Hydrogen sulfide dysfunction in metabolic syndrome-associated vascular complications involves cGMP regulation through soluble guanylyl cyclase persulfidation.

Here, by using in vitro and ex vivo approaches, we elucidate the impairment of the hydrogen sulfide (H2S) pathway in vascular complications associated with metabolic syndrome (MetS). In the in vitro model simulating hyperlipidemic/hyperglycemic conditions, we observe significant hallmarks of endothelial dysfunction, including eNOS/NO signaling impairment, ROS overproduction, and a reduction in CSE-derived H2S. Transitioning to an ex vivo model using db/db mice, a genetic MetS model, we identify a downregulation of CBS and CSE expression in aorta, coupled with a diminished L-cysteine-induced vasorelaxation. Molecular mechanisms of eNOS/NO signaling impairment, dissected using pharmacological and molecular approaches, indicate an altered eNOS/Cav-1 ratio, along with reduced Ach- and Iso-induced vasorelaxation and increased L-NIO-induced contraction. In vivo treatment with the H2S donor Erucin ameliorates vascular dysfunction observed in db/db mice without impacting eNOS, further highlighting a specific action on smooth muscle component rather than the endothelium. Analyzing the NO signaling pathway in db/db mice aortas, reduced cGMP levels were detected, implicating a defective sGC/cGMP signaling. In vivo Erucin administration restores cGMP content. This beneficial effect involves an increased sGC activity, due to enzyme persulfidation observed in sGC overexpressed cells, coupled with PDE5 inhibition. In conclusion, our study demonstrates a pivotal role of reduced cGMP levels in impaired vasorelaxation in a murine model of MetS involving an impairment of both H2S and NO signaling. Exogenous H2S supplementation through Erucin represents a promising alternative in MetS therapy, targeting smooth muscle cells and supporting the importance of lifestyle and nutrition in managing MetS.

Development of inhalable hyaluronan/mannitol composite dry powders for flucytosine repositioning in local therapy of lung infections.

Flucytosine (5-fluorocytosine, 5-FC) is a fluorinated analogue of cytosine currently approved for the systemic treatment of fungal infections, which has recently demonstrated a very promising antivirulence activity against the bacterial pathogen Pseudomonas aeruginosa. In this work, we propose novel inhalable hyaluronic acid (HA)/mannitol composite dry powders for repositioning 5-FC in the local treatment of lung infections, including those affecting cystic fibrosis (CF) patients. Different dry powders were produced in one-step by spray-drying. Powder composition and process conditions were selected after in depth formulation studies aimed at selecting the 5-FC/HA/mannitol formulation with convenient aerosolization properties and drug release profile in simulated lung fluids. The optimized 5-FC/HA/mannitol powder for inhalation (HyaMan_FC#3) was effectively delivered from different breath-activated dry powder inhalers (DPI) already available to CF patients. Nevertheless, the aerodynamic assessment of fine particles suggested that the developed formulation well fit with a low-resistance DPI. HyaMan_FC#3 inhibited the growth of the fungus Candida albicans and the production of the virulence factor pyoverdine by P. aeruginosa at 5-FC concentrations that did not affect the viability of both wild type (16HBE14o-) and CF (CFBE41o-) human bronchial epithelial cells. Finally, pharmacokinetics of HyaMan_FC#3 inhalation powder and 5-FC solution after intratracheal administration in rats were compared. In vivo results clearly demonstrated that, when formulated as dry powder, 5-FC levels in both bronchoalveolar lavage fluid and lung tissue were significantly higher and sustained over time as compared to those obtained with the 5-FC solution. Of note, when the same 5-FC amount was administered intravenously, no significant drug amount was found in the lung at each time point from the injection. To realize a 5-FC lung concentration similar to that obtained by using HyaMan_FC#3, a 6-fold higher dose of 5-FC should be administered intravenously. Taken together, our data demonstrate the feasibility to deliver 5-FC by the pulmonary route likely avoiding/reducing the well-known side effects associated to the high systemic 5-FC doses currently used in humans. Furthermore, our results highlight that an appropriate formulation design can improve the persistence of the drug at lungs, where microorganisms causing severe infections are located.

OS064. Contribute of the L-cysteine/ H2S pathway in placenta homeostasisin hypertensive disorders.

INTRODUCTION: Hydrogen sulfide (H2S) is considered the third endogenous gas transmitter besides nitric oxide and carbon monoxide [1]. It is produced from L-cysteine or L-methionine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). H2S is involved in the control of vascular homeostasis, having either relaxant or contractant effect on smooth muscle cells. The H2S involvement in rat and human intrauterine tissues has also been shown [2]. OBJECTIVES: The aim of our study was to investigate the L-cysteine/ H2S pathway in rat and human placenta in hypertensive state. METHODS: Placental samples were collected from spontaneous hypertensive rats (SHR) and normotensive rat (Wistar Kyoto; WKY). In parallel, placental samples were collected from 10 pre-eclamptic women and 5 controls after caesarean sections. Pre-eclamptic women were divided into two subgroups: Group1 (women who developed Early Preeclampsia, n=4); Group2 (women who developed Late Preeclampsia, n=6). The expression of CBS and CSE was evaluated in sample tissues by Western blotting analysis. The enzymatic activity was assessed in basal and stimulated (L- cysteine) condiction by a colorimetric assay. Statistical analysis was performed by using Student's t test. P<0,05 was considered as statistically significant. RESULTS: The expression of CBS and CSE in placenta of SHR rats were significantly reduced (p<0.05) compared to WKY. The H2S production resulted significantly (p<0,05) lower in SHR than WKY rats. In human placenta, the basal H2S production was similar in the three groups; interestingly the H2S production by adding L-cysteine, was higher in Late Preeclampsia compared to control group. CONCLUSION: H2S was produced in rat and human placenta. CBS and CSE, the enzymes involved in the production of H2S, were down-regulated in SHR rats and, as a consequence the H2S production was significantly reduced. Starting from these data, we tried to analyze the role of hydrogen sulfide in preeclampsia to assess the contribute of this gas transmitter in the development of this condition. Unexpectedly, preliminary data demonstrated that in women developing Late Preeclampsia there was an higher production of H2S after stimulation with L-cysteine, not revealed in Early Preeclampsia or in healthy control group. Our results indicated that the L-cysteine/H2S pathway could contribute to the development of preeclampsia condition.

Lowered superoxide dismutase in highly metastatic B16 melanoma cells.

Superoxide dismutases (SOD), which are enzymes scavenging the superoxide radical, were studied in two variant lines of the B16 melanoma: B16F1 with low metastatic potential and B16F10 with high metastatic potential. SOD activity was measured by a method utilizing reduction in the chemiluminescence of luminol. Using cell free extracts it was shown that the highly metastatic B16F10 cell line has a SOD activity lower (20.70 +/- 3.07) units/mg protein, n = 8, than that of the less metastatic B16F1 cell line (81.38 +/- 6.78) units/mg protein, n = 8. Acrylamide gel electrophoresis suggested that Mn-SOD activity is higher in B16F1 cells.

Xanthine oxidase activity in intestinal tumors induced by 1,2-dimethylhydrazine dihydrochloride.

1,2-Dimethylhydrazine-induced large bowel tumors in adult male rats contained significantly lower levels of xanthine oxidase and xanthine dehydrogenase activities when compared to levels in normal intestinal tissue. Xanthine dehydrogenase activity in the blood serum of DMH-treated rats was significantly higher than that of the control animals.

Effect of copper on blood serum xanthine dehydrogenase in rats given D-L-ethionine.

Hepatic lesions by D-L-ethionine in rats produce a significant increase in the blood serum xanthine dehydrogenase activity. Cupric acetate is a potent inhibitor (Ki = 3.42 X 10(-6) M) of the xanthine dehydrogenase activity. Ethionine protects against the inhibitory effect of cupric acetate by formation of a copper complex with behaviour different from that of free ethionine.

Efect of cold environment on the rat serum xanthine dehydrigenase in carbon tetrachloride poisoning

Cold acclimatization (2-5-C) not less than 20 days increases the blood serum xanthine dehydrogenase in normal as in CCl4 poisoned rats. The enzyme of the liver remains unchanged

Serum and liver xanthine oxidase activity in tumor bearing rats and mice.

Liver X.O. activity does not change significantly in tumor bearers although it is known that in this case there is a severe protein deficiency (hypoproteinosis). A decrease in the blood serum X.D. activity was observed in tumor bearing rats but no change occurred in the level of the plasmatic protein. It was suggested that the fall in the blood serum enzyme can be due to factors not correlated with the protein deficiency due to the animals inanition.

Kinetic studies of the blood serum xanthine dehydrogenase inhibition by alloxan (2,4,5,6-tetraoxypyrimidine 5,6-dioxyuracil).

Alloxan is an inhibitor of the enzyme xanthine: NAD+ oxido reductase (E.C.1.2.1.37). Alloxan acts as an electron acceptor and competes "in vitro" with the tetrazolium salt used as electron acceptor in the assay system used for the determination of the dehydrogenase activity of the enzyme. The alloxan inhibition was reverted when phenazine methosulfate (PMS) was added to the system.

Regulation of the kidney xanthine dehydrogenase by glutaminase.

The steady-state concentrations of glutamine, glutamate and ammonia in the kidney cells might regulate the rate of renal xanthine dehydrogenase activity. Both glutamate and glutamine were found to be effective inhibitors of the renal xanthine dehydrogenase activity in vivo. The inhibition by glutamate depends essentially on the glutaminase inhibition.

Serum xanthine dehydrogenase of carbon tetrachloride-induced hepatotoxicity in alloxan-diabetic rats.

Xanthine dehydrogenase activity was determined in blood serum of rats in which diabetes had been induced by alloxan administration. The results show that there is no statistical significance in the difference found for normal and diabetic rats. Alloxan produced an inhibition in the enzyme activity in animals in which a carbon tetrachloride hepatotoxicity had been induced.