Effects of endothelial removal and regeneration on smooth muscle glycosaminoglycan synthesis and growth in rat carotid artery in organ culture.

Segments of rat carotid artery were maintained in serum-free and serum-supplemented media with endothelium both present and substantially removed by air drying. At intervals of 3, 7, and 14 days the synthesis of glycosaminoglycan across the vessel walls was determined by autoradiographic detection of incorporated [3H]glucosamine. In control carotids the typical pattern of incorporation was 40% of label in the intima, consisting of endothelium and subendothelial matrix, 23, 13, and 15% in the three medial layers (M1, M2, M3, respectively), and 9% in the adventitia. During the first week in culture the proportion, and often the amount, of label in M1 increased significantly. Following air drying labeling decreased markedly in M1 but often increased in M2 and M3. By 14 days residual endothelial cells had regenerated, and the pattern of incorporation in the medial layers beneath this new endothelium was the same as for the controls with a high level of labeling in M1. In areas free of endothelium incorporation in M1 remained at a low level. Digestion with chondroitinase ABC and Streptomyces hyaluronidase showed that the changes in M1-labeling levels were due to changes in the amounts of both hyaluronic acid and sulfated glycosaminoglycan, whereas pulse and continuous labeling studies showed that the different labeling levels for the various layers and conditions were due to different rates of synthesis and not degradation. Carotids were also labeled with [3H]thymidine. Control and regenerating endothelia were active in serum-free and serum-supplemented media and had similar mitotic indices. Indices for smooth muscle cells in M1, however, were generally very low and were not affected by the presence or absence of endothelium. We conclude that endothelial removal results in decreased glycosaminoglycan synthesis in the inner media, that mitotically active endothelium correlates with increased glycosaminoglycan synthesis in the inner media, and that these changes occur independently of smooth muscle cell growth.