Glycosaminoglycan composition and biosynthesis in the endothelium-covered neointima of de-endothelialized rabbit aorta

Abstract

The biosynthesis and composition of glycosaminoglycans (GAG) in the endothelium-covered neointima, formed in response to de-endothelialization of the rabbit aorta by a balloon catheter, was examined. The [14C]glucosamine incorporation into GAG during an in vitro incubation with intimal-medial tissue was monitored periodically up to 24 hr. The GAG were isolated after an exhaustive proteolytic digestion with pronase and protease followed by ethanolic precipitation at 4 degrees C. Electrophoretic migration on cellulose acetate paper was compared for identification. The distribution of GAG was determined after a selective enzymatic digestion of isolated GAG using specific enzymes. Heparan sulfates were estimated after nitrous acid treatment. The concentration of GAG was measured spectrophotometrically by forming colored complexes with Alcian blue dye. In addition, the specific activity (dpm/microgram GAG) and the rate of GAG synthesis (ng/mg dry defatted tissue/day) were determined. The results indicate that the rate of GAG synthesis by de-endothelialized neointima (DEA) was twice that of intact aorta (control). In the re-endothelialized neointima (REA), the GAG synthetic rate was three times more than in control. However, the release of GAG into medium from REA accounts for only 25% of the GAG synthesized by this tissue type, and the release from DEA accounts for 60% of the synthesized GAG. Similarly, a threefold increase in the GAG concentration in REA compared to control was found. The relative distribution as chondroitin-6-sulfate (C6S), chondroitin-4-sulfate (C4S), dermatan sulfate (DS), heparan sulfate (HS) and hyaluronic acid (HA) was markedly altered in the injured neointima. There was an increase in chondroitin sulfates (CS) and DS concomitant with a decrease in HS. It is concluded that injury to aortic endothelium induces stimulation of GAG synthesis in the arterial wall. Furthermore, the greater release of GAG from DEA, compared to control and REA, suggests that endothelium may function as a "reverse" barrier in the neointima covered by regenerated endothelium.