[PubMed] [Google Scholar]Petroll WM, Ma L, Kim A, Ly L, Vishwanath M

[PubMed] [Google Scholar]Petroll WM, Ma L, Kim A, Ly L, Vishwanath M. 06: Supplemental Video 6: A keratocyte seeded in 3-D rat tail collagen matrix cultured in Serum-free basal media w. PDGF and 8M BB-94. Horizontal field width = 238 microns. NIHMS577871-supplement-06.mov (1.5M) GUID:?88A51395-1A49-47DB-81E7-4A4970535CC7 07: Supplemented Figure 1. Single plane confocal reflection image of reconstituted ATB-337 rat tail collagen matrix after 4 days of culture in IL-1 with Plasminogen. Owing to collagenolysis of soluble MMPs produced by NRK cells, the matrix had disrupted, shorter collagen fibrils and a lower fibril density, comparing with parallel PDGF group. NIHMS577871-supplement-07.tif (2.7M) GUID:?540F233C-0202-4B4B-BCDD-50C8F4463AB8 ATB-337 08: Supplemented Figure 2. ATB-337 NRK cell migration in 3-D bovine Type I collagen matrices. Each migration index (cell number/1.5mm region around the interface) is the average of triplicate experiments. NIHMS577871-supplement-08.tif (2.3M) GUID:?61411B48-6273-4D50-ABDC-F23535FFA2DE Abstract Previous studies have shown that platelet derived growth factor (PDGF) can stimulate corneal keratocyte spreading and migration within 3-D collagen matrices, without inducing transformation to a contractile, fibroblastic phenotype. The goal of this study was to investigate the role of matrix metalloproteinases (MMPs) in regulating PDGF-induced changes in keratocyte motility and mechanical differentiation. Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-) to maintain their quiescent phenotype. A nested collagen matrix construct was used to assess 3-D cell migration, and a standard collagen matrix model was used to assess cell morphology and cell-mediated matrix contraction. In both cases constructs were cultured in S- supplemented with PDGF, with or without the broad spectrum MMP inhibitors GM6001 or BB-94. After 4 days, f-actin, nuclei and collagen fibrils were imaged using confocal microscopy. To assess sub-cellular mechanical activity (extension and retraction of cell processes), time-lapse DIC imaging was also performed. MT1-MMP expression and MMP-mediated collagen degradation by were also examined. Results exhibited that neither GM6001 nor BB-94 affected corneal keratocyte viability or proliferation in 3-D culture. PDGF stimulated elongation and migration of corneal keratocytes within type I collagen matrices, without causing a loss of their dendritic morphology or inducing formation of intracellular stress fibers. Treatment with GM6001 and BB-94 inhibited PDGF-induced keratocyte spreading and migration. Relatively low levels of keratocyte-induced matrix contraction were also maintained in PDGF, and the amount of PDGF-induced collagen degradation was similar to that observed in S- controls. The collagen degradation pattern was consistent with membrane-associated MMP activity, and keratocytes showed positive staining for MT1-MMP, albeit weak. Both matrix contraction and collagen degradation were ATB-337 reduced by MMP inhibition. For most outcome measures, the inhibitory effect of BB-94 was significantly greater than that of GM6001. Overall, the data demonstrate for the first time that even under conditions in which low levels of contractility and extracellular matrix proteolysis are maintained, MMPs still play an important role in mediating cell spreading and migration within 3-D collagen matrices. This appears to be mediated at least in part by membrane-tethered MMPs, such as MT1-MMP. = ring number, = time interval, N= total number of process segments in ring at time interval (Jester et al., 1994; Lakshman et al., 2010). The number of cells in 3-D collagen matrices cultured with PDGF media increased by 69.4% after 4 days of culture, and neither GM6001, BB-94 or DMSO (vehicle) affected keratocyte proliferation in response to PDGF (= rat tail collagen fibrils acquired by confocal reflection imaging. Previous studies have exhibited that cancer cell migration through rat tail collagen matrices is dependent on MMPs. However, for some types of cancer cells, migration through bovine Rabbit Polyclonal to CBX6 collagen matrices MMP-independent (Sabeh et al., 2009b;.