Reporter: Sudipta Saha, Ph.D.
Assessment of the propensity for vascular events has been based on measurement of risk factors predisposing one to vascular injury. These assessments are based on the strong associations between risk factors such as hypertension, cholesterol levels, smoking, and diabetes which were first described almost a half century ago. 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. Vascular health is now better represented as a balance between ongoing injury and resultant vascular repair, mediated at least in part by circulating endothelial progenitor cells (http://www.ncbi.nlm.nih.gov/pubmed/19124422). Accurate enumeration of circulating endothelial progenitor cells is essential for their potential application as biomarkers of angiogenesis. Different stem cell markers (CD34, CD133) and endothelial cell antigens (KDR/VEGFR-2, CD31) in different flow cytometric protocols are assessed for the purpose of circulating progenitor endothelial cell quantification (http://www.ncbi.nlm.nih.gov/pubmed/20381496). Enumeration of circulating progenitor endothelial cells are used in the assessment of various diseases and physiological states, such as: type 2 diabetes patients with peripheral vascular disease, certain phases during congestive heart failure, acute myocardial infarction, atherosclerosis, cardiovascular disease, physical training, cessation of smoking. Two modern instruments used now-a-days to measure the circulating progenitor endothelial cells are discussed below:
MACSQuant® Analyzer:
Circulating progenitor endothelial cells are defined by co-expression of the markers CD34, CD309 (VEGFR-2/KDR), and CD133, though CD133 expression is lost during maturation to endothelial cells.8-10 Since circulating progenitor endothelial cells are rare in peripheral blood, EPC enumeration protocols are rather extensive and laborious. To obtain reliable enumeration results for these rare cells, the sensitivity of flow cytometric analysis needs to be increased. This has been achieved by magnetic enrichment of circulating progenitor endothelial cells prior to flow cytometric analysis, which reduces the number of events that have to be analyzed. The circulating progenitor endothelial cell Enrichment and Enumeration Kit have been designed for enumeration of circulating progenitor endothelial cells from peripheral blood, cord blood, bone marrow, or leukapheresis products. In combination with magnetic pre-enrichment and flow cytometric analysis on the MACSQuant® Analyzer, this kit overcomes some of the limitations of circulating progenitor endothelial cell analysis and offers a simple and time effective solution for EPC enumeration. The circulating progenitor endothelial cell Enrichment and Enumeration Kit in combination with pre-enrichment and flow cytometric analysis on the MACSQuant Analyzer is an effective method to enumerate circulating progenitor endothelial cells in 10 mL of whole blood. Based on the calculated starting number of cells, the circulating progenitor endothelial cell Express Mode analysis template automatically calculates the absolute number and concentration of circulating progenitor endothelial cells in 10 mL of starting material, i.e., whole blood, bone marrow, cord blood, or leukapheresis products. The MACSQuant Analyzer has the ability to enrich cells using MACS technology. This capability makes the enumeration of circulating progenitor endothelial cells fast and easy. The entire process takes less than 2 hours to perform from blood draw to analyzed data and drastically reduces the time and difficulty of such a protocol by combining magnetic enrichment and flow cytometric analysis in one streamlined experiment (http://www.miltenyibiotec.com/downloads/6760/6764/18602/31184/MQ_ApplicationFlyer_EPC.pdf).
Attune® Acoustic Focusing Cytometer:
In cancer research, circulating progenitor endothelial cells have been suggested as a noninvasive biomarker for angiogenic activity, providing insight into tumor regrowth, resistance to chemotherapy, early recurrence, and metastasis during or after chemotherapy. In healthy individuals, circulating progenitor endothelial cells are reported to be present in very low numbers: 0.01%–0.0001% of all peripheral blood mononuclear cells. Flow cytometry offers the necessary collection and analysis capabilities for detection of circulating progenitor endothelial cells, but is subject to numerous technical challenges. In comparison to traditional hydrodynamic focusing cytometers, the Attune® Acoustic Focusing Cytometer, with its fast acquisition times and increased precision, overcomes the technological hurdles involved in analyzing circulating progenitor endothelial cells. The method includes a number of conventional ways to improve rare-event detection: a blocking step, a viability stain (SYTOX® AADvanced™ Dead Cell Stain), and the use of a dump channel to eliminate unwanted cells and decrease background fluorescence. The challenge of collecting a large enough number of events in a reasonable amount of time is met by using a collection rate of 1,000 μL/min with the Attune® cytometer. This setting enables the collection of more than 4,000,000 live white blood cell (WBC) events in just 35 minutes; the acquisition time using a traditional hydrodynamic focusing cytometer would be 10–12 times longer, close to 6 hours. Furthermore, this method delivers additional time savings by eliminating wash steps to avoid sample loss and employing a simpler sample preparation method. (http://zh.invitrogen.com/etc/medialib/files/Cell-Analysis/PDFs.Par.54318.File.tmp/CO24210-Human-CEC_cancer.pdf)
Dr. Saha,
Thank you for this important post. In addition to cEPC relevance to cardiovascular disease, as you write, “In cancer research, circulating progenitor endothelial cells have been suggested as a noninvasive biomarker for angiogenic activity, providing insight into tumor regrowth, resistance to chemotherapy, early recurrence, and metastasis during or after chemotherapy.”
This post will become part of a series of posts on cEPC. Please see the following post which provided just the bibliography of a post of mine which will be out next week. If in this list you find ONE paper of interest or more than one, for you to continue BUILDING domain knowledge on this very important topic, then please, go ahead and define a post based on excerpts from that list.
Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk http://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk-2/
Please see all the Categories of Research I have checked on for this post and the tags
Thanks for the comments ……
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This is very insightful. There is no doubt that there is the bias you refer to. 42 years ago, when I was postdocing in biochemistry/enzymology before completing my residency in pathology, I knew that there were very influential mambers of the faculty, who also had large programs, and attracted exceptional students. My mentor, it was said (although he was a great writer), could draft a project on toilet paper and call the NIH. It can’t be true, but it was a time in our history preceding a great explosion. It is bizarre for me to read now about eNOS and iNOS, and about CaMKII-á, â, ã, ä – isoenzymes. They were overlooked during the search for the genome, so intermediary metabolism took a back seat. But the work on protein conformation, and on the mechanism of action of enzymes and ligand and coenzyme was just out there, and became more important with the research on signaling pathways. The work on the mechanism of pyridine nucleotide isoenzymes preceded the work by Burton Sobel on the MB isoenzyme in heart. The Vietnam War cut into the funding, and it has actually declined linearly since.
A few years later, I was an Associate Professor at a new Medical School and I submitted a proposal that was reviewed by the Chairman of Pharmacology, who was a former Director of NSF. He thought it was good enough. I was a pathologist and it went to a Biochemistry Review Committee. It was approved, but not funded. The verdict was that I would not be able to carry out the studies needed, and they would have approached it differently. A thousand young investigators are out there now with similar letters. I was told that the Department Chairmen have to build up their faculty. It’s harder now than then. So I filed for and received 3 patents based on my work at the suggestion of my brother-in-law. When I took it to Boehringer-Mannheim, they were actually clueless.