Albert Ferro

The lipid and non-lipid effects of statins.

The 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors, more commonly known as statins, are a class of drug widely used for the treatment of hypercholesterolaemia in patients with established cardiovascular disease as well as those at high risk of developing atherosclerosis. Their predominant action is to reduce circulating levels of low-density lipoprotein (LDL) cholesterol; to a smaller degree, they also increase high-density lipoprotein (HDL) cholesterol and reduce triglyceride concentrations. In recent years, however, there has been an increasing body of evidence that their effects on lipid profile cannot fully account for their cardiovascular protective actions: their beneficial effects are too rapid to be easily explained by their relatively slow effects on atherogenesis and too large to be accounted for by their relatively small effects on plaque regression. Experimental models have revealed that statins exert a variety of other cardiovascular effects, which would be predicted to be of clinical benefit: they possess anti-inflammatory properties, as evidenced by their ability to reduce the accumulation of inflammatory cells in atherosclerotic plaques; they inhibit vascular smooth muscle cell proliferation, a key event in atherogenesis; they inhibit platelet function, thereby limiting both atherosclerosis and superadded thrombosis; and they improve vascular endothelial function, largely through augmentation of nitric oxide (NO) generation. The relative importance of the lipid- and non-lipid-related effects of the statins in the clinical situation remains the subject of much continuing research.

Activation of nitric oxide synthase by β2‐adrenoceptors in human umbilical vein endothelium in vitro

Some animal studies suggest that β‐adrenoceptor‐mediated vasorelaxation is in part mediated through nitric oxide (NO) release. Furthermore, in humans, we have recently shown that forearm blood flow is increased by infusion of β2‐adrenergic agonists into the brachial artery, and the nitric oxide synthase (NOS) inhibitor NG‐monomethyl‐L‐arginine (L‐NMMA) inhibits this response. The purpose of the present study was to determine whether stimulation of human umbilical vein endothelial β‐adrenoceptors causes vasorelaxation and nitric oxide generation, and whether this might be mediated by cyclic adenosine‐3′,5′‐monophosphate (cyclic AMP). Vasorelaxant responses were determined in umbilical vein rings to the nonselective β‐adrenergic agonist isoprenaline and to the cyclic AMP analogue dibutyryl cyclic AMP, following precontraction with prostaglandin F2α. NOS activity was measured in cultured human umbilical vein endothelial cells (HUVEC) by the conversion of [3H]‐L‐arginine to [3H]‐L‐citrulline, and adenylyl cyclase activity by the conversion of [α‐32P]‐ATP to [32P]‐cyclic AMP. Isoprenaline relaxed umbilical vein rings, and this vasorelaxation was abolished by β2‐ (but not β1‐) adrenergic blockage, and by endothelium removal or 1 mM L‐NMMA. In addition, vasorelaxant responses to dibutyryl cyclic AMP were inhibited by 1 mM L‐NMMA, with a reduction in Emax from 90.0±9.3% to 50.5±9.9% (P<0.05). Isoprenaline 1 μM increased NOS activity in HUVEC (34.0±5.9% above basal, P<0.001). Furthermore, isoprenaline increased adenylyl cyclase activity in a concentration‐dependent manner; this response was inhibited by β2 (but not β1‐) adrenergic blockade. Forskolin 1 μM and dibutyryl cyclic AMP 1 mM each increased NOS activity in HUVEC, to a degree similar to isoprenaline 1 μM. The increase in L‐arginine to L‐citrulline conversion observed with each agent was abolished by co‐incubation with NOS inhibitors. These results indicate that endothelial β2‐adrenergic stimulation and cyclic AMP elevation activate the L‐arginine/NO system, and give rise to vasorelaxation, in human umbilical vein.

Pharmacogenetics of aspirin resistance: a comprehensive systematic review

AIMS The aim was to perform a systematic review of all candidate gene association studies in aspirin resistance. METHODS Electronic databases were searched up until 1 December 2007 for all studies investigating any candidate gene for aspirin resistance in humans. Aspirin resistance was required to have been measured by a standardized laboratory technique to be included in the analysis. RESULTS Within 31 studies, 50 polymorphisms in 11 genes were investigated in 2834 subjects. The PlA1/A2 polymorphism in the GPIIIa platelet receptor was the most frequently investigated, with 19 studies in 1389 subjects. The PlA1/A2 variant was significantly associated with aspirin resistance when measured in healthy subjects [odds ratio (OR) 2.36, 95% confidence interval (CI) 1.24, 4.49; P = 0.009]. Combining genetic data from all studies (comprising both healthy subjects and those with cardiovascular disease) reduced the observed effect size (OR 1.14, 95% CI 0.84, 1.54; P = 0.40). Moreover, the observed effect of PlA1/A2 genotype varied depending on the methodology used for determining aspirin sensitivity/resistance. No significant association was found with aspirin resistance in four other investigated polymorphisms in the COX-1, GPla, P2Y1 or P2Y12 genes. CONCLUSIONS Our data support a genetic association between the PlA1/A2 molecular variant and aspirin resistance in healthy subjects, with the effect diminishing in the presence of cardiovascular disease. The laboratory methodology used influences the detection of aspirin resistance. However, as heterogeneity was significant and our results are based on a limited number of studies, further studies are required to confirm our findings.

Impairment of vascular endothelial nitric oxide synthase activity by advanced glycation end products

Endothelial damage is believed to play a key role in the development of both micro‐ and macrovascular disease in diabetes, and advanced glycation end products (AGEs) may contribute importantly to this. To determine whether glucose‐derived AGEs can cause endothelial dysfunction, we examined the effects of albumin AGE‐modified by glucose (AGE‐Glu) both in vivo, after injection into rabbit femoral artery, and in vitro on rabbit aortic rings and cultured human umbilical vein endothelial cells (HUVEC). Exposure of blood vessels to AGE‐Glu, in vivo and in vitro, inhibited endothelium‐dependent vasorelaxation, whereas unmodified albumin did not. In isolated rabbit aorta, this effect was reversible after AGE‐Glu washout, and the response to the endothelium‐independent vasodilator sodium nitroprusside was unaffected by AGE‐Glu. In HUVEC, AGE‐Glu inhibited endothelial nitric oxide synthase activity, and this was associated with a decrease in serine phosphorylation of this enzyme. Longer term (72 h) incubation decreased HUVEC viability. Use of specific antibodies demonstrated that these effects were mediated by Nε‐(carboxymethyl)lysine (CML), an important AGE found in vivo, and by the AGE‐R1 receptor. Furthermore, these effects all occurred at CML concentrations similar to those found in the plasma of diabetic patients. These results suggest an important role of AGE in the pathogenesis of diabetic vasculopathy.

Platelet-Derived Nitric Oxide Signaling and Regulation

Nitric oxide (NO) exerts important vasodilatory, antiplatelet, antioxidant, antiadhesive, and antiproliferative effects. Although endothelium derived NO has been shown to be of prime importance in cardio- and vasculoprotection, until recently little was known about the role of platelet-derived NO. New evidence suggests that NO synthesized by platelets regulates platelet functions, in particular suppressing platelet activation and intravascular thrombosis. Moreover, platelet NO biosynthesis may be decreased in patients with cardiovascular risk factors or with coronary heart disease, and this may contribute to arterial thrombotic disease in these patients. Here, we review the current state of knowledge as regards the role of platelet-derived NO, both in normal physiology and in cardiovascular disease states, and compare platelet NO signaling and regulation with that in endothelial cells.

Monocyte-platelet interaction induces a pro-inflammatory phenotype in circulating monocytes

Background Activated platelets exert a pro-inflammatory action that can be largely ascribed to their ability to interact with leukocytes and modulate their activity. We hypothesized that platelet activation and consequent formation of monocyte-platelet aggregates (MPA) induces a pro-inflammatory phenotype in circulating monocytes. Methodology/Principal Findings CD62P+ platelets and MPA were measured, and monocytes characterized, by whole blood flow cytometry in healthy subjects, before and two days after receiving influenza immunization. Three monocytic subsets were identified: CD14+CD16−, CD14highCD16+and CD14lowCD16+. The increase in high sensitivity C-reactive protein post-immunization was accompanied by increased platelet activation and MPA formation (25.02±12.57 vs 41.48±16.81; p = 0.01), along with enhancement of circulating CD14highCD16+ cells (4.7±3.6 vs 10.4±4.8; p = 0.003), their percentage being linearly related to levels of CD62P+-platelets (r2 = 0.4347; p = 0.0008). In separate in vitro experiments, co-incubation of CD14+CD16− cells, isolated from healthy donor subjects, with autologous platelets gave rise to up-regulation of CD16 on monocytes as compared with those maintained in medium alone (% change in CD14+CD16+ cells following 48 h co-incubation of monocytes with platelets was +106±51% vs monocytes in medium alone; p<0.001). This effect correlated directly with degree of MPA formation (r2 = 0.7731; p<0.0001) and was associated with increased monocyte adhesion to endothelial cells. P-selectin glycoprotein ligand-1 (PSGL-1) blocking antibody, which abrogates MPA formation, abolished these effects, as did the cyclooxygenase (COX)-2 selective inhibitor NS-398, aspirin and the EP1/EP2-selective antagonist AH6809. Conclusions/Significance These data suggest that MPA formation, as occurs in the blood under pro-inflammatory conditions, expands the pool of circulating CD14highCD16+ monocytes in a COX-2 dependent manner, and these monocytes exhibit increased adhesion to endothelium. Our findings delineate a novel mechanism underlying the pro-inflammatory effect of platelet activation.

Reaction of Carnosine with Aged Proteins

Cellular aging is often associated with an increase in protein carbonyl groups arising from oxidation‐ and glycation‐related phenomena and suppressed proteasome activity. These “aged” polypeptides may either be degraded by 20S proteasomes or cross‐link to form structures intractable to proteolysis and inhibitory to proteasome activity. Carnosine (β‐alanyl‐l‐histidine) is present at surprisingly high levels (up to 20 mM) in muscle and nervous tissues in many animals, especially long‐lived species. Carnosine can delay senescence in cultured human fibroblasts and reverse the senescent phenotype, restoring a more juvenile appearance. As better antioxidants/free‐radical scavengers than carnosine do not demonstrate these antisenescent effects, additional properties of carnosine must contribute to its antisenescent activity. Having shown that carnosine can react with protein carbonyls, thereby generating “carnosinylated” polypeptides using model systems, we propose that similar adducts are generated in senescent cells exposed to carnosine. Polypeptide‐carnosine adducts have been recently detected in beef products that are relatively rich in carnosine, and carnosines reaction with carbonyl functions generated during amino acid deamidation has also been described. Growth of cultured human fibroblasts with carnosine stimulated proteolysis of long‐labeled proteins as the cells approached their “Hayflick limit,” consistent with the idea that carnosine ameliorates the senescence‐associated proteolytic decline. We also find that carnosine suppresses induction of heme‐oxygenase‐1 activity following exposure of human endothelial cells to a glycated protein. The antisenescent activity of the spin‐trap agent α‐phenyl‐N‐t‐butylnitrone (PBN) towards cultured human fibroblasts resides in N‐t‐butyl‐hydroxylamine, its hydrolysis product. As hydroxylamines are reactive towards aldehydes and ketones, the antisenescent activity of N‐t‐butyl‐hydroxylamine and other hydroxylamines may be mediated, at least in part, by reactivity towards macromolecular carbonyls, analogous to that proposed for carnosine.

Nitric oxide-dependent β2-adrenergic dilatation of rat aorta is mediated through activation of both protein kinase A and Akt

Vasorelaxation to β2‐adrenoceptor stimulation occurs through both endothelium‐dependent and endothelium‐independent mechanisms, and the former is mediated through Ca2+‐independent activation of endothelial‐type nitric oxide synthase (NOS‐3). Since Ca2+‐independent NOS‐3 activation may occur through its serine phosphorylation via protein kinase A (PKA) or Akt, we determined the PKA and Akt dependency of β2‐adrenergic relaxation of rat aorta. Rat aortic rings were pre‐incubated with the PKA inhibitor H‐89 (10−7 M), the phosphatidylinositol 3‐kinase (PI3K) inhibitor wortmannin (5 × 10−7 M), Akt inhibitor (10−5 M), or vehicle, in the absence or presence of the NOS inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME, 10−4 M). Rings were then contracted with phenylephrine (10−7 M), and concentration–relaxation responses determined to the β2‐adrenoceptor agonist albuterol. Rings exhibited a concentration‐dependent relaxation to albuterol: pEC50 6.9±0.2, Emax 88.2±4.0%. L‐NAME attenuated Emax to 60.2±3.5% (P<0.001). In the presence of L‐NAME, wortmannin or Akt inhibitor did not influence albuterol responses, whereas H‐89 reduced Emax further, to 27.5±2.2% (P<0.001). In the absence of L‐NAME, Emax to albuterol was reduced by H‐89, wortmannin or Akt inhibitor, to 56.2±2.2, 56.0±1.6 and 55.4±1.8%, respectively (P<0.001 for each); the combinations H‐89 plus wortmannin or H‐89 plus Akt inhibitor reduced Emax further still. Western blotting of NOS‐3 immunoprecipitates from rat aortas confirmed that albuterol increased serine phosphorylation of NOS‐3, and this increase was attenuated by H‐89 or Akt inhibitor. Our results indicate that β2‐adrenoceptor stimulation relaxes rat aorta through both NO‐dependent and independent mechanisms. The latter is predominantly PKA‐mediated, whereas the former occurs through both PKA and PI3K/Akt activation.

Nitric oxide signalling in the regulation of cardiovascular and platelet function

Nitric oxide (NO) exerts important protective actions on the cardiovascular system. Generated from L-arginine by the action of endothelial (or type 3) nitric oxide synthase (NOS3), NO regulates vascular tone in humans and causes endothelium-dependent vasodilation. Additionally endothelium-derived NO exerts antioxidant, antiproliferative and anti-inflammatory properties, thus playing an important role in inhibiting the atherosclerotic process. With regard to effects on platelet function, NO produced by both endothelial cells and platelets has important antithrombotic effects by decreasing platelet activation, a phenomenon which contributes importantly to the thrombotic tendency which accompanies a variety of cardiovascular disease states. Additionally, by inhibiting platelet activation, NO prevents heterotypic aggregation between platelets and monocytes, thereby reducing monocyte-platelet aggregates in the circulation which are believed to play an important pathophysiological role in the initiation and progression of atherosclerosis. New therapeutic interventions aimed at improving NO availability have been investigated in animal as well as in vitro studies and show considerable promise, but it remains to be seen whether such therapies will be equally efficacious in humans clinically.

β2-Adrenoceptors Activate Nitric Oxide Synthase in Human Platelets

Nitric oxide (NO), generated by platelets through stimulation of nitric oxide synthase (NOS), limits platelet adhesion and aggregation after a prothrombotic stimulus. Platelet beta-adrenoceptors (betaARs) mediate inhibition of aggregation, but no direct link has been shown between these receptors and platelet adhesion or NO production. We examined NOS activity in human platelets from the conversion of L-[(3)H]-arginine to L-[(3)H]-citrulline, after betaAR stimulation or cAMP elevation. Basal NOS activity was 0.11+/-0.03 pmol L-citrulline/10(8) platelets. The betaAR agonist isoproterenol 1 micromol/L and the adenylyl cyclase activator forskolin 1 micromol/L each increased NOS activity, to 0.26+/-0.04 and 0.23+/-0.03 pmol L-citrulline/10(8) platelets, respectively (P<0.01 for each). Both responses were abolished by the adenylyl cyclase inhibitor SQ22536 50 micromol/L. NOS activation by isoproterenol or forskolin was not associated with a change in intracellular Ca(2+). In functional studies, isoproterenol inhibited U46619-induced platelet aggregation in a concentration-dependent manner, but this effect was not significantly diminished by NOS inhibition. In contrast, thrombin-stimulated platelet adhesion to cultured human umbilical vein endothelial cell monolayers was inhibited by isoproterenol, and this effect was abolished by NOS inhibition (1.3+/-0.2% versus 2.6+/-0.2% respectively; P<0.001). Effects of isoproterenol on NOS activity, platelet aggregation, and adhesion were mediated exclusively through beta(2)ARs, as determined by coincubation with betaAR subtype-selective antagonists. We conclude that beta(2)ARs activate platelet NOS by increasing cAMP, and that this activation is Ca(2+)-independent. beta(2)ARs may contribute to modulation of platelet aggregation and adhesion to endothelium, and our findings suggest that activation of the L-arginine/NO system mediates the effects of beta(2)ARs on adhesion but not aggregation.