André Heinen

CXCR7 is an active component of SDF-1 signalling in astrocytes and Schwann cells

The alternative SDF-1 (stromal cell derived factor-1) receptor, CXCR7, has been suggested to act as either a scavenger of extracellular SDF-1 or a modulator of the primary SDF-1 receptor, CXCR4. CXCR7, however, also directly affects the function of various tumor-cell types. Here, we demonstrate that CXCR7 is an active component of SDF-1 signalling in astrocytes and Schwann cells. Cultured cortical astrocytes and peripheral nerve Schwann cells exhibit comparable total and cell-surface levels of expression of both SDF-1 receptors. Stimulation of astrocytes with SDF-1 resulted in the temporary activation of Erk1/2, Akt and PKCζ/λ, but not p38 and PKCα/β. Schwann cells showed SDF-1-induced activation of Erk1/2, Akt and p38, but not PKCα/β and PKCζ/λ. The respective signalling pattern remained fully inducible in astrocytes from CXCR4-deficient mice, but was abrogated following depletion of astrocytic CXCR7 by RNAi. In Schwann cells, RNAi-mediated depletion of either CXCR4 or CXCR7 silenced SDF-1 signalling. The findings of the astrocytic receptor-depletion experiments were reproduced by CXCR7 antagonist CCX754, but not by CXCR4 antagonist AMD3100, both of which abolished astrocytic SDF-1 signalling. Further underlining the functional importance of CXCR7 signalling in glial cells, we show that the mitogenic effects of SDF-1 on both glial cell types are impaired upon depleting CXCR7.

Effect of acute hyperglycaemia and diabetes mellitus with and without short-term insulin treatment on myocardial ischaemic late preconditioning in the rabbit heart in vivo.

Diabetes mellitus (DM) and the resulting hyperglycaemia may interfere with the cardioprotective effect of ischaemic late preconditioning (LPC). Therefore, we investigated the effect of acute hyperglycaemia (part 1) and the effect of alloxan-induced DM with or without short-term insulin treatment (part 2) on LPC. Rabbits, chronically instrumented with a coronary artery occluder, were subjected to 30xa0min coronary artery occlusion and 2xa0h reperfusion (I/R) and infarct size (IS) was assessed. In part 1, four groups were studied. Controls were not treated further. LPC induced by a 5-min period of myocardial ischaemia 24xa0h before I/R reduced IS from 42±14 (controls) to 22±8% of the area at risk. Hyperglycaemia (600xa0mg dl−1 by dextrose infusion, H600) before and during the 30xa0min ischaemia tended to increase IS (57±16%, P=0.14 vs. controls) and blocked cardioprotection by LPC (H600+LPC, 59±19%, P=1.0 vs. H600, P=0.0003 vs. LPC). In part 2, LPC reduced infarct size from 43±13% (control) to 23±10% (P=0.003). In diabetic animals, IS was 39±11%, and cardioprotection by LPC could not be elicited (DM+LPC, 41±16%, P=0.02 vs. LPC). Short-term insulin treatment (I, 90xa0min before I/R, blood glucose <150xa0mg dl−1) did not restore the cardioprotective effects of LPC (DM+I, 42±15%; DM+LPC+I, 40±10%, P=0.03 vs. LPC). It is concluded that acute hyperglycaemia and DM block the cardioprotection induced by LPC in rabbits and that the cardioprotection is not restored by short-term insulin treatment.

p57kip2 is dynamically regulated in experimental autoimmune encephalomyelitis and interferes with oligodendroglial maturation

The mechanisms preventing efficient remyelination in the adult mammalian central nervous system after demyelinating inflammatory diseases, such as multiple sclerosis, are largely unknown. Partial remyelination occurs in early disease stages, but repair capacity diminishes over time and with disease progression. We describe a potent candidate for the negative regulation of oligodendroglial differentiation that may underlie failure to remyelinate. The p57kip2 gene is dynamically regulated in the spinal cord during MOG-induced experimental autoimmune encephalomyelitis. Transient down-regulation indicated that it is a negative regulator of post-mitotic oligodendroglial differentiation. We then applied short hairpin RNA-mediated gene suppression to cultured oligodendroglial precursor cells and demonstrated that down-regulation of p57kip2 accelerates morphological maturation and promotes myelin expression. We also provide evidence that p57kip2 interacts with LIMK-1, implying that p57kip2 affects cytoskeletal dynamics during oligodendroglial maturation. These data suggest that sustained down-regulation of p57kip2 is important for oligodendroglial maturation and open perspectives for future therapeutic approaches to overcome the endogenous remyelination blockade in multiple sclerosis.

Remote ischemic preconditioning preserves Connexin 43 phosphorylation in the rat heart in vivo

BackgroundRemote ischemic preconditioning (RIPC) protects the heart from ischemia and reperfusion (I/R) injury. The underlying molecular mechanisms are unclear. It has been demonstrated that Connexin 43 (Cx43) is critically involved in cardioprotective interventions including classical ischemic preconditioning. In the present study we investigated the influence of RIPC on the expression patterns of Cx43 after I/R in the rat heart in vivo.MethodsMale Wistar rats were subjected to 35xa0min regional myocardial ischemia followed by 2xa0h reperfusion with or without 4xa0cycles of 5xa0minutes bilateral hind limb ischemia and reperfusion (RIPC), to RIPC without ischemia or underwent no intervention (Sham). Infarct size was measured by TTC staining. The myocardium was divided into area at risk (AAR) and area not at risk (non AAR). Expression of Cx43-mRNA and protein was analyzed by qPCR and Western Blot analysis, respectively. Localization of Cx43 was visualized by confocal immunofluorescence staining.ResultsRIPC reduced the infarct size (I/R: 73u2009±u20095% vs. RIPC I/R: 34u2009±u200914%, pu2009<u20090.05). Expression of Cx43 mRNA did not differ between groups. I/R caused a strong decrease of relative Cx43 protein expression in the AAR that was partly abolished by RIPC. Furthermore, RIPC decreased the level of ischemia-induced dephosphorylation of Cx43. Confocal immunofluorescence staining showed that I/R caused a loss of the Cx43 signal at the intercalated discs, while the Cx43 signal at the intercalated discs was partly sustained after RIPC.ConclusionPreservation of Cx43 protein expression and phosphorylation after RIPC might protect the rat heart in vivo.

Wheat germ agglutinin staining as a suitable method for detection and quantification of fibrosis in cardiac tissue after myocardial infarction

The quantification of fibrotic tissue is an important task in the analysis of cardiac remodeling. The use of established fibrosis staining techniques is limited on frozen cardiac tissue sections due to a reduced color contrast compared to paraffin embedded sections. We therefore used FITC-labeled wheat germ agglutinin (WGA), which marks fibrotic tissue in comparable quality as the established picrosirius red (SR) staining, for the staining of post myocardial infarction scar tissue. The fibrosis amount was quantified in a histogram-based approach using the non-commercial image processing program ImageJ. Our results clearly demonstrate that WGA-FITC is a suitable marker for cardiac fibrosis in frozen tissue sections. In combination with the histogram-based analysis, this new quantification approach is i) easy and fast to perform; ii) suitable for raw frozen tissue sections; and iii) allows the use of additional antibodies in co-immunostaining.

Additive protective effects of late and early ischaemic preconditioning are mediated by the opening of KATP channels in vivo.

Abstract. We investigated whether a combination of ischaemic late preconditioning (LPC) and ischaemic early preconditioning (EPC) induces additive myocardial protection in vivo, and the role of ATP-sensitive K (KATP) channels in ischaemic LPC and in LPC+EPC. Sixty rabbits were divided into seven groups. Anaesthetized animals were subjected to 30xa0min of coronary artery occlusion and 120xa0min of reperfusion (I/R). Controls (CON, n=9) were not preconditioned. LPC (n=10) was induced in conscious rabbits by a 5-min period of myocardial ischaemia 24xa0h before I/R. The KATP channel blocker 5-hydroxydecanoate (5-HD, 5xa0mg/kg) was given 10xa0min before I/R with (LPC+5-HD, n=9) or without LPC (5-HD, n=8). EPC (n=8) was induced by a 5-min period of myocardial ischaemia 10xa0min before I/R. Animals received LPC and EPC without (LPC+EPC, n=8) or with 5-HD (LPC+EPC+5-HD, n=8). LPC reduced infarct size (IS, triphenyltetrazolium staining) from 57±11% (MW±SD, CON) of the area at risk to 31±19% (LPC, P=0.004). 5-HD did not affect IS (5-HD: 60±12%, P=0.002 versus LPC), but abolished the cardioprotective effects of LPC (LPC+5-HD: 62±18%, P=0.001 versus LPC). EPC reduced IS to 18±8%. Additional LPC led to a further reduction to 8±4% (LPC+EPC, n=8; P=0.005 versus EPC; P=0.004 versus LPC). 5-HD abolished this additional cardioprotective effect of LPC+EPC (LPC+EPC+5-HD, n=8; 46±11%, P≤0.001 versus LPC+EPC). We conclude that the combination of ischaemic LPC and EPC induces additive cardioprotection. KATP channel opening mediates the cardioprotective effects of ischaemic LPC and LPC+EPC.

Histone methyltransferase enhancer of zeste homolog 2 regulates Schwann cell differentiation.

Epigenetic control is crucial for the differentiation of a variety of cells including oligodendrocytes, the myelinating glial cells of the central nervous system. However, studies about the implication of epigenetic factors in peripheral nervous system maturation are just emerging. Here, we demonstrate for the first time the impact of a histone methyltransferase, encoded by the enhancer of zeste homolog 2 (EZH2) gene, on Schwann cell differentiation. In sciatic nerves, EZH2 expression was found in Schwann cells and to peak perinatally. Suppression of EZH2 expression in cultured primary rat Schwann cells reduced the length of cell processes. These morphological changes were accompanied by widespread alterations in the gene expression pattern, including downregulation of myelin genes and induction of p57kip2, which we have recently identified as an intrinsic inhibitory regulator of Schwann cell maturation. In addition, we show that EZH2 suppression in dorsal root ganglion cocultures interferes with in vitro myelination. Chromatin immunoprecipitation analysis revealed binding of EZH2 at the p57kip2 promoter and reduction of histone H3K27 trimethylation upon gene suppression. EZH2 suppression‐dependent effects on morphology and myelin genes could be reversed by concomitant suppression of p57kip2, indicating that p57kip2 is a downstream effector of EZH2. Furthermore, we describe Hes5 as transcriptional repressor of myelin genes in Schwann cells, which was induced upon EZH2 suppression and downregulated in p57kip2‐suppressed Schwann cells. Therefore, we have identified a molecular link between histone methylation and control of Schwann cell differentiation and demonstrate that this epigenetic mechanism is crucial for glial differentiation to proceed.

Negative Regulators of Schwann Cell Differentiation—Novel Targets for Peripheral Nerve Therapies?

As myelinating glial cells of the peripheral nervous system, Schwann cells wrap around axons and thereby provide insulation, acceleration of electric signal propagation, and axonal protection and maintenance. Schwann cells are main effectors for regeneration in a variety of peripheral neuropathic conditions, including inherited, inflammatory, toxic, and diabetic neuropathies, as well as traumatic injuries to peripheral nerve fibers. Due to their high differentiation plasticity, these cells can respond to injury and disease by myelin sheath degradation, dedifferentiation into an immature Schwann cell-like phenotype, proliferation, and remyelination of sprouting axons. In doing so, they can support and promote axonal regrowth and target tissue innervation. Developmental differentiation as well as regenerative de- and redifferentiation are tightly controlled by a balance of positive and negative regulators of Schwann cell maturation. Since misregulated expression of such negative regulators is potentially involved in inefficient or failed regeneration, we will provide an overview about recent work revealing the complex interactions between extrinsic and intrinsic signals in the inhibition of Schwann cell differentiation.

Fingolimod induces the transition to a nerve regeneration promoting Schwann cell phenotype

Successful regeneration of injured peripheral nerves is mainly attributed to the plastic behavior of Schwann cells. Upon loss of axons, these cells trans-differentiate into regeneration promoting repair cells which provide trophic support to regrowing axons. Among others, activation of cJun was revealed to be involved in this process, initiating the stereotypic pattern of Schwann cell phenotype alterations during Wallerian degeneration. Nevertheless, the ability of Schwann cells to adapt and therefore the nerves potential to regenerate can be limited in particular after long term denervation or in neuropathies leading to incomplete regeneration only and thus emphasizing the need for novel therapeutic approaches. Here we stimulated primary neonatal and adult rat Schwann cells with Fingolimod/FTY720P and investigated its impact on the regeneration promoting phenotype. FTY720P activated a number of de-differentiation markers including cJun and interfered with maturation marker and myelin expression. Functionally, FTY720P treated Schwann cells upregulated growth factor expression and these cells enhanced dorsal root ganglion neurite outgrowth on inhibitory substrates. Our results therefore provide strong evidence that FTY720P application supports the generation of a repair promoting cellular phenotype and suggest that Fingolimod could be used as treatment for peripheral nerve injuries and diseases.

Oligodendroglial Maturation Is Dependent on Intracellular Protein Shuttling

Multiple sclerosis is an autoimmune disease of the CNS resulting in degeneration of myelin sheaths and loss of oligodendrocytes, which means that protection and electrical insulation of axons and rapid signal propagation are impaired, leading to axonal damage and permanent disabilities. Partial replacement of lost oligodendrocytes and remyelination can occur as a result of activation and recruitment of resident oligodendroglial precursor cells. However, the overall remyelination capacity remains inefficient because precursor cells often fail to generate new oligodendrocytes. Increasing evidence points to the existence of several molecular inhibitors that act on these cells and interfere with their cellular maturation. The p57kip2 gene encodes one such potent inhibitor of oligodendroglial differentiation and this study sheds light on the underlying mode of action. We found that subcellular distribution of the p57kip2 protein changed during differentiation of rat, mouse, and human oligodendroglial cells both in vivo and in vitro. Nuclear export of p57kip2 was correlated with promoted myelin expression, higher morphological phenotypes, and enhanced myelination in vitro. In contrast, nuclear accumulation of p57kip2 resulted in blocked oligodendroglial differentiation. Experimental evidence suggests that the inhibitory role of p57kip2 depends on specific interactions with binding proteins such as LIMK-1, CDK2, Mash1, and Hes5 either by controlling their site of action or their activity. Because functional restoration in demyelinating diseases critically depends on the successful generation of oligodendroglial cells, a therapeutic need that is currently unmet, the regulatory mechanism described here might be of particular interest for identifying suitable drug targets and devising novel therapeutic approaches.