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Keywords:

  • obesity;
  • plasminogen activator inhibitor-1;
  • stroke

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Summary. Background: It is widely accepted that obesity is a risk factor for ischemic heart disease, but the association with stroke is less clear. Adipose tissue is an important source of plasminogen activator inhibitor-1 (PAI-1), the main inhibitor of plasminogen activation. Objective: To test the hypothesis that elevated PAI-1 levels associated with obesity negatively affect the outcome of thrombotic ischemic stroke. Methods: Middle cerebral artery (MCA) occlusion was induced photochemically in mice with nutritionally induced or genetically determined obesity and their lean counterparts. Results: The MCA occlusion time (to obtain complete occlusion) was significantly shorter in obese (nutritionally induced) than in lean wild-type (WT) C57Bl/6 mice, whereas the infarct size was significantly larger and intracranial hemorrhage (ICH) was enhanced (all < 0.05). Similar observations were made in genetically obese ob/ob mice, as compared to lean WT littermates. In both strains, obesity was associated with markedly elevated circulating PAI-1 levels, probably originating from the fat tissue. In contrast, PAI-1-deficient lean and obese mice did not display significant differences in MCA occlusion time, infarct volume or ICH. Conclusions: Plasminogen activator inhibitor-1 may play a functional role in the deleterious effect of obesity on the outcome of thrombotic ischemic stroke in mice.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Stroke is a major cause of death and disability in Western societies, and several risk factors for ischemic stroke have been recognized [1]. It is increasingly being accepted that obesity is a modifiable risk factor for ischemic heart disease [2], but the association between obesity and stroke is less well established [3]. Several prospective studies have shown an increased risk for stroke with increasing body mass index (BMI) [4–13], whereas others have found no association [14–17]. Some studies suggested that abdominal obesity (measured by raised waist-to-hip ratio) rather than general obesity (measured by raised BMI) is associated with increased risk for stroke [18,19], whereas others found general and abdominal obesity to be independent risk factors [5,9,13,20]. A prospective cohort study of male US physicians followed up for 12.5 years found that BMI ≥ 30 kg m–2 was associated with enhanced relative risk of ischemic stroke, and that each unit increase in BMI was associated with a 6% increase in the relative risk [5]. A recent large random population study involving urban Swedish middle-aged men followed up for 28 years revealed that increased BMI was associated with an increased risk for total and ischemic stroke, but not with hemorrhagic stroke [20]. A prospective cohort study among women participating in the Women’s Health Study reached the same conclusion [21]. The finding that adipose tissue is an important source of elevated circulating plasminogen activator inhibitor-1 (PAI-1) levels [22] suggests that this may be related to the increased risk for stroke. In the present study, we have used murine models of nutritionally induced or genetically determined obesity to study the effect of obesity on the outcome of thrombotic ischemic stroke. With the use of gene-deficient mice, we have also studied a potential role of PAI-1, the main physiologic inhibitor of plasminogen activation and fibrinolysis [23,24].

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Animals and reagents

Male wild-type (WT) mice (100% C57Bl/6) and homozygous PAI-1-deficient (PAI-1–/–) mice (81.25% C57Bl/6; 18.75% 129SV/ev) were obtained as described elsewhere [25], and were generated in the K.U. Leuven animal facility. Genetically obese Lep/ob mice (100% C57Bl/6) were initially purchased from Charles River Laboratories (Wilmington, MA, USA); homozygous leptin-deficient ob/ob mice and WT littermates were obtained from heterozygous breeding pairs. Mice were kept in microisolation cages on a 12-h day/night cycle, and fed ad libitum. Five-week-old WT and PAI-1–/– mice were kept for 15 weeks on a standard fat diet (SFD) [kM-04-k12 (Muracon, Carfil, Oud-Turnhout, Belgium), containing 13% kcal as fat, with a caloric value of 10.9 kJ g–1] or on a high-fat diet (HFD) [TD 88137 (Harlan Taklad Zeist, the Netherlands), containing 42% kcal as fat, with a caloric value of 20.1 kJ g–1]. In addition, 5-week-old male ob/ob and corresponding WT mice were kept on SFD for 15 weeks. Three days before the thrombosis experiment, blood was collected after overnight fasting from the retro-orbital sinus on trisodium citrate (final concentration 0.01 m); plasma was obtained by centrifugation and stored at –20 °C. Inguinal s.c. and epididymal (gonadal) fat pads, as well as liver and spleen, were removed and weighed. All animal experiments were approved by the local ethical committee (K.U. Leuven P03112), and performed in accordance with the guiding principles of the International Society on Thrombosis and Haemostasis [26].

Photochemically induced middle cerebral artery (MCA) thrombosis model

Photochemically induced thrombosis was produced as described elsewhere [27]. Briefly, anesthesia was induced with 2% isoflurane, the rectal temperature was maintained at 37 °C, and a catheter (2FG; SIMS Portex Limited, Kent, UK) was placed in the left jugular vein for the administration of Rose Bengal (Sigma, St Louis, MI, USA). The temporal muscle was dissected, the skull was exposed, and a 1.5-mm opening was made over the MCA. Photo-illumination of green light (wavelength 540 nm) was achieved with a xenon lamp (model L-4887; Hamamatsu Photonics, Hamamatsu, Japan) with heat-absorbing and green filters, via an optic fiber with a focus of 1 mm, placed on the opening in the skull. Rose Bengal (10 mg kg–1) was injected, and photo-illumination was performed for 10 min, after which the temporal muscle and skin were replaced. For ob/ob mice, the dose of Rose Bengal was adjusted to the higher blood volume, as measured following injection of 125I-labeled bovine serum albumin [28]. The MCA occlusion time (from the start of light exposure until the flow in the MCA is stopped) was monitored by observation in real time under the microscope.

After 24 h, the animals were anesthetized with sodium pentobarbital (500 mg kg–1; Abbott Laboratories, North Chicago, IL, USA) and decapitated. The brain was sectioned in 1-mm segments and photographed before and after staining with 2% 2,3,5-triphenyltetrazolium chloride. The intracranial hemorrhage (ICH) size and infarct volume were determined by planimetry as the sum of the hemorrhage areas in each section before staining and of the unstained areas after staining, respectively [29,30]. Edema was defined as the difference in volume between the ipsilateral and contralateral hemispheres [31].

Assays

Total cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol and triglyceride levels were evaluated with routine clinical assays. Plasma PAI-1 antigen levels were measured with a specific enzyme-linked immunosorbent assay [32].

Statistical analysis

Data are given as means ± SEM. Statistical significance for differences between groups was analyzed by non-parametric t-testing (Mann–Whitney). Correlations were analyzed according to the Spearman non-parametric correlation analysis. Significance was set at < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Effect of nutritionally induced obesity on thrombotic ischemic stroke

High-fat diet feeding of C57Bl/6 WT mice for 15 weeks resulted in significantly higher total body weights and both s.c. and gonadal fat mass as compared to SFD feeding (Table 1). Obese mice also displayed significantly higher levels of cholesterol than their lean counterparts. Circulating PAI-1 levels were significantly elevated in obese mice (12 ± 2.3 ng mL–1 vs. 2.0 ± 0.12 ng mL–1 for lean mice; < 0.0005).

Table 1.   Adipose tissue and organ weights and metabolic parameters of wild-type (WT) and PAI-1-deficient (PAI-1–/–) mice with nutritionally induced obesity and of genetically obese (ob/ob) and corresponding WT mice
 WTPAI-1–/–Lep/ob
Lean (= 10)Obese (= 9)Lean (= 11)Obese (= 9)WT (= 12)ob/ob (= 9)
  1. HDL, high-density lipoprotein; LDL, low-density lipoprotein; ND, not determined.

  2. Data are mean ± SEM of n experiments.

  3. *< 0.05, **< 0.005, ***< 0.0005.

Body weight (g)31 ± 1.243 ± 0.8***30 ± 1.140 ± 1.3***29 ± 0.753 ± 1.9***
S.c. fat (mg)460 ± 711490 ± 41***340 ± 491180 ± 150***190 ± 142870 ± 210***
Gonadal fat (mg)750 ± 1201920 ± 63***550 ± 981860 ± 150***330 ± 353180 ± 200***
Liver (mg)1210 ± 903160 ± 330***1040 ± 581890 ± 210**1140 ± 742950 ± 290***
Spleen (mg)79 ± 8.0140 ± 8.3**130 ± 9.0200 ± 26*100 ± 1797 ± 18
Total cholesterol (mg dL–1)100 ± 4.8270 ± 18***120 ± 7.8250 ± 29**110 ± 7.4180 ± 17**
HDL cholesterol (mg dL–1)91 ± 4.2220 ± 12***100 ± 8.0220 ± 22**93 ± 6.6160 ± 15**
LDL cholesterol (mg dL–1)ND30 ± 5.225 ± 1521 ± 6.715 ± 1.426 ± 3.3*
Triglycerides (mg dL–1)74 ± 9.482 ± 6.950 ± 7.144 ± 7.945 ± 6.874 ± 12*

In the photochemically induced thrombosis model (Fig. 1), the MCA occlusion time was significantly shorter in the obese (= 9) than in the lean (= 9) mice (160 ± 11 s vs. 195 ± 5.5 s; < 0.05). The total damage size was significantly larger in the obese mice (32 ± 2.1 mm3 vs. 24 ± 0.9 mm3 for lean mice; < 0.005), as a result of larger damaged areas both in the cortical (23 ± 1.4 mm3 vs. 19 ± 1.1 mm3; = 0.05) and in the subcortical (9.2 ± 1.3 mm3 vs. 5.2 ± 0.5 mm3; < 0.05) regions. Thus, the total damaged area in the hemisphere amounted to 25% ± 1.7% for obese mice and 19% ± 0.9% for lean mice (< 0.01). Edema was comparable for obese and lean mice in the cortical region (6.9 ± 0.7 mm3 vs. 7.3 ± 1.3 mm3; = 0.77), but larger in the subcortical region of obese mice (8.0 ± 1.0 mm3 vs. 4.1 ± 1.1 mm3; < 0.05), yielding somewhat higher values for total edema in obese as compared to lean mice (15 ± 1.5 mm3 vs. 12 ± 1.4 mm3, = 0.11), corresponding to areas of 6.2% ± 0.7% vs. 4.7% ± 0.5% (= 0.10) in the hemisphere. ICH amounted to 4.9 ± 0.9 mm3 for obese mice as compared to 2.7 ± 0.4 mm3 for lean mice (< 0.05) (Fig. 1).

image

Figure 1.  Photochemically induced middle cerebral artery thrombosis model. Occlusion time (A), infarct size (B) and intracerebral hemorrhage (C) following photochemically induced thrombotic ischemic stroke in lean and obese wild-type (WT) or plasminogen activator inhibitor-1 (PAI-1)-deficient (PAI-1–/–) mice, and in obese (ob/ob) and corresponding WT Lep/ob mice. Data are mean ± SEM. *< 0.05 and **< 0.01 vs. corresponding lean or WT mice.

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Effect of genetically determined obesity on thrombotic ischemic stroke

Standard fat diet feeding of ob/ob mice resulted in significantly higher total body weight and both s.c. and gonadal fat mass, as compared to WT littermate mice (Table 1). Ob/ob mice also displayed significantly higher levels of cholesterol. Circulating PAI-1 levels were significantly elevated in ob/ob mice as compared to lean WT littermates (7.4 ± 1.2 ng mL–1 vs. 2.6 ± 0.65 ng mL–1; < 0.01).

Photochemically induced thrombosis resulted in MCA occlusion times of 172 ± 12 s vs. 208 ± 5.4 s (< 0.01) for ob/ob (= 9) and WT (= 12) mice, respectively. The total infarct size was significantly larger in the ob/ob mice (30 ± 2.9 mm3 vs. 23 ± 1.5 mm3 for lean WT mice; < 0.05), mainly as a result of more brain damage in the cortical region (21 ± 1.7 mm3 vs. 16 ± 1.0 mm3; < 0.01), but not in the subcortical region (8.4 ± 1.3 mm3 vs. 7.3 ± 1.0 mm3; = 0.50). Thus, the total damaged area in the hemisphere amounted to 26% ± 2.5% for ob/ob mice and 18% ± 1.2% for WT mice (= 0.01). Edema was comparable for ob/ob and WT mice, both in the cortical (5.8 ± 0.9 mm3 vs. 7.2 ± 0.9 mm3; = 0.30) and in the subcortical (3.8 ± 0.7 mm3 vs. 4.0 ± 1.4 mm3; = 0.90) regions, yielding comparable values for total edema (9.5 ± 1.1 mm3 vs. 11 ± 1.8 mm3; = 0.50), corresponding to areas of 4.3% ± 0.5% vs. 4.7% ± 0.8% (= 0.67) in the hemisphere. ICH was significantly more pronounced for ob/ob than for lean WT mice (5.0 ± 0.9 mm3 vs. 2.7 ± 0.4 mm3, < 0.05) (Fig. 1).

Effect of PAI-1 deficiency on thrombotic ischemic stroke

When PAI-1–/– mice were kept on HFD for 15 weeks, they also developed significantly higher total body weight, as well as s.c. and gonadal fat mass, than when kept on SFD (Table 1). Photochemically induced thrombosis resulted in comparable MCA occlusion times for PAI–/– mice on HFD (= 9) or SFD (= 11) (154 ± 5.4 s vs. 174 ± 9.2 s; = 0.10) (Fig. 1). The damage size in the cortical (22 ± 0.9 mm3 vs. 18 ± 2.1 mm3; = 0.10) and subcortical (8.7 ± 0.9 mm3 vs. 8.9 ± 1.4 mm3; = 0.91) regions was comparable for PAI-1–/– mice on HFD or SFD, resulting in comparable total damage size (31 ± 1.8 mm3 vs. 27 ± 2.8 mm3; = 0.24), and damaged area (24% ± 1.3% vs. 21% ± 2.1%; = 0.25). Also, edema in the cortical (12 ± 0.9 mm3 vs. 8.5 ± 2.1 mm3; = 0.13) and subcortical (6.2 ± 1.3 mm3 vs. 5.2 ± 1.0 mm3; = 0.55) regions was comparable, yielding comparable total edema zones (18 ± 1.6 mm3 vs. 14 ± 2.4 mm3; = 0.13), corresponding to areas of 7.5% ± 0.7% vs. 5.4% ± 0.9% (= 0.08) in the hemisphere, for PAI-1–/– mice on HFD or SFD respectively. ICH was not significantly different for PAI-1–/– mice on HFD or SFD (5.0 ± 1.2 mm3 vs. 4.1 ± 1.1 mm3; = 0.60) (Fig. 1).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The mechanism(s) underlying the apparent increased risk of obese subjects for developing ischemic stroke remain enigmatic. Recent findings suggest a role of hypertension, diabetes and cholesterol levels as mediators in the link between obesity and stroke, but also suggest an association that is independent of established risk factors [20]. Previously, associations have been suggested with the metabolic syndrome, insulin resistance, inflammation and hemostasis [33–36]. Development of obesity and metabolic syndrome is associated with enhanced PAI-1 levels, and adipose tissue is an important source of circulating PAI-1 [22].

In the present study, we report that both nutritionally induced and genetically determined obesity in mice have a deleterious effect on thrombotic ischemic stroke, as revealed by shorter MCA occlusion times, larger infarcts and more severe ICH. Obesity in both models is associated with markedly enhanced circulating levels of PAI-1, possibly originating from the fat tissue. It was indeed shown previously that adipose tissue is an important contributor to the elevated plasma PAI-1 levels in obese mice [36]. A weak negative correlation was observed between PAI-1 levels and the MCA occlusion times (= 0.38, = 0.06), and a weak positive correlation was observed between PAI-1 levels and the infarct size (= 0.36, = 0.08).

The hypothesis that these high PAI-1 levels may play a functional role in a worse outcome of stroke is supported by our findings that obese and lean PAI-1–/– mice respond similarly to thrombotic ischemic stroke. The small increase in brain damage in PAI-1–/– mice may indicate that other factors besides PAI-1–/– also play a role. It is conceivable that enhanced cholesterol levels in obese mice play a role in the poor outcome [20]. In obese animals, we have indeed observed significantly elevated cholesterol levels, as also reported previously [37]. The finding that cholesterol levels are also enhanced in obese as compared to lean PAI-1–/– mice indicates that they are not induced by PAI-1, but are associated with the obesity status. Ribo et al. [38] have previously reported that hyperglycemia, such as is seen in obese patients, may also be a modulator of the outcome in ischemic stroke patients treated with tissue-type plasminogen activator (t-PA), accelerating brain damage. In our model, high PAI-1 levels (low fibrinolytic activity) are compatible with shorter MCA occlusion times (time between onset of light exposure and flow arrest). Furthermore, the occlusion in this model is platelet-rich, and would depend less on fibrinolytic activity [27,40]. We have not measured recanalization times, which may be prolonged in the presence of high PAI-1 levels. In patients treated with t-PA, high plasma PAI-1 levels have indeed been associated with low revascularization rates of the MCA occlusion [39].

The suggestion that PAI-1 may play a functional role in the development of ischemic stroke is in line with a previous study in the same model, reporting that high PAI-1 levels enhanced the infarct volume because of a more severe ischemic insult and reperfusion injury as a result of delayed recanalization of the thrombotically occluded MCA [40]. Furthermore, intracerebroventricular injection of PAI-1 in this model reduced infarct volume in WT mice but not in t-PA-deficient mice, suggesting that this may be due to a suppression of the neurotoxicity of t-PA [40]. These data corroborate the findings that high plasma PAI-1 levels represent a risk factor for thrombotic ischemic stroke [41], and that PAI-1 is induced in the ischemic brain area [42,43].

The present study does not allow direct comparison of infarct volumes between WT and PAI-1–/– mice on either SFD or HFD, as the genetic backgrounds of these strains are not identical and their cerebrovascular anatomies may be different. Furthermore, it cannot be excluded that compensatory mechanisms related to differences in vessel architecture between genotypes contribute to the fact that stroke damage in PAI-1–/– mice was not affected by obesity, in contrast to other genotypes. We have not measured blood pressures in our experiments, and thus cannot exclude the possibility that obese PAI-1–/– mice could have lower blood pressure, which may be protective. It should also be emphasized that the photochemically induced MCA thrombotic occlusion model used in this study is transient in nature. In contrast, a focal cerebral ischemic infarction model in mice produced by ligation of the left MCA, which is permanent in nature, indicated that PAI-1 overexpression reduced infarct volume [40], whereas PAI-1 deficiency increased infarct size [44]. This may be explained by differences between the models in extravasation of PAI-1, affecting t-PA-induced neurotoxicity [45] and blood–brain barrier breakdown [46]. The photochemical injury also induces damage in subcortical regions, because of occlusion of small branches in the striatum.

Taken together, our data thus suggest that elevated PAI-1 levels may contribute to the deleterious outcome of thrombotic ischemic stroke in obese subjects. Specific and effective PAI-1 inhibitors will be required to establish whether this effect of PAI-1 in vivo requires its antiproteolytic activity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Skilful technical assistance by S. Helsen and L. Frederix is gratefully acknowledged.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

This study was supported by the Leducq Foundation (Paris, France; LINAT project) and by grants from the KU Leuven (OT/03/48 and EF/05/13).

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
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