Plasma soluble vascular adhesion protein-1 concentration correlates with arterial stiffness: A cross-sectional study

Archives of Gerontology and Geriatrics 61 (2015) 67–71

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Plasma soluble vascular adhesion protein-1 concentration correlates with arterial stiffness: A cross-sectional study Da-Wei Chen a, Rui-Min Zhao b, Ying Jin a, Jing Zhang a, Chunlei Han c, Shu-Qiang Jiang d, Hai-Fang Zheng d, Jian-Chang Wang a,* a

Geriatric Institute, The General Hospital of the Air Force, PLA (The Chinese People’s Liberation Army), 30# Fucheng Road, Haidian District, Beijing 100142, China Clinical Laboratory Center, The General Hospital of the Air Force, PLA (The Chinese People’s Liberation Army), 30# Fucheng Road, Haidian District, Beijing 100142, China c Turku PET Centre, Turku University Hospital and University of Turku, Turku, Finland d Health Examination Center, the General Hospital of the Air Force, PLA (The Chinese People’s Liberation Army), 30# Fucheng Road, Haidian District, Beijing 100142, China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 December 2014 Received in revised form 2 April 2015 Accepted 20 April 2015 Available online 27 April 2015

Background: Arterial stiffness is related to inflammation, oxidative stress, advanced glycation end products (AGEs), and endothelial dysfunction. Vascular adhesion protein-1 (VAP-1) is both as an adhesion molecule involving in inflammation and as an amine oxidase producing aldehyde and hydrogen peroxide involved in protein cross-linking, oxidative stress and endothelial injury. Objective: We explored the associations of plasma soluble VAP-1 (sVAP-1) with arterial stiffness. Design: Cross-sectional study. Setting: Health Examination Center at the General Hospital of the Air Force in Beijing, China. Subjects: 568 Han Chinese healthy persons living in Beijing (aged 50.7  8.0 years). Methods: sVAP-1 concentration was assessed by enzyme-linked immunosorbent assay. Arterial stiffness was measured as brachial-ankle pulse wave velocity (baPWV) on both left and right sides of the examinees, and the larger and the mean values were recorded. Cardiovascular risk factors were investigated. Results: sVAP-1 was significantly associated with maximal or mean baPWV in subjects of age60 years after adjusting for baPWV-related confounders (b = 36.922, p < 0.05 or b = 32.512, p < 0.05) or after adjusting for all the variables (b = 37.924, p < 0.05 or b = 33.193, p < 0.05), but not in subjects of age <60 years. sVAP-1 had an independent and positive correlation with age (r = 0.222, p < 0.001). Conclusions: Plasma sVAP-1, increased with age, is associated with arterial stiffness in older individuals. VAP-1 may be important mechanism for vascular aging. ß 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Vascular adhesion protein-1 Semicarbazide-sensitive amine oxidase Arterial stiffness Aging

1. Introduction Arterial stiffness is a common change associated with aging (Shirwany & Zou, 2010; Zieman, Melenovsky, & Kass, 2005). There is evidence showing that arterial stiffness is associated with arterial stenosis (Zhang et al., 2011) and can predict the risks of future cardiovascular events such as coronary heart diseases and stroke (Vlachopoulos, Aznaouridis, & Stefanadis, 2010). Arterial

* Corresponding author. Tel.: +86 13331161600; fax: +86 01088410099. E-mail addresses: [email protected], [email protected] (J.-C. Wang). http://dx.doi.org/10.1016/j.archger.2015.04.007 0167-4943/ß 2015 Elsevier Ireland Ltd. All rights reserved.

stiffness can also predict all-cause and cardiovascular mortality (Vlachopoulos et al., 2010). In addition, arterial stiffness is also associated with functional outcome of acute ischemic stroke (Lee, Park, Kim, Kang, & Park, 2014). Some potential mechanisms, such as inflammation, oxidative stress, advanced glycation end products (AGEs), and endothelial dysfunction, are involved in vascular stiffening (Zieman et al., 2005; Lee & Oh, 2010; Ungvari, Kaley, de Cabo, Sonntag, & Csiszar, 2010). Vascular adhesion protein-1 (VAP-1) is a dual-function protein and implicated in vascular damage. VAP-1 can act as an adhesion molecule and is involved in the inflammation (Merinen et al., 2005). VAP-1 also has some semicarbazide-sensitive amine

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oxidase (SSAO) activity. SSAO is a multiple functional enzyme which can catalyze the breakdown of primary amines to aldehyde, hydrogen peroxide, and ammonia (Jalkanen & Salmi, 2008). The main deaminated products of SSAO, formaldehyde and methyglyoxal, can mediate direct cytotoxic damage to endothelial cells (Obata, 2006) or cross-link protein and DNA to generate advance glycated end products (AGEs) (Stolen et al., 2004b). Hydrogen peroxide is a source of oxidative stress. Therefore, we hypothesized that VAP-1 might play an important role in arterial stiffness by multiple mechanisms. VAP-1 is associated with cell membranes, and it is also present as a soluble form (sVAP-1) in plasma (Boomsma, Hut, Bagghoe, van der Houwen, & van den Meiracker, 2005). In this cross-sectional study, we analyzed the associations of plasma sVAP-1 concentration with brachial-ankle pulse wave velocity (baPWV). We found that plasma sVAP-1, positively associated with aging, was an independent risk factor of increased baPWV in subjects of age >55 years. 2. Materials and methods 2.1. Subjects In this cross-sectional study, the subjects who underwent a health examination at the General Hospital of Air Force PLA in Beijing city during 2013–2014, were invited to participate in this study. Eligibility criteria included the following: (1) age 30 years or older; (2) Han Chinese person; (3) living in Beijing more than 10 years; (4) with baPWV data. We excluded the subjects with following diseases since these diseases affected the plasma sVAP-1 concentration (Boomsma et al., 2005): (1) inflammatory liver diseases; (2) end-stage renal disease; (3) inflammatory skin diseases (e.g. psoriasis and atopic eczema); (4) inflammatory nervous system diseases (e.g. multiple sclerosis and Alzheimer’s disease); (5) inflammation-related ocular diseases (e.g. uveitis, age-related macular degeneration); (6) congestive heart failure. Participants provided written informed consent to participate in the study. An illiterate participant was required to provide verbal informed consent, which was recorded. The study was approved by the institutional review committee of Air Force General Hospital. In a preliminary study with a sample size of 40 subjects, the mean and standard deviation (SD) of sVAP-1 were 340.5 ng/mL and 88.5 ng/mL, respectively. Bases on this, assuming a permissible error of 10 ng/mL and a 5% significance level, a sample size of 310 subjects would be needed. A total of 600 subjects was planned in this survey to allow for subgroups analysis and subject exclusions. 2.2. Procedure of health examination Subjects were asked to have an overnight fast and to avoid intensive exercise, overeating and consuming caffeine and alcohol 12 h before the health examination. First, height and weight were measured, and blood pressure (BP) measured two times by an electric sphygmomanometer after 5 min at a supine position. Body mass index (BMI) was calculated as weight (kg)/height (m2). Second, venous blood samples were drawn for analysis of glucose, total cholesterol, triglycerides, low density lipoprotein (LDL) and high density lipoprotein (HDL), creatine at the clinical chemical laboratory center of the General Hospital of Air Force PLA. The glomerular filtration rate (GFR) was estimated by simplified MDRD (modification of diet in renal disease trial) equations. Impaired renal function was defined by GFR <80 mL/min. Plasma samples were stored at 70 ?C for the measurement of sVAP-1 concentration. Third, questionnaires were used to collect information on hypertension, diabetes mellitus, smoking and alcohol drinking. Smokers or alcohol users was defined as regular smoking or alcohol

consumption daily or every week for more than 6 months. Hypertension was defined as taking anti-hypertensive medications or exhibiting an average brachial artery systolic and diastolic BP equal to or greater than 140/90 mmHg. Diabetes mellitus was defined by treatment with insulin, oral anti-diabetic agents, or by a fasting glucose level 7.0 mmol/L. At last, baPWV were measured in noninvasive vascular evaluation laboratory. 2.3. Measurement of plasma sVAP-1 concentration sVAP-1 concentration was assessed by ELISA kit (MedSystem Diagnostic GmbH, Vienna, Austria) according to the manufacturer’s instructions. All analyzed serum samples were diluted 1:100 with a buffer supplied with the kit and then transferred to microtiter plates coated with anti-VAP-1 antibody. After addition of mouse antibody conjugated with biotin, the plate was incubated at room temperature for 2 h on a shaker (300 rounds/min). After incubation, streptavidin conjugated with horseradish peroxidase was added to each vial and after another 1 h of incubation on a shaker (300 rounds/min) the reaction was visualized using tetramethylbenzidine (TMB) reagent and phosphatic acid. Finally, the absorbance for each sample was read at 450 nm, using the Model 450 microplate reader (BIO-RAD Laboratories Inc., USA), and applying a reference wavelength of 620 nm. The linearity (R2) of the standard curves was 0.999–1.000. The coefficient of variation (CV) of the standard curves was 0.3–3.6%. The intra-batch CV was 0–5.4% and inter-batch CV was 0.3–9.5%. The calibration range was from 31.5 ng/mL to 2000 ng/mL. The intraclass correlation coefficients (ICC) for the reliability of sVAP-1 was 0.96(0.92–0.98). 2.4. Measurement of baPWV baPWV was measured by a single technician blinded for subject identification using an automatic device (Colin VP 1000, Omron co., Kyoto, Japan). The distance between the brachium and the ankle for baPWV was calculated by the height of the patient. PWV was calculated as the distance between the brachial and the ankle divided by the time delay between the two arterial points, expressed as centimeters per second. After examinations were performed on both the left and right sides, the larger value and the mean value were used for further analyses. The ICC for the reliabilities of larger value and mean value of baPWV was 0.97(0.89–0.99) and 0.97(0.90–0.99). 2.5. Statistical analysis Categorical variables were reported as the percentage of patients in the subgroup. The distributions of continuous variables were examined by the Kolmogorov–Smirnov test. Continuous variables distributed normally were presented as means and SD. Continuous variables with skewed distribution were analyzed after square root transformation into normal distribution and were presented as medians (interquartile ranges). In univariate analysis, the Student t tests was used for 2 class comparisons and one-way ANOVA for multiple-class comparisons of continuous variables. x2 tests was used for the comparisons of categorical variables. A series of linear regression models were used to analyze the associations of sVAP-1 with larger value and mean value of baPWV by adjusting for no variable, or for only significant variables associated with baPWV, or for all of the variables. The associations of sVAP-1 with baPWV were also analyzed according to age stratification, and the cutoffs of age groups were based on the sum of the two sum of square of the linear regression models (one model above the age cutoff and another model below the age cutoff). Pearson correlation coefficient t test was used for the association of sVAP-1 with age. The statistical significance was defined as

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p  0.05 and all statistical analyses were performed using the SPSS software version 16.0.

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Table 2 Correlations between baPWV and various categorical variables. Maximal baPWV

3. Results

Mean  SD

600 subjects who met the eligibility criteria, were invited to participate in our study. Six subjects declined to participate in this study. Fifteen subjects were excluded for diseases influencing sVAP-1(3 with inflammatory liver diseases, 2 with end-stage renal disease, 2 with psoriasis, 1 of eczema, 2 with Alzheimer’s disease, 3 with age-related macular degeneration, 2 with uveitis). Three plasma samples were lost and eight plasma samples were excluded because of hemolysis and chylemia. In the end, a total of 568 participants (210 females and 358 males) with a mean age of 50.7  8.0 years (range 30–86 years) were analyzed in this study (Supplementary Table 1). As shown in Table 1, both maximal and mean baPWV were associated with sVAP-1. These baPWVs were also associated with age, blood pressure/hypertension, glucose/diabetes mellitus, BMI, total cholesterol, triglycerides, LDL and GFR (Tables 1 and 2). After adjusting for the significant confounding variables related to baPWV or all variables, sVAP-1 became not significantly associated with maximal or mean baPWV (Table 3). When we added an interaction term of age  sVAP-1 into the models with all variables, there was an interaction between age and sVAP-1 on maximal or mean baPWV (b = 1.715, p < 0.001 or b = 1.573, p = 0.001). We further stratified subjects according to different cutoffs of age groups, and the largest sums of square of the linear regression models (0.709 and 0.713 for maximal and mean baPWV respectively) appeared at the cutoff of 60 years. sVAP-1 became significantly associated with maximal or mean baPWV in subjects of age 60 years after adjusting for these baPWV-related confounders, but not in subjects of age <60 years (Table 3). After adjusting for all variables, sVAP-1 was still significantly associated with maximal or mean baPWV in subjects of age 60 years (Table 3). Moreover, as shown in Fig. 1, sVAP-1 had a positive correlation with age (r = 0.283, p < 0.001) and this association was till significant after adjusted for all other variables (r = 0.222, p < 0.001). 4. Discussion Our study showed that plasma sVAP-1 concentration increased with age and was an independent determinant of baPWV in subjects aged 60 years. As we have known, this is the first study to show the association of sVAP-1 concentration with increased baPWV in the older.

Sex Male Female Hypertensiton Yes No Diabetes mellitus Yes No Smoking Yes No Alcohol use Yes No GFR <80 mL/min 80 mL/min

p

r Age Systolic BP Diastolic BP BMI FPG Total cholesterol Triglycerides LDL HDL Creatine SQRT(sVAP-1)

0.584 0.533 0.293 0.198 0.202 0.149 0.112 0.108 0.015 0.073 0.131

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.007 0.010 0.729 0.082 0.002

Mean baPWV r

p 0.557 0.540 0.295 0.186 0.196 0.150 0.109 0.108 0.007 0.074 0.133

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.009 0.010 0.874 0.077 0.001

sVAP-1 was transformed into normal distribution by SQRT (square root). sVAP1 = soluble vascular adhesive protein 1; baPWV = brachial-ankle pulse wave velocity; BP = blood pressure; BMI = body mass index; FPG = Fasting plasma glucose; LDL = low density lipoprotein; HDL = high density lipoprotein.

p

Mean  SD

p

1551  315 1539  349

0.664

1519  299 1508  338

0.700

1744  396 1468  258

<0.001

1703  379 1440  247

<0.001

1704  390 1525  313

<0.001

1666  368 1496  301

<0.001

1574  341 1533  321

0.167

1538  322 1504  310

0.222

1561  329 1538  328

0.415

1528  311 1508  316

0.465

1802  524 1536  313

0.024

1763  494 1505  300

0.021

baPWV = brachial-ankle pulse wave velocity; SD = standard deviation; GFR = glomerular filtration rate.

VAP-1 exists as transmembrane protein expressed on the surface of endothelium, smooth muscle cells and adipocytes, and also as a soluble form in the plasma. Adipocytes and vascular endothelial cells could be the source of sVAP-1. sVAP-1 is probably derived from membrane-bound form by a metalloproteasedependent shedding process. Release form adipose cells is regulated by TNF-a and insulin (Aalto, Havulinna, Jalkanen, Salomaa, & Salmi, 2014; Boomsma et al., 2005; Dunkel et al., 2008). Kurkija¨rvi et al. (1998) first detected sVAP-1 and found that its concentration in patients with inflammatory liver disease were higher than normal individuals. Later, lots of studies demonstrated that elevated levels of sVAP-1 were associated with some other conditions, such as diabetes and its complications, cardiovascular diseases and its mortality, inflammation-related skin, nervous system and ocular diseases, and so on (Aalto et al., 2014; Boomsma et al., 2005; Dunkel et al., 2008).sVAP-1 may play an important role in the arterial stiffness through three mechanisms. First is inflammation. The stability, resilience, and compliance of vascular wall are dependent on the relative contribution of its two prominent scaffolding proteins: collagen and elastin. The relative Table 3 Effect of plasma sVAP-1 concentration on baPWV in multivariable linear regression.

Table 1 Correlations between baPWV and various continuous variables. Maximal baPWV

Mean baPWV

Mean baPWV Crude model Model 1 Model 2 Maximal baPWV Crude model Model 1 Model 2

All (n = 568)

Age <60 years (n = 488)

Age 60 years (n = 80)

b

SE

b

SE

b

SE

15.102 1.24 0.527

4.723*** 3.982 4.077

0.141 4.520 4.239

3.899 3.609 3.703

45.159 32.512 33.193

15.415** 14.201* 14.272*

15.561 0.928 0.412

4.937** 4.179 4.280

0.066 4.524 4.411

4.072 3.711 3.869

48.014 36.922 37.924

16.097** 15.471* 15.533*

sVAP-1 was transformed into normal distribution by square root. Model 1 was adjusted for the variables significantly associated with baPWV (age, hypertension, DM, BMI, Triglyceride, LDL, GFR); Model 2 was adjusted for all the variables (age, sex, smoking, alcohol use, hypertension, DM, BMI, Triglyceride, LDL, GFR). sVAP-1 = soluble vascular adhesion protein 1; baPWV = brachial-ankle pulse wave velocity; DM = diabetes mellitus; BMI = body mass index; LDL = low density lipoproteins; GFR = glomerular filtration rate. * p < 0.05. ** p < 0.01. *** p < 0.001.

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Fig. 1. Correlation of sVAP-1 concentration with age. sVAP-1 concentration was transformed into normal distribution by SQRT (square root) which positively correlated to age (r = 0.286, p < 0.001). This correlation was still significant after adjustment for sex, smoking, alcohol use, hypertension, diabetes, body mass index, triglyceride, low density lipoproteins, and glomerular filtration rate (r = 0.222, p < 0.001). sVAP-1 = soluble vascular adhesive protein 1.

content of these molecules is normally held stable by a slow, but dynamic, process of production and degradation. Dysregulation of this balance, mainly by stimulation of an inflammatory milieu, leads to overproduction of abnormal collagen and diminished quantities of normal elastin, which contribute to vascular stiffness (Zieman et al., 2005). Histological examination of the intima of stiffened vessels reveals infiltration of macrophages and mononuclear cells (Zieman et al., 2005). VAP-1 is an adhesion molecule involved in the rolling, adhesion, and transmigration of lymphocytes, granulocytes and monocytes from the blood into sites of inflammation (Merinen et al., 2005). Current models propose that VAP-1 serves a dual adhesive function by its multiple O- and Nlinked oligosaccharides and by its enzymatic activity (Jalkanen & Salmi, 2008). In addition, the oxidase activity of VAP-1 may have signaling effects and induce expression of E- and P-selectins, intercellular adhesion molecule-1 (ICAM-1) in human endothelial cells involved in the leucocyte extravasation cascade (Jalkanen et al., 2007). Second is AGEs from non-enzymatic protein glycation. AGE-linked collagen is stiffer and less susceptible to hydrolytic turnover (Bailey, 2001; Verzijl et al., 2000). The resilience of elastin molecular is decreased after AGE cross-linking (Konova, Baydanoff, Atanasova, & Velkova, 2004). In the transgenic mouse model overexpressing Vap-1/SSAO activity, there was a higher pulse pressure (PP) than in control littermates (Gokturk et al., 2007). These transgenic mice exhibited an abnormal morphology of elastin in large arteries (e.g., Aorta and renal artery) where the elastic laminae were straight instead of folded due to contraction of the vessel wall in the absence of blood pressure (Go¨ktu¨rk et al., 2013). Electron microscopy showed that the elastic fibers were disorganized in tangled webs between the elastic laminae in the aorta of transgenic mice, whereas the fibers were smoothly arranged along the elastic laminae in control mice (Gokturk et al., 2007). The elevated PP, together with an abnormal elastin structure suggests an increased stiffness of large arteries as a results of an elevated SSAO activity (Kagan & Trackman, 1991) or by production of reactive species, such as formaldehyde or methylglyoxal (Stolen et al., 2004b). Third is vascular smooth muscle cell (VSMC) tone. VSMC tone can be modified by oxidant stress (Gurtner & Burke-Wolin, 1991). Vidrio et al. have proposed that SSAO-mediated hydrogen peroxide production could induce vasoconstriction of resistance vessels and increase vascular tone

(Vidrio et al., 2003). Gokturk et al. (2007) showed that the NO donor sodium nitroprusside gave an immediate decrease in blood pressure in the control mice, but only a minor response in the transgenic mice overexpressing SSAO activity, which suggested that an increase in SSAO activity would counteract the vascular dilation effect from NO. Previous reports showed that both SSAO activity and sVAP-1 concentration positively correlated with age in a Finnish population (Aalto et al., 2014, 2012) and in a Taiwanese population (Li et al., 2011; Wang et al., 2013). Our results further demonstrated that this association was not affected by other factors. There are two possible mechanisms for the observed correlation between sVAP-1 concentration and age. Firstly, abundant experimental and clinical data show that aging is associated with chronic low-grade inflammation (Ungvari et al., 2010). Even in normal healthy aging, there is an up-regulation of inflammatory cytokines, chemokines and adhesion molecules both in laboratory rodents and in primates (Ungvari et al., 2010). In humans, plasma concentration of several inflammatory markers (e.g., sVCAM-1, sICAM-1, sE-selectin) are positively correlated with age, independent of other cardiovascular risk factors (Ungvari et al., 2010). Since sVAP-1 is also an adhesion molecule and involved in inflammation (Merinen et al., 2005), it is logical to assume that sVAP-1 concentration increases with age like other adhesion molecules. Second, previous studies have reported that fasting plasma glucose levels increase with age because of impaired insulin secretion and insulin resistance (Toyoda et al., 2008). Stolen et al. demonstrate that hyperglycemia alone can upregulate sVAP-1 concentration in mice (Stolen et al., 2004a), and Abella et al. have shown that insulin can down-regulate the release of VAP-1 into the culture media of 3T3-L1adipocytes (Abella et al., 2004). In humans, the insulin is a negative regulator (Salmi et al., 2002), and acute and chronic hyperglycemia are positive regulators for plasma sVAP-1 concentration (Li et al., 2009). The relationship between sVAP-1 concentration and glucose/insulin level may be likely due to the insulin-like effects of sVAP-1 concentration (Stolen et al., 2004a). This relationship can explain that sVAP-1 concentration will increase with age to compensate for decrease of insulin secretion and insulin sensitivity. Interestingly, our study showed that sVAP-1 concentration was an independent risk factor for arterial stiffness only in the older participants (age 60 years). Since sVAP-1 is increased with age, it is reasonable that sVAP-1 begin to have an effect on arterial stiffness in older persons who are with enough high sVAP-1 concentration. In addition, PWV may be a more sensitive marker for arterial stiffness in older persons as it shows a concave curvilinear relationship with age (Lee & Oh, 2010). Therefore, the correlation of sVAP-1 concentration with baPWV was not found in younger individuals. We are aware that our study has some inherent limitations. In this study, baPWV, rather than carotid-femoral PWV (cfPWV) which represents the gold standard among the PWV parameters and reflects the stiffness of central artery, was used to assess arterial stiffness. However, recent studies showed that baPWV was significantly and positively associated with cfPWV, and that both PWV values were similarly associated with cardiovascular risk factors and clinical events (Lee et al., 2014). In addition, PWV and the augmentation index (AI) are the 2 major non-invasive methods of assessing arterial stiffness. Recent studies suggest that AI might be a more sensitive marker of arterial stiffness in younger individuals, and PWV is more sensitive in older persons (Lee & Oh, 2010). Since we only measured PWV but not AI in this study and found the correlation of sVAP-1 with arterial stiffness in older persons, it will deserve to study this correlation in younger persons by using AI. This cross-sectional study did not suggest a causal relation between sVAP-1 and arterial stiffness. However, sVAP-1 may affect the arterial stiffness by multiple mechanisms. At last,

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generalization of the results to other country populations may be cautious because all the subjects in our study were Han Chinese long-time living in Beijing. In conclusion, our study showed that plasma sVAP-1 concentration increased with age, and was an independent determinant of arterial stiffness in older individuals. These results suggests that sVAP-1 may be important mechanism for age-associated arterial stiffness (vascular aging). In the further, sVAP-1 may become a target for preventing and treating arterial stiffness. Conflict of interest statement The authors of the present study have no interest which might be perceived as posing a conflict or bias. Acknowledgments This work was supported by grants from the Military Healthcare Committee (12BJZ21). We thank the Clinical Aviation Medicine Research Center of the General Hospital of Air Force, PLA. The studies were conducted within the Centre of Excellence (COE) supported by the Academy of Finland, the University of Turku, the Turku University Hospital, and the A˚bo Akademin University. References Aalto, K., Havulinna, A. S., Jalkanen, S., Salomaa, V., & Salmi, M. (2014). Soluble vascular adhesion protein-1 predicts incident major adverse cardiovascular events and improves reclassification in a Finnish prospective cohort study. Circulation Cardiovascular Genetics, 7(4), 529–535. Aalto, K., Maksimow, M., Juonala, M., Viikari, J., Jula, A., Ka¨ho¨nen, M., et al. (2012). Soluble vascular adhesion protein-1 correlates with cardiovascular risk factors and early atherosclerotic manifestations. Arteriosclerosis, Thrombosis, and Vascular Biology, 32(2), 523–532. Abella, A., Garcı´a-Vicente, S., Viguerie, N., Ros-Baro´, A., Camps, M., Palacı´n, M., et al. (2004). Adipocytes release a soluble form of VAP-1/SSAO by a metalloproteasedependent process and in a regulated manner. Diabetologia, 47(3), 429–438. Bailey, A. J. (2001). Molecular mechanisms of ageing in connective tissues. Mechanisms of Ageing and Development, 122(7), 735–755. Boomsma, F., Hut, H., Bagghoe, U., van der Houwen, A., & van den Meiracker, A. (2005). Semicarbazide-sensitive amine oxidase (SSAO): From cell to circulation. Medical Science Monitor, 11(4), RA122–RA126. Dunkel, P., Gelain, A., Barlocco, D., Haider, N., Gyires, K., Sperla´gh, B., et al. (2008). Semicarbazide-sensitive amine oxidase/vascular adhesion protein 1: Recent developments concerning substrates and inhibitors of a promising therapeutic target. Current Medicinal Chemistry, 15(18), 1827–1839. Gokturk, C., Sugimoto, H., Blomgren, B., Roomans, G. M., Forsberg-Nilsson, K., Oreland, L., et al. (2007). Macrovascular changes in mice overexpressing human semicarbazide-sensitive amine oxidase in smooth muscle cells. American Journal of Hypertension, 20(7), 743–750. Go¨ktu¨rk, C., Nilsson, J., Nordquist, J., Kristensson, M., Svensson, K., So¨derberg, C., et al. (2013). Overexpression of semicarbazide-sensitive amine oxidase in smooth muscle cells leads to an abnormal structure of the aortic elastic laminas. American Journal of Pathology, 163(5), 1921–1928. Gurtner, G. H., & Burke-Wolin, T. (1991). Interactions of oxidant stress and vascular reactivity. American Journal of Pathology, 260(4 Pt 1), L207–L211.

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