Colin J. Schwartz

Increased endothelial cell turnover in areas of in vivo Evans Blue uptake in the pig aorta

Abstract Focal areas of spontaneous increased aortic endothelial permeability to the protein-binding azo dye Evans Blue in vivo have been further characterized in 8–12 week-old normocholesterolaemic pigs by means of Hautchen preparations with [ 3 H]thymidine autoradiography, together with observations on endothelial cell length, cell area, and surface morphology. Endothelial cell turnover was significantly greater in areas of dye accumulation than in contiguous areas showing no dye accumulation. No differences in turnover between cells derived from white areas in the thoracic arch and abdominal aorta were observed. Control studies indicated that the greater labelling index in blue areas was not due to the presence of Evans Blue dye itself. Although both endothelial cell length and area were not measurably different in blue and white areas, endothelial cells derived from areas of dye accumulation appeared rounder and exhibited a less distinct polarity. This study has shown that as early as 8–12 weeks after birth, focal areas can be identified in the macroscopically normal pig aorta which exhibit an increased endothelial labelling index. The increased [ 3 H]thymidine labelling of endothelial cells is considered to reflect increased endothelial regeneration resulting from haemodynamically-induced injury. The findings are discussed in terms of enhanced endothelial permeability, haemodynamic injury, contractility, and the early development of atherosclerosis.

Aortic endothelial permeability to albumin: Focal and regional patterns of uptake and transmural distribution of 131I-albumin in the young pig☆

Abstract Both focal and regional patterns of uptake, and the transmural distribution of 131 I-albumin have been examined in vivo in the macroscopically normal young pig aorta. Uptake of 131 I-albumin was significantly greater in areas of Evans Blue accumulation than in contiguous areas of the aortic arch showing no dye accumulation (white areas), both 2 and 24 hr after the intravenous injection of labeled albumin. Only 3 to 7% of the aortic uptake was associated with the endothelium at 2 and 24 hr, respectively. Regional differences in the uptake of 131 I-albumin were also observed; the uptake in both upper and lower abdominal aortic segments was significantly less than in the aortic arch. The transmural distribution of 131 I activity showed a distinct gradient in each of the three regions of the aorta, namely aortic arch, upper abdominal, and lower abdominal aortic segments. Activity was greatest in the intima and inner media, and least in the outer media. Although the slope of the gradients appeared similar for blue and white areas of the aortic arch, 131 I activity was significantly greater in areas of dye accumulation than in white areas at each level across the aortic wall. Activity in the upper and lower abdominal aortic segments was similar, and significantly less than in the arch at each level across the aortic wall. Albumin influx rates were calculated for blue and white areas from the aortic arch, and for white areas in the upper and lower abdominal aortic segments. In areas of the aortic arch showing no dye accumulation, the calculated influx was 16.3 μg/cm 2 /hr, while in areas of Evans Blue accumulation, the influx was 27.0 μg/cm 2 /hr. Influx in the upper and lower abdominal segments was similar with a value of 9 μg/cm 2 /hr, or half that of the white areas in the aortic arch. These studies have shown that 131 I-albumin crosses the normal aortic endothelium, and that the uptake shows both regional and focal differences. The slopes of the transmural gradients from the intima-media to the outer media are consistent with the entry of albumin from the aortic lumen. Focal differences in 131 I-albumin uptake are interpreted as indicating focal spontaneous areas of increased endothelial permeability, possibly the result of focal hemodynamic injury. The potential significance of these findings in terms of endothelial permeability and atherogenesis is discussed.

Endothelial cell morphology in focal areas of in vivo Evans Blue uptake in the young pig aorta: I. Quantitative light microscopic findings☆

Abstract The morphology of endothelial cells from the pig aorta was examined using silver nitrate-stained Hautchen preparations. Areas of enhanced uptake of the protein-binding azo dye Evans Blue (blue areas) and areas of no dye uptake (white areas) were compared. From representative light photomicrographs the appearance of silver granules, stigmata, stomata and gaps in the silver-stained cell boundaries was examined. The numbers of these structures per cell were counted in both blue and white areas, and cell length and polarity in the two areas were also quantitated. Silver-stained cell boundaries in blue areas appeared consistently thicker and more irregular than in white areas, and cells from the latter were often longer and more tapered. Quantitatively, a significantly greater number of gaps or breaks in the boundary lines was found in blue areas. Over 85% of cells from both areas were aligned within 30° of the long axis of the vessel, and stigmata and stomata occurred with the same frequency in both areas. These and other findings are discussed in terms of the known permeability differences between areas of Evans Blue uptake and areas of no dye uptake.

Focal and regional patterns of uptake and the transmural distribution of 131I-fibrinogen in the pig aorta in vivo

Abstract The focal and regional patterns of uptake, and the transmural distribution of human 131 I-fibrinogen have been examined in the macroscopically normal pig aorta in vivo . Uptake of 131 I-fibrinogen by focal areas of the aortic arch accumulating Evans blue dye (blue areas) was significantly greater than uptake into areas of the arch showing no dye accumulation (white areas) 2 hr after the intravenous injection of labeled fibrinogen. Not more than 5% of the 131 I-fibrinogen was associated with the endothelium. Regional differences in the aortic uptake of 131 I-fibrinogen were also observed; uptake into white areas of upper and lower abdominal aortic segments was significantly less than into white areas from the aortic arch. 131 I-Fibrinogen activity showed a distinct transmural gradient in each of the three regions of the aorta studied, namely, aortic arch and upper and lower abdominal aortic segments. As activity was greatest in the intima and inner media, the slope of the gradients has been interpreted as indicating fibrinogen entry from the endothelial surface. 131 I-Fibrinogen activity was consistently greater in areas of dye accumulation than in white areas at each level across the aortic arch, although the greatest differences were observed in the intima and innermost media. Activity across the aortic wall in both the upper and lower abdominal segments was significantly less than in the arch at each of the levels studied. In the inner 300 μm the activity was less in the lower than in the upper abdominal segments. Fibrinogen influx rates were calculated for blue and white areas from the aortic arch, and for white areas in the upper and lower abdominal aortic segments. In the arch, calculated influx was 0.82±0.13 μg/cm 2 /hr in white areas, and 1.73 ± 0.30 μg/cm 2 /hr in blue areas. In both the upper and lower abdominal aortic segments, influx was approximately half that of white areas in the aortic arch. This study has shown that plasma 131 I-fibrinogen crosses the normal aortic endothelium, and that the uptake shows both regional and focal differences. It is concluded that the presence of fibrinogen or fibrin in early atheromatous lesions may reflect the demonstrated permeability of normal aortic endothelium to circulating fibrinogen.

False aneurysm of left ventricle due to perforation of mitral-aortic intervalvular fibrosa with rupture and cardiac tamponade: Rare complication of infective endocarditis

A case of subacute bacterial endocarditis occurring in a congenital bicuspid aortic valve in a 17 year old boy is presented. The infection secondarily involved the mitral-aortic intervalvular fibrosa, the junctional zone between the mitral and aortic valves. Perforation of this zone, with extension of infection and blood into the epicardial wedge overlying the fibrosa, resulted in the formation of a false aneurysm situated at the base of the left ventricle between the aorta and the left atrium. Rupture of the aneurysm with hemorrhage into the pericardial cavity resulted in death from cardiac tamponade.

Endotoxin-induced vascular endothelial injury and repair: II. Focal injury, en face morphology, [3H]thymidine uptake and circulating endothelial cells in the dog

Abstract In this study we have examined the influence of intravenous Escherichia coli endotoxin on the induction of endothelial injury and repair in the aorta and pulmonary artery of the dog, with special reference to areas of Evans blue dye uptake. The sequential changes were monitored by quantitation of the numbers of circulating endothelial cells in three vascular beds, by incorporation of [3H] thymidine by endothelial cell nuclei, and by light microscopy of silver nitrate-stained Hautchen preparations. Circulating endothelial cells were observed as early as 5 min after endotoxin injection, being most numerous in the pulmonary circulation. By 60 min, most circulating cells had disappeared. In the aorta, uptake of [3H]thymidine by endothelial cell nuclei was maximal in the arch and, in particular, in those areas showing Evans blue dye accumulation. Light microscopy of silver-stained Hautchen preparations from blue areas of the aorta and pulmonary artery revealed foci of intense silver staining, which have been interpreted as indicating damage or denuded endothelium. Blue areas also exhibited darkly staining globular structures of variable size and shape apparently overlying the endothelial surface. Neither the foci of intense silver staining nor the globular structures were observed in specimens from adjoining areas of no dye uptake (white areas), which were essentially similar in appearance to control samples, except that in white areas from endotoxin-treated animals, endothelial cells were more granular, and silver-stained cell boundaries showed less separation. The results confirm the findings of an earlier study in which endotoxin-induced endothelial injury in the rabbit aorta was found to be most severe in the arch. The en face morphologic features of endotoxin-induced endothelial injury are shown to be largely confined to areas of enhanced endothelial permeability to protein as demarcated by in vivo Evans blue dye uptake.

Focal [3h]cholesterol uptake in the pig aorta Part 2. Distribution of [3h]cholesterol across the aortic wall in areas of high and low uptake in vivo☆☆☆

Abstract Focal areas of increased uptake of [3H]cholesterol in vivo by the macroscopically normal young pig aorta have been identified by their ability to accumulate the protein-binding azo dye, Evans blue. The distribution of labelled cholesterol across the aortic wall in areas of dye uptake (blue areas) and no dye uptake (white areas) was determined at intervals from 10 min to 24 h after the injection of label. Labelled free cholesterol represented most of the label in the aorta. It was distributed non-uniformly across the aortic wall, with most label concentrated in the intima and inner media. Labelled cholesterol ester represented only a small proportion of the aortic label and was distributed evenly through the vessel wall. Free cholesterol activity and free cholesterol specific activity were greater in blue areas than in white areas. These differences were confined to the intima and inner media and also the adventitia. Free cholesterol distribution and DNA content and distribution across the aortic wall were similar in blue and white areas. It is suggested that the increased focal uptake of labelled free cholesterol by blue areas reflects a higher free cholesterol turnover in these areas, and that labelled plasma cholesterol enters the pig aorta mainly through the intima in vivo .

The pathologic basis of sudden death

Abstract The innumerable conditions associated with or causing sudden unexpected death are reviewed, with particular attention to the pathologic basis of sudden cardiac death. Diseases of the cardiovascular system account for the majority of sudden deaths, and coronary artery disease is considered to be responsible for most cases of sudden cardiac death. It was emphasized that a proportion of sudden cardiac deaths exhibit no significant myocradial or coronary artery pathology which might reasonably account for death, and the validity of including such cases in the category of sudden death due to ischemic heart disease has been questioned. It is suggested that the category, sudden unexpected death due to ischemic heart disease be more carefully and critically defined for future epidemiological studies, and that alternate pathophysiological mechanisms which might account for sudden cardiac death be rigorously sought and studied. The potential importance of pathologic lesions in the conduction system has been discussed, and it is recommended that an examination of the conduction system and its blood supply become a routine in all cases of sudden death. Additionally, the role of microvascular disease, including coronary micro-embolism has been stressed as a potential mechanism for sudden cardiac death, and the need for research studies in this area has been emphasized.

Patterns of aortic evans blue uptake in vivo and in vitro

Patterns of aortic uptake of the protein-binding azo dye, Evans blue, have been studied both in vitro and in vivo in young (8–12 week) and older (6 month) pigs. In vitro patterns of blueing were independent of the composition of the various incubation media employed, and were similar for young and older animals. Dye uptake patterns in vivo were likewise similar for young and older pigs, but differed markedly from the patterns observed in vitro. Altered tissue viability did not appear to account for the observed in vivo and in vitro differences. It is likely that hemodynamic factors determine the focal patterns of dye uptake in vivo. The factor or factors responsible for the in vitro dye uptake pattern remain uncertain, and injury occurring during aortic removal cannot be excluded. These studies have shown that the characteristic in vivo pattern of aortic Evans blue uptake is not reproduced in vitro, and for this reason, further characterization of the structural and metabolic features of areas of Evans blue uptake will necessitate the use of the in vivo blueing model.

Influence of experimental aortic coarctation on the pattern of aortic Evans Blue uptake in vivo

Abstract The influence of aortic coarctation on the pattern of Evans Blue dye uptake by the aorta has been examined. Modification of aortic haemodynamics associated with the experimental production of aortic coarctation resulted in three changes in the dye-uptake pattern; first, an increased area of dye uptake in the aortic arch proximal to the coarctation; second, decreased bluing distal to the coarctation; and third, the appearance of new small areas of dye uptake a short distance below the coarctation. The latter were considered as early jet lesions, probably the result of a direct jet injury to the endothelium. The area of dye uptake proximal to the coarctation was greatly increased when the coarctation was tight (90% reduction in aortic cross-sectional area), but was not modified appreciably when the degree of narrowing was less. Reduction of the spontaneous focal bluing pattern distal to the coarctation was observed in the presence of both tight and loose coarctations. The relationship of these modifications of aortic Evans Blue uptake to the altered haemodynamics associated with aortic coarctation, endothelial injury, and permeability are discussed. It is concluded that the haemodynamic changes associated with aortic coarctation are capable of modifying the aortic uptake patterns of the protein-binding azo dye Evans Blue. It is suggested that the spontaneous focal patterns of aortic Evans Blue uptake might also be determined by focal haemodynamic factors.