Ito cell activity do in later step.

Development of altered hepatocyte foci by separate and combined treatments with radiation and diethylnitrosamine in neonatal rats

To establish an in vivo radiation carcinogenesis model using glutathione S-transferase placental form positive (GST-P+) hepatic foci, newborn rats were irradiated once by 0.5 Gy and 2 Gy of gamma ray or 0.15 Gy and 0.6 Gy of neutron with or without 0.05% phenobarbital (PB). When the rats were sacrificed at the 12th or 21st week, the incidence of GST-P+ foci induction by radiation alone was very low. The neutron was more sensitive than the gamma ray at week 12 and the reverse phenomenon was observed in the groups at week 21. PB combination showed an increased incidence of GST-P+ foci in gamma ray irradiated groups. The neutron irradiation combined with PB did not show any significant difference compared with the corresponding PB untreated groups. We also investigated the combined effect of diethylnitrosamine (DEN) and 0.75 Gy of gamma ray irradiation. Intraperitoneal injection of 0.15 mumol/g body weight of DEN at 1 hour after gamma ray irradiation showed significantly increased the number and area of GST-P+ foci compared with those of DEN alone or DEN at 1 hour before gamma radiation (P < 0.001). From these data, after more defined experiments, an in vivo radiation carcinogenesis model will be established by radiation alone or a combination of radiation and carcinogens.

Lung adenoma development and NK activity in mice treated with multiple carcinogens

A wide-spectrum initiation model was investigated in mice. Sequential treatments with diethylnitrosamine, urethane and N-methylnitrosourea, with or without a promoter, phenobarbital, resulted in tumor formation in the lungs in 85-90% of animals, but did not produce any tumorous lesions in other organs. The lung tumors were adenomas and the mean number of adenomas was 2.2-2.6 per mouse. Phenobarbital combination had no additive effect on lung tumor incidence and multiplicity. Splenic NK cell activity showed inconsistent increment in the carcinogen plus phenobarbital-treated group during the experiment (P less than 0.05).

Inhibitory effects of biochanin A on mouse lung tumor induced by benzo(a)pyrene

Biochanin A, an isoflavone compound, is reported to have an inhibitory effect on benzo(a)pyrene [B(a)P] metabolism. We examined the modifying effect of biochanin A on in vivo carcinogenesis using a mouse lung tumor model. As carcinogens, a single subcutaneous injection of 0.5mg of B(a)P was given within 24 hours after birth. The test groups were injected with 0.125mg of biochanin A in 0.1ml DMSO by i.p. 3 times a week for 6 weeks after weaning. All mice were sacrificed at week 9 and the incidence and multiplicity of lung tumors were examined. Concomitant administration of biochanin A showed a significant inhibitory effect on the incidence of tumor-bearing mice (12.5%, P<0.01), as well as the mean number of tumors (0.13, P<0.001), compared with the group treated with B(a)P alone in which the incidence was 57.1% and the mean number was 1.0. These results suggest that biochanin A has inhibitory potential on the development of mouse lung tumor induced by B(a)P.

Effects of biochanic A on mouse lung tumor and lymphocyte proliferative / 대한암학회지

No abstract available.