[Treatment expectations of multimodal pain therapy inpatients]. / Behandlungserwartungen bei Patienten einer stationären multimodalen Schmerztherapie.

BACKGROUND: Apart from rehabilitation research, there have been no studies regarding the expectations of patients with chronic back pain in terms of inpatient multimodal pain therapy. The aim of this naturalistic longitudinal study is to explore treatment expectations, their fulfilment, and influence on the treatment success of inpatient multimodal pain therapy. METHODS: This study included 118 patients with chronic back pain who were physically examined and assessed for their psychological comorbidity. They were interviewed pre and post pain therapy. Treatment expectations were recorded via the questionnaire for assessing rehabilitational expectations and motivations (FREM-17), and further variables via the Pain Disability Index (PDI, german version) and the german Hospital Anxiety and Depression Scale (HADS-D). RESULTS: The results show that treatment expectations have an impact on therapy success or failure. In particular, patients' expectations of coping with illness and recovery could be met by inpatient multimodal pain therapy, whereas health and pension-related expectations remained unfulfilled. In addition to the treatment expectations, the therapy result was primarily determined by the patient's ability to perform before the therapy. CONCLUSIONS: From the clinical side, treatment expectations should be explored and checked for feasibility to avoid patient disappointment.

A FeFET with a novel MFMFIS gate stack: towards energy-efficient and ultrafast NVMs for neuromorphic computing.

The discovery of ferroelectricity in the fluorite structure based hafnium oxide (HfO2) material sparked major efforts for reviving the ferroelectric field effect transistor (FeFET) memory concept. A Novel metal-ferroelectric-metal-ferroelectric-insulator-semiconductor (MFMFIS) FeFET memory is reported based on dual ferroelectric integration as an MFM and MFIS in a single gate stack using Si-doped Hafnium oxide (HSO) ferroelectric (FE) material. The MFMFIS top and bottom electrode contacts, dual HSO based ferroelectric layers, and tailored MFM to MFIS area ratio (AR-TB) provide a flexible stack structure tuning for improving the FeFET performance. The AR-TB tuning shows a tradeoff between the MFM voltage increase and the weaker FET Si channel inversion, particularly notable in the drain saturation currentID(sat)when the AR-TB ratio decreases. Dual HSO ferroelectric layer integration enables a maximized memory window (MW) and dynamic control of its size by tuning the MFM to MFIS switching contribution through the AR-TB change. The stack structure control via the AR-TB tuning shows further merits in terms of a low voltage switching for a saturated MW size, an extremely linear at wide dynamic range of the current update, as well as high symmetry in the long term synaptic potentiation and depression. The MFMFIS stack reliability is reported in terms of the switching variability, temperature dependence, endurance, and retention. The MFMFIS concept is thoroughly discussed revealing profound insights on the optimal MFMFIS stack structure control for enhancing the FeFET memory performance.

Low-Magnetic-Field Regime of a Gate-Defined Constriction in High-Mobility Graphene.

We report on the evolution of the coherent electronic transport through a gate-defined constriction in a high-mobility graphene device from ballistic transport to quantum Hall regime upon increasing the magnetic field. At a low field, the conductance exhibits Fabry-Pérot resonances resulting from the npn cavities formed beneath the top-gated regions. Above a critical field B* corresponding to the cyclotron radius equal to the npn cavity length, Fabry-Pérot resonances vanish, and snake trajectories are guided through the constriction with a characteristic set of conductance oscillations. Increasing further the magnetic field allows us to probe the Landau level spectrum in the constriction and unveil distortions due to the combination of confinement and deconfinement of Landau levels in a saddle potential. These observations are confirmed by numerical calculations.

Overexpression of Cx43 in cells of the myocardial scar: Correction of post-infarct arrhythmias through heterotypic cell-cell coupling.

Ventricular tachycardia (VT) is the most common and potentially lethal complication following myocardial infarction (MI). Biological correction of the conduction inhomogeneity that underlies re-entry could be a major advance in infarction therapy. As minimal increases in conduction of infarcted tissue markedly influence VT susceptibility, we reasoned that enhanced propagation of the electrical signal between non-excitable cells within a resolving infarct might comprise a simple means to decrease post-infarction arrhythmia risk. We therefore tested lentivirus-mediated delivery of the gap-junction protein Connexin 43 (Cx43) into acute myocardial lesions. Cx43 was expressed in (myo)fibroblasts and CD45+ cells within the scar and provided prominent and long lasting arrhythmia protection in vivo. Optical mapping of Cx43 injected hearts revealed enhanced conduction velocity within the scar, indicating Cx43-mediated electrical coupling between myocytes and (myo)fibroblasts. Thus, Cx43 gene therapy, by direct in vivo transduction of non-cardiomyocytes, comprises a simple and clinically applicable biological therapy that markedly reduces post-infarction VT.

Improved heart repair upon myocardial infarction: Combination of magnetic nanoparticles and tailored magnets strongly increases engraftment of myocytes.

Cell replacement in the heart is considered a promising strategy for the treatment of post-infarct heart failure. Direct intramyocardial injection of cells proved to be the most effective application route, however, engraftment rates are very low (<5%) strongly hampering its efficacy. Herein we combine magnetic nanoparticle (MNP) loading of EGFP labeled embryonic cardiomyocytes (eCM) and embryonic stem cell-derived cardiomyocytes (ES-CM) with application of custom designed magnets to enhance their short and long-term engraftment. To optimize cellular MNP uptake and magnetic force within the infarct area, first numerical simulations and experiments were performed in vitro. All tested cell types could be loaded efficiently with SOMag5-MNP (200 pg/cell) without toxic side effects. Application of a 1.3 T magnet at 5 mm distance from the heart for 10 min enhanced engraftment of both eCM and ES-CM by approximately 7 fold at 2 weeks and 3.4 fold (eCM) at 8 weeks after treatment respectively and also strongly improved left ventricular function at all time points. As underlying mechanisms we found that application of the magnetic field prevented the initial dramatic loss of cells via the injection channel. In addition, grafted eCM displayed higher proliferation and lower apoptosis rates. Electron microscopy revealed better differentiation of engrafted eCM, formation of cell to cell contacts and more physiological matrix formation in magnet-treated grafts. These results were corroborated by gene expression data. Thus, combination of MNP-loaded cells and magnet-application strongly increases long-term engraftment of cells addressing a major shortcoming of cardiomyoplasty.

Tunable transmission of quantum Hall edge channels with full degeneracy lifting in split-gated graphene devices.

Charge carriers in the quantum Hall regime propagate via one-dimensional conducting channels that form along the edges of a two-dimensional electron gas. Controlling their transmission through a gate-tunable constriction, also called quantum point contact, is fundamental for many coherent transport experiments. However, in graphene, tailoring a constriction with electrostatic gates remains challenging due to the formation of p-n junctions below gate electrodes along which electron and hole edge channels co-propagate and mix, short circuiting the constriction. Here we show that this electron-hole mixing is drastically reduced in high-mobility graphene van der Waals heterostructures thanks to the full degeneracy lifting of the Landau levels, enabling quantum point contact operation with full channel pinch-off. We demonstrate gate-tunable selective transmission of integer and fractional quantum Hall edge channels through the quantum point contact. This gate control of edge channels opens the door to quantum Hall interferometry and electron quantum optics experiments in the integer and fractional quantum Hall regimes of graphene.

Abrogation of Gap Junctional Communication in ES Cells Results in a Disruption of Primitive Endoderm Formation in Embryoid Bodies.

Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co-expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here, we describe the generation of Cx43/Cx45-double deficient mouse ES cells to achieve almost complete breakdown of GJIC. Cre-loxP induced deletion of both, Cx43 and Cx45, results in a block of differentiation in embryoid bodies (EBs) without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC-incompetent ES cells fail to form primitive endoderm in EB cultures, representing the inductive key step of further differentiation events. Lentiviral overexpression of either Cx43 or Cx45 in Cx43/45 mutants rescued the observed phenotype, confirming the specificity and indicating a partially redundant function of both connexins. Upon differentiation GJIC-incompetent ES cells exhibit a strikingly altered subcellular localization pattern of the transcription factor NFATc3. Control EBs exhibit significantly more activated NFATc3 in cellular nuclei than mutant EBs suggesting that Cx-mediated communication is needed for synchronized NFAT activation to induce orchestrated primitive endoderm formation. Moreover, pharmacological inhibition of NFATc3 activation by Cyclosporin A, a well-described inhibitor of calcineurin, phenocopies the loss of GJIC in control cells. Stem Cells 2017;35:859-871.

Improvement of vascular function by magnetic nanoparticle-assisted circumferential gene transfer into the native endothelium.

Gene therapy is a promising approach for chronic disorders that require continuous treatment such as cardiovascular disease. Overexpression of vasoprotective genes has generated encouraging results in animal models, but not in clinical trials. One major problem in humans is the delivery of sufficient amounts of genetic vectors to the endothelium which is impeded by blood flow, whereas prolonged stop-flow conditions impose the risk of ischemia. In the current study we have therefore developed a strategy for the efficient circumferential lentiviral gene transfer in the native endothelium under constant flow conditions. For that purpose we perfused vessels that were exposed to specially designed magnetic fields with complexes of lentivirus and magnetic nanoparticles thereby enabling overexpression of therapeutic genes such as endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF). This treatment enhanced NO and VEGF production in the transduced endothelium and resulted in a reduction of vascular tone and increased angiogenesis. Thus, the combination of MNPs with magnetic fields is an innovative strategy for site-specific and efficient vascular gene therapy.

Vascular Repair by Circumferential Cell Therapy Using Magnetic Nanoparticles and Tailored Magnets.

Cardiovascular disease is often caused by endothelial cell (EC) dysfunction and atherosclerotic plaque formation at predilection sites. Also surgical procedures of plaque removal cause irreversible damage to the EC layer, inducing impairment of vascular function and restenosis. In the current study we have examined a potentially curative approach by radially symmetric re-endothelialization of vessels after their mechanical denudation. For this purpose a combination of nanotechnology with gene and cell therapy was applied to site-specifically re-endothelialize and restore vascular function. We have used complexes of lentiviral vectors and magnetic nanoparticles (MNPs) to overexpress the vasoprotective gene endothelial nitric oxide synthase (eNOS) in ECs. The MNP-loaded and eNOS-overexpressing cells were magnetic, and by magnetic fields they could be positioned at the vascular wall in a radially symmetric fashion even under flow conditions. We demonstrate that the treated vessels displayed enhanced eNOS expression and activity. Moreover, isometric force measurements revealed that EC replacement with eNOS-overexpressing cells restored endothelial function after vascular injury in eNOS(-/-) mice ex and in vivo. Thus, the combination of MNP-based gene and cell therapy with custom-made magnetic fields enables circumferential re-endothelialization of vessels and improvement of vascular function.

Anti-necrotic and cardioprotective effects of a cytosolic renin isoform under ischemia-related conditions.

UNLABELLED: In the heart, secretory renin promotes hypertrophy, apoptosis, necrosis, fibrosis, and cardiac failure through angiotensin generation from angiotensinogen. Thus, inhibitors of the renin-angiotensin system are among the most potent drugs in the treatment of cardiac failure. Renin transcripts have been identified encoding a renin isoform with unknown targets and unknown functions that are localized to the cytosol and mitochondria. We hypothesize that this isoform, in contrast to secretory renin, exerts cardioprotective effects in an angiotensin-independent manner. Cells overexpressing cytosolic renin were generated by transfection or obtained from CX(exon2-9)renin transgenic rats. Overexpression of cytosolic renin reduced the rate of necrosis in H9c2 cardiomyoblasts and in primary cardiomyocytes after glucose depletion. These effects were not mediated by angiotensin generation since an inhibitor of renin activity did not influence the in vitro effects. siRNA-mediated knockdown of endogenous cytosolic renin increased the rate of necrosis and aggravated the pro-necrotic effects of glucose depletion. Isolated perfused hearts obtained from transgenic rats overexpressing cytosolic renin exhibited a 50% reduction of infarct size after ischemia-reperfusion injury. Cytosolic renin is essential for survival, both under basal conditions and during glucose starvation. The protective effects are angiotensin-independent and contrary to the known actions of secretory renin. KEY MESSAGES: A cytosolic isoform of renin with unknown functions is expressed in the heart. Cytosolic renin diminishes ischemia induced damage to the heart. The protective effects of cytosolic renin contradict the known function of secretory renin. The effects of cytosolic renin are not mediated via angiotensin generation. Renin-binding protein is a potential target for cytosolic renin.

Direct lentivirus injection for fast and efficient gene transfer into brown and beige adipose tissue.

Brown adipose tissue is a special type of fat contributing to energy expenditure in human newborns and adults. Moreover, subcutaneous white adipose tissue has a high capacity to adapt an energy-consuming, brown-like/beige phenotype. Here, we developed an easy to handle and fast to accomplish method to efficiently transfer genes into brown and beige fat pads in vivo. Lentiviral vectors are directly injected into the target fat pad of anesthetized mice through a small incision using a modified, small needle connected to a microsyringe, which is well suited for infiltration of adipose tissues. Expression of the target gene can be detected in brown/beige fat one week after injection. The method can be applied within minutes to efficiently deliver transgenes into subcutaneous adipose tissues. Thus, this protocol allows for studying genes of interest in a timely manner in murine brown/beige fat and could potentially lead to new gene therapies for obesity.

Experimental TLR4 inhibition improves intestinal microcirculation in endotoxemic rats.

INTRODUCTION: Toll like receptor 4 (TLR4) represents a critical cellular link for endotoxin-induced pathology. The aim of this study was to evaluate the potential role of TLR4 inhibition on the intestinal microcirculation during experimental endotoxemia. MATERIALS AND METHODS: The intestinal microcirculation was studied by intravital microscopy in four groups of Lewis rats (n=10 per group): healthy controls (CON group), endotoxemic animals (15mg/kg lipopolysaccharide, LPS group), endotoxemic animals treated with a TLR4 antagonist (1mg/kg CRX-526, LPS+CRX526 group), and controls treated with CRX-526 (C-CRX526 group). Plasma samples were obtained for cytokine measurements at the end of the experiments. RESULTS: Endotoxemia significantly increased leukocyte adhesion in intestinal submucosal venules (e.g., V1 venules: CON 20.4±6.5n/mm(2), LPS 237.5±36.2n/mm(2), p<0.05) and reduced capillary perfusion of the intestinal wall (e.g., longitudinal muscular layer: CON 112.5±5.9cm/cm(2), LPS 71.3±11.0cm/cm(2), p<0.05) at 2h. TLR4 inhibition significantly reduced endotoxemia-associated leukocyte adhesion (V1 venules: 104.3±7.8n/mm(2)) and improved capillary perfusion (longitudinal muscular layer: 111.0±12.3cm/cm(2)). Cytokine release was not significantly affected. CONCLUSIONS: The TLR4 pathway may be a target in clinical Gram-negative sepsis since administration of the TLR4 antagonist CRX-526 improved intestinal microcirculation parameters in experimental endotoxemia.

Non-small cell lung cancer cell survival crucially depends on functional insulin receptors.

Insulin plays an important role as a growth factor and its contribution to tumor proliferation is intensely discussed. It acts via the cognate insulin receptor (IR) but can also activate the IGF1 receptor (IGF1R). Apart from increasing proliferation, insulin might have additional effects in lung cancer. Therefore, we investigated insulin action and effects of IR knockdown (KD) in three (NCI-H292, NCI-H226 and NCI-H460) independent non-small cell lung cancer (NSCLC) cell lines. All lung cancer lines studied were found to express IR, albeit with marked differences in the ratio of the two variants IR-A and IR-B. Insulin activated the classical signaling pathway with IR autophosphorylation and Akt phosphorylation. Moreover, activation of MAPK was observed in H292 cells, accompanied by enhanced proliferation. Lentiviral shRNA IR KD caused strong decrease in survival of all three lines, indicating that the effects of insulin in lung cancer go beyond enhancing proliferation. Unspecific effects were ruled out by employing further shRNAs and different insulin-responsive cells (human pre-adipocytes) for comparison. Caspase assays demonstrated that IR KD strongly induced apoptosis in these lung cancer cells, providing the physiological basis of the rapid cell loss. In search for the underlying mechanism, we analyzed alterations in the gene expression profile in response to IR KD. A strong induction of certain cytokines (e.g. IL20 and tumour necrosis factor) became obvious and it turned out that these cytokines trigger apoptosis in the NSCLC cells tested. This indicates a novel role of IR in tumor cell survival via suppression of pro-apoptotic cytokines.

Efficient and graded gene expression in glia and neurons of primary cerebellar cultures transduced by lentiviral vectors.

Lentiviral vectors are valuable tools to express genes of interest in living animals and stem cell cultures. The use of promoters in lentiviral constructs has been successfully used to drive gene expression in particular cell types including neurons and glia of the central nervous system in vivo. However, their suitability in cell culture is less well documented. In this paper, we describe lentiviral vectors containing neuronal promoters of the murine stem cell virus, of the synapsin 1 gene, the tubulin alpha 1 gene, and the calmodulin kinase II gene, and the glial promoter of the glial fibrillary acidic protein gene to drive reporter gene expression in primary dissociated cerebellar cell cultures and in slice cultures. While the glial promoter was highly specific for glia, the neuronal promoters were active in neurons and glia of dissociated cultures to a comparable extent. In slice cultures, neuronal and glial promoters demonstrated higher, but not absolute selectivity for particular cell types. In addition, the promoters allowed for an efficient and graded expression of genes in dissociated cultures. By using selected combinations of vectors, it was also possible to drive the expression of two genes in one cell type with high efficiency. A gene of interest in combination with a reporter gene can thus be expressed in a graded manner to reveal gene function in a rather short time and in a complex cellular environment.

Inhibition of serotonin transporter expression by C/EBPß in LPS-activated macrophage cells (HD11).

Serotonin (5-hydroxytryptamine; 5-HT) transporter (5-HTT) is involved in inflammation and the stress response. In this study, we examined the regulation of 5-HTT expression in macrophage HD11 cells in response to bacterial LPS. Long-term exposure of cells to LPS (6-18 h) produced a decrease in 5-HTT mRNA expression. Accordingly, reduced 5-HTT activity measured by 5-HT uptake was also observed in LPS-treated HD11 cells. Moreover, LPS treatment, as well as co-transfection with an expression vector encoding the chicken CCAAT/enhancer binding protein beta (C/EBPß), resulted in inhibition of 5-HTT promoter activity. Indeed, sequence analysis revealed several C/EBPß binding motifs in the upstream region of the 5-HTT gene, which specifically interacted with C/EBPß both in an in vitro band shift assay and in living HD11 cells. The C/EBPß binding was activated in cells treated with LPS. The role of C/EBPß in LPS inhibition of 5-HTT expression was further confirmed by small interfering RNA interference, which demonstrated that knockdown of endogenous C/EBPß attenuated the inhibition of 5-HTT expression in LPS-treated cells. Taken together, the results suggest that C/EBPß plays a critical role in regulating the 5-HTT gene in macrophages in response to pro-inflammatory stimuli.

Adenosine receptor activation improves microcirculation in experimental intestinal ischemia/reperfusion.

Gut ischemia and reperfusion (IR), e.g. in small bowel transplantation or during resuscitation, may result in severe impairment of the intestinal microcirculation. Potential sequelae are mucosal damage, loss of intestinal barrier function, bacterial translocation, systemic inflammation, multiple organ failure and death. We hypothesized a protective role for extracellular adenosine signalling in intestinal IR injury. Using intravital microscopy we investigated the effects of the adenosine receptor (AR) agonist NECA (5'-N-ethyl carboxamide adenosine) on leukocyte-endothelial interactions and capillary perfusion in the intestinal microcirculation following intestinal IR. Six groups of Lewis rats (n = 44) were studied: control, NECA (5'-N-ethyl carboxamide adenosine), IR (30 minutes of intestinal ischemia, 2 hours of reperfusion), IR + NECA, IR + NECA + MRS1754 (A(2B)AR antagonist), IR + NECA + DPCPX (A(1)AR antagonist). All substances were administered i.v. immediately after declamping of the superior mesenteric artery. Intravital microscopy was performed after 2 hours of reperfusion. Following IR we observed a significant increase of leukocyte adhesion in the intestinal submucosal venules and a reduced capillary perfusion within the muscular layers. NECA reduced leukocyte activation and improved capillary perfusion significantly. Administration of A(2B)AR antagonist completely reversed the NECA effect, whereas A(1)AR inhibition only partially abolished the action of NECA. The data support the hypothesis that adenosine signalling is involved in intestinal IR injury. A(2B)AR may be more important than A(1)AR because A(2B)AR inhibition by MRS1754 completely reversed the effect of the adenosine receptor agonist NECA.

A functional variant in the 5'-flanking region of the chicken serotonin transporter gene is associated with increased body weight and locomotor activity.

In this study, we identified a polymorphism in the 5'-flanking region of the chicken serotonin transporter (5-HTT) gene. Sequencing analysis revealed that in comparison with the wild-type variant (W), a deleted variant (D) is generated by deletion of four nucleotides (5'-AATT-3') and a single nucleotide change (A→T). Using a polyacrylamide gel electrophoresis system, we found that the 360-bp DNA fragment containing the W variant with the wild-type sequence 5'-AATTAATT-3' shows intrinsic DNA curvature while the 356-bp fragment containing the D variant lacking the four base pairs AATT is not curved. Quantitative real-time RT-PCR and ELISA demonstrated that the expression of 5-HTT in D/D chickens was higher than that in W/W and W/D chickens. In addition, transient transfection experiments with chloramphenicol acetyltransferase reporter gene constructs revealed increased 5-HTT promoter activity mediated by the D variant and a silencer activity of the W variant. Interestingly, females and males with D/D genotype showed significant greater increase in body weight from 6 weeks and 16 weeks of age, respectively, and higher body mass index. Moreover, we found that D/D chickens of both genders were physically more active than W/W and W/D chickens.

Analysis of gastrin-releasing peptide gene and gastrin-releasing peptide receptor gene in patients with agoraphobia.

A gastrin-releasing peptide receptor (GRPR) knock-out mouse model provided evidence that the gastrin-releasing peptide (GRP) and its neural circuitry operate as a negative feedback-loop regulating fear, suggesting a novel candidate mechanism contributing to individual differences in fear-conditioning and associated psychiatric disorders such as agoraphobia with/without panic disorder. Studies in humans, however, provided inconclusive evidence on the association of GRP and GRPR variations in agoraphobia with/without panic disorder. Based on these findings, we investigated whether GRP and GRPR variants are associated with agoraphobia. Mental disorders were assessed via the Munich-Composite International Diagnostic Interview (M-CIDI) in 95 patients with agoraphobia with/without panic disorder and 119 controls without any mental disorders. A complete sequence analysis of GRP and GRPR was performed in all participants. We found no association of 16 GRP and 7 GRPR variants with agoraphobia with/without panic disorder.

Distinct CD11b+-monocyte subsets accelerate endothelial cell recovery after acute and chronic endothelial cell damage.

BACKGROUND: Endothelial cell recovery requires replenishment of primary cells from the endothelial lineage. However, recent evidence suggests that cells of the innate immune system enhance endothelial regeneration. METHODS AND RESULTS: Focusing on mature CD11b+-monocytes, we analyzed the fate and the effect of transfused CD11b+-monocytes after endothelial injury in vivo. CD11b-diphtheria-toxin-receptor-mice--a mouse model in which administration of diphtheria toxin selectively eliminates endogenous monocytes and macrophages--were treated with WT-derived CD11b+-monocytes from age-matched mice. CD11b+-monocytes improved endothelium-dependent vasoreactivity after 7 days while transfusion of WT-derived CD11b--cells had no beneficial effect on endothelial function. In ApoE-/--CD11b-DTR-mice with a hypercholesterolemia-induced chronic endothelial injury transfusion of WT-derived CD11b+-monocytes stimulated by interferon-γ (IFNγ) decreased endothelial function, whereas interleukin-4-stimulated (IL4) monocytes had no detectable effect on vascular function. Bioluminescent imaging revealed restriction of transfused CD11b+-monocytes to the endothelial injury site in CD11b-DTR-mice depleted of endogenous monocytes. In vitro co-culture experiments revealed significantly enhanced regeneration properties of human endothelial outgrowth cells (EOCs) when cultured with preconditioned-media (PCM) or monocytes of IL4-stimulated-subsets compared to the effects of IFNγ-stimulated monocytes. CONCLUSION: CD11b+-monocytes play an important role in endothelial cell recovery after endothelial injury by homing to the site of vascular injury, enhancing reendothelialization and improving endothelial function. In vitro experiments suggest that IL4-stimulated monocytes enhance EOC regeneration properties most likely by paracrine induction of proliferation and cellular promotion of differentiation. These results underline novel insights in the biology of endothelial regeneration and provide additional information for the treatment of vascular dysfunction.

Decoding signaling and function of the orphan G protein-coupled receptor GPR17 with a small-molecule agonist.

Replacement of the lost myelin sheath is a therapeutic goal for treating demyelinating diseases of the central nervous system (CNS), such as multiple sclerosis (MS). The G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) GPR17, which is phylogenetically closely related to receptors of the "purinergic cluster," has emerged as a modulator of CNS myelination. However, whether GPR17-mediated signaling positively or negatively regulates this critical process is unresolved. We identified a small-molecule agonist, MDL29,951, that selectively activated GPR17 even in a complex environment of endogenous purinergic receptors in primary oligodendrocytes. MDL29,951-stimulated GPR17 engaged the entire set of intracellular adaptor proteins for GPCRs: G proteins of the Gα(i), Gα(s), and Gα(q) subfamily, as well as ß-arrestins. This was visualized as alterations in the concentrations of cyclic adenosine monophosphate and inositol phosphate, increased Ca²âº flux, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), as well as multifeatured cell activation recorded with label-free dynamic mass redistribution and impedance biosensors. MDL29,951 inhibited the maturation of primary oligodendrocytes from heterozygous but not GPR17 knockout mice in culture, as well as in cerebellar slices from 4-day-old wild-type mice. Because GPCRs are attractive targets for therapeutic intervention, inhibiting GPR17 emerges as therapeutic strategy to relieve the oligodendrocyte maturation block and promote myelin repair in MS.