Gravitational Experimental Platform for Animal Models, a New Platform at ESA's Terrestrial Facilities to Study the Effects of Micro- and Hypergravity on Aquatic and Rodent Animal Models.

Using rotors to expose animals to different levels of hypergravity is an efficient means of understanding how altered gravity affects physiological functions, interactions between physiological systems and animal development. Furthermore, rotors can be used to prepare space experiments, e.g., conducting hypergravity experiments to demonstrate the feasibility of a study before its implementation and to complement inflight experiments by comparing the effects of micro- and hypergravity. In this paper, we present a new platform called the Gravitational Experimental Platform for Animal Models (GEPAM), which has been part of European Space Agency (ESA)'s portfolio of ground-based facilities since 2020, to study the effects of altered gravity on aquatic animal models (amphibian embryos/tadpoles) and mice. This platform comprises rotors for hypergravity exposure (three aquatic rotors and one rodent rotor) and models to simulate microgravity (cages for mouse hindlimb unloading and a random positioning machine (RPM)). Four species of amphibians can be used at present. All murine strains can be used and are maintained in a specific pathogen-free area. This platform is surrounded by numerous facilities for sample preparation and analysis using state-of-the-art techniques. Finally, we illustrate how GEPAM can contribute to the understanding of molecular and cellular mechanisms and the identification of countermeasures.

S-Nitrosothiols as potential therapeutics to induce a mobilizable vascular store of nitric oxide to counteract endothelial dysfunction.

Endothelial dysfunction predisposing to cardiovascular diseases is defined as an imbalance in the production of vasodilating factors, such as nitric oxide (NO), and vasoconstrictive factors. To insure its physiological role, NO, a radical with very short half-life, requires to be stored and transported to its action site. S-nitrosothiols (RSNOs) like S-nitrosoglutathione (GSNO) represent the main form of NO storage within the vasculature. The NO store formed by RSNOs is still bioavailable to trigger vasorelaxation. In this way, RSNOs are an emerging class of NO donors with a potential to restore NO bioavailability within cardiovascular disorders. The aim of this study was to compare S-nitrosothiols ability, formed of peptide (glutathione) like the physiologic GSNO or derived from amino acids (cysteine, valine) like the synthetics S-nitroso-N-acetylcysteine (NACNO) and S-nitroso-N-acetylpenicillamine (SNAP), respectively, to produce a vascular store of NO either in endothelium-intact or endothelium-removed aortae in order to evaluate whether RSNOs can be used as therapeutics to compensate endothelial dysfunction. Sodium nitroprusside (SNP), a marketed drug already in clinics, was used as a non-RSNO NO-donor. Endothelium-intact or endothelium-removed aortae, isolated from normotensive Wistar rats, were exposed to RSNOs or SNP. Then, NO-derived (NOx) species, representing the NO store inside the vascular wall, were quantified using the diaminonaphthalene probe coupled to mercuric ions. The bioavailability of the NO store and its ability to induce vasodilation was tested using N-acetylcysteine, then its ability to counteract vasoconstriction was challenged using phenylephrine (PHE). All the studied RSNOs were able to generate a NO store materialized by a three to five times increase in NOx species inside aortae. NACNO was the most potent RSNO to produce a vascular NO store bioavailable for vasorelaxation and the most efficient to induce vascular hyporeactivity to PHE in endothelium-removed aortae. GSNO and SNAP were equivalent and more efficient than SNP. In endothelium-intact aortae, the NO store was also formed whereas it seemed less available for vasorelaxation and did not influence PHE-induced vasoconstriction. In conclusion, RSNOs - NACNO in a better extent - are able to restore NO bioavailability as a functional NO store within the vessel wall, especially when the endothelium is removed. This was associated with a hyporeactivity to the vasoconstrictive agent phenylephrine. Treatment with RSNOs could present a benefit to restore NO-dependent functions in pathological states associated with injured endothelium.

S-nitrosoglutathione inhibits cerebrovascular angiotensin II-dependent and -independent AT1 receptor responses: A possible role of S-nitrosation.

BACKGROUND AND PURPOSE: Angiotensin II (AngII) and NO regulate the cerebral circulation. AngII AT1 receptors exert ligand-dependent and ligand-independent (myogenic tone [MT]) vasoconstriction of cerebral vessels. NO induces post-translational modifications of proteins such as S-nitrosation (redox modification of cysteine residues). In cultured cells, S-nitrosation decreases AngII's affinity for the AT1 receptor. The present work evaluated the functional consequences of S-nitrosation on both AngII-dependent and AngII-independent cerebrovascular responses. EXPERIMENTAL APPROACH: S-Nitrosation was induced in rat isolated middle cerebral arteries by pretreatment with the NO donors, S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP). Agonist-dependent activation of AT1 receptors was evaluated by obtaining concentration-response curves to AngII. Ligand-independent activation of AT1 receptors was evaluated by calculating MT (active vs. passive diameter) at pressures ranging from 20 to 200 mmHg in the presence or not of a selective AT1 receptor inverse agonist. KEY RESULTS: GSNO or SNP completely abolished the AngII-dependent AT1 receptor-mediated vasoconstriction of cerebral arteries. GSNO had no impact on responses to other vasoconstrictors sharing (phenylephrine, U46619) or not (5-HT) the same signalling pathway. MT was reduced by GSNO, and the addition of losartan did not further decrease MT, suggesting that GSNO blocks AT1 receptor-dependent MT. Ascorbate (which reduces S-nitrosated compounds) restored the response to AngII but not the soluble GC inhibitor ODQ, suggesting that these effects are mediated by S-nitrosation rather than by S-nitrosylation. CONCLUSIONS AND IMPLICATIONS: In rat middle cerebral arteries, GSNO pretreatment specifically affects the AT1 receptor and reduces both AngII-dependent and AngII-independent activation, most likely through AT1 receptor S-nitrosation.

Synthesis and evaluation of new designed multiple ligands directed towards both peroxisome proliferator-activated receptor-γ and angiotensin II type 1 receptor.

Because of the complex biological networks, many pathologic disorders fail to be treated with a molecule directed towards a single target. Thus, combination therapies are often necessary, but they have many drawbacks. An alternative consists in building molecules intended to interact with multiple targets, called designed multiple ligands. We followed such a strategy in order to treat metabolic syndrome, by setting up molecules directed towards both type 1 angiotensin II (AT1) receptor and peroxisome proliferator-activated receptor-γ (PPAR-γ). For this purpose, many molecules were prepared by merging both pharmacophores following three different strategies. Their ability to activate PPAR-γ and to block AT1 receptors were evaluated in vitro. This strategy led to the preparation of many new PPAR-γ activating and AT1 blocking molecules. Among them, some exhibited both activities, highlighting the convenience of this approach.

No answer to the lack of specificity: mouse monoclonal antibody targeting the angiotensin II type 1 receptor AT1 fails to recognize its target.

Numerous antibodies targeting G protein-coupled receptors (GPCRs) have been described as non-specific among the polyclonal antibodies against angiotensin II type 1 receptor (AT1). We have tested the newly developed AT1 receptor mouse monoclonal antibody for its specificity. Human embryonic kidney (HEK293) cells, which do not endogenously express AT1 receptor, were transfected in order to overexpress a fluorescently labeled enhanced green fluorescent protein (EGFP)-tagged human AT1 receptor. Western blot and immunofluorescence assays were performed to test the specificity of the Santa Cruz monoclonal antibody sc-57036. These results were compared to the ones obtained with the polyclonal sc-1173 anti-AT1 receptor antibodies that have already been described as non-specific. While the positive controls using GFP antibodies detected the EGFP-tagged AT1 receptor, both polyclonal and monoclonal anti-AT1 receptor antibodies failed to specifically recognize the corresponding band by Western blot, as similar bands were revealed in either transfected or non-transfected cells. It also failed to detect AT1 receptor in immunofluorescence experiments. The lack of target recognition of the monoclonal AT1 receptor antibody in our experimental conditions suggests that this antibody could give misleading results such as misidentification of the protein. To our knowledge, no specific antibodies targeting AT1 receptors have been developed so far and the field is thus in need of new technical developments.

Intestinal absorption of S-nitrosothiols: Permeability and transport mechanisms.

S-Nitrosothiols, a class of NO donors, demonstrate potential benefits for cardiovascular diseases. Drugs for such chronic diseases require long term administration preferentially through the oral route. However, the absorption of S-nitrosothiols by the intestine, which is the first limiting barrier for their vascular bioavailability, is rarely evaluated. Using an in vitro model of intestinal barrier, based on human cells, the present work aimed at elucidating the mechanisms of intestinal transport (passive or active, paracellular or transcellular pathway) and at predicting the absorption site of three S-nitrosothiols: S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-l-cysteine (NACNO) and S-nitroso-N-acetyl-d-penicillamine (SNAP). These S-nitrosothiols include different skeletons carrying the nitroso group, which confer different physico-chemical characteristics and biological activities (antioxidant and anti-inflammatory). According to the values of apparent permeability coefficient, the three S-nitrosothiols belong to the medium class of permeability. The evaluation of the bidirectional apparent permeability demonstrated a passive diffusion of the three S-nitrosothiols. GSNO and NACNO preferentially cross the intestinal barrier though the transcellular pathway, while SNAP followed both the trans- and paracellular pathways. Finally, the permeability of NACNO was favoured at pH 6.4, which is close to the pH of the jejunal part of the intestine. Through this study, we determined the absorption mechanisms of S-nitrosothiols and postulated that they can be administrated through the oral route.

Aging and hypertension decrease endothelial NO-related dilating function and gamma-glutamyl transferase activity but not S-nitrosoglutathione-induced aortic vasodilation.

S-nitrosoglutathione (GSNO), which is involved in the transport and the storage of NO, induces vasorelaxation. It requires gamma-glutamyl transferase (GGT), an enzyme present on the endothelium, to transfer NO into the cell. We evaluated whether aging and hypertension, which induce NO-related dilating dysfunction, are associated with decreased vascular GGT activity and modify the vasorelaxant effect of GSNO. Thoracic aortic rings isolated from male spontaneous hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) aged 20-22 (adult) or 57-60 weeks (mature) were preconstricted with phenylephrine, then submitted to concentration-vasorelaxant response curves (maximal response: Emax ; pD2 ) to GSNO and carbachol (the latter to measure NO-related dilating function). GGT activity was measured using chromogenic substrate. Both aging and hypertension lowered Emax values for carbachol (Emax -8% in adult SHR, -42% in mature SHR vs. age-matched WKY, page and phypertension  < 0.05) demonstrating NO-related dilating dysfunction. Aortic GGT activity also decreased with aging and hypertension (-22% in adult and -75%, reaching 3 nmol/min/g of tissue, in mature SHR vs. 12 in age-matched WKY and 23 in adult WKY, page and phypertension  < 0.05). The pD2 values of GSNO were similar in mature SHR and WKY but higher in adult SHR (pinteraction  < 0.05). Aging in hypertensive rats decreased NO-related vasorelaxant function and vascular GGT activity, but did not lower the vasorelaxant response to GSNO. This opens perspectives for GSNO-based therapeutics restoring nitric oxide bioavailability and vascular protection in a context of endothelial dysfunction.

In vivo and in silico evaluation of a new nitric oxide donor, S,S'-dinitrosobucillamine.

PURPOSE: In a previous work, we have synthetized a new dinitrosothiol, i.e. S,S'-dinitrosobucillamine BUC(NO)2 combining S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO) in its structure. When exposed to isolated aorta, we observed a 1.5-fold increase of •NO content and a more potent vasorelaxation (1 log higher pD2) compared to NACNO and SNAP alone or combined (Dahboul et al., 2014). In the present study, we analyzed the thermodynamics and kinetics for the release of •NO through computational modeling techniques and correlated it to plasma assays. Then BUC(NO)2 was administered in vivo to rats, assuming it will induce higher and/or longer hypotensive effects than its two constitutive S-mononitrosothiols. METHODS: Free energies for the release of •NO entities have been computed at the density functional theory level assuming an implicit model for the aqueous environment. Degradation products of BUC(NO)2 were evaluated in vitro under heating and oxidizing conditions using HPLC coupled with tandem mass spectrometry (MS/MS). Plasma from rats were spiked with RSNO and kinetics of RSNO degradation was measured using the classical Griess-Saville method. Blood pressure was measured in awake male Wistar rats using telemetry (n = 5, each as its own control, 48 h wash-out periods between subcutaneous injections under transient isoflurane anesthesia, random order: 7 mL/kg vehicle, 3.5, 7, 14 µmol/kg SNAP, NACNO, BUC(NO)2 and an equimolar mixture of SNAP + NACNO in order to mimic the number of •NO contained in BUC(NO)2). Variations of mean (ΔMAP, reflecting arterial dilation) and pulse arterial pressures (ΔPAP, indirectly reflecting venodilation, used to determine effect duration) vs. baseline were recorded for 4 h. RESULTS: Computational modeling highlights the fact that the release of the first •NO radical in BUC(NO)2 requires a free energy which is intermediate between the values obtained for SNAP and NACNO. However, the release of the second •NO radical is significantly favored by the concerted formation of an intramolecular disulfide bond. The corresponding oxidized compound was also characterized as related substance obtained under degradation conditions. The in vitro degradation rate of BUC(NO)2 was significantly greater than for the other RSNO. For equivalent low and medium •NO-load, BUC(NO)2 produced a hypotension identical to NACNO, SNAP and the equimolar mixture of SNAP + NACNO, but its effect was greater at higher doses (-62 ± 8 and -47 ± 14 mmHg, maximum ΔMAP for BUC(NO)2 and SNAP + NACNO, respectively). Its duration of effect on PAP (-50%) lasted from 35 to 95 min, i.e. shorter than for the other RSNO (from 90 to 135 min for the mixture SNAP + NACNO). CONCLUSION: A faster metabolism explains the abilities of BUC(NO)2 to release higher amounts of •NO and to induce larger hypotension but shorter-lasting effects than those induced by the SNAP + NACNO mixture, despite an equivalent •NO-load.

Regulation of protein function by S-nitrosation and S-glutathionylation: processes and targets in cardiovascular pathophysiology.

Decades of chemical, biochemical and pathophysiological research have established the relevance of post-translational protein modifications induced by processes related to oxidative stress, with critical reflections on cellular signal transduction pathways. A great deal of the so-called 'redox regulation' of cell function is in fact mediated through reactions promoted by reactive oxygen and nitrogen species on more or less specific aminoacid residues in proteins, at various levels within the cell machinery. Modifications involving cysteine residues have received most attention, due to the critical roles they play in determining the structure/function correlates in proteins. The peculiar reactivity of these residues results in two major classes of modifications, with incorporation of NO moieties (S-nitrosation, leading to formation of protein S-nitrosothiols) or binding of low molecular weight thiols (S-thionylation, i.e. in particular S-glutathionylation, S-cysteinylglycinylation and S-cysteinylation). A wide array of proteins have been thus analyzed in detail as far as their susceptibility to either modification or both, and the resulting functional changes have been described in a number of experimental settings. The present review aims to provide an update of available knowledge in the field, with a special focus on the respective (sometimes competing and antagonistic) roles played by protein S-nitrosations and S-thionylations in biochemical and cellular processes specifically pertaining to pathogenesis of cardiovascular diseases.

Polymer nanocomposites enhance S-nitrosoglutathione intestinal absorption and promote the formation of releasable nitric oxide stores in rat aorta.

Alginate/chitosan nanocomposite particles (GSNO-acNCPs), i.e. S-nitrosoglutathione (GSNO) loaded polymeric nanoparticles incorporated into an alginate and chitosan matrix, were developed to increase the effective GSNO loading capacity, a nitric oxide (NO) donor, and to sustain its release from the intestine following oral administration. Compared with free GSNO and GSNO loaded nanoparticles, GSNO-acNCPs promoted 2.7-fold GSNO permeation through a model of intestinal barrier (Caco-2 cells). After oral administration to Wistar rats, GSNO-acNCPs promoted NO storage into the aorta during at least 17h, as highlighted by (i) a long-lasting hyporeactivity to phenylephrine (decrease in maximum vasoconstrictive effect of aortic rings) and (ii) N-acetylcysteine (a thiol which can displace NO from tissues)-induced vasodilation of aorxxtic rings preconstricted with phenylephrine. In conclusion, GSNO-acNCPs enhance GSNO intestinal absorption and promote the formation of releasable NO stores into the rat aorta. GSNO-acNCPs are promising carriers for chronic oral application devoted to the treatment of cardiovascular diseases.

Reduced Activity of the Aortic Gamma-Glutamyltransferase Does Not Decrease S-Nitrosoglutathione Induced Vasorelaxation of Rat Aortic Rings.

Aims: Gamma-glutamyl transferase (GGT), an enzyme present on the endothelium, is involved in the release of nitric oxide (NO) from S-nitrosoglutathione (GSNO) and in the GSNO-induced vasodilation. Endogenous GSNO is a physiological storage form of NO in tissues while exogenous GSNO is an interesting candidate for compensating for the decreased NO bioavailability occurring during cardiovascular diseases. We investigated in a rat model of human hypertension, the spontaneous hypertensive rat (SHR), submitted or not to high salt diet, whether a decreased vascular GGT activity modifies the vasorelaxant effect of GSNO. Methods: Thoracic aortic rings isolated from male SHR and Wistar Kyoto rats (WKY) aged 20-22 weeks-submitted or not for 8 weeks to a high salt diet (1% w/v NaCl in drinking water) were pre-constricted with phenylephrine then submitted to concentration-vasorelaxant response curves (maximal response: Emax; pD2) to carbachol or sodium nitroprusside to evaluate endothelial dependent or independent NO-induced vasodilation, or GSNO (exogenous NO vasodilation depending from the endothelial GGT activity). GGT activity was measured using a chromogenic substrate in aortic homogenates. Its role in GSNO-induced relaxation was assessed following inhibition of the enzyme activity (serine-borate complex). That of protein disulfide isomerase (PDI), another redox sensitive enzyme involved in GSNO metabolism, was assessed following inhibition with bacitracin. Results: Aortic GGT activity (18-23 µmol/min/mg of tissue in adult WKY) decreased by 33% in SHR and 45% in SHR with high salt diet. Emax and pD2 for sodium nitroprusside were similar in all groups. Emax for carbachol decreased by -14%, reflecting slight endothelial NO-dependent dysfunction. The GSNO curve was slightly shifted to the left in SHR and in SHR with high salt diet, showing a small enhanced sensitivity to GSNO. Involvements of GGT, as that of PDI, in the GSNO effects were similar in all groups (pD2 for GSNO -0.5 to -1.5 following enzymatic inhibition). Conclusion: Hypertension is associated with a decreased aortic GGT activity without decreasing the vasorelaxant effects of GSNO, whose bioactivity may be supplemented through the alternative enzymatic activity of PDI.

In Situ Microparticles Loaded with S-Nitrosoglutathione Protect from Stroke.

Treatment of stroke, especially during the first hours or days, is still lacking. S-nitrosoglutathione (GSNO), a cerebroprotective agent with short life time, may help if administered early with a sustain delivery while avoiding intensive reduction in blood pressure. We developed in situ forming implants (biocompatible biodegradable copolymer) and microparticles (same polymer and solvent emulsified with an external oily phase) of GSNO to lengthen its effects and allow cerebroprotection after a single subcutaneous administration to Wistar rats. Arterial pressure was recorded for 3 days (telemetry, n = 14), whole-blood platelet aggregation up to 13 days (aggregometry, n = 58), and neurological score, cerebral infarct size and edema volume for 2 days after obstruction of the middle cerebral artery by autologous blood clots (n = 30). GSNO-loaded formulations (30 mg/kg) induced a slighter and longer hypotension (-10 vs. -56 ± 6 mmHg mean arterial pressure, 18 h vs. 40 min) than free GSNO at the same dose. The change in pulse pressure (-50%) lasted even up to 42 h for microparticles. GSNO-loaded formulations (30 mg/kg) prevented the transient 24 h hyper-aggregability observed with free GSNO and 7.5 mg/kg-loaded formulations. When injected 2 h after stroke, GSNO-loaded microparticles (30 mg/kg) reduced neurological score at 24 (-62%) and 48 h (-75%) vs. empty microparticles and free GSNO 7.5 mg/kg and, compared to free GSNO, divided infarct size by 10 and edema volume by 8 at 48 h. Corresponding implants reduced infarct size and edema volume by 2.5 to 3 times. The longer (at least 2 days) but slight effects on arterial pressures show sustained delivery of GSNO-loaded formulations (30 mg/kg), which prevent transient platelet hyper-responsiveness and afford cerebroprotection against the consequences of stroke. In conclusion, in situ GSNO-loaded formulations are promising candidates for the treatment of stroke.

Impact of short-term treatment with telmisartan on cerebral arterial remodeling in SHR.

BACKGROUND AND PURPOSE: Chronic hypertension decreases internal diameter of cerebral arteries and arterioles. We recently showed that short-term treatment with the angiotensin II receptor blocker telmisartan restored baseline internal diameter of small cerebral arterioles in spontaneously hypertensive rats (SHR), via reversal of structural remodeling and inhibition of the angiotensin II vasoconstrictor response. As larger arteries also participate in the regulation of cerebral circulation, we evaluated whether similar short-term treatment affects middle cerebral arteries of SHR. METHODS: Baseline internal diameters of pressurised middle cerebral arteries from SHR and their respective controls, Wistar Kyoto rats (WKY) and responses to angiotensin II were studied in a small vessel arteriograph. Pressure myogenic curves and passive internal diameters were measured following EDTA deactivation, and elastic modulus from stress-strain relationships. RESULTS: Active baseline internal diameter was 23% lower in SHR compared to WKY, passive internal diameter (EDTA) 28% lower and elastic modulus unchanged. Pressure myogenic curves were shifted to higher pressure values in SHR. Telmisartan lowered blood pressure but had no effect on baseline internal diameter nor on structural remodeling (passive internal diameter and elastic modulus remained unchanged compared to SHR). Telmisartan shifted the pressure myogenic curve to lower pressure values than SHR. CONCLUSION: In the middle cerebral arteries of SHR, short-term treatment with telmisartan had no effect on structural remodeling and did not restore baseline internal diameter, but allowed myogenic tone to adapt towards lower pressure values.

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

S-nitrosothiols (RSNO) are considered as potential drugs for delivering nitric oxide (NO) or related species in cardiovascular disorders associated with decrease in NO bioavailability. We have synthesized a new RSNO, i.e. S,S'-dinitrosobucillamine (BUC(NO)2), which combines in its structure two S-mononitrosothiols, S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO). Synthesized BUC(NO)2 was structurally characterized using high-performance liquid chromatography/mass spectrometry (HPLC/MS), (1)H nuclear magnetic resonance ((1)H NMR), infrared (IR) and UV-visible spectroscopies, and thermal analysis; resulting data are consistent with the expected structure. The vasorelaxant effect of BUC(NO)2 was evaluated using isolated rat aortic rings and compared to SNAP, NACNO, and to an equimolar mixture of NACNO plus SNAP in order to mimic the number of NO contained in a BUC(NO)2 molecule. BUC(NO)2 (pD2=7.8±0.1) was more potent in vasorelaxation than NACNO (pD2=6.4±0.2), SNAP (pD2=6.7±0.1) and the mixture of SNAP plus NACNO (pD2=6.7±0.2). The release of NO from BUC(NO)2 was 6-fold that of the basal value and significantly higher than the release of NO from the SNAP plus NACNO mixture (4-fold increase versus basal value). Finally, the role of protein disulfide isomerase (PDI) in BUC(NO)2 metabolism was investigated. Vasorelaxant effect (pD2=6.8±0.2) and NO release decreased in the presence of a PDI inhibitor (both P<0.05 versus BUC(NO)2). In conclusion, BUC(NO)2 releases a larger amount of NO into the aorta, partially through PDI activation, and induces vasorelaxation at lower concentrations than other RSNO previously reported.

Are in situ formulations the keys for the therapeutic future of S-nitrosothiols?

S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) were formulated into in situ forming implants (ISI) and microparticles (ISM) using PLGA and either N-methyl-2-pyrrolidone (NMP) or triacetin. Physicochemical characterization was carried out, including the study of matrix structure and degradation. A strong correlation between drug hydrophobicity and the in vitro release profiles was observed: whatever the formulation, GSNO and SNAP were completely released after ca. 1 day and 1 week, respectively. Then, selected formulations (i.e., SNAP-loaded NMP formulations) demonstrated the ability to sustain the vasodilation effect of SNAP, as shown by monitoring the arterial pressure (telemetry) of Wistar rats after subcutaneous injection. Both ISI and ISM injections resulted in a 3-fold extended decrease in pulse arterial pressure compared with the unloaded drug, without significant decrease in the mean arterial pressure. Hence, the results emphasize the suitability of these formulations as drug delivery systems for S-nitrosothiols, widening their therapeutic potential.

Differential effects of short-term treatment with two AT1 receptor blockers on diameter of pial arterioles in SHR.

Chronic treatment with angiotensin receptor blockers is largely accepted for protecting cerebral circulation during hypertension, but beneficial effects of short-term treatments are questionable, as highlighted by the recent SCAST trial. We compared the impact of 10 days treatment with candesartan (as SCAST) versus telmisartan (previously described to reverse arteriolar remodeling, chronic treatment) on pial arterioles of spontaneously hypertensive rats (SHR). We explored whether PPAR-gamma agonist activity or AT(1) receptor blockade are involved in their differential effects. In the first study, 4-month-old male SHR were treated with telmisartan (TELMI, 2 mg/kg per day) or candesartan cilexetil (CANDE, 10 mg/kg per day) and compared to vehicle treated SHR and normotensive WKY. In a second study, SHR were treated with CANDE, pioglitazone (a PPAR-gamma agonist, PIO 2.5 mg/kg per day) or CANDE+PIO, compared to TELMI. Internal diameter of pial arterioles (ID, cranial window) was measured at baseline, during hemorrhage-induced hypotension, or following suffusion of Ang II (10(-6) mol/L) or EDTA inactivation of smooth muscle cells (passive ID). PPAR-gamma and eNOS (target gene of PPAR-gamma) mRNA were evaluated in brain microvessels. For similar antihypertensive effects, TELMI (+44% versus SHR), but not CANDE, increased baseline ID. During hemorrhage, ID in TELMI group was similar to WKY, while ID in SHR and CANDE remained lower. In the second study, TELMI (+36%, versus SHR) and CANDE+PIO (+43%) increased baseline ID, but not CANDE or PIO alone. TELMI (-66%) and CANDE+PIO (-69%), but neither CANDE nor PIO alone, decreased Ang II-induced vasoconstriction. CANDE+PIO, but not CANDE, increased passive ID. In both studies, PPAR-gamma and eNOS expressions were higher in TELMI than CANDE. Short-term treatment with TELMI, but not with CANDE, reverses narrowing of pial arteriolar ID in SHR. This may involve PPAR-gamma related mechanisms, since CANDE+PIO treatment induced similar effects, and a better blockade of AT(1) receptors.

Endothelial γ-glutamyltransferase contributes to the vasorelaxant effect of S-nitrosoglutathione in rat aorta.

S-nitrosoglutathione (GSNO) involved in storage and transport of nitric oxide ((•)NO) plays an important role in vascular homeostasis. Breakdown of GSNO can be catalyzed by γ-glutamyltransferase (GGT). We investigated whether vascular GGT influences the vasorelaxant effect of GSNO in isolated rat aorta. Histochemical localization of GGT and measurement of its activity were performed by using chromogenic substrates in sections and in aorta homogenates, respectively. The role of GGT in GSNO metabolism was evaluated by measuring GSNO consumption rate (absorbance decay at 334 nm), (•)NO release was visualized and quantified with the fluorescent probe 4,5-diaminofluorescein diacetate. The vasorelaxant effect of GSNO was assayed using isolated rat aortic rings (in the presence or absence of endothelium). The role of GGT was assessed by stimulating enzyme activity with cosubstrate glycylglycine, as well as using two independent inhibitors, competitive serine borate complex and non-competitive acivicin. Specific GGT activity was histochemically localized in the endothelium. Consumption of GSNO and release of free (•)NO decreased and increased in presence of serine borate complex and glycylglycine, respectively. In vasorelaxation experiments with endothelium-intact aorta, the half maximal effective concentration of GSNO (EC50 = 3.2 ± 0.5.10(-7) M) increased in the presence of the two distinct GGT inhibitors, serine borate complex (1.6 ± 0.2.10(-6) M) and acivicin (8.3 ± 0.6.10(-7) M), while it decreased with glycylglycine (4.7 ± 0.9.10(-8) M). In endothelium-denuded aorta, EC(50) for GSNO alone increased to 2.3 ± 0.3.10(-6) M, with no change in the presence of serine borate complex. These data demonstrate the important role of endothelial GGT activity in mediating the vasorelaxant effect of GSNO in rat aorta under physiological conditions. Because therapeutic treatments based on GSNO are presently under development, this endothelium-dependent mechanism involved in the vascular effects of GSNO should be taken into account in a pharmacological perspective.

Formulation, characterization and pharmacokinetic studies of coenzyme Q10 PUFA's nanoemulsions.

Coenzyme Q(10) (CoQ(10)) is an insoluble antioxidant molecule with great biological value but exhibit poor bioavailability. To improve the bioavailability of CoQ(10), we have proposed to formulate a nanoemulsion consisting of salmon oil, salmon lecithin, CoQ(10) and water. A commercial oily mixture, based on soybean oil and CoQ(10), was used for comparison, as well as a second oily mixture, composed of salmon lecithin, salmon oil and CoQ(10). Salmon oil and salmon lecithin were used as sources of polyunsaturated fatty acids (PUFA). The maximum solubility of CoQ(10) in salmon oil was 81.30 ± 0.08 mg/mL at 37 °C. Mean droplets size of the control and CoQ(10) nanoemulsions was 164 and 167 nm, respectively. The nanoemulsion was stable during 30 days at 25 °C. Bioavailability was evaluated as the area under the curve of CoQ(10) plasma concentration in male Wistar rats following oral administration of the three formulations of CoQ(10). The nanoemulsion increases at twice the bioavailability of CoQ(10) than conventional oily formulations regardless the nature of used fatty acids (soybean and salmon oils). Prepared nanoemulsion represents a vectorization of both LC-PUFAs and CoQ(10). That could be an interesting way to increase the absorption of these two bioactive molecules with natural low availability.

High salt intake abolishes AT(2)-mediated vasodilation of pial arterioles in rats.

BACKGROUND: Angiotensin II (Ang II) induces constriction (AT(1)) and dilation (AT(2) receptors) of cerebral arterioles. High sodium intake induces changes in receptors expression and loss of AT(2)-mediated vasodilation in extracerebral vessels. We investigated whether high salt modifies the AT(2)-mediated response of cerebral arterioles. METHODS: Three-month-old male Wistar rats received drinking water supplemented or not with 1% NaCl. We measured at day 4 or 30 plasma aldosterone concentration, AT receptors expression (brain microvessels, western blot, RT-qPCR), internal diameter of pial arterioles (cranial window) following suffusion with Ang II (10(-6) mol/l, or 10(-8) mol/l + losartan 10(-5) mol/l), serotonin (5-HT, 10(-6) mol/l), sodium nitroprusside (10(-5) mol/l) and adenosine diphosphate (ADP, 10(-4) mol/l). RESULTS: High salt did not modify arterial pressure, baseline arteriolar diameter, vasoconstriction to Ang II or 5-HT, nor vasodilation to SNP. High salt lowered plasma aldosterone concentration (d4 138 ± 71 not significant vs. control 338 ± 73; d30 150 ± 21 P < 0.05 vs. control 517 ± 79 µmol/l). AT receptors mRNA did not change while protein level of AT(2) receptors decreased at d4 (64 ± 9% of control, P < 0.05). AT(2)-mediated vasodilation (control d4; d30 8 ± 2; 5 ± 2%) was abolished at d4 (-2 ± 2%, P < 0.05) and reversed to vasoconstriction at d30 (-7 ± 2%, P < 0.05). ADP-induced vasodilation is abolished at d30 (2 ± 2, P < 0.05 vs. control 19 ± 4%). CONCLUSION: High salt specifically abolishes AT(2)-mediated vasodilation, immediately, via decreased level of AT(2) receptor protein, and after 30 days, in association with abolition of endothelial vasodilation. Such loss of AT(2)-mediated vasodilation may be deleterious in case of stroke.

Plasma volume and arterial stiffness in the cardiac alterations associated with long-term high sodium feeding in rats.

BACKGROUND: Rats fed an early and long-term high-salt diet (HS, NaCl 8%) developed significant cardiovascular hypertrophy without major changes in blood pressure. The mechanism of this cardiac hypertrophy has not been yet elucidated. METHODS: In the present work, we assessed the influence of volume overload and arterial stiffness on the structural and functional cardiac changes induced by a high salt feeding from weaning to 5 months of age in Sprague-Dawley rats. RESULTS: Cardiac hypertrophy in HS rats was associated with clear augmentation in the size of left ventricular (LV) cardiomyocyte as compared with rats fed regular diet (NS). Echocardiography revealed a marked increase in relative wall thickness. Of note, no alteration of global and regional systolic and diastolic function was detected in HS rats. High sodium consumption was associated with a slight increase in aortic mean and pulse pressure (PP) without effect on pulse wave velocity (PWV) and elastic modulus. Plasma volume and central venous pressure were higher in HS than NS rats. Whereas plasma endothelin level was twofold higher in HS than in NS rats, LV endothelin level was similar in both groups. Treatment by the endothelin receptors blocker bosentan had no detectable effect on the changes induced by HS diet. CONCLUSIONS: High sodium intake was associated with concentric cardiac hypertrophy without change of systolic and diastolic function. Aortic rigidity was not a determinant of cardiac hypertrophy. Beside a likely direct effect of sodium on cardiovascular system the slight increase in arterial pressure and plasma volume play a role.