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Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response
Published on: September 15, 2017
Quantifying endothelial damage by digital droplet polymerase chain reaction (PCR) of endothelial cell-free DNA in
Tiphaine Ruggeri1,2, Gertrud Wiedemann1,2, Noëlia Schärz1,2
1Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, Bern, Switzerland.
Insights
Endothelial cell damage in COVID-19 patients was measured using a novel assay. Elevated cell-free DNA from endothelial cells correlated with disease severity, indicating microvascular damage.
Area of Science:
- Cardiovascular Biology
- Molecular Diagnostics
- Infectious Diseases
Background:
- COVID-19 (caused by SARS-CoV-2) induces systemic inflammation and organ dysfunction.
- Microvascular complications, linked to endothelial cell issues and immunothrombosis, are key in COVID-19.
- Cell-free DNA (cfDNA) release occurs upon cell activation/death; endothelial cfDNA may indicate microvascular damage severity.
Purpose of the Study:
- Develop a specific assay for endothelial cell-derived DNA.
- Quantify microvascular damage in COVID-19 patients.
Main Methods:
- Developed a methylation-specific digital droplet PCR assay.
- Targeted the NOS3 gene promoter to measure endothelial cfDNA.
- Monitored COVID-19 patients longitudinally (inclusion, day 11, day 28).
Main Results:
- Endothelial cfDNA levels increased with COVID-19 severity.
- Severe COVID-19 patients had the highest endothelial cfDNA.
- Mild cases showed normal levels; moderate cases had transiently elevated levels; severe cases had persistently high levels.
Conclusions:
- Demonstrated endothelial cell damage in COVID-19 patients.
- Developed a specific assay for endothelial cfDNA.
- Endothelial cfDNA levels correlate with COVID-19 disease severity.
Background:
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.
Objectives:
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.
Methods:
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.
Results:
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.
Conclusion:
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.
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