Pre-clinical safety assessment of biotechnologically produced lacto-N-tetraose (LNT).

Lacto-N-tetraose (LNT) is a human milk oligosaccharide with average concentrations ranging from 0.74 to 1.07 g/L in breastmilk, depending on the lactation stage. In this study, the preclinical safety of LNT produced by the Escherichia coli K-12 E2083 production strain was assessed. LNT was negative in both the bacterial reverse mutation assay and the in vitro micronucleus assay, demonstrating the absence of genotoxic potential for this substance. In the OECD 408 guideline compliant 90-day oral toxicity study rat, LNT did not induce any adverse effects in any treatment group up to and including the highest dose tested, and no LOAEL could be determined. Therefore, the no-observed-adverse effect level (NOAEL) is set at the highest dose level tested, i.e. a dietary level of 5 % (w/w), corresponding to ≥2856 mg/kg bw/day and ≥3253 mg/kg bw/day for males and females, respectively. This might be an underestimation of the NOAEL, caused by the range of dose levels tested. The results obtained in the current study are in good agreement with available data generated using other biotechnologically produced LNT batches and therefore support its safe use as a food ingredient.

Gram-scale chemical synthesis of galactosyllactoses and their impact on infant gut microbiota in vitro.

Galactooligosaccharides (GOS) are widely used as a supplement in infant nutrition to mimic the beneficial effects found in prebiotic human milk oligosaccharides (HMOs). However, the complexity of the GOS mixture makes it challenging to ascertain which of the GOS components contribute most to their health benefits. Galactosyllactoses (GLs) are lactose-based trisaccharides containing a ß-galactopyranosyl residue at the 3'-position (3'galactosyllactose, 3'-GL), 4'-position (4'-galactosyllactose, 4'-GL), or the 6'-position (6'-galactosyllactose, 6'-GL). These GLs are of particular interest as they are present in both GOS mixtures and human milk at early stages of lactation. However, research on the potential health benefits of these individual GLs has been limited. Gram quantities are needed to assess their health benefits but these GLs are not readily available at this scale. In this study, we report the gram-scale chemical synthesis of 3'-GL, 4'-GL, and 6'-GL. All three galactosyllactoses were obtained on a gram scale in good purity from cheap and commercially available lactose. Furthermore, in vitro incubation of GLs with infant faecal microbiota demonstrates that the GLs were able to increase the abundance of Bifidobacterium and stimulate short chain fatty acid production.

Linking human milk oligosaccharide metabolism and early life gut microbiota: bifidobacteria and beyond.

SUMMARYHuman milk oligosaccharides (HMOs) are complex, multi-functional glycans present in human breast milk. They represent an intricate mix of heterogeneous structures which reach the infant intestine in an intact form as they resist gastrointestinal digestion. Therefore, they confer a multitude of benefits, directly and/or indirectly, to the developing neonate. Certain bifidobacterial species, being among the earliest gut colonizers of breast-fed infants, have an adapted functional capacity to metabolize various HMO structures. This ability is typically observed in infant-associated bifidobacteria, as opposed to bifidobacteria associated with a mature microbiota. In recent years, information has been gleaned regarding how these infant-associated bifidobacteria as well as certain other taxa are able to assimilate HMOs, including the mechanistic strategies enabling their acquisition and consumption. Additionally, complex metabolic interactions occur between microbes facilitated by HMOs, including the utilization of breakdown products released from HMO degradation. Interest in HMO-mediated changes in microbial composition and function has been the focal point of numerous studies, in recent times fueled by the availability of individual biosynthetic HMOs, some of which are now commonly included in infant formula. In this review, we outline the main HMO assimilatory and catabolic strategies employed by infant-associated bifidobacteria, discuss other taxa that exhibit breast milk glycan degradation capacity, and cover HMO-supported cross-feeding interactions and related metabolites that have been described thus far.

Early-Life Gut Health Indicators and Reported Prevalence of Infant Functional Constipation by Healthcare Professionals.

A healthy gut during early childhood is important. However, it seems that there are no standard indicators used to assess it. Healthcare professionals (HCPs) were asked via an electronic survey question about gut health indicators (GHIs) for infants and toddlers, in addition to an estimated prevalence of infant's functional constipation (FC) and its management. HCPs from eight countries participated in the survey (Russia (66.0%, 1449), Indonesia (11.0%, 242), Malaysia (6.0%, 132), Mexico (5.7%, 125), KSA (5.1%, 113), Turkey (3.0%, 66), Hong Kong (2.2%, 49), and Singapore (1.0%, 23)). The 2199 participating respondents were further classified into three continents (Asia (20.2%), Europe (68.8%), and others (11.0%)). Most of them were pediatricians (80.3%), followed by pediatric gastroenterologists (7.0%), general practitioners (6.4%), and others (6.3%). The top three preferred GHIs were similar for infants and toddlers: an absence of gastrointestinal (GI) symptoms, effective digestion/absorption as assessed by normal growth, and a general feeling of well-being. The absence of GI-related infection was the least preferred indicator. Most of the respondents reported the prevalence of FC among infants was less than 5%, with the peak incidence between the ages of 3 and 6 months. The reported choices of intervention to manage FC in infants were a change to a specific nutritional solution from a standard formula (40.2%), parental reassurance (31.7%), and lactulose (17.0%). Conclusion: The HCPs in the eight countries preferred the absence of GI symptoms, normal growth for effective digestion and absorption, and general well-being as the gut health indicators in infants and toddlers. The reported prevalence of FC in infants was less than 5%.

Reported Prevalence and Nutritional Management of Functional Constipation among Young Children from Healthcare Professionals in Eight Countries across Asia, Europe and Latin America.

Background: The prevalence of functional constipation (FC) among children varies widely. A survey among healthcare professionals (HCPs) was conducted to better understand the HCP-reported prevalence and (nutritional) management of FC in children 12−36 months old. Methods: An anonymous e-survey using SurveyMonkey was disseminated via emails or WhatsApp among HCPs in eight countries/regions. Results: Data from 2199 respondents were analyzed. The majority of the respondents (65.9%) were from Russia, followed by other countries (Indonesia (11.0%), Malaysia (6.0%)), Mexico, KSA (5.1% (5.7%), Turkey (3.0%), Hong Kong (2.2%), Singapore (1.1%)). In total, 80% of the respondents (n = 1759) were pediatricians. The prevalence of FC in toddlers was reported at less than 5% by 43% of the respondents. Overall, 40% of the respondents reported using ROME IV criteria in > 70% of the cases to diagnose FC, while 11% never uses Rome IV. History of painful defecation and defecations < 2 x/week are the two most important criteria for diagnosing FC. In total, 33% of the respondents reported changing the standard formula to a specific nutritional solution, accompanied by parental reassurance. Conclusion: The most reported prevalence of FC in toddlers in this survey was less than five percent. ROME IV criteria are frequently used for establishing the diagnosis. Nutritional management is preferred over pharmacological treatment in managing FC.

An Evaluation of the Mechanisms of Galacto-Oligosaccharide (GOS)-Induced IgE Cross-Linking on Basophils in GOS Allergy.

The prebiotics, galacto-oligosaccharides (GOS), are small carbohydrate molecules with 1-7 galactose units linked to glucose and have been shown to trigger IgE-mediated anaphylaxis in some cases following ingestion. It is still an unresolved question of how GOS cross-links IgE on basophils. In this study, we examined whether human galectins, a class of lectins that bind specifically to ß-galactoside carbohydrates, are involved in GOS-induced basophil activation. Basophil activation test to GOS and control allergen, Blomia tropicalis (Blo t) extract were performed in the presence or absence of four sugar-based galectin inhibitors (lactose, thiodigalactoside [TDG], TD139, and GB1107) and one peptide-based inhibitor, G3-C12. Results showed that TD139, GB1107, and G3-C12 did not display a specific inhibitory effect on GOS-induced basophil activation as compared to control allergen. An inhibitory effect of lactose and TDG on GOS-induced basophil activation was observed and varied between subjects with up to 100% inhibition at low doses of GOS. The results of competitive ELISA suggest that the inhibitory effects of high dose lactose and TDG on the basophil activation is likely due to the cross-reactivity of GOS-specific IgE to lactose and TDG. Basophil activation is performed using purified basophils suggested that cell surface receptors on other blood cells were not required to induce basophil activation. In conclusion, our results suggest that GOS, a low molecular weight sugar, is able to cross-link IgE independently.

A randomized controlled trial of different young child formulas on upper respiratory and gastrointestinal tract infections in Chinese toddlers.

BACKGROUND: Bioactive proteins and human milk oligosaccharides (HMOs), important ingredients in breast milk, that protect against infections are lacking in young child formula (YCF). This study investigated the effects of new YCFs on respiratory and gastrointestinal infections in toddlers. METHODS: Four hundred and sixty one healthy Chinese children aged 1-2.5 years were recruited in this randomized, controlled, double-blind, parallel-group clinical trial of different YCFs. They were randomly assigned to either standard milk formula (YCF-Ref) or one of three new YCFs containing bioactive proteins and/or the HMO 2'-fucosyllactose (2'-FL) and/or milk fat for six months. Primary outcomes were incidence of upper respiratory tract infection (URTI) and duration of gastrointestinal tract infections (GITI). RESULTS: There were no significant between-group differences in primary outcomes. For secondary outcomes, subjects receiving 2'-FL-supplemented YCF had longer URTI. Subjects receiving YCF supplemented with milk fat and intact bioactive proteins, and 2'-FL at levels found in breast milk, had more GITI episodes and shorter time to first GITI but similar effects on URTI duration than YCF-Ref recipients. No effects on URTI and GITI were observed in toddlers receiving YCF with bioactive proteins at lower levels than breast milk. Occurrence of adverse events and anthropometry were similar in all groups. CONCLUSIONS: All three YCFs supplemented with different combinations of intact bioactive proteins, 2'-FL, and milk fat are safe in toddlers. No difference is detected among YCFs on URTI incidence and GITI duration. Further studies are needed to verify these findings especially in infants who may benefit most from the immune-boosting effects of bioactive proteins and HMOs.

Modular synthesis and immunological evaluation of suspected allergenic galactooligosaccharides.

Galactooligosaccharides (GOS) are widely used in the food industry as prebiotics and in very rare cases, can lead to an allergic reaction. Due to the microheterogeneity of GOS it is very difficult to extract pure and well defined oligosaccharides to establish which component is responsible for the observed allergenicity. Herein, we report the chemical synthesis of a suspected allergen 4PX and three closely related oligosaccharides based on a modular approach. The fact that synthesized 4PX and a regioisomer did not cause basophil activation in subjects with confirmed GOS-allergy excludes both tetrasaccharides as key-epitopes in GOS-allergenicity in Singapore.

Dietary galacto-oligosaccharides prevent airway eosinophilia and hyperresponsiveness in a murine house dust mite-induced asthma model.

BACKGROUND: Allergic asthma is strongly associated with the exposure to house dust mite (HDM) and is characterized by eosinophilic pulmonary inflammation and airway hyperresponsiveness (AHR). Recently, there is an increased interest in using dietary oligosaccharides, also known as prebiotics, as a novel strategy to prevent the development of, or reduce, symptoms of allergy. AIM: We investigated the preventive capacity of dietary galacto-oligosaccharides (GOS) compared to an intra-airway therapeutic treatment with budesonide on the development of HDM-induced allergic asthma in mice. METHODS: BALB/c mice were intranasally sensitized with 1 µg HDM on day 0 followed by daily intranasal challenge with PBS or 10 µg HDM on days 7 to 11. Two weeks prior to the first sensitization and throughout the experiment mice were fed a control diet or a diet containing 1% GOS. Reference mice were oropharyngeally instilled with budesonide (500 µg/kg) on days 7, 9, 11, and 13, while being fed the control diet. On day 14, AHR was measured by nebulizing increasing doses of methacholine into the airways. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lungs were collected. RESULTS: Sensitization and challenge with HDM resulted in AHR. In contrast to budesonide, dietary intervention with 1% GOS prevented the development of AHR. HDM sensitization and challenge resulted in a significant increase in BALF leukocytes numbers, which was suppressed by budesonide treatment and dietary intervention with 1% GOS. Moreover, HDM sensitization and challenge resulted in significantly enhanced concentrations of IL-6, CCL17, IL-33, CCL5 and IL-13 in lung tissue. Both dietary intervention with 1% GOS or budesonide treatment significantly decreased the HDM-induced increased concentrations of CCL5 and IL-13 in lung tissue, while budesonide also reduced the HDM-enhanced concentrations of IL-6 and CCL17 in lung tissue. CONCLUSION: Not only did dietary intervention with 1% GOS during sensitization and challenge prevent the induction of airway eosinophilia and Th2-related cytokine and chemokine concentrations in the lung equally effective as budesonide treatment, it also prevented AHR development in HDM-allergic mice. GOS might be useful for the prevention and/or treatment of symptoms in asthmatic disease.

Leukocyte cathepsin C deficiency attenuates atherosclerotic lesion progression by selective tuning of innate and adaptive immune responses.

OBJECTIVE: The protein degrading activity of cathepsin C (CatC), combined with its role in leukocyte granule activation, suggests a contribution of this cystein protease in atherosclerosis. However, no experimental data are available to validate this concept. APPROACH AND RESULTS: CatC gene and protein expression were increased in ruptured versus advanced stable human carotid artery lesions. To assess causal involvement of CatC in plaque progression and stability, we generated LDLr(-/-)//CatC(-/-) chimeras by bone marrow transplantation. CatC(-/-) chimeras presented attenuated plaque burden in carotids, descending aorta, aortic arch and root, at both the early and advanced plaque stage. CatC was abundantly expressed by plaque macrophages and foam cells. CatC expression and activity were dramatically downregulated in plaques of CatC(-/-) chimeras, supporting a hematopoietic origin of plaque CatC. Our studies unveiled an unexpected feedback of CatC deficiency on macrophage activation programs and T helper cell differentiation in as much as that CatC expression was upregulated in M1 macrophages, whereas its deficiency led to combined M2 (in vitro) and Th2 polarization (in vivo). CONCLUSIONS: Our data implicate CatC has a role in the selective tuning of innate and adaptive immune responses, relevant to a chronic immune disease, such as atherosclerosis.

Chemokine-like receptor 1 deficiency does not affect the development of insulin resistance and nonalcoholic fatty liver disease in mice.

The adipokine chemerin and its receptor, chemokine-like receptor 1 (Cmklr1), are associated with insulin resistance and nonalcoholic fatty liver disease (NAFLD), which covers a broad spectrum of liver diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). It is possible that chemerin and/or Cmklr1 exert their effects on these disorders through inflammation, but so far the data have been controversial. To gain further insight into this matter, we studied the effect of whole-body Cmklr1 deficiency on insulin resistance and NAFLD. In view of the primary role of macrophages in hepatic inflammation, we also transplanted bone marrow from Cmklr1 knock-out (Cmklr1-/-) mice and wild type (WT) mice into low-density lipoprotein receptor knock-out (Ldlr-/-) mice, a mouse model for NASH. All mice were fed a high fat, high cholesterol diet containing 21% fat from milk butter and 0.2% cholesterol for 12 weeks. Insulin resistance was assessed by an oral glucose tolerance test, an insulin tolerance test, and by measurement of plasma glucose and insulin levels. Liver pathology was determined by measuring hepatic inflammation, fibrosis, lipid accumulation and the NAFLD activity score (NAS). Whole-body Cmklr1 deficiency did not affect body weight gain or food intake. In addition, we observed no differences between WT and Cmklr1-/- mice for hepatic inflammatory and fibrotic gene expression, immune cell infiltration, lipid accumulation or NAS. In line with this, we detected no differences in insulin resistance. In concordance with whole-body Cmklr1 deficiency, the absence of Cmklr1 in bone marrow-derived cells in Ldlr-/- mice did not affect their insulin resistance or liver pathology. Our results indicate that Cmklr1 is not involved in the pathogenesis of insulin resistance or NAFLD. Thus, we recommend that the associations reported between Cmklr1 and insulin resistance or NAFLD should be interpreted with caution.

In vivo knockdown of TAK1 accelerates bone marrow proliferation/differentiation and induces systemic inflammation.

TAK1 (TGF-ß Activated Kinase 1) is a MAPK kinase kinase, which activates the p38- and JNK-MAPK and NF-κB pathways downstream of receptors such as Toll-Like-, cytokine- and T-cell and B-cell receptors. Representing such an important node in the pro-inflammatory signal-transduction network, the function of TAK1 has been studied extensively. TAK1 knock-out mice are embryonic lethal, while conditional knock-out mice demonstrated either a pro- or anti-inflammatory function. To study the function of TAK1 protein in the adult immune system, we generated and characterized a transgenic mouse expressing TAK1 shRNA under the control of a doxycycline-inducible promoter. Following treatment of TAK-1 shRNA transgenic mice with doxycycline an effective knockdown of TAK1 protein levels was observed in lymphoid organs and cells in the peritoneal cavity (>50% down regulation). TAK1 knockdown resulted in significant changes in leukocyte populations in blood, bone marrow, spleen and peritoneal cavity. Upon TAK1 knockdown mice demonstrated splenomegaly, signs of systemic inflammation (increased levels of circulating cytokines and increase in cellularity of the B-cell areas and in germinal center development in the follicles) and degenerative changes in heart, kidneys and liver. Not surprisingly, TAK1-Tg mice treated with LPS or anti-CD3 antibodies showed enhanced cytokine/chemokine secretion. Finally, analysis of progenitor cells in the bone marrow upon doxycycline treatment showed increased proliferation and differentiation of myeloid progenitor cells. Given the similarity of the phenotype with TGF-ß genetic models, our data suggest that in our model the function of TAK1 in TGF-ß signal-transduction is overruling its function in pro-inflammatory signaling.

Anaphylaxis to cow's milk formula containing short-chain galacto-oligosaccharide.

BACKGROUND: On the basis of the proven prebiotic effects of oligosaccharides in cow's milk formula (CMF) in infants, CMFs are supplemented with oligosaccharides. OBJECTIVE: We present a series of 5 cases of cow's milk-tolerant but atopic patients with a history of respiratory allergies. All had anaphylaxis after the ingestion of CMF supplemented with short-chain galacto-oligosaccharide (scGOS). The allergen trigger was investigated. METHODS: Clinical histories were collated. Skin prick tests (SPTs) and basophil activation tests (BATs) were carried out with the eliciting CMF that triggered anaphylaxis, with or without supplemented prebiotics (scGOS) and with scGOS fractions containing oligosaccharides of different chain lengths. RESULTS: The median age of presentation was 6 years (range, 5-38 years). Anaphylaxis occurred within 30 minutes of the first known exposure to CMF supplemented with prebiotics in all patients. Only 1 patient was subjected to oral challenge, which resulted in an anaphylactic reaction. All patients demonstrated IgE sensitization through SPTs and BATs to scGOS and fractions of scGOS containing 3 sugar units or greater but not to cow's milk or long-chain fructo-oligosaccharide. Eight child control subjects tolerant to regular ingestion of scGOS-supplemented CMF and 1 adult volunteer were found to have negative results to scGOS through SPTs and BATs. In addition, in vitro BATs with donor basophils sensitized with sera from 2 of the 3 reported cases showed reactions to scGOS. The scGOS-induced basophil activation was inhibited in the presence of wortmannin, a phosphatidylinositol 3-kinase inhibitor. CONCLUSIONS: This study describes an unusual form of IgE-mediated anaphylaxis triggered by low-molecular-weight oligosaccharides in scGOS. The primary sensitizer for this phenomenon requires further investigation.

Btk levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice.

On antigen binding by the B-cell receptor (BCR), B cells up-regulate protein expression of the key downstream signaling molecule Bruton tyrosine kinase (Btk), but the effects of Btk up-regulation on B-cell function are unknown. Here, we show that transgenic mice overexpressing Btk specifically in B cells spontaneously formed germinal centers and manifested increased plasma cell numbers, leading to antinuclear autoantibody production and systemic lupus erythematosus (SLE)-like autoimmune pathology affecting kidneys, lungs, and salivary glands. Autoimmunity was fully dependent on Btk kinase activity, because Btk inhibitor treatment (PCI-32765) could normalize B-cell activation and differentiation, and because autoantibodies were absent in Btk transgenic mice overexpressing a kinase inactive Btk mutant. B cells overexpressing wild-type Btk were selectively hyperresponsive to BCR stimulation and showed enhanced Ca(2+) influx, nuclear factor (NF)-κB activation, resistance to Fas-mediated apoptosis, and defective elimination of selfreactive B cells in vivo. These findings unravel a crucial role for Btk in setting the threshold for B-cell activation and counterselection of autoreactive B cells, making Btk an attractive therapeutic target in systemic autoimmune disease such as SLE. The finding of in vivo pathology associated with Btk overexpression may have important implications for the development of gene therapy strategies for X-linked agammaglobulinemia, the immunodeficiency associated with mutations in BTK.

Cannabinoid Receptor 2 Deficiency in Haematopoietic cells Aggravates Early Atherosclerosis in LDL Receptor Deficient Mice.

OBJECTIVE: The cannabinoid receptor 2 (CB2) has been implicated to play a role in various inflammatory processes. Since atherosclerosis is currently considered a chronic inflammatory disease, we studied the effect of haematopoietic CB2 deficiency on atherosclerosis development. METHODS AND RESULTS: To investigate the effect of CB2 deficiency in immune cells on atherogenesis in vivo, a bone marrow transplantation was performed in irradiated LDL receptor deficient mice (LDLr(-/-)), using CB2 deficient (CB2(-/-)) or wildtype (WT) donor mice. After 12 weeks on a high fat-high cholesterol diet, en face analysis showed that atherosclerosis in the aortic arch was significantly increased in CB2(-/-) transplanted animals (6.40 ± 3.21%) as compared to WT transplanted mice (3.85 ± 1.61%). Although the total lesion area in the aortic root was not significantly different between WT and CB2(-/-) transplanted mice (0.45 ± 0.13 mm(2) and 0.51 ± 0.17 mm(2), respectively), CB2(-/-) transplanted mice showed a significantly larger plaque area (0.13 ± 0.07 mm(2)) than WT transplanted mice (0.08 ± 0.05 mm(2)) in the aortic valve in which atherogenesis is in an earlier stage than in the other aortic valves. CONCLUSIONS: Lack of endocannabinoid signaling via the CB2 receptor aggravates early atherosclerosis development in LDLr(-/-) mice, suggesting that CB2 specific activation may prevent the development of atherosclerosis.

Apolipoprotein C-I binds free fatty acids and reduces their intracellular esterification.

Mice that overexpress human apolipoprotein C-I (apoC-I) homozygously (APOC1(+/+) mice) are protected against obesity and show cutaneous abnormalities. Although these effects can result from our previous observation that apoC-I inhibits FFA generation by LPL, we have also found that apoC-I impairs the uptake of a FFA analog in adipose tissue. In this study, we tested the hypothesis that apoC-I interferes with cellular FFA uptake independent of LPL activity. The cutaneous abnormalities of APOC1(+/+) mice were not affected after transplantation to wild-type mice, indicating that locally produced apoC-I prevents lipid entry into the skin. Subsequent in vitro studies with apoC-I-deficient versus wild-type macrophages revealed that apoC-I reduced the cell association and subsequent esterification of [(3)H]oleic acid by approximately 35% (P < 0.05). We speculated that apoC-I binds FFA extracellularly, thereby preventing cell association of FFA. We showed that apoC-I was indeed able to mediate the binding of oleic acid to otherwise protein-free VLDL-like emulsion particles involving electrostatic interaction. We conclude that apoC-I binds FFA in the circulation, thereby reducing the availability of FFA for uptake by cells. This mechanism can serve as an additional mechanism behind the resistance to obesity and the cutaneous abnormalities of APOC1(+/+) mice.

Rosuvastatin reduces plasma lipids by inhibiting VLDL production and enhancing hepatobiliary lipid excretion in ApoE*3-leiden mice.

The present study was designed to investigate the lipid-lowering properties and mechanisms of action of a new HMG-CoA reductase inhibitor, rosuvastatin, in female ApoE*3-Leiden transgenic mice. Mice received a high fat/cholesterol (HFC) diet containing either rosuvastatin (0 [control], 0.00125%, 0.0025%, or 0.005% [w/w]) or 0.05% (w/w) lovastatin. The highest dose of rosuvastatin reduced plasma cholesterol and triglyceride levels by 39% and 42%, respectively, compared with the HFC control. Lovastatin had no effect on plasma cholesterol and triglyceride levels. In ApoE*3-Leiden mice on a chow diet, rosuvastatin (0.005% [w/w]) decreased plasma cholesterol levels by 35% without having an effect on triglyceride levels. On a chow diet, expression of genes involved in cholesterol biosynthesis and uptake in the liver was increased by rosuvastatin. Further mechanistic studies in HFC-fed mice showed that rosuvastatin treatment resulted in decreased hepatic VLDL-triglyceride and VLDL-apolipoprotein B production. VLDL lipid composition remained unchanged, indicating a reduction in the number of VLDL particles secreted. Lipolytic activity and expression of genes involved in cholesterol and triglyceride synthesis and beta-oxidation of fatty acids in the liver were not affected by rosuvastatin treatment, and hepatic lipid content did not change. However, activity of hepatic diacylglycerol acyltransferase was significantly decreased by 25% after rosuvastatin treatment. Moreover, biliary excretion of cholesterol, phospholipids, and bile acids was increased during treatment. The results indicate that rosuvastatin treatment in ApoE*3-Leiden mice on a HFC diet leads to redistribution of cholesterol and triglycerides in the body, both by reduced hepatic VLDL production and triglyceride synthesis and by enhanced hepatobiliary removal of cholesterol, bile acids, and phospholipids, resulting in substantial reductions in plasma cholesterol and triglyceride levels.

Differential effects of amlodipine and atorvastatin treatment and their combination on atherosclerosis in ApoE*3-Leiden transgenic mice.

This study was designed to investigate the potential antiatherosclerotic effects of the calcium antagonist amlodipine as compared with the HMG-CoA reductase inhibitor atorvastatin and the combination of both in ApoE*3-Leiden transgenic mice. Four groups of 15 ApoE*3-Leiden mice were put on a high-cholesterol diet. One group received 0.002% (wt/wt) amlodipine in the diet, which had no effect on plasma cholesterol levels. Another group received 0.01% (wt/wt) atorvastatin, resulting in a decrease of plasma cholesterol by 50% by a reduction in very low density lipoprotein production. The combination group received both amlodipine and atorvastatin. After 28 weeks, atherosclerosis in the aortic root was quantified. Treatment with amlodipine had no significant effect on atherosclerotic lesion area, whereas atorvastatin markedly reduced atherosclerosis by 77% compared with the control group. Atorvastatin also reduced inflammation markers. The combination of amlodipine and atorvastatin tended to reduce lesion area by 61% compared with the atorvastatin-only group; however, this effect did not reach statistical significance. Amlodipine treatment significantly reduced calcification in the lesions, whereas atorvastatin alone had no effect. The combination of amlodipine and atorvastatin resulted in a near absence of calcium deposits in the lesions. This study demonstrates that amlodipine treatment alone does not significantly reduce atherosclerotic lesion development. Atorvastatin was shown to have strong antiatherosclerotic effects, and cotreatment with amlodipine may potentiate the antiatherosclerotic effect of atorvastatin.