AMP-Activated Protein Kinase: An Ubiquitous Signaling Pathway With Key Roles in the Cardiovascular System

AMP-Activated Protein Kinase: An Ubiquitous Signaling Pathway With Key Roles in the Cardiovascular System

Ian P. Salt, D. Grahame Hardie

Structure of human AMP-activated protein kinase (AMPK; α1β2γ1 complex).8 The model was created in space-filling mode using PyMol v1.7.4.2 with the co-ordinates in PDB file 4RER and with color coding similar to that in Figure 1. The heterotrimer was crystallized in the presence of β-cyclodextrin, which occupies the glycogen-binding site, staurosporine, which occupies the active site, and AMP, which occupies sites 1, 3, and 4 on the γ subunit (sites 1 and 4 are round the back in this view). Although Thr172 was phosphorylated, it is not visible in this view but lies in the cleft between the kinase domain C lobe and the β-CTD, just over the right-hand shoulder of the C-lobe. ADaM indicates Allosteric Drug and Metabolite; AID, autoinhibitory domain; CBM, carbohydrate-binding module; CBS1, cystathionine beta-synthase motif-1, etc; CTD, C-terminal domain; and NTD, N-terminal domain. [Powerpoint File]

Autophagy and Mitophagy in Cardiovascular Disease

Autophagy and Mitophagy in Cardiovascular Disease

José Manuel Bravo-San Pedro, Guido Kroemer, Lorenzo Galluzzi

Detrimental effects of autophagy or mitophagy inhibition on cardiovascular health. Multiple genetic interventions that limit autophagic or mitophagic flux at the whole-body level or in specific compartments of the cardiovascular system have been associated with the spontaneous development of cardiovascular disorders. AP indicates autophagosome; APL, autophagolysosome; Atg5, autophagy related 5; Bnip3l, BCL2 interacting protein 3 like; Dnml1, dynamin 1 like; Fbxo32, F-box protein 32; L, lysosome; Lamp2, lysosomal-associated membrane protein 2; Mfn1, mitofusin 1; Park2, Parkinson disease (autosomal recessive, juvenile) 2, parkin; Pink1, PTEN-induced putative kinase 1; Sirt6, sirtuin 6; Tfrc, transferrin receptor; and Trp53, transformation-related protein 53. [Powerpoint File]

Extracellular Vesicles in Metabolic Syndrome

Extracellular Vesicles in Metabolic Syndrome

M. Carmen Martínez, Ramaroson Andriantsitohaina

Extracellular vesicles (EVs) participate in the development of atherosclerotic plaque. EVMP from smooth muscle cells (pink) induce endothelial dysfunction and macrophage infiltration in the vessel wall through the reactive oxygen species (ROS) production and p38 activation in endothelial cells. EVEXO derived from dendritic cells (blue) increase endothelial inflammation by activation of nuclear factor-κB (NF-κB) pathway and increasing expression of proinflammatory molecules, including vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM1), and E-selectin. [Powerpoint File]

Extracellular Vesicles in Angiogenesis

Extracellular Vesicles in Angiogenesis

Dilyana Todorova, Stéphanie Simoncini, Romaric Lacroix, Florence Sabatier, Françoise Dignat-George

Mechanisms involved in the modulation of Angiogenesis by platelet-derived extracellular vesicles (EVs). Platelet-derived EVs contain various growth factors and chemokines that induce proangiogenic signaling in endothelial cell (EC). Spingosine-1-phosphate (S1P1), present on the EV surface, induces PI3K activation and, together with VEGF and bFGF, promotes angiogenesis. The EVs released by platelets stimulate the proangiogenic potential of circulating angiogenic cells by increasing their expression of both membrane molecules and soluble factors. Platelet-derived EVs can inhibit angiogenesis by transferring the p22phox and gp91 subunits of NADPH oxidase and increasing the oxidative stress in EC. ? indicates that the exact content of the EVs is not reported; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; HGF, hepatocyte growth factor; NADPH, nicotinamide adenine dinucleotide phosphate; PI3K, phosphoinositide 3-kinase; RANTES, regulated on activation, normal T-cell–expressed and secreted; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor; and VEGFR, vascular endothelial growth factor receptor. [Powerpoint FIle]

Extracellular Vesicles in Angiogenesis

Extracellular Vesicles in Angiogenesis

Dilyana Todorova, Stéphanie Simoncini, Romaric Lacroix, Florence Sabatier, Françoise Dignat-George

Mechanisms involved in the modulation of Angiogenesis by endothelial cell (EC)–derived extracellular vesicles (EVs). EC release EVs rich in micro-RNA such as miR-214 and miR-126, that are transferred to recipient EC and induce proangiogenic signaling. EVs contain functional matrix metalloproteinases that facilitate angiogenesis through the degradation of components of the extracellular matrix. Dll4 is transferred to EC by the EVs and induces Notch receptor internalization and tip cell formation. EVs bear, at their surface, a tissue factor that interacts with β1 integrin and induces Rac1-ERK1/2-ETS1 signaling, leading to the increased secretion of CCL2. EVs transport the complex uPA/uPAR, which stimulates angiogenesis through plasmin generation. The phosphatidylserine present on the surface of the EVs interacts with CD36 and induces Fyn kinase signaling, which leads to increased oxidative stress and the inhibition of angiogenesis. ATM indicates ataxia telangiectasia mutated; CCl2, chemokine c-c motif ligand 2; Dll4, Delta-like 4; ECM, extracellular matrix; ERK1/2, extracellular signal-related kinase 1 and 2; ETS1, avian erythroblastosis virus E26 homolog-1; IL-3R, interleukin-3 receptor; MMPs, matrix metalloproteinases; NotchR, Notch receptor; PS, phosphatidylserine; Rac1, Ras-related C3 botulinum toxin substrate 1; ROS, reactive oxygen species; TF, tissue factor; TIMPS, tissue inhibitor of metalloproteinases; uPA, urokinase plasminogen activator; and uPAR, urokinase plasminogen activator receptor. [Powerpoint File]

Extracellular Vesicles in Metabolic Syndrome

Extracellular Vesicles in Metabolic Syndrome

M. Carmen Martínez, Ramaroson Andriantsitohaina

Effects of extracellular vesicles (EVs) on blood vessel. EVMP from endothelial cells (dark pink) transfer miR-503 to pericytes and subsequently inhibit vascular endothelial growth factor (VEGF) expression, resulting in decreased migration and proliferation. EVEXO from smooth muscle cells (pink) induce downregulation of LC3 II, ATG5, and Beclin-1 expression in endothelial cells. EVEXO from macrophages (green) evoke intercellular adhesion molecule 1 (ICAM1) overexpression in endothelial cells and reduce level of miR-17. Also, macrophage foam cell–derived EVs favor both migration and adhesion of vascular smooth muscle cells by activating ERK (extracellular signal-regulated kinase) and Akt (protein kinase B/AKT) pathways and by transfer integrins β1 and α5 into vascular smooth muscle cells. EVMP from metabolic syndrome (MetS) patients act on smooth muscle cells and induce overexpression of inducible nitric oxide synthase (iNOS) and monocyte chemoattractant molecule (MCP)-1, leading to vascular hyporeactivity. Also, these EVMP directly act on endothelial cells evoking reduced nitric oxide (NO) production, enhanced cytosolic and mitochondrial reactive oxygen species (ROS) production, and unfolding protein response (UPR). All effects of EVMP from MetS patients are mediated by the interaction Fas/FasL. [Powerpoint File]

Extracellular Vesicles in Cardiovascular Disease: Potential Applications in Diagnosis, Prognosis, and Epidemiology

Extracellular Vesicles in Cardiovascular Disease: Potential Applications in Diagnosis, Prognosis, and Epidemiology

Felix Jansen, Georg Nickenig, Nikos Werner

Extracellular vesicles (EVs) as biomarker in different stages of coronary artery disease. Levels of circulating EVs are detectable in plasma of healthy subjects and are elevated in patients with cardiovascular risk factors or already present cardiovascular diseases. This figure summarizes EV surface markers from clinical studies, which showed increased circulating levels of endothelial-, platelet-, white blood cell–, and red blood cell–derived EVs in different stages of coronary artery disease from patients at risk to acute coronary syndromes (Illustration credit: Ben Smith). [Powerpoint File]

Methodological Guidelines to Study Extracellular Vesicles

Methodological Guidelines to Study Extracellular Vesicles

Frank A.W. Coumans, Alain R. Brisson, Edit I. Buzas, Françoise Dignat-George, Esther E.E. Drees, Samir El-Andaloussi, Costanza Emanueli, Aleksandra Gasecka, An Hendrix, Andrew F. Hill, Romaric Lacroix, Yi Lee, Ton G. van Leeuwen, Nigel Mackman, Imre Mäger, John P. Nolan, Edwin van der Pol, D. Michiel Pegtel, Susmita Sahoo, Pia R.M. Siljander, Guus Sturk, Olivier de Wever, Rienk Nieuwland

Working principle of common methods to isolate extracellular vesicles (EVs). Separation is based on size, density, and immunophenotype. Straight brackets: isolated EVs; yellow: soluble components; and blue: buffer. A, In differential centrifugation, separation is based on size, and large EVs (gray) collect earlier at the bottom of the tube and at lower g forces than small EVs (green). The soluble components are not affected by centrifugation, but non-EV particles such as lipoproteins and protein aggregates may copellet with EVs. B, In density gradient centrifugation, separation is based on density, and EVs will travel to their equilibrium density. Non-EV particles such as lipoproteins may coelute with EVs because of similar density or interaction. The soluble components with a high density relative to the gradient will collect at the bottom of the tube. C, Size exclusion chromatography uses a porous matrix (dotted circles) that separates on size. Soluble components and particles smaller than the size cutoff enter the porous matrix temporarily, whereas EVs and particles larger than the size cutoff do not enter the porous matrix. As a result, EVs and particles larger than the size cutoff elute before the soluble components and particles smaller than the size cut-off. D, In ultrafiltration, soluble proteins and particles smaller than the size cutoff (≈105 kDa) are pushed through the filter, and the EVs are collected at the filter. E, In immunocapture assays, EVs are captured based on their immunophenotype. EVs are captured using an monoclonal antibody (mAb) directed against an antigen exposed on the targeted (green) EVs only. F, In precipitation, addition of a precipitating agent induces clumping of EVs, non-EV particles, and soluble proteins. The clumps will sediment, and sedimentation can be accelerated by centrifugation. [Powerpoint File]

Atrial Fibrillation Epidemiology, Pathophysiology, and Clinical Outcomes

Atrial Fibrillation: Epidemiology, Pathophysiology, and Clinical Outcomes

Laila Staerk, Jason A. Sherer, Darae Ko, Emelia J. Benjamin, Robert H. Helm

Atrial fibrillation (AF) risk factors (RFs) induce structural and histopathologic changes to the atrium that are characterized by fibrosis, inflammation, and cellular and molecular changes. Such changes increase susceptibility to AF. Persistent AF further induces electric and structural remodeling that promotes perpetuation of AF. AF also may lead to the development of additional AF risk factors that further alters the atrial substrate. Finally, AF is associated with several clinical outcomes. *There are limited data supporting the association. BMI, body mass index; ERP, effective refractory period; HF, heart failure; IL, interleukin; MI, myocardial infarction; OSA, obstructive sleep apnea; SEE, systemic embolism event; TNF, tumor necrosis factor; and VTE, venous thromboembolism. [Powerpoint File]

Engineering Cardiac Muscle Tissue: A Maturating Field of Research

Engineering Cardiac Muscle Tissue: A Maturating Field of Research

Florian Weinberger, Ingra Mannhardt, Thomas Eschenhagen

Histological structure of native mouse and rat heart (longitudinal mouse heart in A; cross section of rat heart in B), engineered heart tissue (EHT) from neonatal rat heart cells (longitudinal in C, E, cross section in D), or human pluripotent stem cell–derived cardiomyocytes (F–J). A, staining of actinin (green). B, Staining of sarcolemma with wheat germ agglutinin (white in B). C and D, Staining of actinin (red in C and I), F-actin (green in D and J), or actinin and troponin T (green/red in H) and nuclei in blue (all). E, Hematoxylin and eosin–stained paraffin section. A, Reprinted from Mearini et al135 with permission of the publisher. Copyright © 2014, Macmillan Publishers Limited. B, Reprinted from Bensley et al136 with permission. Copyright © 2016, The Authors. C, D, I, and J, Reprinted from Jackman et al35 with permission of the publisher. Copyright © 2016, Elsevier; and from Eschenhagen et al134 (E) with permission of the publisher. Copyright © 2012, the American Physiological Society. F, Reprinted from Hirt et al36 with permission of the publisher. Copyright © 2014, Elsevier. G, Reprinted from Mannhardt et al78 with permission. Copyright © 2016, The Authors. H, Riegler et al93 with permission of the publisher. Copyright © 2015, American Heart Association. [Powerpoint File]

Engineering Cardiac Muscle Tissue: A Maturating Field of Research

Engineering Cardiac Muscle Tissue: A Maturating Field of Research

Florian Weinberger, Ingra Mannhardt, Thomas Eschenhagen

Plane engineered heart tissue (EHT) on Velcro-covered rods (Eschenhagen et al2, A), ring EHTs (Zimmermann et al24, B), fibrin-based mini-EHT on PDMS (polydimethylsiloxane) racks (Hansen et al25, C), cardiac micro tissues (CMT) on fluorescent pillars (Boudou et al34, D), cardiobundles on PDMS frame (Jackman et al35, Copyright © 2016, Elsevier; E), micro heart muscle (Huebsch et al81, Copyright © 2016, The Authors; F), cardiac biowires (Nunes et al131, Copyright © 2013, Nature Publishing Group; G), cardiac patch (Bian et al133, H). Please note that scale bars are only representative, and sketches might not match the exact dimensions. Graphics in A, B, and D were modified from Eschenhagen et al134 with permission of the publisher. Copyright © 2012, the American Physiological Society. [Powerpoint File]

Critical Issues for the Translation of Cardioprotection

Critical Issues for the Translation of Cardioprotection

Gerd Heusch

Lack of robust preclinical data, lack of phase II dosing and timing studies, and too loose inclusion criteria in clinical trials contribute to poor translation. Adapted from Rossello and Yellon6 with permission of the publisher. Copyright ©2016, American Heart Association, Inc. Authorization for this adaptation has been obtained both from the owner of the copyright in the original work and from the owner of copyright in the translation or adaptation. TIMI indicates thrombolysis in myocardial infarction. [Powerpoint File]

Cell Therapy Trials in Congenital Heart Disease

Cell Therapy Trials in Congenital Heart Disease

Hidemasa Oh

Preclinical study of intracoronary cardiosphere-derived cell (CDC) infusion in a rat model of right heart failure. A–K, Pulmonary artery (PA) banding was created to induce pressure overload right heart failure in rats (weighing 250–300 g). The left thorax was opened to expose the pulmonary artery. A silk suture was tied tightly around an 18-gauge needle alongside the pulmonary artery, followed by a rapid removal of the needle to leave the pulmonary artery constricted in the lumen equal to the diameter of the needle. Intracoronary infusion was performed 4 weeks after pulmonary artery banding. The ascending aorta and the pulmonary artery were occluded with a snare twice for a 20-s interval, 10 min apart, during which time rats received an infusion of CDCs or vehicle into the aortic root directly. Animals were euthanized at 4 weeks after treatment to obtain immuno-histological data. Masson-trichrome staining and hematoxylin and eosin (H&E) staining are shown. One month after pulmonary artery banding, significant right ventricular hypertrophy and fibrosis were observed. Note that CDC treatment reduced cardiac fibrosis, but not hypertrophy, one month after infusion (F and I). Bars, 2 mm in A to C; 20 μm in D to I. J, Animals were separated into 4 groups: (1) sham-operated animals (n=12), (2) rats subjected to pulmonary artery banding for 4 weeks with vehicle treatment (n=12), and (3 and 4) rats subjected to banding for 4 weeks with 2 doses of CDC infusion (0.5×105 or 1×105 cells: n=12 in each). Cardiac fibrosis induced by right ventricle pressure overload was measured. CDC infusion significantly reduced the fibrotic area in a cell dose-dependent manner. K, CDC treatment did not affect the diameter of myocytes, which are mechanically enlarged by pressure overload. L, Rat CDCs were infected by lentiviral vectors harboring human cytomegalovirus promoter-driven LacZ reporter gene and were subjected to intracoronary transfer into rats 4 weeks after pulmonary artery banding. Clear CDC engraftment could be detected along the endocardium and surrounding capillary vessels where the cells had been injected. M, Cardiomyocytes were stained with α-sarcomeric actin (red). Newly regenerated LacZ-positive cardiac muscle cells could be detected by β-galactosidase staining (green). Substantial cardiomyocyte regeneration was verified 4 weeks after CDC delivery. Bars, 50 μm. N, Engrafted LacZ-positive CDCs were evaluated by X-gal staining and corrected by the number of total nuclei appreciated within the respective area. Fibrotic and nonfibrotic areas were determined by Masson-trichrome staining derived from serial sections. O, Differentiated cardiomyocytes after CDC infusion were verified by the cells coexpressing both α-sarcomeric actin (red) and β-galactosidase (LacZ, green). The frequency of the cells coexpressing α-sarcomeric actin among the β-galactosidase–positive cells is shown. Data are expressed as the means (SD). [Powerpoint File]

Bioresorbable Scaffold: The Emerging Reality and Future Directions

Bioresorbable Scaffold: The Emerging Reality and Future Directions

Yohei Sotomi, Yoshinobu Onuma, Carlos Collet, Erhan Tenekecioglu, Renu Virmani, Neal S. Kleiman, Patrick W. Serruys

Strut thickness and platelet activation. The thick protruding strut disrupts the laminar flow and induces flow disturbances, and thereby endothelial shear stress (ESS) microgradients (upper panel). The shear microgradients can induce the formation of stabilized discoid platelet aggregates, the size of which is directly regulated by the magnitude and spatial distribution of the gradient.72,73 Shear microgradient–dependent platelet aggregation requires 3 principal features: shear acceleration phase, peak shear phase, and shear deceleration phase. During shear acceleration, platelets in the central regions of blood flow exposed to laminar flow (constant physiological shear) are suddenly accelerated through the shear microgradient. During the peak shear phase, a proportion of the discoid platelets that are accelerated into the peak shear zone adhere to exposed thrombogenic surfaces through platelet membrane glycoprotein (GP) Ib/IX/V. Exposure of these platelets to elevated hemodynamic drag leads to the extrusion of thin filamentous membrane tethers. Membrane tether formation initiates discoid platelet adhesion with the thrombogenic surface and also facilitates the recruitment of discoid platelets into the downstream deceleration zone. During the shear deceleration phase, platelets transitioning into the flow deceleration zone experience decreasing hemodynamic drag forces. Reduced shear within this zone progressively favors the formation of integrin αIIbβ3 adhesion contacts. Integrin αIIbβ3 engagement is associated with low-frequency calcium spikes that trigger tether restructuring, leading to the stabilization of discoid platelet aggregates. Ongoing discoid platelet recruitment drives the propagation of the thrombus in the downstream deceleration zone, which may in turn amplify the shear microgradient and promote further platelet aggregation. Thus, the shear microgradients caused by the thick struts induce platelet aggregation, formation of microthrombi with potential embolization, and micromyocardial necrosis (so-called a nidus of thrombus). The magnitude of flow disturbance depends on the degree of protrusion of the strut into the lumen. Therefore, thin struts could be a potential solution for the less flow disturbance and thus less thrombogenic status (lower panel). There is another cascade of von Willebrand factor (VWF)/GPIb activation, namely agglutination-elicited GPIb signaling.73 In contrast to shear stress–induced GPIb-elicited signaling, agglutination-elicited GPIb signaling that activates integrin αIIbβ3 requires thromboxane A2 (TXA2). Agglutination-elicited TXA2 production is independent of Ca2+ influx and mobilization of internal Ca2+ stores. [Powerpoint File]

Bioresorbable Scaffold: The Emerging Reality and Future Directions

Bioresorbable Scaffold: The Emerging Reality and Future Directions

Yohei Sotomi, Yoshinobu Onuma, Carlos Collet, Erhan Tenekecioglu, Renu Virmani, Neal S. Kleiman, Patrick W. Serruys

Design and optical coherence tomography (OCT) appearance of first and next generation bioresorbable scaffolds (BRSs). [Powerpoint File]