The lack of effective therapies to limit neurovascular injury in ischemic retinopathy is a major clinical problem. This study aimed to examine the role of ureohydrolase enzyme, arginase 1 (A1), in retinal ischemia-reperfusion (IR) injury. A1 competes with nitric oxide synthase (NOS) for their common substrate l-arginine. A1-mediated l-arginine depletion reduces nitric oxide (NO) formation by NOS leading to vascular dysfunction when endothelial NOS is involved but prevents inflammatory injury when inducible NOS is involved. Studies were performed using wild-type (WT) mice, global A1+/− knockout (KO), endothelial-specific A1 KO, and myeloid-specific A1 KO mice subjected to retinal IR injury. Global as well as myeloid-specific A1 KO mice showed worsened IR-induced neuronal loss and retinal thinning. Deletion of A1 in endothelial cells had no effect, while treatment with PEGylated (PEG) A1 improved neuronal survival in WT mice. In addition, A1+/− KO mice showed worsened vascular injury manifested by increased acellular capillaries. Western blotting analysis of retinal tissue showed increased inflammatory and necroptotic markers with A1 deletion. In vitro experiments showed that macrophages lacking A1 exhibit increased inflammatory response upon LPS stimulation. PEG-A1 treatment dampened this inflammatory response and decreased the LPS-induced metabolic reprogramming. Moreover, intravitreal injection of A1 KO macrophages or systemic macrophage depletion with clodronate liposomes increased neuronal loss after IR injury. These results demonstrate that A1 reduces IR injury-induced retinal neurovascular degeneration via dampening macrophage inflammatory responses. Increasing A1 offers a novel strategy for limiting neurovascular injury and promoting macrophage-mediated repair.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disorder of cardiomyocyte intercalated disk proteins causing sudden death. Heterozygous mutations of the desmosomal protein plakophilin-2 (PKP-2) are the commonest genetic cause of ARVC. Abnormal gap junction connexin43 expression has been reported in autosomal dominant forms of ARVC (Naxos and Carvajal disease) caused by homozygous mutations of desmosomal plakoglobin and desmoplakin. In tissue culture, suppression of PKP-2 results in decreased expression of connexin43. We sought to characterize the expression and localization of connexin43 in patients with ARVC secondary to heterozygous PKP-2 mutations. Complete PKP-2 gene sequencing of 27 ARVC patients was utilized to identify mutant genotypes. Endomyocardial biopsies of identified carriers were then assessed by immunofluorescence to visualize intercalated disk proteins. N-cadherin was targeted to highlight intercalated disks, followed by counterstaining for PKP-2 or connexin43 using confocal double immunofluorescence microscopy. Immunofluorescence was quantified using an Adobe® Photoshop protocol, and colocalization coefficients were determined. PKP-2 siRNA experiments were performed in mouse cardiomyocyte (HL1) cell culture with Western blot analysis to assess connexin43 expression following PKP-2 suppression. Missense and frameshift mutations of the PKP-2 gene were found in four patients with biopsy material available for analysis. Immunofluorescent studies showed PKP-2 localization to the intercalated disk despite mutations, but associated with decreased connexin43 expression and abnormal colocalization. PKP-2 siRNA in HL1 culture confirmed decreased connexin43 expression. Reduced connexin43 expression and localization to the intercalated disk occurs in heterozygous human PKP-2 mutations, potentially explaining the delayed conduction and propensity to develop arrhythmias seen in this disease.

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Retinal degenerative diseases are major causes of untreatable blindness worldwide and efficacious treatments for these diseases are sorely needed. A novel target for treatment of retinal disease is the transmembrane protein Sigma 1 Receptor (Sig1R). This enigmatic protein is an evolutionary isolate with no known homology to any other protein. Sig1R was originally thought to be an opioid receptor. That notion has been dispelled and more recent pharmacological and molecular studies suggest that it is a pluripotent modulator with a number of biological functions, many of which are relevant to retinal disease. This review provides an overview of the discovery of Sig1R and early pharmacologic studies that led to the cloning of the Sig1R gene and eventual elucidation of its crystal structure. Studies of Sig1R in the eye were not reported until the late 1990s, but since that time there has been increasing interest in the potential role of Sig1R as a target for retinal disease. Studies have focused on elucidating the mechanism(s) of Sig1R function in retina including calcium regulation, modulation of oxidative stress, ion channel regulation and molecular chaperone activity. Mechanistic studies have been performed in isolated retinal cells, such as Müller glial cells, microglial cells, optic nerve head astrocytes and retinal ganglion cells as well as in the intact retina. Several compelling studies have provided evidence of powerful in vivo neuroprotective effects against ganglion cell loss as well as photoreceptor cell loss. Also described are studies that have examined retinal structure/function in various models of retinal disease in which Sig1R is absent and reveal that these phenotypes are accelerated compared to retinas of animals that express Sig1R. The collective evidence from analysis of studies over the past 20 years is that Sig1R plays a key role in modulating retinal cellular stress and that it holds great promise as a target in retinal neurodegenerative disease.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a myocardial disease characterized by fibro-fatty replacement of myocardium in the right ventricular free wall and frequently results in life-threatening ventricular arrhythmias and sudden cardiac death. A heterozygous missense mutation in the transmembrane protein 43 (TMEM43) gene, p.S358L, has been genetically identified to cause autosomal dominant ARVC type 5 in a founder population from the island of Newfoundland, Canada. Little is known about the function of the TMEM43 protein or how it leads to the pathogenesis of ARVC. We sought to determine the distribution of TMEM43 and the effect of the p.S358L mutation on the expression and distribution of various intercalated (IC) disc proteins as well as functional effects on IC disc gap junction dye transfer and conduction velocity in cell culture. Through Western blot analysis, transmission electron microscopy (TEM), immunofluorescence (IF), and electrophysiological analysis, our results showed that the stable expression of p.S358L mutation in the HL-1 cardiac cell line resulted in decreased Zonula Occludens (ZO-1) expression and the loss of ZO-1 localization to cell-cell junctions. Junctional Plakoglobin (JUP) and α-catenin proteins were redistributed to the cytoplasm with decreased localization to cell-cell junctions. Connexin-43 (Cx43) phosphorylation was altered, and there was reduced gap junction dye transfer and conduction velocity in mutant TMEM43-transfected cells. These observations suggest that expression of the p.S358L mutant of TMEM43 found in ARVC type 5 may affect localization of proteins involved in conduction, alter gap junction function and reduce conduction velocity in cardiac tissue.

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This study evaluated the effects of elevated homocysteine (Hcy) on the oxidative stress response in retinal Müller glial cells. Elevated Hcy has been implicated in retinal diseases including glaucoma and optic neuropathy, which are characterized by retinal ganglion cell (RGC) loss. To understand the mechanisms of Hcy-induced RGC loss, in vitro and in vivo models have been utilized. In vitro isolated RGCs are quite sensitive to elevated Hcy levels, while in vivo murine models of hyperhomocysteinemia (HHcy) demonstrate a more modest RGC loss (∼20%) over a period of many months. This differential response to Hcy between isolated cells and the intact retina suggests that the retinal milieu invokes mechanisms that buffer excess Hcy. Oxidative stress has been implicated as a mechanism of Hcy-induced neuron loss and NRF2 is a transcription factor that plays a major role in regulating cytoprotective responses to oxidative stress. In the present study we investigated whether HHcy upregulates NRF2-mediated stress responses in Müller cells, the chief retinal glial cell responsible for providing trophic support to retinal neurons. Primary Müller cells were exposed to L-Hcy-thiolactone [50μM-10mM] and assessed for viability, reactive oxygen species (ROS), and glutathione (GSH) levels. Gene/protein levels of Nrf2 and levels of NRF2-regulated antioxidants (NQO1, CAT, SOD2, HMOX1, GPX1) were assessed in Hcy-exposed Müller cells. Unlike isolated RGCs, isolated Müller cells are viable over a wide range of Hcy concentrations [50 μM - 1 mM]. Moreover, when exposed to elevated Hcy, Müller cells demonstrate decreased oxidative stress and decreased ROS levels. GSH levels increased by ∼20% within 24 h exposure to Hcy. Molecular analyses revealed 2-fold increase in Nrf2 expression. Expression of antioxidant genes Nqo1, Cat, Sod2, Hmox1, Gpx1 increased significantly. The consequences of Hcy exposure were evaluated also in Müller cells harvested from Nrf2 mice. In contrast to WT Müller cells, in which oxidative stress decreased upon exposure to Hcy, the Nrf2 Müller cells showed a significant increase in oxidative stress. Our data suggest that at least during early stages of Hhcy, a cytoprotective response may be in place, mediated in part by NRF2 in Müller cells.

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