Increased MMP activity in curved geometries disrupts the endothelial cell glycocalyx creating a proinflammatory environment.

Wall shear stress gradients (WSSGs) induce an inflammatory phenotype in endothelial cells (ECs) which is hypothesized to be mediated by mechanotransduction through the EC glycocalyx (GCX). We used a three-dimensional in vitro cell culture model with a 180o curved geometry to investigate if WSSGs created by curvature can cause EC inflammation and disruption of the GCX. The hydrodynamics of the model elicited a morphological response in ECs as well as a pattern of leukocyte adhesion towards the inner wall of curvature that was attenuated with enzymatic removal of GCX components. GCX degradation was also observed in regions of curvature which corresponded to increased activity of MMPs. Together, these results support the hypothesis that the EC GCX is involved in mechanotransduction of WSSGs and that components of the GCX are regulated by MMP activity in regions of curvature.

Glycocalyx Degradation Induces a Proinflammatory Phenotype and Increased Leukocyte Adhesion in Cultured Endothelial Cells under Flow.

Leukocyte adhesion to the endothelium is an early step in the pathogenesis of atherosclerosis. Effective adhesion requires the binding of leukocytes to their cognate receptors on the surface of endothelial cells. The glycocalyx covers the surface of endothelial cells and is important in the mechanotransduction of shear stress. This study aimed to identify the molecular mechanisms underlying the role of the glycocalyx in leukocyte adhesion under flow. We performed experiments using 3-D cell culture models, exposing human abdominal aortic endothelial cells to steady laminar shear stress (10 dynes/cm2 for 24 hours). We found that with the enzymatic degradation of the glycocalyx, endothelial cells developed a proinflammatory phenotype when exposed to uniform steady shear stress leading to an increase in leukocyte adhesion. Our results show an up-regulation of ICAM-1 with degradation compared to non-degraded controls (3-fold increase, p<0.05) and we attribute this effect to a de-regulation in NF-κB activity in response to flow. These results suggest that the glycocalyx is not solely a physical barrier to adhesion but rather plays an important role in governing the phenotype of endothelial cells, a key determinant in leukocyte adhesion. We provide evidence for how the destabilization of this structure may be an early and defining feature in the initiation of atherosclerosis.

Misfolded SOD1 associated with motor neuron mitochondria alters mitochondrial shape and distribution prior to clinical onset.

Mutations in superoxide dismutase (SOD1) are causative for inherited amyotrophic lateral sclerosis. A proportion of SOD1 mutant protein is misfolded onto the cytoplasmic face of mitochondria in one or more spinal cord cell types. By construction of mice in which mitochondrially targeted enhanced green fluorescent protein is selectively expressed in motor neurons, we demonstrate that axonal mitochondria of motor neurons are primary in vivo targets for misfolded SOD1. Mutant SOD1 alters axonal mitochondrial morphology and distribution, with dismutase active SOD1 causing mitochondrial clustering at the proximal side of Schmidt-Lanterman incisures within motor axons and dismutase inactive SOD1 producing aberrantly elongated axonal mitochondria beginning pre-symptomatically and increasing in severity as disease progresses. Somal mitochondria are altered by mutant SOD1, with loss of the characteristic cylindrical, networked morphology and its replacement by a less elongated, more spherical shape. These data indicate that mutant SOD1 binding to mitochondria disrupts normal mitochondrial distribution and size homeostasis as early pathogenic features of SOD1 mutant-mediated ALS.

TAR DNA-binding protein 43 (TDP-43) regulates stress granule dynamics via differential regulation of G3BP and TIA-1.

TAR deoxyribonucleic acid-binding protein 43 (TDP-43) is a multifunctional protein with roles in transcription, pre-messenger ribonucleic acid (mRNA) splicing, mRNA stability and transport. TDP-43 interacts with other heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP A2, via its C-terminus and several hnRNP family members are involved in the cellular stress response. This relationship led us to investigate the role of TDP-43 in cellular stress. Our results demonstrate that TDP-43 and hnRNP A2 are localized to stress granules (SGs), following oxidative stress, heat shock and exposure to thapsigargin. TDP-43 contributes to both the assembly and maintenance of SGs in response to oxidative stress and differentially regulates key SGs components, including TIA-1 and G3BP. The controlled aggregation of TIA-1 is disrupted in the absence of TDP-43 resulting in slowed SG formation. In addition, TDP-43 regulates the levels of G3BP mRNA, a SG nucleating factor. The disease-associated mutation TDP-43(R361S) is a loss-of-function mutation with regards to SG formation and confers alterations in levels of G3BP and TIA-1. In contrast, a second mutation TDP-43(D169G) does not impact this pathway. Thus, mutations in TDP-43 are mechanistically divergent. Finally, the cellular function of TDP-43 extends beyond splicing and places TDP-43 as a participant of the central cellular response to stress and an active player in RNA storage.

Altered neuronal densities in sexually dimorphic structures: comparable effects from perinatal magnetic fields with nitric oxide synthase inhibitors and postnatal hypoxia.

Cytometry of the neuronal density within four sexually dimorphic nuclei was completed for adult rats that had been perinatally exposed to 0.5Hz, 5-10nT magnetic fields or sham conditions while their mothers drank tap water containing the nitric oxide synthase (NOS) inhibitor L-NAME or only tap water. One week after birth the rats were rendered hypoxic for 1 min or served as controls. Exposures to either the magnetic field or the NOS inhibitor reduced the numbers of neurons within the bed nucleus of the stria terminalis by about 25%, whereas exposure to either the hypoxia or magnetic fields resulted in comparable decreases in cell numbers within the ventromedial nucleus (dorsomedial part). For comparison males had 15% fewer neurons in these nuclei compared to females. The effect sizes for the interactions involving the perinatal exposure for 8 days to the magnetic fields were comparable to the magnitudes of those associated with 1 min of hypoxia 1 week postnatally. These results show the sensitivity of specific structures of the developing brain to interactions between subtle environmental variables.