Grazia Maugeri

Parkin modulates expression of HIF-1α and HIF-3α during hypoxia in gliobastoma-derived cell lines in vitro

Mutation of the Parkin gene causes an autosomal recessive juvenile-onset form of Parkinson’s disease. However, recently, it has been also linked to a wide variety of malignancies, including glioblastoma multiforme (GBM). In this pathology, Parkin exhibits a tumor suppressor role by mitigating the proliferation rate in both in vitro and in vivo models. However, Parkin involvement in the hypoxic process has not as yet been investigated. GBM is the most common and aggressive primary brain tumor in adults and is characterized by hypoxic areas. The low oxygen supply causes the expression of hypoxia-inducible factors (HIFs) leading to an accumulation of pro-angiogenic factors and tumoral invasiveness. We assess the relationship between Parkin and two HIFs expressed during hypoxic conditions, namely HIF-1α and HIF-3α. Our data show that Parkin is downregulated under hypoxia and that it interferes with HIF expression based on cellular oxygen tension. These results suggest a role for the involvement of Parkin in GBM, although further studies will be needed to understand the mechanism by which it modulates HIF-1α and HIF-3α expression.

PACAP and VIP Inhibit HIF-1α-Mediated VEGF Expression in a Model of Diabetic Macular Edema

Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) exert a protective role against retinal injuries, including diabetic macular edema (DME). The macular damage is induced by hyperglycemia, which damages vessels supplying blood to the retina and induces hypoxia. The microenvironmental changes stimulate the expression of hypoxia‐inducible factors (HIFs), which promote the choroidal endothelial cell transmigration across the retinal pigmented epithelium (RPE) into neurosensory retina, where they proliferate into new vessels under stimulation of the vascular endothelial growth factor (VEGF). In the present study, we have investigated whether PACAP and VIP prevent retinal damage by modulating the expression of HIFs, VEGF, and its receptors. In accord to our hypothesis, we have shown that both peptides are able to significantly reduce HIF‐1α and increase HIF‐3α expression in ARPE‐19 cells exposed to hyperglycemic/hypoxic insult. This effect is also related to a reduction of VEGF and its receptors expression. Moreover, both peptides also reduce the activation of p38 mitogen‐activated protein kinase (MAPK), a pro‐apoptotic signaling pathway, which is activated by VEGFR‐1 and 2 receptors. In conclusion, our study has further elucidated the protective role performed by PACAP and VIP, against the harmful combined effect of hyperglycemia/hypoxia characterizing the DME microenvironment. J. Cell. Physiol. 232: 1209–1215, 2017.

PACAP and VIP Inhibit the Invasiveness of Glioblastoma Cells Exposed to Hypoxia through the Regulation of HIFs and EGFR Expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) through the binding of vasoactive intestinal peptide receptors (VIPRs), perform a wide variety of effects in human cancers, including glioblastoma multiforme (GBM). This tumor is characterized by extensive areas of hypoxia, which triggers the expression of hypoxia-inducible factors (HIFs). HIFs not only mediate angiogenesis but also tumor cell migration and invasion. Furthermore, HIFs activation is linked to epidermal growth factor receptor (EGFR) overexpression. Previous studies have shown that VIP interferes with the invasive nature of gliomas by regulating cell migration. However, the role of VIP family members in GBM infiltration under low oxygen tension has not been clarified yet. Therefore, in the present study we have investigated, for the first time, the molecular mechanisms involved in the anti-invasive effect of PACAP or VIP in U87MG glioblastoma cells exposed to hypoxia induced by treatment with desferrioxamine (DFX). The results suggest that either PACAP or VIP exert an anti-infiltrative effect under low oxygen tension by modulating HIFs and EGFR expression, key elements involved in cell migration and angiogenesis. These peptides act through the inhibition of PI3K/Akt and MAPK/ERK signaling pathways, which are known to have a crucial role in HIFs regulation.

Expression profile of parkin isoforms in human gliomas

Mutations of parkin gene are not restricted to familial forms of Parkinsonism but they also occur in a wide variety of malignancies including gliomas. Parkin over-expression reduces glioma cells proliferation and analysis of its expression is predictive for the survival outcome of patients with glioma. To date have been identified 21 parkin alternative splice variants. However, most of the studies have focused their attention exclusively on full-length protein. In the present study, the expression profile of parkin isoforms in different grades of astrocytomas was analyzed for the first time, in order to evaluate their involvement in this malignancy. Furthermore, to investigate their role in cellular processes, their expression in three glioblastoma cell lines was analyzed following treatment with the proteasome inhibitor MG132, or induction of mitophagy with CCCP, or after serum deprivation. Results suggested that H20, H1 and H5 isoforms are always expressed in tumors both in vivo and in vitro models. Therefore, these isoforms might be used as specific biomarkers to develop a prognostic tool for brain tumors.

Expression pattern of parkin isoforms in lung adenocarcinomas

The parkin gene has been shown to be genetically altered in a wide variety of human tumors including lung cancer. Although many parkin splice variants have been identified, to date, most of the studies have only been focused on originally cloned isoforms. In this work, for the first time, the expression profile of parkin isoforms in human lung adenocarcinomas has been analyzed to identify their involvement in lung cancer. Their contribution in some biological conditions, such as proteasomal degradation or mitophagy or cell death, has been analyzed in human lung cells. The expression profile of parkin isoforms has been investigated in paraffin-embedded samples of human lung adenocarcinomas by using Western blot analysis. Their expression has also been evaluated in human lung adenocarcinoma and in human normal bronchial epithelial cell lines following treatment with a proteasome inhibitor or mitochondrial depolarizing agent, or in serum starvation. Parkin proteins were detected on blot by using two antibodies, AbI and AbII, which recognize different domains of originally cloned parkin. Furthermore, parkin immunolocalization has been visualized in both cell lines by using immunofluorescence analysis. Results have shown that H1 and/or H5, H14, H4 and/or H8 and/or H17 and H3 and/or H12 isoforms are expressed in human lung adenocarcinomas. Some of them are also present in A549 cell line, whereas they are absent or faintly expressed in BEAS-2B cells. Furthermore, their expression changed after treatment. Human lung adenocarcinomas express different parkin isoforms, which might represent markers of malignancy and could be linked to specific biological functions.

Nap Interferes with Hypoxia-Inducible Factors and VEGF Expression in Retina of Diabetic Rats

The retinal microvascular damage is a complication of diabetic retinopathy (DR). Hyperglycemia and hypoxia are responsible of aberrant vessel’s proliferation. The cellular response to hypoxia is mediated through activation of hypoxia-inducible factors (HIFs). Among these, HIF-1α modulates expression of its target gene, VEGF, whose upregulation controls the angiogenic event during DR development. In a previous study, we have demonstrated that a small peptide, NAP, is able to protect retina from hyperglycemic insult. Here, we have demonstrated that its intraocular administration in a rat model of diabetic retinopathy has reduced expression of HIF-1α, HIF-2α, and VEGF by increasing HIF-3α levels. These data have been also confirmed by immunolocalization study by confocal microscopy. Although these evidences need to be further deepened to understand the molecular mechanism involved in the protective NAP action, the present data suggest that this small peptide may be effective to prevent the development of this ocular pathology.

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.

HYDROTESTOLACTONE LOWERS SERUM OESTRADIOL AND PRL LEVELS IN NORMAL MEN: EVIDENCE OF A ROLE OF OESTRADIOL IN PRL SECRETION*

The effect on serum PRL levels of lowering serum oestradiol (E2) concentration by short‐term administration of an aromatase activity inhibitor, hydrotestolactone (HT), was studied in six healthy male subjects. After HT administration serum E2 levels decreased from 68 ± 5±8 to 26 ± 2±5 pmol/1 (mean ± SE, P < 0±05). These E2 changes were accompanied by a significant decrease in mean 2‐h PRL levels from 11±2 ± 2±1 to 6±5 ± 1±6 ng/ml mean ± SE, P < 0±05). The evaluation of individual percentage change from basal concentrations showed a varying decrease in all subjects. These findings suggest that under physiological conditions E2 may be one of the factors which control blood PRL concentrations in men.

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.