Hypoxia regulates RhoA and Wnt/ß-catenin signaling in a context-dependent way to control re-differentiation of chondrocytes.

Cartilage tissue is avascular and hypoxic which regulates chondrocyte phenotype via stabilization of HIFs. Here, we investigated the role of hypoxia and HIFs in regulation of Rho and canonical Wnt signaling in chondrocytes. Our data demonstrates that hypoxia controls the expression of RhoA in chondrocytes in a context-dependent manner on the culturing conditions. Within a 3D microenvironment, hypoxia suppresses RhoA on which hypoxia-driven expression of chondrogenic markers depends. Conversely, hypoxia leads to upregulation of RhoA in chondrocytes on 2D with a failure in re-expression of chondrogenic markers. Similarly to RhoA, hypoxic regulation of Wnt/ß-catenin signaling depends on the microenvironment. Hypoxia downregulates ß-catenin within 3D hydrogels whereas it causes a potent increase on 2D. Hypoxia-induced suppression of canonical Wnt signaling in 3D contributes to the promotion of chondrogenic phenotype as induction of Wnt signaling abrogates the hypoxic re-differentiation of chondrocytes. Inhibiting Wnt/ß-catenin signaling via stabilization of Axin2 leads to a synergistic enhancement of hypoxia-induced expression of chondrogenic markers. The effects of hypoxia on Rho and Wnt/ß-catenin signaling are HIF-dependent as stabilizing HIFs under normoxia revealed similar effects on chondrocytes. The study reveals important insights on hypoxic signaling of chondrocytes and how hypoxia regulates cellular mechanisms depending on the cellular microenvironment.

Mixing at the microscale: Power input in shaken microtiter plates.

Power input and local energy dissipation are crucial parameters for the engineering characterization of mixing and fluid dynamics at the microscale. Since hydrodynamic stress is solely dependent on the maximum power input, we adapted the clay/polymer method to obtain flock destruction kinetics in six-, 24-, and 96-well microtiter plates on orbital shakers. We also determined the specific power input using calorimetry and found that the power input is at the same order of magnitude for the six- and 96-well plates and the laboratory-scale stirred tank reactor, with 40 to 90 W/m3 (Re' = 180 to 440), 40 to 140 W/m3 (Re' = 320 to 640), and 30 to 50 W/m3 (Re = 4000 to 8500), respectively. All of these values are significantly below 450 to 2100 W/m3 determined for the pilot-scale reactor. The hydrodynamic stress differs significantly between the different formats of MTPs, as the 96-well plates showed very low shear stress on the shaker with a shaking amplitude of 3 mm. Thus, the transfer of mixing conditions from the microtiter plate to small-scale and pilot-scale reactors must be undertaken with care. Our findings, especially the power input determined by the calorimetric method, show that the hydrodynamic conditions in laboratory- and pilot-scale reactors cannot be reached.

Pumpkin seed extract: Cell growth inhibition of hyperplastic and cancer cells, independent of steroid hormone receptors.

Pumpkin seeds have been known in folk medicine as remedy for kidney, bladder and prostate disorders since centuries. Nevertheless, pumpkin research provides insufficient data to back up traditional beliefs of ethnomedical practice. The bioactivity of a hydro-ethanolic extract of pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, was investigated. As pumpkin seed extracts are standardized to cucurbitin, this compound was also tested. Transactivational activity was evaluated for human androgen receptor, estrogen receptor and progesterone receptor with in vitro yeast assays. Cell viability tests with prostate cancer cells, breast cancer cells, colorectal adenocarcinoma cells and a hyperplastic cell line from benign prostate hyperplasia tissue were performed. As model for non-hyperplastic cells, effects on cell viability were tested with a human dermal fibroblast cell line (HDF-5). No transactivational activity was found for human androgen receptor, estrogen receptor and progesterone receptor, for both, extract and cucurbitin. A cell growth inhibition of ~40-50% was observed for all cell lines, with the exception of HDF-5, which showed with ~20% much lower cell growth inhibition. Given the receptor status of some cell lines, a steroid-hormone receptor independent growth inhibiting effect can be assumed. The cell growth inhibition for fast growing cells together with the cell growth inhibition of prostate-, breast- and colon cancer cells corroborates the ethnomedical use of pumpkin seeds for a treatment of benign prostate hyperplasia. Moreover, due to the lack of androgenic activity, pumpkin seed applications can be regarded as safe for the prostate.

Red clover isoflavone metabolite bioavailability is decreased after fructooligosaccharide supplementation.

BACKGROUND: Red clover is an important source of isoflavones; which has been made commercially available as dietary supplements for the treatment of menopausal symptoms. Bioavailability and metabolism of these red clover isoflavones (RCI) have not been studied in detail. Fructooligosaccharides (FOS) stimulate the growth of intestinal bacteria and play an important role in the formation of certain isoflavone metabolites, such as equol and O-desmethylangolensin. OBJECTIVE: To determine the bioavailability of RCI metabolites and analyse whether FOS supplementation could influence their bioavailability. METHODS: Seventeen healthy adults were enrolled in the study carried out in two periods. In the first, compound bioavailability was determined after consumption of 80 mg of RCI (MF11RCE). In the second, a 6-day supplementation of 2×3000 mg/day of FOS was administered before isoflavone consumption. RESULTS: Biochanin A and formononetin were rapidly absorbed and both reached maximum concentrations at an average of 5-7h. Demethylation was a major reaction in the metabolic pathway. Daidzein serum level peaked after about 12.6h. Supplementation with FOS led to a significant decrease in the bioavailability of daidzein, dihydroformononetin, dihydrogenistein and dihydrodaidzein. An increase in equol production was also observed which did not reach statistical significance (p>0.05). CONCLUSION: This study is the first to provide detailed data on RCI bioavailability in humans and determine no influence of FOS yet a trend toward increased equol production. More research is warranted involving a greater sample size.

Eriosema laurentii De Wild (Leguminosae) methanol extract has estrogenic properties and prevents menopausal symptoms in ovariectomized Wistar rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Eriosema laurentii De Wild (Leguminosae) is a medicinal plant used in West and Central Africa for different diseases. In Cameroon, this plant is used as a treatment for infertility, and various gynecological and menopausal complaints. However, despite this use as a natural remedy, the biological activity of Eriosema laurentii has not been studied until now. AIM OF STUDY: In order to determine the potential use of this plant in gynecological conditions/disorders, we evaluated the estrogenic properties of a methanol extract of its aerial parts and its ability to prevent different menopausal health problems induced by bilateral oophorectomy. MATERIAL AND METHODS: Two approaches were used. In vitro, recombinant yeast systems were applied, featuring either the respective human receptors (ERα, AR, and PR) or into chromosome III integrated human aryl hydrocarbon receptor (AhR) and the respective reporter plasmid. In vivo, the investigation was carried out using the 3 days uterotrophic assay and 9 weeks oral treatment in ovariectomized rats. RESULTS: The results showed that the methanol extract of the aerial parts of Eriosema laurentii transactivated the estrogen receptor-α and displayed AhR agonistic activity but was neither androgenic nor progesteronic. In rats, the extract did not induce endometrium proliferation either in the 3-day or the 9-week treatment regimens, but induced vaginal stratification and cornification, prevented loss of femur bone mass, increased high density lipoprotein cholesterol (HDL-C), and reduced total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), TC/HDL-C ratio, LDL-C/HDL-C ratio and the atherogenic index of plasma (AIP). CONCLUSION: These results suggest that the methanol extract of the aerial parts of Eriosema laurentii does not seem to have an undesirable influence on the endometrium but might prevent vaginal dryness and bone mass loss and improve the lipid profile.

Red clover extract: a source for substances that activate peroxisome proliferator-activated receptor alpha and ameliorate the cytokine secretion profile of lipopolysaccharide-stimulated macrophages.

OBJECTIVE: Inflammation and hyperlipidemia or dyslipidemia contribute to an increased risk of atherosclerosis and cardiovascular disease. Cardiovascular disease represents one of the major causes of premature death worldwide. The activation of peroxisome proliferator-activated receptor (PPAR) alpha, a drug target for hyperlipidemia and dyslipidemia, leads to an improved blood lipid profile. In this study, we determined the putative anti-inflammatory and PPARalpha stimulatory activities of red clover, an alternative to the classic hormone therapy used currently to treat menopausal symptoms. METHODS: Lipopolysaccharide-induced macrophages were used as a model for anti-inflammatory activity, and a chimeric GAL4-PPARalpha system was used as a model for putative hypolipidemic activity. RESULTS: Red clover extract and the isoflavones genistein and biochanin A were moderate PPARalpha activators. Daidzein only slightly activated PPARalpha, but its metabolite 6-hydroxydaidzein exerted a much higher PPARalpha activity. Similarly, the metabolite 3'-hydroxygenistein achieved higher activation efficiency than its precursor, genistein, did. In lipopolysaccharide-stimulated macrophages, red clover extract and its compounds reduced the secretion of proinflammatory cytokines, interleukin-6 and tumor necrosis factor alpha, increased the secretion of the anti-inflammatory interleukin-10, and/or reduced the expression of nuclear factor-kappaB, inducible nitric oxide synthase, and/or cyclooxygenase 2. Tumor necrosis factor alpha production was most efficiently reduced by biochanin A and genistein. Interleukin-6 levels were most efficiently reduced by genistein and equol. CONCLUSIONS: Owing to its PPARalpha activation and modulation of the secreted cytokine profile, red clover extract is a putative candidate for preventing atherosclerosis and, thus, cardiovascular disease.