Diabetes-Associated Biobanking: More Topical Than Ever?

The increasing prevalence of diabetes and its accompanying long-term complications, as well as the associated economic burden, calls for a rapid clinical translation of biomedical research to better valid the physiological relevance of the findings from basic research. To meet this condition, the Collaborative Research Center (CRC) 1118 has established the first nationwide diabetes-specific Biomaterialbank (BMB) that permanently preserves solid and liquid specimen retro- and prospectively at the Institute of Pathology and Department of Endocrinology of the University Hospital Heidelberg. The main purpose of this BMB is to collect, preserve, characterize and provide human diabetic specimen to researchers investigating the role of reactive metabolites (RM) as cause of diabetic late complications. In this review we discuss the urgent need to support translational and clinical research projects by making use of diabetic solid and liquid specimen and provide an insight into the organization and general conditions of biobanking procedures which are pivotal to guaranteeing high-quality human biomaterial. In light of diabetes-tailored biobanking, we describe our newly initiated activities and introduce the diverse technology platforms that can be used for the investigation of promising molecular targets pertinent for diabetes. With this article we demonstrate that the preservation of rare specimen is also particularly relevant in the non-neoplastic field and contributes to basic investigation, promotes comprehensive scientific data and fortifies the sustainability for diabetes research. In addition, the increased understanding of how metabolic imbalance triggers diabetes onset and progression and favors diabetic late symptoms might hold some promise for future innovative diagnostic and/ or therapeutic applications, eventually adding to the improvement of patient care.

ELMO1 protects renal structure and ultrafiltration in kidney development and under diabetic conditions.

Engulfment and cell motility 1 (ELMO1) functions as a guanine exchange factor for Rac1 and was recently found to protect endothelial cells from apoptosis. Genome wide association studies suggest that polymorphisms within human elmo1 act as a potential contributing factor for the development of diabetic nephropathy. Yet, the function of ELMO1 with respect to the glomerulus and how this protein contributes to renal pathology was unknown. Thus, this study aimed to identify the role played by ELMO1 in renal development in zebrafish, under hyperglycaemic conditions, and in diabetic nephropathy patients. In zebrafish, hyperglycaemia did not alter renal ELMO1 expression. However, hyperglycaemia leads to pathophysiological and functional alterations within the pronephros, which could be rescued via ELMO1 overexpression. Zebrafish ELMO1 crispants exhibited a renal pathophysiology due to increased apoptosis which could be rescued by the inhibition of apoptosis. In human samples, immunohistochemical staining of ELMO1 in nondiabetic, diabetic and polycystic kidneys localized ELMO1 in glomerular podocytes and in the tubules. However, ELMO1 was not specifically or distinctly regulated under either one of the disease conditions. Collectively, these results highlight ELMO1 as an important factor for glomerular protection and renal cell survival via decreasing apoptosis, especially under diabetic conditions.

Fibrinogen scaffolds with immunomodulatory properties promote in vivo bone regeneration.

The hypothesis behind this work is that fibrinogen (Fg), classically considered a pro-inflammatory protein, can promote bone repair/regeneration. Injury and biomaterial implantation naturally lead to an inflammatory response, which should be under control, but not necessarily minimized. Herein, porous scaffolds entirely constituted of Fg (Fg-3D) were implanted in a femoral rat bone defect and investigated at two important time points, addressing the bone regenerative process and the local and systemic immune responses, both crucial to elucidate the mechanisms of tissue remodelling. Fg-3D led to early infiltration of granulation tissue (6 days post-implantation), followed by bone defect closure, including periosteum repair (8 weeks post-injury). In the acute inflammatory phase (6 days) local gene expression analysis revealed significant increases of pro-inflammatory cytokines IL-6 and IL-8, when compared with non-operated animals. This correlated with modified proportions of systemic immune cell populations, namely increased T cells and decreased B, NK and NKT lymphocytes and myeloid cell, including the Mac-1+ (CD18+/CD11b+) subpopulation. At 8 weeks, Fg-3D led to decreased plasma levels of IL-1ß and increased TGF-ß1. Thus, our data supports the hypothesis, establishing a link between bone repair induced by Fg-3D and the immune response. In this sense, Fg-3D scaffolds may be considered immunomodulatory biomaterials.

Differential Regulation of SOX9 Protein During Chondrogenesis of Induced Pluripotent Stem Cells Versus Mesenchymal Stromal Cells: A Shortcoming for Cartilage Formation.

Induced pluripotent stem cells (iPSCs) are an attractive cell source for cartilage regeneration, but current in vitro chondrogenic differentiation protocols yield insufficient results. In search for shortcomings of iPSC chondrogenesis, this study investigated whether SOX9 protein was adequately regulated during multiphase chondrogenic differentiation of two human iPSC lines in a comparable manner like during mesenchymal stromal cell (MSC) chondrogenesis. Upon generation of intermediate mesenchymal progenitor cells (iMPCs), SOX9 was induced and reached variable protein levels compared to MSCs. Along with an altered condensation behavior, iMPC cartilage formation was less robust compared to MSCs and better in the iMPC line with higher SOX9 protein levels. Despite efficient Smad-2/3 phosphorylation, TGF-ß-driven chondrogenic stimulation downregulated SOX9 protein in iMPCs rather than increasing levels like in MSCs. Chondrogenesis was further improved by cotreatment with TGF-ß + BMP-4, which appeared to shorten the duration of the SOX9 protein decline. However, this was insufficient to overcome heterogenic outcome and came at the expense of undesired hypertrophy. In iMPCs, but not MSCs, high levels of the SOX9-antagonizing hsa-miR-145 correlated with low SOX9 protein quantity. Thus, considerable iMPC heterogeneity with variable SOX9 protein levels, an altered condensation pattern, and low early SOX9 inducibility appeared as critical shortcomings of iPSC chondrogenesis. We suggest consistent quality of intermediate cell populations with high SOX9 protein induction as important indicators to obtain robust cartilage formation from iPSCs. The impact of this study is the identification of a SOX9 protein regulation opposite to MSC chondrogenesis that will now enable a selective adaptation of the currently limited protocols to the specific needs of iPSCs.

Stage-Specific miRs in Chondrocyte Maturation: Differentiation-Dependent and Hypertrophy-Related miR Clusters and the miR-181 Family.

Human mesenchymal stromal cells (hMSC) differentiating toward the chondrogenic lineage recapitulate successive phases of embryonic chondrocyte maturation developing from progenitor cells to hypertrophic chondrocytes. Osteoarthritic cartilage is characterized by an alteration in chondrocyte metabolism and upregulation of hypertrophic differentiation markers. A number of studies point toward a functional role for microRNAs (miRs) in controlling chondrocyte differentiation and development of osteoarthritis (OA). However, information on miRs that may regulate a specific phase of chondrocyte maturation, especially hypertrophy, is lacking. We here aimed to unravel miR profiles modulated during chondrogenesis of hMSC to obtain new differentiation markers and potential new targets relevant for differentiation outcome and OA development. hMSC were subjected to transforming growth factor-ß (TGF-ß)-driven chondrogenesis and miR profiles were determined by microarray analysis at distinct developmental time points. Expression of selected miRs was compared to cultures lacking chondrogenesis and to redifferentiated nonhypertrophic articular chondrocytes. Among 1349 probed miRs, 553 were expressed and 169 (31%) were significantly regulated during chondrogenesis. Hierarchical clustering identified specific miR expression patterns representative for MSC, prechondrocytes, chondroblasts, chondrocytes, and hypertrophic chondrocytes, respectively. Regulation of miR-181 family members allowed discrimination of successive differentiation stages. Levels of several miRs, including miR-23b, miR-140, miR-181, and miR-210 positively correlated with successful chondrocyte formation. Hypertrophic MSC-derived chondrocytes and nonhypertrophic articular chondrocytes showed differential expression of miR-181a, miR-210, and miR-31, but not miR-148a implicated in COL10A1-regulation. We conclude that the here identified stage-dependent miR clusters may have imperative functions during chondrocyte differentiation providing novel diagnostic tools and targets of potential relevance for OA development.

AMP-activated protein kinase is activated by non-steroidal anti-inflammatory drugs.

AMP-activated kinase (AMPK) is a cellular energy sensor, which is activated in stages of increased adenosine triphosphate (ATP) consumption. Its activation has been associated with a number of beneficial effects such as decrease of inflammatory processes and inhibition of disease progression of diabetes and obesity. A recent study suggested that salicylate, the active metabolite of the non-steroidal anti-inflammatory drug (NSAID) acetyl-salicylic acid (aspirin), is able to activate AMPK pharmacologically. This observation raised the question whether or not other NSAIDs might also act as AMPK activators and whether this action might contribute to their cyclooxygenase (COX)-independent anti-inflammatory properties. In this study, we investigated mouse and human neuronal cells and liver tissue of mice after treatment with various NSAIDs. Our results showed that the non-selective acidic NSAIDs ibuprofen and diclofenac induced AMPK activation similar to aspirin while the COX-2 selective drug etoricoxib and the non-opioid analgesic paracetamol, both drugs have no acidic structure, failed to activate AMPK. In conclusion, our results revealed that AMPK can be activated by specific non-steroidal anti-inflammatory drugs such as salicylic acid, ibuprofen or diclofenac possibly depending on the acidic structure of the drugs. AMPK might therefore contribute to their antinociceptive and anti-inflammatory properties.

TANK-binding kinase 1 (TBK1) modulates inflammatory hyperalgesia by regulating MAP kinases and NF-κB dependent genes.

BACKGROUND: TANK-binding kinase (TBK1) is a non-canonical IκB kinase (IKK) involved in the regulation of type I interferons and of NF-κB signal transduction. It is activated by viral infections and inflammatory mediators and has therefore been associated with viral diseases, obesity, and rheumatoid arthritis. Its role in pain has not been investigated so far. Due to the important roles of NF-κB, classical IκB Kinases and the IKK-related kinase, IKKε, in inflammatory nociception, we hypothesized that TBK1, which is suggested to form a complex with IKKε under certain conditions, might also alter the inflammatory nociceptive response. METHODS: We investigated TBK1 expression and regulation in "pain-relevant" tissues of C57BL/6 mice by immunofluorescence, quantitative PCR, and Western blot analysis. Furthermore, nociceptive responses and the underlying signal transduction pathways were assessed using TBK1(-/-) mice in two models of inflammatory nociception. RESULTS: Our data show that TBK1 is expressed and regulated in the spinal cord after peripheral nociceptive stimulation and that a deletion of TBK1 alleviated the inflammatory hyperalgesia in mice while motor function and acute nociception were not altered. TBK1-mediated effects are at least partially mediated by regulation of NF-κB dependent COX-2 induction but also by alteration of expression of c-fos via modulation of MAP kinases as shown in the spinal cord of mice and in cell culture experiments. CONCLUSION: We suggest that TBK1 exerts pronociceptive effects in inflammatory nociception which are due to both modulation of NF-κB dependent genes and regulation of MAPKs and c-fos. Inhibition of TBK1 might therefore constitute a novel effective tool for analgesic therapy.

LPS inhibits caspase 3-dependent apoptosis in RAW264.7 macrophages induced by the AMPK activator AICAR.

AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase 3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner. In conclusion, our results revealed that AMPK activation is not only associated with positive effects but might also contribute to risk factors by disturbing important features of macrophages. The fact that LPS is able to restore AMPK-associated apoptosis might indicate an important role of TLR4 agonists in preventing unfavorable cell death of immune cells.

Activation of the AMP-activated protein kinase reduces inflammatory nociception.

UNLABELLED: The activation of the adenosine monophosphate (AMP)-activated kinase (AMPK) has been associated with beneficial effects such as improvement of hyperglycemic states in diabetes as well as reduction of obesity and inflammatory processes. Recent studies provide evidence for a further role of AMPK in models of acute and neuropathic pain. In this study, we investigated the impact of AMPK on inflammatory nociception. Using 5-amino-1-ß-d-ribofuranosyl-imidazole-4-carboxamide (AICAR) and metformin as AMPK activators, we observed anti-inflammatory and antinociceptive effects in 2 models of inflammatory nociception. The effects were similar to those observed with the standard analgesic ibuprofen. The mechanism appears to be based on regulation of the AMPKα2 subunit of the kinase because AMPKα2 knockout mice showed increased nociceptive responses that could not be reversed by the AMPK activators. On the molecular level, antinociceptive effects are at least partially mediated by reduced activation of different MAP-kinases in the spinal cord and a subsequent decrease in pain-relevant induction of c-fos, which constitutes a reliable marker of elevated activity in spinal cord neurons following peripheral noxious stimulation. In summary, our results indicate that activation of AMPKα2 might represent a novel therapeutic option for the treatment of inflammation-associated pain, providing analgesia with fewer unwanted side effects. PERSPECTIVE: AMPK activation is associated with beneficial effects on diabetes and obesity. In addition, we have shown analgesic properties of pharmacologic AMPK activation in inflammatory nociception, indicating that AMPK might serve as a novel therapeutic target in pain with fewer unwanted side effects.

Novel findings in pain processing pathways: implications for miRNAs as future therapeutic targets.

miRNAs are small noncoding RNAs that are important players in development, as well as in a number of physiological and pathophysiological processes. Due to their regulatory role in protein expression, it has been assumed that they are associated with peripheral and central sensitization mechanisms in the nervous system after nociceptive insults. However, the study of miRNAs in pain has emerged only recently. First reports mostly focused on miRNA regulations in different pain states while studies examining the functional role of individual miRNAs are only now arising. In this review, the authors summarize the current knowledge and progress in miRNA research in pain and discuss their potential role as therapeutic antinociceptive targets.

Modulation of central nervous system-specific microRNA-124a alters the inflammatory response in the formalin test in mice.

microRNAs (miRNAs) are small noncoding RNAs that have been linked to a number of disease-related signal transduction pathways. Several studies indicate that they are also involved in nociception. It is not clear, however, which miRNAs are important and which genes are modulated by miRNA-associated mechanisms. This study focuses on the regulation and function of the central nervous system (CNS)-specific miRNA-124a in the spinal cord of mice in a formalin model of inflammatory nociception. miRNA-124a is constitutively expressed in the spinal cord of mice, particularly in neurons of the dorsal horn. Peripheral noxious stimulation with formalin led to significant down-regulation of its expression. Knock-down of miRNA-124a by intravenous administration of a specific miRNA-124a inhibitor further increased the nociceptive behavior associated with an upregulation of the pain-relevant miRNA-124a target MeCP2 and proinflammatory marker genes. In contrast, administration of a miRNA-124a mimic counteracted these effects and decreased nociception by down-regulation of the target gene. In conclusion, our results indicate that miRNA-124a is involved in inflammatory nociception by regulation of relevant target proteins and might therefore constitute a novel target for anti-inflammatory therapy.

Altered gene expression in the prefrontal cortex of young rats induced by the ADHD drug atomoxetine.

Atomoxetine (ATX), a selective norepinephrine reuptake inhibitor, is a non-stimulant approved for the treatment of attention deficit/hyperactivity disorder (ADHD). Little is known about the molecular basis for its therapeutic effect. The objective of this animal study was to determine alterations in gene expression patterns in the prefrontal cortex after long-term administration of atomoxetine. Rats were treated for 21 days during childhood and early adolescent stages of development with a once-daily oral application of 0.05 g/kg atomoxetine, which resulted in plasma levels similar to those described in children. A whole genome RNA-microarray of rat prefrontal cortical gene expression after administration of atomoxetine versus sterile water revealed an mRNA increase in 114 genes (≥2-fold) while 11 genes were down-regulated (≤0.5-fold). By applying quantitative real-time PCR (qRT-PCR) and Western Blot we confirmed a significant increase in the expression of GABA A receptor subunits as well as ubiquinol-cytochrome c reductase complex core protein 2 (Uqcrc2). SNAP-25 (synaptosomal-associated protein of 25 kDa), which is an ADHD candidate gene and an important vesicle protein involved in axonal growth, synaptic plasticity and regulation of neurotransmitter release was also significantly upregulated on RNA- and protein level after atomoxetine treatment. In summary, we could show that long-term treatment with the ADHD drug atomoxetine induces the regulation of several genes in the prefrontal cortex of young rats. Especially the increased expression of SNAP-25 and GABA-A receptor subunits may indicate additional active therapeutic mechanisms for atomoxetine.

NADPH oxidase-4 maintains neuropathic pain after peripheral nerve injury.

Reactive oxygen species (ROS) contribute to sensitization of pain pathways during neuropathic pain, but little is known about the primary sources of ROS production and how ROS mediate pain sensitization. Here, we show that the NADPH oxidase isoform Nox4, a major ROS source in somatic cells, is expressed in a subset of nonpeptidergic nociceptors and myelinated dorsal root ganglia neurons. Mice lacking Nox4 demonstrated a substantially reduced late-phase neuropathic pain behavior after peripheral nerve injury. The loss of Nox4 markedly attenuated injury-induced ROS production and dysmyelination processes of peripheral nerves. Moreover, persisting neuropathic pain behavior was inhibited after tamoxifen-induced deletion of Nox4 in adult transgenic mice. Our results suggest that Nox4 essentially contributes to nociceptive processing in neuropathic pain states. Accordingly, inhibition of Nox4 may provide a novel therapeutic modality for the treatment of neuropathic pain.

The p21-activated kinase PAK 5 is involved in formalin-induced nociception through regulation of MAP-kinase signaling and formalin-specific receptors.

p21-activated kinases (PAKs) are involved in signal cascades relevant for nociceptive processing and neuropathic pain. Particularly, the recently described group B PAKs 4, 5 and 6 regulate MAP-kinases and the rearrangement of the actin cytoskeleton, both of which have been linked to pain processing. However, a specific role of these PAKs in nociception has not yet been demonstrated. We found PAK 4, 5 and 6 expression in pain-relevant tissues in peripheral and CNS. Since viable knock-out mice only exist for the PAK isoform 5, we further assessed the impact of this PAK on acute and chronic pain using different behavioral models in mice. PAK 5 knock-out mice showed normal acute nociception and did not differ from wild type mice in their neuropathic pain behavior. However, the nociceptive response in formalin-induced paw inflammation was significantly reduced in knock-out mice associated with inhibition of MAP-kinase activation and a decreased number of formalin-induced c-Fos positive neurons in the spinal cord. Furthermore, in isolated neurons, we found a significantly reduced calcium response after stimulation of TRPA1-channels in PAK 5(-/-)- compared to PAK 5(+/+)-cells. Our results indicate that PAK 5 is involved in formalin-induced inflammatory nociception through regulation of MAPK-induced c-Fos-activation and formalin-specific TRP-channels.

The protein kinase IKKε is a potential target for the treatment of inflammatory hyperalgesia.

Inhibitor-κB kinase ε (IKKε) was only recently identified as an enzyme with high homology to the classical I-κB kinase subunits, IKKα and IKKß. Despite this similarity, it is mainly discussed as a repressor of viral infections by modulating type I IFNs. However, in vitro studies also showed that IKKε plays a role in the regulation of NF-κB activity, but the distinct mechanisms of IKKε-mediated NF-κB activation are not clear. Given the paramount role of NF-κB in inflammation, we investigated the regulation and function of IKKε in models of inflammatory hyperalgesia in mice. We found that IKKε was abundantly expressed in nociceptive neurons in the spinal cord and in dorsal root ganglia. IKKε mRNA and protein levels rapidly increased in spinal cord and dorsal root ganglia during hind paw inflammation evoked by injection of zymosan or formalin. IKKε knockout mice showed normal nociceptive responses to acute heat or mechanical stimulation. However, in inflammatory pain models, IKKε-deficient mice exhibited a significantly reduced nociceptive behavior in comparison with wild type mice, indicating that IKKε contributed to the development of inflammatory hyperalgesia. Antinociceptive effects were associated with reduced activation of NF-κB and attenuated NF-κB-dependent induction of cyclooxygenase-2, inducible NO synthase, and metalloproteinase-9. In contrast, IRF-3, which is an important IKKε target in viral infections, was not regulated after inflammatory nociceptive stimulation. Therefore, we concluded that IKKε modulates inflammatory nociceptive sensitivity by activation of NF-κB-dependent gene transcription and may be useful as a therapeutic target in the treatment of inflammatory pain.