Basic FGF Research Articles

A Xeno-Free Culturing Protocol for Pluripotent Stem Cell-Derived Oligodendrocyte Precursor Cell Production

To show that human embryonic stem cells (hESCs) can be efficiently differentiated into oligodendrocyte precursor cells (OPCs) in a xeno-free medium with a specific medium supplement and specific human recombinant growth factors. The xeno-free OPC-differentiation medium for pluripotent stem cells was developed by using StemPro® neural stem cell xeno-free medium supplement together with human recombinant growth factors SHH, PDGF-AA, IGF-1, EGF, basic FGF and CNTF, in addition to RA, T3, human laminin and ascorbic acid. We analyzed the differentiated hESC-derived OPCs and oligodendrocytes with quantitative real-time (RT)-PCR, RT-PCR, flow cytometry and immunocytochemistry, and we performed NG2-positive selection for OPC cultures with fluorescence-activated cell sorting. Based on quantitative RT-PCR analysis, OPCs after 9 weeks of differentiation in xeno-free medium expressed OLIG2, SOX10 and NKX2.2 at elevated levels compared with control conditions. According to the flow cytometric analysis, the cells expressed A2B5 (>70%) and NG2 (40-60%) at 5 weeks time point whereas maturing oligodendrocytes expressed O4 (60-80%) at 11 weeks time point. In addition, hESC-derived OPC populations were purified based on NG2-positive selection using fluorescence-activated cell sorting. NG2-positive OPC populations survived and differentiated further into O4 expressing oligodendrocytes in xeno-free medium, and the sorted cell populations were free from pluripotent Tra1-81 and Oct-4 -positive cells. This study confirms that the xeno-free culturing method can support the differentiation and purification of hESC-derived OPC populations and provides an initial step toward safe cell graft production for the future clinical applications.

Signal Integration and Coincidence Detection in the Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase (ERK) Cascade: CONCOMITANT ACTIVATION OF RECEPTOR TYROSINE KINASES AND OF LRP-1 LEADS TO SUSTAINED ERK PHOSPHORYLATION VIA DOWN-REGULATION OF DUAL SPECIFICITY PHOSPHATASES (DUSP1 AND -6)

Diverse stimuli can feed into the MAPK/ERK cascade; this includes receptor tyrosine kinases, G protein-coupled receptors, integrins, and scavenger receptors (LDL receptor-related protein (LRP)). Here, we investigated the consequence of concomitant occupancy of the receptor tyrosine kinases (by EGF, basic FGF, VEGF, etc.) and of LRP family members (by LDL or lactoferrin). The simultaneous stimulation of a receptor tyrosine kinase by its cognate ligand and of LRP-1 (by lactoferrin or LDL) resulted in sustained activation of ERK, which was redirected to the cytoplasm. Accordingly, elevated levels of active cytosolic ERK were translated into accelerated adhesion to vitronectin. The sustained ERK response was seen in several cell types, but it was absent in cells deficient in LRP-1 (but not in cells lacking the LDL receptor). This response was also contingent on the presence of urokinase (uPA) and its receptor (uPAR), because it was absent in uPA−/− and uPAR−/− fibroblasts. Combined stimulation of the EGF receptor and of LRP-1 delayed nuclear accumulation of phosphorylated ERK. This shift in favor of cytosolic accumulation of phospho-ERK was accounted for by enhanced proteasomal degradation of dual specificity phosphatases DUSP1 and DUSP6, which precluded dephosphorylation of cytosolic ERK. These observations demonstrate that the ERK cascade can act as a coincidence detector to decode the simultaneous engagement of a receptor tyrosine kinase and of LRP-1 and as a signal integrator that encodes this information in a spatially and temporally distinct biological signal. In addition, the findings provide an explanation of why chronic elevation of LRP-1 ligands (e.g. PAI-1) can predispose to cancer.

BMP9 Protects Septal Neurons from Axotomy-Evoked Loss of Cholinergic Phenotype

BackgroundCholinergic projection from the septum to the hippocampus is crucial for normal cognitive function and degeneration of cells and nerve fibers within the septohippocampal pathway contributes to the pathophysiology of Alzheimer's disease. Bone morphogenetic protein (BMP) 9 is a cholinergic differentiating factor during development both in vivo and in vitro.Methodology/Principal FindingsTo determine whether BMP9 could protect the adult cholinergic septohippocampal pathway from axotomy-evoked loss of the cholinergic phenotype, we performed unilateral fimbria-fornix transection in mice and treated them with a continuous intracerebroventricular infusion of BMP9 for six days. The number of choline acetyltransferase (CHAT)-positive cells was reduced by 50% in the medial septal nucleus ipsilateral to the lesion as compared to the intact, contralateral side, and BMP9 infusion prevented this loss in a dose-dependent manner. Moreover, BMP9 prevented most of the decline of hippocampal acetylcholine levels ipsilateral to the lesion, and markedly increased CHAT, choline transporter CHT, NGF receptors p75 (NGFR-p75) and TrkA (NTRK1), and NGF protein content in both the lesioned and unlesioned hippocampi. In addition, BMP9 infusion reduced bilaterally hippocampal levels of basic FGF (FGF2) protein.Conclusions/SignificanceThese data indicate that BMP9 administration can prevent lesion-evoked impairment of the cholinergic septohippocampal neurons in adult mice and, by inducing NGF, establishes a trophic environment for these cells.

Basic fibroblast growth factor inhibits mineralization but induces neuronal differentiation by human dental pulp stem cells through a FGFR and PLCγ signaling pathway

Basic fibroblast growth factor (basic FGF) has pivotal roles in the function of various cell types. Here, we report the effects of basic FGF in the regulation of dental pulp stem cell (DPSC) behaviors including maintaining stemness and directing differentiation. Cells isolated from human dental pulp tissues exhibited stem cell properties including the expression of mRNA markers for embryonic and mesenchymal stem cells, the expression of Stro-1, and the multipotential differentiation. Basic FGF stimulated colony-forming units of DPSCs and up-regulated the expression of the embryonic stem cell markers; Oct4, Rex-1, and Nanog. Moreover, osteogenic medium containing basic FGF inhibited alkaline phosphatase enzymatic activity and mineralization of DPSCs. On the contrary, basic FGF appeared to be an influential growth factor in the neurogenic differentiation of DPSCs. In the presence of basic FGF, increased DPSCs neurosphere size and the up-regulation of neurogenic markers were noted. Inhibitors of FGFR or PLCγ were able to ablate the basic FGF-induced neuronal differentiation of DPSCs. Taken together, these results suggest basic FGF may be involved in the mechanisms controlling DPSCs cell fate decisions.

Basic FGF Promotes Proliferation of Ovarian Granulosa Cells in the Laying Chickens Via FGFR1 and PKC Pathway

The proliferating effect of basic fibroblast growth factor (bFGF) on granulosa cells from ovarian pre-hierarchical follicles was evaluated in the laying chickens. Expression of bFGF receptor 1 (FGFR1) from small yellow follicles was determined by immunohistochemistry and RT-PCR. The FGFR1 protein and mRNA were intensively expressed in the granulosa layer. After 8- to 24-h treatment with bFGF (0.1-100 ng/ml), the proliferation of cultured granulosa cells was remarkably enhanced in a dose- and time-dependent manner. The FGFR1 antagonist SU5402 inhibited bFGF-induced cell proliferation. This stimulating effect was further confirmed by 5-bromo-2-deoxyuridine incorporation and terminal transferase dUTP nick end-labelling assay. Immunocytochemistry of protein kinases A (PKA) and C (PKC) showed that the pro-proliferation action of bFGF predominantly activated PKC expression. Meanwhile, the bFGF-induced cell proliferation was significantly promoted by PKC activator PMA and inhibited by PKC inhibitor H(7) (p < 0.05). In addition, the bFGF-elicited cell proliferation was accompanied with increased mRNA expression of the cell cycle-regulating genes including cyclins D1 and E1, cyclin-dependent kinases 2 and 6. In conclusion, bFGF promoted the proliferation of ovarian granulosa cells through binding with FGFR1 and involving PKC pathway in the pre-hierarchical follicles of the laying chickens.

Enhancement of in vitro human tubulogenesis by endothelial cell-derived factors: implications for in vivo tubular regeneration after injury

Renal proximal tubular epithelium can regenerate after various insults. To examine whether the tubular repair process is regulated by surrounding peritubular capillaries, we established an in vitro human tubulogenesis model that mimics in vivo tubular regeneration after injury. In this model, HGF, a potent renotropic factor, dose dependently induced tubular structures in human renal proximal tubular epithelial cells cultured in gels. Consistent with regenerating tubular cells after injury, HGF-induced tubular structures expressed a developmental gene, Pax-2, and a mesenchymal marker, vimentin, and formed a lumen with aquaporin-1 expression. Electron microscopic analysis showed the presence of microvilli on the apical site of the lumen, suggesting that these structures are morphologically equivalent to renal tubules in vivo. When cocultured with human umbilical vein endothelial cells (HUVEC), HGF-induced tubular formation was significantly enhanced. This could not be reproduced by the addition of VEGF, basic FGF, or PDGF. Protein array revealed that HUVEC produced various matrix metalloproteinases (MMPs). The stimulatory effects of coculture with HUVEC or HUVEC-derived conditional medium were almost completely abolished by addition of the tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-2. These data suggest that endothelial cell-derived factors including MMPs play a critical role in tubulogenesis and imply a potential role of peritubular capillary endothelium as a source of factor(s) required for tubular recovery after injury.

Polyethylene Glycol (PEG)-induced Mouse Model of Choroidal Neovascularization

In this study, we describe a new method for inducing choroidal neovascularization (CNV) in C57BL/6 mice, an animal model of wet age-related macular degeneration (AMD). AMD is a disease that causes central blindness in humans. We injected PEG-8 subretinally in different doses (0.125-2 mg) to induce CNV. After PEG-8 injection, we examined CNV at several time points (days 3-42). We also used Western blotting, immunohistochemistry, and ELISA to examine the complement component C3 split products, C9, VEGF, TGF-β2, and basic FGF. As early as day 1 after treatment, we found that a single subretinal injection of 1 mg of PEG-8 increased the C3 split products and the C9, TGF-β2, and basic FGF levels in the retinal pigment epithelium-choroid tissue. By day 3 after PEG-8 injection, the intraocular activation of the complement system caused induction and progression of CNV, including new vessels penetrating the Bruch's membrane. At day 5 after PEG-8 injection, we observed a fully developed CNV and retinal degeneration. Thus, in this study, we present a new, inexpensive, and accelerated mouse model of CNV that may be useful to study AMD.

Abstract 5057: HuGAL-F2, a humanized monoclonal antibody to Fibroblast Growth Factor 2, effectively inhibits the growth of hepatocellular carcinoma xenografts

Abstract FGF2 (also known as basic FGF; bFGF) is a potent mitogenic and angiogenic growth factor believed to play a role in various tumors, especially hepatocellular carcinoma (HCC). In this study, we explored the therapeutic potential of the HuGAL-F2 humanized monoclonal antibody (mAb) derived from our neutralizing anti-FGF2 mAb, GAL-F2. The GAL-F2 mAb was previously shown to recognize a unique epitope on human FGF2 in a competitive binding assay with various other anti-FGF2 mAbs. By testing several FGF2-FGF1 chimeric proteins followed by alanine scanning mutagenesis of FGF2, we demonstrated that FGF2 residues F40 and M151 are critical for GAL-F2 binding and that residues R31, Y33, K55 and S152 are also part of the epitope. Using well-established humanization techniques, GAL-F2 was humanized to create HuGAL-F2, which binds FGF2 with at least as high affinity as GAL-F2. HuGAL-F2 also had similar activity to GAL-F2 in various bioassays such as inhibition of FGF2-induced proliferation of Mv 1 Lu epithelial cells and HUVEC cells. Hepatomas are characterized by neovascularization, and angiogenesis plays a key role in their growth. Thus, we determined the levels of two angiogenic factors, FGF2 and VEGF, secreted from several HCC cell lines (HEP-G2, HLE, HLF, SK-HEP-1, and SMMC-7721) using ELISA. All these cell lines secreted significant levels of FGF2 and VEGF. To demonstrate that FGF2 indeed plays an important role in HCC tumor growth and to investigate the therapeutic potential of HuGAL-F2, we tested the efficacy of HuGAL-F2 in xenograft tumor models of the HCC lines, SMMC-7721 and HEP-G2. HuGAL-F2, administered i.p. at 5 mg/kg twice per week, significantly inhibited the growth of established xenografts of both these cell lines. Moreover, in the HEP-G2 model, GAL-F2 and HuGAL-F2 were equally effective as sorafenib (Nexavar) and the A4.6.1 anti-VEGF mouse precursor mAb of bevacizumab (Avastin) and had strong additive activity with each of these agents. We conclude that FGF2 is a promising molecular target, and that HuGAL-F2 is a viable clinical candidate for the treatment of HCC and possibly other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5057. doi:10.1158/1538-7445.AM2011-5057

Abstract 5056: HuGAL-FR21, a humanized monoclonal antibody to Fibroblast Growth Factor Receptor 2, effectively inhibits the growth of gastric tumor xenografts

Abstract The family of 22 fibroblast growth factors include potent mitogenic and angiogenic factors believed to play a role in the growth of many tumors. The various FGFs bind to only four tyrosine kinase signaling receptors, Fibroblast Growth Factor Receptors 1-4 (FGFR1-4). FGFR2 is a major cellular receptor for FGF2 (basic FGF), FGF7 (keratinocyte growth factor, KGF) and FGF10; and mutation or overexpression of FGFR2 is associated with a number of cancers, notably endometrial cancer and gastric carcinoma. In this study, we explored the therapeutic potential of the HuGAL-FR21 humanized monoclonal antibody (mAb) derived from our blocking anti-FGFR2 mAb, GAL-FR21. We previously showed that GAL-FR21 binds to only the FGFR2IIIb isoform of FGFR2, which is expressed on epithelial tissues and thus more commonly on carcinomas. Using well-established humanization techniques, GAL-FR21 was humanized to create HuGAL-FR21, which binds human FGFR2IIIb with at least as high affinity as GAL-FR21. HuGAL-FR21, like GAL-FR21, also bound well to monkey and mouse FGFR2IIIb, facilitating study of this mAb in animal models. In functional assays, HuGAL-FR21 inhibited binding of FGF2, FGF7 and FGF10 to FGFR2IIIb as completely as GAL-FR21. And HuGAL-FR21 inhibited FGF7 or FGF10-induced proliferation of the Ba/F3 cells transfected with FGFR2IIIb, actually better than GAL-FR21. Finally, HuGAL-FR21 inhibited phosphorylation of cellular FGFR2 induced by FGF7 and FGF10 as well as GAL-FR21. There are two major types of gastric cancer, well-differentiated and poorly differentiated, and published results show that many poorly differentiated tumors overexpress FGFR2, which is in fact the oncogenic driver for these tumors. We therefore tested the ability of HuGAL-FR21 to inhibit growth of xenografts of two cell lines, SNU-16 and OCUM-2M, derived from FGFR2-overexpressing, poorly differentiated gastric tumors. HuGAL-FR21, administered i.p. at 2.5 or 5 mg/kg twice per week, strongly inhibited the growth of established xenografts of both these cell lines. We conclude that FGFR2 is a promising molecular target, and that HuGAL-FR21 is a viable clinical candidate for the treatment of gastric and possibly other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5056. doi:10.1158/1538-7445.AM2011-5056

Nuclear Orphan Receptor TLX Induces Oct-3/4 for the Survival and Maintenance of Adult Hippocampal Progenitors upon Hypoxia

Hypoxia promotes neural stem cell proliferation, the mechanism of which is poorly understood. Here, we have identified the nuclear orphan receptor TLX as a mediator for proliferation and pluripotency of neural progenitors upon hypoxia. We found an enhanced early protein expression of TLX under hypoxia potentiating sustained proliferation of neural progenitors. Moreover, TLX induction upon hypoxia in differentiating conditions leads to proliferation and a stem cell-like phenotype, along with coexpression of neural stem cell markers. Following hypoxia, TLX is recruited to the Oct-3/4 proximal promoter, augmenting the gene transcription and promoting progenitor proliferation and pluripotency. Knockdown of Oct-3/4 significantly reduced TLX-mediated proliferation, highlighting their interdependence in regulating the progenitor pool. Additionally, TLX synergizes with basic FGF to sustain cell viability upon hypoxia, since the knockdown of TLX along with the withdrawal of growth factor results in cell death. This can be attributed to the activation of Akt signaling pathway by TLX, the depletion of which results in reduced proliferation of progenitor cells. Cumulatively, the data presented here demonstrate a new role for TLX in neural stem cell proliferation and pluripotency upon hypoxia.

Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition

Cell therapy with bone marrow multipotential stromal cells (MSCs) represents a promising approach to promote wound healing and tissue regeneration. MSCs expanded in vitro lose early progenitors with differentiation and therapeutic potentials under normoxic condition, whereas hypoxic condition promotes MSC self-renewal through preserving colony forming early progenitors and maintaining undifferentiated phenotypes. Hypoxia inducible factor (HIF) pathway is a crucial signaling pathway activated in hypoxic condition. We evaluated the roles of HIFs in MSC differentiation, colony formation, and paracrine activity under hypoxic condition. Hypoxic condition reversibly decreased osteogenic and adipogenic differentiation. Decrease of osteogenic differentiation depended on HIF pathway; whereas decrease of adipogenic differentiation depended on the activation of unfolded protein response (UPR), but not HIFs. Hypoxia-mediated increase of MSC colony formation was not HIF-dependent also. Hypoxic exposure increased secretion of VEGF, HGF, and basic FGF in a HIF-dependent manner. These findings suggest that HIF has a limited, but pivotal role in enhancing MSC self-renewal and growth factor secretions under hypoxic condition.

Longterm cultures of the aged human RPE do not maintain epithelial morphology and high transepithelial resistance

Dear Editor, Visual rehabilitation achieved with peripheral RPE autografts in exudative AMD is only modest. Moreover, complication rates using the popular RPE–choroid patch technique remain relatively high. Tissue-engineered transplants might improve the outcome of RPE transplantation. A culturing step could place cells on a substrate prior to transplantation, thereby optimizing delivery and long-term function [1]. This would require sufficient cellular plasticity (i.e., response to environmental stimuli) to achieve a well-differentiated, functional graft. While culture plasticity was confirmed for fetal cells or RPE cell lines [2, 3], only sparse information is available for aged RPE [4]. We studied the influence of culture conditions on select differentiation characteristics in long-term primary RPE cultures from ten human donors above age 55, and compared them to a 23-year-old and two fetal donors (20 and 21 weeks of gestation respectively). The main outcome measures at 6 weeks post-confluence (PC) were morphology and transepithelial resistance (TER), an electrophysiological method for assessing tight junctions build-up. Eyes were preserved within 6 hours of death. Adult primary RPE cultures (aRPE) were initiated from: (1) mechanically scraped RPE, or (2) collagenase pretreated (1 mg/ml for 40 minutes, CLS I, Worthington Biochemical Corp, Lakewood, NJ, USA) and mechanically scraped RPE, or (3) fetal (fRPE) and adult cultures started from floater passages [2]. Cells were grown on permeable polyester inserts (Transwells, Corning Inc., Corning, NY, USA) in modified Hu & Bok media [2], containing 2% calf serum, basic FGF (5 ng/ml), L-alanyl-L-glutamine (2 mM), Albumax (200 μg/ml), and antibiotics/antimycotics. RPE culture purity was confirmed by a uniform pancytokeratin staining. Experimental matrices analyzed the influence of above harvesting methods, seeding density, laminin coating (2 μg/cm), and media composition (2% vs 10% serum until confluence, or calcium switch at confluence, or 5 mM vs 25 mM glucose) on above outcome measures. Morphology was monitored weekly in all and transepithelial resistance (TER) in all conditions, except the laminin matrix (i.e., seven of ten aged donors). Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, FL, USA, April 30–May 4 2006.

Basic fibroblast growth factor enhances osteogenic and chondrogenic differentiation of human bone marrow mesenchymal stem cells in coral scaffold constructs

Temporomandibular joint (TMJ) disorders are commonly occurring degenerative joint diseases that require surgical replacement of the mandibular condyle in severe cases. Transplantation of tissue-engineered mandibular condyle constructs may solve some of the current surgical limitations to TMJ repair. We evaluated the feasibility of mandibular condyle constructs engineered from human bone marrow-derived mesenchymal cells (BMSCs). Specifically, human BMSCs were transfected with basic FGF (bFGF) gene-encoding plasmids and induced to differentiate into osteoblasts and chondroblasts. The cells were seeded onto mandibular condyle-shaped porous coral scaffolds and evaluated for osteogenic/chondrogenic differentiation, cell proliferation, collagen deposition and tissue vascularization. Transfected human BMSCs expressed bFGF and were highly proliferative. Osteogenesis was irregular, showing neovascularization around new bone tissue. There was no evidence of bilayered osteochondral tissue present in normal articulating surfaces. Collagen deposition, characteristic of bone and cartilage, was observed. Subcutaneous transplantation of seeded coral/hydrogel hyaluran constructs into nude mice resulted in bone formation and collagen type I and type II deposition. Neovascularization was observed around newly formed bone tissue; bFGF expression was detected in implanted constructs seeded with bFGF expressing hBMSCs. This report demonstrates that engineered porous coral constructs using bFGF gene-transfected human BMSCs may be a feasible option for surgical transplantation in TMJ repair.

Friedreich's Ataxia Induced Pluripotent Stem Cells Model Intergenerational GAA⋅TTC Triplet Repeat Instability

The inherited neurodegenerative disease Friedreich's ataxia (FRDA) is caused by GAA⋅TTC triplet repeat hyperexpansions within the first intron of the FXN gene, encoding the mitochondrial protein frataxin. Long GAA⋅TTC repeats cause heterochromatin-mediated gene silencing and loss of frataxin in affected individuals. We report the derivation of induced pluripotent stem cells (iPSCs) from FRDA patient fibroblasts by transcription factor reprogramming. FXN gene repression is maintained in the iPSCs, as are the global gene expression signatures reflecting the human disease. GAA⋅TTC repeats uniquely in FXN in the iPSCs exhibit repeat instability similar to patient families, where they expand and/or contract with discrete changes in length between generations. The mismatch repair enzyme MSH2, implicated in repeat instability in other triplet repeat diseases, is highly expressed in pluripotent cells and occupies FXN intron 1, and shRNA silencing ofMSH2 impedes repeat expansion, providing a possible molecular explanation for repeat expansion in FRDA.

Generation of Induced Pluripotent Stem Cells in Rabbits: POTENTIAL EXPERIMENTAL MODELS FOR HUMAN REGENERATIVE MEDICINE

Human induced pluripotent stem (iPS) cells have the potential to establish a new field of promising regenerative medicine. Therefore, the safety and the efficiency of iPS-derived cells must be tested rigorously using appropriate animal models before human trials can commence. Here, we report the establishment of rabbit iPS cells as the first human-type iPS cells generated from a small laboratory animal species. Using lentiviral vectors, four human reprogramming genes (c-MYC, KLF4, SOX2, and OCT3/4) were introduced successfully into adult rabbit liver and stomach cells. The resulting rabbit iPS cells closely resembled human iPS cells; they formed flattened colonies with sharp edges and proliferated indefinitely in the presence of basic FGF. They expressed the endogenous pluripotency markers c-MYC, KLF4, SOX2, OCT3/4, and NANOG, whereas the introduced human genes were completely silenced. Using in vitro differentiating conditions, rabbit iPS cells readily differentiated into ectoderm, mesoderm, and endoderm. They also formed teratomas containing a variety of tissues of all three germ layers in immunodeficient mice. Thus, the rabbit iPS cells fulfilled all of the requirements for the acquisition of the fully reprogrammed state, showing high similarity to their embryonic stem cell counterparts we generated recently. However, their global gene expression analysis revealed a slight but rigid difference between these two types of rabbit pluripotent stem cells. The rabbit model should enable us to compare iPS cells and embryonic stem cells under the same standardized conditions in evaluating their ultimate feasibility for pluripotent cell-based regenerative medicine in humans.

Immunohistochemical study of the growth factors, aFGF, bFGF, PDGF-AB, VEGF-A and its receptor (Flk-1) during arteriogenesis

Growth factors are viewed as main arteriogenic stimulators for collateral vessel growth. However, the information about their native expression and distribution in collateral vessels is still limited. This study was designed to profile expression of acidic and basic FGF, platelet-derived growth factor (PDGF-AB) and vascular endothelial growth factor (VEGF-A) and its receptor, fetal liver kinase-1 (Flk-1) during arteriogenesis by confocal immunofluorescence in both dog ameroid constrictor model and rabbit arteriovenous shunt model of arteriogenesis. We found that: (1) in normal arteries (NA) in dog heart, aFGF, bFGF, and PDGF-AB all were mainly expressed in endothelial cells (EC) and media smooth muscle cells (SMC), but the expression of aFGF was very weak, with those of the other two being moderate; (2) in collateral arteries (CAs), aFGF, bFGF, and PDGF-AB all were significantly upregulated (P < 0.05); they were present in all the layers of the vascular wall and were 2.1, 1.7, and 1.9 times higher than that in NA, respectively; and (3) in NA in rabbit hind limb, VEGF-A was absent, Flk-1 was only weakly present in endothelial cells, but in one week CAs VEGF-A and Flk-1 were significantly increased in both shunt and ligation sides; this was more evident in the shunt-side CAs, 2.3, and 2 times higher than that in the ligation side, respectively. In conclusion, our data demonstrate for the first time that growth factors, aFGF, bFGF, and PDGF-AB are significantly upregulated in collateral vessels in dog heart, and enhanced VEGF-A and its receptor, Flk-1, are associated with rapid and lasting increased shear stress. These findings suggest that endogenous production of growth factors could be an important factor promoting collateral vessel growth.

Serum levels of VEGF and bFGF in hypoxic patients with exacerbated COPD

Hypoxia frequently complicates the course of chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the two most potent angiogenic factors and may play a role in adaptation to hypoxia. The aims of the study were to assess the serum levels of VEGF and bFGF and to evaluate their mutual relationship in hypoxic patients with exacerbated COPD. The study group consisted of 50 hypoxic (PaO(2) 53 mmHg) patients with exacerbated COPD. Control groups were 30 stable COPD patients with PaO(2) 70 mmHg, and 30 healthy blood donors. The serum concentrations of VEGF and bFGF were measured using commercial enzyme-linked immunoassay kits. Patients with exacerbated COPD had significantly higher serum VEGF levels (1,089.16 +/- 1,128.03 pg/mL) compared to those with stable COPD (197.68 +/- 178.06 pg/mL) (p < 0.0001) and healthy blood donor group (257.69 +/- 170.4 pg/mL) (p < 0.0001). Serum bFGF levels were significantly higher in the exacerbated COPD group (6.15 +/- 2.56 pg/mL) compared to control groups (p = 0.0001). Basic FGF was undetectable in the stable COPD and blood donor groups. Since VEGF and bFGF correlated significantly with the majority of factors investigated in COPD patients, multivariate analysis was performed. According to the step-wise regression analysis, VEGF was best determined by PaO(2), WBC and IL-6. Basic FGF was best determined by PaO(2) and pH. The highly significant, simple correlation between VEGF and bFGF was lost in multivariate analysis. This suggests that their correlation is not independent, but due to factors that remain in the model after step-wise regression. These are essentially linked to the level of hypoxia. Results of our study suggest that VEGF and bFGF production is stimulated in hypoxic patients with exacerbated COPD. Elevated levels of VEGF and bFGF may activate the process of neoangiogenesis, which may lead to increased perfusion and an improvement in tissue oxygenation in this group of patients.

Plasma biomarker analysis of a phase I trial of ASA404 (vadimezan) in combination with paclitaxel and carboplatin in Japanese patients with non-small cell lung cancer (NSCLC).

e18035 Background: ASA404 (vadimezan, DMXAA) is a flavonoid, non-tubulin-binding tumor-vascular disrupting agent (Tumor- VDA) that causes irreversible tumor vasculature disruption and extensive hemorrhagic necrosis of the tumor core. In combination with paclitaxel and carboplatin (P/C), ASA404 increased the median overall survival of previously untreated NSCLC patients from 8.8 to 14.0 months in a phase II clinical trial. Methods: A phase I trial assessed the safety profile and tolerability of three dose levels of ASA404 (600, 1,200, and 1,800 mg/m2) administered in combination with P/C (P, 200 mg/m2; C, AUC 6) every 3 weeks in Japanese patients with NSCLC. A panel of plasma biomarkers of angiogenesis and cytokines were evaluated to investigate the pharmacodynamic effect and mechanism of action (MOA) of ASA404. Plasma VEGF and basic FGF (bFGF) were measured by multiplex assays using the Meso-Scale Discovery platform. Plasma IL-8 and macrophage chemotactic protein (MCP)1 were analyzed by Luminex beads platform. Plasma 5-hydroxyindole-3-acetic acid (5HIAA), a by-product of platelet activation and putative biomarker for ASA404's vascular disruption activity, was analyzed by tandem mass spectrometry. Results: Four-hours after ASA404 infusion at Cycle 1 Day 1, plasma 5HIAA levels increased in a dose-dependent manner by 15%, 111%, or 137% compared with pre-ASA404 treatment level in the 600 (n = 3), 1,200 (n = 6) or 1,800 (n = 6) mg/m2 groups, respectively. Plasma VEGF levels decreased by 5 to 50% after 4 h and 24 h of ASA404 infusion. Plasma bFGF levels decreased by 55 to 88% after 24 h ASA404 infusion in the three ASA404 dose groups. Furthermore, over 50% induction of plasma IL-8 and MCP-1 were observed after 24 h infusion of ASA404. Conclusions: ASA404 caused dose-dependent induction of plasma 5HIAA levels. Unlike anti-angiogenic compounds such as sunitinib, sorafenib or bevacizumab which upregulate systemic VEGF, ASA404 did not appear to cause a systemic VEGF elevation. Induction of plasma inflammatory cytokines (IL-8, MCP-1) after ASA404 treatment may represent ASA404 MOA and amplify the direct antivascular effect of ASA404. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis

Mycophenolate mofetil modifies kidney tubular injury and Foxp3 + regulatory T cell trafficking during recovery from experimental ischemia–reperfusion

Lymphocytes participate in the early pathogenesis of ischemia–reperfusion injury (IRI) in kidney; however, their role during repair is largely unknown. Recent data have shown that Foxp3 + regulatory T cells (Tregs) traffic into kidney during healing from IRI and directly participate in repair. Since lymphocyte-targeting therapy is currently administered to prevent rejection during recovery from IRI in renal transplants, we hypothesized that mycophenolate mofetil (MMF) would alter Treg trafficking and kidney repair. C57BL/6 J and T cell deficient mice underwent unilateral clamping of renal pedicle for 45 min, followed by reperfusion, and were sacrificed at day 10. Mice were treated with saline (C) or MMF (100 mg/kg) i.p. daily starting at day 2 until sacrifice ( n = 5–12/group). MMF worsened kidney tubular damage compared to C at 10 days (cortex and outer medulla: p < 0.05) in wild-type mice; tubular apoptotic index was increased in cortex in MMF group as well ( p = 0.01). MMF reduced the total number of kidney-infiltrating mononuclear cells ( p < 0.001 versus C) and the percentages of TCRβ +CD4 + and TCRβ +CD8 + T cells ( p < 0.01), but not natural killer (NK), NKT or B lymphocytes. MMF specifically reduced kidney Foxp3 + Tregs (0.82 ± 0.11% versus 1.75 ± 0.17%, p < 0.05). Tubular proliferative index and tissue levels of basic FGF were increased in MMF group ( p < 0.05), IL-10 and IL-6 were decreased ( p < 0.05). To evaluate if MMF effect occurred through non-lymphocytic cells, T cell deficient mice were treated with MMF. Tubular injury in T cell deficient mice was not affected by MMF treatment, though MMF-treated animals had increased VEGF and decreased PDGF-BB protein tissue levels compared to controls ( p < 0.05). Thus, MMF modifies the structural, epithelial proliferative and inflammatory response during healing, likely through effects on T cells and possibly Tregs. Kidney repair after IRI can be altered by agents that target lymphocytes.

Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells.

Although adipose tissue is an expandable and readily attainable source of proliferating, multipotent stem cells, its potential for use in regenerative medicine has not been extensively explored. Here we report that adult human and mouse adipose-derived stem cells can be reprogrammed to induced pluripotent stem (iPS) cells with substantially higher efficiencies than those reported for human and mouse fibroblasts. Unexpectedly, both human and mouse iPS cells can be obtained in feeder-free conditions. We discovered that adipose-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF, TGFbeta, fibronectin, and vitronectin and can serve as feeders for both autologous and heterologous pluripotent cells. These results demonstrate a great potential for adipose-derived cells in regenerative therapeutics and as a model for studying the molecular mechanisms of feeder-free iPS generation and maintenance.