Cellular Physiology and Biochemistry
Cell Physiol Biochem 2013;32:1798-1807 DOI: 10.1159/000356613 Published online: December 13, 2013
© 2013 S. Karger AG, Basel www.karger.com/cpb
Jing et al.: November OPN SNP and Stroke Prognosis Accepted: 11, Serum 2013 Levels Confer Ischemic 1421-9778/13/0326-1798$38.00/0 This is an Open Access article licensed under the terms of the Creative Commons AttributionNonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.
Original Paper
OPN Gene Polymorphism and the Serum OPN Levels Confer the Susceptibility and Prognosis of Ischemic Stroke in Chinese Patients Meng Jinga Baoxin Lib Xuwei Houc Jayaram Shobad Changyu Lia Hongsheng Lianga Xiangtong Zhanga Enzhong Liua Baofeng Yangb Xiangxi Menga Department of Neurosurgery, the 1st Affiliated Hospital of Harbin Medical University, Harbin, Department of Pharmacology, Harbin Medical University, Harbin, P.R. China; cDepartment of Cardiology, Hangzhou First Municipal Hospital and Hangzhou Hospital, Nanjing Medical University, Hangzhou, China; dSangolli Nursing Home 270, Mahalakshmi Layout, Bangalore, India a
b
Key Words Osteopontin • Ischemic stroke • Susceptibility • Prognosis • Chinese Abstract Aim: To investigate the association of Osteopontin (OPN) gene polymorphism and serum thrombin-cleaved OPN level with the susceptibility to ischemic stroke (IS) and its prognosis. Methods: A total of 377 patients with IS and 551 healthy individuals were recruited. The OPN gene polymorphisms at −156 G>GG, −443 C>T and -66 T>G were genotyped. Serum fulllength and the thrombin-cleaved OPN were determined. Results: We found that only the -443 C>T polymorphism was significantly associated with the susceptibility to IS. The -443 CC represented a near 2 time higher risk for IS incidence than TT carriers. Also, the -443 CC genotype had significantly poorer outcome and they significantly had higher occurrence for bad recovery as determined by modified Rankin Scale (mRS) (OR=2.18, p=0.043) and Barthel Index (BI) (OR=2.12, p=0.05). The mean serum thrombin-cleaved OPN level in IS group were significantly higher than that in control group. ROC analysis showed that the thrombincleaved OPN level (cut-off value, 166.8 ng/ml) can discriminate IS patients from controls with a specificity of 86.3% and a sensitivity of 57.7%. The serum thrombin-cleaved OPN was significantly associated with the clinical outcome at 12 months after discharge from hospital. Conclusion: These results suggest that the -443 C>T polymorphism of OPN gene and serum thrombin-cleaved OPN can be used as a biomarker for the susceptibility and prognosis of IS patients. Copyright © 2013 S. Karger AG, Basel
Xiangxi Meng
Department of Neurosurgery, the 1st Affiliated Hospital of Harbin Medical University 23 Youzheng Str. Nangang District, 150001, Harbin (P.R. China) Tel. +86 451-85555109, Fax +86 451-85555109, E-Mail [email protected]
1798
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2013;32:1798-1807 DOI: 10.1159/000356613 Published online: December 13, 2013
© 2013 S. Karger AG, Basel www.karger.com/cpb
Jing et al.: OPN SNP and Serum Levels Confer Ischemic Stroke Prognosis
Introduction
Stroke is a leading cause of death and adult disability worldwide [1-3]. More than 80% of stroke cases are ischemic stroke (IS) in origin. Many environmental factors, including age, obesity, hypertension, inflammation, smoking, diabetes mellitus (DM) and lipid disorders, have been established as risk factors for IS. In addition, previous studies also suggest that genetic background can determine the predisposition to IS [4, 5-10]. Osteopontin (OPN) is a matricellular protein participating in many physiological and pathologic processes including wound healing, bone turnover, tumor genesis, inflammation, and immune responses [11-14]. OPN mediates the lateral migration of neuroblasts from the subventricular zone after focal cerebral ischemia [15]. A recent study suggests that OPN as a novel biomarker for atherothrombosis in patients with ischemic stroke [16]. OPN levels are increased in IS patients with worse prognosis, suggesting OPN may predict the long-term outcome of IS [17]. The expression of OPN is significantly influenced by its genetic polymorphisms [18]. Several polymorphisms in the human OPN encoding gene have been identified in different populations, of which the -66 T>G (rs28357094), -156 G>GG (rs17524488), and -443 C>T (rs11730582) polymorphisms were mostly studied. These OPN gene polymorphisms have been reported to be associated with inflammatory disease e.g. systemic lupus erythematosus [19], chronic hepatitis C [20], lupus nephritis [21] and large artery atherosclerosis [22]. However, the association of OPN gene polymorphisms with susceptibility to IS remains largely unknown. In the present study, we enrolled IS patients and healthy controls to investigate the role of OPN gene polymorphism in determining the susceptibility to IS. In addition, the association of OPN gene polymorphism with the clinical outcome of IS was also investigated in this study. Materials and Methods
Recruitment We consecutively enrolled 560 patients in our hospital from May 2009 to May 2011 and eventually 377 patients who met the enrollment criteria were included. The diagnosis of IS was made by CT or MRI scans of the brain. All patients were recruited within 24 hours after initial stroke symptom onset. All patients had clinical signs consistent with the World Health Organization definition of stroke [23]. The Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria was used to classify the stoke subtype [24]. Only patients with small vessel disease (SVD, n=253) and large vessel disease (LVD, n=124) were included in this study. Patients with recurrent stroke, hemorrhagic stroke, liver and kidney disorders, chronic inflammatory diseases and tumors of all types were excluded from this study. A control group comprising of 551 age and sex-matched healthy individuals were recruited as controls. All controls were free from arteriosclerosis in the carotid bifurcation by ultrasound screening and did not take medications. The study was approved by the institutional ethical committee of our hospital. Informed consent was obtained from all subjects before the collection of information. Demographic and clinical covariates The demographic data and clinical parameters such as presence of hypertension, diabetes, smoking status, the body mass index (BMI) were obtained. The serum total cholesterol (TC), triglyceride (TG), Low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C), serum creatinine (sCr) and C-reaction protein (CRP) were measured. Stroke severity and outcome evaluation Stroke severity at admission was measured using the National Institutes of Health Stroke Scale (NIHSS; scores ranging 0–42 with higher scores indicating more severe pathology) [25]. At 12 months, the physical disability was measured with the Barthel Index (BI; scores ranging 0–100) [26]. Patients with BI score equal or less than 85 were assigned to bad recovery; those with BI score greater than 85 were assigned to good
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Cellular Physiology and Biochemistry
Cell Physiol Biochem 2013;32:1798-1807 DOI: 10.1159/000356613 Published online: December 13, 2013
© 2013 S. Karger AG, Basel www.karger.com/cpb
Jing et al.: OPN SNP and Serum Levels Confer Ischemic Stroke Prognosis
recovery. Functional outcome were measured by using modified Rankin scale (mRS) [27]. Patients with mRS score (0-1) were assigned to good recovery group; those with mRS score (2-6) were assigned to bad recovery group.
OPN gene polymorphisms DNA was extracted from peripheral whole blood using a Qiagen DNA Isolation Kit (Qiagen, Valencia, CA, USA). Three single nucleotide polymorphisms on the promoter region of OPN gene, including -66 T>G (rs28357094), -156 G>GG (rs17524488), and -443 C>T (rs11730582), were determined using TaqMan 5′ allelic discrimination assay. It was performed using commercially available kit Assays-on-Demand™ SNP genotyping products (Applied Biosystems, Foster City, CA). Serum OPN level detection The levels of the full length and the thrombin-cleaved OPN in serum were determined by capture enzyme-linked immunosorbent assay (ELISA) according to the protocol provided by the manufacturer (Calbiochem, San Diego, CA). The sensitivity for OPN was 3.33 ng/ml with an intra- and inter-assay coefficients of variation (CV) being T polymorphism and the serum thrombin-cleaved OPN to predict IS risk and prognosis. OPN has recently emerged as a key factor in both vascular remodeling and the development of atherosclerosis [29-33]. OPN is highly expressed in atherosclerotic plaques [34]. Transgenic mice overexpressing OPN display medial thickening and neointimal formation in the carotid artery [35]. Clinically, the concentration of OPN within the endarterectomy specimens removed from symptomatic atherosclerosis patients, suggesting that OPN is involved in the atherosclerotic plaque stability [36]. In essential hypertensive patients, plasma OPN levels were positively correlated with mean intima-media thickness (IMT) and negatively with mean diastolic and systolic flow velocities, suggesting that OPN plays a role in the development of atherosclerosis [30]. Plasma OPN levels have been shown to be correlated with the angiographic severity of coronary artery disease (CAD) [12]. In the context of IS, one previous study have shown a large increase in the expression of OPN mRNA in the ischemic tissues following transient focal ischemia in rats [37]. In IS rat model, up-regulation of OPN was observed in the ischemic brain. Blockade of OPN activity in the ischemic brain significantly decreased focal ischemia-induced lateral migration of neuroblasts, indicating that OPN plays a significant role in post-ischemic lateral migration of neuroblasts [15]. Collectively, these studies suggest that OPN may serve as a potential marker of cardiovascular and cerebrovascular diseases. In this clinical study, we found that the serum thrombin-cleaved OPN levels are closely associated with the occurrence and outcome of IS. As far as we know, this is the first study reporting the role of OPN gene polymorphism in a clinical setting. OPN exists either as a full-length molecule or as proteolytic fragments. The thrombincleaved OPN is a unique N-terminal fragment of OPN produced by thrombin proteolysis 6 amino acids C-terminal of the Role of Arg-Gly-Asp (RGD) domain. We found that only the thrombin-cleaved OPN had the above mentioned effect and not the full-length OPN. In contrast, most of the cited studies did not specify the isotopes of OPN in their studies. The OPN encoding gene is mapped on human chromosome 4q21-q25 and polymorphisms in the OPN gene promoter can affect its transcriptional activity [38]. A previous study explored the possible role of −443 C>T polymorphism for OPN expression in melanoma cells. The authors found that the -443 CC genotype had higher levels of OPN mRNA compared with other allelic variants and -443 C>T variants might influence the OPN mRNA levels via binding of c-Myb transcription factor [39]. The role of OPN gene polymorphism in cardiovascular conditions has been previously reported. An association of the human -66 G>T promoter variant with carotid artery intima-media thickness was reported in Caucasian adults [40]. The OPN -443 C>T was associated with increased intima-media thickness in stroke patients [41]. In this study, we found that the -443 CC represented a near 2 time higher chance for IS incidence than TT carriers. Also, the -443 CC genotype had significantly poorer outcome and they had significantly higher risk for bad recovery. Some limitation in this study should be addressed. Firstly, this is a single center based study, which enrolled Chinese patients in north China area, especially Liaoning, Jilin and Heilongjiang provinces. The association of OPN gene polymorphism with IS need to be duplicated in other ethnic populations. Secondly, this is a primary observational study in a clinical setting. Studies exploring the effect on endothelial function, thrombus formation and the stability of carotid AS plaque are in process to better elucidate the mechanism under which the OPN polymorphisms and OPN protein affect IS susceptibility and prognosis. Acknowledgements
This work was supported by grants from Foundation of the 1st Affliated Hospital of Harbin Medical University (No B08-010), Scientific Research fund of Heilongjiang Provincial Health Department. (No 2009-035) and Scientific Research fund of Heilongjiang Provincial Education Department (No 12511261).
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Cell Physiol Biochem 2013;32:1798-1807 DOI: 10.1159/000356613 Published online: December 13, 2013
© 2013 S. Karger AG, Basel www.karger.com/cpb
Jing et al.: OPN SNP and Serum Levels Confer Ischemic Stroke Prognosis
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Cell Physiol Biochem 2013;32:1798-1807 DOI: 10.1159/000356613 Published online: December 13, 2013
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Jing et al.: OPN SNP and Serum Levels Confer Ischemic Stroke Prognosis
Mochida S, Hashimoto M, Matsui A, Naito M, Inao M, Nagoshi S, Nagano M, Egashira T, Mishiro S, Fujiwara K: Genetic polymorphims in promoter region of osteopontin gene may be a marker reflecting hepatitis activity in chronic hepatitis C patients. Biochem Biophys Res Commun 2004;313:1079-1085. Miyazaki T, Ono M, Qu WM, Zhang MC, Mori S, Nakatsuru S, Nakamura Y, Sawasaki T, Endo Y, Nose M: Implication of allelic polymorphism of osteopontin in the development of lupus nephritis in MRL/lpr mice. Eur J Immunol 2005;35:1510-1520. Kim Y, Lee C: Haplotype analysis revealed a genetic influence of osteopontin on large artery atherosclerosis. J Biomed Sci 2008;15:529-533. Farooq MU, Chaudhry AH, Amin K, Majid A: The WHO STEPwise Approach to Stroke Surveillance. J Coll Physicians Surg Pak 2008;18:665. Fure B, Wyller TB, Thommessen B: TOAST criteria applied in acute ischemic stroke. Acta Neurol Scand 2005;112:254-258. Kasner SE, Chalela JA, Luciano JM, Cucchiara BL, Raps EC, McGarvey ML, Conroy MB, Localio AR: Reliability and validity of estimating the NIH stroke scale score from medical records. Stroke 1999;30:1534-1537. Mahoney FI, Barthel DW: Functional Evaluation: The Barthel Index. Md State Med J 1965;14:61-65. Coletta EM, Murphy JB: Physical and functional assessment of the elderly stroke patient. Am Fam Physician 1994;49:1777-1785. Hasegawa M, Nakoshi Y, Iino T, Sudo A, Segawa T, Maeda M, Yoshida T, Uchida A: Thrombin-cleaved osteopontin in synovial fluid of subjects with rheumatoid arthritis. J Rheumatol 2009;36:240-245. Jono S: [Osteopontin and atherosclerosis]. Nihon Rinsho 2005;63:635-640. Kurata M, Okura T, Watanabe S, Fukuoka T, Higaki J: Osteopontin and carotid atherosclerosis in patients with essential hypertension. Clin Sci (Lond) 2006;111:319-324. Hou X, Appleby N, Fuentes T, Longo LD, Bailey LL, Hasaniya N, Kearns-Jonker M: Isolation, Characterization, and Spatial Distribution of Cardiac Progenitor Cells in the Sheep Heart. J Clin Exp Cardiolog 2012;S6. Hou XW, Son J, Wang Y, Ru YX, Lian Q, Majiti W, Amazouzi A, Zhou YL, Wang PX, Han ZC: Granulocyte colonystimulating factor reduces cardiomyocyte apoptosis and improves cardiac function in adriamycin-induced cardiomyopathy in rats. Cardiovasc Drugs Ther 2006;20:85-91. Xu H, Hou X, Wang N, Hui B, Jin J, Yun S, Wang X, He X, He J, Zhang S, Han Y: Gender-specific effect of estrogen receptor-1 gene polymorphisms in coronary artery disease and its angiographic severity in Chinese population. Clin Chim Acta 2008;395:130-133. O'Brien ER, Garvin MR, Stewart DK, Hinohara T, Simpson JB, Schwartz SM, Giachelli CM: Osteopontin is synthesized by macrophage, smooth muscle, and endothelial cells in primary and restenotic human coronary atherosclerotic plaques. Arterioscler Thromb 1994;14:1648-1656. Matsui Y, Rittling SR, Okamoto H, Inobe M, Jia N, Shimizu T, Akino M, Sugawara T, Morimoto J, Kimura C, Kon S, Denhardt D, Kitabatake A, Uede T: Osteopontin deficiency attenuates atherosclerosis in female apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2003;23:1029-1034. Golledge J, McCann M, Mangan S, Lam A, Karan M: Osteoprotegerin and osteopontin are expressed at high concentrations within symptomatic carotid atherosclerosis. Stroke 2004;35:1636-1641. Wang X, Louden C, Yue TL, Ellison JA, Barone FC, Solleveld HA, Feuerstein GZ: Delayed expression of osteopontin after focal stroke in the rat. J Neurosci 1998;18:2075-2083. Giacopelli F, Marciano R, Pistorio A, Catarsi P, Canini S, Karsenty G, Ravazzolo R: Polymorphisms in the osteopontin promoter affect its transcriptional activity. Physiol Genomics 2004;20:87-96. Schultz J, Lorenz P, Ibrahim SM, Kundt G, Gross G, Kunz M: The functional -443T/C osteopontin promoter polymorphism influences osteopontin gene expression in melanoma cells via binding of c-Myb transcription factor. Mol Carcinog 2009;48:14-23. de las Fuentes L, Gu CC, Mathews SJ, Reagan JL, Ruthmann NP, Waggoner AD, Lai CF, Towler DA, DavilaRoman VG: Osteopontin promoter polymorphism is associated with increased carotid intima-media thickness. J Am Soc Echocardiogr 2008;21:954-960. Brenner D, Labreuche J, Touboul PJ, Schmidt-Petersen K, Poirier O, Perret C, Schonfelder J, Combadiere C, Lathrop M, Cambien F, Brand-Herrmann SM, Amarenco P: Cytokine polymorphisms associated with carotid intima-media thickness in stroke patients. Stroke 2006;37:1691-1696.
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Copyright: S. Karger AG, Basel 2013. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Cell Physiol Biochem 2013;32:1798-1807 DOI: 10.1159/000356613 Published online: December 13, 2013
© 2013 S. Karger AG, Basel www.karger.com/cpb
Jing et al.: November OPN SNP and Stroke Prognosis Accepted: 11, Serum 2013 Levels Confer Ischemic 1421-9778/13/0326-1798$38.00/0 This is an Open Access article licensed under the terms of the Creative Commons AttributionNonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.
Original Paper
OPN Gene Polymorphism and the Serum OPN Levels Confer the Susceptibility and Prognosis of Ischemic Stroke in Chinese Patients Meng Jinga Baoxin Lib Xuwei Houc Jayaram Shobad Changyu Lia Hongsheng Lianga Xiangtong Zhanga Enzhong Liua Baofeng Yangb Xiangxi Menga Department of Neurosurgery, the 1st Affiliated Hospital of Harbin Medical University, Harbin, Department of Pharmacology, Harbin Medical University, Harbin, P.R. China; cDepartment of Cardiology, Hangzhou First Municipal Hospital and Hangzhou Hospital, Nanjing Medical University, Hangzhou, China; dSangolli Nursing Home 270, Mahalakshmi Layout, Bangalore, India a
b
Key Words Osteopontin • Ischemic stroke • Susceptibility • Prognosis • Chinese Abstract Aim: To investigate the association of Osteopontin (OPN) gene polymorphism and serum thrombin-cleaved OPN level with the susceptibility to ischemic stroke (IS) and its prognosis. Methods: A total of 377 patients with IS and 551 healthy individuals were recruited. The OPN gene polymorphisms at −156 G>GG, −443 C>T and -66 T>G were genotyped. Serum fulllength and the thrombin-cleaved OPN were determined. Results: We found that only the -443 C>T polymorphism was significantly associated with the susceptibility to IS. The -443 CC represented a near 2 time higher risk for IS incidence than TT carriers. Also, the -443 CC genotype had significantly poorer outcome and they significantly had higher occurrence for bad recovery as determined by modified Rankin Scale (mRS) (OR=2.18, p=0.043) and Barthel Index (BI) (OR=2.12, p=0.05). The mean serum thrombin-cleaved OPN level in IS group were significantly higher than that in control group. ROC analysis showed that the thrombincleaved OPN level (cut-off value, 166.8 ng/ml) can discriminate IS patients from controls with a specificity of 86.3% and a sensitivity of 57.7%. The serum thrombin-cleaved OPN was significantly associated with the clinical outcome at 12 months after discharge from hospital. Conclusion: These results suggest that the -443 C>T polymorphism of OPN gene and serum thrombin-cleaved OPN can be used as a biomarker for the susceptibility and prognosis of IS patients. Copyright © 2013 S. Karger AG, Basel
Xiangxi Meng
Department of Neurosurgery, the 1st Affiliated Hospital of Harbin Medical University 23 Youzheng Str. Nangang District, 150001, Harbin (P.R. China) Tel. +86 451-85555109, Fax +86 451-85555109, E-Mail [email protected]
1798
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2013;32:1798-1807 DOI: 10.1159/000356613 Published online: December 13, 2013
© 2013 S. Karger AG, Basel www.karger.com/cpb
Jing et al.: OPN SNP and Serum Levels Confer Ischemic Stroke Prognosis
Introduction
Stroke is a leading cause of death and adult disability worldwide [1-3]. More than 80% of stroke cases are ischemic stroke (IS) in origin. Many environmental factors, including age, obesity, hypertension, inflammation, smoking, diabetes mellitus (DM) and lipid disorders, have been established as risk factors for IS. In addition, previous studies also suggest that genetic background can determine the predisposition to IS [4, 5-10]. Osteopontin (OPN) is a matricellular protein participating in many physiological and pathologic processes including wound healing, bone turnover, tumor genesis, inflammation, and immune responses [11-14]. OPN mediates the lateral migration of neuroblasts from the subventricular zone after focal cerebral ischemia [15]. A recent study suggests that OPN as a novel biomarker for atherothrombosis in patients with ischemic stroke [16]. OPN levels are increased in IS patients with worse prognosis, suggesting OPN may predict the long-term outcome of IS [17]. The expression of OPN is significantly influenced by its genetic polymorphisms [18]. Several polymorphisms in the human OPN encoding gene have been identified in different populations, of which the -66 T>G (rs28357094), -156 G>GG (rs17524488), and -443 C>T (rs11730582) polymorphisms were mostly studied. These OPN gene polymorphisms have been reported to be associated with inflammatory disease e.g. systemic lupus erythematosus [19], chronic hepatitis C [20], lupus nephritis [21] and large artery atherosclerosis [22]. However, the association of OPN gene polymorphisms with susceptibility to IS remains largely unknown. In the present study, we enrolled IS patients and healthy controls to investigate the role of OPN gene polymorphism in determining the susceptibility to IS. In addition, the association of OPN gene polymorphism with the clinical outcome of IS was also investigated in this study. Materials and Methods
Recruitment We consecutively enrolled 560 patients in our hospital from May 2009 to May 2011 and eventually 377 patients who met the enrollment criteria were included. The diagnosis of IS was made by CT or MRI scans of the brain. All patients were recruited within 24 hours after initial stroke symptom onset. All patients had clinical signs consistent with the World Health Organization definition of stroke [23]. The Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria was used to classify the stoke subtype [24]. Only patients with small vessel disease (SVD, n=253) and large vessel disease (LVD, n=124) were included in this study. Patients with recurrent stroke, hemorrhagic stroke, liver and kidney disorders, chronic inflammatory diseases and tumors of all types were excluded from this study. A control group comprising of 551 age and sex-matched healthy individuals were recruited as controls. All controls were free from arteriosclerosis in the carotid bifurcation by ultrasound screening and did not take medications. The study was approved by the institutional ethical committee of our hospital. Informed consent was obtained from all subjects before the collection of information. Demographic and clinical covariates The demographic data and clinical parameters such as presence of hypertension, diabetes, smoking status, the body mass index (BMI) were obtained. The serum total cholesterol (TC), triglyceride (TG), Low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C), serum creatinine (sCr) and C-reaction protein (CRP) were measured. Stroke severity and outcome evaluation Stroke severity at admission was measured using the National Institutes of Health Stroke Scale (NIHSS; scores ranging 0–42 with higher scores indicating more severe pathology) [25]. At 12 months, the physical disability was measured with the Barthel Index (BI; scores ranging 0–100) [26]. Patients with BI score equal or less than 85 were assigned to bad recovery; those with BI score greater than 85 were assigned to good
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Cellular Physiology and Biochemistry
Cell Physiol Biochem 2013;32:1798-1807 DOI: 10.1159/000356613 Published online: December 13, 2013
© 2013 S. Karger AG, Basel www.karger.com/cpb
Jing et al.: OPN SNP and Serum Levels Confer Ischemic Stroke Prognosis
recovery. Functional outcome were measured by using modified Rankin scale (mRS) [27]. Patients with mRS score (0-1) were assigned to good recovery group; those with mRS score (2-6) were assigned to bad recovery group.
OPN gene polymorphisms DNA was extracted from peripheral whole blood using a Qiagen DNA Isolation Kit (Qiagen, Valencia, CA, USA). Three single nucleotide polymorphisms on the promoter region of OPN gene, including -66 T>G (rs28357094), -156 G>GG (rs17524488), and -443 C>T (rs11730582), were determined using TaqMan 5′ allelic discrimination assay. It was performed using commercially available kit Assays-on-Demand™ SNP genotyping products (Applied Biosystems, Foster City, CA). Serum OPN level detection The levels of the full length and the thrombin-cleaved OPN in serum were determined by capture enzyme-linked immunosorbent assay (ELISA) according to the protocol provided by the manufacturer (Calbiochem, San Diego, CA). The sensitivity for OPN was 3.33 ng/ml with an intra- and inter-assay coefficients of variation (CV) being T polymorphism and the serum thrombin-cleaved OPN to predict IS risk and prognosis. OPN has recently emerged as a key factor in both vascular remodeling and the development of atherosclerosis [29-33]. OPN is highly expressed in atherosclerotic plaques [34]. Transgenic mice overexpressing OPN display medial thickening and neointimal formation in the carotid artery [35]. Clinically, the concentration of OPN within the endarterectomy specimens removed from symptomatic atherosclerosis patients, suggesting that OPN is involved in the atherosclerotic plaque stability [36]. In essential hypertensive patients, plasma OPN levels were positively correlated with mean intima-media thickness (IMT) and negatively with mean diastolic and systolic flow velocities, suggesting that OPN plays a role in the development of atherosclerosis [30]. Plasma OPN levels have been shown to be correlated with the angiographic severity of coronary artery disease (CAD) [12]. In the context of IS, one previous study have shown a large increase in the expression of OPN mRNA in the ischemic tissues following transient focal ischemia in rats [37]. In IS rat model, up-regulation of OPN was observed in the ischemic brain. Blockade of OPN activity in the ischemic brain significantly decreased focal ischemia-induced lateral migration of neuroblasts, indicating that OPN plays a significant role in post-ischemic lateral migration of neuroblasts [15]. Collectively, these studies suggest that OPN may serve as a potential marker of cardiovascular and cerebrovascular diseases. In this clinical study, we found that the serum thrombin-cleaved OPN levels are closely associated with the occurrence and outcome of IS. As far as we know, this is the first study reporting the role of OPN gene polymorphism in a clinical setting. OPN exists either as a full-length molecule or as proteolytic fragments. The thrombincleaved OPN is a unique N-terminal fragment of OPN produced by thrombin proteolysis 6 amino acids C-terminal of the Role of Arg-Gly-Asp (RGD) domain. We found that only the thrombin-cleaved OPN had the above mentioned effect and not the full-length OPN. In contrast, most of the cited studies did not specify the isotopes of OPN in their studies. The OPN encoding gene is mapped on human chromosome 4q21-q25 and polymorphisms in the OPN gene promoter can affect its transcriptional activity [38]. A previous study explored the possible role of −443 C>T polymorphism for OPN expression in melanoma cells. The authors found that the -443 CC genotype had higher levels of OPN mRNA compared with other allelic variants and -443 C>T variants might influence the OPN mRNA levels via binding of c-Myb transcription factor [39]. The role of OPN gene polymorphism in cardiovascular conditions has been previously reported. An association of the human -66 G>T promoter variant with carotid artery intima-media thickness was reported in Caucasian adults [40]. The OPN -443 C>T was associated with increased intima-media thickness in stroke patients [41]. In this study, we found that the -443 CC represented a near 2 time higher chance for IS incidence than TT carriers. Also, the -443 CC genotype had significantly poorer outcome and they had significantly higher risk for bad recovery. Some limitation in this study should be addressed. Firstly, this is a single center based study, which enrolled Chinese patients in north China area, especially Liaoning, Jilin and Heilongjiang provinces. The association of OPN gene polymorphism with IS need to be duplicated in other ethnic populations. Secondly, this is a primary observational study in a clinical setting. Studies exploring the effect on endothelial function, thrombus formation and the stability of carotid AS plaque are in process to better elucidate the mechanism under which the OPN polymorphisms and OPN protein affect IS susceptibility and prognosis. Acknowledgements
This work was supported by grants from Foundation of the 1st Affliated Hospital of Harbin Medical University (No B08-010), Scientific Research fund of Heilongjiang Provincial Health Department. (No 2009-035) and Scientific Research fund of Heilongjiang Provincial Education Department (No 12511261).
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Cell Physiol Biochem 2013;32:1798-1807 DOI: 10.1159/000356613 Published online: December 13, 2013
© 2013 S. Karger AG, Basel www.karger.com/cpb
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