Effect of dicarbonyl modification of fibronectin on retinal capillary pericytes.
OBJECTIVE
To determine effects of alpha-dicarbonyl modification of an extracellular matrix protein on retinal capillary pericyte attachment and viability.


METHODS
Primary cultures of bovine retinal pericytes (BRPs) were seeded on either normal fibronectin (FN) or FN modified by methylglyoxal (MGO) and glyoxal (GO). Apoptosis was measured by flow cytometry along with caspase-3 activity. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and Akt/PKB were evaluated by Western blot analysis. Cellular glutathione and reactive oxygen species were measured. alphaB-crystallin was measured by Western blot analysis and, to determine its role in apoptosis, experiments were conducted using BRPs that were transiently transfected with alphaB-crystallin.


RESULTS
Cultures seeded on MGO- or GO-modified FN showed a significant reduction in the number of viable cells, an increase in the number of apoptotic cells, and increased caspase-3 activity, which correlated with the extent of FN modification. Pericytes seeded on either type of modified FN showed phosphorylation of p38 MAPK and dephosphorylation of Akt/PKB. Cultures seeded on dicarbonyl-modified FN had reduced glutathione and increased levels of reactive oxygen species compared with those on a normal matrix. Cells on the altered matrices had reduced alphaB-crystallin levels as well. Transient transfection of rat alphaB-crystallin into BRPs significantly reduced the apoptosis triggered by alpha-dicarbonyl-modified FN.


CONCLUSIONS
These observations indicate that modification of FN by alpha-dicarbonyl compounds triggers apoptosis through a combination of increased oxidative stress and reduction of alphaB-crystallin. This mechanism may contribute to loss of pericytes in diabetic retinopathy and contribute to the resultant vascular lesions.
Liu
BingFen
B
Department of Ophthalmology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Bhat
Manjunatha
M
Padival
Anoop K
AK
Smith
Dawn G
DG
Nagaraj
Ram H
RH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002196:Capillaries; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005353:Fibronectins / Q000494:pharmacology*; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism; D006037:Glyoxal / Q000494:pharmacology*; D018613:Microscopy, Confocal; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009842:Oligopeptides; D020286:Pericytes / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011765:Pyruvaldehyde / Q000494:pharmacology*; D012171:Retinal Vessels / Q000378:metabolism / Q000473:pathology*; D014162:Transfection; D038203:alpha-Crystallin B Chain / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D005353:Fibronectins; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D038203:alpha-Crystallin B Chain; D006037:Glyoxal; D011765:Pyruvaldehyde; C046617:arginyl-glycyl-aspartyl-serine; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione