Endothelial dysfunction markers and immune response indices in cosmonauts’ blood after long-duration space flights

www.nature.com/npjmgrav ARTICLE OPEN Endothelial dysfunction markers and immune response indices in cosmonauts’ blood after long-duration space flights D. S. Kuzichkin 1 ✉, I. A. Nichiporuk 1, O. A. Zhuravleva O. V. Kutko 1, V. A. Shmarov 1 and S. A. Ponomarev 1 1 , A. A. Markin1, M. P. Rykova 1 , T. V. Zhuravleva 1 , A. A. Sadova 1 , 1234567890():,; Space flight factors are known to cause a malfunction in the human immune system and lead to damage to blood vessels. The hemostatic function of endothelium during space missions and its interaction with human immunity has not been determined so far. In this work, we investigated the markers of endothelial activation and damage (plasma concentrations of soluble thrombomodulin fraction (sTM), von Willebrand factor (vWF), highly sensitive C-reactive protein (hs-CRP)), as well as the level of D-dimer and compared them to the immunological parameters characterizing the state of human humoral and cellular immunity. The immune status of long-duration ISS crewmembers was assessed by whole-blood testing, and comprehensive postflight immune assessment included the analysis of leukocyte distribution. Flow cytometry was applied to determine the absolute counts and the percentage of lymphocyte subsets: B cells (CD19+), T cells (CD3+, CD3+CD4+, CD3+CD8+), NK cells (CD3−CD16+CD56+, CD11b+CD56+), and activated subsets (CD3+CD25+ and CD3+HLA-DR+). The in vitro basal cytokine production was investigated in whole blood cell culture. The cytokines IFN-gamma, IL-1-beta, IL-4, IL-6, IL-10, IL-18, and TNF-alpha were measured in plasma and the 24-h supernatants by a sensitive enzyme-linked immunosorbent assay. A significant increase in the plasma levels of vWF and hs-CRP and a decrease in the concentration of sTM after spaceflights were detected. Divergent changes in the parameters characterizing the state of the immune system were observed. We propose that the changes revealed may lead to an increase in the procoagulant activity of blood plasma, suppression of protein C activation and thrombin inhibition, as well as to an increase in the adhesive-aggregate potential of platelets, especially in case of changes in the rheological characteristics of blood flow during re-adaptation to ground conditions. We also speculate that the immune system might play an important role in vessel damage during long-duration missions. npj Microgravity (2022)8:46 ; https://doi.org/10.1038/s41526-022-00237-0 INTRODUCTION It is known that the effect of space flight factors on the human body can lead to damage, activation, and increasing dysfunction of endothelium1. It has been shown that the number of endothelial cells circulating in the blood increases after 2 months of head-down tilt bed-rest2. Signs of cytoskeleton damage increased permeability of cell membranes, and a decrease in the proliferative activity of cultured endothelial cells after orbital flight were revealed3. However, the function of the endothelium consisting in regulating the blood aggregate state under the influence of space flight factors has not been practically studied. It is common knowledge that factors of space flight can damage blood vessels and affect the blood hemostasis system4, increasing the risk of thrombophilia and, in some cases, leading to the appearance of occlusive thrombosis during orbital flight5. We selected two specific markers of endothelial hemostatic potential for research, namely, von Willebrand factor (vWF) and thrombomodulin (TM), as well as highly sensitive C-reactive protein (hsCRP) as an additional marker of endothelial dysfunction6. vWF is a multimeric glycoprotein present in blood plasma, endothelial cells, megakaryocytes, and platelets. vWF is constantly secreted into the bloodstream by the endothelial cells’ Weibel–Palade bodies and by the platelets’α-granules during activation. The pool of circulating vWF consists of multimers of various sizes, from several dimers to high-molecular multimers, which contain 11–20 dimers. The former mainly serve as carriers (apoenzymes) of the factor VIII coagulation cascade, protecting it from proteolysis7, and the latter mediates platelet adhesion and aggregation8. TM is synthesized mainly in endothelial cells and is expressed on their membrane. It regulates the activity of thrombin and physiological anticoagulant protein C9. The presence of soluble TM fragments in plasma is sometimes considered a sign of endothelial damage, although it is also functional in plasma: it serves as a cofactor for thrombin binding and protein C activation10 and mediates antifibrinolytic activity11. Hs-CRP in the form of a protein made of identical subunits forming a cyclic pentamer is synthesized by hepatocytes, adipocytes, and atherosclerotic plaques. It enhances the synthesis of reactive oxygen species, binds to oxidized particles of lowdensity lipoproteins (LDL), and stimulates their absorption by macrophages, contributing to an increase in the production of adhesion molecules (ICAM, VCAM, E-selectin, MCP-1). It induces the secretion of monocyte tissue factor (TF) and inhibits the production of plasminogen activator I inhibitor (PAI–I) and tissue plasminogen activator (TPA). This protein has a direct effect on the release of proinflammatory cytokines from leukocytes, thereby disrupting the vasoreactivity of the endothelium and causing instability of atherosclerotic plaques, which, in turn, leads to an increased risk of thrombosis12. The aim of this work was to study the levels of vWF, TM, and hsCRP in cosmonauts’ blood plasma after long-term orbital expeditions to the International Space Station and to compare the results to the parameters characterize the state of the immune system which can be involved in into endothelial dysfunction. State Scientific Center of the Russian Federation - Institute for Biomedical Problems of the Russian Academy of Sciences, Moscow, Russia. ✉email: 1 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA D.S. Kuzichkin et al. 1234567890():,; 2 Fig. 1 The concentrations of the blood plasma parameters of cosmonauts before the mission (Baseline) and after space flight completion (Days +1 and +7). a von Willebrand factor, b thrombomodulin, c highly sensitive C-reactive protein, and d D-dimer. The center lines represent the median, the bounds of boxes represent the 25th percentile and 75th percentile (Q1 and Q2 correspondingly), the bonds of whiskers represent the minimum and the maximum values, the triangles within the diagrams are the raw data for particular volunteers, p values were obtained using the Wilcoxon’s test. RESULTS AND DISCUSSION The dynamics of the parameters in the post-flight period Parameters of vWF, TM, and hs-CRP in the dynamics of pre- and postflight examinations of cosmonauts are shown in Figs. 1–3. The concentrations of vWF in analyzed blood plasma samples were significantly increased by (p = 0.015) on the first day after the flight and by ( (...truncated)