Management of polyamine pools and the regulation of ornithine decarboxylase

The management of polyamine synthesis and polyamine pools differs fundamentally from that of most other small molecular-weight endproducts. The polyamines are vital to growth and important cellular functions, but they are toxic in excess. I argue here that their multivalent cationic character, leading to binding to cell constituents, precludes fluent feedback inhibition of synthesis. This has led to the development of elaborate alternative regulatory mechanisms controlling ornithine decarboxylase, the key initial enzyme of the pathway. Poorly regulated polyamine synthesis and the toxicity of polyamines impose upon cells a need to control uptake and to dispose of excess polyamines. Recent data on polyamine transport suggest unorthodox mechanisms of accomplishing these functions.     

Improvement of plant abiotic stress tolerance through modulation of the polyamine pathway FA简

Polyamines （mainly putrescine （Put）, spermidine （Spd）, and spermine （Spin）） have been widely found in a range of physiological processes and in almost all diverse environ- mental stresses. In various plant species, abiotic stresses modulated the accumulation of polyamines and related gene expression. Studies using loss-of-function mutants and transgenic overexpression plants modulating polyamine metabolic pathways confirmed protective roles of polyamines during plant abiotic stress responses, and indicated the possibility to improve plant tolerance through genetic manipulation of the polyamine pathway. Additionally, puta- tive mechanisms of polyamines involved in plant abiotic stress tolerance were thoroughly discussed and crosstalks among polyamine, abscisic acid, and nitric oxide in plant responses to abiotic stress were emphasized. Special attention was paid to the interaction between polyamine and reactive oxygen species, ion channels, amino acid and carbon metabolism, and other adaptive responses. Further studies are needed to elucidate the polyamine signaling pathway, especially polyamine-regulated downstream tar- gets and the connections between polyamines and other stress responsive molecules.     

Improvement of plant abiotic stress tolerance through modulation of the polyamine pathway

Polyamines(mainly putrescine(Put),spermidine(Spd),and spermine(Spm))have been widely found in a range of physiological processes and in almost all diverse environmental stresses.In various plant species,abiotic stresses modulated the accumulation of polyamines and related gene expression.Studies using loss-of-function mutants and transgenic overexpression plants modulating polyamine metabolic pathways confirmed protective roles of polyamines during plant abiotic stress responses,and indicated the possibility to improve plant tolerance through genetic manipulation of the polyamine pathway.Additionally,putative mechanisms of polyamines involved in plant abiotic stress tolerance were thoroughly discussed and crosstalks among polyamine,abscisic acid,and nitric oxide in plant responses to abiotic stress were emphasized.Special attention was paid to the interaction between polyamine and reactive oxygen species,ion channels,amino acid and carbon metabolism,and other adaptive responses.Further studies are needed to elucidate the polyamine signaling pathway,especially polyamine-regulated downstream targets and the connections between polyamines and other stress responsive molecules.     

Control of plant disease by perturbation of fungal polyamine metabolism

The diamine putrescine and the polyamines spermidine and spermine are ubiquitous in nature and are essential for cell proliferation. Since polyamine biosynthesis in plants can start from either ornithine or arginine, while fungal polyamine biosynthesis appears to utilise only the ornithine route, it was suggested that specific inhibition of fungal polyamine biosynthesis should be lethal. Indeed, inhibitors of polyamine biosynthesis, e.g. the ornithine decarboxylase inhibitor α-difluoromethylornithine, have been shown to inhibit fungal growth in vitro and to control fungal infections on a variety of plants under glasshouse and field conditions. It is now known that polyamine analogues can perturb polyamine metabolism leading to powerful antiproliferative effects in cancer cells. This paper reviews the results of a research programme focused on the synthesis and evaluation of putrescine analogues as novel fungicides. A number of aliphatic, alicyclic and cyclic diamines have been shown to possess considerable fungicidal activity, but although many of these compounds perturb polyamine metabolism in fungal cells, such changes are not considered sufficient to account for the observed antifungal effects. More recent work on spermidine analogues is also described.     

Hormone-independent polyamine metabolism of squamous cell carcinoma of the prostate

The levels of polyamines and their synthesizing enzymes in squamous cell carcinoma of prostate implanted in intact as well as castrated male rats were determined after certain hormonal manipulations. The tumour was found to grow with an identical rate in non-castrated and castrated rats. Polyamine content and activities of polyamine synthesizing enzymes in the tumour were found to be much lower compared to their values in ventral prostate. Moreover, the levels of these parameters were comparable in tumours whether implanted in non-castrated or gonadectomized animals. The sequential analyses of putrescine and spermidine and activities of L-ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase of tumours at different time intervals showed a significant reduction in their levels at 30 days compared to 10 days post implantation in non-castrated as well as castrated rats. Daily intramuscular administration of tumour-bearing intact or castrated animals with testosterone (50 micrograms/g), beta-estradiol (2 micrograms/g) or cyproterone (12.5 micrograms/g) for 10 days did not influence polyamine metabolism in tumour tissue. However, either beta-estradiol and cyproterone treatments or castration were found to decrease polyamine synthesis in ventral prostate. At the same time, the testosterone replacement therapy did not allow polyamine levels or activities of polyamine synthesizing enzymes to decline in the ventral prostate of castrated rats. Our results demonstrated that contrary to ventral prostate, the polyamine metabolism in squamous cell carcinoma of prostate is independent of hormonal control. The loss of hormonal sensitivity of polyamine metabolism in the prostatic tumour could be the result of qualitative changes that occurred during transformation.     

An imbalance in autophagy contributes to retinal damage in a rat model of oxygen-induced retinopathy

In retinopathy of prematurity (ROP), the abnormal retinal neovascularization is often accompanied by retinal neuronal dysfunction. Here, a rat model of oxygen-induced retinopathy (OIR), which mimics the ROP disease, was used to investigate changes in the expression of key mediators of autophagy and markers of cell death in the rat retina. In addition, rats were treated from birth to postnatal day 14 and 18 with 3-methyladenine (3-MA), an inhibitor of autophagy. Immunoblot and immunofluorescence analysis demonstrated that autophagic mechanisms are dysregulated in the retina of OIR rats and indicated a possible correlation between autophagy and necroptosis, but not apoptosis. We found that 3-MA acts predominantly by reducing autophagic and necroptotic markers in the OIR retinas, having no effects on apoptotic markers. However, 3-MA does not ameliorate retinal function, which results compromised in this model. Taken together, these results revealed the crucial role of autophagy in retinal cells of OIR rats. Thus, inhibiting autophagy may be viewed as a putative strategy to counteract ROP.     

RhoB antibody alters retinal vascularization in models of murine retinopathy

Neovascularization in cancer or retinopathy is driven by pathological changes that foster abnormal sprouting of endothelial cells. Mouse genetic studies indicate that the stress-induced small GTPase RhoB is dispensable for normal physiology but required for pathogenic angiogenesis. In diabetic retinopathy, retinopathy of prematurity (ROP) or age-related wet macular degeneration (AMD), progressive pathologic anatomic changes and ischemia foster neovascularization are characterized by abnormal sprouting of endothelial cells. This process is driven by the angiogenic growth factor VEGF, which induces and supports the formation of new blood vessels. While injectable biologics targeting VEGF have been used to treat these pathological conditions, many patients respond poorly, prompting interest in other types of mechanism-based therapy. Here we report the preclinical efficacy of a monoclonal antibody that specifically targets RhoB, a signaling molecule that is genetically dispensable for normal physiology but required for pathogenic retinal angiogenesis. In murine models of proliferative retinal angiogenesis or oxygen-induced retinopathy, administering a monoclonal RhoB antibody (7F7) was sufficient to block neoangiogenesis or avascular pathology, respectively. Our findings offer preclinical proof of concept for antibody targeting of RhoB to limit diabetic retinopathy, ROP or wet AMD and perhaps other diseases of neovasculogenesis such as hemangioma or hemangiosarcoma nonresponsive to existing therapies.     

Arginase activity in frog urinary bladder epithelial cells and its involvement in regulation of nitric oxide production

The activity of arginase converting arginine into ornithine and urea is of particular interest among many factors regulating NO production in the cells. It is known that by competing with NO-synthase for common substrate (arginine), arginase can affect NO synthesis. In the present work, properties of arginase from the common frog Rana temporaria L. urinary bladder epithelial cells containing the NO-synthase were characterized, and possible contribution of arginase to regulation of NO production by epithelial cells was studied. It has been shown that the enzyme has temperature optimum in the range of 55–60°C, K _M for arginine 23 mM, and V _max about 10 nmole urea/mg of protein/min, and its activity was efficiently inhibited by (S)-(2-boronoethyl)-L-cysteine (BEC), an inhibitor of arginase, at concentrations from 10^?6 to 10^?4 M. The comparison of arginase activity in various frog tissues revealed the following pattern: liver > kidney > brain > urinary bladder (epithelium) > heart > testis. The arginase activity in isolated urinary bladder epithelial cells was 3 times higher that in the intact urinary bladder wall. To evaluate the role of arginase in regulation of NO production, the epithelial cells were cultivated in the media L-15 or 199 containing different amounts of arginine; the concentration of NO₂ ^?, the stable NO metabolites, was de-termined in the cultural fluid after 18–20 h of cell incubation. The vast majority of the produced nitrites are associated with NOS activity, as L-NAME, the NO inhibitor, decreased their accumulation by 77.1% in the L-15 medium and by 80% in the 199 medium. BEC (10^?4 M) increased nitrite production by 18.0% ± 2.7% in the L-15 medium and by 24.4% ± 3.5% in the 199 medium (p < 0.05). The obtained data indicate a relatively high activity of arginase in the frog urinary bladder epithelium and its involvement in regulation of NO production.     

Effects of Ri TL-DNA from Agrobacterium rhizogenes and the inhibitors of polyamine synthesis on growth, floral development, sexual organogenesis and polyamine metabolism in tobacco

Our recent work associated the phenotypic alterations caused by the Ri TL-DNA (root-inducing, left-hand, transferred DNA) from Agrobacterium rhizogenes with a general decrease in the accumulation of polyamines and their derivatives and showed that irreversible and specific inhibition of putrescine biosynthesis causes a phenotype similar to that attributed to the Ri TL-DNA. In this review we report recent data supporting a correlation between the degree of expression of the transformed phenotype due to the root-inducing, left-hand, transferred DNA and inhibition of polyamine accumulation, strongly suggesting that genes carried by the root-inducing, transferred DNA may act through interference with polyamine production via the ornithine pathway.     

The retinal renin-angiotensin system: roles of angiotensin II and aldosterone

In the present review we examine the experimental and clinical evidence for the presence of a local renin-angiotensin system within the retina. Interest in a pathogenic role for the renin-angiotensin system in retinal disease originally stemmed from observations that components of the pathway were elevated in retina during the development of certain retinal pathologies. Since then, our knowledge about the contribution of the RAS to retinal disease has greatly expanded. We discuss the known functions of the renin-angiotensin system in retinopathy of prematurity and diabetic retinopathy. This includes the promotion of retinal neovascularization, inflammation, oxidative stress and neuronal and glial dysfunction. The contribution of specific components of the renin-angiotensin system is evaluated with a particular focus on angiotensin II and aldosterone and their cognate receptors. The therapeutic utility of inhibiting key components of the renin-angiotensin system is complex, but may hold promise for the prevention and improvement of vision threatening diseases.     

Therapeutic Effect of Liposomal Superoxide Dismutase in an Animal Model of Retinopathy of Prematurity

A newborn rat model of retinopathy of prematurity was used to test the hypothesis that a lack of superoxide dismutase contributes to the retinal vaso-attenuation seen during exposure of the animals to hyperoxic conditions. To determine the endogenous superoxide dismutase activity of the retina under hyperoxic conditions, litters of albino rats were placed in either constant 80% ambient oxygen (constant hyperoxia), or placed in 21% oxygen (room air) immediately after birth. Every other day, for 14 days, several rat pups were sacrificed and their retinas removed for the determination of total superoxide dismutase (SOD) activity and manganese-associated SOD activity. An attempt was made to increase retinal SOD activity by intraperitoneal administration of exogenous SOD encapsulated in polyethylene glycol-modified liposomes. Additional litters were exposed to the same oxygen treatments and supplemented twice daily with either liposome-encapsulated superoxide dismutase in saline or liposomes containing saline without SOD. Animals were sacrificed at various time points for the determination of total superoxide dismutase activity and computer-assisted analysis of vessel density and avascular area. Animals raised in an atmosphere of constant 80% oxygen had significantly reduced levels of retinal superoxide dismutase activity through 6 days of life when compared to their room air-raised littermates. At 6 days of age, daily supplementation with liposome-encapsulated SOD had significantly increased retinal superoxide dismutase activity and reduced oxygen-induced vaso-attenuation as evidenced by increased vessel density and decreased avascular area, when compared to littermates exposed to constant hyperoxia that received control liposomes. Superoxide dismutase had no adverse effects on any of the animals regardless of treatment. Tracing experiments demonstrated that liposomes entered the retina and were found in cells morphologically resembling mi-croglia. Delivery of SOD to the retina via long-circulating liposomes proved beneficial, suggesting that restoration and/or supplementation of endogenous antioxidants in oxygen-damaged retinal tissue is a potentially valuable therapeutic strategy.     

A new perspective on ornithine decarboxylase regulation: prevention of polyamine toxicity is the overriding theme

The polyamines are essential cellular components for growth. Control of a key regulated enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), as a function of growth, is an area of intense interest. A unique regulatory property of ODC is the short half-life of the protein, which has been suggested to be an important factor in rapid activation of polyamine biosynthesis after cells are mitogenically stimulated. In this paper, it is argued that the biological significance of the short half-life of ODC is unrelated to the rate of its induction to a new steady state by growth factors, which is in fact limited by the relatively long half-life of the ODC mRNA. Instead, I suggest that the rapid turnover of ODC protein becomes of significance when cells cease growth and expeditious downregulation of the enzyme is important in preventing polyamine overproduction, which would result in cytotoxicity in the arrested cells. Although mitogenic activation of ODC expression has been studied extensively, there is very little known about the mechanisms controlling downregulation of polyamine biosynthesis during the arrest of animal cell growth. These considerations suggest that this would be a fertile area of future inquiry.     

Overexpression of S‐adenosyl‐l‐methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism

S‐adenosyl‐l ‐methionine (SAM) synthetase is the key enzyme involved in the biosynthesis of SAM, which serves as a common precursor for polyamines (PAs) and ethylene. A SAM synthetase cDNA (SlSAMS1) was introduced into the tomato genome using the Agrobacterium tumefaciens transformation method. Transgenic plants overexpressing SlSAMS1 exhibited a significant increase in tolerance to alkali stress and maintained nutrient balance, higher photosynthetic capacity and lower oxidative stress compared with WT lines. Both in vivo and in vitro experiments indicated that the function of SlSAMS1 mainly depended on the accumulation of Spd and Spm in the transgenic lines. A grafting experiment showed that rootstocks from SlSAMS1‐overexpressing plants provided a stronger root system, increased PAs accumulation, essential elements absorption, and decreased Na⁺ absorption in the scions under alkali stress. As a result, fruit set and yield were significantly enhanced. To our knowledge, this is the first report to provide evidence that SlSAMS1 positively regulates tomato tolerance to alkali stress and plays a major role in modulating polyamine metabolism, resulting in maintainability of nutrient and ROS balance.     

Coupling of the polyamine and iron metabolism pathways in the regulation of proliferation: Mechanistic links to alterations in key polyamine biosynthetic and catabolic enzymes

Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N¹-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced ³H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the “reprogramming” of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation.     

Beta‐lapachone inhibits pathological retinal neovascularization in oxygen‐induced retinopathy via regulation of HIF‐1α

Retinal neovascularization in retinopathy of prematurity (ROP) is the most common cause of blindness for children. Despite evidence that hypoxia inducible factor (HIF)‐1α ‐VEGF axis is associated with the pathogenesis of ROP, the inhibitors of HIF‐1α have not been established as a therapeutic target in the control of ROP pathophysiology. We investigated the hypothesis that degradation of HIF‐1α as a master regulator of angiogenesis in hypoxic condition, using β‐lapachone, would confer protection against hypoxia‐induced retinopathy without affecting physiological vascular development in mice with oxygen‐induced retinopathy (OIR), an animal model of ROP. The effects of β‐lapachone were examined after intraocular injection in mice with OIR. Intraocular administration of β‐lapachone resulted in significant reduction in hypoxia‐induced retinal neovascularization without retinal toxicity or perturbation of developmental retinal angiogenesis. Our results demonstrate that HIF‐1α–mediated VEGF expression in OIR is associated with pathological neovascularization, not physiological angiogenesis. Thus, strategies blocking HIF‐1α in the developing eye in the pathological hypoxia could serve as a novel therapeutic target for ROP.     

Overexpression of miR-181a-5p inhibits retinal neovascularization through endocan and the ERK1/2 signaling pathway

Retinal neovascularization (RNV) is a common pathological feature of angiogenesis-related retinopathy. Endocan inhibition has previously been reported to suppress RNV in oxygen-induced retinopathy (OIR); however, its molecular mechanisms remain to be elucidated. Here, we investigated the role and mechanism of endocan in OIR. We established an OIR mouse model and detected aberrant endocan overexpression in OIR mouse retinas. Endocan inhibition through small interfering RNA or a neutralizing antibody inhibited vascular endothelial growth factor-induced cell survival, cell proliferation, and tube formation in human retinal endothelial cells in vitro and reduced the RNV area in vivo. Using RNA sequencing, a luciferase reporter assay, and bioinformatics analyses, we identified endocan as a microRNA-181a-5p target gene. The antiangiogenic effect of miR-181a-5p on RNV was verified by intravitreal injection, and we showed that this involved the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling pathway. Collectively, our data demonstrate that miR-181a-5p/endocan regulates retinal angiogenesis through the ERK1/2 signaling pathway and might represent an attractive therapeutic strategy for RNV.     

Elevation of GM2 ganglioside during ethanol-induced apoptotic neurodegeneration in the developing mouse brain

GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate staining mainly in activated microglia, in which it partially overlapped with staining for LAMP1, a late endosomal/lysosomal marker. Also, there was weaker neuronal staining, which partially co-localized with complex IV, a mitochondrial marker, and was augmented in cleaved caspase 3-positive neurons. In contrast, the control brain showed only faint and diffuse GM2 staining in neurons. Incubation of isolated brain mitochondria with GM2 in vitro induced cytochrome c release in a manner similar to that of GD3 ganglioside. Because ethanol is known to trigger mitochondria-mediated apoptosis with cytochrome c release and caspase 3 activation in the 7-day-old mouse brain, the GM2 elevation in mitochondria may be relevant to neuroapoptosis. Subsequently, activated microglia accumulated GM2, indicating a close relationship between GM2 and ethanol-induced neurodegeneration.     

Bone marrow mesenchymal stem cells modified by angiogenin-1 promotes tissue repair in mice with oxygen-induced retinopathy of prematurity by promoting retinal stem cell proliferation and differentiation

Retinopathy has become one of the major factors that lead to blindness worldwide. Although many clinical therapies are concerned about such disease, most of them focus on symptoms alleviation. In this study, we aim to investigate whether coculture retinal stem cells (RSCs) with bone marrow mesenchymal stem cells transfected with angiogenin-1 (Ang-1-BMSCs) affects the damaged retinal tissue of oxygen-induced retinopathy of prematurity (OIR-ROP) mice. After OIR-ROP mouse model establishment, Ang-1-BMSCs, RSCs, and OIR-ROP retinal tissues were cocultured in a a transwell chamber. RSCs proliferation and the expression of Ang-1, insulin-like growth factor-1 (IGF-1) in the supernatant of RSCs, as well as β-tubulin and protein kinase C (PKC) expression were evaluated. Finally, the repair of OIR-ROP mice retinal tissues was observed by injecting Ang-1-BMSCs + RSCs. In the OIR-ROP mouse model, RSCs cocultured with OIR-ROP retinal tissues could be induced to differentiate into cells expressing β-tubulin and PKC and promote the expression of Ang-1 and IGF-1. coculture of Ang-1-BMSCs further enhanced the proliferation and differentiation of RSCs by promoting the expression of Ang-1 and IGF-1. Coculture of RSCs + Ang-1-BMSCs induced differentiation of Ang-1-BMSCs through interaction among intercellular factors and restored the damaged retinal tissue of OIR-ROP mice. Collectively, our study provided evidence that coculture of Ang-1-BMSCs and RSCs could promote the proliferation and differentiation of RSCs and improve the treatment for the damaged retina tissue of OIR-ROP mice.