In vitro Treatment with cis-[Ru(H-dcbpy - ) 2 (Cl)(NO)] Improves the Endothelial Function in Aortic Rings with Endothelial Dysfunction

- Purpose: The ruthenium complex cis -[Ru(H-dcbpy - ) 2 (Cl)(NO)] (DCBPY) is a nitric oxide (NO) donor and studies suggested that the ruthenium compounds can inactivate O 2-. The aim of this study is to test if DCBPY can revert and/or prevent the endothelial dysfunction. Methods: Normotensive (2K) and hypertensive (2K-1C) wistar rats were used. To vascular reactivity study, thoracic aortas were isolated, rings with intact endothelium were incubated with: DCBPY: 0.1; 1 and 10μM, DCBPY plus hydroxocobalin (NO scavenger) or tempol during 30 minutes, and concentration effect curves to acetylcholine were performed. The potency values (pD2) and maximum effect (ME) were analyzed. The O 2 - was generated using hypoxantine xantine oxidase and the reduction of cytochrome c, NO consumption by O 2- and the effect in avoid NO consumption was measured. Results: In 2K-1C DCBPY at 0.1; 1 or 10μM improved the relaxation endothelium dependent induced by acetylcholine in aortic rings compared to control 2K-1C, and also improved ME. In rings from 2K incubation with DCBPY (0.1; 1.0 and 10 μM) did not change pD2 or ME. Incubation with 0.1 μM of DCBPY plus hydroxocobalamin did not modify the potency and ME in 2K-1C compared to DCBPY (0.1 μM). DCBPY and SOD inhibits the reduction of cytochrome c and inhibited the NO consumption by O 2- , showing that O 2- has been removed from the solution. Conclusion: Our results suggest that DCBPY at a lower concentration (0.1 µM) is not an NO generator, but can inactivate superoxide and improves the endothelial function.

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Nitric oxide is involved in multiple physiological and pathophysiological processes which has promoted a large amount of research involved to development of drugs that are able to modulate NO concentration in biological environment, for therapeutic purpose [7], including NO donors.
NO donors are pharmacological active substances that in-vivo or in-vitro release NO. Organic nitrates (eg, glyceryltrinitrate GTN) and sodium nitroprusside (SNP) have been used as therapeutic agents, which presents an important vasodilation effect [8]. However, despite its vasodilation effect, GNT causes headache and tolerance, while SNP releases highly cytotoxic cyanide [9][10].
Exogenous NO donors agents based on ruthenium-derived metal nitrosyl complexes have been developed as strategy to reduce side effects and cytotoxicity. They have not displayed any toxic effects and are able to induce vascular relaxation and decrease blood pressure in normotensive and hypertensive rats [11][12][13][14][15]. Rodrigues et al [16] showed that the compound cis-[Ru(H-dcbpy _ )2(Cl)(NO)] (DCBPY) is an NO generator that promotes relaxation of rat aortic rings by increasing the cytosolic NO concentration and reduce the cytosolic Ca 2+ concentration [Ca 2+ ]c in rat aortic rings.
Hypertension is associated with impaired endothelial function that includes reduced production and/or NO release. Previous studies have reported that in aortas from renal ________________________________________ hypertensive rats, the NO-dependent relaxation endothelium dependent [17][18] or endotheliumindependent is impaired, due to several factors such as caveolae number alteration [19,14], increased production of superoxide anion among others [20][21]. An antioxidant action as well as superoxide anion scavenger action has been attributed to ruthenium compounds [22]. Thus, a compound that could remove superoxide anion and release NO would be desired to improve endothelial function in hypertension.
In this context, this study tested the hypothesis that the ruthenium DCBPY is able to revert the endothelial dysfunction by inactivate superoxide anion in aortas from hypertensive rats. (Ru-NO2 -) after its solubilization at pH 7.4 [16]. The conversion was complete after 150 min. Thus, the Ru-NO2 -(DCBPY) complex was employed in the present work.

Experimental animals
Experimental protocols followed standards and policies of the Animal Care and Use Committee of the Federal University of São Carlos, and was approved by this committee (Protocol number: 012/13). Renovascular hypertension was induced in rats following the two kidney one clip (2K-1C) Goldblatt model [23]. Briefly, male Wistar rats (180-200 g) were anesthetized with tribromoethanol (2.5 mg/kg, i.p.) and after a midline laparotomy a silver clip with an internal diameter of 0.20 mm was placed around the left renal artery. Normotensive two-kidney rats (2K) were only submitted to laparotomy. Animals were maintained on standard rat chow with a 12 h light/dark cycle and given free access to both food (standard rat chow) and water. The systolic blood pressure (SBP) was measured weekly in nonanesthetized animals by an indirect tail-cuff method (MLT125R pulse transducer/pressure cuff coupled to the PowerLab 4/S analog-to-digital converter; AD Instruments Pty Ltd., Castle Hill, Australia) and rats were considered hypertensive when the SBP was higher than 160 mmH, six weeks after surgery.

Vascular reactivity study
Rats were killed by decapitation six weeks after surgery, and the thoracic aortas were isolated and placed in a Krebs solution. Aortas were carefully dissected and mounted as ring preparations (≅4 mm in length) and placed in bath chambers (5 mL) containing Krebs solution at 37° C, continuously bubbled with 95% O2 and 5% CO2, pH 7.4, in an isometric Mulvany-Halpern myograph (model 610 DMT-USA, Marietta, GA) and recorded by a PowerLab8/SP data acquisition system (AD Instruments Pty Ltd., Colorado Springs, CO). The aortic rings were submitted to a tension of 1.5 g, which was readjusted every 15 min during a 60 min equilibration period before addition of the given drug. Endothelial integrity was assessed by the degree of relaxation induced by 1μmol/L acetylcholine in the presence of contractile tone induced by phenylephrine (0.1μmol/l). The ring was discarded if relaxation with acetylcholine was lower than 80% in 2K and 60% in 2K-1C rat aortas. After the endothelial integrity test, aortic rings were pre-contracted with phenylephrine (0.1μM). When the plateau was reached, concentration-effect curves to acetylcholine (0.1nM to 0.1mM) were constructed in the 2K and 2K-1C aortic rings. The potency (pD2) and the maximum relaxant effect (ME) were evaluated. Aortic rings from 2K and 2K-1C received different treatment for 30 min, including: DCBPY (at concentrations: 0.1; 1.0 and 10µM); DETA-NO (0.1 µM); tempol (1mM). In addition, aortic rings were incubated for 10 min with hydroxicobalamin (100 µM) followed by addition of DCBPY (0.1 µM). After incubation period, aortic rings were washed 3 times to remove drugs and concentration-effect curves to acetylcholine were constructed in aortic rings, pre-contracted with phenylephrine (0.1μM). Control responses were obtained in experiments where DCBPY, DETA-NO and tempol were replaced by PBS.

Detection of NOº by electrode
An electrode selective for NO coupled with a permeable gas membrane was employed (Apollo 4000-Free Radical Analyzer, World Precision Instrument, Sarasota, FL, USA). The electrode was suspended in phosphate buffer solution at pH 7.4 (2 ml) and kept at 37° C, under constant stirring. The electrode was calibrated by using an NO donor PROLI NONOate. The NO donor diethyltryamine NONOate (DETA/NO) was selected for the assays because it can spontaneously release NO in a pH-dependent manner. The NONOate was prepared in basic solution 10 mM NaOH, in order to maintain its stability. DETA/NO (100 µM) was added to the PBS solution and the experiments were initiated when the current was stable, as verified on the recording (data not included).
To the superoxide (O2 -)-scavenging effect of DCBPY, O2was generated at 37º C by using 500 µM hypoxantine (HX) and 5mU xantine oxidase (XO) (Figure 9). To this end, HX was firstly added to the solution, followed by DETA/NO (100 µM). When the current reached a stable value, XO was added, in order to promote consumption of NO, and then DCBPY (12.5, 25, or 50 µM) or SOD (10, 20, or 30 µM) was added, to consume the superoxide.
The formation of O2was spectrophotometrically detected by measuring the reduction of cytochrome c at 550 nm. The inhibition of the reduction of cytochrome c was determined in the presence of DCBPY (1.25, 2.5, 12.5, or 25 µM) or SOD (20 µM). The difference in absorbance (AU) was quantified, and the latter value was employed for statistical analysis.
Cell Culture HUVEC were culture in DMEM (Dulbecco Modified Earle's balanced salt solution) medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 μg/ml). Cells were grown in humidified incubator containing 5% CO2 at 37° C. For NO production experiments, cells were washed and treated with 0.1 μM of DCBPY or DETA-NO for 30 min. NO production was determined using DAF-2 DA cell membrane-permeable NO sensitive fluorescent dye, which converts to DAF-2 and reacts with NO to form a triazole form fluorescente DAF-2T [24]. The increase in fluorescence intensity was monitored with a fluorescence microplate reader (SpectraMax GeminiXS, Molecular Devices) at 485 nm excitation and 515 nm emission for 4 hours at 37° C. Alternatively, NO production was examined in DAF-stained HUVEC in fluorescence microscope (Axiovert, Zeiss, Oberkochen, Germany) at 40X objective lens magnification. The fluorescence intensity (FI) value were obtained from 3 different experiments (n=3) to all protocols performed.

STATISTICAL ANALYSIS
Data are expressed as mean ± S.E.M. In each set of experiments, n indicates the number of rats studied. Statistical analysis of the results was performed by using GraphPad Prism version 3.0. Statistical significance was tested by one-way ANOVA followed by Newman-Keuls post hoc analysis. Values of p < 0.05 were considered significant Drugs and chemicals Acetylcholine, phenylephrine, DETA-NO, TEMPOL, hydroxocobalamin, PROLI NONOate, were purchased from Sigma-Aldrich (St.Louis, MO, USA).

Nitric oxide, superoxide detection and interaction between nitric oxide and superoxide.
To evaluate if DCBPY releases NO in endothelial cells over 30 min (which corresponds to the effects observed in aortic rings treatment), HUVEC were treated with 0.1μM of DCBPY and DETA-NO. The latter used as control of spontaneous NO donor. Figure 6A shows that DCBPY did not change in NO concentration during the time monitored (PBS: 4.32 ± 0.18 FI, n=3; compared to incubation with 0.1 µM of DCBPY: 4.43 ± 0.31 FI, n=4). However, DETA-NO showed an increase in NO in relation to its respective control (6.20 ± 0.21 FI, n=3, P<0.05). Nitric oxide production was further confirmed by fluorescence microscopy images also indicated the accumulation of DAF-2T product as green spots in HUVEC ( Figure 6B). The potential effect of DCBPY with respect to scavenging superoxide was examined by means of the cytochrome c assay. The production of O2was verified by reduction of cytochrome c (93.32 x 10 -3 ± 6.10 x 10 -3 AU, n=4), (Figure 7).  The effect of the interaction between superoxide and NO was investigated. A steady state concentration of NO can be detected over time in a solution containing DETA-NO. The addition of XO (2.70 ± 0.02 µM, n=10) evokes consumption of the NO released from DETA-NO, as predicted by the consumption of NO by O2 - (Figure 8). The DCBPY compound avoids the consumption of NO in a DCBPY-concentrationdependent manner (12.5 µM: 0.92 ± 0.02 µM, n=3; 25 µM: 1.49 ± 0.05 µM, n=4; 50 µM: 3.50 ± 0.12 µM, n=4, p<0.001) ( Figure 8C). SOD also prevents the consumption of NO in an SODconcentration-dependent manner (10 µM: 0.57 ± 0.02 µM, n=3; 20 µM: 1.06 ± 0.03 µM, n=3; 30 µM: 1.36 ± 0.1 µM, n=4, p<0.001) ( Figure 8C). To make sure that in the presence of XO/HX there is no NO release from Ru-NO2 -, the same experiment was conducted in the absence of DETA-NO, and no release of NO was verified, as can be verified in the Figure 8B.

DISCUSSION
The main finding of the present manuscript was that the DCPBY aortic treatment improves the relaxation endothelium dependent in aortic rings with endothelium dysfunction, which is not induced by NO release.
Our results have shown that the endotheliumdependent relaxation induced by acetylcholine is impaired in aortic rings from hypertensive rats (2K-1C). These results are in accordance with previous investigation [25,26,27,14]. In endothelial cells, muscarinic receptor activation by acetylcholine induces NO production by increase cytosolic Ca 2+ concentration with consecutive activation of NO synthase (NOS) enzyme. The NO produced in endothelial cells migrates to smooth muscle cells and activate the soluble guanylyl cyclase (sGC) enzyme, producing cyclic GMP (cGMP), which activates cGMP-dependent protein kinase (PGK), inducing a decrease in cytosolic Ca 2+concentration with vascular relaxation [28].
Produced in low concentrations, NO in the blood is rapidly converted to nitrate and nitrite after 10 seconds of its formation, [29]. This short half-life is even more shortened in various  cardiovascular diseases, as in the hypertension, in which oxidative stress reduces the NO availability. Several models of hypertension stimulate the deficiency in the production / availability of NO, among other alterations that are typical of endothelial dysfunction [30].
The aortic rings treatment with 0.1µM of DCBPY improved the relaxation endothelium dependent induced by acetylcholine just in hypertensive aortic rings. Also, the aortic rings treatment from hypertensive rats with 0.1µM of DETA/NO improved the relaxation endotheliumdependent, suggesting that a NO donor can improve this response. In order to evaluate if DCBPY effect is due to NO release, we have used a NO scavenger (hydroxocobalamin) together with DCBPY. Our results have suggested that this effect is not due to NO release, considering that hydroxocobalamin did not cancel the DCBPY effect. In addition, our results in HUVECs cells confirm that at concentration 0.1µM (incubation for 30 min) the compound DCBPY does not release NO. In previous publication [14] it was verified that the relaxation induced by DCPPY in aortic rings was abolished in the presence of hydroxocobalamin, indicating that hydroxocobalamin is able to scavenge NO release from DCBPY.
Reactive oxygen species (ROS) are important molecules regulating numerous physiological processes. However, their overproduction is harmful as these species could participate in several pathological processes. ROS could contribute to the mechanism of hypertension and are responsible for the cardiovascular diseases and atherosclerosis [31] it increases formation of superoxide radicals, lipid peroxidation and protein oxidation, which can directly promote damage of cellular organelles and DNA [32][33]. The anion superoxide (O2 -) is an important member of the ROS family, which has been found in elevated concentration in the vascular smooth muscle cells from renal hypertensive rats [21,20]. The NO bioavailability could be significantly reduced in the presence of O2because NO reacts with O2to form peroxynitrite (ONOO -) [34]. It has been suggested that ruthenium complex can react with O2and inactivate or scavenge this ROS [22]. Thus, our results suggest that the improvement in endothelial function verified in response to DCBPY treatment is related to O2inactivation, considering that this improvement was verified just in hypertensive aortic rings. In positive control, with tempol (SOD mimetic) aortic treatment, the endothelial function improvement also was verified just in hypertensive aortic rings, suggesting that the same mechanism is present in response to DCBPY and tempol. The next step was to evaluate the potential action of DCBPY with respect to scavenging superoxide, by means of the cytochrome c assay. Our results suggested that DCBPY inactivate O2 -in a concentration dependent manner. In addition, the effect of DCBPY on NO consumption by O2was evaluated. Our results indicated that DCBPY prevents the NO consumption by O2in a DCBPY-concentration-dependent manner. Taken together, our results indicate that DCBPY inactivates O2 -.
In conclusion, our results suggest that the DCBPY at a lower concentration (0.1 µM) is not a NO generator, but it can inactivate superoxide in aortic rings and improves the endothelial function.