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Circulating Endothelial Progenitors Cells (cEPCs) as Biomarkers

Posted in Atherogenic Processes & Pathology, Best evidence, Biomarkers & Medical Diagnostics, Biomedical Measurement Science, Bone marrow derived cells, Cardiovascular Research, Chemical Genetics, Circulating Progenitor Cells, Curation, Cytoskeleton, Diagnostic Immunology, Discovery process, Endothelial cells, Experimental validation, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Genomic Testing: Methodology for Diagnosis, Immunodiagnostics, Interviews with Scientific Leaders, Metabolomics, Nitric Oxide in Health and Disease, Nutrition, Origins of Cardiovascular Disease, Population Health Management, Nutrition and Phytochemistry, Proteomics, Technology Advance Assessment of, Translational Effectiveness, Translational Research, Translational Science, Umbilical cord cells, tagged CEC subtypes, cell function correlation, cell identification by labelling, Circulating Progenitor Endothelial Cells, endothelial cell, Endothelial progenitor cell, increased number of CECs, ischemic dardiac disease, myocardial infarction, target cell blockers on January 14, 2014| Leave a Comment »

Circulating Endothelial Progenitors Cells (cEPCs) as Biomarkers

Article Curator: Larry H. Bernstein, MD, FCAP

and

Topic Curator: Aviva Lev-Ari, PhD, RN

Article ID #105: Circulating Endothelial Progenitors Cells (cEPCs) as Biomarkers. Published on 1/14/2014

WordCloud Image Produced by Adam Tubman

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.

CECs were found both as individual cells and as aggregates.
CEC numbers were higher in MI patients compared with healthy controls.
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