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RATIONALE - Atherosclerosis is, in part, caused by immune and inflammatory cell infiltration into the vascular wall, leading to enhanced inflammation and lipid accumulation in the aortic endothelium. Understanding the molecular mechanisms underlying this disease is critical for the development of new therapies. Our recent studies demonstrate that epsins, a family of ubiquitin-binding endocytic adaptors, are critical regulators of atherogenicity. Given the fundamental contribution lesion macrophages make to fuel atherosclerosis, whether and how myeloid-specific epsins promote atherogenesis is an open and significant question.
OBJECTIVE - We will determine the role of myeloid-specific epsins in regulating lesion macrophage function during atherosclerosis.
METHODS AND RESULTS - We engineered myeloid cell-specific epsins double knockout mice (LysM-DKO) on an ApoE background. On Western diet, these mice exhibited marked decrease in atherosclerotic lesion formation, diminished immune and inflammatory cell content in aortas, and reduced necrotic core content but increased smooth muscle cell content in aortic root sections. Epsins deficiency hindered foam cell formation and suppressed proinflammatory macrophage phenotype but increased efferocytosis and anti-inflammatory macrophage phenotype in primary macrophages. Mechanistically, we show that epsin loss specifically increased total and surface levels of LRP-1 (LDLR [low-density lipoprotein receptor]-related protein 1), an efferocytosis receptor with antiatherosclerotic properties. We further show that epsin and LRP-1 interact via epsin's ubiquitin-interacting motif domain. ox-LDL (oxidized LDL) treatment increased LRP-1 ubiquitination, subsequent binding to epsin, and its internalization from the cell surface, suggesting that epsins promote the ubiquitin-dependent internalization and downregulation of LRP-1. Crossing ApoE/LysM-DKO mice onto an LRP-1 heterozygous background restored, in part, atherosclerosis, suggesting that epsin-mediated LRP-1 downregulation in macrophages plays a pivotal role in propelling atherogenesis.
CONCLUSIONS - Myeloid epsins promote atherogenesis by facilitating proinflammatory macrophage recruitment and inhibiting efferocytosis in part by downregulating LRP-1, implicating that targeting epsins in macrophages may serve as a novel therapeutic strategy to treat atherosclerosis.

Infection with Helicobacter pylori is one of the strongest risk factors for development of gastric cancer. Although these bacteria infect approximately half of the world's population, only a small fraction of infected individuals develops gastric malignancies. Interactions between host and bacterial virulence factors are complex and interrelated, making it difficult to elucidate specific processes associated with H. pylori-induced tumorigenesis. In this study, we found that H. pylori inhibits p14ARF tumor suppressor by inducing its degradation. This effect was found to be strain-specific. Downregulation of p14ARF induced by H. pylori leads to inhibition of autophagy in a p53-independent manner in infected cells. We identified TRIP12 protein as E3 ubiquitin ligase that is upregulated by H. pylori, inducing ubiquitination and subsequent degradation of p14ARF protein. Using isogenic H. pylori mutants, we found that induction of TRIP12 is mediated by bacterial virulence factor CagA. Increased expression of TRIP12 protein was found in infected gastric epithelial cells in vitro and human gastric mucosa of H. pylori-infected individuals. In conclusion, our data demonstrate a new mechanism of ARF inhibition that may affect host-bacteria interactions and facilitate tumorigenic transformation in the stomach.

Neuroinflammation is consistently found in many neurological disorders, but whether or not the inflammatory response independently affects neuronal network properties is poorly understood. Here, we report that intracerebroventricular injection of the prototypical inflammatory molecule lipopolysaccharide (LPS) in rats triggered a strong and long-lasting inflammatory response in hippocampal microglia associated with a concomitant upregulation of Toll-like receptor (TLR4) in pyramidal and hilar neurons. This, in turn, was associated with a significant reduction of the dendritic hyperpolarization-activated cyclic AMP-gated channel type 1 (HCN1) protein level while Kv4.2 channels were unaltered as assessed by western blot. Immunohistochemistry confirmed the HCN1 decrease in CA1 pyramidal neurons and showed that these changes were associated with a reduction of TRIP8b, an auxiliary subunit for HCN channels implicated in channel subcellular localization and trafficking. At the physiological level, this effect translated into a 50% decrease in HCN1-mediated currents (I) measured in the distal dendrites of hippocampal CA1 pyramidal cells. At the functional level, the band-pass-filtering properties of dendrites in the theta frequency range (4-12 Hz) and their temporal summation properties were compromised. We conclude that neuroinflammation can independently trigger an acquired channelopathy in CA1 pyramidal cell dendrites that alters their integrative properties. By directly changing cellular function, this phenomenon may participate in the phenotypic expression of various brain diseases.

Estrogen receptor-α positive (ERα+) breast cancer accounts for approximately 70-80% of the nearly 25,0000 new cases of breast cancer diagnosed in the US each year. Endocrine-targeted therapies (those that block ERα activity) serve as the first line of treatment in most cases. Despite the proven benefit of endocrine therapies, however, ERα+ breast tumors can develop resistance to endocrine therapy, causing disease progression or relapse, particularly in the metastatic setting. Anti-apoptotic Bcl-2 family proteins enhance breast tumor cell survival, often promoting resistance to targeted therapies, including endocrine therapies. Herein, we investigated whether blockade of anti-apoptotic Bcl-2 family proteins could sensitize luminal breast cancers to anti-estrogen treatment. We used long-term estrogen deprivation (LTED) of human ERα+ breast cancer cell lines, an established model of sustained treatment with and acquired resistance to aromatase inhibitors (AIs), in combination with Bcl-2/Bcl-xL inhibition (ABT-263), finding that ABT-263 induced only limited tumor cell killing in LTED-selected cells in culture and in vivo. Interestingly, expression and activity of the Bcl-2-related factor Mcl-1 was increased in LTED cells. Genetic Mcl-1 ablation induced apoptosis in LTED-selected cells, and potently increased their sensitivity to ABT-263. Increased expression and activity of Mcl-1 was similarly seen in clinical breast tumor specimens treated with AI + the selective estrogen receptor downregulator fulvestrant. Delivery of Mcl-1 siRNA loaded into polymeric nanoparticles (MCL1 si-NPs) decreased Mcl-1 expression in LTED-selected and fulvestrant-treated cells, increasing tumor cell death and blocking tumor cell growth. These findings suggest that Mcl-1 upregulation in response to anti-estrogen treatment enhances tumor cell survival, decreasing response to therapeutic treatments. Therefore, strategies blocking Mcl-1 expression or activity used in combination with endocrine therapies would enhance tumor cell death.

OBJECTIVE - Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-mesenchymal states and is underexpressed in epithelial malignancy. However, the functional impact of BVES loss on tumourigenesis is unknown. Here we define the in vivo role of BVES in colitis-associated cancer (CAC), its cellular function and its relevance to patients with IBD.
DESIGN - We determined promoter methylation status using an Infinium HumanMethylation450 array screen of patients with UC with and without CAC. We also measured mRNA levels in a tissue microarray consisting of normal colons and CAC samples. and wild-type mice (controls) were administered azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce tumour formation. Last, we used a yeast two-hybrid screen to identify BVES interactors and performed mechanistic studies in multiple cell lines to define how BVES reduces c-Myc levels.
RESULTS - mRNA was reduced in tumours from patients with CAC via promoter hypermethylation. Importantly, promoter hypermethylation was concurrently present in distant non-malignant-appearing mucosa. As seen in human patients, was underexpressed in experimental inflammatory carcinogenesis, and mice had increased tumour multiplicity and degree of dysplasia after AOM/DSS administration. Molecular analysis of tumours revealed Wnt activation and increased c-Myc levels. Mechanistically, we identified a new signalling pathway whereby BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, to mediate c-Myc destruction.
CONCLUSION - Loss of BVES promotes inflammatory tumourigenesis through dysregulation of Wnt signalling and the oncogene c-Myc. promoter methylation status may serve as a CAC biomarker.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

BACKGROUND AND AIMS - c-Myc is highly expressed in pancreatic multipotent progenitor cells (MPC) and in pancreatic cancer. The transition from MPC to unipotent acinar progenitors is associated with c-Myc downregulation; a role for c-Myc in this process, and its possible relationship to a role in cancer, has not been established.
DESIGN - Using coimmunoprecipitation assays, we demonstrate that c-Myc and Ptf1a interact. Using reverse transcriptase qPCR, western blot and immunofluorescence, we show the erosion of the acinar programme. To analyse the genomic distribution of c-Myc and Ptf1a and the global transcriptomic profile, we used ChIP-seq and RNA-seq, respectively; validation was performed with ChIP-qPCR and RT-qPCR. Lineage-tracing experiments were used to follow the effect of c-Myc overexpression in preacinar cells on acinar differentiation.
RESULTS - c-Myc binds and represses the transcriptional activity of Ptf1a c-Myc overexpression in preacinar cells leads to a massive erosion of differentiation. In adult mice: (1) c-Myc binds to Ptf1a, and Tcf3 is downregulated; (2) Ptf1a and c-Myc display partially overlapping chromatin occupancy but do not bind the same E-boxes; (3) at the proximal promoter of genes coding for digestive enzymes, we find reduced PTF1 binding and increased levels of repressive chromatin marks and PRC2 complex components. Lineage tracing of committed acinar precursors reveals that c-Myc overexpression does not restore multipotency but allows the persistence of a preacinar-like cell population. In addition, mutant KRas can lead to c-Myc overexpression and acinar dysregulation.
CONCLUSIONS - c-Myc repression during development is crucial for the maturation of preacinar cells, and c-Myc overexpression can contribute to pancreatic carcinogenesis through the induction of a dedifferentiated state.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

The objective of the present study was to assess the effect of elevating epoxygenated fatty acids on retinal vascular inflammation. To stimulate inflammation we utilized TNFα, a potent pro-inflammatory mediator that is elevated in the serum and vitreous of diabetic patients. In TNFα-stimulated primary human retinal microvascular endothelial cells, total levels of epoxyeicosatrienoic acids (EETs), but not epoxydocosapentaenoic acids (EDPs), were significantly decreased. Exogenous addition of 11,12-EET or 19,20-EDP when combined with 12-(3-adamantane-1-yl-ureido)-dodecanoic acid (AUDA), an inhibitor of epoxide hydrolysis, inhibited VCAM-1 and ICAM-1 expression and protein levels; conversely the diol product of 19,20-EDP hydrolysis, 19,20-DHDP, induced VCAM1 and ICAM1 expression. 11,12-EET and 19,20-EDP also inhibited leukocyte adherence to human retinal microvascular endothelial cell monolayers and leukostasis in an acute mouse model of retinal inflammation. Our results indicate that this inhibition may be mediated through an indirect effect on NFκB activation. This is the first study demonstrating a direct comparison of EET and EDP on vascular inflammatory endpoints, and we have confirmed a comparable efficacy from each isomer, suggesting a similar mechanism of action. Taken together, these data establish that epoxygenated fatty acid elevation will inhibit early pathology related to TNFα-induced inflammation in retinal vascular diseases.

Recent studies have shown that circular RNAs (circRNAs) are abundant, widely expressed in mammals, and can display cell-type specific expression. However, how production of circRNAs is regulated and their precise biological function remains largely unknown. To study how circRNAs might be regulated during colorectal cancer progression, we used three isogenic colon cancer cell lines that differ only in KRAS mutation status. Cellular RNAs from the parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically-matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only) were analyzed using RNA-Seq. We developed a bioinformatics pipeline to identify and evaluate circRNA candidates from RNA-Seq data. Hundreds of high-quality circRNA candidates were identified in each cell line. Remarkably, circRNAs were significantly down-regulated at a global level in DLD-1 and DKO-1 cells compared to DKs-8 cells, indicating a widespread effect of mutant KRAS on circRNA abundance. This finding was confirmed in two independent colon cancer cell lines HCT116 (KRAS mutant) and HKe3 (KRAS WT). In all three cell lines, circRNAs were also found in secreted extracellular-vesicles, and circRNAs were more abundant in exosomes than cells. Our results suggest that circRNAs may serve as promising cancer biomarkers.

Chronic inflammation contributes to the development of various forms of cancer. The polyamine catabolic enzyme spermine oxidase (SMOX) is induced in chronic inflammatory conditions, including Helicobacter pylori-associated gastritis, where its production of hydrogen peroxide contributes to DNA damage and subsequent tumorigenesis. MicroRNA expression levels are also altered in inflammatory conditions; specifically, the tumor suppressor miR-124 becomes silenced by DNA methylation. We sought to determine if this repression of miR-124 is associated with elevated SMOX activity and concluded that miR-124 is indeed a negative regulator of SMOX. In gastric adenocarcinoma cells harboring highly methylated and silenced mir-124 gene loci, 5-azacytidine treatment allowed miR-124 re-expression and decreased SMOX expression. Overexpression of an exogenous miR-124-3p mimic repressed SMOX mRNA and protein expression as well as HO production by >50% within 24 h. Reporter assays indicated that direct interaction of miR-124 with the 3'-untranslated region of SMOX mRNA contributes to this negative regulation. Importantly, overexpression of miR-124 before infection with H. pylori prevented the induction of SMOX believed to contribute to inflammation-associated tumorigenesis. Compelling human in vivo data from H. pylori-positive gastritis tissues indicated that the mir-124 gene loci are more heavily methylated in a Colombian population characterized by elevated SMOX expression and a high risk for gastric cancer. Furthermore, the degree of mir-124 methylation significantly correlated with SMOX expression throughout the population. These results indicate a protective role for miR-124 through the inhibition of SMOX-mediated DNA damage in the etiology of H. pylori-associated gastric cancer.

BACKGROUND - Cardiovascular disease burden and treatment patterns among patients with familial hypercholesterolemia (FH) in the United States remain poorly described. In 2013, the FH Foundation launched the Cascade Screening for Awareness and Detection (CASCADE) of FH Registry to address this knowledge gap.
METHODS AND RESULTS - We conducted a cross-sectional analysis of 1295 adults with heterozygous FH enrolled in the CASCADE-FH Registry from 11 US lipid clinics. Median age at initiation of lipid-lowering therapy was 39 years, and median age at FH diagnosis was 47 years. Prevalent coronary heart disease was reported in 36% of patients, and 61% exhibited 1 or more modifiable risk factors. Median untreated low-density lipoprotein cholesterol (LDL-C) was 239 mg/dL. At enrollment, median LDL-C was 141 mg/dL; 42% of patients were taking high-intensity statin therapy and 45% received >1 LDL-lowering medication. Among FH patients receiving LDL-lowering medication(s), 25% achieved an LDL-C <100 mg/dL and 41% achieved a ≥50% LDL-C reduction. Factors associated with prevalent coronary heart disease included diabetes mellitus (adjusted odds ratio 1.74; 95% confidence interval 1.08-2.82) and hypertension (2.48; 1.92-3.21). Factors associated with a ≥50% LDL-C reduction from untreated levels included high-intensity statin use (7.33; 1.86-28.86) and use of >1 LDL-lowering medication (1.80; 1.34-2.41).
CONCLUSIONS - FH patients in the CASCADE-FH Registry are diagnosed late in life and often do not achieve adequate LDL-C lowering, despite a high prevalence of coronary heart disease and risk factors. These findings highlight the need for earlier diagnosis of FH and initiation of lipid-lowering therapy, more consistent use of guideline-recommended LDL-lowering therapy, and comprehensive management of traditional coronary heart disease risk factors.
© 2016 American Heart Association, Inc.