FGF2 Translationally Induced by Hypoxia Is Involved in Negative and Positive Feedback Loops with HIF-1α

BACKGROUND: Fibroblast growth factor 2 (FGF2) is a major angiogenic factor involved in angiogenesis and arteriogenesis, however the regulation of its expression during these processes is poorly documented. FGF2 mRNA contains an internal ribosome entry site (IRES), a translational regulator expected to allow mRNA expression during cellular stress. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we have developed a skin ischemia model in transgenic mice expressing a reporter transgene under the control of the FGF2 IRES. The results reveal that FGF2 is induced at the protein level during ischemia, concomitant with HIF-1alpha induction and a decrease in FGF2 mRNA. In addition, the FGF2 IRES is strongly activated under these ischemic conditions associated with hypoxia, whereas cap-dependent translation is repressed by 4E-BP hypophosphorylation. We also show that up-regulation of FGF2 protein expression in response to hypoxia correlates with the increase of FGF2 IRES activity in vitro, in human retinoblasts 911. The use of siRNAs targeting HIF or FGF2 indicates that FGF2 and HIF-1alpha reciprocally regulate their expression/accumulation, by a negative feedback loop in early hypoxia, followed by a positive feedback loop in late hypoxia. CONCLUSION/SIGNIFICANCE: FGF2 expression is up-regulated in vivo and in vitro in response to hypoxia. Strikingly, this up-regulation is not transcriptional. It seems to occur by an IRES-dependent mechanism, revealing new mechanistic aspects of the hypoxic response. In addition, our data show that FGF2 interacts with HIF-1alpha in a unique crosstalk, with distinct stages in early and late hypoxia. These data reveal the physiological importance of IRES-dependent translation during hypoxic stress and underline the complexity of the cellular response to hypoxia, suggesting a novel role of FGF2 in the regulation of HIF-1alpha during the induction of angiogenesis

The impact of the Fungus-Host-Microbiota interplay upon Candida albicans infections: current knowledge and new perspectives

This is the final version. Available on open access from Oxford University Press via the DOI in this recordCandida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients

The use of mesenchymal stem cells for cartilage repair and regeneration: a systematic review.

BACKGROUND: The management of articular cartilage defects presents many clinical challenges due to its avascular, aneural and alymphatic nature. Bone marrow stimulation techniques, such as microfracture, are the most frequently used method in clinical practice however the resulting mixed fibrocartilage tissue which is inferior to native hyaline cartilage. Other methods have shown promise but are far from perfect. There is an unmet need and growing interest in regenerative medicine and tissue engineering to improve the outcome for patients requiring cartilage repair. Many published reviews on cartilage repair only list human clinical trials, underestimating the wealth of basic sciences and animal studies that are precursors to future research. We therefore set out to perform a systematic review of the literature to assess the translation of stem cell therapy to explore what research had been carried out at each of the stages of translation from bench-top (in vitro), animal (pre-clinical) and human studies (clinical) and assemble an evidence-based cascade for the responsible introduction of stem cell therapy for cartilage defects. This review was conducted in accordance to PRISMA guidelines using CINHAL, MEDLINE, EMBASE, Scopus and Web of Knowledge databases from 1st January 1900 to 30th June 2015. In total, there were 2880 studies identified of which 252 studies were included for analysis (100 articles for in vitro studies, 111 studies for animal studies; and 31 studies for human studies). There was a huge variance in cell source in pre-clinical studies both of terms of animal used, location of harvest (fat, marrow, blood or synovium) and allogeneicity. The use of scaffolds, growth factors, number of cell passages and number of cells used was hugely heterogeneous. SHORT CONCLUSIONS: This review offers a comprehensive assessment of the evidence behind the translation of basic science to the clinical practice of cartilage repair. It has revealed a lack of connectivity between the in vitro, pre-clinical and human data and a patchwork quilt of synergistic evidence. Drivers for progress in this space are largely driven by patient demand, surgeon inquisition and a regulatory framework that is learning at the same pace as new developments take place

Translation of remote control regenerative technologies for bone repair

The role of biomechanical stimuli, or mechanotransduction, in normal bone homeostasis and repair is understood to facilitate effective osteogenesis of mesenchymal stem cells (MSCs) in vitro. Mechanotransduction has been integrated into a multitude of in vitro bone tissue engineering strategies and provides an effective means of controlling cell behaviour towards therapeutic outcomes. However, the delivery of mechanical stimuli to exogenous MSC populations, post implantation, poses a significant translational hurdle. Here, we describe an innovative bio-magnetic strategy, MICA, where magnetic nanoparticles (MNPs) are used to remotely deliver mechanical stimuli to the mechano-receptor, TREK-1, resulting in activation and downstream signalling via an external magnetic array. In these studies, we have translated MICA to a pre-clinical ovine model of bone injury to evaluate functional bone repair. We describe the development of a magnetic array capable of in vivo MNP manipulation and subsequent osteogenesis at equivalent field strengths in vitro. We further demonstrate that the viability of MICA-activated MSCs in vivo is unaffected 48 hrs post implantation. We present evidence to support early accelerated repair and preliminary enhanced bone growth in MICA-activated defects within individuals compared to internal controls. The variability in donor responses to MICA-activation was evaluated in vitro revealing that donors with poor osteogenic potential were most improved by MICA-activation. Our results demonstrate a clear relationship between responders to MICA in vitro and in vivo. These unique experiments offer exciting clinical applications for cell-based therapies as a practical in vivo source of dynamic loading, in real-time, in the absence of pharmacological agents

Outcomes of cartilage repair techniques for chondral injury in the hip-a systematic review.

OBJECTIVE/PURPOSE: The aim of the study was to assess the options of treatment and their related outcomes for chondral injuries in the hip based on the available evidence whilst highlighting new and innovative techniques. METHODS: A systematic review of the literature from PubMed (Medline), EMBASE, Google Scholar, British Nursing Index (BNI), Cumulative Index to Nursing and Allied Health Literature (CINAHL) and Allied and Complementary Medicine Database (AMED) was undertaken from their inception to March 2017 using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Clinical outcome studies, prospective/retrospective case series and case reports that described the outcome of cartilage repair technique for the chondral injury in the hip were included. Studies on total hip replacement, animal studies, basic studies, trial protocols and review articles were excluded. RESULTS: The systematic review found 21 relevant papers with 596 hips. Over 80% of the included studies were published in or after 2010. Most studies were case series or case reports (18 studies, 85.7%). Arthroscopy was used in 11 studies (52.4%). The minimum follow-up period was six months. Mean age of the participants was 37.2 years; 93.5% of patients had cartilage injuries of the acetabulum and 6.5% of them had injuries of the femoral head. Amongst the 11 techniques described in the systematic review, autologous matrix-induced chondrogenesis, osteochondral autograft transplantation and microfracture were the three frequently reported techniques. CONCLUSION: Over ten different techniques are available for cartilage repair in the hip, and most of them have good short- to medium-term outcomes. However, there are no robust comparative studies to assess superiority of one technique over another, and further research is required in this arena

Lysine Deacetylase Substrate Selectivity: Distinct Interaction Surfaces Drive Positive and Negative Selection for Residues Following Acetyllysine

Lysine acetylation is a post-translational modification that is reversed by lysine deacetylases (KDACs). The goal of this work was to identify determinants of substrate specificity for KDACs, focusing on short-range interactions occurring with residues immediately following the acetyllysine. Using a fluorescence-based in vitro assay, we determined the activity for each enzyme with a limited panel of derivative substrate peptides, revealing a distinct reactivity profile for each enzyme. We mapped the interaction surface for KDAC6, KDAC8, and KDAC1 with the +1 and +2 substrate residues (with respect to acetyllysine) based on enzyme–substrate interaction pairs observed in molecular dynamics simulations. Characteristic residues in each KDAC interact preferentially with particular substrate residues and correlate with either enhanced or inhibited activity. Although nonpolar aromatic residues generally enhanced activity with all KDACs, the manner in which each enzyme interacted with these residues is distinct. Furthermore, each KDAC has distinctive interactions that correlate with lower activity, primarily ionic in nature. KDAC8 exhibited the most diverse and widest range of effects, while KDAC6 was sensitive only to the +1 position and KDAC1 selectivity was primarily driven by negative selection. The substrate preferences were validated for KDAC6 and KDAC8 using a set of peptides derived from known acetylated proteins. Overall, we determined how KDAC6, KDAC8, and KDAC1 achieve substrate specificity with residues following the acetyllysine. These new insights into KDAC specificity will be critical for identifying novel substrates of particular KDACs, designing KDAC-specific inhibitors, and demonstrate a general framework for understanding substrate specificity for other enzyme classes

Endogenous expression of inactive lysine deacetylases reveals deacetylation-dependent cellular mechanisms.

Acetylation of lysine residues is an important and common post-translational regulatory mechanism occurring on thousands of non-histone proteins. Lysine deacetylases (KDACs or HDACs) are a family of enzymes responsible for removing acetylation. To identify the biological mechanisms regulated by individual KDACs, we created HT1080 cell lines containing chromosomal point mutations, which endogenously express either KDAC6 or KDAC8 having single inactivated catalytic domain. Engineered HT1080 cells expressing inactive KDA6 or KDAC8 domains remained viable and exhibited enhanced acetylation on known substrate proteins. RNA-seq analysis revealed that many changes in gene expression were observed when KDACs were inactivated, and that these gene sets differed significantly from knockdown and knockout cell lines. Using GO ontology, we identified several critical biological processes associated specifically with catalytic activity and others attributable to non-catalytic interactions. Treatment of wild-type cells with KDAC-specific inhibitors Tubastatin A and PCI-34051 resulted in gene expression changes distinct from those of the engineered cell lines, validating this approach as a tool for evaluating in-cell inhibitor specificity and identifying off-target effects of KDAC inhibitors. Probing the functions of specific KDAC domains using these cell lines is not equivalent to doing so using previously existing methods and provides novel insight into the catalytic functions of individual KDACs by investigating the molecular and cellular changes upon genetic inactivation