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K gene polymorphism and probable vascular dementia in humans
Neuroscience Letters 326 (2002) 171–174 www.elsevier.com/locate/neulet
Association between intercellular adhesion molecule-1 E/K gene polymorphism and probable vascular dementia in humans Roberto Pola a,b,*, Andrea Flex a,c, Eleonora Gaetani a,c, Pierangelo Papaleo c, Demetrio De Martini c, Laura Gerardino c, Michele Serricchio c, Paolo Pola a,c, Roberto Bernabei b a
Laboratory of Vascular Biology and Genetics, A. Gemelli University Hospital, Universita` Cattolica del Sacro Cuore School of Medicine, Rome, Italy b Department of Geriatric Medicine, A. Gemelli University Hospital, Universita` Cattolica del Sacro Cuore School of Medicine, Rome, Italy c Department of Internal Medicine, A. Gemelli University Hospital, Universita` Cattolica del Sacro Cuore School of Medicine, Rome, Italy Received 7 January 2002; received in revised form 19 March 2002; accepted 22 March 2002
Abstract Intercellular adhesion molecule-1 (ICAM-1) is implicated in the pathogenesis of ischemic cardiovascular disorders, including cerebral ischemia. A common polymorphism of the ICAM-1 gene (K469E) has been recently reported. In this case-control study, we evaluated the association between this polymorphism and vascular dementia (VD) by studying 107 patients affected by probable VD and 115 age- and sex-matched controls. The frequency of the EE genotype was significantly higher in VD patients than controls (P ¼ 0:009). Logistic regression analysis indicated that the presence of the EE genotype significantly increased the risk of VD (odds ratio 3.25, P ¼ 0:024). Our findings support the hypothesis that ICAM-1 plays a role in the physiopathology of ischemic cerebrovascular disorders and suggest that genetic polymorphisms of ICAM-1 might be clinically important in the development and progression of neurodegenerative diseases. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Probable vascular dementia; Cerebral ischemia; Inflammation; Intercellular adhesion molecule-1; Gene polymorphism
Vascular dementia (VD) is the second most common cause of cognitive deterioration after Alzheimer’s disease (AD). It occurs more often in persons who have hypertension and/or diabetes mellitus or who abuse tobacco. However, the mechanisms for clinical cognitive deterioration in patients with cerebral ischemia are not completely understood. Several recent observations hint at the role of inflammatory processes in the development of brain ischemia and multi-infarct cognitive impairment, as demonstrated by the accumulation of inflammatory cells and mediators in the ischemic brain [5]. Intercellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein involved in cell adhesion and acts as a cell surface ligand for members of the leukocyte integrin family [17]. The interaction of leukocyte integrins with endothelial ICAM-
* Corresponding author. Istituto di Patologia Speciale Medica, A. Gemelli University Hospital, Universita` Cattolica del Sacro Cuore, L.go A. Gemelli 8, 00168 Rome, Italy. Tel.: 139-0630154518; fax: 139-06-35500486. E-mail address: [email protected] (R. Pola).
1 leads to leukocyte adherence, transendothelial migration and cell activation [17], which are fundamental for the recruitment of blood monocytes to the intima, the transformation of macrophages into foam cells, and the development of atherosclerosis [15]. A role for ICAM-1 in carotid atherosclerosis and cerebral ischemia has been described [5,10,19]. These data are consistent with the observation that the plasma levels of ICAM-1 may be a marker for coronary atherosclerosis [8] and for the development of acute coronary syndromes, particularly unstable angina [12]. In addition, elevated serum levels of soluble (s)ICAM-1 have been found in patients after an acute coronary event. Recently, the K469E polymorphism of the ICAM-1 gene has been described [18]. In this study, we investigated the association between VD and ICAM-1 E/K gene polymorphism. First, we used the NINDS-AIREN criteria (impairment of memory and two or more cognitive domains, clinical and neuroradiologic evidence of cerebrovascular disease, and a relationship between the two disorders, inferred by onset of
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dementia within 3 months following a recognized stroke or abrupt and stepwise deterioration of cognitive functions) [14] to identify patients with probable VD among subjects consecutively admitted to the Department of Medicine of the “A. Gemelli” University Hospital of Rome, Italy, from November 1, 2000 to May 31, 2001. The Hachinski ischemic score (HIS) [7] was also used to aid in distinguishing between probable VD and dementia from other neurological disorders, such as AD. Then, patients with probable VD who had clinical evidence of repeated strokes, focal neurological signs and symptoms, and the presence of multiple territorial infarcts of the brain by CT scan evaluation were enrolled in the study. Finally, the studied population consisted of 107 patients affected by probable VD (47.7% men; mean age 76.3 years; SD 8.6). Control subjects were 115 individuals (47.8% men; mean age 76.1 years; SD 6.4) admitted to the same department in the same period of time, but without dementia and with no history and clinical and instrumental evidence of ischemic cerebral events. Controls had no relationship with cases and no family history of dementia. The cognitive function of control subjects was evaluated by a team of neurologists by formal neuro-psychological testing and mini-mental state examination (MMSE) [6]. In addition, all control patients underwent brain imaging evaluation by CT scan, which did not show the presence of ischemic lesions nor cerebral atrophy. All subjects with AD, suspected mixed dementia (VD and AD), or a family history of AD were excluded in this study. Subjects affected by tumors, chronic inflammatory diseases, and autoimmune diseases were excluded as well. All subjects were Caucasians from central and southern Italy and belonged to independent pedigrees. Informed consent was obtained from all patients. The study protocol was accepted by the Ethics Committee of our University Hospital. Genomic DNAs were assayed with polymerase chain reaction (PCR) for the detection of the ICAM-1 gene, using the published primer set 5 0 -GGAACCCATTGCCCGAGC-3 0 (sense primer) and 5 0 -GGTGAGGATTGCATTAGGTC-3 0 (antisense primer). PCR reaction, containing 20 pmol of each primer, 0.2 mmol/l of dNTPs (Boehringer GmbH, Mannheim, Germany), 1.5 mmol/l MgCl2, and 2.5 units of AmpliTaq polymerase (Perkin-Elmer, Cetus, Norwalk, CT) in a final volume of 20 ml, was performed in a GeneAmp PCR System 9700 (Perkin-Elmer) with an initial denaturation step of 7 min at 96 8C and a final extension step of 7 min at 72 8C. The following thermal profile was repeated for 35 cycles: denaturation for 35 s at 96 8C, annealing for 1 min at 64 8C and extension for 1 min 35 s at 72 8C. The amplified sequence was digested by BstU I restriction enzyme (New England BioLabs, Beverly, MA) at 60 8C for 4 h. The digested products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining. The EE genotype corresponded to the contemporary presence of 223 and 87 bp fragments. The EK genotype corresponded to the contemporary presence of 223, 136, and 87 bp fragments. The KK genotype corresponded to a 223 bp fragment.
Table 1 Demographic and clinical data in VD patients and controls
Age (years ^ SD) Sex M Hypertension Hypercholesterolemia Diabetes History of CAD History of PAOD Smoking (current) Smoking (former) a
Patients
Controls
P value
76.3 ^ 8.6 51 (47.7) a 77 (71.9) 36 (33.6) 31 (28.9) 43 (40.1) 29 (27.1) 9 (8.40) 22 (20.5)
76.1 ^ 6.4 55 (47.8) 59 (51.3) 39 (33.9) 21 (18.2) 30 (26.0) 13 (11.3) 13 (11.3) 29 (25.20)
0.80 0.98 0.002 0.96 0.06 0.02 0.002 0.47 0.41
Absolute numbers with % of total in parentheses.
Demographic and clinical data between groups were compared by x 2 test and by t-test. Genotype and allele frequencies were compared by x 2 test. Odds ratios were calculated with 95% CI. To estimate the association between genotype and probable VD, a logistic regression model was used (STATA software). Statistical significance was established at P , 0:05. Table 1 shows the demographic and clinical data of VD and control subjects. There were no significant differences between groups in terms of age, sex, hypercholesterolemia, and smoking habit. Diabetes was more frequent in patients than controls, but this difference did not reach statistical significance (P ¼ 0:06). Hypertension was found in 71.9% of VD subjects and 51.3% of controls (P ¼ 0:002). A history of coronary artery disease (CAD) was reported in 40.1% of VD patients and 26.0% of controls (P ¼ 0:02). A history of peripheral artery disease (PAOD) was more present in VD patients as well (27.1 vs. 11.3%, P ¼ 0:002). The distribution of ICAM-1 genotypes and alleles in cases and controls is shown in Table 2. Genotypes were in Hardy–Weinberg equilibrium. The frequency of the EE genotype in patients with dementia was significantly higher than in controls (P ¼ 0:009). In contrast, the frequency of the KK genotype was 2.4 times higher in controls than in subjects with dementia (P ¼ 0:03). Similarly, allele distribution was significantly different between the two groups (P ¼ 0:004). A logistic analysis (Table 3) showed that the EE genotype is an independent risk factor for probable VD Table 2 ICAM-1 genotype and allele distribution between groups Patients E/E a E/K K/K Ec K a b c
29 (27.1) b 58 (54.2) 20 (18.7) 116 (54.2) 98 (45.8)
Controls
P value
15 (13.0) 64 (55.6) 36 (31.4)
0.009 0.83 0.03
94 (40.9) 136 (59.1)
0.004
Genotype distribution. Numbers in parentheses are % of total. Allele distribution.
R. Pola et al. / Neuroscience Letters 326 (2002) 171–174 Table 3 Risk factors of probable VD based on logistic regression analysis
E/E genotype K/K genotype Diabetes PAOD Sex M Age Hypertension Hypercholesterolemia History of CAD Smoking (current) Smoking (former)
Odds ratio
95% CI
P value
3.2 0.5 3.4 4.7 0.7 1.0 1.6 1.1 1.1 0.1 0.7
1.16–9.04 0.17–1.29 1.38–8.34 1.71–12.9 0.30–1.75 0.96–1.08 0.69–3.72 0.46–2.54 0.45–2.54 0.01–0.92 0.27–2.10
0.024 0.144 0.007 0.003 0.481 0.461 0.262 0.843 0.862 0.041 0.597
in our population. Patients carrying the EE genotype have a risk more than three times higher than KK homozygous patients of developing VD (odds ratio 3.2 (1.16–9.04), P ¼ 0:024). Recent reports have demonstrated in mice that the deficiency of several CAMs (ICAM-1, P-selectin, CD18) reduces the development of atherosclerotic fatty streaks after a high-fat, high-cholesterol diet [2,11]. On the other hand, clinical studies have demonstrated that, in apparently healthy men, elevated baseline levels of sICAM-1 are associated with increased risk of future myocardial infarction [13]. In addition, in animal models of permanent occlusion of the middle cerebral artery, levels of ICAM-1 mRNA increase significantly in the ischemic cortex at 3 h, and remain elevated for up to 5 days, suggesting that adhesion molecules on brain capillary endothelium play an important role in leukocyte migration into ischemic tissue [19]. Connolly et al. [4] have studied the potential role of ICAM-1 in the pathogenesis of stroke, showing cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion. In addition, ICAM-1 has been found to induce expression of several other proinflammatory cytokines, such as IL-1 alpha, IL-1 beta, IL-6, and TNF alpha, specifically in astrocytes [9]. Although the importance of ICAM-1 protein in various kinds of cardiac and vascular ischemic disorders has been extensively demonstrated [3,5,8,10,12,19], no data are available about the role played in these pathologic conditions by common genetic polymorphisms of ICAM-1. The present study is the first evaluating the association between a polymorphism of the ICAM-1 gene and an ischemic cardiovascular disease, such as VD. In our population, we found that the distribution of alleles and genotypes of ICAM-1 E/ K gene polymorphism is significantly different between probable VD patients and controls. Our control population consisted of subjects with no dementia and without a history and clinical and radiological evidence of ischemic cerebral events. The allele and genotype distribution that we found in this control group is consistent with the results reported in previous studies evaluating ICAM-1 E/K gene polymorphism in the Italian population [1,16]. At univariate analysis,
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the E allele and EE genotype are significantly more frequent in patients with probable VD, while the K allele and KK genotype are significantly more frequent in controls. These data suggest that the presence of the E allele is a risk factor for probable VD and the K allele is protective for the disease. At multivariate analysis, we found that subjects with the EE genotype have a risk 3.2 times higher than subjects with the KK genotype of developing VD. This study has some potential limitations. Our patients have a higher incidence of CAD and PAOD than controls (Table 1), suggesting that these subjects have a severe atherosclerotic disease involving several organs and systems. Therefore, the ICAM-1 gene polymorphism might be associated with ischemic vascular disorders in general. Likewise, it is possible that the observed association is with cerebral ischemia only or cerebral ischemia and dementia. In conclusion, we demonstrate an association between the ICAM-1 K469E gene polymorphism and probable VD in an Italian population. These results confirm a role for ICAM-1 in the pathophysiology of cerebrovascular diseases and indicate that genetic polymorphisms of ICAM-1 might be clinically important. These data also suggest that antiinflammatory therapy might be useful in the prevention and treatment of VD. [1] Boiardi, L., Salvarani, C., Casali, B., Olivieri, I., Ciancio, G., Cantini, F., Salvi, F., Malatesta, R., Govoni, M., Trotta, F., Filippini, D., Paolazzi, G., Nicoli, D., Farnetti, E. and Macchioni, L., Intercellular adhesion molecule-1 gene polymorphisms in Behcet’s disease, J. Rheumatol., 28 (2001) 1283–1287. [2] Bourdillon, M.C., Poston, R.N., Covacho, C., Chignier, E., Bricca, G. and McGregor, J.L., ICAM-1 deficiency reduces atherosclerotic lesions in double-knockout mice ApoE (2/2)/ICAM-1(2/2) fed a fat or chow diet, Arterioscler. Thromb. Vasc. Biol., 20 (2000) 2630–2635. [3] Buja, L.M. and Willerson, J.T., Role of inflammation in coronary plaque disruption, Circulation, 89 (1994) 503– 505. [4] Connolly Jr., E.S., Winfree, C.J., Springer, T.A., Naka, Y., Liao, H., Yan, S.D., Stern, D.M., Solomon, R.A., GutierrezRamos, J.C. and Pinsky, D.J., Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion. Role of neutrophil adhesion in the pathogenesis of stroke, J. Clin. Invest., 97 (1996) 209–216. [5] del Zoppo, G., Ginis, I., Hallembeck, J.M., Iadecola, C., Wang, X. and Feuerstein, G.Z., Inflammation and stroke: putative role for cytokine adhesion molecules and iNOS in brain response to ischemia, Brain Pathol., 10 (2000) 95– 112. [6] Folstein, M.F., Folstein, S.E. and McHugh, P.R., Mini-mental state: a practical method for grading the cognitive state of patients for the clinician, J. Psychiatr. Res., 12 (1975) 189– 198. [7] Hachinski, V.C., Iliff, L.D., Zilhka, E., Du Boulay, G.H., McAllister, V.L., Marshall, J., Russell, R.W. and Symon, L., Cerebral blood flow in dementia, Arch. Neurol., 32 (1975) 632– 637. [8] Ikata, J., Wakatsuki, T., Oishi, Y., Oki, T. and Ito, S., Leukocyte counts and concentration of soluble adhesion molecules as predictors of coronary atherosclerosis, Coron. Artery Dis., 11 (2000) 445–449.
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[9] Lee, S.J., Drabik, K., Van Wagoner, N.J., Lee, S., Choi, C., Dong, Y. and Benveniste, E.N., ICAM-1 induced expression of proinflammatory cytokines in astrocytes: involvement of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathway, J. Immunol., 165 (2000) 4658–4666. [10] Mile, J., Parodi, J.C., Fernandez Alonso, G., Barone, A., Beigelman, R., Ferreira, L.M., Arrigoni, G. and Matturri, L., Carotid atherosclerosis. Immunocytochemical analysis of the vascular and cellular composition in endarterectomies, Cardiologia, 41 (1996) 535–542. [11] Nageh, M.F., Sandberg, E.T., Marotti, K.R., Lin, A.H., Melchior, E.P., Bullard, D.C. and Beaudet, A.L., Deficiency of inflammatory cell adhesion molecules protects against atherosclerosis in mice, Arterioscler. Thromb. Vasc. Biol., 17 (1997) 1517–1520. [12] O’Malley, T., Ludlam, C.A., Riemermsa, R.A. and Fox, K.A., Early increase level of soluble inter-cellular adhesion molecule-1 (sICAM-1); potential risk factor for the acute coronary syndrome, Eur. Heart J., 22 (2001) 1226–1234. [13] Ridker, P.M., Hennekens, C.H., Roitman-Johnson, B., Stampfer, M.J. and Allen, J., Plasma concentration of soluble intercellular adhesion molecule-1 and risks of future
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myocardial infarction in apparently healthy men, Lancet, 351 (1998) 88–92. Roman, G.C., Tatemichi, T.K., Erkinjuntti, T., Cummings, J.L., Masdeu, J.C., Garcia, J.H., Amaducci, L., Orgogozo, J.M., Brun, A. and Hofman, A., Vascular dementia: diagnostic criteria for research studies, Neurology, 43 (1993) 259–260. Ross, R., Atherosclerosis: an inflammatory disease, N. Engl. J. Med., 340 (1999) 115–126. Salvarani, C., Casali, B., Boiardi, L., Ranzi, A., Macchioni, P., Nicoli, D., Farnetti, E., Brini, M. and Portioli, I., Intercellular adhesion molecule-1 gene polymorphism in polymyalgia rheumatica/giant cell arteritis: association with disease risk and severity, J. Rheumatol., 27 (2000) 1215–1221. Springer, T.A., Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration, Annu. Rev. Physiol., 57 (1995) 827–872. Vora, D.K., Rosenbloom, G.L., Beaudet, A.L. and Cottingham, R.W., Polymorphisms and linkage analysis for ICAM-1 and the selectin gene cluster, Genomics, 21 (1994) 473–477. Wang, X. and Feuerstein, G.Z., Induced expression of adhesion molecules following focal brain ischemia, J. Neurotrauma, 12 (1995) 825–832.
Association between intercellular adhesion molecule-1 E/K gene polymorphism and probable vascular dementia in humans Roberto Pola a,b,*, Andrea Flex a,c, Eleonora Gaetani a,c, Pierangelo Papaleo c, Demetrio De Martini c, Laura Gerardino c, Michele Serricchio c, Paolo Pola a,c, Roberto Bernabei b a
Laboratory of Vascular Biology and Genetics, A. Gemelli University Hospital, Universita` Cattolica del Sacro Cuore School of Medicine, Rome, Italy b Department of Geriatric Medicine, A. Gemelli University Hospital, Universita` Cattolica del Sacro Cuore School of Medicine, Rome, Italy c Department of Internal Medicine, A. Gemelli University Hospital, Universita` Cattolica del Sacro Cuore School of Medicine, Rome, Italy Received 7 January 2002; received in revised form 19 March 2002; accepted 22 March 2002
Abstract Intercellular adhesion molecule-1 (ICAM-1) is implicated in the pathogenesis of ischemic cardiovascular disorders, including cerebral ischemia. A common polymorphism of the ICAM-1 gene (K469E) has been recently reported. In this case-control study, we evaluated the association between this polymorphism and vascular dementia (VD) by studying 107 patients affected by probable VD and 115 age- and sex-matched controls. The frequency of the EE genotype was significantly higher in VD patients than controls (P ¼ 0:009). Logistic regression analysis indicated that the presence of the EE genotype significantly increased the risk of VD (odds ratio 3.25, P ¼ 0:024). Our findings support the hypothesis that ICAM-1 plays a role in the physiopathology of ischemic cerebrovascular disorders and suggest that genetic polymorphisms of ICAM-1 might be clinically important in the development and progression of neurodegenerative diseases. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Probable vascular dementia; Cerebral ischemia; Inflammation; Intercellular adhesion molecule-1; Gene polymorphism
Vascular dementia (VD) is the second most common cause of cognitive deterioration after Alzheimer’s disease (AD). It occurs more often in persons who have hypertension and/or diabetes mellitus or who abuse tobacco. However, the mechanisms for clinical cognitive deterioration in patients with cerebral ischemia are not completely understood. Several recent observations hint at the role of inflammatory processes in the development of brain ischemia and multi-infarct cognitive impairment, as demonstrated by the accumulation of inflammatory cells and mediators in the ischemic brain [5]. Intercellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein involved in cell adhesion and acts as a cell surface ligand for members of the leukocyte integrin family [17]. The interaction of leukocyte integrins with endothelial ICAM-
* Corresponding author. Istituto di Patologia Speciale Medica, A. Gemelli University Hospital, Universita` Cattolica del Sacro Cuore, L.go A. Gemelli 8, 00168 Rome, Italy. Tel.: 139-0630154518; fax: 139-06-35500486. E-mail address: [email protected] (R. Pola).
1 leads to leukocyte adherence, transendothelial migration and cell activation [17], which are fundamental for the recruitment of blood monocytes to the intima, the transformation of macrophages into foam cells, and the development of atherosclerosis [15]. A role for ICAM-1 in carotid atherosclerosis and cerebral ischemia has been described [5,10,19]. These data are consistent with the observation that the plasma levels of ICAM-1 may be a marker for coronary atherosclerosis [8] and for the development of acute coronary syndromes, particularly unstable angina [12]. In addition, elevated serum levels of soluble (s)ICAM-1 have been found in patients after an acute coronary event. Recently, the K469E polymorphism of the ICAM-1 gene has been described [18]. In this study, we investigated the association between VD and ICAM-1 E/K gene polymorphism. First, we used the NINDS-AIREN criteria (impairment of memory and two or more cognitive domains, clinical and neuroradiologic evidence of cerebrovascular disease, and a relationship between the two disorders, inferred by onset of
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 33 7- 3
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R. Pola et al. / Neuroscience Letters 326 (2002) 171–174
dementia within 3 months following a recognized stroke or abrupt and stepwise deterioration of cognitive functions) [14] to identify patients with probable VD among subjects consecutively admitted to the Department of Medicine of the “A. Gemelli” University Hospital of Rome, Italy, from November 1, 2000 to May 31, 2001. The Hachinski ischemic score (HIS) [7] was also used to aid in distinguishing between probable VD and dementia from other neurological disorders, such as AD. Then, patients with probable VD who had clinical evidence of repeated strokes, focal neurological signs and symptoms, and the presence of multiple territorial infarcts of the brain by CT scan evaluation were enrolled in the study. Finally, the studied population consisted of 107 patients affected by probable VD (47.7% men; mean age 76.3 years; SD 8.6). Control subjects were 115 individuals (47.8% men; mean age 76.1 years; SD 6.4) admitted to the same department in the same period of time, but without dementia and with no history and clinical and instrumental evidence of ischemic cerebral events. Controls had no relationship with cases and no family history of dementia. The cognitive function of control subjects was evaluated by a team of neurologists by formal neuro-psychological testing and mini-mental state examination (MMSE) [6]. In addition, all control patients underwent brain imaging evaluation by CT scan, which did not show the presence of ischemic lesions nor cerebral atrophy. All subjects with AD, suspected mixed dementia (VD and AD), or a family history of AD were excluded in this study. Subjects affected by tumors, chronic inflammatory diseases, and autoimmune diseases were excluded as well. All subjects were Caucasians from central and southern Italy and belonged to independent pedigrees. Informed consent was obtained from all patients. The study protocol was accepted by the Ethics Committee of our University Hospital. Genomic DNAs were assayed with polymerase chain reaction (PCR) for the detection of the ICAM-1 gene, using the published primer set 5 0 -GGAACCCATTGCCCGAGC-3 0 (sense primer) and 5 0 -GGTGAGGATTGCATTAGGTC-3 0 (antisense primer). PCR reaction, containing 20 pmol of each primer, 0.2 mmol/l of dNTPs (Boehringer GmbH, Mannheim, Germany), 1.5 mmol/l MgCl2, and 2.5 units of AmpliTaq polymerase (Perkin-Elmer, Cetus, Norwalk, CT) in a final volume of 20 ml, was performed in a GeneAmp PCR System 9700 (Perkin-Elmer) with an initial denaturation step of 7 min at 96 8C and a final extension step of 7 min at 72 8C. The following thermal profile was repeated for 35 cycles: denaturation for 35 s at 96 8C, annealing for 1 min at 64 8C and extension for 1 min 35 s at 72 8C. The amplified sequence was digested by BstU I restriction enzyme (New England BioLabs, Beverly, MA) at 60 8C for 4 h. The digested products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining. The EE genotype corresponded to the contemporary presence of 223 and 87 bp fragments. The EK genotype corresponded to the contemporary presence of 223, 136, and 87 bp fragments. The KK genotype corresponded to a 223 bp fragment.
Table 1 Demographic and clinical data in VD patients and controls
Age (years ^ SD) Sex M Hypertension Hypercholesterolemia Diabetes History of CAD History of PAOD Smoking (current) Smoking (former) a
Patients
Controls
P value
76.3 ^ 8.6 51 (47.7) a 77 (71.9) 36 (33.6) 31 (28.9) 43 (40.1) 29 (27.1) 9 (8.40) 22 (20.5)
76.1 ^ 6.4 55 (47.8) 59 (51.3) 39 (33.9) 21 (18.2) 30 (26.0) 13 (11.3) 13 (11.3) 29 (25.20)
0.80 0.98 0.002 0.96 0.06 0.02 0.002 0.47 0.41
Absolute numbers with % of total in parentheses.
Demographic and clinical data between groups were compared by x 2 test and by t-test. Genotype and allele frequencies were compared by x 2 test. Odds ratios were calculated with 95% CI. To estimate the association between genotype and probable VD, a logistic regression model was used (STATA software). Statistical significance was established at P , 0:05. Table 1 shows the demographic and clinical data of VD and control subjects. There were no significant differences between groups in terms of age, sex, hypercholesterolemia, and smoking habit. Diabetes was more frequent in patients than controls, but this difference did not reach statistical significance (P ¼ 0:06). Hypertension was found in 71.9% of VD subjects and 51.3% of controls (P ¼ 0:002). A history of coronary artery disease (CAD) was reported in 40.1% of VD patients and 26.0% of controls (P ¼ 0:02). A history of peripheral artery disease (PAOD) was more present in VD patients as well (27.1 vs. 11.3%, P ¼ 0:002). The distribution of ICAM-1 genotypes and alleles in cases and controls is shown in Table 2. Genotypes were in Hardy–Weinberg equilibrium. The frequency of the EE genotype in patients with dementia was significantly higher than in controls (P ¼ 0:009). In contrast, the frequency of the KK genotype was 2.4 times higher in controls than in subjects with dementia (P ¼ 0:03). Similarly, allele distribution was significantly different between the two groups (P ¼ 0:004). A logistic analysis (Table 3) showed that the EE genotype is an independent risk factor for probable VD Table 2 ICAM-1 genotype and allele distribution between groups Patients E/E a E/K K/K Ec K a b c
29 (27.1) b 58 (54.2) 20 (18.7) 116 (54.2) 98 (45.8)
Controls
P value
15 (13.0) 64 (55.6) 36 (31.4)
0.009 0.83 0.03
94 (40.9) 136 (59.1)
0.004
Genotype distribution. Numbers in parentheses are % of total. Allele distribution.
R. Pola et al. / Neuroscience Letters 326 (2002) 171–174 Table 3 Risk factors of probable VD based on logistic regression analysis
E/E genotype K/K genotype Diabetes PAOD Sex M Age Hypertension Hypercholesterolemia History of CAD Smoking (current) Smoking (former)
Odds ratio
95% CI
P value
3.2 0.5 3.4 4.7 0.7 1.0 1.6 1.1 1.1 0.1 0.7
1.16–9.04 0.17–1.29 1.38–8.34 1.71–12.9 0.30–1.75 0.96–1.08 0.69–3.72 0.46–2.54 0.45–2.54 0.01–0.92 0.27–2.10
0.024 0.144 0.007 0.003 0.481 0.461 0.262 0.843 0.862 0.041 0.597
in our population. Patients carrying the EE genotype have a risk more than three times higher than KK homozygous patients of developing VD (odds ratio 3.2 (1.16–9.04), P ¼ 0:024). Recent reports have demonstrated in mice that the deficiency of several CAMs (ICAM-1, P-selectin, CD18) reduces the development of atherosclerotic fatty streaks after a high-fat, high-cholesterol diet [2,11]. On the other hand, clinical studies have demonstrated that, in apparently healthy men, elevated baseline levels of sICAM-1 are associated with increased risk of future myocardial infarction [13]. In addition, in animal models of permanent occlusion of the middle cerebral artery, levels of ICAM-1 mRNA increase significantly in the ischemic cortex at 3 h, and remain elevated for up to 5 days, suggesting that adhesion molecules on brain capillary endothelium play an important role in leukocyte migration into ischemic tissue [19]. Connolly et al. [4] have studied the potential role of ICAM-1 in the pathogenesis of stroke, showing cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion. In addition, ICAM-1 has been found to induce expression of several other proinflammatory cytokines, such as IL-1 alpha, IL-1 beta, IL-6, and TNF alpha, specifically in astrocytes [9]. Although the importance of ICAM-1 protein in various kinds of cardiac and vascular ischemic disorders has been extensively demonstrated [3,5,8,10,12,19], no data are available about the role played in these pathologic conditions by common genetic polymorphisms of ICAM-1. The present study is the first evaluating the association between a polymorphism of the ICAM-1 gene and an ischemic cardiovascular disease, such as VD. In our population, we found that the distribution of alleles and genotypes of ICAM-1 E/ K gene polymorphism is significantly different between probable VD patients and controls. Our control population consisted of subjects with no dementia and without a history and clinical and radiological evidence of ischemic cerebral events. The allele and genotype distribution that we found in this control group is consistent with the results reported in previous studies evaluating ICAM-1 E/K gene polymorphism in the Italian population [1,16]. At univariate analysis,
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the E allele and EE genotype are significantly more frequent in patients with probable VD, while the K allele and KK genotype are significantly more frequent in controls. These data suggest that the presence of the E allele is a risk factor for probable VD and the K allele is protective for the disease. At multivariate analysis, we found that subjects with the EE genotype have a risk 3.2 times higher than subjects with the KK genotype of developing VD. This study has some potential limitations. Our patients have a higher incidence of CAD and PAOD than controls (Table 1), suggesting that these subjects have a severe atherosclerotic disease involving several organs and systems. Therefore, the ICAM-1 gene polymorphism might be associated with ischemic vascular disorders in general. Likewise, it is possible that the observed association is with cerebral ischemia only or cerebral ischemia and dementia. In conclusion, we demonstrate an association between the ICAM-1 K469E gene polymorphism and probable VD in an Italian population. These results confirm a role for ICAM-1 in the pathophysiology of cerebrovascular diseases and indicate that genetic polymorphisms of ICAM-1 might be clinically important. These data also suggest that antiinflammatory therapy might be useful in the prevention and treatment of VD. [1] Boiardi, L., Salvarani, C., Casali, B., Olivieri, I., Ciancio, G., Cantini, F., Salvi, F., Malatesta, R., Govoni, M., Trotta, F., Filippini, D., Paolazzi, G., Nicoli, D., Farnetti, E. and Macchioni, L., Intercellular adhesion molecule-1 gene polymorphisms in Behcet’s disease, J. Rheumatol., 28 (2001) 1283–1287. [2] Bourdillon, M.C., Poston, R.N., Covacho, C., Chignier, E., Bricca, G. and McGregor, J.L., ICAM-1 deficiency reduces atherosclerotic lesions in double-knockout mice ApoE (2/2)/ICAM-1(2/2) fed a fat or chow diet, Arterioscler. Thromb. Vasc. Biol., 20 (2000) 2630–2635. [3] Buja, L.M. and Willerson, J.T., Role of inflammation in coronary plaque disruption, Circulation, 89 (1994) 503– 505. [4] Connolly Jr., E.S., Winfree, C.J., Springer, T.A., Naka, Y., Liao, H., Yan, S.D., Stern, D.M., Solomon, R.A., GutierrezRamos, J.C. and Pinsky, D.J., Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion. Role of neutrophil adhesion in the pathogenesis of stroke, J. Clin. Invest., 97 (1996) 209–216. [5] del Zoppo, G., Ginis, I., Hallembeck, J.M., Iadecola, C., Wang, X. and Feuerstein, G.Z., Inflammation and stroke: putative role for cytokine adhesion molecules and iNOS in brain response to ischemia, Brain Pathol., 10 (2000) 95– 112. [6] Folstein, M.F., Folstein, S.E. and McHugh, P.R., Mini-mental state: a practical method for grading the cognitive state of patients for the clinician, J. Psychiatr. Res., 12 (1975) 189– 198. [7] Hachinski, V.C., Iliff, L.D., Zilhka, E., Du Boulay, G.H., McAllister, V.L., Marshall, J., Russell, R.W. and Symon, L., Cerebral blood flow in dementia, Arch. Neurol., 32 (1975) 632– 637. [8] Ikata, J., Wakatsuki, T., Oishi, Y., Oki, T. and Ito, S., Leukocyte counts and concentration of soluble adhesion molecules as predictors of coronary atherosclerosis, Coron. Artery Dis., 11 (2000) 445–449.
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