Paul F. Davis

Distribution of type VIII collagen in tissues: an immunohistochemical study.

Type VIII collagen was first detected as a secretion product of diverse endothelial cell cultures, including those derived from aorta, arteries and veins. Initial studies of its tissue distribution (using a monoclonal antibody) showed it to be present in a restricted number of tissues and failed to find it in the vasculature. Recently, type VIII collagen was shown (using monospecific polyclonal antibodies) to be a component of large blood vessels with predominant localization in the subendothelium. We applied an improved immunofluorescence technique using these antibodies to define the tissue distribution of type VIII collagen. We show that it is a component of arterioles and venules in muscle, heart, kidney, spleen, liver and lung and is also found in connective tissue layers around hair follicles, around nerve bundles in muscle, in the dura of the optic nerve, in cornea and sclera, and in the perichondrium of cartilaginous tissues. This collagen variant appears to have a wider distribution than originally assumed. It is a macromolecular component of the subendothelium, possibly a constituent of the vascular intimal basement membrane.

An improved immunofluorescence techniquefor the histological examination of blood vessel tissue

Autofluorescence of elastic fibres in blood vessel samples is a common interference with the specific fluorescence of FITC-conjugated antibodies. Counterstaining with eriochrome black T changed the yellow-green colour of elastic fibres to dark red, thus turning a disturbing feature into a useful reference background. A second counterstain, p-phenylenediamine, visualized cell nuclei as an amber colour. To demonstrate the improvement of this staining technique, cryosections from blood vessel samples, derived from control veins, arteries and experimental aneurysms of different ages (15 to 99 month old) in 5 sheep, were stained with antibodies against procollagen III, collagen type IV, laminin, and nidogen. The specific distribution of these connective tissue components could now be related to the location of the elastic fibres and the cells (cell nuclei).

Scanning electron microscopic study of hemodynamically induced tears in the internal elastic lamina of rabbit arteries

&NA; A scanning electron microscopic investigation of tears in the internal elastic lamina of the afferent artery of experimental arteriovenous fistulae was undertaken to determine their site of initiation and to elucidate their nature. Carotid‐jugular arteriovenous fistulae and contralateral common carotid arteriotomies were performed in rabbits sacrificed from two to 63 days postoperatively. Following de‐endothelialization, scanning electron microscopy of the internal elastic lamina revealed predominantly transverse straight tears with sharp margins. Even in chronic fistulae the tears retained their sharp margins despite their propagation. No evidence of repair was observed. The tears commenced by rupture of the elastic trabeculae traversing the fenestrae. Enzymatic digestion of the internal elastic lamina did not resemble the hemodynamically induced tears and no evidence of elastolytic enzyme activity was detected. The tears suggest that the abnormally stressed elastic tissue has undergone some structural alteration leading to a reduction in its tensile strength.

Immunolocalization of type VIII collagen in vascular tissue.

Type VIII collagen was first detected in the culture medium of aortic endothelial cells. Subsequently its synthesis by a number of other cell lines was demonstrated. Its presence in vascular tissue is reported here. It is a component of sheep aorta and carotid artery but could not be demonstrated in the jugular vein. It is principally localized in the subendothelial region but this can only be demonstrated after pretreatment of the tissue with proteases. Thus type VIII collagen is a constituent of blood vessels.

Salt-soluble collagen and elastin in the human aorta and pulmonary artery

Collagen and elastin, the major structural components of blood vessels, have a very low turnover. In disease, this rate may be increased and an elevation of the tissue concentration of the soluble degradation fragments might be anticipated. In this preliminary study the concentration of extractable collagen and elastin in the aorta and pulmonary artery of eight human subjects postmortem was determined. The proportion of pulmonary artery collagen and elastin that was soluble was generally either equal to or greater than that in the abdominal aorta. The fraction of collagen that was salt extractable was larger than the soluble elastin fraction.

Type VI collagen in experimental atherosclerosis

Diffuse intercellular immunofluorescence staining of type VI collagen was found in the experimentally thickened vascular wall and in control blood vessel tissues as well, superimposed by more intense staining around basement membranes. While the basement membrane staining disappeared in advanced mural thickenings, the diffusely distributed network of type VI collagen remained.

The biochemical composition of haemodynamically stressed vascular tissue III. The collagen composition of experimental arteriovenous fistulae

The external jugular vein and the common carotid artery were anastomosed in 8 sheep. Morphological changes similar to those observed in human atherosclerosis are produced particularly in the venous tissue. There is a significant increase in the collagen content of the dilated mid-segment of the experimental vein. Also there is a positive correlation between post-operative age and the amount of collagen, especially in the distal region of the artery and in the proximal and middle regions of the vein. In both the control and experimental veins the amino acid composition of collagen was very similar. However, the arterial preparations did not exhibit the same degree of purity. Most of the collagen was the type I variant (approx. 80% in arteries and 70% in veins). In the sham-operated veins about 30% of the collagen was type III but it was nearer 17% in the control arteries. Whilst experimental and control arteries had similar proportions of type III collagen, there was a 30% reduction in the experimental veins. Although there was no significant correlation between the proportion of any of the genetic types of collagen and the post-operative age of the animals, there was a strong correlation for the increment in type I in all regions of the experimental vein as a function of time.

Reversed-phase liquid chromatography of elastin peptides.

Soluble fragments of elastin are frequently present in biological tissue in small amounts. Because of their hydrophobic character, these peptides are not well resolved by a number of conventional techniques. However, their separation should be possible by reversed-phase chromatography. A wide range of columns, gradients and solvents were evaluated. Two systems are described. One was a C18 liganded silica column eluted isocratically by gravity flow. Some degree of size fractionation was achieved with larger peptides being eluted with methanol and smaller ones with isopropanol. The second system uses a pressurized elution from another C18 ligand column. A concave gradient of trifluoroacetic acid-acetonitrile with a decreasing acetonitrile concentration was optimal. Similar resolution of peptides produced by a variety of digestion methods was obtained with the lower-molecular-mass peptides eluting in the middle of the gradient.

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.

The collagenous protein with elastin crosslinks from Descemet's membrane is not related to type VIII collagen.

A collagen-like insoluble protein containing the elastin cross-links (desmosine and isodesmosine) has been isolated from Descemets membrane. Recently type VIII collagen (endothelial collagen) has been shown to be a major constituent of this membrane. Biochemical studies suggest that these two proteins are unrelated. The cyanogen bromide peptide maps show negligible similarity. Antiserum raised against oxalic acid digests of elastin (alpha-elastin) did not react against an oxalic acid digests of type VIII collagen but did show some reaction against the cross-linked preparation. Immunofluorescent localization has demonstrated the presence of type VIII collagen in trachea but a desmosine cross-linked collagen could not be isolated from this tissue.