Markers of subclinical atherosclerosis in premenopausal women with vitamin D deficiency and effect of vitamin D replacement

Atherosclerosis 237 (2014) 784e789

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Markers of subclinical atherosclerosis in premenopausal women with vitamin D deficiency and effect of vitamin D replacement Kadri Murat Gurses a, *, Lale Tokgozoglu a, Muhammed Ulvi Yalcin a, Duygu Kocyigit a, Muhammet Dural a, Hande Canpinar b, Hikmet Yorgun a, Mehmet Levent Sahiner a, Ergun Baris Kaya a, Safak Akin c, Alper Gurlek c, Dicle Guc b, Kudret Aytemir a a b c

Department of Cardiology, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey Cancer Institute Basic Oncology Department, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey Department of Internal Medicine, Section of Endocrinology and Metabolism, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 August 2014 Received in revised form 5 October 2014 Accepted 17 October 2014 Available online 29 October 2014

Background: Recent studies have revealed a relationship between vitamin D deficiency and atherosclerosis. This study aims to investigate the impact of vitamin D deficiency and replacement on markers of subclinical atherosclerosis in young premenopausal women in whom vitamin D deficiency is prevalent. Methods: Thirty-one premenopausal vitamin D deficient women and 27 age and gender-matched control subjects were enrolled in this study. Markers of subclinical atherosclerosis including carotid intima-media thickness (cIMT), flow-mediated dilatation (FMD), endothelial progenitor cell (EPC) count and cytokine levels were determined at baseline. All measurements were repeated at 6-month follow-up in vitamin D-deficient subjects after vitamin D replacement. Results: Vitamin D deficient premenopausal women had lower FMD (9.9 ± 1.3 vs. 13.8 ± 1.7%, p < 0.001) and EPC counts at baseline. This population also had lower IL-10 and higher IL-17 levels. A 6-month vitamin D replacement therapy resulted in a significant increase in FMD (9.9 ± 1.3 vs. 11.4 ± 1.4%, p < 0.001) and EPC counts. Furthermore, cytokine profile shifted toward a more anti-inflammatory phenotype including elevated IL-10 and decreased IL-17 levels. cIMT was not different between patient and control groups and did not change following vitamin D replacement. Change in 25(OH)D and IL-17 levels were independent predictors of the change in FMD measurements following vitamin D replacement. Conclusion: This study demonstrates that endothelial function is impaired in otherwise healthy vitamin D deficient young premenopausal women and improves with 6-month replacement therapy. Immune-modulatory effects of vitamin D may, at least partly, be responsible for its beneficial effects on vascular health. © 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Vitamin D Subclinical atherosclerosis Endothelial function Endothelial progenitor cell count Inflammation

1. Introduction Vitamin D is a secosteroid hormone, which plays a crucial role in mineral homeostasis and skeletal health [1]. Vitamin D has also been implicated in a wide range of systemic diseases, including autoimmune disorders, cancer, diabetes mellitus and cardiovascular diseases [2]. Previous studies have demonstrated an association of vitamin D deficiency with cardiovascular mortality, atherosclerosis and atherosclerotic risk factors; however the exact mechanism underlying this relationship is still unclear [3]. Serum 25-hydroxyvitamin D [25(OH)D] level is considered as the best indicator of vitamin D status [1]. Although there is a lack of

* Corresponding author. E-mail address: [email protected] (K.M. Gurses). http://dx.doi.org/10.1016/j.atherosclerosis.2014.10.096 0021-9150/© 2014 Elsevier Ireland Ltd. All rights reserved.

universally accepted definition for vitamin D deficiency, recent guidelines define vitamin D deficiency and insufficiency as serum 25(OH)D < 20 ng/mL and 21e29 ng/mL, respectively [4]. According to this definition, one billion people worldwide and 36% of healthy young adults in the United States have vitamin D deficiency [4]. Vitamin D deficiency is more prevalent among young women in Middle Eastern populations. A previous study in Turkey reported that 54.3% of premenopausal women have vitamin D deficiency [5]. However, the impact of vitamin D deficiency on current and forthcoming vascular health status in these young premenopausal women has not been investigated yet. Cardiovascular disease is a global health problem with high morbidity and mortality rates in women. In Turkey, 44.6% of deaths in women have been reported to be caused by cardiovascular diseases [6]. Similar mortality rates associated with cardiovascular diseases in women have been stated in epidemiological studies

K.M. Gurses et al. / Atherosclerosis 237 (2014) 784e789

performed in countries with similar dietary and geographical features including Greece (49.30%) and Italy (42.68%) [7]. Identifying the risk factors is of vital importance for taking preventive measures to lower associated mortality rates. Therefore, in this study, we aimed to investigate the effects of vitamin D deficiency and replacement on markers of subclinical atherosclerosis including carotid intima-media thickness (cIMT), flow-mediated dilatation (FMD), circulating endothelial progenitor cell (EPC) count and cytokine profile in healthy premenopausal women. 2. Methods 2.1. Study population 31 premenopausal women (age: 33.1 ± 5.7 years) who were diagnosed with vitamin D deficiency and scheduled for vitamin D replacement according to 2011 Guideline of Endocrine Society [4] and 27 control subjects (age: 34.1 ± 7.6 years) were included in this study between June 2012eJanuary 2013. Patients with atherosclerotic vascular disease, major risk factors for atherosclerotic diseases (diabetes mellitus, hypertension, dyslipidemia, smoking, family history for premature atherosclerosis), chronic inflammatory disease, malignancy, use of drugs affecting endothelial progenitor cell count or function (statin, angiotensin- converting enzyme inhibitor (ACEi)/ angiotensin receptor blocker (ARB), nitrate, calcium channel blocker (CCB), oral contraceptive), acute or chronic infections were excluded. The study was approved by the local ethics committee of Baskent University Faculty of Medicine (Reference No. KA 12/159) and was carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. 2.2. Study protocol Medical history and detailed physical examination findings including height, weight, waist circumference and blood pressure (BP) were recorded for all patients. In addition to routine complete blood cell count and biochemistry tests, endothelial functions were assessed with FMD; cIMT was measured with ultrasonography and left ventricular systolic and diastolic functions, chamber dimensions and valvular functions were evaluated with transthoracic echocardiography in the study population. Furthermore, EPC (CD34þ/KDRþ and CD133þ/KDRþ) count was determined by flow cytometry and cytokine (IFN-g, IL-10, IL-13 and IL-17) levels were measured with enzyme-linked immunosorbent assay (ELISA) methods in the Basic Oncology Department. Patients with a serum concentration of 25(OH)D < 20 ng/mL were defined as vitamin D deficient. Vitamin D3 treatment with 50.000 IU/week for 8 weeks and maintenance dose of 1.500e2.000 IU/day were administered to vitamin D deficient patients in accordance with the 2011 Guideline of Endocrine Society [4]. Target serum 25(OH)D level following replacement was identified as 30 ng/mL. Subjects who underwent vitamin D replacement were re-evaluated for the same parameters at the sixth month.

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ranges were stated as 10e60 ng/mL in winter and 20e120 ng/mL in summer in the manufacturer's instructions. 2.4. Vascular endothelial function Vascular endothelial function was evaluated with FMD of the brachial artery. All subjects were required to fast and avoid smoking, alcohol or caffeine consumption for at least 8 h prior to the FMD measurement. Furthermore, it was confirmed that the patients did not receive any medication that could affect endothelial function in the last week prior to the measurements. Considering that the study population was all of female gender and hormones could have an effect on vascular function, all measurements were taken in the follicular phase of the menstrual cycle. All measurements were performed in a quiet room at room temperature of 20e25  C, by a single experienced cardiologist blinded to the patients' diagnosis and clinical characteristics. Intra-observer variability testing for repeated FMD measurements from 20 subjects revealed a correlation coefficient of 0.90 (p < 0.001). Ultrasound scans were performed with a high-resolution ultrasonographic scanner (Vivid S6; GE Healthcare, Horten, Norway) equipped with a 10-MHz linear-array transducer found in the Hacettepe University Department of Cardiology. The right brachial artery was scanned over a longitudinal section approximately 2 cm above the antecubital fossa. The electrocardiogram was monitored continuously. After measurement of the baseline brachial artery diameter, which was calculated as the mean of 3 end-diastolic brachial artery diameter measurements, the cuff of a sphygmomanometer was placed on the upper antecubital fossa. The pneumatic tourniquet was held inflated around the forearm at a pressure of 50 mmHg above the subject's systolic blood pressure for 5 min. The arterial diameter was measured for five times with 1 min intervals after rapid deflation of the pneumatic tourniquet and the largest end-diastolic diameter was used to assess FMD. FMD values were defined as the percentage change of the brachial artery diameter following reactive hyperemia compared with the baseline. 2.5. Carotid intima-media thickness Carotid intima-media thickness (cIMT) was defined as the distance between the mediaeadventitia interface and the lumeneintima interface and measurement was performed using a high-resolution ultrasonographic scanner (Vivid S6; GE Healthcare, Horten, Norway) equipped with an 10-MHz linear-array transducer found in the Hacettepe University Department of Cardiology. Measurements were taken following at least 8 h of fasting in a quiet room at room temperature by an experienced cardiologist blinded to the patients' diagnosis and clinical characteristics. Patients were examined in the supine position, with the head slightly hyperextensed and turned away from the side that was being scanned at 30e45 . cIMT was measured at the far wall of the right and left common carotid arteries 10e20 mm proximal to the carotid bulb. Measurements were taken on each side, and the mean of the greatest cIMT obtained on each side was reported.

2.3. Biochemical analysis of 25(OH)D

2.6. Endothelial progenitor cell count

Venous blood samples were obtained from all participants in the morning following overnight fasting and analyzed in the Hacettepe University Biochemistry Laboratories. Plasma 25(OH)D levels were measured with the high pressure liquid chromatography device (Shimadzu LC-20AT) by chromatographic methods using the Immuchrom (Immuchrom GmbH, Hessen, Germany) 25(OH)D kit. The analytical detection limit for the kit was 2.3 ng/mL. Reference

Peripheral venous blood samples were taken into the citrate containing tubes for the isolation of EPCs. Peripheral blood mononuclear cells (PBMCs) were isolated from these samples by Ficoll Histopaque density centrifugation (Histopaque-1077, SigmaeAldrich, St. Louis, MO, USA). Cell suspensions were labeled with CD34FITC (Clone 561, Biolegend), CD133PE (AC133, mACS, Miltenyl Biotec) and CD309 PerCP (Clone HICDR-1, Biolegend)

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antibodies in order to identify endothelial cell progenitors and Mouse IgG1 PerCP (MCPC-21, Biolegend), Mouse IgGaFITC (MOPc173, Biolegend) and Mouse IgG2a PE (MOPC-173, Biolegend) as isotype-matched antibodies. Cells labeled with specific antibodies were analyzed by flow cytometry (Beckman Coulter EPICS XL MCL, USA) in which 500.000 events were counted. The absolute number of cells expressing CD34þ/KDRþ and CD133þ/KDR þ per 500.000 events in the lymphocyte gate was calculated.

used, respectively. Spearman correlation analysis was used to demonstrate the relationship between changes in parameters following replacement. Multiple linear regressions were performed to determine predictors of DFMD. Statistical analyses were performed using SPSS statistical software (version 21.0; SPSS Inc., Chicago, Illinois, USA). A two-tailed p < 0.05 was considered statistically significant. 3. Results

2.7. Serum cytokine levels 3.1. Study population Serum samples collected from the patients were store at 800 C until used. Serum cytokine levels, IFN-g (Aviva Systems Biology, San Diego, CA, USA), IL-10 (Aviva Systems Biology, San Diego, CA, USA), IL-13 (e Bioscience, San Diego, CA, USA) and IL-17 (Aviva Systems Biology, San Diego, CA, USA) were analyzed by ELISA. The assay was performed according to the manufacturer's instructions. 2.8. Statistical analysis Normally distributed parameters were presented as mean ± standard deviation and skewed parameters were expressed as median (interquartile range). Descriptive data were presented as frequencies (number and percentage) and compared using chisquare test. Univariate analyses were performed on continuous variables with the use of the independent student's t-test for normally distributed variables and the ManneWhitney U test for nonnormally distributed data. For comparing continuous variables (values before and after replacement) with normal and non-normal distribution; dependent sample's t-test and Wilcoxon tests were

Table 1 Baseline characteristics of the study population. Control group (n ¼ 27) Clinical parameters Age (years) 34.10 BMI (kg/m2) 23.50 Waist circumference 77.80 (cm) Systolic blood pressure 109.10 (mmHg) Diastolic blood pressure 67.40 (mmHg) Biochemical parameters Total cholesterol 4.83 (mmol/L) LDL cholesterol 2.79 (mmol/L) HDL cholesterol 1.60 (mmol/L) Triglyceride (mmol/L) 0.95 Fasting blood glucose 84.20 (mg/dL) TSH (mIU/mL) 1.78 Creatinine (mg/dL) 0.67 Albumin (mg/dL) 4.40 Calcium (mg/dL) 9.60 Phosphorus (mg/dL) 3.57 WBC count (X103/mL) 6.51 25(OH)D (ng/mL) 34.40 Echocardiographic parameters End-diastolic diameter 4.53 (cm) Ejection fraction (%) 68.10

3.2. Effects of vitamin D replacement Vitamin D deficient group (n ¼ 31)

p value

± 7.60 ± 2.30 ± 7.10

33.10 ± 5.70 22.80 ± 3.10 77.00 ± 8.60

0.597 0.280 0.686

± 9.80

113.20 ± 11.20

0.228

± 6.60

70.70 ± 7.40

0.152

± 0.67

4.78 ± 0.93

0.774

± 0.59

2.74 ± 0.88

0.785

± 0.28

1.63 ± 0.36

0.757

± 0.51 ± 8.80

0.93 ± 0.51 85.50 ± 10.50

0.623 0.596

± ± ± ± ± ± ±

1.93 0.65 4.50 9.50 3.46 6.30 10.60

± ± ± ± ± ± ±

0.936 0.495 0.152 0.140 0.398 0.469 <0.001*

0.75 0.06 0.28 0.38 0.32 1.11 10.30

Baseline clinical, biochemical and echocardiographic parameters of the groups are presented in Table 1. Patient and control groups were similar with respect to age, body mass index (BMI), waist circumference, left ventricular systolic function and systolic and diastolic BP (Table 1, all p > 0.05). Furthermore, there were no significant differences in baseline renal functions, white blood cell (WBC) counts and serum lipid, fasting blood glucose, thyroid stimulating hormone (TSH), calcium and phosphorus levels between two groups (Table 1, all p > 0.05). Baseline serum 25(OH)D levels were lower in the patient group compared with the control group (p < 0.001). Baseline measurements for cIMT, FMD, EPC counts and cytokine levels are presented in Table 2. Baseline cIMT, IL-13 and IFN-g levels were similar between the two groups (all p > 0.05). On the other hand, baseline FMD measurements for the vitamin D deficient group were significantly lower when compared to the controls (p < 0.001). CD34þ/KDRþ and CD133þ/KDR þ EPC counts and IL-10 levels were also significantly lower in the patient group (all p < 0.001). IL-17 levels were found to be significantly higher in the patient group (p < 0.001).

1.00 0.11 0.22 0.16 0.61 1.12 4.70

± 0.24

4.46 ± 0.29

0.294

± 3.00

68.40 ± 3.20

0.774

25(OH)D, 25-hydroxy vitamin D; BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TSH, thyroid-stimulating hormone; WBC, white blood cell. *p < 0.05.

Effects of vitamin D replacement in the patient group are presented in Table 3. At 6 months follow-up, patients who received vitamin D replacement therapy had a significantly increased serum 25(OH)D levels (p < 0.001). Vitamin D replacement did not have significant effect on BMI, left ventricular systolic function, blood pressure, renal function, WBC count and serum levels of lipids, fasting blood glucose, TSH, calcium and phosphorus (all p > 0.05). Carotid intima-media thickness (cIMT) measurements and IFNg levels did not change significantly following vitamin D replacement (all p > 0.05). There was a significant increase in FMD values (p < 0.001) and both CD34þ/KDRþ (p < 0.001) and CD133þ/KDRþ Table 2 Baseline parameters related to subclinical atherosclerosis and cytokine profile. Control group (n ¼ 27)

Vitamin D deficient group (n ¼ 31)

Parameters related to subclinical atherosclerosis cIMT (mm) 0.44 ± 0.02 0.45 ± 0.02 FMD (%) 13.80 ± 1.70 9.90 ± 1.30 CD34þ/KDR þ EPCs 64.50 ± 17.10 26.20 ± 20.50 (/mL) 49.40 ± 23.10 27.40 ± 18.50 CD133þ/KDR þ EPCs (/mL) Parameters related to cytokine profile IL- 10 (pg/mL) 15.78 ± 2.10 10.99 ± 4.15 IL-13 (pg/mL) 1.40 (0.10e4.56) 1.40 (0.08e3.45) IL-17 (pg/mL) 5.64 ± 2.99 11.76 ± 4.99 IFN-g (pg/mL) 3.39 (1.10e13.31) 3.93 (1.70e14.30)

p value

0.275 <0.001* <0.001* <0.001*

<0.001* 0.258 <0.001* 0.307

cIMT, carotid intima-media thickness; EPC, endothelial progenitor cell; FMD, flowmediated dilatation; IFN, interferon; IL, interleukin. *p < 0.05.

K.M. Gurses et al. / Atherosclerosis 237 (2014) 784e789 Table 3 Effects of vitamin D replacement on baseline characteristics of the patient group. Patient group: Before replacement (n ¼ 31)

Patient group: After replacement (n ¼ 31)

Clinical parameters BMI (kg/m2) 22.80 ± 3.10 22.70 ± 3.00 Waist circumference (cm) 77.00 ± 8.60 76.70 ± 8.80 Systolic blood pressure 113.20 ± 11.20 111.50 ± 10.50 (mmHg) Diastolic blood pressure 70.70 ± 7.40 68.90 ± 7.00 (mmHg) Biochemical parameters Total cholesterol (mmol/L) 4.78 ± 0.93 4.76 ± 0.78 LDL cholesterol (mmol/L) 2.74 ± 0.88 2.72 ± 0.70 HDL cholesterol (mmol/L) 1.63 ± 0.36 1.68 ± 0.31 Triglyceride (mmol/L) 0.93 ± 0.51 0.92 ± 0.54 Fasting blood glucose 85.50 ± 10.50 85.00 ± 8.00 (mg/dL) TSH (mIU/mL) 1.93 ± 1.00 1.97 ± 0.65 Creatinine (mg/dL) 0.65 ± 0.11 0.64 ± 0.10 Albumin (mg/dL) 4.50 ± 0.22 4.50 ± 0.23 Calcium (mg/dL) 9.50 ± 0.16 9.60 ± 0.35 Phosphorus (mg/dL) 3.46 ± 0.61 3.56 ± 0.47 WBC count (x103/mL) 6.30 ± 1.12 6.47 ± 1.02 25(OH)D (ng/mL) 10.60 ± 4.70 31.40 ± 8.60 Echocardiographic parameters End-diastolic diameter (cm) 4.46 ± 0.29 4.47 ± 0.29 Ejection fraction (%) 68.40 ± 3.20 68.40 ± 2.80 Parameters related to subclinical atherosclerosis cIMT (mm) 0.45 ± 0.02 0.44 ± 0.02 FMD (%) 9.90 ± 1.30 11.40 ± 1.40 CD34þ/KDR þ EPCs (/mL) 26.20 ± 20.50 40.50 ± 19.20 CD133þ/KDR þ EPCs (/mL) 27.40 ± 18.50 33.90 ± 18.20 Parameters related to cytokine profile IL- 10 (pg/mL) 10.99 ± 4.15 14.33 ± 3.20 IL-13 (pg/mL) 1.40 (0.08e3.45) 1.55 (0.30e3.84) IL-17 (pg/mL) 11.76 ± 4.99 6.95 ± 3.56 IFN-g (pg/mL) 3.93 (1.70e14.30) 3.74 (1.49e14.30)

p value

0.778 0.756 0.344 0.237

0.981 0.842 0.632 0.732 0.687 0.501 0.344 0.518 0.334 0.222 0.359 <0.001* 0.784 0.827 0.157 <0.001* <0.001* 0.025* <0.001* 0.022* <0.001* 0.383

25(OH)D, 25-hydroxy vitamin D; BMI, body mass index; cIMT, carotid intima-media thickness; EPC, endothelial progenitor cell; FMD, flow-mediated dilatation; HDL, high-density lipoprotein; IFN, interferon; IL, interleukin; LDL, low-density lipoprotein; TSH, thyroid-stimulating hormone; WBC, white blood cell. *p < 0.05.

(p ¼ 0.025) EPC counts after treatment. Cytokine balance in vitamin D deficient patients shifted toward a more anti-inflammatory phenotype following replacement therapy that IL-10 (p < 0.001) and IL-13 (p ¼ 0.022) levels increased, whereas IL-17 (p < 0.001) levels decreased significantly. 3.3. Correlation analyses Univariate and multivariate correlation analyses were done to determine the factors influencing endothelial function after treatment and data regarding these analyses is presented in Table 4. Change in FMD measurements was positively correlated with the change in 25(OH)D levels (r ¼ 0.811, p < 0.001), number of CD34þ/ KDRþ (r ¼ 0.801, p < 0.001) and CD133þ/KDRþ (r ¼ 0.631, p < 0.001) EPCs, IL-10 (r ¼ 0.645, p < 0.001) and IL-13 (r ¼ 0.485, p ¼ 0.006) levels, whereas it was negatively correlated with the change in IL-17 levels (r ¼ 0.793, p < 0.001) (Fig. 1). Multivariate linear regression model revealed that the change in 25(OH)D levels (b ¼ 0.024, p ¼ 0.016) and IL-17 levels (b ¼ 0.122, p < 0.001) independently predicted the change in FMD measurements following vitamin D replacement. 4. Discussion The results of this study show that vitamin D deficient premenopausal women had impaired endothelial function reflected by

787

Table 4 Correlation of change in FMD with the change in baseline characteristics after replacement in the vitamin D deficient group. Univariate linear regression analysis

Multivariate linear regression analysis

DFMD

r

p value

b

D25(OH)D DTotal cholesterol DLDL DHDL DTriglyceride DFasting blood glucose DTSH DAlbumin DCreatinine DCalcium DPhosphorus DWBC count DcIMT DIL-13 D IL-10 D IL-17 DIFNg D CD34þ/KDR þ EPCs D CD133þ/KDR þ EPCs

0.811 0.047 0.087 0.063 0.277 0.206 0.025 0.258 0.087 0.086 0.361 0.125 0.173 0.485 0.645 0.793 0.290 0.801 0.631

<0.001* 0.803 0.642 0.736 0.132 0.266 0.894 0.161 0.643 0.646 0.046* 0.503 0.351 0.006* <0.001* <0.001* 0.114 <0.001* <0.001*

p value 0.024

0.016*

0.076

0.300

0.021

0.784

0.066

0.399

0.034 0.030 0.122 0.023 0.077 0.017

0.802 0.792 <0.001* 0.746 0.601 0.931

25(OH)D, 25-hydroxy vitamin D; cIMT, carotid intima-media thickness; EPC, endothelial progenitor cell; FMD, flow-mediated dilatation; HDL, high-density lipoprotein; IFN, interferon; IL, interleukin; LDL, low-density lipoprotein; TSH, thyroid-stimulating hormone; WBC, white blood cell. *p < 0.05.

lower FMD; CD34þ/KDRþ and CD133þ/KDR þ EPC counts. This population also had a pro-inflammatory cytokine profile determined with lower IL-10 and higher IL-17 levels. A 6-month vitamin D supplementation in accordance with the 2011 Guideline of Endocrine Society resulted in a significant improvement in vitamin D status accompanied with amelioration of endothelial function demonstrated by an increase in FMD; CD34þ/KDRþ and CD133þ/ KDR þ EPC counts. Furthermore, cytokine profile shifted toward a more anti-inflammatory phenotype including elevated IL-10 and IL-13 and decreased IL-17 levels. cIMT was not different between patient and control groups and did not change following vitamin D replacement in the vitamin D deficient group. Change in 25(OH)D and IL-17 levels were independent predictors of the change in FMD measurements following vitamin D replacement. To the best of our knowledge, this is the first study revealing the effects of vitamin D deficiency and replacement on vascular function and cytokine balance in this population. Impact of vitamin D deficiency on development of atherosclerosis has been demonstrated in several epidemiological studies [8,9] and has been further validated in animal models [10,11]. Various mechanisms have been proposed to clarify the relationship between vitamin D and atherosclerosis [12]. Among them, some have pointed out that vitamin D deficiency may have an indirect effect via other cardiovascular risk factors [13,14] and reninangiotensin-aldosterone system [15]; whereas the others have indicated a direct role via cardiovascular and immune system cells expressing vitamin D receptors (VDR) [12]. Impairment in endothelial function is known to be the initial step in development of atherosclerosis [16]. Various studies have demonstrated that lower vitamin D levels were associated with impaired endothelial function [17,18]. However, regarding the impact of vitamin D replacement on vascular function, contradictory data have been obtained in studies performed on vitamin D deficient populations with different baseline characteristics in terms of gender, race and co-morbidities [18e21]. In our study, 31 vitamin D deficient otherwise healthy premenopausal women had impaired endothelial function reflected by lower FMD compared to

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Fig. 1. Correlation of change in FMD with changes in 25(OH)D (panel A) and IL-17 levels (panel B).

age and gender matched control group. A 6-month vitamin D supplementation resulted in amelioration of endothelial function demonstrated by an increase in FMD accompanied with an increase in EPC counts and pro-inflammatory shift in cytokine profile. Studies investigating the effects of vitamin D replacement on endothelial function in high risk patients, such as type 2 diabetic patients, have failed to demonstrate beneficial effects [19,21]. In these studies, approximately 90% of patients were reported to use renin-angiotensin-aldosteron system blockers and/or statins. These drugs are known to have an impact on endothelial functions via mechanisms [22,23] which are responsible for the substantial effects of vitamin D on vascular status, including promotion of nitric oxide formation and inhibition of anti-inflammatory and oxidative stress [24,25]. Furthermore, Ott et al. have shown that adverse effects of vitamin D deficiency on renal vasculature could be alleviated with rosuvastatin therapy [26]. Therefore, use of these drugs may have concealed the impact of vitamin D replacement on endothelial function in the forementioned studies [19,21]. Circulating EPCs are proposed to exert an important function as an endogenous repair mechanism to maintain the integrity of the endothelial layer, which is crucial to prevent atherosclerotic lesion development and thrombotic complications [27]. Cianciolo et al. have reported that EPCs expressed VDRs and this expression was dependent on the inflammatory status of the patient [28]. Few studies have evaluated the relation between EPC count and vitamin D status. Mikirova et al. [29] and Yiu et al. [17] have shown positive correlation between 25(OH)D levels and CD34þ/KDRþ and CD133þ/KDR þ EPC counts, respectively. Yiu et al. failed to demonstrate a beneficial effect of vitamin D replacement on EPC count in a type 2 diabetic population [21]. In our study, vitamin D deficient otherwise healthy premenopausal women had lower CD34þ/KDRþ and CD133þ/KDR þ EPC counts compared to age and gender matched control group. A 6-month vitamin D supplementation significantly increased CD34þ/KDRþ and CD133þ/ KDR þ EPC counts. Inflammation is regarded to have a key role in endothelial dysfunction and development of atherosclerosis [30,31]. Immunemodulatory effects of vitamin D have been suggested to contribute to its beneficial effects on atherosclerotic process [3]. Previous studies have shown that vitamin D and its metabolites have downregulated the expression of pro-inflammatory cytokines

and upregulated anti-inflammatory cytokine expression in immune cells [32,33]. In our study, vitamin D deficient premenopausal women had lower IL-10 and higher IL-17 levels. A 6-month vitamin D supplementation resulted in an elevation in IL-10 and IL13 and decline in IL-17 levels. Increase in serum IL-10 level, which is known to be associated with limited oxidative stress in the endothelium [34] and improved mobilization, survival and functionality of EPCs [35], was also found to be correlated with the improvement in endothelial function similar with a previous study [36]. In our study, decrease in IL-17 levels was found to be an independent predictor of the change in FMD values. IL-17 may have an indirect effect on endothelial function via inducing release of other pro-inflammatory cytokines including IL-6 and IL-1b [37]. In addition, Nguyen et al. have shown that IL-17 increased Rho-kinase activity which inhibits e-NOS and impairs endothelial function [38]. Ranganathan et al. [39] have demonstrated that elevated levels of IL-17 in vitamin D deficient rheumatoid arthritis patients were associated with endothelial dysfunction. cIMT measurement is a widely accepted tool in the assessment of subclinical atherosclerosis. However, increase in cIMT is a relatively late finding of atherosclerotic process compared to the impairment in endothelial function [40]. Relationship between vitamin D status and cIMT is not clear yet. Although Targher et al. [41] and Reis et al. [42] reported an inverse relationship between 25(OH)D level and cIMT; recent studies with larger study population have not confirmed these findings [43e45]. In our study, cIMT was not different between patient and control groups and did not change following vitamin D replacement in the vitamin D deficient group. This data is compatible with the results of recent studies. Our young study population with very low cardiovascular risk status may also have an impact on these results, since increased cIMT is not expected as a marker of atherosclerosis in this population. This study has some limitations. First, this study is not a randomized placebo-controlled study. Second, functional and proliferative properties of EPCs are not measured. 5. Conclusion This study demonstrates endothelial function is impaired in otherwise healthy vitamin D deficient young premenopausal women and improves with 6-month replacement therapy.

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