TNF-α inhibits trophoblast integration into endothelial cellular networks.

Preeclampsia has been linked to shallow trophoblast invasion and failure of uterine spiral artery transformation. Interaction between trophoblast cells and maternal uterine endothelium is critically important for this remodelling. The aim of our study was to investigate the effect of TNF-α on the interactions of trophoblast-derived JEG-3 cells into capillary-like cellular networks. We have employed an in vitro trophoblast-endothelial cell co-culture model to quantify trophoblast integration into endothelial cellular networks and to investigate the effects of TNF-α. Controlled co-cultures were also treated with anti-TNF-α antibody (5 µg/ml) to specifically block the effect of TNF-α. The invasion was evaluated by performing quantitative PCR (Q-PCR) to analyse gene expression of matrix metalloproteinases-2 (MMP-2), MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1, integrins (α(1)ß(1) and α(6)ß(4)), plasminogen activator inhibitor (PAI)-1, E-cadherin and VE-cadherin. JEG-3 cell integration into endothelial networks was significantly inhibited by exogenous TNF-α. The inhibition was observed in the range of 0.2-5 ng/ml, to a maximum 56% inhibition at the highest concentration. This inhibition was reversed by anti-TNF-α antibody. Q-PCR analysis showed that mRNA expression of integrins α(1)ß(1) and MMP-2 was significantly decreased. VE-cadherin mRNA expression was significantly up-regulated (32-80%, p < 0.01) but its protein concentration in the cell lysates was significantly reduced (20-45%, p < 0.01). PAI-1, MMP-9, TIMP-1 and E-cadherin were not affected. In conclusion, TNF-α can inhibit trophoblast-like cells (JEG-3) integration into maternal endothelial cellular networks, and this process involves the inhibition of MMP-2 and a failure of integrins switch from α(6)ß(4) to α(1)ß(1.) These molecular correlations reflect the changes identified in human preeclampsia.

The effects of obesity and non-pharmacological weight loss on vascular and ventricular function and structure.

AIMS: The mechanisms by which obesity confers increased cardiovascular risk and the effects of moderate weight loss on cardiovascular health are incompletely understood. We sought to characterize the preclinical changes in cardiac and vascular health that accompany obesity and the influence of lifestyle modification on these parameters. METHODS: Preclinical markers of vasculopathy in resistance vessels and conduit arteries and left ventricular structure and function were assessed in 39 obese subjects (BMI > 30 kg/m(2)) and 11 healthy weight controls. The influence of serum on cellular adhesion molecule (CAM) expression on human endothelial cells was studied ex vivo in a subgroup of 13 obese and nine healthy weight subjects. These analyses were repeated in all 17 of the obese subjects who complied with 4-9 months of lifestyle modification treatment (six with weight loss >5% and 11 with weight loss <5%). RESULTS: Compared with healthy weight controls, obese subjects had decreased peak hyperaemic forearm blood flow (p = 0.015), increased carotid intima-media thickness (p = 0.009), increased left ventricular wall thickness and volume and evidence of systolic and diastolic dysfunction as assessed using tissue Doppler imaging (S', p = 0.09; E'/A', p = 0.02), and serum from obese subjects increased the intercellular CAM-1 expression on human endothelial cells (p = 0.009). However, arterial endothelial function assessed by flow-mediated dilatation was not altered (p = 0.99). Lifestyle modification treatment resulted in potentially beneficial changes in fibrinogen (p = 0.003), HDL cholesterol (p = 0.05) and soluble vascular CAM-1 (p = 0.06). In subjects with weight loss greater than 5% of body weight, there was also a decrease in low-level inflammation (high-sensitivity C-reactive protein, p = 0.05), lipid peroxidation (thiobarbituric acid-reactive substances, p = 0.05) and triglycerides (p = 0.07). CONCLUSIONS: Obesity is associated with widespread alterations in cardiac and vascular structure and function. Moderate short-term weight loss by lifestyle modification results in some beneficial changes in serum profile; however, these are not accompanied by significant alterations to either cardiac or vascular structure and function.

Adenosine prevents neutrophil adhesion to human endothelial cells after hypoxia/reoxygenation.

BACKGROUND: Neutrophil adhesion to vascular endothelium has been implicated in the pathogenesis of myocardial injury after ischaemia/reperfusion (IR) and the "no-reflow" phenomenon. Adenosine and sodium-nitroprusside (SNP) have been used clinically to ameliorate this injury. We set out to establish a human cellular model for the study of IR and to evaluate the effects of adenosine and SNP on neutrophil adhesion in vitro. METHODS: Cultured human umbilical vein endothelial cells (HUVEC) were exposed to hypoxia (5% CO2, 95% N2) or normoxia (room air, 5% CO2) for 2 h, followed by reoxygenation for 30 min (IR condition). Human neutrophils were then added together with adenosine (50 microM), SNP (10 microM) or no additive (control). After incubation for 1 h, neutrophil adhesion to endothelial cells was quantified via automated cell counts. The experiment was repeated with the adenosine treatment alone, with and without the addition of the adenosine A2A receptor blocker ZM-241385. RESULTS: Compared with baseline neutrophil adhesion after normoxia, hypoxia followed by reoxygenation increased adhesion to 189+/-43% (p=0.01), but this effect was prevented by the addition of adenosine (109+/-17%, p=NS compared to control conditions). SNP did not affect the increased adhesion caused by hypoxia (166+/-25%, p=NS). The addition of ZM-241385 did not inhibit the effect of adenosine on neutrophil adhesion after hypoxia/reoxygenation. CONCLUSIONS: Exposure of human endothelial cells to hypoxia/reoxygenation causes increased neutrophil adhesion. This effect is prevented by adenosine, but not mediated by the A2A receptor. SNP does not prevent neutrophil adhesion after IR in vitro.

Reperfusion injury in the human forearm is mild and not attenuated by short-term ischaemic preconditioning.

1. Ischaemia-reperfusion (IR) injury is an important contributor to tissue damage and has been shown to be attenuated by preconditioning (PC) in some animal models. A recent report has suggested that the forearm can be used for the study of this phenomenon in humans. We aimed to reproduce and further characterize this model. 2. Healthy young adult volunteers (mean (+/-SEM) age 32+/-6 years) were studied on two occasions. During one visit, IR alone was induced by 10 min of upper arm cuff occlusion, whereas on another occasion a PC stimulus (three 3 min cuff inflations) preceded IR. Endothelial function in the ischaemic arm was assessed by measuring arterial flow-mediated dilatation (FMD) and by calculation of forearm blood flow at baseline and 15 and 60 min after IR. Systemic venous blood was sampled from the non-ischaemic arm at baseline, after PC and at 2, 15 and 30 min after IR to assess neutrophil/leucocyte (CD11b) and platelet (bound glycoprotein IIb/IIIa and fibrinogen) activation, as well as numbers of platelet-leucocyte complexes, which were determined by flow cytometry. Because of a lack of measurable effects, the IR experiment was repeated with 20 min ischaemia in six subjects. 3. Five females and eight males completed the study. Flow-mediated dilatation was significantly impaired 30 min after IR (4.1 vs 6.2% at baseline; P<0.05);however, this was not significantly attenuated by ischaemic PC (FMD reduction at 30 min compared with baseline was 2.1+/-0.5% with IR alone and 2.6+/-1.4% with IR after PC; NS). No significant effect was seen on the number of platelet-leucocyte aggregates or on white cell or platelet activation after IR alone or after IR with PC (P>0.6 for all comparisons). Similar results were obtained in six subjects studied subjected to 20 min ischaemia. 4. In conclusion, in healthy young adults, brief periods of skeletal muscle ischaemia lead to arterial endothelial dysfunction, but no significant platelet or white cell activation. Preconditioning does not attenuate this effect on the endothelium. Further experiments with longer ischaemia times and varying PC stimuli may be necessary to produce measurable effects; however, this may prove difficult in conscious human subjects.

Endothelin-1 plus oxidized low-density lipoprotein, but neither alone, increase human monocyte adhesion to endothelial cells.

Endothelin-1 is a potent vasoconstrictor and mitogenic peptide that is implicated in the atherosclerosis of apolipoprotein E-deficient mice and may promote atherogenesis in humans. We hypothesized that endothelin-1 might promote the adhesion of monocytes to endothelial cells, a key early event in atherosclerosis. We investigated the adhesion of primary human monocytes (isolated by elutriation) to human umbilical vein endothelial cell cultures after incubation with endothelin-1 (0.1 and 0.01 nM; approximately physiological concentrations), copper-oxidized low-density lipoprotein (LDL) (0.1 mg/ml) and a combination of the two. After a 4 h incubation with 0.1 or 0.01 nM endothelin-1 combined with oxidized LDL, adhesion was increased to 120+/-4% (P<0.001 compared with control) and 118+/-4% (P<0.002) respectively, whereas neither substance alone increased adhesion (92-104% of control values; not significant). Neither endothelin receptor A blockade nor co-incubation with anti-fibronectin antibody inhibited the pro-adhesive effects of endothelin-1 plus oxidized LDL (115+/-7% and 115+/-3% of control compared with 120+/-4% respectively; not significant). Endothelial cell expression of intercellular adhesion molecule-1, vascular adhesion molecule-1 and E-selectin were unchanged throughout the experiment. Therefore physiological concentrations of endothelin-1 and oxidized LDL may act synergistically to increase the adhesion of human monocytes to endothelial cells, contributing in part to the observed pro-atherogenic effects of endothelin-1.

Androgen receptor expression is greater in macrophages from male than from female donors. A sex difference with implications for atherogenesis.

BACKGROUND: Male sex is an independent risk factor for the extent and severity of atherosclerosis. The influence of androgens on foam cell formation, a key event in atherogenesis, has not yet been investigated. METHODS AND RESULTS: Primary human monocytes were allowed to differentiate into macrophages. RNA was then extracted from healthy male-donor (n=8) and premenopausal female-donor (n=8) macrophages, and message for the androgen receptor (AR) was examined by RT-PCR. There was a significantly higher level of AR mRNA in macrophages isolated from men than in those from women (0.64+/-0.06 versus 0.15+/-0.02 amol/microgram total RNA; P<0.001). AR mRNA levels were similar in macrophages from postmenopausal and premenopausal women (P=0.16). The functional consequence of this sex difference was then explored. Lipid-loading studies were performed on male (n=9) macrophages treated with the androgen dihydrotestosterone (DHT) and/or the AR antagonist hydroxyflutamide. These showed that DHT caused a dose-dependent and receptor-mediated increase in macrophage cholesteryl ester content (109+/-10%, 117+/-3%, and 120+/-4% for 4, 40, and 400 nmol/L DHT, respectively, as a percentage of control, P=0.002; 95+/-8% for DHT with hydroxyflutamide, P=0.58 versus controls). By contrast, there was no significant effect of androgen on lipid loading in female-donor macrophages (P>0.2 versus controls). CONCLUSIONS: Sex differences in androgen-mediated macrophage lipid loading may contribute to the greater prevalence and severity of atherosclerosis in men.

Estrogen and progesterone reduce lipid accumulation in human monocyte-derived macrophages: a sex-specific effect.

BACKGROUND: Males have an earlier onset and greater prevalence of clinical atherosclerosis than age-matched females, which is consistent with an atheroprotective effect of the female sex steroids, estrogen and progesterone. We therefore examined the effects of estrogen and progesterone on human foam cell formation, a key early event in atherogenesis. METHODS AND RESULTS: Monocytes from healthy female and male donors were obtained from white cell concentrates and allowed to differentiate into macrophages over 10 days. These human monocyte-derived macrophages (MDMs) were exposed to either control (0.1% vol/vol ethanol) or estrogen or progesterone treatment on days 3 through 10. Lipid loading was achieved on days 8 through 10 by incubation with acetylated LDL. Lipid from the MDMs was then extracted for analysis of cholesteryl ester (CE) content. 17beta-Estradiol at both physiological (2 nmol/L) and supraphysiological (20 and 200 nmol/L) concentrations produced a significant reduction in macrophage CE content (88+/-3%, 88+/-2%, and 85+/-4%, respectively; P<0.02 compared with control). Physiological and supraphysiological levels of progesterone (2, 10, and 200 nmol/L) produced an even more dramatic reduction in CE content (74+/-9%, 56+/-10%, and 65+/-8%, respectively; P<0.002 compared with control). This effect could be abrogated by coincubation with the progesterone receptor antagonist RU486. Neither estrogen nor progesterone produced a reduction in lipid loading in male-donor-derived MDMs. Detailed lipid trafficking studies demonstrated that both estrogen and progesterone altered macrophage uptake and/or processing of modified LDL. CONCLUSIONS: Physiological levels of estrogen and progesterone are associated with a female-sex-specific reduction in human macrophage lipid loading, which is consistent with an atheroprotective effect.