Hypoxia inducible factors expression in lung adenocarcinoma cells

Lung adenocarcinoma is one of the most deadly malignancies with a low sur- vival rate.A typical characteristic of this tumor is angiogenesis which stimulates its growth. It is generate following hypoxia that induces activation of the hypoxia-inducible factors (HIFs) including HIF-1α, HIF-1β, HIF-2α, HIF-2β and HIF-3α. Previous studies have demonstrated the expression of these factors in lung adenocarcinomas [1-3]. In the present work we have analyzed their temporal expression profile in lung adenocarcinomas cells A549 by comparing it to that of normal bronchial epithelial cell lines BEAS-2B, during hypoxia with deferoxamine (DFX). This stressor induces a significant, time dependent, reduction of viabilityin both cell lines but more evident in BEAS-2B as shown by MTT analysis. Expression profile of HIFs members was assessed by Western blot analysis. During hypoxia HIF-1α expression increased in both cell lines, with a peak after 6h to 48h and then decreased significantly at 72h following treatment with DFX. HIF-1β levels reached a peak after 72h of treatment in both A549 and BEAS-2B cells, whereas HIF-2βsignificantly increases at 6h in A549 and at 72h in BEAS-2B of hypoxia. HIF-3α expression levels were inversely linked to those of HIF-1α in A549 while this correlation was absent in BEAS-2B. These data were also visualized by immunofluoerescence analysis. The present results have confirmed the involvement of HIFs members in lung cancer

Effect of NAP in diabetic retinopathy

Diabetic retinopathy (DR) is a microvascular complication of diabetes leading to vision loss. Hypoxic/hyperglycaemic microenvironment is responsible of outer blood retinal barrier integrity impairment and uncontrolled vascularization typical of this pathology. Activation the hypoxia-inducible factors (HIFs) conduce to aberrant expression of some target genes such as vascular endothelial growth factor (VEGF). Many studies have showed the protective role of a small peptide, known as NAP, in counteract retinal damage induced by different insults (Gozes et al., 2004). In particular, we have previously demonstrated that a single intraocular dose of this peptide is able to protect retina from hyperglycaemic insult (Scuderi et al., 2014). However, the involvement of NAP in the modulation of HIFs and their downstream target genes has not been identified yet. In this work, we have instigated its effect on HIFs /VEGF system both in vitro and in vivo models of DR. Results have demonstrated that NAP treatment prevents outer BRB breakdown comprising human retinal pigmented epithelial cells (ARPE-19) grown in transwell supports and exposed to high glucose (HG) and low oxygen tension by adding desferoxamine mesylate salt (DFX). Peptide administration also reduced HIF1α /HIF2α, VEGF/VEGFRs and increased HIF3α expression in cells cultured in HG/DFX. Moreover, it reduced apoptotic cells rate by modulating BAX and Bcl2 expression, two genes involved in programmed death. Furthermore, NAP intraocular administration in STZ-induced diabetic rat reduced retinal expression of HIF-1α, HIF-2α and VEGF by increasing HIF-3α levels. These data have been also confirmed by immunolocalization analysis detected through confocal microscopy showing the different distribution of these factors in retinal layers following hyperglycaemic insult. Our data suggest that this small peptide may be efficacious in counteract retinal damages during DR

Role of nicotine during diabetic macular edema development

Diabetic macular edema (DME) represents the major cause of visual loss in diabetes patients. It is characterized by retinal thickening in the macular area due to breakdown of the blood-retinal barrier (BRB) [1]. By altering blood vessels supplying retina, hyperglycemia triggers tissue hypoxia. The primary response to latter is mediated by hypoxia-inducible factors (HIFs) which in turn promote vascular endothelial growth factor (VEGF) expression. The most important psychoactive compound in cigarette smoke, nicotine (NT), binds nicotinic cholinergic receptors (nAchRs) which are widely distributed in several human tissues, including retinal pigmented epithelium (RPE) [2]. Until now, little is known about risk factors linked to cigarette smoke inducing DME development. In the present study, we have evaluated NT effect in an in vitro model of outer BRB following exposure to hyperglycemic/hypoxic insult mimicking DME microenvironment. Our results have suggested that NT deeply impacts on outer BRB integrity by increasing its permeability. To investigate the molecular mechanisms involved in negative effect of this compound, we have analyzed HIF/VEGF system in cells exposed to hyperglycemic/hypoxic damage. NT treatment induced upregulation of HIF-1α/HIF-2α, VEGF mediated through activation of MAPK/ERK1/2 pathway. In conclusion, all this data have suggested a unfavorable role of this psychoactive agent in smokers DME affected

Parkin interferes with hypoxia-inducible factors expression in glioblastoma cells

Parkin, also known as PARK2, is one of the largest genes in the human genome encoding for an E3 ubiquitin ligase. Its mutation cause a form of autosomal recessive juvenile-onset of Parkinson’s disease, but recently it has been linked in a wide variety of malignancies, including glioblastoma multiforme (GBM) (1). This is the most common and aggressive primary brain tumor in adults, characterized by hypoxic areas. The insufficient oxygen supply causes expression of hypoxia-inducible factors (HIF) which induce tumor growth and vascularization. In particular, in this condition increases expression of HIF-1α which has been correlated with angiogenesis, glucose metabolism and poor prognosis in glioblastoma. Instead HIF-3α is a negative regulator of HIF-1 α. In the present study we investigated if parkin interferes with such HIFs expression during hypoxic event. Parkin knockdown in glioblastoma cells induces a significant increase of HIF-1α expression in normoxia, whereas during hypoxia the absence of parkin abolishes its expression. HIF-3α expression significantly decreases both in normoxic or hypoxic condition following parkin silencing. These data have also been confirmed by immunofluorescence analysis. These results suggest that parkin is implicated in HIF regulation, therefore its modulation might be considered in GBM progressio

Ameliorative effect of VIP family members on blood retinal barrier breakdown in diabetic macular edema

Diabetic macular edema (DME) is one of the main complications of diabetic retinopathy [1]. This pathology is owed to impairment of the blood-retinal barrier (BRB) [2]. Many factors, such as hypoxia, contribute to barrier dysfunction and progression of the disease. Low oxygen tension is one of the main events involved in the formation of new blood vessels that characterize the typical uncontrolled angiogenesis in proliferative stage of diabetic retinopathy. In the last decades, various studies have focused their attention on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in the pathophysiology of DME. However, the effects of these peptides in maintaining the integrity of the BRB exposed to hypoxia remains to be elucidated. In the present work we have studied, for the first time, the effect of these peptides on outer BRB integrity following hypoxic insult in an experimental model of DME. To this end, we have used the human retinal pigment epithelial cells (ARPE-19) to test the effect of both peptides on cellular permeability, transepithelial electrical resistance, tight junctions expression and hypoxia-induced apoptosis. Results have demonstrated that both PACAP and VIP are able to rescue the integrity of cell monolayer during the hypoxic event, minimizing apoptotic damages induced by low tissue oxygen tension through the activation of phosphoinositide 3 kinase /Akt and mammalian mitogen activated protein kinase/Erk kinase signaling pathways. Furthermore, these peptides modulate the expression of vascular endothelial growth factor which is one of the downstream transcription factor activated during the hypoxic process. In conclusion, we have demonstrated that PACAP and VIP are able to counteract the damage induced by hypoxia on BRB through the modulation of hypoxia inducible factors expression

Correlation between expression profile of Wilms tumor 1 gene isoforms and neuroblastoma grade malignancy

Wilms tumor 1 gene (WT1) is expressed in neuroblastoma (NB) which represents the most aggressive extracranial pediatric tumor. This latter may transform into a more benign form such as ganglioneuroblastoma and ganglioneuroma or progress into a highly aggressive metastatic cancer with a poor survival rate. WT1 acts as tumor suppressor gene in NB by inducing the maturation in a less invasive mass. To date, it has been identified 13 mRNA WT1 variants encoding 13 proteins, however, most of the studies have focused their attention exclusively on isoform of ~49 kDa molecular weight (1, 2). In the present study, we have analyzed, the expression profile of WT1 isoforms, in undifferentiated and all-trans retinoic acid (RA) differentiated NB cells in order to evaluate their involvement in tumor malignancy. Results have shown that different isoforms are expressed both in untreated and RA treated NB cells. Their expression is significantly increased in RA treated cells, suggesting that WT1 isoforms are inversely related to NB malignancy grade. In accord to this hypothesis, WT1 isoforms and nestin expression are inversely related in undifferentiated and RA treated cells. Furthermore, the inhibition of the two signalling pathways specifically involved in differentiation of NB, PI3K/Akt and MAPK/ERK respectively, trigger an overexpression of all WT1 isoforms. In conclusion, these data suggest that overexpression of WT1 isoforms might promote trans-differentiation of NB into a more benign tumor such as ganglioneuroblastoma or ganglioneuroma

Effects of Different Continuous Aerobic Training Protocols in a Heterozygous Mouse Model of Niemann-Pick Type C Disease

The positive effects of physical activity on cognitive functions are widely known. Aerobic training is known to promote the expression of neurotrophins, thus inducing an increase in the development and survival of neurons, as well as enhancing synaptic plasticity. Based on this evidence, in the present study, we analyze the effects of two different types of aerobic training, progressive continuous (PC) and varying continuous (VC), on synaptic and muscular plasticity in heterozygous mice carrying the genetic mutation for Niemann-Pick type C disease. We also analyze the effects on synaptic plasticity by extracellular recordings in vitro in mouse hippocampal slices, while the morphological structure of muscle tissue was studied by transmission electron microscopy. Our results show a modulation of synaptic plasticity that varies according to the type of training protocol used, and only the VC protocol administered twice a week, has a significantly positive effect on long-term potentiation. On the contrary, ultrastructural analysis of muscle tissue shows an improvement in cellular conditions in all trained mice. These results confirm the beneficial effects of exercise on quality of life, supporting the hypothesis that physical activity could represent an alternative therapeutic strategy for patients with Niemann-Pick type C disease

Interleukin-1 family members in the retina of streptozotocin-injected rats

Diabetic retinopathy (DR) is one of the most common complications of diabetes. It has been demonstrated that pro-inflammatory cytokines are increased in diabetic retina, including interleukin-1β (IL-1β) (Joussen et al., 2001), suggesting that this cytokine might play an important role in the pathogenesis of DR (Kowluru and Odenbach, 2004). The principal components of the IL-1 family are two secreted factors, IL-1α and IL-1β, two transmembrane receptors (IL-1RI and IL-1RII), and a natural antagonist receptor of IL-1 function (IL-1Ra). To date the molecular mechanisms mediated by IL-1 family members in DR have not been fully characterized. In the present study, to explore the role of IL-1, we analyzed the expression and distribution of IL-1α and IL-1β and relative receptors in a model in vivo of DR. Diabetes was induced in adult rats by intraperitoneal injection of streptozotocin (60 mg/kg). Protein expression and distribution of IL-1 members and relative receptors were analyzed in retinas of nondiabetic and diabetic rats three weeks after induction of diabetes by Western blot and confocal microscopy analyses. Hyperglycemia induced a significant increased in IL-1β protein expression levels and distribution as compared to nondiabetic animals. Specifically, IL-1β retinal immunoreactivity was found not only in the rod and cone layer (RCL), but also in the outer plexiform layer (OPL), inner plexiform (IPL) and in the ganglion cell layer (GCL) of diabetic rats. IL-1α transcription levels were unchanged in the retinas of both animal groups. Consistent with expression studies, IL-1α localization did not differ between diabetic and nondiabetic rats. IL-1RI, IL-1RII and IL-1Ra expression was significantly increased in the retina of diabetic rats when compared to controls. Accordingly, IL-1RI positiveness was thoroughly increased in all retinal layers of diabetic rats, while no evident changes were apparent for IL-1RII, which was localized in the RCL layer and in outer nuclear layer (ONL) of both diabetic and nondiabetic rats. These finding point to IL-1 family members as key elements in the pro-inflammatory cascade after hyperglycemia-induced retinal damage, and therefore support the implementation of novel therapeutic strategies aimed at reducing IL-1 production for the treatment of DR

Dopamine D3 receptor knockout mice exhibit increased hippocampal cAMP response element binding protein (CREB) following acquisition of passive avoidance memory

The dopamine D3 (D3R) receptor seems to be implicated in synaptic plasticity and memory-related processes as identified by several pharmacological and behavioral approaches (Laszy et al., 2005; Swant 2008). In a previous study we have shown that D3Rs mediate an inhibitory effect on learning, since D3R knockout (D3-/-) mice display enhanced cognitive performance in the single trial step-through passive avoidance task (PA) as compared to WTs (Micale et al., 2010; D’Amico et al., 2013). Formation of new memories is known to require de novo synthesis of proteins related to synaptic function, possibly through the activation of a number of signaling pathways including the mitogen-activated protein kinases (MAPKs), protein kinase B (namely Akt) and the activation of nuclear transcription factors such as the cAMP response element binding protein (CREB). However, no clear indications have yet been provided regarding the specific involvement of D3Rs in the activation of these signaling cascades after acquisition of PA. Therefore, in the present study we assessed whether activity/phosphorylation levels of several MAPKs, Akt and CREB were differentially affected by PA in both wild-type (WT) and D3-/- mice. Animals were divided into four groups: naive, unconditioned stimulus trained (USTA), conditioned stimulus trained (CSTA) and conditioned (CA) animals. Phosphorylation of MAPKs, including extracellular signal-regulated kinase 1/2 (ERK 1/2), c-Jun-N-terminal kinase (JNK) and p38, as well as of Akt and CREB were assessed by immunoblotting and immunohistochemical analyses. Results showed that acquisition of PA induced a significant increase in hippocampal pCREB levels both in WT and D3-/- mice. However, the extent of PA-driven increase in pCREB levels was significantly higher in mice lacking D3Rs. Similarly, hippocampal pERK 1/2 were further augmented in D3-/- mice subjected to PA as compared to trained WTs. JNK and p38 phosphorylation was not affected neither by PA nor by genetic background. Finally, a significantly increased Akt activation was observed only when comparing naïve WT and D3-/- mice, but not in response to PA acquisition. This result suggests that the Akt signaling cascade is influenced by the absence of the receptor, but only under basal conditions. In conclusion, the data here presented supports the notion that D3Rs might modulate CREB phosphorylation after acquisition of PA, probably via activation of the ERK signaling pathway