J Mol Neurosci DOI 10.1007/s12031-014-0291-x
ApoE-Deficient Promotes Blood–Brain Barrier Disruption in Experimental Autoimmune Encephalomyelitis via Alteration of MMP-9 Minghua Zheng & Junjie Wei & Yulan Tang & Chengcheng Yang & Yunfei Wei & Xiaoduan Yin & Qianqian Liu
Received: 2 March 2014 / Accepted: 18 March 2014 # Springer Science+Business Media New York 2014
Abstract Disruption of the blood–brain barrier (BBB) is a surrogate marker of acute inflammatory lesions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Data from experiments suggest that apolipoprotein E (ApoE) plays an important role in the antiinflammatory and immunological process in MS/EAE. Recent researches have shown that lack of ApoE leads to loss of cerebrovascular integrity and BBB breakdown causing neuronal injury. Cerebrovascular effects of ApoE might be another important element resulting to more susceptibility to MS/EAE. However, there is no direct evidence that ApoE dependently contributes to maintaining BBB integrity in EAE. In this study, we induced EAE in ApoE−/− mice and wild-type mice. During EAE, our results show that lack of ApoE increased the Evan’s blue (EB) permeability of BBB. Furthermore, deficiency of ApoE upregulated MMP-9 expression activity but decreased the expression of endothelial cell tight junction integral proteins claudin-5 and occludin. Our result also suggests that the protective role of ApoE in EAE by maintaining BBB integrity could be another interesting therapeutic target at MS/EAE. Keywords Apolipoprotein E . ApoE . Matrix metalloproteinase-9 . MMP-9 . Blood–brain barrier . BBB . Experimental autoimmune encephalomyelitis . EAE M. Zheng and J. Wei contributed equally. M. Zheng : Y. Tang (*) : C. Yang : Y. Wei : X. Yin : Q. Liu Department of Neurology, The First Affiliated Hospital, Guangxi Medical University, Nanning, Guangxi, China e-mail: [email protected] J. Wei Department of Neurology, The People’s Hospital Of Guangxi Zhuang Autonomous Region, No 6, Shuangyong Road, Nanning, Guangxi, China
Introduction Multiple sclerosis (MS) is a common chronic inflammatory disease caused by autoreactive T cells that respond to the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE), a murine model of MS, has been widely used to study the pathophysiology of MS (Steinman and Zamvil 2006). However, their etiology to date remains elusive. As previously shown, blood–brain barrier (BBB) damage leads to immune cell infiltration into the CNS which is the predominant pathogenic mechanism in the early-stage form of MS and EAE (Alvarez et al. 2011; Errede et al. 2012). In peripheral tissues, apolipoprotein E (ApoE) is synthesized by the liver and secreted into the circulation. In the CNS, it is the primary apolipoprotein and mainly produced by astrocytes (Gaudreault et al. 2012). There are three isoforms of ApoE in human, namely, ApoE2, ApoE3, and ApoE4. ApoE4 is a major genetic risk factor for Alzheimer disease and poor neurological outcome after traumatic brain injury, cerebral hemorrhage, and other neuropathological disorders (Zlokovic 2013). In the current study, we intended to evaluate the role of ApoE in CNS injury repair. At first, ApoE resembling high-density lipoproteins regulates cholesterol and lipid metabolism in the CNS (Hauser et al. 2011). Secondly, studies have shown that ApoE plays an important component of the immune system, including inhibiting lymphocyte proliferation, modulating the functions of macrophages, and reducing oxidative stress (Lee et al. 2004; Zhang et al. 2010). Finally, research found that ApoE is associated with cerebrovascular integrity. Lack of ApoE promoting BBB integrity damage has been demonstrated in animal models, such as brain injury (Methia et al. 2001), hyperlipidemia, and/or atherosclerosis, and aged mice (Grinberg and Thal 2010; Badaut et al. 2012). Our previous study demonstrated that lack of ApoE will cause increased infiltration of inflammatory cells in the CNS of EAE
J Mol Neurosci
(Wei et al. 2013), suggesting that ApoE deficiency may increase BBB permeability. Data from experiments suggested that BBB dysfunction resulting from ApoE deficiency might lead to more susceptibility to EAE, but ApoE influencing the BBB by what mechanism has not been shown in EAE. The BBB is formed and maintained by endothelial cells and corresponding tight junctions (TJ) formed by claudins and occludins in collaboration with pericytes, microglial cells, macrophages, and astrocytes (Abbott et al. 2006; Arima et al. 2013). Matrix metalloproteinase (MMPs) as a family of zinc-binding proteolytic enzymes, the activation is a critical event during BBB disruption (Adibhatla and Hatcher 2008; Roe et al. 2012). Among the MMPs, MMP-9 is the key enzyme for degrading type IV collagen, laminin, and fibronectin, which are the major components of the basement membrane of BBB (Chaturvedi and Kaczmarek 2013). Reports also show that expression of matrix-degrading MMP-9 is changed in the EAE brain and that increased MMP-9 plays a significant role in BBB dysfunction (Liuzzi et al. 2002; Opdenakker and Van Damme 2011). Tissue inhibitors of metalloproteinase 1 (TIMP-1) is a crucial endogenous inhibitor of MMP-9. The imbalance between MMP-9 and TIMP-1 could induce the disruption of BBB, thus being implicated in multiple diseases (Rosenberg et al. 1998). In this report, we hypothesized that ApoE contributes to the loss of barrier integrity in EAE by increasing expression and activity of MMP-9. To test this hypothesis, we measured the efficacy of ApoE on the BBB of EAE induced by myelin oligodendrocyte glycoprotein 35–55 amino acid peptide (MOG35-55) in ApoE−/− mice and wild-type mice.
Materials and Methods Mice Six to seven-week female ApoE−/− mice on a C57BL/6 background and C57BL/6 mice weighing 17–22 g were purchased from the Institute of Laboratory Animal, Chinese Academy of Medical Sciences (Beijing, China). These mice were fed a regular diet and housed in a constant-temperature (24 °C) room in the Experimental Animal Center of Guangxi Medical University. Experiments were carried out according to the guidelines of the Animal Ethical Review Committee of the First Affiliated Hospital of Guangxi Medical University. EAE Induction EAE was induced by subcutaneous injection with 200 μg MOG35–55 (Sangon Biotech, China) dissolved in an emulsion of 50 μl of complete Freund’s adjuvant (Sigma, USA) containing 0.4 mg of heat-killed Mycobacterium tuberculosis H37Ra (Difco, USA) and 50 μl phosphate buffered saline
(PBS). On the day of immunization (day 0) and 48 h later (day 2), every mouse was injected with 200 ng pertussis toxin (Sigma, USA) in 100 μl PBS. The progression of EAE was evaluated daily by following a five-point standardized rating of clinical symptoms: 0, no signs; 1, loss of tail tonicity; 2, flaccid tail; 3, ataxia and/or paresis of hind limbs; 4, complete paralysis of hind limbs; 5, moribund or death. The onset was defined as the day each mouse firstly displayed clinical score ≥1. Histopathology and Immunohistochemistry On day 21 after immunization, mice (n=4, each group) were killed for evaluation of the degree of histopathological lesions of EAE. At time of death, mice were perfused with 4 % paraformaldehyde in PBS. Brain tissue were harvested, postfixed in 4 % formaldehyde for 24 h; 4-μm-thick transverse sections were prepared and stained with hematoxylineosin (HE) for inflammatory infiltration as well as immunohistochemistry using antibodies with anti-MMP-9 (1:500, ab104686; Abcam) and anti-TIMP-1(1:500, ab86482; Abcam) The antibody-bound sections were visualized with 3,3-diaminobenzidine (DAB) (Boster, China) and hematoxylin counterstain. The images of stained sections were examined by Olympus PX53 microscope with a DP72 digital camera attached. Determination of BBB Permeability The integrity of BBB was detected by quantitative measurement for Evan’s blue (EB) content at day 21. Mice (n=5, each group) were injected with 100 μl EB (EB, 4 % in PBS, Sigma) through the tail vein. Two hours after injection, mice were perfused with saline to remove intravascular EB dye. Brains were rapidly removed, and each sample was weighed, then homogenized in 2-ml formamide (1:20w/v) and incubated at 60 °C overnight. The homogenate was centrifuged for 30 min at 1,000g, and the EB content in the supernatant was measured at 610 nm for absorbance of spectrophotometer. EB was expressed as micrograms per gram of brain tissue against a standard curve. Real-Time PCR Analysis The total RNA was isolated from brain tissues using TRIZOL Reagent (Invitrogen, USA) and then was treated for reverse transcription using a RT reagent kit with genomic DNA Eraser (Takara, Japan), according to the manufacturer’s protocol. Real-time PCR was performed using SYBR® Premix Ex TaqTMII (Tli RNase H Plus, Takara) on LightCycler 480 II system (Roche, Germany). The overall 20 μl reaction by adding RNase-free dH2O 6 μl, the primer sense 1 μl, antisense sequences 1 μl, Premix Ex TaqTMII 10 μl, and 2 μl
J Mol Neurosci
template. Cycling conditions included a predegeneration step of 30 s at 95 °C, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The primers for RT-PCR are MMP-9 forward, 5′-CAATCCTTGCAATGTGGATG-3′; reverse 5′-AGTAAG GAAGGGGCCCTGTA-3′; TIMP-1 forward, 5′-GTCACT CTCCAGTTTGCAAG-3′; reverse 5′-GACCACCTTATA CCAGCGTT-3′;occludin forward, 5′-CTCAAGTGGCAT AGATGTGGAAG-3′; reverse 5′-CTGTACCGAGGCTGCC TGAA-3′; claudin-5 forward, 5′-GTCGGGTGAGCATTCA GTCTTTAG-3′; reverse 5′-GAAGGCAGTCACCTGC CACA-3′; β-actin forward, 5′-AATTCCATCATGAAGT GTGA-3′; and reverse 5′-ACTCCTGCTTGCTGATCCAC-3′. The relative mRNA of each target gene was normalized by β-actin as the internal control. Western Blot Analysis On day 25 after immunization, mice from different groups (n=5–6, respectively) were decapitated and brains were rapidly removed. Total protein was isolated from brain tissue using RIPA lysis buffer (Beyotime, Haimen, China). Equal amount (20 μg) of protein was loaded on 10 % SDSpolyacrylamide gels. Protein was transferred onto PVDF membrane (Bio-Rad). The membranes were blocked with a buffer containing 5 % non-fat milk for 1 h. Afterward, the membranes were incubated with polyclonal anti-MMP-9 (1:500, ab104686; Abcam), monoclonal anti-TIMP-1 (1:1,000, ab86482; Abcam), monoclonal anti-Occludin (1:1500, ab167161; Abcam), polyclonal anti-Claudin-5 (1:500, ab53765; Abcam), and GAPDH (1:1000, AG019, Beyotime, Haimen, China), overnight respectively on ice. Then, they were washed with TBST buffer and incubated with 1:10,000 anti-Rabbit antibody (Li-Cor Biosciences) for 1 h. The membranes were scanned by using the Odyssey (042-0618-06; KPL, Washington, USA). Intensity of bands for densitometric analysis was determined by the Odyssey program. Density values were normalized to those of GAPDH. Gelatin Zymography for MMP-9 To determine the effect of ApoE on the protease activity of the MMP-9, Gelatin zymography was performed. This method was used to measure enzyme activity as a complement to Western blot protein expression data collected as described above. Equal amount of protein samples (20 μg/lane) was loaded and separated by 10 % Tris-glycine gel with 0.1 % gelatin as substrate. MMP-9 activity was quantified via standard densitometry.
SEM. The significance of differences was evaluated by using Student’s t test or one-way analysis of variance (ANOVA). A value of P
ApoE-Deficient Promotes Blood–Brain Barrier Disruption in Experimental Autoimmune Encephalomyelitis via Alteration of MMP-9 Minghua Zheng & Junjie Wei & Yulan Tang & Chengcheng Yang & Yunfei Wei & Xiaoduan Yin & Qianqian Liu
Received: 2 March 2014 / Accepted: 18 March 2014 # Springer Science+Business Media New York 2014
Abstract Disruption of the blood–brain barrier (BBB) is a surrogate marker of acute inflammatory lesions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Data from experiments suggest that apolipoprotein E (ApoE) plays an important role in the antiinflammatory and immunological process in MS/EAE. Recent researches have shown that lack of ApoE leads to loss of cerebrovascular integrity and BBB breakdown causing neuronal injury. Cerebrovascular effects of ApoE might be another important element resulting to more susceptibility to MS/EAE. However, there is no direct evidence that ApoE dependently contributes to maintaining BBB integrity in EAE. In this study, we induced EAE in ApoE−/− mice and wild-type mice. During EAE, our results show that lack of ApoE increased the Evan’s blue (EB) permeability of BBB. Furthermore, deficiency of ApoE upregulated MMP-9 expression activity but decreased the expression of endothelial cell tight junction integral proteins claudin-5 and occludin. Our result also suggests that the protective role of ApoE in EAE by maintaining BBB integrity could be another interesting therapeutic target at MS/EAE. Keywords Apolipoprotein E . ApoE . Matrix metalloproteinase-9 . MMP-9 . Blood–brain barrier . BBB . Experimental autoimmune encephalomyelitis . EAE M. Zheng and J. Wei contributed equally. M. Zheng : Y. Tang (*) : C. Yang : Y. Wei : X. Yin : Q. Liu Department of Neurology, The First Affiliated Hospital, Guangxi Medical University, Nanning, Guangxi, China e-mail: [email protected] J. Wei Department of Neurology, The People’s Hospital Of Guangxi Zhuang Autonomous Region, No 6, Shuangyong Road, Nanning, Guangxi, China
Introduction Multiple sclerosis (MS) is a common chronic inflammatory disease caused by autoreactive T cells that respond to the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE), a murine model of MS, has been widely used to study the pathophysiology of MS (Steinman and Zamvil 2006). However, their etiology to date remains elusive. As previously shown, blood–brain barrier (BBB) damage leads to immune cell infiltration into the CNS which is the predominant pathogenic mechanism in the early-stage form of MS and EAE (Alvarez et al. 2011; Errede et al. 2012). In peripheral tissues, apolipoprotein E (ApoE) is synthesized by the liver and secreted into the circulation. In the CNS, it is the primary apolipoprotein and mainly produced by astrocytes (Gaudreault et al. 2012). There are three isoforms of ApoE in human, namely, ApoE2, ApoE3, and ApoE4. ApoE4 is a major genetic risk factor for Alzheimer disease and poor neurological outcome after traumatic brain injury, cerebral hemorrhage, and other neuropathological disorders (Zlokovic 2013). In the current study, we intended to evaluate the role of ApoE in CNS injury repair. At first, ApoE resembling high-density lipoproteins regulates cholesterol and lipid metabolism in the CNS (Hauser et al. 2011). Secondly, studies have shown that ApoE plays an important component of the immune system, including inhibiting lymphocyte proliferation, modulating the functions of macrophages, and reducing oxidative stress (Lee et al. 2004; Zhang et al. 2010). Finally, research found that ApoE is associated with cerebrovascular integrity. Lack of ApoE promoting BBB integrity damage has been demonstrated in animal models, such as brain injury (Methia et al. 2001), hyperlipidemia, and/or atherosclerosis, and aged mice (Grinberg and Thal 2010; Badaut et al. 2012). Our previous study demonstrated that lack of ApoE will cause increased infiltration of inflammatory cells in the CNS of EAE
J Mol Neurosci
(Wei et al. 2013), suggesting that ApoE deficiency may increase BBB permeability. Data from experiments suggested that BBB dysfunction resulting from ApoE deficiency might lead to more susceptibility to EAE, but ApoE influencing the BBB by what mechanism has not been shown in EAE. The BBB is formed and maintained by endothelial cells and corresponding tight junctions (TJ) formed by claudins and occludins in collaboration with pericytes, microglial cells, macrophages, and astrocytes (Abbott et al. 2006; Arima et al. 2013). Matrix metalloproteinase (MMPs) as a family of zinc-binding proteolytic enzymes, the activation is a critical event during BBB disruption (Adibhatla and Hatcher 2008; Roe et al. 2012). Among the MMPs, MMP-9 is the key enzyme for degrading type IV collagen, laminin, and fibronectin, which are the major components of the basement membrane of BBB (Chaturvedi and Kaczmarek 2013). Reports also show that expression of matrix-degrading MMP-9 is changed in the EAE brain and that increased MMP-9 plays a significant role in BBB dysfunction (Liuzzi et al. 2002; Opdenakker and Van Damme 2011). Tissue inhibitors of metalloproteinase 1 (TIMP-1) is a crucial endogenous inhibitor of MMP-9. The imbalance between MMP-9 and TIMP-1 could induce the disruption of BBB, thus being implicated in multiple diseases (Rosenberg et al. 1998). In this report, we hypothesized that ApoE contributes to the loss of barrier integrity in EAE by increasing expression and activity of MMP-9. To test this hypothesis, we measured the efficacy of ApoE on the BBB of EAE induced by myelin oligodendrocyte glycoprotein 35–55 amino acid peptide (MOG35-55) in ApoE−/− mice and wild-type mice.
Materials and Methods Mice Six to seven-week female ApoE−/− mice on a C57BL/6 background and C57BL/6 mice weighing 17–22 g were purchased from the Institute of Laboratory Animal, Chinese Academy of Medical Sciences (Beijing, China). These mice were fed a regular diet and housed in a constant-temperature (24 °C) room in the Experimental Animal Center of Guangxi Medical University. Experiments were carried out according to the guidelines of the Animal Ethical Review Committee of the First Affiliated Hospital of Guangxi Medical University. EAE Induction EAE was induced by subcutaneous injection with 200 μg MOG35–55 (Sangon Biotech, China) dissolved in an emulsion of 50 μl of complete Freund’s adjuvant (Sigma, USA) containing 0.4 mg of heat-killed Mycobacterium tuberculosis H37Ra (Difco, USA) and 50 μl phosphate buffered saline
(PBS). On the day of immunization (day 0) and 48 h later (day 2), every mouse was injected with 200 ng pertussis toxin (Sigma, USA) in 100 μl PBS. The progression of EAE was evaluated daily by following a five-point standardized rating of clinical symptoms: 0, no signs; 1, loss of tail tonicity; 2, flaccid tail; 3, ataxia and/or paresis of hind limbs; 4, complete paralysis of hind limbs; 5, moribund or death. The onset was defined as the day each mouse firstly displayed clinical score ≥1. Histopathology and Immunohistochemistry On day 21 after immunization, mice (n=4, each group) were killed for evaluation of the degree of histopathological lesions of EAE. At time of death, mice were perfused with 4 % paraformaldehyde in PBS. Brain tissue were harvested, postfixed in 4 % formaldehyde for 24 h; 4-μm-thick transverse sections were prepared and stained with hematoxylineosin (HE) for inflammatory infiltration as well as immunohistochemistry using antibodies with anti-MMP-9 (1:500, ab104686; Abcam) and anti-TIMP-1(1:500, ab86482; Abcam) The antibody-bound sections were visualized with 3,3-diaminobenzidine (DAB) (Boster, China) and hematoxylin counterstain. The images of stained sections were examined by Olympus PX53 microscope with a DP72 digital camera attached. Determination of BBB Permeability The integrity of BBB was detected by quantitative measurement for Evan’s blue (EB) content at day 21. Mice (n=5, each group) were injected with 100 μl EB (EB, 4 % in PBS, Sigma) through the tail vein. Two hours after injection, mice were perfused with saline to remove intravascular EB dye. Brains were rapidly removed, and each sample was weighed, then homogenized in 2-ml formamide (1:20w/v) and incubated at 60 °C overnight. The homogenate was centrifuged for 30 min at 1,000g, and the EB content in the supernatant was measured at 610 nm for absorbance of spectrophotometer. EB was expressed as micrograms per gram of brain tissue against a standard curve. Real-Time PCR Analysis The total RNA was isolated from brain tissues using TRIZOL Reagent (Invitrogen, USA) and then was treated for reverse transcription using a RT reagent kit with genomic DNA Eraser (Takara, Japan), according to the manufacturer’s protocol. Real-time PCR was performed using SYBR® Premix Ex TaqTMII (Tli RNase H Plus, Takara) on LightCycler 480 II system (Roche, Germany). The overall 20 μl reaction by adding RNase-free dH2O 6 μl, the primer sense 1 μl, antisense sequences 1 μl, Premix Ex TaqTMII 10 μl, and 2 μl
J Mol Neurosci
template. Cycling conditions included a predegeneration step of 30 s at 95 °C, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The primers for RT-PCR are MMP-9 forward, 5′-CAATCCTTGCAATGTGGATG-3′; reverse 5′-AGTAAG GAAGGGGCCCTGTA-3′; TIMP-1 forward, 5′-GTCACT CTCCAGTTTGCAAG-3′; reverse 5′-GACCACCTTATA CCAGCGTT-3′;occludin forward, 5′-CTCAAGTGGCAT AGATGTGGAAG-3′; reverse 5′-CTGTACCGAGGCTGCC TGAA-3′; claudin-5 forward, 5′-GTCGGGTGAGCATTCA GTCTTTAG-3′; reverse 5′-GAAGGCAGTCACCTGC CACA-3′; β-actin forward, 5′-AATTCCATCATGAAGT GTGA-3′; and reverse 5′-ACTCCTGCTTGCTGATCCAC-3′. The relative mRNA of each target gene was normalized by β-actin as the internal control. Western Blot Analysis On day 25 after immunization, mice from different groups (n=5–6, respectively) were decapitated and brains were rapidly removed. Total protein was isolated from brain tissue using RIPA lysis buffer (Beyotime, Haimen, China). Equal amount (20 μg) of protein was loaded on 10 % SDSpolyacrylamide gels. Protein was transferred onto PVDF membrane (Bio-Rad). The membranes were blocked with a buffer containing 5 % non-fat milk for 1 h. Afterward, the membranes were incubated with polyclonal anti-MMP-9 (1:500, ab104686; Abcam), monoclonal anti-TIMP-1 (1:1,000, ab86482; Abcam), monoclonal anti-Occludin (1:1500, ab167161; Abcam), polyclonal anti-Claudin-5 (1:500, ab53765; Abcam), and GAPDH (1:1000, AG019, Beyotime, Haimen, China), overnight respectively on ice. Then, they were washed with TBST buffer and incubated with 1:10,000 anti-Rabbit antibody (Li-Cor Biosciences) for 1 h. The membranes were scanned by using the Odyssey (042-0618-06; KPL, Washington, USA). Intensity of bands for densitometric analysis was determined by the Odyssey program. Density values were normalized to those of GAPDH. Gelatin Zymography for MMP-9 To determine the effect of ApoE on the protease activity of the MMP-9, Gelatin zymography was performed. This method was used to measure enzyme activity as a complement to Western blot protein expression data collected as described above. Equal amount of protein samples (20 μg/lane) was loaded and separated by 10 % Tris-glycine gel with 0.1 % gelatin as substrate. MMP-9 activity was quantified via standard densitometry.
SEM. The significance of differences was evaluated by using Student’s t test or one-way analysis of variance (ANOVA). A value of P
