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

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.

INTRODUCTION

Emerging diseases are a frequent threat to global health, with a high potential for the development of epidemic and pandemic outbreaks. The world is currently afflicted by coronavirus disease 2019 (COVID-19), a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1, 2). Clinical consequences of COVID-19 are intense and diffuse lung injury, leading to respiratory failure and, in more serious situations, death (3). Severe cases of COVID-19 are associated with immune response dysfunction and high levels of cytokines, such as interleukins (IL), especially IL-1 and IL-6 (4, 5). This systemic cytokine storm is responsible for target organ injury and damage (6), including extrapulmonary damage, as seen in patients with COVID-19. In this context, viral genetic material has already been found in the central nervous, gastrointestinal, cardiorenal, and other systems (7).

Patients with chronic diseases, such as diabetes, obesity, and hypertension, over 60 yr of age, and pregnant women are considered at risk for COVID-19 complications (8, 9). Because of changes in cardiorespiratory and immune function, pregnancy is often considered a condition of high susceptibility to viral infections, especially those that affect the respiratory system, including SARS-CoV-2 (10–12). SARS-CoV-2 infection during pregnancy induces unique inflammatory responses at the maternal-fetal interface, increasing maternal vulnerability to an intensive care unit stay or mechanical ventilation (13) besides increasing the risk of fetal (14) and maternal (15) death. Umbilical cord and placental infected cells include the syncytiotrophoblasts and cytotrophoblasts in the villi, stromal, perivascular, endothelial, and vascular smooth muscle cells (16, 17).

COVID-19 infection has a negative impact on the cytokine profile of pregnant women, varying according to the trimesters of pregnancy (18). Increased levels of cytokines, mainly proinflammatory cytokines, are correlated with the severity of the disease and predispose to miscarriage or premature birth (19, 20). The cytokine storm caused by SARS-CoV-2 culminates in widespread inflammation in pregnant women and, under these conditions, several cells produce free radicals in excess, such as reactive oxygen species (ROS), contributing to the pathogenesis of COVID-19 and various associated diseases, including cardiovascular disease (21, 22).

ROS are intracellular signaling molecules that modulate several cellular responses, including the activity of proteins that regulate cardiac function and vascular tone (23). Sources of ROS in various diseases, including cardiovascular diseases, include NADPH oxidase, xanthine oxidase, endoplasmic reticulum, and mitochondrial respiratory chain, among others (24). Specifically, in vascular smooth muscle cells, excess ROS reduces the bioavailability of nitric oxide, an important vasodilator agent, and activates contractile kinases, such as mitogen-activated protein kinases (ERK, JNK, and p38) and myosin light chain-associated kinases (MLCK, RhoA/Rho, and MYPT-1) (25, 26). This set of actions favors smooth muscle hyperreactivity and vascular dysfunction.

Proinflammatory cytokines activate pro-oxidant enzymes, increasing ROS generation (27, 28). IL-6, for example, activates the enzyme NADPH oxidase in endothelial and vascular smooth muscle cells, increasing the formation of superoxide anion and hydrogen peroxide (29, 30). In the COVID-19 setting, elevated serum IL-6 levels are linked to worse prognosis and deteriorating clinical outcomes (31). COVID-19 patients with elevated levels of IL-6 are prone to adverse cardiac events (32).

It is unclear how circulating IL-6 in severe COVID-19 patients might affect gestational health, particularly concerning the function of the umbilical cord. Thus, this study tested the hypothesis that serum IL-6 in women with severe COVID-19 causes umbilical cord artery dysfunction by increasing ROS generation and by activating redox-sensitive proteins.

MATERIALS AND METHODS

Human Samples

The Brazilian National Committee for Ethics in Research (CONEP) approved all procedures performed in the study (CONEP CAAE: 30816620.0.0000.5440 and 24891819.0.1001.8155). This investigation followed the principles of the Helsinki declaration as well as resolution no. 466/2012 from the Brazilian Ministry of Health for research involving humans. Informed consent was obtained from all participants or their next of kin before inclusion in the present investigation.

The patient population of the present study consisted of 1) 15 nonpregnant women hospitalized in the Hospital das Clínicas de Ribeirão Preto (Ribeirao Preto Medical School) with a reverse transcription-polymerase chain reaction (RT-PCR) of nasopharyngeal samples positive for SARS-CoV-2 infection; 2) a control group with 13 healthy and nonpregnant individuals of the same sex; and 3) 15 pregnant women hospitalized for childbirth in the Hospital das Clínicas de Jataí. The healthy nonpregnant and pregnant women tested negative for SARS-CoV-2 infection.

Blood from COVID-19 patients was collected on the first day of admission to the intensive care unit of Hospital das Clínicas de Ribeirão Preto, between the period from May 2020 to January 2021. For clinical classification of COVID-19 severity, we used the World Health Organization Clinical Progression Scale (33). Patients that required intensive care unit admission and mechanical ventilation or oxygen therapy by noninvasive ventilation or high-flow nasal cannula were classified as having severe disease. Blood from healthy and nonpregnant individuals was collected after an 8-h fast. Blood samples were collected in tubes without anticoagulant to obtain the serum. Samples were stored at −80°C for later analysis. Table 1 summarizes clinical information and biochemical parameters for patients with COVID-19 and healthy subjects.

Table 1.

Baseline characteristics of healthy women and women with COVID-19

	Healthy (n = 13)	COVID-19 (n = 15)	P Value
Age, yr (interquartile range)	28 (20–35)	32 (20–35)	>0.05a
Hypertension, %	0	20	<0.05b
Obesity, %	0	13.3	<0.05b
Diabetes, %	0	13.3	<0.05b
Symptom days (means ± SD)		14 ± 3
Hospitalization days (means ± SD)		28 ± 7
Mechanical ventilation, %		100
Kidney injury, %		10
Mortality rate, %		20
D-dimer, mg/dL (interquartile range)	0.8 (0.4–1.0)	1.4 (1.1–3.6)	<0.05a
Fibrinogen, mg/dL (interquartile range)	250 (200–355)	802 (659–847)	<0.05a
$\text{P}{\text{a}}_{{\text{O}}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ (interquartile range)		280 (210–360)
SOFA score (interquartile range)		3 (2–4)
SAPS-3 (interquartile range)		45 (34–50)

Criteria for defining hypertension, obesity, diabetes, and kidney injury, respectively, were high blood pressure (>120/80 mmHg), BMI (>24 kg/m²), high fasting blood glucose (>100 mg/dL), and high levels of serum creatinine (>1.3 mg/dL). P < 0.05, comparison between healthy and critically ill individuals with COVID-19. BMI, body mass index; COVID-19, coronavirus disease 2019; $\text{P}{\text{a}}_{{\text{O}}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ ratio, the ratio between arterial oxygen partial pressure ($\text{P}{\text{a}}_{{\text{O}}_2}$ in mmHg) and fractional inspired oxygen; SOFA, sequential assessment of organ failure; SAPS-3, simplified acute physiology score III. ^aStudent’s t test; ^bχ² test.

Pregnant women included in the study were selected based on the following pre-established inclusion criteria: prenatal follow-up from the beginning of pregnancy, live newborn, newborn without fetal malformation on ultrasound and/or postnatal examination, gestational age between 32 and 41 wk, normotensive pregnant women with a maximum blood pressure level of 120/80 mmHg maintained throughout pregnancy and a gestational body mass index (BMI) between 23.7 and 29.2 kg/m².

ELISA Assay

Serum samples collected by venous puncture from healthy women and patients with severe COVID-19 were analyzed for circulating cytokines [IL-6 (Cat. No. DY206), tumor necrosis factor-α (TNF-α, Cat. No. DY210), IL-1β (Cat. No. DY201), interferon-γ (IFN-γ, Cat. No. DY285B), and IL-10 (Cat. No. DY217B)] by enzyme-linked immunosorbent assay (ELISA) using Human DuoSet ELISA (R&D Systems, Minneapolis, MO). The samples were kept at −80°C until the test was performed. The assays were performed in duplicate according to the manufacturer's instructions. Values are expressed in pg/mL.

Vascular Reactivity

Umbilical cord arteries were placed in a Petri dish containing modified Krebs-Henseleit nutrient solution at 4°C, with the following composition (in mM): NaCl, 113; KCl, 4.7; KH₂PO₄, 1.1; MgSO₄, 1.1; NaHCO₃, 25; NaNa EDTA, 0.03; CaCl₂, 2.5; dextrose, 5.5. The median portion of the artery was divided into four rings of 4 mm in length. After incubations, the rings were mounted in an organ bath system to study the isometric force generation, connected to a signal transducer (Letica Scientific Instruments, Barcelona, ESP). The organ bath vats contained a modified Krebs-Henseleit nutrient solution, aerated with 95% O₂ and 5% CO₂, and heated to 37°C. The ring preparations remained under basal tension of 3 g for 60 min (min) for stabilization, with changes in the nutrient solution and tension adjustment every 15 min. After the stabilization period, vascular integrity was tested by evaluating the contraction of potassium chloride (KCl, 120 mM), after 10 min of stimulation, which was followed by a new stabilization until the basal tension resumed. Concentration-effect curves for serotonin (0.3 nM to 0.1 mM) and Y27632 (Rho kinase inhibitor, 0.3 nM to 0.1 mM) were performed in vascular segments with intact endothelium, incubated with serum from healthy women or women who developed severe COVID-19 [10% vol/vol for 24 h (h)]. Each arterial segment was incubated with serum from only one healthy or COVID-19-positive patient. Contraction values were corrected for the ring length. Serotonin-induced contractions were also determined after incubation with tocilizumab (recombinant humanized monoclonal antibody that acts as an IL-6 receptor antagonist, 100 µg/mL for 30 min, Actemra, Roche), tiron (superoxide anion scavenger, 1 µM, for 30 min, Santa Cruz; Cat. No. sc-253699), ML171 (Nox 1 inhibitor, 1 µM, for 30 min, Tocris; Cat. No. 4653) and Y27632 (1 µM, for 30 min, Tocris; Cat. No. 1254). Incubations with pharmacological agents were performed 30 min before 24 h serum incubated period. All incubations were performed in 12-well plates containing Krebs-Henseleit nutrient solution, in a CO₂ incubator at 37°C and 5% CO₂. Each vascular preparation was tested with a single agent. All agents were diluted in Krebs-Henseleit nutrient solution, which was the vehicle considered.

Measurement of Reactive Oxygen Species

Umbilical cord arteries were maintained in Krebs-Henseleit solution and incubated as previously described. At the end of the incubations, in 96-well plates, the arteries were incubated with the dihydroethidine probe (DHE, 5 µM for 30 min at 37°C, Biotrend; Cat. No. 17084-50) and washed with phosphate-buffered saline (PBS). For each sample, the assay was performed in duplicate. Analyzes were performed by fluorimetry, using Flexstation 3 (Molecular Devices, San Jose, CA), at excitation and emission wavelengths of 358 and 461 nm, respectively. Results are expressed as fluorescence intensity/mg protein. The extracted proteins were quantified by the Bradford method (34).

Quantitative Real-Time Reverse Transcription-PCR

Total RNA was isolated from umbilical cord arteries using TRIzol. RNA was treated with DNAse I (1 U/μL) and 2 μg of RNA was reverse-transcribed in a reaction containing oligo dT (High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher; Cat. No. 4368814). mRNA levels were quantified in triplicate by qPCR (StepOnePlus Life Technologies, Foster City). Specific primers (TaqMan) for real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) were as follows: Nox 1 (Hs01071088_m1), Nox 4 (Hs01379108_m1), and β-actin (Hs01060665_g1), purchased from Life Technologies. qPCR cycling conditions included 10 min at 95°C, followed by 40 cycles at 95°C for 15 s, 60°C for 1 min, and 72°C for 60 s. Dissociation curve analysis confirmed that signals corresponded to unique amplicons. Specific mRNA expression levels were normalized relative to β-actin mRNA levels using the comparative ΔΔCt method.

Lucigenin-Enhanced Chemiluminescence

Lucigenin-derived chemiluminescence was used to determine NADPH oxidase activity. Umbilical cord arteries were incubated as previously described. Briefly, incubated umbilical cord arteries were washed with ice-cold PBS and homogenized in lysis buffer (20 mM KH₂PO₄, 1 mM EGTA, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, and 1 mM PMSF). Fifty microliter of the sample was added to a suspension containing 175 μL of assay buffer (50 mM KH₂PO₄, 1 mM EGTA, and 150 mM sucrose) and lucigenin (5 μM, Sigma-Aldrich; Cat. No. M8010). Chemiluminescence was measured with a luminometer (Orion II Berthold, Pforzheim, GER) before and after stimulation with NADPH (100 µM, Sigma-Aldrich; Cat. No. N7265). A buffer blank was subtracted from each reading. The results are expressed as a fold change in arbitrary units per milligram of protein.

Rho Kinase Activity

Umbilical cord arteries were incubated as previously described. At the end of the incubations, Rho kinase activity was measured with a Rho Kinase Activity Assay Kit (Cell Biolabs; Cat. No. STA-416). Briefly, samples were lysed and added to the 96-well plate and incubated for 60 min under shaking. Diluted anti-phospho-MYPT1 (Thr⁶⁹⁶) was added and incubated for 1 h on an orbital shaker. Subsequently, 100 μL of the diluted horseradish peroxidase-conjugated secondary antibody was added to each well and incubated for 1 h. After the addition of the stop solution, the absorbance of each microwell was read in a spectrophotometer using the 450 nm wavelength.

Western Blot

Umbilical cord arteries were frozen in liquid nitrogen and homogenized in a lysis buffer (50 mM Tris·HCl, 150 mM NaCl, 1% Nonidet P40, 1 mM EGTA, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 1 μg/mL aprotinin, 1 mM sodium orthovanadate, 1 mM PMSF, and 1 mM sodium fluoride). The extracted proteins were quantified by the Bradford method. Proteins (40 μg) were separated by electrophoresis on 12% polyacrylamide gel and transferred onto nitrocellulose membranes. Nonspecific binding sites were blocked with 5% bovine serum albumin in tris-buffered saline containing 0.1% tween 20 (for 1 h at 24°C). Membranes were incubated with antibodies (at the indicated dilutions) overnight at 4°C. Antibodies were used as follows: anti-p-RhoA^Ser188 (1:1,000 dilution; Santa Cruz; Cat. No. sc-32954) and anti-βactin (1:10,000 dilution; Sigma-Aldrich; Cat. No. A3854). After incubation with a secondary antibody (1:5,000 dilution; Abcam; Cat. No. ab6721), signals were obtained by chemiluminescence and quantified densitometrically.

Data and Statistical Analyses

Data were assessed for normality with the Shapiro–Wilk test. Medians and interquartile range values were used for the normally distributed descriptive parameters. Student’s t test were performed for comparing the median values between the groups. Categorical variables were presented with numbers and percentages. χ² test was used to compare categorical variables. The individual concentration-effect curves were fitted into a curve by nonlinear regression. The curves were analyzed by repeated measures Two-way ANOVA with Bonferroni posttest. pD₂ (defined as the negative logarithm of the EC₅₀ values) and maximal response (E_max) were compared by one-way ANOVA with Bonferroni posttest. The results of the molecular experiments were analyzed by Student’s t test or one-way ANOVA, followed by the Bonferroni posttest. Outliers were analyzed and removed by Grubb’s test (α = 0.05). The Prism software, version 9.0 (GraphPad Software, Inc., San. Diego, CA) was used to analyze these parameters as well as to fit the sigmoidal curves. Data are presented as means ± standard error of the mean. The acceptable level of significance was P < 0.05.

RESULTS

A total of 43 women were included in the present study: 15 women of childbearing age who tested positive for COVID-19 and developed the severe form of the disease, 13 healthy women of childbearing age in the control group, and 15 healthy pregnant women. The use of mechanical ventilation, the $\text{P}{\text{a}}_{{\text{O}}_2}$/$\text{F}{\text{I}}_{{\text{O}}_2}$ ratio, and D-dimer concentrations were used to identify the severe form of the disease. In all analyses, the clinical profile of women with COVID-19 was potentially critical (Table 1).

Women who developed severe COVID-19 had significantly higher serum levels of IL-6 and IFN-γ compared with healthy women (Fig. 1, A and B). TNF-α and IL-1β levels did not differ between groups (Figs. 1, C and D). Furthermore, women who developed severe COVID-19 had significantly lower serum levels of IL-10 compared with healthy women (Fig. 1E).

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.

Rings of human umbilical cord arteries from 15 healthy pregnant women were incubated with serum from healthy pregnant women, healthy nonpregnant women, and nonpregnant women with severe COVID-19. There were no significant changes in the serotonin-induced contractile response in rings incubated with serum from healthy pregnant women (Supplemental Fig. S1) and healthy women. However, rings incubated with serum from women with severe COVID-19 showed increased serotonin-induced contractile responses (Fig. 2A, Table 2). The levels of contraction in rings incubated with serum from healthy women and control rings, which were free from any stimuli, were similar. In the presence of the antibody that blocks the IL-6 receptor, tocilizumab; that is, by blocking IL-6-mediated signaling, the reactivity of the arterial segment incubated with serum from women with severe COVID-19 resembled the reactivity of control rings and segments incubated with serum from healthy women (Fig. 2B, Table 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.

Table 2.

E_max and pD₂ values of serotonin-induced contraction in umbilical cord arteries incubated with serum from healthy women and women with severe COVID-19 in the presence of vehicle, tocilizumab, and tiron

	E max	pD2
Control	2.53 ± 0.08	7.01 ± 0.09
Healthy	2.41 ± 0.09	7.15 ± 0.12
COVID-19	3.28 ± 0.11*	7.96 ± 0.08*
COVID-19 + TZ	2.40 ± 0.09#	7.31 ± 0.09#
COVID-19 + tiron	2.37 ± 0.12#	7.29 ± 0.10#

Values are expressed as means ± SE (n = 13–15). Control group is formed by rings of umbilical arteries from healthy women free of any stimulus. One-way ANOVA: *P < 0.05 vs. Control; #P < 0.05 vs. COVID-19. COVID-19, coronavirus disease 2019; E_max, maximal response; pD₂, sensitivity; tiron, superoxide anion scavenger; TZ, tocilizumab (IL-6 receptor antagonist).

Considering that IL-6 is a potent inducer of ROS, we evaluated the reactivity of umbilical cord arteries incubated with serum from women with severe COVID-19 in the presence of vehicle and tiron, a superoxide anion scavenger. Tiron prevented increased contractions to serotonin induced by serum from women with severe COVID-19 (Fig. 2C, Table 2).

We next determined ROS generation by the DHE probe. Umbilical arteries incubated with serum from women with severe COVID-19 exhibited increased ROS generation when compared with control arteries (in the absence of any stimuli). Tocilizumab and tiron prevented increased ROS generation induced by serum from women with severe COVID-19 (Fig. 2D).

We then investigated the potential source of ROS. Umbilical arteries incubated with serum from women with severe COVID-19 showed increased Nox 1, but not Nox4 gene expression (Fig. 3, A and B) compared with control or arteries incubated with serum from healthy women. In addition, tocilizumab prevented the increased Nox 1 gene expression. ML171, a Nox 1 inhibitor, prevented increased contractile responses to serotonin in rings incubated with serum from women with severe COVID-19 (Fig. 3C, Table 3). Lucigenin-derived chemiluminescence was used to determine NADPH oxidase activity in umbilical arteries. Serum from women with severe COVID-19 increased NADPH-dependent ROS generation. This increase was prevented by tocilizumab and ML171 (Fig. 3D).

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.

Table 3.

E_max and pD₂ values of serotonin-induced contraction in umbilical cord arteries incubated with serum from healthy women and women with severe COVID-19 in the presence of vehicle and ML171

	E max	pD2
Control	2.33 ± 0.12	7.16 ± 0.12
COVID-19	3.27 ± 0.09*	8.03 ± 0.09*
COVID-19 + ML171	2.68 ± 0.09#	7.40 ± 0.11#

Values are expressed as means ± SE (n = 13–15). Control group is formed by rings of umbilical arteries from healthy women free of any stimulus. One-way ANOVA: *P < 0.05 vs. Control; #P < 0.05 vs. COVID-19. COVID-19, coronavirus disease 2019; E_max, maximal response; pD₂, sensitivity; ML171, Nox1 inhibitor.

To determine mechanisms by which IL-6-derived ROS induces umbilical cord arteries dysfunction, vascular reactivity was evaluated in the presence of Y27632, an inhibitor of the redox-sensitive protein Rho kinase, which potentiates vasoconstriction. The Rho kinase inhibitor abrogated the increased vasoconstriction to serotonin induced by serum from women with severe COVID-19 (Fig. 4A, Table 4). Arteries incubated with serum from women with severe COVID-19 exhibited increased sensitivity to Rho kinase inhibition compared with control or healthy women-derived serum (Fig. 4B, Table 5).

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-RhoA^Ser188. 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.

Table 4.

E_max and pD₂ values of serotonin-induced contraction in umbilical cord arteries incubated with serum from healthy women and women with severe COVID-19 in the presence of vehicle and Y27632

	E max	pD2
Control	2.41 ± 0.09	7.01 ± 0.09
COVID-19	3.14 ± 0.08*	7.96 ± 0.08*
COVID-19 + Y27632	2.54 ± 0.11#	7.31 ± 0.09#

Values are expressed as means ± SE (n = 13–15). Control group is formed by rings of umbilical arteries from healthy women free of any stimulus. One-way ANOVA: *P < 0.05 vs. Control; #P < 0.05 vs. COVID-19. COVID-19, coronavirus disease 2019; E_max, maximal response; pD₂, sensitivity; Y27632, Rho kinase inhibitor.

Table 5.

E_max and pD₂ values of Y27632-induced relaxation in umbilical cord arteries incubated with serum from healthy women and women with severe COVID-19

	E max	pD2
Control	91.5 ± 3.7	6.89 ± 0.09
Healthy	92.2 ± 2.3	7.01 ± 0.08
COVID-19	90.1 ± 2.5	7.97 ± 0.09*

Values are expressed as means ± SE (n = 13–15). Control group is formed by rings of umbilical arteries from healthy women free of any stimulus. One-way ANOVA: *P < 0.05 vs. Control. COVID-19, coronavirus disease 2019; E_max, maximal response; pD₂, sensitivity.

Rho kinase activity was inferred by its phosphorylated form and by an enzymatic assay. Umbilical arteries incubated with serum from women with severe COVID-19 exhibited increased Rho kinase phosphorylation, compared with control arteries and arteries incubated with serum from healthy women (Fig. 4C, Supplemental Fig. S2). Furthermore, serum from women with severe COVID-19 increased Rho activity in umbilical arteries. Tocilizumab and tiron prevented this increase (Fig. 4D), supporting that IL-6 and ROS are key to the increased Rho kinase activity induced by the serum of women with severe COVID-19.

DISCUSSION

Several reports on the clinical characteristics of patients with COVID-19 have described the presence of bidirectional interaction between COVID-19 and the cardiovascular system, with more severe forms of the disease being associated with circulatory damage and multiple organ failure. This study shows that serum from women with severe COVID-19 promotes umbilical cord artery dysfunction. Vascular dysfunction is linked to proinflammatory cytokines, especially IL-6, redox imbalance, and overactivation of the Rho kinase pathway.

The impact of COVID-19 on the health of pregnant women and fetal development is still unclear. However, pregnant women are more likely to develop severe manifestations of COVID-19 (35). In addition, the discovery of more contagious and dangerous variants and the reporting of more severe manifestations of the disease in individuals with cardiovascular and metabolic risks make pregnant women with obesity, diabetes, or hypertension more susceptible to developing severe COVID-19. During pregnancy, COVID-19 also negatively affects the health of the fetus. A recent meta-analysis study showed that ∼8.8% of newborns had a positive PCR or serologic test, indicating SARS-CoV-2 infection. In addition, a quarter of babies born to COVID-19-positive mothers developed symptoms such as fever, tachypnea, and shortness of breath (36).

For the reasons described and in a situation similar to severe COVID-19, we propose to evaluate the function of the umbilical arteries, which connect the mother to the fetus, allowing the exchanges necessary for fetal development (37). This study shows that serum from women with severe COVID-19 promotes umbilical artery hyperreactivity. Recently, our group demonstrated that in severe COVID-19, vascular function is directly affected. Endothelial cells incubated with serum from COVID-19 patients exhibit shedding of the glycocalyx, increasing the possibility of thrombotic events and procoagulant effects (38). In addition, directly, SARS-CoV-2 infection impairs mitochondrial function and activates inflammatory signaling by toll-like receptor 9 in endothelial cells (39).

Elevated levels of IL-6 are observed in severe cases of COVID-19, being considered one of the main actors of the famous cytokine storm (40). In this study, tocilizumab, a monoclonal antibody against the IL-6 receptor, prevented the vascular hyperreactivity induced by the serum of women with severe COVID-19. In a recent study, the inhibition of IL-6 by tocilizumab improved vascular function and arterial elastic properties, leading to a greater increase in effective cardiovascular work in patients with severe COVID-19 (41).

The cytokine storm caused by SARS-CoV-2 results in the excessive production of ROS. This fact, associated with the reduction of antioxidant defenses, establishes the condition of oxidative stress, which contributes to the pathogenesis of several diseases, including cardiovascular diseases (42). Tocilizumab reduced ROS production caused by the serum of women with severe COVID-19. Furthermore, using a superoxide anion scavenger, we confirmed the participation of ROS in vascular hyperreactivity. In fact, in the vascular system, ROS promotes inflammation, hypertrophy, fibrosis, activation of contractile pathways, and reduction of relaxing pathways (24, 25).

There are few studies linking NADPH oxidase activity with COVID-19 pathogenesis. It is possible that ROS, via NADPH oxidase, plays a prominent role in the cardiovascular complications associated with COVID-19. For example, Nox 2 activation is associated with thrombotic events in patients with severe COVID-19 (43). We demonstrate in this study that serum from women with severe COVID-19 increases the expression and activity of Nox 1 and that these events are caused by IL-6. The mechanisms by which IL-6 leads to increased expression and activity of Nox 1 are still unclear. However, in vascular cells under experimental conditions of hyperglycemia and senescence, IL-6 increases Nox 1 dependent on the transcription factor NFκB (44).

The RhoA/Rho kinase pathway is a potential target for increased ROS production, derived from IL-6 activity, and a potential contributor to the increased contraction in arteries incubated with serum from women with severe COVID-19. ROS generation stimulates Rho translocation and Rho kinase activation, which leads to the inhibition of myosin light chain phosphatase, resulting in smooth muscle contraction in the endothelium-denuded aorta (26, 45). In this study, the Rho kinase inhibitor Y27632 prevented the increased contractions to serotonin. Y27632-induced relaxation provided additional evidence that serum from women with COVID-19 increased RhoA/Rho kinase activity since umbilical arteries incubated in this condition were more sensitive to Y27632-induced relaxation. The increase in Rho kinase activity was abolished by tocilizumab and tiron, indicating that IL-6 and ROS contribute to the increased activity of this kinase. Umbilical arteries of pregnant women with preeclampsia show hyperreactivity to 8-iso prostaglandin E2. This effect is prevented by the Rho kinase inhibitor, Y27632 (46).

As discussed in a recent review, Rho kinase inhibitors appear to be potentially effective in preventing and treating complications in severe COVID-19. It is possible that its beneficial effects may be mediated via the modulation of the immune system and protection of respiratory tract cells (47). All together, these data indicate a great potential for IL-6 and Rho kinase inhibitors in the therapy of umbilical and cardiovascular disorders in severe COVID-19 during pregnancy.

An analysis of the function of the umbilical arteries of pregnant women with severe COVID-19 would enormously add to the present study. However, this specific population is scarce, making it difficult to evaluate how the disease affects pregnancy. To overcome this limitation, we recruited women of childbearing age who developed severe COVID-19 and healthy pregnant women and addressed the effects of their serum on umbilical artery reactivity. The incubation of umbilical arteries with serum from women with severe COVID-19 allowed us to identify potential mechanisms that occur in pregnancy-associated COVID-19 and that may account for pregnancy complications in this condition.

While this study expands upon existing data sets, it has some limitations, such as the fact that serum samples were collected during the early period of the pandemic, and the number of samples collected was limited. In the period in which the samples were recruited, there were no distinctions or knowledge about the possible variants of the SARS-CoV-2 virus. Therefore, we believe that the Gamma P.1 variant is major in the selected individuals. It is possible that other variants, with different degrees of pathogenicity, may generate different magnitudes of effects on umbilical cord reactivity. Furthermore, donors were recruited before vaccination started, so the potential effect that vaccines could have on the umbilical arteries’ response should be also evaluated in future assays. Similarly, serums were also obtained from healthy and nonpregnant individuals from whom no information was provided due to data protection assignments. Future studies may determine whether the response seen in our study would be different depending on the serum donor profile.

Perspectives and Significance

As the massive impact of the pandemic COVID-19 proceeded, there were growing concerns about the future of studies involving the cardiovascular impact caused by this condition. Likely, the future everyday practice will also have a significant impact on cardiovascular disease prevalence. In fact, we are still facing an increasing number of myocardial infarction rates and postmyocardial infarct complications in those patients who had COVID-19, and also severe manifestations of COVID-19 in pregnant women. The logical tragic consequence of this scenario might be a superior incidence of morbidity that will need chronic care. Acknowledging the important consequences of the disease is important. We here provide a perspective on two aspects strictly related to COVID-19 pathophysiology: the multiple implications for vascular physiology, which contribute to the pathogenesis of several cardiovascular diseases, and the indirect ones related to the impact on fetal development and newborn health. Integrating our knowledge of the biological processes impacted by the virus with clinical findings might significantly improve our understanding of the potential mechanisms underlying COVID-19 complications, covering the way toward the development of preventative and therapeutic solutions. Mechanistically, the interaction between circulating proinflammatory cytokines, especially IL-6, and the redox imbalance is likely to have a central role in disease pathogenesis (Fig. 5), especially in cardiovascular manifestations of this disease, and this interaction is a potential target for the prevention and treatment of COVID-19.

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.

DATA AVAILABILITY

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

SUPPLEMENTAL DATA

Supplemental Figs. S1 and S2: https://doi.org/10.6084/m9.figshare.21964712.v1.

GRANTS

This study was funded by the National Council for Scientific and Technological Development (CNPq), under Grant Agreement number 433898/2018-06 to R.M.C. and Sao Paulo Research Foundation (FAPESP), under Grant Agreement number 2013/08216-2 to R.C.T. (Center for Research in Inflammatory Diseases; CRID).

DISCLOSURES

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.

AUTHOR CONTRIBUTIONS

C.R.A., R.C.T., N.S.L., and R.M.C. conceived and designed research; C.R.A., J.F.L., M.R.M., J.V.A., A.E.S.C., L.C.B.C., C.D.A-M., M.A.-M.,, C.B., P.L.-J., R.C.T., N.S.L., and R.M.C. performed experiments; C.R.A., J.F.L., M.R.M., J.V.A., A.E.S.C., L.C.B.C., C.D.A-M., M.A.-M., C.B., P.L.-J., R.C.T., N.S.L., and R.M.C. analyzed data; C.R.A., N.S.L., and R.M.C. interpreted results of experiments; C.R.A. and R.M.C. prepared figures; C.R.A., N.S.L., and R.M.C. drafted manuscript; C.R.A., R.C.T., N.S.L., and R.M.C. edited and revised manuscript; C.R.A., R.C.T., N.S.L., and R.M.C. approved final version of manuscript.