RESUMEN
A combined experimental and mathematical model of intermittent hypoxia (IH) conditioned engineered tissue was used to characterize the effects of IH on the formation of in vitro vascular networks. Results showed that the frequency of hypoxic oscillations has pronounced influence on the vascular response of endothelial cells and fibroblasts.
Asunto(s)
Vasos Sanguíneos/citología , Hipoxia/metabolismo , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/metabolismo , Proliferación Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Sangre Fetal/citología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Modelos Biológicos , Oxígeno/metabolismo , Oxígeno/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Metastasis is the cause of over 90% of all human cancer deaths. Early steps in the metastatic process include: the formation of new blood vessels, the initiation of epithelial-mesenchymal transition (EMT), and the mobilization of tumor cells into the circulation. There are ongoing efforts to replicate the physiological landscape of human tumor tissue using three-dimensional in vitro culture models; however, few systems are able to capture the full range of authentic, complex in vivo events such as neovascularization and intravasation. Here we introduce the Prevascularized Tumor (PVT) model to investigate early events of solid tumor progression. PVT spheroids are composed of endothelial and tumor cells, and are embedded in a fibrin matrix containing fibroblasts. The PVT model facilitates two mechanisms of vessel formation: robust sprouting angiogenesis into the matrix, and contiguous vascularization within the spheroid. Furthermore, the PVT model enables the intravasation of tumor cells that is enhanced under low oxygen conditions and is also dependent on the key EMT transcription factor Slug. The PVT model provides a significant advance in the mimicry of human tumors in vitro, and may improve investigation and targeting of events in the metastatic process.
Asunto(s)
Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Fibrina/metabolismo , Hipoxia/metabolismo , Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Western Blotting , Línea Celular Tumoral , Humanos , Técnicas In Vitro , Microscopía Fluorescente , ARN Interferente Pequeño/farmacología , Factores de Transcripción de la Familia Snail , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismoRESUMEN
The Snail family of zinc-finger transcription factors are evolutionarily conserved proteins that control processes requiring cell movement. Specifically, they regulate epithelial-to-mesenchymal transitions (EMT) where an epithelial cell severs intercellular junctions, degrades basement membrane and becomes a migratory, mesenchymal-like cell. Interestingly, Slug expression has been observed in angiogenic endothelial cells (EC) in vivo, suggesting that angiogenic sprouting may share common attributes with EMT. Here, we demonstrate that sprouting EC in vitro express both Slug and Snail, and that siRNA-mediated knockdown of either inhibits sprouting and migration in multiple in vitro angiogenesis assays. We find that expression of MT1-MMP, but not of VE-Cadherin, is regulated by Slug and that loss of sprouting as a consequence of reduced Slug expression can be reversed by lentiviral-mediated re-expression of MT1-MMP. Activity of MMP2 and MMP9 are also affected by Slug expression, likely through MT1-MMP. Importantly, we find enhanced expression of Slug in EC in human colorectal cancer samples compared with normal colon tissue, suggesting a role for Slug in pathological angiogenesis. In summary, these data implicate Slug as an important regulator of sprouting angiogenesis, particularly in pathological settings.
Asunto(s)
Factores de Transcripción/metabolismo , Células Cultivadas , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Técnica del Anticuerpo Fluorescente , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inmunohistoquímica , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Metilcelulosa/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción de la Familia SnailRESUMEN
Engineered tissue constructs are limited in size, and thus clinical relevance, when diffusion is the primary mode of oxygen transport. Understanding the extent of oxygen diffusion and cellular consumption is necessary for the design of engineered tissues, particularly those intended for implantation into hypoxic wound sites. This study presents a combined experimental and computation model to predict design constraints for cellularized fibrin tissues subjected to a step change in the oxygen concentration to simulate transplantation. Nonsteady state analysis of oxygen diffusion and consumption was used to estimate the diffusion coefficient of oxygen (mean±SD, 1.7×10(-9)±8.4×10(-11) m(2)/s) in fibrin hydrogels as well as the Michaelis-Menten parameters, Vmax (1.3×10(-17)±9.2×10(-19) mol·cell(-1)·s(-1)), and Km (8.0×10(-3)±3.5×0(-3) mol/m(3)), of normal human lung fibroblasts. Nondimensionalization of the governing diffusion-reaction equation enabled the creation of a single dimensionless parameter, the Thiele modulus (φ), which encompasses the combined effects of oxygen diffusion, consumption, and tissue dimensions. Tissue thickness is the design parameter with the most pronounced influence on the distribution of oxygen within the system. Additionally, tissues designed such that φ<1 achieve a near spatially uniform and adequate oxygen concentration following the step change. Understanding and optimizing the Thiele modulus will improve the design of engineered tissue implants.