Scavenger Receptor Class-A Has a Central Role in Cerebral Ischemia

Abstract

The innate immune response is involved in the pathophysiology of cerebral ischemia–reperfusion (I/R) injury. Recent evidence suggests that scavenger receptors have a role in the induction of innate immunity. In this study, we examined the role of scavenger receptor A (SR-A) in focal cerebral I/R injury. Both SR-A^−/− mice (n=10) and age-matched wild-type (WT) mice (n=9) were subjected to focal cerebral ischemia (60 minutes), followed by reperfusion (for 24 hours). Infarct size was determined by TTC (triphenyltetrazolium chloride) staining. The morphology of neurons in the brain sections was examined by Nissl's staining. Activation of intracellular signaling was analyzed by western blot. Cerebral infarct size in SR-A^−/− mice was significantly reduced by 63.9% compared with WT mice after cerebral I/R. In SR-A^−/− mice, there was less neuronal damage in the hippocampus compared with WT mice. Levels of FasL, Fas, FADD, caspase-3 activity, and terminal deoynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling-positive apoptotic cells were significantly increased in WT mice after cerebral I/R, but not in SR-A^−/− mice. Cerebral I/R increased nuclear factor-κB activation in WT mice, but not in SR-A^−/− mice. These data suggest that SR-A has a central role in cerebral I/R injury and that suppression of SR-A may be a useful approach for ameliorating brain injury in stroke patients.

Keywords

apoptosis cerebral ischemia–reperfusion inflammatory response innate immunity scavenger receptor SR-A

Introduction

A growing body of evidence suggests that innate immune and inflammatory responses are involved in brain ischemia–reperfusion (I/R) injury (Jordan et al, 2008; Brea et al, 2009). Indeed, we along with others have shown that modulation of innate immune responses significantly attenuated cerebral I/R injury (Caso et al, 2007; Tang et al, 2007; Hua et al, 2007b, 2008, 2009). However, the mechanisms by which innate immune and inflammatory responses are activated in the ischemic brain remain unclear.

Pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), have a critical role in the induction of innate immunity and inflammatory responses (Medzhitov et al, 1997). Toll-like receptor-mediated signaling predominately activates nuclear factor-kappaB (NF-κB), which is an important transcription factor controlling innate immune and inflammatory cytokine gene expressions (Medzhitov et al, 1997). We, along with others, have shown that TLR4 deficiency protects the brain from cerebral ischemic injury (Caso et al, 2007; Tang et al, 2007; Hua et al, 2007b, 2008, 2009).

The macrophage scavenger receptor A (SR-A also known as CD204) was initially discovered because of its ability to bind and internalize modified low-density lipoprotein (Goldstein et al, 1979). Subsequently, SR-A has been shown to recognize and clear modified host components, apoptotic cells, and pathogens (Platt and Gordon, 2001; Greaves and Gordon, 2009). Numerous studies have shown that SR-A has a critical role in the induction of innate immune and inflammatory responses by recognition of exogenous pathogen-associated molecular patterns and endogenous ligands (Zhu et al, 2001; Hollifield et al, 2007; Limmon et al, 2008). However, the intracellular signaling mediated by SR-A is still unclear. Most studies have indicated that SR-A does not directly transduce a signal into the cell (Cotena et al, 2004), because the intracellular domain of SR-A lacks signaling motifs (Bowdish and Gordon, 2009). Several other studies have suggested that SR-A intracellular domains can be phosphorylated, which may facilitate the interaction of the SR-A transmembrane domain with intracellular signaling components (Fong and Li, 1999; Hsu et al, 2001; Nikolic et al, 2007; Todt et al, 2008). Recent evidence also suggests that SR-A is a coreceptor for TLRs to facilitate innate immune recognition and response, resulting in an overexuberant response (Seimon et al, 2006). For example, TLR ligands synergize with SR-A to mediate bacterial phagocytosis (Amiel et al, 2009), induce SR-A expression (Xu et al, 2007), and promote SR-A binding to the TLR4 ligand, lipopolysaccharide (Xu et al, 2007). Scavenger receptor A interacts with TLR4 to promote a proinflammatory, proapoptotic phenotype in lipopolysaccharide-exposed macrophages (Seimon et al, 2006). In addition, SR-A suppresses prosurvival signaling pathways, such as interferon regulatory factor-3-mediated interferon-β production (Seimon et al, 2006). In contrast, SR-A ligands trigger apoptosis in the endoplasmic reticulum-stressed macrophages by cooperating with TLR4 (Seimon et al, 2006) and serve as a negative regulator of TLR4 in mediating immune responses (Yi et al, 2009). Collectively, these data suggest that SR-A could contribute to cerebral ischemic injury by promoting the activation of innate immune and inflammatory responses (Zhu et al, 2001; Hollifield et al, 2007; Limmon et al, 2008), by acting as a coreceptor to TLR4 (Seimon et al, 2006), and by suppressing the prosurvival signaling pathway (Seimon et al, 2006).

In this study, we examined the role of SR-A in focal cerebral I/R injury. We observed that SR-A deficiency significantly protected the brain from focal cerebral ischemic injury. This is the first report to show that SR-A contributes to brain damage caused by ischemic stroke. Our data suggest that suppression of SR-A could be a strategy for prevention and therapy in stroke patients.

Materials and methods

Animals

Breeding pairs of macrophage SR-A-deficient mice on the C57BL/6J background were provided by Dr Siamon Gordon (Sir William Dunn School of Pathology, Oxford University) (Cotena et al, 2004). A breeding colony was established and maintained in the Division of Laboratory Animal Resources, Quillen College of Medicine, ETSU (East Tennessee State University). Male wild-type (WT) C57BL mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). The experiments outlined in this article conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85–23, revised 1996). The animal care and experimental protocols were approved by the ETSU Committee on Animal Care.

Focal Cerebral Ischemia–Reperfusion

Focal cerebral I/R was induced by occlusion of the middle cerebral artery on the left side as described in our previous studies (Hua et al, 2008, 2009). Briefly, mice were subjected to anesthesia by 5.0% isoflurane and anesthesia was maintained by inhalation of 1.5% to 2% isoflurane driven by 100% oxygen flow. Mice were ventilated (110 breaths per minute with volume 0.5 mL), and body temperature was regulated at 37.0°C by surface water heating. After skin incision, the left common carotid artery, the external carotid artery, and the internal carotid artery were carefully exposed. Microvascular aneurysm clips were applied to the left common carotid artery and the internal carotid artery. A coated 6–0 filament (6023PK, Doccol, Redlands, CA, USA) was introduced into an arteriotomy hole, fed distally into the internal carotid artery. After the internal carotid artery clamp was removed, the filament was advanced 11 mm from the carotid bifurcation, and focal cerebral ischemia started. After ischemia for 60 minutes, the filament and the common carotid artery clamp were gently removed (reperfusion starts). The collar suture at the base of the external carotid artery stump was tightened. The skin was closed, anesthesia discontinued, and the animal was allowed to recover in prewarmed cages. Control mice underwent a neck dissection and coagulation of the external carotid artery, but no occlusion of the middle cerebral artery.

Examination of Infarct Size

Infarct size was determined as described previously (Hua et al, 2008, 2009). After ischemia (60 minutes), followed by reperfusion (24 hours), mice were killed and perfused with ice-cold phosphate-buffered saline through the ascending aorta. Brains were removed and sectioned coronally into 2 mm-thick slices. The slices were stained with 2% TTC (triphenyltetrazolium chloride) solution at 37°C for 15 minutes, followed by fixation with 10% formalin neutral buffer solution (pH 7.4). The infarct areas were traced and quantified using an image-analysis system. Unstained areas (pale color) were defined as ischemic lesions. The areas of infarction and the areas of both hemispheres were calculated for each brain slice. An edema index was calculated by dividing the total volume of the left hemisphere by the total volume of the right hemisphere. The actual infarct volume adjusted for edema was calculated by dividing the infarct volume by the edema index. Infarct volumes are expressed as percentage of the total brain volume ± s.e.m.

Evaluation of Neuronal Damage in Hippocampal Formation

Neuronal damage in brain sections were determined by Nissl's method as described in our previous studies (Hua et al, 2008, 2009). Paraffin sections cut in the coronal plane at ∼1.5 mm behind the bregma with a thickness of 7 μm were deparaffinized and then stained with 0.1% cresyl violet for 2 minutes. The sections were evaluated using light microscopy.

Western Blots

Cellular proteins were prepared from the brain tissues and immunoblots were performed as described previously (Li et al, 2003; Hua et al, 2007a, b , 2008, 2009). The cellular proteins were separated by SDS-PAGE and transferred onto Hybond ECL membranes (Amersham Pharmacia, Piscataway, NJ, USA). The ECL membranes were incubated with the appropriate primary antibody (anti-phospho-Jun N-terminal kinase (JNK), anti-JNK, anti-phospho-MKK4/7, anti-MKK4/7, anti-phospho-IκBα (Cell Signaling Technology, Beverly, MA, USA), anti-Fas, anti-FasL, and anti-Fas-associated protein with death domain (FADD) (Santa Cruz Biotechnology, Santa Cruz, CA, USA)), respectively, followed by incubation with peroxidase-conjugated secondary antibodies (Cell Signaling Technology). The signals were detected using the ECL system (Amersham Pharmacia). To control for lane loading, the same membranes were probed with anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase, Biodesign, Saco, ME, USA) after being washed with the stripping buffer. The signals were quantified by scanning densitometry using a Bio-Image Analysis System (Bio-Rad, Hercules, CA, USA). The results from each experimental group were expressed as relative integrated intensity compared with that of control hearts measured at the same time.

Electrophoretic Mobility Shift Assay

Nuclear proteins were isolated from ischemic cerebral hemispheres as described previously (Li et al, 2003; Hua et al, 2007a, b , 2008, 2009). Nuclear factor-κB binding activity was determined by electrophoretic mobility shift assay as described in our previous studies. Nuclear factor-κB binding activity was examined by electrophoretic mobility shift assay in a 15 μL binding reaction mixture containing 15 μg of nuclear proteins and 35 fmol of [γ-³²P]-labeled double-stranded NF-κB consensus oligonucleotide. A supershift assay using antibodies to p65 and p50 was performed to confirm NF-κB binding specificity and subunits as described previously (Li et al, 2003).

Caspase-3 Activity Assay

Caspase-3 activity in the brain tissue was measured using a Caspase-Glo assay kit (Promega, Madison, WI, USA) according to the manufacturer's protocol.

Terminal Deoynucleotidyl Transferase-Mediated 2′-Deoxyuridine 5′-Triphosphate-Biotin Nick End Labeling Analysis

Terminal deoynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining was performed using the In Situ Cell Death Detection Kit (Promega) according to the manufacturer's protocol. The number of total cells and TUNEL-positive cells were counted throughout borders between infarction and normal areas in the fields of the hippocampus (Hua et al, 2007b). The number of TUNEL-positive cells was expressed as the percentage of total counted cells.

Statistical Analysis

Data are expressed as mean ± s.e. Comparisons of data between groups were made using one-way ANOVA (analysis of variance), and Tukey's procedure for multiple range tests was performed. A P-value < 0.05 was considered significant.

Results

SR-A Deficiency Reduced Cerebral Infarct Size After Ischemia–Reperfusion

We have previously shown that TLR4 deficiency attenuated cerebral infarction after I/R (Hua et al, 2007b, 2009). Scavenger receptor A is considered to be a PRR (Zhu et al, 2001; Hollifield et al, 2007; Limmon et al, 2008); therefore, we examined whether SR-A is involved in cerebral I/R injury. As shown in Figure 1, focal cerebral I/R resulted in large infarcts in WT mice. Scavenger receptor A–deficient mice show significantly reduced cerebral infarct volumes (64.2%), when compared with WT I/R mice (6.85% ± 1.91% versus 19.14% ± 2.85%, P < 0.05).

Figure 1

Reduced cerebral infarct size in SR-A^−/− mice after I/R injury. SR-A-deficient (n = 10) and C57BL (WT) mice (n = 9) were subjected to focal cerebral ischemia (60 minutes), followed by reperfusion (24 hours). Infarct size was determined by TTC staining and expressed as the percentage of actual infarct size volume in the total cerebral volume. Representative brain sections stained with TTC are shown at the top. ^#P < 0.05 compared with WT. I/R, ischemia–reperfusion; SR-A, scavenger receptor A; TTC, triphenyltetrazolium chloride; WT, wild type.

SR-A Deficiency Attenuated Neuronal Damage in Hippocampal Formation After Cerebral Ischemia–Reperfusion

We examined neuronal damage in hippocampal formation after cerebral I/R. Nissl's staining showed neuronal damage in the CA1 field of the hippocampal formation in WT-I/R mice characterized by numerous, often confluent zones of shrunken cell bodies accompanied by shrunken and pyknotic nuclei (Figure 2). Similar changes were observed in the dentate gyrus and CA4. In contrast, the neurons in the CA1, CA4, and DG fields in SR-A-deficient mice brain showed less neuronal damage and the morphology was well preserved (Figure 2).

Figure 2

SR-A deficiency attenuated neuronal damage in HF after cerebral I/R. SR-A-deficient (n = 6) and C57BL (WT) mice (n = 6) were subjected to focal cerebral ischemia (60 minutes), followed by reperfusion (24 hours). Sham surgically operated mice (n = 4 per group) served as sham control. Brain sections were stained with 0.1% cresyl violet. The hippocampal neurons in the CA1 field showed extreme changes in WT mice, with abnormal nuclei and neuronal shrinkage. In contrast, the neurons in this field showed less neuronal damage and the morphology of neurons was well preserved in the SR-A-deficient mice. HF, hippocampal formation; I/R, ischemia–reperfusion; SR-A, scavenger receptor A; WT, wild type.

Ischemia–Reperfusion-Induced Nuclear Factor-κB Binding Activity is Prevented by SR-A Deficiency

Nuclear factor-κB is an important transcription factor that regulates inflammatory cytokine gene expression (Medzhitov et al, 1997). Inhibition of NF-κB binding activity has been shown to significantly attenuate cerebral ischemic injury (Schneider et al, 1999a, b ). We examined I/R-induced NF-κB binding activity in WT- and SR-A-deficient mice. As shown in Figure 3A, cerebral I/R significantly increased NF-κB binding activity by 46.1%, compared with sham control (1.52 ± 0.09 versus 1.04 ± 0.141, P < 0.05). However, NF-κB binding activity in SR-A-deficient mice was not increased after cerebral I/R and was significantly lower than that observed in WT mice (1.04 ± 0.05 versus 1.52 ± 0.09, P < 0.05). The Supershift assay showed the specificity of NF-κB binding activity with p65 and p50 subunits (Figure 3B).

Figure 3

I/R-increased IRAK and IκBα phosphorylation and NF-κB binding activity are effectively prevented in SR-A deficiency. SR-A-deficient (n = 6) and WT mice (n = 6) were subjected to focal cerebral ischemia (45 minutes)/reperfusion (6 hours). Sham surgically operated mice served as sham control (n = 4 per group). Nuclear and cytoplasmic proteins were isolated from brain tissues for analysis of NF-κB binding activity by EMSA (A), supershift assay (B), and for examination of phospho-IRAK (C) and phospho-IκBα (D) by western blot. EMSA, electrophoretic mobility shift assay; I/R: ischemia–reperfusion; IRAK, interleukin receptor-associated kinase; NF-κB, nuclear factor-κB; S, sham; SR-A, scavenger receptor A; WT, wild type;. *P < 0.05 compared with indicated groups.

Ischemia–Reperfusion-Induced Phosphorylation of IRAK and IκBα is Prevented in the Brain Tissue of SR-A-Deficient Mice

In the NF-κB activation pathway, interleukin receptor-associated kinase (IRAK) and IκBα phosphorylation are important upstream regulators of NF-κB activation and nuclear translocation (Medzhitov et al, 1997). Therefore, we examined IRAK and IκBα phosphorylation in the brain tissues after cerebral I/R. Figures 3C and 3D show that the levels of p-IRAK and p-IκBα were significantly increased by 54.0% (1.09 ± 0.04 versus 0.71 ± 0.03) and 128.7% (1.76 ± 0.17 versus 0.77 ± 0.25), respectively, in WT mice after cerebral I/R compared with sham control. In contrast, cerebral I/R did not increase IRAK and IκBα phosphorylation in the brain tissues of SR-A-deficient mice. In fact, levels of p-IRAK (0.67 ± 0.05 versus 1.09 ± 0.04) and p-IκBα (0.70 ± 0.06 versus 1.76 ± 0.17) in SR-A^−/− brains were significantly lower than that in WT mice after I/R.

Ischemia–Reperfusion-Associated Increases in FasL, Fas, and FADD are not Observed in the Brain Tissue from SR-A-Deficient Mice

The Fas-mediated apoptotic signaling pathway has an important role in cerebral ischemic injury (Dzietko et al, 2008). Inhibition of apoptotic signaling pathways has been shown to protect the brain from ischemic injury (Dzietko et al, 2008). We examined the effect of SR-A deficiency on the Fas-mediated apoptotic signaling pathway after cerebral I/R. As shown in Figures 4A–4C, cerebral I/R significantly increased the levels of Fas, FasL, and FADD in the WT brain tissues by 33.3, 49.6, and 52.2%, respectively, when compared with sham control. In striking contrast, SR-A deficiency prevented I/R-induced Fas, FasL, and FADD in the brain tissues (Figures 4A–4C).

Figure 4

I/R-increased FasL, Fas, and FADD are attenuated by SR-A deficiency. SR-A-deficient (n = 6) and WT mice (n = 6) were subjected to focal cerebral ischemia (45 minutes)/reperfusion (6 hours). Sham surgically operated mice served as sham control (n = 4 per group). Cytoplasmic proteins were isolated from brain tissues for western blot analysis of FasL (A), Fas (B), and FADD (C) expression. *P < 0.05 compared with indicated groups. I/R, ischemia–reperfusion; S, sham; SR-A, scavenger receptor A; WT, wild type.

SR-A Deficiency Effectively Prevented Ischemia–Reperfusion-Induced MKK4 and JNK Phosphorylation in the Brain Tissues

Activation of the JNK signaling pathway has been shown to contribute to cerebral ischemic injury (Gao et al, 2005). In addition, ligation of SR-A activates the JNK signaling pathway (Ricci et al, 2004). MKK4 is an upstream regulator of JNK (Gao et al, 2005). We examined the effects of SR-A on MKK4 and JNK phosphorylation in the brain tissues after cerebral I/R. As expected, cerebral I/R increased the levels of phosphorylated-JNK by 58.7% and phosphorylated Mkk4 by 57.7%, when compared with WT sham controls (Figures 5A and 5B). In striking contrast, the levels of p-JNK and p-MKK4 were essentially normal in SR-A-deficient mice after cerebral I/R.

Figure 5

SR-A deficiency prevented I/R-increased MKK4 and JNK phosphorylation in the brain. SR-A-deficient (n = 6) and WT mice (n = 6) were subjected to focal cerebral ischemia (45 minutes)/reperfusion (6 hours). Sham surgically operated mice served as sham control (n = 4 per group). Cytoplasmic proteins were isolated from brain tissues for western blot analysis of MKK4/7 (A) and JNK (B) phosphorylation. *P < 0.05 compared with indicated groups. I/R, ischemia–reperfusion; JNK, Jun N-terminal kinase; S, sham; SR-A, scavenger receptor A; WT, wild type.

Attenuation of Ischemia–Reperfusion Induced Brain Caspase-3 Activity in SR-A-Deficient Mice

We examined caspase-3 activity in the brain tissues after cerebral I/R. Figure 6A shows that I/R increased caspase-3 activity by 57.9% in WT mice compared with sham control. In contrast, cerebral I/R did not increase caspase-3 activity in SR-A-deficient mice compared with sham control mice. The TUNEL assay indicated that I/R significantly increased the number of apoptotic brain cells, when compared with the sham control (45.6% ± 4.34% versus 21.4% ± 2.1%), which is consistent with caspase-3 activity. However, apoptotic cells were significantly reduced in the brain tissues of SR-A^−/− mice after cerebral I/R compared with WT mice (Figure 6B).

Figure 6

Attenuation of I/R induced brain caspase-3 activity in SR-A-deficient mice. SR-A-deficient (n = 6) and WT mice (n = 6) were subjected to focal cerebral ischemia (45 minutes)/reperfusion (6 hours). Sham surgically operated mice served as sham control (n = 4 per group). (A) Cytoplasmic proteins were isolated from brain tissues for analysis of caspase-3 activity. (B) TUNEL assay was performed on the brain sections. *P < 0.05 compared with indicated groups. I/R, ischemia–reperfusion; S, sham; SR-A, scavenger receptor A; TUNEL, terminal deoynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling; WT, wild type.

Discussion

This study provides compelling evidence that the macrophage SR-A contributes to the pathophysiology of cerebral ischemic injury. In particular, we observed that mice deficient in SR-A showed dramatically smaller brain infarcts in response to early cerebral I/R injury, implying that SR-A mediates, in part, brain I/R pathology. Our data indicate that SR-A deficiency attenuated cerebral I/R-activated apoptotic signaling pathways and NF-KB binding activity, both of which are known to contribute to cerebral ischemic injury. When considered as a whole, these data suggest that SR-A has a critical role in early cerebral ischemic injury. The data also suggest that SR-A may be an important target for preventing and treating cerebral ischemic injury.

The SR-A is a trimeric, type II membrane glycoprotein that was initially described on the basis of its ability to bind and internalize modified low-density lipoprotein (Goldstein et al, 1979). The SR-A expression is principally on macrophages and dendritic cells and can be induced in the endothelium and smooth muscle cells in atherosclerotic plaques (Krieger, 1997; Yi et al, 2009). Interestingly, Matsumoto et al (1990) reported the presence of SR-A in the human brain in 1990. Since then, SR-A has been detected in the microglia (Grewal et al, 1997; El Koury et al, 1998), in senile plaques of brain tissue from patients with Alzheimer's disease (Christie et al, 1996), and on Mato's fluorescent granular perithelial perivascular macrophages in the normal brain tissue (Mato et al, 1996). We observed in this study that SR-A deficiency significantly reduced infarction after cerebral I/R, suggesting that SR-A, that is expressed on the microglia and Mato cells, could mediate pathophysiological response to cerebral I/R injury. Cho et al (2005) (Kim et al, 2008; Kunz et al, 2008) recently reported that the class B scavenger receptor (CD36) has a role in brain ischemic injury. CD36 is a membrane glycoprotein that is found on many cell types, including platelets, endothelial cells, macrophages, adipocytes, and microglia, and has been implicated in a host of normal and disease processes (Silverstein and Febbraio, 2009). Mice deficient in CD36 were partially protected from experimental brain ischemic injury (Cho et al, 2005; Kim et al, 2008; Kunz et al, 2008). The mechanism of protection was shown to be due to a decreased inflammatory response (Silverstein and Febbraio, 2009).

Scavenger receptor A has recently been shown to act as a PRR and has an important role in the induction of innate immune and inflammatory responses (Zhu et al, 2001; Hollifield et al, 2007; Limmon et al, 2008). For example, SR-A can recognize several pathogen-associated molecular patterns, such as lipopolysaccharide, lipoteichoic acid, bacterial CpG DNA, double-stranded RNA, and yeast zymosan/β-glucans (Zhu et al, 2001; Mukhopadhyay and Gordon, 2004; Hollifield et al, 2007; Limmon et al, 2008; Areschoug and Gordon, 2008). The contribution of the innate immune and inflammatory responses to cerebral I/R has been well demonstrated (Jordan et al, 2008; Brea et al, 2009). Cerebral ischemia activates innate immunity and triggers inflammatory responses through the activation of TLR-mediated signaling pathways (Caso et al, 2007; Tang et al, 2007; Hua et al, 2007b, 2008, 2009). We, along with other investigators, have shown that TLR4 contributes to cerebral ischemic injury (Caso et al, 2007; Tang et al, 2007; Hua et al, 2007b, 2008, 2009). Toll-like receptor 4 deficiency protects the brain against cerebral ischemic injury by inhibition of NF-κB binding activity, resulting in the down-regulation of inflammatory responses (Caso et al, 2007; Tang et al, 2007; Hua et al, 2007b, 2008, 2009). Interestingly, recent studies have suggested that SR-A can act as a coreceptor with TLR4 in modulating the inflammatory response to TLR agonists (Seimon et al, 2006; Amiel et al, 2009). For example, TLR-specific stimuli synergized with SR-A to mediate bacterial phagocytosis (Amiel et al, 2009). Toll-like receptor ligands dramatically induced SR-A expression and promoted macrophages to bind and internalize TLR4 ligand through SR-A (Xu et al, 2007). On the other hand, SR-A ligands triggered apoptosis in the endoplasmic reticulum-stressed macrophages by cooperating with TLR4 (Seimon et al, 2006) and may serve as a physiologic negative regulator of TLR4-mediated immune responses (Yi et al, 2009). Collectively, SR-A may contribute to cerebral I/R injury by deleterious augmentation of innate immune and inflammatory challenges.

An increasing body of evidence indicates that focal and global cerebral ischemia induce apoptosis, which is an active process (Gao et al, 2005; Dzietko et al, 2008). Death receptor signaling molecules such as FasL and TRAIL have been implicated in ischemia-induced neuronal apoptosis. Fas-mediated neuronal apoptosis during cerebral I/R is deleterious (Dzietko et al, 2008). Mice that lack functional Fas and FasL are protected from ischemic neuronal injury (Graham et al, 2004). We observed in this study that cerebral I/R significantly increased the levels of FasL and Fas in the brain tissues of WT mice. Caspase-3 activity and apoptotic cells in the brains of WT mice were significantly increased after cerebral I/R. However, SR-A deficiency significantly prevented cerebral I/R-increased Fas and FasL levels in the brain tissues. The caspase-3 activity and apoptotic cells in the brains were significantly attenuated in SR-A-deficient mice after cerebral I/R. Our observations suggest that SR-A may promote the Fas/FasL-mediated apoptotic signaling pathway after cerebral I/R. However, it is unclear how SR-A promotes Fas/FasL-mediated apoptotic signaling during cerebral I/R. Scavenger receptor A has been considered as a coreceptor in the induction of innate immune and inflammatory responses (Seimon et al, 2006); therefore, it could be possible that SR-A may cooperate with Fas in activating the apoptotic signaling pathway during cerebral I/R.

Activation of the JNK pathway has a role in cerebral ischemic injury through the apoptotic pathway (Gao et al, 2005). Specific inhibition of JNK activation has been shown to attenuate cerebral ischemic injury (Gao et al, 2005). We observed that SR-A deficiency attenuated cerebral I/R-increased phosphorylation of MKK4/7 and JNK. Attenuation of JNK activation after cerebral I/R could be an additional mechanism by which SR-A deficiency prevents activation of the apoptotic signaling pathway after cerebral I/R. It has been shown that stimulation of SR-A by its ligands will activate the JNK signaling pathway (Ricci et al, 2004). Collectively, SR-A could be a target for preventing apoptosis during cerebral I/R.

Nuclear factor-κB is an important transcription factor, which regulates the expression of genes associated with immune and inflammatory responses. Activation of NF-κB has been shown to contribute to cerebral ischemic injury (Schneider et al, 1999a; Stephenson et al, 2000). For example, inhibition of NF-κB binding activity showed a therapeutic effect on experimental cerebral I/R injury (Schneider et al, 1999a, b ). We have reported that TLR4 deficiency protects against cerebral ischemic injury (Hua et al, 2007b, 2008, 2009). Nuclear factor-κB binding activity in the particularly ischemia-sensitive hippocampal formation was significantly attenuated in TLR4-deficient mice after cerebral I/R compared with WT mice (Hua et al, 2007b, 2008, 2009). In this study, we observed that SR-A deficiency significantly attenuated cerebral I/R-in-creased NF-κB activation. Although we do not know the mechanism by which deficiency of SR-A attenuated NF-κB activation after cerebral I/R, recent studies have shown a cooperation between SR-A and TLR4 (Seimon et al, 2006; Amiel et al, 2009; Yi et al, 2009). Therefore, activation of SR-A by its ligands during I/R may trigger TLR4 to activate the NF-κB pathway. In addition, SR-A is now considered to be a PRR which can recognize several ligands, such as double-stranded RNA (Limmon et al, 2008), CpG DNA (Zhu et al, 2001), apoptotic cells, and unknown endogenous ligands, which may be released from stressed or damaged cells after I/R, resulting in the activation of NF-κB through ligation of TLRs. We observed that NF-κB binding activity was increased by 46.1% after cerebral I/R in this study, which is consistent with our previous report (Hua et al, 2009). However, we observed in a global brain ischemia (12 minutes)/reperfusion (24 hours) that NF-κB binding activity was significantly increased by 91% (Hua et al, 2007b). These data suggest that the time of ischemia and the proportion of dead tissue could affect the degree of NF-κB binding activity in a cerebral I/R model.

In summary, SR-A has a central role in the pathophysiology of cerebral ischemic injury. The mechanisms involve prevention of I/R-increased activation of apoptotic signaling and NF-κB binding activity. The data show that SR-A mediates activation of inflammatory signaling and apoptosis in ischemic stroke, both of which contribute to cerebral injury. These data also suggest that modulation of SR-A activity may be a useful approach for ameliorating brain injury in stroke patients.

Footnotes

The authors declare no conflict of interest.

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Scavenger Receptor Class-A Has a Central Role in Cerebral Ischemia–Reperfusion Injury

Abstract

Keywords

Introduction

Materials and methods

Animals

Focal Cerebral Ischemia–Reperfusion

Examination of Infarct Size

Evaluation of Neuronal Damage in Hippocampal Formation

Western Blots

Electrophoretic Mobility Shift Assay

Caspase-3 Activity Assay

Terminal Deoynucleotidyl Transferase-Mediated 2′-Deoxyuridine 5′-Triphosphate-Biotin Nick End Labeling Analysis

Statistical Analysis

Results

SR-A Deficiency Reduced Cerebral Infarct Size After Ischemia–Reperfusion

SR-A Deficiency Attenuated Neuronal Damage in Hippocampal Formation After Cerebral Ischemia–Reperfusion

Ischemia–Reperfusion-Induced Nuclear Factor-κB Binding Activity is Prevented by SR-A Deficiency

Ischemia–Reperfusion-Induced Phosphorylation of IRAK and IκBα is Prevented in the Brain Tissue of SR-A-Deficient Mice

Ischemia–Reperfusion-Associated Increases in FasL, Fas, and FADD are not Observed in the Brain Tissue from SR-A-Deficient Mice

SR-A Deficiency Effectively Prevented Ischemia–Reperfusion-Induced MKK4 and JNK Phosphorylation in the Brain Tissues

Attenuation of Ischemia–Reperfusion Induced Brain Caspase-3 Activity in SR-A-Deficient Mice

Discussion

Footnotes

References