Vitamin D and Cardiovascular Disease, with Emphasis on Hypertension, Atherosclerosis, and Heart Failure.

Vitamin D deficiency is the most common nutritional deficiency, affecting almost one billion people worldwide. Vitamin D is mostly known for its role in intestinal calcium absorption and bone mineralization. However, the observation of seasonal changes in blood pressure and the subsequent identification of vitamin D receptor (VDR) and 1α-hydroxylase in cardiomyocytes, as well as endothelial and vascular smooth muscle cells, implicated a role of vitamin D in the cardiovascular system. Animal studies provided compelling evidence that vitamin D signaling is essential for cardiovascular integrity, especially for the regulation of vascular tone and as an antifibrotic and antihypertrophic signaling pathway in the heart. In addition, observational studies reported an association between vitamin D deficiency and risk of hypertension, atherosclerosis, and heart failure. However, recent clinical intervention studies failed to prove the causal relationship between vitamin D supplementation and beneficial effects on cardiovascular health. In this review, we aim to highlight our current understanding of the role of vitamin D in the cardiovascular system and to find potential explanations for the large discrepancies between the outcome of experimental studies and clinical intervention trials.

The PPARα Agonist Fenofibrate Improves the Musculoskeletal Effects of Exercise in Ovariectomized Rats.

The musculoskeletal effects of exercise are attenuated by estrogen deficiency. The peroxisome proliferator-activated receptor-α agonist fenofibrate exerts beneficial effects in bone and muscle. We therefore examined whether fenofibrate could enhance the musculoskeletal training response during estrogen deficiency. We investigated the combined effects of 8 weeks of fenofibrate and jumping exercise in ovariectomized (OVX) Sprague Dawley rats. Female rats were allocated to a sham-operated group and four OVX groups; fenofibrate (OVX-Fen), exercise (OVX-Ex), combined fenofibrate and exercise (OVX-FenEx), and a control group (OVX-Ctr) (n = 12/group). Fenofibrate (90 mg/kg/d) or methylcellulose was given by gavage. The combination of exercise and fenofibrate resulted in enhanced femoral bone mineral density (BMD) and improved bone microarchitecture compared with fenofibrate alone as well as increased trabecular BMD compared with OVX-Ctr. These effects were not seen in the OVX-Ex group. Femoral BMD was normalized in both exercise groups relative to sham and increased more in all intervention groups compared with OVX-Ctr. A higher plasma level of the bone formation marker type 1 collagen amino propeptide was observed in the OVX-Fen and OVX-FenEx groups compared with controls. Lean mass and soleus muscle weight were higher in the OVX-FenEx group than in the OVX-Ctr group, which coincided with lower mRNA levels of Atrogin1. These results suggest that peroxisome proliferator-activated receptor-α activation improves the musculoskeletal effects of exercise during estrogen deficiency.

Skeletal effects of a gastrin receptor antagonist in H+/K+ATPase beta subunit KO mice.

Epidemiological studies suggest an increased fracture risk in patients taking proton pump inhibitors (PPIs) for long term. The underlying mechanism, however, has been disputed. By binding to the gastric proton pump, PPIs inhibit gastric acid secretion. We have previously shown that proton pump (H(+)/K(+)ATPase beta subunit) KO mice exhibit reduced bone mineral density (BMD) and inferior bone strength compared with WT mice. Patients using PPIs as well as these KO mice exhibit gastric hypoacidity, and subsequently increased serum concentrations of the hormone gastrin. In this study, we wanted to examine whether inhibition of the gastrin/CCK2 receptor influences bone quality in these mice. KO and WT mice were given either the gastrin/CCK2 receptor antagonist netazepide dissolved in polyethylene glycol (PEG) or only PEG for 1year. We found significantly lower bone mineral content and BMD, as well as inferior bone microarchitecture in KO mice compared with WT. Biomechanical properties by three-point bending test also proved inferior in KO mice. KO mice receiving netazepide exhibited significantly higher cortical thickness, cortical area fraction, trabecular thickness and trabecular BMD by micro-CT compared with the control group. Three-point bending test also showed higher Young's modulus of elasticity in the netazepide KO group compared with control mice. In conclusion, we observed that the gastrin receptor antagonist netazepide slightly improved bone quality in this mouse model, suggesting that hypergastrinemia may contribute to deteriorated bone quality during acid inhibition.

Roux-en-Y gastric bypass surgery but not vertical sleeve gastrectomy decreases bone mass in male rats.

The most effective treatment for obesity is bariatric surgery. However, there is increasing concern that bariatric surgery can cause nutrient deficiencies that translate into metabolic bone disease. Whether this is true for all surgery types is not yet clear. We therefore investigated the effects of 2 commonly applied bariatric surgeries (Roux-en-Y gastric bypass [RYGB] and vertical sleeve gastrectomy) on energy and bone metabolism in rats 60 days after surgery. Both surgeries resulted in similar reductions of body weight, body fat, and food intake. Glucose tolerance was improved to a similar extent after both surgeries and was accompanied by increased postprandial secretion of glucose-dependent insulinotropic peptide. Using microcomputed tomography, we found that, relative to sham-operated rats, bone volume was significantly reduced after RYGB but not vertical sleeve gastrectomy. RYGB rats also had markedly reduced lipid absorption from the intestine and significantly lower serum 25-hydroxyvitamin D and calcium levels. Importantly, dietary supplementation with calcium and vitamin D could not fully rescue the reduced bone volume after RYGB surgery. Both surgeries resulted in a significant increase in stomach pH, which may have worsened the malabsorption in RYGB rats. Our findings suggest that bone loss in RYGB rats is not exclusively driven by calcium and vitamin D malabsorption but also by additional factors that may not be rescuable by dietary supplementation. These data point toward important similarities and differences between bariatric procedures that should be considered in clinical settings as guidance for which procedure will be best for specific patient populations.

Gender- and dose-related effects of cyclosporin A on hepatic and bone metabolism.

Previous data have shown gender-related differences in the skeletal effects of the immunosuppressive drug cyclosporin A (CsA) in rats. To test the hypothesis that the gender-related skeletal effects of CsA are caused by gender-specific metabolism of this drug, we treated aged male and female sham-operated, gonadectomized (GX) as well as sex hormone-supplemented GX rats with 5 mg/kg CsA three times per week for 2 months, and analyzed the bone phenotype as well as the concentrations of CsA and its major metabolites AM1, AM1c, AM9, and AM4N in blood, urine, and liver tissue. CsA treatment induced high turnover osteopenia in males, but not females. Male rats showed several-fold higher CsA and CsA metabolite blood levels compared with females. Renal clearance data revealed that CsA undergoes selective tubular reabsorption in male, but not female rats. However, a mathematical modeling approach demonstrated that the higher CsA blood levels in males were almost exclusively caused by a 6-fold lower hepatic clearance rate compared with females. In addition, we subcutaneously treated female rats with up to 6-fold higher doses of CsA. Similar to males, high dose CsA induced high turnover osteopenia in female rats. Our data show that the gender-related differences in the skeletal effects of CsA are caused by a higher hepatic clearance rate for CsA in female compared to male rats, and not by a differential skeletal response to CsA. Moreover, our study indicates that CsA blood levels of ≤200 ng/ml measured by HPLC do not induce high turnover osteopenia in aged rats.

Long-term marginal zinc supply is not detrimental to the skeleton of aged female rats.

In this experiment, we investigated the long-term effects of a marginal zinc (Zn) supply on bone metabolism in aged rats. Nine-mo-old female Fischer-344 rats were divided into 8 weight-matched groups of 8 rats each. All rats were adapted for 1 mo to restrictive feeding (7.5 g/d) of a purified diet containing 8 g/kg sodium phytate and 64 mg/kg Zn. Control rats were pair-fed throughout the experiment. During the 1-mo depletion phase, controls received the Zn-replete diet with 64 mg/kg Zn, whereas Zn-deficient rats were fed the same diet with 2.2 mg/kg Zn. The depletion phase was followed by a 3-mo marginal phase in which the rats fed the diet with 2.2 mg/kg Zn received an additional daily Zn supplement of 75 microg Zn/rat by gavage. In the following 2-mo repletion phase, a marginal group was switched to the Zn-replete diet, while the other groups were maintained on marginal Zn supply or on the Zn-replete diet. Zn depletion and marginal Zn reduced serum and bone Zn and serum alkaline phosphatase activity. Zn repletion normalized serum Zn. However, apart from subtle changes in bone mineralization density distribution, Zn deficiency was not associated with detrimental effects on bone mineral density, turnover, architecture, or biomechanics relative to control rats at any time point. Our data suggest that Zn does not play an essential role in bone metabolism in aged rats and cast doubt on the hypthosis that Zn deficiency is a risk factor for osteoporosis.

Cortical bone loss in androgen-deficient aged male rats is mainly caused by increased endocortical bone remodeling.

INTRODUCTION: Hypogonadism is considered to be one of the major risk factors for osteoporosis in men. Here, we sequentially studied the effects of androgen deficiency on cortical bone in aged orchiectomy (ORX) rats. MATERIALS AND METHODS: One hundred seventy 13-mo-old male Fischer-344 rats were either ORX or sham-operated. After in vivo fluorochrome labeling, groups of 8-15 SHAM and ORX rats each were killed at 2 wk and 1, 2, 3, 4, 6, and 9 mo after surgery. To examine the effects of testosterone replacement therapy, 9-mo-old ORX rats were supplemented with testosterone undecanoate at a weekly dose of 6 mg/kg for 4 mo. Cortical bone changes in the tibial shaft were monitored by pQCT analysis and by bone histomorphometry. RESULTS: SHAM rats did not show age-related bone loss at the tibial diaphysis. pQCT analysis and bone histomorphometry showed cortical bone osteopenia in ORX rats, beginning from 2 mo after surgery until the end of the study. Androgen deficiency induced a sustained decrease in periosteal bone formation during the first 4 mo after ORX. However, although periosteal expansion of the tibial shaft tended to be slower in ORX rats compared with SHAM controls, the reduction in total cross-sectional area in ORX animals reached statistical significance only at 4 mo after surgery. The major mechanism for cortical bone loss in aged ORX rats was a progressive expansion of the marrow cavity, which was associated with an initial increase in endocortical eroded perimeter at 1 and 2 mo after surgery, followed by a sustained increase in endocortical bone formation until the end of the study. All these changes were prevented in aged ORX rats receiving testosterone supplementation in an insulin-like growth factor system-independent fashion. CONCLUSIONS: We conclude that androgen deficiency-induced cortical bone loss in aged, nongrowing rats is mainly caused by augmented endocortical bone remodeling.

Long-term sensitivity of uterus and hypothalamus/pituitary axis to 17beta-estradiol is higher than that of bone in rats.

UNLABELLED: We examined the long-term sensitivity of uterus and bone to low-dose 17beta-estradiol in a 4-month experiment in OVX rats and found that a dose of estradiol that fully protected against uterine atrophy did not protect against bone loss. Our results suggest higher estrogen sensitivity of the uterus compared with bone. INTRODUCTION: Estrogen is essential for the function of reproductive tissues and for the normal acquisition and maintenance of bone mass in females. This study was designed to examine the long-term sensitivity of the uterus and bone to low-dose estrogen. MATERIALS AND METHODS: In preliminary experiments, we determined the lowest subcutaneous dose of 17beta-estradiol able to fully protect against uterine atrophy in ovariectomized (OVX) rats. This dose was found to be 1.5 microg/kg, given five times per week. Subsequently, groups of sham-operated (SHAM) or OVX 6-month-old rats (n = 8 each) were subcutaneously injected with vehicle or 1.5 microg/kg 17beta-estradiol five times per week. All animals were killed 4 months after surgery. Serum osteocalcin and urinary deoxypyridinoline were measured as biochemical markers of bone turnover. Bones were analyzed by bone histomorphometry and pQCT. RESULTS AND CONCLUSIONS: Our study clearly showed that a dose of estradiol that restores physiological estradiol serum levels, fully maintains uterine weight in OVX rats at the SHAM control level, and suppresses serum follicle-stimulating hormone (FSH) by 67% relative to OVX vehicle controls does not provide significant protection against OVX-induced bone loss at different cancellous and cortical bone sites. We conclude that the long-term sensitivity of the uterus and the hypothalamus/pituitary axis to 17beta-estradiol is higher than that of bone in rats.

Prevention of bone loss in ovariectomized rats by combined treatment with risedronate and 1alpha,25-dihydroxyvitamin D3.

Bisphosphonates inhibit bone loss through inhibition of osteoclast-mediated bone resorption. At low doses, vitamin D metabolites can prevent bone loss in models of osteopenia in rats by an antiresorptive effect, while at high doses they also stimulate osteoblast activity and show an anabolic effect. Therefore, combined therapy with bisphosphonates and vitamin D analogs might be expected to be more effective than either treatment alone. It was the aim of this study to compare the efficacy of risedronate and of the naturally occurring vitamin D hormone 1alpha,25-dihydroxyvitamin D3 (calcitriol), alone and in combination, for the prevention of ovariectomy-induced bone loss in rats. One hundred ten female 4-month-old Sprague-Dawley rats were used for this experiment. Ninety rats were bilaterally ovariectomized (OVX), 10 rats were sham-operated (SHAM), and 10 rats were killed at the time of surgery as a baseline control. Groups of rats (10 rats/group) received vehicle or daily doses of 0.1 mg or 0.5 mg of risedronate or 0.05 microg or 0.1 microg of calcitriol/kg body weight, alone and in combination. Both compounds were administered orally via gavage, commencing on the day after surgery. Although estrogen deficiency-induced bone loss was prevented by individual prophylactic administration of risedronate or calcitriol, OVX rats treated with a combination of risedronate and calcitriol had higher bone mineral density (BMD), cancellous bone area (B.Ar), and bone strength in long bones and vertebrae compared with rats receiving risedronate alone. Furthermore, calcitriol enhanced the suppressive effects of risedronate on osteoclast number and partially counteracted the suppressive effects of risedronate on bone formation and histomorphometric indices of osteoblast team performance. Risedronate did not reduce the anabolic effect of calcitriol, and at the high dose it normalized hypercalcemia in calcitriol-treated OVX rats. Therefore, this study in OVX rats suggests that combined therapy with bisphosphonates and vitamin D analogs may offer advantages over the treatment with bisphosphonates or vitamin D analogs alone.