Lymphatic Endothelial Cell Adhesion Molecule Expression In Vitro Lymphangiogenesis

Objective To examine expression of adhesion molecules PECAM-1, ICAM-3,VCAM-1 and CD44 on the lymphatic endothelium of the tissues and the culturedLECs. To investigate roles of endothelial adhesion molecules in proliferation,migration and tube formation of LECs by establishing three-dimensional model oflymphangiogenesis in vitro. Method The canine jejunum and the skin wereremoved and the frozen sections were prepared. Expression of the adhesion moleculeson the lymphatic endothelium was examined using immunohistochemical staining.LECs of canine thoracic ducts were isolated and incubated with MEM containing20% FBS. Adhesion molecules on the LECs were labeled using immunofluorescentstaining, and the stained cells were viewed by confocal laser scanning microscope.The OD of adhesion molecules was determined with an image analysis system. TheOD in control group was compared with that in TNF-α and LPS groups. Theendothelial cells stimulated with TNF-α or LPS were treated with the blockingantibodies against PECAM-1, ICAM-3 and CD44 respectively. The proliferation andmigration of cells were determined with cell counting. The three-dimensional collagengel model of lymphangiogenesis was prepared, and then the tube formation wasviewed after endothelial PECAM-1, ICAM-3 or CD44 was blocked. The length andarea of the tubes formed from LECs were measured and their characteristics wereexamined with a transmission electron microscope. Result PECAM-1 and ICAM-3were expressed on the lymphatic endothelium of the intestine and skin. Theexpression of PECAM-1, ICAM-3 and CD44 was positive on the cultured LECs. Theexpression of VCAM-1 was negative. Compared with control group, the OD ofPECAM-1 and ICAM-3 was not changed and that of CD44 was reduced in TNF-αgroup. In LPS group, the OD of PECAM-1, ICAM-3 and CD44 were not changed.VCAM-1 was expressed weakly on TNF- α or LPS-stimulated cells. In the control,TNF-α and LPS groups, the proliferated cells reduced after PECAM-1 or CD44 of thecells was blocked. However, there were no significant changes in the numbers ofproliferated cells when ICAM-3 of the cells was blocked. The percentage of themigrated cells decreased and the length and area of the tubes reduced after PECAM-1,ICAM-3 and CD44 of the cells were blocked respectively. In the semi-ultrathin andultrathin sections, the tube structures formed from LECs were similar to lymphaticcapillary. Conclusion Expression of adhesion molecules on the lymphatic 6复旦大学博士学位论文endothelium of the tissues and the cultured LECs is not consistent. Expression of theadhesion molecules on the cultured LECs is not uniform after the cells are stimulatedwith TNF-α or LPS. PECAM-1, ICAM-3 and CD44 are involved inlymphangiogenesis that includes proliferation, migration and tube formation ofendothelial cells.