Mechanism of a flow-gated angiogenesis switch: early signaling events at cell–matrix and cell–cell junctions

A bias towards angiogenesis from the venous circulation has long been known, but its cause remains unclear. Here we explore the possibility that high interstitial pressure in tumors and the resultant net filtration pressure gradient that would induce flow from the interstitium into the venous circulation or lymphatics could also be an important mechanical regulator of angiogenesis. The objective of this study was to test the hypothesis that basal-to-apical (B–A) transendothelial flow promotes angiogenesis and to investigate potential mechanisms. Macro- and microvascular endothelial monolayers were cultured on type I collagen gels in a microfluidic cell culture device and subjected to apical-to-basal (A–B) and B–A transendothelial flows. Samples were perfusion fixed and analyzed for morphological responses, localization and degree of phosphorylation of certain signaling proteins. Application of B–A, but not A–B flow, to cultured endothelial monolayers was found to promote capillary morphogenesis and resulted in distinct localization patterns of VE-cadherin and increased FAK phosphorylation. These results suggest that B–A flow triggers the transition of vascular endothelial cells from a quiescent to invasive phenotype and that the flow-mediated response involves signaling at cell–cell and cell–matrix interfaces. These results support the hypothesis that transendothelial pressure gradients resulting in B–A flow promotes sprouting angiogenesis and are consistent with early observations that tumor angiogenesis occurs from the venous side of the circulation.National Institute for Biomedical Imaging and Bioengineering (U.S.) (EB003805)National Science Foundation (U.S.) (STC CBET-0939511)National Science Foundation (U.S.). Office of Emerging Frontiers in Research and Innovation (0735997

Human Vascular Tissue Models Formed from Human Induced Pluripotent Stem Cell Derived Endothelial Cells

Here we describe a strategy to model blood vessel development using a well-defined induced pluripotent stem cell-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats.National Institutes of Health (U.S.) (NIH 1UH2 TR000506-01)National Institutes of Health (U.S.) (3UH2 TR000506-02S1)National Institutes of Health (U.S.) (T32 HL007936-12)National Institutes of Health (U.S.) (RO1 HL093282)National Institutes of Health (U.S.) (R21 EB016381-01

Microfluidic-based three dimensional cell culture for studies of biophysical and biochemical regulation of endothelial function

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (p. 210-229).New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigations. The ultimate challenge lies in replicating the native cell/tissue environment ex vivo. Certain key features of living tissues such as the three dimensionality, biophysical and biochemical microenvironment cannot be readily replicated in traditional culture platforms. Moreover, the capability for multi-parameter manipulation, on a single platform, with the optical resolution to monitor the dynamics of individual cells or small populations is lacking. In this thesis, we developed a novel multiparameter microfluidic-based cell culture platform. The system permits 2D or 3D culture of cells on/in biologically-derived or synthetic hydrogel scaffolds and allows for controlled flow rates, pressure and concentration gradients while directly visualizing cellular response. In addition to the realtime and post-fixation imaging using optical microscopy, methods were developed to extend post-fixation analysis to transmission electron microscopy (TEM). The platform was subsequently used to demonstrate for the first time, two microfluidicbased 3D in vitro assays with direct relevance to tumor development and glaucoma. For the first assay, biochemical induced sprouting was demonstrated. Endothelial cells sprout from an intact monolayer to form multicellular capillary-like structures. Furthermore, using time-lapse microscopy the cellular dynamics during sprouting angiogenesis were observed with great detail, showing tip cell dynamics, cell division events and lumen formation. Of particular relevance to tissue engineering community, we demonstrated that endothelial cells when cultured for several days can assemble into vascular networks with open, perfusable lumen. Using this new system, we present novel findings and results supporting a potential mechanism for flow-mediated mechanical regulation of angiogenesis by transendothelial fluid flow. We demonstrate that flow direction is sufficient to define an angiogenic ON or OFF state. The balance is tipped by forces generated at mechano-sensitive cell-matrix adhesions involving FAK-mediated signaling. These results provide one explanation for the bias towards angiogenesis occurring from the venous side of the circulation. For the second assay, an aqueous humor (AH) outflow model was developed. Subsequent proof-of-concept experiments confirmed its capability for studying the role of the inner wall endothelium in the regulation of AH outflow dynamics.by Vernella V. V. Vickerman.Ph.D

An Expandable, Inducible Hemangioblast State Regulated by Fibroblast Growth Factor

During development, the hematopoietic and vascular lineages are thought to descend from common mesodermal progenitors called hemangioblasts. Here we identify six transcription factors, Gata2, Lmo2, Mycn, Pitx2, Sox17, and Tal1, that “trap” murine cells in a proliferative state and endow them with a hemangioblast potential. These “expandable” hemangioblasts (eHBs) are capable, once released from the control of the ectopic factors, to give rise to functional endothelial cells, multilineage hematopoietic cells, and smooth muscle cells. The eHBs can be derived from embryonic stem cells, from fetal liver cells, or poorly from fibroblasts. The eHBs reveal a central role for fibroblast growth factor, which not only promotes their expansion, but also facilitates their ability to give rise to endothelial cells and leukocytes, but not erythrocytes. This study serves as a demonstration that ephemeral progenitor states can be harnessed in vitro, enabling the creation of tractable progenitor cell lines

Transport-mediated angiogenesis in 3D epithelial coculture

Increasing interest has focused on capturing the complexity of tissues and organs in vitro as models of human pathophysiological processes. In particular, a need exists for a model that can investigate the interactions in three dimensions (3D) between epithelial tissues and a microvascular network since vascularization is vital for reconstructing functional tissues in vitro. Here, we implement a microfluidic platform to analyze angiogenesis in 3D cultures of rat primary hepatocytes and rat/human microvascular endothelial cells (rMVECs/hMVECs). Liver and vascular cells were cultured on each sidewall of a collagen gel scaffold between two microfluidic channels under static or flow conditions. Morphogenesis of 3D hepatocyte cultures was found to depend on diffusion and convection across the nascent tissue. Furthermore, rMVECs formed 3D capillary-like structures that extended across an intervening gel to the hepatocyte tissues in hepatocyte-rMVEC coculture while they formed 2D sheet-like structures in rMVEC monoculture. In addition, diffusion of fluorescent dextran across the gel scaffold was analyzed, demonstrating that secreted proteins from the hepatocytes and MVECs can be exchanged across the gel scaffold by diffusional transport. The experimental approach described here is useful more generally for investigating microvascular networks within 3D engineered tissues with multiple cell types in vitro.—Sudo, R., Chung, S., Zervantonakis, I. K., Vickerman, V., Toshimitsu, Y., Griffith, L. G., Kamm, R. D. Transport-mediated angiogenesis in 3D epithelial coculture

Biomechanical Regulation of Endothelium-dependent Events Critical for Adaptive Remodeling*S⃞

Alterations in hemodynamic shear stress acting on the vascular endothelium are critical for adaptive arterial remodeling. The molecular mechanisms regulating this process, however, remain largely uncharacterized. Here, we sought to define the responses evoked in endothelial cells exposed to shear stress waveforms characteristic of coronary collateral vessels and the subsequent paracrine effects on smooth muscle cells. A lumped parameter model of the human coronary collateral circulation was used to simulate normal and adaptive remodeling coronary collateral shear stress waveforms. These waveforms were then applied to cultured human endothelial cells (EC), and the resulting differences in EC gene expression were assessed by genome-wide transcriptional profiling to identify genes distinctly regulated by collateral flow. Analysis of these transcriptional programs identified several genes to be differentially regulated by collateral flow, including genes important for endothelium-smooth muscle interactions. In particular, the transcription factor KLF2 was up-regulated by the adaptive remodeling coronary collateral waveform, and several of its downstream targets displayed the expected modulation, including the down-regulation of connective tissue growth factor. To assess the effect of endothelial KLF2 expression on smooth muscle cell migration, a three-dimensional microfluidic assay was developed. Using this three-dimensional system, we showed that KLF2-expressing EC co-cultured with SMC significantly reduce SMC migration compared with control EC and that this reduction can be rescued by the addition of exogenous connective tissue growth factor. Collectively, these results demonstrate that collateral flow evokes distinct EC gene expression profiles and functional phenotypes that subsequently influence vascular events important for adaptive remodeling