David A. Ingram

Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells

From the paradigm shifting observations of Harvey, Malpighi, and van Leeuwenhoek, blood vessels have become recognized as distinct and dynamic tissue entities that merge with the heart to form a closed circulatory system.1 Vessel structures are comprised predominantly of a luminal layer of endothelial cells that is surrounded by some form of basement membrane, and mural cells (pericytes or vascular smooth muscle cells) that make up the vessel wall. In larger more complex vessel structures the vessel wall is composed of a complex interwoven matrix with nerve components. Understanding the cellular and molecular basis for the formation, remodeling, repair, and regeneration of the vasculature have been and continue to be popular areas for investigation. The endothelium has become a particularly scrutinized cell population with the recognition that these cells may play important roles in maintaining vascular homeostasis and in the pathogenesis of a variety of diseases.2 Although it has been known for several decades that some shed or extruded endothelial cells enter the circulation as apparent contaminants in the human blood stream,3 only more recent technologies have permitted the identification of not only senescent sloughed endothelial cells,4 but also endothelial progenitor cells (EPCs), which have been purported to represent a normal component of the formed elements of circulating blood5 and play roles in disease pathogenesis.6–9 Most citations refer to an article published in 1997 in which Asahara and colleagues isolated, characterized, and examined the in vivo function of putative EPCs from human peripheral blood as a major impetus for generating interest in the field.10 This seminal article presented some evidence to consider emergence of a new paradigm for the process of neovascularization in the form of postnatal vasculogenesis. Since publication of that article, interest in circulating endothelial cells, and particularly EPCs, has soared, …

Endothelial progenitor cells: identity defined?

•  Introduction •  The proof‐of‐concept in vivo: the cell, the read‐out and the animal model ‐  The CEPC: Still a putative cell ‐  Do CEPCs play an essential role in vascular (patho)physiology? ‐  The in vivo read‐out and animal model •  EPCs defined in vitro: the achilles heel in EPC biology ‐  EOCs and EC‐like cells ‐  What are potential caveats with in vitro defined cells? ‐  The search for the EOC precursor: lessons from embryonic development ‐  Do EOCs derive from an immature CEPC? ‐  Do EOCs derive from high proliferative vessel wall ECs? ‐  CEPCs and CECs: Different cells having the same identity? •  Summary

Nf1-Dependent Tumors Require a Microenvironment Containing Nf1+/−- and c-kit-Dependent Bone Marrow

Interactions between tumorigenic cells and their surrounding microenvironment are critical for tumor progression yet remain incompletely understood. Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a common genetic disorder characterized by complex tumors called neurofibromas. Genetic studies indicate that biallelic loss of Nf1 is required in the tumorigenic cell of origin in the embryonic Schwann cell lineage. However, in the physiologic state, Schwann cell loss of heterozygosity is not sufficient for neurofibroma formation and Nf1 haploinsufficiency in at least one additional nonneoplastic lineage is required for tumor progression. Here, we establish that Nf1 heterozygosity of bone marrow-derived cells in the tumor microenvironment is sufficient to allow neurofibroma progression in the context of Schwann cell Nf1 deficiency. Further, genetic or pharmacologic attenuation of c-kit signaling in Nf1+/- hematopoietic cells diminishes neurofibroma initiation and progression. Finally, these studies implicate mast cells as critical mediators of tumor initiation.

Robust functional vascular network formation in vivo by cooperation of adipose progenitor and endothelial cells.

Rapid induction and maintenance of blood flow through new vascular networks is essential for successfully treating ischemic tissues and maintaining function of engineered neo-organs. We have previously shown that human endothelial progenitor cells (EPCs) form functioning vessels in mice, but these are limited in number and persistence; and also that human adipose stromal cells (ASCs) are multipotent cells with pericytic properties which can stabilize vascular assembly in vitro. In this study, we tested whether ASCs would cooperate with EPCs to coassemble vessels in in vivo implants. Collagen implants containing EPCs, ASCs, or a 4:1 mixture of both were placed subcutaneously into NOD/SCID mice. After a range of time periods, constructs were explanted and evaluated with regard to vascular network assembly and cell fate; and heterotypic cell interactions were explored by targeted molecular perturbations. The density and complexity of vascular networks formed by the synergistic dual-cell system was many-fold higher than found in implants containing either ASCs or EPCs alone. Coimplantation of ASCs and EPCs with either pancreatic islets or adipocytes produced neoorgans populated by these parenchymal cells, as well as by chimeric human vessels conducting flow. This study is the first to demonstrate prompt and consistent assembly of a vascular network by human ASCs and endothelial cells and vascularization by these cells of parenchymal cells in implants. Mixture of these 2 readily available, nontransformed human cell types provides a practical approach to tissue engineering, therapeutic revascularization, and in vivo studies of human vasculogenesis.

Periostin Is Required for Maturation and Extracellular Matrix Stabilization of Noncardiomyocyte Lineages of the Heart

The secreted periostin protein, which marks mesenchymal cells in endocardial cushions following epithelial–mesenchymal transformation and in mature valves following remodeling, is a putative valvulogenesis target molecule. Indeed, periostin is expressed throughout cardiovascular morphogenesis and in all 4 adult mice valves (annulus and leaflets). Additionally, periostin is expressed throughout the fibrous cardiac skeleton and endocardial cushions in the developing heart but is absent from both normal and/or pathological mouse cardiomyocytes. Periostin (perilacZ) knockout mice exhibit viable valve disease, with neonatal lethality in a minority and latent disease with leaflet abnormalities in the viable majority. Surviving perilacZ-null leaflets are truncated, contain ectopic cardiomyocytes and smooth muscle, misexpress the cartilage proteoglycan aggrecan, demonstrate disorganized matrix stratification, and exhibit reduced transforming growth factor-&bgr; signaling. Neonatal perilacZ nulls that die (14%) display additional defects, including leaflet discontinuities, delamination defects, and deposition of acellular extracellular matrix. Assessment of collagen production, 3D lattice formation ability, and transforming growth factor-&bgr; responsiveness indicate periostin-deficient fibroblasts are unable to support normal valvular remodeling and establishment of a mature cardiac skeleton. Furthermore, pediatric stenotic bicuspid aortic valves that have lost normal extracellular matrix trilaminar stratification have greatly reduced periostin. This suggests that loss of periostin results in inappropriate differentiation of mesenchymal cushion cells and valvular abnormalities via a transforming growth factor-&bgr;–dependent pathway during establishment of the mature heart. Thus, perilacZ knockouts provide a new model of viable latent valve disease.

Suppression of Hepatocyte Growth Factor Production Impairs the Ability of Adipose‐Derived Stem Cells to Promote Ischemic Tissue Revascularization

The use of adipose‐derived stem/stromal cells (ASCs) for promoting repair of tissues is a promising potential therapy, but the mechanisms of their action are not fully understood. We and others previously demonstrated accelerated reperfusion and tissue salvage by ASCs in peripheral ischemia models and have shown that ASCs secrete physiologically relevant levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor. The specific contribution of HGF to ASC potency was determined by silencing HGF expression. RNA interference was used to downregulate HGF expression. A dual‐cassette lentiviral construct expressing green fluorescent protein (GFP) and either a small hairpin RNA specifically targeted to HGF mRNA (shHGF) or an inactive control sequence (shCtrl) were used to stably transduce ASCs (ASC‐shHGF and ASC‐shCtrl, respectively). Transduced ASC‐shHGF secreted >80% less HGF, which led to a reduced ability to promote survival, proliferation, and migration of mature and progenitor endothelial cells in vitro. ASC‐shHGF were also significantly impaired, compared with ASC‐shCtrl, in their ability to promote reperfusion in a mouse hindlimb ischemia model. The diminished ability of ASCs with silenced HGF to promote reperfusion of ischemic tissues was reflected by reduced densities of capillaries in reperfused tissues. In addition, fewer GFP+ cells were detected at 3 weeks in ischemic limbs of mice treated with ASC‐shHGF compared with those treated with ASC‐shCtrl. These results indicate that production of HGF is important for the potency of ASCs. This finding directly supports the emerging concept that local factor secretion by donor cells is a key element of cell‐based therapies.

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.

Early Pulmonary Vascular Disease in Preterm Infants at Risk for Bronchopulmonary Dysplasia

RATIONALE Pulmonary hypertension (PH) is associated with poor outcomes among preterm infants with bronchopulmonary dysplasia (BPD), but whether early signs of pulmonary vascular disease are associated with the subsequent development of BPD or PH at 36 weeks post-menstrual age (PMA) is unknown. OBJECTIVES To prospectively evaluate the relationship of early echocardiogram signs of pulmonary vascular disease in preterm infants to the subsequent development of BPD and late PH (at 36 wk PMA). METHODS Prospectively enrolled preterm infants with birthweights 500-1,250 g underwent echocardiogram evaluations at 7 days of age (early) and 36 weeks PMA (late). Clinical and echocardiographic data were analyzed to identify early risk factors for BPD and late PH. MEASUREMENTS AND MAIN RESULTS A total of 277 preterm infants completed echocardiogram and BPD assessments at 36 weeks PMA. The median gestational age at birth and birthweight of the infants were 27 weeks and 909 g, respectively. Early PH was identified in 42% of infants, and 14% were diagnosed with late PH. Early PH was a risk factor for increased BPD severity (relative risk, 1.12; 95% confidence interval, 1.03-1.23) and late PH (relative risk, 2.85; 95% confidence interval, 1.28-6.33). Infants with late PH had greater duration of oxygen therapy and increased mortality in the first year of life (P < 0.05). CONCLUSIONS Early pulmonary vascular disease is associated with the development of BPD and with late PH in preterm infants. Echocardiograms at 7 days of age may be a useful tool to identify infants at high risk for BPD and PH.

Premature senescence of highly proliferative endothelial progenitor cells is induced by tumor necrosis factor-α via the p38 mitogen-activated protein kinase pathway

Senescence of endothelial cells increases with systemic aging and is thought to contribute to the development of atherosclerosis. Cell therapy with highly proliferative endothelial progenitor cells (EPCs) is an emerging therapeutic option to promote endothelial regeneration, but little is known about their senescence and their vulnerability to inflammatory stressors. We therefore studied the senescence of proliferative human EPCs and investigated the effects of the proinflammatory cytokine tumor necrosis factor‐α (TNF‐α) on their senescence. Human EPCs had a significantly lower rate of senescence at baseline, compared with that of mature endothelial cells. However, EPCs up‐regulated the expression of the senescence‐associated cell cycle arrest protein p16INK4aand markedly increased measured senescence levels when exposed to chronic TNF‐α treatment. Analysis of telomere length showed that the increases in senescence were not related to changes in telomere length. Inhibition of the p38 mitogen‐activated protein kinase pathway blocked the induction of p16INK4a and cellular senescence. In conclusion, highly proliferative EPCs have a low rate of intrinsic senescence but are vulnerable to premature senescence induction by chronic proinflammatory stimulation. These findings will lead to a better understanding of physiological endothelial regeneration as well as to targeted therapies with the aim of promoting endothelial regeneration through endothelial progenitor cells.— Zhang, Y., Herbert, B.‐S., Rajashekhar, G., Ingram, D. A., Yoder, M. C., Clauss, M., Rehman. J. Premature senescence of highly proliferative endothelial progenitor cells is induced by tumor necrosis factor‐α via the p38 mitogen‐activated protein kinase pathway. FASEBJ. 23, 1358–1365 (2009)

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.