Julie A. Mund

Flow Cytometric Identification and Functional Characterization of Immature and Mature Circulating Endothelial Cells

Objective—We sought to identify and characterize 2 distinct populations of bona fide circulating endothelial cells, including the endothelial colony-forming cell (ECFC), by polychromatic flow cytometry (PFC), colony assays, immunomagnetic selection, and electron microscopy. Methods and Results—Mononuclear cells from human umbilical cord blood and peripheral blood were analyzed using our recently published PFC protocol. A population of cells containing both ECFCs and mature circulating endothelial cells was determined by varying expressions of CD34, CD31, and CD146 but not AC133 and CD45. After immunomagnetic separation, these cells failed to form hematopoietic colonies, yet clonogenic endothelial colonies with proliferative potential were obtained, thus verifying their identity as ECFCs. The frequency of ECFCs were increased in cord blood and were extremely rare in the peripheral blood of healthy adults. We also detected another mature endothelial cell population in the circulation that was apoptotic. Finally, when comparing this new protocol with a prior method, we determined that the present protocol identifies circulating endothelial cells, whereas the earlier protocol identified extracellular vesicles. Conclusion—Two populations of circulating endothelial cells, including the functionally characterized ECFC, are now identifiable in human cord blood and peripheral blood by PFC.

Endothelial progenitor cells and cardiovascular cell-based therapies

Since their initial discovery more than a decade ago, bone marrow (BM)-derived circulating endothelial progenitor cells (EPC) have been reported to play a role in postnatal vasculogenesis through vessel regeneration and remodeling. These cells have been reported to mobilize into the blood stream in response to vascular injury, and differentiate into cells expressing a host of endothelial cell (EC) markers in vitro. Because of demonstrable regenerative capacity in animal models of human disease, EPC are thought to represent a novel treatment option for problematic cardiovascular conditions such as myocardial infarction (MI) and peripheral vascular disease (PVD). Various studies have been performed to test the clinical efficacy of EPC in patients with cardiovascular disease (CVD), including the mobilization of EPC with pharmacologic agents in patients with heart disease, and harvesting of cells from the circulation and BM for autologous reinfusion in affected patients. The outcomes of these trials have been mixed and not as robust as predicted from the animal models, partly because of the variation in the definition of human EPC and the resulting heterogeneity in cell populations used in the treatments. This review will decipher a number of published studies that have been conducted to examine cell therapies for treatment of CVD, will attempt to explain why efficacy of treatment with putative EPC has been inconsistent, and predict which aspects of these trials may need to be redesigned for future successful treatment of CVD.

Application of polychromatic flow cytometry to identify novel subsets of circulating cells with angiogenic potential

Defining whether human circulating proangiogenic cells represent a subset of the hematopoietic system and express CD45 or are hematopoietic derivatives that do not express CD45 (and are called endothelial progenitor cells) remains controversial. We have previously developed a polychromatic flow cytometry (PFC) protocol to isolate subsets of hematopoietic cells and we now identify the circulating pool of CD34+CD45dim cells representing functional circulating hematopoietic stem and progenitor cells (CHSPCs) that can be separated on the basis of AC133 expression and report that the AC133+ subset of the CHSPCs enhances the growth of tumor blood vessels in vivo in immunodeficient mice. In addition, the ratio of AC133+ proangiogenic CHSPCs to AC133− nonangiogenic CHSPCs unambiguously correlates with the severity of the clinical state of patients with peripheral arterial disease. In sum, a PFC protocol validated via in vitro and in vivo analyses, can be used to interrogate the roles of human hematopoietic elements in the growth and maintenance of the vasculature.

Identification of Endothelial Cells and Progenitor Cell Subsets in Human Peripheral Blood

An assay for circulating cell subsets in human peripheral blood by flow cytometry is used as a biomarker to determine cardiovascular disease risk and tumor responsiveness to chemotherapy since endothelial progenitor cells (EPCs) function in vasculogenesis and angiogenesis. Despite analytical advances in polychromatic flow cytometry (PFC), conventional approaches are routinely utilized to enumerate and isolate EPCs, which has led to varied results in clinical studies, potential cellular misidentification, and thus a lack of a plausible biological explanation for how purported EPCs function. Herein, a reproducible PFC protocol is provided to identify a rare circulating endothelial colony‐forming cell (ECFC) with proliferative potential, along with a population of circulating progenitor cells (CPCs) in which the ratio analysis distinguishes between healthy and disease populations. In sum, a reliable PFC protocol, which can be used to investigate the roles of human hematopoietic and endothelial elements in the growth and maintenance of the vasculature, is described. Curr. Protoc. Cytom. 52:9.33.1‐9.33.11.

Endothelial Abnormalities in Adolescents with Type 1 Diabetes: A Biomarker for Vascular Sequelae?

OBJECTIVE To evaluate whether counts of circulating colony forming unit-endothelial cells (CFU-ECs), cells co-expressing CD34, CD133, and CD31 (CD34+CD133+CD31+), and CD34+CD45- cells are altered in adolescents with type 1 diabetes and if the changes in counts correlate with endothelial dysfunction. STUDY DESIGN Adolescents with diabetes (ages 18 to 22 years) and race- and sex-matched control subjects were studied. We assessed circulating CFU-ECs, using colony assays, and CD34+CD133+CD31+ and CD34+CD45- cells, using poly-chromatic flow cytometry. CFU-ECs and CD34+CD133+CD31+ are hematopoietic-derived progenitors that inversely correlate with cardiovascular risk in adults. CD34+CD45- cells are enriched for endothelial cells with robust vasculogenic potential. Vascular reactivity was tested by laser Doppler iontophoresis. RESULTS Subjects with diabetes had lower CD34+CD133+CD31+ cells, a trend toward reduced CFU-ECs, and increased CD34+CD45- cells compared with control subjects. Endothelium-dependent vasodilation was impaired in subjects with diabetes, which correlated with reductions in circulating CD34+CD133+CD31+ cells. CONCLUSIONS Long-term sequelae of type 1 diabetes include vasculopathies. Endothelial progenitor cells promote vascular health by facilitating endothelial integrity and function. Lower CD34+CD133+CD31+ cells may be a harbinger of future macrovascular disease risk. Higher circulating CD34+CD45- cells may reflect ongoing endothelial damage. These cells are potential biomarkers to guide therapeutic interventions to enhance endothelial function and to prevent progression to overt vascular disease.

The ontogeny of endothelial progenitor cells through flow cytometry

Purpose of reviewSince the discovery of endothelial progenitor cells (EPCs), there have been conflicting reports as to the precise phenotypic identity, and thus an accurate description of the function of these cells in disease pathology is lacking. This review will detail the protocols that have been published within 2010 to help decipher the true identity of the various cells that have been reported as EPCs in numerous clinical trials. Recent findingsThroughout 2010, three protocols have been published alleging to identify EPCs, yet only one provides a true nonhematopoietic origin for a cell that is classified as an EPC. In addition to the protocols published to try to establish a consensus definition, 10 studies involving EPCs across disease pathologies were published with various degrees of correlation to disease phenotype and cellular level. SummaryA true phenotypic definition of a circulating EPC capable of becoming an endothelial colony forming cell with proliferative potential has been given. It is now time the EPC field drops this ambiguous term (i.e. EPCs), as many studies purporting to measure EPCs are in fact still quantifying cells of a hematopoietic origin. It is necessary for cross study comparisons that a uniform phenotypic definition be adhered to when using the term EPC.

Polychromatic flow cytometry identifies novel subsets of circulating cells with angiogenic potential in pediatric solid tumors

Pediatric solid tumors depend upon angiogenesis for their growth and metastases. A new polychromatic flow cytometry (PFC) protocol has revealed circulating cells of hematopoietic and endothelial lineages from the peripheral blood (PB) of healthy individuals, and has defined the different cell types involved in the growth of tumor vasculature that are critical in angiogenesis.

Gestational diabetes induces alterations in the function of neonatal endothelial colony-forming cells.

Background:Children born to mothers with gestational diabetes mellitus (GDM) experience increased risk of developing hypertension, type 2 diabetes mellitus, and obesity. Disrupted function of endothelial colony-forming cells (ECFCs) may contribute to this enhanced risk. The goal of this study was to determine whether cord blood ECFCs from GDM pregnancies exhibit altered functionality.Methods:ECFCs isolated from the cord blood of control and GDM pregnancies were assessed for proliferation, senescence, and Matrigel network formation. The requirement for p38MAPK in hyperglycemia-induced senescence was determined using inhibition and overexpression studies.Results:GDM-exposed ECFCs were more proliferative than control ECFCs. However, GDM-exposed ECFCs exhibited decreased network-forming ability in Matrigel. Aging of ECFCs by serial passaging led to increased senescence and reduced proliferation of GDM-exposed ECFCs. ECFCs from GDM pregnancies were resistant to hyperglycemia-induced senescence compared with those from controls. In response to hyperglycemia, control ECFCs activated p38MAPK, which was required for hyperglycemia-induced senescence. In contrast, GDM-exposed ECFCs showed no change in p38MAPK activation under equivalent conditions.Conclusion:Intrauterine exposure of ECFCs to GDM induces unique phenotypic alterations. The resistance of GDM-exposed ECFCs to hyperglycemia-induced senescence and decreased p38MAPK activation suggest that these progenitor cells have undergone changes that induce tolerance to a hyperglycemic environment.

Synthesis and mechanistic studies of a novel homoisoflavanone inhibitor of endothelial cell growth.

Preventing pathological ocular angiogenesis is key to treating retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. At present there is no small molecule drug on the market to target this process and hence there is a pressing need for developing novel small molecules that can replace or complement the present surgical and biologic therapies for these neovascular eye diseases. Previously, an antiangiogenic homoisoflavanone was isolated from the bulb of a medicinal orchid, Cremastra appendiculata. In this study, we present the synthesis of a novel homoisoflavanone isomer of this compound. Our compound, SH-11052, has antiproliferative activity against human umbilical vein endothelial cells, and also against more ocular disease-relevant human retinal microvascular endothelial cells (HRECs). Tube formation and cell cycle progression of HRECs were inhibited by SH-11052, but the compound did not induce apoptosis at effective concentrations. SH-11052 also decreased TNF-α induced p38 MAPK phosphorylation in these cells. Intriguingly, SH-11052 blocked TNF-α induced IκB-α degradation, and therefore decreased NF-κB nuclear translocation. It decreased the expression of NF-κB target genes and the pro-angiogenic or pro-inflammatory markers VCAM-1, CCL2, IL8, and PTGS2. In addition SH-11052 inhibited VEGF induced activation of Akt but not VEGF receptor autophosphorylation. Based on these results we propose that SH-11052 inhibits inflammation induced angiogenesis by blocking both TNF-α and VEGF mediated pathways, two major pathways involved in pathological angiogenesis. Synthesis of this novel homoisoflavanone opens the door to structure-activity relationship studies of this class of compound and further evaluation of its mechanism and potential to complement existing antiangiogenic drugs.

Neurofibromin-deficient myeloid cells are critical mediators of aneurysm formation in vivo

Background— Neurofibromatosis type 1 (NF1) is a genetic disorder resulting from mutations in the NF1 tumor suppressor gene. Neurofibromin, the protein product of NF1, functions as a negative regulator of Ras activity in circulating hematopoietic and vascular wall cells, which are critical for maintaining vessel wall homeostasis. NF1 patients have evidence of chronic inflammation resulting in the development of premature cardiovascular disease, including arterial aneurysms, which may manifest as sudden death. However, the molecular pathogenesis of NF1 aneurysm formation is unknown. Method and Results— With the use of an angiotensin II–induced aneurysm model, we demonstrate that heterozygous inactivation of Nf1 (Nf1+/–) enhanced aneurysm formation with myeloid cell infiltration and increased oxidative stress in the vessel wall. Using lineage-restricted transgenic mice, we show that loss of a single Nf1 allele in myeloid cells is sufficient to recapitulate the Nf1+/– aneurysm phenotype in vivo. Finally, oral administration of simvastatin or the antioxidant apocynin reduced aneurysm formation in Nf1+/– mice. Conclusion— These data provide genetic and pharmacological evidence that Nf1+/– myeloid cells are the cellular triggers for aneurysm formation in a novel model of NF1 vasculopathy and provide a potential therapeutic target.