Reactive Oxygen Species Modulate the Barrier Function of the Human Glomerular Endothelial Glycocalyx

Reactive oxygen species (ROS) play a key role in the pathogenesis of proteinuria in glomerular diseases like diabetic nephropathy. Glomerular endothelial cell (GEnC) glycocalyx covers the luminal aspect of the glomerular capillary wall and makes an important contribution to the glomerular barrier. ROS are known to depolymerise glycosaminoglycan (GAG) chains of proteoglycans, which are crucial for the barrier function of GEnC glycocalyx. The aim of this study is to investigate the direct effects of ROS on the structure and function of GEnC glycocalyx using conditionally immortalised human GEnC. ROS were generated by exogenous hydrogen peroxide. Biosynthesis and cleavage of GAG chains was analyzed by radiolabelling (S³⁵ and ³H-glucosamine). GAG chains were quantified on GEnC surface and in the cell supernatant using liquid chromatography and immunofluorescence techniques. Barrier properties were estimated by measuring trans-endothelial passage of albumin. ROS caused a significant loss of WGA lectin and heparan sulphate staining from the surface of GEnC. This lead to an increase in trans-endothelial albumin passage. The latter could be inhibited by catalase and superoxide dismutase. The effect of ROS on GEnC was not mediated via the GAG biosynthetic pathway. Quantification of radiolabelled GAG fractions in the supernatant confirmed that ROS directly caused shedding of HS GAG. This finding is clinically relevant and suggests a mechanism by which ROS may cause proteinuria in clinical conditions associated with high oxidative stress.     

Critical Structure Sparing in Stereotactic Ablative Radiotherapy for Central Lung Lesions: Helical Tomotherapy vs. Volumetric Modulated Arc Therapy

Background

Helical tomotherapy (HT) and volumetric modulated arc therapy (VMAT) are both advanced techniques of delivering intensity-modulated radiotherapy (IMRT). Here, we conduct a study to compare HT and partial-arc VMAT in their ability to spare organs at risk (OARs) when stereotactic ablative radiotherapy (SABR) is delivered to treat centrally located early stage non-small-cell lung cancer or lung metastases.

Methods

12 patients with centrally located lung lesions were randomly chosen. HT, 2 & 8 arc (Smart Arc, Pinnacle v9.0) plans were generated to deliver 70 Gy in 10 fractions to the planning target volume (PTV). Target and OAR dose parameters were compared. Each technique’s ability to meet dose constraints was further investigated.

Results

HT and VMAT plans generated essentially equivalent PTV coverage and dose conformality indices, while a trend for improved dose homogeneity by increasing from 2 to 8 arcs was observed with VMAT. Increasing the number of arcs with VMAT also led to some improvement in OAR sparing. After normalizing to OAR dose constraints, HT was found to be superior to 2 or 8-arc VMAT for optimal OAR sparing (meeting all the dose constraints) (p = 0.0004). All dose constraints were met in HT plans. Increasing from 2 to 8 arcs could not help achieve optimal OAR sparing for 4 patients. 2/4 of them had 3 immediately adjacent structures.

Conclusion

HT appears to be superior to VMAT in OAR sparing mainly in cases which require conformal dose avoidance of multiple immediately adjacent OARs. For such cases, increasing the number of arcs in VMAT cannot significantly improve OAR sparing.     

ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N₃-ATP) and 8-azidoadenosine 5′-monophosphate (8-N₃-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N₃-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N₃-ATP at ATP-binding site 2. The adenylate kinase active center probe P¹,P⁵-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.     

Subclinical Thyroid Dysfunction and Cognitive Decline in Old Age

Background

Subclinical thyroid dysfunction has been implicated as a risk factor for cognitive decline in old age, but results are inconsistent. We investigated the association between subclinical thyroid dysfunction and cognitive decline in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER).

Methods

Prospective longitudinal study of men and women aged 70–82 years with pre-existing vascular disease or more than one risk factor to develop this condition (N = 5,154). Participants taking antithyroid medications, thyroid hormone supplementation and/or amiodarone were excluded. Thyroid function was measured at baseline: subclinical hyper- and hypothyroidism were defined as thyroid stimulating hormones (TSH) <0.45 mU/L or >4.50 mU/L respectively, with normal levels of free thyroxine (FT4). Cognitive performance was tested at baseline and at four subsequent time points during a mean follow-up of 3 years, using five neuropsychological performance tests.

Results

Subclinical hyperthyroidism and hypothyroidism were found in 65 and 161 participants, respectively. We found no consistent association of subclinical hyper- or hypothyroidism with altered cognitive performance compared to euthyroid participants on the individual cognitive tests. Similarly, there was no association with rate of cognitive decline during follow-up.

Conclusion

We found no consistent evidence that subclinical hyper- or hypothyroidism contribute to cognitive impairment or decline in old age. Although our data are not in support of treatment of subclinical thyroid dysfunction to prevent cognitive dysfunction in later life, only large randomized controlled trials can provide definitive evidence.     

Structure and function insights garnered from in silico modeling of the thrombospondin type-1 domain-containing 7A antigen

The thrombospondin type-1 domain containing 7A (THSD7A) protein is known to be one of the antigens responsible for the autoimmune disorder idiopathic membranous nephropathy. The structure of this antigen is currently unsolved experimentally. Here we present a homology model of the extracellular portion of the THSD7A antigen. The structure was evaluated for folding patterns, epitope site prediction, and function was predicted. Results show that this protein contains 21 extracellular domains and with the exception of the first two domains, has a regular repeating pattern of TSP-1-like followed by F-spondin-like domains. Our results indicate the presence of a novel Trp-ladder sequence of WxxxxW in the TSP-1-like domains. Of the 21 domains, 18 were shown to have epitope binding sites as predicted by epitopia. Several of the F-spondin-like domains have insertions in the canonical TSP fold, most notably the coiled coil region in domain 4, which may be utilized in protein-protein binding interactions, suggesting that this protein functions as a heparan sulfate binding site.     

VEGF receptor 2/‐3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/‐3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/‐3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.     

Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer

Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. Whereas AAV serotype 2-mediated gene transfer to muscle has partially replaced factor IX deficiency in hemophilia patients, its ability to mediate gene transfer to the lungs for cystic fibrosis is hindered by lack of apical receptors. However, AAV serotype 5 infects human airway epithelia from the lumenal surface. We found that in contrast to AAV2, the apical membrane of airway epithelia contains abundant high affinity receptors for AAV5. Binding and gene transfer with AAV5 was abolished by genetic or enzymatic removal of sialic acid from the cell surface. Furthermore, binding and gene transfer to airway epithelia was competed by lectins that specifically bind 2,3-linked sialic acid. These observations suggest that 2,3-linked sialic acid is either a receptor for AAV5 or it is a necessary component of a receptor complex. Further elucidation of the receptor for this virus should enhance understanding of parvovirus biology and expand the therapeutic targets for AAV vectors.