Sanne Verbruggen

Feed-forward signaling by membrane-bound ligand receptor circuit: the case of NOTCH DELTA-like 4 ligand in endothelial cells.

The DELTA like-4 ligand (DLL4) belongs to the highly conserved NOTCH family and is specifically expressed in the endothelium. DLL4 regulates crucial processes in vascular growth, including endothelial cell (EC) sprouting and arterial specification. Its expression is increased by VEGF-A. In the present study, we show that VEGF-induced DLL4 expression depends on NOTCH activation. VEGF-induced DLL4 expression was prevented by the blockage of NOTCH signaling with γ-secretase or ADAM inhibitors in human cardiac microvascular ECs. Similar to VEGF-A, recombinant DLL4 itself stimulated NOTCH signaling and resulted in up-regulation of DLL4, suggesting a positive feed-forward mechanism. These effects were abrogated by NOTCH inhibitors but not by inhibition of VEGF signaling. NOTCH activation alone suffices to induce DLL4 expression as illustrated by the positive effect of NOTCH intracellular domain (NICD)-1 or -4 overexpression. To discriminate between NICD/RBP-Jκ and FOXC2-regulated DLL4 expression, DLL4 promoter activity was assessed in promoter deletion experiments. NICD induced promoter activity was dependent on RBP-Jκ site but independent of the FOXC2 binding site. Accordingly, constitutively active FOXC2 did not affect DLL4 expression. The notion that the positive feed-forward mechanism might propagate NOTCH activation to neighboring ECs was supported by our observation that DLL4-eGFP-transfected ECs induced DLL4 expression in nontransfected cells in their vicinity. In summary, our data provide evidence for a mechanism by which VEGF or ligand-induced NOTCH signaling up-regulates DLL4 through a positive feed-forward mechanism. By this mechanism, DLL4 could propagate its own expression and enable synchronization of NOTCH expression and signaling between ECs.

Soluble Jagged-1 Inhibits Neointima Formation by Attenuating Notch-Herp2 Signaling

Objective—Notch has been implicated in neointima formation as reflected by increased Notch/Jagged expression on vascular injury and the promigratory effect of Notch signaling on smooth muscle cells. Soluble Jagged-1 (sJag1) has been shown to inhibit Notch signaling in vitro; however, its capacity to suppress neointima formation remains unknown. Methods and Results—Balloon injury of rat carotid arteries induced Notch1, Notch3, and Jagged-1 expression at days 3 and 14 postinjury. Notch signaling was activated as shown by increased expression of the Notch target gene Herp2. Adenoviral sJag1 (Ad-sJag1) transfection reduced neointima formation in carotid artery and enhanced reendothelialization, whereas adenoviral full-length Jagged-1 (Ad-Fl-Jag1) or LacZ had no effect. Injury-induced Herp2 expression was absent in vessels treated with Ad-sJag1. Consistently, Herp2 expression was reduced in Ad-sJag1-infected or recombinant sJag1 –treated coronary artery smooth muscle cells (CASMCs). Ad-sJag1 had no effect on human umbilical endothelial cell behavior, but it significantly reduced proliferation and migration of CASMCs. Overexpression of Herp2 in sJag1-treated CASMCs rescued the migratory and proliferative capacity in vitro. Conclusion—Our results demonstrate that sJag1 can inhibit neointima formation after balloon injury by decreasing smooth muscle cell proliferation and migration through interference with Notch-Herp2 signaling.

Alternatives for large-scale production of cultured beef: A review

Cultured beef is a method where stem cells from skeletal muscle of cows are cultured in vitro to gain edible muscle tissue. For large-scale production of cultured beef, the culture technique needs to become more efficient than todays 2-dimensional (2D) standard technique that was used to make the first cultured hamburger. Options for efficient large-scale production of stem cells are to culture cells on microcarriers, either in suspension or in a packed bed bioreactor, or to culture aggregated cells in suspension. We discuss the pros and cons of these systems as well as the possibilities to use the systems for tissue culture. Either of the production systems needs to be optimized to achieve an efficient production of cultured beef. It is anticipated that the optimization of large-scale cell culture as performed for other stem cells can be translated into successful protocols for bovine satellite cells resulting in resource and cost efficient cultured beef.

ADAM10 and ADAM17 have opposite roles during sprouting angiogenesis

During angiogenesis, endothelial tip cells start sprouting and express delta-like 4 (DLL4) downstream of vascular endothelial growth factor (VEGF). DLL4 subsequently activates Notch in the adjacent stalk cells suppressing sprouting. VEGF also activates A disintegrin and metalloproteases (ADAMs) that induce Notch ectodomain shedding. Although two major ADAMs, i.e. ADAM10 and ADAM17, have been implicated in Notch-signalling activation, their apparent different roles in angiogenesis have not been fully understood yet. The objective of this study was to determine the roles of ADAM10 and ADAM17 activity in angiogenesis. In mouse retinas, ADAM10 or γ-secretase inhibition induced vascular sprouting and density in vivo, whereas attenuation of both ADAM10 and ADAM17 activity produced the opposite phenotype. Retinal blood vessel analysis in ADAM17 hypomorphic mice confirmed the requirement for ADAM17 activity in angiogenesis. However, ADAM17 inhibition did not phenocopy blood vessel increase by Notch blockage. These observations suggest that ADAM17 regulates other fundamental players during angiogenesis besides Notch, which were not affected by ADAM10. By means of an angiogenesis proteome assay, we found that ADAM17 inhibition induced the expression of a naturally occurring inhibitor of angiogenesis Thrombospondin 1 (TSP1), whereas ADAM10 inhibition did not. Accordingly, ADAM17 overexpression downregulated TSP1 expression, and the TSP1 inhibitor LSKL rescued angiogenesis in the tube formation assay downstream of VEGF in the presence of ADAM17 inhibition. Finally, genetic and pharmacological ADAM17 blockade resulted in increased TSP1 expression in mouse retina. Altogether, our results show that ADAM10 and ADAM17 have opposite effects on sprouting angiogenesis that may be unrelated to Notch signalling and involves differentially expressed anti-angiogenic proteins such as TSP1.

Erratum to: CXCL1 promotes arteriogenesis through enhanced monocyte recruitment into the peri-collateral space.

Aims The mechanisms of monocyte recruitment to arteriogenic collaterals are largely unknown. We investigated the role of chemokine (C-X-C-motif) ligand 1 (CXCL1) and its cognate receptor, chemokine (C-X-C-motif) receptor 2 (CXCR2) in arteriogenesis. Methods and results After femoral artery ligation in Sprague–Dawley rats, either native collaterals were harvested or placebo, CXCL1 or CXCR2 blocker was administered via an osmopump. Perfusion recovery was measured with Laser Doppler, leukocyte populations were analyzed by fluorescence-activated cell sorting, and hind limb sections were stained for macrophage marker cluster of differentiation 68 (CD68). In vitro, fluorescent CXCL1 or human acute monocytic leukemia cell line (THP-1) monocytic cells were flown over shear-stressed endothelium. CXCL1 mRNA expression in collaterals was dramatically upregulated already 1 h after ligation (ratio ligated/sham 5.73). CD68 mRNA was upregulated from 12 h until 3 days after ligation (peak ratio ligated/sham 2.65). CXCL1 treatment augmented perfusion recovery at 3 and 7 days (p \ 0.05) after ligation, and a significant increase in the number of peri-collateral macrophages was evident concomitantly (p \ 0.05). Conversely, CXCR2 antagonist treatment caused a decrease in perfusion recovery both at 7 and 10 days postligation (p = 0.01) and also significantly reduced the number of pericollateral macrophages (p \ 0.05). In vitro, CXCL1 tethered to and was taken up by endothelial cells under shear stress conditions and enhanced THP-1 adherence compared to control (p \ 0.05). In contrast, CXCR2 antagonist compromised THP-1 adherence to endothelial cells (p \ 0.05). Conclusion CXCL1 presented on the luminal endothelial surface leads to an increase in the number of peri-collateral macrophages, thus improving the arteriogenic response after arterial ligation.

Bovine myoblast cell production in a microcarriers-based system

For several tissue engineering applications, in particular food products, scaling up culture of mammalian cells is a necessary task. The prevailing method for large scale cell culture is the stirred tank bioreactor where anchor dependent cells are grown on microcarriers suspended in medium. We use a spinner flask system with cells grown on microcarriers to optimize the growth of bovine myoblasts. Freshly isolated primary cells were seeded on microcarriers (Synthemax®, CellBIND® and Cytodex® 1 MCs). In this study, we provide proof of principle that bovine myoblasts can be cultured on microcarriers. No major differences were observed between the three tested microcarriers, except that sparsely populated beads were more common with CellBIND® and Synthemax® II beads suggesting a slower initiation of exponential growth than on Cytodex®. We also provide direct evidence that bovine myoblasts display bead-to-bead transfer. A remarkable pick up of growth was observed by adding new MCs. Bovine myoblasts seem to behave like human mesenchymal stem cells. Thus, our results provide valuable data to further develop and scale-up the production of bovine myoblasts as a prerequisite for efficient and cost-effective development of cultured meat. Applicability to other anchorage dependent cells can extend the importance of these results to cell culture for medical tissue engineering or cell therapy.