# Quantifying endothelial damage by digital droplet polymerase chain reaction (PCR) of endothelial cell-free DNA in COVID-19 patients

## TL;DR

This study shows that measuring endothelial cell-free DNA in blood can reveal microvascular damage severity in COVID-19 patients.

## Contribution

A methylation-specific digital droplet PCR assay was developed to quantify endothelial cell-derived DNA in COVID-19 patients.

## Key findings

- Endothelial cell-free DNA levels increased with disease severity in COVID-19 patients.
- Severe cases showed persistently high endothelial cell-free DNA levels over time.
- Mild cases had endothelial DNA levels similar to healthy controls with no significant changes.

## Abstract

COVID-19, caused by SARS-CoV-2, triggers severe systemic inflammation and multiple organ dysfunction. Microvascular complications, potentially arising from endothelial cell infection and/or immunothrombosis, play a central role in the disease's pathophysiology. Upon cell activation and/or cell death, cells release cell-free DNA (cfDNA) into the circulation, and cfDNA derived specifically from endothelial cells may serve as a marker of microvascular damage severity.

In this study, we aimed to develop an assay to specifically measure endothelial cell-derived DNA as a marker of microvascular damage in COVID-19 patients.

In this study, we developed a methylation-specific digital droplet polymerase chain reaction assay targeting the promoter of the NOS3 gene to quantify circulating endothelial cell-derived cfDNA in COVID-19 patients followed longitudinally at inclusion, day 11, and day 28.

Total cfDNA and endothelial-specific cfDNA levels significantly increased with COVID-19 disease severity, with the highest levels in patients with severe COVID-19. Notably, patients with mild COVID-19 showed endothelial cfDNA levels comparable to those of healthy controls, and levels remained stable from inclusion through day 28. In contrast, patients with moderate disease severity showed significantly elevated endothelial cfDNA levels compared with controls, which declined over time. Patients with severe COVID-19 displayed persistently high endothelial cfDNA levels throughout the observation period.

Using a digital droplet polymerase chain reaction assay specific for cfDNA from endothelial cells, we demonstrated endothelial cell damage in patients with COVID-19 that correlated with disease severity.

•COVID-19 may cause microvascular injury by damaging endothelial cells.•We monitored endothelial cell-free DNA in COVID-19 patients across 3 time points.•Endothelial cell-free DNA rose with increasing disease severity and remained high in severe cases.•The findings show that endothelial injury closely reflects COVID-19 severity and disease progression.

COVID-19 may cause microvascular injury by damaging endothelial cells.

We monitored endothelial cell-free DNA in COVID-19 patients across 3 time points.

Endothelial cell-free DNA rose with increasing disease severity and remained high in severe cases.

The findings show that endothelial injury closely reflects COVID-19 severity and disease progression.