Ezh2 emerges as an epigenetic checkpoint regulator during monocyte differentiation limiting cardiac dysfunction post-MI.

Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during alternative immunoregulatory M2-like macrophage polarization; known to impact cardiac repair after Myocardial Infarction (MI). We hypothesized that EZH2, responsible for H3K27 methylation, could act as an epigenetic checkpoint regulator during this process. We demonstrate for the first time an ectopic EZH2, and putative, cytoplasmic inactive localization of the epigenetic enzyme, during monocyte differentiation into M2 macrophages in vitro as well as in immunomodulatory cardiac macrophages in vivo in the post-MI acute inflammatory phase. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, resolves H3K27 methylation of bivalent gene promoters, thus enhancing their expression to promote human monocyte repair functions. In line with this protective effect, GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction in female mice post-MI in vivo. In conclusion, our study reveals that pharmacological epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.

In Utero Alcohol Exposure Impairs Retinal Angiogenesis and the Microvessel-Associated Positioning of Calretinin Interneurons.

In addition to brain disorders, which constitute a devastating consequence of prenatal alcohol exposure (PAE), eye development is also significantly affected. Given that the retina is a readily accessible part of the central nervous system, a better understanding of the impact of ethanol on retinal development might provide ophthalmological landmarks helpful for early diagnosis of fetal alcohol syndrome. This study aimed to provide a fine morphometric and cellular characterization of the development of retinal microvasculature and neurovascular interactions in a mouse model of fetal alcohol spectrum disorder (FASD). The data revealed that PAE impaired superficial vascular plexus development. In particular, progression of the vascular migration front was significantly decreased in PAE retinas, supporting a delay in plexus progression. Moreover, a significant decrease in the vessel density and number of perforating vessels was quantified in PAE mice, supporting less angiogenesis. The present study provides also the first evidence of a close interaction between migrating calretinin-positive interneurons and perforating microvessels in the inner nuclear layer of the developing retina. This neurovascular association was significantly impaired by PAE. Moreover, projections of amacrine cells were abnormally distributed and densified in stratum S1 and S2. In humans, comparison of a five-month-old control infant with a three-month-old alcohol-exposed case revealed a similar mispositioning of calretinin-positive interneurons. This opens new research avenues regarding a neurovascular contribution in the deleterious effects of alcohol in the developing retina and support that ophthalmological examination could become a promising approach for early detection of alcohol-exposed infants presenting with neurovascular brain defects.

Regulation and impact of cardiac lymphangiogenesis in pressure-overload-induced heart failure.

AIMS: Lymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphangiogenesis in non-ischaemic cardiomyopathy following pressure-overload remains to be determined. Here, we investigated cardiac lymphangiogenesis following transversal aortic constriction (TAC) in C57Bl/6 and Balb/c mice, and in end-stage HF patients. METHODS AND RESULTS: Cardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, oedema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice. We found that VEGFR3-signalling was essential to prevent cardiac lymphatic rarefaction after TAC in C57Bl/6 mice. While anti-VEGFR3-induced lymphatic rarefaction did not significantly aggravate myocardial oedema post-TAC, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, whereas inhibition of lymphangiogenesis did not aggravate interstitial fibrosis, it increased perivascular fibrosis and accelerated development of left ventricular (LV) dilation and dysfunction. In clinical HF samples, cardiac lymphatic density tended to increase, although lymphatic sizes decreased, notably in patients with dilated cardiomyopathy. Similarly, comparing C57Bl/6 and Balb/c mice, lymphatic remodelling post-TAC was linked to LV dilation rather than to hypertrophy. The striking lymphangiogenesis in Balb/c was associated with reduced cardiac levels of macrophages, B cells, and perivascular fibrosis at 8 weeks post-TAC, as compared with C57Bl/6 mice that displayed weak lymphangiogenesis. Surprisingly, however, it did not suffice to resolve myocardial oedema, nor prevent HF development. CONCLUSIONS: We demonstrate for the first time that endogenous lymphangiogenesis limits TAC-induced cardiac inflammation and perivascular fibrosis, delaying HF development in C57Bl/6 but not in Balb/c mice. While the functional impact of lymphatic remodelling remains to be determined in HF patients, our findings suggest that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.

Mechanical regulation of early vertebrate embryogenesis.

Embryonic cells grow in environments that provide a plethora of physical cues, including mechanical forces that shape the development of the entire embryo. Despite their prevalence, the role of these forces in embryonic development and their integration with chemical signals have been mostly neglected, and scrutiny in modern molecular embryology tilted, instead, towards the dissection of molecular pathways involved in cell fate determination and patterning. It is now possible to investigate how mechanical signals induce downstream genetic regulatory networks to regulate key developmental processes in the embryo. Here, we review the insights into mechanical control of early vertebrate development, including the role of forces in tissue patterning and embryonic axis formation. We also highlight recent in vitro approaches using individual embryonic stem cells and self-organizing multicellular models of human embryos, which have been instrumental in expanding our understanding of how mechanics tune cell fate and cellular rearrangements during human embryonic development.

Implication of folate deficiency in CYP2U1 loss of function.

Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders. Understanding of their pathogenic mechanisms remains sparse, and therapeutic options are lacking. We characterized a mouse model lacking the Cyp2u1 gene, loss of which is known to be involved in a complex form of these diseases in humans. We showed that this model partially recapitulated the clinical and biochemical phenotypes of patients. Using electron microscopy, lipidomic, and proteomic studies, we identified vitamin B2 as a substrate of the CYP2U1 enzyme, as well as coenzyme Q, neopterin, and IFN-α levels as putative biomarkers in mice and fluids obtained from the largest series of CYP2U1-mutated patients reported so far. We also confirmed brain calcifications as a potential biomarker in patients. Our results suggest that CYP2U1 deficiency disrupts mitochondrial function and impacts proper neurodevelopment, which could be prevented by folate supplementation in our mouse model, followed by a neurodegenerative process altering multiple neuronal and extraneuronal tissues.

Novel Graphene Electrode for Retinal Implants: An in vivo Biocompatibility Study.

Evaluating biocompatibility is a core essential step to introducing a new material as a candidate for brain-machine interfaces. Foreign body reactions often result in glial scars that can impede the performance of the interface. Having a high conductivity and large electrochemical window, graphene is a candidate material for electrical stimulation with retinal prosthesis. In this study, non-functional devices consisting of chemical vapor deposition (CVD) graphene embedded onto polyimide/SU-8 substrates were fabricated for a biocompatibility study. The devices were implanted beneath the retina of blind P23H rats. Implants were monitored by optical coherence tomography (OCT) and eye fundus which indicated a high stability in vivo up to 3 months before histology studies were done. Microglial reconstruction through confocal imaging illustrates that the presence of graphene on polyimide reduced the number of microglial cells in the retina compared to polyimide alone, thereby indicating a high biocompatibility. This study highlights an interesting approach to assess material biocompatibility in a tissue model of central nervous system, the retina, which is easily accessed optically and surgically.

A detailed in-vitro study of Retinal pigment epithelium's growth as seen from the perspective of impedance spectroscopy analysis.

In this paper, we present a method to assess growth and maturation phases of the Retinal Pigment Epithelium (RPE) in-vitro at the cell layer level using impedance spectroscopy measurements on platinum electrodes. We extracted relevant parameters from an electrical circuit model fitted with the measured spectra. Based on microscopic imaging, the growth state of an independent culture developing in the same conditions is used as reference. We show that the confluence point is identified from a graphical analysis of the spectra transition as well as by observing a reconstructed parameter representing the average capacitance of the cell layer. More generally, this work presents a detailed investigation on how cell culture's state relates with either model parameter analysis or with graphical analysis of the measured spectra over a wide frequency band. While applied to the RPE, this work is also suitable for the study of any kind of monolayer epithelial cells growth.

Impedance spectroscopy study of the retinal pigment epithelium: Application to the monitoring of blue light exposure effect on A2E-loaded in-vitro cell cultures.

In age-related macular degeneration, the retinal pigment epithelium can be damaged by light acting on photosensitizers like N-retinylidene-N-retinylethanolamine (A2E). In this paper, the underlying cellular mechanism of lesion at the cell layer scale is analyzed by impedance spectroscopy. Retinal pigment epithelium (RPE) cells are cultured on top of custom-made electrodes capable of taking impedance measurements, with the help of a custom-made electronic setup but without the use of any chemical markers. An incubator is used to house the cells growing on the electrodes. An electrical model circuit is presented and linked to the constituents of the cell layer in which various electrical elements have been defined including a constant phase element (CPE) associated to the interface between the cell layer and the electrolyte. Their values are extracted from the fitted model of the measured impedance spectra. In this paper, we first investigate which parameters of the model can be analyzed independently. In that way, the parameter's evolution is examined with respect to two different targeted changes of the epithelium: 1. degradation of tight junctions between cells by extracellular calcium sequestration with Ethylenediaminetetraacetic acid (EDTA); 2. application of high amplitude short length electric field pulses. Based on the results obtained showing a clear relation between the model and the physiological state of the cell layer, the same procedure is applied to blue light exposure experiment. When A2E-loaded cells are exposed to blue light, the model parameters indicate, as expected, a clear degradation of the cell layer opposed to a relative stability of the not loaded ones.

Behavioural responses to a photovoltaic subretinal prosthesis implanted in non-human primates.

Retinal dystrophies and age-related macular degeneration related to photoreceptor degeneration can cause blindness. In blind patients, although the electrical activation of the residual retinal circuit can provide useful artificial visual perception, the resolutions of current retinal prostheses have been limited either by large electrodes or small numbers of pixels. Here we report the evaluation, in three awake non-human primates, of a previously reported near-infrared-light-sensitive photovoltaic subretinal prosthesis. We show that multipixel stimulation of the prosthesis within radiation safety limits enabled eye tracking in the animals, that they responded to stimulations directed at the implant with repeated saccades and that the implant-induced responses were present two years after device implantation. Our findings pave the way for the clinical evaluation of the prosthesis in patients affected by dry atrophic age-related macular degeneration.

Protuberant Electrode Structures for Subretinal Electrical Stimulation: Modeling, Fabrication and in vivo Evaluation.

Many neural interfaces used for therapeutic applications are based on extracellular electrical stimulation to control cell polarization and thus functional activity. Amongst them, retinal implants have been designed to restore visual perception in blind patients affected by photoreceptor degeneration diseases, such as age-related macular degeneration (AMD) or retinitis pigmentosa (RP). While designing such a neural interface, several aspects must be taken into account, like the stimulation efficiency related to the current distribution within the tissue, the bio-interface optimization to improve resolution and tissue integration, and the material biocompatibility associated with long-term aging. In this study, we investigate the use of original microelectrode geometries for subretinal stimulation. The proposed structures combine the use of 3D wells with protuberant mushroom shaped electrode structures in the bottom, implemented on a flexible substrate that allows the in vivo implantation of the devices. These 3D microelectrode structures were first modeled using finite element analysis. Then, a specific microfabrication process compatible with flexible implants was developed to create the 3D microelectrode structures. These structures were tested in vivo to check the adaptation of the retinal tissue to them. Finally, preliminary in vivo stimulation experiments were performed.

Viscoelasticity of model interphase chromosomes.

We investigated the viscoelastic response of model interphase chromosomes by tracking the three-dimensional motion of hundreds of dispersed Brownian particles of sizes ranging from the thickness of the chromatin fiber up to slightly above the mesh size of the chromatin solution. In agreement with previous computational studies on polymer solutions and melts, we found that the large-time behaviour of the diffusion coefficient and the experienced viscosity of moving particles as functions of particle size deviate from the traditional Stokes-Einstein relation and agree with a recent scaling theory of diffusion of non-sticky particles in polymer solutions. Interestingly, we found that at short times large particles are temporarily "caged" by chromatin spatial constraints, which thus form effective domains whose sizes match remarkably well with recent experimental results for micro-tracers inside interphase nuclei. Finally, by employing a known mathematical relation between the time mean-square displacement of tracked particles and the complex shear modulus of the surrounding solution, we calculated the elastic and viscous moduli of interphase chromosomes.