Christian Opländer

Whole Body UVA Irradiation Lowers Systemic Blood Pressure by Release of Nitric Oxide From Intracutaneous Photolabile Nitric Oxide Derivates

Rationale: Human skin contains photolabile nitric oxide derivates like nitrite and S-nitroso thiols, which after UVA irradiation, decompose and lead to the formation of vasoactive NO. Objective: Here, we investigated whether whole body UVA irradiation influences the blood pressure of healthy volunteers because of cutaneous nonenzymatic NO formation. Methods and Results: As detected by chemoluminescence detection or by electron paramagnetic resonance spectroscopy in vitro with human skin specimens, UVA illumination (25 J/cm2) significantly increased the intradermal levels of free NO. In addition, UVA enhanced dermal S-nitrosothiols 2.3-fold, and the subfraction of dermal S-nitrosoalbumin 2.9-fold. In vivo, in healthy volunteers creamed with a skin cream containing isotopically labeled 15N-nitrite, whole body UVA irradiation (20 J/cm2) induced significant levels of 15N-labeled S-nitrosothiols in the blood plasma of light exposed subjects, as detected by cavity leak out spectroscopy. Furthermore, whole body UVA irradiation caused a rapid, significant decrease, lasting up to 60 minutes, in systolic and diastolic blood pressure of healthy volunteers by 11±2% at 30 minutes after UVA exposure. The decrease in blood pressure strongly correlated (R2=0.74) with enhanced plasma concentration of nitrosated species, as detected by a chemiluminescence assay, with increased forearm blood flow (+26±7%), with increased flow mediated vasodilation of the brachial artery (+68±22%), and with decreased forearm vascular resistance (−28±7%). Conclusions: UVA irradiation of human skin caused a significant drop in blood pressure even at moderate UVA doses. The effects were attributed to UVA induced release of NO from cutaneous photolabile NO derivates.

Effects of blue light irradiation on human dermal fibroblasts

Previous studies have reported that separately from UV-radiation also blue light influences cellular physiology in different cell types. However, little is known about the blue light action spectrum. The purpose of this study was to investigate effects of blue light at distinct wavelengths (410, 420, 453, 480 nm) emitted by well defined light-emitting-diodes on viability, proliferation and antioxidative capacity of human dermal fibroblasts. We found that irradiation with blue light (410, 420 nm) led to intracellular oxidative stress and toxic effects in a dose and wavelength dependent manner. No toxicity was observed using light at 453 nm and 480 nm. Furthermore, blue light (410, 420, 453 nm) at low doses reduced the antioxidative capacity of fibroblasts. At non-toxic doses, irradiations at 410, 420 and 453 nm reduced proliferation indicating a higher susceptibility of proliferating fibroblasts to blue light. Our results show that blue light at different wavelengths may induce varying degrees of intracellular oxidative stress with different physiological outcome, which could contribute to premature skin photoaging. On the other hand, the use of blue light due to its antiproliferative and toxic properties may represent a new approach in treatment and prevention of keloids, hypertrophic scars and fibrotic skin diseases.

The topical use of non-thermal dielectric barrier discharge (DBD): Nitric oxide related effects on human skin

Dielectric barrier discharge (DBD) devices generate air plasma above the skin containing active and reactive species including nitric oxide (NO). Since NO plays an essential role in skin physiology, a topical application of NO by plasma may be useful in the treatment of skin infections, impaired microcirculation and wound healing. Thus, after safety assessments of plasma treatment using human skin specimen and substitutes, NO-penetration through the epidermis, the loading of skin tissue with NO-derivates in vitro and the effects on human skin in vivo were determined. After the plasma treatment (0-60 min) of skin specimen or reconstructed epidermis no damaging effects were found (TUNEL/MTT). By Franz diffusion cell experiments plasma-induced NO penetration through epidermis and dermal enrichment with NO related species (nitrite 6-fold, nitrate 7-fold, nitrosothiols 30-fold) were observed. Furthermore, skin surface was acidified (~pH 2.7) by plasma treatment (90 s). Plasma application on the forearms of volunteers increased microcirculation fourfold in 1-2 mm and twofold in 6-8 mm depth in the treated skin areas. Regarding the NO-loading effects, skin acidification and increase in dermal microcirculation, plasma devices represent promising tools against chronic/infected wounds. However, efficacy of plasma treatment needs to be quantified in further studies and clinical trials.

Mechanism and biological relevance of blue-light (420-453 nm)-induced nonenzymatic nitric oxide generation from photolabile nitric oxide derivates in human skin in vitro and in vivo.

Human skin contains photolabile nitric oxide (NO) derivates such as nitrite and S-nitrosothiols, which upon UVA radiation decompose under high-output NO formation and exert NO-specific biological responses such as increased local blood flow or reduced blood pressure. To avoid the injurious effects of UVA radiation, we here investigated the mechanism and biological relevance of blue-light (420-453 nm)-induced nonenzymatic NO generation from photolabile nitric oxide derivates in human skin in vitro and in vivo. As quantified by chemiluminescence detection (CLD), at physiological pH blue light at 420 or 453 nm induced a significant NO formation from S-nitrosoalbumin and also from aqueous nitrite solutions by a to-date not entirely identified Cu(1+)-dependent mechanism. As detected by electron paramagnetic resonance spectrometry in vitro with human skin specimens, blue light irradiation significantly increased the intradermal levels of free NO. As detected by CLD in vivo in healthy volunteers, irradiation of human skin with blue light induced a significant emanation of NO from the irradiated skin area as well as a significant translocation of NO from the skin surface into the underlying tissue. In parallel, blue light irradiation caused a rapid and significant rise in local cutaneous blood flow as detected noninvasively by using micro-light-guide spectrophotometry. Irradiation of human skin with moderate doses of blue light caused a significant increase in enzyme-independent cutaneous NO formation as well as NO-dependent local biological responses, i.e., increased blood flow. The effects were attributed to blue-light-induced release of NO from cutaneous photolabile NO derivates. Thus, in contrast to UVA, blue-light-induced NO generation might be therapeutically used in the treatment of systemic and local hemodynamic disorders that are based on impaired physiological NO production or bioavailability.

Non-enzymatic NO production in human skin: Effect of UVA on cutaneous NO stores

Nitric oxide (NO(*)) in human skin has been under investigation since first reports of NOS expression in skin tissue in 1992. NO(*) plays a key role in the dermal response to external stimuli such as heat, ultraviolet (UV) light, or infection, and in healing of abrasions, lesions or burns. Recently, a range of non-enzymatic pathways for NO(*) release has been identified, mostly in the context of systemic blood flow. In this article we consider the non-enzymatic formation of NO(*) in human skin tissues. Significant quantities of NO() are continuously released from human skin into the ambient air. This release can be significantly enhanced by photolysis of endogenous NO() stores under UVA. In addition, we give the first estimate of the basal enzymatic NO(*) production in healthy human skin.

Zinc regulates iNOS-derived nitric oxide formation in endothelial cells

Aberrant production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of endothelial dysfunction and vascular disease. Mechanisms responsible for the fine-tuning of iNOS activity in inflammation are still not fully understood. Zinc is an important structural element of NOS enzymes and is known to inhibit its catalytical activity. In this study we aimed to investigate the effects of zinc on iNOS activity and expression in endothelial cells. We found that zinc down-regulated the expression of iNOS (mRNA+protein) and decreased cytokine-mediated activation of the iNOS promoter. Zinc-mediated regulation of iNOS expression was due to inhibition of NF-κB transactivation activity, as determined by a decrease in both NF-κB-driven luciferase reporter activity and expression of NF-κB target genes, including cyclooxygenase 2 and IL-1β. However, zinc did not affect NF-κB translocation into the nucleus, as assessed by Western blot analysis of nuclear and cytoplasmic fractions. Taken together our results demonstrate that zinc limits iNOS-derived high output NO production in endothelial cells by inhibiting NF-κB-dependent iNOS expression, pointing to a role of zinc as a regulator of iNOS activity in inflammation.

Dermal Application of Nitric Oxide In Vivo: Kinetics, Biological Responses, and Therapeutic Potential in Humans

Many local hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). Previous findings point to a therapeutic potential of dermal NO application in the treatment of hemodynamic disorders, but no reliable data are available on the mechanisms, kinetics, or biological responses relating to cutaneous exposure to NO in humans in vivo. Here we show that, owing to its excellent diffusion capacity, cutaneously applied NO rapidly penetrates the epidermal barrier in significant amounts, strongly enriching skin tissue and blood plasma with its vasoactive derivates. In parallel, it significantly increased vasodilatation and blood flow and reduced thrombocyte aggregation capacity. Data presented here for the first time show that, in humans, dermal application of NO has strong potential for use in the therapy of local hemodynamic disorders arising from insufficient availability of NO or its bioactive derivates.

Non-Thermal Dielectric Barrier Discharge (DBD) Effects on Proliferation and Differentiation of Human Fibroblasts Are Primary Mediated by Hydrogen Peroxide

The proliferation of fibroblasts and myofibroblast differentiation are crucial in wound healing and wound closure. Impaired wound healing is often correlated with chronic bacterial contamination of the wound area. A new promising approach to overcome wound contamination, particularly infection with antibiotic-resistant pathogens, is the topical treatment with non-thermal “cold” atmospheric plasma (CAP). Dielectric barrier discharge (DBD) devices generate CAP containing active and reactive species, which have antibacterial effects but also may affect treated tissue/cells. Moreover, DBD treatment acidifies wound fluids and leads to an accumulation of hydrogen peroxide (H2O2) and nitric oxide products, such as nitrite and nitrate, in the wound. Thus, in this paper, we addressed the question of whether DBD-induced chemical changes may interfere with wound healing-relevant cell parameters such as viability, proliferation and myofibroblast differentiation of primary human fibroblasts. DBD treatment of 250 μl buffered saline (PBS) led to a treatment time-dependent acidification (pH 6.7; 300 s) and coincidently accumulation of nitrite (~300 μM), nitrate (~1 mM) and H2O2 (~200 μM). Fibroblast viability was reduced by single DBD treatments (60–300 s; ~77–66%) or exposure to freshly DBD-treated PBS (60–300 s; ~75–55%), accompanied by prolonged proliferation inhibition of the remaining cells. In addition, the total number of myofibroblasts was reduced, whereas in contrast, the myofibroblast frequency was significantly increased 12 days after DBD treatment or exposure to DBD-treated PBS. Control experiments mimicking DBD treatment indicate that plasma-generated H2O2 was mainly responsible for the decreased proliferation and differentiation, but not for DBD-induced toxicity. In conclusion, apart from antibacterial effects, DBD/CAP may mediate biological processes, for example, wound healing by accumulation of H2O2. Therefore, a clinical DBD treatment must be well-balanced in order to avoid possible unwanted side effects such as a delayed healing process.

Characterization of novel nitrite-based nitric oxide generating delivery systems for topical dermal application

Topical application of nitric oxide (NO) has been shown to exert beneficial effects in the therapy of chronic wounds, impaired microcirculation, and skin infections. Nitrite acidified by ascorbic acid has been widely used in many studies as NO-donor system, unfortunately with inflammatory and toxic effects on the treated skin due to unregulated excessive NO generation, low pH and possible toxic side products. Here we describe an essentially modified nitrite based NO generating system that avoid the mentioned unwanted side effects on human skin by using a pH-stable acetate/acetic acid buffer with a skin neutral pH of 5.5 and sodium ascorbate. In order to overcome the shortcoming of lower NO yields due to the higher pH-value and low nitrite concentrations, we have determined additionally the influence of copper ions. To investigate the influence of different NO release and penetration kinetics on NO-induced toxicity, we have developed a fibroblast assay using cell culture plates with gas permeable bottoms. The results show clearly that the donor system can achieve a sustained NO generation without generating high peaks. Furthermore, the presence of Cu(2+) ions enhances manifold NO generation of pH/ascorbate-induced nitrite decomposition, a mechanism comprising the reduction of Cu(2+) ions to Cu(1+) by ascorbate. Finally, we have found that apart from the NO dose the NO release kinetics had a significant influence of cell toxicity. Thus, application of comparable NO amounts within a time interval of 600s led to the development of variable cell toxicities, which predominantly depended on the NO concentration values generated in the first 200s. In summary, we here describe a novel nitrite-based NO-donor system that can provide well defined NO concentrations at skin neutral pH-values for side effect poor topical dermal application, i.e. in the therapy of chronic wounds and impaired microcirculation.

A new method for sustained generation of ultra-pure nitric oxide-containing gas mixtures via controlled UVA-photolysis of nitrite solutions

Exogenous gaseous nitric oxide (gNO) is an FDA approved drug for treatment of a variety of human pathologies like Persistent Pulmonary Hypertension in neonates and premature babies, skin lesions and fungal dermatophyte infections. Substantial disadvantages of current gNO-based therapies are the high therapy costs, high storage costs of the gas cylinders, and the rapid contamination of compressed NO gases with various decomposition products. Here we describe a new, very simple, and inexpensive photolytic generator of uncontaminated NO-containing gas mixtures at therapeutic concentrations. The new method bases on UVA-induced and redox-assisted decomposition of nitrite ions in aqueous solutions. NO formation via UVA-induced photolysis of nitrite is accompanied by an OH radical-dependent production of NO(2) that beside its toxic character additionally strongly reduces the NO yield by consuming NO in its reaction to N(2)O(3). During the UVA-induced photodecomposition process both, inhibition of NO(2) formation or NO(2) depletion by antioxidants hinders the NO-consuming reaction with NO(2) and ensured a maximal purity and maximal yield of NO-containing gas mixtures. Therefore, NO-containing gas mixtures generated by the described method are suitable for medical applications like inhalation or gassing of chronic non-healing wounds. Control of temperature, UVA intensity and composition of the reaction mixture allows facile control over the final NO level in the carrier gas over a wide concentration range. We demonstrate the sustained and stable release of NO over a wide dynamic range (10-5000 ppm NO) for many hours. The method avoids contamination-prone long time storage of NO gas. As such, it appears particularly relevant for applications involving the additional presence of oxygen (e.g. inhalation).