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Cardiovascular Research 2002 56(2):293-302; doi:10.1016/S0008-6363(02)00538-2
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Serotonin receptor blockade improves distal perfusion after lower limb ischemia in the fatty Zucker rat

Philip Janiaka,*, Pierre Lainéea, Yoann Grataloupa, Charles-Edouard Luytb, Jean-Pierre Bidouarda, Jean-Baptiste Michelb, Steve E O’Connora and Jean-Marc Herberta

aCardiovascular-Thrombosis Department, Sanofi-Synthélabo Research, 1 Avenue Pierre Brossolette, 91385 Chilly-Mazarin Cedex, France
bINSERM U460, CHU Xavier Bichat, 75018 Paris, France

philip.janiak{at}sanofi-synthelabo.com

* Corresponding author. Tel.: +33-1-6979-7833; fax: +33-1-6979-7238.

Received 15 April 2002; accepted 18 June 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Lower limb perfusion following arterial occlusion depends on the recruitment of collateral vessels. Blood flow through these collateral vessels may be jeopardized by hypersensitivity to vasoconstriction by serotonin (5-HT), as has been reported during hyperlipidemia and diabetes. Therefore the aim of this study was to determine the benefits of chronic treatment with SL65.0472, a mixed 5-HT1B/5-HT2A receptor antagonist, on lower limb ischemia in fatty fa/fa Zucker rats, a strain characterized by obesity, diabetes and hyperlipidemia. Comparison was made with lean control fa/+ Zucker rats. Methods: SL65.0472 (3 mg/kg/day, n=16) or its vehicle (n=20) were administered orally for 13 days to male fatty fa/fa Zucker rats submitted to lower limb ischemia. Hindlimb ischemia was induced unilaterally by resection of the left femoral and external iliac arteries and embolization of the left internal iliac artery with microspheres. Distal perfusion was measured under mild anesthesia by laser Doppler imaging of both feet. The perfusion deficit ({Delta}%) was calculated before and 3, 7 and 14 days after induction of hindlimb ischemia. Twenty-four hours after the last administration of SL65.0472, muscular partial oxygen pressure, iliac blood flows, maximal vasodilatory reserve and the vasoreactivity to 5-HT were measured in both limbs. Results: Chronic administration of SL65.0472 improved the distal perfusion from day 3. At day 14, the deficit of perfusion was limited to –36±7% in SL65.0472-treated animals vs. –70±6% in the vehicle-treated group (P<0.001) and was accompanied by a significant improvement of partial oxygen pressure in the ischemic limb (SL65.0472: 10.4±3.9 mmHg vs. vehicle: 3.5±1.1 mmHg, P<0.05). Maximal vasodilatory reserve tended to increase from 2.2±0.4 ml/min in the vehicle-treated group to 3.8±0.6 ml/min after SL65.0472. SL65.0472 markedly reduced 5-HT-mediated vasoconstriction, which was enhanced in the hypoperfused limb, without altering arterial pressure. Induction of hindlimb ischemia led to the overexpression of both 5-HT1B and 5-HT2A receptors only in the hypoperfused skeletal muscle as assessed by semi-quantitative RT-PCR. Conclusion: These results suggest that the recruitment of collateral vessels after the induction of hindlimb ischemia is significantly impaired in obese fa/fa Zucker rats due to a persistent vasoconstriction mediated by 5-HT and involving stimulation of 5-HT1B and/or 5-HT2A receptors.

KEYWORDS Collateral circulation; Diabetes; Ischemia; Serotonine (5HT); Vasoconstriction/dilation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Atherosclerosis, resulting in progressive occlusion of major limb arteries and reduced distal perfusion, underlies the pathogenesis of peripheral arterial disease (PAD). The prevalence of PAD is at least five times higher in the diabetic population than in non-diabetics [1]. Despite the advances made in the areas of therapeutic angiogenesis and percutaneous revascularization techniques, limb salvage and relief of ischemic pain cannot be achieved in many patients with diffuse PAD. The severity of hindlimb ischemia in patients with PAD depends in part upon the ability to mobilize collateral vessels. Recruitment of collateral vessels could be compromised by an excessive vasoconstrictor reactivity to 5-HT, which has been documented in various models of hindlimb ischemia [2–5]. Although plasma 5-HT concentrations have been found to be raised in patients with PAD or diabetes [6,7], no study, as yet, has shown that chronic blockade of vasoconstrictor serotonin receptors would improve the outcome in a model of hindlimb ischemia. 5-HT-induced vasoconstriction involves at least two receptor subtypes, termed 5-HT1B and 5-HT2A, the relative contribution of which varies according to species, age, metabolic status, endothelial function and presence of atherosclerosis lesions [8–10].

The aim of this study was to determine the benefits of SL65.0472, a mixed 5-HT1B/5-HT2A receptor antagonist [11,12], on lower limb ischemia in fatty fa/fa Zucker rats, a strain characterized by obesity, hyperglycemia, hyperinsulinemia and hyperlipidemia. The obese Zucker strain with type II diabetes was chosen in order to mimic as closely as possible the clinical situation, diabetes, in which chronic limb ischemia is more susceptible to occur than in the general population. For comparison purposes, the same experimental protocol was also conducted in lean fa/+ Zucker rats. We speculated that induction of hindlimb ischemia in a chronic hyperglycemic and hyperlipidemic context would favour a deleterious contribution of endogenous 5-HT to perfusion of the limb muscles.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Model of hindlimb ischemia
In order to have the possibility to adjust finely the severity and the persistence of hindlimb ischemia according to the genetic background of the obese fa/fa Zucker rats, we have developed a new rat model of hindlimb ischemia induced by combining excision of the femoral and external iliac arteries and embolization of the internal iliac artery by microspheres [13]. This study was performed in accordance with the European Community Standards on the Care and Use of Laboratory Animals and approved by the Animal Care and Use committee of Sanofi-Synthelabo Recherche. Preliminary experiments performed in our laboratory indicated that in rats, even after surgical excision of the femoral and external iliac arteries, pedal blood flow monitored by laser Doppler perfusion imaging showed a rapid recovery and returned to pre-ischemic values by day 14. Others have shown that combined ligation of common iliac and femoral arteries failed to induce a persistent perfusion defect in the rat [14,15]. In rats, mobilization of collateral vessels which originate from patent proximal arteries allows a rapid recovery of the distal perfusion pressure and hence the relief of ischemia. Therefore to achieve a sustained ischemia, microembolization of the vascular limb bed by injection of microspheres was required. In addition, microembolization of the muscular territory combined with resection of a major artery mimics more closely the pathological changes found in patients with PAD, in whom the etiology does not result solely from the acute occlusion of a large proximal artery.

Thirty-six fatty obese fa/fa and 15 lean fa/+ 13-week-old Zucker rats (Iffa-Creddo, L’arbresle, France) were used for this study. The animals were anesthetized with sodium pentobarbital 60 mg/kg i.p. An incision in the left inguinal area was performed and the external iliac and femoral arteries were dissected free from the beginning of the external iliac artery (i.e. the termination of the common iliac artery) to the bifurcation of the saphenous and popliteal arteries. In order to isolate the vascular territory supplied by the left internal iliac artery, the common iliac artery was clamped temporarily, and a catheter was inserted retrogradely through the external iliac artery and placed near the proximal origin of the internal iliac artery. Microspheres (diameter 170 µm; Cytodex 2, Amersham, Les Ulis, France) were suspended in a saline solution by vortex and ultrasonication just prior to injection. Six thousand microspheres were injected in the internal iliac artery through the catheter. After microembolization, the catheter and the clamp were removed, all branches of the femoral and external iliac arteries were isolated and cautherized, the femoral and external iliac arteries were ligated and excised and finally the skin was sutured.

Hindlimb ischemia was detected immediately by blueing and cooling of the skin of the foot and confirmed by laser Doppler perfusion imaging measurements (LDPI, see below). Rats were then returned to individual cages for recovery and follow up. Rats were examined daily in order to identify distal necrosis. Such observation led to the sacrifice of the animal by pentobarbital injection and was used to grade the severity of hindlimb ischemia.

2.2 Effects of SL65.0472 on hindlimb ischemia
Fatty fa/fa Zucker rats were divided in two groups to receive orally once a day either SL65.0472 (3 mg/kg/day) (n=16) or its vehicle (n=20). In parallel, lean fa/+ Zucker rats underwent the same treatment with SL65.0472 (3 mg/kg/day) (n=7) or its vehicle (n=8). Treatment was begun just prior to the surgery and lasted 14 days.

2.2.1 Perfusion and hemodynamic measurements
Serial evaluations of foot skin perfusion were performed by LDPI (PIM II, Lisca AB, Sweden). This technique was previously described in mice to quantify the deficit of perfusion as an index of distal ischemia [16]. Under light anesthesia (ketamine 50 mg/kg s.c. and xylazine 10 mg/kg s.c.), both normal and ischemic feet were scanned, mean perfusion scores (MPS) were obtained for each foot to calculate the distal perfusion ratio (DPR: (ischemic MPS/control MPS)x100). For each animal, this non-invasive procedure was performed before and just after the surgery, and repeated 3, 7 and 14 days later.

On day 14, rats were anesthetized with ketamine and xylazine as described above for LDPI measurement, and then were instrumented for an extended hemodynamic study. A catheter was introduced into the right jugular vein to allow prolonged anesthesia (pentobarbital 1 mg/kg/min i.v.) and another into the right carotid artery to measure mean arterial pressure (MAP) and to determine heart rate (HR). Iliac bifurcations were exposed after a median laparotomy and common iliac arteries were both dissected free in order to place Doppler flow probes (20 MHz—0.8 to 1.3 mm diameter, Triton, USA). Control and ischemic iliac blood flows (IBF) were calculated from velocity according to the standard formula: BF=1.25xdiameter squared (mm2)xDoppler shift (kHz). Maximal vasodilatory reserve was assessed by measuring the maximal iliac flow increase after a 30-s abdominal aortic occlusion performed with a silk suture above the iliac bifurcation.

To further complete the characterization of hindlimb ischemia, muscular oxygen partial pressure (PmO2) was measured in both limbs with electrochemical microprobes (Licox pO2, GMS mbH, Mielkendorf, Germany) inserted in the posterior part of the calf muscle by means of a 16G needle. PmO2 was determined after a 20-min stabilization period and expressed in mmHg.

In order to ensure that anesthesia did not interfere with hemodynamic measurements, values were discarded if either mean arterial pressure was below 50 mmHg, or PmO2 in the control limb was below 15 mmHg, or right basal iliac flow was below 1.5 ml/min.

2.2.2 Vascular reactivity to 5-HT
Vascular reactivity to 5-HT (30 µg/kg) was determined in each group after intravenous injection into the jugular vein. MAP, control IBF and ischemic IBF were monitored. To avoid interference of cardiac reflexes in the interpretation of the vascular response to 5-HT, atropine (1 mg/kg i.v.) and propranolol (1 mg/kg i.v.) were administered prior to administration of 5-HT.

2.2.3 Biochemical parameters
Plasma glucose, cholesterol and triglyceride levels were determined in obese fa/fa and lean fa/+ Zucker rats after chronic treatment with SL65.0472 or its vehicle.

2.3 Quantification of 5-HT1B and 5-HT2A receptors by RT-PCR
For RT-PCR analysis, 15 obese fa/fa Zucker rats were operated and sacrificed 3, 7 and 14 days after induction of ischemia (n=5 per group).

The anterior tibialis muscles were excised and snap-frozen in liquid nitrogen. Total muscle RNA was extracted using a previously described procedure [17]. The quality of isolated mRNA was verified by gel electrophoresis. 5-HT1B-receptor, 5-HT2A-receptor and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression were quantified in muscles by comparative RT-PCR. One µg of total RNA was primed with 1 µg oligo (dT) (Pharmacia Biotech, Les Ulis, France) and reverse transcribed (Gibco BRL Life Technologies, Cergy-Pontoise, France). The cDNA was amplified by PCR using specific oligonucleotide primers for rat 5-HT1B-receptor, 5-HT2A-receptor and GAPDH (Table 1). Double-strand cDNAs were synthesized and amplified. The amplification was carried out in a DNA thermal cycler (Techne, Elancourt, France) using 35 cycles for the 5-HT1B-receptor (94 °C, 30 s, 58 °C, 1 min, 72 °C, 1 min), 35 cycles for the 5-HT2A-receptor (94 °C, 30 s; 54 °C, 1 min; 72 °C, 1 min), and 21 cycles for GAPDH (94 °C, 30 s; 55 °C, 1 min; 72 °C, 1 min).


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  		Table 1 Primer sequences used for RT-PCR receptors expression

 
PCR fragments were analyzed by 8% polyacrylamide gel electrophoresis and revealed by ethidium bromide staining. Bands were cut out, dissolved in periodic acid, and counted using a RackBeta liquid scintillation counter. PCR amplification was verified to be exponential, and the amplification products were proportional to the sample input. 5-HT1B-receptor and 5-HT2A-receptor mRNA expressions were calculated by normalizing to GAPDH mRNA.

2.4 Drugs
Drugs used were obtained as follows: serotonin and propranolol from Sigma (Saint-Quentin Fallavier, France), pentobarbital from Sanofi Santé Animale (Libourne, France), ketamine from Merial (Lyon, France), xylazine from Bayer Pharma (Puteaux, France), atropine from Aguettant Laboratory (Lyon, France). SL65.0472 was synthesized by Sanofi-Synthelabo Research (Chilly Mazarin, France).

2.5 Statistics
Parameters were reported either as absolute or percent change between ischemic and control legs and expressed as mean±S.E.M. Parameters involving both limbs or different time points were compared by two-way analysis of variance with repeated measurements followed by a Dunett test when appropriate. Other parameters were compared by one-way analysis of variance followed by a Dunett test. Survival rates were compared by means of a LogRank test. All the analyses were performed with SAS software (version 6.09) under VMS environment with a significance threshold fixed at 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Perfusion and hemodynamic measurements
Distal hindlimb perfusion decreased dramatically immediately after surgery with a DPR less than –90% in all groups. The recovery of pedal perfusion following the surgical procedure was similar between fatty fa/fa and lean fa/+ Zucker rats treated with vehicle during the first week of the follow-up. However at day 14, vehicle-treated fa/+ Zucker rats showed a higher DPR than the fa/fa Zucker rats (–52±11 vs. –70±6%, P<0.05). In fatty fa/fa Zucker rats, SL65.0472 accelerated significantly the recovery of the pedal perfusion from day 3 after the induction of hindlimb ischemia (Fig. 1), and tended to reduce the incidence of foot necrosis to 13% versus 20% in the vehicle-treated group (ns). In contrast, in lean fa/+ Zucker rats, characterized by a less severe hindlimb ischemia, SL65.0472 did not alter the time-course of DPR recovery (Fig. 1).


Figure 1
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  		Fig. 1 Distal perfusion ratios (DPR) are expressed as percentage variations of ischemic compared to control (non-ischemic) distal perfusion measurements. DPR was markedly depressed in obese fa/fa Zucker rats. In this strain, chronic treatment with SL65.0472 (3 mg/kg/day, p.o.) improved DPR significantly as early as 3 days after surgery. SL65.0472 did not modify DPR in lean fa/+ Zucker rats. *P<0.05 between vehicle and SL65.0472-treated groups.

 
Reflecting distal hypoperfusion, PmO2 was reduced in ischemic limbs in both strains of rats. This reduction was significantly more profound in fatty fa/fa than in lean fa/+ Zucker rats (Fig. 2). Chronic treatment with SL65.0472 improved significantly PmO2 only in the ischemic limbs of obese fa/fa Zucker rats, reaching values similar to those observed in ischemic limbs of lean fa/+ Zucker rats (Fig. 2).


Figure 2
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  		Fig. 2 Muscular oxygen partial pressures (PmO2) were decreased in the ischemic limb of both obese fa/fa and lean fa/+ Zucker rats. SL65.0472 restored significantly PmO2 in ischemic limb of fatty fa/fa Zucker rats only. #P<0.05 between control and ischemic limb of the same treatment group. *P<0.05 between vehicle and SL65.0472-treated groups of the same strain.

 
MAP and HR were not altered by chronic treatment with SL65.0472 (Table 2). However, these hemodynamic parameters were lower in obese fa/fa compared to lean fa/+ Zucker rats due possibly to a different sensitivity to anesthesia.


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  		Table 2 Mean arterial pressure (MAP), heart rate (HR), basal control (BCF) and ischemic (BIF), maximal control (MCF) and ischemic (MIF) iliac blood flows in obese fa/fa and lean fa/+ Zucker rats were not modified by SL65.0472

 
Although basal iliac blood flows were not modified by SL65.0472 in either control or ischemic limb (Table 2), the maximal vasodilatory reserve assessed by reactive hyperemia after transient aortic occlusion was significantly increased by SL65.0472 only in the ischemic limb of obese fa/fa Zucker rats and not in the control lean strain (Fig. 3).


Figure 3
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  		Fig. 3 Maximal vasodilatory reserve, as assessed by reactive hyperemia, was still significantly reduced in the ischemic limbs 2 weeks after surgery. Chronic treatment with SL65.0472 (3 mg/kg/day, p.o.) improved significantly the maximal vasodilatory capacity in the ischemic limb of obese fa/fa Zucker rats. #P<0.05 between control and ischemic limb of the same treatment group. *P<0.05 between vehicle and SL65.0472 groups of the same strain.

 
3.2 Vascular reactivity to 5-HT
In both obese and lean Zucker strains, intravenous injection of 5-HT (30 µg/kg) resulted in a marked rise in MAP reaching +101±5% and +94±4% in fa/fa and fa/+ Zucker rats, respectively. In animals chronically receiving SL65.0472, this pressor response was totally abolished with a residual change in MAP of +2±0% and +7±2% in fa/fa and fa/+ Zucker rats, respectively, and unmasking a secondary hypotension ({Delta}%MAP: –22±2%, P<0.05 in fa/fa Zucker rats and –23±3%, P<0.05 in lean fa/+ Zucker rats).

At the iliac blood flow level, 5-HT produced initially a strong vasoconstriction (Fig. 4), which was enhanced in the ischemic limb compared to control normally perfused limb in both Zucker strains. This fall in iliac blood flow was followed by a slight dilatation which was more pronounced in the non-ischemic control leg and reached statistical significance only in fatty fa/fa Zucker rats. The modest secondary increase in iliac blood flow change was impaired in the ischemic leg as compared to the control leg ({Delta}%IBF: +9±2 vs. +24±3%, P<0.05, respectively) in obese fa/fa Zucker rats, but not significantly in lean fa/+ Zucker rats ({Delta}%IBF: +11±3 vs. +20±3%, ns, respectively). SL65.0472 markedly inhibited the reduction in iliac blood flow caused by 5-HT (Fig. 4) and enhanced the secondary vasodilatation to 5-HT significantly in fa/fa Zucker rats only on both ischemic (+31±6%, P<0.05) and control legs (+53±4%, P<0.05).


Figure 4
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  		Fig. 4 Maximal variations of iliac blood flows after bolus injection of 5-HT (30 µg/kg i.v.) in lean fa/+ (a) and obese fa/fa Zucker (b) rats. Exogenous 5-HT produced a biphasic response on iliac blood flow. Vasoconstriction to 5-HT was enhanced in the ischemic limbs of both Zucker strains. This primary fall in iliac blood flow caused by 5-HT was markedly reduced by SL65.0472 treatment. The secondary blood flow increase was exacerbated after SL65.0472 in obese fa/fa Zucker rats on both legs. #P<0.05 between control and ischemic limb of the same treated group. *P<0.05 between vehicle and SL65.0472 groups of the same strain.

 
3.3 Biochemical parameters and body weight
Obese fa/fa Zucker rats showed a significant increase in plasma glucose and triglyceride levels as compared to lean fa/+ Zucker rats. These metabolic parameters were not modified by chronic administration of SL65.0472 (Table 3). Plasma cholesterol levels were slightly elevated in obese fa/fa Zucker rats, although this elevation did not reach statistical significance and was not altered by SL65.0472. SL65.0472 treatment did not change body weight in either obese fa/fa or lean fa/+ Zucker rats.


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  		Table 3 Effects of SL65.0472 on body weight and plasma glucose, cholesterol and triglycerides level in obese fa/fa and lean fa/+ Zucker rats

 
3.4 Quantification of 5-HT1B and 5-HT2A receptors by RT-PCR
Semi-quantitative measurements of 5-HT1B and 5-HT2A receptor mRNA expression by RT-PCR indicated higher expression of 5-HT2A than 5-HT1B receptors in the skeletal limb muscle of obese fa/fa Zucker rats (Fig. 5). Induction of hindlimb ischemia increased significantly the expression of both 5-HT receptor subtypes in the hypoperfused skeletal muscle, whereas it remained unchanged with time in the normal control muscles. Overexpression of 5-HT1B receptor mRNA was significant from day 3 after the surgical procedure leading to hindlimb ischemia and remained elevated for both 5-HT receptor subtypes until day 14.


Figure 5
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  		Fig. 5 Expression of 5-HT1B and 5-HT2A receptors mRNA as assessed by RT-PCR in control and ischemic muscles of obese fa/fa Zucker rats over the time course of hindlimb ischemia. Induction of hindlimb ischemia promoted the expression of both 5-HT1B and 5-HT2A receptors’ mRNA in ischemic skeletal muscles. *P<0.05 between control and ischemic limb of obese fa/fa Zucker rats.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Although atherosclerosis is the primary cause of PAD, diabetes remains a major risk factor for coronary and peripheral arterial diseases [18]. Diabetes has been suggested to alter the compensatory mechanisms which normally limit the ischemic consequences of arterial insufficiency [19]. These mechanisms include arteriogenesis of ischemic territories and mobilization of existing collateral vessels. In diabetic patients, endogenous arteriogenesis is impaired as a result of reduced production of growth factors, and collateral-related perfusion is jeopardized by an abnormal vascular reactivity due to a sustained endothelial dysfunction and an oversensitivity to vasoconstricting agents such as 5-HT [2–5,19]. It is notable that in diabetic patients platelet reactivity is enhanced and plasma 5-HT levels are elevated [6,7]. These changes are probably secondary to diabetes-induced glycosylation of membrane proteins. The glycosylated structures may become antigenic and trap immunoglobulins, thus promoting platelet aggregation and endothelial damage [1] and leading to the prothrombotic microangiopathic changes found in diabetes. Activated blood platelets may affect tissue perfusion by releasing various vasoactive products such as 5-HT, thromboxane A2, ADP, prostaglandins, further impairing blood supply in the collateral-dependent ischemic hindlimb.

SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno[3,2-c]pyrin-4-yl) piperazin-1-yl]ethyl]-1,2-dihydroquinoline-acetamide) is a mixed orally active 5-HT1B/5-HT2A receptor antagonist, with a long duration of action and a limited cerebral penetration [11,12]. In vivo, SL65.0472 antagonizes vasoconstriction induced by activation of 5-HT1B and 5-HT2A receptors and inhibits thrombus formation in various models of arterial thrombosis via its 5-HT2A-receptor antagonist component [12,20].

To determine whether chronic blockade of 5-HT1B and 5-HT2A receptors would improve hindlimb ischemia in diabetic animals, obese fa/fa Zucker rats were treated chronically by SL65.0472 after surgical induction of lower limb arterial hypoperfusion. Resection of the left femoral and external iliac arteries and embolization of the left internal iliac artery with microspheres caused an immediate and profound impairment of pedal perfusion followed by a slow and incomplete recovery over days, reflecting a sustained critical hindlimb ischemia. After 2 weeks, the deficit of perfusion remained greater in obese fa/fa Zucker rats than in lean controls. These observations are in agreement with previous reports indicating that diabetic mice showed a reduced capacity to recover from hindlimb ischemia as compared to normoglycemic control mice [19]. Chronic administration of SL65.0472 markedly improved the pedal perfusion altered by hindlimb ischemia in obese fa/fa Zucker rats but not in lean fa/+ Zucker rats, the latter showing a greater natural ability to recover from arterial hypoperfusion. The absence of a beneficial effect on perfusion in lean fa/+ Zucker rats treated with SL65.0472 suggests that endogenous 5-HT does not contribute significantly to the pathogenesis of hindlimb ischemia in animals with no metabolic disorders. This observation fits with previous clinical reports indicating that plasma 5-HT levels are elevated in diabetic patients [6,7], these latter being more susceptible to develop PAD [1]. Improved perfusion recovery by SL65.0472 appeared as early as day 3 after induction of hindlimb ischemia, and remained effective over the time course of the experiment in obese fa/fa Zucker rats. Neither increased body weight nor the hyperglycemia and dyslipidemia which characterize obese fa/fa Zucker rats were changed by SL65.0472 treatment.

The severity of hindlimb ischemia was associated with the appearance of foot necrosis in obese fa/fa Zucker rats, which affected 20% of the animals in the control group. This observation supports again the clinical relevance of this model. Patients with advanced PAD suffer generally from complex disturbances of skin microcirculation, which lead classically to lesion formation, for example, in the toes [1]. These skin lesions rarely heal by themselves, but rather progress to necrosis and gangrene. Chronic treatment with SL65.0472 tended to diminish the occurrence of foot necrosis in our study, although this effect was not statistically significant.

The beneficial effects of SL65.0472 were confirmed after 2 weeks by measuring the muscular oxygen partial pressure and by completing the peripheral hemodynamic assessment. As compared to the vehicle-treated groups, a treatment with SL65.0472 did not change MAP and HR in both Zucker rat strains. Basal iliac blood flow was still significantly reduced in the lower limb 2 weeks after surgery, and apparently was not improved by SL65.0472 even in obese fa/fa Zucker rats, a result which contrasts with the beneficial effects shown by LDPI measurements. This discrepancy might result from the different anatomical sites and protocols used for the two hemodynamic measurements. Under physiological conditions, the common iliac artery is the main vessel ensuring foot perfusion. However in hindlimb ischemia, pedal perfusion is maintained not only by the common iliac artery, but also in part from the recruitment of existing collaterals and the opening of arterial shunts originating from patent proximal arteries such as caudal tail and vertebral arteries [21]. Therefore, monitoring basal iliac blood flow is not sufficient to fully characterize the deficit of perfusion detected at the foot level or to investigate the benefits of a pharmacological treatment. To further characterize the effects of SL65.0472 on hindlimb perfusion, maximal vasodilatory reserve was assessed by measuring reactive hyperemia. Maximal vasodilatory reserve was significantly augmented by SL65.0472 treatment in obese Zucker rats only. Thus, the beneficial effects of chronic antagonism of 5-HT1B and 5-HT2A receptors on hindlimb ischemia was manifest as improvements in pedal perfusion, muscular partial oxygen pressure and maximal vasodilatory reserve, with no change in body weight and metabolic profile.

Intravenous bolus injection of 5-HT (30 µg/kg) produced a significant increase in MAP associated with biphasic changes in iliac blood flows, a strong initial vasoconstriction followed by a modest dilatation. The fall in iliac blood flow induced by 5-HT was greater in the ischemic limb than in the normal contralateral limb. This preferential vasoconstriction of ischemic vasculature by 5-HT seemed to be more accentuated in obese fa/fa than in lean fa/+ Zucker rats, although it did not reach statistical significance. High sensitivity of collateral vessels to 5-HT-induced vasoconstriction has been previously described in various species including dogs, rabbits, cats and rats [2–5]. Pressor and vasoconstrictor effects of 5-HT were markedly reduced in both Zucker strains after chronic administration of SL65.0472, whereas the secondary vasodilatation to 5-HT was potentiated (probably as a consequence of inhibition of the initial vasoconstriction). Two major receptor subtypes, 5-HT1B and 5-HT2A, located on vascular smooth muscle cells, are involved in the vasoconstriction caused by 5-HT, the relative contribution of which varies with the species, the vessels, the disease state and the individual [8–10]. For example, in human atherosclerotic coronary arteries, complete inhibition of the vasoconstriction evoked by 5-HT required systematically a dual blockade of both these 5-HT receptor subtypes [8,9]. In nondiseased coronary arteries 5-HT promotes vasodilatation through an endothelium-dependent mechanism and produces an increase in blood flow. Endothelial dysfunction caused by atherosclerotic lesions, hyperlipidemia or diabetes unmasks the vasoconstrictor properties of 5-HT. In obese fa/fa Zucker rats, endothelium-dependent dilation as assessed by hypoxia is impaired in skeletal muscle arterioles [22], which may contribute to the magnitude of the vasoconstrictor response to 5-HT in the ischemic limb. In order to investigate whether the marked reduction in iliac blood flow caused by 5-HT administration to the ischemic limb could be attributed in part to an upregulation of 5-HT1B and/or 5-HT2A receptors, expression of both 5-HT receptor mRNAs were determined by RT-PCR in the ischemic and control tibialis anterior muscle at 3, 7 and 14 days after surgery in obese fa/fa Zucker rats. Before induction of hindlimb ischemia, 5-HT2A receptor expression predominated over 5-HT1B receptors. Induction of chronic hindlimb ischemia caused a rapid and sustained upregulation in the expression of both 5-HT receptors’ mRNA. In contrast no modification in the expression of these receptors could be detected in the normally perfused limb muscle. In view of these results, it can be speculated that enhanced vasoconstriction to exogenous 5-HT in ischemic hindlimb could be attributed in part to an upregulation of both vasoconstrictor 5-HT receptors, 5-HT1B and 5-HT2A. On the other hand, we cannot exclude the possibility that chronic ischemia could promote endothelial dysfunction which would further unbalance the vascular response to 5-HT towards vasoconstriction.

We believe that the beneficial effects of SL65.0472 on hindlimb perfusion following ischemia are likely to be due to improved mobilization of existing collateral vessels, following inhibition of 5-HT-induced vasoconstriction, thus resulting in increased blood flow. Given the inability of SL65.0472 to restore the deficit of perfusion in non-obese Zucker rats, it seems improbable that arteriogenesis contributes to its mechanism of action. The high sensitivity of collateral vessels to the vasoconstrictor effects of 5-HT is well established in ischemia models from several different species [2,3,5]. These effects are blocked by serotonin receptor antagonists such as ketanserin and naftidrofuryl. Furthermore, this class of pharmacological agents also inhibits collateral vessel vasoconstriction induced by platelet activation or endothelial injury [3,4], thus suggesting a role for endogenous serotonin in situations which mimic athero-thrombotic vascular disease. Evidence for a contribution of endogenous serotonin to collateral vessel constriction is not limited to animal models because clinical studies performed in patients with atherosclerotic peripheral or coronary artery disease have demonstrated that administration of serotonin receptor antagonists (ketanserin [23], sarpogrelate [24]) produce collateral vessel vasodilation. Besides inhibition of 5-HT-induced vasoconstriction [11,12], SL65.0472 via 5-HT2A receptor antagonism, has been shown to inhibit platelet aggregation and arterial thrombosis [20]. However, it seems unlikely that its antithrombotic activity could contribute to its beneficial effect on hindlimb ischemia as no evidence of arterial thrombosis causing reduction of distal perfusion has been reported in this model. Altogether these results suggest that the metabolic disorders found in obese fa/fa Zucker rats have a negative impact on the recruitment of collaterals, and therefore the recovery from hindlimb ischemia, and involve the participation of endogenous 5-HT. Other metabolic abnormalities such as hypercholesterolemia have been reported to promote elevation of plasma levels of 5-HT and hypersensitivity to vasoconstriction by 5-HT. For instance, cholesterol-fed rabbits showed increased plasma 5-HT levels, and chronic blockade of 5-HT2A receptors in these animals improved muscle oxygenation in the ischemic limb [25].

This study demonstrates a beneficial effect of SL65.0472, a 5-HT1B/5-HT2A receptor antagonist, in a rat model hindlimb ischemia associated with obesity and type II diabetes, thus indicating a detrimental role of endogenous 5-HT in our experimental conditions. These results suggest that SL65.0472 could be of clinical interest for the treatment of hindlimb ischemia in patients suffering from peripheral obstructive arterial diseases.

Time for primary review 24 days.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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