Spatial Metabolomics Identifies LPC(18:0) and LPA(18:1) in Advanced Atheroma With Translation to Plasma for Cardiovascular Risk Estimation.

BACKGROUND: The metabolic alterations occurring within the arterial architecture during atherosclerosis development remain poorly understood, let alone those particular to each arterial tunica. We aimed first to identify, in a spatially resolved manner, the specific metabolic changes in plaque, media, adventitia, and cardiac tissue between control and atherosclerotic murine aortas. Second, we assessed their translatability to human tissue and plasma for cardiovascular risk estimation. METHODS: In this observational study, mass spectrometry imaging (MSI) was applied to identify region-specific metabolic differences between atherosclerotic (n=11) and control (n=11) aortas from low-density lipoprotein receptor-deficient mice, via histology-guided virtual microdissection. Early and advanced plaques were compared within the same atherosclerotic animals. Progression metabolites were further analyzed by MSI in 9 human atherosclerotic carotids and by targeted mass spectrometry in human plasma from subjects with elective coronary artery bypass grafting (cardiovascular risk group, n=27) and a control group (n=27). RESULTS: MSI identified 362 local metabolic alterations in atherosclerotic mice (log2 fold-change ≥1.5; P≤0.05). The lipid composition of cardiac tissue is altered during atherosclerosis development and presents a generalized accumulation of glycerophospholipids, except for lysolipids. Lysolipids (among other glycerophospholipids) were found at elevated levels in all 3 arterial layers of atherosclerotic aortas. LPC(18:0) (lysophosphatidylcholine; P=0.024) and LPA(18:1) (lysophosphatidic acid; P=0.025) were found to be significantly elevated in advanced plaques as compared with mouse-matched early plaques. Higher levels of both lipid species were also observed in fibrosis-rich areas of advanced- versus early-stage human samples. They were found to be significantly reduced in human plasma from subjects with elective coronary artery bypass grafting (P<0.001 and P=0.031, respectively), with LPC(18:0) showing significant association with cardiovascular risk (odds ratio, 0.479 [95% CI, 0.225-0.883]; P=0.032) and diagnostic potential (area under the curve, 0.778 [95% CI, 0.638-0.917]). CONCLUSIONS: An altered phospholipid metabolism occurs in atherosclerosis, affecting both the aorta and the adjacent heart tissue. Plaque-progression lipids LPC(18:0) and LPA(18:1), as identified by MSI on tissue, reflect cardiovascular risk in human plasma.

NKG2A genetic deletion promotes human primary NK cell anti-tumor responses better than an anti-NKG2A monoclonal antibody.

Natural killer (NK) cells eliminate infected or cancer cells via their cytotoxic capacity. NKG2A is an inhibitory receptor on NK cells and cancer cells often overexpress its ligand HLA-E to evade NK cell surveillance. Given the successes of immune checkpoint blockade in cancer therapy, NKG2A is an interesting novel target. However, anti-NKG2A antibodies have shown limited clinical response. In the pursuit of enhancing NK cell-mediated anti-tumor responses, we devised a Cas9-based strategy to delete KLRC1, encoding NKG2A, in human primary NK cells. Our approach involved electroporation of KLRC1-targeting Cas9 ribonucleoprotein resulting in effective ablation of NKG2A expression. Compared with anti-NKG2A antibody blockade, NKG2AKO NK cells exhibited enhanced activation, reduced suppressive signaling, and elevated expression of key transcription factors. NKG2AKO NK cells overcame inhibition from HLA-E, significantly boosting NK cell activity against solid and hematologic cancer cells. We validated this efficacy across multiple cell lines, a xenograft mouse model, and primary human leukemic cells. Combining NKG2A knockout with antibody coating of tumor cells further enhanced cytotoxicity through ADCC. Thus, we provide a comprehensive comparison of inhibition of the NKG2A pathway using genetic ablation and antibodies and provide novel insight in the observed differences in molecular mechanisms, which can be translated to enhance adoptive NK cell immunotherapy.

Protective role of chaperone-mediated autophagy against atherosclerosis.

Chaperone-mediated autophagy (CMA) contributes to regulation of energy homeostasis by timely degradation of enzymes involved in glucose and lipid metabolism. Here, we report reduced CMA activity in vascular smooth muscle cells and macrophages in murine and human arteries in response to atherosclerotic challenges. We show that in vivo genetic blockage of CMA worsens atherosclerotic pathology through both systemic and cell-autonomous changes in vascular smooth muscle cells and macrophages, the two main cell types involved in atherogenesis. CMA deficiency promotes dedifferentiation of vascular smooth muscle cells and a proinflammatory state in macrophages. Conversely, a genetic mouse model with up-regulated CMA shows lower vulnerability to proatherosclerotic challenges. We propose that CMA could be an attractive therapeutic target against cardiovascular diseases.

Neuronal distribution in colorectal cancer: associations with clinicopathological parameters and survival.

Over the last years, insights in the cancer neuroscience field increased rapidly and a potential role for neurons in colorectal carcinogenesis has been recognized. However, knowledge on the neuronal distribution, subtypes, origin and associations with clinicopathological characteristics in human studies is sparse. In this study, colorectal tumor tissues from the Netherlands Cohort Study on diet and cancer (n=490) and an in-cohort validation population (n=529) were immunohistochemically stained for the pan-neuronal markers neurofilament (NF) and protein gene product 9.5 (PGP9.5) to study the association between neuronal marker expression and clinicopathological characteristics. In addition, tumor and healthy colon tissue were stained for neuronal subtype markers and their immunoreactivity in colorectal cancer (CRC) stroma was analyzed. NF and PGP9.5 positive nerve fibers were found within the tumor stroma and were mostly characterized by the neuronal subtype markers vasoactive intestinal protein (VIP) and neuronal nitric oxide synthase (nNOS), suggesting that inhibitory neurons are the most prominent neuronal subtype in CRC. NF and PGP9.5 protein expression were not consistently associated with tumor stage, sublocation, differentiation grade and median survival. NF immunoreactivity was associated with a worse CRC-specific survival in the study cohort (p=0.025), independent of other prognostic factors (HR=2.31; 95% CI 1.33-4.03; p=0.003), but these results were not observed in the in-cohort validation group. PGP9.5 on the other hand, was associated with a worse CRC-specific survival in the in-cohort validation (p=0.046) but not in the study population. This effect disappeared in multivariate analyses (HR=0.81; 95% CI 0.50-1.32; p=0.393) indicating that this effect was dependent on other prognostic factors. This study demonstrates that the tumor stroma of CRC patients mainly harbors inhibitory neurons and that NF as a single marker is significantly associated with a poorer CRC-specific survival in the study cohort but necessitates future validation.

M6229 Protects against Extracellular-Histone-Induced Liver Injury, Kidney Dysfunction, and Mortality in a Rat Model of Acute Hyperinflammation.

Extracellular histones have been shown to act as DAMPs in a variety of inflammatory diseases. Moreover, they have the ability to induce cell death. In this study, we show that M6229, a low-anticoagulant fraction of unfractionated heparin (UFH), rescues rats that were challenged by continuous infusion of calf thymus histones at a rate of 25 mg histones/kg/h. Histone infusion by itself induced hepatic and homeostatic dysfunction characterized by elevated activity of hepatic enzymes (ASAT and ALAT) and serum lactate levels as well as by a renal dysfunction, which contributed to the significantly increased mortality rate. M6229 was able to restore normal levels of both hepatic and renal parameters at 3 and 9 mg M6229/kg/h and prevented mortality of the animals. We conclude that M6229 is a promising therapeutic agent to treat histone-mediated disease.

Transcription factor NKX2-1 drives serine and glycine synthesis addiction in cancer.

BACKGROUND: One-third of cancers activate endogenous synthesis of serine/glycine, and can become addicted to this pathway to sustain proliferation and survival. Mechanisms driving this metabolic rewiring remain largely unknown. METHODS: NKX2-1 overexpressing and NKX2-1 knockdown/knockout T-cell leukaemia and lung cancer cell line models were established to study metabolic rewiring using ChIP-qPCR, immunoblotting, mass spectrometry, and proliferation and invasion assays. Findings and therapeutic relevance were validated in mouse models and confirmed in patient datasets. RESULTS: Exploring T-cell leukaemia, lung cancer and neuroendocrine prostate cancer patient datasets highlighted the transcription factor NKX2-1 as putative driver of serine/glycine metabolism. We demonstrate that transcription factor NKX2-1 binds and transcriptionally upregulates serine/glycine synthesis enzyme genes, enabling NKX2-1 expressing cells to proliferate and invade in serine/glycine-depleted conditions. NKX2-1 driven serine/glycine synthesis generates nucleotides and redox molecules, and is associated with an altered cellular lipidome and methylome. Accordingly, NKX2-1 tumour-bearing mice display enhanced tumour aggressiveness associated with systemic metabolic rewiring. Therapeutically, NKX2-1-expressing cancer cells are more sensitive to serine/glycine conversion inhibition by repurposed anti-depressant sertraline, and to etoposide chemotherapy. CONCLUSION: Collectively, we identify NKX2-1 as a novel transcriptional regulator of serine/glycine synthesis addiction across cancers, revealing a therapeutic vulnerability of NKX2-1-driven cancers. Transcription factor NKX2-1 fuels cancer cell proliferation and survival by hyperactivating serine/glycine synthesis, highlighting this pathway as a novel therapeutic target in NKX2-1-positive cancers.

Adipocytes control hematopoiesis and inflammation through CD40 signaling.

The co-stimulatory CD40-CD40L dyad plays an important role in chronic inflammatory diseases associated with aging. Although CD40 is mainly expressed by immune cells, CD40 is also present on adipocytes. We aimed to delineate the role of adipocyte CD40 in the aging hematopoietic system and evaluated the effects of adipocyte CD40 deficiency on cardiometabolic diseases. Adult adipocyte CD40-deficient mice (AdiCD40KO) mice had a decrease in bone marrow hematopoietic stem cells (Lin-Sca+cKit+, LSK) and common lymphoid progenitors, which was associated with increased bone marrow adiposity and T-cell activation, along with elevated plasma corticosterone levels, a phenotype that became more pronounced with age. Atherosclerotic AdiCD40koApoE-/- (CD40AKO) mice also displayed changes in the LSK population, showing increased myeloid and lymphoid multipotent progenitors, and augmented corticosterone levels. Increased T-cell activation could be observed in bone marrow, spleen, and adipose tissue, while the numbers of B cells were decreased. Although atherosclerosis was reduced in CD40AKO mice, plaques contained more activated T cells and larger necrotic cores. Analysis of peripheral adipose tissue in a diet-induced model of obesity revealed that obese AdiCD40KO mice had increased T-cell activation in adipose tissue and lymphoid organs, but decreased weight gain and improved insulin sensitivity, along with increased fat oxidation. In conclusion, adipocyte CD40 plays an important role in maintaining immune cell homeostasis in bone marrow during aging and chronic inflammatory diseases, particularly of the lymphoid populations. Although adipocyte CD40 deficiency reduces atherosclerosis burden and ameliorates diet-induced obesity, the accompanying T-cell activation may eventually aggravate cardiometabolic diseases.

CARMN Loss Regulates Smooth Muscle Cells and Accelerates Atherosclerosis in Mice.

[Figure: see text].

Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2.

The N-Myc Downstream-Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4-/- ) CRC models and an indirect co-culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4-/- ENS cell secretome, which is enriched for Nidogen-1 (Nid1) and Fibulin-2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS-derived Nidogen-1 and Fibulin-2 enhance colorectal carcinogenesis.

A Proinflammatory Gut Microbiota Increases Systemic Inflammation and Accelerates Atherosclerosis.

RATIONALE: Several studies have suggested a role for the gut microbiota in inflammation and atherogenesis. A causal relation relationship between gut microbiota, inflammation, and atherosclerosis has not been explored previously. OBJECTIVE: Here, we investigated whether a proinflammatory microbiota from Caspase1-/- ( Casp1-/-) mice accelerates atherogenesis in Ldlr-/- mice. METHOD AND RESULTS: We treated female Ldlr-/- mice with antibiotics and subsequently transplanted them with fecal microbiota from Casp1-/- mice based on a cohousing approach. Autologous transplantation of fecal microbiota of Ldlr-/- mice served as control. Mice were cohoused for 8 or 13 weeks and fed chow or high-fat cholesterol-rich diet. Fecal samples were collected, and factors related to inflammation, metabolism, intestinal health, and atherosclerotic phenotypes were measured. Unweighted Unifrac distances of 16S rDNA (ribosomal DNA) sequences confirmed the introduction of the Casp1-/- and Ldlr-/- microbiota into Ldlr-/- mice (referred to as Ldlr-/-( Casp1-/-) or Ldlr-/-( Ldlr-/-) mice). Analysis of atherosclerotic lesion size in the aortic root demonstrated a significant 29% increase in plaque size in 13-week high-fat cholesterol-fed Ldlr-/-( Casp1-/-) mice compared with Ldlr-/-( Ldlr-/-) mice. We found increased numbers of circulating monocytes and neutrophils and elevated proinflammatory cytokine levels in plasma in high-fat cholesterol-fed Ldlr-/-( Casp1-/-) compared with Ldlr-/-( Ldlr-/-) mice. Neutrophil accumulation in the aortic root of Ldlr-/-( Casp1-/-) mice was enhanced compared with Ldlr-/-( Ldlr-/-) mice. 16S-rDNA-encoding sequence analysis in feces identified a significant reduction in the short-chain fatty acid-producing taxonomies Akkermansia, Christensenellaceae, Clostridium, and Odoribacter in Ldlr-/-( Casp1-/-) mice. Consistent with these findings, cumulative concentrations of the anti-inflammatory short-chain fatty acids propionate, acetate and butyrate in the cecum were significantly reduced in 13-week high-fat cholesterol-fed Ldlr-/-( Casp1-/-) compared with Ldlr-/-( Ldlr-/-) mice. CONCLUSIONS: Introduction of the proinflammatory Casp1-/- microbiota into Ldlr-/- mice enhances systemic inflammation and accelerates atherogenesis.

Atherothrombosis model by silencing of protein C in APOE*3-Leiden.CETP transgenic mice.

Murine atherosclerosis models are key for investigation of atherosclerosis pathophysiology and drug development. However, they do not feature spontaneous atherothrombosis as a final stage of atherosclerosis. Transgenic mice expressing both the human mutant apolipoprotein E form APOE*3-Leiden and human cholesteryl ester transfer protein (CETP), i.e. APOE*3-Leiden.CETP mice, feature a moderate hyperlipoproteinemia and atherosclerosis phenotype. In contrast to apolipoprotein E deficient (Apoe-/-) mice, APOE*3-Leiden.CETP mice respond well to lipid-lowering and anti-atherosclerotic drugs. The aim of the study was to investigate whether silencing of anticoagulant Protein C (Proc) allows APOE*3-Leiden.CETP mice to feature thrombosis as a final stage of atherosclerosis. Female APOE*3-Leiden.CETP mice were fed a Western-type diet to induce advanced atherosclerosis, followed by an injection with a small interfering RNA targeting Proc (siProc). Presence of atherosclerosis and atherothrombosis was determined by histologic analysis of the aortic root. Atherosclerosis severity in the aortic root area of APOE*3-Leiden.CETP mice varied from type "0" (no lesions) to type "V" lesions (advanced and complex lesions). Atherothrombosis following siProc injection was observed for 4 out of 21 APOE*3-Leiden.CETP mice (19% incidence). The atherothrombosis presented as large, organized, fibrin- and leukocyte-rich thrombi on top of advanced (type "V") atherosclerotic plaques in the aortic root. This atherothrombosis was comparable in appearance and incidence as previously reported for Apoe-/- mice with a more severe atherosclerosis (19% incidence). APOE*3-Leiden.CETP mice with modest hyperlipidemia and atherosclerosis can develop atherothrombosis upon transient Proc-silencing. This further extends the use of these mice as a test model for lipid-lowering and anti-atherosclerotic drugs.

Inhibition of atherogenesis by the COP9 signalosome subunit 5 in vivo.

Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neural precursor cell-expressed, developmentally down-regulated 8 (NEDD8) moieties from cullins (thus termed "deNEDDylase") and a subunit of the cullin-RING E3 ligase-regulating COP9 signalosome complex, attenuates proinflammatory NF-κB signaling. We previously showed that CSN5 is up-regulated in human atherosclerotic arteries. Here, we investigated the role of CSN5 in atherogenesis in vivo by using mice with myeloid-specific Csn5 deletion. Genetic deletion of Csn5 in Apoe-/- mice markedly exacerbated atherosclerotic lesion formation. This was broadly observed in aortic root, arch, and total aorta of male mice, whereas the effect was less pronounced and site-specific in females. Mechanistically, Csn5 KO potentiated NF-κB signaling and proinflammatory cytokine expression in macrophages, whereas HIF-1α levels were reduced. Inversely, inhibition of NEDDylation by MLN4924 blocked proinflammatory gene expression and NF-κB activation while enhancing HIF-1α levels and the expression of M2 marker Arginase 1 in inflammatory-elicited macrophages. MLN4924 further attenuated the expression of chemokines and adhesion molecules in endothelial cells and reduced NF-κB activation and monocyte arrest on activated endothelium in vitro. In vivo, MLN4924 reduced LPS-induced inflammation, favored an antiinflammatory macrophage phenotype, and decreased the progression of early atherosclerotic lesions in mice. On the contrary, MLN4924 treatment increased neutrophil and monocyte counts in blood and had no net effect on the progression of more advanced lesions. Our data show that CSN5 is atheroprotective. We conclude that MLN4924 may be useful in preventing early atherogenesis, whereas selectively promoting CSN5-mediated deNEDDylation may be beneficial in all stages of atherosclerosis.

Deficiency of the T cell regulator Casitas B-cell lymphoma-B aggravates atherosclerosis by inducing CD8+ T cell-mediated macrophage death.

Aims: The E3-ligase CBL-B (Casitas B-cell lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis. Methods and results: The expression of CBL-B in human atherosclerotic plaques was lower in advanced lesions compared with initial lesions and correlated inversely with necrotic core area. Twenty weeks old Cblb-/-Apoe-/- mice showed a significant increase in plaque area in the aortic arch, where initial plaques were present. In the aortic root, a site containing advanced plaques, lesion area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages due to increased apoptosis, larger necrotic cores, and more CD8+ T cells. Cblb-/-Apoe-/- macrophages exhibited enhanced migration and increased cytokine production and lipid uptake. Casitas B-cell lymphoma-B deficiency increased CD8+ T cell numbers, which were protected against apoptosis and regulatory T cell-mediated suppression. IFNγ and granzyme B production was enhanced in Cblb-/-Apoe-/- CD8+ T cells, which provoked macrophage killing. Depletion of CD8+ T cells in Cblb-/-Apoe-/- bone marrow chimeras rescued the phenotype, indicating that CBL-B controls atherosclerosis mainly through its function in CD8+ T cells. Conclusion: Casitas B-cell lymphoma-B expression in human plaques decreases during the progression of atherosclerosis. As an important regulator of immune responses in experimental atherosclerosis, CBL-B hampers macrophage recruitment and activation during initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques.

Inactivation of the E3 Ubiquitin Ligase IDOL Attenuates Diet-Induced Obesity and Metabolic Dysfunction in Mice.

Objective- The E3 ubiquitin ligase IDOL (inducible degrader of the LDLR [LDL (low-density lipoprotein) receptor]) is a post-transcriptional regulator of LDLR abundance. Model systems and human genetics support a role for IDOL in regulating circulating LDL levels. Whether IDOL plays a broader metabolic role and affects development of metabolic syndrome-associated comorbidities is unknown. Approach and Results- We studied WT (wild type) and Idol(-/-) (Idol-KO) mice in 2 models: physiological aging and diet-induced obesity. In both models, deletion of Idol protected mice from metabolic dysfunction. On a Western-type diet, Idol loss resulted in decreased circulating levels of cholesterol, triglycerides, glucose, and insulin. This was accompanied by protection from weight gain in short- and long-term dietary challenges, which could be attributed to reduced hepatosteatosis and fat mass in Idol-KO mice. Although feeding and intestinal fat uptake were unchanged in Idol-KO mice, their brown adipose tissue was protected from lipid accumulation and had elevated expression of UCP1 (uncoupling protein 1) and TH (tyrosine hydroxylase). Indirect calorimetry indicated a marked increase in locomotion and suggested a trend toward increased cumulative energy expenditure and fat oxidation. An increase in in vivo clearance of reconstituted lipoprotein particles in Idol-KO mice may sustain this energetic demand. In the BXD mouse genetic reference population, hepatic Idol expression correlates with multiple metabolic parameters, thus providing support for findings in the Idol-KO mice. Conclusions- Our study uncovers an unrecognized role for Idol in regulation of whole body metabolism in physiological aging and on a Western-type diet. These findings support Idol inhibition as a therapeutic strategy to target multiple metabolic syndrome-associated comorbidities.

Constitutive CD40 Signaling in Dendritic Cells Limits Atherosclerosis by Provoking Inflammatory Bowel Disease and Ensuing Cholesterol Malabsorption.

The costimulatory molecule CD40 is a major driver of atherosclerosis. It is expressed on a wide variety of cell types, including mature dendritic cells (DCs), and is required for optimal T-cell activation and expansion. It remains undetermined whether and how CD40 on DCs impacts the pathogenesis of atherosclerosis. Here, the effects of constitutively active CD40 in DCs on atherosclerosis were examined using low-density lipoprotein-deficient (Ldlr-/-) bone marrow chimeras that express a transgene containing an engineered latent membrane protein 1 (LMP)/CD40 fusion protein conferring constitutive CD40 signaling under control of the DC-specific CD11c promoter (DC-LMP1/CD40). As expected, DC-LMP1/CD40/Ldlr-/- chimeras (DC-LMP1/CD40) showed increased antigen-presenting capacity of DCs and increased T-cell numbers. However, the mice developed extensive neutrophilia compared to CD40wt/Ldlr-/- (CD40wt) chimeras. Despite overt T-cell expansion and neutrophilia, a reduction in conventional DC frequency and a dramatic (approximately 80%) reduction in atherosclerosis was observed. Further analyses revealed that cholesterol and triglyceride levels had decreased by 37% and 60%, respectively, in DC-LMP1/CD40 chimeras. Moreover, DC-LMP1/CD40 chimeras developed inflammatory bowel disease characterized by massive transmural influx of leukocytes and lymphocytes, resulting in villous degeneration and lipid malabsorption. Constitutive activation of CD40 in DCs results in inflammation of the gastrointestinal tract, thereby impairing lipid uptake, which consequently results in attenuated atherosclerosis.

Comparability of histological outcomes in rats and humans in a hernia model.

BACKGROUND: Hernia repair is one of the most frequently performed operations. In search of the ideal mesh for hernia repair, animal research is required. Although rats are most often used in experimental mesh experiments, no correlation with clinical findings in humans has ever been shown. Therefore, the aim of our study was to investigate whether adhesion formation and foreign body reactions to meshes in rats are comparable with the reactions in humans. MATERIALS AND METHODS: A fixed type of mesh was implanted intraperitoneally in a group of 10 rats and 10 patients undergoing elective, temporary stoma formation. In case of the latter, meshes were placed around the stoma. After a follow-up period of 12 wk in rats and after a median follow-up of 6 mo in humans, samples of the mesh were collected. Adhesion assessments were performed, and (immuno-) histochemical evaluation was performed by a specialized experimental pathologist and an experienced clinical pathologist. RESULTS: After the follow-up period, adhesion formation did not differ significantly between rats and humans. Moreover, general inflammation scores were comparable, although granulocytes and giant cells were more present in rats, compared with humans. On the other hand, the presence of fibrosis was more evident in humans compared with rats. CONCLUSIONS: To our knowledge, this is the first study, which showed that a specific animal model, namely a rat model, correlates with adhesion formation and the foreign body reaction to meshes in humans. It can be recommended to use rats in future experimental mesh for incisional hernia research.

Silencing of Anticoagulant Protein C Evokes Low-Incident but Spontaneous Atherothrombosis in Apolipoprotein E-Deficient Mice-Brief Report.

OBJECTIVE: Murine atherosclerosis models do not spontaneously develop atherothrombotic complications. We investigated whether disruption of natural anticoagulation allows preexisting atherosclerotic plaques to progress toward an atherothrombotic phenotype. APPROACH AND RESULTS: On lowering of plasma protein C levels with small interfering RNA (siProc) in 8-week Western-type diet-fed atherosclerotic apolipoprotein E-deficient mice, 1 out of 4 mice displayed a large, organized, and fibrin- and leukocyte-rich thrombus on top of an advanced atherosclerotic plaque located in the aortic root. Although again at low incidence (3 in 25), comparable thrombi at the same location were observed during a second independent experiment in 9-week Western-type diet-fed apolipoprotein E-deficient mice. Mice with thrombi on their atherosclerotic plaques did not show other abnormalities and had equally lowered plasma protein C levels as siProc-treated apolipoprotein E-deficient mice without thrombi. Fibrinogen and thrombin-antithrombin concentrations and blood platelet numbers were also comparable, and plaques in siProc mice with thrombi had a similar composition and size as plaques in siProc mice without thrombi. Seven out of 25 siProc mice featured clots in the left atrium of the heart. CONCLUSIONS: Our findings indicate that small interfering RNA-mediated silencing of protein C in apolipoprotein E-deficient mice creates a condition that allows the occurrence of spontaneous atherothrombosis, albeit at a low incidence. Lowering natural anticoagulation in atherosclerosis models may help to discover factors that increase atherothrombotic complications.

Inhibition of CD40-TRAF6 interactions by the small molecule inhibitor 6877002 reduces neuroinflammation.

BACKGROUND: The influx of leukocytes into the central nervous system (CNS) is a key hallmark of the chronic neuro-inflammatory disease multiple sclerosis (MS). Strategies that aim to inhibit leukocyte migration across the blood-brain barrier (BBB) are therefore regarded as promising therapeutic approaches to combat MS. As the CD40L-CD40 dyad signals via TNF receptor-associated factor 6 (TRAF6) in myeloid cells to induce inflammation and leukocyte trafficking, we explored the hypothesis that specific inhibition of CD40-TRAF6 interactions can ameliorate neuro-inflammation. METHODS: Human monocytes were treated with a small molecule inhibitor (SMI) of CD40-TRAF6 interactions (6877002), and migration capacity across human brain endothelial cells was measured. To test the therapeutic potential of the CD40-TRAF6-blocking SMI under neuro-inflammatory conditions in vivo, Lewis rats and C57BL/6J mice were subjected to acute experimental autoimmune encephalomyelitis (EAE) and treated with SMI 6877002 for 6 days (rats) or 3 weeks (mice). RESULTS: We here show that a SMI of CD40-TRAF6 interactions (6877002) strongly and dose-dependently reduces trans-endothelial migration of human monocytes. Moreover, upon SMI treatment, monocytes displayed a decreased production of ROS, tumor necrosis factor (TNF), and interleukin (IL)-6, whereas the production of the anti-inflammatory cytokine IL-10 was increased. Disease severity of EAE was reduced upon SMI treatment in rats, but not in mice. However, a significant reduction in monocyte-derived macrophages, but not in T cells, that had infiltrated the CNS was eminent in both models. CONCLUSIONS: Together, our results indicate that SMI-mediated inhibition of the CD40-TRAF6 pathway skews human monocytes towards anti-inflammatory cells with reduced trans-endothelial migration capacity, and is able to reduce CNS-infiltrated monocyte-derived macrophages during neuro-inflammation, but minimally ameliorates EAE disease severity. We therefore conclude that SMI-mediated inhibition of the CD40-TRAF6 pathway may represent a beneficial treatment strategy to reduce monocyte recruitment and macrophage activation in the CNS and has the potential to be used as a co-treatment to combat MS.

Low-Dose Lipopolysaccharide Causes Biliary Injury by Blood Biliary Barrier Impairment in a Rat Hepatic Ischemia/Reperfusion Model.

This study explored whether bacterial endotoxins, in the form of lipopolysaccharides (LPS), could have an injurious effect on the biliary tract in conjunction with ischemia. A total of 64 rats were randomly assigned to 4 groups: sham operation (sham group), 1 mg/kg LPS intraperitoneal (LPS group), hepatic ischemia/reperfusion (IR; IR group), and IR combined with LPS (IR+LPS group). Following 1 or 6 hours of reperfusion, serum liver tests, bile duct histology, immunofluorescence microscopy (zonula occludens-1 [ZO-1]), bile composition (bile salts, phospholipids, lactate dehydrogenase), hepatic gene expression (bile salt transporters and inflammatory mediators), as well as serum and biliary cytokine concentrations were quantified and compared between the study groups. In addition, the integrity of the blood biliary barrier (BBB) was assayed in vivo using horseradish peroxidase (HRP). LPS administration induced severe small bile duct injury following 6 hours of reperfusion. Furthermore, total bile salts and bilirubin concentrations in serum were increased in the LPS groups compared with sham controls (LPS, + 3.3-fold and +1.9-fold; IR+LPS, + 3.8-fold and +1.7-fold, respectively). The BBB was impaired in the LPS groups as evidenced by elevated levels of HRP in bile (+4.9-fold), and decreased expression of claudin 1 (-6.7-fold) and claudin 3 (-3.6-fold). LPS was found to be a potent inducer of small bile duct injury following hepatic ischemia and 6 hours of reperfusion. This injury was associated with increased permeability of the BBB and impaired hepatic bile salt clearance. Liver Transplantation 23 194-206 2017 AASLD.

Impact of tissue adhesives on the prevention of anastomotic leakage of colonic anastomoses: an in vivo study.

BACKGROUND: Tissue adhesives (TA) may be useful to strengthen colorectal anastomoses, thereby preventing anastomotic leakage (AL). Previous studies have identified cyanoacrylate (CA) TAs as the most promising colonic anastomotic sealants. This study investigates the protective effects of sealing colonic anastomoses with various CAs. MATERIALS AND METHODS: Fifty-five Wistar rats underwent laparotomy and transection of the proximal colon. An anastomosis was created with 4 interrupted sutures followed by either application of Histoacryl Flexible, Omnex, Glubran 2, or no TA seal. An additional control group was included with a 12-suture anastomosis and no TA seal. After 7 days, the rats were sacrificed and scored for the presence of AL as the main outcome. Secondary outcomes were the occurrence of bowel obstruction, adhesions, and anastomotic bursting pressure. Histological evaluation was performed. RESULTS: The highest AL rate was found in the Glubran 2 group (7/11), followed by the 4-sutures group without TA (5/11), and the Omnex group (5/11). Histoacryl Flexible showed the lowest AL rate (2/11). In the control group, only one rat showed signs of AL. Histologically, the highest influx of inflammatory cells was found in the 4-suture group without TA and for Omnex and Glubran 2. Histoacryl Flexible caused more mature collagen deposition when compared to the other TA groups. CONCLUSIONS: Histoacryl Flexible showed the lowest leakage rate compared to the other TA groups and to the 4-suture control group. Glubran 2 showed the highest AL rate and a high inflammatory response. Histoacryl Flexible was associated with the presence of more mature collagen and seems to promote anastomotic healing.