Interstitial Fibroblasts Enhance Capillary Morphogenesis In Dense 3-D Matrices

摘要: Numerous fibrotic pathologies adversely affect angiogenesis, yet the effects of increased ECM density associated with these pathologies remain unclear. To study this phenomenon, we have utilized a 3-D, fibrin-based in vitro angiogenesis assay in which fibroblasts (FBs) are plated on top of a hydrogel ~2 mm away from HUVEC-coated microcarrier beads polymerized within the matrix. Increasing fibrin density from 2.5 to 10 mg/ml results in a three-fold reduction in capillary network formation. However, distributing FBs throughout the matrix eliminates this inhibitory effect; the number of vessel segments and total network length are enhanced in all conditions independent of matrix density. The interstitial FBs do not adopt an enhanced myofibroblastic phenotype nor extensively remodel the matrix, as shown by insignificant changes in the levels of characteristic markers (ie alpha-SMA, MMP2/9, fibronectin ED-A, collagen). Alternatively, we hypothesized that increasing fibrin density restricts the ability of fibroblast-derived angiogenic factors to reach the HUVECs by diffusion. Using a novel method developed to image bulk diffusion of FITC-dextran (avg. MWs of 10, 40, 70, and 150 kDa) through the matrices, we demonstrate an order of magnitude reduction in the diffusion coefficient with a 4-fold increase in fibrin density. We conclude that diffusion restrictions imposed by fibrotic matrices are a primary factor in inhibiting angiogenesis, a conclusion supported by the enhanced morphogenesis observed when a source of angiogenic regulators is distributed throughout the matrix