Chronic homocysteine exposure upregulates endothelial adhesion molecules and mediates leukocyte: endothelial cell interactions under flow conditions.

AIMS: Homocysteine upregulates expression of adhesion molecules on endothelial cells which recruits leukocytes and initiates atherosclerosis. Endothelial cells in hyperhomocysteinemic patients are continuously exposed to high levels of homocysteine. This study exposed adult endothelial cells and endothelial cells from immune naïve foetal tissue to homocysteine chronically and studied effects on cellular adhesion molecule expression under static and flow conditions. METHODS: Human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) were cultured in medium containing 1 mM dl-homocysteine or l-cysteine for 5-9 days. Proliferation was assessed. Cells were subjected to flowing neutrophils and numbers of tethered, rolled fixed and transmigrated neutrophils on endothelial cells were counted and compared to controls. Immunofluorescence staining with antibodies against Intercellular adhesion molecule-1 (ICAM-1), E-selectin and P-selectin were used to quantify expression. RESULTS: Chronic treatment with 1 mM homocysteine inhibited proliferation of HUVEC and HSVEC. Homocysteine treated cells showed significantly increased expression of ICAM-1, E-selectin and to a lesser extent P-selectin. In both cell types, homocysteine significantly increased interactions between neutrophils and endothelial cells under flow conditions (p < 0.05) while cysteine had no effect. CONCLUSION: Endothelial cells from adult and immune naïve foetal tissue showed similar responses to chronic treatment with homocysteine.

Regulation of the synthesis of lipoprotein lipase in adipose tissue by dexamethasone.

The incorporation of [3H]leucine into lipoprotein lipase during incubations of rat epididymal fat-bodies in vitro was significantly stimulated by dexamethasone, whereas total protein synthesis was unaffected. The stimulation by dexamethasone required the presence of insulin. The results suggest that dexamethasone, in the presence of insulin, may specifically induce lipoprotein lipase synthesis in adipose tissue.

Degradation of lipoprotein lipase in rat adipose tissue.

Pulse-chase studies have shown that the lipoprotein lipase protein of rat epididymal fat bodies is apparently rapidly degraded (43% in 3 h) during incubation at 37 degrees C under conditions where little degradation of the total adipose tissue protein is taking place.

Neutrophils and the endothelium in post-ischemic alterations in skeletal muscle blood flow.

Thromboxane A2, leukotriene B4, and NE are all released from ischemic muscle during reperfusion. Thromboxane A2 levels peaked at 10 min and this potent vasoconstrictor may be responsible for low reflow. Neutrophil elastase levels did not rise until 240 min of reperfusion, following that of leukotriene B4 at 120 min, indicating that neutrophil recruitment and activation is a relatively late event following revascularization. In conclusion, it would appear that endothelial factors have a significant role in the vasomotor changes that account for low reflow. Equally altered neutrophil function almost certainly contributes to the final development of reperfusion injury.

Profile of eicosanoids produced by human saphenous vein endothelial cells and the effect of dietary fatty acids.

Human saphenous vein endothelial cells (HSVECs) derived from primary cultures of adult human veins constitute an excellent in vitro model for studying human endothelial metabolism. In this study we report the (14)C-labelled prostanoid profile of HSVECs under resting and stimulated conditions and the effect of the n-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid on them. Results indicate that HSVECs while under resting conditions produce mainly prostaglandin F(2alpha)(PGF(2alpha)). After stimulation with calcium ionophore A23187, the cells were found to synthesise PGI(2), PGE(2)and PGF(2alpha) as major products and thromboxane B(2)and PGD(2)as minor products. Production of (14)C-labelled hydroxyeicosatetraenoic acids was not detected. Eicosapentaenoic acid was found to inhibit basal and stimulated prostanoid production whereas docosahexaenoic acid inhibited basal but strongly increased stimulated prostanoid production. These results may offer the basis for further studies aiming to investigate targets for pharmacological intervention in inflammatory conditions.

The effect of 5,6-dimethylxanthenone-4-acetic acid on tumour necrosis factor production by human immune cells.

5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA) is a potent anti-tumour agent which is undergoing early clinical evaluation. It was developed as an improved analogue of flavone acetic acid (FAA) which failed in clinical trial although it had impressive anti-tumour activity in mice. It has been postulated that one possible reason for the clinical failure of FAA was its inability to induce cytokines, especially tumour necrosis factor (TNF) in humans whereas this was seen as an important component of its mechanism of action in mice. Previous studies have demonstrated that 5,6-MeXAA induces mRNA for TNF in the human HL-60 cell line although the protein was not detected. This study has demonstrated mRNA for TNF alpha by RT-PCR in human and murine immune cells incubated with either 5,6-MeXAA, FAA or in culture medium alone. Using a bioassay technique 5,6-MeXAA and FAA were shown to induce TNF production in vitro by murine macrophages but TNF was not detected when human or murine peripheral blood mononuclear cells were stimulated with these agents. A small but significant production of TNF was seen in the HL-60 cell line after 5,6-MeXAA treatment suggesting 5,6-MeXAA can directly stimulate human cells to produce TNF albeit at low levels.

Morphological and immunological observations on the effects of hexadecylphosphocholine (HPC) in nude mice bearing MT-1 breast cancer xenografts.

Hexadecylphosphocholine (HPC) is active against experimental and clinical breast cancer in vivo but the precise mechanisms involved are not fully understood. Studies in this and other laboratories have demonstrated that HPC has very significant activity against MT-1 human mammary xenografts in nude mice and preliminary studies have indicated immunomodulatory effects. The aims of this study were to investigate the influence of HPC on immune cell populations in nude mice bearing MT-1 xenografts and the effects of HPC on MT-1 xenograft vascularisation. After treatment, significant increases in the number of cells were observed in the spleen paracortex and cortex and the follicles were more developed compared with lymph nodes from untreated mice. Immune cell populations in spleens from untreated MT-1 tumour bearing nude mice were compared with those from HPC treated mice. After HPC treatment, increases in the macrophage and T cell populations as well as T cell subsets were observed in spleens. Histological examination of treated tumours showed the presence of giant cells and large lytic areas. Immunostaining revealed increases in endothelial cells (p < 0.005) associated with massive infiltration of M phi, T cells and B cells. The results suggest that HPC affects the development of immune cells in the secondary immune tissues of nude mice. An increase in tumour vasculature appears to be accompanied by infiltration of immune cells into the tumour.

Effect of homocysteine on cytokine production by human endothelial cells and monocytes.

BACKGROUND: Hyperhomocysteinaemia is an independent risk factor in the development of cardiovascular disease. Although homocysteine has been shown to affect endothelial cell function, the mechanisms by which it induces disease states are still poorly understood. Here, we report the ability of homocysteine to influence inflammatory cytokine/chemokine production by human saphenous vein endothelial cells, peripheral blood monocytes and monocyte-derived macrophages. METHODS: Human saphenous vein endothelial cells, peripheral blood monocytes and monocyte-derived macrophages were treated with homocysteine (0.1-5 mmol/L) for 4 and/or 24h. Tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and IL-8 production was measured in the cell culture media using commercially available enzyme-linked immunosorbent assays. RESULTS: Interleukin-6 production by human saphenous vein endothelial cells was significantly stimulated following a 24-h treatment with homocysteine, whilst IL-8 concentrations were inhibited after both 4- and 24-h treatments. Homocysteine was also found to stimulate IL-1beta production by human peripheral blood monocytes and TNF-alpha production by monocyte-derived macrophages. CONCLUSIONS: Overall, results from this study suggest that homocysteine alters the profile of cytokine/chemokine production by endothelial cells and macrophages. This altered profile may be important in the inflammatory events that initiate or enhance the development of atherosclerotic lesions.

Purification and characterization of rat adipose tissue lipoprotein lipase.

Lipoprotein lipase (EC 3.1.1.34) extracted from adipose tissue of glucose-fed rats with 5 mM-sodium barbital, pH 7.5, containing 20% (v/v) glycerol and 0.1% (v/v) Triton X-100, was partially purified by affinity chromatography on heparin linked to Sepharose 4B. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the partially purified enzyme preparation revealed the presence of two major Coomassie-staining bands (mol.wts. 62 000 and 56 000) as well as a number of minor bands. Treatment of partially purified enzyme with [1,3-3H]di-isopropyl fluorophosphate resulted in the incorporation of radiolabel into the band of mol.wt. 56 000, but not into the band of mol.wt. 62 000. Both the amount of the 56 000-mol.wt. polypeptide and the incorporation of [1,3-3H]di-isopropyl fluorophosphate into this band were greatly reduced in the enzyme preparations isolated from adipose tissue of 48 h-starved rats. whereas the amount of the 62 000-mol.wt. polypeptide was unaffected by starvation. Purification of lipoprotein lipase from adipose tissue of glucose-fed rats was also carried out using affinity chromatography on Sepharose 4B linked to heparin with low affinity for antithrombin-III. This procedure resulted in the presence of a single band of mol.wt. 56 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These results suggest that the polypeptide of mol.wt. 56 000 corresponds to the subunit of lipoprotein lipase, whereas the 62 000-mol.wt. polypeptide probably represents antithrombin-III.

Effects of glucose and insulin on the activation of lipoprotin lipase and on protein-synthesis in rat adipose tissue.

Glucose, and certain sugars that can readily be converted to glucose 6-phosphate, bring about an activation of adipose-tissue lipoprotein lipase when epididymal fat-bodies from starved rats are incubated in the presence of cycloheximide. Other substrates do not support the activation. If the tissue is preincubated in the presence of cycloheximide for longer than 2h, the ability of added glucose to activate the enzyme is lost. On the other hand, the addition of glucose still brings about an increase in lipoprotein lipase activity after preincubation in the absence of cycloheximide for as long as 4h. The magnitude of the increase in enzyme activity brought about by the addition of glucose is increased when protein synthesis is stimulated during the preincubation period by insulin. The results are interpreted in terms of the existence in adipose tissue of a proenzyme pool of lipoprotein lipase that is normally maintained by protein synthesis and that is converted to complete enzyme of higher specific activity by a process that specifically requires glucose.

Role of neutrophil depletion and elastase inhibition in modifying skeletal muscle reperfusion injury.

This study investigated the effect of neutrophil depletion and neutrophil elastase inhibition on the severity of skeletal muscle reperfusion injury. In a rodent model, indices (experimental/normal limb) of gastrocnemius muscle viability (histochemical staining), oedema (wet:dry weight ratio) and myeloperoxidase content (neutrophil recruitment) were assessed in normal (no ischaemia), ischaemic (6-h unilateral hindlimb ischaemia), control (6-h ischaemia and 4-h reperfusion), neutrophil-depleted rats (given antineutrophil serum) and rats receiving the neutrophil elastase inhibitor Elafin. Neutrophil recruitment muscle infarction and oedema did not occur in normal limbs, or in those subjected to ischaemia without reperfusion. In contrast increased muscle myeloperoxidase levels (P < 0.001), muscle infarction (P < 0.01) and oedema (P < 0.001) all occurred in the reperfused limbs of control animals compared with those of normal and ischaemic rats. Antineutrophil serum and Elafin both reduced neutrophil recruitment during reperfusion (P < 0.001 and P < 0.01 respectively) and muscle viability was preserved. Reperfusion oedema still occurred however, suggesting that altered endothelial permeability is mediated by factors other than neutrophils.

Role of neutrophil-endothelial adhesion in skeletal muscle reperfusion injury.

During postischaemic revascularization neutrophil-endothelial adhesion in the skeletal muscle microcirculation, promoted by the neutrophil adhesion molecule Mac-1, may impair muscle blood flow and release oxygen free radicals and proteolytic enzymes which causes further tissue injury. This study has assessed the effect of an anti-Mac-1 monoclonal antibody on the severity of skeletal muscle injury in a rat model of 6-h hindlimb ischaemia and 4-h reperfusion. In control animals a sustained impairment of muscle perfusion was associated with neutrophil sequestration, muscle infarction and muscle oedema (P < 0.001 versus normal rats). In contrast, intravenous administration of anti-Mac-1 monoclonal antibody before revascularization prevented neutrophil recruitment, reduced muscle necrosis and improved postischaemic muscle perfusion at 120 and 240 min (not significantly different from normal), thus confirming that neutrophils are important cellular mediators of skeletal muscle reperfusion injury. Monoclonal antibodies targeting neutrophil adhesion molecules may, therefore, have a role in the prevention of this complication of limb revascularization.