Anthrax Toxin Receptor 1 Is Essential for Arteriogenesis in a Mouse Model of Hindlimb Ischemia.

Anthrax toxin receptor 1/tumor endothelial marker 8 (Antxr1 or TEM8) is up-regulated in tumor vasculature and serves as a receptor for anthrax toxin, but its physiologic function is unclear. The objective of this study was to evaluate the role of Antxr1 in arteriogenesis. The role of Antxr1 in arteriogenesis was tested by measuring gene expression and immunohistochemistry in a mouse model of hindlimb ischemia using wild-type and ANTXR1(-/-) mice. Additional tests were performed by measuring gene expression in in vitro models of fluid shear stress and hypoxia, as well as in human muscle tissues obtained from patients having peripheral artery disease. We observed that Antxr1 expression transiently increased in ischemic tissues following femoral artery ligation and that its expression was necessary for arteriogenesis. In the absence of Antxr1, the mean arterial lumen area in ischemic tissues decreased. Antxr1 mRNA and protein expression was positively regulated by fluid shear stress, but not by hypoxia. Furthermore, Antxr1 expression was elevated in human peripheral artery disease requiring lower extremity bypass surgery. These findings demonstrate an essential physiologic role for Antxr1 in arteriogenesis and peripheral artery disease, with important implications for managing ischemia and other arteriogenesis-dependent vascular diseases.

Feedback and hormonal regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase: the concept of cholesterol buffering capacity.

Regulation of the expression of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by the major end product of the biosynthetic pathway, cholesterol, and by various hormones is critical to maintaining constant serum and tissue cholesterol levels in the face of an ever-changing external environment. The ability to downregulate this enzyme provides a means to buffer the body against the serum cholesterol-raising action of dietary cholesterol. The higher the basal expression of hepatic HMG-CoA reductase, the greater the "cholesterol buffering capacity" and the greater the resistance to dietary cholesterol. This review focuses on the mechanisms of feedback and hormonal regulation of HMG-CoA reductase in intact animals rather than in cultured cells and presents the evidence that leads to the proposal that regulation of hepatic HMG-CoA reductase acts as a cholesterol buffer. Recent studies with animals have shown that feedback regulation of hepatic HMG-CoA reductase occurs at the level of translation in addition to transcription. The translational efficiency of HMG-CoA reductase mRNA is diminished through the action of dietary cholesterol. Oxylanosterols appear to be involved in this translational regulation. Feedback regulation by dietary cholesterol does not appear to involve changes in the state of phosphorylation of hepatic HMG-CoA reductase or in the rate of degradation of this enzyme. Several hormones act to alter the expression of hepatic HMG-CoA reductase in animals. These include insulin, glucagon, glucocorticoids, thyroid hormone and estrogen. Insulin stimulates HMG-CoA reductase activity likely by increasing the rate of transcription, whereas glucagon acts by opposing this effect. Hepatic HMG-CoA reductase activity undergoes a significant diurnal variation due to changes in the level of immunoreactive protein primarily mediated by changes in insulin and glucagon levels. Thyroid hormone increases hepatic HMG-CoA reductase levels by acting to increase both transcription and stability of the mRNA. Glucocorticoids act to decrease hepatic HMG-CoA reductase expression by destabilizing reductase mRNA. Estrogen acts to increase hepatic HMG-CoA reductase activity primarily by stabilizing the mRNA. Deficiencies in those hormones that act to increase hepatic HMG-CoA reductase gene expression lead to elevations in serum cholesterol levels. High basal expression of hepatic HMG-CoA reductase, whether due to genetic or hormonal factors, appears to result in greater cholesterol buffering capacity and thus increased resistance to dietary cholesterol.

Effects of 15-oxa-32-vinyl-lanost-8-ene-3 beta,32 diol on the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and low density lipoprotein receptor in rat liver.

The mechanisms by which oxylanosterols regulate expression of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and lower serum cholesterol levels were examined by using a novel nonmetabolizable oxylanosterol mimic, 15-oxa-32-vinyl-lanost-8-ene-3 beta, 32 diol (DMP 565). This compound, unlike other nonmetabolizable oxylanosterols, is not a substrate for lanosterol 14 alpha-methyl demethylase. Feeding rats a diet supplemented with 0.02% DMP 565 markedly decreased HMG-CoA reductase immunoreactive protein and enzyme activity levels without affecting mRNA levels. The rate of reductase protein degradation was unaffected. However, the rate of translation was reduced to less than 20% of control. Thus, DMP 565 appears to regulate hepatic HMG-CoA reductase gene expression primarily at the level of translation. The pronounced inhibition of HMG-CoA reductase by DMP 565 resulted in a compensatory increase in the functioning of the hepatic low density lipoprotein (LDL) receptor, possibly by increased cycling, as evidenced by a marked increase in the rate of degradation of the LDL receptor. The half-life of the receptor was decreased from over 7 h to only 1 h in animals receiving DMP 565. This increase in the rate of degradation occurred without a change in the steady state level of the receptor. Addition of dietary cholesterol attenuated the increased turnover of the LDL receptor. These effects on the hepatic LDL receptor have also been observed with HMG-CoA reductase inhibitors (G. C. Ness et al., 1996, Arch. Biochem, Biophys. 325, 242-248). However, the effect of DMP 565 on the rate of degradation of the hepatic LDL receptor was of a greater magnitude when equal doses of the drugs were used. These regulatory actions of DMP 565 provide, in part, an explanation for the observed hypocholesterolemic action of this compound.

Effects of L-triiodothyronine and the thyromimetic L-94901 on serum lipoprotein levels and hepatic low-density lipoprotein receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and apo A-I gene expression.

The mechanisms by which thyroid hormone (triiodothyronine (T3)) and a thyromimetic, 2-amino-3-(3,5-dibromo-4-[4-hydroxy-3-(6-oxo-1,6-dihydro-pyridazin -3-ylmethyl)-phenoxyl]-phenyl)propionic acid (L-94901), lower plasma low density lipoprotein (LDL) cholesterol and raise plasma high density lipoprotein (HDL) cholesterol levels was investigated in thyroidectomized and sham-operated rats. Thyroidectomy resulted in a 77% increase in plasma LDL cholesterol, a 60% decrease in plasma triglycerides, and a modest reduction in HDL cholesterol. Daily oral dosing with T3 (10-170 nmol/kg) or L94901 (100-1000 nmol/kg) for 7 days decreased plasma LDL cholesterol in thyroidectomized rats by 60-80%, respectively. This reduction in LDL cholesterol was accompanied by a dose-dependent increase in HDL cholesterol levels of up to 60%. Thus, the ratio of LDL to HDL was decreased from 1.01 to 0.12 after treatment with L-94901 and to 0.25 after dosing with T3. In sham-operated animals, T3 and L-94901 lowered LDL cholesterol by 61 and 46%, respectively, and increased HDL cholesterol by 25 and 53%, respectively. Immunoblotting analysis of liver membranes prepared from thyroidectomized or sham-operated rats demonstrated that LDL receptor protein levels were increased by up to eight-fold. Northern blotting analysis revealed similar large increases in hepatic LDL receptor mRNA levels that accounted for the increases in LDL receptor protein levels. Hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA, protein, and activity were increased 2- to 3-fold. The T3- and L-94901-mediated increases in serum HDL levels were associated with 2- to 3-fold increases in apo A-I mRNA levels. In contrast with most other hypocholesterolemic agents, T3 and L-94901 significantly increase HDL cholesterol levels in addition to decreasing LDL cholesterol levels due to induction of hepatic apo A-I and LDL receptor gene expression.

Dietary cholesterol regulates hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression in rats primarily at the level of translation.

The level of gene expression at which dietary cholesterol exerts feedback regulation on hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was investigated using young male Sprague-Dawley rats. Previous studies suggested that this regulation might be exerted posttranscriptionally. Thus, possible regulation at the levels of catalytic efficiency, protein turnover, and translation was investigated. To examine possible regulation at the level of catalytic efficiency, rats were placed on chow diets supplemented with 2% cholesterol and the rates of decline in hepatic HMG-CoA reductase activity and immunoreactive protein levels were determined. Both decreased slowly over a 72-h period. The catalytic efficiency did not change. These observations are inconsistent with phosphorylation-dephosphorylation or thiol-disulfide interchange as possible mechanisms. The possibility that dietary cholesterol might act by increasing the rate of turnover of HMG-CoA reductase protein was examined by determining the half-life of the enzyme in livers from rats consuming chow or chow supplemented with 2% cholesterol. The half-life of HMG-CoA reductase protein was not decreased in the animals receiving cholesterol, thus ruling out this possibility. Regulation at the level of translation was investigated by measuring the rate of HMG-CoA reductase protein synthesis in liver slices using [35S]methionine and [35S]cysteine. It was found that the rate of synthesis was reduced by over 80% in liver slices from rats fed a diet supplemented with 2% cholesterol. Similar results were obtained with liver slices from rats given mevalonolactone, which supplies both sterol and nonsterol endproducts. These results indicate that cholesterol regulates hepatic HMG-CoA reductase gene expression in rats primarily at the level of translation.

Compensatory responses to inhibition of hepatic squalene synthase.

The mechanism by which depletion of hepatic cholesterol levels, achieved by inhibition of squalene synthase, alters hepatic LDL receptor, HMG-CoA reductase, and cholesterol 7alpha-hydroxylase gene expression was investigated by measuring transcription rates, mRNA stability, rates of translation, translational efficiency, and levels of sterol response element binding proteins. It was found that the transcription of both hepatic LDL receptor and HMG-CoA reductase were increased about twofold. The increase in LDL receptor transcription occurred within 2 h after giving 2 mg/kg zaragozic acid A, a potent inhibitor of squalene synthase. This preceded the increase in transcription of HMG-CoA reductase that occurred at 4 h. Increases in the stability of both of these mRNAs were also observed. These changes account for the increases in LDL receptor and HMG-CoA reductase mRNA levels previously observed. The rate of transcription of hepatic cholesterol 7alpha-hydroxylase was decreased to about 25% of control within 3 h after administration of zaragozic acid A, which correlates with the decrease in this mRNA. The rates of translation, as determined by pulse labeling, of both hepatic HMG-CoA reductase and LDL receptor were increased two- to threefold. The translational efficiency of these two mRNAs was also increased as judged by polysome profile analysis. There was an increase in mRNA associated with the heaviest polysome fraction and a decrease in that associated with monosomes. No significant change was observed in the levels of sterol response element binding protein 2, the form that mediates induced transcription, in response to zaragozic acid A treatment, indicating that this protein might not be involved in mediating the observed transcriptional changes. An increase in sterol response element binding protein -1 was observed 30 min after giving zaragozic acid A. The results suggest that compensatory responses to depletion of squalene-derived products involve alterations in the rates of transcription, mRNA stability, and translational of key proteins involved in cholesterol homeostasis.

Atorvastatin action involves diminished recovery of hepatic HMG-CoA reductase activity.

The effects of atorvastatin on the expression of the hepatic HMG-CoA reductase and LDL receptor genes were investigated in rats. Like the other statins, atorvastatin increased the rate of degradation and presumably cycling of the hepatic LDL receptor. In atorvastatin-treated rats, the half-life of the receptor was decreased by over 60%. Hepatic HMG-CoA reductase mRNA levels were increased about 3-fold by feeding a diet containing 0.04% atorvastatin while reductase protein levels were increased by as much as 700-fold. Apparent HMG-CoA reductase activity was not increased as much as protein levels. Washing experiments revealed that atorvastatin is more difficult to remove from microsomes than lovastatin. The results support the conclusion that the potent hypocholesterolemic action of atorvastatin involves decreased hepatic VLDL production due to effective inhibition of in vivo cholesterol biosynthesis resulting from diminished recovery of HMG-CoA reductase activity following drug treatment.

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors unmask cryptic regulatory mechanisms.

The possibility that potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may alter the mechanisms by which dietary cholesterol and farnesol regulate this gene was investigated by comparing the regulatory responses of rats maintained on diets with or without 0.04% Lovastatin supplementation to dietary cholesterol. It was found that the rate of hepatic HMG-CoA reductase transcription was significantly decreased by dietary cholesterol in animals fed Lovastatin-supplemented diets, whereas animals maintained on a normal chow diet showed no decrease in the rate of transcription. The levels of reductase mRNA were decreased to about 10% of controls in Lovastatin-supplemented animals in response to dietary cholesterol but not affected in nonsupplemented animals. Administration of farnesol, reputed to be the nonsterol regulator of reductase, to rats maintained on a diet containing Lovastatin decreased hepatic HMG-CoA reductase protein by 30% and the half-life of reductase immunoreactive protein to 4.0 h, which is close to that observed in chow-fed animals. In contrast, farnesol treatment does not affect the turnover rate of reductase protein in rats fed a normal chow diet. These results suggest that potent inhibitors of HMG-CoA reductase may unmask transcriptional regulation by dietary cholesterol and accelerated degradation of the reductase by the putative nonsterol regulator farnesol.

Translational regulation of hepatic HMG-CoA reductase by dietary cholesterol.

The question of whether dietary cholesterol exerts feedback regulation on hepatic HMG-CoA reductase at the level of translation was examined by performing polysome profile analysis. Liver polysomes from rats fed 2% cholesterol in their diets for 3 days were compared with those isolated from rats fed a normal chow diet. Northern blotting analysis of the individual fractions revealed that cholesterol feeding reduced the portion of HMG-CoA reductase mRNA associated with translationally active polysomes by over 50% and progressively increased the percentage of reductase mRNA present in the monosomal fractions. In the lightest monosomal fraction over 10 times as much reductase mRNA was present in samples from cholesterol animals as compared to controls. These findings indicate that dietary cholesterol exerts significant feedback regulation on hepatic HMG-CoA reductase at the translational level.

Smith-Lemli-Opitz syndrome produced in rats with AY 9944 treated by intravenous injection of lipoprotein cholesterol.

A limitation to treating Smith-Lemli-Opitz infants by giving dietary cholesterol is their impaired ability to absorb cholesterol due to a deficiency of bile acids. Since intravenously administered lipoprotein cholesterol should not require bile acids for uptake into tissues, we tested the effects of this form of cholesterol on tissue cholesterol and 7-dehydrocholesterol levels in an animal model of SLO, created by feeding rats 0.02% AY 9944. Intravenous administration of 15 mg of bovine cholesterol supertrate twice daily increased serum cholesterol levels from 11 to over 250 mg/dl. This treatment increased liver cholesterol levels from 309 to over 900 micrograms/g and lowered hepatic 7-dehydrocholesterol levels from 1546 to 909 micrograms/g. A combination of iv cholesterol and 2% dietary cholesterol was most effective as it raised hepatic cholesterol levels to 1950 micrograms/g, which is 50% above normal. 7-Dehydrocholesterol levels were decreased to 760 micrograms/g. Similar responses were seen for heart, lung, kidney, and testes. Brain sterol levels were not significantly affected. AY 9944 caused a modest increase in hepatic HMG-CoA reductase activity. Administration of dietary cholesterol together with iv cholesterol lowered hepatic HMG-CoA reductase activity to barely detectable levels. The data indicate that the combination of iv and dietary cholesterol was most effective in raising cholesterol levels, lowering 7-dehydrocholesterol levels, and inhibiting de novo cholesterol biosynthesis.

The diurnal variation of hepatic HMG-CoA reductase activity is due to changes in the level of immunoreactive protein.

The diurnal variation in levels of hepatic HMG-CoA reductase immunoreactive protein and enzyme activity were determined in rats. Immunoreactive protein levels changed together with enzyme activity. Thus the catalytic efficiency of HMG-CoA reductase was not significantly changed. The data suggest that the diurnal variation in hepatic HMG-CoA reductase activity is due to changes in enzyme protein levels rather than changes in phosphorylation state of the enzyme, for example, which would cause changes in catalytic efficiency.

Ecdysteroid titers during postembryonic development of Dermanyssus gallinae (Acari: Dermanyssidae).

Ecdysteroid titers in methanolic extracts of protonymphal and deutonymphal stages of the chicken mite, Dermanyssus gallinae (De Geer), were measured using radioimmunoassay. Highest titers of ecdysteroids were present 24 h after feeding. Further studies using high-performance liquid chromatography radioimmunoassay showed that ecdysteroid immunopositive material had the same retention time as 20-hydroxyecdysone and ecdysone. Ecdysteroid conjugates appear to be present in polar and apolar fractions.

A preliminary study of the translocation of aldicarb across the duck eggshell.

The duck eggshell has the reputation of being more permeable than that of the domestic hen. If this is true, the developing embryo could be at greater risk from xenobiotic agents, since toxicants picked up on the feathers could be transferred to the embryo during incubation. This study looked for such an effect on the developing embryo after the application of aldicarb to the eggshell. At 72 hr, the eggs were painted with 3, 7, 11, or 15 microM aldicarb in 500 microliters water. The eggs were then incubated to Day 24. The gross morphological measurements were then recorded. A similar study was made using domestic hen eggs; these were treated after 36 hr incubation and incubated to Day 17. Direct injection into the yolk sac of both species was used for further comparison. There was a statistically significant reduction (P less than 0.01) in the middle web toe length with 11 and 15 microM aldicarb and the tarsometatarsus length with 7, 11, and 15 microM. Compared with the duck control group, the group given 15 microM aldicarb had reductions of approximately 8% in the tarsometatarsus and approximately 9% in the middle web toe. No statistically significant changes were produced in the chick embryos.

Maternal alcoholism and anorexia nervosa: a possible association?

The use of modern epidemiological and biostatistical methods has been lacking in the research literature on anorexia nervosa. The present study utilized a retrospective case control design in which 40 cases of anorexia nervosa were matched to two separate control series, one being population based and one being clinic based. Odds ratio of 4.00 (p less than 0.006) and 7.67 (p less than 0.001) were obtained for family history of alcoholism and family history of psychological disorders, respectively, among the families of anorexia nervosa cases. The implications of this result for treatment planning and implementation are discussed.

Patterns of mental development among young, middle-class Jamaican children.

This study looked at the performance on the Griffiths Mental Development Scales of 149 middle-class children, aged 6-60 months, from three suburban neighborhoods in Kingston, Jamaica. Their mean DQs were higher than the English standardisation sample. The high scores below 2 years were similar to those reported from black children elsewhere. After 2 years the scores fell into the general range reported for middle-class children from other countries. The children had relatively high hearing and speech scores and low performance scores. Girls scored higher than boys, but birth order and maternal age were not associated with DQs.

Warfarin and the grey squirrel.

The anticoagulant warfarin [3-(alpha-acetonylbenzyl)-4-hydroxycoumarin] has been used for controlling the grey squirrel Sciurus carolinensis in Great Britain. Before legislation was passed to allow for its use in Ireland, it was felt that more information on its effectiveness was required. Since the small mammal population of Ireland is low, any toxic effects on the native mammal population could greatly deplete the wildlife in Ireland. No accurate assessment of the toxicity of warfarin for the squirrel appears in the literature. The squirrel were dosed with warfarin at four different concentrations, 6.0, 1.25, 0.5, 0.01 mg per kg of body weight, by stomach tube, in order to find the least effective dose. The prothrombin times (PT) were monitored as an assessment of the action of the drug. The control prothrombin time and the standard deviation of the mean was 13.83 +/- 3.64. Animals that were treated with warfarin showed an elevated PT even at the lowest dose by the 7th day of treatment. The PT was found to be in excess of 30 s and this was statistically significant, p = 0.05. Within the group receiving 0.5 mg/kg one animal showed a very significant initial rise in PT, but after 22 days of daily dosing, the PT decreased to within the normal range of the controls. Despite continuous dosing for a total of 26 days, it remained within this range.