Tibor Zemplenyi

Adaptation to arterial wall hypoxia demonstrated in vivo with oxygen microcathodes.

Iliofemoral arteries of 9 rabbits were balloon de-endothelialized resulting in subintimal thickening. Contrary to expectation, enzyme and lactate determinations did not indicate arterial wall hypoxia when compared with arteries of 10 control rabbits. The explanation came from in vivo measurement of oxygen tension profiles across the de-endothelialized and control femoral arteries and from the subsequent histological findings. They showed that the impaired oxygen supply of the de-endothelialized arteries with subintimal thickening was counteracted by a centripetal oxygenation of the arterial wall obviously induced by proliferation of newly formed nutrient vessels in the adventitia. Such adaptation is an important mechanism against hypoxia induced by arterial injury and may be an essential protective factor in atherogenesis.

Light- and electron-microscopic characteristics of arterial smooth muscle cell cultures subjected to hypoxia or carbon monoxide

Smooth muscle cells from the tunica media of piglet aortae grown under hypoxic conditions undergo the following changes: First, they become modified by partial loss of myofilaments and proliferation of organelles, which are characteristics of young primitive cells. Second, an increase in number of pinocytotic vesicles at and below the cell surface, indicating increased extracellular uptake of material, can be detected. This is followed by accumulation of Oil Red O positive intracytoplasmic granules and vacuoles as well as the subsequent formation of mount-like protrusions. The latter consist of a core of extracellular material and necrotic debris covered with a cap of viable cells. A third feature of the cells subjected to hypoxia is a conspicuous rise in the number of lysosomes. This is considered to be a manifestation of a defense mechanism of the cells to remove undesirable material from cytoplasm. Cells exposed to an atmosphere rich in carbon monoxide exhibit basically the same alterations as those grown under hypoxic conditions; however, formation of mound-like aggregates is less prominent, while the rise in the number of lysosomes is more evident than in the hypoxic cells. The above alterations are similar to changes observed in smooth muscle cells of rabbit with experimental atherosclerosis. It is suggested that whereever the arterial smooth muscle cell is subjected to adverse conditions basically the same mechanism, consisting of dedifferentiation, increased permeability and lysosomal defense reaction, takes place.

Arterial enzymes and their relation to atherosclerosis in pigeons

Abstract Comparison of metabolic processes between the atherosclerosis-resistant Show Racer (SR) and susceptible White Carneau (WC) pigeon strains offers an opportunity to study factors which may predispose to atherosclerosis. The activity of several glycolytic enzymes, Krebs cycle enzymes, enzymes of the ATP cycle and of the glycerol phosphate shuttle was studied in SR and WC arteries. In 4–6 yr old pigeons the activity of lipoamide dehydrogenase and malate dehydrogenase is significantly lower in WC than SR arteries. The differences are not the result of aging or atherosclerosis, because they were also detected in arteries of very young 5–8 wk old pigeons. Furthermore, the arteries of the young pigeons revealed a significantly higher activity of two glycolytic enzymes, namely phosphofructokinase and aldolase, in the WC arteries as compared with SR arteries. The results in the young birds indicate that the differences between the two pigeon strains are of an inherited (genetic?) nature. It is suggested that low activity of lipoamide and malate dehydrogenases slows down the Krebs cycle and leads to low citrate and ATP production. The latter factor is an essential part of the feedback control adjustments that regulate the efficiency of glycolysis via phosphofructokinase. Increased dependence of the WC arteries on glycolysis appears to facilitate the development of atherosclerosis in these birds, and the mechanism may be similar to the mechanism by which tissue hypoxia induces lipid accumulation and connective tissue alterations in the arterial wall. An additional finding in these studies is the higher activity in female than male arteries of phosphofructokinase, aldolase, isocitrate dehydrogenase, glycerokinase, ATPase and creatine phosphokinase.

Metabolic intermediates, enzymes and lysosomal activity in aortas of spontaneously hypertensive rats☆

Metabolic intermediate levels, glycolytic and Krebs cycle enzyme activities and lysosomal acid hydrolase activities were measured in aortas of spontaneously hypertensive (SHR) versus normotensive (WKY) rats. In the hypertensive aortas the level of lactate, the ratio of lactate to glucose and of lactate to malate was higher in the SHR than WKY aortas. In the hypertensive aortas the obvious shift of metabolism toward higher rate of glycolysis was associated with decreased activity of malate dehydrogenase and espically of lipoamide dehydrogenase. The latter is an essential compoenent of the alpha-ketoglutarate and pyruvate dehydrogenase enzyme complexes and it appears that these complexes are among the sites of arterialmetavolism which are primarily altered by the elevated blood pressure, resulting in increased production of lactate. The activity of the marker lysosomal enzyme N-acetyl-beta-glucosaminidase was unequivocally elevated in the hypertensive aortas. The activity of beta-glucuronidase exhibited incogruous differences between the SHR and WKY aortas and the activity of aortic acid phosphatase did not differ in the two rat strains. The results are discussed in relation to arterial injury, permeability, and atherogenesis.

The induction of atherosclerotic plaque-like mounds in cultures of aortic smooth muscle cells

SummarySmooth muscle cells harvested from the tunica media of piglet aortae were maintained in continuous culture for 10 months. When grown in the presence of 95% air and 5% CO2 they maintained a mature morphology as evaluated ultrastructurally. As these populations became confluent, the cells became oriented parallel to each other. When grown in the presence of 4% O2, 91% N2, and 5% CO2, this polarized pattern was disrupted. Focal areas of lipid accumulation were observed, succeeded by mound formation at these sites. The mound stained positive with PAS, aldehyde fuchsin, and oil red O. They were surrounded by 2—4 layers of intact cells. The centers of the mounds were composed of extracellular material and cell debris.

Inherited depression of arterial lipoamide dehydrogenase activity associated with susceptibility to atherosclerosis in pigeons.

The activity of lipoamide dehydrogenase (E.C.1.6.4.3) was measured in arterial homogenates from very young pigeons (5-8 weeks old) known to differ in their susceptibility to atherosclerosis. The activity of the arterial enzyme was significantly lower in the atherosclerosis-susceptible White Carneau pigeons than it was in the atherosclerosis-resistant Show Racer pigeons. Lipoamide dehydrogenase is a component of the pyruvate dehydrogenase and a-ketoglutarate multienzyme complexes. The first complex catalyzes the conversion of pyruvate to oxaloacetate via acetyl-CoA, and this reaction represents a crucial link between glycolysis and the Krebs cycle. The second complex is essential for the oxidative breakdown of carbohydrates, fats, and amino acids via the Krebs cycle. Reduced activity of these complexes, resulting from low activity of lipoamide dehydrogenase, favors reduction of pyruvate to lactate and a shift to glycolysis. This situation is in accord with other results obtained in avian and human arteries which appear to indicate a higher rate of glycolysis in atherosclerosis-susceptible and atherosclerotic arteries. It appears that the increased dependence of the White Carneau arteries on glycolysis, suggested by the reduced lipoamide dehydrogenase activity, facilitates the development of atherosclerosis in this pigeon strain.

Chemical sympathectomy by 6-hydroxydopamine and arterial enzymes and lactate in the rabbit

Abstract The effect of 6-hydroxydopamine (6-OHDA) on the activity of arterial enzymes and lactate was investigated in rabbits. It is known that 6-OHDA exerts a specific damaging effect on the adrenergic nerve endings and produces gangliopetal degeneration, thus eliminating the adrenergic effect pathway. Eight weeks following chemical sympathectomy malate dehydrogenase, isocitrate dehydrogenase, lipoamide dehydrogenase, and creatine phosphokinase exhibited significantly lower activity in the iliofemoral arteries of the 6-OHDA-treated animals as compared to those of the control rabbits. A direct inhibition of the activity of the enzymes by 6-OHDA can be reasonably excluded on the basis of in vitro studies. The aortas of the 6-OHDA-treated animals revealed an increase of pyruvate kinase only, without any significant change in activity of the other enzymes studied. The results dealing with enzymes in the iliofemoral arteries resemble the findings obtained previously in injured or atherosclerotic or “preatherosclerotic” human and animal arteries. The lactate level in the abdominal aortas, which are structurally similar to the iliofemoral arteries, was significantly higher in the 6-OHDA-treated animals than in the control rabbits. It appears that alterations of arterial innervation produce a metabolic pattern which may contribute to increased susceptibility to atherosclerosis.

Aortic enzymes and lactate in high altitude-raised and cholesterol-fed rabbits

Fourteen male rabbits born at elevation 4000 ft (first experimental series) were transferred at age of 2 months to elevation 12470 ft and raised there for 18 weeks. Half of the animals remained on a commercial rabbit chow (group H) while the other half was on the same diet supplemented with cholesterol (group C). Eight male rabbits raised at sea level served as controls (group S). Intima-media homogenates from the thoracic aortas were assayed for lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lipoamide dehydrogenase, pyruvate kinase (PK), phosphofructokinase (PFK) and the lysosomal hydrolases beta-glucuronidase and N-acetyl-beta-glucosaminidase (NAGA). Aortic lactate and glucose were also measured. Thirty-two male rabbits (second experimental series) were subdivided into 4 groups. Rabbits were fed a cholesterol-supplemented diet not only at high altitude (8 rabbits matching group C) but also 8 animals raised at sea level. The degree of atherosclerosis in the aortas of these 4 groups was assessed by measuring the aortic cholesterol contents. Plasma cholesterol was also determined. In the aortas of the rabbits of group H the activity of PK was significantly elevated, and the activity of the lysosomal hydrolases significantly decreased compared with aortas of group S rabbits. There was no difference in the other enzyme activities or in the aortic glucose and lactate content of these groups. Cholesterol feeding of the animals of group C resulted in a significantly increased activity of the lysosomal hydrolases as well as of LDH and PK. The lipid analyses (second experimental series) revealed a trend to a lower concentration of aortic cholesterol in the high altitude than in the sea level animals, both fed a cholesterol diet, in spite of the higher plasma cholesterol concentrations in the high altitude animals. The low aortic lysosomal hydrolase activities in the high altitude rabbits are in accord with their comparatively lower susceptibility to experimental atherosclerosis. This metabolic feature may be due to a lower degree of exposure of these aortas to injurious factors, such as infections or lower blood pressure. The elevated activity of PK without increased lactate content in group H animals seems to parallel the well-known general adaptation of the organism to high altitude hypoxia, and does not indicate a metabolic switch toward anaerobic glycolysis.

Elevation of arterial phosphofructokinase activity associated with susceptibility to atherosclerosis in pigeons

The activity of phosphofructokinase (PFK, 2.7.1.11) was measured in arteries of very young (5-8 week old) pigeons known to differ in susceptibility to atherosclerosis. The activity of the arterial enzyme was significantly higher in the atherosclerosis-susceptible White Carneau (WC) pigeons than in the resistant Show Racers (SR). The difference was significant whether enzyme activity was calculated on the basis of extract protein, DNA content or fat-free dry weight. In the White Carneau arteries the activity of the enzyme was higher in the female than the male pigeons. PFK is a key regulatory enzyme of glycolysis and is subject to fine control adjustments. A low ATP/ADP ratio and a fall in citrate concentration, as for example, induced by hypoxia, are meditors of a feedback mechanism leading to a rise in PFK activity and enhancement of glycolysis for energy production. This mechanism appears to be the cause of the higher PFK activity in the WC arteries, because related studies indicate impaired Krebs cycle activity in these vessels. It is suggested that the increased dependence of the WC arteries on glycolysis facilitates the development of atherosclerosis in this pigeon strain and that the mechanism is similar to the mechanism by which tissue hypoxia causes lipid accumulation and connective tissue alterations in the arterial wall.

The effect of combined deendothelialization and hypercholesterolemia on some arterial lysosomal and glycolytic enzymes and lactate in rabbits

In eight New Zealand white male rabbits the abdominal aorta and one iliofemoral artery was balloon deendothelialized (group A). After 2 weeks they were kept for 6 weeks on a high cholesterol diet together with eight unoperated rabbits (group B). Eight more rabbits were kept on a commercial diet only (group C). The degree of atherosclerosis was much higher in the deendothelialized Group A vessels than in the uninjured group B vessels. The activity of lactate dehydrogenase and of the rate-limiting glycolytic pyruvate kinase was significantly increased and the activity of lipoamide dehydrogenase decreased in the group A aortas. In the iliofemoral arteries a similar but statistically insignificant tendency was detected. There was no significant difference, however, in aortic lactate between the three groups. Thus, local hypoxia did not significantly contribute to the high degree of atherosclerosis in the group A animals in spite of the enzyme activity differences. Previous experience of the authors, using arterial microcathode pO2 measurements, indicates that following deendothelialization an adaptive proliferation of nutrient vessels and increased arterial oxygenation takes place. The average activity of the lysosomal N-acetyl-beta-glucosaminidase was five times and that of beta-glucuronidase, seven times higher in the Group A than Group B aortas; in the iliofemoral arteries the differences were even larger. The huge elevation of these hydrolases, which are involved in glycosaminoglycan catabolism, provides indirect indication that accumulation of glycosaminoglycans and possibly their ability to form complexes with apoB-containing lipoproteins played a major role in the much increased degree of atherosclerotic lesions in the Group A rabbits.