Endothelial cell polarization and chemotaxis in a microfluidic device
ABSTRACT The directed migration of endothelial cells is an early and critical step in angiogenesis, or new blood vessel formation. In this study, the polarization and chemotaxis of human umbilical vein endothelial cells (HUVEC) in response to quantified gradients of vascular endothelial growth factor (VEGF) were examined. To accomplish this, a microfluidic device was designed and fabricated to generate stable concentration gradients of biomolecules in a cell culture chamber while minimizing the fluid shear stress experienced by the cells. Finite element simulation of the device geometry produced excellent agreement with the observed VEGF concentration distribution, which was found to be stable across multiple hours. This device is expected to have wide applicability in the study of shear-sensitive cells such as HUVEC and non-adherent cell types as well as in the study of migration through three-dimensional matrices. HUVEC were observed to chemotax towards higher VEGF concentrations across the entire range of concentrations studied (18-32 ng mL(-1)) when the concentration gradient was 14 ng mL(-1) mm(-1). In contrast, shallow gradients (2 ng mL(-1) mm(-1)) across the same concentration range were unable to induce HUVEC chemotaxis. Furthermore, while all HUVEC exposed to elevated VEGF levels (both in steep and shallow gradients) displayed an increased number of filopodia, only chemotaxing HUVEC displayed an asymmetric distribution of filopodia, with enhanced numbers of protrusions present along the leading edge. These results suggest a two-part requirement to induce VEGF chemotaxis: the VEGF absolute concentration enhances the total number of filopodia extended while the VEGF gradient steepness induces filopodia localization, cell polarization, and subsequent directed migration.
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ABSTRACT: Vasculogenesis, the assembly of the first vascular network, is an intriguing developmental process that yields the first functional organ system of the embryo. In addition to being a fundamental part of embryonic development, vasculogenic processes also have medical importance. To explain the organizational principles behind vascular patterning, we must understand how morphogenesis of tissue level structures can be controlled through cell behavior patterns that, in turn, are determined by biochemical signal transduction processes. Mathematical analyses and computer simulations can help conceptualize how to bridge organizational levels and thus help in evaluating hypotheses regarding the formation of vascular networks. Here, we discuss the ideas that have been proposed to explain the formation of the first vascular pattern: cell motility guided by extracellular matrix alignment (contact guidance), chemotaxis guided by paracrine and autocrine morphogens, and sprouting guided by cell-cell contacts.Birth Defects Research Part C Embryo Today Reviews 06/2012; 96(2):153-62. DOI:10.1002/bdrc.21010 · 3.87 Impact Factor
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ABSTRACT: Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for directional cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF, indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions, suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner.Cell 03/2012; 148(5):973-87. DOI:10.1016/j.cell.2011.12.034 · 33.12 Impact Factor
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ABSTRACT: A key challenge in tissue engineering is overcoming cell death in the scaffold interior due to the limited diffusion of oxygen and nutrients therein. We here hypothesize that immobilizing a gradient of a growth/survival factor from the periphery to the center of a porous scaffold would guide endothelial cells into the interior of the scaffold, thus overcoming a necrotic core. Proteins were immobilized by one of three methods on porous collagen scaffolds for cardiovascular tissue engineering. The proteins were first activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/sulfo N-hydroxysuccinimide and then applied to the scaffold by one of three methods to establish the gradient: perfusion (the flow method), use of a source and a sink (the source-sink method) or by injecting 5 μl of the solution at the center of the scaffold (point source method). Due to the high reproducibility and ease of application of the point source method it was further used for VEGF-165 gradient formation, where an ~2 ng ml(-1) mm(-1) gradient was formed in a radial direction across a scaffold, 12 mm in diameter and 2.5mm thick. More endothelial cells were guided by the VEGF-165 gradient deep into the center of the scaffold compared with both uniformly immobilized VEGF-165 (with the same total VEGF concentration) and VEGF-free controls. All scaffolds (including the controls) yielded the same number of cells, but notably the VEGF-165 gradient scaffolds demonstrated a higher cell density in the centre. Thus we concluded that the VEGF-165 gradient promoted the migration, but not proliferation, of cells into the scaffold. These gradient scaffolds provide the foundation for future in vivo tissue engineering studies.Acta biomaterialia 05/2011; 7(8):3027-35. DOI:10.1016/j.actbio.2011.05.002 · 5.68 Impact Factor