Comparison of the pro-inflammatory potential of monocytes from healthy adults and those with peripheral arterial disease using an in vitro culture model.

We adapted a monocyte:endothelial cell co-culture model to investigate the pro-inflammatory potential of monocytes from patients with peripheral arterial disease (PAD). Isolated monocytes were cultured with human umbilical vein endothelial cells (HUVEC) for 24h, after which the ability of the HUVEC to recruit flowing neutrophils was tested. Development of a usable protocol required comparisons of primary HUVEC with cells that had been passaged and/or frozen and thawed, evaluation of optimal culture media and comparison of monocytes from freshly drawn and stored blood. We found, for instance, that expansion of HUVEC was assisted by inclusion of hydrocortisone, but this agent was withdrawn before the test phase because it reduced responses of HUVEC. Using the optimal practical protocol, we found great variation in the ability of monocytes from different donors to cause neutrophil adhesion. Slightly more ( approximately 20%) monocytes from patients with PAD adhered to HUVEC than monocytes from healthy controls, and the monocytes from PAD patients induced approximately 70% greater subsequent adhesion of neutrophils. Thus, we developed a functional model of inflammatory potential usable in clinically-related studies and found that patients with PAD had circulating monocytes with greater than normal ability to activate endothelial cells.

Effect of vitamin E supplementation on circulating cell adhesion molecules pre- and post-coronary angioplasty.

The soluble adhesion molecules P-selectin (sP-selectin) and intercellular adhesion molecule-1 (sICAM-1) are derived from platelets and endothelial cells. Circulating concentrations of these soluble adhesion molecules are raised in patients with atherosclerosis and following percutaneous transluminal coronary angioplasty (PTCA). We have investigated the effects of vitamin E supplements (800 IU/day) on circulating plasma ICAM-1 and P-selectin levels pre- and post-PTCA. Patients, randomized to group, were pre-treated with vitamin E or placebo (soybean oil) for 1 month before routine PTCA. Plasma sICAM-1 and sP-selectin were measured by enzyme-linked immunosorbent assay on blood taken immediately pre- and post-PTCA. Total protein and alpha-tocopherol were measured on the same samples. Plasma alpha-tocopherol concentrations increased in patients receiving vitamin E: 19.1 (1.5) [mean (standard error of the mean, SEM)] mg/mL post-PTCA versus 13.9 (0.6) mg/mL pre-PTCA (n=23; P<0.01). Plasma sP-selectin and sICAM-1 levels were not significantly increased following PTCA in the vitamin E group. Pre-angioplasty mean (SEM) plasma sP-selectin concentration in the vitamin E group was 8.83 (0.97) ng/mg protein; the corresponding mean post-angioplasty value was 9.34 (0.89) ng/mg protein (P=0.85). The mean (SEM) pre-angioplasty sICAM-1 concentration in this group was 2.18 (0.24) ng/mg protein, and was 2.20 (0.23) ng/mg protein following angioplasty (P = 0.84). In the placebo group (n = 24) there was a significant increase in mean (SEM) sP-selectin concentration following angioplasty, from 7.48 (0.73) to 9.70 (0.78) ng/mg protein (P<0.05). The change (mean, SEM) in plasma sP-selectin concentration following angioplasty was significantly greater for the placebo group [2.22 (0.50) ng/mg protein] than for the group receiving vitamin E [0.50 (0.50) ng/mg protein] (P<0.02). This difference remained significant (P<0.05) even after adjustment for pre-angioplasty P-selectin concentrations. Mean (SEM) plasma sICAM-1 concentrations remained unchanged following angioplasty [pre-angioplasty: 2.16 (0.20) ng/mg protein; post-angioplasty: 1.97 (0.13) ng/mg protein]. Vitamin E may therefore limit platelet or endothelial activation during PTCA.

Mononuclear cell adhesion to collagen ex vivo is related to pulse pressure in elderly subjects.

Mononuclear cells and platelets are intimately involved in the pathogenesis and complications of cardiovascular disease. Platelet activation has been reported in hypertension, though the activation-state of monocytes has received less attention. In this study the adhesiveness of monocytes and platelets was assessed and any relationship between the adhesive properties of these cellular elements and plasma levels of soluble adhesion molecules and blood pressure parameters determined. Fifty six elderly volunteers, of whom 32 were classified hypertensive (daytime SBP > or = 135 mmHg), underwent 24 h blood pressure monitoring, assessment of monocyte and platelet adhesion and measurement of the plasma soluble adhesion molecules ICAM-1, L-selectin, E-selectin and vWF. In the elderly hypertensive subjects, monocyte adhesion to collagen coated (P < 0.05) and tissue culture plastic microwells (P < 0.05) was significantly elevated and circulating levels of soluble ICAM-1 (P < 0.01) and soluble E-selectin (P < 0.05) were significantly raised compared to their normotensive counterparts. A significant correlation was found to exist between monocyte adhesion to collagen and daytime pulse pressure (r = 0.39, P < 0.01) and also between plasma levels of soluble E-selectin and clinic DBP (r = 0.40, P < 0.001). The increased monocyte adhesion witnessed in hypertensive subjects and with increasing pulse pressure may contribute to the increased risk of cardiovascular disease in hypertension. Whether this increased adhesiveness is a property of the monocytes. or reflects endothelial cell activation, remains to be determined.

Cholesterol standardized plasma vitamin E levels are reduced in patients with severe angina pectoris.

Vitamin E, the major lipid soluble plasma antioxidant, has been reported to be reduced in patients with coronary atherosclerosis. We have measured the levels of plasma alpha-tocopherol (the predominant form of plasma vitamin E) in 128 patients with different reported degrees of angina. Patients with mild to moderate angina (grades I or II (CSS score)) (n = 64), and patients with severe angina (grades III and IV) (n = 64) were recruited from Cardiology Clinics in the U. K. Healthy controls (n = 33) and patients with hyperlipidaemia (n = 28) were also recruited. The groups of patients with angina did not differ significantly for mean age (58 +/- 1.0 years vs. 59 +/- 1.0 years, respectively); sex distribution (the M:F ratio was 48 : 16 and 46 : 18 for the respective groups); or prevalence of smoking (12% vs. 9%), or hypertension (19% vs. 33%). Total cholesterol levels were higher in the group with severe angina (5.9 +/- 0.16 mmol/l vs. 5.3 +/- 0.13 mmol/l P < 0.05). Absolute levels of plasma vitamin E were not significantly different between the angina subgroups (12.9 +/- 0.40 mg/l for the mild-moderate angina group vs. 12.5 +/- 0.51 mg/l for the severely affected group), but were positively correlated with plasma cholesterol concentrations in each case (P < 0.001). The ratio between plasma vitamin E: total cholesterol was significantly lower in the patients with severe angina (mean 2.20 +/- 0.09 mg/mmol) vs. a mean value of 2.46 +/- 0. 08 mg/m mol in the mildly affected group (P < 0.05). The plasma vitamin E: total cholesterol ratio in patients with severe angina was also significantly lower (P < 0.05) compared to either healthy controls with comparable total cholesterol levels (n = 33), or hypercholesterolaemic subjects (n = 28) without symptomatic coronary disease (mean ratios were 2.69 +/- 0.40 mg/mmol and 2.74 +/- 0.68 mg/mmol, respectively). Vitamin E has previously been demonstrated to protect endothelial function in the presence of hypercholesterolaemia, possibly by preserving nitric oxide bio-activity. It also inhibits LDL oxidation. Hence, a high plasma vitamin E: total cholesterol ratio may be associated with an amelioration of angina.

Effects of vitamin E on human platelet and mononuclear cell responses in vitro.

Recent epidemiological studies have provided evidence supporting the potential benefits of antioxidants in coronary prevention. We have investigated the effects of vitamin E on platelets, monocytes and endothelial cells in vitro. Pre-incubation of platelets with vitamin E inhibited subsequent thrombin- (P < 0.05, n = 5), collagen- (P < 0. 0001, n = 5) and ADP-(P < 0.05, n = 4) induced platelet aggregation measured using a microtitre plate method, or conventional aggregometry. The adhesion of thrombin-activated platelets to collagen was also inhibited by vitamin E (P < 0.05, n = 8), but not by vitamin C (P > 0.05, n = 8); nor was the adhesion of unstimulated platelets significantly affected (P > 0.05, n = 8). Pre-incubation of monocytes with vitamin E inhibited their subsequent adhesion to plastic (P < 0.05, n = 9), and was also associated with an 18% reduction in adhesion to EA.hy 926 endothelial cells (n = 8), although this failed to reach statistical significance. Pre-incubation of the endothelial cells with vitamin E also significantly reduced subsequent mononuclear cell adhesion by 56% (P < 0.05, n = 3).

Relationship of internalized racism to abdominal obesity and blood pressure in Afro-Caribbean women.

Racism is associated with increased psychosocial stress and blood pressure in blacks. However, little is known of the relationship of racism to other features of insulin resistance syndrome. This study examined the relationship of internalized racism to abdominal obesity and elevated blood pressure in a population of black Caribbean women aged 20-55 years. One hundred thirty-three randomly selected women from the island of Barbados comprised the study sample. Data collected included anthropometric and blood pressure measurements, and information about internalized racism, anxiety, and depression. The stress measures including anxiety, depression, and internalized racism were significantly correlated with waist circumference (r = .25, r = .21, and r = .25). After adjusting for age, education, anxiety, and depression, internalized racism remained significantly correlated with waist circumference. The odds ratio associated with development of abdominal obesity among those with high internalized racism (OR = 2.4 [95% CI, 1.1,5.3]) was significant (P < .05) after adjusting for age, education, and body mass index. Blood pressure was not independently related to internalized racism. Studies comparing black-white differences in diseases for which abdominal obesity is a risk factor (eg, diabetes and cardiovascular disease) should take into account the potential role of internalized racism in defining the differences between ethnic groups.

Dietary vitamin E supplementation inhibits thrombin-induced platelet aggregation, but not monocyte adhesiveness, in patients with hypercholesterolaemia.

Several recent studies have indicated the possible beneficial effects of antioxidants, specifically vitamin E, in primary and secondary coronary prevention. These studies suggest that a diet enriched in vitamin E is insufficient to have a significant protective effect, whereas supplements, in excess of 200 international units (IU) per day, are efficacious in preventing coronary disease in both men and women. The mechanisms by which vitamin E may exert its protection are uncertain, but, vitamin E is lipophilic and has been shown to inhibit the oxidative modification of low density lipoprotein (LDL), a process thought to be of crucial importance in atherogenesis. We have also previously shown that alpha-tocopherol (the biologically most potent isomer of vitamin E) has important direct effects on vascular endothelial and smooth muscle cells. In the present study we have investigated the effects of oral supplements of vitamin E (400 IU per day) on platelet and mononuclear cell function in patients with hypercholesterolaemia. We found that although vitamin E supplementation had no significant effect on mononuclear cell adhesion ex vivo, it had a significant effect on the thrombin-induced platelet aggregation (P < 0.01; ANOVA): 6 weeks after starting the vitamin E supplements, the mean EC50 for thrombin-induced aggregation increased 132% (P < 0.05; paired t-test) compared to treatment with placebo. The effects of vitamin E on platelet function may, in part, explain its anti-atherogenic properties.

Insulin-like growth factor-I modulates monocyte adhesion to EAhy 926 endothelial cells.

IGF-I is a ubiquitous growth factor, found in platelets and elaborated by many other cell types. It is thought to be involved in several pathophysiological processes including embryonic development, angiogenesis and wound healing. We report that the adherence of human peripheral blood monocytes to an endothelial cell line (EAhy 926) is inhibited in a dose and time-dependent manner by pre-incubating the endothelial cells with IGF-I (P < 0.001). Monocyte adhesion was inhibited 17.9 +/- 1.9% by IGF-I at a dose of 1000 ng/ml (P < 0.01). In contrast, IGF-I had no significant effect on monocyte adherence to plastic. The inhibitory effects of IGF-I were reversed by co-incubating the endothelial cells with the nitric oxide synthase inhibitor, L-NAME. These data suggest that the effects of IGF-I are mediated by the release of nitric oxide from the endothelial cells.