PRODUCTION OF TYPE-IV COLLAGEN AND 72 KDA GELATINASE BY HUMAN ENDOTHELIAL-CELLS CULTURED IN HIGH GLUCOSE - EFFECTS OF A PROTEIN-KINASE-C INHIBITOR, GF-109203X

Since diabetic microangiopathy and macroangiopathy are characterized b y type IV collagen accumulation in vascular basement membranes, it was of interest to study type IV collagen production and type IV collagen ase secretion by endothelial cells (EC) cultured in high glucose and t o evaluate the role of protein kinase C (PKC) activation in the altera tions induced by high glucose. Primary cultures of human umbilical vei n EC were exposed to high glucose concentration for 3 days at the begi nning of confluence. The number of EC decreased with glucose concentra tion from 5 to 50 mM. At 16.7 mM glucose concentration, the amount of type IV collagen, determined by a two-step ELISA, increased in the cul ture supernatant and in the insoluble fraction associated with the ext racellular matrix and cells; proline incorporation was more markedly e levated in the collagenous than in the total proteins of the culture s upenatant and of the extracellular matrix and cell extracts. Gelatin z ymography of the culture supernatant showed that EC mainly produce a 7 2-kDa gelatinase known to degrade type IV collagen. At 16.7 mM glucose concentration, total gelatinase activity per millilitre of culture su pernatant was reduced and the 72-kDa gelatinase activity measured on t he zymogram scan was lowered. When EC were exposed to 16.7 mM glucose, the specific PKC inhibitor GF 109203X corrected the increases in type IV collagen concentration and in proline incorporation into the colla genous or total proteins present in the culture supernatant or in the extract of the insoluble fraction, including the extracellular matrix and cells. Our results show that soluble and insoluble type IV collage n accumulation by EC cultured at high glucose concentration is not onl y associated with increased synthesis of the collagenous and total pro teins but also with decreased total 72-kDa gelatinase activity in the extracellular fluid. The observed effects of GF 109203X are in favour of the involvement of PKC activation in the type IV collagen accumulat ion.