Stimulating effect of biologically modified low density lipoproteins on ADP-induced aggregation of washed platelets persists in absence of specific binding

Oxidized low density lipoproteins are closely associated with atherosclerosis and also might be directly involved in thrombosis because they have been shown to mediate a stimulating effect on human platelets. In this work, we used biologically modified low density lipoproteins (i.e., low density lipoproteins sufficiently oxidized to show specificity for the macrophage scavenger receptor system) to examine if specific binding of the oxidized apolipoprotein moiety to the platelet surface is a prerequisite for the platelet-stimulating effects reported by other authors. We find that biologically modified low density lipoproteins show specific binding to human platelets (K_d=5.83±0.4 μg/mL, 3850±620 sites/platelet) and strongly augment both ADP- and thrombin-induced aggregation of washed platelets. Maleylated albumin, an antagonist of oxidized low density lipoproteins binding to all currently classified scavenger receptors, is able to reduce platelet oxidized low density lipoproteins binding to background levels. Nevertheless, maleylated albumin is not able to exert any kind of normalizing effect on the augmented ADP-induced aggregation response observed in the presence of biologically modified low density lipoproteins. From these data, we conclude that specific binding of oxidatively modified apolipoprotein B to the platelet surface is not essential to the process of platelet stimulation. Therefore, we conclude that these stimulating effects may be mediated by unidentified compounds formed in the lipid phase of the lipoproteins.     

Oxidized high-density lipoprotein induces neuron death

High-density lipoprotein (HDL) exists within the brain and is highly vulnerable to oxidative modifications. The focus of the present study was to determine the effect of HDL and oxidized HDL (oxHDL) upon neurons, astrocytes, and microglia. Administration of highly oxidized HDL, but not native, minimally, or moderately modified HDL resulted in a dose- and time-dependent increase in oxidative stress and death of cultured rat embryonic neurons. Astrocyte and microglia cultures treated with highly oxidized HDL displayed increased reactive oxygen species formation but no toxicity. Application of oxHDL exacerbated oxidative stress and neuron death induced by beta-amyloid peptide. Studies using pharmacological inhibitors implicate the involvement of calcium and reactive oxygen species in oxHDL-induced neuronal loss. Neural cells expressing increased levels of BCL-2 had decreased levels of oxidative stress and neuron death following exposure to oxHDL. Together, these data demonstrate that oxHDL increases oxidative stress in neurons, astrocytes, and microglia which ultimately culminate in neuron death.     

Modified forms of low density lipoprotein affect platelet aggregation

Modified forms of low density lipoprotein (LDL) unlike native LDL can lead to macrophage cholesterol accumulation and foam cell formation. Since platelets interact with both lipoprotein and macrophages in the atherosclerotic plaque, the present study was designed to analyze the effect of modified LDL on washed human platelet composition and aggregation. Platelet aggregation was increased following 2 h of incubation with native LDL. Phospholipase C modified LDL and hepatic lipase modified LDL but not acetyl LDL further increased collagen induced platelet aggregation in a dose dependent manner by up to 15% (p less than 0.01). Oxidized LDL, however, demonstrated 25% reduction in both collagen and ADP induced platelet aggregation in comparison to the effect of native LDL. Platelet aggregation was found to be directly related to changes in platelet phospholipid content whereas platelet cholesterol content was similarly affected by all lipoproteins. Platelet cholesterol/phospholipid ratio was directly related to platelet aggregation. Our study thus demonstrates that modified forms of LDL significantly affect platelet lipid composition and function and if similar interactions occur in vivo it might also affect the atherogenic process.     

Agmatine induces glutamate release and cell death in cultured rat cerebellar granule neurons

We investigated the effect of agmatine on cell viability of rat cerebellar granule neurons in a high-K+ (27.5 mM) medium. Exposure of cultured rat cerebellar granule neurons to agmatine (200-800 microM) resulted in a significant decrease in cell viability. Agmatine-induced neuronal death began to occur 6-12 h after addition, and gradually progressed. The agmatine neurotoxicity was attenuated by N-methyl-D-aspartate (NMDA) receptor antagonists and by enzymatic degradation of L-glutamate with glutamic pyruvic transaminase. Furthermore, a significant increase in extracellular L-glutamate concentration was detected before cell death occurred. In addition, agmatine-induced glutamate release and cell death were both blocked by pretreatment with botulinum toxin C, which is known to specifically inhibit the exocytosis. The agmatine neurotoxicity was not observed when extracellular K+ concentration was lower (10 mM). These results suggest that agmatine induces glutamate release through the exocytosis and thereby causes NMDA receptor-mediated neuronal death in conditions in which extracellular K+ concentrations are elevated.     

Basic fibroblast growth factor rescues CNS neurons from cell death caused by high oxygen atmosphere in culture

In the present study, we cultured rat CNS neurons and tested the neurotrophic support provided by basic fibroblast growth factor (bFGF) to prevent the oxygen-induced neuronal cell death. When rat basal forebrain (septum and vertical limb of diagonal band of Broca) cells of embryonic day 20 were cultured in a serum-free medium containing 5 microM cytosine arabinoside in a 50% oxygen atmosphere, the neuronal cells, which were immunostained by an anti-microtubule-associated protein 2 (MAP2) antibody, gradually died after 1 day in culture. After 3.5 days in culture, only 2-5% of neuronal cells survived. This oxygen-induced cell death of cultured basal forebrain neurons was reversed by the addition of bFGF at a concentration of 100 ng/ml. This cell-saving effect was dose-dependent, and the ED50 value was 12 ng/ml. Nerve growth factor (NGF) and insulin-like growth factor II could not prevent cell death. The activity of choline acetyltransferase was also maintained when bFGF was present in the basal forebrain culture. Viable astroglial cells, which were immunostained by an anti-glial fibrillary acidic protein, accounted for a few percent of the total number of cells after 3 days in culture both with and without 100 ng/ml of bFGF. The survival-enhancing effect of bFGF was observed not only in basal forebrain neurons but also in neocortical and hippocampal neurons. However, the sensitivity to oxygen toxicity of cultured neurons from the 3 CNS regions varied greatly. The neocortical neurons were the most sensitive to oxidative stress, while the hippocampal neurons were the most resistant. These results suggest that bFGF plays an important role in saving neuronal cells from oxidative stress during their long life without division.     

Depolarization or glutamate receptor activation blocks apoptotic cell death of cultured cerebellar granule neurons

Cerebellar granule neurons can be readily maintained in culture if depolarized with high concentrations of K⁺ or subtoxic concentrations of various excitatory amino acids. We now report that these depolarizing stimuli promote cerebellar granule neuron survival by blocking their programmed death via apoptosis. Cerebellar granule neurons maintained in depolarizing conditions and then changed to non-depolarizing conditions, exhibit the morphological and biochemical features of apoptosis, including cytoplasmic blebbing, condensation and aggregation of nuclear chromatin internucleosomal DNA fragmentation. Inhibitors of RNA or protein synthesis greatly attenuate cell death induced by non-depolarizing culture conditions. In contrast, cerebellar granule neurons, when exposed to fresh serum-containing medium or to high concentrations of glutamate, exhibit a delayed-type of neurotoxicity which is non-apoptotic in nature. Given the actions of excitatory amino acid receptor agonists in preventing apoptosis of cultured cerebellar granule neurons, we hypothesize that the functional innervation of postmigratory granule neurons during cerebellar development may prevent further elimination of these neurons by blocking their programmed death.     

GPNMB ameliorates mutant TDP‐43‐induced motor neuron cell death

Glycoprotein nonmetastatic melanoma protein B (GPNMB) aggregates are observed in the spinal cord of amyotrophic lateral sclerosis (ALS) patients, but the detailed localization is still unclear. Mutations of transactive response DNA binding protein 43kDa (TDP‐43) are associated with neurodegenerative diseases including ALS. In this study, we evaluated the localization of GPNMB aggregates in the spinal cord of ALS patients and the effect of GPNMB against mutant TDP‐43 induced motor neuron cell death. GPNMB aggregates were not localized in the glial fibrillary acidic protein (GFAP)‐positive astrocyte and ionized calcium binding adaptor molecule‐1 (Iba1)‐positive microglia. GPNMB aggregates were localized in the microtubule‐associated protein 2 (MAP‐2)‐positive neuron and neurofilament H non‐phosphorylated (SMI‐32)‐positive neuron, and these were co‐localized with TDP‐43 aggregates in the spinal cord of ALS patients. Mock or TDP‐43 (WT, M337V, and A315T) plasmids were transfected into mouse motor neuron cells (NSC34). The expression level of GPNMB was increased by transfection of mutant TDP‐43 plasmids. Recombinant GPNMB ameliorated motor neuron cell death induced by transfection of mutant TDP‐43 plasmids and serum‐free stress. Furthermore, the expression of phosphorylated ERK1/2 and phosphorylated Akt were decreased by this stress, and these expressions were increased by recombinant GPNMB. These results indicate that GPNMB has protective effects against mutant TDP‐43 stress via activating the ERK1/2 and Akt pathways, and GPNMB may be a therapeutic target for TDP‐43 proteinopathy in familial and sporadic ALS. © 2016 Wiley Periodicals, Inc.     

Oxidized low‐density lipoprotein (oxLDL)‐induced cell death in dorsal root gangion cell cultures depends not on the lectin‐like oxLDL receptor‐1 but on the toll‐like receptor‐4

DRG cells have been found to undergo apoptosis and necrosis after oxidized low‐density lipoprotein (oxLDL) stimulation in vitro. However, the mechanism of oxLDL‐induced DRG cell death is unclear. For this reason, we studied the expression of two potential oxLDL receptors: lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) and toll‐like receptor‐4 (TLR4) in dorsal root ganglion (DRG) cell cultures from postnatal rats. Cells were cultivated with and without oxLDL. In oxLDL‐treated DRG cell cultures, the increase of cleaved caspase‐3 protein was observed as a sign of enhanced apoptosis. Untreated and oxLDL‐treated DRG cell cultures expressed LOX‐1 and TLR4 at similar levels. The LOX‐1 expression remained unchanged after receptor blockade. However, the inhibition of LOX‐1 caused a significant increase of cleaved caspase‐3 and a decrease of TLR4 levels. The TLR4‐inhibited DRG cell cultures lacked changes in LOX‐1 expression for all experimental groups. The inhibition of TLR4 caused activation of jun N‐terminal kinase (JNK) and a significant decrease of cleaved caspase‐3 but did not change the TLR4 level. We conclude that LOX‐1 and TLR4 are expressed in cultivated rat DRG cells and that the oxLDL‐induced cell death in DRG cell cultures does not depend on the LOX‐1 but on the TLR4. © 2009 Wiley‐Liss, Inc.     

Age-related changes in cell density and the proliferation rate of olfactory ensheathing cells in the lamina propria of postnatal mouse olfactory mucosa

We investigated age-related changes in the distribution and proliferation of olfactory ensheathing cells (OECs) in postnatal mouse olfactory mucosa. In contrast to reported data on other glial cell types in the peripheral and central nervous systems, OEC cell density in the olfactory nerve bundles in the lamina propria remained almost constant from 10 days through 16 months of age. Electron microscopy of the nerve bundles revealed that axon packing density also was constant during that period. These findings suggest that the ratio of the number of OECs to the unit length of the olfactory neuron axons ensheathed by them does not change markedly throughout the lifetime of mice in an undisturbed condition. By contrast, OEC proliferative density decreased rapidly in the 10-day to 1-month-old period, showing a significant difference, and for the rest of life remained at low level, similar to previous values reported for other glial cell types.