Endothelial Progenitor Cells in Breast Cancer

Editorial

Identifying the molecular phenotype of renal progenitor cells

Although many of the molecular interactions in kidney development are now well understood, the molecules involved in the specification of the metanephric mesenchyme from surrounding intermediate mesoderm and, hence, the formation of the renal progenitor population are poorly characterized. In this study, cDNA microarrays were used to identify genes enriched in the murine embryonic day 10.5 (E10.5) uninduced metanephric mesenchyme, the renal progenitor population, in comparison with more rostral derivatives of the intermediate mesoderm. Microarray data were analyzed using R statistical software to determine accurately genes differentially expressed between these populations. Microarray outliers were biologically verified, and the spatial expression pattern of these genes at E10.5 and subsequent stages of early kidney development was determined by RNA in situ hybridization. This approach identified 21 genes preferentially expressed by the E10.5 metanephric mesenchyme, including Ewing sarcoma homolog, 14-3-3 theta, retinoic acid receptor-alpha, stearoyl-CoA desaturase 2, CD24, and cadherin-11, that may be important in formation of renal progenitor cells. Cell surface proteins such as CD24 and cadherin-11 that were strongly and specifically expressed in the uninduced metanephric mesenchyme and mark the renal progenitor population may prove useful in the purification of renal progenitor cells by FACS. These findings may assist in the isolation and characterization of potential renal stem cells for use in cellular therapies for kidney disease.

Functional maturation of isolated neural progenitor cells from the adult rat hippocampus

Although neural progenitor cells (NPCs) may provide a source of new neurons to alleviate neural trauma, little is known about their electrical properties as they differentiate. We have previously shown that single NPCs from the adult rat hippocampus can be cloned in the presence of heparan sulphate chains purified from the hippocampus, and that these cells can be pushed into a proliferative phenotype with the mitogen FGF2 [Chipperfield, H., Bedi, K.S., Cool, S.M. & Nurcombe, V. (2002) Int. J. Dev. Biol., 46, 661-670]. In this study, the active and passive electrical properties of both undifferentiated and differentiated adult hippocampal NPCs, from 0 to 12 days in vitro as single-cell preparations, were investigated. Sparsely plated, undifferentiated NPCs had a resting membrane potential of approximate to -90 mV and were electrically inexcitable. In > 70%, ATP and benzoylbenzoyl-ATP evoked an inward current and membrane depolarization, whereas acetylcholine, noradrenaline, glutamate and GABA had no detectable effect. In Fura-2-loaded undifferentiated NPCs, ATP and benzoylbenzoyl-ATP evoked a transient increase in the intracellular free Ca2+ concentration, which was dependent on extracellular Ca2+ and was inhibited reversibly by pyridoxalphosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS), a P2 receptor antagonist. After differentiation, NPC-derived neurons became electrically excitable, expressing voltage-dependent TTX-sensitive Na+ channels, low- and high-voltage-activated Ca2+ channels and delayed-rectifier K+ channels. Differentiated cells also possessed functional glutamate, GABA, glycine and purinergic (P2X) receptors. Appearance of voltage-dependent and ligand-gated ion channels appears to be an important early step in the differentiation of NPCs.

Fibrosis correlates with a ductular reaction in hepatitis C: Roles of impaired replication, progenitor cells and steatosis

The mechanisms for progressive fibrosis and exacerbation by steatosis in patients with chronic hepatitis C (HCV) are still unknown. We hypothesized that proliferative blockade in HCV-infected and steatotic hepatocytes results in the default activation of hepatic progenitor cells (HPC), capable of differentiating into both biliary and hepatocyte lineages, and that the resultant ductular reaction promotes portal fibrosis. To study this concept, 115 liver biopsy specimens from subjects with HCV were scored for steatosis, inflammation, and fibrosis. Biliary epithelium and HPC were decorated by cytokeratin 7 immunoperoxidase, and the replicative state of hepatocytes was assessed by p21 and Ki-67 immunohistochemistry. A ductular reaction at the portal interface was common. There was a highly significant correlation between the area of ductular reaction and fibrosis stage (r = 0.453, P < .0001), which remained independently associated after multivariate analysis. HPC numbers also correlated with fibrosis (r = 0.544, P < .0001) and the ductular area (r = 0.624, P < .0001). Moreover, steatosis correlated with greater HPC proliferation (r = 0.372, P = .0004) and ductular reaction (r = 0.374, P < .0001) but was not an obligate feature. Impaired hepatocyte replication by p21 expression was independently associated with HPC expansion (P = .002) and increased with the body mass index (P < .001) and lobular inflammation (P = .005). In conclusion, the strong correlation between portal fibrosis and a periportal ductular reaction with HPC expansion, the exacerbation by steatosis, and the associations with impaired hepatocyte replication suggest that an altered regeneration pathway drives the ductular reaction. We believe this triggers fibrosis at the portal tract interface. This may be a stereotyped response of importance in other chronic liver diseases.

The adult mouse hippocampal progenitor is neurogenic but not a stem cell

The aim of this investigation was to characterize the proliferative precursor cells in the adult mouse hippocampal region. Given that a very large number of new hippocampal cells are generated over the lifetime of an animal, it is predicted that a neural stem cell is ultimately responsible for maintaining this genesis. Although it is generally accepted that a proliferative precursor resides within the hippocampus, contradictory reports exist regarding the classification of this cell. Is it a true stem cell or a more limited progenitor? Using a strict functional definition of a neural stem cell and a number of in vitro assays, we report that the resident hippocampal precursor is a progenitor capable of proliferation and multipotential differentiation but is unable to self-renew and thus proliferate indefinitely. Furthermore, the mitogen FGF-2 stimulates proliferation of these cells to a greater extent than epidermal growth factor ( EGF). In addition, we found that BDNF was essential for the production of neurons from the hippocampal progenitor cells, being required during proliferation to trigger neuronal fate. In contrast, a bona fide neural stem cell was identified in the lateral wall of the lateral ventricle surrounding the hippocampus. Interestingly, EGF proved to be the stronger mitogenic factor for this cell, which was clearly a different precursor from the resident hippocampal progenitor. These results suggest that the stem cell ultimately responsible for adult hippocampal neurogenesis resides outside the hippocampus, producing progenitor cells that migrate into the neurogenic zones and proliferate to produce new neurons and glia.

Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hernatopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen 1, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis. (c) 2005 Elsevier Inc. All rights reserved.

Characterization of neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain

Many studies have demonstrated a role for netrin-1-deleted in colorectal cancer (DCC) interactions in both axon guidance and neuronal migration. Neogenin, a member of the DCC receptor family, has recently been shown to be a chemorepulsive axon guidance receptor for the repulsive guidance molecule (RGM) family of guidance cues [Rajagopalan S, Deitinghoff L, Davis D, Conrad S, Skutella T, Chedotal A, Mueller B, Strittmatter S (2004) Neogenin mediates the action of repulsive guidance molecule. Nat Cell Biol 6:755-762]. Here we show that neogenin is present on neural progenitors, including neurogenic radial glia, in the embryonic mouse forebrain suggesting that neogenin expression is a hallmark of neural progenitor populations. Neogenin-positive progenitors were isolated from embryonic day 14.5 forebrain using flow cytometry and cultured as neurospheres. Neogenin-positive progenitors gave rise to neurospheres displaying a high proliferative and neurogenic potential. In contrast, neogenin-negative forebrain cells did not produce long-term neurosphere cultures and did not possess a significant neurogenic potential. These observations argue strongly for a role for neogenin in neural progenitor biology. In addition, we also observed neogenin on parvalbumin- and calbindin-positive interneuron neuroblasts that were migrating through the medial and lateral ganglionic eminences, suggesting a role for neogenin in tangential migration. Therefore, neogenin may be a multi-functional receptor regulating both progenitor activity and neuroblast migration in the embryonic forebrain. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

Neural progenitor number is regulated by nuclear factor-kappa B p65 and p50 subunit-dependent proliferation rather than cell survival

The number of cells generated by a proliferating stem or precursor cell can be influenced both by proliferation and by the degree of cell death/survival of the progeny generated. In this study, the extent to which cell survival controls progenitor number was examined by comparing the growth characteristics of neurosphere cultures derived from mice lacking genes for the death inducing Bcl-2 homologue Hara Kiri (Hrk), apoptosis-associated protein 1 (Apaf1), or the prosurvival nuclear factor-kappa B (NF kappa B) subunits p65, p50, or c-rel. We found no evidence that Hrk or Apaf1, and by inference the mitochondrial cell death pathway, are involved in regulating the number of neurosphere-derived progeny. However, we identified the p65p50 NF kappa B dimer as being required for the normal growth and expansion of neurosphere cultures. Genetic loss of both p65 and p50 NF kappa B subunits resulted in a reduced number of progeny but an increased proportion of neurons. No effect on cell survival was observed. This suggests that the number and fate of neural progenitor cells are more strongly regulated by cell cycle control than survival. (c) 2005 Wiley-Liss, Inc.

Nitric oxide-dependent bone marrow progenitor mobilization by carbon monoxide enhances endothelial repair after vascular injury

Carbon monoxide (CO) has emerged as a vascular homeostatic molecule that prevents balloon angioplasty-induced stenosis via antiproliferative effects on vascular smooth muscle cells. The effects of CO on reendothelialization have not been evaluated.

Directing migration of endothelial progenitor cells with applied DC electric fields

Naturally-occurring, endogenous electric fields (EFs) have been detected at skin wounds, damaged tissue sites and vasculature. Applied EFs guide migration of many types of cells, including endothelial cells to migrate directionally. Homing of endothelial progenitor cells (EPCs) to an injury site is important for repair of vasculature and also for angiogenesis. However, it has not been reported whether EPCs respond to applied EFs. Aiming to explore the possibility to use electric stimulation to regulate the progenitor cells and angiogenesis, we tested the effects of direct-current (DC) EFs on EPCs. We first used immunofluorescence to confirm the expression of endothelial progenitor markers in three lines of EPCs. We then cultured the progenitor cells in EFs. Using time-lapse video microscopy, we demonstrated that an applied DC EF directs migration of the EPCs toward the cathode. The progenitor cells also align and elongate in an EF. Inhibition of vascular endothelial growth factor (VEGF) receptor signaling completely abolished the EF-induced directional migration of the progenitor cells. We conclude that EFs are an effective signal that guides EPC migration through VEGF receptor signaling in vitro. Applied EFs may be used to control behaviors of EPCs in tissue engineering, in homing of EPCs to wounds and to an injury site in the vasculature.

The development and growth of tissues derived from cranial neural crest and primitive mesoderm is dependent on the ligation status of retinoic acid receptor γ:evidence that retinoic acid receptor γ functions to maintain stem/progenitor cells in the absence of retinoic acid

Retinoic acid (RA) signaling is important to normal development. However, the function of the different RA receptors (RARs)-RARα, RARβ, and RARγ-is as yet unclear. We have used wild-type and transgenic zebrafish to examine the role of RARγ. Treatment of zebrafish embryos with an RARγ-specific agonist reduced somite formation and axial length, which was associated with a loss of hoxb13a expression and less-clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist also disrupted formation of tissues arising from cranial neural crest, including cranial bones and anterior neural ganglia. There was a loss of Sox 9-immunopositive neural crest stem/progenitor cells in the same anterior regions. Pectoral fin outgrowth was blocked by RARγ agonist treatment. However, there was no loss of Tbx-5-immunopositive lateral plate mesodermal stem/progenitor cells and the block was reversed by agonist washout or by cotreatment with an RARγ antagonist. Regeneration of the caudal fin was also blocked by RARγ agonist treatment, which was associated with a loss of canonical Wnt signaling. This regenerative response was restored by agonist washout or cotreatment with the RARγ antagonist. These findings suggest that RARγ plays an essential role in maintaining stem/progenitor cells during embryonic development and tissue regeneration when the receptor is in its nonligated state.

Rad18 confers hematopoietic progenitor cell DNA damage tolerance independently of the Fanconi Anemia pathway in vivo.

In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18(-/-) mice. Moreover, primary Rad18(-/-) mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18(-/-) HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18(-/-) mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting.