Increased sensitivity of Apolipoprotein E knockout mice to swainsonine dependent immunomodulation.

The mechanisms that mediate accelerated atherosclerosis in autoimmune diseases remain unclear. One common mechanism that has been documented in autoimmune diseases and atherosclerosis is formation of hypoglycosyalted N-glycans on the cell surface. In this study we tested the effects of swainsonine, a class II α-mannosidase inhibitor which results in formation of hypoglycosylated N-glycans, on atherogenesis and immune cell dynamics in the atheroprone and hypercholesterolemic ApoE -/- mouse. Wild type or ApoE-/- mice (8 weeks of age) were fed a normal chow diet and administered swainsonine via the drinking water for 8 weeks at which time, atherosclerosis, and systemic markers of markers of inflammation were evaluated. Interestingly, no change in the rate of atherosclerosis development was observed in ApoE -/- mice treated with swainsonine. However, swainsonine significantly increased the number of peripheral blood leukocytes in ApoE -/- mice, with trends toward similar increases in swainsonine treated wild type mice noted. Assessment of leukocyte subsets using specific markers of all major blood lineages indicated that the increase in circulating leukocytes was due to the elevated number of progenitor cells. Consistent with swainsonine having a greater effect in ApoE -/- vs. wild type mice, increases in circulating inflammatory markers (IgA, IgG and chemokines) were observed in the former. Collectively, these data demonstrate that predisposition of ApoE -/- mice to vascular disease is associated with sensitization to the immunomodulatory effects of swainsonine and indicate that changes in N-glycans may provide a mechanism linking autoimmunity to atherogenesis.

The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells.

BACKGROUND: The endoplasmic reticulum (ER) is involved in Ca(2+) signaling and protein processing. Accumulation of unfolded proteins following ER Ca(2+) depletion triggers the ER stress response (ERSR), which facilitates protein folding and removal of damaged proteins and can induce cell death. Unfolded proteins bind to chaperones, such as the glucose-regulated protein (GRP)78 and cause the release of GRP78-repressed proteins executing ERSR. METHODS: Several glioma cell lines and primary astrocytes were used to analyze ERSR using standard western blots, reverse transcription-PCR, viability assays, and single cell Ca(2+) imaging. RESULTS: ERSR induction with thapsigargin results in a more intense ERSR associated with a larger loss of ER Ca(2+), activation of ER-associated caspases (4/12) and caspase 3, and a higher rate of malignant glioma cell death than in normal glial cells. Malignant glioma cells have higher levels of protein synthesis and expression of the translocon (a component of the ribosomal complex, guiding protein entry in the ER), the activity of which is associated with the loss of ER Ca(2+). Our experiments confirm increased expression of the translocon in malignant glioma cells. In addition, blockade of the ribosome-translocon complex with agents differently affecting translocon Ca(2+) permeability causes opposite effects on ERSR deployment and death of malignant glioma cells. CONCLUSIONS: Excessive ER Ca(2+) loss due to translocon activity appears to be responsible for the enhancement of ERSR, leading to the death of glioma cells. The results reveal a characteristic of malignant glioma cells that could be exploited to develop new therapeutic strategies to treat incurable glial malignancies.

Chlorine gas exposure disrupts nitric oxide homeostasis in the pulmonary vasculature.

Exposure to chlorine (Cl2) gas during industrial accidents or chemical warfare leads to significant airway and distal lung epithelial injury that continues post exposure. While lung epithelial injury is prevalent, relatively little is known about whether Cl2 gas also promotes injury to the pulmonary vasculature. To determine this, rats were subjected to a sub-lethal Cl2 gas exposure (400 ppm, 30 min) and then brought back to room air. Pulmonary arteries (PA) were isolated from rats at various times post-exposure and contractile (phenylephrine) and nitric oxide (NO)-dependent vasodilation (acetylcholine and mahmanonoate) responses measured ex vivo. PA contractility did not change, however significant inhibition of NO-dependent vasodilation was observed that was maximal at 24-48 h post exposure. Superoxide dismutase restored NO-dependent vasodilation suggesting a role for increased superoxide formation. This was supported by ∼2-fold increase in superoxide formation (measured using 2-hydroethidine oxidation to 2-OH-E+) from PA isolated from Cl2 exposed rats. We next measured PA pressures in anesthetized rats. Surprisingly, PA pressures were significantly (∼4 mmHg) lower in rats that had been exposed to Cl2 gas 24 h earlier suggesting that deficit in NO-signaling observed in isolated PA experiments did not manifest as increased PA pressures in vivo. Administration of the iNOS selective inhibitor 1400W, restored PA pressures to normal in Cl2 exposed, but not control rats suggesting that any deficit in NO-signaling due to increased superoxide formation in the PA, is offset by increased NO-formation from iNOS. These data indicate that disruption of endogenous NO-signaling mechanisms that maintain PA tone is an important aspect of post-Cl2 gas exposure toxicity.

Heterogenic endothelial responses to inflammation: role for differential N-glycosylation and vascular bed of origin.

BACKGROUND: Endothelial cell responses during inflammation are heterogeneous and key for selectivity in how leukocytes hone in on specific sites and why vascular diseases are highly bed specific. However, mechanisms for this specificity remain unclear. METHODS AND RESULTS: Here, we exposed human endothelial cells isolated from 5 systemic arterial beds from 1 donor (to overcome donor-to-donor genetic/epigenetic differences), the umbilical vein, and pulmonary microvasculature to TNF-α, LPS, and IL-1ß and assessed acute (ERK1/2 and p65) and chronic (ICAM-1, VCAM-1 total and surface expression) signaling responses and assessed changes in surface N-glycans and monocyte adhesion. Significant diversity in responses was evident by disparate changes in ERK1/2 and p65 NF-κB phosphorylation, which varied up to 5-fold between different cells and in temporal and magnitude differences in ICAM-1 and VCAM-1 expression (maximal VCAM-1 induction typically being observed by 4 hours, whereas ICAM-1 expression was increased further at 24 hours relative to 4 hours). N-glycan profiles both basally and with stimulation were also bed specific, with hypoglycosylated N-glycans correlating with increased THP-1 monocyte adhesion. Differences in surface N-glycan expression tracked with dynamic up- or downregulation of α-mannosidase activity during inflammation. CONCLUSIONS: These results demonstrate a critical role for the vascular bed of origin in controlling endothelial responses and function to inflammatory stimuli and suggest that bed-specific expression of N-linked sugars may provide a signature for select leukocyte recruitment.

Decreased hematopoietic progenitor cell mobilization in pearl mice.

Neutropenia is common to both Hermansky-Pudlak syndrome type 2 and canine cyclic hematopoiesis (CH) which are caused by mutations in the AP3B1 gene. The purpose of this study was to determine if pearl mice were neutropenic. Complete blood counts (CBCs) and bone marrow differential counts, colony forming unit (CFU) assay, bone marrow lineage negative (lin(-)), Sca(+) and c-kit(+) cells (LSK cells), bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were compared in C57Bl6 (Bl/6) and pearl mice. Stress granulopoiesis was evaluated following 200 mg/kg cyclophosphamide or 1 mg/kg bortezomib administration and by limiting dilution bone marrow transplantation. The CBCs and CFUs were determined in Bl/6 and pearl mice following AMD3100 or granulocyte colony-stimulating factor (G-CSF) administration. Pearl mice were not neutropenic and did not have cyclic neutropenia. Bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were similar in pearl and Bl/6 mice. The numbers of CFU-G, CFU-GEMM, and LSK cells were increased moderately in pearl mice. Stress granulopoiesis was similar in Bl/6 and pearl mice. CFU assays and CBCs performed on Bl/6 and pearl mice administered AMD3100 resulted in similar results. However, normal mice administered G-CSF had higher peripheral blood neutrophil counts and greater CFU numbers compared with pearl mice. Unlike patients with HPS-2 and dogs with CH, pearl mice did not have neutropenia or CH but had decreased hematopoietic progenitor cell and granulocyte mobilization in response to G-CSF.

A pilot feasibility study of daily rTMS to modify corticospinal excitability during lower limb immobilization.

Short term immobilization of the lower limb is associated with increased corticospinal excitability at 24 hours post cast removal. We wondered whether daily stimulation of the motor cortex might decrease brain reorganization during casting. We tested the feasibility of this approach. Using transcranial magnetic stimulation (TMS), resting motor threshold and recruitment curves were obtained at baseline in 6 healthy participants who then had leg casts placed for 10 days. On 7 of the 10 days subjects received 20 minutes of 1 Hz repetitive TMS (rTMS). TMS measures were then recorded immediately after and 24 hours post cast removal. Four of 6 subjects completed the study. At the group level there were no changes in excitability following cast removal. At the individual level, two participants did not show any change, 1 participant had higher and one lower excitability 24 hours after cast removal. Daily rTMS over motor cortex is feasible during casting and may modify neuroplastic changes occurring during limb disuse. A prospective double blind study is warranted to test whether daily rTMS might improve outcome in subjects undergoing casting, and perhaps in other forms of limb disuse such as those following brain injury or weightlessness in space flight.