- Email: [email protected]
Relationship between HLA molecules and late restenosis after coronary stent placement
Hasan Kudat b,⇑, Mustafa Ozcan a, Tufan Tükek b, Ahmet Bilge Sözen b, Vakur Akkaya b, Fatma Oguz c, Yalçın Seyhun c a
Department of Cardiology, Istanbul Medical Faculty, Istanbul University, Istanbul; b Department of Internal Medicine, Istanbul Medical Faculty, Istanbul University, 34390 Istanbul; c Department of Medical Biology, Istanbul Medical Faculty, Istanbul University, Istanbul
a,b,c
Turkey
Objective: The objective of this study is to confirm whether there is relation between the human leucocyte antigen (HLA) locus and restenosis after percutaneous coronary intervention (PCI) holds in our patient population and whether it can be useful as a prognostic factor. Methods: We examined the HLA phenotypes in 46 consecutive patients (39 men, 7 women, mean age of 57 ± 9 years) who had successful stent implantation in the coronary artery. Selective coronary arteriography was performed 6 months after coronary stenting to assess the presence of restenosis. The HLA phenotyping was performed for HLA-A,-B,-C antigens by Terasaki microlymphocytotoxicity technique and for HLA-DR alleles with PCR-SSP technique. Results: Restenosis(R+) was present in 12 (26.1%) patients (11 men, 1 woman, mean age of 57 ± 10 years). For HLA Class I antigens frequency of HLA-B62 and HLA-CW2 antigen was slightly higher in restenotic patients but did not reach statistical significance. For HLA-DR alleles restenotic patients had higher frequencies for HLA-DRB1⁄01(R+ %25, R %14.7), and HLA-DR11(R+ %41.7, R %20.6), without reaching statistical significance and lower frequencies for DR7(R+ %0, R %17.6) and D13(R+%8.3, R %32.4) and HLA-DR53 (R+ %25, R %35.3) without reaching statistical significance. Conclusion: In conclusion, results show that there was no relationship between the development of restenosis and HLA-subtypes. Ó 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Keywords: Restenosis, HLA, Stent implantation
I
n light of increasing numbers of patients being treated with multiple percutaneous coronary interventions (PCI), in-stent restenosis is a mounting problem [1]. There are two lines of research on the subject, the first being the development of stents with lower incidence of restenosis, such as
drug-eluting stents [2] and the second, inextricably wound up with the first is to reach an understanding of restenosis, its predictors and risk factors. Identification of markers indicating a higher risk of restenosis could be helpful in individualizing treatment.
Received 26 September 2011; accepted 5 October 2011. Available online 18 October 2011
⇑ Corresponding author. Tel.: +90 212 5246102. E-mail address: [email protected] (H. Kudat).
P.O. Box 2925 Riyadh – 11461KSA Tel: +966 1 2520088 ext 40151 Fax: +966 1 2520718 Email: [email protected] URL: www.sha.org.sa
1016–7315 Ó 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of King Saud University. URL: www.ksu.edu.sa doi:10.1016/j.jsha.2011.10.001
FULL LENGTH ARTICLE
Relationship between HLA molecules and late restenosis after coronary stent placement
86 FULL LENGTH ARTICLE
KUDAT ET AL HLA AND CORONARY RESTENOSIS
Various factors, like inflammation [3–5], release of cytokines and growth factors into the vessel wall [6], intimal migration and hypertrophy of the smooth muscle cells [7–9] are implicated in the development of neointimal hyperplasia, the cause of the restenotic process after stent implantation. As not all patients with stent implantation develop late restenosis, the restenotic process may be a genetically determined immune response. Human leukocyte antigens (HLA) are a major histocompatibility complex and play an important role in immune responses to human disease. There are few studies on the putative influence of HLA systems on the inflammatory response after stent implantation. Watanabe et al [10] reported a significant relation between restenosis and HLA-C locus after balloon angioplasty and indicated that HLA-type may be a prognostic factor. The objective of this study is to confirm whether there is relation between the human leucocyte antigen (HLA) locus and restenosis after percutaneous transluminal coronary angioplasty (PTCA) holds in our patient population and whether it can be useful as a prognostic factor.
Material and method Patient population and study design We examined the HLA phenotypes in 46 consecutive patients (39 men, 7 women, mean age. 57 ± 9 years) who had de novo stabile, significant coronary stenosis (>70% luminal stenosis) and had a successful stent implantation into the coronary artery. We excluded from the present study; [1] patients who presented with acute myocardial infarction or unstable angina, because these are pathophysiologically characterized by plaque instability and thrombus formation; [5] failed PTCA patients; [3] aortocoronary artery bypass patients, having previous stent implantation. All were asked to undergo control angiography and clinical follow-up at six months. The study was approved by the appropriate institutional committees, and patients gave informed consent. This investigation conforms with the principles outlined in the Declaration of Helsinki.
Coronary arteriographic evaluation and stent placement Coronary arteriography and stent placement were performed via the femoral artery by the standard method. Control coronary arteriography was performed six months after coronary stenting to assess the presence of restenosis regardless of
J Saudi Heart Assoc 2012;24:85–88
Table 1. Clinical characteristics of the study population. R(+) n = 12 R( ) n = 34 P value Age (years) Sex (male/female) Diabetes mellitus(%) Hypertension(%) Smoking(%) Total cholesterol (mg/dl) Triglycerides (mg/dl) HDL-cholesterol (mg/dl) LDL-cholesterol (mg/dl)
57 ± 9 11/1 5(%) 9 (%) 8(%) 211 ± 29
57 ± 10 28/6 8(%) 27(%) 23(%) 222 ± 46
NS NS NS NS NS NS
164 ± 81
201 ± 117
NS
43 ± 8
41 ± 11
NS
139 ± 19
144 ± 42
NS
Medications ACEI/AT2 Calcium channel blocker Antiaggregants Nitrates Statine use
26% 80% 100% 40% 100%
29% 83% 100% 45% 100%
NS NS NS NS NS
Vessels disease LAD Cx RCA Mean stenosis (%)
10 1 1 80.3 ± 10.8
20 6 6 84.7 ± 9.24
NS NS NS NS
R: Restenosis, NS: not significant, LAD: Left anterior descending, Cx: Circumflex artery, RCA: Right coronary artery.
the actual clinical presence or absence of ischemia. Among the 12 patients with restenosis 8 (66%) had clinical and noninvasive laboratory findings of reversible ischemia. Patients’ demographic data are shown in Table 1. Coronary angiograms were evaluated visually as reported in previous studies by at least two cardiologists. Restenosis was defined as at least 50% stenosis on the control arteriography [1].
HLA typing All patients were typed at the Department of Medical Biology, Istanbul Medical Faculty which has accreditation to perform clinical HLA typing by the European Federation of Immunogenetics. All samples were typed at the HLA-DRB1/3/4/ loci by the sequence-specific primer (PCR-SSP) method at low resolution [11]. This typing method is the molecular equivalent of serological HLA-DR typing and identifies all two-digit specificities accurately including the superspecificities DR52 and DR53 encoded by the DRB3 and DRB4 genes, respectively. PCR was performed on a 9700 thermal cycle (PE Biosystems, CA). Class I antigens were investigated using Terasaki microlymphocytotoxicity assay. In serological method, a 144-well plate containing HLA-A and HLA-B and HLA-C antibodies were used [12].
KUDAT ET AL HLA AND CORONARY RESTENOSIS
Table 2. Number of subjects possessing each HLA-DRB1 allele and HLA-DRB1 marker frequencies (%) in patients. DRB1/B3/ B4/B5
Restenosis Restenosis absent P value present N = 12 N = 34
DRB1⁄01 DRB1⁄03 DRB1⁄04 DRB1⁄07 DRB1⁄11(5)a DRB1⁄12 (5)a DRB1⁄13 (6)a DRB1⁄14 (6)a DRB1⁄15(2)a DRB1⁄16 (2)a DRB5(51)a DRB3(52)a DRB4(53)a
3 (25%) 1 (11.8%) 4 (33.3%) 0 (0%) 5 (41.7%) 0 (0%) 1(8.3%) 0 (0%) 4 (33.3%) 0 (0%) 3 (25%) 5 (41.7%) 3 (25%)
5 (14.7%) 4 (11.8%) 9 (26.5%) 6 (17.6%) 7 (20.6%) 2 (5.9%) 11 (32.4%) 4 (11.8%) 3 (8.8%) 8 (23.5%) 7 (20%) 16 (47.1%) 12 (35.3%)
0.4 1 0.7 0.3 0.3 1 0.1 0.5 0.06 0.09 0.7 1 1
a Numbers in brackets in the HLA-DRB1 column denote the broad groups of DRBI specificities.
Statistical analysis Statistical analysis was carried out with Statistical Package for Social Sciences for Windows version 10.0 (SPSS Inc, Chicago, USA). All nonnumeric variables are reported as frequency and percentage, and all numeric variables are reported as mean ± SD. The comparison of the means of two numerical parameters was done with Student’s t-test. Comparisons of the frequencies of HLA subtypes and the presence and absence of restenosis were done with v2 test. For 22 contingency tables Yate’s correction was done. When assumptions were violated for 22 contingency tables Fisher’s exact test was used. As the number of patients were small all comparisons with p value <0.1 were included in a logistic regression test to check their significance. P value less than 0.05 was accepted as significant.
Results As shown in Table 1 risk factors such as age, plasma lipid levels, blood pressure, diabetes and smoking were not significantly different between patients with and without restenosis. Restenosis was present in 12 (26.1%) patients (11 man, 1 woman, mean age 57 ± 10 years). There was no significant difference between frequencies of HLA-A group antigens for patients with or without restenosis. Among the HLA-B and HLA-C locus antigens frequency of HLAB62 and HLA-CW2 antigen was slightly higher in restenosic patients but did not reach statistical significance. Findings for HLA-DRB1 allele and HLA-DRB1 marker frequencies (%) in patients are given on Table 2. The frequencies of HLA-DRB⁄01(R+ %25,
87
R %14.7, p = NS), and HLA-DRB1⁄11(R+ %41.7, R %20.6, p = NS) were more frequent in the restenotic group without reaching statistical significance. Whereas DRB1⁄07(R+ %0, R %17.6, p = NS) and DRB1⁄13 (R+ %8.3, R %32.4, p = NS) and HLA-DRB4(53) (R+ %25, R %35.3, p = NS) alleles were higher in non-restenotic patients but it also did not reach statistical significance.
Discussion Restenosis is the most important long term prognostic factor after coronary stent implantation. Conventional risk factors associated with atherosclerotic processes are known to be factors in the development of stent restenosis, however it is also a fact that restenosis can occur in patients in whom risk factors are rigorously controlled [1]. It is known that diabetes, multiple stents and final minimal lumen diameter are strong predictors of restenosis [1]. In our study there was no difference for risk factors among patients who had or did not have restenosis. It is generally accepted that stent restenosis within the first 6 months is related to vascular smooth muscle cell reactive proliferation (neointimal hyperplasia) and increased extracellular matrix [13–15]. Stent implantation and injury caused by the balloon triggers an inflammatory reaction in the vessel wall. Inflammatory cells are activated after vascular injury [5,15]. These cells release some mediators that facilitate smooth muscle cells migration and proliferation into the arterial intima [7–9], cytokine and growth factor release may result in the development of restenosis [6]. The inhibition of inflammation and neointimal hyperplasia in balloon injury animal models with IL 10 (a monocyte deactivator) [16], and blockage of b2 integrin Mac-1 activity (a leucocyte adhesion molecule) either with antibodies [17] or in Mac-1 knock out rats [18] supports the idea that inflammation is crucial in the development of stent restenosis. It was also reported that nonspecific systemic stimulation of the immune system concurrently with arterial vascular injury facilitated neointimal formation and increased restenosis [13]. MHC (major histocompatibility complex) molecules bind peptides derived from pathogens and self-antigens in an intracellular processing compartment and transport these peptides to the cell surface for recognition by T cells. It is possible that individual polymorphism in antigen processing will affect its specificity, resulting in different sets
FULL LENGTH ARTICLE
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of peptides derived from the same antigen being presented to T cells in different individuals regardless of their HLA genotype. The presence of natural killer cells in atherosclerotic plaques may imply that HLA gene locus may play a role in the atherosclerotic process [19,20]. McKay et al suggested that IgG antibodies to HLA class I antigen may help predict the risk of restenosis after PCI in their study [21]. The association of HLA-C locus with restenosis after balloon angioplasty reported by Watanabe et al may imply that HLA antigens may have a prognostic role in the development of restenosis. They used the serology method for tissue typing but this method is too often hindered by a lack of discrimination due to antibody cross reactivity or a lack of useful typing reagents. DNA based tissue typing methods such as PCR-SSP have been used successfully to improve the quality of HLA typing [11,21]. In our study, we used serology for HLA Class I antigens and PCR-SSP technique for HLA-DRB, however we found no relation between HLA antigens and the development of restenosis or we were unable to confirm the previous result. The results of these studies must be interpreted with great caution as numerous different extrinsic factors are observed in these studies which might contribute to restenosis differently. Furthermore, these studies are of relatively small sample size and exhibit wide confidence intervals. On the other hand, one must take into account that it is likely that multiple factors are involved in restenosis. In conclusion, results show that there was no relationship between development of restenosis and certain HLA-types suggesting that HLA typing is not a predictor for restenosis.
J Saudi Heart Assoc 2012;24:85–88
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14] [15]
Conflict of Interest Statement
[16]
The authors have no conflicts of interest to declare. [17]
References [1] Kastrati A, Schomig A, Elezi S, Schuhlen H, Dirschinger J, Hadamitzky M, et al.. Predictive factors of restenosis after coronary stent placement. J Am Coll Cardiol 1997;30:1428–36. [2] Marzocchi A, Saia F, Piovaccari G, Manari A, Aurier E, Benassi A, Cremonesi A, Percoco G, Varani E, Magnavacchi P, Guastaroba P, Grilli R, Maresta A. Longterm safety and efficacy of drug-eluting stents: two-year results of the REAL (REgistro AngiopLastiche dell’Emilia Romagna) multicenter registry. Circulation 2007;115(25):3181–8. [3] Hancock WW, Adams DH, Wyner LR, Sayegh MH, Karnovsky MJ. CD4+ mononuclear cells induce cytokine expression, vascular smooth muscle cell proliferation, and
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arterial occlusion after endothelial injury. Am J Pathol 1994;145:1008–14. Okamoto E, Couse T, De Leon H, Vinten-Johansen J, Goodman RB, Scott NA, et al.. Perivascular inflammation after balloon angioplasty of porcine coronary arteries. Circulation 2001;104:2228–35. Serrano Jr CV, Ramires JA, Venturinelli M, Arie S, D’Amico E, Zweier JL, et al.. Coronary angioplasty results in leukocyte and platelet activation with adhesion molecule expression Evidence of inflammatory responses in coronary angioplasty. J Am Coll Cardiol 1997;29:1276–83. Kornowski R, Hong MK, Tio FO, Bramwell O, Wu H, Leon MB. In-stent restenosis: contributions of inflammatory responses and arterial injury to neointimal hyperplasia. J Am Coll Cardiol 1998;31:224–30. Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res 1994;75:539–45. Jawien A, Bowen-Pope DF, Lindner V, Schwartz SM, Clowes AW. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest 1992;89:507–11. Libby P, Schwartz D, Brogi E, Tanaka H, Clinton SK. A cascade model for restenosis. A special case of atherosclerosis progression. Circulation 1992;86(Suppl. 6):11147–52. Watanabe Y, Yamada N, Yokoi H, Fujiwara Y, Mokuno H, Daida H, et al.. Relationship between HLA-C locus and restenosis after coronary artery balloon angioplasty. JAMA 1997;277:983–4. Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 h: an alternative to serological DR typing in clinical practice including donor–recipient matching in cadaveric transplantation. Tissue Antigens 1992;39:225. Terasaki PI, McClelland JD, Park MS, McCurdy B. 1973 Microdroplet Lymphocyte Cytotoxicity Test, Manuel of Tissue Techniques. Washington, DC, US Government Printing Office, DHEW publication No (NIH) 74-545, p54. Danenberg HD, Welt FG, Walker M 3rd, Seifert P, Toegel GS, Edelman ER. Systemic inflammation induced by lipopolysaccharide increases neointimal formation after balloon and stent injury in rabbits. Circulation 2002;105:2917–22. Inoue S, Koyama H, Miyata T, Shigematsu H. Pathogenetic heterogeneity of in-stent lesion formation in human peripheral arterial disease. J Vasc Surg 2002;35:672–8. Wilensky RL, March KL, Gradus-Pizlo I, Sandusky G, Fineberg N, Hathaway DR. Vascular injury, repair, and restenosis after percutaneous transluminal angioplasty in the atherosclerotic rabbit. Circulation 1995;92:2995–3005. Feldman LJ, Aguirre L, Ziol M, Bridou JP, Nevo N, Michel JB, et al.. Interleukin-10 inhibits intimal hyperplasia after angioplasty or stent implantation in hypercholesterolemic rabbits. Circulation 2000;101:908–16. Rogers C, Edelman ER, Simon DI. A mAb to the beta2leukocyte integrin Mac-1 (CD11b/CD18) reduces intimal thickening after angioplasty or stent implantation in rabbits. Proc Natl Acad Sci U S A 1998;95:10134–9. Simon DI, Dhen Z, Seifert P, Edelman ER, Ballantyne CM, Rogers C. Decreased neointimal formation in Mac-1( / ) mice reveals a role for inflammation in vascular repair after angioplasty. J Clin Invest 2000;105:293–300. Falk K, Rotzschke O, Grahovac B, Schendel D, Stevanovic S, Gnau V, et al.. Allele-specific peptide ligand motifs of HLA-C molecules. Proc Natl Acad Sci USA 1993;90:12005–9. Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis 1986;6:131–8. Bunce M, Yong NT, Welsh KI. Molecular HLA typing–the brave new world. Transplantation 1997;64:1505.
Department of Cardiology, Istanbul Medical Faculty, Istanbul University, Istanbul; b Department of Internal Medicine, Istanbul Medical Faculty, Istanbul University, 34390 Istanbul; c Department of Medical Biology, Istanbul Medical Faculty, Istanbul University, Istanbul
a,b,c
Turkey
Objective: The objective of this study is to confirm whether there is relation between the human leucocyte antigen (HLA) locus and restenosis after percutaneous coronary intervention (PCI) holds in our patient population and whether it can be useful as a prognostic factor. Methods: We examined the HLA phenotypes in 46 consecutive patients (39 men, 7 women, mean age of 57 ± 9 years) who had successful stent implantation in the coronary artery. Selective coronary arteriography was performed 6 months after coronary stenting to assess the presence of restenosis. The HLA phenotyping was performed for HLA-A,-B,-C antigens by Terasaki microlymphocytotoxicity technique and for HLA-DR alleles with PCR-SSP technique. Results: Restenosis(R+) was present in 12 (26.1%) patients (11 men, 1 woman, mean age of 57 ± 10 years). For HLA Class I antigens frequency of HLA-B62 and HLA-CW2 antigen was slightly higher in restenotic patients but did not reach statistical significance. For HLA-DR alleles restenotic patients had higher frequencies for HLA-DRB1⁄01(R+ %25, R %14.7), and HLA-DR11(R+ %41.7, R %20.6), without reaching statistical significance and lower frequencies for DR7(R+ %0, R %17.6) and D13(R+%8.3, R %32.4) and HLA-DR53 (R+ %25, R %35.3) without reaching statistical significance. Conclusion: In conclusion, results show that there was no relationship between the development of restenosis and HLA-subtypes. Ó 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Keywords: Restenosis, HLA, Stent implantation
I
n light of increasing numbers of patients being treated with multiple percutaneous coronary interventions (PCI), in-stent restenosis is a mounting problem [1]. There are two lines of research on the subject, the first being the development of stents with lower incidence of restenosis, such as
drug-eluting stents [2] and the second, inextricably wound up with the first is to reach an understanding of restenosis, its predictors and risk factors. Identification of markers indicating a higher risk of restenosis could be helpful in individualizing treatment.
Received 26 September 2011; accepted 5 October 2011. Available online 18 October 2011
⇑ Corresponding author. Tel.: +90 212 5246102. E-mail address: [email protected] (H. Kudat).
P.O. Box 2925 Riyadh – 11461KSA Tel: +966 1 2520088 ext 40151 Fax: +966 1 2520718 Email: [email protected] URL: www.sha.org.sa
1016–7315 Ó 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of King Saud University. URL: www.ksu.edu.sa doi:10.1016/j.jsha.2011.10.001
FULL LENGTH ARTICLE
Relationship between HLA molecules and late restenosis after coronary stent placement
86 FULL LENGTH ARTICLE
KUDAT ET AL HLA AND CORONARY RESTENOSIS
Various factors, like inflammation [3–5], release of cytokines and growth factors into the vessel wall [6], intimal migration and hypertrophy of the smooth muscle cells [7–9] are implicated in the development of neointimal hyperplasia, the cause of the restenotic process after stent implantation. As not all patients with stent implantation develop late restenosis, the restenotic process may be a genetically determined immune response. Human leukocyte antigens (HLA) are a major histocompatibility complex and play an important role in immune responses to human disease. There are few studies on the putative influence of HLA systems on the inflammatory response after stent implantation. Watanabe et al [10] reported a significant relation between restenosis and HLA-C locus after balloon angioplasty and indicated that HLA-type may be a prognostic factor. The objective of this study is to confirm whether there is relation between the human leucocyte antigen (HLA) locus and restenosis after percutaneous transluminal coronary angioplasty (PTCA) holds in our patient population and whether it can be useful as a prognostic factor.
Material and method Patient population and study design We examined the HLA phenotypes in 46 consecutive patients (39 men, 7 women, mean age. 57 ± 9 years) who had de novo stabile, significant coronary stenosis (>70% luminal stenosis) and had a successful stent implantation into the coronary artery. We excluded from the present study; [1] patients who presented with acute myocardial infarction or unstable angina, because these are pathophysiologically characterized by plaque instability and thrombus formation; [5] failed PTCA patients; [3] aortocoronary artery bypass patients, having previous stent implantation. All were asked to undergo control angiography and clinical follow-up at six months. The study was approved by the appropriate institutional committees, and patients gave informed consent. This investigation conforms with the principles outlined in the Declaration of Helsinki.
Coronary arteriographic evaluation and stent placement Coronary arteriography and stent placement were performed via the femoral artery by the standard method. Control coronary arteriography was performed six months after coronary stenting to assess the presence of restenosis regardless of
J Saudi Heart Assoc 2012;24:85–88
Table 1. Clinical characteristics of the study population. R(+) n = 12 R( ) n = 34 P value Age (years) Sex (male/female) Diabetes mellitus(%) Hypertension(%) Smoking(%) Total cholesterol (mg/dl) Triglycerides (mg/dl) HDL-cholesterol (mg/dl) LDL-cholesterol (mg/dl)
57 ± 9 11/1 5(%) 9 (%) 8(%) 211 ± 29
57 ± 10 28/6 8(%) 27(%) 23(%) 222 ± 46
NS NS NS NS NS NS
164 ± 81
201 ± 117
NS
43 ± 8
41 ± 11
NS
139 ± 19
144 ± 42
NS
Medications ACEI/AT2 Calcium channel blocker Antiaggregants Nitrates Statine use
26% 80% 100% 40% 100%
29% 83% 100% 45% 100%
NS NS NS NS NS
Vessels disease LAD Cx RCA Mean stenosis (%)
10 1 1 80.3 ± 10.8
20 6 6 84.7 ± 9.24
NS NS NS NS
R: Restenosis, NS: not significant, LAD: Left anterior descending, Cx: Circumflex artery, RCA: Right coronary artery.
the actual clinical presence or absence of ischemia. Among the 12 patients with restenosis 8 (66%) had clinical and noninvasive laboratory findings of reversible ischemia. Patients’ demographic data are shown in Table 1. Coronary angiograms were evaluated visually as reported in previous studies by at least two cardiologists. Restenosis was defined as at least 50% stenosis on the control arteriography [1].
HLA typing All patients were typed at the Department of Medical Biology, Istanbul Medical Faculty which has accreditation to perform clinical HLA typing by the European Federation of Immunogenetics. All samples were typed at the HLA-DRB1/3/4/ loci by the sequence-specific primer (PCR-SSP) method at low resolution [11]. This typing method is the molecular equivalent of serological HLA-DR typing and identifies all two-digit specificities accurately including the superspecificities DR52 and DR53 encoded by the DRB3 and DRB4 genes, respectively. PCR was performed on a 9700 thermal cycle (PE Biosystems, CA). Class I antigens were investigated using Terasaki microlymphocytotoxicity assay. In serological method, a 144-well plate containing HLA-A and HLA-B and HLA-C antibodies were used [12].
KUDAT ET AL HLA AND CORONARY RESTENOSIS
Table 2. Number of subjects possessing each HLA-DRB1 allele and HLA-DRB1 marker frequencies (%) in patients. DRB1/B3/ B4/B5
Restenosis Restenosis absent P value present N = 12 N = 34
DRB1⁄01 DRB1⁄03 DRB1⁄04 DRB1⁄07 DRB1⁄11(5)a DRB1⁄12 (5)a DRB1⁄13 (6)a DRB1⁄14 (6)a DRB1⁄15(2)a DRB1⁄16 (2)a DRB5(51)a DRB3(52)a DRB4(53)a
3 (25%) 1 (11.8%) 4 (33.3%) 0 (0%) 5 (41.7%) 0 (0%) 1(8.3%) 0 (0%) 4 (33.3%) 0 (0%) 3 (25%) 5 (41.7%) 3 (25%)
5 (14.7%) 4 (11.8%) 9 (26.5%) 6 (17.6%) 7 (20.6%) 2 (5.9%) 11 (32.4%) 4 (11.8%) 3 (8.8%) 8 (23.5%) 7 (20%) 16 (47.1%) 12 (35.3%)
0.4 1 0.7 0.3 0.3 1 0.1 0.5 0.06 0.09 0.7 1 1
a Numbers in brackets in the HLA-DRB1 column denote the broad groups of DRBI specificities.
Statistical analysis Statistical analysis was carried out with Statistical Package for Social Sciences for Windows version 10.0 (SPSS Inc, Chicago, USA). All nonnumeric variables are reported as frequency and percentage, and all numeric variables are reported as mean ± SD. The comparison of the means of two numerical parameters was done with Student’s t-test. Comparisons of the frequencies of HLA subtypes and the presence and absence of restenosis were done with v2 test. For 22 contingency tables Yate’s correction was done. When assumptions were violated for 22 contingency tables Fisher’s exact test was used. As the number of patients were small all comparisons with p value <0.1 were included in a logistic regression test to check their significance. P value less than 0.05 was accepted as significant.
Results As shown in Table 1 risk factors such as age, plasma lipid levels, blood pressure, diabetes and smoking were not significantly different between patients with and without restenosis. Restenosis was present in 12 (26.1%) patients (11 man, 1 woman, mean age 57 ± 10 years). There was no significant difference between frequencies of HLA-A group antigens for patients with or without restenosis. Among the HLA-B and HLA-C locus antigens frequency of HLAB62 and HLA-CW2 antigen was slightly higher in restenosic patients but did not reach statistical significance. Findings for HLA-DRB1 allele and HLA-DRB1 marker frequencies (%) in patients are given on Table 2. The frequencies of HLA-DRB⁄01(R+ %25,
87
R %14.7, p = NS), and HLA-DRB1⁄11(R+ %41.7, R %20.6, p = NS) were more frequent in the restenotic group without reaching statistical significance. Whereas DRB1⁄07(R+ %0, R %17.6, p = NS) and DRB1⁄13 (R+ %8.3, R %32.4, p = NS) and HLA-DRB4(53) (R+ %25, R %35.3, p = NS) alleles were higher in non-restenotic patients but it also did not reach statistical significance.
Discussion Restenosis is the most important long term prognostic factor after coronary stent implantation. Conventional risk factors associated with atherosclerotic processes are known to be factors in the development of stent restenosis, however it is also a fact that restenosis can occur in patients in whom risk factors are rigorously controlled [1]. It is known that diabetes, multiple stents and final minimal lumen diameter are strong predictors of restenosis [1]. In our study there was no difference for risk factors among patients who had or did not have restenosis. It is generally accepted that stent restenosis within the first 6 months is related to vascular smooth muscle cell reactive proliferation (neointimal hyperplasia) and increased extracellular matrix [13–15]. Stent implantation and injury caused by the balloon triggers an inflammatory reaction in the vessel wall. Inflammatory cells are activated after vascular injury [5,15]. These cells release some mediators that facilitate smooth muscle cells migration and proliferation into the arterial intima [7–9], cytokine and growth factor release may result in the development of restenosis [6]. The inhibition of inflammation and neointimal hyperplasia in balloon injury animal models with IL 10 (a monocyte deactivator) [16], and blockage of b2 integrin Mac-1 activity (a leucocyte adhesion molecule) either with antibodies [17] or in Mac-1 knock out rats [18] supports the idea that inflammation is crucial in the development of stent restenosis. It was also reported that nonspecific systemic stimulation of the immune system concurrently with arterial vascular injury facilitated neointimal formation and increased restenosis [13]. MHC (major histocompatibility complex) molecules bind peptides derived from pathogens and self-antigens in an intracellular processing compartment and transport these peptides to the cell surface for recognition by T cells. It is possible that individual polymorphism in antigen processing will affect its specificity, resulting in different sets
FULL LENGTH ARTICLE
J Saudi Heart Assoc 2012;24:85–88
88 FULL LENGTH ARTICLE
KUDAT ET AL HLA AND CORONARY RESTENOSIS
of peptides derived from the same antigen being presented to T cells in different individuals regardless of their HLA genotype. The presence of natural killer cells in atherosclerotic plaques may imply that HLA gene locus may play a role in the atherosclerotic process [19,20]. McKay et al suggested that IgG antibodies to HLA class I antigen may help predict the risk of restenosis after PCI in their study [21]. The association of HLA-C locus with restenosis after balloon angioplasty reported by Watanabe et al may imply that HLA antigens may have a prognostic role in the development of restenosis. They used the serology method for tissue typing but this method is too often hindered by a lack of discrimination due to antibody cross reactivity or a lack of useful typing reagents. DNA based tissue typing methods such as PCR-SSP have been used successfully to improve the quality of HLA typing [11,21]. In our study, we used serology for HLA Class I antigens and PCR-SSP technique for HLA-DRB, however we found no relation between HLA antigens and the development of restenosis or we were unable to confirm the previous result. The results of these studies must be interpreted with great caution as numerous different extrinsic factors are observed in these studies which might contribute to restenosis differently. Furthermore, these studies are of relatively small sample size and exhibit wide confidence intervals. On the other hand, one must take into account that it is likely that multiple factors are involved in restenosis. In conclusion, results show that there was no relationship between development of restenosis and certain HLA-types suggesting that HLA typing is not a predictor for restenosis.
J Saudi Heart Assoc 2012;24:85–88
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Conflict of Interest Statement
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The authors have no conflicts of interest to declare. [17]
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