Nerve growth factor (NGF) promotes angiogenesis in the quail chorioallantoic membrane.

Angiogenesis, the formation of new blood vessels, is tightly regulated by growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). The authors hypothesize that nerve growth factor (NGF), a well known neurotrophin, may play a direct angiogenic role. To test this hypothesis, the authors measured the effects of NGF on the natural vascularization of the quail chorioallantoic membrane (CAM). The angiogenic effect of NGF was compared to that of human recombinant VEGF165 (rhVEGF) and basic FGF (rhbFGF). In comparison to phosphate-buffered saline-treated controls, NGFs from different biological sources (mouse, viper, and cobra) increased the rate of angiogenesis in a dose-dependent fashion from 0.5 to 5 microg. For quantitative morphometry, grayscale images of the blood vessels end points of the CAM arteries were binarized for visualization and skeletonized for quantization by fractal analysis. In mouse NGF-treated embryos the fractal dimension (Df), indicative of arterial vessel length and density, increased to 1.266 +/- 0.021 compared to 1.131 +/- 0.018 (p < .001) for control embryos. This effect was similar to that of 0.5 microg rhVEGF (1.290 +/- 0.021, p < .001) and 1.5 microg rhbFGF (1.264 +/- 0.028, p < .001). The mouse NGF-induced angiogenic effect was blocked by 1 microM K252a (1.149 +/- 0.018, p < .001), an antagonist of the NGF/trkA receptor, but not by 1 microM SU-5416 (1.263 +/- 0.029, p < .001), the VEGF/Flk1 receptor antagonist, indicating a direct, selective angiogenic effect of NGF via quail embryo trkA receptor activation. These results confirm previous observations that NGF has angiogenic activity and suggest that this neurotrophin may also play an important role in the cardiovascular system, besides its well-known effects in the nervous system. The angiogenic properties of NGF may be beneficial in engineering new blood vessels and for developing novel antiangiogenesis therapies for cancer.     

Cholinergic basal forebrain structures are involved in the mediation of the arousal effect of noradrenaline

Cholinergic basal forebrain structures are implicated in cortical arousal and regulation of the sleep–wake cycle. Cholinergic neurones are innervated by noradrenergic terminals, noradrenaline excites them via alpha‐1 receptors and microinjection of noradrenaline into the basal forebrain enhances wakefulness. However, it is not known to what extent the cholinergic versus non‐cholinergic basal forebrain projection neurones contribute to the arousing effects of noradrenaline. To elucidate the roles of cholinergic basal forebrain structures we administered methoxamine, an alpha‐1‐adrenergic agonist into the basal forebrain, in intact animals and again after selective destruction of the basal forebrain cholinergic cells by 192 IgG‐saporin. In eight male Han–Wistar rats implanted with electroencephalogram/electromyogram electrodes, a microdialysis probe targeted into the basal forebrain was perfused with artificial cerebrospinal fluid for 6 h on a baseline day, and with cerebrospinal fluid in the first and with methoxamine in the second 3‐h period of the subsequent day. The sleep–wake activity was recorded for 24 h on both days. Saporin was then injected into the basal forebrain and 2 weeks later the same experimental schedule (with cerebrospinal fluid and methoxamine) was repeated. In the intact animals, methoxamine exhibited a robust arousing effect and non‐rapid eye movement (NREM) and REM sleep was suppressed. Lesioning of the basal forebrain cholinergic neurones abolished almost completely the NREM sleep‐suppressing effect of methoxamine, whereas the REM sleep‐suppressing effect remained intact. Thus, the basal forebrain cholinergic neurones mediate, at least in part, cortical arousal and non‐REM sleep‐suppression, but they are not involved in the REM sleep‐suppressing effects of noradrenaline.     

Enhanced Survival and Neurite Network Formation of Human Umbilical Cord Blood Neuronal Progenitors in Three-Dimensional Collagen Constructs

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.     

Rolipram, an Antidepressant That Increases the Availability of cAMP, Transiently Enhances Wakefulness in Rats

A study was carried out on the effects on sleep of rolipram, an antidepressant that increases the availability of cAMP by inhibiting a phosphodiesterase isoenzyme. Rats were treated with rolipram (0.1 or 1 mg/kg) twice a day (at light and dark onset) for 11 days, after a chronic period of injection of physiological saline for habituation purposes. The sleep–wake activity was recorded for 12 h following the injection at light onset on the baseline day (physiological saline), on rolipram days 1, 5, and 11, and also on day 12, when physiological saline was injected again (withdrawal day). The high (1 mg/kg) dose of rolipram enhanced wakefulness (W) in postinjection h 1 on day 1 of rolipram treatment. After administration of 0.1 mg/kg rolipram, only a tendency to an increase in W was noted. The promotion of W might be attributed, at least in part, to an increased release of noradrenaline due to a cAMP-mediated stimulation of tyrosine hydroxylase.     

Endothelial Lined Skeletal Muscle Ventricles: Open and Percutaneous Seeding Techniques

Twelve bilateral skeletal muscle ventricles (SMVs) were constructed in six dogs by wrapping each latissimus dorsi muscle around a cylindrical, plastic mandrel (volume 30 cc). After 6 to 10 weeks, five dogs had one of their SMVs seeded with allogeneic cultured canine endothelial cells (8 times 10⁶ cells/pouch) via an open technique, while the contralateral SMV was seeded by percutaneous injection of cells into the space around the mandrel. After 1 week, the SMVs were excised. Viable, adherent endothelial cells were present in all seeded pouches; this was confirmed via fluorescent microscopy with several endothelial cell markers: KLH-2, dilacetylated low-density lipoprotein and antibodies to von Willebrand factor. The inner lining of the SMVs were also examined with scanning and transmission electron microscopy; the highest concentration of cells were seen at the apex where a continuous endothelial monolayer was observed. No significant difference in the distribution or the morphology of the endothelial lining was noted between the open and percutaneous seeding techniques. These data show that SMVs can be seeded with an endothelial monolayer using both open and percutaneous techniques.     

Atherosclerosis and glomerulosclerosis in WHHL rabbits and obese Zucker rats

The aorta and the kidney of 12 month old hyperlipidemic WHHL and obese Zucker rats, were examined morphologically. The WHHL developed severe and premature atherosclerosis but did not develop glomerulosclerosis. In contrast, the Zucker rats did not manifest atherosclerosis of the aorta, but developed glomerulosclerosis. These two animal models could be useful in understanding the roles of heterogeneous lipoprotein particles, genetic susceptibility, hemodynamic stress, and mesangial interactions with lipoproteins in the development of glomerulosclerosis.     

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 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.     

Novel methods for delivery of cell-based therapies

BACKGROUND: Pulmonary hypoplasia (PH) is found in 15% to 20% of all neonatal autopsies, accounting for 2850 deaths yearly. Development of engineered tissue substitutes that could functionally restore damaged tissue remains a unique opportunity for biotechnology. Recently, we isolated and characterized murine fetal pulmonary cells (FPC) and engineered 3-D pulmonary tissue constructs in vitro. Our goal is to devise a reliable and reproducible method for delivering FPC into a live animal model of PH. MATERIALS AND METHODS: Three methods of delivery were explored: intraoral, intratracheal, and intrapulmonary injection. Adult Swiss Webster mice were anesthetized and fluorescent labeled microspheres (20 microm diameter) were delivered by intraoral and intratracheal injection. Subsequently, labeled FPC (Cell Tracker, CMTPX; Molecular Probes, Eugene, OR) were delivered by the same methods. In addition, direct transpleural intrapulmonary injection of FPC was performed. Outcome analysis included survival, reproducibility, diffuse versus confined location of the injected substance, and adequacy of delivery. Routine histological examination, fluorescent microscopy, and immunostaining were performed. RESULTS: Microspheres: We demonstrated reproducible, diffuse instillation via tracheotomy into the distal alveoli. Intraoral delivery appeared less reliable compared to direct intratracheal injection. FPC: Intratracheal injection was a reliable method of delivery. Labeled FPC showed transepithelial migration after 7 d of in vivo culture. Intrapulmonary injection led to local accumulation of cells in sites of injection. CONCLUSIONS: We demonstrate that delivery of FPC is feasible with intratracheal injection giving the most reliable, diffuse delivery throughout the lung. This represents the first step toward translational research with site-specific delivery for a cell-based therapeutic approach toward PH and similar pulmonary diseases.