Cationic LNP-formulated mRNA expressing Tie2-agonist in the lung endothelium prevents pulmonary vascular leakage.

Dysfunction of endothelial cells (ECs) lining the inner surface of blood vessels are causative for a number of diseases. Hence, the ability to therapeutically modulate gene expression within ECs is of high therapeutic value in treating diseases such as those associated with lung edema. mRNAs formulated with lipid nanoparticles (LNPs) have emerged as a new drug modality to induce transient protein expression for modulating disease-relevant signal transduction pathways. In the study presented here, we tested the effect of a novel synthetic, nucleoside-modified mRNA encoding COMP-Ang1 (mRNA-76) formulated into a cationic LNP on attenuating inflammation-induced vascular leakage. After intravenous injection, the respective mRNA was found to be delivered almost exclusively to the ECs of the lung, while sparing other vascular beds and bypassing the liver. The mode of action of mRNA-76, such as its activation of the Tie2 signal transduction pathway, was tested by pharmacological studies in vitro and in vivo in respective mouse models. mRNA-76 was found to prevent lung vascular leakage/lung edema as well as neutrophil infiltration in a lipopolysaccharide-challenging model.

Development of a multiplex microsphere immunoassay for the detection of antibodies against highly pathogenic viruses in human and animal serum samples.

Surveillance of highly pathogenic viruses circulating in both human and animal populations is crucial to unveil endemic infections and potential zoonotic reservoirs. Monitoring the burden of disease by serological assay could be used as an early warning system for imminent outbreaks as an increased seroprevalance often precedes larger outbreaks. However, the multitude of highly pathogenic viruses necessitates the need to identify specific antibodies against several targets from both humans as well as from potential reservoir animals such as bats. In order to address this, we have developed a broadly reactive multiplex microsphere immunoassay (MMIA) for the detection of antibodies against several highly pathogenic viruses from both humans and animals. To this aim, nucleoproteins (NP) of Ebola virus (EBOV), Marburg virus (MARV) and nucleocapsid proteins (NP) of Crimean-Congo haemorrhagic fever virus, Rift Valley fever virus and Dobrava-Belgrade hantavirus were employed in a 5-plex assay for IgG detection. After optimisation, specific binding to each respective NP was shown by testing sera from humans and non-human primates with known infection status. The usefulness of our assay for serosurveillance was shown by determining the immune response against the NP antigens in a panel of 129 human serum samples collected in Guinea between 2011 and 2012 in comparison to a panel of 88 sera from the German blood bank. We found good agreement between our MMIA and commercial or in-house reference methods by ELISA or IIFT with statistically significant higher binding to both EBOV NP and MARV NP coupled microspheres in the Guinea panel. Finally, the MMIA was successfully adapted to detect antibodies from bats that had been inoculated with EBOV- and MARV- virus-like particles, highlighting the versatility of this technique and potentially enabling the monitoring of wildlife as well as human populations with this assay. We were thus able to develop and validate a sensitive and broadly reactive high-throughput serological assay which could be used as a screening tool to detect antibodies against several highly pathogenic viruses.

Nutritional and health attributes of milk and milk imitations.

PURPOSE: Modern food technology allows designing products aiming to simulate and replace traditional food. In affluent societies there is a rising tendency to consume foods derived from plants including milk imitations or plant drinks based on cereals, nuts, legumes, oil seeds or other plant families. Herein we review production and composition of such drinks, summarize consumers' motivations to change from milk to plant drinks and highlight nutritional and health implications of consuming plant drinks instead of milk, in particular if non-fortified and if consumed by infants, children, adolescents and the elderly. RESULTS: Whereas the macronutrient concentrations of some plant drinks (soy) may approach in some cases (protein) that of cow's milk, the nutritional quality of most plant drinks, e.g., the biological value of protein and the presence and amount of vitamins and essential minerals with high bioavailability does not. If cow's milk is exchanged for non-fortified and non-supplemented plant drinks consumers may risk deficiencies of calcium, zinc, iodine, vitamins B2, B12, D, A, and indispensable amino acids, particularly in infants and toddlers who traditionally consume significant portions of milk. The vegetable nature, appearance and taste of such plant drinks may be appealing to adult consumers and be chosen for adding variety to the menu. However, in young children fed exclusively such plant drinks severe metabolic disturbances may occur. CONCLUSION: Parents, dietitians, physicians and consumers should be aware of such potential risks, if non-fortified plant drinks are consumed instead of milk.

Biological fractionation of stable Ca isotopes in Göttingen minipigs as a physiological model for Ca homeostasis in humans.

In order to investigate fractionation of calcium (Ca) isotopes in vertebrates as a diagnostic tool to detect Ca metabolism dysfunction we analyzed the Ca isotopic composition (δ(44/40)Ca = [((44)Ca/(40)Ca)sample/((44)Ca/(40)Ca)reference]-1) of diet, faeces, blood, bones and urine from Göttingen minipigs, an animal model for human physiology. Samples of three groups were investigated: 1. control group (Con), 2. group with glucocorticosteroid induced osteoporosis (GIO) and 3. group with Ca and vitamin D deficiency induced osteomalacia (-CaD). In contrast to Con and GIO whose average δ(44/40)Cafaeces values (0.39 ± 0.13‰ and 0.28 ± 0.08‰, respectively) tend to be lower than their diet (0.47 ± 0.02‰), δ(44/40)Cafaeces of -CaD (-0.27 ± 0.21‰) was significantly lower than their δ(44/40)Cadiet (0.37 ± 0.03‰), but also lower than δ(44/40)Cafaeces of Con and GIO. We suggest that the low δ(44/40)Cafaeces of -CaD might be due to the contribution of isotopically light Ca from gastrointestinal fluids during gut passage. Assuming that this endogenous Ca source is a common physiologic feature, a fractionation during Ca absorption is also required for explaining δ(44/40)Cafaeces of Con and GIO. The δ(44/40)Caurine of all groups are high (>2.0‰) reflecting preferential renal reabsorption of light Ca isotopes. In Göttingen minipigs we found a Ca isotope fractionation between blood and bones (Δ(44/40)Cablood-bone) of 0.68 ± 0.15‰.

Modulation of vitamin d status and dietary calcium affects bone mineral density and mineral metabolism in göttingen minipigs.

Calcium and vitamin D deficiency impairs bone health and may cause rickets in children and osteomalacia in adults. Large animal models are useful to study experimental osteopathies and associated metabolic changes. We intended to modulate vitamin D status and induce nutritional osteomalacia in minipigs. The control group (n = 9) was fed a semisynthetic reference diet with 6 g calcium and 6,500 IU vitamin D3/kg and the experimental group (n = 10) the same diet but with only 2 g calcium/kg and without vitamin D. After 15 months, the deficient animals were in negative calcium balance, having lost bone mineral density significantly (means ± SEM) with -51.2 ± 14.7 mg/cm(3) in contrast to controls (-2.3 ± 11.8 mg/cm(3)), whose calcium balance remained positive. Their osteoid surface was significantly higher, typical of osteomalacia. Their plasma 25(OH)D dropped significantly from 60.1 ± 11.4 nmol/L to 15.3 ± 3.4 nmol/L within 10 months, whereas that of the control group on the reference diet rose. Urinary phosphorus excretion and plasma 1,25-dihydroxyvitamin D concentrations were significantly higher and final plasma calcium significantly lower than in controls. We conclude that the minipig is a promising large animal model to induce nutritional osteomalacia and to study the time course of hypovitaminosis D and associated functional effects.

The role of bone morphogenetic protein-5 (BMP-5) in human nephrosclerosis.

BACKGROUND: Bone morphogenetic protein-5 (BMP-5) has been shown to be essential for nephrogenesis. Its role in adult kidney and in patients with hypertensive nephrosclerosis is still unknown. METHODS: BMP-5 expression was evaluated by immunostaining and real-time PCR in tissue samples from normal and nephrosclerotic human kidneys. The impact of transforming growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α) and angiotensin-II (AT-II) on expression of BMP-5 and its receptors was quantified in proximal tubular cells (HK-2). Functional characteristics of BMP-5 were evaluated by testing its influence on TGF-ß-induced epithelial-to-mesenchymal transition (EMT), TNF-α-induced apoptosis of HK-2 cells and inflammatory cell infiltration. RESULTS: BMP-5 expression was localized in tubular epithelial cells and significantly decreased in nephrosclerotic kidneys. Stimulation of HK-2 cells with TGF-ß, TNF-α and AT-II resulted in a significant decreased expression of BMP-5 and its receptors. BMP-5 attenuated TGF-ß-induced EMT, TNF-α-induced apoptosis and migration of mononuclear cells. CONCLUSIONS: BMP-5 is expressed in the tubuli of adult kidneys. Its decreased expression in nephrosclerosis along with its regenerative capabilities in HK-2 cells may point to a protective role in hypertensive nephrosclerosis.

Bone morphogenetic protein (BMP)-7 expression is decreased in human hypertensive nephrosclerosis.

BACKGROUND: Bone Morphogenetic Protein (BMP)-7 is protective in different animal models of acute and chronic kidney disease. Its role in human kidneys, and in particular hypertensive nephrosclerosis, has thus far not been described. METHODS: BMP-7 mRNA was quantified using real-time PCR and localised by immunostaining in tissue samples from normal and nephrosclerotic human kidneys. The impact of angiotensin (AT)-II and the AT-II receptor antagonist telmisartan on BMP-7 mRNA levels and phosphorylated Smad 1/5/8 (pSmad 1/5/8) expression was quantified in proximal tubular cells (HK-2). Functional characteristics of BMP-7 were evaluated by testing its influence on TGF-ß induced epithelial-to-mesenchymal transition (EMT), expression of TGF-ß receptor type I (TGF-ßRI) and phosphorylated Smad 2 (pSmad 2) as well as on TNF-α induced apoptosis of proximal tubular cells. RESULTS: BMP-7 was predominantly found in the epithelia of the distal tubule and the collecting duct and was less abundant in proximal tubular cells. In sclerotic kidneys, BMP-7 was significantly decreased as demonstrated by real-time PCR and immunostaining. AT-II stimulation in HK-2 cells led to a significant decrease of BMP-7 and pSmad 1/5/8, which was partially ameliorated upon co-incubation with telmisartan. Only high concentrations of BMP-7 (100 ng/ml) were able to reverse TNF-α-induced apoptosis and TGF-ß-induced EMT in human proximal tubule cells possibly due to a decreased expression of TGF-ßRI. In addition, BMP-7 was able to reverse TGF-ß-induced phosphorylation of Smad 2. CONCLUSIONS: The findings suggest a protective role for BMP-7 by counteracting the TGF-ß and TNF-α-induced negative effects. The reduced expression of BMP-7 in patients with hypertensive nephrosclerosis may imply loss of protection and regenerative potential necessary to counter the disease.

RNA interference for therapy in the vascular endothelium.

RNA interference (RNAi) has become an indispensable tool for loss of function analysis in today's basic cell biological research, and is currently being utilized for developing novel therapeutic methods. Systemic administration of synthetic small interfering RNA (siRNA) into the bloodstream is a feasible route for targeting the vascular endothelium or peripheral blood cells. The vascular endothelium can be considered an organ by itself, involved in many pathophysiological processes. Here, we aim to summarize current strategies of using RNAi for analysis of gene function in endothelial (in vitro loss-of-function analysis) and of exploiting RNAi for therapeutic purposes in order to suppress disrupted gene expression ("RNAi therapeutics"). With respect to the latter, we will summarize recent experimental concepts as well as ongoing therapeutic applications of RNAi mediated suppression of gene expression modulating angiogenic processes in cancer and retinopathies. We will further discuss the opportunities, prospects, challenges and potential fallbacks as well as the present strategies for the realization of "RNAi therapeutics" in combating vascular diseases.

Predictors of complications in therapeutic plasma exchange.

Plasma exchange (PE) is used for blood purification to modulate proteins involved in pathological processes. As the number of patients receiving PE treatment and the heterogeneity of the underlying diseases is steadily increasing, we evaluated the most frequent complications and analyzed causes leading to adverse reactions. 883 PE procedures in 113 patients between the years 2000 to 2006 were retrospectively analyzed with respect to complications. Additionally, underlying diseases and settings of PE procedure were analyzed to identify high-risk patients and respective PE settings. A total of 226 adverse reactions were recorded (25.6% of all PE procedures). Most complications were mild (n = 121, 13.7%) or moderate (n = 98, 11.0%). In seven cases (n = 7, 0.7%), severe, life-threatening adverse events were induced by PE either due to severe allergic reactions (n = 4, 0.5%) or to sepsis (n = 3, 0.3%). Patients with neurologic diseases had a significantly higher risk to develop complications compared to those with internal diseases (P = 0.013). This was due to a higher rate of PE associated adverse events (in particular hypotension) and complications associated with vascular access. Among patients from internal medicine those with hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) had the highest risk to develop complications. Patients with neurological diseases compared to those with medical conditions and patients with HUS/TTP compared to those with other diseases had a higher risk to develop complications. However, severe adverse events are rare. Thus, PE seems to be a safe and recommendable procedure.

Galacto-oligosaccharides are bifidogenic and safe at weaning: a double-blind randomized multicenter study.

OBJECTIVES: The primary objective of this study was to determine the bifidogenic effect of galacto-oligosaccharides (GOS) in a follow-on formula and the effects on other intestinal bacteria. Secondary objectives were the effects on stool characteristics, growth, and general well-being. PARTICIPANTS AND METHODS: In a multicenter, double-blind study, 159 healthy infants, formula-fed at enrollment (at 4-6 months), were randomized to an experimental follow-on formula supplemented with 5 g/L (GOS) (77 infants), or to a standard follow-on formula (control, 82 infants). Infants were evaluated at enrollment (study day 1 = sd1), after 6 weeks (study day 2 = sd2), and after an additional 12 weeks (study day 3 = sd3). At each study day, a fresh stool sample for the bacterial counts was collected, and the growth parameters were measured. At sd2, urinary specimens were collected for the evaluation of urinary osmolarity. RESULTS: At sd2 and sd3, the GOS group had a higher median number (colony-forming units per gram of stool) of bifidobacteria than did the control group (sd2 GOS 9.2 x 10(9) vs control 4.4 x 10(9), P = 0.012); (sd3 GOS 7.2 x 10(9) vs control 2.4 x 10(9), P = 0.027). Other bacteria did not show any significant differences between the 2 groups at all study days. The GOS produced softer stools but had no effect on stool frequency. The urinary osmolarity (mOsm/L) at sd2 was comparable in both groups. Supplementation had no influence on the incidence of gastrointestinal side effects or on the growth of the infants. CONCLUSIONS: These data indicate that the addition of GOS (5 g/L) to a follow-on formula positively influences the bifidobacteria flora and the stool consistency in infants during the supplementation period at weaning. No local or systemic side effects were recorded.

Short-term effect of bedtime consumption of fermented milk supplemented with calcium, inulin-type fructans and caseinphosphopeptides on bone metabolism in healthy, postmenopausal women.

BACKGROUND: Milk products are good sources of calcium and their consumption may reduce bone resorption and thus contribute to prevent bone loss. AIM OF THE STUDY: We tested the hypothesis that bedtime consumption of fermented milk supplemented with calcium inhibits the nocturnally enhanced bone resorption more markedly than fermented milk alone, and postulated that this effect was most pronounced when calcium absorption enhancers were added. METHODS: In a controlled, parallel, double-blind intervention study over 2 weeks we investigated the short-term effects of two fermented milks supplemented with calcium from milk minerals (f-milk + Ca, n = 28) or calcium from milk minerals, inulin-type fructans and caseinphosphopeptides (f-milk + Ca + ITF + CPP; n = 29) on calcium and bone metabolism in healthy, postmenopausal women, and compared them with the effect of a fermented control milk without supplements (f-milk, n = 28). At bedtime 175 ml/d of either test milk was consumed. Fasting blood samples and 48 h-urine were collected at baseline and at the end of the intervention. Urine was divided into a pooled daytime and nighttime fraction. Multifactorial ANOVA was performed. RESULTS: Fermented milk independent of a supplement (n = 85) reduced the nocturnal excretion of deoxypyridinoline, a marker of bone resorption, from 11.73 +/- 0.54 before to 9.57 +/- 0.54 micromol/mol creatinine at the end of the intervention (P = 0.005). No effect was seen in the daytime fraction. Differences between the three milks (n = 28 resp. 29) were not significant. Fermented milk reduced bone alkaline phosphatase, a marker of bone formation, from 25.03 +/- 2.08 to 18.96 +/- 2.08 U/l, with no difference between these groups either. Fermented milk increased the nocturnal but not daytime urinary excretion of calcium and phosphorus. The effects on calcium and phosphorus excretion were mainly due to the group supplemented with Ca + ITF + CPP. CONCLUSION: Bedtime consumption of fermented milk reduced the nocturnal bone resorption by decelerating its turnover. Supplemented calcium from milk mineral had no additional effect unless the absorption enhancers ITF + CPP were added. A stimulated intestinal calcium absorption may be assumed, since urinary calcium excretion increased at a constant bone resorption.

Bioactive peptides and proteins from foods: indication for health effects.

Some dietary proteins cause specific effects going beyond nutrient supply. A number of proteins seem to act directly in the intestine, such as IGFs, lactoferrin and immunoglobulins. Many substances, however, are peptides encrypted in intact molecules and are released from their encrypted position by enzymes during gastrointestinal transit or by fermentation or ripening during food processing. Among food-derived bioactive proteins and peptides from plants and animals, those obtained from milk are known in particular. Numerous effects have been described after in vitro and animal trials for bioactive proteins and peptides, such as immunomodulating, antihypertensive, osteoprotective, antilipemic, opiate, antioxidative and antimicrobial. This article reviews the current knowledge of the existence of bioactive proteins and of in vitro bioactivity and the present evidence of health effects exerted by such substances or products containing bioactive compounds. For example, there is evidence for the antihypertensive effects of milk products fermented with Lactobacillus helveticus containing the tripeptides IPP and VPP, which inhibit angiotensin converting enzyme, and for osteoprotective effects by milk basic protein. There is less profound evidence on the immunomodulating effects of lactoferrin and postprandial triglyceride reduction by a hydrolysate of bovine hemoglobin.

Inulin and oligofructose and mineral metabolism: the evidence from animal trials.

Nondigestible oligosaccharides have been shown to increase the absorption of several minerals (calcium, magnesium, in some cases phosphorus) and trace elements (mainly copper, iron, zinc). Inulin-type fructans including oligofructose and fructooligosaccharides derived from sucrose by enzymatic transfructosylation are the best investigated food ingredients in this respect. The stimulation of absorption was more pronounced when the demand for calcium was high, i.e., in animals in the rapid growing stage and in animals with impaired calcium absorption because of either ovariectomy or gastrectomy. Even a small stimulation of calcium absorption increased the mineral accumulation in the skeleton because of its persisting effect over months. Inulin-type fructans stimulated mineral absorption and bone mineral accretion when combined with probiotic lactobacilli and in the presence of antibiotics. Direct comparison of different inulin-type fructans revealed a more pronounced effect by inulin or a mixture of long-chain inulin and oligofructose than by oligofructose alone. Mechanisms on how inulin-type fructans mediate this effect include acidification of the intestinal lumen by short-chain fatty acids increasing solubility of minerals in the gut, enlargement of the absorption surface, increased expression of calcium-binding proteins mainly in the large intestine, modulated expression of bone-relevant cytokines, suppression of bone resorption, increased bioavailability of phytoestrogens, and, via stimulation of beneficial commensal microorganisms, increase of calcium uptake by enterocytes. Under certain conditions, inulin-type fructans may improve mineral absorption by their impact on the amelioration of gut health including stabilization of the intestinal flora and reduction of inflammation. The abundance of reports indicate that inulin-type fructans are promising substances that could help to improve the supply with available calcium in human nutrition and by this contribute to bone health.

Prebiotics, probiotics, and synbiotics affect mineral absorption, bone mineral content, and bone structure.

Several studies in animals and humans have shown positive effects of nondigestible oligosaccharides (NDO) on mineral absorption and metabolism and bone composition and architecture. These include inulin, oligofructose, fructooligosaccharides, galactooligosaccharides, soybean oligosaccharide, and also resistant starches, sugar alcohols, and difructose anhydride. A positive outcome of dietary prebiotics is promoted by a high dietary calcium content up to a threshold level and an optimum amount and composition of supplemented prebiotics. There might be an optimum composition of fructooligosaccharides with different chain lengths (synergy products). The efficacy of dietary prebiotics depends on chronological age, physiological age, menopausal status, and calcium absorption capacity. There is evidence for an independent probiotic effect on facilitating mineral absorption. Synbiotics, i.e., a combination of probiotics and prebiotics, can induce additional effects. Whether a low content of habitual NDO would augment the effect of dietary prebiotics or synbiotics remains to be studied. The underlying mechanisms are manifold: increased solubility of minerals because of increased bacterial production of short-chain fatty acids, which is promoted by the greater supply of substrate; an enlargement of the absorption surface by promoting proliferation of enterocytes mediated by bacterial fermentation products, predominantly lactate and butyrate; increased expression of calcium-binding proteins; improvement of gut health; degradation of mineral complexing phytic acid; release of bone-modulating factors such as phytoestrogens from foods; stabilization of the intestinal flora and ecology, also in the presence of antibiotics; stabilization of the intestinal mucus; and impact of modulating growth factors such as polyamines. In conclusion, prebiotics are the most promising but also best investigated substances with respect to a bone-health-promoting potential, compared with probiotics and synbiotics. The results are more prominent in animal models, where more studies have been performed, than in human studies, where experimental conditions are more difficult to control.

Extracellular matrix changes in knee joint cartilage following bone-active drug treatment.

Certain drugs or treatments that are known to affect bone quality or integrity might have side effects on the extracellular matrix of articular cartilage. We investigated the effects of vitamin D and calcium deficiency, estrogen deficiency, and hypercortisolism alone or in combination with bisphosphonates or sodium fluoride in an animal model, viz., the Göttingen miniature pig (n=29). The articular cartilage from knee joints was analyzed for its content of glycosaminoglycans (GAGs, as macromolecules responsible for the elasticity of articular cartilage) by a spectrometric method with dimethylene blue chloride. In cryo- or paraffin sections, alkaline phosphatase (AP, as an enzyme indicating mineralization or reorganization of articular cartilage matrix) was localized by enzyme histochemistry, and positive cells were counted, whereas differently sulfated GAGs were stained histochemically. A significant decrease in GAG content was measured in ovariectomized and long-term glucocorticoid-treated animals compared with untreated animals. In the glucocorticoid/sodium fluoride group, GAGs were significantly diminished, and significantly fewer AP-positive chondrocytes were counted compared with the control. GAG content was slightly higher, and significantly more AP-positive chondrocytes were counted in short-term glucocorticoid-treated animals then in the control group. GAGs, as part of proteoglycans, are responsible for the water-storage capacity that gives articular cartilage its unique property of elasticity. Thus, ovariectomy and long-term glucocorticoid therapy, especially when combined with sodium fluoride, have detrimental effects on this tissue.

The role of vascular endothelial growth factor in glucocorticoid-induced bone loss: evaluation in a minipig model.

Vascular endothelial growth factor (VEGF) has been recently shown to play an important role during endochondral bone formation in hypertrophic cartilage remodeling, ossification, and angiogenesis. To our knowledge there are no previous studies investigating the role of VEGF in osteoporosis. We hypothesized that VEGF expression in bone would be reduced under glucocorticoid (GC) treatment and tested this in a minipig model. As part of a larger study, 17 primiparous sows (Göttingen minipig) were allocated to two experimental groups when they were 15 months old: a control group (n = 9) and a group receiving GC treatment for 15 months (n = 8). All animals were fed a semisynthetic diet until they were sacrificed. The GC group received prednisolone orally at a daily dose of 1 mg/kg body wt for 8 weeks and thereafter 0.5 mg/kg body wt. VEGF levels in lumbar vertebrae were measured by enzyme-linked immunosorbent assay (ELISA) and verified by Western blot analysis. VEGF and its receptors (VEGFR) were localized by immunohistochemistry. Expression of VEGF-mRNA was analyzed by reverse transcription-polymerase chain reaction. VEGF protein was quantified in supernatants of cultivated osteoblasts by ELISA. Spinal bone mineral density was assessed in vivo by quantitative computed tomography. Expression of cyclooxygenase-2 (COX-2) protein was investigated by immunohistochemistry. High VEGF concentrations were measured in normal lumbar vertebrae whereas VEGF concentrations were 60% lower (P < 0.0001) in GC-treated minipigs. VEGF levels were associated (r = 0.7) with rates of spinal trabecular bone loss, which differed significantly (P < 0.0013) between controls (-0.47 +/- 2.2% SEM) and GC-treated minipigs (-12.8 +/- 2.3% SEM). Osteoblasts were immunopositive for VEGF. VEGF receptors VEGFR-2 (KDR, flk-1) and VEGFR-1 (flt-1) could be immunostained on osteoclasts and osteoblasts. VEGF-mRNA and protein were detectable in the lumbar vertebrae of all animals. The expression of COX-2 protein was decreased in GC-treated animals. VEGF is produced in osteoblasts and its concentration is decreased in GC-treated animals as well as in osteoblasts exposed to GC. Since reductions in VEGF concentrations correlate with parallel measurement of bone mineral density in GC-treated minipigs we hypothesize that VEGF may be an important modulating factor for bone remodeling, specifically in GC-induced osteoporosis. GC inhibit COX-2 and hence prostaglandin E2 (PGE2) production. Since PGE2 is able to increase VEGF synthesis, this may be the link between GC and VEGF decrease.

Effect of oligofructose or dietary calcium on repeated calcium and phosphorus balances, bone mineralization and trabecular structure in ovariectomized rats*.

We investigated the effects of dietary oligofructose and Ca on bone structure in ovariectomized rats, using microradiography and histomorphometry. Ninety-six animals were allocated to seven experimental groups: G1, sham-operated; G2-G7, ovariectomized. Semi-purified diets containing 5 g Ca/kg (recommended content) without oligofructose (G1, G2) or with 25, 50 or 100 g oligofructose/kg (G3, G4, G5) or 10 g Ca/kg (high content) without oligofructose (G6) or with 50 g oligofructose/kg (G7) were fed for 16 weeks. At the recommended level of Ca, high oligofructose (G5) increased femur mineral levels in ovariectomized rats, while medium oligofructose did so at high Ca. Increasing Ca in the absence of oligofructose did not increase femur mineral content. Trabecular bone area (%) analysed in the tibia was 10.3 (sem 1.2) (G1), 7.7 (sem 0.6) (G2), 9.3 (sem 0.7) (G3), 9.4 (sem 1.0) (G4), 9.5 (sem 0.7) (G5), 10.2 (sem 0.8) (G6), and 12.6 (sem 0.8) (G7). At the recommended level of Ca, 25 g oligofructose/kg prevented loss of trabecular area due to increased trabecular thickness, while 50 or 100 g oligofructose/kg increased trabecular perimeter. At high Ca, oligofructose prevented loss of bone area due to increased trabecular number but similar thickness (G7 v. G6). When Ca was raised in the presence of oligofructose (G7), trabecular area and cortical thickness were highest, while loss of trabecular connectivity was lowest of all groups. At the same time, lumbar vertebra Ca was higher; 44.0 (sem 0.8) (G7) compared with 41.6 (sem 0.8) (G2), 41.4 (sem 0.7) (G4), and 40.5 (sem 1.0) mg (G6). We conclude that ovariectomy-induced loss of bone structure in the tibia was prevented but with different trabecular architecture, depending on whether dietary Ca was increased, oligofructose was incorporated, or both. Oligofructose was most effective when dietary Ca was high.

Inulin, oligofructose and mineral metabolism - experimental data and mechanism.

Numerous investigations performed in animal models in the past 10 years have shown repeatedly that non-digestible oligosaccharides (NDO), such as inulin, oligofructose or transgalacto-oligosaccharides (TOS), stimulate mineral absorption, mainly calcium and magnesium. Long-term beneficial effects on bone health have been indicated by accumulation of bone mineral content in growing rats or prevention of bone loss in ovariectomized rats. However, bone mineral content or density are not necessarily associated with bone quality. In recent studies both oligofructose and calcium prevented loss of trabecular bone area induced by oestrogen deficiency, this, however, occurred at different trabecular shapes. The effects of NDO on mineral metabolism may be based on the enhancement of passive and active mineral transport across the intestinal epithelium, mediated by an increase in certain metabolites of the intestinal flora and a reduction of pH. The possible impact of short-chain fatty acids, butyrate in particular, and of polyamines on the stimulation of mineral absorption capacity, and the interaction of oligofructose and antibiotics is discussed.

The effect of estrogens and dietary calcium deficiency on the extracellular matrix of articular cartilage in Göttingen miniature pigs.

Clinical observations have suggested that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis (OA). However, positive and negative associations between the incidence of OA and serum estrogen concentrations have been reported. In contrast to this, osteoporosis is regarded as a disease with a strong estrogen-dependent component. Moreover, there is an interaction between estrogen and calcium deficiency: calcium supplementation potentiates the effect of estrogen therapy. The present study was designed to investigate how estrogen deficiency affects the articular cartilage depending on calcium supply. The distribution of different types of glycosaminoglycans and collagens can be used as an indicator for extracellular matrix changes induced by estrogen deficiency. Different levels of dietary calcium were therefore fed to intact and ovariectomized Göttingen miniature pigs for one year before articular cartilage was harvested. The histochemical staining for heavy sulfated glycosaminoglycans in the extracellular matrix of ovariectomized miniature pigs, especially of those fed with a low calcium diet, was stronger in comparison to intact animals. In intact animals type II-collagen was immunodetected in all zones of unmineralized and mineralized articular cartilage, while immunostaining for this protein was negative to weak in the deep radiated fiber zone of ovariectomized minipigs. These results suggest that the synthesis of heavy sulfated glycosaminoglycans and immunohistochemically detectable type II-collagen is possibly influenced by estrogen deficiency. In conclusion, under estrogen deficiency, the extracellular matrix of articular cartilage underwent similar changes to those observed in physiologically aging cartilage where keratan sulfate is increased as a heavy sulfated glycosaminoglycan.