Abstract
Hyaluronan (HA) in the ocular trabecular meshwork (TM) is a critical modulator of aqueous humor outflow. Individual HA strands in the pericellular matrix can coalesce to form cable-like structures, which have different functional properties. Here, we investigated HA structural configuration by TM cells in response to various stimuli known to stimulate extracellular matrix (ECM) remodeling. In addition, the effects of HA cable induction on aqueous outflow resistance was determined. Primary TM cell cultures grown on tissue culture-treated plastic were treated for 12-48 h with TNFα, IL-1α, or TGFβ2. TM cells grown on silicone membranes were subject to mechanical stretch, which induces synthesis and activation of ECM proteolytic enzymes. HA structural configuration was investigated by HA binding protein (HAbp) staining and confocal microscopy. HAbp-labeled cables were induced by TNFα, TGFβ2 and mechanical stretch, but not by IL-1α. HA synthase (HAS) gene expression was quantitated by quantitative RT-PCR and HA concentration was measured by ELISA assay. By quantitative RT-PCR, HAS-1, -2, and -3 genes were differentially up-regulated and showed temporal differences in response to each treatment. HA concentration was increased in the media by TNFα, TGFβ2 and IL-1α, but mechanical stretch decreased pericellular HA concentrations. Immunofluorescence and Western immunoblotting were used to investigate the distribution and protein levels of the HA-binding proteins, tumor necrosis factor-stimulated gene-6 (TSG-6) and inter-α-inhibitor (IαI). Western immunoblotting showed that TSG-6 and IαI were increased by TNFα, TGFβ2 and IL-1α, but mechanical stretch reduced their levels. The underlying substrate appears to affect the identity of IαI·TSG-6·HA complexes since different complexes were detected when TM cells were grown on a silicone substrate compared to a rigid plastic surface. Porcine anterior segments were perfused with 10 μg/ml polyinosinic:polycytidylic acid (polyI:C), a potent inducer of HA cables, and outflow rates were monitored for 72 h. PolyI:C had no significant effect on outflow resistance in porcine anterior segments perfused at physiological pressure. Collectively, HAS gene expression, HA concentration and configuration are differentially modified in response to several treatments that induce ECM remodeling in TM cells. In ocular TM cells, our data suggests that the most important determinant of HA cable formation appears to be the ratio of HA chains produced by the different HAS genes. However, the act of rearranging pericellular HA into cable-like structures does not appear to influence aqueous outflow resistance.
| Original language | English (US) |
|---|---|
| Article number | 6733 |
| Pages (from-to) | 97-107 |
| Number of pages | 11 |
| Journal | Experimental Eye Research |
| Volume | 139 |
| DOIs | |
| State | Published - 2015 |
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Keywords
- Aqueous outflow
- Extracellular matrix
- Glaucoma
- Hyaluronan
- Trabecular meshwork
ASJC Scopus subject areas
- Ophthalmology
- Sensory Systems
- Cellular and Molecular Neuroscience
Cite this
Hyaluronan cable formation by ocular trabecular meshwork cells. / Sun, Ying Ying; Keller, Kate.
In: Experimental Eye Research, Vol. 139, 6733, 2015, p. 97-107.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Hyaluronan cable formation by ocular trabecular meshwork cells
AU - Sun, Ying Ying
AU - Keller, Kate
PY - 2015
Y1 - 2015
N2 - Hyaluronan (HA) in the ocular trabecular meshwork (TM) is a critical modulator of aqueous humor outflow. Individual HA strands in the pericellular matrix can coalesce to form cable-like structures, which have different functional properties. Here, we investigated HA structural configuration by TM cells in response to various stimuli known to stimulate extracellular matrix (ECM) remodeling. In addition, the effects of HA cable induction on aqueous outflow resistance was determined. Primary TM cell cultures grown on tissue culture-treated plastic were treated for 12-48 h with TNFα, IL-1α, or TGFβ2. TM cells grown on silicone membranes were subject to mechanical stretch, which induces synthesis and activation of ECM proteolytic enzymes. HA structural configuration was investigated by HA binding protein (HAbp) staining and confocal microscopy. HAbp-labeled cables were induced by TNFα, TGFβ2 and mechanical stretch, but not by IL-1α. HA synthase (HAS) gene expression was quantitated by quantitative RT-PCR and HA concentration was measured by ELISA assay. By quantitative RT-PCR, HAS-1, -2, and -3 genes were differentially up-regulated and showed temporal differences in response to each treatment. HA concentration was increased in the media by TNFα, TGFβ2 and IL-1α, but mechanical stretch decreased pericellular HA concentrations. Immunofluorescence and Western immunoblotting were used to investigate the distribution and protein levels of the HA-binding proteins, tumor necrosis factor-stimulated gene-6 (TSG-6) and inter-α-inhibitor (IαI). Western immunoblotting showed that TSG-6 and IαI were increased by TNFα, TGFβ2 and IL-1α, but mechanical stretch reduced their levels. The underlying substrate appears to affect the identity of IαI·TSG-6·HA complexes since different complexes were detected when TM cells were grown on a silicone substrate compared to a rigid plastic surface. Porcine anterior segments were perfused with 10 μg/ml polyinosinic:polycytidylic acid (polyI:C), a potent inducer of HA cables, and outflow rates were monitored for 72 h. PolyI:C had no significant effect on outflow resistance in porcine anterior segments perfused at physiological pressure. Collectively, HAS gene expression, HA concentration and configuration are differentially modified in response to several treatments that induce ECM remodeling in TM cells. In ocular TM cells, our data suggests that the most important determinant of HA cable formation appears to be the ratio of HA chains produced by the different HAS genes. However, the act of rearranging pericellular HA into cable-like structures does not appear to influence aqueous outflow resistance.
AB - Hyaluronan (HA) in the ocular trabecular meshwork (TM) is a critical modulator of aqueous humor outflow. Individual HA strands in the pericellular matrix can coalesce to form cable-like structures, which have different functional properties. Here, we investigated HA structural configuration by TM cells in response to various stimuli known to stimulate extracellular matrix (ECM) remodeling. In addition, the effects of HA cable induction on aqueous outflow resistance was determined. Primary TM cell cultures grown on tissue culture-treated plastic were treated for 12-48 h with TNFα, IL-1α, or TGFβ2. TM cells grown on silicone membranes were subject to mechanical stretch, which induces synthesis and activation of ECM proteolytic enzymes. HA structural configuration was investigated by HA binding protein (HAbp) staining and confocal microscopy. HAbp-labeled cables were induced by TNFα, TGFβ2 and mechanical stretch, but not by IL-1α. HA synthase (HAS) gene expression was quantitated by quantitative RT-PCR and HA concentration was measured by ELISA assay. By quantitative RT-PCR, HAS-1, -2, and -3 genes were differentially up-regulated and showed temporal differences in response to each treatment. HA concentration was increased in the media by TNFα, TGFβ2 and IL-1α, but mechanical stretch decreased pericellular HA concentrations. Immunofluorescence and Western immunoblotting were used to investigate the distribution and protein levels of the HA-binding proteins, tumor necrosis factor-stimulated gene-6 (TSG-6) and inter-α-inhibitor (IαI). Western immunoblotting showed that TSG-6 and IαI were increased by TNFα, TGFβ2 and IL-1α, but mechanical stretch reduced their levels. The underlying substrate appears to affect the identity of IαI·TSG-6·HA complexes since different complexes were detected when TM cells were grown on a silicone substrate compared to a rigid plastic surface. Porcine anterior segments were perfused with 10 μg/ml polyinosinic:polycytidylic acid (polyI:C), a potent inducer of HA cables, and outflow rates were monitored for 72 h. PolyI:C had no significant effect on outflow resistance in porcine anterior segments perfused at physiological pressure. Collectively, HAS gene expression, HA concentration and configuration are differentially modified in response to several treatments that induce ECM remodeling in TM cells. In ocular TM cells, our data suggests that the most important determinant of HA cable formation appears to be the ratio of HA chains produced by the different HAS genes. However, the act of rearranging pericellular HA into cable-like structures does not appear to influence aqueous outflow resistance.
KW - Aqueous outflow
KW - Extracellular matrix
KW - Glaucoma
KW - Hyaluronan
KW - Trabecular meshwork
UR - http://www.scopus.com/inward/record.url?scp=84939166153&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84939166153&partnerID=8YFLogxK
U2 - 10.1016/j.exer.2015.07.018
DO - 10.1016/j.exer.2015.07.018
M3 - Article
C2 - 26247678
AN - SCOPUS:84939166153
VL - 139
SP - 97
EP - 107
JO - Experimental Eye Research
JF - Experimental Eye Research
SN - 0014-4835
M1 - 6733
ER -