Molecular Mechanisms and Therapeutic Implications of Human Pericyte-like Adipose-Derived Mesenchymal Stem Cells in an In Vitro Model of Diabetic Retinopathy.

The blood-retinal barrier (BRB) is strongly compromised in diabetic retinopathy (DR) due to the detachment of pericytes (PCs) from retinal microvessels, resulting in increased permeability and impairment of the BRB. Western blots, immunofluorescence and ELISA were performed on adipose mesenchymal stem cells (ASCs) and pericyte-like (P)-ASCs by co-cultured human retinal endothelial cells (HRECs) under hyperglycemic conditions (HG), as a model of DR. Our results demonstrated that: (a) platelet-derived growth factor receptor (PDGFR) and its activated form were more highly expressed in monocultured P-ASCs than in ASCs, and this expression increased when co-cultured with HRECs under high glucose conditions (HG); (b) the transcription factor Nrf2 was more expressed in the cytoplasmic fraction of ASCs and in the P-ASC nuclear fraction, under normal glucose and, even more, under HG conditions; (c) cytosolic phospholipase A2 activity and prostaglandin E2 release, stimulated by HG, were significantly reduced in P-ASCs co-cultured with HRECs; (d) HO-1 protein content was significantly higher in HG-P-ASCs/HRECs than P-ASCs/HRECs; and (e) VEGF-A levels in media from HG-co-cultures were reduced in P-ASCs/HRECs with respect to ASCs/HRECs. The data obtained highlighted the potential of autologous differentiated ASCs in future clinical applications based on cell therapy to counteract the damage induced by DR.

Protective Effects of Human Pericyte-like Adipose-Derived Mesenchymal Stem Cells on Human Retinal Endothelial Cells in an In Vitro Model of Diabetic Retinopathy: Evidence for Autologous Cell Therapy.

Diabetic retinopathy (DR) is characterized by morphologic and metabolic alterations in endothelial cells (ECs) and pericytes (PCs) of the blood-retinal barrier (BRB). The loss of interendothelial junctions, increased vascular permeability, microaneurysms, and finally, EC detachment are the main features of DR. In this scenario, a pivotal role is played by the extensive loss of PCs. Based on previous results, the aim of this study was to assess possible beneficial effects exerted by adipose mesenchymal stem cells (ASCs) and their pericyte-like differentiated phenotype (P-ASCs) on human retinal endothelial cells (HRECs) in high glucose conditions (25 mM glucose, HG). P-ASCs were more able to preserve BRB integrity than ASCs in terms of (a) increased transendothelial electrical resistance (TEER); (b) increased expression of adherens junction and tight junction proteins (VE-cadherin and ZO-1); (c) reduction in mRNA levels of inflammatory cytokines TNF-α, IL-1ß, and MMP-9; (d) reduction in the angiogenic factor VEGF and in fibrotic TGF-ß1. Moreover, P-ASCs counteracted the HG-induced activation of the pro-inflammatory phospho-ERK1/2/phospho-cPLA2/COX-2 pathway. Finally, crosstalk between HRECs and ASCs or P-ASCs based on the PDGF-B/PDGFR-ß axis at the mRNA level is described herein. Thus, P-ASCs might be considered valuable candidates for therapeutic approaches aimed at countering BRB disruption in DR.

Antioxidant Activity of Cyanidin-3-O-Glucoside and Verbascoside in an in Vitro Model of Diabetic Retinopathy.

BACKGROUND: Reactive oxygen species (ROS) accumulation plays a pivotal role in the onset of cell damage induced by hyperglycemia and represents one of the major factors in the pathogenesis of diabetic retinopathy. In this study, we tested the antioxidants cyanidin-3-O-glucoside (C3G) and verbascoside (Verb) in the protection of retinal endothelium against glucose toxicity "in vitro". METHODS: Increasing amounts (5-50 µM) of C3G, Verb or the combination of both compounds were tested in Human Retinal Endothelial Cells (HREC) grown with normal glucose (5 mM, NG) or high glucose (25 mM, HG). RESULTS: Reduced cell viability and enhanced ROS levels (evaluated by MTT and H2DCFDA assays, respectively) in HG-stimulated HREC were restored by C3G and Verb in a dose-dependent manner, achieving the maximum protection in the presence of both compounds. Moreover, co-treatment with C3G and Verb worked better than each single molecule alone in the prevention of the disruption of blood-retinal-barrier-like properties by HG in a confluent HREC monolayer, as assessed by trans endothelial electrical resistance (TEER) and Na-Fluorescein permeability assays. Accordingly, C3G and Verb together also better counteracted the HG-induced down-regulation of the tight junction membrane proteins Zonula Occludens-1 and VE-Cadherin evaluated by immunocytochemical and Western blot analyses. CONCLUSIONS: In conclusion, our data indicate that C3G and Verb could efficiently protect the retinal endothelium against high glucose damage.

Pericytes of Stria Vascularis Are Targets of Cisplatin-Induced Ototoxicity: New Insights into the Molecular Mechanisms Involved in Blood-Labyrinth Barrier Breakdown.

The stria vascularis (SV) contributes to cochlear homeostasis and consists of three layers, one of which contains the blood-labyrinthic barrier (BLB), with a large number of bovine cochlear pericytes (BCPs). Cisplatin is a chemotherapeutic drug that can damage the SV and cause hearing loss. In this study, cell viability, proliferation rate, cytotoxicity and reactive oxygen species production were evaluated. The protein content of phospho-extracellular signal-regulated kinases (ERK) 1/2, total ERK 1/2, phospho-cytosolic phospholipase A2 (cPLA2), total cPLA2 and cyclooxygenase 2 (COX-2) and the release of prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) from BCPs were analyzed. Finally, the protective effect of platelet-derived growth factor (PDGF-BB) on BCPs treated with cisplatin was investigated. Cisplatin reduced viability and proliferation, activated ERK 1/2, cPLA2 and COX-2 expression and increased PGE2 and VEGF release; these effects were reversed by Dexamethasone. The presence of PDGF-BB during the treatment with cisplatin significantly increased the proliferation rate. No studies on cell regeneration in ear tissue evaluated the effect of the PDGF/Dex combination. The aim of this study was to investigate the effects of cisplatin on cochlear pericytes and propose new otoprotective agents aimed at preventing the reduction of their vitality and thus maintaining the BLB structure.

Melatonin loaded hybrid nanomedicine: DoE approach, optimization and in vitro study on diabetic retinopathy model.

Melatonin (MEL) is a pleiotropic neurohormone of increasing interest as a neuroprotective agent in ocular diseases. Improving the mucoadhesiveness is a proposed strategy to increase the bioavailability of topical formulations. Herein, the design and optimization of MEL-loaded lipid-polymer hybrid nanoparticles (mel-LPHNs) using Design of Experiment (DoE) was performed. LPHNs consisted of PLGA-PEG polymer nanoparticles coated with a cationic lipid-shell. The optimized nanomedicine showed suitable size for ophthalmic administration (189.4 nm; PDI 0.260) with a positive surface charge (+39.8 mV), high encapsulation efficiency (79.8 %), suitable pH and osmolarity values, good mucoadhesive properties and a controlled release profile. Differential Scanning Calorimetry and Fourier-Transform Infrared Spectroscopy confirmed the encapsulation of melatonin in the systems and the interaction between lipids and polymer matrix. Biological evaluation in an in vitro model of diabetic retinopathy demonstrated enhanced neuroprotective and antioxidant activities of mel-LPHNs, compared to melatonin aqueous solution at the same concentration (0.1 and 1 µM). A modified Draize test was performed to assess the ocular tolerability of the formulation showing no signs of irritation. To the best our knowledge, this study reported for the first time the development of mel-LPHNs, a novel and safe hybrid platform suitable for the topical management of retinal diseases.

Vitamin D3 preserves blood retinal barrier integrity in an in vitro model of diabetic retinopathy.

The impairment of the blood retinal barrier (BRB) represents one of the main features of diabetic retinopathy, a secondary microvascular complication of diabetes. Hyperglycemia is a triggering factor of vascular cells damage in diabetic retinopathy. The aim of this study was to assess the effects of vitamin D3 on BRB protection, and to investigate its regulatory role on inflammatory pathways. We challenged human retinal endothelial cells with high glucose (HG) levels. We found that vitamin D3 attenuates cell damage elicited by HG, maintaining cell viability and reducing the expression of inflammatory cytokines such as IL-1ß and ICAM-1. Furthermore, we showed that vitamin D3 preserved the BRB integrity as demonstrated by trans-endothelial electrical resistance, permeability assay, and cell junction morphology and quantification (ZO-1 and VE-cadherin). In conclusion this in vitro study provided new insights on the retinal protective role of vitamin D3, particularly as regard as the early phase of diabetic retinopathy, characterized by BRB breakdown and inflammation.

Histological and immunohistochemical study of gilthead seabream tongue from the early stage of development: TRPV4 potential roles.

BACKGROUND: Taste buds, the morphofunctional units for taste perception, transduce gustatory stimuli using G protein-coupled receptors and a complex arrangement of ion channels, among which TRPV4, a member of the TRP superfamily. Studies on taste buds development on gilthead seabream are unknown, and the TRPV4 expression on fish taste cells studies were conducted only on zebrafish. METHODS: In our study, we have investigated the histological features of the gilthead seabream tongue dorsal surface from the earliest stage of development using Masson trichrome with aniline blue staining. Additionally, the TRPV4 expression pattern was studied by means of immunohistochemical labeling and quantitative RT-PCR. RESULTS: We have recorded for the first time on gilthead seabream lingual dorsal surface the presence of, stage-specific, three types of taste buds: type I, type II and type III in larvae, juveniles and adults respectively. At 40 days post-hatching, taste buds were mature-looking. TRPV4 expression was detected in a subpopulation of taste cells of larvae, juveniles, and adults. Furthermore, TRPV4 was expressed in the basal epithelial cells of the tongue at the larvae and juvenile stage, while this expression pattern was more diffused within all the epithelial cell layers in the adult. CONCLUSION: Our findings presume a taste sensory role of TRPV4 in the three stage-specific taste buds and oral epithelia of gilthead seabream. In addition to its sensory role on the epithelial cell layers, we hypothesize that TRPV4 is implicated in epithelial cells differentiation and membrane protection.

ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells.

BACKGROUND: Adipose-derived stem cells (ASCs) have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability, high proliferation rate, multipotent differentiation ability and low immunogenicity. In this respect, they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease. In ocular diseases involving the retina, various cell types may be affected, such as Müller cells, astrocytes, photoreceptors and retinal pigment epithelium (RPE), which plays a fundamental role in the homeostasis of retinal tissue, by secreting a variety of growth factors that support retinal cells. AIM: To test ASC neural differentiation using conditioned medium (CM) from an RPE cell line (ARPE-19). METHODS: ASCs were isolated from adipose tissue, harvested from the subcutaneous region of healthy donors undergoing liposuction procedures. Four ASC culture conditions were investigated: ASCs cultured in basal Dulbecco's Modified Eagle Medium (DMEM); ASCs cultured in serum-free DMEM; ASCs cultured in serum-free DMEM/F12; and ASCs cultured in a CM from ARPE-19, a spontaneously arising cell line with a normal karyotype derived from a human RPE. Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1, 4 and 8 d of culture. At the same time points, ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers: Nestin, neuronal specific enolase (NSE), protein gene product (PGP) 9.5, and glial fibrillary acidic protein (GFAP). RESULTS: Depending on the culture medium, ASC proliferation rate and viability showed some significant differences. Overall, less dense populations were observed in serum-free cultures, except for ASCs cultured in ARPE-19 serum-free CM. Moreover, a different cell morphology was seen in these cultures after 8 d of treatment, with more elongated cells, often showing cytoplasmic ramifications. Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation. In fact, basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM. Specifically, neural marker overexpression was more marked at 8 d. The most evident increase was observed for NSE and GFAP, a modest increase was observed for nestin, and less relevant changes were observed for PGP9.5. CONCLUSION: The presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina.

Targeting the miRNA-155/TNFSF10 network restrains inflammatory response in the retina in a mouse model of Alzheimer's disease.

Age-related disorders, such as Alzheimer's disease (AD) and age-related macular degeneration (AMD) share common features such as amyloid-ß (Aß) protein accumulation. Retinal deposition of Aß aggregates in AMD patients has suggested a potential link between AMD and AD. In the present study, we analyzed the expression pattern of a focused set of miRNAs, previously found to be involved in both AD and AMD, in the retina of a triple transgenic mouse model of AD (3xTg-AD) at different time-points. Several miRNAs were differentially expressed in the retina of 3xTg-AD mice, compared to the retina of age-matched wild-type (WT) mice. In particular, bioinformatic analysis revealed that miR-155 had a central role in miRNA-gene network stability, regulating several pathways, including apoptotic and inflammatory signaling pathways modulated by TNF-related apoptosis-inducing ligand (TNFSF10). We showed that chronic treatment of 3xTg-AD mice with an anti-TNFSF10 monoclonal antibody was able to inhibit the retinal expression of miR-155, which inversely correlated with the expression of its molecular target SOCS-1. Moreover, the fine-tuned mechanism related to TNFSF10 immunoneutralization was tightly linked to modulation of TNFSF10 itself and its death receptor TNFRSF10B, along with cytokine production by microglia, reactive gliosis, and specific AD-related neuropathological hallmarks (i.e., Aß deposition and Tau phosphorylation) in the retina of 3xTg-AD mice. In conclusion, immunoneutralization of TNFSF10 significantly preserved the retinal tissue in 3xTg-AD mice, suggesting its potential therapeutic application in retinal degenerative disorders.

Haloperidol Metabolite II Valproate Ester (S)-(-)-MRJF22: Preliminary Studies as a Potential Multifunctional Agent Against Uveal Melanoma.

Increased angiogenesis and vascular endothelial growth factor (VEGF) levels contribute to higher metastasis and mortality in uveal melanoma (UM), an aggressive malignancy of the eye in adults. (±)-MRJF22, a prodrug of the sigma (σ) ligand haloperidol metabolite II conjugated with the histone deacetylase (HDAC) inhibitor valproic acid, has previously demonstrated a promising antiangiogenic activity. Herein, the asymmetric synthesis of (R)-(+)-MRJF22 and (S)-(-)-MRJF22 was performed to investigate their contribution to (±)-MRJF22 antiangiogenic effects in human retinal endothelial cells (HREC) and to assess their therapeutic potential in primary human uveal melanoma (UM) 92-1 cell line. While both enantiomers displayed almost identical capabilities to reduce cell viability than the racemic mixture, (S)-(-)-MRJF22 exhibited the highest antimigratory effects in endothelial and tumor cells. Given the fundamental contribution of cell motility to cancer progression, (S)-(-)-MRJF22 may represent a promising candidate for novel antimetastatic therapy in patients with UM.

Potential therapeutic applications of mesenchymal stem cells for the treatment of eye diseases.

Stem cell-based treatments have been extensively explored in the last few decades to develop therapeutic strategies aimed at providing effective alternatives for those human pathologies in which surgical or pharmacological therapies produce limited effects. Among stem cells of different sources, mesenchymal stem cells (MSCs) offer several advantages, such as the absence of ethical concerns, easy harvesting, low immunogenicity and reduced tumorigenesis risks. Other than a multipotent differentiation ability, MSCs can release extracellular vesicles conveying proteins, mRNA and microRNA. Thanks to these properties, new therapeutic approaches have been designed for the treatment of various pathologies, including ocular diseases. In this review, the use of different MSCs and different administration strategies are described for the treatment of diabetic retinopathy, glaucoma, and retinitis pigmentosa. In a large number of investigations, positive results have been obtained by in vitro experiments and by MSC administration in animal models. Most authors agree that beneficial effects are likely related to MSC paracrine activity. Based on these considerations, many clinical trials have already been carried out. Overall, although some adverse effects have been described, promising outcomes are reported. It can be assumed that in the near future, safer and more effective protocols will be developed for more numerous clinical applications to improve the quality of life of patients affected by eye diseases.

Glucose-impaired Corneal Re-epithelialization Is Promoted by a Novel Derivate of Dimethyl Fumarate.

Glucose induces corneal epithelial dysfunctions characterized by delayed wound repair. Nuclear erythroid 2-related factor 2 (Nrf2) mediates cell protection mechanisms even through the Heme oxygenase-1 (HO-1) up-regulation. Here, we synthesized new HO-1 inducers by modifying dimethyl fumarate (DMF) and used docking studies to select VP13/126 as a promising compound with the best binding energy to Kelch-like ECH-associated protein 1 (keap1), which is the the regulator of Nrf2 nuclear translocation. We verified if VP13/126 protects SIRC cells from hyperglycemia compared to DMF. SIRC were cultured in normal (5 mM) or high glucose (25 mM, HG) in presence of DMF (1-25 µM) or VP13/126 (0.1-5 µM) with or without ERK1/2 inhibitor PD98059 (15 µM). VP13/126 was more effective than DMF in the prevention of HG-induced reduction of cell viability and proliferation. Reduction of wound closure induced by HG was similarly counteracted by 1 µM VP13/126 and 10 µM DMF. VP13/126 strongly increased phospho/total ERK1/2 and restored HO-1 protein in HG-treated SIRC; these effects are completely counteracted by PD98059. Moreover, high-content screening analysis showed a higher rate of Nrf2 nuclear translocation induced by VP13/126 than DMF in HG-stimulated SIRC. These data indicate that VP13/126 exerts remarkable pro-survival properties in HG-stimulated SIRC, promoting the Nrf2/HO-1 axis.

Effects of High Glucose Concentration on Pericyte-Like Differentiated Human Adipose-Derived Mesenchymal Stem Cells.

A pericyte-like differentiation of human adipose-derived mesenchymal stem cells (ASCs) was tested in in vitro experiments for possible therapeutic applications in cases of diabetic retinopathy (DR) to replace irreversibly lost pericytes. For this purpose, pericyte-like ASCs were obtained after their growth in a specific pericyte medium. They were then cultured in high glucose conditions to mimic the altered microenvironment of a diabetic eye. Several parameters were monitored, especially those particularly affected by disease progression: cell proliferation, viability and migration ability; reactive oxygen species (ROS) production; inflammation-related cytokines and angiogenic factors. Overall, encouraging results were obtained. In fact, even after glucose addition, ASCs pre-cultured in the pericyte medium (pmASCs) showed high proliferation rate, viability and migration ability. A considerable increase in mRNA expression levels of the anti-inflammatory cytokines transforming growth factor-ß1 (TGF-ß1) and interleukin-10 (IL-10) was observed, associated with reduction in ROS production, and mRNA expression of pro-inflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), and angiogenic factors. Finally, a pmASC-induced better organization of tube-like formation by retinal endothelial cells was observed in three-dimensional co-culture. The pericyte-like ASCs obtained in these experiments represent a valuable tool for the treatment of retinal damages occurring in diabetic patients.

The Anti-Inflammatory Effect of the ß1-Adrenergic Receptor Antagonist Metoprolol on High Glucose Treated Human Microvascular Retinal Endothelial Cells.

Hyperglycemia-induced impairment of the blood-retinal barrier represents the main pathological event in diabetic retinopathy that is elicited by a reduced cellular response to an accumulation of reactive oxygen species (ROS) and increased inflammation. The purpose of the study was to evaluate whether the selective ß1-adrenoreceptor (ß1-AR) antagonist metoprolol could modulate the inflammatory response to hyperglycemic conditions. For this purpose, human retinal endothelial cells (HREC) were treated with normal (5 mM) or high glucose (25 mM, HG) in the presence of metoprolol (10 µM), epinephrine (1 µM), or both compounds. Metoprolol prevented both the HG-induced reduction of cell viability (MTT assays) and the modulation of the angiogenic potential of HREC (tube formation assays) reducing the TNF-α, IL-1ß, and VEGF mRNA levels (qRT-PCR). Moreover, metoprolol prevented the increase in phospho-ERK1/2, phospho-cPLA2, COX2, and protein levels (Western blot) as well as counteracting the translocation of ERK1/2 and cPLA2 (high-content screening). Metoprolol reduced ROS accumulation in HG-stimulated HREC by activating the anti-oxidative cellular response mediated by the Keap1/Nrf2/HO-1 pathway. In conclusion, metoprolol exerted a dual effect on HG-stimulated HREC, decreasing the activation of the pro-inflammatory ERK1/2/cPLA2/COX2 axis, and counteracting ROS accumulation by activating the Keap1/Nrf2/HO-1 pathway.

Pericyte-like differentiation of human adipose-derived mesenchymal stem cells: An in vitro study.

BACKGROUND: Adipose-derived mesenchymal stem cells (ASCs) are characterized by long-term self-renewal and a high proliferation rate. Under adequate conditions, they may differentiate into cells belonging to mesodermal, endodermal or ectodermal lineages. Pericytes support endothelial cells and play an important role in stabilizing the vessel wall at the microcirculation level. The loss of pericytes, as occurs in diabetic retinopathy, results in a breakdown of the blood-retina barrier (BRB) and infiltration of inflammatory cells. In this context, the use of pericyte-like differentiated ASCs may represent a valuable therapeutic strategy for restoring BRB damage. AIM: To test in vitro strategies to obtain pericyte-like differentiation of human ASCs (hASCs). METHODS: Different culture conditions were tested: hASCs cultured in a basal medium supplemented with transforming growth factor ß1; and hASCs cultured in a specific pericyte medium (PM-hASCs). In a further sample, pericyte growth supplement was omitted from the PM. In addition, cultures of human retinal pericytes (hRPCs) were used for comparison. Pericyte-like differentiation of hASCs was tested by immunocytochemical staining and western blotting to evaluate the expression of α-smooth muscle actin (α-SMA) and neural/glial antigen 2 (NG2). Interactions between human retinal endothelial cells (hRECs) and different groups of hASCs were investigated in co-culture experiments. In these cases, the expression of typical junctional proteins such as vascular endothelial-Cadherin, zonula occludens-1 and Occludin were assessed in hRECs. In an in vitro model of the BRB, values of trans-endothelial electrical resistance were measured when hRECs were co-cultured with various groups of pretreated hASCs. The values observed were compared with co-cultures of hRECs and hRPCs as well as with cultures of hRECs alone. Three-dimensional co-cultures of hRECs and hRPCs or pericyte-like hASCs in Matrigel were designed to assess their reciprocal localization. RESULTS: After 3-6 d of culture, α-SMA and NG2 immunocytochemistry showed that the closest pericyte-like phenotype was observed when hASCs were cultured in Pericyte Medium (PM-hASCs). In particular, α-SMA immunoreactivity, already visible at the basal level in pericytes and ASCs, was strongly increased only when transforming growth factor was added to the culture medium. NG2 expression, almost undetectable in most conditions, was substantially increased only in PM-hASCs. Immunocytochemical results were confirmed by western blot analysis. The presence of pericyte growth supplement seems to increase NG2 expression rather than α-SMA, in agreement with its role in maintaining pericytes in the proliferative state. In co-culture experiments, immunoreactivity of vascular endothelial-Cadherin, zonula occludens-1 and Occludin was considerably increased in hRECs when hRPCs or PM-hASCs were also present. Supporting results were found by trans-endothelial electrical resistance measurements, gathered at 3 and 6 d of co-culture. The highest resistance values were obtained when hRECs were co-cultured with hRPCs or PM-hASCs. The pericyte-like phenotype of PM-hASCs was also confirmed in three-dimensional co-cultures in Matrigel, where PM-hASCs and hRPCs similarly localized around the tubular formations made by hRECs. CONCLUSION: PM-hASCs seem able to strengthen the intercellular junctions between hRECs, likely reinforcing the BRB; thus, hASC-based therapeutic approaches may be developed to restore the integrity of retinal microcirculation.

Activation of the VEGF-A/ERK/PLA2 Axis Mediates Early Retinal Endothelial Cell Damage Induced by High Glucose: New Insight from an In Vitro Model of Diabetic Retinopathy.

Early blood retinal barrier (BRB) dysfunction induced by hyperglycemia was related to increased pro-inflammatory activity of phospholipase A2 (PLA2) and the upregulation of vascular endothelial growth factor A (VEGF-A). Here, we tested the role of VEGF-A in high glucose (HG)-induced damage of human retinal endothelial cells (HRECs) mediated by Ca++-dependent (cPLA2) and Ca++-independent (iPLA2) PLA2s. HRECs were treated with normal glucose (5 mM, NG) or high glucose (25 mM, HG) for 48 h with or without the VEGF-trap Aflibercept (Afl, 40 µg/mL), the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3; 15 µM), the iPLA2 inhibitor bromoenol lactone (BEL; 5 µM), or VEGF-A (80 ng/mL). Both Afl and AACOCF3 prevented HG-induced damage (MTT and LDH release), impairment of angiogenic potential (tube-formation), and expression of VEGF-A mRNA. Furthermore, Afl counteracted HG-induced increase of phospho-ERK and phospho-cPLA2 (immunoblot). VEGF-A in HG-medium increased glucose toxicity, through upregulation of phospho-ERK, phospho-cPLA2, and iPLA2 (about 55%, 45%, and 50%, respectively); immunocytochemistry confirmed the activation of these proteins. cPLA2 knockdown by siRNA entirely prevented cell damage induced by HG or by HG plus VEGF-A, while iPLA2 knockdown produced a milder protective effect. These data indicate that VEGF-A mediates the early glucose-induced damage in retinal endothelium through the involvement of ERK1/2/PLA2 axis activation.

Uveal Melanoma Cells Elicit Retinal Pericyte Phenotypical and Biochemical Changes in an in Vitro Model of Coculture.

Vascular pericytes are an important cellular component in the tumor microenvironment, however, their role in supporting cancer invasion is poorly understood. We hypothesized that PDGF-BB could be involved in the transition of human retinal pericytes (HRPC) in cancer-activated fibroblasts (CAF), induced by the 92.1 uveal melanoma (UM) cell line. In our model system, HRPC were conditioned by co-culturing with 92.1UM for 6 days (cHRPC), in the presence or absence of imatinib, to block PDGF receptor-ß (PDGFRß). The effects of the treatments were tested by wound healing assay, proliferation assay, RT-PCR, high-content screening, Western blot analysis, and invasion assay. Results showed profound changes in cHRPC shape, with increased proliferation and motility, reduction of NG2 and increase of TGF-ß1, α-SMA, vimentin, and FSP-1 protein levels, modulation of PDGF isoform mRNA levels, phospho-PDGFRß, and PDGFRß, as well as phospho-STAT3 increases. A reduction of IL-1ß and IFNγ and an increase in TNFα, IL10, and TGF-ß1, CXCL11, CCL18, and VEGF mRNA in cHRPC were found. Imatinib was effective in preventing all the 92.1UM-induced changes. Moreover, cHRPC elicited a significant increase of 92.1UM cell invasion and active MMP9 protein levels. Our data suggest that retinal microvascular pericytes could promote 92.1UM growth through the acquisition of the CAF phenotype.

Altered dopamine D3 receptor gene expression in MAM model of schizophrenia is reversed by peripubertal cannabidiol treatment.

Gestational methylazoxymethanol acetate (MAM) treatment produces offspring with adult phenotype relevant to schizophrenia, including positive- and negative-like symptoms, cognitive deficits, dopaminergic dysfunction, structural and functional abnormalities. Here we show that adult rats prenatally treated with MAM at gestational day 17 display significant increase in dopamine D3 receptor (D3) mRNA expression in prefrontal cortex (PFC), hippocampus and nucleus accumbens, accompanied by increased expression of dopamine D2 receptor (D2) mRNA exclusively in the PFC. Furthermore, a significant change in the blood perfusion at the level of the circle of Willis and hippocampus, paralleled by the enlargement of lateral ventricles, was also detected by magnetic resonance imaging (MRI) techniques. Peripubertal treatment with the non-euphoric phytocannabinoid cannabidiol (30 mg/kg) from postnatal day (PND) 19 to PND 39 was able to reverse in MAM exposed rats: i) the up-regulation of the dopamine D3 receptor mRNA (only partially prevented by haloperidol 0.6 mg/kg/day); and ii) the regional blood flow changes in MAM exposed rats. Molecular modelling predicted that cannabidiol could bind preferentially to dopamine D3 receptor, where it may act as a partial agonist according to conformation of ionic-lock, which is highly conserved in GPCRs. In summary, our results demonstrate that the mRNA expression of both dopamine D2 and D3 receptors is altered in the MAM model; however only the transcript levels of D3 are affected by cannabidiol treatment, likely suggesting that this gene might not only contribute to the schizophrenia symptoms but also represent an unexplored target for the antipsychotic activity of cannabidiol.

Pericytes in Microvessels: From "Mural" Function to Brain and Retina Regeneration.

Pericytes are branched cells located in the wall of capillary blood vessels that are found throughout the body, embedded within the microvascular basement membrane and wrapping endothelial cells, with which they establish a strong physical contact. Pericytes regulate angiogenesis, vessel stabilization, and contribute to the formation of both the blood-brain and blood-retina barriers by Angiopoietin-1/Tie-2, platelet derived growth factor (PDGF) and transforming growth factor (TGF) signaling pathways, regulating pericyte-endothelial cell communication. Human pericytes that have been cultured for a long period give rise to multilineage progenitor cells and exhibit mesenchymal stem cell (MSC) features. We focused our attention on the roles of pericytes in brain and ocular diseases. In particular, pericyte involvement in brain ischemia, brain tumors, diabetic retinopathy, and uveal melanoma is described. Several molecules, such as adenosine and nitric oxide, are responsible for pericyte shrinkage during ischemia-reperfusion. Anti-inflammatory molecules, such as IL-10, TGFß, and MHC-II, which are increased in glioblastoma-activated pericytes, are responsible for tumor growth. As regards the eye, pericytes play a role not only in ocular vessel stabilization, but also as a stem cell niche that contributes to regenerative processes in diabetic retinopathy. Moreover, pericytes participate in melanoma cell extravasation and the genetic ablation of the PDGF receptor reduces the number of pericytes and aberrant tumor microvessel formation with important implications for therapy efficacy. Thanks to their MSC features, pericytes could be considered excellent candidates to promote nervous tissue repair and for regenerative medicine.

Blood-retinal barrier protection against high glucose damage: The role of P2X7 receptor.

Blood retinal barrier (BRB) breakdown is a hallmark of diabetic retinopathy, whose occurrence in early or later phases of the disease has not yet been completely clarified. Recent evidence suggests that hyperglycemia induces activation of the P2X7 receptor (P2X7R) leading to pericyte cell death. We herein investigated the role of P2X7R on retinal endothelial cells viability and expression of tight- and adherens-junctions following high glucose (HG) exposure. We found that HG elicited P2X7R activation and expression and release of the pro-inflammatory cytokine IL-1ß in human retinal endothelial cells (HRECs). Furthermore, HG exposure caused a decrease in HRECs viability and a damage of the BRB. JNJ47965567, a P2X7R antagonist, protected HRECs from HG-induced damage (LDH release) and preserved the BRB, as shown by transendothelial electrical resistance and cell junction morphology (ZO-1, claudin-5 and VE-cadherin). Moreover, JNJ47965567 treatment significantly decreased IL-1ß expression and release, elicited by HG. These data indicate that P2X7R plays an important role to regulate BRB integrity, in particular the block of this receptor was useful to counteract the damage elicited by HG in HRECs, and warranting further clinical evaluation of P2X7R antagonists for the treatment of diabetic macular edema.