ToxSci Advance Access originally published online on October 15, 2008
Toxicological Sciences 2009 107(1):298-305; doi:10.1093/toxsci/kfn218
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An Increased Regional Blood Flow Precedes Mesenteric Inflammation in Rats Treated by a Phosphodiesterase 4 Inhibitor
* Université François-Rabelais de Tours, Centre National de la Recherche Scientifique Formation de Recherche en Evolution 3092, F-37200 Tours, France
Drug Safety R&D, Pfizer, Z.I. Pocé-sur-Cisse, BP 159, F-37401, Amboise, France
1 To whom correspondence should be addressed at Université François-Rabelais de Tours, Centre National de la Recherche Scientifique Formation de Recherche en Evolution 3092 and Department of Pharmacology, Faculty of Pharmacy, 31 Avenue Monge, F-37200 Tours, France. Fax: +33-2-47-36-72-07. E-mail: jean-louis.freslon{at}univ-tours.fr.
Received July 25, 2008; accepted October 7, 2008
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ABSTRACT |
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The study was undertaken to assess the hemodynamic effects induced by a single dose of the phosphodiesterase 4 (PDE4) inhibitor, CI-1044, which is known to cause mesenteric vascular alterations in rats. In the present study, an administration of 160 mg/kg of CI-1044 caused perivascular and interstitial inflammation, with infiltrates of admixed neutrophils and macrophages but without evidence of vascular necrosis (ileum, 15/20 rats; duodenum + jejunum, 7/20 rats). Four hours after administration, blood pressure was decreased (– 13%). A fluorescent microsphere technique demonstrated that, in these conditions, cardiac output was doubled (+ 100%) and total peripheral resistance was decreased (– 54%). The largest increases in blood flow were measured in the duodenum (+ 101%), in the jejunum (+ 110%), and in the ileum (+ 192%). Therefore, the mesentery was the most sensitive organ affected by the drug and, within this area, parts with the highest incidence of vascular alteration were those which had shown the highest increase in flow. In addition, isolated precontracted mesenteric resistance arteries dissected from untreated animals were fully relaxed when incubated with increasing concentrations of CI-1044 up to 2.5 x 10–5M. At this latter concentration, contractile abilities and sensitivities to the physiological agonist noradrenaline (NA) and to the thromboxane analogue U46619 [GenBank] were significantly attenuated (– 28 and – 27%, respectively). This effect could lead to a decreased response to NA and possibly to other agonists in vivo consistent with the vasodilation observed with the microsphere technique. These data provide evidence that the PDE4 inhibitor CI-1044 induces changes of vascular tone that could lead to histological alterations in the mesenteric area.
Key Words: drug-induced vascular injury; mesenteric artery; PDE4 inhibitor; mesenteric blood flow; vasoreactivity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Phosphodiesterases (PDEs), a superfamilly of enzymes degrading cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, are the targets of specific therapeutic agents (Beavo, 1995
; Bender and Beavo, 2006; Lugnier, 2006). PDE4 is the major cAMP-metabolizing enzyme, and it is expressed in inflammatory, immune, and smooth muscle cells (Barnette, 1999; Lugnier, 2006). PDE4 inhibitors are therefore considered to be potential treatments for diseases with both inflammatory and contractile components such as asthma and chronic obstructive pulmonary disease (Boswell-Smith et al., 2006; Lugnier, 2006; Zhang et al., 2005). They may also be promising as memory-enhancing agents and even as antidepressants (Bender and Beavo, 2006). However, several PDE4 inhibitors induced vascular lesions in various organs during preclinical studies. It has been shown that the mesenteric arterial bed is a primary toxicity target organ in rats (Larson et al., 1996; Mecklenburg et al., 2006; Robertson et al., 2001; Slim et al., 2002; 2003; Zhang et al., 2006). Histopathological evaluations of rats treated with PDE4 inhibitors presented mixed inflammatory cell infiltration as well as hemorrhage and segmental medial necrosis which may be transmural or circumferential depending on the severity of the lesion (Dietsch et al., 2006; Slim et al., 2003).
A review of the literature indicates that at least three major mechanistic pathways can contribute to the development of drug-induced vascular injury: (1) biomechanical injury following changes in shear and/or wall stress, (2) toxicity following direct pharmacological and/or chemical compromise, and (3) injury via immunological and/or inflammatory mechanisms, with possible overlap between these mechanisms (Expert Working Group on Drug-Induced Vascular Injury, Kerns et al., 2005). With regards to the first hypothesis, it has been postulated that lesions develop because of marked vasodilation, increased blood flow, decreased shear stress, and increase of wall tension. In the rat, indirect evidence linking increases in localized mesenteric blood flow and development of lesions in the mesenteric area has been proposed (Kerns et al., 1989). However, direct measurements of early and/or sustained alterations in regional blood flow are still lacking (Kerns et al., 2005)
Therefore, the aim of this study was to determine in vivo in the rat the effects of a selective inhibitor of PDE4, CI-1044, on regional blood flow, with a focus on the changes in the mesenteric bed. A previous time course study performed with a dose of 160 mg/kg of CI-1044 showed changes in gene expression from 2 h postdosing, whereas inflammatory mesenteric artery lesions were first detected by histology 16 h postdosing (Dagues et al., 2007a,b). To evaluate variations of systemic and regional hemodynamic parameters in the rat following CI-1044 administration, we used the fluorescent microsphere (FM) technique, which has been fully validated in this species (Gervais et al., 1999; Hakkinen et al., 1995; Sampaio et al., 2003).
In order to further investigate the mechanisms involved in the CI-1044-induced changes in mesenteric blood flow, functional studies in isolated arteries dissected from different vascular beds (aorta, femoral and mesenteric resistance arteries) from nontreated rats were performed. Vasomotor effects of CI-1044 used at a concentration relevant to that observed in vivo in treated animals were also assessed in mesenteric resistance arteries dissected from nontreated animals and precontracted with either the physiological agonist noradrenaline (NA) or a thromboxane analogue, U46619 [GenBank] .
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MATERIALS AND METHODS |
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Animals.
Male Sprague-Dawley rats (Charles River, L'Arbresle, France), 7–2 weeks old, were used. The animals were housed in polyethylene cages in an environment with a controlled temperature of 22°C ± 1°C, a constant humidity of 55 ± 10%, and a 12-h light-dark cycle. Animals had free access to a standard diet (AO3) from SAFE (Epinay sur Orge, France) and tap water ad libitum. The rats were acclimatized for at least 1 week prior experiments and were randomly assigned into different groups. Protocols were approved by Drug Safety R&D Pfizer animal ethical committee.
Chemicals and solutions.
CI-1044 [(R)-N-[9-amino-3,4,6,7-tetrahydro-4-oxo-1-phenylpyrrolo[3,2,1-j,k][1,4] benzodiazepin-3-yl]-3-pyridinecarboxamide], a selective inhibitor of PDE4 with a purity of 98.5%, was obtained from Drug Safety R&D Pfizer (Amboise, France). IC50 (µM) of CI-1044 for the PDE4 isozyme was 0.5 ± 0.2, whereas it was greater than 100µM for PDE3, PDE1, and PDE5 (Burnouf et al., 2000).
For in vivo studies, CI-1044-treated rats received a single oral dose of 160 mg/kg of the drug in suspension in methylcellulose (0.5%) at a volume of 10 ml/kg. Control rats received the same volume of methylcellulose. This dose of CI-1044 was determined from a previous dose-ranging study where three doses were used in the same conditions: 80 mg/kg, which induced a minimal perivascular inflammation in 4/20 animals; 160 mg/kg, which induced both perivascular and interstitial minimal inflammation in 17/20 animals; and 320 mg/kg, which produced the same types of inflammation in 19/20 animals. From these observations, it was decided to retain a mid dose—160 mg/kg—since it affected a high proportion of the animals.
For in vitro studies, CI-1044 was dissolved in dimethyl sulfoxide (DMSO) and then diluted in physiological salt solution (PSS) with the following composition (mM): NaCl 119; KCl 4.7; CaCl2 2.5; MgSO4 1.2; NaHCO3 25; KH2PO4 1.2; glucose 5.5. U46619 [GenBank] , a thromboxane A2 analogue, purchased from Spi-Bio (Montigny Le Bretonneaux, France) was first dissolved in ethanol and then diluted in water. Other reagents of analytical grade were obtained from Sigma-Aldrich (Saint Quentin, France) and dissolved in water.
Histopathology.
Microscopic examination was conducted 16 h postdosing in a separate group of animals composed of 10 control rats and 20 rats treated with CI 1044 at 160 mg/kg. This measurement period was retained since histological alterations were only observed from 16 h after treatment (Dagues et al., 2007b). Heart, skeletal muscle, and mesenteric bed were prepared for evidence of treatment-related changes. On the basis of macroscopical examinations, the mesenteric bed was divided into two parts: one part composed of vessels irrigating the proximal intestinal tract (duodenum + jejunum) and another part composed of vessels irrigating the distal intestinal tract (ileum). Organs were rinsed with saline (NaCl 0.9%), fixed in 10% formalin solution, processed, and embedded in paraffin wax. In all, 4- to 5-µm thick sections were cut and stained with hemalun-eosin and Masson's trichrome and then microscopically examined.
Determination of systemic and regional hemodynamic parameters.
Systemic and regional hemodynamic parameters were determined 2, 4, and 16 h after a single oral administration of vehicle (control) or CI-1044 at the dose of 160 mg/kg (treated). These measurement times were determined from a previous pharmacokinetic study showing that, in the same treatment conditions, plasma concentration of CI-1044 increased from 0 to 4 h postdosing and then it was sustained up to 16 h postdosing. Therefore, in the present study, the measurement time 2 h postdosing was chosen in the ascending phase of the plasma concentration, the 4-h time was at the beginning of the plateau phase, and the 16-h time point was in the plateau phase, at a time when the vascular inflammation could be detected by histopathology (data not shown). Rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) 10 min before the time of measurement. Body temperature monitored with a rectal probe was stabilized at approximately 35°C–36°C. A Teflon catheter connected to a pressure transducer was placed in the left femoral artery to record the blood pressure signal and heart rate (PowerLab ADInstruments, Castle Hill, Australia). The right femoral artery was cannulated and connected to a withdrawal pump (Harvard Apparatus, Les Ulis, France). For administration of FMs, a catheter was inserted into the left ventricle via the right carotid artery. The positioning of the cannula in the left ventricle was confirmed by blood pressure recording.
Systemic hemodynamics and regional blood flow were determined with the reference sample method using 15-µm FMs (ID-TECH-BIOSEB, Chaville, France) as previously reported (Gervais et al., 1999; Raab et al., 1999). Briefly, polystyrene microspheres dyed with orange color were used. To prevent aggregation, microspheres suspended in 0.02% Tween 80 were mixed for 1 min, followed by 20-s sonication. A volume (0.5 ml) containing 200,000 FMs were infused into the left ventricle over a 10-s period and with 0.3 ml of saline over an additional 10-s period. To calculate the blood flow, arterial blood was withdrawn from the right femoral artery at a rate of 0.85 ml/min (reference flow [RF]) in a syringe anticoagulated by adding sodium citrate 4% (wt/vol). Withdrawal was started 10 s before the microsphere injection and continued for a total time of 90 s. At the end of the experiments, the rat was euthanized with an overdose of sodium pentobarbitone and tissues (kidneys, heart, brain, stomach, testis, epididymis, skeletal muscle of inferior limb, and mesenteric bed) were dissected. The mesenteric bed was divided in to three parts: (1) irrigating the duodenum (Mes-Duo), (2) the jejunum (Mes-Jej), and (3) the ileum (Mes-Ile). Tissues were weighed and placed in individual vials (Kunstoff und Metallprodukte, Kappel-Grafenhausen, Germany). Tissues and reference blood samples were digested using a 4 N aqueous KOH solution containing 2% Tween 80. To avoid KOH crystallization, 1.5 ml of isopropanol was added before the digestion period for 24 h at room temperature. Tissues with a higher percentage of fat were stored for 1 week in 20 ml of 1 N hydrochloric acid. After a 5-min immersion in phosphate buffer to neutralize the hydrochloric acid, digestion of these tissues was performed. At the end of the digestion period, samples were filtered and rinsed with 20 ml of phosphate buffer (174mM). The dye was extracted with 2 ml of the organic solvent Cellosolve. The fluorescence intensity of the dye was measured using a spectrofluorimeter (Spectramax Gemini XS; Molecular Devices Ltd, Wokingham GB, 490 and 530 nm).
Mean arterial pressure (MAP, in mm Hg) was calculated as MAP = (SAP + 2 DAP)/3 with SAP and DAP as the systolic and diastolic arterial pressures in mm Hg, respectively. Cardiac output (CO, in ml/min) was calculated as CO = RF (0.85 ml/min) x TF/RF, where TF is total injected fluorescence and RF the fluorescence of the reference blood sample. Regional blood flow (Qo in ml/min/g of organ) was calculated for each organ or part of organ according to the formula Qo = 0.85 ml/min x organ fluorescence/RF x organ weight (g). The cardiac index (CI, in ml/min/kg) was calculated as CI = CO/animal body weight (kg). The total peripheral resistance (TPR, in mm Hg.min.kg/ml) was calculated as MAP/CI.
Adequate mixing of microspheres in the circulation was verified by the absence of significant difference between values of blood flow measured in the right compared to the left kidney (data not shown, see also Table 1).
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In vitro effects of CI-1044 in arteries.
These experiments were performed in preparations dissected from a group of nontreated rats. After anesthesia (sodium pentobarbital, 60 mg/kg, i.p.) and heparinization (500 UI/kg, i.v.), thoracic aortic rings, femoral artery segment (small side branch that feed into the gracilis muscle), and mesenteric bed irrigating the ileum were removed and immediately placed in PSS. After dissection of surrounding tissue, segments (length 1–2 mm) of aorta, femoral, and second-order mesenteric resistance arteries were mounted in a Mulvany-Halpern myograph (Multi Myograph system 610M; JP Trading, Aarhus, Danemark) and were equilibrated for 1 h at 37°C in PSS bubbled with 5% CO2–95% O2. Aortic rings were submitted to a 2-g basal tension. Femoral and mesenteric resistance arteries were stretched for determination of the normalized internal diameter, that is, the diameter the vessel would have in situ when relaxed and under a transmural pressure of 100 mm Hg (Mulvany and Halpern, 1977
). In the present study, the normalized diameter of the mesenteric arteries was 360 ± 8 µm (n = 115) Then, measurements of isometric force (mN/mm) were performed according to the methods described by Mulvany and Halpern (1977). The functional integrity of the endothelium was confirmed routinely by the presence of at least 70% relaxation in response to 10–6M acetylcholine in arteries precontracted with 10–6M U46619
[GenBank]
. In a first set of experiments, aortic rings and femoral and mesenteric resistance arteries preparations were first precontracted with U46619 [GenBank] at 9.10–8M for aortae and 10–6M for femoral and mesenteric arteries. Preparations were then submitted to increasing concentrations from 10–9 to 10–4M of CI-1044 dissolved in DMSO. It was checked that the final concentration of DMSO (0.5%) did not significantly modify the level of the precontraction to U46619. [GenBank] Cumulative concentration-response curves (CCRCs) to CI-1044 were constructed, and relaxations were expressed as percentage of the U46619 [GenBank] -induced precontraction.
In a second set of experiments, functional studies were performed in mesenteric resistance arteries incubated with CI-1044 at the concentration of 2.5 x 10–5M. This concentration was determined from the previous pharmacokinetic study already mentioned, where the plasma concentration of 2.5 x 10–5M was observed during the plateau phase between 4 and 16 h after the administration of CI-1044 (data not shown). To examine the effect of CI-1044 on the contractile response, CCRC to NA and U46619 [GenBank] were determined in the presence of 2.5 x 10–5M CI-1044.
EC50 values were obtained from individual concentration-response relationship by fitting experimental data to a sigmoidal equation using Origin 6.0 (Microcal Software, Northampton, MA). The sensitivity to agonist was assessed by pD2 = – log EC50 (M) and maximal relaxation (Rmax as percentage of U46619 [GenBank] -induced precontraction).
Data and statistical analysis.
Data were reported as the mean ± SD. For each variable, statistical analysis was performed with a one-way ANOVA, the factor being treatment followed by intergroup pairwise comparisons with Bonferroni post hoc test when significant differences were detected. CCRCs were compared by two-way ANOVA with the two factors being treatment and concentration followed by intergroup pairwise comparisons with Bonferroni post hoc test. Differences were considered significant when p < 0.05. Values of "n" represent number of preparations.
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RESULTS |
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Macroscopic and Microscopic Effects of a CI-1044 Treatment
There was no macroscopic lesion observed in the control animals. In the treated rats, 16 h after a single administration of CI-1044 at the dose of 160 mg/kg, yellowish contents of the stomach and/or intestine (18/20 rats treated with CI-1044) and a vasodilatation of abdominal viscera (3/20) were noted. The other macroscopical findings were recorded in the epididymis with red areas (2/20) and in the gastric mucosa with crateriform lesions (1/20).
The histologic evaluation was performed for three organs: mesentery, heart, and skeletal muscle. No inflammation either perivascular or interstitial was observed in control animals. In treated animals, CI-1044-related microscopic findings were localized only in the mesentery. They consisted of multifocal inflammatory infiltrates, mostly in perivascular location, around variably sized vessels (arteries and arterioles, veins and venules, and capillaries) in 15/20 animals (Fig. 1B, arrows). When the lesions were more extensive, the inflammation tended to be interstitial (in the mesenteric, adipose tissue) in 11/20 animals. Infiltrates were composed of admixed neutrophils and macrophages, with a few lymphocytes and fibroblasts. Perivascular inflammatory lesions were more frequent in the distal (ileum, 15/20 animals) compared to the proximal part of the mesentery (duodenum + jejunum, 7/20 animals). There was no evidence of vascular necrosis 16 h postdosing (Fig. 1), and this was in agreement with previous studies, for which vascular necrosis occurred after 2 days of treatment (Dagues et al., 2007a).
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Effect of an Oral Administration of CI-1044 on Systemic and Regional Hemodynamics
The mean arterial blood pressure was significantly decreased (– 13%, p < 0.05) 4 h after a single administration of 160 mg/kg CI-1044 whereas no modification was observed 2 and 16 h postdosing (Fig. 2A).
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The CI was increased (+ 100%, p < 0.05) 4 h postdosing and was stable at 2 and 16 h postdosing (Fig. 2B), whereas the heart rate was not modified (
420 beats/min for the control groups, 428 ± 11 beats/min, 425 ± 16 beats/min, and 419 ± 15 beats/min 2, 4, and 16 h postdosing, respectively). TPR was decreased (– 54%, p < 0.05) 4 h after the CI-1044 administration. Even if a trend to a return to control values was observed 16 h postdosing, the reduction (– 20%) was still significant (p < 0.05) (Fig. 2C).
Effect of an Oral Administration of CI-1044 on Regional Blood Flow
Blood flow in the three parts of the mesenteric bed was increased 2 h postdosing. This variation was only statistically significant for the Mes-Duo area at this time. This increase was more important 4 h postdosing reaching 101, 110, and 192% for the Mes-Duo, Mes-Jej, and Mes-Ile, respectively (p < 0.05). However, this increase was not maintained since the blood flow 16 h postdosing returned almost to the control values (Fig. 3).
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Blood flow in the kidneys and in the epididymis was only increased 4 h postdosing without modification at 2 and 16 h (Table 1).
Blood flow in the heart, brain, stomach, testicles, and skeletal muscle was not modified by the treatment (Table 1).
In Vitro Effects of CI-1044 in Isolated Arteries
Vasorelaxant effect of CI-1044 in arteries from various vascular beds.
CI-1044 induced a concentration-dependent relaxation of precontracted preparations of aorta, femoral, and mesenteric resistance arteries (Fig. 4). This relaxation occurred at lower concentrations of CI-1044 for the mesenteric resistance arteries compared to aorta and femoral artery as shown by the significant (p < 0.05) leftward shift of the CCRC and the significant (p < 0.05) lower values of pD2 (6.0 ± 0.1 for mesenteric resistance arteries vs. 5.0 ± 0.2 and 4.9 ± 0.1 for aorta and femoral artery, respectively). Therefore, the mesenteric vasculature exhibited a greater sensitivity to the in vitro relaxing effects of CI-1044.
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Effect of CI-1044 on agonist-induced vasoconstriction.
The functional effect of CI-1044 on contractile responses of mesenteric resistance arteries to NA and U46619 [GenBank] are shown in Figures 5A and 5B, respectively.
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NA induced a concentration-dependent contraction of mesenteric resistance arteries, which was significantly attenuated from 4.0 ± 0.3 to 2.9 ± 0.3 mN/mm (– 28%, p < 0.001) when rings were exposed to CI-1044 (2.5 x 10–5M) 20 min before and throughout the duration of the CCRC. This was accompanied by a decrease in pD2 (6.1 ± 0.1 to 5.4 ± 0.1, p < 0.01, for control and treated groups, respectively).
The same phenomenon was observed when arteries were challenged with the thromboxane analog U46619 [GenBank] (Fig. 5B). CI-1044 significantly decreased the maximal tension induced by U46619 [GenBank] from 3.8 ± 0.4 to 2.8 ± 0.3 mN/mm (– 26%, p < 0.05). It also shifted to the right the CCRC, showing a decrease in the sensitivity to the agonist (pD2 7.4 ± 0.1 and 6.3 ± 0.1, p < 0.01 for control and treated groups, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Our results showed (1) a significant vasodilatation in rats treated with the PDE4 inhibitor, CI-1044, at a single dose of 160 mg/kg, (2) that the mesenteric vascular bed, which is the most strongly affected area by the vasodilator effect of CI-1044 4 h postdosing, is affected by both perivascular and interstitial inflammation 16 h following dosing, with the distal part being more affected than the proximal part of the intestine, and (3) that this vasodilatation can be related to the inhibition by CI-1044 of the vasoconstrictor response to the physiological agonist NA and possibly to other agonists.
The most significant change in systemic hemodynamics was a decrease in total vascular peripheral resistances showing that the CI-1044 administration was associated with a vasodilatation. This effect could explain the decrease of the MAP and the increase of the CI since heart rate was unchanged. The absence of tachycardia in response to the hypotensive effect of CI-1044 may be related to the possible depressing effect of sodium pentobarbitone (Smith and Hutchins, 1980).
The mesenteric vascular bed was prone to the vasodilatation induced by CI-1044 administration since the most important elevations in regional blood flow were observed in the mesenteric area: approximately 100%—in the vessels irrigating the duodenum and the jejunum—and approximately 200%—in the vessels irrigating the ileum. Supporting these in vivo data, we show for the first time that isolated mesenteric resistance arteries exhibit the highest sensitivity to the vasodilating effects of CI-1044 compared to aorta and femoral artery.
The histological alterations observed in this study are in agreement with previous data showing inflammatory changes only 16 h after CI-1044 administration (Dagues et al., 2007a,b). After a single administration of another PDE4 inhibitor, BYK 169171, an absence of morphological alteration in the mesenteric area was reported (Mecklenburg et al., 2006). However, the number of rats (2) submitted to the protocol could have reduced the possibilities to detect inflammatory lesions. On the other hand, after a chronic administration of BYK 169171, the same group found a moderate to marked inflammatory process in the mesentery, with the doubling of small blood vessels diameter.
It is also interesting to note that the most important increase in mesenteric blood flow was observed in the vessels irrigating the distal part of the intestine which also present a higher incidence of vascular lesions. This observation suggests that the increase in flow induced by CI-1044 would be a contributive factor to the apparition of vascular lesions. Our results also showed an increased blood flow in the epididymis, which is an area in which perivascular inflammation was also found (Dagues, personal communication).
The vasodilatation at 4 h postdose preceded the apparition of multifocal perivascular inflammatory infiltrates in the mesenteric vasculature (16 h postdose). This vasodilatation may contribute to the delayed apparition of the inflammatory vascular injuries via different mechanisms including variations of shear stress. In a gene expression profile study with CI-1044, an upregulation of genes associated with inflammation (coding for TNF-
, IL6, C3, IL18) and genes coding for proteins involved in vascular tone regulation, such as the prostacyclin synthase, were reported (Dagues et al., 2007a). It is interesting to note that prostacyclin synthase has been shown to be increased when human endothelial cells were exposed to an elevation in shear stress (Chen et al., 2003; Okahara et al., 1998), a phenomenon which appears when arteries are submitted to an increase in blood flow as observed in the present study.
The significant vasodilatation observed in the mesenteric vascular bed prompted us to investigate the possible mechanisms involved in this effect. We used third-order mesenteric arteries which can considered as feed but also as resistance arteries since they play a significant role in the control of intestinal blood flow (Fenger-Gron et al., 1997; Mulvany and Aalkjaer, 1990). The tone of this vascular bed and, consequently, its resistance is strongly dependent on the sympathetic system via NA release (Fenger-Gron et al., 1997; Furness and Marshall, 1974). Our results clearly show that incubation of mesenteric arteries segments isolated from untreated animals with CI-1044 at the concentration of 2.5 x 10–5M, relevant to in vivo conditions, impaired the vasoconstriction induced by NA as shown by the reduced contractile abilities and sensitivities (expressed as pD2) to the agonist. Therefore, these in vitro results lead us to propose that a reduced reactivity to contractile effects of NA probably prevails in the in vivo conditions. Since mesenteric blood flow is largely under the influence of NA release by sympathetic transmission, this could explain the significant increases in blood flow observed in the mesenteric area. Furthermore, similar results were observed using another contractile agent, the thromboxane analogue U46619.
[GenBank]
In vascular smooth muscle, this substance induces a strong contractile response which is due to various activation processes: extracellular calcium entry, protein kinase C, tyrosine kinase, and Rho kinase activation (Ding and Murray, 2005; Huang et al., 2004). We propose that the inhibitory effects of CI-1044 that we observed against NA and U46619
[GenBank]
could possibly develop against any other contractile substance present in the vascular wall (e.g., endothelin, which exhibits the same transduction pathways (Rubanyi and Polokoff, 1994).
In conclusion, this is the first in vivo demonstration of an increased mesenteric blood flow after the administration of a PDE4 inhibitor, CI-1044, which can be explained by the in vitro vascular effects of the drug. Even if hemodynamic alterations may not be the sole event underlying the vascular toxicity of PDE4 inhibitors, these phenomena could play a role in the development of vasculopathy.
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FUNDING |
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Pfizer's Drug Safety R&D (DSRD) (to C.B.); a partnership grant between Université François-Rabelais and Pfizer’s DSRD (Contrat Industriel de Formation par la Recherche to S.K.).
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