An unbalanced monocyte polarisation in peripheral blood and bone marrow of patients with type 2 diabetes has an impact on microangiopathy.

AIM/HYPOTHESIS: Monocytes/macrophages play important roles in adipose and vascular tissues and can be polarised as inflammatory M1 or anti-inflammatory M2. We sought to analyse monocyte polarisation status in type 2 diabetes, which is characterised by chronic inflammation. METHODS: We enrolled 60 individuals without diabetes and 53 patients with type 2 diabetes. We quantified standard monocyte subsets defined by cluster of differentiation (CD)14 and CD16. In addition, based on the phenotype of polarised macrophages in vitro, we characterised and quantified more definite M1 (CD68(+)CCR2(+)) and M2 (CX3CR1(+)CD206(+)/CD163(+)) monocytes. We also analysed bone marrow (BM) samples and the effects of granulocyte-colony stimulating factor (G-CSF) stimulation in diabetic and control individuals. RESULTS: We found no alterations in standard monocyte subsets (classical, intermediate and non-classical) when comparing groups. For validation of M1 and M2 phenotypes, we observed that M2 were enriched in non-classical monocytes and had lower TNF-α content, higher LDL scavenging and lower transendothelial migratory capacity than M1. Diabetic patients displayed an imbalanced M1/M2 ratio compared with the control group, attributable to a reduction in M2. The M1/M2 ratio was directly correlated with waist circumference and HbA1c and, among diabetic patients, M2 reduction and M1/M2 increase were associated with microangiopathy. A decrease in M2 was also found in the BM from diabetic patients, with a relative M2 excess compared with the bloodstream. BM stimulation with G-CSF mobilised M2 macrophages in diabetic but not in healthy individuals. CONCLUSIONS/INTERPRETATION: We show that type 2 diabetes markedly reduces anti-inflammatory M2 monocytes through a dysregulation in bone-marrow function. This defect may have a negative impact on microangiopathy.

Lipid and non-lipid effects of statins.

Long- and short-term trials with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have demonstrated significant reductions in cardiovascular events in patients with and without history of coronary heart disease. Statins are well-established low-density lipoprotein (LDL)-lowering agents, but their clinical benefit is believed to result from a number of lipid and non-lipid effects beyond LDL lowering, including a rise in plasma high-density lipoprotein levels. Beyond improving the lipid profile, statins have additional non-lipid effects including benefit on endothelial function, inflammatory mediators, intima-media thickening, prothombotic factors that ultimately result in plaque stabilization. These effects arise through the inhibition of several mevalonate-derived metabolites other than cholesterol itself, which are involved in the control of different cellular functions. Although statins represent the gold standard in the prevention and treatment of coronary heart disease, combination therapy with other lipid-lowering drugs, as well as novel therapeutic indications, may increase their therapeutic potential.

Phytoestrogens: pharmacological and therapeutic perspectives.

Phytoestrogens exert different estrogen receptor-dependent and -independent pharmacological actions. They share with estrogens several structural features and show greater affinity for the newly described estrogen receptor-beta. Many hope that phytoestrogens can exert the cardioprotective, anti-osteoporotic and other beneficial effects of the estrogens used in hormone replacement therapy in postmenopausal women without adversely affecting the risk of thrombosis and the incidence of breast and uterine cancers. Although there are many positive indications that phytoestrogens can fulfil this role, it remains to be proven: controlled interventional studies are lacking, and many questions remain unanswered. This review analyzes, on the basis of available experimental and epidemiological studies, the pros and cons of phytoestrogen use and describes the potential tissue targets and mechanisms of action of phytoestrogens.

Gender differences and antioxidant treatment affect aortic reactivity in short-term diabetic rats.

Diabetes is associated with gender-specific macrovascular complications arising from increased oxidant stress in the vascular wall. In this study, male and female rats were treated with two structurally unrelated drugs sharing antioxidant properties, lercanidipine and Leucoselect (both 3 mg/kg/day), for 1 week starting 1 day after streptozotocin-diabetes induction. Concentration-response curves to L-nitroarginine methylester (L-NAME), superoxide dismutase and acetylcholine in aortic rings showed significantly greater nitric oxide-mediated relaxation in female compared with male non-diabetic rats. Diabetes increased contractility to noradrenaline and L-NAME in both genders, whereas relaxation to acetylcholine and iloprost were significantly attenuated in females only. Treatment with lercanidipine and Leucoselect restored, at least in part, responses to noradrenaline, acetylcholine and iloprost without affecting those to L-NAME and sodium nitroprusside. Unexpectedly, both drugs impaired superoxide dismutase response in female tissues. In conclusion, female rat aorta is markedly exposed to short-term diabetic vascular injury, which may be prevented by antioxidant treatment.

ATP binding cassette transporter ABCA1 modulates the secretion of apolipoprotein E from human monocyte-derived macrophages.

Apolipoprotein E (apoE) produced by macrophages in the arterial wall protects against atherosclerosis, but the regulation of its secretion by these cells is poorly understood. Here we investigated the contribution of the adenosine triphosphate binding cassette transporters ABCA1 and ABC8 to the secretion of apoE from either primary human monocyte-derived macrophages (HMDM) or human THP1 macrophages. During incubations of up to 6 h, apoE secretion from both THP1 macrophages and HMDM was stimulated by 8-Br-cAMP, which activates ABCA1 expression. The putative ABCA1 inhibitor glyburide and antisense oligonucleotides directed against ABCA1 mRNA significantly reduced apoE secretion from THP1 macrophages and HMDM. Antisense oligonucleotides directed against ABC8 mRNA also inhibited apoE secretion, although this inhibition was less pronounced and consistent than in the case of ABCA1. ApoE secretion from HMDM of ABCA1-deficient patients with Tangier disease was also decreased. ApoE mRNA expression was not affected by inhibition of ABCA1 or ABC8 in normal HMDM or the lack of functional ABCA1 in HMDM from Tangier disease patients. Inhibition of ABCA1 in HMDM prevented the occurrence of anti-apoE-immunoreactive granular structures in the plasma membrane. We conclude that ABCA1 and, to a lesser extent, ABC8 both promote secretion of apoE from human macrophages.

Direct effects of estrogen on the vessel wall.

The understanding of the biological effects of estrogen on the vessel wall has improved dramatically since the discovery of estrogen receptors (ERs). Most, but not all estrogen-mediated effects in blood vessels are thought to be mediated by ERs. Two major ER subclasses have been characterized so far: the ERalpha and the more recently described ERbeta. This review will primarily focus on a new perspective that highlights ERs as essential mediators of the vascular effects of estrogen. In view of the rising research interest in this area, it can be also expected that tissue- and ER subclass-selective agonists and antagonists will be developed over the next few years, thus providing invaluable tools for pharmacological and clinical applications.

The influence of sex hormones on vascular responses in the aorta of streptozotocin-diabetic male rats.

Diabetes mellitus reduces gender-related differences in the prevalence of cardiovascular disease by fading the vascular protective effects afforded by estrogen in females. However, the impact of estrogen treatment on and the contribution of androgens to vascular function in vessels from male diabetics are largely unknown. We investigated the effects of androgen deficiency and in vivo estrogen treatment by assessing the responsiveness to a number of vasoactive agents and the formation of eicosanoid mediators in aortic rings from intact and castrated streptozotocin-diabetic rats which had been implanted with 17beta-estradiol (E2) or its vehicle for 5 days. Castration was found to attenuate contractility to noradrenaline, to enhance tone-related release of NO, as shown by curves for N-methyl-L-arginine and superoxide dismutase (SOD), and to increase endothelium-dependent relaxation to carbachol and histamine, compared with intact animals. Smooth muscle sensitivity to exogenous NO and platelet thromboxane A2 production were unchanged but prostacyclin release by aortic tissue dropped by about 40% following castration. Treatment with E2 to intact animals still attenuated contractility to noradrenaline and potentiated relaxation to SOD and histamine but affected no other parameters. In contrast, when E2 was administered to castrated animals, responses to SOD, carbachol and histamine were significantly impaired. Thus, androgen deprivation appears to improve vascular function in male diabetic rats, whereas E2 treatment exerts some beneficial effects in intact, but not in castrated animals. Our findings therefore provide new insights into the role of sex hormones in the development of diabetic vascular complications.

Diabetes influences the effect of 17beta-estradiol on mechanical responses of rat urethra and detrusor strips.

Estrogen deficiency is one of the factors involved in the stress incontinence in postmenopausal women, and estrogens have been used clinically in the treatment of urinary disorders during menopause. Sex hormones seem to be also involved in the diabetic changes of urinary bladder and urethra, because ovariectomy causes an increase in the micturition of streptozotocin-diabetic rats. In the present study diabetic and healthy female rats were used to investigate the effect of 17beta-estradiol on mechanical contractions to norepinephrine and to KCI and relaxations to ATP on isolated proximal urethral preparations as well as on contractions to ACh, ATP and KCl on detrusor smooth muscle strips. The data were compared with those obtained in OVX animals, with or without estradiol replacement. The present study showed that ovariectomy decreased the responses to ATP, NE and KCl in urethral preparations, and responses to ATP, ACh and KCl in bladder strips from both healthy and diabetic rats. Diabetes appeared to potentiate the effect of ovariectomy in both tissues. Estrogen replacement was able to recover functional responses in urethras of healthy rats. In diabetic rats, this treatment partially restored ATP-induced responses in both tissues, almost completely restored those to NE in urethra and those to ACh in bladder. This study clearly indicated that abnormalities of urethra and bladder function caused by ovariectomy can be restored by estrogen treatment also in diabetic animals, at least at an early stage of disease.

Androgen deprivation, estrogen treatment and vascular function in male rat aorta.

The beneficial effects of estrogen on arterial function in women are well established, whereas studies concerning the vascular role of androgens have produced conflicting results. In the present study, we examined the effects of androgen deprivation and of estrogen treatment on vascular responses in male rats. Vascular reactivity was studied in aortic rings excised from intact and castrated rats, which had been implanted with capsules containing either 17beta-estradiol (E2) or its vehicle for 5 days. Contractile responses to noradrenaline were potentiated by castration and by E2 treatment. Concentration-response curves for N-methyl-L-arginine and superoxide dismutase indicated that the tone-related release of NO increased in tissues from castrated, compared with intact rats, but was not affected by E2 treatment. Endothelium-dependent relaxation elicited by carbachol and histamine were not altered by castration and were attenuated by E2 in preparations from intact, but not from castrated rats. Moreover, aortic prostacyclin release dropped by about 40% after E2 treatment in tissues from both intact and castrated animals. Similarly, smooth muscle sensitivity to NO significantly decreased following castration and E2 treatment, as assessed by responses to sodium nitroprusside. Finally, no differences among groups were detected in platelet thromboxane A2 production. Thus, vascular responses in male rats were not improved by androgen deprivation alone or by E2 treatment, whose effects differed in the presence or absence of androgens. These findings provide evidence for the gender specificity of the vascular effects of estrogen and may be consistent with a beneficial role of physiologic levels of male sex hormones in arterial function.

Diabetes abolishes the vascular protective effects of estrogen in female rats.

Estrogen is known to exert a protective effect against cardiovascular disease. However, women with diabetes have three times the risk as compared with age-matched non-diabetic women. Our previous study on aortic rings of ovariectomized (OVX) female rats treated with 17-beta-estradiol (E2) demonstrated that the beneficial effect of estrogen is related to the basal release of NO from endothelial cells. In the present study, in order to understand why estrogen protection is abolished in diabetes, we tested vascular responses in OVX, streptozotocin-diabetic female rats and their non-diabetic controls receiving or not E2 replacement. Concentration-response curves to norepinephrine (NE) showed attenuation of the contractile response in E2-treated diabetic, with respect to non-diabetic preparations. This response was further impaired in diabetic, E2-deprived rats. The basal release of NO, as evaluated by concentration-related responses to N(G)-methyl-L-arginine acetate in NE-precontracted aortic rings, was found to be impaired in E2-treated diabetic rats, no further effect being induced by E2 deprivation. The endothelium-dependent relaxation produced by carbachol did not change between groups, whereas the relaxation produced by histamine was enhanced by both diabetes and E2 deprivation. However, E2 treatment counteracted the response to histamine only in preparations from non-diabetic animals. Finally, the relaxation induced by sodium nitroprusside, an endothelium-independent relaxant agent, was comparable between groups. These findings suggest that the lack of protective effects of estrogen in diabetes may be mainly ascribed to the failure of estrogen to reverse the impaired basal release of NO and the abnormal relaxation to histamine, which are observed in the aorta of diabetic rats.

Differential effects of lovastatin on the trafficking of endogenous and lipoprotein-derived cholesterol in human monocyte-derived macrophages.

Lovastatin has been shown to reduce cholesterol esterification in cholesterol-loaded human macrophages. Surprisingly, in nonloaded macrophages, lovastatin produces the opposite effect, lowering free cholesterol and increasing cholesteryl ester levels, as measured by high-performance liquid chromatography. In cholesterol-loaded cells, lovastatin reduced the cholesteryl esters of unsaturated but not those of saturated fatty acids. In nonloaded cells, by contrast, the cholesteryl esters of unsaturated fatty acids tended to increase after lovastatin treatment. Total (free plus esterified) cellular cholesterol content in nonloaded cells fell by 18% with 12-micromol/L lovastatin treatment but did not change in cholesterol-loaded cells. Lovastatin had no effect on the binding or uptake of acetylated low density lipoprotein, acyl coenzyme A:cholesterol acyltransferase (ACAT) activity, the secretion of [3H]cholesterol into the medium, or lysosomal hydrolysis of cholesteryl esters. Apolipoprotein (apo) E mRNA levels increased but apoE secretion into the medium decreased with lovastatin treatment in both cholesterol-loaded and nonloaded cells. Cholesterol of exogenous origin has been shown to pass via the cell membrane before its esterification by ACAT. We postulate that this is not the case for endogenous cholesterol, which may have direct access to ACAT. Our findings therefore suggest that lovastatin hinders the delivery of intracellular cholesterol to the plasma membrane, resulting in increased free cholesterol and lower levels of cholesteryl ester in cholesterol-loaded cells. In nonloaded cells, virtually all cholesterol is of endogenous origin and is normally translocated to the cell membrane. Lovastatin prevents this process, thus shunting newly synthesized cholesterol toward esterification and leading to an increase in the concentration of cholesteryl esters, even in the face of a drop in total and free cholesterol levels. Intracellular apoE may play a role in this process.

Platelet activation supports the development of venous thrombosis in hyperlipidemic rats.

This investigation sought to determine how different components of the hemostatic system affect the development of venous thrombosis in rats displaying hyperlipidemia, either on a genetic basis or secondary to metabolic disorders. On employing an experimental model of collagen-triggered venous thrombosis, both spontaneously hyperlipidemic (Yoshida strain) and streptozotocin-induced diabetic rats generated about 2.3-fold greater thrombi than normolipidemic controls. This was associated with significant platelet activation, as revealed by increased levels of serum thromboxane B2 in diabetics (1.5-fold) as well as in Yoshida (8-fold) rats, in comparison with controls. In contrast, ex vivo total fibrinolytic activity, as measured by euglobulin lysis time, did not differ between normo- and hyperlipidemic or diabetic animals. Plasminogen activator inhibitor activity was lower in both Yoshida and diabetic rats than in controls. However, tissue-type plasminogen activator activity was differently affected by the genetic or the diabetes-related hyperlipidemia, showing significantly lower values in Yoshida (-26%), but significantly higher values in diabetic rats (+29%) than in normolipidemic controls. We conclude that platelet activation, rather than consistent modifications of the fibrinolytic system, is likely to influence the enhanced thrombus development associated with primary or secondary forms of hyperlipidemia.

Phenotype-dependent differences in apolipoprotein E metabolism and in cholesterol homeostasis in human monocyte-derived macrophages.

In this study, we investigated the impact of the common apoE polymorphism on apoE metabolism and cholesterol homeostasis in monocyte-derived macrophages isolated from E2/2, E3/3, and E4/4 subjects. Unloaded cells of all genotypes contained similar amounts of free cholesterol, cholesteryl ester, and apoE mRNA. E3/3 cells secreted 77 and 30% more apoE than E2/2 or E4/4 cells, respectively. Pulse-chase studies confirmed that the apoE secretion rate was greatest in E3/3 and least in E2/2 cells and showed that a portion of apoE2, but not apoE3 or apoE4, was degraded intracellularly. Surface binding of apoE was greatest in E4/4 cells, as revealed by heparinase treatment. On cholesterol loading with acetylated LDL, apoE mRNA levels and protein secretion rose most in E4/4 and least in E2/2 cells. Cholesterol and cholesteryl ester content, however, rose most in E2/2 and least in E3/3 cells. Incubations with 3H-cholesterol-labeled acetylated LDL revealed that E2/2 cells were most efficient at secreting cholesterol. The greatest reuptake of 3H-cholesterol-rich particles was from E4/4 macrophage- conditioned media. Thus, E2/2 macrophages, despite a low apoE secretion rate, are protected from cholesterol storage by apoE-mediated cholesterol efflux. In E3/3 macrophages, cholesterol accumulation is lessened by a high basal apoE secretion rate. E4/4 macrophages secrete the most apoE but lack effective net cholesterol efflux due to enhanced surface binding and reuptake of cholesterol-rich particles.

Downregulation of the selectin ligand-producing fucosyltransferases Fuc-TIV and Fuc-TVII during foam cell formation in monocyte-derived macrophages.

Identification of genes expressed during foam cell formation is important for understanding the molecular basis of atherosclerosis. We used polymerase chain reaction (PCR)-based differential display to isolate differentially expressed cDNA species in foam cells induced by incubation of human monocyte-derived macrophages in the presence of acetylated or oxidized LDL. This led to identification of a 306-bp cDNA with 100% homology to type IV fucosyltransferase (Fuc-TIV), which was downregulated by factors of 20 and 3 in acetylated LDL- and oxidized LDL-loaded macrophages, respectively. This enzyme is sufficient for the expression of Lewis X and sialyl Lewis X, carbohydrate adhesion molecules that bind to receptors of the selectin family. Expression of a second fucosyltransferase (Fuc-TVII) that synthesizes sialyl Lewis X but not Lewis X was shown by quantitative reverse transcription-PCR to also be reduced, by 40% and 20% in acetylated LDL- and oxidized LDL-loaded macrophages, respectively. alpha-(1,3)-Fucosyltransferase enzyme activity was reduced in lysates from both acetylated LDL- and oxidized LDL-loaded cells. Analysis by flow cytometry showed reduced expression of the CD15 (corresponding to Lewis X) and CD15s (sialyl Lewis X) antigens on the surface of cells loaded with either acetylated or oxidized LDL. Transformation of macrophages into foam cells results in reduced expression of selectin-binding ligands on the surface of such cells.

An improved method for quantification of cholesterol and cholesteryl esters in human monocyte-derived macrophages by high performance liquid chromatography with identification of unassigned cholesteryl ester species by means of secondary ion mass spectrometry.

The measurement of cholesteryl esters in human monocyte-derived macrophages using previously described high performance liquid chromatography methods is hampered by the presence in these cells of large amounts of triglycerides. We present a simple reversed phase high performance liquid chromatography protocol for quantification of cholesterol and cholesteryl esters in human monocyte/macrophages or other triglyceride-rich cells. Our method requires only lipid extraction and hydrolysis of triglycerides using a solution of ethanolic potassium hydroxide and is of sufficient sensitivity to allow measurement in 10(5) cells. Use of this protocol led to the isolation of eight previously unassigned cholesteryl ester peaks comprising 16% of the total cholesteryl ester content of human monocyte-derived macrophages. Using time-of-light secondary ion mass spectrometry and synthesized authentic standards, seven of these peaks were found to comprise cholesterol esterified with polyunsaturated n-3 (omega 3) (cholesteryl eicosapentaenoate, docosatrienoate, docosapentaenoate, and docosahexaenoate) and n-6 (omega 6) (cholesteryl docosatetraenoate, eicosadienoate, and eicosatrienoate) fatty acids. The remaining peak was shown to be the cholesteryl ester of n-7 (omega 7) palmitoleic acid by comparison with a commercially available standard. The identification of all the cholesteryl esters in cholesterol-loaded human monocyte-derived macrophages will assist future studies of lipid metabolism in these cells.

Differential effects of low- and high-dose estrogen treatments on vascular responses in female rats.

In an attempt to study the mechanisms by which estrogens affect vascular responses, we utilized aortic preparations from intact and ovariectomized female rats receiving low- and high-dose subcutaneous estrogen treatments. Oil-treated, as well as male rats, served as controls. In ovariectomized females, low-dose 17-beta-estradiol injections (5 microg/kg daily for two days) affected the basal release of nitric oxide, as evaluated by concentration-related curves to superoxide dismutase and N(G)-Methyl-L-arginine acetate, which was found to be greater in 17-beta-estradiol-treated females compared to oil-treated females or males. Conversely, the nitric oxide-related vascular relaxation evoked by acetylcholine and sodium nitroprusside was unchanged. Prostacyclin production was also evaluated. Aortic rings from ovariectomized 17-beta-estradiol-treated females released significantly more prostacyclin than those from oil-treated females. These results point out a possible role for nitric oxide and prostacyclin in the vascular protection brought about by physiological levels of estrogens. When intact females were treated with high doses of ethynilestradiol (100 microg/Kg daily for one month), a component of contraceptive pills, either the basal release of nitric oxide, or acetylcholine-induced relaxation underwent a significant decrease. Likewise, the relaxant responses to sodium nitroprusside were impaired in the aortic rings obtained from ethynilestradiol-treated animals when compared to controls. Similarly, the amount of prostacyclin released from aortic tissues obtained from ethynilestradiol-treated animals was significantly reduced. These results may provide a possible explanation for the higher incidence of cardiovascular disease in women who take contraceptive preparations containing high doses of estrogens.

Novel lipid-lowering properties of Vaccinium myrtillus L. leaves, a traditional antidiabetic treatment, in several models of rat dyslipidaemia: a comparison with ciprofibrate.

Vaccinium myrtillus L. (blueberry) leaf infusions are traditionally used as a folk medicine treatment of diabetes. To further define this therapeutical action, a dried hydroalcoholic extract of the leaf was administered orally to streptozotocin-diabetic rats for 4 days. Plasma glucose levels were consistently found to drop by about 26% at two different stages of diabetes. Unexpectedly, plasma triglyceride (TG) were also decreased by 39% following treatment. Subsequent to the latter observation, possible lipid-lowering properties of the extract were investigated on other models of hyperlipidaemia and ciprofibrate, a well-established hypolipidaemic drug, was used as a reference compound. Both drug reduced TG levels of rats on hyperlipidaemic diet in a dose-dependent fashion. When administered at single doses over the same experimental period, blueberry and ciprofibrate were effective in lowering TG concentrations in ethanol-treated normolipidaemic animals and in genetically hyperlipidaemic Yoshida rats. Unlike ciprofibrate, however, blueberry failed to prevent the rise in plasma TG elicited by fructose and did not affect free fatty acid levels in any of the above experimental conditions. In rats treated with Triton WR-1339, blueberry feeding induced an hypolipidaemic activity one hour after injection but proved to be ineffective at later time points, thus suggesting that its hypolipidaemic action may reflect improved TG-rich lipoprotein catabolism. In addition, ciprofibrate and the extract were tested for antithrombotic activity using a collagen-triggered model of venous thrombosis in diabetic and Yoshida rats. Only ciprofibrate, however, significantly reduced thrombus formation in diabetics, possibly because of its effects on free fatty acid metabolism, whereas no effect was observed in Yoshida rats. In conclusion, the present findings indicate that active consituent(s) of Vaccinium myrtillus L. leaves may prove potentially useful for treatment of dyslipidaemiae associated with impaired TG-rich lipoprotein clearance.

Antithrombotic activity of nicardipine in spontaneously hypertensive rats.

Calcium metabolism appears to be altered in human and experimental hypertension, which represents an important risk factor for thrombotic events. We investigated the possible effect of the calcium antagonist nicardipine on a model of experimental venous thrombosis in spontaneously hypertensive rats (SHR). Thrombus formation was highly enhanced in SHR with respect to normotensive Wistar Kyoto rats (WKY). Nicardipine, when administered orally (10 mg kg-1) at a single dose or once a day for three days, completely counteracted the increase in thrombus size caused by hypertension. Furthermore, a significant rise in prostacyclin production from aortic tissue [19.2 +/- 1.5 vs 13.2 +/- 2.4 ng (mg dry tissue)-1], associated with a fall in thromboxane A2 release from activated platelets (328.3 +/- 74.6 vs 705.0 +/- 88.1 ng ml-1), was observed in nicardipine-treated SHR. Plasma triglyceride and free fatty acid levels were also lowered by drug administration. Our results suggest that the actions of nicardipine on calcium metabolism result in antithrombotic effect through an increased availability of vasodilating eicosanoids in vessel walls and through a reduced amount of prothrombotic agents (thromboxane, free fatty acids).