blister and sphingosine-1-phosphate

Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

Topics: Actins; Aminoquinolines; Animals; Antigens, CD; Cadherins; Capillary Permeability; Cell Membrane Permeability; Cells, Cultured; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Human Umbilical Vein Endothelial Cells; Humans; Lysophospholipids; Male; Mesentery; Microscopy, Confocal; Microscopy, Fluorescence; Myosin Light Chains; Phosphorylation; Pseudopodia; Pyrimidines; rac1 GTP-Binding Protein; Rats, Sprague-Dawley; rhoA GTP-Binding Protein; Sphingosine; Thrombin; Time-Lapse Imaging; Venules

Three-dimensional (3D) cell-matrix cultures provide a useful model to analyze and dissect the structural, functional, and mechanical aspects of cell-matrix interactions and motile behavior important for cell and tissue morphogenesis. In the current studies we tested the effects of serum and physiological growth factors on the morphogenetic behavior of human fibroblasts cultured on the surfaces of 3D collagen matrices. Fibroblasts in medium containing serum contracted into clusters, whereas cells in medium containing platelet-derived growth factor (PDGF) were observed to migrate as individuals. The clustering activity of serum appeared to depend on lysophosphatidic acid, required cell contraction based on inhibition by blocking Rho kinase or myosin II, and was reversed upon switching to PDGF. Oncogenic Ras transformed human fibroblasts did not exhibit serum-stimulated cell clustering. Our findings emphasize the importance of cell-specific promigratory and procontractile growth factor environments in the differential regulation of cell motile function and cell morphogenesis.

Topics: Actins; Amides; Cell Aggregation; Cell Culture Techniques; Cell Line, Transformed; Cell Movement; Cell Shape; Collagen; Enzyme Inhibitors; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Intercellular Signaling Peptides and Proteins; Lysophospholipids; Male; Myosin Type II; Oncogene Protein p21(ras); Platelet-Derived Growth Factor; Pyridines; rho-Associated Kinases; Serum; Sphingosine; Vinculin

Previous studies suggest that Dupuytren's disease is caused by fibroblast and myofibroblast contractility within Dupuytren's nodules; however, the stimulus for cell contractility is unknown. Sphingosine-1-phosphate (S1P) is a serum-derived lysophospholipid mediator that enhances cell contractility by activating the S1P receptor, S1P(2). It is hypothesized that S1P stimulates Dupuytren's fibroblast contractility through S1P(2) activation of non-muscle myosin II (NMMII). This investigation examined the role of S1P and NMMII activation in Dupuytren's disease progression and suggests potential targets for treatment.. We enmeshed Dupuytren's fibroblasts into fibroblast-populated collagen lattices (FPCLs) and assayed S1P-stimulated FPCL contraction in the presence of the S1P(2) receptor inhibitor JTE-013, the Rho kinase inhibitor Y-27632, the myosin light chain kinase inhibitor ML-7, and the NMMII inhibitor blebbistatin. Tissues from Dupuytren's fascia (n = 10) and normal palmar fascia (n = 10) were immunostained for NMMIIA and NMMIIB.. Sphingosine-1-phosphate stimulated FPCL contraction in a dose-dependent manner. Inhibition of S1P(2) and NMMII prevented S1P-stimulated FPCL contraction. Rho kinase and myosin light chain kinase inhibited both S1P and control FPCL contraction. Dupuytren's nodule fibroblasts robustly expressed NMMIIA and NMMIIB, compared with quiescent-appearing cords and normal palmar fascia.. Sphingosine-1-phosphate promotes Dupuytren's fibroblast contractility through S1P(2), which stimulates activation of NMMII. NMMII isoforms are ubiquitously expressed throughout Dupuytren's nodules, which suggests that nodule fibroblasts are primed to respond to S1P stimulation to cause contracture formation. S1P-promoted activation of NMMII may be a target for disease treatment.

Topics: Cell Line; Collagen; Disease Progression; Dupuytren Contracture; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Immunoenzyme Techniques; Lysophospholipids; Muscle Contraction; Muscle, Smooth; Myosin Type II; Sphingosine