Previous work has shown that sphingosine 1-phosphate (S1P) decreases outflow facility in perfused porcine eyes while dramatically increasing giant vacuole density in the inner wall of the aqueous plexus, with no obvious changes in the trabecular meshwork (TM). Due to known effects of S1P on cell-cell junction assembly in vascular endothelia, we hypothesized that S1P would decrease outflow facility in human eyes by increasing the complexity of cell-cell junctions in Schlemm’s canal (SC) inner wall endothelia. Perfusion of enucleated post mortem human eyes at 8 mmHg constant pressure in the presence or absence of 5 µM S1P showed that S1P decreased outflow facility by 36 ± 20% (n = 10 pairs; p=0.0004); an effect likely mediated by activation of S1P1 and/or S1P3 receptor subtypes, which were found to be the principal S1P receptors expressed by both TM and SC cells by RT-PCR, confocal immunofluorescence microscopy and western blot analyses. Examination of SC’s inner wall using confocal microscopy revealed no consistent differences in VE-cadherin, β-catenin, phosphotyrosine or filamentous actin abundance/distribution between S1P-treated eyes and controls. Moreover, morphological inspection of conventional outflow tissues by light and scanning electron microscopy showed no significant differences between S1P-treated and control eyes, particularly in giant vacuole density. Thus, unlike the situation in porcine eyes, we did not observe changes in inner wall morphology in human eyes treated with S1P, despite a significant and immediate decrease in outflow facility in both species. Regardless, S1P receptor antagonists represent novel therapeutic prospects for ocular hypertension in humans.
Purpose Ocular pulse decreases outflow facility of perfused anterior segments. However, the mechanism by which conventional outflow tissues respond to cyclic intraocular pressure oscillations is unknown. The purpose of the present study was to examine responses of trabecular meshwork (TM) cells to cyclic biomechanical stress in the presence and absence of compounds known to affect cell contractility. Methods To model flow in the juxtacanalicular region of the TM and to measure changes in transendothelial flow, human TM cell monolayers on permeable filters were perfused at a constant flow rate until reaching a stable baseline pressure and then were exposed to cyclic stress with an average amplitude of 2.7 mm Hg peak to peak at a 1-Hz frequency for 2 hours in the presence or absence of compounds known to affect cell contractility (isoproterenol, Y27632, pilocarpine, and nifedipine). Pressure was recorded continuously. Immunocytochemistry staining was used to determine filamentous actin stress fiber content, whereas Western blot analysis was used to measure the extent of myosin light chain (p-MLC) phosphorylation and ratio of filamentous to globular actin. Results Human TM cells respond to cyclic pressure oscillations by increasing mean intrachamber pressure (decreasing hydraulic conductivity) (126.13% ± 2.4%; P < 0.05), a response blocked in the presence of Y27632, a rho-kinase inhibitor (101.35 ± 0.59; P = 0.234), but not isoproterenol, pilocarpine, or nifedipine. Although mechanical stress appeared to have no effect, Y27632 decreased phosphorylated myosin light chain, filamentous/globular actin ratio, and stress fiber formation in TM cells. Conclusions Human TM cells respond to cyclic mechanical stress by increasing intrachamber pressure. Pulse-mediated effects are blocked by Y27632, implicating a role for Rho-kinase-mediated signaling and cellular contractility in ocular pulse-associated changes in outflow facility.
Results obtained in this study support the hypothesis that S1P increases actomyosin organization at the SC cell cortex and promotes intercellular junctions at the level of the inner wall of SC to increase transendothelial resistance and in part explains the S1P-induced decrease of outflow facility in organ culture.
BackgroundMaintaining proper adhesion between neighboring cells depends on the ability of cells to mechanically respond to tension at cell-cell junctions through the actin cytoskeleton. Thus, identifying the molecules involved in responding to cell tension would provide insight into the maintenance, regulation, and breakdown of cell-cell junctions during various biological processes. Vinculin, an actin-binding protein that associates with the cadherin complex, is recruited to cell-cell contacts under increased tension in a myosin II-dependent manner. However, the precise role of vinculin at force-bearing cell-cell junctions and how myosin II activity alters the recruitment of vinculin at quiescent cell-cell contacts have not been demonstrated.ResultsWe generated vinculin knockdown cells using shRNA specific to vinculin and MDCK epithelial cells. These vinculin-deficient MDCK cells form smaller cell clusters in a suspension than wild-type cells. In wound healing assays, GFP-vinculin accumulated at cell-cell junctions along the wound edge while vinculin-deficient cells displayed a slower wound closure rate compared to vinculin-expressing cells. In the presence of blebbistatin (myosin II inhibitor), vinculin localization at quiescent cell-cell contacts was unaffected while in the presence of jasplakinolide (F-actin stabilizer), vinculin recruitment increased in mature MDCK cell monolayers.ConclusionThese results demonstrate that vinculin plays an active role at adherens junctions under increased tension at cell-cell contacts where vinculin recruitment occurs in a myosin II activity-dependent manner, whereas vinculin recruitment to the quiescent cell-cell junctions depends on F-actin stabilization.
Elevated intraocular pressure is the main risk factor in primary open-angle glaucoma, involving an increased resistance to aqueous humor outflow in the juxtacanalicular region of the conventional outflow pathway which includes the trabecular meshwork (TM) and the inner wall of Schlemm's canal (SC). Previously, sphingosine-1-phosphate (S1P) was shown to decrease outflow facility in porcine and human eyes, thus increasing outflow resistance and intraocular pressure. Owing to S1P's known effect of increasing barrier function in endothelial cells and the robust expression of the S1P₁ receptor on the inner wall of SC, we hypothesized that S1P₁ receptor activation promotes junction formation and decreases outflow facility. The effects of subtype-specific S1P receptor compounds were tested in human and porcine whole-eye perfusions and human primary cultures of SC and TM cells to determine the receptor responsible for S1P effects on outflow resistance. The S1P₁-specific agonist SEW2871 failed to both mimic S1P effects in paired human eye perfusions, as well as increase myosin light chain (MLC) phosphorylation in cell culture, a prominent outcome in S1P-treated SC and TM cells. In contrast, the S1P₂ antagonist JTE-013, but not the S1P₁ or S1P₁,₃ antagonists, blocked the S1P-promoted increase in MLC phosphorylation. Moreover, JTE-013 prevented S1P-induced decrease in outflow facility in perfused human eyes (P < 0.05, n = 6 pairs). Similarly, porcine eyes perfused with JTE-013 + S1P did not differ from eyes with JTE-013 alone (P = 0.53, n = 3). These results demonstrate that S1P₂ , and not S1P₁ or S1P₃, receptor activation increases conventional outflow resistance and is a potential target to regulate intraocular pressure.
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