Gut microbiota regulate hepatic von Willebrand factor synthesis and arterial thrombus formation via Toll-like receptor-2.

The symbiotic gut microbiota play pivotal roles in host physiology and the development of cardiovascular diseases, but the microbiota-triggered pattern recognition signaling mechanisms that impact thrombosis are poorly defined. In this article, we show that germ-free (GF) and Toll-like receptor-2 (Tlr2)-deficient mice have reduced thrombus growth after carotid artery injury relative to conventionally raised controls. GF Tlr2-/- and wild-type (WT) mice were indistinguishable, but colonization with microbiota restored a significant difference in thrombus growth between the genotypes. We identify reduced plasma levels of von Willebrand factor (VWF) and reduced VWF synthesis, specifically in he…

The gut microbiota instructs the hepatic endothelial cell transcriptome

Summary The gut microbiota affects remote organ functions but its impact on organotypic endothelial cell (EC) transcriptomes remains unexplored. The liver endothelium encounters microbiota-derived signals and metabolites via the portal circulation. To pinpoint how gut commensals affect the hepatic sinusoidal endothelium, a magnetic cell sorting protocol, combined with fluorescence-activated cell sorting, was used to isolate hepatic sinusoidal ECs from germ-free (GF) and conventionally raised (CONV-R) mice for transcriptome analysis by RNA sequencing. This resulted in a comprehensive map of microbiota-regulated hepatic EC-specific transcriptome profiles. Gene Ontology analysis revealed that …

Contribution of the commensal microbiota to atherosclerosis and arterial thrombosis

The commensal gut microbiota is an environmental factor that has been implicated in the development of cardiovascular disease. The development of atherosclerotic lesions is largely influenced not only by the microbial-associated molecular patterns of the gut microbiota but also by the meta-organismal trimethylamine N-oxide pathway. Recent studies have described a role for the gut microbiota in platelet activation and arterial thrombosis. This review summarizes the results from gnotobiotic mouse models and clinical data that linked microbiota-induced pattern recognition receptor signalling with atherogenesis. Based on recent insights, we here provide an overview of how the gut microbiota cou…

Evaluation of blood collection methods and anticoagulants for platelet function analyses on C57BL/6J laboratory mice

The exploration of thrombotic mechanisms relies on the application of blood collection methods from laboratory mice with a minimal pre-activation of platelets and the clotting system. So far, very little is known on how the blood collection method and the anticoagulant used influence pre-activation of mouse platelets and coagulation. To determine the most suitable blood collection method, we systematically compared blood collection by heart puncture,

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension…

Microbiota-Derived Trimethylamine

The Microbiota Promotes Arterial Thrombosis in Low-Density Lipoprotein Receptor-Deficient Mice

Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr−/− mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions.

Gut Microbiota Restricts NETosis in Acute Mesenteric Ischemia-Reperfusion Injury.

Objective: Recruitment of neutrophils and formation of neutrophil extracellular traps (NETs) contribute to lethality in acute mesenteric infarction. To study the impact of the gut microbiota in acute mesenteric infarction, we used gnotobiotic mouse models to investigate whether gut commensals prime the reactivity of neutrophils towards formation of neutrophil extracellular traps (NETosis). Approach and Results: We applied a mesenteric ischemia-reperfusion (I/R) injury model to germ-free (GF) and colonized C57BL/6J mice. By intravital imaging, we quantified leukocyte adherence and NET formation in I/R-injured mesenteric venules. Colonization with gut microbiota or monocolonization with Esch…

The Interplay between Nutrition, Innate Immunity, and the Commensal Microbiota in Adaptive Intestinal Morphogenesis

The gastrointestinal tract is a functionally and anatomically segmented organ that is colonized by microbial communities from birth. While the genetics of mouse gut development is increasingly understood, how nutritional factors and the commensal gut microbiota act in concert to shape tissue organization and morphology of this rapidly renewing organ remains enigmatic. Here, we provide an overview of embryonic mouse gut development, with a focus on the intestinal vasculature and the enteric nervous system. We review how nutrition and the gut microbiota affect the adaptation of cellular and morphologic properties of the intestine, and how these processes are interconnected with innate immunit…

Germ-free housing conditions do not affect aortic root and aortic arch lesion size of late atherosclerotic low-density lipoprotein receptor-deficient mice

The microbiota has been linked to the development of atherosclerosis, but the functional impact of these resident bacteria on the lesion size and cellular composition of atherosclerotic plaques in the aorta has never been experimentally addressed with the germ-free low-density lipoprotein receptor-deficient (Ldlr(-/-)) mouse atherosclerosis model. Here, we report that 16 weeks of high-fat diet (HFD) feeding of hypercholesterolemicLdlr(-/-)mice at germ-free (GF) housing conditions did not impact relative aortic root plaque size, macrophage content, and necrotic core area. Likewise, we did not find changes in the relative aortic arch lesion size. However, late atherosclerotic GFLdlr(-/-)mice …

Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology

In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to …

The Commensal Microbiota Enhances ADP-Triggered Integrin αIIbβ3 Activation and von Willebrand Factor-Mediated Platelet Deposition to Type I Collagen

The commensal microbiota is a recognized enhancer of arterial thrombus growth. While several studies have demonstrated the prothrombotic role of the gut microbiota, the molecular mechanisms promoting arterial thrombus growth are still under debate. Here, we demonstrate that germ-free (GF) mice, which from birth lack colonization with a gut microbiota, show diminished static deposition of washed platelets to type I collagen compared with their conventionally raised (CONV-R) counterparts. Flow cytometry experiments revealed that platelets from GF mice show diminished activation of the integrin αIIbβ3 (glycoprotein IIbIIIa) when activated by the platelet agonist adenosine diphosphate (ADP). Fu…