Platelets Do Not Alter Flow-Mediated Dilation or Arterial Conduction in vivo.

The aim of this study was to investigate whether platelets contribute to shear stress and vascular conductance in the iliac vascular bed in vivo. Flow-mediated dilation of pig iliac was induced by downstream injection of acetylcholine (50 µg), and separately, conductance (ΔF/ΔP) was calculated. This was carried out before and after removal of 1 L of arterial blood in 240 mL increments, and each 240 mL was spun in a centrifuge (1,500 rcf for 7 min); platelet-rich plasma was replaced with equal volume of heparinised saline and reinjected. The circulating platelet count fell from 369 × 109/L (n = 5) to 165 × 109/L (p = 0.01; n = 4; Student's unpaired t). An increase in flow led to an increase in the iliac diameter by 0.49 ± 0.03 mm (mean ± SEM) before platelet reduction and 0.55 ± 0.05 mm after (p = 0.36, Student's paired t, n = 5); the change in arterial conductance was also not significantly affected by platelet reduction, control: 1.44 ± 0.34 mL/min/mm Hg, after platelet reduction: 1.39 ± 0.04 mm (p = 0.55, Student's paired t, n = 4). Therefore, platelets do not contribute to shear stress or conductance in vivo.

The vascular glycocalyx is not a mechanosensor in conduit arteries in the anesthetized pig.

BACKGROUND: The role of the glycocalyx as the endothelial sensor of an increase in blood flow was assessed in the iliac artery in vivo. METHODS: Acetylcholine-induced flow mediated dilation was evaluated before and after vascular glycocalyx disruption. This was accomplished by exposing the iliac lumen to the chemotactic agent fMLP (1 µM; n = 6 pigs), concomitant heparinase III (100 mU ml-1) and hyaluronidase (14 mg ml-1) (n = 4), and neuraminidase (140 mU ml-1; n = 5), for 20 min in separate iliac artery preparations. Only one lumen intervention per iliac was conducted. RESULTS: For the heparinase III + hyaluronidase experiment, the iliac diameter increased by an average of 0.54 ± 0.11 mm before and 0.45 ± 0.03 mm after the enzymes (P = 0.42; paired Student's t test). The iliac diameter increased by 0.31 ± 0.02 mm before and 0.29 ± 0.07 mm after fMLP exposure (P = 0.7) and the diameter increased by 0.54 ± 0.11 mm before and 0.54 ± 0.09 mm after neuraminidase exposure (P = 0.98). In all cases, the shear stress changes before and after lumen exposure were not significantly different to each other. CONCLUSION: There was no significant reduction in flow mediated dilation of the iliac in response to any of the interventions conducted. Therefore, the vascular endothelial glycocalyx as whole is not required for flow mediated dilation in conduit arteries in the intact animal.

Small and intermediate Ca2+-sensitive K+ channels do not play a role in vascular conductance during resting blood flow in the anaesthetised pig.

Flow-induced dilation in resistance arteries is mediated by endothelium-dependent hyperpolarisation via small and intermediate conducting Ca2+ sensitive K+ channels. The aim of the current study was to assess the effect of blocking both channels, using the toxins apamin and charybdotoxin, on flow-induced dilation in a conduit artery and vascular conductance. Experiments were carried out on the iliac and its vascular bed in anaesthetised pigs (n = 4). Flow-induced dilation and vascular conductance (∆F/∆P) were assessed before and after administration of toxins intra-arterially (i.a.) at 50 µg kg-1. Iliac diameter increased from baseline to 2.39 ± 0.4 mm before and 2.09 ± 0.46 mm after toxin administration, which was not significantly different (P = 0.63, Student's paired t test). Control conductance was 1.49 ± 0.27 ml min-1 mmHg-1 (P < 0.00001, ANOVA), and 1.53 ± 0.18 ml min-1 mmHg-1 (P < 0.00001, ANOVA) in the presence of the toxins which was not significantly different (P = 0.93 homogeneity of regression analysis). There was a small but significant increase in mean arterial pressure after the toxins were administered, from 74 ± 5 to 80 ± 9 mmHg (P = 0.03, Student's paired t test); but all other measured parameters were not significantly affected. Small- and intermediate-conducting Ca2+-sensitive K+ channels are not involved in flow-mediated dilation in conduit arteries and do not play a role in resistance vessel diameter maintenance at resting blood flow.

Lipidic dispersion to reduce food dependent oral bioavailability of fenofibrate: In vitro, in vivo and in silico assessments.

Novel formulations that overcome the solubility limitations of poorly water soluble drugs (PWSD) are becoming ever more critical to a drug development process inundated with these compounds. There is a clear need for developing bio-enabling formulation approaches to improve oral bioavailability for PWSD, but also to establish a range of predictive in vitro and in silico biopharmaceutics based tools for guiding formulation design and forecasting in vivo effects. The dual aim of this study was to examine the potential for a novel lipid based formulation, termed a lipidic dispersion, to enhance fasted state oral bioavailability of fenofibrate, while also assessing the predictive ability of biorelevant in vitro and in silico testing. Formulation as a lipidic dispersion improved both dissolution and solubilisation of fenofibrate through a combination of altered solid state characteristics and incorporation of solubilising lipidic excipients. These changes resulted in an increased rate of absorption and increased maximal plasma concentrations compared to a commercial, micronised product (Lipantil® Micro) in a pig model. Combination of biorelevant in vitro measurements with in silico physiologically based pharmacokinetic (PBPK) modelling resulted in an accurate prediction of formulation performance and forecasts a reduction in food effects on fenofibrate bioavailability through maximising its fasted state dissolution.

Intraluminal hyperglycaemia causes conduit and resistance artery dilatation and inhibits vascular autoregulation in the anaesthetised pig.

The effect of intraluminal hyperglycaemia was investigated in the iliac artery of 11 anaesthetised pigs. Following isolation of a test segment, hyperglycaemic blood (40 mmol·L(-1)) caused a significant dilatation of the artery of 167 ± 208 µm (mean ± SD; n = 6, P = 0.031). Dilatations were reduced by N(G)-nitro-l-arginine methyl esther (250 µg·mL(-1)) from 145 ± 199 to 38 ± 5 µm), but this was not statistically significant (n = 6, P = 0.18). Intra-arterial infusions of d-glucose (20-40 mmol·L(-1)·min(-1)), during graded constrictions, caused statistically significant increases in blood flow (n = 11, P = 0.0013). Vasodilatation was confirmed by measurements of the ratio of immediate pressure steps to flow steps (∂P/∂F) during the graded obstruction experiments, showing a decrease in instantaneous vascular resistance from a control of 0.62 ± 0.30 to 0.33 ± 0.34 mm Hg·mL(-1)·min(-1) (n = 7, P = 0.016). Autoregulation was assessed from the slopes of the plots of steady-state flow versus pressure. There were significant increases in the slope from 2.32 ± 1.03 to 5.88 ± 5.60 mL·min(-1)·(mm Hg)(-1) (n = 7, P = 0.0078), indicating significant impairment of autoregulation. In conclusion, luminal hyperglycaemia relaxes both arterial and resistance vessel smooth muscle.

A novel lipid-based solid dispersion for enhancing oral bioavailability of Lycopene--in vivo evaluation using a pig model.

Lycopene is a potent anti-oxidant, which has been widely reported for its potential benefits at reducing the risks of certain types of cancer e.g. prostate cancer. The oral bioavailability of this highly lipophilic carotenoid is low and highly influenced by the extent of intestinal lymphatic uptake. The aim of this study was to develop an optimised formulation, which allows for efficient absorption following oral administration. A self-emulsifying drug delivery system (SEDDS) and solid dispersion of Lycopene were developed initially. Subsequently, a novel lipid based solid dispersion (LBSD) was designed. Processing via a solid dispersion approach was found to alter the solid state characteristics of Lycopene, as determined by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The bioavailability of Lycopene was significantly increased after oral administration of LBSD to fasted pigs, relative to the commercial product (Lycovit(®)). A clear distinction in terms of Cmax and AUC was observed between Lycovit(®) and LBSD. In conclusion, a novel LBSD formulation was developed to enhance the oral bioavailability of the model lipophilic compound, Lycopene, by enhancing dissolution in the gastrointestinal tract and promoting intestinal lymphatic uptake utilising digestible lipid excipients.

What is the mechanism of flow-mediated arterial dilatation.

The present review attempts to explain the controversies concerning the mechanism of shear stress-mediated arterial dilatation, commonly called flow-mediated arterial dilatation (FMD). Flow-mediated dilatation occurs in an artery when the blood flow to the organ supplied by the artery is increased. There are two hypotheses regarding the stimulus for FMD: (i) a wave of endothelial and smooth muscle hyperpolarization, conducted in a retrograde fashion from the vasodilated peripheral vascular bed towards the relevant conduit artery; and (ii) an increase in shear stress sensed by the endothelial cells. The latter hypothesis is associated with two further postulates concerning the method of mechanotransduction of the shear stress stimulus: (i) direct transmission from endothelial cell cytoskeleton to the vascular smooth muscle to induce dilatation; and (ii) indirect transmission to the endothelial cell cytoskeleton via the glycocalyx. The virtues and inconsistencies of these hypotheses are discussed. The first hypothesis is excluded because a vasodilated peripheral vascular bed does not cause dilation of the upstream conduit artery if an increase in flow within the conduit artery is prevented and because FMD is completely blocked by inhibition of nitric oxide synthase (NOS). It is probable that the stimulus is an increase in shear stress between the blood and the adjacent layer of the arterial wall, the glycocalyx. Ultimately, a change in the endothelial cell cytoskeleton is the likely event that leads to activation of NOS and this activation does not occur without a functioning glycocalyx.

The effect of alphaxalone-alphadolone, propofol, and pentobarbitone anaesthesia on the ß-endorphin and ACTH response to haemorrhage in the pig.

In the literature there appears to be variability in reported levels of certain hormones during haemorrhage, specifically adrenocorticotrophic hormone (ACTH) and ß-endorphin. It is possible that this variability may be due to the choice of anaesthetic. Therefore, the effect of 3 common research-only anaesthetic agents (alphaxalone-alphadolone, propofol, and pentobarbitone) on ACTH and ß-endorphin levels during haemorrhage was assessed in pigs. Animals were divided into 3 groups: group I received alphaxalone-alphadolone (n = 5), group II received propofol (n = 6), and group III received pentobarbitone (n = 6). Pigs were subjected to a continuous fixed-volume haemorrhage under one of the above anaesthetics while being mechanically ventilated. ACTH and ß-endorphin levels increased significantly during haemorrhage under propofol and pentobarbitone anaesthesia but not with alphaxalone-alphadolone. For ACTH there was no significant difference between the groups, whereas for ß-endorphin there was a significant difference between the propofol- and pentobarbitone-anaesthetized pigs. The increase in heart rate during haemorrhage was significantly different between the alphaxalone-alphadolone and propofol as well as between the propofol and pentobarbitone groups. The drop in blood pressure was only significantly different between the alphaxalone-alphadolone- and propofol-anaesthetized pigs. These results indicate that the choice of anaesthetic agent can affect the hormone response to haemorrhage and may account for the variable hormone levels reported in the published literature to date.

Effect of clazosentan, a selective endothelin A receptor antagonist, and tezosentan, a dual endothelin A/B antagonist, on pulsatile shear stress induced constriction of the iliac in the anaesthetized pig.

1. The effects of changes in mean and pulsatile shear stress on the diameter of the iliac of the anaesthetized pig were investigated in the presence of clazosentan and tezosentan. 2. A total of 17 pigs were used. Mean shear stress was increased by infusing acetylcholine downstream (2-20 µg/min) through the deep femoral artery. Pulsatile shear stress was enhanced first by injecting varying volumes (1-10 mL) of calcium gluconate (stock 10 mg/mL) directly into the left ventricle. Second, by electrical stimulation of the left sympathetic nerves to the heart (1-16 Hz, 4 min duration, supramaximal voltage). 3. An increase in mean shear stress induced a vasodilation that was not altered significantly by the selective endothelin A antagonist clazosentan (10 mg/kg i.v.). Similarly, the vasoconstriction induced by an increase in pulsatile shear stress brought about by either calcium gluconate injections or left sympathetic nerve stimulation was unaffected by clazosentan. However, tezosentan (10 mg/kg i.v.), significantly attenuated the vasoconstriction induced by an increase in pulsatile shear stress. 4. In conclusion, an increase in pulsatile shear stress causes vasoconstriction of the pig iliac artery, which is attenuated by dual endothelin receptor antagonism, but not by specific endothelin A blockade.