Experimental evaluation of hyaluronidase activity in combination with specific drugs applied in clinical techniques of interventional pain management and local anaesthesia.

BACKGROUND: Hyaluronidase is an enzyme additive used in local anaesthesia and interventional pain reducing procedures such as adhesiolysis of epidural scar tissue after spinal surgery. Only a limited number of studies describe the influence of drugs on hyaluronidase activity. Postulated effects and effectiveness of hyaluronidase are only based on clinical observations. OBJECTIVE: The aim of this study is to investigate the influence of the combined drugs on the activity of hyaluronidase under standardized conditions and to verify the effectiveness of the enzyme. DESIGN: An ELISA-based microtiter-technique is used to evaluate the activity of hyaluronidase in combination with local anaesthetics, corticosteroids, NaCl 10%, and iodinated contrast media. METHODS: Microtiter plates were coated with biotinylated hyaluronate and incubated with hyaluronidase in combination with the above-mentioned drugs. The activity of hyaluronidase was determined by an avidin-peroxidase-based procedure using an ELISA reader. Incubations were carried out at room temperature as well as at 37 degrees C. RESULTS: The data show that drugs affect the activity of hyaluronidase in different ways. Iodinated contrast media, NaCl (10%), and the absence of corticosteroids reduce hyaluronidase activity. In contrast, higher activities were detected at a lower NaCl concentration (0.9%). We cannot attribute a significant influence to local anaesthetics. CONCLUSIONS: Hyaluronidase is effective in all combinations with drugs. To get the maximum effect calculated use of accompanying drugs is necessary.

Erythropoietin improves functional and histological recovery of traumatized skeletal muscle tissue.

Apart from its hematopoietic effect, erythropoietin (EPO) is known as pleiotropic cytokine with anti-inflammatory and anti-apoptotic properties. Here, we evaluated for the first time the EPO-dependent regeneration capacity in an in vivo rat model of skeletal muscle trauma. A myoblast cell line was used to study the effect of EPO on serum deprivation-induced cell apoptosis in vitro. A crush injury was performed to the left soleus muscle in 80 rats treated with either EPO or saline. Muscle recovery was assessed by analysis of contraction capacities. Intravital microscopy, BrdU/laminin double immunohistochemistry and cleaved caspase-3 immunohistochemistry of muscle tissue on days 1, 7, 14, and 42 posttrauma served for assessment of local microcirculation, tissue integrity, and cell proliferation. Serum deprivation-induced myoblast apoptosis of 23.9 +/- 1.5% was reduced by EPO to 17.2 +/- 0.8%. Contraction force analysis in the EPO-treated animals revealed significantly improved muscle strength with 10-20% higher values of twitch and tetanic forces over the 42-day observation period. EPO-treated muscle tissue displayed improved functional capillary density as well as reduced leukocytic response and consecutively macromolecular leakage over day 14. Concomitantly, muscle histology showed significantly increased numbers of BrdU-positive satellite cells and interstitial cells as well as slightly lower counts of cleaved caspase-3-positive interstitial cells. EPO results in faster and better regeneration of skeletal muscle tissue after severe trauma and goes along with improved microcirculation. Thus, EPO, a compound established as clinically safe, may represent a promising therapeutic option to optimize the posttraumatic course of muscle tissue healing.

Erythropoietin receptor-mediated Egr-1 activation: structural requirements and functional implications.

The transcription factor Egr-1 is encoded by an immediate early response gene and has been shown to be a key regulator in the induction of apoptosis, mitogenesis and differentiation. It is rapidly induced by different stimuli including the glycoprotein hormone erythropoietin. In this report, we analyse the role of different erythropoietin receptor substructures for the activation of Egr-1 and the functional consequences of Egr-1 overexpression in the erythroleukemic cell line ELM-I-1. The investigation of receptor variants revealed that the activity of JAK2 and the phosphorylation of receptor tyrosine residues are essential preconditions for the ability to target Egr-1. Furthermore, we observed a close correlation of the abilities of receptors to activate the Ras-MAPK pathway and Egr-1. Using mass spectrometry we identified the Ras-GTPase-activating protein-SH3-domain-binding protein 1 (G3BP-1), a component of the Ras network of proteins, as an Egr-1 interacting protein in EPO stimulated ELM-I-1 cells. The overexpression of Egr-1 in these cells resulted in an enhanced rate of spontaneous erythroid differentiation.

Profiling of early gene expression induced by erythropoietin receptor structural variants.

The development of erythroid progenitor cells is triggered via the expression of the erythropoietin receptor (EPOR) and its activation by erythropoietin. The function of the resulting receptor complex depends critically on the presence of activated JAK2, and the complex contains a large number of signaling molecules recruited to eight phosphorylated tyrosine residues. Studies using mutant receptor forms have demonstrated that truncated receptors lacking all tyrosines are able to support red blood cell development with low efficiency, whereas add-back mutants containing either Tyr343 or Tyr479 reconstitute EPOR signaling and erythropoiesis in vivo. To study the contribution of tyrosines to receptor function, we analyzed the activation of essential signaling pathways and early gene induction promoted by different receptor structural variants using human epidermal growth factor receptor/murine EPOR hybrids. In our experiments, receptors lacking all tyrosine residues or the JAK2-binding site did not induce mitogenic and anti-apoptotic signaling, whereas add-back mutant receptors containing single tyrosine residues (Try343 and Tyr479) supported the activation of these functions efficiently. Profiling of early gene expression using cDNA array hybridization revealed that (i) the high redundancy in the activation of signaling pathways is continued at the level of transcription; (ii) the expression of many genes targeted by the wild-type receptor is not supported by add-back mutants; and (iii) a small set of genes are exclusively induced by add-back receptors. We report the identification of several early genes that have not been implicated in the EPOR-dependent response so far.

Relative quantification of erythropoietin receptor-dependent phosphoproteins using in-gel 18O-labeling and tandem mass spectrometry.

On examining different proteomics approaches for the investigation of structure-function relationships of erythropoietin (EPO) receptor signaling, it was found that two-dimensional gel electrophoresis/mass spectrometry procedures are clearly limited in their ability to detect low-expressed signaling proteins. Instead it was found that a strategy involving anti-phosphotyrosine immunoprecipitation, one-dimensional gel electrophoresis (1DE), and capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) provides the sensitivity required for identification of signaling proteins. In the present work the immunoprecipitation/1DE/LC/MS approach was combined with an in-gel 18O-labeling technique to analyze EPO receptor-dependent proteins. Identification and relative quantification of more than 180 EPO receptor-dependent proteins were achieved directly based on the in-gel 18O-labeling approach.

Phosphoprotein profiling of erythropoietin receptor- dependent pathways using different proteomic strategies.

Proteomic techniques provide new tools for the global analysis of protein profiles but also for the investigation of specific protein functions. The analysis of signaling cascades has traditionally been performed by the determination of enzymatic or transcription factor activities representing a certain pathway. Functional proteomics now allows more comprehensive approaches to study cellular responses induced during ligand/receptor interactions. In this study we evaluated proteomic strategies for the investigation of structure-function relationships in the erythropoietin receptor signalling complex. After expression of epidermal growth factor/erythropoietin receptor mutant molecules in an identical cellular background we characterized their potential to induce cellular activities. Using this system we focused our efforts on post-translational modifications of signalling proteins reflecting a substantial part of receptor-dependent signaling events. Although tyrosine phosphorylated proteins were enriched by immunoprecipitation the analysis using the classical approach combining two-dimensional gel electrophoresis and identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry revealed that low expressed signaling proteins cannot be detected by this technique. An alternative strategy using one-dimensional gel separation of phosphoproteins and liquid chromatography-tandem mass spectrometry, however, allowed us to identify multiple proteins involved in intracellular signalling representing already established pathways but also proteins which have not been linked to EPO-induced signaling so far. This approach offers the potential to extend functional proteomic studies to complex signaling processes.

Opposite effects of inhibitors of mitogen-activated protein kinase pathways on the egr-1 and beta-globin expression in erythropoietin-responsive murine erythroleukemia cells.

The effect of erythropoietin (Epo) on the expression of mitogen-activated protein kinase (MAPK) target genes egr-1 and c-fos was investigated in Epo-responsive murine erythroblastic cell line ELM-I-1. Epo induced a transient rise in egr-1 mRNA without a similar effect on c-fos expression. The induction of egr-1 correlated with a rapid ERK1/2 phosphorylation and was prevented with MEK1/2 inhibitors PD 98059 and UO126. The p38 inhibitor SB 203580 enhanced ERK1/2 phosphorylation and egr-1 mRNA levels. Longer incubations of ELM-I-1 cells with Epo revealed a second later phase of increase in egr-1 expression which was also prevented by MEK1/2 inhibitors, whereas SB 203580 had a stimulatory effect. In contrast, the beta-globin mRNA production was enhanced in the presence of PD 98059 and UO126 and reduced by SB 203580. The results suggest a regulatory role of egr-1 expression in Epo signal transduction and provide pharmacological evidence for the negative modulation of differentiation-specific gene expression by the ERK1/2 pathway in murine erythroleukemia cells.

Differential regulation of SOCS genes in normal and transformed erythroid cells.

The SOCS family of genes are negative regulators of cytokine signalling with SOCS-1 displaying tumor suppressor activity. SOCS-1, CIS and SOCS-3 have been implicated in the regulation of red blood cell production. In this study, a detailed examination was conducted on the expression patterns of these three SOCS family members in normal erythroid progenitors and a panel of erythroleukemic cell lines. Unexpectedly, differences in SOCS gene expression were observed during maturation of normal red cell progenitors, viz changes to CIS were inversely related to the alterations of SOCS-1 and SOCS-3. Similarly, these SOCS genes were differentially expressed in transformed erythoid cells - erythroleukemic cells immortalized at an immature stage of differentiation expressed SOCS-1 and SOCS-3 mRNA constitutively, whereas in more mature cell lines SOCS-1 and CIS were induced only after exposure to erythropoietin (Epo). Significantly, when ectopic expression of the tyrosine kinase Lyn was used to promote differentiation of immature cell lines, constitutive expression of SOCS-1 and SOCS-3 was completely suppressed. Modulation of intracellular signalling via mutated Epo receptors in mature erythroleukemic lines also highlighted different responses by the three SOCS family members. Close scrutiny of SOCS-1 revealed that, despite large increases in mRNA levels, the activity of the promoter did not alter after erythropoietin stimulation; in addition, erythroid cells from SOCS-1-/- mice displayed increased sensitivity to Epo. These observations indicate complex, stage-specific regulation of SOCS genes during normal erythroid maturation and in erythroleukemic cells.