Vitamin D deficiency induces high blood pressure and accelerates atherosclerosis in mice

Abstract

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.

Figure 1. Blood pressure is reversibly increased in vitamin D deficient LDLR^−/− mice.

Non-invasive systolic (SBP) and diastolic blood pressure (DBP) in LDLR^−/− mice on vitamin D-sufficient (black) or –deficient (white) diet (A) at baseline (n_suf = 12, n_def = 17), (B) after high fat diet (HFD) (n_suf = 13, n_def = 13), and (C) after 1 year on chow (n_suf = 10, n_def = 9). (D) Serum renin activity at baseline and after HFD (pooled samples of 10 animals per group). (E) Urinary sodium excretion at baseline and after HFD (baseline n_suf = 7, n_def = 5, HFD n_suf = 10, n_def = 13). (F) Blood pressure 6 weeks after returning vitamin D-deficient mice to a -sufficient diet (replacement: gray) (n_def = 7, n_replaced = 8). (G) Serum renin activity after returning vitamin D-deficient mice to a -sufficient diet (replacement: gray) (n_def = 3, n_replaced = 7). Data is expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.0001.

Figure 2. Vitamin D deficiency increases atherosclerosis.

(A) Representative en face aortae from vitamin D-sufficient or -deficient LDLR^−/− mice. (B–D) Quantitative analysis of atherosclerotic lesion area in the aortic arch, thoracic and abdominal aorta for ApoE^−/− (n_suf = 12, n_def = 13) and LDLR−/− (n_suf = 12, n_def = 13) after HFD as well as LDLR^−/− mice after 1 year on chow (n_suf = 8, n_def = 7). Individual vitamin D-sufficient mice are represented as black squares and vitamin D-deficient as white circles. Median value indicated for each group. *p<0.05, **p<0.01, ***p<0.0001.

Figure 3. Vitamin D deficiency increases foam cell formation by altering macrophage lipid metabolism in ApoE^−/− mice.

Peritoneal macrophages were harvested from ApoE^−/− mice after vitamin D –sufficient (black) or –deficient (white) HFD. (A) Representative Oil-Red-O stain. (B–D) Total cholesterol, free cholesterol, and triglyceride content (n = 3 per group). (E–F) Dil-oxLDL cholesterol uptake (n = 4 per group) and ApoAI-stimulated and HDL-stimulated cholesterol efflux (n = 4 per group). Data expressed as mean ± SEM. *p<0.05.

Figure 4. Vitamin D deficiency promotes a pro-atherogenic M2 macrophage phenotype.

Peritoneal macrophages from ApoE^−/− mice after vitamin D-sufficient or –deficient HFD were assessed by flow cytometry for (A) Cell surface markers for M1 and M2 phenotype (CCR7: gray, CD86: black, MR: dots, CD163: white) and (B) Macrophage phenotype ratio calculated from flow cytometry analysis to assess M1 vs. M2 predominance (vitamin D-sufficient: black, vitamin D-deficient: white). From the aortic root of vitamin D-sufficient (top) and -deficient (bottom) animals after 8 weeks on HFD, (C) Represenative image of double immunofluorescent staining for CCR7 (M1, green), MR (M2, green), and ADRP (red). Scale bar represents 50 µm. (D) Quantification of CCR7 immunofluorescent staining as a percentage of total atherosclerotic plaque area, (E) Co-localization (yellow) of CCR7 and MR with ADRP as a percentage of ADRP-positive area (n = 3 per group for all). Data expressed as mean ± SEM. *p<0.05, **p<0.01.

Figure 5. Macrophage ER stress is increased in vitamin D deficiency.

ApoE^−/− mice were assessed after vitamin D-sufficient (black) or –deficient (white) HFD. (A) Western blot and (B) quantification of ER stress protein expression (n = 4 per group) in unstimulated peritoneal macrophages. (C) Triple immunofluorescent staining of the aortic root of vitamin D –sufficient (top) or –deficient (bottom) for DAPI (blue), CHOP (red), and MOMA (green). Data expressed as mean ± SEM. *p<0.05, **p<0.01, ***p≤0.0001.

Figure 6. Suppression of ER stress improves cholesterol handling and reduces atherosclerosis in vitamin D-deficient mice.

ApoE^−/− mice were assessed after vitamin D-deficient HFD with and without PBA treatment. (A) Western blot for ER stress protein expression in peritoneal macrophages (n = 4 per group). (B) Quantification of atherosclerotic lesion area for control saline-treated (white circles) and PBA-treated (gray triangles) mice (n = 10 per group). (C–D) Macrophage total cholesterol and triglyceride content (n = 6 per group). (E) Macrophage phenotype ratio based on flow cytometry analysis of cell surface markers (n = 6 per group). (F–G) Dil-oxLDL cholesterol uptake (n = 4 per group) and HDL-stimulated cholesterol efflux (n = 6 per group). Vitamin D-deficient data is shown in white and PBA treatment in gray. Data expressed as mean ± SEM. *p<0.05, **p<0.01, ***p≤0.0001.