key: cord-0879814-nycanol5
authors: Dupont, Vincent; Kanagaratnam, Lukshe; Goury, Antoine; Poitevin, Gaël; Bard, Mathieu; Julien, Gauthier; Bonnivard, Michel; Champenois, Vanessa; Noel, Violaine; Mourvillier, Bruno; Nguyen, Philippe
title: Excess soluble fms-like tyrosine kinase 1 correlates with endothelial dysfunction and organ failure in critically ill COVID-19 patients
date: 2020-07-16
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1007
sha: deacd6e6db4905fc0382af587bc4873bfd5d6462
doc_id: 879814
cord_uid: nycanol5

Excess soluble fms-like tyrosine kinase 1 (sFlt-1), a soluble inhibitor of the vascular endothelial growth factor pathway, has been demonstrated to promote endothelial dysfunction. Here we demonstrate that sFlt-1 plasma levels correlate with respiratory symptoms severity, expression of endothelial dysfunction biomarker and incidence of organ failure in COVID-19 patients.

M a n u s c r i p t 3 Background Acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for a global pandemic by causing a spectrum of phenotypes varying from asymptomatic presentation to acute respiratory distress syndrome (ARDS) requiring admission in intensive care unit (ICU). The underlying mechanisms explaining why some coronavirus disease 2019 (COVID-19) patients develop lifethreatening symptoms, while others do not, remain incompletely elucidated. Several arguments indicate that the most severe forms of COVID-19 may be related to an endothelial injury [1, 2] .

The soluble fms-like tyrosine kinase 1 (sFlt-1) is a splice variant of the receptor 1 for Vascular Endothelial Growth Factor A (VEGF-A) that lacks the cytoplasmic and transmembrane domains. By binding to its circulating ligand with high-affinity, sFlt-1 inhibits the VEGF-A pathway and impairs endothelial cell homeostasis [3] . Overexpression of sFlt-1 has been well demonstrated to promote endothelial dysfunction, notably during preeclampsia [4] .

The objectives of this study were to compare admission sFlt-1 levels in COVID-19 patients with mild to moderate and severe symptoms, and to analyze sFlt-1 levels in critically-ill COVID-19 patients with or without organ failure.

A c c e p t e d M a n u s c r i p t 4

Patients samples were collected from our local COVID-19 biobank: a single-center prospective cohort of adult patients hospitalized in University Hospital of Reims (northeastern 

All samples were drawn in EDTA tubes, centrifuged at 1,500 x g for 15 minutes at room temperature, then frozen at -80°C within one hour of collection. Quantikine ELISA kits and quantitative controls were purchased to perform sFlt-1 and sVCAM-1 measurements (R&D systems, Minneapolis, MN, USA).

Quantitative variables are reported as the median (interquartile range) and qualitative data as number and percentage. SFlt-1 plasma levels in COVID-19 patients with mild to moderate or severe symptoms were compared using Mann Whitney test. Correlation between sFlt-1 and sVCAM-1 levels was studied using Spearman's correlation test. SFlt-1 levels in critically ill COVID-19 patients with or without organ failure were compared using the Mann Whitney test. Repeated measures correlation coefficient was calculated to assess the correlation between sFlt-1, sVCAM-1 and SOFA scores taking into account measures at day 0, 3, 7 and 14. A p value < 0.05 was considered statistically significant. All analyses were performed using XLSTAT version 2020.1.1 and R version 3.6.1 A c c e p t e d M a n u s c r i p t 6

All subjects provided written informed consent to participate in the study. This study was approved by ethics committee (CPP EST-III 20.04.13, April 23 th 2020) and was registered in Clinicaltrial.gov (NCT04394195).

We included 46 severe and 10 mild to moderate COVID-19 patients. Among severe patients, and SOFA scores (r=0.23; p=0.15) over time.

Innovative investigations are direly needed to advance our understanding of the most severe forms of COVID-19, and ultimately develop new therapeutic paradigms that offer safe and effective alternatives to ICU care. We focused specifically on sFlt-1, and report that high sFlt-1 circulating levels are associated with severe COVID-19 phenotype. Moreover, we found a correlation between sFlt-1 and the endothelial dysfunction biomarker sVCAM-1 in critically ill COVID-19 patients at ICU admission.

SARS-CoV-2 binds to, and downregulates Angiotensin-Converting-Enzyme 2 (ACE2), which leads to an increase in angiotensin II bioavailability [5] . The interaction between angiotensin II and its receptor AT1 has been found to promote sFlt-1 upregulation during hypoxia [6] . Moreover, animal models have previously demonstrated that excess sFlt-1 reduces the phosphorylation of endothelial nitric oxide (NO) synthase, which leads to a decreased NO formation, and an increase in oxidative stress and angiotensin sensitivity [7] . Interestingly, patients at higher risk of developing severe phenotypes of COVID-19 (with hypertension, obesity and diabetes) are well known to exhibit chronically lower NO bioavailability [8, 9] .

Our results suggest an association between sFlt-1 upregulation and organ failure occurrence in critically-ill COVID-19 patients. High sFlt-1 levels have been previously reported in patients with bacterial sepsis. Similarly, biological markers of sepsis-associated endothelial dysfunction and sepsisinduced immunosuppression have also been noticed in COVID-19 patients [10] . Taken together, these data suggest that the disease mechanism of COVID-19 involves common pathogenic processes A c c e p t e d M a n u s c r i p t 8 with bacterial sepsis, and support the hypothesis of a viral sepsis [11] . However, compared with previous reports of bacterial sepsis, we noticed drastically higher sFlt-1 levels in COVID-19 patients, despite the absence of bacterial coinfection [12] .

While admission sFlt-1 levels were associated with severe respiratory symptoms and both sVCAM-1 levels and the incidence of organ failure during ICU stay, our results did not find any correlation between sFlt-1, sVCAM-1 and SOFA score over the time in critically-ill patients. The fact that no further correlation was found over time may suggest that, if available, a potential therapeutic intervention should be considered in the early stages of the natural disease evolution.

While our results need to be replicated in larger cohorts, this study may be clinically relevant because excess sFlt-1 associated endothelial dysfunction is potentially reversible using a phosphodiesterase-5 inhibitor. The ability of the FDA approved sildenafil to increase cGMP levels and enhance NO signaling makes it of particular interest for patients at high risk of developing severe COVID-19 [7] . Both its safety and efficacy remain to be established in COVID-19.

In conclusion, our findings strongly suggest that excess sFlt-1 could be an important determinant of COVID-19 associated endothelial and organ dysfunction. A c c e p t e d M a n u s c r i p t 13 Figure 1 

COVID-19, ACE2, and the cardiovascular consequences

Endothelial cell infection and endotheliitis in COVID-19

Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1): a dual regulator for angiogenesis

Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia

SARS and MERS: recent insights into emerging coronaviruses

Angiotensin II induces soluble fms-Like tyrosine kinase-1 release via calcineurin signaling pathway in pregnancy

Soluble fms-like tyrosine kinase 1 promotes angiotensin II sensitivity in preeclampsia

Vascular inflammation, insulin resistance, and reduced nitric oxide production precede the onset of peripheral insulin resistance

Nitric oxide in hypertension

Clinical features of patients infected with novel coronavirus in Wuhan

SARS-Cov-2 and viral sepsis: observations and hypothesis

The association of endothelial cell signaling, severity of illness, and organ dysfunction in sepsis

We thank the patients and their families who made this study possible; Maryse Strady and Clotilde Fauvet for samples management; Grace Stockton for English language proofreading.

None.

The authors declare no competing interests.A c c e p t e d M a n u s c r i p t 10