title: High glucose-induced altered basement membrane composition and structure increases trans-endothelial permeability: implications for diabetic retinopathy.
abstract: OBJECTIVE
  The following study was designed to investigate early biosynthetic and ultrastructural changes that alter functional properties of the basement membrane (BM) and affect vascular permeability in diabetic retinopathy.
  
  
  METHODS
  To determine whether altered matrix synthesis affects cell monolayer permeability, rat retinal endothelial cells (RRECs) were grown for 4 days to confluency in normal (N, 5 mM) or high glucose (HG, 30 mM) medium on transwell inserts and subjected to an in vitro cell monolayer permeability assay. Inserts were cut out and viewed under a transmission electron microscope to assess extracellular matrix (ECM) accumulation and cell morphology. In parallel cell cultures, fibronectin and collagen IV protein expression were determined using Western Blot analysis.
  
  
  RESULTS
  Electron microscopic analysis of cells exposed to short-term HG showed no difference in inter-endothelial cell tight junctions (TJs) or in the number of vesicles or coated pits compared to those of normal cells. However, ECM accumulation underlying HG cells was significantly increased compared to that of cells grown in N medium (139 ± 7% of control, p = 0.04), with areas of focal thickening. Western blot analysis showed increased fibronectin and collagen IV expression (152 ± 24% of control, p = 0.01; 146 ± 16% of control, p = 0.02, respectively) in cells grown in HG compared to those grown in N medium. Cell monolayers grown in HG exhibited increased permeability to FITC-dextran compared to cells grown in N medium (134 ± 15% of control, p = 0.02).
  
  
  CONCLUSIONS
  High glucose-induced excess ECM accumulation and altered composition underlies structural and functional changes that allow increased permeability. This finding provides evidence for the first time that the thickened vascular basement membrane contributes to the development of excess permeability seen in diabetic retinopathy.
authors.affiliation: Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.
authors.forename: Argyrios
authors.initials: A
authors.lastname: Chronopoulos
authors.forename: Kyle
authors.initials: K
authors.lastname: Trudeau
authors.forename: Sumon
authors.initials: S
authors.lastname: Roy
authors.forename: Hu
authors.initials: H
authors.lastname: Huang
authors.forename: Stanley A
authors.initials: SA
authors.lastname: Vinores
authors.forename: Sayon
authors.initials: S
authors.lastname: Roy
mesh_terms: D000818:Animals; D001485:Basement Membrane / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D015153:Blotting, Western; D002199:Capillary Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000378:metabolism; D003911:Dextrans / Q000378:metabolism; D003930:Diabetic Retinopathy / Q000378:metabolism* / Q000473:pathology; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism* / Q000648:ultrastructure; D005353:Fibronectins / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D051381:Rats; D012171:Retinal Vessels / Q000166:cytology; D013549:Sweetening Agents / Q000494:pharmacology*; D019108:Tight Junctions; D015854:Up-Regulation
chemical_list: D024141:Collagen Type IV; D003911:Dextrans; D005353:Fibronectins; D013549:Sweetening Agents; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate; D005947:Glucose