Cocoa flavanols lower vascular arginase activity in human endothelial cells in vitro and in erythrocytes in vivo.

The availability of l-arginine can be a rate-limiting factor for cellular NO production by nitric oxide synthases (NOS). Arginase competes with NOS for l-arginine as the common substrate. Increased arginase activity has been linked to low NO levels, and an inhibition of arginase activity has been reported to improve endothelium-dependent vasorelaxation. Based on the above, we hypothesized that an increase in the circulating NO pool following flavanol consumption could be correlated with decreased arginase activity. To test this hypothesis we (a) investigated the effects of (-)-epicatechin and its structurally related metabolites on endothelial arginase expression and activity in vitro; (b) evaluated the effects of dietary flavanol-rich cocoa on kidney arginase activity in vivo; and (c) assessed human erythrocyte arginase activity following flavanol-rich cocoa beverage consumption in a double-blind intervention study with cross-over design. The results demonstrate that cocoa flavanols lower arginase-2 mRNA expression and activity in HUVEC. Dietary intervention with flavanol-rich cocoa caused diminished arginase activity in rat kidney and, erythrocyte arginase activity was lowered in healthy humans following consumption of a high flavanol beverage in vivo.

Limited availability of L-arginine increases DNA-binding activity of NF-kappaB and contributes to regulation of iNOS expression.

The impact of nutrients on gene expression can be mediated by the availability of amino acids. The aim of this study is to examine the effect of limited availability of L: -arginine on the DNA-binding activity of NF-kappaB, a dominant transcription factor in inflammation, and the consequence for the expression pattern of inducible nitric oxide synthase (iNOS) in murine keratinocytes. Low availability of L: -arginine leads to activation and increased DNA-binding activity of NF-kappaB and induction of iNOS messenger RNA (mRNA) in the absence of cytokines, but not to translation into iNOS protein. Cytokine challenge at low L: -arginine also enhances iNOS mRNA expression, but translation into iNOS protein is diminished, leading to lowered nitric oxide production. The decrease in iNOS protein expression is mediated by the phosphorylation of the translation initiation factor eIF2alpha subunit, a key regulator of cellular translation. In contrast, the mRNA expression of the NF-kappaB-dependent genes IL-1alpha and cationic amino acid transporter-2 (CAT-2) are not affected by the availability of L-arginine. These results demonstrate that the availability of L: -arginine can play a role in the control of gene expression by augmenting the DNA-binding activity of NF-kappaB, which can affect the initiation and progression of dermal inflammation.

Transforming growth factor-beta1 enhances the antifibrinolytic and prothrombotic state of growing endothelial cells in a cell cycle-specific manner.

Cell cycle-dependent modulation of protein expression may influence the balance of antithrombotic and prothrombotic properties of endothelial cells. In the present study, we examined the regulation of prothrombotic and antithrombotic molecules by transforming growth factor-beta1 (TGF-beta1) during distinct phases of the cell cycle in human umbilical vein endothelial cells. In the absence of TGF-beta1, the expression of thrombomodulin, the plasminogen activators u-PA and t-PA, and their inhibitor PAI-1 was significantly increased in the S/G2 compared to the G1 phase. Treatment of endothelial cells with TGF-beta1, however, resulted in elevated expression of PAI-1 specifically in the S/G2 phase, while t-PA and u-PA increased to the same extent in both the G1 and S/G2 phase. These findings demonstrate that the expression of a subset of hemostatically relevant proteins is regulated during endothelial cell cycle and that TGF-beta1 can differentially modulate cell cycle-controlled protein expression.

Solar-simulated radiation induces secretion of IL-6 and production of isoprostanes in human skin in vivo.

Cytokines represent an integral part of the large group of mediators involved in dermal inflammation. In this in vivo study, ultraviolet light which is one of the major environmental factors affecting cytokine release patterns in the skin was employed. The effects of repeated versus one-time irradiation with solar-simulated ultraviolet light was studied regarding the secretion of proinflammatory cytokines with a Bio-Plex cytokine assay using suction blisters as a model for localized inflammatory processes. The IL-6 concentration increased markedly after 24 h in skin areas irradiated with a twofold minimal erythemal dose (MED) compared to areas challenged repeatedly with 0.3 MED. In addition, we investigated the concentration of 8-isoprostane in the suction blister fluid as a marker of lipid peroxidation due to a UV-induced increase in free radical production. 8-Isoprostane was increased immediately after treatment but declined after 24 h with the exception of the skin area exposed to 2 MED. The differential expression and release of cytokines, and the extent of oxidative damage might therefore depend on the dose and regimen of exposure to solar-simulated radiation.

Impact of Mycoplasma hyorhinis infection on L-arginine metabolism: differential regulation of the human and murine iNOS gene.

Infection with mycoplasma is a common problem in cell cultures, with Mycoplasma hyorhinis being the predominant species. Here we investigate the effect of M. hyorhinis infection on L-arginine metabolism, with focus on iNOS-mediated NO synthesis in murine keratinocytes and the human colon cancer cell line DLD-1. iNOS and arginase are L-arginine-metabolizing enzymes involved in the regulation of inflammatory processes, with NO contributing to innate immunity. In murine cells, M. hyorhinis infection enhances cytokine-induced iNOS expression and augments iNOS activity, whereas in the absence of cytokines it causes de novo induction of iNOS mRNA without subsequent translation into iNOS protein. In turn, arginase-1 mRNA expression is diminished in M. hyorhinis-infected murine keratinocytes, resulting in decreased arginase activity. One of the underlying upstream mechanisms is NF-kappaB activation. In contrast, in human cells neither iNOS mRNA nor protein expression is affected by M. hyorhinis infection, but NO synthesis is enhanced, which may be caused by increased L-arginine import. This demonstrates that infection with M. hyorhinis leads to different effects on gene regulation of the murine and human iNOS gene. Our study underlines the importance of routine checking of cell cultures for mycoplasma contamination, particularly in studies on NO-mediated effects or inflammatory processes.

Arginase-1 overexpression induces cationic amino acid transporter-1 in psoriasis.

Regulated uptake of extracellular l-arginine by cationic amino acid transporters (CATs) is required for inducible nitric oxide synthase and arginase activity. Both enzymes were recently recognized as important in the pathophysiology of psoriasis because of their contribution to epidermal hyperproliferation. We here characterize the expression pattern of CATs in psoriatic skin compared to healthy skin. CAT-1 mRNA expression was strongly upregulated in lesional and nonlesional areas of psoriatic skin compared to healthy skin, whereas expression of CAT-2A and the inducible isoform CAT-2B was unaltered in psoriatic skin. Furthermore, we tested the hypothesis that arginase-1 overexpression regulates CAT expression via intracellular l-arginine concentration. In in vitro experiments with arginase-1 overexpressing HaCaT cells, CAT-1 mRNA expression was increased. Likewise, this occurs in l-arginine-starved HaCaT cells. Both CAT-2 isoforms were not affected. Arginase-1 overexpression limits the synthesis of NO at physiological, but not supraphysiological, l-arginine levels. Plasma l-arginine concentration was diminished in psoriasis patients and the arginase product l-ornithine was significantly increased compared to healthy controls. In summary, arginase-1 overexpression leads to upregulated CAT-1 expression in psoriatic skin, which is due to lowered intracellular l-arginine levels and limits NO synthesis at physiological l-arginine concentrations.

The role of iNOS in chronic inflammatory processes in vivo: is it damage-promoting, protective, or active at all?

The expression of the inducible nitric oxide synthase (iNOS) is one of the direct consequences of an inflammatory process. Early studies have focused on the potential toxicity of the ensuing high-output NO-synthesis serving as a means to eliminate pathogens or tumor cells but also contributing to local tissue destruction during chronic inflammation. More recently, however, data are accumulating on a protective effect of high-output NO synthesis and - equally important - on a gene-regulatory function that helps to mount a protective stress response and simultaneously aids in down-regulating the proinflammatory response. These findings appear to contrast to the often observed sustained iNOS-expression during chronic inflammatory diseases, as for instance in Psoriasis vulgaris and other conditions with a chronic Th1-like reactivity. We here pose the question as to whether the iNOS is really active in these diseases. We review the data accumulated on iNOS expression in chronic diseases. We also report on the various factors that potentially interfere with proper NO formation by the expressed enzyme. We also highlight the recent findings of how, why and where evidences emerge that impeded NO formation contributes to chronic disease processes and finally present details on our current understanding of such abnormally low NO synthesis and its contribution to the pathophysiological processes of the human proinflammatory skin disease Psoriasis vulgaris.

UVB radiation-mediated expression of inducible nitric oxide synthase activity and the augmenting role of co-induced TNF-alpha in human skin endothelial cells.

Nitric oxide (NO) plays a pivotal role in ultraviolet radiation-induced inflammation in human skin. We had earlier reported on the inducible nitric oxide synthase (iNOS) inducing activity of UVA radiation. We now demonstrate that UVB-exposure induces expression of the iNOS in vessel endothelia of normal human skin and in cultured human dermal endothelial cells (HUDEC), although by a molecular mechanism different from UVA-mediated induction. With HUDEC, UVB induces iNOS expression and leads to significant enzyme activities, although at app. 5-fold lower levels than can be achieved with proinflammatory cytokines. In contrast to our earlier observation with UVA, cytokine-challenge combined with simultaneous UVB-exposure had no additive effects on iNOS expression nor activity. Interestingly, a time-delay between UVB-irradiation and cytokine-challenge enhances endothelial iNOS enzyme activity 2.5-fold over cytokines activation only. This time-dependent effect strongly correlates with UVB-induced endothelial TNF-alpha expression. In HUDEC addition of TNF-alpha results in enhanced expression of the inducible arginine transporter system CAT-2 essential for substrate supply and thus iNOS activity. In summary, UVB induces iNOS mRNA and enzyme activity in HUDEC. Moreover, UVB augments CAT-2 expression through a TNF-alpha- dependent mechanism which essentially contributes to increased iNOS activity.

The presence of nitrite during UVA irradiation protects from apoptosis.

Nitrite occurs ubiquitously in biological fluids such as blood and sweat, representing an oxidation product of nitric oxide. Nitrite has been associated with a variety of adverse effects such as mutagenicity, carcinogenesis, and toxicity. In contrast, here we demonstrate that the presence of nitrite, but not nitrate, during irradiation of endothelial cells in culture exerts a potent and concentration-dependent protection against UVA-induced apoptotic cell death. Protection is half-maximal at a concentration of 3 mM, and complete rescue is observed at 10 mM. Nitrite-mediated protection is mediated via inhibition of lipid peroxidation in a similar manner as seen with butylated hydroxytoluene, a known inhibitor of lipid peroxidation. Interestingly, nitrite-mediated protection is completely abolished by coincubation with the NO scavenger cPTIO. Using electron paramagnetic resonance (EPR) spectroscopy or Faraday modulation spectroscopy, we directly prove UVA-induced NO formation in solutions containing nitrite. In conclusion, evidence is presented that nitrite represents a protective agent against UVA-induced apoptosis due to photodecomposition of nitrite and subsequent formation of NO.

The importance of cationic amino acid transporter expression in human skin.

Inducible nitric oxide synthase and arginase activities are acknowledged as important players in human skin epidermal function. For proper enzyme function the substrate availability of L-arginine for both enzymes and thus its transport across the cell membrane via the y+-system (also named cationic amino acid transporters) is critical. Here, we examine the expression of cationic amino acid transporters and their functional role in modulating inducible nitric oxide synthase and arginase activities in human skin and primary keratinocytes, fibroblasts and endothelial cells as well as their impact on keratinocyte proliferation. Skin biopsies were found to express constitutively both cationic amino acid transporter-1 and cationic amino acid transporter-2 mRNA, an expression pattern known to occur in hepatocytes and muscle cells only. To determine the cellular components expressing cationic amino acid transporter, we analyzed the expression patterns in the different human skin cell types in vitro, i.e., in fibroblasts, dermal endothelial cells, and keratinocytes as well as in the HaCaT cell line. An ubiquitous cationic amino acid transporter-1 mRNA expression was found in all cells, whereas constitutive cationic amino acid transporter-2 mRNA expression occurs in resident keratinocytes and dermal endothelial cells only. De novo induction of cationic amino acid transporter-2 and inducible nitric oxide synthase by proinflammatory cytokines was seen in fibroblasts and HaCaT. Competitive inhibition of the cationic amino acid transporter-mediated L-arginine transport by culturing primary human keratinocytes in the presence of increased L-lysine concentration led to decreased inducible nitric oxide synthase and arginase activities with a concomitant significant decrease in keratinocyte proliferation. In summary, our results demonstrate that human keratinocytes constitutively express cationic amino acid transporters 1 and 2 and that cationic amino acid transporter mediated L-arginine influx, is essential for both inducible nitric oxide synthase and arginase enzyme activities, which in turn modulate proliferation and differentiation of human epidermal skin cells.

Critical role of L-arginine in endothelial cell survival during oxidative stress.

BACKGROUND: Oxidative damage of vascular endothelium represents an important initiation step in the development of atherosclerosis. Recently, we reported about protection of inducible nitric oxide synthase (iNOS)-derived high-output NO in endothelial cells. Because iNOS activity critically depends on the availability of its substrate l-arginine, the present study aims at elucidating iNOS-mediated effects on H2O2-induced apoptosis of cytokine-activated rat aortic endothelial cells (AECs) subject to medium l-arginine concentrations. METHODS AND RESULTS: In cytokine-activated AECs, iNOS activity was found to be half-maximal at 60 micromol/L arginine, which represents the medium serum level in rats but also in humans. Maximal activity is seen at and above 200 micromol/L arginine. Activated cells grown in the absence of arginine with minimal iNOS activity are highly sensitive toward H2O2-induced apoptosis, and increases in medium arginine concentrations result in increased cell survival. Moreover, competition experiments show that iNOS activity is completely dependent on cationic amino acid transporter-mediated arginine uptake. We also find that the arginine-dependent protection includes inhibition of endothelial lipid peroxidation and increases in the expression of vasoprotective stress response genes. CONCLUSIONS: Our data demonstrate that arginine concentrations corresponding to physiological serum levels do not allow for optimal endothelial iNOS activity. Thus, decreases in systemic arginine concentrations, or locally within atherosclerotic plaques, will impair the endothelial iNOS-mediated stress response and will significantly increase the risk of endothelial dysfunction.

Specific iNOS-targeted antisense knockdown in endothelial cells.

The inhibition of inducible nitric oxide synthase (iNOS) expression via antisense oligonucleotides (AS-ODN) may represent a highly specific tool. Endothelial cells (EC) represent prime candidate cells for in vivo application, and we therefore aimed at optimizing this technique for effectiveness and specificity in primary nontransformed rat EC. EC or L929 fibroblasts were incubated with iNOS-specific ODN optimizing all experimental steps. We find that ODN uptake, as analyzed by fluorescence microscopy and labeled ODN, was absolutely dependent on vehicle presence, and among the vehicles tested, Lipofectin displayed negligible toxicity and good uptake. In addition, omission of serum was also essential, a factor that might limit its use in vivo. Moreover, intranuclear accumulation of AS-ODN appeared crucial for successive inhibition. The impact of ODN on iNOS mRNA, protein, and enzyme activity was specific and resulted in >95% inhibition of protein formation. In conclusion, in this article we provide a protocol for an optimized AS-mediated knockdown, representing a specific and efficient instrument for blocking of iNOS formation and allowing for studying the impact of iNOS expression on endothelial function. We also expose application problems of this technique when working in inflammatory conditions.

Arginase 1 overexpression in psoriasis: limitation of inducible nitric oxide synthase activity as a molecular mechanism for keratinocyte hyperproliferation.

Abnormal proliferation of keratinocytes in the skin appears crucial to the pathogenesis of psoriasis, but the underlying mechanisms remain unknown. Nitric oxide (NO), released from keratinocytes at high concentrations, is considered a key inhibitor of cellular proliferation and inducer of differentiation in vitro. Although high-output NO synthesis is suggested by the expression of inducible NO synthase (iNOS) mRNA and protein in psoriasis lesions, the pronounced hyperproliferation of psoriatic keratinocytes may indicate that iNOS activity is too low to effectively deliver anti-proliferative NO concentrations. Here we show that arginase 1 (ARG1), which substantially participates in the regulation of iNOS activity by competing for the common substrate L-arginine, is highly overexpressed in the hyperproliferative psoriatic epidermis and is co-expressed with iNOS. Expression of L-arginine transporter molecules is found to be normal. Treatment of primary cultured keratinocytes with Th1-cytokines, as present in a psoriatic environment, leads to de novo expression of iNOS but concomitantly a significant down-regulation of ARG1. Persistent ARG1 overexpression in psoriasis lesions, therefore, may represent a disease-associated deviation from normal expression patterns. Furthermore, the culturing of activated keratinocytes in the presence of an ARG inhibitor results in a twofold increase in nitrite accumulation providing evidence for an L-arginine substrate competition in human keratinocytes. High-output NO synthesis is indeed associated with a significant decrease in cellular proliferation as shown by down-regulation of Ki67 expression in cultured keratinocytes but also in short-term organ cultures of normal human skin. In summary, our data demonstrate for the first time a link between a human inflammatory skin disease, limited iNOS activity, and ARG1 overexpression. This link may have substantial implications for the pathophysiology of psoriasis and the development of new treatment strategies.