The American Journal of Pathology

This Month in AJP

2013-03-25

The following highlights summarize research articles that are published in the current issue of The American Journal of Pathology.

Going Forward with Genetics: Recent Technological Advances and Forward Genetics in Mice

Eva Marie Y. Moresco, Xiaohong Li, Bruce Beutler — 2013-05-01

Forward genetic analysis is an unbiased approach for identifying genes essential to defined biological phenomena. When applied to mice, it is one of the most powerful methods to facilitate understanding of the genetic basis of human biology and disease. The speed at which disease-causing mutations can be identified in mutagenized mice has been markedly increased by recent advances in DNA sequencing technology. Creating and analyzing mutant phenotypes may therefore become rate-limiting in forward genetic experimentation. We review the forward genetic approach and its future in the context of recent technological advances, in particular massively parallel DNA sequencing, induced pluripotent stem cells, and haploid embryonic stem cells.

Biological Relevance of Inflammation and Oxidative Stress in the Pathogenesis of Arterial Diseases

David P. Hajjar, Antonio M. Gotto — 2013-05-01

Over the past three decades, age-adjusted rates of cardiovascular morbidity and mortality have fallen in the United States, but the prevalence of obesity and associated metabolic disorders has risen dramatically. Recent studies have begun to unravel the complex linkages between adipose and vascular tissues that may accelerate the development of atherosclerosis in the context of obesity. Experimental models indicate that inflammation and oxidative stress, which mutually amplify each other within the vasculature and in visceral fat, are key processes that drive the initiation, progression, and subsequent rupture of the atherosclerotic lesion. Emerging research is further elucidating the contributions made by chemokines and their receptors, adipokines, and miRNAs to arterial disease. Translation of these basic science findings to clinical applications represents a tantalizing possibility for reducing the global burden of obesity-associated atherosclerosis and other cardiovascular diseases.

The Emerging Roles of HTRA1 in Musculoskeletal Disease

André Nicki Tiaden, Peter James Richards — 2013-03-13

High-temperature requirement serine protease A1 (HTRA1) is one of four known proteases belonging to the broadly conserved family of HTRA proteins. Although it was originally considered as representing an important modulator of tumorigenesis, an increasing number of reports have suggested that its influence on human disease may extend beyond cancer. HTRA1 has the capacity to degrade numerous extracellular matrix proteins, and as such, its potential involvement in diseases of the musculoskeletal system has been gaining increased attention. Musculoskeletal disease constitutes a wide variety of degenerative conditions that can manifest themselves in different ways such as joint and back pain, as well as deficiencies in skeletal bone quality, and ultimately result in significant suffering and reduced quality of life. Convincing data now exist to support a detrimental role for HTRA1 in the pathogenesis of joint and intervertebral disk degeneration. However, the function of HTRA1 in other closely related musculoskeletal diseases affecting bone and muscle remains unclear and largely unexplored. To help set the stage for future research, we discuss here some of the recent advances in our understanding of the role played by HTRA1 in musculoskeletal pathology.

Revealing the Mechanism of Tissue Damage Due to Tobacco Use: Finally, a Smoking Gun?

Philip Furmanski — 2013-03-13

This Commentary highlights the article by Li et al that presents a compelling case for a mechanism by which tobacco smoke extract (TSE) induces damage to the extracellular matrix, a key element in the pathogenesis of tobacco-related disease.

R132C IDH1 Mutations Are Found in Spindle Cell Hemangiomas and Not in Other Vascular Tumors or Malformations

2013-02-27

Spindle cell hemangioma (SCH) is a rare, benign vascular tumor of the dermis and subcutis. The lesions can be multifocal and are overrepresented in Maffucci syndrome, in which patients also have multiple enchondromas. Somatic mosaic R132C IDH1 hotspot mutations were recently identified in Maffucci syndrome. We evaluated the presence of mutations in solitary and multiple SCHs in patients without multiple enchondromas and tested a range of other vascular lesions that enter into the differential diagnosis. The R132C IDH1 mutation was identified by hydrolysis probes assay and confirmed by Sanger sequencing in 18 of 28 (64%) SCHs; of the 10 negative cases, 2 harbored a mutation in IDH2 (R172T and R172M) by Sanger sequencing. None of 154 other vascular malformations and tumors harbored an IDH1 R132C mutation, and R132H IDH1 mutations were absent in all 182 cases. All 16 SCHs examined by immunohistochemistry were negative for expression of HIF-1α. In conclusion, 20 of 28 (71%) SCHs harbored mutations in exon 4 of IDH1 or IDH2. Given that mutations were absent in 154 other vascular lesions, the mutation seems to be highly specific for SCH. The mutation does not induce expression of HIF-1α in SCH, and therefore the exact mechanism by which mutations in IDH1 or IDH2 lead to vascular tumorigenesis remains to be established.

Absence of B Cells Does Not Compromise Intramembranous Bone Formation during Healing in a Tibial Injury Model

2013-03-15

Previous studies have generated conflicting results regarding the contribution of B cells to bone formation during physiology and repair. Here, we have investigated the role of B cells in osteoblast-mediated intramembranous anabolic bone modeling. Immunohistochemistry for CD45 receptor expression indicated that B cells had no propensity or aversion for endosteal regions or sites of bone modeling and/or remodeling in wild-type mice. In the endocortical diaphyseal region, quantitative immunohistology demonstrated that young wild-type and B-cell deficient mice had similar amounts of osteocalcin+ osteoblast bone modeling surface. The degree of osteoblast-associated osteomac canopy was also comparable in these mice inferring that bone modeling cellular units were preserved in the absence of B cells. In a tibial injury model, only rare CD45 receptor positive B cells were located within areas of high anabolic activity, including minimal association with osterix+ osteoblast-lineage committed mesenchymal cells in wild-type mice. Quantitative immunohistology demonstrated that collagen type I matrix deposition and macrophage and osteoclast distribution within the injury site were not compromised by the absence of B cells. Overall, osteoblast distribution during normal growth and bone healing via intramembranous ossification proceeded normally in the absence of B cells. These observations support that in vivo, these lymphoid cells have minimal influence, or at most, make redundant contributions to osteoblast function during anabolic bone modeling via intramembranous mechanisms.

Voluntary Physical Activity Protects from Susceptibility to Skeletal Muscle Contraction–Induced Injury But Worsens Heart Function in mdx Mice

2013-03-06

It is well known that inactivity/activity influences skeletal muscle physiological characteristics. However, the effects of inactivity/activity on muscle weakness and increased susceptibility to muscle contraction–induced injury have not been extensively studied in mdx mice, a murine model of Duchenne muscular dystrophy with dystrophin deficiency. In the present study, we demonstrate that inactivity (ie, leg immobilization) worsened the muscle weakness and the susceptibility to contraction-induced injury in mdx mice. Inactivity also mimicked these two dystrophic features in wild-type mice. In contrast, we demonstrate that these parameters can be improved by activity (ie, voluntary wheel running) in mdx mice. Biochemical analyses indicate that the changes induced by inactivity/activity were not related to fiber-type transition but were associated with altered expression of different genes involved in fiber growth (GDF8), structure (Actg1), and calcium homeostasis (Stim1 and Jph1). However, activity reduced left ventricular function (ie, ejection and shortening fractions) in mdx, but not C57, mice. Altogether, our study suggests that muscle weakness and susceptibility to contraction-induced injury in dystrophic muscle could be attributable, at least in part, to inactivity. It also suggests that activity exerts a beneficial effect on dystrophic skeletal muscle but not on the heart.

SPLUNC1/BPIFA1 Contributes to Pulmonary Host Defense against Klebsiella pneumoniae Respiratory Infection

2013-03-18

Epithelial host defense proteins comprise a critical component of the pulmonary innate immune response to infection. The short palate, lung, nasal epithelium clone (PLUNC) 1 (SPLUNC1) protein is a member of the bactericidal/permeability-increasing (BPI) fold-containing (BPIF) protein family, sharing structural similarities with BPI-like proteins. SPLUNC1 is a 25 kDa secretory protein that is expressed in nasal, oropharyngeal, and lung epithelia, and has been implicated in airway host defense against Pseudomonas aeruginosa and other organisms. SPLUNC1 is reported to have surfactant properties, which may contribute to anti-biofilm defenses. The objective of this study was to assess the importance of SPLUNC1 surfactant activity in airway epithelial secretions and to explore its biological relevance in the context of a bacterial infection model. Using cultured airway epithelia, we confirmed that SPLUNC1 is critically important for maintenance of low surface tension in airway fluids. Furthermore, we demonstrated that recombinant SPLUNC1 (rSPLUNC1) significantly inhibited Klebsiella pneumoniae biofilm formation on airway epithelia. We subsequently found that Splunc1−/− mice were significantly more susceptible to infection with K. pneumoniae, confirming the likely in vivo relevance of this anti-biofilm effect. Our data indicate that SPLUNC1 is a crucial component of mucosal innate immune defense against pulmonary infection by a relevant airway pathogen, and provide further support for the novel hypothesis that SPLUNC1 protein prevents bacterial biofilm formation through its ability to modulate surface tension of airway fluids.

Glomerular Endothelial Surface Layer Acts as a Barrier against Albumin Filtration

2013-03-20

Glomerular endothelium is highly fenestrated, and its contribution to glomerular barrier function is the subject of debate. In recent years, a polysaccharide-rich endothelial surface layer (ESL) has been postulated to act as a filtration barrier for large molecules, such as albumin. To test this hypothesis, we disturbed the ESL in C57Bl/6 mice using long-term hyaluronidase infusion for 4 weeks and monitored albumin passage using immunolabeling and correlative light-electron microscopy that allows for complete and integral assessment of glomerular albumin passage. ESL ultrastructure was visualized by transmission electron microscopy using cupromeronic blue and by localization of ESL binding lectins using confocal microscopy. We demonstrate that glomerular fenestrae are filled with dense negatively charged polysaccharide structures that are largely removed in the presence of circulating hyaluronidase, leaving the polysaccharide surfaces of other glomerular cells intact. Both retention of cationic ferritin in the glomerular basement membrane and systemic blood pressure were unaltered. Enzyme treatment, however, induced albumin passage across the endothelium in 90% of glomeruli, whereas this could not be observed in controls. Yet, there was no net albuminuria due to binding and uptake of filtered albumin by the podocytes and parietal epithelium. ESL structure and function completely recovered within 4 weeks on cessation of hyaluronidase infusion. Thus, the polyanionic ESL component, hyaluronan, is a key component of the glomerular endothelial protein permeability barrier.

A Lymphocyte-Dependent Mode of Action for Imatinib Mesylate in Experimental Pulmonary Hypertension

2013-03-20

The capacity of imatinib mesylate to reverse established pulmonary arterial hypertension (PAH) has been attributed to a reduction in pulmonary arterial muscularization via inhibition of platelet-derived growth factor receptor-β on vascular smooth muscle cells. However, there is also a significant immunomodulatory component to the action of imatinib that may account for its efficacy in PAH. We found that monocrotaline-induced pulmonary hypertension was associated with a significant decrease in pulmonary natural killer (NK) cells and T lymphocytes and the accumulation of macrophages in the lungs of F344 rats. The prevention of pulmonary hypertension by imatinib blocked these changes in pulmonary leukocyte content and induced elevations in pulmonary interferon-γ, tumor necrosis factor α, and IL-10, corresponding to the enhanced activity of splenic NK cells ex vivo. Treatment with anti–asialo GM1 antiserum (ASGM1), which ablated circulating NK cells and depleted T cells, eliminated the therapeutic benefit of imatinib. ASGM1-treated animals also exhibited significant pulmonary arteriolar muscularization in response to monocrotaline challenge compared with immunocompetent controls despite daily imatinib administration to both groups. In the athymic rat, imatinib decreased right ventricular hypertrophy and pulmonary arteriolar muscularization in monocrotaline-challenged animals versus saline-treated controls but did not prevent pulmonary macrophage accumulation or the development of pulmonary hypertension. These data demonstrate that the immunomodulatory effects of imatinib are critical to its therapeutic action in experimental PAH.

Novel Proteolytic Microvesicles Released from Human Macrophages after Exposure to Tobacco Smoke

Chun-Jun Li, Yu Liu, Yan Chen, Demin Yu, Kevin Jon Williams, Ming-Lin Liu — 2013-03-13

Cigarette smoking damages the extracellular matrix in a variety of locations, leading to atherosclerotic plaque instability and emphysematous lung destruction, but the underlying mechanisms remain poorly understood. Here, we sought to determine whether exposure of human macrophages, a key participant in extracellular matrix damage, to tobacco smoke extract (TSE) induces the release of microvesicles (MVs; or microparticles) with proteolytic activity; the major proteases involved; and the cellular mechanisms that might mediate their generation. We found that MVs released from TSE-exposed macrophages carry substantial gelatinolytic and collagenolytic activities that surprisingly can be predominantly attributed to a single transmembrane protease of the matrix metalloproteinase (MMP) superfamily (namely, MMP14). Flow cytometric counts revealed that exposure of human macrophages to TSE for 20 hours more than quadrupled their production of MMP14-positive MVs (control, 1112 ± 231; TSE-induced, 5823 ± 2192 MMP14-positive MVs/μL of conditioned medium; means ± SEM; n = 6; P < 0.01). Our results indicate that the production of these MVs by human macrophages relies on a series of regulated steps that include activation of two mitogen-activated protein kinases (MAPKs, i.e., the Jun N-terminal kinase and p38 MAPK), and then MAPK-dependent induction and maturation of cellular MMP14, a remarkable accumulation of MMP14 into nascent plasma membrane blebs, and finally caspase- and MAPK-dependent apoptosis and apoptotic microvesicle generation. Proteolytically active MVs induced by tobacco smoke may be novel mediators of clinical important matrix destruction in smokers.

Membrane-Type Matrix Metalloproteinase 1 Regulates Trophoblast Functions and Is Reduced in Fetal Growth Restriction

2013-03-07

Fetal growth restriction (FGR) results from placental insufficiency to adequately supply the fetus. This insufficiency involves impaired cytotrophoblast functions, including reduced migration and invasion, proliferation, and syncytium formation. Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a key enzyme in these cellular processes. MT1-MMP exists in various forms: a 63-kDa proenzyme is synthesized as primary translation product, which is cleaved into a 57-kDa membrane-anchored active form. We hypothesized that reduced placental MT1-MMP in FGR impairs trophoblast functions. MT1-MMP mRNA and active enzyme was quantified in placentas from FGR and age-matched control pregnancies. MT1-MMP protein was localized in first-trimester and term placentas. Putative MT1-MMP functions in trophoblasts were determined using two blocking antibodies for measuring migration and proliferation, as well as fusion of primary trophoblasts and trophoblast-derived cells. MT1-MMP was expressed predominantly in the syncytiotrophoblast and the villous and extravillous cytotrophoblasts. In FGR placentas, levels of MT1-MMP mRNA and of active MT1-MMP protein were reduced (−34.2%, P < 0.05, and −21.5%, P < 0.01, respectively), compared with age-matched controls. MT1-MMP–blocking antibodies diminished migration, proliferation, and trophoblast fusion. We conclude that reduced placental MT1-MMP in FGR may contribute to the impaired trophoblast functions associated with this pathology.

FAK-Related Nonkinase Is a Multifunctional Negative Regulator of Pulmonary Fibrosis

2013-03-15

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease whose underlying molecular mechanisms are largely unknown. Herein, we show that focal adhesion kinase–related nonkinase (FRNK) plays a key role in limiting the development of lung fibrosis. Loss of FRNK function in vivo leads to increased lung fibrosis in an experimental mouse model. The increase in lung fibrosis is confirmed at the histological, biochemical, and physiological levels. Concordantly, loss of FRNK function results in increased fibroblast migration and myofibroblast differentiation and activation of signaling proteins that drive these phenotypes. FRNK-deficient murine lung fibroblasts also have an increased capacity to produce and contract matrix proteins. Restoration of FRNK expression in vivo and in vitro reverses these profibrotic phenotypes. These data demonstrate the multiple antifibrotic actions of FRNK. More important, FRNK expression is down-regulated in human IPF, and down-regulation of FRNK in normal human lung fibroblasts recapitulates the profibrotic phenotype seen in FRNK-deficient cells. The effect of loss and gain of FRNK in the experimental model, when taken together with its down-regulation in human IPF, suggests that FRNK acts as an endogenous negative regulator of lung fibrosis by repressing multiple profibrotic responses.

Reduced Expression of 15-Hydroxy Prostaglandin Dehydrogenase in Chorion during Labor Is Associated with Decreased PRB and Increased PRA and GR Expression

Yuan Li, Ping He, Qianqian Sun, Jie Liu, Lu Gao, Xingji You, Hang Gu, Xin Ni — 2013-03-18

The chorion laeve controls the levels of active prostaglandins within the uterus by NAD-dependent 15-hydroxy prostaglandin dehydrogenase (PGDH). The expression of PGDH in chorion is modulated by glucocorticoids and progesterone. In this study, we investigated glucocorticoid receptor (GR) and progesterone receptor A and B (PRA and PRB) in the regulation of PGDH expression in chorion, and we determined whether reduced PGDH expression in chorion during labor is associated with the changes in GR and PR expression by real-time RT-PCR and Western blot analysis. Dexamethasone (DEX) inhibited PGDH expression whereas progesterone stimulated PGDH expression in chorionic trophoblasts. DEX suppressed PGDH expression in GR overexpression and PR knockdown cells. The inhibitory effect of DEX did not occur in GR knockdown cells. Progesterone inhibited PGDH in GR overexpression and PR knockdown cells and it stimulated PGDH in PRB overexpression cells whereas it suppressed PGDH in PRA overexpression cells. Knockdown of c-Jun resulted in a loss of progesterone- and DEX-induced effects. PGDH was down-regulated in chorion tissues during labor. PRB was decreased whereas PRA and GR were increased in chorion during labor. Glucocorticoids inhibit PGDH expression via GR in chorionic trophoblasts. Progesterone enhances PGDH expression through PRB, whereas it inhibits PGDH expression via GR and PRA. Decreased PGDH expression is associated with increased GR and PRA, although decreased PRB, in chorion during labor.

Bifidobacteria Stabilize Claudins at Tight Junctions and Prevent Intestinal Barrier Dysfunction in Mouse Necrotizing Enterocolitis

2013-03-07

Whether intestinal barrier disruption precedes or is the consequence of intestinal injury in necrotizing enterocolitis (NEC) remains unknown. Using a neonatal mouse NEC model, we examined the changes in intestinal permeability and specific tight-junction (TJ) proteins preceding NEC and asked whether these changes are prevented by administration of Bifidobacterium infantis, a probiotic known to decrease NEC incidence in humans. Compared with dam-fed controls, pups submitted to the NEC protocol developed i) significantly increased intestinal permeability at 12 and 24 hours (as assessed by 70-kDa fluorescein isothiocyanate–dextran transmucosal flux); ii) occludin and claudin 4 internalization at 12 hours (as assessed by immunofluorescence and low-density membrane fraction immunoblotting); iii) increased claudin 2 expression at 6 hours and decreased claudin 4 and 7 expression at 24 hours; and iv) increased claudin 2 protein at 48 hours. Similar results were seen in human NEC, with claudin 2 protein increased. In mice, administration of B. infantis micro-organisms attenuated increases in intestinal permeability, preserved claudin 4 and occludin localization at TJs, and decreased NEC incidence. Thus, an increase in intestinal permeability precedes NEC and is associated with internalization of claudin 4 and occludin. Administration of B. infantis prevents these changes and reduces NEC incidence. The beneficial effect of B. infantis is, at least in part, due to its TJ and barrier-preserving properties.

Antifibrotic Effects of a Recombinant Adeno-Associated Virus Carrying Small Interfering RNA Targeting TIMP-1 in Rat Liver Fibrosis

2013-03-13

Elevated tissue inhibitor of metalloproteinase 1 (TIMP-1) expression contributes to excess production of extracellular matrix in liver fibrosis. Herein, we constructed a recombinant adeno-associated virus (rAAV) carrying siRNA of the TIMP-1 gene (rAAV/siRNA–TIMP-1) and investigated its effects on liver fibrosis in rats. Two models of rat liver fibrosis, the carbon tetrachloride and bile duct ligation models, were treated with rAAV/siRNA–TIMP-1. In the carbon tetrachloride model, rAAV/siRNA–TIMP-1 administration attenuated fibrosis severity, as determined by histologic analysis of hepatic collagen accumulation, hydroxyproline content, and concentrations of types I and III collagen in livers and sera. Levels of mRNA and active matrix metalloproteinase (MMP) 13 were elevated, whereas levels of mRNA and active MMP-2 were decreased. Moreover, a marked decrease was noted in the expression of α-smooth muscle actin, a biomarker of activated hepatic stellate cells (HSCs), and transforming growth factor-β1, critical for the development of liver fibrosis. Similarly, rAAV/siRNA–TIMP-1 treatment significantly alleviated bile duct ligation–induced liver fibrosis. Furthermore, this treatment dramatically suppressed TIMP-1 expression in HSCs from both model rats. These data indicate that the administration of rAAV/siRNA–TIMP-1 attenuated liver fibrosis by directly elevating the function of MMP-13 and diminishing activated HSCs. It also resulted in indirect decreased expression of type I collagen, MMP-2, and transforming growth factor-β1. In conclusion, rAAV/siRNA–TIMP-1 may be an effective antifibrotic gene therapy agent.

Knockout of Ste20-Like Proline/Alanine-Rich Kinase (SPAK) Attenuates Intestinal Inflammation in Mice

2013-03-15

Inflammatory bowel diseases are characterized by epithelial barrier disruption and alterations in immune regulation. Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but the underlying mechanisms need to be defined. Herein, SPAK knockout (KO) C57BL/6 mice exhibited significant increases in intestinal transepithelial resistance, a marked decrease in paracellular permeability to fluorescence isothiocyanate–dextran, and altered apical side tight junction sodium ion selectivity, compared with wild-type mice. Furthermore, the expression of junction protein, claudin-2, decreased. In contrast, expressions of occludin, E-cadherin, β-catenin, and claudin-5 increased significantly, whereas no obvious change of claudin-1, claudin-4, zonula occludens protein 1, and zonula occludens protein 2 expressions was observed. In murine models of colitis induced by dextran sulfate sodium and trinitrobenzene sulfuric acid, KO mice were more tolerant than wild-type mice, as demonstrated by colonoscopy features, histological characteristics, and myeloperoxidase activities. Consistent with these findings, KO mice showed increased IL-10 levels and decreased proinflammatory cytokine secretion, ameliorated bacterial translocation on treatment with dextran sulfate sodium, and regulation of with no lysine (WNK) kinase activity. Together, these features may reduce epithelial permeability. In conclusion, SPAK deficiency increases intestinal innate immune homeostasis, which is important for control or attenuation of pathological responses in inflammatory bowel diseases.

miR-101 Inhibits Cholangiocarcinoma Angiogenesis through Targeting Vascular Endothelial Growth Factor (VEGF)

Jinqiang Zhang, Chang Han, Hanqing Zhu, Kyoungsub Song, Tong Wu — 2013-05-01

Recent evidence has suggested an important role of miRNAs in liver biology and diseases, although the implication of miRNAs in cholangiocarcinoma remains to be defined further. This study was designed to examine the biological function and molecular mechanism of miR-101 in cholangiocarcinogenesis and tumor progression. In situ hybridization and quantitative RT-PCR were performed to determine the expression of miR-101 in human cholangiocarcinoma tissues and cell lines. Compared with noncancerous biliary epithelial cells, the expression of miR-101 is decreased in 43.5% of human cholangiocarcinoma specimens and in all three cholangiocarcinoma cell lines used in this study. Forced overexpression of miR-101 significantly inhibited cholangiocarcinoma growth in severe combined immunodeficiency mice. miR-101-overexpressed xenograft tumor tissues showed decreased capillary densities and decreased levels of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2). The VEGF and COX-2 mRNAs were identified as the bona fide targets of miR-101 in cholangiocarcinoma cells by both computational analysis and experimental assays. miR-101 inhibits cholangiocarcinoma angiogenesis by direct targeting of VEGF mRNA 3′untranslated region and by repression of VEGF gene transcription through inhibition of COX-2. This study established a novel tumor-suppressor role of miR-101 in cholangiocarcinoma and it suggests the possibility of targeting miR-101 and related signaling pathways for future therapy.

Interaction between CX3CL1 and CX3CR1 Regulates Vasculitis Induced by Immune Complex Deposition

Sohshi Morimura, Makoto Sugaya, Shinichi Sato — 2013-03-07

A type III hypersensitivity reaction induced by an immune complex, such as leukocytoclastic vasculitis, is mediated by inflammatory cell infiltration that is highly regulated by multiple adhesion molecules. CX3CL1, a ligand for CX3C chemokine receptor 1 (CX3CR1), has recently been identified as a key mediator of leukocyte adhesion that functions without the recruitment of integrins or selectin-mediated rolling. To elucidate the role of CX3CL1 and CX3CR1 in the development of leukocytoclastic vasculitis, the cutaneous and peritoneal reverse Arthus reactions, classic experimental models for immune complex–mediated tissue injury, were examined in mice lacking CX3CR1. CX3CL1 expression in sera and lesional skin of patients with polyarteritis nodosa (PN) and healthy controls was also examined. Edema and hemorrhage were significantly reduced in CX3CR1−/− mice compared with wild-type mice. Infiltration of neutrophils and mast cells and expression of IL-6 and tumor necrosis factor-α were also decreased in CX3CR1−/− mice. CX3CL1 was expressed in endothelial cells during the cutaneous reverse Arthus reactions. Furthermore, serum CX3CL1 levels were significantly higher in patients with PN than in healthy controls. Endothelial cells in lesional skin of patients with PN strongly expressed CX3CL1. These results suggest that interactions between CX3CL1 and CX3CR1 may contribute to the development of leukocytoclastic vasculitis by regulating neutrophil and mast cell recruitment and cytokine expression.

Role and Regulation of PDGFRα Signaling in Liver Development and Regeneration

Prince K. Awuah, Kari N. Nejak-Bowen, Satdarshan P.S. Monga — 2013-03-25

Aberrant platelet-derived growth factor receptor-α (PDGFRα) signaling is evident in a subset of hepatocellular cancers (HCCs). However, its role and regulation in hepatic physiology remains elusive. In the current study, we examined PDGFRα signaling in liver development and regeneration. We identified notable PDGFRα activation in hepatic morphogenesis that, when interrupted by PDGFRα-blocking antibody, led to decreased hepatoblast proliferation and survival in embryonic liver cultures. We also identified temporal PDGFRα overexpression, which is regulated by epidermal growth factor (EGF) and tumor necrosis factor α, and its activation at 3 to 24 hours after partial hepatectomy. Through generation of hepatocyte-specific PDGFRA knockout (KO) mice that lack an overt phenotype, we show that absent PDGFRα compromises extracelluar signal-regulated kinases and AKT activation 3 hours after partial hepatectomy, which, however, is alleviated by temporal compensatory increases in the EGF receptor (EGFR) and the hepatocyte growth factor receptor (Met) expression and activation along with rebound activation of extracellular signal-regulated kinases and AKT at 24 hours. These untimely increases in EGFR and Met allow for normal hepatocyte proliferation at 48 hours in KO, which, however, are aberrantly prolonged up to 72 hours. Intriguingly, such compensation also was visible in primary KO hepatocyte cultures but not in HCC cells after siRNA-mediated PDGFRα knockdown. Thus, temporal activation of PDGFRα in liver development is important in hepatic morphogenesis. In liver regeneration, despite increased signaling, PDGFRα is dispensable owing to EGFR and Met compensation, which is unique to normal hepatocytes but not HCC cells.

Ranibizumab Is a Potential Prophylaxis for Proliferative Vitreoretinopathy, a Nonangiogenic Blinding Disease

2013-04-11

Proliferative vitreoretinopathy (PVR) exemplifies a disease that is difficult to predict, lacks effective treatment options, and substantially reduces the quality of life of an individual. Surgery to correct a rhegmatogenous retinal detachment fails primarily because of PVR. Likely mediators of PVR are growth factors in vitreous, which stimulate cells within and behind the retina as an inevitable consequence of a breached retina. Three classes of growth factors [vascular endothelial growth factor A (VEGF-A), platelet-derived growth factors (PDGFs), and non-PDGFs (growth factors outside of the PDGF family)] are relevant to PVR pathogenesis because they act on PDGF receptor α, which is required for experimental PVR and is associated with this disease in humans. We discovered that ranibizumab (a clinically approved agent that neutralizes VEGF-A) reduced the bioactivity of vitreous from patients and experimental animals with PVR, and protected rabbits from developing disease. The apparent mechanism of ranibizumab action involved derepressing PDGFs, which, at the concentrations present in PVR vitreous, inhibited non–PDGF-mediated activation of PDGF receptor α. These preclinical findings suggest that available approaches to neutralize VEGF-A are prophylactic for PVR, and that anti–VEGF-based therapies may be effective for managing more than angiogenesis- and edema-driven pathological conditions.

Amelioration of Experimental Autoimmune Encephalomyelitis by the Quinoline-3-Carboxamide Paquinimod: Reduced Priming of Proinflammatory Effector CD4+ T Cells

2013-03-18

Quinoline-3-carboxamide compounds (Q compounds) have demonstrated efficacy in treating autoimmune disease in both humans and mice. However, the mode of action of these compounds is poorly understood. Here, we show that preventive treatment with the Q compound paquinimod (ABR-215757) during the first 5 days after induction of experimental autoimmune encephalomyelitis is sufficient to significantly ameliorate disease symptoms. Parallel cell-depletion experiments demonstrated that Ly6Chi inflammatory monocytes play an essential role in this phase. The paquinimod-induced amelioration correlated with reduced priming of antigen-specific CD4+ T cells and reduced frequency of IFN-γ– and IL-17–producing cells in draining lymph nodes. Importantly, the treatment did not inhibit T-cell division per se. In mice with established experimental autoimmune encephalomyelitis, the numbers of Ly6Chi CD115+ inflammatory monocytes and CD11b+CD11c+ dendritic cells (DCs) were reduced in spleen, but not in bone marrow or draining lymph nodes of treated mice. Inflammatory monocyte–derived DCs and CD4+ T cells were also reduced in the brain. In contrast, there was no decrease in DC subsets previously shown to be critical for effector CD4+ T-cell development in lymph nodes. Taken together, these data indicate that preventive treatment with paquinimod ameliorates experimental autoimmune encephalomyelitis by reducing effector T-cell priming and, on prolonged treatment, displays a selective effect by decreasing distinct subpopulations of splenic CD11b+ myeloid cells.

Progressive Reactive Lymphoid Connective Tissue Disease and Development of Autoantibodies in Scavenger Receptor A5–Deficient Mice

Juha Risto Matias Ojala, Timo Pikkarainen, Göran Elmberger, Karl Tryggvason — 2013-03-18

Scavenger receptor A5 (SCARA5) is a member of the class A scavenger receptors, with most similarity to SCARA1 (SR-A) and SCARA2 (MARCO), which are primarily expressed by macrophages and dendritic cells, in which they participate in clearance of various polyanionic macromolecules, pollution particles, and pathogens. The biological role of SCARA5 has been unknown. Herein, we show that SCARA5 is an endocytotic receptor whose ligand repertoire includes the typical scavenger receptor ligands, whole bacteria, and purified Gram-negative bacterial lipopolysaccharide. In contrast to expression of SCARA1 and SCARA2 in immune cells, SCARA5 is found in a subset of fibroblast-like cells in the interstitial stroma of most organs, with additional expression in the epithelial cells of testis and choroid plexus. SCARA5-null mice develop with age lymphoid cell accumulation in many organs, in particular the lungs, and show decreased endocytotic function in fibroblasts. Furthermore, about one-third of the mice develop antinuclear antibodies. These disturbances are reminiscent of those found in many human autoimmune connective tissue disorders, which suggests that defects in fibroblast SCARA5 can underlie some forms of autoimmune disease.

Kruppel-Like Factor 2 Is a Transcriptional Regulator of Chronic and Acute Inflammation

2013-03-15

Although myeloid cell activation is requisite for an optimal innate immune response, this process must be tightly controlled to prevent collateral host tissue damage. Kruppel-like factor 2 (KLF2) is a potent regulator of myeloid cell proinflammatory activation. As an approximately 30% to 50% reduction in KLF2 levels has been observed in human subjects with acute or chronic inflammatory disorders, we studied the biological response to inflammation in KLF2+/− mice. Herein, we show that partial deficiency of KLF2 modulates the in vivo response to acute (sepsis) and subacute (skin) inflammatory challenge. Mechanistically, we link the anti-inflammatory effects of KLF2 to the inhibition of NF-κB transcriptional activity. Collectively, the observations provide biologically relevant insights into KLF2-mediated modulation of these inflammatory processes that could potentially be manipulated for therapeutic gain.

Visfatin Enhances the Production of Cathelicidin Antimicrobial Peptide, Human β-Defensin-2, Human β-Defensin-3, and S100A7 in Human Keratinocytes and Their Orthologs in Murine Imiquimod-Induced Psoriatic Skin

2013-03-18

Psoriasis, a chronic inflammatory dermatosis, is frequently associated with metabolic disorders, suggesting that adipokines are involved in its pathogenesis. We recently reported that the adipokine visfatin activates NF-κB and STAT3 in keratinocytes. Antimicrobial peptide expression is enhanced in psoriatic lesions and may promote disease development. Here, we investigated the effects of visfatin on antimicrobial peptide expression. In vitro, visfatin enhanced basal and tumor necrosis factor-α (TNF-α)–induced mRNA expression and secretion of cathelicidin antimicrobial peptide (CAMP), and enhanced TNF-α–induced human β-defensin-2 (hBD-2), hBD-3, and S100A7 mRNA expression and secretion in human keratinocytes. siRNAs targeting CCAAT/enhancer-binding protein-α (C/EBPα) suppressed visfatin-induced and visfatin plus TNF-α–induced CAMP production. siRNAs targeting NF-κB p65 and STAT3 suppressed visfatin plus TNF-α–induced hBD-2 and S100A7 production. siRNAs targeting c-Jun and STAT3 suppressed visfatin plus TNF-α–induced hBD-3 production. Visfatin and/or TNF-α enhanced C/EBP transcriptional activity and C/EBPα phosphorylation, which were suppressed by p38 mitogen-activated protein kinase (MAPK) inhibition. Visfatin and/or TNF-α induced p38 MAPK phosphorylation. Visfatin increased mRNA and protein expression of CAMP, hBD-2, hBD-3, and S100A7 orthologs in murine imiquimod-treated skin, mimicking psoriasis. In conclusion, visfatin enhances CAMP, hBD-2, hBD-3, and S100A7 production in human keratinocytes and their orthologs in murine imiquimod-treated psoriatic skin. Visfatin may potentiate the development of psoriasis via antimicrobial peptides.

Calsyntenin-3 C-Terminal Fragment Accumulates in Dystrophic Neurites Surrounding Aβ Plaques in Tg2576 Mouse and Alzheimer Disease Brains: Its Neurotoxic Role in Mediating Dystrophic Neurite Formation

Yoko Uchida, Fujiya Gomi, Shigeo Murayama, Hiroshi Takahashi — 2013-03-15

Dystrophic neurites surrounding β-amyloid (Aβ) plaques precede neuronal death in Alzheimer disease. These neuritic alterations may be one of the initial stages for synaptic loss and dysfunction. However, intracellular pathways that cause local disruption of neuronal processes by Aβ remain to be fully elucidated. The identification of Aβ-induced genes that mediate neuritic pathology would provide considerable insight into the mechanisms of Alzheimer’s disease. Previously, we reported that selective up-regulation of calsyntenin-3 (Cst-3) by Aβ and accumulation of neurotoxic Cst-3 in dystrophic neurites surrounding Aβ plaques may lead to local disruption of these neurites. Like amyloid precursor protein, Cst-3 undergoes two-step proteolytic processing: the primary cleavage with α-secretase generates an N-terminal ectodomain and a C-terminal fragment (CTF). The CTF is subsequently cleaved into p3 peptide and an intracellular domain via γ-secretase. It would be interesting to know whether accumulated Cst-3 in dystrophic neurites surrounding Aβ plaques is the full-length version or a CTF. Herein, we show that the CTF but not full-length Cst-3 accumulated in dystrophic neurites surrounding Aβ plaques in Tg2576 mouse and Alzheimer disease brains. In vitro experiments with Cst-3 fragments have revealed that only the CTF resulted in acceleration of neuronal death. These results indicate that accumulation of the neurotoxic CTF in neurites surrounding Aβ plaques may lead to local disruption of neuronal processes and development of dystrophic neurites.

Loss of Aquaporin 9 Expression Adversely Affects the Survival of Retinal Ganglion Cells

Akiko Miki, Akiyasu Kanamori, Akira Negi, Maiko Naka, Makoto Nakamura — 2013-03-18

Aquaporin 9 (AQP9), an aquaglyceroporin belonging to the AQP water channel family, is permeable not only to water but also to noncharged solutes such as lactate. In neurons, lactate presumably acts as an energy substrate and as a source of NADH (the reduced form of nicotinamide adenine dinucleotide), a scavenger of reactive oxygen species (ROS). We previously reported that retinal ganglion cells (RGCs) express AQP9 and that elevated intraocular pressure reduces AQP9 expression and increases death of neurons in the retinal ganglion cell layer of rodents. In the present study, we investigated the association of AQP9 expression with serum deprivation–induced death of RGC-5 cells and with death of neurons in the rat retinal ganglion cell layer after optic nerve transection (ONT). The effect of AQP9 RNA interference on serum deprivation–induced apoptosis, ROS accumulation, and the NAD+/NADH ratio in RGC-5 cells was examined. Both serum deprivation and ONT significantly reduced AQP9 protein expression in RGCs and increased the rate of RGC death. Retinal AQP9 gene expression also declined after ONT. Down-regulation of AQP9 significantly increased apoptosis, ROS accumulation, and the NAD+/NADH ratio in the RGC-5 cells. These findings suggest that AQP9 loss adversely affects survival of RGCs, at least partly because of decreased transport of lactate as a substrate for energy and/or ROS scavenger.

The Bradykinin B1 Receptor Regulates Aβ Deposition and Neuroinflammation in Tg-SwDI Mice

2013-03-07

The deposition of amyloid-β peptides (Aβ) in the cerebral vasculature, a condition known as cerebral amyloid angiopathy, is increasingly recognized as an important component leading to intracerebral hemorrhage, neuroinflammation, and cognitive impairment in Alzheimer disease (AD) and related disorders. Recent studies demonstrated a role for the bradykinin B1 receptor (B1R) in cognitive deficits induced by Aβ in mice; however, its involvement in AD and cerebral amyloid angiopathy is poorly understood. Herein, we investigated the effect of B1R inhibition on AD-like neuroinflammation and amyloidosis using the transgenic mouse model (Tg-SwDI). B1R expression was found to be up-regulated in brains of Tg-SwDI mice, specifically in the vasculature, neurons, and astrocytes. Notably, administration of the B1R antagonist, R715, to 8-month-old Tg-SwDI mice for 8 weeks resulted in higher Aβ40 levels and increased thioflavin S–positive fibrillar Aβ deposition. Moreover, blockage of B1R inhibited neuroinflammation, as evidenced by the decreased accumulation of activated microglia and reactive astrocytes, diminished NF-κB activation, and reduced cytokine and chemokine levels. Together, our results indicate that B1R activation plays an important role in limiting the accumulation of Aβ in AD-like brain, likely through the regulation of activated glial cell accumulation and release of pro-inflammatory mediators. Therefore, the modulation of the receptor may represent a novel therapeutic approach for AD.

Blocking the Interaction between Apolipoprotein E and Aβ Reduces Intraneuronal Accumulation of Aβ and Inhibits Synaptic Degeneration

2013-03-13

Accumulation of β-amyloid (Aβ) in the brain is a key event in Alzheimer disease pathogenesis. Apolipoprotein (Apo) E is a lipid carrier protein secreted by astrocytes, which shows inherent affinity for Aβ and has been implicated in the receptor-mediated Aβ uptake by neurons. To characterize ApoE involvement in the intraneuronal Aβ accumulation and to investigate whether blocking the ApoE/Aβ interaction could reduce intraneuronal Aβ buildup, we used a noncontact neuronal-astrocytic co-culture system, where synthetic Aβ peptides were added into the media without or with cotreatment with Aβ12-28P, which is a nontoxic peptide antagonist of ApoE/Aβ binding. Compared with neurons cultured alone, intraneuronal Aβ content was significantly increased in neurons co-cultured with wild-type but not with ApoE knockout (KO) astrocytes. Neurons co-cultured with astrocytes also showed impaired intraneuronal degradation of Aβ, increased level of intraneuronal Aβ oligomers, and marked down-regulation of several synaptic proteins. Aβ12-28P treatment significantly reduced intraneuronal Aβ accumulation, including Aβ oligomer level, and inhibited loss of synaptic proteins. Furthermore, we showed significantly reduced intraneuronal Aβ accumulation in APPSW/PS1dE9/ApoE KO mice compared with APPSW/PS1dE9/ApoE targeted replacement mice that expressed various human ApoE isoforms. Data from our co-culture and in vivo experiments indicate an essential role of ApoE in the mechanism of intraneuronal Aβ accumulation and provide evidence that ApoE/Aβ binding antagonists can effectively prevent this process.

Embryonic Stem Cell–Based Modeling of Tau Pathology in Human Neurons

2013-03-13

Alterations in the microtubule (MT)–associated protein, tau, have emerged as a pivotal phenomenon in several neurodegenerative disorders, including frontotemporal dementia and Alzheimer's disease. Although compelling lines of evidence from various experimental models suggest that hyperphosphorylation and conformational changes of tau can cause its aggregation into filaments, the actual tau species and effective mechanisms that conspire to trigger the degeneration of human neurons remain obscure. Herein, we explored whether human embryonic stem cell–derived neural stem cells can be exploited to study consequences of an overexpression of 2N4R tau (two normal N-terminal and four MT-binding domains; n-tau) versus pseudohyperphosphorylated tau (p-tau) directly in human neurons. Given the involvement of tau in MT integrity and cellular homeostasis, we focused on the effects of both tau variants on subcellular transport and neuronal survival. By using inducible lentiviral overexpression, we show that p-tau, but not n-tau, readily leads to an MC-1–positive protein conformation and impaired mitochondrial transport. Although these alterations do not induce cell death under standard culture conditions, p-tau–expressing neurons cultured under non–redox-protected conditions undergo degeneration with formation of axonal varicosities sequestering transported proteins and progressive neuronal cell death. Our data support a causative link between the phosphorylation and conformational state of tau, microtubuli-based transport, and the vulnerability of human neurons to oxidative stress. They further depict human embryonic stem cell–derived neurons as a useful experimental model for studying tau-associated cellular alterations in an authentic human system.

Aspirin-Triggered Lipoxin A4 Stimulates Alternative Activation of Microglia and Reduces Alzheimer Disease–Like Pathology in Mice

2013-03-18

Microglia play an essential role in innate immunity, homeostasis, and neurotropic support in the central nervous system. In Alzheimer disease (AD), these cells may affect disease progression by modulating the buildup of β-amyloid (Aβ) or releasing proinflammatory cytokines and neurotoxic substances. Discovering agents capable of increasing Aβ uptake by phagocytic cells is of potential therapeutic interest for AD. Lipoxin A4 (LXA4) is an endogenous lipid mediator with potent anti-inflammatory properties directly involved in inflammatory resolution, an active process essential for appropriate host responses, tissue protection, and the return to homeostasis. Herein, we demonstrate that aspirin-triggered LXA4 (15 μg/kg) s.c., twice a day, reduced NF-κB activation and levels of proinflammatory cytokines and chemokines, as well as increased levels of anti-inflammatory IL-10 and transforming growth factor-β. Such changes in the cerebral milieu resulted in recruitment of microglia in an alternative phenotype, as characterized by the up-regulation of YM1 and arginase-1 and the down-regulation of inducible nitric oxide synthase expression. Microglia in an alternative phenotype–positive cells demonstrated improved phagocytic function, promoting clearance of Aβ deposits and ultimately leading to reduction in synaptotoxicity and improvement in cognition. Our data indicate that activating LXA4 signaling may represent a novel therapeutic approach for AD.

Possible Role of Mural Cell–Covered Mature Blood Vessels in Inducing Drug Resistance in Cancer-Initiating Cells

2013-03-13

Cancer recurrence has been suggested to be induced by residual cancer-initiating cells (CICs) or cancer stem cells (CSCs) after chemotherapy. Moreover, it is possible that CICs/CSCs acquire more aggressive behavior after therapy as shown by invasion and metastasis. In the cancer microenvironment, CICs/CSCs may localize in a specific area, the so-called stem cell niche, and isolation of this niche is important to elucidate the molecular mechanism of how CICs/CSCs acquire malignancy. We analyzed whether CICs acquire drug resistance after cancer drug treatment in a tumor cell allograft model in which we could identify and isolate living CICs by detecting a higher level of transcriptional activity of the PSF1 gene promoter. In our models using Lewis lung carcinoma (LLC) mouse lung cancer and colon26 mouse colon cancer cell lines, we found that CICs in both tumors acquired drug resistance after cancer drug treatment. Interestingly, response to the anticancer drug was quite different between LLC and colon26 original tumors (ie, the proportion of CICs in LLC tumors increased but in colon26 tumors the proportion decreased). We found that CICs frequently localized near mature blood vessels in which endothelial cells were covered with mural cells and that the incidence of mature blood vessels in LLC tumors was four times higher than in colon26 tumors. These results suggest a relationship between mature blood vessels and CIC drug resistance.

Inhibition of ATP Citrate Lyase Induces an Anticancer Effect via Reactive Oxygen Species: AMPK as a Predictive Biomarker for Therapeutic Impact

2013-03-18

De novo lipogenesis is activated in most cancers. Inhibition of ATP citrate lyase (ACLY), the enzyme that catalyzes the first step of de novo lipogenesis, leads to growth suppression and apoptosis in a subset of human cancer cells. Herein, we found that ACLY depletion increases the level of intracellular reactive oxygen species (ROS), whereas addition of an antioxidant reduced ROS and attenuated the anticancer effect. ACLY depletion or exogenous hydrogen peroxide induces phosphorylation of AMP-activated protein kinase (p-AMPK), a crucial regulator of lipid metabolism, independently of energy status. Analysis of various cancer cell lines revealed that cancer cells with a higher susceptibility to ACLY depletion have lower levels of basal ROS and p-AMPK. Mitochondrial-deficient ρ0 cells retained high levels of ROS and p-AMPK and were resistant to ACLY depletion, whereas the replenishment of normal mitochondrial DNA reduced the levels of ROS and p-AMPK and restored the sensitivity to ACLY depletion, indicating that low basal levels of mitochondrial ROS are critical for the anticancer effect of ACLY depletion. Finally, p-AMPK levels were significantly correlated to the levels of oxidative DNA damage in colon cancer tissues, suggesting that p-AMPK reflects cellular ROS levels in vitro and in vivo. Together, these data suggest that ACLY inhibition exerts an anticancer effect via increased ROS, and p-AMPK could be a predictive biomarker for its therapeutic outcome.

Decreased Tumorigenesis and Mortality from Bladder Cancer in Mice Lacking Urothelial Androgen Receptor

2013-03-13

Much fewer mice lacking androgen receptor (AR) in the entire body develop bladder cancer (BCa). However, the role of urothelial AR (Uro-AR) in BCa development remains unclear. In the present study, we generated mice that lacked only Uro-AR (Uro-AR−/y) to develop BCa by using the carcinogen BBN [N-butyl-N-(4-hydroxybutyl)-nitrosamine] and found that Uro-AR−/y mice had a lower incidence of BCa and a higher survival rate than did their wild-type (WT; Uro-AR+/y) littermates. In vitro assay also demonstrated that Uro-AR facilitates the neoplastic transformation of normal urothelial cells to carcinoma. IHC staining exhibited less DNA damage, with much higher expression of p53 and its downstream target protein PNCA in Uro-AR−/y than that found in WT urothelium, which suggests that Uro-AR may modulate bladder tumorigenesis through p53-PCNA DNA repair signaling. Indeed, Uro-AR−/y mice with the transgene, simian vacuolating virus 40 T (SV40T), in the urothelium (Uro-SV40T-AR−/y) had a similar incidence of BCa as did their WT littermates (Uro-SV40T-AR+/y), and p53 was inactivated by SV40T in both genotypes. Use of the AR degradation enhancer ASC-J9 led to suppression of bladder tumorigenesis, with few adverse effects in the BBN-induced BCa mouse model. Together, these results provide the first direct in vivo evidence that Uro-AR has an important role in promoting bladder tumorigenesis and BCa progression. Targeting AR with ASC-J9 may provide a novel approach to suppress BCa initiation.

Galectin-3 Accelerates M2 Macrophage Infiltration and Angiogenesis in Tumors

2013-03-13

It is widely accepted that robust invasion of tumor-associated macrophages resembling M2 macrophage correlates with disease aggressiveness by affecting cancer cell invasion, metastasis, and angiogenesis. Many chemokines that induce migration of macrophages have been identified during inflammatory responses; however, further precise analysis of macrophage migration in the tumor microenvironment is required. Here, we analyzed the function of galectin-3 (Gal-3; gene LGALS3, alias Gal3) for macrophage chemotaxis using Gal3−/− mice as hosts, and a tumor allograft model. We engineered a concentration gradient of Gal-3 produced by the tumor. In this model, we found that macrophage infiltration was enhanced in tumors developing in these Gal3−/− mice relative to the Gal3+/+ animals. This was accompanied by enhanced tumor angiogenesis and tumor growth in Gal3−/− mice. We found that macrophages of the M2 phenotype were dominant in infiltrates in the Gal3−/− mice and that they expressed only low levels of Gal-3. Gal3 knockdown by siRNA in macrophages resulted in enhanced chemotaxis. These data suggest that M2-like macrophages migrate into the tumor along a Gal-3 gradient and that high-level Gal-3 expression in the tumor results in acceleration of angiogenesis and tumor growth. Therefore, Gal-3 could be a potential target for the development of new treatments to inhibit tumor growth.

Protective Roles of Epithelial Cells in the Survival of Adult T-Cell Leukemia/Lymphoma Cells

2013-03-07

Adult T-cell leukemia/lymphoma (ATL) is a highly invasive and intractable T-cell malignancy caused by human T-cell leukemia virus-1 infection. We demonstrate herein that normal tissue–derived epithelial cells (NECs) exert protective effects on the survival of leukemic cells, which may partially account for high resistance to antileukemic therapies in patients with ATL. Viral gene–silenced, ATL-derived cell lines (ATL cells) dramatically escaped from histone deacetylase inhibitor–induced apoptosis by direct co-culture with NECs. Adhesions to NECs suppressed p21Cip1 expression and increased a proportion of resting G0/G1 phase cells in trichostatin A (TSA)–treated ATL cells. ATL cells adhering to NECs down-regulated CD25 expression and enhanced vimentin expression, suggesting that most ATL cells acquired a quiescent state by cell-cell interactions with NECs. ATL cells adhering to NECs displayed highly elevated expression of the cancer stem cell marker CD44. Blockade of CD44 signaling diminished the NEC-conferred resistance of ATL cells to TSA-induced apoptosis. Co-culture with NECs also suppressed the expression of NKG2D ligands on TSA-treated ATL cells, resulting in decreased natural killer cell–mediated cytotoxicity. Combined evidence suggests that interactions with normal epithelial cells augment the resistance of ATL cells to TSA-induced apoptosis and facilitate immune evasion by ATL cells.

Surfactant Protein A Suppresses Lung Cancer Progression by Regulating the Polarization of Tumor-Associated Macrophages

2013-03-15

Surfactant protein A (SP-A) is a large multimeric protein found in the lungs. In addition to its immunoregulatory function in infectious respiratory diseases, SP-A is also used as a marker of lung adenocarcinoma. Despite the finding that SP-A expression levels in cancer cells has a relationship with patient prognosis, the function of SP-A in lung cancer progression is unknown. We investigated the role of SP-A in lung cancer progression by introducing the SP-A gene into human lung adenocarcinoma cell lines. SP-A gene transduction suppressed the progression of tumor in subcutaneous xenograft or lung metastasis mouse models. Immunohistochemical analysis showed that the number of M1 antitumor tumor-associated macrophages (TAMs) was increased and the number of M2 tumor-promoting TAMs was not changed in the tumor tissue produced by SP-A–expressing cells. In addition, natural killer (NK) cells were also increased and activated in the SP-A–expressing tumor. Moreover, SP-A did not inhibit tumor progression in mice depleted of NK cells. Taking into account that SP-A did not directly activate NK cells, these results suggest that SP-A inhibited lung cancer progression by recruiting and activating NK cells via controlling the polarization of TAMs.

Skp2 Deficiency Inhibits Chemical Skin Tumorigenesis Independent of p27Kip1 Accumulation

2013-03-07

S-phase kinase-associated protein 2 (Skp2) functions as the receptor component of the Skp–Cullin–F-box complex and is implicated in the degradation of several cell cycle regulators, such as p21Cip1, p27Kip1, p57Kip2, and cyclin E. Numerous studies in human and experimental tumors have demonstrated low p27Kip1 levels and elevated Skp2 expression. However, a direct association between the inverse correlation of Skp2 and p27Kip1 with tumorigenesis has not been demonstrated. Herein, we provide evidence that skin tumorigenesis is inhibited in Skp2−/− mice. An analysis of mouse keratinocytes indicates that increased p27Kip1 levels in Skp2−/− epidermis cause reduced cell proliferation that is alleviated in the epidermis from Skp2−/−/p27−/− compound mice. In contrast, we establish that a p27Kip1 deficiency does not overturn the reduced skin tumorigenesis experienced by Skp2−/− mice. In addition, Skp2−/− epidermis exhibits an accumulation of p53-cofactor CBP/p300 that is associated with elevated apoptosis in hair follicles and decreased skin tumorigenesis. We conclude that p27Kip1 accumulation is responsible for the hypoplasia observed in normal tissues of Skp2−/− mice but does not have a preponderant function in reducing skin tumorigenesis.

Epstein-Barr Virus–Encoded miR-BART20-5p Inhibits T-bet Translation with Secondary Suppression of p53 in Invasive Nasal NK/T-Cell Lymphoma

Ting-Chu Lin, Ting-Yun Liu, Su-Ming Hsu, Chung-Wu Lin — 2013-05-01

Nasal NK/T-cell lymphoma (NNL) is an Epstein-Barr virus (EBV)-associated lymphoma derived from cytotoxic NK or T cells of the nasal mucosa. NNLs are noninvasive in the earliest stage, and become invasive with disease progression. The EBV encodes at least 44 miRNAs, whose functions in the pathogenesis of NNL are mostly unknown. We evaluated the levels of 39 EBV-encoded miRNAs with quantitative real-time RT-PCR in a series of 20 noninvasive NNLs and 20 invasive NNLs. miR-BART20-5p was associated most strongly with invasion (P ≤ 0.001), and lack of T-bet, the master transcription factor for cytotoxic NK cells. However, we identified T-bet (official symbol, TBX21) transcripts in T-bet–negative NNLs, implying a block in the translation of T-bet by miR-BART20-5p. In co-transfection experiments, miR-BART20-5p inhibited T-bet translation in both non-Hodgkin and Hodgkin lymphoma cell lines. Endogenous mir-BART20-5p also inhibited translation of T-bet in EBV-infected YT lymphoma cells of NK-cell origin. Induction of T-bet in YT cells up-regulated p53, leading to increased sensitivity in response to doxorubicin. Finally, YT cells transplanted into severe combined immunodeficiency mice had an invasive behavior. Taken together, we conclude that EBV-encoded miR-BART20-5p inhibits T-bet translation with secondary suppression of p53 in invasive nasal NK/T-cell lymphoma. An antagomir to miR-BART20-5p might be an effective therapeutic agent through induction of T-bet and p53.

miR-92a Inhibits Peritoneal Dissemination of Ovarian Cancer Cells by Inhibiting Integrin α5 Expression

2013-03-18

Ovarian cancer is characterized by widespread peritoneal dissemination and ascites and has a cure rate of only 30%. As has been previously reported, integrin α5 plays a key role in the peritoneal dissemination of ovarian cancer. Our aim was to identify a new miRNA that regulates integrin α5 expression and analyze the therapeutic potential of targeting this miRNA. By using an IHC analysis, we proved that high integrin α5 expression correlates with a poor prognosis in Japanese patients with International Federation of Gynecology and Obstetrics stage III ovarian cancer. Based on an miRNA algorithm search, we identified hsa-mir-92a (miR-92a) as a candidate. The level of miR-92a expression was significantly inversely correlated with ITGA5 expression in various cancer cells. Transfection of precursor miR-92a reduced integrin α5 expression in ovarian cancer cells, which was accompanied by the inhibition of cancer cell adhesion, invasion, and proliferation. miR-92a overexpression reduced the luciferase activity of the ITGA5 3′-untranslated region, suggesting that ITGA5 mRNA is a direct target of miR-92a. In in vivo ovarian cancer xenografts, the enforced expression of miR-92a in HeyA-8 cells suppressed peritoneal dissemination. Although we still have a long way to go before an effective and nontoxic miRNA-based cancer therapy can be introduced into the clinic, the inhibition of integrin α5 expression by targeting miR-92a needs to be explored further for future applications in ovarian cancer treatment.

RANKL Synthesized by Both Stromal Cells and Cancer Cells Plays a Crucial Role in Osteoclastic Bone Resorption Induced by Oral Cancer

2013-03-18

The molecular mechanisms underlying bone destruction by invading oral cancer are not well understood. Using IHC, we demonstrated that receptor activator of nuclear factor-κB ligand (RANKL)–positive fibroblasts and cancer cells were located at sites of bone invasion in human oral cancers. HSC3 and HO-1-N-1, human oral cancer cell lines, expressed RANKL and stimulated Rankl expression in the UAMS-32 murine osteoblastic cell line. We discriminated the roles of RANKL synthesized by stromal cells and cancer cells in cancer-associated bone resorption by using species-specific RANKL antibodies against murine RANKL and human RANKL, respectively. Osteoclastogenesis induced by the conditioned medium of HSC3 and HO-1-N-1 cells in a co-culture of murine bone marrow cells and UAMS-32 cells was inhibited by the addition of antibodies against either mouse or human RANKL. HSC3-induced bone destruction was greatly inhibited by the administration of anti-mouse RANKL antibody in a xenograft model. HO-1-N-1–induced bone destruction was inhibited by the administration of either anti-mouse or anti-human RANKL antibody. Bone destruction induced by the transplantation of human RANKL-overexpressing cells (HSC3-R2) was greatly inhibited by the injection of anti-human RANKL antibody. The present study revealed that RANKL produced by both stromal and cancer cells is involved in oral cancer–induced osteoclastic bone resorption. These results provide important information for understanding the cellular and molecular basis of cancer-associated bone destruction and the mechanism of action underlying RANKL antibody (denosumab) therapy.

Expression of Myoferlin in Human and Murine Carcinoma Tumors: Role in Membrane Repair, Cell Proliferation, and Tumorigenesis

Cleo Leung, Carol Yu, Michelle I. Lin, Cristina Tognon, Pascal Bernatchez — 2013-03-18

Cancer cells are often characterized by high proliferation rates, a consequence of increased mitotic signaling coupled with unchecked cellular growth. We recently demonstrated that vascular endothelial cells unexpectedly express ferlins, a family of muscle-specific proteins capable of regulating the fusion of lipid patches to the plasma membrane, and that these highly regulated membrane fusion events are essential to endothelial cell proliferation and homeostasis. Here, we show that human and mouse breast cancer cell lines also express myoferlin at various levels, and that the processes of transformation, epithelial–mesenchymal transition, and metastasis do not appear to have any effect on myoferlin expression in vitro. In vivo, we observed that solid mouse and human carcinoma tissues also express high levels of myoferlin protein. Loss-of-function studies performed in mice revealed that myoferlin gene knockdown can attenuate cancer cell proliferation in vitro and decrease tumor burden, and that accelerated tumor cell growth appears to rely on intact myoferlin-dependent membrane repair and signaling under exponential growth conditions. To our knowledge, these data provide the first evidence of myoferlin expression in solid human and mouse tumors. We have thus identified a novel membrane repair process that likely helps sustain the high growth rates characteristic of tumors, and we suggest that interfering with normal myoferlin expression and/or membrane repair and remodeling may provide therapeutically relevant antiproliferative effects.

Peptide Inhibitor of NF-κB Translocation Ameliorates Experimental Atherosclerosis

2013-04-18

Atherosclerosis is a chronic inflammatory disease of the arterial wall. NF-κB is a major regulator of inflammation that controls the expression of many genes involved in atherogenesis. Activated NF-κB was detected in human atherosclerotic plaques, and modulation of NF-κB inflammatory activity limits disease progression in mice. Herein, we investigate the anti-inflammatory and atheroprotective effects of a cell-permeable peptide containing the NF-κB nuclear localization sequence (NLS). In vascular smooth muscle cells and macrophages, NLS peptide specifically blocked the importin α–mediated nuclear import of NF-κB and prevented lipopolysaccharide-induced pro-inflammatory gene expression, cell migration, and oxidative stress. In experimental atherosclerosis (apolipoprotein E–knockout mice fed a high-fat diet), i.p., 0.13 μmol/day NLS peptide administration for 5 weeks attenuated NF-κB activation in atherosclerotic plaques. NLS peptide significantly inhibited lesion development at both early (age 10 weeks) and advanced (age 28 weeks) stages of atherosclerosis in mice, without affecting serum lipid levels. Plaques from NLS-treated mice contained fewer macrophages of pro-inflammatory M1 subtype than those from respective untreated controls. By contrast, the relative smooth muscle cell and collagen content was increased, indicating a more stable plaque phenotype. NLS peptide also attenuated pro-inflammatory gene expression and oxidative stress in aortic lesions. Our study demonstrates that targeting NF-κB nuclear translocation hampers inflammation and atherosclerosis development and identifies cell-permeable NLS peptide as a potential anti-atherosclerotic agent.

Side-Specific Endothelial-Dependent Regulation of Aortic Valve Calcification: Interplay of Hemodynamics and Nitric Oxide Signaling

2013-03-13

Arterial endothelial cells maintain vascular homeostasis and vessel tone in part through the secretion of nitric oxide (NO). In this study, we determined how aortic valve endothelial cells (VEC) regulate aortic valve interstitial cell (VIC) phenotype and matrix calcification through NO. Using an anchored in vitro collagen hydrogel culture system, we demonstrate that three-dimensionally cultured porcine VIC do not calcify in osteogenic medium unless under mechanical stress. Co-culture with porcine VEC, however, significantly attenuated VIC calcification through inhibition of myofibroblastic activation, osteogenic differentiation, and calcium deposition. Incubation with the NO donor DETA-NO inhibited VIC osteogenic differentiation and matrix calcification, whereas incubation with the NO blocker l-NAME augmented calcification even in 3D VIC–VEC co-culture. Aortic VEC, but not VIC, expressed endothelial NO synthase (eNOS) in both porcine and human valves, which was reduced in osteogenic medium. eNOS expression was reduced in calcified human aortic valves in a side-specific manner. Porcine leaflets exposed to the soluble guanylyl cyclase inhibitor ODQ increased osteocalcin and α-smooth muscle actin expression. Finally, side-specific shear stress applied to porcine aortic valve leaflet endothelial surfaces increased cGMP production in VEC. Valve endothelial-derived NO is a natural inhibitor of the early phases of valve calcification and therefore may be an important regulator of valve homeostasis and pathology.

Reverse Apolipoprotein A-I Mimetic Peptide R-D4F Inhibits Neointimal Formation following Carotid Artery Ligation in Mice

2013-03-18

The ApoA-I mimetic peptide D-4F has demonstrated potent atheroprotective actions in vivo and in vitro. We investigated the effect of R-D4F (ie, the D-4F peptide with reverse order of amino acids) on intimal hyperplasia after vascular injury in a mouse model of carotid artery ligation. Adult male C57BL/6J mice were pretreated intraperitoneally with vehicle, D-4F (1 mg/kg), or R-D4F (1 mg/kg or 5 mg/kg) daily for 3 days; the mice were then subjected to left carotid artery ligation. All treatments were continued for 28 days after surgery. Neither D-4F nor R-D4F treatment affected serum lipid levels. Morphometric analysis showed that the occluded vessels had significant neointimal formation, compared with the uninjured arteries in vehicle-treated mice. Like the D-4F treatment, R-D4F treatment significantly (P < 0.05) inhibited intimal hyperplasia (−42%), local neutrophil and macrophage infiltration, and mRNA expression of the proinflammatory mediator monocyte chemotactic protein 1 (−55%) and vascular cell adhesion protein 1 (−53%), compared with vehicle. Furthermore, the vasoprotective effect of high-dose R-D4F was significantly enhanced, compared with the low dose. In cultured mouse RAW 264.7 macrophages, pretreatment with R-D4F also effectively inhibited lipopolysaccharide-induced leukocyte integrin CD11b expression, a key molecule for leukocyte infiltration. Taken together, these results suggest that R-D4F has significant anti-inflammatory features and facilitates prevention of neointimal formation after vascular injury in mice.

Editorial Board

2013-05-01

The American Journal of Pathology

This Month in AJP

Going Forward with Genetics: Recent Technological Advances and Forward Genetics in Mice

Biological Relevance of Inflammation and Oxidative Stress in the Pathogenesis of Arterial Diseases

The Emerging Roles of HTRA1 in Musculoskeletal Disease

Revealing the Mechanism of Tissue Damage Due to Tobacco Use: Finally, a Smoking Gun?

R132C IDH1 Mutations Are Found in Spindle Cell Hemangiomas and Not in Other Vascular Tumors or Malformations

Absence of B Cells Does Not Compromise Intramembranous Bone Formation during Healing in a Tibial Injury Model

Voluntary Physical Activity Protects from Susceptibility to Skeletal Muscle Contraction–Induced Injury But Worsens Heart Function in mdx Mice

SPLUNC1/BPIFA1 Contributes to Pulmonary Host Defense against Klebsiella pneumoniae Respiratory Infection

Glomerular Endothelial Surface Layer Acts as a Barrier against Albumin Filtration

A Lymphocyte-Dependent Mode of Action for Imatinib Mesylate in Experimental Pulmonary Hypertension

Novel Proteolytic Microvesicles Released from Human Macrophages after Exposure to Tobacco Smoke

Membrane-Type Matrix Metalloproteinase 1 Regulates Trophoblast Functions and Is Reduced in Fetal Growth Restriction

FAK-Related Nonkinase Is a Multifunctional Negative Regulator of Pulmonary Fibrosis

Reduced Expression of 15-Hydroxy Prostaglandin Dehydrogenase in Chorion during Labor Is Associated with Decreased PRB and Increased PRA and GR Expression

Bifidobacteria Stabilize Claudins at Tight Junctions and Prevent Intestinal Barrier Dysfunction in Mouse Necrotizing Enterocolitis

Antifibrotic Effects of a Recombinant Adeno-Associated Virus Carrying Small Interfering RNA Targeting TIMP-1 in Rat Liver Fibrosis

Knockout of Ste20-Like Proline/Alanine-Rich Kinase (SPAK) Attenuates Intestinal Inflammation in Mice

miR-101 Inhibits Cholangiocarcinoma Angiogenesis through Targeting Vascular Endothelial Growth Factor (VEGF)

Interaction between CX3CL1 and CX3CR1 Regulates Vasculitis Induced by Immune Complex Deposition

Role and Regulation of PDGFRα Signaling in Liver Development and Regeneration

Ranibizumab Is a Potential Prophylaxis for Proliferative Vitreoretinopathy, a Nonangiogenic Blinding Disease

Amelioration of Experimental Autoimmune Encephalomyelitis by the Quinoline-3-Carboxamide Paquinimod: Reduced Priming of Proinflammatory Effector CD4+ T Cells

Progressive Reactive Lymphoid Connective Tissue Disease and Development of Autoantibodies in Scavenger Receptor A5–Deficient Mice

Kruppel-Like Factor 2 Is a Transcriptional Regulator of Chronic and Acute Inflammation

Visfatin Enhances the Production of Cathelicidin Antimicrobial Peptide, Human β-Defensin-2, Human β-Defensin-3, and S100A7 in Human Keratinocytes and Their Orthologs in Murine Imiquimod-Induced Psoriatic Skin

Calsyntenin-3 C-Terminal Fragment Accumulates in Dystrophic Neurites Surrounding Aβ Plaques in Tg2576 Mouse and Alzheimer Disease Brains: Its Neurotoxic Role in Mediating Dystrophic Neurite Formation

Loss of Aquaporin 9 Expression Adversely Affects the Survival of Retinal Ganglion Cells

The Bradykinin B1 Receptor Regulates Aβ Deposition and Neuroinflammation in Tg-SwDI Mice

Blocking the Interaction between Apolipoprotein E and Aβ Reduces Intraneuronal Accumulation of Aβ and Inhibits Synaptic Degeneration

Embryonic Stem Cell–Based Modeling of Tau Pathology in Human Neurons

Aspirin-Triggered Lipoxin A4 Stimulates Alternative Activation of Microglia and Reduces Alzheimer Disease–Like Pathology in Mice

Possible Role of Mural Cell–Covered Mature Blood Vessels in Inducing Drug Resistance in Cancer-Initiating Cells

Inhibition of ATP Citrate Lyase Induces an Anticancer Effect via Reactive Oxygen Species: AMPK as a Predictive Biomarker for Therapeutic Impact

Decreased Tumorigenesis and Mortality from Bladder Cancer in Mice Lacking Urothelial Androgen Receptor

Galectin-3 Accelerates M2 Macrophage Infiltration and Angiogenesis in Tumors

Protective Roles of Epithelial Cells in the Survival of Adult T-Cell Leukemia/Lymphoma Cells

Surfactant Protein A Suppresses Lung Cancer Progression by Regulating the Polarization of Tumor-Associated Macrophages

Skp2 Deficiency Inhibits Chemical Skin Tumorigenesis Independent of p27Kip1 Accumulation

Epstein-Barr Virus–Encoded miR-BART20-5p Inhibits T-bet Translation with Secondary Suppression of p53 in Invasive Nasal NK/T-Cell Lymphoma

miR-92a Inhibits Peritoneal Dissemination of Ovarian Cancer Cells by Inhibiting Integrin α5 Expression

RANKL Synthesized by Both Stromal Cells and Cancer Cells Plays a Crucial Role in Osteoclastic Bone Resorption Induced by Oral Cancer

Expression of Myoferlin in Human and Murine Carcinoma Tumors: Role in Membrane Repair, Cell Proliferation, and Tumorigenesis

Peptide Inhibitor of NF-κB Translocation Ameliorates Experimental Atherosclerosis

Side-Specific Endothelial-Dependent Regulation of Aortic Valve Calcification: Interplay of Hemodynamics and Nitric Oxide Signaling

Reverse Apolipoprotein A-I Mimetic Peptide R-D4F Inhibits Neointimal Formation following Carotid Artery Ligation in Mice

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