The ability to predict endothelial cell migration rates may aid in

The ability to predict endothelial cell migration rates may aid in the design of biomaterials that endothelialize following implantation. rates. In immortalized endothelial cells expression of proteins that inhibit Akt Src and Rac (PTEN CSK and is enhanced by VEGF physiologic levels of fluid shear stress but S1P has almost no effect on cells stimulated with both VEGF and shear stress.15 INTS6 Furthermore the migration response to S1P is different in blood plasma than in cell culture medium and is affected by the density and identity of adhesion ligands.2 47 Engineering methods may help deconvolute the migration response of endothelial cells in the presence of a myriad of stimulatory factors. The application of systematic network analysis may aid in the design of devices by allowing useful predictions of the nonlinear migration response resulting from stimulation with combinations of factors. For example knowledge of the activation states of all relevant growth factor receptors or integrins might allow construction of a predictive model.37 39 However the number of potentially relevant receptors is quite large and thus difficult to characterize experimentally. An alternative approach is to look downstream of cell-surface activation at signaling events immediately distal to receptor activation since receptor-mediated signals tend to converge on a few common pathways. RhoGTPases (Rho Rac and Cdc42) greatly impact endothelial cell migration 10 with activation of Rac-GTP strongly correlated with endothelial hapto- and chemotaxis.41 42 However different variants of constitutively active Rac (V12 vs. L61) have opposite effects on endothelial cell migration.14 42 In neutrophils very low levels of Rac-GTP significantly decrease chemotaxis 11 while a decrease in Rac activity of approximately 30% serves as a switch between random and persistent migration in both fibroblasts and endothelial cells.35 At the other extreme high levels of Rac-GTP inhibit cell migration e.g. during cell spreading.36 Optimal levels of Rac activity may be needed for rapid Ispinesib (SB-715992) cell migration but the presence of a complex nonlinear relationship may mean that Rac alone will be difficult to use as a predictor of cell migration rates. We hypothesized that Rac Akt and Src activity together may yield better predictions of cell migration as Akt Src and Rac are particularly important in S1P-mediated cell responses.12 25 Akt and Src family kinases are activated in endothelial cells by fluid shear stress receptor tyrosine kinases G protein-coupled receptors and focal adhesions.20 21 40 45 In essence these factors together may better reflect the Ispinesib (SB-715992) overall activation state of multiple growth factor receptors mechano-receptors G protein-coupled receptors and integrins that as a whole drive the cell migration response. To study endothelial cell migration as a multivariate function of Akt Src and Rac activities we collected an extensive biochemical and biophysical dataset Ispinesib (SB-715992) across a range of cellular perturbations. Since direct knockdown of Akt Src or Rac could lead Ispinesib (SB-715992) to very low levels of activation that might not be relevant in highly Ispinesib (SB-715992) stimulated migrating cells we decreased expression of inhibitory accessory proteins resulting in increased basal levels of active Akt Src and Rac. The accessory proteins targeted with shRNA were phosphatase and homolog deleted on chromosome ten (PTEN) c-terminal Src kinase (CSK) and = 4-9). For validation experiments cells were plated in 24-well tissue culture plates for 24 h. After serum starvation for 4 h cells were pre-treated with LY294002 (Calbiochem 0 = 4 biological replicates 16 wounds total per condition). The distance traveled by the wound edge was quantified with ImageJ. For long-term wounding assays (24 h) cells were first serum starved (0.1% FBS) for 12 h and then stimulated with low serum medium (0.1% FBS) 100 nM S1P or complete growth medium. A scrape wound was made using a P1000 plastic pipette tip. After rinsing micrographs of the wounds were captured using a ×4 objective and the cells were cultured in a CO2-rich environment for 24 h. Micrographs of the same wound area were captured after 24 h and the number of cells that migrated into the area was counted and is reported as a cell density. Rac Activity Assay (ELISA-based) The concentration of Rac-GTP was measured using the Rac GLISA assay (Cytoskeleton Inc) per manufacturer’s protocol. The optimum lysate concentration for the endothelial cells used in this study was determined to be 0.75 mg/mL. All.