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.

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.

Inflammation and mitochondria in the pathogenesis of chronic Chagas disease cardiomyopathy.

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, is a neglected disease affecting around 6 million people. About 30% of CD patients develop chronic Chagas disease cardiomyopathy (CCC), an inflammatory cardiomyopathy that occurs decades after the initial infection, while most infected patients (60%) remain asymptomatic in the so-called indeterminate form (IF). Death results from heart failure or arrhythmia in a subset of CCC patients. Myocardial fibrosis, inflammation, and mitochondrial dysfunction are involved in the arrhythmia substrate and triggering events. Survival in CCC is worse than in other cardiomyopathies, which may be linked to a Th1-T cell rich myocarditis with abundant interferon (IFN)-γ and tumor necrosis factor (TNF)-α, selectively lower levels of mitochondrial energy metabolism enzymes in the heart, and reduced levels of high-energy phosphate, indicating poor adenosine triphosphate (ATP) production. IFN-γ and TNF-α signaling, which are constitutively upregulated in CD patients, negatively affect mitochondrial function in cardiomyocytes, recapitulating findings in CCC heart tissue. Genetic studies such as whole-exome sequencing (WES) in nuclear families with multiple CCC/IF cases has disclosed rare heterozygous pathogenic variants in mitochondrial and inflammatory genes segregating in CCC cases. In this minireview, we summarized studies showing how IFN-γ and TNF-α affect cell energy generation, mitochondrial health, and redox homeostasis in cardiomyocytes, in addition to human CD and mitochondria. We hypothesize that cytokine-induced mitochondrial dysfunction in genetically predisposed patients may be the underlying cause of CCC severity and we believe this mechanism may have a bearing on other inflammatory cardiomyopathies.

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.