Melania Olivieri

Aflibercept, bevacizumab and ranibizumab prevent glucose-induced damage in human retinal pericytes in vitro, through a PLA2/COX-2/VEGF-A pathway.

Diabetic retinopathy, a major cause of vision loss, is currently treated with anti-VEGF agents. Here we tested two hypotheses: (i) high glucose damages retinal pericytes, the cell layer surrounding endothelial cells, via VEGF induction, which may be counteracted by anti-VEGFs and (ii) activation of PLA2/COX-2 pathway by high glucose might be upstream and/or downstream of VEGF in perycites, as previously observed in endothelial cells. Human retinal pericytes were treated with high glucose (25mM) for 48h and/or anti-VEGFs (40μg/ml aflibercept, 25μg/ml bevacizumab, 10μg/ml ranibizumab). All anti-VEGFs significantly prevented high glucose-induced cell damage (assessed by LDH release) and improved cell viability (assessed by MTT and Evans blue). High glucose-induced VEGF-A expression, as detected both at mRNA (qPCR) and protein (ELISA) level, while receptor (VEGFR1 and VEGFR2) expression, detected in control condition, was unaffected by treatments. High glucose induced also activation of PLA2/COX-2 pathway, as revealed by increased phosphorylation of cPLA2, COX-2 expression and PGE2 release. Treatment with cPLA2 (50μM AACOCF3) and COX-2 (5μM NS-392) inhibitors prevented both cell damage and VEGF-A induced by high glucose. Finally, challenge with exogenous VEGF-A (10ng/ml) induced VEGF-A expression, while anti-VEGFs reduced VEGF-A expression induced by either high glucose or exogenous VEGF-A. These data indicate that high glucose directly damages pericytes through activation of PLA2/COX-2/VEGF-A pathway. Furthermore, a kind of feed-forward loop between cPLA2/COX-2/PG axis and VEGF appears to operate in this system. Thus, anti-VEGFs afford protection of pericytes from high glucose by inhibiting this loop.

Anti-angiogenic Therapy in Cancer: Downsides and New Pivots for Precision Medicine

Primary solid tumors originate close to pre-existing tissue vasculature, initially growing along such tissue blood vessels, and this phenomenon is important for the metastatic potential which frequently occurs in highly vascularized tissues. Unfortunately, preclinic and clinic anti-angiogenic approaches have not been very successful, and multiple factors have been found to contribute to toxicity and tumor resistance. Moreover, tumors can highlight intrinsic or acquired resistances, or show adaptation to the VEGF-targeted therapies. Furthermore, different mechanisms of vascularization, activation of alternative signaling pathways, and increased tumor aggressiveness make this context even more complex. On the other hand, it has to be considered that the transitional restoration of normal, not fenestrated, microvessels allows the drug to reach the tumor and act with the maximum efficiency. However, these effects are time-limited and different, depending on the various types of cancer, and clearly define a specific “normalization window.” So, new horizons in the therapeutic approaches consist on the treatment of the tumor with pro- (instead of anti-) angiogenic therapies, which could strengthen a network of well-structured blood vessels that facilitate the transport of the drug.

Classical VEGF, Notch and Ang signalling in cancer angiogenesis, alternative approaches and future directions (Review)

Angiogenesis is the formation of new vessels starting from pre-existing vasculature. Tumour environment is characterized by ‘aberrant angiogenesis’, whose main features are tortuous and permeable blood vessels, heterogeneous both in their structure and in efficiency of perfusion and very different from normal vessels. Therapeutic strategies targeting the three pathways chiefly involved in tumour angiogenesis, VEGF, Notch and Ang signalling, have been identified to block the vascular supply to the tumour. However, phenomena of toxicity, development of primary and secondary resistance and hypoxia significantly blunted the effects of anti-angiogenic drugs in several tumour types. Thus, different strategies aimed to overcome these problems are imperative. The focus of the present review was some principal ‘alternative’ approaches to classic antiangiogenic therapies, including the cyclooxygenase-2 (COX-2) blockade, the use of oligonucleotide complementary to the miRNA to compete with the mRNA target (antimiRs) and the inhibition of matrix metalloproteinases (MMPs). The role of blood soluble VEGFA as a predictive biomarker during antiangiogenic therapy in gastric, ovarian and colorectal cancer was also examined.

Molecular Mechanisms Mediating Antiangiogenic Action of the Urokinase Receptor-Derived Peptide UPARANT in Human Retinal Endothelial Cells

Purpose To investigate the molecular mechanisms of the antiangiogenic activity of UPARANT, an antagonist of the urokinase-type plasminogen activator receptor (uPAR), on primary human retinal endothelial cells (HREC) as a model of in vitro angiogenesis. Methods The antiangiogenic activity of UPARANT was evaluated on endothelial cell migration, invasion, and tube formation. Human REC were further analyzed for viability, transendothelial electrical resistance (TEER), and tight junction (TJ) expression at the protein and mRNA levels. Vascular endothelial growth factor-related signaling molecules were also analyzed by Western and northern blots. Results UPARANT inhibited in a dose-dependent fashion HREC motility, invasion, and tube formation stimulated by VEGF-A, in a range of doses (1-100 nM) that had no effect on cell viability and proliferation. UPARANT also prevented the loss of permeability induced by VEGF-A, restoring normal TEER values and TJ protein expression. At the molecular level, UPARANT inhibited VEGFR-2 and STAT3 phosphorylation, thus decreasing VEGF and hypoxia-inducible factor 1-alpha expression, finally resulting in decreased activation of MEK/ERK, JNK, p38, and AKT signaling proteins. Conclusions These findings indicate that UPARANT exerts its antiangiogenic effects through the inhibition of the downstream signaling activated by angiogenic factors such as VEGF-A.

Role of cytosolic and calcium independent phospholipases A2 in insulin secretion impairment of INS-1E cells infected by S. aureus

Cytosolic PLA2 (cPLA2) and Ca2+‐independent PLA2 (iPLA2) play a significant role in insulin β‐cells secretion. Bacterial infections may be responsible of the onset of diabetes. The mechanism by which Staphylococcus aureus infection of INS‐1 cells alters glucose‐induced insulin secretion has been examined. After acute infection, insulin secretion and PLA2 activities significantly increased. Moreover, increased expressions of phospho‐cPLA2, phospho‐PKCα and phospho‐ERK 1/2 were observed. Chronic infection causes a decrease in insulin release and a significant increase of iPLA2 and COX‐2 protein expression. Moreover, insulin secretion in infected cells could be restored using specific siRNAs against iPLA2 isoform and specific COX‐2 inhibitor.

Gabapentin Attenuates Ocular Inflammation: In vitro and In vivo Studies

To investigate the effects of gabapentin, a structural analog of γ-amino butyric acid (GABA), on the inflammatory response of lipopolysaccharide (LPS)-stimulated rabbit corneal cells (SIRC) and on endotoxin-induced uveitis (EIU) in rabbits. We investigated the LPS-induced expression of several inflammatory mediators, such as TNF-α, IL-1β, cPLA2, COX-2, and PGE2 in the SIRC cells with or without gabapentin treatment. Gabapentin treatment significantly (p < 0.05) attenuated cytokines production, cPLA2 activation, COX-2 expression, and PGE2 levels in SIRC. EIU was induced by an intraocular injection of 0.1 μg of LPS in albino rabbit eye. After 7 and 24 h from LPS injection clinical signs of ocular inflammation were examined by slit lamp with or without topical treatment of 0.5% gabapentin. Tears, aqueous, cornea, conjunctiva, and iris-ciliary body were collected and inflammatory biomarkers assessed. Topical treatment with gabapentin significantly (p < 0.05) reduced clinical signs and biomarkers of inflammation compared with the LPS group both at 7 and 24 h. In conclusion, the results generated in the present study suggest that ophthalmic formulation based on gabapentin may be useful in the treatment of inflammatory conditions associated to ocular pain such as uveitis, and that clinical studies to evaluate this possibility may be warranted.

Blood-Brain Barrier in a Haemophilus influenzae Type a In Vitro Infection: Role of Adenosine Receptors A2A and A2B

The blood–brain barrier (BBB) is mainly made up of tightly connected microvascular endothelial cells (BMECs), surrounded by pericytes (BMPCs) which regulate BBB tightness by providing soluble factors that control endothelial proliferation. Haemophilus influenzae type a (Hia) is able to reach the BBB, crossing it, thus causing meningitis. In this study, by using an in vitro model of BBB, performed with human BMECs and human BMPCs in co-culture, we demonstrated that, after Hia infection, the number of hBMPCs decreased whereas the number of hBMECs increased in comparison with non-infected cells. SEM and TEM images showed that Hia was able to enter hBMECs and reduce TEER and VE-cadherin expression. When the cells were infected in presence of SCH58261 and PSB603 but not DPCPX, an increase in TEER values was observed thus demonstrating that A2A and A2B adenosine receptors play a key role in BBB dysfunction. These results were confirmed by the use of adenosine receptor agonists CGS21680, CCPA, and NECA. In infected co-cultures cAMP and VEGF increased and TEER reduction was counter-balanced by VEGF-R1 or VEGF-R2 antibodies. Moreover, the phosphorylated CREB and Rho-A significantly increased in infected hBMECs and hBMPCs and the presence of SCH58261 and PSB603 significantly abrogated the phosphorylation. In conclusion, this study demonstrated that the infection stimulated A2A and A2B adenosine receptors in hBMECs and hBMPCs thus inducing the pericytes to release large amounts of VEGF. The latter could be responsible for both, pericyte detachment and endothelial cell proliferation, thus provoking BBB impairment.

Age-Related Dry Eye Lactoferrin and Lactobionic Acid

Dry eye is the most prominent pathology among those involving the ocular surface: a decrease of the aqueous (less frequent) or the lipid (more frequent) component of the tear film is the cause of the diminished stability of tears that is observed in this pathology. Dry eye shows a clear distribution linked to both sex (being more frequent among women) and age (increasing with aging). Therefore, specific treatments taking into account the etiology of the disease would be desired. The role of lactoferrin and its functional mimetic lactobionic acid are reported here as a possible remedy for age-related dry eye.

Phenotypic characterization of the SIRC (Statens Seruminstitut Rabbit Cornea) cell line reveals a mixed epithelial and fibroblastic nature

The aim of the present study was to investigate, in the Statens Seruminstitut Rabbit Cornea (SIRC) cell line, the presence of epithelial and fibroblastic markers, comparing their levels with those of the human Retinal Pigmented Epithelial (ARPE-19) cell line, and the Human Keratocyte (HK) cell line, respectively. SIRC cells, often described as of epithelial origin, are used as a corneal epithelial barrier model to study the permeability of ophthalmic drugs. However, they show a morphology that is more consistent with a fibroblastic cell phenotype, similar to corneal keratocytes. Our comparative analyses of cell type specific markers demonstrated that SIRC do not express cytokeratins 19 and 16 (typical of ARPE-19) and cytokeratin 9 (typical of HK); they do express cytokeratins 3 and 18 common to all three cell lines, and cytokeratin 12 typical of ARPE-19. Tight junction proteins were absent in HK, and lower in SIRC than in ARPE-19. All cell lines expressed the markers lumican and vimentin, with SIRC expressing intermediate levels between HK and ARPE-19; alpha-SMA was highly expressed in all lines. These markers, considered typical of fibroblasts, can be, however, expressed by epithelial cells during wound healing. These results might suggest that long-term in vitro cultivation of cell lines leads to a derangement of their specific phenotype, most likely due to genetic and epigenetic factors. This could be the reason why SIRC cells came to exhibit a hybrid nature between epithelial and fibroblastic cells.

N-hydroxymethylglycinate with EDTA is an efficient eye drop preservative with very low toxicity: an in vitro comparative study

Abstract Purpose: Preservatives are used in multi-dose ophthalmic topical medications in order to prevent contamination by bacteria and fungi. However, prolonged use of preserved eye drops, as it may happen in dry eye or glaucoma, may damage cells of the ocular surface. Therefore, an important goal is to find preservatives with low toxicity which are mild to host cells, still able to prevent drug contamination so to maintain their sterility and efficacy. Hence, aim of this study has been to compare the relative toxicity on a rabbit corneal cell line of a new preservative, made by the association of N-hydroxy-methyl-glycinate (NIG) with disodium-ethylene diamine tetra-acetate (EDTA), with other known and widely used eye-drops preservatives. Materials and methods: Rabbit corneal cells (SIRC) were tested either in 96-well plates or in suspension culture. Treatments with preservatives (used at known bacteriostatic concentrations) included: benzalkonium chloride (BAK), polyquaternium-1 (PQ-1), sodium perborate (SP: NaBO3 * H2O), and NIG ± EDTA at different concentrations (0.001% and 0.002%), and different treatment times (from 30 minutes to 120 hours). At the end of treatment, cell survival was evaluated by a specific spectrophotometric method through the metabolic conversion of MTT [3-(4,5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide] into formazan crystals. Results: Almost no cell toxicity was evident for NIG and SP at either concentration (0.001% or 0.002%), while a low toxicity was observed for PQ-1 (62% at the highest dose at 120 hours). BAK, as expected, showed the highest toxicity (60–80% at 30 minutes, and over 90% from eight hours onward). EDTA 0.1% alone or in combination with NIG 0.002%, showed no toxicity at 24 hours, and even resulted in cell growth promotion (46% and 38%, respectively), after 48 hours of treatment. Conclusions: These data show that the new preservative NIG/EDTA, at doses known to have effective antimicrobial properties, has a very low toxicity on corneal cells, and so it can be safely used in multi-dose eye drops.