Active Serum Vitamin D Levels Are Inversely Correlated With Coronary Calcification
Background Arterial calcification is a common feature of atherosclerosis, occurring in >90% of angiographically significant lesions. Recent evidence from this and other studies suggests that development of atherosclerotic calcification is similar to osteogenesis; thus, we undertook the current investigation on the potential role of osteoregulatory factors in arterial calcification.
Methods and Results We studied two human populations (173 subjects) at high and moderate risk for coronary heart disease and assessed them for associations between vascular calcification and serum levels of the osteoregulatory molecules osteocalcin, parathyroid hormone, and 1α,25-dihydroxyvitamin D3 (1,25-vitamin D). Our results revealed that 1,25-vitamin D levels are inversely correlated with the extent of vascular calcification in both groups. No correlations were found between extent of calcification and levels of osteocalcin or parathyroid hormone.
Conclusions These data suggest a possible role for vitamin D in the development of vascular calcification. Vitamin D is also known to be important in bone mineralization; thus, 1,25-vitamin D may be one factor to explain the long observed association between osteoporosis and vascular calcification.
Calcification is a nearly universal feature of atherosclerosis.123 Almost all angiographically significant atherosclerotic lesions are calcified,4 and the presence of calcium within coronary vasculature has been associated with several adverse clinical events including dissection during angioplasty,5 increased risk of myocardial infarction,6 and poorer 5-year survival.7 Atherosclerotic calcification has long been considered an unregulated, end-stage process that is simply a result of cell death; emerging evidence, however, suggests that calcification occurs much earlier in atherosclerosis than previously believed,89 and far from being an unregulated process, likely involves complex, regulated mechanisms. We hypothesized that atherosclerotic calcification is in many ways similar to skeletal osteogenesis, and recent evidence from our laboratory and others supports this premise.101112131415 These similarities raise the possibility that osteoregulatory molecules involved in bone development may also be involved in development of arterial calcification and that systemic agents such as calcium and vitamin D that are prescribed to enhance deposition of calcium in bones may also have an impact on deposition of calcium in the vascular wall.
To examine the potential role of osteoregulatory factors on development of vascular calcification, we studied two human populations at risk for coronary heart disease and assessed the relation between serum levels of osteoregulatory molecules and the extent of vascular calcification. Levels of osteocalcin, PTH, and 1,25-vitamin D were compared with extent of vascular calcification as detected by EBCT. Of these osteoregulatory factors, only 1,25-vitamin D showed a significant association with vascular calcification, and quite unexpectedly, this was a negative correlation revealing that higher serum 1,25-vitamin D levels were associated with lower amounts of vascular calcification. These data suggest a potential role for endogenous 1,25-vitamin D in inhibition of vascular calcification.
1,25-Vitamin D is known to regulate deposition of calcium in the axial skeleton, and the current data suggest it may regulate deposition of calcium in the vascular wall as well. This may be one factor to explain the long observed association between osteoporosis and vascular calcification.16171819202122 These results also emphasize the importance of understanding the effect of vitamin D supplements, which are widely prescribed for the treatment of osteoporosis, on the occurrence of vascular calcification.
Two groups of human subjects were studied and analyzed separately, both in accordance with institutional guidelines and with institutional review board approval. The Southbay HW group consists of a population of asymptomatic individuals with a ≥5% 4-year risk of developing CHD according to Framingham Study estimates.23 Additional entry criteria for the HW group included age of >45 years and absence of symptoms of CHD. Exclusionary criteria for the current study included known malignancy, renal failure, or hyperparathyroidism. All entered participants answered a detailed questionnaire, underwent EBCT for quantification of coronary calcification, and had blood collected for biochemical analyses.
The FH group consists of patients homozygous for FH, all with a very high risk of developing CHD, and most with symptoms or documentation of atherosclerotic heart disease by angiography at the time of study. This group was assembled from patients treated at the Clinical Center of the National Institutes of Health.24 All subjects ≥13 years old for whom EBCT studies were available were included in the present study and underwent collection of blood for biochemical analyses.
The degree of association between the dependent variables, calcium mass and calcium score, and the independent variables, sex, age, serum cholesterol levels, serum osteocalcin, serum PTH, and serum 1,25-vitamin D, was measured using the Pearson correlation coefficient and stepwise multiple linear regression analyses. The calcium values were normalized by logarithmic transformation. When calculating regression equations, cases with Studentized residual of >2 or <−2 were weighted 0.5.
EBCT scans for the HW group were performed and analyzed at the St. John’s Cardiovascular Research Center at Harbor-UCLA Medical Center using an Imatron C-100 scanner. EBCT scans for the FH group were performed and analyzed at the National Institutes of Health, NHLBI, using an Imatron C-100XL scanner. For both groups, all scans used an image acquisition time of 100 msec, gated to 80% of the ECG RR interval. Scan parameters were 130 kV and 625 mA; images were reconstructed to a 512×512 matrix with a 26-cm reconstruction circle; and calcification was defined as an area of ≥1 mm2 at any site with density values of ≥130 Hounsfield units using densitometric region-of-interest software (in which a region of interest is placed around each lesion and the peak density recorded). Images of the entire heart were acquired in 20 contiguous slices, cephalad to caudad, and calcification was quantified either as calcium mass (HW group) or calcium score (FH group) as described below. Both groups had identical scans for coronary calcification with supplementary data (40 contiguous slices cephalad to caudad) obtained in the FH group to image the entire aorta as well as the coronary arteries. Aortic calcification data were analyzed separately.
Calcium score for each artery is calculated as follows: Calcium Score=[Sum of (Suprathreshold Area×N)]×T/3, where N is a density index with a value of 1 through 4 based on a truncated peak CT number (a measure of density with a range of 130 to 499); and T is the slice thickness.
Calcium mass is calculated as follows: Calcium Mass=T×(Sum of Area×[Mean−Mean′])/Slope, where T is the slice thickness; Area is the suprathreshold area involving at least eight contiguous pixel; Mean is the mean CT number of that area (density); Mean′ is the mean CT number of baseline (adjacent cardiac blood pool); and Slope is the slope of the calibration line calculated from the calcium phantom incorporated into each scan.
Both indices represent the product of slice thickness, the sum of areas exceeding a minimum density, and their density values. The differences are that calcium score is based on a truncated value of peak density, whereas the calcium mass is based on the mean density. In addition, calcium mass is converted to milligram units using a conversion factor based on a calibration phantom of known calcium mass included in each scan.
Whole blood was collected at the respective study sites by venipuncture in the fasting state; the serum was separated by centrifugation and then stored at −80°C. Frozen serum samples were transferred to the host site for analysis. Radioassays were performed on serum for the presence of PTH, osteocalcin, and 1,25-vitamin D (all reagents were from Nichols Laboratories). Both the PTH and osteocalcin assays are two-site immunoradiometric “sandwich” assays. The antibodies used in the PTH assay were goat polyclonal antibodies directed against the amino-terminal PTH 1-34 and the midregion and carboxyl-terminal PTH 39-84. The osteocalcin assay uses polyclonal goat antibodies directed against the 20-36 region of the osteocalcin peptide and the 1-19 region of the osteocalcin peptide. The 1,25-vitamin D assay is a radioreceptor assay using the vitamin D binding protein and tritiated 1,25-vitamin D. All samples were assayed in duplicate, and any pair that differed by >20% was reassayed. If sufficient serum was not available, the subject was excluded from analysis. The mean coefficients of variation for the assays were 2.89±2.18 for PTH, 5.67±6.03 for osteocalcin, and 5.40±4.93 for 1,25-vitamin D.
Predictors of Vascular Calcification in HW Group
One-hundred sixty consecutive HW subjects whose blood was collected during the period December 1993 through June 1994 were analyzed. Seven of these were ultimately excluded according to our predetermined exclusionary criteria. The clinical characteristics of the remaining subset are presented in Table 1.
In the HW group, only one variable was significantly associated with total coronary calcification, and unexpectedly, this was a negative association between serum 1,25-vitamin D levels and coronary calcification (r=−.18; P=.024) (Fig 1). In univariate analysis, no other variable, including PTH, osteocalcin, total cholesterol, LDL cholesterol, Framingham Study risk, or age, revealed a significant association with coronary calcification. Although age did not significantly correlate in univariate analysis, in multivariate analysis, age did slightly but significantly contribute to the variation in coronary calcification when adjusted for 1,25-vitamin D levels. The estimated regression equation considering age and 1,25-vitamin D simultaneously is: ln(Calcium Mass)=0.677+0.094 (Age)−0.053 (1,25-vitamin D).
Because of the possibility that 1,25-vitamin D levels might be affected by either daily UV exposure (time of year) or oral supplementation, we performed analyses addressing these variables. Serum 1,25-vitamin D levels did not vary by the time of year of the blood draw or by ingestion of oral vitamin D supplements. The mean 1,25-vitamin D level of patients tested in winter months was 39.4±12.6 versus 40.7±12.1 pg/mL for patients tested in the summer. The mean 1,25-vitamin D level of patients reporting ingestion of oral vitamin D supplements was 39.7±12.3 versus 40.0±12.9 for patients reporting no vitamin D supplementation.
Predictors of Vascular Calcification in FH Group
In the FH group, three variables were significantly associated with vascular calcification. Both total and LDL cholesterol levels were positively associated with coronary calcification (Fig 2, A and B), with correlation coefficients of .68 (P=.01) and .67 (P=.01), respectively. Total and LDL cholesterol levels were also strongly correlated with each other (r=.94), as expected. Again, as in the HW group, serum 1,25-vitamin D unexpectedly revealed a significant negative association with coronary calcification in the FH group, with a correlation coefficient of −.57 (P=.05) (Fig 3A). The estimated regression equation for 1,25-vitamin D is: ln(Calcium Score)=8.52−0.056 (1,25-vitamin D). Furthermore, when total vascular calcification (coronary plus aortic calcification) was analyzed, this same negative correlation persisted (r=−.60; P=.04) (Fig 3B). Neither HDL cholesterol, age, osteocalcin, nor PTH showed any significant associations with vascular calcification in this group; however, the small sample size does not allow us to definitively conclude that any of these variables are, in fact, not associated with vascular calcium deposition.
Biochemical Differences Between HW and FH Groups
A summary of the biochemical analyses is presented in Table 2. The mean levels of the three osteoregulatory molecules studied—osteocalcin, PTH, and 1,25-vitamin D—did not differ significantly between the HW and the FH groups, and the vast majority of values for both groups fell within normal limits. Although the mean PTH value is higher in the HW group, the difference is not statistically significant and is most likely accounted for by the well known observation that PTH values tend to rise with increasing age in both men and women.25
Vascular calcification is widespread, occurring in >90% of patients with CHD. Growing evidence supports the hypothesis that vascular calcification develops by mechanisms similar to bone formation; thus, we undertook the current investigation to determine the role of systemic osteoregulatory factors on development of vascular calcification. The surprising finding of these studies is that lower serum 1,25-vitamin D levels, which have been shown by other investigators to be associated with lower levels of bone calcification,26 are associated with higher levels of vascular calcification.
In the current studies, both the HW and FH groups manifested significant negative associations between serum 1,25-vitamin D levels and amount of vascular calcification; however, the strength of this association differed between the two groups (r=−.18 for the HW group and r=−.57 for the FH group). Several potential differences between the groups may explain this (see Table 1). First, the HW group consists of a heterogeneous population of asymptomatic individuals who although at high risk for CHD relative to the general population, are nevertheless at significantly lower risk than the FH group. The HW subjects were selected on the basis of absence of documented CHD at the time of the study, whereas in the FH group, most had angiographically documented CHD at the time of the study. Thus, in the FH group, the presence of atherosclerosis was nearly eliminated as a variable, whereas in the HW group, there presumably was much greater heterogeneity. Second, the FH group is very unique in that the predominate etiology of vascular disease in this group is the marked elevation of LDL cholesterol. In the HW group, as in the general population, the etiology of vascular disease is multifactorial without one dominant factor. Furthermore, all of the FH subjects had detectable vascular calcification, but only 73% of the HW subjects had detectable coronary calcification.
Another possible explanation for the quantitative differences in correlation between the groups might be the different indices used to assess coronary calcification (calcium mass versus calcium score). Although these indices were shown to have no significant differences in reliability,27 they incorporate slightly different corrective factors, which may influence the degree of correlation. The most likely effect of the corrective factors, however, would be a reduction in correlation when the calcium score was used due to the coarser density scale, yet in the group for which this index was used (the FH group), there was a higher correlation coefficient, indicating that the strength of correlation may be underestimated for the FH group.
The actions of 1,25-vitamin D are directed toward maintaining serum calcium homeostasis28 through control of osteoblast function and osteoclast differentiation of monocytes. In addition, 1,25-vitamin D is the only known stimulator of intestinal calcium absorption, an effect thought to account for most of its value in the prevention of osteoporosis.
In vivo, 1,25-vitamin D may be either a potent stimulator of bone resorption or a potent stimulator of bone mineralization, depending on the physiological state.29 For example, in vitamin D–replete animals (based on levels of the storage form, 25-hydroxyvitamin D), active vitamin D stimulates bone resorption, whereas in vitamin D–depleted animals, it stimulates bone mineralization.
In addition, oral consumption of vitamin D does not directly relate to levels of 1,25-vitamin D because of the strict physiological regulation of the activating enzymes 25-hydroxylase and 1-α-hydroxylase.30 Thus, the finding of previous investigators that high doses of oral vitamin D3 induce severe vascular calcification in animals3132333435 does not address the relationship of vascular calcification to serum levels of 1,25-vitamin D and is not inconsistent with our results. Indeed, it is reported that patients using continuous ambulatory peritoneal dialysis develop vascular calcification when treated with oral vitamin D3 but not when treated with 1,25-vitamin D.36
There are a number of possible explanations for the observed inverse relation between serum 1,25-vitamin D levels and vascular calcification. The consistency and statistical significance of the observation between the two different study groups render chance unlikely. One potential confounding factor, age, is also unlikely to explain the relationship. Although age is generally associated with increased vascular calcification over a broad range, it has not been associated with higher or lower levels of 1,25-vitamin D. In addition, the inverse relationship between 1,25-vitamin D and vascular calcification remained significant after adjustment for age in both study groups. Another potential confounding effect is that the association of vascular calcification with atherosclerosis may link it to renovascular disease, which may result in decreased 1-α-hydroxylase activity in the kidney and therefore less conversion of 25-hydroxyvitamin D to its active form, 1,25-vitamin D. Such effects would be minimized in this study because subjects diagnosed with renal failure were excluded from analysis.
Known cellular and molecular effects of 1,25-vitamin D provide several potential biological mechanisms for the relation. One possibility is that 1,25-vitamin D directly inhibits vascular calcification. Previous studies of bone cell cultures have shown both positive and negative effects of 1,25-vitamin D on osteoblastic cell functions depending on the concentration and stage of cell differentiation at treatment.37 Such effects may be mediated through vitamin D receptor binding to vitamin D response elements in the promoters for various calcification-related genes. For example, there are inhibitory vitamin D response elements in the promoters for PTH-related peptide,38 collagen I,39 and bone sialoprotein genes.40 The presence of two functional vitamin D–responsive elements in the promoter for the 24-hydroxylase gene, the gene product of which converts the storage form of vitamin D to inactive 24,25-dihydroxyvitamin D, suggests that 1,25-vitamin D may enhance the metabolism of its own precursor.41
Adding to the complexity of this phenomenon, macrophages in atherosclerotic lesions associated with vascular calcification may express 1-α-hydroxylase activity, producing 1,25-vitamin D.4243 In addition, some macrophages may share the osteoclastic capacity for phagocytic removal of calcium mineral from the artery wall, and such resorption would provide a source of serum calcium and potentially reduce activation of vitamin D.
The similarities between vascular and skeletal calcification, and now the finding that levels of the systemic osteoregulatory factor 1,25-vitamin D are inversely correlated with vascular calcification, call attention to the possibility that vascular calcification results from a systemic derangement of calcium metabolism. Interestingly, a significant association between osteoporosis and vascular calcification has been noted by several investigators, beginning as early as 1946,16171819202122 showing an inverse relationship between the amount of calcium in the axial skeleton and that in the vascular tree. The mechanism of this relationship has not been determined.
Public health education is widely used to increase dietary and supplemental calcium and vitamin D consumption, yet the potential effects on vascular calcification are unknown. In the current investigation, we have shown that serum 1,25-vitamin D levels are associated with lower levels of vascular calcification, supporting the possibility that the process is regulated. These findings may stimulate a new paradigm for the mechanism of soft tissue calcification in general.
Selected Abbreviations and Acronyms
|1,25-vitamin D||=||1α,25-dihydroxyvitamin D3|
|EBCT||=||electron beam computed tomography|
|CHD||=||coronary heart disease|
|HW Group||FH Group|
|Mean age, y||63.3±7.1||29.5±10.6|
|Sex, % F/M||14/86||69/31|
|CHD symptoms, %||0||63|
|Documented CHD, %||0||73|
|Vascular calcification, %||73||100|
|Mean total cholesterol, mg/dL||226||804|
|Mean LDL cholesterol, mg/dL||139||691|
|Mean HDL cholesterol, mg/dL||ND||32.6|
|HW Group||FH Group|
|PTH, pg/mL||40.3±28.9||24.1 ±4.2|
|1,25-Vitamin D, pg/mL||40.1±13.0||34.5±9.9|
This work was supported in part by NIH grants MO1-RR-00865 and HL-30568, Streisand Fund of the Lincy Foundation, Stein-Oppenheimer Award, and Oberkotter Foundation. Dr Watson is a Merck Fellow of the American College of Cardiology. We are grateful for the technical assistance of Patricia Wilsey, the Clinical Research Center Laboratory of UCLA, and the Department of Biostatistics at UCLA.
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