Extraction and analysis of glycosaminoglycans from intima-media of single rabbit aortae: effect of balloon catheter de-endothelialization on the content and profile of glycosaminoglycans
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A novel procedure is described for extracting, and simultaneously 3H-labelling, glycosaminoglycans from the intima-media of a single rabbit aorta. The procedure was used to compare contents and types of glycosaminoglycans isolated from uninjured (control) aortae, and partially re-endothelialized aortae at 11 weeks after de-endothelialization by a balloon catheter. Briefly, the isolated delipidated tissue was digested in 0.8 M NaOH containing NaB3H4 at room temperature. The neutralized digest was then degraded by a non-specific proteinase during dialysis. The 3H-labelled glycosaminoglycan fraction was recovered after a gel filtration step. The yield (10.5 micrograms of glycosaminoglycan/mg of dry, delipidated tissue) was within the range reported previously for rabbit aorta. Although the de-endothelialized (DEA) and re-endothelialized areas (REA) of all injured aortae contained a significantly thickened intima, the glycosaminoglycan concentration (DEA, 11.2 micrograms/mg; REA, 11.6 micrograms/mg) did not differ significantly from that of control aorta. The profile of [3H]glycosaminoglycan types was determined by the serial use of glycosaminoglycan-selective methods: greater than 86% of 3H was released as small molecular weight products by this analytical scheme. The glycosaminoglycan profile for control tissue compared well with several previous reports. Compared with control aortae, both DEA and REA contained relatively less chondroitin sulphate, whereas DEA contained more hyaluronic acid and REA contained more heparan sulphate. Future use of this procedure will improve measurement of the changes to the extracellular matrix which take place after injury to the vessel wall and which may precede atherogenesis.