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1.  Binge Alcohol Consumption Aggravates Oxidative Stress and Promotes Pathogenesis of NASH from Obesity-Induced Simple Steatosis 
Molecular Medicine  2014;20(1):490-502.
The pathogenesis of nonalcoholic steatohepatitis (NASH) is a two-stage process in which steatosis is the “first hit” and an unknown “second hit.” We hypothesized that “a binge” could be a “second hit” to develop NASH from obesity-induced simple steatosis. Thirty-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) rats were administered 10 mL of 10% ethanol orally for 5, 3, 2, and 1 d/wk for 3 consecutive weeks. As control, male Otsuka Long-Evans Tokushima (OLET) rats were administered the same amount of alcohol. Various biochemical parameters of obesity, steatosis and NASH were monitored in serum and liver specimens in untreated and ethanol-treated rats. The liver sections were evaluated for histopathological alterations of NASH and stained for cytochrome P-4502E1 (CYP2E1) and 4-hydroxy-nonenal (4-HNE). Simple steatosis, hyperinsulinemia, hyperglycemia, insulin resistance, hypertriglycemia and marked increases in hepatic CYP2E1 and 4-HNE were present in 30-wk-old untreated OLETF rats. Massive steatohepatitis with hepatocyte ballooning was observed in the livers of all OLETF rats treated with ethanol. Serum and hepatic triglyceride levels as well as tumor necrosis factor (TNF)-α mRNA were markedly increased in all ethanol-treated OLETF rats. Staining for CYP2E1 and 4-NHE demonstrated marked increases in the hepatic tissue of all the groups of OLETF rats treated with ethanol compared with OLET rats. Our data demonstrated that “a binge” serves as a “second hit” for development of NASH from obesity-induced simple steatosis through aggravation of oxidative stress. The enhanced levels of CYP2E1 and increased oxidative stress in obesity play a significant role in this process.
doi:10.2119/molmed.2014.00048
PMCID: PMC4277551  PMID: 25121719
2.  Leukocyte Activity in Patients with ST-Segment Elevation Acute Myocardial Infarction Treated with Anakinra 
Molecular Medicine  2014;20(1):486-589.
Anakinra, the recombinant form of the human interleukin (IL)-1 receptor antagonist, blunts the acute systemic inflammatory response in patients with ST-segment elevation myocardial infarction (STEMI), by determining a fall in peripheral blood leukocyte and plasma C-reactive protein levels. The aim of the present study was to determine the effects of anakinra on the activity of leukocytes measured ex vivo. Blood was collected 72 h after admission in 17 patients enrolled in the Virginia Commonwealth University - Anakirna Remodeling Trial (2) (VCU-ART2) and randomly treated with anakinra (N = 7) or placebo (N = 10). Whole blood was cultured at 37°C for 24 h to measure spontaneous production of IL-6 or stimulated with Escherichia coli lipopolysaccharide (LPS) for toll-like receptor (TLR)-4 or heat-killed Staphylococcus epidermidis (SE) for TLR-2 activation. The cultures of anakinra-treated patients produced significantly less IL-6 spontaneously (71 pg/mL [27–114]) compared with placebo-treated patients (290 pg/mL [211–617], p = 0.005). LPS- or SE-induced IL-6 production, on the other hand, was not statistically different between anakinra-versus placebo-treated patients (344 pg/mL [94–560] versus 370 pg/mL [306–991], p = 0.32 for LPS, and 484 pg/mL [77–612] versus 615 pg/mL [413–871], p = 0.31 for SE, respectively). IL-1 blockade with anakinra in STEMI patients results in reduced spontaneous leukocyte activity ex vivo without impairing the responsiveness to bacterial stimuli.
doi:10.2119/molmed.2014.00121
PMCID: PMC4277550  PMID: 25180626
3.  A Journey in Science: Early Lessons from the Hemoglobin Field 
Molecular Medicine  2014;20(1):478-485.
Real innovations in medicine and science are historic and singular; the stories behind each occurrence are precious. At Molecular Medicine we have established the Anthony Cerami Award in Translational Medicine to document and preserve these histories. The monographs recount the seminal events as told in the voice of the original investigators who provided the crucial early insight. These essays capture the essence of discovery, chronicling the birth of ideas that created new fields of research; and launched trajectories that persisted and ultimately influenced how disease is prevented, diagnosed, and treated. In this volume, the Cerami Award Monograph is by David J Weatherall, Founder, Weatherall Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital. A visionary in the field of hemoglobin, this is the story of Professor Weatherall’s scientific journey.
doi:10.2119/molmed.2014.00093
PMCID: PMC4277548  PMID: 25548947
4.  Cell Death and DAMPs in Acute Pancreatitis 
Molecular Medicine  2014;20(1):466-477.
Cell death and inflammation are key pathologic responses of acute pancreatitis (AP), the leading cause of hospital admissions for gastrointestinal disorders. It is becoming increasingly clear that damage-associated molecular pattern molecules (DAMPs) play an important role in the pathogenesis of AP by linking local tissue damage to systemic inflammation syndrome. Endogenous DAMPs released from dead, dying or injured cells initiate and extend sterile inflammation via specific pattern recognition receptors. Inhibition of the release and activity of DAMPs (for example, high mobility group box 1, DNA, histones and adenosine triphosphate) provides significant protection against experimental AP. Moreover, increased serum levels of DAMPs in patients with AP correlate with disease severity. These findings provide novel insight into the mechanism, diagnosis and management of AP. DAMPs might be an attractive therapeutic target in AP.
doi:10.2119/molmed.2014.00117
PMCID: PMC4277549  PMID: 25105302
5.  Regulation of Autophagy by α1-Antitrypsin: “A Foe of a Foe Is a Friend” 
Molecular Medicine  2014;20(1):417-426.
Autophagy is involved in both the cell protective and the cell death process but its mechanism is largely unknown. The present work unravels a novel intracellular mechanism by which the serpin α1-antitrypsin (AAT) acts as a novel negative regulator of autophagic cell death. For the first time, the role of intracellularly synthesized AAT, other than in liver cells, is demonstrated. Autophagic cell death was induced by N-α-tosyl-l-phenylalanine chloromethyl ketone (TPCK) and tamoxifen. By utilizing a fluorescently tagged TPCK analog, AAT was “fished out” (pulled out) as a TPCK intracellular protein target. The interaction was further verified by competition binding experiments. Both inducers caused downregulation of AAT expression associated with activation of trypsin-like proteases. Furthermore, silencing AAT by siRNA induced autophagic cell death. Moreover, AAT administration to cultured cells prevented autophagic cell death. This new mechanism could have implications in the treatment of diseases by the regulation of AAT levels in which autophagy has a detrimental function. Furthermore, the results imply that the high synthesis of endogenous AAT by cancer cells could provide a novel resistance mechanism of cancer against autophagic cell death.
doi:10.2119/molmed.2014.00054
PMCID: PMC4212011  PMID: 25105300
6.  Human Genetic Evidence for Involvement of CD137 in Atherosclerosis 
Molecular Medicine  2014;20(1):456-465.
Atherosclerosis is an inflammatory disease and the main cause of cardiovascular disease. Inflammation promotes plaque instability and clinical disease, such as myocardial infarction, stroke and peripheral vascular disease. Subclinical atherosclerosis begins with thickening of the arterial intimal layer, and increased intima-media thickness (IMT) in the carotid artery is a widely used measurement of subclinical atherosclerosis. Activation of CD137 (tumor necrosis factor receptor super family 9) promotes inflammation and disease development in murine atherosclerosis. CD137 is expressed in human atherosclerosis, but its role is largely unknown. This study uses a genetic approach to investigate CD137 in human atherosclerotic disease. In publicly available data on genotype and gene expression from the HapMap project, the minor T allele of rs2453021, a single nucleotide polymorphism in CD137, was significantly associated with CD137 gene expression. In the PROCARDIS and Wellcome Trust Case Control Consortium (WTCCC) cohorts of 13,029 cases and controls, no significant association was detected between the minor T allele of rs2453021 and risk for coronary artery disease or myocardial infarction. However, in the IMPROVE multicenter study of 3,418 individuals, the minor T allele of rs2453021 was associated with increased IMT of the common carotid artery (CCA), as measured by ultrasonography, with presence of plaque in CCA and with increased incidence of adverse noncardiac vascular events. Taken together, this study shows that the minor T allele of rs2453021 is associated with increased IMT in the CCA and increased risk of incident noncardiac vascular events, thus providing the first human genetic evidence for involvement of CD137 in atherosclerosis.
doi:10.2119/molmed.2014.00004
PMCID: PMC4212009  PMID: 25032953
7.  Adoptive Transfer of Heme Oxygenase-1 (HO-1)–Modified Macrophages Rescues the Nuclear Factor Erythroid 2–Related Factor (Nrf2) Antiinflammatory Phenotype in Liver Ischemia/Reperfusion Injury 
Molecular Medicine  2014;20(1):448-455.
Macrophages are instrumental in the pathophysiology of liver ischemia/reperfusion injury (IRI). Although Nrf2 regulates macrophage-specific heme oxygenase-1 (HO-1) antioxidant defense, it remains unknown whether HO-1 induction might rescue macrophage Nrf2-dependent antiinflammatory functions. This study explores the mechanisms by which the Nrf2–HO-1 axis regulates sterile hepatic inflammation responses after adoptive transfer of ex vivo modified HO-1 overexpressing bone marrow–derived macrophages (BMMs). Livers in Nrf2-deficient mice preconditioned with Ad-HO-1 BMMs, but not Ad-β-Gal-BMMs, ameliorated liver IRI (at 6 h of reperfusion after 90 min of warm ischemia), evidenced by improved hepatocellular function (serum alanine aminotransferase [sALT] levels) and preserved hepatic architecture (Suzuki histological score). Treatment with Ad-HO-1 BMMs decreased neutrophil accumulation, proinflammatory mediators and hepatocellular necrosis/apoptosis in ischemic livers. Moreover, Ad-HO-1 transfection of Nrf2-deficient BMMs suppressed M1 (Nos2+) while promoting the M2 (Mrc-1/Arg-1+) phenotype. Unlike in controls, Ad-HO-1 BMMs increased the expression of Notch1, Hes1, phosphorylation of Stat3 and Akt in IR-stressed Nrf2-deficient livers as well as in lipopolysaccharide (LPS)-stimulated BMMs. Thus, adoptive transfer of ex vivo generated Ad-HO-1 BMMs rescued Nrf2-dependent antiinflammatory phenotype by promoting Notch1/Hes1/Stat3 signaling and reprogramming macrophages toward the M2 phenotype. These findings provide the rationale for a novel clinically attractive strategy to manage IR liver inflammation/damage.
doi:10.2119/molmed.2014.00103
PMCID: PMC4212016  PMID: 25014792
8.  Chemokine (C-X-C Motif) Receptor 4 and Atypical Chemokine Receptor 3 Regulate Vascular α1-Adrenergic Receptor Function 
Molecular Medicine  2014;20(1):435-447.
Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α1-adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α1-AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α1-AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca2+ channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α1-AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α1-AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasoreactivity and blood pressure.
doi:10.2119/molmed.2014.00101
PMCID: PMC4212013  PMID: 25032954
9.  The Role of Estrogen Receptor β in Prostate Cancer 
Molecular Medicine  2014;20(1):427-434.
Although androgen receptor (AR) signaling is the main molecular tool regulating growth and function of the prostate gland, estrogen receptor β (ERβ) is involved in the differentiation of prostatic epithelial cells and numerous antiproliferative actions on prostate cancer cells. However, ERβ splice variants have been associated with prostate cancer initiation and progression mechanisms. ERβ is promising as an anticancer therapy and in the prevention of prostate cancer. Herein, we review the recent experimental findings of ERβ signaling in the prostate.
doi:10.2119/molmed.2014.00105
PMCID: PMC4212017  PMID: 25032955
10.  Protease Activated Receptor-1 Deficiency Diminishes Bleomycin-Induced Skin Fibrosis 
Molecular Medicine  2014;20(1):410-416.
Accumulating evidence shows that protease-activated receptor-1 (PAR-1) plays an important role in the development of fibrosis, including lung fibrosis. However, whether PAR-1 also plays a role in the development of skin fibrosis remains elusive. The aim of this study was to determine the role of PAR-1 in the development of skin fibrosis. To explore possible mechanisms by which PAR-1 could play a role, human dermal fibroblasts and keratinocytes were stimulated with specific PAR-1 agonists or antagonists. To investigate the role of PAR-1 in skin fibrosis, we subjected wild-type and PAR-1-deficient mice to a model of bleomycin-induced skin fibrosis. PAR-1 activation leads to increased proliferation and extra cellular matrix (ECM) production, but not migration of human dermal fibroblasts (HDF) in vitro. Moreover, transforming growth factor (TGF)-β production was increased in keratinocytes upon PAR-1 activation, but not in HDF. The loss of PAR-1 in vivo significantly attenuated bleomycin-induced skin fibrosis. The bleomycin-induced increase in dermal thickness and ECM production was reduced significantly in PAR-1-deficient mice compared with wild-type mice. Moreover, TGF-β expression and the number of proliferating fibroblasts were reduced in PAR-1-deficient mice although the difference did not reach statistical significance. This study demonstrates that PAR-1 contributes to the development of skin fibrosis and we suggest that PAR-1 potentiates the fibrotic response mainly by inducing fibroblast proliferation and ECM production.
doi:10.2119/molmed.2014.00027
PMCID: PMC4212012  PMID: 24842054
11.  Biologic Treatment of Mild and Moderate Intervertebral Disc Degeneration 
Molecular Medicine  2014;20(1):400-409.
Disc degeneration is the most common cause of back pain in adults and has enormous socioeconomic implications. Conservative management is ineffective in most cases, and results of surgical treatment have not yet reached desirable standards. Biologic treatment options are an alternative to the above conventional management and have become very attractive in recent years. The present review highlights the currently available biologic treatment options in mild and moderate disc degeneration, where a potential for regeneration still exists. Biologic treatment options include protein-based and cell-based therapies. Protein-based therapies involve administration of biologic factors into the intervertebral disc to enhance matrix synthesis, delay degeneration or impede inflammation. These factors can be delivered by an intradiscal injection, alone or in combination with cells or tissue scaffolds and by gene therapy. Cell-based therapies comprise treatment strategies that aim to either replace necrotic or apoptotic cells, or minimize cell death. Cell-based therapies are more appropriate in moderate stages of degenerated disc disease, when cell population is diminished; therefore, the effect of administration of growth factors would be insufficient. Although clinical application of biologic treatments is far from being an everyday practice, the existing studies demonstrate promising results that will allow the future design of more sophisticated methods of biologic intervention to treat intervertebral disc degeneration.
doi:10.2119/molmed.2014.00145
PMCID: PMC4212014  PMID: 25171110
12.  Lapatinib Antagonizes Multidrug Resistance–Associated Protein 1–Mediated Multidrug Resistance by Inhibiting Its Transport Function 
Molecular Medicine  2014;20(1):390-399.
Lapatinib, a tyrosine kinase inhibitor, is used in the treatment of advanced or metastatic breast cancer overexpressing human epidermal receptor 2 (HER2). Lapatinib can modulate the function of ATP-binding cassette (ABC) transporters (ABCB1 and ABCG2), which are the major mechanism responsible for multidrug resistance (MDR) in cancer. In this study, we investigated the effect of lapatinib on multidrug resistance–associated protein 1 (MRP1 [ABCC1]), MRP2 (ABCC2), MRP4 (ABCC4) and lung relative resistance protein (LRP) drug efflux pumps. We demonstrated that lapatinib could enhance the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells in vitro and in vivo, but no effect in MRP2-, MPR4- and LRP-overexpressing cells. Furthermore, lapatinib significantly increased the accumulation of rhodamine 123 (Rho123) and doxorubicin (DOX) in MRP1-overexpressing cells. However, lapatinib did not alter the protein or mRNA expression levels of MRP1. Further studies showed that the level of phosphorylation of AKT and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) were not altered at the indicated concentrations of lapatinib. In conclusion, lapatinib enhanced the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells by inhibiting MRP1 transport function without altering the level of AKT or ERK1/2 phosphorylation. These findings will encourage the clinical research of lapatinib combined with conventional chemotherapeutic drugs in MRP1-overexpressing cancer patients.
doi:10.2119/molmed.2014.00059
PMCID: PMC4212010  PMID: 25105301
13.  A Journey in Science: Medical Scientist in Translation 
Molecular Medicine  2014;20(1):381-389.
Real innovations in medicine and science are historic and singular; the stories behind each occurrence are precious. At Molecular Medicine we have established the Anthony Cerami Award in Translational Medicine to document and preserve these histories. The monographs recount the seminal events as told in the voice of the original investigators who provided the crucial early insight. These essays capture the essence of discovery, chronicling the birth of ideas that created new fields of research; and launched trajectories that persisted and ultimately influenced how disease is prevented, diagnosed and treated. In this volume, the Cerami Award Monograph is by Göran K Hansson, MD, PhD, Karolinska Institute. A visionary in the field of cardiovascular research, this is the story of Dr. Hansson’s scientific journey.
doi:10.2119/molmed.2014.00092
PMCID: PMC4212015  PMID: 25356751
14.  Decreased Langerhans Cell Responses to IL-36γ: Altered Innate Immunity in Patients with Recurrent Respiratory Papillomatosis 
Molecular Medicine  2014;20(1):372-380.
Recurrent respiratory papillomatosis (RRP) is a rare, chronic disease caused by human papillomaviruses (HPVs) types 6 and 11 that is characterized by the polarization of adaptive immune responses that support persistent HPV infection. Respiratory papillomas express elevated mRNA levels of IL-36γ, a proinflammatory cytokine in comparison to autologous clinically normal laryngeal tissues; however there is no evidence of inflammation in these lesions. Consistent with this, respiratory papillomas do not contain TH1-like CD4+ T-cells or cytotoxic CD8+ T-cells, but instead contain a predominance of TH2-like and T regulatory cells (Tregs). In addition, papillomas also are infiltrated with immature Langerhans cells (iLCs). In this study, we show that papilloma cells express IL-36γ protein, and that human keratinocytes transduced with HPV11 have reduced IL-36γ secretion. We now provide the first evidence that peripheral blood-derived iLCs respond to IL-36γ by expressing inflammatory cytokines and chemokines. When stimulated with IL-36γ, iLCs from patients with RRP had lower expression levels of the TH2-like chemokine CCL-20 as compared with controls. Patients’ iLCs also had decreased steady state levels of CCL-1, which is a proinflammatory chemokine. Moreover, CCL-1 levels in iLCs inversely correlated with the severity of RRP. The combined decrease of TH1- and a TH2-like chemokines by iLCs from patients could have consequences in the priming of IFN-γ expression by CD8+ T-cells. Taken together, our results suggest that, in RRP, there is a defect in the proinflammatory innate immune responses made by iLCs in response to IL-36γ. The consequence of this defect may lead to persistent HPV infection by failing to support an effective HPV-specific, TH1-like and/or Tc1-like adaptive response, thus resulting in the predominant TH2-like and/or Treg micromilieu present in papillomas.
doi:10.2119/molmed.2014.00098
PMCID: PMC4153838  PMID: 24950037
15.  Activation of Poly(ADP-Ribose) Polymerase-1 Delays Wound Healing by Regulating Keratinocyte Migration and Production of Inflammatory Mediators 
Molecular Medicine  2014;20(1):363-371.
Poly(ADP-ribosyl)ation (PARylation) is a protein modification reaction regulating various diverse cellular functions ranging from metabolism, DNA repair and transcription to cell death. We set out to investigate the role of PARylation in wound healing, a highly complex process involving various cellular and humoral factors. We found that topically applied poly[ADP-ribose] polymerase (PARP) inhibitors 3-aminobenzamide and PJ-34 accelerated wound closure in a mouse model of excision wounding. Moreover, wounds also closed faster in PARP-1 knockout mice as compared with wild-type littermates. Immunofluorescent staining for poly(ADP-ribose) (PAR) indicated increased PAR synthesis in scattered cells of the wound bed. Expression of interleukin (IL)-6, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase and matrix metalloproteinase-9 was lower in the wounds of PARP-1 knockout mice as compared with control, and expression of IL-1β, cyclooxygenase-2, TIMP-1 and -2 also were affected. The level of nitrotyrosine (a marker of nitrating stress) was lower in the wounds of PARP-1 knockout animals as compared with controls. In vitro scratch assays revealed significantly faster migration of keratinocytes treated with 3-aminobenzamide or PJ34 as compared with control cells. These data suggest that PARylation by PARP-1 slows down the wound healing process by increasing the production of inflammatory mediators and nitrating stress and by slowing the migration of keratinocytes.
doi:10.2119/molmed.2014.00130
PMCID: PMC4153841  PMID: 25014793
16.  High Mobility Group Box 1 (HMGB1) Phenotypic Role Revealed with Stress 
Molecular Medicine  2014;20(1):359-362.
High mobility group box 1 (HMGB1) is an evolutionarily ancient protein that is present in one form or another in all eukaryotes. It fundamentally resides in the nucleus but translocates to the cytosol with stress and is subsequently released into the extracellular space. HMGB1 global knockout mice exhibit lethal hypoglycemia, whereas tissues and cells from conditional knockout or knock-in mice are born alive without apparent significant functional deficit. An aberrant response to targeted stress in the liver, pancreas, heart or myeloid cells is consistent with a protective role for HMGB1 in sustaining nuclear homeostasis and enabling other stress responses, including autophagy. Under some conditions, HMGB1 is not required for liver and heart function. Many challenges remain with respect to understanding the multiple roles of HMGB1 in health and disease.
doi:10.2119/molmed.2014.00063
PMCID: PMC4153844  PMID: 24937773
17.  α7 Nicotinic Acetylcholine Receptor Signaling Inhibits Inflammasome Activation by Preventing Mitochondrial DNA Release 
Molecular Medicine  2014;20(1):350-358.
The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Cellular stress is associated with changes in immunity and activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, a key component of innate immunity. Here we show that α7 nicotinic acetylcholine receptor (α7 nAchR)-signaling inhibits inflammasome activation and prevents release of mitochondrial DNA, an NLRP3 ligand. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibits inflammasome activation, whereas genetic deletion of α7 nAchR significantly enhances inflammasome activation. Acetylcholine accumulates in macrophage cytoplasm after adenosine triphosphate (ATP) stimulation in an α7 nAchR-independent manner. Acetylcholine significantly attenuated calcium or hydrogen oxide–induced mitochondrial damage and mitochondrial DNA release. Together, these findings reveal a novel neurotransmitter-mediated signaling pathway: acetylcholine translocates into the cytoplasm of immune cells during inflammation and inhibits NLRP3 inflammasome activation by preventing mitochondrial DNA release.
doi:10.2119/molmed.2013.00117
PMCID: PMC4153835  PMID: 24849809
18.  Maternal Magnesium Deficiency in Mice Leads to Maternal Metabolic Dysfunction and Altered Lipid Metabolism with Fetal Growth Restriction 
Molecular Medicine  2014;20(1):332-340.
Inadequate magnesium (Mg) intake is a widespread problem, with over 50% of women of reproductive age consuming less than the Recommended Dietary Allowance (RDA). Because pregnancy increases the requirement for Mg and the beneficial effects of magnesium sulfate for preeclampsia/eclampsia and fetal neuroprotection are well described, we examined the outcomes of Mg deficiency during pregnancy. Briefly, pregnant Swiss Webster mice were fed either control or Mg-deficient diets starting on gestational day (GD) 6 through euthanasia on GD17. Mg-deficient dams had significantly reduced weight gain and higher plasma adipokines, in the absence of inflammation. Livers of Mg-deficient dams had significantly higher saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) and lower polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) (P < 0.0001) and arachidonic acid (AA) (P < 0.0001). Mechanistically, Mg deficiency was accompanied by enhanced desaturase and elongase mRNA expression in maternal livers along with higher circulating insulin and glucose concentrations (P < 0.05) and increased mRNA expression of Srebf1 and Chrebp, regulators of fatty acid synthesis (P < 0.05). Fetal pups exposed to Mg deficiency were growth-restricted and exhibited reduced survival. Mg-deficient fetal livers showed lower MUFAs and higher PUFAs, with lower desaturase and elongase mRNA expression than controls. In addition, DHA concentrations were lower in Mg-deficient fetal brains (P < 0.05). These results indicate that Mg deficiency during pregnancy influences both maternal and fetal fatty acid metabolism, fetal growth and fetal survival, and support better understanding maternal Mg status before and during pregnancy.
doi:10.2119/molmed.2014.00137
PMCID: PMC4153836  PMID: 25025397
19.  Immunochip Identifies Novel, and Replicates Known, Genetic Risk Loci for Rheumatoid Arthritis in Black South Africans 
Molecular Medicine  2014;20(1):341-349.
The aim of this study was to identify genetic variants associated with rheumatoid arthritis (RA) risk in black South Africans. Black South African RA patients (n = 263) were compared with healthy controls (n = 374). Genotyping was performed using the Immunochip, and four-digit high-resolution human leukocyte antigen (HLA) typing was performed by DNA sequencing of exon 2. Standard quality control measures were implemented on the data. The strongest associations were in the intergenic region between the HLA-DRB1 and HLA-DQA1 loci. After conditioning on HLA-DRB1 alleles, the effect in the rest of the extended major histocompatibility (MHC) diminished. Non-HLA single nucleotide polymorphisms (SNPs) in the intergenic regions LOC389203|RBPJ, LOC100131131|IL1R1, KIAA1919|REV3L, LOC643749|TRAF3IP2, and SNPs in the intron and untranslated regions (UTR) of IRF1 and the intronic region of ICOS and KIAA1542 showed association with RA (p < 5 × 10−5). Of the SNPs previously associated with RA in Caucasians, one SNP, rs874040, locating to the intergenic region LOC389203|RBPJ was replicated in this study. None of the variants in the PTPN22 gene was significantly associated. The seropositive subgroups showed similar results to the overall cohort. The effects observed across the HLA region are most likely due to HLA-DRB1, and secondary effects in the extended MHC cannot be detected. Seven non-HLA loci are associated with RA in black South Africans. Similar to Caucasians, the intergenic region between LOC38920 and RBPJ is associated with RA in this population. The strong association of the R620W variant of the PTPN22 gene with RA in Caucasians was not replicated since this variant was monomorphic in our study, but other SNP variants of the PTPN22 gene were also not associated with RA in black South Africans, suggesting that this locus does not play a major role in RA in this population.
doi:10.2119/molmed.2014.00097
PMCID: PMC4153842  PMID: 25014791
20.  Letter from Incoming Editor in Chief of Molecular Medicine, Dr. Betty Diamond 
Molecular Medicine  2014;20(1):331.
doi:10.2119/molmed.2014.00150
PMCID: PMC4153839  PMID: 25356560
22.  Modulation of Poly(ADP-Ribose) Polymerase-1 (PARP-1)-Mediated Oxidative Cell Injury by Ring Finger Protein 146 (RNF146) in Cardiac Myocytes 
Molecular Medicine  2014;20(1):313-328.
Poly(ADP-ribose) polymerase-1 (PARP-1) activation is a hallmark of oxidative stress–induced cellular injury that can lead to energetic failure and necrotic cell death via depleting the cellular nicotinamide adenine dinucleotide (NAD+) and ATP pools. Pharmacological PARP-1 inhibition or genetic PARP-1 deficiency exert protective effects in multiple models of cardiomyocyte injury. However, the connection between nuclear PARP-1 activation and depletion of the cytoplasmic and mitochondrial energy pools is poorly understood. By using cultured rat cardiomyocytes, here we report that ring finger protein 146 (RNF146), a cytoplasmic E3-ubiquitin ligase, acts as a direct interactor of PARP-1. Overexpression of RNF146 exerts protection against oxidant-induced cell death, whereas PARP-1–mediated cellular injury is augmented after RNF146 silencing. RNF146 translocates to the nucleus upon PARP-1 activation, triggering the exit of PARP-1 from the nucleus, followed by rapid degradation of both proteins. PARP-1 and RNF146 degradation occurs in the early phase of myocardial ischemia-reperfusion injury; it precedes the induction of heat shock protein expression. Taken together, PARP-1 release from the nucleus and its rapid degradation represent newly identified steps of the necrotic cell death program induced by oxidative stress. These steps are controlled by the ubiquitin-proteasome pathway protein RNF146. The current results shed new light on the mechanism of necrotic cell death. RNF146 may represent a distinct target for experimental therapeutic intervention of oxidant-mediated cardiac injury.
doi:10.2119/molmed.2014.00102
PMCID: PMC4153837  PMID: 24842055
23.  Low-Dose T3 Replacement Restores Depressed Cardiac T3 Levels, Preserves Coronary Microvasculature and Attenuates Cardiac Dysfunction in Experimental Diabetes Mellitus 
Molecular Medicine  2014;20(1):302-312.
Thyroid dysfunction is common in individuals with diabetes mellitus (DM) and may contribute to the associated cardiac dysfunction. However, little is known about the extent and pathophysiological consequences of low thyroid conditions on the heart in DM. DM was induced in adult female Sprague Dawley (SD) rats by injection of nicotinamide (N; 200 mg/kg) followed by streptozotocin (STZ; 65 mg/kg). One month after STZ/N, rats were randomized to the following groups (N = 10/group): STZ/N or STZ/N + 0.03 μg/mL T3; age-matched vehicle-treated rats served as nondiabetic controls (C). After 2 months of T3 treatment (3 months post-DM induction), left ventricular (LV) function was assessed by echocardiography and LV pressure measurements. Despite normal serum thyroid hormone (TH) levels, STZ/N treatment resulted in reductions in myocardial tissue content of THs (T3 and T4: 39% and 17% reduction versus C, respectively). Tissue hypothyroidism in the DM hearts was associated with increased DIO3 deiodinase (which converts THs to inactive metabolites) altered TH transporter expression, reexpression of the fetal gene phenotype, reduced arteriolar resistance vessel density, and diminished cardiac function. Low-dose T3 replacement largely restored cardiac tissue TH levels (T3 and T4: 43% and 10% increase versus STZ/N, respectively), improved cardiac function, reversed fetal gene expression and preserved the arteriolar resistance vessel network without causing overt symptoms of hyperthyroidism. We conclude that cardiac dysfunction in chronic DM may be associated with tissue hypothyroidism despite normal serum TH levels. Low-dose T3 replacement appears to be a safe and effective adjunct therapy to attenuate and/or reverse cardiac remodeling and dysfunction induced by experimental DM.
doi:10.2119/molmed.2013.00040
PMCID: PMC4153843  PMID: 24960246
24.  Chronic Lymphocytic Leukemia Cells in a Lymph Node Microenvironment Depict Molecular Signature Associated with an Aggressive Disease 
Molecular Medicine  2014;20(1):290-301.
Chronic lymphocytic leukemia (CLL) cells survive longer in vivo than in vitro, suggesting that the tissue microenvironment provides prosurvival signals to tumor cells. Primary and secondary lymphoid tissues are involved in the pathogenesis of CLL, and the role of these tissue microenvironments has not been explored completely. To elucidate host–tumor interactions, we performed gene expression profiling (GEP) of purified CLL cells from peripheral blood (PB; n = 20), bone marrow (BM; n = 18), and lymph node (LN; n = 15) and validated key pathway genes by real-time polymerase chain reaction, immunohistochemistry and/or TCL1 trans-genic mice. Gene signatures representing several pathways critical for survival and activation of B cells were altered in CLL cells from different tissue compartments. Molecules associated with the B-cell receptor (BCR), B cell–activating factor/a proliferation-inducing ligand (BAFF/APRIL), nuclear factor (NF)-κB pathway and immune suppression signature were enriched in LN-CLL, suggesting LNs as the primary site for tumor growth. Immune suppression genes may help LN-CLL cells to modulate antigen-presenting and T-cell behavior to suppress antitumor activity. PB CLL cells overexpressed chemokine receptors, and their cognate ligands were enriched in LN and BM, suggesting that a chemokine gradient instructs B cells to migrate toward LN or BM. Of several chemokine ligands, the expression of CCL3 was associated with poor prognostic factors. The BM gene signature was enriched with antiapoptotic, cytoskeleton and adhesion molecules. Interestingly, PB cells from lymphadenopathy patients shared GEP with LN cells. In Eμ-TCL1 transgenic mice (the mouse model of the disease), a high percentage of leukemic cells from the lymphoid compartment express key BCR and NF-κB molecules. Together, our findings demonstrate that the lymphoid microenvironment promotes survival, proliferation and progression of CLL cells via chronic activation of BCR, BAFF/APRIL and NF-κB activation while suppressing the immune response.
doi:10.2119/molmed.2012.00303
PMCID: PMC4107103  PMID: 24800836
25.  Human Myocardium Releases Heat Shock Protein 27 (HSP27) after Global Ischemia: The Proinflammatory Effect of Extracellular HSP27 through Toll-like Receptor (TLR)-2 and TLR4 
Molecular Medicine  2014;20(1):280-289.
The myocardial inflammatory response contributes to cardiac functional injury associated with heart surgery obligating global ischemia/reperfusion (I/R). Toll-like receptors (TLRs) play an important role in the mechanism underlying myocardial I/R injury. The aim of this study was to examine the release of small constitutive heat shock proteins (HSPs) from human and mouse myocardium after global ischemia and examine the role of extracellular small HSP in myocardial injury. HSP27 release was assessed by enzyme-linked immunosorbent assay. Anti-HSP27 was applied to evaluate the role of extracellular HSP27 in the postischemic inflammatory response and functional injury in mouse hearts. Isolated hearts and cultured coronary vascular endothelial cells were exposed to recombinant HSP27 to determine its effect on proinflammatory signaling and production of proinflammatory mediators. HSP27 levels were markedly elevated in coronary sinus blood of patients and in coronary effluent of mouse hearts after global ischemia. Neutralizing extracellular HSP27 suppressed myocardial nuclear factor (NF)-κB activation and interleukin (IL)-6 production and improved cardiac function in mouse hearts. Perfusion of HSP27 to mouse hearts induced NF-κB activation and IL-6 production and depressed contractility. Further, recombinant HSP27 induced NF-κB phosphorylation and upregulated monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 production in both human and mouse coronary vascular endothelial cells. TLR2 knockout (KO) or TLR4 mutation abolished NF-κB phosphorylation and reduced MCP-1 and ICAM-1 production induced by extracellular HSP27 in endothelial cells. In conclusion, these results show that the myocardium releases HSP27 after global ischemia and that extracellular HSP27 is proinflammatory and contributes to the inflammatory mechanism of myocardial functional injury. Both TLR2 and TLR4 are involved in mediating the proinflammatory effect of extracellular HSP27.
doi:10.2119/molmed.2014.00058
PMCID: PMC4107099  PMID: 24918749

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