Mitigation of human iris angiogenesis through uPAR/LRP-1 interaction antagonism in an organotypic ex vivo model.

Rubeosis Iridis (RI) is characterized by an increase in neovascularization and inflammation factors in the iris. During angiogenesis, the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a pivotal role in extracellular matrix remodeling, where uPAR regulates endothelial cell migration and proliferation through assembly with transmembrane receptors. Here, in the context of hypoxia-induced angiogenesis, the uPA/uPAR system blockage was investigated by using UPARANT in a novel ex vivo human iris organotypic angiogenesis assay. The effects of uPA/uPAR system antagonism in the humanized model of ocular pathologic angiogenesis were analyzed by sprouting angiogenesis and protein assays (western, dot blots, and co-immunoprecipitation) and correlated to vascular endothelial growth factor (VEGF) inhibition. Phosphoprotein and co-immunoprecipitation assay illustrated an unidentified antagonism of UPARANT in the interaction of uPAR with the low-density lipoprotein receptor-related protein-1 (LRP-1), resulting in inhibition of ß-catenin-mediated angiogenesis in this model. The effects of uPA/uPAR system inhibition were focal to endothelial cells ex vivo. Comparison between human iris endothelial cells and human retinal endothelial revealed an endothelial-specific mechanism of ß-catenin-mediated angiogenesis inhibited by uPA/uPAR system blockage and not by VEGF inhibition. Collectively, these findings broaden the understanding of the effects of the uPA/uPAR system antagonism in the context of angiogenesis, revealing non-canonical ß-catenin downstream effects mediated by LRP-1/uPAR interaction.

The Methyltransferase Smyd1 Mediates LPS-Triggered Up-Regulation of IL-6 in Endothelial Cells.

The lysine methyltransferase Smyd1 with its characteristic catalytic SET-domain is highly enriched in the embryonic heart and skeletal muscles, participating in cardiomyogenesis, sarcomere assembly and chromatin remodeling. Recently, significant Smyd1 levels were discovered in endothelial cells (ECs) that responded to inflammatory cytokines. Based on these biochemical properties, we hypothesized that Smyd1 is involved in inflammation-triggered signaling in ECs and therefore, investigated its role within the LPS-induced signaling cascade. Human endothelial cells (HUVECs and EA.hy926 cells) responded to LPS stimulation with higher intrinsic Smyd1 expression. By transfection with expression vectors containing gene inserts encoding either intact Smyd1, a catalytically inactive Smyd1-mutant or Smyd1-specific siRNAs, we show that Smyd1 contributes to LPS-triggered expression and secretion of IL-6 in EA.hy926 cells. Further molecular analysis revealed this process to be based on two signaling pathways: Smyd1 increased the activity of NF-κB and promoted the trimethylation of lysine-4 of histone-3 (H3K4me3) within the IL-6 promoter, as shown by ChIP-RT-qPCR combined with IL-6-promoter-driven luciferase reporter gene assays. In summary, our experimental analysis revealed that LPS-binding to ECs leads to the up-regulation of Smyd1 expression to transduce the signal for IL-6 up-regulation via activation of the established NF-κB pathway as well as via epigenetic trimethylation of H3K4.

Structural Microangiopathies in Skeletal Muscle Related to Systemic Vascular Pathologies in Humans.

It is unclear how microangiopathic changes in skeletal muscle vary among systemic vascular pathologies. We therefore analyzed the capillary fine structure in skeletal muscle from patients with arterial hypertension (HYPT), diabetes mellitus type 2 (T2DM) or intermittent claudication - peripheral arterial disease (IC/PAD). Tablet-based image analysis (TBIA) was carried out to largely re-evaluate 5,000 transmission electron micrographs of capillaries from 126 vastus lateralis biopsies of 75 individuals (HYPT, T2DM or IC/PAD patients as well as healthy individuals before and after endurance exercise training) used in previous morphometric studies, but assessed using stereological counting grids of different sizes. Serial block-face scanning electron microscopy (SBFSEM) of mouse skeletal muscle was used for validation of the particular fine structural events observed in human biopsies. The peri-capillary basement membrane (BM) was 38.5 and 45.5% thicker (P < 0.05) in T2DM and IC/PAD patients than in the other groups. A 17.7-39.6% lower (P < 0.05) index for intraluminal endothelial cell (EC) surface enlargement by projections was exclusively found in T2DM patients by TBIA morphometry. The proportion of capillaries with disrupted BM between pericytes (PC) and EC was higher (P < 0.05) in HYPT (33.2%) and T2DM (38.7%) patients than in the control group. Empty EC-sockets were more frequent (P < 0.05) in the three patient groups (20.6% in HYPT, 27.1% in T2DM, 30.0% in IC/PAD) than in the healthy individuals. SBFSEM confirmed that EC-sockets may exhibit close proximity to the capillary lumen. Our comparative morphometric analysis demonstrated that structural arrangement of skeletal muscle capillaries is more affected in T2DM than in HYPT or IC/PAD, although some similar elements of remodeling were found. The increased frequency of empty EC-sockets in the three patient groups indicates that the PC-EC interaction is commonly disturbed in these systemic vascular pathologies.

ZNF580 - a brake on Interleukin-6.

BACKGROUND: Zinc finger protein 580 (ZNF580) was reported to modulate angiogenesis, endothelial homeostasis and blood pressure control. ZNF580 regulated genes include VEGF-A and IL-8. However, it is unknown if ZNF580 could play a role during inflammation. The aim of this study was to find out if ZNF580 affects the expression of IL-6, if it occurs in monocytic cells and responds to inflammatory mediators. RESULTS: Overexpression of ZNF580 reduced LPS-induced promotor activity of IL-6. Consistently, overexpression of ZNF580 reduced by half the LPS-induced expression of IL-6. ZNF580 was strongly expressed in the nucleus of MonoMac6, a human monocytic cell line. LPS-stimulated IL-6 secretion increased when ZNF580 was suppressed with siRNA. After stimulation of MonoMac6 with LPS for 24 h, ZNF580 negatively correlated with the amount of secreted IL-6. In response to LPS, ZNF580 was increased within the first 8 h, followed by a marked decrease after 16 h. This decrease coincided with sustained IL-6 production. CONCLUSION: This study demonstrated that ZNF580 inhibits LPS-induced expression of IL-6. ZNF580 was highly expressed in monocytic cells and therefore may contribute to the modulation of its IL-6 production, at least in response to LPS. This suggests cooperation between ZNF580 and NFκB, which could play a role during sepsis.