Hadar Arien-Zakay

Interactions between the cells of the immune and nervous system: neurotrophins as neuroprotection mediators in CNS injury.

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.

Neuroprotection by cord blood neural progenitors involves antioxidants, neurotrophic and angiogenic factors

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.

Neuroprotection by NGF in the PC12 In Vitro OGD Model

Abstract: Neurodegenerative disorders and chronic disability due to stroke in the brain or spinal cord afflict a large sector of the population. To investigate the mechanism involved in ischemic stroke and to develop neuroprotective drugs/therapies, in vivo and in vitro, pharmacological models are needed. To investigate the cellular and molecular neuroprotective mechanisms of nerve growth factor (NGF), a member of the nervous system neurotrophin family of growth factors, under ischemia, we used an oxygen‐glucose‐deprivation (OGD) device and pheochromocytoma PC12 cells exposed to a paradigm of ischemic insult. Pretreatment of the cultures with 50 ng/mL of NGF, 18 h prior to OGD insult, conferred 30% of neuroprotection. Time‐course experiments showed marked activation of the ERK, JNK, and p‐38 MAPK isoforms during the OGD phase, but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL of NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD‐induced activation of JNK 1, and 20% and 50% attenuation of OGD‐induced activation of p‐38 α and β, respectively. The effect of NGF on gene expression in the PC12 ischemic model using Affymatrix Rat DNA‐Microarray technology indicates that only 6% of the genes are differentially regulated (induced/suppressed) by OGD insult and/or NGF. These findings support the notion that pretreatment with NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms and differential gene expression. This ischemic model may be useful to investigate molecular mechanisms of OGD‐induced neurotoxicity and NGF‐induced neuroprotection, and to generate novel therapeutic concepts for stroke treatment.

Tissue regeneration potential in human umbilical cord blood

Regenerative medicine is the process of creating functional tissue with the aid of stem cells, to repair loss of organ function. Possible targets for regenerative medicine include orthopaedic, cardiac, hepatic, pancreatic and central nervous system (CNS) applications. Umbilical cord blood (CB) has established itself as a legitimate source for haematopoietic stem cell transplantation. It is also considered an accessible and less immunogenic source for mesenchymal, unrestricted somatic and for other stem cells with pluri/multipotent properties. The latter are capable of differentiating into a wide variety of cell types including bone, cartilage, cardiomyocytes and neural. They also possess protective abilities that may contribute to tissue repair even if in vitro differentiation is excluded. In view of the absence of treatment for many devastating diseases, the elucidation of non-haematopoietic applications for CB will facilitate the development of pioneering relevant cell therapy approaches. This review focusses on current studies using human CB-derived cells for regenerative medicine.

Neuroprotective effects of nimodipine and nifedipine in the NGF-differentiated PC12 cells exposed to oxygen-glucose deprivation or trophic withdrawal

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.

Nerve Growth Factor-Induced Protection of Brain Capillary Endothelial Cells Exposed to Oxygen―Glucose Deprivation Involves Attenuation of Erk Phosphorylation

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 p75NTR 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.

Neuroprotection by human umbilical cord blood- derived progenitors in ischemic brain injuries

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.

Human placental eXpanded (PLX) mesenchymal-like adherent stromal cells confer neuroprotection to nerve growth factor (NGF)-differentiated PC12 cells exposed to ischemia by secretion of IL-6 and VEGF.

Mesenchymal stem cells are potent candidates in stroke therapy due to their ability to secrete protective anti-inflammatory cytokines and growth factors. We investigated the neuroprotective effects of human placental mesenchymal-like adherent stromal cells (PLX) using an established ischemic model of nerve growth factor (NGF)-differentiated pheochromocytoma PC12 cells exposed to oxygen and glucose deprivation (OGD) followed by reperfusion. Under optimal conditions, 2 × 10⁵ PLX cells, added in a trans-well system, conferred 30-60% neuroprotection to PC12 cells subjected to ischemic insult. PC12 cell death, measured by LDH release, was reduced by PLX cells or by conditioned medium derived from PLX cells exposed to ischemia, suggesting the active release of factorial components. Since neuroprotection is a prominent function of the cytokine IL-6 and the angiogenic factor VEGF165, we measured their secretion using selective ELISA of the cells under ischemic or normoxic conditions. IL-6 and VEGF165 secretion by co-culture of PC12 and PLX cells was significantly higher under ischemic compared to normoxic conditions. Exogenous supplementation of 10 ng/ml each of IL-6 and VEGF165 to insulted PC12 cells conferred neuroprotection, reminiscent of the neuroprotective effect of PLX cells or their conditioned medium. Growth factors as well as co-culture conditioned medium effects were reduced by 70% and 20% upon pretreatment with 240 ng/ml Semaxanib (anti VEGF165) and/or 400 ng/ml neutralizing anti IL-6 antibody, respectively. Therefore, PLX-induced neuroprotection in ischemic PC12 cells may be partially explained by IL-6 and VEGF165 secretion. These findings may also account for the therapeutic effects seen in clinical trials after treatment with these cells.

Pharmacological aspects of Vipera xantina palestinae venom.

In Israel, Vipera xantina palestinae (V.x.p.) is the most common venomous snake, accounting for several hundred cases of envenomation in humans and domestic animals every year, with a mortality rate of 0.5 to 2%. In this review we will briefly address the research developments relevant to our present understanding of the structure and function of V.x.p. venom with emphasis on venom disintegrins. Venom proteomics indicated the presence of four families of pharmacologically active compounds: (i) neurotoxins; (ii) hemorrhagins; (iii) angioneurin growth factors; and (iv) different types of integrin inhibitors. Viperistatin, a α1β1selective KTS disintegrin and VP12, a α2β1 selective C-type lectin were discovered. These snake venom proteins represent promising tools for research and development of novel collagen receptor selective drugs. These discoveries are also relevant for future improvement of antivenom therapy towards V.x.p. envenomation.

Transient signaling of Erk1/2, Akt and PLCγ induced by nerve growth factor in brain capillary endothelial cells

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 PLCgamma 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.