Abstract 25 Cord Blood Mononuclear Cells and Cord Blood Plasma Alter Human Cardiac Endothelial Cell Angiogenic Sprouting and Growth In Vitro

Abstract

Abstract Introduction The mononuclear cell fraction of human placental cord blood (CBMNC) comprises immune cells, hematopoietic, mesenchymal and other stem or progenitor cells. CBMNCs are proposed to afford therapeutic benefit based on their paracrine release of multiple cytokines, growth factors and other proteins that stimulate signalling for wound healing, including attenuation of inflammation and fibrosis. Whether CBMNCs augment angiogenic sprouting and tube formation by endothelial cells in human ventricular myocardium is not well characterised. Objectives The aim of this study was to investigate the in vitro influence of CBMNCs on endothelial cell angiogenic-like growth. Methods A tube formation assay for human donor primary ventricular myocardium microvascular endothelial cells (HMVECs), (3 female donors, age 48-50 yrs, 3 replicates per treatment group) was established using Matrigel-coated 96-well plates (62,500-78,125 cells/cm2). Cells from passage 5-8 were used for all experiments.. Angiogenic processes, as characterized by endothelial sprouting followed by tube extension into honeycomb-like mesh structures, were imaged over 24 hours. HMVECs were treated with CBMNCs at a concentration of 1:10 (MNC:HMVEC) or cord blood plasma (1μg/μL) at 0h, 3h, or 6h after the HMVECs were seeded. The cells were visualized by phase contrast microscope and images (4x magnification) were analysed using the Angiogenesis Analyzer plugin in FIJI ImageJ. Size exclusion chromatography and multiplex ELISAs were performed to determine the presence of analytes in media collected from each well. Results CBMNC-treated HMVECs exhibited greater average total elongated branching length and more tube-like structured meshes, compared to untreated controls. MMP-1, MMP-2, MMP-9, MMP-10, TIMP-1, TIMP-2, angiopoietin-2, EGF, endoglin, endothelin-1, FGF-2, follistatin, HB-EGF, IL-8, PLGF, VEGF-A, and VEGF-C were detected in extracellular media during in vitro tube formation. With CBMNC treatment, only MMP-9 was increased by 475.94%, whereas angiopoietin-2, follistatin, and PLGF levels were decreased by 27.90%, 15.90%, and 26.18% respectively. In contrast, CB plasma-treated HMVECs did not form tube sprouts, instead an intense endothelial cell proliferation was observed which was associated with increased MMP-2, MMP-9, endothelin-1 and IL-8 levels with decreased angiopoietin-2, HB-EGF, PLGF, and endoglin levels. Discussion CBMNC treatment enhanced pro-angiogenic processes in HMVECs in vitro through increased branching and network formation, whereas CB plasma evoked cell proliferation.