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Mapping molecular differences and extracellular matrix gene expression in segmental outflow pathways of the human ocular trabecular meshwork.

Vranka JA, Bradley JM, Yang YF, Keller KE, Acott TS - PLoS ONE (2015)

Bottom Line: Several collagen genes (COL16A1, COL4A2, COL6A1 and 2) and MMPs (1, 2, 3) were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions.Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences.These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway.

View Article: PubMed Central - PubMed

Affiliation: Casey Eye Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd, Portland, Oregon, United States of America.

ABSTRACT
Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, and lowering IOP remains the only effective treatment for glaucoma. The trabecular meshwork (TM) in the anterior chamber of the eye regulates IOP by generating resistance to aqueous humor outflow. Aqueous humor outflow is segmental, but molecular differences between high and low outflow regions of the TM are poorly understood. In this study, flow regions of the TM were characterized using fluorescent tracers and PCR arrays. Anterior segments from human donor eyes were perfused at physiological pressure in an ex vivo organ culture system. Fluorescently-labeled microspheres of various sizes were perfused into anterior segments to label flow regions. Actively perfused microspheres were segmentally distributed, whereas microspheres soaked passively into anterior segments uniformly labeled the TM and surrounding tissues with no apparent segmentation. Cell-tracker quantum dots (20 nm) were localized to the outer uveal and corneoscleral TM, whereas larger, modified microspheres (200 nm) localized throughout the TM layers and Schlemm's canal. Distribution of fluorescent tracers demonstrated a variable labeling pattern on both a macro- and micro-scale. Quantitative PCR arrays allowed identification of a variety of extracellular matrix genes differentially expressed in high and low flow regions of the TM. Several collagen genes (COL16A1, COL4A2, COL6A1 and 2) and MMPs (1, 2, 3) were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions. Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences. These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway. New insight into the molecular mechanisms of segmental aqueous outflow may aid in the design and delivery of improved treatments for glaucoma patients.

No MeSH data available.


Related in: MedlinePlus

Quantitative PCR array of TM from high flow regions in comparison with low flow regions.Human TM’s were dissected from perfused anterior segments, RNA was isolated, reverse transcribed, and measured using the human extracellular matrix and adhesion molecule quantitative PCR array. Fold change gene expression is shown as either enriched in high flow regions (values greater than 1.0) or enriched in low flow regions (values less than 1.0). All fold changes greater than 1.5 fold and less than 0.5 were considered to be biologically significant. SAM (significance analysis of microarrays), version 4.01, with 4 biological replicates, was used to determine statistically significant fold gene expression changes for HF regions in comparison with LF regions. The raw data using the delta delta Ct values for HF and LF regions was normalized, then subjected to SAM analysis. All fold change genes shown here were determined to be statistically significant by SAM, and biologically significant, by our 1.5 and 0.5 fold criteria. Error bars are the s.e.m.; n = 4 high flow and low flow regions from 4 individual donor eyes.
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pone.0122483.g006: Quantitative PCR array of TM from high flow regions in comparison with low flow regions.Human TM’s were dissected from perfused anterior segments, RNA was isolated, reverse transcribed, and measured using the human extracellular matrix and adhesion molecule quantitative PCR array. Fold change gene expression is shown as either enriched in high flow regions (values greater than 1.0) or enriched in low flow regions (values less than 1.0). All fold changes greater than 1.5 fold and less than 0.5 were considered to be biologically significant. SAM (significance analysis of microarrays), version 4.01, with 4 biological replicates, was used to determine statistically significant fold gene expression changes for HF regions in comparison with LF regions. The raw data using the delta delta Ct values for HF and LF regions was normalized, then subjected to SAM analysis. All fold change genes shown here were determined to be statistically significant by SAM, and biologically significant, by our 1.5 and 0.5 fold criteria. Error bars are the s.e.m.; n = 4 high flow and low flow regions from 4 individual donor eyes.

Mentions: ECM is thought to be the source of outflow resistance so we hypothesized that ECM gene expression may be different between regions, which may provide clues as to genes that are important to facilitate aqueous humor outflow. In order to determine molecular differences in ECM genes in high and low flow regions of the TM, RNA was extracted and ECM and adhesion gene quantitative PCR arrays were performed (Fig 6). Fold change values are represented graphically and show gene changes in high flow regions in comparison with low flow regions. The collagen genes COL1A1, COL4A2, COL6A1, COL6A2, and COL16A1, were all more enriched in high flow regions, whereas COL15A1 was enriched in low flow regions. The matrix metalloproteinase (MMP) genes MMP1, 2, 3 and 12 were enriched in high flow regions. Conversely, MMP16 was relatively enriched in the low flow regions. Additional differences were seen with the matricellular gene SPARC which was enriched in high flow regions, whereas osteopontin (SPP1) was enriched in low flow regions. Other gene families such as the integrins (ITG) and laminins (LAM) also showed variable expression with some members enriched in high flow regions and others in low flow regions. Thus, there are distinct molecular differences in ECM gene expression levels in high and low flow regions of the TM.


Mapping molecular differences and extracellular matrix gene expression in segmental outflow pathways of the human ocular trabecular meshwork.

Vranka JA, Bradley JM, Yang YF, Keller KE, Acott TS - PLoS ONE (2015)

Quantitative PCR array of TM from high flow regions in comparison with low flow regions.Human TM’s were dissected from perfused anterior segments, RNA was isolated, reverse transcribed, and measured using the human extracellular matrix and adhesion molecule quantitative PCR array. Fold change gene expression is shown as either enriched in high flow regions (values greater than 1.0) or enriched in low flow regions (values less than 1.0). All fold changes greater than 1.5 fold and less than 0.5 were considered to be biologically significant. SAM (significance analysis of microarrays), version 4.01, with 4 biological replicates, was used to determine statistically significant fold gene expression changes for HF regions in comparison with LF regions. The raw data using the delta delta Ct values for HF and LF regions was normalized, then subjected to SAM analysis. All fold change genes shown here were determined to be statistically significant by SAM, and biologically significant, by our 1.5 and 0.5 fold criteria. Error bars are the s.e.m.; n = 4 high flow and low flow regions from 4 individual donor eyes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380331&req=5

pone.0122483.g006: Quantitative PCR array of TM from high flow regions in comparison with low flow regions.Human TM’s were dissected from perfused anterior segments, RNA was isolated, reverse transcribed, and measured using the human extracellular matrix and adhesion molecule quantitative PCR array. Fold change gene expression is shown as either enriched in high flow regions (values greater than 1.0) or enriched in low flow regions (values less than 1.0). All fold changes greater than 1.5 fold and less than 0.5 were considered to be biologically significant. SAM (significance analysis of microarrays), version 4.01, with 4 biological replicates, was used to determine statistically significant fold gene expression changes for HF regions in comparison with LF regions. The raw data using the delta delta Ct values for HF and LF regions was normalized, then subjected to SAM analysis. All fold change genes shown here were determined to be statistically significant by SAM, and biologically significant, by our 1.5 and 0.5 fold criteria. Error bars are the s.e.m.; n = 4 high flow and low flow regions from 4 individual donor eyes.
Mentions: ECM is thought to be the source of outflow resistance so we hypothesized that ECM gene expression may be different between regions, which may provide clues as to genes that are important to facilitate aqueous humor outflow. In order to determine molecular differences in ECM genes in high and low flow regions of the TM, RNA was extracted and ECM and adhesion gene quantitative PCR arrays were performed (Fig 6). Fold change values are represented graphically and show gene changes in high flow regions in comparison with low flow regions. The collagen genes COL1A1, COL4A2, COL6A1, COL6A2, and COL16A1, were all more enriched in high flow regions, whereas COL15A1 was enriched in low flow regions. The matrix metalloproteinase (MMP) genes MMP1, 2, 3 and 12 were enriched in high flow regions. Conversely, MMP16 was relatively enriched in the low flow regions. Additional differences were seen with the matricellular gene SPARC which was enriched in high flow regions, whereas osteopontin (SPP1) was enriched in low flow regions. Other gene families such as the integrins (ITG) and laminins (LAM) also showed variable expression with some members enriched in high flow regions and others in low flow regions. Thus, there are distinct molecular differences in ECM gene expression levels in high and low flow regions of the TM.

Bottom Line: Several collagen genes (COL16A1, COL4A2, COL6A1 and 2) and MMPs (1, 2, 3) were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions.Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences.These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway.

View Article: PubMed Central - PubMed

Affiliation: Casey Eye Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd, Portland, Oregon, United States of America.

ABSTRACT
Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, and lowering IOP remains the only effective treatment for glaucoma. The trabecular meshwork (TM) in the anterior chamber of the eye regulates IOP by generating resistance to aqueous humor outflow. Aqueous humor outflow is segmental, but molecular differences between high and low outflow regions of the TM are poorly understood. In this study, flow regions of the TM were characterized using fluorescent tracers and PCR arrays. Anterior segments from human donor eyes were perfused at physiological pressure in an ex vivo organ culture system. Fluorescently-labeled microspheres of various sizes were perfused into anterior segments to label flow regions. Actively perfused microspheres were segmentally distributed, whereas microspheres soaked passively into anterior segments uniformly labeled the TM and surrounding tissues with no apparent segmentation. Cell-tracker quantum dots (20 nm) were localized to the outer uveal and corneoscleral TM, whereas larger, modified microspheres (200 nm) localized throughout the TM layers and Schlemm's canal. Distribution of fluorescent tracers demonstrated a variable labeling pattern on both a macro- and micro-scale. Quantitative PCR arrays allowed identification of a variety of extracellular matrix genes differentially expressed in high and low flow regions of the TM. Several collagen genes (COL16A1, COL4A2, COL6A1 and 2) and MMPs (1, 2, 3) were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions. Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences. These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway. New insight into the molecular mechanisms of segmental aqueous outflow may aid in the design and delivery of improved treatments for glaucoma patients.

No MeSH data available.


Related in: MedlinePlus