Lamellipodial motility in wounded endothelial cells exposed to physiologic flow is associated with different patterns of beta1-integrin and vinculin localization.
Integrins- and cytoskeletal-associated focal adhesion proteins may participate in the process of endothelial wound closure, but their relationship in these wounds and in the presence of shear forces has not been defined. The goal in this study was to test the hypotheses that (1) modulation of beta(1)-integrin in human coronary artery endothelial cells (HCAEC) would alter endothelial wound closure under shear stress, and (2) beta(1)-integrin association with vinculin would be necessary for mediating this closure. HCAEC monolayers were pre-conditioned to attain alignment by shearing at 12 dynes/cm(2) for 18 h in a parallel-plate flow chamber. Subsequently, they were divided into three groups: (a) control, (b) treated with anti-beta(1)-integrin adhesion blocking antibody, or (c) treated with anti-beta(1)-integrin adhesion promoting antibody. Next, the monolayers were wounded with a metal spatula, and re-sheared at 20 dynes/cm(2) or left static. Time-lapse imaging and deconvolution microscopy were then performed for 3 h. Immunocytochemistry for beta(1)-integrin expression and vinculin was performed on all wounded monolayers. Under shear stress, vinculin localized to the ends of stress fibers, while beta(1)-integrin took on an intracellular macroaggregate appearance. Treatment with anti-beta(1)-integrin adhesion blocking antibody enhanced wound closure, left the vinculin staining at the lamellipodial tips unchanged, but was associated with beta(1)-integrin staining at the lateral cell edges. Treatment with the anti-beta(1)-integrin adhesion promoting antibody retarded wound closure, increased vinculin staining at cell-cell junctions, and was associated with a fibrillar pattern of beta(1)-integrin staining. Modulation of beta(1)-integrin and changes in beta(1)-integrin and vinculin localization may further our understanding of laminar shear stress-induced endothelial repair in the coronary circulation.
Albuquerque
Maria Luiza C
ML
Critical Care Laboratory of Vascular Research, Division of Critical Care Medicine (Children's Memorial Hospital), Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA. malbuquerque@northwestern.edu
Flozak
Annette S
AS
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D005434:Flow Cytometry; D018056:Hemorheology; D006801:Humans; D007150:Immunohistochemistry; D039641:Integrin beta Chains / Q000378:metabolism; D019012:Integrin beta1 / Q000276:immunology / Q000378:metabolism*; D039661:Integrin beta3 / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D011554:Pseudopodia / Q000502:physiology*; D013314:Stress, Mechanical; D016596:Vinculin / Q000378:metabolism*; D014945:Wound Healing / Q000187:drug effects / Q000502:physiology
D000911:Antibodies, Monoclonal; D039641:Integrin beta Chains; D019012:Integrin beta1; D039661:Integrin beta3; D021122:Protein Subunits; C084344:integrin beta5; D016596:Vinculin