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

Abstract

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.

Anaphylaxis: Mediators, Biomarkers, and Microenvironments

“…Complement, contact and coagulation systems are involved in the pathophysiology of anaphylaxis [61,115]. These molecular cascades are in a constant crosstalk and in continuous amplification and inhibition loops keeping their effect balanced during an inflammatory situation.…”

“…Endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) interact with each other and govern the vascular pathophysiological processes of anaphylactic reactions [27,115].…”

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

“…In this context, the wide endothelium surface would also be contributing differentially to the pathophysiology of the anaphylaxis. The endothelium arises as an important organ-cell like in anaphylaxis playing a role not only in the control of fluids and the vascular tone but also as an activation surface for the coagulation, contact, and complement systems ( 44 , 162 , 163 ). Moreover, ECs release relevant anaphylactic mediators such as NO, although it is likely that other molecules (presumably cytokines or interleukins) could also be released contributing to the pool of mediators in the reaction.…”

“…Additionally, in acute inflammatory situations, as it has been observed in COVID-19 disease, ECs contribute as effector cells to the cytokine storm ( 161 ). In addition, the endothelium actively participates in the activation of the coagulation, contact, and complement in anaphylaxis ( 44 , 162 , 163 ). Therefore, its study is a hot topic and of great importance for the treatment of several pathologies such as anaphylaxis.…”

State-of-the-Art on Biomarkers for Anaphylaxis in Obstetrics

“…Activation of the coagulation/fibrinolytic system usually appears together with the activation of the complement system, which is considered a triggering mechanism in severe anaphylaxis. C3a, C4a and C5a (referred classically as anaphylatoxins) are considered biomarkers of complement activation [ 98 , 99 , 100 ].…”