Frank P. Bell

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.

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.

Effect of dietary di-2-ethylhexyl phthalate on lipid biosynthesis in selected tissues from the rat, in vitro.

Di-2-ethylhexyl phthalate (DEHP), a plasticizer commonly used in the production of polyvinyl chloride plastics, has become an environmental pollutant. At the present time, the biological significance of phthalates in the environment is unknown. In the present studies, we observed that addition of DEHP to a stock diet of rats resulted in marked effects on incorporation of14C-acetate into lipid by liver and kidney slices; other organs, such as heart, testes, and aorta were unaffected. Incorporation of14C-acetate into total lipid of liver (dpm/mg wet wt) from rats, fed 0.5% or 1.0% DEHP for 10 or 18 days, respectively, was decreased to ca. 50% of control values. The decreased incorporation into liver lipid is not attributable to any one lipid fraction, inasmuch as incorporation into the phospholipid, sterol+diglyceride, free fatty acid, triglyceride, and sterol ester+hydrocarbon fractions was decreased 30–70% with respect to controls. In addition, the percent distribution of14C-acetate among the individual phospholipids was ca. 25% lower in phosphatidyl choline of the DEHP-fed rats. In rats fed 0.5% DEHP, incorporation of14C-acetate into total lipid of kidney was similar to control values, but incorporation into the triglyceride and sterol ester+hydrocarbon fraction was decreased 30–40%, whereas incorporation into the sterol+diglyceride fraction was increased 38%. Livers from DEHP-fed rats were ca. 20% larger than livers from control rats and, at the 0.%% level of DEHP feeding, testes wts were elevated; no significant changes were noted in wts of spleen, heart, aorta, kidney, or body wt gains in rats fed DEHP. These studies emphasize a subtle toxicity of phthalate esters not previously reported and emphasize the need for further biochemical studies to evaluate the effect of phthalates on biological systems.

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.

Inhibition of hepatic sterol and squalene biosynthesis in rats fed di-2-ethylhexyl phthalate

Di-2-ethylhexyl phthalate (DEHP), a commonly used plasticizer, was found to be an inhibitor of the biosynthesis of hepatic nonsaponifiable lipids in the rat. The addition of DEHP at levels of 0.5% or 1.0% to a stock diet of rats resulted in a decreased conversion of acetate-1-14C and mevalonate-5-3H into squalene, C30 sterols, and C27 sterols by liver minces or slices, in vitro. In studies conducted with 0.5% DEHP feeding from 2 to 11 days, the degree of inhibition was found to increase with the duration of DEHP feeding; the inhibition of3H-mevalonate conversion to squalene and sterols developed more slowly, being reduced to ca. 70% of control values in 11 days, whereas14C-acetate conversion was reduced to ca. 35% of control values during the same period.3H-mevalonate conversion to sterols and squalene was, however, found to be suppressable to the same extent as14C-acetate conversion when diets containing 1.0% DEHP were fed for 18 days. The inhibitory effect of dietary DEHP on sterol and squalene biosynthesis from14C-acetate and3H-mevalonate by rat liver preparations is unlikely to be accounted for by the negative feedback of cholesterol secondary to hepatic sterol accumulation since, in these studies, hepatic total lipid and hepatic total sterol levels were simialr in control and DEHP-fed rats.

Differing patterns of cholesterol accumulation and 3H-cholesterol influx in areas of the cholesterol-fed pig aorta identified by evans blue dye☆

Abstract In this study we have examined the possible focal and regional nature of aortic cholesterol accumulation in pigs receiving a cholesterol-containing diet for 6 to 16 weeks. Using uptake of the protein-binding azo dye Evans Blue as a marker for focal areas of increased aortic endothelial permeability in vivo , we have found a significantly greater intimal accumulation of cholesterol in areas of dye uptake (blue areas) than in contiguous areas of the thoracic aorta showing no dye uptake (white areas). This focal difference in cholesterol accumulation was confined to the intima alone, and was not observed in either intima-media, or medial preparations. No measurable cholesterol accumulation was observed in the intima of blue relative to white areas in the absence of cholesterol feeding. [ 3 H]cholesterol was administered 72 hr before death to four pigs receiving a cholesterol diet for 6 weeks. Unesterified cholesterol radioactivity was found to be 60–70% of the total activity in intimal or medial tissue. For each site studied (thoracic blue, thoracic white, and abdominal white), intimal cholesterol radioactivity was significantly greater than in the underlying media. Unesterified and esterified cholesterol radioactivity in intimal blue areas was significantly greater than the respective activities in the intima from thoracic white or abdominal white areas. Cholesterol accumulation in the intima, but not in the intima-media alone, was found to exhibit a regional difference as well, with significantly greater accumulation in the thoracic than abdominal aortic segments. This regional pattern was also reflected in the 3 H unesterified and esterified cholesterol activity in the intimal preparations from the thoracic and abdominal aortic segments. This study has shown that aortic cholesterol accumulation is not homogeneous. The regional and focal difference in cholesterol accumulation observed may reflect local hemodynamic effects. The implications of these findings in terms of endothelial permeability to lipoproteins, and the pathogenesis of atherosclerois are discussed.

Lipid Composition and Metabolism of Thromboatherosclerotic Lesions Produced by Continued Endothelial Damage in Normal Rabbits

Thromboatherosclerotic and fibrous lesions were produced by endothelial damage with polyethylene catheters inserted into the aortas in rabbits on a normal diet. Two weeks after insertion of the catheters, the concentration of both free cholesterol and cholesteryl ester in the thromboatherosclerotic lesions was significantly greater than that in the adjacent normal intima. A further increase in the concentration of free cholesterol and particularly of cholesteryl ester occurred during the remainder of the 4-month study period. Gas-liquid chromatography indicated that the raised thromboatherosclerotic lesions contained more cholesteryl oleate and less cholesteryl linoleate than did either the normal intima or the fibrous lesions. The incorporation of [1−14C] oleic acid into combined lipid in the aortas incubated in vitro showed that, by 2 weeks, two to three times more oleic acid had been incorporated into cholesteryl ester in the thromboatherosclerotic raised lesions than in the normal intima. A similar increase was demonstrated at 2 and 4 months. When [32P] phosphate was used as a precursor, incorporation into lecithin was higher and incorporation into phosphatidyl inositol was lower in the raised lesions than they were in the normal intima. Fibrous lesions did not differ significantly from the adjacent normal intima in their incorporation of either of these precursors into lipid. Therefore, the accumulation of cholesteryl ester in the thromboatherosclerotic lesions resulted in part from synthesis in the arterial wall, which was stimulated by the prominent platelet components of the lesions.

Focal differences in lipid metabolism of the young pig aorta Synthesis from [1 −14c]acetate and [u−14c]glucose in vitro☆

Abstract In the pig, focal areas of in vivo aortic Evans Blue accumulation (blue areas) have previously been found to show greater [3H]cholesterol uptake than areas showing no dye accumulation (white areas) 21,25 . The present experiments were undertaken to examine lipid synthesis in these blue and white areas in vitro . Blue areas showed a significantly greater incorporation of [1 − 14 C]acetate into total lipid than adjacent white areas, an increase which was demonstrable in all neutral lipid and phospholipid fractions. Additionally, blue and white areas differed in the percentage distribution of incorporated labelled acetate among the different lipid classes. In blue areas a significantly greater proportion of label appeared in phospholipid, which was attribut able to a greater percentage of label incorporated in phosphatidyl choline, and sphingomyelin. Blue and white areas showed no significant difference in the incorporation of [U− 14 C]glucose into neutral lipid and phospholipid. Further, there was no difference in the percentage distribution of label among individual lipid fractions. The major proportion of [1− 14 C]acetate incorporated appeared in the fatty acid moiety of glyceride lipid, while the major proportion of [U− 14 C]glucose incorporated appeared in the glycerol moiety of glyceride lipid.

Effect of dietary di-2-ethylhexyl phthalate on oxidation of14C-palmitoyl CoA by mitochondria from mammalian heart and liver

Oxidation of [1-14C] palmitoyl CoA by heart and liver mitochondria from rats fed dietary di-2-ethylhexyl phthalate (DEHP) was investigated in vitro. Oxidation of14C-palmitoyl CoA to14CO2 increased two- to threefold in hepatic mitochondria from rats fed 0.1% DEHP for 2 to 3 days; this increase appeared to be a maximum response since similar data were obtained using hepatic mitochondria from rats receiving 0.5% or 1.0% DEHP in the diet. The response of hepatic mitochondria to DEHP was found to continue throughout the duration of 35-day trials in which 1.0% DEHP was fed. In contrast to hepatic mitochondria, the oxidation of14C-palmitoyl CoA by heart mitochondria decreased ca. 40% upon addition of 0.1% or 0.5% DEHP to the diet; this effect of DEHP on heart mitochondria was not sustained beyond ca. 8 days of DEHP feeding. Limited studies were also performed in rabbits and pigs. Oxidation of14C-palmitoyl CoA was increased ca. twofold in hepatic mitochondria from rabbits fed 1% dietary DEHP for 12 days and in hepatic mitochondria from pigs that received 5 doses of DEHP (0.8g/kg) at 12-hr intervals; the oxidation14C-palmitoyl CoA by heart mitochondria from these same animals was unchanged in the rabbit but increased an average of 37% in the pig. DEHP feeding to rats was associated with increased yields of hepatic mitochondrial protein; standardized preparations of heart mitochondria were not similarly affected.

Cholesterol exchange between microsomal, mitochondrial and erythrocyte membranes and its enhancement by cytosol

1. Cholesterol exchanges between isolated rat liver microsomes and mitochondria and between erythrocytes and microsomes or mitochondria during incubation in vitro. The exchange process is temperature dependent and is no accompanied by a net movement of sterol. 2. cholesterol exchange between the membranes was enhanced by the addition of 105 000 x g supernatant fraction (S105) from rat liver. The degree to which sterol exchange was enhanced was dependent on the amount of this supernatant fraction present in the incubation. 3. enhancement of sterol exchange was not observed with heated S105 fraction, but activity was retained after dialysis or aging at 10 degrees C; these results suggest the presence of a cholesterol-exchange protein in the cytosol from rat liver.