Christie M. Orschell

Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors

Background—Endothelial progenitor cells (EPCs) have been isolated from peripheral blood and can enhance angiogenesis after infusion into host animals. It is not known whether the proangiogenic effects are a result of such events as endothelial differentiation and subsequent proliferation of EPCs or secondary to secretion of angiogenic growth factors. Methods and Results—Human EPCs were isolated as previously described, and their phenotypes were confirmed by uptake of acetylated LDL and binding of ulex-lectin. EPC proliferation and surface marker expression were analyzed by flow cytometry, and conditioned medium was assayed for growth factors. The majority of EPCs expressed monocyte/macrophage markers such as CD14 (95.7±0.3%), Mac-1 (57.6±13.5%), and CD11c (90.8±4.9%). A much lower percentage of cells expressed the specific endothelial marker VE-cadherin (5.2±0.7%) or stem/progenitor-cell markers AC133 (0.16±0.05%) and c-kit (1.3±0.7%). Compared with circulating monocytes, cultured EPCs showed upregulation of monocyte activation and macrophage differentiation markers. EPCs did not demonstrate any significant proliferation but did secrete the angiogenic growth factors vascular endothelial growth factor, hepatocyte growth factor, granulocyte colony–stimulating factor, and granulocyte-macrophage colony–stimulating factor. Conclusions—Our findings suggest that acetylated LDL(+)ulex-lectin(+) cells, commonly referred to as EPCs, do not proliferate but release potent proangiogenic growth factors. The majority of acetylated LDL(+)ulex-lectin(+) cells are derived from monocyte/macrophages. The findings of low proliferation and endothelial differentiation suggest that their angiogenic effects are most likely mediated by growth factor secretion. These findings may allow for development of novel angiogenic therapies relying on secreted growth factors or on recruitment of endogenous monocytes/macrophages to sites of ischemia.

Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist

Improving approaches for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is clinically important because increased numbers of these cells are needed for enhanced transplantation. Chemokine stromal cell derived factor-1 (also known as CXCL12) is believed to be involved in retention of HSCs and HPCs in bone marrow. AMD3100, a selective antagonist of CXCL12 that binds to its receptor, CXCR4, was evaluated in murine and human systems for mobilizing capacity, alone and in combination with granulocyte colony-stimulating factor (G-CSF). AMD3100 induced rapid mobilization of mouse and human HPCs and synergistically augmented G-CSF–induced mobilization of HPCs. AMD3100 also mobilized murine long-term repopulating (LTR) cells that engrafted primary and secondary lethally-irradiated mice, and human CD34+ cells that can repopulate nonobese diabetic-severe combined immunodeficiency (SCID) mice. AMD3100 synergized with G-CSF to mobilize murine LTR cells and human SCID repopulating cells (SRCs). Human CD34+ cells isolated after treatment with G-CSF plus AMD3100 expressed a phenotype that was characteristic of highly engrafting mouse HSCs. Synergy of AMD3100 and G-CSF in mobilization was due to enhanced numbers and perhaps other characteristics of the mobilized cells. These results support the hypothesis that the CXCL12-CXCR4 axis is involved in marrow retention of HSCs and HPCs, and demonstrate the clinical potential of AMD3100 for HSC mobilization.

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).

Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer

Purpose: These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how. Experimental Design: Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)–positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. Results: ER-positive estradiol-dependent lines (IC50, 12-24 μmol/L) and ER-negative (IC50, 45 μmol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-α levels notably in ER-positive lines. Ritonavir causes G1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D1 but not cyclin E, and depletes phosphorylated Rb and Ser473 Akt. Ritonavir induces apoptosis independent of G1 arrest, inhibiting growth of cells that have passed the G1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum Cmax of 22 ± 8 μmol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (KD, 7.8 μmol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90α short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. Conclusions: Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.

Establishing a murine model of the hematopoietic syndrome of the acute radiation syndrome.

Abstract The authors have developed a murine model of the Hematopoietic Syndrome of the Acute Radiation Syndrome (H-ARS) for efficacy testing of medical countermeasures (MCM) against radiation according to the FDA Animal Rule. Ten- to 12-wk-old male and female C57BL/6 mice were exposed to the LD50/30–LD70/30 dose of total body irradiation (TBI, 137Cs, 0.62–0.67 Gy min−1) in the morning hours when mice were determined to be most radiosensitive, and they were assessed for 30-d survival and mean survival time (MST). Antibiotics were delivered in drinking water on days 4–30 post-TBI at a concentration based on the amount of water that lethally-irradiated mice were found to consume. The fluoroquinolones, ciprofloxacin and levofloxacin, as well as the tetracycline doxycycline, and aminoglycoside neomycin, all significantly increased MST of decedent mice, while ciprofloxacin (p = 0.061) and doxycycline + neomycin (p = 0.005) showed at least some efficacy to increase 30-d survival. Blood sampling (30 &mgr;L/mouse every fifth day) was found to negatively impact 30-d survival. Histopathology of tissues harvested from nonmoribund mice showed expected effects of lethal irradiation, while moribund mice were largely septicemic with a preponderance of enteric organisms. Kinetics of loss and recovery of peripheral blood cells in untreated mice and those treated with two MCM, granulocyte-colony stimulating factor and Amifostine further characterized and validated this model for use in screening studies and pivotal efficacy studies of candidate MCM for licensure to treat irradiated individuals suffering from H-ARS.

Prevention and mitigation of acute radiation syndrome in mice by synthetic lipopeptide agonists of toll-like receptor 2 (TLR2)

Bacterial lipoproteins (BLP) induce innate immune responses in mammals by activating heterodimeric receptor complexes containing Toll-like receptor 2 (TLR2). TLR2 signaling results in nuclear factor-kappaB (NF-κB)-dependent upregulation of anti-apoptotic factors, anti-oxidants and cytokines, all of which have been implicated in radiation protection. Here we demonstrate that synthetic lipopeptides (sLP) that mimic the structure of naturally occurring mycoplasmal BLP significantly increase mouse survival following lethal total body irradiation (TBI) when administered between 48 hours before and 24 hours after irradiation. The TBI dose ranges against which sLP are effective indicate that sLP primarily impact the hematopoietic (HP) component of acute radiation syndrome. Indeed, sLP treatment accelerated recovery of bone marrow (BM) and spleen cellularity and ameliorated thrombocytopenia of irradiated mice. sLP did not improve survival of irradiated TLR2-knockout mice, confirming that sLP-mediated radioprotection requires TLR2. However, sLP was radioprotective in chimeric mice containing TLR2-null BM on a wild type background, indicating that radioprotection of the HP system by sLP is, at least in part, indirect and initiated in non-BM cells. sLP injection resulted in strong transient induction of multiple cytokines with known roles in hematopoiesis, including granulocyte colony-stimulating factor (G-CSF), keratinocyte chemoattractant (KC) and interleukin-6 (IL-6). sLP-induced cytokines, particularly G-CSF, are likely mediators of the radioprotective/mitigative activity of sLP. This study illustrates the strong potential of LP-based TLR2 agonists for anti-radiation prophylaxis and therapy in defense and medical scenarios.

A role for the fanconi anemia C protein in maintaining the DNA damage induced G2 checkpoint

Fanconi anemia (FA) is a complex, heterogeneous genetic disorder composed of at least 11 complementation groups. The FA proteins have recently been found to functionally interact with the cell cycle regulatory proteins ATM and BRCA1; however, the function of the FA proteins in cell cycle control remains incompletely understood. Here we show that the Fanconi anemia complementation group C protein (Fancc) is necessary for proper function of the DNA damage-induced G2/M checkpoint in vitro and in vivo. Despite apparently normal induction of the G2/M checkpoint after ionizing radiation, murine and human cells lacking functional FANCC did not maintain the G2 checkpoint as compared with wild-type cells. The increased rate of mitotic entry seen in Fancc–/–mouse embryo fibroblasts correlated with decreased inhibitory phosphorylation of cdc2 kinase on tyrosine 15. An increased inability to maintain the DNA damage-induced G2 checkpoint was observed in Fancc –/–; Trp53 –/–cells compared with Fancc –/–cells, indicating that Fancc and p53 cooperated to maintain the G2 checkpoint. In contrast, genetic disruption of both Fancc and Atm did not cooperate in the G2 checkpoint. These data indicate that Fancc and p53 in separate pathways converge to regulate the G2 checkpoint. Finally, fibroblasts lacking FANCD2 were found to have a G2 checkpoint phenotype similar to FANCC-deficient cells, indicating that FANCD2, which is activated by the FA complex, was also required to maintain the G2 checkpoint. Because a proper checkpoint function is critical for the maintenance of genomic stability and is intricately related to the function and integrity of the DNA repair process, these data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.

Long-term hematopoietic stem cell damage in a murine model of the hematopoietic syndrome of the acute radiation syndrome.

Abstract Residual bone marrow damage (RBMD) persists for years following exposure to radiation and is believed to be due to decreased self-renewal potential of radiation-damaged hematopoietic stem cells (HSC). Current literature has examined primarily sublethal doses of radiation and time points within a few months of exposure. In this study, the authors examined RBMD in mice surviving lethal doses of total body ionizing irradiation (TBI) in a murine model of the Hematopoietic Syndrome of the Acute Radiation Syndrome (H-ARS). Survivors were analyzed at various time points up to 19 mo post-TBI for hematopoietic function. The competitive bone marrow (BM) repopulating potential of 150 purified c-Kit+ Sca-1+ lineage- CD150+ cells (KSLCD150+) remained severely deficient throughout the study compared to KSLCD150+ cells from non-TBI age-matched controls. The minimal engraftment from these TBI HSCs is predominantly myeloid, with minimal production of lymphocytes both in vitro and in vivo. All classes of blood cells as well as BM cellularity were significantly decreased in TBI mice, especially at later time points as mice aged. Primitive BM hematopoietic cells (KSLCD150+) displayed significantly increased cell cycling in TBI mice at all time points, which may be a physiological attempt to maintain HSC numbers in the post-irradiation state. Taken together, these data suggest that the increased cycling among primitive hematopoietic cells in survivors of lethal radiation may contribute to long-term HSC exhaustion and subsequent RBMD, exacerbated by the added insult of aging at later time points.

Recovery from hematopoietic injury by modulating prostaglandin E2 signaling post-irradiation

While high dose total body irradiation (TBI) is used therapeutically, the proliferation of nuclear weapons, increasing use of nuclear power, and worldwide radical terrorism underscore the need to develop countermeasures to a radiological mass casualty event. The hematopoietic syndrome of the acute radiation syndrome (HS-ARS) results from severe compromise to the hematopoietic system, including lymphocytopenia, neutropenia, thrombocytopenia, and possible death from infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow hematopoietic stem cells (HSC) and progenitor cells (HPC) may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E(2) (PGE(2)) has been shown to have pleiotropic effects on hematopoiesis, acting to inhibit apoptosis and promote self-renewal of HSC, while inhibiting HPC proliferation. We assessed the radio-mitigating potential of modulating PGE(2) signaling in a mouse model of HS-ARS. Treatment with the PGE(2) analog 16,16 dimethyl PGE(2) (dmPGE(2)) 6h post-irradiation or inhibition of PGE(2) synthesis via delayed administration of the non-steroidal anti-inflammatory drug (NSAID) Meloxicam resulted in increased survival of lethally irradiated mice. Both early dmPGE(2) and delayed Meloxicam treatment were associated with increased HPC activity 35days following irradiation, demonstrating enhanced recovery of hematopoiesis. Our results define two different treatment modalities that are highly effective and safe to administer, and can be readily available.

Pyk2 regulates megakaryocyte-induced increases in osteoblast number and bone formation

Preclinical and clinical evidence from megakaryocyte (MK)‐related diseases suggests that MKs play a significant role in maintaining bone homeostasis. Findings from our laboratories reveal that MKs significantly increase osteoblast (OB) number through direct MK‐OB contact and the activation of integrins. We, therefore, examined the role of Pyk2, a tyrosine kinase known to be regulated downstream of integrins, in the MK‐mediated enhancement of OBs. When OBs were co‐cultured with MKs, total Pyk2 levels in OBs were significantly enhanced primarily because of increased Pyk2 gene transcription. Additionally, p53 and Mdm2 were both decreased in OBs upon MK stimulation, which would be permissive of cell cycle entry. We then demonstrated that OB number was markedly reduced when Pyk2−/− OBs, as opposed to wild‐type (WT) OBs, were co‐cultured with MKs. We also determined that MKs inhibit OB differentiation in the presence and absence of Pyk2 expression. Finally, given that MK‐replete spleen cells from GATA‐1–deficient mice can robustly stimulate OB proliferation and bone formation in WT mice, we adoptively transferred spleen cells from these mice into Pyk2−/− recipient mice. Importantly, GATA‐1–deficient spleen cells failed to stimulate an increase in bone formation in Pyk2−/− mice, suggesting in vivo the important role of Pyk2 in the MK‐induced increase in bone volume. Further understanding of the signaling pathways involved in the MK‐mediated enhancement of OB number and bone formation will facilitate the development of novel anabolic therapies to treat bone loss diseases.