Grace Wang

Inhibition of MicroRNA 195 Prevents Apoptosis and Multiple-Organ Injury in Mouse Models of Sepsis

BACKGROUND MicroRNAs (miRs) are a class of short RNA molecules, which negatively regulate gene expression. The levels of circulating miR-15 family members are elevated in septic patients and may be associated with septic death. This study investigated whether inhibition of miR-195, a member of the miR-15 family, provided beneficial effects in sepsis. METHODS AND RESULTS Sepsis was induced by injection of feces into the peritoneum in mice. miR-195 was upregulated in the lung and liver of septic mice. Silencing of miR-195 increased the protein levels of BCL-2, Sirt1, and Pim-1; prevented apoptosis; reduced liver and lung injury; and improved the survival in septic mice. Silencing of miR-195 provided similar protection in lipopolysaccharide-induced endotoxemic mice. In endothelial cells, upregulation of miR-195 induced apoptosis, and inhibition of miR-195 prevented lipopolysaccharide-induced apoptosis. miR-195 repressed expression of its protein targets, BCL-2, Sirt1, and Pim-1. Furthermore, overexpression of Pim-1 prevented apoptosis induced by lipopolysaccharide and miR-195 mimic. Inhibition of Pim-1 attenuated the protective effects of miR-195 silencing in septic mice. CONCLUSIONS Silencing of miR-195 reduced multiple-organ injury and improved the survival in sepsis, and the protective effects of miR-195 inhibition were associated with upregulation of Bcl-2, Sirt1, and Pim-1. Thus, inhibition of miR-195 may represent a new therapeutic approach for sepsis.

Calpain-1 induces endoplasmic reticulum stress in promoting cardiomyocyte apoptosis following hypoxia/reoxygenation

Both calpain activation and endoplasmic reticulum (ER) stress are implicated in ischemic heart injury. However, the role of calpain in ER stress remains largely elusive. This study investigated whether calpain activation causes ER stress, thereby mediating cardiomyocyte apoptosis in an in vitro model of hypoxia/re-oxygenation (H/R). In neonatal mouse cardiomyocytes and rat cardiomyocyte-like H9c2 cells, up-regulation of calpain-1 sufficiently induced ER stress, c-Jun N-terminal protein kinase1/2 (JNK1/2) activation and apoptosis. Inhibition of ER stress or JNK1/2 prevented apoptosis induced by calpain-1. In an in vitro model of H/R-induced injury in cardiomyocytes, H/R was induced by a 24-hour hypoxia followed by a 24-hour re-oxygenation. H/R activated calpain-1, induced ER stress and JNK1/2 activation, and triggered apoptosis. Inhibition of calpain and ER stress blocked JNK1/2 activation and prevented H/R-induced apoptosis. Furthermore, blockade of JNK1/2 signaling inhibited apoptosis following H/R. The role of calpain in ER stress was also demonstrated in an in vivo model of ischemia/reperfusion using transgenic mice over-expressing calpastatin. In summary, calpain-1 induces ER stress and JNK1/2 activation, thereby mediating apoptosis in cardiomyocytes. Accordingly, inhibition of calpain prevents ER stress, JNK1/2 activation and apoptosis in H/R-induced cardiomyocytes. Thus, ER stress/JNK1/2 activation may represent an important mechanism linking calpain-1 to ischemic injury.

Administration of nicotinamide riboside prevents oxidative stress and organ injury in sepsis

Aims Sepsis‐caused multiple organ failure remains the major cause of morbidity and mortality in intensive care units. Nicotinamide riboside (NR) is a precursor of nicotinamide adenine dinucleotide (NAD+), which is important in regulating oxidative stress. This study investigated whether administration of NR prevented oxidative stress and organ injury in sepsis. Methods Mouse sepsis models were induced by injection of lipopolysaccharides (LPS) or feces‐injection‐in‐peritoneum. NR was given before sepsis onset. Cultured macrophages and endothelial cells were incubated with various agents. Results Administration of NR elevated the NAD+ levels, and elicited a reduction of oxidative stress, inflammation and caspase‐3 activity in lung and heart tissues, which correlated with attenuation of pulmonary microvascular permeability and myocardial dysfunction, leading to less mortality in sepsis models. These protective effects of NR were associated with decreased levels of plasma high mobility group box‐1 (HMGB1) in septic mice. Consistently, pre‐treatment of macrophages with NR increased NAD+ content and reduced HMGB1 release upon LPS stimulation. NR also prevented reactive oxygen species (ROS) production and apoptosis in endothelial cells induced by a conditioned‐medium collected from LPS‐treated macrophages. Furthermore, inhibition of SIRT1 by EX527 offset the negative effects of NR on HMGB1 release in macrophages, and ROS and apoptosis in endothelial cells. Conclusions Administration of NR prevents lung and heart injury, and improves the survival in sepsis, likely by inhibiting HMGB1 release and oxidative stress via the NAD+/SIRT1 signaling. Given NR has been used as a health supplement, it may be a useful agent to prevent organ injury in sepsis. Graphical abstract Figure. No Caption available. HighlightsAdministration of nicotinamide riboside prevents oxidative stress in sepsis.Nicotinamide riboside reduces organ injury and mortality in septic mice.Nicotinamide riboside inhibits HMGB1 release under septic conditions.Nicotinamide riboside prevents septic organ injury via NAD+/Sirt1 signaling.

Stromal Cell-Derived Factor-1 Mediates Cardiac Allograft Tolerance Induced by Human Endometrial Regenerative Cell-Based Therapy

Endometrial regenerative cells (ERCs) are mesenchymal‐like stromal cells, and their therapeutic potential has been tested in the prevention of renal ischemic reperfusion injury, acute liver injury, ulcerative colitis, and immunosuppression. However, their potential in the induction of transplant tolerance has not been investigated. The present study was undertaken to investigate the efficacy of ERCs in inducing cardiac allograft tolerance and the function of stromal cell‐derived factor‐1 (SDF‐1) in the ERC‐mediated immunoregulation. The inhibitory efficacy of human ERCs in the presence or absence of rapamycin was examined in both mouse cardiac allograft models between BALB/c (H‐2d) donors and C57BL/6 (H‐2b) recipients and in vitro cocultured splenocytes. AMD3100 was used to inhibit the function of SDF‐1. Intragraft antibody (IgG and IgM) deposition and immune cell (CD4+ and CD8+) infiltration were measured by immunohistochemical staining, and splenocyte phenotypes were determined by fluorescence‐activated cell sorting analysis. The results showed that ERC‐based therapy induced donor‐specific allograft tolerance, and functionally inhibiting SDF‐1 resulted in severe allograft rejection. The negative effects of inhibiting SDF‐1 on allograft survival were correlated with increased levels of intragraft antibodies and infiltrating immune cells, and also with reduced levels of regulatory immune cells including MHC class IIlowCD86lowCD40lowdendritic cells, CD68+CD206+macrophages, CD4+CD25+Foxp3+T cells, and CD1dhighCD5highCD83lowIL‐10highB cells both in vivo and in vitro. These data showed that human ERC‐based therapy induces cardiac allograft tolerance in mice, which is associated with SDF‐1 activity, suggesting that SDF‐1 mediates the immunosuppression of ERC‐based therapy for the induction of transplant tolerance. Stem Cells Translational Medicine 2017;6:1997–2008

Genetic Confirmation Rate in Clinically Suspected Maturity-Onset Diabetes of the Young.

OBJECTIVES Maturity-onset diabetes of the young (MODY) is the most common form of monogenic diabetes, reportedly accounting for 2% to 5% of all cases of diabetes. In samples from Canadian patients referred for molecular genetic confirmation of a clinically suspected MODY, we determined the prevalence of likely disease-causing DNA variants in known MODY genes. METHODS Between 1999 and 2015, our centre received requests from colleagues for DNA sequencing of 96 samples from unrelated Canadian patients with clinically suspected MODY. Prior to 2012, we used Sanger sequencing, and since 2012 we have used targeted next-generation sequencing. RESULTS Of 96 samples received, 39 (40.6%) had a likely rare causal variant in 1 of 8 known MODY genes. Of these, 20 (51.3%) and 19 (48.7%) were diagnosed by Sanger and targeted next-generation sequencing, respectively. The 39 mutation-positive samples had 1 of 39 rare variants, of which the majority were in genes encoding either glucokinase (GCK, or MODY2) or hepatocyte nuclear factor 1-alpha (HNF1A, or MODY3). Furthermore, 12 (30.8%) of the detected rare variants had been unreported previously but were likely to have been clinically significant according to standard bioinformatic methods. An additional 6 samples had rare variants in MODY genes that were of uncertain clinical significance. CONCLUSIONS The findings suggest that clinical suspicion for MODY has a diagnostic yield of ~40% at the molecular level. Confirmatory genetic testing in patients suspected to have MODY allows for definitive diagnoses which, in turn, may guide management and provide rationales for screening other family members presymptomatically.

Borinic Acid-Catalyzed, Regioselective Ring Opening of 3,4-Epoxy Alcohols

Diarylborinic acids (Ar2BOH) catalyze the C3-selective ring opening of 3,4-epoxy alcohols with aniline, dialkylamine and arenethiol nucleophiles. The regiochemical outcome is consistent with a catalytic tethering mechanism in which the borinic acid interacts with both the electrophile and the nucleophile. The rate acceleration resulting from this induced intramolecularity effect is sufficient to overcome steric biases that would otherwise favor C4-selective opening of the substituted epoxy alcohols.

Skin Allografting Activates Anti-tumor Immunity and Suppresses Growth of Colon Cancer in Mice

INTRODUCTION: The tumor cells could escape from the immune elimination through the immunoediting mechanisms including the generation of immunosuppressive or immunoregulative cells. By contrast, allograft transplantation could activate the immune system and induce a strong allogenic response. The aim of this study was to investigate the efficacy of allogenic skin transplantation in the inhibition of tumor growth through the activation of allogenic immune response. METHODS: Full-thickness skin transplantation was performed from C57BL/6 (H-2b) donors to BALB/c (H-2d) recipients that were receiving subcutaneous injection of isogenic CT26 colon cancer cells (2 × 106 cells) at the same time. The tumor size and pathological changes, cell populations and cytokine profiles were evaluated at day 14 post-transplantation. RESULTS: The results showed that as compared to non-transplant group, the allogenic immune response in the skin-grafting group inhibited the growth of tumors, which was significantly associated with increased numbers of intra-tumor infiltrating lymphocytes, increased populations of CD11c+MHC-classII+CD86+ DCs, CD3+CD4+ T cells, CD3+CD8+ T cells, and CD19+ B cells, as well as decreased percentage of CD4+CD25+Foxp3+ T cells in the spleens. In addition, the levels of serum IgM and IgG, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were significantly higher within the tumor in skin transplant groups than that in non-transplant group. CONCLUSIONS: Allogenic skin transplantation suppresses the tumor growth through activating the allogenic immune response, and it may provide a new immunotherapy option for the clinical refractory tumor treatment.

B7-H1 Expression Is Required for Human Endometrial Regenerative Cells in the Prevention of Transplant Vasculopathy in Mice

Vasculopathy is one of the primary pathological changes in chronic rejection of vascularized allograft transplantation. Endometrial regenerative cells (ERCs) are mesenchymal-like stromal cells with immunosuppressive effect. B7-H1 is a negative costimulator that mediates active immune suppression. The aim of this study was to investigate the requirement of B7-H1 in the immunoregulation of ERCs in preventing transplant vasculopathy of aorta allografts. The results showed that B7-H1 expression on ERCs was upregulated by IFN-γ in a dose-dependent manner and it was required for ERCs to inhibit the proliferation of peripheral blood mononuclear cells (PBMCs) in vitro. ERCs could alleviate transplant vasculopathy, as the intimal growth of transplanted aorta was limited, and the preventive effects were correlated with an increase in the percentages of CD11c+MHC class IIlowCD86low dendritic cells, CD68+CD206+ macrophages, and CD4+CD25+Foxp3+ T cells, as well as a decrease in the percentages of CD68+ macrophages, CD3+CD4+ T cells, CD3+CD8+ T cells, and donor-reactive IgM and IgG antibodies. Moreover, overexpression of B7-H1 by IFN-γ can promote the immunosuppressive effect of ERCs. These results suggest that overexpression of B7-H1 stimulated by IFN-γ is required for ERCs to prevent the transplant vasculopathy, and this study provides a theoretical basis for the future clinical use of human ERCs.