Anna Freyschuss

Antioxidant treatment inhibits the development of intimal thickening after balloon injury of the aorta in hypercholesterolemic rabbits.

The effect of the antioxidant butylated hydroxytoluene (BHT) on the accumulation of intimal smooth muscle cells (SMC) and development of intimal thickening after balloon catheter injury of the aorta were studied in rabbits with dietary-induced hyperlipidemia. Two sets of New Zealand White rabbits (eight rabbits in each group) were fed either 0.25% cholesterol or 0.25% cholesterol/1% BHT for a total of 6 wk. Serum lipid levels did not differ between the two groups. 3 wk after the start of the study, a balloon injury of the aorta was performed, after which the rabbits were kept on their respective diets for another 3 wk. After this period of time, the rabbits were killed and their aortas were investigated. The BHT-treated rabbits had only one fourth of the intimal thickness (P < 0.0001) and half the number of SMC/mm intima (P < 0.001), as compared to the rabbits fed only cholesterol. There was also a lower number of macrophages in the BHT-treated group. T lymphocytes were present in the intima of cholesterol-fed rabbits, whereas no such cells could be identified in the BHT-fed animals. There were significantly lower levels of autooxidation products of cholesterol (7-oxocholesterol, cholesterol-5,6-epoxide, and 7 beta-hydroxycholesterol) in the aortas of BHT-treated rabbits, P < 0.001. In conclusion, the antioxidant BHT effectively inhibited the accumulation of intimal SMC and the development of intimal thickening of the aorta in hypercholesterolemic rabbits after a balloon catheter-induced injury. These results indicate that antioxidants may modify intimal response to injury.

Effect of ascorbic acid on microcirculation in patients with Type II diabetes: a randomized placebo-controlled cross-over study

Manifestations of vascular disease, including microvascular changes, constitute the major part of the morbidity and mortality in diabetic patients. Oxidative stress has been suggested to play an important role in the vascular dysfunction of diabetic patients. Furthermore, epidemiological observations indicate a beneficial effect of an increased dietary intake of antioxidants. The present study tested the hypothesis that the antioxidant ascorbic acid influences microcirculatory function in patients with Type II diabetes. Patients with Type II diabetes were treated with 1 g of ascorbic acid three times a day for 2 weeks in a randomized placebo-controlled double-blind cross-over design. Microvascular reactivity was assessed by vital capillaroscopy and PRH (post-occlusive reactive hyperaemia). hs-CRP (high-sensitivity C-reactive protein), IL-6 (interleukin-6), IL-1ra (interleukin-1 receptor antagonist) and ox-LDL (oxidized low-density lipoprotein) were analysed. The results showed no significant change in microvascular reactivity assessed after 2 weeks of ascorbic acid treatment. TtP (time to peak) was 12.0+/-3.3 s before and 11.2+/-3.5 s after ascorbic acid (n=17). In comparison, TtP was 11.5+/-2.9 s before and 10.6+/-2.8 s after placebo (not significant). IL-1ra, IL-6, hs-CRP and ox-LDL did not change significantly after ascorbic acid, neither as absolute or relative values. In conclusion, in contrast with some studies reported previously, we could not demonstrate an effect of continuous oral treatment with ascorbic acid on microvascular reactivity assessed at the level of individual capillaries. Furthermore, we found no indication of an effect on inflammatory cytokines or ox-LDL.

Vitamin C Reduces Cholesterol-Induced Microcirculatory Changes in Rabbits

The microcirculation was studied for 10 weeks in untreated rabbits (n = 12) and in rabbits treated with vitamin C in their drinking water (0.5 g/d; n = 6), a 1% cholesterol diet (n = 12), or a combination of the two treatments (n = 11). The studies were performed by direct intravital microscopic imaging of the conjunctiva of both eyes to evaluate blood flow velocity, microvessel diameter, and microhemorheologic conditions. As we reported previously, changes occurred in all of the aforementioned variables as a consequence of cholesterol feeding. After 3 and 6 weeks of feeding, there was a marked and significant (P < .0001) decrease in blood flow velocity in third-order arterioles, which was accompanied by stasis and erythrocyte aggregation in the smaller conjunctival vessels. When cholesterol treatment was combined with vitamin C, blood flow was almost identical to that of controls and significantly (P < .0001) higher than that of rabbits treated with cholesterol alone. All other changes were also significantly reduced by the addition of vitamin C treatment to the cholesterol diet. Cholesterol-treated rabbits developed macroscopic arterial lesions that were not significantly reduced by vitamin C treatment. Neither circulating oxysterol levels nor atheromas were reduced by vitamin C treatment, which also had no significant effect on lipid or circulating vitamin E levels. We have previously shown that the lipid-soluble antioxidant BHT is able to prevent both cholesterol-induced microcirculatory changes and the development of arterial lesions in rabbits. This phenomenon is compatible with a critical oxidation step occurring in the lipid phase that is common to both processes. The finding that microcirculatory changes can be prevented by a water-soluble antioxidant is compatible with a role for water-soluble oxidants in this context. The possibility is discussed that vitamin C might also be important for the microcirculation in humans.

The antioxidant butylated hydroxytoluene prevents early cholesterol-induced microcirculatory changes in rabbits.

Microcirculation was studied during 10 wk in untreated rabbits (n = 13) and in rabbits treated with dietary addition of 1% cholesterol (n = 13), 1% cholesterol + 1% of the antioxidant BHT (butylated hydroxytoluene) (n = 11), or 1% BHT (n = 5). The studies were performed by direct intravital microscopic imaging of the left and right conjunctivae with the use of a stereo microscope and a high resolution television camera. Microvessel diameter, erythrocyte flow velocity, and microhemorheologic conditions were evaluated quantitatively via a computer-assisted digital image processing system. Significant and marked changes occurred in all the above variables as a consequence of cholesterol feeding. After 3 wk of feeding there was a dramatic decrease (approximately 30%) in blood flow velocity in arterioli of the third order (P < 0.0001), accompanied by aggregation of cells in 40-50% of the smaller conjunctival vessels (P < 0.0001). These changes were enhanced further during the following 7 wk of treatment. All the above changes in the microcirculation were markedly reduced by the addition of BHT treatment. The diameter of the above arterioli decreased in the purely cholesterol-fed group (P < 0.005), whereas this did not occur in the group fed both cholesterol and BHT. In rabbits fed BHT in the absence of cholesterol, there was no significant effect on any assessed microcirculatory variable. In conclusion, the results demonstrate that the antioxidant BHT prevented early cholesterol-induced microcirculatory changes. This prevention occurred in the absence of a reduction of blood lipid levels. The results provide strong support for the hypothesis that a considerable part of the effects on microcirculation in hypercholesterolemia may be due to cholesterol-induced oxidations and not to cholesterol itself. The results are discussed in relation to the previously demonstrated antiatherogenic effect of BHT and the possible use of antioxidants in the therapy and prophylaxis of atherosclerosis.

On the anti-atherogenic effect of the antioxidant BHT in cholesterol-fed rabbits: inverse relation between serum triglycerides and atheromatous lesions

We have shown that inclusion of the antioxidant butylated hydroxytoluene (BHT) in the diet protects against development of atherosclerotic lesions in cholesterol-fed rabbits. In parallel, BHT treatment results in increased plasma triglyceride levels. The present study explores the relationship between the triglyceride-inducing and protective effects of BHT in two different studies. The combined material contains 22 rabbits fed cholesterol and 18 rabbits fed cholesterol in combination with 1% BHT. In the BHT group there was an inverse relationship between triglyceride exposure/cholesterol exposure and extent of lesions with r=0.74 (P=0.0005). Our results show that increased triglyceride exposure parallels the anti-atherogenic effect of BHT. There was no significant correlation between atheromatosis and serum BHT levels. beta-very low density lipoprotein (beta-VLDL) from cholesterol and BHT animals was triglyceride-enriched and smaller compared to beta-VLDL from cholesterol-fed animals, but there was no significant association between the anti-atherogenic effect of BHT and particle size or apolipoprotein pattern of LDL or beta-VLDL. LDL isolated from rabbits treated with cholesterol and BHT was less sensitive to oxidative modification than LDL isolated from rabbits treated with cholesterol only. In conclusion, our results demonstrate that the degree of triglyceride exposure may be an important modulator of the anti-atherogenic effect of an antioxidant.

Microvascular Reactivity in Response to Smoking and Oral Antioxidants in Humans

Please cite this paper as: Henriksson, Diczfalusy and Freyschuss (2012). Microvascular Reactivity in Response to Smoking and Oral Antioxidants in Humans. Microcirculation 19(1), 86–93.

Post-occlusive reactive hyperemia in single nutritive capillaries of the nail fold: methodological considerations

Endothelial function at the arterial level has been extensively assessed by a noninvasive method using flow-mediated dilatation (FMD) of the brachial artery. Early disturbances have been found in patient groups prone to later development of manifest macrovascular atherosclerosis. A possible non-invasive means of studying blood-flow regulation and function at the microcirculatory level is through videophotometric capillaroscopy. The most stable variable in such an investigation is the time-to-peak (TtP) flow after a brief arterial occlusion. The short-term reproducibility of such assessments is excellent but the coefficient of variation (CV) in long-term studies is reported to be in the order of slightly less than 20%. The aim of the present methodological study was to evaluate different sources of variations in such microcirculatory assessments in order to be able to propose design recommendations that minimize the number of patients and recordings needed to achieve sufficient statistical power in longitudinal studies. We used a symmetric design with 144 recordings of TtP after a one-minute arterial occlusion in healthy volunteers. We did six occlusions each time in the capillaries of two fingers on each occasion, and repeated the procedure three times with an interval of at least one week between each investigation. All recordings were analyzed off-line using a cross-correlation technique with the Capiflow ® system. Each analysis was performed at least three times, giving a total of slightly less than 500 assessments. In our material (n= 10) TtP had a mean of 6.3 s (95% confidence interval 5.2 - 7.4). The correlation between repeated measurements in a single capillary during a single session was r>0.91 (CV 6%). The between-finger CV was 8% (r= 0.84). The CV of measurements between different days was about 20% when single measurements were compared. However, the CV decreased to less than 13% when the mean of at least two time-to-peak assessments on each occasion was used. In conclusion, the methodological error including day-to-day variation could be minimized using the mean of at least two repeated assessments of post-occlusive hyperemia at each time point in a longitudinal study. This finding should be taken into consideration in the design of future longitudinal studies.

Micro‐ and Macrocirculatory Effects of Apheresis in Patients withFamilial Hyperlipidemia

High lipid levels have been shown to coexist with a disturbedvascular endothelial function. Thus, acute lipid lowering couldbe expected to improve vascular reactivity. Patients with familialhyperlipidemia (FH) have substantial and rapid decreases in lipidlevels after low‐density lipoprotein apheresis. Thus, an opportunityexists to study acute effects of lipid lowering on vascular reactivity.Flow mediated vasodilation (FMD) is commonly used to assess endothelialfunction in conduit vessels, whereas vital capillary microscopyduring the post‐reactive hyperemia (PRH) phase is used to assessresponse at the microvascular level. It is not known whether endothelialdysfunction is a general phenomenon in all kinds of vessels or ifthere are differences between micro‐ and macrovessels. In the presentstudy, we were not able to demonstrate an improved vascular responseafter acute lipid lowering in FH patients. This was true both inthe microcirculation (PRH) and in the conduit vessels (FMD). However,the two methods gave concordant results, demonstrating disturbedvascular reactivity in the patients as compared with healthy subjects.We have found no previous report with parallel assessments at themicrocirculatory and macrocirculatory levels.

Immediate Effect of Passive Smoking on Microcirculatory Flow

Exposure to SHS, as by passive smoking, seems to increase the incidence of cardiovascular events. It has been shown that active smoking of a single cigarette causes an immediate and significant decrease in microcirculatory blood flow velocity, whereas the acute effects of exposure to SHS on microcirculatory flow have as yet not been demonstrated.

Preserved microcirculatory response to acute estrogen not reflected by exercise capacity

Background. To assess the acute effect of a single dose of 10 mg oral micronized 17β‐estradiol on microcirculation in postmenopausal women with and without coronary artery disease and its potential influence on exercise capacity. Methods. Postmenopausal women (n=11) with coronary artery disease had symptoms of ischemic heart disease and at least 1 mm ST depression at exercise. Microcirculation was examined by vital microscopy, with and without the acute administration of estrogen in a placebo‐controlled cross‐over design. Exercise test was performed on bicycle. The microcirculatory findings were contrasted to those in 14 healthy postmenopausal women. Results. 17β‐Estradiol in serum and blood flow velocity increased significantly after acute oral estrogen administration both in women with coronary artery disease (p<0.001) and in healthy women (p<0.0001), with no significant difference between the two groups. No effect on exercise capacity or ST depression at exercise was detected. Conclusions. Previously reported data that a single dose of estrogen administered to postmenopausal women results in positive effects on exercise was not reproduced. An increased peripheral microvascular flow velocity was detected in women with coronary artery disease and this increase was not accompanied by an increased exercise capacity.