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Posts Tagged ‘cell function correlation’


Brain Biobank and studies of disease structure correlates

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Unveiling Psychiatric Diseases

Researchers create neuropsychiatric cellular biobank

Image: iStock/mstroz
Image: iStock/mstroz
Researchers from Harvard Medical School and Massachusetts General Hospital have completed the first stage of an important collaboration aimed at understanding the intricate variables of neuropsychiatric disease—something that currently eludes clinicians and scientists.

The research team, led by Isaac Kohane at HMS and Roy Perlis at Mass General, has created a neuropsychiatric cellular biobank—one of the largest in the world.

It contains induced pluripotent stem cells, or iPSCs, derived from skin cells taken from 100 people with neuropsychiatric diseases such as schizophrenia, bipolar disorder and major depression, and from 50 people without neuropsychiatric illness.

In addition, a detailed profile of each patient, obtained from hours of in-person assessment as well as from electronic medical records, is matched to each cell sample.

As a result, the scientific community can now for the first time access cells representing a broad swath of neuropsychiatric illness. This enables researchers to correlate molecular data with clinical information in areas such as variability of drug reactions between patients. The ultimate goal is to help treat, with greater precision, conditions that often elude effective management.

The cell collection and generation was led by investigators at Mass General, who in collaboration with Kohane and his team are working to characterize the cell lines at a molecular level. The cell repository, funded by the National Institutes of Health, is housed at Rutgers University.

“This biobank, in its current form, is only the beginning,” said Perlis, director of the MGH Psychiatry Center for Experimental Drugs and Diagnostics and HMS associate professor of psychiatry. “By next year we’ll have cells from a total of four hundred patients, with additional clinical detail and additional cell types that we will share with investigators.”

A current major limitation to understanding brain diseases is the inability to access brain biopsies on living patients. As a result, researchers typically study blood cells from patients or examine post-mortem tissue. This is in stark contrast with diseases such as cancer, for which there are many existing repositories of highly characterized cells from patients.

The new biobank offers a way to push beyond this limitation.

 

A Big Step Forward

While the biobank is already a boon to the scientific community, researchers at MGH and the HMS Department of Biomedical Informatics will be adding additional layers of molecular data to all of the cell samples. This information will include whole genome sequencing and transcriptomic and epigenetic profiling of brain cells made from the stem cell lines.

Collaborators in the HMS Department of Neurobiology, led by Michael Greenberg, department chair and Nathan Marsh Pusey Professor of Neurobiology,  will also work to examine characteristics of other types of neurons derived from these stem cells.

“This can potentially alter the entire way we look at and diagnose many neuropsychiatric conditions,” said Perlis.

One example may be to understand how the cellular responses to medication correspond to the patient’s documented responses, comparing in vitro with in vivo. “This would be a big step forward in bringing precision medicine to psychiatry,” Perlis said.

“It’s important to recall that in the field of genomics, we didn’t find interesting connections to disease until we had large enough samples to really investigate these complex conditions,” said Kohane, chair of the HMS Department of Biomedical Informatics.

“Our hypothesis is that here we will require far fewer patients,” he said. “By measuring the molecular functioning of the cells of each patient rather than only their genetic risk, and combining that all that’s known of these people in terms of treatment response and cognitive function, we will discover a great deal of valuable information about these conditions.”

Added Perlis, “In the early days of genetics, there were frequent false positives because we were studying so few people. We’re hoping to avoid the same problem in making cellular models, by ensuring that we have a sufficient number of cell lines to be confident in reporting differences between patient groups.”

The generation of stem cell lines and characterization of patients and brain cell lines is funded jointly by the the National Institute of Mental Health, the National Human Genome Research Institute and a grant from the Centers of Excellence in Genomic Science program.

 

On C.T.E. and Athletes, Science Remains in Its Infancy

Se Hoon ChoiYoung Hye KimMatthias Hebisch, et al.

http://www.nature.com/articles/nature13800.epdf

Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles1. The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau2, 3. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology4, 5. Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles6, 7, 8, 9, 10, 11. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

 

 

Figure 2: Robust increases of extracellular amyloid-β deposits in 3D-differentiated hNPCs with FAD mutations.close

Robust increases of extracellular amyloid-[bgr] deposits in 3D-differentiated hNPCs with FAD mutations.

a, Thin-layer 3D culture protocol. HC, histochemistry; IF, immunofluorescence; IHC, immunohistochemistry. b, Amyloid-β deposits in 6-week differentiated control and FAD ReN cells in 3D Matrigel (green, GFP; blue, 3D6; scale bar, …

 

Stem Cell-Based Spinal Cord Repair Enables Robust Corticospinal Regeneration

 

Novel use of EPR spectroscopy to study in vivo protein structure

http://www.news-medical.net/whitepaper/20160315/Novel-use-of-EPR-spectroscopy-to-study-in-vivo-protein-structure.aspx

α-synuclein

α-synuclein is a protein found abundantly throughout the brain. It is present mainly at the neuron ends where it is thought to play a role in ensuring the supply of synaptic vesicles in presynaptic terminals, which are required for the release of neurotransmitters to relay signals between neurons. It is critical for normal brain function.

However, α-synuclein is also the primary protein component of the cerebral amyloid deposits characteristic of Parkinson’s disease and its precursor is found in the amyloid plaques of Alzheimer’s disease. Although α-synuclein is present in all areas of the brain, these disease-state amyloid plaques only arise in distinct areas.

Alpha-synuclein protein. May play role in Parkinson’s and Alzheimer’s disease.  © molekuul.be / Shutterstock.com

Imaging of isolated samples of α-synuclein in vitro indicate that it does not have the precise 3D folded structure usually associated with proteins. It is therefore classed as an intrinsically disordered protein. However, it was not known whether the protein also lacked a precise structure in vivo.

There have been reports that it can form helical tetramers. Since the 3D structure of a biological protein is usually precisely matched to the specific function it performs, knowing the structure of α-synuclein within a living cell will help elucidate its role and may also improve understanding of the disease states with which it is associated.

If α-synuclein remains disordered in vivo, it may be possible for the protein to achieve different structures, and have different properties, depending on its surroundings.

Techniques for determining protein structure

It has long been known that elucidating the structure of a protein at an atomic level is fundamental for understanding its normal function and behavior. Furthermore, such knowledge can also facilitate the development of targeted drug treatments. Unfortunately, observing the atomic structure of a protein in vivo is not straightforward.

X-ray diffraction is the technique usually adopted for visualizing structures at atomic resolution, but this requires crystals of the molecule to be produced and this cannot be done without separating the molecules of interest from their natural environment. Such processes can modify the protein from its usual state and, particularly with complex structures, such effects are difficult to predict.

The development of nuclear magnetic resonance (NMR) spectroscopy improved the situation by making it possible for molecules to be analyzed under in vivo conditions, i.e. same pH, temperature and ionic concentration.

More recently, increases in the sensitivity of NMR and the use of isotope labelling have enabled determinations of the atomic level structure and dynamics of proteins to be determined within living cells1. NMR has been used to determine the structure of a bacterial protein within living cells2 but it is difficult to achieve sufficient quantities of the required protein within mammalian cells and to keep the cells alive for NMR imaging to be conducted.

Electron paramagnetic resonance (EPR) spectroscopy for determining protein structure

Recently, researchers have managed to overcome these obstacles by using in-cell NMR and electron paramagnetic resonance (EPR) spectroscopy. EPR spectroscopy is a technique that is similar to NMR spectroscopy in that it is based on the measurement and interpretation of the energy differences between excited and relaxed molecular states.

In EPR spectroscopy it is electrons that are excited, whereas in NMR signals are created through the spinning of atomic nuclei. EPR was developed to measure radicals and metal complexes, but has also been utilized to study the dynamic organization of lipids in biological membranes3.

EPR has now been used for the first time in protein structure investigations and has provided atomic-resolution information on the structure of α-synuclein in living mammalians4,5.

Bacterial forms of the α-synuclein protein labelled with 15N isotopes were introduced into five types of mammalian cell using electroporation. Concentrations of α-synuclein close to those found in vivo were achieved and the 15N isotopes allowed the protein to be clearly defined from other cellular components by NMR. The conformation of the protein was then determined using electron paramagnetic resonance (EPR).

The results showed that within living mammalian cells α-synuclein remains as a disordered and highly dynamic monomer. Different intracellular environments did not induce major conformational changes.

Summary

The novel use of EPR spectroscopy has resolved the mystery surrounding the in vivo conformation of α-synuclein. It showed that α-synuclein maintains its disordered monomeric form under physiological cell conditions. It has been demonstrated for the first time that even in crowded intracellular environments α-synuclein does not form oligomers, showing that intrinsic structural disorder can be sustained within mammalian cells.

References

  1. Freedberg DI and Selenko P. Live cell NMR Annu. Rev. Biophys. 2014;43:171–192.
  2. Sakakibara D, et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 2009;458:102–105.
  3. Yashroy RC. Magnetic resonance studies of dynamic organisation of lipids in chloroplast membranes. Journal of Biosciences 1990;15(4):281.
  4. Alderson TA and Bax AD. Parkinson’s Disease. Disorder in the court. Nature 2016; doi:10.1038/nature16871.
  5. Theillet FX, et al. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 2016; doi:10.1038/nature16531.

 

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Circulating Endothelial Progenitors Cells (cEPCs) as Biomarkers

Article Curator: Larry H. Bernstein, MD, FCAP

and

Topic Curator: Aviva Lev-Ari, PhD, RN

Circulating progenitor cells have gained much interest rapidly in the past year primarily in identification of damaged tissue that has turnover of cells that are identifiable in the circulation.  This has to require a sensitivity for identification at one or two logs lower than circulating hematopoietic cells.  I mention this untested view only because cells of the circulation are detected routinely by automated hematology instruments like those of Beckman-Coulter and Siemens, with graphical presentation of results.  The Sysmex also reports immature granulocytes that are a small percent of the neutrophil count.  In the evaluation of leukemias, flow cytometry has been used for years, but require a preparative step.  Cell types have been identified by acidic and basic dye stains to identify basophilic, acidophilic and neutrophilic granulocyte series, and by size of the cell population, and nuclear features, differentiating mature and nucleated red cells, the granulocyte series, monocytes and lymphocytes, as well as platelets (aggregation gives an underestimate of platelet count).  But to detect cancer cells or damaged endothelial cells, the number of cells in the circulation requires and antibody to the surface with a visualizable ligand attached to an antibody for identification.  Visualization could be by a fluorophor, or perhaps a luciferase reaction.  Here are two articles that identify circulating endothelial cells, making them suitable for biomarkers of cardiovascular injury.  Whether they can detect early predictive ischemia, or frank AMI needs investigation.  The concept of piecemeal necrosis in the heart may be applicable to cardiomyocyte injury that is found unexpectedly at autopsy as “silent infarct”.

Circulating endothelial progenitors–cells as biomarkers

Rosenzweig, Anthony
N Engl J Med. 2005 Sep 8;353(10):1055-7

Comment on

Circulating endothelial progenitor cells and cardiovascular outcomes

[N Engl J Med. 2005]  PMID: 16148292 [PubMed – indexed for MEDLINE]

Endothelial injury and dysfunction are thought to be critical events in the  pathogenesis of atherosclerosis. Thus,

  • understanding the mechanisms that  maintain and restore endothelial function
    • may have important clinical  implications.

A series of clinical and basic studies prompted by the discovery 

  • of bone marrow derived endothelial progenitor cells1 have
  • provided insights into these processes and
    • opened a door to the development of new therapeutic approaches.

Growing evidence suggests that bone marrow derived endothelial progenitor cells circulate in the blood and

  • play an important role in the formation of new blood vessels as well as
  • contribute to vascular homeostasis in the adult.

Circulating endothelial progenitor cells were initially identified

  • through their expression of CD34
    (a surface marker common to hematopoietic stem cells and mature endothelial cells)
  • and vascular endothelial cell growth-factor receptor 2
    (VEGFR2 or kinase-domain related [KDR] receptor),

but not of other markers seen on fully differentiated endothelial cells.1

Subsequent studies have also used other identifiers, such as

  • the stem-cell marker CD133, and
  • functional assays, including
    • the ability to form endothelial colonies.

Endothelial progenitor cells defined in these ways probably represent

  • a heterogeneous population, which,
  • in combination with the lack of a consensual definition,

complicates the interpretation of work in this field.

Nevertheless, numerous studies in animals have shown that endothelial  progenitor cells can integrate into new and existing blood vessels.2,3,4
Intravenous injection of cytokine-mobilized human endothelial progenitor cells

  • improved myocardial neoangiogenesis and
  • the recovery of functioning in a rat model of infarction.3

Repeated injection of bone marrow derived cells in a mouse model of atherosclerosis

  • reduced the rate of plaque formation without altering serum lipids levels, and
  • donor endothelial progenitor cells could subsequently be identified in the recipient’s blood vessels.4

Previous clinical studies have shown that

  • traditional risk factors for coronary atherosclerosis
  • are associated with low levels of circulating endothelial progenitor cells,5 whereas
  • protective interventions, including statin therapy6 and exercise,7
    • appear to increase the supply of these cells.

Hill et al. found that even in healthy volunteers,

  • levels of endothelial progenitor cells were inversely correlated with the Framingham risk score and
  • actually appeared to predict vascular function better than the Framingham risk score.5

Together, these data suggest that circulating endothelial progenitor cells may participate

  • not only in forming new blood vessels
  • but also in maintaining the integrity and function of vascular  endothelium,

thereby mitigating disease processes such as atherosclerosis.

In this issue of the Journal, Werner and colleagues have further advanced our understanding of the clinical implications of endothelial progenitor cells.8 Endothelial progenitor cells were quantitated in 519 patients with coronary artery disease who

  • were followed for one year after undergoing catheterization.

Patients with higher levels of endothelial progenitor cells had

  • a reduced risk of death from cardiovascular causes and of
  • the composite end point of major cardiovascular events.

These relationships were preserved even

  • after adjustment for traditional risk factors and prognostic variables.

A similar relationship was seen

  • whether endothelial progenitor cells were  identified by virtue of expression
    either of CD34 and KDR or of CD133 or
  • because of their ability to form endothelial colonies,

further strengthening the authors’ conclusions. Repeated catheterization was not performed in this  cohort, so

  • we do not know whether the reduction in clinical events reflected a slowed progression of atherosclerosis or some other clinical effect.

A  dissociation between anatomical measures of atherosclerosis and clinical events has been well documented in other settings.

Although this study is consistent with prior work suggesting that circulating endothelial progenitor cells may play a protective role in vascular homeostasis, other explanations

  • for the association between endothelial progenitor number and outcome remain possible.

Changes in the number of endothelial progenitor cells and

  • in clinical events might reflect a common underlying etiology,
      • rather than a causal relation.

For example, a defect in the production of nitric oxide, which plays an important role

  • in both the mobilization of endothelial progenitor cells9 and blood-vessel function, might account for both observations.

Similarly, the number of endothelial progenitor cells

  • may mirror a person’s regenerative capacity more broadly and
  • predict clinical events on that basis.

Even if endothelial progenitor cells are mechanistically linked to clinical cardiovascular events,

  • such clinical studies do not distinguish between the possibility
  • that the protection is mediated through the integration of endothelial  progenitor cells into blood vessels and

its possible mediation by other  mechanisms, such as the

  • paracrine benefits of endothelial progenitor  cell secreted products.

Although such questions will undoubtedly continue to provide fertile ground  for fundamental investigation,

  • the report by Werner and colleagues has more  immediate clinical implications.

First, it suggests that circulating cell  populations may represent a new class of biomarkers

  • that naturally integrate  diverse genetic and environmental effects,
  • thereby providing robust  physiological and prognostic insights.

Second, in the context of coronary  disease, the study shows that

  • the number of endothelial progenitor cells is an independent predictor of hard clinical outcomes.

As with other biomarkers, a demonstration of clinical usefulness will ultimately require

  • the examination of other patient populations, as well as
  • a demonstration that clinical therapy can be guided and enhanced by this information.

Finally, the increased risk associated with reduced levels of endothelial progenitor cells

  • supports the growing interest in the therapeutic potential of enhancing the level of these cells.

The most dramatic extension of this line of reasoning involves

transferring  bone marrow or peripheral blood cells that are likely to include endothelial  progenitor cells to patients with coronary artery disease. Although it would be premature to judge the clinical success of these strategies, early trials, including one randomized (though incompletely blinded) trial, have suggested

  • at least short-term functional benefits of intracoronary infusion of bone marrow cells after acute infarction.10

Trials are planned to address more definitively the potential benefits of such cells

  • in the settings of acute infarction and chronic ischemic cardiomyopathy.

Such efforts would be aided substantially by the identification of specific markers as well as

  • an improved understanding of the role of subtypes of endothelial progenitor cells and
  • of the mechanisms by which they work.

Ironically, the data presented by Werner and colleagues in combination with work showing

  • the impaired functioning of endothelial progenitor cells in high-risk patients5 suggest
  • that the patients most in need of endothelial progenitor cells may be
      • those who are least able to donate them for autologous transplantation.

Whether these limitations can be overcome through

  • ex vivo expansion or  genetic modification of endothelial progenitor cells is unclear.

In addition to possible cell-based therapies, work on endothelial progenitor cells provides yet another rationale

  • for redoubling efforts to comply with established therapeutic guidelines,
  • including lifestyle modifications and the use of statin therapy,
      • both of which appear to enhance the number of circulating endothelial progenitor cells.

Whether there will be a downside to enhancing the number and function of  endothelial progenitor cells remains unclear,

  • although obvious concerns  include exacerbating conditions that are characterized by adverse vessel  formation,
    • such as diabetic retinopathy and tumor angiogenesis.

Small studies have suggested an association between high levels of circulating endothelial progenitor cells and the risk of certain cancers, such as multiple myeloma.11 Moreover, studies in animals show that

  • bone marrow derived endothelial progenitors participate in tumor angiogenesis, thereby
      • enhancing tumor growth.12

In the study by Werner and colleagues,

  • the number of deaths from cardiovascular causes among patients with high levels of endothelial progenitor cells
  • was substantially lower than that among patients with lower levels of these cells,
  • without a reduction in the risk of death overall.8

Although this finding could raise the specter of a counterbalancing adverse effect of endothelial progenitor cells,

  • there was no apparent pattern in the deaths due to other causes,
  • and no deaths from cancer were noted in this population.

It is possible that as we learn more about the biology of endothelial progenitor cells, there may be opportunities

  • to target vessel formation more specifically.

In addition, therapeutic strategies

  • tailored to individualized risk will undoubtedly help in practice.

For example, in the study by Werner et al.,

  • patients in the group with the lowest baseline levels of endothelial progenitor cells
  • had a risk of death from cardiovascular causes of 8.3 percent during one year of follow-up,
  • suggesting that the benefits of enhancing the function and number of endothelial progenitor cells
      • may well outweigh the risks in such high-risk populations.

Additional studies will be necessary to address these questions definitively. Larger studies

  • of longer duration performed in different cohorts will be required to determine fully
    • the clinical usefulness of endothelial progenitor cells as a biomarker.

Rigorous interventional studies will indicate

  • whether levels of endothelial progenitor cells can be used to guide therapy and
  • whether cell transfer has a role in augmenting the levels of these cells.

Basic-science studies should help guide these clinical efforts by

  • further defining the desirable subpopulations of endothelial progenitor cells and
  • the mechanisms by which they mediate their effects.

By establishing a connection between circulating endothelial progenitor cells and hard clinical end points, Werner and colleagues

  • provide a potent stimulus for clinical and basic studies to address these important issues.

Source Information

From the Program in Cardiovascular Gene Therapy, Massachusetts General  Hospital, and Harvard Medical School ― both in Boston.

References

Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor  endothelial cells for angiogenesis. Science 1997;275:964-967.

Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced  mobilization of bone marrow-derived endothelial progenitor cells for  neovascularization. Nat Med 1999;5:434-438.

Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001; 7: 430-436.

Rauscher FM, Goldschmidt-Clermont PJ, Davis BH, et al. Aging, progenitor cell exhaustion, and atherosclerosis. Circulation 2003; 108: 457-463.

Hill JM, Zalos G, Halcox JPJ, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348:593-600.

Vasa M, Fichtlscherer S, Adler K, et al. Increase in circulating endothelial  progenitor cells by statin therapy in patients with stable coronary artery  disease. Circulation 2001; 103: 2885-2890.

Laufs U, Werner N, Link A, et al. Physical training increases endothelial  progenitor cells, inhibits neointima formation, and enhances angiogenesis.  Circulation 2004; 109: 220-226.

Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 2005; 353: 999-1007.

Aicher A, Heeschen C, Mildner-Rihm C, et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med  2003; 9: 1370-1376.

Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow  cell transfer after myocardial infarction: the BOOST randomised controlled  clinical trial. Lancet 2004; 364: 141-148.

Zhang H, Vakil V, Braunstein M, et al. Circulating endothelial progenitor cells in multiple myeloma: implications and significance. Blood 2005; 105: 3286-3294.

Lyden D, Hattori K, Dias S, et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 2001;7:1194-1201.

Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction.

Kelly Bethel, Madelyn S Luttgen, Samir Damani, Anand Kolatkar, Rachelle Lamy, Mohsen Sabouri-Ghomi, Sarah Topol, Eric J Topol, Peter Kuhn

Physical Biology (Impact Factor: 2.62). 01/2014; 11(1):016002. http://dx.doi.org/10.1088/1478-3975/11/1/016002
Source: PubMed

Elevated levels of circulating endothelial cells (CECs) occur in response to various pathological conditions including myocardial infarction (MI). Here, we adapted

  • a fluid phase biopsy technology platform that successfully detects circulating tumor cells in the blood of cancer patients (HD-CTC assay),
  • to create a high-definition circulating endothelial cell (HD-CEC) assay for the detection and characterization of CECs.

Peripheral blood samples were collected from 79 MI patients, 25 healthy controls and six patients undergoing vascular surgery (VS). CECs were defined

  • by positive staining for DAPI, CD146 and von Willebrand Factor
  • and negative staining for CD45.

In addition, CECs exhibited distinct morphological features that

  • enable differentiation from surrounding white blood cells.
  1. CECs were found both as individual cells and as aggregates.
  2. CEC numbers were higher in MI patients compared with healthy controls.
  3. VS patients had lower CEC counts when compared with MI patients

but were not different from healthy controls.

Both HD-CEC and CellSearch® assays could discriminate

  • MI patients from healthy controls with comparable accuracy

but the HD-CEC assay exhibited

  • higher specificity while maintaining high sensitivity.

Our HD-CEC assay may be used as a robust diagnostic biomarker in MI patients.

MicroRNA function in endothelial cells

Solving the mystery of an unknown target gene using microRNA Target Site Blockers
Dr. Virginie Mattot
Dr. Virgine Mattot works in the team “Angiogenesis, endothelium activation and Cancer” directed by Dr. Fabrice Soncin at the Institut de Biologie de Lille in France where she studies the roles played by microRNAs in endothelial cells during physiological and pathological processes such as angiogenesis or endothelium activation. She has been using Target Site Blockers to investigate the role of microRNAs on putative targets which functions are yet unknown.
What is the main focus of the research conducted in your lab?
We are studying endothelial cell functions with a particular interest
  • in angiogenesis and endothelium activation during physiological and tumoral vascular development.
How did your research lead to the study of microRNAs?
A few years ago, we identified in my team
  • a new endothelial cell-specific gene which harbors a microRNA in its intronic sequence.

We have since been working on understanding

  • the functions of both this new gene and
  • its intronic microRNA in endothelial cells

What is the aim of your current project?

While we were searching for the functions of the intronic microRNA,
  • we identified an unknown gene as a putative target.
The aim of my project was to investigate if this unknown gene was actually a genuine target and
  • if regulation of this gene by the microRNA was involved in endothelial cell function.
We had already characterized the endothelial cell phenotype associated with the inhibition of our intronic microRNA.
We then used miRCURY LNA™ Target Site Blockers to demonstrate
  • that the expression of this unknown gene is actually controlled by this microRNA.
Further, we also demonstrated that the microRNA regulates
  • specific endothelial cell properties through regulation of this unknown gene.
How did you perform the experiments and analyze the results?
LNA™ enhanced target site blockers (TSB) for our microRNA were designed by Exiqon.
We transfected the TSBs into endothelial cells using our standard procedure and
  • analysed the induced phenotype.
As a control for these experiments, a mutated version of the TSB was designed by Exiqon and
  • transfected into endothelial cells.
We first verified that this TSB was functional by
  • analyzing the expression of the miRNA target
      • against which the TSB was directed in transfected cells.
Finally, we showed that the TSB induced similar phenotypes as those found when we inhibited the microRNA in the same cells. 
What were some specific challenges in your experiments and how did you overcome them?
The fact that the target gene for our microRNA was unknown was a major challenge. Without specific available tools, like antibodies,
  • it becomes difficult to demonstrate the effect of the microRNA on the gene in question and
  • to show that the unknown gene is indeed responsible for the functions of the microRNA.
However through the use of specific target site blockers, we were able to demonstrate
  • that this unknown gene was associated with the phenotype observed
    • when the microRNA was inhibited in endothelial cells.
How do you feel about your results so far?
We are very pleased with the results of the TSB experiments and
  • altogether these results demonstrate that our miRNA of interest
  • is functional in endothelial cells
    • through the regulation of a target gene with a previously unknown role.
What do you find to be the main benefits/advantage of the LNA™ microRNA target site blockers from Exiqon?
Target Site Blockers are efficient tools to demonstrate the

  • specific involvement of putative microRNA targets
  • in the function played by this microRNA.
The use of LNA™ allows the design of short oligonucleotides that are very specific and easy to work with. 
What would be your advice to colleagues about getting started with microRNA functional analysis?
In order to address the role played by a microRNA,
  • it is essential to perform both gain and loss of functions experiments.
What are the next steps in the current project and how do you plan to perform them?
We plan to use microRNA inhibitor libraries to identify
  • more microRNAs specifically involved in the processes that we currently study.
When and where will be hear /read more about your studies?
We are currently in the process of submitting a manuscript regarding the function of my microRNA of interest.

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