Posts Tagged: cardiovascular diseases

Reactive Oxygen Species in Metabolic and Inflammatory Signaling

Reactive Oxygen Species in Metabolic and Inflammatory Signaling

Steven J. Forrester, Daniel S. Kikuchi, Marina S. Hernandes, Qian Xu, Kathy K. Griendling

Cytosolic reactive oxygen species (ROS) production and regulation of cytosolic metabolic pathways. Cytosolic ROS are formed most notably through NOX (nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) activity and influence metabolic processes including glycolysis and downstream oxidative phosphorylation, pentose phosphate pathway activity, and autophagy. ACC indicates acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; ATM, ataxia-telangiectasia mutated; CAMKKβ, Ca2+/calmodulin-dependent protein kinase β; CoA, coenzyme A; CRAC, calcium release–activated channel; ER, endoplasmic reticulum; GLUT1, glucose transporter 1; HIF1α, hypoxia-inducible factor 1α; HK, hexokinase; IP3R, inositol 1,4,5-trisphosphate receptor; LKB1, liver kinase B1; mTOR, mammalian target of rapamycin; NADPH, nicotinamide adenine dinucleotide phosphate; NOS, nitric oxide synthase; OONO, peroxynitrite; OxPL, oxidized phospholipid; PDK-1, phosphoinositide-dependent kinase-1; PFK, phosphofructo kinase; PI3K, phosphoinositide 3-kinase; PKM2, pyruvate kinase 2; PPP, pentose phosphate pathway; STIM1, stromal interaction molecule 1; and TSC2, tuberous sclerosis protein 2. [Powerpoint File]

Reactive Oxygen Species in Metabolic and Inflammatory Signaling

Reactive Oxygen Species in Metabolic and Inflammatory Signaling

Steven J. Forrester, Daniel S. Kikuchi, Marina S. Hernandes, Qian Xu, Kathy K. Griendling

Peroxisomal reactive oxygen species (ROS) and metabolism. Peroxisomal ROS are produced as by-products of enzymatic reactions within β-oxidation, polyamine synthesis, d-amino acid deamination, and hypoxanthine oxidation and have been found to be key regulators of pexophagy. ACOX indicates acyl-coenzyme A (CoA) oxidases; ATM, ataxia-telangiectasia mutated; CAT, catalase; CoA, coenzyme A; DAO, d-amino acid oxidase; GPX, glutathione peroxidase; LC3, light chain 3; mTOR, mammalian target of rapamycin; PAO, polyamine oxidase; PEX, peroxin; PRDX, peroxiredoxin; SOD, superoxide dismutase; TSC1, tuberous sclerosis protein 1; ULK1, uncoordinated 51-like kinase 1; and XAO, xanthine oxidase. [Powerpoint File]

 

Reactive Oxygen Species in Metabolic and Inflammatory Signaling

Reactive Oxygen Species in Metabolic and Inflammatory Signaling

Steven J. Forrester, Daniel S. Kikuchi, Marina S. Hernandes, Qian Xu, Kathy K. Griendling

Endoplasmic reticulum and reactive oxygen species (ROS). The endoplasmic reticulum (ER) is highly sensitive to redox status, and altered ROS signaling can influence protein folding, Ca2+ release, and mitochondrial respiration. ATF6 indicates activating transcription factor 6; CAMKKII, Ca2+/calmodulin-dependent protein kinase II; Cyto, cytoplasm; ER, endoplasmic reticulum; ERO1, ER oxidoreductin 1; GPX, glutathione peroxidase; GRP78, glucose-regulated protein, 78 kDa; GSH, glutathione; GSSG, glutathione disulfide; IMS, intermembrane space; IP3R, inositol 1,4,5-trisphosphate receptor; IRE1α, inositol-requiring enzyme 1α; MCU, mitochondrial calcium uniporter; NOX, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; OxPL, oxidized phospholipid; p66shc, SHC-transforming protein 1 isoform p66; PDI, protein disulfide isomerase; PERK, protein kinase R (PKR)–like endoplasmic reticulum kinase; and Prdx, peroxiredoxin [Powerpoint File]

New Insights Into the Role of mTOR Signaling in the Cardiovascular System

New Insights Into the Role of mTOR Signaling in the Cardiovascular System

Sebastiano Sciarretta, Maurizio Forte, Giacomo Frati, Junichi Sadoshima

Schematic overview of the upstream signaling modulators of mTORC1 (mTOR [mechanistic target of rapamycin] complex 1) and mTORC2. Signaling network regulating mTORC1 and mTORC2 activity. Arrows indicate the effects of different conditions or cellular events on mTOR activity. Red arrows represent maladaptive input signals, whereas green arrows represent adaptive input signals. Akt indicates protein kinase B; AMPK, adenosine monophosphate activated protein kinase; CASTOR 1/2, cellular arginine sensor for mTORC1 1/2; ERK1/2, extracellular signal–regulated kinase 1/2; GATOR 1/2, GAP activity toward Rags 1/2; GSK-3β, glycogen synthase kinase-3β; IKKβ, inhibitor of NF-κB kinase-β; PI3K, phosphoinositide 3 kinase; PRAS40, proline-rich Akt substrate 40; Rag, Ras-related GTPase; Raptor, regulatory-associated protein of mTOR; REDD1, regulated in development and DNA damage responses 1; Rheb, Ras homolog enriched in brain; and TSC1/2, tuberous sclerosis protein 1/2. [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]

Gut Microbiota in Cardiovascular Health and Disease

Gut Microbiota in Cardiovascular Health and Disease

W.H. Wilson Tang, Takeshi Kitai, Stanley L. Hazen

Gut microbiota and possible molecular pathways linked to cardiovascular and cardiometabolic diseases. BAT indicates brown adipose tissue; FXR, farnesoid X receptor; GLP, glucagon-like peptide; GPR, G-protein–coupled receptor; LPS, lipopolysaccharide; PYY, peptide YY; TLR, toll-like receptor; TMA, trimethylamine; and TMAO, trimethylamine N-oxide. [Powerpoint File]

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Thomas Doetschman, Teodora Georgieva

Cas9 RNA-guided nuclease system. Schematic representation of the Streptococcus pyogenes Cas9 nuclease (green) targeted to genomic DNA by a single-guide RNA (sgRNA) consisting of an ≈20-nt guide sequence (blue) and a scaffold (red). The guide sequence is directly upstream of the protospacer adjacent motif (PAM), NGG (orange circles). Cas9 mediates a double-strand DNA break (DSB) ≈3 bp upstream of the PAM (red triangles). The break is repaired by 1 of 2 mechanisms: nonhomologous end joining (NHEJ) that creates random insertions or deletions at the target site or homology-directed repair (HDR). Two types of template can be used for HDR: small single-stranded DNA (ssDNA) oligonucleotide donor with short 60- 70-bp homology arms and a linear or circular dsDNA plasmid with long homology arms of 1 to 3 kb. [Powerpoint File]

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Thomas Doetschman, Teodora Georgieva

Large deletions, marker insertions, and single-nucleotide polymorphism (SNP) insertions. A, Multiple exons can be deleted by nonhomologous end joining (NHEJ) by designing single-guide RNAs (sgRNAs) that flank the region to be deleted. In the presence of Cas9 double-strand DNA breaks (DSBs) will occur resulting in small indels at the site of end joining. The double bar at the sgRNA:gene hybridization site represents the DSB caused by Cas9. Cas9 can be introduced as either mRNA or recombinant protein. B, Marker gene insertion can be achieved through homology-directed repair (HDR) in which the homologous regions are introduced in a plasmid as would be done by gene targeting. C, A chromosomal region deletion has been done using HDR in which 2 sgRNAs were designed at each end of the region to be deleted. The homology template was an single-strand DNA (ssDNA) oligo with homology at each end of the region to be deleted. D, SNP insertion using HDR with ssDNA oligo as homology template. [Powerpoint File]

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Thomas Doetschman, Teodora Georgieva

High-throughput screens. A, High-throughput gene knockout screens in which a lentiviral single-guide RNA (sgRNA) library for multiple sites in the early coding region of thousands of genes results in cell clones in which each single gene is knocked out by nonhomologous end joining repair in which indels occur. Several clones will represent each gene. A screen for a specific phenotypic outcome, such as drug resistance or altered growth, will identify genes involved in those processes. B, High-throughput screen for long noncoding RNAs (lncRNAs). A lentiviral library of paired sgRNAs, each pair of which flanks an individual lncRNA, is transduced into cells along with Cas9. Several sgRNA pairs (color matched) are designed for each lncRNA. Phenotyping clones with similar characteristics will identify lncRNAs associated with that phenotype. C, High-throughput regulatory region screen. A lentiviral library consisting of sgRNAs for tiling at ≈10-bp intervals in the regulatory region of multiple genes can be used to identify regulatory regions for each gene. A similar approach has been used to make a library of cells in which a guide RNA (gRNA or CRISPR RNA) is inserted into a modified Rosa locus in which the hairpin RNA (or tracrRNA) is adjacent to the gRNA insertion site thereby generating a sgRNA for each cell. ssDNA indicates single-strand DNA. [Powerpoint File]

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease

Thomas Doetschman, Teodora Georgieva

CRISPR-mediated modulation of gene expression. A, CRISPR interference system with dCas9 (dead, nuclease-deficient Cas9 colored in red) fused to the negative transcriptional effector KRAB (Krüppel-associated box of the Kox1 gene, colored red). A lentiviral library of single-guide RNAs for tiling at gene regulatory regions can identify enhancer elements and their location flanking the transcriptional start site (TSS) that affect gene expression. B, CRISPR activation system with dCas9 fused to a tethered string of an antibody epitope. Cotransduction of an epitope-specific antibody fused to multiple Herpes virus transcriptional activation genes VP16 will bind to the string of epitopes bringing multiple VP16s to the transcriptional machinery, thereby activating the gene. VP64 is a complex of 4 VP16 molecules. Figure derived from Figure 5A from Tanenbaum et al. [Powerpoint File]

Novel Risk Markers and Risk Assessments for Cardiovascular Disease

Novel Risk Markers and Risk Assessments for Cardiovascular Disease

Mark R. Thomas, Gregory Y.H. Lip

Acute coronary syndromes. A, Foam cells (derived from macrophages [Ma]) and lymphocytes have a central role in the development of a lipid-rich atherosclerotic plaque with a necrotic core (NC). Rupture or erosion of an atherosclerotic plaque triggers platelet (P) adhesion to subendothelial components, resulting in the formation of an occlusive thrombus, which also recruits monocytes (M) and neutrophils (N). Platelet–leukocyte interactions cause the release of proinflammatory cytokines and recruited neutrophils also release neutrophil extracellular traps. B, Myocardial ischemia, caused by coronary artery obstruction, leads to the recruitment of neutrophils and monocytes toward chemokines. Leukocyte adhesion molecules then mediate transmigration of leukocytes. Monocytes may then differentiate into Ma, alongside Ma that are already resident within myocardial tissue. Fibroblasts (F) proliferate and differentiate into myofibroblasts (MF). C, Impaired myocardial contractility and hemodynamics results in myocardial stretch, leading to consequent renal disturbances. [Powerpoint File]

Novel Risk Markers and Risk Assessments for Cardiovascular Disease

Novel Risk Markers and Risk Assessments for Cardiovascular Disease

Mark R. Thomas, Gregory Y.H. Lip

Heart failure. A, Myocardial injury, which may be triggered by a variety of insults, can lead to (B) myocardial necrosis, systemic inflammation, and infiltration of leukocytes, predominantly neutrophils (N), driven by chemokines and cytokines. N release their granule contents, thereby exerting oxidative stress and phagocytose necrotic cells and dead cardiomyocytes (DC) in conjunction with activated macrophages (Ma). C, A subsequent transition toward a reparative phase involves downregulation of the inflammatory response, release of anti-inflammatory cytokines, such as interleukin-10, and proliferation of monocytes (Mo) and lymphocytes (L). Fibroblasts (F) proliferate and differentiate in to myofibroblasts (MF), which promote collagen production and fibrosis, mediated in particular by transforming growth factor-β. D, The subsequent formation of a collagen (C)-rich scar maintains structural integrity of the myocardium at the expense of contractility and electric conductivity. [Powerpoint File]

Novel Risk Markers and Risk Assessments for Cardiovascular Disease

Novel Risk Markers and Risk Assessments for Cardiovascular Disease

Mark R. Thomas, Gregory Y.H. Lip

Atrial fibrillation (AF). The pathophysiology of AF is complex. Atrial fibrosis and electric abnormalities, including abnormal calcium homeostasis and ion-channel dysfunction, play a particularly prominent role in precipitating AF, whereas inflammation and oxidative stress reinforce pathological changes in myocardial structure. After the onset of AF, reduced blood flow through the atria predisposes toward thrombosis. The formation of an atrial thrombus, with subsequent embolization to the brain, is one of the most important causes of stroke, which may have devastating consequences. It is well-recognized that thrombosis may not purely be related to stasis of blood within the atria, but likely also reflects multiple clinical risk factors for stroke, which are particularly common in patients with AF. [Powerpoint File]

Circulating Noncoding RNAs as Biomarkers of Cardiovascular Disease and Injury

Circulating Noncoding RNAs as Biomarkers of Cardiovascular Disease and Injury

Janika Viereck, Thomas Thum

Biogenesis and function of microRNAs (miRNAs). MiRNAs are transcribed from longer precursors with protein or other noncoding gene sequences and further processed via 2 endonucleolytic processing steps. Mature miRNAs associate with Argonaute proteins (Ago2) forming the RNA-induced silencing complex (miRISC). Within this complex, miRNAs recognize their target sequence and block their expression by translational repression or degradation. MiRNAs can be released or actively secreted into the extracellular space and circulatory system stabilized in vesicles or proteinous binding partners. DGCR8, DiGeorge syndrome critical region 8; TRBP, transactivation-responsive RNA-binding protein. [Powerpoint File]