Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation

The biogenesis of autophagosomes depends on the conjugation of Atg8-like proteins with phosphatidylethanolamine. Atg8 processing by the cysteine protease Atg4 is required for its covalent linkage to phosphatidylethanolamine, but it is also necessary for Atg8 deconjugation from this lipid to release it from membranes. How these two cleavage steps are coordinated is unknown. Here we show that phosphorylation by Atg1 inhibits Atg4 function, an event that appears to exclusively occur at the site of autophagosome biogenesis. These results are consistent with a model where the Atg8-phosphatidylethanolamine pool essential for autophagosome formation is protected at least in part by Atg4 phosphorylation by Atg1 while newly synthesized cytoplasmic Atg8 remains susceptible to constitutive Atg4 processing

Microvascular Networks From Endothelial Cells and Mesenchymal Stromal Cells From Adipose Tissue and Bone Marrow: A Comparison

A promising approach to overcome hypoxic conditions in tissue engineered constructs is to use the potential of endothelial cells (EC) to form networks in vitro when co-cultured with a supporting cell type in a 3D environment. Adipose tissue-derived stromal cells (ASC) as well as bone marrow-derived stromal cells (BMSC) have been shown to support vessel formation of EC in vitro, but only very few studies compared the angiogenic potential of both cell types using the same model. Here, we aimed at investigating the ability of ASC and BMSC to induce network formation of EC in a co-culture model in fibrin. While vascular structures of BMSC and EC remained stable over the course of 3 weeks, ASC-EC co-cultures developed more junctions and higher network density within the same time frame. Both co-cultures showed positive staining for neural glial antigen 2 (NG2) and basal lamina proteins. This indicates that vessels matured and were surrounded by perivascular cells as well as matrix molecules involved in stabilization. Gene expression analysis revealed a significant increase of vascular endothelial growth factor (VEGF) expression in ASC-EC co-culture compared to BMSC-EC co-culture. These observations were donor-independent and highlight the importance of organotypic cell sources for vascular tissue engineering

A microarray analysis of two distinct lymphatic endothelial cell populations

We have recently identified lymphatic endothelial cells (LECs) to form two morphologically different populations, exhibiting significantly different surface protein expression levels of podoplanin, a major surface marker for this cell type. In vitro shockwave treatment (IVSWT) of LECs resulted in enrichment of the podoplaninhigh cell population and was accompanied by markedly increased cell proliferation, as well as 2D and 3D migration. Gene expression profiles of these distinct populations were established using Affymetrix microarray analyses. Here we provide additional details about our dataset (NCBI GEO accession number GSE62510) and describe how we analyzed the data to identify differently expressed genes in these two LEC populations

Microvascular networks from endothelial cells and mesenchymal stromal cells from adipose tissue and bone marrow: A comparison

A promising approach to overcome hypoxic conditions in tissue engineered constructs is to use the potential of endothelial cells (EC) to form networks in vitro when co-cultured with a supporting cell type in a 3D environment. Adipose tissue-derived stromal cells (ASC) as well as bone marrow-derived stromal cells (BMSC) have been shown to support vessel formation of EC in vitro, but only very few studies compared the angiogenic potential of both cell types using the same model. Here, we aimed at investigating the ability of ASC and BMSC to induce network formation of EC in a co-culture model in fibrin. While vascular structures of BMSC and EC remained stable over the course of 3 weeks, ASC-EC co-cultures developed more junctions and higher network density within the same time frame. Both co-cultures showed positive staining for neural glial antigen 2 (NG2) and basal lamina proteins. This indicates that vessels matured and were surrounded by perivascular cells as well as matrix molecules involved in stabilization. Gene expression analysis revealed a significant increase of vascular endothelial growth factor (VEGF) expression in ASC-EC co-culture compared to BMSC-EC co-culture. These observations were donor-independent and highlight the importance of organotypic cell sources for vascular tissue engineering

Microvascular networks from endothelial cells and mesenchymal stromal cells from adipose tissue and bone marrow:A comparison

\u3cp\u3eA promising approach to overcome hypoxic conditions in tissue engineered constructs is to use the potential of endothelial cells (EC) to form networks in vitro when co-cultured with a supporting cell type in a 3D environment. Adipose tissue-derived stromal cells (ASC) as well as bone marrow-derived stromal cells (BMSC) have been shown to support vessel formation of EC in vitro, but only very few studies compared the angiogenic potential of both cell types using the same model. Here, we aimed at investigating the ability of ASC and BMSC to induce network formation of EC in a co-culture model in fibrin. While vascular structures of BMSC and EC remained stable over the course of 3 weeks, ASC-EC co-cultures developed more junctions and higher network density within the same time frame. Both co-cultures showed positive staining for neural glial antigen 2 (NG2) and basal lamina proteins. This indicates that vessels matured and were surrounded by perivascular cells as well as matrix molecules involved in stabilization. Gene expression analysis revealed a significant increase of vascular endothelial growth factor (VEGF) expression in ASC-EC co-culture compared to BMSC-EC co-culture. These observations were donor-independent and highlight the importance of organotypic cell sources for vascular tissue engineering.\u3c/p\u3

Scientific Reports / Photobiomodulation (PBM) promotes angiogenesis in-vitro and in chick embryo chorioallantoic membrane model

The application of light in various therapeutic settings known as Photobiomodulation (PBM) is well established. Indications are the improvement of wound healing and tissue regeneration, scarring, and perfusion as well as pain therapy. Tissue perfusion is mandatory for successful wound healing. Nevertheless, there is a lack of mechanistic studies. We investigate the potential effect of PBM from light emitting diodes (LED) at 635nm, 80mW/cm, 24J/cm on angiogenesis in a two-part study: 1.) Investigation of the effect of PBM on the proliferation of endothelial cells and on vasculogenesis in a co-culture model of endothelial cells and stem cells. 2.) Investigation of the influence of PBM at chick egg chorioallantoic membrane (CAM) assays with fresh human skin xenografts. In both study phases, we observed a stimulating effect of PBM at 635nm; in part 1: for proliferation of HUVEC (human umbilical vein endothelial cells) (2583312859 versus 6300235760 cells/well, p<0.05, for cellular network formation (2.12.1 versus 4.63.5, p<0.05) and for less cell compactness p=0.01; in part 2: for the increase of number of vessel junctions per ROI (region of interest) (15.92.6 versus 20.85.4, p<0.05). Our results suggest significant promotion of angiogenesis by PBM at 635nm in vitro and in vivo.(VLID)472520

Medical 3D printing with polyjet technology: effect of material type and printing orientation on printability, surface structure and cytotoxicity

Abstract Due to its high printing resolution and ability to print multiple materials simultaneously, inkjet technology has found wide application in medicine. However, the biological safety of 3D-printed objects is not always guaranteed due to residues of uncured resins or support materials and must therefore be verified. The aim of this study was to evaluate the quality of standard assessment methods for determining the quality and properties of polyjet-printed scaffolds in terms of their dimensional accuracy, surface topography, and cytotoxic potential. Standardized 3D-printed samples were produced in two printing orientations (horizontal or vertical). Printing accuracy and surface roughness was assessed by size measurements, VR-5200 3D optical profilometer dimensional analysis, and scanning electron microscopy. Cytotoxicity tests were performed with a representative cell line (L929) in a comparative laboratory study. Individual experiments were performed with primary cells from clinically relevant tissues and with a Toxdent cytotoxicity assay. Dimensional measurements of printed discs indicated high print accuracy and reproducibility. Print accuracy was highest when specimens were printed in horizontal direction. In all cytotoxicity tests, the estimated mean cell viability was well above 70% (p < 0.0001) regardless of material and printing direction, confirming the low cytotoxicity of the final 3D-printed objects. Graphical Abstrac

Additional file 1 of Medical 3D printing with polyjet technology: effect of material type and printing orientation on printability, surface structure and cytotoxicity

Additional file 1

Gene expression profile of sorted subpopulations.

<p>(A) Transcripts with more than two-fold stronger expression in one of the respective populations (B) Heatmap visualization of 3 replicates showing log₂-transformed gene expression of podoplanin high and podoplanin low LECs. Affymetrix.CEL files were mas5 normalized. Log2-transformed expression values were normalized centred to the median of the 25 plotted transcripts.</p