Nitric oxide synthase expression and activity in the tight-skin mouse model of fibrosis.

OBJECTIVES: Nitric oxide (*NO) is an important physiological signalling molecule and a potent vasodilator. We have previously demonstrated abnormal *NO metabolism in the plasma of patients with systemic sclerosis (SSc; scleroderma), a disease that features vascular dysfunction as well as collagen overproduction and fibrosis. The aim of the present study was to examine nitric oxide synthase (NOS) expression and activity and assess the potential role of antioxidants in the scleroderma-like syndrome of the tight-skin 1 (TSK-1/+) mouse, an experimental animal model for fibrosis. METHODS: Skin, lung or plasma was taken from TSK-1/+ (n = 15) and wild-type (WT; n = 12) littermate mice. Type 1 collagen, endothelial NOS (eNOS), haemoxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein and gene expression were determined by western blot and reverse transcriptase-polymerase chain reaction. eNOS expression was further determined by immunohistochemistry. NOS activity was evaluated by conversion of [14C] L-arginine to [14C] L-citrulline. Levels of circulating plasma nitrite/nitrate (NO(x)) were also measured. Total antioxidant activity was evaluated by ABTS+ production (ABTS = 2,2'-azino-bis-[3-ethylbenz-thiazoline-6-sulphonic acid). RESULTS: In the skin, eNOS was present in the epidermal layer, hair follicles and also in the endothelial cells lining the blood vessels. Expression of both the eNOS protein and gene was significantly reduced in TSK-1/+ skin tissue, while type 1 collagen protein was elevated compared with WT. Furthermore, there was decreased NOS activity in TSK-1/+ skin tissue; however, there was no measurable difference in plasma NO(x). Correspondingly, the protective antioxidant enzyme HO-1 and the associated transcription factor Nrf2 showed reduced protein and gene expression levels in TSK-1/+ skin, while there was also less total antioxidant activity. In TSK-1/+ lung tissue, however, we observed no difference in collagen protein expression, *NO metabolism or HO-1 expression and total antioxidant activity compared with WT. CONCLUSIONS: The findings suggest that there is also abnormal *NO metabolism in the TSK-1/+ mouse model of fibrosis, particularly in the skin, while expression and activity of protective antioxidants are reduced. The TSK-1/+ mouse may also be useful for testing treatments that target vascular endothelial cell function in patients with SSc.

The solubility of cholesterol and its exchange between membranes.

It has been proposed that exchange between membrane cholesterol pools occurs by desorption of molecules into the aqueous environment rather than by formation of a transitory collision complex between the membranes. The rate of exchange is likely to be determined by the rate of dissociation of cholesterol from the membrane bilayer and by the concentration of cholesterol monomers or aggregates of cholesterol molecules in solution. The aim of this study was to measure the effects of agents known to increase cholesterol exchange rates on cholesterol solubility, critical micellar concentration and on the activation energy of exchange. A comparison was also made with regard to these parameters, of the exchange of cholesterol to that of 4-cholesten-3-one, another steroid which exchanges more rapidly than cholesterol. Acetone and dimethylsulphoxide increased cholesterol exchange between liposomes and erythrocytes, but only modestly increased the apparent solubility of cholesterol in saline and had no effect on the activation energy of the exchange process. However, acetone and dimethylsulphoxide increased the critical micellar concentration of the cholesterol 3-fold, although tetraethylammonium iodide, which had a smaller effect on exchange, did not. 4-Cholesten-3-one had a lower solubility and critical micellar concentration than that of cholesterol, but had the same activation energy for exchange. It is concluded that the apparent solubility of steroid aggregates are unlikely to determine the rate of exchange, but that agents which substantially increase exchange also increase the critical micellar concentration. The low critical micellar concentration of cholestenone suggests that the actual monomer concentration in an exchange system is low and that the rate of dissociation of the molecules from the liposomes must determine the exchange rate. This is not reflected in the activation energy measurements since these are a composite of all the elements of the exchange process.

The effect of lipid peroxidation and lipolysis on the ability of lipoproteins to influence thromboplastin activity.

High, low and very low density lipoproteins and lipoprotein (a) were prepared from porcine serum. The apolipoprotein components of the lipoproteins were then isolated and resuspended in soybean lecithin. Apolipoprotein B was also resuspended in lipids more representative of those found in LDL and VLDL. Lipid peroxidation was induced in samples of all the lipoproteins and reconstituted apolipoproteins by incubation with either Cu2+ ions or hedgehog 15-lipoxygenase. Furthermore, aliquots of the samples were incubated with a mixture of lipases. The effect of native preparations and the treated samples on the procoagulant activity of thromboplastin was examined. Native HDL, apo A-II, native LDL, reconstituted LDL and apo B inhibited thromboplastin activity, whereas native VLDL and reconstituted VLDL enhanced this activity. While the ability of HDL and apolipoprotein A-II to inhibit thromboplastin was unaltered by either Cu2+ oxidation, lipoxygenase oxidation or lipolysis, VLDL and particles resembling VLDL, which acted cooperatively with thromboplastin lost their activating potential. On the other hand, LDL and particles resembling LDL changed from being inhibitory to enhancing the thromboplastin activity following oxidation, but not after lipolysis. Apolipoprotein B fragments obtained by mild digestion of this protein, expressed an inhibitory effect towards thromboplastin, while extensive degradation of the protein reduced its inhibitory potential. It is suggested that modifications of lipoproteins in vivo can lead to a hypercoagulable state by modulation of the cofactor activity of thromboplastin to factor VII.

Evidence for a water-soluble intermediate in exchange of cholesterol between membranes.

The mechanism of inter-membrane cholesterol exchange has been a matter of some debate. Evidence from kinetic studies indicates that cholesterol must transfer to and from membranes in a water-soluble form. In this study attempts have been made to demonstrate that this occurs using either dialysis membranes or a barrierless multiphase polymer system to physically separate the membranes. In both systems small amounts of cholesterol were seen to transfer from one membrane pool to another using both liposomes and erythrocyte membranes as donors or acceptors. The cholesterol transfer was shown to be independent of the movement of other membrane components. The amount of transfer observed was limited by the physical properties of the systems employed. The barrier to cholesterol transfer in the dialysis membrane system is primarily the pore size of the membrane, while in the multiphase polymer system the transfer was limited by the viscosity of the medium and the distance between the lower and upper phases containing the membranes. Nevertheless, the results provide evidence that cholesterol transfer is by a dissociation of molecules from membranes into the aqueous medium and does not require the formation of a collision complex between the membranes.

The incorporation of cholesterol into inner mitochondrial membranes and its effect on lipid phase transition.

Incubations of rat liver inner mitochondrial membranes with liposomes prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol resulted in a considerable enrichment of the cholesterol composition of these membranes. This enrichment is not accompanied by an alteration in the membrane phospholipid content or fatty acid composition. The exogenous cholesterol appears to be integrated into the membrane structure because it has effects consistent with the known properties of this sterol in other natural and artificial membrane systems. Differential scanning calorimetry on both intact membranes and extracted lipids showed that as the ratio of cholesterol to phospholipid was increased, the endotherm corresponding to the lipid phase transition was reduced. Freeze-fracture electron microscopy of the native membranes showed that intramembranous particles are randomly distributed above the phase transition temperature. Below this temperature large smooth areas, believed to correspond to lipid in the gel state from which proteins have been excluded, can be observed. In the presence of high concentrations of cholesterol the fracture faces observed below the lipid transition temperature show no regions of phase segregation, and observation consistent with previous studies using pure lipids where cholesterol was observed to prevent the lipid undergoing a cooperative phase transition. The results are discussed in terms of the observed low concentrations of cholesterol in normal liver inner mitochondrial membranes and the distribution of cholesterol within the liver cells.

The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+-K+ pump in erythrocytes.

cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the Na+-K+ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the Na+-K+ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the Na+-K+ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the Na+-K+ pump. This difference in sensitivity to membrane lipids order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.

Changes in the secondary structure of apolipoprotein B-100 after Cu2+-catalysed oxidation of human low-density lipoproteins monitored by Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectra have been obtained of human low-density lipoproteins (LDL) in H2O and 2H2O buffers. The absorption bands are assigned to vibrations of the lipid and apolipoprotein B-100 components. The analysis of second-derivative spectra allowed an assignment of individual protein bands to alpha-helical, random, coil or beta-structure and beta-turn conformations. Changes in the FTIR spectra after Cu2+-catalysed oxidation of the LDL particles indicate that the structure of apolipoprotein B-100 becomes less ordered, with some alterations of alpha-helical and beta-turn conformation. The main beta-structure absorption at 1620 cm-1 is unaffected by oxidation. Taking into account the resistance to oxidation and the slow H-2H exchange it is suggested that the beta-structure is hidden from external factors whereas other structures are mostly present on the surface of the LDL particle. Oxidation affects mainly the surface region of apolipoprotein B-100 and leads to a structural rearrangement which consequently changes the receptor specificity of the LDL.

Alterations in the structure of apolipoprotein B-100 determine the behaviour of LDL towards thromboplastin.

Apolipoprotein B-100 acts as an inhibitor of thromboplastin activity independently of the tissue factor pathway inhibitor (TFPI) associated with plasma lipoproteins. Analysis of the primary structure of Apo B-100 showed a higher than expected occurrence of lysine groups in the receptor-binding region. In order to demonstrate the participation of lysine groups of Apo B-100 in the inhibition of thromboplastin, thromboplastin and Apo B-100 were incubated together in the presence of poly-L-lysine, poly-L-arginine, lysine and arginine monomers. The inhibition of thromboplastin by Apo B-100 was completely suppressed in the presence of poly-L-lysine. Poly-L-arginine was found to be less effective and neither lysine or arginine monomers had any significant effect on the inhibitory effect of Apo B-100. Alterations in the structure of Apo B-100 reconstituted in lipid vesicles resembling LDL, brought about by lipid peroxidation and lipid loading were examined by means of Fourier transform infra-red spectroscopy. It was found that, upon oxidation without the addition of cupric ions, the apolipoprotein attains a more exposed conformation with an increase in alpha-helical structure. This increase occurred at the expense of beta-structure. On lipid loading, an increase in beta-structure at the expense of the alpha-helix, was demonstrated. It is therefore proposed that the variable action of LDL towards thromboplastin derives from alterations in the secondary structure of the Apo B-100, particularly the receptor-binding region.

Phosphatidylserine-dependent adhesion of T cells to endothelial cells.

Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.

The modulation of ferryl myoglobin formation and its oxidative effects on low density lipoproteins by nitric oxide.

This study has investigated the interactions between nitric oxide and haem protein radicals. The results demonstrate that nitric oxide interacts with activated ferryl myoglobin species with reduction to metmyoglobin, but the extent and duration of the reduction depends on the relative concentrations of nitric oxide and hydrogen peroxide. Ferryl myoglobin has a much greater relative potential for oxidising polyunsaturated fatty acid side chains in low density lipoproteins than in cell membranes. The peroxidative response can be modulated by nitric oxide: ferryl myoglobin-mediated peroxidation of LDL may be enhanced or suppressed by nitric oxide depending on the relative concentrations of NO and hydrogen peroxide.

Low-density lipoproteins increase intracellular calcium in aequorin-loaded platelets.

Low-density lipoproteins activate isolated human platelets. The mechanism of this activation is unknown, but may involve increased phosphoinositide turnover. We have examined the effect of low-density lipoproteins on intracellular calcium concentrations in platelets loaded with the photoprotein aequorin. The lipoproteins induced concentration-dependent increases in intracellular calcium, associated with shape change and aggregation. These responses could be partially inhibited by the removal of extracellular calcium and by pre-incubation with acetylsalicylic acid. They were also antagonised by agents which increase cellular concentrations of cyclic adenosine and guanosine monophosphates. It is not clear whether the platelet-lipoprotein interaction involves a 'classical' lipoprotein receptor.

The nitration of platelet cytosolic proteins during agonist-induced activation of platelets.

The nitration of protein tyrosine residues by peroxynitrous acid has been associated with pathological conditions. Here it is shown, using a sensitive competitive enzyme-linked immunosorbent assay and immunoblotting for nitrotyrosine, that spontaneous nitration of specific proteins occurs during a physiological process, the activation of platelets by collagen. One of the main proteins nitrated is vasodilator-stimulated phosphoprotein. Endogenous synthesis of nitric oxide and activity of cyclo-oxygenase were required for the nitration of tyrosine. The nitration was mimicked by addition of peroxynitrite to unstimulated platelets, although the level of nitrotyrosine formation was greater and its distribution among the proteins was less specific.

Modification of tissue factor by peroxynitrite influences its procoagulant activity.

Peroxynitrite, a reactive oxidising species resulting from a reaction between nitric oxide and the superoxide anion, modifies proteins by nitration of certain amino acids such as tyrosine. Tissue factor (TF), a transmembrane protein, is expressed on cells under inflammatory conditions and initiates the coagulation cascade. The extracellular domain of TF is rich in tyrosine. Exposure of recombinant TF and cellular TF to peroxynitrite was associated with a reduction in procoagulant activity. This was accompanied by an elevated level of nitrotyrosine residues. Peroxynitrite may have a protective role by attenuation of the thrombotic properties of TF.

The role of the C-terminal domain in the inhibitory functions of tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) inhibits the activity of coagulation factors VIIa and Xa through Kunitz domains, thereby inhibiting the activity of tissue factor. However, it has been shown that the C-terminal of this inhibitor is essential for the maximal anticoagulant activity of TFPI. We have investigated the endogenous ability of the C-terminal of TFPI to influence coagulation. A synthetic peptide corresponding to residues 254-265 within the C-terminal of TFPI was prepared and shown to be capable of inhibiting tissue factor pathway by preventing the activation of factor VII. Mutational analysis of the peptide revealed the identity of the key lysine residues.

Fatty acid metabolism in adipose tissue of aging mice after direct tracer injection into fat pads.

We have examined previously reported age-related defects in triglyceride synthesis from [1-14C]palmitate in adipose tissue of mice. Three techniques were used: in vitro, using adipocytes isolated from epididymal fat pads of young and old mice; and in vivo, using two new methods to measure free fatty acid (FFA) esterification by adipose tissue (direct injection of labeled palmitate-albumin complexes in large or small volumes into the extracellular spaces of the epididymal or inguinal fat pads of young and old mice). When the entire fat pad was filled with tracer we no longer observed heterogeneous labeling of adipocytes in epididymal fat pads that occurred in an earlier study in which an in vivo-in vitro method has been used. Free fatty acids were converted to triacyglycerol faster by adipocytes of large cells from older animal than by those of small cells from young mice; when the cell sizes of young and old mice were approximately equal, then the rates of FFA esterification were the same in young and old adipocytes. When FFA was injected as a small bolus the fractional rates of FFA disappearance and of FFA incorporation into triacylglycerol in the different fat pads, observed during a 60-min period, were the same (about 5 min or less) regardless of the region of the fat pad studied (distal or proximal epididymal fat pad), the type of fat pad (epididymal or inguinal), or the age of the mice (12-92 weeks). Other potential applications of the direct injection technique for studying FFA metabolism and structure-function in adipose tissue in vivo are discussed. Our findings, coupled with the earlier study in which labeled FFA was added to the outside of fat pads, indicate that, in adipose tissue of old mice, there exist barriers comprising mesothelial cells, collagenous structures, and/or the outer layer of adipocytes in fat pads, that interfere in the transport of FFA to the interior adipocytes when FFA is added outside the fat pad. This age-related defect may be circumvented by injecting tracer directly into the interstitial fluid compartment.

Moderate supplementation with natural alpha-tocopherol decreases platelet aggregation and low-density lipoprotein oxidation.

Previous studies have shown that oral administration of 300 mg alpha-tocopherol/day to healthy volunteers decreases platelet function and enhances their sensitivity to the platelet inhibitor, prostaglandin E(1), when full dose-response curves to a range of agonist concentrations are made. In this study, the effects of oral doses of natural alpha-tocopherol (75, 200 and 400 IU/day) were studied in order to determine whether the same effects might be achieved with lower intakes of vitamin E and whether inhibition is related to the platelet levels of the antioxidant in platelet membranes. Twenty two subjects undertook the supplementation regime, divided into three units of 2 weeks, each cycling through each of the dosages. The results show that uptake of vitamin E by the platelets was optimal at 75 IU/day, correlating with the maximal influence on platelet aggregation and platelet responsiveness to inhibition by PGE1, increased supplemental levels exerting no greater effects.

Probucol and other antioxidants prevent the inhibition of endothelium-dependent relaxation by low density lipoproteins.

Hypercholesterolaemia and atherosclerosis impair responses to endothelium-derived nitric oxide (EDRF) in human and animal coronary arteries, a dysfunction that correlates with elevated low density lipoproteins (LDL). Previous studies show that native LDL immediately and reversibly inhibit acetylcholine-evoked EDRF responses in rabbit aortic ring precontracted with noradrenaline or serotonin whereas Cu(2+)-oxidised LDL (oxLDL) inhibit relaxations after 30 min with a potency that varies with the donor. We now show that antioxidants, probucol (10 microM) and ascorbic acid (100 microM) in vitro, prevent the inhibition by native LDL, indicating that this effect involves free radicals. As expected, the antioxidants had no influence on the inhibition by oxLDL. Superoxide dismutase appeared to have no effect on the inhibition by native or oxLDL. The oral administration of probucol to selected volunteers also prevented the inhibition of relaxation by their native LDL. These preparations showed a diminished susceptibility to oxidation and their oxLDL caused a markedly reduced and always reversible inhibition of relaxation compared to the potent and sometimes irreversible inhibition prior to administration of the drug. We conclude that antioxidants such as probucol reduce the formation of free radicals and the oxidative modification of LDL that lead to the impairment of EDRF responses and may prevent this same dysfunction in hypercholesterolaemia and atherosclerosis.

The influence of antioxidant nutrients on platelet function in healthy volunteers.

There is mounting evidence that antioxidants may help to prevent coronary heart disease and modulate some thrombotic events such a platelet adhesion. However, the effects of antioxidant supplementation on platelet function in vivo are controversial. A double-blind, randomised, placebo-controlled study was performed on 40 healthy volunteers (20-50 years) supplemented daily with vitamin E (300 mg), vitamin C (250 mg) or beta-carotene (15 mg) for 8 weeks. Platelet function was assessed by platelet aggregation induced by ADP, arachidonic acid or collagen, platelet responsiveness to the inhibitor PGE1, beta-thromboglobulin release and ATP secretion. Supplementation with vitamin E resulted in a significant increase in platelet alpha-tocopherol level (+68%) reflecting closely the increase in plasma alpha-tocopherol level (+69%). Platelet function was significantly decreased by vitamin E as revealed by the decreased platelet aggregation in response to ADP and arachidonic acid, the increased sensitivity to inhibition by PGE1, the decreased plasma beta-thromboglobulin concentration and the decreased ATP secretion. Supplementation with vitamin C did not affect platelet function significantly although a trend towards a decreased platelet aggregability and an increased sensitivity to the inhibitor PGE1 were observed. No significant changes in platelet function occurred after supplementation with beta-carotene. In conclusion, supplementation of healthy volunteers with vitamin E decreased platelet function whereas supplementation with vitamin C or beta-carotene had no significant effects.

The increased sensitivity of platelets to prostacyclin in marathon runners.

Platelet-rich plasma was obtained 24 hr after the race ended from athletes who ran in the London marathon. The platelets were only marginally less sensitive to adrenaline than were those of non-runners using conventional aggregation tests. However, the runners' platelets were much more sensitive to inhibition by prostacyclin, a prostaglandin synthesized by endothelial cells. It appeared that this effect was due to a greater activity in the platelets of the membrane-bound adenylate cyclase enzyme which generates intracellular cyclic AMP. Cyclic AMP production is known to be stimulated by prostacyclin and to cause the inhibition of platelet aggregation. The results indicate another possible protective effect of exercise against cardiovascular disease which is independent of the known changes in lipoprotein concentrations previously observed in athletes.

The effect of lanosterol on platelet aggregation in human platelets.

It has been reported that lanosterol can sensitize isolated rat platelets to agonists such as ADP and thrombin (4). The purpose of this paper was to determine whether lanosterol had similar effects on human platelets and whether this was achieved by changes in membrane fluidity. Lanosterol did increase the sensitivity of human platelets, particularly to adrenaline and ADP at concentrations as low as 5 mg.L-1 when added from solutions in ethanol. At similar concentrations cholesterol, 4-cholesten-3-one or ethynyloestradiol had either no effect or were inhibitory. Measurement of membrane fluidity with diphenylhexatriene indicated that lanosterol did not affect membrane fluidity. Incubation of platelets with [4C]-mevalonic acid gave rise to a very small incorporation into lanosterol, squalene and farnesol. Sudden activation of the platelets did not accelerate lanosterol synthesis during or after platelet aggregation. It was concluded that lanosterol could only influence platelet behaviour if it came from the plasma. However the concentration of the steroid in both platelets and plasma is ten fold less than that required to sensitise the platelets.