title: Fibronectin fragments promote human retinal endothelial cell adhesion and proliferation and ERK activation through alpha5beta1 integrin and PI 3-kinase.
abstract: OBJECTIVE
  Extracellular matrix degradation is associated with neovascularization in diabetic retinas. Fibronectin fragments (Fn-fs) are generated during vascular remodeling. The effects of cellular fibronectin (Fn) and selected Fn-fs on adhesion, proliferation, and signal transduction in human retinal endothelial cells (HRECs) were characterized.
  
  
  METHODS
  Relative quantitative RT-PCR, flow cytometry, and immunocytochemistry determined integrin expression on HRECs. Adhesion was evaluated by coating plastic with Fn or Fn-fs of 45, 70, 110, or 120 kDa, and MTT conversion was used to measure proliferation and survival. Peptide inhibitors and blocking antibodies determined adhesive sites and integrins used for adhesion. Pharmacologic inhibitors and Western analyses were used to evaluate intracellular signaling.
  
  
  RESULTS
  HRECs produced significant levels of alpha(2), alpha(3), alpha(5), alpha(v), beta(1), beta(3), and beta(5) integrin subunit mRNA. Flow cytometry of surface integrin expression revealed high levels of alpha(3), alpha(5), and beta(1) and lower levels of alpha(1), alpha(v), beta(3), and beta(5). These results were confirmed by immunocytochemistry. For adhesion to Fn and Fn-fs. the alpha(5)beta(1) integrin was essential. Pharmacologic inhibitors of PI 3-kinase blocked adhesion to Fn and Fn-fs, whereas the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD98059 blocked phosphorylation. The 110- and 120-kDa Fn-fs showed a concentration-dependent increase in proliferation, whereas 500 ng of the 70 kDa Fn-f-induced proliferation. Addition of III1-C, a matrix assembly domain, increased the proliferative effect of these Fn-fs.
  
  
  CONCLUSIONS
  Fn and its Fn-fs modulate HREC adhesion and proliferation through signal-transduction pathways involving coupling of the alpha(5)beta(1) integrin through PI 3-kinase. Mitogenic signals for endothelial cells from degraded extracellular matrix may contribute to the development of diabetic retinopathy.
authors.affiliation: Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida, USA.
authors.forename: Sylvia H
authors.initials: SH
authors.lastname: Wilson
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Ljubimov
authors.forename: Alex O
authors.initials: AO
authors.lastname: Morla
authors.forename: Sergio
authors.initials: S
authors.lastname: Caballero
authors.forename: Lynn C
authors.initials: LC
authors.lastname: Shaw
authors.forename: Polyxenie E
authors.initials: PE
authors.lastname: Spoerri
authors.forename: Roy W
authors.initials: RW
authors.lastname: Tarnuzzer
authors.forename: Maria B
authors.initials: MB
authors.lastname: Grant
mesh_terms: D019138:Antibodies, Blocking / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005353:Fibronectins / Q000494:pharmacology*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D012171:Retinal Vessels / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D019138:Antibodies, Blocking; D004791:Enzyme Inhibitors; D005353:Fibronectins; D039081:Integrin alpha5beta1; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases