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. 2023 Apr 1;324(4):R435-R445.
doi: 10.1152/ajpregu.00154.2022. Epub 2023 Feb 3.

Inhibition of IL-6 signaling prevents serum-induced umbilical cord artery dysfunction from patients with severe COVID-19

Cellyne R Almeida  1 , Júlia F Lima  1 , Mirele R Machado  2 , Juliano V Alves  2 , Ariel E S Couto  3 , Ligia C B Campos  3 , Carolina D Avila-Mesquita  3 , Maria Auxiliadora-Martins  3 , Christiane Becari  3 , Paulo Louzada-Júnior  4 , Rita C Tostes  2 , Núbia S Lobato  1 , Rafael M Costa  1   2
Affiliations

Inhibition of IL-6 signaling prevents serum-induced umbilical cord artery dysfunction from patients with severe COVID-19

Cellyne R Almeida et al. Am J Physiol Regul Integr Comp Physiol. .

Abstract

Coronavirus disease 2019 (COVID-19) infection has a negative impact on the cytokine profile of pregnant women. Increased levels of proinflammatory cytokines seem to be correlated with the severity of the disease, in addition to predisposing to miscarriage or premature birth. Proinflammatory cytokines increase the generation of reactive oxygen species (ROS). It is unclear how interleukin-6 (IL-6) found in the circulation of patients with severe COVID-19 might affect gestational health, particularly concerning umbilical cord function. This study tested the hypothesis that IL-6 present in the circulation of women with severe COVID-19 causes umbilical cord artery dysfunction by increasing ROS generation and activating redox-sensitive proteins. Umbilical cord arteries were incubated with serum from healthy women and women with severe COVID-19. Vascular function was assessed using concentration-effect curves to serotonin in the presence or absence of pharmacological agents, such as tocilizumab (antibody against the IL-6 receptor), tiron (ROS scavenger), ML171 (Nox1 inhibitor), and Y27632 (Rho kinase inhibitor). ROS generation was assessed by the dihydroethidine probe and Rho kinase activity by an enzymatic assay. Umbilical arteries exposed to serum from women with severe COVID-19 were hyperreactive to serotonin. This effect was abolished in the presence of tocilizumab, tiron, ML171, and Y27632. In addition, serum from women with severe COVID-19 increased Nox1-dependent ROS generation and Rho kinase activity. Increased Rho kinase activity was abolished by tocilizumab and tiron. Serum cytokines in women with severe COVID-19 promote umbilical artery dysfunction. IL-6 is key to Nox-linked vascular oxidative stress and activation of the Rho kinase pathway.

Keywords: COVID-19; IL-6; Rho kinase; reactive oxygen species; umbilical cord arteries.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Rita Tostes is an editor of American Journal of Physiology-Regulatory, Integrative and Comparative Physiology and was not involved and did not have access to information regarding the peer-review process or final disposition of this article. An alternate editor oversaw the peer-review and decision-making process for this article.

Figures

Figure 1.
Figure 1.
Women with severe COVID-19 exhibit high concentrations of proinflammatory cytokines. The figures show in pg/mL the circulating levels of IL-6 (A), IFN-γ (B), TNF-α (C), IL-1β (D), and IL-10 (E) in the serum of healthy women and women with severe COVID-19. Values represent means ± SE (n = 13–15). Student’s t test. COVID-19, coronavirus disease 2019; NS, not significant.
Figure 2.
Figure 2.
IL-6 and reactive oxygen species contribute to hyperreactivity of umbilical cord arteries incubated with serum from women with severe COVID-19. Concentration-effect curves to serotonin in human umbilical cord arteries from healthy pregnant women. The rings were incubated with serum from healthy women and women with severe COVID-19 (A, both at a concentration of 10% vol/vol for 24 h), in the presence of vehicle or tocilizumab (B, 100 µg/mL for 30 min) and tiron (C, 1 µM, for 30 min). D: in fluorescence intensity, the products derived from the oxidation of dihydroethidine (DHE), resulting from the reactive oxygen species generation. Control group is formed by rings of umbilical arteries free of any stimuli. Values represent means ± SE (n = 10–15). ANOVA test. *P < 0.05 vs. Control. COVID-19, coronavirus disease 2019; TZ, tocilizumab.
Figure 3.
Figure 3.
Nox 1 contributes to reactive oxygen species generation and hyperreactivity of umbilical cord arteries incubated with serum from women with severe COVID-19. mRNA expression of Nox 1 (A) and Nox 4 (B). Concentration-effect curves to serotonin in human umbilical cord arteries from healthy pregnant women. The rings were incubated with serum from women with severe COVID-19 (at a concentration of 10% vol/vol for 24 h), in the presence of vehicle or ML171 (1 µM, for 30 min; C). D: in relative luminance units, the products derived from the NADPH oxidase. Control group is formed by rings of umbilical arteries free of any stimuli. Values represent means ± SE (n = 6–10). ANOVA test. *P < 0.05 vs. Control. COVID-19, coronavirus disease 2019; TZ, tocilizumab.
Figure 4.
Figure 4.
Rho kinase is involved in the hyperreactivity of umbilical cord arteries incubated with serum from women with severe COVID-19. Concentration-effect curves to serotonin (A) and Y27632 (B) in human umbilical cord arteries from healthy pregnant women. The rings were incubated with serum from healthy women and women with severe COVID-19 (both at a concentration of 10% vol/vol for 24 h), in the presence of vehicle or Y27632 (1 µM, for 30 min). C: representative western blot in the upper panels, with quantitative analysis in the lower panels to p-RhoASer188. D: Rho kinase activity. Control group is formed by rings of umbilical arteries free of any stimuli. Values represent means ± SE (n = 10–15). ANOVA test. *P < 0.05 vs. Control. COVID-19, coronavirus disease 2019; TZ, tocilizumab.
Figure 5.
Figure 5.
Representative diagram with findings of the present study. Serum from women who developed severe COVID-19 promotes umbilical artery dysfunction associated with vascular smooth muscle hypercontractility by increased IL-6 levels and consequently increased Nox1-dependent ROS generation and activation of the Rho kinase pathway. COVID-19, coronavirus disease 2019; IL-6, interleukin 6; ROS, reactive oxygen species; VSM, vascular smooth muscle.
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