Direct observation and quantitative analysis of spatiotemporal dynamics of individual living monocytes during transendothelial migration

• Published in: Atherosclerosis 2004-11, Vol.177 (1), p.19-27
• Main Authors: Hashimoto, Ken, Kataoka, Noriyuki, Nakamura, Emi, Asahara, Hiroko, Ogasawara, Yasuo, Tsujioka, Katsuhiko, Kajiya, Fumihiko
• Format: Article
• Language: English
• Subjects: 3D-live-single cell imaging; Atherosclerosis; Atherosclerosis (general aspects, experimental research); Biological and medical sciences; Blood and lymphatic vessels; Cardiology. Vascular system; Cardiovascular system; Cell Movement; Cells, Cultured; Endothelium; Endothelium, Vascular - cytology; Humans; Interaction of monocyte and endothelial cell; Investigative techniques of hemodynamics; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Monocyte transendothelial migration; Monocytes - cytology; Pneumology; Pulmonary hypertension. Acute cor pulmonale. Pulmonary embolism. Pulmonary vascular diseases
• ISSN: 0021-9150; 1879-1484
• DOI: 10.1016/j.atherosclerosis.2004.06.016

To visualize and quantitatively analyze spatiotemporal dynamics of individual living monocytes during transendothelial migration (TEM). We developed an in vitro new experimental system using confocal laser scanning microscope with following two improvements: (1) ultra thin collagen gel layer (30–50 μm thick) constructed under human umbilical vein endothelial cell layer for three-dimensional observation with high magnification; (2) appropriate fluorescent labeling of living monocytes and endothelial cells to keep highest cell activity. Individual monocytes behaved quite diversely. Approximately 70% of adhered monocytes directionally crawled to intercellular junction, and started invasion. Time from adhesion to start of invasion was 8.6 ± 5.4 min (mean ± S.D., n = 61 monocytes). Approximately 80% of such invading monocytes completed TEM, but remaining 20% of once invading monocytes hesitated transmigration, and returned onto the endothelial surface. Time from start to finish of invasion was 6.3 ± 3.2 min (mean ± S.D., n = 53 monocytes). Using our collagen gel-based newly-developed system, we visualized and quantitatively analyzed detailed spatiotemporal, three-dimensional dynamics of individual living monocytes during TEM. We revealed that monocytes encountered at least two hurdles, at starting invasion, and leaving endothelium, to achieve complete TEM. Approximately 56% (80% of 70% of adhered monocytes) passed both hurdles.