Bioactive Nerve Growth Factor Research Articles

Sustained local delivery of bioactive nerve growth factor in the central nervous system via tunable diblock copolypeptide hydrogel depots

Biomaterial vehicles that can provide sustained, site-specific molecular delivery in the central nervous system (CNS) have potential for therapeutic and investigative applications. Here, we present in vitro and in vivo proof of principle tests of diblock copolypeptide hydrogels (DCH) to serve as depots for sustained local release of protein effector molecules. We tested two DCH, K180L20 and E180L20, previously shown to self-assemble into biocompatible, biodegradable deposits that persist four to eight weeks after injection into mouse forebrain. In vitro tests demonstrated sustained release from dialysis cassettes of the representative protein, lysozyme, dissolved in K180L20 or E180L20 hydrogels. Release time in vitro varied in relation to DCH charge and mechanical properties, and ionic strength of the media. To evaluate bioactive protein delivery in vivo, we used nerve growth factor (NGF) and measured the size of mouse forebrain cholinergic neurons, which respond to NGF with cellular hypertrophy. For in vivo tests, the storage modulus of DCH depots was tuned to just below that of CNS tissue. In comparison with NGF injected in buffer, depots of NGF dissolved in either K180L20 or E180L20 provided significantly longer delivery of NGF bioactivity, maintaining hypertrophy of local forebrain cholinergic neurons for at least 4 weeks and inducing hypertrophy a further distance away (up to 5 mm) from injection sites. These findings show that depots of DCH injected into CNS can provide sustained delivery within the blood–brain barrier of a bioactive protein growth factor that exerts a predicted, quantifiable effect on local cells over a prolonged subacute time.

Preparation and evaluation of NGF-microsphere conduits for regeneration of defective nerves

Objectives: To fabricate nerve growth factor (NGF) microsphere conduits and evaluate their promotional effects on regeneration of defective nerves.Methods: NGF microspheres containing ovalbumin were prepared using a double-emulsion method. The total amount of NGF was determined by enzyme-linked immunosorbent assay, and bioactive NGF was quantified by the PC12 cell line method; the NGF bioactivity present during the release period was monitored. Conduits made of polylacticacid were added to the NGF microspheres to bridge and repair the nerve injury. The sciatic nerve of Wistar rats was used to evaluate nerve-regeneration efficacy.Results: NGF microspheres were spherical, with smooth and compact surfaces. The fabrication yield, encapsulation efficiency, and drug loading of the microspheres were 58·3, 29·72, and 0·003%, respectively. The monitoring of NGF bioactivity remaining during the release period showed that ∼40% of the original bioactivity was kept on the twenty-eighth day and 10% left on the ninetieth day. Polylacticacid conduits are lowly antigenic, porous (facilitates oxygen diffusion), and able to prevent long-term compression. After 15 days, the length of nerve regenerated from NGF-microsphere conduits was statistically different from that of the control groups (P<0·05). NGF microspheres can accelerate the early nerve-regeneration rate, although the rate was not as good as that caused by autografts. Three months later, the recovery rate of the regenerated nerve collected from NGF-microsphere conduits showed lower values of nerve-conduction velocity, muscular tension, and muscle weight. However, poorer rates of regeneration were observed in the self-mutilation foot compared with those of the control group. The control group had no statistical difference from the sodium chloride and NGF-solution conduits, the best in the autograft group.Discussion: A long-term promoting effect of microsphere-bound exogenous NGF on the functional repair of peripheral nerves could not be confirmed, although the nerve-regeneration rate was rapid in the early stage. Better methods are needed to incorporate protein-release systems into nerve conduits to improve functional recovery in patients with injured nerves.

Ionically cross-linked chitosan microspheres for controlled release of bioactive nerve growth factor

Controlled release of neurotrophic factors to target tissue via microsphere-based delivery systems is critical for the treatment strategies of diverse neurodegenerative disorders. The present study aims to investigate the feasibility of the controlled release of bioactive nerve growth factor (NGF) with ionically cross-linked chitosan microspheres (NGF–CMSs). The microspheres were prepared by the emulsion-ionic cross-linking method with sodium tripolyphosphate (STPP) as an ionic cross-linking agent. The size and distribution of the microspheres, SEM images, Fourier transform infra red spectroscopy (FT-IR), encapsulation efficiency, in vitro release tests and bioactivity assay were subsequently evaluated. We found that the microspheres had relatively rough surfaces with mean sizes between 20 and 31μm. FT-IR results provided evidence of ionic interaction between amino groups and phosphoric groups of chitosan and STPP. The NGF encapsulation efficiency ranged from 63% to 88% depending on the concentration of STPP. The in vitro release profiles of NGF from NGF–CMSs were influenced by the concentration of STPP. NGF–CMSs which were cross-linked with higher concentration of STPP showed slower but sustained release of NGF. In addition, the released NGF from NGF–CMSs was capable of maintaining the viability of PC12 cells, as well as promoting their differentiation. Taken together, our findings suggest that NGF–CMSs are capable of releasing bioactive NGF over 7 days, thus having potential application in nerve injury repair.

Inducible Nerve Growth Factor Delivery for Peripheral Nerve Regeneration In Vivo

HEK-293 cells can be genetically modified to release and regulate nerve growth factor (NGF) in vitro. The aim of this study was to evaluate the impact of this NGF delivery system on peripheral nerve regeneration in vivo. HEK-293 cells were transfected with an ecdysone receptor, NGF cDNA, and herpes simplex virus-thymidine kinase suicide vector. NGF production is induced by ponasterone A and stopped by ganciclovir. A 13-mm sciatic nerve gap was bridged with Silastic conduits in 120 nude rats, and transfected HEK-293 cells were added, induced, and boostered to secrete bioactive NGF. The induction of the cell line and additional booster with ponasterone A demonstrated significantly higher levels of bioactive NGF, enhanced macroscopic nerve growth, improved functional recovery, and histologic regeneration when compared with control groups after 7, 14, and 21 days, and 2 and 4 months. The treatment with ganciclovir resulted in suppression of the NGF production and decreased functional and histologic outcomes. Transfected HEK-293 cells can be regulated to inducibly produce bioactive NGF in vivo over prolonged periods. This tissue-engineered nerve construct including the NGF delivery system is able to improve peripheral nerve regeneration and functional recovery and appears to be superior to nerve isografts.

Long-term Delivery of Nerve Growth Factor by Encapsulated Cell Biodelivery in the Göttingen Minipig Basal Forebrain

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

A novel nerve guidance conduit with sustained release of NGF enhances sciatic nerve regeneration

Based on diffusion of nerve growth factor (NGF) from degradable material, a novel nerve guidance conduit (NGC) with sustained NGF release was prepared by embedding NGF into a degradable RGD-modified composite which has good cell affinity and biocompatibility. In vitro, the NGF release was quantified using enzyme-linked immunosorbent assay method, and the bioactivity of released NGF was examined by PC-12 cell bioassay. In vivo, the performance of the NGF-containing conduit was examined using rat sciatic nerve defect model. The experimental results show that the NGF release process is prolonged over 30 days, and at least some degree of NGF bioactivity is preserved after the fabrication process. Due to the promoting effect of bioactive released NGF on nerve regeneration, the performance of NGC for nerve repair is improved significantly. The results of this study indicate that the sustained NGF release system is suitable for peripheral nerve repair.

Fas ligand acts as a counter-receptor in Schwann cells and induces the secretion of bioactive nerve growth factor

Fas ligand (FasL) is best known for its role in apoptosis. Membrane-bound FasL can signal in FasL-bearing cells, a process known as reverse signalling. The biological and functional consequences of FasL reverse signalling in Schwann cells were studied. FasL engagement induced the secretion of soluble mediator(s) that stimulated neurite growth in PC12 cells, NGF secretion, and NGF mRNA levels. ERK1/2 and Src phosphorylation was rapidly increased and inhibition of their activation affected NGF synthesis and release. FasL can therefore act as a signal-transducing molecule in Schwann cells, leading to the secretion of NGF, and may contribute to peripheral nerve regeneration.

Monocytes deliver bioactive nerve growth factor through a brain capillary endothelial cell-monolayer in vitro and counteract degeneration of cholinergic neurons

Alzheimer's disease is an age-dependent brain disorder, characterized by progressive memory deficits and cognitive decline and loss of cholinergic neurons. Nerve growth factor (NGF) is the most potent protein to protect cholinergic neurons against degeneration. However, problems of delivery to the brain limit the therapeutical use of NGF. The aim of the present study was to test, if primary rat monocytes can be loaded with recombinant NGF and pass an in vitro monolayer of brain capillary endothelial cells (BCEC), release NGF, and support the cholinergic neurons in an organotypic brain slice model. Monocytes were isolated from rat blood by negative magnetic selection, loaded with recombinant NGF using Bioporter™. The monocytes adhered and migrated through an in vitro rat BCEC-monolayer. NGF released at the basolateral side counteracted degeneration of cholinergic basal nucleus of Meynert neurons. In conclusion, our present study shows a proof-of-principle, that primary monocytes secreting NGF might be useful tools to deliver NGF into the brain, however, further in vivo studies are necessary.

Trophically and topographically functionalized silk fibroin nerve conduits for guided peripheral nerve regeneration

Artificial nerve conduits (NC) can be used as an alternative to autologous nerve grafts to enhance the repair of small nerve gaps. Current NC lack adequate molecular and structural functionalities. Thus, we developed silk fibroin NC (SF NC) that were loaded with glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) and topographically functionalized with aligned and non-aligned SF nanofibers. The SF NC were produced from fully functionalized SF membranes on which initial experiments were performed. DRG (dorsal root ganglions) sensory neurons and spinal cord (SpC) motor neurons, both from chicken embryos, exhibited an augmented length and rate of axonal outgrowth parallel to the aligned nanofibers. In addition, glial cells from DRG proliferated and migrated in close association and even slightly ahead of the outgrowing axons. On the contrary, axonal and glial growth was slower and randomly oriented on non-aligned nanofibers. The DRG and SpC explants were also inserted into the lumen of the finished SF NC. The unidirectional orientation of axo-glial outgrowth from the explants evidenced the preservation of the trophic and topographical functionalities in the SF NC. Bioactive GDNF and NGF were released in vitro from SF NC over 4 weeks. Thus, the developed functionalized SF NC hold promise to enhance functional recovery of injured peripheral nerves.

Controlled nerve growth factor release from multi-ply alginate/chitosan-based nerve conduits

The delivery kinetics of growth factors has been suggested to play an important role in the regeneration of peripheral nerves following axotomy. In this context, we designed a nerve conduit (NC) with adjustable release kinetics of nerve growth factor (NGF). A multi-ply system was designed where NC consisting of a polyelectrolyte alginate/chitosan complex was coated with layers of poly(lactide-co-glycolide) (PLGA) to control the release of embedded NGF. Prior to assessing the in vitro NGF release from NC, various release test media, with and without stabilizers for NGF, were evaluated to ensure adequate quantification of NGF by ELISA. Citrate (pH 5.0) and acetate (pH 5.5) buffered saline solutions containing 0.05% Tween 20 yielded the most reliable results for ELISA active NGF. The in vitro release experiments revealed that the best results in terms of reproducibility and release control were achieved when the NGF was embedded between two PLGA layers and the ends of the NC tightly sealed by the PLGA coatings. The release kinetics could be efficiently adjusted by accommodating NGF at different radial locations within the NC. A sustained release of bioactive NGF in the low nanogram per day range was obtained for at least 15 days. In conclusion, the developed multi-ply NGF loaded NC is considered a suitable candidate for future implantation studies to gain insight into the relationship between local growth factor availability and nerve regeneration.

Maintaining bioactivity of NGF for controlled release from PLGA using PEG

The goal of this work was to investigate methods to retain bioactivity of nerve growth factor (NGF) after encapsulation in poly(lactic-co-glycolic acid) (PLGA) discs for controlled release. Poly(ethylene glycol) (PEG) was chosen as a porogen not only to control the release rate of NGF but also because it has been used to help maintain bioactivity of molecules in organic solvents. NGF and PEG were encapsulated in PLGA via standard dissolution-evaporation techniques with methylene chloride as the solvent. Morita et al. (Pharm Res 2000; 17:1367-1373) indicated that colyophilization of PEG and bioactive factors before exposure to organic solvents increased the retention of bioactivity. Therefore, various PEG:NGF mass ratios were colyophilized before encapsulation within PLGA to examine retained activity of NGF. When PEG was not colyophilized before encapsulation, NGF activity was lost during the fabrication process. In contrast, colyophilization of PEG and NGF supported retention of NGF activity during the entire fabrication process. The amount of PEG encapsulated was the dominating factor in the rate of NGF release regardless of the fabrication method. These results demonstrate the usefulness of PEG in both acting as a porogen to modulate release and aiding in the retention of activity of NGF. This process may be extended to other methods to enhance activity of growth factors after exposure to organic solvents.

Herpes Simplex Virus-Thymidine Kinase–Based Suicide Gene Therapy as a “Molecular Switch Off” for Nerve Growth Factor Production In Vitro

Tissue-engineered constructs offer a new hope to patients suffering from functional impairment after nerve injury. An effort has been made to focus on delivery, regulation, and "molecular shutoff" of nerve growth factor (NGF) in tissue-engineered constructs. We have previously demonstrated that human embryonic kidney (HEK-293) cells can be genetically modified to secrete NGF at varying time points upon up regulation with Ponasterone A (PonA) both in vitro and in vivo. In the present study, HEK-293 cells that stably and inducibly produce NGF were further stably transfected with herpes simplex virus-thymidine kinase gene as a suicide gene (hNGF-EcR-293-TK) in order to shut off the NGF secretion and kill the cells upon treatment with ganciclovir (GCV). These cells following induction with PonA secreted NGF levels of 6659.2 +/- 489.4 pg/mL at day 10 postbooster dose at day 5, which was significantly higher than the control noninduced cells. The NGF secreted by these cells was bioactive as determined by a rat adrenal pheochromocytoma (PC-12) cell bioassay. Treatment of these cells with GCV significantly reduced the NGF levels to 645.3 +/- 16.2 pg/mL at day 10 and live cell numbers dropped to 7.95 x 10(3) +/- 278 compared to 2.73 x 10(5) +/- 6.1 x 10(4). GCV-treated cell media when transferred to the PC-12 cell bioassay demonstrated less than 10% cells differentiating into neurite-like extensions. We conclude that hNGF-EcR-293-TK cells can inducibly secrete bioactive NGF when treated with the inducing agent and can also be killed upon treatment with GCV. This double-gene transfection for gene expression and molecular shutoff mechanism will be a useful tool in tissue-engineered nerve constructs.

Controlled release of nerve growth factor from fibrin gel

Nerve growth factor (NGF) is known to promote the axonal regeneration in injured nerve system. Delivery of NGF for a long period in a controlled manner may enhance the regeneration efficacy. In this study, we investigated whether NGF can be released from fibrin gel for a long period in a controlled manner. We also investigated whether sustained delivery of NGF using fibrin gel can enhance the efficacy of NGF in vitro. The addition of heparin to fibrin gel decreased the rate of NGF release from the fibrin gel. As the concentrations of thrombin and fibrinogen in fibrin gel increased, the NGF release rate decreased significantly, and the initial release burst decreased. NGF was released for up to 14 days in vitro. The bioactivity of NGF released from fibrin gel was assessed by morphological changes of pheochromocytoma (PC12) cells cultured in the presence of NGF-containing fibrin gel. NGF released from fibrin gel exhibited significantly higher degrees of PC12 cell viability and differentiation than NGF added in a free form daily into the culture medium. This study demonstrates that fibrin gel can release NGF in a sustained, controlled manner and in a bioactive form.

Nerve growth factor expression by PLG-mediated lipofection

Biomaterials capable of efficient gene delivery provide a fundamental tool for basic and applied research models, such as promoting neural regeneration. We developed a system for the encapsulation and sustained release of plasmid DNA complexed with a cationic lipid and investigated their efficacy using in vitro models of neurite outgrowth. Sustained lipoplex release was obtained for up to 50 days, with rates controlled by the fabrication conditions. Released lipoplexes retained their activity, transfecting 48.2±8.3% of NIH3T3 cells with luciferase activity of 3.97×10 7 RLU/mg. Expression of nerve growth factor (NGF) was employed in two models of neurite outgrowth: PC12 and primary dorsal root ganglia (DRG) co-culture. Polymer-mediated lipofection of PC12 produced bioactive NGF, eliciting robust neurite outgrowth. An EGFP/NGF dual-expression vector identified transfected cells (GFP-positive) while neurite outgrowth verified NGF secretion. A co-culture model examined the ability of NGF secretion by an accessory cell population to stimulate DRG neurite outgrowth. Polymer-mediated transfection of HEK293T with an NGF-encoding plasmid induced outgrowth by DRG neurons. This system could be fabricated as implants or nerve guidance conduits to support cellular and tissue regeneration. Combining this physical support with the ability to locally express neurotrophic factors will potentiate regeneration in nerve injury and disease models.

Nerve Growth Factor Expression Response to Induction Agent Booster Dosing in Transfected Human Embryonic Kidney Cells

To assess whether nerve growth factor (NGF) expression would respond to booster dosing with the inducing agent ponasterone A, human embryonic kidney cells (HEK-293) were transfected with human NGF cDNA. Cells were cultured for 5 days in media with or without ponasterone A. On day 5, controls received a ponasterone A media replacement, whereas experimental groups received ponasterone A booster media replacement. NGF protein expression bioactivity was assessed using a PC-12 cell bioassay and the concentration of secreted NGF was quantified using NGF enzyme-linked immunosorbent assay. Cells with and without ponasterone A were left for 5 days without changing the medium. On day 5, the supernatants were collected and flash-frozen for enzyme-linked immunosorbent assay. The ponasterone A-positive and -negative booster medium was replaced in the appropriate wells. Supernatants were collected from the wells at 2, 4, and 6 days after the booster dose and removal of original supernatant. The medium was flash-frozen for enzyme-linked immunosorbent assay (1.5 ml), and the remaining 500 mul was transferred to PC-12 cells seeded onto 12-well plates to determine NGF bioactivity. All experiments were performed in quadruplicate. NGF production was measured daily by enzyme-linked immunosorbent assay over a 6-day period after the ponasterone A booster to a maximal release of 1233 +/- 130 pg/ml at day 6 (11 days after original induction). Maximal NGF production per 10(3) cells was 2.5 +/- 0.61 pg at day 6. Bioactivity was determined by percentage differentiation (per 100 cells counted) at 26, 52, and 98 percent for ponasterone A-treated wells on 2, 4, and 6 days after booster dosing (7, 9, and 11 days after induction), respectively. PC-12 cell differentiation was not visualized in the ponasterone A-negative control wells. Human NGF-EcR-293 cells can inducibly secrete bioactive NGF when exposed to the induction agent ponasterone A. Furthermore, repeated bioactive NGF expression peaks beyond that previously demonstrated can be achieved using induction agent booster dosing, indicating the ability to regulate the system over an extended period.

In Vivo Induction and Delivery of Nerve Growth Factor, Using HEK-293 Cells

Tissue-engineering strategies offer hope to patients facing functional impairment after nerve injury. We have previously demonstrated that HEK-293 cells can release nerve growth factor (NGF) in vitro, using an inducible system of expression. In this study, our objective was to assess the efficacy of the NGF delivery system in vivo, using nude rats. HEK-293 cells were transfected with human NGF cDNA. Ponasterone A (PonA) was used as the inducing agent. NGF collection chambers were implanted subcutaneously in nude rats. Sealed chambers were filled with one of the following: (1) DMEM, (2) untransfected 293 cells (EcR-293) plus PonA, (3) untransfected EcR-293 without PonA, (4) transfected 293 cells (hNGF-EcR-293) plus PonA, or (5) transfected hNGF-EcR-293 without PonA. Chambers were aspirated 24, 48, and 120 h postimplantation. NGF secretion was analyzed in the following ways: (1) NGF protein expression bioactivity was assessed in a PC-12 cell bioassay, and (2) the concentration of secreted NGF was quantified by NGF ELISA. NGF quantification by ELISA reached a maximal release of 12.9 +/- 3.57 ng/mL at 120 h. PC-12 cells exposed to media from induced transfected HEK-293 cell chambers demonstrated higher levels of differentiation compared with controls. We conclude that hNGF-EcR-293 cells can inducibly secrete bioactive NGF when exposed to the induction agent PonA. This regulated delivery system can secrete bioactive NGF for up to 5 days in vivo. We believe this regulated delivery system will be useful for tissue-engineered nerve constructs.

Human embryonic kidney cells (HEK-293 cells): characterization and dose-response relationship for modulated release of nerve growth factor for nerve regeneration.

The development of engineered constructs to bridge nerve gaps may hold the key to improved functional outcomes in the repair of injured peripheral nerves. These constructs must be rendered bioactive by providing the growth factors required for successful peripheral nerve regeneration. Previous studies demonstrated that harvested human and rat dermal fibroblasts could be genetically engineered to release nerve growth factor (NGF) both in vitro and in vivo. The use of fibroblasts, however, has the potential to cause scarring, and the expression of NGF from those cells was transient. To overcome these potential difficulties, human embryonic kidney cells were modified for use with the ecdysone-inducible mammalian expression system. These cells (hNGF-EcR-293) have been engineered and regulated to secrete human NGF in response to the ecdysone analogue ponasterone A. HEK-293 cells were transfected with human NGF cDNA with the ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, Calif.). Stable clones were then selected. Ponasterone A, an analogue of ecdysone, was used as the inducing agent. The secretion of NGF into the medium was analyzed with two different methods. After 24 hours of exposure to the inducing agent, cell medium was transferred to PC-12 cells seeded in 12-well plates, for determination of whether the secreted NGF was bioactive. Medium from untreated or ponasterone A-treated hNGF-EcR-293 cells was deemed bioactive on the basis of its ability to induce PC-12 cell differentiation. The concentrations of secreted NGF were also quantified with an enzyme-linked immunosorbent assay, in triplicate. NGF production was measured in successive samples of the same medium during a 9-day period, with maximal release of 9.05 +/- 2.6 ng/ml at day 9. Maximal NGF production was 8.46 +/- 2.1 pg/10(3) cells at day 9. These levels were statistically significantly different from levels in noninduced samples (p <or= 0.05). Differences in NGF secretion with the three different concentrations of ponasterone A (1, 2, and 3 microM) were not statistically significant. PC-12 cells exposed to medium from induced transfected HEK-293 cells demonstrated markedly higher levels of differentiation, compared with control levels, indicating bioactive protein secretion. It was demonstrated that this regulated delivery system could secrete bioactive NGF for up to 9 days and might be useful for in vivo applications. This regulated delivery system should be useful for tissue-engineered nerve constructs.

An autocrine function of nerve growth factor for cell cycle regulation of vascular endothelial cells

Nerve growth factor (NGF) regulates maintenance, survival, and function of not only neuronal cells but also various kinds of non-neuronal cells. Here we clearly demonstrated that mouse aortic endothelial cells (AEC) produced bioactive NGF, and the production was enhanced by a proinflammatory cytokine, interleukin (IL)-1β. AEC expressed both high affinity (TrkA) and low affinity (p75 NGFR) receptors for NGF. Exogenously added NGF induced rapid phosphorylation of TrkA tyrosine kinase. Addition of anti-NGF neutralizing antibody resulted in an increase in the proportion of AEC in S and G 2/M phases and in a hypodiploid range. Since the vascular endothelium plays a pivotal role in inflammatory conditions, these results strongly suggest that NGF, whose production is enhanced at the affected site, may contribute to maintenance, survival, and function of vascular endothelial cells by autocrine and/or paracrine mechanisms.

Human dermal microvascular endothelial cells produce nerve growth factor: implications for wound repair.

Following cutaneous injury, sensory nerves regenerate into the dermis and epidermis. Tissues that are innervated by sensory nerves synthesize neurotrophins such as nerve growth factor (NGF). The close anatomic proximity of nerves and capillaries throughout the skin suggests that mutual regulation may exist between nerve fibers and microvascular endothelial cells (MECs) during wound repair. Release of the neuropeptide substance P by sensory nerves induces endothelial cell rounding, capillary leak, and cytokine upregulation. We propose that dermal endothelial cells produce neurotrophins required for nerve fiber maintenance and regeneration. In this study, we demonstrate that substance P stimulates NGF messenger RNA expression by cultured human dermal MECs. Likewise, enzyme-linked immunosorbant assay demonstrated that conditioned medium from cultured dermal MECs contains NGF. NGF bioactivity in the supemates was verified by conditioned medium-induced clonal rat pheochromocytoma (PC-12) cell differentiation. This activity was inhibited by anti-NGF antibodies. Therefore, we have demonstrated that substance P, an inflammatory neuropeptide released by sensory nerve fibers, induces endothelial cells to produce NGF. Our data suggest that MECs may be unrecognized contributors to nerve regeneration after cutaneous injury.

Neurotrophins in cultured cells from periodontal tissues.

We review the basic functions of neurotrophins and their receptors and discuss the expression and functions of neurotrophins and their specific receptors based on recent data using cultured cells from human periodontal tissues. Neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) play crucial roles in the differentiation and survival of neural cells. Neurotrophins activate 2 different receptor classes: the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases (TrkA, TrkB, and TrkC) and the p75 receptor, a member of the tumor necrosis factor receptor superfamily. Neurotrophins regulate both cell death and cell survival through activations of Trk receptors and/or p75 neurotrophin receptor. It has been reported that neurotrophins are also produced from non-neuronal cells, such as leukocytes, osteoblasts, or fibroblasts, and act in many other ways on non-neuronal cells. Neurotrophin expression during bone fracture healing is especially interesting, and neurotrophins are now implicated in hard tissue regeneration. It is well known that neurotrophins and their receptors are expressed in tooth development. Recent studies have found that neurotrophins and Trk receptors are expressed in mouse osteoblastic cell lines. Human periodontal ligament cells, human gingival fibroblasts, and human gingival keratinocytes expressed mRNA for NGF and TrkA. The secretion of bioactive NGF peptides from human periodontal ligament cells and human gingival keratinocytes was confirmed by bioassay using PC12 cells (rat adrenal pheochromocytoma cells). The expression of NGF and TrkA.mRNA was regulated by interleukin (IL)-1beta. NGF increased DNA synthesis and expressions of mRNA for bone-related proteins, alkaline phosphatase, and osteopontin in human periodontal ligament cells. Neurotrophins and Trk receptors expressed in human periodontal tissue may contribute to regeneration as well as innervation of periodontal tissue through local autocrine and paracrine pathways. Recent data suggest that some functions of neurotrophins and Trk receptors relate to periodontal disease and periodontal tissue regeneration. However, in vivo studies will be required to clarify the roles of neurotrophins and their receptors, including p75, in periodontal disease and periodontal tissue regeneration.