Dexamethasone increases pigment epithelium-derived factor in perfused human eyes.

PURPOSE: To investigate the effects of dexamethasone (DEX) on pigment epithelium-derived factor (PEDF) cDNA and secreted protein in human trabecular meshwork (TM). METHODS: Anterior segment organ cultures were perfused with 0.1 microM DEX (OD) and vehicle (OS). Primary human TM cells (HTM) were treated with DEX under similar conditions. PEDF mRNA and secreted PEDF protein were quantitated by RT-PCR and Western blot. RESULTS: PEDF mRNA and secreted PEDF protein levels were significantly higher in DEX over vehicle-treated cultures. In contrast, DEX decreased the activity of a 92-kDa gelatinolytic zymogen in organ culture effluents. CONCLUSION: DEX action in the human TM might include a PEDF-mediating pathway.

Versican-thrombospondin-1 binding in vitro and colocalization in microfibrils induced by inflammation on vascular smooth muscle cells.

We identified a specific interaction between two secreted proteins, thrombospondin-1 and versican, that is induced during a toll-like receptor-3-dependent inflammatory response in vascular smooth muscle cells. Thrombospondin-1 binding to versican is modulated by divalent cations. This interaction is mediated by interaction of the G1 domain of versican with the N-module of thrombospondin-1 but only weakly with the corresponding N-terminal region of thrombospondin-2. The G1 domain of versican contains two Link modules, which are known to mediate TNFalpha-stimulated gene-6 protein binding to thrombospondin-1, and the related G1 domain of aggrecan is also recognized by thrombospondin-1. Therefore, thrombospondin-1 interacts with three members of the Link-containing hyaladherin family. On the surface of poly-I:C-stimulated vascular smooth muscle cells, versican organizes into fibrillar structures that contain elastin but are largely distinct from those formed by hyaluronan. Endogenous and exogenously added thrombospondin-1 incorporates into these structures. Binding of exogenous thrombospondin-1 to these structures, to purified versican and to its G1 domain is potently inhibited by heparin. At higher concentrations, exogenous thrombospondin-1 delays the poly-I:C induced formation of structures containing versican and elastin, suggesting that thrombospondin-1 negatively modulates this component of a vascular smooth muscle inflammatory response.

Thrombospondin-1 inhibits endothelial cell responses to nitric oxide in a cGMP-dependent manner.

Redox signaling plays an important role in the positive regulation of angiogenesis by vascular endothelial growth factor, but its role in signal transduction by angiogenesis inhibitors is less clear. Using muscle explants in 3D culture, we found that explants from mice lacking the angiogenesis inhibitor thrombospondin-1 (TSP1) exhibit exaggerated angiogenic responses to an exogenous NO donor, which could be reversed by providing exogenous TSP1. To define the basis for inhibition by TSP1, we examined the effects of TSP1 on several proangiogenic responses of endothelial cells to NO. NO has a biphasic effect on endothelial cell proliferation. The positive effect at low doses of NO is sensitive to inhibition of cGMP signaling and picomolar concentrations of TSP1. NO stimulates both directed (chemotactic) and random (chemokinetic) motility of endothelial cells in a cGMP-dependent manner. TSP1 potently inhibits chemotaxis stimulated by NO. Low doses of NO also stimulate adhesion of endothelial cells on type I collagen in a cGMP-dependent manner. TSP1 potently inhibits this response both upstream and downstream of cGMP. NO-stimulated endothelial cell responses are inhibited by recombinant type 1 repeats of TSP1 and a CD36 agonist antibody but not by the N-terminal portion of TSP1, suggesting that CD36 or a related receptor mediates these effects. These results demonstrate a potent antagonism between TSP1 and proangiogenic signaling downstream of NO. Further elucidation of this inhibitory signaling pathway may identify new molecular targets to regulate pathological angiogenesis.

Micropatterning neuronal cells on polyelectrolyte multilayers.

This paper describes an approach to adhere retinal cells on micropatterned polyelectrolyte multilayer (PEM) lines adsorbed on poly(dimethylsiloxane) (PDMS) surfaces using microfluidic networks. PEMs were patterned on flat, oxidized PDMS surfaces by sequentially flowing polyions through a microchannel network that was placed in contact with the PDMS surface. Polyethyleneimine (PEI) and poly(allylamine hydrochloride) (PAH) were the polyions used as the top layer cellular adhesion material. The microfluidic network was lifted off after the patterning was completed and retinal cells were seeded on the PEM/PDMS surfaces. The traditional practice of using blocking agents to prevent the adhesion of cells on unpatterned areas was avoided by allowing the PDMS surface to return to its uncharged state after the patterning was completed. The adhesion of rat retinal cells on the patterned PEMs was observed 5 h after seeding. Cell viability and morphology on the patterned PEMs were assayed. These materials proved to be nontoxic to the cells used in this study regardless of the number of stacked PEM layers. Phalloidin staining of the cytoskeleton revealed no apparent morphological differences in retinal cells compared with those plated on polystyrene or the larger regions of PEI and PAH; however, cells were relatively more elongated when cultured on the PEM lines. Cell-to-cell communication between cells on adjacent PEM lines was observed as interconnecting tubes containing actin that were a few hundred nanometers in diameter and up to 55 microm in length. This approach provides a simple, fast, and inexpensive method of patterning cells onto micrometer-scale features.