The alpha1(VIII) and alpha2(VIII) collagen chains form two distinct homotrimeric proteins in vivo.

The short chain collagen variant, type VIII, is considered to be comprised of two distinct gene products, the alpha1 and alpha2 polypeptide chains. However, recent in vitro translation studies suggest that these chains can form homotrimers. We report here data from biochemical, immunohistochemical and molecular biological experiments, which together provide evidence that alpha1 and alpha2 polypeptides of type VIII collagen exist as homotrimers in cells and tissues. High-performance liquid chromatographic separation of type VIII collagen isolated from Descemet's membrane consistently demonstrated equimolar quantities of the two chains (alpha1:alpha2 1. 03+/-0.02 (S.E.M.); n=41). The availability of highly specific antibodies for the two polypeptides has assisted the in vivo characterisation of type VIII collagen. Immunoprecipitation of trimeric type VIII collagen from Descemet's membrane with purified anti-alpha1(VIII) and anti-alpha2(VIII) yielded fractions that contained only the alpha1(VIII) and alpha2(VIII) chains, respectively. Cultured human mesangial cells synthesised both polypeptides, but the alpha1(VIII) chain was found exclusively in the cell pellet, while the media contained only the alpha2(VIII) chain. The RNA from human mesangial cells and cornea showed message for both chains. However, in peritoneal fibroblast and mesothelial cell RNA, only alpha1(VIII) mRNA was detectable, demonstrating that the transcription of these two genes was not always co-ordinated. Immunohistochemistry showed that both polypeptides were present in cornea, optic nerve, aorta and umbilical cord but did not always co-localise. These results indicate the alpha1(VIII) and alpha2(VIII) chains preferentially form pepsin-resistant, homotrimeric molecules and so can exist as two distinct proteins.

Mast cells and type VIII collagen in human diabetic nephropathy.

Renal injury in diabetes mellitus is associated with progressive interstitial fibrosis and extracellular matrix accumulation. However, the phenotypes of cells forming the interstitial infiltrate in diabetic nephropathy have not been precisely defined. There is increasing evidence for the association of mast cells with angiogenesis, chronic inflammatory conditions and fibrosis. We have recently shown that human mast cells can produce the non-fibrillar short chain type VIII collagen in vivo. Using immunohistochemistry, in situ hybridisation and reverse transcriptase-polymerase chain reaction, we examined the contribution of mast cells and type VIII collagen to the fibrotic changes occurring in biopsy-proven diabetic nephropathy. We observed that the number of interstitial mast cells was significantly increased in diabetic nephropathy compared with normal kidney tissue. In specimens from diabetic subjects, intense immunohistochemical staining for type VIII collagen was detected in mast cells, on periglomerular fibres and in perivascular and interstitial sites. The expression of type VIII collagen in periglomerular and interstitial sites coincided with that of alpha smooth muscle actin, a marker for myofibroblastic differentiation mRNA for type VIII collagen was detected by reverse transcriptase-polymerase chain reaction in diabetic nephropathy and in a human mast cell line. By in situ hybridisation the transcripts for type VIII collagen were localised to renal mast cells. The increased number of mast cells and the elevated type VIII collagen deposition in human diabetic nephropathy provides a potential link between the extracellular matrix accumulation and the fibrosis observed in this condition.

Cleavage of type VIII collagen by human neutrophil elastase.

In this report, the susceptibility of type VIII collagen to human neutrophil elastase is compared to other extracellular matrix components. Type X collagen is degraded to specific fragments at a substrate to enzyme ratio of 5:1 after 20 h at room temperature, but type VIII collagen is almost completely degraded after only 4 h incubation at a substrate to enzyme ratio of 50:1 and partly degraded after only 15 min. Laminin, merosin and types I, III, IV and V collagen exhibit no susceptibility to neutrophil elastase under the latter conditions, while fibronectin is degraded.

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 Descemet's 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.

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.

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.

Scanning electron microscopic investigation of the afferent arteries of experimental femoral arteriovenous fistulae in rabbits.

Scanning electron microscopy of the intimal surface of anastomosed arteries of experimental femoral arteriovenous fistulae in rabbits was conducted to determine whether the muscular femoral artery differed from the elastic common carotid artery in its response to the hemodynamic stress of the arteriovenous shunt. Control femoral arteriotomies were performed in an additional five rabbits. The animals were sacrificed at varying intervals from 2 days to more than nine months postoperatively. Within two days postoperatively transverse and longitudinal tears involving the internal elastic lamina and also the endothelium appeared in the afferent artery as far proximal to the fistula as the lower abdominal aorta. Tears distal to the fistula were fewer and later in appearance. The tears healed rapidly. The response as demonstrated by this technique was similar to that of the common carotid artery of carotid-jugular fistulae except that tears appeared earlier postoperatively with longitudinal as well as transverse disposition. Similar tears were found in four of the five control arteriotomies in the vicinity of the suture or at the site of clamping. The experiments reveal the readiness with which hemodynamic stress induces intimal tears.

X-ray microanalysis of mineralized matrix vesicles of experimental saccular aneurysms.

An energy dispersive X-ray microanalytical study was designed to examine the mineral deposits in matrix vesicles found in the walls of experimental aneurysms from two rabbits (103 and 1071 days postoperatively) and two sheep aneurysms (234 and 1202 days postoperatively). The freeze-substitution technique was adopted for use to retain inorganic ions in situ. Numerous, various sized extracellular electron-dense structures, believed to be matrix vesicles were observed. Size histograms for the mineralized vesicles showed that the proportion of smaller vesicles was higher in the older animals. A total of 370 vesicles were analyzed. Calcium and phosphorus with small amounts of magnesium were identified. No particular calcium phosphate mineral was dominant with the mean Ca/P molar ratio for all animals falling in the 1.1-1.2 range. Correlation coefficients for interrelationships between calcium, phosphorus, magnesium, and size were weak except for calcium vs phosphorus which was close to one, consistent with some type of calcium phosphate being the major constituent of the mineralized vesicles. Smaller electron-dense particles, probably mitochondrial granules were seen in some smooth muscle cells; a small number were analyzed and contained calcium and phosphorus (mean Ca/P molar ratio of 0.86) but no magnesium.

Haemodynamically-induced intimal tears in experimental U-shaped arterial loops as seen by scanning electron microscopy.

Experimentally-induced U-shaped carotid loops, simulating arterial tortuosities and kinks were examined by scanning electron microscopy to seek flow-induced changes in the intimal surface. In 14 New Zealand white rabbits carotid arterial transplants were fashioned into U-shaped loops by microvascular surgery. The rabbits were sacrificed from 4 to 226 days post-operatively. Tears in the internal elastic lamina occurred in all loops from 5 days post-operatively and were predominantly transverse and localised about the greater curvature of the bends of each loop. Though initially denuded, all tears appeared endothelialised after 6 days, coalescing later as they increased in size and extent. In older animals only islands of wrinkled internal elastic lamina remained at those sites. Endothelial cells in the tears were small, numerous and polyhedral with raised nuclei. The lesser curvature of the three bends in the loops displayed some irregular wrinkling of the internal elastic lamina. In the three oldest animals a few longitudinal tears were observed on the lesser curvature of the main bend. The specific localization of intimal tears supports the concept that they were hemodynamically induced.

Salt soluble cross-linked elastin: formation and composition of fibers.

Cross-linked elastin has been isolated from the salt extract of sheep vascular tissue by means of hydrophobic interaction chromatography on a column of decyl-agarose. Dialysis of the dimethylformamide and sodium dodecyl sulphate column eluates against distilled water produced a precipitate that was fibrous and that resembled insoluble elastin fibers. As judged by amino acid analyses and SDS-polyacrylamide gel electrophoresis, this precipitation resulted in further purification of the soluble cross-linked elastin. Similar chromatography and precipitation of oxalic acid solubilized cross-linked elastin (alpha-elastin) produced identical results.

Scanning electron-microscopic study of experimentally induced intimal tears in rabbit arteries.

A scanning electron-microscopic study of the intimal surface of anastomosed arteries of experimental arteriovenous fistulae in 20 rabbits was conducted to determine the effect of altered haemodynamics on the arterial wall. Control arteriotomies were performed on 12 animals on the contralateral common carotid arteries. The rabbits were killed from 1 to 447 days postoperatively. Transverse intimal tears involving the internal elastic lamina developed in both proximal and distal segments of the anastomosed artery as early as 5 days postoperatively. Some were covered with thrombus and attenuated endothelial cell remnants, but all tears became endothelialised by 7 days. Endothelial cells in the floor of the tears had prominent nuclei and were smaller and more plentiful than the normal intima but, in longstanding fistulae, they resumed a more elongated spindle shape. Tears similar to those in the experimental arteries were found in most control arteriotomies, being confined to the suture zone. The results reveal the profound effect of altered haemodynamics on the arterial wall.

Scanning electron-microscopic study of the inner surface of experimental aneurysms in rabbits.

A scanning electron-microscopic study of the inner surface of experimental saccular aneurysms in rabbits was conducted to demonstrate the effects of haemodynamics on the aneurysmal wall. Animals with aneurysms were killed from 2 to 83 weeks postoperatively, and the controls from 7 to 51 weeks. In both series, re-endothelialization was complete in approximately 2 weeks. In control tissue endothelium was normal, and sutures caused mild distortion of the luminal surface. In the aneurysms, there were tracts of spindle-shaped cells, often arranged in whorl-like patterns and interspersed with regions of polyhedral cells with thickened intercellular borders, prominent stomata and numerous microvilli. In some areas, long filamentous intercellular processes were prominent. The few jet lesions seen were covered with polyhedral cells and were less complex than those in arteriovenous fistulae. Ridges, caused by elevated subendothelial connective tissues were crescentic or circular, outlining depressions and craters, as if delineating vortices. The carotid artery opposite the aneurysm exhibited endothelium similar to normal tissue, except in older aneurysms where corrugations caused by the internal elastic lamina were completely or partially absent. The experiments demonstrate the effect of intra-aneurysmal haemodynamics on the topography of the endothelium and subendothelial tissues of the sac wall, and support the concept that endothelial topography in the vascular system under physiological conditions is haemodynamically determined.

Scanning electron-microscopic study of the anastomosed vein of arteriovenous fistulae.

A scanning electron-microscopic study of experimentally-induced arteriovenous fistulae in rabbits was conducted to investigate the haemodynamically-induced endothelial changes caused by the shunt. Animals were examined from 5 to 696 days postoperatively. The initial endothelial denudation in the vicinity of the anastomosis was rapidly repaired and by 15-20 days re-endotheliazation was complete. Severe surface changes were found in the region of greatest turbulence, opposite the shunt. Changes characteristically consisted of craters, scallops, mounds and ridges, fused together to give a moonscape-like appearance. This area, termed the "jet lesion", was covered with polygonal endothelial cells with thickened, distorted and often highly-crenated margins. The margin of the fistula, where shear stress was expected to be high, exhibited flattened, elongated, generally fusiform endothelial cells with serrated margins. They were aligned parallel to the blood flow, whereas those in the "jet lesion"were polygonal and randomly orientated. These profound changes in the endothelium and the underlying connective tissues, observed as early as 15 days postoperatively, revealed the extent and severity of the effect of haemodynamics on the wall.