Embryonic vitamin D deficiency programs hematopoietic stem cells to induce type 2 diabetes.

Environmental factors may alter the fetal genome to cause metabolic diseases. It is unknown whether embryonic immune cell programming impacts the risk of type 2 diabetes in later life. We demonstrate that transplantation of fetal hematopoietic stem cells (HSCs) made vitamin D deficient in utero induce diabetes in vitamin D-sufficient mice. Vitamin D deficiency epigenetically suppresses Jarid2 expression and activates the Mef2/PGC1a pathway in HSCs, which persists in recipient bone marrow, resulting in adipose macrophage infiltration. These macrophages secrete miR106-5p, which promotes adipose insulin resistance by repressing PIK3 catalytic and regulatory subunits and down-regulating AKT signaling. Vitamin D-deficient monocytes from human cord blood have comparable Jarid2/Mef2/PGC1a expression changes and secrete miR-106b-5p, causing adipocyte insulin resistance. These findings suggest that vitamin D deficiency during development has epigenetic consequences impacting the systemic metabolic milieu.

Highlights from the 24th workshop on vitamin D in Austin, September 2022.

The 24th Workshop on Vitamin D was held September 7-9, 2022 in Austin, Texas and covered a wide diversity of research in the vitamin D field from across the globe. Here, we summarize the meeting, individual sessions, awards and presentations given.

Non-classical Vitamin D Actions for Renal Protection.

Chronic Kidney Disease (CKD), a disorder that affects 11% of the world's population, is characterized by an acceleration in skeletal, immune, renal, and cardiovascular aging that increases the risk of cardiovascular mortality by 10- to 20-fold, compared to that in individuals with normal renal function. For more than two decades, the progressive impairment in renal capacity to maintain normal circulating levels of the hormonal form of vitamin D (1,25-dihydroxyvitamin D or calcitriol) was considered the main contributor to the reduced survival of CKD patients. Accordingly, calcitriol administration was the treatment of choice to attenuate the progression of secondary hyperparathyroidism (SHPT) and its adverse impact on bone health and vascular calcification. The development of calcitriol analogs, designed to mitigate the resistance to calcitriol suppression of PTH associated with CKD progression, demonstrated survival benefits unrelated to the control of SHPT or skeletal health. The exhaustive search for the pathophysiology behind survival benefits associated with active vitamin D analogs has identified novel anti-inflammatory, anti-hypertensive, anti-aging actions of the vitamin D endocrine system. A major paradigm shift regarding the use of calcitriol or active vitamin D analogs to improve survival in CKD patients emerged upon demonstration of a high prevalence of vitamin D (not calcitriol) deficiency at all stages of CKD and, more significantly, that maintaining serum levels of the calcitriol precursor, 25(OH)vitamin D, above 23 ng/ml delayed CKD progression. The cause of vitamin D deficiency in CKD, however, is unclear since vitamin D bioactivation to 25(OH)D occurs mostly at the liver. Importantly, neither calcitriol nor its analogs can correct vitamin D deficiency. The goals of this chapter are to present our current understanding of the pathogenesis of vitamin D deficiency in CKD and of the causal link between defective vitamin D bioactivation to calcitriol and the onset of molecular pathways that promote CKD progression independently of the degree of SHPT. An understanding of these mechanisms will highlight the need for identification of novel sensitive biomarkers to assess the efficacy of interventions with vitamin D and/or calcitriol(analogs) to ameliorate CKD progression in a PTH-independent manner.

Immunity and Hypertension.

Hypertension is the primary cause of cardiovascular mortality. Despite multiple existing treatments, only half of those with the disease achieve adequate control. Therefore, understanding the mechanisms causing hypertension is essential for the development of novel therapies. Many studies demonstrate that immune cell infiltration of the vessel wall, kidney and central nervous system, as well as their counterparts of oxidative stress, the renal renin-angiotensin system (RAS) and sympathetic tone play a critical role in the development of hypertension. Genetically modified mice lacking components of innate and/or adaptive immunity confirm the importance of chronic inflammation in hypertension and its complications. Depletion of immune cells improves endothelial function, decreases oxidative stress, reduces vascular tone and prevents renal interstitial infiltrates, sodium retention and kidney damage. Moreover, the ablation of microglia or central nervous system perivascular macrophages reduces RAS-induced inflammation and prevents sympathetic nervous system activation and hypertension. Therefore, understanding immune cell functioning and their interactions with tissues that regulate hypertensive responses may be the future of novel antihypertensive therapies.

Improving HbA1c with Glucose Self-Monitoring in Diabetic Patients with EpxDiabetes, a Phone Call and Text Message-Based Telemedicine Platform: A Randomized Controlled Trial.

Background:We conducted a randomized controlled trial of EpxDiabetes, a novel digital health intervention as an adjunct therapy to reduce HbA1c and fasting blood glucose (FBG) among patients with type 2 diabetes mellitus (T2DM). In addition, we examined the effect of social determinants of health on our system.Methods:Sixty-five (n = 65) patients were randomized at a primary care clinic. Self-reported FBG data were collected by EpxDiabetes automated phone calls or text messages. Only intervention group responses were shared with providers, facilitating follow-up and bidirectional communication. ΔHbA1c and ΔFBG were analyzed after 6 months.Results:There was an absolute HbA1c reduction of 0.69% in the intervention group (95% confidence interval [CI], -1.41 to 0.02) and an absolute reduction of 0.03% in the control group (95% CI, -0.88 to 0.82). For those with baseline HbA1c >8%, HbA1c decreased significantly by 1.17% in the intervention group (95% CI, -1.90 to -0.44), and decreased by 0.02% in the control group (95% CI, -0.99 to 0.94). FBG decreased in the intervention group by 21.6 mg/dL (95% CI, -37.56 to -5.639), and increased 13.0 mg/dL in the control group (95% CI, -47.67 to 73.69). Engagement (proportion responding to ≥25% of texts or calls over 4 weeks) was 58% for the intervention group (95% CI, 0.373-0.627) and 48% for the control group (95% CI, 0.296-0.621). Smoking, number of comorbidities, and response rate were significant predictors of ΔHbA1c.Conclusions:EpxDiabetes helps to reduce HbA1c in patients with uncontrolled T2DM and fosters patient-provider communication; it has definite merit as an adjunct therapy in diabetes management. Future work will focus on improving the acceptability of the system and implementation on a larger scale trial.

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 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 patient's 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.

Improving Glycemic Control With a Standardized Text-Message and Phone-Based Intervention: A Community Implementation.

BACKGROUND: Type II diabetes mellitus (T2DM) presents a major disease burden in the United States. Outpatient glycemic control among patients with T2DM remains difficult. Telemedicine shows great potential as an adjunct therapy to aid in glycemic control in real-world settings. OBJECTIVE: We aimed to explore the effectiveness of EpxDiabetes, a novel digital health intervention, in improving hemoglobin A1c (HbA1c) and fasting blood glucose (FBG) among patients with uncontrolled diabetes. METHODS: We recruited 396 patients from a community clinic in St. Louis, Missouri, from a database of patients diagnosed with T2DM and with a most recent HbA1c >7% as part of a quality improvement project. An automated call or text-messaging system was used to monitor patient-reported FBG. If determined to be elevated, care managers were notified by email, text, or electronic medical record alert. Participants self-reported their FBG data by replying to EpxDiabetes automated phone calls or text messages. Data were subsequently analyzed, triaged, and shared with providers to enable appropriate follow-up and care plan adjustments. Absolute HbA1c reduction, patient engagement, and absolute patient-reported FBG reduction were examined at approximately 6 months post implementation. RESULTS: EpxDiabetes had an average 95.6% patient response rate to messages at least once per month and an average 71.1% response rate to messages at least once per week. Subsequent HbA1c drop with EpxDiabetes use over 4 months was -1.15% (95% CI -1.58 to -0.71) for patients with HbA1c >8% at baseline compared to the change in HbA1c over 4 months prior to the implementation of EpxDiabetes of only -0.005 points (95% CI -0.28 to 0.27), P=.0018. CONCLUSIONS: EpxDiabetes may help reduce HbA1c in patients with high HbA1c baselines (>8%). The intervention demonstrates high patient engagement sustainable for at least 6 months.

Adiposity and Cardiometabolic Risk in Children With and Without Antipsychotic Drug Treatment.

CONTEXT: Pediatric obesity is common, particularly in children treated with antipsychotic medications. Antipsychotic exposure can increase cardiometabolic risk by increasing adiposity, and possibly via other adiposity-independent pathways. OBJECTIVE: The objectives were to characterize relationships of adiposity with intrahepatic triglyceride (IHTG) content and carotid intima media thickness (CIMT) in children with and without antipsychotic drug treatment, and to explore whether vitamin D alters any effects in these relationships. DESIGN: This was a cross-sectional case-control study. SETTING: The setting was an academic medical center. PATIENTS OR OTHER PARTICIPANTS: Participants were 44 children (ages, 6-19 y): 25 cases treated with antipsychotic and other psychotropic drug therapies and 19 untreated controls, frequency-matched on age, gender, and body mass index. MAIN OUTCOME MEASURES: Main outcome measures were dual-energy x-ray absorptiometry percentage body fat (DEXA %fat), IHTG measured by magnetic resonance spectroscopy, and CIMT measured by ultrasonography. Fasting blood glucose, insulin, lipids, C-reactive protein, and liver enzymes were also evaluated. RESULTS: There were no significant differences between cases and controls on measures of IHTG, CIMT, or DEXA %fat. In combined crude and adjusted analyses, DEXA %fat predicted IHTG (R(2) = 0.30) but not CIMT. Low levels of vitamin D were associated with larger effects of DEXA %fat on IHTG. CONCLUSION: In treated and untreated children alike, adiposity is a significant predictor of liver fat content. This relationship was altered by low vitamin D level. These results suggest a modifiable pathway to hepatic steatosis. Further research is needed to test the hypothesis that children with high adiposity and low vitamin D have particularly increased risks for the development of fatty liver.

Deletion of macrophage Vitamin D receptor promotes insulin resistance and monocyte cholesterol transport to accelerate atherosclerosis in mice.

Intense effort has been devoted to understanding predisposition to chronic systemic inflammation because it contributes to cardiometabolic disease. We demonstrate that deletion of the macrophage vitamin D receptor (VDR) in mice (KODMAC) is sufficient to induce insulin resistance by promoting M2 macrophage accumulation in the liver as well as increasing cytokine secretion and hepatic glucose production. Moreover, VDR deletion increases atherosclerosis by enabling lipid-laden M2 monocytes to adhere, migrate, and carry cholesterol into the atherosclerotic plaque and by increasing macrophage cholesterol uptake and esterification. Increased foam cell formation results from lack of VDR-SERCA2b interaction, causing SERCA dysfunction, activation of ER stress-CaMKII-JNKp-PPARγ signaling, and induction of the scavenger receptors CD36 and SR-A1. Bone marrow transplant of VDR-expressing cells into KODMAC mice improved insulin sensitivity, suppressed atherosclerosis, and decreased foam cell formation. The immunomodulatory effects of vitamin D in macrophages are thus critical in diet-induced insulin resistance and atherosclerosis in mice.

25(OH) vitamin D suppresses macrophage adhesion and migration by downregulation of ER stress and scavenger receptor A1 in type 2 diabetes.

Cardiovascular disease (CVD) is the leading cause of mortality in patients with type 2 diabetes mellitus (T2DM). Vitamin D deficiency is not only more prevalent in diabetics but also doubles the risk of developing CVD. However, it is unknown whether 25-hydroxy vitamin D [25(OH)D3] replacement slows monocyte adhesion and migration, critical mechanisms involved in atherosclerosis progression. In this study, monocytes from vitamin D-deficient diabetic patients were cultured either in the patient's serum or in vitamin D-deficient media with or without 25(OH)D3 treatment. Adding 25(OH)D3 to monocytes cultured in vitamin D-deficient serum or media decreased monocyte adhesion to fibronectin and migration stimulated by monocyte chemotactic protein 1 (MCP-1). Accordingly, 25(OH)D3 decreased adhesion marker ß1- and ß2-integrin expression and migration receptor chemokine (C-C motif) receptor 2 (CCR2) expression. 25(OH)D3 treatment downregulated monocyte endoplasmic reticulum (ER) stress and scavenger receptor class A, type 1 (SR-A1) expression. The absence of SR-A1 prevented the increased macrophage adhesion and migration induced by vitamin D deficiency. Moreover, the absence of SR-A1 prevented the induction of adhesion and migration and expression of their associated membrane receptors by Thapsigargin, an ER stress inducer. These results identify cellular activation of monocyte/macrophage vitamin D signaling through 25(OH)D3 as a potential mechanism that could modulate adhesion and migration in diabetic subjects. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

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

BACKGROUND: Coronary 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/DESIGN: The 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. DISCUSSION: This 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.

Hypotension due to Kir6.1 gain-of-function in vascular smooth muscle.

BACKGROUND: KATP channels, assembled from pore-forming (Kir6.1 or Kir6.2) and regulatory (SUR1 or SUR2) subunits, link metabolism to excitability. Loss of Kir6.2 results in hypoglycemia and hyperinsulinemia, whereas loss of Kir6.1 causes Prinzmetal angina-like symptoms in mice. Conversely, overactivity of Kir6.2 induces neonatal diabetes in mice and humans, but consequences of Kir6.1 overactivity are unknown. METHODS AND RESULTS: We generated transgenic mice expressing wild-type (WT), ATP-insensitive Kir6.1 [Gly343Asp] (GD), and ATP-insensitive Kir6.1 [Gly343Asp,Gln53Arg] (GD-QR) subunits, under Cre-recombinase control. Expression was induced in smooth muscle cells by crossing with smooth muscle myosin heavy chain promoter-driven tamoxifen-inducible Cre-recombinase (SMMHC-Cre-ER) mice. Three weeks after tamoxifen induction, we assessed blood pressure in anesthetized and conscious animals, as well as contractility of mesenteric artery smooth muscle and KATP currents in isolated mesenteric artery myocytes. Both systolic and diastolic blood pressures were significantly reduced in GD and GD-QR mice but normal in mice expressing the WT transgene and elevated in Kir6.1 knockout mice as well as in mice expressing dominant-negative Kir6.1 [AAA] in smooth muscle. Contractile response of isolated GD-QR mesenteric arteries was blunted relative to WT controls, but nitroprusside relaxation was unaffected. Basal KATP conductance and pinacidil-activated conductance were elevated in GD but not in WT myocytes. CONCLUSIONS: KATP overactivity in vascular muscle can lead directly to reduced vascular contractility and lower blood pressure. We predict that gain of vascular KATP function in humans would lead to a chronic vasodilatory phenotype, as indeed has recently been demonstrated in Cantu syndrome.

1,25(OH)2 vitamin D suppresses macrophage migration and reverses atherogenic cholesterol metabolism in type 2 diabetic patients.

Reduced monocyte infiltration into the vessel wall and increased macrophage cholesterol efflux are critical components in atherosclerotic plaque regression. During inflammation, monocyte chemotactic protein 1 (MCP-1) signaling activation and cholesterol deposition in macrophages induce endoplasmic reticulum (ER) stress, which promotes an increased inflammatory response. Increased macrophage ER stress shifts macrophages into an M2 macrophage phenotype with increased cholesterol uptake and deposition. In type 2 diabetes, a population with elevated baseline risk of cardiovascular disease (CVD), vitamin D deficiency doubles that risk. We have found that 1,25-dihydroxy vitamin D [1,25(OH)2D] prevents foam cell formation during macrophage differentiation by suppressing ER stress. However, it is unknown whether suppression of ER stress by 1,25(OH)2D decreases monocyte infiltration and reverses atherogenic cholesterol metabolism in previously differentiated, vitamin D-deplete macrophages. We collected peripheral monocytes from type 2 diabetic patients and differentiated them into macrophages under vitamin D-deplete or 1,25(OH)2D-supplemented conditions. 1,25(OH)2D supplementation suppressed macrophage migration in response to MCP-1 and mRNA expression of chemokine (C-C motif) receptor 2 (CCR2), the MCP-1 receptor, compared to vitamin D-deplete cells. Furthermore, inhibition of ER stress with phenyl butyric acid resulted in similar effects even in vitamin D-deplete cells, while induction of ER stress with Thapsigargin under 1,25(OH)2D-supplemented conditions increased macrophage migration and CCR2 expression, suggesting that the effects of vitamin D on migration are mediated through ER stress suppression. To determine whether the detrimental pattern of macrophage cholesterol metabolism in vitamin D depletion is reversible, we assessed cholesterol uptake in macrophages differentiated under vitamin D-deplete conditions as described above, then supplemented with 1,25(OH)2D or maintained in vitamin D-deplete conditions. Cholesterol uptake was decreased in 1,25(OH)2D-supplemented compared to vitamin D-deplete cells, suggesting slowed cholesterol deposition with active vitamin D. 1,25(OH)2D supplementation also suppressed cholesteryl ester formation and enhanced cholesterol efflux in M2 macrophages compared to vitamin D-deplete cells, suggesting facilitation of cholesterol egress in the presence of 1,25(OH)2D. We thus provide further evidence that active vitamin D is an ER stress reliever that may have a role in atherosclerotic plaque regression. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

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.

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, ß(1)-integrin, and ß(2)-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)(2)D(3) 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.

Endoplasmic reticulum stress controls M2 macrophage differentiation and foam cell formation.

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.

Enhanced hepatic apoA-I secretion and peripheral efflux of cholesterol and phospholipid in CD36 null mice.

CD36 facilitates oxidized low density lipoprotein uptake and is implicated in development of atherosclerotic lesions. CD36 also binds unmodified high and very low density lipoproteins (HDL, VLDL) but its role in the metabolism of these particles is unclear. Several polymorphisms in the CD36 gene were recently shown to associate with serum HDL cholesterol. To gain insight into potential mechanisms for these associations we examined HDL metabolism in CD36 null (CD36(-/-)) mice. Feeding CD36(-/-) mice a high cholesterol diet significantly increased serum HDL, cholesterol and phospholipids, as compared to wild type mice. HDL apolipoproteins apoA-I and apoA-IV were increased and shifted to higher density HDL fractions suggesting altered particle maturation. Clearance of dual-labeled HDL was unchanged in CD36(-/-) mice and cholesterol uptake from HDL or LDL by isolated CD36(-/-) hepatocytes was unaltered. However, CD36(-/-) hepatocytes had higher cholesterol and phospholipid efflux rates. In addition, expression and secretion of apoA-I and apoA-IV were increased reflecting enhanced PXR. Similar to hepatocytes, cholesterol and phospholipid efflux were enhanced in CD36(-/-) macrophages without changes in protein levels of ABCA1, ABCG1 or SR-B1. However, biotinylation assays showed increased surface ABCA1 localization in CD36(-/-) cells. In conclusion, CD36 influences reverse cholesterol transport and hepatic ApoA-I production. Both pathways are enhanced in CD36 deficiency, increasing HDL concentrations, which suggests the potential benefit of CD36 inhibition.

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.