Clonal Endotheliopoiesis in Patients with Carotid Atherosclerosis

Introduction: It has been recognized that certain somatic mutations causing clonal hematopoiesis in hematological malignancies, increase with age in the normal population. This condition is termed as CHIP (Clonal hematopoiesis of indeterminate potential). Population studies demonstrated that CHIP mutations in peripheral blood cells (PBC) elevate the risk of atherosclerotic cardiovascular diseases. Animal studies have shown that monocytes derived from the CHIP-mutant clones caused excessive inflammatory responses, increased necrotic core formation, and significantly enlarged the size of atherosclerotic plaques. Although the role of CHIP mutations in atherosclerosis is increasingly recognized, there are no studies on somatic mutations affecting endothelial cells (EC) and their impact on endothelial functions and atherosclerosis. In this study, we aimed to evaluate the somatic clonal mutations in EC from atheroma material extracted from patients with symptomatic carotid artery atherosclerosis.

Methods: Thirty consecutive patients who developed carotid artery stenosis due to atherosclerosis and underwent carotid endarterectomy were included in the study. None of the patients had hematological or solid malignancies. Atheroma tissue, venous blood, and buccal swab samples of the patients were collected. The patient's atherosclerotic risk factors, personal information, and comorbidities were recorded. CD31⁺CD45^- EC were isolated from the carotid atheroma samples through flow cytometry. DNA isolated from EC, PBC, and oral epithelial cells was examined using whole exome sequencing (WES), followed by bioinformatics analysis to investigate somatic mutations.

Results: Among the 30 patients included in the study, the male/female ratio was 22/8, and the median age was 70 (range 58-84). Seventeen patients were diagnosed with diabetes mellitus and 22 with hypertension. All patients had atherosclerotic lesions of varying degrees in other arteries as well, and 14 patients had undergone coronary stenting and/or bypass surgery. After WES and filtering for variant allele frequency (VAF) ratio>2%, 12 different CHIP mutations were identified in the atherosclerotic carotid EC of 10 patients. To determine if the CHIP mutations detected in the EC of 10 patients were somatic, oral epithelial cells were evaluated using WES. PBCs were also assessed to evaluate the relationship of the mutations with hematopoietic stem cells. Four distinct CHIP mutations found in 4 patients were present in both EC and PBC but not in oral epithelial cells, indicating that these were somatic mutations. The somatic CHIP mutations (NSD1, rs1763371836, C>A, G; CCND2, rs1864224655, A>C; BCORL1, rs1929229770, C>T, and ETV6, rs139975161, G>A) found in EC affect genes that regulate cell proliferation and differentiation. These are missense mutations with VAF ratios ranging from 38% to 97%. When the data of the 10 patients who underwent WES for EC, PBC, and oral epithelial cells were filtered for VAF≤ 2%, different combinations of somatic CHIP mutations were found in the EC and/or PBC of all 10 patients.

Conclusions: Our data indicate that somatic clonal mutations develop in atherosclerotic EC similarly to those in hematopoietic cells. We defined this condition as ‘clonal endotheliopoiesis’. The second important finding of our study is that the specific somatic mutations we identified in EC were also found in PBC at similar VAF ratios. This supports the idea that hemangioblast-like hematopoietic stem cells persist postnatally and are the source of these somatic mutations. The third important finding of our study is that, when considering low (≤2%) VAF ratios, multiple somatic clonal mutations were present in various combinations in the EC and/or PBC of all 10 patients examined. We propose that atherosclerosis should be considered as a manifestation of chronic myeloid neoplasia. According to our study, EC in atherosclerosis are not innocent bystanders but active players whose functions are altered by somatic mutations. Understanding the effects of these somatic mutations in EC and their roles in atherosclerosis could enable targeted therapy approaches for atherosclerosis, similar to those used in hematological malignancies.