| 1. |
- Fischer, Silvia, et al.
(författare)
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Extracellular RNA Liberates Tumor Necrosis Factor-alpha to Promote Tumor Cell Trafficking and Progression
- 2013
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 73:16, s. 5080-5089
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular RNA (eRNA) released from injured cells promotes tissue permeability, thrombosis, and inflammation in vitro and in vivo, and RNase1 pretreatment can reduce all these effects. In this study, we investigated the role of the eRNA/RNase1 system in tumor progression and metastasis. Under quiescent and stimulatory conditions, tumor cells released much higher levels of endogenous extracellular RNA (eRNA) than nontumor cells. In glioblastomas, eRNA was detected at higher levels in tumors than nontumor tissue. eRNA induced tumor cells to adhere to and migrate through human cerebral microvascular endothelial cells (HCMEC/D3), in a manner requiring activation of VEGF signaling. In addition, eRNA liberated TNF-alpha from macrophages in a manner requiring activation of the TNF-alpha-converting enzyme TACE. Accordingly, supernatants derived from eRNA-treated macrophages enhanced tumor cell adhesion to HCMEC/D3. TNF-alpha release evoked by eRNA relied upon signaling activation of mitogen-activated protein kinases and the NF-kappa B pathway. In subcutaneous xenograft models of human cancer, administration of RNase1 but not DNase decreased tumor volume and weight. Taken together, these results suggest that eRNA released from tumor cells has the capacity to promote tumor cell invasion through endothelial barriers by both direct and indirect mechanisms, including through a mechanism involving TNF-alpha release from tumor-infiltrating monocytes/macrophages. Our findings establish a crucial role for eRNA in driving tumor progression, and they suggest applications for extracellular RNase1 as an antiinvasive regimen for cancer treatment.
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| 2. |
- Heiss, Maximilian, et al.
(författare)
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Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro
- 2015
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 29:7, s. 3076-3084
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Tidskriftsartikel (refereegranskat)abstract
- Given the need for robust and cost-efficient in vitro models to study angiogenesis and reproducibly analyze potential pro-and antiangiogenic compounds in preclinical studies, we developed a 3-dimensional in vitro angiogenesis assay that is based on collagen gel-embedded, size-defined spheroids generated from cultured human umbilical vein endothelial cells (HUVECs). Despite its wide distribution, limitations, sensitivity, robustness, and improvements, the capacity of this assay for functional screening purposes has not been elucidated thus far. By using time-lapse video microscopy, we show that tip cells lead the formation of capillary-like and partially lumenized sprouts originating from the spheroids. Angiogenic sprouting from spheroids generated from 5 different primary cultured human endothelial cell types was induced by physiologic concentrations of vascular endothelial cell growth factor 165. Based on this assay system, we determined the capacity of 880 approved drugs to interfere with or boost angiogenic sprouting, thereby assessing their putative angiogenesis-related side effects or novel applications. However, although this assay allowed for a rapid and reproducible determination of functional IC50 values of individual compounds, the sprouting results were partially affected by the HUVEC passage number and donor variability. To overcome this limitation, immortalized HUVECs (iHUVECs) showing a more homogenous response in terms of proliferation and sprouting over multiple population doublings were used in the course of this study. Collectively, the spheroid-based angiogenesis assay provides a sensitive and versatile tool to study the impact of pro-and antiangiogenic determinants on multiple steps of the angiogenic cascade. It is compatible with different endothelial cell types and allows use of iHUVECs to improve its overall robustness.
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| 3. |
- Korhonen, Emilia A., et al.
(författare)
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Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression
- 2022
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Ingår i: Journal of Clinical Investigation. - : American Society for Clinical Investigation. - 0021-9738 .- 1558-8238. ; 132:15
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Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C???induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110?? subunit or with small -molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C???induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.
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| 4. |
- Nowak-Sliwinska, Patrycja, et al.
(författare)
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Consensus guidelines for the use and interpretation of angiogenesis assays
- 2018
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Ingår i: Angiogenesis. - : Springer. - 0969-6970 .- 1573-7209. ; 21:3, s. 425-532
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Forskningsöversikt (refereegranskat)abstract
- The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.
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| 5. |
- Roth, Lise, et al.
(författare)
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Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation
- 2016
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 9:425
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Tidskriftsartikel (refereegranskat)abstract
- Neuropilin-1 (NRP1) regulates developmental and pathological angiogenesis, arteriogenesis, and vascular permeability, acting as a coreceptor for semaphorin 3A (Sema3A) and the 165-amino acid isoform of vascular endothelial growth factor A (VEGF-A(165)). NRP1 is also the receptor for the CendR peptides, a class of cell-and tissue-penetrating peptides with a specific R-x-x-R carboxyl-terminal motif. Because the cytoplasmic domain of NRP1 lacks catalytic activity, NRP1 is mainly thought to act through the recruitment and binding to other receptors. We report here that the NRP1 intracellular domain mediates vascular permeability. Stimulation with VEGF-A(165), a ligand-blocking antibody, and a CendR peptide led to NRP1 accumulation at cell-cell contacts in endothelial cell monolayers, increased cellular permeability in vitro and vascular leakage in vivo. Biochemical analyses, VEGF receptor-2 (VEGFR-2) silencing, and the use of a specific VEGFR blocker established that the effects induced by the CendR peptide and the antibody were independent of VEGFR-2. Moreover, leakage assays in mice expressing a mutant NRP1 lacking the cytoplasmic domain revealed that this domain was required for NRP1-induced vascular permeability in vivo. Hence, these data define a vascular permeability pathway mediated by NRP1 but independent of VEGFR-2 activation.
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| 6. |
- Tisch, Nathalie, et al.
(författare)
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Caspase-8 modulates physiological and pathological angiogenesis during retina development
- 2019
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Ingår i: Journal of Clinical Investigation. - : AMER SOC CLINICAL INVESTIGATION INC. - 0021-9738 .- 1558-8238. ; 129:12, s. 5092-5107
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Tidskriftsartikel (refereegranskat)abstract
- During developmental angiogenesis, blood vessels grow and remodel to ultimately build a hierarchical vascular network. Whether, how, cell death signaling molecules contribute to blood vessel formation is still not well understood. Caspase-8 (Casp-8), a key protease in the extrinsic cell death-signaling pathway, regulates cell death via both apoptosis and necroptosis. Here, we show that expression of Casp-8 in endothelial cells (ECs) is required for proper postnatal retina angiogenesis. EC-specific Casp-8-KO pups (Casp-8(ECKO)) showed reduced retina angiogenesis, as the loss of Casp-8 reduced EC proliferation, sprouting, and migration independently of its cell death function. Instead, the loss of Casp-8 caused hyperactivation of p38 MAPK downstream of receptor-interacting serine/threonine protein kinase 3 (RIPK3) and destabilization of vascular endothelial cadherin (VE-cadherin) at EC junctions. In a mouse model of oxygen-induced retinopathy (OIR) resembling retinopathy of prematurity (ROP), loss of Casp-8 in ECs was beneficial, as pathological neovascularization was reduced in Casp-8ECKO pups. Taking these data together, we show that Casp-8 acts in a cell death-independent manner in ECs to regulate the formation of the retina vasculature and that Casp-8 in ECs is mechanistically involved in the pathophysiology of ROP.
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| 7. |
- Veikkola, Tanja, et al.
(författare)
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Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function.
- 2003
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 17:14
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Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial cells are characterized by a high degree of functional and phenotypic plasticity, which is controlled both by their pericellular microenvironment and their intracellular gene expression programs. To gain further insight into the mechanisms regulating the endothelial cell phenotype, we have compared the responses of lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs) to vascular endothelial growth factors (VEGFs). VEGFR-3-specific signals are sufficient for LEC but not BEC proliferation, as shown by the ability of the specific ligand VEGF-C156S to stimulate cell cycle entry only in LECs. On the other hand, we found that VEGFR-3 stimulation did not induce LEC cell shape changes typical of VEGFR-2-stimulated LECs, indicating receptor-specific differences in the cytoskeletal responses. Genes induced via VEGFR-2 also differed between BECs and LECs: angiopoietin-2 (Ang-2) was induced via VEGFR-2 in BECs and LECs, but the smooth muscle cell (SMC) chemoattractant BMP-2 was induced only in BECs. Both BECs and LECs were able to promote SMC chemotaxis, but contact with SMCs led to down-regulation of VEGFR-3 expression in BECs in a 3-dimensional coculture system. This was consistent with the finding that VEGFR-3 is down-regulated in vivo at sites of endothelial cell-pericyte/smooth muscle cell contacts. Collectively, these data show intrinsic cell-specific differences of BEC and LEC responses to VEGFs and identify a pericellular regulatory mechanism for VEGFR-3 down-regulation in endothelial cells.
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