Effects of aflibercept and bevacizumab on cell viability, cell metabolism and inflammation in hypoxic human Müller cells.

Anti-VEGF (vascular endothelial growth factor) drugs such as aflibercept (AFL) and bevacizumab (BVZ) inhibit pathological neo-angiogenesis and vascular permeability in retinal vascular diseases. As cytokines and growth factors are produced by Müller glial cells under stressful and pathological conditions, we evaluated the in vitro effect of AFL (Eylea®, 0.5 mg/mL) and BVZ (Avastin®, 0.5 mg/mL) on cell viability/metabolism, and cytokine/growth factor production by Müller cells (MIO-M1) under cobalt chloride (CoCl2)-induced hypoxia after 24h, 48h and 72h. Cell viability/metabolism were analyzed by Trypan Blue and MTT assays and cytokine/growth factors in supernatants by Luminex xMAP-based multiplex bead-based immunoassay. Cell viability increased with AFL at 48h and 72h and decreased with BVZ or hypoxia at 24h. BVZ-treated cells showed lower cell viability than AFL at all exposure times. Cell metabolism increased with AFL but decreased with BVZ (72h) and hypoxia (48h and72h). As expected, AFL and BVZ decreased VEGF levels. AFL increased PDGF-BB, IL-6 and TNF-α (24h) and BVZ increased PDGF-BB (72h). Hypoxia reduced IL-1ß, -6, -8, TNF-α and PDGF-BB at 24h, and its suppressive effect was more prominent than AFL (EGF, PDGF-BB, IL-1ß, IL-6, IL-8, and TNF-α) and BVZ (PDGF-BB and IL-6) effects. Hypoxia increased bFGF levels at 48h and 72h, even when combined with anti-VEGFs. However, the stimulatory effect of BVZ predominated over hypoxia for IL-8 and TNF-α (24h), as well as for IL-1ß (72h). Thus, AFL and BVZ exhibit distinct exposure times effects on MIO-M1 cells viability, metabolism, and cytokines/growth factors. Hypoxia and BVZ decreased MIO-M1 cell viability/metabolism, whereas AFL likely induced gliosis. Hypoxia resulted in immunosuppression, and BVZ stimulated inflammation in hypoxic MIO-M1 cells. These findings highlight the complexity of the cellular response as well as the interplay between anti-VEGF treatments and the hypoxic microenvironment.

Inhibition of Rho kinase (ROCK) impairs cytoskeletal contractility in human Müller glial cells without effects on cell viability, migration, and extracellular matrix production.

The epiretinal membrane is a fibrocontractile tissue that forms on the inner surface of the retina, causing visual impairment ranging from mild to severe, and even retinal detachment. Müller glial cells actively participate in the formation of this membrane. Current research is constantly seeking for new therapeutic approaches that aim to prevent or treat cellular dysfunctions involved in the progression of this common fibrosis condition. The Rho GTPases signaling pathway regulates several processes associated with the epiretinal membrane, such as cell proliferation, migration, and contraction. Rho kinase (ROCK), an effector of the RhoA GTPase, is an interesting potential therapeutic target. This study aimed to evaluate the effects of a ROCK inhibitor (Y27632) on human Müller cells viability, growth, cytoskeletal organization, expression of extracellular matrix components, myofibroblast differentiation, migration, and contractility. Müller cells of the MIO-M1 lineage were cultured and treated for different periods with the inhibitor. Viability was evaluated by MTT assay and trypan blue exclusion method, and growth was evaluated by growth curve and BrdU incorporation assay. The actin cytoskeleton was stained with fluorescent phalloidin, intermediate filaments and microtubules were analyzed with immunofluorescence for vimentin and α-tubulin. Gene and protein expression of collagens I and V, laminin and fibronectin were evaluated by rt-PCR and immunofluorescence. Chemotactic and spontaneous cell migration were studied by transwell assay and time-lapse observation of live cells, respectively. Cell contractility was assessed by collagen gel contraction assay. The results showed that ROCK inhibition by Y27632 did not affect cell viability, but decreased cell growth and proliferation after 72 h. There was a change in cell morphology and organization of F-actin, with a reduction in the cell body, disappearance of stress fibers and formation of long, branched cell extensions. Microtubules and vimentin filaments were also affected, possibly because of F-actin alterations. The inhibitor also reduced gene expression and immunoreactivity of smooth muscle α-actin, a marker of myofibroblasts. The expression of extracellular matrix components was not affected by the inhibitor. Chemotactic cell migration showed no significant changes, while cell contractility was substantially reduced. No spontaneous migration of MIO-M1 cells was observed. In conclusion, pharmacological inhibition of ROCK in Müller cells could be a potentially promising approach to treat epiretinal membranes by preventing cell proliferation, contractility and transdifferentiation, without affecting cell viability.

High glucose impairs human periodontal ligament cells migration through lowered microRNAs 221 and 222.

OBJECTIVE: To investigate the effects of miR-221 and miR-222 and high glucose on human periodontal ligament (PL) cells morphology, cytoskeleton, adhesion, and migration. BACKGROUND: Chronic hyperglycemia is common in uncontrolled diabetes mellitus (DM) and plays a central role in long-term DM complications, such as impaired periodontal healing. We have previously shown that high glucose increases apoptosis of human PL cells by inhibiting miR-221 and miR-222 and consequently augmenting their target caspase-3. However, other effects of miR-221/222 downregulation on PL cells are still unknown. METHODS: Cells from young humans' premolar teeth were cultured for 7 days under 5 or 30 mM glucose. Directional and spontaneous migration on fibronectin were studied using transwell and time-lapse assays, respectively. F-actin staining was employed to study cell morphology and the actin cytoskeleton. MiR-221 and miR-222 were inhibited using antagomiRs, and their expressions were evaluated by real-time RT-PCR. RESULTS: High glucose inhibited PL cells early adhesion, spreading, and migration on fibronectin. Cells exposed to high glucose showed reduced polarization, velocity, and directionality. They formed several simultaneous unstable and short-lived protrusions, suggesting impairment of adhesion maturation. MiR-221 and miR-222 inhibition also reduced migration, decreasing cell directionality but not significantly cell velocity. After miR-221 and miR-222 downregulation cells showed morphological resemblance with cells exposed to high glucose. CONCLUSION: High glucose impairs human PL cells migration potentially through a mechanism involving reduction of microRNA-221 and microRNA-222 expression. These effects may contribute to the impairment of periodontal healing, especially after surgery and during guided regeneration therapies.

Blockade of the TGF-ß pathway by galunisertib inhibits the glial-mesenchymal transition in Müller glial cells.

Aging increases the risks for developing fibrocontractile membranes on the retina, which causes significant macular distortion, as in the idiopathic epiretinal membrane (iERM). Retinal Müller glial cells are components of these membranes and may play a key role in the iERM pathogenesis. The transforming growth factor-ß (TGF-ß) induces Müller cell transdifferentiation into myofibroblast, reducing glial cell markers (glutamine synthetase, GS, and glial fibrillary acidic protein, GFAP) and increasing α-smooth muscle actin (α-SMA). Our aim was to investigate the effect of the TGF-ß inhibitor galunisertib (LY2157299) on the glial-mesenchymal transition and contraction of Müller cells. MIO-M1 human Müller cells were treated with TGF-ß1 (10 ng/mL), galunisertib (5, 10 and 20 µM) and TGF-ß1+galunisertib for 24h and 48h. Galunisertib cytotoxicity was analyzed by MTT and trypan blue, and TGF-ß1 blockade by phospho-SMAD3 immunofluorescence. Caspase-3 (cell death indicator), GS, GFAP and α-SMA expression was examined by immunofluorescence, Western blotting, and qPCR analysis. Cell contractility was determined by collagen gel contraction assay with Müller cells incorporated. Galunisertib did not show cytotoxicity at the concentrations evaluated and maintained the Müller cells phenotype, ensuring the GS expression. Galunisertib inhibited the TGF-ß1 pathway by decreasing phospho-SMAD3 immunoreactivity, attenuated the α-SMA expression, and prevented the contraction of Müller cells in collagen gel. Although more studies are needed, in vitro assays suggest that galunisertib may be a potential candidate to attenuate the formation of fibrocontractile membranes and prevent retinal detachment and consequent loss of vision.

Cellular components of the idiopathic epiretinal membrane.

Idiopathic epiretinal membrane (iERM) is a fibrocellular proliferation on the inner surface of the retina, which leads to decreased visual acuity and even central visual loss. As iERM is associated to advanced age and posterior vitreous detachment, a higher prevalence is expected with increasing life expectancy and aging of the global population. Although various cell types of retinal and extra-retinal origin have been described in iERMs (Müller glial cells, astrocytes, hyalocytes, retinal pigment epithelium cells, myofibroblasts, and fibroblasts), myofibroblasts have a central role in collagen production and contractile activity. Thus, myofibroblast differentiation is considered a key event for the iERM formation and progression, and fibroblasts, Müller glial cells, hyalocytes, and retinal pigment epithelium have been identified as myofibroblast precursors. On the other side, the different cell types synthesize growth factors, cytokines, and extracellular matrix, which have a crucial role in ERM pathogenesis. In the present review, the major cellular components and their functions are summarized, and their possible roles in the iERM formation are discussed. By exploring in detail the cellular and molecular aspects of iERM, we seek to contribute for better understanding of this fibrotic disease and the origin of myofibroblasts, which may eventually drive to more targeted therapeutic approaches.

Let-7, Lin28 and Hmga2 Expression in Ciliary Epithelium and Retinal Progenitor Cells.

Purpose: Ciliary epithelium (CE) of adult mammalian eyes contains quiescent retinal progenitor/stem cells that generate neurospheres in vitro and differentiate into retinal neurons. This ability doesn't evolve efficiently probably because of regulatory mechanisms, such as microRNAs (miRNAs) that control pluripotent, progenitor, and differentiation genes. Here we investigate the presence of Let-7 miRNAs and its regulator and target, Lin28 and Hmga2, in CE cells from neurospheres, newborns, and adult tissues. Methods: Newborn and adult rats CE cells were dissected into pigmented and nonpigmented epithelium (PE and NPE). Newborn PE cells were cultured with growth factors to form neurospheres and we analyzed Let-7, Lin28a, and Hmga2 expression. During the neurospheres formation, we added chemically modified single-stranded oligonucleotides designed to bind and inhibit or mimic endogenous mature Let-7b and Let-7c. After seven days in culture, we analyzed neurospheres size, number and expression of Let-7, Lin28, and Hmga2. Results: Let-7 miRNAs were expressed at low rates in newborn CE cells with significant increase in adult tissues, with higher levels on NPE cells, that does not present the stem cells reprogramming ability. The Lin28a and Hmga2 protein and transcripts were more expressed in newborns than adults cells, opposed to Let-7. Neurospheres presented higher Lin28 and Hmga2 expression than newborn and adult, but similar Let-7 than newborns. Let-7b inhibitor upregulated Hmga2 expression, whereas Let-7c mimics upregulated Lin28 and downregulated Hmga2. Conclusions: This study shows the dynamic of Lin28-Let-7-Hmga regulatory axis in CE cells. These components may develop different roles during neurospheres formation and postnatal CE cells.

Age-related increase of let-7 family microRNA in rat retina and vitreous.

Vitreous alterations occur from early stages and continue through the normal aging, with gradual lamellae formation and the appearance of liquefied spaces, which eventually leads to complications, such as retinal tear, retinal detachment, and intravitreal hemorrhage. The aim of the present study was to investigate the expression of let-7 miRNA family in the vitreous and retina in newborn (1-3- day-old), young adult (2-month-old), and aging (12-month-old) rats, as well as their role as regulators of vitreous components. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression. Our results showed detection of all investigated let-7 isoforms (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f and let-7i) in the retina and vitreous. Although most let-7 members were significantly upregulated in the vitreous during development, only let-7b, let-7c, and let-7e followed this same expression pattern in the retina. Let-7b and -7c increased in aging vitreous as well, and were expressed in vitro by Müller glial cells and their extracellular vesicles. Moreover, let-7 targeted hyaluronan synthase 2 (Has2) mRNA, a synthesizing enzyme of hyaluronan. These observations indicate that let-7 function is important during retina and vitreous development, and that isoforms of let-7 increased with aging, potentially modulating hyaluronan content.

Distribution of rods and cones in the red-eared turtle retina (Trachemys scripta elegans).

We have identified the photoreceptors of Trachemys scripta elegans, an intensely studied species that is a model for color vision work. To recognize and count the different photoreceptor types, we labeled them with a combination of morphological and immunohistochemistry markers. The counts for the determination of the density of each photoreceptor type were made in wholemount retinas. The percentages found for each cone type were 29, 23, 21, 12, and 6%, respectively, for L (both types), double, M, S, and ultraviolet cones. The cones were found to be organized horizontally in a visual streak, a linear region with a higher density of photoreceptors that ends temporally in the periphery and more centrally in the nasal side. This region of high density of photoreceptors was not symmetrical along its extension; there was a region with conspicuous central density peaks in the temporal area, suggestive of an area centralis. We also observed a dorsoventral asymmetry in photoreceptor density, with greater density in the ventral region. This asymmetry was observed in cones and rods, but it was more pronounced in the rods. Our results corroborate and extend the findings of previous work in the literature describing the retinal photoreceptors of T. s. elegans and their spatial organization. The higher cone density within the visual streak reflects increased spatial resolution and its existence suggests the possibility of binocular vision. It is remarkable that within this region the entire potential for color vision is also present.

Functional regulation of neuronal nitric oxide synthase expression and activity in the rat retina.

In the nervous system within physiological conditions, nitric oxide (NO) production depends on the activity of nitric oxide synthases (NOSs), and particularly on the expression of the neuronal isoform (nNOS). In the sensory systems, the role of NO is poorly understood. In this study, we identified nNOS-positive cells in the inner nuclear layer (INL) of the rat retina, with distinct characteristics such as somata size, immunolabeling level and location. Employing mathematical cluster analysis, we determined that nNOS amacrine cells are formed by two distinct populations. We next investigated the molecular identity of these cells, which did not show colocalization with calbindin (CB), choline acetyltransferase (ChAT), parvalbumin (PV) or protein kinase C (PKC), and only partial colocalization with calretinin (CR), revealing the accumulation of nNOS in specific amacrine cell populations. To access the functional, circuitry-related roles of these cells, we performed experiments after adaptation to different ambient light conditions. After 24h of dark-adaptation, we detected a subtle, yet statistically significant decrease in nNOS transcript levels, which returned to steady-state levels after 24h of normal light-dark cycle, revealing that nNOS expression is governed by ambient light conditions. Employing electron paramagnetic resonance (EPR), we demonstrated that dark-adaptation decreases NO production in the retina. Furthermore, nNOS accumulation changed in the dark-adapted retinas, with a general reduction in the inner plexiform layer. Finally, computational analysis based on clustering techniques revealed that dark-adaptation differently affected both types of nNOS-positive amacrine cells. Taken together, our data disclosed functional regulation of nNOS expression and activity, disclosing new circuitry-related roles of nNOS-positive cells. More importantly, this study indicated unsuspected roles for NO in the sensory systems, particularly related to adaptation to ambient demands.

Rho GTPases control ciliary epithelium cells proliferation and progenitor profile induction in vivo.

PURPOSE: Rho GTPases play a central role in actin-based cytoskeleton reorganization and regulate multiple signaling pathways that control gene transcription, cell survival, and proliferation. We investigated the effect of Rho GTPases on cell cycle regulation and progenitor genes expression on mouse ciliary epithelium (CE), a potential source of progenitor/stem cells in the adult retina. METHODS: Rho GTPases were activated by intraocular injection of lysophosphatidic acid and inactivated by Clostridium difficile Toxin A (general Rho GTPase inhibitor), NSC23766 (Rac1 activation inhibitor), or Y27632 (Rho-associated protein kinase [ROCK] inhibitor). Thereafter, we assayed for RhoA, RhoB, and Rac1 protein localization in CE cells. Proliferation was examined by the expression levels of cell cycle regulators p27(kip), p16(INK4a), and Ki67 and the effects on progenitors by determining the changes in Pax6 and Chx10 progenitor markers expression. RESULTS: All GTPases investigated were expressed in mouse CE cells. Activation increased the coexpression of Pax6 and Chx10, but had no significant effect on the proliferation of CE cells. In contrast, Rho GTPases inactivation increased cell proliferation and potentiated the proliferative effect of growth factors. Specific inactivation of Rac1 or ROCK increased the levels of Ki67 and decreased the expression of the cell cycle inhibitors p27(kip) and p16(INK4a). CONCLUSIONS: This study reports that Rho GTPase modulation (activation and inactivation) controls the expression of retinal progenitor genes and proliferation, respectively, in the adult ciliary epithelial progenitor/stem cells of rodent eyes. The modulation of these two different mechanisms (proliferation and reprogramming) may provide a potential new approach in retinal repair.

Jararhagin, a snake venom metalloprotease-disintegrin, activates the Rac1 GTPase and stimulates neurite outgrowth in neuroblastoma cells.

It has been shown previously that the snake venom metalloprotease-disintegrin jararhagin stimulates cell migration and cytoskeletal rearrangement, independently of its effects on cellular adhesion but possibly associated with the activation of small GTP-binding proteins from the Rho family [Costa, E.P., Santos, M.F., 2004. Toxicon 44(8), 861-870.] Here we show that jararhagin stimulates spreading, actin dynamics and neurite outgrowth in neuroblastoma cells, and that this effect is accompanied by the translocation of the Rac1 small GTPase to the membrane fraction, suggesting its activation. Stimulation of neurite outgrowth was observed within minutes and was dependent on the proteolytic activity of the toxin. These results suggest that jararhagin may stimulate neuronal differentiation, being a potential tool for neuronal regeneration studies.

Expression of connexins 36, 43, and 45 during postnatal development of the mouse retina.

Gap junction channels formed by connexins (Cx) may play essential roles in some processes that occur during retinal development, such as apoptosis and calcium wave spread. The present study was undertaken to determine the distribution pattern of Cx36, Cx43, and Cx45 by immunofluorescence, as well as their gene expression levels by quantitative PCR during postnatal development of the mouse retina. Our results showed an increased expression of neuronal Cx36 from P1 until P10, when this Cx reached adult levels, and it was mainly distributed in the outer and inner plexiform layers. In turn, Cx43 was almost absent in retinal progenitor cells at P1, it became more prominent in glial cell processes about P10, and did not change until adulthood. Double-labeling studies in situ and in vitro with antivimentin, a Müller cell marker, confirmed that Cx43 was expressed by these cells. In addition, quantitative PCR showed that Cx43 and vimentin shared very similar temporal expression patterns. Finally, in contrast to Cx36 and Cx43, Cx45 mRNA was strongly down-regulated during development. In early postnatal days, Cx45 was seen ubiquitously distributed throughout the retina in cells undergoing proliferation and differentiation, as well in differentiated neurons. In adult retina, this protein had a more restricted distribution both in neurons and glial cells, as confirmed in situ and in vitro. In conclusion, we observed a distinct temporal expression pattern for Cx36, Cx43, and Cx45, which is probably related to particular roles in retinal function and maintenance of homeostasis during development of the mouse retina.

Prolonged dark adaptation changes connexin expression in the mouse retina.

In the retina, ambient light levels influence the cell coupling provided by gap junction (GJ) channels, to compensate the visual function for various lighting conditions. However, the effects of ambient light levels on expression of connexins (Cx), the proteins that form the GJ channels, are poorly understood. In the present study, we first determined whether gene expression of specific Cx (Cx26, Cx31.1, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, and Cx57) was affected by prolonged dark adaptation. Cx mRNA relative levels were determined in mouse retinas dark adapted for 3 hr, 1 day, and 7 days by using quantitative real-time PCR. Transcript levels of some Cx were repressed after 3 hr (Cx57), 1 day (Cx45), or 7 days (Cx36 and Cx43) of dark adaptation; others were increased after 1 day (Cx50) or 7 days (Cx31.1 and Cx37); and two of them (Cx26 and Cx40) were not significantly altered. The second aim was to determine whether prolonged dark adaptation affects protein expression of two important Cx in retina: neuronal Cx36 and glial Cx43. We were able to demonstrate that important changes in protein distribution and expression also took place in retina during long-term dark adaptation. Given their localization, the specific alterations in Cx expression may reflect their distinct response to ambient light levels.

Small GTP-binding protein RhoB is expressed in glial Müller cells in the vertebrate retina.

Among several small Rho GTPases observed in the chick retina, RhoB was transiently expressed during development and mainly present in glial Müller cells in the adult. The aim of this study was to compare the distribution of RhoB in the chick and mouse adult retinas and to study its potential role in the maintenance of cell morphology. The distribution of RhoB was studied in situ and pure Müller cell cultures were submitted to Clostridium difficile toxin A and lysophosphatidic acid (LPA) treatment in order to inactivate and activate Rho proteins, respectively. Cell morphology, F-actin arrangement, RhoB, and vimentin distribution were studied by immunofluorescence and confocal microscopy. The results showed that, in both species, all vimentin-containing cells also expressed RhoB in situ and in vitro. Toxin A promoted cell rounding and detachment due to actin depolymerization, changing the distribution of RhoB only in chick cells. In serum-starved cells LPA stimulated actin polymerization and cell spreading, but only in chick cells was RhoB distribution recruited to expanding cellular processes and newly formed focal adhesions. These data suggest that, although RhoB is expressed by Müller cells in chick and mouse, its role in the maintenance of cellular morphology and regulation may be different. In addition, we show that RhoB may be an interesting Müller cell marker in the adult retina.

Protecting RNA in fixed tissue: an alternative method for LCM users.

RNA degradation is a major drawback in most common fixation protocols in techniques that require both RNA integrity and preserved morphology, such as laser capture microdissection (LCM) followed by RT-PCR. Moreover, RNA isolation kits especially developed for LCM samples are very expensive. Our aim was to determine an easy protocol that ideally must provide an acceptable morphology, allow proper laser capture of selected cells and improve RNA yield and quality. In this study, retinas were dissected, briefly incubated in a RNA preservative and fixed in 2% paraformaldehyde before being cut on a cryostat. LCM was carried out in retinal sections for immediate RNA isolation, by using TRIzol common protocol with minor modifications. Real-time PCR was performed next in order to compare availability of RNA from samples submitted to different protocols. The use of the RNA preservative followed by a fast fixation did not jeopardize tissue morphology, allowing microdissection of selected cells, combined to minor modifications in usual RNA isolation procedures, significantly improved RNA yield and quality. Furthermore, only LCM samples submitted to our protocol provided amplifiable mRNA, as determined by real-time PCR. Taken together, the combination of the described procedures resulted in a reliable alternative for LCM users.