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Prefacing the e-Book Epilogue: Metabolic Genomics and Pharmaceutics

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Prefacing the e-Book Epilogue: Metabolic Genomics and Pharmaceutics

Author and Curator: Larry H. Bernstein, MD, FCAP

 

Adieu, adieu, adieu …

Sound of Music

Snoopy – Charlie happiness

This work has been a coming to terms with my scientific and medical end of career balancing in a difficult time after retiring, but it has been rewarding.  In the clinical laboratories, radiology, anesthesiology, and in pharmacy, there has been some significant progress in support of surgical, gynecological, developmental, medical practices, and even neuroscience directed disciplines, as well as epidemiology over a period of half a century.  Even then, cancer and neurological diseases have been most difficult because the scientific basic research has either not yet uncovered a framework, or because that framework has proved to be multidimensional.  In the clinical laboratory sciences, there has been enormous progress in instrumental analysis, with the recent opening of molecular methods not yet prepared for routine clinical use, which will be a very great challenge to the profession, which has seen the development of large sample volume, multianalite, high-throughput, low-cost support emerging for decades.  The capabilities now underway will also enrrich the the capabilities of the anatomic pathology suite and the capabilities of 3-dimensional radiological examination.  In both pathology and radiology, we have seen the division of the fields into major subspecialties.  The development of the electronic health record had to take lessons from the first developments in the separate developments of laboratory, radiology, and pharmacy health record systems, to which were added, full cardiology monitoring systems.  These have been unintegrated.  This made it difficult to bring forth a suitable patient health record because the information needed to support decision-making by practitioners was in separate “silos”.  The mathematical methods that are being applied to the -OMICS sciences, can be brought to bear on the simplification and amplification of the clinicians’ ability to make decisions with near “errorless” discrimination, still allowing for an element of “art” in carrying out the history, physical examination, and knowledge unique to every patient.

We are at this time opening a very large, complex, study of biology in relationship to the human condition.  This will require sufficient resources to be invested in the development of these for a better society, which I suspect, will go on beyond the life of my grandchildren.  Hopefully, the long-term dangers of climate change will be controlled in that time.  As a society, or as a group of interdependent societies, we have no long term interest in continuing self-destructive behaviors that have predominated in the history of mankind.  I now top off these discussions with some further elucidation of what lies before us.

Metabolomics and systems pharmacology: why and how to model the human metabolic network for drug discovery

Douglas B. Kell and Royston Goodacre
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK
Drug Discovery Today Feb 2014;19(2)  http://dx.doi.org/10.1016/j.drudis.2013.07.014

Metabolism represents the ‘sharp end’ of systems biology,

• because changes in metabolite concentrations
• are necessarily amplified relative to
• changes in the transcriptome, proteome and enzyme activities,
• which can be modulated by drugs.

To understand such behaviour, we therefore need
(and increasingly have)

• reliable consensus (community) models of the human metabolic network
• that include the important transporters.

Small molecule ‘drug’ transporters are in fact metabolite transporters,

• because drugs bear structural similarities to metabolites known
• from the network reconstructions and from measurements of the metabolome.

Recon2 represents the present state-of-the-art human metabolic
network reconstruction; it can predict inter alia:

1. the effects of inborn errors of metabolism;
2. which metabolites are exometabolites, and
3. how metabolism varies between tissues and cellular compartments.

Even these qualitative network models are not yet complete. As our
understanding improves so do we recognize more clearly the need for a systems (poly)pharmacology.

Modelling biochemical networks – why we do so
There are at least four types of reasons as to why one would wish to model a biochemical network:

1. Assessing whether the model is accurate, in the sense that it
   reflects – or can be made to reflect – known experimental facts.
2. Establishing what changes in the model would improve the
   consistency of its behaviour with experimental observations
   and improved predictability, such as with respect to metabolite
   concentrations or fluxes.
3. Analyzing the model, typically by some form of sensitivity
   analysis, to understand which parts of the system contribute
   most to some desired functional properties of interest.
4. Hypothesis generation and testing, enabling one to analyse
   rapidly the effects of manipulating experimental conditions in
   the model without having to perform complex and costly
   experiments (or to restrict the number that are performed).

In particular, it is normally considerably cheaper to perform
studies of metabolic networks in silico before trying a smaller
number of possibilities experimentally; indeed for combinatorial
reasons it is often the only approach possible. Although
our focus here is on drug discovery, similar principles apply to the
modification of biochemical networks for purposes of ‘industrial’
or ‘white’ biotechnology.
Why we choose to model metabolic networks more than

• transcriptomic or proteomic networks

comes from the recognition – made particularly clear

• by workers in the field of metabolic control analysis

– that, although changes in the activities of individual enzymes tend to have

• rather small effects on metabolic fluxes,
• they can and do have very large effects on metabolite concentrations (i.e. the metabolome).

Modelling biochemical networks – how we do so

Although one could seek to understand the

1. time-dependent spatial distribution of signalling and metabolic substances within indivi
   dual cellular compartments and
2. while spatially discriminating analytical methods such as Raman spectroscopy and
   mass spectrometry do exist for the analysis of drugs in situ,

• the commonest type of modelling, as in the spread of substances in
  ecosystems,
• assumes ‘fully mixed’ compartments and thus ‘pools’ of metabolites.

Although an approximation, this ‘bulk’ modelling will be necessary for complex ecosystems such as humans where, in addition to the need for tissue- and cell-specific models, microbial communities inhabit this superorganism and the
gut serves as a source for nutrients courtesy of these symbionts.

Topology and stoichiometry of metabolic networks as major constraints on fluxes
Given their topology, which admits a wide range of parameters for
delivering the same output effects and thereby reflects biological
robustness,

• metabolic networks have two especially important constraints that assist their accurate modelling:

(i) the conservation of mass and charge, and
(ii) stoichiometric and thermodynamic constraints.

These are tighter constraints than apply to signalling networks.

New developments in modelling the human metabolic network
Since 2007, several groups have been developing improved but nonidentical models of the human metabolic network at a generalised level and in tissue-specific forms. Following a similar community-driven strategy in Saccharomyces cerevisiae, surprisingly similar to humans, and in Salmonella typhimurium,

we focus in particular on a recent consensus paper that provides a highly curated and semantically annotated model of the human metabolic network, termed

• Recon2 (http://humanmetabolism.org/).

In this work, a substantial number of the major groups active in this area came together to provide a carefully and manually constructed/curated network, consisting of some 1789 enzyme-encoding genes, 7440 reactions and 2626 unique metabolites distributed over eight cellular compartments.  A variety of dead-end metabolites and blocked reactions remain (essentially orphans and widows). But Recon2 was able to

• account for some 235 inborn errors of metabolism,
• a variety of metabolic ‘tasks’ (defined as a non-zero flux through a reaction or through a pathway leading to the production of a metabolite Q from a metabolite P).
• filtering based on expression profiling allowed the construction of 65 cell-type-specific models.
• Excreted or exometabolites are an interesting set of metabolites,
• and Recon2 could predict successfully a substantial fraction of those

Role of transporters in metabolic fluxes

The uptake and excretion of metabolites between cells and their macrocompartments

• requires specific transporters and in the order of one third of ‘metabolic’ enzymes,
• and indeed of membrane proteins, are in fact transporters or equivalent.

What is of particular interest (to drug discovery), based on their structural similarities, is the increasing recognition (Fig. 3) that pharmaceutical drugs also

• get into and out of cells by ‘hitchhiking’ on such transporters, and not –

to any significant extent –

• by passing through phospholipid bilayer portions
  of cellular membranes.

This makes drug discovery even more a problem of systems biology than of biophysics.

role of solute carriers and other transporters in cellular drug uptake

Two views of the role of solute carriers and other transporters in cellular drug uptake. (a) A more traditional view in which all so-called ‘passive’drug uptake occurs through any unperturbed bilayer portion of membrane that might be present.
(b) A view in which the overwhelming fraction of drug is taken up via solute transporters or other carriers that are normally used for the uptake of intermediary metabolites. Noting that the protein:lipid ratio of biomembranes is typically 3:1 to 1:1 and that proteins vary in mass and density (a typical density is 1.37 g/ml) as does their extension, for example, normal to the ca. 4.5 nm lipid bilayer region, the figure attempts to portray a section of a membrane with realistic or typical sizes and amounts of proteins and lipids. Typical protein areas when viewed normal to the membrane are 30%, membranes are rather more ‘mosaic’ than ‘fluid’ and there is some evidence that there might be no genuinely ‘free’ bulk lipids (typical phospholipid masses are 750 Da) in biomembranes that are uninfluenced by proteins. Also shown is a typical drug: atorvastatin (LipitorW) – with a molecular mass of 558.64 Da – for size comparison purposes. If proteins are modelled as
cylinders, a cylinder with a diameter of 3.6 nm and a length of 6 nm has a molecular mass of ca. 50 kDa. Note of course that in a ‘static’ picture we cannot show the dynamics of either phospholipid chains or lipid or protein diffusion.

‘Newly discovered’ metabolites and/or their roles

To illustrate the ‘unfinished’ nature even of Recon2, which concentrates on the metabolites created via enzymes encoded in the human genome, and leaving aside the more exotic metabolites of drugs and foodstuffs and the ‘secondary’ metabolites of microorganisms, there are several examples of interesting ‘new’ (i.e. more or less recently recognised) human metabolites or roles thereof that are worth highlighting, often from studies seeking biomarkers of various diseases – for caveats of biomarker discovery, which is not a topic that we are covering here, and the need for appropriate experimental design. In addition, classes of metabolites not well represented in Recon2 are oxidised molecules such as those caused by nonenzymatic reaction of metabolites with free radicals such as the hydroxyl radical generated by unliganded iron. There is also significant interest in using methods of determining small molecules such as those in the
metabolome (inter alia) for assessing the ‘exposome’, in other words all the potentially polluting agents to which an
individual has been exposed.

Recently discovered effects of metabolites on enzymes 

Another combinatorial problem reflects the fact that in molecular enzymology it is not normally realistic to assess every possible metabolite to determine whether it is an effector (i.e.activator or inhibitor) of the enzyme under study. Typical proteins are highly promiscuous and there is increasing evidence for the comparative promiscuity of metabolites
and pharmaceutical drugs. Certainly the contribution of individual small effects of multiple parameter changes can have substantial effects on the potential flux through an overall pathway, which makes ‘bottom up’ modelling an inexact science. Even merely mimicking the vivo (in Escherichia coli) concentrations of K+, Na+, Mg2+, phosphate, glutamate, sulphate and Cl significantly modulated the activities of several enzymes tested relative to the ‘usual’ assay conditions. Consequently, we need to be alive to the possibility of many (potentially major) interactions of which we are as yet ignorant. One class of example relates to the effects of the very widespread post-translational modification on metabolic
enzyme activities.

A recent and important discovery (Fig. 4) is that a single transcriptome experiment, serving as a surrogate for fluxes through individual steps, provides a huge constraint on possible models, and predicts in a numerically tractable way and
with much improved accuracy the fluxes to exometabolites without the need for such a variable ‘biomass’ term. Other recent and related strategies that exploit modern advances in ‘omics and network biology to limit the search space in constraint-based metabolic modelling.

Fig 4. Workflow for expression-profile-constrained metabolic flux estimation

1. Genome-scale metabolic model with gene-protein-reaction relationships
2. Map absolute gene expression levels to reactions
3. Maximise correlation between absolute gene expression and metabolic flux
4. Predict fluxes to exometabolites
5. Compare predicted with experimental fluxes to exometabolites

Drug Discovery Today

The steps in a workflow that uses constraints based on (i) metabolic network stoichiometry and chemical reaction properties (both encoded in the model) plus, and (ii) absolute (RNA-Seq) transcript expression profiles to enable the
accurate modelling of pathway and exometabolite fluxes. .

Concluding remarks – the role of metabolomics in systems pharmacology

What is becoming increasingly clear, as we recognize that to understand living organisms in health and disease we must treat them as systems, is that we must bring together our knowledge of the topologies and kinetics of metabolic networks with our knowledge of the metabolite concentrations (i.e. metabolomes) and fluxes. Because of the huge constraints imposed on metabolism by reaction stoichiometries, mass conservation and thermodynamics, comparatively few well-chosen ‘omics measurements might be needed to do this reliably (Fig. 4). Indeed, a similar approach exploiting constraints has come to the fore in denovo protein folding and interaction studies.

What this leads us to in drug discovery is the need to develop and exploit a ‘systems pharmacology’ where multiple binding targets are chosen purposely and simultaneously. Along with other measures such as phenotypic screening, and the integrating of the full suite of e-science approaches, one can anticipate considerable improvements in the rate of discovery of safe and effective drugs.

Metabolomics: the apogee of the omics trilogy
Gary J.!Patti, Oscar Yanes and Gary Siuzdak

Metabolites, the chemical entities that are transformed during metabolism, provide a functional readout of cellular biochemistry. With emerging technologies in mass spectrometry, thousands of metabolites can now be
quantitatively measured from minimal amounts of biological material, which has thereby enabled systems-level analyses. By performing global metabolite profiling, also known as untargeted metabolomics, new discoveries linking cellular pathways to biological mechanism are being revealed and are shaping our understanding of cell biology, physiology and medicine.

Metabolites are small molecules that are chemically transformed during metabolism and, as such, they provide a functional readout of cellular state. Unlike genes and proteins, the functions of which are subject to epigenetic regulation and posttranslational modifications, respectively, metabolites serve as direct signatures of biochemical activity and are therefore easier to correlate with phenotype. In this context, metabolite profiling, or metabolomics, has become a powerful approach that has been widely adopted for clinical diagnostics.

The field of metabolomics has made remarkable progress within the past decade and has implemented new tools that have offered mechanistic insights by allowing for the correlation of biochemical changes with phenotype.

In this Innovation article, we first define and differentiate between the targeted and untargeted approaches to metabolomics. We then highlight the value of untargeted metabolomics in particular and outline a guide to performing such studies. Finally, we describe selected applications of un targeted metabolomics and discuss their potential in cell biology.

• metabolites serve as direct signatures of biochemical activity

1. In some instances, it may be of interest to examine a defined set of metabolites by using a targeted approach.
2. In other cases, an untargeted or global approach may be taken in which as many metabolites as possible are measured and compared between samples without bias.
3. Ultimately, the number and chemical composition of metabolites to be studied is a defining attribute of any metabolomic experiment and shapes experimental design with respect to sample preparation and choice of instrumentation.

The targeted and untargeted workflow for LC/MS-based metabolomics.

a | In the triple quadrupole (QqQ)-based targeted metabolomic workflow, standard compounds for the metabolites of interest are first used to set up selected reaction monitoring methods. Here, optimal instrument voltages are determined and response curves are generated for absolute quantification. After the targeted methods have been established
on the basis of standard metabolites, metabolites are extracted from tissues, biofluids or cell cultures and analysed. The data output provides quantification only of those metabolites for which standard methods have been built.

b | In the untargeted metabolomic workflow, metabolites are first isolated from biological samples and subsequently analysed by liquid chromatography followed by mass spectrometry (LC/MS). After data acquisition, the results are processed by using bioinformatic software such as XCMS to perform nonlinear retention time alignment and identify peaks that are changing between the groups of samples measured. The m/z value s for the peaks of interest are searched in metabolite databases to obtain putative identifications. Putative identifications are then confirmed
by comparing tandem mass spectrometry (MS/MS) data and retention time data to that of standard compounds. The untargeted workflow is global in scope and outputs data related to comprehensive cellular metabolism.

Metabolic Biomarker and Kinase Drug Target Discovery in Cancer Using Stable Isotope-Based Dynamic Metabolic Profiling (SIDMAP)

László G. Boros1*, Daniel J. Brackett2 and George G. Harrigan3
1UCLA School of Medicine, Harbor-UCLA Research and Education Institute, Torrance, CA. 2Department of Surgery, University of Oklahoma Health Sciences Center & VA Medical Center, Oklahoma City, OK, 3Global High Throughput
Screening (HTS), Pharmacia Corporation, Chesterfield, MO.
Current Cancer Drug Targets, 2003, 3, 447-455.

Tumor cells respond to growth signals by the activation of protein kinases, altered gene expression and significant modifications in substrate flow and redistribution among biosynthetic pathways. This results in a proliferating phenotype
with altered cellular function. These transformed cells exhibit unique anabolic characteristics, which includes increased and preferential utilization of glucose through the non-oxidative steps of the pentose cycle for nucleic acid synthesis but limited denovo fatty acid synthesis and TCA cycle glucose oxidation. This primarily nonoxidative anabolic profile reflects an undifferentiated highly proliferative aneuploid cell phenotype and serves as a reliable metabolic biomarker to determine cell proliferation rate and the level of cell transformation/differentiation in response to drug treatment. Novel drugs effective in particular cancers exert their anti-proliferative effects by inducing significant reversions of a few specific non-oxidative anabolic pathways. Here we present evidence that cell transformation of various mechanisms is sustained by a unique
disproportional substrate distribution between the two branches of the pentose cycle for nucleic acid synthesis, glycolysis and the TCA cycle for fatty acid synthesis and glucose oxidation. This can be demonstrated by the broad labeling and unique specificity of [1,2-13C2]glucose to trace a large number of metabolites in the metabolome. Stable isotope-based dynamic metabolic profiles (SIDMAP) serve the drug discovery process by providing a powerful new tool that integrates the metabolome into a functional genomics approach to developing new drugs. It can be used in screening kinases and their metabolic targets, which can therefore be more efficiently characterized, speeding up and improving drug testing, approval and labeling processes by saving trial and error type study costs in drug testing.

Navigating the HumanMetabolome for Biomarker Identification and Design of Pharmaceutical Molecules

Irene Kouskoumvekaki and Gianni Panagiotou
Department of Systems Biology, Center for Biological Sequence Analysis, Building 208, Technical University of Denmark, Lyngby, Denmark
Hindawi Publishing Corporation  Journal of Biomedicine and Biotechnology 2011, Article ID 525497, 19 pages
http://dx.doi.org:/10.1155/2011/525497

Metabolomics is a rapidly evolving discipline that involves the systematic study of endogenous small molecules that characterize the metabolic pathways of biological systems. The study of metabolism at a global level has the potential to contribute significantly to biomedical research, clinical medical practice, as well as drug discovery. In this paper, we present the most up-to-date metabolite and metabolic pathway resources, and we summarize the statistical, and machine-learning tools used for the analysis of data from clinical metabolomics.

Through specific applications on cancer, diabetes, neurological and other diseases, we demonstrate how these tools can facilitate diagnosis and identification of potential biomarkers for use within disease diagnosis. Additionally, we
discuss the increasing importance of the integration of metabolomics data in drug discovery. On a case-study based on the Human Metabolome Database (HMDB) and the Chinese Natural Product Database (CNPD), we demonstrate the close relatedness of the two data sets of compounds, and we further illustrate how structural similarity with human metabolites could assist in the design of novel pharmaceuticals and the elucidation of the molecular mechanisms of medicinal plants.

Metabolites are the byproducts of metabolism, which is itself the process of converting food energy to mechanical energy
or heat. Experts believe there are at least 3,000 metabolites that are essential for normal growth and development (primary metabolites) and thousands more unidentified (around 20,000, compared to an estimated 30,000 genes and 100,000 proteins) that are not essential for growth and development (secondary metabolites) but could represent prognostic, diagnostic, and surrogate markers for a disease state and a deeper understanding of mechanisms of disease.

Metabolomics, the study of metabolism at the global level, has the potential to contribute significantly to biomedical
research, and ultimately to clinical medical practice. It is a close counterpart to the genome, the transcriptome and the proteome. Metabolomics, genomics, proteomics, and other “-omics” grew out of the Human Genome Project, a massive research effort that began in the mid-1990s and culminated in 2003 with a complete mapping of all the genes in the human body. When discussing the clinical advantages of metabolomics, scientists point to the “real-world” assessment
of patient physiology that the metabolome provides since it can be regarded as the end-point of the “-omics” cascade. Other functional genomics technologies do not necessarily predict drug effects, toxicological response, or disease states at the phenotype but merely indicate the potential cause for phenotypical response. Metabolomics can bridge this information gap. The identification and measurement of metabolite profile dynamics of host changes provides the closest link to the various phenotypic responses. Thus it is clear that the global mapping of metabolic signatures pre- and postdrug treatment is a promising approach to identify possible functional relationships between medication and medical phenotype.

Human Metabolome Database (HMDB). Focusing on quantitative, analytic, or molecular scale information about
metabolites, the enzymes and transporters associated with them, as well as disease related properties the HMDB represents the most complete bioinformatics and chemoinformatics medical information database. It contains records for
thousands of endogenous metabolites identified by literature surveys (PubMed, OMIM, OMMBID, text books), data
mining (KEGG, Metlin, BioCyc) or experimental analyses performed on urine, blood, and cerebrospinal fluid samples.
The annotation effort is aided by chemical parameter calculators and protein annotation tools originally developed for
DrugBank.

A key feature that distinguishes the HMDB from other metabolic resources is its extensive support for higher level database searching and selecting functions. More than 175 hand-drawn-zoomable, fully hyperlinked human
metabolic pathway maps can be found in HMDB and all these maps are quite specific to human metabolism and
explicitly show the subcellular compartments where specific reactions are known to take place. As an equivalent to
BLAST the HMDB contains a structure similarity search tool for chemical structures and users may sketch or
paste a SMILES string of a query compound into the Chem-Query window. Submitting the query launches a
structure similarity search tool that looks for common substructures from the query compound that match the
HMDB’s metabolite database. The wealth of information and especially the extensive linkage to metabolic diseases
to normal and abnormal metabolite concentration ranges, to mutation/SNP data and to the genes, enzymes, reactions
and pathways associated with many diseases of interest makes the HMDB one the most valuable tool in the hands
of clinical chemists, nutritionists, physicians and medical geneticists.

Metabolomics in Drug Discovery and Polypharmacology Studies

Drug molecules generally act on specific targets at the cellular level, and upon binding to the receptors, they exert
a desirable alteration of the cellular activities, regarded as the pharmaceutical effect. Current drug discovery depends
largely on ransom screening, either high-throughput screening (HTS) in vitro, or virtual screening (VS) in silico. Because
the number of available compounds is huge, several druglikeness filters are proposed to reduce the number of compounds that need to be evaluated. The ability to effectively predict if a chemical compound is “drug-like” or “nondruglike” is, thus, a valuable tool in the design, optimization, and selection of drug candidates for development. Druglikeness is a general descriptor of the potential of a small molecule to become a drug. It is not a unified descriptor
but a global property of a compound processing many specific characteristics such as good solubility, membrane
permeability, half-life, and having a pharmacophore pattern to interact specifically with a target protein. These
characteristics can be reflected as molecular descriptors such as molecular weight, log P, the number of hydrogen bond
donors, the number of hydrogen-bond acceptors, the number of rotatable bonds, the number of rigid bonds, the
number of rings in a molecule, and so forth.

Metabolomics for the Study of Polypharmacology of Natural Compounds

Internationally, there is a growing and sustained interest from both pharmaceutical companies and public in medicine
from natural sources. For the public, natural medicine represent a holistic approach to disease treatment, with
potentially less side effects than conventional medicine. For the pharmaceutical companies, bioactive natural products
constitute attractive drug leads, as they have been optimized in a long-term natural selection process for optimal interaction with biomolecules. To promote the ecological survival of plants, structures of secondary products have evolved to interact with molecular targets affecting the cells, tissues and physiological functions in competing microorganisms,
plants, and animals. In this, respect, some plant secondary products may exert their action by resembling endogenous
metabolites, ligands, hormones, signal transduction molecules, or neurotransmitters and thus have beneficial
effects on humans.

Future Perspectives

Metabolomics, the study of metabolism at the global level, is moving to exciting directions.With the development ofmore
sensitive and advanced instrumentation and computational tools for data interpretation in the physiological context,
metabolomics have the potential to impact our understanding of molecular mechanisms of diseases. A state-of-theart
metabolomics study requires knowledge in many areas and especially at the interface of chemistry, biology, and
computer science. High-quality samples, improvements in automated metabolite identification, complete coverage of
the human metabolome, establishment of spectral databases of metabolites and associated biochemical identities, innovative experimental designs to best address a hypothesis, as well as novel computational tools to handle metabolomics data are critical hurdles that must be overcome to drive the inclusion of metabolomics in all steps of drug discovery and drug development. The examples presented above demonstrated that metabolite profiles reflect both environmental and genetic influences in patients and reveal new links between metabolites and diseases providing needed prognostic,diagnostic, and surrogate biomarkers. The integration of these signatures with other omic technologies is of utmost importance to characterize the entire spectrum of malignant phenotype.

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Leaders in Pharmaceutical Business Intelligence Announced New Cardiovascular Series of e-Books at SACHS Associates 14th Annual Biotech In Europe Forum

Posted in Abdominal Aorta, Academic Publishing, Artificial Heart, BiVAD, CABG, Cancer Surgery of the Heart, Cardiac & Vascular Repair Tools Subsegment, Cardiac and Cardiovascular Surgical Procedures, Cardiovascular Pharmacogenomics, Carotid Artery, Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Pulmonary Arterial Hypertension (PAH), Clinical Diagnostics, Coagulation Therapy and Internal Bleeding, Conference Coverage with Social Media, CT, Curation, Curation methodology, De novo synthesis, Dialectic, Discovery process, Evolutionary cognition, Exec Compensation in the Cardiac & Vascular Repair Tools Subsegment, Experimental validation, Explanatory, Frontiers in Cardiology and Cardiovascular Disorders, Heart Transplant, Heart-Lung Transplant, Historical relevance, Mechanical Assist Devices: LVAD, Mitral Valve: Repair and Replacement, PCI, Peripheral Arterial Disease & Peripheral Vascular Surgery, Renal Denervation, RVAD, TAVI vs Open Heart Surgery, Technology Advance Assessment of, Thoracic Aorta, tagged Aviva Lev-Ari, Biology, Cardiovascular disease, Cardiovascular Disorders, ebook, health, Heart disease, Heart Failure, Hypertension, Larry H. Bernstein, medical breakthrough, medical education, nitric oxide, Personalized medicine, research on September 27, 2014| Leave a Comment »

Leaders in Pharmaceutical Business Intelligence Announced New Cardiovascular Series of e-Books at SACHS Associates 14th Annual Biotech In Europe Forum

Reporter: Aviva Lev-Ari, PhD, RN

Volume II can be found at http://pharmaceuticalintelligence.com/biomed-e-books/series-a-e-books-on-cardiovascular-diseases/volume-two-cardiovascular-original-research-cases-in-methodology-design-for-content-co-curation/

Volume III can be found at http://pharmaceuticalintelligence.com/biomed-e-books/series-a-e-books-on-cardiovascular-diseases/volume-three-etiologies-of-cardiovascular-diseases-epigenetics-genetics-genomics/

Volume IV can be found at http://pharmaceuticalintelligence.com/biomed-e-books/series-a-e-books-on-cardiovascular-diseases/volume-four-therapeutic-promise-cvd-regenerative-translational-medicine/

 

 

Please see Further Titles at

http://pharmaceuticalintelligence.com/biomed-e-books/

Please see Further Information on the Sachs Associates 14th Annual Biotech in Europe Forum for Global Investing & Partnering at:

http://pharmaceuticalintelligence.com/2014/03/25/14th-annual-biotech-in-europe-forum-for-global-partnering-investment-930-1012014-%E2%80%A2-congress-center-basel-sachs-associates-london/

AND

http://www.sachsforum.com/basel14/index.html

ON TWITTER Follow at

@SachsAssociates

#Sachs14thBEF

@pharma_BI

@AVIVA1950 

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Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1

Posted in Academic Publishing, Acute Myocardial Infarction, Advanced Drug Manufacturing Technology, Alzheimer's Disease, Atherogenic Processes & Pathology, Autologous Cell Therapy, Automated Cell Processing, Bio Instrumentation in Experimental Life Sciences Research, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Biomedical Measurement Science, Bone Disease and Musculoskeletal Disease, Bone marrow derived cells, Ca2+ triggered activation, Ca2+ triggered activation, Cachexia, Calcium Signaling, Calmodulin, Calmodulin Kinase and Contraction, Cardiac & Vascular Repair Tools Subsegment, Cardiac and Cardiovascular Surgical Procedures, Cardiac muscle regeneration, Cardiomyopathy, Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Pulmonary Arterial Hypertension (PAH), Circulating Progenitor Cells, Coagulation Therapy and Internal Bleeding, Competition in regenerative stem cell science, Computational Biology/Systems and Bioinformatics, Curation, Cytoskeleton, Diabetes Mellitus, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Drug Delivery Platform Technology, Drug Toxicity, Electrophysiology, Endothelial cells, Epigenetics and Cardiovascular Risks, Etiology, Evolutionary cognition, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Genomic Expression, Health Economics and Outcomes Research, Human Immune System in Health and in Disease, International Global Work in Pharmaceutical, Interviews with Scientific Leaders, Medical and Population Genetics, Medical Device Therapies for Altzheimer’s Disease, Medical Devices R&D Investment, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Metabolomics, Molecular Genetics & Pharmaceutical, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Na-K transport, Na-K-ATPase, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Nutrition, Nutritional Supplements: Atherogenesis, lipid metabolism, Origins of Cardiovascular Disease, Oxidative phosphorylation, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmacologic toxicities, Pharmacotherapy of Cardiovascular Disease, Phosphorylation, PKC, Population Health Management, Genetics & Pharmaceutical, Population Health Management, Nutrition and Phytochemistry, Proteomics, Regenerative Biology and Medicine, S-nitrosylation, Signaling, Skeletal muscle regeneration, Statistical Methods for Research Evaluation, Stem Cells for Regenerative Medicine, Stents & Tools, Synaptic vesicle, Systemic Inflammatory Response Related Disorders, Technology Advance Assessment of, Translational Effectiveness, Translational Science, Ubiquitinylation, Water Transporters, tagged bioengineering, Cardiovascular Disorders, computational biology, Coronary artery disease, DNA Sequencing, functional proteomics, gene expression, gene silencing, genetic engineering, Heart Failure, Human Genome Project, Hypertension, MicroRNA, mtRNA, myocardial infarction, nitric oxide, Nitric oxide synthase, Personalized medicine, protein modifications, Proteomics, reverse engineering, RNA, signaling pathways, Stem cell, synbiology on April 28, 2014| Leave a Comment »

Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1

Author and Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

 

Part 1 of Volume 4 in the e-series A: Cardiovascular Diseases and Translational Medicine, provides a foundation for grasping a rapidly developing surging scientific endeavor that is transcending laboratory hypothesis testing and providing guidelines to:

• Target genomes and multiple nucleotide sequences involved in either coding or in regulation that might have an impact on complex diseases, not necessarily genetic in nature.
• Target signaling pathways that are demonstrably maladjusted, activated or suppressed in many common and complex diseases, or in their progression.
• Enable a reduction in failure due to toxicities in the later stages of clinical drug trials as a result of this science-based understanding.
• Enable a reduction in complications from the improvement of machanical devices that have already had an impact on the practice of interventional procedures in cardiology, cardiac surgery, and radiological imaging, as well as improving laboratory diagnostics at the molecular level.
• Enable the discovery of new drugs in the continuing emergence of drug resistance.
• Enable the construction of critical pathways and better guidelines for patient management based on population outcomes data, that will be critically dependent on computational methods and large data-bases.

What has been presented can be essentially viewed in the following Table:

 

Summary Table for TM – Part 1

 

 

 

There are some developments that deserve additional development:

1. The importance of mitochondrial function in the activity state of the mitochondria in cellular work (combustion) is understood, and impairments of function are identified in diseases of muscle, cardiac contraction, nerve conduction, ion transport, water balance, and the cytoskeleton – beyond the disordered metabolism in cancer.  A more detailed explanation of the energetics that was elucidated based on the electron transport chain might also be in order.

2. The processes that are enabling a more full application of technology to a host of problems in the environment we live in and in disease modification is growing rapidly, and will change the face of medicine and its allied health sciences.

 

Electron Transport and Bioenergetics

Deferred for metabolomics topic

Synthetic Biology

Introduction to Synthetic Biology and Metabolic Engineering

Kristala L. J. Prather: Part-1    <iBiology > iBioSeminars > Biophysics & Chemical Biology >

http://www.ibiology.org Lecturers generously donate their time to prepare these lectures. The project is funded by NSF and NIGMS, and is supported by the ASCB and HHMI.
Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”.

http://synthetic_biology/Introduction_to_Synthetic_Biology_and_Metabolic_Engineering/Kristala_ L.J._Prather/E2/iBiology.htm#/sthash.71kZ0ofr.dpuf

Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching.  Learn more about how Kris became a scientist at

http://science360.gov/obj/video/753bb4fa-aafb-4315-848e-51066ed9799a/finding-way-kristala-l-jones-prather-phd 

Prather 1: Synthetic Biology and Metabolic Engineering  2/6/14IntroductionLecture Overview In the first part of her lecture, Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”. The key material in building these machines is synthetic DNA. Synthetic DNA can be added in different combinations to biological hosts, such as bacteria, turning them into chemical factories that can produce small molecules of choice. In Part 2, Prather describes how her lab used design principles to engineer E. coli that produce glucaric acid from glucose. Glucaric acid is not naturally produced in bacteria, so Prather and her colleagues “bioprospected” enzymes from other organisms and expressed them in E. coli to build the needed enzymatic pathway. Prather walks us through the many steps of optimizing the timing, localization and levels of enzyme expression to produce the greatest yield. Speaker Bio: Kristala Jones Prather received her S.B. degree from the Massachusetts Institute of Technology and her PhD at the University of California, Berkeley both in chemical engineering. Upon graduation, Prather joined the Merck Research Labs for 4 years before returning to academia. Prather is now an Associate Professor of Chemical Engineering at MIT and an investigator with the multi-university Synthetic Biology Engineering Reseach Center (SynBERC). Her lab designs and constructs novel synthetic pathways in microorganisms converting them into tiny factories for the production of small molecules. Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching.

VIEW VIDEOS

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=0

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=12

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=74

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=129

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk#t=168

https://www.youtube.com/watch?feature=player_embedded&v=ndThuqVumAk

 

David Bartel: micro-RNAs

 

I. Introduction to microRNAs

https://www.youtube.com/watch?feature=player_embedded&v=dupzE66J8u4#t=0

– See more at: http://www.ibiology.org/ibioseminars/genetics-gene-regulation/david-bartel-part-1.html#sthash.nGGr1tIt.dpuf

II. Regulatory Effects of Mammalian microRNAs

https://www.youtube.com/watch?feature=player_embedded&v=TcZK_A_wcWQ#t=0

https://www.youtube.com/watch?feature=player_embedded&v=TcZK_A_wcWQ

Calcium Cycling in Synthetic and Contractile Phasic or Tonic Vascular Smooth Muscle Cells

in INTECH
Current Basic and Pathological Approaches to
the Function of Muscle Cells and Tissues – From Molecules to HumansLarissa Lipskaia, Isabelle Limon, Regis Bobe and Roger Hajjar
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/48240

1. Introduction
Calcium ions (Ca ) are present in low concentrations in the cytosol (~100 nM) and in high concentrations (in mM range) in both the extracellular medium and intracellular stores (mainly sarco/endo/plasmic reticulum, SR). This differential allows the calcium ion messenger that carries information
as diverse as contraction, metabolism, apoptosis, proliferation and/or hypertrophic growth. The mechanisms responsible for generating a Ca signal greatly differ from one cell type to another.

In the different types of vascular smooth muscle cells (VSMC), enormous variations do exist with regard to the mechanisms responsible for generating Ca signal. In each VSMC phenotype (synthetic/proliferating and contractile [1], tonic or phasic), the Ca signaling system is adapted to its particular function and is due to the specific patterns of expression and regulation of Ca.
For instance, in contractile VSMCs, the initiation of contractile events is driven by mem- brane depolarization; and the principal entry-point for extracellular Ca is the voltage-operated L-type calcium channel (LTCC). In contrast, in synthetic/proliferating VSMCs, the principal way-in for extracellular Ca is the store-operated calcium (SOC) channel.
Whatever the cell type, the calcium signal consists of  limited elevations of cytosolic free calcium ions in time and space. The calcium pump, sarco/endoplasmic reticulum Ca ATPase (SERCA), has a critical role in determining the frequency of SR Ca release by upload into the sarcoplasmic
sensitivity of  SR calcium channels, Ryanodin Receptor, RyR and Inositol tri-Phosphate Receptor, IP3R.
Synthetic VSMCs have a fibroblast appearance, proliferate readily, and synthesize increased levels of various extracellular matrix components, particularly fibronectin, collagen types I and III, and tropoelastin [1].
Contractile VSMCs have a muscle-like or spindle-shaped appearance and well-developed contractile apparatus resulting from the expression and intracellular accumulation of thick and thin muscle filaments [1].

Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs

 

Figure 1. Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs.

Left panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Contractile re-sponse is initiated by extracellular Ca influx due to activation of Receptor Operated Ca (through phosphoinositol-coupled receptor) or to activation of L-Type Calcium channels (through an increase in luminal pressure). Small increase of cytosolic due IP3 binding to IP3R (puff) or RyR activation by LTCC or ROC-dependent Ca influx leads to large SR Ca IP3R or RyR clusters (“Ca -induced Ca SR calcium pumps (both SERCA2a and SERCA2b are expressed in quiescent VSMCs), maintaining high concentration of cytosolic Ca and setting the sensitivity of RyR or IP3R for the next spike.
Contraction of VSMCs occurs during oscillatory Ca transient.
Middle panel: schematic representa tion of atherosclerotic vessel wall. Contractile VSMC are located in the media layer, synthetic VSMC are located in sub-endothelial intima.
Right panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Agonist binding to phosphoinositol-coupled receptor leads to the activation of IP3R resulting in large increase in cytosolic Ca calcium pumps (only SERCA2b, having low turnover and low affinity to Ca depletion leads to translocation of SR Ca sensor STIM1 towards PM, resulting in extracellular Ca influx though opening of Store Operated Channel (CRAC). Resulted steady state Ca transient is critical for activation of proliferation-related transcription factors ‘NFAT).
Abbreviations: PLC – phospholipase C; PM – plasma membrane; PP2B – Ca /calmodulin-activated protein phosphatase 2B (calcineurin); ROC- receptor activated channel; IP3 – inositol-1,4,5-trisphosphate, IP3R – inositol-1,4,5- trisphosphate receptor; RyR – ryanodine receptor; NFAT – nuclear factor of activated T-lymphocytes; VSMC – vascular smooth muscle cells; SERCA – sarco(endo)plasmic reticulum Ca sarcoplasmic reticulum.

 

Time for New DNA Synthesis and Sequencing Cost Curves

By Rob Carlson

I’ll start with the productivity plot, as this one isn’t new. For a discussion of the substantial performance increase in sequencing compared to Moore’s Law, as well as the difficulty of finding this data, please see this post. If nothing else, keep two features of the plot in mind: 1) the consistency of the pace of Moore’s Law and 2) the inconsistency and pace of sequencing productivity. Illumina appears to be the primary driver, and beneficiary, of improvements in productivity at the moment, especially if you are looking at share prices. It looks like the recently announced NextSeq and Hiseq instruments will provide substantially higher productivities (hand waving, I would say the next datum will come in another order of magnitude higher), but I think I need a bit more data before officially putting another point on the plot.

 

cost-of-oligo-and-gene-synthesis

Illumina’s instruments are now responsible for such a high percentage of sequencing output that the company is effectively setting prices for the entire industry. Illumina is being pushed by competition to increase performance, but this does not necessarily translate into lower prices. It doesn’t behoove Illumina to drop prices at this point, and we won’t see any substantial decrease until a serious competitor shows up and starts threatening Illumina’s market share. The absence of real competition is the primary reason sequencing prices have flattened out over the last couple of data points.

Note that the oligo prices above are for column-based synthesis, and that oligos synthesized on arrays are much less expensive. However, array synthesis comes with the usual caveat that the quality is generally lower, unless you are getting your DNA from Agilent, which probably means you are getting your dsDNA from Gen9.

Note also that the distinction between the price of oligos and the price of double-stranded sDNA is becoming less useful. Whether you are ordering from Life/Thermo or from your local academic facility, the cost of producing oligos is now, in most cases, independent of their length. That’s because the cost of capital (including rent, insurance, labor, etc) is now more significant than the cost of goods. Consequently, the price reflects the cost of capital rather than the cost of goods. Moreover, the cost of the columns, reagents, and shipping tubes is certainly more than the cost of the atoms in the sDNA you are ostensibly paying for. Once you get into longer oligos (substantially larger than 50-mers) this relationship breaks down and the sDNA is more expensive. But, at this point in time, most people aren’t going to use longer oligos to assemble genes unless they have a tricky job that doesn’t work using short oligos.

Looking forward, I suspect oligos aren’t going to get much cheaper unless someone sorts out how to either 1) replace the requisite human labor and thereby reduce the cost of capital, or 2) finally replace the phosphoramidite chemistry that the industry relies upon.

IDT’s gBlocks come at prices that are constant across quite substantial ranges in length. Moreover, part of the decrease in price for these products is embedded in the fact that you are buying smaller chunks of DNA that you then must assemble and integrate into your organism of choice.

Someone who has purchased and assembled an absolutely enormous amount of sDNA over the last decade, suggested that if prices fell by another order of magnitude, he could switch completely to outsourced assembly. This is a potentially interesting “tipping point”. However, what this person really needs is sDNA integrated in a particular way into a particular genome operating in a particular host. The integration and testing of the new genome in the host organism is where most of the cost is. Given the wide variety of emerging applications, and the growing array of hosts/chassis, it isn’t clear that any given technology or firm will be able to provide arbitrary synthetic sequences incorporated into arbitrary hosts.

 TrackBack URL: http://www.synthesis.cc/cgi-bin/mt/mt-t.cgi/397

 

Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products

28 Nov 2013 | PR Web

Dr. Jon Rowley and Dr. Uplaksh Kumar, Co-Founders of RoosterBio, Inc., a newly formed biotech startup located in Frederick, are paving the way for even more innovation in the rapidly growing fields of Synthetic Biology and Regenerative Medicine. Synthetic Biology combines engineering principles with basic science to build biological products, including regenerative medicines and cellular therapies. Regenerative medicine is a broad definition for innovative medical therapies that will enable the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. Regenerative therapies that are in clinical trials today may enable repair of damaged heart muscle following heart attack, replacement of skin for burn victims, restoration of movement after spinal cord injury, regeneration of pancreatic tissue for insulin production in diabetics and provide new treatments for Parkinson’s and Alzheimer’s diseases, to name just a few applications.

While the potential of the field is promising, the pace of development has been slow. One main reason for this is that the living cells required for these therapies are cost-prohibitive and not supplied at volumes that support many research and product development efforts. RoosterBio will manufacture large quantities of standardized primary cells at high quality and low cost, which will quicken the pace of scientific discovery and translation to the clinic. “Our goal is to accelerate the development of products that incorporate living cells by providing abundant, affordable and high quality materials to researchers that are developing and commercializing these regenerative technologies” says Dr. Rowley

 

Life at the Speed of Light

http://kcpw.org/?powerpress_pinw=92027-podcast

NHMU Lecture featuring – J. Craig Venter, Ph.D.
Founder, Chairman, and CEO – J. Craig Venter Institute; Co-Founder and CEO, Synthetic Genomics Inc.

J. Craig Venter, Ph.D., is Founder, Chairman, and CEO of the J. Craig Venter Institute (JVCI), a not-for-profit, research organization dedicated to human, microbial, plant, synthetic and environmental research. He is also Co-Founder and CEO of Synthetic Genomics Inc. (SGI), a privately-held company dedicated to commercializing genomic-driven solutions to address global needs.

In 1998, Dr. Venter founded Celera Genomics to sequence the human genome using new tools and techniques he and his team developed.  This research culminated with the February 2001 publication of the human genome in the journal, Science. Dr. Venter and his team at JVCI continue to blaze new trails in genomics.  They have sequenced and a created a bacterial cell constructed with synthetic DNA,  putting humankind at the threshold of a new phase of biological research.  Whereas, we could  previously read the genetic code (sequencing genomes), we can now write the genetic code for designing new species.

The science of synthetic genomics will have a profound impact on society, including new methods for chemical and energy production, human health and medical advances, clean water, and new food and nutritional products. One of the most prolific scientists of the 21st century for his numerous pioneering advances in genomics,  he  guides us through this emerging field, detailing its origins, current challenges, and the potential positive advances.

His work on synthetic biology truly embodies the theme of “pushing the boundaries of life.”  Essentially, Venter is seeking to “write the software of life” to create microbes designed by humans rather than only through evolution. The potential benefits and risks of this new technology are enormous. It also requires us to examine, both scientifically and philosophically, the question of “What is life?”

J Craig Venter wants to digitize DNA and transmit the signal to teleport organisms

http://pharmaceuticalintelligence.com/2013/11/01/j-craig-venter-wants-to-digitize-dna-and-transmit-the-signal-to-teleport-organisms/

2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

http://pharmaceuticalintelligence.com/2013/02/11/2013-genomics-the-era-beyond-the-sequencing-human-genome-francis-collins-craig-venter-eric-lander-et-al/

Human Longevity Inc (HLI) – $70M in Financing of Venter’s New Integrative Omics and Clinical Bioinformatics

http://pharmaceuticalintelligence.com/2014/03/05/human-longevity-inc-hli-70m-in-financing-of-venters-new-integrative-omics-and-clinical-bioinformatics/

 

 

Where Will the Century of Biology Lead Us?

By Randall Mayes

A technology trend analyst offers an overview of synthetic biology, its potential applications, obstacles to its development, and prospects for public approval.

• In addition to boosting the economy, synthetic biology projects currently in development could have profound implications for the future of manufacturing, sustainability, and medicine.
• Before society can fully reap the benefits of synthetic biology, however, the field requires development and faces a series of hurdles in the process. Do researchers have the scientific know-how and technical capabilities to develop the field?

Biology + Engineering = Synthetic Biology

Bioengineers aim to build synthetic biological systems using compatible standardized parts that behave predictably. Bioengineers synthesize DNA parts—oligonucleotides composed of 50–100 base pairs—which make specialized components that ultimately make a biological system. As biology becomes a true engineering discipline, bioengineers will create genomes using mass-produced modular units similar to the microelectronics and computer industries.

Currently, bioengineering projects cost millions of dollars and take years to develop products. For synthetic biology to become a Schumpeterian revolution, smaller companies will need to be able to afford to use bioengineering concepts for industrial applications. This will require standardized and automated processes.

A major challenge to developing synthetic biology is the complexity of biological systems. When bioengineers assemble synthetic parts, they must prevent cross talk between signals in other biological pathways. Until researchers better understand these undesired interactions that nature has already worked out, applications such as gene therapy will have unwanted side effects. Scientists do not fully understand the effects of environmental and developmental interaction on gene expression. Currently, bioengineers must repeatedly use trial and error to create predictable systems.

Similar to physics, synthetic biology requires the ability to model systems and quantify relationships between variables in biological systems at the molecular level.

The second major challenge to ensuring the success of synthetic biology is the development of enabling technologies. With genomes having billions of nucleotides, this requires fast, powerful, and cost-efficient computers. Moore’s law, named for Intel co-founder Gordon Moore, posits that computing power progresses at a predictable rate and that the number of components in integrated circuits doubles each year until its limits are reached. Since Moore’s prediction, computer power has increased at an exponential rate while pricing has declined.

DNA sequencers and synthesizers are necessary to identify genes and make synthetic DNA sequences. Bioengineer Robert Carlson calculated that the capabilities of DNA sequencers and synthesizers have followed a pattern similar to computing. This pattern, referred to as the Carlson Curve, projects that scientists are approaching the ability to sequence a human genome for $1,000, perhaps in 2020. Carlson calculated that the costs of reading and writing new genes and genomes are falling by a factor of two every 18–24 months. (see recent Carlson comment on requirement to read and write for a variety of limiting  conditions).

Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products

http://pharmaceuticalintelligence.com/2013/11/28/startup-to-strengthen-synthetic-biology-and-regenerative-medicine-industries-with-cutting-edge-cell-products/

Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

http://pharmaceuticalintelligence.com/2013/05/17/synthetic-biology-on-advanced-genome-interpretation-for-gene-variants-and-pathways-what-is-the-genetic-base-of-atherosclerosis-and-loss-of-arterial-elasticity-with-aging/

Synthesizing Synthetic Biology: PLOS Collections

http://pharmaceuticalintelligence.com/2012/08/17/synthesizing-synthetic-biology-plos-collections/

Capturing ten-color ultrasharp images of synthetic DNA structures resembling numerals 0 to 9

http://pharmaceuticalintelligence.com/2014/02/05/capturing-ten-color-ultrasharp-images-of-synthetic-dna-structures-resembling-numerals-0-to-9/

Silencing Cancers with Synthetic siRNAs

http://pharmaceuticalintelligence.com/2013/12/09/silencing-cancers-with-synthetic-sirnas/

Genomics Now—and Beyond the Bubble

Futurists have touted the twenty-first century as the century of biology based primarily on the promise of genomics. Medical researchers aim to use variations within genes as biomarkers for diseases, personalized treatments, and drug responses. Currently, we are experiencing a genomics bubble, but with advances in understanding biological complexity and the development of enabling technologies, synthetic biology is reviving optimism in many fields, particularly medicine.

BY MICHAEL BROOKS    17 APR, 2014     http://www.newstatesman.com/

Michael Brooks holds a PhD in quantum physics. He writes a weekly science column for the New Statesman, and his most recent book is The Secret Anarchy of Science.

The basic idea is that we take an organism – a bacterium, say – and re-engineer its genome so that it does something different. You might, for instance, make it ingest carbon dioxide from the atmosphere, process it and excrete crude oil.

That project is still under construction, but others, such as using synthesised DNA for data storage, have already been achieved. As evolution has proved, DNA is an extraordinarily stable medium that can preserve information for millions of years. In 2012, the Harvard geneticist George Church proved its potential by taking a book he had written, encoding it in a synthesised strand of DNA, and then making DNA sequencing machines read it back to him.

When we first started achieving such things it was costly and time-consuming and demanded extraordinary resources, such as those available to the millionaire biologist Craig Venter. Venter’s team spent most of the past two decades and tens of millions of dollars creating the first artificial organism, nicknamed “Synthia”. Using computer programs and robots that process the necessary chemicals, the team rebuilt the genome of the bacterium Mycoplasma mycoides from scratch. They also inserted a few watermarks and puzzles into the DNA sequence, partly as an identifying measure for safety’s sake, but mostly as a publicity stunt.

What they didn’t do was redesign the genome to do anything interesting. When the synthetic genome was inserted into an eviscerated bacterial cell, the new organism behaved exactly the same as its natural counterpart. Nevertheless, that Synthia, as Venter put it at the press conference to announce the research in 2010, was “the first self-replicating species we’ve had on the planet whose parent is a computer” made it a standout achievement.

Today, however, we have entered another era in synthetic biology and Venter faces stiff competition. The Steve Jobs to Venter’s Bill Gates is Jef Boeke, who researches yeast genetics at New York University.

Boeke wanted to redesign the yeast genome so that he could strip out various parts to see what they did. Because it took a private company a year to complete just a small part of the task, at a cost of $50,000, he realised he should go open-source. By teaching an undergraduate course on how to build a genome and teaming up with institutions all over the world, he has assembled a skilled workforce that, tinkering together, has made a synthetic chromosome for baker’s yeast.

 

Stepping into DIYbio and Synthetic Biology at ScienceHack

Posted April 22, 2014 by Heather McGaw and Kyrie Vala-Webb

We got a crash course on genetics and protein pathways, and then set out to design and build our own pathways using both the “Genomikon: Violacein Factory” kit and Synbiota platform. With Synbiota’s software, we dragged and dropped the enzymes to create the sequence that we were then going to build out. After a process of sketching ideas, mocking up pathways, and writing hypotheses, we were ready to start building!

The night stretched long, and at midnight we were forced to vacate the school. Not quite finished, we loaded our delicate bacteria, incubator, and boxes of gloves onto the bus and headed back to complete our bacterial transformation in one of our hotel rooms. Jammed in between the beds and the mini-fridge, we heat-shocked our bacteria in the hotel ice bucket. It was a surreal moment.

While waiting for our bacteria, we held an “unconference” where we explored bioethics, security and risk related to synthetic biology, 3D printing on Mars, patterns in juggling (with live demonstration!), and even did a Google Hangout with Rob Carlson. Every few hours, we would excitedly check in on our bacteria, looking for bacterial colonies and the purple hue characteristic of violacein.

Most impressive was the wildly successful and seamless integration of a diverse set of people: in a matter of hours, we were transformed from individual experts and practitioners in assorted fields into cohesive and passionate teams of DIY biologists and science hackers. The ability of everyone to connect and learn was a powerful experience, and over the course of just one weekend we were able to challenge each other and grow.

Returning to work on Monday, we were hungry for more. We wanted to find a way to bring the excitement and energy from the weekend into the studio and into the projects we’re working on. It struck us that there are strong parallels between design and DIYbio, and we knew there was an opportunity to bring some of the scientific approaches and curiosity into our studio.

 

 

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ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery

Posted in CABG, Cardiac and Cardiovascular Surgical Procedures, tagged Cardiovascular Disorders, Coronary artery bypass surgery, Coronary artery disease, Heart disease, William Nugent on November 5, 2013| Leave a Comment »

Reporter: Aviva Lev-Ari, PhD, RN

We decided to include ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery in this Scientific Journal as a Reference resource to all our Experts, Authors, Writers.

Example of the Guidelines in use in this Journal:

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?

http://pharmaceuticalintelligence.com/2013/11/04/mitral-valve-repair-who-is-a-candidate-for-a-non-ablative-fully-non-invasive-procedure/

SOURCE

ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery

http://circ.ahajournals.org/content/100/13/1464.long

• ACC/AHA Practice Guidelines

ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery: Executive Summary and Recommendations

A Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Revise the 1991 Guidelines for Coronary Artery Bypass Graft Surgery)

1. Committee Members

1. Kim A. Eagle, MD, FACC, Cochair;
2. Robert A. Guyton, MD, FACC, Cochair;
3. Ravin Davidoff, MB, BCh, FACC;
4. Gordon A. Ewy, MD, FACC;
5. James Fonger, MD;
6. Timothy J. Gardner, MD, FACC;
7. John Parker Gott, MD, FACC;
8. Howard C. Herrmann, MD, FACC;
9. Robert A. Marlow, MD, MA, FAAFP;
10. William Nugent, MD;
11. Gerald T. O’Connor, PhD, DSc;
12. Thomas A. Orszulak, MD;
13. Richard E. Rieselbach, MD, BS, FACP;
14. William L. Winters, MD, FACC;
15. Salim Yusuf, MB, BS, PhD

1. Task Force Members

1. Raymond J. Gibbons, MD, FACC, Chair;
2. Joseph S. Alpert, MD, FACC;
3. Kim A. Eagle, MD, FACC;
4. Timothy J. Gardner, MD, FACC;
5. Arthur Garson Jr, MD, MPH, FACC;
6. Gabriel Gregoratos, MD, FACC;
7. Richard O. Russell, MD, FACC;
8. Thomas J. Ryan, MD, FACC;
9. Sidney C. Smith Jr, MD, FACC

Key Words:

• ACC/AHA Practice Guidelines
• bypass
• angioplasty
• morbidity
• mortality
• revascularization
• risk factors

Next Section

I. Introduction

The American College of Cardiology/American Heart Association (ACC/AHA) Task Force on Practice Guidelines was formed to make recommendations regarding the appropriate use of diagnostic tests and therapies for patients with known or suspected cardiovascular disease. Coronary artery bypass graft (CABG) surgery is among the most common operations performed in the world and accounts for more resources expended in cardiovascular medicine than any other single procedure. Since the original Guidelines were published in 1991, there has been considerable evolution in the surgical approach to coronary disease, and at the same time there have been advances in preventive, medical, and percutaneous catheter approaches to therapy. These revised guidelines are based on a computerized search of the English literature since 1989, a manual search of final articles, and expert opinion.

As with other ACC/AHA guidelines, this document uses ACC/AHA classifications I, II, and III as summarized below:

Class I: Conditions for which there is evidence and/or general agreement that a given procedure or treatment is useful and effective.

Class II: Conditions for which there is conflicting evidence and/or a divergence of opinion about the usefulness or efficacy of a procedure.

Class IIa: Weight of evidence/opinion is in favor of usefulness/efficacy.

Class IIb: Usefulness/efficacy is less well established by evidence/opinion.

Class III: Conditions for which there is evidence and/or general agreement that the procedure/treatment is not useful/effective and in some cases may be harmful.

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II. Outcomes

A. Hospital Outcomes

Seven core variables (priority of operation, age, prior heart surgery, sex, left ventricular [LV] ejection fraction [EF], percent stenosis of the left main coronary artery, and number of major coronary arteries with significant stenoses) are the most consistent predictors of mortality after coronary artery surgery. The greatest risk is correlated with the urgency of operation, advanced age, and 1 or more prior coronary bypass surgeries. Additional variables that are related to mortality include coronary angioplasty during index admission; recent myocardial infarction (MI); history of angina, ventricular arrhythmias, congestive heart failure, or mitral regurgitation; and comorbidities such as diabetes, cerebrovascular disease, peripheral vascular disease, chronic obstructive pulmonary disease, and renal dysfunction. Table 1⇓ shows a method by which key patient variables can be used to predict an individual patient’s operative risk of death, stroke, or mediastinitis.

B. Morbidity Associated With Bypass Surgery

1. Neurological Events

Neurological impairment after bypass surgery may be attributable to hypoxia, emboli, hemorrhage, and/or metabolic abnormalities. Postoperative neurological deficits have been divided into 2 types: type 1, associated with major, focal neurological deficits, stupor, or coma; and type 2, in which deterioration in intellectual function is evident. Adverse cerebral outcomes are observed in ≈6% of patients after bypass surgery and are equally divided between type 1 and type 2 deficits. Predictors of cerebral complications after bypass surgery include advanced age and a history of hypertension. Particular predictors of type 1 deficits include proximal aortic atherosclerosis as defined by the surgeon at operation, history of prior neurological disease, use of the intra-aortic balloon pump, diabetes, hypertension, unstable angina, and increased age. Predictors of type 2 deficits include a history of excess alcohol consumption; dysrhythmias, including atrial fibrillation; hypertension; prior bypass surgery; peripheral vascular disease; and congestive heart failure. Estimation of a patient’s risk for postoperative stroke can be calculated from Table 1⇓.

2. Mediastinitis

Deep sternal wound infection occurs in 1% to 4% of patients after bypass surgery and carries a mortality of ≈25%. Predictors of this complication include obesity, reoperation, use of both internal mammary arteries at surgery, duration and complexity of surgery, and diabetes. An individual patient’s risk of postoperative mediastinitis can be estimated from Table 1⇓.

3. Renal Dysfunction

Postoperative renal dysfunction occurs in as many as 8% of patients. Among patients who develop postoperative renal dysfunction (defined as a postoperative serum creatinine level >2.0 mg/dL or an increase in baseline creatinine level of >0.7 mg/dL), 18% require dialysis. Overall mortality among patients who develop postoperative renal dysfunction is 19% and approaches two thirds among patients requiring dialysis. Predictors of renal dysfunction include advanced age, a history of moderate or severe congestive heart failure, prior bypass surgery, type 1 diabetes, and prior renal disease. Table 2⇓ can be used to estimate the risk for an individual patient. Patients with advanced preoperative renal dysfunction who undergo CABG surgery have an extraordinarily high rate of requiring postoperative dialysis. Among patients with a preoperative creatinine level >2.5 mg/dL, 40% to 50% require hemodialysis.

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Table 2.

Risk of Postoperative Renal Dysfunction (PRD) After Coronary Artery Bypass Graft Surgery

C. Long-Term Outcomes

Predictors of poor long-term survival after bypass surgery include advanced age, poor LVEF, diabetes, number of diseased vessels, and female sex. In some studies, additional predictors include angina class, hypertension, prior MI, renal dysfunction, and clinical congestive heart failure. Predictors of the recurrence of angina, late MI, or any cardiac event also include obesity and lack of use of an internal mammary artery, as well as those factors identified above. Of these events, the return of angina is the most common and is primarily related to late vein-graft atherosclerosis and occlusion.

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III. Comparison of Medical Therapy Versus Surgical Revascularization

The comparison of medical therapy with coronary surgical revascularization is primarily based on randomized, clinical trials and large registries. Although clinical trials have provided valuable insights, there are limitations to their interpretation in the current era. Patient selection had primarily included individuals ≤65 years of age, very few included large cohorts of women, and for the most part, the studies evaluated patients at low risk who were clinically stable. In addition, because the studies were done in the late 1970s and early 1980s, only 1 of the trials used arterial grafts, and even that trial had no arterial grafts in 86% of patients. Newer modalities of cardioprotection during cardiopulmonary bypass were not used, nor were minimally invasive or off-bypass techniques. Finally, medical therapy was not optimized in the trials. Lipid-lowering therapy had not yet become standard, aspirin was not widely used, and β-blockers were used in just half of the patients. Angiotensin-converting enzyme inhibitors were not being routinely used in patients with congestive heart failure or dilated cardiomyopathy. Accordingly, although the clinical trials have provided important insights, their interpretation must be viewed with caution, given the evolution in all types of coronary therapies.

For the most part, stratification of patients in the trials was based on the number of vessels with anatomically significant disease, whether or not the major epicardial obstruction was proximal, and the extent of LV dysfunction as determined by global EF. The end point of the trials was primarily survival.

Overview: Randomized Trials

There were 3 major, randomized trials and several smaller ones. A collaborative meta-analysis of 7 trials with a total enrollment of 2649 patients has allowed comparison of outcomes at 5 and 10 years (Tables 3⇓, 4⇓, and 5⇓ and the Figure⇓). Among all patients, the extension survival of CABG surgical patients compared with medically treated patients was 4.3 months at 10 years of follow-up. The benefit of CABG compared with medical therapy in various clinical subsets is presented below.

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Figure 1.

Extension of survival after 10 years of follow-up in various subgroups of patients, from a meta-analysis of 7 randomized studies. LV indicates left ventricular; VA, Veterans Administration.

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Table 3.

Total Mortality at 5 and 10 Years

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Table 4.

Subgroup Results at 5 Years

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Table 5.

Subgroup Analysis of 5-Year Mortality by Risk Stratum

1. Left Main Coronary Artery Disease

The trials defined significant left main coronary artery stenosis as a >50% reduction in lumen diameter. Median survival for surgically treated patients was 13.3 years versus 6.6 years in medically treated patients. Left main equivalent disease (≥70% stenosis in both the proximal left anterior descending [LAD] and proximal left circumflex arteries) appeared to behave similarly to true left main coronary artery disease. Median survival for surgical patients was 13.1 years versus 6.2 years for medically assigned patients. The benefit of surgery for left main coronary artery disease patients continued well beyond 10 years. By 15 years, it was estimated that two thirds of patients originally assigned to medical therapy and who survived would have had surgery. The 15-year cumulative survival for left main coronary artery disease patients having CABG surgery was 44% versus 31% for medical patients.

2. Three-Vessel Disease

If one defines 3-vessel disease as stenosis of 50% or more in all 3 major coronary territories, the overall extension of survival was 7 months in CABG patients compared with medically treated patients. Patients with class III or IV angina, those with more proximal and severe LAD stenosis, those with worse LV function, and/or those with more positive stress tests derived more benefit from surgery.

3. Proximal LAD Disease

In patients with severe, proximal LAD stenosis, the relative risk reduction due to bypass surgery compared with medical therapy was 42% at 5 years and 22% at 10 years. This was even more striking in patients with depressed LV function.

4. LV Function

In patients with mildly to moderately depressed LV function, the poorer the LV function, the greater was the potential advantage of CABG surgery. Although the relative benefit was similar, the absolute benefit was greater because of the high-risk profile of these patients.

5. Symptoms and Quality of Life

Improvement in symptoms and quality of life after bypass surgery parallels the outcome data regarding survival. Beyond survival, bypass surgery may be indicated to alleviate symptoms of angina above and beyond medical therapy or to reduce the incidence of nonfatal complications like MI, congestive heart failure, and hospitalization. Registry studies have shown a reduction in late MI among highest-risk patients, such as those with 3-vessel disease, and/or those with severe angina. In pooled analyses, a benefit on the incidence of MI was not evident. This result likely reflected an early increase in MI perioperatively after CABG, which was balanced by fewer MIs over the long term among CABG recipients. Antianginal medications were required less frequently after bypass surgery. At 5 years, two thirds of bypass patients were symptom-free compared with 38% of medically assigned patients. By 10 years, however, these differences were no longer significant. This result is related to the attrition of vein grafts in the bypass group as well as crossover of medically assigned patients to bypass surgery.

6. Loss of Benefit of Surgery

After 10 to 12 years of follow-up, there was a tendency for the bypass surgery and medical therapy curves to converge, in regard to both survival as well as nonfatal outcomes. This convergence is due to a number of factors. First, the reduced life expectancy of patients with coronary disease (regardless of treatment) leads to a steady attrition. Second, the increased event rate in the late follow-up period of surgically assigned patients was likely related to the progression of native coronary disease and graft disease over time. Finally, medically assigned patients crossed over to surgery late, thus allowing the highest-risk medically assigned patients to gain from the benefit of surgery later in the course of follow-up. By 10 years, 37% to 50% of medically assigned patients had crossed over to surgery. Tables 3⇑, 4⇑, and 5⇑ and the Figure⇑ provide estimates of long-term outcomes among patients randomized in the trials. These tables and the Figure⇑ can be used to estimate the general survival expectations in various anatomic categories.

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IV. Comparison of Bypass Surgery With Percutaneous Revascularization

The results of a number of randomized, clinical trials comparing angioplasty and bypass surgery have been published. The trials excluded patients in whom survival had already been shown to be longer with bypass surgery than with medical therapy. Also, none of the trials was sufficiently large to detect relatively modest differences in survival between the 2 techniques. Most of the trials did not have a long-term follow-up, ie, 5 to 10 years, and therefore were unable to provide clear inferences regarding long-term benefit of the 2 techniques in similar populations. Also, and perhaps most notably, only ≈5% of screened patients with multivessel disease at enrolling institutions were included in the trials. Half of the patients approached were ineligible owing to left main coronary artery disease, insufficient symptoms, or other reasons. Even among a large group of patients with multivessel disease suitable for enrollment, only half were actually randomized. It appeared that physicians elected not to enroll many patients with 3-vessel disease in the trials but rather refer them for bypass surgery, whereas patients with 2-vessel disease tended to be referred for angioplasty rather than be enrolled in the trials.

Overall, procedural complications were low for both procedures but tended to be higher with CABG surgery (Table 6⇓). For patients randomized to angioplasty, CABG was needed in ≈6% during the index hospitalization and in nearly 20% by 1 year. The initial cost and length of stay were lower for angioplasty than for CABG. Patients having angioplasty returned to work sooner and were able to exercise more at 1 month. The extent of revascularization achieved by bypass surgery was generally higher than with angioplasty. Long-term survival was difficult to evaluate owing to the short period of follow-up and the small sample size of the trials. However, for the Bypass Angioplasty Revascularization Investigation (BARI) trial, bypass patients had a 5-year survival of 89.3% compared with 86.3% for angioplasty. Secondary analysis revealed that in treated diabetic patients in the BARI trials, CABG led to significantly superior survival compared with percutaneous transluminal coronary angioplasty (PTCA). However, this finding was not evident in other trials. In long-term follow-up, the most striking difference was the 4- to 10-fold-higher likelihood of reintervention after initial PTCA. Quality of life, physical activity, employment, and cost were similar by 3 to 5 years after both procedures. The BARI trial suggested higher mortality associated with PTCA in several high-risk groups, including those with diabetes, unstable angina, and/or non–Q wave MI, and in patients with heart failure.

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Table 6.

CABG vs PTCA: Randomized Controlled Trials

An analysis of registries generally shows data similar to those of the trials. However, a recent analysis of ≈60 000 patients who were treated in New York State in the early 1990s provides a 3-year survival analysis of patients undergoing CABG and PTCA. After adjustment for various covariates, bypass surgery in the New York State registry experience was associated with longer survival in patients with severe proximal LAD stenosis and/or 3-vessel disease. Contrariwise, patients with 1-vessel disease not involving the proximal LAD had improved survival with PTCA. Table 7⇓ summarizes survival data from the New York State registry with respect to various cohorts of patients undergoing angioplasty or bypass surgery. These data can be used to estimate 3-year survival expectations for patients with various anatomic features.

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Table 7.

Three-Year Survival by Treatment in Each Anatomic Subgroup

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V. Management Strategies

Reduction of Perioperative Mortality and Morbidity

1. Reducing the Risk of Type 1 Brain Injury After CABG

Postoperative neurological complications represent 1 of the most devastating consequences of CABG surgery. Type 1 injury, in which a significant, permanent, neurological injury is sustained, occurs in ≈3% of patients overall and is responsible for a 21% mortality.

Atherosclerotic Ascending Aorta

An important predictor of this complication is the surgeon’s identification of a severely atherosclerotic, ascending aorta before or during the bypass operation. Perioperative atheroembolism from aortic plaque is thought to be responsible for approximately one third of strokes after CABG. Atherosclerosis of the ascending aorta is strongly related to increased age. Thus, stroke risk is particularly increased in patients beyond 75 to 80 years of age. Preoperative, noninvasive testing to identify high-risk patients has variable accuracy. Computed tomography identifies the most severely involved aortas but underestimates mild or moderate involvement. Transesophageal echocardiography is useful for aortic arch examination, but examination of the ascending aorta may be limited by the intervening trachea. Intraoperative assessment with epiaortic imaging is superior to both methods. Intraoperative palpation underestimates the high-risk aorta. The highest-risk aortic pattern is a protruding or mobile aortic arch plaque. An aggressive approach to the management of patients with severely diseased ascending aortas identified by intraoperative echocardiographic imaging reduces the risk of postoperative stroke. For patients with aortic walls ≤3 mm thick, standard treatment is used. For aortas >3 mm thick, the cannulation, clamp, or proximal anastomotic sites may be changed, or a no-clamp, fibrillatory arrest strategy may be used. For high-risk patients with multiple or circumferential involvement or those with extensive middle ascending aortic involvement, replacement of the ascending aorta under hypothermic circulatory arrest may be indicated. Alternatively, a combined approach with off-bypass, in situ internal mammary grafting to the LAD and percutaneous coronary intervention to treat other vessel stenoses has conceptual merit.

Atrial Fibrillation and Stroke

Chronic atrial fibrillation is a hazard for perioperative stroke. Intraoperative surgical manipulation or spontaneous resumption of sinus rhythm during the early postoperative period may lead to embolism of a left atrial clot. One approach to reduce this risk is the performance of preoperative, transesophageal echocardiography. The absence of a left atrial clot would suggest that the operation may proceed with acceptable risk. For elective patients, if a left atrial clot is identified, 3 to 4 weeks of anticoagulation therapy followed by restudy and then subsequent surgery is reasonable. Few clinical trial data are available to assist clinicians in this circumstance.

New-onset postoperative atrial fibrillation occurs in ≈30% of post-CABG patients, particularly on the second and third postoperative days, and is associated with a 2- to 3-fold increased risk of postoperative stroke. Risk factors include advanced age, chronic obstructive pulmonary disease, proximal right coronary disease, prolonged operation, atrial ischemia, and withdrawal of β-blockers. The role of anticoagulants in patients who develop post-CABG atrial fibrillation is unclear. Aggressive anticoagulation and cardioversion may reduce the neurological complications associated with this arrhythmia. Early cardioversion within 24 hours of the onset of atrial fibrillation can probably be performed safely without anticoagulation. However, persistence of the arrhythmia beyond this time argues for the use of oral anticoagulants to reduce stroke risk in patients who remain in atrial fibrillation and/or in those for whom later cardioversion is planned.

Recent MI, LV Thrombus, and Stroke

Patients with a recent, anterior MI and residual wall-motion abnormality are at increased risk for the development of an LV mural thrombus and its potential for embolization. For patients undergoing surgical revascularization after sustaining an anterior MI, preoperative screening with echocardiography may be appropriate to identify the presence of a clot. Detection of an acute LV mural thrombus may call for long-term anticoagulation and reevaluation by echocardiography to ensure resolution or organization of the thrombus before coronary bypass surgery. Additionally, 3 to 6 months of anticoagulation therapy is appropriate for patients with persistent, anterior wall–motion abnormalities after coronary bypass surgery.

Recent, Antecedent Cerebrovascular Event

A recent, preoperative cerebrovascular accident represents a situation in which delaying surgery may reduce the perioperative neurological risk. In particular, evidence of a hemorrhagic component based on computed tomographic scan identifies high risk for the extension of neurological damage with cardiopulmonary bypass. It is generally believed that a delay of 4 weeks or more after a cerebrovascular accident is prudent, if coronary anatomy and symptoms permit, before proceeding with CABG.

Carotid Disease and Neurological Risk Reduction

Hemodynamically significant carotid stenoses are thought to be responsible for up to 30% of early postoperative strokes. The trend for coronary surgery to be performed in an increasingly elderly population and the increasing prevalence of carotid disease in this same group of patients underscore the importance of this issue. Perioperative stroke risk is thought to be <2% when carotid stenoses are <50%, 10% when stenoses are 50% to 80%, and 11% to 19% in patients with stenoses >80%. Patients with untreated, bilateral, high-grade stenoses and/or occlusions have a 20% chance of stroke. Carotid endarterectomy for patients with high-grade stenosis is generally done preceding or coincident with coronary bypass surgery and, with proper teamwork in high-volume centers, is associated with a low risk for both short- and long-term neurological sequelae. Carotid endarterectomy performed in this fashion carries a low mortality (3.5%) and reduces early postoperative stroke risk to <4%, with a concomitant 5-year freedom from stroke of 88% to 96%.

The decision about who should undergo preoperative carotid screening is controversial. Predictors of important carotid stenosis include advanced age, female sex, known peripheral vascular disease, previous transient ischemic attack or stroke, a history of smoking, and left main coronary artery disease. Many centers screen all patients >65 years old. Patients with left main coronary disease are often screened, as are those with a previous transient ischemic attack or stroke. Preoperative central nervous system symptoms suggestive of vertebral basilar insufficiency should lead to an evaluation before elective CABG.

When surgery of both carotid and coronary disease is planned, the most common approach is to perform the operation in a staged manner, in which the patient first has carotid surgery followed by coronary bypass in 1 to 5 days. Alternatively, especially if the patient has compelling cardiac symptoms or coronary anatomy, the operations may be performed during a single period of anesthesia, with the carotid endarterectomy immediately preceding coronary bypass. Neither strategy has been established as being superior. Stroke risk is increased if a reversed-stage procedure is used, in which the coronary bypass operation precedes the carotid endarterectomy by ≥1 day.

2. Reducing the Risk of Type 2 Brain Injury

Type 2 neurological complications are seen in ≈3% of patients and are correlated with a 10% risk of postoperative death, with 40% of patients requiring additional care in a transitional facility after hospital discharge. Microembolization is thought to be a major contributor to the postoperative cerebral dysfunction after CABG. The release of microemboli during extracorporeal circulation, involving small gaseous or lipid emboli, may be responsible. The use of a 40-μm arterial-line filter on the heart-lung machine circuit and routine use of membrane oxygenators rather than bubble oxygenators may reduce such neurological injury. Additional maneuvers to reduce type 2 neurological injury include the maintenance of steady, cerebral blood flow during cardiopulmonary bypass, avoidance of cerebral hyperthermia during and after cardiopulmonary bypass, meticulous control of perioperative hyperglycemia, and avoidance and limitation of postoperative cerebral edema.

3. Reducing the Risk of Perioperative Myocardial Dysfunction

Protection in Patients With Normal LV Function

There is no universally applicable myocardial protection technique. Among patients with preserved preoperative cardiac function, no strong argument can currently be made for warm versus cold and crystalloid versus blood cardioplegia. However, certain techniques may offer a wider margin of safety for special patient subsets.

Myocardial Protection for Acutely Depressed Cardiac Function

Several studies have suggested that blood cardioplegia (compared with crystalloid) may offer a greater margin of safety during CABG performed on patients with acute coronary occlusion, failed angioplasty, urgent revascularization for unstable angina, and/or chronically impaired LV function.

Protection for Chronically Depressed LV Function

The use of a prophylactic intra-aortic balloon pump as an adjunct to myocardial protection may reduce mortality in patients having CABG in the setting of severe LV dysfunction (eg, LVEF <0.25). Placement of the intra-aortic balloon pump immediately before operation appears to be as effective as placement on the day preceding bypass surgery.

Adjuncts to Myocardial Protection

Although it is widely appreciated that use of the internal mammary artery leads to improved long-term survival after coronary bypass surgery, it has also been documented that use of the internal mammary artery influences operative mortality itself. Thus, internal mammary artery use should be encouraged in the elderly, emergent, or acutely ischemic patient and other patient groups.

Inferior Infarct With Right Ventricular Involvement

An acutely infarcted right ventricle is at great risk for severe, postoperative dysfunction and predisposes the patient to a higher postoperative mortality. During operation, loss of the pericardial constraint may lead to acute dilatation of the dysfunctional right ventricle, which then fails to recover even with optimal myocardial protection and revascularization. The best defense against right ventricular dysfunction is its recognition during preoperative evaluation. When possible, CABG should be delayed for ≥4 weeks to allow the right ventricle to recover.

4. Reducing the Systemic Consequences of Cardiopulmonary Bypass

A variety of measures have been tried to reduce the systemic consequences of cardiopulmonary bypass, which elicits a diffuse inflammatory response that may cause transient or prolonged multisystem organ dysfunction. Administration of corticosteroids before cardiopulmonary bypass may reduce complement activation and release of proinflammatory cytokines. Proper timing and duration of corticosteroid application are incompletely resolved. The administration of the serine protease inhibitor aprotinin may attenuate complement activation and cytokine release during extracorporeal circulation. Unfortunately, aprotinin is relatively expensive. Another method to reduce the inflammatory response is perioperative leukocyte depletion through hematologic filtration.

5. Reducing the Risk of Perioperative Infections

Several methods exist to reduce the risk of wound infections in patients undergoing CABG. These begin with interval reporting to individual surgeons regarding their respective wound infection rates and adherence to sterile operative techniques. Additional strategies include skin preparation with topical antiseptics, clipping rather than shaving the skin, avoidance of hair removal, reduction of operating room traffic, laminar-flow ventilation, shorter operation, minimization of electrocautery, avoidance of bone wax, use of double-glove barrier techniques for the operating room team, and routine use of a pleural pericardial flap. Aggressive, perioperative glucose control in diabetics through the use of continuous, intravenous insulin infusion reduces perioperative hyperglycemia and its associated infection risk. Avoidance of homologous blood transfusions after CABG may reduce the risk of both viral and bacterial infections. This is due to an immunosuppressive effect of transfusion. Leukodepletion of transfused blood also reduces this effect. This can be accomplished by regional blood blanks at the time of donation or at the bedside by use of a transfusion filter.

Preoperative antibiotic administration reduces the risk of postoperative infection 5-fold. Efficacy is dependent on adequate drug tissue levels before microbial exposure. Cephalosporins are currently the agents of choice. Table 8⇓ identifies appropriate choices, doses, and routes of therapy. A 1-day course of intravenous antimicrobials is as effective as 48 hours or more. Therapy should be administered within 30 minutes of incision and again in the operating room if the operation exceeds 3 hours. Many centers deliver antibiotics just before incision. One fail-safe method is to have the anesthesiologist administer the cephalosporin after induction but before skin incision. If deep sternal wound infection does occur, aggressive surgical debridement and early vascularized muscle flap coverage are the most effective methods for treatment, along with long-term systemic antibiotics.

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Table 8.

Prophylactic Antimicrobials for Coronary Artery Bypass Graft Surgery

6. Prevention of Postoperative Dysrhythmias

Postoperative atrial fibrillation increases the length of stay, cost, and most important, the risk of stroke. Atrial fibrillation occurs in up to 30% of patients, usually on the second or third postoperative day. Methods to avoid atrial fibrillation are several. First, withdrawal of preoperative β-blockers in the postoperative period doubles the risk of atrial fibrillation after CABG. Thus, early reinitiation of β-blockers is critical for avoidance of this complication. Virtually every study of patients receiving β-blockers prophylactically has shown benefit in lowering the frequency of atrial fibrillation. Most have used the drug in the postoperative period, but greater benefit may occur if β-blockade is begun before the operation. More recently, small studies of propafenone, sotalol, and amiodarone have also shown effectiveness in reducing the risk of postoperative atrial fibrillation. Table 9⇓provides a review of pharmacological approaches in the randomized trials. Digoxin and calcium channel blockers have no consistent benefit for preventing atrial fibrillation after CABG, although they are frequently used to control its rate after it does occur. Currently, the routine preoperative or early postoperative administration of β-blockers is considered standard therapy to reduce the risk of atrial fibrillation after CABG.

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Table 9.

Pharmacological Strategies for Prevention of Atrial Fibrillation (AF) After Coronary Artery Bypass Graft Surgery

7. Strategies to Reduce Perioperative Bleeding and Transfusion Risk

Transfusion Risk

Despite the increasing safety of homologous blood transfusion, concerns surrounding viral transmission during transfusion remain. Currently, the risks are likely very low and have been estimated to be 1/493 000 for human immunodeficiency virus, 1/641 000 for human T-cell lymphotrophic virus, 1/103 000 for hepatitis C virus, and 1/63 000 for hepatitis B virus.

Perioperative Bleeding

Risk factors for blood transfusion after CABG include advanced age, low preoperative red blood cell volume, preoperative aspirin therapy, urgent operation, duration of cardiopulmonary bypass, recent thrombolytic therapy, reoperation, and differences in heparin management. Institutional protocols that establish minimum thresholds for transfusion lead to a reduced number of units transfused and the percentage of patients requiring blood. Additional strategies can reduce the transfusion requirement after CABG. For stable patients, aspirin and other antiplatelet drugs may be discontinued 7 days before elective CABG. Aprotinin, a serum protease inhibitor with antifibrinolytic activity, also decreases postoperative blood loss and transfusion requirements in high-risk patients. Although there has been some concern that aprotinin may reduce early graft patency, recent studies have failed to document this effect. Routine use of aprotinin is limited by its high cost. Multidisciplinary approaches to conserve blood in single institutions appear to be effective.

For patients without exclusions, such as low hemoglobin values, heart failure, unstable angina, left main coronary artery disease, or advanced anginal symptoms, self-donation of 1 to 3 units of red blood cells over 30 days before operation reduces the need for homologous transfusion during or after operation. Donation immediately before cardiopulmonary bypass yields a higher platelet and hemoglobin count compared with simple hemodilution without pre–cardiopulmonary bypass blood harvesting.

8. Antiplatelet Therapy for Saphenous Vein Graft Patency

Aspirin significantly reduces vein graft closure during the first postoperative year. The aspirin should be started within 24 hours after surgery because its benefit on saphenous vein graft patency is lost when begun later. Dosing regimens from as little as 100 mg/d to as much as 325 mg TID appear to be efficacious. Ticlopidine offers no advantage over aspirin but is an alternative in truly aspirin-allergic patients. Life-threatening neutropenia is a rare but recognized side effect. Clopidogrel offers the potential for fewer side effects compared with ticlopidine as an alternative in aspirin-allergic patients. Its incidence of severe leukopenia is rare.

9. Pharmacological Management of Hyperlipidemia

Aggressive treatment of hypercholesterolemia reduces progression of atherosclerotic vein graft disease in patients after bypass surgery. Statin therapy has been shown to reduce saphenous vein graft disease progression over the ensuing years after bypass. Patients with unknown low-density lipoprotein (LDL) cholesterol levels after bypass should have cholesterol levels determined and treated pharmacologically if the LDL exceeds 100 mg/dL. Patients with treated LDL cholesterol should have their low-fat diet and cholesterol-lowering medications continued after bypass surgery to reduce subsequent graft attrition. Data regarding the benefit of cholesterol lowering after bypass surgery are most supported by studies that have used HMG CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors, particularly targeting LDL levels to <100 mg/dL.

10. Hormonal Manipulation

While observational studies have suggested that hormone replacement therapy in postmenopausal women leads to a reduction in all-cause mortality, a recent, randomized trial for secondary coronary prevention failed to show a beneficial effect on the overall rate of coronary events. Thus, hormone replacement therapy should be considered in postmenopausal women after bypass when, in the physician’s judgment, the potential coronary benefit is not offset by an increased risk of uterine or breast cancer.

11. Smoking Cessation

Smoking cessation is the single, most important risk-modification goal after CABG in patients who smoke. Smoking cessation leads to less recurrent angina, improved physical function, fewer admissions, maintenance of employment, and improved survival. Treatment individualized to the patient is crucial. Depression may be an important complicating factor and should be approached with behavioral and drug therapy. Nicotine replacement with a transdermal patch, nasal spray, gum, or inhaler is beneficial. A sustained-release form of bupropion, an antidepressant similar to selective serotonin reuptake inhibitors, reduces the nicotine craving and anxiety of smokers who quit. All smokers should receive educational counseling and be offered smoking cessation therapy after CABG (Table 10⇓).

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Table 10.

Proven Management Strategies to Reduce Perioperative and Late Morbidity and Mortality

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Table 1.

12. Cardiac Rehabilitation

Cardiac rehabilitation, including early ambulation during hospitalization, outpatient prescriptive exercise, family education, and dietary and sexual counseling, has been shown to improve outcomes after CABG. The benefits include better physical mobility and perceived health. A higher proportion of rehabilitated patients are working at 3 years after CABG. The benefits of rehabilitation extend to the elderly and to women. Cardiac rehabilitation reinforces pharmacological therapy and smoking cessation and should be offered to all eligible patients after CABG.

13. Emotional Dysfunction and Psychosocial Considerations

Lack of social participation and low religious strength are independent predictors of death in elderly patients undergoing CABG. Although controversial, the high prevalence of depression after bypass surgery may reflect a high prevalence preoperatively. Cardiac rehabilitation has a highly beneficial effect in patients who are moderately or severely depressed. Evaluation of social supports and attempts to identify and treat underlying depression should be part of routine post-CABG care.

14. Rapid Sustained Recovery After Operation

Rapid recovery and early discharge are standard goals after CABG. The shortest in-hospital postoperative stays are followed by the fewest rehospitalizations. Important components of “fast-track” care are careful patient selection, patient and family education, early extubation, prophylactic antiarrhythmic therapy, dietary considerations, early ambulation, early outpatient telephone follow-up, and careful coordination with other physicians and healthcare providers.

15. Communication Between Caregivers

Maintenance of appropriate and timely communication between treating physicians regarding care of the patient is crucial. When possible, the primary care physician should follow up the patient during the perioperative course. The referral physician needs to provide clear, written reports of the findings and recommendations to the primary care physician, including discharge medications and dosages along with long-term goals.

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VI. Impact of Evolving Technology

A. Less-Invasive Coronary Bypass Surgery

Technical modifications of CABG have been developed to decrease the morbidity of the operation, either by using limited incision or by eliminating cardiopulmonary bypass. Currently, “less-invasive” CABG surgery can be divided into 3 categories: (1) off-bypass CABG performed through a median sternotomy with a smaller skin incision, (2) minimally invasive direct CABG (MID-CAB) performed through a left anterior thoracotomy without cardiopulmonary bypass, and (3) port-access CABG with femoral-to-femoral cardiopulmonary bypass and cardioplegic arrest with limited incision.

Off-bypass coronary surgery is performed on a beating heart after reduction of cardiac motion with a variety of pharmacological and mechanical devices. These include slowing the heart with β-blockers and calcium channel blockers and use of a mechanical stabilizing device to isolate and stabilize the target vessel. Retraction techniques may elevate the heart to allow access to vessels on the lateral and inferior surfaces of the heart. Because this technique generally uses a median sternotomy, its primary benefit is the avoidance of cardiopulmonary bypass, not a less extensive incision.

MID-CAB refers to bypass surgery without median sternotomy and without the use of cardiopulmonary bypass. Generally, this is performed with a small left anterior thoracotomy, exposing the heart through the fourth intercostal interspace with access to the LAD and diagonal branches and occasionally, the anterior marginal vessels. The right coronary artery can be approached by using a right anterior thoracotomy. MID-CAB procedures are generally performed on only 1 or 2 coronary targets. Observational studies have suggested that MID-CAB is associated with a reduced average length of stay and an earlier return to work. Although initial reports of 2-year actuarial and event-free survival are encouraging, the data must be viewed with caution. Because the number of anastomoses performed on a beating heart is usually 1 or occasionally 2, the potential long-term effects of incomplete revascularization are unknown.

The closed-chest, port-access, video-assisted CABG operation uses cardiopulmonary bypass and cardioplegia of a globally arrested heart. Vascular access for cardiopulmonary bypass is achieved via the femoral artery and vein. A triple-lumen catheter with an inflatable balloon at its distal end is used to achieve endovascular aortic occlusion, cardioplegia delivery, and LV decompression. With cardiopulmonary bypass and cardioplegic arrest, CABG can be performed with video assistance on a still and decompressed heart through several small ports. In comparison with the MID-CAB, port access allows access to different areas of the heart, thus facilitating more complete revascularization, and the motionless heart may allow a more accurate anastomosis. Compared with conventional CABG, median sternotomy is avoided. However, potential morbidity of the port-access operation includes multiple wounds at port sites, the limited thoracotomy, and the groin dissection for femoral-femoral bypass. Vigorous scrutiny of the long-term benefits versus risks of port access is required.

B. Arterial and Alternate Conduits

Another area of evolving technology is the use of arterial and alternate conduits. The 5-year patency of coronary artery–vein bypass grafts is 74%, and at 10 years, just 41%. Contrariwise, patency rates of the internal mammary artery implanted into the LAD are as high as 83% at 10 years. As a consequence of improved patency, patients receiving an LAD graft with an internal mammary artery have improved survival compared with patients receiving only vein grafts. Currently, routine use of the left internal mammary artery for LAD grafting with supplemental saphenous vein grafts to other coronary lesions is generally accepted as a standard grafting method. The use of bilateral internal mammary arteries appears to be safe and efficacious. However, there is a higher rate of deep sternal wound infection when both internal mammary arteries are used. This is particularly true for patients with obesity and diabetes and perhaps for those requiring prolonged ventilatory support. The benefits of bilateral internal mammary artery use include lower rates of recurrent angina, MI, and need for reoperation and a trend for better survival. Recently, the radial artery has been used more frequently as a conduit for coronary bypass surgery. Five-year patency appears to be in the range of 85% (compared with nearly 90% for the internal mammary graft). In patients for whom mammary artery, radial artery, and standard vein conduits are unavailable, the in situ right gastroepiploic artery, the inferior epigastric free artery graft, and either lesser saphenous or upper-extremity vein conduits have been used. Long-term patency of these alternative grafts has not been extensively studied.

C. Percutaneous Technology

Technological improvements in percutaneous coronary angioplasty have included the introduction of new devices and improved medical therapy of patients in whom angioplasty is performed. The most notable improvement has been the introduction of intracoronary stents that have reduced late restenosis and the frequency with which emergency bypass surgery is required after PTCA. Intracoronary stents have been used to treat saphenous vein graft stenosis in patients with previous CABG. However, stented patients still have an ≈25% combined rate of death, MI, need for repeat CABG, or re-revascularization of the target vessel. For some patients, hybrid procedures may be the best choice, such as the combined use of CABG surgery and coronary angioplasty. Such an approach is relevant to the patient whose ascending aorta is involved with severe atherosclerosis, for which the implantation of free vein grafts or arterial grafts leads to risk for atheroembolism. In such a patient, the use of in situ internal mammary artery grafting without cardiopulmonary bypass combined with additional coronary angioplasty in other diseased vessels represents a strategy to provide complete revascularization without the concomitant risks of cardiopulmonary bypass and/or manipulation of the ascending aorta.

D. Transmyocardial Revascularization

A fourth area that is rapidly evolving is transmyocardial revascularization. The use of transmyocardial laser revascularization has generally been performed surgically for patients with severe angina refractory to medical therapy and who are not suitable candidates for standard surgical revascularization, PTCA, or heart transplant. While several studies have suggested improvement in angina severity with transmyocardial laser revascularization, the mechanism by which angina improves and the overall benefit on long-term angina and/or survival await further clarification.

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VII. Special Patient Subsets

A. Elderly Patients

Elderly patients being considered for CABG have a higher average risk for mortality and morbidity in a direct relation to age, LV function, extent of coronary disease, and comorbid conditions and whether the procedure is urgent, emergent, or a reoperation. Nonetheless, functional recovery and sustained improvement in the quality of life can be achieved in the majority of such patients. The patient and physician together must explore the potential benefits of improved quality of life with the attendant risks of surgery versus alternative therapies that take into account baseline functional capacities and patient preferences. Age alone should not be a contraindication to CABG if it is thought that long-term benefits outweigh the procedural risk.

B. Women

A number of earlier reports had suggested that female sex was an independent risk factor for mortality and morbidity after CABG. More recent studies have suggested that women on average have a disadvantageous, preoperative clinical profile that accounts for much of this perceived difference. Thus, the issue is not necessarily sex itself but the comorbid conditions that are particularly associated with the later age at which women present for coronary surgery. Thus, CABG should not be delayed in or denied to women who have appropriate indications.

C. Diabetic Patients

Coronary heart disease is the leading cause of death among adult diabetics and accounts for 3 times as many deaths among diabetics as among nondiabetics. While CABG carries an increased morbidity and mortality in diabetics, data suggest that in appropriate candidates, the absolute risk reduction provided by successful revascularization remains high. The BARI trial suggested that diabetics with multivessel coronary disease derived advantage from bypass surgery compared with angioplasty. Several of the other randomized trials, albeit with smaller numbers of patients, failed to show this trend. Diabetics who are candidates for renal transplantation have a particularly high incidence of coronary artery disease, even in the absence of symptoms or signs. In appropriate candidates, CABG appears to offer morbidity and mortality benefit in such patients.

D. Patients With Chronic Obstructive Pulmonary Disease

Because CABG is associated with variable degrees of postoperative respiratory insufficiency, it is important to identify patients at particular risk for pulmonary complications. The intent is to treat reversible problems that may contribute to respiratory insufficiency in high-risk patients, with the hope of avoiding prolonged periods of mechanical ventilation after CABG. High-risk patients often benefit from preoperative antibiotics, bronchodilator therapy, a period of cessation from smoking, perioperative incentive spirometry, deep-breathing exercises, and chest physiotherapy. If pulmonary venous congestion or pleural effusions are identified, diuresis often improves lung performance.

Although preoperative spirometry directed to identifying patients with a low (eg, <1 L) 1-second forced expiratory volume has been used by some to qualify or disqualify candidates for CABG, clinical evaluation of lung function is likely as important if not more so. Patients with advanced chronic obstructive pulmonary disease are at particular risk for postoperative arrhythmias that may be fatal. While moderate to severe degrees of obstructive pulmonary disease represent a significant risk factor for early mortality and morbidity after CABG, it is also true that with careful preoperative assessment and treatment of the underlying pulmonary abnormality, many such patients are successfully carried through the operative procedure.

E. Patients With End-Stage Renal Disease

Coronary artery disease is the most important cause of mortality in patients with end-stage renal disease. Many of such patients have diabetes and other coronary risk factors, including hypertension, myocardial dysfunction, abnormal lipids, anemia, and increased plasma homocysteine levels. Although patients on chronic dialysis are at higher risk when undergoing coronary angioplasty or bypass, they are at even higher risk with conservative medical management. Thus, in patients with modest reductions in LV function, significant left main or 3-vessel disease, and/or unstable angina, coronary revascularization can lead to relief of coronary symptoms, improvement in overall functional status, and improved long-term survival in this select high-risk patient population.

F. Reoperative Patients

Operative survival and long-term benefit of reoperative CABG are distinctly inferior to first-time operations. Patients undergoing repeated CABG have higher rates of postoperative bleeding, perioperative MI, and neurological and pulmonary complications. Nevertheless, reasonable 5- and 10-year survival rates after reoperation for coronary disease can be achieved, and the operation is appropriate if the severity of symptoms and anticipated benefit justify the immediate risk. Data suggest that the need for reoperation is less common in patients undergoing internal mammary artery grafting to the LAD. More recently, short-term follow-up studies suggest that patients undergoing multiple arterial grafts have even lower rates of reoperation. These early results are consistent with the known superior graft patency of arterial conduits compared with vein grafts.

G. Concomitant Peripheral Vascular Disease

The presence of clinical and subclinical peripheral vascular disease is a strong predictor of increased hospital and long-term mortality rates in patients undergoing CABG. However, the absolute benefit offered by coronary revascularization is elevated in patients with peripheral vascular disease, particularly those with 3-vessel coronary disease, more advanced angina, and/or a depressed LVEF. Excess perioperative mortality in such patients is related to an increased incidence of heart failure and dysrhythmias rather than peripheral arterial complications.

H. Poor LV Function

Patients with severe LV dysfunction have increased perioperative and long-term mortality compared with patients with normal LV function. However, studies suggest that the beneficial effects of myocardial revascularization in patients with ischemic heart disease and severe LV dysfunction are sizeable when compared with medically treated patients of similar status in terms of symptom relief, exercise tolerance, and survival.

I. CABG in Acute Coronary Syndromes

Coronary bypass surgery offers a survival advantage compared with medical therapy in patients with unstable angina and LV dysfunction, particularly in the presence of 3-vessel disease. However, the risk of bypass surgery in patients with unstable or postinfarction angina or early after non–Q wave infarction and during acute MI is increased severalfold compared with patients with stable angina. Although this risk is not necessarily higher than that with medical therapy, it has led to the argument to consider angioplasty or to delay CABG in such patients if medical stabilization can be easily accomplished.

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VIII. Institutional and Operator Competence

Studies suggest that mortality after CABG is higher when carried out in institutions that annually perform fewer than a minimum number of cases. Similar conclusions have been drawn regarding individual surgeons’ volumes. This observation strengthens the argument for careful outcome tracking and supports the monitoring of institutions or individuals who annually perform <100 cases. It is also true that there is a wide variation in risk-adjusted mortality rates in low-volume situations. Thus, some institutions and practitioners maintain excellent outcomes despite relatively low volumes.

Outcome reporting in the form of risk-adjusted mortality rates after bypass has been effective in reducing mortality rates nationwide. Public release of hospital and physician-specific mortality rates has not been shown to drive this improvement and has failed to effectively guide consumers or alter physician referral patterns.

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IX. Cost-Effectiveness of Bypass Surgery

A variety of studies of CABG have found the technique to be cost-effective in patients for whom survival and/or symptomatic benefit is demonstrable. Within these subsets, the cost-effectiveness of CABG compares favorably with that of other accepted medical therapies.

When compared with PTCA, the initial hospital cost of CABG is significantly higher. However, by 5 years, the cumulative cost of PTCA compared with initial surgical therapy is within 5% of CABG, or a difference of <$3000. Observational studies showing a poorer survival effect of PTCA in patients with more advanced disease suggest that there may be a significant cost gradient for PTCA as the extent of disease increases, which is not apparent for coronary bypass surgery.

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X. Indications

A. Indications for CABG in Asymptomatic or Mild Angina

Class I

1. Significant left main coronary artery stenosis.

2. Left main equivalent: significant (≥70%) stenosis of proximal LAD and proximal left circumflex artery.

3. Three-vessel disease. (Survival benefit is greater in patients with abnormal LV function; eg, with an EF <0.50.)

Class IIa

1. Proximal LAD stenosis with 1- or 2-vessel disease.*¹

Class IIb

1. One- or 2-vessel disease not involving the proximal LAD.†²

Class III

See text.

B. Indications for CABG in Stable Angina

Class I

1. Significant left main coronary artery stenosis.

2. Left main equivalent: significant (≥70%) stenosis of proximal LAD and proximal left circumflex artery.

3. Three-vessel disease. (Survival benefit is greater when LVEF is <0.50.)

4. Two-vessel disease with significant proximal LAD stenosis and either EF <0.50 or demonstrable ischemia on noninvasive testing.

5. One- or 2-vessel coronary artery disease without significant proximal LAD stenosis, but with a large area of viable myocardium and high-risk criteria on noninvasive testing.

6. Disabling angina despite maximal medical therapy, when surgery can be performed with acceptable risk. If angina is not typical, objective evidence of ischemia should be obtained.

Class IIa

1. Proximal LAD stenosis with 1-vessel disease.*¹

2. One- or 2-vessel coronary artery disease without significant proximal LAD stenosis, but with a moderate area of viable myocardium and demonstrable ischemia on noninvasive testing.

Class III

1. One- or 2-vessel disease not involving significant proximal LAD stenosis, in patients (1) who have mild symptoms that are unlikely due to myocardial ischemia or have not received an adequate trial of medical therapy and (A) have only a small area of viable myocardium or (B) have no demonstrable ischemia on noninvasive testing.

2. Borderline coronary stenoses (50% to 60% diameter in locations other than the left main coronary artery) and no demonstrable ischemia on noninvasive testing.

3. Insignificant (<50% diameter) coronary stenosis.

C. Indications for CABG in Unstable Angina/Non–Q Wave MI

Class I

1. Significant left main coronary artery stenosis.

2. Left main equivalent: significant (≥70%) stenosis of proximal LAD and proximal left circumflex artery.

3. Ongoing ischemia not responsive to maximal nonsurgical therapy.

Class IIa

1. Proximal LAD stenosis with 1- or 2-vessel disease.*¹

Class IIb

2. One- or 2-vessel disease not involving the proximal LAD.†²

Class III

See text.

D. Indications for CABG in ST-Segment Elevation (Q-Wave) MI

Class I

None.

Class IIa

1. Ongoing ischemia/infarction not responsive to maximal nonsurgical therapy.

Class IIb

1. Progressive LV pump failure with coronary stenosis compromising viable myocardium outside the initial infarct area.

2. Primary reperfusion in the early hours (≤6 to 12 hours) of an evolving ST-segment elevation MI.

Class III

1. Primary reperfusion late (≥12 hours) in evolving ST-segment elevation MI without ongoing ischemia.

E. Indications for CABG in Poor LV Function

Class I

1. Significant left main coronary artery stenosis.

2. Left main equivalent: significant (≥70%) stenosis of proximal LAD and proximal left circumflex artery.

3. Proximal LAD stenosis with 2- or 3-vessel disease.

Class IIa

1. Poor LV function with significant viable, noncontracting, revascularizable myocardium without any of the aforementioned anatomic patterns.

Class III

1. Poor LV function without evidence of intermittent ischemia and without evidence of significant revascularizable, viable myocardium.

F. Indications for CABG in Life-Threatening Ventricular Arrhythmias

Class I

1. Left main coronary artery stenosis.

2. Three-vessel coronary disease.

Class IIa

1. Bypassable 1- or 2-vessel disease causing life-threatening ventricular arrhythmias.‡³

2. Proximal LAD disease with 1- or 2-vessel disease.‡³

Class III

1. Ventricular tachycardia with scar and no evidence of ischemia.

G. Indications for CABG After Failed PTCA

Class I

1. Ongoing ischemia or threatened occlusion with significant myocardium at risk.

2. Hemodynamic compromise.

Class IIa

1. Foreign body in crucial anatomic position.

2. Hemodynamic compromise in patients with impairment of coagulation system and without previous sternotomy.

Class IIb

1. Hemodynamic compromise in patients with impairment of coagulation system and with previous sternotomy.

Class III

1. Absence of ischemia.

2. Inability to revascularize owing to target anatomy or no-reflow state.

H. Indications for CABG in Patients With Previous CABG

Class I

1. Disabling angina despite maximal noninvasive therapy. (If angina is not typical, then objective evidence of ischemia should be obtained.)

Class IIa

1. Bypassable distal vessel(s) with a large area of threatened myocardium on noninvasive studies.

Class IIb

1. Ischemia in the non-LAD distribution with a patent internal mammary graft to the LAD supplying functioning myocardium and without an aggressive attempt at medical management and/or percutaneous revascularization.

Class III

See text.

Previous Section

Footnotes

• “ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery: Executive Summary and Recommendations: A Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Revise the 1991 Guidelines for Coronary Artery Bypass Graft Surgery)” was approved by the American College of Cardiology Board of Trustees in March 1999 and by the American Heart Association Science Advisory and Coordinating Committee in July 1999.

• When citing this document, the American College of Cardiology and the American Heart Association request that the following citation format be used: Eagle KA, Guyton RA, Davidoff R, Ewy GA, Fonger J, Gardner TJ, Gott JP, Herrmann HC, Marlow RA, Nugent W, O’Connor GT, Orszulak TA, Rieselbach RE, Winters WL, Yusuf S. ACC/AHA guidelines for coronary artery bypass graft surgery: executive summary and recommendations: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Revise the 1991 Guidelines for Coronary Artery Bypass Graft Surgery). Circulation. 1999;100:1464-1480.

• This document is available on the World Wide Web sites of the American College of Cardiology (www.acc.org) and the American Heart Association (www.americanheart.org). A single reprint of the executive summary and recommendations is available by calling 800-242-8721 (US only) or writing the American Heart Association, Public Information, 7272 Greenville Ave, Dallas, TX 75231-4596. Ask for reprint No. 71-0173. To obtain a reprint of the complete guidelines published in the October 1999 issue of the Journal of the American College of Cardiology, ask for reprint No. 71-0174. To purchase additional reprints (specify version and reprint number): up to 999 copies, call 800-611-6083 (US only) or fax 413-665-2671; 1000 or more copies, call 214-706-1466, fax 214-691-6342, or . To make photocopies for personal or educational use, call the Copyright Clearance Center, 978-750-8400.

• ↵1 Becomes Class I if extensive ischemia documented by noninvasive study and/or an LVEF <0.50.

• ↵2 If a large area of viable myocardium and high-risk criteria on noninvasive testing, becomes Class I.

• ↵3 Becomes Class I if arrhythmia is resuscitated sudden cardiac death or sustained ventricular tachycardia.

• CPB indicates cardiopulmonary bypass.

• Data taken from (1) Townsend TR, Reitz BA, Bilker WB, Bartlett JG. Clinical trial of cefamandole, cefazolin, and cefuroxime for antibiotic prophylaxis in cardiac operations. J Thorac Cardiovasc Surg. 1993;106:664–670. (2) Antimicrobial prophylaxis in surgery. Med Lett Drugs Ther. 1997;39:97–101. (3) Vuorisalo S, Pokela R, Syrjala H. Comparison of vancomycin and cefuroxime for infection prophylaxis in coronary artery bypass surgery. Infect Control Hosp Epidemiol. 1998;19:234–239. (4) Romanelli VA, Howie MB, Myerowitz PD, Zvara DA, Rezaei A, Jackman DL, Sinclair DS, McSweeny TD. Intraoperative and postoperative effects of vancomycin administration in cardiac surgery patients: a prospective, double-blind, randomized trial. Crit Care Med. 1993;21:1124–1131.

• Copyright © 1999 by American Heart Association

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SOURCE

http://circ.ahajournals.org/content/100/13/1464.long

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Progenitor Cell Transplant for MI and Cardiogenesis

Posted in Cardiac & Vascular Repair Tools Subsegment, Cardiovascular Research, Chemical Genetics, Diagnostic Immunology, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Innovation in Immunology Diagnostics, Pre-Clinical Animal Model Development, Stem Cells for Regenerative Medicine, Technology Transfer: Biotech and Pharmaceutical, tagged Adult stem cell, Aviva Lev-Ari, Cardiac muscle, Cardiogenesis, cardiomyocyte, cardiomyocyte transformation, cardiosphere, Cardiovascular Disorders, Cardiovascular Research, cell migration, Coronary artery disease, fibro-myocyte, gene expression, Larry H. Bernstein, myocardial infarct, myocardial infarction, nanotechnology, Progenitor cell, progenitor cells, regeneration, sheet mounted cells, Stem cell, transplant on October 28, 2013| Leave a Comment »

Progenitor Cell Transplant for MI and Cardiogenesis  (Part 1

Author and Curator: Larry H. Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

This article is Part I of a review of three perspectives on stem cell transplantation onto a substantial size of infarcted myocardium to generate cardiogenesis in tissue that is composed of both repair fibroblasts and cardiomyocytes, after essentially nontransmural myocardial infarct.

Progenitor Cell Transplant for MI and Cardiogenesis (Part 1)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013/10/28/progenitor-cell-transplant-for-mi-and-cardiogenesis/

Source of Stem Cells to Ameliorate Damage Myocardium (Part 2)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013-10-29/larryhbern/Source_of_Stem_Cells_to_Ameliorate_ Damaged_Myocardium/

An Acellular 3-Dimensional Collagen Scaffold Induces Neo-angiogenesis
 (Part 3)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2013-10-29/larryhbern/An_Acellular_3-Dimensional_Collagen_Scaffold _Induces_Neo-angiogenesis/

The same approach is considered for stroke in one of these studies.  These are issues that need to be considered

1. Adult stem cells
2. Umbilical cord tissue sourced cells
3. Sheets of stem cells
4. Available arterial supply at the margins
5. Infarct diameter
6. Depth of ischemic necrosis
7. Distribution of stroke pressure
8. Stroke volume
9. Mean Arterial Pressure (MAP)
10. Location of infarct
11. Ratio of myocytes to fibrocytes
12. Coexisting heart disease and, or
13. Comorbidities predisposing to cardiovascular disease, hypertension
14. Inflammatory reaction against the graft

Transplantation of cardiac progenitor cell sheet onto infarcted heart promotes cardiogenesis and improves function

L Zakharova1, D Mastroeni1, N Mutlu1, M Molina1, S Goldman2,3, E Diethrich4, and MA Gaballa1*

1Center for Cardiovascular Research, Banner Sun Health Research Institute, Sun City, AZ; 2Cardiology Section, Southern Arizona VA Health Care System, and 3Department of Internal Medicine, The University of Arizona, Tucson, AZ; and 4Arizona Heart Institute, Phoenix, AZ

Cardiovascular Research (2010) 87, 40–49   http://dx.doi.org/10.1093/cvr/cvq027

Abstract

Aims

Cell-based therapy for myocardial infarction (MI) holds great promise; however, the ideal cell type and delivery system have not been established. Obstacles in the field are the massive cell death after direct injection and the small percentage of surviving cells differentiating into cardiomyocytes. To overcome these challenges we designed a novel study to deliver cardiac progenitor cells as a cell sheet.

Methods and results

Cell sheets composed of rat or human cardiac progenitor cells (cardiospheres), and cardiac stromal cells were transplanted onto the infarcted myocardium after coronary artery ligation in rats. Three weeks later, transplanted cells survived, proliferated, and differentiated into cardiomyocytes (14.6 ± 4.7%). Cell sheet transplantation suppressed cardiac wall thinning and increased capillary density (194 ± 20 vs. 97 ± 24 per mm2, P < 0.05) compared with the untreated MI. Cell migration from the sheet was observed along the necrotic trails within the infarcted area. The migrated cells were located in the vicinity of stromal-derived factor (SDF-1) released from the injured myocardium, and about 20% of these cells expressed CXCR4, suggesting that the SDF-1/CXCR4 axis plays, at least, a role in cell migration. Transplantation of cell sheets resulted in a preservation of cardiac contractile function after MI, as was shown by a greater ejection fraction and lower left ventricular end diastolic pressure compared with untreated MI.

Conclusion

The scaffold-free cardiosphere-derived cell sheet approach seeks to efficiently deliver cells and increase cell survival.These transplanted cells effectively rescue myocardium function after infarction by promoting not only neovascular-ization but also inducing a significant level of cardiomyogenesis

Keywords  Myocardial infarction • Cardiac progenitor cells • Cardiospheres • Cardiac regeneration • Contractility

Introduction

Despite advances in cardiac treatment after myocardial infarction (MI), congestive heart failure remains the number one killer world-wide. MI results in an irreversible loss of functional cardiomyocytes followed by scar tissue formation. To date, heart transplant remains the gold standard for treatment of end-stage heart failure, a procedure which will always be limited by the availability of a donor heart. Hence, developing a new form of therapy is vital.

A number of adult non-cardiac progenitor cells have been tested for myocardial regeneration, including skeletal myoblasts,1 bone-marrow2, and endothelial progenitor cells.3,4 In addition, several cardiac resident stem cell populations have been characterized based on the expression of stem cell marker proteins.5–8 Among these, the c-Kit+ population has been reported to promote myocardial repair.5,9 Recently, an ex vivo method to expand cardiac-derived progenitor cells from human myocardial biopsies and murine hearts was developed.10 Using this approach, undifferentiated cells (or cardiospheres) grow as self-adherent clusters from postnatal atrium or ventricular biopsy specimens.11

To date, the most common technique for cell delivery is direct injection into the infarcted myocardium.12 This approach is inefficient because more than 90% of the delivered cells die by apoptosis and only a small number of the survived cells differentiated into cardiomyocytes.13 An alternative approach to cell delivery is a biodegradable scaffold-based engineered tissue.14,15 This approach has the clear advantage in creating tissue patches of different shapes and sizes and in creating a beating heart by decellularization technology.16 Advances are being made to overcome the issue of small patch thickness and to minimize possible toxicity of the degraded substances from the scaffold.15 Recently, scaffold-free cell sheets were created from fibroblasts, mesenchymal cells, or neonatal myocytes.17,18 Transplantation of these sheets resulted in a limited improvement in cardiac function due to induced neovascularization and angiogenesis through secretion of angiogenic factors.17–19 However, few of those progenitor cells have differentiated into cardiomyocytes.17 The need to improve cardiac contractile function suggests focusing on cells with higher potential to differentiate to cardiomyocytes with an improved delivery method.

In the present study, we report a cell-based therapeutic strategy that surpasses limitation inherent in previously used methodologies. We have created a scaffold-free sheet composed of cardiac progenitor cells (cardiospheres) incorporated into a layer of cardiac stromal cells. The progenitor cells survived when transplanted as a cell sheet onto the infarcted area, improved cardiac contractile functions, and supported recovery of damaged myocardium by promoting not only vascularization but also a significant level of cardiomyogenesis. We also showed that cells from a sheet can be recruited to the site of injury driven, at least partially, by the stromal-derived factor (SDF-1) gradient.

Methods

Detailed methods are provided in the Supplementary Methods

Animals

Three-month-old Sprague Dawley male rats were used. Rats were randomly placed into four groups:

(1) sham-operated rats, n = 12;

(2) MI, n = 12;

(3) MI treated with rat sheet, n = 10; and

(4) MI treated with human sheet, n = 10.

Myocardial infarction

MI was created by the ligation of the left coronary artery.20 Animals were intubated and ventilated using a small animal ventilator (Harvard Apparatus). A left thoracotomy was performed via the third intercostal rib, and the left coronary artery was ligated. The extent of infarct was verified by measuring the area at risk: heart was perfused with PBS containing 4 mg/mL Evans Blue as previously described by our laboratory.20 The area at risk was estimated by recording the size of the under-perfused (pale-colored) area of myocardium (see Supplementary material online, Figure S1). Only animals with an area at risk >30% were used in the present study. Post-mortem infarct size was measured using triphenyl tetrazolium chloride staining as previously described by our laboratory.20

Isolation of cardiosphere-forming cells

Cardiospheres were generated as described10 from atrial tissues obtained from:

(1) human atrial resection samples obtained from patients (aged from 53 to 73 years old) undergoing cardiac bypass surgery at Arizonam Heart Hospital (Phoenix, AZ) in compliance with Institutional Review Board protocol (n = 10),

(2) 3-month-old SD rats (n = 10). Briefly, tissues were cut into 1–2 mm3 pieces and tissue fragments were cultured ‘as explants’ in a complete explants medium for 4 weeks (Supplementary Methods).

Cell sheet preparation, labelling, handling, and transplantation

Cardiosphere-forming cells (CFCs) combined with cardiac stromal cells were seeded on double-coated plates (poly-L-lysine and collagen type IV from human placenta) in cardiosphere growing medium (Supplementary Methods). The sheets created from the same cell donors were divided into two groups,

one for transplantation and the other for characterization by immunostaining and RT–PCR (Supplementary Methods).

Prior to transplantation, rat cell sheets were labelled with 2 mM 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine, DiI, for tracking transplanted cells in rat host myocardium (Molecular Probes, Eugene, OR). Sheets created using human cells were transplanted unlabelled. Sheets were gently peeled off the collagen-coated plate and folded twice to form four layers. The entire sheet with 200 ml of media was

• gently aspirated into the pipette tip,
• transferred to the supporting polycarbonate filter (Costar) and
• spread off by adding media drops on the sheet (Figure 2A).

Polycarbonate filter was used as a flexible mechanical support for cell sheet to facilitate handling during the transplantation. Immediately after LAD occlusion, the cell sheet was transplanted onto the infarcted area, allowed to adhere to the ventricle for 5–7 min, and the filter was removed before closing the chest (Figure 2A).

Cardiac function

Three weeks after MI, closed-chest in vivo cardiac function was measured using a Millar pressure conductance catheter system (Millar Instruments, Houston, TX) (Supplementary Methods).

Cell sheet survival, engraftment, and cell migration

Rat host myocardium and cell sheet composition after transplantation were characterized by immunostaining (Supplementary Methods). Rat-originated cells were traced by DiI, while human-originated cells were identified by immunostaining with anti-human nuclei or human lamin antibodies.

1. To assess sheet-originated cardiomyocytes within the host myocardium, the number of cells positive for both human nuclei and myosin heavy chain (MHC) (human sheet); or both DiI and MHC (rat sheet) were counted.
2. To assess sheet-originated capillaries within the rat host myocardium, the number of cells positive for both human nuclei and von Willebrand factor (vWf) (human sheet); or both DiI and vWf (rat sheet) were counted. Cells were counted in five microscopic fields within cell sheet and area of infarct (n = 5). The number of cells expressing specific markers was normalized to the total number of cells determined by 40,6-diamidino-2-phenylindole staining of the nuclei DNA.
3. To assess the survival of transplanted cells, sections were stained with Ki-67 antibody followed by fluorescent detection and caspase 3 primary antibodies followed by DAB detection (Supplementary Methods).
4. To evaluate human sheet engraftment, sections were stained with human lamin antibody followed by fluorescent detection (Supplementary Methods).
5. Rat host inflammatory response to the transplanted human cell sheet 21 days after transplantation was evaluated by counting tissue mononuclear phagocytes and neutrophils (Supplementary Methods).

Imaging

Images were captured using Olympus IX70 confocal microscope (Olympus Corp, Tokyo, Japan) equipped with argon and krypton lasers or Olympus IX-51 epifluorescence microscope using excitation/emission maximum filters: 490/520 nm, 570 /595 nm, and 355 /465 nm. Images were processed using DP2-BSW software (Olympus Corp).

Statistics

All data are represented as mean ± SE Significance (P < 0.05) was deter-mined using ANOVA (StatView).

Results

Generation of cardiospheres

Cardiospheres were generated from atrial tissue explants. After 7–14 days in culture, a layer of stromal cells arose from the attached explants (Supplementary material online, Figure S2a). CFCs, small phase-bright single cells, emerged from explants and bedded down on the stromal cell layer (Supplementary material online, Figure S2b).

• After 4 weeks, single CFCs, as well as cardiospheres (spherical colonies generated from CFCs) were observed (Supplementary material online, Figure S2c).

Cellular characteristics of cardiospheres in vitro

Immunocytochemical analysis of dissociated cardiospheres revealed that

• 30% of cells were c-Kitþ indicating that the CFCs maintain multi-potency. About
• 22 and 28% of cells expressed a, b-MHC and cardiac troponin I, respectively.

These cells represent an immature cardiomyocyte population because they were smaller (10–15 pm in length vs. 60–80 pm for mature cardiomyocytes) and no organized structure of MHC was detected. Furthermore

• 17% of the cells expressed a-smooth muscle actin (SMA) and
• 6% were positive for vimentin,
  • both are mesenchymal cell markers (Supplementary material online, Figure S3a and b).
• Less then 5% of cells were positive for endothelial cell marker; vWf.

Cell characteristics of human cardiospheres are similar to those from rat tissues (Supplementary material online, Figure S3c).

Cardiospheres were further characterized based on the expression of c-Kit antigen. RT–PCR analysis was performed on both c-Kitþ and c-Kit2 subsets isolated from re-suspended cardiospheres. KDR, kinase domain protein receptor, was recently identified as a marker for cardiovascular lineage progenitors in differentiating embryonic stem cells.21 Here, we found that

• the c-Kitþ cells were also Nkx2.5 and GATA4-positive, but were low or negative for KDR (Supplementary material online, Figure S3d). In contrast,
• c-Kit2 cells strongly expressed KDR and GATA4, but were negative for Nkx2.5.
• Both c-Kitþ and c-Kit2 subsets did not express Isl1, a marker for multipotent secondary heart field progenitors.22

Characteristics of cell sheet prior to transplantation

The cell sheet is a layer of cardiac stromal cells in which the cardiospheres were incorporated at a frequency of 21 ± 0.5 spheres per 100,000 viable cells (Figure 1A). The average diameter of cardiospheres within a sheet was 0.13 ± 0.02 mm and their average area was 0.2 ± 0.06 mm2 (Figure 1A). After sheets were peeled off the plate, it exhibited a heterogeneous thickness ranging from 0.05– 0.1 mm (n 1/4 10), H&E staining (Figure1B) and Masson’s Trichrome staining (Figure 1C) of the sheet sections revealed tissue-like organized structures composed of muscle tissue intertwined with streaks of collagen with no necrotic core. Based on the immunostaining results, sheet compiled of several cell types including

• SMAþ cardiac stromal cells (50%),
• MHCþ cardiomyocytes (20%), and
• vWfþ endothelial cells (10%) (Figure 1D and E).
• 15% of the sheet-forming cells were c-Kitþ suggesting the cells multipotency (Figure 1E).
• Cells within the sheet expressed gap-junction protein C43, an indicator of electromechanical coupling between cells (Figure 1D).
• 40% of cells were positive for the proliferation marker Ki-67 suggesting an active cell cycle state (Figure 1D, middle panel).

Human sheet expressed genes

1. known to be upregulated in undifferentiated cardiovascular progenitors such as c-Kit and KDR;
2. cardiac transcription factors Nkx2.5 and GATA4; genes related to adhesion, cell homing, and
3. migration such as ICAM (intercellular adhesion molecule), CXCR4 (receptor for SDF-1), and
4. matrix metalloprotease 2 (MMP2).

No expression of Isl1 was detected in human sheet (Figure 1F).

Figure 1 Cell sheet characteristics. (A) Fully formed cell sheet. Arrow indicates integrated cardiosphere. (B) H&E staining; pink colour (arrowhead) indicates cytosol and blue (arrows) indicates nuclear stain. Note that there is no necrotic core within the cell sheet. (C) Masson’s Trichrome staining of sheet section. Arrowhead indicates collagen deposition within the sheet. (D and E) Sheet sections were labelled with antibodies against following markers: (D) vWf (green), Ki-67 (green), C43 (green); (E) c-Kit (green), MHC (red), SMA (red) as indicated on top of each panel. Nuclei were labelled with blue fluorescence of 40,6-diamidino-2-phenylindole (DAPI). (F) Gene expression analysis of the cell sheet. Scale bars, 200 pm (A) or 50 pm (B–E).

Cell sheet survival and proliferation

Two approaches were used to track transplanted cells in the host myocardium.

• rat cell sheets were labelled with red fluorescent dye, DiI, prior to the transplantation.
• the sheet created from human cells (human sheet) were identified in rat host myocardium by immunostaining with human nuclei antibodies.

DiI-labelling together with trichrome staining showed engraftment of the cardiosphere-derived cell sheet to the infarcted myocardium (Figure 2B–D). In vivo sheets grew into a stratum with heterogeneous thickness ranging from 0.1–0.5 mm over native tissue. The percentage of Ki-67þ cells within the sheet was 37.5 ± 6.5 (Figure 2F) whereas host tissue was mostly negative (except for the vasculature).

To assess the viability of transplanted cells, the heart sections were stained with the apoptosis marker, caspase 3. A low level of caspase 3 was detected within the sheet, suggesting that the majority of transplanted cells survived after transplantation (Figure 2G).

Figure 2 Transplantation and growth of cell sheet after transplantation.

(A) Sheet transplantation onto infarcted heart. Detached cell sheet on six-well plate (left); cell sheet folded on filter (middle); and transplanted onto left ventricle (right). Scale bar 2 mm. DiI-labelled cell sheets grafted above MI area at day 3

(B) and day 21

(C) after transplantation.

(D) LV section of untreated MI rat at day 21 showing no significant red fluorescence background.

Bottom row (B–D) demonstrates the enlargement of box-selected area of corresponding top panels.

(E) Similar sections stained with Masson’s Trichrome. Section of rat (F) or human (G) sheet treated rat at day 21 after MI.

(F) Section was stained with antibody against Ki-67 (green). Cell sheet was pre-labelled with DiI (red). Nuclei stained with blue fluorescence of DAPI.

(G) Section was double stained with human nuclei (blue) and caspase 3 (brown, arrows) antibodies and counterstained with eosin.

Asterisks (**) indicate cell sheet area. Scale bars 200 mm (B–D, top row), 100 mm (B–D, bottom row, and E) or 50 mm (F, G).

Identification of inflammatory response

Twenty-one days after transplantation of human cell sheet, inflammatory response of rat host was examined. Transplantation of human sheet on infarcted rats reduced the number of mononuclear phagocytes (ED1-like positive cells) compared with untreated MI control (Supplementary material online, Figure S4a–e and l). In addition, the number of neutrophils was similar in both control untreated MI and sheet-treated sections (Supplementary material online, Figure S4f–k and m). These data suggest that at 21 days post transplantation, human cell sheet was not associated with significant infiltration of host immune cells.

Cell sheet engraftment and migration

Development of new vasculature was determined in cardiac tissue sections by co-localization of DiI labelling and vWf staining (Figure 3C). Three weeks after transplantation, the capillary density of ischaemic myocardium in the sheet-treated group significantly increased compared with MI animals (194 ± 20 vs. 97 ± 24 per mm2, P < 0.05, Figure 3A and B). The capillaries originated from the sheet ranged in diameter from 10 to 40 jim (n 1/4 30). A gradient in capillary density was observed with higher density in the sheet area which was decreased towards underlying infarcted myocardium. Mature blood vessels were identified within the sheet area and in the underlying myocardium in close proximity to the sheet evident by vWf and SMA double staining (Figure 3D).

Figure 3 Neovascularization of infarcted wall. (A) Frozen tissue sections stained with vWf antibody (green). LV section of control (sham), infarcted (MI), and MI treated with cell sheet (sheet) rats. Scale bar, 100 jim. (B) Capillary density decreased in the MI compared with sham (*P < 0.05) and improved after cell sheet treatment (#P < 0.05). (C) Neovascularization within cell sheet area was recognized by co-localization of DiI- (red) and vWf (green) staining. Scale bar 100 jim. (D) Mature blood vessels (arrows) were identified by co-localization of SMA (red) and vWf (green) staining. Scale bar 50 jim.

Furthermore, 3 weeks after transplantation, a large number of labelled human nuclei positive or DiI-labelled cells were detected deep within the infarcted area indicating cell migration from the epicardial surface to the infarct (Figure 4A, B, and D). Minor or no migration was detected when the cell sheet was transplanted onto non-infarcted myocardium, sham control (Figure 4C). To evaluate engraftment of sheet-originated cells, sections were labelled with anti-human nuclear lamin antibody. Quantification of engraftment was performed using two approaches: fluorescence intensity and cell counting. Fluorescence intensity of the signal was analysed and compared for different areas of myocardium (Figure 4E–J). Since the transplanted sheets are created by human cells and are stained with human nuclear lamin-labelled with green fluorescence, the signal intensity of the sheet is set to 100% (100% of cells are lamin-positive). Myocardial area with no or limited number of labelled cells had the lowest level of fluorescence signal (13%, or 3.2 ± 1.4% of total number of cells), while

1. the area where the cell migrated from the sheet to the infarcted myocardium had higher signal intensity (47%, or 11.9 ± 1.7% of total number of cells), indicating a higher number of sheet-originated cells are engrafted in the infarcted area.) (Figure 4K and L).
2. Migrated cells were positive for KDR (Supplementary material online, Figure S5).

Figure 4 Engraftment quantification of cells migrated from the sheet into the infarcted area of MI. Animals were treated with rat (A) or human (B–F) sheets. Cardiomyocytes were labelled with MHC antibody (A, green or B, red). Rat sheet-originated cells were identified with DiI-labelling, red (A). Arrows indicate the track of migrating cells. Human sheet-originated cells were identified by immunostaining with human nuclei antibody followed by secondary antibodies conjugated with either Alexa 488 (B, E and F, green) or AP (C, D, blue). No migration was detected when the cell sheet was transplanted onto non-infarcted myocardium (C). Heart sections were counterstained with eosin, pink (C–D). Higher magnification of area selected in the box is presented (D, right). Immunofluorescence of sheet (green) grafted to the myocardium surface (E) or cells migrated to the infarction area (F). Fluorescence profiles acrossthe cell sheet itself(G, box 1), area underlying cell sheet (I, box 2) and infarction areawith migrated cells (F, box 3). Mean fluorescence intensityofthe grafted human (K) cells was determined by outlining the region of interest (ROI) and subtracting the background fluorescence for the same region. Fluorescence intensity was normalized to the area of ROI (ii 1/4 6). (L) Percent engraftment was defined as number of lamin-positive cells divided by total number of cells per ROI. ‘M’, myocardium,’S’ sheet, ‘I’ infarction. Scale bars 100 mm (A–C, D, left, E and F), or 50 mm (D, right).

To elucidate a possible mechanism of cell migration, sections were stained to detect SDF1 and its unique receptor CXCR4. The migration patterns of cells from the sheet coincided with SDF-1 expression. Within 3 days after MI, SDF-1 was expressed in the injured myocardium (Figure 5A). At 3 weeks after MI and sheet transplantation, SDF-1 was co-localized with the migrated labelled cells (Figure 5B). PCR analysis revealed CXCR4 expression in cell sheet before transplantation (Figure 1F). However, after transplantation only a fraction of migrated cells expressed CXCR4 (Figure 5C).

Figure 5 Migration of sheet-originated cells into the infarcted area. Confocal images of MI animals treated with sheets from rats (A and B) or human (C). SDF1 (green) was detected at border zone of the infarct at day 3 (A) and day 21 (B). Rat sheet-originated cells were identified with DiI-labelling (red). Note co-localization of DiI-positive sheet-originated cells with SDF1 at 21 days after MI (B). Human cells were identified by immunostaining with human nuclei antibody, red, (C). Note human cells that migrated to the area of infarct express CXCR4 (green) (C). Scale bar, 200 mm (A, B) or 50 mm (C). ‘M’, myocardium, ‘S’ sheet, ‘I’ infarct.

3.7 Cardiac regeneration

The differentiation of migrating cells into cardiomyocytes was evident by the co-localization of MHC staining with either human nuclei (Figure 6A) or DiI (Figure 6B and C). In contrast to the immature cardiomyocyte-like cells within the pre-transplanted cell sheet, the migrated and newly differentiated cells within the myocardium were about 30–50 mm in size and co-expressed C43 (see Supplementary material online, Figure S6). Cardiomyogenesis within the infarcted myocardium was observed in the sheets created from either rat or human cells.

Figure 6 Cardiac regeneration. Sections of MI animals treated with human (A) or rat (B, C) sheets. Human sheet was identified by immunostaining with human nuclei antibody (green). Section was double-stained with MHC (red) antibody. Newly formed cardiomyocytes was identified by co-localization of human nuclei and MHC (yellow, arrow). (B) Rat sheet-originated cells were identified by DiI labelling (red). Section was double-stained with MHC (green) antibody. Newly formed cardiomyocytes were detected by co-localization of DiI with MHC (yellow, arrows). (C) Higher magnification of area selected in the boxes (B). Scale bars 200 mm (B), or 20 mm (A, C). ‘M’, myocardium, ‘S’ sheet, ‘I’ infarct.

Cell sheet improved cardiac contractile function and retarded LV remodelling after MI

Closed-chest in vivo cardiac function was derived from left ventricle (LV) pressure–volume loops (PV loops), which were measured using a solid-state Millar conductance catheter system. MI resulted in a characteristic decline in LV systolic parameters and an increase in diastolic parameters (Table 1). Cell sheet treatment improved both systolic and diastolic parameters (Table 1). Specifically, load-dependent parameters of systolic function: ejection fraction (EF), dP/dTmax, and cardiac index (CI) were decreased in MI rats and increased towards sham control with the cell sheet treatment (Table 1). Diastolic function parameters, dP/dTmin, relaxation constant (Tau), EDV, and EDP were increased in the MI rats and returned towards sham control parameters after sheet treatment (Table 1). However, load-independent systolic function, Emax, was decreased after MI. Treatment with human sheet improved Emax, while treatment with rat sheet had no effect (Table 1). Treatment with either rat or human sheets retarded LV remodelling; as such that it increased the ratio of anteriolateral wall thickness/LV inner diameter (t/Di) and wall thickness/LV outer diameter (t/Do) (see Supplementary material online, Table S3). However, human sheets appear to further improve LV remodelling compared with rat sheets as indicated by increased ratio of wall thickness to ventricular diameter and decreased both EDV and EDP (Table 1 and see Supplementary material online, Table S3).

Table 1 Hemodynamic parameters

Discussion

The majority of the cardiac progenitor cells delivered using our scaffold-free cell sheet survived after transplantation onto the infarcted heart. A significant percentage of transplanted cells migrated from the cell sheet to the site of infarction and differentiated into car-diomyocytes and vasculature leading to improving cardiac contractile function and retarding LV remodelling. Thus, delivery of cardiac progenitor cells together with cardiac mesenchymal cells in a form of scaffold-free cell sheet is an effective approach for cardiac regeneration after MI.

Consistent with previous studies,5,11 here we showed that cardio-spheres are composed of multipotent precursors, which have the capacity to differentiate to cardiomyocytes and other cardiac cell types. When we fractioned cardiospheres based on c-Kit expression, we identified two subsets: Kitþ /KDR2/low/Nkx2.5þ and Kit2/KDRþ/ Nkx2.52(Supplementary material online, Figure S3d), which are likely reflecting cardiac and vascular progenitors.20

In the present study, delivery of cardiac progenitor cells as a cell sheet facilitates cell survival after transplantation. Necrotic cores, commonly observed in tissue engineered patches,23,24 are absent in cardiosphere sheets prior to transplantation (Figure 1B and C). Poor cell survival is caused by multiple processes such as: ischemia from the lack of vasculature and anoikis due to cell detachment from sub-strate.25 A possible mechanism of cell survival within the sheet is the induction of neo-vessels soon after transplantation due to the presence of endothelial cells within the sheet before transplantation (Figure 10). The cell sheet continued to grow in vivo (Figure 2B and C), suppressed cardiac wall thinning, and prevented LV remodelling at 21 days after transplantation (see Supplementary material online, Table S3). This maybe due to the induction of neovascularization (Figure 3), which may prevents ischemia-induced cell death (Figure 2G). Another likely mechanism of cell survival is that the cells within the scaffold-free sheet maintained cell-to-cell adhesion16 as shown by ICAM expression (Figure 1F). The cells also exhibit C43-positive junctions (Figure 10, see Supplementary material online, Figure S6), which may facilitate electromechanical coupling between the transplanted cells and the native myocardium.

We observed cell migration from the sheet to the infarcted myocardium (Figure 4A and B, E and F), which may be facilitated by the strong expression of MMP2 in the cell sheet (Figure 1F). Although, the mechanism of cardiac progenitor cell migration remains unclear, previous observations showed that SDF-1 is upregulated after MI and plays a role in bone-marrow and cardiac stem cell migration.26,27 Our data suggest that SDF-1-CXCR4 axis plays, at least in part, a role in cardiac progenitor cell migration from cell sheet to the infarcted myocardium. This conclusion is based on the following observations: (1) cell sheet expresses CXCR4 prior to transplantation (Figure 1F), (2) migrated cells are located in the vicinity of SDF-1 release (Figure 5A and B), and (3) about 20% of migrated cells expressed CXCR4. Note, not all the migrated cells expressed CXCR4 suggesting other mechanisms are involved in cell migration (Figure 5C).

Here we report that implanting cardiosphere-generated cell sheet onto infarcted myocardium not only improved vascularization but also promoted cardiogenesis within the infarcted area (Figure 6). A larger number of newly formed cardiomyocytes were found deep within the infarct compared with the cell sheet periphery. Notably the transplantation of the cell sheet resulted in a significant improvement of the cardiac contractile function after MI, as was shown by an increase of EF and decrease of LV end diastolic pressure (Table 1).

The beneficial effect of cell sheet is, in part, due to the presence of a large number of activated cardiac mesenchymal stromal cells (myofibroblasts) within the sheet. Myofibroblasts are known to provide a mechanical support for grafted cells, facilitating contraction28 and to induce neovascularization through the release of cytokines.17 In addition, mesenchymal cells are uniquely immunotolerant. In xenograft models unmatched mesenchymal cells transplanted to the heart of immunocompetent rats were shown to suppress host immune response29 presumably due to inhibition of T-cell activation.30 Consistently with previous study from our laboratory,31 here, we demonstrated host tolerance to the cell sheet 21 days after MI. Finally, phase II and III clinical trials are currently undergoing in which allogeneic MSCs are used to treat MI in patients (Osiris Therapeutic, Inc.).

In summary, our results show that cardiac progenitor cells can be delivered as a cell sheet, composed of a layer of cardiac stromal cells impregnated with cardiospheres. After transplantation, cells from the cell sheet migrated to the infarct, partially driven by SDF-1 gradient, and differentiated into cardiomyocytes and vasculature. Transplantation of cell sheet was associated with prevention of LV remodelling, reconstitution of cardiac mass, reversal of wall thinning, and significant improvement in cardiac contractile function after MI. Our data also suggest that strategies, which utilize undigested cells, intact cell–cell interactions, and combined cell types such as our scaffold-free cell sheet should be considered in designing effective cell therapy.

References

Fuchs JR, Nasseri BA, Vacanti JP, Fauza DO. Postnatal myocardial augmentation with skeletal myoblast-based fetal tissue engineering. Surgery 2006;140:100–107.

Orlic D, Kajstura J, Chimenti S, Bodine DM, Leri A, Anversa P. Bone marrow stem cells regenerate infarcted myocardium. Pediatr Transplant 2003;7(Suppl. 3):86–88.

Kawamoto A, Tkebuchava T, Yamaguchi J, Nishimura H, Yoon YS, Milliken C et al. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 2003;107:461–468.

Iwasaki H, Kawamoto A, Ishikawa M, Oyamada A, Nakamori S, Nishimura H et al. Dose-dependent contribution of CD34-positive cell transplantation to concurrent vasculogenesis and cardiomyogenesis for functional regenerative recovery after myocardial infarction. Circulation 2006;113:1311–1325.

Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114: 763–776.

Oh H, Bradfute SB, Gallardo TD, Nakamura T, Gaussin V, Mishina Y et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci USA 2003;100:12313–12318.

Laugwitz KL, Moretti A, Lam J, Gruber P, Chen Y, Woodard S et al. Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 2005;433: 647–653.

Pfister O, Mouquet F, Jain M, Summer R, Helmes M, Fine A et al. CD31- but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res 2005;97:52–61.

Dawn B, Stein AB, Urbanek K, Rota M, Whang B, Rastaldo R et al. Cardiac stem cells delivered intravascularly traverse the vessel barrier, regenerate infarcted myocardium, and improve cardiac function. Proc Natl Acad Sci USA 2005;102:3766–3771.

 

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