Tissue distribution of factor VIII gene expression in vivo--a closer look.

Previous studies have shown that factor VIII (FVIII) is expressed by multiple tissues. However, little is known about its cellular origin or its level of expression in different organs. In the present study, we examined FVIII gene expression in different tissues on a quantitative basis. Most of the tissues, especially liver and kidney, expressed high levels of FVIII mRNA compared to their level of expression of other hemostatic proteins, including von Willebrand factor (VWF). This was unexpected since FVIII is a trace protein. In situ hybridization analysis confirmed that liver and kidney were rich in FVIII mRNA. In the liver, a clear hybridization signal was detected in cells lining the sinusoids. FVIII mRNA analysis of purified liver cells confirmed the expression of FVIII mRNA by sinusoidal endothelial cells and Kupffer cells. Low but significant levels of FVIII mRNA were also detected in the hepatocytes. VWF mRNA was not detectable in these cells. Similarly, immunohistochemical staining of liver tissue revealed that FVIII protein is primarily associated with sinusoidal cells. VWF protein was predominantly located in the endothelium of larger vessels. In the kidney, FVIII synthesis was localized to the glomeruli and to tubular epithelial cells. Taken together, these results suggest that besides hepatocytes, non-parenchymal cells (e.g. sinusoidal endothelial cells) contribute to FVIII synthesis. VWF synthesis is primarily confined to extra-hepatic tissues.

Oxidized LDL and lysophosphatidylcholine stimulate plasminogen activator inhibitor-1 expression in vascular smooth muscle cells.

Plasminogen activator inhibitor-1 (PAI-1) functions as an important regulator of fibrinolysis by inhibiting both tissue-type and urokinase-type plasminogen activator. PAI-1 is produced by smooth muscle cells (SMCs) in atherosclerotic arteries, but the mechanisms responsible for induction of PAI-1 in SMCs are less well understood. In cultured human aortic SMCs, PAI-1 mRNA expression and protein secretion were increased after incubation with oxidized low-density lipoprotein (LDL) and the lipid peroxidation product lysophosphatidylcholine, whereas the effects of native LDL on PAI-1 production and release were more variable and did not reach statistical significance. The effect of LDL on arterial expression of PAI-1 in vivo was also studied in an animal model. Intravenous injection of human LDL in Sprague-Dawley rats resulted in accumulation of apolipoprotein B in the aorta within 12 hours as assessed by immunohistochemical testing. Epitopes specific for oxidized LDL began to develop in the aorta 12 hours after injection of LDL and peaked at 24 hours; this peak was accompanied by intense expression of PAI-1 immunoreactivity in the media. Also, increased aortic expression of PAI-1 mRNA after LDL injection was detected by using in situ hybridization. The transcription factor activator protein-1, which is known to bind to the promoter of the PAI-1 gene, was activated in the aortic wall 24 hours after LDL injection as assessed by electrophoretic mobility shift assay. Pretreatment of LDL with the antioxidant probucol decreased expression of oxidized LDL and PAI-1 immunoreactivity and activator protein-1 induction in the aorta but did not affect expression of apolipoprotein B immunoreactivity. These findings demonstrate that LDL oxidation enhances secretion of PAI-1 from cultured SMCs and that a similar mechanism may be involved in vascular expression of PAI-1.

Active oxygen species and lysophosphatidylcholine are involved in oxidized low density lipoprotein activation of smooth muscle cell DNA synthesis.

It has recently been shown that oxidative modification of LDL enhances the mitogenic effect of LDL on smooth muscle cell (SMC) DNA synthesis. However, because of its complex chemical structure, the mitogenic components have not been well characterized. Exposure of LDL to the oxidant Cu2+ is followed by a rapid accumulation of peroxides that peaks after 8 to 12 hours and a conversion of the phospholipid phosphatidylcholine into lysophosphatidylcholine that continues for up to 48 hours. Most of the mitogenic activity is formed during the first 4 hours of oxidation. Both superoxide dismutase and catalase effectively inhibit the mitogenic activity of oxidized LDL, suggesting involvement of reactive oxygen intermediates. In the presence of 1% serum, low concentrations of hydrogen peroxide activated SMC DNA synthesis in a dose-dependent manner, with a maximal effect at a concentration of 200 mumol/L, whereas higher concentrations were inhibitory. Lysophosphatidylcholine also enhanced SMC DNA synthesis, with a maximal stimulation at a concentration of 10 mumol/L. Oxysterols, which also accumulate in oxidized LDL, effectively inhibited DNA synthesis. These results demonstrate that oxidation of LDL is associated with formation of several substances affecting the growth of SMCs. Among these substances, low levels of reactive oxygen intermediates and lysophosphatidylcholine stimulate DNA synthesis, whereas at a higher concentration they, as well as oxysterols, are inhibitory.

Lipid oxidation and atherosclerosis.

Hypercholesterolemia, and in particular high levels of low density lipoprotein (LDL), is a well established risk factor for development of coronary heart disease (CHD), but the biological mechanisms by which LDL promote formation of atherosclerotic plaques are still poorly understood. During the last decade several lines of evidence have suggested that oxidative modification of LDL is a key process in this respect. Oxidation of LDL has been found to increase its uptake in macrophages and lead to formation of macrophage foam cells. Other studies have indicated that oxidized LDL may induce vascular inflammation and even give rise to autoimmune reactions in the vascular wall. These findings have it made important to investigate the possible role of LDL oxidation in CHD also in clinical studies and the initial results of such studies support the notion that oxidation of LDL also may be of clinical relevance. In a group of young post-myocardial infarction patients the in vitro susceptibility of LDL to oxidative modification was found to be significantly related to the severity of coronary atherosclerosis as assessed by angiography. In another study presence of antibodies against oxidized LDL was found to be associated with increased progression of carotid disease. Should these findings be confirmed in larger patient groups and LDL oxidation established as a key factor in the development of atherosclerosis this would have a considerable impact on future strategies for prevention and treatment of coronary heart disease.

Protective role of endogenous pulmonary glutathione and other sulfhydryl compounds against lung damage by alkylating agents. Investigations with 4-ipomeanol in the rat.

Because endogenous glutathione is known to participate in the detoxification of highly reactive, hepatotoxic drug metabolites, we studied the role of this substance in the pulmonary toxicity of 4-ipomeanol [1-(3-furyl)-4-hydroxypentanone] in rats. 4-Ipomeanol was an appropriate model for these studies since previous investigations have indicated that an alkylating metabolite, formed in situ, is responsible for selective lung damage by 4-ipomeanol. Toxic doses of 4-ipomeanol preferentially depleted rat lung glutathione. Pretreatment of animals with piperonyl butoxide, an inhibitor of the metabolic activation of 4-ipomeanol, prevented both the depletion of lung glutathione and the pulmonary toxicity of 4-ipomeanol. Prior depletion of lung glutathione by diethylmaleate increased both the pulmonary covalent binding and the toxicity of 4-ipomeanol, whereas administration of cysteine and cysteamine decreased both the covalent binding and the toxicity. These in vivo studies, in conjunction with previous in vitro studies which showed inhibitory effects of sulfhydryl compounds on the covalent binding of 4-ipomeanol, are consistent with the view that pulmonary glutathione plays a protective role against pulmonary alkylation and lung toxicity by 4-ipomeanol, probably by reacting with the toxic metabolite(s) to form nontoxic conjugate(s). Pulmonary glutathione may similarly provide protection against other electrophilic drugs or metabolites that can damage the lungs.

Acute pulmonary injury in rats by nitrofurantoin and modification by vitamin E, dietary fat, and oxygen.

The subcutaneous administration of nitrofurantoin to rats caused severe pulmonary damage, characterized by edema, congestion, and hemorrhage. The acute lethality of the drug was greater in rats fed vitamin E-deficient diets high in polyunsaturated fats as compared to rats fed the NIH open-formula diet. The survival times of vitamin E-deficient rats were increased if such animals were fed diets supplemented with vitamin E and/or diets containing saturated fat (lard) for 3 weeks before administration of nitrofurantoin. The toxicity of nitrofurantoin was enhanced in both the rats deficient in vitamin E and in those given vitamin E supplements and exposed to O2-enriched atmospheres. These results, in conjunction with previous metabolic studies in vitro showing redox cycling and O2 activation in rat lung microsomes in the presence of nitrofurantoin, illustrate certain similarities with the lung-toxic herbicide, paraquat, and raise the question of whether the 2 agents may be capable of damaging lungs by a common mechanism.