Multiple studies in humans have demonstrated an association between vitamin D deficiency and hypertension and atherosclerosis. Several studies in humans indicate that replacement with vitamin D orally or by UVB reduces blood pressure. However, it has never been demonstrated that nutritional vitamin D deficiency contributes to increased blood pressure and atherosclerosis progression. Here, we demonstrate that diet-induced vitamin D deficiency increased blood pressure in mice through activation of the RAS and that vitamin D supplementation reversed this hemodynamic change by suppression of RAS activation. Furthermore, activation of macrophage ER stress by vitamin D deficiency accelerated atherosclerosis by inducing a predominance of M2 macrophages in the plaque, characterized by increased cholesterol uptake and foam cell formation. In contrast, suppression of macrophage ER stress despite vitamin D-deficient diet induced an M1-predominant macrophage profile with decreased cholesterol uptake and decreased atherosclerosis without modifying blood pressure, suggesting that multiple mechanisms are involved in the effects of vitamin D on cardiovascular disease and its risk factors.
In humans, meta-analysis of several large prospective cohorts totaling more than 1800 patients found an increased relative risk (1.76) of incident hypertension in those with 25(OH)D level <50 nmol/L (20 ng/mL) compared to those >75 nmol/L (30 ng/mL) 
. In animal models, vitamin D downregulates renin gene promoter activity independently of its effects on calcium metabolism 
. Mice lacking the VDR exhibit hypertension and cardiac hypertrophy due to increased renin expression and plasma angiotensin II production 
. However, it is unclear whether the less severe phenotype conferred by nutritional vitamin D deficiency affects the RAS. In this study, we confirmed in multiple mouse models that vitamin D deficiency induced hypertension by activation of the systemic RAS, facilitating sodium retention. Interestingly, after prolonged vitamin D deficiency in animals on a high fat diet, differences in sodium retention resolved despite continued RAS activation, suggesting possible aldosterone escape similar to what occurs in patients with primary hyperaldosteronism 
. We also demonstrated that replacement of vitamin D reversed these changes, which is consistent with the results of several other interventional trials in animals and humans. In vitamin D-sufficient hypertensive rats, oral administration of vitamin D3
decreased blood pressure and improved endothelial cell-dependent vasodilatation 
. In an 8-week treatment study consisting of oral calcium and vitamin D3
replacement in elderly non-diabetic women with vitamin D deficiency, plasma 25(OH)D levels increased to ≥62 nmol/L (25 ng/mL) and SBP decreased significantly by 13 mmHg compared with the calcium-treated control group 
. UVB exposure by skin tanning sessions increased plasma 25(OH)D levels to 100 nmol/L (40 ng/mL) and decreased blood pressure in mildly hypertensive normoglycemic patients 
. Despite mixed results from additional interventional trials 
, our study supports the data suggesting that vitamin D replacement could be an antihypertensive therapy and, importantly, suggests that vitamin D deficiency itself is a causal factor in the development of hypertension through activation of the RAS.
Data linking vitamin D deficiency and atherosclerosis comes from large epidemiological and clinical studies 
. A prospective study of the Framingham Offspring indicates that low vitamin D levels increase CVD risk. In middle-aged Framingham volunteers with hypertension (HTN), low 25(OH)D levels (≤15 ng/ml) increased the risk of CVD by 60% during a follow-up of 5.4 years 
. Further evidence among individuals with T2DM in NHANES III showed that low 25(OH)D levels nearly double (OR 1.70) the likelihood of developing CVD compared with normal 25(OH)D levels 
. In LDLR−/−
mice, the absence of VDR signaling (LDLR−/−
) accelerates atherosclerosis in the ascending aorta after 8 weeks of high fat diet, possibly by local activation of the RAS in macrophages 
. Furthermore, LDLR−/−
mice fed a low vitamin D diet have more calcification of lesions in the aortic root and higher expression of osteogenic factors than mice fed a high vitamin D diet 
. In this study using multiple murine models of diet-induced atherosclerosis, we found that vitamin D deficiency caused accelerated atherosclerosis by increasing macrophage cholesterol uptake and foam cell formation without a compensatory increase in cholesterol efflux. We previously found that in vitro, active 1,25(OH)2
D suppresses macrophage cholesterol uptake and foam cell formation by downregulation of scavenger receptor CD36 and SRA-1 expression 
. In contrast, the macrophages in this analysis were exposed to low 25(OH)D but normal 1,25(OH)2
D levels in vivo and still had increased macrophage cholesterol deposition, suggesting that local production of active vitamin D from plasma 25(OH)D within the macrophage may be a key component of the effects of vitamin D on macrophage cholesterol deposition and foam cell formation and could explain the link between atherosclerosis and vitamin D deficiency in humans.
It is known that hemodynamic features play a major role in the localization of atherosclerotic lesions within the vascular tree. In areas such as the aortic arch, renal arteries, and the iliac bifurcation, the low shear stress and high turbulence from vessel curvature or branching induce hemodynamic disturbances that accelerate atherosclerotic development 
. However, hemodynamic changes are not necessarily causative, but may simply increase susceptibility to atherosclerosis in areas of turbulent flow, while additional insults such as hypertension or hyperlipidemia induced by HFD are required for atherosclerosis to develop in areas of low turbulence. In mouse models of angiotensin II–induced hypertension or renal artery stenosis, hypertension and hyperlipidemia are synergistic to worsen atherosclerosis in both the ascending and descending aorta 
. In this study of diet-induced vitamin D deficiency, mice in both backgrounds also showed increased blood pressure and accelerated atherosclerosis in all aortic segments, but this effect was the most severe in the proximal aorta. Similarly, absence of the VDR (LDLR−/−
) in mice increases blood pressure and accelerates atherosclerosis 
. Interestingly, 1 year-old LDLR−/−
mice fed vitamin D-deficient chow diet showed higher blood pressure, but increased atherosclerosis only in the proximal aorta and not in the thoracic or abdominal segments, similar to previous studies in chow fed LDLR−/−
, supporting the concept that other insults must be present, such as hyperlipidemia, to work synergistically with the effects of vitamin D deficiency (hypertension, immunomodulation, macrophage cholesterol deposition) to induce atherosclerosis in aortic areas of low turbulence.
Current literature regarding atherosclerotic plaque progression suggests that M1 macrophages are more pathogenic with a pro-inflammatory profile while M2 macrophages contribute to tissue repair 
. However, considering the complexity of the cytokine milieu and the changes during different stages of plaque evolution, the current inflammatory paradigm may be too simplistic. Previous studies have demonstrated that macrophages can shift their differentiated phenotype back and forth from M1 to M2 under various environmental conditions during plaque evolution 
. In chow-fed ApoE−/−
mice, lesion-infiltrated macrophages of young mice exhibit predominantly the M2 phenotype, while M1 macrophages are dominant in more advanced lesions of aged mice 
. In models of plaque regression, induction of the M1 migration marker CCR7 in macrophages facilitates their egression to the lymph nodes 
, while antibodies against CCR7 ligands inhibit macrophage egression, establishing a functional role for CCR7 in plaque regression 
. In contrast, activated M2 macrophages display a reduced capacity to handle cholesterol with increased cholesterol uptake and decreased cholesterol efflux 
, metabolic changes associated with inhibition of macrophage migration 
. Therefore, discovering the environmental conditions that regulate macrophage phenotype differentiation is critical to understanding atherosclerotic plaque evolution and regression 
. Recently, we found in diabetic patients that 1,25(OH)2
D shifts M2-differentiated macrophages to M1-predominant cells with decreased foam cell formation 
suggesting vitamin D as a key factor in atherosclerosis development; however, it is unclear if these macrophage phenotypic characteristics affect atherosclerosis progression in vivo. In this study, we demonstrate in multiple mouse models of diet-induced atherosclerosis that vitamin D status is critical to the development of atherosclerosis. Vitamin D deficiency accelerated atherosclerosis by promoting the differentiation of macrophages into the M2 subtype with high cholesterol deposition and increased cholesterol uptake. These findings suggest that vitamin D deficiency accelerates atherosclerosis by shifting plaque macrophages toward a subtype with increased cholesterol deposition and lower expression of membrane receptors that facilitate plaque egression.
Vascular ER stress is present during multiple stages of atherosclerosis development 
. In mouse models of diet-induced atherosclerosis, multiple mechanisms of decreasing ER stress, including knockout of ER stress protein CHOP or suppression with PBA, prevent the development of atherosclerosis 
. Chronic activation of CHOP triggers macrophage apoptosis and atherosclerosis plaque instability 
. We recently demonstrated in diabetic patients that ER stress is a key regulator of macrophage differentiation and cholesterol deposition. ER stress is required to generate the M2 phenotype through a JNK-PPARγ-dependent pathway and increases expression of scavenger receptors CD36 and SR-A1 to increase foam cell formation 
. Active vitamin D suppresses ER stress to prevent monocyte adhesion and shift M2-differentiated macrophages to M1-predominant cells with decreased foam cell formation 
, suggesting that ER stress is a critical link between cholesterol metabolism and macrophage phenotype. However, the relationship between nutritional vitamin D status and ER stress activation in vivo and the development of atherosclerosis is unknown. In this study, we found that vitamin D-deficient mice had increased atherosclerosis with plaque macrophage ER stress signaling activation and an M2-predominant phenotype when compared to vitamin D-sufficient mice. Furthermore, suppression of ER stress by PBA reduced aortic arch and thoracic atherosclerosis by decreasing macrophage cholesterol deposition, suppressing cholesterol uptake, and shifting the macrophage phenotype from an M2- to an M1-predominance despite vitamin D-deficiency. Interestingly, suppression of ER stress with PBA did not alter the hypertension induced by vitamin D deficiency or atherosclerosis in the abdominal aorta. Taken together, these data suggest that nutritional vitamin D works through a complex interplay of multiple mechanisms to improve atherosclerosis and its risk factors beyond that of ER stress activation in the vessel wall.
In summary, this work provides evidence that vitamin D deficiency is a causative factor of hypertension by stimulation of the RAS, which is reversible with vitamin D replacement. Additionally, vitamin D deficiency increased atherosclerosis by profoundly modulating the macrophage phenotype within the atherosclerotic plaque through activation of ER stress. Increased macrophage ER stress accelerated atherosclerosis by inducing a predominance of M2 macrophages, characterized by increased cholesterol uptake and foam cell formation. Interestingly, suppression of ER stress by a chemical chaperone promoted an anti-atherogenic macrophage phenotype and prevented vitamin D deficiency-induced atherosclerosis without affecting blood pressure. Thus, we suggest that vitamin D deficiency acts through multiple mechanisms, including activation of 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. New interventional trials from our laboratory and others evaluating the influence of vitamin D replacement on atherosclerosis progression are underway.