Peroxisome proliferator-activated receptor gamma agonist rosiglitazone attenuates oxyhemoglobin-induced Toll-like receptor 4 expression in vascular smooth muscle cells

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Peroxisome proliferator-activated receptor gamma agonist rosiglitazone attenuates oxyhemoglobin-induced Toll-like receptor 4 expression in vascular smooth muscle cells Yi Wu, Xu-Dong Zhao, Zong Zhuang, Ya-Jun Xue, Hui-Lin Cheng, Hong-Xia Yin, Ji-Xin Shi⁎ Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province, China

A R T I C LE I N FO

AB S T R A C T

Article history:

Inflammation and immune response have been implicated in the pathogenesis of cerebral

Accepted 27 January 2010

vasospasm after subarachnoid hemorrhage (SAH). Recently, increased TLR4 expression has

Available online 2 February 2010

been associated with the development of cerebral vasospasm in a rabbit model of SAH. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists, effective inhibitors of

Keywords:

TLR4 activation, may modulate the vasospasm progression via their anti-inflammation

PPAR-gamma

effects. We investigate whether the blood component oxyhemoglobin (OxyHb) can induce

Vasospasm

the expression of Toll-like receptor (TLR) 4 in vascular smooth muscle cells (VSMCs), and

Toll-like receptor 4

evaluate the modulatory effects of PPARγ agonist rosiglitazone on OxyHb-induced

Inflammation

inflammation in VSMCs. Cultured VSMCs incubated with or without rosiglitazone were

Subarachnoid hemorrhage

exposed to OxyHb at 10 μM for up to 48 h. Expression of TLR4 was assessed by

Vascular smooth cell

immunocytochemistry and Western blot analysis. Production of tumor necrosis factor α (TNF-α) in conditioned medium were quantified by ELISA. A marked increase of TLR4 production and TNF-α release was observed at 48 h after cells were treated with OxyHb. Rosiglitazone reduced TLR4 immunocytochemistry staining and protein production significantly in VSMCs. A specific antagonist for PPARγ, GW9662, could reverse the antiinflammatory effects of rosiglitazone. The results demonstrated that OxyHb exposure could induce TLR4 activation in cultured VSMCs. Rosiglitazone suppressed TLR4 expression and cytokine release via the activation of PPARγ and may have a therapeutic potential for the treatment of vasospasm following SAH. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is the leading cause of mortality and morbidity following aneurysm rupture. Despite that significant progress has been made in understanding the underlying pathophysiology of cerebral vasospasm and developing treatment paradigms,

however, the exact molecular mechanism is obscure and cerebral vasospasm remains a major contributor to poor outcome following SAH. In recent years, inflammation and immunity have been demonstrated to be a critical pathway in vasospasm development. Many activated cytokines, adhesion molecules, transcription factors and a series of signaling pathways including MAPK, JNK, and JAK–STAT pathway are

⁎ Corresponding author. Fax: + 86 25 84817581. E-mail address: [email protected] (J.-X. Shi). 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.01.073

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reported to be involved (Osuka et al., 2006; Sasaki et al., 2004; Wakade et al., 2009; Yatsushige et al., 2005; Zhou et al., 2007a). Oxyhemoglobin (OxyHb) is a major component of blood that has been proven to be the principal cause of cerebral vasospasm and delayed neurological deficits following aneurysmal subarachnoid hemorrhage. The possible mechanisms, according to reports, can be divided into several aspects: (1) OxyHb acts a scavenger of NO, which deprives the NO of cerebral arteries and result in vasoconstriction (Schwartz et al., 2000); (2) OxyHb inhibits the activity of K+ channel, induces R-type voltage dependent Ca2+ channel (VDCC) expression and causes the opening of VDCC (Ishiguro et al., 2006; Ishiguro et al., 2008; Jewell et al., 2004; Link et al., 2008); (3) OxyHb activates the Rho/Rho kinase pathway and protein kinase C (Wickman et al., 2003); and (4) oxidation of OxyHb to MetHb generates reactive oxygen species (ROS) and induces MAPK activation (Ayer and Zhang, 2008; Zubkov et al., 2001). Toll-like receptor (TLR) family is a family of pattern recognition receptors responsible for recognizing microbial components and endogenous ligands. Activation of the TLRs could initiate innate immune responses (Akira et al., 2006; Kirk and Bazan, 2005). Recent evidence indicates that TLR4 has been implicated in inflammatory diseases of the central nervous system (CNS) including infectious diseases, neurodegenerative diseases and trauma (Kielian, 2009; Prinz et al., 2006; Suh et al., 2009; Tang et al., 2007). Our previous study also demonstrated that elevated expression of TLR4 was detected in the arterial wall in a rabbit experimental SAH model (Zhou et al., 2007b). Therefore, modulating the activation of TLR4 and holding its favorable effects for the host may be a feasible strategy for minimizing the tissue damage that originated from inflammatory responses. Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear hormone receptor superfamily that plays an important role in the regulation of inflammatory and immune reactions (Daynes and Jones, 2002; Lee et al., 2008). A rapidly expanding literature on the subject showed that PPARγ agonists could attenuate the damage produced by stroke, infection, trauma and neurodegenerative diseases in the CNS (Luo et al., 2006; Schintu et al., 2009; Yi et al., 2008). Their neuroprotective effects in those inflammatory diseases may be correlated with the suppression of a key proinflammatory transcription factor Nuclear Factor kappa B (NF-κB) (Dehmer et al., 2004; Pereira et al., 2005), upregulation of antioxidase (Collino et al., 2006), inhibition of proinflammatory mediators (Storer et al., 2005), and prevention of monocytes for their migration, adhesion and infiltration (Ramirez et al., 2008). TLR4 activation is thought to be connected with the development of cerebral vasospasm (Zhou et al., 2007b), and other studies have documented that selective PPARγ agonists are effective inhibitors of TLR4 activation both in vitro and in vivo. (Dasu et al., 2009; Gurley et al., 2008; Ji et al., 2009). It may be reasonable to postulate that PPARγ agonists could negatively regulate inflammatory responses through TLR4 pathway and play neuroprotective effects following SAH. In addition, the role of TLR4 in oxyhemoglobin-induced inflammation in rat vascular smooth muscle cells (VSMCs) is unclear. The purpose of our study is to examine the expression of TLR4 in OxyHb-treated VSMCs and evaluate the modulatory effects of PPARγ agonist rosiglitazone on OxyHb-induced inflammation in VSMCs.

2.

103

Results

2.1. Effects of PPARγ activator and inhibitor on OxyHb-induced immunocytochemical staining for TLR4 in VSMCs To determine whether the PPARγ agonist could modulate TLR4 activation in the background of SAH, the effect of rosiglitazone on immunocytochemical staining for TLR4 in VSMCs in response to OxyHb was examined. A few staining of TLR4 was observed in VSMCs in control group. Compared with that, the intensity of TLR4 staining was found significantly increased in VSMCs 48 h after OxyHb treatment. In contrast, cells treated with rosiglitazone displayed a significant decrease of TLR4 staining induced by OxyHb. However, cotreatment with GW9662 could reverse the anti-inflammatory effects of rosiglitazone and raise the immunoreactivity of TLR4 again (Figs. 1A–D). Quantitative analysis showed a low TLR4 immunoreactivity in control group with the average optical density (AOD) of 0.039 ± 0.002. TLR4 immunoreactivity was increased in OxyHb group, with an AOD of 0.120 ± 0.019, which is appropriately 3 times higher than that of control (P < 0.01). Significant difference was also observed between the rosiglitazone group (AOD = 0.052 ± 0.006) and OxyHb group (P < 0.01). Pretreating with GW9662 bringing the values of AOD in positive staining cells to get close to those measured in OxyHb group (AOD = 0.104 ± 0.02). There is no significant difference in TLR4 staining between rosiglitazone group and control group. (Fig. 1E).

2.2. PPARγ agonist rosiglitazone downregulated TLR4 protein expression in VSMCs following OxyHb treatment To detect the changes of TLR4 protein production in VSMCs, Western blot analysis was performed. A significant increase in TLR4 expression was noted after cells were treated for 48 h with OxyHb (P < 0.01). The PPARγ agonist rosiglitazone was found to reduce TLR4 expression dramatically in VSMCs in response to OxyHb (P < 0.01). Thus no significant difference in the production of TLR4 was found between rosiglitazone group and control group. Whereas, GW9662, the PPARγ-specific antagonist, could completely abolish the TLR4-inhibiting effect of rosiglitazone (Figs. 2A and B).

2.3. Ability of rosiglitazone to modulate the cytokine release in VSMCs after OxyHb treatment To evaluate whether the PPARγ agonist may modulate the inflammatory process through the regulation of cytokine secretion, the levels of TNF-α in conditioned medium were analyzed. A marked increase of TNF-α release was found in conditioned medium from VSMCs at 48 h after OxyHb treatment (P < 0.01). In agreement with the results observed in immunocytochemistry and Western blot, a significant decline of TNF-α production was also detected in rosiglitazone group in comparison with that of OxyHb group (P < 0.01). Compared with OxyHb group, there was no significant decrease in production of TNF-α in cells pretreated with GW9662 + rosiglitazone (Fig. 3).

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Fig. 1 – Effects of rosiglitazone on OxyHb-induced immunohistochemical staining for TLR4 in VSMCs. VSMCs were incubated with DMEM in the presence of vehicle (DMSO) or 10 μM OxyHb for 48 h (A and B). The cells were treated with 10 μM rosiglitazone for 1 h before OxyHb treatment (C). The cells were treated with 10 μM GW9662 for 30 min before rosiglitazone and OxyHb treatment (D). The intensity of TLR4 immunostaining was significantly increased in OxyHb-treated cells compared with that of control group. There is significant difference in the staining between VSMCs treated with OxyHb alone versus treated with OxyHb + rosiglitazone. Whereas, the TLR4-inhibiting effect of rosiglitazone was completely abolished by PPARγ-specific antagonist GW9662 (E). Bars represent the mean ± SD (n = 6, each group). **P < 0.01 compared with cells incubated under the indicated conditions, by one-way ANOVA with Fischer's LSD post hoc comparison.

3.

Discussion

The present work is consistent with our previous findings in a rabbit SAH model that received the intracisternal injection of autologous whole blood. In basilar arterial wall from a rabbit SAH, the elevated expression of TLR4 was detected and peaked on day 3 and 5, displaying a parallel time course to the development of cerebral vasospasm. In the present study, we demonstrated both qualitative and quantitative evidence that oxyhemoglobin can induce TLR4 activation and downstream proinflammatory cytokine production in cultured VSMCs. We also reported the VSMCs pretreated with PPARγ agonist rosiglitazone exhibited a significant reduction of TLR4 expression and cytokine release in response to OxyHb. Furthermore, the anti-inflammatory effect of rosiglitazone was abolished by

GW9662, a blocker of PPARγ. These current findings provide evidence for the involvement of TLR4 in OxyHb-mediated inflammatory activation in VSMCs, which may contribute to the progression of cerebral vasospasm after SAH. Results also reveal the key role of PPARγ in negative regulation of inflammatory responses through TLR4 signaling. Animal studies show that injecting adequate amounts of blood into the cerebrospinal fluid can reproduce cerebral vasospasm identical with that occurring in humans (Vatter et al., 2006). OxyHb, a major ingredient of blood, has been proved to be the key causative agent affecting smooth muscle contraction and leading to cerebral vasospasm after SAH (Macdonald and Weir, 1991). Rapidly increased evidence indicates that inflammation and immunity play an important role in the development of SAH-induced cerebral vasospasm. Various kinds of inflammatory constituents including leukocytes, cytokines,

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Fig. 2 – Effects of rosiglitazone on OxyHb-induced TLR4 protein production in VSMCs. (A) VSMCs were treated with vehicle or 10 μM rosiglitazone for 1 h, or 10 μM GW9662 for 30 min before rosiglitazone incubation, and stimulated with OxyHb for 48 h. Cells were harvested, and the protein level of TLR4 was measured by Western blot. (B) Quantitative analysis of the Western blot results for TLR4. It showed that expression of TLR4 in rosiglitazone group was significantly reduced compared with that in OxyHb group. Whereas, the TLR4-inhibiting effect of rosiglitazone was completely abolished by GW9662. Results were expressed as the mean ± SD of three experiments done in triplicate (n = 3, each group). **P < 0.01 compared with cells incubated under the indicated conditions, by one-way ANOVA with Fischer's LSD post hoc comparison.

adhesion molecules, matrix metalloproteinases, chemokines and transcription factors have been reported to be implicated in the pathogenesis of cerebral vasospasm (Dumont et al., 2003; Lu et al., 2009; McGirt et al., 2002). Additionally, numerous antiinflammatory agents targeting inflammatory cascade have been tested in animal SAH model for cerebral vasospasm therapy and achieved notable effects (Clatterbuck et al., 2003; Pradilla et al., 2005; Yatsushige et al., 2005). However, the efficacy of these agents has not yet been supported in clinical studies, which prevents their application to clinical pharmacological treatment for SAH (Singhal et al., 2005; van den Bergh et al., 2006). It is probably because the exact role of inflammation in the pathophysiology of vasospasm is still obscure, thus further research is required to comprehend the underlying mechanisms of inflammatory activation in cerebral artery following SAH and cerebral vasospasm.

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Fig. 3 – Effects of rosiglitazone on OxyHb-induced TNF-α secretion in VSMCs. VSMCs were treated with vehicle or 10 μM rosiglitazone for 1 h, or 10 μM GW9662 for 30 min before rosiglitazone incubation, and stimulated with OxyHb for 48 h. Supernatants were harvested, and TNF-α concentration were quantified by ELISA. The results indicated that rosiglitazone could effectively suppress VSMC production of TNF-α induced by OxyHb. Bars represent the mean ± SD (n = 6, each group). **P < 0.01 compared with cells incubated under the indicated conditions, by one-way ANOVA with Fischer's LSD post hoc comparison.

Recently, TLR4 has been identified as a key component of innate-immunity and a suspected contributor to the initiation and aggravation of vascular pathologies. Expanding evidence support a role of TLR4 signaling in the regulation of vascular inflammation and in the development of vessel wall damage (Coenen et al., 2009; Deng et al., 2009). Meanwhile, it was suggested that vascular smooth muscle may be a crucial target site for inflammatory stimuli within the vessel wall (Coenen et al., 2009; Jimenez et al., 2005). Elevated expression of TLR4 in VSMCs was observed in response to irritation elicited by various causative agents (Lin et al., 2006; Shinohara et al., 2007). Further, the activation of TLR4 signaling could induce a proinflammatory phenotype in VSMCs and play an active role in vascular inflammation through the release of chemokines and proinflammatory cytokines (Patel et al., 2006; Yang et al., 2005a,b). The present study demonstrated that the expression of TLR4 and downstream cytokine TNF-α in VSMCs was also enhanced by OxyHb exposure, which may correlate with inflammatory activation on the artery wall and contribute to vascular smooth muscle contraction after SAH. Despite being well known initially as a key regulator of lipid and glucose metabolism, PPARγ has also been extensively documented recently to have a role in regulating inflammatory responses. Ligands for PPARγ have been shown to mediate negative effects on inflammation and immune responses (Clark, 2002; Daynes and Jones, 2002). The therapeutic effects of PPARγ agonists have been testified in many different neuroinflammatory diseases, such as cerebral ischemia (Luo et al., 2006), multiple sclerosis (Schmidt et al., 2004), Alzheimer's disease (Xu et al., 2008), as well as traumatic brain

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and spinal cord injury (Stahel et al., 2008). Furthermore, PPARgamma plays critical roles in the vasculature. PPAR-gamma activation inhibits vascular smooth muscle cells proliferation, migration, and vascular remodeling associated with atherosclerosis (Meredith et al., 2009). It is now believed that PPARγ agonists are effective inhibitors of TLR4 activation both in vitro and in vivo (Dasu et al., 2009; Gurley et al., 2008). Moreover, it was found recently that rosiglitazone regulates angiotensin IIinduced inflammatory responses in VSMCs through the TLR4dependent signaling pathway (Ji et al., 2009). The present results showed that rosiglitazone significantly reduced OxyHb-mediated TLR4 expression and TNF-α release in VSMCs. It may indicate that rosiglitazone exerts its neuroprotective effects by suppressing TLR4 signaling activation in smooth muscle to prevent the aggravation of vascular inflammation and the development of vasospasm associated with SAH. Although rosiglitazone has several PPARγ-independent effects, we believe its anti-inflammatory effects were mediated, at least in part, through PPARγ activation, because the negative effects of rosiglitazone were effectively counteracted by the PPARγ antagonist GW9662. Our data showed that the ability of attenuating inflammation by rosiglitazone in VSMCs was dependent on the PPARγ pathway. In conclusion, the present study demonstrated the upregulation of TLR4 expression in VSMCs following OxyHb exposure. PPARγ agonist, rosiglitazone, could inhibit the OxyHb-induced inflammatory responses in VSMCs by blocking TLR4 activation and cytokine release. Moreover, attenuation of inflammation by rosiglitazone in VSMCs was dependent on PPARγ activation. These findings suggested that PPARγ agonists may have a therapeutic potential for the treatment of cerebral vasospasm following SAH, while the precise role of PPARγ in vasospasm needs further research.

4.

Experimental procedures

4.1.

Primary VSMC culture

Primary VSMCs were isolated from the aortic arch of male Sprague Dawley rats (3 to 4 weeks of age) by the explant method. Cells were propagated in Dulbecco's modified Eagle's medium (DMEM, Invitrogen Corp., Carlsbad, CA, USA) containing 20% Fetal bovine serum (FBS, Invitrogen Corp.), 100 U/mL penicillin, and 100 U/mL streptomycin. The medium was changed every 3 days. After reaching a subconfluent state, cells were subsequently subcultured at a split ratio of 1:2. Subcultured cells from passages 4 to 6 were used. VSMCs were verified by their characteristic hills-and-valleys appearance and >98% positive immunostaining of α-actin. VSMCs were seeded into 6-well or 96-well plates at 2 × 105 or 2 × 104 cells/well, respectively, and incubated overnight. The next day, the cells were treated with OxyHb (Bomei biocompany, Hefei, China) at a concentration of 10 μM. After 48 h of OxyHb incubation, the cells and the supernatants of culture media were collected and stored at − 80 °C until tested for western blot or enzyme-linked immunosorbent assays (ELISA) respectively.

4.2.

PPARγ induction and inhibition

To evaluate the involvement of PPARγ in the activation of TLR4, PPARγ agonist rosiglitazone (10 μM, Cayman Chemical Co., Ann Arbor, MI, USA) was added 1 h before OxyHb incubation (PPARγ group). Meanwhile, to determine whether the effects of rosiglitazone on TLR4 expression is dependent on PPARγ activation, the specific PPARγ antagonist, GW9662 (10 μM, Sigma-Aldrich, Inc., Saint Luis, MO, USA) was added into the culture medium 30 min before rosiglitazone incubation in some groups (GW9662 group). In control or OxyHb group, untreated cells and OxyHb-treated cells received equal volumes of dimethyl sulfoxide (DMSO, Sigma-Aldrich, Inc.) (vehicle for rosiglitazone and GW9662).

4.3.

Immunocytochemical examination

VSMCs were plated on glass coverslips in 6-well plates at a density of 2 × 105 cells/well and incubated overnight. Then cells were treated as previously described. After 48 h incubation, VSMCs grown on the coverslips were fixed with ice-cold 4% (wt./vol.) paraformaldehyde for 20 min. After washing three times in PBS for 10 min, coverslips were blocked by 40 min incubation in 10% goat serum at room temperature. Subsequently, cells were incubated at 4 °C overnight with rabbit antiTLR4 antibody (diluted in 1:50, Bioworld Technology, Inc., Minneapolis, MN, USA), followed by a 10-min wash in PBS. Coverslips were incubated with horseradish peroxidase (HRP)conjugated goat anti-rabbit IgG (1:500 dilution, Santa Cruz Biotechnology, Inc., California, USA) for 60 min at room temperature, and then DAB was used as chromogen. Coverslips incubated in the absence of primary antibody were used as negative control. Finally, coverslips were mounted on glass slides with mounting medium. Six views were selected randomly for each slide and observed under a light microscope (×200). Then the average optical densities of positive staining cells in six views were regarded as the data for each sample and measured to evaluate the activation of TLR4 in VSMCs. To analyze the optical densities, Image Pro-Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) was used.

4.4.

Western blot analysis

Culture medium was removed and washed twice with chilled (4 °C) PBS. The cells were quickly scraped and collected by centrifugation and then stored at −80 °C until they were assayed. Cells were lysed in 20 mM Tris–HCl (pH 8.0), 4 M NaCl, 0.5 M EDTA. Protein concentration in the supernatant was determined by the Bradford assay (Bio-Rad Lab, Hercules, CA, USA). Equal amounts of protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. The membranes were blocked with 3% (wt./vol.) low-fat milk and then incubated overnight at 4 °C with primary antibodies for TLR4 at the dilution of 1:500. The β-actin (diluted in 1:6000, Sigma-Aldrich, Inc.) was used as a loading control. After the membranes were washed and further incubated with HRPconjugated secondary antibody at 1:400 for 2 h, the blotted protein bands were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham, Arlington Heights, IL,

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USA). Optical densities of the resulting bands were quantified using Glyko Bandscan software (Glyko, Novato, CA, USA).

4.5.

Measurement of cytokine

VSMCs were seeded in 96-well plates at a density of 2 × 105 cells/well and then treated as previously described. After 48 h incubation, the conditioned media from each group were centrifuged to remove the detached cells, and the supernatants were collected for appliance. Protein levels of TNF-α (rat TNF-α ELISA, Bender MedSystems, Vienna, Austria) were quantified in supernatants using ELISA Kit according to the manufacturer's instructions (level of sensitivity = 11.2 pg/mL).

4.6.

Statistical analysis

All results were presented as the mean ± SD. The responses of control and treated VSMCs were compared by one-way ANOVA. Fischer's LSD post hoc test was used for multiple comparisons. A probability value of less than 0.05 was considered statistically significant.

Acknowledgments We thank Dr. Honglin Yin for his technical assistance. This work was supported by grants from the Jinling Hospital of China.

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