title: Isolation, culture, and characterisation of MHC class II-positive microvascular endothelial cells from the human heart.
abstract: Immunological studies of human endothelial cells have been performed almost entirely on endothelial cells (EC) derived from foetal (umbilical vein) or adult (aorta) large vessels. Immunologically these EC have in common the fact that they are negative for major histocompatibility complex (MHC) class II antigens (HLA-DR), in contrast to microvascular cells within the heart which are constitutively positive for HLA-DR antigens. Here we describe a simple method for isolating microvascular EC from chunks of adult human ventricle using antibody (anti-HLA-DR) or Ulex lectin-coated magnetic beads. Cytospins of primary isolates reveal the majority of cells binding the beads are CD31+ve, CD36+ve, and HLA-DR+ve. After 14 weeks in culture, 19% of cells remained HLA-DR+ve but CD36 expression had disappeared. Cultures after 4-7 passages were analysed by flow cytometry and immunocytochemical staining of cytospins with mAbs against conventional endothelial antigens (EN4, PECAM-1, vWF, uptake of acetylated LDL) and nonendothelial antigens (CD45, cytokeratin, alpha-actin, desmin, MHC class I and class II). The observation that all the cells remained positive for CD31 and did stain strongly for cytokeratin confirmed their endothelial origin. There was some persistent, albeit low level, expression of HLA-DR which was upregulated by interferon-gamma, showing kinetics of induction similar to that found on large vessel EC. These microvascular cells should be very useful for those wishing to compare microvascular with macrovascular EC and for immunological studies of heart endothelial cells.
authors.affiliation: Heart Science Centre, National Heart and Lung Institute at Harefield Hospital, Middlesex, United Kingdom.
authors.forename: R M
authors.initials: RM
authors.lastname: McDouall
authors.forename: M
authors.initials: M
authors.lastname: Yacoub
authors.forename: M L
authors.initials: ML
authors.lastname: Rose
mesh_terms: D000328:Adult; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D018955:CD36 Antigens / Q000378:metabolism; D002232:Carbocyanines; D015815:Cell Adhesion Molecules / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000276:immunology* / Q000378:metabolism; D005456:Fluorescent Dyes; D006684:HLA-DR Antigens / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007633:Keratins / Q000378:metabolism; D008833:Microcirculation / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D009206:Myocardium / Q000166:cytology* / Q000276:immunology* / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011994:Recombinant Proteins; D013997:Time Factors
chemical_list: D015214:Antigens, Differentiation, Myelomonocytic; D018955:CD36 Antigens; D002232:Carbocyanines; D015815:Cell Adhesion Molecules; D005456:Fluorescent Dyes; D006684:HLA-DR Antigens; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011994:Recombinant Proteins; C024286:3,3'-dioctadecylindocarbocyanine; D007633:Keratins; D007371:Interferon-gamma