Enhancement of bone regeneration with the accordion technique via HIF‐1α/VEGF activation in a rat distraction osteogenesis model

Axial micromotion of bone fragments promotes callus formation and bone healing during the process of distraction osteogenesis (DO). This study investigated the effects of the combined axial compression and distraction (accordion) technique on bone regeneration in rat DO model. Male Sprague–Dawley rats (n = 62) underwent right tibial transverse osteotomy and were randomly divided into four groups after lengthening: control (no manipulation) and three experimental groups assigned on the basis of the period of accordion manoeuvres in the consolidation phase (Groups 1, 2, and 3 with accordion technique applied at Weeks 1, 3, and 5, respectively). Animals were terminated at 1 week after each accordion phase (i.e., Weeks 2, 4, and 6). Callus formation was monitored by X‐ray radiography; new bone quality was evaluated by microcomputed tomography, histological analysis, and mechanical testing. Serum levels of hypoxia‐inducible factor (HIF)‐1α and vascular endothelial growth factor (VEGF) were measured. Callus formation after accordion manoeuvre at Week 3 (Group 2) increased significantly over time of consolidation. The microcomputed tomography and mechanical analysis revealed Group 2 had more newly formed bone and superior mechanical properties in contrast to the other groups at termination. Histomorphological and immunohistochemical analyses confirmed a greater degree of osteogenesis and angiogenesis corresponding to increased serum levels of HIF‐1α and VEGF in Group 2. The accordion technique was effective in promoting bone consolidation via activation of HIF‐1α/VEGF during DO. The accordion technique may be used in the middle phase of bone consolidation to promote bone formation in patients undergoing DO treatment.     

Effects of variation in the human α2A‐ and α2C‐adrenoceptor genes on cognitive tasks and pain perception

Background: The mechanisms underlying interindividual variability in pain perception and cognitive responses are undefined but highly heritable. α_2C‐ and α_2A‐adrenergic receptors regulate noradrenergic activity and are important mediators of pain perception and analgesia. We hypothesized that common genetic variants in these genes, particularly the ADRA2C 322–325 deletion variant, affect pain perception or cognitive responses. Methods: We studied 73 healthy subjects (37 Caucasians and 36 African–Americans) aged 25.4 ± 4.6 years. Pain response to a cold pressor test was measured using a 10 cm visual analog scale and again on the next day, after three infusions of the selective α₂‐agonist dexmedetomidine. Standardized cognitive tests were administered at baseline and after each infusion. The contribution of ADRA2C deletion genotype, dexmedetomidine concentration, and other covariates to pain perception and cognitive responses was determined using multiple linear regression models. Secondary analysis examined the effects of ADRA2A and other ADRA2C variants on pain perception. Results: ADRA2C Del homozygotes had higher pain scores in response to cold at baseline (6.3 ± 1.8 cm) and after dexmedetomidine (5.6 ± 2.2 cm) than insertion allele carriers (4.6 ± 2.1 cm [baseline] and 3.8 ± 1.9 cm [after dexmedetomidine]; adjusted P‐values = 0.019 and 0.004, respectively). Cognitive responses were unrelated to ADRA2C Ins/Del genotype. None of the other ADRA2A and ADRA2C variants was significantly related to cold pain sensitivity before dexmedetomidine; after dexmedetomidine, ADRA2A rs1800038 was marginally associated (P = 0.03). Conclusion: The common ADRA2C del322–325 variant affected pain perception before and after dexmedetomidine but did not affect other cognitive responses, suggesting that it contributes to interindividual variability in pain perception.     

Effect of chemical immobilization of SDF‐1α into muscle‐derived scaffolds on angiogenesis and muscle progenitor recruitment

The availability of three‐dimensional bioactive scaffolds with enhanced angiogenic capacity that have the capability to recruit tissue specific resident progenitors is of great importance for the regeneration of impaired skeletal muscle. Here, we have investigated whether introduction of chemoattractant factors to tissue specific extracellular matrix promotes cellular behaviour in vitro as well as muscle progenitor recruitment and vascularization in vivo. We developed an interconnective macroporous sponge from decellularized skeletal muscle with maintained biochemical traits of the intact muscle. SDF‐1α, a potent cell homing factor involved in muscle repair, was physically adsorbed or chemically immobilized in these muscle‐derived sponges. The immobilized sponges showed significantly higher SDF‐1α conjugation efficiency along with improved metabolism and infiltration of muscle‐derived stem cells in vitro, and thus generated uniform cellular constructs. In vivo, femoral muscle implantation in rats revealed a negligible immune response in all scaffold groups. We observed enhanced engraftment, neovascularization, and infiltration of CXCR4⁺ cells in the immobilized‐SDF‐1α sponge compared with nonimmobilized controls. Although Pax7⁺ cells identified adjacent to the immobilized‐SDF‐1α implantation site, other factors appear to be necessary for efficient penetration of Pax7⁺ cells into the sponge. These findings suggest that immobilization of cell homing factors via chemical mediators can result in recruitment of cells to the microenvironment with subsequent improvement in angiogenesis.     

Antibacterial and β‐Lactamase Inhibitory Activity of Monocyclic β‐Lactams

Due to the widespread emergence of resistant bacterial strains, an urgent need for the development of new antibacterial agents with novel modes of action has emerged. The discovery of naturally occurring monocyclic β‐lactams in the late 1970s, mainly active against aerobic Gram‐negative bacteria, has introduced a new approach in the design and development of novel antibacterial β‐lactam agents. The main goal was the derivatization of the azetidin‐2‐one core in order to improve their antibacterial potency, broaden their spectrum of activity, and enhance their β‐lactamase stability. In that respect, our review covers the updates in the field of monocyclic β‐lactam antibiotics during the last three decades, taking into account an extensive collection of references. An overview of the relationships between the structural features of these monocyclic β‐lactams, classified according to their N‐substituent, and the associated antibacterial or β‐lactamase inhibitory activities is provided. The different paragraphs disclose a number of well‐established classes of compounds, such as monobactams, monosulfactams, monocarbams, monophosphams, nocardicins, as well as other known representative classes. Moreover, this review draws attention to some less common but, nevertheless, possibly important types of monocyclic β‐lactams and concludes by highlighting the recent developments on siderophore‐conjugated classes of monocyclic β‐lactams.     

Enterococcus faecalis sir2‐like gene enhances aerobic metabolism of themselves and mitochondrial respiration of mammal cells to bring about improving metabolic syndrome through the PGC‐1α pathway

Recent studies showed that probiotics could improve metabolic syndrome, making the identification of factors affecting metabolic control more important than ever. The mammalian sirtuin protein family has received much attention for its regulatory role, especially in various mitochondrial ATP, glucose, and lipid metabolic pathways. However, compared with the mammalian sirtuin protein family, the function of prokaryotic sir2 protein is much less known. We studied the effects of probiotics sir2 protein on cell energy metabolize pathway, which showed that deletion of Enterococcus faecalis sir2 inhibited the aerobic oxidation of bacteria and increased the bacterial fermentation. The study of EF‐sir2 (sir2 protein of E. faecalis) role of molecular targets demonstrated that deacetylation of EF‐sir2 was via Rho upregulating in E. faecalis. When transfected into HEK293T cells, EF‐sir2 could significantly facilitate aerobic oxidation of glucose, enhance the respiration to generate more ATP, and cause upregulation of NRF1 target gene. Then, we found EF‐sir2 could increase activity of PGC‐1α by deacetylation and PGC‐1α inhibition decreased the expression of NRF1 target gene. Finally, we demonstrated that EF‐sir2 could significantly improve the metabolic index of mammalian cells through insulin resistanced model in vitro and metabolic syndrome rat model in vivo. Our results first revealed that prokaryotic sir2 genes affect the molecular mechanism of cellular metabolism and the regulatory of cell homeostasis in prokaryotic and mammalian cells, suggesting that EF‐sir2 has a positive regulatory effect on metabolic disturbance and may be used for the prevention and treatment of pathological processes related to metabolic syndrome.     

Physical activity,skeletal muscle β-adrenoceptor changes and oxidative metabolism in experimental chronic heart failure†

Summary— In chronic heart failure (CHF), changes in sympathetic nervous activity and skeletal muscle metabolism contribute to a limitation in the capacity for exercise. The aim of this study was to investigate the potential relationships between physical deconditioning, skeletal muscle β-adrenoceptor (β-AR) characteristics and muscle metabolic changes in rats with coronary ligation-induced experimental CHF. Muscle β-AR and norepinephrine levels were assessed in rats with CHF that had been treated with propranolol at 28 mg/kg/day and compared with rats with CHF that had not been treated and those that had undergone sham operations. The soleus muscle was investigated because of its predominantly oxidative fibre-type composition. Measurements of spontaneous locomotion activity were carried out using telemetry. After 85 days, muscle energetic phosphate levels were assessed using ³¹P-magnetic resonance spectroscopy. The phosphocreatine resynthesis rate was decreased in the untreated CHF rats (15 ± 3 vs 33 ± 5 mmol L^?1 min^?1 in the sham-operated rats, p < 0.05), but this had been partially reversed in the rats given propranolol (22 ± 3 mmol L^?1 min^?1, non-significant (NS) when compared with the sham-operated rats). Spontaneous activity did not differ among the three groups of animals. Soleus β-adrenoceptor density was decreased in rats with CHF (8.8 ± 3.0 fM/mg of protein vs 22.0 ± 7.0 fM/mg of protein in the sham-operated rats, p < 0.05) and normalized in the propranolol-treated rats (31.9 ± 7.0 fM/mg of protein, NS vs the sham-operated rats; p < 0.05 vs the untreated rats with CHF). Unchanged spontaneous activity in the rats with CHF suggests that physical deconditioning could not account for the muscle metabolic changes. Changes in skeletal muscle energy metabolism were accompanied by changes in β-AR density, occurring in typically oxidative β-AR-rich muscles, reversible after β-blocker therapy and therefore suggestive of β-AR downregulation.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

Retinoic acid improves recovery after nephrectomy and decreases renal TGF‐β1 expression. Gender‐related effects

End‐stage renal disease is a cause for death worldwide. Renal transplant is a therapeutic alternative, restricted by the scant number of donors. Function of the donor kidney is under risk of adverse circumstances such as fibrosis, where profibrotic effect of transforming growth factor beta 1 (TGF‐β1) plays a key role. Efforts to diminish risks of damage in the remnant kidney of the donor are required. Vitamin A represents one alternative. It has beneficial effects on some nephropathies, mainly those related to oxidative stress. It also participates in normal intrauterine renal development. We studied the effect of all‐trans retinoic acid (ATRA), active form of vitamin A, on postnephrectomy compensatory growth, in male or female rats. Compensatory growth and renal function were evaluated on four experimental groups: Control without treatment (CTL), ATRA‐treated intact rats (CTL + RA), nephrectomized rats (NFX), and ATRA‐treated nephrectomized rats (NFX + RA). We evaluated glomerular function (inulin clearance), tubular function (fractional excretions of sodium and potassium), and urinary flow. Renal mass was also estimated. In ATRA‐treated animals, compensatory growth was higher than in nephrectomized rats without treatment. Hyperfiltration after nephrectomy was less intense in ATRA‐treated female than in male rats. In tubular functions, effect of ATRA was more evident in female than in male rats. Glomerular expression of TGF‐β1 was lower in ATRA‐treated animals than in controls. ATRA reduced intensity and duration of compensatory changes after nephrectomy, improving recovery.     

Magnetic labeling of pancreatic β‐cells modulates the glucose‐ and insulin‐induced phosphorylation of ERK1/2 and AKT

This study was undertaken to investigate the effect of a magnetic resonance imaging (MRI) contrast agent, superparamagnetic iron oxide nanoparticle (SPIO), on signal transduction by glucose and insulin in pancreatic β‐cells. INS‐1 cells were labeled in culture medium containing clinically approved SPIO for 24 h. Labeled and unlabeled cells were stimulated with glucose (25 mM) or insulin (0.1–1 µM) for 12 h. The phosphorylation of extracellular signal‐regulated kinase1/2 (ERK1/2) and protein kinase B (AKT) and intracellular insulin protein levels were assessed by Western blotting. After labeling with increasing amounts of SPIO, cytotoxicity was not observed, yet the intracellular iron concentration increased in a dose‐dependent manner. SPIO labeling (200 µg Fe ml^?1) induced a significant increase in ERK1/2 and AKT phosphorylation (labeled vs unlabeled, p < 0.05), but significantly reduced the glucose‐stimulated phosphorylation of ERK1/2 and AKT and insulin‐stimulated phosphorylation of AKT (labeled vs unlabeled, p < 0.05). The level of intracellular insulin protein was found to be lower in labeled cells than unlabeled cells (labeled vs unlabeled, p < 0.05). This study demonstrates that SPIO labeling alters some fundamental functional variables, at least in INS‐1 cells, through modulation of the glucose‐ or insulin‐induced activation of ERK1/2 and AKT, which leads to insulin biosynthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

A role for adhesion and degranulation‐promoting adapter protein in collagen‐induced platelet activation mediated via integrin α2β1

Summary. Background: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α₂β₁. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates α_IIbβ₃ in platelets and α_Lβ₂ in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α₂β₁. Methods: Using ADAP^?/? mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP^?/? platelets. However, aggregation and signaling induced by collagen‐related peptide (CRP), a GPVI‐selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α₂β₁‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α₂β₁, was reduced in ADAP^?/? platelets. An impedance‐based label‐free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non‐fluorescent differential‐interference contrast microscopy, which revealed reduced filpodia formation in ADAP^?/? platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen‐binding integrin α₂β₁. In addition, we found that ADAP^?/? mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild‐type animals. This may reflect increased removal of platelets from the circulation.     

A new optical imaging probe targeting αVβ3 integrin in glioblastoma xenografts

α_Vβ₃ Integrins are a widely recognized target for in vivo molecular imaging of pathological conditions such as inflammation, cancer and rheumatoid arthritis. We have evaluated the sensitivity of a new, near‐infrared fluorescence (NIRF), RGD cyclic probe (DA364) in noninvasive detection of α_Vβ₃ integrin‐overexpressing tumors. DA364's binding affinity for α_Vβ₃ integrin was first evaluated in vitro. Human α_Vβ₃ integrin‐positive, U‐87 MG glioblastoma cells were then xenografted in nude mice, and DA364 was injected intravenously (i.v.) to evaluate its in vivo distribution, specificity and sensitivity in comparison with a commercially available probe. DA364 bound α_Vβ₃ integrin on U‐87 MG cells with high affinity and specificity, both in vitro and in vivo. This binding specificity was corroborated by the strong inhibition of its tumor uptake induced by nonfluorescent, cyclic‐RGD peptides. Ex vivo analysis showed that DA364 accumulated at the tumor site, whereas very low levels were detected in liver and spleen. In conclusion, DA364 allows sensitive and specific detection of transplantable glioblastoma by NIRF imaging, and is thus a promising candidate for the elaboration of imaging and therapeutic probes for α_Vβ₃ integrin‐overexpressing tumors. Copyright © 2011 John Wiley & Sons, Ltd.     

Resveratrol counteracts IL‐1β‐mediated impairment of extracellular matrix deposition in 3D articular chondrocyte constructs

When aiming at cell‐based therapies in osteoarthritis (OA), proinflammatory conditions mediated by cytokines such as IL‐1β need to be considered. In recent studies, the phytoalexin resveratrol (RSV) has exhibited potent anti‐inflammatory properties. However, long‐term effects on 3D cartilaginous constructs under inflammatory conditions with regard to tissue quality, especially extracellular matrix (ECM) composition, have remained unexplored. Therefore, we employed long‐term model cultures for cell‐based therapies in an in vitro OA environment and evaluated effects of RSV. Pellet constructs made from expanded porcine articular chondrocytes were cultured with either IL‐1β (1–10 ng/ml) or RSV (50 μM) alone, or a cotreatment with both agents. Treatments were applied for 14 days, either directly after pellet formation or after a preculture period of 7 days. Culture with IL‐1β (10 ng/ml) decreased pellet size and DNA amount and severely compromised glycosaminoglycan (GAG) and collagen content. Cotreatment with RSV distinctly counteracted the proinflammatory catabolism and led to partial rescue of the ECM composition in both culture systems, with especially strong effects on GAG. Marked MMP13 expression was detected in IL‐1β‐treated pellets, but none upon RSV cotreatment. Expression of collagen type I was increased upon IL‐1β treatment and still observed when adding RSV, whereas collagen type X, indicating hypertrophy, was detected exclusively in pellets treated with RSV alone. In conclusion, RSV can counteract IL‐1β‐mediated degradation and distinctly improve cartilaginous ECM deposition in 3D long‐term inflammatory cultures. Nevertheless, potential hypertrophic effects should be taken into account when considering RSV as cotreatment for articular cartilage repair techniques.