Self-regulation of TNF- α Induces Dysfunction of Endothelial Colony-forming Cells from Patients with Venous Thromboembolic Disease

Background. Endothelial colony-forming cells (ECFCs) contribute to postnatal vasculo-genesis. In venous thromboembolic disease (VTD), they are functionally abnormal and produce high concentrations of TNF-alpha.Objective. To analyze the TNF-alpha signaling pathway and its relationship with the ex-pression of cell-cycle regulators.Methods. Mononuclear cells (MNCs) were collected from the peripheral blood of 20 healthy human volunteers (controls) and 30 patients with VTD matched by age (20 -50 years) and sex to obtain ECFCs. We analyzed the relative quantification of the gene tran-scripts of TNF, NFkB1, PLAU, HMOX1, GSS, eNOS, CDKN1A, and CDKN1B through quantitative RT-PCR (qRT-PCR assays). Identification of NF -KB and activated targets of each pathway: NF -KB (Ser536); IKB alpha (Ser32/Ser36); p38 (Thr180/Tyr182) JNK (Thr183/Tyr185), p53 and cell-cycle regulators: p16, p18, p21, p27, p57, Cyclin D, Cyclin E, Cyclin A, Cyclin B, CDK2, CDK4; cell-cycle status was determined by KI-67 and 7-AAD. Cells were analyzed with flow cytometry and the FlowJo vX software.Results. In ECFCs from VTD patients, TNF-alpha receptor and NFkB were overexpressed and hyper-phosphorylated; eNOS and HMOX1 were down-regulated; cell-cycle regula-tors (p53, p18, p21) were elevated. In addition, the cell cycle was locked in the G2 phase.Conclusions. Our results strongly suggest that these molecular alterations in the pathway of TNF-alpha and cell cycle regulation induce endothelial dysfunction, reduced proliferation potential and vascular regeneration, and consequently, the occurrence of new thrombotic events. (c) 2022 Instituto Mexicano del Seguro Social (IMSS). Published by Elsevier Inc. All rights reserved.