1. IGF2 and IGF1R identified as novel tip cell genes in primary microvascular endothelial cell monolayersMarchien G. Dallinga, Bahar Yetkin-Arik, Richelle P. Kayser, Ilse M.C. Vogels, Patrycja Nowak-Sliwinska, Arjan W. Griffioen, Cornelis J. F. van Noorden, Ingeborg Klaassen, Reinier O. Schlingemann, 2018, original scientific article Abstract: Tip cells, the leading cells of angiogenic sprouts, were identified in cultures of human umbilical vein endothelial cells (HUVECs) by using CD34 as a marker. Here, we show that tip cells are also present in primary human microvascular endothelial cells (hMVECs), a more relevant endothelial cell type for angiogenesis. By means of flow cytometry, immunocytochemistry, and qPCR, it is shown that endothelial cell cultures contain a dynamic population of CD34+ cells with many hallmarks of tip cells, including filopodia-like extensions, elevated mRNA levels of known tip cell genes, and responsiveness to stimulation with VEGF and inhibition by DLL4. Furthermore, we demonstrate that our in vitro tip cell model can be exploited to investigate cellular and molecular mechanisms in tip cells and to discover novel targets for anti-angiogenesis therapy in patients. Small interfering RNA (siRNA) was used to knockdown gene expression of the known tip cell genes angiopoietin 2 (ANGPT2) and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), which resulted in similar effects on tip cells and sprouting as compared to inhibition of tip cells in vivo. Finally, we identified two novel tip cell-specific genes in CD34+ tip cells in vitro: insulin-like growth factor 2 (IGF2) and IGF-1-receptor (IGF1R). Knockdown of these genes resulted in a significant decrease in the fraction of tip cells and in the extent of sprouting in vitro and in vivo. In conclusion, this study shows that by using our in vitro tip cell model, two novel essential tip cells genes are identified.
Keywords: Angiogenesis, tip cells, CD34, IGF2, endothelial cells, cultured cells, endothelial growth factors
Published in DiRROS: 24.07.2024; Views: 136; Downloads: 115
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2. IGF-binding proteins 3 and 4 are regulators of sprouting angiogenesisMarchien G. Dallinga, Yasmin I. Habani, Richelle P. Kayser, Cornelis J. F. van Noorden, Ingeborg Klaassen, Reinier O. Schlingemann, 2020, original scientific article Abstract: Purpose
We have previously identified insulin-like growth factor 2 (IGF2) and insulin-like growth factor 1 receptor (IGF1R) as essential proteins for tip cell maintenance and sprouting angiogenesis. In this study, we aim to identify other IGF family members involved in endothelial sprouting angiogenesis.
Methods
Effects on sprouting were analyzed in human umbilical vein endothelial cells (HUVECs) using the spheroid-based sprouting model, and were quantified as mean number of sprouts per spheroid and average sprout length. RNA silencing technology was used to knockdown gene expression. Recombinant forms of the ligands (IGF1 and IGF2, insulin) and the IGF-binding proteins (IGFBP) 3 and 4 were used to induce excess effects. Effects on the tip cell phenotype were analyzed by measuring the fraction of CD34+ tip cells using flow cytometry and immunohistochemistry in a 3D angiogenesis model. Experiments were performed in the presence and absence of serum.
Results
Knockdown of IGF2 inhibited sprouting in HUVECs, in particular when cultured in the absence of serum, suggesting that components in serum influence the signaling of IGF2 in angiogenesis in vitro. We then determined the effects of IGFBP3 and IGFBP4, which are both present in serum, on IGF2-IGF1R signaling in sprouting angiogenesis in the absence of serum: knockdown of IGFBP3 significantly reduced sprouting angiogenesis, whereas knockdown of IGFBP4 resulted in increased sprouting angiogenesis in both flow cytometry analysis and immunohistochemical analysis of the 3D angiogenesis model. Other IGF family members except INSR did not affect IGF2-IGF1R signaling.
Conclusions
Serum components and IGF binding proteins regulate IGF2 effects on sprouting angiogenesis. Whereas IGFBP3 acts as co-factor for IGF2-IGF1R binding, IGFBP4 inhibits IGF2 signaling.
Keywords: Angiogenesis, tip cells, IGF2, IGF binding proteins, endothelial cells, cultured cells, endothelial growth factors
Published in DiRROS: 23.07.2024; Views: 112; Downloads: 128
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3. Cysteine cathepsins, stefins and extracellular matrix degradation during invasion of transformed human breast cell linesIrena Zajc, Aleš Bervar, Tamara Lah Turnšek, 2006, original scientific article Abstract: Background. Human breast cellular model, comprising four cell lines originating from spontaneously immortalized human breast epithelial MCF10A cell line, its c-Ha-ras transfectant, MCF10AT, and two tumourigenic derivatives, cultured from two sequential mouse xenographs, MCF10AT-Ca1a and MCF10AT-Ca1d, were used to compare the relative protein concentration of cathepsins and stefins in single cells. Methods. The relative protein concentration of cathepsins and stefins in single cells was analysed by confocal microscopy, and compared to their protein expression in cell homogenates. Results. The most invasive, MCF10AT cell line contained several fold higher protein concentration of cathepsin B and increased levels of stefins, but similar levels of cathepsin L, compared with the parental MCF10A cells. This was associated with five fold higher endocytosis of Matrigel-DQ-collagen IV (DQC) and a simultaneous increase in signal overlap between DQC and cathepsin L as well as DQC and stefin B, but a decrease in that of DQC and cathepsin B overlap in the MCF10AT cells. Simultaneously, increased signal overlaps between both cathepsins and between cathepsins-stefins pairs, were observed in this cell line. Conclusions. These results suggest that the increased collagen endocytosis and degradation in theinvasive phenotype significantly affect also the subcellular localization of cysteine cathepsins and stefins. Based on these and the reports of other authors, we hypothesize that the intracellular degradation may also be assoeiated with cathepsin L, whereas cathepsin B in the ras transformed breastcells is involved in both, the intracellular and pericellular degradation of extracellular matrix during cell migration and invasion.
Keywords: breast neoplasms, tumor cells cultured, neoplasms invasiveness, cathepsins, extracellular matrix
Published in DiRROS: 15.02.2024; Views: 344; Downloads: 83
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5. Natural porcine interferon gamma (PoIFN gamma)Bratko Filipič, Sonja Rozman, Katarina Carlsson, Avrelija Cencič, 1993, original scientific article Abstract: The natural porcine motogen induced gamma interferon (PoIFN gamma) was studied and compared with the human inerferons gamma (HuIFN gamma) and alpha (HuIFN alpha). A comparison was performed by the following criteria: pH 2.0 and 56 C stability, molecular weight, dot-blot reactions and cross reactivity (neutralisation index). The biological activity of porcine and human interferons in vitro (antiviral and antiproliferative) was correlated on humannontransformed (HEF) and transformed cells (FL).
Keywords: interferon type II-analysis, cells, cultured-drug effects
Published in DiRROS: 10.01.2024; Views: 309; Downloads: 97
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