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Supplementary Methods from High Plasma Levels and Effective Lymphatic Uptake of Docetaxel in an Orally Available Nanotransporter Formulation

Docetaxel, an efficient chemotherapeutic drug, exhibits low and variable oral bioavailability due to the active efflux by P-glycoprotein (P-gp) and more so to CYP3A4 gut metabolism. Using a spray-drying technique, docetaxel was incorporated in PLGA [poly(lactic-co-glycolic acid)] nanocapsules (NC) which were embedded in entero-coated microparticles. An oral administration of the NC formulation elicited a higher absolute bioavailability than both a docetaxel solution (276%) and a free docetaxel NC formulation (400%) injected intravenously, a 5-mg/kg dose. The batches (B) I and II NC formulations elicited Cmax values that were 1,735% and 2,254%, respectively; higher than the Cmax value of the oral docetaxel solution combined with blank microparticles, a 10-mg/kg dose. No significant difference in AUC (area under curve) was observed between the batches. These unexpected results can be explained only if the pharmacokinetics of docetaxel had been modified. It was shown that NCs released from the microparticles penetrated the enterocytes, bypassing P-gp; apparently circumventing gut metabolism and accumulating within the lymphatic system from where both intact or biodegraded NCs and free docetaxel were progressively released into the circulation as plausibly supported by the fluorescent imaging results. Furthermore, the circulating docetaxel in plasma was unencapsulated and circulated either in free form or bound to albumin. Both free docetaxel NCs and microparticles exhibited in vitro efficacy on WRC 256 cells suggesting that the activity of docetaxel was not altered. This delivery concept has potential for clinical translation, perhaps allowing docetaxel chemotherapy to be switched from intravenous to oral delivery. Cancer Res; 71(8); 3018–28. ©2011 AACR.

There is a clear unmet clinical need for novel biotechnology-based therapeutic approaches to lung repair and/or replacement, such as tissue engineering of whole bioengineered lungs. Recent studies have demonstrated the feasibility of decellularizing the whole organ by removal of all its cellular components, thus leaving behind the extracellular matrix as a complex three-dimensional (3D) biomimetic scaffold. Implantation of decellularized lung scaffolds (DLS), which were recellularized with patient-specific lung (progenitor) cells, is deemed the ultimate alternative to lung transplantation. Preclinical studies demonstrated that, upon implantation in rodent models, bioengineered lungs that were recellularized with airway and vascular cells were capable of gas exchange for up to 14 days. However, the long-term applicability of this concept is thwarted in part by the failure of current approaches to reconstruct a physiologically functional, quiescent endothelium lining the entire vascular tree of reseeded lung scaffolds, as inferred from the occurrence of hemorrhage into the airway compartment and thrombosis in the vasculature in vivo. In this review, we explore the idea that successful whole lung bioengineering will critically depend on 1) preserving and/or reestablishing the integrity of the subendothelial basement membrane, especially of the ultrathin respiratory membrane separating airways and capillaries, during and following decellularization and 2) restoring vascular physiological functionality including the barrier function and quiescence of the endothelial lining following reseeding of the vascular compartment. We posit that physiological reconstitution of the pulmonary vascular tree in its entirety will significantly promote the clinical translation of the next generation of bioengineered whole lungs.

Mesenchymal stem cells (MSCs) of different biological sources are in Phase 1 clinical trials and are being considered for Phase 2 therapy of lung disorders, and lung (progenitor) cells derived from pluripotent stem cells (SCs) are under development in preclinical animal models.PubMed.gov and ClinicalTrials.gov.There is consensus about the therapeutic potential of transplanted SCs, mainly MSCs, primarily involves paracrine 'bystander' effects that confer protection of the epithelial and endothelial linings of the lung caused by inflammation and/or fibrosis and lead to increased survival in animal models. Clinical trials of Phase 1 indicate safety and suggest that the efficacy of SC therapy in patients with various lung diseases will require a higher dosage than previously evaluated.A growing interest in the re-epithelialization and re-endothelialization of damaged lung tissue involves the putative pulmonary differentiation of exogenous MSCs. Currently, it is not clear whether or not the observed regeneration of distal airways/vasculature is derived from lung-resident and/or transplanted SCs.Important topics under investigation include optimization of the cell source with a decrease in cell population heterogeneity characterized by defined markers, route of delivery for effective treatment, potential dose and therapeutic protocol of SC application, development of quantitative assays and biomarkers of lung disease and repair, and the potential use of tissue engineered lung.Ability of MSCs to differentiate into epithelial cells of the lung, use of autologous induced pluripotent SCs (iPSCs) derived from the patients, complete biochemical characterization of the secretome of SCs used for therapy, and the incorporation of simultaneous and/or subsequent treatment with drugs which also aid in lung repair and regeneration.Although safety of MSC-based cell therapy was proved in Phase 1, efficacy, long-term survival and preservation of lung respiratory function need to be further evaluated, cautioning against hastily translating SCs therapy from animal models of lung injury to clinical trials of patients with lung disorders.

We investigated the effects of hypoxia on spontaneous (SP)- and activin A (AA)-induced definitive endoderm (DE) differentiation of mouse embryonic stem cells (mESCs) and their subsequent differentiation into distal pulmonary epithelial cells. SP differentiation for 6 days of mESCs toward endoderm at hypoxia of 1% O2, but not at 3% or 21% (normoxia), increased the expression of Sox17 and Foxa2 by 31- and 63-fold above maintenance culture, respectively. Treatment of mESCs with 20 ng/mL AA for 6 days under hypoxia further increased the expression of DE marker genes Sox17, Foxa2, and Cxcr4 by 501-, 1,483-, and 126-fold above maintenance cultures, respectively. Transient exposure to hypoxia, as short as 24 h, was sufficient to enhance AA-induced endoderm formation. The involvement of hypoxia-inducible factor (HIF)-1α and reactive oxygen species (ROS) in the AA-induced endoderm enrichment was assessed using HIF-1α(-/-) mESCs and the ROS scavenger N-acetylcysteine (NAC). Under SP conditions, HIF-1α(-/-) mESCs failed to increase the expression of endodermal marker genes but rather shifted toward ectoderm. Hypoxia induced only a marginal potentiation of AA-induced endoderm differentiation in HIF-1α(-/-) mESCs. Treatment of mESCs with AA and NAC led to a dose-dependent decrease in Sox17 and Foxa2 expression. In addition, the duration of exposure to hypoxia in the course of a recently reported lung differentiation protocol resulted in differentially enhanced expression of distal lung epithelial cell marker genes aquaporin 5 (Aqp5), surfactant protein C (Sftpc), and secretoglobin 1a1 (Scgb1a1) for alveolar epithelium type I, type II, and club cells, respectively. Our study is the first to show the effects of in vitro hypoxia on efficient formation of DE and lung lineages. We suggest that the extent of hypoxia and careful timing may be important components of in vitro differentiation bioprocesses for the differential generation of distal lung epithelial cells from pluripotent progenitors.

Conditioned media (CM) of transformed cells, such as the human lung-derived A549 cells, is a useful tool for directing differentiation of embryonic stem cells (ESCs). Previous work indicates that A549-CM induced pulmonary differentiation of mouse ESCs (mESCs). In this study, we compared the effects of A549-CM treatment on the differentiation of mESCs organized in monolayer or embryoid bodies. We analyzed the cultures treated with A549-CM using specific lineage markers by quantitative polymerase chain reaction (qPCR) and lineage-focused PCR arrays and demonstrated heterogeneous CM-induced differentiation. We then constructed bioinformatics-based gene networks to establish correlations between the upregulated lineage-specific genes and proteins in the A549-CM identified by proteomic analysis. Network analysis supported the phenotypic and genotypic heterogeneic differentiation of mESCs into multiple cell lineages via enriched stemness, cardiovascular, neuronal, and lung development gene ontologies (GOs). The significant enrichment toward lung ontologies was specific for treatment with A549-CM, but not CM of liver (HepG2) and pancreas (Capan-1) cells. Based on network analysis, we identified laminin alpha5, prosaposin, lamin A/C, dickkopf homolog 1, clusterin, and calreticulin as the most relevant proteins related to the enrichment of lung GOs. We validated the effects of laminin isoforms on mESC differentiation in vitro and found enriched differential induction of surfactant protein gene expression. Our data suggest that A549-CM can be used for identifying secreted proteins for the heterogeneous mixed-lineage differentiation of mESCs toward a variety of lung-relevant cells. Such a heterogeneous cell population will be required for the in vitro generation of complex lung tissue and mixed cell populations for regenerative pulmonary therapy.

Repopulation of decellularized lung scaffolds (DLS) is limited due to alterations in the repertoire and ratios of the residual extracellular matrix (ECM) proteins, characterized by e.g., the retention of type I collagen and loss of glycoproteins. We hypothesized that pre-treatment of decellularized matrices with defined ECM proteins, which match the repertoire of integrin receptors expressed by the cells to be seeded (e.g., embryonic stem cells) can increase the efficacy of the reseeding process. To test this hypothesis, we first determined the integrin receptors profile of mouse embryonic stem cells (mESCs). Mouse ESCs express α3, α5, α6, α9 and β1, but not α1, α2 and α4 integrin subunits, as established by Western blotting and adhesion to laminin and fibronectin, but not to collagens type I and IV. Reseeding of DLS with mESCs was inefficient (6.9 ± 0.5%), but was significantly enhanced (2.3 ± 0.1 fold) by pre-treating the scaffolds with media conditioned by A549 human lung adenocarcinoma cells, which we found to contain ~5 μg/ml laminin. Furthermore, pre-treatment with A549-conditioned media resulted in a significantly more uniform distribution of the seeded mESCs throughout the engineered organ as compared to untreated DLS. Our study may advance whole lung engineering by stressing the importance of matching the integrin receptor repertoire of the seeded cells and the cell binding motifs of DLS.

In this study, we present the applicability of imaging epidermal growth factor (EGF) receptor levels in preclinical models of COLO205 carcinoma cells in vitro, mice with orthotopic tumors and ex vivo colorectal tumor biopsies, using EGF-labeled with IRDye800CW (EGF-NIR). The near infrared (NIR) bio-imaging of COLO205 cultures indicated specific and selective binding, reflecting EGF receptors levels. In vivo imaging of tumors in mice showed that the highest signal/background ratio between tumor and adjacent tissue was achieved 48 hours post-injection. Dissected colorectal cancer tissues from different patients demonstrated ex vivo specific imaging using the NIR bio-imaging platform of the heterogeneous distributed EGF receptors. Moreover, in the adjacent gastrointestinal tissue of the same patients, which by Western blotting was demonstrated as EGF receptor negative, no labeling with EGF-NIR probe was detected. Present results support the concept of tumor imaging by measuring EGF receptor levels using EGF-NIR probe. This platform is advantageous for EGF receptor bio-imaging of the NCI-60 recommended panel of tumor cell lines including 6–9 colorectal cell lines, since it avoids radioactive probes and is appropriate for use in the clinical setting using NIR technologies in a real-time manner.

Umbilical cord blood (CB) stem cells have been proposed for cell-based therapeutic applications for diverse diseases of the CNS. We hypothesized that tissue-engineering strategies may extend the efficacy of these approaches by improving the long-term viability and function of stem cell-derived neuronal progenitors. To test our hypothesis, we explored the survival and differentiation of human CB-derived neuronal progenitors (HUCBNP) in a three-dimensional (3D) collagen construct. In contrast to two-dimensional culture conditions, the cells survived in 3D for an extended period of time of more than 2 months. Under 3D conditions, HUCBNP underwent spontaneous neuronal differentiation, which was further enhanced by treatment with neuronal conditioned medium (CM) and nerve growth factor (NGF). Neurite outgrowth, quantified by assessing the fractal dimension (D f) of the complex neuronal networks, was significantly enhanced under 3D conditions in the presence of CM/NGF, concomitant with a reduced expression of the early neuronal marker nestin (1.9-fold), and increased levels of mature neuronal markers such as MAP-2 (3.6-fold), β-tubulin (1.5-fold), and neuronal specific enolase (6.6-fold) and the appearance of the synaptic marker synaptophysin. To assess the feasibility for clinical usage, HUCBNP were also isolated from frozen CB samples and cultured under 3D conditions. The data indicate the essential complete preservation of neurotrophic (survival) and neurotropic (neurite outgrowth) properties. In conclusion, 3D culture conditions are proposed as an essential step for both maintenance of CB neuronal progenitors in vitro and for investigating specific features of neuronal differentiation towards future use in regenerative therapy.

The goal of this study was to compare the neuroprotective properties of the l -type Ca2+ channel blockers, nimodipine and nifedipine, using nerve growth factor (NGF)-differentiated PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) and trophic withdrawal-induced cell death. Nimodipine (1–100 μM) conferred 65 ± 13% neuroprotection upon exposure to OGD and 35 ± 6% neuroprotection towards different trophic withdrawal-induced cell death measured by lactate dehydrogenase and caspase 3 activities. The time window of nimodipine conferred neuroprotection was detected during the first 5 h but not at longer OGD exposures. Nifedipine (1–100 μM), to a lower potency than nimodipine, conferred 30–55 ± 8% neuroprotection towards OGD in PC12 cells and 29 ± 5% in rat hypocampal slices, and 10 ± 3% neuroprotection at 100 μM towards trophic withdrawal-induced PC12 cell death. The ability to demonstrate that nimodipine conferred neuroprotection in a narrow therapeutic time-window indicates that the OGD PC12 model mimics the in vivo models and therefore suitable for neuroprotective drug discovery and development.

A new liposome-based near-infrared probe that combines both imaging and targeting abilities was developed for application in medical imaging. The near-infrared fluorescent molecule indocyanine green (ICG), and the cetuximab monoclonal antibody for epidermal growth factor receptor (EGFR) were attached to liposomes by passive adsorption. It was found that ICG molecules adsorbed to the liposomes are more fluorescent than free ICG and have a larger quantum yield. Cetuximab-adsorbed fluorescent liposomes preserved EGFR recognition, as is evident from internalization and selective binding to A431 colon carcinoma cells overexpressing EGFR. The binding of cetuximab-targeted fluorescent liposomes to A431 compared with IEC-6 cells (normal enterocytes expressing physiological EGFR levels) was greater by a factor of 3.5, ensuring imaging abilities with available fluorescent equipment. Due to relatively high quantum yield and specific tumor cell-recognizing ability, this technology deserves further in vivo evaluation for imaging and diagnostic purposes. From the Clinical Editor A new liposome-based near-infrared probe combining both imaging and targeting abilities is reported. Due to relatively high quantum yield and EGFR-expressing tumor cell specificity, this technology deserves further in vivo evaluation for imaging and diagnostic purposes.

Human umbilical cord blood (HUCB), a valuable source for cell therapy of hematologic diseases, was recently found to confer neuroprotection in animal models of brain injury. To explore the neuroprotective properties of HUCB in an ischemic environment, we studied a unique sub-population of collagen-adherent cells. In the presence of nerve growth factor (NGF) and interferon-gamma, these cells differentiate in vitro into a neuronal phenotype (termed HUCBNPs). Using a closed-head injury brain trauma model, we recently demonstrated neuroprotective effects of HUCB upon their local injection into the brain. Our in-vitro studies indicated that upon co-culture with ischemia exposed PC12 cells, HUCBNP conferred about 30% neuroprotection, as assessed by decreased lactate dehydrogenase (LDH) release and inhibition of caspase-3 activity. HUCBNP decreased by 95% the level of free radicals in the insulted-PC12 cells, which was in correlation with the appearance of antioxidants and NGF in the medium. HUCBNP-induced neuroprotection also correlated with changes in NGF mRNA expression levels, a process which was blocked by K252a, an antagonist of the NGF-neurotrophin receptor TrkA. To further extend the possible contribution of NGF-mediated protective role, we investigated its neuroprotective mechanisms using PC12 cells and brain capillary endothelial cells exposed to ischemic insult. Pretreatment of the cultures with NGF conferred neuroprotection in a dose- and time-dependent fashion. Time course experiments indicated marked activation of mitogen activated protein kinasas (MAPKs) during the ischemic insult, while treatment of the cultures with NGF inhibited them to different degrees. It appears that both neuronal and endothelial compartments may be protected by NGF under ischemic conditions. Therefore, in future neuroprotection studies, we propose to address additional components of the neurovascular unit such as astrocytes and pericytes in mono-, co- and multi-culture setups. In conclusion, our findings indicate that HUCBNP-induced neuroprotection involves antioxidant(s) and NGF, which by paracrine and/or autocrine route, affects all components of the neurovascular unit.

Docetaxel, an efficient chemotherapeutic drug, exhibits low and variable oral bioavailability due to the active efflux by P-glycoprotein (P-gp) and more so to CYP3A4 gut metabolism. Using a spray-drying technique, docetaxel was incorporated in PLGA [poly(lactic-co-glycolic acid)] nanocapsules (NC) which were embedded in entero-coated microparticles. An oral administration of the NC formulation elicited a higher absolute bioavailability than both a docetaxel solution (276%) and a free docetaxel NC formulation (400%) injected intravenously, a 5-mg/kg dose. The batches (B) I and II NC formulations elicited Cmax values that were 1,735% and 2,254%, respectively; higher than the Cmax value of the oral docetaxel solution combined with blank microparticles, a 10-mg/kg dose. No significant difference in AUC (area under curve) was observed between the batches. These unexpected results can be explained only if the pharmacokinetics of docetaxel had been modified. It was shown that NCs released from the microparticles penetrated the enterocytes, bypassing P-gp; apparently circumventing gut metabolism and accumulating within the lymphatic system from where both intact or biodegraded NCs and free docetaxel were progressively released into the circulation as plausibly supported by the fluorescent imaging results. Furthermore, the circulating docetaxel in plasma was unencapsulated and circulated either in free form or bound to albumin. Both free docetaxel NCs and microparticles exhibited in vitro efficacy on WRC 256 cells suggesting that the activity of docetaxel was not altered. This delivery concept has potential for clinical translation, perhaps allowing docetaxel chemotherapy to be switched from intravenous to oral delivery. Cancer Res; 71(8); 3018–28. ©2011 AACR.

The use of stem cells for reparative medicine was first proposed more than three decades ago. Hematopoietic stem cells from bone marrow, peripheral blood and human umbilical cord blood (CB) have gained major use for treatment of hematological indications. CB, however, is also a source of cells capable of differentiating into various non-hematopoietic cell types, including neural cells. Several animal model reports have shown that CB cells may be used for treatment of neurological injuries. This review summarizes the information available on the origin of CB-derived neuronal cells and the mechanisms proposed to explain their action. The potential use of stem/progenitor cells for treatment of ischemic brain injuries is discussed. Issues that remain to be resolved at the present stage of preclinical trials are addressed.

The tuftelin protein isoforms undergo post-translation modifications, and are ubiquitously expressed in various tissues in embryos, adults, and tumors. Developmental and pathological studies suggested an apparent correlation between oxygen deprivation and tuftelin expression. The aim of the study was therefore to investigate the effect of a pathological insult (hypoxia) and a physiological growth factor (NGF), which antagonistically regulate HIF1 expression, on tuftelin expression using the neuronal PC12 cell model. In the present study, we first demonstrated the expression of tuftelin in PC12 cells, providing an experimental system to investigate the pathophysiological role of tuftelin. Furthermore, we demonstrated the induction of tuftelin during hypoxia by oxygen deprivation and during chemical hypoxia by cobalt chloride. Down-regulation of HIF1α mRNA blocked hypoxia-induced HIF1α expression, and reduced by 89% hypoxia-induced tuftelin expression. In mice, intraperitoneal injection of cobalt chloride significantly induced tuftelin mRNA and protein expression in the brain. During NGF-mediated PC12 differentiation, tuftelin expression was significantly induced in correlation with neurite outgrowth. This induction was partially blocked by K252a, a selective antagonist of the NGF receptor TrkA, indicating the involvement of the TrkA-signaling pathways in tuftelin induction by NGF. Revealing the physiological role of tuftelin will clarify mechanisms related to the “hypoxic genome,” and NGF-induced neurotrophic and angiogenic effects. J. Cell. Physiol. 226: 165–172, 2010. © 2010 Wiley-Liss, Inc.

Nerve growth factor (NGF) supports the survival and differentiation of sympathetic and sensory neurons and is also mitogenic for a variety of tumors. K252a, an antagonist of NGF receptor TrkA, was previously used as a pharmacological tool to study NGF actions and as a lead compound for developing anti-tumor drugs. Since recently, NGF was characterized as an angiogenic factor, we sought to investigate the angiostatic properties of K252a on endothelial cells (ECs). For this purpose, we used a murine brain microcapillary ECs model in which we found autocrine release of NGF in the culture medium and activation of TrkA receptor-induced downstream signaling molecules Erk1/2, Akt, and PLCgamma. In this model, we demonstrated the angiostatic property of K252a based on its ability to affect several important angiogenic steps. K252a, but not its cell membrane impermeable analogue K252b at 100 nM: (i) inhibited the proliferation of the ECs by 45 +/- 9%; (ii) reduced by 70 +/- 4% the migration of the ECs measured in a wound-closure model; (iii) reduced by 29 +/- 9% the formation of tube-like structures of the ECs cultured on Matrigel; (iv) stimulated by 100 +/- 25% the collagen deposition by the ECs, a process responsible for the increased endothelial barrier functions expressed by 22 +/- 5% reduction of paracellular permeability and by 17 +/- 3% elevation of transendothelial electrical resistance. These data suggest that NGF/TrkA may represent a target for the development of novel, K252a-derived multikinase inhibitors drugs with anti-tumor and angiostatic dual activities.

Endothelial cells (ECs) from different vascular beds not only display common characteristics but are also quite heterogeneous in terms of expression and secretion of neuro-angiogenic factors, which may help explain some of their distinct physiological roles. We investigated by RT-PCR the gene expression, by PC12 bioassay the neurotropic activity, and by ELISAs the levels of NGF and FGF-2 using conditioned medium collected from cultures of ECs derived from myocardial and cerebral capillaries. While NGF was expressed and released by both cell types, FGF-2 was expressed and released solely by the brain but not heart ECs. Oxygen-glucose deprivation (ischemic) insult blocked NGF secretion from heart and brain ECs and inhibited by 70% the secretion of FGF-2 from brain ECs. We propose that the differential expression of NGF and FGF-2 in heart and brain EC cultures reflect heterogeneity on demand of the microcapillary components and the surrounding microenvironment for a proper tissue-specific homeostasis.

Nerve growth factor (NGF) was recently characterized as an angiogenic factor inducing proliferation, migration, and capillary sprouting in endothelial cells (ECs) of different vascular beds. While NGF neuroprotective effects on neurons were described, its survival-inducing effects on brain capillary ECs were not yet addressed. Using a model of oxygen-glucose deprivation (OGD) followed by reoxygenation, we demonstrated that NGF conferred protection in brain capillary ECs. These cells express TrkA and p75(NTR) receptors and respond to NGF by stimulation of Erk1/2 phosphorylation and stimulation of proliferation and migration. The NGF protective effect was dose-dependent, inhibited by NGF/TrkA antagonist, K252a, and required presence of NGF during both OGD and reoxygenation phases while the major protective effect was related to decreased cell death during the reoxygenation phase. A causal relationship was found between NGF-induced protection and attenuation of OGD-induced Erk1/2 phosphorylation, supporting the death-promoting role of insult-induced Erk1/2 phosphorylation in the brain capillary ECs. These results emphasize the importance of NGF in the process of EC survival in response to ischemic injury and suggest fine-tuning regulation of Erk1/2 phosphorylation, extending the neuroprotective impact of NGF from sympathetic neuroendocrine cells to brain capillary ECs as the other element in the neurovascular tandem.

Background. Renal cell carcinoma (RCC) is considered resistant to ionizing radiation. Recently, the extracellular matrix (ECM) has been shown to play a role in both drug resistance and radiation resistance (RR). While fibronectin has been extensively investigated in the context of RR, the role of type I collagen [col(I)], a principal constituent of the ECM in tumour metastases, in RR of RCC is unknown. Methods. RCC cell adhesion to matrix was studied via precoating a variety of ECM glycoproteins onto plates. Cancer cell apoptosis and cell cycle were evaluated with flow cytometry using annexin V and propidium iodide stains, respectively. Activation of cellular survival signalling was analysed with western blots, and specific molecular inhibitors were correspondingly employed to block signalling. Hypoxia (< 1%) was induced via N 2 /CO 2 gas flow in a specialized chamber. Results. While adherence to col(I) enhanced RCC cell proliferation in general, col(I) and fibronectin, but not fibrinogen, could confer specific anti-apoptotic RR to RCC cells. The radioprotective effect of col(I) was maintained during both hypoxia/reoxygenation and normoxia conditions. In contrast to intact col(I), micronized col(I), lacking the natural fibrillar structure, was not radioprotective. The effect of col(I) in RCC cells is mediated via attenuation of apoptosis rather than cell cycle redistribution, involving the PI3 kinase/Akt pathway but not the MAP kinase pathway. Conclusions. Adherence to col(I) appears to be a relevant environmental cue enhancing RR in RCC cells, Akt dependently. Our results support inhibition of the PI3-kinase/Akt pathway as a radiosensitizing approach.

Human umbilical cord blood (HUCB) provides a source of progenitors for cell therapy. We isolated and characterized an HUCB-derived population of progenitors (HUCBNP), differentiated toward neuronal phenotype by human neuroblastoma-conditioning medium (CM) and nerve growth factor (NGF), which have been found to confer neuroprotection toward hypoxia-mediated neuronal injury. This study investigated whether interferon-gamma (IFN-gamma) contributes to HUCBNP differentiation. IFN-gamma was detected in the CM used for the induction of differentiation of HUCBNP and a neutralizing antibody of IFN-gamma significantly inhibited either IFN-gamma or CM-induced differentiation. Transcriptome analysis of CM-differentiated HUCBNP, identified 86 genes as highly upregulated, among them 25 were IFN-induced (such as 2',5'-oligoadenylate synthetase 1 and 2, IFN-induced protein and transmembrane proteins, STAT1 (IFN-gamma-receptor signal transducer and activator of transcription) and chemokine C-X-C motif ligand 5). Treatment of HUCBNP with human recombinant IFN-gamma, inhibited cell proliferation in a dose-dependent manner. IFN-gamma (1-100 ng/ml) enhanced neuronal differentiation, expressed by neurite outgrowths and increased expression of the neuronal markers beta-tubulin III, microtubule-associated protein 2, neuronal nuclei, neurofilament M and neuronal-specific enolase. IFN-gamma additively cooperated with NGF to induce the differentiation of HUCBNP. These data indicate that IFN-gamma promotes neuronal differentiation of HUCB-derived progenitors, proposing its use in future protocols towards cell therapy.

Myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction. The complexity of the disease and its treatments make MG patients particularly susceptible to adverse effects of drugs. MG is not a painful condition; however, as pain management armamentarium includes drugs from diverse pharmacological groups and with potential for drug-drug interactions, managing pain in patients with MG can be challenging. The underlying disease and the concomitant medications of each patient must be considered and the analgesic treatment individualized. This review presents an update on the various aspects of pain pharmacotherapy in patients with MG, focusing primarily on medications used to treat chronic pain. Drugs discussed are opioids, nonsteroidal anti-inflammatory drugs, antidepressants, anticonvulsants, muscle relaxants, benzodiazepines, intravenous magnesium, and local anesthetics. Drug interactions with agents used for MG treatment (acethylcholinesterase inhibitors, corticosteroids, immunosuppressants) and plasmapheresis are discussed. The clinical usefulness and limitations of each of the drug classes and agents are described.

Pardaxin is a fish toxin belonging to the alpha-helical, pore-forming peptide family, used in toxicological and biophysical research to study toxin-cell and -lipid-artificial membranes interactions. We investigated the membrane interaction of two pardaxin analogues using a colorimetric phospholipid/polydiacetylene biomimetic assay. In this assay, polydiacetylene undergoes visible, concentration dependent, blue-red transformation induced through interactions of pardaxins with the vesicle membrane. Pardaxins P4 and P5, are composed of 33 amino acids, but differ in a single amino acid substitution at the carboxy-terminal (G31 to D31, respectively) known to decrease the pore forming activity. Addition of pardaxins in the colorimetric assay induced dose-dependent color transitions with different kinetics. The colorimetric analysis could distinguish between different pardaxins-membrane interaction profiles, suggesting bilayer surface association for P4 and vesicle membrane penetration for P5. The colorimetric assay could distinguish between pardaxins membrane interaction profiles although circular dichroism spectra of vesicle-interacting pardaxins did not indicate a significant difference in the secondary structure between these two toxin analogues. The colorimetric platform utilized in the present report represents a useful assay with general applications for studying membrane interactions of peptides in general and pore-forming toxins in particular, and may become an important tool for evaluating quantitative toxin structure-activity relationship.

There is a paucity of quantitative methods for evaluating the morphological differentiation of neuronal cells in a three-dimensional (3-D) system to assist in quality control of neural tissue engineering constructs for use in reparative medicine. Neuronal cells tend to aggregate in the 3-D scaffolds, hindering the application of two-dimensional (2-D) morphological methods to quantitate neuronal differentiation. To address this problem, we developed a stable transfectant green fluorescence protein (GFP)-PC12 neuronal cell model, in which the differentiation process in 3-D can be monitored with high sensitivity by fluorescence microscopy. Under 2-D conditions, the green cells showed collagen adherence, round morphology, proliferation properties, expression of the nerve growth factor (NGF) receptors TrkA and p75(NTR), stimulation of extracellular signal-regulated kinase phosphorylation by NGF and were able to differentiate in a dose-dependent manner upon NGF treatment, like wild-type (wt)-PC12 cells. When grown within 3-D collagen gels, upon NGF treatment, the GFP-PC12 cells differentiated, expressing long neurite outgrowths. We describe here a new validated method to measure NGF-induced differentiation in 3-D. Having properties similar to those of wt-PC12 and an ability to grow and differentiate in 3-D structures, these highly visualized GFP-expressing PC12 cells may serve as an ideal model for investigating various aspects of differentiation to serve in neural engineering.

Plasma endothelin-1 levels rise in diabetes and after exposure to contrast media suggesting a role in progressive diabetic and acute radiocontrast nephropathies. Here we studied individual and combined effects of streptozotocin-induced diabetes and contrast media on renal endothelin converting enzyme-1 levels in the rat. In vivo, medullary (but not cortical) endothelin converting enzyme protein gradually increased 4 to 5-fold following the induction of diabetes or after the administration of contrast media but rose 15-fold when diabetic rats were given contrast media. Changes in mRNA expression paralleled those of the protein. Immunohistochemistry confirmed that increased tubular and endothelial cell endothelin converting enzyme-1 were most pronounced in the medulla. In vitro, endothelin-1 levels increased 3-fold following incubation of endothelial cells with media high in glucose or with contrast and 4-fold with their combination. Endothelin converting enzyme-1 protein and mRNA expression changed in a similar pattern while prepro endothelin-1 mRNA increased with each insult but not in an additive way. Our study shows that diabetes and contrast media up-regulate renal medullary endothelin converting enzyme-1 expression and synthesis.

In the accompanying study, the authors presented phosphometabolite patterns of endothelial cells grown under three-dimensional (3D) conditions using (31)P magnetic resonance spectroscopy (MRS). Here the authors describe the effect of nonsteroidal anti-inflammatory drugs (NSAIDs), using this enabling platform technology, which is relevant for evaluating drug effects in tissue-engineered endothelial constructs. Treatment with indomethacin significantly changed the phosphometabolite fingerprint in this endothelial model, by, respectively, increasing (81%) and decreasing (42%) glycerophosphocholine (GPC) and phosphomonoesters (PM). Furthermore, a safer approach using a NSAID prodrug was also demonstrated in this study with a indomethacin phospholipid-derived prodrug (DP-155). Like the parental drug, DP-155 increased and decreased the levels of GPC and PM by 100% and 20%, respectively. These changes represent useful biomarkers to monitor NSAID effects on endothelized tissue-engineered constructs for the purpose of controlling endothelial cell survival and inflammation upon implantation.

PC12 clones are established neuronal models to investigate mechanisms involved in the cross-talk between the neurotrophins and drugs. Chronic treatment of PC12 cells with the glucocorticoid agonist drug, dexamethasone (Dex), elicited a 50% decrease in the selective binding of 125 I-NGF along with a reduction in the NGF receptor p75 NTR mRNA and protein levels, suggesting a transcriptional mechanism. This down regulation of p75 NTR was antagonized by the glu- cocorticoid type II receptor (GR-2), RU-38486 but not by the minerallocorticoid receptor, RU-28318, antagonists. This process was associated with increased autophosphorylation of the NGF receptor, TrkA. Chronic treatment of PC12 with Dex abolished the NGF-induced proliferation of the cells after 20 hours and inhibited by 45% the neurite elongation after 96 hours. RU-38486 blocked Dex-induced shift of PC12 cells from dopaminergic to noradrenergic phenotype. Dex- induced down regulation of p75 NTR receptor is mediated by GR-2 and is correlated with disruption of NGF induced prolif

Direct interaction of α9β1 integrin with nerve growth factor (NGF) has been previously reported to induce pro-proliferative and pro-survival activities of non-neuronal cells. We investigated participation of p75(NTR) in α9β1 integrin-dependent cellular response to NGF stimulation. Using selective transfection of glioma cell lines with these receptors, we showed a strong, cation-independent association of α9 integrin subunit with p75(NTR) on the cellular membrane by selective immunoprecipitation experiments. The presence of the α9/p75(NTR) complex increases NGF-dependent cell adhesion, proliferation and migration. Other integrin subunits including β1 were not found in complex with p75(NTR). FRET analysis indicated that p75(NTR) and α9 integrin subunit are not closely associated through their cytoplasmic domains, most probably because of the molecular interference with other cytoplasmic proteins such as paxillin. Interaction of α9β1 integrin with another ligand, VCAM-1 was not modulated by the p75(NTR). α9/p75(NTR) complex elevated NGF-dependent activation of MAPK Erk1/2 arty for integrin that may create active complexes with other types of receptors belonging to the TNF superfamily.

Background Cysteine-rich secretory protein (CRISP) is present in majority of vertebrate including human. The physiological role of this protein is not characterized. We report that a CRISP isolated from Echis carinatus sochureki venom (ES-CRISP) inhibits angiogenesis. Methods The anti-angiogenic activity of purified ES-CRISP from snake venom was investigated in vitro using endothelial cells assays such as proliferation, migration and tube formation in Matrigel, as well as in vivo in quail embryonic CAM system. The modulatory effect of ES-CRISP on the expression of major angiogenesis factors and activation of angiogenesis pathways was tested by qRT-PCR and Western blot. Results The amino acid sequence of ES-CRISP was found highly similar to other members of this snake venom protein family, and shares over 50% identity with human CRISP-3. ES-CRISP supported adhesion to endothelial cells, although it was also internalized into the cytoplasm in a granule-like manner. It blocked EC proliferation, migration and tube formation in Matrigel. In the embryonic quail CAM system, ES-CRISP abolished neovascularization process induced by exogenous growth factors (bFGF, vpVEGF) and by developing gliomas. CRISP modulates the expression of several factors at the mRNA level, which were characterized as regulators of angiogenesis and blocked activation of MAPK Erk1/2 induced by VEGF. Conclusions ES-CRISP was characterized as a negative regulator of the angiogenesis, by direct interaction with endothelial cells. General significance The presented work may lead to the development of novel angiostatic therapy, as well as contribute to the identification of the physiological relevance of this functionally uncharacterized protein.

Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor (EGF) receptors (EGFR) during the neuronal differentiation of PC12 cells. This process was characterized by a progressive decrease in EGFR level, as measured by (125)I-EGF binding and Scatchard analysis, tyrosine phosphorylation, Western blotting, and bio-imaging using EGF-labeled with a near-infrared probe. Differentiation of the cells with NGF for 5-7 days produces a 95 % reduction in the amount of (35)S-methionine-labeled EGFR. This down-regulation does not occur in PC12-nnr5 cells, which lack the TrkA NGF receptor but is reconstituted in these cells upon their stable transfection with TrkA. The process of NGF-induced EGFR down-regulation was inhibited by K252a, a TrkA antagonist and by anti-TrkA antibodies but not by Thx-B, a blocker of the interaction of NGF with p75(NTR) receptors. NGF-induced (heterologous) down-regulation, but not EGF-induced (homologous) down-regulation of EGFR, was blocked in Ras-deficient PC12 cells. NGF treatment for 5-7 days of PC12 cells, grown in suspension or in 3D collagen gels, induces down-regulation of EGFR independent of neurite outgrowth. The messenger RNA (mRNA) for EGFR decreased in a comparable fashion. This process was correlated temporally with a decrease in the transcription of the EGFR gene. Treatment with NGF also increased the cellular content of GCF2, a putative inhibitory transcription factor of the EGFR gene. The temporal increase in GCF2, like the decrease in the EGFR mRNA, was not seen in TrkA deficient PC12 cells nor in cells expressing dominant-negative Ras. The results suggest that NGF-induced down-regulation of the EGFR is under transcriptional control, is TrkA and Ras-dependent, may involve transcriptional repression by GCF2, and independent of mechanisms that lead to NGF-induced neurite outgrowth in PC12cells. This heterologous down-regulation of EGFR would appear to be an efficient mean of desensitizing the neuron to proliferative stimuli, thereby representing a safety latch for initiating and sustaining NGF-induced neuronal differentiation.

One of the challenges in regenerative medicine is the development of novel biodegradable materials to build scaffolds that will support multiple cell types for tissue engineering. Here we describe the preparation, characterization, and cytocompatibility of homo- and hetero-polyesters of α-hydroxy amino acid derivatives with or without lactic acid conjugation. The polymers were prepared by a direct condensation method and characterized using gel permeation chromatography, (1)H-nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, optical activity, and solubility. The surface charge of the polymers was evaluated using zeta potential measurements. The polymers were coated onto glass cover slips followed by characterization using nano-surface profiler, thin film reflectometry, and atomic force microscopy (AFM). Their interaction with endothelial and neuronal cells was assessed using adhesion, proliferation, and differentiation assays. Of the characterized polymers, Poly-HOVal-LA, but not Poly-(D)HOPhe, significantly augmented nerve growth factor (NGF)-induced neuronal differentiation of the PC12 pheochromcytoma cells. In contrast, Poly-HOLeu increased by 20% the adhesion of endothelial cells, but did not affect PC12 cell differentiation. NGF-induced Erk1/2 phosphorylation in PC12 cells grown on the different polymers was similar to the effect observed for cells cultured on collagen type I. While no significant association could be established between charge and the differentiative/proliferative properties of the polymers, AFM analysis indicated augmentation of NGF-induced neuronal differentiation on smooth polymer surfaces. We conclude that overall selective cytocompatibility and bioactivity might render α-hydroxy amino acid polymers useful as extracellular matrix-mimicking materials for tissue engineering.

Stem cells play a major role in human development, and postnatal tissues homeostasis, and repair. Therefore, they are considered as significant building blocks for tissue engineering and regenerative medicine applications. Stem cells receive important cues from their natural microenvironment, the niche, which determine each stem cell’s behavior and fate decision. The stem cell niche is composed of physico-chemical and cellular elements, which could originate from local or distant tissues, or from sources external to the body. In addition to the niche, the behavior of stem cells may be explained by their normal process of development. Therefore, an understanding of stem cells niche, the initial stages of development and sources and origins of stem cells is necessary to facilitate the design of protocols for the use of stem cells in tissue engineering and regenerative medicine. When these protocols involve in vitro manipulation of stem cells, the elements of niche components are usually used as the major players including soluble factors, extracellular matrix components, physical factors, and cells from other lineages. Also, the recently defined concepts of transdifferentiation and reprogramming have opened new possibilities for generation of further cell sources, especially from autologous tissues. There are, however, still many gaps in the knowledge of in vivo and in vitro stem cell biology, and in understanding the mechanisms of a number of diseases which will benefit from stem cell therapy. Filling these gaps will permit the progression from stem cells research to clinical applications.

The phospholipid composition of red blood cells (RBC) from 32 haloperidol-treated schizophrenic patients, classified according to the positive and negative syndrome scale (PANSS) as showing either predominantly positive or predominantly negative symptoms, was determined and compared with that of normal controls. While the levels of phosphatidylcholine and phosphatidylserine were similar in all three groups, sphingomyelin (SM) and phosphatidylethanolamine (PE) were, respectively, increased and decreased in RBCs of schizophrenic patients. In both patient groups, the SM/PE ratios correlated directly with the PANSS negative symptom scale scores and inversely with the positive symptom scale scores. However, the inverse changes in the contents of SM and PE were much more expressed in the negative group. It is suggested that a main source of that difference is a higher activity of the polyunsaturated acid-selective phospholipase A(2) in the negative syndrome patients than in the positive syndrome and control groups.

Nerve growth factor (NGF) has been reported to play an important role in physiological and pathological angiogenesis. Based on these observations, we hypothesized that NGF may induce the formation of functional blood vessels in a hindlimb ischemic rabbit model. Hindlimb ischemia was induced in 34 rabbits bilaterally by endovascular embolization of femoral arteries. On the 7th, 14th, and 20th postembolization days, NGF was injected intramuscularly, in 1 ischemic limb, and vehicle was injected in the contralateral control limb. On the 40th day, newly developed collateral vessels (diameter >500 μm) were quantified by transauricular intraarterial subtraction angiography. Perfusion analysis of an in vivo dynamic computed tomography study was performed to the limbs to investigate the hemodynamic recovery of the distal ischemic tissues. Functional estimation of limb perfusion showed a statistically significant increase of blood flow and blood volume for NGF. However, the increase of the collateral vessels was not detectable angiographically, providing evidence for the existence of a NGF-stimulated capillary angiogenic network but not increase of arteriogenesis. The combination of NGF with either tropomyosin-related kinase type A or vascular endothelial growth factor receptor 2 antagonists abolished the NGF-induced hemodynamic recovery. These findings provide new insights into understanding the involvement of NGF in vascular formation and its applications in therapeutic angiogenesis.

Nerve growth factor (NGF), a member of the neurotrophin family of growth factors, plays a crucial function in the nervous system [1] and is very important in the cross talk between the nervous and cardiovascular systems [2]. Growing evidence has suggested a critical role for NGF during cardiovascular development. NGF is an essential factor in the formation of the heart and a critical regulator of vascular development. Postnatally, NGF controls the survival of blood vessel endothelial cells, vascular smooth muscle cells, and heart cardiomyocytes, and regulates angiogenesis and vasculogenesis by autocrine and paracrine mechanisms [3]. While many studies have established the role of NGF in cardiac pathophysiology [4], the hypothesis that NGF plays an important cardioprotective role in myocardial ischemia/reperfusion injury (IRI) has not yet been explored. To determine the effects of NGF on myocardial IRI, NGF was administered intravenously (IV) 15 min before ischemia to rats, in a series of experiments (n=6 per group) for finding the dose-dependent effect (0.015–15 μg/kg) and for the assessment of the optimal dose. To determine the mechanisms involved, the rats were injected with inhibitors of phosphatidylinositol-3 kinase (PI3K) (wortmannin, 15 μg/kg) or nitric oxide synthase (NOS) [N(G)-monomethyl-l-arginine (L-NMA), 15 mg/kg] 15 min before the injection of NGF (0.15 μg/kg) – conditions previously reported by us to inhibit these signaling pathways in this rat model [5, 6]. The principal end point was infarct size expressed either as a percentage of the area at risk (AAR) or the left ventricular area (LVA). The infarct size was 60%±3% of the AAR (Figure 1A) and 30%±2% of the LVA (Figure 1B). A bell-shaped dose-response reduction in infarct size was seen with NGF treatment, 0.15 μg/kg being the optimally effective dose. The hearts of treated rats had an infarct size of 24.9%±1.8% of the AAR and 13.8%±1% of the LVA, which are approximately 58.5% and 54% reduction in infarct size compared with the control, respectively (Figure 1A,B). Heart rate and blood pressure were monitored throughout the procedure, and there were no significant differences in basal hemodynamic parameters between groups. Mean arterial pressures decreased during ischemia and reperfusion by 35%–45% in all groups [7]; however, there was no significant difference between the groups (Figure 1C,D). Figure 1 NGF-induced cardioprotection (A, B) and hemodynamic values of mean arterial pressure (C) and heart rate (D) in a rat myocardial ischemia/reperfusion model PI3K is an important mediator of cardioprotection [8]. To determine whether NGF-induced cardioprotection is mediated by this pathway, we injected IV, before IRI, wortmannin, a PI3K inhibitor, alone or in combination with NGF. Wortmannin abolished the cardioprotective effect of NGF by 50% but had no effect when given alone. Increased NO produced by activating NOS protects the heart against IRI [9]. To determine whether NGF-induced cardioprotection is mediated by this pathway, we injected IV, before IRI, L-NMA, a general NOS inhibitor. L-NMA abolished the cardioprotective effect of NGF by 50% but had no effect when given alone. The major finding of this study is that a single treatment with NGF before ischemia significantly reduced the heart infarct size associated with IRI. In addition, we found that inhibition of the signaling molecules PI3K and NOS significantly abrogated the NGF-induced cardioprotective effect, proposing their involvement in the mechanism of action of NGF. It is known that heart cells secrete NGF and express its receptors p75NTR and trkA. The activation of these receptors in cardiomyocytes triggers a prosurvival effect involving the PI3K/Akt pathway [10]. It is also established that NGF, by activation of its trkA receptors, activates the Ras-Erk pathway responsible for the induction of NOS in sympathetic neurons [11] involved in NGF-induced neuroprotective effects [12]. Therefore, it is reasonable to propose that the NGF-induced cardio-protective effect in the rat heart exposed to IRI is mediated by trkA activation of PI3K/NOS. We recognize that the contributions of these signaling pathways were primarily examined with kinase inhibitors, which is not without problems. However, by showing that the Akt phosphorylation and endothelial NOS (eNOS) regulation of cGMP production are inhibited by wortmannin and L-NMA [5, 6], we believe that this supports the circumstantial evidence that PI3K/Akt and eNOS are involved in NGF cardioprotection, primarily suggesting that NGF modulates cardiomyocyte ischemic tolerance: PI3K and NOS are the major modulators of cardiomyocyte ischemic tolerance [13, 14]. NGF is present in infarcted human heart, and mice with infarcted heart treated with an antibody against NGF showed cardiomyocyte apoptosis and worsened cardiac function [15], further stressing the important cardioprotective role of NGF in the heart. Following myocardial infarction (MI), NGF levels initially increase and thereafter decline, emphasizing its cardioprotective, neurotrophic, and neurotropic sympathetic pleiotropic effects in the heart [4]. The mechanism behind the ability of NGF to upregulate the cardioprotective pathways is currently unknown. We do not know if the cardioprotective effect of NGF is a reflection of its direct prosurvival effect on heart cardiomyocytes [10], indirectly mediated by remote IRI preconditioning [16] due to sympathetic and/or sensory nerves and/or induced by myocardium microcapillary endothelial [17] and/or smooth muscle cell activation. Future studies should address the role of endogenous NGF on ischemic tolerance and specifically its role in the different forms of preconditioning. Whatever the mechanism, the present results are the first to demonstrate that a single treatment with NGF confers immediate protection against injury caused by ischemia/reperfusion in the heart, proposing a potential therapeutic role for NGF in MI. The potential impact of the modulation of cardiac ischemic tolerance by NGF can be manifold: increasing ischemic tolerance, and particularly tolerance to reperfusion, can serve as an important therapeutic target. Furthermore, interactions with NGF of existing drugs and endogenous agents should be studied: if exogenous drugs, such as noradrenaline infusion, reduce pericardial NGF or its effect [18], then they may reduce ischemic tolerance, thereby increasing the risk of ischemic events, leading to occult cardiotoxicity [19].

Novel strategies that target the epidermal growth factor receptor (EGFR) have led to the clinical development of monoclonal antibodies, which treat metastatic colorectal cancer (mCRC) but only subgroups of patients with increased wild type KRAS and EGFR gene copy, respond to these agents. Furthermore, resistance to EGFR blockade inevitably occurred, making future therapy difficult. Novel bio-imaging (BOI) methods may assist in quantization of EGFR in mCRC tissue thus complementing the immunohistochemistry methodology, in guiding the future treatment of these patients. The aim of the present study was to explore the usefulness of near infrared-labeled EGF (EGF-NIR) for bio-imaging of CRC using in vitro and in vivo orthotopic tumor CRC models and ex vivo human CRC tissues. We describe the preparation and characterization of EGF-NIR and investigate binding, using BOI of a panel of CRC cell culture models resembling heterogeneity of human CRC tissues. EGF-NIR was specifically and selectively bound by EGFR expressing CRC cells, the intensity of EGF-NIR signal to background ratio (SBR) reflected EGFR levels, dose-response and time course imaging experiments provided optimal conditions for quantization of EGFR levels by BOI. EGF-NIR imaging of mice with HT-29 orthotopic CRC tumor indicated that EGF-NIR is more slowly cleared from the tumor and the highest SBR between tumor and normal adjacent tissue was achieved two days post-injection. Furthermore, images of dissected tissues demonstrated accumulation of EGF-NIR in the tumor and liver. EGF-NIR specifically and strongly labeled EGFR positive human CRC tissues while adjacent CRC tissue and EGFR negative tissues expressed weak NIR signals. This study emphasizes the use of EGF-NIR for preclinical studies. Combined with other methods, EGF-NIR could provide an additional bio-imaging specific tool in the standardization of measurements of EGFR expression in CRC tissues.

The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9β1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9β1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.

Tuftelin, an acidic protein, thought to play a role in the initial stages of ectodermal enamel mineralization, has since been detected in mesenchymal-derived tissues. During bone/cartilage development and regeneration, mesenchymal stem cells (MSCs) undergo an avascular period in a hypoxic environment, involving induction of hypoxia-inducible factor 1-alpha (HIF-1-alpha), a key component in this process. In the present study we investigated, in a mouse mesenchymal C3H10T1/2 stem cell model, the hypothesis that oxygen stress modulates tuftelin 1 expression in relation to HIF-1-alpha (Hif1a), in a mouse mesenchymal C3H10T1/2 stem cell model. The results of the present study showed a biphasic induction of tuftelin, similar to the pattern of HIF-1-alpha expression, in MSCs subjected to a hypoxic insult of 1% O(2) through a period of 2-24 h. Immunocytochemistry analysis of the cells exposed to hypoxic insult for 2-24 h revealed the same biphasic pattern of tuftelin protein expression. Tuftelin localization appears to be mainly in the cytoplasm, and concentrated at the perinuclear region of the cells by 24 h of hypoxic insult. Based on our previous studies using the neuronal PC12 cell model, in which tuftelin induction was mediated by Hif1a, we propose that tuftelin is a member of oxygen-sensitive genes and implicated in the adaptive mechanisms regulating MSC function.

Stem cells have an extremely high potential to treat many devastating diseases, including neuronal injuries. Albeit the need for human neuronal stem cells, their quantities are very limited by relying on early human embryos as the main source. Therefore, progenitors of other origins, such as human umbilical cord blood (CB) are being considered. In the last decade, various populations isolated from the CB were reported to differentiate in vitro towards a neural phenotype. The conditions to induce the cell differentiation are not conclusive and may include addition of chemicals, cytokines and growth factors, including the nerve growth factor (NGF). Some CB cells were found to express the TrkANGF receptor, suggesting an endogenous role for this growth factor also in the CB environment. The ability of CB and derived stem cell populations to protect against neurological deficits was shown, both in vitro and in vivo, in models of ischemic brain injuries. In rodent models of stroke, heatstroke, brain trauma and brain damage at birth, CB cells either by intravenous injection or intrastriatal transplantation, were found to reduce the infarct size and the neurological deficits caused by the injury. The restorative effects of CB were suggested to be mediated by mechanisms other than cell replacement. Some of the proposed mechanisms involve reduced inflammation, nerve fiber reorganization by trophic actions, increased cell survival and enhanced angiogenesis. Furthermore, treatment with CB was found to have a therapeutic window of days compared with the present 36 hour window for the treatment of stroke with clinically available tools such as recombinant tissue plasminogen activator. Considering the encouraging results with whole CB and derived cells transplantation in ischemic injury models and since CB is widely available and have been used clinically, they may be an excellent source of cells for treatment of human brain ischemic disorders.

The increasing exposure to environmental neurotoxicants in the last decades caused serious health problems in the world population. Some of the neurotoxic agents are being used in agriculture and household such as insecticides and rodenticides and others are of natural origin like snake and scorpion venoms. Additional group of harmful substances is the chemical warfare agents including nerve and blistering agents that are known for their disastrous effects on neuronal tissues. The present paper presents a combination of epidemiological/clinical and molecular approaches for investigating the effect of certain groups of neurotoxicants on a variety of pathologies. The work of Finkelstein and coworkers describes epidemiological and clinical studies on acute and chronic organophosphate (OP)-induced neurotoxicity in certain populations in Israel. They mainly investigated the neurotoxic effects of low-level long-term exposure to OP in agricultural areas but also dealt with acute exposures as well. A molecular approach to OP mechanism of neuronal injury was described by Milatovic and coworkers. They demonstrated OP-induced oxidative injury in pyramidal neurons in the CA1 hippocampal area and its suppression by antioxidants. Lecht and coworkers described the novel snake venom angioneurins as important mediators of the physiological cross-talk between the cardiovascular and nervous systems. They also showed that under certain conditions these angioneurins may induce pathologies such as tumor development or disruption of the vascular barrier function during envenomation. Additional mechanistic/therapeutic approach was presented by Brodsky, Rosengarten, Proscura, Shapira and Wormser. They developed a novel anti-inflammatory peptide that reduced skin irritation induced by heat and sulfur mustard (SM) stimuli. Since SM causes neuropsychiatric symptoms and alterations in neurological functions this peptide may serve as a potential treatment of neuronal injuries caused by environmental neurotoxicants. These reviews highlight different aspects of neurotoxicity, addressing epidemiology and mechanisms of toxicity; and identifying novel potential therapies.

Cumulative evidences suggest that nerve growth factor (NGF) promotes angiogenic effects such as proliferation and migration of endothelial cells (ECs) from different vascular beds, induces capillary sprouting in chorioallantoic membrane and improves in vivo vascularization in a hind-limb ischemic model. In the present study, we sought to investigate the signaling properties of NGF in a microcapillary ECs model compared to those of a neuronal model. NGF-induced phosphorylation of signaling molecules Erk1/2, Akt and PLCγ were measured using Western blotting and compared between mouse NGF (mNGF) and snake venom NGF analogues. NGFs-induced signaling was TrkA mediated as evident by inhibition with the TrkA antagonist K252a. NGF and its analogues-induced signaling in ECs were characterized by a transient effect in contrast to a prolonged stimulation in neuronal cells. The potency of mouse, cobra and viper NGFs to induce Erk1/2 phosphorylation in ECs was higher than in neurons. In ECs, mNGF exhibited the highest efficacy of stimulation of Erk1/2 phosphorylation, followed by viper and cobra NGFs. The efficacy of stimulation of Erk1/2 phosphorylation measured with neurons was opposite from that in ECs. NGF-induced temporal signaling differences between ECs and neurons may explain the dual vascular and neurotrophic effects of this growth factor.

Very few studies describe endothelial cell (EC) properties under three-dimensional (3D) conditions using (31)P magnetic resonance spectroscopy (MRS). The authors developed a model in which living ECs growing in Matrigel threads (3D conditions) for 5 days are monitored by (31)P MRS, providing the fingerprint of the major EC phosphometabolites. Organic extracts of membranal phospholipids were also analyzed by (31)P MRS. For comparison and as a model for two-dimensional (2D) tissue culture conditions, (31)P MRS spectra of aqueous extracts of EC phosphometabolites grown under 2D conditions were also evaluated. The phosphometabolites fingerprint of the cells cultured under 3D was significantly different from that of ECs maintained under 2D. Moreover, the pattern of phosphometabolites was affected by coculture with C6-glioma cells and upon treatment with valproic acid, which is under clinical investigation as an antioangiogenic anticancer drug. The major effects were modulation of (i) energy metabolism intermediates such as phosphocreatine, (ii) precursors of phospholipids such as phosphomonoesters, and (iii) degradation products of phospholipids such as glycerophosphocholine. This endothelial model will be usefull as an enabling platform technology for tissue engineering.

Human umbilical cord blood (HUCB) is a valuable source for cell therapy since it confers neuroprotection in stroke animal models. However, the responsible sub-populations remain to be established and the mechanisms involved are unknown. To explore HUCB neuroprotective properties in a PC12 cell-based ischemic neuronal model, we used an HUCB mononuclear-enriched population of collagen-adherent cells, which can be differentiated in vitro into a neuronal phenotype (HUCBNP). Upon co-culture with insulted-PC12 cells, HUCBNP conferred approximately 30% neuroprotection, as evaluated by decreased lactate dehydrogenase and caspase-3 activities. HUCBNP decreased by 95% the level of free radicals in the insulted-PC12 cells, in correlation with the appearance of antioxidants, as measured by changes in the oxidation-reduction potential of the medium using cyclic-voltammetry. An increased level of nerve growth factor (NGF), vascular endothelial growth factor and basic fibroblast growth factor in the co-culture medium was temporally correlated with a -medium neuroprotection effect, which was partially abolished by heat denaturation. HUCBNP-induced neuroprotection was correlated with changes in gene expression of these neurotrophic factors, while blocked by K252a, an antagonist of the TrkA/NGF receptor. These findings indicate that HUCBNP-induced neuroprotection involves antioxidant(s) and neurotrophic factors, which, by paracrine and/or autocrine interactions between the insulted-PC12 and the HUCBNP cells, conferred neuroprotection.

Parkinson's disease (PD) is a progressive neurodegenerative, dopamine deficiency disorder. The main therapeutic strategies for PD treatment relies on dopamine precursors (levodopa), inhibition of dopamine metabolism (monoamine oxidase [MAO] B and catechol-O-methyl transferase inhibitors), and dopamine receptor agonists. Recently, a novel selective and irreversible MAO B propargylamine inhibitor rasagiline (N-propargyl-1-R-aminoindan, Azilect((R))) was approved for PD therapy. In contrast to selegiline, the prototype of MAO B inhibitors, rasagiline is not metabolized to potentially toxic amphetamine metabolites. The oral bioavailability of rasagiline is 35%, it reaches T(max) after 0.5-1 hours and its half-life is 1.5-3.5 hours. Rasagiline undergoes extensive hepatic metabolism primarily by cytochrome P450 type 1A2 (CYP1A2). Rasagiline is initiated at 1 mg once-daily dosage as monotherapy in early PD patients and at 0.5-1 mg once-daily as adjunctive to levodopa in advanced PD patients. Rasagiline treatment was not associated with "cheese effect" and up to 20 mg per day was well tolerated. In PD patients with hepatic impairment, rasagiline dosage should be carefully adjusted. Rasagiline should not be administered with other MAO inhibitors and co-administration with certain antidepressants and opioids should be avoided. Although further clinical evidence is needed on the neuroprotective effects of rasagiline in PD patients, this drug provides an additional tool for PD therapy.

The integrin alpha9beta1 is a multifunctional receptor that interacts with a variety of ligands including vascular cell adhesion molecule 1, tenascin C and osteopontin. We found that this integrin is a receptor for nerve growth factor (NGF) and two other neurotrophins, brain-derived neurotrophic factor and NT3, using a cell adhesion assay with the alpha9SW480 cell line. Interaction of alpha9beta1 with NGF was confirmed in an ELISA assay by direct binding to purified integrin. alpha9beta1 integrin binds to neurotrophins in a manner similar to another common neurotrophin receptor, p75(NTR) (NGFR), although alpha9beta1 activity is correlated with induction of pro-survival and pro-proliferative signaling cascades. This property of alpha9beta1 resembles the interaction of NGF with a high affinity receptor, TrkA, however, this integrin shows a low affinity for NGF. NGF induces chemotaxis of cells expressing alpha9beta1 and their proliferation. Moreover, alpha9beta1 integrin is a signaling receptor for NGF, which activates the MAPK (Erk1/2) pathway. The alpha9beta1-dependent chemotactic ability of NGF appears to result from the activation of paxillin.

Inflammatory processes in the central nervous system (CNS) are considered neurotoxic, although recent studies suggest that they also can be beneficial and confer neuroprotection (neuroprotective autoimmunity). Cells from the immune system have been detected in CNS injury and found to produce and secrete a variety of neurotrophins such as NGF, BDNF, NT-3 and NT-4/5, and to express (similarly to neuronal cells), members of the tyrosine kinase (Trk) receptor family such as TrkA, TrkB and TrkC. Indeed, autocrine and paracrine interactions are observed at the site of CNS injury, resulting in a variety of homologic-heterologic modulations of immune and neuronal cell function. The end result of the inflammatory process, neurotoxicity and/or neuroprotection, is a function of the fine balance between the two cellular systems, i.e., of the complex signaling relationships between anti-inflammatory neuroprotective factors (neurotrophins and other chemical mediators) and proinflammatory neurotoxic factors (TNF, free radicals, certain cytokines, etc.). Autoimmune neuroprotection is a novel therapeutic approach aimed at shifting the balance between the immune and neuronal cells towards survival pathways in a variety of CNS injuries. This review focuses on data supporting this concept and its future therapeutical implications for optic nerve injury and multiple sclerosis.