American Journal Of Pathology (Am J Pathol)

Publications in this journal

• Article: Primary Epiphyseal Arteriopathy in a Mouse Model of Steroid-Induced Osteonecrosis.

  Laura J Janke, Chengcheng Liu, Peter Vogel, Jitesh Kawedia, Kelli L Boyd, Amy J Funk, Mary V Relling
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  ABSTRACT: Patients undergoing glucocorticoid therapy for a variety of disorders, including autoimmune diseases and hematological malignancies, are at risk of developing osteonecrosis. Despite extensive research in both patients and animal models, the underlying pathogenesis remains unclear. Proposed inciting mechanisms include intravascular thrombotic occlusion, marrow fat hypertrophy, osteocyte and/or endothelial cell apoptosis, hypercoagulability, and vasoconstriction of specific arteries and arterioles supplying bone. Our laboratory has developed a model of steroid-induced osteonecrosis in Balb/cJ mice which reflects clinically relevant exposures to glucocorticoids in which treated mice develop osteonecrosis of the distal femoral epiphysis when administered 4 to 8 mg/L dexamethasone in drinking water for 6 weeks. We identified lesions in arterioles supplying this area, with the mildest occurring in knees without any evidence of osteonecrosis. However, arteriopathy was more common among mice that did versus did not develop osteonecrosis (P < 0.0001); in mice with osteonecrosis, the associated vessels showed transmural necrosis and thickening of the vessel wall progressing to the point of luminal obstruction. In the most severe cases of osteonecrosis, end-stage lesions consisted of fully occluded vessels with marrow and bone necrosis involving the entire epiphysis. We propose that a primary arteriopathy is the initiating event in the genesis of steroid-induced osteonecrosis and provides a basis for future investigation of this disease process.
  American Journal Of Pathology 05/2013;
• Article: Interleukin-6 Mediates the Platelet Abnormalities and Thrombogenesis Associated with Experimental Colitis.

  Elena Y Senchenkova, Shunsuke Komoto, Janice Russell, Lidiana D Almeida-Paula, Li-Sue Yan, Songlin Zhang, D Neil Granger
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  ABSTRACT: Clinical studies and animal experimentation have shown that colonic inflammation is associated with an increased number and reactivity of platelets, coagulation abnormalities, and enhanced thrombus formation. The objective of this study was to define the contribution of IL-6 to the thrombocytosis, exaggerated agonist-induced platelet aggregation, and enhanced extra-intestinal thrombosis that occur during experimental colitis. The number of mature and immature platelets, platelet life span, thrombin-induced platelet aggregation response, and light/dye-induced thrombus formation in cremaster muscle arterioles were measured in wild-type (WT) and IL-6-deficient (IL-6(-/-)) mice with dextran sodium sulfate (DSS)-induced colitis. DSS colitis in WT mice was associated with thrombocytosis with an elevated number of both mature and immature platelets and no change in platelet life span. The thrombocytosis response was absent in IL-6(-/-) mice. DSS treatment also enhanced the platelet aggregation response to thrombin and accelerated thrombus development in WT mice, but not in IL-6(-/-) mice. Exogenous IL-6 administered to WT mice elicited a dose-dependent enhancement of thrombus formation. These findings indicate that IL-6 mediates the thrombocytosis, platelet hyperreactivity, and accelerated thrombus development associated with experimental colitis. The IL-6-dependent colitis-induced thrombocytosis appears to result from an enhancement of thrombopoiesis because platelet life span is unchanged.
  American Journal Of Pathology 05/2013;
• Article: Tamoxifen Elicits Atheroprotection through Estrogen Receptor α AF-1 But Does Not Accelerate Reendothelialization.

  Coralie Fontaine, Anne Abot, Audrey Billon-Galés, Gilles Fouriot, Hortense Bergès, Etienne Grunenwald, Alexia Vinel, Marie-Cécile Valera, Pierre Gourdy, Jean-François Arnal
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  ABSTRACT: Based on both experimental and clinical data, tamoxifen has been proposed to have cardiovascular benefits, although the mechanism(s) contributing to that protective effect are still poorly understood. In vitro experiments demonstrated that tamoxifen elicits its transcriptional effect through estrogen receptor (ER) α, but other targets can participate in its actions. However, although tamoxifen selectively activates the activating function (AF)-1 of ERα, we recently showed that this ERα subfunction is dispensable for the atheroprotective action of 17β-estradiol (E2), the main ligand of ERs. The goal of the present work is to determine to which extent ERα and its AF-1 mediate the vasculoprotective action of tamoxifen. Our data confirm that tamoxifen exerts an atheroprotective action on LDL-r(-/-) female mice, but, in contrast to E2, it fails to accelerate reendothelialization after carotid electric injury. Tamoxifen and E2 elicit differences in gene expression profiles in the mouse aorta. Finally, the atheroprotective action of tamoxifen is abrogated in ERα(-/-)LDL-r(-/-) mice and in LDL-r(-/-) mice selectively deficient in ERαAF-1 (ERαAF-1(0/0)LDL-r(-/-)). Our results demonstrate, for the first time to our knowledge, that tamoxifen mediates its actions in vivo through the selective activation of ERαAF-1, which is sufficient to prevent atheroma, but not to accelerate endothelial healing.
  American Journal Of Pathology 05/2013;
• Article: Absence of SERPINB6A Causes Sensorineural Hearing Loss with Multiple Histopathologies in the Mouse Inner Ear.

  Justin Tan, Monica D Prakash, Dion Kaiserman, Phillip I Bird
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  ABSTRACT: A homozygous mutation of SERPINB6, a gene encoding an intracellular protease inhibitor, has recently been associated with post-lingual, autosomal-recessive, nonsyndromic hearing loss in humans (DFNB91). Herein, we describe the physiological changes underlying SERPINB6 deficiency by analyzing mutant mice in which the orthologous gene is replaced by enhanced green fluorescent protein. SERPINB6A is present in the neurosensory epithelium, lateral wall, and spiral limbus of the cochlea, with highest levels in the inner and outer hair cells of the organ of Corti, cells lining the inner sulcus, and supporting cells distributed along the epithelial gap junction layer to the outer sulcus. Measurements of hearing thresholds in these mice demonstrated age-related hearing loss in all homozygous-null, but not heterozygous, mice. Hearing impairment was first detected at 3 weeks of age, affecting only high frequencies before spreading to other frequencies as the mice aged. The defect is associated with progressive cellular degeneration within the cochlea. This begins with the hair cells, then involves the primary auditory neurons, and, finally, the fibrocytes in the lateral wall. These findings establish these mutant mice as a suitable model system to elucidate how SERPINB6 deficiency causes deafness in humans.
  American Journal Of Pathology 05/2013;
• Article: Chronic Estradiol Exposure Is a Direct Mitogen for Insulin/EGF-Primed Endometrial Cells and Drives PTEN Loss-Induced Hyperplasic Growth.

  Nuria Eritja, Cristina Mirantes, David Llobet, Andree Yeramian, Laura Bergadà, Mari A Dosil, Mónica Domingo, Xavier Matias-Guiu, Xavier Dolcet
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  ABSTRACT: Loss of tumor-suppressor PTEN is the most common alteration in endometrial carcinoma. However, the relationship between loss of PTEN, growth factors [eg, insulin/insulin-like growth factor (IGF)-1], epidermal growth factor (EGF), and hyperestrogenism in the development of endometrial carcinoma is still controversial. By using three-dimensional (3D) cultures of PTEN(+/+) and PTEN(+/-) endometrial epithelial cells, we investigated the effects of EGF, insulin/IGF, and estradiol in endometrial cell proliferation. We have previously demonstrated that 3D cultures of endometrial cells require EGF and insulin/IGF to proliferate. Herein, we demonstrate that, in the presence of EGF and insulin/IGF, chronic estradiol treatment directly induces proliferation of 3D cultures. Moreover, we show that the mitogenic effects of estradiol require the presence of insulin/IGF and EGF, because withdrawal of such factors completely abolishes estradiol-induced proliferation. In the presence of EGF and insulin/IGF, PTEN(+/-) and PTEN(+/+) spheroids display a similar rate of proliferation. However, the addition of estradiol causes an exaggerated proliferation of PTEN(+/-) cultures, leading to formation of complex structures, such as those observed in endometrial hyperplasia or carcinoma. In summary, we demonstrate that EGF and insulin/IGF prime endometrial epithelial cells to direct the mitogenic effects of estradiol. Furthermore, PTEN deficiency results in enhanced responsiveness to this combination, leading to the development of hyperplasia of endometrial cells in culture.
  American Journal Of Pathology 05/2013;
• Article: Combination of Oxidative Stress and FOXM1 Inhibitors Induces Apoptosis in Cancer Cells and Inhibits Xenograft Tumor Growth.

  Marianna Halasi, Bulbul Pandit, Ming Wang, Veronique Nogueira, Nissim Hay, Andrei L Gartel
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  ABSTRACT: Tumor cells accumulate high level of reactive oxygen species (ROS) because they are metabolically more active than healthy cells. Elevated ROS levels increase tumorigenecity but also render cancer cells more vulnerable to oxidative stress than healthy cells. The oncogenic transcription factor Forkhead Box M1 (FOXM1), which is overexpressed in a wide range of human cancers, was reported to protect cancer cells from the adverse effects of oxidative stress by up-regulating the expression of scavenger enzymes. We therefore hypothesized that the combination of FOXM1 ablation and ROS inducers could selectively eradicate cancer cells. We show that RNA interface-mediated knockdown of FOXM1 further elevates intracellular ROS levels and increases sensitivity of cancer cells to ROS-mediated cell death after treatment with ROS inducers. We also report that the combination of ROS inducers with FOXM1/proteasome inhibitors induces robust apoptosis in different human cancer cells. In addition, we report evidence that the FOXM1/proteasome inhibitor bortezomib in combination with the ROS inducer β-phenylethyl isothiocyanate efficiently inhibits the growth of breast tumor xenografts in nude mice. We conclude that the combination of ROS inducers and FOXM1 inhibitors could be used as a therapeutic strategy to selectively eliminate cancer cells.
  American Journal Of Pathology 05/2013;
• Article: Spdef Null Mice Lack Conjunctival Goblet Cells and Provide a Model of Dry Eye.

  Christina Kaiser Marko, Balaraj B Menon, Gang Chen, Jeffrey A Whitsett, Hans Clevers, Ilene K Gipson
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  ABSTRACT: Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef(-/-) mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef(-/-) mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef(-/-) mice revealed down-regulation of goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Up-regulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and proinflammatory genes (Il1-α, Il-1β, Tnf-α), all of which are up-regulated in dry eye. Interestingly, four Wnt pathway genes were down-regulated. SPDEF expression was significantly decreased in the conjunctival epithelium of Sjögren syndrome patients with dry eye and decreased goblet cell mucin expression. These data demonstrate that Spdef is required for conjunctival goblet cell differentiation and down-regulation of SPDEF may play a role in human dry eye with goblet cell loss. Spdef(-/-) mice have an ocular surface phenotype similar to that in moderate dry eye, providing a new, more convenient model for the disease.
  American Journal Of Pathology 05/2013;
• Article: Positioning Ganglioside D3 as an Immunotherapeutic Target in Lymphangioleiomyomatosis.

  Emily R Gilbert, Jonathan M Eby, Adam M Hammer, Jared S Klarquist, David G Christensen, Allison J Barfuss, Raymond E Boissy, Maria M Picken, Robert B Love, Daniel F Dilling, I Caroline Le Poole
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  ABSTRACT: Tumors that develop in lymphangioleiomyomatosis (LAM) as a consequence of biallelic loss of TSC1 or TSC2 gene function express melanoma differentiation antigens. However, the percentage of LAM cells expressing these melanosomal antigens is limited. Here, we report the overexpression of ganglioside D3 (GD3) in LAM. GD3 is a tumor-associated antigen otherwise found in melanoma and neuroendocrine tumors; normal expression is largely restricted to neuronal cells in the brain. We also observed markedly reduced serum antibody titers to GD3, which may allow for a population of GD3-expressing LAM cells to expand within patients. This is supported by the demonstrated sensitivity of cultured LAM cells to complement mediated cytotoxicity via GD3 antibodies. GD3 can serve as a natural killer T (NKT) cell antigen when presented on CD1d molecules expressed on professional antigen-presenting cells. Although CD1d-expressing monocyte derivatives were present in situ, enhanced NKT-cell recruitment to LAM lung was not observed. Cultured LAM cells retained surface expression of GD3 over several passages and also expressed CD1d, implying that infiltrating NKT cells can be directly cytotoxic toward LAM lung lesions. Immunization with antibodies to GD3 may thus be therapeutic in LAM, and enhancement of existing NKT-cell infiltration may be effective to further improve antitumor responses. Overall, we hereby establish GD3 as a suitable target for immunotherapy of LAM.
  American Journal Of Pathology 05/2013;
• Article: FAM190A Deficiency Creates a Cell Division Defect.

  Kalpesh Patel, Francesca Scrimieri, Soma Ghosh, Jun Zhong, Min-Sik Kim, Yunzhao R Ren, Richard A Morgan, Christine A Iacobuzio-Donahue, Akhilesh Pandey, Scott E Kern
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  ABSTRACT: Like the p16, SMAD4, and RB1 genes, FAM190A lies at a consensus site of homogeneous genomic deletions in human cancer. FAM190A transcripts in 40% of cancers also contain in-frame deletions of evolutionarily conserved exons. Its gene function was unknown. We found an internal deletion of the FAM190A gene in a pancreatic cancer having prominent focal multinuclearity. The experimental knockdown of FAM190A expression by shRNA caused focal cytokinesis defects, multipolar mitosis, and multinuclearity as observed in time-lapse microscopy. FAM190A was localized to the γ-tubulin ring complex of early mitosis and to the midbody in late cytokinesis by immunofluorescence assay and was present in the nuclear fraction of unsynchronized cells by immunoblot. FAM190A interacted with EXOC1 and Ndel1, which function in cytoskeletal organization and the cell division cycle. Levels of FAM190A protein peaked 12 hours after release from thymidine block, corresponding to M-phase. Slower-migrating phosphorylated forms accumulated toward M-phase and disappeared after release from a mitotic block and before cytokinesis. Studies of FAM190A alterations may provide mechanistic insights into mitotic dysregulation and multinuclearity in cancer. We propose that FAM190A is a regulator or structural component required for normal mitosis and that both the rare truncating mutations and common in-frame deletion alteration of FAM190A may contribute to the chromosomal instability of cancer.
  American Journal Of Pathology 05/2013;
• Article: Frequent Genetic Alterations in EGFR and HER2 Driven Pathways in Breast Cancer Brain Metastases.

  Ina Hohensee, Katrin Lamszus, Sabine Riethdorf, Sönke Meyer-Staeckling, Markus Glatzel, Jakob Matschke, Isabell Witzel, Manfred Westphal, Burkhard Brandt, Volkmar Müller, Klaus Pantel, Harriet Wikman
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  ABSTRACT: Current standard systemic therapies for treating breast cancer patients with brain metastases are inefficient. Targeted therapies against human epidermal growth factor receptors are of clinical interest because of their alteration in a subset of breast cancers (BCs). We analyzed copy number, mutation status, and protein expression of epidermal growth factor receptor (EGFR), HER2, phosphatase and tensin homologue, deleted on chromosome 10 (PTEN), and catalytic subunit of phosphoinositide 3-kinase (PIK3CA) in 110 ductal carcinoma in situ, primary tumor, and metastatic BC samples. Alterations in EGFR, HER2, and PTEN, alone or in combination, were found in a significantly larger fraction of breast cancer brain metastases tumor tissue compared with samples from primary tumors with good prognosis, bone relapse, or other distant metastases (all P < 0.05). Primary tumor patients with a subsequent brain relapse showed almost equally high frequencies of especially EGFR and PTEN alteration as the breast cancer brain metastases patients. PIK3CA was not associated with an increased risk of brain metastases. Genetic alterations in both EGFR and PTEN were especially common in triple-negative breast cancer patients and rarely was seen among HER2-positive patients. In conclusion, we identified two independent high-risk primary BC subgroups for developing brain metastases, represented by genetic alterations in either HER2 or EGFR/PTEN-driven pathways. In contrast, none of these pathways was associated with an increased risk of bone metastasis. These findings highlight the importance of both pathways as possible targets in the treatment of brain metastases in breast cancer.
  American Journal Of Pathology 05/2013;
• Article: Ultrasound-Guided Intracardiac Injection: An Improvement for Quantitative Brain Colonization Assays.

  Lukxmi Balathasan, John S Beech, Ruth J Muschel
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  ABSTRACT: Brain metastasis is a frequent occurrence in patients with cancer, with devastating consequences. The current animal models for brain metastasis are highly variable, leading to a need for improved in vivo models that recapitulate the clinical disease. Herein, we describe an experimental brain metastasis model that uses ultrasound guidance to perform intracardiac injections. This method is easy to perform, giving consistent and quantitative results. Demonstrating the utility of this method, we have assessed a variety of metastatic cell lines for their ability to develop into brain metastases. Those cell lines that were competent at brain colonization could be detected in the brain vasculature 4 hours after intracardiac injection, and a few adherent cells persisted until colonization occurred. In contrast, those cell lines that were deficient in brain colonization were infrequently found 4 hours after introduction into the arterial circulation and were not detected at later time points. All of these cells were capable of brain colonization after intraparenchymal injection. We propose that adherence to the brain vasculature may be the key limiting step that determines the ability of a cancer cell to form brain metastases successfully. Identifying brain endothelium-specific adhesion molecules may enable development of screening modalities to detect brain-colonizing cancer cells and therapies to prevent these metastatic cells from seeding the brain.
  American Journal Of Pathology 05/2013;
• Article: Regulation of Lung Injury and Fibrosis by p53-Mediated Changes in Urokinase and Plasminogen Activator Inhibitor-1.

  Yashodhar P Bhandary, Shwetha K Shetty, Amarnath S Marudamuthu, Hong-Long Ji, Pierre F Neuenschwander, Vijay Boggaram, Gilbert F Morris, Jian Fu, Steven Idell, Sreerama Shetty
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  ABSTRACT: Alveolar type II epithelial cell (ATII) apoptosis and proliferation of mesenchymal cells are the hallmarks of idiopathic pulmonary fibrosis, a devastating disease of unknown cause characterized by alveolar epithelial injury and progressive fibrosis. We used a mouse model of bleomycin (BLM)-induced lung injury to understand the involvement of p53-mediated changes in urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) levels in the regulation of alveolar epithelial injury. We found marked induction of p53 in ATII cells from mice exposed to BLM. Transgenic mice expressing transcriptionally inactive dominant negative p53 in ATII cells showed augmented apoptosis, whereas those deficient in p53 resisted BLM-induced ATII cell apoptosis. Inhibition of p53 transcription failed to suppress PAI-1 or induce uPA mRNA in BLM-treated ATII cells. ATII cells from mice with BLM injury showed augmented binding of p53 to uPA, uPA receptor (uPAR), and PAI-1 mRNA. p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions neither interfered with p53 DNA binding activity nor p53-mediated promoter transactivation. However, increased expression of p53 binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions in ATII cells suppressed PAI-1 and induced uPA after BLM treatment, leading to inhibition of ATII cell apoptosis and pulmonary fibrosis. Our findings indicate that disruption of p53-fibrinolytic system cross talk may serve as a novel intervention strategy to prevent lung injury and pulmonary fibrosis.
  American Journal Of Pathology 05/2013;
• Article: Pharmacologic Inhibition of IκB Kinase Activates Immediate Hypersensitivity Reactions in Mice.

  Dai Miyazaki, Sachiko Mihara, Koudai Inata, Shin-Ichi Sasaki, Takeshi Tominaga, Keiko Yakura, Waka Ishida, Atsuki Fukushima, Yoshitsugu Inoue
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  ABSTRACT: Pharmacologic inhibitors of IκB kinase (IKK), especially IKK-β, have been developed to treat inflammatory diseases. However, their interactions with components of the NF-κB pathways are not fully known in allergic diseases. To examine whether IKK is involved in immediate hypersensitivity reactions and to determine whether counterregulatory mechanisms in the NF-κB activation system were active, we examined the role played by IKK components on mast cell degranulation using a murine ocular immediate hypersensitivity reaction model. Pharmacologic inhibition of IKK in mice caused paradoxical aggravation of the mast cell-mediated immediate hypersensitivity reaction and up-regulation in the expression of inflammatory cytokines. Downstream analyses showed that B-cell deficiency or treatment by IL-1 receptor antagonist corrected the aberrant activation of tissue-resident mast cells, which would indicate contribution by activated B cells. Analyses of co-cultures of tissue-resident mast cells showed the contribution of activated B cells to activation of mast cells and secretion of inflammatory cytokines. Aberrant activation of the NF-κB promoter in isolated B cells was induced exclusively by IKK-β inhibition and was negated by ablating IKK-α. Aggravated mast cell degranulation by pharmacologic IKK inhibition in the murine immediate hypersensitivity reaction was corrected by B-cell-targeted inhibition of IKK-α. Thus, IKK-β limits B-cell-mediated mast cell activation and inflammatory cytokine induction in immediate hypersensitivity by counterbalancing the activity of IKK-α.
  American Journal Of Pathology 05/2013;
• Article: Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex.

  Vishakha S Bhave, Wendy Mars, Shashikiran Donthamsetty, Xiyue Zhang, Langzhu Tan, Jianhua Luo, William C Bowen, George K Michalopoulos
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  ABSTRACT: Previous studies from our laboratory have found glypican 3 (GPC3) as a negative regulator of growth. CD81 was found to be a binding partner for GPC3, and its expression and co-localization with GPC3 increased at the end of hepatocyte proliferation. However, the mechanisms through which these two molecules might regulate liver regeneration are not known. We tested the hypothesis that GPC3 down-regulates the hedgehog (HH) signaling pathway by competing with patched-1 for HH binding. We found decreased GPC3-Indian HH binding at peak proliferation in mice followed by increase in glioblastoma 1 protein (effector of HH signaling). We performed a yeast two-hybrid assay and identified hematopoietically expressed homeobox (Hhex, a known transcriptional repressor) as a binding partner for CD81. We tested the hypothesis that Hhex binding to CD81 keeps it outside the nucleus. However, when GPC3 binds to CD81, CD81-Hhex binding decreases, resulting in nuclear translocation of Hhex and transcriptional repression. In support of this, we found decreased GPC3-CD81 binding at hepatocyte proliferation peak, increased CD81- binding, and decreased nuclear Hhex. GPC3 transgenic mice were used as an additional tool to test our hypothesis. Overall, our data suggest that GPC3 down-regulates cell proliferation by binding to HH and down-regulating the HH signaling pathway and binding with CD81, thus making it unavailable to bind to Hhex and causing its nuclear translocation.
  American Journal Of Pathology 05/2013;
• Article: Mineralization/Anti-Mineralization Networks in the Skin and Vascular Connective Tissues.

  Qiaoli Li, Jouni Uitto
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  ABSTRACT: Ectopic mineralization has been linked to several common clinical conditions with considerable morbidity and mortality. The mineralization processes, both metastatic and dystrophic, affect the skin and vascular connective tissues. There are several contributing metabolic and environmental factors that make uncovering of the precise pathomechanisms of these acquired disorders exceedingly difficult. Several relatively rare heritable disorders share phenotypic manifestations similar to those in common conditions, and, consequently, they serve as genetically controlled model systems to study the details of the mineralization process in peripheral tissues. This overview will highlight diseases with mineral deposition in the skin and vascular connective tissues, as exemplified by familial tumoral calcinosis, pseudoxanthoma elasticum, generalized arterial calcification of infancy, and arterial calcification due to CD73 deficiency. These diseases, and their corresponding mouse models, provide insight into the pathomechanisms of soft tissue mineralization and point to the existence of intricate mineralization/anti-mineralization networks in these tissues. This information is critical for understanding the pathomechanistic details of different mineralization disorders, and it has provided the perspective to develop pharmacological approaches to counteract the consequences of ectopic mineralization.
  American Journal Of Pathology 05/2013;
• Article: STAT5A/B Gene Locus Undergoes Amplification during Human Prostate Cancer Progression.

  Bassem R Haddad, Lei Gu, Tuomas Mirtti, Ayush Dagvadorj, Paraskevi Vogiatzi, David T Hoang, Renu Bajaj, Benjamin Leiby, Elyse Ellsworth, Shauna Blackmon, [......], Adam Ertel, Chengbao Liu, Hallgeir Rui, Tapio Visakorpi, Lukas Bubendorf, Costas D Lallas, Edouard J Trabulsi, Peter McCue, Leonard Gomella, Marja T Nevalainen
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  ABSTRACT: The molecular mechanisms underlying progression of prostate cancer (PCa) to castrate-resistant (CR) and metastatic disease are poorly understood. Our previous mechanistic work shows that inhibition of transcription factor Stat5 by multiple alternative methods induces extensive rapid apoptotic death of Stat5-positive PCa cells in vitro and inhibits PCa xenograft tumor growth in nude mice. Furthermore, STAT5A/B induces invasive behavior of PCa cells in vitro and in vivo, suggesting involvement of STAT5A/B in PCa progression. Nuclear STAT5A/B protein levels are increased in high-grade PCas, CR PCas, and distant metastases, and high nuclear STAT5A/B expression predicts early disease recurrence and PCa-specific death in clinical PCas. Based on these findings, STAT5A/B represents a therapeutic target protein for advanced PCa. The mechanisms underlying increased Stat5 protein levels in PCa are unclear. Herein, we demonstrate amplification at the STAT5A/B gene locus in a significant fraction of clinical PCa specimens. STAT5A/B gene amplification was more frequently found in PCas of high histologic grades and in CR distant metastases. Quantitative in situ analysis revealed that STAT5A/B gene amplification was associated with increased STAT5A/B protein expression in PCa. Functional studies showed that increased STAT5A/B copy numbers conferred growth advantage in PCa cells in vitro and as xenograft tumors in vivo. The work presented herein provides the first evidence of somatic STAT5A/B gene amplification in clinical PCas.
  American Journal Of Pathology 05/2013;
• Article: The PPARγ Agonist Efatutazone Increases the Spectrum of Well-Differentiated Mammary Cancer Subtypes Initiated by Loss of Full-Length BRCA1 in Association with TP53 Haploinsufficiency.

  Rebecca E Nakles, Bhaskar V S Kallakury, Priscilla A Furth
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  ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have anticancer activity and influence cell differentiation. We examined the impact of the selective PPARγ agonist efatutazone on mammary cancer pathogenesis in a mouse model of BRCA1 mutation. Mice with conditional loss of full-length BRCA1 targeted to mammary epithelial cells in association with germline TP53 insufficiency were treated with efatutazone through the diet starting at age 4 months and were euthanized at age 12 months or when palpable tumor reached 1 cm(3). Although treatment did not reduce percentage of mice developing invasive cancer, it significantly reduced prevalence of noninvasive cancer and total number of cancers per mouse and increased prevalence of well-differentiated cancer subtypes not usually seen in this mouse model. Invasive cancers from controls were uniformly estrogen receptor α negative and undifferentiated, whereas well-differentiated estrogen receptor α-positive papillary invasive cancers appeared in efatutazone-treated mice. Expression levels of phosphorylated AKT and CDK6 were significantly reduced in the cancers developing in efatutazone-treated mice. Efatutazone treatment reduced rates of mammary epithelial cell proliferation and development of hyperplastic alveolar nodules and increased expression levels of the PPARγ target genes Adfp, Fabp4, and Pdhk4 in preneoplastic mammary tissue. Intervention efatutazone treatment in mice with BRCA1 deficiency altered mammary cancer development by promoting development of differentiated invasive cancer and reducing prevalence of noninvasive cancer and preneoplastic disease.
  American Journal Of Pathology 05/2013;
• Article: Functional Interaction of Osteogenic Transcription Factors Runx2 and Vdr in Transcriptional Regulation of Opn during Soft Tissue Calcification.

  Ann-Kathrin Sowa, Frank J Kaiser, Juliane Eckhold, Thorsten Kessler, Redouane Aherrahrou, Sandra Wrobel, Piotr M Kaczmarek, Lars Doehring, Heribert Schunkert, Jeanette Erdmann, Zouhair Aherrahrou
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  ABSTRACT: Loss of Abcc6 gene expression was identified to be responsible for dystrophic calcification of the heart (DCC) or vessels after acute injury in several strains of laboratory mice. This calcification shares features with osteogenesis and may involve osteogenic factors. Tissue expression of osteopontin (Opn) and 11 osteogenic transcription factors were studied in vivo in mouse models for DCC and in vitro using luciferase reporter gene assays. Compared with DCC-resistant C57BL/6 mice, a significant increase in Opn transcription was demonstrated in necrotic lesions of both DCC-susceptible C3H/He and B6.C3H(Dyscalc1) congenic mice at day 3 after injury. Significant increases in gene expression were also demonstrated for the transcription factors runt domain-containing transcription factor 2 (Runx2), vitamin D receptor (Vdr), SRY (sex-determining region Y)-box 9 protein, and Nfkb1 in C3H/He mice versus C57BL/6 controls. However, only Runx2 remained significantly increased in the B6.C3H(Dyscalc1) congenic mice, which carry only the Dyscalc1 locus with functional Abcc6 deletion on a C57BL/6 genetic background. Luciferase assay use increased Opn promoter activity, which was demonstrated after overexpression of Runx2. A poly-T stretch insertion was identified to stabilize the binding of Runx2, thus significantly enhancing Opn promoter activity. This Runx2-mediated activation was further enhanced by cotransfection with Vdr. Our data suggest a key role of Runx2 in the regulation of Opn in a model of cardiovascular calcification and demonstrate a synergistic cooperation of Runx2 and Vdr.
  American Journal Of Pathology 05/2013;
• Article: 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
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  ABSTRACT: 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.
  American Journal Of Pathology 05/2013; 182(5):1865-75.
• Article: Biological relevance of inflammation and oxidative stress in the pathogenesis of arterial diseases.

  David P Hajjar, Antonio M Gotto
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  ABSTRACT: 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.
  American Journal Of Pathology 05/2013; 182(5):1474-81.