Laura E. Mead

In Vitro Hyperglycemia or a Diabetic Intrauterine Environment Reduces Neonatal Endothelial Colony-Forming Cell Numbers and Function

OBJECTIVE—Emerging data demonstrate that maternal diabetes has long-term health consequences for offspring, including the development of hypertension. In adults, circulating endothelial progenitor cells (EPCs) participate in vascular repair, and EPC numbers and function inversely correlate with the risk of developing vascular disease. Therefore, our objectives were to determine whether hyperglycemia or exposure to a diabetic intrauterine environment alters EPC function. RESEARCH DESIGN AND METHODS—We used well-established clonogenic endothelial colony-forming cell (ECFC) assays and murine transplantation experiments to examine human vasculogenesis. RESULTS—Both in vitro hyperglycemia and a diabetic intrauterine environment reduced ECFC colony formation, self-renewal capacity, and capillary-like tube formation in matrigel. This cellular phenotype was linked to premature senescence and reduced proliferation. Further, cord blood ECFCs from diabetic pregnancies formed fewer chimeric vessels de novo after transplantation into immunodeficient mice compared with neonatal ECFCs harvested from uncomplicated pregnancies. CONCLUSIONS—Collectively, these data demonstrate that hyperglycemia or exposure to a diabetic intrauterine environment diminishes neonatal ECFC function both in vitro and in vivo, providing potential mechanistic insights into the long-term cardiovascular complications observed in newborns of diabetic pregnancies.

Clonogenic Endothelial Progenitor Cells Are Sensitive to Oxidative Stress

Endothelial progenitor cells (EPCs) circulate in the peripheral blood and reside in blood vessel walls. A hierarchy of EPCs exists where progenitors can be discriminated based on their clonogenic potential. EPCs are exposed to oxidative stress during vascular injury as residents of blood vessel walls or as circulating cells homing to sites of neovascularization. Given the links between oxidative injury, endothelial cell dysfunction, and vascular disease, we tested whether EPCs were sensitive to oxidative stress using newly developed clonogenic assays. Strikingly, in contrast to previous reports, we demonstrate that the most proliferative EPCs (high proliferative potential‐endothelial colony‐forming cells and low proliferative potential‐endothelial colony‐forming cells) had decreased clonogenic capacity after oxidant treatment. In addition, EPCs exhibited increased apoptosis and diminished tube‐forming ability in vitro and in vivo in response to oxidative stress, which was directly linked to activation of a redox‐dependent stress‐induced kinase pathway. Thus, this study provides novel insights into the effect of oxidative stress on EPCs. Furthermore, this report outlines a framework for understanding how oxidative injury leads to vascular disease and potentially limits the efficacy of transplantation of EPCs into ischemic tissues enriched for reactive oxygen species and oxidized metabolites.

Isolation and Characterization of Endothelial Progenitor Cells from Human Blood

Circulating endothelial progenitor cells (EPCs) in adult human peripheral blood were originally identified in 1997 by Asahara et al., which challenged the paradigm that vasculogenesis is a process restricted to embryonic development. Since their original identification, EPCs have been extensively studied as biomarkers to assess the risk of cardiovascular disease in human subjects and as a potential cell therapeutic for vascular regeneration. Endothelial colony-forming cells (ECFCs), which are a subtype of EPCs, were recently identified from circulating adult and human umbilical cord blood. In contrast to other types of EPCs, which display various monocyte/macrophage phenotypes and functions, ECFCs are characterized by robust proliferative potential, secondary and tertiary colony formation upon replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In this unit, we describe detailed methodologies for isolation and characterization of ECFCs from both human peripheral and umbilical cord blood.

Endothelial colony forming cells and mesenchymal stem cells are enriched at different gestational ages in human umbilical cord blood.

: Endothelial progenitor cells (EPCs) are used for angiogenic therapies and as biomarkers of cardiovascular disease. Human umbilical cord blood (UCB) is a rich source of endothelial colony forming cells (ECFCs), which are EPCs with robust proliferative potential that may be useful for clinical vascular regeneration. Previous studies show that hematopoietic progenitor cells are increased in premature UCB compared with term controls. Based on this paradigm, we hypothesized that premature UCB would be an enriched source of ECFCs. Thirty-nine UCB samples were obtained from premature infants (24–37 wk gestational age (GA)) and term controls. ECFC colonies were enumerated, clonally isolated, and identified by expression of endothelial cell surface antigens and functional analysis. GA of 33–36 wk UCB yielded predominantly ECFC colonies at equivalent numbers to term infants. UCB from 24 to 28 wk GA infants had significantly fewer ECFCs. Surprisingly, 24–28 wk GA UCB yielded predominantly mesenchymal stem cell (MSC) colonies, capable of differentiating into adipocytes, chondrocytes, and osteocytes. MSCs were rarely identified in 37–40 wk GA UCB. These studies demonstrate that circulating MSCs and ECFCs appear at different GA in the human UCB, and that 24–28 wk GA UCB may be a novel source of MSCs for therapeutic use in human diseases.

Cord Blood Stem and Progenitor Cells

Cord blood has served as a source of hematopoietic stem and progenitor cells for successful repopulation of the blood cell system in patients with malignant and nonmalignant disorders. It was information on these rare immature cells in cord blood that led to the first use of cord blood for transplantation. Further information on these cells and how they can be manipulated both in vitro and in vivo will likely enhance the utility and broadness of applicability of cord blood for treatment of human disease. This chapter reviews information on the clinical and biological properties of hematopoietic stem and progenitor cells, as well as the biology of endothelial progenitor cells, and serves as a source for the methods used to detect and quantitate these important functional cells. Specifically, methods are presented for enumerating human cord blood myeloid progenitor cells, including granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM or CFU-Mix) progenitors, and their replating potential; hematopoietic stem cells, as assessed in vitro for long-term culture-initiating cells (LTC-ICs), cobblestone area-forming cells (CAFCs), and myeloid-lymphoid-initiating cells (ML-ICs), and as assessed in vivo for nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mouse repopulating cells (SRCs); and high and low proliferative potential endothelial progenitor cells (EPCs).

Nf1+/− mice have increased neointima formation via hyperactivation of a Gleevec sensitive molecular pathway

Neurofibromatosis type I (NF1) is a genetic disorder caused by mutations in the NF1 tumor suppressor gene. Neurofibromin is encoded by NF1 and functions as a negative regulator of Ras activity. Somatic mutations in the residual normal NF1 allele within cancers of NF1 patients is consistent with NF1 functioning as a tumor-suppressor. However, the prevalent non-malignant manifestations of NF1, including learning and bone disorders emphasize the importance of dissecting the cellular and biochemical effects of NF1 haploinsufficiency in multiple cell lineages. One of the least studied complications of NF1 involves cardiovascular disorders, including arterial occlusions that result in cerebral and visceral infarcts. NF1 vasculopathy is characterized by vascular smooth muscle cell (VSMC) accumulation in the intima area of vessels resulting in lumen occlusion. We recently showed that Nf1 haploinsufficiency increases VSMC proliferation and migration via hyperactivation of the Ras-Erk pathway, which is a signaling axis directly linked to neointima formation in diverse animal models of vasculopathy. Given this observation, we tested whether heterozygosity of Nf1 would lead to vaso-occlusive disease in genetically engineered mice in vivo. Strikingly, Nf1+/- mice have increased neointima formation, excessive vessel wall cell proliferation and Erk activation after vascular injury in vivo. Further, this effect is directly dependent on a Gleevec sensitive molecular pathway. Therefore, these studies establish an Nf1 model of vasculopathy, which mirrors features of human NF1 vaso-occlusive disease, identifies a potential therapeutic target and provides a platform to further dissect the effect of Nf1 haploinsufficiency in cardiovascular disease.

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.

Production of the endocannabinoids anandamide and 2-arachidonoylglycerol by endothelial progenitor cells

Recent studies have highlighted the importance of paracrine growth factors as mediators of pro‐angiogenic effects by endothelial progenitor cells (EPCs), but little is known about the release of lipid‐based factors like endocannabinoids by EPCs. In the current study, the release of the endocannabinoids anandamide and 2‐arachidonoylglycerol by distinct human EPC sub‐types was measured using HPLC/tandem mass‐spectrometry. Anandamide release was highest by adult blood colony‐forming EPCs at baseline and they also demonstrated increased 2‐arachidonoylglycerol release with TNF‐α stimulation. Treatment of mature endothelial cells with endocannabinoids significantly reduced the induction of the pro‐inflammatory adhesion molecule CD106 (VCAM‐1) by TNF‐α.

Distinct contribution of human cord blood‐derived endothelial colony forming cells to liver and gut in a fetal sheep model

Although the vasculogenic potential of circulating and cord blood (CB)‐derived endothelial colony‐forming cells (ECFC) has been demonstrated in vitro and in vivo, little is known about the inherent biologic ability of these cells to home to different organs and contribute to tissue‐specific cell populations. Here we used a fetal sheep model of in utero transplantation to investigate and compare the intrinsic ability of human CB‐derived ECFC to migrate to the liver and to the intestine, and to define ECFCs intrinsic ability to integrate and contribute to the cytoarchitecture of these same organs. ECFCs were transplanted by an intraperitoneal or intrahepatic route (IH) into fetal sheep at concentrations ranging from 1.1‐2.6 × 106 cells/fetus. Recipients were evaluated at 85 days posttransplant for donor (human) cells using flow cytometry and confocal microscopy. We found that, regardless of the route of injection, and despite the IH delivery of ECFC, the overall liver engraftment was low, but a significant percentage of cells were located in the perivascular regions and retained the expression of hallmark endothelial makers. By contrast, ECFC migrated preferentially to the intestinal crypt region and contributed significantly to the myofibroblast population. Furthermore, ECFC expressing CD133 and CD117 lodged in areas where endogenous cells expressed those same phenotypes. Conclusion: ECFC inherently constitute a potential source of cells for the treatment of intestinal diseases, but strategies to increase the numbers of ECFC persisting within the hepatic parenchyma are needed in order to enhance ECFC therapeutic potential for this organ. (HEPATOLOGY 2012;56:1086–1096)

15 The Effects of Hyperglycemia on Newborn Endothelial Progenitor Cells.

Purpose of Study: Diabetes Mellitus (DM) is the most prevalent risk factor for acquiring vascular disease. Since optimal glycemic control delays onset and progression of vascular morbidities, hyperglycemia is hypothesized to be a key pathogenic factor for vasculopathies. Vascular integrity requires endothelial repair and angiogenesis. Recently, circulating endothelial progenitor cells (EPCs) were shown to have a critical role in promoting endothelial repair and angiogenesis. Given that infants exposed to a diabetic intrauterine environment have an increased risk of DM and vascular disease, we hypothesize that hyperglycemia alters neonatal EPC function. Thus, our aims were to examine the effect of hyperglycemia on newborn EPC clonogenic capacity, proliferative potential, apoptosis, senescence, and capillary tube formation.Methods Used: EPCs were isolated from multiple cord blood donors as described (Ingram et al, Blood, 104:2752, 2004) and subjected to euglycemia or hyperglycemia (10-100 mM dextrose) before conducting cellular and functional assays. Limiting dilution assays assessed EPC colony formation. Single cell assays quantitated the proportion of single EPCs capable of dividing. Proliferation potential was also assessed by population doubling studies. Apoptosis was evaluated using a TUNEL assay while senescence was examined using an acidic β-galactosidase method. Matrigel assays assessed capillary tube forming ability.Summary of Results: Hyperglycemia (10-100 mM) resulted in reduced EPC colony formation compared to controls (n=3, p<0.05). Hyperglycemia led to decreased EPC proliferative potential as reflected by a decrease in the number of single EPC cells dividing (n=96 single cells) and a lower cumulative population doubling level. In addition, increased apoptosis was detected after a 24 or 48 hour exposure to high dextrose concentrations (50mM and 100mM) compared to controls (n=3, p< 0.003). Senescence assays are underway, and preliminary matrigel assays suggest that hyperglycemia treated EPCs exhibit reduced capillary tube formation.Conclusion: These data demonstrate that neonatal EPCs have decreased clonogenic potential after hyperglycemia treatment. The mechanisms for the observed decrease in clonogenic capacity may include an increase in both apoptosis and senescence together with a decrease in proliferation. Collectively, these observations may have implications for the pathogenesis of vascular disease in diabetic patients as well as in infants exposed to a diabetic intrauterine environment.