Coronary Vascular Endothelium Research Articles

PRODUCTION OF GROWTH FACTORS AND DESQUAMATION OF ENDOTHELIOCYTES IN THE HEART IN ISCHEMIC CARDIOMYOPATHY

Highlights Dysregulation of angiogenesis may be the pathogenetic factor of ischemic cardiomyopathy (ICMP). Aim. To determine the content of growth factors and desquamated endothelial cells (DEC) in the blood from the coronary sinus and ulnar vein in association with the number of progenitor endothelial cells (PEC) in the blood from the ulnar vein in patients with coronary heart disease (CHD), suffering and not suffering from ICMP.Methods. The study included 30 patients with ICMР and 22 patients with CHD, and 18 healthy donors. The content of DEC (CD45–CD146+) was determined in blood from the cubital vein (peripheral) and coronary sinus, and the content of DEC (CD14+CD34+VEGFR2+) was determined in peripheral blood by flow cytometry (antibodies “BD Biosciences”, USA). The concentrations of VEGF-A, VEGF-B, PDGF, SDF-1, SCF, FGF-1, TGF-β1 in blood plasma from both locations were evaluated by multiplex analysis (set “Cloud-Clone Corp.”, USA).Results. The content of DEC in peripheral blood was elevated in patients with CHD of both groups, and in patients with ICMP in sinus blood was higher than in peripheral. At the same time, in patients with CHD without cardiomyopathy, an excess of PEC and SDF-1 in the blood from the ulnar vein was established in combination with an increase in the concentration of PDGF and a decrease in the content of VEGF-B in the blood from the coronary sinus relative to the parameters of systemic blood flow. In patients with ICMP, these changes were not detected, but there was an increase in the concentration of TGF-β1 in sinus blood compared with peripheral blood. Regardless of the presence of ICMP, the concentration of SCF, FGF-1, VEGF-A in the blood from the ulnar vein corresponded to the norm and that in sinus blood; the content of VEGF-A in the coronary bloodstream exceeded its systemic level.Conclusion. In patients with ICMP, desquamation of the coronary vascular endothelium is enhanced against the background of violations of its repair processes due to insufficient (relative to CHD without cardiomyopathy) mobilization of PEC from the bone marrow due to the absence of an excess of SDF-1 in the blood and their insufficient homing into the myocardium due to weak PDGF production in the heart.

Guidelines for the Prevention of Symptomatic Cardiovascular Disease, Based upon the Presence of Coronary Artery Calcified Plaque—Provided by the Society for the Prevention of Symptomatic Heart Disease

Introduction: The epidemic of atherosclerotic cardiovascular disease in the Western World accounts for the majority of morbidity and mortality in adults. This disease is on the increase, and previous recommendations using “risk equations” to prescribe preventive treatment have not been successful in reducing the prevalence of cardiovascular disease. Furthermore, compliance with statin medication has been limited, with approximately 50% of individuals being non-compliant within five years. This situation is unacceptable since atherosclerosis is both preventable and reversible. Methods: The guidelines presented in this article utilize coronary artery calcium scanning as the basis for preventative therapeutic decisions and identifying the presence of asymptomatic cardiovascular disease. This radiographic technique is superior to “risk equations” in predicting future cardiovascular events. It provides a comprehensive assessment of the lifelong insults to the coronary artery vascular endothelium and the resulting inflammation. Coronary artery calcium scanning is widely available, inexpensive, safe, and reproducible. It has the major advantage of increasing treatment compliance in patients with positive coronary artery calcium scores. Results: All suggested guidelines are supported by published scientific data. Citations are provided to allow the reader to obtain further information. The authors are available for further consultation. Each guideline provides specific recommendations that the primary caregiver can discuss with the patient. Patient involvement in decision-making is strongly recommended. Both treatment costs and adverse effects are minimal. Conclusion: It is anticipated that the early identification of asymptomatic cardiovascular disease and its aggressive treatment will result in regression of subclinical atherosclerosis. Adoption of these guidelines will stop the epidemic of symptomatic heart disease and result in healthier and more satisfied patients.

Coronary vasodilation mediated by T cells expressing choline acetyltransferase

CD4+ T cells expressing choline acetyltransferase (ChAT) have recently been shown to cause a drop in systemic blood pressure when infused into mice. The aim of this study was to determine if ChAT-expressing T cells could regulate coronary vascular reactivity. Preconstricted segments of epicardial and intramyocardial porcine coronary arteries relaxed in response to Jurkat T cells (JT) that overexpressed ChAT (JTChAT cells). The efficacy of the JTChAT cells was similar in epicardial and intramyocardial vessels with a maximum dilator response to 3 × 105 cells/mL of 38.0 ± 6.7% and 38.7 ± 7.25%, respectively. In contrast, nontransfected JT cells elicited a weak dilator response, followed by a weak contraction. The response of JTChAT cells was dependent on the presence of the endothelial cells. In addition, the response could be significantly reduced by Nω-nitro-l-arginine methyl ester (l-NAME) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) in the presence of indomethacin. JTChAT cells, but not JT cells, increased the expression of phosphorylated endothelial nitric oxide synthase (eNOS). JTChAT cells contained significantly greater levels of acetylcholine compared with JT cells; however, the nonselective muscarinic antagonist atropine and the M1 receptor antagonist pirenzepine both failed to block the dilator effect of JTChAT cells. Exogenously added acetylcholine induced only a weak relaxation (∼10%) at low concentrations, which became a contractile response at higher concentrations. These data illustrate the capacity for cells that express ChAT to regulate coronary vascular reactivity, via mechanisms that are dependent on interaction with the endothelium and in part mediated by the release of nitric oxide.NEW & NOTEWORTHY This study shows ChAT-expressing T cells can induce vasodilation of the blood vessel in the coronary circulation and that this effect relies on a direct interaction between T cells and the coronary vascular endothelium. The study establishes a potential immunomodulatory role for T cells in the coronary circulation. The present findings offer an additional possibility that a deficiency of ChAT-expressing T cells could contribute to reduced coronary blood flow and ischemic events in the myocardium.

Identification of cardiac hemo-vascular precursors and their requirement of sphingosine-1-phosphate receptor 1 for heart development

The cardiac endothelium plays a crucial role in the development of a functional heart. However, the precise identification of the endocardial precursors and the mechanisms they require for their role in heart morphogenesis are not well understood. Using in vivo and in vitro cell fate tracing concomitant with specific cell ablation and embryonic heart transplantation studies, we identified a unique set of precursors which possess hemogenic functions and express the stem cell leukemia (SCL) gene driven by its 5′ enhancer. These hemo-vascular precursors give rise to the endocardium, atrioventricular cushions and coronary vascular endothelium. Furthermore, deletion of the sphingosine-1-phosphate receptor 1 (S1P1) in these precursors leads to ventricular non-compaction cardiomyopathy, a poorly understood condition leading to heart failure and early mortality. Thus, we identified a distinctive population of hemo-vascular precursors which require S1P1 to exert their functions and are essential for cardiac morphogenesis.

A novel role of CDX1 in embryonic epicardial development.

The molecular mechanism that regulates epicardial development has yet to be understood. In this study, we explored the function of CDX1, a Caudal-related family member, in epicardial epithelial-to-mesenchymal transition (EMT) and in the migration and the differentiation of epicardium-derived progenitors into vascular smooth muscle cells. We detected a transient expression of CDX1 in murine embryonic hearts at 11.5 days post coitum (dpc). Using a doxycycline-inducible CDX1 mouse model, primary epicardium, and ex vivo heart culture, we further demonstrated that ectopic expression of CDX1 promoted epicardial EMT. In addition, a low-dose CDX1 induction led to enhanced migration and differentiation of epicardium-derived cells into α-SMA+ vascular smooth muscles. In contrast, either continued high-level induction of CDX1 or CDX1 deficiency attenuated the ability of epicardium-derived cells to migrate and to mature into smooth muscles induced by TGF-β1. Further RNA-seq analyses showed that CDX1 induction altered the transcript levels of genes involved in neuronal development, angiogenesis, and cell adhesions required for EMT. Our data have revealed a previously undefined role of CDX1 during epicardial development, and suggest that transient expression of CDX1 promotes epicardial EMT, whereas subsequent down-regulation of CDX1 after 11.5 dpc in mice is necessary for further subepicardial invasion of EPDCs and contribution to coronary vascular endothelium or smooth muscle cells.

2 IL-1B Secretion in Coronary Vascular Endothelium is Mediated by Neutrophil Serine Protease and is Independent of caspase-1

Endothelial cells (ECs) are critically involved in the pathogenesis of atherosclerosis by producing inflammatory mediators, including interleukin-1 beta (IL-1β). However, its mechanism of externalisation is yet to be elucidated. This study investigates the effect of neutrophil elastase (NE) on ECs in terms of IL-1β release and explores the underlying mechanism. Human coronary artery endothelial cells (HCAEC) were treated with a combination of tumour necrosis factor-alpha and interleukin-1 alpha then incubated with NE for 2 or 6 h. ELISA, western blotting, flow cytometry and live cell imaging were used. Paraffin-embedded sections of aortic sinus lesions of apoe-/- mice were immunostained for NE and VWF (Von Willebrand factor). NE is predominantly expressed in ECs in experimental atherosclerosis. In vitro , NE caused significant IL-1β release into supernatants after 6h (579 ± 90 vs. 80 ± 19 pg/ml in control; n = 4, p < 0.0001). The release was via microparticle shedding which is significantly attenuated by neutrophil elastase inhibitor III (62.64 ± 10 vs. 579.3 ± 90 pg/ml in NE-primed cells; n = 3, p<0.05); but, the levels of IL-1β in the supernatant or lysate did not change in the presence of caspase-1 inhibitor. NE enters ECs, cleaves proIL-1β (31kDa) inside cells resulting in release of active isoforms of IL-1β in microparticles. No remarkable effects were seen on NALP3 or caspase-1 expression in EC lysates. This is the first description of the secretion of IL-1β by microvesicle shedding from ECs which is caspase-1 independent. NE is detected in the endothelium of murine atherosclerotic plaques and, therefore, this is a plausible mechanism to promote local IL-1β release in the vasculature.

Heterogeneity of endothelial cell phenotype within and amongst conduit vessels of the swine vasculature

The purpose of this study was to investigate the extent of endothelial cell phenotypic heterogeneity throughout the swine vasculature, with a focus on the conduit vessels of the arterial and venous circulations. We tested the hypothesis that atheroprone arteries exhibit higher expression of markers of inflammation and oxidative stress than do veins and atheroresistant arteries. The study sample included tissues from 79 castrated, male swine. Immediately after the animals were killed, endothelial cells were mechanically scraped from isolated segments of the thoracic and abdominal aorta, carotid, brachial, femoral and renal arteries, and the vein regionally associated with each of these vessels, as well as the internal mammary and right coronary arteries. Cells were also taken from two regions of the aortic arch contrasted by atheroprone versus atherosusceptible haemodynamics. Endothelial cell phenotype was assessed by either immunoblotting or quantitative real-time PCR for a host of both pro- and anti-atherogenic markers (e.g. endothelial nitric oxide synthase, p67phox, cyclo-oxygenase-1 and superoxide dismutase 1). Marked heterogeneity across the vasculature was observed in the expression of both pro- and anti-atherogenic markers, at both the protein and transcriptional levels. In particular, the coronary vascular endothelium expressed higher levels of the oxidative stress marker p67phox (P < 0.05 versus other arteries). In addition, differential expression of endothelial nitric oxide synthase and KLF4 was evident between atheroprone and atherosusceptible regions of the aorta, while expression of endothelial nitric oxide synthase, KLF2, KLF4 and cyclo-oxygenase-1 was lower in both areas of the aortic arch compared with the internal mammary artery. Conduit arteries typically expressed higher levels of both pro- and anti-atherogenic markers relative to their associated veins. We show, for the first time, that endothelial cell phenotype is variable within vessels, across six major vascular territories, and between the arterial and venous circulations. Importantly, even straight vessel segments from systemic conduit arteries (e.g. brachial and carotid arteries) exhibited regional phenotypic heterogeneity; a finding not expected on the basis of local haemodynamic forces alone.

Distinct Compartments of the Proepicardial Organ Give Rise to Coronary Vascular Endothelial Cells

The proepicardial organ is an important transient structure that contributes cells to various cardiac lineages. However, its contribution to the coronary endothelium has been disputed, with conflicting data arising in chick and mouse. Here we resolve this conflict by identifying two proepicardial markers, Scleraxis (Scx) and Semaphorin3D (Sema3D), that genetically delineate heretofore uncharacterized proepicardial subcompartments. In contrast to previously fate-mapped Tbx18/WT-1-expressing cells that give rise to vascular smooth muscle, Scx- and Sema3D-expressing proepicardial cells give rise to coronary vascular endothelium both in vivo and in vitro. Furthermore, Sema3D(+) and Scx(+) proepicardial cells contribute to the early sinus venosus and cardiac endocardium, respectively, two tissues linked to vascular endothelial formation at later stages. Taken together, our studies demonstrate that the proepicardial organ is a molecularly compartmentalized structure, reconciling prior chick and mouse data and providing a more complete understanding of the progenitor populations that establish the coronary vascular endothelium.

Editorial Comment: Myocardial protection with postconditioning in cardiac surgery: the importance of the model

In this issue of the European Journal of Cardio-Thoracic Surgery, Maruyama and Chambers [1] pose the question of whether postconditioning (POC) enhances the protection provided by normothermic St Thomas’ number 2 (ST2) cardioplegia solution. This is an excellent question, posed by a highly productive group, and whether POC provides protection in addition to that exerted by cardioplegia is a question that has been asked by others [2]. Maruyama and Chambers found that a POC algorithm applied after increasing durations of arrest induced by a single infusion of warm St Thomas’ number 2 (ST2) cardioplegia did not enhance the primary end points of recovery of contractile function or reduce infarct size relative to that observed after arrest and reperfusion alone. It is important that the intervention targets the end points or relevant mechanisms. POC has been shown to target mechanisms impacting these two end points; the improvement in global contractile function is likely through the reduction in infarct size since no effect has been demonstrated on contractile function per se. POC did not enhance the postcardioplegic outcomes at any duration of ischaemia, but was only effective when the magnesium concentration of ST2 cardioplegia solution was reduced to zero. Maruyama and Chambers conclude that POC has limited efficacy after arrest with ST2 cardioplegia solution, and there is likely a magnesium-sensitive component of POC protection. POC is defined as reperfusion interrupted by brief periods of ischaemia applied during the opening minutes of reflow [3]. In cardiac surgery, POC can be applied by directly clamping and declamping the aorta, as performed by Luo et al. [4], or by iteratively infusing and halting the cardioplegia solution using a dedicated delivery system (Quest Medical Inc.’ MPS, Terumo Cardiovascular Systems System 1 Master/Follower, etc). In contrast to cardioplegia solutions and adjunct strategies such as hypothermia that are designed to reduce the severity of ischaemia that, in part, also determines the severity of reperfusion injury, POC exclusively targets reperfusion injury. Conceptually, both cardioplegia and POC could reduce post-ischaemic injury. If the cardioplegia solution and related strategies successfully limit ischaemic injury, then there is little subsequent reperfusion injury on which POC may exert protection. If, on the other hand, the majority of injury occurs during ischaemia with or without cardioplegia, there may be little room for protection from reperfusion injury, and again POC may not play a role as in the 45 and 60 min ischaemia-only group (Figure 2). It was important for the authors to show that the POC algorithm was effective in improving functional recovery and reducing infarct size, at least after 30 min of normothermic ischaemia alone (no cardioplegia). However, the observation that POC did not improve functional recovery or reduce infarct size does not necessarily represent a failure or loss of cardioprotection, but rather represents a limitation of that algorithm to stimulate the triggers and cardioprotective pathways [5] that impact the effectors of POC; this phenomenon has been called the ‘threshold’ of protection [6]. When a threshold is encountered, whereby protection is no longer observed after longer durations of ischaemia, increasing the number of postconditioning cycles has been shown to re-establish cardioprotection. The authors may have encountered such a threshold at the 45 and 60 min of index ischaemia in the present study. This should be determined in further studies. The model often profoundly affects the outcomes of a study. In the Langendorff isolated perfused, non-working (not ejecting and performing external work) heart preparation, there may be fundamental species differences in biology, metabolic differences that effect the time course of ischaemic injury and efficacy of interventions. Whether the injury occurs primarily during ischaemia or during reperfusion is a fundamental question in this study. ST2 cardioplegia solution was used at 37°C, which differs from its use clinically, and is not consistent with clinical studies testing the efficacy of POC after hypothermic cardioplegia. Tables 2 and 3 show left ventricular end-diastolic pressures >30 mmHg at the end of ischaemia (0 min reperfusion), suggesting that contracture has already occurred to some extent during ischaemia with or without cardioplegia; contracture is associated with the onset of irreversible injury at the end of ischaemia that would not be amenable to reperfusion therapy such as POC. In addition, the isolated perfused heart preparation inherently lacks components of the inflammatory response, i.e. plasma-borne inflammatory cells, proteases and cytokines that contribute to reperfusion injury. POC has been shown to inhibit neutrophil accumulation, activation of the coronary vascular endothelium,

Prediction of Coronary Conduit Artery Endothelial Cell Phenotype by Phenotypic Assessment in the Systemic Vasculature

Human studies have established a correlation between endothelial vasomotor function assessed at the brachial and coronary arteries. This relationship spurred widespread investigation of brachial artery vasomotor function and, more recently, the isolation and phenotypic analysis of endothelial cells harvested invasively from the brachial artery (presumably with the application of understanding the coronary vascular endothelium). However, notwithstanding the correlation between vasomotor function in brachial and coronary arteries, the degree to which markers of endothelial cell phenotype correlate between systemic and coronary arteries is unknown. PURPOSE: Determine the degree to which endothelial cell phenotype assessed in peripheral conduit vessels is predictive of phenotype in the coronary artery endothelium in swine. METHODS: Isolated segments of thoracic and abdominal aorta, carotid, brachial, femoral, renal, internal mammary, and right coronary arteries were harvested at the time of sacrifice from 19 swine. Vessels were placed in a dissection buffer, cleaned of excess connective tissue, cut longitudinally, and the endothelium was mechanically scraped yielding an endothelial enriched sample of vascular cells. Samples were subsequently probed for eNOS, HSP90, p67phox, SOD1 and SOD3 protein expression via immunobloting. RESULTS: Expression of eNOS, HSP90, SOD1, and SOD3 was on average well correlated (p < 0.05 vs. coronary artery for each vessel) between systemic conduit arteries and the right coronary artery (mean rho value = 0.71 ± 0.02, 0.83 ± 0.02, 0.72 ± 0.02, and 0.91 ± 0.01 for eNOS, HSP90, SOD1, and SOD3, respectively). In contrast, expression of p67phox in systemic conduit artery endothelium was not correlated to that in the right coronary artery (mean rho value = 0.24 ± 0.04, p > 0.05 vs. coronary artery for each vessel). CONCLUSION: We conclude that endothelial cell protein expression assessed in peripheral conduit arteries is predictive of expression levels in the conduit coronary arteries in the cases of some (e.g., eNOS, HSP90, SOD1 and SOD3) but not all (e.g., p67phox) markers of endothelial cell phenotype. Supported by: P01-HL052490 (M.H.L.) and T32-AR048523 (G.H.S. and J.P.).

The Multidimensional Physiological Responses to Postconditioning

Reperfusion is the definitive treatment to reduce infarct size and other manifestations of postischemic injury. However, reperfusion contributes to postischemic injury, and, therefore, reperfusion therapies do not achieve the optimal salvage of myocardium. Other tissues as well undergo injury after reperfusion, notably, the coronary vascular endothelium. Postconditioning has been shown to have salubrious effects on different tissue types within the heart (cardiomyocytes, endothelium) and to protect against various pathologic processes, including necrosis, apoptosis, contractile dysfunction, arrhythmias, and microvascular injury or "no-reflow." The mechanisms by which postconditioning alters the pathophysiology of reperfusion injury is exceedingly complex and involves physiological mechanisms (e.g., delaying re-alkalinization of tissue pH, triggering release of autacoids, and opening and closing of various channels) and molecular mechanisms (activation of kinases) that affect cellular and subcellular targets or effectors. The physiologic responses to postconditioning are not isolated or mutually exclusive, but are interactive, with one response affecting another in an integrated manner. This integrated response on multiple targets differs from the monotherapy approach by drugs that have failed to reduce reperfusion injury on a consistent basis and may underlie the efficacy of this therapeutic approach across species and in human trials.

Substrate availability for both endothelial nitric oxide synthase (eNOS) and glucose‐6‐phosphate dehydrogenase (G6PD) result in enzyme dysfunction as a result of ischemia/reperfusion injury: the role of NADPH in endothelial dysfunction.

Ischemia/Reperfusion (IR) injury causes dysfunction in the coronary vascular endothelium. Endothelial nitric oxide synthase is a critical enzyme involved in endothelial function. Previously, we showed endothelial dysfunction occurs without direct damage to eNOS, but rather via depletion of the essential eNOS cofactor, tetrahydrobiopterin (BH4). Repletion of BH4 lost during IR partially restored endothelial function. Recently, we have found that 30 min of IR injury also results in the depletion of the eNOS substrate NADPH, to ~47% of pre‐injury levels. In isolated hearts repletion of NADPH restored endothelial function (as measured by coronary flow) to near pre‐ischemic levels, highlighting the significance the IR‐induced decrease in NADPH. Our current work shows levels of NADPH are altered throughout IR injury, rising ~20% above control values at 10 min ischemia (increasing from 49.2±2.2 to 61.2±3.5 nmoles/gram tissue), and then decreasing to below baseline (31.8±7.8 nmoles/gram tissue) 20 min post‐ischemia. Glucose‐6‐phosphate dehydrogenase (G6PD) is the rate limiting enzyme in the production of NADPH. We observed that while total G6PD activity is not changed during IR injury, it is limited by substrate availability. In conclusion, the modulation of NADPH throughout IR injury, partially due to a change in substrate availability of G6PD, gives new insights to the intracellular response to IR injury.

Nitric oxide and coronary vascular endothelium adaptations in hypertension

This review highlights a number of nitric oxide (NO)-related mechanisms that contribute to coronary vascular function and that are likely affected by hypertension and thus become important clinically as potential considerations in prevention, diagnosis, and treatment of coronary complications of hypertension. Coronary vascular resistance is elevated in hypertension in part due to impaired endothelium-dependent function of coronary arteries. Several lines of evidence suggest that other NO synthase isoforms and dilators other than NO may compensate for impairments in endothelial NO synthase (eNOS) to protect coronary artery function, and that NO-dependent function of coronary blood vessels depends on the position of the vessel in the vascular tree. Adaptations in NOS isoforms in the coronary circulation to hypertension are not well described so the compensatory relationship between these and eNOS in hypertensive vessels is not clear. It is important to understand potential functional consequences of these adaptations as they will impact the efficacy of treatments designed to control hypertension and coronary vascular disease. Polymorphisms of the eNOS gene result in significant associations with incidence of hypertension, although mechanistic details linking the polymorphisms with alterations in coronary vasomotor responses and adaptations to hypertension are not established. This understanding should be developed in order to better predict those individuals at the highest risk for coronary vascular complications of hypertension. Greater endothelium-dependent dilation observed in female coronary arteries is likely related to endothelial Ca(2+) control and eNOS expression and activity. In hypertension models, the coronary vasculature has not been studied extensively to establish mechanisms for sex differences in NO-dependent function. Genomic and nongenomic effects of estrogen on eNOS and direct and indirect antioxidant activities of estrogen are discussed as potential mechanisms of interest in coronary circulation that could have implications for sex- and estrogen status-dependent therapy for hypertension and coronary dysfunction. The current review identifies some important basic knowledge gaps and speculates on the potential clinical relevance of hypertension adaptations in factors regulating coronary NO function.

Abstract 4881: Depletion of the Endothelial Nitric Oxide Synthase (eNOS) Substrate NADPH Triggers Endothelial Dysfunction in the Post-Ischemic Heart: Role of Glucose 6 Phosphate Dehydrogenase (G6PD)

Reperfusion injury causes dysfunction in the coronary vascular endothelium; however, current understanding of this process is incomplete. Delineation of the mechanisms involved is critical for advancing treatment of myocardial infarction. Previously, we showed that endothelial dysfunction is triggered by loss of the eNOS cofactor, tetrahydrobiopterin (BH 4 ), and BH 4 repletion partially restored endothelial-dependent vasodilation. We hypothesize that the oxidative stress induced by ischemia/reperfusion (I/R) injury may also result in loss of the essential eNOS substrate NADPH, which is also utilized for BH 4 resynthesis, thus impairing eNOS function. We observe in the isolated rat heart that NADPH declines by ~70% after 30 min I and 30 min R (from 0.41±0.02 to 0.13±0.04 μ M/gram tissue, by HPLC, n=4, p&lt;0.01). The importance of this NADPH depletion was demonstrated by the marked rescue of coronary flow, with 80.0±4.0% increase, following NADPH repletion (175 μ M liposomal formulation infused for 10 min) in hearts subjected to 30 min of I and R. Further studies were performed to understand the mechanism of NADPH depletion. In the presence of oxidative stress, NADPH is rapidly oxidized and glucose 6 phosphate dehydrogenase (G6PD), is required for its regeneration. We observe that myocardial G6PD activity was decreased ~43% during ischemia (Table ), but this activity partially recovered after 30 min R. Interestingly, with addition of excess exogenous glucose-6-phosphate (G6P) the activity did not significantly change from control levels after 30 min of I and after 30 min R only a modest 20% fall was seen (Table ). Thus, the ability of G6PD to resynthesize NADPH remained largely intact but was limited by lack of its substrate G6P. In conclusion, we found that repletion of NADPH post ischemia results in a near total restoration of endothelial dependent coronary flow, and that ischemic depletion of G6P may be partially responsible for the limited bioavailability of NADPH. Glucose-6-Phosphate Dehydrogenase Enzyme Activity

Hypoxia‐induced expression of bradykinin type‐2 receptors in endothelial cells triggers NO production, cell migration, and angiogenesis

Bradykinin receptors are differentially expressed in the coronary vascular endothelium of rat and human hearts during the pathogenesis of heart failure, but the mechanisms responsible for this regulation have remained vague. Here we show by quantitative real-time PCR, Western blot analysis, and immunohistochemistry, that hypoxia triggers the expression of bradykinin type-2 receptors (BK-2Rs) in cultured human coronary artery endothelial cells (HCAECs), in isolated rat cardiac microvascular endothelial cells (RCMECs), and in rat hearts subjected to ligation of the left anterior descending coronary artery. Mild hypoxia (5% O(2)) induced a fourfold temporal increase in BK-2R mRNA expression in HCAECs, which was also observed at the protein level, whereas severe hypoxia (1% O(2)) slightly inhibited the mRNA expression of BK-2Rs. In addition, HOE-140, a BK-2R antagonist, inhibited mRNA and protein expression of BK-2Rs. The BK-2Rs induced by mild hypoxia were biologically active, that is, capable of inducing intracellular production of nitric oxide (NO) upon activation of HCAECs with bradykinin (BK), a response attenuated by HOE-140. In rat hearts recovering from myocardial infarction, BK-2Rs were upregulated in the endothelium of vessels forming at the border zone between fibrotic scar tissue and healthy myocardium. Furthermore, in an in vitro wound-healing assay, RCMEC migration was increased under mild hypoxic culture conditions in the presence of BK and was attenuated with HOE-140. Our present results show that mild hypoxia triggers a temporal expression of functional BK-2Rs in human and rat endothelial cells and support a role for BK-2Rs in hypoxia-induced angiogenesis.

Exogenous nitric oxide requires an endothelial glycocalyx to prevent postischemic coronary vascular leak in guinea pig hearts

IntroductionPostischemic injury to the coronary vascular endothelium, in particular to the endothelial glycocalyx, may provoke fluid extravasation. Shedding of the glycocalyx is triggered by redox stress encountered during reperfusion and should be alleviated by the radical scavenger nitric oxide (NO). The objective of this study was to investigate the effect of exogenous administration of NO during reperfusion on both coronary endothelial glycocalyx and vascular integrity.MethodsIsolated guinea pig hearts were subjected to 15 minutes of warm global ischemia followed by 20 minutes of reperfusion in the absence (Control group) and presence (NO group) of 4 μM NO. In further experiments, the endothelial glycocalyx was enzymatically degraded by means of heparinase followed by reperfusion without (HEP group) and with NO (HEP+NO group).ResultsIschemia and reperfusion severely damaged the endothelial glycocalyx. Shedding of heparan sulfate and damage assessed by electron microscopy were less in the presence of NO. Compared with baseline, coronary fluid extravasation increased after ischemia in the Control, HEP, and HEP+NO groups but remained almost unchanged in the NO group. Tissue edema was significantly attenuated in this group. Coronary vascular resistance rose by 25% to 30% during reperfusion, but not when NO was applied, irrespective of the state of the glycocalyx. Acute postischemic myocardial release of lactate was comparable in the four groups, whereas release of adenine nucleotide catabolites was reduced 42% by NO. The coronary venous level of uric acid, a potent antioxidant and scavenger of peroxynitrite, paradoxically decreased during postischemic infusion of NO.ConclusionThe cardioprotective effect of NO in postischemic reperfusion includes prevention of coronary vascular leak and interstitial edema and a tendency to forestall both no-reflow and degradation of the endothelial glycocalyx.

Inoprotection: The Perioperative Role of Levosimendan

Levosimendan is emerging as a novel cardioprotective inotrope. Levosimendan augments myocardial contractility by sensitising contractile myofilaments to calcium without increasing myosin adenosine triphosphatase activity or oxygen consumption. Levosimendan activates cellular adenosine triphosphate-dependent potassium channels, a mechanism which is postulated to protect cells from ischaemia in a manner similar to ischaemic preconditioning. Levosimendan may therefore protect the ischaemic myocardium during ischaemia-reperfusion as well as improve the contractile function of the heart. Adenosine triphosphate-dependent potassium channel activation by levosimendan may also be protective in other tissues, such as coronary vascular endothelium, kidney and brain. Clinical trials in patients with decompensated heart failure and myocardial ischaemia show levosimendan to improve haemodynamic performance and potentially improve survival. This paper reviews the known pharmacology of levosimendan, the clinical experience with the drug to date and the potential use of levosimendan as a cardioprotective agent during surgery.

Gene expression profiling of cardiovascular disease markers in response to hormone therapy in post-menopausal coronary artery vascular endothelium

Hormone therapies (HT) have been associated with increased risk of cardiovascular events resulting in the early termination of clinical trials. Because endothelial dysfunction has been clinically demonstrated in coronary arteries of symptomatic patients, specific markers for endothelial dysfunction that are sensitive to female hormones need to be identified. Our objective was to study the markers associated with cardiovascular disease in endothelial cells.

Endothelial Dysfunction in Postmenopausal Women and Hypertension

The endothelium is a major regulator of homeostasis and exerts a number of vasoprotective effects, such as vasodilation, inhibition of inflammatory responses and suppression of smooth muscle cell growth. Dysfunction of the endothelium thus causes reduction or abolition of these vasoprotective effects. Factors that lead to endothelial dysfunction (ED) include a reduction in nitric oxide (NO) production, increased oxidative stress and a decrease in NO bioavailability, whereas endothelium-derived contracting factors are increased. This imbalance leads to an impairment of endothelium-dependent vasodilation, which represents the functional characteristic of ED. Moreover, ED also comprises a specific state of ‘endothelial activation’, which is characterized by a proinflammatory, proliferative and procoagulatory milieu that favors all stages of atherogenesis [1,2]. Hence, ED it is considered a systemic disorder and a key variable in the pathogenesis of atherosclerosis and its complications. Current evidence suggests that endothelial status is not determined solely by the individual risk-factor burden but, rather, may be regarded as an integrated index of all atherogenic and atheroprotective factors present in an individual. ED reflects a vascular phenotype prone to atherogenesis, and may therefore serve as a marker of the inherent atherosclerotic risk in an individual. In line with this hypothesis, dysfunction of either the coronary or peripheral vascular endothelium was shown to constitute an independent predictor of cardiovascular events, providing valuable prognostic information additional to that derived from conventional risk-factor assessment [3].

Recruitment of New Cells into the Postnatal Heart

Establishment of the circulatory system occurs very early in development to support the rapid growth of the embryo. Therefore, the heart is the first functional organ to be formed during both avian and mammalian development. Historically, cardiac development has been considered to occur only during embryogenesis from cell sources located within the primordial structures that generate the myocardium and associated coronary vascular endothelium and smooth muscle and cardiac fibroblasts. Recently, however, contribution to the cardiac structures has been demonstrated to occur during embryonic development from extracardiac sources, like the anterior heart field, raising questions as to whether cardiogenesis may be an ongoing process that extends into adult life. In this brief article, we describe the contribution of circulating adult bone marrow hematopoietic stem cells to the cardiac cell populations and the potential regulation of their differentiation by the extracellular matrix protein, periostin.