Combinatorial Design of Slippery, Nitric Oxide-Releasing Surfaces Incorporating Copper Nanoparticles for Blood-Contacting Devices.

Epidemiology, etiology, and diagnosis of hospital-acquired pneumonia and ventilator-associated pneumonia in Asian countries

Background: Numerous biomaterials are developed to mitigate untoward effects of blood exposure to foreign surfaces on blood-contacting medical devices, such as extracorporeal life support (ECLS); however, limited testing is performed using clinical blood flow conditions and device components. A liquid infused nitric oxide-releasing (LiNORel) material was developed to prevent surface protein adsorption while eluting nitric oxide (NO) to minimize platelet activation and aggregation. The study objective was to assess LiNORel applied to full-scale ECLS circuit tubing during 6 h ex vivo circulation of porcine blood at a clinically relevant flow rate. We hypothesized that LiNORel reduces thrombus deposition and platelet sequestration versus unmodified tubing. Methods: Heparinized blood (0.75 U/mL) was collected from mechanically ventilated swine (n=9, 45-55kg) with ARDS following polytrauma in an unrelated multi-day ICU study. Blood was divided into circuits with the following (500 mL/circuit): CTRL – unmodified tubing; LI – tubing with liquid infused surface modification; NO – tubing with NOrelease modification; LiNORel – tubing with liquid infused and NO-release modifications. Circuits included a blood reservoir, centrifugal pump, and 2 tubing segments (1 m, 3/8” ID). Circulation was performed for 6 h at 1.5 L/min flow rate. Heparin was sparingly administered to maintain activated clotting times (ACT) of 125-160s. Data included: heparin administered, blood gas, methemoglobin (MetHb) as an indicator of NO toxicity, pump revolutions per minute. At baseline, 3- and 6 h, a blood panel was performed to assess platelet count/activation, thromboelastography, PT, aPTT, fibrinogen, D-dimer, von Willebrand factor (vWF) activity and plasma free hemoglobin. Post-circulation thrombus deposition was analyzed. Data were analyzed for within-group time-dependent changes and between group differences. All tests were two-sided (p<0.05 for significance). Results: Circuits remained patent in all groups with ACT in range. Numerically (n.s.), CTRL received more heparin (100±49 U) to maintain ACT (NO=50±32 U, LI=33±27, LiNORel=47±33 U). There was no group difference in blood gases and MetHb. Numerically, reduction in platelet count at 6 h was greatest in CTRL and least in LiNORel (n.s., Fig 1A). Activated platelets were elevated at 6 h in CTRL vs NO (p=0.013) and LiNORel (p=0.020) (Fig 1B). Numerically, procoagulant platelets were elevated in CTRL (Fig 1C). No group differences in thromboelastography, PT, aPTT and fibrinogen were observed. D-dimer was elevated at 6 h in CTRL (p=0.007) and LI (p=0.027) groups. vWF was elevated at 6 h in CTRL (p=0.001). Clot deposition was minimal and not different between groups. Conclusions: LiNORel reduced platelet activation in this model which utilized a clinically relevant blood flow rate and full-scale circulation tubing for partial lung support. Additional testing in vivo for multi-day duration is ongoing in our laboratory. Figure 1. Platelet count (A), concentration of activated platelets indicated by expression of P-selectin (B) and concentration of procoagulant platelets indicated by phosphatidyl serine expression (C) during 6-hours ex vivo circulation. Circuit tubing consisted of Control (CTRL) – uncoated standard tubing; Nitric Oxide (NO) – tubing with embedded nitric oxide donor; Liquid Infused (LI) – tubing swelled with non-adhesive lubricant layer; Liquid-infused Nitric Oxide Release (LiNORel) – tubing with embedded nitric oxide donor and non-adhesive lubricant layer combination coating. †Indicates significant difference between LiNORel and CTRL. ‡Indicates significant difference between NO and CTRL. *Indicates significant difference within NO group from baseline (BL). **Indicates significant difference within CTRL group from BL. ***Indicates significant difference within LiNORel group from BL. ****Indicates significant difference within LI group from BL. All tests two-sided with p<0.05 for significance.

Cardiac tissue undergoes changes during ischemia-reperfusion (I-R) that compromise its normal function. Cell death is one of the consequences of such damage, as well as diminution in nitric oxide (NO) content. This signaling molecule regulates the function of the cardiovascular system through dependent and independent effects of cyclic guanosine monophosphate (cGMP). The independent cGMP pathway involves post-translational modification of proteins by S-nitrosylation. Studies in vitro have shown that NO inhibits the activity of caspases and calpains through S-nitrosylation of a cysteine located in their catalytic site, so we propose to elucidate if the regulatory mechanisms of NO are related with changes in S-nitrosylation of cell death proteins in the ischemic-reperfused myocardium. We used two compounds that increase the levels of NO by different mechanisms: Prolame, an amino-estrogenic compound with antiplatelet and anticoagulant effects that induces the increase of NO levels in vivo by activating the endothelial nitric oxide synthase (eNOS) and that has not been tested as a potential inhibitor of apoptosis. On the other hand, S-Nitroso-N-acetylpenicillamine (SNAP), a synthetic NO donor that has been shown to decrease cell death after inducing hypoxia-reoxygenation in cell cultures. Main experimental groups were Control, I-R, I-R+Prolame and I-R+SNAP. Additional groups were used to evaluate the NO action pathways. Contractile function represented as heart rate and ventricular pressure was evaluated in a Langendorff system. Infarct size was measured with 2,3,5-triphenyltetrazolium chloride stain. NO content was determined indirectly by measuring nitrite levels with the Griess reaction and cGMP content was measured by Enzyme-Linked ImmunoSorbent Assay. DNA integrity was evaluated by DNA laddering visualized on an agarose gel and by Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling assay. Activities of caspase-3, caspase-8, caspase-9 and calpain-1 were evaluated spectrophotometrically and the content of caspase-3 and calpain-1 by western blot. S-nitrosylation of caspase-3 and calpain-1 was evaluated by labeling S-nitrosylated cysteines. Our results show that both Prolame and SNAP increased NO content and improved functional recovery in post-ischemic hearts. cGMP-dependent and S-nitrosylation pathways were activated in both groups, but the cGMP-independent pathway was preferentially activated by SNAP, which induced higher levels of NO than Prolame. Although SNAP effectively diminished the activity of all the proteases, a correlative link between the activity of these proteases and S-nitrosylation was not fully established.

72-Hour in vivo evaluation of nitric oxide generating artificial lung gas exchange fibers in sheep

Nitric Oxide (NO) is a second messenger related to development and (a)biotic stress responses in plants. We have studied the role of NO in signaling during plant defense responses upon xylanase elicitation. Treatment of tomato cell cultures with the fungal elicitor xylanase resulted in a rapid and dose-dependent NO accumulation. We have demonstrated that NO is required for the production of the lipid second messenger phosphatidic acid (PA) via the activation of the phospholipase C (PLC) and diacylglycerol kinase (DGK) pathway. Defense-related responses downstream of PA were studied. PA and, correspondingly, xylanase were shown to induce reactive oxygen species production. Scavenging of NO or inhibition of either the PLC or the DGK enzyme diminished xylanase-induced reactive oxygen species production. Xylanase-induced PLDbeta1 and PR1 mRNA levels decreased when NO or PA production were compromised. Finally, we have shown that NO and PA are involved in the induction of cell death by xylanase. Treatment with NO scavenger cPTIO, PLC inhibitor U73122, or DGK inhibitor R59022 diminished xylanase-induced cell death. On the basis of biochemical and pharmacological experimental results, we have shown that PLC/DGK-derived PA represents a novel downstream component of NO signaling cascade during plant defense.

S-nitrosothiols have been implicated as intermediary transducers of nitric oxide bioactivity; however, the mechanisms by which these compounds affect cellular functions have not been fully established. In this study, we have examined the effect of S-nitrosothiol transport on intracellular thiol status and upon the activity of a target protein (caspase-3), in bovine aortic endothelial cells. We have previously demonstrated that the specific transport of amino acid-based S-nitrosothiols (S-nitroso-L-cysteine and S-nitrosohomocysteine) occurs via amino acid transport system L to generate high levels of intracellular protein S-nitrosothiols (Zhang, Y., and Hogg, N. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 7891-7896). In this study, we demonstrate that the transport of S-nitrosothiols is essential for these compounds to affect intracellular thiol levels and to modify intracellular protein activity. Importantly, the ability of these compounds to affect intracellular processes occurs independently of nitric oxide formation. These findings suggest that the major action of these compounds is not to liberate nitric oxide in the extracellular space but to be specifically transported into cells where they are able to modify cellular functions through nitric oxide-independent mechanisms.

Staphylococcus (S.) aureus is an important causative agent of wound infections with increasing incidence in the past decades. Specifically, the emergence of methicillin-resistant S. aureus (MRSA) causes serious problems, especially in nosocomial infections. Therefore, there is an urgent need to develop of alternative or supportive antimicrobial therapeutic modalities to meet these challenges. Purified compounds from hops have previously shown promising antimicrobial effects against MRSA isolates in vitro. In this study, purified beta-acids from hops were tested for their potential antimicrobial and healing properties using a porcine model of wounds infected by MRSA. The results show highly significant antimicrobial effects of the active substance in both the powder and Ambiderman-based application forms compared to both no-treatment control and treatment with Framycoin. Moreover, the macroscopic evaluation of the wounds during the treatment using the standardized Wound Healing Continuum indicated positive effects of the beta-acids on the overall wound healing. This is further supported by the microscopic data, which showed a clear improvement of the inflammatory parameters in the wounds treated by beta-acids. Thus, using the porcine model, we demonstrate significant therapeutic effects of hops compounds in the management of wounds infected by MRSA. Beta-acids from hops, therefore, represent a suitable candidate for the treatment of non-responsive nosocomial tissue infections by MRSA.

Nitric Oxide Inhibits Neonatal Hepatocyte Oxidative Metabolism

This study tested the hypothesis that exogenous nitric oxide (NO) inhibits basal release of NO in isolated rat aortic rings and in vivo. Thoracic aortic rings were suspended in organ chambers with Krebs-Henseleit solution. In untreated rings, the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) markedly increased basal vascular tone by 34.6 +/- 5.2% of maximal force produced by 100 nM thromboxane A2 mimetic U-46619, indicating a basal release of NO. Other rings were pretreated with the exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP) for 20 min and then washed free of drug. In these rings, L-NAME-induced vasoconstriction was significantly attenuated in a concentration-dependent manner (from 34.6 +/- 5.2 to 25.7 +/- 2.9% at SNAP = 0.5 microM, 15.2 +/- 3.1% at 1 microM, and 11.9 +/- 2.5% at 5 microM), while having no effect on NO-independent phenylephrine-induced vasoconstriction (35.4 +/- 4.7 untreated vs. 41.3 +/- 4.3% SNAP pretreated, not significant). In addition, the nonnitrosylated parent molecule of SNAP, acetylpenicillamine, had no effect on the vasoconstriction induced by L-NAME. In the in vivo studies in anesthetized rats, L-NAME caused significant hypertensive responses (34 +/- 4-mmHg increase in mean arterial blood pressure). Subvasoactive doses of SNAP attenuated these hypertensive responses in a dose-dependent manner (20 +/- 3-mmHg increase with 10 micrograms/kg SNAP pretreatment and 16 +/- 4-mmHg increase with 20 micrograms/kg SNAP pretreatment), but any dose of acetylpenicillamine studied had no effect. Coadministration of superoxide dismutase and SNAP significantly potentiated the inhibitory effect of the NO donor on vasocontraction responses to L-NAME. Furthermore, SNAP did not attenuate the hypertensive responses to phenylephrine. These results indicate that exogenous NO significantly inhibits basal NO release both in vitro and in vivo, suggesting that NO plays an important negative-feedback regulatory role under physiological conditions.

S,S′-dinitrosobucillamine, a new nitric oxide donor, induces a better vasorelaxation than other S-nitrosothiols

Na+K+-ATPase is an important enzyme serving vital functions in various mammalian tissues, including the intestine. We have previously documented that endotoxin (LPS) and nitric oxide (NO) can induce enterocyte injury in vitro. To examine whether alterations Na+,K+-ATPase activity might be involved in LPS- or NO-induced enterocyte dysfunction, we carried out four series of experiments. The first set of experiments documented that LPS decreases IEC-6 Na+,K+-ATPase activity at concentrations as low as 0.10 microg/ml. The second set of experiments tested whether exposure of IEC-6 cells to the exogenous NO donor, S-Nitroso-N-acetylpenicillamine (SNAP), would decrease IEC-6 Na+,K+-ATPase activity. The results of these experiments documented that SNAP significantly decreased IEC-6 Na+,K+-ATPase activity in a dose-dependent fashion at a threshold inhibitory concentration of 0.1 mM, and there was an inverse correlation between Na+,K+-ATPase activity and NO concentrations in the medium. Since enterocytes contain iNOS, and LPS can increase iNOS activity, the third set of experiments examined the relationship between LPS-induced inhibition of Na+),K+-ATPase activity and NO production by the IEC-6 cells. These results showed that LPS increased IEC-6 NO production in both a dose- and time-dependent fashion and an inverse correlation existed between LPS-induced NO production and decreased Na+,K+-ATPase activity. Addition of the NOS inhibitor, L-NNA, prevented the LPS-induced decrease in Na+,K+ATPase activity, suggesting that NO is involved in the decrease of Na+,K+-ATPase activity observed in the IEC-6 cells incubated with LPS. One mechanism by which the increased NO concentrations could have contributed to the decrease in Na+,K+ATPase activity, after the addition of LPS or SNAP, is via the production of peroxynitrite during the reaction of NO with superoxide. This notion was supported by studies showing that SNAP- and LPS-induced decreases in IEC-6 Na+,K+-ATPase activity could be blocked by adding superoxide dismutase to the medium. The last set of experiments tested whether the inhibition of Na+,K+-ATPase activity with the specific Na+,K+-ATPase inhibitor ouabain would increase the permeability of an IEC-6 monolayer. IEC-6 monolayer permeability was increased by ouabain, but only at a high concentration. In conclusion, these studies indicate that LPS or the NO donor, SNAP, inhibit Na+,K+-ATPase activity and this inhibition is at least partly related to peroxynitrite production. These studies also suggest that LPS-induced NO production by the IEC-6 cells decreases IEC-6 Na+,K+-ATPase activity in an autocrine fashion.

We hypothesized that angiotensin subtype-2 receptor (AT(2)R) inhibits renal renin biosynthesis in young rats via nitric oxide (NO). We monitored changes in renal NO, cGMP, renal renin content (RRC), and ANG II in 4-wk-old rats in response to low sodium (LNa(+)) intake alone and combined with 8-h direct renal cortical administration of AT(1) receptor blocker valsartan (VAL), AT(2)R blocker PD123319 (PD), NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME), NO donor S-nitroso-N-acetyl penicillamine (SNAP), or guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,2-alpha] quinoxaline-1-one (ODQ). In addition, we monitored renal endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in response to VAL or PD. LNa(+), VAL, PD, l-NAME, and ODQ increased RRC, ANG II, and renin mRNA. PD and l-NAME decreased NO and cGMP, while SNAP reduced RRC, ANG II, renin mRNA, and reversed the effects of PD. PD also reduced eNOS and nNOS protein and mRNA. Combined treatment with PD, l-NAME, or ODQ and VAL reversed the effects of VAL and caused further increase in RRC, ANG II, renin mRNA, and protein. ODQ reversed the effects of SNAP. These data demonstrate that the renal AT(2) receptor decreases renal renin biosynthesis and ANG II production in young rats. Reversal of the PD effects by SNAP and SNAP effects by ODQ confirms that NO and cGMP mediate the AT(2) receptor inhibition of renal renin production.

Objective To investigate the activation mechanism of matrix metalloproteinase-13 zymogen (pro-MMP-13) induced by nitric oxide (NO). Methods Human chondrosarcoma cells (SW1353) were grouped and treated with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP), SNAP + NO scavenger oxyhemoglobin (OxyHb), and SNAP + tissue inhibitor of metalloproteinase material -2 (TIMP-2) respectively. After stimulation, matrix metalloproteinase -13 ( a-MMP-13 ) expression level was detected. Besides, the expression levels of MT1- MMP were detected after SW1353 cells stimulated with SNAP and SNAP + OxyHb, respectively. At last, the activities of the recombinant MMP-13 (r-MMP-13) were detected after r-MMP-13 stimulated with SNAP, recombinant MT1-MMP (r-MT1-MMP) and r-MT1-MMP + TIMP-2,respectively. Results SNAP increased human chondrocytes a-MMP-13 expression, OxyHb and TIMP-2 inhibited a- MMP- 13 expression. SNAP also increased the expression of MT1- MMP levels, OxyHb inhibited the expression of MT1-MMP. r-MT1-MMP activated the r-MMP-13, but SNAP did not, TIMP-2 inhibited the activity of r-MMP-13 induced by r-MT1-MMP. Conclusions NO can not directly activate pro-MMP13 by the role of S-nitroso-(S nitrosylation). MT1-MMP mediates the activiation of pro-MMP-13 induced by NO. Key words: Nitric oxide; Matrix metalloproteinase- 13; Activation

Nitricoxide (NO) has many important physiological functions, includingits ability to inhibit platelet activation and serve as potent antimicrobialagent. The multiple roles of NO in vivo have led to great interestin the development of biomaterials that can deliver NO for specificbiomedical applications. Herein, we report a simple solvent impregnationtechnique to incorporate a nontoxic NO donor, S-nitroso-N-acetylpenicillamine (SNAP), into a more biocompatiblebiomedical grade polymer, CarboSil 20 80A. The resulting polymer-crystalcomposite material yields a very stable, long-term NO release biomaterial.The SNAP impregnation process is carefully characterized and optimized,and it is shown that SNAP crystal formation occurs in the bulk ofthe polymer after solvent evaporation. LC-MS results demonstrate thatmore than 70% of NO release from this new composite material originatesfrom the SNAP embedded CarboSil phase, and not from the SNAP speciesleaching out into the soaking solution. Catheters prepared with CarboSiland then impregnated with 15 wt % SNAP provide a controlled NO releaseover a 14 d period at physiologically relevant fluxes and are shownto significantly reduce long-term (14 day) bacterial biofilm formationagainst Staphylococcus epidermidis and Pseudonomasaeruginosa in a CDC bioreactor model. After 7 h of catheterimplantation in the jugular veins of rabbit, the SNAP CarboSil cathetersexhibit a 96% reduction in thrombus area (0.03 ± 0.01 cm2/catheter) compared to the controls (0.84 ± 0.19 cm2/catheter) (n = 3). These results suggestthat SNAP impregnated CarboSil can become an attractive new biomaterialfor use in preparing intravascular catheters and other implanted medicaldevices.

Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis-mediated disorders. Because nitric oxide (NO) is known to play a key role in various vascular diseases, the present study was undertaken to determine the role of NO in angiogenesis-mediated processes using the NO donor, S-nitroso N-acetyl penicillamine (SNAP) and S-nitroso N-acetyl glutathione (SNAG). The antiangiogenic efficacy of these NO donors was examined using in vivo and in vitro model systems. The in vitro studies demonstrated the ability of SNAP to inhibit cytokine fibroblast growth factor (FGF2)-stimulated tube formation and serum-induced cell proliferation. The inhibitory effect on cell proliferation by SNAP concentrations above the millimolar range was associated with significant shifts in the concentration of NO metabolites. Furthermore, using the mouse Matrigel implant model and the chick chorioallantoic membrane (CAM) models, SNAP demonstrated maximal inhibitory efficacy (85-95% inhibition) of cytokine (FGF2)-induced neovascularization in both in vivo models. SNAP and SNAG resulted in 85% inhibition of FGF2-induced neovascularization in the mouse Matrigel model when given at 5 mg/kg/day infusion in minipumps during 14 days and 87% inhibition of angiogenesis induced by FGF2 in the CAM when administered a single dose of 50 microg. Thus, NO donors might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or neovascular, ocular, and inflammatory diseases.