The microenvironment in the Hirschsprung's disease gut supports myenteric plexus growth.

INTRODUCTION: The transplantation of neural crest derived stem cells (NCSC) is a potent alternative for the treatment of Hirschsprung's disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. Influences of HSCR-smooth-muscle-protein extracts upon isolated myenteric plexus cells, dissociated dorsal root ganglia and NCSC were studied in vitro to investigate the quality of this microenvironment effects. METHODS: Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) and transforming-growth-factor-ß-1 (TGFß-1) concentration were measured in the homogenates from the individual segment using ELISA. Myenteric plexus and dissociated dorsal root ganglia (DRG) cultures, as well as NCSCs were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival, and branching was evaluated. RESULTS AND CONCLUSIONS: The amount of the factors varied considerably between the individual segments and also from patient to patient. Four major expression patterns could be detected. While all extracts tested lead to a significant increase in neurite outgrowth compared to the control, extracts from proximal segments tended to have more prominent effects. In one experiment, extracts from all individual segments of a single patient were tested. Neurite outgrowth, neuronal survival, and branching pattern varied from segment to segment, but all HSCR-muscle-protein extracts increased neuronal survival and network formation. Smooth muscle protein from aganglionic bowel supports the survival and outgrowth of myenteric neurons and NCSCs and is so an appropriate target for neural stem cell treatment.

Smooth muscle proteins from Hirschsprung's disease facilitates stem cell differentiation.

BACKGROUND AND AIMS: The transplantation of neural crest derived stem cells (NCSC's) is a potent alternative for the treatment of Hirschsprung's disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. To investigate the quality of this microenvironment, effects of HSCR-smooth-muscle-protein extracts upon NCSC's were studied in vitro. METHODS: Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) concentration was measured in the homogenates from the individual segment using ELISA. NCSC's were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival and branching was evaluated. RESULTS: The amount of the factors varied considerably between the proximal and distal segments, and also from patient to patient. While extracts from proximal segments tended to have more prominent effects, all HSCR-muscle-protein extracts increased neuronal survival and network formation. CONCLUSION: Muscle protein from aganglionic bowel supports the survival and outgrowth of NCSC's and is so an appropriate target for neural stem cell treatment.

Identification of structural elements in Nox1 and Nox4 controlling localization and activity.

Nox NADPH oxidases differ in their mode of activation, subcellular localization, and physiological function. Nox1 releases superoxide anions (O(2)(-)) and depends on cytosolic activator proteins, whereas Nox4 extracellularly releases hydrogen peroxide (H(2)O(2)), and its activity does not require cotransfection of additional proteins. We constructed chimeric proteins consisting of Nox1 and Nox4 expressed in HEK293 cells. When the cytosolic tail of Nox4 was fused with the transmembrane part of Nox1, Nox1 became constitutively active. The reciprocal construct was inactive, suggesting that cytosolic subunit-dependent activation requires elements in the transmembrane loops. By TIRF-microscopy, Nox1 was observed in the plasma membrane, whereas Nox4 colocalized with proteins of the endoplasmic reticulum. Fusion proteins of Myc and Nox revealed that the N-terminal part of Nox1 but not Nox4 is cleaved. When the potential signal peptide of Nox4 was inserted into Nox1, plasma-membrane localization was lost, and the protein was retained in vesicle-like structures below the plasma membrane. The potential signal peptide of Nox1 failed to translocate Nox4 to the plasma membrane but switched the extracellularly detectable ROS from H(2)O(2) to O(2)(-). Thus, the very N-terminal part of Nox proteins determines subcellular localization and the ROS type released, whereas the cytosolic tail regulates activity.

Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant.

A large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, relied on data obtained with the inhibitor apocynin (4'-hydroxy-3'methoxyacetophenone). Because the mode of action of apocynin, however, is elusive, we determined its mechanism of inhibition on vascular NADPH oxidases. In HEK293 cells overexpressing NADPH oxidase isoforms (Nox1, Nox2, or Nox4), apocynin failed to inhibit superoxide anion generation detected by lucigenin chemiluminescence. In contrast, apocynin interfered with the detection of reactive oxygen species in assay systems selective for hydrogen peroxide or hydroxyl radicals. Importantly, apocynin interfered directly with the detection of peroxides but not superoxide, if generated by xanthine/xanthine oxidase or nonenzymatic systems. In leukocytes, apocynin is a prodrug that is activated by myeloperoxidase, a process that results in the formation of apocynin dimers. Endothelial cells and smooth muscle cells failed to form these dimers and, therefore, are not able to activate apocynin. Dimer formation was, however, observed in Nox-overexpressing HEK293 cells when myeloperoxidase was supplemented. As a consequence, apocynin should only inhibit NADPH oxidase in leukocytes, whereas in vascular cells, the compound could act as an antioxidant. Indeed, in vascular smooth muscle cells, the activation of the redox-sensitive kinases p38-mitogen-activate protein kinase, Akt, and extracellular signal-regulated kinase 1/2 by hydrogen peroxide and by the intracellular radical generator menadione was prevented in the presence of apocynin. These observations indicate that apocynin predominantly acts as an antioxidant in endothelial cells and vascular smooth muscle cells and should not be used as an NADPH oxidase inhibitor in vascular systems.