Low serum 25-hydroxyvitamin D levels are associated with increased cardiovascular morbidity and mortality

ABSTRACT Background There is controversy about the association between vitamin D and cardiovascular disease (CVD). This article aims to explore the association of serum 25-hydroxyvitaminD (25 OHD) with the risk of CVD. Methods PubMed, EMBASE, Web of Science database, OVID, and Cochrane Library databases (last updated in August 2022) were systematically searched. The relationship between 25OHD and the risk of CVD was assessed by using the 95% confidence intervals (CI) and hazard ratio (HR). The effect model was selected by the size of heterogeneity. Results The meta-analysis included 40 cohort studies that contained 652352 samples. The pooled results showed that a decreased level of 25OHD was associated with an increased relative risk of total CVD events (HR = 1.35, 95% CI: 1.26–1.43). Furthermore, the results also showed that a decreased circulating 25OHD level was associated with an increased mortality of CVD (HR = 1.43, 95% CI: 1.30–1.57) and incidence of CVD (HR = 1.26, 95% CI: 1.16–1.36), especially an increased risk of heart failure (HF) (HR = 1.38, 95% CI: 1.2–1.6), myocardial infarction (MI) (HR = 1.28, 95% CI: 1.13–1.44) and coronary heart disease (CHD) (HR = 1.28, 95% CI: 1.1–1.49). Conclusions The current meta-analysis shows that reduced serum 25OHD concentrations is not only associated with increased total cardiovascular events and cardiovascular mortality, but also with increased risk of HF, MI, and CHD. Limitations The underlying mechanism still needs to be explored further, and well-designed RCTs are needed to confirm the role of vitamin D in the occurrence and development of CVD.


Introduction
The prevalence of vitamin D insufficiency is high in both developed and developing countries and over half of the world population has inadequate vitamin D levels (serum 25-hydroxyvitaminD) [1][2][3]. Further,  is an accepted measure of vitamin D status [4,5].
Not only does vitamin D play an important role in bone metabolism, research in recent decades has shown that vitamin D also plays a non-negligible role in the pathogenesis of extra-skeletal chronic diseases, such as hypertension, diabetes, cancer, metabolic syndrome, and mortality [6][7][8][9][10]. In particular, vitamin D deficiency has an effect on the risk of cardiovascular morbidity and mortality [5,11].
It is well known that cardiovascular disease (CVD) is a globally recognized serious public health event and its morbidity and mortality are increasing every year [12,13]. The evidence for the link between vitamin D and the risk of CVD disease has been inconclusive. In fact, some studies have shown an inverse association between serum 25OHD levels and CVD risk [5], and recent studies have shown an inverse J-shaped association between the two factors [14]. However, some meta-analysis studies have reported controversial or invalid results for the link between serum 25OHD and CVDs [15].
If a causal link between low vitamin D levels and CVD is detected, it would have great public health significance. Therefore, this study aimed to explore the relationship between 25OHD levels and CVD morbidity and mortality in the population.

Data sources and search strategy
We systematically searched the EMBASE, PubMed, OVID, Web of Science, and Cochrane Library databases (last updated in August 2022). The following Medical Subject Headings (MeSH) search terms and keywords were used: '25-hydroxyvitamin D' or '25(OH)D' or 'cholecalciferol' and 'heart failure (HF)' or 'myocardial infarction (MI)' or 'ischemic heart disease (IHD)' or 'coronary heart disease (CHD)' or 'cardiovascular disease' or 'cardiovascular events' and 'mortality' or 'incidence.' (3) circulating 25-hydroxyvitamin D. Exclusion criteria were as follows: (1) animal experiment, letter, case reports, editorials, literature reviews, and comments; (2) cross-sectional studies and case-control studies; (3) studies that did not use CVD incidence or CVD mortality as an endpoint; (4) HR and 95% CIs adjusted for confounders were not provided.

Data extraction and quality assessment
Two reviewers (WL and DX) extracted the data and assessed the accuracy. The following information was extracted for each study: study characteristics (author's name, year of publication, sex, samples, age, and country), follow-up duration, method of 25OHD measurement, value of adjusted HR values and their 95% CI, adjustment confounders, cardiovascular outcome, and levels of circulating 25OHD.
The quality of studies was assessed according to the Newcastle-Ottawa scale. This scale, ranging from 0 to 9 assigns scores for selection, comparability, and outcome. Studies with a rating of seven points or higher were considered to be of high quality. Disagreements between the two reviewers were resolved by discussion with other researchers until a consensus was reached.

Statistical analysis
We converted the HR values and 95% CI in each study using their natural logarithms. The natural logarithms of HR values and 95% CI were used to assess the relationship between 25OHD and the incidence or mortality of CVD.
Heterogeneity was assessed by the I 2 test and Cochran's Q, with I 2 > 50% and p < 0.01 indicating significant heterogeneity. If the heterogeneity was significant, the random-effect model was adopted; otherwise, the fixed-effect model was applied.
Sensitivity analysis examined the impact of each study by eliminating individual studies and assessing the influence on cumulative risk estimates. Egger's linear regression was used to provide statistical proof for existence of publication bias. Statistical analysis was performed using STATA, version 14.0. The two-tailed P value was less than 0.05, and the difference was statistically significant.

25OHD and the risk of CVD events
Each included study reported the HR and 95% CI between serum 25OHD and cardiovascular outcomes. The effect of 25OHD on total CVD events was calculated using the HR adjusted for confounders. Here, HR (95% CI) for the highest vs. lowest categories of 25OHD was used. In general, a decreased level of 25OHD was associated with an increased relative risk of total CVD events (HR = 1.35, 95% CI: 1.26-1.43) and the heterogeneity was significant (I 2 = 65.5%, p < 0.001) (Figure 2), low circulating 25OHD levels increased the risk of CVD by 35%.
The meta-analysis showed that a decreased circulating 25OHD levels was also associated with increased risk of HF (HR = 1.38, 95% CI: 1. To explore the main source of heterogeneity, we performed a subgroup analysis, and it showed the following findings: gender  Subgroup analysis meta-regression methods revealed significant evidence of heterogeneity between subgroups stratified by follow-up time, gender, method of 25OHD measurement and age (Table 2, Figure S1-9).

Sensitivity analyses and publication bias
Sensitivity analyses were carried out by eliminating one study at a time. In these analyses, there was no great fluctuation in the HR [1.35 (1.25-1.45)] ( Figure S10).
We conducted Egger's linear regression. The P value of Egger's test was 0.232 (Table S3), and the funnel plot is shown in Figure S11. There was no indication of publication bias.

Discussion
The results of the meta-analysis, which included 652,352 participants from 40 cohort studies , showed a negative association between the serum 25OHD concentration and the risk of CVD events. A decreased level of 25OHD was not only associated with an increased risk of CVD morbidity, but also with increased CVD mortality. Furthermore, the meta-analysis also suggested that a decreased level of 25OHD was associated with increased risk of HF, MI, and CHD.
Most cohort studies found a significant inverse association between 25OHD levels and CVD mortality, and it was consistent with previous reviews [56][57][58]. Interestingly, our meta-analysis also revealed a significant inverse relationship between the 25OHD status and CVD incidence, especially HF, MI, and CHD incidence. This was not reflected in previous reviews [5,11].
A retrospective, observational, nested case-control study showed that in patients with vitamin D deficiency and no history of MI, those with 25OHD levels of 30 ng/mL or more had lower rates of MI events than those with 25OHD levels of 21-29 ng/mL and patients with ≤ levels of 20 ng/mL [59]. In addition, in a nonlinear Mendelian analysis conducted in the British Biobank, there were 44,519 CVD cases and 251,269 controls. In that study, using a large-scale genetic design, serum 25OH D concentrations were measured using 35 proven genome-wide significant variants. It also showed evidence of a causal effect of vitamin D deficiency on cardiovascular health. There was an L-type association between genetically predicted serum 25OH D and CVD risk (P nonlinear = 0.007), in which CVD risk initially decreased sharply with increasing concentrations, and it tended to be stable at about 50 nmol/ L [60]. Another secondary analysis suggested that daily vitamin D supplementation to maintain serum 25(OH) D levels was a promising approach to reducing the risk of diabetes in adult prediabetic patients [61].
Although data from numerous studies, including this metaanalysis, suggest a strong link between vitamin D and CVD, it is important to note that there are residual confounders in observational studies, and randomized clinical trials (RCTs) have failed to demonstrate causation. Randomized controlled trials suggested that vitamin D supplementation did not reduce cardiovascular events [62,63]. In the latest randomized controlled meta-analysis, vitamin D supplementation was not associated with a reduction in major cardiovascular adverse events (mortality from MI, stroke, and CVDs), or all-cause mortality. Studies have shown that vitamin D supplements do not provide cardiovascular protection [62]. Although an RCT is the gold-standard method for establishing causality, its potential benefit in capturing vitamin D supplementation is being questioned, as most trials included individuals who have been supplemented with vitamin D. Given that vitamin D testing and supplements were readily available, this makes baseline 25OHD concentrations too high; and contamination and non-blindness in the placebo group made the situation worse [60,64]. A low vitamin D dose and the fact that the results did not reach 25OHD concentrations in the analyzed individuals may also have contributed to the result bias [62].
In recent decades, the relationship between vitamin D deficiency and cardiovascular events has received great public health interest because vitamin D is a very modifiable risk factor for CVD events. To our knowledge, 25OHD is converted to its active form 1,25-dihydroxyvitamin D in the kidney by the action of 1-alpha-hydroxylase and it exerts its biological effects through the vitamin D receptor (VDR). Vitamin D receptor and 1-α-hydroxylase are expressed in dozens of human target tissues, including endothelial cells, vascular smooth muscle cells and cardiomyocytes [65,66]. In the heart, it exhibits anti-cardiac hypertrophy, and modulates extracellular matrix turnover and myocardial contractility [67]. It also acts as a negative regulator of the renin-angiotensin-aldosterone system [68], alters endothelial function through its antioxidant and anti-inflammatory effects [69], and may reduce vascular calcification [67]. Even in normal or near-normal coronary arteries, vitamin D deficiency is associated with endothelial dysfunction, reduced coronary flow, and subclinical atherosclerosis [70]. Furthermore, vitamin D deficiency is associated with several cardiovascular risk factors [71,72]. Through the regulation of renin and angiotensin II synthesis, vitamin D deficiency increases the production of reactive oxygen species and G proteins, leading to inhibition of pathways required for intracellular glucose transport; thus, resulting in leading to abnormal insulin function and the development of metabolic syndrome [73]. The direct effect of vitamin D on smooth muscle proliferation may contribute to its effects on cardiovascular health [74]. The present meta-analysis has the following advantages: First, this study assessed more comprehensive outcomes than previous meta-analyses, including MI, HF, CHD, and cardiovascular mortality and corresponding subgroup analysis. Second, the meta-analysis included a large number of participants (n = 652,352). Third, with the combined data of adjusted potential confounding factors, it could minimize the confounding factors, thus, the conclusions based on this analysis are likely to be valid and could improve the accuracy of risk assessment. Fourth, all included studies were high-quality cohort studies, which further increased the reliability of the conclusions obtained. Serum 25OHD is known to be the primary biomarker for assessing the vitamin D status [4]. Measurements of dietary vitamin D intake are less objective than laboratory measurements of 25OHD [4,75], and in this meta-analysis we used serum 25OHD as a measure of exposure.
However, the present meta-analysis has the following limitations: First, as with any observational study, an association does not imply causation. Second, we could not rule out residual confounding due to unmeasured confounding factors in CVD. Although the included studies were adjusted for multiple potential confounders, the possibility of residual confounding due to features that affect the risk of CVD could not be ruled out. Third, heterogeneity was evident in this meta-analysis, possibly due to differences in follow-up duration, age, gender, method of 25OHD measurement and the well-known variability in serum 25OHD assay data. Fourth, our meta-analysis included studies were medium-to high-quality cohort studies, most of which were prospective, but there were still 5 retrospective cohort studies. Finally, dose-response effects of vitamin D on CVD morbidity and mortality were not assessed because the number of participants in each 25OHD dose subgroup and the corresponding number of CVD events and deaths was not available from the original text.
In conclusion, the current meta-analysis shows that reduced serum 25OHD concentration was not only associated with increased the total cardiovascular events and cardiovascular mortality, but also with increased risk of HF, MI, and CHD. The underlying mechanism still needs to be explored further, and well-designed RCTs are needed to confirm the role of vitamin D in the occurrence and development of CVD.

Funding
This paper was not funded.

Declaration interests
No potential conflict of interest was reported by the author(s).

Reviewer disclosures
A reviewer on this manuscript has disclosed that they have receive funding from Bio-Tech Pharmacal, Inc. (Fayetteville, AR). Peer reviewers on this manuscript have no other relevant financial relationships or otherwise to disclose.

Author contributions
WL and DX had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: WL, DX and JZ. Acquisition of data: YZ and QY Analysis and interpretation of data: All authors.