Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

Background aimsTransplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media.MethodsThe effects of prolonged hypoxic culture on growth and pro-angiogenic properties were investigated using human ASC cultured at 1%, 5% and 21% oxygen. The effect of trypsinization on the expression of pro-angiogenic genes was also determined.ResultsTrypsinization induced up-regulation of the vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) genes independent of oxygen concentration. The expression of VEGF and IGF-1 was up-regulated in ASC cultured at 1% oxygen for 13 days compared with 4 days. The VEGF concentration in ASC-conditioned media was higher after prolonged hypoxic culture compared with short-term culture, while the IGF-1 and chemokine (CXC motif) ligand 12 (CXCL12) concentrations were unchanged. The VEGF receptor blocker SU5416 abolished angiogenesis in a cultured rat aortic ring model. Media from cells exposed to hypoxia increased angiogenesis, an effect that was dependent on factors other than just the VEGF concentration in the added media.ConclusionsOptimization of the angiogenic potential of stem cell-based therapy in the treatment of vascular disease is important. We have demonstrated that prolonged hypoxic culture and trypsinization augment the therapeutic angiogenic potential of ASC.     

EGF receptor inhibitors increase ErbB3 mRNA and protein levels in breast cancer cells

The potential benefits of drugs directly targeting the ErbB receptors for cancer therapy have led to an extensive development within this field. However, the clinical effects of ErbB receptor-targeting drugs in cancer treatment are limited due to a high frequency of resistance. It has been reported that, when inhibiting the epidermal growth factor receptor (EGFR) with the tyrosine kinase inhibitor gefitinib, increased activation of ErbB3 via MET, or by re-localization of ErbB3 mediates cell survival. Here we show further evidence that members of the ErbB receptor family facilitate resistance to EGFR inhibitor treatment in ErbB2 overexpressing breast cancer cells. We found that gefitinib treatment increased ErbB3 expression, both at protein and mRNA levels. ErbB3 expression was upregulated not only by gefitinib but also by a panel of different EGFR inhibitors, suggesting that inhibition of EGFR in general affects ErbB3 expression. In addition, we found that gefitinib treatment increased ErbB2 expression levels while EGFR inhibitors decreased the activity of ErbB2. Concentrations of gefitinib that decreased phospho-ErbB2 reversely increased ErbB3 levels. We further examined changes induced by gefitinib treatment on mRNA levels of the most common genes known to be involved in breast cancer. As expected, we found that gefitinib downregulated genes whose functions were linked to cellular proliferation, such as Ki-67, topoisomerase II alpha and cyclins, and surprisingly downregulated gene expression of FAS which is involved in apoptotic signaling. Together, our data strongly suggest that resistance to EGFR inhibitors may result from the compensation of other family members and that combinations of anti-cancer drugs are required to increase the sensitivity of these treatments.     

Rapid exchange of metal between Zn(7)-metallothionein-3 and amyloid-β peptide promotes amyloid-related structural changes

Metal ions, especially Zn(2+) and Cu(2+), are implemented in the neuropathogenesis of Alzheimer's disease (AD) by modulating the aggregation of amyloid-β peptides (Aβ). Also, Cu(2+) may promote AD neurotoxicity through production of reactive oxygen species (ROS). Impaired metal ion homeostasis is most likely the underlying cause of aberrant metal-Aβ interaction. Thus, focusing on the body's natural protective mechanisms is an attractive therapeutic strategy for AD. The metalloprotein metallothionein-3 (MT-3) prevents Cu-Aβ-mediated cytotoxicity by a Zn-Cu exchange that terminates ROS production. Key questions about the metal exchange mechanisms remain unanswered, e.g., whether an Aβ-metal-MT-3 complex is formed. We studied the exchange of metal between Aβ and Zn(7)-MT-3 by a combination of spectroscopy (absorption, fluorescence, thioflavin T assay, and nuclear magnetic resonance) and transmission electron microscopy. We found that the metal exchange occurs via free Cu(2+) and that an Aβ-metal-MT-3 complex is not formed. This means that the metal exchange does not require specific recognition between Aβ and Zn(7)-MT-3. Also, we found that the metal exchange caused amyloid-related structural and morphological changes in the resulting Zn-Aβ aggregates. A detailed model of the metal exchange mechanism is presented. This model could potentially be important in developing therapeutics with metal-protein attenuating properties in AD.     

Dual PPARalpha /gamma activation provides enhanced improvement of insulin sensitivity and glycemic control in ZDF rats

Improvement of insulin sensitivity and lipid and glucose metabolism by coactivation of both nuclear peroxisome proliferator-activated receptor (PPAR)gamma and PPARalpha potentially provides beneficial effects over existing PPARgamma and alpha preferential drugs, respectively, in treatment of type 2 diabetes. We examined the effects of the dual PPARalpha/gamma agonist ragaglitazar on hyperglycemia and whole body insulin sensitivity in early and late diabetes stages in Zucker diabetic fatty (ZDF) rats and compared them with treatment with the PPARgamma preferential agonist rosiglitazone. Despite normalization of hyperglycemia and Hb A(1c) and reduction of plasma triglycerides by both compounds in both prevention and early intervention studies, ragaglitazar treatment resulted in overall reduced circulating insulin and improved insulin sensitivity to a greater extent than after treatment with rosiglitazone. In late-intervention therapy, ragaglitazar reduced Hb A(1c) by 2.3% compared with 1.1% by rosiglitazone. Improvement of insulin sensitivity caused by the dual PPARalpha/gamma agonist ragaglitazar seemed to have beneficial impact over that of the PPARgamma-preferential activator rosiglitazone on glycemic control in frankly diabetic ZDF rats.     

TRP channels: an overview

The TRP ("transient receptor potential") family of ion channels now comprises more than 30 cation channels, most of which are permeable for Ca2+, and some also for Mg2+. On the basis of sequence homology, the TRP family can be divided in seven main subfamilies: the TRPC ('Canonical') family, the TRPV ('Vanilloid') family, the TRPM ('Melastatin') family, the TRPP ('Polycystin') family, the TRPML ('Mucolipin') family, the TRPA ('Ankyrin') family, and the TRPN ('NOMPC') family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data on the roles of TRPs in a variety of tissues and species, including mammals, insects, and yeast. The present review summarizes the most pertinent recent evidence regarding the structural and functional properties of TRP channels, focusing on the regulation and physiology of mammalian TRPs.     

Sensory nerve inactivation by resiniferatoxin improves insulin sensitivity in male obese Zucker rats

Recent studies have suggested that sensory nerves may influence insulin secretion and action. The present study investigated the effects of resiniferatoxin (RTX) inactivation of sensory nerves (desensitization) on oral glucose tolerance, insulin secretion and whole body insulin sensitivity in the glucose intolerant, hyperinsulinemic, and insulin-resistant obese Zucker rat. After RTX treatment (0.05 mg/kg RTX sc given at ages 8, 10, and 12 wk), fasting plasma insulin was reduced (P < 0.0005), and oral glucose tolerance was improved (P < 0.005). Pancreas perfusion showed that baseline insulin secretion (7 mM glucose) was lower in RTX-treated rats (P = 0.01). Insulin secretory responsiveness to 20 mM glucose was enhanced in the perfused pancreas of RTX-treated rats (P < 0.005) but unaffected in stimulated, isolated pancreatic islets. At the peak of spontaneous insulin resistance in the obese Zucker rat, insulin sensitivity was substantially improved after RTX treatment, as evidenced by higher glucose infusion rates (GIR) required to maintain euglycemia during a hyperinsulinemic euglycemic (5 mU.kg(-1).min(-1)) clamp (GIR(60-120min): 5.97 +/- 0.62 vs. 11.65 +/- 0.83 mg.kg(-1).min(-1) in RTX-treated rats, P = 0.003). In conclusion, RTX treatment and, hence, sensory nerve desensitization of adult male obese Zucker rats improved oral glucose tolerance by enhancing insulin secretion, and, in particular, by improving insulin sensitivity.     

Isotope labeling and microautoradiography of active heterotrophic bacteria on the basis of assimilation of 14CO(2)

Most heterotrophic bacteria assimilate CO(2) in various carboxylation reactions during biosynthesis. In this study, assimilation of (14)CO(2) by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient (14)CO(2) during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of (14)C-labeled organic substrates. Experiments with E. coli showed that (14)CO(2) was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO(2)-MAR, was evaluated by targeting metabolic active filamentous bacteria, including "Candidatus Microthrix parvicella" in activated sludge. "Ca. Microthrix parvicella" was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [(14)C]oleic acid. However, the new HetCO(2)-MAR approach indicated that "Ca. Microthrix parvicella," did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO(2)(-), whereas the addition of O(2) or NO(3)(-) initiated growth, as indicated by detectable (14)CO(2) assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO(2)-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO(2)-MAR results were supported by stable isotope analysis of (13)C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of (13)CO(2). In conclusion, the novel HetCO(2)-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.     

Identity and ecophysiology of uncultured actinobacterial polyphosphate-accumulating organisms in full-scale enhanced biological phosphorus removal plants

Microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAO) in a full-scale enhanced biological phosphorus removal (EBPR) plant. The results showed that, in addition to uncultured Rhodocyclus-related PAO, two morphotypes hybridizing with gene probes for the gram-positive Actinobacteria were also actively involved in uptake of orthophosphate (Pi). Clone library analysis and further investigations by MAR-FISH using two new oligonucleotide probes revealed that both morphotypes, cocci in clusters of tetrads and short rods in clumps, were relatively closely related to the genus Tetrasphaera within the family Intrasporangiaceae of the Actinobacteria (93 to 98% similarity in their 16S rRNA genes). FISH analysis of the community biomass in the treatment plant investigated showed that the short rods (targeted by probe Actino-658) were the most abundant (12% of all Bacteria hybridizing with general bacterial probes), while the cocci in tetrads (targeted by probe Actino-221) made up 7%. Both morphotypes took up P(i) aerobically only if, in a previous anaerobic phase, they had taken up organic matter from wastewater or a mixture of amino acids. They could not take up short-chain fatty acids (e.g., acetate), glucose, or ethanol under anaerobic or aerobic conditions. The storage compound produced during the anaerobic period was not polyhydroxyalkanoates, as for Rhodocyclus-related PAO, and its identity is still unknown. Growth and uptake of Pi took place in the presence of oxygen and nitrate but not nitrite, indicating a lack of denitrifying ability. A survey of the occurrence of these actinobacterial PAO in 10 full-scale EBPR plants revealed that both morphotypes were widely present, and in several plants more abundant than the Rhodocyclus-related PAO, thus playing a very important role in the EBPR process.     

QTL mapping of vernalization response in perennial ryegrass (Lolium perenne L.) reveals co-location with an orthologue of wheat VRN1

The objective of this study was to map quantitative trait loci (QTL) for the vernalization response in perennial ryegrass (Lolium perenne L.). The mapping population consisted of 184 F₂ genotypes produced from a cross between one genotype of a synthetic perennial ryegrass variety Veyo and one genotype from the perennial ryegrass ecotype Falster. Veyo and Falster were chosen among four different populations because of their contrasting vernalization requirements. In total, five QTL for the vernalization response, measured as days to heading, were identified and mapped to linkage groups (LG) LG2, LG4, LG6 and LG7. Individually, these QTL explained between 5.4 and 28.0% of the total phenotypic variation. The overall contribution of these five QTL was 80% of the total phenotypic variation. A putative orthologue of Triticum monococcum VRN1 was amplified from genomic DNA from perennial ryegrass. PCR fragments covering the proximal part of the promoter and the 5 end of the orthologue were subsequently PCR-amplified from both parents of the mapping population and shown to possess 95% DNA sequence identity to VRN1. Several polymorphisms were identified between Veyo and Falster in this fragment of the putative VRN1 orthologue. A CAPS marker, vrn-1, was developed and found to co-segregate with a major QTL on LG4 for the vernalization response. This indicates that the CAPS marker vrn-1 could be located in an orthologous gene of the wheat VRN1.