Amy E. Riek

1,25(OH)2 Vitamin D Inhibits Foam Cell Formation and Suppresses Macrophage Cholesterol Uptake in Patients With Type 2 Diabetes Mellitus

Background— Cardiovascular disease is the leading cause of death among those with diabetes mellitus. Vitamin D deficiency is associated with an increased risk of cardiovascular disease in this population. To determine the mechanism by which vitamin D deficiency mediates accelerated cardiovascular disease in patients with diabetes mellitus, we investigated the effects of active vitamin D on macrophage cholesterol deposition. Methods and Results— We obtained macrophages from 76 obese, diabetic, hypertensive patients with vitamin D deficiency (25-hydroxyvitamin D <80 nmol/L; group A) and 4 control groups: obese, diabetic, hypertensive patients with normal vitamin D (group B; n=15); obese, nondiabetic, hypertensive patients with vitamin D deficiency (group C; n=25); and nonobese, nondiabetic, nonhypertensive patients with vitamin D deficiency (group D; n=10) or sufficiency (group E; n=10). Macrophages from the same patients in all groups were cultured in vitamin D—deficient or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] –supplemented media and exposed to modified low-density lipoprotein cholesterol. 1,25(OH)2D3 suppressed foam cell formation by reducing acetylated or oxidized low-density lipoprotein cholesterol uptake in diabetic subjects only. Conversely, deletion of the vitamin D receptor in macrophages from diabetic patients accelerated foam cell formation induced by modified LDL. 1,25(OH)2D3 downregulation of c-Jun N-terminal kinase activation reduced peroxisome proliferated–activated receptor-γ expression, suppressed CD36 expression, and prevented oxidized low-density lipoprotein–derived cholesterol uptake. In addition, 1,25(OH)2D3 suppression of macrophage endoplasmic reticulum stress improved insulin signaling, downregulated SR-A1 expression, and prevented oxidized and acetylated low-density lipoprotein–derived cholesterol uptake. Conclusion— These results identify reduced vitamin D receptor signaling as a potential mechanism underlying increased foam cell formation and accelerated cardiovascular disease in diabetic subjects.

Implications of vitamin D deficiency in pregnancy and lactation

Vitamin D is an essential fat soluble vitamin and a key modulator of calcium metabolism in children and adults. Because calcium demands increase in the third trimester of pregnancy, vitamin D status becomes crucial for maternal health, fetal skeletal growth, and optimal maternal and fetal outcomes. Vitamin D deficiency is common in pregnant women (5-50%) and in breastfed infants (10-56%), despite the widespread use of prenatal vitamins, because these are inadequate to maintain normal vitamin D levels (>or=32 ng/mL). Adverse health outcomes such as preeclampsia, low birthweight, neonatal hypocalcemia, poor postnatal growth, bone fragility, and increased incidence of autoimmune diseases have been linked to low vitamin D levels during pregnancy and infancy. Studies are underway to establish the recommended daily doses of vitamin D in pregnant women. This review discusses vitamin D metabolism and the implications of vitamin D deficiency in pregnancy and lactation.

Endoplasmic Reticulum Stress Controls M2 Macrophage Differentiation and Foam Cell Formation

Background: The interplay of lipid signaling with macrophage phenotype is critical for vascular disease progression. Results: ER stress links scavenger receptor signaling to macrophage phenotype and foam cell formation through a JNK- and PPARγ-dependent pathway. Conclusion: ER stress is a functional switch controlling macrophage phenotype and cellular cholesterol content. Significance: Suppression of ER stress is a potential therapeutic target to reduce atherosclerosis progression. Macrophages are essential in atherosclerosis progression, but regulation of the M1 versus M2 phenotype and their role in cholesterol deposition are unclear. We demonstrate that endoplasmic reticulum (ER) stress is a key regulator of macrophage differentiation and cholesterol deposition. Macrophages from diabetic patients were classically or alternatively stimulated and then exposed to oxidized LDL. Alternative stimulation into M2 macrophages lead to increased foam cell formation by inducing scavenger receptor CD36 and SR-A1 expression. ER stress induced by alternative stimulation was necessary to generate the M2 phenotype through JNK activation and increased PPARγ expression. The absence of CD36 or SR-A1 signaling independently of modified cholesterol uptake decreased ER stress and prevented the M2 differentiation typically induced by alternative stimulation. Moreover, suppression of ER stress shifted differentiated M2 macrophages toward an M1 phenotype and subsequently suppressed foam cell formation by increasing HDL- and apoA-1-induced cholesterol efflux indicating suppression of macrophage ER stress as a potential therapy for atherosclerosis.

Vitamin D Deficiency Induces High Blood Pressure and Accelerates Atherosclerosis in Mice

Multiple epidemiological studies link vitamin D deficiency to increased cardiovascular disease (CVD), but causality and possible mechanisms underlying these associations are not established. To clarify the role of vitamin D-deficiency in CVD in vivo, we generated mouse models of diet-induced vitamin D deficiency in two backgrounds (LDL receptor- and ApoE-null mice) that resemble humans with diet-induced hypertension and atherosclerosis. Mice were fed vitamin D-deficient or -sufficient chow for 6 weeks and then switched to high fat (HF) vitamin D-deficient or –sufficient diet for 8–10 weeks. Mice with diet-induced vitamin D deficiency showed increased systolic and diastolic blood pressure, high plasma renin, and decreased urinary sodium excretion. Hypertension was reversed and renin was suppressed by returning chow-fed vitamin D-deficient mice to vitamin D-sufficient chow diet for 6 weeks. On a HF diet, vitamin D-deficient mice had ∼2-fold greater atherosclerosis in the aortic arch and ∼2–8-fold greater atherosclerosis in the thoracic and abdominal aorta compared to vitamin D-sufficient mice. In the aortic root, HF-fed vitamin D-deficient mice had increased macrophage infiltration with increased fat accumulation and endoplasmic reticulum (ER) stress activation, but a lower prevalence of the M1 macrophage phenotype within atherosclerotic plaques. Similarly, peritoneal macrophages from vitamin D-deficient mice displayed an M2-predominant phenotype with increased foam cell formation and ER stress. Treatment of vitamin D-deficient mice with the ER stress reliever PBA during HF feeding suppressed atherosclerosis, decreased peritoneal macrophage foam cell formation, and downregulated ER stress proteins without changing blood pressure. Thus, we suggest that vitamin D deficiency activates both the renin angiotensin system and macrophage ER stress to contribute to the development of hypertension and accelerated atherosclerosis, highlighting vitamin D replacement as a potential therapy to reduce blood pressure and atherosclerosis.

Vitamin D Suppression of Endoplasmic Reticulum Stress Promotes an Antiatherogenic Monocyte/Macrophage Phenotype in Type 2 Diabetic Patients

Background: Interactions between environmental conditions and monocyte phenotype are critical for the development of vascular complications in diabetes. Results: Modulation of ER stress by vitamin D controls monocyte/macrophage phenotype and vascular adhesion. Conclusion: Vitamin D is a natural ER stress reliever that promotes an anti-inflammatory monocyte/macrophage phenotype. Significance: Vitamin D is a potential therapy to reduce vascular complications in diabetics. Cardiovascular disease is the leading cause of morbidity/mortality in patients with type 2 diabetes mellitus (T2DM), but there is a lack of knowledge about the mechanism(s) of increased atherosclerosis in these patients. In patients with T2DM, the prevalence of 25-hydroxy vitamin D (25(OH)D) deficiency is almost twice that for nondiabetics and doubles the relative risk of developing cardiovascular disease compared with diabetic patients with normal 25(OH)D. We tested the hypothesis that monocytes from vitamin D-deficient subjects will have a proatherogenic phenotype compared with vitamin D-sufficient subjects in 43 patients with T2DM. Serum 25(OH)D level inversely correlated with monocyte adhesion to endothelial cells even after adjustment for demographic and comorbidity characteristics. Vitamin D-sufficient patients (≥30 ng/ml 25(OH)D) had lower monocyte endoplasmic reticulum (ER) stress, a predominance of M1 over M2 macrophage membrane receptors, and decreased mRNA expression of monocyte adhesion molecules PSGL-1, β1-integrin, and β2-integrin compared with patients with 25(OH)D levels of <30 ng/ml. In vitamin D-deficient macrophages, activation of ER stress increased adhesion and adhesion molecule expression and induced an M2-predominant phenotype. Moreover, adding 1,25(OH)2D3 to vitamin D-deficient macrophages shifted their phenotype toward an M1-predominant phenotype with suppressed adhesion. Conversely, deletion of the vitamin D receptor in macrophages from diabetic patients activated ER stress, accelerated adhesion, and increased adhesion molecule expression. The absence of ER stress protein CCAAT enhancer-binding protein homologous protein suppressed monocyte adhesion, adhesion molecule expression, and the M2-predominant phenotype induced by vitamin D deficiency. Thus, vitamin D is a natural ER stress reliever that induced an antiatherogenic monocyte/macrophage phenotype.

Vitamin D regulates macrophage cholesterol metabolism in diabetes.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with type 2 diabetes mellitus (T2DM). In type 2 diabetics, the prevalence of vitamin D deficiency is 20% higher than in non-diabetics, and low vitamin D levels nearly double the relative risk of developing CVD compared to diabetic patients with normal vitamin D levels. However, the mechanism(s) by which vitamin D deficiency leads to an increased susceptibility to atherosclerosis in these patients is unknown. We studied the effects of vitamin D replacement on macrophage cholesterol metabolism and foam cell formation in obese, hypertensive diabetics and non-diabetic controls. We found that 1,25-dihydroxy vitamin D3 [1,25(OH)2D3] suppressed foam cell formation by reducing acetylated low density lipoprotein (AcLDL) and oxidized low density lipoprotein (oxLDL) cholesterol uptake in diabetics only. 1,25(OH)2D3 downregulation of c-Jun N-terminal kinase activation reduced PPARgamma and CD36 expression, and prevented oxLDL-derived cholesterol uptake. In addition, 1,25(OH)2D3 suppression of macrophage endoplasmic reticulum stress improved insulin signaling, downregulated SR-A1 expression, and prevented oxLDL- and AcLDL-derived cholesterol uptake. The results of this research reveal novel insights into the mechanisms linking vitamin D signaling to foam cell formation in diabetics and suggest a potential new therapeutic target to reduce cardiovascular risk in this population.

The St. Louis African American health-heart study: methodology for the study of cardiovascular disease and depression in young-old African Americans

BackgroundCoronary artery disease (CAD) is a major cause of death and disability worldwide. Depression has complex bidirectional adverse associations with CAD, although the mechanisms mediating these relationships remain unclear. Compared to European Americans, African Americans (AAs) have higher rates of morbidity and mortality from CAD. Although depression is common in AAs, its role in the development and features of CAD in this group has not been well examined. This project hypothesizes that the relationships between depression and CAD can be explained by common physiological pathways and gene-environment interactions. Thus, the primary aims of this ongoing project are to: a) determine the prevalence of CAD and depression phenotypes in a population-based sample of community-dwelling older AAs; b) examine the relationships between CAD and depression phenotypes in this population; and c) evaluate genetic variants from serotoninP and inflammatory pathways to discover potential gene-depression interactions that contribute significantly to the presence of CAD in AAs.Methods/designThe St. Louis African American Health (AAH) cohort is a population-based panel study of community-dwelling AAs born in 1936–1950 (inclusive) who have been followed from 2000/2001 through 2010. The AAH-Heart study group is a subset of AAH participants recruited in 2009–11 to examine the inter-relationships between depression and CAD in this population. State-of-the-art CAD phenotyping is based on cardiovascular characterizations (coronary artery calcium, carotid intima-media thickness, cardiac structure and function, and autonomic function). Depression phenotyping is based on standardized questionnaires and detailed interviews. Single nucleotide polymorphisms of selected genes in inflammatory and serotonin-signaling pathways are being examined to provide information for investigating potential gene-depression interactions as modifiers of CAD traits. Information from the parent AAH study is being used to provide population-based prevalence estimates. Inflammatory and other biomarkers provide information about potential pathways.DiscussionThis population-based investigation will provide valuable information on the prevalence of both depression and CAD phenotypes in this population. The study will examine interactions between depression and genetic variants as modulators of CAD, with the intent of detecting mechanistic pathways linking these diseases to identify potential therapeutic targets. Analytic results will be reported as they become available.

Vitamin D3 supplementation decreases a unique circulating monocyte cholesterol pool in patients with type 2 diabetes

Cross-sectional studies indicate consistent associations between low 25(OH)D concentration and increased risk of cardiovascular disease (CVD), but results of randomized control trials (RCTs) are mixed. However, the majority of the RCTs do not focus on type 2 diabetics, potentially obscuring the effects of vitamin D in this population. In vitro 1,25(OH)2D3 downregulates macrophage cholesterol deposition, but the in vivo effects are unknown. To explore potential mechanisms of the effects of vitamin D on CVD risk in patients with type 2 diabetes, we isolated monocytes in a subset of 26 patients from our RCT of diabetics with baseline serum 25(OH)D <25ng/mL randomized to vitamin D3 4000 IU/day or placebo for 4 months. Upon enrollment, the mean 25(OH)D level was 17ng/mL, which increased to 36ng/mL after vitamin D and remained unchanged in the placebo group. Before randomization, groups demonstrated similar mean hemoglobin A1c and plasma lipids levels, none of which was significantly altered by vitamin D supplementation. Moreover, assessment of oxidized LDL uptake in monocytes cultured in the patients own serum before vs. after treatment resulted in >50% reduction in the vitamin D group with no change in the placebo group. This was mediated through suppression of endoplasmic reticulum stress and scavenger receptor CD36 protein expression. The reduction in monocyte cholesterol uptake was reflected in a 19% decrease in total monocyte cholesterol content. Interestingly, cross-sectional analysis of circulating monocytes from vitamin D-deficient vs. sufficient diabetic patients revealed 8-fold higher cholesteryl ester content, confirming the capacity of these monocytes to uptake and carry cholesterol in the circulation. This study identifies a unique circulating cholesterol pool within monocytes that is modulated by vitamin D and has the potential to contribute to CVD in type 2 diabetes.

Deletion of JNK2 prevents vitamin-D-deficiency-induced hypertension and atherosclerosis in mice

The c-Jun N-terminal kinase 2 (JNK2) signaling pathway contributes to inflammation and plays a key role in the development of obesity-induced insulin resistance and cardiovascular disease. Macrophages are key cells implicated in these metabolic abnormalities. Active vitamin D downregulates macrophage JNK activation, suppressing oxidized LDL cholesterol uptake and foam cell formation and promoting an anti-inflammatory phenotype. To determine whether deletion of JNK2 prevents high blood pressure and atherosclerosis known to be induced by vitamin D deficiency in mice, we generated mice with knockout of JNK2 in a background susceptible to diet-induced atherosclerosis (LDLR-/-). JNK2-/- LDLR-/- and LDLR-/- control mice were fed vitamin D-deficient chow for 8 weeks followed by vitamin D-deficient high fat diet (HFD) for 10 weeks and assessed before and after HFD. There was no difference in fasting glucose, cholesterol, triglycerides, or free fatty acid levels. However, JNK2-/- mice, despite vitamin D-deficient diet, had 20-30mmHg lower systolic (SBP) and diastolic (DBP) blood pressure before HFD compared to control mice fed vitamin D-deficient diets, with persistent SBP differences after HFD. Moreover, deletion of JNK2 reduced HFD-induced atherosclerosis by 30% in the proximal aorta when compared to control mice fed vitamin D-deficient diets. We have previously shown that peritoneal macrophages obtained from LDLR-/- mice fed vitamin D-deficient HFD diets have higher foam cell formation compared to those from mice on vitamin D-sufficient HFD. The increased total cellular cholesterol and modified cholesterol uptake in macrophages from mice on vitamin D-deficient HFD were blunted by deletion of JNK2. These data suggest that JNK2 signaling activation is necessary for the atherosclerosis and hypertension induced by vitamin D deficiency.

Vitamin D and the Cardiovascular System

Abstract Vitamin D has recently been shown not only to be important for bone and calcium metabolism but also for homeostasis of critical tissues involved in vascular disease. The vitamin D receptor (VDR) and the 1α-hydroxylase enzyme are present in critical cells implicated in the development of vascular disease. Vitamin D influences multiple mechanisms to decrease vascular inflammation; it suppresses the renin-angiotensin system, promotes endothelial nitric oxide release, decreases vascular inflammatory markers and cholesterol deposition, and imbues immune cells with antiinflammatory properties. Studies in mouse models of diet-induced insulin resistance show that vitamin D deficiency or VDR deletion promotes renin-dependent hypertension and accelerates atherosclerosis. However, the effects of vitamin D supplementation on blood pressure and atherosclerosis have been mixed and depend on baseline vitamin D status, dose and vitamin D compound administered, and animal model. Human observational studies indicate consistent associations between low 25(OH)D levels and increased cardiovascular disease, but the effects of vitamin D supplementation for prevention are conflicting and study design limitations preclude adequate conclusions.