November 2011
Volume 52, Issue 12
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Physiology and Pharmacology  |   November 2011
In Vivo Gene Expression Profiling of Retina Postintravitreal Injections of Dexamethasone and Triamcinolone at Clinically Relevant Time Points for Patient Care
Author Affiliations & Notes
  • Zeljka Smit-McBride
    From the Vitreoretinal Research Laboratory, Department of Ophthalmology, University of California Davis, Davis, California.
  • Sara P. Modjtahedi
    From the Vitreoretinal Research Laboratory, Department of Ophthalmology, University of California Davis, Davis, California.
  • Christopher T. Cessna
    From the Vitreoretinal Research Laboratory, Department of Ophthalmology, University of California Davis, Davis, California.
  • David G. Telander
    From the Vitreoretinal Research Laboratory, Department of Ophthalmology, University of California Davis, Davis, California.
  • Leonard M. Hjelmeland
    From the Vitreoretinal Research Laboratory, Department of Ophthalmology, University of California Davis, Davis, California.
  • Lawrence S. Morse
    From the Vitreoretinal Research Laboratory, Department of Ophthalmology, University of California Davis, Davis, California.
  • Corresponding author: Lawrence S. Morse, Vitreoretinal Research Laboratory, UC Davis Department of Ophthalmology, 4860 Y Street, Suite 2400, Sacramento, CA 95817; lsmorse@ucdavis.edu
Investigative Ophthalmology & Visual Science November 2011, Vol.52, 8965-8978. doi:10.1167/iovs.10-7084
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      Zeljka Smit-McBride, Sara P. Modjtahedi, Christopher T. Cessna, David G. Telander, Leonard M. Hjelmeland, Lawrence S. Morse; In Vivo Gene Expression Profiling of Retina Postintravitreal Injections of Dexamethasone and Triamcinolone at Clinically Relevant Time Points for Patient Care. Invest. Ophthalmol. Vis. Sci. 2011;52(12):8965-8978. doi: 10.1167/iovs.10-7084.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To identify retinal genes and their relevant expression pathways affected by intravitreal injections of dexamethasone (Dex) and triamcinolone acetonide (TAA) in mice at clinically relevant time points for patient care.

Methods.: Differential gene expressions of over 34,000 well-characterized mouse genes, in the retinas of 6-week-old C57BL/6J mice, were analyzed after intravitreal steroid injections at 1 week and 1 month time points, using mouse genome microarrays. The data were analyzed using commercial microarray analysis software for biologically relevant changes in gene expression pathways.

Results.: A common gene pathway, with differentially activated genes for both steroids and time points, was “Semaphorin Signaling in Neurons,” a member of the “Axonal Guidance Signaling System.” At 1 week postinjection a common theme was activation of genes expressed in retinal glial cells, tumor necrosis factor-α, and transforming growth factor-β signaling pathways and upregulation of stress response proteins (Serpina3n, Cebpd), as well as neuropeptide signaling somatostatin receptor (Sstr2). Unique for Dex was the upregulation of acute phase proteins (Gfap, Cp, Edn2) as well as Plexna2, a semaphorin signaling receptor, whereas EphrinB receptor ephexin 1 (Argef15) was downregulated. Folate signaling appears to be unique for TAA at 1 week (Folh1, Cubn), whereas aryl-hydrocarbon receptor signaling might be important for both steroids at 1 month postinjection.

Conclusions.: Understanding the molecular and genetic effects of intraocular steroid treatments is of clinical relevance. This in vivo study has elucidated several genes and pathways that are potentially altering the neuroprotective/neurodegenerative balance between glial and retinal ganglion cells during intravitreal steroid treatment.

Intravitreal administration of dexamethasone (Dex) and triamcinolone (TAA) is commonly used in clinical practice to treat underlying inflammatory changes in a wide range of retinal pathologies, including diabetes, uveitis, and choroidal neovascularization. Despite their established therapeutic benefit, side effects such as cataract formation and ocular hypertension/glaucoma raise concerns. 1 Clinical decision-making could be greatly enhanced with a better appreciation of the differential gene expression induced by intravitreal steroids. In this study we examined the differential gene expression effects of intravitreal injections of Dex and TAA in C57BL/6J mouse neurosensory retinal tissue in vivo, at clinically relevant time points of 1 week and 1 month postinjection. The dosages used in mice were comparable to the doses that are commonly used in intravitreal injections in humans (0.1 mg of Dex in 1 mL of vitreous and 1 mg of TAA in 1 mL of vitreous). Microarray analysis was used to identify common and unique changes in gene expression and activated pathways between the two steroids at two clinically relevant time points. Our findings suggest a delicate balance between protective and deleterious effects of the genes and pathways that might be causing beneficial anti-inflammatory and neuroprotective responses, as well as those that could be causing neuronal damage of the retina as a result of the glial cell inflammatory response. 
Methods
Animals
All research was conducted in compliance with the ARVO Statement for the use of animals in ophthalmic and vision research. We used male C57BL/6J mice (Jax-West, Sacramento, CA) for all experiments. For each time point (1 week and 1 month) we had three groups of animals with four animals per group, 6 to 8 weeks of age. 
Mouse Anesthesia
Ketamine (1 mL, 100 mg/mL), 0.1 mL of xylazine (100 mg/mL), and 8.9 mL of sterile water were mixed to prepare a final solution for injection. Animals were anesthetized by injecting 0.1 mL/10 g body weight intraperitoneally. 
Intravitreal Injections
Intravitreal injections were performed transconjunctivally in anesthetized C57BL/6J mice using a syringe (Hamilton Co., Reno, NV) equipped with a 33-gauge needle, delivering 2 μL of solution at day 0. In group 1, control group, animals received intravitreal injections of physiologic saline solution (PSS), group 2: TAA (20 μg), group 3: Dex (2 μg). 
RNA Isolation
At 1 week and 1 month postinjection, mice from each group were euthanized, using a CO2 chamber, and the eyes were harvested for retinal tissue using published procedures. 2 Dissected retinas were immediately placed in 300 μL RNA stabilization reagent (RNAlater; Qiagen Inc., Valencia, CA) and stored at −20°C. Retinas were next homogenized using a 1-mL syringe and an 18-gauge needle. An isolation kit (RNeasy; Qiagen) was used to isolate total RNA from the tissue. RNA samples were run on a microfluidics chip–based platform (BioAnalyzer RNA Nano 6000; Agilent Technologies, Santa Clara, CA) to assess quality and quantity. Of the four samples, the three having the highest quality (RNA Integrity Number [RIN] ≥ 7 value) were labeled as probes for the microarrays (Affymetrix GeneChip; Affymetrix, Santa Clara, CA). 
Microarray Probe Labeling
RNA samples were labeled using 0.100 μg of total RNA, following the manufacturer's protocol for small sample (Gene-Chip Two-cycle Target Labeling Assay; Affymetrix). 2 After labeling, probes were hybridized to microarrays (Affymetrix Mouse Genome 430 2.0 GeneChip Array). This array contains 45,101 probe sets, analyzing the expression of 39,000 transcripts and variants from over 34,000 well-characterized mouse genes. Hybridization was performed in the University of California Davis Genome Center Microarray Core Facility using a standard procedure (Affymetrix). The total data set included 18 microarrays (GeneChip; Affymetrix). 
Data Normalization
The data sets were analyzed with gene expression analysis software (GeneSpringGX 11; Agilent Technologies), using the robust multiarray analysis (RMA) algorithm. Raw .cel files were imported into the analysis software GeneSpringGX 11 and the RMA summarization algorithm was applied with chip–chip normalization using baseline to median of all samples. Probe sets were filtered to their raw signal intensity values (36,326 of 45,101 passed the filter), with the criterion that 100% of the samples in any one of six conditions must have values between the 20th and 100th percentiles in the data. 
Statistical Analysis
One-way ANOVA was used to identify statistically significant genes at a significance level of P ≤ 0.01. The Benjamini–Hochberg post hoc correction method was too conservative for microarray results with the limited number of biological replicas such as this. No probe sets met this significance level of testing. Therefore, a different set of stringency requirements was applied to reduce the risk of false positives. To identify biologically relevant gene expression changes for each of the time point/treatment conditions, Volcano plot analysis 3 was performed, using a combination of unpaired t-test and asymptotic P value computation. The Volcano plot is a scatter plot of the fold-change versus the P value (in −log10 P scale). It is commonly used to simultaneously depict the P value and the fold-change for gene selection cutoff schemes in microarray data. It may be viewed as a summary of “statistical” significance and “biological” significance over a large number of genes. We used the standard approach of using a P value (P ≤ 0.05) as the primary criterion followed by fold change (−1.5 ≥ FC ≥ 1.5) as the secondary criterion to select differentially expressed genes. This approach ensures control of false-positive error and preserves the desired biological significance. 4  
The data discussed in this publication have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus 5,6 and are accessible through GEO Series accession number GSE30701 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30701). (See Supplementary Material, which contains the complete list of the differentially expressed genes identified by ANOVA at P ≤ 0.01 and Volcano plot at [−1.5 ≥ FC ≥ 1.5], P ≤ 0.05.) 
Bioinformatics/Pathways Analysis
Biological pathway analysis was performed using pathway analysis software (Ingenuity Pathway Analysis [IPA]; Ingenuity Systems, Redwood City, CA). The lists of candidate genes identified by statistical analysis were uploaded to pathway analysis software (IPA), and a core analysis algorithm was applied to provide a list of activated pathways and gene networks with the highest degree of significance. 
Quantitative PCR
Quantitative PCR (qPCR) was performed as a second method of validation of candidate genes. All gene expression assays (Taqman; Applied Biosystems, Foster City, CA) were conducted in the Real-time PCR Research and Diagnostic Core Facility at University of California Davis. 
Results
Genes with Significantly Altered Expression Changes
Statistical analysis by one-way ANOVA identified 1558 probe sets differentially expressed at P ≤ 0.01 when the whole 18-chip sample set was considered. One-way ANOVA analysis for each of the time points separately identified 462 genes at 1 week and 597 genes at 1 month that were differentially expressed at P ≤ 0.01. Comparison of treatment versus control for each time point using an unpaired t-test identified a set of common genes similarly regulated for both steroids as well as a set of unique genes differentially regulated by each steroid. Figures 1A–D represent Venn diagrams detailing the numbers of genes in each category. A list of common genes and the 25 most interesting unique genes for each steroid/time point/regulation group, are presented in Tables 1 2 3 4 56. The full lists of differentially expressed genes at P ≤ 0.01 and P ≤ 0.05 and −1.5 ≥ FC ≥ 1.5 can be found in the Supplementary Materials
Figure 1.
 
One-way ANOVA for each of the time points separately identified 462 genes at 1 week and 597 genes at 1 month that were differentially expressed at P ≤ 0.01. Venn diagrams of Dex versus control compared with TAA versus control identify common and unique genes for each steroid at P ≤ 0.01 at 1 week (A) and 1 month (B). The lower set of Venn diagrams identify the number of genes per each category that satisfy a criterium of −1.5 ≥ FC ≥ 1.5 and P ≤ 0.05 at 1 week (C) and 1 month (D).
Figure 1.
 
One-way ANOVA for each of the time points separately identified 462 genes at 1 week and 597 genes at 1 month that were differentially expressed at P ≤ 0.01. Venn diagrams of Dex versus control compared with TAA versus control identify common and unique genes for each steroid at P ≤ 0.01 at 1 week (A) and 1 month (B). The lower set of Venn diagrams identify the number of genes per each category that satisfy a criterium of −1.5 ≥ FC ≥ 1.5 and P ≤ 0.05 at 1 week (C) and 1 month (D).
Table 1.
 
Common Genes for Dex and TAA at 1 Week: Upregulated and Downregulated
Table 1.
 
Common Genes for Dex and TAA at 1 Week: Upregulated and Downregulated
Probe Set ID Gene Symbol Dex/1 wk TAA/1 wk
FC* P FC‡ P § Gene Title
Upregulated
1419100_at Serpina3n 3.40 0.023 7.88 0.036 Ser (or Cys) peptidase inhibitor, clade A, mem.3N
1423233_at Cebpd 2.22 0.001 1.53 0.005 CAAT/enhancer binding protein (C/EBP), delta
1456605_at
1436193_at Man1c1 1.38 0.003 1.12 0.007 Mannosidase, alpha, class 1C, member 1
1456380_x_at Cnn3 1.26 0.002 1.22 0.006 Calponin 3, acidic /// similar to calponin 3, acidic
1454960_at Smad3 1.25 0.007 1.10 0.004 MAD homolog 3 (Drosophila)
1438111_at Pvrl1 1.22 0.006 1.24 0.010 Poliovirus receptor-related 1 (Pvrl1), mRNA
1439273_at Ripk1 1.20 0.009 −1.19 0.001 Receptor (TNFRSF)-interacting serine-threonine kinase 1
1424440_at Mrps6 1.17 0.008 1.17 0.009 Mitochondrial ribosomal protein S6
1434561_at Asxl1 1.13 0.008 −1.16 0.009 Additional sex combs like 1 (Drosophila)
1422256_at Sstr2 1.12 0.006 1.21 0.002 Somatostatin receptor 2
Downregulated
1435126_at Dusp15 −1.21 0.009 −1.17 0.009 Dual specificity phosphatase-like 15
1457823_at Cyr61 −1.18 0.003 −1.18 0.002 Cysteine rich protein 61
1432260_at Grp39 −1.14 0.004 −1.24 0.008 G protein-coupled receptor 39
1452388_at Hspa1a −1.12 0.003 −1.20 0.003 Heat shock protein 1A
1460402_at Brpf1 −1.07 0.001 −1.14 0.008 Bromodomain and PHD finger containing 1
Table 2.
 
Unique Genes for Dex at 1 Week: Upregulated and Downregulated
Table 2.
 
Unique Genes for Dex at 1 Week: Upregulated and Downregulated
No. Probe Set ID Gene Symbol Dex/1 wk Gene Title
FC* P
Upregulated
1 1449161_at Edn2 3.76 0.013 Endothelin 2
2 1456940_at Slc43a2 2.29 0.009 Solute carrier family 43, member 2
3 1455393_at Cp 2.19 0.000 Ceruloplasmin
4 1450650_at Myo10 1.96 0.007 Myosin X
5 1438037_at Herc5 1.82 0.009 Hect domain and RLD 5
6 1426509_s_at Gfap 1.74 0.002 Glial fibrillary acidic protein
7 1429772_at Plxna2 1.67 0.016 Plexin A2
1453286_at 0.002
8 1437422_at Sema5a 1.60 0.029 Semaphorin 5A
9 1459928_at Lcor 1.60 0.008 Ligand-dependent nuclear receptor corepressor
10 1416776_at Crym 1.56 0.004 Crystallin, mu
11 1450716_at Adamts1 1.54 0.005 A disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1
12 1418133_at Bcl3 1.53 0.005 B-cell leukemia/lymphoma 3
13 1423760_at Cd44 1.53 0.001 CD44 antigen
14 1428114_at Slc14a1 1.50 0.006 Solute carrier family 14 (urea transporter), member 1
15 1419665_a_at Nupr1 1.46 0.000 Nuclear protein 1
16 1418448_at Rras 1.45 0.009 Harvey rat sarcoma oncogene, subgroup R
17 1460700_at Stat3 1.43 0.006 Signal transducer and activator of transcription 3
18 1417128_at Plekho1 1.24 0.009 Pleckstrin homology domain containing, family O, member 1
19 1450923_at Tgfb2 1.22 0.007 Transforming growth factor, beta 2
20 1449865_at Sema3a 1.21 0.001 Sema domain, immunoglobulin domain (Ig), (semaphorin) 3A
21 1455374_at Kcnj3 1.17 0.007 Potassium inwardly-rectifying channel, subfamily J, member 3
22 1428967_at Igf1r 1.17 0.006 Insulin-like growth factor 1 receptor
23 1425869_a_at Psen2 1.15 0.009 Presenilin 2
24 1434387_at Itfg3 1.15 0.002 Integrin alpha FG-GAP repeat containing 3
Downregulated
1 1457172_at Arhgef15 −1.68 0.048 Rho guanine nucleotide exchange factor (GEF) 15
2 1429504_at Rnpc3 −1.55 0.049 RNA-binding region (RNP1, RRM) containing 3
3 1438841_s_at Arg2 −1.41 0.005 Arginase type II
4 1417925_at Ccl22 −1.33 0.007 Chemokine (C-C motif) ligand 22
5 1417443_at Fam151a −1.33 0.008 Family with sequence similarity 151, member A
6 1429781_s_at Ccdc39 −1.32 0.009 Coiled-coil domain containing 39
7 1435064_a_at Tmem27 −1.32 0.008 Transmembrane protein 27
8 1435158_at Rbm12b −1.31 0.003 RNA-binding motif protein 12B
9 1436799_at Enox1 −1.31 0.002 Ecto-NOX disulfide-thiol exchanger 1
10 1418640_at Sirt1 −1.30 0.007 Sirtuin 1 (silent mating type information regulation 2, homolog) 1 (S. cerevisiae)
11 1427126_at Hspa1b −1.30 0.004 Heat shock protein 1B
12 1436994_a_at Hist1h1c −1.30 0.004 Histone cluster 1, H1c
13 1452054_at Ube2w −1.29 0.008 Ubiquitin-conjugating enzyme E2W (putative)
14 1425589_at Hsd17b13 −1.26 0.009 Hydroxysteroid (17-beta) dehydrogenase 13
15 1444816_at Olfr75-ps1 −1.25 0.001 V1 olfactory receptor protein
16 1425370_a_at Erg −1.24 0.007 Avian erythroblastosis virus E-26 (v-ets) oncogene related
17 1447234_s_at Snx6 −1.23 0.005 Sorting nexin 6
18 1417625_s_at Cxcr7 −1.18 0.005 Chemokine (C-X-C motif) receptor 7
19 1422029_at Ccl20 −1.15 0.008 Chemokine (C-C motif) ligand 20
20 1449399_a_at Il1b −1.14 0.003 Interleukin 1 beta
21 1425530_a_at Stx3 −1.14 0.003 Syntaxin 3
22 1419307_at Tnfrsf13c −1.13 0.010 Tumor necrosis factor receptor superfamily, member 13c
23 1422446_x_at Ins2 −1.13 0.002 Insulin II
24 1424647_at Gabrp −1.12 0.007 gamma-aminobutyric acid (GABA) A receptor, pi
25 1449734_s_at Bbs4 −1.08 0.005 Bardet-Biedl syndrome 4 (human)
Table 3.
 
Unique Genes for TAA at 1 Week: Upregulated and Downregulated
Table 3.
 
Unique Genes for TAA at 1 Week: Upregulated and Downregulated
No. Probe Set ID Gene Symbol TAA/1 wk Gene Title
FC* P
Upregulated
1 1450154_at Folh1 3.65 0.007 Folate hydrolase
2 1452975_at Agxt2l1 2.70 0.009 Alanine-glyoxylate aminotransferase 2-like 1
3 1424714_at Aldoc 2.38 0.039 Aldolase C, fructose-bisphosphate
4 1434202_a_at Fam107a 2.22 0.034 Family with sequence similarity 107, member A
5 1428352_at Arrdc2 2.03 0.029 Arrestin domain containing 2
6 1424825_a_at Glycam1 1.97 0.007 Glycosylation-dependent cell adhesion molecule 1
7 1427355_at Calca 1.94 0.004 Calcitonin/calcitonin-related polypeptide, alpha
8 1427883_a_at Col3a1 1.86 0.005 Collagen, type III, alpha 1
9 1438643_at Camk1d 1.84 0.047 Calcium/calmodulin-dependent protein kinase ID, mRNA
10 1437060_at Olfm4 1.55 0.023 Olfactomedin 4
11 1432543_a_at Klfl3 1.53 0.011 Kruppel-like factor 13
12 1435888_at Egfr 1.52 0.027 Epidermal growth factor receptor
13 1419040_at Cyp2d22 1.51 0.009 Cytochrome P450, family 2, subfamily d, polypeptide 22
14 1454780_at Galntl4 1.42 0.005 UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase
15 1416166_a_at Prdx4 1.42 0.002 Peroxiredoxin 4
16 1454642_a_at Commd3 1.40 0.002 COMM domain containing 3
17 1448136_at Enpp2 1.39 0.001 Ectonucleotide pyrophosphatase/phosphodiesterase 2
18 1417366_s_at Calm1 1.39 0.001 Calmodulin 1
19 1430798_x_at Mrpl15 1.38 0.004 Mitochondrial ribosomal protein L15
20 1458487_at Klf3 1.38 0.002 Kruppel-like factor 3 (basic)
21 1454886_x_at Trim9 1.37 0.010 Tripartite motif-containing 9
22 1455712_at Hist3h2a 1.36 0.001 Histone cluster 3, H2a
23 1421740_at Gnas 1.34 0.004 GNAS (guanine nucleotide binding protein, α stimulating) complex locus
24 1417948_s_at Ilf2 1.33 0.009 Interleukin enhancer binding factor 2
25 1431795_a_at Sema3b 1.17 0.003 Sema domain, immunoglobulin domain (Ig), (semaphorin) 3B
Downregulated
1 1456418_at Kcnj13 −2.00 0.001 Potassium inwardly-rectifying channel, subfamily J, member 13
2 1453385_at Xiap −1.74 0.015 X-linked inhibitor of apoptosis
3 1426990_at Cubn −1.74 0.006 Cubilin (intrinsic factor-cobalamin receptor)
4 1435064_a_at Tmem27 −1.62 0.014 Transmembrane protein 27
5 1431725_at Fmn2 −1.62 0.046 Formin 2
6 1457145_at Plekhg4 −1.60 0.048 Pleckstrin homology domain containing, family G (with RhoGef domain), member 4
7 1427749_at Galnt3 −1.52 0.000 UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 3
8 1433599_at Baz1a −1.47 0.006 Bromodomain adjacent to zinc finger domain 1A
9 1425519_a_at Cd74 −1.40 0.003 CD74 antigen
10 1443183_at Huwe1 −1.40 0.010 HECT, UBA, and WWE domain containing 1
11 1419191_at Hipk3 −1.40 0.007 Homeodomain interacting protein kinase 3
12 1440081_at Cep192 −1.36 0.009 Premature mRNA for mKIAA1569 protein
13 1446516_at Bcl7c −1.35 0.005 B-cell CLL/lymphoma 7C
14 1421138_a_at Pkib −1.34 0.001 Protein kinase inhibitor beta, cAMP-dependent, testis specific
15 1448994_at Sp1 −1.32 0.008 Trans-acting transcription factor 1
16 1437385_at Ccbe1 −1.32 0.010 Collagen and calcium binding EGF domains 1
17 1450332_s_at Fmo5 −1.31 0.009 Flavin containing monooxygenase 5
18 1436826_at Tmtc3 −1.30 0.006 Transmembrane and tetratricopeptide repeat containing 3
19 1434787_at Arf3 −1.30 0.007 ADP-ribosylation factor 3, mRNA
20 1423199_at Brd3 −1.29 0.006 Bromodomain containing 3
21 1443609_s_at Syvn1 −1.28 0.004 Synovial apoptosis inhibitor 1, synoviolin
22 1433737_at Uhmk1 −1.28 0.004 U2AF homology motif (UHM) kinase 1
23 1439477_at Ube2b −1.28 0.006 Ubiquitin-conjugating enzyme E2B, RAD6 homology (S. cerevisiae)
24 1424553_at Hhatl −1.26 0.003 Hedgehog acyltransferase-like
24 1431239_at Nono −1.27 0.004 Non-POU-domain-containing, octamer binding protein
25 1448389_at Wdr5 −1.26 0.006 WD repeat domain 5
25 1441982_at Taf7 −1.27 0.003 TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated factor
Table 4.
 
Common Genes for Dex and TAA at 1 Month: Upregulated and Downregulated
Table 4.
 
Common Genes for Dex and TAA at 1 Month: Upregulated and Downregulated
Probe Set ID Gene Symbol Dex/1 mo TAA/1 mo Gene Title
FC* P FC‡ P §
Upregulated
1434201_at Chrdl1 1.71 0.016 1.68 0.007 Chordin-like 1
1423424_at Zic3 1.65 0.021 1.78 0.017 Zinc finger protein of the cerebellum 3
1457472_at Gigyf2 1.55 0.048 1.60 0.008 GRB10 interacting GYF protein 2
1422631_at Ahr 1.37 0.008 1.25 0.005 Aryl-hydrocarbon receptor
1457790_at Asb3 1.17 0.006 1.18 0.008 Ankyrin repeat and SOCS box-containing 3
1450784_at Reck 1.15 3.01E-06 1.27 3.06E-05 Reversion-inducing-cysteine-rich protein with kazal motifs
Downregulated
1418284_at Vps72 −1.69 9.96E-04 −1.29 0.001 Vacuolar protein sorting 72 (yeast)
1435857_s_at Aplp1 −1.61 0.012 −1.60 0.003 Amyloid beta (A4) precursor-like protein 1
1436659_at Dclk1 −1.61 0.018 −1.61 0.046 Doublecortin-like kinase 1
1455143_at Nlgn2 −1.56 7.80E-04 −1.44 0.008 Neuroligin 2
1417628_at Supt6h −1.54 0.002 −1.24 0.004 Suppressor of Ty 6 homolog (S. cerevisiae)
1434635_at Rph3a −1.42 0.007 −1.32 5.91E-04 Rabphilin 3A
1449381_a_at Pacsin1 −1.38 6.60E-04 −1.31 0.004 Protein kinase C and casein kinase substrate in neurons 1
1424552_at Casp8 −1.35 0.002 −1.24 4.96E-04 Caspase 8
1426540_at Endod1 −1.35 3.42E-05 −1.15 0.005 Endonuclease domain containing 1
1429876_at Supt7l −1.24 0.001 −1.29 5.06E-04 Suppressor of Ty 7 (S. cerevisiae)-like
1423047_at Tollip −1.22 0.008 −1.15 0.010 Toll interacting protein
1450435_at L1cam −1.22 0.003 −1.15 0.009 L1 cell adhesion molecule
1427218_at Klhl11 −1.21 0.001 −1.21 5.80E-04 Kelch-like 11 (Drosophila)
1431811_a_at Fbxo34 −1.18 0.003 −1.29 0.002 F-box protein 34
1431973_at ‘Sep6’ −1.17 0.004 −1.27 1.11E-04 Septin 6
1423975_s_at Numa1 −1.17 0.009 −1.20 0.004 Nuclear mitotic apparatus protein 1
1448116_at Uba1 −1.15 2.47E-04 −1.09 0.006 Ubiquitin-like modifier activating enzyme 1
1457167_at Med14 −1.14 0.008 −1.17 0.003 Mediator complex subunit 14
1441829_s_at Akap10 −1.13 0.004 −1.11 0.009 A kinase (PRKA) anchor protein 10
1420853_at Sdc3 −1.11 0.009 −1.18 0.006 Syndecan 3
1421040_a_at Gsta2 −1.09 0.008 −1.21 0.002 Glutathione S-transferase, alpha 2 (Yc2)
Table 5.
 
Unique Genes for Dex at 1 Month: Upregulated and Downregulated
Table 5.
 
Unique Genes for Dex at 1 Month: Upregulated and Downregulated
No. Probe Set ID Gene Symbol Dex/1 mo Gene Title
FC* P
Upregulated
1 1422535_at Ccne2 1.81 0.018 Cyclin E2
2 1416422_a_at Ssb 1.78 0.036 Sjogren syndrome antigen B
3 1443930_at Zfp811 1.65 0.005 Zinc finger protein 811
4 1427388_at Lrrc2 1.60 0.003 Leucine rich repeat containing 2
5 1457566_at Zfp677 1.50 0.004 Zinc finger protein 677
6 1415903_at Slc38a1 1.43 0.002 Solute carrier family 38, member 1
7 1459157_at Stox2 1.43 0.009 CDNA clone IMAGE: 9053762
8 1424135_at Rspry1 1.42 0.005 Ring finger and SPRY domain containing 1
9 1439283_at Osbpl9 1.40 0.008 Nardilysin, N-arginine dibasic convertase, NRD convertase 1, mRNA
10 1456359_at Ppwd1 1.40 0.007 Peptidylprolyl isomerase domain and WD repeat containing 1
11 1449294_at Mrps15 1.40 0.004 Mitochondrial ribosomal protein S15
12 1425012_at Gng5 1.39 0.007 Guanine nucleotide binding protein (G protein), gamma 5, mRNA
13 1439401_x_at Ppp2r5e 1.36 0.005 Protein phosphatase 2, regulatory subunit B (B56), epsilon isoform
14 1420044_at Osbpl9 1.35 0.001 Oxysterol binding protein-like 9
15 1425731_at Ankrd24 1.34 0.008 Ankyrin repeat domain 24
16 1418318_at Rnfl28 1.31 0.003 Ring finger protein 128
17 1434808_at Palb2 1.30 0.004 Partner and localizer of BRCA2
18 1415946_at Pigq 1.30 0.003 Phosphatidylinositol glycan anchor biosynthesis, class Q
19 1428252_at Chmp2b 1.29 0.006 Chromatin modifying protein 2B
20 1418726_a_at Tnnt2 1.27 0.004 Troponin T2, cardiac
21 1418332_a_at Agtpbp1 1.27 0.009 ATP/GTP binding protein 1
22 1426741_a_at Fastkd2 1.26 0.007 FAST kinase domains 2
23 1422043_at Tsc1 1.25 0.003 Tuberous sclerosis 1
24 1459642_at Adam23 1.24 0.003 A disintegrin and metallopeptidase domain 23 (Adam23), mRNA
25 1445243_at D10Ertd533e 1.24 0.007 DNA segment, Chr 10, ERATO Doi 533, expressed
Downregulated
1 1440142_s_at Gfap −2.56 0.007 Glial fibrillary acidic protein
2 1448883_at Lgmn −2.12 0.007 Legumain
3 1451663_a_at Trim3 −2.03 0.002 Tripartite motif-containing 3
4 1428850_x_at Cd99 −1.89 0.043 CD99
5 1417050_at C1qtnf4 −1.83 0.003 C1q and tumor necrosis factor-related protein 4
6 1428584_a_at Haghl −1.80 0.009 Hydroxyacylglutathione hydrolase-like
7 1427481_a_at Atp1a3 −1.77 0.007 ATPase, Na+/K+ transporting, alpha 3 polypeptide
8 1434707_at Sbf1 −1.76 0.001 SET binding factor 1
9 1427004_at Fbxo2 −1.71 0.002 F-box protein 2
10 1427754_a_at Dnm1 −1.67 0.005 Dynamin 1
11 1418252_at Padi2 −1.67 0.000 Peptidyl arginine deiminase, type II
12 1417440_at Arid1a −1.66 0.000 AT rich interactive domain 1A (SWI-like)
13 1434849_at Tspyl2 −1.66 0.004 TSPY-like 2
14 1427288_at Apba2 −1.62 0.013 Amyloid beta (A4) precursor protein-binding, family A, member 2
15 1455269_a_at Coro1a −1.55 0.009 Coronin, actin binding protein 1A
16 1428813_a_at Caly −1.54 0.006 Calcyon neuron-specific vesicular protein
17 1451129_at Calb2 −1.53 0.004 Calbindin 2
18 1424137_at Gprin1 −1.51 0.002 G protein-regulated inducer of neurite outgrowth 1
19 1423892_at Apbb1 −1.50 0.004 Amyloid beta (A4) precursor protein-binding, family B, member 1
20 1416853_at Ncdn −1.46 0.003 Neurochondrin
21 1436137_at Slc6a17 −1.45 0.001 Solute carrier family 6 (neurotransmitter transporter), member 17
22 1448366_at Stx1a −1.43 0.007 Syntaxin 1A (brain)
23 1456759_at Lrrc4c −1.43 0.002 Leucine rich repeat containing 4C, mRNA
24 1429518_at Faim2 −1.43 0.007 Fas apoptotic inhibitory molecule 2
25 1434489_at Elmo3 −1.41 0.007 Engulfment and cell motility 3, ced-12 homolog (C. elegans)
26 1421887_a_at Aplp2 −1.39 0.008 Amyloid beta (A4) precursor-like protein 2
27 1448110_at Sema4a −1.29 0.010 Sema domain, immunoglobulin domain (Ig), (semaphorin) 4A
Table 6.
 
Unique Genes for TAA at 1 Month: Upregulated and Downregulated
Table 6.
 
Unique Genes for TAA at 1 Month: Upregulated and Downregulated
No. Probe Set ID Gene Symbol TAA/1 mo Gene Title
FC* P
Upregulated
1 1451119_a_at Fbln1 2.73 0.005 Fibulin 1
2 1417580_s_at Selenbp1 2.13 0.025 Selenium binding protein 1
3 1451791_at Tfpi 1.89 0.009 Tissue factor pathway inhibitor
4 1443163_at Slc39a2 1.74 0.006 Solute carrier family 39 (zinc transporter), member 2
5 1434201_at Chrdl1 1.68 0.007 Chordin-like 1
6 1429117_at Tradd 1.67 0.013 TNFRSF1A-associated via death domain
7 1457472_at Gigyf2 1.60 0.008 GRB10 interacting GYF protein 2
8 1417319_at Pvrl3 1.55 0.007 Poliovirus receptor-related 3
9 1440166_x_at Htr1d 1.54 0.008 5-Hydroxytryptamine (serotonin) receptor 1D
10 1436728_s_at Rtel1 1.53 0.002 Regulator of telomere elongation helicase 1
11 1427256_at Vcan 1.52 0.027 Versican
12 1430170_at Bbs10 1.52 0.002 Bardet-Biedl syndrome 10 (human)
13 1425460_at Mtmr2 1.50 0.001 Myotubularin-related protein 2
14 1431295_a_at Stx18 1.46 0.005 Syntaxin 18
15 1420930_s_at Ctnnal1 1.45 0.007 Catenin (cadherin-associated protein), alpha-like 1
16 1460107_at Fam154b 1.43 0.009 Family with sequence similarity 154, member B
17 1419306_at Ccdc 96 1.42 0.000 Coiled-coil domain containing 96
18 1428800_a_at Pus7l 1.41 0.002 Pseudouridylate sunthase 7 homolog (S. cerevisiae)-like
19 1417188_s_at Ube2k 1.41 0.007 Ubiquitin-conjugating enzyme E2K (UBC1 homolog, yeast)
20 1453752_at Rpl17 1.39 0.008 Ribosomal protein L17
21 1451193_x_at Ttc4 1.39 0.007 Tetratricopeptide repeat domain 4
22 1434203_at Fam107a 1.39 0.008 Family with sequence similarity 107, member A
23 1440936_at Serac1 1.38 0.003 Serine active site containing 1
24 1423336_at Orc4l 1.38 0.000 Origin recognition complex, subunit 4-like (S. cerevisiae)
25 1440383_at Dclre1b 1.37 0.008 DNA cross-link repair 1B, PSO2 homolog (S. cerevisiae)
Downregulated
1 1437581_at Zfp800 −3.50 0.010 Zinc finger protein 800
2 1421085_at Rs1 −3.15 0.004 Retinoschisis (X-linked, juvenile) 1 (human)
3 1438363_at Pnmal2 −2.00 0.002 PNMA-like 2
4 1451847_s_at Arid4b −1.93 0.004 AT-rich interactive domain 4B (RBP1-like)
5 1421955_a_at Nedd4 −1.90 0.001 Neural precursor cell expressed, developmentally downregulated 4
6 1438040_a_at Hsp90b1 −1.77 0.000 Heat shock protein 90, beta (Grp94), member 1
7 1417029_a_at Trim2 −1.72 0.007 Tripartite motif-containing 2
8 1445418_at Smg6 −1.68 0.008 Smg-6 homolog, nonsense-mediated mRNA decay factor (C. elegans)
9 1452322_a_at Brwd1 −1.64 0.002 Bromodomain and WD repeat domain containing 1
10 1419550_a_at Stk39 −1.61 0.003 Serine/threonine kinase 39, STE20/SPS1 homolog (yeast)
11 1435857_s_at Aplp1 −1.60 0.003 Amyloid beta (A4) precursor-like protein 1
12 1419650_at Zfr −1.59 0.002 Zinc finger RNA binding protein
13 1458685_at Garnl1 −1.58 0.007 GTPase activating RANGAP domain-like 1
14 1448235_s_at Hmgl1l −1.58 0.008 High mobility group box 1-like
15 1416134_at Aplp1 −1.57 0.008 Amyloid beta (A4) precursor-like protein 1
16 1421361_at Grk1 −1.57 0.004 G protein-coupled receptor kinase 1 /// similar to rhodopsin kinase
17 1456281_at Fbxl18 −1.57 0.001 F-box and leucine-rich repeat protein 18
18 1436904_at Med13 −1.53 0.008 Mediator complex subunit 13
19 1432269_a_at Sh3kbp1 −1.52 0.003 SH3-domain kinase binding protein 1
20 1437201_at Lrrc4c −1.49 0.002 Leucine-rich repeat containing 4C
21 1424287_at Prkx −1.48 0.003 Protein kinase, X-linked
22 1418514_at Mtf2 −1.47 0.002 Metal response element binding transcription factor 2
23 1417832_at Smc1a −1.46 0.004 Structural maintenance of chromosomes 1A
24 1457432_at Prox1 −1.45 0.008 Prospero-related homeobox 1
25 1437648_at Pcyt1b −1.45 0.002 Phosphate cytidyltransferase 1, choline, beta isoform
Independent Validation of Data by qPCR
Independent qPCR validation of representative differentially expressed genes was performed using commercial gene expression assays (TaqMan; Applied Biosystems). The fold changes and their direction were confirmed for the chosen sets of genes. Data are presented in Table 7
Table 7.
 
Comparison of Microarray (Affymetrix) Fold Change Data versus qPCR (Taqman) Fold Change Data
Table 7.
 
Comparison of Microarray (Affymetrix) Fold Change Data versus qPCR (Taqman) Fold Change Data
Gene Fold Change Comparisons
Microarray qPCR
Dex/1 wk (Affy) Dex/1 wk (Taq)
Serpina3n 3.40 5.96
Gfap 1.74 2.07
Plxna2 1.67 2.12
Sema5a 1.60 1.73
Gjc1 1.50 2.08
Gene TAA/1 wk (Affy) TAA/1 wk (Taq)
Serpina3n 7.80 31.24
Gfap 2.29 3.66
Folh1 3.65 3.09
Gene Dex/1 mo (Affy) Dex/1 mo (Taq)
Serpina3n −3.20 −2.62
Gfap −2.56 −2.53
Gene TAA/1 mo (Affy) TAA/1 mo (Taq)
Gfap −1.58 −1.69
Folh1 1.44 1.23
Biological Network and Pathway Analysis of Significantly Changed Genes
Biological network/pathway analysis was performed with pathway analysis software (IPA) to identify integral biological pathways that are commonly and uniquely affected by the two steroids at early and late time points. ANOVA of the whole set identified 1558 probe sets differentially expressed at P ≤ 0.01. From this data set the most interesting candidate pathway is “Semaphorin Signaling in Neurons,” a member of the “Axonal Guidance Signaling System.” The top canonical pathways (identified by IPA) are represented in Table 8
Table 8.
 
Top Canonical Pathways Generated by Ingenuity Pathway Analysis
Table 8.
 
Top Canonical Pathways Generated by Ingenuity Pathway Analysis
Figure 2 represents modification of the “Semaphorin Signaling in Neurons” canonical pathway overlaid with expression data from each of the time point/steroid combinations. Color coding shows the dynamics of the gene expression changes in each steroid/time point combination: red represents upregulation and green downregulation. 
Figure 2.
 
Semaphorin Signaling in Neurons. Axonal Guidance Signaling Pathway (Semaphorin Signaling boxed) (A). Semaphorin Signaling in Dex at 1 week (B), TAA at 1 week (C), Dex at 1 month (D), and TAA at 1 month (E). Red: upregulated genes; green: downregulated genes.
Figure 2.
 
Semaphorin Signaling in Neurons. Axonal Guidance Signaling Pathway (Semaphorin Signaling boxed) (A). Semaphorin Signaling in Dex at 1 week (B), TAA at 1 week (C), Dex at 1 month (D), and TAA at 1 month (E). Red: upregulated genes; green: downregulated genes.
Gene Network Analysis of Significantly Changed Genes
Although pathway analysis shows vertical flow of signaling from the cellular membrane toward the nucleus and DNA, gene network analysis preferentially shows horizontal gene interactions and highlights cross-talk between different gene expression pathways. The top five gene networks for each of the steroids at both time points observed are presented in Table 9. All steroid/time point combinations have one network in common: “Cell-to-Cell Signaling and Interaction.” In Figure 3, the “Cell-to-Cell Signaling and Interaction Network” is overlaid with information on how its genes/gene complexes feed into some top canonical pathways (“Glucocorticoid Receptor Signaling,” “Acute Phase Response Signaling,” “Semaphorin Signaling,” etc.) and many functional categories (ophthalmic disorders, activation of neuroglia, anti-inflammatory response of macrophages, etc.). To take a closer look at changes in gene expression of the genes making up that network, see Figure 3. Color coding shows the dynamics of the gene expression changes in each steroid/time point combination: red represents upregulation and green downregulation. 
Table 9.
 
IPA: Top Five Networks Generated with Ingenuity Pathway Analysis for Each of the Steroids at Both Time Points
Table 9.
 
IPA: Top Five Networks Generated with Ingenuity Pathway Analysis for Each of the Steroids at Both Time Points
ID Associated Network Functions Score
1 Posttranslational Modification; Cell-to-Cell Signaling and Interaction; Cellular Assembly and Organization 45
2 Cell Morphology; Cellular Development; Cancer 42
3 Cancer; Molecular Transport; RNA Trafficking 40
4 DNA Replication, Recombination, and Repair; Nucleic Acid Metabolism; Small Molecule Biochemistry 35
5 Connective Tissue Disorders; Organismal Injury and Abnormalities; Amino Acid Metabolism 33
Figure 3.
 
Cell-to-Cell Signaling and Interaction Network for Dex and TAA overlaid with Diseases and Canonical Pathways (A), Dex at 1 week (B), TAA at 1 week (C), Dex at 1 month (D), and TAA at 1 month (E). Red: upregulated genes; green: downregulated genes.
Figure 3.
 
Cell-to-Cell Signaling and Interaction Network for Dex and TAA overlaid with Diseases and Canonical Pathways (A), Dex at 1 week (B), TAA at 1 week (C), Dex at 1 month (D), and TAA at 1 month (E). Red: upregulated genes; green: downregulated genes.
Candidate Genes and Pathways for Retinal Neuroprotection/Neurodegeneration Induced by Steroids
Genes Common for Dex and TAA: 1 Week.
Upregulated >1.5-fold were two genes for stress response proteins: Serpina3n, a murine ortholog of antichymotrypsin, and Cebpd. Moderately upregulated were genes with a role in immune response and apoptosis (Smad3, a member of TGF-β receptor signaling and Ripk1, a member of the TNF-α signaling cascade), cell adhesion, and retina development in camera-type eye (Pvrl1), as well as somatostatin receptor (Sstr2), a G-protein–coupled receptor for the neuropeptide signaling pathway, and part of the cellular response to glucocorticoid stimulation (Table 1). 
Genes Unique for Dex: 1 Week.
A common theme was activation of retinal glia macrophages and activation of genes that are regulated by transforming growth factor-α, nuclear factor-κB, and tumor protein p53. Upregulated (fold change: FC ≥ 1.5) were genes for several acute-phase proteins (Gfap, Cp, Edn2, Crym, Soc3) and apoptosis-related genes (Bcl3, Nupr1). The top canonical pathways identified by pathway analysis (IPA) were “Glioma Invasiveness Signaling” and “VEGF Signaling Pathway” (Table 8B). Upregulated were also members of “Semaphorin Signaling in Neurons,” Plxna2 and Sema5a. The strongest downregulation was observed with EphrinB receptor (Argef15), a negative regulator of excitatory synapses formation, 7 and several chemokine signaling molecules (Ccl22, Cxcr7, Ccl20) (Table 2). 
Genes Unique for TAA: 1 Week.
Two members of the folate pathway were disregulated: Folh1, a member of “One Carbon Pool by Folate,” and Cubn, a gene locus for albuminuria. 8 Also upregulated were genes from the immune and inflammatory response (Glycam1, Calca), dopamine metabolism, and signaling (Cyp2d22, Gnas), as well as a positive regulator of vascular endothelial growth factor (VEGF) (Gata4). Strongly downregulated genes were Kcnj13, a potassium ion channel gene whose mutations cause autosomal-dominant snowflake vitreoretinal degeneration, 9,10 and Galnt3, a regulator of FGF7 and matrix metalloproteinase expression, 11 whose mutation causes eyelid calcification. 12 Downregulated were several genes involved with DNA repair (Ube2b, Nono, Fancd2, Pohl), regulation of apoptosis (Xiap, CD74, Bcl7c, Syvn1, Lpar1), angiogenesis (Ccbe1), and synaptic transmission (Dlgap1, Fancd2), as well as Plekhg4, a member of the semaphorin signaling pathway. One of the top pathways unique for TAA identified was “VDR/RXR activation”. 
Genes Common for Dex and TAA: 1 Month.
Strongly upregulated were the genes for the aryl-hydrocarbon (AhR) receptor, involved in signal transduction in response to xenobiotic stimulus, stress, regulator of cell cycle and apoptosis, and Zn-finger protein of the cerebellum (Zic3). Downregulated were genes involved in nervous system development and regulation of synaptic transmission (L1cam, Casp8, Pacsin1, Nlgn2), as well as amyloid beta (A4) precursor family members (Apbb1 and Aplp2, and Aplp1 in TAA), involved in photoreceptor cell morphogenesis and response to light stimulus, visual learning, and axonal guidance. 
Genes Unique for Dex: 1 Month.
One of the top upregulated genes is SNAT1, a gene with an abundant expression in Müller cells, and important in glutamine transport in the retina. 13 Several genes with a role in visual perception, photoreceptor, and nervous system development were upregulated (Agtpbp1, Tsc1). Downregulated were Gfap, a marker of glial cell activation; Arid1a, a member of the glucocorticoid signaling pathway; and several genes with a role in neural development, synaptic plasticity, and neurotransmission (Lrrc4c, Ncdn, Gprin1, and Slc6a17). One of the top canonical pathways unique for Dex was “Huntington's Disease Signaling” and “mTOR Signaling.” Downregulated was Syntaxin1, a vesicle-mediated transport protein and SNARE protein, also known as a marker for amacrine cells. 14 16  
Genes Unique for TAA: 1 Month.
Several genes with a role in visual perception, photoreceptor maintenance, and retinal homeostasis were disregulated at 1 month with TAA. Upregulated were Chrdl1 and Pvrl3, which are involved in retina morphogenesis in camera-type eyes and Bardet–Biedl syndrome 10. Downregulated were genes for neural precursor protein Nedd4, member of “IGF-1, and glucocorticoid receptor signaling”; Lrrc4c, a regulator of axonogenesis; Prox1, which is involved in optic placode formation involved in camera-type eyes; Cobl, involved in neural tube closure; and Rorb, with a role in visual perception and eye photoreceptor cell development. The gap junction protein 1 was upregulated, as well as Vcan, mutations of which have been associated with VCAN-related vitreoretinopathy, which includes Wagner syndrome and erosive vitreoretinopathy. 17 Two of the top canonical signaling pathways identified were “IGF-1 Signaling” and “Glioma Signaling.” 
Discussion
Although intraocular steroids have a profound anti-inflammatory clinical effect, several complications are common, including cataract formation and steroid-induced glaucoma. 18 20 The results of our study highlight this clinical dilemma: whereas glucocorticoids alter the expression of a multitude of anti-inflammatory and neuroprotective genes, they also upregulate immune response genes, likely in glial cells, that cause damage to retinal neural tissue. Studies on steroid-induced glaucoma have shown that TAA and Dex induce dose-dependent, differential gene profiles in human trabecular meshwork (TM) cells, revealed by microarray technology. 18 21 Today, the TM epithelium is considered to be the target tissue and the myocilin gene, expressed mainly in TM, the top candidate gene for the development of steroid-induced glaucoma, through the proposed mechanism of increased intraocular pressure (IOP). 22 Although an elevated IOP is a significant risk factor for the development of glaucoma, it is important to note that sight-threatening effects of glaucoma are not necessarily coming from increased IOP but from retinal and optic nerve neurodegeneration. There are examples of patients that have normal pressure glaucoma, as well as patients with no retinal damage that have chronically high IOP. 23 25 Even in a DBA/2J mouse, a model for secondary angle-closure glaucoma, it has been shown that hypertension is not the only causative factor in glaucomatous optic neuropathy. 26 The mechanisms leading to retinal ganglion cell death are not fully understood and, recently, attention has been shifted to the role of autoimmunity in glaucoma pathogenesis. 27 30 Examples of immune system involvement with glaucoma include increased autoantibodies in glaucoma patients, 31 altered populations of T cells, 32 glial cell activation in the optic nerve and retina, 33 and activation of antigen presentation, seen as increased expression of the major histocompatibility complex class II by lamina cribrosa astrocytes. 34 Recently, complement factor C1q has been implicated in neuronal death in the DBA/2J mouse glaucoma model 35 and has shown increased expression in glaucomatous retina. 36 It has been shown that increased synthesis and deposition of complement factors C1q and C3 within the retina are induced by elevated IOP. 37  
Candidate Genes and Pathways for Neuroprotection/Neurodegeneration
Upregulation of several acute phase proteins at 1 week postinjection could potentially be quite damaging to the retina. This set of genes includes Serpina3n, a murine ortholog of antichymotrypsin, Gfap, Cebpd, Cp, Edn2, Bcl3, CD44, and Rras. Antichymotrypsin is upregulated during photoreceptor degeneration in the Bbs4-knockout mouse model. 38 It is also induced by inflammatory stimuli in ocular tissue in endotoxin-induced uveitis in rats to prevent degenerative proteolysis, 39 and in the pineal gland as a result of peripheral nerve injury, suggesting its involvement during early events after nerve injury and repair. 40 Photoreceptor-derived endothelin 2 (Edn2) has been reported to be a general stress signaling molecule to Müller cells after photoreceptor injury. 41 Similar findings of activation of acute-phase genes were reported by Zhu et al. 42 in their microarray study of differentially induced genes in the inferior and superior retina after hyperoxic shock, suggesting steroid-induced activation of oxidative response genes. 
Since one of the common unwelcome side effects of intravitreal steroid administration is glaucoma, we wanted to determine whether any of the differentially expressed genes that we identified are identical or related to the genes identified by microarray analysis of DBA/J mice, a mouse glaucoma model. 43,44 We found several genes with a common pattern of differential expression in our steroid-injected mice and DBA/2J mice. Steele et al. 43 found upregulation of Lcn2, Gfap, Serpina3n, Edn2, Cp, Olfml3, and downregulation of Selenbp1 at 8 months vs. 3 months in the DBA/2J retinas. In the survey of genes preceding occurrence of increased IOP, Fan et al. 44 identified an increase in Edn2, Lcn2, Gfap, Sst, and GstM1, and decrease in Bcl2, and Camk4 expression. In our data set we see upregulation of Serpina3n after injection of either steroid at 1 week, Edn2, Cp, GstM1, and Gfap in Dex at 1 week, Lcn2 in TAA at 1 week, and upregulation of Selenbp1 TAA at 1 month. So, it remains to be seen whether upregulation of these genes has a protective or damaging effect on retinal and optic nerve degeneration, as well as glaucoma development. 
Our results point out a few signaling pathways involved at the clinically relevant time points of 1 week and 1 month postinjection. 
Semaphorin Signaling in Retinal Diseases
One of the top pathways identified using pathway analysis of the ANOVA-identified genes at a statistical level (P ≤ 0.01) was “Semaphorin Signaling in Neurons.” Semaphorins are signaling molecules, secreted and/or membrane bound, that act as axonal growth cone guidance molecules. They are expressed on glial cells, whereas semaphorin receptors, plexins, neuropilins, and integrins are expressed on the neurons. 45 Besides their role in axonal guidance, semaphorins also have a major role in immune function. Figure 2 displays the activation and differential regulation of genes for semaphorins and their receptors from the associated pathway at each steroid/time point. At 1 week after Dex treatment we observed upregulation of genes for ligands Sema3, Sema4, and Sema5a on the glial side, and Plexins A and A2 receptors on the neuronal side. One week after TAA injection we observed upregulation of Sema4 and Sema3, whereas Plexins A and A2 (Plekhg4) were downregulated. At 1 month postinjection Sema4 and Sema3, and plexins are downregulated with Dex, whereas in TAA upregulation of Sema3 is still observed. 
Semaphorin 4A is highly expressed in brain and retina, and has an important function in the developing visual system. This is demonstrated in the mouse knockout of Sema4A, which develops severe retinal degeneration. 46 Acting as a cue for neuronal guidance, semaphorin 3A has been found to be secreted by ischemic neurons in the avascular retina in response to the proinflammatory cytokine IL-1β, thus preventing vascular regeneration in proliferative retinopathies. 47 The role of Sema3E in eye therapy is currently being debated in the literature: both inhibition of semaphorin 3E 48 as well as intravitreal application 49 of semaphorin 3E are proposed strategies for therapeutic anti-angiogenesis. In later work, semaphorin 3E-Plexin D1 signaling has been shown to initiate a signaling pathway that normalizes angiogenic directionality in both developing retinas and ischemic retinopathy. In that work a new paradigm has been suggested for vascular regeneration therapy based on the regulatory interplay of astrocyte-derived VEGF, which normally promotes PlexinD1 expression in growing blood vessels, and neuron-derived semaphorin 3E signals to PlexinD1, which counteract VEGF-induced filopodial projections. Intravitreal administration of semaphorin 3E selectively suppressed extraretinal vascular outgrowth without affecting the desired regeneration of the retinal vasculature. Sema5A, upregulated 1 week after Dex injection, has been shown to promote angiogenesis by increasing endothelial cell proliferation, migration, and decreasing apoptosis in vitro. 50 Semaphorin 5A has also been shown to represent a bifunctional axonal guidance cue for mammalian midbrain neurons as well as axial motoneurons in vivo. 51 Activation of the semaphorin 4D–plexinB signaling complex produces an acute collapse of axonal growth cones in retinal neurons. 52  
Sema7A is expressed by components of the glial scar, such as reactive astrocytes, and in general appears to be a marker for astrocytic activation. 53 However, we do not see any involvement of this semaphorin with our steroid intravitreal injections. We do see activation of a gene for another glial scar protein, glial fibrillary acidic protein (Gfap), which is also a member of the semaphorin pathway. This protein is considered an exclusive marker for Müller cells in the retina and to the retinal injury response. 54 Multiple disorders are associated with improper Gfap regulation leading to “glial scarring.” The scar acts as a barrier to neuronal growth and prevents neural regeneration, 55 such as in proliferative vitreoretinopathy in the mouse 56 and Alexander disease. 57 This protein also has a neuroprotective role and increases regeneration of neurons after an injury. 58 Gfap is reported to decrease dramatically in response to acute infection or neurodegeneration. 59 Our data show that Gfap is upregulated by both Dex and TAA at 1 week, and downregulated at week 4. Another member of the semaphorin pathway, gene for X-linked inhibitor of apoptosis, Xiap, has been shown to increase survival of transplanted rod precursors in a degenerating host retina. 60 Xiap is downregulated by steroids at 1 week, and upregulated at the 1 month time point. 
Folate Signaling and Retinal Diseases
Folate signaling appears to play a significant role, especially in TAA-injected animals. Folate, a water-soluble vitamin essential for the synthesis of DNA, RNA, and proteins, is required for cell survival. Folate deficiency has deleterious consequences on the retina. Folate deficiencies can precipitate accumulation of homocysteine, which has been implicated in retinal diseases such as maculopathy, open-angle glaucoma, and diabetic retinopathy. 61 One week after TAA injection, folate hydrolase gene (Folh1), a member of the “One Carbon Pool by Folate” pathway was significantly upregulated. The overexpression of Folh1 has been associated with retinal degeneration in Canavan disease. 62 Cubilin (Cubn), another member of the folate pathway and a gene locus for albuminuria, 8 is downregulated with TAA at 1 week time point. 
Somatostatin Receptor Signaling
At 1 week postinjection both Dex and TAA treatments show small, but statistically significant (P ≤ 0.01) upregulation of somatostatin receptor 2 gene (Sstr2). Somatostatin is expressed on amacrine cells and serves as a neuromodulator in the CNS and retina. Sstr2 expression has been localized to photoreceptors and rod bipolar cells of the retina, and it has been shown via a mouse Sst2 knockout that somatostatin signaling regulates glutamate release in the retina via somatostatin receptor 2. 63 It has been suggested that Sstr2 has an antiangiogenic role in the retina, 64 and thus its upregulation, in our case, might be a result of the protective effect of steroids against inflammation. 
Aryl-Hydrocarbon Receptor Signaling
Common for both steroids at 1 month postinjection was the upregulation of AhR at P ≤ 0.01. It has been shown that AhR has a regulatory role for small heat shock proteins induced in the retina and other tissues of the eye. 65 Several human retinal dystrophies such as Leber congenital amaurosis, juvenile retinitis pigmentosa, and dominant cone–rod dystrophy are mapped to the AhR pathway. 66,67 Members of the AhR family play critical roles in a broad range of biological functions, including regulation of circadian rhythm, neurogenesis, hypoxia response, and drug metabolism. Aryl-hydrocarbon receptor mediates these effects through interactions with various proteins: retinoblastoma, transforming growth factor-β, estrogen receptor, and nuclear factor-κB. 68,69 Proteins related to AhR are of particular interest, because there are several currently available drugs that target these molecules. 70  
Because neurodegeneration of retinal ganglion cells and retinal tissue in glaucoma leads to vision loss, understanding the molecular and genetic effects of intraocular steroid treatments may be important in reducing clinical complications. Our focus in data interpretation was on genes with known neuroprotective versus neurodegenerative effects and/or known role in ophthalmic or neurodegenerative diseases. We have identified several genes and pathways that appear to be affected by intravitreal steroid injections. Perhaps modulation of some of these potential targets in combination with steroid intravitreal therapy will, in the future, lead to reduced side effects. 
Why do different steroids show a difference in subsets of genes that they activate? There are several published whole genome analyses comparing two or three related steroids in TM human cells in vitro showing common and unique sets of genes for each steroid. 18,20,21 To our knowledge, this is the first such work done in vivo in mouse retinas. A study on structure–function analysis was recently published by Cidlowski and colleagues 71 that revealed 9-fluoro and 17-hydroxy groups on the steroid significantly impact glucocorticoid receptor (GR) distribution. Since Dex and TAA differ at this position, this might be a partial explanation of those differences. Even a slight conformational difference of the GR, which results from binding of different steroid ligands, such as Dex and TAA, can be multiplied through combinatorial action of steroid receptors with coreceptors and other members of transcriptional machinery, thus having an effect on the level of activation/repression of particular genes, or changing the target genes all together. In addition, Cidlowski 72 has very nicely reviewed the complexity and diversity of the glucocorticoid receptors in human tissue. All that complexity has to be taken into account in any attempt to explain the complex and differential response that we observed for Dex and TAA gene expression in retinal tissue. 
Our data show that different glucocorticoid steroids manifest similar yet distinct effects on inflammation, neuroprotection/neurodegeneration, cytokine expression, and oxidative stress pathways that may have important clinical relevance for treatment of specific retinal disorders. These genes and pathways are common to a variety of retinal disorders where intravitreal steroid treatments have been used, including complications from age-related macular degeneration, diabetic retinopathy, venous occlusive disease, and retinal degenerations. A better understanding of the specific targets of steroids and their effects on gene expression in the retina may have important clinical implications for better management of different retinal disorders in the near future. 
Supplementary Materials
Table st1, PDF - Table st1, PDF 
Table st2, PDF - Table st2, PDF 
Footnotes
 Supported in part by an unrestricted grant from Research to Prevent Blindness (LSM, DGT, LMH), UC Davis Innovative Development Award (ZSM), Allergan Research grant (LSM, ZSM, LMH), and Allergan Horizon grant (SPM).
Footnotes
 Disclosure: Z. Smit-McBride, None; S.P. Modjtahedi, None; C.T. Cessna, None; D.G. Telander, None; L.M. Hjelmeland, None; L.S. Morse, None
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Figure 1.
 
One-way ANOVA for each of the time points separately identified 462 genes at 1 week and 597 genes at 1 month that were differentially expressed at P ≤ 0.01. Venn diagrams of Dex versus control compared with TAA versus control identify common and unique genes for each steroid at P ≤ 0.01 at 1 week (A) and 1 month (B). The lower set of Venn diagrams identify the number of genes per each category that satisfy a criterium of −1.5 ≥ FC ≥ 1.5 and P ≤ 0.05 at 1 week (C) and 1 month (D).
Figure 1.
 
One-way ANOVA for each of the time points separately identified 462 genes at 1 week and 597 genes at 1 month that were differentially expressed at P ≤ 0.01. Venn diagrams of Dex versus control compared with TAA versus control identify common and unique genes for each steroid at P ≤ 0.01 at 1 week (A) and 1 month (B). The lower set of Venn diagrams identify the number of genes per each category that satisfy a criterium of −1.5 ≥ FC ≥ 1.5 and P ≤ 0.05 at 1 week (C) and 1 month (D).
Figure 2.
 
Semaphorin Signaling in Neurons. Axonal Guidance Signaling Pathway (Semaphorin Signaling boxed) (A). Semaphorin Signaling in Dex at 1 week (B), TAA at 1 week (C), Dex at 1 month (D), and TAA at 1 month (E). Red: upregulated genes; green: downregulated genes.
Figure 2.
 
Semaphorin Signaling in Neurons. Axonal Guidance Signaling Pathway (Semaphorin Signaling boxed) (A). Semaphorin Signaling in Dex at 1 week (B), TAA at 1 week (C), Dex at 1 month (D), and TAA at 1 month (E). Red: upregulated genes; green: downregulated genes.
Figure 3.
 
Cell-to-Cell Signaling and Interaction Network for Dex and TAA overlaid with Diseases and Canonical Pathways (A), Dex at 1 week (B), TAA at 1 week (C), Dex at 1 month (D), and TAA at 1 month (E). Red: upregulated genes; green: downregulated genes.
Figure 3.
 
Cell-to-Cell Signaling and Interaction Network for Dex and TAA overlaid with Diseases and Canonical Pathways (A), Dex at 1 week (B), TAA at 1 week (C), Dex at 1 month (D), and TAA at 1 month (E). Red: upregulated genes; green: downregulated genes.
Table 1.
 
Common Genes for Dex and TAA at 1 Week: Upregulated and Downregulated
Table 1.
 
Common Genes for Dex and TAA at 1 Week: Upregulated and Downregulated
Probe Set ID Gene Symbol Dex/1 wk TAA/1 wk
FC* P FC‡ P § Gene Title
Upregulated
1419100_at Serpina3n 3.40 0.023 7.88 0.036 Ser (or Cys) peptidase inhibitor, clade A, mem.3N
1423233_at Cebpd 2.22 0.001 1.53 0.005 CAAT/enhancer binding protein (C/EBP), delta
1456605_at
1436193_at Man1c1 1.38 0.003 1.12 0.007 Mannosidase, alpha, class 1C, member 1
1456380_x_at Cnn3 1.26 0.002 1.22 0.006 Calponin 3, acidic /// similar to calponin 3, acidic
1454960_at Smad3 1.25 0.007 1.10 0.004 MAD homolog 3 (Drosophila)
1438111_at Pvrl1 1.22 0.006 1.24 0.010 Poliovirus receptor-related 1 (Pvrl1), mRNA
1439273_at Ripk1 1.20 0.009 −1.19 0.001 Receptor (TNFRSF)-interacting serine-threonine kinase 1
1424440_at Mrps6 1.17 0.008 1.17 0.009 Mitochondrial ribosomal protein S6
1434561_at Asxl1 1.13 0.008 −1.16 0.009 Additional sex combs like 1 (Drosophila)
1422256_at Sstr2 1.12 0.006 1.21 0.002 Somatostatin receptor 2
Downregulated
1435126_at Dusp15 −1.21 0.009 −1.17 0.009 Dual specificity phosphatase-like 15
1457823_at Cyr61 −1.18 0.003 −1.18 0.002 Cysteine rich protein 61
1432260_at Grp39 −1.14 0.004 −1.24 0.008 G protein-coupled receptor 39
1452388_at Hspa1a −1.12 0.003 −1.20 0.003 Heat shock protein 1A
1460402_at Brpf1 −1.07 0.001 −1.14 0.008 Bromodomain and PHD finger containing 1
Table 2.
 
Unique Genes for Dex at 1 Week: Upregulated and Downregulated
Table 2.
 
Unique Genes for Dex at 1 Week: Upregulated and Downregulated
No. Probe Set ID Gene Symbol Dex/1 wk Gene Title
FC* P
Upregulated
1 1449161_at Edn2 3.76 0.013 Endothelin 2
2 1456940_at Slc43a2 2.29 0.009 Solute carrier family 43, member 2
3 1455393_at Cp 2.19 0.000 Ceruloplasmin
4 1450650_at Myo10 1.96 0.007 Myosin X
5 1438037_at Herc5 1.82 0.009 Hect domain and RLD 5
6 1426509_s_at Gfap 1.74 0.002 Glial fibrillary acidic protein
7 1429772_at Plxna2 1.67 0.016 Plexin A2
1453286_at 0.002
8 1437422_at Sema5a 1.60 0.029 Semaphorin 5A
9 1459928_at Lcor 1.60 0.008 Ligand-dependent nuclear receptor corepressor
10 1416776_at Crym 1.56 0.004 Crystallin, mu
11 1450716_at Adamts1 1.54 0.005 A disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1
12 1418133_at Bcl3 1.53 0.005 B-cell leukemia/lymphoma 3
13 1423760_at Cd44 1.53 0.001 CD44 antigen
14 1428114_at Slc14a1 1.50 0.006 Solute carrier family 14 (urea transporter), member 1
15 1419665_a_at Nupr1 1.46 0.000 Nuclear protein 1
16 1418448_at Rras 1.45 0.009 Harvey rat sarcoma oncogene, subgroup R
17 1460700_at Stat3 1.43 0.006 Signal transducer and activator of transcription 3
18 1417128_at Plekho1 1.24 0.009 Pleckstrin homology domain containing, family O, member 1
19 1450923_at Tgfb2 1.22 0.007 Transforming growth factor, beta 2
20 1449865_at Sema3a 1.21 0.001 Sema domain, immunoglobulin domain (Ig), (semaphorin) 3A
21 1455374_at Kcnj3 1.17 0.007 Potassium inwardly-rectifying channel, subfamily J, member 3
22 1428967_at Igf1r 1.17 0.006 Insulin-like growth factor 1 receptor
23 1425869_a_at Psen2 1.15 0.009 Presenilin 2
24 1434387_at Itfg3 1.15 0.002 Integrin alpha FG-GAP repeat containing 3
Downregulated
1 1457172_at Arhgef15 −1.68 0.048 Rho guanine nucleotide exchange factor (GEF) 15
2 1429504_at Rnpc3 −1.55 0.049 RNA-binding region (RNP1, RRM) containing 3
3 1438841_s_at Arg2 −1.41 0.005 Arginase type II
4 1417925_at Ccl22 −1.33 0.007 Chemokine (C-C motif) ligand 22
5 1417443_at Fam151a −1.33 0.008 Family with sequence similarity 151, member A
6 1429781_s_at Ccdc39 −1.32 0.009 Coiled-coil domain containing 39
7 1435064_a_at Tmem27 −1.32 0.008 Transmembrane protein 27
8 1435158_at Rbm12b −1.31 0.003 RNA-binding motif protein 12B
9 1436799_at Enox1 −1.31 0.002 Ecto-NOX disulfide-thiol exchanger 1
10 1418640_at Sirt1 −1.30 0.007 Sirtuin 1 (silent mating type information regulation 2, homolog) 1 (S. cerevisiae)
11 1427126_at Hspa1b −1.30 0.004 Heat shock protein 1B
12 1436994_a_at Hist1h1c −1.30 0.004 Histone cluster 1, H1c
13 1452054_at Ube2w −1.29 0.008 Ubiquitin-conjugating enzyme E2W (putative)
14 1425589_at Hsd17b13 −1.26 0.009 Hydroxysteroid (17-beta) dehydrogenase 13
15 1444816_at Olfr75-ps1 −1.25 0.001 V1 olfactory receptor protein
16 1425370_a_at Erg −1.24 0.007 Avian erythroblastosis virus E-26 (v-ets) oncogene related
17 1447234_s_at Snx6 −1.23 0.005 Sorting nexin 6
18 1417625_s_at Cxcr7 −1.18 0.005 Chemokine (C-X-C motif) receptor 7
19 1422029_at Ccl20 −1.15 0.008 Chemokine (C-C motif) ligand 20
20 1449399_a_at Il1b −1.14 0.003 Interleukin 1 beta
21 1425530_a_at Stx3 −1.14 0.003 Syntaxin 3
22 1419307_at Tnfrsf13c −1.13 0.010 Tumor necrosis factor receptor superfamily, member 13c
23 1422446_x_at Ins2 −1.13 0.002 Insulin II
24 1424647_at Gabrp −1.12 0.007 gamma-aminobutyric acid (GABA) A receptor, pi
25 1449734_s_at Bbs4 −1.08 0.005 Bardet-Biedl syndrome 4 (human)
Table 3.
 
Unique Genes for TAA at 1 Week: Upregulated and Downregulated
Table 3.
 
Unique Genes for TAA at 1 Week: Upregulated and Downregulated
No. Probe Set ID Gene Symbol TAA/1 wk Gene Title
FC* P
Upregulated
1 1450154_at Folh1 3.65 0.007 Folate hydrolase
2 1452975_at Agxt2l1 2.70 0.009 Alanine-glyoxylate aminotransferase 2-like 1
3 1424714_at Aldoc 2.38 0.039 Aldolase C, fructose-bisphosphate
4 1434202_a_at Fam107a 2.22 0.034 Family with sequence similarity 107, member A
5 1428352_at Arrdc2 2.03 0.029 Arrestin domain containing 2
6 1424825_a_at Glycam1 1.97 0.007 Glycosylation-dependent cell adhesion molecule 1
7 1427355_at Calca 1.94 0.004 Calcitonin/calcitonin-related polypeptide, alpha
8 1427883_a_at Col3a1 1.86 0.005 Collagen, type III, alpha 1
9 1438643_at Camk1d 1.84 0.047 Calcium/calmodulin-dependent protein kinase ID, mRNA
10 1437060_at Olfm4 1.55 0.023 Olfactomedin 4
11 1432543_a_at Klfl3 1.53 0.011 Kruppel-like factor 13
12 1435888_at Egfr 1.52 0.027 Epidermal growth factor receptor
13 1419040_at Cyp2d22 1.51 0.009 Cytochrome P450, family 2, subfamily d, polypeptide 22
14 1454780_at Galntl4 1.42 0.005 UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase
15 1416166_a_at Prdx4 1.42 0.002 Peroxiredoxin 4
16 1454642_a_at Commd3 1.40 0.002 COMM domain containing 3
17 1448136_at Enpp2 1.39 0.001 Ectonucleotide pyrophosphatase/phosphodiesterase 2
18 1417366_s_at Calm1 1.39 0.001 Calmodulin 1
19 1430798_x_at Mrpl15 1.38 0.004 Mitochondrial ribosomal protein L15
20 1458487_at Klf3 1.38 0.002 Kruppel-like factor 3 (basic)
21 1454886_x_at Trim9 1.37 0.010 Tripartite motif-containing 9
22 1455712_at Hist3h2a 1.36 0.001 Histone cluster 3, H2a
23 1421740_at Gnas 1.34 0.004 GNAS (guanine nucleotide binding protein, α stimulating) complex locus
24 1417948_s_at Ilf2 1.33 0.009 Interleukin enhancer binding factor 2
25 1431795_a_at Sema3b 1.17 0.003 Sema domain, immunoglobulin domain (Ig), (semaphorin) 3B
Downregulated
1 1456418_at Kcnj13 −2.00 0.001 Potassium inwardly-rectifying channel, subfamily J, member 13
2 1453385_at Xiap −1.74 0.015 X-linked inhibitor of apoptosis
3 1426990_at Cubn −1.74 0.006 Cubilin (intrinsic factor-cobalamin receptor)
4 1435064_a_at Tmem27 −1.62 0.014 Transmembrane protein 27
5 1431725_at Fmn2 −1.62 0.046 Formin 2
6 1457145_at Plekhg4 −1.60 0.048 Pleckstrin homology domain containing, family G (with RhoGef domain), member 4
7 1427749_at Galnt3 −1.52 0.000 UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 3
8 1433599_at Baz1a −1.47 0.006 Bromodomain adjacent to zinc finger domain 1A
9 1425519_a_at Cd74 −1.40 0.003 CD74 antigen
10 1443183_at Huwe1 −1.40 0.010 HECT, UBA, and WWE domain containing 1
11 1419191_at Hipk3 −1.40 0.007 Homeodomain interacting protein kinase 3
12 1440081_at Cep192 −1.36 0.009 Premature mRNA for mKIAA1569 protein
13 1446516_at Bcl7c −1.35 0.005 B-cell CLL/lymphoma 7C
14 1421138_a_at Pkib −1.34 0.001 Protein kinase inhibitor beta, cAMP-dependent, testis specific
15 1448994_at Sp1 −1.32 0.008 Trans-acting transcription factor 1
16 1437385_at Ccbe1 −1.32 0.010 Collagen and calcium binding EGF domains 1
17 1450332_s_at Fmo5 −1.31 0.009 Flavin containing monooxygenase 5
18 1436826_at Tmtc3 −1.30 0.006 Transmembrane and tetratricopeptide repeat containing 3
19 1434787_at Arf3 −1.30 0.007 ADP-ribosylation factor 3, mRNA
20 1423199_at Brd3 −1.29 0.006 Bromodomain containing 3
21 1443609_s_at Syvn1 −1.28 0.004 Synovial apoptosis inhibitor 1, synoviolin
22 1433737_at Uhmk1 −1.28 0.004 U2AF homology motif (UHM) kinase 1
23 1439477_at Ube2b −1.28 0.006 Ubiquitin-conjugating enzyme E2B, RAD6 homology (S. cerevisiae)
24 1424553_at Hhatl −1.26 0.003 Hedgehog acyltransferase-like
24 1431239_at Nono −1.27 0.004 Non-POU-domain-containing, octamer binding protein
25 1448389_at Wdr5 −1.26 0.006 WD repeat domain 5
25 1441982_at Taf7 −1.27 0.003 TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated factor
Table 4.
 
Common Genes for Dex and TAA at 1 Month: Upregulated and Downregulated
Table 4.
 
Common Genes for Dex and TAA at 1 Month: Upregulated and Downregulated
Probe Set ID Gene Symbol Dex/1 mo TAA/1 mo Gene Title
FC* P FC‡ P §
Upregulated
1434201_at Chrdl1 1.71 0.016 1.68 0.007 Chordin-like 1
1423424_at Zic3 1.65 0.021 1.78 0.017 Zinc finger protein of the cerebellum 3
1457472_at Gigyf2 1.55 0.048 1.60 0.008 GRB10 interacting GYF protein 2
1422631_at Ahr 1.37 0.008 1.25 0.005 Aryl-hydrocarbon receptor
1457790_at Asb3 1.17 0.006 1.18 0.008 Ankyrin repeat and SOCS box-containing 3
1450784_at Reck 1.15 3.01E-06 1.27 3.06E-05 Reversion-inducing-cysteine-rich protein with kazal motifs
Downregulated
1418284_at Vps72 −1.69 9.96E-04 −1.29 0.001 Vacuolar protein sorting 72 (yeast)
1435857_s_at Aplp1 −1.61 0.012 −1.60 0.003 Amyloid beta (A4) precursor-like protein 1
1436659_at Dclk1 −1.61 0.018 −1.61 0.046 Doublecortin-like kinase 1
1455143_at Nlgn2 −1.56 7.80E-04 −1.44 0.008 Neuroligin 2
1417628_at Supt6h −1.54 0.002 −1.24 0.004 Suppressor of Ty 6 homolog (S. cerevisiae)
1434635_at Rph3a −1.42 0.007 −1.32 5.91E-04 Rabphilin 3A
1449381_a_at Pacsin1 −1.38 6.60E-04 −1.31 0.004 Protein kinase C and casein kinase substrate in neurons 1
1424552_at Casp8 −1.35 0.002 −1.24 4.96E-04 Caspase 8
1426540_at Endod1 −1.35 3.42E-05 −1.15 0.005 Endonuclease domain containing 1
1429876_at Supt7l −1.24 0.001 −1.29 5.06E-04 Suppressor of Ty 7 (S. cerevisiae)-like
1423047_at Tollip −1.22 0.008 −1.15 0.010 Toll interacting protein
1450435_at L1cam −1.22 0.003 −1.15 0.009 L1 cell adhesion molecule
1427218_at Klhl11 −1.21 0.001 −1.21 5.80E-04 Kelch-like 11 (Drosophila)
1431811_a_at Fbxo34 −1.18 0.003 −1.29 0.002 F-box protein 34
1431973_at ‘Sep6’ −1.17 0.004 −1.27 1.11E-04 Septin 6
1423975_s_at Numa1 −1.17 0.009 −1.20 0.004 Nuclear mitotic apparatus protein 1
1448116_at Uba1 −1.15 2.47E-04 −1.09 0.006 Ubiquitin-like modifier activating enzyme 1
1457167_at Med14 −1.14 0.008 −1.17 0.003 Mediator complex subunit 14
1441829_s_at Akap10 −1.13 0.004 −1.11 0.009 A kinase (PRKA) anchor protein 10
1420853_at Sdc3 −1.11 0.009 −1.18 0.006 Syndecan 3
1421040_a_at Gsta2 −1.09 0.008 −1.21 0.002 Glutathione S-transferase, alpha 2 (Yc2)
Table 5.
 
Unique Genes for Dex at 1 Month: Upregulated and Downregulated
Table 5.
 
Unique Genes for Dex at 1 Month: Upregulated and Downregulated
No. Probe Set ID Gene Symbol Dex/1 mo Gene Title
FC* P
Upregulated
1 1422535_at Ccne2 1.81 0.018 Cyclin E2
2 1416422_a_at Ssb 1.78 0.036 Sjogren syndrome antigen B
3 1443930_at Zfp811 1.65 0.005 Zinc finger protein 811
4 1427388_at Lrrc2 1.60 0.003 Leucine rich repeat containing 2
5 1457566_at Zfp677 1.50 0.004 Zinc finger protein 677
6 1415903_at Slc38a1 1.43 0.002 Solute carrier family 38, member 1
7 1459157_at Stox2 1.43 0.009 CDNA clone IMAGE: 9053762
8 1424135_at Rspry1 1.42 0.005 Ring finger and SPRY domain containing 1
9 1439283_at Osbpl9 1.40 0.008 Nardilysin, N-arginine dibasic convertase, NRD convertase 1, mRNA
10 1456359_at Ppwd1 1.40 0.007 Peptidylprolyl isomerase domain and WD repeat containing 1
11 1449294_at Mrps15 1.40 0.004 Mitochondrial ribosomal protein S15
12 1425012_at Gng5 1.39 0.007 Guanine nucleotide binding protein (G protein), gamma 5, mRNA
13 1439401_x_at Ppp2r5e 1.36 0.005 Protein phosphatase 2, regulatory subunit B (B56), epsilon isoform
14 1420044_at Osbpl9 1.35 0.001 Oxysterol binding protein-like 9
15 1425731_at Ankrd24 1.34 0.008 Ankyrin repeat domain 24
16 1418318_at Rnfl28 1.31 0.003 Ring finger protein 128
17 1434808_at Palb2 1.30 0.004 Partner and localizer of BRCA2
18 1415946_at Pigq 1.30 0.003 Phosphatidylinositol glycan anchor biosynthesis, class Q
19 1428252_at Chmp2b 1.29 0.006 Chromatin modifying protein 2B
20 1418726_a_at Tnnt2 1.27 0.004 Troponin T2, cardiac
21 1418332_a_at Agtpbp1 1.27 0.009 ATP/GTP binding protein 1
22 1426741_a_at Fastkd2 1.26 0.007 FAST kinase domains 2
23 1422043_at Tsc1 1.25 0.003 Tuberous sclerosis 1
24 1459642_at Adam23 1.24 0.003 A disintegrin and metallopeptidase domain 23 (Adam23), mRNA
25 1445243_at D10Ertd533e 1.24 0.007 DNA segment, Chr 10, ERATO Doi 533, expressed
Downregulated
1 1440142_s_at Gfap −2.56 0.007 Glial fibrillary acidic protein
2 1448883_at Lgmn −2.12 0.007 Legumain
3 1451663_a_at Trim3 −2.03 0.002 Tripartite motif-containing 3
4 1428850_x_at Cd99 −1.89 0.043 CD99
5 1417050_at C1qtnf4 −1.83 0.003 C1q and tumor necrosis factor-related protein 4
6 1428584_a_at Haghl −1.80 0.009 Hydroxyacylglutathione hydrolase-like
7 1427481_a_at Atp1a3 −1.77 0.007 ATPase, Na+/K+ transporting, alpha 3 polypeptide
8 1434707_at Sbf1 −1.76 0.001 SET binding factor 1
9 1427004_at Fbxo2 −1.71 0.002 F-box protein 2
10 1427754_a_at Dnm1 −1.67 0.005 Dynamin 1
11 1418252_at Padi2 −1.67 0.000 Peptidyl arginine deiminase, type II
12 1417440_at Arid1a −1.66 0.000 AT rich interactive domain 1A (SWI-like)
13 1434849_at Tspyl2 −1.66 0.004 TSPY-like 2
14 1427288_at Apba2 −1.62 0.013 Amyloid beta (A4) precursor protein-binding, family A, member 2
15 1455269_a_at Coro1a −1.55 0.009 Coronin, actin binding protein 1A
16 1428813_a_at Caly −1.54 0.006 Calcyon neuron-specific vesicular protein
17 1451129_at Calb2 −1.53 0.004 Calbindin 2
18 1424137_at Gprin1 −1.51 0.002 G protein-regulated inducer of neurite outgrowth 1
19 1423892_at Apbb1 −1.50 0.004 Amyloid beta (A4) precursor protein-binding, family B, member 1
20 1416853_at Ncdn −1.46 0.003 Neurochondrin
21 1436137_at Slc6a17 −1.45 0.001 Solute carrier family 6 (neurotransmitter transporter), member 17
22 1448366_at Stx1a −1.43 0.007 Syntaxin 1A (brain)
23 1456759_at Lrrc4c −1.43 0.002 Leucine rich repeat containing 4C, mRNA
24 1429518_at Faim2 −1.43 0.007 Fas apoptotic inhibitory molecule 2
25 1434489_at Elmo3 −1.41 0.007 Engulfment and cell motility 3, ced-12 homolog (C. elegans)
26 1421887_a_at Aplp2 −1.39 0.008 Amyloid beta (A4) precursor-like protein 2
27 1448110_at Sema4a −1.29 0.010 Sema domain, immunoglobulin domain (Ig), (semaphorin) 4A
Table 6.
 
Unique Genes for TAA at 1 Month: Upregulated and Downregulated
Table 6.
 
Unique Genes for TAA at 1 Month: Upregulated and Downregulated
No. Probe Set ID Gene Symbol TAA/1 mo Gene Title
FC* P
Upregulated
1 1451119_a_at Fbln1 2.73 0.005 Fibulin 1
2 1417580_s_at Selenbp1 2.13 0.025 Selenium binding protein 1
3 1451791_at Tfpi 1.89 0.009 Tissue factor pathway inhibitor
4 1443163_at Slc39a2 1.74 0.006 Solute carrier family 39 (zinc transporter), member 2
5 1434201_at Chrdl1 1.68 0.007 Chordin-like 1
6 1429117_at Tradd 1.67 0.013 TNFRSF1A-associated via death domain
7 1457472_at Gigyf2 1.60 0.008 GRB10 interacting GYF protein 2
8 1417319_at Pvrl3 1.55 0.007 Poliovirus receptor-related 3
9 1440166_x_at Htr1d 1.54 0.008 5-Hydroxytryptamine (serotonin) receptor 1D
10 1436728_s_at Rtel1 1.53 0.002 Regulator of telomere elongation helicase 1
11 1427256_at Vcan 1.52 0.027 Versican
12 1430170_at Bbs10 1.52 0.002 Bardet-Biedl syndrome 10 (human)
13 1425460_at Mtmr2 1.50 0.001 Myotubularin-related protein 2
14 1431295_a_at Stx18 1.46 0.005 Syntaxin 18
15 1420930_s_at Ctnnal1 1.45 0.007 Catenin (cadherin-associated protein), alpha-like 1
16 1460107_at Fam154b 1.43 0.009 Family with sequence similarity 154, member B
17 1419306_at Ccdc 96 1.42 0.000 Coiled-coil domain containing 96
18 1428800_a_at Pus7l 1.41 0.002 Pseudouridylate sunthase 7 homolog (S. cerevisiae)-like
19 1417188_s_at Ube2k 1.41 0.007 Ubiquitin-conjugating enzyme E2K (UBC1 homolog, yeast)
20 1453752_at Rpl17 1.39 0.008 Ribosomal protein L17
21 1451193_x_at Ttc4 1.39 0.007 Tetratricopeptide repeat domain 4
22 1434203_at Fam107a 1.39 0.008 Family with sequence similarity 107, member A
23 1440936_at Serac1 1.38 0.003 Serine active site containing 1
24 1423336_at Orc4l 1.38 0.000 Origin recognition complex, subunit 4-like (S. cerevisiae)
25 1440383_at Dclre1b 1.37 0.008 DNA cross-link repair 1B, PSO2 homolog (S. cerevisiae)
Downregulated
1 1437581_at Zfp800 −3.50 0.010 Zinc finger protein 800
2 1421085_at Rs1 −3.15 0.004 Retinoschisis (X-linked, juvenile) 1 (human)
3 1438363_at Pnmal2 −2.00 0.002 PNMA-like 2
4 1451847_s_at Arid4b −1.93 0.004 AT-rich interactive domain 4B (RBP1-like)
5 1421955_a_at Nedd4 −1.90 0.001 Neural precursor cell expressed, developmentally downregulated 4
6 1438040_a_at Hsp90b1 −1.77 0.000 Heat shock protein 90, beta (Grp94), member 1
7 1417029_a_at Trim2 −1.72 0.007 Tripartite motif-containing 2
8 1445418_at Smg6 −1.68 0.008 Smg-6 homolog, nonsense-mediated mRNA decay factor (C. elegans)
9 1452322_a_at Brwd1 −1.64 0.002 Bromodomain and WD repeat domain containing 1
10 1419550_a_at Stk39 −1.61 0.003 Serine/threonine kinase 39, STE20/SPS1 homolog (yeast)
11 1435857_s_at Aplp1 −1.60 0.003 Amyloid beta (A4) precursor-like protein 1
12 1419650_at Zfr −1.59 0.002 Zinc finger RNA binding protein
13 1458685_at Garnl1 −1.58 0.007 GTPase activating RANGAP domain-like 1
14 1448235_s_at Hmgl1l −1.58 0.008 High mobility group box 1-like
15 1416134_at Aplp1 −1.57 0.008 Amyloid beta (A4) precursor-like protein 1
16 1421361_at Grk1 −1.57 0.004 G protein-coupled receptor kinase 1 /// similar to rhodopsin kinase
17 1456281_at Fbxl18 −1.57 0.001 F-box and leucine-rich repeat protein 18
18 1436904_at Med13 −1.53 0.008 Mediator complex subunit 13
19 1432269_a_at Sh3kbp1 −1.52 0.003 SH3-domain kinase binding protein 1
20 1437201_at Lrrc4c −1.49 0.002 Leucine-rich repeat containing 4C
21 1424287_at Prkx −1.48 0.003 Protein kinase, X-linked
22 1418514_at Mtf2 −1.47 0.002 Metal response element binding transcription factor 2
23 1417832_at Smc1a −1.46 0.004 Structural maintenance of chromosomes 1A
24 1457432_at Prox1 −1.45 0.008 Prospero-related homeobox 1
25 1437648_at Pcyt1b −1.45 0.002 Phosphate cytidyltransferase 1, choline, beta isoform
Table 7.
 
Comparison of Microarray (Affymetrix) Fold Change Data versus qPCR (Taqman) Fold Change Data
Table 7.
 
Comparison of Microarray (Affymetrix) Fold Change Data versus qPCR (Taqman) Fold Change Data
Gene Fold Change Comparisons
Microarray qPCR
Dex/1 wk (Affy) Dex/1 wk (Taq)
Serpina3n 3.40 5.96
Gfap 1.74 2.07
Plxna2 1.67 2.12
Sema5a 1.60 1.73
Gjc1 1.50 2.08
Gene TAA/1 wk (Affy) TAA/1 wk (Taq)
Serpina3n 7.80 31.24
Gfap 2.29 3.66
Folh1 3.65 3.09
Gene Dex/1 mo (Affy) Dex/1 mo (Taq)
Serpina3n −3.20 −2.62
Gfap −2.56 −2.53
Gene TAA/1 mo (Affy) TAA/1 mo (Taq)
Gfap −1.58 −1.69
Folh1 1.44 1.23
Table 8.
 
Top Canonical Pathways Generated by Ingenuity Pathway Analysis
Table 8.
 
Top Canonical Pathways Generated by Ingenuity Pathway Analysis
Table 9.
 
IPA: Top Five Networks Generated with Ingenuity Pathway Analysis for Each of the Steroids at Both Time Points
Table 9.
 
IPA: Top Five Networks Generated with Ingenuity Pathway Analysis for Each of the Steroids at Both Time Points
ID Associated Network Functions Score
1 Posttranslational Modification; Cell-to-Cell Signaling and Interaction; Cellular Assembly and Organization 45
2 Cell Morphology; Cellular Development; Cancer 42
3 Cancer; Molecular Transport; RNA Trafficking 40
4 DNA Replication, Recombination, and Repair; Nucleic Acid Metabolism; Small Molecule Biochemistry 35
5 Connective Tissue Disorders; Organismal Injury and Abnormalities; Amino Acid Metabolism 33
Table st1, PDF
Table st2, PDF
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