The secretome of endothelial progenitor cells promotes brain endothelial cell activity through PI3-kinase and MAP-kinase

BACKGROUND Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. METHODS Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. RESULTS Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. CONCLUSION The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects.

Cell cycle reactivation of cochlear progenitor cells in neonatal FUCCI mice by a GSK3 small molecule inhibitor.

Due to the lack of regenerative capacity of the mammalian auditory epithelium, sensory hair cell loss results in permanent hearing deficit. Nevertheless, a population of tissue resident stem/progenitor cells has been recently described. Identification of methods to trigger their activity could lead to exploitation of their potential therapeutically. Here we validate the use of transgenic mice reporting cell cycle progression (FUCCI), and stemness (Lgr5-GFP), as a valuable tool to identify regulators of cell cycle re-entry of supporting cells within the auditory epithelium. The small molecule compound CHIR99021 was used to inhibit GSK3 activity. This led to a significant increase in the fraction of proliferating sphere-forming cells, labeled by the FUCCI markers and in the percentage of Lgr5-GFP + cells, as well as a selective increase in the fraction of S-G2-M cells in the Lgr5 + population. Using whole mount cultures of the organ of Corti we detected a statistically significant increment in the fraction of proliferating Sox2 supporting cells after CHIR99021 treatment, but only rarely appearance of novel MyoVIIa+/Edu + hair cells. In conclusion, these tools provide a robust mean to identify novel regulators of auditory organ regeneration and to clarify the contribution of stem cell activity.

Increased sensitivity to apoptotic stimuli in c-abl-deficient progenitor B-cell lines

The protooncogene c-abl encodes a nonreceptor tyrosine kinase whose cellular function is unknown. To study the possible involvement of c-Abl in proliferation, differentiation, and cell cycle regulation of early B cells, long-term lymphoid bone marrow cultures were established from c-abl-deficient mice and their wild-type littermates. Interleukin 7-dependent progenitor B-cell clones and lines expressing B220 and CD43 could be generated from both mutant and wild-type mice. The mutant and wild-type lines displayed no difference in their proliferative capacity as measured by thymidine incorporation in response to various concentrations of interleukin 7. Similarly, c-abl deficiency did not interfere with the ability of mutant clones to differentiate into surface IgM-positive cells in vitro. Analysis of cultures after growth factor deprivation, however, revealed a strikingly accelerated rate of cell death in c-abl mutant cells, due to apoptosis as confirmed by terminal deoxynucleotidyltransferase-mediated UTP nick end labeling analysis. Furthermore, a greater susceptibility to apoptotic cell death in c-abl mutant cells was also observed after glucocorticoid treatment. These results suggest that mutant c-Abl renders the B-cell progenitors more sensitive to apoptosis, and may account for some of the phenotypes observed in c-abl-deficient animals.

Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow

The adhesive mechanisms allowing hematopoietic progenitor cells (HPC) homing to the bone marrow (BM) after BM transplantation are poorly understood. We investigated the role of endothelial selectins and vascular cell adhesion molecule-1 (VCAM-1) in this process. Lethally irradiated recipient mice deficient in both P-and E-selectins (P/E−/−), reconstituted with minimal numbers (≤5 × 104) of wild-type BM cells, poorly survived the procedure compared with wild-type recipients. Excess mortality in P/E−/− mice, after a lethal dose of irradiation, was likely caused by a defect of HPC homing. Indeed, we observed that the recruitment of HPC to the BM was reduced in P/E−/− animals, either splenectomized or spleen-intact. Homing into the BM of P/E−/− recipient mice was further compromised when a function-blocking VCAM-1 antibody was administered. Circulating HPC, 14 hr after transplantation, were greatly increased in P/E−/− mice treated with anti-VCAM-1 compared with P/E−/− mice treated with just IgG or wild-type mice treated with either anti-VCAM-1 or IgG. Our results indicate that endothelial selectins play an important role in HPC homing to the BM. Optimal recruitment of HPC after lethal doses of irradiation requires the combined action of both selectins and VCAM-1 expressed on endothelium of the BM.

Identification of an early T cell progenitor for a pathway of T cell maturation in the bone marrow

We have identified a rare (≈0.05–0.1%) population of cells (Thy-1hiCD16+CD44hiCD2−TCRαβ−B220−Mac-1−NK1.1−) in the adult mouse bone marrow that generates CD4+ and CD8+ TCRαβ+ T cells after tissue culture for 48 hr in the presence of Ly5 congenic marrow cells. The essential stages in the maturation of the progenitors were determined; the stages included an early transition from CD2−CD16+CD44hiTCRαβ− to CD2+CD16int/−CD44int/−TCRαβ− cells, and a later transition to CD4+CD8+TCRαβ+ double-positive T cells that rapidly generate the CD4+ and CD8+ single-positive T cells. The maturation of the progenitors is almost completely arrested at the CD2+TCRαβ− stage by the presence of mature T cells at the initiation of cultures. This alternate pathway is supported by the marrow microenvironment; it recapitulates critical intermediary steps in intrathymic T cell maturation.

Identity of adenylyl cyclase isoform determines the rate of cell cycle progression in NIH 3T3 cells

Cell cycle progression is regulated by cAMP in several cell types. Cellular cAMP levels depend on the activity of different adenylyl cyclases (ACs), which have varied signal-receiving capabilities. The role of individual ACs in regulating proliferative responses was investigated. Native NIH 3T3 cells contain AC6, an isoform that is inhibited by a variety of signals. Proliferation of exogenous AC6-expressing cells was the same as in control cells. In contrast, expression of AC2, an isoform stimulated by protein kinase C (PKC), resulted in inhibition of cell cycle progression and increased doubling time. In AC2-expressing cells, platelet-derived growth factor (PDGF) elevated cAMP levels in a PKC-dependent manner. PDGF stimulation of mitogen-activated protein kinases 1 and 2 (MAPK 1,2), DNA synthesis, and cyclin D1 expression was reduced in AC2-expressing cells as compared with control cells. Dominant negative protein kinase A relieved the AC2 inhibition of PDGF-induced DNA synthesis. Expression of AC2 also blocked H-ras-induced transformation of NIH 3T3 cells. These observations indicate that, because AC2 is stimulated by PKC, it can be activated by PDGF concurrently with the stimulation of MAPK 1,2. The elevation in cAMP results in inhibition of signal flow from the PDGF receptor to MAPK 1,2 and a significant reduction in the proliferative response to PDGF. Thus, the molecular identity and signal receiving capability of the AC isoforms in a cell could be important for proliferative homeostasis.

Regulated expression of P210 Bcr-Abl during embryonic stem cell differentiation stimulates multipotential progenitor expansion and myeloid cell fate

P210 Bcr-Abl is an activated tyrosine kinase oncogene encoded by the Philadelphia chromosome associated with human chronic myelogenous leukemia (CML). The disease represents a clonal disorder arising in the pluripotent hematopoietic stem cell. During the chronic phase, patients present with a dramatic expansion of myeloid cells and a mild anemia. Retroviral gene transfer and transgenic expression in rodents have demonstrated the ability of Bcr-Abl to induce various types of leukemia. However, study of human CML or rodent models has not determined the direct and immediate effects of Bcr-Abl on hematopoietic cells from those requiring secondary genetic or epigenetic changes selected during the pathogenic process. We utilized tetracycline-regulated expression of Bcr-Abl from a promoter engineered for robust expression in primitive stem cells through multilineage blood cell development in combination with the in vitro differentiation of embryonal stem cells into hematopoietic elements. Our results demonstrate that Bcr-Abl expression alone is sufficient to increase the number of multipotent and myeloid lineage committed progenitors in a dose-dependent manner while suppressing the development of committed erythroid progenitors. These effects are reversible upon extinguishing Bcr-Abl expression. These findings are consistent with Bcr-Abl being the sole genetic change needed for the establishment of the chronic phase of CML and provide a powerful system for the analysis of any genetic change that alters cell growth and lineage choices of the hematopoietic stem cell.

Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: identity with the receptor that binds to the globular "heads" of C1q (gC1q-R).

High molecular weight kininogen (HK) and factor XII are known to bind to human umbilical vein endothelial cells (HUVEC) in a zinc-dependent and saturable manner indicating that HUVEC express specific binding site(s) for those proteins. However, identification and immunochemical characterization of the putative receptor site(s) has not been previously accomplished. In this report, we have identified a cell surface glycoprotein that is a likely candidate for the HK binding site on HUVECs. When solubilized HUVEC membranes were subjected to an HK-affinity column in the presence or absence of 50 microM ZnCl2 and the bound membrane proteins eluted, a single major protein peak was obtained only in the presence of zinc. SDS/PAGE analysis and silver staining of the protein peak revealed this protein to be 33 kDa and partial sequence analysis matched the NH2 terminus of gC1q-R, a membrane glycoprotein that binds to the globular "heads" of C1q. Two other minor proteins of approximately 70 kDa and 45 kDa were also obtained. Upon analysis by Western blotting, the 33-kDa band was found to react with several monoclonal antibodies (mAbs) recognizing different epitopes on gC1q-R. Ligand and dot blot analyses revealed zinc-dependent binding of biotinylated HK as well as biotinylated factor XII to the isolated 33-kDa HUVEC molecule as well as recombinant gC1q-R. In addition, binding of 125I-HK to HUVEC cells was inhibited by selected monoclonal anti-gC1q-R antibodies. C1q, however, did not inhibit 125I-HK binding to HUVEC nor did those monoclonals known to inhibit C1q binding to gC1q-R. Taken together, the data suggest that HK (and factor XII) bind to HUVECs via a 33-kDa cell surface glycoprotein that appears to be identical to gC1q-R but interact with a site on gC1q-R distinct from that which binds C1q.

Identification of a unique membrane-bound molecule on a hemopoietic stem cell line and on multipotent progenitor cells.

Hemopoietic stem cells are a distinct population of cells that can differentiate into multilineages of hemopoietic cells and have long-term repopulation capability. A few membrane-bound molecules have been found to be preferentially, but not uniquely, present on the surface of these primitive cells. We report here the identification of a unique 105-kDa glycoprotein on the surface of hemopoietic stem cell line BL3. This molecule, recognized by the absorbed antiserum, is not present on the surface of myeloid progenitors 32D and FDC-P1 cells, EL4 T cells, and NIH 3T3 fibroblasts. This antiserum can also be used to block the proliferation of BL3 cells even in the presence of mitogen-stimulated spleen cell conditioned medium, which is known to have a stimulating activity on BL3 cells. It can also inhibit development of in vitro, fetal liver cell-derived multilineage colonies, but not other types of colonies, and of in vivo bone marrow cell-derived colony-forming unit spleen foci. These data suggest that gp105 plays an important role in hemopoietic stem cell differentiation.

Characterization of a new murine retinal cell line (MU-PH1) with glial, progenitor and photoreceptor characteristics

Unlike fish and amphibians, mammals do not regenerate retinal neurons throughout life. However, neurogenic potential may be conserved in adult mammal retina and it is necessary to identify the factors that regulate retinal progenitor cells (RPC) proliferative capacity to scope their therapeutic potential. Müller cells can be progenitors for retinal neuronal cells and can play an essential role in the restoration of visual function after retinal injury. Some members of the Toll-like receptor (TLR) family, TLR2, TLR3 and TLR4, are related to progenitor cells proliferation. Müller cells are important in retinal regeneration and stable cell lines are useful for the study of retinal stem cell biology. Our purpose was to obtain a Müller-derived cell line with progenitor characteristics and potential interest in regeneration processes. We obtained and characterized a murine Müller-derived cell line (MU-PH1), which proliferates indefinitely in vitro. Our results show that (i) MU-PH1 cells expresses the Müller cell markers Vimentin, S-100, glutamine synthetase and the progenitor and stem cell markers Nestin, Abcg2, Ascl1, α-tubulin and β-III-tubulin, whereas lacks the expression of CRALBP, GFAP, Chx10, Pax6 and Notch1 markers; (ii) MU-PH1 cell line stably express the photoreceptor markers recoverin, transducin, rhodopsin, blue and red/green opsins and also melanopsin; (iii) the presence of opsins was confirmed by the recording of intracellular free calcium levels during light stimulation; (iv) MU-PH1 cell line also expresses the melatonin MT1 and MT2 receptors; (v) MU-PH1 cells express TLR1, 2, 4 and 6 mRNA; (vi) MU-PH1 express TLR2 at cell surface level; (vii) Candida albicans increases TLR2 and TLR6 mRNA expression; (viii) C. albicans or TLR selective agonists (Pam(3)CysSK(4), LPS) did not elicit morphological changes nor TNF-α secretion; (ix) C. albicans and Pam(3)CysSK(4) augmented MU-PH1 neurospheres formation in a statistically significant manner. Our results indicate that MU-PH1 cell line could be of great interest both as a photoreceptor model and in retinal regeneration approaches and that TLR2 may also play a role in retinal cell proliferation.