Arterial injury by cholesterol oxidation products causes endothelial dysfunction and arterial wall cholesterol accumulation

Cholesterol oxidation products (ChOx) have been reported to cause acute vascular injury in vivo; however, the pharmacokinetics of ChOx after administration and the mechanisms by which they cause chronic vascular injury are not well understood. To further study the pharmacokinetics and atherogenic properties of ChOx, New Zealand White rabbits were injected intravenously (70 mg per injection, 20 injections per animal) with a ChOx mixture having a composition similar to that found in vivo during a 70-day period. Total ChOx concentrations in plasma peaked almost immediately after a single injection, declined rapidly, and returned to preinjection levels in 2 hours. After multiple injections, the ChOx concentrations rose gradually to levels 2- to 3-fold above baseline levels, increasing mostly in the cholesteryl ester fraction of LDL and VLDL. Rabbit serum and the isolated LDL/VLDL fraction containing elevated ChOx concentrations were cytotoxic to V79 fibroblasts and rabbit aortic endothelial cells. At the time of killing, cholesterol levels in the aortas from ChOx-injected rabbits were significantly elevated despite the fact that plasma cholesterol levels remained in the normal range. In addition, aortas from the ChOx-injected rabbits retained more 125I-labeled horseradish peroxidase, measured 20 minutes after intravenous injection. Transmural concentration profiles across the arterial wall also showed increased horseradish peroxidase accumulation in the inner half of the media from the thoracic aorta in ChOx-injected rabbits. In conclusion, ChOx injection resulted in accumulation of circulating ChOx and induced increased vascular permeability and accumulation of lipids and macromolecules. This study reveals that even under normocholesterolemic conditions, ChOx can cause endothelial dysfunction, increased macromolecular permeability, and increased cholesterol accumulation, parameters believed to be involved in the development of early atherosclerotic lesions.     

Watanabe rabbits with heritable hypercholesterolaemia: a model of atherosclerosis

Many factors play important roles in the development of atherosclerotic lesions. The leading risk factor for atherosclerosis is familial hypercholesterolaemia (FH). FH is a genetic disease characterized by a deficiency of receptors for low density lipoprotein (LDL) on the plasmalemma of endothelial cells, a high level of serum LDL, and early development of atherosclerosis and skin xanthoma. Watanabe and colleagues have developed a line of rabbits with unprovoked hypercholesterolaemia, increased blood level of LDL, pronounced atherosclerosis and skin xanthoma. These Watanabe Heritable Hyperlipidaemic (WHHL) rabbits possess an inheritable mutation of one gene, similar to that in human FH. The morphogenesis of atherosclerosis in patients with FH is characterized by multifocal deposit of lipids in the stromal cells of thymus, spleen, skin, interstitial and parenchymatous cells of kidneys and the presence of some single foam cells in aorta. The manifestation of atherosclerotic lesions in WHHL rabbits increases progressively with age but the presence of atherosclerotic lesions in newborn WHHL rabbits suggest that the process may commence in utero. Moreover, the main mass of plasma cholesterol in WHHL rabbits is first found in LDL and to a lesser degree in lipoproteins of intermediate density. This is contrary to diet-induced atherosclerosis in rabbits where the main mass of serum cholesterol is found in very low density beta-lipoproteins. Thus the distribution of cholesterol among lipoprotein fractions differs from that in WHHL rabbits. Atherosclerotic damage of arteries in WHHL rabbits goes through several stages. During the progression of intimal damage, lipid and foam cell deposits are found in the internal surface together with developing plaques and increased content of lipids in the tunica media. Calcification often follows this process. The main factors initiating atherosclerosis in WHHL rabbits are adhesion of leukocytes and platelets to endothelial cells and the accumulation of lipids in the aortic wall. The deposits of lipids in macrophages and intimal smooth muscle cells in WHHL rabbits occurs mostly at the expense of cytoplasmic neutral lipid particles with some accumulation in lysosomes. Hypertension as a risk factor increases the area of atherosclerotic damage in all arterial vessels in WHHL rabbits, particularly in the thoracic and abdominal aorta. Morphogenesis of the development of atherosclerosis in WHHL and diet-induced atherosclerosis in rabbits was similar, but differs from rats with heritable hypercholesterolaemia. Damage or loss of endothelial cells can predispose the atherosclerotic vessels to vasospasm and can leave vessels unprotected against vasoconstrictor stimuli. The development of the WHHL model has not only given insight into the mechanisms of development of familial hypercholesterolaemia but has also provided a model for assessing various therapeutic approaches for the prevention and treatment of atherosclerosis.     

Atherosclerotic risks from chemicals: part I. Toxicological observations and mechanisms of atherosclerosis

Atherosclerosis is a common disease, primarily of the large arteries, that begins in childhood and progresses with advancing age. Atherosclerosis leads to coronary heart disease, the major cause of death in the United States. Several risk factors affect atherosclerosis, but high LDL cholesterol is the most important risk factor. In addition, high levels of lipoprotein (a) appear to be associated with increased atherosclerosis and myocardial infarction. The level of lipoprotein (a) is genetically determined and is not affected by diet or exercise. Studies on the pathogenesis of atherosclerosis suggest that several steps are involved, including endothelial injury, increased arterial permeability to plasma lipoproteins, smooth muscle cell proliferation, and platelet aggregation. Atherosclerotic plaques are benign neoplasms of the arterial wall that result from the monoclonal proliferation of a single mutated smooth muscle cell. Abnormal proliferation of smooth muscle cells is the key event in the initiation and progression of atherosclerosis. Endothelial injury is another major contributory factor. Many factors associated with an increased risk of cancer are also associated with atherosclerosis. Cancer and atherosclerosis go through the same stages of initiation, promotion, and complication. Both inflammatory and immune reactions play important roles in the progressions of the two diseases. Smooth muscle cells and endothelial cells produce and respond to several cytokines and growth factors, which may influence the initiation, progression, and complication of the atherosclerotic lesions. Many studies have shown that the production of nitric oxide is decreased in atherosclerosis-reduction in the bioavailability of nitric oxide in the arterial wall may lead to leukocyte adhesion and platelet aggregation. It should be noted additionally, nitric oxide is a mutagenic agent involved in the origin of neoplastic diseases. Atherosclerotic plaques express genes for products not found in the normal arterial wall. As with carcinogenesis, there may be more than one mechanism that promotes atherosclerotic lesions and there may be common mechanistic similarities between the two diseases. The purpose of this study is to establish an exploratory scientific hypothesis that will permit the use of standardized toxicological test data to evaluate different chemicals. The companion paper that follows will use a method of relative toxicological potencies to develop tentative risk coefficients based on relative potency. These papers, in combination, provide both a conceptual and a quantitative hypothesis that can be tested with data from forthcoming epidemiological studies or animal test models.     

Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development

The subendothelial aggregation and retention of low density lipoprotein (LDL) are key events in atherogenesis, but the mechanisms in vivo are not known. Previous studies have shown that treatment of LDL with bacterial sphingomyelinase (SMase) in vitro leads to the formation of lesion-like LDL aggregates that become retained on extracellular matrix and stimulate macrophage foam cell formation. In addition, aggregated human lesional LDL, but not unaggregated lesional LDL or plasma LDL, shows evidence of hydrolysis by an arterial wall SMase in vivo, and several arterial wall cell types secrete a SMase (S-SMase). S-SMase, however, has a sharp acid pH optimum using a standard in vitro SM-micelle assay. Thus, a critical issue regarding the potential role of S-SMase in atherogenesis is whether the enzyme can hydrolyze lipoprotein-SM, particularly at neutral pH. We now show that S-SMase can hydrolyze and aggregate native plasma LDL at pH 5.5 but not at pH 7.4. Remarkably, LDL modified by oxidation, treatment with phospholipase A2, or enrichment with apolipoprotein CIII, which are modifications associated with increased atherogenesis, is hydrolyzed readily by S-SMase at pH 7.4. In addition, lipoproteins from the plasma of apolipoprotein E knock-out mice, which develop extensive atherosclerosis, are highly susceptible to hydrolysis and aggregation by S-SMase at pH 7.4; a high SM:PC ratio in these lipoproteins appears to be an important factor in their susceptibility to S-SMase. Most importantly, LDL extracted from human atherosclerotic lesions, which is enriched in sphingomyelin compared with plasma LDL, is hydrolyzed by S-SMase at pH 7.4 10-fold more than same donor plasma LDL, suggesting that LDL is modified in the arterial wall to increase its susceptibility to S-SMase. In summary, atherogenic lipoproteins are excellent substrates for S-SMase, even at neutral pH, making this enzyme a leading candidate for the arterial wall SMase that hydrolyzes LDL-SM and causes subendothelial LDL aggregation.     

Lipoprotein lipase increases lipoprotein binding to the artery wall and increases endothelial layer permeability by formation of lipolysis products

Mechanisms responsible for the accumulation of low-density lipoprotein (LDL) were investigated in a new model, the perfused hamster aorta. To do this, we developed a method to study LDL flux in real time in individually perfused arteries; each artery served as its own control. Using quantitative fluorescence microscopy, the rates of LDL accumulation and efflux were separately determined. Perfusion of arteries with buffer plus lipoprotein lipase (LpL) increased LDL accumulation 5-fold (0.1 +/- 0.03 mV/min [control] versus 0.5 +/- 0.05 mV/min [LpL]) by increasing LDL retention in the artery wall. This effect was blocked by heparin and monoclonal antibodies directed against the amino-terminal region of apolipoprotein B (apo B). This suggests that specific regions of apo B are involved in LDL accumulation within arteries. Also, the effect of hydrolysis of triglyceride-rich lipoproteins on endothelial barrier function was studied. We compared endothelial layer permeability using a water-soluble reference molecule, fluorescently labeled dextran. When LpL was added to hypertriglyceridemic plasma, dextran accumulation within the artery wall increased > 4-fold (0.024 +/- 0.01 mV/min [control] versus 0.098 +/- 0.05 mV/min [LpL]). Under the same conditions, LpL increased LDL accumulation approximately 3-fold (0.016 +/- 0.003 mV/min [control] versus 0.047 +/- 0.013 mV/min [LpL]). Rapid efflux of LDL from the artery wall indicated that increased endothelial layer permeability was the primary mechanism during periods of increased lipolysis. Our data demonstrate two LpL-mediated effects that may increase the amount of LDL in the artery wall. These findings may pertain to the observed relationship between increased postprandial lipemia and atherosclerosis.     

Homocysteine, a risk factor of atherosclerosis

Homocysteine is a sulphurated amino acid which, at high plasma concentrations, predisposes to thrombosis and induces focal arteriosclerosis. These characteristics have been established both in patients with homocystinuria, a genetic disease in which homocysteine accumulates in the blood, and in animals submitted to intravenous infusions of this amino acid. Many recent publications have addressed the problem of whether mild increases in plasma homocysteine predisposed to the development of the usual forms of atherosclerosis. Transverse epidemiological studies have established a correlation between homocysteine levels and atherosclerosis at all its vascular localisations, coronary, carotid and lower limb. Multivariate analysis in several prospective studies have shown plasma homocysteine to be an independent risk factor for cerebrovascular accidents and myocardial infarction. Causes of mild increases in plasma homocysteine are usually dietetic deficiencies in folic acid, vitamin B6 or B12, or genetic by mutation of the methylene-tetrahydrofolate reductase. Renal failure is also associated with a high risk in plasma homocysteine levels. However, the toxicity of homocysteine to the arterial wall at slightly elevated concentration remains speculative.     

Health related quality of life outcomes in patients treated for metastatic kidney cancer: a pilot study

The use of the immuno-suppressant cyclosporine A (CsA) after transplantation has been associated with less favorable plasma lipid profiles, which may contribute to the high incidence of cardiovascular morbidity and mortality in transplant recipients. Recent studies have suggested that oxidative modification of LDL plays an important role in the initiation and progression of atherosclerosis. It has also been demonstrated that CsA may facilitate lipid peroxidation in vitro and in vivo. Therefore, we determined several parameters of LDL oxidizability in renal transplant recipients who were switched from CsA to azathioprine (AZA)-based immunosuppressive treatment. The susceptibility of LDL to in vitro oxidation, LDL particle size, plasma titers of IgG and IgM antibodies against oxidized LDL and plasma LDL subclass patterns in 19 renal transplant recipients were determined during CsA treatment and 16 weeks after these patients were converted to AZA treatment. In addition, mean arterial pressure was recorded, and glomerular filtration rate and renal blood flow were estimated from the clearance of radiolabeled thalamate and hippurate. After conversion, the plasma concentrations of total cholesterol, LDL cholesterol and triglyceride decreased, while plasma HDL cholesterol did not change. During CsA therapy plasma LDL was significantly more susceptible to in vitro oxidation than during AZA, as reflected by a longer lag phase during in vitro oxidation (98.9 +/- 24.3 vs. 114.7 +/- 17.3 min, P = 0.031). In addition, the LDL size increased (236.5 +/- 7.3 vs. 240.7 +/- 6.8 nm, P = 0.00001), and the titers of IgM- and IgG-autoantibodies against oxidized LDL decreased significantly after patients were converted from CsA to AZA. The more atherogenic LDL subclass pattern B was present in 13 out of 19 patients during CsA. In five patients, pattern B changed into pattern A after conversion. The subclass B pattern was maintained in eight patients and subclass A pattern in six patients. In all patients the lag time of in vitro LDL oxidation increased, although the biggest changes were found in those patients in whom the LDL subclass changed from pattern B to pattern A. Mean arterial pressure decreased and renal function improved significantly after conversion. No correlation between parameters of lipid peroxidation and changes in blood pressure or renal function upon conversion, underlying renal disease, time since transplantation, or antihypertensive treatment was found. Our study demonstrates that treatment with CsA increases the susceptibility of LDL to in vitro oxidation, and also enhances the oxidation of LDL in vivo. In addition, conversion to AZA results in a more favorable lipid profile, which in combination with a lower arterial pressure and better renal function may decrease the risk for atherosclerosis. These factors may account for the cardiovascular complications during CsA treatment after organ transplantation, and also when CsA is used for other diseases.     

Effect of environmental tobacco smoke on LDL accumulation in the artery wall

BACKGROUND: Previous research has shown that exposure to environmental tobacco smoke (ETS) increases the risk of atherosclerosis. To test the hypothesis that exposure to ETS increases LDL accumulation in the artery wall, we developed a model to measure the rate of LDL accumulation in individually perfused rat carotid arteries after the artery had been perfused with plasma taken from rats exposed to ETS (ETS-plasma). METHODS AND RESULTS: Rats were exposed to ETS in a chamber in which steady-state sidestream smoke was continuously circulating. After exposure, blood from the animals was collected. Carotid arteries from unexposed rats were perfused first with normal plasma containing fluorescently labeled LDL. Then the same arteries (10 arteries from five rats) were perfused with ETS-plasma plus fluorescently labeled LDL. Photometric measurements were made during perfusion of the arteries with fluorescently labeled LDL, and rate of LDL accumulation (mV/min) and lumen volume (mV) (volume of fluorescently labeled LDL solution) were determined. Perfusion with ETS-plasma increased the rate of LDL accumulation (mean +/- SEM, 6.9 +/- 1.8 mV/min) compared with control (1.6 +/- 0.40 mV/min, P < or = .02). LDL accumulation was primarily dependent on LDL interaction with ETS-plasma rather than the interaction of ETS-plasma with the artery wall. Also, ETS-plasma significantly increased lumen volume (43.3 +/- 5.1 mV) compared with control (35.1 +/- 4.4 mV, P < or = .005). CONCLUSIONS: Exposure to ETS acutely increased LDL accumulation in perfused arteries. Repeated exposure to ETS may represent important early events in atherogenesis.     

In vivo transfer of lipoprotein(a) into human atherosclerotic carotid arterial intima

The aim of this study was to compare the atherogenic potential of lipoprotein(a) [Lp(a)] and LDL by measuring the intimal clearance of these two plasma lipoproteins in the atherosclerotic intima of the human carotid artery in vivo. Autologous 131I-Lp(a) and 125I-LDL were mixed and reinjected intravenously 3 hours before elective surgical removal of the arterial intima in four patients. The intimal clearance of Lp(a) and LDL was 229+/-48 and 405+/-127 nL/cm2 per hour, respectively (paired t test; P=.12). The mass accumulation of Lp(a) (114+/-32 ng/cm2 per hour) was on average one 15th that of LDL (paired t test; P=.06), mainly reflecting a low plasma concentration of Lp(a) compared with LDL in the human subjects studied. In accordance with our previous observation in rabbits, there was a positive association between the intimal clearance of LDL and that of Lp(a) (r=.97, P=.03). Accordingly, high plasma levels of Lp(a) may share with LDL the potential for causing lipid accumulation in the arterial intima in humans.     

Atherosclerosis, with special reference to vitamin E

The relationship between atherosclerosis and fat soluble vitamin, especially vitamin E is reviewed on the basis of oxidised modification of low density lipoprotein (LDL). Data now support the notion that the oxidised LDL is present in the blood and arterial wall and antioxidant drugs such as probucol and vitamin E, beta-carotene, may prevent the progression of atherosclerosis. LDL alpha-tocopherol levels are generally correlated to the plasma concentrations and supplementation with alpha-tocopherol increases its content in LDL. There is a significant correlation between the LDL alpha-tocopherol level and the resistance to oxidative modification. Epidemiological data also shows the relation between low levels of plasma vitamin E and the increased incidence of coronary heart disease. Clinical application of vitamin E should be clarified in detail to inhibit the progression of atherosclerosis.     

A partial estrogen receptor agonist with strong antiatherogenic properties without noticeable effect on reproductive tissue in cholesterol-fed female and male rabbits

Estrogen replacement therapy retards the development of cardiovascular disease and osteoporosis in postmenopausal women. However, long-term unopposed use increases the risk of cancer in endometrium and possibly in breast. The racemic compound ormeloxifene, widely used in India as an antifertility agent, is a partial estrogen receptor agonist with antiosteoporotic properties. The present study was undertaken to investigate the effect of the L-enantiomer (levormeloxifene) and the d-enantiomer (d-ormeloxifene) on the development of atherosclerosis. In a short-term experiment (6 weeks), 4 x 10 ovariectomized female rabbits were fed a 0.25% cholesterol-enriched diet and the effect on plasma cholesterol levels was studied. In a long-term experiment (13 weeks), 4 x 15 ovariectomized female and 4 x 15 shamoperated male rabbits were maintained at a similar plasma cholesterol level of 25 mmol/L and the effect on undamaged and balloon-injured arterial wall was studied. In both experiments, the rabbits were treated with levormeloxifene, d-ormeloxifene, 17 beta-estradiol, or placebo, respectively. In the short-term experiment, levormeloxifene, in contrast to d-ormeloxifene, significantly reduced plasma cholesterol by 30% compared with the placebo group. In the long-term experiment, levormeloxifene, in contrast to d-ormeloxifene, significantly reduced atherosclerosis by 50% in the undamaged arterial wall of both female and male rabbits. Because these rabbits were cholesterol-clamped, the antiatherogenic effect was not mediated via plasma cholesterol lowering. Like estrogen, levormeloxifene did not inhibit atherosclerosis in the endothelium-denuded site of aorta. The antiatherogenic effects of levormeloxifene were thus similar to those of estrogen, but produced in the absence of any noticeable estrogenic effect on uterine or testicular tissue.     

A high concentration of melatonin inhibits in vitro LDL peroxidation but not oxidized LDL toxicity toward cultured endothelial cells

The pineal hormone, melatonin, was recently found to be a potent free scavenger for hydroxyl and peroxyl radicals. Melatonin also inhibits neuronal and thymocyte damage due to oxidative stress. Atherosclerosis development is mediated by low-density lipoprotein (LDL) oxidation and the endocytosis of oxidized LDL by resident macrophages in the subendothelial vascular wall. Furthermore, the cytotoxic effect of oxidized LDL increases atherogenicity. The goal of this study was to compare the antioxidant activities of melatonin and vitamin E against in vitro LDL oxidation and their cytoprotective actions against oxidized LDL-induced endothelial cell toxicity. An attempt at loading LDL with melatonin by incubating human plasma with an ethanolic melatonin solution gave only low protection against Cu2+-induced LDL oxidation in comparison with vitamin E and gave no detectable incorporation of melatonin into LDL, measured by high-performance liquid chromatography (HPLC) coupled to UV detection. High concentrations of melatonin (10-100 microM) added to the oxidative medium induced a clear inhibition of Cu2+-induced LDL oxidation, characterized as an increase in the lag-phase duration of conjugated diene formation and decreases in the maximal rate of the propagation phase and in the maximal amount of conjugated diene formation. Determination of the median efficacious dose (ED50) of melatonin and vitamin E by their ability to increase lag-phase duration showed that melatonin was less active than vitamin E (ED50, 79 vs. 10 microM, respectively). Melatonin was also less active than vitamin E in limiting the formation of thiobarbituric acid-reactive substances (TBARS) and LDL fluorescence intensity increase in the medium during Cu2+-induced LDL oxidation. Cu2+-induced LDL oxidation in the presence of 100 microM melatonin produced oxidized LDLs that were less recognizable for the scavenger receptors of J774 macrophages than were untreated LDLs. Vitamin E, 10 microM, was more active than 100 microM melatonin in inhibiting LDL oxidation and the resulting lipoprotein alterations leading to binding internalization and degradation by the J774 macrophages. Vitamin E, 100 microM, inhibited the pursuit of the oxidation of oxidized LDL mediated by bovine aortic endothelial cells (BAECs) in a culture medium containing Cu2+, whereas 100 microM melatonin had no antioxidant effect. Melatonin, 100 microM, as well as 100 microM vitamin E inhibited intracellular TBARS formation during the incubation of BAECs with highly oxidized LDL but had no influence on the increase in glutathione (GSH) concentration during this lengthy exposure (16 h) of BAECs to highly oxidized LDL. During this period, the same dose of vitamin E but not of melatonin tended to limit the decrease in adenosine triphosphate (ATP) concentration. Vitamin E, 100 microM, did not significantly reduce cellular lactate dehydrogenase (LDH) release in the culture medium during the incubation of oxidized LDL with BAECs, whereas 100 microM melatonin dramatically increased this release. These data show that melatonin is less active than vitamin E in inhibiting in vitro LDL oxidation and does not inhibit the cytotoxicity of oxidized LDL toward cultured endothelial cells. The concentrations necessary to inhibit LDL oxidation are far beyond those found in human plasma (100 microM vs. 100 pM). Therefore our results indicate that the pineal hormone melatonin per se appears to have little antiatherogenic property in the in vitro oxidation of LDL and the cytoprotective action against the toxicity of oxidized LDL. Nevertheless, in vivo LDL oxidation takes place in the subendothelium of the artery wall, and nothing is known about the concentration of melatonin or its catabolites in this environment.     

Antiatherosclerotic and antioxidative effects of captopril in apolipoprotein E-deficient mice

The effect of the angiotensin-converting enzyme (ACE) inhibitor, captopril, on the development of atherosclerosis was determined in the apolipoprotein (apo) E-deficient mice. These mice develop severe hypercholesterolemia and extensive atherosclerotic lesions on chow diet, similar to those found in humans. Furthermore, in these mice, accelerated atherosclerosis is associated with increased plasma lipid peroxidation, a phenomenon that may play a crucial role in the buildup of the atherosclerotic lesions. Mice received either placebo or 50 mg/kg/day of captopril. After 12 weeks of treatment, captopril reduced the aortic-lesion area by 70% compared with that of the placebo-treated group. Captopril also increased the resistance of low-density lipoprotein (LDL) to CuSO4-induced oxidative stress, as shown by a significant reduction in the LDL content of malondialdehyde (MDA) by 30%, as well as by the prolongation of the lag time required for LDL oxidation from 55 min in the placebo-treated mice to 70 min in the captopril-treated mice, and reduction of the maximum LDL oxidation at 150 min by 35%. In vitro studies demonstrated that preincubation of LDL with captopril, inhibited the onset of CuSO4-induced LDL peroxidation up to 120 min, and reduced the LDL content of MDA by 90%. We conclude that captopril attenuates atherosclerosis in the apo E-deficient mice, and this phenomenon may be related to its inhibitory effect on the plasma LDL oxidation.     

Homocysteine: a new risk factor for atherosclerosis

The accumulating evidence for the role of homocysteine as a risk factor for atherosclerosis is persuasive. A high plasma homocysteine concentration induces pathologic changes in the arterial wall and thus is strongly associated with an increased risk of atherosclerosis, manifested as cardiovascular, cerebrovascular and peripheral vascular events. Studies are being conducted to determine whether lowering homocysteine levels prevents occlusive events. At present, testing for elevated homocysteine concentrations should be considered in patients with premature atherosclerosis or a strong family history of atherosclerosis, since hyperhomocysteinemia is a common risk factor in these patients. Treatment of hyperhomocysteinemia is straightforward and associated with minimal risk. This disorder is usually correctable with vitamin supplements containing folic acid.     

Effects of continuous infusion of endothelin-1 in pregnant sheep

Plasma concentration of endothelin-1, a potent vasoconstrictor produced by the vascular endothelium, has been observed to be significantly increased in a number of pathophysiological states, including preeclampsia. In the present study we have evaluated the effects of elevated plasma endothelin-1 in pregnant sheep by continuous exogenous endothelin-1 administration. Nine pregnant ewes (110+/-5 days' gestation) were instrumented for measurements of maternal mean arterial pressure, renal blood flow, and uterine blood flow. After recovery, endothelin-1 was infused intravenously for 4 hours at a dose that was adjusted to raise mean arterial pressure by approximately 20 mm Hg by the end of the first hour (range 5 to 20 ng/kg per minute). Mean arterial pressure, renal blood flow, uterine blood flow, urinary protein excretion, hematocrit, and plasma endothelin-1 concentration were measured hourly, and renal and uterine vascular resistances were calculated. Endothelin-1 produced significant increases (% change from baseline at t=4 hours) in mean arterial pressure (45+/-8%), renal vascular resistance (353+/-66 %), and uterine vascular resistance (59+/-21%). Endothelin-1 also increased microvascular permeability both systemically and within the kidney, as suggested by marked increases in hematocrit (0.27+/-0.01 to 0.32+/-0.01) and urinary protein concentration (0.95+/-0.1 to 7.9+/-3.2 mg/mL per mg creatinine). There was a highly significant correlation (P<.0001) between plasma endothelin-1 and mean arterial pressure, renal vascular resistance, uterine vascular resistance, hematocrit, and urinary protein content in all sheep studied. In addition, plasma endothelin-1 corresponded well with the time course of the changes in cardiovascular parameters and urinary protein excretion observed. These results provide evidence to suggest that elevation of circulating endothelin-1 in pregnant sheep can produce cardiovascular and hemodynamic changes that in many ways resemble the human disease preeclampsia. This supports the hypothesis that endothelial cell damage and/or dysfunction that is associated with increased production of endothelin-1 could directly contribute to the progression of preeclampsia.     

Gender differences in intima-media permeability to low-density lipoprotein at atherosclerosis-prone aortic sites in rabbits. Lack of effect of 17 beta-estradiol

Premenopausal women are protected from coronary heart disease, and premenopausal nonhuman primates are protected from atherosclerosis, the underlying cause of coronary heart disease. Estrogen is thought to account for this protection in females, and part of this protection is independent of the effects on risk factors, including lipoprotein levels. This study considered the hypothesis that reduced intima-media permeability to low-density lipoproteins (LDL) may account for the protection from atherosclerosis and coronary heart disease in premenopausal females and that this effect might be mediated by estrogen. Intima-media permeability to LDL was determined in male and female rabbits made hypercholesterolemic by feeding them 0.5% cholesterol for 8 days. The diet of half of the female rabbits was supplemented with 17 beta-estradiol (4 mg/d) during cholesterol feeding and the preceding 4 weeks. Estrogen treatment in the female rabbits did not influence the intima-media permeability to LDL. However, intima-media permeability to LDL for branch sites of the abdominal aorta and aortic arch (regions highly susceptible to atherosclerosis) was 43% and 38% lower, respectively, in male rabbits than in female rabbits: (2.93 +/- 0.39 microL/h/g, (n = 8), vs 6.28 +/- 0.86 microL/h/g, (n = 16), P < .001, and 4.69 +/- 0.28 microL/h/g, (n = 8) vs 7.57 +/- 0.75 microL/h/g, (n = 16), P < .02). In contrast, intima-media permeability to LDL in 7 of 8 aortic sites relatively resistant to atherosclerosis did not differ between male and female rabbits. These data suggest that the protection from atherosclerosis associated with female sex and estrogen is mediated by mechanism(s) other than reduction in intima-media permeability to LDL.     

Antiphospholipid antibodies and atherosclerosis

The family of antiphospholipid antibodies includes antibodies binding to cardiolipin in serological test for syphilis, antibodies prolonging the clotting time in lupus anticoagulant test, antibodies reacting with plasma phospholipid-binding proteins, such as beta 2-glycoprotein I and prothrombin, and antibodies binding to oxidized low-density lipoprotein (LDL). Antiphospholipid antibodies are traditionally associated with arterial and venous thrombosis in patients with primary or secondary antiphospholipid syndrome. The recent studies, especially those on patients with myocardial infarction, extend the concept of antiphospholipid antibodies, and suggest that they play a role also in atherosclerosis. Based on the clinical studies and immunological findings, it seems that the differences in the specificity of antiphospholipid antibodies may reflect to their pathogenetic mechanisms and, finally, to their clinical consequences. The present review suggests that antibodies to oxidized LDL may not interfere directly with blood coagulation, but seem to have importance in the inflammation of the vessel wall in atherosclerosis and in vasculitis. Instead, antibodies to beta 2-glycoprotein I and to prothrombin show a closer association with thrombosis. It is possible that in the atherosclerotic plaque, the plasma proteins, such as beta 2-glycoprotein I or prothrombin, are bound to the endothelial surface and antibodies to cryptic epitopes revealed in these proteins are induced. These antibodies may contribute to the formation of atherosclerotic thrombosis by changing the balance of haemostasis toward hypercoagulative state.     

The role of reactive oxygen species in atherosclerosis

Reactive oxygen species (ROS) are probably not only unintended, toxic side-products of oxygen metabolism in mammalian cells, they also have several important physiologic functions including antimicrobial killing, regulation of cellular proliferation and growth, and regulation of vascular tone. ROS are generated within the vessel wall by several mechanisms, including a vascular type of a NAD(P)H oxidase. ROS formation can be stimulated by mechanical stress, environmental factors, the peptide angiotensin II, cytokines, native low-density lipoproteins (LDL), and in the presence of catalytic metal ions. Their ability to modify LDL, react with endothelial-derived nitric oxide subsequently forming peroxynitrite, and amplify the expression of various genes important for leukocyte recruitment within the arterial wall are the basis of the oxidant injury theory of atherosclerosis. In animal studies, antioxidant therapy (probucol, butylated hydroxytoluene, N', N'-diphenylenediamide, vitamin E, superoxide dismutase) have been successfully used to prevent fatty streak formation, and to restore impaired nitric oxide-dependent vasorelaxation. In man, antioxidant therapy (e.g., supplementation with vitamin E) clearly increased the resistance of LDL to oxidative modification. Case-controlled and prospective clinical studies suggest a relation between baseline antioxidant plasma levels and/or antioxidant supplementation and risk of cardiovascular events. In one secondary prevention trial (randomized, blinded, placebo-controlled), vitamin E supplementation reduced significantly the risk for non-fatal myocardial infarctions. Before general recommendations can be made, results of further large-scale trials should be awaited.     

Stimulation of immune suppressive CD34+ cells from normal bone marrow by Lewis lung carcinoma tumors

Oxidative modification of LDL may occur via mechanisms, which are either dependent or independent of lipid peroxidation. Peroxidation of lipids in LDL, either initiated by radicals or catalysed by myeloperoxidase, results in the generation of aldehydes which substitute lysine residues in the apolipoprotein B-100 moiety and thus in the generation of oxidised LDL. Phospholipase activity, prostaglandin synthesis and platelet adhesion/activation are associated with the release of aldehydes which induce oxidative modifications of LDL in the absence of lipid peroxidation and thus in the generation of malondialdehyde-modified LDL. Recently, we have demonstrated an association between coronary artery disease and increased plasma levels of oxidised LDL. The increase of circulating oxidised LDL is most probably due to backdiffusion of oxidised LDL from the atherosclerotic arterial wall in the blood and is independent of plaque instability. Indeed, plasma levels of oxidised LDL were very similar in patients with stable coronary artery disease and in patients with acute coronary syndromes. Acute coronary syndromes were, however, associated with increased release of malondialdehyde-modified LDL that was independent of necrosis of myocardial cells. Indeed, plasma levels of malondialdehyde-modified LDL were very similar in patients with unstable angina and patients with acute myocardial infarction, in contrast with levels of troponin I which were significantly higher in acute myocardial infarction patients. These data suggest that oxidised LDL is rather a marker of coronary atherosclerosis whereas malondialdehyde-modified LDL is rather a marker of plaque instability and atherothrombosis. At present, in the absence of prospective studies, the causative role of oxidatively modified LDL in atherothrombosis is, however, not established.     

Impairment of endothelial and subendothelial sites by a circulating plasma factor associated with minimal change disease

BACKGROUND: The pathogenesis of albuminuria in minimal change disease (MCD) is unknown. A human plasma factor (denoted as 100KF) is able to induce minimal change-like glomerular alterations, i.e. loss of glomerular sialoglycoproteins and decreased expression of glomerular ecto-ATPase, following in vitro incubation with kidney tissue. In addition, increased (selective) permeability for plasma proteins occurs after perfusion of 100KF into the rat kidney ex vivo. As in kidney tissue from subjects with minimal change disease, subendothelial injury has also been observed, i.e. reduced anionic sites in the lamina rara interna, the question was raised whether injury induced by the plasma factor 100KF involves vascular endothelium or subendothelial matrix of the glomerular capillary wall. METHODS: Permeability studies were carried out by using confluent endothelial monolayers (HUVEC) cultured on a standard two-compartment system. The permeability for a macromolecular marker (horse radish peroxidase) was tested following incubation of the monolayers with either the native plasma factor 100KF or the control factor (heat-inactivated 100KF), in combination with histochemical evaluation of the cells for ecto-ATPase expression. Also quantification of glomerular anionic sites at the ultrastructural level was carried out, after ex vivo perfusion of 100KF or control factor into rat kidneys. RESULTS: The plasma factor 100KF is able to increase the permeability of human endothelial monolayers for macromolecules in a dose-dependent manner (relative increase 122.4 +/- 24, 178.4 +/- 34 and 236.1 +/- 58% after preincubation with 0.05; 0.5 and 1.5 mg/ml 100KF respectively), concomitant with induction of minimal change-like histochemical alterations such as reduced expression of ecto-ATPase. The number of anionic sites in the lamina rara interna of the glomerular capillary wall is significantly diminished following perfusion with the plasma factor 100KF versus control factor (7.58 +/- 1.60 versus 12.57 +/- 2.05 per 1000 nm; P < or = 0.02); in contrast to the lamina rara externa (22.71 +/- 3.15 versus 22.27 +/- 2.92 per 1000 nm; statistically not significant). CONCLUSIONS: Endothelial cells and subendothelial matrix along the glomerular filtration barrier may be considered as target structures for the plasma factor 100KF, leading to initial minimal change-like alterations associated with glomerular albumin leakage.