Pathophysiological, Cellular, and Molecular Events of the Vascular System in Anaphylaxis.

Anaphylaxis is a systemic hypersensitivity reaction that can be life threatening. Mechanistically, it results from the immune activation and release of a variety of mediators that give rise to the signs and symptoms of this pathological event. For years, most of the research in anaphylaxis has focused on the contribution of the immune component. However, approaches that shed light on the participation of other cellular and molecular agents are necessary. Among them, the vascular niche receives the various signals (e.g., histamine) that elicit the range of anaphylactic events. Cardiovascular manifestations such as increased vascular permeability, vasodilation, hypotension, vasoconstriction, and cardiac alterations are crucial in the pathophysiology of anaphylaxis and are highly involved to the development of the most severe cases. Specifically, the endothelium, vascular smooth muscle cells, and their molecular signaling outcomes play an essential role downstream of the immune reaction. Therefore, in this review, we synthesized the vascular changes observed during anaphylaxis as well as its cellular and molecular components. As the risk of anaphylaxis exists both in clinical procedures and in routine life, increasing our knowledge of the vascular physiology and their molecular mechanism will enable us to improve the clinical management and how to treat or prevent anaphylaxis. Key Message: Anaphylaxis, the most severe allergic reaction, involves a variety of immune and non-immune molecular signals that give rise to its pathophysiological manifestations. Importantly, the vascular system is engaged in processes relevant to anaphylactic events such as increased vascular permeability, vasodilation, hypotension, vasoconstriction, and decreased cardiac output. The novelty of this review focuses on the fact that new studies will greatly improve the understanding of anaphylaxis when viewed from a vascular molecular angle and specifically from the endothelium. This knowledge will improve therapeutic options to treat or prevent anaphylaxis.

Metabolic Alterations Identified in Urine, Plasma and Aortic Smooth Muscle Cells Reflect Cardiovascular Risk in Patients with Programmed Coronary Artery Bypass Grafting.

Atherosclerosis is the predominant pathology associated to premature deaths due to cardiovascular disease. However, early intervention based on a personalized diagnosis of cardiovascular risk is very limited. We have previously identified metabolic alterations during atherosclerosis development in a rabbit model and in subjects suffering from an acute coronary syndrome. Here we aim to identify specific metabolic signatures which may set the basis for novel tools aiding cardiovascular risk diagnosis in clinical practice. In a cohort of subjects with programmed coronary artery bypass grafting (CABG), we have performed liquid chromatography and targeted mass spectrometry analysis in urine and plasma. The role of vascular smooth muscle cells from human aorta (HA-VSMCs) was also investigated by analyzing the intra and extracellular metabolites in response to a pro-atherosclerotic stimulus. Statistically significant variation was considered if p value < 0.05 (Mann-Whitney test). Urinary trimethylamine N-oxide (TMAO), arabitol and spermidine showed higher levels in the CVrisk group compared with a control group; while glutamine and pantothenate showed lower levels. The same trend was found for plasma TMAO and glutamine. Plasma choline, acetylcholine and valine were also decreased in CVrisk group, while pyruvate was found increased. In the secretome of HA-VSMCs, TMAO, pantothenate, glycerophosphocholine, glutathion, spermidine and acetylcholine increased after pro-atherosclerotic stimulus, while secreted glutamine decreased. At intracellular level, TMAO, pantothenate and glycerophosphocholine increased with stimulation. Observed metabolic deregulations pointed to an inflammatory response together with a deregulation of oxidative stress counteraction.

Proteomic and Biological Analysis of an In Vitro Human Endothelial System in Response to Drug Anaphylaxis.

Anaphylaxis is a life-threatening systemic hypersensitivity reaction. During anaphylaxis, mediator release by effector cells causes endothelial barrier breakdown, increasing vascular permeability and leakage of fluids, which may lead to tissue edema. Although endothelial cells (ECs) are key players in this context, scant attention has been paid to the molecular analysis of the vascular system, and further analyses of this cell type are necessary, especially in humans. The protein expression pattern of human microvascular ECs was analyzed in response to sera from anaphylactic patients (EC-anaphylaxis) and sera from non-allergic subjects (EC-control) after 2 hours of contact. Firstly, a differential quantitative proteomic analysis of the protein extracts was performed by mass spectrometry using an isobaric labeling method. Second, the coordinated behavior of the identified proteins was analyzed using systems biology analysis (SBA). The proteome of the EC-anaphylaxis system showed 7,707 proteins, of which 1,069 were found to be significantly altered between the EC-control and EC-anaphylaxis groups (p-value < 0.05). Among them, a subproteome of 47 proteins presented a high rate of change (|ΔZq| ≥ 3). This panel offers an endothelial snapshot of the anaphylactic reaction. Those proteins with the highest individual changes in abundance were hemoglobin subunits and structural support proteins. The interacting network analysis of this altered subproteome revealed that the coagulation and complement systems are the main biological processes altered in the EC-anaphylactic system. The comprehensive SBA resulted in 5,512 functional subcategories (biological processes), 57 of which were significantly altered between EC-control and EC-anaphylaxis. The complement system, once again, was observed as the main process altered in the EC system created with serum from anaphylactic patients. Findings of the current study further our understanding of the underlying pathophysiological mechanisms operating in anaphylactic reactions. New target proteins and relevant signaling pathways operating in the in vitro endothelial-serum system have been identified. Interestingly, our results offer a protein overview of the micro-EC-anaphylaxis environment. The relevance of the coagulation, fibrinolytic, contact and complement systems in human anaphylaxis is described. Additionally, the untargeted high-throughput analysis used here is a novel approach that reveals new pathways in the study of the endothelial niche in anaphylaxis.

In Vitro Investigation of Vascular Permeability in Endothelial Cells from Human Artery, Vein and Lung Microvessels at Steady-State and Anaphylactic Conditions.

Human anaphylactic reactions largely involve an increase in vascular permeability, which is mainly controlled by endothelial cells (ECs). Due to the acute and serious nature of human anaphylaxis, in vivo studies of blood vessels must be replaced or supplemented with in vitro models. Therefore, we used a macromolecular tracer assay (MMTA) to investigate the EC permeability of three phenotypes of human ECs: artery (HAECs), vein (HSVECs) and microvessels from lung (HMLECs). ECs were stimulated with two fast-acting anaphylactic mediators (histamine and platelet-activating factor (PAF)) and one longer-lasting mediator (thrombin). At steady-state conditions, HSVEC monolayers were the most permeable and HMLEC the least (15.8% and 8.3% after 60 min, respectively). No response was found in ECs from artery or vein to any stimuli. ECs from microvessels reacted to stimulation with thrombin and also demonstrated a tendency of increased permeability for PAF. There was no reaction for histamine. This was not caused by missing receptor expression, as all three EC phenotypes expressed receptors for both PAF and histamine. The scarce response to fast-acting mediators illustrates that the MMTA is not suitable for investigating EC permeability to anaphylactic mediators.

The TNF-like weak inducer of the apoptosis/fibroblast growth factor-inducible molecule 14 axis mediates histamine and platelet-activating factor-induced subcutaneous vascular leakage and anaphylactic shock.

BACKGROUND: Anaphylaxis includes mast cell (MC) activation, but less is known about downstream mechanisms (ie, vascular permeability controlled by endothelial cells [ECs]). The TNF-like weak inducer of apoptosis (TWEAK) and its sole receptor, fibroblast growth factor-inducible molecule 14 (Fn14), belong to the TNF superfamily and are involved in proinflammatory responses. OBJECTIVE: We sought to investigate the role of TWEAK/Fn14 axis in anaphylaxis. METHODS: In vivo vascular permeability and mouse models of passive systemic anaphylaxis (PSA) and active systemic anaphylaxis were applied to wild-type (WT), TWEAK- and Fn14-deficient mice (TWEAK-/- and Fn14-/-, respectively). Primary bone marrow-derived mast cells (BMMCs) and ECs from WT and Fn14-/- or TWEAK-/- mice were studied. The TWEAK/Fn14 axis was also investigated in human samples. RESULTS: Mice with PSA and active systemic anaphylaxis had increased Fn14 and TWEAK expression in lung tissues and increased serum soluble TWEAK concentrations. TWEAK and Fn14 deficiencies prevent PSA-related symptoms, resulting in resistance to decreased body temperature, less severe reactions, and maintained physical activity. Numbers of MCs after PSA are similar between genotypes in different tissue regions, such as ear skin and the trachea, tongue, peritoneum, lungs, and bone marrow. Moreover, in vitro studies revealed no differences in degranulation or mediator release between WT and Fn14-/- BMMCs after IgE-FcεRI stimulation. In vivo and in vitro histamine and platelet-activating factor administration increases Fn14 receptor expression in lungs and ECs. Moreover, Fn14 deficiency in ECs maintained in vitro impermeability when stimulated by mediators or activated BMMCs but not by TWEAK-/- BMMCs, indicating that Fn14 is crucial for endothelial barrier function. TWEAK/Fn14 deletion or TWEAK-blocking antibody prevented histamine/platelet-activating factor-induced vascular subcutaneous permeability. Circulating soluble TWEAK levels were increased in patients with anaphylaxis, and plasma from those patients increased Fn14 expression in ECs. CONCLUSION: The TWEAK/Fn14 axis participates in anaphylactic reactions. Inhibition of TWEAK/Fn14 interaction could be efficacious in anaphylaxis therapy.

Interaction of Alt a 1 with SLC22A17 in the airway mucosa.

BACKGROUND: Despite all the efforts made up to now, the reasons that facilitate a protein becoming an allergen have not been elucidated yet. Alt a 1 protein is the major fungal allergen responsible for chronic asthma, but little is known about its immunological activity. Our main purpose was to investigate the ligand-dependent interactions of Alt a 1 in the human airway epithelium. METHODS: Alt a 1 with and without its ligand (holo- and apo- forms) was incubated with the pulmonary epithelial monolayer model, Calu-3 cells. Allergen transport and cytokine production were measured. Pull-down and immunofluorescence assays were employed to identify the receptor of Alt a 1 using the epithelial cell model and mouse tissues. Receptor-allergen-ligand interactions were analyzed by computational modeling. RESULTS: The holo-form could activate human monocytes, PBMCs, and polarized airway epithelial (Calu-3) cell lines. The allergen was also transported through the monolayer, without any alteration of the epithelial integrity (TEER). Alt a 1 also induced the production of proinflammatory IL8 and specific epithelial cytokines (IL33 and IL25) by Calu-3 cells. The interaction between epithelial cells and holo-Alt a 1 was found to be mediated by the SLC22A17 receptor, and its recognition of Alt a 1 was explained in structural terms. CONCLUSIONS: Our findings identified the Alt a 1 ligand as a central player in the interaction of the allergen with airway mucosa, shedding light into its potential role in the immunological response, while unveiling its potential as a new target for therapy intervention.