Ibuprofen inhibits migration and proliferation of human coronary artery smooth muscle cells by inducing a differentiated phenotype: role of peroxisome proliferator-activated receptor y

Objectives: The search for agents that are capable of preventing restenosis and reduce the risk of late thrombosis is of utmost importance. In this study we aim to evaluate the in vitro effects of ibuprofen on proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) and on human coronary artery endothelial cells (HCAECs) migration. Methods: Cell proliferation was evaluated by direct cell counting using trypan blue exclusion. Cell migration was assessed by wound healing “scratch” assay and by time lapse video-microscopy. Protein expression was assessed by immunoblotting, and morphological changes were studied by immunocytochemistry. The involvement of the PPARγ pathway was studied with the selective agonist troglitazone, and the use of highly selective antagonists of PPARγ such as PGF2α and GW9662. Results: We demonstrate that ibuprofen inhibits proliferation and migration of HCASMCs and induces a switch in HCASMCs towards a differentiated and contractile phenotype, and that these effects are mediated through the PPARγ pathway. Importantly we also show that the effects of ibuprofen are cell type specific as it does not affect migration and proliferation of endothelial cells. Conclusions: Taken together, our results suggest that ibuprofen could be an effective drug for the development of novel drug eluting stents, which could lead reduced rates of restenosis and potentially other complications of DES stent implantation.

Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function

There has been much recent interest in the cardiovascular benefits of dietary isoflavones. The aim of the present in vitro studies was to investigate potential anti-thrombogenic and anti-atherogenic effects of the isoflavones genistein and daidzein in platelets, macrophages and endothelial cells. Pre-treatment with either isoflavone inhibited collagen-induced platelet aggregation in a dose-dependent manner. In a macrophage cell line (RAW 264-7) activated with interferon gamma plus lipopolysaccharide, both isoflavones were found to inhibit NO production and tumour necrosis factor alpha (TNF-alpha) secretion dose-dependently, but they did not affect mRNA levels for inducible nitric oxide synthase and cyclo-oxygenase-2. Both isoflavones also dose-dependently decreased monocyte chemoattractant protein-1 secretion induced by TNF-alpha in human umbilical vein endothelial cells. Compared with daidzein, genistein exerted greater inhibitory effects for all parameters studied. The present data contributes to our knowledge on the molecular mechanisms by which isoflavones may protect against coronary artery disease. Further studies are required to determine whether the effects of isoflavones observed in the current in vitro studies are relevant to the aetiology of coronary artery disease in vivo.

Meal ingestion provokes entry of lipoproteins containing fat from the previous meal: possible metabolic implications

Background: Prolonged and exaggerated postprandial plasma triacylglycerol (TAG) concentrations are considered as an independent risk factor for coronary artery disease. Western populations eat many meals at regular intervals, and can be in a postprandial state for at least 17h of a 24h period. After consuming 2 meals an early plasma TAG peak has been observed after the second meal, the origin of which is unclear. Aim of the study: To test the hypothesis that the early TAG peak observed following sequential meals was of intestinal origin and represented fat derived from the previous meal. Methods: Postprandial plasma lipaemic responses of 17 healthy postmenopausal women were studied by giving a test breakfast followed by a lunch. Watermiscible retinyl palmitate (RP) was added to the breakfast, but not the lunch test meal. Plasma TAG, retinyl esters (RE) and apo B-48 were determined for a 10h period following breakfast. Results: In response to the test meals, RE, apo B-48 and TAG showed multiple peaks. Despite omission of RP from the lunch, RE showed an early peak response after ingestion of lunch in 15 of 17 subjects. The peak response after lunch of all three markers appeared significantly earlier compared with their respective peak responses after the breakfast (P < 0.0001). The area of RE response after lunch was significantly correlated with the RE lipaemic response to the breakfast (r = 0.67; P < 0.004) and to the fasting TAG concentration (r = 0.48; P < 0.05). Conclusions: Since the lunch did not contain RP, the distinctive second influx of RE after lunch was believed to have originated from the breakfast. This, together with the fact that all three markers showed an earlier response to the lunch than the breakfast, supports the view that ingestion of a second meal provokes entry of fat from the previous meal, from an as yet unidentified site (gut, enterocytes, lymph). The results indicate that the degree of TAG "storage" from previous meals might be a function of TAG tolerance and provide a possible site of regulation of the entry of fat into the systemic circulation.

Triglyceride-rich lipoproteins inhibit cholesterol efflux to apolipoprotein (apo) A1 from human macrophage foam cells

High circulating levels of triglyceride-rich lipoproteins (TGRL) represent an independent risk factor for coronary artery disease. Here, we show that TGRL inhibit the efflux of cholesterol from 'foam cell' macrophages to lipid-poor apolipoprotein (apo) A1, and may thereby inhibit arterial reverse cholesterol transport and promote the formation of atherosclerotic lesions. Human (THP-1) monocyte-derived macrophages were pre-incubated (48h) with acetylated low-density lipoprotein (AcLDL) to provide a foam cell model of cholesterol efflux to apoA1. Pre-incubation of macrophage 'foam cells' with TGRL (0-200 mug/ml, 0-24 h) inhibited the efflux of exogenously radiolabelled ([H-3]), endogenously synthesised ([C-14]) and cellular cholesterol mass to lipid-poor apoA1, but not control medium, during a (subsequent) efflux period. This inhibition is dependent upon the length of prior exposure to, and concentration of, TGRL employed, but is independent of changes in intracellular triglyceride accumulation or turnover of the cholesteryl ester pool. Despite the negative impact of TGRL on cholesterol efflux, major proteins involved in this process-namely apoE, ABCA1, SR-B1 and caveolin-1-were unaffected by TGRL pre-incubation, suggesting that exposure to these lipoproteins inhibits an alternate, and possibly novel, anti-atherogenic pathway. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Moderate fish-oil supplementation reverses low-platelet, long-chain n-3 polyunsaturated fatty acid status and reduces plasma triacylglycerol concentrations in British Indo-Asians

Background: The mechanisms involved in the increased mortality from coronary artery disease in British Indo-Asians are not well understood. Objectives: This study aimed to investigate whether British Indo-Asian Sikhs have higher plasma triacylglycerol concentrations, lower platelet phospholipid levels, and lower dietary intakes of long-chain n-3 polyunsaturated fatty acids (PUFAs) than do age- and weight-matched Europeans and whether moderate dietary fish-oil intake can reverse these differences. Design: A randomized, double-blind, placebo-controlled, parallel, fish-oil intervention study was performed. After a 2-wk run-in period, 44 Europeans and 40 Indo-Asian Sikhs were randomly assigned to receive either 4.0 g fish oil [1.5 g eicosapentaenoic acid (EPA) and 1.0 g docosahexaenoic acid (DHA)] or 4.0 g olive oil (control) daily for 12 wk. Results: At baseline, the Indo-Asians had significantly higher plasma triacylglycerol, small dense LDL, apolipoprotein B, and dietary and platelet phospholipid n-6 PUFA values and significantly lower long-chain n-3 PUFAs (EPA and DHA) than did the Europeans. A significant decrease in plasma triacylglycerol, plasma apolipoprotein B-48, and platelet phospholipid arachidonic acid concentrations and a significant increase in plasma HDL concentrations and platelet phospholipid EPA and DHA levels were observed after fish-oil supplementation. No significant effect of ethnicity on the responses to fish-oil supplementation was observed. Conclusions: Moderate fish-oil supplementation contributes to a reversal of lipid abnormalities and low n-3 PUFA levels in Indo-Asians and should be considered as an important, yet simple, dietary manipulation to reduce CAD risk in Indo-Asians with an atherogenic lipoprotein phenotype.

Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function

There has been much recent interest in the cardiovascular benefits of dietary isoflavones. The aim of the present in vitro studies was to investigate potential anti-thrombogenic and anti-atherogenic effects of the isoflavones genistein and daidzein in platelets, macrophages and endothelial cells. Pre-treatment with either isoflavone inhibited collagen-induced platelet aggregation in a dose-dependent manner. In a macrophage cell line (RAW 264-7) activated with interferon gamma plus lipopolysaccharide, both isoflavones were found to inhibit NO production and tumour necrosis factor alpha (TNF-alpha) secretion dose-dependently, but they did not affect mRNA levels for inducible nitric oxide synthase and cyclo-oxygenase-2. Both isoflavones also dose-dependently decreased monocyte chemoattractant protein-1 secretion induced by TNF-alpha in human umbilical vein endothelial cells. Compared with daidzein, genistein exerted greater inhibitory effects for all parameters studied. The present data contributes to our knowledge on the molecular mechanisms by which isoflavones may protect against coronary artery disease. Further studies are required to determine whether the effects of isoflavones observed in the current in vitro studies are relevant to the aetiology of coronary artery disease in vivo.

Molecular mechanisms by which dietary isoflavones potentially prevent atherosclerosis

Dietary isoflavones are currently receiving much attention because of their potential role in preventing coronary artery disease and other chronic diseases. Accumulating evidence from cell culture and laboratory animal experiments indicates that isoflavones have the potential to prevent or delay atherogenesis. Suggested mechanisms of action include: a reduction in low-density lipoprotein (LDL) cholesterol and a potential reduction in the susceptibility of the LDL particle to oxidation; (2) an improvement in vascular reactivity; (3) an inhibition of pro-inflammatory cytokines, cell adhesion proteins and nitric oxide (NO) production; and (4) an inhibition of platelet aggregation. These mechanisms are consistent with the epidemiological evidence that a high consumption of isoflavone-rich soy products is associated with a reduced incidence of coronary artery disease. Biological effects of isoflavones are dependent on many factors, including dose consumed, duration of use, protein-binding affinity, and an individual's metabolism or intrinsic oestrogenic state. Further clinical studies are necessary to determine the potential health effects of isoflavones in specific population groups as we currently know little about age-related differences in exposure to these compounds and there are few guidelines on optimal dose for cardiovascular health benefits.

Increased n-6 polyunsaturated fatty acids do not attenuate the effects of long-chain n-3 polyunsaturated fatty acids on insulin sensitivity or triacylglycerol reduction in Indian Asians

Background: Indian Asians in Western countries have a higher rate of coronary artery disease than do the indigenous white populations, and this higher rate may be influenced by a dietary imbalance of n-6 and n-3 polyunsaturated fatty acids (PUFAs). Objective: The objective of the study was to test the hypothesis that a high background dietary intake of n-6 PUFA attenuates the effects of fish-oil supplementation on insulin sensitivity and associated blood lipids of the metabolic syndrome. Design: Twenty-nine Indian Asian men were recruited to participate in a 12-wk dietary intervention trial. Volunteers were randomly assigned to receive either a moderate or a high n-6 PUFA diet featuring modified oils and spreads over a 6-wk period. After this 6-wk period, both groups were supplemented with 4.0 g fish oil/d (2.5 g eicosapentaenoic acid + docosahexaenoic acid) for an additional 6 wk in combination with the dietary treatment. Volunteers participated in a postprandial study and an insulin sensitivity test after the 6-wk dietary intervention and again after the fish-oil supplementation period. Results: There was no significant time X treatment interaction for blood lipids or insulin action after dietary intervention with the moderate or high n-6 PUFA diets in combination with fish oil. After the 6-wk period of fish oil supplementation, fasting and postprandial plasma triacylglycerol concentrations decreased significantly. Conclusion: The background dietary n-6 PUFA concentration did not modulate the effect of fish-oil supplementation on blood lipids or measures of insulin sensitivity in this ethnic group.

Reprogramming the cell cycle machinery to treat cardiovascular disease

Coronary artery disease is one of the most common heart pathologies. Restriction of blood flow to the heart by atherosclerotic lesions, leading to angina pectoris and myocardial infarction, damages the heart, resulting in impaired cardiac function. Damaged myocardium is replaced by scar tissue since surviving cardiomyocytes are unable to proliferate to replace lost heart tissue. Although narrowing of the coronary arteries can be treated successfully using coronary revascularisation procedures, re-occlusion of the treated vessels remains a significant clinical problem. Cell cycle control mechanisms are key in both the impaired cardiac repair by surviving cardiomyocytes and re-narrowing of treated vessels by maladaptive proliferation of vascular smooth muscle cells. Strategies targeting the cell cycle machinery in the heart and vasculature offer promise both for the improvement of cardiac repair following MI and the prevention of restenosis and bypass graft failure following revascularisation procedures.

Differences in postprandial lipaemic response between Northern and Southern Europeans

Postprandial lipaemic responses to two test meals were investigated in 30 Northern (15 British and 15 Irish), and 30 Southern (Greeks from Crete) healthy male Europeans. The meals were a saturated fatty acid (SFA) meal, which resembled the fatty acid composition of an average UK diet, and a monounsaturated fatty acid (MUFA) meal in which the fat consisted of olive oil. Habitual diets of the two groups differed, with higher total fat, (P < 0.03) and MUFA (P < 0.0001) and lower polyunsaturated fatty acid (PUFA) (P < 0.0001) intakes in Southern than Northern Europeans. Levels of total MUFA (P < 0.02) and oleic acid (P < 0.004) were also higher in adipose tissue of Southern in comparison to Northern Europeans. In both European groups there were no significant differences in postprandial triglyceride response between the two meal types, SFA or MUFA. However, Northern and Southern Europeans showed significant differences in their patterns of postprandial response in plasma triglycerides (P < 0.0001), apolipoprotein B-48 (P < 0.0001), NEFA (P < 0.0001), insulin (P < 0.0007), and factor VII activity (P-0.03). In the case of NEFA, areas under the response curve were higher following the SFA than the MUFA meal for both groups, (P < 0.003) and were greater in Southern than Northern Europeans (P < 0.002) and apo B-48 responses were lower (P < 0.005). Some of these differences may reflect differences in fasting levels since fasting apolipoprotein B-48 levels were lower (P < 0.01) and fasting NEFA (P < 0.02) and insulin (P < 0.005) were higher in the Southern than in the Northern Europeans. In addition, 9 h postprandial post-heparin lipoprotein lipase activity was lower in the Southern than in the Northern Europeans (P < 0.0006). This is the first report of differences in postprandial lipid, factor VII and insulin responses in Southern and Northern Europeans which may be of importance in explaining the different susceptibilities of these two populations to risk of coronary artery disease.

Limitations of the MRL mouse as a model for cardiac regeneration

Objective Myocardial repair following injury in mammals is restricted such that damaged areas are replaced by scar tissue, impairing cardiac function. MRL mice exhibit exceptional regenerative healing in an ear punch wound model. Some myocardial repair with restoration of heart function has also been reported following cryoinjury. Increased cardiomyocyte proliferation and a foetal liver stem cell population were implicated. We investigated molecular mechanisms facilitating myocardial repair in MRL mice to identify potential therapeutic targets in non-regenerative species. Methods Expressions of specific cell-cycle regulators that might account for regeneration (CDKs 1, 2, 4 and 6; cyclins A, E, D1 and B1; p21, p27 and E2F5) were compared by immunoblotting in MRL and control C57BL/6 ventricles during development. Flow cytometry was used to investigate stem cell populations in livers from foetal mice, and infarct sizes were compared in coronary artery-ligated and sham-treated MRL and C57BL/6 adult mice. Key findings No differences in the expressions of cell cycle regulators were observed between the two strains. Expressions of CD34+Sca1+ckit-, CD34+Sca1+ckit+ and CD34+Sca1-ckit+ increased in livers from C57BL/6 vs MRL mice. No differences were observed in infarct sizes, levels of fibrosis, Ki67 staining or cardiac function between MRL and C57BL/6 mice. Conclusions No intrinsic differences were observed in cell cycle control molecules or stem cell populations between MRL and control C57BL mouse hearts. Pathophysiologically relevant ischaemic injury is not repaired more efficiently in MRL myocardium, questioning the use of the MRL mouse as a reliable model for cardiac regeneration in response to pathophysiologically relevant forms of injury.

Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease

A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the NO-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Employing immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-3H-Arginine to L-3H-Citrulline in a Ca2+/Calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform the activity of which is compromised in patients with coronary artery disease.

Conditional transgenic expression of fibroblast growth factor 9 in the adult mouse heart reduces heart failure mortality after myocardial infarction

BACKGROUND: Fibroblast growth factor 9 (FGF9) is secreted from bone marrow cells, which have been shown to improve systolic function after myocardial infarction (MI) in a clinical trial. FGF9 promotes cardiac vascularization during embryonic development but is only weakly expressed in the adult heart. METHODS AND RESULTS: We used a tetracycline-responsive binary transgene system based on the α-myosin heavy chain promoter to test whether conditional expression of FGF9 in the adult myocardium supports adaptation after MI. In sham-operated mice, transgenic FGF9 stimulated left ventricular hypertrophy with microvessel expansion and preserved systolic and diastolic function. After coronary artery ligation, transgenic FGF9 enhanced hypertrophy of the noninfarcted left ventricular myocardium with increased microvessel density, reduced interstitial fibrosis, attenuated fetal gene expression, and improved systolic function. Heart failure mortality after MI was markedly reduced by transgenic FGF9, whereas rupture rates were not affected. Adenoviral FGF9 gene transfer after MI similarly promoted left ventricular hypertrophy with improved systolic function and reduced heart failure mortality. Mechanistically, FGF9 stimulated proliferation and network formation of endothelial cells but induced no direct hypertrophic effects in neonatal or adult rat cardiomyocytes in vitro. FGF9-stimulated endothelial cell supernatants, however, induced cardiomyocyte hypertrophy via paracrine release of bone morphogenetic protein 6. In accord with this observation, expression of bone morphogenetic protein 6 and phosphorylation of its downstream targets SMAD1/5 were increased in the myocardium of FGF9 transgenic mice. CONCLUSIONS: Conditional expression of FGF9 promotes myocardial vascularization and hypertrophy with enhanced systolic function and reduced heart failure mortality after MI. These observations suggest a previously unrecognized therapeutic potential for FGF9 after MI.

Antithrombotic actions of statins involve PECAM-1 signalling

Statins are widely prescribed cholesterol-lowering drugs that are a first-line treatment for coronary artery disease and atherosclerosis, reducing the incidence of thrombotic events such as myocardial infarction and stroke. Statins have been shown to reduce platelet activation, although the mechanism(s) through which this occurs is unclear. Since several of the characteristic effects of statins on platelets are shared with those elicited by the inhibitory platelet adhesion receptor PECAM-1, we investigated a potential connection between the influence of statins on platelet function and PECAM-1 signalling. Statins were found to inhibit a range of platelet functional responses and thrombus formation in vitro and in vivo. Notably, these effects of statins on platelet function in vitro and in vivo were diminished in PECAM-1-/- platelets. Activation of PECAM-1 signalling results in its tyrosine phosphorylation, the recruitment and activation of tyrosine phosphatase SHP-2, the subsequent binding of phosphoinositol 3-kinase (PI3-K) and diminished PI3-K signalling. Statins resulted in the stimulation of these events, leading to the inhibition of Akt activation. Together, these data provides evidence for a fundamental role of PECAM-1 in the inhibitory effects of statins on platelet activation, which may explain some of the pleiotropic actions of these drugs.

Association of low adiponectin levels with the metabolic syndrome--the Chennai Urban Rural Epidemiology Study (CURES-4)

The aim of the study was to assess the relation of adiponectin levels with the metabolic syndrome in Asian Indians, a high-risk group for diabetes and premature coronary artery disease. The study was conducted on 100 (50 men and 50 women) type 2 diabetic subjects and 100 age and sex matched subjects with normal glucose tolerance selected from the Chennai Urban Rural Epidemiology Study, an ongoing population study in Chennai in southern India. Metabolic syndrome was defined using modified Adult Treatment Panel III (ATPIII) guidelines. Adiponectin values were significantly lower in diabetic subjects (men: 5.2 vs 8.3 microg/mL, P=.00l; women: 7.6 vs 11.1 microg/mL, P<.00l) and those with the metabolic syndrome (men: 5.0 vs 6.8 microg/mL, P=.01; women: 6.5 vs 9.9 microg/mL, P=.001) compared with those without. Linear regression analysis revealed adiponectin to be associated with body mass index (P<.05), waist circumference (P<.01), fasting plasma glucose (P=.001), glycated hemoglobin (P<.001), triglycerides (P<.00l), high-density lipoprotein (HDL) cholesterol (P<.001), cholesterol/HDL ratio (P<.00l), and insulin resistance measured by homeostasis assessment model (P<.00l). Factor analysis identified 2 factors: factor 1, negatively loaded with adiponectin and HDL cholesterol and positively loaded with triglycerides, waist circumference, and insulin resistance measured by homeostasis assessment model; and factor 2, with a positive loading of waist circumference and systolic and diastolic blood pressure. Logistic regression analysis revealed adiponectin to be negatively associated with metabolic syndrome (odds ratio [OR], 0.365; P<.001) even after adjusting for age (OR, 0.344; P<.00l), sex (OR, 0.293; P<.001), and body mass index (OR, 0.292; P<.00l). Lower adiponectin levels are associated with the metabolic syndrome per se and several of its components, particularly, diabetes, insulin resistance, and dyslipidemia in this urban south Indian population.