Chad L. Barber

Autocrine VEGF signaling is required for vascular homeostasis.

Vascular endothelial growth factor (VEGF) is essential for developmental and pathological angiogenesis. Here we show that in the absence of any pathological insult, autocrine VEGF is required for the homeostasis of blood vessels in the adult. Genetic deletion of vegf specifically in the endothelial lineage leads to progressive endothelial degeneration and sudden death in 55% of mutant mice by 25 weeks of age. The phenotype is manifested without detectable changes in the total levels of VEGF mRNA or protein, indicating that paracrine VEGF could not compensate for the absence of endothelial VEGF. Furthermore, wild-type, but not VEGF null, endothelial cells showed phosphorylation of VEGFR2 in the absence of exogenous VEGF. Activation of the receptor in wild-type cells was suppressed by small molecule antagonists but not by extracellular blockade of VEGF. These results reveal a cell-autonomous VEGF signaling pathway that holds significance for vascular homeostasis but is dispensable for the angiogenic cascade.

The Ever-Elusive Endothelial Progenitor Cell: Identities, Functions and Clinical Implications

The concept of an Endothelial Progenitor Cell (EPC) that participates in adult angiogenesis is less than a decade old, yet it has received a great deal of attention due to its potential for cell-based clinical therapies in many pathologies. However, controversy remains as to the identity of this bone marrow-derived cell type and its ability to give rise to new endothelium in the adult. Reports on the contribution of EPCs to new vessels in ischemic tissue or tumors vary widely, ranging from 80–90% to negligible. As researchers hone their ability to identify, isolate, and expand these cells by their markers and functionality, mounting evidence suggests that they might constitute multiple, but related cell types. At least two general phenotypes have emerged from studies of bone marrow-derived cells contributing to angiogenesis: one that incorporates into the endothelial wall directly contributing to vascular expansion and another that is able to home to neovessels, but it locates behind the endothelial wall. Nonetheless, experimental evidence indicates that this second cell type supports the viability of newly formed vessels and thus it is equally relevant to neovascular growth. As our understanding of neovascularization in pathologic states expands, a more clear definition of the multiple cellular components required for the process will shed light into new models of therapeutic intervention. The identification of a cell type that could be isolated, expanded and infused into a patient would be very useful for promoting angiogenesis in ischemia, myocardial infarct and other pathologies.

Endothelial Progenitors: A Consensus Statement on Nomenclature

Endothelial progenitor cell (EPC) nomenclature remains ambiguous and there is a general lack of concordance in the stem cell field with many distinct cell subtypes continually grouped under the term “EPC.” It would be highly advantageous to agree on standards to confirm an endothelial progenitor phenotype and this should include detailed immunophenotyping, potency assays, and clear separation from hematopoietic angiogenic cells which are not endothelial progenitors. In this review, we seek to discourage the indiscriminate use of “EPCs,” and instead propose precise terminology based on defining cellular phenotype and function. Endothelial colony forming cells and myeloid angiogenic cells are examples of two distinct and well‐defined cell types that have been considered EPCs because they both promote vascular repair, albeit by completely different mechanisms of action. It is acknowledged that scientific nomenclature should be a dynamic process driven by technological and conceptual advances; ergo the ongoing “EPC” nomenclature ought not to be permanent and should become more precise in the light of strong scientific evidence. This is especially important as these cells become recognized for their role in vascular repair in health and disease and, in some cases, progress toward use in cell therapy. Stem Cells Translational Medicine 2017;6:1316–1320

Reduced production of B-1–specified common lymphoid progenitors results in diminished potential of adult marrow to generate B-1 cells

B-1 B cells have been proposed to be preferentially generated from fetal progenitors, but this view is challenged by studies concluding that B-1 production is sustained throughout adult life. To address this controversy, we compared the efficiency with which hematopoietic stem cells (HSCs) and common lymphoid progenitors (CLPs) from neonates and adults generated B-1 cells in vivo and developed a clonal in vitro assay to quantify B-1 progenitor production from CLPs. Adult HSCs and CLPs generated fewer B-1 cells in vivo compared with their neonatal counterparts, a finding corroborated by the clonal studies that showed that the CLP compartment includes B-1– and B-2–specified subpopulations and that the former cells decrease in number after birth. Together, these data indicate that B-1 lymphopoiesis is not sustained at constant levels throughout life and define a heretofore unappreciated developmental heterogeneity within the CLP compartment.

Induction of radioprotective peroxiredoxin‐I by ionizing irradiation

Results of this study indicate a radioprotective effect of peroxiredoxin‐I. Peroxiredoxin‐I is an antioxidant that scavenges hydroperoxides, whereas reactive oxygen species are the main mediators of ionizing radiation toxicity. We hypothesized that peroxiredoxin‐I might be induced by cellular exposure to radiation and act to protect them against its cytotoxic effects. Western blot and Northern blot analyses were used to assess peroxiredoxin‐I protein and mRNA expression. Rat C6 glioma cells were engineered to overexpress sense or antisense human peroxiredoxin‐I using retroviral vectors. Clonogenic cell survival was used to assess radiosensitivities of the engineered cells. Ionizing radiation induced peroxiredoxin‐I protein and mRNA expression in human HT29 colon cancer and rat C6 glioma cells in a dose‐ and time‐dependent manner over a 24 hr period. To determine the effect of peroxiredoxin‐I on radiation responses, C6 glioma cells were engineered to overexpress sense or antisense human peroxiredoxin‐I. In clonogenic assays, cells overexpressing peroxiredoxin‐I were more radioresistant. Cells transduced with antisense peroxiredoxin‐I were marginally more sensitive to radiation toxicity. Irradiation can induce peroxiredoxin‐I expression, and the increased peroxiredoxin‐I may protect cells from further radiation damage. These results suggest that protection by peroxiredoxin‐I may play an important role in the survival of glioma and colon cancer cells in patients undergoing radiation therapy.

Notch1 regulates angio-supportive bone marrow–derived cells in mice: relevance to chemoresistance

Host responses to chemotherapy can induce resistance mechanisms that facilitate tumor regrowth. To determine the contribution of bone marrow-derived cells (BMDCs), we exposed tumor-bearing mice to chemotherapeutic agents and evaluated the influx and contribution of a genetically traceable subpopulation of BMDCs (vascular endothelial-cadherin-Cre-enhanced yellow fluorescent protein [VE-Cad-Cre-EYFP]). Treatment of tumor-bearing mice with different chemotherapeutics resulted in a three- to 10-fold increase in the influx of VE-Cad-Cre-EYFP. This enhanced influx was accompanied by a significant increase in angiogenesis. Expression profile analysis revealed a progressive change in the EYFP population with loss of endothelial markers and an increase in mononuclear markers. In the tumor, 2 specific populations of VE-Cad-Cre-EYFP BMDCs were identified: Gr1⁺/CD11b⁺ and Tie2high/platelet endothelial cell adhesion moleculelow cells, both located in perivascular areas. A common signature of the EYFP population that exits the bone marrow is an increase in Notch. Inducible inactivation of Notch in the EYFP⁺ BMDCs impaired homing of these BMDCs to the tumor. Importantly, Notch deletion reduced therapy-enhanced angiogenesis, and was associated with an increased antitumor effect of the chemotherapy. These findings revealed the functional significance of a specific population of supportive BMDCs in response to chemotherapeutics and uncovered a new potential strategy to enhance anticancer therapy.

Radiation‐induced posttranscriptional control of M6P/IGF2r expression in breast cancer cell lines

The mannose 6‐phosphate/insulin‐like growth factor 2 receptor (M6P/IGF2r), a member of the IGF axis of growth factors, is a negative regulator of cell growth and a putative tumor suppressor gene. Regulation of M6P/IGF2r levels is critical in breast physiology; low expression is associated with various aspects of breast cancer. We have found that ionizing radiation induces the rapid expression of M6P/IGF2r in a dose‐dependent manner in MCF7 human breast cancer cells. We show that this increase is mediated, at least in part, by a stabilization of M6P/IGF2r transcripts by radiation in both ER positive (MCF7 and T47D) and ER negative (MDA‐MB‐231) breast cancer cell lines. It is probable, therefore, that posttranscriptional dysregulation of M6P/IGF2r is a contributing mechanism in breast cancer development and breast cancer response to therapy. This is a novel find that underscores the importance of posttranscriptional control of radiation‐induced gene expression—a phenomenon that has often been paradigmatically attributed to transcriptional control.

Developmental relationships between B-1 and B-2 progenitors

Comment on: Barber CL, et al. Proc Natl Acad Sci USA 2011; In press.

A Dose-Dependent Decrease in the Fraction of Cases Harboring M6P/IGF2R Mutations in Hepatocellular Carcinomas from the Atomic Bomb Survivors

Abstract Iwamoto, K. S., Yano, S., Barber, C. L., MacPhee, D. G., and Tokuoka, S. A Dose-Dependent Decrease in the Fraction of Cases Harboring M6P/IGF2R Mutations in Hepatocellular Carcinomas from the Atomic Bomb Survivors. Radiat. Res. 166, 870–876 (2006). The risk for hepatocellular carcinoma (HCC) development is significantly heightened in the atomic bomb survivors, but the mechanism is unclear. We have previously reported finding a radiation dose-dependent increase in HCCs with TP53 mutations from the survivors. We now show that, in the same HCC samples, the frequency of 3′-untranslated region (3′UTR) mutations in M6P/IGF2R, a candidate HCC tumor suppressor gene, decreases with dose (P = 0.0091), implying a radiation dose-dependent negative selection of cells harboring such mutations. The fact that they were in the 3′UTR implicates changes in transcript stability rather than in protein function as the mechanism. Moreover, these M6P/IGF2R 3′UTR mutations and the TP53 mutations detected previously were mutually exclusive in most of the tumors, suggesting two independent pathways to HCC development, with the TP53 pathway being more favored with increasing radiation dose than the M6P/IGF2R pathway. These results suggest that tumors attributable to radiation may be genotypically different from tumors of other etiologies and hence may provide a way of distinguishing radiation-induced cancers from “background” cancers—a shift from the current paradigm.

Abstract 1022: Notch regulates the egression of angio-supportive bone marrow-derived cells after chemotherapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction Our understanding of angiogenesis has evolved from a simple endothelial-centric model into a multi-faceted process in which bone marrow-derived cells (BMDCs) are found to support the vasculature. BMDCs can act in an immediate “seek and repair” manner in response to various types of therapy, presumably to support tissue regeneration. This can induce an angiogenesis rebound after therapy and thereby impair therapy efficacy. Method By transplanting the bone marrow from the transgenic tamoxifen-inducible VE-cadherin Cre ERT2/EYFP (CIVE) mice into wild type, lethally irradiated, C57 Bl/6 mice, followed by tamoxifen treatment of the recipient mice, we created a mouse with YFP+ bone marrow-derived VE-Cadherin expressing cells (0.3% of the bone marrow). Allowing us to specifically track this lineage of BMDCs. In a second set of experiments, we used the CIVE-EYFP-notch-KO mice as bone marrow-donors. After tamoxifen induction, LLC cells were implanted subcutaneously and after eight days mice were either treated with vehicle control, cisplatin or paclitaxel. Tumor growth was measured and tumors were analyzed by FACS analysis and immunohistochemistry. Results Eight days after treatment with chemotherapy a significant increase in endothelial cells was seen in the tumor compared to the untreated tumors (p<0.01). This correlated with a rapid re-growth of the treated tumors six days after treatment initiation. This treatment-enhanced angiogenesis was accompanied by a clear increase in YFP+ cells as soon as 1 day after chemotherapy for 0.1 to 0.3% (p<0.05). The influx of YFP+ cells was specific for the tumor tissue, as no YFP+ cells were detected in control organs like lung, liver and spleen. Two populations of YFP+ cells were identified in the tumor, a macrophage-like population (Gr1/CD11b+) and an endothelial-like population (PECAM+). The YFP+ cells in the tumor were found in close association with the tumor vessels as per confocal microscopy. FACS analysis showed that in the untreated tumors housed 0.45% of all PECAM+ cells were YFP+, however, this value increased to 1-3% after chemotherapy. Transcriptional analysis of the YFP+ cells revealed high levels of Notch in this population. Using the notch KO mice as bone marrow donors resulted in a defective homing of the YFP+ cells to the tumor which completely prevented the therapy-enhanced angiogenesis and significantly enhanced anti-tumor effect of the chemotherapy. Conclusion In summary, our study identifies an important role for the BMD-VE-cadherin expressing cells in supporting angiogenesis, especially in response to chemotherapy. It reveals the critical role of notch signaling in the ability of these cells to egress the bone marrow and home to the tumor. Preventing the contribution of these angio-supportive cells significantly enhanced therapy efficacy and provides a new strategy to enhance anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1022. doi:1538-7445.AM2012-1022