History-Introduction

Prior to 1997, endothelial progenitor cells (EPCs) were thought to exist only in embryos and were initially known as angioblasts (embryonic EPCs).  These cells were derived from extra-embryonic mesenchyme cells that differentiate into mature endothelial cells, and aid in the formation of primitive blood vessels; angiogenesis (Cogle 2004).   However in 1997, Asahara and colleagues discovered the presence of adult bone marrow-derived EPCs in human peripheral blood (Asahara 1997).  EPCs can also be found in the umbilical cord blood (Murohara 2000). 

Through research on avian embryonic development, the hierarchy of embryonic and adult EPCs proposed endothelial and hematopoietic cells are derived from one common precursor cell, the hemangioblast (His 1900, Murray 1932, Sabin 1920, Wagner 1980).  The origin of the hemangioblast is the mesoderm germ layer of epiblasts, which asymmetrically divide to form angioblasts or pluripotent hematopoietic stem cells (Cogle 2004) (Figure 1).  However, accumulating evidence indicates that hemangioblasts exist in adults (Bailey 2003, Bailey 2004, Cogle 2004, Gunsilius 2000, Loges 2004).  The identity of the EPCs in adults is an ongoing venture since CD133+ cells from mobilized peripheral blood can differentiate into hematopoietic or endothelial cells (Loges 2004).  The co-existence of EPCs and hemangioblasts in adults strongly suggest their contribution to the maintenance and repair of the vascular and hematopoietic systems.  Moreover, adult-human hemangioblasts will aid in the development of new therapies, in the treatment of disease, and to the further understanding of EPCs (Cogle 2004).

EPCs are defined as progenitor cells (monopotent), in contrast to other stem cells that are pluripotent.  Despite the monopentency of EPCs, they nonetheless possess stem cell properties, such as self-renewal.  EPCs have the ability to differentiate into mature endothelial cells and have been shown to transdifferentiate into smooth muscle cells in vitro (Frid 2002).  The vascular zone niche of the bone marrow is the main residence of EPCs. From the bone marrow, EPCs can home to other organs where they elicit neovascularization, cardiac regeneration, vessel repair and aid in tumor growth.
Phenotypically, human EPCs express CD34, CD133 (also referred as AC133 and human prominin-1), vascular endothelial growth factor receptor-2 (VEGFR-2) and KDR/ Flk-1.  (Figure 2)  Since surface markers could be downregulated, they should not be used as definitive markers of adult EPCs (Dao 2003, Hristov 2004, Zammaretti 2005).  For example, EPCs found in the peripheral blood are positive for CD133, but can also be negative for CD133, which can be a benchmark for EPC maturing into endothelial-like cells (Hristov 2003).  EPCs located in the peripheral blood are often referred to as Circulating Endothelial Cells (CECs).  It was found that CECs can be identified by specific markers in peripheral blood as CD34+, CD146+, CD105+, CD11b- (Zhang 2005).

Location

EPCs are found in the bone marrow, peripheral blood, umbilical cord blood and vessel walls.  Umbilical cord blood is a rich source of EPCs and contains high levels of CD133+ and CD34+ HSCs similar to the peripheral blood from adults (Ingram 2004) that can differentiate into endothelial cells ex vivo (Murohara 2000).  An example to show the high levels of EPC in the cord blood was made by comparing the number of endothelial cell colonies derived from the umbilical cord blood with the adult peripheral blood. The results showed that EPC-derived ECs have high levels of telomerase activity giving it the ability to replicate into secondary and tertiary colonies. This expansion is known as 100 population doublings.

Most recently, EPCs have been identified within vessel walls, specifically human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs).  The biggest finding with this discovery is that HUVECs and HAECs are considered to be mature endothelial cells without great differentiation ability (Ingram 2005).  However, Ingram and partners were able to complete 40 population doublings in vitro of HUVECs and HAECs offering insight into the clonogenic ability of these cells (Ingram 2005).

Studies have shown that EPCs can come from another source: myeloid cells.  These cells are inside the peripheral blood and have the ability to transdifferentiate into endothelial lineage. Current studies have suggested that these monocyte-derived endothelial progenitor cells seem to have a less proliferation capability than EPCs derived from the adult HSCs or cord blood.  On the other hand, these different cell types have a comparable ability to contribute most neovascularization.  It can then either be concluded that proliferation capacity is not important in vivo, or that these monocyte-derived EPCs produce growth factors to help them balance their inability to proliferation well (Hur 2004, Kalka 2000, Urbich 2003).

Despite the challenges today of improving patient health using neovascularization, it is necessary to identify and understand the use of EPC functional markers from different sources (Figure 2) that control the transformation of non-HSC derived cells to mature endothelial cells.  By identifying these growth factors in other cells, it may be possible to form mature endothelial cells when treating people with vascular diseases or acute myocardial infraction.

Homing Properties

Vasculogenesis is the process of forming new blood vessels. Various type of cells are involved in this process, including smooth muscle cells, pericyte cells, stromal cells, and adult EPCs, and act in a symbiotic, paracrine manner.  Since most of the blood vessels form during the fetal stage, embryonic vasculogenesis is more understood than adult vasculogenesis.  In adults, the rate of endothelial cell turnover is very low and the frequency of EPCs in circulating blood is also low, representing only ~0.01% of all the cells (Zammaretti 2005).

Within the bone marrow (BM) niche, the EPCs are in a quiescent state.  When the body is developing new blood vessels (such as during the developmental stage), or affected by injury, EPCs are activated and migrate into the vascular zone of the BM where proliferation is increased.  Various injuries such as ischemia, atherosclerotic lesions, traumatic wound, tumor angiogenesis (Christoph 2000, Gill 2001, Henrich 2005, Urbich 2003), and heart infarction (Kocher 2003) cause the frequency of EPCs in the peripheral blood to  increase up to 50-fold (Gill 2001).  This mechanism appears to be mediated by the upregulation of VEGF in the periphery, which plays a crucial role in vasculogenesis, angiogenesis, and EPC kinetics (Satoshi 2005).  VEGF and its receptor (VEGFR) also promote proliferation, differentiation and chemotaxis.  Other molecules that have been shown to facilitate EPC mobilization from the bone marrow to the periphery are: angiopoietin-1, fibroblast growth factor, stromal cell-derived factor-1 (SDF-1), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hydroxymethylglutaryl–coenzyme A (HMG-CoA) synthase inhibitor (Satoshi, 2005), erythropoietin (EPO) (Anagnostou 1994), peroxisome proliferator-activated receptor-gamma agonists (Wang 2004), 17ß-Estradiol (E2) together with fibroblast growth factor-2 (FGF2) (Fontaine 2006), and reactive oxygen species (ROS) such as nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) and Rac1 (Moldovan 2006). 

The upregulation of VEGF activates the endothelial nitric oxide synthase (eNOS) pathway, which is synthesized by the stromal cells.  The activation of eNOS upregulates matrix metalloproteinase-9 on EPCs (Aicher 2003).  Metalloproteinase-9 transforms membrane-bound Kit ligand into soluble Kit ligand and enables c-kit+ EPCs or hematopoietic stem cells to migrate tothe vascular region of the bone marrow (Histrov 2003).  Additionally, EPCs express protease cathepsin L (CathL) (Urbich 2005), plasminogen activators, heparinases, chymases, and tryptases (Luttun 2000, Jackson 2002, Pepper 2001).  These types of proteases are important for matrix degradation and invasion by EPCs.

VEGF has to be regulated by other mechanisms.  In one proposed mechanism, expression of TGF-ß1 (transforming growth factor-beta 1) by smooth muscle cells upregulates VEGF and the VEGF receptor (VEGFR) (Zhu 2005).  Through the presence of TGF- ß1, EPC cell adhesion is promoted with the formation of extracellular matrix (ECM) at the site of vasculogenesis.  In another mechanism, IL-8, a cytokine involved in inflammation, was shown to upregulate VEGF.  A recent murine study found IL-8 to increase the expression of vascular cell adhesion molecule-1 (VCAM-1), which promotes adhesion and homing (Editorial 2004). 

After activation, EPCs require some form of guidance mechanism to migrate and interact with the ECM at the site of vasculogenesis.  One study shows that ephB2 and ephB4 enhances SDF-1/CXCR4's signaling and chemotaxis, which orchestrates the movement of EPCs to the specific site (Ombretta 2006).  When the EPCs contact with ECM, they also interact with the integrins, including alpha-v-beta-3 and alpha-v-beta-5 integrins that are presented on the ECM (Hood 2002, Hynes 2002).  The integrins' roles in upregulation or downregulation of angiogenesis remain to be seen.  There are also additional soluble factors that may inhibit EPC vasculogenesis, such as C-reactive protein (CRP), which has been shown to be an indicator of vascular death (Verma 2004).  It was shown that the presence of CRP decreases the angiogenic potential of EPCs and upregulates EPC apoptosis by negatively affecting the endothelial nitric oxide synthase (eNOS) expression.

Positive Role of EPCs in Medicine

Potential therapeutic use of EPCs in ischemic disease is dynamic and promising. In a mouse induced ischemia model, EPCs were mobilized from the BM to the peripheral blood where they home to the target tissue via cytokine and chemokine signaling in order to promote neovascularization (Asahara 1999).  In fact, circulating EPCs may soon be a predictor of ischemic outcome.  Measuring CD34+KDR+ (kinase insert domain receptor) and CD133+ cells in a population of patients with acute myocardial function can predict their survival rate and possibly even their rate of recovery.  Their study divided such patients into three groups (Low, Medium, High) of circulating EPCs quantitatively.  Patients with the highest amount of EPCs after a major cardiac event had the best survival rate, whereas the patients with the lowest circulating EPCs had the poorest outcome (Werner 2005).   

In addition to being a predictor of ischemic outcome, recent research suggests that EPC levels in the circulation have prognostic value for individuals with hypertension.  It is theorized by van Zonneveld and Rabelink that patients at risk for cardiovascular disease have reduced levels of circulating EPCs with reduced function (van Zonneveld 2006).  Their research also cites that the prediction of a cardiovascular event may be predicted by the circulating levels of CD34+/KDR+ cells (van Zonneveld 2006).   However, a study by Guven et al suggested that the levels of EPCs are increased in patients with significant coronary artery disease (Guven 2006).  Thus, the true prognostic value of EPC level in the peripheral blood is unclear, but should offer additional insight into the importance that EPCs play in vascular health.

By promoting new blood vessel formation in ischemic tissue, severity of the disease and recovery can be lessened and improved, respectively.  Restoring blood flow as soon as possible is paramount, and the therapeutic use of EPCs can accomplish this.  One such study demonstrates not only the therapeutic potential, but also the feasibility of introducing EPCs via catherdization in a myocardial ischemic event.  Using autologous EPCs or EPCs derived from the patient are injected directly to the site of ischemia resulted in enhanced neovascularization of the myocardium of male Hsd:RH-rnu (athymic nude) rats, improving tissue repair in chronic myocardial ischemia (Kawamoto 2001).

There is also evidence of EPCs transdifferentiating into both smooth muscle cells and cardiomyocytes. This is of great clinical importance in regards to not only support vascular repair, but also cardiac regeneration (Badorff 2003). Although EPCs are not currently used in everyday clinical medicine, it is evidently clear that being able to mobilize more EPCs in the event of an ischemic event is paramount to survival and recovery. Several studies also suggest that aerobic exercise and smoking affect one’s ability to mobilize their own EPCs.  Aerobic exercise will increase this ability to mobilize EPCs and contribute to greater repair of ischemic tissue.  Smoking, however, will do just the opposite.  In fact, cigarette smoking is associated with a reduced number of EPCs together with an important impairment of EPC differentiation and functional activities (Michaud 2005).

Negative Role of EPCs in Medicine

EPCs can play a lot of negative roles in the body, and the most prominent one in being cancer-assisting cells.  Tumors need a lot of blood and nutrients in order to grow, proliferate, and metastasize.  In order to do so, they need more blood vessels, either via neoangiogenesis or angiogenesis.  Since EPCs are involved in blood vessels formation, tumors will look for ways to recruit EPCs.  Research confirms that EPCs participate in neovascularization and contribute to tumor angiogenesis (Mancuso 2001, Haruchika 2003, Takeshi 2002).  Tumors will release various cytokines and chemokines, most notably VEGF, angiopoeitin (Hattori 2001), G-CSF (Takeshi 2002), and SDF-1 (Yamaguchi 2003).  Because EPCs have receptors for these various cytokines and chemokines, including VEGFR-2 for VEGF and CXCR-4 for SDF-1, they will migrate to the region to initiate blood vessels formation.  Once they arrive at the site, EPCs attract more EPCs to the site by releasing more VEGF, HGF, G-SCF, and GM-CSF (Rehman 2003), in addition to sprouting blood vessels, forming cellular clusters, and developing functional microvasculatures (Ribatti 2004).  Thus, the vicious cycle of tumor growth, proliferation, and metastasis continues.

Malignant tumor growth results in neoplastic tissue hypoxia. This could result in the mobilization of EPCs via cytokine production and through activation of the transcription factor, hypoxia-inducible factor 1 (HIF-1) (Takahashi 1999, Manalo 2005).  EPCs have been implicated in various cancers and disorders because of their blood vessel forming property, including diabetic retinopathies, rheumatoid arthritis, psoriasis, atherosclerosis, lymphomas, hepatocellular carcinoma (Ferrara 2005), non-small cell lung cancer (Hilbe 2004), colon cancers (Gupta 2005), breast cancer (Naik 2006), infantile hemangioma (Yu 2004), and multiple myelomas (Zhang 2005). Inhibition of VEGF receptors on the EPCs will impair mobilization of EPCs to tumors, resulting in cutting off their blood supply and therefore retarding the growth of certain tumors (Ferrara 2005).  In addition, because SDF-1/CXCR-4 is involved in activation, migration, signaling, and overall act as a guidance mechanism, blocking SDF-1/CXCR-4 should limit tumor growth (Burger 2006).

Low levels of circulating EPCs have been linked to atherosclerosis, partly due to shortening of telomere in old age (Rauscher 2003), coronary artery disease (Powell 2005), cerebrovascular disease (Ghani 2005), and rheumatoid arthritis (Herbrig 2006). EPCs that have impaired proliferation, adhesion, and incorporation into vascular structures have been found in patients with type II diabetes mellitus (Tepper 2002). Another disease associated with a dysfunction in EPCs is Alzheimer’s disease.  It turns out that brain endothelial cells undergo cellular and biochemical changes, thereby releasing neurotoxic factors that contribute to neuronal cell loss in Alzheimer’s disease (Vagnucci 2003).  Statin therapy and exercise can be used together to increase the supply of healthy, functional EPCs, thereby reducing the risk of atherosclerosis and other associated diseases (Dimmeler 2001).  Risks of enhancing the number and function of EPCs include adverse vessel formation or progression of cancer.  However, the benefits of such therapies may outweigh the risks in high-risk populations.

The Future of EPCs

EPCs will become a central player in cancer therapeutics.  They provide an important functional role in neovascularization and lymphangiogenesis.  How do these EPCs contribute to neoplasm metastasis? Do EPCs contribute to leukemia or other blood disorders?  Can understanding EPCs lineage and development hold therapeutic potential for anti-angiogenesis and anti-tumorgenesis?  Research is being conducted on how EPCs are recruited to the tumor microenvironment and affect late carcinogenesis, whereby the tumor vessels promote their own growth and amplification (Spring 2005).

EPCs may also become instrumental in wound healing.  If harnessed into a successful and cost effective therapeutic agent, EPCs could speed surgical recovery time thereby decreasing rehabilitation time, hospital stay, and length of infection exposure.  Wound healing potential is attributed to growth factors and cytokines secreted by EPCs in damaged tissues that accelerate wound re-epithelization (Suh 2005).  Additionally, EPCs recruit monocytes and macrophages that are known to play a pivotal role in wound healing to injury sites (Suh 2005).

Soon after the discovery of adult EPCs, the fight against heart disease, especially ischemia, became a bright and hopeful future.  Cardiovascular disease, along with cancer, is the major cause of death in the United States of America (for more information see www.nih.gov: National Heart, Lung and Blood Institute and National Cancer Institute).  Although EPCs have had a dramatic impact on the way cardiovascular research is conducted, further understanding is required.  For example: What are their unequivocal molecular identities?  How do circulating EPCs aid endothelial cell repair, neovascularization, and cardiac regeneration?  How can all the different source-derived EPCs (i.e. umbilical cord blood-, HSC-) correlate to heart disease and tumor growth?  These types of questions are being addressed in animal models and clinical trials.  Clinical trials, essential due to the slight differences between animals and humans (i.e. surface markers and different culture conditions), will provide further insight into how EPCs will benefit both healthy and diseased patients.

The utilization of EPCs provides promising avenues for prognostic potential, genetic therapy and tissue engineering (Dzau 2005, Garmy-Susini 2005, Hristov 2004, Melo 2005, Ribatti 2005, Zammaretti 2005).  Moreover, the need for ex-vivo expansion of EPCs is critical for cell-therapeutic application and in general, regenerative medicine (Ishikawa 2004).  As of late 2006, there are 20 clinical trials conducting research on the use of EPCs according to ClinicalTrials.gov.  Research is looking into the link between EPCs and liver disease, obesity, colon cancer, kidney disease, diabetes, and breast cancer.  For the latest trials, visit:  www.clinicaltrials.gov and search for “Endothelial progenitor cells”.  

Figure 1.  Origin and fate of embryonic endothelial progenitor cells (EPCs).

Figure 2.  Hypothesized origin, fate and contributors of adult-human endothelial progenitor cells derived from bone marrow with key surface markers.  Umbilical cord blood is a source for EPCs.  Myeloid cells within peripherial blood can transdifferentiate into EPCs.  Hematopoietic stem cells (HSCs) in bone marrow can dedifferentiate to form hemangioblast.  Final maturity of EPCs is endothelial cells.  Mesenchymal stem cells, adipose tissue-derived cells, cardiac progenitor cells, and neuro-progenitor cells can all form mature endothelial cells.

Acknowledgements
  This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, UMDNJ:  Tamunotonye Briggs, Krista D. Buono, Michael Mitchel, Chimaobi Odumuko, Elisa Wolenuk, (in alphabetical order, 2005).
  Teaching Assistant:  Raghav Murthy
  Updated Fall 2006 by Jason Aptaker, Fenwick Garvey and John Le (in alphabetical order).
  Teaching Assistant (Fall 2006):  Krista Buono

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