Posttransplantation cyclophosphamide mediates effective reconstitution of memory B cells after allogeneic hematopoietic cell transplantation.

OBJECTIVES: Impaired B-cell reconstitution after allogeneic hematopoietic cell transplantation (allo-HCT) contributes to the pathogenesis of chronic graft-versus-host disease (cGVHD). Therefore, methods to consistently achieve effective B cell lymphogenesis are required. We assessed the long-term effects of posttransplantation cyclophosphamide (PTCy) use on immune reconstitution in clinical settings, an emerging strategy to suppress allogeneic immunological inflammation early after allo-HCT and prevent subsequent GVHD. METHODS: We comprehensively analyzed peripheral immune cell subsets and measured serum immunoglobulin G (IgG) or cytokine levels in 39 patients who survived for >1 year after allo-HCT. RESULTS: The absolute counts of B1 and IgM memory B cells were significantly lower in patients with severe cGVHD than in those without. The absolute count and percentage (among total CD19+ B cells) of switched memory B cells and serum IgG levels were significantly higher in patients transplanted with PTCy than in those transplanted with conventional GVHD prophylaxis. Interestingly, increased percentages of switched memory B cells and serum IgG levels were observed only in patients transplanted with PTCy and not in those transplanted with umbilical cord blood. CONCLUSIONS: PTCy administration can mediate favorable memory B-cell reconstitution long after allo-HCT and may therefore suppress cGVHD.

Pharmacological activation of SERCA ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive muscular weakness because of the loss of dystrophin. Extracellular Ca2+ flows into the cytoplasm through membrane tears in dystrophin-deficient myofibers, which leads to muscle contracture and necrosis. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) takes up cytosolic Ca2+ into the sarcoplasmic reticulum, but its activity is decreased in dystrophic muscle. Here, we show that an allosteric SERCA activator, CDN1163, ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice. The administration of CDN1163 prevented exercise-induced muscular damage and restored mitochondrial function. In addition, treatment with CDN1163 for 7 weeks enhanced muscular strength and reduced muscular degeneration and fibrosis in mdx mice. Our findings provide preclinical proof-of-concept evidence that pharmacological activation of SERCA could be a promising therapeutic strategy for DMD. Moreover, CDN1163 improved muscular strength surprisingly in wild-type mice, which may pave the new way for the treatment of muscular dysfunction.

Deficiency and overexpression of Rtl1 in the mouse cause distinct muscle abnormalities related to Temple and Kagami-Ogata syndromes.

Temple and Kagami-Ogata syndromes are genomic imprinting diseases caused by maternal and paternal duplication of human chromosome 14, respectively. They exhibit different postnatal muscle-related symptoms as well as prenatal placental problems. Using the mouse models for these syndromes, it has been demonstrated that retrotransposon gag like 1 [Rtl1, also known as paternally expressed 11 (Peg11)] located in the mouse orthologous imprinted region is responsible for the prenatal placental problems because it is an essential placental gene for maintenance of fetal capillary network during gestation. However, the causative imprinted gene for the postnatal muscle-related symptoms remains unknown. Here, we demonstrate that Rtl1 also plays an important role in fetal/neonatal skeletal muscle development: its deletion and overproduction in mice lead to neonatal lethality associated with severe but distinct skeletal muscle defects, similar to those of Temple and Kagami-Ogata syndromes, respectively. Thus, it is strongly suggested that RTL1 is the major gene responsible for the muscle defects in addition to the placental defects in these two genomic imprinting diseases. This is the first example of an LTR retrotransposon-derived gene specific to eutherians contributing to eutherian skeletal muscle development.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy.

The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. Although a variety of experimental therapies are in development, achieving effective treatments for DMD remains exceptionally challenging, not least because the pathological consequences of dystrophin loss are incompletely understood. Here we have performed proteome profiling in tibialis anterior muscles from two murine DMD models (mdx and mdx52) at three ages (8, 16, and 80 weeks of age), all n = 3. High-resolution isoelectric focusing liquid chromatography-tandem MS (HiRIEF-LC-MS/MS) was used to quantify the expression of 4974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to WT (C57BL/6) controls at each age. Furthermore, 1795 proteins were differentially expressed when samples were pooled across ages and dystrophic strains. These included numerous proteins associated with the extracellular matrix and muscle function that have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators, which together are indicative of cross-talk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Upregulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was upregulated during, but not required for, the differentiation of C2C12 myoblasts suggesting that this protein may affect muscle regeneration. This study provides novel insights into mutation-independent proteomic signatures characteristic of the dystrophic phenotype and its progression with aging.

Viral isolation analysis of SARS-CoV-2 from clinical specimens of COVID-19 patients.

Genetic testing using reverse transcriptase real-time polymerase chain reaction (rRT-PCR) is the mainstay of diagnosis of COVID-19. However, it has not been fully investigated whether infectious viruses are contained in SARS-CoV-2 genome-positive specimens examined using the rRT-PCR test. In this study, we examined the correlation between the threshold Cycle (Ct) value obtained from the rRT-PCR test and virus isolation in cultured cells, using 533 consecutive clinical specimens of COVID-19 patients. The virus was isolated from specimens with a Ct value of less than 30 cycles, and the lower the Ct value, the more efficient the isolation rate. A cytopathic effect due to herpes simplex virus type 1 contamination was observed in one sample with a Ct value of 35 cycles. In a comparison of VeroE6/TMPRSS2 cells and VeroE6 cells used for virus isolation, VeroE6/TMPRSS2 cells isolated the virus 1.7 times more efficiently than VeroE6 cells. There was no significant difference between the two cells in the mean Ct value of the detectable sample. In conclusion, Lower Ct values in the PCR test were associated with higher virus isolation rates, and VeroE6/TMPRSS2 cells were able to isolate viruses more efficiently than VeroE6 cells.

[A Case of Pulmonary Pleomorphic Carcinoma in a Hemodialysis Patient Treated with Pembrolizumab].

The patient was a 67-year-old male undergoing maintenance hemodialysis due to chronic renal failure caused by diabetic nephropathy. A left upper lobe resection was carried out for non-small cell lung cancer of the left upper lobe. It was histologically confirmed as pleomorphic carcinoma pT3N0M0, Stage ⅡB. He suffered a relapse with multiple metastases occurring in both lungs 3 months following surgery. The PD-L1 tumor proportion score(TPS)was 90%, indicating a high level of expression; 200mg of pembrolizumab was administered every 3 weeks on non-dialysis days. Two courses of administration achieved a partial response. A total of 17 courses were administered until discontinuation due to drug-induced lung injury.

Periostin Is Required for the Maintenance of Muscle Fibers during Muscle Regeneration.

Skeletal muscle regeneration is a well-organized process that requires remodeling of the extracellular matrix (ECM). In this study, we revealed the protective role of periostin, a matricellular protein that binds to several ECM proteins during muscle regeneration. In intact muscle, periostin was localized at the neuromuscular junction, muscle spindle, and myotendinous junction, which are connection sites between muscle fibers and nerves or tendons. During muscle regeneration, periostin exhibited robustly increased expression and localization at the interstitial space. Periostin-null mice showed decreased muscle weight due to the loss of muscle fibers during repeated muscle regeneration. Cultured muscle progenitor cells from periostin-null mice showed no deficiencies in their proliferation, differentiation, and the expression of Pax7, MyoD, and myogenin, suggesting that the loss of muscle fibers in periostin-null mice was not due to the impaired function of muscle stem/progenitor cells. Periostin-null mice displayed a decreased number of CD31-positive blood vessels during muscle regeneration, suggesting that the decreased nutritional supply from blood vessels was the cause of muscle fiber loss in periostin-null mice. These results highlight the novel role of periostin in maintaining muscle mass during muscle regeneration.

The nSMase2/Smpd3 gene modulates the severity of muscular dystrophy and the emotional stress response in mdx mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive, degenerative muscular disorder and cognitive dysfunction caused by mutations in the dystrophin gene. It is characterized by excess inflammatory responses in the muscle and repeated degeneration and regeneration cycles. Neutral sphingomyelinase 2/sphingomyelin phosphodiesterase 3 (nSMase2/Smpd3) hydrolyzes sphingomyelin in lipid rafts. This protein thus modulates inflammatory responses, cell survival or apoptosis pathways, and the secretion of extracellular vesicles in a Ca2+-dependent manner. However, its roles in dystrophic pathology have not yet been clarified. METHODS: To investigate the effects of the loss of nSMase2/Smpd3 on dystrophic muscles and its role in the abnormal behavior observed in DMD patients, we generated mdx mice lacking the nSMase2/Smpd3 gene (mdx:Smpd3 double knockout [DKO] mice). RESULTS: Young mdx:Smpd3 DKO mice exhibited reduced muscular degeneration and decreased inflammation responses, but later on they showed exacerbated muscular necrosis. In addition, the abnormal stress response displayed by mdx mice was improved in the mdx:Smpd3 DKO mice, with the recovery of brain-derived neurotrophic factor (Bdnf) expression in the hippocampus. CONCLUSIONS: nSMase2/Smpd3-modulated lipid raft integrity is a potential therapeutic target for DMD.

Glycerophospholipid profile alterations are associated with murine muscle-wasting phenotype.

INTRODUCTION: Phospholipids are essential components of cellular membranes and are closely associated with cellular functions, but relationships involving skeletal muscle phospholipid profiles and their physiological phenotypes have remained unclear. METHODS: We carried out comprehensive phospholipid analyses using liquid chromatography-tandem mass spectrometry to determine the phospholipid profiles of skeletal muscles derived from muscle-wasting mouse models, including denervated and Duchenne muscular dystrophy mouse models (mdx) as well as rescued mdx mice expressing truncated dystrophin. RESULTS: Consistent phosphatidylcholine and phosphatidylethanolamine alterations in skeletal muscles isolated from denervated and mdx mice were observed. Notably, the levels of these phospholipids binding polyunsaturated fatty acids were reduced in denervated and mdx muscles. Moreover, rescuing the mdx pathology by expressing truncated dystrophin led to the restoration of phospholipid profiles. DISCUSSION: Our findings support the hypothesis that phospholipid profiles of the skeletal muscle may be associated with skeletal muscle function.

Exons 45-55 Skipping Using Mutation-Tailored Cocktails of Antisense Morpholinos in the DMD Gene.

Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO cocktail sets for exons 45-55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder or asymptomatic individuals with an in-frame exons 45-55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient muscle cells significantly induced exons 45-55 skipping with removing 3, 8, or 10 exons and dystrophin restoration as represented by western blotting. In vivo skipping of the maximum 11 human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45-55 skipping and treat over 65% of DMD patients carrying out-of-frame or in-frame deletions.

Efficacy of Multi-exon Skipping Treatment in Duchenne Muscular Dystrophy Dog Model Neonates.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

iNOS is not responsible for RyR1 S-nitrosylation in mdx mice with truncated dystrophin.

BACKGROUND: Previous research indicated that nitric oxide synthase (NOS) is the key molecule for S-nitrosylation of ryanodine receptor 1 (RyR1) in DMD model mice (mdx mice) and that both neuronal NOS (nNOS) and inducible NOS (iNOS) might contribute to the reaction because nNOS is mislocalized in the cytoplasm and iNOS expression is higher in mdx mice. We investigated the effect of iNOS on RyR1 S-nitrosylation in mdx mice and whether transgenic expression of truncated dystrophin reduced iNOS expression in mdx mice or not. METHODS: Three- to 4-month-old C57BL/6 J, mdx, and transgenic mdx mice expressing exon 45-55-deleted human dystrophin (Tg/mdx mice) were used. We also generated two double mutant mice, mdx iNOS KO and Tg/mdx iNOS KO to reveal the iNOS contribution to RyR1 S-nitrosylation. nNOS and iNOS expression levels in skeletal muscle of these mice were assessed by immunohistochemistry (IHC), qRT-PCR, and Western blotting. Total NOS activity was measured by a citrulline assay. A biotin-switch method was used for detection of RyR1 S-nitrosylation. Statistical differences were assessed by one-way ANOVA with Tukey-Kramer post-hoc analysis. RESULTS: mdx and mdx iNOS KO mice showed the same level of RyR1 S-nitrosylation. Total NOS activity was not changed in mdx iNOS KO mice compared with mdx mice. iNOS expression was undetectable in Tg/mdx mice expressing exon 45-55-deleted human dystrophin, but the level of RyR1 S-nitrosylation was the same in mdx and Tg/mdx mice. CONCLUSION: Similar levels of RyR1 S-nitrosylation and total NOS activity in mdx and mdx iNOS KO demonstrated that the proportion of iNOS in total NOS activity was low, even in mdx mice. Exon 45-55-deleted dystrophin reduced the expression level of iNOS, but it did not correct the RyR1 S-nitrosylation. These results indicate that iNOS was not involved in RyR1 S-nitrosylation in mdx and Tg/mdx mice muscles.

Effects of systemic multiexon skipping with peptide-conjugated morpholinos in the heart of a dog model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by an absence of the dystrophin protein in bodywide muscles, including the heart. Cardiomyopathy is a leading cause of death in DMD. Exon skipping via synthetic phosphorodiamidate morpholino oligomers (PMOs) represents one of the most promising therapeutic options, yet PMOs have shown very little efficacy in cardiac muscle. To increase therapeutic potency in cardiac muscle, we tested a next-generation morpholino: arginine-rich, cell-penetrating peptide-conjugated PMOs (PPMOs) in the canine X-linked muscular dystrophy in Japan (CXMDJ) dog model of DMD. A PPMO cocktail designed to skip dystrophin exons 6 and 8 was injected intramuscularly, intracoronarily, or intravenously into CXMDJ dogs. Intravenous injections with PPMOs restored dystrophin expression in the myocardium and cardiac Purkinje fibers, as well as skeletal muscles. Vacuole degeneration of cardiac Purkinje fibers, as seen in DMD patients, was ameliorated in PPMO-treated dogs. Although symptoms and functions in skeletal muscle were not ameliorated by i.v. treatment, electrocardiogram abnormalities (increased Q-amplitude and Q/R ratio) were improved in CXMDJ dogs after intracoronary or i.v. administration. No obvious evidence of toxicity was found in blood tests throughout the monitoring period of one or four systemic treatments with the PPMO cocktail (12 mg/kg/injection). The present study reports the rescue of dystrophin expression and recovery of the conduction system in the heart of dystrophic dogs by PPMO-mediated multiexon skipping. We demonstrate that rescued dystrophin expression in the Purkinje fibers leads to the improvement/prevention of cardiac conduction abnormalities in the dystrophic heart.

Amelioration of intracellular Ca2+ regulation by exon-45 skipping in Duchenne muscular dystrophy-induced pluripotent stem cell-derived cardiomyocytes.

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by frameshift mutations in the DMD gene. DMD involves cardiac muscle, and the presence of ventricular arrhythmias or congestive failure is critical for prognosis. Several novel therapeutic approaches are being evaluated in ongoing clinical trials. Among them, exon-skipping therapy to correct frameshift mutations with antisense oligonucleotides is promising; however, their therapeutic efficacies on cardiac muscle in vivo remain unknown. In this study, we established induced-pluripotent stem cells (iPSCs) from T cells from a DMD patient carrying a DMD-exon 46-55 deletion, differentiated the iPSCs into cardiomyocytes, and treated them with phosphorodiamidate morpholino oligomers. The efficiency of exon-45 skipping increased in a dose-dependent manner and enabled restoration of the DMD gene product, dystrophin. Further, Ca2+-imaging analysis showed a decreased number of arrhythmic cells and improved transient Ca2+ signaling after exon skipping. Thus, exon-45 skipping may be effective for cardiac involvement in DMD patients harboring the DMD-exon 46-55 deletion.

Long-term, open-label, phase 3 study of rasagiline in Japanese patients with early Parkinson's disease.

Rasagiline is a monoamine oxidase B inhibitor with demonstrated efficacy and safety in patients with Parkinson's disease (PD). We recently conducted the first randomized, double-blind, placebo-controlled trial of rasagiline in Japanese patients with early PD and now report the results of its open-label extension (clinicaltrials.gov, NCT02337751). In the double-blind trial, patients aged 30-79 years with PD diagnosis within 5 years and Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) Part II + Part III total score ≥ 14 were randomized to placebo or rasagiline 1 mg/day for 26 weeks. Of 210 patients who completed the randomized trial, 198 (95 placebo, 103 rasagiline) entered the extension and received rasagiline 1 mg/day for 26 weeks. Analyses included patients who received rasagiline anytime during double-blind and/or extension periods; mean (standard deviation) treatment duration was 169.6 (39.57) and 316.5 (88.89) days in placebo-rasagiline (n = 95) and rasagiline-rasagiline (n = 117) groups, respectively. The incidence of treatment-emergent adverse events (TEAEs; primary outcome) was 53.7% and 77.8% in the placebo-rasagiline and rasagiline-rasagiline groups, respectively. Drug-related TEAEs occurred in 24.2% and 49.6% of patients and serious TEAEs occurred in four (two drug related) and six (one drug related) patients in the placebo-rasagiline and rasagiline-rasagiline groups, respectively. The mean change in MDS-UPDRS Part II + III total score from baseline (before rasagiline) was - 2.8 points in both the placebo-rasagiline (mean [95% confidence interval] - 2.8 [- 4.05, - 1.59]) and rasagiline-rasagiline (- 2.8 [- 4.57, - 1.01]) groups. In conclusion, up to 52 weeks, rasagiline was well tolerated with sustained motor symptom improvement, supporting its use in Japanese patients with early PD.

Long-term safety and efficacy of adjunctive rasagiline in levodopa-treated Japanese patients with Parkinson's disease.

Rasagiline is a monoamine oxidase type-B inhibitor in development in Japan for Parkinson's disease (PD). This open-label study evaluated the long-term safety and efficacy of rasagiline in Japanese patients with PD receiving levodopa. Patients were aged 30-79 years and had wearing-off or weakened effect. Patients received rasagiline 1 mg/day for 52 weeks. The primary objective was to evaluate safety. Secondary endpoints included MDS-UPDRS Part II and Part III total scores (ON-state) and change from baseline in mean daily OFF-time. An additional endpoint was the Parkinson's Disease Questionnaire-39 (PDQ-39) Summary Index (SI) score. In total, 222 patients were enrolled; 52.3% had wearing-off phenomena. Treatment-emergent adverse events (TEAEs) were mostly mild or moderate and occurred in 83.3% of patients; 63.1% had drug-related TEAEs; and 21.2% had TEAEs resulting in discontinuation. Fall (16.7%), nasopharyngitis (14.0%), and dyskinesia (10.8%) were the most frequent TEAEs. Serious TEAEs were reported in 17.6% of patients, and led to discontinuation in 9.5%. At week 52 (last-observation-carried forward), the mean change from baseline in MDS-UPDRS Part III total score (ON-state) was - 7.6; the mean change from baseline in daily OFF-time was - 0.89 h in patients with wearing-off phenomena at the start of the run-in period. The mean change from baseline in PDQ-39 SI was - 0.64. No major safety issues were observed during this 52-week trial of rasagiline as an adjunct to levodopa in Japanese patients. Mean changes in MDS-UPDRS scores and daily OFF-time suggested that adjunctive rasagiline treatment with levodopa was efficacious, with efficacy maintained for at least 52 weeks.

Synthesis of 2'-O-(N-methylcarbamoylethyl) 5-methyl-2-thiouridine and its application to splice-switching oligonucleotides.

The effect of 2'-O-(N-methylcarbamoyl)ethyl (MCE) modification on splice-switching oligonucleotides (SSO) was systematically evaluated. The incorporation of five MCE nucleotides at the 5'-termini of SSOs effectively improved the splice switching effect. In addition, the incorporation of 2'-O-(N-methylcarbamoylethyl)-5-methyl-2-thiouridine (s2TMCE), a duplex-stabilizing nucleotide with an MCE modification, into SSOs further improved splice switching. These SSOs may be useful for the treatment of genetic diseases associated with splicing errors.

Reactive oxygen species upregulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells.

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

Vascular abnormalities in the placenta of Chst14-/- fetuses: implications in the pathophysiology of perinatal lethality of the murine model and vascular lesions in human CHST14/D4ST1 deficiency.

Collagen is one of the most important components of the extracellular matrix that is involved in the strength of tissues, cell adhesion and cell proliferation. Mutations in several collagen and post-translational modification enzyme genes cause Ehlers-Danlos syndrome (EDS) characterized by joint and skin hyperextensibility as well as fragility of various organs. Carbohydrate sulfotransferase 14/dermatan 4-O-sulfotransferase-1 (CHST14/D4ST1) is a critical enzyme for biosynthesis of dermatan sulfate, a side chain of various proteoglycans including biglycan that regulates collagen fibrils through their interaction. Mutations in CHST14 were found to cause a new form of EDS, named musculocontractural type EDS (mcEDS-CHST14). Large subcutaneous hematomas are one of the most serious complications accompanied by decreased quality of life and potential lethality. In this study, Chst14 gene-deleted mice were expected to be an animal model of the vascular abnormalities of mcEDS-CHST14. However, only limited numbers of adult mice were generated because of perinatal lethality in most Chst14 gene-deleted homozygote (Chst14-/-) mice. Therefore, we investigated the placentas of these fetuses. The placentas of Chst14-/- fetuses showed a reduced weight, alterations in the vascular structure, and ischemic and/or necrotic-like changes. Electron microscopy demonstrated an abnormal structure of the basement membrane of capillaries in the placental villus. These findings suggest that Chst14 is essential for placental vascular development and perinatal survival of fetuses. Furthermore, placentas of Chst14-/- fetuses could be a useful model for vascular manifestations in mcEDS-CHST14, such as the large subcutaneous hematomas.