DOI: 10.1111/eci.12240

ORIGINAL ARTICLE Homocysteine levels are inversely associated with capillary density in men, not in premenopausal women *, Ed C. Eringa§, Nienke J. Wijnstok*,†, Henk J. Blom¶, Jacqueline M. Hornstra*, Trynke Hoekstra†,‡, Erik H. Serne †,‡ * Jos W. R. Twisk and Yvo M. Smulders *Department of Internal and Vascular Medicine, Institute for Cardiovascular Research (IcaR-VU), VU University Medical Centre, Amsterdam, the Netherlands, †Department of Health Sciences, Faculty of Earth and Life Sciences, EMGO Institute for Health and Care Research, VU University Amsterdam, Amsterdam, the Netherlands, ‡Department of Epidemiology and Biostatistics, VU University Medical Centre, Amsterdam, the Netherlands, §Department of Physiology, Institute for Cardiovascular Research (IcaR-VU), VU University Medical Centre, Amsterdam, the Netherlands, ¶Metabolic Unit, Department of Clinical Chemistry, VU University Medical Centre, Amsterdam, the Netherlands

ABSTRACT Background Homocysteine is an independent predictor of cardiovascular risk. The mechanisms underlying this link are not fully elucidated. Whereas the role of vascular dysfunction in conduit arteries is extensively studied, the role of the microcirculation in this relationship is largely unexplored. We assessed the relationship between homocysteine levels and microvascular structure and function in a healthy, population-based cohort. Materials and methods We cross-sectionally studied 260 participants (aged 42 years, 47% men) of the Amsterdam Growth and Health Longitudinal Study. Nailfold videocapillaroscopy was used to assess capillary density at baseline, during venous occlusion and during peak reactive hyperaemia. The relationship between tertiles of homocysteine and microvascular outcomes was evaluated using linear regression analyses, with adjustment for BMI and blood pressure. Stratified analyses were performed for men and women. Results In men, we observed a negative, nonlinear relationship between homocysteine and baseline capillary density, showing a lower capillary density in the highest tertile of homocysteine [adjusted B 8
65 capillaries/ mm2 (95%-CI: 16
05 to 1
25); P = 0
02]. In women, no significant associations were found between homocysteine and microvascular outcomes. Conclusions In men, higher homocysteine levels are associated with a reduction in basal perfusion of skin capillaries. This finding provides a novel potential explanation for how homocysteine influences cardiovascular disease risk. Keywords Capillaroscopy, cardiovascular diseases, homocysteine, microcirculation. Eur J Clin Invest 2014; 44 (3): 333–340

Introduction Moderately increased homocysteine levels are independently associated with an increased risk of myocardial infarction and stroke [1]. The mechanisms underlying this association, however, are obscure. Moreover, randomized clinical trials have cast doubt on the potential of homocysteine-lowering therapy to reduce cardiovascular risk [2,3]. Of note, the follow-up period of these trials was usually limited to a period of 3–5 years, precluding conclusions regarding potential longer-term benefits. Moreover, most of these trials were conducted in subjects with advanced or established cardiovascular disease. Thus, novel paradigms on the association between homocysteine and vascular disease are clearly needed [4–6].

A relatively understudied domain of the human vascular system is the microcirculation, which consists of arterioles, capillaries and venules. Disturbances in structure and/or function of the human microcirculation have been implicated in the pathogenesis of hypertension, insulin resistance and disturbed tissue perfusion [7–9]. Hence, the association between microvascular perturbations and cardiovascular disease (CVD) is largely indirect, as these perturbations contribute to CVD via the pathogenesis of risk factors, rather than having a direct effect on atherosclerosis or thrombosis. Consequently, the lag time between the occurrence of microvascular perturbations and CVD is conceivably longer than that of conventional risk factors and CVD. From a mechanistic point of view, an association between hyperhomocysteinaemia and the

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microcirculation is plausible. Homocysteine itself, as well as its associated components of one-carbon metabolism, has been reported to exert its detrimental effects via oxidative stress, endothelial dysfunction, decreased bioavailability of nitric oxide (NO) and smooth muscle cell proliferation, all of which bear relevance for microvascular structure and function [10–13]. It follows that an association between hyperhomocysteinaemia and microvascular structure and function may be clinically relevant, particularly for long-term cardiovascular risk and impaired organ perfusion. Previous studies on this association in humans have been scarce, but suggest a link between homocysteine levels and microvascular complications such as retinopathy and encephalopathy [14,15]. Animal studies support the concept of homocysteine causing microvascular perturbations [16–20], but extrapolation of these findings to the human situation is troublesome. We hypothesize that higher homocysteine levels are associated with impaired microvascular structure and/or function. We address this hypothesis in a population-based study in healthy, middle-aged adults.

Materials and methods Study design and subjects We performed a cross-sectional study using data from the Amsterdam Growth and Health Longitudinal Study (AGAHLS), an observational cohort study which started in 1977 with 450 children at the age of 13. The initial goals of the study were to describe the natural development of growth, health and lifestyle and further to investigate associations between health and lifestyle. Details of the AGAHLS are described elsewhere [21]. In the most recent measurement round (2006), at the age of 42, subjects were invited for microvascular measurements and additional laboratory tests, including homocysteine. Microvascular measurements were performed in 344 subjects. Participants using cardiovascular-related medication (drugs for rate/ rhythm control, cholesterol and blood pressure lowering) were excluded. All female subjects were premenopausal. Inclusion criteria were availability of homocysteine levels and availability of at least one of three microvascular outcomes. The study was approved by the medical ethical committee of the VU University Medical Centre, and all subjects gave their written informed consent.

Procedures Microvascular function and structure were assessed by nailfold videocapillaroscopy (1009, Capiscope
; KK technologies, Devon, UK). Capillaries were visualized in the nailfold of the dorsal skin of the third finger with a system magnification of

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1009, linked to a personal computer. With this technique, all capillaries that are erythrocyte perfused are visualized. For all subjects, two separate fields of 1 mm2 were recorded on videotape. Per field, three measures were taken. We defined three microvascular outcomes, as described previously: baseline capillary density, venous occlusion and peak reactive hyperaemia [22]. Baseline capillary density was defined as the number of capillaries that were continuously perfused during 15 s in resting state. Peak reactive hyperaemia was assessed by counting the maximal number of capillaries perfused after 4 min of arterial occlusion (cuff inflated at 300 mmHg). Venous occlusion was defined as the maximal number of perfused capillaries after 1 min of venous occlusion (cuff inflated at 50 mmHg), exposing a maximal number of capillaries [23,24]. All measurements were separated with a 5-min rest period. Capillary density is reported as the number of capillaries per mm2. Restricting conditions to measure microvascular function in participants were a minimum hand temperature of 28 °C, fasting state and at least 30 min rest in a temperature-controlled room before the measurement, as this could possibly influence microvascular perfusion. If hand temperature dropped below 28 °C before or during the measurement, subjects were excluded from further analyses. Reproducibility of the microvascular parameters was tested using intraclass correlation coefficients (ICC’s). In our hands, intraobserver ICC’s of capillary density counts during baseline and after 4 min of arterial occlusion were 0.97 and 0.96, and interobserver ICC’s were 0.86 and 0.90, respectively.

Homocysteine measurements Homocysteine in plasma was measured by the Abbott IMx fluorescence polarization immunoassay (IMx; Abbott Laboratories, Abbott Park, IL, USA). Intra- and interassay coefficients of variation were < 2% and 4%, respectively.

Statistical analyses As both homocysteine (the central determinant) [25] and skin microvascular parameters (the dependent variable) [22] are gender dependent, we performed stratified analyses for men and women. For comparing means between men and women, independent sample T-tests or Mann–Whitney U-tests were used, as appropriate. Linear regression analysis was used to analyse the relationship between homocysteine levels and microvascular structure and function. To improve the interpretability of the results of the regression analyses, homocysteine levels were divided into tertiles which were entered in the model as two dummy variables, the lowest tertile being the reference group. Two models were employed separately for each microvascular outcome: a crude model in which the tertiles of homocysteine

ª 2014 Stichting European Society for Clinical Investigation Journal Foundation

HYPERHOMOCYSTEINAEMIA AND CAPILLARY DENSITY

Results

*

120

* p < 0·05

*

100

Capillaries/mm2

were related to the microvascular outcome and an adjusted model including body mass index and systolic blood pressure, which are known determinants of microvascular function/ structure. A P-value of ≤ 0
5 was considered significant. All statistical analyses were performed with SASW Statistics 18.0 (SPSS Inc., Chicago, IL, USA).

80 60

Men

40

Women

20

Microvascular measurements were performed in 344 subjects, of whom 275 were included based on availability of homocysteine and technically successful microvascular measurements. Another 15 subjects were excluded due to the use of cardiovascular medication. The characteristics of the remaining 260 participants are shown in Table 1. In accordance with the literature [25], higher homocysteine levels were found in men (P < 0
01). Moderate hyperhomocysteinaemia (16–30 lM) was present in 24 subjects. Intermediate hyperhomocysteinaemia (31–100 lM) was present in only five subjects, all men. In addition, men were characterized by a less favourable cardiovascular risk profile as assessed by the parameters reported in Table 1. Microvascular outcomes are presented in Fig 1. The number of perfused capillaries during both venous occlusion and peak reactive hyperaemia was significantly higher in women than in men (P < 0
01 and P < 0
05 respectively), as we reported previously [22].

0

Baseline capillary density

Figure 2 illustrates baseline capillary density in tertiles of fasting plasma homocysteine in (a) men and (b) women and shows a gradual fall in baseline capillary density with increasing homocysteine levels in men, but not in women. Similar trends of lower capillary counts in men with higher homocysteine levels were observed for venous occlusion and peak reactive hyperaemia, but with a different pattern than for baseline capillary density (Table 2).

2

BMI (kg/m ) WHR Smokers (%)

121

139

42

42

25
1  3
0

23
9  3
3

0
89 (0
85–0
93) 18

14

122  13

110  13

DBP (mmHg)

73  8

68  8

Homocysteine (lM)

12
8 (10
9–14
9)

10
3 (8
7–11
7)

Total cholesterol (mM)

5
1  0
9

4
9  0
8

HDL cholesterol (mM)

1
5  0
3

1
9  0
4

Triglycerides (mM)

1
1 (0
7–1
5)

0
8 (0
6–1
1)

105
7  8
7

*

91
3  9
1

* p < 0·05

60 50 40 30 0

0
76 (0
74–0
80)

SBP (mmHg)

Creatinine (lM)

Baseline capillary density (capillaries/mm2)

Age (years)

Women

8·0 - 11·2

11·2 - 14·3

14·3 - 57·2

Homocysteine (mmol/L)

(b) Baseline capillary density (capillaries/mm2)

N

Men

Peak reactive hyperemia

Figure 1 Microvascular measurements (mean and SD) for men and women.

(a)

Table 1 Baseline characteristics

Venous occlusion

60 50 40 30 0

6·0 - 9·2

9·2 - 11·1

11·3 - 22·5

Homocysteine (mmol/L) BMI, body mass index; WHR, waist-to-hip ratio; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein. Continuous variables are expressed as mean  standard deviation or median (interquartile range), depending on the normality of the distribution.

Figure 2 Baseline capillary density (mean and SD) for tertiles of homocysteine in (a) men and (b) women.

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Table 2 Microvascular measurements in men and women divided into tertiles of homocysteine Men

Women

Tertile 1

45
5  15
6

41
1  15
5

Tertile 2

39
0  18
7

41
8  14
2

Tertile 3

37
3  14
4

41
2  14
3

Tertile 1

83
9  20
3

87
3  23
9

Tertile 2

74
9  26
6

87
8  20
1

Tertile 3

78
4  20
5

88
0  21
1

Tertile 1

80
9  19
1

81
4  24
6

Tertile 2

69
3  25
0

82
2  21
0

Tertile 3

74
0  20
4

83
6  20
7

Baseline capillary density

Venous occlusion

Peak reactive hyperaemia

Data are expressed in number of capillaries/mm2 and as mean  standard deviation.

The results of regression analyses with homocysteine as the independent and microvascular measurements as dependent variables are shown in Table 3. In men, the highest tertile of homocysteine is associated with a lower baseline capillary density compared with the reference tertile (model A). Adjustment for BMI and blood pressure did not affect the association (model B), neither did adjustment for smoking (data not shown). For venous occlusion and peak reactive hyperaemia, a similar, but nonsignificant pattern of lower capillary counts in men with higher homocysteine levels was found. In women, no significant associations were found between homocysteine and microvascular outcomes using regression analyses.

Discussion The salient finding of this study is that in men, but not in women, homocysteine is inversely associated with the number of perfused skin capillaries. The clinical relevance of this result should be discussed in the context of our current knowledge of the human microcirculation. As microvascular disease is implicated in the pathogenesis of high blood pressure, insulin resistance and organ failure [8,9], it is conceivable that homocysteine impacts on the risk of developing these conditions via its effect on the microcirculation. Although studies are limited, there are indeed indications that moderate hyperhomocysteinaemia increases the risk of developing both hypertension [26–28] and insulin resistance

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[29–31]. In addition, homocysteine may play a role in the development of microvascular complications in these conditions [32]. As a consequence of the indirect nature of the possible association between homocysteine and CVD risk, the lag time between (treatment of) hyperhomocysteinaemia and (reduction in) CVD may conceivably be longer than that of conventional risk factors. Another implication involves the microcirculation as a determinant of tissue perfusion. Cutaneous microvascular function is used as a surrogate for generalized microvascular function. Indeed, correlations between coronary and skin microvascular function have been reported [33,34], and recent work also demonstrates a close association between skin and muscle microvascular function [35]. The previously described association between homocysteine and, for example, coronary flow reserve [36,37] may possibly be explained by our finding of an association between homocysteine and the microcirculation. The few human studies on skin microcirculation, which is often used as a model for systemic microcirculation [35,38], support the existence of an association between homocysteine levels and microvascular dysfunction measured by laser Doppler flowmetry [39,40]. In these studies, however, acute experimental hyperhomocysteinaemia was used which, in contrast with our study, does not reflect the biological condition of hyperhomocysteinaemia. Another large population-based, nonexperimental study showed an association between homocysteine and narrowing of retinal microvessels in elderly subjects [15]. Thus, in general, previous studies, using different approaches, support the existence of an association between homocysteine and microvascular perturbations. However, our study is the first to show such an association with a nonexperimental set-up and in relatively young and healthy subjects. A particular finding of this study is the gender difference in the relationship between homocysteine and the microvasculature. The slightly higher BMI and blood pressure, which are known for their association with the microcirculation, could not explain the observed association between homocysteine and capillary density in men. Gender differences may possibly be explained by protective effects of oestrogens against homocysteine-induced vascular dysfunction [19,41,42] and thus do not automatically imply postmenopausal women, which were not included in our study. Homocysteine was significantly correlated with baseline capillary density only. We did not observe a significant association with peak reactive hyperaemia, which is commonly interpreted as a more functional characteristic of microvascular health. Also, venous occlusion, which is believed to mostly represent the structurally available amount of skin capillaries, was not significantly associated with homocysteine. Baseline capillary density may represent functional changes and may be

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Table 3 Regression analyses Men B

Women 95% CI

P

B

95% CI

P

Baseline capillary density Model A Tertile 2

6
66

14
03 to 0
72

0
076

0
59

5
54 to 6
73

0
85

Tertile 3

8
47

15
79 to

0
024

0
06

6
07 to 6
19

0
99

Tertile 2

6
87

14
33 to 0
58

0
070

0
94

5
26 to 7
14

0
77

Tertile 3

8
65

16
05 to

0
022

0
16

6
01 to 6
34

0
96

Tertile 2

9
39

19
96 to 1
19

0
081

0
27

8
91 to 9
46

0
95

Tertile 3

6
08

16
34 to 4
18

0
243

0
51

8
63 to 9
65

0
91

Tertile 2

9
38

20
05 to 1
29

0
084

0
37

8
95 to 9
69

0
94

Tertile 3

5
93

16
30 to 4
43

0
259

0
68

8
56 to 9
91

0
89

21
91 to

0
017

0
65

8
62 to 9
93

0
89

17
43 to 2
15

0
125

2
09

7
18 to 11
37

0
66

21
93 to

0
018

1
26

8
09 to 9
93

0
79

0
141

2
60

6
71 to 11
90

0
58

1
14

Model B

1
25

Venous occlusion Model A

Model B

Peak reactive hyperaemia Model A Tertile 2

12
06

Tertile 3

7
64

2
21

Model B Tertile 2

12
00

Tertile 3

7
39

2
07

17
27 to 2
50

Dependent variable: homocysteine; Tertile 1 was used as reference category; Model A: crude model; Model B: adjusted for BMI and SBP.

a very early vascular abnormality in homocysteine-associated vascular perturbation. In the development of hypertension, for example, reduced baseline capillary density has been demonstrated in an early stage of disease [43]. Detrimental effects of homocysteine on capillary density could be explained by homocysteine-induced changes in the capillary bed itself, as well as vascular changes upstream, particularly in precapillary arterioles. In the literature, several possible underlying mechanisms are described. Firstly, homocysteine-induced endothelial dysfunction and (arteriolar) smooth muscle cell dysfunction might play a role. Impaired flow-mediated vasodilation has been shown in chronic and acute hyperhomocysteinaemia [44,45]. Higher levels of homocysteine have also been associated with impaired endothelium-independent vasodilation [11]. On a cellular level, homocysteine-induced endothelial dysfunction can be explained by a reduced production or bioavailability of

vasoactive substances such as NO and endothelium-derived hyperpolarizing factor [46–48]. Secondly, structural rather than functional changes in precapillary arterioles may contribute to capillary rarefaction. There is evidence that homocysteine augments smooth muscle cell proliferation [49,50] and vascular remodelling [51]. This would be compatible with a previous study, suggesting that homocysteine levels are associated with retinal arteriolar narrowing [15]. Finally, reduced angiogenesis may contribute to capillary rarefaction. Experimental animal studies show impaired angiogenesis in diet-induced hyperhomocysteinaemia. The postulated underlying mechanisms include reduced NO availability, oxidative stress and inhibited endothelial cell proliferation and migration [52–54]. A limitation of the present study is its cross-sectional nature. It is, however, one of the largest existing studies addressing the human microcirculation. Another limitation is that

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metabolites related to homocysteine and B vitamins were not measured. A previous study showed that S-adenosylmethionine and folate, both important in homocysteine metabolism, were even stronger correlates of vascular function than homocysteine itself [11], suggesting an underestimation of the relationship between components of homocysteine metabolism and the microcirculation in the present study. In conclusion, we found an association between homocysteine and microvascular function. In addition, our study suggests a gender difference in this association. Our findings provide a new potential mechanism by which homocysteine affects CVD risk.

Perspectives Previous studies have consistently shown that mild-to-moderate hyperhomocysteinaemia is an independent predictor of cardiovascular disease. However, the resulting hypothesis that homocysteine is a causal contributor to cardiovascular disease has been questioned since the negative results of homocysteinelowering intervention trials. Hence, novel paradigms are needed. Although causality cannot be established in our crosssectional study, the observed association between homocysteine and capillary density in healthy, middle-aged men suggests that homocysteine is related to adverse changes in the microvasculature in what could be an early stage of cardiovascular disease. In addition, our study suggests a gender difference in the relationship between homocysteine and the microcirculation.

Sources of funding E.C. Eringa is supported by the Netherlands Organisation for Scientific Research (Grant 916.76.179) and the Netherlands Foundation for Cardiovascular Excellence. E.H. Serne is supported by a fellowship from The Netherlands Heart Foundation (Grant no. 2010T041).

Conflict of interest None.

Address Department of Internal and Vascular Medicine, Institute for Cardiovascular Research (IcaR-VU), VU University Medical Centre, De Boelelaan 1117, 1081 HV, Amsterdam, the Netherlands (J. M. Hornstra, E. H. Serne, N. J. Wijnstok, Y. M. Smulders); Department of Health Sciences, Faculty of Earth and Life Sciences, EMGO Institute for Health and Care Research, De Boelelaan 1085, 1081 HV, VU University Amsterdam, Amsterdam, the Netherlands (T. Hoekstra, N. J. Wijnstok, J. W. R.Twisk); Department of Physiology, Institute for Cardiovascular Research (IcaR-VU), van der Boechorststraat 7, 1081 BT,

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VU University Medical Centre, Amsterdam, the Netherlands (E. C. Eringa); Metabolic Unit, Department of Clinical Chemistry, De Boelelaan 1117, 1081 HV, VU University Medical Centre, Amsterdam, the Netherlands (H. J. Blom). Correspondence to: Jacqueline Hornstra, De Boelelaan 1117, room 4A35, 1081 HV Amsterdam, the Netherlands. e-mail: [email protected] Received 5 April 2013; accepted 7 January 2014 References 1 Homocysteine Studies Collaboration. Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis. JAMA 2002;288:2015–22. 2 Clarke R, Halsey J, Bennett D, Lewington S. Homocysteine and vascular disease: review of published results of the homocysteinelowering trials. J Inherit Metab Dis 2011;34:83–91. 3 Zhou YH, Tang JY, Wu MJ, Lu J, Wei X, Qin YY et al. Effect of folic acid supplementation on cardiovascular outcomes: a systematic review and meta-analysis. PLoS ONE 2011;6:e25142. 4 Joseph J, Handy DE, Loscalzo J. Quo vadis: whither homocysteine research? Cardiovasc Toxicol 2009;9:53–63. 5 Loscalzo J. Homocysteine trials–clear outcomes for complex reasons. N Engl J Med 2006;354:1629–32. 6 Smulders YM, Blom HJ. The homocysteine controversy. J Inherit Metab Dis 2011;34:93–9. 7 De Boer MP, Meijer RI, Wijnstok NJ, Jonk AM, Houben AJ, Stehouwer CD et al. Microvascular dysfunction: a potential mechanism in the pathogenesis of obesity-associated insulin resistance and hypertension. Microcirculation 2012;19:5–18. 8 Levy BI, Schiffrin EL, Mourad JJ, Agostini D, Vicaut E, Safar ME et al. Impaired tissue perfusion: a pathology common to hypertension, obesity, and diabetes mellitus. Circulation 2008;118:968–76. 9 Serne EH, de Jongh RT, Eringa EC, IJzerman RG, Stehouwer CD. Microvascular dysfunction: a potential pathophysiological role in the metabolic syndrome. Hypertension 2007;50:204–11. 10 Moat SJ, Lang D, McDowell IF, Clarke ZL, Madhavan AK, Lewis MJ et al. Folate, homocysteine, endothelial function and cardiovascular disease. J Nutr Biochem 2004;15:64–79. 11 Spijkerman AM, Smulders YM, Kostense PJ, Henry RM, Becker A, Teerlink T et al. S-adenosylmethionine and 5methyltetrahydrofolate are associated with endothelial function after controlling for confounding by homocysteine: the Hoorn Study. Arterioscler Thromb Vasc Biol 2005;25:778–84. 12 van Guldener C, Stehouwer CD. Hyperhomocysteinemia, vascular pathology, and endothelial dysfunction. Semin Thromb Hemost 2000;26:281–9. 13 Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med 1998;338:1042–50. 14 Bertsch T, Mielke O, Holy S, Zimmer W, Casarin W, Aufenanger J et al. Homocysteine in cerebrovascular disease: an independent risk factor for subcortical vascular encephalopathy. Clin Chem Lab Med 2001;39:721–4. 15 Van Hecke MV, Dekker JM, Nijpels G, Teerlink T, Jakobs C, Stolk RP et al. Homocysteine, S-adenosylmethionine and S-adenosylhomocysteine are associated with retinal

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