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IGF-1 Signaling via the PI3K/Akt Pathway Confers Neuroprotection in Human Retinal Pigment Epithelial Cells Exposed to Sodium Nitroprusside Insult
Abstract
The pathological increase in the levels of the second messenger nitric oxide (NO) in the vitreous cavity and retina leads to injury and cell death of the retinal pigment epithelium (RPE) cells and eventually may contribute to the occurrence and development of diabetic retinopathy. In this study, we developed a cellular model of retinopathy using D407 cells (a human RPE cell line) exposed to sodium nitroprusside (SNP) and investigated the protective effect of the insulin-like growth factor-1 (IGF-1) towards this insult. Cell death and apoptosis were examined by the methyl thiazolyl tetrazolium assay and Hoechst staining, respectively. Specific inhibitors were used and phosphorylation of relevant signaling proteins was determined by Western blotting. SNP, in a concentration-dependent fashion, increased the production of reactive oxygen species (ROS) and lipid peroxidation process causing cell death by apoptosis of D407 cells. IGF-1, in a time- and dose-dependent manner, conferred protection towards SNP-mediated insult. Both phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinase (MAPK) were activated by IGF-1 in relation to the protective effect. Blockade of the PI3K/Akt pathway abolished the protective effect of IGF-1 whereas inhibition of the MAPK pathway was ineffective. SNP decreased the phosphorylation of Akt in the cells while IGF-1 reversed this inhibitory effect. These results indicate that the protective effect of IGF-1 on D407 exposed to SNP insult is mediated by the PI3K/Akt pathway. This proposal may be exploited in the clinic to improve the viability of insulted retinal cells for maintaining physiological vision.
... The binding of IGF-1 to its receptors causes the phosphorylation/activation of IGF-1R, and subsequently activates downstream signal transduction [24]. The phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways are two major pathways that mediate the effects of IGF-1/IGF-1R [25][26][27]. Recent studies have suggested that the level of circulating IGF-1 is higher in patients with pheochromocytoma [28], glioma [29], breast cancer or prostate cancer [30]. IGF-1R is also seen highly activated in tumor cells [31]. ...
... Cell proliferation was evaluated by the MTT assay [25]. In brief, PC12 cells were seeded in a 96-well plate. ...
The insulin like growth factor 1 (IGF-1) and its receptor (IGF-1R) facilitate tumor proliferation and progression. Tanshinone IIA (TSN) is an active diterpene quinone isolated from the roots of the herbal plant Salvia miltiorrhiza. TSN inhibits the proliferation of various types of cancer cells but its role in the IGF-1R-induced proliferation of pheochromocytoma (PC12) cells and the potential mechanisms are largely unknown. This study aims to investigate the anti-proliferative effect of TSN in PC12 cells and its role on IGF-1R signaling transduction. PC12 cells were treated with IGF-1 with or without TSN, methyl thiazolytetrazolium (MTT) assay, and cell counting kit-8 and flow cytometry were used to evaluate the proliferation of PC12 cells. The role of TSN on the apoptosis of PC12 cells were detected by flow cytometry as well. The effects of TSN and IGF-1 on the phosphorylation of IGF-1R, protein kinase B (Akt), extracellular-signal related kinase 1/2 (ERK1/2) and other downstream targets were analyzed by Western blotting analysis. Our results showed that IGF-1 promoted the growth of PC12 cells in a dose-dependent manner and increased the phosphorylation of IGF-1R, whereas TSN attenuated the effect of IGF-1. Interestingly, TSN did not induce cell apoptosis in PC12 cells. Moreover, TSN attenuated the phosphorylation of Akt and ERK1/2 induced by IGF-1, and the phosphorylation of glycogen synthase kinase-3β, forkhead box O3a (FOXO3a) and c-Raf were also inhibited by TSN. Furthermore, TSN inhibited cell growth induced by IGF-1 and blocked the activation of IGF-1R in SH-SY5Y cells. Taken together, TSN has an inhibitory effect on the proliferation of PC12 cells via down-regulation of the phosphorylated IGF-1R and its downstream signaling.
... As we had previously shown an increase in retinal IGF-1 and its mRNA in another mouse model (βB1-Norrin) with Norrin overexpression 12 , we investigated the retinal IGF-1 mRNA levels in DBA/2J/Pax6-Norrin mice and found them to be twice as high (p < 0.05) as those in DBA/2J littermates (Fig. 4A). IGF-1 has neuroprotective properties that are mediated through PI3K/Akt signaling [35][36][37] . By immunohistochemistry of retinae from two-month-old mice, a specific signal for phosphorylated AKT (pAKT) was observed in the outer nuclear layer of DBA/2J mice, whereas pAKT immunoreactivity was weak in the ganglion cell layer (Fig. 4C). ...
... In a previous study, we observed that Norrin induces the expression of IGF-1 in both Müller cells and microvascular endothelial cells via the activation of canonical Wnt/β-catenin signaling 12 , and it appears reasonable to assume that the same holds true for DBA/2J/Pax6-Norrin mice. IGF-1 is well known for its neuroprotective properties that are mediated through PI3K/Akt signaling [35][36][37]51 . Consequently, IGF-1-induced high levels of pAKT are likely an essential component of Norrin's protective effects against glaucomatous damage in DBA/2J/Pax6-Norrin mice. ...
Norrin is a secreted signaling molecule activating the Wnt/β-catenin pathway. Since Norrin protects retinal neurons from experimental acute injury, we were interested to learn if Norrin attenuates chronic damage of retinal ganglion cells (RGC) and their axons in a mouse model of glaucoma. Transgenic mice overexpressing Norrin in the retina (Pax6-Norrin) were generated and crossed with DBA/2J mice with hereditary glaucoma and optic nerve axonal degeneration. One-year old DBA/2J/Pax6-Norrin animals had significantly more surviving optic nerve axons than their DBA/2J littermates. The protective effect correlated with an increase in insulin-like growth factor (IGF)-1 mRNA and an enhanced Akt phosphorylation in DBA/2J/Pax6-Norrin mice. Both mouse strains developed an increase in intraocular pressure during the second half of the first year and marked degenerative changes in chamber angle, ciliary body and iris structure. The degenerations were slightly attenuated in the chamber angle of DBA/2J/Pax6-Norrin mice, which showed a β-catenin increase in the trabecular meshwork. We conclude that high levels of Norrin and the subsequent constitutive activation of Wnt/β-catenin signaling in RGC protect from glaucomatous axonal damage via IGF-1 causing increased activity of PI3K-Akt signaling. Our results identify components of a protective signaling network preventing degeneration of optic nerve axons in glaucoma.
... Intracellular ROS accumulation was measured using H2D-CF-DA (Wang et al., 2015a). Briefly, after treatment, RGC-5 cells were washed and then stained with 10 μM H2DCF-DA (Sigma-Aldrich) in serum-free medium for 30 minutes at 37°C in the dark. ...
... MDA reacts with thiobarbituric acid (TBA) to produce a fluorescent product (Wang et al., 2015a) that can be measured using a pectrofluorometer microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 535 nm. Therefore, RGC-5 cells in 6-well plates were exposed to 100 μM PA with or without 50 ng/mL NGF pretreatment and cultured to more than 90% confluence. ...
Accumulating evidence supports an important role for nerve growth factor (NGF) in diabetic retinopathy. We hypothesized that NGF has a protective effect on rat retinal ganglion RGC-5 cells injured by palmitic acid (PA), a metabolic factor implicated in the development of diabetes and its complications. Our results show that PA exposure caused apoptosis of RGC-5 cells, while NGF protected against PA insult in a concentration-dependent manner. Additionally, NGF significantly attenuated the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in RGC-5 cells. Pathway inhibitor tests showed that the protective effect of NGF was completely reversed by LY294002 (PI3K inhibitor), Akt VIII inhibitor, and PD98059 (ERK1/2 inhibitor). Western blot analysis revealed that NGF induced the phosphorylation of Akt/FoxO1 and ERK1/2 and reversed the PA-evoked reduction in the levels of these proteins. These results indicate that NGF protects RGC-5 cells against PA-induced injury through anti-oxidation and inhibition of apoptosis by modulation of the PI3K/Akt and ERK1/2 signaling pathways.
... Western blot experiments were performed as described previously (Wang et al. 2013a;b;Wang et al. 2015a), with some modifications. Briefly, after the treatments described in the BCell Treatment^section above, the cells were washed twice with cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (KeyGen Biotech., Nanjing, China) with protease inhibitor cocktail and phosphatase inhibitors. ...
... Cells with condensed chromatin or fragmented nuclei were scored as apoptotic. For each Hoechst experiment, at least 500 cells in eight random fields were collected and quantified, and the percentage of apoptotic cells was calculated (apoptotic cells/ total cells × 100) (Wang et al. 2015a). ...
The selective serotonin reuptake inhibitor fluoxetine is neuroprotective in several brain injury models. It is commonly used to treat major depressive disorder and related conditions, but its mechanism of action remains incompletely understood. Activation of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FoxO3a) and protein kinase A/cAMP-response element binding protein (PKA/CREB) signaling pathways has been strongly implicated in the pathogenesis of depression and might be the downstream target of fluoxetine. Here, we used PC12 cells exposed to corticosterone (CORT) to study the neuroprotective effects of fluoxetine and the involvement of the PI3K/Akt/FoxO3a and PKA/CREB signaling pathways. Our results show that CORT reduced PC12 cells viability by 70 %, and that fluoxetine showed a concentration-dependent neuroprotective effect. Neuroprotective effects of fluoxetine were abolished by inhibition of PI3K, Akt, and PKA using LY294002, KRX-0401, and H89, respectively. Treatment of PC12 cells with fluoxetine resulted in increased phosphorylation of Akt, FoxO3a, and CREB. Fluoxetine also dose-dependently rescued the phosphorylation levels of Akt, FoxO3a, and CREB, following administration of CORT (from 99 to 110, 56 to 170, 80 to 170 %, respectively). In addition, inhibition of PKA and PI3K/Akt resulted in decreased levels of p-CREB, p-Akt, and p-FoxO3a in the presence of fluoxetine. Furthermore, fluoxetine reversed CORT-induced upregulation of p53-upregulated modulator of apoptosis (Puma) and Bcl-2-interacting mediator of cell death (Bim) via the PI3K/Akt/FoxO3a signaling pathway. H89 treatment reversed the effect of fluoxetine on the mRNA level of brain-derived neurotrophic factor, which was decreased in the presence of CORT. Our data indicate that fluoxetine elicited neuroprotection toward CORT-induced cell death that involves dual regulation from PI3K/Akt/FoxO3a and PKA/CREB pathways.
... 57 They found that intravitreal IGF-1 injections were able to promote survival in RPE cells suffering from light injury and maintain a-and b-wave electroactivity. 57 Wang et al and Liao et al reported similar survival benefits to RPE cells supplemented with IGF-1 compared with controls when exposed to stressors such as nitroprusside 58 and amiodarone. 59 In studying retinal endothelial cells, He et al noted that a quinic analog (KZ-41) promoted survival via the kinase domains of IGF-1R and its downstream MAPK pathway effects. ...
Purpose:
FDA approval of teprotumumab for thyroid eye disease in January 2020 reinforced interest in the pharmacologic potential of insulin-like growth factor-1 (IGF-1) and its receptor, IGF-1R. Despite recent approval and adaptation for ophthalmic use, IGF-1R inhibitors are not a new therapeutic class. In 1986, Yamashita described aIR3, a monoclonal antibody to IGF-1R (anti-IGF-1R), that inhibited the effect of IGF-1 on growth hormone release. Given the widespread presence of IGF-1R, interrupting this receptor can lead to systemic physiologic effects, some adverse. We aim to review what is known about IGF-1/IGF-1R in the eye and consider the possible local side effects, unintended consequences, and potential uses of this medication class.
Methods:
A PubMed database search utilizing the keywords "insulin-like growth factor-1, eye, inhibitor, antibody, side effect" was performed to identify publications discussing IGF-1 in the human eye from January 2011 to August 2021. Criteria for acceptance included studies discussing human subjects or human tissue specifically related to the eye.
Results:
Out of a total of 230 articles, 47 were organized in 3 subject groups for discussion: thyroid-associated orbitopathy, cornea and the ocular surface, and the retina and neovascularization. Review of the literature demonstrated that IGF-1 affects growth and development of the eye, epithelial proliferation, retinal angiogenesis, inflammation, and is associated with thyroid-associated orbitopathy.
Conclusions:
IGF-1R exists throughout in the human body, including the cornea, retina, and orbit. Research regarding ocular effects of IGF-1/IGF-1R outside thyroid eye disease is limited. Carefully designed studies and clinical assessments of patients undergoing treatment with anti-IGF-1R may identify ocular side effects and foster consideration of the role of anti-IGF-1R in ocular therapeutics. Given the increasing use of anti-IGF-1R antibodies, understanding their ocular effects, side effects, and potential systemic implications for use in disease is critical.
... In NMDA-induced excitotoxicity (autophagy cell death) of cultured hippocampal neurons, IGF-1 pre-treatment conferred neuroprotection, dependent on the PI3K-AKT-mTOR signaling pathway [77][78][79]. Considering that: i. the cellular effects of IGF-I are mediated by the insulin receptor substrate (IRS) proteins [80]; ii. IRS2 deficiency impairs activation of the NR2B subunit of NMDA receptors; and iii. ...
Insulin-like growth factor-1 (IGF-1) and its binding proteins and receptors are widely expressed in the central nervous system (CNS), proposing IGF-1-induced neurotrophic actions in normal growth, development, and maintenance. However, while there is convincing evidence that the IGF-1 system has specific endocrine roles in the CNS, the concept is emerging that IGF-I might be also important in disorders such as ischemic stroke, brain trauma, Alzheimer’s disease, epilepsy, etc., by inducing neuroprotective effects towards glutamate-mediated excitotoxic signaling pathways. Research in rodent models has demonstrated rescue of pathophysiological and behavioral abnormalities when IGF-1 was administered by different routes, and several clinical studies have shown safety and promise of efficacy in neurological disorders of the CNS. Focusing on the relationship between IGF-1-induced neuroprotection and glutamate-induced excitatory neurotoxicity, this review addresses the research progress in the field, intending to provide a rationale for using IGF-I clinically to confer neuroprotective therapy towards neurological diseases with glutamate excitotoxicity as a common pathological pathway.
... Interestingly, we found that phosphorylation of Akt is further up-regulated by IGF-1 in Cav-1 depleted ARPE-19 cells. It is well known that IGF-1 preserves RPE cells from damage via the activation of the PI3K/Akt signaling pathway [12,23,24]. Therefore, our results suggest that depletion of Cav-1 may improve the protective effects of IGF-1 on RPE cells. ...
The insulin-like growth factor 1 (IGF-1) stimulates expression and secretion of vascular endothelial growth factor-A (VEGF-A), the main actor in ocular neovascularization, by RPE cells. Activity of IGF-1 is regulated by interaction between its receptor and Caveolin-1 (Cav-1), the main component of caveolae. The aim of this study was to investigate whether modulation of Cav-1 expression affects synthesis and secretion of VEGF-A. ARPE-19 cells were transfected with small interfering RNA for Cav-1 (si-Cav-1) and with control siRNA (si-CTR) and stimulated with IGF-1. We found that down-regulation of Cav-1 did not affect activation of IGF-1R but regulated in an opposite manner the phosphorylation of Akt and Erk1/2. Moreover, we found that IGF-1 increased mRNA levels of VEGF-A in both si-CTR and in si-Cav-1 ARPE-19 cells and that Cav-1 silencing significantly reduced basal and IGF-1-stimulated VEGF-A release. Then we investigated the response of the microvascular endothelial cell line HMEC-1 to secretory products of ARPE-19 cells by evaluating wound healing closure, finding that conditioned media from si-Cav-1-ARPE-19 cells reduced endothelial cell migration rate. These data demonstrate that Cav-1 regulates secretion of VEGF-A, and that the depletion of Cav-1 reduces IGF-1 induced VEGF-A secretion in ARPE-19 cells and the migratory potential of their secretory products.
... The RPE is a pigmented monolayer which plays a key role in maintaining the function of photoreceptors including nutrient transport, phagocytosis of photoreceptor outer segments, and regeneration of visual pigments [5][6][7][8][9]. The degeneration of RPE has been considered an initial event of AMD [10]. ...
Age-related macular degeneration (AMD) is a major cause of severe and irreversible vision loss with limited effective therapies. Diminished autophagy and increased oxidative damage caused by ROS in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of AMD, and strategies aimed at enhancing autophagy are likely to protect these cells from oxidative damage. We have previously shown that berberine (BBR), an isoquinoline alkaloid isolated from Chinese herbs, was able to protect human RPE cells from H2O2-induced oxidative damage through AMPK activation. However, the precise mechanisms behind this protective effect remain unclear. Given the essential role of AMPK in autophagy activation, we postulated that BBR may confer protection against H2O2-induced oxidative damage by stimulating AMPK-dependent autophagy. Our results showed that BBR was able to induce autophagy in D407 cells, whereas autophagy inhibitor PIKIII or silencing of LC3B blocked the protective effect of BBR. Further analysis showed that BBR activated the AMPK/mTOR/ULK1 signaling pathways and that both pharmacological and genetic inhibitions of the AMPK pathway abolished the autophagy-stimulating effect of BBR. Similar results were obtained in primary cultured human RPE cells. Taken together, these results demonstrate that BBR is able to stimulate autophagy in D407 cells via the activation of AMPK pathway and that its protective effect against H2O2-induced oxidative damage relies on its autophagy-modulatory effect. Our findings also provide evidence to support the potential application of BBR in preventing and treating AMD.
... Insulin-like growth factor 1 receptor (IGF-1R) signaling mediated by IGF-1 is an important pathway for the survival and maintenance of multiple cell types within the central nervous system [12]. Our previous studies showed that IGF-1 can protect neuronal cells from various insults [13,14]. The physiological function of IGF-1 is mainly mediated by its highly-affinity receptors [15]. ...
Glutamate-induced neurotoxicity is involved in various neuronal diseases, such as Alzheimer’s disease. We have previously reported that glutamate attenuated the survival signaling of insulin-like growth factor-1 (IGF-1) by N-methyl-D-aspartate receptors (NMDARs) in cultured cortical neurons, which is viewed as a novel mechanism of glutamate-induced neurotoxicity. However, the phosphorylation sites of IGF-1 receptor (IGF-1R) affected by glutamate remain to be elucidated, and importantly, which subtype of NMDARs plays a major role in attenuating the prosurvival effect of IGF-1 is still unknown. In the present study, glutamate was found to attenuate the tyrosine phosphorylation of the IGF-1R and the prosurvival effect of IGF-1 in primary cultured cortical neurons. NMDAR inhibitors, MK801 and AP-5, blocked the inhibitory effect of glutamate on the phosphorylation of IGF-1R and increased cell survival, while DNQX, LY341495, and CPCCOEt had no effect. Interestingly, we found that glutamate decreased the phosphorylation of tyrosine residues 1131, 1135/1136, 1250/1251, and 1316, while it had no effect on tyrosine 950 in cortical neurons. Moreover, using specific antagonists and siRNA to downregulate individual NMDAR subunits, we found that the activation of NR2B-containing NMDARs was essential for glutamate to inhibit IGF-1 signaling. These findings indicate that the glutamate-induced attenuation of IGF-1 signaling is mediated by NR2B-containing NMDARs. Our study also proposes a novel mechanism of altering neurotrophic factor signaling by the activation of NMDARs.
... The IGF-1-AKT1-mTOR signalling pathway is considered to regulate cell proliferation, protein synthesis and the development of muscle in animals (Murgia et al. 2000;Orellana et al. 2012). Previous studies showed that muscle growth slowed and muscle-fibre area was reduced in IGF-1null mice (Hay and Sonenberg 2004), whereas IGF-1 activated the PI3K-AKT pathway to induce hypertrophy of muscle fibres (Wang et al. 2015). AKT, as a signal gene, activated mTOR to promote protein synthesis and cause muscle fibre hypertrophy. ...
The objective of the present study was to investigate the effect of nutritional restriction during the suckling period on growth performance, carcass traits and meat quality of fattening pigs. A one-way experimental design was used. In total, 24 male pigs of normal birthweight (1.54 ± 0.05 kg) were randomly allocated at 7 days of age to three groups: control, fed ad libitum; 30% nutritional restriction (Re30%); and 60% nutritional restriction (Re60%). The Re30% and Re60% groups showed decreased average daily gain, average daily feed intake and feed conversion ratio from Day 7 to Day 28. After Day 28, differences in these parameters were observed only in the Re60% group relative to the control. With regard to hormone levels, the Re60% group showed decreased serum concentration of insulin-like growth factor-1 at Day 28 and increased serum concentration of growth hormone at Day 147. Furthermore, the Re60% group had decreased carcass weight, ham weight and dressing percentage, and increased carcass lean percentage relative to the control, as well as lower cross-sectional area and myofibre diameter of muscle. The Re60% group had lower levels of myosin heavy chain (MyHC) IIx and MyHC IIb mRNA and a higher percentage of MyHC I and MyHC IIa mRNA in longissimus dorsi muscle than the Re30% group. In conclusion, nutritional restriction during the suckling period decreased weaning weight, with post-weaning growth performance, carcass traits and myofibre type affected in the Re60% group rather than Re30% group.
... Cells were co-transfected with pmirGLO-NNT-AS1-wild type (wt)/mutated type (mut) reporter plasmids and miR-203 mimic through Lipofectamine 2000 (Invitrogen). Additionally, according the previous studies [39][40][41] and the target gene prediction website (http://www.targetscan. org/vert_72), we found the potential targets of miR-203 in PI3K/AKT pathway and EMT, which were insulinlike growth factor type 1 receptor (IGF1R) and zinc finger E-box binding homeobox 1 (ZEB1), respectively. ...
Background:
Cholangiocarcinoma (CCA) is a serious malignant tumor. Long non-coding RNA NNT-AS1 (NNT-AS1) takes crucial roles in several tumors. So, we planned to research the roles and underlying mechanism of NNT-AS1 in CCA.
Results:
NNT-AS1 overexpression was appeared in CCA tissues and cell lines. Proliferation was promoted by NNT-AS1 overexpression in CCLP1 and TFK1 cells. Besides, NNT-AS1 overexpression reduced E-cadherin level and raised levels of N-cadherin, vimentin, Snail and Slug. However, the opposite trend was occurred by NNT-AS1 knockdown. Further, NNT-AS1 overexpression promoted phosphatidylinositol 3 kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK)1/2 pathways. MiR-203 was sponged by NNT-AS1 and miR-203 mimic reversed the above promoting effects of NNT-AS1. Additionally, insulin-like growth factor type 1 receptor (IGF1R) and zinc finger E-box binding homeobox 1 (ZEB1) were two potential targets of miR-203.
Conclusion:
NNT-AS1 promoted proliferation, EMT and PI3K/AKT and ERK1/2 pathways in CCLP1 and TFK1 cells through down-regulating miR-203.
Methods:
CCLP1 and TFK1 cells were co-transfected with pcDNA-NNT-AS1 and miR-203 mimic. Bromodeoxyuridine (BrdU), flow cytometry, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot were employed to detect roles and mechanism of NNT-AS1. Interaction between NNT-AS1 and miR-203 or miR-203 and target genes was examined through luciferase activity experiment.
... Similarly, we found that CARB treatment increased mRNA expression of both PI3K and Akt in the retina, suggesting that CARB attenuated diabetic-induced neuronal loss via activation of NGF/PI3K/Akt. Additionally, one in vitro study documented that insulin like growth factor-1 signaling, through the PI3K/Akt pathway, affords neuroprotection against an insult of sodium nitroprusside in human retinal pigment epithelial cells (Wang et al., 2015). ...
Aim: Diabetic retinopathy causes loss of vision in adults at working-age. Few therapeutic options are available for treatment of diabetic retinopathy. Carbamazepine (CARB), a widely used antiepileptic drug, was recently accounted for its neuroprotective effect. Nerve growth factor (NGF) activates various cascades among which, PI3K/Akt/mTOR pathway has a vital action in NGF-mediated neuronal differentiation and survival. This study evaluated the effect of CARB in the treatment of diabetic retina and unveiled some of the underlying molecular mechanisms. Main Methods: Alloxan diabetes model was induced in 36 albino well-acclimatized mice. After establishment of the diabetic model in 9 weeks, mice were assigned to treatment groups: (1) saline, (2) alloxan-diabetic, (3 and 4) alloxan+CARB (25 or 50 mg per kg p.o) for 4 weeks. After completion of the therapeutic period, mice were sacrificed and eyeballs were enucleated. Retinal levels of NGF and PI3K/Akt were assessed using real-time polymerase chain reaction. Further, total and phosphorylated TrKA, PI3K, Akt, mTOR as well as Caspase-3 were measured by Western blot analysis. Key Findings: Histopathological examination demonstrated that CARB attenuated vacuolization and restored normal thickness and organization of retinal cell layers. In addition, CARB increased pTrKA/TrKA ratio and ameliorated diabetes-induced
... For example, insulin-like growth factor-1 is an endogenous growth factor proposed to act through the PI3K/Akt pathway to inhibit RPE cell death to reduce/delay the development of DR. Exogenous administration of insulin-like growth factor-1 in a model of NO toxicity characteristic of some patients with DR prevents RPE cell death, likely through activation of the PI3K/Akt pathway [90]. Moreover, vascular endothelial growth factor (VEGF)-A may promote RPE cell survival through the Akt pathway in the presence of oxidative stress [91]. ...
The significance of oxidative stress in the development of chronic neurodegenerative diseases of the retina has become increasingly apparent in recent years. Reactive oxygen species (ROS) are free radicals produced at low levels as a result of normal cellular metabolism that are ultimately metabolized and detoxified by endogenous and exogenous mechanisms. In the presence of oxidative cellular stress, ROS are produced in excess, resulting in cellular injury and death and ultimately leading to tissue and organ dysfunction. Recent studies have investigated the role of excess ROS in the pathogenesis and development of chronic neurodegenerative diseases of the retina including glaucoma, diabetic retinopathy, and age-related macular degeneration. Findings from these studies are promising insofar as they provide clear rationales for innovative treatment and prevention strategies of these prevalent and disabling diseases where currently therapeutic options are limited. Here, we briefly outline recent developments that have contributed to our understanding of the role of ROS in the pathogenesis of chronic neurodegenerative diseases of the retina. We then examine and analyze the peer-reviewed evidence in support of ROS as targets for therapy development in the area of chronic neurodegeneration of the retina.
... The STRING analysis showed that all the proteins already mentioned are involved in different pathways concerning neuroprotection, neurodifferentiation and/or axonal growth. Of these, the most relevant are some signaling pathways, namely PI3K-AKT [60][61][62], Wnt [63][64][65], mTOR [66][67][68], VEGF [69][70][71] and Notch [72,73], and also other processes such as Hsp70 protein binding [74,75], axonogenesis and axon guidance. ...
Mesenchymal stem cells (MSCs), and within them adipose tissue derived stem cells (ASCs), have been shown to have therapeutic effects on central nervous system (CNS) cell populations. Such effects have been mostly attributed to soluble factors, as well as vesicles, present in their secretome. Yet, little is known about the impact that MSC passaging might have in the secretion therapeutic profile. Our aim was to show how human ASCs (hASCs) passage number influences the effect of their secretome in neuronal survival, differentiation and axonal growth. For this purpose, post-natal rat hippocampal primary cultures, human neural progenitor cell (hNPCs) cultures and dorsal root ganglia (DRGs) explants were incubated with secretome, collected as conditioned media (CM), obtained from hASCs in P3, P6, P9 and P12. Results showed no differences when comparing percentages of MAP-2 positive cells (a mature neuronal marker) in neuronal cultures or hNPCs, after incubation with hASCs secretome from different passages. The same was observed regarding DRG neurite outgrowth. In order to characterize the secretomes obtained from different passages, a proteomic analysis was performed, revealing that its composition did not vary significantly with passage number P3 to P12. Results allowed us to identify several key proteins, such as pigment epithelium derived factor (PEDF), DJ-1, interleucin-6 (IL-6) and galectin, all of which have already proven to play neuroprotective and neurodifferentiating roles. Proteins that promote neurite outgrowth were also found present, such as semaphorin 7A and glypican-1. We conclude that cellular passaging does not influence significantly hASCs's secretome properties especially their ability to support post-natal neuronal survival, induce neurodifferentiation and promote axonal growth.
... IGF1 influences SSC propagation in conjunction with GDNF, it cannot influence SSC proliferation independently. IGF1 influences synthesis of DNA and acts through IGF1R and AKT signaling pathway, instead of MAP kinases employed by GDNF (Kubota et al., 2004;Wang et al., 2015). ...
... Different approaches have been investigated in an attempt to block the deleterious effects of Ab and to lessen the progression of this disease. Insulin-like growth factor-I (IGF-I) acts as a survival factor in the brain through activation of the Akt pathway ( Wang et al., 2015). Some neurodegenerative conditions have reduced levels of IGF-I in plasma and brain, both in humans ( Torres-Alema´nAlema´n et al., 1998) and in AD experimental models ( Busiguina et al., 2000), suggesting that this growth factor may be implicated in the etiopathogenesis of AD. ...
Somatostatin (SRIF), a neuropeptide highly distributed in the hippocampus and involved in learning and memory, is markedly reduced in the brain of Alzheimer's disease patients. The effects of insulin-like growth factor-I (IGF-I) against β amyloid (Aβ)-induced neuronal death and associated cognitive disorders have been extensively reported in experimental models of this disease. Here, we examined the effect of IGF-I on the hippocampal somatostatinergic system in Aβ-treated rats and the molecular mechanisms associated with changes in this peptidergic system. Intracerebroventricular Aβ25-35 administration during 14 days (300 pmol/day) to male rats increased Aβ25-35 levels and cell death and markedly reduced SRIF and SRIF receptor 2 levels in the hippocampus. These deleterious effects were associated with reduced Akt and cAMP response element-binding protein (CREB) phosphorylation and activation of c-Jun N-terminal kinase (JNK). Subcutaneous IGF-I co-administration (50 µg/kg/day) reduced hippocampal Aβ25-35 levels, cell death and JNK activation. In addition, IGF-I prevented the reduction in the components of the somatostatinergic system affected by Aβ infusion. Its co-administration also augmented protein kinase A (PKA) activity, as well as Akt and CREB phosphorylation. These results suggest that IGF-I co-administration may have protective effects on the hippocampal somatostatinergic system against Aβ insult through up-regulation of PKA activity and Akt and CREB phosphorylation.
... The protective effects of the GHRH agonist, MR-409, could be direct and/or indirect. GHRH-mediated rescue of IGF1, IGFBP-3, GLP-1, and GH could indirectly contribute to the observed neuroprotective effects during the early stages of DR (30)(31)(32)(33)(34)(35)(36)(37). Of particular interest are our results on the expression pattern of SST, which was boosted above control levels by the GHRH agonist, MR-409, and was further down-regulated below control levels in response to the antagonist, MIA-602 (SI Appendix, Fig. S3D). ...
Significance
The studies described here are relevant to the cure of diabetic retinopathy, a leading cause of blindness with currently limited therapeutic options. Here we provided evidence showing that agonists of growth hormone-releasing hormone (GHRH) can significantly diminish retinal neurovascular injury characterizing the early stages of diabetic retinopathy through antioxidant and anti-inflammatory effects. The results of the presented studies provide information on the potential therapeutic effects of GHRH agonists and shed light on the role of hypothalamic hormones in retinal physiology and their effect on visual disorders. In addition, our findings suggest protective effects of GHRH analogs in other disease conditions affecting retinal neuronal cells and, possibly, other nonretinal neurons.
... IGF-1 binds to these receptors which initiates its auto phosphorylation, which further triggers its intrinsic tyrosine kinases, resulting in the phosphorylation of many intracellular substrates, followed by activation of various downstream signaling pathways such as the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2) [14]. IGF-1 confers neuroprotection via these signaling cascades against multiple abuses such as potassium deprivation, serum deprivation, SNP toxicity, cerebral ischemia, and glutamate excitotoxicity [15][16][17][18]. IGF-1 is gaining increasing attention as a potential therapeutic agent for the treatment of neurodegenerative disorders. ...
Impairment of insulin-like growth factor I (IGF-I) signaling plays an important role in the development of neurodegeneration. In the present study, we investigated the effect of H2O2 on the survival signaling of IGF-1 and its underlying mechanisms in human neuronal cells SH-SY5Y. Our results showed that IGF-1 promoted cell survival and stimulated phosphorylation of IGF-1R as well as its downstream targets like AKT and ERK1/2 in these cells. Meanwhile, these effects of IGF-1 were abolished by H2O2 at 200μM concentration which did not cause any significant toxicity to cells itself in our experiments. Moreover, studies using various glutamate receptor subtype antagonists displayed that N-methyl-D -aspartate (NMDA) receptor antagonist dizocilpine maleate (MK-801) blocked the effects of H2O2, whereas other glutamate receptor subtype antagonists, such as non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), metabolic glutamate receptor antagonists LY341495 and CPCCOEt, had no effect. Further studies revealed that NR2B-containing NMDARs are responsible for these effects as its effects were blocked by pharmacological inhibitor Ro25-698 or specific siRNA for NR2B, but not NR2A. Finally, our data also showed that Ca2+ influx contributes to the effects of H2O2. Similar results were obtained in primary cultured cortical neurons. Taken together, the results from the present study suggested that H2O2 attenuated IGF-1R tyrosine phosphorylation and its survival signaling properties via NR2B containing NMDA receptors and Ca2+ influx in SH-SY5Y cells. Therefore, NMDAR antagonists, especially NR2B-selective ones, combined with IGF-1 may serve as an alternative therapeutic agent for oxidative stress related neurodegenerative disease.
... We have previously reported that IGF-1 promotes the survival of various cell types in which cell death is initiated by serum deprivation. The cellular protective action of IGF-1 involves the PI3K/Akt signaling pathway, although the full characterization including upstream mediators and downstream effectors and targets have not been fully elucidated [29][30][31][32][33]. ...
Amiodarone (AM) is the most effective antiarrhythmic agent currently available. However, clinical application of AM is limited by its serious toxic adverse effects including optic neuropathy. The purpose of this study was to explore the effects of AM and to assess if insulin-like growth factor-1 (IGF-1) could protect retinal neuronal cells from AM-induced apoptosis, and to determine the molecular mechanisms underlying the effects. Accordingly, the phosphorylation/activation of Akt and FoxO3a were analyzed by Western blot while the possible pathways involved in the protection of IGF-1 were investigated by application of various pathway inhibitors. The full electroretinogram (FERG) was used to evaluate in vivo effect of AM and IGF-1 on rat retinal physiological functions. Our results showed that AM concentration dependently caused an apoptosis of RGC-5 cells, while IGF-1 protected RGC-5 cells against this effect by AM. The protective effect of IGF-1 was reversed by PI3K inhibitors LY294002 and wortmannin as well as the Akt inhibitor VIII. AM decreased p-Akt and p-FoxO3a while increased the nuclear localization of FoxO3a in the RGC-5 cells. IGF-1 reversed the effect of AM on the p-Akt and p-FoxO3a and the nuclear translocation of FoxO3a. Similar results were obtained in primary cultured retinal ganglia cells. Furthermore, FERG in vivo recording in rats showed that AM decreased a-wave and b-wave of FERG while IGF-1 reversed the effects of AM. These data show that AM induced apoptosis of retinal neuronal cells via inhibiting the PI3K/Akt/FoxO3a pathway while IGF-1 protected RGC-5 cells against AM-induced cell apoptosis by stimulating this pathway.
... Furthermore, IGF-1 mediated activation of the PI3K/AKT pathway has been shown to inhibit caspase-3 activation [15]. Additionally, the protective effect of IGF-1 was abolished by blocking the PI3K/Akt pathway [39]. Alternatively, as stated above excess glutamate could adversely affect IGF-1mediated neuroprotection [8,40]. ...
Glutamate that accumulates in injured brain tissue has been shown to hinder the neuroprotection rendered by insulin-like growth factor-1 (IGF-1). However, its role in attenuating the neuroprotective effect of IGF-1 in the hypoxic retina is unknown and the current study was aimed at elucidating this. One-day-old Wistar rats were exposed to hypoxia for 2 h and the retinas were studied at 3 h to 14 days after exposure. Following hypoxia, the concentrations of glutamate and IGF-1 were significantly increased over control values in the immature retina and in cultured retinal ganglion cells (RGCs). In addition to IGF-1, the relative expression of insulin receptor substrate-1 (IRS1) phosphorylated at the tyrosine residue (p-IRS1tyr), phosphorylated protein kinase B (p-AKT) and phosphorylated protein kinase A (p-PKA), which are involved in IGF-1 signalling, was also studied in hypoxic retinas and in cultured RGCs. Glutamate-mediated inhibition of the IGF-1 pathway in hypoxic RGCs was evident with a reduced expression of p-IRS1tyr and p-AKT and an increased expression of p-PKA. However, the addition of exogenous IGF-1 reversed this. Glutamate enables the phosphorylation of IRS1 at the serine residue (p-IRS1ser) through a PKA-dependent pathway. The increased expression of p-IRS1ser and its increased association with IGF-1 receptors in hypoxic RGCs suggested a possible interference by glutamate with the IGF-1 pathway. Moreover, there was increased caspase-3/7 activity in hypoxic RGCs. These results suggest that glutamate, by blocking IGF-1-mediated neuroprotection, could cause the apoptosis of RGCs in the hypoxic neonatal retina.
... PI3K/Akt signaling pathways mediate cell death or survival, differentiation, and apoptosis responses [40,41]. PC12 and SH-SY5Y cells exposed to cadmium showed reduction in PTEN, resulting in increased PI3K/Akt signaling. ...
L-Theanine is an amino acid derivative from green tea. The present work was aimed at the effect of L-theanine on neuron-like rat pheochromocytoma (PC12) cells stimulated with cadmium chloride. Treatment with L-theanine before cadmium exposure increased cell viability; the experiments of Annexin V/PI staining indicated that L-theanine inhibited cadmium-induced cell apoptosis. Meanwhile, L-theanine decreased ROS production and protected from cadmium-induced disruption of mitochondrial transmembrane potential. Compared with cadmium-treated cells, L-theanine could also decrease the ratio of Bax/Bcl-2, as well as the level of cleaved caspase-9, caspase-3 and poly(ADP-ribose) polymerase. Furthermore, L-theanine depresses cadmium-induced up regulation of phosphorylations of PI3K/Akt, MAPK ERK1/2, and JNK signaling. These data suggest that L-theanine pretreatment reduces severity of cadmium toxicity probably via antioxidant action. Therefore, it may be concluded that L-theanine could be exploited for prevention of cadmium-induced diseases.
In the early stages of diabetic retinopathy (DR), diabetes-related hyperglycemia directly inhibits the AKT signaling pathway by increasing oxidative stress or inhibiting growth factor expression, which leads to retinal cell apoptosis, nerve proliferation and fundus microvascular disease. However, due to compensatory vascular hyperplasia in the late stage of DR, the vascular endothelial growth factor (VEGF)/phosphatidylinositol 3 kinase (PI3K)/AKT cascade is activated, resulting in opposite levels of AKT regulation compared with the early stage. Studies have shown that many factors, including insulin, insulin-like growth factor-1 (IGF-1), VEGF and others, can regulate the AKT pathway. Disruption of the insulin pathway decreases AKT activation. IGF-1 downregulation decreases the activation of AKT in DR, which abrogates the neuroprotective effect, upregulates VEGF expression and thus induces neovascularization. Although inhibiting VEGF is the main treatment for neovascularization in DR, excessive inhibition may lead to apoptosis in inner retinal neurons. AKT pathway substrates, including mammalian target of rapamycin (mTOR), forkhead box O (FOXO), glycogen synthase kinase-3 (GSK-3)/nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear factor kappa-B (NF-κB), are a research focus. mTOR inhibitors can delay or prevent retinal microangiopathy, whereas low mTOR activity can decrease retinal protein synthesis. Inactivated AKT fails to inhibit FOXO and thus causes apoptosis. The GSK-3/Nrf2 cascade regulates oxidation and inflammation in DR. NF-κB is activated in diabetic retinas and is involved in inflammation and apoptosis. Many pathways or vital activities, such as the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and mitogen-activated protein kinase (MAPK) signaling pathways, interact with the AKT pathway to influence DR development. Numerous regulatory methods can simultaneously impact the AKT pathway and other pathways, and it is essential to consider both the connections and interactions between these pathways. In this review, we summarize changes in the AKT signaling pathway in DR and targeted drugs based on these potential sites.
Bone marrow-derived mesenchymal stem cells (BMSCs) have therapeutic potential for spinal cord injury (SCI). We have shown that insulin-like growth factor 1 (IGF-1) enhances the cellular proliferation and survivability of BMSCs-derived neural progenitor cells (NPCs) by downregulating miR-22-3p. However, the functional application of BMSCs-derived NPCs has not been investigated fully. In this study, we demonstrate that knockdown of endogenous miR-22-3p in BMSCs-derived NPCs upregulates Akt1 expression, leading to enhanced cellular proliferation. RNASeq analysis reveals 3,513 differentially expressed genes in NPCs. The upregulated genes in NPCs enrich the gene ontology term associated with nervous system development. Terminally differentiated NPCs generate cells with neuronal-like morphology and phenotypes. Transplantation of NPCs in the SCI rat model results in better recovery in locomotor and sensory functions 4 weeks after transplantation. Altogether, the result of this study demonstrate that NPCs derived with IGF-1 supplementation could be differentiated into functional neural lineage cells and are optimal for stem cell therapy in SCI.
Emerging research has suggested the anticancer potential of tanshinone IIA, the bioactive ingredient isolated from the traditional Chinese herb Salvia miltiorrhiza. However, the molecular mechanism of sodium tanshinone IIA sulfonate (STS) antilung cancer effect is not very clear. In this study, our purpose is to investigate the roles of STS and elongation factor‐2 kinase (eEF‐2K) in regulating the proliferation, migration, and invasion of A549 cells and explore the implicated pathways. We found that STS suppressed A549 cell survival and proliferation in a time‐ and xdose‐dependent manner. Knockdown of eEF‐2K and treatment with STS synergistically exerted antiproliferative, ‐migratory, and ‐invasive effects on A549 cells. These effects were caused by attenuation of the extracellular signal‐regulated kinase (ERK) pathway via inhibition of tissue transglutaminase (TG2). In summary, the inhibition of eEF‐2K synergizes with STS treatment, exerting anticancer effects on lung adenocarcinoma cells through the TG2/ERK signaling pathway, which provides a potential therapeutic target for treating lung adenocarcinoma.
Background:
Pathologic ocular neovascularization in retinopathy of prematurity (ROP) and other proliferative retinopathies are characterized by dysregulation of vascular endothelial growth factor-A (VEGF-A). A study of Vegfa isoform expression during oxygen-induced ischemic retinopathy (OIR) may enhance our understanding of Vegf dysregulation.
Methods:
Following induction of OIR, immunohistochemistry and polymerase chain reaction (PCR) was performed on room air (RA) and OIR mice.
Results:
Total Vegfa messenger RNA (mRNA) expression was stable in RA mice, but increased in OIR mice with a peak at postnatal day 17 (P17), before returning to RA levels. Vegfa164a expression was similar in both OIR and RA mice at P10 (Phase 1 OIR), but 2.4-fold higher in OIR mice compared to RA mice at P16 (Phase 2 OIR). At P10, Vegfa164b mRNA was similar in OIR vs RA mice, but was expressed 2.5-fold higher in OIR mice compared to RA mice at P16. At P10 and P16, Vegfr2/Vegfr1 expression was increased in OIR mice compared to RA mice. Increased activation of microglia was seen in OIR mice.
Conclusions:
Vegfa164a, Vegfa164b, and Vegfr1 were overexpressed in OIR mice, leading to abnormal signaling and angiogenesis. Further studies of mechanisms of Vegf dysregulation may lead to novel therapies for ROP and other proliferative retinopathies.
Impact:
Vegfa164 has two major isoforms, a proangiogenic, Vegfa164a, and an antiangiogenic, Vegfa164b, with opposing receptors, inhibitory Vegfr1, and stimulatory Vegfr2, but their role in OIR is unclear. In Phase 1 OIR, both isoforms and receptors are expressed similarly. In Phase 2 OIR, both isoforms are overexpressed, with an increased ratio of inhibitory Vegfr1. Modulation of angiogenesis by Vegf regulation enables pruning of excess angiogenesis during physiology, but results in ineffective angiogenesis during OIR. Knowledge of VEGF dysregulation may have novel therapeutic implications in the management of ROP and retinal proliferative diseases.
Regenerative medicine strategies rely on exogenous cell transplantation and/or endogenous cell stimulation. Biomaterials can help to increase the regenerative potential of cells and biomolecules by controlling transplanted cell fate and provide a local, sustained release of biomolecules. In this review, we describe the use of a hyaluronan/methylcellulose (HAMC)-based hydrogel as a delivery vehicle to the brain, spinal cord, and retina to promote cellular survival and tissue repair. We discuss various controlled release strategies to prolong the delivery of factors for neuroprotection. The versatility of this hydrogel for a diversity of applications highlights its potential to enhance cell- and biomolecule-based treatment strategies.
Background and purpose:
Amiodarone (AM) is one of the most effective anti-arrhythmic drugs available. However, clinical applications of AM are limited by its toxic side effects including optic toxicity. The purpose of this study was to investigate the toxic effect of AM on D407 cells (a human Retinal Pigmented Epithelial cell line) and the mechanisms of the protective effect of insulin-like growth factor-1 (IGF-1).
Experimental approach:
The involvement of the kinases, Akt and Erk, was analyzed by Western blot. Intracellular accumulation of ROS was measured using fluorophotometric quantitation. A pharmacological approach with inhibitors was used to investigate the pathways involved in the protective action of IGF-1.
Key results:
AM concentration-dependently augmented the production of ROS, lipid peroxidation, and apoptosis in D407 cells. IGF-1 time- and concentration-dependently reversed the effects of AM and rescued D407 cells from AM mediated toxicity. AM inhibited the activation/phosphorylation of Akt without apparent effect on Erk, while IGF-1 reversed the inhibitory effect of AM on the activation of Akt. Moreover, IGF-1 failed to suppress AM-induced cytotoxicity in the presence of the PI3K/Akt inhibitor LY294002 suggesting the direct involvement of the PI3K/Akt pathway. Furthermore, rat FERG in vivo recording showed that AM decreased a-wave and b-wave of FERG while IGF-1 reversed the effects of AM. Immunocytochemistry showed that vitreous IGF-1 injections promoted the survival of RPE cells in rat retina and increased the expression of RPE65 in RPE cells from AM injury.
Conclusion and implications:
These results indicate that IGF-1 is able to protect RPE cells from AM mediated injury via the PI3K/Akt pathway in vivo and in vitro. Our findings suggest the potential use of IGF-1 as a protectant drug for the prevention and treatment of AM-induced optic toxicity.
Alveolar epithelial cell (AEC) apoptosis and inadequate repair resulting from 'exaggerated' lung aging and mitochondrial dysfunction are critical determinants promoting lung fibrosis. α-Klotho, which is an anti-aging molecule that is expressed predominantly in the kidney and secreted in the blood, can protect lung epithelial cells against hyperoxia-induced apoptosis. We reasoned that Klotho protects AEC exposed to oxidative stress in part by maintaining mitochondrial DNA (mtDNA) integrity and mitigating apoptosis. We find that Klotho levels are decreased in both serum and alveolar type II (AT2) cells from asbestos-exposed mice. We show that oxidative stress reduces AEC Klotho mRNA and protein expression whereas Klotho over-expression is protective while Klotho silencing augments AEC mtDNA damage. Compared to wild-type, Klotho heterozygous hypomorphic allele (kl/+) mice have increased asbestos-induced lung fibrosis due in part to increased AT2 cell mtDNA damage. Notably, we demonstrate that serum Klotho levels are reduced in WT but not mitochondrial catalase over-expressing (MCAT) mice 3 weeks following exposure to asbestos and that EUK-134, a MnSOD / catalase mimetic, mitigates oxidant-induced reductions in AEC Klotho expression. Using pharmacologic and genetic silencing studies, we show that Klotho attenuates oxidant-induced AEC mtDNA damage and apoptosis via mechanisms dependent upon AKT activation arising from upstream fibroblast growth factor receptor 1 (FGFR1) activation. Our findings suggest that Klotho preserves AEC mtDNA integrity in the setting of oxidative stress necessary for preventing apoptosis and asbestos-induced lung fibrosis. We reason that strategies aimed at augmenting AEC Klotho levels may be an innovative approach for mitigating age-related lung diseases.
Endothelin-1 (ET-1) is a vasoactive peptide produced by activated astrocytes and microglia and is implicated in initiating and sustaining reactive gliosis in neurodegenerative diseases. We have previously suggested that ET-1 can play a role in the pathophysiology of amyotrophic lateral sclerosis (ALS). Indeed, we reported that this peptide is abundantly expressed in reactive astrocytes in the spinal cord of SOD1-G93A mice and ALS patients and exerts a toxic effect on motor neurons (MNs) in an in vitro model of mixed spinal cord cultures enriched with reactive astrocytes. Here, we explored the possible mechanisms underlying the toxic effect of ET-1 on cultured MNs. We show that ET-1 toxicity is not directly caused by oxidative stress or activation of cyclooxygenase-2 but requires the synthesis of nitric oxide and is mediated by a reduced activation of the phosphoinositide 3-kinase pathway. Furthermore, we observed that ET-1 is also toxic for microglia, although its effect on MNs is independent of the presence of this type of glial cells. Our study confirms that ET-1 may contribute to MN death and corroborates the view that the modulation of ET-1 signaling might be a therapeutic strategy to slow down MN degeneration in ALS.
Poor cell survival in vitro and in vivo is one of the key challenges in tissue engineering. Pro-survival therapeutic proteins, such as insulin-like growth factor-1 (IGF-1), can promote cell viability but require controlled delivery systems due to their short half-lives and rapid clearance. Biocompatible materials are commonly used for drug-delivery platforms or to encapsulate cells for increased viability, but few materials have been used for both applications simultaneously. In this work we present a dual-use platform. A blend of hyaluronan (HA) and methylcellulose (MC), known to promote cell survival, was covalently modified with Src homology 3 (SH3)-binding peptides and demonstrated tunable, affinity-based release of the pro-survival fusion protein SH3-IGF-1. The material also significantly increased the viability of retinal pigment epithelium cells (RPE) in anchorage-independent conditions. This novel platform is applicable to a broad range of cells and protein therapeutics and is a promising drug delivery/cell transplantation strategy to increase the viability of both exogenous and endogenous cells in tissue engineering applications.
Hypoxia-induced retinal ganglion cell (RGC) apoptosis has been implicated in many optic neuropathies. Insulin-like growth factor-1 (IGF-1) is important in maintaining neuronal survival, proliferation, and differentiation. The purpose of this study is to explore whether IGF-1 can protect RGCs from hypoxia-induced apoptosis and to determine the precise mechanisms that regulate this process.
Purified RGC cultures were obtained from the retinas of neonatal Sprague Dawley (SD) rats using a two-step panning method. Primary cultured RGCs were cultured in a closed hypoxic chamber (5% O2, 5% CO2, and 90% N2) for 12 h with or without IGF-1. The degree of apoptosis in the RGCs was detected by caspase-3 expression and TUNEL and JC-1 staining assays. The expression and phosphorylation of protein kinase B (Akt), p44/42 mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase-1/2 [Erk-1/2]), Bad, and caspase-3 was investigated with immunoblot analysis.
Hypoxia induces apoptosis in primary Sprague Dawley rat RGCs, as detected by caspase-3 expression and TUNEL and JC-1 staining assays, and that IGF-1 treatment could significantly reduce this effect in RGCs. Interestingly, pretreatment of RGCs with AG1024 (an IGF-1 inhibitor), U0126 (an Erk-1/2 inhibitor), and LY294002 (an Akt inhibitor) markedly attenuated the effects of IGF-1 treatment. Furthermore, western blot analysis suggested that the Erk-1/2 and Akt signaling pathways play a role in the protective effects of IGF-1 on RGCs exposed to hypoxia.
These data indicate that IGF-1 can protect primary cultured RGCs against hypoxia-induced apoptosis via the Erk-1/2 and Akt signaling pathways, suggesting that IGF-1 treatment is a potential therapeutic approach for treating hypoxia-induced neurodegeneration in the retina.
The transcription factor FoxG1 regulates neurogenesis in the embryonic telencephalon as well as a number of other neurodevelopmental processes. While FoxG1 continues to be expressed in neurons postnatally and through adulthood, its role in fully differentiated neurons is not known. The current study demonstrates that FoxG1 promotes the survival of postmitotic neurons. In cerebellar granule neurons primed to undergo apoptosis, FoxG1 expression is reduced. Ectopic expression of FoxG1 blocks neuronal death, whereas suppression of its expression induces death in otherwise healthy neurons. The first 36 residues of FoxG1 are necessary for its survival-promoting effect, while the C-terminal half of the protein is dispensable. Mutation of Asp219, a residue necessary for DNA binding, abrogates survival promotion by FoxG1. Survival promotion is also eliminated by mutation of Thr271, a residue phosphorylated by Akt. Pharmacological inhibition of Akt blocks the survival effects of wild-type FoxG1 but not forms in which Thr271 is replaced with phosphomimetic residues. Treatment of neurons with IGF-1, a neurotrophic factor that promotes neuronal survival by activating Akt, prevents the apoptosis-associated downregulation of FoxG1 expression. Moreover, the overexpression of dominant-negative forms of FoxG1 blocks the ability of IGF-1 to maintain neuronal survival suggesting that FoxG1 is a downstream mediator of IGF-1/Akt signaling. Our study identifies a new and important function for FoxG1 in differentiated neurons.
Impairing intracellular signaling induced by survival factors and excess glutamate have recently been suggested to play important role in neurodegenerative processes. However, the underlying mechanism(s) and interrelationships between these factors mostly remain to be established. In the present study, we show that glutamate attenuates the tyrosine phosphorylation of the insulin-like growth factor-1 (IGF-1) receptor and the survival effect of IGF-1 (100 nm) in hippocampal cultured neurons. Pretreatment of cultured hippocampal neurons with glutamate concentration dependently inhibited the tyrosine phosphorylation of IGF-1 receptors as well as that of IRS-1 and Shc, two IGF-1 receptor adapter proteins. The effect of glutamate was also evident on the phosphorylation of Akt, as well as its upstream kinase PI3K/PDK1 and downstream targets, GSK3beta and FOXO3a. The inhibitory effect of glutamate (1 mm) was blocked by antagonists of the N-methyl-d-aspartate (NMDA) receptor, including MK801 (20 microm) and AP5 (100 microm), but not by blockers of other ionotropic or metabotropic glutamate receptor sub-types demonstrating the involvement of the NMDA receptor. This hypothesis is supported further by the observation that treatment with NMDA concentration dependently inhibited the activation and phosphorylation of IGF-1 receptors and downstream targets induced by IGF-1 (100 nm). These findings demonstrate that glutamate can block the effect of IGF-1 by decreasing IGF-1 receptor signaling and responsiveness, hence attenuating the survival properties of this trophic factor in neuronal cells. Our results also suggest a novel mechanism by which glutamate can reduce cell viability and induce neurotoxicity.
The objective of this study was to determine whether endogenous nitric oxide (NO) inhibits leukocyte adhesion to vascular endothelium. This was accomplished by superfusing a cat mesenteric preparation with inhibitors of NO production, NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME), and observing single (30-microns diameter) venules by intravital video microscopy. Thirty minutes into the superfusion period the number of adherent and emigrated leukocytes, the erythrocyte velocity, and the venular diameter were measured; venular blood flow and shear rate were calculated from the measured parameters. The contribution of the leukocyte adhesion glycoprotein CD11/CD18 was determined using the CD18-specific monoclonal antibody IB4. Both inhibitors of NO production increased leukocyte adherence more than 15-fold. Leukocyte emigration was also enhanced, whereas venular shear rate was reduced by nearly half. Antibody IB4 abolished the leukocyte adhesion induced by L-NMMA and L-NAME. Incubation of isolated cat neutrophils with L-NMMA, but not L-NAME, resulted in direct upregulation of CD11/CD18 as assessed by flow cytometry. Decrements in venular shear rate induced by partial occlusion of the superior mesenteric artery in untreated animals revealed that only a minor component of L-NAME-induced leukocyte adhesion was shear rate-dependent. The L-NAME-induced adhesion was inhibited by L-arginine but not D-arginine. These data suggest that endothelium-derived NO may be an important endogenous modulator of leukocyte adherence and that impairment of NO production results in a pattern of leukocyte adhesion and emigration that is characteristic of acute inflammation.
Osteopenia has been ascribed to diabetics without residual insulin secretion and high insulin requirement. However, it is not known if this is partially due to disturbances in the IGF system, which is a key regulator of bone cell function. To address this question, we performed a cross-sectional study measuring serum levels of IGF-I, IGF-binding protein-1 (IGFBP-1), IGFBP-3, IGFBP-4 and IGFBP-5 by specific immunoassays in 52 adults with Type 1 (n=27) and Type 2 (n=25) diabetes mellitus and 100 age- and sex-matched healthy blood donors. In the diabetic patients, we further determined serum levels of proinsulin, intact parathyroid hormone (PTH), 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and several biochemical bone markers, including osteocalcin (OSC), bone alkaline phosphatase (B-ALP), carboxy-terminal propeptide of type I procollagen (PICP), and type I collagen cross-linked carboxy-terminal telopeptide (ICTP). Urinary albumin excretion was ascertained as a marker of diabetic nephropathy. Bone mineral density (BMD) of hip and lumbar spine was determined by dual-energy X-ray absorptiometry. Data are presented as means+/-s.e.m. Differences between the experimental groups were determined by performing a one-way analysis of variance (ANOVA), followed by Newman-Keuls test. Correlations between variables were assessed using univariate linear regression analysis and partial correlation analysis. Type 1 diabetics showed significantly lower IGF-I (119+/-8 ng/ml) and IGFBP-3 (2590+/-104 ng/ml) but higher IGFBP-1 levels (38+/-10 ng/ml) compared with Type 2 patients (170+/-13, 2910+/-118, 11+/-3 respectively; P<0.05) or healthy controls (169+/-5, 4620+/-192, 3.5+/-0.4 respectively; P<0.01). IGFBP-5 levels were markedly lower in both diabetic groups (Type 1, 228+/-9; Type 2, 242+/-11 ng/ml) than in controls (460+/-7 ng/ml,P<0. 01), whereas IGFBP-4 levels were similar in diabetics and controls. IGF-I correlated positively with IGFBP-3 and IGFBP-5 and negatively with IGFBP-1 and IGFBP-4 in all subjects. Type 1 patients showed a lower BMD of hip (83+/-2 %, Z-score) and lumbar spine (93+/-2 %) than Type 2 diabetics (93+/-5 %, 101+/-5 % respectively), reaching significance in the female subgroups (P<0.05). In Type 1 patients, BMD of hip correlated negatively with IGFBP-1 (r=-0.34, P<0.05) and IGFBP-4 (r=-0.3, P<0.05) but positively with IGFBP-5 (r=0.37, P<0. 05), which was independent of age, diabetes duration, height, weight and body mass index, as assessed by partial correlation analysis. Furthermore, biochemical markers indicating bone loss (ICTP) and increased bone turnover (PTH, OSC) correlated positively with IGFBP-1 and IGFBP-4 but negatively with IGF-I, IGFBP-3 and IGFBP-5, while the opposite was observed with bone formation markers (PICP, B-ALP) and vitamin D3 metabolites. In 20 Type 2 patients in whom immunoreactive proinsulin could be detected, significant positive correlations were found between proinsulin and BMD of hip (r=0.63, P<0.005), IGF-I (r=0.59, P<0.01) as well as IGFBP-3 (r=0.49, P<0.05). Type 1 and Type 2 patients with macroalbuminuria showed a lower BMD of hip, lower IGFBP-5 but higher IGFBP-4 levels, suggesting that diabetic nephropathy may contribute to bone loss by a disturbed IGF system. In conclusion, the findings of this study support the hypothesis that the imbalance between individual IGF system components and the lack of endogenous proinsulin may contribute to the lower BMD in Type 1 diabetics.
The ability of insulin to protect neurons from apoptosis was examined in differentiated R28 cells, a neural cell line derived
from the neonatal rat retina. Apoptosis was induced by serum deprivation, and the number of pyknotic cells was counted. p53
and Akt were examined by immunoblotting after serum deprivation and insulin treatment, and caspase-3 activation was examined
by immunocytochemistry. Serum deprivation for 24 h caused ∼20% of R28 cells to undergo apoptosis, detected by both pyknosis
and activation of caspase-3. 10 nm insulin maximally reduced the amount of apoptosis with a similar potency as 1.3 nm (10 ng/ml) insulin-like growth factor 1, which acted as a positive control. Insulin induced serine phosphorylation of Akt,
through the phosphatidylinositol (PI) 3-kinase pathway. Inhibition of PI 3-kinase with wortmannin or LY294002 blocked the
ability of insulin to rescue the cells from apoptosis. SN50, a peptide inhibitor of NF-κB nuclear translocation, blocked the
rescue effect of insulin, but neither insulin or serum deprivation induced phosphorylation of IκB. These results suggest that
insulin is a survival factor for retinal neurons by activating the PI 3-kinase/Akt pathway and by reducing caspase-3 activation.
The rescue effect of insulin does not appear to be mediated by NF-κB or p53. These data suggest that insulin provides trophic
support for retinal neurons through a PI 3-kinase/Akt-dependent pathway.
Little is known about interventions that may prevent predegenerative changes in the diabetic retina. This study tested the hypothesis that immediate, systemic treatment with an insulin-like growth factor (IGF)-1 analog can prevent abnormal accumulations of type 1 IGF receptor, and phospho-Akt (Thr 308) immunoreactivity in predegenerative retinas of streptozotocin (STZ) diabetic rats. Type 1 IGF receptor immunoreactivity increased approximately 3-fold in both inner nuclear layer (INL) and ganglion cell layer (GCL) in retinas from STZ rats versus nondiabetic controls. Phospho-Akt (Thr 308) immunoreactivity increased 5-fold in GCL and 8-fold in INL of STZ rat retinas. In all cases, immunoreactive cells were significantly reduced in STZ des(1-3)IGF-1-treated versus STZ rats. Preliminary results suggested that vascular endothelial growth factor (VEGF) levels may also be reduced. Hyperglycemia/failure of weight gain in diabetic rats continued despite systemic des(1-3)IGF-1. These data show that an IGF-1 analog can prevent early retinal biochemical abnormalities implicated in the progression of diabetic retinopathy, despite ongoing hyperglycemia.
To investigate the presence of somatostatin and its receptors (sst(1-5) receptors) and their possible involvement in the regulation of nitric oxide (NO) production in human RPE cell cultures.
Human RPE cells (D407) were used for all studies performed. Somatostatin levels were detected by radioimmunoassay, and RT-PCR and immunocytochemistry studies were performed to identify the somatostatin receptors (sst1-sst5). Radioligand binding assays were also performed examining the ability of certain somatostatin ligands (sst1, sst2, sst5) to compete for [125I]Tyr11 somatostatin binding. The presence of NO synthase in the cultures was assayed with NADPH-diaphorase cytochemistry, and RT-PCR, and NO levels were assessed by examining the production of its stable metabolites NO2- and NO3- (NOx-).
SRIF was detected in a concentration of 0.56 +/- 0.13 picomoles/mg protein. sst1, sst2, and sst5 mRNAs were detected, yet only sst2B and sst5 immunoreactivity was observed in human RPE cell cultures. sst1- and sst5- but not sst2-selective ligands displaced the specific [125I]Tyr11 somatostatin binding to RPE cell membranes. NADPH-diaphorase stain and iNOS mRNA were detected. SRIF and the sst2-selective analogue MK678 increased the levels of NOx- in a concentration-dependent manner. This increase was blocked by the sst2 antagonist CYN-154806 (Ac-4NO2-Phe-c(dCys-Tyr-dTrp-Lys-Thr-Cys)-dTyr-NH2).
These results demonstrate the presence of somatostatin, and its receptors sst1, sst2B, and sst5 in human RPE cells and suggest an autocrine or paracrine role for somatostatin. Somatostatin's ability to regulate NO production, by activating sst2 receptors, provides a functional role of somatostatin in the RPE.
To examine insulin-like growth factor (IGF)-1 stimulation of expression of hypoxia inducible factor (HIF)-1 alpha and secretion of vascular endothelial growth factor (VEGF) and IGF binding protein (IGFBP)-3 in human retinal pigment epithelial (RPE) cell line D407.
D407 cells cultured in dishes or Transwell inserts were treated with cobalt chloride or varying doses of IGF-1. Whole cell lysates were assayed by immunoblot for HIF-1 alpha expression, whereas conditioned medium was TCA precipitated and assayed by immunoblot for VEGF and ligand blot for IGFBP-3. Cells grown on coverslips were similarly treated and probed with antibodies to HIF-1 alpha, VEGF, and IGFBP-3, and visualized by epifluorescence microscopy. Cells grown on Transwell inserts were probed with antibodies to the Na(+)/K(+)-ATPase alpha-1 subunit and either the alpha or beta subunits of the IGF-1 receptor and visualized in Z-section using confocal microscopy.
Immunoblot analysis of whole cell lysates from IGF-1-treated D407 cells revealed the upregulation of HIF-1 alpha protein. Epifluorescence microscopy demonstrated a positive correlation between HIF-1 alpha expression and nuclear localization, VEGF and IGFBP-3 synthesis and export, and IGF-1 action. Western and ligand blot analyses of RPE cell-conditioned medium indicated that IGF-1 induced increases in VEGF and IGFBP-3 secretion. Cells grown on Transwell inserts exhibited constitutive apical secretion of VEGF and IGFBP-3, which increased on apical or basolateral treatment with IGF-1. Confocal analysis of Transwell-cultured D407 cells confirmed the apical localization of the Na(+)/K(+)-ATPase alpha-1 subunit, characteristic of polarized RPE, with IGF-1 receptor alpha and beta subunits exhibiting a nonpolarized distribution.
IGF-1 stimulates increased HIF-1 alpha expression as well as VEGF and IGFBP-3 secretion in D407 cells. Similar to their in vivo counterparts, D407 cells maintain reversed epithelial polarity. Apical secretion of VEGF and IGFBP-3 increases in response to either apical or basolateral IGF-1 stimulation consistent with the nonpolarized distribution of IGF-1 receptors.
We examined the endothelial nitric oxide (eNOS) gene polymorphisms to assess its possible association with diabetic retinopathy and macular edema.
A total of 226 patients with type 2 diabetes and 186 healthy subjects were studied. Type 2 diabetic patients consisted of 110 patients without retinopathy, 46 patients with nonproliferative diabetic retinopathy, and 71 patients with proliferative diabetic retinopathy. Diabetic macular edema was present in 48 patients. Three polymorphisms of the eNOS gene were determined: T-786C in the promoter region, 27-bp repeat in intron 4, and Glu298Asp in exon 7.
Close linkage disequilibrium was observed between the T-786C polymorphism and the 27-bp repeat, as has been previously reported, but Glu298Asp was not in linkage disequilibrium with the other two polymorphisms. The eNOS gene polymorphisms were not significantly associated with the presence of retinopathy or with retinopathy severity or type 2 diabetes itself. However, by both association study and multiple logistic regression analysis, the T-786C and 27-bp repeat polymorphisms were significantly associated with a risk of developing macular edema with the -786C allele and the "a" allele increasing the risk.
The present study suggests that the eNOS gene is a novel genetic risk factor for diabetic macular edema. The eNOS gene polymorphisms may contribute to the development of macular edema by impairing basal eNOS expression and resulting in the breakdown of the blood-retina barrier.
Insulin-like growth factors I and II (IGF-I and IGF-II) have an ancient origin and play essential roles in fundamental biological processes. Although IGFs are principally known for their roles in regulating cell growth and survival, their ability to influence cell motility is just as significant. In the past 20 years, research has provided indisputable evidence for the regulatory role of IGFs in the migration of various cell types. Cell migration is crucial for reproduction, development, and tissue regeneration; IGFs play an important role in coordinating these processes. Moreover, studies continue to uncover the IGFs' role in stimulating cancer cell migration, invasion and metastasis. This review surveys current knowledge on the cell migration-modulating properties of IGFs and the biochemical pathways by which these peptides regulate cell movement in both physiological and pathological conditions.
Neurotrophins signal via Trk tyrosine kinase receptors. Nerve growth factor (NGF) is the cognate ligand for TrkA, the brain-derived neurotrophic factor for TrkB, and NT-3 for TrkC. NT-3 also binds TrkA as a lower affinity heterologous ligand. Because neurotrophin-3 (NT-3) interactions with TrkA are biologically relevant, we aimed to define the TrkA "hot spot" functional docking sites of NT-3. The Trk extracellular domain consists of two cysteine-rich subdomains (D1 and D3), flanking a leucine-rich subdomain (D2), and two immunoglobulin-like subdomains IgC1(D4) and IgC2(D5). Previously, the D5 subdomain was defined as the primary ligand-binding site of neurotrophins for their cognate receptors (e.g. NGF binds and activates through TRKA-D5 hot spots). Here binding studies with truncated and chimeric extracellular subdomains show that TRKA-D5 also includes an NT-3 docking and activation hot spot (site 1), and competition studies show that the NGF and NT-3 hot spots on TRKA-D5 are distinct but partially overlapping. In addition, ligand binding studies provide evidence for an NT-3-binding/allosteric site on TRKA-D4 (site 2). NT-3 docking on sites 1 and/or 2 partially blocks NGF binding. Functional survival studies showed that sites 1 and 2 regulate TrkA activation. NT-3 docking on both sites 1 and 2 affords full agonism, which can be additive with NGF activation of Trk. However, NT-3 docking solely on site 1 is partially agonistic but noncompetitively antagonizes NGF binding and activation of Trk. This study demonstrates that Trk signaling is more complex than previously thought because it involves several receptor subdomains and hot spots.
How HIV-1 enter into the eyes remains obscure. We postulated that HIV-1 Tat protein can alter the expression of specific tight-junction proteins and disturb the blood retinal barrier, and contributes to HIV trafficking into the eyes. This study is to determine the effects of HIV-1 Tat proteins on the barrier function and tight-junction protein expression of retinal pigment epithelial cell (RPE).
A human RPE cell line (D407) cultured on microporous filter-supports was used. After treating with HIV-1 Tat protein, transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of the RPE cells to sodium fluorescein was measured. The expressions of the occludin and claudins were determined by real-time polymerase chain reaction, immunofluorescence, and Western blot analysis. Activation of ERK1/2 was detected by Western blot analysis with specific antiphospho protein antibodies. NF-kappaB DNA binding activity was determined by transcription factor assay. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in barrier destruction of RPE cells exposed to HIV-1 Tat.
Treating cultured human retinal pigment epithelial cells with 100 nM Tat for 24 hours increased the permeability and decreased the TER of the epithelial monolayer. HIV-1 Tat also disrupted and downregulated the tight-junction proteins claudin-1, claudin-3, and claudin-4 in these cells, whereas claudin-2 was upregulated, and the expression of occludin was unaffected. HIV-1 Tat protein also induced activation of ERK1/2 and NF-kappaB. HIV-1 Tat protein induced barrier destruction, changes in expression of TJs, and activation of ERK1/2 and NF-kappaB were abrogated by inhibitor of ERK1/2 and NF-kappaB.
HIV-1 Tat protein causes increases in the paracellular permeability of RPE cells in vitro concomitant with changes in expression of certain transmembrane proteins associated with the tight junction. The effects of HIV-1 Tat on barrier function of the RPE may be mediated by ERK MAPK and NF-kappaB activation, which may represent potential targets for novel therapeutic approaches for the retinopathy induced by HIV infection.
Peripheral neuropathy is a particularly debilitating complication of both type 1 and type 2 diabetes characterized by sensory and motor neuron damage and decreased circulating levels of insulin-like growth factor 1 (IGF-1). Quite often, an early hyperalgesia is followed by hypoalgesia and muscle weakness. Hypoalgesia can lead to significant morbidity for which there is no current treatment. Hyperglycemic, streptozotocin (STZ)-induced rodent models reproduce these symptoms. We investigated whether increasing systemic IGF-1 could improve neuronal function in hyper- and hypoalgesic STZ-treated mice. Increased circulating levels of IGF-1 were achieved by delivering a plasmid or adeno-associated viral (AAV) vector bearing mouse IGF-1 to the liver. Treating mice in the hyperalgesia stage prevented later hypoalgesia. Treating mice in the hypoalgesia stage reversed existing hypoalgesia. This latter effect could be seen by merely restoring IGF-1 serum levels to normalcy, which was possible to achieve by IGF-1 gene therapy or insulin treatment. Sensory nerve functional correction was seen to be correlated with attenuated Schwann cell vacuolization and demyelination in peripheral sensory nerve fibers. A further increase in serum IGF-1 levels with gene therapy also improved motor function, consistent with the observed prevention of both muscle atrophy and peripheral motor nerve fiber demyelination. These results suggest that the restoration of systemic levels of IGF-1 may prove to be a highly effective therapeutic modality for treating diabetic peripheral neuropathy.
Osteopenia has been ascribed to diabetics without residual insulin secretion and high insulin requirement. However, it is not known if this is partially due to disturbances in the IGF system, which is a key regulator of bone cell function. To address this question, we performed a cross-sectional study measuring serum levels of IGF-I, IGF-binding protein-1 (IGFBP-1), IGFBP-3, IGFBP-4 and IGFBP-5 by specific immunoassays in 52 adults with Type 1 (n=27) and Type 2 (n=25) diabetes mellitus and 100 age- and sex-matched healthy blood donors. In the diabetic patients, we further determined serum levels of proinsulin, intact parathyroid hormone (PTH), 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and several biochemical bone markers, including osteocalcin (OSC), bone alkaline phosphatase (B-ALP), carboxy-terminal propeptide of type I procollagen (PICP), and type I collagen cross-linked carboxy-terminal telopeptide (ICTP). Urinary albumin excretion was ascertained as a marker of diabetic nephropathy. Bone mineral density (BMD) of hip and lumbar spine was determined by dual-energy X-ray absorptiometry. Data are presented as means+/-s.e.m. Differences between the experimental groups were determined by performing a one-way analysis of variance (ANOVA), followed by Newman-Keuls test. Correlations between variables were assessed using univariate linear regression analysis and partial correlation analysis. Type 1 diabetics showed significantly lower IGF-I (119+/-8 ng/ml) and IGFBP-3 (2590+/-104 ng/ml) but higher IGFBP-1 levels (38+/-10 ng/ml) compared with Type 2 patients (170+/-13, 2910+/-118, 11+/-3 respectively; P<0.05) or healthy controls (169+/-5, 4620+/-192, 3.5+/-0.4 respectively; P<0.01). IGFBP-5 levels were markedly lower in both diabetic groups (Type 1, 228+/-9; Type 2, 242+/-11 ng/ml) than in controls (460+/-7 ng/ml,P<0. 01), whereas IGFBP-4 levels were similar in diabetics and controls. IGF-I correlated positively with IGFBP-3 and IGFBP-5 and negatively with IGFBP-1 and IGFBP-4 in all subjects. Type 1 patients showed a lower BMD of hip (83+/-2 %, Z-score) and lumbar spine (93+/-2 %) than Type 2 diabetics (93+/-5 %, 101+/-5 % respectively), reaching significance in the female subgroups (P<0.05). In Type 1 patients, BMD of hip correlated negatively with IGFBP-1 (r=-0.34, P<0.05) and IGFBP-4 (r=-0.3, P<0.05) but positively with IGFBP-5 (r=0.37, P<0. 05), which was independent of age, diabetes duration, height, weight and body mass index, as assessed by partial correlation analysis. Furthermore, biochemical markers indicating bone loss (ICTP) and increased bone turnover (PTH, OSC) correlated positively with IGFBP-1 and IGFBP-4 but negatively with IGF-I, IGFBP-3 and IGFBP-5, while the opposite was observed with bone formation markers (PICP, B-ALP) and vitamin D3 metabolites. In 20 Type 2 patients in whom immunoreactive proinsulin could be detected, significant positive correlations were found between proinsulin and BMD of hip (r=0.63, P<0.005), IGF-I (r=0.59, P<0.01) as well as IGFBP-3 (r=0.49, P<0.05). Type 1 and Type 2 patients with macroalbuminuria showed a lower BMD of hip, lower IGFBP-5 but higher IGFBP-4 levels, suggesting that diabetic nephropathy may contribute to bone loss by a disturbed IGF system. In conclusion, the findings of this study support the hypothesis that the imbalance between individual IGF system components and the lack of endogenous proinsulin may contribute to the lower BMD in Type 1 diabetics.
purpose. To investigate the presence of somatostatin and its receptors (sst1–5 receptors) and their possible involvement in the regulation of nitric oxide (NO) production in human RPE cell cultures.
methods. Human RPE cells (D407) were used for all studies performed. Somatostatin levels were detected by radioimmunoassay, and RT-PCR and immunocytochemistry studies were performed to identify the somatostatin receptors (sst1-sst5). Radioligand binding assays were also performed examining the ability of certain somatostatin ligands (sst1, sst2, sst5) to compete for [¹²⁵I]Tyr¹¹ somatostatin binding. The presence of NO synthase in the cultures was assayed with NADPH-diaphorase cytochemistry, and RT-PCR, and NO levels were assessed by examining the production of its stable metabolites NO2⁻ and NO3⁻ (NOx⁻).
results. SRIF was detected in a concentration of 0.56 ± 0.13 picomoles/mg protein. sst1, sst2, and sst5 mRNAs were detected, yet only sst2B and sst5 immunoreactivity was observed in human RPE cell cultures. sst1- and sst5- but not sst2-selective ligands displaced the specific [¹²⁵I]Tyr¹¹ somatostatin binding to RPE cell membranes. NADPH-diaphorase stain and iNOS mRNA were detected. SRIF and the sst2-selective analogue MK678 increased the levels of NOx⁻ in a concentration-dependent manner. This increase was blocked by the sst2 antagonist CYN-154806 (Ac-4NO2-Phe-c(dCys-Tyr-dTrp-Lys-Thr-Cys)-dTyr-NH2).
conclusions. These results demonstrate the presence of somatostatin, and its receptors sst1, sst2B, and sst5 in human RPE cells and suggest an autocrine or paracrine role for somatostatin. Somatostatin’s ability to regulate NO production, by activating sst2 receptors, provides a functional role of somatostatin in the RPE.
Purpose:
Diabetic retinopathy (DR) is associated with nitrosative stress. The purpose of this study was to evaluate the beneficial effects of S-nitrosoglutathione (GSNO) eye drop treatment on an experimental model of DR.
Methods:
Diabetes (DM) was induced in spontaneously hypertensive rats (SHR). Treated animals received GSNO eye drop (900 nM or 10 μM) twice daily in both eyes for 20 days. The mechanisms of GSNO effects were evaluated in human RPE cell line (ARPE-19).
Results:
In animals with DM, GSNO decreased inducible nitric oxide synthase (iNOS) expression and prevented tyrosine nitration formation, ameliorating glial dysfunction measured with glial fibrillary acidic protein, resulting in improved retinal function. In contrast, in nondiabetic animals, GSNO induced oxidative/nitrosative stress in tissue resulting in impaired retinal function. Nitrosative stress was present markedly in the RPE layer accompanied by c-wave dysfunction. In vitro study showed that treatment with GSNO under high glucose condition counteracted nitrosative stress due to iNOS downregulation by S-glutathionylation, and not by prevention of decreased GSNO and reduced glutathione levels. This posttranslational modification probably was promoted by the release of oxidized glutathione through GSNO denitrosylation via GSNO-R. In contrast, in the normal glucose condition, GSNO treatment promoted nitrosative stress by NO formation.
Conclusions:
In this study, a new therapeutic modality (GSNO eye drop) targeting nitrosative stress by redox posttranslational modification of iNOS was efficient against early damage in the retina due to experimental DR. The present work showed the potential clinical implications of balancing the S-nitrosoglutathione/glutathione system in treating DR.
Cathepsin S plays multiple roles in MHC class II antigen presentation, extracellular matrix degradation, angiogenesis, and tumorogenesis. Our previous study revealed that targeting cathepsin S could induced cellular cytotoxicity and reduce cell viability. For the current study, we further investigated the molecular executioner is responsible for targeting cathepsin S-induced cell death and its association with autophagy. Distinct from regulation of the classic autophagy pathway by reactive oxygen species (ROS), we demonstrated that autophagy to be the genuine regulator of early ROS production. The molecular silencing of autophagy-dependent ATG genes (ATG5, ATG7, and LC3) and the pharmacologic inhibition of autophagy with 3-MA and wortmannin reduced ROS production significantly. In addition, xanthine oxidase (XO), which is up-regulated by autophagy, is required for early ROS production, oxidative DNA damage, and consequent cell death. Autophagy inhibition suppresses the upregulation of XO, which is induced by cathepsin S inhibition, resulting in reduced ROS generation, DNA damage, and cell death. Collectively, our study reveals a non-canonical molecular pathway in which, after the inhibition of cathepsin S, autophagy induces early ROS production for oxidative DNA damage and cell death through XO.
Insulin-like growth factor-1 (IGF-1) is a trophic factor promoting cell survival by activating phosphatidylinositol 3-kinase (PI3K)/Akt kinase pathway. FKHRL1, a member of the Forkhead family of transcription factors possibly involved in cell cycle and apoptosis, is a downstream target of Akt in fibroblasts. However, very little information is available concerning neurons. We report herein that IGF-1 rapidly induced the phosphorylation of endogenous FKHRL1 in hippocampal neurons. The PI3K/Akt kinase pathway mediates this action, as evidenced by the use of different kinase inhibitors, the expression of constitutively active Akt, and in vitro kinase assay. IGF-1 blocked the nuclear translocation of FKHRL1 in hippocampal neurons and promoted survival in parallel to the phosphorylation of Akt and FKHRL1. Similarly, the expression of constitutively active Akt in PC-12 cells increased the phosphorylation of FKHRL1 and promoted survival, whereas the expression of kinase dead Akt attenuated IGF-1–mediated survival of PC-12 cells. Moreover, the overexpression of wild-type FKHRL1 and its nonphosphorylated mutant induced apoptosis in cultured hippocampal neurons. Interestingly, IGF-1 and PI3-kinase inhibitors have no significant effect on the cell cycle inhibitor p27kip1 in hippocampal neurons. This finding suggests that in contrast to fibroblasts, FKHRL1 is unlikely to be involved in cell cycle in neurons. Taken together, these data reveal that endogenous FKHRL1 is a downstream substrate of PI3K/Akt in IGF-1 receptor signaling in hippocampal neurons and suggest that the phosphorylation of this transcription factor may play an important role in the neuronal survival properties of IGF-1.
Insulin-like growth factor-1 (IGF-1) is essential for the development of nervous system and locates at many cell types. Relatively, little is known about IGF-1 expression on the brain microvascular endothelial cells (BMECs). For in vivo study, we examined the expressions of IGF-1 and IGFBP-2 after focal cerebral ischemia for 12 hours, 24 hours, 3 days and 7 days utilizing a permanent middle cerebral artery occlusion (MCAO) model in rats. For in vitro study, the levels of IGF-1 and insulin-like growth factors binding protein 2 (IGFBP-2) in the cultured media or primarily cultured BMECs injured by glucose deprivation (OGD) were examined. Then we elucidated the protective effects of IGF-1 on cortical neurons injured by OGD and the possible mechanism. In addition, we detected the effect of BMECs conditioned media (BMECs-CM) on insulin-like growth factor-1 receptor (IGF-1R) expression in neurons. The results showed that IGF-1 increased in serum and brain tissue, while IGFBP-2 decreased in brain tissue of MCAO-injured rats. In primarily cultured BMECs, the expressions of IGF-1 and IGFBP-2 were all significantly higher under OGD conditions in culture. IGF-1 administration improved the neuron viability upon normoxia or OGD and upregulated the p-Akt expression. This effect was reversed by the LY294002, a specific inhibitor of the PI3K/Akt signaling pathway. Furthermore, conditioned medium from OGD-induced BMECs remarkably suppressed neuron viability and the expression of IGF-1R simultaneously. These data demonstrate that future therapeutic strategies that prioritize the early recovery of IGF-1 system in BMECs might be a promising therapeutic intervention in ischemic injury. This article is protected by copyright. All rights reserved.
Ultraviolet (UV)-induced damage plays a major role in ocular diseases, such as cataracts and retinal degeneration. UVB may also cause retinal phototoxicity and photic retinopathy. In this study, we explored the effects of UVB on the cell cycle and the role of silent mating type information regulation 2 homolog 1 (SIRT1) in the UVB-induced damage. UVB dose-dependently suppressed the growth of retinal pigment epithelial (RPE) cells by activating the phosphatidylinositol 3-kinase (PI3K) pathway and triggering cell cycle arrest at the S phase. SIRT1, an NAD-dependent histone deacetylase, is involved in multiple biological processes, such as the stress response and the regulation of the cell cycle. However, its role in the effects of UVB on RPE cells is unclear. We showed that UVB down-regulates SIRT1 expression in a dose-dependent manner. Resveratrol, an SIRT1 activator, prevented the UVB-induced damage by inhibiting AKT and ERK phosphorylation. A specific PI3K inhibitor attenuated the UVB-induced ERK1/2 and p53 phosphorylation. Finally, UVB activated the PI3K/AKT/ERK pathway by reducing the expression of SIRT1 in ARPE-19 cells. Our study, therefore, illustrated the molecular mechanisms of UVB-induced phototoxicity and damage of RPE cells. SIRT1 and resveratrol may be significant regulators, protecting against UVB-induced injury.
Rationale
Antidepressants could exert neuroprotective effects against various insults and the antidepressant-like effect may result from its neuroprotective effects. The phosphatidylinositol-3-kinase/protein kinase B/Forkhead box O3 (PI3K/Akt/FoxO3a) pathway is a key signaling pathway in mediating cell survival. However, no information is available regarding the interaction of FoxO3a and antidepressants.
Objectives
PC12 cells treated with corticosterone were used as a model to study the protective effect of venlafaxine and underlying mechanisms.
Methods
Methyl thiazolyl tetrazolium (MTT) assay, Hoechst staining, and the observation of FoxO3a subcellular location were used to study the protective effect of venlafaxine against cell damage caused by corticosterone. Pretreatments with various pathway inhibitors were used to investigate the possible pathways involved in the protection of venlafaxine. The phosphorylation of Akt and FoxO3a was analyzed by Western blot.
Results
Corticosterone decreased the phosphorylation of Akt and FoxO3a and led to the nuclear localization of FoxO3a and the apoptosis of PC12 cells. Venlafaxine concentration-dependently protected PC12 cells against corticosterone. The protective effect of venlafaxine was reversed by LY294002 and wortmannin, two PI3K inhibitors, and Akt inhibitor VIII, whereas mitogen-activated protein kinase kinase (MAPK kinase) inhibitor PD98059 and the p38 MAPK inhibitor PD160316 had no effect. Western blot analyses showed that venlafaxine induced the phosphorylation of Akt and FoxO3a by the PI3K/Akt pathway and reversed the reduction of the phosphorylated Akt and FoxO3a, and the nuclear translocation of Foxo3a induced by corticosterone.
Conclusions
Venlafaxine protects PC12 cells against corticosterone-induced cell death by modulating the activity of the PI3K/Akt/FoxO3a pathway.
Insulin-like growth factors I and II (IGF I and II) are polypeptides with both growth-promoting and insulin-like metabolic effects16,27. Immunoreactive IGF I is present in the retina14 and both IGF I and II are present in vitreal fluid12. The type I and type II IGF receptors are also localized within the neural retina3,15,20,33. The presence of IGFs and IGF receptors within the eye suggests a possible growth-promoting effect of IGFs on ocular tissues. IGF may enter the eye from the blood or, alternatively, arise from an ocular cell type which synthesizes and secretes IGF. IGF I and II mRNA synthesis in scleral cells13 and IGF I synthesis in rat retina14 suggests endogenous IGF production in the eye. We hypothesized that IGFs and IGF receptors are synthesized by one ocular cell type, the retinal pigment-epithelium (RPE). As a first step in studying IGF production by the RPE, we analyzed expression of the IGF and IGF receptor genes by cultured human RPE cells. Using Northern analysis, RNase protection and reverse-transcriptase polymerase chain reaction (RT-PCR), we found that cultured RPE cells synthesize mRNA for IGF I and the type II IGF receptors.
We tested 3-nitro-4-((4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)methyl) benzoylguanidine tartrate (TG-6) which is combinated of two known cardioprotective agents cariporide and trimetazidine, whether additively to reduce ischemia-reperfusion injury in rats. Using models of in vitro perfusion (Langendorff system) and in vivo open chest left anterior descending coronary artery ligation causing ischemia-reperfusion injury. We also used Fura-2 to measure the cytosolic Ca²⁺ concentrations ([Ca²⁺]i) in cardiomyocytes, western blot analysis the protein expression of Kv1.4, Kv4.2, Kv4.3 in myocardial ischemia-reperfusion rats. TG-6 improved the cardiac function in both in vivo and in vitro models, lowered Lactate Dehydrogenase (LDH), Creatine Kinase (CK), Malodialdehyed (MDA) activity while enhanced Superoxide Dismutase (SOD) activity. High dose of TG-6 improved the hypoxia injury of cardiomyocytes induced by sodium dithionite (Na₂S₂O₄), enhanced the viability and decreased the [Ca²⁺]i. It also down-regulated the expression of Kv1.4 and increased the expression of Kv4.2 and Kv4.3, so it might through regulating the expression of the transient outward potassium current (Ito) to improve the cardiac function.
In this study, we found an imbalance between stress-mediated and survival signaling and elevated apoptotic markers in retinal pigment epithelium (RPE) from diabetic patients. Since fenofibric acid (FA) treatment reduces the progression of diabetic retinopathy (DR), we investigated the effect of hyperglycemia and hypoxia, two components of the diabetic milieu, on stress, apoptosis, and survival pathways in ARPE-19 cells (immortalized human RPE cell line) and whether FA is able to prevent the deleterious effects induced by these conditions. ARPE-19 cells cultured in high-glucose (HG) medium or under hypoxia (1% oxygen)-induced phosphorylation of the stress-activated kinases JNK and p38 MAPK. This effect was increased by the combination of both conditions. Likewise, hyperglycemia and hypoxia triggered the phosphorylation of the endoplasmic reticulum (ER) stress markers PERK and eIF2α and the induction of the pro-apoptotic transcription factor CHOP. Under these experimental conditions, reactive oxygen species (ROS) were elevated and the integrity of tight junctions was disrupted. Conversely, ARPE-19 cells treated with FA were protected against these deleterious effects induced by hyperglycemia and hypoxia. FA increased insulin-like growth factor I receptor (IGF-IR)-mediated survival signaling in cells cultured under hyperglycemia and hypoxia, thereby suppressing caspase-3 activation and down-regulation of BclxL. Moreover, FA increased LC3-II, an autophagy marker. In conclusion, our results demonstrated that FA elicits a dual protective effect in RPE by down-regulation of stress-mediated signaling and induction of autophagy and survival pathways. These molecular mechanisms could be involved in the beneficial effects of fenofibrate reported in clinical trials.
Neuronal activity leads to arteriole dilation and increased blood flow in retinal vessels. This response, termed functional hyperemia, is diminished in the retinas of diabetic patients, possibly contributing to the development of diabetic retinopathy. The mechanism responsible for this loss is unknown. Here we show that light-evoked arteriole dilation was reduced by 58% in a streptozotocin-induced rat model of type 1 diabetes. Functional hyperemia is believed to be mediated by glial cells and we found that glial-evoked vasodilation was reduced by 60% in diabetic animals. The diabetic retinas showed neither a decrease in the thickness of the retinal layers nor an increase in neuronal loss, although signs of early glial reactivity and an upregulation of inducible nitric oxide synthase (iNOS) were detected. Inhibition of iNOS restored both light- and glial-evoked dilations to control levels. These findings suggest that high NO levels resulting from iNOS upregulation alters glial control of vessel diameter and may underlie the loss of functional hyperemia observed in diabetic retinopathy. Restoring functional hyperemia by iNOS inhibition may limit the progression of retinopathy in diabetic patients.
Apelin is an endogenous ligand for the angiotensin-1-like receptor APJ. Because apelin has been reported to regulate angiogenesis, the authors searched for associations between apelin and proliferative diabetic retinopathy.
The study included 55 patients undergoing vitrectomy for proliferative diabetic retinopathy (study group) and 34 patients undergoing vitrectomy for idiopathic preretinal membranes or macular hole (control group). Using enzyme-linked immunosorbent assay, the authors measured the concentrations of apelin and vascular endothelial growth factor (VEGF) in the vitreous and plasma. The expression of apelin and angiotensin-1-like receptor APJ in the excised membranes was examined by fluorescence immunostaining and semiquantitative reverse transcription polymerase chain reaction.
Vitreous concentrations of apelin were significantly higher in the study group than in the control group (P = 0.005), whereas plasma concentrations of apelin did not vary significantly (P = 0.66). The vitreous concentrations (P < 0.001) and the plasma concentrations (P = 0.03) of VEGF were significantly higher in the study group than in the control group. Neither the vitreous concentrations of apelin and VEGF (P = 0.47) nor the plasma concentrations of apelin and VEGF (P = 0.19) were significantly associated with each other. In the fibrovascular membranes of the study group, colocalization of the endothelial markers CD31 with the markers for apelin and colocalization of the endothelial markers CD31 and APJ was observed. Expression of apelin mRNA (P = 0.03), APJ mRNA (P = 0.02), and VEGF mRNA (P < 0.01) was significantly higher in fibrovascular proliferative diabetic retinopathy membranes than in idiopathic epiretinal membranes.
The apelin/APJ system may be involved in retinal neovascularization during the development of proliferative diabetic retinopathy.
Antipsychotic drug treatment may be associated with common and problematic sexual dysfunction, especially impotence, which can diminish quality of life and lead to treatment noncompliance. Nitric oxide synthase (NOS) is an important cellular modulator of erectile function. We have therefore investigated the effect of antipsychotic drug on activity and gene expression of NOS in rat penile tissues. The activity of constitutive NOS was significantly suppressed below control by a 21 days administration of 1 mg/kg haloperidol, which also significantly decreased expression of endothelial NOS (eNOS) and neural NOS mRNA. Risperidone at 0.5 mg/kg also reduced eNOS mRNA expression. Haloperidol or risperidone did not change gene expression and activity of inducible NOS (iNOS). Quetiapine significantly increased activity and mRNA expression of iNOS with 20 and 40 mg/kg doses. These preliminary results have important implications for enhancing our understanding of mechanisms by which antipsychotic drugs induce sexual dysfunction.
The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin-like growth factor I (IGF-I) immunoreactivity that co-elutes with authentic human IGF-I in HPLC analyses. Cultured human RPE cells synthesize and release IGF-I, raising the possibility that the RPE serves as a source of IPM IGF-I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF-I receptors with alpha-subunits of 120 and 138 kDa, respectively. They thus appear to be of the "brain" (in photoreceptors) and "peripheral" (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF-BP) that specifically binds IGF-I and IGF-II. The IPM-BP is visualized as a single radiographic band by both ligand blot and affinity cross-linking procedures. With enzymes specific for removing N- and O-linked oligosaccharides, the IPM-BP was found to contain O- but not N-linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM-BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF-I and IGF-BP in the IPM, together with the presence of IGF-I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine-paracrine system.
Oxidative stress and oxidative damage to tissues are common end points of chronic diseases, such as atherosclerosis, diabetes, and rheumatoid arthritis. The question addressed in this review is whether increased oxidative stress has a primary role in the pathogenesis of diabetic complications or whether it is a secondary indicator of end-stage tissue damage in diabetes. The increase in glycoxidation and lipoxidation products in plasma and tissue proteins suggests that oxidative stress is increased in diabetes. However, some of these products, such as 3-deoxyglucosone adducts to lysine and arginine residues, are formed independent of oxidation chemistry. Elevated levels of oxidizable substrates may also explain the increase in glycoxidation and lipoxidation products in tissue proteins, without the necessity of invoking an increase in oxidative stress. Further, age-adjusted levels of oxidized amino acids, a more direct indicator of oxidative stress, are not increased in skin collagen in diabetes. We propose that the increased chemical modification of proteins by carbohydrates and lipids in diabetes is the result of overload on metabolic pathways involved in detoxification of reactive carbonyl species, leading to a general increase in steady-state levels of reactive carbonyl compounds formed by both oxidative and nonoxidative reactions. The increase in glycoxidation and lipoxidation of tissue proteins in diabetes may therefore be viewed as the result of increased carbonyl stress. The distinction between oxidative and carbonyl stress is discussed along with the therapeutic implications of this difference.
Recent studies have suggested a role for neurotrophic substances in the pathogenesis and treatment of diabetic neuropathy. In this study, the effect of insulin-like growth factor I (IGF-I) on diabetic sympathetic autonomic neuropathy was examined in an experimental streptozotocin-induced diabetic rat model. Two months of IGF-I treatment of chronically diabetic rats with established neuroaxonal dystrophy (the neuropathological hallmark of the disease) involving the superior mesenteric ganglion and ileal mesenteric nerves resulted in nearly complete normalization of the frequency of neuroaxonal dystrophy in both sites without altering the severity of diabetes. Treatment with low-dose insulin (to control for the transient glucose-lowering effects of IGF-I) failed to affect the frequency of ganglionic or mesenteric nerve neuroaxonal dystrophy or the severity of diabetes. The striking improvement in the severity of diabetic autonomic neuropathy shown with IGF-I treatment in these studies and the fidelity of the rat model to findings in diabetic human sympathetic ganglia provide promise for the development of new clinical therapeutic strategies.
Recently, increasing interest has been directed toward the role of leukocytes in microvascular disorders including diabetic retinopathy because of their large cell volume, high cytoplasmic rigidity, natural tendency to stick to the vascular endothelium, and capacity to generate toxic superoxide radicals and proteolytic enzymes. Leukocytes in diabetes are reported to be less deformable and more activated, and may be involved in capillary non-perfusion, endothelial cell damage, and vascular leakage in the retinal microcirculation. In fact, histological studies show many capillary occlusions by leukocytes and capillary dropout or degeneration associated with leukocytes in the diabetic retina. Serial acridine orange leukocyte fluorography and fluorescein angiography studies also identify trapped leukocytes directly associated with areas of downstream non-perfusion in the diabetic retinal microcirculation. More recent studies suggest that adhesion molecules may mediate retinal leukocyte stasis (leukostasis) in diabetes and a reduction in the leukostasis by anti-adhesion antibodies can suppress retinal vascular leakage. In addition, some agents inhibiting leukostasis are reported to improve retinal abnormalities induced by diabetes. Thus, leukostasis in the retinal microcirculation can be a new promising target in the treatment of diabetic retinopathy.
Exercise appears to increase reactive oxygen species, which can result in damage to cells. Exercise results in increased amounts of malondialdehyde in blood and pentane in breath; both serve as indirect indicators of lipid peroxidation. However, not all studies report increases; these equivocal results may be due to the large intersubject variability in response or the nonspecificity of the assays. Some studies have reported that supplementation with vitamins C and E, other antioxidants, or antioxidant mixtures can reduce symptoms or indicators of oxidative stress as a result of exercise. However, these supplements appear to have no beneficial effect on performance. Exercise training seems to reduce the oxidative stress of exercise, such that trained athletes show less evidence of lipid peroxidation for a given bout of exercise and an enhanced defense system in relation to untrained subjects. Whether the body's natural antioxidant defense system is sufficient to counteract the increase in reactive oxygen species with exercise or whether additional exogenous supplements are needed is not known, although trained athletes who received antioxidant supplements show evidence of reduced oxidative stress. Until research fully substantiates that the long-term use of antioxidants is safe and effective, the prudent recommendation for physically active individuals is to ingest a diet rich in antioxidants.
Vision is dependent on proper function of several intraocular structures. Immune responses to eliminate invading pathogens from the eye may threat vision by causing damage to these structures. Therefore, immunological defence of the eye should be carefully balanced between efficacy and maintenance of functional integrity. The eye is equipped with several regulatory mechanisms to prevent certain immune and inflammatory responses and is, therefore, regarded as an immune privileged site. The retinal pigment epithelium (RPE) contributes to the immune privileged status of the eye as part of the blood-eye barrier and by the secretion of immunosuppressive factors inside the eye. RPE cells, however, may also play an important role in the development of immune and inflammatory responses in the posterior part of the eye. During the last decade it has become clear that RPE cells are highly sensitive to a variety of inflammatory cytokines. Under inflammatory conditions, RPE cells produce a myriad of cytokines that may activate the resident ocular cells or attract and activate leukocytes. Cytokine stimulation of RPE cells causes profound effects, including nitric oxide secretion, cell surface expression of MHC class II and adhesion molecules and abrogation of barrier function. This article provides a comprehensive review of the literature concerning RPE cells and cytokines.
In the search for neuroprotective factors in Huntington's disease, we found that insulin growth factor 1 via activation of the serine/threonine kinase Akt/PKB is able to inhibit neuronal death specifically induced by mutant huntingtin containing an expanded polyglutamine stretch. The IGF-1/Akt pathway has a dual effect on huntingtin-induced toxicity, since activation of this pathway also results in a decrease in the formation of intranuclear inclusions of mutant huntingtin. We demonstrate that huntingtin is a substrate of Akt and that phosphorylation of huntingtin by Akt is crucial to mediate the neuroprotective effects of IGF-1. Finally, we show that Akt is altered in Huntington's disease patients. Taken together, these results support a potential role of the Akt pathway in Huntington's disease.
Insulin-like growth factor-1 (IGF-1) is a trophic factor promoting cell survival by activating phosphatidylinositol 3-kinase (PI3K)/Akt kinase pathway. FKHRL1, a member of the Forkhead family of transcription factors possibly involved in cell cycle and apoptosis, is a downstream target of Akt in fibroblasts. However, very little information is available concerning neurons. We report herein that IGF-1 rapidly induced the phosphorylation of endogenous FKHRL1 in hippocampal neurons. The PI3K/Akt kinase pathway mediates this action, as evidenced by the use of different kinase inhibitors, the expression of constitutively active Akt, and in vitro kinase assay. IGF-1 blocked the nuclear translocation of FKHRL1 in hippocampal neurons and promoted survival in parallel to the phosphorylation of Akt and FKHRL1. Similarly, the expression of constitutively active Akt in PC-12 cells increased the phosphorylation of FKHRL1 and promoted survival, whereas the expression of kinase dead Akt attenuated IGF-1-mediated survival of PC-12 cells. Moreover, the overexpression of wild-type FKHRL1 and its nonphosphorylated mutant induced apoptosis in cultured hippocampal neurons. Interestingly, IGF-1 and PI3-kinase inhibitors have no significant effect on the cell cycle inhibitor p27kip1 in hippocampal neurons. This finding suggests that in contrast to fibroblasts, FKHRL1 is unlikely to be involved in cell cycle in neurons. Taken together, these data reveal that endogenous FKHRL1 is a downstream substrate of PI3K/Akt in IGF-1 receptor signaling in hippocampal neurons and suggest that the phosphorylation of this transcription factor may play an important role in the neuronal survival properties of IGF-1.
To investigate the relationship between insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in the vitreous fluid of diabetic patients with proliferative diabetic retinopathy (PDR).
Observational case-control study.
In a prospective study, 37 consecutive diabetic patients with PDR (14 type I and 23 type II diabetes mellitus) in whom a vitrectomy was performed were compared with 21 nondiabetic patients with other conditions requiring vitrectomy (control group). Free IGF-I and VEGF were measured by ELISA.
Vitreal levels of both free IGF-1 and VEGF were higher in diabetic patients with PDR than in control subjects (P <.01, and P <.0001, respectively). After adjusting for total intravitreous protein concentration, VEGF (ng/mg of proteins) remained significantly higher in diabetic patients with PDR than in the control group (P <.0001), whereas free IGF-I (ng/mg of proteins) was lower in diabetic patients than in control subjects (P <.0001). The vitreous concentrations of VEGF were higher in patients with active PDR than in patients with quiescent PDR (P <.005), whereas vitreous free IGF-I was not related to PDR activity. Finally, we did not observe a correlation between the vitreal levels of free IGF-I and VEGF.
We conclude that free IGF-I and VEGF are both increased, but not related, within the vitreous fluid of diabetic patients with PDR. In addition, our results support the current concept that VEGF is directly involved in the pathogenesis of PDR, whereas the precise role of free IGF-I remains to be established.
The three papers in this symposium are based on presentations to an RSM meeting on the Diabetic Eye, held on 9 April 2003. The matter is particularly topical because the National Service Framework for Diabetes calls for a high-quality retinal screening programme. After a review of the various ophthalmic conditions likely to be encountered in diabetic patients (A Negi, S A Vernon) we proceed to the most important, diabetic retinopathy, with a discussion of screening methods (D M Squirrell, J F Talbot) and an account of laser treatments (J G F Dowler). Colour versions of the clinical photographs are available online [www.jrsm.org]. Publication was coordinated by Professor Susan Lightman, of Moorfields Eye Hospital, London, UK.
FoxO1, a member of the FoxO subfamily of forkhead transcription factors, is an important target for insulin and growth factor signaling in the regulation of metabolism, cell cycle and proliferation, and survival in peripheral tissues. However, its role in the central nervous system is mostly unknown. In this study, we examined the effect of neurotrophic factors on nuclear/cytoplasmic shuttling of FoxO1. We showed that insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF) potently induced the nuclear exclusion of FoxO1-green fluorescent protein (GFP) while neurotrophin (NT)-3 and NT-4 were much weaker and brain-derived neurotrophic factor (BDNF) failed to induce FoxO1 translocation in PC12 cells. FoxO1 translocation was inhibited by LY294002, a well-established PI3K/Akt kinase inhibitor. Moreover, FoxO1 was phosphorylated at Thr24 and Ser256 residues by the above neurotrophic factors, with the exception of BDNF. Triple mutant FoxO1, in which three Akt/PKB phosphorylation sites (Thr24, Ser256 and Ser319) were mutated to alanine, resulted in the complete nuclear targeting of the expressed FoxO1-GFP fusion protein in the presence of the above neurotrophic factors in both PC12 cells and cultured hippocampal and cortical neurons. Taken together, these findings demonstrate that neurotrophic factors are able to regulate nuclear/cytoplasmic shuttling of FoxO1 via the PI3K/Akt pathway in neuronal cells.
Diabetic retinopathy can result in apoptotic cell death of retinal neurons, as well as significant visual loss. It is further known that insulin-like growth factor (IGF) levels are reduced in diabetes and that IGF-I can prevent cell death in many cell types. In this study, we tested the hypothesis that systemic treatment with IGF-I could inhibit death of neuroretinal cells in diabetic rats by examining the expression of proapoptotic markers. In diabetic rat retina, the number of TUNEL-immunoreactive cells increased approximately sixfold in the photoreceptor layer (P<.001) and eightfold in the inner nuclear layer (INL; P<.001); phospho-Akt (p-Akt; Thr 308) immunoreactivity increased eightfold in the ganglion cell layer (GCL; P<.001) and threefold in the INL (P<.01). Subcutaneous IGF-I treatment significantly reduced the number of TUNEL (P<.001) and p-Akt immunoreactive retinal cells (P<.05) in diabetic rats approximately to the level of the nondiabetic group. Qualitative results showed that caspase-3 and BAD immunoreactivities were also elevated in diabetes and reduced in IGF-I-treated animals. Elevated TUNEL and p-Akt immunoreactivities were localized to distinct cell layers in the retina of diabetic rats. Early intervention with systemic IGF-I reduced the presence of proapoptotic markers indicative of neuroretinal cell death, despite ongoing hyperglycemia and weight loss. The eye is a special sensory organ, and these data show that cell loss in the nervous system, even in uncontrolled diabetes, can be prevented by IGF-I administration.
Free insulin growth factor-I and vascular endothelial growth factor in the vitreous fluid of patients with proliferative diabetic retinopa-thy IGF-1-induced VEGF and IGFBP-3 secretion correlates with increased HIF-1 alpha expression and activity in retinal pigment epithelial cell line D407
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