Immune System, Inflammation and Autoantigens in Wet Age-Related Macular Degeneration: Pathological Significance and Therapeutic Importance.

Wet age-related macular degeneration (wAMD) is a chronic inflammation-associated neurodegenerative disease affecting the posterior part of the eye in the aging population. Aging results in the reduced functionality of cells and tissues, including the cells of the retina. Initiators of a chronic inflammatory and pathologic state in wAMD may be a result of the accumulation of inevitable metabolic injuries associated with the maintenance of tissue homeostasis from a young age to over 50. Apart from this, risk factors like smoking, genetic predisposition, and failure to repair the injuries that occur, alongside attempts to rescue the hypoxic outer retina may also contribute to the pathogenesis. Aging of the immune system (immunosenescence) and a compromised outer blood retinal barrier (BRB) result in the exposure of the privileged milieu of the retina to the systemic immune system, further increasing the severity of the disease. When immune-privileged sites like the retina are under pathological stress, certain age- and disease-related conditions may necessitate assistance from cells distant from the resident ones to help restore the functionality of the tissue. As a necessary part of tissue repair, inflammation is a major response to disease and recruits immune cells to the site of damage. We suspect that the specific reparative inflammatory responses are controlled by an autoantigen-T cell-mediated mechanism, a process that may be hindered in wAMD.

CD73 controls ocular adenosine levels and protects retina from light-induced phototoxicity.

ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5'-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39high/CD73low processes and forming local "purinergic junctions" with CD39low/CD73- neuronal cell bodies and CD39high/CD73- retinal blood vessels. The relevance of the CD73-adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells.

Compatibility of intravitreally applied epidermal growth factor and amphiregulin.

INTRODUCTION: To examine the compatibility of intravitreally injected epidermal growth factor (EGF) and amphiregulin as EGF family member. METHODS: Four rabbits (age: 4 months; body weight: 2.5 kg) received three intravitreal injections of EGF (100 ng) uniocularly in monthly intervals and underwent ocular photography, tonometry, biometry, and optical coherence tomography. After sacrificing the rabbits, the globes were histomorphometrically examined. In a second study part, eyes of 22 guinea pigs (age: 2-3 weeks) received two intravitreal administrations of amphiregulin (10 ng) or phosphate buffered solution (PBS) in 10-day interval, or were left untouched. Ten days after the second injection, the guinea pigs were sacrificed, the enucleated eyes underwent histological and immune-histological examinations. RESULTS: The rabbit eyes with EGF injections versus the contralateral untouched eyes did not show significant differences in intraocular pressure (7.5 ± 2.4 mmHg vs. 6.8 ± 2.2 mmHg; P = 0.66), retinal thickness (158 ± 5 µm vs. 158 ± 3 µm; P = 1.0), cell counts in the retinal ganglion cell layer (3.3 ± 1.7 cells/150 µm vs. 3.0 ± 1.4 cells/150 µm; P = 0.83), inner nuclear layer (46.4 ± 23.2 cells/150 µm vs. 39.6 ± 6.4 cells/150 µm; P = 0.61), and outer nuclear layer (215 ± 108 cells/150 µm vs. 202 ± 47 cells/150 µm; P = 0.83), or any apoptotic retinal cells. The guinea pig eyes injected with amphiregulin versus eyes with PBS injections did not differ (P = 0.72) in the degree of microglial activation, and both groups did not differ from untouched eyes in number of apoptotic retinal cells and retinal gliosis. CONCLUSIONS: Intravitreal applications of EGF (100 ng) in rabbits nor intravitreal applications of amphiregulin (10 ng) in guinea pigs led to intraocular specific inflammation or any observed intraocular destructive effect. The findings support the notion of a compatibility of intraocular applied EGF and amphiregulin.

Polyamine flux analysis by determination of heavy isotope incorporation from 13C, 15N-enriched amino acids into polyamines by LC-MS/MS.

The study of polyamine flux, i.e. the circulating flow of polyamines through the interconnected biosynthetic and catabolic pathways, is of considerable interest because of the established links between the polyamine metabolism and many diseases, such as cancer and diabetes. To study polyamine flux in detail, a novel method based on following the label incorporation from the (13)C, (15)N-labeled polyamine precursors, arginine, methionine and ornithine, into polyamines by LC-MS/MS was implemented. This methodology was tested on three distinct cell lines with different spermidine/spermine-N (1)-acetyltransferase (SSAT) expression levels, i.e. non-transgenic, transgenic and knockout. These trials allowed the identification of the critical conditions for the successful polyamine flux measurement, such as the functional time frame of label incorporation, until plateau phase with the selected precursor is reached. The novel LC-MS/MS-based method for polyamine flux overcame the limitations of previous existing methodologies, with baseline separation of the different polyamine species and the exact quantification of the incorporated label. Moreover, the obtained results clearly show that the increased SSAT expression is associated with accelerated polyamine flux.

Continuous oxidative stress due to activation of polyamine catabolism accelerates aging and protects against hepatotoxic insults.

Enhanced polyamine catabolism via polyamine acetylation-oxidation elevates the oxidative stress in an organism due to increased production of reactive oxygen species (ROS). We studied a transgenic mouse line overexpressing the rate limiting enzyme in the polyamine catabolism, spermidine/spermine N (1)-acetyltransferase (SSAT) that is characterized by increased putrescine and decreased spermidine and spermine pools. In order to protect the mice from the chronic oxidative stress produced by the activation of polyamine catabolism, the hepatic expression of the transcription factor p53 was found threefold elevated in the transgenic mice. In addition, the prolonged activation of p53 accelerated the aging of transgenic mice and reduced their lifespan (50%). Aging was associated with decreased antioxidant enzyme activities. In the transgenic mice the activities of catalase and Cu, Zn-superoxide dismutase (SOD) were 42 and 23% reduced respectively, while the expression of CYP450 2E1 was 60% decreased and oxidative stress measured as protein carbonyl content was tenfold elevated. In the transgenic mice, the age-related repression of the different antioxidant enzymes served as a protection against the hepatotoxic effects of carbon tetrachloride and thioacetamide.

Activated polyamine catabolism leads to low cholesterol levels by enhancing bile acid synthesis.

Transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N(1)-acetyltransferase (SSAT) have significantly reduced plasma total cholesterol levels. In our study, we show that low cholesterol levels were attributable to enhanced bile acid synthesis in combination with reduced cholesterol absorption. Hepatic cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme catalyzing the conversion of cholesterol to bile acids, plays an important role in the removal of excess cholesterol from the body. We suggest that by reducing activity of Akt activated polyamine catabolism increased the stability and activity of peroxisome proliferator-activated receptor gamma co-activator 1alpha, the critical activator of CYP7A1. This is supported by our finding that the treatment with SSAT activator, N (1) ,N(11)-diethylnorspermine, reduced significantly the amount of phosphorylated (active) Akt in HepG2 cells. In summary, activated-polyamine catabolism is a novel mechanism to regulate bile acid synthesis. Therefore, polyamine catabolism could be a potential therapeutic target to control hepatic CYP7A1 expression.