Production and utilization of growth factors related to fibroblast growth factor by embryonal carcinoma cells and their differentiated cells

Previous studies have established that embryonal carcinoma (EC) cells produce several different growth factors, but express few, if any, receptors for epidermal growth factor, platelet-derived growth factor, or transforming growth factor type-beta. In this study, the production and utilization of fibroblast growth factor (FGF) by EC cells and their differentiated cells were investigated. We have determined that EC cells produce a heat-labile, heparin-binding factor that competes with FGF for binding to membrane receptors and appears to be immunologically related to FGF. The same or a similar factor is produced by three different EC cell lines, including a multipotent human EC cell line. However, production of this factor is apparently reduced when each EC cell line differentiates. Unlike the parental EC cells, the differentiated cells respond to FGF by growth stimulation and the growth responses to FGF correlate with increased binding of FGF. Although the binding data indicate that both the EC cells and their differentiated cells exhibit high affinity receptors for FGF, the differentiated cells express these receptors at levels approximately 10-fold higher. These findings suggest that the FGF-related growth factor could influence the growth of EC cells or their differentiated cells.     

Identification and characterization of polypeptide growth factors secreted by murine embryonal carcinoma cells

Undifferentiated P19 and PC13 murine embryonal carcinoma (EC) cells have been analyzed for their ability to secrete polypeptide growth factors. This has been carried out by a combination of specific bioassays and the use of biochemical and immunological detection methods. Both P19 and PC13 EC cells secrete a platelet-derived growth factor (PDGF)-like growth factor, a type beta transforming growth factor, and insulin-like growth factors. In addition, PC13 EC cells secrete a heparin-binding growth factor functionally related to fibroblast growth factor, while P19 EC cells secrete transforming growth factor-alpha. This is the first demonstration for secretion of transforming growth factor-alpha by an equivalent of early embryonic cells. The possible paracrine growth stimulating effects of these growth factors have been tested on differentiated derivatives of P19 EC cells, corresponding to all three germ layers. The differences in growth factor production by various embryonal carcinoma cells are discussed in relation to the developmental origin of these cell lines.     

MT1-MMP down-regulates the glucose 6-phosphate transporter expression in marrow stromal cells: a molecular link between pro-MMP-2 activation, chemotaxis, and cell survival

Bone marrow-derived stromal cells (BMSC) are avidly recruited by experimental vascularizing tumors, which implies that they must respond to tumor-derived growth factor cues. In fact, BMSC chemotaxis and cell survival are regulated, in part, by the membrane type-1 matrix metalloproteinase (MT1-MMP), an MMP also involved in pro-MMP-2 activation and in degradation of the extracellular matrix (ECM). Given that impaired chemotaxis was recently observed in bone marrow cells isolated from a glucose 6-phosphate transporter-deficient (G6PT-/-) mouse model, we sought to investigate the potential MT1-MMP/G6PT signaling axis in BMSC. We show that MT1-MMP-mediated activation of pro-MMP-2 by concanavalin A (ConA) correlated with an increase in the sub-G1 cell cycle phase as well as with cell necrosis, indicative of a decrease in BMSC survival. BMSC isolated from Egr-1-/- mouse or MT1-MMP gene silencing in BMSC with small interfering RNA (siMT1-MMP) antagonized both the ConA-mediated activation of pro-MMP-2 and the induction of cell necrosis. Overexpression of recombinant full-length MT1-MMP triggered necrosis and this was signaled through the cytoplasmic domain of MT1-MMP. ConA inhibited both the gene and protein expression of G6PT, while overexpression of recombinant G6PT inhibited MT1-MMP-mediated pro-MMP-2 activation but could not rescue BMSC from ConA-induced cell necrosis. Cell chemotaxis in response to the tumorigenic growth factor sphingosine 1-phosphate was significantly abrogated in siMT1-MMP BMSC and in chlorogenic acid-treated BMSC. Altogether, we provide evidence for an MT1-MMP/G6PT signaling axis that regulates BMSC survival, ECM degradation, and mobilization. This may lead to optimized clinical applications that use BMSC as a platform for the systemic delivery of therapeutic or anti-cancer recombinant proteins in vivo.     

CXCR4 and matrix metalloproteinase-2 are involved in mesenchymal stromal cell homing and engraftment to tumors

Background aimsBone marrow-derived mesenchymal stromal cells (BMSC) have been shown to migrate to injury, ischemia and tumor microenvironments. The mechanisms by which mesenchymal stromal cells (MSC) migrate across endothelium and home to the target tissues are not yet fully understood.MethodsWe used rat BMSC to investigate the molecular mechanisms involved in their tropism to tumors in vitro and in vivo.ResultsBMSC were shown to migrate toward four different tumor cells in vitro, and home to both subcutaneous and lung metastatic prostate tumor models in vivo. Gene expression profiles of MSC exposed to conditioned medium (CM) of various tumor cells were compared and revealed that matrix metalloproteinase-2 (MMP-2) expression in BMSC was downregulated after 24 h exposure to tumor CM. Chemokine (C–X–C motif) Receptor 4 (CXCR4) upregulation was also found in BMSC after 24 h exposure to tumor CM. Exposure to tumor cell CM enhanced migration of BMSC toward tumor cells. Stromal Cell-Derived Factor (SDF-1) inhibitor AMD3100 and MMP-2 inhibitor partly abolished the BMSC migration toward tumor cells in vitro.ConclusionsThese results suggest that the CXCR4 and MMP-2 are involved in the multistep migration processes of BMSC tropism to tumors.     

Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function

Pharmacological focal adhesion kinase (FAK) inhibition prevents tumor growth and metastasis, via actions on both tumor and stromal cells. In this paper, we show that vascular endothelial cadherin (VEC) tyrosine (Y) 658 is a target of FAK in tumor-associated endothelial cells (ECs). Conditional kinase-dead FAK knockin within ECs inhibited recombinant vascular endothelial growth factor (VEGF-A) and tumor-induced VEC-Y658 phosphorylation in vivo. Adherence of VEGF-expressing tumor cells to ECs triggered FAK-dependent VEC-Y658 phosphorylation. Both FAK inhibition and VEC-Y658F mutation within ECs prevented VEGF-initiated paracellular permeability and tumor cell transmigration across EC barriers. In mice, EC FAK inhibition prevented VEGF-dependent tumor cell extravasation and melanoma dermal to lung metastasis without affecting primary tumor growth. As pharmacological c-Src or FAK inhibition prevents VEGF-stimulated c-Src and FAK translocation to EC adherens junctions, but FAK inhibition does not alter c-Src activation, our experiments identify EC FAK as a key intermediate between c-Src and the regulation of EC barrier function controlling tumor metastasis.     

Production of PDGF-like growth factors by embryonal carcinoma cells and binding of PDGF to their endoderm-like differentiated cells

In this report, we demonstrate that F9 and PC-13 embryonal carcinoma (EC) cells do not bind significant amounts of platelet-derived growth factor (PDGF), whereas the endoderm-like differentiated cells derived from EC cells do. The F9-differentiated cells exhibit approximately 8300 receptors per cell, with an apparent dissociation constant of 30 pM. Two endoderm-like cell lines, PSA-5E and PYS-2, also bind PDGF and exhibit approximately 4800 and 23,500 receptors per cell, respectively. The lack of PDGF binding by the parental EC cells is consistent with their release of a factor(s) that is closely related to PDGF. This factor(s) competes with PDGF for binding to membrane receptors and is recognized by antibodies raised against PDGF. However, this factor(s) does not appear to be antigenically identical to PDGF. We also show that production of this PDGF-like factor(s) is reduced more than 90% when F9 EC cells differentiate into cells that bind PDGF. Thus, our findings indicate that EC cells release a factor(s) that should be capable of binding to their differentiated cells. This raises the possibility that PDGF, or a closely related factor, can influence cell proliferation and/or cell behavior of early embryonic cells.     

Intra- and extracellular signaling by endothelial neuregulin-1

Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.     

Endothelial cells are activated by angiopoeitin-1 gene transfer and produce coordinated sprouting in vitro and arteriogenesis in vivo

RATIONAL AND OBJECTIVES: Activation of fully differentiated vascular cells using angiogenic genes can lead to phenotypic changes resulting in formation of new blood vessels. We tested whether Ang-1 gene transfer to endothelial cells (EC) activates these cells. METHODS AND RESULTS: EC and SMC were transduced using retroviral or adenoviral vectors to produce Ang-1 or vascular endothelial growth factor (VEGF). EC Tie-2 receptor was phosphorilated by autologous secretion of Ang-1. Transduced EC and SMC sprouting capacity was tested using collagen embedded spheroids assay and capacity to produce arteriogenesis was tested in a hind limb model of ischemia. EC expressing Ang-1 in the presence of SMC expressing VEGF exhibited high levels of sprouting of the two cell types. Flow and numbers of arteries were increased after transduced cells implantation in vivo. CONCLUSIONS: Autologous secretion of Ang-1 by transduced EC resulted in Tie-2 activation and in the presence of SMC expressing VEGF resulted in coordinated sprouting in vitro and increase in flow and number of arteries in vivo.     

Safrole oxide induced neuronal differentiation of rat bone-marrow mesenchymal stem cells by elevating Hsp70

In a previous study, we found that at low concentrations, safrole oxide (SFO) could induce vascular endothelial cell (VEC) transdifferentiation into neuron-like cells; however, whether SFO could induce bone-marrow mesenchymal stem cell (BMSC) neural differentiation was unknown. Here, we found that SFO could effectively induce BMSC neural differentiation in the presence of serum and fibroblast growth factor 2 and did not affect cell viability at low concentrations. The levels of neuron-specific enolase and neurofilament-L were increased greatly, but that of glial fibrillary acidic protein was absent with SFO treatment for 48 h. Furthermore, SFO could increase the level of heat shock protein 70 (Hsp70), an important factor in neuronal differentiation. Knockdown of Hsp70 by its small interfering RNA blocked SFO-induced BMSC differentiation. Thus, SFO is a novel inducer of BMSC differentiation to neuron-like cells and Hsp70 is implicated in the differentiation process. We provide a new tool for obtaining neuron-like cells from BMSCs and for further investigating the new effect of Hsp70 on BMSC neuronal differentiation.     

Cerebral vascular endothelial cells are effective targets for in vitro lysis by encephalitogenic T lymphocytes.

Lysis of cerebral vascular endothelial cells (EC) by CD4-positive, myelin basic protein-specific encephalitogenic T cell lines was investigated. Unstimulated EC were not lysed, but culture in the presence of murine rIFN-gamma resulted in the expression of class II MHC (Ia) molecules and the concomitant ability to function as effective target cells for lysis. The possible requirement for Ia molecules was further demonstrated by antibody-blocking experiments. Lysis of EC targets also required the presence of specific Ag (myelin basic protein); PPD-specific T cell lines also lysed the PPD-pulsed EC. In all cases, lysis was directly proportional to E:T ratios. In addition, continuous passage of T cell lines resulted in the concomitant loss of encephalitogenicity and ability to affect EC lysis, indicating a possible relationship between these two factors. These results demonstrate that CD4+ T cells interact with cerebral vascular EC. It is suggested that such interactions may be important in the pathogenesis of diseases involving migrations of these cells across the blood-brain barrier.     

Neural differentiation ability of mesenchymal stromal cells from bone marrow and adipose tissue: a comparative study

Background aimsThe characteristics, such as morphologic and phenotypic characteristics and neural transdifferentiation ability, of mesenchymal stromal cells (MSC) derived from different origins have yet to be reported for cases isolated from the same individual.MethodsThe proliferation capacity, secretion ability of neurotrophins (NT) and neural differentiation ability in rat MSC isolated from bone marrow (BMSC) and adipose tissue (ADSC) were compared from the same animal.ResultsThe ADSC had a significantly higher proliferation capacity than BMSC according to cell cycle and cumulative population doubling analyses. The proportion of cells expressing neural markers was greater in differentiated ADSC than in differentiated BMSC. Furthermore, the single neurosphere derived from ADSC showed stronger expansion and differentiation abilities than that derived from BMSC. The findings from Western blot lent further support to the immunocytochemical data. The mRNA and protein levels of nerve growth factor (NGF) and brain-derived growth factor (BDNF) expressed in ADSC were significantly higher than those in BMSC at different stages before and following induction.ConclusionsOur study suggests that the proliferation ability of ADSC is superior to that of BMSC. Furthermore, differentiated ADSC expressed higher percentages of neural markers. As one possible alternative source of BMSC, ADSC may have wide potential for treating central nervous system (CNS) diseases.     

Expression of vascular endothelial growth factor receptor 1 in bone marrow-derived mesenchymal cells is dependent on hypoxia-inducible factor 1

Bone marrow-derived cells are recruited to sites of ischemia, where they promote tissue vascularization. This response is dependent upon the expression of vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1), which mediates cell migration in response to VEGF or placental growth factor (PLGF). In this study, we found that exposure of cultured mouse bone marrow-derived mesenchymal stromal cells (MSC) to hypoxia or an adenovirus encoding a constitutively active form of hypoxia-inducible factor 1 (HIF-1) induced VEGFR1 mRNA and protein expression and promoted ex vivo migration in response to VEGF or PLGF. MSC in which HIF-1 activity was inhibited by a dominant negative or RNA interference approach expressed markedly reduced levels of VEGFR1 and failed to migrate or activate AKT in response to VEGF or PLGF. Thus, loss-of-function and gain-of-function approaches demonstrated that HIF-1 activity is necessary and sufficient for basal and hypoxia-induced VEGFR1 expression in bone marrow-derived MSC.     

Neuroprotective and growth-promoting effects of bone marrow stromal cells after cervical spinal cord injury in adult rats

Background aimsBone marrow stromal cells (BMSC) have been shown to provide neuroprotection after transplantation into the injured central nervous system. The present study investigated whether adult rat BMSC differentiated along a Schwann cell lineage could increase production of trophic factors and support neuronal survival and axonal regeneration after transplantation into the injured spinal cord.MethodsAfter cervical C4 hemi-section, 5-bromo-2-deoxyuridine (BrdU)/green fluorescent protein (GFP)-labeled BMSC were injected into the lateral funiculus at 1 mm rostral and caudal to the lesion site. Spinal cords were analyzed 2–13 weeks after transplantation.Results and ConclusionsTreatment of native BMSC with Schwann cell-differentiating factors significantly increased production of brain-derived neurotrophic factor in vitro. Transplanted undifferentiated and differentiated BMSC remained at the injection sites, and in the trauma zone were often associated with neurofilament-positive fibers and increased levels of vascular endothelial growth factor. BMSC promoted extensive in-growth of serotonin-positive raphaespinal axons and calcitonin gene-related peptide (CGRP)-positive dorsal root sensory axons into the trauma zone, and significantly attenuated astroglial and microglial cell reactions, but induced aberrant sprouting of CGRP-immunoreactive axons in Rexed's lamina III. Differentiated BMSC provided neuroprotection for axotomized rubrospinal neurons and increased the density of rubrospinal axons in the dorsolateral funiculus rostral to the injury site. The present results suggest that BMSC induced along the Schwann cell lineage increase expression of trophic factors and have neuroprotective and growth-promoting effects after spinal cord injury.     

Characterization of human embryonal carcinoma cell lines derived from testicular germ-cell tumors

Four human embryonal carcinoma (EC) cell lines (ITO, NEC 8, NEC 14, NEC 15) derived independently from testicular germ-cell tumors were established in vitro. In their xenografted tumor tissues, all of them exhibited histological characteristics consistent with EC. The cell-biological characterization of these human EC cell lines was investigated with reference to well-known murine EC cell lines. This included examination of their morphology, growth, tumorigenic potential, karyotype, cell-aggregate formation, HLA expression, large glycopeptides, AFP and HCG production, plasminogen-activator secretion, and LDH profiles. Three (ITO, NEC 14, NEC 15) of these human EC cell lines shared cell-biological characteristics consistent with typical EC, but one of them (NEC 8) differed from the others with respect to its rapid growth, high tumorigenic potential, formation of solid cell aggregates, and less differentiated, solid histological pattern. Thus, it is suggested that there are several developmentally different types of human EC cells. The relationship between the properties of these human EC cell lines and their differentiation potential is discussed.     

Endothelial cell regulation by transforming growth factor-beta.

Pronounced changes including growth inhibition, increased matrix deposition and suppression of cell-associated proteolytic activity, take place in endothelial cells (EC) upon the application of TGF-beta. Interrelationships between these effects have shed some light on the mechanism of action of TGF-beta and on its role in regulating EC function vis-a-vis angiogenesis. For instance, preliminary evidence has indicated that increased levels of certain matrix components may be partly responsible for the antiproliferative action of TGF-beta. In addition, TGF-beta and bFGF have opposing effects on cellular proteolytic balance which may contribute to the antagonistic effect that TGF-beta has on bFGF-induced EC growth and possibly to the anti-angiogenic effect exerted by TGF-beta under certain circumstances. Of particular interest in this regard is the fact that physical contact between EC and vascular mural cells in EC:mural cell cocultures has been found to generate active TGF-beta, thus further implicating TGF-beta in the maintenance of the quiescent, differentiated aggregation of EC as found in vascular structures in vivo. While more information is needed to define what, if any role TGF-beta plays in endothelial differentiation, it is to be noted that many of the cellular and biochemical processes affected by TGF-beta are linked to differentiation. It is therefore possible that the growth inhibition of EC by TGF-beta primes them for differentiation and/or is critical for the maintenance of a differentiated state.     

Multipotent Adult Progenitor Cells (MAPC) contribute to hepatocarcinoma neovasculature

The use of stem cells as a vehicle of therapeutic genes is an attractive approach for the development of new antitumoral strategies based on gene therapy. The aim of our study was to assess the potential of bone marrow-derived Multipotent Adult Progenitor Cells (rMAPCs) to differentiate in vitro and in vivo into endothelial cells and to be recruited to areas of tumor vasculogenesis. In vitro, rMAPCs obtained from Buffalo rats differentiated into cells expressing endothelial markers and demonstrated functional endothelial capacity. Intravenous injection of undifferentiated rMAPC transduced with a lentivirus expressing GFP in an orthotopic rat model of hepatocellular carcinoma, resulted in tumor recruitment of the injected cells and in vivo differentiation into endothelial cells in the tumor area with contribution to vasculogenesis. In summary, our results suggest that rMAPCs can be efficiently recruited by vascularized tumors and differentiate to endothelium and thus may represent a useful vehicle for delivery of therapeutic genes to sites of active tumor neovascularization.     

Human tumor cells are selectively inhibited by colicins

The activity in vitro of four types of colicins (A, E1, E3, U) against one human standard fibroblast line and against 11 human tumor-cell lines carrying defined mutations of the p53 gene was quantified by MTT (tetrazolium bromide) assay. Flow cytometry showed that the pore-forming colicins A, E1 and U affected the cell cycle of 5 of these cell lines. Colicins E3 and U did not show any distinct inhibitory effects on the cell lines, while colicins E1 and especially A inhibited the growth of all of them (with one exception concerning colicin E1). Colicin E1 inhibited the growth of the tumor lines by 17-40% and standard fibroblasts MRC5 by 11%. Colicin A exhibited a differentiated 16-56% inhibition, the growth of standard fibroblasts being inhibited by 36%. In three of the lines, colicins A and E1 increased the number of cells in the G1 phase (by 12-58%) and in apoptosis (by 7-58%). These results correlated with the data from sensitivity assays. Hence, the inhibitory effect of colicins on eukaryotic cells in cell-selective, colicin-specific and can be considered to be cytotoxic.