Effects Of Nerve Growth Factor Research Articles

Effects of the nerve growth factor and its carrier protein on the inflammatory response from human monocytes.

The nerve growth factor (NGF) has been previously shown to be involved in cellular proliferation, differentiation, survival, or wound healing. This factor displays a variety of biological effects that yet remain to be explored. Previous data on cell lines show a pro-inflammatory role of NGF on monocytes. The objective of the study was to investigate the pro-inflammatory effect of NGF, using a model of fresh human monocytes. Monocytes obtained from PBMC were exposed to NGF at various concentrations. Alternatively, monocytes were exposed to BSA, the NGF carrier protein without the NGF. Gene expression and cytokine release in the supernatant were monitored. We found that NGF increased the expression of pro-inflammatory, chemotactic, and remodeling genes such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and C-X-C motif ligand (CXCL)8. The protein levels of CXCL8 and matrix metalloproteinase (MMP)-9 were also increased in the cell supernatants following NGF exposure. BSA alone was found to drive part of this response, bringing nuance to the inflammatory potential of the NGF. These data suggest that NGF is able to enhance monocyte inflammatory responses once cells are stimulated with another signal but is possibly not able to directly activate it. This could have implications for example in patients with bacterial infections, where NGF could worsen the local inflammation by over-activating immune cells.

Comparison of the effects of EGF, FGF-b, and NGF on the proliferation, migration, and reprogramming of primary rat Müller cells.

During the healing process of full-thickness macular holes (FTMHs), the closure and recovery of the hole depend on the migration, proliferation, and activation of Müller cells to promote the closure of holes and restoration of the photosensitive layer. In this study, we investigated the ability of the epidermal growth factor (EGF), fibroblast growth factor-basic (FGF-b), and nerve growth factor (NGF) to influence this process by regulating proliferation, migration, and reprogramming of primary rat Müller cells. Cell proliferation was measured using CCK8 [2- (2-Methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5- (2,4-disulfophenyl)-2H-tetrazolium Sodium Salt] colorimetric assays and EdU [5-Ethynyl-2'-deoxyuridine] assays over 48 h. Cell migration was measured using scratch-wound assays and transwell migration assays over 48 h. In addition, we conducted Western blot assays and immunofluorescence assays on cells that were specially treated for 1, 3, and 5 days for cell reprogramming. The percentage of EdU-positive cells in Nestin-positive have also been tested by co-immunofluorescence (Co-IF) staining. EGF and FGF-b significantly promoted the proliferation of Müller cells (p < 0.05) at a concentration of 0-50 ng/mL, but NGF did not (p > 0.05), compared to untreated controls. Exogenous FGF-b and EGF promote the reprogramming of primary rat Müller cells, significantly enhancing the neural stem cell marker Nestin after stimulation on the 1st, 3rd, and 5th days, respectively. The expression of Müller cell marker Vimentin was significantly (p < 0.05) reduced during this period compared to the control group. However, there was no significant difference between the NGF and control groups. Furthermore, the EGF group expressed stronger Nestin expression than the SCM group. The Co-IF staining showed that early 50% of activated cells came from newly proliferating cells on the 5th day. These observations suggest that FGF-b can promote the activation of Müller cells in a short time and enhance the possessive features of neural stem cells, while EGF may act for a longer period of time. This may further the understanding of growth factor therapy in treating FTMHs, and Müller glia may be promising candidates for cell replacement therapy.

NGF contributes to activities of acid-sensing ion channels in dorsal root ganglion neurons of male rats with experimental peripheral artery disease.

A feature of peripheral artery diseases (PAD) includes limb ischemia/reperfusion (I/R) and ischemia. Both I/R and ischemia amplify muscle afferent nerve-activated reflex sympathetic nervous and blood pressure responses (termed as exercise pressor reflex). Nevertheless, the underlying mechanisms responsible for the exaggerated autonomic responses in PAD are undetermined. Previous studies suggest that acid-sensing ion channels (ASICs) in muscle dorsal root ganglion (DRG) play a leading role in regulating the exercise pressor reflex in PAD. Thus, we determined if signaling pathways of nerve growth factor (NGF) contribute to the activities of ASICs in muscle DRG neurons of PAD. In particular, we examined ASIC1a and ASIC3 currents in isolectin B4 -negative muscle DRG neurons, a distinct subpopulation depending on NGF for survival. Hindlimb I/R and ischemia were obtained in male rats. In results, femoral artery occlusion increased the levels of NGF and NGF-stimulated TrkA receptor in DRGs, whereas they led to upregulation of ASIC3 but not ASIC1a. In addition, application of NGF onto DRG neurons increased the density of ASIC3 currents and the effect of NGF was significantly attenuated by TrkA antagonist GW441756. Moreover, the enhancing effect of NGF on the density of ASIC3-like currents was decreased by the respective inhibition of intracellular signaling pathways, namely JNK and NF-κB, by antagonists SP600125 and PDTC. Our results suggest contribution of NGF to the activities of ASIC3 currents via JNK and NF-κB signaling pathways in association with the exercise pressor reflex in experimental PAD.

SİNİR BÜYÜME FAKTÖRÜ İLE FARKLILAŞTIRILMIŞ PC12 HÜCRELERİNİN MORFOMETRİK VE FLORESANS ANALİZİ

Objective&#x0D; PC12 is a rat pheochromocytoma cell line. These&#x0D; cells characteristically undergo differentiation when&#x0D; cultured with nerve growth factor (NGF). Depending&#x0D; on the dose of NGF, the length of neurite extensions&#x0D; changes. Thanks to this differentiation property,&#x0D; the cells are used in neuroscience and in modeling&#x0D; pathophysiological diseases such as Alzheimer's,&#x0D; Parkinson's, and Amyotrophic Lateral Sclerosis.&#x0D; However, literature studies showing the effect of NGF&#x0D; on neurite extensions formed in PC12 cells are very&#x0D; limited. This study aimed to investigate the effect of&#x0D; NGF on neurite extensions and cell viability depending&#x0D; on dose and incubation time.&#x0D; Materials and Methods&#x0D; In this study, PC12 cells were incubated with 50 ng/ml&#x0D; and 100 ng/ml NGF for 3, 6 and 7 days. The lengths&#x0D; of neurite outgrowths and dead cell ratios were&#x0D; calculated in incubated cells.&#x0D; Results&#x0D; The results showed that the length of neurite&#x0D; extensions and dead cell ratio increased depending&#x0D; on NGF doses and incubation time. When NGF&#x0D; incubation times were compared, no difference was&#x0D; found between 50 ng/ml NGF 6 days and 100 ng/ml&#x0D; NGF 3 days groups.&#x0D; Conclusion&#x0D; When the dead cell ratios and sizes of neurite&#x0D; extensions in the experimental groups are evaluated,&#x0D; it is thought that 100 ng/ml NGF and 3 days incubation&#x0D; time parameters are ideal for PC12 cell differentiation.

Nerve Growth Factor, Antimicrobial Peptides and Chemotherapy: Glioblastoma Combination Therapy to Improve Their Efficacy.

Glioblastoma (GBM) is an aggressive and lethal malignancy of the central nervous system with a median survival rate of 15 months. We investigated the combined anticancer effects of nerve growth factor (NGF), cathelicidin (LL-37), and protegrin-1 (PG-1) with chemotherapy (temozolomide, doxorubicin, carboplatin, cisplatin, and etoposide) in the glioblastoma U251 cell line to overcome the limitations of conventional chemotherapy and to guarantee specific treatments to succeed. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to study cell viability and to determine the cytotoxic effects of NGF, LL-37, and PG-1 and their combination with chemotherapy in U251 cells. Synergism or antagonism was determined using the combination index (CI) method. Caspase-3 activity was evaluated spectrophotometrically using a caspase-3 activity assay kit. Apoptosis was analyzed with flow cytometry using propidium iodide (PI) and YO-PRO-1. NGF and the peptides showed a strong cytotoxic effect on U251 glioma cells in the MTT test (IC50 0.0214, 3.1, and 26.1 μM, respectively) compared to chemotherapy. The combination of PG-1 + etoposide had a synergistic effect on apoptosis of U251 glioma cells. It should be noted that the cells were in the early and late stages of apoptosis, respectively, compared with the control cells. The caspase-3 activation analysis revealed that the caspase-3 level was not significantly (p > 0.05) increased in U251 cells following PG-1 with etoposide treatment compared with that in the untreated cells, suggesting that the combination of PG-1 and etoposide may induce caspase-independent apoptosis in U251 cells. NGF, LL-37, and PG-1 represent promising drug candidates as the treatment regimen for GBM. Furthermore, the synergistic efficacy of the combined protocol using PG-1 and etoposide may overcome some of the typical limitations of the conventional therapeutic protocols, thus representing a promising approach for GBM therapy.

Topical Nerve Growth Factor (NGF) restores electrophysiological alterations in the Ins2Akita mouse model of diabetic retinopathy

People suffering from diabetes mellitus commonly have to face diabetic retinopathy (DR), an eye disease characterized by early retinal neurodegeneration and microvascular damage, progressively leading to sight loss. The Ins2Akita (Akita) diabetic mouse presents the characteristics of DR and experimental drugs can be tested on this model to check their efficacy before going to the clinic. Topical administration of Nerve Growth Factor (NGF) has been recently demonstrated to prevent DR in the Akita mouse, reverting the thinning of retinal layers and protecting the retinal ganglion cells (RGCs) from death. In this study, we characterize the effects of topical NGF on neuroretina function, quantified with the electroretinogram (ERG). In particular, we show that NGF can ameliorate RGC conduction in the retina of Akita mice, which correlates with a recovery of retinal nerve fiber plus ganglion cell layer (RNFL-GCL) structure. Overall, our preclinical results highlight that topical administration of NGF could be a promising therapeutic approach for DR, being capable of exerting a beneficial impact on retinal functionality.

MASH1 induces neuron transdifferentiation of adrenal medulla chromaffin cells.

Nerve growth factor (NGF) induces neuron transdifferentiation of adrenal medulla chromaffin cells (AMCCs) and consequently downregulates the secretion of epinephrine (EPI), which may be involved in the pathogenesis of bronchial asthma. Mammalian achaete scute-homologous 1 (MASH1), a key regulator of neurogenesis in the nervous system, has been proved to be elevated in AMCCs with neuron transdifferentiation in vivo. This study aims to explore the role of MASH1 in the process of neuron transdifferentiation of AMCCs and the mechanisms. Rat AMCCs were isolated and cultured. AMCCs were transfected with siMASH1 or MASH1 overexpression plasmid, then were stimulated with NGF and/or dexamethasone, PD98059 (a MAPK kinase-1 inhibitor) for 48 hours. Morphological changes were observed using light and electron microscope. Phenylethanolamine-N-methyltransferase (PNMT, the key enzyme for epinephrine synthesis) and tyrosine hydroxylase were detected by immunofluorescence. Western blotting was used to test the protein levels of PNMT, MASH1, peripherin (neuronal markers), extracellular regulated protein kinases (ERK), phosphorylated extracellular regulated protein kinases (pERK), and JMJD3. Real-time RT-PCR was applied to analyze the mRNA levels of MASH1 and JMJD3. EPI levels in the cellular supernatant were measured using ELISA. Cells with both tyrosine hydroxylase and PNMT positive by immunofluorescence were proved to be AMCCs. Exposure to NGF, AMCCs exhibited neurite-like processes concomitant with increases in pERK/ERK, peripherin, and MASH1 levels (all P<0.05). Additionally, impairment of endocrine phenotype was proved by a signifcant decrease in the PNMT level and the secretion of EPI from AMCCs (all P<0.01). MASH1 interference reversed the effect of NGF, causing increases in the levels of PNMT and EPI, conversely reduced the peripherin level and cell processes (all P<0.01). MASH1 overexpression significantly increased the number of cell processes and peripherin level, while decreased the levels of PNMT and EPI (all P<0.01). Compared with the NGF group, the levels of MASH1, JMJD3 protein and mRNA in AMCCs in the NGF+PD98059 group were decreased (all P<0.05). After treatment with PD98059 and dexamethasone, the effect of NGF on promoting the transdifferentiation of AMCCs was inhibited, and the number of cell processes and EPI levels were decreased (both P<0.05). In addition, the activity of the pERK/MASH1 pathway activated by NGF was also inhibited. MASH1 is the key factor in neuron transdifferentiation of AMCCs. NGF-induced neuron transdifferentiation is probably mediated via pERK/MASH1 signaling.

Reduced levels of NGF shift astrocytes toward a neurotoxic phenotype.

Nerve growth factor (NGF) is critical for neuronal physiology during development and adulthood. Despite the well-recognized effect of NGF on neurons, less is known about whether NGF can actually affect other cell types in the central nervous system (CNS). In this work, we show that astrocytes are susceptible to changes in ambient levels of NGF. First, we observe that interfering with NGF signaling in vivo via the constitutive expression of an antiNGF antibody induces astrocytic atrophy. A similar asthenic phenotype is encountered in an uncleavable proNGF transgenic mouse model (TgproNGF#72), effectively increasing the brain proNGF levels. To examine whether this effect on astrocytes is cell-autonomous, we cultured wild-type primary astrocytes in the presence of antiNGF antibodies, uncovering that a short incubation period is sufficient to potently and rapidly trigger calcium oscillations. Acute induction of calcium oscillations by antiNGF antibodies is followed by progressive morphological changes similar to those observed in antiNGF AD11 mice. Conversely, incubation with mature NGF has no effect on either calcium activity nor on astrocytic morphology. At longer timescales, transcriptomic analysis revealed that NGF-deprived astrocytes acquire a proinflammatory profile. In particular, antiNGF-treated astrocytes show upregulation of neurotoxic transcripts and downregulation of neuroprotective mRNAs. Consistent with that data, culturing wild-type neurons in the presence of NGF-deprived astrocytes leads to neuronal cell death. Finally, we report that in both awake and anesthetized mice, astrocytes in layer I of the motor cortex respond with an increase in calcium activity to acute NGF inhibition using either NGF-neutralizing antibodies or a TrkA-Fc NGF scavenger. Moreover, in vivo calcium imaging in the cortex of the 5xFAD neurodegeneration mouse model shows an increased level of spontaneous calcium activity in astrocytes, which is significantly reduced after acute administration of NGF. In conclusion, we unveil a novel neurotoxic mechanism driven by astrocytes, triggered by their sensing and reacting to changes in the levels of ambient NGF.

Increased levels of nerve growth factor accompany oxidative load in recurrent pregnancy loss. Machine learning applied to FT-Raman spectra study.

The presented article is focused on developing and validating an efficient, credible, minimally invasive technique based on spectral signatures of blood serum samples in patients with diagnosed recurrent pregnancy loss (RPL) versus healthy individuals who were followed at the Gynecology department. A total of 120 participants, RPL disease (n = 60) and healthy individuals (n = 60), participated in the study. First, we investigated the effect of circulating nerve growth factor (NGF) in RPL and healthy groups. To show NGF's effect, we measured the level of oxidative loads such as Total Antioxidant Level (TAS), Total Oxidant Level (TOS), and Oxidative Stress Index (OSI) with Beckman Coulter AU system and biochemical assays. We find a correlation between oxidative load and NGF level. Oxidative load mainly causes structural changes in the blood. Therefore, we obtained Raman measurements of the participant's serum. Then we selected two Raman regions, 800 and 1800cm-1, and between 2700cm-1 and 3000cm-1, to see chemical changes. We noted that Raman spectra obtained for RPL and healthy women differed. The findings confirm that the imbalance between reactive oxygen species and antioxidants has important implications for the pathogenesis of RPL and that NGF levels accompany the level of oxidative load in the RPL state. Biomolecular structure and composition were determined using Raman spectroscopy and machine learning methods, and the correlation of these parameters was studied alongside machine learning technologies to advance toward clinical translation. Here we determined and validated the development of instrumentation for the Analysis of RPL patients' serum that can differentiate from control individuals with an accuracy of 100% using the Raman region corresponding to structural changes. Furthermore, this study found a correlation between traditional biochemical parameters and Raman data. This suggests that Raman spectroscopy is a sensitive tool for detecting biochemical changes in serum caused by RPL or other diseases.

ProNGF Expression and Targeting in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most lethal adult brain cancer. Temozolomide (TMZ), the standard chemotherapeutic drug used in GBM, has limited benefit and alternate therapies are needed to improve GBM treatment. Nerve growth factor (NGF) and its precursor proNGF are increasingly recognized as stimulators of human tumor progression. The expression and stimulatory effect of NGF on GBM cell growth has previously been reported, but the status of proNGF in GBM is unreported. In this study, we have investigated proNGF expression and biological activity in GBM. A clinical cohort of GBM (n = 72) and low-grade glioma (n = 20) was analyzed by immunohistochemistry for proNGF and digital quantification. ProNGF expression was significantly increased in GBM compared to low grade gliomas and proNGF was also detected in patient plasma samples. ProNGF was also detected in most GBM cell lines by Western blotting. Although anti-proNGF blocking antibodies inhibited cell growth in GBM cells with methylated MGMT gene promoter, targeting proNGF could not potentiate the efficacy of TMZ. In subcutaneous xenograft of human GBM cells, anti-proNGF antibodies slightly reduced tumor volume but had no impact on TMZ efficacy. In conclusion, this data reveals that proNGF is overexpressed in GBM and can stimulate cancer cell growth. The potential of proNGF as a clinical biomarker and therapeutic target warrants further investigations.

Effects of NGF and Photobiomodulation Therapy on Crush Nerve Injury and Fracture Healing: A Stereological and Histopathological Study in an Animal Model.

A stereological and histopathological study in an animal model. This study explores the effects of the nerve growth factor and photobiomodulation therapy on the damaged nerve tissue and fracture healing. A total of 24 rabbits were divided into 4 groups: control group (n = 5), nerve growth factor (NGF) group (n = 7), photobiomodulation (PBMT) group (n = 6), and nerve growth factor and photobiomodulation therapy (NGF+PBMT) group (n = 6). The vertical fracture was performed between the mental foramen and the first premolar, and the mental nerve was crushed for 30 seconds with a standard serrated clamp with a force of approximately 50N in all groups. The control group received an isotonic solution (.02mL, .09% NaCl) to the operation site locally. The NGF group received 1μg human NGF-β/.9% .2mL NaCl solution for 7 days locally. The PBMT group received PBMT treatment (GaAlAs laser, 810nm, .3W, 18J/cm2) every 48 hours for 14 sessions following the surgery. The NGF+PBMT group received both NGF and PBMT treatment as described above. After 28 days, the bone tissues and mental nerves from all groups were harvested and histologically and stereologically analyzed. According to the stereological results, the volume of the new vessel and the volume of the new bone were significantly higher in the PBMT group than in other groups (P < .001). According to the histopathological examinations, higher myelinated axons were observed in experimental groups than in the control group. As a result, PBMT has beneficial effects on bone regeneration. Based on the light microscopic evaluation, more regenerated axon populations were observed in the NGF group than in the PBMT and PBMT + NGF groups in terms of myelinated axon content.

NGF and the Male Reproductive System: Potential Clinical Applications in Infertility.

Infertility is a worldwide health issue defined by the World Health Organization (WHO) as the inability to establish a pregnancy after 12 months or more of regular and unprotected sexual intercourse. Male infertility etiology can be related to either congenital or acquired factors. The therapeutical approach to male infertility depends on the underlying causes and includes medical and surgical treatments. In recent studies, the potential role of nerve growth factor (NGF) in male reproductive physiology has been proposed. It has been hypothesized that neurotrophins might be involved in testis morphogenesis and regulation of several aspects of spermatogenesis. Moreover, it has been shown that NGF exerts its role on gonadotropin-releasing hormone (GnRH) neurons through the activation of the PKC/p-ERK1/2/p-CREB cascade, which leads to the activation of hypothalamic cells and the consequent activation of hypothalamus-pituitary-gonadal axis (HPG) with the secretion of GnRH. Lastly, it has been shown that the physiology of mature sperm is affected by both exogenous and endogenous NGF. The NGF impact on the HPG axis and its effect on GnRH neurons might be exploited in the therapy of male hypogonadism or used as a protective strategy against gonadal dysfunction related to chemotherapeutic agents. Moreover, the improving effect of NGF on sperm motility and vitality could be useful to enhance assisted reproduction outcomes. NGF could be supplemented to cryopreserved sperm samples to counteract the oxidative stress induced by the frozen and thawing processes. Indeed, the potential clinical applications of NGF in male infertility treatment have been discussed.

The effects of painless nerve growth factor on human microglia polarization.

Previous studies in the rat suggest that microglial cells represent a potential druggable target for nerve growth factor (NGF) in the brain. The painless human Nerve Growth Factor (hNGFp) is a recombinant mutated form of human nerve growth factor (hNGF) that shows identical neurotrophic and neuroprotective properties of wild-type NGF but displays at least 10-fold lower algogenic activity. From the pharmacological point of view, hNGFp is a biased tropomyosin receptor kinase A (TrkA) agonist and displays a significantly lower affinity for the p75 neurotrophin receptor (p75NTR). This study aimed to evaluate the expression of TrkA and p75NTR NGF receptors in two different human microglia cell lines, and to investigate the effects of hNGFp and wild-type NGF (NGF) on L-arginine metabolism, taken as a marker of microglia polarization. Both NGF receptors are expressed in human microglia cell lines and are effective in transducing signals triggered by NGF and hNGFp. The latter and, to a lesser extent, NGF inhibit cytokine-stimulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in these cells. Conversely NGF but not hNGFp stimulates arginase-mediated urea production.

NGF regulates sertoli cell growth and prevents LPS-induced junction protein damage via PI3K/AKT/NFκB signaling

Damage to Sertoli cell junction proteins caused by inflammation can lead to male infertility. Nerve growth factor (NGF) plays an important role in reproductive and inflammatory disease; however, whether and how NGF regulates Sertoli cell function remains unclear. Here, we aimed to assess the effect of NGF on the growth of Sertoli cells isolated from the testes of dairy goats and evaluate if NGF has a protective effect on these cells. We confirmed that Sertoli cell viability, proliferation, and ATP content increased following NGF treatment. In addition, qPCR results suggested that Sertoli cell apoptosis was inhibited after NGF treatment. To investigate the protective effect of NGF on Sertoli cells under pathological inflammatory conditions, LPS was used to induce inflammatory response in Sertoli cells. Post-treatment, the entangled filamentous pseudopodia of the cells loosened and no longer spanned adjacent cells. The expression of several junction proteins (ZO-1, occludin, CX-43, β-catenin, and N-cadherin), which was down-regulated after inflammatory response induction, was restored following NGF treatment. LPS-induced changes in cytotoxicity and transepithelial electrical resistance were reversed and the intercellular connections became tighter after NGF treatment. We further demonstrated that NGF prevented the inflammatory response of Sertoli cells via the PI3K/AKT/NFκB signaling pathway, similar to the effect of the PI3K-inhibitor, LY294002, which is modified by the PI3K activator, 740Y–P. These results provide insights for devising strategies for protecting the male reproductive system and curing or preventing associated pathological conditions.

Nerve Growth Factor (NGF) Encourages the Neuroinvasive Potential of Pancreatic Cancer Cells by Activating the Warburg Effect and Promoting Tumor Derived Exosomal miRNA-21 Expression.

Background It has been reported that signaling from the nerve growth factor (NGF) pathway associated with peripheral nerves is able to contribute to perineural invasion (PNI) of pancreatic cancer (PC). Nevertheless, the underlying mechanism by which NGF leads to PNI remained poorly understood. Methods Western blotting was employed to determine NGF level in PC and paracarcinoma tissues and in PC cell lines as well as pancreatic ductal epithelial cells. MiaPaCa-2 and CFPAC-1 cells were treated with 100 ng/ml of NGF or the NGF inhibitor Tanezumab for 24 h, CCK-8 and Transwell assays were employed to test cell proliferation, invasion, and migration, respectively. TrkA expression was knocked down in MiaPaCa-2 and dorsal root ganglion (DRG) cells treated with NGF to determine its effect on the Warburg effect. To reveal that the NGF-TrkA signaling pathway was closely associated with PC PNI, in vitro neuroinvasion model was established by using MiaPaCa-2 cells via coculturing DRG cells in Matrigel. Further, exosomes were extracted from PC cells and identified by examining the levels of specific markers for exosomes. Then RT-qPCR was applied to test miR-21-5p level in tumor derived exosomal (TDE-miR-21-5p). RIP assay was performed to validate NGF and miR-21 binding ability in MiaPaCa-2 cells. Rescue experiments were performed by using coprocessing of Tanezumab and miR-21-5p mimic on MiaPaCa-2 cells, followed by coculture with DRG cells. Subsequently, we used a model of neuroinvasion in nude mice to assess the effect of NGF in vivo on tumor nerve invasion as well as on nociceptive transmission. Results NGF level was preeminently higher in PC tissues and cell lines than in paracarcinoma tissues and normal pancreatic epithelial cell lines. NGF promoted MiaPaCa-2 and CFPAC-1 cell invasion and migration, while Tanezumab treatment showed the opposite results. Besides, NGF binding to TrkA receptors encouraged the intracellular Warburg effect in PC and DRG cells. TrkA blocking-up could restrain NGF induced PC cell migration and neural invasion. Mechanistically, NGF could upregulate TDE-miR-21-5p levels, and DRG cells took up TDE to activate the Warburg effect and stimulate nociceptor gene expression. miR-21-5p inhibitor could abolish the facilitative effect of NGF on PNI in MiaPaCa-2 cells. In vivo tumorigenesis experiments, Tanezumab markedly alleviated nerve invasion of PC cells as well as relieved nociceptive conduction in animal models. Conclusions These findings displayed that NGF/TrkA encouraged the neuroinvasive potential of PC cells by activating the Warburg effect in DRG cells through upregulation of TDE-miR-21-5p expression.

NGF Prevents Loss of TrkA/VEGFR2 Cells, and VEGF Isoform Dysregulation in the Retina of Adult Diabetic Rats.

Among the factors involved in diabetic retinopathy (DR), nerve growth factor (NGF) and vascular endothelial growth factor A (VEGFA) have been shown to affect both neuronal survival and vascular function, suggesting that their crosstalk might influence DR outcomes. To address this question, the administration of eye drops containing NGF (ed-NGF) to adult Sprague Dawley rats receiving streptozotocin (STZ) intraperitoneal injection was used as an experimental paradigm to investigate NGF modulation of VEGFA and its receptor VEGFR2 expression. We show that ed-NGF treatment prevents the histological and vascular alterations in STZ retina, VEGFR2 expression decreased in GCL and INL, and preserved the co-expression of VEGFR2 and NGF-tropomyosin-related kinase A (TrkA) receptor in retinal ganglion cells (RGCs). The WB analysis confirmed the NGF effect on VEGFR2 expression and activation, and showed a recovery of VEGF isoform dysregulation by suppressing STZ-induced VEGFA121 expression. Reduction in inflammatory and pro-apoptotic intracellular signals were also found in STZ+NGF retina. These findings suggest that ed-NGF administration might favor neuroretina protection, and in turn counteract the vascular impairment by regulating VEGFR2 and/or VEGFA isoform expression during the early stages of the disease. The possibility that an increase in the NGF availability might contribute to the switch from the proangiogenic/apoptotic to the neuroprotective action of VEGF is discussed.

Topical nerve growth factor prevents neurodegenerative and vascular stages of diabetic retinopathy.

Specific and effective preventive treatment for diabetic retinopathy (DR) is presently unavailable, mostly because the early stages of the complication have been, until recently, poorly understood. The recent demonstration that the vascular phase of DR is preceded and possibly caused by the neurodegeneration of retinal ganglion cells suggests that DR could, at least theoretically, be prevented through an early neuroprotective approach. The aims of our study were to clarify the natural history of diabetes-driven retinal neurodegeneration and to verify the possibility to prevent DR using topical nerve growth factor (NGF). The results of the study show that retinal neurodegeneration, characterized by the loss of retinal ganglion cells represents a relatively early phenomenon of diabetes (between 5 and 16 weeks of age), which tends to be self-limiting in the long run. Neurodegeneration is followed by the development of DR-related vascular dysfunctions, as confirmed by the development of acellular capillaries and the loss of retinal pericytes. Both retinal neurodegeneration and subsequent vascular dysfunction can be successfully prevented by topical NGF administration. These findings suggest that: 1) The first stage of DR consists in a self-limiting retinal neurodegeneration 2) The demonstrated effectiveness of topical NGF in the prevention of DR could be rapidly translated into clinical practice.

Evaluation of clinical and histological effects of KGF-2 and NGF on corneal wound healing in an experimental alkali burn rabbit model

Endogenously produced peptide growth factors such as keratinocyte growth factor-2 (KGF-2) and nerve growth factor (NGF) play a key role in the natural corneal wound healing process. However, this self-healing ability of the corneal tissue is often impaired in cases of severe corneal damage, as in corneal alkali injuries. In the present study, we investigated the clinical and histopathological effects of topical recombinant human keratinocyte growth factor-2 and nerve growth factor treatments in a rabbit model of corneal alkali burn. After induction of an alkali burn, 24 rabbits were divided equally into three groups: control group, KGF-2 group, and NGF group. Clinical parameters including epithelial healing, opacification, neovascularization and central corneal thickness were evaluated on the first (D1), seventh (D7) and fourteenth (D14) days after injury. Corneal histology was performed using hematoxylin/eosin (H&E) and Masson's Trichrome stains. Immunohistochemical staining for matrix metalloproteinase-2 (MMP-2), MMP-9 and transforming growth factor-β (TGF-β) was performed. On D14, the percentage of epithelial defect and opacity were significantly less in the KGF-2 and NGF groups compared to the control group (p < 0.05). There was no significant difference between the groups in central corneal thickness. In the evaluation of neovascularization on D14, the NGF group was significantly less vascularized than the control group (p = 0.011). Histological examination showed a significant increase in stromal edema and inflammation in the control group compared to both treatment groups (p < 0.05). There was also a significant difference between the NGF and control groups in histological evaluation of epithelial repair and vascularization (p < 0.05). When immunoreactivity of MMP-2, MMP-9 and TGF-β was examined, there was a significant increase in the control group compared to the NGF group (p < 0.05). Taken together, both NGF and KGF-2 treatments were effective for early re-epithelialization and decrease in inflammation, opacity and neovascularization after corneal alkali burn. The inhibitory effect of NGF treatment on chemical-induced neovascularization was found to be superior to KGF-2 treatment.

Anticancer Effect of Cathelicidin LL-37, Protegrin PG-1, Nerve Growth Factor NGF, and Temozolomide: Impact on the Mitochondrial Metabolism, Clonogenic Potential, and Migration of Human U251 Glioma Cells.

Glioblastoma (GBM) is one of the most aggressive and lethal malignancy of the central nervous system. Temozolomide is the standard of care for gliomas, frequently results in resistance to drug and tumor recurrence. Therefore, further research is required for the development of effective drugs in order to guarantee specific treatments to succeed. The aim of current study was to investigate the effects of nerve growth factor (NGF), human cathelicidin (LL-37), protegrin-1 (PG-1), and temozolomide on bioenergetic function of mitochondria, clonogenicity, and migration of human U251 glioma cells. Colony formation assay was used to test the ability of the glioma cells to form colonies in vitro. The U251 glioma cells migration was evaluated using wound-healing assay. To study the mitochondrial metabolism in glioma cells we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) using a Seahorse XF cell Mito stress test kit and Seahorse XF cell Glycolysis stress kit, respectively. We revealed that LL-37, NGF, and TMZ show strong anti-tumorigenic activity on GMB. LL-37 (4 μM), TMZ (155 μM), and NGF (7.55 × 10−3 μM) inhibited 43.9%–60.3%, 73.5%–81.3%, 66.2% the clonogenicity of glioma U251 cells for 1–2 days, respectively. LL-37 (4 μM), and NGF (7.55 × 10−3 μM) inhibited the migration of U251 glioma cells on the third and fourth days. TMZ also inhibited the migration of human glioma U251 cells over 1–3 days. In contrast, PG-1 (16 μM) stimulated the migration of U251 glioma cells on the second, fourth, and sixth days. Anti-mitogenic and anti-migration activities of NGF, LL-37, and TMZ maybe are relation to their capacity to reduce the basal OCR, ATP-synthetase, and maximal respiration of mitochondria in human glioma U251 cells. Glycolysis, glycolytic capacity and glycolytic spare in glioma U251 cells haven`t been changed under the effect of NGF, LL-37, PG-1, and TMZ in regard to control level. Thus, LL-37 and NGF inhibit migration and clonogenicity of U251 glioma cells, which may indicate that these compounds have anti-mitogenic and anti-migration effects on human glioma cells. The study of the mechanisms of these effects may contribute in the future to the use of NGF and LL-37 as therapeutic agents for gliomas.

Effect of Coriander on H2O2-induced Oxidative Stress in PC12 Cells

Objective: Coriander (Coriandrum sativum), a member of the Apiaceae family of plants, is traditionally used as a spice and medicinal plant which has anti-aging, anti-inflammatory, and anti-anxiety effects and is used for the treatment of diabetes. The present study aimed to investigate the effects of coriander on H2O2-induced neurotoxicity in PC12 cells. Methods: PC12 cells were cultured in Dulbecco’s modified Eagle’s medium, and the effects of various treatments were analyzed using cytotoxicity assay, morphological evaluation of neurite outgrowth, and western blotting. Results: The cytotoxicity assay showed increased levels of LDH in cells treated above 10-5 M H2O2, however there was no significant change in the coriander administration group. Morphological evaluation showed significantly increased neurite outgrowth in the nerve growth factor (NGF) treatment group after 72 h. Treatment of NGF-stimulated cells with H2O2 reversed the effect of NGF on neurite outgrowth, which returned to the levels of the control group. Simultaneous administration of H2O2, NGF and 0.1 or 0.01 µg/mL of coriander significantly increased neurite outgrowth after 72 h. Western blot showed that phosphorylated ERK levels were significantly increased in the NGF group compared with H2O2+NGF group, and no significant differences were found compared to the H2O2+NGF+0.1 µg/mL coriander administration group. Conclusion: Coriander reversed the reduction in neurite outgrowth following treatment of NGF-stimulated PC12 cells with H2O2. This suggests that coriander may help prevent oxidative stress-induced neurodegeneration.