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Reprogramming Cell Fate

Posted in Biological Networks, Gene Regulation and Evolution, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, International Global Work in Pharmaceutical, Liver & Digestive Diseases Research, Metabolomics, Molecular Genetics & Pharmaceutical, Nutrigenomics, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , , , , , on March 2, 2013| 4 Comments »

Reprogramming Cell Fate

 

Reporter: Larry H.Bernstein, MD, FCAP

Kathy Liszewski: reporting Gordon Conference “Reprogramming Cell Fate” meeting
M. Azim Surani, Ph.D., Univ Cambridge
Source unknown: June 21, 2012;32(11)
They report two critical steps both of which are needed for exploring epigenetic reprogramming.  While females have two X chromosomes ,
  • the inactivation of one is necessary for cell differentiation.
  • Only after epigenetic reprogramming of the X chromosome can pluripotency be acquired.

Pluripotent stem cells can generate – any fetal or adult cell type but

    • don’t develop into a complete organism.
Pioneer transcription factors take the lead in – facilitating cellular reprogramming – and responses to environmental cues.
Multicellular organisms consist of
  • functionally distinct cellular types
  • produced by differential activation of gene expression.
They seek out and bind specific regulatory sequences in DNA, even though DNA is coated with and condensed into a thick fiber of chromatin.
The pioneer factor, discovered by Prof. KS Zaret at UPenn SOM in 1996, endows the competence for gene activity,
  • being among the first transcription factors to
  • engage and pry open the target sites in chromatin.
FoxA factors, expressed in the foregut endoderm of the mouse,are necessary for
  • induction of the liver program.
    •  nearly one-third of the DNA sites bound by FoxA in the adult liver occur near silent genes.
organ regeneration example from induced plurip...

organ regeneration example from induced pluripotent stem cells(iPS cell) (Photo credit: Wikipedia)

English: Pathway of stem cell differentiation

English: Pathway of stem cell differentiation (Photo credit: Wikipedia)

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What is the Future for Genomics in Clinical Medicine?

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Infectious Disease & New Antibiotic Targets, International Global Work in Pharmaceutical, Interviews with Scientific Leaders, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Scientist: Career considerations, Stem Cells for Regenerative Medicine, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , on February 17, 2013| 5 Comments »

What is the Future for Genomics in Clinical Medicine?

Author and Curator: Larry H Bernstein, MD, FCAP

 

Introduction

This is the last in a series of articles looking at the past and future of the genome revolution.  It is a revolution indeed that has had a beginning with the first phase discovery leading to the Watson-Crick model, the second phase leading to the completion of the Human Genome Project, a third phase in elaboration of ENCODE.  But we are entering a fourth phase, not so designated, except that it leads to designing a path to the patient clinical experience.
What is most remarkable on this journey, which has little to show in treatment results at this time, is that the boundary between metabolism and genomics is breaking down.  The reality is that we are a magnificent “magical” experience in evolutionary time, functioning in a bioenvironment, put rogether like a truly complex machine, and with interacting parts.  What are those parts – organelles, a genetic message that may be constrained and it may be modified based on chemical structure, feedback, crosstalk, and signaling pathways.  This brings in diet as a source of essential nutrients, exercise as a method for delay of structural loss (not in excess), stress oxidation, repair mechanisms, and an entirely unexpected impact of this knowledge on pharmacotherapy.  I illustrate this with some very new observations.

Gutenberg Redone

The first is a recent talk on how genomic medicine has constructed a novel version of the “printing press”, that led us out of the dark ages.

Topol_splash_image

In our series The Creative Destruction of Medicine, I’m trying to get into critical aspects of how we can Schumpeter or reboot the future of healthcare by leveraging the big innovations that are occurring in the digital world, including digital medicine.

We have this big thing about evidence-based medicine and, of course, the sanctimonious randomized, placebo-controlled clinical trial. Well, that’s great if one can do that, but often we’re talking about needing thousands, if not tens of thousands, of patients for these types of clinical trials. And things are changing so fast with respect to medicine and, for example, genomically guided interventions that it’s going to become increasingly difficult to justify these very large clinical trials.

For example, there was a drug trial for melanoma and the mutation of BRAF, which is the gene that is found in about 60% of people with malignant melanoma. When that trial was done, there was a placebo control, and there was a big ethical charge asking whether it is justifiable to have a body count. This was a matched drug for the biology underpinning metastatic melanoma, which is essentially a fatal condition within 1 year, and researchers were giving some individuals a placebo.

The next observation is a progression of what he have already learned. The genome has a role is cellular regulation that we could not have dreamed of 25 years ago, or less. The role is far more than just the translation of a message from DNA to RNA to construction of proteins, lipoproteins, cellular and organelle structures, and more than a regulation of glycosidic and glycolytic pathways, and under the influence of endocrine and apocrine interactions. Despite what we have learned, the strength of inter-molecular interactions, strong and weak chemical bonds, essential for 3-D folding, we know little about the importance of trace metals that have key roles in catalysis and because of their orbital structures, are essential for organic-inorganic interplay. This will not be coming soon because we know almost nothing about the intracellular, interstitial, and intrvesicular distributions and how they affect the metabolic – truly metabolic events.

I shall however, use some new information that gives real cause for joy.

Reprogramming Alters Cells’ Fate

Kathy Liszewski
Gordon Conference  Report: June 21, 2012;32(11)
New and emerging strategies were showcased at Gordon Conference’s recent “Reprogramming Cell Fate” meeting. For example, cutting-edge studies described how only a handful of key transcription factors were needed to entirely reprogram cells.
M. Azim Surani, Ph.D., Marshall-Walton professor at the Gurdon Institute, University of Cambridge, U.K., is examining cellular reprogramming in a mouse model. Epiblast stem cells are derived from the early-stage embryonic stage after implantation of blastocysts, about six days into development, and retain the potential to undergo reversion to embryonic stem cells (ESCs) or to PGCs.”  They report two critical steps both of which are needed for exploring epigenetic reprogramming.  “Although there are two X chromosomes in females, the inactivation of one is necessary for cell differentiation. Only after epigenetic reprogramming of the X chromosome can pluripotency be acquired. Pluripotent stem cells can generate any fetal or adult cell type but are not capable of developing into a complete organism.”
The second read-out is the activation of Oct4, a key transcription factor involved in ESC development. The expression of Oct4 in epiSCs requires its proximal enhancer.  Dr. Surani said that their cell-based system demonstrates how a systematic analysis can be performed to analyze how other key genes contribute to the many-faceted events involved in reprogramming the germline.
Reprogramming Expressway
A number of other recent studies have shown the importance of Oct4 for self-renewal of undifferentiated ESCs. It is sufficient to induce pluripotency in neural tissues and somatic cells, among others. The expression of Oct4 must be tightly regulated to control cellular differentiation. But, Oct4 is much more than a simple regulator of pluripotency, according to Hans R. Schöler, Ph.D., professor in the department of cell and developmental biology at the Max Planck Institute for Molecular Biomedicine.
Oct4 has a critical role in committing pluripotent cells into the somatic cellular pathway. When embryonic stem cells overexpress Oct4, they undergo rapid differentiation and then lose their ability for pluripotency. Other studies have shown that Oct4 expression in somatic cells reprograms them for transformation into a particular germ cell layer and also gives rise to induced pluripotent stem cells (iPSCs) under specific culture conditions.
Oct4 is the gatekeeper into and out of the reprogramming expressway. By modifying experimental conditions, Oct4 plus additional factors can induce formation of iPSCs, epiblast stem cells, neural cells, or cardiac cells. Dr. Schöler suggests that Oct4 a potentially key factor not only for inducing iPSCs but also for transdifferention.  “Therapeutic applications might eventually focus less on pluripotency and more on multipotency, especially if one can dedifferentiate cells within the same lineage. Although fibroblasts are from a different germ layer, we recently showed that adding a cocktail of transcription factors induces mouse fibroblasts to directly acquire a neural stem cell identity.
Stem cell diagram illustrates a human fetus st...

Stem cell diagram illustrates a human fetus stem cell and possible uses on the circulatory, nervous, and immune systems. (Photo credit: Wikipedia)

English: Embryonic Stem Cells. (A) shows hESCs...

English: Embryonic Stem Cells. (A) shows hESCs. (B) shows neurons derived from hESCs. (Photo credit: Wikipedia)

Transforming growth factor beta (TGF-β) is a s...

Transforming growth factor beta (TGF-β) is a secreted protein that controls proliferation, cellular differentiation, and other functions in most cells. http://en.wikipedia.org/wiki/TGFbeta (Photo credit: Wikipedia)

Pioneer Transcription Factors

Pioneer transcription factors take the lead in facilitating cellular reprogramming and responses to environmental cues. Multicellular organisms consist of functionally distinct cellular types produced by differential activation of gene expression. They seek out and bind specific regulatory sequences in DNA. Even though DNA is coated with and condensed into a thick fiber of chromatin. The pioneer factor, discovered by Prof. KS Zaret at UPenn SOM in 1996, he says, endows the competence for gene activity, being among the first transcription factors to engage and pry open the target sites in chromatin.
FoxA factors, expressed in the foregut endoderm of the mouse,are necessary for induction of the liver program. They found that nearly one-third of the DNA sites bound by FoxA in the adult liver occur near silent genes

A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication

ME Hubbi, K Shitiz, DM Gilkes, S Rey,….GL Semenza. Johns Hopkins University SOM
Sci. Signal 2013; 6(262) 10pgs. [DOI: 10.1126/scisignal.2003417]   http:dx.doi.org/10.1126/scisignal.2003417

http://SciSignal.com/A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication/

Many of the cellular responses to reduced O2 availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the mini-chromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. The binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. To whom correspondence should be addressed. E-mail: gsemenza@jhmi.edu
Citation: M. E. Hubbi, Kshitiz, D. M. Gilkes, S. Rey, C. C. Wong, W. Luo, D.-H. Kim, C. V. Dang, A. Levchenko, G. L. Semenza, A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication. Sci. Signal. 6, ra10 (2013).

Identification of a Candidate Therapeutic Autophagy-inducing Peptide

Nature 2013;494(7436).    http://nature.com/Identification_of_a_candidate_therapeutic_autophagy-inducing_peptide/   http://www.ncbi.nlm.nih.gov/pubmed/23364696
http://www.readcube.com/articles/10.1038/nature11866

Beth Levine and colleagues have constructed a cell-permeable peptide derived from part of an autophagy protein called beclin 1. This peptide is a potent inducer of autophagy in mammalian cells and in vivo in mice and was effective in the clearance of several viruses including chikungunya virus, West Nile virus and HIV-1.

Could this small autophagy-inducing peptide may be effective in the prevention and treatment of human diseases?

PR-Set7 Is a Nucleosome-Specific Methyltransferase that Modifies Lysine 20 of

Histone H4 and Is Associated with Silent Chromatin

K Nishioka, JC Rice, K Sarma, H Erdjument-Bromage, …, D Reinberg.   Molecular Cell, Vol. 9, 1201–1213, June, 2002, Copyright 2002 by Cell Press   http://www.cell.com/molecular-cell/abstract/S1097-2765(02)00548-8

http://www.sciencedirect.com/science/article/pii/S1097276502005488           http://www.ncbi.nlm.nih.gov/pubmed/12086618
http://www.cienciavida.cl/publications/b46e8d324fa4aefa771c4d6ece4d2e27_PR-Set7_Is_a_Nucleosome-Specific.pdf

We have purified a human histone H4 lysine 20methyl-transferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second in-star larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions polytene chromosomes, including chromocenter euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

Next-Generation Sequencing vs. Microarrays

Shawn C. Baker, Ph.D., CSO of BlueSEQ
GEN Feb 2013
With recent advancements and a radical decline in sequencing costs, the popularity of next generation sequencing (NGS) has skyrocketed. As costs become less prohibitive and methods become simpler and more widespread, researchers are choosing NGS over microarrays for more of their genomic applications. The immense number of journal articles citing NGS technologies it looks like NGS is no longer just for the early adopters. Once thought of as cost prohibitive and technically out of reach, NGS has become a mainstream option for many laboratories, allowing researchers to generate more complete and scientifically accurate data than previously possible with microarrays.

Gene Expression

Researchers have been eager to use NGS for gene expression experiments for a detailed look at the transcriptome. Arrays suffer from fundamental ‘design bias’ —they only return results from those regions for which probes have been designed. The various RNA-Seq methods cover all aspects of the transcriptome without any a priori knowledge of it, allowing for the analysis of such things as novel transcripts, splice junctions and noncoding RNAs. Despite NGS advancements, expression arrays are still cheaper and easier when processing large numbers of samples (e.g., hundreds to thousands).
Methylation
While NGS unquestionably provides a more complete picture of the methylome, whole genome methods are still quite expensive. To reduce costs and increase throughput, some researchers are using targeted methods, which only look at a portion of the methylome. Because details of exactly how methylation impacts the genome and transcriptome are still being investigated, many researchers find a combination of NGS for discovery and microarrays for rapid profiling.

Diagnostics

They are interested in ease of use, consistent results, and regulatory approval, which microarrays offer. With NGS, there’s always the possibility of revealing something new and unexpected. Clinicians aren’t prepared for the extra information NGS offers. But the power and potential cost savings of NGS-based diagnostics is alluring, leading to their cautious adoption for certain tests such as non-invasive prenatal testing.
Cytogenetics
Perhaps the application that has made the least progress in transitioning to NGS is cytogenetics. Researchers and clinicians, who are used to using older technologies such as karyotyping, are just now starting to embrace microarrays. NGS has the potential to offer even higher resolution and a more comprehensive view of the genome, but it currently comes at a substantially higher price due to the greater sequencing depth. While dropping prices and maturing technology are causing NGS to make headway in becoming the technology of choice for a wide range of applications, the transition away from microarrays is a long and varied one. Different applications have different requirements, so researchers need to carefully weigh their options when making the choice to switch to a new technology or platform. Regardless of which technology they choose, genomic researchers have never had more options.

Sequencing Hones In on Targets

Greg Crowther, Ph.D.

GEN Feb 2013

Cliff Han, PhD, team leader at the Joint Genome Institute in the Los Alamo National Lab, was one of a number of scientists who made presentations regarding target enrichment at the “Sequencing, Finishing, and Analysis in the Future” (SFAF) conference in Santa Fe, which was co-sponsored by the Los Alamos National Laboratory and DOE Joint Genome Institute. One of the main challenges is that of target enrichment: the selective sequencing of genomic or transcriptomic regions. The polymerase chain reaction (PCR) can be considered the original target-enrichment technique and continues to be useful in contexts such as genome finishing. “One target set is the unique gaps—the gaps in the unique sequence regions. Another is to enrich the repetitive sequences…ribosomal RNA regions, which together are about 5 kb or 6 kb.” The unique-sequence gaps targeted for PCR with 40-nucleotide primers complementary to sequences adjacent to the gaps, did not yield the several-hundred-fold enrichment expected based on previously published work. “We got a maximum of 70-fold enrichment and generally in the dozens of fold of enrichment,” noted Dr. Han.

“We enrich the genome, put the enriched fragments onto the Pacific Biosciences sequencer, and sequence the repeats,” continued Dr. Han. “In many parts of the sequence there will be a unique sequence anchored at one or both ends of it, and that will help us to link these scaffolds together.” This work, while promising, will remain unpublished for now, as the Joint Genome Institute has shifted its resources to other projects.
At the SFAF conference Dr. Jones focused on going beyond basic target enrichment and described new tools for more efficient NGS research. “Hybridization methods are flexible and have multiple stop-start sites, and you can capture very large sizes, but they require library prep,” said Jennifer Carter Jones, Ph.D., a genomics field applications scientist at Agilent. “With PCR-based methods, you have to design PCR primers and you’re doing multiplexed PCR, so it’s limited in the size that you can target. But the workflow is quick because there’s no library preparation; you’re just doing PCR.” She discussed Agilent’s recently acquired HaloPlex technology, a hybrid system that includes both a hybridization step and a PCR step. Because no library preparation is required, sequencing results can be obtained in about six hours, making it suitable for clinical uses. However, the hybridization step allows capture of targets of up to 5 megabases—longer than purely PCR-based methods can deliver. The Agilent talk also provided details on the applications of SureSelect, the company’s hybridization technology, to Methyl-Seq and RNA-Seq research. With this technology, 120-mer baits hybridize to targets, then are pulled down with streptavidin-coated magnetic beads.
These are selections from the SFAF conference, which is expected to be a boost to work on the microbiome, and lead to infectious disease therapeutic approaches.

Summary

We have finished a breathtaking ride through the genomic universe in several sessions.  This has been a thorough review of genomic structure and function in cellular regulation.  The items that have been discussed and can be studied in detail include:

  1.  the classical model of the DNA structure
  2. the role of ubiquitinylation in managing cellular function and in autophagy, mitophagy, macrophagy, and protein degradation
  3. the nature of the tight folding of the chromatin in the nucleus
  4. intramolecular bonds and short distance hydrophobic and hydrophilic interactions
  5. trace metals in molecular structure
  6. nuclear to membrane interactions
  7. the importance of the Human Genome Project followed by Encode
  8. the Fractal nature of chromosome structure
  9. the oligomeric formation of short sequences and single nucletide polymorphisms (SNPs)and the potential to identify drug targets
  10. Enzymatic components of gene regulation (ligase, kinases, phosphatases)
  11. Methods of computational analysis in genomics
  12. Methods of sequencing that have become more accurate and are dropping in cost
  13. Chromatin remodeling
  14. Triplex and quadruplex models not possible to construct at the time of Watson-Crick
  15. sequencing errors
  16. propagation of errors
  17. oxidative stress and its expected and unintended effects
  18. origins of cardiovascular disease
  19. starvation and effect on protein loss
  20. ribosomal damage and repair
  21. mitochondrial damage and repair
  22. miscoding and mutational changes
  23. personalized medicine
  24. Genomics to the clinics
  25. Pharmacotherapy horizons
  26. driver mutations
  27. induced pluripotential embryonic stem cell (iPSCs)
  28. The association of key targets with disease
  29. The real possibility of moving genomic information to the bedside
  30. Requirements for the next generation of electronic health record to enable item 29

Related articles

Other Related articles on this Open Access Online Scientific Journal, include the following:

http://pharmaceuticalintelligence.com/2013/01/14/oogonial-stem-cells-purified-a-view-towards-the-future-of-reproductive-biology/   SSaha

http://pharmaceuticalintelligence.com/2012/10/22/blood-vessel-generating-stem-cells-discovered/ RSaxena

http://pharmaceuticalintelligence.com/2012/08/22/a-possible-light-by-stem-cell-therapy-in-painful-dark-of-osteoarthritis-kartogenin-a-small-molecule-differentiates-stem-cells-to-chondrocyte-healthy-cartilage-cells/   ASarkar and RSaxena

http://pharmaceuticalintelligence.com/2012/08/07/human-embryonic-pluripotent-stem-cells-and-healing-post-myocardial-infarction/    LHB

http://pharmaceuticalintelligence.com/2013/02/03/genome-wide-detection-of-single-nucleotide-and-copy-number-variation-of-a-single-human-cell/  SJWilliams

http://pharmaceuticalintelligence.com/2013/01/09/gene-therapy-into-healthy-heart-muscle-reprogramming-scar-tissue-in-damaged-hearts/ ALev-Ari

http://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/  SJWilliams

http://pharmaceuticalintelligence.com/2012/12/09/naotech-therapy-for-breast-cancer/  TBarliya

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Human embryonic pluripotent stem cells and healing post-myocardial infarction

Posted in Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Origins of Cardiovascular Disease, Pharmacotherapy of Cardiovascular Disease, Resident-cell-based, tagged , , , , , , , , , , , , , on August 7, 2012| 7 Comments »

Human embryonic pluripotent stem cells and healing post-myocardial infarction

Curator: Larry H. Bernstein, MD, FCAP
I present a followup based on several recent posts related to the promise of using induced human pluripotent stem cells for repair of ischemia damaged myocardium postinfarct and related effect of heart failure (HF).  There has been a change in the concept of cardiovascular risk related to the emergent knowledge of the biology underlying oxidative stress.  The more recent discovery of the relationship between ongoing inflammation and clinical outcomes has led to a variety of blood-based assays which may impart additional knowledge about an individual’s propensity for future cardiovascular events (1). Vascular injury and repair are significantly mediated by circulating endothelial progenitor cells (1).  Circulating progenitor endothelial cells are defined by co-expression of the markers CD34, CD309 (VEGFR-2/KDR) which are measured by pre-enrichment flow cytometry with specific identification of cell markers (CD34, CD133) and endothelial cell antigens (KDR/VEGFR-2, CD31) (2), used in the assessment of various diseases and physiological states.  Improvements in flow cytometry include the Attune® cytometer, which enables the collection of more than 4,000,000 live white blood cell (WBC) events in just 35 minutes (3). Using these methods of analyses, it became evident that circulating endothelial progenitor cells have angiogenic potential.

Activators and inhibitors have been tested for their ability to modulate angiogenesis in early phase clinical trials, and in the case of anti-Flk1 antibodies clinical utility has been demonstrated for anti-tumor strategies (4). Extending this concept further, we pose that just as the progenitor role invoked for angiogenesis, transcriptional networks and interactions are involved in the morphogenesis of the developing vertebrate heart. The identities of crucial regulators involved in defined events in cardio-genesis are being uncovered at a rapid rate. Tissue development and regeneration involve tightly coordinated and integrated processes: selective proliferation of resident stem and precursor cells, differentiation into target somatic cell type, and spatial morphological organization. (4, 5, 6). However, our ability to cross the divide between knowledge and change has not been easy, as reported by Aviva Lev-Ari (7).  In a two-day-old mouse, a heart attack causes active stem cells to grow new heart cells; a few months later, the heart is mostly repaired. But in an adult mouse, recovery from such an attack leads to classic after-effects: scar tissue, permanent loss of function and life-threatening arrhythmias (7, 8).

Myocardial cell replacement therapies are hampered by a paucity of sources for human cardiomyocytes and by the expected immune rejection of allogeneic cell grafts. The success using dermal fibroblasts from HF patients reprogrammed by retroviral delivery of Oct4, Sox2, and Klf4 or by using an excisable polycistronic lentiviral vector resulted in HF-hiPSCs induced to differentiate into cardiomyocytes (HF-hiPSC-CMs)(9). Multi-electrode array recordings revealed adequate responses to stimulation.  Further study with in vivo transplantation in the rat heart revealed the ability of the HF-hiPSC-CMs to engraft, survive, and structurally integrate with host cardiomyocytes and within 48 hours the tissues were beating together. Human-induced pluripotent stem cells thus can be established from patients with advanced heart failure and coaxed to differentiate into cardiomyocytes, which can integrate with host cardiac tissue (10).  The success of the approach rests on modifying the myocardial electro-physiological substrate using cell grafts genetically engineered to express specific ionic channels (11). The expressed potassium channels alter the local myocardial electrophysiological properties by reducing cardiac automaticity and prolonging refractoriness.  The key feature involves reprogramming a patient’s own skin cells by delivering three genes followed by a small molecule called valproic acid to the cell nucleus (12).

An alternative approach avoiding the caveats of limited graft survival, is to stimulate a resident source, restricted homing to the site of injury and host immune rejection (13). Thymosin β4 restores vascular potential to adult epicardial-derived progenitor cells with injury.  Specifically, it activates adult progenitors to re-express a key embryonic epicardial gene, Wilm’s tumour 1 (Wt1).  It was inferred that embryonic reprogramming would mobilize this cell population and differentiation would give rise to de novo cardiomyocytes. Delivery of Tβ4, in conjunction with GMT (an acronym for three genes that normally guide embryonic heart development), into the damaged region resulted in reduction of scar area and improvement in cardiac function compared to GMT or Tβ4 alone. Thymosin-beta4 facilitates cardiac repair after infarction by promoting cell migration and myocyte survival. Additionally, the tetra peptide Ac-SDKP was reported to reduce left ventricular fibrosis in hypertensive rats, reverse fibrosis and inflammation in rats with MI, and stimulate both in vitro and in vivo angiogenesis. Effects of Ac-SDKP, such as the enhancement of angiogenesis and the decrease in inflammation and collagenase activity, are similar to those described for thymosin-beta4. However, there are conflicting reports (14-18).

There are other studies that show promise.  There has been the first infusion of stem cells into the coronary artery (19). This result was at least as effective as intramyocardial injection in limiting LV remodeling and improving both regional and global LV function. The intracoronary route appears to be superior in terms of uniformity of cell distribution, myocyte regeneration, and amount of viable tissue in the risk region. Another finds that down regulation of leukocyte HIF-1? Expression resulted in decreased recruitment of WBC to the sites of inflammation and improvement in cardiac function following MI (20).  Irradiated 6-to 8-week-old C57/BL6J mice received 50 000 GFP HIF-1? or scramble siRNA transfected hematopoietic stem cells. Down regulation of HIF-1? suppressed WBC cytokine receptors CCR1,-2, and-4, which are necessary for WBC mobilization and recruitment to inflammatory cytokines following MI.  There also have been cited limitations to success in older patients (21). The findings suggest that coronary artery disease and cardiac remodeling in chronic ischemia has a significant negative correlation between the age of the patient and the number of migrated ckit-positive cells.

Lymphocytes infiltrate and react with ischemia damaged heart tissue, which can impair proper tissue healing.  In a study with isoproterenol induced myocardial necrosis TNF-α, IFN-γ and CCL-5, but not FOXP3 + expression, was increased in draining lymph nodes, indicating that the observed lymphocyte population that proliferated in response to cardiac components presented a pro-inflammatory and pro-fibrotic profile.  The group was rendered tolerant by myocardial gavage and expressed cardiac FOXP3 + earlier than did the control group, and showed a milder inflammatory infiltrate, lower MMP-9 expression, less collagen deposition, and improved cardiac performance when compared to animals that received only isoproterenol administration (22).  Patients with acute myocardial infarction show high circulating levels of neuropeptide Substance P (SP) and NK1-positive cells that co express Progenitor Cell (PC) antigen, such as CD34, KDR, and CXCR4. Moreover, NK1-expressing PC is abundant in infarcted hearts, highlighting the role of SP in reparative neovascularization (23). Do CD4 + T cells become activated and influence wound healing after experimental MI?   To study the role of CD4 + T cells in wound healing and remodeling, CD4 + T-cell- deficient mice (CD4 knockout [KO], MHCII) and T-cell receptor-transgenic OT-II. Within the infarcted myocardium, CD4 KO mice displayed higher total numbers of leukocytes and proinflammatory monocytes (18.3±3.0 104/mg WT versus 75.7±17.0 10 4/mg CD4 KO, P<0.05), and MHCII and OT-II mice displayed significantly greater mortality. Collagen matrix formation in the infarct zone was severely disturbed in CD4 KO and MHCII mice, as well as in OT-II mice (24).

Thus, it appears that CD4T cells become activated after MI and facilitate wound healing of the myocardium. Inflammation and immune responses are integral components in he healing process after myocardial infarction. Importantly, dendritic cell (DC) infiltration occurs in the infarcted heart.  In concert with the previous two studies, DC-ablated infarcts had enhanced monocyte/ macrophage recruitment. Among these cells, marked infiltration of proinflammatory Ly6C high monocytes and F4/80 + CD206 – M1 macrophages and, conversely, impaired recruitment of anti-inflammatory Ly6C low monocytes and F4/80 + CD206 + M2 macrophages in the infarcted myocardium were identified in the DC-ablated group compared with the control group (25). Thus, the DC is a potent immunoprotective regulator during the post-infarction healing process via its control of monocyte/macrophage homeostasis.  Despite the recent successes, there are a number of interlocking and possibly explanatory processes to control in the mix.

What about medical therapies?  Here too there is a factor in engaging eNOS or iNOS activity as detailed in the presentation by Aviva Lev-Ari (26).  60–70% of major cardiovascular events cannot be prevented with current approaches focused on LDL, such as statin therapy, and low HDL levels are particularly common in males with early-onset atherosclerosis.  She makes the point that there is compelling evidence that HDL is not solely a marker of lower risk of cardiovascular disease but instead is a mediator of vascular health.

Aviva Lev-Ari examines the phytoestrogen, Genistein, and other drugs. Genistein acutely stimulates Nitric Oxide synthesis in vascular Endothelial cells by a cyclic adenosine 5′-monophosphate-dependent mechanism (Liu et al., 2004). The intracellular signaling pathways for activation of eNOS by genistein were shown independent of PI3K/Akt or ERK/MAPK but depended on the cAMP/PKA cascade. In addition, the genistein action on eNOS was not inhibited by an ER antagonist and was unrelated to tyrosine kinase inhibition. Furthermore, genistein has antiatherogenic effects and inhibits proliferation of vascular endothelial and smooth muscle cells, and in vitro studies suggest a protective role of genistein in the vasculature.  In Liu et al., (2004) study, genistein acted directly on BAECs and HUVECs to activate eNOS and NO production through nongenomic mechanisms in whole vascular endothelial cells.  In addition, 5-hydroxytryptamine evokes endothelial nitric oxide synthase activation.  In this example, eNOS co-localizes with PECAM-1, but not with VE-cadherin and plakoglobin at the intercellular junctions of the endothelium.

Finally, activation of endothelial nitric oxide synthase (eNOS) resulted in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells.  The responses were effectively blocked by a 5-HT1B receptor antagonist, a 5-HT1B/5-HT2 receptor antagonist, and eNOS selective antagonists, L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO). This lends credence to a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT.  Finally, a third-generation ß-blocker augments vascular Nitric Oxide release. Nebivolol increases vascular NO productionby causing a rise in endothelial free [Ca2+]i and endothelial NO synthase–dependent NO production. It is a ß1-selective adrenergic receptor antagonist with proposed nitric oxide (NO)–mediated vasodilating properties. Nevertheless, it appears that not nebivolol, but its metabolites augment NO production (Broeders et al., 2000).  These findings reveal new insights into interaction with eNOS in vascular therapy: [1] new indications for TDZs as stimulators of eNOS; [2] new indications for beta blockers as NO stimulant. Nebivolol is a vasodilator, thus functions as an antihypertensive.

References:

1.  Saha S. Innovations in Bio-instrumentation for Measurement of Circulating Progenitor Endothelial Cells in Human Blood.  Pharma Intell. July 8, 2012. http://pharmaceuticalintelligence.com/2012/07/08/innovations-in-bio-instrumentation-for-measurement-of-circulating-progenitor-endothelial-cells-in-human-blood/

(http://www.ncbi.nlm.nih.gov/pubmed/19124422)

2.  Ibid (http://www.ncbi.nlm.nih.gov/pubmed/20381496).

3.  Ibid (http://zh.invitrogen.com/etc/medialib/files/Cell-Analysis/PDFs.Par.54318.File.tmp/CO24210-Human-CEC_cancer.pdf)

4. Saha S. Endothelial Differentiation and Morphogenesis of Cardiac Precursors. Pharma Intelligence. July 17, 2012.

5. Ibid (http://circres.ahajournals.org/content/90/5/509.full).

6. Ibid (http://www.ncbi.nlm.nih.gov/pubmed/22669846).

7.  Aviva-Lev-Ari.  Stem cells create new heart cells in baby mice, but not in adults, study shows.Aug 3, 2012. Pharma Intelligence.

8.  Krishna Ramanujan http://www.news.cornell.edu/stories/July12/HeartStemCells.html

9. Saha S. Human Embryonic-Derived Cardiac Progenitor Cells for Myocardial Repair.  Pharma Intelligence. Aug 1, 2012.

10.  Zwi-Dantsis L, Huber I, Habib M, Winterstern A, (..), Gepstein L. Derivation and cardiomyocyte differentiation of induced pluripotent stem cells from heart failure patients. Eur Heart J. 2012 May 22. [Epub ahead of print]  (VBL RX, Inc. Tel Aviv, http://www.vblrx.com).

11.  Yankelson L, Feld Y, Bressler-Stramer T, Itzhaki I,(..), Gepstein L. Cell therapy for modification of the myocardial electrophysiological substrate. Circulation. 2008 Feb 12; 117(6):720-31. Epub 2008 Jan 22.

12.  Huber I, Itzhaki I, Caspi O, Arbel G, (..), Gepstein L. Identification and selection of cardiomyocytes during human embryonic stem cell differentiation. FASEB J. 2007 Aug; 21(10):2551-63. Epub 2007 Apr 13.

13.  Aviva Lev-Ari. Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair. Pharma Intelligence. April 30, 2012.

14.  Ibid. Shrivastava S, Srivastava D, Olson EN, DiMaio JM, Bock-Marquette I.

Ann N Y Acad Sci. 2010 Apr; 1194:87-96.

15.  Ibid.  Smart N, Risebro CA, Clark JE, Ehler E, (..), Riley PR, Thymosin beta4 facilitates epicardial neovascularization of the injured adult heart.  Ann N Y Acad Sci. 2010 Apr;1194:97-104

16.   Ibid. Smart N, Bollini S, Dubé KN, Vieira JM, (..) Riley PR. Nature. 2011 Jun 8; 474(7353):640-4 

17.   Ibid. Zhou B, Honor LB, Ma Q, Oh JH, (..), Pu WT. Thymosin beta 4 treatment after myocardial infarction does not reprogram epicardial cells into cardiomyocytes.  J Mol Cell Cardiol. 2012 Jan; 52(1):43-7. Epub 2011 Aug 26.

18.   Ibid. Scientists Report that Process of Converting Non-Beating Heart Cells into Functional, Beating Heart Cells is Enhanced Using Thymosin Beta 4 in Conjunction with Gene Therapy.  Regenerx Biopharmaceuticals, Inc. Nature. Apr. 18, 2012

19.  Li, Q., Guo, Y., Ou, Q., Chen, N., (…), Bolli, R. Intracoronary administration of cardiac stem cells in mice: A new, improved technique for cell therapy in murine models.  Basic Research in Cardiology 2011; 106 (5), pp. 849-864.

20. Dong, F., Khalil, M.,Kiedrowski, M.,O’Connor, C.,(..) ,Penn, M.S. Critical role for leukocyte hypoxia inducible factor-1α expression in post-myocardial infarction left ventricular remodeling.  Circulation Research   2010; 106 (3) , pp. 601-610

21. Aghila Rani, K.G.,Jayakumar, K.,Sarma, P.S., Kartha, C.C. Clinical determinants of ckit-positive cardiac cell yield in coronary disease. Asian Cardiovascular and Thoracic Annals 2009; 17 (2), pp. 139-142.

22. Ramos, G.C., Dalbó, S., Leite, D.P.,Goldfeder, E. ,(..), Assreuy, J. The autoimmune nature of post-infarct myocardial healing: Oral tolerance to cardiac antigens as a novel strategy to improve cardiac healing. Autoimmunity 2012; 45 (3), pp. 233-244.

23.  Amadesi, S., Reni, C., Katare, R., Meloni, M., (…),Madeddu, P. Role for substance P-based nociceptive signaling in progenitor cell activation and angiogenesis during ischemia in mice and in human subjects. Circulation 2012; 125 (14) , pp. 1774-1786.

24. Hofmann, U.,Beyersdorf, N.,Weirather, J.,Podolskaya, A.(..), Frantz, S. Activation of CD4 + T lymphocytes improves wound healing and survival after experimental myocardial infarction in mice. Circulation 2012; 125 (13) , pp. 1652-1663.

25. Anzai, A., Anzai, T.,Nagai, S., Maekawa, Y., (…), Fukuda, K. Regulatory role of dendritic cells in postinfarction healing and left ventricular remodeling. Circulation 2012; 125 (10), pp. 1234-1245

26. Lev-Ari A. Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production. Pharma Intelligence. July 19, 2012.

27.  Ibid. Li AC, Binder CJ, Gutierrez A, Brown KK, (..), Glass CK. Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPAR-alpha, Beta/delta, and gamma.  J Clin Invest 2004; 114:1564-1576.

28.  Ibid. Broeders MAW, Doevendans PA, Bekkers BCAM, (…), van der Zee R. Nebivolol: A Third-Generation ß-Blocker That Augments Vascular Nitric Oxide Release, Endothelial ß2-Adrenergic Receptor–Mediated Nitric Oxide Production. Circulation 2000; 102:677.

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