Hindawi Publishing Corporation International Journal of Endocrinology Volume 2014, Article ID 703696, 6 pages http://dx.doi.org/10.1155/2014/703696
Research Article Plasma Antimicrobial Peptide LL-37 Level Is Inversely Associated with HDL Cholesterol Level in Patients with Type 2 Diabetes Mellitus Shu Meguro,1 Masuomi Tomita,2 Takeshi Katsuki,2 Kiyoe Kato,2 Henpiru Oh,3 Akira Ainai,4 Ryo Ito,4 Toshihide Kawai,1 Hiroshi Itoh,1 and Hideki Hasegawa4 1
Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan 2 Department of Internal Medicine, Saiseikai Central Hospital, 1-4-17 Mita, Minato-ku, Tokyo 108-0073, Japan 3 Minami-Aoyama Home Clinic, 7-5-2 Minami Aoyama, Minato-ku, Tokyo 107-0062, Japan 4 Department of Pathology, National Institute of Infectious Disease, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-0052, Japan Correspondence should be addressed to Shu Meguro; [email protected] Received 7 January 2014; Revised 17 February 2014; Accepted 17 February 2014; Published 26 March 2014 Academic Editor: Faustino R. P´erez-L´opez Copyright © 2014 Shu Meguro et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction. Relation between atherosclerosis and innate immunity has attracted attention. As the antimicrobial peptide, LL-37, could have an important role in atherosclerosis, we supposed that there could be a meaningful association of plasma LL-37 level with risk factors for cardiovascular disease in subjects with type 2 diabetes mellitus. Materials and Methods. We evaluated plasma LL-37 level and other clinical markers in Japanese subjects with type 2 diabetes mellitus (𝑛 = 133, 115 men and 18 women; age 64.7 ± 11.5 years; HbA1c 8.1 ± 1.6%). Plasma level of LL-37 was measured by ELISA. Results. Mean plasma LL-37 level was 71.2 ± 22.3 ng/mL. Plasma LL-37 level showed significant correlations with HDL cholesterol (𝑟 = −0.450, 𝑃 < 0.01), triglyceride (𝑟 = 0.445, 𝑃 < 0.01), and high sensitive C-reactive protein (𝑟 = 0.316, 𝑃 < 0.01) but no significant correlation with age, body mass index, HbA1c, estimated glomerular filtration rate, 25-hydroxyvitamin D, or vitamin D binding protein. Multiple linear regression analysis showed significant correlations of plasma LL-37 level with HDL cholesterol (𝛽 = −0.411, 𝑃 < 0.01) and high sensitive C-reactive protein (𝛽 = 0.193, 𝑃 < 0.05). Conclusion. Plasma LL-37 level was positively correlated with inflammatory markers and negatively correlated with HDL cholesterol in patients with type 2 diabetes mellitus.
1. Introduction Antimicrobial peptides are peptides that can kill viruses, fungi, bacteria, and other microbes [1]. They are produced in most multicellular organisms and expressed on epithelial surfaces and within circulating white cells, constitutively or in response to stimuli such as tissue injury, through interleukin-1 and other cytokines or microbial components such as lipopolysaccharide (LPS). Among such peptides, which play important roles in the innate immune system, LL37, also known as cathelicidin, is one of the most studied. In addition to its anti-infective activities, LL-37 stimulates local angiogenesis, acts synergistically with the epidermal growth factor receptor to promote epithelial growth, and attracts
monocytes and neutrophils through formyl peptide receptors on these cells. In this way the peptide helps orchestrate the inflammatory process [2–4]. Innate immunity is important for primary defense not only against antimicrobial infections, but also against atherosclerosis via nonspecific inflammatory processes [5]. Involvement of LL-37 in the atherosclerotic process was suggested by previous reports. Increased transcription of LL37 was demonstrated in human atherosclerotic plaques [6], and the presence of LL-37 in human atherosclerotic lesions obtained at autopsy was shown by immunohistochemical study and it induced death of vascular smooth muscle cells [7]. A recent study in a rodent model showed that cathelicidin-related antimicrobial peptide (CRAMP: mouse
2 cathelicidin equivalent to LL-37 in human) and Apo E double knockout mice exhibited reduced atherosclerotic lesions compared to Apo E knockout mice [8]. Type 2 diabetes mellitus is a worldwide epidemic and increases macrovascular and microvascular risk [9, 10]. Residual risk for cardiovascular disease still exists in spite of widely adopted statin treatment. As one of the explanations for the residual risk of atherosclerosis, chronic subclinical inflammation is a candidate [11]. We therefore suppose that analysis of plasma LL-37 level, which is an important player in the innate immune system, in subjects with type 2 diabetes mellitus may be meaningful in regard to its association with risk factors for cardiovascular disease.
2. Materials and Methods The present study was conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from each subject after a full explanation of the purpose, nature, and risk of all procedures used. The protocol was approved by the ethical review committees of Saiseikai Central Hospital, Tokyo, Japan. We originally aimed this study to observe the association between pandemic influenza virus A/H1N1pdm09 infection and plasma 25-hydroxyvitamin D [25(OH)D] and/or plasma LL-37 level in subjects with type 2 diabetes mellitus who were considered as high risk for influenza infection. Subjects were recruited in routine outpatient clinics for their diabetes managements. Patients with hematological disorders, liver cirrhosis, malignant disease, or active gastrointestinal disease were not enrolled in this study. Patients with active inflammatory or infectious disease were excluded by their comorbidities, clinical symptoms, and body temperatures. As plasma 25(OH)D level is influenced by their renal function, we actively enrolled subjects with nephropathy to obtain equivalent numbers of subjects with each clinical stage of diabetic nephropathy according to their laboratory data at the consent to evaluate the effect of advanced stages of diabetic nephropathy. As a result, we measured plasma LL37, 25(OH)D, vitamin D binding protein (VDBP), and other clinical markers in Japanese subjects with type 2 diabetes mellitus (𝑛 = 135, 117 men and 18 women; age 64.7 ± 11.5 years; HbA1c 8.1 ± 1.6%) who attended the outpatient clinic of Saiseikai Central Hospital between September 2009 and February 2010. After obtaining written informed consent, blood samples in an overnight fasting state were drawn at the next visit to the usual outpatient clinic. Routine blood analysis was performed by the hospital laboratory immediately after blood sampling. After centrifugation, a part of the plasma was preserved at −80∘ C until further analysis. Assays of fasting plasma glucose (FPG), total cholesterol, HDL cholesterol, triglyceride, serum creatinine level, and several other biochemical parameters were performed with autoanalyzers. HbA1c level was determined by high-performance liquid chromatography (Arkray Inc., Kyoto, Japan) according to the recommended method by the Japan Diabetes Society at that time and converted to the National Glycohemoglobin Standardization
International Journal of Endocrinology Program (NGSP) value [12]. Microalbuminuria was defined as an albumin creatinine ratio (ACR) of 30 to 300 mg/g Cr and macroalbuminuria as ACR of more than 300 mg/g Cr in spot urinalysis. Estimated glomerular filtration rate (eGFR) (mL/min/1.73 m2 ) was calculated using the equation provided by the Japanese Society of Nephrology as 194 × Cr−1.094 × Age−0.287 (with further multiplication by 0.739 for female subjects) [13]. Plasma levels of LL-37, 25(OH)D, and VDBP were measured by ELISA using a human LL-37 ELISA kit (Hycult Biotechnology, Uden, Netherlands), 25(OH)vitamin D direct ELISA kit (Immundiagnostik AG, Bensheim, Germany), and vitamin D binding protein ELISA kit (Immundiagnostik AG, Bensheim, Germany), respectively, according to the manufacturers’ manuals, at the laboratory of the National Institute of Infectious Diseases. Hypertension was defined as systolic blood pressure >140 mmHg, diastolic blood pressure >90 mmHg, or the prescription of antihypertensive medication. Dyslipidemia was defined as LDL cholesterol >3.63 mmol/L, triglyceride >1.72 mmol/L, HDL cholesterol
Research Article Plasma Antimicrobial Peptide LL-37 Level Is Inversely Associated with HDL Cholesterol Level in Patients with Type 2 Diabetes Mellitus Shu Meguro,1 Masuomi Tomita,2 Takeshi Katsuki,2 Kiyoe Kato,2 Henpiru Oh,3 Akira Ainai,4 Ryo Ito,4 Toshihide Kawai,1 Hiroshi Itoh,1 and Hideki Hasegawa4 1
Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan 2 Department of Internal Medicine, Saiseikai Central Hospital, 1-4-17 Mita, Minato-ku, Tokyo 108-0073, Japan 3 Minami-Aoyama Home Clinic, 7-5-2 Minami Aoyama, Minato-ku, Tokyo 107-0062, Japan 4 Department of Pathology, National Institute of Infectious Disease, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-0052, Japan Correspondence should be addressed to Shu Meguro; [email protected] Received 7 January 2014; Revised 17 February 2014; Accepted 17 February 2014; Published 26 March 2014 Academic Editor: Faustino R. P´erez-L´opez Copyright © 2014 Shu Meguro et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction. Relation between atherosclerosis and innate immunity has attracted attention. As the antimicrobial peptide, LL-37, could have an important role in atherosclerosis, we supposed that there could be a meaningful association of plasma LL-37 level with risk factors for cardiovascular disease in subjects with type 2 diabetes mellitus. Materials and Methods. We evaluated plasma LL-37 level and other clinical markers in Japanese subjects with type 2 diabetes mellitus (𝑛 = 133, 115 men and 18 women; age 64.7 ± 11.5 years; HbA1c 8.1 ± 1.6%). Plasma level of LL-37 was measured by ELISA. Results. Mean plasma LL-37 level was 71.2 ± 22.3 ng/mL. Plasma LL-37 level showed significant correlations with HDL cholesterol (𝑟 = −0.450, 𝑃 < 0.01), triglyceride (𝑟 = 0.445, 𝑃 < 0.01), and high sensitive C-reactive protein (𝑟 = 0.316, 𝑃 < 0.01) but no significant correlation with age, body mass index, HbA1c, estimated glomerular filtration rate, 25-hydroxyvitamin D, or vitamin D binding protein. Multiple linear regression analysis showed significant correlations of plasma LL-37 level with HDL cholesterol (𝛽 = −0.411, 𝑃 < 0.01) and high sensitive C-reactive protein (𝛽 = 0.193, 𝑃 < 0.05). Conclusion. Plasma LL-37 level was positively correlated with inflammatory markers and negatively correlated with HDL cholesterol in patients with type 2 diabetes mellitus.
1. Introduction Antimicrobial peptides are peptides that can kill viruses, fungi, bacteria, and other microbes [1]. They are produced in most multicellular organisms and expressed on epithelial surfaces and within circulating white cells, constitutively or in response to stimuli such as tissue injury, through interleukin-1 and other cytokines or microbial components such as lipopolysaccharide (LPS). Among such peptides, which play important roles in the innate immune system, LL37, also known as cathelicidin, is one of the most studied. In addition to its anti-infective activities, LL-37 stimulates local angiogenesis, acts synergistically with the epidermal growth factor receptor to promote epithelial growth, and attracts
monocytes and neutrophils through formyl peptide receptors on these cells. In this way the peptide helps orchestrate the inflammatory process [2–4]. Innate immunity is important for primary defense not only against antimicrobial infections, but also against atherosclerosis via nonspecific inflammatory processes [5]. Involvement of LL-37 in the atherosclerotic process was suggested by previous reports. Increased transcription of LL37 was demonstrated in human atherosclerotic plaques [6], and the presence of LL-37 in human atherosclerotic lesions obtained at autopsy was shown by immunohistochemical study and it induced death of vascular smooth muscle cells [7]. A recent study in a rodent model showed that cathelicidin-related antimicrobial peptide (CRAMP: mouse
2 cathelicidin equivalent to LL-37 in human) and Apo E double knockout mice exhibited reduced atherosclerotic lesions compared to Apo E knockout mice [8]. Type 2 diabetes mellitus is a worldwide epidemic and increases macrovascular and microvascular risk [9, 10]. Residual risk for cardiovascular disease still exists in spite of widely adopted statin treatment. As one of the explanations for the residual risk of atherosclerosis, chronic subclinical inflammation is a candidate [11]. We therefore suppose that analysis of plasma LL-37 level, which is an important player in the innate immune system, in subjects with type 2 diabetes mellitus may be meaningful in regard to its association with risk factors for cardiovascular disease.
2. Materials and Methods The present study was conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from each subject after a full explanation of the purpose, nature, and risk of all procedures used. The protocol was approved by the ethical review committees of Saiseikai Central Hospital, Tokyo, Japan. We originally aimed this study to observe the association between pandemic influenza virus A/H1N1pdm09 infection and plasma 25-hydroxyvitamin D [25(OH)D] and/or plasma LL-37 level in subjects with type 2 diabetes mellitus who were considered as high risk for influenza infection. Subjects were recruited in routine outpatient clinics for their diabetes managements. Patients with hematological disorders, liver cirrhosis, malignant disease, or active gastrointestinal disease were not enrolled in this study. Patients with active inflammatory or infectious disease were excluded by their comorbidities, clinical symptoms, and body temperatures. As plasma 25(OH)D level is influenced by their renal function, we actively enrolled subjects with nephropathy to obtain equivalent numbers of subjects with each clinical stage of diabetic nephropathy according to their laboratory data at the consent to evaluate the effect of advanced stages of diabetic nephropathy. As a result, we measured plasma LL37, 25(OH)D, vitamin D binding protein (VDBP), and other clinical markers in Japanese subjects with type 2 diabetes mellitus (𝑛 = 135, 117 men and 18 women; age 64.7 ± 11.5 years; HbA1c 8.1 ± 1.6%) who attended the outpatient clinic of Saiseikai Central Hospital between September 2009 and February 2010. After obtaining written informed consent, blood samples in an overnight fasting state were drawn at the next visit to the usual outpatient clinic. Routine blood analysis was performed by the hospital laboratory immediately after blood sampling. After centrifugation, a part of the plasma was preserved at −80∘ C until further analysis. Assays of fasting plasma glucose (FPG), total cholesterol, HDL cholesterol, triglyceride, serum creatinine level, and several other biochemical parameters were performed with autoanalyzers. HbA1c level was determined by high-performance liquid chromatography (Arkray Inc., Kyoto, Japan) according to the recommended method by the Japan Diabetes Society at that time and converted to the National Glycohemoglobin Standardization
International Journal of Endocrinology Program (NGSP) value [12]. Microalbuminuria was defined as an albumin creatinine ratio (ACR) of 30 to 300 mg/g Cr and macroalbuminuria as ACR of more than 300 mg/g Cr in spot urinalysis. Estimated glomerular filtration rate (eGFR) (mL/min/1.73 m2 ) was calculated using the equation provided by the Japanese Society of Nephrology as 194 × Cr−1.094 × Age−0.287 (with further multiplication by 0.739 for female subjects) [13]. Plasma levels of LL-37, 25(OH)D, and VDBP were measured by ELISA using a human LL-37 ELISA kit (Hycult Biotechnology, Uden, Netherlands), 25(OH)vitamin D direct ELISA kit (Immundiagnostik AG, Bensheim, Germany), and vitamin D binding protein ELISA kit (Immundiagnostik AG, Bensheim, Germany), respectively, according to the manufacturers’ manuals, at the laboratory of the National Institute of Infectious Diseases. Hypertension was defined as systolic blood pressure >140 mmHg, diastolic blood pressure >90 mmHg, or the prescription of antihypertensive medication. Dyslipidemia was defined as LDL cholesterol >3.63 mmol/L, triglyceride >1.72 mmol/L, HDL cholesterol
