Hypercholesterolemic Research Articles

Peripheral blood mononuclear cell production of interleukin-8 and IL-8-dependent neutrophil function in hypercholesterolemic patients

Interleukin-8 is a cytokine produced by mononuclear cells that is involved in polymorphonuclear neutrophil leukocyte (PMN) recruitment and activation. Several studies have previously demonstrated a leukocyte activation during hypercholesterolemia and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been found to play a role in the prevention of atherothrombotic disease. The purpose of this study was to determine interleukin-8 (IL-8) mRNA expression and ex vivo production from peripheral blood mononuclear cells (PBMCs) and IL-8-dependent PMN activation of hypercholesterolemic (HC) patients with respect to normocholesterolemic (NC) subjects. Using Northern blot analysis, we found a four- and threefold increase in the amount of IL-8 transcript in PBMC from HC patients, in unstimulated and LPS stimulated cultures, respectively. A specific immunoassay showed a correspondingly significant increase of IL-8 immunoactivity in the conditioned medium of PBMC from HC subjects as compared with controls (unstimulated PBMC: 15±4 vs. 4.2±3 ng/ml; P<0.0001; LPS stimulated PBMC: 65.3±8 vs. 36.6±9 ng/ml; P<0.0001). PMN of HC patients stimulated with IL-8 showed a reduced elastase release with respect to NC subjects before physiological granule release after f-Met-Leu-Phe (fMLP) treatment. These results indicate an upregulation of the IL-8 system in dyslipidemic patients and provide evidence for ongoing in vivo IL-8-dependent PMN activation during hypercholesterolemia.

Reperfusion-induced changes in capillary perfusion and filtration: effects of hypercholesterolemia.

Fluid filtration rate (J(v)/S) and red blood cell velocity (V(RBC)) in individual mesenteric capillaries of normocholesterolemic (NC) and hypercholesterolemic (HC) rats were measured before and after ischemia and reperfusion (I/R). In NC rats, a correlation was found between baseline J(v)/S and the percent of the feeding arteriole length that was paired (<15 micrometer) with a postcapillary venule (A-V pairing), but not in the HC group. Additionally, in NC rats only, a correlation was found between baseline V(RBC) and A-V pairing. In capillaries in which A-V pairing was substantial (>20%), V(RBC) dropped after reperfusion in the HC group (54% of baseline; P < 0.05), but not in the NC group (79%). The decrease in V(RBC) in HC rats could be attenuated by a P-selectin antibody (PB1.3). PB1.3 was also able to attenuate the increase in I/R-induced capillary J(v)/S in HC rats (median increase = 1.26-fold vs. 1.53-fold without PB1.3). These data suggest a role for A-V pairing in capillary perfusion in NC rats and a potential role for P-selectin in I/R-induced microvascular dysfunction in HC rats.

Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase.

Hypercholesterolemia is a central pathogenic factor of endothelial dysfunction caused in part by an impairment of endothelial nitric oxide (NO) production through mechanisms that remain poorly characterized. The activity of the endothelial isoform of NO synthase (eNOS) was recently shown to be modulated by its reciprocal interactions with the stimulatory Ca2+-calmodulin complex and the inhibitory protein caveolin. We examined whether hypercholesterolemia may reduce NO production through alteration of this regulatory equilibrium. Bovine aortic endothelial cells were cultured in the presence of serum obtained from normocholesterolemic (NC) or hypercholesterolemic (HC) human volunteers. Exposure of endothelial cells to the HC serum upregulated caveolin abundance without any measurable effect on eNOS protein levels. This effect of HC serum was associated with an impairment of basal NO release paralleled by an increase in inhibitory caveolin-eNOS complex formation. Similar treatment with HC serum significantly attenuated the NO production stimulated by the calcium ionophore A23187. Accordingly, higher calmodulin levels were required to disrupt the enhanced caveolin-eNOS heterocomplex from HC serum-treated cells. Finally, cell exposure to the low-density lipoprotein (LDL) fraction alone dose-dependently reproduced the inhibition of basal and stimulated NO release, as well as the upregulation of caveolin expression and its heterocomplex formation with eNOS, which were unaffected by cotreatment with antioxidants. Together, our data establish a new mechanism for the cholesterol-induced impairment of NO production through the modulation of caveolin abundance in endothelial cells, a mechanism that may participate in the pathogenesis of endothelial dysfunction and the proatherogenic effects of hypercholesterolemia.

Increased metabolism of peroxynitrite-modified [125I] beta-VLDL by monocytes from hypercholesterolemic rabbits.

The metabolism (uptake and degradation) of peroxynitrite-modified beta-VLDL and native beta-VLDL labeled with [125I]iodine was studied in monocytes isolated from peripheral blood of hypercholesterolemic (HC) and normolipidemic rabbits (NC). The peroxynitrite-modified beta-VLDL uptake and degradation was up to 2-fold higher in monocytes from HC rabbits than monocytes from NC rabbits. In addition, monocytes from HC rabbits took up and degraded significantly more peroxynitrite-modified beta-VLDL than native beta-VLDL. In contrast, monocytes from NC rabbits took up both lipoproteins in a similar rate and degraded more native beta-VLDL than peroxynitrite-modified beta-VLDL. Our results suggest that hypercholesterolemia can affect monocyte metabolism enhancing foam cell formation by increasing the influx of peroxynitrite-modified beta-VLDL.

Erythrocyte antioxidant status in asymptomatic hypercholesterolemic men.

An imbalance between antioxidant and oxidant-generating systems leading to an oxidative stress has already been proposed in the pathogenesis of atherosclerosis. In the present study we investigated the antioxidant status in 60 asymptomatic hypercholesterolemic (HC) men compared with 48 normocholesterolemic (NC) men. Hypercholesterolemic subjects had a significantly lower red blood cell vitamin E (vit E-RBC) content in spite of their normal total plasma and HDL vitamin E concentrations. Activities of erythrocyte superoxide dismutase and glutathione peroxidase were not significantly different between groups. We also determined the resistance of RBCs to an oxidative stress by determining the extent of hemolysis induced by a water-soluble azo-compound. This resistance was significantly decreased in HC men compared with NC subjects. These results demonstrate an altered antioxidant status of RBC in asymptomatic HC men associated with an increased erythrocyte susceptibility to an oxidative stress. The measure of the vitamin E content in RBC might be the most sensitive parameter for evidencing early oxidative stress which does not need an adaptation of enzymatic protective systems.

Age-related changes in cardiovascular disease risk factors of hypercholesterolemic children

Objective: To describe the age-related changes in cardiovascular disease risk factors in young, hypercholesterolemic (HC) children. Methods: Hypercholesterolemic ( n = 227) and nonhypercholesterolemic (NHC) ( n = 80) children between the ages of 4 and 10 years were identified. Height, weight, skin-fold and blood pressure measurements, and total cholesterol levels were measured. The HC group also had insulin levels evaluated. The groups were compared by analysis of variance. Simple Spearman correlations evaluated the associations between factors within each group. Results: The HC and NHC groups had similar mean ages, heights, and weights, both contained 51% girls, and all were white subjects. Percent weight-for-height median, and biceps, triceps, suprailiac and subscapular skin-fold measurements were all larger for the HC group. A significant age interaction demonstrated that the HC group's larger suprailiac and sum of skin-fold measures were expressed in the 8.0- to 9.9-year-old children, but not the 4.0- to 5.9-year-olds. For both groups, systolic blood pressure was associated with the measures of adiposity. For the HC group, insulin levels were also associated with adiposity. Conclusions: These results suggest that: (1) children with HC have greater body fat, (2) the expression of the hypercholesterolemia precedes the expression of increased body fat, (3) body fat increases with age, and (4) altered insulin and blood pressure levels are expressed in association with the increased body fat in children with HC. Confirmation with longitudinal data is necessary. (J Pediatr 1998;132:414-20)

Down-regulation of endothelin-B receptor sites in cavernosal tissue of hypercholesterolaemic rabbits.

To investigate the density and distribution of endothelin-1 (ET-1) and endothelin receptor subtypes in cavernosal tissue of healthy New Zealand White (NZW) rabbits (controls) and to assess any changes in a genetic model of hypercholesterolaemia (the Watanabe rabbit). Penises were excised from six hypercholesterolaemic (HC) rabbits 6 months after birth. Low- and high-resolution autoradiography was performed on cavernosal sections using radioligands for ET-1, endothelin A (ETA) and endothelin B (ETB) receptors, and the autoradiographs analysed densitometrically. The results were compared with those from six age-matched control rabbits. Immunohistochemical localization of ET-1-like immunoreactivity was also performed on adjacent cavernosal sections. ET-1, ETA and ETB receptor binding sites were primarily localized to the smooth muscle cells of the corpus cavernosum and the endothelium lining the cavernosal spaces. There was a significant decrease in ETB receptor binding sites in cavernosal tissue from HC rabbits when compared to age-matched healthy controls. The findings suggest that ET-1 may have a role in the pathophysiology of erectile dysfunction in HC. These effects may partly be due to enhanced vasoconstrictor actions and smooth muscle cell proliferation, consequent on a reduction in endothelial ETB receptors.

Binding and degradation of native and acetylated low density lipoproteins by monocyte derived macrophages of normal and hypercholesterolemic rabbits

Low density lipoprotein (LDL) was isolated from normal rabbits and was modified with acetic anhydride. Blood monocyte derived macrophages from normal and hypercholesterolemic (HC) rabbits were cultured, and on the 8th day the cells were incubated with native and modified LDL to study their binding and degradation. Macrophages from both normal and hypercholesterolemic rabbits express a limited number of receptors for normal plasma LDL whereas they exhibit a large number of receptors for acetyl LDL. There was no significant difference between binding and degradation of acetyl LDL by normal or hypercholesterolemic cells. However, binding and degradation of native LDL by monocytes of hypercholesterolemic cells were significantly less (p<0.0001) in comparison to binding and degradation of native LDL by normal cells indicating that there is a feedback inhibitory pathway in the cell that inhibits the synthesis of LDL receptors in the presence of excess LDL.

Long-term Cholesterol-Lowering Effects of 4 Fat-Restricted Diets in Hypercholesterolemic and Combined Hyperlipidemic Men

The long-term effect of aggressively vs moderately fat-restricted diets has not been studied extensively in free-living subjects with different types of hyperlipidemia. To compare the cholesterol-lowering effects of 4 levels of dietary fat intake restriction after 1 year. Randomized, parallel, comparison trial. Male employees of a large industry. A total of 444 men had low-density lipoprotein cholesterol (LDL-C) levels above the 75th age-specific percentile. Subjects with triglyceride (TG) levels less than the 75th age-specific percentile were defined as hypercholesterolemic (HC) and those with TG levels at or above the 75th age-specific percentile were defined as combined hyperlipidemic (CHL). Hypercholesterolemic subjects were randomized to diets 1, 2, 3, and 4 taught to contain 30%, 26%, 22%, and 18% fat, and the CHL subjects were randomized to diets 1, 2, and 3. All 4 diets were taught to subjects and spouses or partners in 8 weekly 2-hour classes. Plasma lipoprotein levels after 1 year. Fat intake after 1 year declined from a mean of 34% to 36% of energy to 27%, 26%, 25%, and 22% in the 4 HC diet groups and 28%, 26%, and 25% in the 3 CHL diet groups. Mean+/-SD percent LDL-C reductions were 5.3%+/-16.2%, 13.4%+/-12.6%, 8.4%+/-11.2%, and 13.0%+/-15.7% in the HC diet groups and 7.0%+/-16.2%, 2.8%+/-15.8%, and 4.6%+/-13.5% in the CHL diet groups (P<.01 in all but 1 instance). Apoprotein B levels decreased 8.6%, 10.7%, 4.3%, and 5.3% in the HC groups and 14.6%, 11.4%, and 9.9% in the CHL groups (P<.05-.01 in each instance). Triglyceride levels increased significantly in subjects following HC diets 3 and 4, 21.7% and 38.7% (P<.05 and .01), but not in any CHL subjects. High-density lipoprotein cholesterol decreased 2.8% and 3.2% in subjects on HC diets 3 and 4, respectively (P<.05 in both cases). After 1 year, moderate restriction of dietary fat intake attains meaningful and sustained LDL-C reductions in HC subjects and apoprotein B reductions in both HC and CHL subjects. More extreme restriction of fat intake offers little further advantage in HC or CHL subjects and potentially undesirable effects in HC subjects.

Plasma clearance and biodistribution of oxidatively modified 99mTc-beta-VLDL in rabbits.

The biodistribution and removal from plasma (measured as fractional clearance rate, FCR, per hour) of native and oxidatively modified 99mtechnetium-labeled beta-very low density lipoprotein (99mTc-beta-VLDL) were investigated in hypercholesterolemic (HC) and control (C) three-month old New Zealand rabbits. The intracellular accumulation of beta-VLDL labeled with 99mTc was studied in vitro in THP-1 cells and monocyte-derived macrophages isolated from rabbits. After intravenous injection into C rabbits, copper-oxidized beta-VLDL (99mTc-ox-beta-VLDL) was cleared from the circulation faster (0.362 +/- 0.070/h) than native beta-VLDL (99mTc-nat-beta-VLDL, 0.241 +/- 0.070/h). In contrast, the FCR of 99mTc-ox-beta-VLDL in HC rabbits was lower (0.100 +/- 0.048/h) than that of 99mTc-nat-beta-VLDL (0.163 +/- 0.043/h). The hepatic uptake of radiolabeled lipoproteins was lower in HC rabbits (0.114 +/- 0.071% injected dose/g tissue for 99mTc-nat-beta-VLDL and 0.116 +/- 0.057% injected dose/g tissue for 99mTc-ox-beta-VLDL) than in C rabbits (0.301 +/- 0.113% injected dose/g tissue for 99mTc-nat-beta-VLDL and 0.305 +/- 0.149% injected dose/g tissue for 99mTc-ox-beta-VLDL). The uptake of 99mTc-nat-beta-VLDL and 99mTc-ox-beta-VLDL by atherosclerotic aorta lesions isolated from HC rabbits (99mTc-nat-beta-VLDL: 0.033 +/- 0.012% injected dose/g tissue and 99mTc-ox-beta-VLDL: 0.039 +/- 0.017% injected dose/g tissue) was higher in comparison to that of non-atherosclerotic aortas from C rabbits (99mTc-nat-beta-VLDL: 0.023 +/- 0.010% injected dose/g tissue and 99mTc-ox-beta-VLDL: 0.019 +/- 0.010% injected dose/g tissue). However, 99mTc-nat-beta-VLDL and 99mTc-ox-beta-VLDL were taken up by atherosclerotic lesions at similar rates. In vitro studies showed that both monocyte-derived macrophages isolated from rabbits and THP-1 macrophages significantly internalized more 99mTc-ox-beta-VLDL than 99mTc-nat-beta-VLDL. These results indicate that in cholesterol-fed rabbits 99mTc-ox-beta-VLDL is slowly cleared from plasma and accumulates in atherosclerotic lesions. However, although the extent of in vitro uptake of 99mTc-ox-beta-VLDL by macrophages was high, the in vivo accumulation of this radiolabeled lipoprotein by atherosclerotic lesions did not differ from that of 99mTc-nat-beta-VLDL.

DIRECT ANTIATHEROGENIC ACTIVITY OF ISRADIPINE AND LACIDIPINE ON NEOINTIMAL LESIONS INDUCED BY PERIVASCULAR MANIPULATION IN RABBITS

Thein vivoantiatherogenic activity of two calcium antagonists of the dihydropyridine class (isradipine and lacidipine) was investigated in a new experimental model. The proliferative lesion induced in the rabbit carotid artery was obtained by positioning a hollow silastic collar around the vessel. The neointimal formation was determined by measuring cross sectional thickness of intimal (I) and medial (M) tissue of fixed arteries obtained 14 days after collar placement. The effectiveness in inhibiting neointimal formation was assessed for isradipine (0·5, 1 and 4 mg kg−1day−1) in normocholesterolemic (NC) animals and for lacidipine (1, 3, and 10 mg kg−1day−1) in hypercholesterolemic (HC) rabbits. In NC control animals a neointimal formation was clearly detectable (I/M 0·53±0·18,n=5). In isradipine-treated groups I/M ratios were significantly decreased (0·15±0·03, 0·12±0·02, 0.1±0.02 for the 0·5, 1 and 4 mg kg−1day−1doses respectively). In HC rabbits the administration of cholesterol 1% mixed with food and drug treatment started either 60 days before collar insertion (pretreated group, HC60) or on the same day (non pretreated group, HC15) of the collar placement. Only the pharmacological pretreatment was effective in reducing neointimal formation (0·47±0·02, 0·4±0·09, and 0·32±0·02 for dose 1, 3 and 10 mg kg−1day−1vs1·1±0·14 in control animals). The inhibition of neointimal hyperplasia was much less evident in nonpretreated animals (0·7±0·15, 0·6±0·18 and 0·43±0·08 for dose 1, 3, and 10 mg kg−1day−1vs0·72±0·2 in control animals).These results suggest a direct antiatherosclerotic effect of isradipine and lacidipine on neointimal hyperplasia induced in NC and HC pretreated rabbits independently of modulation of risk factors such as hypercholesterolemia and/or hypertension.

EXERCISE ALTERS LIPID CONCENTRATIONS BUT NOT CETP ACTIVITY IN HYPER-AND NORMOCHOLESTEROLEMIC MEN 32

To study the effects of exercise on cholesteryl ester transfer protein activity (CETPa) and on selected blood lipid concentrations, blood samples were obtained from 13 untrained, hypercholesterolemic (HC) and 12 normocholesterolemic (NC) men of similar age and weight (combined [horizontal bar over]x ± SEM = 44 ± 1 yr and 86 ± 4 kg, respectively) at four time points; before (PRE), then immediately (IPE), 24 h, and 48 h after treadmill exercise (70% ˙VO2max, 500 kcal). Concentrations were adjusted for plasma volume changes. RESULTS. The table shows significant effects (p < 0.05) from 2 (GROUP) × 4 (TIME) ANOVA with repeats on TIME; no interactions were significant (values are [horizontal bar over]x ± SEM; * = group difference; row means with similar superscripts not different; lipid concentrations in mg/dL; CETPa in% cholesterol ester transferred / 4 h). Table CONCLUSIONS: 1) CETPa is not altered by exercise; 2) despite higher CETPa in HC men, postexercise changes in lipid concentrations do not differ from those in NC men of similar age and weight.

Plasma and erythrocyte vitamin E content in asymptomatic hypercholesterolemic subjects

The present study was designed to assess plasma and erythrocyte vitamin E concentrations in 57 asymptomatic hypercholesterolemic (HC) men compared with 56 normocholesterolemic (NC) men. Vitamin E concentrations were determined by using a reversed-phase HPLC method. Compared with NC subjects, HC men had a significantly lower red blood cell (RBC) vitamin E content in spite of their normal plasma vitamin E concentration. This study demonstrates that total plasma vitamin E concentration is not a suitable predictor of cell vitamin E status and suggests an abnormal transfer of tocopherol between plasma and RBCs in HC men. Moreover, the RBCs of HC men were more susceptible to a peroxidative stress. The strong correlation between RBC susceptibility to oxidation and RBC vitamin E content suggests that the low RBC vitamin E content found in HC men has physiological consequences on the RBC oxidation.

Hypolipidemic effect of taurine in stroke-prone spontaneously hypertensive rats.

The hypolipidemic and antiatherosclerotic effects of taurine were investigated in genetically hypertensive rats: strokeprone spontaneously hypertensive rats (SHRSP). SHRSP were fed a hypercholesterolemic (HC) diet supplemented with 3% taurine for 50 days, and serum cholesterol was monitored. Cholesterol content and enzymatic activity responsible for cholesterol synthesis and metabolism were also determined in the liver, aorta, and intestine. Taurine prevented increases in the cholesterol level of the serum, liver, and aorta induced by a HC diet. Severe fat deposits of the mesenteric arteries induced by a HC diet were improved by the taurine treatment, showing the hypolipidemic and antiatherosclerotic effects of taurine. Taurine enhanced the activity of cholesterol 7 alpha-hydroxylase, a rate-limiting enzyme of bile acid synthesis, and stimulated bile acid production. These results suggest that taurine stimulates bile acid synthesis, which is closely related to the enhancement of cholesterol 7 alpha-hydroxylase activity, and thereby reduces serum cholesterol. In addition, a decrease in the intestinal acyl CoA:cholesterol acyltransferase activity by taurine suggests that the inhibition of cholesterol absorption may also be related to the hypolipidemic effect of taurine, in part.

Diet-induced hypercholesterolemia increases endothelin-1 release by aortic endothelial cells.

Our previous studies demonstrated the morphological heterogeneity of endothelial cells (ECs) and the emergence of large multinucleated ECs in human and animal atherosclerotic lesions. To investigate the functional alteration of ECs in diet-induced atherosclerosis, immunoreactive endothelin-1 (irET-1) release by ECs of the rabbit aorta was correlated with scanning electron microscopy. Rabbits were fed a cholesterol diet for 12 weeks: by scanning electron microscopy, the area of ECs in the aorta increased in the hypercholesterolemic (HC) group as atherosclerosis progressed. Cultured ECs of the HC group released significantly more irET-1 than ECs of the control. The plasma irET-1 level was also elevated in the HC group. The results obtained suggest that accelerated secretion of ET-1 by ECs contributes to the development of atherosclerotic vascular lesions.

Hypolipidemic and Antiatherosclerotic Effects of a Novel ACAT Inhibitor, HL-004, in Stroke-Prone Spontaneously Hypertensive Rats

The hypolipidemic and antiatherosclerotic effects of a novel acyl CoA: cholesterol acyltransferase (ACAT) inhibitor, HL-004, were studied in stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were administered 0.01-0.09% HL-004 mixed in a hypercholesterolemic (HC) diet for 50 days. HL-004 reduced the cholesterol and triglyceride levels in serum, as well as those in the liver, small intestine, and aorta, in a dose-dependent manner. HC diet-induced severe fat deposition in the mesenteric arteries, which is characteristic of SHRSP, was also decreased by HL-004. The ACAT activity of the small intestine and liver was decreased by HL-004. In particular, liver ACAT activity was significantly low in SHRSP given 0.09% HL-004, compared to that of normal animals. These results suggest that HL-004 is a systemic ACAT inhibitor and that the ACAT inhibition in the intestine, liver, and aorta is involved in the hypolipidemic and antiatherosclerotic effects of HL-004.

In Vitro Responses of Human Peripheral Small Arteries in Hypercholesterolemia and Effects of Therapy

Studies in both animals and humans with raised lipid levels have demonstrated abnormalities in vascular function usually manifested by an impairment in endothelium-dependent vasorelaxation. This is believed to be an early event in atheroma formation. There are few data on the effects on vascular function in humans of lowering serum lipids. We conducted a study to investigate the effects of cholesterol reduction on the in vitro function of human peripheral small arteries in middle-aged patients with hypercholesterolemia. Subcutaneous gluteal fat biopsies were taken from 18 hypercholesterolemic (HC) patients (mean +/- SEM serum total cholesterol, 9.7 +/- 0.57 mmol/L) and 16 age- and sex-matched control subjects (mean cholesterol, 4.69 +/- 0.18 mmol/L). Subcutaneous small arteries (internal diameter, < 330 microns) were dissected and mounted on a wire myograph for isometric tension measurements. The HC patients showed impaired relaxation to acetylcholine (10(-9) to 10(-6) mol/L) after preconstriction with the thromboxane A2 analogue U46619 (10(-6) mol/L, mean maximum relaxation, 42.9 +/- 5.4%) compared with control subjects (85.7 +/- 4.0%, P < .00001). Incubation with the nitric oxide substrate L-arginine (3 mmol/L) improved the endothelium-dependent vasorelaxation response to acetylcholine (70.9 +/- 6.0%, P < .01) in patients but not in control subjects. Also, there was a smaller but significant difference in responses to the endothelium-independent agent sodium nitroprusside (10(-9) to 10(-6) mol/L) between the HC group (mean maximum relaxation, 76.9 +/- 6.0%) and the control subjects (89.7 +/- 6%; P < .01). A total of 10 patients had a second gluteal skin biopsy and repeat functional studies after successful cholesterol-lowering therapy after a mean period of 9.9 +/- 4.7 months. A significant reduction in total and LDL cholesterol was achieved (5.29 +/- 0.2 and 3.23 +/- 0.21 mmol/L, respectively; P < .001). This restored vasorelaxation to control values in response to both acetylcholine (mean maximum relaxation, 83.3 +/- 3.8%; P < .0001) and sodium nitroprusside (87.9 +/- 4.8%, P < .01). Although both groups were normotensive, there were significantly higher blood pressures in the HC group compared with control subjects (139 +/- 4.1 versus 123 +/- 3.0 mm Hg systolic, P < .01; 84 +/- 1.3 versus 75 +/- 2.2 mm Hg diastolic, P < .01). There was no difference in initial blood pressures between the entire group of 18 and the 10 patients who had repeat biopsies. The blood pressures fell to control values after cholesterol reduction (129.33 +/- 4.93 mm Hg systolic and 72.33 +/- 2.93 diastolic mm Hg, P < .02 relative to pretreatment values). These results demonstrate abnormalities of both endothelium-dependent and -independent relaxation in human peripheral small arteries that are normalized with effective lipid lowering. The changes in blood pressure may have been secondary to the improvement in vascular function.

765-4 Adhesiveness of Mononuclear Cells is Increased in Hypercholesterolemic Humans, and Reduced by the NO Precursor

Enhanced interaction of mononuclear cells with the endothelium is the first observable event in the development of atherosclerosis. Hypercholesterolemia reduces vascular nitric oxide activity. This dysfunction may promote endothelial monocyte interaction, as NO is a potent inhibitor of adhesion. We have previously shown that dietary L-arginine (Arg) supplementation in hypercholesterolemic rabbits restores NO activity and inhibits monocyte-endothelial cell interaction, in association with a reduction in atherogenesis. Accordingly we developed a functional binding assay to assess the adhesiveness of human mononuclear cells, so as to determine the effects of hypercholesterolemia and L-arginine therapy. We found a 50% increase in adhesion of mononuclear cells (MNC) from hypercholesterolemic (HC) subjects to cultured endothelial cells ex vivo compared to a normocholesterolemic control population (p &lt; 0.0001, n = 20). Increased MNC adhesion was reversed to normal levels by preincubation of the MNC suspension with 10 -5 M sodium nitroprusside (164.4 ± 8.7% vs 97.5 ± 6.9%, P &lt; 0.0005, n = 7), while L-nitroarginine and Arg did not have an effect in vitro . In a double-blinded placebo-controlled study, oral Arg (8.4 g/day) was administered to 7 HC subjects. Over a course of two weeks, this treatment abolished the increased adhesion (158.5 ± 10.9% vs 103.5 ± 4.9% vs 100 ± 5.2%, HCvs Arg vs Control. p &lt; 0.005), while MNC adhesion remained significantly elevated in placebo-treated subjects. The adhesiveness of human mononuclear cells is increased by hypercholesterolemia. The increase in adhesiveness is reversed in vitro by NO donors, and reversed in vivo by treatment with the NO precursor L-arginine.

797-3 Restoration of Thromboresistance in Injured Aortic Segments by a Novel Nitric Oxide Donor

Endothelium-derived nitric oxide (NO) is one of the principal mediators of thromboresistance in intact blood vessels. To determine whether repletion of NO would restore thromboresistance in injured arterial segments, we investigated the effect of a novel short-lived NO donor — NO adduct of N,N’-dimethylhexanediamine (DMHD/NO) on arterial injury-induced thrombosis in an ex vivo rabbit carotid-jugular arteriovenous shunt model. Segments of porcine aortic subendothelium (endothelium-deficient but intact internal elastic lamina) and media, simulating mild and deep arterial injury, respectively, were placed in a Badimon perfusion chamber and exposed to flowing arterial blood from hypercholesterolemic (HC) rabbits (cholesterol of 431 ± 187 mg/dL, n = 5) for 10 minutes at a shear rate of 1700/s (typical of mild coronary stenosis). Thrombus volume was quantified in control and treated segments by morphometry of histological sections. Treatment consisted of DMHD/NO (0.01 μM) and phosphate buffered saline as control. Thrombus formation was minimal in subendothelium (8 ± 4μm3/μm2, of exposed area), whereas media developed large thrombi (78 ± 15μm3/μm2 (p = 0.001). DMHD/NO strikingly reduced thrombus volume in media by 85 ± 5% to 15 ± 6μm3/μm2, thereby effectively reversing thrombus formation to that in subendothelium. Infusion of saline had no effect on thrombus formation. Mean arterial pressure fell by about 3% after infusion of DMHD/NO. In this ex vivo model of arterial thrombosis 1) deeply injured-arterial segments (media) were highly thrombogenic and mildly injure-darterial segments (subendothelium) were relatively thromboresistant, and 2) DMHD/NO, a novel nitric oxide donor, effectively restored thromboresistance in deeply injured-arterial segments without producing significant systemic hemodynamic effects.

Binding of vascular anticoagulant alpha (annexin V) to the aortic intima of the hypercholesterolemic rabbit. An autoradiographic study.

Qualitative ultrastructural autoradiography was used to study the binding of the vascular anticoagulant alpha (annexin V) to normal and atherosclerotic (AS) rabbit aortic intima. Recombinant annexin V was labelled with 125I by the Iodogen method. Rabbits were fed a hypercholesterolemic (HC) diet up to 10 months. After laparotomy and exsanguination, the aortae were perfused with dilutions of 125I-annexin V (I-AV) for 10-15 min, either on ice or at 22 degrees C and then perfusion-fixed with aldehydes. Fragments of the labelled aortae were used for en face contact autoradiography, followed by Sudan Black staining of intimal lipid. Specimens were also included in Epon and sectioned for light- and electron-microscopic autoradiography. The binding of I-AV was increased on the AS aortae as compared with the normal ones, with an apparent preference for the lesioned areas. Microscopically, I-AV was found at the luminal front of aortic intima, on endothelial cells (EC), on macrophage foam cells, and on their disrupted remnants. The presence of the AV binding sites (reportedly known to interact with high affinity with phosphatidylserine) in the rabbit AS aortic intima, together with other known procoagulant conditions, may contribute to the initiation of coagulation events into the lesioned vascular wall, and may offer a rationale for the use of annexin V as an anticoagulant drug.