Showing papers in "Cardiovascular Research in 2013"

The NLRP3 inflammasome is up-regulated in cardiac fibroblasts and mediates myocardial ischaemia–reperfusion injury

Abstract: Aims N ucleotide-binding oligomerization domain- L ike R eceptor with a P yrin domain 3 (NLRP3) is considered necessary for initiating a profound sterile inflammatory response. NLRP3 forms multi-protein complexes with A poptosis-associated S peck-like protein containing a C aspase recruitment domain (ASC) and Caspase-1, which activate pro-interleukin-1β (IL-1β) and pro-IL-18. The role of NLRP3 in cardiac cells is not known. Thus, we investigated the expression and function of NLRP3 during myocardial ischaemia. Methods and results Myocardial infarction (MI) was induced in adult C57BL/6 mice and Wistar rats by ligation of the coronary artery. A marked increase in NLRP3, IL-1β, and IL-18 mRNA expression was found in the left ventricle after MI, primarily located to myocardial fibroblasts. In vitro studies in cells from adult mice showed that myocardial fibroblasts released IL-1β and IL-18 when primed with lipopolysaccharide and subsequently exposed to the danger signal adenosine triphosphate, a molecule released after tissue damage during MI. When hearts were isolated from NLRP3-deficient mice, perfused and subjected to global ischaemia and reperfusion, a marked improvement of cardiac function and reduction of hypoxic damage was found compared with wild-type hearts. This was not observed in ASC-deficient hearts, potentially reflecting a protective role of other ASC-dependent inflammasomes or inflammasome-independent effects of NLRP3. Conclusion This study shows that the NLRP3 inflammasome is up-regulated in myocardial fibroblasts post-MI, and may be a significant contributor to infarct size development during ischaemia–reperfusion.

Does low and oscillatory wall shear stress correlate spatially with early atherosclerosis? A systematic review

Abstract: Low and oscillatory wall shear stress is widely assumed to play a key role in the initiation and development of atherosclerosis. Indeed, some studies have relied on the low shear theory when developing diagnostic and treatment strategies for cardiovascular disease. We wished to ascertain if this consensus is justified by published data. We performed a systematic review of papers that compare the localization of atherosclerotic lesions with the distribution of haemodynamic indicators calculated using computational fluid dynamics. The review showed that although many articles claim their results conform to the theory, it has been interpreted in different ways: a range of metrics has been used to characterize the distribution of disease, and they have been compared with a range of haemodynamic factors. Several studies, including all of those making systematic point-by-point comparisons of shear and disease, failed to find the expected relation. The various pre- and post-processing techniques used by different groups have reduced the range of shears over which correlations were sought, and in some cases are mutually incompatible. Finally, only a subset of the known patterns of disease has been investigated. The evidence for the low/oscillatory shear theory is less robust than commonly assumed. Longitudinal studies starting from the healthy state, or the collection of average flow metrics derived from large numbers of healthy vessels, both in conjunction with point-by-point comparisons using appropriate statistical techniques, will be necessary to improve our understanding of the relation between blood flow and atherogenesis.

Biological responses in stented arteries

Abstract: Vascular walls change their dimension and mechanical properties in response to injury such as balloon angioplasty and endovascular stent implantation. Placement of bare metal stents induces neointimal proliferation/restenosis which progresses through different phases of repair with time involving a cascade of cellular reactions. These phases just like wound healing comprise distinct steps consisting of thrombosis, inflammation, proliferation, and migration followed by remodelling. It is noteworthy that animals show a rapid progression of healing after stent deployment compared with man. During stenting, endothelial cells are partially to completely destroyed or crushed along with medial wall injury and stretching promoting activation of platelets, and thrombus formation accompanied by inflammatory reaction. Macrophages and platelets play a central role through the release of cytokines and growth factors that induce vascular smooth muscle cell accumulation within the intima. Smooth muscle cells undergo complex phenotypic changes including migration and proliferation from the media towards the intima, and transition from a contractile to a synthetic phenotype; the molecular mechanisms responsible for this change are highlighted in this review. Since studies in animals and man show that smooth muscle cells play a dominant role in restenosis, drugs like rapamycin and paclitaxel have been coated on stent with polymers to allow local slow release of drugs, which have resulted in dramatic reduction of restenosis that was once the Achilles' heel of interventional cardiologists.

MicroRNA-containing microvesicles regulating inflammation in association with atherosclerotic disease

Abstract: In addition to intracellular organelles, eukaryotic cells contain extracellular organelles which are released, or shed, into the microenvironment. In practice, most human studies have examined mixed populations containing both exosomes and shedding microvesicles (also called ectosomes or microparticles); only a few studies have rigorously distinguished between the two. Accordingly, in this review, exosomes and shedding microvesicles are collectively called microvesicles. The first aim of this review was to discuss the role of microvesicles in cell-to-cell communication in general and in specific interactions between cells in chronic inflammation associated with atherosclerotic disease. Hereby, we focused on cell-specific microvesicles derived from platelets, endothelial cells and monocyte and monocyte-derived cells. The latter were also found to be associated with inflammation in obesity and type 2 diabetes prior to atherosclerotic disease, and cancer. Our second aim was to discuss specific changes in microvesicle content in relation with inflammation associated with metabolic and atherosclerotic disease, and cancer. Because many studies supported the putative diagnostic value of microRNAs, we emphasized therein changes in microRNA content rather than protein or lipid content. The most interesting microRNAs in inflammatory microvesicles in association with metabolic and cardiovascular diseases were found to be the let-7 family, miR-17/92 family, miR-21, miR-29, miR-126, miR-133, miR-146, and miR-155. These data warrant further investigation of the potential of microvesicles as putative biomarkers and as novel carriers for the cell-specific transfer of microRNAs and other therapeutic agents.

Arginase as a potential target in the treatment of cardiovascular disease: reversal of arginine steal?

Abstract: Functional integrity of the vascular endothelium is of fundamental importance for normal vascular function. A key factor regulating endothelial function is the bioavailability of nitric oxide (NO). Recently, the enzyme arginase has emerged as an important regulator of NO production by competing for l-arginine, which is a substrate for both arginase and NO synthase. Increased activity of arginase may reduce the availability of l-arginine for NO synthase, thus reducing NO production, increasing formation of reactive oxygen species, and leading ultimately to endothelial dysfunction. Increased activity and expression of arginase have been demonstrated in several pathological cardiovascular conditions, including hypertension, pulmonary arterial hypertension, atherosclerosis, myocardial ischaemia, congestive heart failure, and vascular dysfunction in diabetes mellitus. Experimental studies have demonstrated that inhibition of arginase under these conditions increases NO bioavailability, reduces oxidative stress, improves vascular function, and protects against ischaemia-reperfusion injury. Initial clinical interventional studies are also promising. The purpose of this review is to discuss the role of arginase in cardiovascular pathologies, its contribution to the development of several cardiovascular disease states and the feasibility of using arginase inhibition as a therapeutic strategy.

The atherosusceptible endothelium: endothelial phenotypes in complex haemodynamic shear stress regions in vivo

Abstract: Atherosclerosis initiates at predictable focal sites and develops to a spatially regional disease with limited distribution. There is compelling evidence that links haemodynamics to the localized origin of atherosclerotic lesions. Arterial flow in vivo is unsteady, dynamically complex, and regionally variable. Sites susceptible to atherosclerosis near arterial branches and curves are associated with regions of disturbed blood flow that contain repetitive phases of flow reversal resulting in steep multidirectional temporal and spatial gradients of wall shear stresses. Endothelium in atherosusceptible regions relative to protected sites shows activation of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), the altered expression of pro-inflammatory Nuclear Factor kappa B (NFκB) and oxidant/antioxidant pathways, and low expression of major protective factors, notably endothelial nitric oxide synthase and Kruppel-like Factors KLF2 and KLF4. At some atherosusceptible locations, reactive oxygen species levels are significantly elevated. Here we describe flow-related phenotypes identified in steady-state in vivo and outline some of the molecular mechanisms that may contribute to pre-lesional atherosusceptibility as deduced from complementary cell experiments in vitro. We conclude that disturbed flow is a significant local risk factor for atherosclerosis that induces a chronic low-level inflammatory state, an adaptive response to ensure continued function at the expense of increased susceptibility to atherogenesis. Surprisingly, when challenged by short-term hypercholesterolaemia in vivo, atherosusceptible endothelial phenotype was resistant to greater pro-inflammatory expression, suggesting that sustained hyperlipidaemia is required to overcome these protective characteristics.

Translating cardioprotection for patient benefit: position paper from the Working Group of Cellular Biology of the Heart of the European Society of Cardiology

Abstract: Coronary heart disease (CHD) is the leading cause of death and disability worldwide. Despite current therapy, the morbidity and mortality for patients with CHD remains significant. The most important manifestations of CHD arise from acute myocardial ischaemia-reperfusion injury (IRI) in terms of cardiomyocyte death and its long-term consequences. As such, new therapeutic interventions are required to protect the heart against the detrimental effects of acute IRI and improve clinical outcomes. Although a large number of cardioprotective therapies discovered in pre-clinical studies have been investigated in CHD patients, few have been translated into the clinical setting, and a significant number of these have failed to show any benefit in terms of reduced myocardial infarction and improved clinical outcomes. Because of this, there is currently no effective therapy for protecting the heart against the detrimental effects of acute IRI in patients with CHD. One major factor for this lack of success in translating cardioprotective therapies into the clinical setting can be attributed to problems with the clinical study design. Many of these clinical studies have not taken into consideration the important data provided from previously published pre-clinical and clinical studies. The overall aim of this ESC Working Group Cellular Biology of the Heart Position Paper is to provide recommendations for optimizing the design of clinical cardioprotection studies, which should hopefully result in new and effective therapeutic interventions for the future benefit of CHD patients.

Interleukin-17 causes Rho-kinase-mediated endothelial dysfunction and hypertension

Abstract: Elevated levels of pro-inflammatory cytokine interleukin-17A (IL-17) are associated with hypertensive autoimmune diseases; however, the connection between IL-17 and hypertension is unknown. We hypothesized that IL-17 increases blood pressure by decreasing endothelial nitric oxide production. Methods and results Acute treatment of endothelial cells with IL-17 caused a significant increase in phosphorylation of the inhibitory endothelial nitric oxide (NO) synthase residue threonine 495 (eNOS Thr495). Of the kinases known to phosphor- ylate eNOS Thr495, only inhibition of Rho-kinase prevented the IL-17-induced increase. IL-17 caused a threefold increase in the Rho-kinase activator RhoA, and this was prevented by an IL-17 neutralizing antibody. In isolated mouse aortas, IL-17 significantly increased eNOS Thr495 phosphorylation, induced RhoA expression, and decreased NO-dependent relaxation responses, all of which were prevented by either an IL-17 neutralizing antibody or inhib- ition of Rho-kinase. In mice, IL-17 treatment for 1 week significantly increased systolic blood pressure and this was associated with decreased aortic NO-dependent relaxation responses, increased eNOS Thr495 phosphorylation, and increased RhoA expression. Inhibition of Rho-kinase prevented the hypertension caused by IL-17. Conclusion These data demonstrate that IL-17 activates RhoA/Rho-kinase leading to endothelial dysfunction and hypertension. Inhibitors of IL-17 or Rho-kinase may prove useful as anti-hypertensive drugs in IL-17-associated autoimmune

Biomechanical factors in the biology of aortic wall and aortic valve diseases.

Abstract: The biomechanical factors that result from the haemodynamic load on the cardiovascular system are a common denominator of several vascular pathologies. Thickening and calcification of the aortic valve will lead to reduced opening and the development of left ventricular outflow obstruction, referred to as aortic valve stenosis. The most common pathology of the aorta is the formation of an aneurysm, morphologically defined as a progressive dilatation of a vessel segment by more than 50% of its normal diameter. The aortic valve is exposed to both haemodynamic forces and structural leaflet deformation as it opens and closes with each heartbeat to assure unidirectional flow from the left ventricle to the aorta. The arterial pressure is translated into tension-dominated mechanical wall stress in the aorta. In addition, stress and strain are related through the aortic stiffness. Furthermore, blood flow over the valvular and vascular endothelial layer induces wall shear stress. Several pathophysiological processes of aortic valve stenosis and aortic aneurysms, such as macromolecule transport, gene expression alterations, cell death pathways, calcification, inflammation, and neoangiogenesis directly depend on biomechanical factors.

Deranged myofilament phosphorylation and function in experimental heart failure with preserved ejection fraction.

Abstract: Aims Heart failure (HF) with preserved ejection fraction (HFpEF) is a major cause of morbidity and mortality. Key alterations in HFpEF include increased left ventricular (LV) stiffness and abnormal relaxation. We hypothesized that myofilament protein phosphorylation and function are deranged in experimental HFpEF vs . normal myocardium. Such alterations may involve the giant elastic protein titin, which contributes decisively to LV stiffness. Methods and results LV tissue samples were procured from normal dogs (CTRL) and old dogs with hypertension-induced LV hypertrophy and diastolic dysfunction (OHT/HFpEF). We quantified the expression and phosphorylation of myofilament proteins, including all-titin and site-specific titin phosphorylation, and assessed the expression/activity of major protein kinases (PKs) and phosphatases (PPs), myofilament calcium sensitivity (pCa50), and passive tension (Fpassive) of isolated permeabilized cardiomyocytes. In OHT vs . CTRL hearts, protein kinase-G (PKG) activity was decreased, whereas PKCα activity and PP1/PP2a expression were increased. Cardiac MyBPC, TnT, TnI and MLC2 were less phosphorylated and pCa50 was increased in OHT vs . CTRL. The titin N2BA (compliant) to N2B (stiff) isoform-expression ratio was lowered in OHT. Hypophosphorylation in OHT was detected for all-titin and at serines S4010/S4099 within titin-N2Bus, whereas S11878 within proline, glutamate, valine, and lysine (PEVK)-titin was hyperphosphorylated. Cardiomyocyte Fpassive was elevated in OHT, but could be normalized by PKG or PKA, but not PKCα, treatment. Conclusions This patient-mimicking HFpEF model is characterized by titin stiffening through altered isoform composition and phosphorylation, both contributing to increased LV stiffness. Hypophosphorylation of myofilament proteins and increased calcium sensitivity suggest that functional impairment at the sarcomere level may be an early event in HFpEF.

High glucose condition increases NADPH oxidase activity in endothelial microparticles that promote vascular inflammation

Abstract: Diabetes is a major risk factor for cardiovascular diseases. Circulating endothelial microparticles (EMP) are increased in diabetic patients, but their potential contribution in atherogenesis is unclear. We sought to determine the role of EMP derived under high glucose conditions in the development of atherosclerosis. Methods and results EMP were generated from human coronary endothelial cells (HCAEC) exposed to high glucose concentrations in order to mimic diabetic conditions. These EMP were defined as 'injured' EMP (iEMP) and their effects were compared with EMP generated from 'healthy' untreated HCAEC. iEMP injection significantly impaired endothelial function in ApoE 2/2 mice compared with EMP and vehicle treatment. Immunofluorescent experiments showed increased macrophage infiltration and adhesion protein expression in atherosclerotic lesions of iEMP-treated ApoE 2/2 mice compared with controls. To further investigate the underlying mechanism of iEMP-induced vascular inflammation, additional in vitro experiments were performed. iEMP, but not EMP, induced activation of HCAEC in a time- and dose-dependent manner and increased monocyte adhesion. Further experiments demonstrated that iEMP induced activation of HCAEC by phosphorylation of p38 into its biologically active form phospho-p38. Inhibition of p38 activation abrogated iEMP-dependent induction of adhesion proteins and monocyte adhesion on HCAEC. Moreover, we could demonstrate that iEMP show increased NADPH oxidase activity and contain significantly higher level of reactive oxygen species (ROS) than EMP. iEMP triggered ROS production in HCAEC and thereby activate p38 in an ROS-dependent manner. Conclusion High glucose condition increases NADPH oxidase activity in endothelial microparticles that amplify endothelial in- flammation and impair endothelial function by promoting activation of the endothelium. These findings provide new insights into the pathogenesis of diabetes-associated atherosclerosis.

Flow detection and calcium signalling in vascular endothelial cells.

Abstract: Blood vessels alter their morphology and function in response to changes in blood flow, and their responses are based on blood flow detection by the vascular endothelium. Endothelial cells (ECs) covering the inner surface of blood vessels sense shear stress generated by flowing blood and transmit the signal into the interior of the cell, which evokes a cellular response. The EC response to shear stress is closely linked to the regulation of vascular tone, blood coagulation and fibrinolysis, angiogenesis, and vascular remodelling, and it plays an important role in maintaining the homoeostasis of the circulatory system. Impairment of the EC response to shear stress leads to the development of vascular diseases such as hypertension, thrombosis, aneurysms, and atherosclerosis. Rapid progress has been made in elucidating shear stress mechanotransduction by using in vitro methods that apply controlled levels of shear stress to cultured ECs in fluid-dynamically designed flow-loading devices. The results have revealed that shear stress is converted into intracellular biochemical signals that are mediated by a variety of membrane molecules and microdomains, including ion channels, receptors, G-proteins, adhesion molecules, the cytoskeleton, caveolae, the glycocalyx, and primary cilia, and that multiple downstream signalling pathways become activated almost simultaneously. Nevertheless, neither the shear-stress-sensing mechanisms nor the sensor molecules that initially sense shear stress are yet known. Their identification would contribute to a better understanding of the pathophysiology of the vascular diseases that occur in a blood flow-dependent manner and to the development of new treatments for them.

Emerging mechanisms of T-tubule remodelling in heart failure

Abstract: Cardiac excitation–contraction coupling occurs primarily at the sites of transverse (T)-tubule/sarcoplasmic reticulum junctions The orderly T-tubule network guarantees the instantaneous excitation and synchronous activation of nearly all Ca2+ release sites throughout the large ventricular myocyte Because of the critical roles played by T-tubules and the array of channels and transporters localized to the T-tubule membrane network, T-tubule architecture has recently become an area of considerable research interest in the cardiovascular field This review will focus on the current knowledge regarding normal T-tubule structure and function in the heart, T-tubule remodelling in the transition from compensated hypertrophy to heart failure, and the impact of T-tubule remodelling on myocyte Ca2+ handling function In the last section, we discuss the molecular mechanisms underlying T-tubule remodelling in heart disease

SIRT6 protects human endothelial cells from DNA damage, telomere dysfunction and senescence

Abstract: Aims Although endothelial cell senescence is known to play an important role in the development of cardiovascular pathologies, mechanisms that attenuate this process have not been extensively investigated. The aim of this study was to investigate whether SIRT6, a member of the sirtuin family of NAD+-dependent protein deacetylases/ADP-ribosyltransferases, protects endothelial cells from premature senescence and dysfunction, and if so which is its mode of action. Methods and results mRNA expression analysis demonstrated comparable levels of SIRT1 and SIRT6 transcripts in endothelial cells derived from different vascular beds and significantly higher levels of SIRT6 in these cells relative to those in haematopoietic progenitor cells. SIRT6 depletion by RNA interference in human umbilical vein endothelial cells (HUVEC) and aortic endothelial cells reduced cell proliferation, increased the fraction of senescence-associated-β-galactosidase-positive cells, and diminished the ability of the cells to form tubule networks on Matrigel. Further examination of SIRT6-depleted HUVEC demonstrated higher intercellular-adhesion molecule-1 (ICAM-1) and plasminogen-activator inhibitor-1 mRNA, lower levels of endothelial nitric oxide synthase mRNA and protein, higher ICAM-1 surface expression, and up-regulation of p21. Fluorescence microscopy of SIRT6-depleted HUVEC stained with anti-phospho-histone H2A.X and anti-telomere-repeat-binding-factor-1 antibodies showed evidence of increased nuclear DNA damage and the formation of telomere dysfunction-induced foci. Conclusion This work demonstrates that the presence of SIRT6 in endothelial cells confers protection from telomere and genomic DNA damage, thus preventing a decrease in replicative capacity and the onset of premature senescence. These findings suggest that SIRT6 may be important to maintain endothelial homeostatic functions and delay vascular ageing.

Early afterdepolarizations in cardiac myocytes: beyond reduced repolarization reserve

Abstract: Early afterdepolarizations (EADs) are secondary voltage depolarizations during the repolarizing phase of the action potential, which can cause lethal cardiac arrhythmias. The occurrence of EADs requires a reduction in outward current and/or an increase in inward current, a condition called reduced repolarization reserve. However, this generalized condition is not sufficient for EAD genesis and does not explain the voltage oscillations manifesting as EADs. Here, we summarize recent progress that uses dynamical theory to build on and advance our understanding of EADs beyond the concept of repolarization reserve, towards the goal of developing a holistic and integrative view of EADs and their role in arrhythmogenesis. We first introduce concepts from nonlinear dynamics that are relevant to EADs, namely, Hopf bifurcation leading to oscillations and basin of attraction of an equilibrium or oscillatory state. We then present a theory of phase-2 EADs in nonlinear dynamics, which includes the formation of quasi-equilibrium states at the plateau voltage, their stabilities, and the bifurcations leading to and terminating the oscillations. This theory shows that the L-type calcium channel plays a unique role in causing the nonlinear dynamical behaviours necessary for EADs. We also summarize different mechanisms of phase-3 EADs. Based on the dynamical theory, we discuss the roles of each of the major ionic currents in the genesis of EADs, and potential therapeutic targets.

Increased expression of microRNA-146a decreases myocardial ischaemia/reperfusion injury

Abstract: Aims We have reported that either toll-like receptor 4 deficiency (TLR4−/−) or TLR2 modulation protects against myocardial ischaemia/reperfusion (I/R) injury. The mechanisms involve attenuation of I/R-induced nuclear factor KappaB (NF-κB) activation. MicroRNA-146a (miR-146a) has been reported to target interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), resulting in inhibiting NF-κB activation. This study examined the role of microRNA-146a in myocardial I/R injury. Methods and results We constructed lentivirus expressing miR-146a (LmiR-146a). LmiR-146a was transfected into mouse hearts through the right common carotid artery. The lentivirus vector (LmiR-Con) served as vector control. Untransfected mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (60 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by triphenyltetrazolium chloride (TTC) staining. In separate experiments, the hearts were subjected to ischaemia (60 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography prior to I/R, 3 and 7 days after myocardial I/R. LmiR-146a transfection significantly decreased I/R-induced myocardial infarct size by 55% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). LmiR-146a transfection attenuated I/R-induced myocardial apoptosis and caspase-3/7 and -8 activities. LmiR-146a transfection suppresses IRAK1 and TRAF6 expression in the myocardium. In addition, transfection of LmiR-146a prevented I/R-induced NF-κB activation and inflammatory cytokine production. Conclusions MicroRNA-146a protects the myocardium from I/R injury. The mechanisms may involve attenuation of NF-κB activation and inflammatory cytokine production by suppressing IRAK1 and TRAF6.

L-type calcium channel targeting and local signalling in cardiac myocytes.

Abstract: In the heart, Ca2+ influx via CaV1.2 L-type calcium channels (LTCCs) is a multi-functional signal that triggers muscle contraction, controls action potential duration, and regulates gene expression. The use of LTCC Ca2+ as a multi-dimensional signalling molecule in the heart is complicated by several aspects of cardiac physiology. Cytosolic Ca2+ continuously cycles between ∼100 nM and ∼1 μM with each heartbeat due to Ca2+ linked signalling from LTCCs to ryanodine receptors. This rapid cycling raises the question as to how cardiac myocytes distinguish the Ca2+ fluxes originating through L-type channels that are dedicated to contraction from Ca2+ fluxes originating from other L-type channels that are used for non-contraction-related signalling. In general, disparate Ca2+ sources in cardiac myocytes such as current through differently localized LTCCs as well as from IP3 receptors can signal selectively to Ca2+-dependent effectors in local microdomains that can be impervious to the cytoplasmic Ca2+ transients that drive contraction. A particular challenge for diversified signalling via cardiac LTCCs is that they are voltage-gated and, therefore, open and presumably flood their microdomains with Ca2+ with each action potential. Thus spatial localization of Cav1.2 channels to different types of microdomains of the ventricular cardiomyocyte membrane as well as the existence of particular macromolecular complexes in each Cav1.2 microdomain are important to effect different types of Cav1.2 signalling. In this review we examine aspects of Cav1.2 structure, targeting and signalling in two specialized membrane microdomains—transverse tubules and caveolae.

Derivation and maturation of synthetic and contractile vascular smooth muscle cells from human pluripotent stem cells

Abstract: Embryonic vascular smooth muscle cells (vSMCs) have a synthetic phenotype (Syn-vSMC), but in adults, they commit to the mature contractile phenotype (Con-vSMC). Con-vSMCs differ from Syn-vSMC derivatives in condensed morphology, prominent filamentous cytoskeleton proteins, elastin production and assembly elastin, low proliferation, numerous active caveolae, enlarged endoplasmic reticulum, ample stress fibers and bundles, as well as high contractility. The human pluripotent stem cell-derivatives can differentiate into a desired phenotype. Differentiation can be controlled by appropriate concentrations of relevant factors. Growth in high serum with platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 induces the Syn-SMC phenotype with increased extracellular matrix protein expression and reduced expression of contractile proteins. Serum starvation and PDGF-BB deprivation causes maturation towards the Con-vSMC phenotype. When transplanted subcutaneously into nude mice, the human Con-vSMCs aligned next to the host's growing functional vasculature, with occasional circumferential wrapping and vascular tube narrowing.

MicroRNAs in flow-dependent vascular remodelling.

Abstract: Changes in haemodynamic forces in the vascular system result in an altered expression of miRs, which play important gene-regulatory roles by pairing to the mRNAs of protein-coding genes to fine-tune post-transcriptional repression. The development and structure of blood vessels are highly adapted to haemodynamic forces, such as shear stress, cyclic stretch, and circumferential wall stress, generated by the conductance of blood. Thus, fluctuations in shear stress contribute to miR-regulated differential gene expression in endothelial cells (ECs), which is essential for maintenance of vascular physiology. Several microRNAs have been identified that are induced by high shear stress mediating an atheroprotective role, such as miR-10a, miR-19a, miR-23b, miR-101, and miR-143/145. While changes in the expression profile of miR-21 and miR-92a by high shear stress are associated with an atheroprotective function, low shear stress-induced expression of miR-21, miR-92a, and miR-663 results in a pathological EC phenotype. MiR-155 fulfils pleiotropic functions in different regions of vasculature, when exposed to different modes of shear stress. Thus, changes in shear stress result in differential expression of numerous miRs, triggering the balance between susceptibility and resistance to cardiovascular diseases. Further elucidating the regulation of miRs by flow may allow future clinical applications of miRs as diagnostic and therapeutic tools.

MicroRNA-638 is highly expressed in human vascular smooth muscle cells and inhibits PDGF-BB-induced cell proliferation and migration through targeting orphan nuclear receptor NOR1

Abstract: Aims Aberrant vascular smooth muscle cell (VSMC) proliferation and migration contribute significantly to the development of vascular pathologies, such as atherosclerosis and restenosis. MicroRNAs have recently emerged as critical modulators in cellular processes and the purpose of this study is to identify novel miRNA regulators implicated in human aortic VSMC proliferation and migration. Methods and results To identify miRNAs that are differentially expressed in human VSMCs, we performed miRNA microarray analysis in human aortic smooth muscle cells (SMCs) at different time points after platelet-derived growth factor (PDGF) stimulation. Here, we identified microRNA-638 (miR-638) as a transcript that was one of the most significantly down-regulated in human VSMCs after PDGF stimulation. Furthermore, we confirmed, by Quantitative RT–PCR, that miR-638 is highly expressed in human VSMCs, and its expression is markedly down-regulated in a dose- and time-dependent manner upon PDGF treatment. Consistent with a critical role in SMC proliferation, we found that miR-638 expression was significantly up-regulated in human VSMCs cultured in differentiation medium, a condition that inhibits SMC proliferation. Furthermore, we identified the orphan nuclear receptor NOR1 as a downstream target gene product of miR-638 and down-regulation of NOR1 is critical for miR-638-mediated inhibitory effects on PDGF-induced cyclin D1 expression, cell proliferation, and migration in human aortic SMCs. Conclusion These results indicate that miR-638 is a key molecule in regulating human VSMC proliferation and migration by targeting the NOR1/cyclin D pathway and suggest that specific modulation of miR-638 in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.

The arrhythmogenic consequences of increasing late INa in the cardiomyocyte

Abstract: This review presents the roles of cardiac sodium channel NaV1.5 late current (late INa) in generation of arrhythmic activity. The assumption of the authors is that proper Na(+) channel function is necessary to the maintenance of the transmembrane electrochemical gradient of Na(+) and regulation of cardiac electrical activity. Myocyte Na(+) channels' openings during the brief action potential upstroke contribute to peak INa and initiate excitation-contraction coupling. Openings of Na(+) channels outside the upstroke contribute to late INa, a depolarizing current that persists throughout the action potential plateau. The small, physiological late INa does not appear to be critical for normal electrical or contractile function in the heart. Late INa does, however, reduce the net repolarizing current, prolongs action potential duration, and increases cellular Na(+) loading. An increase of late INa, due to acquired conditions (e.g. heart failure) or inherited Na(+) channelopathies, facilitates the formation of early and delayed afterpolarizations and triggered arrhythmias, spontaneous diastolic depolarization, and cellular Ca(2+) loading. These in turn increase the spatial and temporal dispersion of repolarization time and may lead to reentrant arrhythmias.

TGF-β signalling and reactive oxygen species drive fibrosis and matrix remodelling in myxomatous mitral valves

Abstract: Aims Myxomatous mitral valve disease (MMVD) is associated with leaflet thickening, fibrosis, matrix remodelling, and leaflet prolapse. Molecular mechanisms contributing to MMVD, however, remain poorly understood. We tested the hypothesis that increased transforming growth factor-β (TGF-β) signalling and reactive oxygen species (ROS) are major contributors to pro-fibrotic gene expression in human and mouse mitral valves. Methods and results Using qRT–PCR, we found that increased expression of TGF-β1 in mitral valves from humans with MMVD ( n = 24) was associated with increased expression of connective tissue growth factor (CTGF) and matrix metalloproteinase 2 (MMP2). Increased levels of phospho-SMAD2/3 (western blotting) and expression of SMAD-specific E3 ubiquitin-protein ligases (SMURF) 1 and 2 (qRT–PCR) suggested that TGF-β1 signalling occurred through canonical signalling cascades. Oxidative stress (dihydroethidium staining) was increased in human MMVD tissue and associated with increases in NAD(P)H oxidase catalytic subunits (Nox) 2 and 4, occurring despite increases in superoxide dismutase 1 (SOD1). In mitral valves from SOD1-deficient mice, expression of CTGF, MMP2, Nox2, and Nox4 was significantly increased, suggesting that ROS can independently activate pro-fibrotic and matrix remodelling gene expression patterns. Furthermore, treatment of mouse mitral valve interstitial cells with cell permeable antioxidants attenuated TGF-β1-induced pro-fibrotic and matrix remodelling gene expression in vitro . Conclusion Activation of canonical TGF-β signalling is a major contributor to fibrosis and matrix remodelling in MMVD, and is amplified by increases in oxidative stress. Treatments aimed at reducing TGF-β activation and oxidative stress in early MMVD may slow progression of MMVD.

Osteopontin-mediated myocardial fibrosis in heart failure: a role for lysyl oxidase?

Abstract: Aims We investigated whether the pro-fibrotic matricellular protein osteopontin (OPN) is associated with the enzymes involved in the extracellular synthesis of fibril-forming collagen type I (i.e. procollagen C-proteinase, PCP) and its cross-linking to form insoluble fibrils (i.e. lysyl oxidase, LOX) in heart failure (HF) of hypertensive origin. Methods and results OPN, PCP, and LOX were assessed by histochemical and molecular methods in the myocardium of 21 patients with hypertensive heart disease (HHD) and HF. Whereas the myocardial expression of OPN was very scarce in control hearts ( n = 10), it was highly expressed in HF patients ( P < 0.0001). OPN was directly correlated with LOX ( r = 0.460, P = 0.041), insoluble collagen ( r = 0.534, P = 0.015), pulmonary capillary wedge pressure ( r = 0.558; P = 0.009), and left-ventricular (LV) chamber stiffness ( r = 0.458, P = 0.037), and inversely correlated with LV ejection fraction ( r = −0.513, P = 0.017) in all patients. OPN did not correlate with PCP and other parameters assessing collagen synthesis by fibroblasts or degradation by matrix metalloproteinases. In vitro studies showed that OPN significantly ( P < 0.05) increases the expression and activity of LOX in human cardiac and dermal fibroblasts. Conclusion An excess of OPN is associated with increased LOX and insoluble collagen, as well as with LV stiffness and systolic dysfunction in patients with HHD and HF. In addition, OPN up-regulates LOX in human fibroblasts. It is suggested that the OPN–LOX axis might facilitate the formation of insoluble collagen (i.e. stiff and resistant to degradation) and the subsequent alteration in LV mechanical properties and function in patients with HHD and HF.

FoxO1 is crucial for sustaining cardiomyocyte metabolism and cell survival.

Abstract: Diabetic cardiomyopathy is a term used to describe cardiac muscle damage-induced heart failure. Multiple structural and biochemical reasons have been suggested to induce this disorder. The most prominent feature of the diabetic myocardium is attenuated insulin signalling that reduces survival kinases (Akt), potentially switching on protein targets like FoxOs, initiators of cell death. FoxO1, a prominent member of the forkhead box family and subfamily O of transcription factors and produced from the FKHR gene, is involved in regulating metabolism, cell proliferation, oxidative stress response, immune homeostasis, pluripotency in embryonic stem cells, and cell death. In this review we describe distinctive functions of FoxOs, specifically FoxO1 under conditions of nutrient excess, insulin resistance and diabetes, and its manipulation to restore metabolic equilibrium to limit cardiac damage due to cell death. Because FoxO1 helps cardiac tissue to combat a variety of stress stimuli, it could be a major determinant in regulating diabetic cardiomyopathy. In this regard, we highlight studies from our group and others who illustrate how cardiac tissue-specific FoxO1 deletion protects the heart against cardiomyopathy and how its down-regulation in endothelial tissue could prevent against atherosclerotic plaques. In addition, we also describe studies that show FoxO1's beneficial qualities by highlighting their role in inducing anti-oxidant, autophagic, and anti-apoptotic genes under stress conditions of ischaemia-reperfusion and myocardial infarction. Thus, the aforementioned FoxO1 traits could be useful in curbing cardiac tissue-specific impairment of function following diabetes.

The effects of stenting on shear stress: relevance to endothelial injury and repair

Abstract: Stent deployment following balloon angioplasty is used routinely to treat coronary artery disease. These interventions cause damage and loss of endothelial cells (EC), and thus promote in-stent thrombosis and restenosis. Injured arteries are repaired (intrinsically) by locally derived EC and by circulating endothelial progenitor cells which migrate and proliferate to re-populate denuded regions. However, re-endothelialization is not always complete and often dysfunctional. Moreover, the molecular and biomechanical mechanisms that control EC repair and function in stented segments are poorly understood. Here, we propose that stents modify endothelial repair processes, in part, by altering fluid shear stress, a mechanical force that influences EC migration and proliferation. A more detailed understanding of the biomechanical processes that control endothelial healing would provide a platform for the development of novel therapeutic approaches to minimize damage and promote vascular repair in stented arteries.

Pressure-overload-induced heart failure induces a selective reduction in glucose oxidation at physiological afterload

Abstract: Aims Development of heart failure is known to be associated with changes in energy substrate metabolism. Information on the changes in energy substrate metabolism that occur in heart failure is limited and results vary depending on the methods employed. Our aim is to characterize the changes in energy substrate metabolism associated with pressure overload and ischaemia–reperfusion (I/R) injury. Methods and results We used transverse aortic constriction (TAC) in mice to induce pressure overload-induced heart failure. Metabolic rates were measured in isolated working hearts perfused at physiological afterload (80 mmHg) using 3H- or 14C-labelled substrates. As a result of pressure-overload injury, murine hearts exhibited: (i) hypertrophy, systolic, and diastolic dysfunctions; (ii) reduction in LV work, (iii) reduced rates of glucose and lactate oxidations, with no change in glycolysis or fatty acid oxidation and a small decrease in triacylglycerol oxidation, and (iv) increased phosphorylation of AMPK and a reduction in malonyl-CoA levels. Sham hearts produced more acetyl CoA from carbohydrates than from fats, whereas TAC hearts showed a reverse trend. I/R in sham group produced a metabolic switch analogous to the TAC-induced shift to fatty acid oxidation, whereas I/R in TAC hearts greatly exacerbated the existing imbalance, and was associated with a poorer recovery during reperfusion. Conclusions Pressure overload-induced heart failure and I/R shift the preference of substrate oxidation from glucose and lactate to fatty acid due to a selective reduction in carbohydrate oxidation. Normalizing the balance between metabolic substrate utilization may alleviate pressure-overload-induced heart failure and ischaemia.

Rac1 signalling mediates doxorubicin-induced cardiotoxicity through both reactive oxygen species-dependent and -independent pathways

Abstract: Aims Doxorubicin causes damage to the heart, often leading to irreversible cardiomyopathy, which is fatal. Reactive oxygen species (ROS) or oxidative stress is involved in cardiomyocyte death, contributing to doxorubicin-induced cardiotoxicity. This study investigated the role of Rac1, an important subunit of NADPH oxidase, in doxorubicin-induced cardiotoxicity and the underlying mechanisms. Methods and results In a mouse model of acute doxorubicin-induced cardiotoxicity, cardiomyocyte-specific deletion of Rac1 inhibited NADPH oxidase activation and ROS production, prevented cardiac cell death, and improved myocardial function in Rac1 knockout mice. Therapeutic administration of the specific Rac1 inhibitor NSC23766 achieved similar cardio-protective effects in doxorubicin-stimulated mice. In rat cardiomyoblasts (H9c2 cells) and cultured neonatal mouse cardiomyocytes, Rac1 inhibition attenuated apoptosis as evidenced by decreases in caspase-3 activity and DNA fragmentation in response to doxorubicin, which correlated with a reduction in ROS production and down-regulation of p53 acetylation and histone H2AX phosphorylation. In contrast, overexpression of Rac1 enhanced apoptosis. Doxorubicin also inhibited the activity of classical histone deacetylases (HDAC), which was preserved by Rac1 inhibition and further decreased by Rac1 overexpression. Interestingly, scavenging ROS mitigated apoptosis but did not change HDAC activity and p53 acetylation stimulated by doxorubicin, suggesting both ROS-dependent and -independent pathways are involved in Rac1-mediated cardiotoxicity. Furthermore, the HDAC inhibitor trichostatin A enhanced apoptosis, p53 acetylation and H2AX phosphorylation in doxorubicin-treated cardiomyocytes. Conclusions Rac1 signalling contributes to doxorubicin-induced cardiotoxicity through both a ROS-dependent mechanism and ROS-independent HDAC/p53 signalling in cardiomyocytes. Thus, inhibition of Rac1 may be a useful therapy for doxorubicin-induced cardiotoxicity.

Differential changes in titin domain phosphorylation increase myofilament stiffness in failing human hearts

Abstract: Aims Titin-based myofilament stiffness is defined by the expression levels of the cardiac titin-isoforms, N2B and N2BA, and by phosphorylation of the elastic titin domains N2-B unique sequence (N2-Bus) and PEVK. Phosphorylation of the N2-Bus by cGMP-dependent protein kinase (PKG) or cAMP-dependent protein kinase (PKA) decreases titin stiffness, whereas phosphorylation of the PEVK-domain by PKC increases it. We aimed to identify specific sites within the N2-Bus phosphorylated by PKA and PKG and to determine whether differential changes in titin domain phosphorylation could affect passive stiffness in human failing hearts. Methods and results Using mass spectrometry, we identified seven partly conserved PKA/PKG-targeted phosphorylation motifs in human and rat N2-Bus. Polyclonal antibodies to pSer4185, pSer4010, and pSer4099 in the N2-Bus, and to pSer11878 in the PEVK-region were used to quantify titin-domain phosphorylation by western blot analyses of a set of human donor and failing hearts with similar titin-isoform composition. Passive tension determined in skinned human myocardial fibre preparations was significantly increased in failing compared with donor hearts, notably at shorter sarcomere lengths where titin contributes most to total passive tension. Phosphorylation of Ser4185, Ser4010, and Ser4099 in the N2-Bus was significantly reduced in failing hearts, whereas phosphorylation of Ser11878 in the PEVK-region was increased compared with donor hearts. Conclusion We conclude that hypo-phosphorylation of the N2-Bus and hyper-phosphorylation of the PEVK domain can act complementary to elevate passive tension in failing human hearts. Differential changes in titin-domain phosphorylation may be important to fine-tune passive myocardial stiffness and diastolic function of the heart.

Toll-like receptor 3 and 4 signalling through the TRIF and TRAM adaptors in haematopoietic cells promotes atherosclerosis

Abstract: Aims Members of the Toll-like receptor (TLR) family initiate innate immune responses and were recently shown to play a role in atherosclerosis. However, the mechanisms that link TLR ligation to vascular inflammation and atherogenesis remain unclear. To identify which signalling pathways downstream of TLRs in immune cells are pro-atherogenic, we analysed the role of the TLR-specific adaptors MyD88 adaptor-like (MAL), TRIF-related adaptor molecule (TRAM), and TIR-domain-containing adaptor-inducing interferon-β (TRIF) in atherosclerosis. Methods and results Using a bone-marrow transplantation strategy into low-density lipoprotein receptor-deficient ( Ldlr−/− ) mice, we could specifically study the absence of the TLR adaptors in immune cells. We showed that haematopoietic deficiency of TRAM and TRIF, but not MAL, reduces atherosclerosis without affecting cholesterol metabolism. This was mediated by decreased aortic inflammation, indicated by lower aortic levels of pro-inflammatory mediators, and reduced influx of macrophages and T cells. Furthermore, by studying Tlr3−/− chimeric Ldlr−/− mice, we found that deleting TLR3 in immune cells significantly reduced both aortic inflammation and atherosclerotic burden. Conclusions By studying hypercholesterolaemic mice with defects in TLR-signalling adaptors, we demonstrated that deleting either TRAM or TRIF in immune cells is sufficient to attenuate vessel inflammation and protect against atherosclerosis. In addition, these adaptors elicit partly different sets of inflammatory mediators and can independently inhibit the disease process. Furthermore, we identify TLR3 as a pro-atherogenic receptor in haematopoietic immune cells. The identification of these pro-atherogenic pathways downstream of TLR3 and TLR4 contributes to a better understanding of TLRs and their signalling pathways in the pathogenesis of atherosclerosis.

Oxidative stress modulates vascular smooth muscle cell phenotype via CTGF in thoracic aortic aneurysm

Abstract: Dissection and rupture of the ascending aorta are life-threatening conditions resulting in 80% mortality. Ascending aortic replacement in patients presenting with thoracic aortic aneurysm (TAA) is determined by metric measurement. However, a significant number of dissections occur outside of the parameters suggested by the current guidelines. We investigate the correlation among altered haemodynamic condition, oxidative stress, and vascular smooth muscle cell (VSMC) phenotype in controlling tissue homoeostasis. Methods and results We demonstrate using finite element analysis (FEA) based on computed tomography geometries that TAA patients have higher wall stress in the ascending aorta than non-dilated patients. We also show that altered haemodynamic conditions are associated with increased levels of reactive oxygen species (ROS), direct regulators of the VSMC phenotype in the microregional area of the ascending aorta. Usinginvitro andexvivo studies on human tissues, we show that ROS accumu- lation correlates with media layer degeneration and increased connective tissue growth factor (CTGF) expression, which modulate the synthetic VSMC phenotype. Results were validated by a murine model of TAA (C57BL/6J) based on Angio- tensin II infusion showing that medial thickening and luminal expansion of the proximal aorta is associated with the VSMC synthetic phenotype as seen in human specimens. Conclusions Increased peak wall stress correlates with change in VSMC towards a synthetic phenotype mediated by ROS accumula- tion via CTGF. Understanding the molecular mechanisms that regulate VSMC towards a synthetic phenotype could unveil new regulatory pathways of aortic homoeostasis and impact the risk-stratification tool for patients at risk of aortic dis- section and rupture.