Letters to the Editor
337
Altered atherosclerotic-related gene expression signature in circulating mononuclear leukocytes from hypercholesterolemic patients with low HDL cholesterol levels D. de Gonzalo-Calvo a, V. Llorente-Cortés a,⁎, J. Orbe b, J.A. Páramo b, L. Badimon a a b
Cardiovascular Research Center, CSIC-ICCC, IIB-Sant Pau, Barcelona, Spain Laboratory of Atherothrombosis, Division of Cardiovascular Sciences, CIMA, Av. Pio XII, 55, 31008 Pamplona, Navarra, Spain
a r t i c l e
i n f o
Article history: Received 5 March 2014 Accepted 9 March 2014 Available online 16 March 2014 Keywords: Atherosclerosis CD36 CD40 Hypercholesterolemia High-density lipoprotein cholesterol Mononuclear leukocytes
HDL exerts atheroprotective effects through modulation of the immune system physiology [1,2]. An abnormal function of immune cells including mononuclear leukocytes has been associated with the pathogenesis of cardiovascular disease (CVD) and their complications [3,4]. Nevertheless, the impact of blood HDL-C levels on leukocyte functionality in populations at high cardiovascular risk has not been previously explored. An additional approach seems to be necessary. The purpose of the current investigation was to compare Peripheral Blood Mononuclear Cells (PBMCs) atherosclerotic-related gene expression in hypercholesterolemic patients with optimal and low HDL-C levels. A total of 75 subjects over 30 years old were recruited at the time of attending the outpatient clinic for vascular risk assessment at the University Clinic of Navarra, Spain (Table 1S, Supplementary content). Attending to current cardiovascular guidelines, hypercholesterolemia was defined as: total cholesterol ≥ 200 mg/dL and/or LDL-C ≥ 130 mg/dL. Low HDL-C levels were defined as HDL-C ≤ 40 mg/dL for males and HDLC ≤ 50 mg/dL for females. Exclusion criteria were the presence of severely impaired renal function, chronic inflammatory conditions, and administration of anti-inflammatory, antithrombotic or hormonal therapy in the previous 2 weeks. Patients with significant acute infection, according to clinical criteria applied by the attending physician, were also excluded. The institutional ethics committee approved this study, and written informed consent was obtained from all patients. PBMCs were isolated from the blood using the Ficoll separation method. Total RNA was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany) as specified by the manufacturer's instructions. Gene
expression analyses were performed at mRNA level by TaqMan LowDensity Array (TLDA). Pre-designed TaqMan probe and primer sets for target genes were chosen from an on-line catalog (Applied Biosystems, Foster City, CA, USA). Genes were chosen based on literature review and previous data from our group (Table 2S, Supplementary content). Multiple logistic regression analyses were performed to explore the associations between HDL-C levels and PBMC gene expression in hypercholesterolemic patients. Previous investigations have demonstrated that TG- and cholesterol-rich lipoproteins other than HDL have a regulatory effect on immune cell gene expression [5]. To determine the influence of these lipid parameters on the associations between HDL-C levels and PBMC gene expression, we made an analysis in duplicate adjusting by non-HDL-C or TG levels. The logistic regression models showed an inverse and close association between HDL-C levels and PBMC CD36, CD40 and IL4 gene expression in models adjusted by age, sex and non-HDL-C levels and PBMC CD36 and CD40 gene expression in models adjusted by age, sex and TG levels (Table 3S, Supplementary content). Since the model adjusted by age and sex showed a significant association between IL4 expression and low HDL-C level in hypercholesterolemia, these data suggest that this relation depends, at least in part, on TG levels. Previous studies suggested a link between statins and HDL-C levels [6]. We thus included this covariable in the logistic regression models. For all non-HDL-C and TG models, the associations between gene expression and HDL-C levels remained unchanged including statin treatment (Table 3S, Supplementary content). Importantly, many investigations have reported an altered gene expression of circulating leukocytes in relation with conditions linked to CVD [7,8]. Therefore, we explored whether the association between low HDL-C levels and PBMC gene expression in hypercholesterolemic patients was influenced by subclinical femoral atherosclerosis, hypertension or type 2 diabetes. Our results show that the relation between HDL-C and CD36/CD40 mRNA levels remains statistically significant even after adjusting by age, sex, non-HDL-C or TG levels, statin intake and prevalence of subclinical femoral atherosclerosis, hypertension or type 2 diabetes (Tables 1 and 2). In contrast, the association with IL4 expression, observed in models adjusted by nonHDL-C levels and hypertension or type 2 diabetes, was lost in a model adjusted by subclinical femoral atherosclerosis (Tables 1 and 2). These results support previous unpublished data from our group showing an inverse association between PBMC IL4 mRNA levels and the presence
Table 1 Association between expression levels of each analyzed gene and low circulating HDL-C in hypercholesterolemia depending on non-HDL cholesterol level. Model 1
Model 2
Model 3
Genes
OR (95% CI)
p-Value
OR (95% CI)
p-Value
OR (95% CI)
p-Value
CD36 CD40 IL4
1.051 (1.006–1.098) 1.198 (1.002–1.431) 1.194 (0.991–1.439)
0.025a 0.047a 0.062
1.048 (1.007–1.090) 1.166 (1.008–1.348) 1.259 (1.004–1.580)
0.022a 0.038a 0.046a
1.052 (1.009–1.098) 1.163 (1.010–1.339) 1.247 (1.009–1.542)
0.017a 0.036a 0.041a
Model 1: Adjusted for age, sex, non-HDL cholesterol level, statin intake and prevalence of subclinical femoral atherosclerosis; Model 2: Adjusted for age, sex, non-HDL cholesterol level, statin intake and hypertension prevalence; Model 3: Adjusted for age, sex, non-HDL cholesterol level, statin intake and type 2 diabetes prevalence. OR: Odds Ratio, CI: Confidence Interval. a Statistically significant.
⁎ Corresponding author at: Cardiovascular Research Center, CSIC-ICCC, Hospital de la Santa Creu i Sant Pau, Sant Antoni Mª Claret, 167, 08025 Barcelona, Spain. Tel.: + 34 935565888; fax: + 34 935565559. E-mail address: [email protected] (V. Llorente-Cortés).
338
Letters to the Editor
Table 2 Association between expression levels of each analyzed gene and low circulating HDL-C in hypercholesterolemia depending on triglyceride level. Model 4
Model 5
Model 6
Genes
OR (95% CI)
p-Value
OR (95% CI)
p-Value
OR (95% CI)
p-Value
CD36 CD40 IL4
1.054 (1.005–1.104) 1.227 (1.013–1.486) 1.169 (0.972–1.406)
0.029a 0.036a 0.097
1.048 (1.005–1.094) 1.181 (1.006–1.387) 1.226 (0.984–1.527)
0.029a 0.043a 0.070
1.052 (1.007–1.099) 1.183 (1.009–1.387) 1.226 (0.992–1.514)
0.023a 0.039a 0.059
Model 4: Adjusted for age, sex, TG level, statin intake and prevalence of subclinical femoral atherosclerosis; Model 5: Adjusted for age, sex, TG level, statin intake and hypertension prevalence; Model 6: Adjusted for age, sex, TG level, statin intake and type 2 diabetes prevalence. OR: Odds Ratio, CI: Confidence Interval. a Statistically significant.
of femoral atheroma plaque. In summary, our results show an inverse, stable and robust association between plasma HDL-C levels and PBMC CD36/CD40 mRNA levels in hypercholesterolemic patients. In order to identify the biological mechanisms, pathways and functions of identified genes, functional in silico analysis was developed. Genes associated with low circulating HDL-C levels in hypercholesterolemia (p b 0.10) for models adjusted by age, sex, non HDL-C levels or TG levels, statin intake and prevalence of subclinical femoral atherosclerosis, hypertension or type 2 diabetes were subjected to Ingenuity Pathway Analysis (Ingenuity Systems, www.ingenuity.com). The pathways associated with the genes considered in the functional analyses were related to immune cell activation (Table 3). Network analysis, together with CD36/CD40 results, points to an altered inflammatory and immune phenotype of circulating mononuclear leukocytes from hypercholesterolemic patients with low HDL-C levels. Owing to the hypercholesterolemic background of the study population, current results are unique and not comparable with investigations in subjects with normal cholesterol levels. However, our results are in line with previous works focused on other study populations. An inverse correlation between inflammation and lipid metabolism gene expression and HDL-C concentration was observed in circulating leukocytes from healthy subjects [9]. Monocytes and monocyte-derived macrophages from subjects with low HDL-C levels are in an activated pro-inflammatory state compared to their age- and sex-matched controls [10]. A small but consistent inverse association between lipoprotein transport and lipid metabolism gene expression and HDL-C concentration was also described in the Framingham Heart Study [11]. In addition, although the association between gene expression and HDL-C levels in hypercholesterolemia reported here does not necessarily imply causality, the described biological functions of HDL in vitro and in vivo [12,13], as well as the benefits of administration or overexpression of HDL-related proteins [14], support the immunomodulatory role of HDL on circulating mononuclear leukocytes. Our study provides important data on the relationship between plasma lipid levels, atherogenic risk factors and leukocyte functionality, one of the main effectors of the atherogenic process. We conclude that low blood HDL-C levels in patients with hypercholesterolemia, without differences in other cardiovascular risk factors, are intimately associated with an altered immune cell physiology, even after adjusting for relevant confounding factors. Our results point to a dysregulated functional state of circulating mononuclear leukocytes of subjects at cardiovascular risk. These findings are significant for understanding the molecular basis of pro-atherogenic lipid profiles and investigating healthy intervention strategies for the amelioration or mitigation of its consequences. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ijcard.2014.03.042.
0167-5273/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2014.03.042
Table 3 Functional and pathway analysis. Top scoring networks for each model Model 1
Model 2 Model 3 Model 4
Model 5 Model 6
Cell-to-cell signaling and interaction, hematological system development and function, cellular development Cellular development, cellular growth and proliferation, hematological system development and function Cellular development, cellular growth and proliferation, hematological system development and function Cell-to-cell signaling and interaction, hematological system development and function, cellular development Infectious disease, cell-to-cell signaling and interaction, hematological system development and function Cell-to-cell signaling and interaction, infectious disease, cellular movement
Common canonical pathways for all the models T helper cell differentiation Communication between innate and adaptive immune cells
References [1] Khera AV, Cuchel M, de la Llera-Moya M, et al. Cholesterol efflux capacity, highdensity lipoprotein function, and atherosclerosis. N Engl J Med 2011;364:127–35. [2] Norata GD, Pirillo A, Ammirati E, Catapano AL. Emerging role of high density lipoproteins as a player in the immune system. Atherosclerosis 2012;220:11–21. [3] Sala F, Cutuli L, Grigore L, et al. Prevalence of classical CD14++/CD16− but not of intermediate CD14++/CD16 + monocytes in hypoalphalipoproteinemia. Int J Cardiol 2013;168:2886–9. [4] Swirski FK, Nahrendorf M. Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure. Science 2013;339:161–6. [5] Alipour A, van Oostrom AJ, Izraeljan A, et al. Leukocyte activation by triglyceriderich lipoproteins. Arterioscler Thromb Vasc Biol 2008;28:792–7. [6] Ansell BJ. Targeting the anti-inflammatory effects of high-density lipoprotein. Am J Cardiol 2007;100:n3–9. [7] Moore DF, Li H, Jeffries N, et al. Using peripheral blood mononuclear cells to determine a gene expression profile of acute ischemic stroke: a pilot investigation. Circulation 2005;111:212–21. [8] Timofeeva AV, Goryunova LE, Khaspekov GL, et al. Altered gene expression pattern in peripheral blood leukocytes from patients with arterial hypertension. Ann N Y Acad Sci 2006;1091:319–35. [9] Ma J, Dempsey AA, Stamatiou D, Marshall KW, Liew CC. Identifying leukocyte gene expression patterns associated with plasma lipid levels in human subjects. Atherosclerosis 2007;191:63–72. [10] Sarov-Blat L, Kiss RS, Haidar B, et al. Predominance of a proinflammatory phenotype in monocyte-derived macrophages from subjects with low plasma HDL-cholesterol. Arterioscler Thromb Vasc Biol 2007;27:1115–22. [11] Joehanes R, Johnson AD, Barb JJ, et al. Gene expression analysis of whole blood, peripheral blood mononuclear cells, and lymphoblastoid cell lines from the Framingham Heart Study. Physiol Genomics 2012;44:59–75. [12] Murphy AJ, Woollard KJ, Hoang A, et al. High-density lipoprotein reduces the human monocyte inflammatory response. Arterioscler Thromb Vasc Biol 2008;28:2071–7. [13] Yvan-Charvet L, Pagler T, Gautier EL, et al. ATP-binding cassette transporters and HDL suppress hematopoietic stem cell proliferation. Science 2010;328:1689–93. [14] Ibañez B, Vilahur G, Cimmino G, et al. Rapid change in plaque size, composition, and molecular footprint after recombinant apolipoprotein A-I Milano (ETC-216) administration: magnetic resonance imaging study in an experimental model of atherosclerosis. J Am Coll Cardiol 2008;51:1104–9.
337
Altered atherosclerotic-related gene expression signature in circulating mononuclear leukocytes from hypercholesterolemic patients with low HDL cholesterol levels D. de Gonzalo-Calvo a, V. Llorente-Cortés a,⁎, J. Orbe b, J.A. Páramo b, L. Badimon a a b
Cardiovascular Research Center, CSIC-ICCC, IIB-Sant Pau, Barcelona, Spain Laboratory of Atherothrombosis, Division of Cardiovascular Sciences, CIMA, Av. Pio XII, 55, 31008 Pamplona, Navarra, Spain
a r t i c l e
i n f o
Article history: Received 5 March 2014 Accepted 9 March 2014 Available online 16 March 2014 Keywords: Atherosclerosis CD36 CD40 Hypercholesterolemia High-density lipoprotein cholesterol Mononuclear leukocytes
HDL exerts atheroprotective effects through modulation of the immune system physiology [1,2]. An abnormal function of immune cells including mononuclear leukocytes has been associated with the pathogenesis of cardiovascular disease (CVD) and their complications [3,4]. Nevertheless, the impact of blood HDL-C levels on leukocyte functionality in populations at high cardiovascular risk has not been previously explored. An additional approach seems to be necessary. The purpose of the current investigation was to compare Peripheral Blood Mononuclear Cells (PBMCs) atherosclerotic-related gene expression in hypercholesterolemic patients with optimal and low HDL-C levels. A total of 75 subjects over 30 years old were recruited at the time of attending the outpatient clinic for vascular risk assessment at the University Clinic of Navarra, Spain (Table 1S, Supplementary content). Attending to current cardiovascular guidelines, hypercholesterolemia was defined as: total cholesterol ≥ 200 mg/dL and/or LDL-C ≥ 130 mg/dL. Low HDL-C levels were defined as HDL-C ≤ 40 mg/dL for males and HDLC ≤ 50 mg/dL for females. Exclusion criteria were the presence of severely impaired renal function, chronic inflammatory conditions, and administration of anti-inflammatory, antithrombotic or hormonal therapy in the previous 2 weeks. Patients with significant acute infection, according to clinical criteria applied by the attending physician, were also excluded. The institutional ethics committee approved this study, and written informed consent was obtained from all patients. PBMCs were isolated from the blood using the Ficoll separation method. Total RNA was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany) as specified by the manufacturer's instructions. Gene
expression analyses were performed at mRNA level by TaqMan LowDensity Array (TLDA). Pre-designed TaqMan probe and primer sets for target genes were chosen from an on-line catalog (Applied Biosystems, Foster City, CA, USA). Genes were chosen based on literature review and previous data from our group (Table 2S, Supplementary content). Multiple logistic regression analyses were performed to explore the associations between HDL-C levels and PBMC gene expression in hypercholesterolemic patients. Previous investigations have demonstrated that TG- and cholesterol-rich lipoproteins other than HDL have a regulatory effect on immune cell gene expression [5]. To determine the influence of these lipid parameters on the associations between HDL-C levels and PBMC gene expression, we made an analysis in duplicate adjusting by non-HDL-C or TG levels. The logistic regression models showed an inverse and close association between HDL-C levels and PBMC CD36, CD40 and IL4 gene expression in models adjusted by age, sex and non-HDL-C levels and PBMC CD36 and CD40 gene expression in models adjusted by age, sex and TG levels (Table 3S, Supplementary content). Since the model adjusted by age and sex showed a significant association between IL4 expression and low HDL-C level in hypercholesterolemia, these data suggest that this relation depends, at least in part, on TG levels. Previous studies suggested a link between statins and HDL-C levels [6]. We thus included this covariable in the logistic regression models. For all non-HDL-C and TG models, the associations between gene expression and HDL-C levels remained unchanged including statin treatment (Table 3S, Supplementary content). Importantly, many investigations have reported an altered gene expression of circulating leukocytes in relation with conditions linked to CVD [7,8]. Therefore, we explored whether the association between low HDL-C levels and PBMC gene expression in hypercholesterolemic patients was influenced by subclinical femoral atherosclerosis, hypertension or type 2 diabetes. Our results show that the relation between HDL-C and CD36/CD40 mRNA levels remains statistically significant even after adjusting by age, sex, non-HDL-C or TG levels, statin intake and prevalence of subclinical femoral atherosclerosis, hypertension or type 2 diabetes (Tables 1 and 2). In contrast, the association with IL4 expression, observed in models adjusted by nonHDL-C levels and hypertension or type 2 diabetes, was lost in a model adjusted by subclinical femoral atherosclerosis (Tables 1 and 2). These results support previous unpublished data from our group showing an inverse association between PBMC IL4 mRNA levels and the presence
Table 1 Association between expression levels of each analyzed gene and low circulating HDL-C in hypercholesterolemia depending on non-HDL cholesterol level. Model 1
Model 2
Model 3
Genes
OR (95% CI)
p-Value
OR (95% CI)
p-Value
OR (95% CI)
p-Value
CD36 CD40 IL4
1.051 (1.006–1.098) 1.198 (1.002–1.431) 1.194 (0.991–1.439)
0.025a 0.047a 0.062
1.048 (1.007–1.090) 1.166 (1.008–1.348) 1.259 (1.004–1.580)
0.022a 0.038a 0.046a
1.052 (1.009–1.098) 1.163 (1.010–1.339) 1.247 (1.009–1.542)
0.017a 0.036a 0.041a
Model 1: Adjusted for age, sex, non-HDL cholesterol level, statin intake and prevalence of subclinical femoral atherosclerosis; Model 2: Adjusted for age, sex, non-HDL cholesterol level, statin intake and hypertension prevalence; Model 3: Adjusted for age, sex, non-HDL cholesterol level, statin intake and type 2 diabetes prevalence. OR: Odds Ratio, CI: Confidence Interval. a Statistically significant.
⁎ Corresponding author at: Cardiovascular Research Center, CSIC-ICCC, Hospital de la Santa Creu i Sant Pau, Sant Antoni Mª Claret, 167, 08025 Barcelona, Spain. Tel.: + 34 935565888; fax: + 34 935565559. E-mail address: [email protected] (V. Llorente-Cortés).
338
Letters to the Editor
Table 2 Association between expression levels of each analyzed gene and low circulating HDL-C in hypercholesterolemia depending on triglyceride level. Model 4
Model 5
Model 6
Genes
OR (95% CI)
p-Value
OR (95% CI)
p-Value
OR (95% CI)
p-Value
CD36 CD40 IL4
1.054 (1.005–1.104) 1.227 (1.013–1.486) 1.169 (0.972–1.406)
0.029a 0.036a 0.097
1.048 (1.005–1.094) 1.181 (1.006–1.387) 1.226 (0.984–1.527)
0.029a 0.043a 0.070
1.052 (1.007–1.099) 1.183 (1.009–1.387) 1.226 (0.992–1.514)
0.023a 0.039a 0.059
Model 4: Adjusted for age, sex, TG level, statin intake and prevalence of subclinical femoral atherosclerosis; Model 5: Adjusted for age, sex, TG level, statin intake and hypertension prevalence; Model 6: Adjusted for age, sex, TG level, statin intake and type 2 diabetes prevalence. OR: Odds Ratio, CI: Confidence Interval. a Statistically significant.
of femoral atheroma plaque. In summary, our results show an inverse, stable and robust association between plasma HDL-C levels and PBMC CD36/CD40 mRNA levels in hypercholesterolemic patients. In order to identify the biological mechanisms, pathways and functions of identified genes, functional in silico analysis was developed. Genes associated with low circulating HDL-C levels in hypercholesterolemia (p b 0.10) for models adjusted by age, sex, non HDL-C levels or TG levels, statin intake and prevalence of subclinical femoral atherosclerosis, hypertension or type 2 diabetes were subjected to Ingenuity Pathway Analysis (Ingenuity Systems, www.ingenuity.com). The pathways associated with the genes considered in the functional analyses were related to immune cell activation (Table 3). Network analysis, together with CD36/CD40 results, points to an altered inflammatory and immune phenotype of circulating mononuclear leukocytes from hypercholesterolemic patients with low HDL-C levels. Owing to the hypercholesterolemic background of the study population, current results are unique and not comparable with investigations in subjects with normal cholesterol levels. However, our results are in line with previous works focused on other study populations. An inverse correlation between inflammation and lipid metabolism gene expression and HDL-C concentration was observed in circulating leukocytes from healthy subjects [9]. Monocytes and monocyte-derived macrophages from subjects with low HDL-C levels are in an activated pro-inflammatory state compared to their age- and sex-matched controls [10]. A small but consistent inverse association between lipoprotein transport and lipid metabolism gene expression and HDL-C concentration was also described in the Framingham Heart Study [11]. In addition, although the association between gene expression and HDL-C levels in hypercholesterolemia reported here does not necessarily imply causality, the described biological functions of HDL in vitro and in vivo [12,13], as well as the benefits of administration or overexpression of HDL-related proteins [14], support the immunomodulatory role of HDL on circulating mononuclear leukocytes. Our study provides important data on the relationship between plasma lipid levels, atherogenic risk factors and leukocyte functionality, one of the main effectors of the atherogenic process. We conclude that low blood HDL-C levels in patients with hypercholesterolemia, without differences in other cardiovascular risk factors, are intimately associated with an altered immune cell physiology, even after adjusting for relevant confounding factors. Our results point to a dysregulated functional state of circulating mononuclear leukocytes of subjects at cardiovascular risk. These findings are significant for understanding the molecular basis of pro-atherogenic lipid profiles and investigating healthy intervention strategies for the amelioration or mitigation of its consequences. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ijcard.2014.03.042.
0167-5273/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2014.03.042
Table 3 Functional and pathway analysis. Top scoring networks for each model Model 1
Model 2 Model 3 Model 4
Model 5 Model 6
Cell-to-cell signaling and interaction, hematological system development and function, cellular development Cellular development, cellular growth and proliferation, hematological system development and function Cellular development, cellular growth and proliferation, hematological system development and function Cell-to-cell signaling and interaction, hematological system development and function, cellular development Infectious disease, cell-to-cell signaling and interaction, hematological system development and function Cell-to-cell signaling and interaction, infectious disease, cellular movement
Common canonical pathways for all the models T helper cell differentiation Communication between innate and adaptive immune cells
References [1] Khera AV, Cuchel M, de la Llera-Moya M, et al. Cholesterol efflux capacity, highdensity lipoprotein function, and atherosclerosis. N Engl J Med 2011;364:127–35. [2] Norata GD, Pirillo A, Ammirati E, Catapano AL. Emerging role of high density lipoproteins as a player in the immune system. Atherosclerosis 2012;220:11–21. [3] Sala F, Cutuli L, Grigore L, et al. Prevalence of classical CD14++/CD16− but not of intermediate CD14++/CD16 + monocytes in hypoalphalipoproteinemia. Int J Cardiol 2013;168:2886–9. [4] Swirski FK, Nahrendorf M. Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure. Science 2013;339:161–6. [5] Alipour A, van Oostrom AJ, Izraeljan A, et al. Leukocyte activation by triglyceriderich lipoproteins. Arterioscler Thromb Vasc Biol 2008;28:792–7. [6] Ansell BJ. Targeting the anti-inflammatory effects of high-density lipoprotein. Am J Cardiol 2007;100:n3–9. [7] Moore DF, Li H, Jeffries N, et al. Using peripheral blood mononuclear cells to determine a gene expression profile of acute ischemic stroke: a pilot investigation. Circulation 2005;111:212–21. [8] Timofeeva AV, Goryunova LE, Khaspekov GL, et al. Altered gene expression pattern in peripheral blood leukocytes from patients with arterial hypertension. Ann N Y Acad Sci 2006;1091:319–35. [9] Ma J, Dempsey AA, Stamatiou D, Marshall KW, Liew CC. Identifying leukocyte gene expression patterns associated with plasma lipid levels in human subjects. Atherosclerosis 2007;191:63–72. [10] Sarov-Blat L, Kiss RS, Haidar B, et al. Predominance of a proinflammatory phenotype in monocyte-derived macrophages from subjects with low plasma HDL-cholesterol. Arterioscler Thromb Vasc Biol 2007;27:1115–22. [11] Joehanes R, Johnson AD, Barb JJ, et al. Gene expression analysis of whole blood, peripheral blood mononuclear cells, and lymphoblastoid cell lines from the Framingham Heart Study. Physiol Genomics 2012;44:59–75. [12] Murphy AJ, Woollard KJ, Hoang A, et al. High-density lipoprotein reduces the human monocyte inflammatory response. Arterioscler Thromb Vasc Biol 2008;28:2071–7. [13] Yvan-Charvet L, Pagler T, Gautier EL, et al. ATP-binding cassette transporters and HDL suppress hematopoietic stem cell proliferation. Science 2010;328:1689–93. [14] Ibañez B, Vilahur G, Cimmino G, et al. Rapid change in plaque size, composition, and molecular footprint after recombinant apolipoprotein A-I Milano (ETC-216) administration: magnetic resonance imaging study in an experimental model of atherosclerosis. J Am Coll Cardiol 2008;51:1104–9.
