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: 137; Downloads: 120
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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: 131
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3. Release of growth factors after mechanical and chemical pleurodesis for treatment of malignant pleural effusion : a randomized control studyAljaž Hojski, Maja Leitgeb, Anton Crnjac, 2015, original scientific article Abstract: Growth factors are key inducers of fibrosis but can also mediate inflammatory responses resulting in increasing pleural effusion and acute respiratory distress syndrome. The primary aim of the study was to analyse growth factors release after performing chemical and mechanical pleurodesis in the first 48 hours at the patients with malignant pleural effusion. The secondary endpoints were to evaluate the effectiveness of the both pleurodeses, symptoms release and the quality of life of patients after the treatment. Patients and methods. A prospective randomized study included 36 consecutive female patients with breast carcinoma and malignant pleural effusion in an intention-to-treat analysis. We treated 18 patients by means of thoracoscopic mechanical pleurodesis and 18 patients by chemical pleurodesis with talcum applied over a chest tube. We gathered the pleural fluid and serum samples in the following 48 hours under a dedicated protocol and tested them for growth factors levels. A quality of life and visual analogue pain score surveys were also performed. Results. Median measured serum vascular endothelial growth factor (VEGF) level after chemical pleurodesis was 930.68 pg/ml (95% CI: 388.22-4656.65) and after mechanical pleurodesis 808.54 pg/ml. (95% CI: 463.20-1235.13) (p = 0.103). Median pleural levels of transforming growth factor (TGF) ß1 were higher after performing mechanical pleurodesis (4814.00 pg/ml [95% CI: 2726.51-7292.94]) when compared to those after performing chemical pleurodesis (1976.50 pg/ml [95% CI: 1659.82-5136.26]) (p = 0.078). We observed similar results for fibroblast growth factor (FGF) ß; the serum level was higher after mechanical pleurodesis (30.45 pg/ml [95% CI: 20.40-59.42]), compared to those after chemical pleurodesis (13.39 pg/ml [95% CI: 5.04-74.60]) (p = 0.076). Mechanical pleurodesis was equally effective as chemical pleurodesis in terms of hospital stay, pleural effusion re-accumulation, requiring of additional thoracentesis, median overall survival, but, it shortened the mean thoracic drainage duration (p = 0.030) and resulted in a higher symptoms release and in a better quality of life (p = 0.047). Conclusions. We recorded an increase in serum VEGF levels after chemical pleurodesis, however on the contrary, an increase in the pleural fluid level of TGF%1 and FGF%] after mechanical pleurodesis with respect to compared group. Although the differences did not reach statistical significance, VEGF, TGFß1 and FGFß remain the most interesting parameters for future research. Considering the mechanisms of growth factors action, we conclude that in our study group mechanical pleurodesis might be more efficient in terms of growth factors release, thoracic drainage duration and resulted in a higher symptoms release and in a better quality of life than chemical pleurodesis.
Keywords: malignant pleural effusion, pleurodesis, growth factors, quality of life
Published in DiRROS: 23.04.2024; Views: 378; Downloads: 328
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4. Engineered combinatorial cell device for wound healing and bone regenerationLucija Kadunc, Duško Lainšček, Rok Gašperšič, Petra Sušjan, Uroš Kovačič, Miha Butinar, Boris Turk, Roman Jerala, Iva Hafner Bratkovič, 2023, original scientific article Abstract: Growth factors are the key regulators that promote tissue regeneration and healing processes. While the effects of individual growth factors are well documented, a combination of multiple secreted growth factors underlies stem cell–mediated regeneration. To avoid the potential dangers and laborintensive individual approach of stem cell therapy while maintaining their regeneration-promoting effects based on multiple secreted growth factors, we engineered a “mix-and-match” combinatorial platform based on a library of cell lines producing growth factors. Treatment with a combination of growth factors secreted by engineered mammalian cells was more efficient than with individual growth factors or even stem cell–conditioned medium in a gap closure assay. Furthermore, we implemented in a mouse model a device for allogenic cell therapy for an in situ production of growth factors, where it improved cutaneous wound healing. Augmented bone regeneration was achieved on calvarial bone defects in rats treated with a cell device secreting IGF, FGF, PDGF, TGF-β, and VEGF. In both in vivo models, the systemic concentration of secreted factors was negligible, demonstrating the local effect of the regeneration device. Finally, we introduced a genetic switch that enables temporal control over combinations of trophic factors released at different stages of regeneration mimicking the maturation of natural wound healing to improve therapy and prevent scar formation.
Keywords: wound healing, bone regeneration, growth factors
Published in DiRROS: 01.06.2023; Views: 712; Downloads: 290
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