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.

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.

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.

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats).

Angiopoietin-4-dependent venous maturation and fluid drainage in the peripheral retina.

The maintenance of fluid homeostasis is necessary for function of the neural retina; however, little is known about the significance of potential fluid management mechanisms. Here, we investigated angiopoietin-4 (Angpt4, also known as Ang3), a poorly characterized ligand for endothelial receptor tyrosine kinase Tie2, in mouse retina model. By using genetic reporter, fate mapping, and in situ hybridization, we found expression in a specific sub-population of astrocytes at the site where venous morphogenesis occurs and that lower oxygen tension, which distinguishes peripheral and venous locations, enhances Angpt4 expression.

Altered glucose-stimulated insulin secretion in a mouse line with activated polyamine catabolism.

Ubiquitous activation of polyamine catabolism has been demonstrated to have protective effects in mice on fat accumulation and insulin sensitivity/glucose tolerance in, both, normal conditions and after a high fat diet. We have analyzed the endocrine pancreas functionality in four months-old male mice overexpressing the rate limiting enzyme in the polyamine catabolism, spermidine/spermine N¹-acetyltransferase (SSAT). The pancreatic SSAT activity was 37-fold elevated in the transgenic mice, which reduced the total pancreatic and islet pools of spermidine (71%) and spermine (69%), and increased putrescine and N¹-acetyl spermidine.

Complex N-acetylation of triethylenetetramine.

Triethylenetetramine (TETA) is an efficient copper chelator that has versatile clinical potential. We have recently shown that spermidine/spermine-N(1)-acetyltransferase (SSAT1), the key polyamine catabolic enzyme, acetylates TETA in vitro. Here, we studied the metabolism of TETA in three different mouse lines: syngenic, SSAT1-overexpressing, and SSAT1-deficient (SSAT1-KO) mice.

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.

The activation of hepatic and muscle polyamine catabolism improves glucose homeostasis.

The mitochondrial biogenesis and energy expenditure regulator, PGC-1α, has been previously reported to be induced in the white adipose tissue (WAT) and liver of mice overexpressing spermidine/spermine N (1)-acetyltransferase (SSAT). The activation of PGC-1α in these mouse lines leads to increased number of mitochondria, improved glucose homeostasis, reduced WAT mass and elevated basal metabolic rate. The constant activation of polyamine catabolism produces a futile cycle that greatly reduces the ATP pools and induces 5'-AMP-activated protein kinase (AMPK), which in turn activates PGC-1α in WAT.

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.

Proteomic analysis of livers from a transgenic mouse line with activated polyamine catabolism.

We have generated a transgenic mouse line that over expresses the rate-controlling enzyme of the polyamine catabolism, spermidine/spermine N (1)-acetyltransferase, under the control of a heavy metal inducible promoter. This line is characterized by a notable increase in SSAT activity in liver, pancreas and kidneys and a moderate increase in the rest of the tissues. SSAT induction results in an enhanced polyamine catabolism manifested as a depletion of spermidine and spermine and an overaccumulation of putrescine in all tissues.

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.

Spermidine is indispensable in differentiation of 3T3-L1 fibroblasts to adipocytes.

Impaired adipogenesis has been shown to predispose to disturbed adipocyte function and development of metabolic abnormalities. Previous studies indicate that polyamines are essential in the adipogenesis in 3T3-L1 fibroblasts. However, the specific roles of individual polyamines during adipogenesis have remained ambiguous as the natural polyamines are readily interconvertible inside the cells.

[Methylated analogues of spermine and spermidine as tools to investigate cellular functions of polyamines and the enzymes of their metabolism].

Biogenic amines spermine and spermidine are essential factors of cellular growth. Polyamine analogues are widely used to investigate and to regulate the enzymes of polyamine metabolism and functions of spermine and spermidine in vitro and in vivo. Recently, it was demonstrated that alpha-methylated derivatives of spermine and spermidine are capable to fulfill key cellular functions of polyamines, moreover in some cases of (R)- and (S)-isomers are actually different.

Role of hypusinated eukaryotic translation initiation factor 5A in polyamine depletion-induced cytostasis.

We have earlier shown that alpha-methylated spermidine and spermine analogues rescue cells from polyamine depletion-induced growth inhibition and maintain pancreatic integrity under severe polyamine deprivation. However, because alpha-methylspermidine can serve as a precursor of hypusine, an integral part of functional eukaryotic translation initiation factor 5A required for cell proliferation, and because alpha, omega-bismethylspermine can be converted to methylspermidine, it is not entirely clear whether the restoration of cell growth is actually attributable to hypusine formed from these polyamine analogues. Here, we have used optically active isomers of methylated spermidine and spermine and show that polyamine depletion-induced acute cytostasis in cultured cells could be reversed by all the isomers of the methylpolyamines irrespective of whether they served or not as precursors of hypusine.

Alpha-methylated polyamines as potential drugs and experimental tools in enzymology.

We describe synthesis of alpha-methylated analogues of the natural polyamines and their use as tools in unraveling polyamine functions. Experiments with alpha-methylated spermidine and spermine revealed that the polyamines are exchangeable in supporting cellular growth. Degradation of the analogues by polyamine oxidase disclosed hidden, aldehyde-guided stereospecificity of the enzyme.

Alpha-methyl polyamines: efficient synthesis and tolerance studies in vivo and in vitro. First evidence for dormant stereospecificity of polyamine oxidase.

Efficient syntheses of metabolically stable alpha-methylspermidine 1, alpha-methylspermine 2, and bis-alpha,alpha'-methylated spermine 3 starting from ethyl 3-aminobutyrate are described. The biological tolerance for these compounds was tested in wild-type mice and transgenic mice carrying the metallothionein promoter-driven spermidine/spermine N(1)-acetyltransferase gene (MT-SSAT). The efficient substitution of natural polyamines by their derivatives was confirmed in vivo with the rats harboring the same MT-SSAT transgene and in vitro with the immortalized fibroblasts derived from these animals.