Showing papers in "Cardiovascular Research in 1999"

Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure

Abstract: Time for primary review 26 days. Soon after the discovery of EDRF (endothelium derived relaxins factor) it became apparent that certain diseases are associated with an impairment of endothelium dependent vasorelaxation. In hypercholesterolemic rabbits and monkeys, vasorelaxation to acetylcholine is almost absent (Fig. 1) or changed into vasoconstriction [1,2]. Similar observations have been made in patients with coronary artery disease [3,4] or risk factors predisposing to atherosclerosis [5]. Likewise, endothelium-dependent vasorelaxation is abnormal in disease states such as heart failure, diabetes and hypertension [6]. In almost all of these disorders, there is a loss of endothelial production and/or bioavailability of NO (nitric oxide, nitrogen monoxide). This alteration of vascular function has been termed ‘endothelial dysfunction’ in the scientific literature. Although this term is widely used, it is quite imprecise. ‘Endothelial dysfunction’ may refer to impairments of important endothelial functions other than vasodilation, including anticoagulant and anti-inflammatory properties of the endothelium [7,8]. Nevertheless, ‘endothelial dysfunction’ has become widely used, and in fact, loss of nitric oxide in these conditions may contribute to alterations of other aspects of vascular function. The mechanisms underlying altered endothelium-dependent vascular relaxation in various disease states are almost certainly multifactorial, and seem to be dependent on the specific pathological condition, its duration, and the vascular bed being studied. Treatment with l-arginine or tetrahydrobiopterin has improved NO-mediated vasodilation in some instances, suggesting that there may be a deficiency of either the substrate for the enzyme NO synthase or one of its critical co-factors. Alterations of endothelial cell signaling may impair appropriate activation of the NO synthase in response to neurohumoral or mechanical stimuli. In very advanced atherosclerosis, expression of NO synthase in the endothelium declines, almost certainly reducing endothelium-dependent vascular relaxation. Finally, there is substantial evidence that in certain disease conditions, NO production … * Corresponding author. Tel.: +1-404-727-3710; fax: +1-404-727-3585 dharr02{at}emory.edu

Electrophysiological remodeling in hypertrophy and heart failure

Abstract: Time for primary review 28 days. Over 2 million Americans suffer from heart failure and more than 200 000 die annually. The incidence is estimated to be 400 000 per year with a prevalence of over 4.5 million, numbers that will increase with the aging of the US population [1]. Despite remarkable improvements in medical therapy the prognosis of patients with myocardial failure remains poor with over 15% of patients dying within 1 year of initial diagnosis and greater than 80% 6 year mortality [2]. Of the deaths in patients with heart failure, up to 50% are sudden and unexpected. The failing heart undergoes a complex series of changes in both myocyte and non-myocyte elements. In an attempt to compensate for the reduction in cardiac function the sympathetic nervous (SNS), renin–angiotensin–aldosterone (RAAS) systems and other neurohumoral mechanisms are activated. The altered signal transduction in heart failure initiates changes in gene expression that produce myocyte hypertrophy. Ultimately the changes in gene expression that initially maintain tissue perfusion prove to be maladaptive, predisposing to further myocyte loss, ventricular chamber remodeling and interstitial hyperplasia resulting in a progressive reduction in force development and impairment of ventricular relaxation. The intrinsic cardiac and peripheral responses to myocardial failure adversely alter the electrophysiology of the heart predisposing patients with heart failure to an increase in arrhythmic death. With progression of heart failure there is an increase in the frequency and complexity of ventricular ectopy [3,4]. Total mortality in heart failure patients correlates with LV function and the presence of complex ventricular ectopy [5–7]. However, there is no clear correlation between SCD and LV function or ventricular ectopy. In fact, data from VHeFT (Veteran’s Administration Heart Failure Trial) and other trials suggest that death is disproportionately sudden in patients with more modest myocardial dysfunction [8] … * Corresponding author. Tel.: +1-410-955-2774; fax: +1-410-955-7953

Enzymatic function of nitric oxide synthases.

Abstract: Nitric oxide (NO) is synthesised from l-arginine by the enzyme NO synthase (NOS). The complex reaction involves the transfer of electrons from NADPH, via the flavins FAD and FMN in the carboxy-terminal reductase domain, to the haem in the amino-terminal oxygenase domain, where the substrate l-arginine is oxidised to l-citrulline and NO. The haem is essential for dimerisation as well as NO production. The pteridine tetrahydrobiopterin (BH4) is a key feature of NOS, affecting dimerisation and electron transfer, although its full role in catalysis remains to be determined. NOS can also catalyse superoxide anion production, depending on substrate and cofactor availability. There are three main isoforms of the enzyme, named neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), which differ in their dependence on Ca2+, as well as in their expression and activities. These unique features give rise to the distinct subcellular localisations and mechanistic features which are responsible for the physiological and pathophysiological roles of each isoform.

The role of neutrophils in myocardial ischemia–reperfusion injury

Abstract: Reperfusion of ischemic myocardium is necessary to salvage tissue from eventual death. However, reperfusion after even brief periods of ischemia is associated with pathologic changes that represent either an acceleration of processes initiated during ischemia per se, or new pathophysiological changes that were initiated after reperfusion. This 'reperfusion injury' shares many characteristics with inflammatory responses in the myocardium. Neutrophils feature prominently in this inflammatory component of postischemic injury. Ischemia-reperfusion prompts a release of oxygen free radicals, cytokines and other proinflammatory mediators that activate both the neutrophils and the coronary vascular endothelium. Activation of these cell types promotes the expression of adhesion molecules on both the neutrophils and endothelium, which recruits neutrophils to the surface of the endothelium and initiate a specific cascade of cell-cell interactions, leading first to adherence of neutrophils to the vascular endothelium, followed later by transendothelial migration and direct interaction with myocytes. This specific series of events is a prerequisite to the phenotypic expression of reperfusion injury, including endothelial dysfunction, microvascular collapse and blood flow defects, myocardial infarction and apoptosis. Pharmacologic therapy can target the various components in this critical series of events. Effective targets for these pharmacologic agents include: (a) inhibiting the release or accumulation of proinflammatory mediators, (b) altering neutrophil or endothelial cell activation and (c) attenuating adhesion molecule expression on endothelium, neutrophils and myocytes. Monoclonal antibodies to adhesion molecules (P-selectin, L-selectin, CD11, CD18), complement fragments and receptors attenuate neutrophil-mediated injury (vascular injury, infarction), but clinical application may encounter limitations due to antigen-antibody reactions with the peptides. Humanized antibodies and non-peptide agents, such as oligosaccharide analogs to sialyl Lewis, may prove effective in this regard. Both nitric oxide and adenosine exhibit broad spectrum effects against neutrophil-mediated events and, therefore, can intervene at several critical points in the ischemic-reperfusion response, and may offer greater benefit than agents that interdict at a single point in the cascade. The understanding of the molecular processes regulating actions of neutrophils in ischemic-reperfusion injury may be applicable to other clinical situations, such as trauma, shock and organ or tissue (i.e. vascular conduits) transplantation.

Ionic mechanisms of electrical remodeling in human atrial fibrillation

Abstract: Objectives: Atrial fibrillation (AF) is associated with a decrease in atrial ERP and ERP adaptation to rate as well as changes in atrial conduction velocity. The cellular changes in repolarization and the underlying ionic mechanisms in human AF are only poorly understood. Methods: Action potentials (AP) and ionic currents were studied with the patch clamp technique in single atrial myocytes from patients in chronic AF and compared to those from patients in stable sinus rhythm (SR). Results: The presence of AF was associated with a marked shortening of the AP duration and a decreased rate response of atrial repolarization. L-type calcium current ( I Ca,L) and the transient outward current ( I to) were both reduced about 70% in AF, whereas an increased steady-state outward current was detectable at test potentials between –30 and 0 mV. The inward rectifier potassium current ( I K1) and the acetylcholine-activated potassium current ( I KACh) were increased in AF at hyperpolarizing potentials. Voltage-dependent inactivation of the fast sodium current ( I Na) was shifted to more positive voltages in AF. Conclusions: AF in humans leads to important changes in atrial potassium and calcium currents that likely contribute to the decrease in APD and APD rate adaptation. These changes contribute to electrical remodeling in AF and are therefore important factors for the perpetuation of the arrhythmia.

Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system

Abstract: Objective: Methylation of the promoter region of the estrogen receptor gene alpha ( ER α ) occurs as a function of age in human colon, and results in inactivation of gene transcription. In this study, we sought to determine whether such age-related methylation occurs in the cardiovascular system, and whether it is associated with atherosclerotic disease. Methods: We used Southern blot analysis to determine the methylation state of the ER α gene in human right atrium, aorta, internal mammary artery, saphenous vein, coronary atherectomy samples, as well as cultured aortic endothelial cells and smooth muscle cells. Results: An age related increase in ER α gene methylation occurs in the right atrium (range 6 to 19%, R =0.36, P <0.05). Significant levels of ER α methylation were detected in both veins and arteries. In addition, ER α gene methylation appears to be increased in coronary atherosclerotic plaques when compared to normal proximal aorta (10±2% versus 4±1%, P <0.01). In endothelial cells explanted from human aorta and grown in vitro, ER α gene methylation remains low. In contrast, cultured aortic smooth muscle cells contain a high level of ER α gene methylation (19–99%). Conclusions: Methylation associated inactivation of the ER α gene in vascular tissue may play a role in atherogenesis and aging of the vascular system. This potentially reversible defect may provide a new target for intervention in heart disease.

Biological significance of endogenous methylarginines that inhibit nitric oxide synthases.

Abstract: The guanidino-methylated arginine analogue N G monomethyl-l-arginine (l-NMMA) has been the standard nitric oxide synthase inhibitor used to evaluate the role of the l-arginine:nitric oxide pathway. However, l-NMMA and other methylated arginine residues are also synthesised in vivo by the action of a family of enzymes known as protein arginine methyltransferases. Proteolysis of proteins containing methylated arginine residues releases free methylarginine residues into the cytosol from where they may pass out of the cell into plasma. Of the three known methylarginine residues produced in mammals only asymmetrically methylated forms (l-NMMA and asymmetric dimethylarginine (ADMA)) but not symmetrically methylated arginine (symmetric dimethylarginine (SDMA)) inhibit nitric oxide synthase (NOS). We and others have proposed that endogenously produced asymmetrically methylated arginines may modulate NO production and that the accumulation of these residues in disease states may contribute to pathology. The activity of the enzyme dimethylarginine dimethylaminohydrolase that metabolises asymmetric methylarginines may be of critical importance in affecting NO pathways in health or disease.

Signal transduction of eNOS activation.

Abstract: Consistent with its classification as a Ca2+/calmodulin-dependent enzyme the constitutive endothelial nitric oxide (NO) synthase (eNOS) can be activated by receptor-dependent and -independent agonists as a consequence of an increase in the intracellular concentration of free Ca2+ ([Ca2+]i) and the association of the Ca2+/calmodulin complex with eNOS. Additional post-translational mechanisms regulate the activity of eNOS, including the interaction of eNOS with caveolin-1, heat shock protein 90 (Hsp90), or membrane phospholipids, as well as enzyme translocation and phosphorylation. In response to fluid shear stress the maintained production of NO by native and cultured endothelial cells is associated with only a transient increase in [Ca2+]i. In the absence of extracellular Ca2+ and in the presence of calmodulin antagonists, shear stress stimulates a maintained production of NO which is insensitive to the removal of extracellular Ca2+, but sensitive to tyrosine kinase inhibitors, Hsp90-binding proteins and phosphatidylinositol 3-kinase inhibitors. A pharmacologically identical activation of eNOS can be induced by protein tyrosine phosphatase inhibitors suggesting that the phosphorylation of eNOS, and possibly that of an associated regulatory protein(s), is crucial for its Ca(2+)-independent activation.

Fibrous cap formation or destruction--the critical importance of vascular smooth muscle cell proliferation, migration and matrix formation.

Abstract: Endothelial activation and infiltration of monocyte macrophages are essential prerequisites for fibrous cap formation, which comprises proliferation and migration of smooth muscle cells and net matrix deposition. Macrophage foam cells and endothelium act as a source of growth factors and chemoattractants for smooth muscle cells. However, growth factors alone do not stimulate smooth muscle cell proliferation or migration. This requires, in addition, the remodelling of the extracellular matrix, at least partly mediated by metalloproteinases. In particular, loss of basement membrane components and contact with the interstitial matrix appears to be required to release a brake on proliferation and migration exerted by the basement membrane. Unless there is a change in the phenotype of macrophages in advanced lesions, it is not clear why fibrous cap destruction rather than formation should take place in macrophage-rich shoulder regions of plaques. Impaired cap formation caused by smooth muscle senescence, mummification and propensity to apoptosis may be as important as increased cap destruction in promoting plaque rupture.

Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm.

Abstract: Objective: We recently demonstrated in our swine model of coronary artery spasm that enhanced myosin light chain (MLC) phosphorylations (both MLC mono- and diphosphorylations) play a central role in the pathogenesis of the spasm. However, the molecular mechanism for and the phosphorylation sites for the enhanced MLC phosphorylations were unknown. In the present study, we addressed these points using hydroxyfasudil, a novel inhibitor of protein kinases, which we found preferentially inhibits Rho-kinase. Methods: The specificity of the inhibitory effects of hydroxyfasudil on Rho-kinase, MLCK, MRCKβ and PKC were examined by kinase assay in vitro. The left porcine coronary artery was chronically treated with interleukin-1β (IL-1β, 2.5 μg). Two weeks after the operation, coronary artery vasomotion was examined both in vivo and in vitro. MLC phosphorylations were examined by Western blot analysis and the sites for the phosphorylations by anti-phosphorylated MLC antibodies that identified the monophosphorylation site as Ser19 and diphophorylation sites as Ser19/Thr18 of MLC. Results: Inhibitory effects of hydroxyfasudil was at least 100 times more potent for Rho-kinase as compared with other protein kinases tested. Intracoronary serotonin (10 μg/kg) caused coronary hyperconstriction at the IL-1β-treated site in vivo, which was dose-dependently inhibited by hydroxyfasudil ( p <0.01). The coronary segment taken from the spastic site also showed hypercontractions to serotonin in vitro, which were again dose-dependently inhibited by hydroxyfasudil ( p <0.01). Western blot analysis showed that MLC monophosphorylation was significantly greater in the spastic segment than in the control segment, while MLC diphosphorylation was noted only at the spastic segment ( p <0.01). The sites for the mono- and diphosphorylated MLC were identified as the monophosphorylated site Ser19 and diphosphorylated sites Ser19/Thr18 of MLC, respectively. Both types of MLC phosphorylations at the spastic segment were markedly inhibited by hydroxyfasudil ( p <0.01). Conclusion: These results indicate that hydroxyfasudil-sensitive Rho-kinase-mediated pathway appears to mediate the enhanced MLC phosphorylations (on Ser19 and Ser19/Thr18 residues) and plays a central role in the pathogenesis of coronary artery spasm.

Angiotensin II-induced superoxide anion generation in human vascular endothelial cells: role of membrane-bound NADH-/NADPH-oxidases.

Abstract: Background: Angiotensin II (ANG II) mediated hypertension accelerates atherosclerosis (AS) and thereby increases the incidence of myocardial infarction (MI). On the other hand, superoxide anion (O2−) is involved in the modification of low density lipoproteins, inhibition of prostacyclin (PGI2) formation and breakdown of nitric oxide. These events finally lead to rapid progression of AS and MI. In the present study, we investigate whether ANG II can induce O2− release from human vascular endothelial cells (HVECs) and the possible mechanisms involved. Methods and Results: The expression of ANG receptors subtype-1 (AT-1) and subtype-2 (AT-2) were identified by using reverse transcription polymerase chain reaction and sequence analysis. The O2− production was dose-dependently increased in HVECs treated with ANG II (10−7–10−9 M) and with a maximum rate after 1 h of incubation. This event was significantly inhibited by pretreatment of cells with the specific AT-1 blocker losartan (10−7 M) and to a lesser extent by the specific AT-2 receptor blocker PD123319 (10−7 M). The combined incubation of both receptor blockers was even more effective. In addition, our lucigenin-enhanced chemiluminescence assay showed that the activity of plasma membrane-bound NADH-/NADPH-oxidases derived from ANG II-treated cells was also significantly increased, this effect was reduced in cells pretreated with losartan or to lesser extent by PD123319. However, the activity of xanthine oxidase remained unchanged in response to ANG II. Furthermore, the basal O2− release from HVECs was inhibited in cells treated with angiotensin-converting enzyme (ACE) inhibitor, Lisinopril (10−6 M), and this event could be reversed by ANG II. Conclusion: ANG II induces O2− release in HVECs via activation of membrane-bound NADH-/NADPH-oxidases, an effect, that is mediated by both AT-1 and AT-2 receptors. This suggests that acceleration of AS and MI in ANG II-mediated hypertension may at least be due to ANG II-induced O2− generation from vascular endothelial cells. In this case, the ACE inhibitors and the ANG receptor antagonists may act as causative “antioxidants”.

Atherosclerotic plaque rupture – pathologic basis of plaque stability and instability

Abstract: Time for primary reveiw 27 days Atherosclerosis continues to be one of the main subjects in pathology research The intriguing complexity of its pathogenesis as well as the importance of its clinical sequelae provide a rationale for this [1] A large number of diseases with totally different clinical presentations are basically atherosclerosis related, and among these, myocardial infarction, stroke, abdominal aneurysms and lower limb ischemia determine to a large extent the morbidity and mortality in Western style populations But, despite this broad spectrum of clinical disease, most of the acute manifestations of atherosclerosis share a common pathogenetic feature: rupture of an atherosclerotic plaque [2–4] Plaque disruptions may vary greatly in extent from tiny fissures or erosions of the plaque surface to deep intimal tears which extend into the soft lipid core of lesions; in all these instances, at least some degree of thrombus formation occurs [5, 6] The abdominal aorta is the arterial site most prominently involved in the process of plaque formation, and also of plaque complications In this large diameter vessel the process of plaque disruption and thrombosis is not ended by luminal occlusion, and may lead to extensive surface ulcerations comprising large areas of the aortic wall, as can be observed in many autopsy cases at older age Apart from the undisputable role of atherosclerosis in abdominal aneurysm formation [7], mural thrombosis leads to a surprisingly low rate of clinically significant complications in these patients, although cholesterol emboli can be regularly found in their kidneys and skin at autopsy Still, it is presently unclear what impact the various biologically active mediators released from eroded aortic surfaces may have on the human body In contrast, in small diameter vessels such as coronary arteries, occlusive thrombosis is a frequent and often fatal complication of plaque … * Corresponding author Tel: +31-20-5665-633; fax: +31-20-914-738; e-mail acvanderwal@amcuvanl

Ionic targets for drug therapy and atrial fibrillation-induced electrical remodeling: insights from a mathematical model

Abstract: Recent advances in molecular electrophysiology have made possible the development of more selective ion channel blockers for therapeutic use. However, more information is needed about the effects of blocking specific channels on repolarization in normal human atrium and in atrial cells of patients with atrial fibrillation (AF). AF-induced electrical remodeling is associated with reductions in transient outward current ( I to), ultrarapid delayed rectifier current ( I Kur), and L-type calcium current ( I Ca,L). Direct evaluation of the results of ion channel depression is limited by the nonspecificity of the available pharmacological probes. Objectives: Using a mathematical model of the human atrial action potential (AP), we aimed to: (1) evaluate the role of ionic abnormalities in producing AP changes characteristic of AF in humans and (2) explore the effects of specific channel blockade on the normal and AF-modified AP (AFAP). Methods: We used our previously developed mathematical model of the normal human atrial AP (NAP) based on directly measured currents. We constructed a model of the AFAP by incorporating experimentally-measured reductions in I to (50%), I Kur (50%), and I Ca,L (70%) current densities observed in AF. Results: The AFAP exhibits the reductions in AP duration (APD) and rate-adaption typical of AF. The reduction in I Ca,L alone can account for most of the morphological features of the AFAP. Inhibition of I to by 90% leads to a reduction in APD measured at −60 mV in both the NAP and AFAP. Inhibition of the rapid component of the delayed rectifier ( I Kr) by 90% slows terminal repolarization of the NAP and AFAP and increases APD by 38% and 34%, respectively. Inhibition of I Kur by 90% slows early repolarization and increases plateau height, activating additional I K and causing no net change in APD at 1 Hz in the NAP. In the presence of AF-induced ionic modifications, I Kur inhibition increases APD by 12%. Combining I Kur and I Kr inhibition under both normal and AF conditions synergistically increases APD. In the NAP, altering the model parameters to reproduce other typical measured AP morphologies can significantly alter the response to K+-channel inhibition. Conclusions: (1) The described abnormalities in I to, I Kur and I Ca,L in AF patients can account for the effects of AF on human AP properties; (2) AP prolongation by I Kur block is limited by increases in plateau height that activate more I K; (3) Blockers of I Kur may be more effective in prolonging APD in patients with AF; 4) Inhibition of both I Kur and I Kr produces supra-additive effects on APD. These observations illustrate the importance of secondary current alterations in the response of the AP to single channel blockade, and have potentially important implications for the development of improved antiarrhythmic drug therapy for AF.

Apoptosis of vascular smooth muscle cells in vascular remodelling and atherosclerotic plaque rupture.

Abstract: Apoptosis (programmed cell death) of vascular smooth muscle cells (VSMCs) has recently been identified as an important process in a variety of human vascular diseases, including atherosclerosis, arterial injury, and restenosis after angioplasty. VSMC apoptosis is regulated by interactions between the local cell-cell and cytokine environment within the arterial wall, and the expression of pro- and anti-apoptotic proteins by the cell, including death receptors, proto-oncogenes and tumour suppressor genes. This review summarises our current knowledge of the occurrence and mechanisms underlying VSMC apoptosis in atherosclerosis and arterial remodelling.

Nitric oxide and the proliferation of vascular smooth muscle cells.

Abstract: Time for primary review 13 days. Vascular smooth muscle cell (VSMC) proliferation is an important component of vessel wall remodelling in response to injury, for example, after angioplasty or vein grafting, and during atherosclerosis formation. Endothelium-derived nitric oxide (NO) production is both a tonic and an induced regulator of blood vessel tone [1–3]. Its function is impaired by atherosclerosis and, more significantly, by atherogenic risk factors, including hypercholesterolaemia, homocysteinaemia, diabetes, smoking and high blood pressure, even before the appearance of overt atherosclerosis [4–6] Endothelial NO production is dysfunctional after balloon injury and in vein grafts at the time when VSMC proliferation and neointima formation is progressing [5,6]. It has been tempting, therefore, to propose a causal relationship between impaired NO production and increased VSMC proliferation. If so, this might explain, in part, the association between endothelial dysfunction and atherogenesis. The primary purpose of this review is to discuss critically the evidence to support such an hypothesis. We will also go on to consider the molecular mechanisms that might underlie the inhibitory effects of NO on VSMC proliferation, with the following important caveats. Firstly, any direct action of NO on an increase in VSMC numbers may be mediated at a variety of levels, for example, on the signal transduction pathways, on energy production or by promoting cell death. Secondly, in the more complex in vivo models, effects of NO on endothelial cells (ECs), platelets and inflammatory cells, rather than directly on VSMCs may be responsible for modulating VSMC proliferation. Thirdly, NO is highly unstable, with a half-life measured in seconds [2,7,8]. It reacts rapidly with oxygen species (O2, O2− and H2O2) to produce, nitrite, nitrate or the highly reactive species, peroxynitrite (ONOO) and with thiol groups to produce nitrosothiols [9]. … * Corresponding author. Tel.: +44-117-928-3154; fax: +44-117-929-9737 j.y.jeremy{at}bristol.ac.uk

Heme oxygenase-carbon monoxide signalling pathway in atherosclerosis: anti-atherogenic actions of bilirubin and carbon monoxide?

Abstract: Atherosclerosis is a major contributor to cardiovascular disease, and genetic disorders of lipoprotein metabolism are recognized risk factors in atherogenesis. The gaseous monoxides nitric oxide (NO) and carbon monoxide (CO), generated within the blood vessel wall, have been identified as important cellular messengers involved in the regulation of vascular smooth muscle tone. Microsomal heme oxygenases degrade heme to biliverdin and CO, and the cytosolic enzyme biliverdin reductase then catalyzes reduction of biliverdin to bilirubin, both powerful chain-breaking antioxidants. Two principal isozymes of heme oxygenase have been identified, a constitutive isoform HO-2 (M(r) approximately 34,000) and an inducible isoform HO-1 (M(r) approximately 32,000), which is expressed at a low basal level in vascular endothelial and smooth muscle cells and is induced by heavy metals, oxidative stress, inflammatory mediators and oxidized low density lipoproteins. Although NO and CO modulate intracellular cGMP levels, platelet aggregation and smooth muscle relaxation, CO has a much lower affinity for soluble guanylyl cyclase than NO. Decreased production or sensitivity to NO in atherosclerosis may be compensated for by an induction of HO-1, with bilirubin acting as a cellular antioxidant and CO as a vasodilator. This review examines the evidence that oxidized low density lipoproteins (LDL), hypoxia and pro-inflammatory cytokines induce HO-1 expression and activity in vascular endothelial and smooth muscle cells, and evaluates the anti-atherogenic potential of the heme oxygenase signalling pathway.

The infarcted myocardium: simply dead tissue, or a lively target for therapeutic interventions.

Abstract: Time for primary review 32 days. It has been known for many years that infarction of the heart induces prominent alterations of cardiac structure. The most apparent is the scarring of the infarct. Structural changes after infarction are, however, not limited to the infarcted area, but also extend into the non-infarcted myocardium. Changes in the non-infarcted myocardium include hypertrophy of the cardiomyocytes, growth of the capillary network, and an increase in interstitial collagen. Cardiac structure is a major determinant of function, which is depressed after myocardial infarction (MI). After infarction, both short term and long term compensatory or regulatory mechanisms are activated. Often these mechanisms also affect cardiac structure. Although activation of these compensatory mechanisms may be beneficial early after infarction, they may have adverse effects, when activation is continued for a longer time. Indeed, pharmacological treatments that block the long term activation of these compensatory mechanisms, like angiotensin converting enzyme inhibitors (ACEI) that block the renin—angiotensin system (RAS), have been shown to improve cardiac function after infarction. Although we know that cardiac function and structure are closely related and do indeed both change after infarction, it is largely unknown what the exact structural component is that causes the reduction in cardiac function after infarction. Also it is not clear which structural component should be targeted for effective pharmacotherapy after infarction. In this review we attempt to clarify the structural alterations after infarction. We and others have focused for many years on the potential importance of changes in the vital non-infarcted myocardium and, indeed, found several alterations in cardiac structure after infarction and effects thereon of drugs that improved cardiac function. However, recent data in animal studies and humans point to the importance of the infarct itself as a potential target for intervention. The infarct appears to be more than … * Corresponding author. Tel.: +31-43-387-6631; fax: +31-43-387-6613 jcl{at}lpat.azm.nl

Nitric oxide and penile erection: is erectile dysfunction another manifestation of vascular disease?

Abstract: There is convincing evidence that the prevalence of erectile dysfunction is increased among men with ischaemic heart disease. This association may be attributed to the fact that both erectile dysfunction and ischaemic heart disease share similar risk factors (e.g. hypertension, dyslipidaemia, diabetes and smoking). Nitric oxide (NO) activity is adversely affected, in penile and vascular tissue, by these risk factors. It is therefore not surprising that a defect in NO activity is thought to play a role in the pathogenesis of both erectile dysfunction and ischaemic heart disease. We consider this evidence and propose that defective NO activity provides a unifying explanation for the association between these two conditions. Further research in this area may improve our understanding of the pathogenesis of cardiovascular diseases as a whole.

Collagen synthesis in atherosclerosis: too much and not enough.

Abstract: Fibrillar collagen is a critical component of atherosclerotic lesions. Uncontrolled collagen accumulation leads to arterial stenosis, while excessive collagen breakdown combined with inadequate synthesis weakens plaques thereby making them prone to rupture. This review discusses cellular sources of collagen synthesis in atherosclerosis, local and systemic factors modulating collagen gene expression, as well as temporal and spatial patterns of collagen production in human and experimental atherosclerotic lesions.

Afterload induced changes in myocardial relaxation: a mechanism for diastolic dysfunction.

Abstract: Background: Diastolic left ventricular (LV) dysfunction manifests as an upward shift of the diastolic pressure–volume relation. One of the possible causes of diastolic LV dysfunction is incomplete myocardial relaxation. It is well known that high afterload slows myocardial relaxation. This contribution investigated to what extent afterload elevation could also affect LV filling pressures including end-diastolic LV pressure (LVP). Methods: Selective, beat-to-beat elevations of afterload were induced in anaesthetised open-chest rabbits ( n =9) by abrupt narrowing of the ascending aorta during the diastole of the preceding heartbeat. This was performed with physiological heart rate and blood pressure. Results: These interventions increased systolic LVP from 90±3 mm Hg at baseline to 103±4, 123±5, 139±5 and 154±6 mm Hg. The last intervention was a total aortic occlusion inducing a first beat isovolumetric contraction. Smaller afterload elevations decreased τ (accelerated LVP fall) and did not elevate diastolic pressure-internal diameter relation (P-ID). Larger afterload elevations increased τ (decelerated LVP fall), induced an upward shift of the diastolic P-ID and increased end-diastolic LVP. Effects of afterload on end-diastolic LVP were correlated with effects on τ ( r =0.89; P <0.01). Incomplete relaxation or load-dependent residual active state appeared to be the mechanism for this diastolic dysfunction. Similar findings were made retrospectively in dogs instrumented with circumferential segment length gauges ( n =16). Conclusions: Diastolic LV dysfunction was induced by elevated afterload in healthy hearts of rabbits and dogs. If this mechanism could be shown to be operative in the failing heart, reversal of diastolic dysfunction should contribute to the beneficial effects of vasodilating and inotropic therapy on pulmonary congestion.

Vascular endothelial growth factor up-regulates nitric oxide synthase expression in endothelial cells

Abstract: Objective: Vascular endothelial growth factor (VEGF), secreted by vascular cells and a variety of tumour cells, is a potent angiogenic factor. Since nitric oxide (NO) seems to play a key role in the VEGF-induced proliferation of endothelial cells, the aim of the present study was to determine whether VEGF stimulates endothelial NO synthase (eNOS) expression and hence results in a maintained increase in NO formation. Methods: Experiments were performed using cultured human umbilical vein endothelial cells (HUVEC) and isolated rat aortic rings. eNOS expression was assessed by Western blotting and RT-PCR analysis. Results: Exposure of either confluent HUVEC or rat aortic rings to VEGF165 significantly increased eNOS mRNA and protein levels. This stimulatory effect of VEGF165 on eNOS expression was associated with an elevation in the basal production of cGMP in HUVEC, and with a leftward shift of the concentration–relaxation curve to acetylcholine in aortic rings. The VEGF-induced increase in eNOS mRNA levels was abolished by tyrosine kinase inhibitors suggesting involvement of a tyrosine kinase-dependent pathway. Since eNOS mRNA levels remained elevated in VEGF-treated cells in the presence of actinomycin D, it is likely that the VEGF-induced up-regulation of eNOS expression may be a consequence of a post-transcriptional effect on eNOS mRNA stability. Conclusion: The results demonstrate that VEGF enhances the expression of eNOS in native and cultured endothelial cells, an effect which may be important in the process of VEGF-induced angiogenesis.

Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen.

Abstract: Objective: Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin α2β1 in platelet activation by collagen. Methods: Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin α2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. Results: (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer α2β1-binding sequence in a repeat Gly-Pro-Pro structure supported α2β1-mediated platelet and HT 1080 cell adhesion and bound α2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize α2β1, but was highly platelet activatory. Conclusions: Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of α2β1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.

Reversal of atrial electrical remodeling following cardioversion of long-standing atrial fibrillation in man

Abstract: Background: In animal studies, atrial fibrillation has been shown to shorten the atrial refractory period and impair its rate adaptation. However, little is known about the effects of chronic atrial fibrillation on atrial electrophysiology and its recovery course in humans. Methods and results: Nineteen patients, mean age 64±14 years, with chronic atrial fibrillation of more than six months duration were included in this study. All of them were successfully converted to sinus rhythm with an external defibrillator. Atrial effective refractory periods at right atrial appendage and distal coronary sinus were determined with five pacing cycle lengths (300, 400, 500, 600 and 700 ms) at 30 min after cardioversion and once a day for four days. The atrial conduction properties, including P wave duration of surface ECG, and right and left atrial conduction times, were also measured at the same time interval. Twenty age-matched patients without a history of atrial tachyarrhythmia were evaluated as controls. In comparison with controls, chronic atrial fibrillation significantly shortened the atrial effective refractory period, impaired its rate adaptation response, especially at distal coronary sinus, and depressed the conduction properties of atria. The atrial conduction properties did not change during the four-day follow-up period; however, the atrial effective refractory period was gradually prolonged and its rate adaptation response improved after restoration of sinus rhythm. Conclusions: In humans, chronic atrial fibrillation significantly shortened the atrial effective refractory period, and impaired its rate adaptation response. Restoration and maintenance of sinus rhythm could reverse these electrophysiological changes.

Activity of cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase in failing and nonfailing human hearts

Abstract: Objectives: A hallmark of human heart failure is prolonged myocardial relaxation. Although the intrinsic mechanism of phospholamban coupling to the Ca2+-ATPase is unaltered in normal and failed human hearts, it remains possible that regulation of phospholamban phosphorylation by cAMP-dependent mechanisms or other second messenger pathways could be perturbed, which may account partially for the observed dysfunctions of the sarcoplasmic reticulum (SR) associated with this disease. Methods: cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase) were characterized initially by DEAE-Sepharose chromatography in hearts from patients with end-stage dilated cardiomyopathy. We measured the activity of PKA and CaM kinase in left ventricular tissue of failing (idiopathic dilated cardiomyopathy; ischemic heart disease) and nonfailing human hearts. Results: Basal PKA activity was not changed between failing and nonfailing hearts. One major peak of CaM kinase activity was detected by DEAE-Sepharose chromatography. CaM kinase activity was increased almost 3-fold in idiopathic dilated cardiomyopathy. In addition, hemodynamical data (left ventricular ejection fraction, cardiac index) from patients suffering from IDC positively correlate with CaM kinase activity. Conclusions: Increased CaM kinase activity in hearts from patients with dilated cardiomyopathy could play a role in the abnormal Ca2+ handling of the SR and heart muscle cell.

The role of corticosteroids in the regulation of vascular tone

Abstract: Time for primary review 31 days. Disease states resulting from excesses of circulating (adreno)corticosteroids include primary hyperaldosteronism, renal artery stenosis, ACTH-secreting tumors, and administration of glucocorticoids for treatment of other diseases. Hypertension is commonly associated with these diseases. Although renal sodium retention and intravascular volume overload contribute to the attendant hypertension, especially early in the course of the disease, a non-renal mechanism (increase in peripheral vascular resistance) is involved in the development and maintenance of hypertension. The concept of non-renal actions of corticosteroids in the development of hypertension stems from a seminal report in which Langford and Snavely [1]demonstrated that deoxycorticosterone acetate raised blood pressure in dogs and rats devoid of renal mass. In addition, corticosteroids in lesser amounts are essential for the maintenance of peripheral vascular resistance in healthy persons. This review details the proposed mechanisms by which corticosteroids maintain and, in excess, enhance vascular tone. If corticosteroids indeed regulate vascular tone, vascular smooth muscle cells (VSMCs), the vasoactive element of the vasculature, should contain specific receptor molecules for corticosteroids. Classically, corticosteroid receptors are considered to be members of the steroid receptor superfamily of ligand-dependent transcription factors [2]. Radioligand binding studies have defined two distinct cytosolic corticosteroid receptors. Type I (mineralocorticoid) receptors bind with greatest affinity to aldosterone, deoxycorticosterone or corticosterone and with less affinity to the synthetic glucocorticoid dexamethasone. In contrast, type II (glucocorticoid) receptors bind with greatest affinity to dexamethasone and with less affinity to aldosterone, deoxycorticosterone or corticosterone. There is significant base sequence homology between these receptors. Corticosteroid receptors possess highly conserved regions that are necessary for ligand binding, receptor dimerization, nuclear translocation, DNA binding and transactivation (recruiting accessory proteins so that transcription will initiate). The genes for the rat [3, 4]and human [5, 6]mineralocorticoid receptor and the human [7]glucocorticoid receptor … * Tel.: +843-792-4122; fax: +843-792-8399; e-mail: ullianme@musc.edu

Ca channels in cardiac myocytes: structure and function in Ca influx and intracellular Ca release

Abstract: There are Ca channels in the plasma membrane and also the sarcoplasmic reticulum (SR) membrane in cardiac myocytes. The relationship between channel structure, associated proteins and function of these Ca channels is discussed. The sarcolemmal Ca channels are crucial both to the basic cellular electrophysiological properties and control of cardiac contractility (via excitation-contraction coupling). The intracellular Ca release channels (or ryanodine receptors) respond to triggering events mediated by sarcolemmal ion currents and are largely responsible for releasing Ca which activates the myofilaments to produce contraction. Several possible mechanisms of excitation-contraction coupling are discussed. The Ca released from the SR can also feedback on several sarcolemmal ion currents and alter action potential configuration as well as contribute to arrhythmogenesis.

Leucocyte recruitment in rupture prone regions of lipid-rich plaques: a prominent role for neovascularization?

Abstract: Objective: Microvessels in atherosclerotic plaques provide an alternative pathway for the recruitment of leucocytes in the lesions. The present study was designed to investigate the potential role of these microvessels in creating vulnerable sites in atherosclerotic plaques. Methods: Thirty-four atherosclerotic plaques were obtained from 25 patients undergoing carotid endartherectomy ( n =16), femoral endartherectomy ( n =6) and aortic surgery ( n =12). Plaques were histologically classified as either lipid-rich (rupture prone, n =21) or fibrous (stable, n =13). Serial cryostat sections were immunohistochemically investigated using monoclonal antibodies against endothelial cells (ULEX-E and F-VIII), vascular endothelial growth factor (VEGF), endothelial adhesion molecules (ICAM-1, VCAM-1, E-Selectin, CD40) and inflammatory cells (macrophages (CD68) and T lymphocytes (CD3). Results: The microvessel density in lipid-rich plaques was significantly increased as compared to fibrous plaques. Most of these vessels were located in the shoulder-region of the plaque and at the base of the atheroma. Microvessels in lipid-rich plaques also expressed increased levels of ICAM-1, VCAM-1, E-Selectin and CD40. Moreover, inflammation was most abundantly present in the proximity of microvessels. VEGF was only observed on vessels and mononuclear cells in lipid-rich plaques, suggesting that this factor may play a role in microvessels formation. Conclusions: Neovascularisation and expression of adhesion molecules by microvessels at sites of vulnerable lipid-rich plaques may sustain the influx of inflammatory cells and hence, could contribute to plaque destabilization.

Cardiac fibrosis and inflammation: interaction with hemodynamic and hormonal factors.

Abstract: It is generally admitted that the pathogenesis of perivascular and interstitial cardiac fibrosis involves the response to two types of stimuli: a hormonal one, mainly involving the renin-angiotensin-aldosterone system and the more recently described endothelin system, and a hemodynamic stimulus, particularly high blood pressure. We propose in the present review a third step which, although not exclusive, interacts with the hormonal and hemodynamic ones, and involves inflammatory mechanisms. Indeed, hypertension is invariably associated with inflammatory cell infiltration either in the intimal part of large vessels or in the adventitial region of arterioles. This has led us to hypothesize that arterial wall cells may trigger the initial communications attracting inflammatory cells to the perivascular region. In this paper, we review the proinflammatory intercellular communications as well as the intracellular signaling which confer an inflammatory phenotype to arteries. In this context, the profibrogenic and proinflammatory effects of hemodynamic overload and peptidergic systems such as angiotensin II and endothelin are considered. The study of the inflammatory process is not without interest, especially in view of the strong modulating effect of the inflammatory mediators both on the inflammatory process itself and on the fibrotic process. The principal and the most potent mediators are reviewed. Finally, the hypothesis that the inflammatory process could be in reality an immune specific process is suggested.

Regulation of basal myocardial function by NO

Abstract: The effects of exogenous and endogenous. NO on myocardial functions such as contraction, relaxation and heart rate have recently gained considerable scientific interest. .NO stimulates myocardial soluble guanylate cyclase to produce cGMP, which activates two major target proteins. A small increase in cGMP levels predominantly inhibits phosphodiesterase III, while high cGMP levels activate cGMP-dependent protein kinase. Accordingly, submicromolar .NO concentrations improve myocardial contraction, while submillimolar .NO concentrations decrease contractility. The latter action includes direct inhibitory .NO effects on ATP synthesis and voltage-gated calcium channels. Overall, the inotropic effects of exogenous .NO are small and probably of minor importance for myocardial contractility. Cardiomyocytes are capable of expressing eNOS and iNOS. Endogenous .NO has effects on myocardial contraction, similar to that of exogenous .NO. Various NOS inhibitors can substantially reduce myocardial contractility in vitro and in vivo, suggesting that basal endogenous .NO production supports myocardial contractility. There is also evidence for a .NO-dependent cardiodepressive effect of cytokines that is mediated by expression of iNOS. This is consistent with the negative inotropic effects of .NO at high concentrations. Cardiodepressive actions of endogenous .NO production may play a role in certain forms of heart failure. Finally, .NO also has an effect on heart rate. Physiologic .NO concentrations can stimulate heart rate by activating the hyperpolarization-activated inward current (If) and this effect decreases at submillimolar .NO concentrations. In summary, physiological concentrations of .NO increase contractility and heart rate under basal conditions, while high .NO concentrations induce the opposite effects.

Myocardial ischaemia and the cardiac nervous system

Abstract: The intrinsic cardiac nervous system has been classically considered to contain only parasympathetic efferent postganglionic neurones which receive inputs from medullary parasympathetic efferent preganglionic neurones In such a view, intrinsic cardiac ganglia act as simple relay stations of parasympathetic efferent neuronal input to the heart, the major autonomic control of the heart purported to reside solely in the brainstem and spinal cord Data collected over the past two decades indicate that processing occurs within the mammalian intrinsic cardiac nervous system which involves afferent neurones, local circuit neurones (interconnecting neurones) as well as both sympathetic and parasympathetic efferent postganglionic neurones As such, intrinsic cardiac ganglionic interactions represent the organ component of the hierarchy of intrathoracic nested feedback control loops which provide rapid and appropriate reflex coordination of efferent autonomic neuronal outflow to the heart In such a concept, the intrinsic cardiac nervous system acts as a distributive processor, integrating parasympathetic and sympathetic efferent centrifugal information to the heart in addition to centripetal information arising from cardiac sensory neurites A number of neurochemicals have been shown to influence the interneuronal interactions which occur within the intrathoracic cardiac nervous system For instance, pharmacological interventions that modify β-adrenergic or angiotensin II receptors affect cardiomyocyte function not only directly, but indirectly by influencing the capacity of intrathoracic neurones to regulate cardiomyocytes Thus, current pharmacological management of heart disease may influence cardiomyocyte function directly as well as indirectly secondary to modifying the cardiac nervous system This review presents a brief summary of developing concepts about the role of the cardiac nervous system in regulating the normal heart In addition, it provides some tentative ideas concerning the importance of this nervous system in cardiac disease states with a view to stimulating further interest in neural control of the heart so that appropriate neurocardiological strategies can be devised for the management of heart disease