Photoactivation of tDodSNO induces localized vasodilation in rats: Metabolically stable S-nitrosothiols can act as targeted nitric oxide donors in vivo.

Nitric oxide (NO) is a key vasodilatory signalling molecule and NO releasing molecules (NO donors) are being examined as potential treatments for many pathologies. The photoresponsive NO donor tert-dodecane S-nitrosothiol (tDodSNO) has been designed to be highly resistant to metabolism; in principle photoactivation of tDodSNO should therefore enable the controlled release of NO in situ via light modulation. To investigate the therapeutic utility of tDodSNO, we tested drug efficacy in Sprague Dawley rats to assess systemic and localised hemodynamic responses under photoactivation, and to confirm drug safety. For comparison, drug action was evaluated alongside the existing NO donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Across a dosing range (0.1-3.0 mg/kg) tDodSNO exerted markedly reduced systemic hypotensive action compared to these standard NO donors, inducing a slight decrease in mean arterial pressure (maximum 14.2 ± 3.0%) without affecting heart rate. Target limb photoactivation of tDodSNO resulted in a substantial localized vasodilatory response, with increases to mean (26.0 ± 7.3%) and maximum (53.2 ± 10.4%) blood flow and decreases to vascular resistance (27.1 ± 3.9%) that were restricted to light exposed tissue. In comparison GSNO and SNP showed variable peripheral effects and were not responsive to photoactivation. tDodSNO did not induce met-Hb formation in blood, or display any signs of toxicity, and was rapidly cleared from the systemic circulation, with no hemodynamic effects detectable 5 min post administration. These data are the first demonstration that drugs based upon a metabolically stable S-nitrosothiol group can be photoactivated in vivo to release NO, and that such agents cause less systemic side effects than existing NO donors. Our data support the use of S-nitrosothiols to enable the spatiotemporal control of NO for therapeutic applications.

SMA-BmobaSNO: an intelligent photoresponsive nitric oxide releasing polymer for drug nanoencapsulation and targeted delivery.

Nitric oxide (NO) is an important biological signalling molecule that acts to vasodilate blood vessels and change the permeability of the blood vessel wall. Due to these cardiovascular actions, co-administering NO with a therapeutic could enhance drug uptake. However current NO donors are not suitable for targeted drug delivery as they systemically release NO. To overcome this limitation we report the development of a smart polymer, SMA-BmobaSNO, designed to release NO in response to a photostimulus. The polymer's NO releasing functionality is an S-nitrosothiol group that, at 10 mg ml-1, is highly resistant to both thermal (t1/216 d) and metabolic (t1/232 h) decomposition, but rapidly brakes down under photoactivation (2700 W m-2, halogen source) to release NO (t1/225 min). Photoresponsive NO release from SMA-BmobaSNO was confirmed in a cardiovascular preparation, where irradiation resulted in a 12-fold decrease in vasorelaxation EC50(from 5.2µM to 420 nM). To demonstrate the polymer's utility for drug delivery we then used SMA-BmobaSNO to fabricate a nanoparticle containing the probe Nile Red (NR). The resulting SMA-BmobaSNO-NR nanoparticle exhibited spherical morphology (180 nm diameter) and sustained NR release (≈20% over 5 d). Targeted delivery was characterised in an abdominal preparation, where photoactivation (450 W m-2) caused localized increases in vasodilation and blood vessel permeability, resulting in a 3-fold increase in NR uptake into photoactivated tissue. Nanoparticles fabricated from SMA-BmobaSNO therefore display highly photoresponsive NO release and can apply the Trojan Horse paradigm by using endogenous NO signalling pathways to smuggle a therapeutic cargo into target tissue.

Quadruply Stranded Metallo-Supramolecular Helicate [Pd2(hextrz)4]4+ Acts as a Molecular Mimic of Cytolytic Peptides.

[Pd2(hextrz)4]4+ is a quadruply stranded helicate, a novel bioinorganic complex designed to mimic the structure and function of proteins due to its high stability and supramolecular size. We have previously reported that [Pd2(hextrz)4]4+ exhibited cytotoxicity toward a range of cell lines, with IC50 values ranging from 3 to 10 µM. Here we demonstrate that [Pd2(hextrz)4]4+ kills cells by forming pores within the cell membrane, a mechanism of cell death analogous to the naturally occurring cytolytic peptides. [Pd2(hextrz)4]4+ induced cell death is characterized by an initial influx of Ca2+, followed by nuclear condensation and mitochondrial swelling. This is accompanied by progressive cell membrane damage that results in the formation of large blebs at the cell surface. This allows the efflux of molecules from the cell leading to loss of cell viability. These data suggest that it may be possible to design metallo-supramolecular complexes to mimic the cytotoxic action of pore forming proteins and peptides and so provide a new class of drug to treat cancer, autoimmune disorders, and microbial infection.

A Dinuclear Platinum(II) N4Py Complex: An Unexpected Coordination Mode For N4Py.

The polypyridyl compound N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine (N4Py) acts as a bridging ligand and coordinates to two Pt(II) ions giving an unexpected diplatinum(II) complex, whose photophysical and anticancer properties were investigated.

Intracellular targeting and pharmacological activity of the superoxide dismutase mimics MnTE-2-PyP5+ and MnTnHex-2-PyP5+ regulated by their porphyrin ring substituents.

Manganese porphyrin-based drugs are potent mimics of the enzyme superoxide dismutase. They exert remarkable efficacy in disease models and are entering clinical trials. Two lead compounds, MnTE-2-PyP(5+) and MnTnHex-2-PyP(5+), have similar catalytic rates, but differ in their alkyl chain substituents (ethyl vs n-hexyl). Herein we demonstrate that these changes in ring substitution impact upon drug intracellular distribution and pharmacological mechanism, with MnTnHex-2-PyP(5+) superior in augmenting menadione toxicity. These findings establish that both catalytic activity and intracellular distribution determine drug action.

Synchrotron radiation induced X-ray emission studies of the antioxidant mechanism of the organoselenium drug ebselen.

Synchrotron radiation induced X-ray emission (SRIXE) spectroscopy was used to map the cellular uptake of the organoselenium-based antioxidant drug ebselen using differentiated ND15 cells as a neuronal model. The cellular SRIXE spectra, acquired using a hard X-ray microprobe beam (12.8-keV), showed a large enhancement of fluorescence at the K(α) line for Se (11.2-keV) following treatment with ebselen (10 µM) at time periods from 60 to 240 min. Drug uptake was quantified and ebselen was shown to induce time-dependent changes in cellular elemental content that were characteristic of oxidative stress with the efflux of K, Cl, and Ca species. The SRIXE cellular Se distribution map revealed that ebselen was predominantly localized to a discreet region of the cell which, by comparison with the K and P elemental maps, is postulated to correspond to the endoplasmic reticulum. On the basis of these findings, it is hypothesized that a major outcome of ebselen redox catalysis is the induction of cellular stress. A mechanism of action of ebselen is proposed that involves the cell responding to drug-induced stress by increasing the expression of antioxidant genes. This hypothesis is supported by the observation that ebselen also regulated the homeostasis of the transition metals Mn, Cu, Fe, and Zn, with increases in transition metal uptake paralleling known induction times for the expression of antioxidant metalloenzymes.

A hydrogen peroxide electrode assay to measure thiol peroxidase activity for organoselenium and organotellurium drugs.

Molecular mimics of the enzyme glutathione peroxidase (GPx) are increasingly being evaluated as redox active drugs. Their molecular mechanism of action parallels that of the native enzyme; however, a major distinction is that GPx mimics can use alternative thiol substrates to glutathione. This generic thiol peroxidase activity implies that it is necessary to assess a GPx mimic's recognition of a range of cellular thiols in order to determine its potential therapeutic effects. We report an electrochemical assay that, by measuring the rate of decrease of the peroxide substrate, allows the activity of GPx mimics to be directly compared against an array of thiols. The derived pseudo zero-order rate constants, k(obs), for representative GPx mimics range between 0 and 6.6 min(-1) and can vary by more than an order of magnitude depending on the thiol electron donor. An additional advantage of the assay is that it enables synergistic interactions between GPx mimics and cellular proteins to be evaluated. Here we report that glutathione disulfide reductase, which is commonly used to evaluate GPx mimic activity, recognizes the GPx mimic ebselen as a substrate, increasing its apparent k(obs). Therefore, reports relying on glutathione disulfide reductase to evaluate GPx mimic activity may exaggerate drug antioxidant action.

Acute Reconstruction of Periorbital Trauma Resulting in Eyelid Anterior Lamella Loss With Simultaneous Full-thickness Skin Grafting and Amniotic Membrane Grafting: A Case Report.

Complex facial lacerations are frequently encountered in the combat environment. Trauma with soft-tissue loss of the periorbital region offers particular challenges in terms of operative reconstruction. Cicatricial changes in the sub-acute phase can lead to eyelid malposition and lagophthalmos. The authors present a novel technique for acute reconstruction of periorbital trauma with eyelid soft-tissue loss with simultaneous full-thickness skin grafting and amniotic membrane grafting. The technique involves standard preparation of the surgical area of injury and infiltration with local anesthetic. Initially, the area of injury is copiously irrigated, and debridement of any necrotic tissue is accomplished. Amniotic membrane grafting is then performed over the defect. Approximately 2 mm × 2 mm full-thickness skin grafts are procured and distributed over the initial amniotic membrane graft. A second amniotic membrane graft is then secured over the skin graft-amniotic membrane graft complex with cyanoacrylate tissue adhesive. A bolstered suture tarsorrhaphy is performed to minimize tissue trauma during the healing process. The operative and postsurgical outcomes were assessed. The graft site healed well without cicatricial changes or lagophthalmos. Peripheral small papillomatous lesions did develop requiring excision for cosmesis, but ultimately the graft site demonstrated appropriate coverage and healthy re-epithelialization over the previous defect. This case demonstrates the viability of simultaneous full-thickness skin grafting with concomitant amniotic membrane grafting for the acute reconstruction of periorbital trauma with eyelid anterior lamella tissue loss. An excellent cosmetic and functional outcome was attained. By providing acute reconstruction, the risk of damage secondary to cicatricial periorbital changes may be avoided.

Long-Term Outcomes After Central Retinal Artery Occlusion Treated Acutely With Hyperbaric Oxygen Therapy: A Case Series.

Purpose: This study assesses the long-term outcomes, including neovascular complications, of central retinal artery occlusion (CRAO) treated acutely with hyperbaric oxygen therapy (HBOT). Methods: Four cases of CRAO treated acutely with HBOT were reviewed. Visual and structural outcomes were reviewed. Ocular complications including neovascularization were assessed and risk factors determined. Results: Two patients with a history of non-insulin dependent diabetes mellitus (NIDDM) developed early-onset ocular neovascularization within 1 month following treatment, with final vision of light perception over 1 year after injury. One patient with NIDDM and 1 patient without NIDDM did not develop ocular neovascularization; both had improvement in final visual acuity to 20/400 and 20/250, respectively. Conclusions: Patients treated acutely with HBOT for CRAO may require more frequent and earlier monitoring for complications, especially in patients with diabetes. Further research is needed to determine the long-term safety and efficacy of HBOT for CRAO, especially in the setting of systemic disease such as diabetes.

Prevalence, Patterns, and Characteristics of Eye Injuries in Professional Mixed Martial Arts.

PURPOSE: To describe the frequency and type of eye injuries in fighters in mixed martial arts (MMA) competition. METHODS: Fight result data were collected from the Nevada Athletic Commission database from 2001 to 2020. Any fighters in a professional mixed martial arts (MMA) contest with an eye injury were included. Main outcome measures included frequency and rate of eye injuries per fight and the types of eye injuries. Secondary outcome measures were gender, laterality, decision type, and length of no-contact recommended. RESULTS: Of the 256 MMA events in the database, 187 events (73.3%) had at least one eye injury. Of a total 2208 fights at these events, there were 363 fighters who sustained 369 eye injuries, with the yearly rate of eye injuries per 100 fighters ranging from 2.56 to 12.22. The most common injuries were eyebrow and eyelid lacerations (n=160, 43%), lacerations around the eye (n=98, 27%), and orbital fractures (n=62, 17%). Most eye injuries were right sided (n=197, 53.3%) and the majority of fighters with eye injuries lost their match (n=228, 62.8%). Fifty-seven fighters were recommended for further ophthalmology clearance after the match. The most common reasons for recommended ophthalmology follow-up was orbital fracture (n=25, 44%) and retinal injury (n=7, 12%). Forty-three fighters received no-contact requirements relating to their injury for an average of 8.9 weeks (range 1-24 weeks). CONCLUSION: Ophthalmic injuries in professional MMA were prevalent, were most often lacerations surrounding the eye, and often accompanied the fighter losing their match.

Posterior Segment Findings in Patients on Extracorporeal Membrane Oxygenation.

Purpose: Extracorporeal membrane oxygenation (ECMO) is an established treatment modality for critically ill patients with cardiopulmonary failure, yet little is known of the ocular pathology in this population. The aim of this study is to characterize the posterior segment findings of ECMO patients. Methods: This study is a retrospective analysis of 20 ECMO patients evaluated by ophthalmology from September 2012 to May 2019 at a level 1 trauma center. Comprehensive examinations assessed for intraocular pathology. Demographic data, exam findings, and mortality were analyzed. Results: The sample size consisted of 20 patients; a majority were male (75%), and mean age was 37.4 years (interquartile range, 26.75-50 years). All patients received ECMO for care of acute respiratory distress syndrome (ARDS). Average duration of ECMO therapy was 9.6 ± 6.5 days. Eleven (55%) patients had acute retinal pathology, including Purtscher-like retinopathy (20%), intraocular hemorrhage (50%), and septic chorioretinitis (bacterial or fungal, 10%). Location of hemorrhage included the retina (40%), vitreous (30%), and optic disc (15%). Sixty percent (n = 12) of patients were unable to provide a subjective history on initial assessment. Ultimately, 5 out of 20 patients (25%) died of systemic illness during their hospital stay. Conclusion: This study demonstrates high rates of retinal pathology, most commonly vitreous and/or retinal hemorrhage alongside a Purtscher-like retinopathy. This is likely secondary to complications of anticoagulation, microthrombi, septicemia, and hemodynamic instability. We found a mortality rate slightly lower than that of prior ECMO studies. Prospective studies with pre-ECMO and post-ECMO fundus photography is warranted for better understanding of these medically complex patients.

Controlled Delivery of Nitric Oxide for Cancer Therapy.

Nitric oxide (NO) is a short-lived, endogenously produced, signaling molecule which plays multiple roles in mammalian physiology. Underproduction of NO is associated with several pathological processes; hence a broad range of NO donors have emerged as potential therapeutics for cardiovascular and respiratory disorders, wound healing, the immune response to infection, and cancer. However, short half-lives, chemical reactivity, rapid systemic clearance, and cytotoxicity have hindered the clinical development of most low molecular weight NO donors. Hence, for controlled NO delivery, there has been extensive effort to design novel NO-releasing biomaterials for tumor targeting. This review covers the effects of NO in cancer biology, NO releasing moieties which can be used for NO delivery, and current advances in the design of NO releasing biomaterials focusing on their applications for tumor therapy.

Encapsulation of tDodSNO generates a photoactivated nitric oxide releasing nanoparticle for localized control of vasodilation and vascular hyperpermeability.

We report the synthesis and characterization of a photoactive nitric oxide (NO) releasing nanoparticle (NP) by encapsulation of the NO donor tert-dodecane S-nitrosothiol (tDodSNO) into a co-polymer of styrene and maleic anhydride (SMA) to afford SMA-tDodSNO. Encapsulation did not affect tDodSNO's stability or NO release profile, but imparted water solubility and protection from degradation reactions with glutathione. Under photoactivation the NP acted as a potent NO donor, with photoactivation acting as a switch to induce localized vasodilation in aortic rings (EC50* 660 nM at 2700 W/m2) and cause vascular hyperpermeability in mesenteric beds (8-fold increase in dye uptake at 1 µM SMA-tDodSNO with 460 W/m2 photoactivation). The NP was markedly superior as a photoactive NO donor in comparison to the S-nitrosothiols GSNO and SNAP, which are commonly used in experimental studies, as well as sodium nitroprusside, a clinically used vasodilator. Future development of this NP may find wide ranging therapeutic applications for treating cardiovascular disease and other disorders related to NO signaling, as well as enhancing macromolecular drug delivery to target organs through selective hyperpermeability. Supporting information describing the biophysical characterization of SMA-tDodSNO is supplied in an accompanying Data in Brief article (Alimoradi et al., doi: 10.1016/j.dib.2018.10.149).

Creutzfeldt-Jakob Disease Presenting with Abducens Nerve Palsy.

Creutzfeldt-Jakob disease (CJD) is a rare neurodegenerative disorder with characteristic clinical and diagnostic features. We describe the unusual case of an elderly man who presented to our ophthalmology clinic with horizontal diplopia secondary to an abducens nerve (cranial nerve six) palsy and was subsequently diagnosed with CJD. Given the non-treatable nature of this disease, ophthalmologic management goals included symptomatic relief and quality of life improvement. Precautions related to the ophthalmologic management of CJD have also been addressed in this case report.

Novel method for measuring S-nitrosothiols using hydrogen sulfide.

Recent advances in techniques that allow sensitive and specific measurement of S-nitrosothiols (RSNOs) have provided evidence for a role for these compounds in various aspects of nitric oxide (NO) biology. The most widely used approach is to couple reaction chemistry that selectively reduces RSNOs by one electron to produce NO, with the sensitive detection of the latter under anaerobic conditions using ozone based chemiluminescence in NO analyzers. Herein, we report a novel reaction that is readily adaptable for commercial NO analyzers that utilizes hydrogen sulfide (H2S), a gas that can reduce RSNO to NO and, analogous to NO, is produced by endogenous metabolism and has effects on diverse biological functions. We discuss factors that affect H2S based methods for RSNO measurement and discuss the potential of H2S as an experimental tool to measure RSNO.

Xanthine oxidase-dependent regulation of hypoxia-inducible factor in cancer cells.

During chemical hypoxia induced by cobalt chloride (CoCl2), hypoxia-inducible factor 1alpha (HIF1-alpha) mediates the induction of a variety of genes including erythropoietin and vascular endothelial growth factor. We used glioma cells with oxidative phosphorylation-dependent (D54-MG) and glycolytic-dependent (U251-MG) phenotypes to monitor HIF1-alpha regulation in association with redox responsiveness to CoCl2 treatment. We showed that CoCl2 increased xanthine oxidase (XO)-derived reactive oxygen species (ROS), which causes accumulation of HIF1-alpha protein in U251-MG cells. Under these conditions, blockade of XO activity by pharmacologic (N-acetyl-L-cysteine or allopurinol) or molecular (by small interfering RNA) approaches significantly attenuated HIF1-alpha expression. Exogenous H2O2 stabilizes HIF1-alpha protein. XO was present in these cells and was the primary source of free radicals. We also showed higher XO activity in cells exposed to CoCl2 compared with cells grown in normoxia. From the experiments shown here, we concluded that ROS were indeed generated in D54-MG cells exposed to CoCl2 but it was unlikely that ROS participated in the hypoxic signal transduction pathways in this cell type. Possibly, cell type-dependent and stimulus-dependent factors may control ROS dependency or redox sensitivity of HIF1-alpha and thus HIF1-alpha activation either directly or by induction of specific signaling cascades. Our findings reveal that XO-derived ROS is a novel and critical component of HIF1-alpha regulation in U251-MG cells, pointing toward a more general role of this transcription factor in tumor progression.

Data characterizing the biophysical and nitric oxide release properties of the tDodSNO - Styrene maleic anhydride nanoparticle SMA-tDodSNO.

Nitric oxide (NO) donor drugs have a range of clinical applications, and are also being developed as therapeutics for the potential treatment of multiple diseases. This article presents data describing the synthesis and characterisation of a novel NO releasing nanoparticle formed by encapsulation of the NO donor tDodSNO into a co-polymer of styrene and maleic acid (SMA) to afford SMA-tDodSNO. The pharmacological activity of SMA-tDodSNO is discussed in our accompanying manuscript "Encapsulation of tDodSNO generates a photoactivated nitric oxide releasing nanoparticle for localized control of vasodilation and vascular hyperpermeability". (Alimoradio et al. [1]).

Anticancer Activity and Cisplatin Binding Ability of Bis-Quinoline and Bis-Isoquinoline Derived [Pd2L4]4+ Metallosupramolecular Cages.

New bis-quinoline (L q) and bis-isoquinoline-based (L iq) ligands have been synthesized, along with their respective homoleptic [Pd2(L q or L iq)4]4+ cages (C q and C iq). The ligands and cages were characterized by 1H, 13C and diffusion ordered (DOSY) NMR spectroscopies, high resolution electrospray ionization mass spectrometry (HR-ESIMS) and in the case of the bis-quinoline cage, X-ray crystallography. The crystal structure of the C q architecture showed that the [Pd2(L q)4]4+ cage formed a twisted meso isomer where the [Pd(quinoline)4]2+ units at either end of the cage architecture adopt the opposite twists (left and right handed). Conversely, Density Functional Theory (DFT) calculations on the C iq cage architecture indicated that a lantern shaped conformation, similar to what has been observed before for related [Pd2(L tripy)4]4+ systems (where L tripy = 2,6-bis(pyridin-3-ylethynyl)pyridine), was generated. The different cage conformations manifest different properties for the isomeric cages. The C iq cage is able to bind, weakly in acetonitrile, the anticancer drug cisplatin whereas the C q architecture shows no interaction with the guest under the same conditions. The kinetic robustness of the two cages in the presence of Cl- nucleophiles was also different. The C iq cage was completely decomposed into free L iq and [Pd(Cl)4]2- within 1 h. However, the C q cage was more long lived and was only fully decomposed after 7 h. The new ligands (L iq and L q) and the Pd(II) cage architectures (C iq and C q) were assessed for their cytotoxic properties against two cancerous cell lines (A549 lung cancer and MDA-MB-231 breast cancer) and one non-cancerous cell line (HDFa skin cells). It was found that L q and C q were both reasonably cytotoxic (IC50S ≈ 0.5 µM) against A549, while C iq was slightly less active (IC50 = 7.4 µM). L iq was not soluble enough to allow the IC50 to be determined against either of the two cancerous cell lines. However, none of the molecules showed any selectivity for the cancer cells, as they were all found to have similar cytotoxicities against HDFa skin cells (IC50 values ranged from 2.6 to 3.0 µM).

The Reactive Sulfur Species Concept: 15 Years On.

Fifteen years ago, in 2001, the concept of "Reactive Sulfur Species" or RSS was advocated as a working hypothesis. Since then various organic as well as inorganic RSS have attracted considerable interest and stimulated many new and often unexpected avenues in research and product development. During this time, it has become apparent that molecules with sulfur-containing functional groups are not just the passive "victims" of oxidative stress or simple conveyors of signals in cells, but can also be stressors in their own right, with pivotal roles in cellular function and homeostasis. Many "exotic" sulfur-based compounds, often of natural origin, have entered the fray in the context of nutrition, ageing, chemoprevention and therapy. In parallel, the field of inorganic RSS has come to the forefront of research, with short-lived yet metabolically important intermediates, such as various sulfur-nitrogen species and polysulfides (Sx2-), playing important roles. Between 2003 and 2005 several breath-taking discoveries emerged characterising unusual sulfur redox states in biology, and since then the truly unique role of sulfur-dependent redox systems has become apparent. Following these discoveries, over the last decade a "hunt" and, more recently, mining for such modifications has begun-and still continues-often in conjunction with new, innovative and complex labelling and analytical methods to capture the (entire) sulfur "redoxome". A key distinction for RSS is that, unlike oxygen or nitrogen, sulfur not only forms a plethora of specific reactive species, but sulfur also targets itself, as sulfur containing molecules, i.e., peptides, proteins and enzymes, preferentially react with RSS. Not surprisingly, today this sulfur-centred redox signalling and control inside the living cell is a burning issue, which has moved on from the predominantly thiol/disulfide biochemistry of the past to a complex labyrinth of interacting signalling and control pathways which involve various sulfur oxidation states, sulfur species and reactions. RSS are omnipresent and, in some instances, are even considered as the true bearers of redox control, perhaps being more important than the Reactive Oxygen Species (ROS) or Reactive Nitrogen Species (RNS) which for decades have dominated the redox field. In other(s) words, in 2017, sulfur redox is "on the rise", and the idea of RSS resonates throughout the Life Sciences. Still, the RSS story isn't over yet. Many RSS are at the heart of "mistaken identities" which urgently require clarification and may even provide the foundations for further scientific revolutions in the years to come. In light of these developments, it is therefore the perfect time to revisit the original hypotheses, to select highlights in the field and to question and eventually update our concept of "Reactive Sulfur Species".

Moderate hypoxia induces xanthine oxidoreductase activity in arterial endothelial cells.

Xanthine oxidoreductase (XOR) activity has been previously noted to be responsive to changes in O2 tension. While prior studies have focused on the extremes (0-3% and 95-100%) of O2 tensions, we report the influence of 10% O2 on endothelial cell XOR, a concentration resembling modest arterial hypoxia commonly found in patients with chronic cardiopulmonary diseases. Exposure of bovine aortic endothelial cells to 10% O2 increased XOR mRNA and protein abundance by 50%. Concomitantly, there was a 3-fold increase in XOR activity, XOR-dependent reactive oxygen species production, and cellular export of active enzyme. Although increases in mRNA and immunoreactive protein levels were observed, inhibition of transcription, translation, or protein degradation did not significantly alter cellular XOR specific activity, suggesting only modest contributions to 10% O2-induced effects. Exposure to 10% O2 did not increase cellular HIF-1alpha protein levels and hypoxia mimics did not alter XOR activity. Treatment of control cells with adenosine resulted in increased XOR activity similar to hypoxia. Exposure to the adenosine receptor agonist NECA increased enzymatic activity 4-fold while 8SPT, an adenosine receptor antagonist, reduced hypoxic induction of XOR activity approximately 50%. Combined, these data reveal that moderate hypoxia significantly enhances endothelial XOR specific activity, release, and XOR-derived reactive oxygen species generation. These effects appear to be mediated in part via adenosine-dependent processes.