Effects of Extracellular Matrix Density and Mesenchymal Stem Cells on Neovascularization In Vivo

Aberrant angiogenesis is common to a variety of diseases in which alterations in tissue mechanical properties also occur. A fundamental understanding of the interdependence of angiogenesis and tissue structural properties may enhance the development of therapeutic strategies. We previously established that increasing extracellular matrix density inhibits capillary morphogenesis in three-dimensional tissues in vitro, and that addition of human mesenchymal stem cells (MSCs) partially rescues a healthy angiogenic phenotype. This study's goal was to investigate if these effects can be recapitulated in vivo. Human umbilical vein endothelial cells, MSCs, or a mixture of both was suspended in fibrin gel precursor solutions of 5, 10, and 15-mg/mL concentrations and injected subcutaneously into SCID mice. Neovascularization was assessed in tissue constructs retrieved at 3, 7, and 21 days by quantifying vessel numbers, perfusion, thickness, maturity, and perivascular collagen deposition. The data show that changing extracellular matrix density inhibits capillary morphogenesis in vivo in a manner consistent with that observed in vitro. Delivery of both human umbilical vein endothelial cells and MSCs produced more robust and mature vessels than delivery of either cell type alone in all tissue concentrations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90460/1/ten-2Etea-2E2010-2E0275.pd

Assessing the Permeability of Engineered Capillary Networks in a 3D Culture

Many pathologies are characterized by poor blood vessel growth and reduced nutrient delivery to the surrounding tissue, introducing a need for tissue engineered blood vessels. Our lab has developed a 3D co-culture method to grow interconnected networks of pericyte-invested capillaries, which can anastamose with host vasculature following implantation to restore blood flow to ischemic tissues. However, if the engineered vessels contain endothelial cells (ECs) that are misaligned or contain wide junctional gaps, they may function improperly and behave more like the pathologic vessels that nourish tumors. The purpose of this study was to test the resistance to permeability of these networks in vitro, grown with different stromal cell types, as a metric of vessel functionality. A fluorescent dextran tracer was used to visualize transport across the endothelium and the pixel intensity was quantified using a customized MATLAB algorithm. In fibroblast-EC co-cultures, the dextran tracer easily penetrated through the vessel wall and permeability was high through the first 5 days of culture, indicative of vessel immaturity. Beyond day 5, dextran accumulated at the periphery of the vessel, with very little transported across the endothelium. Quantitatively, permeability dropped from initial levels of 61% to 39% after 7 days, and to 7% after 2 weeks. When ECs were co-cultured with bone marrow-derived mesenchymal stem cells (MSCs) or adipose-derived stem cells (AdSCs), much tighter control of permeability was achieved. Relative to the EC-fibroblast co-cultures, permeabilities were reduced 41% for the EC-MSC co-cultures and 50% for the EC-AdSC co-cultures after 3 days of culture. By day 14, these permeabilities decreased by 68% and 77% over the EC-fibroblast cultures. Co-cultures containing stem cells exhibit elevated VE-cadherin levels and more prominent EC-EC junctional complexes when compared to cultures containing fibroblasts. These data suggest the stromal cell identity influences the functionality and physiologic relevance of engineered capillary networks