[Show abstract][Hide abstract] ABSTRACT: In vitro endothelial cell organization into capillaries is a long standing challenge of tissue engineering. We recently showed the utility of low level interstitial flow in guiding the organization of endothelial cells through a 3-D fibrin matrix-containing covalently bound vascular endothelial growth factor (VEGF). Here this synergistic phenomenon was extended to explore the effects of matrix composition on in vitro capillary morphogenesis of human blood versus lymphatic endothelial cells (BECs and LECs). Different mixtures of fibrin and collagen were used in conjunction with constant concentrations of matrix-bound VEGF and slow interstitial flow over 10 days. Interestingly, the BECs and LECs each showed a distinct preference in terms of organization for matrix composition: LECs organized the most extensively in a fibrin-only matrix, while BEC organization was optimized in the compliant collagen-containing matrices. Furthermore, the BECs and LECs produced architecturally different structures; while BECs organized in thick, branched networks containing wide lumen, the LECs were elongated into slender, overlapping networks with fine lumen. These data demonstrate the importance of the 3-D matrix composition in facilitating and coordinating BEC and LEC capillary morphogenesis, which is important for in vitro vascularization of engineered tissues.
Biotechnology and Bioengineering 01/2007; 96(1):167-76. DOI:10.1002/bit.21185 · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell organization is largely orchestrated by extracellular gradients of morphogenetic proteins. VEGF, an essential factor for capillary formation, is stored in the extracellular matrix, but the mechanisms by which it and other matrix-bound morphogens are mobilized to form spatial gradients are poorly understood. Here, we suggest an efficient mechanism for morphogen gradient generation by subtle biophysical forces in an in vitro model of capillary morphogenesis. Using a fibrin-bound VEGF variant that is released proteolytically to mimic the in vivo situation, we report that low levels of interstitial flow act synergistically with VEGF to drive endothelial organization, whereas each stimulus alone has very little effect. To help account for this synergy, we show how these slow flows can bias the distribution of cell-secreted proteases, which leads, interestingly, to the creation of an increasing VEGF gradient relative to the cell and skewed in the direction of flow. In contrast, diffusion alone can only account for symmetric, decreasing autocrine gradients. Indeed, branching of capillary structures was biased in the direction of flow only with the combination of VEGF and flow. This work thus demonstrates a general mechanism of morphogen gradient generation and amplification by small ubiquitous mechanical forces that are known to exist in vivo.
Proceedings of the National Academy of Sciences 12/2005; 102(44):15779-84. DOI:10.1073/pnas.0503681102 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Whereas high shearing flows are known to induce endothelial cell remodeling, we show here that very low interstitial flow rates trigger endothelial cell morphogenesis in 3D cultures. Interstitial flow is a functionally critical component of the circulation, and we have recently observed that it plays a regulatory role in lymphangiogenesis; here we investigate interstitial flow as a powerful morphoregulatory stimulant. We exposed both lymphatic and blood endothelial cells (LECs and BECs) to interstitial flow in 3D collagen gels as well as simple shear flow in 2D monolayers. We found that under interstitial flow (average 10 microm/s for 6 days), both cell types underwent drastic morphologic changes from static conditions: LECs formed large vacuoles and long extensions, while BECs formed multicellular branched lumen-containing networks. Under planar shear (20 dyn/cm2 for 24 h), LECs downregulated their cell-cell adhesions compared to BECs but did not differ morphologically; both aligned with flow as expected. The significance of these findings is twofold: first, they identify an important role of interstitial flow for in vitro microvascular organization and stabilization, and second, they demonstrate for the first time notable differences between LEC and BEC response to the biophysical environment, reflecting some of their functional differences in vivo.
Microvascular Research 12/2004; 68(3):258-64. DOI:10.1016/j.mvr.2004.08.002 · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We provide evidence to support our hypothesis that lymphangiogenesis is regulated and organized by interstitial fluid flow. In a mouse model of skin regenerated from an acellular collagen construct (implanted to replace a portion of excised tail skin), our observations of lymphatic regeneration are consistent with fluid channeling and flow organization rather than sprouting. We also show that in a modified model where interstitial flow was allowed to shunt around the construct, lymphatic regeneration was not seen, despite an identical biochemical background.