The American Journal of Pathology

This Month in AJP

2012-01-24

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

Inflammation-Induced Lymph Node Lymphangiogenesis Is Reversible

Viviane Mumprecht, Filip Roudnicky, Michael Detmar — 2011-12-26

The extent of lymph node metastasis is a prognostic indicator of disease progression in many malignancies. Current noninvasive imaging technologies for the clinical assessment of lymph node metastases are based on the detection of cancer cells and commonly suffer from a lack of sensitivity. Recent evidence has indicated that the expansion of lymphatic networks (ie, lymphangiogenesis) within tumor-draining lymph nodes might be the earliest sign of metastasis. Therefore, we recently developed a noninvasive imaging method to visualize lymph node lymphangiogenesis in mice using radiolabeled antibodies against the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) as well as positron emission tomography (PET). This technique, termed anti-LYVE-1 immuno-PET, was found to be very sensitive in the detection of metastasis to the lymph nodes. However, lymphatic vessel expansion to the lymph nodes can also be induced by inflammation, and it is currently unclear whether such vessel expansion is reversed once inflammation has resolved. Detection of residual inflammation-induced lymph node lymphangiogenesis, thus, might hamper the identification of metastasized lymph nodes. In this study, we therefore used a well-established mouse model of inflammation in the skin to investigate whether lymphatic vessels in the lymph nodes regress on resolution of inflammation. Our data reveal that the lymphatic network indeed regresses on the resolution of inflammation and that we can image this process by anti-LYVE-1 immuno-PET.

Host-Derived TGFB1 Deficiency Suppresses Lesion Development in a Mouse Model of Endometriosis

2012-01-03

Transforming growth factor-β1 (TGFB1) is a multifunctional cytokine that is abundant in both endometriotic lesions and the peritoneal fluid in women with endometriosis. However, the role of TGFB1 in the development of endometriosis is as yet undefined. In the present study, we investigated the physiologic function of TGFB1 in endometriotic lesion development, using Tgfb1-null mutant mice on a background of severe combined immunodeficiency. Xenotransplantation of human eutopic endometrial tissue resulted in development of endometriosis-like lesions in 63% of ovariectomized estrogen-supplemented Tgfb1-null mutant mice and in 68% of wild-type control mice. Median lesion weight was reduced by 11-fold in Tgfb1-null mice compared with wild-type control mice, and the fraction of glandular epithelium in lesions from Tgfb1-null mice was reduced by 32% compared with that in control mice. In lesions from Tgfb1-null mice, the relative abundance of both macrophages and α-smooth muscle actin–positive myofibroblasts was reduced by 66% and 47%, respectively. Deficiency of TGFB1 neither altered the percentage of proliferating cells in the epithelial or stromal compartments of the lesions nor affected blood vessel density or vessel size. Observation of this study indicates that host-derived TGFB1 deficiency suppresses endometriotic lesion development and provides proof of principle that targeting TGFB1 signaling pathways in cells that support the survival of ectopic endometrium may be an effective therapeutic approach in women with endometriosis.

MMP-14 Is Expressed in Preeclamptic Placentas and Mediates Release of Soluble Endoglin

2012-02-02

Soluble endoglin is an anti-angiogenic protein that is released from the placenta and contributes to both maternal endothelial dysfunction and the clinical features of severe preeclampsia. The mechanism through which soluble endoglin is released from the placenta is currently unknown; however, recent work in colorectal cancer identified matrix metalloproteinase 14 (MMP-14) as the cleavage protease of endoglin. To determine whether this is also the mechanism responsible for soluble endoglin release in preeclampsia, we investigated the expression of MMP-14 within the placenta and the effects of its inhibition on soluble endoglin release. Placentas were obtained from severe, early onset preeclamptic pregnancies (n = 8) and gestationally matched preterm controls (n = 8). MMP-14 was predominately localized to the syncytiotrophoblast. Results from a proximity ligation assay showed protein interactions between endogenous MMP-14 and endoglin within the preeclamptic placenta. To demonstrate that this interaction produces soluble endoglin, we treated trophoblastic BeWo cells with either a broad-spectrum MMP inhibitor (GM6001) or MMP-14 siRNA. Both treatments produced a decrease in soluble endoglin (P ≤ 0.05). Treatment of mice bearing BeWo xenografts with GM6001 decreased circulating soluble endoglin levels in mouse serum (P ≤ 0.05). These findings indicate that MMP-14 is the likely cleavage protease of endoglin in the setting of preeclampsia. This approach provides a novel method for the development of potential therapeutics to reduce circulating soluble endoglin and ameliorate the clinical features of severe preeclampsia.

Integrative Molecular Profiling Reveals Asparagine Synthetase Is a Target in Castration-Resistant Prostate Cancer

2012-01-13

The identification of new and effective therapeutic targets for the lethal, castration-resistant stage of prostate cancer (CRPC) has been challenging because of both the paucity of adequate frozen tissues and a lack of integrated molecular analysis. Therefore, in this study, we performed a genome-wide analysis of DNA copy number alterations from 34 unique surgical CRPC specimens and 5 xenografts, with matched transcriptomic profiling of 25 specimens. An integrated analysis of these data revealed that the asparagine synthetase (ASNS) gene showed a gain in copy number and was overexpressed at the transcript level. The overexpression of ASNS was validated by analyzing other public CRPC data sets. ASNS protein expression, as detected by reverse-phase protein lysate array, was tightly correlated with gene copy number. In addition, ASNS protein expression, as determined by IHC analysis, was associated with progression to a therapy-resistant disease state in TMAs that included 77 castration-resistant and 40 untreated prostate cancer patient samples. Knockdown of ASNS by small-interfering RNAs in asparagine-deprived media led to growth inhibition in both androgen-responsive (ie, LNCaP) and castration-resistant (ie, C4-2B) prostate cancer cell lines and in cells isolated from a CRPC xenograft (ie, MDA PCa 180-30). Together, our results suggest that ASNS is up-regulated in cases of CRPC and that depletion of asparagine using ASNS inhibitors will be a novel strategy for targeting CRPC cells.

TFF3 Is a Normal Breast Epithelial Protein and Is Associated with Differentiated Phenotype in Early Breast Cancer but Predisposes to Invasion and Metastasis in Advanced Disease

2012-03-01

The trefoil protein TFF3 stimulates invasion and angiogenesis in vitro. To determine whether it has a role in breast tumor metastasis and angiogenesis, its levels were measured by immunohistochemistry in breast tissue with a specific monoclonal antibody raised against human TFF3. TFF3 is expressed in normal breast lobules and ducts, at higher levels in areas of fibrocystic change and papillomas, in all benign breast disease lesions, and in 89% of in situ and in 83% of invasive carcinomas. In well-differentiated tumor cells, TFF3 is concentrated at the luminal edge, whereas in poorly differentiated cells polarity is inverted and expression is directed toward the stroma. Expression was high in well-differentiated tumors and was associated significantly with low histological grade and with estrogen and progesterone receptor expression, accordant with induction of TFF3 mRNA by estrogen in breast cancer cells. Paradoxically, TFF3 expression was associated with muscle, neural, and lymphovascular invasion and the presence and number of involved lymph nodes, and it was an independent predictive marker of lymphovascular invasion and lymph node involvement. Consistent with an angiogenic function, TFF3 expression correlated strongly with microvessel density evaluated with CD31 and CD34. In conclusion, TFF3 is expressed in both the normal and diseased breast. Although associated with features of good prognosis, its profile of expression in invasive cancer is consistent with a role in breast tumor progression and tumor cell dissemination.

Low-Level Expression of miR-375 Correlates with Poor Outcome and Metastasis While Altering the Invasive Properties of Head and Neck Squamous Cell Carcinomas

2012-01-09

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumor's invasive properties.

The NF-κB Subunit c-Rel Stimulates Cardiac Hypertrophy and Fibrosis

2012-01-03

Cardiac remodeling and hypertrophy are the pathological consequences of cardiovascular disease and are correlated with its associated mortality. Activity of the transcription factor NF-κB is increased in the diseased heart; however, our present understanding of how the individual subunits contribute to cardiovascular disease is limited. We assign a new role for the c-Rel subunit as a stimulator of cardiac hypertrophy and fibrosis. We discovered that c-Rel-deficient mice have smaller hearts at birth, as well as during adulthood, and are protected from developing cardiac hypertrophy and fibrosis after chronic angiotensin infusion. Results of both gene expression and cross-linked chromatin immunoprecipitation assay analyses identified transcriptional activators of hypertrophy, myocyte enhancer family, Gata4, and Tbx proteins as Rel gene targets. We suggest that the p50 subunit could limit the prohypertrophic actions of c-Rel in the normal heart, because p50 overexpression in H9c2 cells repressed c-Rel levels and the absence of cardiac p50 was associated with increases in both c-Rel levels and cardiac hypertrophy. We report for the first time that c-Rel is highly expressed and confined to the nuclei of diseased adult human hearts but is restricted to the cytoplasm of normal cardiac tissues. We conclude that c-Rel-dependent signaling is critical for both cardiac remodeling and hypertrophy. Targeting its activities could offer a novel therapeutic strategy to limit the effects of cardiac disease.

Adenovirus-Mediated Gene Transfer of TGF-β1 to the Renal Glomeruli Leads to Proteinuria

2011-12-27

The mechanism of proteinuria in many common kidney diseases involves glomerular hemodynamic effects and local expression of angiogenic, fibrogenic, and vasoactive factors. Transforming growth factor (TGF)-β has been associated with many diseases involving proteinuria and renal fibrosis. TGF-β has been shown to induce podocyte dedifferentiation in vitro, but its in vivo effects on the glomerular filtration barrier are not well described. In this study, we used an adenovirus vector to transfer active TGF-β1 to the glomeruli of rat kidneys. Transient TGF-β1 overexpression induced significant proteinuria, podocyte foot process effacement, nephrin down-regulation, and nephrinuria. The expression of synaptopodin was also significantly down-regulated by TGF-β1. Increased glomerular expression of Snail, suggestive of an in vivo dedifferentiation process, was associated with a loss of podocyte epithelial markers. The expression of angiopoietin-1 and angiopoietin-2 was significantly increased in TGF-β1–transfected glomeruli, and TGF-β1 increased the expression of the angiopoietin receptor, Tie2, in podocyte cell culture. TGF-β1 down-regulated nephrin and synaptopodin expression in podocytes in cell culture; this effect was reversed by the blockade of both angiopoietin and Tie2 activities. These findings suggest that locally produced TGF-β1 can cause podocyte dedifferentiation marked by a loss of synaptopodin, nephrin, and foot process effacement, partly regulated by angiopoietins. This process represents a novel pathway that may explain proteinuria in a variety of common renal diseases.

β1 Integrin Gene Excision in the Adult Murine Cardiac Myocyte Causes Defective Mechanical and Signaling Responses

2012-01-16

How mechanical signals are transmitted in the cardiac myocyte is poorly understood. In this study, we produced a tamoxifen-inducible mouse model in which β1 integrin could be reduced specifically in the adult cardiomyocyte, so that the function of this integrin could be assessed in the postnatal and mechanically stressed heart. The expression of β1 integrin was reduced to 35% of control levels, but function remained normal at baseline. With aortic constriction, the knockout mice survived but had a blunted hypertrophic response. Integrin knockout myocytes, in contrast to controls, showed reduced integrin-linked kinase expression both at baseline and after hemodynamic stress; focal adhesion kinase expression was reduced after stress. Alterations in multiple signaling pathways were detected in the integrin knockout group after acute and chronic hemodynamic stress. Most remarkably, when we challenged the knockout mice with short-term loading, the robust responses of several kinases (extracellular signal–regulated kinase 1/2, p38, and Akt) evident in control mice were essentially abolished in the knockout mice. We also found that reduction of myocyte β1 integrin expression modified adrenergic-mediated signaling through extracellular signal–regulated kinase, p38, and Akt. Reduction of β1 integrin expression in the mature cardiac myocyte leads to a varied response compared with when this protein is reduced during either the embryonic or perinatal period. These results show that β1 integrin expression is required for proper mechanotransductive and adrenergic responses of the adult heart.

Decreased Proteasomal Activity Causes Age-Related Phenotypes and Promotes the Development of Metabolic Abnormalities

2012-01-03

The proteasome is a multicatalytic enzyme complex responsible for the degradation of both normal and damaged proteins. An age-related decline in proteasomal activity has been implicated in various age-related pathologies. The relevance of decreased proteasomal activity to aging and age-related diseases remains unclear, however, because suitable animal models are not available. In the present study, we established a transgenic (Tg) mouse model with decreased proteasomal chymotrypsin-like activity. Tg mice exhibited a shortened life span and developed age-related phenotypes. In Tg mice, polyubiquitinated and oxidized proteins accumulated, and the expression levels of cellular proteins such as Bcl-xL and RNase L were altered. When Tg mice were fed a high-fat diet, they developed more pronounced obesity and hepatic steatosis than did wild-type mice. Consistent with its role in lipid droplet formation, the expression of adipose differentiation-related protein (ADRP) was elevated in the livers of Tg mice. Of note, obesity and hepatic steatosis induced by a high-fat diet were more pronounced in aged than in young wild-type mice, and aged wild-type mice had elevated levels of ADRP, suggesting that the metabolic abnormalities present in Tg mice mimic those in aged mice. Our results provide the first in vivo evidence that decreased proteasomal chymotrypsin-like activity affects longevity and aggravates age-related metabolic disorders, such as obesity and hepatic steatosis.

Cathepsin Cleavage of Sirtuin 1 in Endothelial Progenitor Cells Mediates Stress-Induced Premature Senescence

2012-01-09

Stress-induced premature senescence (SIPS) of endothelial cells (ECs) has emerged as a contributor to global EC dysfunction. One of the cellular abnormalities mechanistically linked to SIPS is lysosomal dysfunction. In this study, we examined the impact of a range of cardiovascular risk factors on the expression of sirtuin 1 (SIRT1), SIPS, and apoptosis, and we documented the role of SIRT1 in reduced EC and endothelial progenitor cell (EPC) viability. These findings were confirmed in mice with selective endothelial SIRT1 knockout. The effects of stressors could be partially mimicked by inducing lysosomal membrane permeabilization or inhibiting autophagy, and were reversed by a cathepsin inhibitor. We provide evidence that SIRT1 is an important substrate of cysteine cathepsins B, S, and L. An antioxidant/peroxynitrite scavenger, ebselen, prevented stress-induced SIRT1 depletion and subversion of autophagy by mitigating lysosomal dysfunction. In conclusion, our data advance the concept of “stem cell aging” by establishing the critical role of lysosomal dysfunction in the development of SIPS through the cathepsin-induced proteolytic cleavage of SIRT1, a mechanism linking cell stress to apoptosis and SIPS. Ebselen potently protects lysosomal membrane integrity, preventing cathepsin-induced cleavage of SIRT 1 in EPCs and blunting SIPS and apoptotic cell death induced by relevant cardiovascular stressors. The proposed mechanism of SIRT1 depletion in stress has all of the attributes of being a paradigm of SIPS of EPCs.

Epithelial Expression of the Cytosolic Retinoid Chaperone Cellular Retinol Binding Protein II Is Essential for in Vivo Imprinting of Local Gut Dendritic Cells by Lumenal Retinoids

2012-01-04

Dendritic cells (DCs) use all-trans retinoic acid (ATRA) to promote characteristic intestinal responses, including Foxp3+ Treg conversion, lymphocyte gut homing molecule expression, and IgA production. How this ability to generate ATRA is conferred to DCs in vivo remains largely unstudied. Here, we observed that among DCs, retinaldehyde dehydrogenase (ALDH1), which catalyzes the conversion of retinal to ATRA, was preferentially expressed by small intestine CD103+ lamina propria (LP) DCs. Retinoids induced LP CD103+ DCs to generate ATRA via ALDH1 activity. Either biliary or dietary retinoids were required to confer ALDH activity to LP DCs in vivo. Cellular retinol-binding protein II (CRBPII), a cytosolic retinoid chaperone that directs enterocyte retinol and retinal metabolism but is redundant to maintain serum retinol, was required to confer ALDH activity to CD103+ LP DCs. CRBPII expression was restricted to small intestine epithelial cells, and ALDH activity in CRBPII−/− DCs was restored by transfer to a wild-type recipient. CD103+ LP DCs from CRBPII−/− mice had a decreased capacity to promote IgA production. Moreover, CD103+ DCs preferentially associated with the small intestine epithelium and LP CD103+ DC ALDH activity, and the ability to promote IgA production was reduced in mice with impaired DC–epithelia associations. These findings demonstrate in vivo roles for the expression of epithelial CRBPII and lumenal retinoids to imprint local gut DCs with an intestinal phenotype.

Chronic Alcohol Exposure Stimulates Adipose Tissue Lipolysis in Mice: Role of Reverse Triglyceride Transport in the Pathogenesis of Alcoholic Steatosis

2012-01-09

Alcohol consumption induces liver steatosis; therefore, this study investigated the possible role of adipose tissue dysfunction in the pathogenesis of alcoholic steatosis. Mice were pair-fed an alcohol or control liquid diet for 8 weeks to evaluate the alcohol effects on lipid metabolism at the adipose tissue–liver axis. Chronic alcohol exposure reduced adipose tissue mass and adipocyte size. Fatty acid release from adipose tissue explants was significantly increased in alcohol-fed mice in association with the activation of adipose triglyceride lipase and hormone-sensitive lipase. Alcohol exposure induced insulin intolerance and inactivated adipose protein phosphatase 1 in association with the up-regulation of phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling 3 (SOCS3). Alcohol exposure up-regulated fatty acid transport proteins and caused lipid accumulation in the liver. To define the mechanistic link between adipose triglyceride loss and hepatic triglyceride gain, mice were first administered heavy water for 5 weeks to label adipose triglycerides with deuterium, and then pair-fed alcohol or control diet for 2 weeks. Deposition of deuterium-labeled adipose triglycerides in the liver was analyzed using Fourier transform ion cyclotron mass spectrometry. Alcohol exposure increased more than a dozen deuterium-labeled triglyceride molecules in the liver by up to 6.3-fold. These data demonstrate for the first time that adipose triglycerides due to alcohol-induced hyperlipolysis are reverse transported and deposited in the liver.

Regulation of Monocyte Chemoattractant Protein-1 Through Angiotensin II Type 1 Receptor in Prostate Cancer

2012-01-09

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is reported to contribute to tumor progression and is regulated by the renin-angiotensin system in hypertensive disease. In this study, we investigated the clinical outcome of MCP-1 expression in patients with prostate cancer (CaP) and the regulation of MCP-1 through angiotensin II (AngII) type 1 receptor (AT1R) in CaP. Specimens were obtained from 138 CaP patients and analyzed by immunostaining for both MCP-1 and macrophages. We investigated the regulation of MCP-1 expression through AT1R both in vivo and in vitro using three human prostate cancer cell lines: LNCaP, C4-2, and C4-2AT6. Specimens with a high Gleason score (≥7) and a high pathological classification (≤pT3), and those with castration-resistant prostate cancer showed significantly higher MCP-1 expression and higher macrophage infiltration than low malignant potential CaP. High MCP-1 expression in CaP correlated significantly with high prostate-specific antigen (PSA) recurrence rates. AngII induced significantly higher MCP-1 levels in C4-2AT6 than in LNCaP, whereas AT1R blockade (ARB) inhibited MCP-1 production via the inhibition of the PI3K/Akt pathway in C4-2AT6. ARB also significantly suppressed MCP-1 expression in C4-2AT6 tumors. Our study is the first to demonstrate that both high MCP-1 expression and high macrophage infiltration in CaP specimens correlate with a high PSA recurrence rate and that ARB inhibits MCP-1 expression through the PI3K/Akt pathway and blocks macrophage infiltration in castration-resistant prostate cancer.

PTEN Regulates PDGF Ligand Switch for β-PDGFR Signaling in Prostate Cancer

2011-12-29

Platelet-derived growth factor (PDGF) family members are potent growth factors that regulate cell proliferation, migration, and transformation. Clinical studies have shown that both PDGF receptor β (β-PDGFR) and its ligand PDGF D are up-regulated in primary prostate cancers and bone metastases, whereas PDGF B, a classic ligand for β-PDGFR, is not frequently detected in clinical samples. In this study, we examined the role of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in the regulation of PDGF expression levels using both a prostate-specific, conditional PTEN-knockout mouse model and mouse prostate epithelial cell lines established from these mice. We found an increase in PDGF D and β-PDGFR expression levels in PTEN-null tumor cells, accompanied by a decrease in PDGF B expression. Among Akt isoforms, increased Akt3 expression was most prominent in mouse PTEN-null cells, and phosphatidylinositol 3-kinase/Akt activity was essential for the maintenance of increased PDGF D and β-PDGFR expression. In vitro deletion of PTEN resulted in a PDGF ligand switch from PDGF B to PDGF D in normal mouse prostate epithelial cells, further demonstrating that PTEN regulates this ligand switch. Similar associations between PTEN status and PDGF isoforms were noted in human prostate cancer cell lines. Taken together, these results suggest a mechanism by which loss of PTEN may promote prostate cancer progression via PDGF D/β-PDGFR signal transduction.

Plasmodium falciparum Histones Induce Endothelial Proinflammatory Response and Barrier Dysfunction

2012-01-18

Plasmodium falciparum is a protozoan parasite of human erythrocytes that causes the most severe form of malaria. Severe P. falciparum infection is associated with endothelial activation and permeability, which are important determinants of the outcome of the infection. How endothelial cells become activated is not fully understood, particularly with regard to the effects of parasite subcomponents. We demonstrated that P. falciparum histones extracted from merozoites (HeH) directly stimulated the production of IL-8 and other inflammatory mediators by primary human dermal microvascular endothelial cells through a signaling pathway that involves Src family kinases and p38 MAPK. The stimulatory effect of HeH and recombinant P. falciparum H3 (PfH3) was abrogated by histone-specific antibodies. The release of nuclear contents on rupture of infected erythrocytes was captured by live cell imaging and confirmed by detecting nucleosomes in the supernatants of parasite cultures. HeH and recombinant parasite histones also induced endothelial permeability through a charge-dependent mechanism that resulted in disruption of junctional protein expression and cell death. Recombinant human activated protein C cleaved HeH and PfH3 and abrogated their proinflammatory effects. Circulating nucleosomes of both human and parasite origin were detected in the plasma of patients with falciparum malaria and correlated positively with disease severity. These results support a pathogenic role for both host- and pathogen-derived histones in P. falciparum-caused malaria.

Chemokine Receptor CCR1 Disruption Limits Renal Damage in a Murine Model of Hemolytic Uremic Syndrome

2011-12-27

Shiga toxin (Stx)–producing Escherichia coli is the main etiological agent that causes hemolytic uremic syndrome (HUS), a microangiopathic disease characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Although direct cytotoxic effects on endothelial cells by Stx are the primary pathogenic event, there is evidence that indicates the inflammatory response mediated by polymorphonuclear neutrophils and monocytes as the key event during HUS development. Because the chemokine receptor CCR1 participates in the pathogenesis of several renal diseases by orchestrating myeloid cell kidney infiltration, we specifically addressed the contribution of CCR1 in a murine model of HUS. We showed that Stx type 2–treated CCR1−/− mice have an increased survival rate associated with less functional and histological renal damage compared with control mice. Stx type 2–triggered neutrophilia and monocytosis and polymorphonuclear neutrophil and monocyte renal infiltration were significantly reduced and delayed in CCR1−/− mice compared with control mice. In addition, the increase of the inflammatory cytokines (tumor necrosis factor-α and IL-6) in plasma was delayed in CCR1−/− mice compared with control mice. These data demonstrate that CCR1 participates in cell recruitment to the kidney and amplification of the inflammatory response that contributes to HUS development. Blockade of CCR1 could be important to the design of future therapies to restrain the inflammatory response involved in the development of HUS.

p47phox Directs Murine Macrophage Cell Fate Decisions

2012-01-04

Macrophage differentiation and function are pivotal for cell survival from infection and involve the processing of microenvironmental signals that determine macrophage cell fate decisions to establish appropriate inflammatory balance. NADPH oxidase 2 (Nox2)–deficient chronic granulomatous disease (CGD) mice that lack the gp91phox (gp91phox−/−) catalytic subunit show high mortality rates compared with wild-type mice when challenged by infection with Listeria monocytogenes (Lm), whereas p47phox-deficient (p47phox−/−) CGD mice show survival rates that are similar to those of wild-type mice. We demonstrate that such survival results from a skewed macrophage differentiation program in p47phox−/− mice that favors the production of higher levels of alternatively activated macrophages (AAMacs) compared with levels of either wild-type or gp91phox−/− mice. Furthermore, the adoptive transfer of AAMacs from p47phox−/− mice can rescue gp91phox−/− mice during primary Lm infection. Key features of the protective function provided by p47phox−/− AAMacs against Lm infection are enhanced production of IL-1α and killing of Lm. Molecular analysis of this process indicates that p47phox−/− macrophages are hyperresponsive to IL-4 and show higher Stat6 phosphorylation levels and signaling coupled to downstream activation of AAMac transcripts in response to IL-4 stimulation. Notably, restoring p47phox protein expression levels reverts the p47phox-dependent AAMac phenotype. Our results indicate that p47phox is a previously unrecognized regulator for IL-4 signaling pathways that are important for macrophage cell fate choice.

Glucocorticoid-Induced Tumor Necrosis Factor Receptor Family-Related Protein Exacerbates Collagen-Induced Arthritis by Enhancing the Expansion of Th17 Cells

2012-01-04

Rheumatoid arthritis (RA), a chronic autoimmune form of inflammatory joint disease, progressively affects multiple joints with pathological changes in the synovia, cartilage, and bone. Numerous studies have suggested a critical role for glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) in the pathogenesis of autoimmune arthritis by modulating both innate and adaptive immune reactions, but the underlying mechanisms by which GITR activation promotes arthritic progression remain largely unclear. In this study, we found that collagen-induced arthritis mice treated with the ligand of GITR (GITRL) displayed an earlier onset of arthritis with a markedly increased severity of arthritic symptoms and joint damage, in which significantly increased Th17 cells in both spleen and draining lymph nodes were observed. Notably, results showed that a marked expansion of Th17 cells with increased RORγt mRNA expression was induced from naïve CD4+ T cells when cultured with GITRL. Consistently, normal mice that were treated with GITRL were found to display a substantial expansion of splenic Th17 cells. Furthermore, we detected elevated serum levels of GITRL in patients with RA, which were positively correlated with an increase in interleukin-17 production. Taken together, the results from this study have revealed a new function of GITRL in exacerbating autoimmune arthritis via the enhancement of the expansion of Th17 cells.

Morphine Decreases Bacterial Phagocytosis by Inhibiting Actin Polymerization through cAMP-, Rac-1-, and p38 MAPK-Dependent Mechanisms

Jana Ninković, Sabita Roy — 2012-01-16

Morphine increases the susceptibility to opportunistic infection by attenuating bacterial clearance through inhibition of Fcγ receptor (FcgR)–mediated phagocytosis. Mechanisms by which morphine inhibits this process remain to be investigated. Actin polymerization is essential for FcgR-mediated internalization; therefore, disruption of the signaling mechanisms involved in this process is detrimental to the phagocytic ability of macrophages. To our knowledge, this study is the first to propose the modulation of actin polymerization and upstream signaling effectors [cAMP, Rac1-GTP, and p38 mitogen-activated protein kinase (MAPK)] as key mechanisms by which morphine leads to inhibition of pathogen clearance. Our results indicate that long-term morphine treatment in vitro and in vivo, through activation of the μ-opioid receptor, leads to an increase in intracellular cAMP, activation of protein kinase A, and inhibition of Rac1-GTPase and p38 MAPK, thereby attenuating actin polymerization and reducing membrane ruffling. Furthermore, because of long-term morphine treatment, FcgR-mediated internalization of opsonized dextran beads is also reduced. Morphine's inhibition of Rac1-GTPase activation is abolished in J774 macrophages transfected with constitutively active pcDNA3-EGFP-Rac1-Q61L plasmid. Dibutyryl-cAMP inhibits, whereas H89 restores, activation of Rac-GTPase and abolishes morphine's inhibitory effect, implicating cAMP as the key effector in morphine's modulation of actin polymerization. These findings indicate that long-term morphine treatment, by increasing intracellular cAMP and activating protein kinase A, leads to inhibition of Rac1-GTPase and p38 MAPK, causing attenuation of actin polymerization, FcgR-mediated phagocytosis, and decreased bacterial clearance.

Interspecies Comparison of Human and Murine Scleroderma Reveals IL-13 and CCL2 as Disease Subset-Specific Targets

2012-01-13

Development of personalized treatment regimens is hampered by lack of insight into how individual animal models reflect subsets of human disease, and autoimmune and inflammatory conditions have proven resistant to such efforts. Scleroderma is a lethal autoimmune disease characterized by fibrosis, with no effective therapy. Comparative gene expression profiling showed that murine sclerodermatous graft-versus-host disease (sclGVHD) approximates an inflammatory subset of scleroderma estimated at 17% to 36% of patients analyzed with diffuse, 28% with limited, and 100% with localized scleroderma. Both sclGVHD and the inflammatory subset demonstrated IL-13 cytokine pathway activation. Host dermal myeloid cells and graft T cells were identified as sources of IL-13 in the model, and genetic deficiency of either IL-13 or IL-4Rα, an IL-13 signal transducer, protected the host from disease. To identify therapeutic targets, we explored the intersection of genes coordinately up-regulated in sclGVHD, the human inflammatory subset, and IL-13–treated fibroblasts; we identified chemokine CCL2 as a potential target. Treatment with anti-CCL2 antibodies prevented sclGVHD. Last, we showed that IL-13 pathway activation in scleroderma patients correlated with clinical skin scores, a marker of disease severity. Thus, an inflammatory subset of scleroderma is driven by IL-13 and may benefit from IL-13 or CCL2 blockade. This approach serves as a model for personalized translational medicine, in which well-characterized animal models are matched to molecularly stratified patient subsets.

JC Virus Intranuclear Inclusions Associated with PML-NBs: Analysis by Electron Microscopy and Structured Illumination Microscopy

Yukiko Shishido-Hara, Shizuko Ichinose, Toshiki Uchihara — 2012-01-24

Progressive multifocal leukoencephalopathy is a fatal demyelinating disorder caused by JC virus infection. JC virus was recently found to target promyelocytic leukemia nuclear bodies (PML-NBs), punctuate domains in the nuclei. Thus, the virus progenies cluster in dots as intranuclear inclusions (ie, as dot-shaped inclusions). In the present study, both the viral major and minor capsid proteins were expressed from polycistronic expression vectors with a powerful promoter, and formation into virus-like particles (VLPs) was examined by electron microscopy. When the upstream regulatory sequence including the agnogene (nt 275 to 490) was present, capsid protein expression was suppressed, but numerous VLPs were efficiently formed with restricted accumulation to PML-NBs. VLPs were uniform, and the cells were severely degraded. In contrast, when the 5′ terminus of the agnogene (nt 275 to 409; 135 bp) was deleted, capsid protein expression was markedly enhanced, but VLPs were more randomly produced in the nucleus outside of PML-NBs. VLPs were pleomorphic, and cell degradation was minimal. JC virus association with PML-NBs was confirmed in human brain tissues by structured illumination microscopy. PML-NBs were shaped in spherical shells, with viral capsid proteins circumscribing the surface. These findings indicate that PML-NBs are intranuclear locations for pathogenic JC virus proliferation. Either the agnogene or its product likely supports efficient progeny production at PML-NBs, leading to subsequent degeneration of host glial cells.

Neural Stem Cell Depletion and CNS Developmental Defects After Enteroviral Infection

2012-01-04

Coxsackieviruses are significant human pathogens causing myocarditis, meningitis, and encephalitis. We previously demonstrated the ability of coxsackievirus B3 (CVB3) to persist within the neonatal central nervous system (CNS) and to target neural stem cells. Given that CVB3 is a cytolytic virus and may therefore damage target cells, we characterized the potential reduction in neurogenesis within the developing brain and the subsequent developmental defects that occurred after the loss of these essential neural stem cells. Neonatal mice were inoculated with a recombinant CVB3 expressing eGFP (eGFP-CVB3), and alterations in neurogenesis and brain development were evaluated over time. We observed a reduction in proliferating cells in CNS neurogenic regions simultaneously with the presence of nestin+ cells undergoing apoptosis. The size of the brain appeared smaller by histology, and a permanent decrease in brain wet weight was observed after eGFP-CVB3 infection. We also observed an inverse relationship between the amount of virus material and brain wet weight up to day 30 postinfection. In addition, signs of astrogliosis and a compaction of the cortical layers were observed at 90 days postinfection. Intriguingly, partial brain wet weight recovery was observed in mice treated with the antiviral drug ribavirin during the persistent stage of infection. Hence, long-term neurological sequelae might be expected after neonatal enteroviral infections, yet antiviral treatment initiated long after the end of acute infection might limit virus-mediated neuropathology.

Growth Defects and Impaired Cognitive–Behavioral Abilities in Mice with Knockout for Eif4h, a Gene Located in the Mouse Homolog of the Williams-Beuren Syndrome Critical Region

2012-01-09

Protein synthesis is a tightly regulated, energy-consuming process. The control of mRNA translation into protein is fundamentally important for the fine-tuning of gene expression; additionally, precise translational control plays a critical role in many cellular processes, including development, cellular growth, proliferation, differentiation, synaptic plasticity, memory, and learning. Eukaryotic translation initiation factor 4h (Eif4h) encodes a protein involved in the process of protein synthesis, at the level of initiation phase. Its human homolog, WBSCR1, maps on 7q11.23, inside the 1.6 Mb region that is commonly deleted in patients affected by the Williams-Beuren syndrome, which is a complex neurodevelopmental disorder characterized by cardiovascular defects, cerebral dysplasias and a peculiar cognitive-behavioral profile. In this study, we generated knockout mice deficient in Eif4h. These mice displayed growth retardation with a significant reduction of body weight that began from the first week of postnatal development. Neuroanatomical profiling results generated by magnetic resonance imaging analysis revealed a smaller brain volume in null mice compared with controls as well as altered brain morphology, where anterior and posterior brain regions were differentially affected. The inactivation of Eif4h also led to a reduction in both the number and complexity of neurons. Behavioral studies revealed severe impairments of fear-related associative learning and memory formation. These alterations suggest that Eif4h might contribute to certain deficits associated with Williams-Beuren syndrome.

Platelet-Derived Growth Factor C Deficiency in C57BL/6 Mice Leads to Abnormal Cerebral Vascularization, Loss of Neuroependymal Integrity, and Ventricular Abnormalities

2012-01-09

Platelet-derived growth factors (PDGFs) and their tyrosine kinase receptors (PDGFRs) are known to play important roles during development of the lungs, central nervous system (CNS), and skeleton and in several diseases. PDGF-C is a ligand for the tyrosine kinase receptor PDGFRα. Mutations in the gene encoding PDGF-C have been linked to clefts of the lip and/or palate in humans, and ablation of PDGF-C in 129/Sv background mice results in death during the perinatal period. In this study, we report that ablation of PDGF-C in C57BL/6 mice results in a milder phenotype than in 129/Sv mice, and we present a phenotypic characterization of PDGF-C deficiency in the adult murine CNS. Multiple congenital defects were observed in the CNS of PDGF-C–null C57BL/6 mice, including cerebral vascular abnormalities with abnormal vascular smooth muscle cell coverage. In vivo imaging of mice deficient in PDGF-C also revealed cerebral ventricular abnormalities, such as asymmetry of the lateral ventricles and hypoplasia of the septum, reminiscent of cavum septum pellucidum in humans. We further noted that PDGF-C–deficient mice displayed a distorted ependymal lining of the lateral ventricles, and we found evidence of misplaced neurons in the ventricular lining. We conclude that PDGF-C plays a critical role in the development of normal cerebral ventricles and neuroependymal integrity as well as in normal cerebral vascularization.

Collagen VI Ablation Retards Brain Tumor Progression Due to Deficits in Assembly of the Vascular Basal Lamina

Weon-Kyoo You, Paolo Bonaldo, William B. Stallcup — 2011-12-26

To investigate the importance of the vascular basal lamina in tumor blood vessel morphogenesis and function, we compared vessel development, vessel function, and progression of B16F10 melanoma tumors in the brains of wild-type and collagen VI-null mice. In 7-day tumors in the absence of collagen VI, the width of the vascular basal lamina was reduced twofold. Although the ablation of collagen VI did not alter the abundance of blood vessels, a detailed analysis of the number of either pericytes or endothelial cells (or pericyte coverage of endothelial cells) showed that collagen VI-dependent defects during the assembly of the basal lamina have negative effects on both pericyte maturation and the sprouting and survival of endothelial cells. As a result of these deficits, vessel patency was reduced by 25%, and vessel leakiness was increased threefold, resulting in a 10-fold increase in tumor hypoxia along with a fourfold increase in hypoxia-inducible factor-1α expression. In 12-day collagen VI-null tumors, vascular endothelial growth factor expression was increased throughout the tumor stroma, in contrast to the predominantly vascular pattern of vascular endothelial growth factor expression in wild-type tumors. Vessel size was correspondingly reduced in 12-day collagen VI-null tumors. Overall, these vascular deficits produced a twofold decrease in tumor volume in collagen VI-null mice, confirming that collagen VI-dependent basal lamina assembly is a critical aspect of vessel development.

ALDH1-Bright Epithelial Ovarian Cancer Cells Are Associated with CD44 Expression, Drug Resistance, and Poor Clinical Outcome

2012-01-04

The role of aldehyde dehydrogenase 1 (ALDH1) as an ovarian cancer stem cell marker and its clinical significance have rarely been explored. We used an Aldefluor assay to isolate ALDH1-bright (ALDH1br) cells from epithelial ovarian cancer cell lines and characterized the properties of the stem cells. ALDH1br cells were enriched in ES-2 (1.3%), TOV-21G (1.0%), and CP70 (1.2%) cells. Both ALDH1br and ALDH1low cells repopulated stem cell heterogeneity, formed spheroids, and grew into tumors in immunocompromised mice, although these processes were more efficient in ALDH1br cells. In the ES-2 and CP70 cells, ALDH1br cells conferred more chemoresistance, and were more enriched in CD44 (by 1.74-fold and 5.18-fold, respectively) than in CD133 (by 1.39-fold and 1.17-fold, respectively), compared with ALDH1low cells. Immunohistochemical staining for ALDH1 on a tissue microarray containing 84 epithelial ovarian cancer samples revealed that patients with higher ALDH1 expression (>50%) had poor overall survival, compared with those with lower ALDH1 (P = 0.004) and yielded an odds ratio of death of 2.43 (95% CI = 1.12 to 5.28) by multivariate analysis. The results did not support ALDH1 alone as an ovarian cancer stem cell marker, but demonstrated that ALDH1 is associated with CD44 expression, chemoresistance, and poor clinical outcome. The use of a combination of ALDH1 with other stem cell markers may help define ovarian cancer stem cells more stringently.

Clinical and Biological Significance of KISS1 Expression in Prostate Cancer

2012-01-09

For men in the United States, prostate cancer (PCa) is the most frequent malignancy and the second leading cause of cancer mortality. The metastatic spread of PCa is responsible for most deaths related to PCa. Although KISS1 functions as a metastasis suppressor in various cancers, its expression levels and functions in PCa development and progression remain undetermined. The goals of this study were to correlate the expression levels of KISS1 in PCas with clinicopathologic characteristics and to assess the biological relevance of KISS1 to the viability and motility of PCa cells. Strong KISS1 staining was detected in benign prostate tissues, but the staining was weaker in primary and metastatic PCas (both P < 0.001, t-test). Furthermore, the low expression levels of KISS1 in PCas correlated with clinical stage (P < 0.01) and with KISS1R expression (P < 0.001). Overexpression of full-length KISS1 in low KISS1-expressing PC-3M cells, but not KFMΔSS, which lacks the secretion signal sequence, induced re-sensitization of cells to anoikis, although it had no effect on either cell proliferation or apoptosis. Overexpression of KISS1 also suppressed steps in the metastatic cascade, including motility and invasiveness. Moreover, cells overexpressing KISS1 were found to enhance chemosensitivity to paclitaxel. Collectively, our data suggest that KISS1 functions as a metastasis suppressor in PCas and may serve as a useful biomarker as well as a therapeutic target for aggressive PCas.

Mutant BRAF Induces DNA Strand Breaks, Activates DNA Damage Response Pathway, and Up-Regulates Glucose Transporter-1 in Nontransformed Epithelial Cells

2012-01-06

Although the oncogenic functions of activating BRAF mutations have been clearly demonstrated in human cancer, their roles in nontransformed epithelial cells remain largely unclear. Investigating the cellular response to the expression of mutant BRAF in nontransformed epithelial cells is fundamental to the understanding of the roles of BRAF in cancer pathogenesis. In this study, we used two nontransformed cyst108 and RK3E epithelial cell lines as models in which to compare the phenotypes of cells expressing BRAFWT and BRAFV600E. We found that transfection of the BRAFV600E, but not the BRAFWT, expression vector suppressed cellular proliferation and induced apoptosis in both cell types. BRAFV600E generated reactive oxygen species, induced DNA double-strand breaks, and caused subsequent DNA damage response as evidenced by an increased number of pCHK2 and γH2AX nuclear foci as well as the up-regulation of pCHK2, p53, and p21. Because BRAF and KRAS (alias Ki-ras) mutations have been correlated with GLUT1 up-regulation, which encodes glucose transporter-1, we demonstrated here that expression of BRAFV600E, but not BRAFWT, was sufficient to up-regulate GLUT1. Taken together, our findings provide new insights into mutant BRAF-induced oncogenic stress that is manifested by DNA damage and growth arrest by activating the pCHK2-p53-p21 pathway in nontransformed cells, while it also confers tumor-promoting phenotypes such as the up-regulation of GLUT1 that contributes to enhanced glucose metabolism that characterizes tumor cells.

Role of ERRF, a Novel ER-Related Nuclear Factor, in the Growth Control of ER-Positive Human Breast Cancer Cells

2012-03-01

Whereas estrogen–estrogen receptor α (ER) signaling plays an important role in breast cancer growth, it is also necessary for the differentiation of normal breast epithelial cells. How this functional conversion occurs, however, remains unknown. Based on a genome-wide sequencing study that identified mutations in several breast cancer genes, we examined some of the genes for mutations, expression levels, and functional effects on cell proliferation and tumorigenesis. We present the data for C1orf64 or ER-related factor (ERRF) from 31 cell lines and 367 primary breast cancer tumors. Whereas mutation of ERRF was infrequent (1 of 79 or 1.3%), its expression was up-regulated in breast cancer, and the up-regulation was more common in lower-stage tumors. In addition, increased ERRF expression was significantly associated with ER and/or progesterone receptor (PR) positivity, which was still valid in human epidermal growth factor receptor 2 (HER2)–negative tumors. In ER-positive tumors, ERRF expression was inversely correlated with HER2 status. Furthermore, higher ERRF protein expression was significantly associated with better disease-free survival and overall survival, particularly in ER- and/or PR-positive and HER2-negative tumors (luminal A subtype). Functionally, knockdown of ERRF in two ER-positive breast cancer cell lines, T-47D and MDA-MB-361, suppressed cell growth in vitro and tumorigenesis in xenograft models. These results suggest that ERRF plays a role in estrogen-ER–mediated growth of breast cancer cells and could, thus, be a potential therapeutic target.

The Nonreceptor-Type Tyrosine Phosphatase PTPN13 Is a Tumor Suppressor Gene in Non–Small Cell Lung Cancer

2012-01-16

The aim of the present work was to identify protein tyrosine phosphatases (PTPs) as novel, candidate tumor suppressor genes in lung cancer. Among the 38 PTPs in the human genome that show specificity for phosphotyrosine, we identified six PTPs by quantitative RT-PCR whose mRNA expression levels were significantly down-regulated in lung cancer–derived cell lines (ie, PTPRE, PTPRF, PTPRU, PTPRK, PTPRD, and PTPN13). After validation in primary samples of non–small cell lung cancer (NSCLC), we selected PTPN13 for further studies. The results presented here demonstrate that PTPN13 is a candidate tumor suppressor gene that is frequently inactivated in NSCLC through the loss of either mRNA and protein expression (64/87, 73%) or somatic mutation (approximately 8%). Loss of PTPN13 expression was apparently due to the loss of one or both copies of the PTPN13 locus at 4q (approximately 26% double deletion and approximately 37% single deletion) but not to promoter methylation. Finally, the manipulation of PTPN13 expression in lung cancer cells (ie, NCI-H292, A549) demonstrated that PTPN13 negatively regulates anchorage-dependent and anchorage-independent growth in vitro and restrains tumorigenicity in vivo, possibly through the control of the tyrosine phosphorylation of both EGFR and HER2. In conclusion, the expression screening of PTPs in lung cancer reported here has identified PTPN13 as a novel candidate tumor suppressor in NSCLC whose loss increases signaling from epidermal growth factor receptor and HER2 tyrosine kinase receptors.

CXCR6 and CCR5 Localize T Lymphocyte Subsets in Nasopharyngeal Carcinoma

2012-01-09

The substantial T lymphocyte infiltrate found in cases of nasopharyngeal carcinoma (NPC) has been implicated in the promotion of both tumor growth and immune escape. Conversely, because malignant NPC cells harbor the Epstein-Barr virus, this tumor is a candidate for virus-specific T cell-based therapies. Preventing the accumulation of tumor-promoting T cells or enhancing the recruitment of tumor-specific cytotoxic T cells offers therapeutic potential. However, the mechanisms involved in T cell recruitment to this tumor are poorly understood. Comparing memory T cell subsets that have naturally infiltrated NPC tissue with their counterparts from matched blood revealed enrichment of CD8+, CD4+, and regulatory T cells expressing the chemokine receptor CXCR6 in tumor tissue. CD8+ and (nonregulatory) CD4+ T cells also were more frequently CCR5+ in tumor than in blood. Ex vivo studies demonstrated that both receptors were functional. CXCL16 and CCL4, unique chemokine ligands for CXCR6 and CCR5, respectively, were expressed by the malignant cells in tumor tissue from the majority of NPC cases, as was another CCR5 ligand, CCL5. The strongest expression of CXCL16 was found on tumor-infiltrating cells. CCL4 was detected on the tumor vasculature in a majority of cases. These findings suggest that CXCR6 and CCR5 play important roles in T cell recruitment and/or retention in NPC and have implications for the pathogenesis and treatment of this tumor.

High Anaplastic Lymphoma Kinase Immunohistochemical Staining in Neuroblastoma and Ganglioneuroblastoma Is an Independent Predictor of Poor Outcome

2011-12-27

Anaplastic lymphoma kinase (ALK) mutations occur in 3% to 11% of neuroblastoma (NBL) cases and are associated with high ALK levels. However, high ALK levels appear to be a mutation-independent hallmark of NBL. Evidence about the prognostic relevance of ALK mutations and ALK tumor positivity in patients with NBL has been inconclusive. In this study, we investigated the prognostic relevance of ALK positivity by IHC and ALK mutation status by PCR sequencing in 71 NBL, 12 ganglioneuroblastoma (GNBL), and 20 ganglioneuroma samples in a multivariate model. ALK mutations were present in 2 of 72 NBL and 2 of 12 GNBL samples, which all contained many ALK-positive cells (>50%). In addition, half of all NBL samples showed ALK positivity in most (>50%) of tumor cells, whereas half of the GNBL showed staining in <20% of the tumor cells. In most ganglioneuroma samples, a low percentage of tumor cells stained positive for ALK, which mainly involved ganglion cells. Higher percentages of ALK-positive cells in NBL and GNBL patient samples correlated with inferior survival in univariate and multivariate analyses with established prognostic factors, such as stage, age, and MYCN status. In conclusion, ALK positivity by IHC is an independent, poor prognostic factor in patients with GNBL and NBL. ALK IHC is an easy test suitable for future risk stratification in patients with NBL and GNBL.

Plexin-B1 and Semaphorin 4D Cooperate to Promote Perineural Invasion in a RhoA/ROK-Dependent Manner

2012-01-16

Perineural invasion (PNI) is a tropism of tumor cells for nerve bundles located in the surrounding stroma. It is a pathological feature observed in certain tumors, referred to as neurotropic malignancies, that severely limits the ability to establish local control of disease and results in pain, recurrent growth, and distant metastases. Despite the importance of PNI as a prognostic indicator, its biological mechanisms are poorly understood. The semaphorins and their receptors, the plexins, compose a family of proteins originally shown to be important in nerve cell adhesion, axon migration, and proper central nervous system development. Emerging evidence has demonstrated that these factors are expressed in tissues outside of the nervous system and represent a widespread signal transduction system that is involved in the regulation of motility and adhesion in different cell types. We believe that the plexins and semaphorins, which are strongly expressed in both axons and many carcinomas, play a role in PNI. In this study, we show that plexin-B1 is overexpressed in tissues and cell lines from neurotropic malignancies and is attracted to nerves that express its ligand, semaphorin 4D, in a Rho/Rho kinase-dependent manner. We also demonstrate that nerves are attracted to tumors through this same system of proteins, suggesting that both plexin-B1 and semaphorin 4D are important in the promotion of PNI.

VEGF-Mediated STAT3 Activation Inhibits Retinal Vascularization by Down-Regulating Local Erythropoietin Expression

2012-01-09

Avascular, hypoxic retina has been postulated to be a source of angiogenic factors that cause aberrant angiogenesis and intravitreal neovascularization (IVNV) in retinopathy of prematurity. Vascular endothelial growth factor (VEGF) is an important factor involved. However, VEGF is also required for normal retinal vascular development, which raises concerns about inhibiting its activity to treat IVNV in retinopathy of prematurity. Therefore, understanding the effects that VEGF has on other factors in the development of avascular retina is important to prevent aberrant angiogenesis and IVNV. Here, we show that STAT3 was activated by increased retinal VEGF in the rat 50/10 oxygen-induced retinopathy model. Phospho-STAT3 colocalized with glutamine synthetase-labeled Müller cells. Inhibition of STAT3 reduced avascular retina and increased retinal erythropoietin (Epo) expression. Epo administered exogenously also reduced avascular retina in the model. In an in vitro study, hypoxia-induced VEGF inhibited Epo gene expression by STAT3 activation in rat Müller cells. The mechanism by which activated STAT3 regulated Epo was by inhibition of Epo promoter activity. Together, these findings show that increased retinal VEGF contributes to avascular retina by regulating retinal Epo expression through Janus kinase/STAT signaling. Our results suggest that rescuing Epo expression in the retina before the development of IVNV may promote normal developmental angiogenesis and, therefore, reduce the stimulus for later pathologic IVNV.

Deficiency of Interleukin-1 Receptor Antagonist Promotes Spontaneous Femoral Artery Aneurysm Formation in Mice

2012-01-13

Femoral artery aneurysms (FAAs) are very rare, and their natural history is not well understood. In this study, we sought to analyze the pathogenesis of inflammatory FAAs in interleukin-1 receptor antagonist–deficient (IL-1Ra−/−) B6 mice. Systolic arterial pressures and plasma lipid levels of IL-1Ra−/− mice and wild-type (WT) mice did not differ significantly. However, IL-1Ra−/− mice spontaneously developed fusiform FAAs. Real-time PCR of 9-month-old IL-1Ra−/− mice revealed significantly increased mRNA levels of IL-1β (6.6-fold), tumor necrosis factor-α (TNF-α) (12.4-fold), and matrix metalloproteinase-9 (6.0-fold) compared with WT mice. Histological analysis revealed numerous inflammatory cells around the FAAs in IL-1Ra−/− mice, and elastin staining showed destruction of both the internal and external elastic lamina in IL-1Ra−/− mice. Afterward, macrophage function was studied. After lipopolysaccharide (1 μg/mL) stimulation, IL-1Ra–deficient macrophages produced much higher levels of TNF-α than those from WT mice. Finally, we performed bone marrow cell transplantation. FAAs with many inflammatory cells in the adventitia were detected in several WT mice that received bone marrow cells from IL-1Ra−/− mice (44%), but not from WT mice (0%). Our study is the first to demonstrate that IL-1Ra deficiency in inflammatory cells disrupts immune system homeostasis and induces inflammatory FAAs in IL-1Ra−/− B6 mice. We believe that these mice will provide much information about the natural history and management of FAAs.

Chlamydophila pneumoniae Infection Induces Alterations in Vascular Contractile Responses

Justin F. Deniset, Thomas E. Hedley, Elena Dibrov, Grant N. Pierce — 2012-01-04

Chlamydophila pneumoniae infection has been associated in previous studies with coronary artery disease. The live bacterium has been detected within atherosclerotic plaques and can induce the structural remodeling of the vessel wall. However, the direct effects of infection on the contractile characteristics of the arteries remain unknown. Left anterior descending coronary arteries isolated from porcine hearts were dissected and placed in culture medium for 72 hours before infection with C. pneumoniae. Contractile responses to high molar KCl and u46619 levels and relaxation responses to bradykinin and sodium nitroprusside were assessed at days 5 and 10 postinfection. C. pneumoniae induced decreases in both KCl- and u46619-induced contractile responses at both time points. The altered contractile responses coincided with a down-regulation of L-type Ca2+ channels at both time points and inositol 1,4,5-triphosphate receptor (IP3R) levels at day 10 postinfection. Infection also induced attenuation of the endothelial-dependent relaxation response to bradykinin at day 10 postinfection. A decrease in endothelial nitric oxide synthase expression levels was noted at day 10 postinfection. Furthermore, an increase in superoxide production combined with an increase in p22phox expression levels was also observed at this time point. These findings indicate that C. pneumoniae infection can directly alter the vascular contractile responses in porcine coronary arteries, providing additional evidence for the role of C. pneumoniae infection in cardiovascular disease.

Promotion of Lymphatic Integrity by Angiopoietin-1/Tie2 Signaling during Inflammation

2011-12-26

The cutaneous lymphatic system plays a major role in tissue fluid homeostasis and inflammation of the skin. Although several lymphangiogenic factors are known to be involved in the formation of lymphatic vessels, the molecular mechanisms that maintain lymphatic integrity and control the functional drainage of interstitial fluid and resolution of inflammation remain unknown. Here we show that angiopoietin-1 (Ang1) enhances lymphatic integrity and function during inflammation. Ang1 transgenic mice under the control of keratin-14 (K14-Ang1) showed attenuated edema formation and inflammation after UV B (UVB) exposure. After UVB irradiation, blood vascular permeability was inhibited in K14-Ang1 mice compared with wild-type (WT) mice. Moreover, lymphatic vessels of WT mice were markedly enlarged and leaky in inflamed skin, whereas K14-Ang1 mice showed relatively contracted lymphatic vessels together with enhanced lymphatic vascularization. Expression of endothelial-specific tight junction molecules claudin-5 and zonula occludens protein 1 (ZO-1) was strongly down-regulated in the inflamed lymphatic vessels of UVB-exposed WT mice, whereas down-regulation of both claudin-5 and ZO-1 was blocked in UVB-exposed K14-Ang1 mice. In vitro studies revealed that the stability of lymphatic endothelial cells was enhanced in the presence of Ang1, presumably via up-regulation of claudin-5, as well as ZO-1. Claudin-5 knockdown markedly increased the permeability of lymphatic endothelial cells. Overall, our data strongly support the idea that Ang1/Tie2 signaling promotes lymphatic integrity by modulating tight junction molecule expression during inflammation.

Tumor Endothelial Cells Acquire Drug Resistance by MDR1 Up-Regulation via VEGF Signaling in Tumor Microenvironment

2012-01-16

Tumor endothelial cells (TECs) are therapeutic targets in anti-angiogenic therapy. Contrary to the traditional assumption, TECs can be genetically abnormal and might also acquire drug resistance. In this study, mouse TECs and normal ECs were isolated to investigate the drug resistance of TECs and the mechanism by which it is acquired. TECs were more resistant to paclitaxel with the up-regulation of multidrug resistance (MDR) 1 mRNA, which encodes the P-glycoprotein, compared with normal ECs. Normal human microvascular ECs were cultured in tumor-conditioned medium (CM) and became more resistant to paclitaxel through MDR1 mRNA up-regulation and nuclear translocation of Y-box–binding protein 1, which is an MDR1 transcription factor. Vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Akt were activated in human microvascular ECs by tumor CM. We observed that tumor CM contained a significantly high level of VEGF. A VEGFR kinase inhibitor, Ki8751, and a phosphatidylinositol 3-kinase–Akt inhibitor, LY294002, blocked tumor CM–induced MDR1 up-regulation. MDR1 up-regulation, via the VEGF-VEGFR pathway in the tumor microenvironment, is one of the mechanisms of drug resistance acquired by TECs. We observed that VEGF secreted from tumors up-regulated MDR1 through the activation of VEGFR2 and Akt. This process is a novel mechanism of the acquisition of drug resistance by TECs in the tumor microenvironment.

Heterogeneity of Tumor Endothelial Cells: Comparison between Tumor Endothelial Cells Isolated from High- and Low-Metastatic Tumors

2012-01-16

An important concept in tumor angiogenesis is that tumor endothelial cells (TECs) are genetically normal and homogeneous. However, we previously reported that TECs differ from normal ECs. Whether the characteristics of TECs derived from different tumors differ remains unknown. To elucidate this, in this study, we isolated two types of TECs from high-metastatic (HM) and low-metastatic (LM) tumors and compared their characteristics. HM tumor–derived TECs (HM-TECs) showed higher proliferative activity and invasive activity than LM tumor–derived TECs (LM-TECs). Moreover, the mRNA expression levels of pro-angiogenic genes, such as vascular endothelial growth factor (VEGF) receptors 1 and 2, VEGF, and hypoxia-inducible factor-1α, were higher in HM-TECs than in LM-TECs. The tumor blood vessels themselves and the surrounding area in HM tumors were exposed to hypoxia. Furthermore, HM-TECs showed higher mRNA expression levels of the stemness-related gene stem cell antigen and the mesenchymal marker CD90 compared with LM-TECs. HM-TECs were spheroid, with a smoother surface and higher circularity in the stem cell spheroid assay. HM-TECs differentiated into osteogenic cells, expressing activated alkaline phosphatase in an osteogenic medium at a higher rate than either LM-TECs or normal ECs. Furthermore, HM-TECs contained more aneuploid cells than LM-TECs. These results indicate that TECs from HM tumors have a more pro-angiogenic phenotype than those from LM tumors.

Co-Regulation of Transcellular and Paracellular Leak Across Microvascular Endothelium by Dynamin and Rac

2011-12-27

Increased permeability of the microvascular endothelium to fluids and proteins is the hallmark of inflammatory conditions such as sepsis. Leakage can occur between (paracellular) or through (transcytosis) endothelial cells, yet little is known about whether these pathways are linked. Understanding the regulation of microvascular permeability is essential for the identification of novel therapies to combat inflammation. We investigated whether transcytosis and paracellular leakage are co-regulated. Using molecular and pharmacologic approaches, we inhibited transcytosis of albumin in primary human microvascular endothelium and measured paracellular permeability. Blockade of transcytosis induced a rapid increase in paracellular leakage that was not explained by decreases in caveolin-1 or increases in activity of nitric oxide synthase. The effect required caveolin-1 but was observed in cells depleted of clathrin, indicating that it was not due to the general inhibition of endocytosis. Inhibiting transcytosis by dynamin blockade increased paracellular leakage concomitantly with the loss of cortical actin from the plasma membrane and the displacement of active Rac from the plasmalemma. Importantly, inhibition of paracellular leakage by sphingosine-1-phosphate, which activates Rac and induces cortical actin, caused a significant increase in transcytosis of albumin in vitro and in an ex vivo whole-lung model. In addition, dominant-negative Rac significantly diminished albumin uptake by endothelia. Our findings indicate that transcytosis and paracellular permeability are co-regulated through a signaling pathway linking dynamin, Rac, and actin.

Corrections

2012-03-01

In the article entitled, “SPARC Oppositely Regulates Inflammation and Fibrosis in Bleomycin-Induced Lung Damage” (Volume 179, pages 3000–3010 of the December 2011 issue of The American Journal of Pathology), several authors' affiliations were listed incorrectly. Patrizia Casalini is a member of the Molecular Targets Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, and Claudio Tripodo is a member of the Department of Health Sciences, Pathology Section, Università degli Studi di Palermo, Palermo, Italy.

Reviewer Acknowledgment

2012-03-01

The Editors gratefully acknowledge the generous assistance of the following reviewers who served The American Journal of Pathology between January 1 and December 31, 2011. Sarki A. Abdulkadir

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2012-03-01

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2012-03-01

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2012-03-01

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This Month in AJP

Inflammation-Induced Lymph Node Lymphangiogenesis Is Reversible

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Integrative Molecular Profiling Reveals Asparagine Synthetase Is a Target in Castration-Resistant Prostate Cancer

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Chemokine Receptor CCR1 Disruption Limits Renal Damage in a Murine Model of Hemolytic Uremic Syndrome

p47phox Directs Murine Macrophage Cell Fate Decisions

Glucocorticoid-Induced Tumor Necrosis Factor Receptor Family-Related Protein Exacerbates Collagen-Induced Arthritis by Enhancing the Expansion of Th17 Cells

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Interspecies Comparison of Human and Murine Scleroderma Reveals IL-13 and CCL2 as Disease Subset-Specific Targets

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Neural Stem Cell Depletion and CNS Developmental Defects After Enteroviral Infection

Growth Defects and Impaired Cognitive–Behavioral Abilities in Mice with Knockout for Eif4h, a Gene Located in the Mouse Homolog of the Williams-Beuren Syndrome Critical Region

Platelet-Derived Growth Factor C Deficiency in C57BL/6 Mice Leads to Abnormal Cerebral Vascularization, Loss of Neuroependymal Integrity, and Ventricular Abnormalities

Collagen VI Ablation Retards Brain Tumor Progression Due to Deficits in Assembly of the Vascular Basal Lamina

ALDH1-Bright Epithelial Ovarian Cancer Cells Are Associated with CD44 Expression, Drug Resistance, and Poor Clinical Outcome

Clinical and Biological Significance of KISS1 Expression in Prostate Cancer

Mutant BRAF Induces DNA Strand Breaks, Activates DNA Damage Response Pathway, and Up-Regulates Glucose Transporter-1 in Nontransformed Epithelial Cells

Role of ERRF, a Novel ER-Related Nuclear Factor, in the Growth Control of ER-Positive Human Breast Cancer Cells

The Nonreceptor-Type Tyrosine Phosphatase PTPN13 Is a Tumor Suppressor Gene in Non–Small Cell Lung Cancer

CXCR6 and CCR5 Localize T Lymphocyte Subsets in Nasopharyngeal Carcinoma

High Anaplastic Lymphoma Kinase Immunohistochemical Staining in Neuroblastoma and Ganglioneuroblastoma Is an Independent Predictor of Poor Outcome

Plexin-B1 and Semaphorin 4D Cooperate to Promote Perineural Invasion in a RhoA/ROK-Dependent Manner

VEGF-Mediated STAT3 Activation Inhibits Retinal Vascularization by Down-Regulating Local Erythropoietin Expression

Deficiency of Interleukin-1 Receptor Antagonist Promotes Spontaneous Femoral Artery Aneurysm Formation in Mice

Chlamydophila pneumoniae Infection Induces Alterations in Vascular Contractile Responses

Promotion of Lymphatic Integrity by Angiopoietin-1/Tie2 Signaling during Inflammation

Tumor Endothelial Cells Acquire Drug Resistance by MDR1 Up-Regulation via VEGF Signaling in Tumor Microenvironment

Heterogeneity of Tumor Endothelial Cells: Comparison between Tumor Endothelial Cells Isolated from High- and Low-Metastatic Tumors

Co-Regulation of Transcellular and Paracellular Leak Across Microvascular Endothelium by Dynamin and Rac

Corrections

Reviewer Acknowledgment

Instructions to Authors

Editorial Board

Table of Contents

Scientific Integrity Policy