Endothelin receptor A antagonism reduces the extent of diffuse axonal injury in a rodent model of traumatic brain injury.

OBJECTIVES: While endothelin-1 and its receptors have traditionally been associated with mediating vasoreactivity, we have recently shown that the vast majority of endothelin receptor A expression following traumatic brain injury is localized within the neuron. While it has been suggested that endothelin receptor A plays a role in influencing neuronal integrity, the significance of neuronally expressed endothelin receptor A remains unclear. One report suggests that endothelin-1 signaling mediates diffuse axonal injury. Therefore, this work sought to determine whether treatment with BQ-123, a selective endothelin receptor A antagonist, diminishes the extent of diffuse axonal injury following trauma. METHODS: A total of 12 male Sprague-Dawley rats (350-400 g) were used in this study. Two groups (n = 6 per group) were generated as follows: sham operation and traumatic brain injury+1·0 mg/kg BQ-123 delivered intravenously 30 minutes prior to the injury. Trauma was induced using a weight acceleration impact device. Animals were terminated 24 or 48 hours after trauma, and a series of six coronal sections through the entire anterior-posterior extent of the corpus callosum were selected from each brain for quantification of diffuse axonal injury by beta-amyloid precursor protein immunostaining. RESULTS: Our data indicated that animals treated with BQ-123 30 minutes prior to trauma showed a significant reduction in diffuse axonal injury in corpus callosum at both 24 and 48 hours post-injury. CONCLUSION: The results show that endothelin receptor A antagonism reduced the extent of diffuse axonal injury, demonstrating a potential influence of the endothelin system on the intra-axonal cascade of molecular events underlying diffuse axonal injury.

Calponin phosphorylation in cerebral cortex microvessels mediates sustained vasoconstriction after brain trauma.

OBJECTIVES: The purpose of this study was to determine the molecular and biochemical changes in the contractile protein, calponin (Cp), which temporally coincide with a previously reported state of sustained contractility following traumatic brain injury (TBI). METHODS: Double immunofluorescence, western analysis and two-dimensional non-equilibrium pH gradient gel electrophoresis (NEPHGE)/SDS-PAGE techniques were utilized to determine both the location and extent of Cp within smooth muscle cells (SM) and the phosphorylation state of Cp following TBI, as induced using a weight drop acceleration impact model. RESULTS: Double immunofluorescence for Cp and SM indicate that following injury, Cp migrates from the cytosol to a location subjacent to the SM membrane. Western analysis revealed a significant increase in Cp protein expression following injury that was maintained up to 48 hours post-injury. Combined Western analysis and NEPHGE indicated that Cp is phosphorylated following TBI. DISCUSSION: Cp migration and phosphorylation may underlie the mechanism for increased vasoreactivity leading to hypoperfusion following TBI.

Neovascularization following traumatic brain injury: possible evidence for both angiogenesis and vasculogenesis.

OBJECTIVE: Our goal was to characterize the angiogenic response following traumatic brain injury (TBI). METHODS: Western analysis for vascular endothelial growth factor (VEGF) expression, double immunofluorescence labeling of endothelium and vascular endothelial growth factor receptor 2 (VEGFR2), bromodioxyuridine (BrdU) incorporation and measurement of capillary density, were all used to determine the temporal angiogenic response following TBI. RESULTS: The angiogenic factors, VEGF and VEGFR2, increase following trauma. Capillary density increases and BrdU incorporation confirm the presence of newly formed vessels up to 48 hours post-injury. DISCUSSION: Our results indicated that following TBI, there is a substantial increase in angiogenesis and based on morphologic characterization of BrdU-positive nuclei within the endothelium, we provide evidence for vasculogenesis following injury.

Regulation of Sam68 activity by small heat shock protein 22.

Sam68 associates with c-Src kinase during mitosis. We previously demonstrated that Sam68 functionally replaces and/or synergizes with HIV-1 Rev in rev response element (RRE)-mediated gene expression and virus production. Furthermore, we reported that knockdown of Sam68 inhibited Rev-mediated RNA export and it is absolutely required for HIV-1 production. In the present study, we identified small heat shock protein, hsp22, as a novel interacting partner of Sam68. Hsp22 binds to Sam68 in vitro and in vivo. Overexpression of hsp22 significantly inhibits Sam68-mediated RRE- as well as CTE (constitutive transport element)-dependent reporter gene expression. Furthermore, exposing 293T cells to heat shock inhibits Sam68/RRE function by virtue of elevating hsp22. The critical domain of hsp22 that interacts with Sam68 resides between amino acids 62 and 133. Our studies provide evidence for the first time that hsp22 specifically binds to Sam68 and modulates its activity, thus playing a role in the post-transcriptional regulation of gene expression.

Effect of exogenous stress on the tissue-cultured mouse lens epithelial cells.

The effects of heat, oxidative and osmotic stress on heat shock proteins (HSP-70(I), HSC-70, and HSP-40 of tissue cultured mouse lens epithelial cells (alphaTN-4) were investigated. The effect of stress on the heat shock transcription factor (HSF-1), and a nuclear matrix protein (NM-60) of alphaTN-4 cells was also examined. Cells were exposed to heat (45 degrees C), oxidative stress (50 mM H(2)0(2)) and osmotic (600 mM medium) shock for 30 min, and then allowed to recover for 18 h in physiological medium. Control cells were maintained at 37 degrees C in an isosmolar medium. Cellular proteins were isolated and fractionated by SDS-PAGE. Western blot was used to determine the levels of HSP and nuclear proteins. Heat stressed cells were also examined, by immunofluorescence, for the specific localization of NM-60 and HSF-1. The results revealed that both NM-60 and HSF-I were present in specific locations in normal and heat-stressed cell nuclei. Nuclei isolated immediately after stress showed localization of fluorescence near the nuclear membrane. When heat stressed cells were allowed to recuperate at 37 degrees C, most of the fluorescence were relocated in discrete areas of the nucleus. These experiments showed that alphaTN-4 cells responded to stress by overexpression of HSP-70(I) and HSP-40. This increase was not present immediately after the end of the stress period, but clearly evident at 18 h of recovery in physiological medium. Immunofluorescent data suggest that heat stress induced the localization of NM-60 and HSF-1 near the nuclear membrane.