Vitamin D and Endothelial Vasodilation in Older Individuals: Data From the PIVUS Study

Vitamin D and endothelial vasodilation in olderindividuals: data from the PIVUS Study

Marcello Maggio, MD, PhD,1,2*, Francesca De Vita, PhD,1, Fulvio Lauretani, MD1,Gian Paolo Ceda, MD,1,2, Elena Volpi, MD, PhD,3,Francesco Giallauria, MD, PhD4,Giuseppe De Cicco, MD5, Chiara Cattabiani, MD6, Håkan Melhus, MD7,Karl Michaëlsson8, Tommy Cederholm, MD9, Lars Lind, MD, PhD10

1 Geriatric Rehabilitation Department, University Hospital of Parma, Via Gramsci, 14, 43126 Parma,Italy.; 2 Department of Clinical and Experimental Medicine, Section of Geriatrics, University of Parma, ViaGramsci, 14, 43126 Parma, Italy.; 3Department of Internal Medicine, Division of Geriatrics, and SealyCenter on Aging, University of Texas Medical Branch, Galveston, 301 University Blvd Galveston, TX77555–0264, USA; 4Department of Clinical Medicine Cardiovascular and Immunological Sciences,University of Naples Federico II, Corso Umberto I, 40, 80138 Napoli, Italy; 5Cardiac Surgery Unit,Community Hospital of Brescia, Piazzale Spedali Civili, 1, 25123 Brescia, Italy; 6Azienda USL Piacenza,Via Taverna, 49, 23121 Piacenza, Italy.; 7 Department of Medical Sciences, Uppsala University, 751 85Uppsala, Sweden.; 8 Department of Surgical Sciences, Uppsala University, Uppsala Science Park, 751 85Uppsala, Sweden.; 9Department of Medicine, Uppsala University Hospital, Uppsala, Uppsala SciencePark, 751 85 Uppsala, Sweden.; 10Department of Public Health and Caring Sciences, Section of ClinicalNutrition and Metabolism, Uppsala University, Uppsala Science Park, 751 85 Uppsala, Sweden.

Context. Vitamin D plays a role in a wide range of extra-skeletal processes, including vascularfunction. Endothelial dysfunction is a predictor of cardiovascular disease especially in older sub-jects. However, the relationship between Vitamin D levels and indexes of endothelial vasodilationhas never been fully addressed in older individuals.

Objective. The objective of this study was to examine the association between vitamin D andendothelial function in a large community-based sample of older subjects.

Methods. This cross-sectional study involved 852 community-dwellers men and women aged 70years from the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS) Study, with com-plete data on vascular function and 25-OH-Vitamin D. We evaluated endothelium-dependentvasodilation (EDV) by invasive forearm technique with acetylcholine, endothelium-independentvasodilation (EIDV) by sodium nitroprussiate, flow-mediated vasodilation (FMD) and the pulsewave analysis (Reflectance Index, RI). Vitamin D levels were measured by chemiluminescence. Weused multivariate regression models adjusted for BMI (Model 1) and for multiple confounders(hsCRP, insulin, total-cholesterol, HDL-cholesterol, LDL-cholesterol, smoking, sex hormones, seasonof blood collection, hypertension, diabetes, cardiovascular medications and diseases, statin usage,plasma calcium and calcium intake, PTH, physical exercise, liver and kidney function tests, albumin)(Model 2).

Results. In women, but not in men, vitamin D levels were positively associated with EIDV in bothmodel 1 (��SE�1.41�0.54, p�0.001), and model 2 (��SE�2.01�0.68, p�0.003).We found nosignificant relationship between vitamin D levels and EDV, FMD and RI in both sexes.

ISSN Print 0021-972X ISSN Online 1945-7197Printed in U.S.A.Copyright © 2014 by the Endocrine SocietyReceived February 24, 2014. Accepted May 30, 2014.

Abbreviations: and Acronyms: EDV, endothelium-dependent vasodilation; EIDV, endothe-lium-independent vasodilation; FMD, flow- mediated vasodilation; RI, Reflectance Index;BMI, body mass index; hsCRP, high-sensitivity C-Reactive Protein; HDL, high-density lipo-protein; LDL, low-density lipoprotein; SHBG, Sex Hormone Binding Globulin; T, Testoster-one; PTH, Parathyroid Hormone; DXA, Dual-energy X-ray Absorptiometry; CABG, Coro-nary Artery Bypass Surgery.

O R I G I N A L A R T I C L E

E n d o c r i n e R e s e a r c h

doi: 10.1210/jc.2014-1536 J Clin Endocrinol Metab jcem.endojournals.org 1

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Conclusions. In older women, but not in men, vitamin D is positively and independently associatedwith EIDV.

In the aging population a physiological gradual decline ofVitamin D levels is frequently observed (1). Vitamin D

deficiency, historically defined and recently recommendedby the Institute of Medicine (IOM) as 25-hydroxyvitaminD (25-OH D) serum levels of less than 50 nmol/L, is acommon condition in older persons affecting 20%–100%of U.S., Canadian, and European elderly men and womenstill living in the community (2–3).

Specific lifestyle factors linked to the advanced age con-cur to determine a poor vitamin D status. These includepoor dietary intake of vitamin D, chronic diseases, insuf-ficient exposure to sunlight, pharmacological treatment,obesity and disability (1).

A suboptimal vitamin D status represents an importantpotential public health issue because it has been associatedwith an increased risk of chronic conditions, cardiovas-cular events, disorders of glucose metabolism, neurode-generative diseases and overall mortality (4).

The consequences of vitamin D deficiency can be ex-plained by the fact that this hormone not only regulatescalcium and phosphate balance and bone structure, butalso plays an important role in a wide range of extraskel-etal biological functions via both genomic and non-genomic signaling (5). In particular, recent evidence sug-gests that Vitamin D could influence vascular biology bymodulating endothelial function and inflammatory status(6). It is well established that endothelial dysfunction is ahallmark of vascular damage and an independent predic-tor of atherosclerosis and cardiovascular events (7).

Observational studies reported that healthy middle-aged 25(OH)D deficient subjects are more prone to de-velop arterial stiffness and endothelial dysfunction (6, 8).A number of biological hypotheses have been formulatedto explain the beneficial effects of Vitamin D at the vas-cular level (6). However, the exact mechanisms need to beclarified. Experimental evidence suggests that Vitamin Dcould affect the vascular wall through its binding to theVitamin D nuclear steroid Receptor (VDR), which is ex-pressed on at least 36 different tissues including cardiacmuscle, vascular smooth muscle, vascular endothelium,and lymphocytes (9–10), In vitro studies in human um-bilical vein endothelial cells have shown that Vitamin Dmetabolites are capable of influencing endothelium-de-pendent vasodilation by increasing nitric oxide synthaseactivity and nitric oxide production (11). 1,25OHD, inparticular, seems to interact with the renin-angiotensin-aldosterone axis (12–13) as well as with vascular endo-thelial growth factor (VEGF) production. Finally, Vitamin

D might also exert antithrombotic effects by reducingplatelet aggregation (14) and altering the production ofproteins involved in regulation of thrombogenesis, eg,thrombomodulin and antithrombin (15).

Despite the potential link between Vitamin D and en-dothelial function, the relationship between vitamin D lev-els and endothelial vasodilation has never been fully ad-dressed in older individuals.

This is a significant issue because older adults are moresusceptible to both vitamin D insufficiency and increasedrisk of cardiovascular diseases (16). Thus, our study wasdesigned to examine the relationship between serum25-OH D and indexes of vascular function using datafrom a unique community-based sample of older men andwomen with complete information on serum 25-OH Dlevels, endothelium-independent vasodilation (EIDV) andendothelium-dependent vasodilation (EDV) assessed bythree different methods: invasive forearm technique withacetylcholine, flow-mediated vasodilation and pulse waveanalysis.

Materials and Methods

PopulationTo address our original hypothesis, we studied participants

aged 70 years from the large population-based Prospective Studyof the Vasculature in Uppsala Seniors (PIVUS) Study.

All subjects aged 70 years and living in the community ofUppsala, Sweden, were eligible. The study population, chosenfrom the registry of community living, was invited in a random-ized order. The subjects received an invitation by letter within 2months of their 70th birthday. Of the 2025 subjects invited, 1016subjects participated (507 men and 509 women), yielding a par-ticipation rate of 50.1%. The baseline investigation started inApril 2001 and was completed in June 2004. From the entiresample of 1016 subjects, we analyzed data of 852 subjects, 428men and 424 women, who had complete information on VitaminD and measures of endothelial function, assessed with three dif-ferent methods.

The study protocol was approved by Ethics Committee of theUniversity of Uppsala. All participants received a detailed de-scription of the purpose and design of the study and signed aninformed participation consent.

Baseline Clinical InvestigationsThe participants were asked to answer a complete question-

naire about their medical history, regular medication, dietaryand smoking habits. Specifically, subjects gave informationabout history of any cardiovascular diagnosis or medication,hypertension (antihypertensive treatment or blood pressure(BP) � 140/90 mmHg), diabetes (antidiabetic treatment includ-ing diet or fasting blood glucose � 6.1 mmol/L), hyperlipidemia

2 Vitamin D and endothelial function in the elderly J Clin Endocrinol Metab


(antihyperlipidemic treatment, Low density lipoprotein (LDL)-cholesterol � 3.5 mmol/L, or serum triglycerides � 1.7 mmol/L).Alcohol, Vitamin D and calcium intake were assessed with a7-day food diary. Participants were instructed in person bytrained dieticians on how to fill in the 7-day precoded

food diary. Daily nutrients intake were calculated using acomputer program and the Swedish National Food Administra-tion database (SLV Database, 1990). Blood pressure was mea-sured with a calibrated mercury sphygmomanometer after atleast 30 minutes of rest, and the average of 3 recordings was used.Standing height was measured to the nearest whole 0.5 cm witha Harpender Stadiometer (Holtain Ltd, Crymych, UK) and bodyweight was measured to the nearest 0.1 kg. Body mass index(BMI) was calculated as body weight/(body height) (2) (kg/m2)and obesity was defined as BMI � 30 kg/m2. Waist circumfer-ence was measured in a standing position midway between thelowest rib and the iliac crest. Exercise habits were divided intofour categories: very light exercise (no sweat) �2 times per week,light exercise 2 times per week, moderate exercise (sweat) 1–2times per week, heavy exercise (sweat) �2 times per week. Be-cause of the known seasonal variability in Vitamin D concen-tration, season for blood collection was considered as categoricalseason variable with 3-month-intervals. Season-categories wereidentified as follows: Winter (time period between December andFebruary), Spring (from March to May), Summer (from June toAugust) and Fall (from September to November).

Laboratory investigationsBlood was collected in the morning, after an overnight fast-

ing. All the routine blood tests were performed on fresh blood.No medication or smoking was allowed after midnight. Plasmacalcium (normal range 2.20–2.50 mmol/l) was measured spec-trophotometrically with a compleximetric method using ortho-cresolphthalein dye binding. Liver function was assessed byplasma alanine aminotransferase (ALT) (ALT, normal range0–40 U/L) and plasma albumin (spectrophotometry using bro-mine cresol purple, normal range 37–48 g/l). Plasma creatininewas analyzed with spectrophotometry using a modified Jaffe’sreaction (normal range 60–106 lmol/l). Creatinine clearance(normal range in men 97– 137 ml/min and in women 88–128ml/min) was calculated using the Cockcroft–Gault formula; formen � 20 years, creatinine clearance � [1.23 X (140-age) �weight]/S-creatinine, and for women � 20 years, creatinineclearance � [1.04 X (140-age) weight]/S-creatinine. Totalplasma cholesterol (normal range 2.6–7.1 mmol/l) and plasmaHDL-cholesterol (normal range 0.8–1.9 mmol/l) were deter-mined by enzymatic assay. Low density lipoprotein cholesterolwas calculated using Friedewald’s formula. High-sensitivity C-reactive protein (CRP) (hsCRP) was measured in serum by latex-enhanced turbidimetry on an Architect Ci8200 analyzer (AbbottLaboratories, Abbott Park, IL).

Hormonal assessmentAll the study samples were measured in one laboratory that

takes part in, and meets the performance targets for the VitaminD External Quality Assessment Scheme (DEQAS). 25-OH D3was measured in serum by chemiluminescence immunoassaytechnology (LIAISON® 25-hydroxyvitamin D Assay, DiaSorin)(17). The coefficient of variation (CV) for interassay analyseswas 18.4% at a 25-OH D level of 39.5 nmol/L and 11.7% at121.25 nmol/L. Severe vitamin D deficiency was defined as se-

rum 25-OH D levels � 25 nmol/L. Vitamin D deficiency wasdefined as serum 25-OH D levels � 50 nmol/L. Vitamin D suf-ficiency was defined as serum 25-OH D levels � 75 nmol/L.Intact plasma Parathyroid Hormone (PTH) (normal range12–65 ng/l) was determined with an immunochemiluminomet-ric assay (Nichol’s Institute, San Juan Capistrano, CA, USA).Serum Sex Hormone Binding Globulin (SHBG), Estradiol (E2)and Testosterone (T) were assessed by chemiluminescence im-munoassay on the immunochemistry platform Access (BeckmanCoulter). The minimal detectable concentrations (MDC) for E2,T and SHBG were 73 pmol/L, 0.35 nmol/L and 2 nmol/L, re-spectively. The CV was � 20% for E2, � 7%, for T and � 6%for SHBG, respectively. Plasma insulin was assayed by usingchemiluminescence (Roche, Basel, Switzerland).

Measures of endothelial functionThree different techniques to evaluate endothelium-depen-

dent vasodilation in the peripheral circulation were simultane-ously used in this large-scale population-based setting. All 3 tech-niques were feasible to perform in a general elderly population.Detailed information has been previously described elsewhere(18).

Briefly: The Forearm blood flow (FBF) was measured by ve-nous occlusion plethysmography (Elektromedicin, Kullavik,Sweden). After evaluation of resting FBF, local intra-arterialdrug-infusions were given over 5 minutes for each dose. Theinfused dosages were 25 and 50 �g/min for acetylcholine (Clin-Alpha, Switzerland) to evaluate EDV and 5 and 10 �g /min forsodium nitroprussiate SNP (Nitropress, Abbot, UK) to evaluateEIDV. EDV mainly reflects vasodilation in forearm resistancearteries. EDV was defined as FBF during infusion of 50 �g/minof Acetylcholine minus resting FBF divided by resting FBF. EIDVwas defined as FBF during infusion of 10 �g /min of SNP minusresting FBF divided by resting FBF. Flow-mediated vasodilation(FMD) was induced by inflation of a pneumatic cuff placedaround the forearm to a pressure at least 50 mmHg above thesubject’s systolic BP for 5 minutes, and defined as the maximalbrachial artery diameter recorded between 30 and 90 secondsfollowing cuff release minus diameter at rest divided by the di-ameter at rest. The brachial artery diameter was measured byexternal B-mode ultrasound imaging 2–3 cm above the elbow(Acuson XP128 with a 10 MHz linear transducer, AcusonMountain View, California, USA) according to the recommen-dations of the International Brachial Artery Task Force (19). ThePulse wave analysis was assessed with a micromanometer tippedprobe (Sphygmocor, Pulse Wave Medical Ltd, Australia) appliedto the surface of the skin overlying the radial artery and theperipheral radial pulse wave was continuously recorded. Themean values of 10 pulse waves were used for analyses. After abaseline recording, terbutaline was subcutaneously adminis-tered (0.25 mg in the upper part of the arm) and a re-evaluationof the pulse wave was performed after 15 and 20 minutes. Themaximal change occurring at either 15 or 20 minutes was usedfor calculations. This technique mainly evaluates vasodilation inresistance arteries. Reflectance index (RI) is the measure assessedby the pulse-wave–based technique.

Assessment of body compositionTotal body fat content was assessed by Dual-energy X-ray

Absorptiometry (DXA) (DPX, Lunar Prodigy, Lunar Corp.,Madison, WI, USA), with the primary outcome total body fat

doi: 10.1210/jc.2014-1536 jcem.endojournals.org 3


mass. In order to evaluate the reproducibility, fifteen subjectswere scanned three times. The coefficient of variation of the DXAmeasurements was calculated to be 1.5% for total fat mass and1.0% for total lean mass (20). The bias associated with DXA fatmeasurement is systematic, with an underestimation of fat con-tent for leaner subjects and an overestimation of fat contentamong obese subjects, but these inaccuracies were less than 2%(21).

Statistical AnalysisCategorical variables were expressed in percentages, and con-

tinuous variables were reported as means (standard deviations)for normally distributed parameters or as medians and inter-quartile ranges (IQR). To approximate normal distributions,log-transformed values for FMD, RI, EDV and EIDV, ALT, cre-atinine clearance, hsCRP, insulin, T and E2, SHBG, PTH, cal-cium intake and total body fat were used in the analysis and backtransformed for data presentation.

The main effects of Vitamin D on endothelial function in bothsexes were tested in a BMI adjusted analysis (Model 1). Theinteraction term sex*Vitamin D was used in a general linearmodel to compare the sex-associated increments in endothelialfunction and Vitamin D between sexes. Since the interactionterm sex*Vitamin D was significant in the relationship betweenVitamin D and EIDV, the models were performed separately formen and women. Parsimonious models obtained by backwardselection from initial fully adjusted models were used to identifyindependent correlates of endothelial function in both sexes(Model 2). Model 2 was adjusted for BMI and other potentialconfounders including hsCRP, creatinine clearance, ALT, albu-min, plasma calcium, calcium intake, PTH, insulin, T, E2,SHBG, total-cholesterol, HDL-cholesterol, LDL-cholesterol,smoke, season of blood collection, diabetes, hypertension, car-diovascular diseases (angina, stroke and myocardial infarction(MI), Coronary Artery Bypass Surgery, CABG), any cardiovas-cular drugs, statin use, and physical exercise. A statistical sig-nificance was set for P � .05. The analyses were performed usingthe SAS statistical package, version 9.1 (SAS Institute Inc., Cary,NC).

Results

Characteristics of the populationThe characteristics of the study population are pre-

sented in Table 1. As expected, women, compared to men,had significantly lower waist circumference (86.8 � 10.8vs 93.9 � 9.9, �0.001), E2 [64 (49–87) vs, 109 (90–132)�0.001], T [0.41 (0.14–0.80) vs12.8 (10.1–15.6),�0.001] and Vitamin D intake (5.30 � 1.85 vs 6.24 �2.35, <0.001) and higher levels of total-cholesterol (5.7 �0.96 vs 5.1 � 0.91, �0.001), HDL-cholesterol (1.7 � 0.4vs 1.4 � 0.4, p��0.001), LDL-cholesterol (3.54 � 0.84vs 3.27 � 0.83, �0.001), SHBG [55.5 (42.1–73.8) vs 42.1(32.4–54.2), P��0.001] and total body fat [26.83(20.99–33.53) vs 22.53 (17.71–27.91), P � .001]. RI wassignificantly different in women than men [-34.8 (-44.3-–25.6) vs –27.9 (-37.1- –10.9)], and this difference was

statistically significant (p��0.001). Alcohol intake (g/d),was available only in 367 out of 428 men and 354 out of424 women. Men had a significantly higher alcohol intakethan women [2.34 (0.65–4.67) vs 1.16 (0.22–2.8),�0.001] (data not shown). Men and women had similarmean serum 25-OH D levels (P � .55).

Interaction sex, Vitamin D and endothelialfunction

By analyzing the interaction between sex and VitaminD we found a statistical significant interaction term re-garding EIDV (Vitamin D*SEX ��SE 1.50 � 0.75, P �

.04). No significant interaction between Vitamin D*SEXwas found for EDV (��SE 0.70 � 1.0, P � .48), FMD(��SE 0.12 � 0.33, P � .70) and RI (��SE –0.02 � 0.05,P � .57).

Vitamin D and endothelial function measures inwomen

After adjustment for BMI, we found a positive rela-tionship between Vitamin D and EIDV in women(��SE � 1.41 � 0.54, P � .001) (Table 2). The relation-ship between Vitamin D and EIDV was maintained afterfurther adjustment for hsCRP, creatinine clearance, ALT,albumin, plasma calcium, calcium intake, insulin, T, E2,SHBG, total-cholesterol, HDL-cholesterol, LDL-choles-terol, hypertension, smoke, season of blood collection,diabetes, any cardiovascular drugs, statin use, calcium in-take, PTH, physical exercise, and cardiovascular diseases(��SE � 2.01 � 0.68, P � .003) (Table 2).

The association was still significant after further ad-justment for total body fat (��SE � 1.98 � 0.66, P �

.003) and vitamin D intake (��SE � 1.47 � 0.72, P � .04)(data not shown). Similarly, in a restricted sample of 721subjects, having complete data on alcohol intake, the in-clusion of this variable in the multivariate regressionmodel did not affect the positive and significant relation-ship between Vitamin D levels and EIDV (��SE � 1.55 �

0.75, P � .004) (data not shown).No significant association was observed between Vita-

min D and EDV, FMD and RI in both model 1 and model2 (Table 2).

Vitamin D and endothelial function measures inmen

In men, we did not detect any significant relationshipbetween Vitamin D and EIDV using Model 1 (��SE �

0.02 � 0.49, P � .95) and Model 2 (��SE�-0.28 � 0.61,P � .65) (Table 3). Similarly, no significant associationwas found between Vitamin D and all measures of endo-thelial function in the BMI and fully adjusted model (Table3). These results were not affected by further adjustment



for total body fat (��SE�-0.18 � 0.58, P � .75) andVitamin D Intake (0.03 � 0.62, P � .95) (data not shown).

Table 1. Characteristics of the Study Population.

Men Women P value*

N 428 424 -Waist

circumference(cm2)

93.9 � 9.9 86.8 � 10.8 <0.001

BMI(kg/m

2)

‡

26.7 � 3.5 26.8 � 4.7 0.85

FMD% †

4.6 [2.4-7.0]

3.6 [2.7-6.4]

0.31

EDV %†

449.9 [298.5-649.6]

478.5 [322.6-696.9]

0.17

EIDV% †

327.8 [226.8-450.5]

334.2 [218.4-478.7]

0.36

� RI %†

�34.8 [-44.3-�25.6]

�27.9 [-37.1-�10.9]

<0.001

Currentsmokersn,(%)

46 (10.8) 51 (12.0) 0.57

TotalCholesterol(mmol/liter)

5.1 � 0.91 5.7 � 0.96 <0.001

HDLCholesterol(mmol/liter)

1.4 � 0.4 1.7 � 0.4 <0.001

LDL-Cholesterol(mmol/liter)

3.27 �0.83

3.54 � 0.84 <0.001

Hypertensionn,(%)

146.3 (22.3) 152.5 (21.9) <0.001

Diabetesn,(%)

41 (9.6) 33 (7.8) 0.38

Anycardiovasculardrugusen,(%)

265 (61.9) 315 (74.3) <0.001

Seasonofbloodcollectionnumbern,(%)§

0.17

Winter 116 (27.1) 98 (23.1)Spring 138 (32.2) 132 (31.1)Summer 55 (12.9) 69 (16.3)Fall 119 (27.8) 125 (29.5)

(Continued )



Table 1. Continued

Men Women P value*

Levelofphysicalactivity

0.52

Physicalactivity:verylight

(%)

51 (12.1) 39 (9.4)

Physicalactivity:light¶(%)

243 (56.1) 262 (61)

Physicalactivity:medium#(%)

96 (22.8) 101 (24.3)

Physicalactivity:hard**(%)

38 (9.0) 22 (5.3)

TotalBodyFat(g/cm2)†

22.53 [17.71-27.91]

26.83 [20.99-33.53]

0.001

ALT(U/liter)†

31 [25-42]

26 [21-35]

0.004

Calcium(mmol/liter)

2.3 � 0.1 2.4 � 0.1 <0.001

Albumin(g/liter)

40.5 � 2.8 40.1 � 2.7 0.10

CreatinineClearance(ml/min)†

83 [73-98]

71 [60-83]

<0.001

Calciumintake(mg/day)†

912.2 [735.4-1102.7]

966.24[718.4-1231.8]

0.02

VitaminDIntake(mcg/day)

6.24 �2.35

5.30 � 1.85 <0.001

(Continued )



Discussion

In a large community-based population of older subjectswe found an independent association between Vitamin Dlevels and EIDV in women but not in men.

Our findings demonstrate a profound link between Vi-tamin D and measures of endothelial independent vaso-dilation, and provide novel insights into the extraskeletaleffects of Vitamin D on the vascular system. To our knowl-edge, this is the first study that has addressed the relation-ship between Vitamin D and endothelial function in a pop-ulation of older individuals with complete information onthe most relevant indexes of endothelial vasodilation andarterial stiffness.

Most of the previous investigations, conducted in smallcohorts of middle-aged subjects, focused on the impact ofVitamin D on surrogate markers of vascular disease (par-

ticularly BP) and noninvasive measures of endothelium-dependent vasodilation (7, 22).

The existing cross-sectional data coming from healthyindividuals and patients with different diseases suggest anassociation between Vitamin D deficiency and an higherrisk of endothelial dysfunction, arterial stiffness and abroad range of cardiovascular disorders and risk factors(2, 3).

Wang and coworkers (23) showed an increased risk ofcardiovascular events in participants without overt car-diovascular disease having 25-OH D concentrations �

37.5 nmol/L. The risk doubled in those participants withthe lowest 25-OH D concentrations. In a recent analysis ofa well-represented study population of 514 subjects aged47 � 13 yrs, Mheid et al (24) demonstrated that lower25-OH D levels were associated with worse endothelium-

Table 1. Continued

Men Women P value*

SHBG(nmol/liter)†

42.1 [32.4-54.2]

55.5 [42.1-73.8]

<0.001

Testosterone(nmol/liter)†

12.8 [10.1-15.6]

0.41 [0.14-0.80]

<0.001

VitaminD(nmol/liter)

58.3 �20.2

57 � 19.4 0.55

Estradiol(pg/ml)†

109 [90-132]

64 [49-87]

<0.001

hsCRP(mg/liter)†

1.2 [0.6-2.3]

1.3 [0.6-2.3]

0.38

Insulin(mlU/liter)†

7.3 [5.2-10.8]

7.3 [5.2-10.2]

0.14

PTH(nmol/liter)†

43.9 [32.4-56.8]

45.2 [34.0-57.7]

0.81

*Data are presented as number of cases (percentage), mean � SD or median and interquartile ranges as appropriate.

† Log transformed values.

‡ Body mass index.

§Season-categories were identified as follows: Winter (time period between December and February), Spring (from March to May), Summer (fromJune to August) and Fall (from September to November).

Very light exercise, no sweat �2 times week.

¶ Light exercise, no sweat �2 times week.

#Moderate exercise, with sweat, �2 times week.

**Heavy exercise, with sweat, �2 times week.

BMI, body mass index; FMD, flow- mediated vasodilation; EDV, endothelium-dependent vasodilation; EIDV, endothelium-independent vasodilation;RI, Reflectance Index; ALT, alanine aminotransferase; SHBG, Sex Hormone Binding Globulin; hsCRP, High-sensitivity C-reactive protein; PTH,Parathyroid Hormone.



dependent brachial artery FMD and arterial stiffness as-sessed by PWV. These results were maintained after ad-justment for age, sex, race, BMI, serum lipid levels,plasma, CRP, and medications. Impaired FMD was alsoreported in chronic diseases such as type 2 diabetes (25).Vitamin D seems to act locally in vascular smooth muscleand endothelial cells through its binding with the VDR. Inthese tissues Vitamin D might modulate the effects of in-flammatory cytokines on the vasculature (26), decreaseendothelial adhesion molecules expression, increase nitricoxide production (12) and reduce platelet aggregation(16, 27).

However, in the present analysis we failed to detect anysignificant association between Vitamin D and EDV,FMD and RI. To explain the discrepancy between our dataand the previous investigations, we suggest that factors

other than Vitamin D might be more relevant in modu-lating endothelial function in older individuals (28).Moreover, as observed for most of the other nuclear re-ceptors, there is a reduction in the number and/or expres-sion of VDR associated with aging (29). In older subjects,low serum levels of Vitamin D may significantly reduceVDR activation and function (30). These observationsprovide evidence to the notion of an age-related relativeinsensitivity of endothelial dependent vasodilation to Vi-tamin D.

Endothelial independent vasodilation, evaluated by in-vasive forearm technique, is considered a reliable markerof the typical age-related changes in vascular structure(loss of elastin fibers, increased collagen fibers and smoothmuscle cells in the media layer) and vascular stiffness (18).This index has been significantly related to the Framing-ham risk score and proinflammatory and prothromboticstates (18, 31). Several lines of evidence suggest that Vi-tamin D may indirectly modulate endothelial-independentfunction by suppressing the renin-angiotensin system andreducing BP (32) and/or decreasing vascular resistance(33).

More interestingly, the findings of the present analysisalso suggest that low Vitamin D is, at least in women, amarker of poor health and overall metabolic status, ratherthan the cause of vascular and physiological disturbances.Low 25-OH D could be the result of vascular diseases anda useful marker in the detection of functional and vasculardamage before its clinical manifestation. This hypothesiswas recently raised in a systematic review documenting adiscrepancy between the existing observational and inter-ventional studies on the role of 25-OH D concentrationsin a wide range of acute and chronic nonskeletal healthdisorders (17). Randomized trials have failed to demon-strate that raising of 25-OH D concentrations can modifythe occurrence or clinical course of vascular disorders(22). Well-designed trials performed in postmenopausalwomen using low (400 IU) (34) and high (2500 IU) dailyVitamin D doses (35), did not show any improvementneither in endothelial dependent vasodilation nor in mea-sures of arterial stiffness. Overall, these data do not sup-port a causal relationship between low Vitamin D statusand a wide range of disorders (17). Interestingly, the re-lationship between Vitamin D and EIDV was independentof hsCRP, and severe cardiovascular and cerebrovasculardiseases, supporting the hypothesis that Vitamin D may bea biomarker of metabolic status rather than inflammatorydiseases.

We also found a specific sex-related influence of Vita-min D on EIDV. These results are confirmed by the sta-tistically significant interaction found between sex andVitamin D for EIDV. To identify potential mediators of

Table 2. Association between Vitamin D andEndothelial Function in Older Women.

Model 1* Model 2 †

� �SE.

p � �SE.

p

EDV 0.49 � 0.68 0.47 0.25 � 0.94 0.79EIDV 1.41 � 0.54 0.001 2.01 � 0.68 0.003FMD �0.05 � 0.009 0.58 �0.01 � 0.01 0.31RI 0.02 � 0.03 0.58 �0.006 � 0.04 0.88

*Adjusted for BMI.

† Adjusted for BMI, hsCRP, creatinine clearance, ALT, albumin,calcium, insulin, total cholesterol, HDL-Cholesterol, LDL-cholesterol,smoking, season of blood collection, Estradiol, Testosterone, Sex-Hormone Binding Globulin, hypertension, diabetes, any cardiovasculardrug, statin usage, calcium intake, PTH, physical exercise, angina,stroke, myocardial infarction.

EDV, endothelium-dependent vasodilation; EIDV, endothelium-independent vasodilation; FMD, flow- mediated vasodilation; RI,Reflectance Index.

Table 3. Associations between Vitamin D andEndothelial Function in Older Men.

Model 1* Model 2 †

� �SE.

p � �SE.

p

EDV �0.02 � 0.68 0.96 0.26 � 0.88 0.77EIDV 0.02 � 0.49 0.95 �0.28 � 0.61 0.65FMD �0.12 � 0.33 0.71 �0.33 � 0.41 0.41RI 0.04 � 0.03 0.23 0.07 � 0.04 0.08

*Adjusted for BMI.

† Adjusted for BMI, hsCRP, creatinine clearance, ALT, albumin,calcium, insulin, total cholesterol, HDL-Cholesterol, LDL-cholesterol,smoking, season of blood collection, Estradiol, Testosterone, Sex-Hormone Binding Globulin, hypertension, diabetes, any cardiovasculardrug, statin usage, calcium intake, PTH, physical exercise, angina,stroke, myocardial infarction.

EDV, endothelium-dependent vasodilation; EIDV, endothelium-independent vasodilation; FMD, flow- mediated vasodilation; RI,Reflectance Index.



this sex-specific association, we determined the role of sexhormones such as T, E2 and the transport protein SHBG.All these variables are known to have different trajectoriesin two sexes across age with older women having lower Tand E2 levels and higher SHBG concentration. However,the inclusion of these covariates in the fully adjusted modeldid not affect the relationship between Vitamin D andEIDV. Another plausible mechanism explaining the dif-ferent results in men and women can be found in the phys-iological and age-related differences in obesity and bodyfat distribution existing between the two sexes (36–37).As expected, in our population men had higher waist cir-cumference than women. We also know that Vitamin D isa fat-soluble Vitamin and lower levels are encountered inobese and diabetic patients (38, 39). To exclude the in-fluence of body fat content, we have also included totalbody fat as confounder, but, again, the association be-tween Vitamin D and EIDV, in women, was still statisti-cally significant.

Limitations and strengthsThe cross-sectional design of the study does not allow

us to establish a cause-effect relationship between VitaminD levels and EIDV in the older female cohort. The sampleincluded in our analysis was comprised of white personsaged 70 years old, most of whom were taking cardiovas-cular drugs. We acknowledge that the study results mightbe affected by the relative small number of study subjects.However, it is not easy to perform invasive methods ofendothelial function assessment especially in such popu-lations of older individuals. To minimize bias, we ac-counted for metabolic and cardiovascular confoundingvariables. However, given the significant number of dif-ferences between female and male participants particu-larly in terms of total, HDL and LDL-cholesterol levels,total body fat, BMI, liver and kidney function, it cannot beexcluded the compounding effects of these variables in therelationship between Vitamin D and EIDV. Moreover,although the Diasorin Liaison immuno-assay, used in thecurrent analysis, is the most common technique in theDEQAS validation program, we acknowledge that othermethodologies such as Liquid chromatography-tandemmass spectrometry (LC-MS/MS) is considered the goldstandard in the assessment of 25-OH D levels. Finally,based on the above mentioned limitations and given thedifficulty to compare our findings with the informationcurrently available in the literature, we cannot exclude thepossibility of the chance finding. Thus, our study-analysiscan be considered a generating hypothesis that needs to beconfirmed in future investigations.

These limitations are offset by the significant strengths.Our study is the first investigating the relationship be-

tween Vitamin D and endothelial function, assessed bythree invasive methods of evaluation, in the only existinglarge-scale, population-based cohort of older individualswith complete information on Vitamin D levels and intakeand other important variables. Finally, all the study sam-ples were measured in one laboratory that met the per-formance targets for the Vitamin D DEQAS (40).

Conclusion

In older women, but not in men, Vitamin D concentrationwas positively and independently associated with endo-thelium-independent vasodilation. Further longitudinalanalyses are needed to delineate the role of Vitamin D insex-related endothelial function and derived diseases andits role as useful marker of early functional vasculardamage.

Acknowledgments

The outstanding work at the endothelium laboratory performedby Nilla Fors, Jan Hall, Kerstin Marttala and Anna Stenborg inthe collection and processing of the data is highly acknowledged.Chemiluminescence kits for hormonal assays were kindly pro-vided by Beckman Coulter.

*Address all correspondence and requests for reprints to:Marcello Maggio, MD, PhD, Department of Clinical and Ex-perimental Medicine Section of Geriatrics, Endocrinology of Ag-ing Unit, University of Parma, Emilia-Romagna Region, viaGramsci 14, 43126 Parma, Italy., Work Telephone number:0039–0521703916 Fax number: 0039–0521987562, .E-mailaddress: [email protected] [email protected].

Disclosure Summary: The authors have nothing to disclose.This work was supported by the Swedish Research Council.

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Vitamin D and Endothelial Vasodilation in Older Individuals: Data From the PIVUS Study