Introduction Thrombin (F2) expression was recently reported in intestinal epithelial cells (IEC) impacting on microbial spatial organization and intestinal injuries. The thrombin receptor protease-activated receptor-1 (PAR1/F2r) is also expressed on IECs and in the context of inflammatory bowel disease, its activation is linked to disorders in gut mucosal healing. Interestingly, the gut microbiota can influence intestinal villus vascularization, required for nutrient harvest, in a tissue factor/PAR1-dependent fashion. Here, we aim to unravel the underlying epithelial-endothelial-crosstalk between epithelial thrombin/PAR1 and small intestinal villus vascularization.

Introduction The gut microbiota has been identified as a dynamic ecosystem impacting the vasculature and remote organ functions. Emerging research demonstrated that gut microbes are involved in modulating cardiovascular diseases (CVD). However, underlying molecular pathways that causally explain the role of the gut microbiota in thrombosis still remain obscure. Thus, we took advantage of established gnotobiotic low-density lipoprotein receptor-deficient (Ldlr-/-) mice to provide new insights on how the commensal gut microbiota, which is influenced by diet, modifies arterial thrombosis.

In this issue of Blood, Kaiser et al(1) pinpoint the glycoprotein IIb (GPIIB)/G alpha(13)/Src tyrosine kinase (c-Src)/14-3-3 zeta signaling axis as a requirement for platelet migration and shed light on how the antineoplastic oral tyrosine kinase inhibitor dasatinib increases the risk of inflammation-associated mucosal hemorrhage.

The gut microbiota influences intestinal barrier integrity through mechanisms that are incompletely understood. Here we show that the commensal microbiota weakens the intestinal barrier by suppressing epithelial neuropilin-1 (NRP1) and Hedgehog (Hh) signaling. Microbial colonization of germ-free mice dampens signaling of the intestinal Hh pathway through epithelial Toll-like receptor (TLR)-2, resulting in decreased epithelial NRP1 protein levels. Following activation via TLR2/TLR6, epithelial NRP1, a positive-feedback regulator of Hh signaling, is lysosomally degraded. Conversely, elevated epithelial NRP1 levels in germ-free mice are associated with a strengthened gut barrier. Functionally, intestinal epithelial cell-specific Nrp1 deficiency (Nrp1ΔIEC) results in decreased Hh pathway activity and a weakened gut barrier. In addition, Nrp1ΔIEC mice have a reduced density of capillary networks in their small intestinal villus structures. Collectively, our results reveal a role for the commensal microbiota and epithelial NRP1 signaling in the regulation of intestinal barrier function through postnatal control of Hh signaling.

In biomedical research, germ-free and gnotobiotic mouse models enable the mechanistic investigation of microbiota-host interactions and their role on (patho)physiology. Throughout any gnotobiotic experiment, standardized and periodic microbiological testing of defined gnotobiotic housing conditions is a key requirement. Here, we review basic principles of germ-free isolator technology, the suitability of various sterilization methods, and the use of sterility testing methods to monitor germ-free mouse colonies. We also discuss their effectiveness and limitations, and share the experience with protocols used in our facility. In addition, possible sources of isolator contamination are discussed and an overview of reported contaminants is provided.

The gut microbiota is increasingly recognized as an actuating variable shaping vascular development and endothelial cell function in the intestinal mucosa but also affecting the microvasculature of remote organs. In the small intestine, colonization with gut microbiota and subsequent activation of innate immune pathways promotes the development of intricate capillary networks and lacteals, influencing the integrity of the gut-vascular barrier as well as nutrient uptake. Since the liver yields most of its blood supply via the portal circulation, the hepatic microcirculation steadily encounters microbiota-derived patterns and active signaling metabolites that induce changes in the organization of the liver sinusoidal endothelium, influencing immune zonation of sinusoids and impacting on metabolic processes. In addition, microbiota-derived signals may affect the vasculature of distant organ systems such as the brain and the eye microvasculature. In recent years, this gut-resident microbial ecosystem was revealed to contribute to the development of several vascular disease phenotypes.

EDITORIAL article Front. Med., 13 July 2022Sec. Hematology https://doi.org/10.3389/fmed.2022.939890

Homo- and heterophilic binding mediated by the immunoglobulin (Ig)-like repeats of cell adhesion molecules play a pivotal role in cell-cell and cell-extracellular matrix interactions. L1CAM is crucial to neuronal differentiation, in both mature and developing nervous systems, and several studies suggest that its functional interactions are mainly mediated by Ig2–Ig2 binding. X-linked mutations in the human L1CAM gene are summarized as L1 diseases, including the most diagnosed CRASH neurodevelopmental syndrome. In silico simulations provided a molecular rationale for CRASH phenotypes resulting from mutations I179S and R184Q in the homophilic binding region of Ig2. A synthetic peptide reproducing such region could both mimic the neuritogenic capacity of L1CAM and rescue neuritogenesis in a cellular model of the CRASH syndrome, where the full L1CAM ectodomain proved ineffective. Presented functional evidence opens the route to the use of L1CAM-derived peptides as biotechnological and therapeutic tools.

The gut microbiota, a part of the environmental exposome, was identified as a dominant modifier of cardiovascular disease (CVD) development. Here, we provide insights on the mechanistic evidence, achieved by studying germ-free mouse models and antibiotic treatment protocols, demonstrating the impact of commensals on CVD and arterial thrombosis. We summarize microbiota-triggered alterations in vascular immune phenotypes and explain how host colonization status can affect vascular inflammation, blood pressure regulation, atherosclerosis and arterial thrombosis. As recent clinical studies and experimental work have associated the diversity of the microbiome with CVD, we provide a comprehensive overview of the various microbiota-derived metabolites and inflammatory pathways that interfere with host physiology to modify the development of CVD and arterial thrombus growth. Thus, we point out how indicator species of the gut microbiome and microbiota-dependent metabolites start to emerge as new diagnostic markers that might turn out useful to prevent and diagnose the progression of CVD.

α-Synuclein (αSyn) is a small disordered protein, highly conserved in vertebrates and involved in the pathogenesis of Parkinson's disease (PD). Indeed, αSyn amyloid aggregates are present in the brain of patients with PD. Although the pathogenic role of αSyn is widely accepted, the physiological function of this protein remains elusive. Beyond the central nervous system, αSyn is expressed in hematopoietic tissue and blood, where platelets are a major cellular host of αSyn. Platelets play a key role in hemostasis and are potently activated by thrombin (αT) through the cleavage of protease-activated receptors. Furthermore, both αT and αSyn could be found in the same spatial environment, i.e. the platelet membrane, as αT binds to and activates platelets that can release αSyn from α-granules and microvesicles. Here, we investigated the possibility that exogenous αSyn could interfere with platelet activation induced by different agonists in vitro. Data obtained from distinct experimental techniques (i.e. multiple electrode aggregometry, rotational thromboelastometry, immunofluorescence microscopy, surface plasmon resonance, and steady-state fluorescence spectroscopy) on whole blood and platelet-rich plasma indicate that exogenous αSyn has mild platelet antiaggregating properties in vitro, acting as a negative regulator of αT-mediated platelet activation by preferentially inhibiting P-selectin expression on platelet surface. We have also shown that both exogenous and endogenous (i.e. cytoplasmic) αSyn preferentially bind to the outer surface of activated platelets. Starting from these findings, a coherent model of the antiplatelet function of αSyn is proposed.

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 immunity. Furthermore, we discuss how nutritional and microbial factors impact the renewal and differentiation of the epithelial lineage, influence the adaptation of capillary networks organized in villus structures, and shape the enteric nervous system and the intestinal smooth muscle layers. Intriguingly, the anatomy of the gut shows remarkable flexibility to nutritional and microbial challenges in the adult organism.

As shown recently, oleic acid (OA) in complex with lactoferrin (LF) causes the death of cancer cells, but no mechanism(s) of that toxicity have been disclosed. In this study, constitutive parameters of the antitumor effect of LF/OA complex were explored. Complex LF/OA was prepared by titrating recombinant human LF with OA. Spectral analysis was used to assess possible structural changes of LF within its complex with OA. Structural features of apo-LF did not change within the complex LF:OA = 1:8, which was toxic for hepatoma 22a cells. Cytotoxicity of the complex LF:OA = 1:8 was tested in cultured hepatoma 22a cells and in fresh erythrocytes. Its anticancer activity was tested in mice carrying hepatoma 22a. In mice injected daily with LF-8OA, the same tumor grew significantly slower. In 20% of animals, the tumors completely resolved. LF alone was less efficient, i.e., the tumor growth index was 0.14 for LF-8OA and 0.63 for LF as compared with 1.0 in the control animals. The results of testing from 48 days after the tumor inoculation showed that the survival rate among LF-8OA-treated animals was 70%, contrary to 0% rate in the control group and among the LF-treated mice. Our data allow us to regard the complex of LF and OA as a promising tool for cancer treatment.

α-Synuclein (αSyn) is a small (140 amino acids) disordered, acidic (pI: 4.7) protein, highly conserved in vertebrates and implicated in the pathogenesis of Parkinson’s disease (PD), a neurodegenerative disease characterized by the deposition of αSyn amyloid fibrils in dopaminergic neurons. Beyond the central nervous system, significant expression of αSyn has also been measured in the blood (~1 μM), where platelets are the main cellular hosts of αSyn. Although the pathological implication of αSyn in PD is widely accepted, the physiological role of blood αSyn is still elusive. Starting from the notion that platelets are either the major cellular reservoir of αSyn in the blood and, concomitantly, act as key players in hemostasis, being activated also by α-thrombin (αT) via cleavage of protease-activated receptors (PARs), we decided to investigate the possibility that αSyn could modulate platelet activation by interfering with the αT-PAR functional axis. Using multiple electrode aggregometry, i.e. a fast and specific platelet-function-testing method, as well as steady-state fluorescence spectroscopy, surface plasmon resonance, and fluorescence microscopy, we show here that monomeric αSyn functions as a negative regulator of αT-mediated platelets activation. αSyn acts either directly, via competitive inhibition of PAR1 activation by αT and TRAP6 agonist, and indirectly, by scavenging αT on the platelet plasma membrane. A simple electrostatic model of αSyn platelet antiaggregating effect is proposed and the possible role of the protein at the interplay of amyloidosis and thrombosis is discussed.

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 several functional processes in the hepatic endothelium were affected. The absence of microbiota influenced the expression of genes involved in cholesterol flux and angiogenesis. Specifically, genes functioning in hepatic endothelial sphingosine metabolism and the sphingosine-1-phosphate pathway showed drastically increased expression in the GF state. Our analyses reveal a prominent role for the microbiota in shaping the transcriptional landscape of the hepatic endothelium.

Aggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1–3% older adults. Fragmented hTTR is the standard composition of amyloid deposits in SSA, but the protease(s) responsible for amyloidogenic fragments generation in vivo is(are) still elusive. Here, we show that subtilisin secreted from Bacillus subtilis , a gut microbiota commensal bacterium, translocates across a simulated intestinal epithelium and cleaves hTTR both in solution and human plasma, generating the amyloidogenic fragment hTTR(59–127), which is also found in SSA amyloids in vivo. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart.