Mimicking Angiogenesis in vitro: Three-dimensional Co-culture of Vascular  Endothelial Cells and Perivascular Cells in Collagen Type I Gels

Auler, Markus; Pitzler, Lena; Pöschl, Ernst; Zhou, Zhigang; Brachvogel, Bent

doi:10.21769/bioprotoc.2247

Cited by 4 publications

(2 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Adapted from the literature ( Auler et al, 2017 ), cells in collagen were fixed with 4% PFA for 2 h before staining. Afterward, the samples were blocked using 5% w/v bovine serum albumin in DPBS for 3 h and subsequently stained with a monoclonal mouse anti-human CD31 primary antibody (1:50) overnight at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

3D-Cultured Vascular-Like Networks Enable Validation of Vascular Disruption Properties of Drugs In Vitro

Yavvari

Laporte

Elomaa

et al. 2022

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

Vascular-disrupting agents are an interesting class of anticancer compounds because of their combined mode of action in preventing new blood vessel formation and disruption of already existing vasculature in the immediate microenvironment of solid tumors. The validation of vascular disruption properties of these drugs in vitro is rarely addressed due to the lack of proper in vitro angiogenesis models comprising mature and long-lived vascular-like networks. We herein report an indirect coculture model of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) to form three-dimensional profuse vascular-like networks. HUVECs embedded and sandwiched in the collagen scaffold were cocultured with HDFs located outside the scaffold. The indirect coculture approach with the vascular endothelial growth factor (VEGF) producing HDFs triggered the formation of progressively maturing lumenized vascular-like networks of endothelial cells within less than 7 days, which have proven to be viably maintained in culture beyond day 21. Molecular weight-dependent Texas red-dextran permeability studies indicated high vascular barrier function of the generated networks. Their longevity allowed us to study the dose-dependent response upon treatment with the three known antiangiogenic and/or vascular disrupting agents brivanib, combretastatin A4 phosphate (CA4P), and 6´-sialylgalactose (SG) via semi-quantitative brightfield and qualitative confocal laser scanning microscopic (CLSM) image analysis. Compared to the reported data on in vivo efficacy of these drugs in terms of antiangiogenic and vascular disrupting effects, we observed similar trends with our 3D model, which are not reflected in conventional in vitro angiogenesis assays. High-vascular disruption under continuous treatment of the matured vascular-like network was observed at concentrations ≥3.5 ng·ml−1 for CA4P and ≥300 nM for brivanib. In contrast, SG failed to induce any significant vascular disruption in vitro. This advanced model of a 3D vascular-like network allows for testing single and combinational antiangiogenic and vascular disrupting effects with optimized dosing and may thus bridge the gap between the in vitro and in vivo experiments in validating hits from high-throughput screening. Moreover, the physiological 3D environment mimicking in vitro assay is not only highly relevant to in vivo studies linked to cancer but also to the field of tissue regeneration.

show abstract

Section: Methodsmentioning

confidence: 99%

3D-Cultured Vascular-Like Networks Enable Validation of Vascular Disruption Properties of Drugs In Vitro

Yavvari

Laporte

Elomaa

et al. 2022

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

show abstract

“…A co-culture of EC + PC resulted in complete closure (100%, P < .0001) with apparent immature tubule vessel formation. 32 Following monoculture studies, we observed the effect of EC and/or PC cell contact on NSC migration by culturing NSC with EC, PC, or EC + PC in a 1:1 cell ratio for co-culture (Figure 1A). In the presence of EC, NSC began to cluster, and interestingly, the clusters (GFP + NSC) began migrating toward the scratched area (46.67% ± 3.80 closure, P < .0001), significantly more than NSC alone (25.05% ± 3.95 closure).…”

Section: Ec Promote Nsc Migration and Clusteringmentioning

confidence: 99%

Endothelial cell secreted metalloproteinase‐2 enhances neural stem cell N‐cadherin expression, clustering, and migration

et al. 2021

View full text Add to dashboard Cite

Neuroblasts have a clustered phenotype critical for their unidirectional migration, which in part is dependent on signaling from microvascular endothelial cells (EC) and pericytes (PC). Diffusible signals secreted by vascular cells have been demonstrated to increase survival, proliferation, and differentiation of subventricular zone resident neural stem cells (NSC); however, the signals that promote the necessary initiating step of NSC clustering are undefined. To investigate the role of vascular cells in promoting NSC clustering and directing migration, we created a 3-D hydrogel that mimics the biomechanics, biochemistry, and architectural complexity of brain tissue. We demonstrate that EC, and not PC, have a crucial role in NSC clustering and migration, further verified through microfluidic chamber systems and traction force microscopy. Ablation of the extended NSC aggregate arm halts aggregate movement, suggesting that clustering is a prerequisite for migration. When cultured with EC, NSC clustering occurs and NSC coincidentally increase their expression of N-cadherin, as compared to NSC cultured alone. NSC-presented N-cadherin expression was increased following exposure to EC secreted metalloproteinase-2 (MMP2).We demonstrate that inhibition of MMP2 prevented NSC N-cadherin surface expression and subsequent NSC clustering, even when NSC were in direct contact with EC. Furthermore, with exogenous activation of EGFR, which serves as a downstream activator of N-cadherin cleavage, NSC form clusters. Our results suggest that EC secretion of MMP2 promotes NSC clustering through N-cadherin expression. The insight gained about the mechanisms by which EC promote NSC migration may enhance NSC therapeutic response to sites of injury. K E Y W O R D S cell clustering, cell migration, N-cadherin, neural stem cell 2 of 17 | MATTA eT Al.

show abstract

Collagens and Collagen-Degrading Enzymes in the Regulation of Angiogenesis

Kanellopoulou

Xanthopoulos

Mikelis

et al. 2022

Matrix Pathobiology and Angiogenesis

View full text Add to dashboard Cite

Mimicking Angiogenesis in vitro: Three-dimensional Co-culture of Vascular Endothelial Cells and Perivascular Cells in Collagen Type I Gels

Cited by 4 publications

References 7 publications

3D-Cultured Vascular-Like Networks Enable Validation of Vascular Disruption Properties of Drugs In Vitro

3D-Cultured Vascular-Like Networks Enable Validation of Vascular Disruption Properties of Drugs In Vitro

Endothelial cell secreted metalloproteinase‐2 enhances neural stem cell N‐cadherin expression, clustering, and migration

Collagens and Collagen-Degrading Enzymes in the Regulation of Angiogenesis

Contact Info

Product

Resources

About