Huakang Wu1, Qizhi Yao, Alan Lumsden, Changyi Chen. 1. Molecular Surgeon Research Center, Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine and the Methodist Hospital, Houston, Texas 77030, USA.
Abstract
BACKGROUND: Heterogeneity of endothelial cells may affect angiogenesis, vascular healing, and cardiovascular disease formation. The objective of this study was to identify and characterize populations of human coronary artery endothelial cells (HCAECs), their gene expression levels, and response to TNF-alpha stimulation. MATERIALS AND METHODS: Commercial HCAECs were cultured with EGM-2 complete medium. Cell population was determined by flow cytometry analysis based on size-scatter distribution. Gene expression for each population with or without TNF-alpha (1 ng/ml) stimulation for 16 h was also studied by flow cytometry analysis with proper gating and fluorescence labeling of antibodies. RESULTS: Two distinguished populations, HCAEC-I (small) and HCAEC-II (large), were identified through all passages (from 2 to 10) during cultures. Both HCAEC-I and HCAEC-II showed more than 90% positive staining for both von Willebrand factor and CD31 and were negative for CD34 and CD4 (less than 2%). HCAEC-I had substantially higher expression of cadherin-5 (71.91% versus 51.07%) than HCAEC-II. HCAEC-I also expressed much higher levels of vascular endothelial growth factor receptor-2 (VEGF-R2) (17.35% versus 0.77%), endothelial nitric oxide synthase (eNOS) (44.64% versus 2.54%), VCAM-1 (6.08% versus 1.18%), E-selectin (18.68% versus 1.42%), CXCR4 (61.05% versus 7.98%), and CCR5 (48.66% versus 1.97%) than HCAEC-II. HCAEC-II, however, had substantially higher expression of P1H12 (93.07% versus 58.73%) and ICAM-1 (94.37% versus 58.62%) than HCAEC-I. Following TNF-alpha stimulation, both populations substantially increased the expression of ICAM-1, VCAM-1, and E-selectin. HCAEC-I, however, had much higher expressions of VEGFR-2, eNOS, CXCR4, and CCR-5 than HACEC-II after TNF-alpha stimulation. CONCLUSIONS: These data demonstrate that commercial HCAECs have two distinguished populations with different gene expression patterns and different responses following TNF-alpha stimulation. This study indicates that the heterogeneity of HCAECs may have biologic or pathologic significances in coronary arteries.
BACKGROUND: Heterogeneity of endothelial cells may affect angiogenesis, vascular healing, and cardiovascular disease formation. The objective of this study was to identify and characterize populations of human coronary artery endothelial cells (HCAECs), their gene expression levels, and response to TNF-alpha stimulation. MATERIALS AND METHODS: Commercial HCAECs were cultured with EGM-2 complete medium. Cell population was determined by flow cytometry analysis based on size-scatter distribution. Gene expression for each population with or without TNF-alpha (1 ng/ml) stimulation for 16 h was also studied by flow cytometry analysis with proper gating and fluorescence labeling of antibodies. RESULTS: Two distinguished populations, HCAEC-I (small) and HCAEC-II (large), were identified through all passages (from 2 to 10) during cultures. Both HCAEC-I and HCAEC-II showed more than 90% positive staining for both von Willebrand factor and CD31 and were negative for CD34 and CD4 (less than 2%). HCAEC-I had substantially higher expression of cadherin-5 (71.91% versus 51.07%) than HCAEC-II. HCAEC-I also expressed much higher levels of vascular endothelial growth factor receptor-2 (VEGF-R2) (17.35% versus 0.77%), endothelial nitric oxide synthase (eNOS) (44.64% versus 2.54%), VCAM-1 (6.08% versus 1.18%), E-selectin (18.68% versus 1.42%), CXCR4 (61.05% versus 7.98%), and CCR5 (48.66% versus 1.97%) than HCAEC-II. HCAEC-II, however, had substantially higher expression of P1H12 (93.07% versus 58.73%) and ICAM-1 (94.37% versus 58.62%) than HCAEC-I. Following TNF-alpha stimulation, both populations substantially increased the expression of ICAM-1, VCAM-1, and E-selectin. HCAEC-I, however, had much higher expressions of VEGFR-2, eNOS, CXCR4, and CCR-5 than HACEC-II after TNF-alpha stimulation. CONCLUSIONS: These data demonstrate that commercial HCAECs have two distinguished populations with different gene expression patterns and different responses following TNF-alpha stimulation. This study indicates that the heterogeneity of HCAECs may have biologic or pathologic significances in coronary arteries.
Authors: A Paez; A R Méndez-Cruz; E Varela; E Rodriguez; J Guevara; L Flores-Romo; L F Montaño; F A Massó Journal: Clin Exp Immunol Date: 2005-09 Impact factor: 4.330