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Lipopolysaccharide-binding protein is associated with arterial stiffness in patients with type 2 diabetes: a cross-sectional study

Lipopolysaccharide-binding protein is associated with arterial stiffness in patients with type 2... Background: Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase reactant that mediates immune responses triggered by LPS. Recent evidence indicates the association of circulating LBP levels with obesity, diabetes, and cardiovascular diseases. In this study, we aimed to investigate the relationship between serum LBP levels and arterial stiffness in patients with type 2 diabetes. Methods: A total of 196 patients with type 2 diabetes, including 101 men and 95 women, were enrolled in this cross- sectional study. Fasting serum LBP levels were determined by enzyme-linked immunosorbent assay. Arterial stiffness was assessed by measuring the aortic pulse wave velocity (PWV ). Results: The mean values of serum LBP and aortic PWV were 18.2 μg/mL and 1194 cm/s, respectively. Serum LBP levels were positively correlated with body mass index, triglycerides, high-sensitivity C-reactive protein, and insulin resistance index and were negatively correlated with high-density lipoprotein cholesterol. They were, however, not significantly correlated with aortic PWV in univariate analyses. Multivariate analysis revealed that serum LBP levels were independently and positively associated with aortic PWV (β = 0.135, p = 0.026) after adjusting for age, sex, body mass index, albumin, high-sensitivity C-reactive protein, and other cardiovascular risk factors. Further analyses revealed that the impact of serum LBP levels on aortic PWV was modified by sex, and the association between serum LBP levels and aortic PWV was found to be significant only in men. Conclusions: Serum LBP levels are associated with arterial stiffness, independent of obesity and traditional cardiovas - cular risk factors, especially in men with type 2 diabetes. This study indicates a potential role of the LPS/LBP-induced innate immunity in the development and progression of arterial stiffness in type 2 diabetes. Keywords: Lipopolysaccharide-binding protein, Arterial stiffness, Pulse wave velocity, Type 2 diabetes derived from Gram-negative bacteria is known to play a Background critical role in triggering the immune and inflammatory The association of chronic inflammation with the patho - responses in vascular cells, leading to atherosclerosis [4]. geneses of obesity, diabetes, and atherosclerosis is well LPS-binding protein (LBP), an acute-phase reactant syn- recognized [1]. Accumulating evidence indicates a link thesized mainly in the liver, binds LPS and initiates the between low-grade inflammation produced by common immune response by presenting LPS to cluster of differ - subclinical or chronic infections and the risk of ath- entiation (CD)14, which in turn interacts with toll-like erosclerosis in humans [2, 3]. Lipopolysaccharide (LPS) receptor (TLR)4 on immune cells [5]. Since LBP is syn- thesized and released into circulation in the presence *Correspondence: [email protected] of LPS, with a relatively long half-life, LBP level is con- Department of Metabolism, Endocrinology and Molecular Medicine, Osaka City University Graduate School of Medicine, 1-4-3, Asahi-machi, sidered a surrogate biomarker for the activation of LPS- Abeno-ku, Osaka 545-8585, Japan induced innate immune responses [6–8]. Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 2 of 8 Several studies in humans recently demonstrated that expiration. Blood was drawn after an overnight fast and serum LBP levels are closely associated with obesity, the biochemical parameters were analyzed using a standard metabolic syndrome, and type 2 diabetes [6, 8–13]. Fur- laboratory method at the Central Clinical Laboratory thermore, serum LBP levels were shown to be a predictor of the Osaka City University Hospital [18, 19]. The esti - of prevalent coronary artery disease [7] and cardiovascu- mated glomerular filtration rate (eGFR) was calculated lar mortality [14], independent of established cardiovas- using the Japanese eGFR equation [20]. Immunoreactive cular risk factors and markers of systemic inflammation. insulin levels were measured for subjects not receiving A recent study also showed that serum LBP levels were insulin therapy (n  =  144) by electro-chemiluminescence positively correlated with subclinical atherosclerosis, as immunoassay [Cobas 8000 (502/602), Roche Diagnostics] assessed by carotid intima-media thickness, in healthy at the Central Clinical Laboratory. Homeostasis model individuals, independent of body mass index (BMI) and assessment of insulin resistance (HOMA-R) was calcu- high-sensitivity C-reactive protein (hs-CRP) level [15]. lated according to the following formula: fasting insu- These studies collectively indicate that serum LBP level lin (μU/mL)  ×  fasting glucose (mg/dL)/405 [21]. Frozen may be a biomarker for atherosclerotic cardiovascu- serum samples were shipped to SRL Inc. (Tokyo, Japan) lar disease and implicate a potential role of the innate and hs-CRP concentrations were measured by means immune mechanisms in the progression of atherosclero- of particle-enhanced immunonephelometry with the sis in humans. Behring nephelometer using N Latex CRP mono reagent. To our knowledge, no previous study has investigated Serum levels of LBP were measured using a commer- the relationship between LBP and arterial stiffness, a cial enzyme-linked immunosorbent assay (HK315-02, well-established surrogate marker for cardiovascular dis- HyCult Biotech Inc., Uden, the Netherlands) as per man- eases [16], in human subjects. Moreover, no study has ufacturer’s instructions. The intra- and inter-assay coef - examined the impact of LBP on subclinical atheroscle- ficients of LBP variation were <5 and <10%, respectively. rosis in patients with comorbid cardiovascular risk fac- tors, including diabetes. Therefore, in the present study, Measurement of arterial stiffness we investigated the association between serum LBP lev- Arterial stiffness was evaluated by measuring PWV in els and arterial stiffness by measuring aortic pulse wave the heart-femoral segment using an automatic waveform velocity (PWV) in patients with type 2 diabetes. analyzer (Model BP-203RPE; Omron Colin Co., Ltd., Tokyo, Japan) as described previously [22, 23]. Reproduc- Methods ibility in the measurements of arterial stiffness was con - Study design and subjects firmed in our previous study, in which the coefficients of We consecutively enrolled 196 patients with type 2 diabe- variation were less than 5% for heart-femoral PWV [22]. tes, including 101 men and 95 women, who were admit- ted to the Diabetes Center of the Osaka City University Statistical analysis Hospital for the purpose of glycemic control, education, Data are expressed as the number (%), mean  ±  stand- and/or evaluation of diabetic complications between July ard deviation (SD), or median (interquartile range) as 2013 and December 2015. Type 2 diabetes was diagnosed appropriate. For comparisons between men and women, based on American Diabetes Association criteria [17]. χ -test, unpaired t-test, or Wilcoxon rank-sum test, was Patients with type 1 diabetes or other types of diabetes performed as appropriate. Skewed parameters, such as were not included in the present study. The following HOMA-R, triglycerides, and hs-CRP were logarithmi- patients were not included in this study: those with acute cally transformed before regression analysis. Simple lin- or chronic infection, chronic inflammatory disease, use ear regression analyses were performed to evaluate the of anti-inflammatory drugs including glucocorticoids, relationship between serum LBP levels or aortic PWV or hematologic or malignant disease, and those who and various clinical variables, including cardiovascular underwent recent surgery within 1 month at the time of risk factors. To explore the association between LBP and entry. A smoker was defined as a current smoker in our aortic PWV, multiple linear regression analyses were per- analyses. formed after adjustment for age, sex, BMI, systolic blood pressure, albumin, eGFR, high-density lipoprotein (HDL) Physical and laboratory measurements cholesterol level, log [hs-CRP], serum LBP level, treat- Blood pressure was determined using an automatic ment with statins, treatment with inhibitors of the renin- sphygmomanometer with a conventional cuff after the angiotensin system (RAS inhibitors), treatment with subject had rested for at least 5 min. Waist circumference calcium-channel blockers, and smoking status. To assess was measured to the nearest centimeter at the level of whether the effect of serum LBP levels on aortic PWV is the umbilicus in a standing position at the end of gentle modified by sex, the interaction term between LBP and Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 3 of 8 sex was inserted into the multiple regression model. A p with statins for dyslipidemia, 72 (36.7%) with RAS inhibi- value of <0.20 was considered significant for interaction tors, and 74 (37.8%) with calcium-channel blockers for effects, as has been used in a previous study [24], and a hypertension. There were significantly more male smok - p value of <0.05 was considered significant for all other ers than female smokers. Serum creatinine levels, but analyses. Statistical analyses were performed by using the not eGFR, were significantly different between men and JMP 10 software program (SAS Institute Inc., Cary, NC, women. Parameters of obesity and insulin resistance, USA). such as BMI, waist circumference, and HOMA-R, were not significantly different between men and women. Tri - Results glycerides levels and diastolic blood pressure were higher, Clinical characteristics, serum LBP levels, and aortic PWV and HDL-cholesterol levels were lower in men than in of the subjects women. The clinical characteristics of the total population, as well Mean  ±  SD value for serum LBP levels of all sub- as of men and women, are shown in Table 1. The subjects jects was 18.2  ±  6.3  μg/mL (range 2.1–36.2  μg/ had a mean age of 61 years, median duration of diabetes mL). Mean  ±  SD value for the aortic PWV was of 10  years, and mean BMI of 27.1  kg/m . One hundred 1194  ±  346  cm/s (range 610–2500  cm/s). Serum LBP sixty-eight subjects (85.7%) were receiving any antihy- levels and aortic PWV were not significantly different perglycemic agents. Eighty (40.8%) subjects were treated between men and women. Table 1 Clinical characteristics, serum LBP levels, and aortic PWV in all subjects as well as in men and women with type 2 diabetes All subjects Men Women p N 196 101 95 – Age (years) 61 ± 14 60 ± 13 62 ± 15 0.346 Duration of diabetes (years) 10 (3–18) 10 (2–20) 10 (5–16) 0.589 BMI (kg/m ) 27.1 ± 6.1 26.9 ± 5.5 27.3 ± 6.7 0.640 Waist circumference (cm) 92 ± 15 93 ± 13 91 ± 16 0.252 Systolic blood pressure (mmHg) 135 ± 20 135 ± 19 135 ± 20 0.962 Diastolic blood pressure (mmHg) 80 ± 12 81 ± 11 77 ± 13 0.022 Smoker n (%) 41 (20.9) 34 (33.7) 7 (7.4) <0.001 Antihyperglycemic agents n (%) 168 (85.7) 86 (85.2) 82 (86.3) 0.816 Statins n (%) 80 (40.8) 38 (37.6) 42 (44.2) 0.348 RAS inhibitors n (%) 72 (36.7) 34 (33.7) 38 (40.0) 0.358 Calcium-channel blockers n (%) 74 (37.8) 35 (34.7) 39 (41.1) 0.356 Albumin (g/dL) 4.0 ± 0.5 4.1 ± 0.6 4.0 ± 0.4 0.205 Creatinine (mg/dL) 0.95 ± 0.58 1.07 ± 0.59 0.82 ± 0.55 0.002 eGFR (mL/min/1.73 m ) 67.3 ± 23.7 66.3 ± 21.8 68.3 ± 25.7 0.548 Fasting glucose (mg/dL) 129 ± 37 127 ± 39 132 ± 34 0.367 HbA1c (%) 8.3 ± 1.9 8.3 ± 2.0 8.3 ± 1.7 0.950 HOMA-R 2.3 (1.5–3.7) 2.2 (1.5–3.4) 2.6 (1.6–3.9) 0.287 Triglycerides (mg/dL) 120 (85–163) 131 (88–176) 106 (76–156) 0.020 HDL-cholesterol (mg/dL) 45 ± 12 42 ± 10 48 ± 14 <0.001 LDL-cholesterol (mg/dL) 116 ± 42 120 ± 49 111 ± 32 0.105 hs-CRP (mg/L) 0.70 (0.27–1.58) 0.65 (0.25–1.12) 0.79 (0.29–1.95) 0.221 LBP (μg/mL) 18.2 ± 6.3 17.7 ± 5.8 18.7 ± 6.7 0.267 Aortic PWV (cm/s) 1194 ± 346 1224 ± 350 1162 ± 340 0.209 Values are expressed as n (%), mean ± SD, or median (interquartile range). p values were determined by using χ -test, unpaired t-test, or Wilcoxon rank-sum test, as appropriate, for comparison between men and women LBP lipopolysaccharide-binding protein, PWV pulse wave velocity, BMI body mass index, RAS renin-angiotensin system, eGFR estimated glomerular filtration rate, HbA1c glycated hemoglobin A1c, HOMA-R homeostasis model assessment of insulin resistance, HDL high-density lipoprotein, LDL low-density lipoprotein, hs-CRP high-sensitivity C-reactive protein N = 144 for all subjects, n = 79 for men, and n = 65 for women not receiving insulin therapy Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 4 of 8 Association between serum LBP levels and cardiovascular Table 2 Correlation between  serum LBP levels or aortic PWV and cardiovascular risk factors risk factors We first examined the association of serum LBP levels Serum LBP levels Aortic PWV with the parameters related to obesity, insulin resist- r p r p ance, and other cardiovascular risk factors by simple lin- ear regression analyses (Table  2). Serum LBP levels were Age −0.126 0.079 0.568 <0.001 significantly correlated with measures of obesity includ - BMI 0.279 <0.001 −0.312 <0.001 ing BMI (r  =  0.279, p  <  0.001) and waist circumference Waist circumference 0.295 <0.001 −0.216 0.003 (r = 0.295, p < 0.001) and with parameters related to insu- Systolic blood pressure 0.082 0.252 0.498 <0.001 lin resistance including HOMA-R (r = 0.257, p = 0.002), Albumin −0.156 0.029 −0.210 0.003 triglycerides (r = 0.234, p < 0.001), and HDL-cholesterol eGFR −0.015 0.838 −0.473 <0.001 levels (r  =  −0.163, p  =  0.020). In addition, serum LBP HbA1c 0.058 0.417 −0.034 0.634 levels were correlated with inflammatory factors such as Log [HOMA-R] 0.257 0.002 −0.279 <0.001 hs-CRP (r =  0.575, p < 0.001) and serum albumin levels Log [triglycerides] 0.234 <0.001 −0.108 0.131 (r = −0.156, p = 0.029) (Table 2). HDL-cholesterol −0.163 0.020 −0.021 0.769 LDL-cholesterol 0.077 0.284 −0.104 0.148 Association of aortic PWV with cardiovascular risk factors Log [hs-CRP] 0.575 <0.001 −0.053 0.462 and serum LBP levels LBP – – 0.065 0.364 Next, we examined the association of aortic PWV with r correlation coefficient determined by simple regression analysis, LBP cardiovascular risk factors and serum LBP levels by sim- lipopolysaccharide-binding protein, PWV pulse wave velocity, BMI body mass index, eGFR estimated glomerular filtration rate, HbA1c glycated hemoglobin ple linear regression analyses (Table  2). Aortic PWV A1c, HOMA-R homeostasis model assessment of insulin resistance, HDL was well correlated with age (r  =  0.568, p  <  0.001), sys- high-density lipoprotein, LDL low-density lipoprotein, hs-CRP high-sensitivity C-reactive protein, PWV pulse wave velocity tolic blood pressure (r  =  0.498, p  <  0.001), and eGFR Subjects excluding those receiving insulin therapy (n = 144) (r  =  −0.473, p  <  0.001). In addition, aortic PWV was found to be negatively correlated with obesity-related parameters, such as BMI (r  =  −0.312, p  <  0.001), waist Table 3 Multiple regression analysis of  the determinants circumference (r  =  −0.216, p  =  0.003), and HOMA-R for aortic PWV (r  =  −0.279, p  <  0.001). Among inflammation-related Model 1 Model 2 parameters, serum albumin levels were negatively cor- related (r  =  −0.210, p  =  0.003), while neither hs-CRP β p β p (r = −0.053, p = 0.462) nor serum LBP levels (r = 0.065, Age (years) 0.344 <0.001 0.327 <0.001 p = 0.364) were significantly correlated with aortic PWV Sex (male = 1, female = 0) −0.117 0.033 −0.158 0.012 in univariate analyses (Table 2). BMI (kg/m ) −0.233 0.001 −0.228 0.007 Systolic blood pressure (mmHg) 0.382 <0.001 0.310 <0.001 Multivariate analyses of the factors associated with aortic Albumin (g/dL) −0.074 0.185 −0.132 0.051 PWV eGFR (mL/min/1.73 m ) −0.113 0.095 −0.150 0.048 To explore whether serum LBP levels have an independ- Log [HOMA-R] – – −0.030 0.692 ent association with arterial stiffness, we performed Log [triglycerides (mg/dL)] −0.092 0.119 −0.105 0.151 multiple regression analyses after adjusting for age; sex; HDL-cholesterol (mg/dL) −0.082 0.180 −0.120 0.086 BMI; systolic blood pressure; albumin; eGFR; log [triglyc- Statins (yes = 1, no = 0) 0.060 0.249 0.101 0.079 erides]; HDL-cholesterol; log [hs-CRP]; use of statins; RAS inhibitors (yes = 1, no = 0) −0.130 0.025 0.180 0.006 use of RAS inhibitors; use of calcium-channel block- Calcium-channel blockers (yes = 1, 0.049 0.412 0.056 0.396 ers; and smoking status (Model 1, Table  3). Aside from no = 0) age (β = 0.344, p < 0.001), BMI (β = −0.233, p = 0.001), Smokers (yes = 1, no = 0) −0.021 0.698 −0.033 0.606 systolic blood pressure (β = 0.382, p < 0.001), and use of Log [hs-CRP (mg/L)] 0.024 0.733 0.005 0.950 RAS inhibitors (β = −0.130, p = 0.025), serum LBP levels LBP (μg/mL) 0.135 0.026 0.125 0.083 were found to be independently and positively associated R 0.585 <0.001 0.631 <0.001 with aortic PWV (β  =  0.135, p  =  0.026). On the other β standard regression coefficient determined by multiple regression analysis, hand, hs-CRP levels were not found to be an independ- R coefficient of determination, PWV pulse wave velocity, BMI body mass ent determinant of aortic PWV (β  =  0.024, p  =  0.733) index, eGFR estimated glomerular filtration rate, HOMA-R homeostasis (Model 1, Table  3). Unlike RAS inhibitors, use of cal- model assessment of insulin resistance, HDL high-density lipoprotein, RAS renin-angiotensin system, hs-CRP high-sensitivity C-reactive protein, LBP cium-channel blockers was not significantly associated lipopolysaccharide-binding protein with aortic PWV (β = 0.049, p = 0.412). The association Subjects excluding those receiving insulin therapy (n = 144) Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 5 of 8 between serum LBP levels and aortic PWV remained cardiovascular mortality [14], independent of established nearly significant (β  =  0.125, p  =  0.083) after further cardiovascular risk factors and inflammatory markers, in adjustment for log [HOMA-R] (Model 2, Table 3). hospital-based cohort studies. Recently, serum LBP lev- els have been shown to be independently associated with Separate correlations between serum LBP levels and aortic carotid intima-media thickness, the most established PWV in men and women morphological surrogate marker for cardiovascular mor- Additionally, we performed an interaction analysis to bidity and mortality [25, 26], after adjusting for age, sex, assess whether sex modified the relationship between BMI, and hs-CRP levels, in healthy subjects [15]. Aortic serum LBP levels and aortic PWV. The analysis indicated PWV has also been established as an independent pre- a potential effect modification by sex on the association dictor of future cardiovascular events and mortality between serum LBP levels and aortic PWV (β = −0.137, [27, 28]. Recent studies have shown a close association p for interaction  =  0.065). Then, we examined the asso - between blood pressure and arterial stiffness as evalu - ciation between serum LBP levels and aortic PWV in ated by brachial-ankle PWV [29] in patients with type 2 men (n  =  101) and women (n  =  95) separately. Serum diabetes, and a predictive value of brachial-ankle PWV LBP levels were found to be positively correlated with on the progression of coronary artery calcification [30] aortic PWV in men (r =  0.242, p  =  0.015), and the cor- The results from the present study are in agreement with relation remained significant (β  = 0.209, p = 0.011) after those of the previous study [15] and indicate a possible adjusting for age; BMI; systolic blood pressure; albumin; role of LBP in the progression of atherosclerosis. Further- eGFR; log [triglycerides]; HDL-cholesterol; log [hs-CRP]; more, this study is the first to report the impact of LBP use of statins; use of RAS inhibitors; use of calcium- on arterial stiffness in patients with type 2 diabetes, who channel blockers, and smoking status. On the contrary, have advanced arterial stiffening [22, 27, 31–33] and ele- no significant correlation was found between serum LBP vated cardiovascular mortality [34]. levels and aortic PWV in women (β = 0.028, p = 0.768). Although not statistically significant, the impact of serum Potential mechanisms underlying the relationship LBP levels on aortic PWV was greater in men (β = 0.146, between LBP and arterial stiffness p = 0.140) than in women (β = −0.020, p = 0.874), after Importantly, in this study, the relationship between further adjustment for log [HOMA-R]. serum LBP levels and aortic PWV was independent of traditional cardiovascular risk factors including age, Discussion obesity, renal dysfunction, hyperglycemia, and dyslipi- The present study demonstrated that serum LBP lev - demia. It is well recognized that low-grade inflammation els are positively associated with arterial stiffness, as is among the major factors involved in arterial stiffness assessed by aortic PWV, in patients with type 2 diabe- in the general population [35] and in patients with type tes. Serum LBP levels were positively correlated with 2 diabetes [33, 36]. Recent evidence indicates that bac- the parameters of obesity, insulin resistance, and inflam - terial endotoxins, or LPS, are an important source of mation in our diabetic subjects, which is in agreement vascular inflammation in atherosclerosis [4]. In  vitro with observations from previous studies of non-diabetic studies have demonstrated that LPS induced expression populations [9, 12, 15]. However, it is noteworthy that of matrix metalloproteinase-9 through the TLR4/nuclear the association between serum LBP levels and aortic factor-κB pathway [37, 38] and stimulated the release of PWV was independent of obesity, inflammation, and proinflammatory cytokines [39] in vascular smooth mus - other traditional cardiovascular risk factors. The results cle cells [37, 39] and endothelial cells [38]. In  vivo stud- further revealed that the association between serum LBP ies also showed that the blockade of LPS signaling by levels and aortic PWV was observed in men, but not in TLR4 antagonists reduced the infiltration of monocytes/ women. To our knowledge, this is the first report to dem - macrophages and expression of interleukin-6 and matrix onstrate the clinical implications of circulating LBP in the metalloproteinase-9 in the atherosclerotic lesions of dia- increased arterial stiffness in type 2 diabetes. betic mice [40]. In humans, prospective population-based studies showed that chronic infection [2] or endotoxemia Clinical association between serum LBP levels and arterial [3] is a strong risk factor of carotid atherosclerosis [2, 3] stiffness and cardiovascular diseases [3]. In a large population- This study clearly demonstrated that serum LBP levels based study, soluble CD14, a mediator for the activation are independently and positively associated with aor- of immune cells by LPS, was independently associated tic PWV in patients with type 2 diabetes. Two previous with aortic PWV, but not with carotid intima-media studies showed that serum LBP levels were a significant thickness [41]. In light of the combined experimental and predictor of prevalent coronary artery disease [7] and clinical evidence, and considering the fact that circulating Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 6 of 8 LBP binds to LPS and promotes the innate immune the present study, these previous studies did not stratify response [5], our data may indicate a critical role of the the data by sex. Several lines of evidence indicate the sex- LPS/LBP-induced inflammation in the pathogenesis of associated difference in inflammatory responses to LPS- arterial stiffness in type 2 diabetes. stimulation in neutrophils in  vitro models [49, 50] and Alternatively, the association between serum LBP in mice in vivo models [51], with increased responses in levels and aortic PWV can be explained by the indirect males than in females. Recent studies have also indicated effects of LBP on aortic PWV via obesity and insulin the sex-associated differences in the gut microbiome, one resistance. Recently, moderately increased LPS in circula- of the major sources of circulating LPS [42], and the sex- tion, or metabolic endotoxemia, in response to a high-fat dependent effects of diet on the gut microbiota in mice diet was shown to trigger obesity and insulin resistance and humans [52]. In a study performed in rats, systemic in mice [42, 43]. Several studies also showed that LBP proinflammatory cytokine levels in response to an oligo - is produced by adipocytes and plays an essential role in fructose-supplemented diet were higher in males than inflammation- and obesity-associated adipose tissue in females [53]. Based on these reports, we can hypoth- dysfunction [44, 45]. The relationship between LBP and esize that men with type 2 diabetes are more prone to the obesity, insulin resistance, and the metabolic syndrome immune response elicited by LPS from the gut micro- has been demonstrated in a number of studies per- biota than are women, leading to increased arterial stiff - formed in humans in both cross-sectional [8, 9, 11, 12] ness in men. To our knowledge, this study is the first to and prospective [10, 13] designs. In agreement with these elucidate the sex-related differences in the association studies, we found that serum LBP levels were positively between LBP and arterial stiffness. correlated with parameters of obesity, insulin resistance, and components of the metabolic syndrome in patients Limitations with type 2 diabetes (Table  2). Considering the evidence This study has several limitations. First, we evaluated that the metabolic syndrome [46] and insulin resistance the endotoxin-induced inflammation by measuring [33] are closely associated with increased arterial stiff - serum LBP levels, but not by a direct measurement of ness, it is conceivable that LBP affected aortic stiffness LPS. However, serum LBP levels, which have been pro- through insulin resistance in our subjects. However, posed as a surrogate marker of LPS-induced immune in our data, the association of obesity and dyslipidemia response in humans, can be simply and stably measured with aortic PWV was less significant compared with that by enzyme-linked immunosorbent assay [8, 10, 11]. We of age, hypertension, renal dysfunction, and serum LBP also did not include the evaluation of the LPS-related fac- levels (Table  3). Moreover, HOMA-R was not indepen- tors other than LBP, such as interleukin-6, soluble CD14, dently associated with aortic PWV and an additional and tumor-necrosis factor-α, that could strengthen our adjustment for log [HOMA-R] did not virtually affect the study. Second, since this was a cross-sectional study, the relationship between LBP and aortic PWV in the mul- causal relationship between LBP and arterial stiffness tivariate model (Model 2, Table  3). Thus, although LPS/ needs to be confirmed by longitudinal and/or interven - LBP-induced immune response is commonly involved tional studies. Third, the subjects were receiving statins in arterial stiffness [41], obesity, and insulin resistance and/or RAS inhibitors, which could have influenced [6, 8–13], our results indicate that the LPS/LBP-induced inflammation, vascular function, and related risk factors. innate immunity independently affects arterial stiffness To minimize the impact of these drugs, the presence of in patients with type 2 diabetes. these treatments was adjusted for in the multiple regres- sion analysis. Fourth, the sample size was too small in Sex‑dependent association between LBP and arterial the sex-stratified analysis to conclude with statistical sig - stiffness nificance the relationship between serum LBP levels and This study further revealed that the effect of serum LBP aortic PWV in men after adjusting for log [HOMA-R]. levels on aortic PWV is modified by sex and that there Finally, because our subjects were hospitalized in a uni- is a significant association between serum LBP levels versity hospital and had inadequate glycemic control, the and aortic PWV in men only. A number of studies have present results may not be generalized to the entire pop- shown sex-dependent association of arterial stiffness with ulation of patients with type 2 diabetes. metabolic risk factors, such as visceral adiposity [18], the metabolic syndrome, and its components [47, 48]. In pre- Conclusions vious studies on the LPS-related factors, sex-adjusted This study clearly demonstrated that serum LBP levels association was observed between LBP/soluble CD14 are independently and positively associated with arte- and aortic stiffness [41], carotid intima-media thickness rial stiffness in patients with type 2 diabetes. Our data [15], and cardiovascular disease [14]. However, unlike indicate a close link between LPS/LBP-induced innate Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 7 of 8 immunity and arterial stiffness in these patients. This Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- study further shows that LBP preferentially affects arte - lished maps and institutional affiliations. rial stiffness in men over women, indicating sex-related differences in the link between LBP and arterial stiffness. Received: 7 March 2017 Accepted: 3 May 2017 Further interventional studies are warranted to clarify whether the reduction of serum LPS levels by antibiot- ics or probiotics would reduce arterial stiffness and the risk of cardiovascular diseases in patients with type 2 References diabetes. 1. Hotamisligil GS. Inflammation and metabolic disorders. Nature. 2006;444:860–7. 2. Kiechl S, Egger G, Mayr M, Wiedermann CJ, Bonora E, Oberhollenzer F, Muggeo M, Xu Q, Wick G, Poewe W, et al. Chronic infections and the risk Abbreviations of carotid atherosclerosis: prospective results from a large population LPS: lipopolysaccharide; LBP: lipopolysaccharide-binding protein; PWV: study. Circulation. 2001;103:1064–70. pulse wave velocity; BMI: body mass index; CD: cluster of differentiation; TLR: 3. Wiedermann CJ, Kiechl S, Dunzendorfer S, Schratzberger P, Egger G, toll-like receptor; hs-CRP: high-sensitivity C-reactive protein; eGFR: estimated Oberhollenzer F, Willeit J. Association of endotoxemia with carotid glomerular filtration rate; HOMA-R: homeostasis model assessment of insulin atherosclerosis and cardiovascular disease: prospective results from the resistance; SD: standard deviation; HbA1c: glycated hemoglobin A1c; HDL: Bruneck Study. J Am Coll Cardiol. 1999;34:1975–81. high-density lipoprotein; LDL: low-density lipoprotein; RAS: renin-angiotensin 4. Stoll LL, Denning GM, Weintraub NL. Potential role of endotoxin as a system. proinflammatory mediator of atherosclerosis. Arterioscler Thromb Vasc Biol. 2004;24:2227–36. Authors’ contributions 5. Triantafilou M, Triantafilou K. Lipopolysaccharide recognition: CD14, TLRs TaS, TM, and AS conceived the study, participated in its design and coordina- and the LPS-activation cluster. Trends Immunol. 2002;23:301–4. tion, and helped in drafting the manuscript. TaS carried out the immuno- 6. Kheirandish-Gozal L, Peris E, Wang Y, Tamae Kakazu M, Khalyfa A, Carreras assays. TaS and TM performed the statistical analyses. TaS, YK, YM, and YY A, Gozal D. Lipopolysaccharide-binding protein plasma levels in children: enrolled patients and performed the vascular examinations. KMot, KMor, SF, effects of obstructive sleep apnea and obesity. J Clin Endocrinol Metab. TeS, ME, and MI contributed to discussions and were involved in either draft- 2014;99:656–63. ing the manuscript or revising it critically. All authors read and approved the 7. Lepper PM, Schumann C, Triantafilou K, Rasche FM, Schuster T, Frank H, final manuscript. Schneider EM, Triantafilou M, von Eynatten M. Association of lipopolysac- charide-binding protein and coronary artery disease in men. J Am Coll Author details Cardiol. 2007;50:25–31. Department of Metabolism, Endocrinology and Molecular Medicine, Osaka 8. Ruiz AG, Casafont F, Crespo J, Cayon A, Mayorga M, Estebanez A, City University Graduate School of Medicine, 1-4-3, Asahi-machi, Abeno-ku, Fernadez-Escalante JC, Pons-Romero F. Lipopolysaccharide-binding Osaka 545-8585, Japan. Department of Vascular Medicine, Osaka City protein plasma levels and liver TNF-α gene expression in obese patients: University Graduate School of Medicine, 1-4-3, Asahi-machi, Abeno-ku, evidence for the potential role of endotoxin in the pathogenesis of non- Osaka 545-8585, Japan. Vascular Science Center for Translational Research, alcoholic steatohepatitis. Obes Surg. 2007;17:1374–80. Osaka City University Graduate School of Medicine, 1-4-3, Asahi-machi, 9. Gonzalez-Quintela A, Alonso M, Campos J, Vizcaino L, Loidi L, Gude F. Abeno-ku, Osaka 545-8585, Japan. Department of Nephrology, Osaka Determinants of serum concentrations of lipopolysaccharide-binding City University Graduate School of Medicine, 1-4-3, Asahi-machi, Abeno-ku, protein (LBP) in the adult population: the role of obesity. PLoS ONE. Osaka 545-8585, Japan. Department of Premier Preventive Medicine, Osaka 2013;8:e54600. City University Graduate School of Medicine, 1-4-3, Asahi-machi, Abeno-ku, 10. Liu X, Lu L, Yao P, Ma Y, Wang F, Jin Q, Ye X, Li H, Hu FB, Sun L, et al. Osaka 545-8585, Japan. Lipopolysaccharide binding protein, obesity status and incidence of metabolic syndrome: a prospective study among middle-aged and older Acknowledgements Chinese. Diabetologia. 2014;57:1834–41. The authors wish to acknowledge the excellent technical assistance of Ms. 11. Moreno-Navarrete JM, Ortega F, Serino M, Luche E, Waget A, Pardo G, Asako Katsuma from the research laboratory of the Department of Vascular Salvador J, Ricart W, Fruhbeck G, Burcelin R, et al. Circulating lipopoly- Medicine, Osaka City University Graduate School of Medicine. saccharide-binding protein (LBP) as a marker of obesity-related insulin resistance. Int J Obes. 2012;36:1442–9. Competing interests 12. Sun L, Yu Z, Ye X, Zou S, Li H, Yu D, Wu H, Chen Y, Dore J, Clement K, The authors declare that they have no competing interests. et al. A marker of endotoxemia is associated with obesity and related metabolic disorders in apparently healthy Chinese. Diabetes Care. Availability of data and materials 2010;33:1925–32. The datasets used and analyzed during the current study are available from 13. Tilves CM, Zmuda JM, Kuipers AL, Nestlerode CS, Evans RW, Bunker CH, the corresponding author on reasonable request. Patrick AL, Miljkovic I. Association of lipopolysaccharide-binding protein with aging-related adiposity change and prediabetes among African Ethics approval and consent to participate ancestry men. Diabetes Care. 2016;39:385–91. This study was performed in accordance with the Declaration of Helsinki 14. Lepper PM, Kleber ME, Grammer TB, Hoffmann K, Dietz S, Winkelmann (1975, as revised in 2013). This study was approved by the Ethics Committee of BR, Boehm BO, Marz W. Lipopolysaccharide-binding protein (LBP) is Osaka City University Graduate School of Medicine (No. 308). 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Lipopolysaccharide-binding protein is associated with arterial stiffness in patients with type 2 diabetes: a cross-sectional study

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2017 The Author(s)
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10.1186/s12933-017-0545-3
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Abstract

Background: Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase reactant that mediates immune responses triggered by LPS. Recent evidence indicates the association of circulating LBP levels with obesity, diabetes, and cardiovascular diseases. In this study, we aimed to investigate the relationship between serum LBP levels and arterial stiffness in patients with type 2 diabetes. Methods: A total of 196 patients with type 2 diabetes, including 101 men and 95 women, were enrolled in this cross- sectional study. Fasting serum LBP levels were determined by enzyme-linked immunosorbent assay. Arterial stiffness was assessed by measuring the aortic pulse wave velocity (PWV ). Results: The mean values of serum LBP and aortic PWV were 18.2 μg/mL and 1194 cm/s, respectively. Serum LBP levels were positively correlated with body mass index, triglycerides, high-sensitivity C-reactive protein, and insulin resistance index and were negatively correlated with high-density lipoprotein cholesterol. They were, however, not significantly correlated with aortic PWV in univariate analyses. Multivariate analysis revealed that serum LBP levels were independently and positively associated with aortic PWV (β = 0.135, p = 0.026) after adjusting for age, sex, body mass index, albumin, high-sensitivity C-reactive protein, and other cardiovascular risk factors. Further analyses revealed that the impact of serum LBP levels on aortic PWV was modified by sex, and the association between serum LBP levels and aortic PWV was found to be significant only in men. Conclusions: Serum LBP levels are associated with arterial stiffness, independent of obesity and traditional cardiovas - cular risk factors, especially in men with type 2 diabetes. This study indicates a potential role of the LPS/LBP-induced innate immunity in the development and progression of arterial stiffness in type 2 diabetes. Keywords: Lipopolysaccharide-binding protein, Arterial stiffness, Pulse wave velocity, Type 2 diabetes derived from Gram-negative bacteria is known to play a Background critical role in triggering the immune and inflammatory The association of chronic inflammation with the patho - responses in vascular cells, leading to atherosclerosis [4]. geneses of obesity, diabetes, and atherosclerosis is well LPS-binding protein (LBP), an acute-phase reactant syn- recognized [1]. Accumulating evidence indicates a link thesized mainly in the liver, binds LPS and initiates the between low-grade inflammation produced by common immune response by presenting LPS to cluster of differ - subclinical or chronic infections and the risk of ath- entiation (CD)14, which in turn interacts with toll-like erosclerosis in humans [2, 3]. Lipopolysaccharide (LPS) receptor (TLR)4 on immune cells [5]. Since LBP is syn- thesized and released into circulation in the presence *Correspondence: [email protected] of LPS, with a relatively long half-life, LBP level is con- Department of Metabolism, Endocrinology and Molecular Medicine, Osaka City University Graduate School of Medicine, 1-4-3, Asahi-machi, sidered a surrogate biomarker for the activation of LPS- Abeno-ku, Osaka 545-8585, Japan induced innate immune responses [6–8]. Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 2 of 8 Several studies in humans recently demonstrated that expiration. Blood was drawn after an overnight fast and serum LBP levels are closely associated with obesity, the biochemical parameters were analyzed using a standard metabolic syndrome, and type 2 diabetes [6, 8–13]. Fur- laboratory method at the Central Clinical Laboratory thermore, serum LBP levels were shown to be a predictor of the Osaka City University Hospital [18, 19]. The esti - of prevalent coronary artery disease [7] and cardiovascu- mated glomerular filtration rate (eGFR) was calculated lar mortality [14], independent of established cardiovas- using the Japanese eGFR equation [20]. Immunoreactive cular risk factors and markers of systemic inflammation. insulin levels were measured for subjects not receiving A recent study also showed that serum LBP levels were insulin therapy (n  =  144) by electro-chemiluminescence positively correlated with subclinical atherosclerosis, as immunoassay [Cobas 8000 (502/602), Roche Diagnostics] assessed by carotid intima-media thickness, in healthy at the Central Clinical Laboratory. Homeostasis model individuals, independent of body mass index (BMI) and assessment of insulin resistance (HOMA-R) was calcu- high-sensitivity C-reactive protein (hs-CRP) level [15]. lated according to the following formula: fasting insu- These studies collectively indicate that serum LBP level lin (μU/mL)  ×  fasting glucose (mg/dL)/405 [21]. Frozen may be a biomarker for atherosclerotic cardiovascu- serum samples were shipped to SRL Inc. (Tokyo, Japan) lar disease and implicate a potential role of the innate and hs-CRP concentrations were measured by means immune mechanisms in the progression of atherosclero- of particle-enhanced immunonephelometry with the sis in humans. Behring nephelometer using N Latex CRP mono reagent. To our knowledge, no previous study has investigated Serum levels of LBP were measured using a commer- the relationship between LBP and arterial stiffness, a cial enzyme-linked immunosorbent assay (HK315-02, well-established surrogate marker for cardiovascular dis- HyCult Biotech Inc., Uden, the Netherlands) as per man- eases [16], in human subjects. Moreover, no study has ufacturer’s instructions. The intra- and inter-assay coef - examined the impact of LBP on subclinical atheroscle- ficients of LBP variation were <5 and <10%, respectively. rosis in patients with comorbid cardiovascular risk fac- tors, including diabetes. Therefore, in the present study, Measurement of arterial stiffness we investigated the association between serum LBP lev- Arterial stiffness was evaluated by measuring PWV in els and arterial stiffness by measuring aortic pulse wave the heart-femoral segment using an automatic waveform velocity (PWV) in patients with type 2 diabetes. analyzer (Model BP-203RPE; Omron Colin Co., Ltd., Tokyo, Japan) as described previously [22, 23]. Reproduc- Methods ibility in the measurements of arterial stiffness was con - Study design and subjects firmed in our previous study, in which the coefficients of We consecutively enrolled 196 patients with type 2 diabe- variation were less than 5% for heart-femoral PWV [22]. tes, including 101 men and 95 women, who were admit- ted to the Diabetes Center of the Osaka City University Statistical analysis Hospital for the purpose of glycemic control, education, Data are expressed as the number (%), mean  ±  stand- and/or evaluation of diabetic complications between July ard deviation (SD), or median (interquartile range) as 2013 and December 2015. Type 2 diabetes was diagnosed appropriate. For comparisons between men and women, based on American Diabetes Association criteria [17]. χ -test, unpaired t-test, or Wilcoxon rank-sum test, was Patients with type 1 diabetes or other types of diabetes performed as appropriate. Skewed parameters, such as were not included in the present study. The following HOMA-R, triglycerides, and hs-CRP were logarithmi- patients were not included in this study: those with acute cally transformed before regression analysis. Simple lin- or chronic infection, chronic inflammatory disease, use ear regression analyses were performed to evaluate the of anti-inflammatory drugs including glucocorticoids, relationship between serum LBP levels or aortic PWV or hematologic or malignant disease, and those who and various clinical variables, including cardiovascular underwent recent surgery within 1 month at the time of risk factors. To explore the association between LBP and entry. A smoker was defined as a current smoker in our aortic PWV, multiple linear regression analyses were per- analyses. formed after adjustment for age, sex, BMI, systolic blood pressure, albumin, eGFR, high-density lipoprotein (HDL) Physical and laboratory measurements cholesterol level, log [hs-CRP], serum LBP level, treat- Blood pressure was determined using an automatic ment with statins, treatment with inhibitors of the renin- sphygmomanometer with a conventional cuff after the angiotensin system (RAS inhibitors), treatment with subject had rested for at least 5 min. Waist circumference calcium-channel blockers, and smoking status. To assess was measured to the nearest centimeter at the level of whether the effect of serum LBP levels on aortic PWV is the umbilicus in a standing position at the end of gentle modified by sex, the interaction term between LBP and Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 3 of 8 sex was inserted into the multiple regression model. A p with statins for dyslipidemia, 72 (36.7%) with RAS inhibi- value of <0.20 was considered significant for interaction tors, and 74 (37.8%) with calcium-channel blockers for effects, as has been used in a previous study [24], and a hypertension. There were significantly more male smok - p value of <0.05 was considered significant for all other ers than female smokers. Serum creatinine levels, but analyses. Statistical analyses were performed by using the not eGFR, were significantly different between men and JMP 10 software program (SAS Institute Inc., Cary, NC, women. Parameters of obesity and insulin resistance, USA). such as BMI, waist circumference, and HOMA-R, were not significantly different between men and women. Tri - Results glycerides levels and diastolic blood pressure were higher, Clinical characteristics, serum LBP levels, and aortic PWV and HDL-cholesterol levels were lower in men than in of the subjects women. The clinical characteristics of the total population, as well Mean  ±  SD value for serum LBP levels of all sub- as of men and women, are shown in Table 1. The subjects jects was 18.2  ±  6.3  μg/mL (range 2.1–36.2  μg/ had a mean age of 61 years, median duration of diabetes mL). Mean  ±  SD value for the aortic PWV was of 10  years, and mean BMI of 27.1  kg/m . One hundred 1194  ±  346  cm/s (range 610–2500  cm/s). Serum LBP sixty-eight subjects (85.7%) were receiving any antihy- levels and aortic PWV were not significantly different perglycemic agents. Eighty (40.8%) subjects were treated between men and women. Table 1 Clinical characteristics, serum LBP levels, and aortic PWV in all subjects as well as in men and women with type 2 diabetes All subjects Men Women p N 196 101 95 – Age (years) 61 ± 14 60 ± 13 62 ± 15 0.346 Duration of diabetes (years) 10 (3–18) 10 (2–20) 10 (5–16) 0.589 BMI (kg/m ) 27.1 ± 6.1 26.9 ± 5.5 27.3 ± 6.7 0.640 Waist circumference (cm) 92 ± 15 93 ± 13 91 ± 16 0.252 Systolic blood pressure (mmHg) 135 ± 20 135 ± 19 135 ± 20 0.962 Diastolic blood pressure (mmHg) 80 ± 12 81 ± 11 77 ± 13 0.022 Smoker n (%) 41 (20.9) 34 (33.7) 7 (7.4) <0.001 Antihyperglycemic agents n (%) 168 (85.7) 86 (85.2) 82 (86.3) 0.816 Statins n (%) 80 (40.8) 38 (37.6) 42 (44.2) 0.348 RAS inhibitors n (%) 72 (36.7) 34 (33.7) 38 (40.0) 0.358 Calcium-channel blockers n (%) 74 (37.8) 35 (34.7) 39 (41.1) 0.356 Albumin (g/dL) 4.0 ± 0.5 4.1 ± 0.6 4.0 ± 0.4 0.205 Creatinine (mg/dL) 0.95 ± 0.58 1.07 ± 0.59 0.82 ± 0.55 0.002 eGFR (mL/min/1.73 m ) 67.3 ± 23.7 66.3 ± 21.8 68.3 ± 25.7 0.548 Fasting glucose (mg/dL) 129 ± 37 127 ± 39 132 ± 34 0.367 HbA1c (%) 8.3 ± 1.9 8.3 ± 2.0 8.3 ± 1.7 0.950 HOMA-R 2.3 (1.5–3.7) 2.2 (1.5–3.4) 2.6 (1.6–3.9) 0.287 Triglycerides (mg/dL) 120 (85–163) 131 (88–176) 106 (76–156) 0.020 HDL-cholesterol (mg/dL) 45 ± 12 42 ± 10 48 ± 14 <0.001 LDL-cholesterol (mg/dL) 116 ± 42 120 ± 49 111 ± 32 0.105 hs-CRP (mg/L) 0.70 (0.27–1.58) 0.65 (0.25–1.12) 0.79 (0.29–1.95) 0.221 LBP (μg/mL) 18.2 ± 6.3 17.7 ± 5.8 18.7 ± 6.7 0.267 Aortic PWV (cm/s) 1194 ± 346 1224 ± 350 1162 ± 340 0.209 Values are expressed as n (%), mean ± SD, or median (interquartile range). p values were determined by using χ -test, unpaired t-test, or Wilcoxon rank-sum test, as appropriate, for comparison between men and women LBP lipopolysaccharide-binding protein, PWV pulse wave velocity, BMI body mass index, RAS renin-angiotensin system, eGFR estimated glomerular filtration rate, HbA1c glycated hemoglobin A1c, HOMA-R homeostasis model assessment of insulin resistance, HDL high-density lipoprotein, LDL low-density lipoprotein, hs-CRP high-sensitivity C-reactive protein N = 144 for all subjects, n = 79 for men, and n = 65 for women not receiving insulin therapy Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 4 of 8 Association between serum LBP levels and cardiovascular Table 2 Correlation between  serum LBP levels or aortic PWV and cardiovascular risk factors risk factors We first examined the association of serum LBP levels Serum LBP levels Aortic PWV with the parameters related to obesity, insulin resist- r p r p ance, and other cardiovascular risk factors by simple lin- ear regression analyses (Table  2). Serum LBP levels were Age −0.126 0.079 0.568 <0.001 significantly correlated with measures of obesity includ - BMI 0.279 <0.001 −0.312 <0.001 ing BMI (r  =  0.279, p  <  0.001) and waist circumference Waist circumference 0.295 <0.001 −0.216 0.003 (r = 0.295, p < 0.001) and with parameters related to insu- Systolic blood pressure 0.082 0.252 0.498 <0.001 lin resistance including HOMA-R (r = 0.257, p = 0.002), Albumin −0.156 0.029 −0.210 0.003 triglycerides (r = 0.234, p < 0.001), and HDL-cholesterol eGFR −0.015 0.838 −0.473 <0.001 levels (r  =  −0.163, p  =  0.020). In addition, serum LBP HbA1c 0.058 0.417 −0.034 0.634 levels were correlated with inflammatory factors such as Log [HOMA-R] 0.257 0.002 −0.279 <0.001 hs-CRP (r =  0.575, p < 0.001) and serum albumin levels Log [triglycerides] 0.234 <0.001 −0.108 0.131 (r = −0.156, p = 0.029) (Table 2). HDL-cholesterol −0.163 0.020 −0.021 0.769 LDL-cholesterol 0.077 0.284 −0.104 0.148 Association of aortic PWV with cardiovascular risk factors Log [hs-CRP] 0.575 <0.001 −0.053 0.462 and serum LBP levels LBP – – 0.065 0.364 Next, we examined the association of aortic PWV with r correlation coefficient determined by simple regression analysis, LBP cardiovascular risk factors and serum LBP levels by sim- lipopolysaccharide-binding protein, PWV pulse wave velocity, BMI body mass index, eGFR estimated glomerular filtration rate, HbA1c glycated hemoglobin ple linear regression analyses (Table  2). Aortic PWV A1c, HOMA-R homeostasis model assessment of insulin resistance, HDL was well correlated with age (r  =  0.568, p  <  0.001), sys- high-density lipoprotein, LDL low-density lipoprotein, hs-CRP high-sensitivity C-reactive protein, PWV pulse wave velocity tolic blood pressure (r  =  0.498, p  <  0.001), and eGFR Subjects excluding those receiving insulin therapy (n = 144) (r  =  −0.473, p  <  0.001). In addition, aortic PWV was found to be negatively correlated with obesity-related parameters, such as BMI (r  =  −0.312, p  <  0.001), waist Table 3 Multiple regression analysis of  the determinants circumference (r  =  −0.216, p  =  0.003), and HOMA-R for aortic PWV (r  =  −0.279, p  <  0.001). Among inflammation-related Model 1 Model 2 parameters, serum albumin levels were negatively cor- related (r  =  −0.210, p  =  0.003), while neither hs-CRP β p β p (r = −0.053, p = 0.462) nor serum LBP levels (r = 0.065, Age (years) 0.344 <0.001 0.327 <0.001 p = 0.364) were significantly correlated with aortic PWV Sex (male = 1, female = 0) −0.117 0.033 −0.158 0.012 in univariate analyses (Table 2). BMI (kg/m ) −0.233 0.001 −0.228 0.007 Systolic blood pressure (mmHg) 0.382 <0.001 0.310 <0.001 Multivariate analyses of the factors associated with aortic Albumin (g/dL) −0.074 0.185 −0.132 0.051 PWV eGFR (mL/min/1.73 m ) −0.113 0.095 −0.150 0.048 To explore whether serum LBP levels have an independ- Log [HOMA-R] – – −0.030 0.692 ent association with arterial stiffness, we performed Log [triglycerides (mg/dL)] −0.092 0.119 −0.105 0.151 multiple regression analyses after adjusting for age; sex; HDL-cholesterol (mg/dL) −0.082 0.180 −0.120 0.086 BMI; systolic blood pressure; albumin; eGFR; log [triglyc- Statins (yes = 1, no = 0) 0.060 0.249 0.101 0.079 erides]; HDL-cholesterol; log [hs-CRP]; use of statins; RAS inhibitors (yes = 1, no = 0) −0.130 0.025 0.180 0.006 use of RAS inhibitors; use of calcium-channel block- Calcium-channel blockers (yes = 1, 0.049 0.412 0.056 0.396 ers; and smoking status (Model 1, Table  3). Aside from no = 0) age (β = 0.344, p < 0.001), BMI (β = −0.233, p = 0.001), Smokers (yes = 1, no = 0) −0.021 0.698 −0.033 0.606 systolic blood pressure (β = 0.382, p < 0.001), and use of Log [hs-CRP (mg/L)] 0.024 0.733 0.005 0.950 RAS inhibitors (β = −0.130, p = 0.025), serum LBP levels LBP (μg/mL) 0.135 0.026 0.125 0.083 were found to be independently and positively associated R 0.585 <0.001 0.631 <0.001 with aortic PWV (β  =  0.135, p  =  0.026). On the other β standard regression coefficient determined by multiple regression analysis, hand, hs-CRP levels were not found to be an independ- R coefficient of determination, PWV pulse wave velocity, BMI body mass ent determinant of aortic PWV (β  =  0.024, p  =  0.733) index, eGFR estimated glomerular filtration rate, HOMA-R homeostasis (Model 1, Table  3). Unlike RAS inhibitors, use of cal- model assessment of insulin resistance, HDL high-density lipoprotein, RAS renin-angiotensin system, hs-CRP high-sensitivity C-reactive protein, LBP cium-channel blockers was not significantly associated lipopolysaccharide-binding protein with aortic PWV (β = 0.049, p = 0.412). The association Subjects excluding those receiving insulin therapy (n = 144) Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 5 of 8 between serum LBP levels and aortic PWV remained cardiovascular mortality [14], independent of established nearly significant (β  =  0.125, p  =  0.083) after further cardiovascular risk factors and inflammatory markers, in adjustment for log [HOMA-R] (Model 2, Table 3). hospital-based cohort studies. Recently, serum LBP lev- els have been shown to be independently associated with Separate correlations between serum LBP levels and aortic carotid intima-media thickness, the most established PWV in men and women morphological surrogate marker for cardiovascular mor- Additionally, we performed an interaction analysis to bidity and mortality [25, 26], after adjusting for age, sex, assess whether sex modified the relationship between BMI, and hs-CRP levels, in healthy subjects [15]. Aortic serum LBP levels and aortic PWV. The analysis indicated PWV has also been established as an independent pre- a potential effect modification by sex on the association dictor of future cardiovascular events and mortality between serum LBP levels and aortic PWV (β = −0.137, [27, 28]. Recent studies have shown a close association p for interaction  =  0.065). Then, we examined the asso - between blood pressure and arterial stiffness as evalu - ciation between serum LBP levels and aortic PWV in ated by brachial-ankle PWV [29] in patients with type 2 men (n  =  101) and women (n  =  95) separately. Serum diabetes, and a predictive value of brachial-ankle PWV LBP levels were found to be positively correlated with on the progression of coronary artery calcification [30] aortic PWV in men (r =  0.242, p  =  0.015), and the cor- The results from the present study are in agreement with relation remained significant (β  = 0.209, p = 0.011) after those of the previous study [15] and indicate a possible adjusting for age; BMI; systolic blood pressure; albumin; role of LBP in the progression of atherosclerosis. Further- eGFR; log [triglycerides]; HDL-cholesterol; log [hs-CRP]; more, this study is the first to report the impact of LBP use of statins; use of RAS inhibitors; use of calcium- on arterial stiffness in patients with type 2 diabetes, who channel blockers, and smoking status. On the contrary, have advanced arterial stiffening [22, 27, 31–33] and ele- no significant correlation was found between serum LBP vated cardiovascular mortality [34]. levels and aortic PWV in women (β = 0.028, p = 0.768). Although not statistically significant, the impact of serum Potential mechanisms underlying the relationship LBP levels on aortic PWV was greater in men (β = 0.146, between LBP and arterial stiffness p = 0.140) than in women (β = −0.020, p = 0.874), after Importantly, in this study, the relationship between further adjustment for log [HOMA-R]. serum LBP levels and aortic PWV was independent of traditional cardiovascular risk factors including age, Discussion obesity, renal dysfunction, hyperglycemia, and dyslipi- The present study demonstrated that serum LBP lev - demia. It is well recognized that low-grade inflammation els are positively associated with arterial stiffness, as is among the major factors involved in arterial stiffness assessed by aortic PWV, in patients with type 2 diabe- in the general population [35] and in patients with type tes. Serum LBP levels were positively correlated with 2 diabetes [33, 36]. Recent evidence indicates that bac- the parameters of obesity, insulin resistance, and inflam - terial endotoxins, or LPS, are an important source of mation in our diabetic subjects, which is in agreement vascular inflammation in atherosclerosis [4]. In  vitro with observations from previous studies of non-diabetic studies have demonstrated that LPS induced expression populations [9, 12, 15]. However, it is noteworthy that of matrix metalloproteinase-9 through the TLR4/nuclear the association between serum LBP levels and aortic factor-κB pathway [37, 38] and stimulated the release of PWV was independent of obesity, inflammation, and proinflammatory cytokines [39] in vascular smooth mus - other traditional cardiovascular risk factors. The results cle cells [37, 39] and endothelial cells [38]. In  vivo stud- further revealed that the association between serum LBP ies also showed that the blockade of LPS signaling by levels and aortic PWV was observed in men, but not in TLR4 antagonists reduced the infiltration of monocytes/ women. To our knowledge, this is the first report to dem - macrophages and expression of interleukin-6 and matrix onstrate the clinical implications of circulating LBP in the metalloproteinase-9 in the atherosclerotic lesions of dia- increased arterial stiffness in type 2 diabetes. betic mice [40]. In humans, prospective population-based studies showed that chronic infection [2] or endotoxemia Clinical association between serum LBP levels and arterial [3] is a strong risk factor of carotid atherosclerosis [2, 3] stiffness and cardiovascular diseases [3]. In a large population- This study clearly demonstrated that serum LBP levels based study, soluble CD14, a mediator for the activation are independently and positively associated with aor- of immune cells by LPS, was independently associated tic PWV in patients with type 2 diabetes. Two previous with aortic PWV, but not with carotid intima-media studies showed that serum LBP levels were a significant thickness [41]. In light of the combined experimental and predictor of prevalent coronary artery disease [7] and clinical evidence, and considering the fact that circulating Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 6 of 8 LBP binds to LPS and promotes the innate immune the present study, these previous studies did not stratify response [5], our data may indicate a critical role of the the data by sex. Several lines of evidence indicate the sex- LPS/LBP-induced inflammation in the pathogenesis of associated difference in inflammatory responses to LPS- arterial stiffness in type 2 diabetes. stimulation in neutrophils in  vitro models [49, 50] and Alternatively, the association between serum LBP in mice in vivo models [51], with increased responses in levels and aortic PWV can be explained by the indirect males than in females. Recent studies have also indicated effects of LBP on aortic PWV via obesity and insulin the sex-associated differences in the gut microbiome, one resistance. Recently, moderately increased LPS in circula- of the major sources of circulating LPS [42], and the sex- tion, or metabolic endotoxemia, in response to a high-fat dependent effects of diet on the gut microbiota in mice diet was shown to trigger obesity and insulin resistance and humans [52]. In a study performed in rats, systemic in mice [42, 43]. Several studies also showed that LBP proinflammatory cytokine levels in response to an oligo - is produced by adipocytes and plays an essential role in fructose-supplemented diet were higher in males than inflammation- and obesity-associated adipose tissue in females [53]. Based on these reports, we can hypoth- dysfunction [44, 45]. The relationship between LBP and esize that men with type 2 diabetes are more prone to the obesity, insulin resistance, and the metabolic syndrome immune response elicited by LPS from the gut micro- has been demonstrated in a number of studies per- biota than are women, leading to increased arterial stiff - formed in humans in both cross-sectional [8, 9, 11, 12] ness in men. To our knowledge, this study is the first to and prospective [10, 13] designs. In agreement with these elucidate the sex-related differences in the association studies, we found that serum LBP levels were positively between LBP and arterial stiffness. correlated with parameters of obesity, insulin resistance, and components of the metabolic syndrome in patients Limitations with type 2 diabetes (Table  2). Considering the evidence This study has several limitations. First, we evaluated that the metabolic syndrome [46] and insulin resistance the endotoxin-induced inflammation by measuring [33] are closely associated with increased arterial stiff - serum LBP levels, but not by a direct measurement of ness, it is conceivable that LBP affected aortic stiffness LPS. However, serum LBP levels, which have been pro- through insulin resistance in our subjects. However, posed as a surrogate marker of LPS-induced immune in our data, the association of obesity and dyslipidemia response in humans, can be simply and stably measured with aortic PWV was less significant compared with that by enzyme-linked immunosorbent assay [8, 10, 11]. We of age, hypertension, renal dysfunction, and serum LBP also did not include the evaluation of the LPS-related fac- levels (Table  3). Moreover, HOMA-R was not indepen- tors other than LBP, such as interleukin-6, soluble CD14, dently associated with aortic PWV and an additional and tumor-necrosis factor-α, that could strengthen our adjustment for log [HOMA-R] did not virtually affect the study. Second, since this was a cross-sectional study, the relationship between LBP and aortic PWV in the mul- causal relationship between LBP and arterial stiffness tivariate model (Model 2, Table  3). Thus, although LPS/ needs to be confirmed by longitudinal and/or interven - LBP-induced immune response is commonly involved tional studies. Third, the subjects were receiving statins in arterial stiffness [41], obesity, and insulin resistance and/or RAS inhibitors, which could have influenced [6, 8–13], our results indicate that the LPS/LBP-induced inflammation, vascular function, and related risk factors. innate immunity independently affects arterial stiffness To minimize the impact of these drugs, the presence of in patients with type 2 diabetes. these treatments was adjusted for in the multiple regres- sion analysis. Fourth, the sample size was too small in Sex‑dependent association between LBP and arterial the sex-stratified analysis to conclude with statistical sig - stiffness nificance the relationship between serum LBP levels and This study further revealed that the effect of serum LBP aortic PWV in men after adjusting for log [HOMA-R]. levels on aortic PWV is modified by sex and that there Finally, because our subjects were hospitalized in a uni- is a significant association between serum LBP levels versity hospital and had inadequate glycemic control, the and aortic PWV in men only. A number of studies have present results may not be generalized to the entire pop- shown sex-dependent association of arterial stiffness with ulation of patients with type 2 diabetes. metabolic risk factors, such as visceral adiposity [18], the metabolic syndrome, and its components [47, 48]. In pre- Conclusions vious studies on the LPS-related factors, sex-adjusted This study clearly demonstrated that serum LBP levels association was observed between LBP/soluble CD14 are independently and positively associated with arte- and aortic stiffness [41], carotid intima-media thickness rial stiffness in patients with type 2 diabetes. Our data [15], and cardiovascular disease [14]. However, unlike indicate a close link between LPS/LBP-induced innate Sakura et al. Cardiovasc Diabetol (2017) 16:62 Page 7 of 8 immunity and arterial stiffness in these patients. This Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- study further shows that LBP preferentially affects arte - lished maps and institutional affiliations. rial stiffness in men over women, indicating sex-related differences in the link between LBP and arterial stiffness. Received: 7 March 2017 Accepted: 3 May 2017 Further interventional studies are warranted to clarify whether the reduction of serum LPS levels by antibiot- ics or probiotics would reduce arterial stiffness and the risk of cardiovascular diseases in patients with type 2 References diabetes. 1. Hotamisligil GS. Inflammation and metabolic disorders. Nature. 2006;444:860–7. 2. Kiechl S, Egger G, Mayr M, Wiedermann CJ, Bonora E, Oberhollenzer F, Muggeo M, Xu Q, Wick G, Poewe W, et al. Chronic infections and the risk Abbreviations of carotid atherosclerosis: prospective results from a large population LPS: lipopolysaccharide; LBP: lipopolysaccharide-binding protein; PWV: study. Circulation. 2001;103:1064–70. pulse wave velocity; BMI: body mass index; CD: cluster of differentiation; TLR: 3. Wiedermann CJ, Kiechl S, Dunzendorfer S, Schratzberger P, Egger G, toll-like receptor; hs-CRP: high-sensitivity C-reactive protein; eGFR: estimated Oberhollenzer F, Willeit J. 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Journal

Cardiovascular Diabetology – Springer Journals

Published: Dec 1, 2017

Keywords: diabetes; angiology; cardiology

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