Identification of Biomarkers for Early Detection of Diabetes Complications by Characterisation of Circulating Endothelial Cells in Human Blood Using Single-cell RNA Sequencing.

There is one particular type of ECs, which are circulating in the blood system called circulating endothelial cells (CECs). The common concept of CECs only refers to mature ECs that have been shed from the vascular wall due to impaired vascular functions caused by e.g. diabetes, and hypertension. Mature CECs have thus been proposed as highly valuable targets for diagnosis, treatment, and prognosis of cardiovascular diseases. In healthy individuals, the number of CECs varies from 0 to 7000 per ml of blood and increases significantly in patients with hypertension, diabetes, and cardiovascular diseases. In addition to mature EC, which circulates as CEC. Immature EC, called "circulating endothelial progenitor cells" (cEPC), is reduced in patients with cardiovascular disorders. Based on the highly heterogeneous phenotype of vascular ECs found within and between tissues, it is not surprising that all currently known surface markers for both mature CEC and cEPC are debatable as these studies did not consider ECs heterogeneity. Proliferative diabetic retinopathy (PDR) is a well-defined diabetes complication caused by ECs dysfunction. This study will examine human blood (from healthy individuals, patients with diabetes and PDR or diabetic maculopathy, or without eye disease) and thereby characterize both CEC and cEPC by single-cell RNA sequencing.

This study aims to discover early CEC biomarkers for diabetes complications exemplified with diabetic eye diseases.

Recruitment and scRNA-seq: Blood samples from healthy individuals and patients with diabetes and PDR, diabetic maculopathy, or without eye disease will be recruited from Steno Diabetes Center Aarhus and the Department of Ophthalmology at Aarhus University Hospital. The PBMCs will be freshly isolated from the blood samples followed by CEC enrichment by Fluorescent-Activated Cell Sorting of CD45-CD31+ live cells. Ethical and GDPR approval has been obtained.

ScRNA-seq of CECs: The CD45-CD31+ cells will be processed immediately to single-cell capture and -barcoded cDNA synthesis using the Next GEM Single Cell 3' GEM kit (10x genomics). NGS libraries will be prepared and sequenced using the MGI2000 sequencers from BGI. GenomeDK will be used to supercomputing clusters followed by scRNA-seq analysis and visualization. Integrative multi-model scRNA-seq analyses: Standard scRNA-seq analyses will be carried out to stratify the differential subtypes of CECs and cEPCs and identify ECs subtypes associated with the development of proliferative diabetic retinopathy and diabetic maculopathy. Integrative analysis of scRNA-seq results aims to discover CECs biomarkers which can be used for early diagnosis, prevention and treatment of diabetes complications.