Daniela Maria Rasà

Caffeine Prevents Blood Retinal Barrier Damage in a Model, In Vitro, of Diabetic Macular Edema.

Diabetic macular edema (DME) is the major cause of vision loss in patients affected by diabetic retinopathy. Hyperglycemia and hypoxia represent the key elements in the progression of these pathologies, leading to breakdown of the blood–retinal barrier (BRB). Caffeine, a psychoactive substance largely consumed in the world, is a nonselective antagonist of adenosine receptors (AR) and it possesses a protective effect in various diseases, including eye pathologies. Here, we have investigated the effect of this substance on BRB integrity following exposure to hyperglycemic/hypoxic insult. Retinal pigmented epithelial cells, ARPE‐19, have been grown on semi‐permeable supports mimicking an experimental model, in vitro, of outer BRB. Caffeine treatment has reduced cell monolayer permeability after exposure to high glucose and desferoxamine as shown by TEER and FITC‐dextran permeability assays. This effect is also mediated through the restoration of membranes tight junction expression, ZO‐1. Moreover, we have demonstrated that caffeine is able to prevent outer BRB damage by inhibiting apoptotic cell death induced by hyperglycemic/hypoxic insult since it downregulates the proapoptotic Bax and upregulates the anti‐apoptotic Bcl‐2 genes. Although further studies are needed to better comprise the beneficial effect of caffeine, we can speculate that it might be used as an innovative drug for DME treatment. J. Cell. Biochem. 118: 2371–2379, 2017.

Modulation of IL-1β and VEGF expression in rat diabetic retinopathy after PACAP administration

HIGHLIGHTSPACAP modulates the inflammatory cytokines IL‐1&bgr; release in STZ‐injected rats.PACAP exogenous administration is able to downregulate VEGF, VEGFR1 and VEGFR2 expression in retina of diabetic rats.Hyperglycaemia and hypoxia concur to trigger a signalling cascade responsible of microvasculature dysfunction. ABSTRACT Diabetic retinopathy (DR) is a microvascular complication of diabetes. Hyperglycemic/hypoxic microenvironment concurs to aberrant angiogenesis characterizing the pathology and activates many downstream target genes including inflammatory cytokines and vasoactive peptides, such as interleukin‐1&bgr; (IL‐1&bgr;) and vascular endothelial growth factor (VEGF). It has been largely demonstrated that pituitary adenylate cyclase‐activating peptide (PACAP) plays a protective effect in DR. In the present study, we investigated the role of PACAP to protect retinal tissue through IL‐1&bgr; and VEGF expression. Diabetes was induced in rats by streptozotocin (STZ) injection, and one week later a single intravitreal injection of 100 &mgr;M PACAP was administrated. Analyses of IL‐1&bgr; and VEGF levels were performed three weeks after diabetes induction. The results demonstrated that a single intraocular administration of PACAP significantly reduced the expression of IL‐1&bgr; in diabetic animals. Moreover, it affects VEGF and its receptors (VEGFRs) levels and interferes with their retinal layers distribution as showed by confocal microscopy analysis. In particular, PACAP treatment downregulates VEGF and VEGFRs that are increasingly expressed in STZ‐treated animals as compared to controls. These results indicate that PACAP plays an important role to attenuate the early phase of DR.

Nap Counteracts Hyperglycemia/Hypoxia Induced Retinal Pigment Epithelial Barrier Breakdown Through Modulation Of HIFs and VEGF Expression

Diabetic macular edema (DME) is a common complication leading to a central vision loss in patients with diabetes. In this eye pathology, the hyperglycaemic/hypoxic microenvironment of pigmented epithelium is responsible for outer blood retinal barrier integrity changes. More recently, we have shown that a small peptide derived from the activity‐dependent neuroprotective protein (ADNP), known as NAP, counteracts damages occurring during progression of diabetic retinopathy by modulating HIFs/VEGF pathway. Here, we have investigated for the first time the role of this peptide on outer blood retinal barrier (BRB) integrity exposed to hyperglycaemic/hypoxic insult mimicking a model in vitro of DME. To characterize NAP role on diseases pathogenesis, we have analyzed its effect on HIFs/VEGF system in human retinal pigmented epithelial cells, ARPE‐19, grown in high glucose and low oxygen tension. The results have shown that NAP prevents outer BRB breakdown by reducing HIF1α/HIF2α, VEGF/VEGFRs, and increasing HIF3α expression, moreover it is able to reduce the percentage of apoptotic cells by modulating the expression of two death related genes, BAX and Bcl2. Further investigations are needed to determine the possible use of NAP in DME treatment.

Trophic effect of PACAP on human corneal endothelium

HighlightsPACAP and its related receptors are expressed in human corneal endothelial cells;.PACAP promotes cell viability and repair of human corneal endothelial lesion;.PACAP improves human corneal endothelium functionality. ABSTRACT Corneas posterior surface includes endothelium maintaining stromal hydration and clarity. Due to their limited proliferative capability, the loss of endothelial cells can outcome in permanent opacity. In the last years, different studies have demonstrated the protective effect of pituitary adenylate cyclase‐activating polypeptide (PACAP) in different ocular diseases. However, its role on human corneal endothelial cells (HCECs) has not been investigated, yet. Here, we have developed a culture protocol to differentiate HCECs from donors cornea. PACAP treatment prevented damage induced by growth factors deprivation of cells grown on transwell supports as revealed by TERR measurements. Moreover, this peptide significantly increased tight junction proteins expression by conferring resistance to endothelial barrier. This effect is also related to promotion of cell viability as demonstrated by MTT assay. Furthermore, PACAP stimulated repairing of corneal endothelium lesion as shown by wound healing analysis. In conclusion, our data suggest that this peptide could represent an important trophic factor in maintaining functionality of human corneal endothelium.

PACAP and VIP regulate hypoxia-inducible factors in neuroblastoma cells exposed to hypoxia

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two related peptides acting as neurotransmitters/neuromodulators in central and peripheral nervous system. They are also involved in cancer showing a controversial role. Particulary, they are implicated in neuroblastoma differentiation (NB). This pediatric tumor can evolve to a malignant metastatic disease or spontaneously regress towards a benign form, known as ganglioneuroblastoma/ganglioneuroma. A negative hallmark of neoplasia progression is represented by hypoxic microenvironment. Low oxygen tension induces activation of hypoxia-inducible factors (HIFs) promoting cells proliferation and metastasis formation. Moreover, HIFs trigger vascular endothelial growth factor (VEGF) release favouring high-risk NB phenotype development. In the present work, we have investigated for the first time, if PACAP and VIP interfere with NB differentiation through modulation of hypoxic/angiogenic process. To this end, we analyzed their effect in malignant undifferentiated and all-trans retinoic acid (RA) differentiated SH-SY5Y cells, representing the benign form of this tumor. Our results have suggested tha both peptides, but predominantly VIP, induce NB differentiation into benign form by regulating HIFs, VEGF and VEGFRs expression and distribution. All these data give new insight regarding PACAP/VIP regulatory role in NB progression.

WT1 Alternative Splicing: Role of Its Isoforms in Neuroblastoma

Wilms tumor 1 (WT1), a tumor suppressor gene, was originally identified in the homonymous renal neoplasm but is also involved in other cancers. Its function is still unclear, since it acts both as a pro- and an anti-oncogene. At least 14 WT1 transcriptional variants have been described; yet most investigations have focused on a small number of isoforms. We describe their structural features and review the evidence of their involvement in cancer with emphasis on neuroblastoma. In future, full characterization of all WT1 isoforms is expected to identify new molecular tumor markers and/or therapeutic targets.

Molecular mechanisms involved in the protective effect of pituitary adenylate cyclase-activating polypeptide in an in vitro model of amyotrophic lateral sclerosis: MAUGERI et al.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. Based on transcriptional profiles of motor cortex samples, in a previous work, we were able to classify two subgroups of sporadic ALS (SALS) patients, named SALS1 and SALS2. A further meta-analysis study has revealed sixteen drug targets commonly deregulated in SALS2 and superoxide dismutase 1 (SOD1) G93A mice. The identified candidate drug targets included pituitary adenylate cyclase-activating polypeptide (PACAP), epidermal growth factor receptor (EGFR) and matrix metallopeptidase-2 (MMP-2). By using a motor neuron-like hybrid cell line (NSC-34) expressing human SOD1 G93A as an in vitro model of ALS, here we investigated the functional correlation among these three genes. Our results have shown that PACAP increases cell viability following serum deprivation. This effect is induced through EGFR transactivation mediated by protein kinase A stimulation. Furthermore, EGFR phosphorylation activates mitogen-activated protein kinases/extracellular signal-regulated kinases 1 and 2 survival signaling pathway and increases MMP-2 expression, significantly reduced by serum starvation. These results suggest that a deeper characterization of mechanisms involved in PACAP/EGFR/MMP-2 axis activation in G93A SOD1 mutated neurons may allow identifying new targets for ALS therapy.

Caffeine inhibits angiogenesis in human glioblastoma cells via HIFs modulation

BACKGROUND Caffeine represents the most used psychoactive drug in the world acting through different mechanisms of action and on several molecular targets. It exerts an anti-cancer role in glioblastoma multiforme (GBM). This neoplasia is characterized by extensive hypoxic foci triggering hypoxia-inducible factors (HIFs) expression. Among these factors, HIF-1α performs a crucial role in the induction of vascular endothelium growth factor (VEGF), a key player in angiogenesis and cell migration. METHODS In this work, we have investigated whether caffeine counteracts GBM progression by modulating hypoxic event. Moreover, we analyzed the activation of phosphoinositide three kinase (PI3K)/Akt and mammalian mitogen activated protein kinase/Erk kinase (MAPK/ERK) signaling cascades. RESULTS Our results have indicated that this psychostimulant drug significantly reduced HIF-1α and VEGF expression in GBM cells exposed to hypoxia. This effect is mediated through inhibition of PI3K/Akt and MAPK/ERK signaling pathways both implied in HIFs regulation. CONCLUSION The present data give new insight into antitumor activity of caffeine during GBM progression.

Nicotine promotes blood retinal barrier damage in a model of human diabetic macular edema

More than 1 billion worlds population actively smokes tobacco containing the bioactive component nicotine (NT). The biological role of this molecule is mediated through the activation of nicotinic cholinergic receptors, widely distributed in various human tissues including retinal pigmented epithelium. The long-term assumption of NT contributes to several diseases development such as diabetic retinopathy. The major complication of this pathology is the diabetic macular edema (DME), characterized by macular area thinning and blood-retinal barrier (BRB) breakdown. Retinal hyperglycemic/hypoxic microenvironment represents one of the main factors favoring DME progression by eliciting the hypoxia-inducible factors (HIFs) expression. The latter induce new vessels formation by stimulating cellular secretion of vascular endothelial growth factor (VEGF). The etiology of DME is multifactorial, but little is known about the risk factors linked to cigarette smoking, in particular to nicotines contribution. In the present study, we have investigated the NT role in a model, in vitro, of DME, by evaluating its effect on outer BRB permeability and HIFs/VEGF expression following exposure to hyperglycemic/hypoxic insult. Our results have demonstrated that this compound alters outer BRB integrity exposed to high glucose and low oxygen pressure microenvironment by upregulating HIF-1α/HIF-2α, VEGF expression and ERK1/2 phosphorylation. These data have suggested that NT may play a negative role in active smokers affected by DME.

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.