title: Glucose-induced fibronectin expression in endothelial cells is mediated by protein kinase C.
abstract: One of the crucial pathophysiological changes in diabetic angiopathy might be the glucose-induced synthesis of components of the extracellular matrix-like fibronectin. This effect has been described for endothelial and mesangial cells. In order to gain further insight into the mechanisms how glucose-induced fibronectin expression is regulated, confluent monolayers of human umbilical vein endothelial cells (HUVEC) were incubated with varying concentrations of D-glucose (5-30 mM) in a time-dependent manner. Using Westernblotting techniques with a monoclonal antibody, a 3 +/- 0.2-fold increase of the 220 kDa-signal corresponding to human fibronectin was visible after an incubation time of 6 h indicating de-novo synthesis. Using incubation times of 6 or 10 days, glucose-mediated fibronectin overexpression was still visible, reaching a maximum at 15 mM D-glucose. The corresponding maximum values were 2.4 +/- 0.3 (6 d) and 2.3 +/- 0.2 (10 d). There was a concomitant glucose-dependent onset of expression of Protein Kinase C (PKC)-isoforms PKC-delta (2.5 +/- 0.3-fold), PKC-epsilon (2 +/- 0.2-fold) and PKC-zeta (1.8 +/- 0.2-fold), which reached a maximum after 12 h and was still visible during long-term culture. Induction of fibronectin expression was also obtained using the PKC-activating phorbolester Phorbol-12-myristat-13-acetat (PMA) which mimicks glucose-induced PKC activation. Glucose-induced fibronectin expression was decreased by the PKC-inhibitor H-7 (1- [5-isoquinolinylsulfonyl]-2-methylpiperazine). These experiments suggest that: 1. Short-term induction of fibronectin expression mediated by glucose is probably PKC-triggered since concomitant induction of PKC-isoforms PKC-delta, PKC-epsilon and PKC-zeta was observed. 2. Long-term incubation with D-glucose leads to an ongoing concentration-dependent coexpression of fibronection and various PKC-isoforms. If glucose-induced, PKC-mediated de-novo synthesis of components of the connective tissue or the extracellular matrix may be important in the pathomechanism of diabetic angiopathy, has to be proven.
authors.affiliation: Medical Clinic, Innenstad, University of Munich, Germany.
authors.forename: H K
authors.initials: HK
authors.lastname: Mueller
authors.forename: U
authors.initials: U
authors.lastname: Fritsche
authors.forename: A
authors.initials: A
authors.lastname: Haslinger
authors.forename: R
authors.initials: R
authors.lastname: Landgraf
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005353:Fibronectins / Q000096:biosynthesis* / Q000302:isolation & purification; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis; D007700:Kinetics; D011493:Protein Kinase C / Q000096:biosynthesis*; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014471:Umbilical Veins; D000099221:Protein Kinase C zeta
chemical_list: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D005353:Fibronectins; D005947:Glucose; D007527:Isoenzymes; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate; D000099221:Protein Kinase C zeta; C497258:PRKCD protein, human; C497255:PRKCE protein, human