Histology and transcriptome insights into the early processes of intestinal anastomotic healing: a rat model.

BACKGROUND: Understanding the early processes underlying intestinal anastomotic healing is crucial to comprehend the pathophysiology of anastomotic leakage. The aim of this study was to assess normal intestinal anastomotic healing and disturbed healing in rats to investigate morphological, cellular and intrinsic molecular changes in the anastomotic tissue. METHOD: Anastomoses were created in two groups of Wistar rats, using four sutures or 12 sutures to mimic anastomotic leakage and anastomotic healing respectively. At 6, 12, 24 hours and 2, 3, 5 and 7 days, anastomotic tissue was assessed macroscopically using the anastomotic complication score and histologically using the modified Ehrlich-Hunt score. Transcriptome analysis was performed to assess differences between anastomotic leakage and anastomotic healing at the first three time points to find affected genes and biological processes. RESULTS: Ninety-eight rats were operated on (49 animals in the anastomotic leakage and 49 in the anastomotic healing group) and seven rats analysed at each time point. None of the animals with 12 sutures developed anastomotic leakage macroscopically, whereas 35 of the 49 animals with four sutures developed anastomotic leakage. Histological analysis showed increasing influx of inflammatory cells up to 3 days in anastomotic healing and up to 7 days in anastomotic leakage, and this increase was significantly higher in anastomotic leakage at 5 (P = 0.041) and 7 days (P = 0.003). Transcriptome analyses revealed large differences between anastomotic leakage and anastomotic healing at 6 and 24 hours, mainly driven by an overall downregulation of genes in anastomotic leakage. CONCLUSION: Transcriptomic analyses revealed large differences between normal and disturbed healing at 6 hours after surgery, which might eventually serve as early-onset biomarkers for anastomotic leakage.

Quantification of carotid plaque composition with a multi-contrast atherosclerosis characterization (MATCH) MRI sequence.

Background and purpose: Carotid atherosclerotic plaques with a large lipid-rich necrotic core (LRNC), intraplaque hemorrhage (IPH), and a thin or ruptured fibrous cap are associated with increased stroke risk. Multi-sequence MRI can be used to quantify carotid atherosclerotic plaque composition. Yet, its clinical implementation is hampered by long scan times and image misregistration. Multi-contrast atherosclerosis characterization (MATCH) overcomes these limitations. This study aims to compare the quantification of plaque composition with MATCH and multi-sequence MRI. Methods: MATCH and multi-sequence MRI were used to image 54 carotid arteries of 27 symptomatic patients with ≥2 mm carotid plaque on a 3.0 T MRI scanner. The following sequence parameters for MATCH were used: repetition time/echo time (TR/TE), 10.1/4.35 ms; field of view, 160 mm × 160 mm × 2 mm; matrix size, 256 × 256; acquired in-plane resolution, 0.63 mm2× 0.63 mm2; number of slices, 18; and flip angles, 8°, 5°, and 10°. Multi-sequence MRI (black-blood pre- and post-contrast T1-weighted, time of flight, and magnetization prepared rapid acquisition gradient echo; acquired in-plane resolution: 0.63 mm2 × 0.63 mm2) was acquired according to consensus recommendations, and image quality was scored (5-point scale). The interobserver agreement in plaque composition quantification was assessed by the intraclass correlation coefficient (ICC). The sensitivity and specificity of MATCH in identifying plaque composition were calculated using multi-sequence MRI as a reference standard. Results: A significantly lower image quality of MATCH compared to that of multi-sequence MRI was observed (p < 0.05). The scan time for MATCH was shorter (7 vs. 40 min). Interobserver agreement in quantifying plaque composition on MATCH images was good to excellent (ICC ≥ 0.77) except for the total volume of calcifications and fibrous tissue that showed moderate agreement (ICC ≥ 0.61). The sensitivity and specificity of detecting plaque components on MATCH were ≥89% and ≥91% for IPH, ≥81% and 85% for LRNC, and ≥71% and ≥32% for calcifications, respectively. Overall, good-to-excellent agreement (ICC ≥ 0.76) of quantifying plaque components on MATCH with multi-sequence MRI as the reference standard was observed except for calcifications (ICC = 0.37-0.38) and fibrous tissue (ICC = 0.59-0.70). Discussion and conclusion: MATCH images can be used to quantify plaque components such as LRNC and IPH but not for calcifications. Although MATCH images showed a lower mean image quality score, short scan time and inherent co-registration are significant advantages.

A Degradable Sustained-Release Drug Delivery System for Bleb-Forming Glaucoma Surgery.

Fibrosis of the filtering bleb is one of the main causes of failure after bleb-forming glaucoma surgery. Intraoperative application of mitomycin C (MMC) is the current gold standard to reduce the fibrotic response. However, MMC is cytotoxic and one-time application is often insufficient. A sustained-release drug delivery system (DDS), loaded with MMC, may be less cytotoxic and equally or more effective. Two degradable (polycaprolactone (PCL) and polylactic-co-glycolic acid (PLGA)) MMC-loaded DDSs are developed. Release kinetics are first assessed in vitro followed by rabbit implants in conjunction with the PRESERFLO MicroShunt. As a control, the MicroShunt is implanted with adjunctive use of a MMC solution. Rabbits are euthanized at postoperative day (POD) 28 and 90. The PLGA and PCL DDSs release (on average) 99% and 75% of MMC, respectively. All groups show functioning blebs until POD 90. Rabbits implanted with a DDS show more inflammation with avascular thin-walled blebs when compared to the control. However, collagen is more loosely arranged. The PLGA DDS shows less inflammation, less foreign body response (FBR), and more complete degradation at POD 90 when compared to the PCL DDS. Further optimization with regard to dosage is required to reduce side effects to the conjunctiva.

The Influence of Design Modifications and Microstructured Surface Topographies on Bleb Survival after Glaucoma Tube Shunt Implantation.

Lowering intraocular pressure (IOP) by placement of a glaucoma shunt is an effective treatment for glaucoma. However, fibrosis of the outflow site can hamper surgical outcome. In this study, the antifibrotic effect of adding an endplate (with or without microstructured surface topographies) to a microshunt made of poly(styrene-block-isobutylene-block-styrene) is investigated. New Zealand white rabbits are implanted with a control implant (without endplate) and modified implants. Afterward, bleb morphology and IOP is recorded for 30 days. After killing of the animals, eyes are collected for histology, Addition of an endplate extended bleb survival, Topography-990 has the longest recorded bleb-survival time. Histology reveals that the addition of an endplate increases the presence of myofibroblasts, macrophages, polymorphonuclear cells, and foreign body giant cells compared to the control. However, an increased capsule thickness and inflammatory response are observed in the groups with surface topographies, The addition of an endplate results in prolonged bleb survival, demonstrating that engineering of the shape of glaucoma implants could prolong bleb functionality. Future research should further elaborate the effect of surface topographies on long-term bleb survival, since an increased presence of pro-fibrotic cells and increased capsule thickness are observed compared to the control.

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.

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.

Pro-Inflammatory Implications of 2-Hydroxypropyl-ß-cyclodextrin Treatment.

Lifestyle- and genetically induced disorders related to disturbances in cholesterol metabolism have shown the detrimental impact of excessive cholesterol levels on a plethora of pathological processes such as inflammation. In this context, two-hydroxypropyl-ß-cyclodextrin (CD) is increasingly considered as a novel pharmacological compound to decrease cellular cholesterol levels due to its ability to increase cholesterol solubility. However, recent findings have reported contra-indicating events after the use of CD questioning the clinical applicability of this compound. Given its potential as a therapeutic compound in metabolic inflammatory diseases, in this study, we evaluated the inflammatory effects of CD administration in the context of cholesterol-induced metabolic inflammation in vivo and in vitro. The inflammatory and cholesterol-depleting effects of CD were first investigated in low-density lipoprotein receptor knockout (Ldlr-/ ) mice that were transplanted with Npc1nih or Npc1wt bone marrow and were fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks, thereby creating an extreme model of lysosomal cholesterol-induced metabolic inflammation. In the final three weeks, these mice received daily injections of either control (saline) or CD subcutaneously. Subsequently, the inflammatory properties of CD were investigated in vitro in two macrophage cell lines and in murine bone marrow-derived macrophages (BMDMs). While CD administration improved cholesterol mobilization outside lysosomes in BMDMs, an overall pro-inflammatory profile was observed after CD treatment, evidenced by increased hepatic inflammation in vivo and a strong increase in cytokine release and inflammatory gene expression in vitro in murine BMDMs and macrophages cell lines. Nevertheless, this CD-induced pro-inflammatory profile was time-dependent, as short term exposure to CD did not result in a pro-inflammatory response in BMDM. While CD exerts desired cholesterol-depleting effects, its inflammatory effect is dependent on the exposure time. As such, using CD in the clinic, especially in a metabolic inflammatory context, should be closely monitored as it may lead to undesired, pro-inflammatory side effects.

Integrative multiomics analysis of human atherosclerosis reveals a serum response factor-driven network associated with intraplaque hemorrhage.

BACKGROUND: While single-omics analyses on human atherosclerotic plaque have been very useful to map stage- or disease-related differences in expression, they only partly capture the array of changes in this tissue and suffer from scale-intrinsic limitations. In order to better identify processes associated with intraplaque hemorrhage and plaque instability, we therefore combined multiple omics into an integrated model. METHODS: In this study, we compared protein and gene makeup of low- versus high-risk atherosclerotic lesion segments from carotid endarterectomy patients, as judged from the absence or presence of intraplaque hemorrhage, respectively. Transcriptomic, proteomic, and peptidomic data of this plaque cohort were aggregated and analyzed by DIABLO, an integrative multivariate classification and feature selection method. RESULTS: We identified a protein-gene associated multiomics model able to segregate stable, nonhemorrhaged from vulnerable, hemorrhaged lesions at high predictive performance (AUC >0.95). The dominant component of this model correlated with αSMA- PDGFRα+ fibroblast-like cell content (p = 2.4E-05) and Arg1+ macrophage content (p = 2.2E-04) and was driven by serum response factor (SRF), possibly in a megakaryoblastic leukemia-1/2 (MKL1/2) dependent manner. Gene set overrepresentation analysis on the selected key features of this model pointed to a clear cardiovascular disease signature, with overrepresentation of extracellular matrix synthesis and organization, focal adhesion, and cholesterol metabolism terms, suggestive of the model's relevance for the plaque vulnerability. Finally, we were able to corroborate the predictive power of the selected features in several independent mRNA and proteomic plaque cohorts. CONCLUSIONS: In conclusion, our integrative omics study has identified an intraplaque hemorrhage-associated cardiovascular signature that provides excellent stratification of low- from high-risk carotid artery plaques in several independent cohorts. Further study revealed suppression of an SRF-regulated disease network, controlling lesion stability, in vulnerable plaque, which can serve as a scaffold for the design of targeted intervention in plaque destabilization.

Anti-Inflammatory Effects of Dietary Plant Stanol Supplementation Are Largely Dependent on the Intake of Cholesterol in a Mouse Model of Metabolic Inflammation.

The prevalence of metabolic disorders characterized by chronic inflammation has been on a sharp rise for decades. As such, tools that address metabolic and inflammatory dysregulation are of great importance. Plant stanols are well-known for reducing intestinal cholesterol absorption and may also have direct anti-inflammatory effects. In this study, our aim was to investigate to what extent the benefits of dietary plant stanol supplementation depend on dietary cholesterol intake in an experimental mouse model for cholesterol-induced metabolic inflammation. Here, we used Ldlr-/- mice transplanted with Npc1nih-derived bone marrow, featuring feature bone marrow-derived immune cells characterized by chronic inflammation induced by lysosomal lipid accumulation. Npc1nih- and Npc1wt-transplanted mice were placed on either a high fat, high cholesterol (HFC) or on a chow diet low in cholesterol, with or without 2% plant stanols supplementation. At the end of the study, the metabolic and inflammatory status of the mice was analyzed. Plant stanol supplementation to the HFC diet reduced liver cholesterol levels and improved lipid metabolism and liver inflammation, particularly in Npc1nih-tp mice. In contrast, plant stanol supplementation to the chow diet did not significantly improve the aforementioned parameters, though similar reductive trends to those in the HFC diet setting were observed regarding liver cholesterol accumulation and liver inflammatory markers. The effects of dietary plant stanol supplementation on dietary cholesterol-induced inflammation are largely dependent on dietary cholesterol intake. Future research should verify whether other models of metabolic inflammation exhibit similar stanol-related effects on inflammation.

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.

Macrophage ATP citrate lyase deficiency stabilizes atherosclerotic plaques.

Macrophages represent a major immune cell population in atherosclerotic plaques and play central role in the progression of this lipid-driven chronic inflammatory disease. Targeting immunometabolism is proposed as a strategy to revert aberrant macrophage activation to improve disease outcome. Here, we show ATP citrate lyase (Acly) to be activated in inflammatory macrophages and human atherosclerotic plaques. We demonstrate that myeloid Acly deficiency induces a stable plaque phenotype characterized by increased collagen deposition and fibrous cap thickness, along with a smaller necrotic core. In-depth functional, lipidomic, and transcriptional characterization indicate deregulated fatty acid and cholesterol biosynthesis and reduced liver X receptor activation within the macrophages in vitro. This results in macrophages that are more prone to undergo apoptosis, whilst maintaining their capacity to phagocytose apoptotic cells. Together, our results indicate that targeting macrophage metabolism improves atherosclerosis outcome and we reveal Acly as a promising therapeutic target to stabilize atherosclerotic plaques.

SIRPα on Mouse B1 Cells Restricts Lymphoid Tissue Migration and Natural Antibody Production.

The inhibitory immunoreceptor SIRPα is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRPα interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRPα on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRPα signaling (SIRPαΔCYT mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRPα signaling in atherosclerosis development. Bone marrow (SIRPαΔCYT>LDLR-/-) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRPα as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRPα as a potential therapeutic target in atherosclerosis.

Myeloid Ezh2 Deficiency Limits Atherosclerosis Development.

Macrophages define a key component of immune cells present in atherosclerotic lesions and are central regulators of the disease. Since epigenetic processes are important in controlling macrophage function, interfering with epigenetic pathways in macrophages might be a novel approach to combat atherosclerosis. Histone H3K27 trimethylation is a repressive histone mark catalyzed by polycomb repressive complex with EZH2 as the catalytic subunit. EZH2 is described to increase macrophage inflammatory responses by supressing the suppressor of cytokine signaling, Socs3. We previously showed that myeloid deletion of Kdm6b, an enzymes that in contrast to EZH2 removes repressive histone H3K27me3 marks, results in advanced atherosclerosis. Because of its opposing function and importance of EZH2 in macrophage inflammatory responses, we here studied the role of myeloid EZH2 in atherosclerosis. A myeloid-specific Ezh2 deficient mouse strain (Ezh2del) was generated (LysM-cre+ x Ezh2fl/fl) and bone marrow from Ezh2del or Ezh2wt mice was transplanted to Ldlr-/- mice which were fed a high fat diet for 9 weeks to study atherosclerosis. Atherosclerotic lesion size was significantly decreased in Ezh2del transplanted mice compared to control. The percentage of macrophages in the atherosclerotic lesion was similar, however neutrophil numbers were lower in Ezh2del transplanted mice. Correspondingly, the migratory capacity of neutrophils was decreased in Ezh2del mice. Moreover, peritoneal Ezh2del foam cells showed a reduction in the inflammatory response with reduced production of nitric oxide, IL-6 and IL-12. In Conclusion, myeloid Ezh2 deficiency impairs neutrophil migration and reduces macrophage foam cell inflammatory responses, both contributing to reduced atherosclerosis.

Peritoneal macrophages have an impaired immune response in obesity which can be reversed by subsequent weight loss.

Introduction: Obesity is recognized as a risk factor for various microbial infections. The immune system, which is affected by obesity, plays an important role in the pathophysiology of these infections and other obesity-related comorbidities. Weight loss is considered the most obvious treatment for obesity. However, multiple studies suggest that the comorbidities of obesity may persist after weight loss. Deregulation of immune cells including adipose tissue macrophages of obese individuals has been extensively studied, but how obesity and subsequent weight loss affect immune cell function outside adipose tissue is not well defined. Research design and methods: Here we investigated the phenotype of non-adipose tissue macrophages by transcriptional characterization of thioglycollate-elicited peritoneal macrophages (PM) from mice with diet-induced obesity and type 2 diabetes (T2D). Subsequently, we defined the characteristics of PMs after weight loss and mimicked a bacterial infection by exposing PMs to lipopolysaccharide. Results and conclusions: In contrast to the proinflammatory phenotype of adipose tissue macrophages in obesity and T2D, we found a deactivated state of PMs in obesity and T2D. Weight loss could reverse this deactivated macrophage phenotype. Anti-inflammatory characteristics of these non-adipose macrophages may explain why patients with obesity and T2D have an impaired immune response against pathogens. Our data also suggest that losing weight restores macrophage function and thus contributes to the reduction of immune-related comorbidities in patients.

Targeting Histone Deacetylases in Myeloid Cells Inhibits Their Maturation and Inflammatory Function With Limited Effects on Atherosclerosis.

Monocytes and macrophages are key drivers in the pathogenesis of inflammatory diseases. Epigenetic targets have been shown to control the transcriptional profile and phenotype of these cells. Since histone deacetylase protein inhibitors demonstrate profound anti-inflammatory activity, we wanted to test whether HDAC inhibition within monocytes and macrophages could be applied to suppress inflammation in vivo. ESM technology conjugates an esterase-sensitive motif (ESM) onto small molecules to allow targeting of cells that express carboxylesterase 1 (CES1), such as mononuclear myeloid cells. This study utilized an ESM-HDAC inhibitor to target monocytes and macrophages in mice in both an acute response model and an atherosclerosis model. We demonstrate that the molecule blocks the maturation of peritoneal macrophages and inhibits pro-inflammatory cytokine production in both models but to a lesser extent in the atherosclerosis model. Despite regulating the inflammatory response, ESM-HDAC528 did not significantly affect plaque size or phenotype, although histological classification of the plaques demonstrated a significant shift to a less severe phenotype. We hereby show that HDAC inhibition in myeloid cells impairs the maturation and activation of peritoneal macrophages but shows limited efficacy in a model of atherosclerosis.

Low human and murine Mcl-1 expression leads to a pro-apoptotic plaque phenotype enriched in giant-cells.

The anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) plays an important role in survival and differentiation of leukocytes, more specifically of neutrophils. Here, we investigated the impact of myeloid Mcl-1 deletion in atherosclerosis. Western type diet fed LDL receptor-deficient mice were transplanted with either wild-type (WT) or LysMCre Mcl-1fl/fl (Mcl-1-/-) bone marrow. Mcl-1 myeloid deletion resulted in enhanced apoptosis and lipid accumulation in atherosclerotic plaques. In vitro, Mcl-1 deficient macrophages also showed increased lipid accumulation, resulting in increased sensitivity to lipid-induced cell death. However, plaque size, necrotic core and macrophage content were similar in Mcl-1-/- compared to WT mice, most likely due to decreased circulating and plaque-residing neutrophils. Interestingly, Mcl-1-/- peritoneal foam cells formed up to 45% more multinucleated giant cells (MGCs) in vitro compared to WT, which concurred with an increased MGC presence in atherosclerotic lesions of Mcl-1-/- mice. Moreover, analysis of human unstable atherosclerotic lesions also revealed a significant inverse correlation between MGC lesion content and Mcl-1 gene expression, coinciding with the mouse data. Taken together, these findings suggest that myeloid Mcl-1 deletion leads to a more apoptotic, lipid and MGC-enriched phenotype. These potentially pro-atherogenic effects are however counteracted by neutropenia in circulation and plaque.

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.

Deficiency of T cell CD40L has minor beneficial effects on obesity-induced metabolic dysfunction.

Objective: Obesity-associated metabolic dysfunction increases the risk of multiple diseases such as type 2 diabetes and cardiovascular disease. The importance of the co-stimulatory CD40-CD40L dyad in diet-induced obesity (DIO), with opposing phenotypes arising when either the receptor (aggravating) or the ligand (protective) is deleted, has been described previously. The functions of CD40 and CD40L are cell type dependent. As co-stimulation via T cell-mediated CD40L is essential for driving inflammation, we here investigate the role of T cell CD40L in DIO. Research design and methods: CD4CreCD40Lfl/fl mice on a C57BL/6 background were generated and subjected to DIO by administration of 15 weeks of high fat diet (HFD). Results: HFD-fed CD4CreCD40Lfl/fl mice had similar weight gain, adipocyte sizes, plasma cholesterol and triglyceride levels as their wild-type (WT) counterparts. Insulin and glucose tolerance were comparable, although CD4CreCD40Lfl/fl mice did have a decreased plasma insulin concentration, suggesting a minor improvement of insulin resistance. Furthermore, although the degree of hepatosteatosis was similar in both genotypes, the gene expression of fatty acid synthase 1 and ATP-citrate lyase had decreased, whereas expression of peroxisome proliferator-activated receptor-α had increased in livers of CD4CreCD40Lfl/fl mice, suggesting decreased hepatic lipid uptake in absence of T cell CD40L.Moreover, CD4CreCD40Lfl/fl mice displayed significantly lower numbers of effector memory CD4+ T cells and regulatory T cells in blood and lymphoid organs compared with WT. However, immune cell composition and inflammatory status of the adipose tissue was similar in CD4CreCD40Lfl/fl and WT mice. Conclusions: T cell CD40L deficiency results in a minor improvement of insulin sensitivity and hepatic steatosis in DIO, despite the strong decrease in effector T cells and regulatory T cells in blood and lymphoid organs. Our data indicate that other CD40L-expressing cell types are more relevant in the pathogenesis of obesity-associated metabolic dysfunction.

Predilection of Low Protein C-induced Spontaneous Atherothrombosis for the Right Coronary Sinus in Apolipoprotein E deficient mice.

Silencing of anticoagulant protein C using RNA interference (siProc) evokes low incident but spontaneous atherothrombosis in the aortic root of apolipoprotein E-deficient (Apoe-/-) mice. The aims of the current study were (1) to analyze if plaque characteristics or circulating factors could be linked to atherothrombosis susceptibility, (2) to increase the incidence of atherothrombosis by transiently increasing blood pressure, and (3) to direct atherothrombosis to an additional predefined vascular site by applying a semi-constrictive collar around the carotid artery. siProc-driven spontaneous atherothrombosis in the aortic root of Apoe-/- mice was reproduced and occurred at an incidence of 23% (9 out of 39 mice), while the incidence of collar-induced atherothrombosis in the carotid artery was 2.6% (1 out of 39 mice). Treatment with phenylephrine, to transiently increase blood pressure, did not increase atherothrombosis in the aortic root of the Apoe-/- mice nor in the carotid arteries with collars. Plaques in the aortic root with an associated thrombus were lower in collagen and macrophage content, and mice with atherothrombosis had significantly more circulating platelets. Plasma protein C, white blood cell counts, total cholesterol, fibrinogen, serum amyloid A, and IL-6 were not different amongst siProc treated mice with or without thrombosis. Remarkably, our data revealed that thrombus formation preferably occurred on plaques in the right coronary sinus of the aortic root. In conclusion, there is a predilection of low protein C-induced spontaneous atherothrombosis in Apoe-/- mice for the right coronary sinus, a process that is associated with an increase in platelets and plaques lower in collagen and macrophage content.