Isolation and purification of extracellular matrix vesicles from blood vessels.

Extracellular membrane-bound vesicles (called matrix vesicles) which occur in abundance in atherosclerotic blood vessels are believed to be associated with lipid accumulation and calcification. A technique has been developed to isolate them from experimental aneurysms in sheep in which they are known to be plentiful. The matrix vesicles were isolated by differential centrifugation following extraction by hypotonic salt solution. Most of the vesicles were pelleted at 30,000g and fell within the size range of matrix vesicles in situ in the aneurysmal wall. Preliminary characterization of the enzymatic activities indicates that many of these vesicles are formed from cell membranes rather than being derived from lysosomes, mitochondria or endoplasmic reticulum. Morphologically they are similar to matrix vesicles of other mineralizing tissues.     

Comparison of nonenzymatic glycosylation of arterial and venous collagens

The extent of nonenzymatic glycosylation of collagen isolated from sheep carotid arteries and jugular veins was compared. It was found that the level of this modification in the arterial collagen was about 2.7 times higher than that in the venous collagen. Arteriovenous fistulae were established between the common carotid artery and external jugular vein on one side only of six sheep. Sham arteriotomy and phlebotomy were performed on the contralateral vessels. Although there was an increase in the concentration of these ketoamine-linked hexoses in all the tissue samples assayed, a difference of between two- and three-fold was maintained between the arterial and venous tissue. The relationship of this finding to the development of vascular complications and to the level of circulating reducing sugar is discussed.     

Distribution of type IV collagen, laminin, nidogen and fibronectin in the haemodynamically stressed vascular wall

Changes in the extracellular matrix of haemodynamically stressed blood vessel walls were studied by immunofluorescence histochemistry in venous-pouch aneurysms fashioned on the site of the common carotid artery of nine sheep. Tissues from the thickened walls of the experimental aneurysms were examined from 11 to 98 months post-operatively for changes in the distribution of the basement membrane components type IV collagen, laminin, nidogen and fibronectin. In the younger aneurysms, there was an increase of the basement membrane components in the thickened area. Very little basement membrane was detected in older aneurysms. Diffuse staining for fibronectin was noted in aneurysms of all ages. Thick deposits of basement membrane material were observed in calcified tissues. The changes in the matrix proteins were similar to alterations occurring during the development of atherosclerosis in human vascular tissue.