François Dupuis

Comparative effects of the angiotensin II receptor blocker, telmisartan, and the angiotensin-converting enzyme inhibitor, ramipril, on cerebrovascular structure in spontaneously hypertensive rats.

Objective Antihypertensive treatment with angiotensin-converting enzyme inhibitors (ACEIs) reverses cerebral arteriolar remodeling, thus restoring dilatation and hence the lower limit of cerebral blood flow (CBF) autoregulation (LLCBF). The objective of this study was to determine whether angiotensin II receptor AT1 blockers (ARBs) produce the same effect. Design We examined the effects of treatment with an ARB [telmisartan (TEL), 1.93 ± 0.04 mg/kg per day] or an ACEI [ramipril (RAM), 1.00 ± 0.02 mg/kg per day] on the cerebral circulation in spontaneously hypertensive rats (SHR). Methods Arteriolar pressure and diameter (cranial window) and CBF (laser Doppler) were measured during stepwise hypotensive hemorrhage, before and after deactivation (ethylenediamine tetraacetic acid), in untreated Wistar–Kyoto (WKY) rats and SHR untreated or treated for 3 months with TEL or RAM in the drinking water. Results Treatment normalized arteriolar internal diameter (SHR, 38 ± 3 μm; TEL, 52 ± 2 μm; RAM, 50 ± 2 μm; WKY, 58 ± 4 μm), essentially by reversing eutrophic inward remodeling, and the LLCBF (SHR, 80 ± 11 mmHg; TEL, 60 ± 4 mmHg; RAM, 71 ± 6 mmHg; WKY, 57 ± 5 mmHg). Conclusion The fact that the ARB (TEL) is as effective as an ACEI (RAM) in reversing cerebral arteriolar remodeling suggests that the cerebrovascular AT1 receptor is an underlying mechanism that promotes hypertensive eutrophic inward remodeling.

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.

Captopril improves cerebrovascular structure and function in old hypertensive rats

1 We examined the effects of an angiotensin‐converting enzyme inhibitor (ACEI), captopril, on cerebral arterioles in young and old spontaneously hypertensive rats (SHR). 2 Animals were anesthetized with sodium pentobarbitone (60 mg kg−1 day−1). We measured cerebral blood flow (CBF, arbitrary units) and cerebral arteriolar internal diameter (ID, μm) prior to and during stepwise hypotension (SH) in 6‐ (WKY‐6) and 15‐month‐old (WKY‐15) Wistar Kyoto rats and in age‐matched SHR that were untreated (SHR‐6 and SHR‐15) or treated for 3 months with captopril (SHR‐6C, 105±2 mg kg−1 day−1 and SHR‐15C, 94±1 mg kg−1 day−1). ID and cross‐sectional area of the vessel wall (CSA) were measured in deactivated (EDTA) cerebral arterioles during a second SH. 3 Captopril decreased the lower limit of CBF autoregulation (61±6 in SHR‐6C and 51±2 in SHR‐15C versus 52±6 in WKY‐6 and 62±7 in WKY‐15 and 83±14 mmHg in SHR‐6 and 120±19 mmHg in SHR‐15; P<0.05) and CSA (510±21 in SHR‐6C and 585±25 in SHR‐15C versus 529±12 in WKY‐6 and 549±20 in WKY‐15 and 644±38 mmHg in SHR‐6 and 704±38 mmHg in SHR‐15; P<0.05). 4 Captopril increased cerebral arteriolar external diameter of SHR (105±5 in SHR‐6C and 94±4 in SHR‐15C vs 125±8 in WKY‐6 and 108±3 in WKY‐15 and 83±2 mmHg in SHR‐6 and 80±2 mmHg in SHR‐15 for a pial arteriolar pressure step of 35–39 mmHg; P<0.05). Captopril attenuated increases in cerebral arteriolar distensibility in young SHR. 5 Thus, ACEIs attenuate eutrophic and hypertrophic inward remodeling of cerebral arterioles in young and old SHR, thus decreasing the lower limit of CBF autoregulation.

Impact of treatment with melatonin on cerebral circulation in old rats.

Melatonin deprival in young rats induces alterations in cerebral arteriolar wall similar to those observed during aging: atrophy and a decrease in distensibility. In this study, we examined the effects of melatonin treatment on cerebral arteriolar structure and distensibility and on the lower limit of cerebral blood flow autoregulation (LLCBF) in old rats. We measured cerebral blood flow (arbitrary unit, laser Doppler, open skull preparation) prior to and during stepwise hypotension (SH) in adult (12/13 months) and old (24/25 months) IcoWI and WAG/Rij male rats. Old rats were untreated or treated for 3 months with melatonin (0.39 (IcoWi) and 0.44 (Wag/Rij) mg kg−1 day−1, drinking water). Stress–strain relationships were determined using cross‐sectional area (CSA, μm2, histometry) and values of arteriolar internal diameter (μm) obtained during a second SH following arteriolar deactivation (EDTA, 67 mmol l−1). Aging induced (a) atrophy of the arteriolar wall in IcoWI (616±20 vs 500±27 μm2, P<0.05) but not in WAG/Rij rats (328±25 vs 341±20 μm2), (b) a decrease in arteriolar wall distensibility and (c) an increase in the LLCBF in both strains (67±10 mmHg in 12‐month‐old vs 95±6 mmHg in 24‐month‐old IcoWi, P<0.05 and 53±2 mmHg in 13‐month‐old vs 67±6 mmHg in 25‐month‐old WAG/Rij). Melatonin treatment induced in IcoWI and WAG/Rij rats (a) hypertrophy of the arteriolar wall (643±34 and 435±25 μm2, respectively), (b) an increase in arteriolar wall distensibility and (c) a decrease in the LLCBF (64±6 and 45±4 mmHg, respectively). Melatonin treatment of old rats induced hypertrophy of the arteriolar wall, prevented the age‐linked decrease in cerebral arteriolar distensibility and decreased the LLCBF.

High salt intake abolishes AT2-mediated vasodilation of pial arterioles in rats

Background Angiotensin II (Ang II) induces constriction (AT1) and dilation (AT2 receptors) of cerebral arterioles. High sodium intake induces changes in receptors expression and loss of AT2-mediated vasodilation in extracerebral vessels. We investigated whether high salt modifies the AT2-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 AT2 receptors decreased at d4 (64 ± 9% of control, P < 0.05). AT2-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 AT2-mediated vasodilation, immediately, via decreased level of AT2 receptor protein, and after 30 days, in association with abolition of endothelial vasodilation. Such loss of AT2-mediated vasodilation may be deleterious in case of stroke.

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.

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.

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 AT1 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 AT1 receptors.

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.

Système rénine-angiotensine-aldostérone : vieux système mais piste stratégique de régulation de la circulation cérébrale

Resume Dans un modele d’hypertension arterielle chronique primaire chez l’Homme, le rat spontanement hypertendu (SHR), l’augmentation chronique de la pression sanguine arteriolaire cerebrale est accompagnee d’un remodelage parietal avec hypertrophie et diminution du diametre arteriolaire. Ce dernier phenomene augmente la resistance cerebrovasculaire et permet ainsi le maintien de l’autoregulation du debit sanguin cerebral global a des niveaux de pression arterielle systemique elevee, mais au prix d’une hypoperfusion accentuee a des pressions arteriolaires faibles telles qu’observees dans l’ischemie cerebrale. En utilisant des inhibiteurs de l’enzyme de conversion de l’angiotensine I (IECAs), des antagonistes des recepteurs AT1 de l’angiotensine II (ARAs) et des antagonistes de l’aldosterone (AAs), nous avons demontre que la normalisation de l’hypertrophie parietale arteriolaire par les IECAs et les ARAs, a savoir une restauration de la valeur observee chez le temoin normotendu, passe par une baisse de la pression sanguine arteriolaire. La normalisation du diametre arteriolaire, par contre, passe par un mecanisme pression-independant qui implique l’aldosterone. Il serait interessant d’evaluer comment ce mecanisme aldosterone-dependant de controle du diametre arteriolaire implique le sodium intracellulaire et son impact sur le metabolisme proteique parietal.