Effects of melatonin on spinal cord injury-induced oxidative damage in mice testis.

This study evaluated the effects of melatonin on spinal cord injury (SCI)-induced oxidative damage in testes. Adult male C57BL/6 mice were randomly divided into sham-, SCI- or melatonin (10 mg/kg, i.p.)-treated SCI groups. To induce SCI, a standard weight-drop method that induced a contusion injury at T10 was used. After 1 week, testicular blood flow velocity was measured using the Laser Doppler Line Scanner. Malondialdehyde (MDA), glutathione (GSH), oxidised glutathione (GSSG) and myeloperoxidase (MPO) were measured in testis homogenates. Microvascular permeability of the testes to Evan's Blue was examined by spectrophotometric and fluorescence microscopic quantitation. The tight junction protein zonula occludens-1 (ZO-1) and occludin in testes were assessed by immunoblot analysis. Melatonin increased the reduced blood flow and decreased SCI-induced permeability of capillaries. MDA levels and MPO activity were elevated in the SCI group compared with shams, which was reversed by melatonin. In contrast, SCI-induced reductions in GSH/GSSG ratio were restored by melatonin. Decreased expression of ZO-1 and occludin was observed, which was attenuated by melatonin. Overall, melatonin treatment protects the testes against oxidative stress damage caused by SCI.

Enhanced matrix metalloproteinases-2 activates aortic endothelial hypermeability, apoptosis and vascular rarefaction in spontaneously hypertensive rat.

Microvascular rarefaction with endothelial cells apoptosis is a common characteristic of various microvascular complications in the spontaneously hypertensive rat (SHR). Elevated levels of proteolytic (e.g. matrix metalloproteinase, MMPs) activity and apoptosis in aortic endothelial cells of SHR were found when compared to its normotensive control. However, the exact mechanisms of microvascular rarefaction and the role of MMPs in this process remain poorly understood. Besides cleavage of VEGFR2 via unbalanced MMPs, we hypothesize that selected cleavage of Beta-Catenin and VE-cadherin by MMPs could induce apoptosis of rat aortic endothelial cells (RAECs) and rarefaction. Primary RAECs were isolated, identified and used in a in-vitro model. Transwell system was used to analyze the permeability of Wistar RAECs, SHR RAECs and SHR RAECs with pretreatment by doxycycline. Qualitative and semi-quantitative analysis of major endothelial adhesion molecules were detected by immunofluorescence technique and Western blot, respectively. MMP-2 activity of SHR RAECs was increased significantly and doxycycline (50 µM) effectively reduced the level of MMP-2 and hyper-permeability in SHR RAECs. SHR RAECs showed enhanced cleavage of VEGFR2, VE-cadherin and B-catenin, which could be prevented by doxycycline (50 µM). Doxycycline (50 µM) attenuated hyper-permeability via decreased MMP-2 by protecting VEGFR2, VE-cadherin, Beta-catenin from cleavage and inhibited the reduction of mitochondrial transmembrane potential (MTP), thus prevented mitochondria-mediated apoptotic signaling and capillary rarefaction in the SHR. It might be a novel insight into the mechanisms of SHR microvascular rarefaction that is independent of pressure but relevant to MMP-2.

N-acetylcysteine improves microcirculatory flow during smoking: new effects of an old drug with possible benefits for smokers.

BACKGROUND: Cigarette smoking provokes marked acute changes in the microcirculatory vasculature, including a reduced blood flow velocity. In accordance with the hypothesis that the reduced blood flow is due to an imbalance between pro-oxidants and oxidants, we recently showed that most of the reduction could be reversed by a high dose of vitamin C. HYPOTHESIS: In the present work we tested the hypothesis that N-acetylcysteine, a mucolyticum and an antioxidant, may have an effect on the smoking-induced changes observed by vital capillary microscopy of the nailfold. METHODS: In all, 37 healthy volunteers of both genders and with varied smoking habits were treated with N-acetylcysteine 200 mg t.i.d. for 2 weeks. In vivo investigation of the microcirculation by capillaroscopy was performed before and after treatment. RESULTS: Treatment with N-acetylcysteine significantly reduced the smoking-induced relative decrease in capillary blood flow velocity in a group of volunteers with varied smoking habits (p = 0.0016). The preventive effect was clearly significant in smokers (p = 0.003). CONCLUSION: Treatment with N-acetylcysteine has a positive impact on microcirculatory flow during smoking, particularly in habitual smokers.

A single high dose of vitamin C counteracts the acute negative effect on microcirculation induced by smoking a cigarette.

Cigarette smoking is associated with marked acute changes in microcirculation including reduced blood flow. We tested the hypothesis that the reduced blood flow velocity is due to the imbalance between prooxidants and antioxidants that occurs as a consequence of smoking and that it can be reduced by an antioxidant. The effect of smoking a single cigarette on nail-fold microcirculation was analyzed in 24 healthy subjects with varying smoking habits. Vital capillary microscopy was used and the blood cell flow velocity in the capillaries was evaluated before and 1-30 min after smoking. Smoking induced a marked decrease in microcirculatory blood flow in 23 of the 24 subjects (40-50% decrease 1-5 min after smoking). This change was reduced by more than 50% in the same subjects after intake of 2 g of vitamin C 2 h before smoking (P < 0.0001 by ANOVA test) with smokers responding similarly to nonsmokers in these experiments. Intake of 1 g of vitamin C had no significant effect on the smoking-induced changes in most of the subjects tested (n = 11). Pretreatment with aspirin had little or no effect on the response to smoking (n = 9). Our results show that treatment with a single high dose of vitamin C can reduce and in some individuals even completely abolish the negative acute effect on microcirculation induced by smoking a single cigarette. This effect of vitamin C is not likely to be mediated by the cyclooxygenase system.

[An animal experiment and clinical investigation on the protective effect of selenium on the microcirculation induced by free radical damaged RBCs].

UNLABELLED: Fluorescent labeling image analysis was used to evaluate the changes in cerebral arteriole and veinlet diameters (D), circulation velocities (FV) and permeability (VP) in rats; while in clinics, a laser-doppler device was used for assessing changes of skins and muscles microcirculation. The results show that in control rats, equal volume perfusion of free radical damaged RBCs resulted in decreases of D and FV significantly but VP was increased, whereas in the case when free radical damaged RBCs were perfused together with selenium, no disturbances in the D and VP were observed with FV even improved. In the human control group, either average skin microcirculative perfusions (ASMP) at 25 degrees C or maximal skin microcirculative perfusions (MSMP) at 42 degrees C were evidently decreased during myocardial ischemia/reperfusion period, while ASMP at 24th hour of post-surgery was kept reducing. At the same time, the changes in muscles microcirculative perfusions (MMP) tended to be similar to the skin, but overloaded than the pre-surgery levels at 24th hour of post-surgery period. In the selenium group before surgery, the ratio of MSMP to ASMP was obviously increased than the control group (3.95 in Se group vs 1.74 in control group, P < 0.05), but did not have significantly differences in ASMP, MSMP and MMP between the two groups during surgery period. RBC deformabilities were not changed. At 24th hour post-surgery, the ASMP were almost restored to pre-surgery levels. However, MMP were still lower than the pre-surgery levels. CONCLUSIONS: (1) free radical damaged RBC perfusion leads to damage of microcirculation; (2) selenium is highly efficient in protecting microcirculation from free radical damaged RBC disturbance; and (3) Oral administration of selenium may improve pre-surgery maximal skin microcirculative perfusion and promote recovery of the worsened skin microcirculation in addition to prevent the occurrence of RBC deformability.

Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-hydroxylase mechanism. Comparison with high density lipoprotein-mediated reverse cholesterol transport.

Cultured macrophages and endothelial cells have been reported to secrete 27-oxygenated metabolites of cholesterol. This mechanism was compared with the classical high density lipoprotein (HDL)-dependent reverse cholesterol transport. Under standard conditions, macrophage preparations had considerably higher capacity to secrete 27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid than had endothelial cells and fibroblasts. Western blotting showed that lung macrophages contained the most sterol 27-hydroxylase protein of the cells tested. The relative amounts of 3beta-hydroxy-5-cholestenoic acid produced by the macrophages were also highest. Macrophages derived from monocytes of patients with sterol 27-hydroxylase deficiency did not secrete 27-oxygenated products, demonstrating that sterol 27-hydroxylase is the critical enzyme for the conversion of cholesterol into the 27-oxygenated steroids. That sterol 27-hydroxylase is responsible not only for 27-hydroxylation of cholesterol but also for the further oxidation of this steroid into 3beta-hydroxy-5-cholestenoic acid was shown with use of tritium-labeled 27-hydroxycholesterol and an inhibitor of sterol 27-hydroxylase. Secretion of 27-oxygenated products by the cultured macrophages as well as the ratio between the alcohol and the acid appeared to be dependent upon total 27-hydroxylase activity, the availability of substrate cholesterol, and the presence of an acceptor for 27-hydroxycholesterol in the medium. With albumin as extracellular acceptor, the major secreted product was 3beta-hydroxy-5-cholestenoic acid. Under such conditions, secretion of labeled 27-oxygenated products was higher than that of labeled cholesterol from lung alveolar macrophages preloaded with [4-14C]cholesterol. With HDL as acceptor, 27-hydroxycholesterol was the major secreted product, and the total secretion of labeled 27-oxygenated products was only about 10% of that of labeled cholesterol. Thus, 27-hydroxycholesterol and cholesterol may compete for HDL-mediated efflux from the cells. The results support the contention that the sterol 27-hydroxylase-mediated elimination of cholesterol is more important in macrophages than in endothelial cells. This mechanism may be an alternative and/or a complement to the classical HDL-mediated reverse cholesterol transport in macrophages, in particular when the concentration of HDL is low.

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.

Dietary cholesterol induces transient changes in plasma nitrate levels in rabbits that are correlated to microcirculatory changes.

Dietary treatment of rabbits with 1% cholesterol resulted in a transient rise in their plasma nitrate levels. After 3 weeks of treatment the nitrate levels were about 50% higher than those of the controls (p<0.005). After 10 weeks of treatment the nitrate levels were similar to those at the start of the study. In accordance with previous work (Xiu et al., J. Clin. Invest., 1994, 93, 2732-2737), the cholesterol treatment let to a decreased blood flow velocity in arterioli of the third order in the conjunctiva, and a decreased diameter of these arterioli. There was a significant correlation between plasma nitrate levels and the two microcirculatory variables (p<0.0001). Nitrate is the major metabolic end product of nitric oxide (NO), and plasma nitrate levels may be used as an index of the endogenous formation of NO. The present results suggest that dietary cholesterol induces a transient increase in the synthesis of NO. Such an increased synthesis may compensate for part of a cholesterol-induced degradation of NO.

Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages.

27-Hydroxycholesterol was found in surprisingly high amounts in atherosclerotic human femoral arteries. When human macrophages were cultured in a medium containing serum, there was a significant transfer of 27-hydroxy-cholesterol and 3 beta-hydroxy-5-cholestenoic acid from the cells into the medium. Sterol 27-hydroxylase (EC 1.14.13.15) is likely to be responsible for formation of the two products as shown by use of immunoblotting, a specific inhibitor, and the 18O-labeling technique. Sterol 27-hydroxylase has the unusual ability to hydroxylate the same methyl group three times to give a carboxylic acid; thus, 3 beta-hydroxy-5-cholestenoic acid is likely to be a direct product of the enzyme. The production of these steroids increased after addition of cholesterol to the culture medium. By using deuterium-labeled cholesterol, it was ascertained that most of the oxidized products were formed from exogenous cholesterol taken up by the cells. 27-Hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid are present in the circulation and are efficiently converted into bile acids in human liver. It is suggested that conversion of cholesterol into 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid represents a general defence mechanism for macrophages and possibly also other peripheral cells exposed to cholesterol. Absence of this defence mechanism may contribute to the premature atherosclerosis known to occur in patients with sterol 27-hydroxylase deficiency (cerebrotendinous xanthomatosis).

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.

Microcirculatory disturbances in AIDS patients--a first report.

The aim of this study is to explore the vascular perturbations associated with AIDS infections as a means of developing strategies to help in the treatment of these patients. Closed-circuit television microscopic observation of the microcirculation in the nail fold was carried out on 11 AIDS patients and 11 healthy European adults. A striking pattern of severe microvascular disturbances was demonstrated in these patients: (i) a suppression of the spontaneous, rhythmic adjustments of capillary blood perfusion, and (ii) damage to the endothelium of the capillary wall. These findings suggest a substantial involvement of the microcirculation in the pathogenetic development of the AIDS syndrome, as well as a close relationship between microvascular perfusion and the immune condition of the organism. The data indicate that the treatment of the AIDS patient should be directed not only toward the inhibition or killing of the virus, but also toward the improvement of microcirculatory perfusion.

[Angiogenesis induced by SP2/0 and HeLa tumor cells].

Cheek pouches of Syrian golden hamsters and transparent access chambers of BALB/C mice were implanted with SP2/0 and HeLa tumor cells separately. Observation was done by continual photo and video from D 1 through the D 10 after implantation. It was found by the development of angiogenesis and the density of microvessels that both kinds of tumor cells could induce angiogenesis. On D 2 after implantation, there appeared leakage and hemorrhage from the microvessels near the tumor cells. On D 3 and D 4, there was an increased density of new capillaries which formed a very fine, tortuous and basketlike vascular plexus of irregular diameter. Most of the new microvessels came from the venules on the edge of the implant mass and they grew toward the tumor cells to penetrate the tumor tissue on D 4 to D 5. On D 7, the density of new microvessels reached the maximum and they began to extend outside the tumor which was surrounded by dense new capillaries. Compound 36 has been proved an effective substance against some tumors in clinical applications in this country. Also proved by our experiments, a drug effective in inhibition of angiogenesis induced by SP2/0 tumor cells.