Objective
The objective of the study was to examine the associations of maternal plasma levels of 25-hydroxyvitamin D [25(OH)D] with angiogenesis and endothelial dysfunction indicators: soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and risk of preeclampsia.
Study Design
In this prospective cohort study (n = 697), maternal plasma 25(OH)D levels were measured at 12-18 and 24-26 weeks; sFlt-1, PlGF, ICAM-1, and VCAM-1 levels were measured at 24-26 weeks.
Results
Maternal PlGF levels were significantly lower in women with 25(OH)D less than 50 nmol/L at 12-18 weeks (median, 449.5 vs 507.9 pg/mL, P = 0.04) and 24-26 weeks (median, 450.4 vs 522.5 pg/mL, P = 0.007). Both maternal 25(OH)D and PlGF levels were inversely associated with the risk of preeclampsia (both P < .05). However, based on a test of interaction, there was no evidence that the association between vitamin D and preeclampsia depended on the level of PlGF.
Conclusion
Maternal vitamin D deficiency is associated with low PlGF levels and increased preeclampsia risk. However, our data do not support the hypothesis that the association between vitamin D deficiency and preeclampsia is mediated by impaired angiogenesis.
Preeclampsia is a pregnancy-specific multisystem disorder characterized by de novo-onset high blood pressure and proteinuria after 20 weeks’ gestation complicates 2-8% of all pregnancies and is a major contributor to maternal and perinatal mortality and morbidities (acute and long term). Despite intensive efforts to delineate the pathophysiology of preeclampsia, neither its etiology nor its pathogenesis has been clearly identified.
Recently epidemiological studies have demonstrated an association between low maternal vitamin D status during pregnancy and the incidence of preeclampsia and suggest that vitamin D deficiency may be an independent risk factor for preeclampsia. However, the underlying mechanisms remain unknown. Vitamin D deficiency is associated with inflammation-linked vascular endothelial dysfunction. Endothelial cell damage or dysfunction appears to be a basic pathophysiological event of the maternal vascular system in women with preeclampsia.
The concept of endothelial dysfunction observed in preeclampsia is supported by the findings of a state of angiogenic disturbance and increased maternal circulating levels of endothelial adhesion molecules. Several biological markers have been used as indicators of endothelial dysfunction. Soluble fms-like tyrosine kinase 1 (sFlt-1) competitively binds to placental growth factor (PlGF) and vascular endothelial growth factor (VEGF), preventing their role in endothelial preservation. The placenta produces sFlt-1 during normal pregnancy, but significantly higher amounts are produced from the hypoxic placenta in preeclamptic pregnancies. The presence of these proteins and their effect on PlGF and VEGF create an angiogenically imbalanced vascular environment that contributes to the endothelial insult occurring in preeclampsia. Soluble adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) have been implicated in the interaction between the trophoblast and the endometrium and can be expressed in a variety of cells during inflammation. ICAM-1 and VCAM-1 are known to be increased in patients with preeclampsia, indicating a pathological role of endothelial cell activation or dysfunction.
The possible mechanisms by which vitamin D could alter the risk of preeclampsia remain to be elucidated. Recently in vitro studies have indicated that vitamin D may improve angiogenesis and inhibit adhesion molecule expression in endothelial cells. However, this information has not been examined in vivo. There is a lack of information in humans as to whether maternal vitamin D status could influence angiogenesis and endothelial dysfunction by altering circulating levels of angiogenic factors or adhesion molecules and result in preeclampsia.
The objective of the present study was to explore the associations between maternal circulating 25-hydroxyvitamin D [25(OH)D] levels, indicators of angiogenesis and endothelial dysfunction (sFlt-1, PlGF, ICAM-1, VCAM-1) and the risk of preeclampsia.
Materials and Methods
Study design
This prospective cohort study was based on a data and biological specimens’ bank generated in the context of a randomized, placebo-controlled international trial of antioxidant supplementation (vitamins C and E) for the prevention of preeclampsia (INTAPP). Canadian INTAPP subjects who were recontacted and consented to participate in a biobank for further research with maternal plasma specimen available at the baseline study visit contributed to this study.
The design and the study population characteristics of this cohort have been described previously. In the INTAPP trial, women were stratified according to the presence or absence of risk factors for preeclampsia. Women were in the high-risk stratum for preeclampsia if they met at least one of the following 4 criteria: (1) prepregnancy chronic hypertension (diastolic blood pressure of 90 mm Hg or greater or the use of an antihypertensive medication before 20 weeks of gestation); (2) prepregnancy diabetes; (3) a multiple pregnancy; or (4) a history of preeclampsia. The remaining women were nulliparae without other risk factors for preeclampsia (low-risk group).
Nonfasting maternal blood samples were collected and banked at 12-18 weeks and 24-26 weeks of gestation. Venous blood was drawn into EDTA tubes, and plasma samples were immediately separated by centrifugation at 500 × g for 10 minutes at 4°C. Plasma samples were rapidly frozen and stored at −80°C for future assays.
The study definitions of gestational hypertension and preeclampsia according to the criteria of the Canadian Hypertension Society Consensus Conference Report have been described previously. Briefly, gestational hypertension was defined as 2 or more readings of diastolic blood pressure of 90 mm Hg or greater taken 4 hours apart but within 72 hours occurring at 20 or more weeks of gestation. Preeclampsia was defined as gestational hypertension with proteinuria. Proteinuria was defined as a urine protein dipstick test 2+ or greater or the urinary excretion of 0.3 g or greater in 24 hours urine collection. Superimposed preeclampsia was defined according to the guidelines of Canadian Hypertension Society Consensus Conference Report.
Laboratory assays
Maternal plasma 25(OH)D levels at 12-18 weeks and 24-26 weeks were measured by a direct, competitive chemiluminescent immunoassay using the DiaSorin LIAISON 25(OH)D TOTAL assay (DiaSorin, Inc, Stillwater, MN) as described previousely. The lower and upper limits of detection for 25(OH)D were 10 nmol/L and 375 nmol/L, respectively. The coefficient of variation (CV) was 5.2-9.2%.
The biomarkers of angiogenesis and endothelial dysfunction, maternal plasma sFlt-1, PlGF, ICAM-1, and VCAM-1 were measured at 24-26 weeks’ gestation. The sFlt-1 level was measured by an automated 2-site chemiluminescent immunometric assay (Beckman-Coulter, Chaska, MN). PlGF concentration was determined by using a solid phase, 2-site fluorommunometric assay on the DELFIA Xpress 6007-0010 platform (PerkinElmer, Turku, Finland). The lower limits of detection of the assay were 5 pg/mL for sFlt-1 and PlGF, and the CVs were less than 7.7%.
Soluble ICAM-1 and soluble VCAM-1 were measured using the Quantikine human immunoassay kit specific for sICAM-1(CD54) and sICAM-1(CD106) (R&D Systems, Minneapolis, MN). The lower limits of detection were 1.6 ng/mL for sICAM-1 and 6.3 ng/mL for sVCAM-1, with CVs in the range of 2.8–6.0%. All assays were run according to the protocol specified by the manufacturer. The individuals performing all study assays were blinded to the participant’s pregnancy outcome.
Statistical analysis
A Spearman correlation analysis was conducted to examine the association between maternal plasma 25(OH)D, sFlt-1, PlGF, ICAM-1, or VCAM-1 concentrations. We explored the maternal plasma sFlt-1, PlGF, ICAM-1, and VCAM-1 levels at 24-26 weeks’ gestation according to maternal vitamin D status at 12-18 weeks and 24-26 weeks of gestation, using the cutoff point of 50 nmol/L, which has been suggested by many experts as the cutoff for vitamin D deficiency. The 50 nmol/L cutoff was consistent with the vitamin D concentration–preeclampsia risk curve observed in our study cohort, as previously reported.
To explore the relationship between maternal sFlt-1, PlGF, ICAM-1, and VCAM-1 levels and vitamin D status, the Wilcoxon rank sum tests were performed for differences in the biomarkers’ concentration in women in the presence and absence of maternal plasma low vitamin D status [25(OH)D less than 50 nmol/L vs 50 nmol/L or greater]. The Wilcoxon rank sum tests were also performed to examine the differences in the biomarkers’ levels between preeclamptic versus nonpreeclamptic women.
Finally, we conducted logistic regression analyses adjusting for the potential confounding factors (maternal age, risk status, smoking, prepregnancy body mass index, and season of blood draw) to estimate the risk (adjusted odds ratio [aOR] with 95% confidence intervals [CIs]) of preeclampsia associated with low vitamin D status. Season of blood draw was defined according to the dates of collection of the specimens: winter if the blood was drawn on November 1 through April 30 (the period of lowest light exposure in Canada); otherwise, it was defined as nonwinter.
A final multivariable logistic model was developed that included biomarkers that were associated with both vitamin D and preeclampsia ( P < .05) to determine whether the inclusion of the biomarker altered the relationship between vitamin D and preeclampsia. We tested for evidence of interaction between 25(OH)D status and PlGF levels by including a multiplicative interaction term in the multivariable logistic model. Two-sided values of P < .05 were considered significant. All analyses were performed using SAS software, version 9.2 (SAS Institute Inc, Cary, NC).
The study was approved by the Research Ethics Committees of the participating hospitals.
Results
There were 697 patients who consented and contributed specimens to the biobank, among whom 32 (4.6 %) developed preeclampsia including 6 superimposed preeclampsia. The characteristics of the study population have been described previously. A strong positive correlation was observed in maternal 25(OH)D concentrations between 12-18 weeks and 24-26 weeks of gestational age windows (r = 0.69, P < .0001). A positive correlation was demonstrated (r = 0.37; P < .0001) between maternal ICAM-1 and VCAM-1. Maternal 25(OH)D levels at 12-18 weeks and 24-26 weeks of gestation were positively correlated with PlGF at 24-26 weeks’ gestation (r = 0.10, P = .01; r = 0.11, P = .01, respectively) ( Table 1 ). Maternal 25(OH)D level at 12-18 weeks’ gestation and ICAM-1 at 24-26 weeks were inversely correlated (r = −0.12; P = .004). However, this association was not observed between maternal levels of 25(OH)D and sFlt-1 or VCAM-1.
| Vitamin D status | sFlt-1 | PlGF | ICAM-1 | VCAM-1 |
|---|---|---|---|---|
| 25(OH)D level at 12-18 weeks’ gestation | ||||
| r | 0.079 | 0.102 | –0.118 | –0.029 |
| P value | .08 | .01 a | .004 a | .48 |
| 25(OH)D level at 24-26 weeks’ gestation | ||||
| r | 0.047 | 0.106 | −0.062 | −0.010 |
| P value | .29 | .01 a | .13 | .81 |
Table 2 presents the associations between maternal plasma vitamin D status (less than 50 nmol/L, 50 nmol/L or greater) at 12-18 weeks and 24-26 weeks of gestation and maternal circulating sFlt-1, PlGF, ICAM-1, VCAM-1 concentrations at 24-26 weeks’ gestation. Maternal plasma PlGF concentrations at 24-26 weeks were significantly lower in the pregnant women whose 25(OH)D levels were less than 50 nmol/L at 12-18 weeks and 24-26 weeks of gestation compared with those were not (median, 449.5 pg/mL vs 507.9 pg/mL, P = .04; 450.4 pg/mL vs 522.5 pg/mL, P = 0.007, respectively). Maternal plasma ICAM-1 concentration at 24-26 weeks was significantly higher in the pregnant women whose 25(OH)D concentrations were less than 50 nmol/L at 12-18 weeks compared with those were not (median, 211.8 ng/mL vs 196.2 ng/mL, P = .02). However, no significant association was observed between vitamin D status and sFlt-1 or VCAM-1 concentrations.
| Biomarkers | Vitamin D status | Median (interquartile range) | Mean ± SD | P value a |
|---|---|---|---|---|
| 12-18 weeks’ gestation | ||||
| sFlt-1, pg/mL | 25(OH)D <50 nmol/L | 2074.4 (1196.6–3332.3) | 2799.3 ± 2371.8 | .06 |
| 25(OH)D ≥50 nmol/L | 2384.6 (1275.2–3987.8) | 3879.8 ± 6147.2 | ||
| PlGF, pg/mL | 25(OH)D <50 nmol/L | 449.5 (277.6–832.1) | 653.8 ± 635.2 | .04 b |
| 25(OH)D ≥50 nmol/L | 507.9 (334.7–933.3) | 754.1 ± 729.3 | ||
| ICAM-1, ng/mL | 25(OH)D <50 nmol/L | 211.8 (173.9–247.2) | 215.9 ± 61.2 | .02 b |
| 25(OH)D ≥50 nmol/L | 196.2 (172.3–234.0) | 204.4 ± 53.3 | ||
| VCAM-1, ng/mL | 25(OH)D <50 nmol/L | 498.8 (411.1–590.7) | 514.2 ± 148.4 | .24 |
| 25(OH)D ≥50 nmol/L | 474.8 (404.5–579.9) | 503.1 ± 143.8 | ||
| 24-26 weeks’ gestation | ||||
| sFlt-1, pg/mL | 25(OH)D <50 nmol/L | 2190.1 (1336.5–3813.4) | 3912.7 ± 7132.2 | .81 |
| 25(OH)D ≥50 nmol/L | 2230.0 (1230.8–3925.9) | 3218.2 ± 3403.5 | ||
| PlGF, pg/mL | 25(OH)D <50 nmol/L | 450.4 (271.6–805.8) | 637.9 ± 638.7 | .007 b |
| 25(OH)D ≥50 nmol/L | 522.5 (344.7–985.0) | 766.7 ± 727.1 | ||
| ICAM-1, ng/mL | 25(OH)D <50 nmol/L | 205.9 (174.1–242.8) | 211.9 ± 55.4 | .18 |
| 25(OH)D ≥50 nmol/L | 197.6 (172.3–234.8) | 206.9 ± 57.5 | ||
| VCAM-1, ng/mL | 25(OH)D <50 nmol/L | 493.2 (407.7–591.0) | 509.9 ± 136.6 | .46 |
| 25(OH)D ≥50 nmol/L | 477.2 (406.1–574.8) | 505.4 ± 150.9 |
a P values in Wilcoxon rank sum tests for differences in biomarker concentrations in women with 25(OH)D less than 50 nmol/L versus 50 nmol/L or greater;
Table 3 presents maternal plasma levels of 25(OH)D and circulating biomarkers in women with preeclampsia vs women without preeclampsia. Maternal PlGF concentration was significantly lower in women with preeclampsia compared with women without preeclampsia (median, 261.6 vs 502.4, P < .0001). Maternal median ICAM-1 concentration was not significantly different in women with preeclampsia compared with women without preeclampsia ( P = .90). Maternal VCAM-1 concentration was significantly higher in women with preeclampsia compared with women without preeclampsia (median, 507.7 vs 483.5, P = .04).
| Variable | Preeclampsia Mean ± SD, median | Nonpreeclampsia Mean ± SD, median | P value a |
|---|---|---|---|
| 25(OH)D, nmol/L | 48.9 ± 16.8, 46.4 | 57.0 ± 19.1, 56.5 | .03 b |
| sFlt-1, pg/mL | 9083.2 ± 16105.4, 2750.4 | 3168.4 ± 3472.1, 2147.1 | .08 |
| PlGF, pg/mL | 374.7 ± 369.4, 261.6 | 733.7 ± 703.8, 502.4 | < .0001 b |
| ICAM-1, ng/mL | 216.9 ± 78.2, 196.8 | 208.6 ± 55.5, 202.7 | .90 |
| VCAM-1, ng/mL | 615.5 ± 256.1, 507.7 | 502.1 ± 136.1, 483.5 | .04 b |
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