Ida Grunnan Fostad

Effect of Biopsy Location and Size on Proliferative Capacity of Ex Vivo Expanded Conjunctival Tissue

PURPOSE To evaluate the effect of location and size of biopsy on phenotype and proliferative capacity of cultured rat conjunctival epithelial cells. METHODS Pieces of conjunctiva were used from six areas: superior and inferior areas of bulbus, fornix, and tarsus of male Sprague-Dawley rats (n = 6). Explants were grown in RPMI 1640 with 10% fetal bovine serum on coverslips for 8 days or assayed for colony-forming efficiency (n = 9). Analysis included immunofluorescence microscopy and outgrowth measurements with ImageJ software. The Mann-Whitney test and Spearmans rank-order correlation test were used. RESULTS Superior (23.9 ± 2.9-fold growth) and inferior (22.4 ± 1.2-fold growth) forniceal tissues yielded significantly more outgrowth with respect to explant size than superior bulbar (13.4 ± 1.9-fold growth; P < 0.05 and P < 0.01, respectively), inferior bulbar (13.6 ± 1.6-fold growth; P = 0.01 and P < 0.01, respectively), and inferior tarsal tissues (14.0 ± 1.3-fold growth; P = 0.01). Outgrowth size correlated positively with explant size (r(s) = 0.54; P < 0.001), whereas explant size correlated negatively with fold growth (r(s) = 0.36; P < 0.001). Superior forniceal cells displayed higher colony-forming efficiency (3.6% ± 0.9%) than superior bulbar (1.1% ± 0.3%; P < 0.05) and inferior bulbar cells (1.6% ± 0.8%; P < 0.05). Percentage of p63+ and PCNA+ cells correlated positively with explant and outgrowth size. CONCLUSIONS Small forniceal conjunctival explants grow the most effectively; however, for transplantation purposes, the loss of p63+ and PCNA+ cells with small explants must be considered.

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed. Materials and Methods Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR. Results Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C. Conclusion HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.

Dry Eye Disease Patients with Xerostomia Report Higher Symptom Load and Have Poorer Meibum Expressibility

The purpose of the study was to investigate if xerostomia (dry mouth) is associated with symptoms and signs of dry eye disease (DED). At the Norwegian Dry Eye Clinic, patients with symptomatic DED with different etiologies were consecutively included in the study. The patients underwent a comprehensive ophthalmological work-up and completed self-questionnaires on symptoms of ocular dryness (Ocular Surface Disease Index [OSDI] and McMonnies Dry Eye Questionnaire) and the Sjögren’s syndrome (SS) questionnaire (SSQ). Three hundred and eighteen patients (52% women and 48% men) with DED were included. Patient demographics were: 0 to 19 years (1%), 20 to 39 (25%), 40 to 59 (34%), 60 to 79 (35%) and 80 to 99 (5%). Xerostomia, defined as “daily symptoms of dry mouth the last three months” (as presented in SSQ) was reported by 23% of the patients. Female sex was more common among patients with xerostomia (81%) than among non-xerostomia patients (44%; P<0.001). Patients with xerostomia (60 ± 15 years) were older than those without xerostomia (51 ± 17; P<0.001). The use of prescription drugs was more prevalent among xerostomia patients (65%) than among non-xerostomia patients (35%; P<0.021; adjusted for age and sex). Patients with xerostomia had a higher OSDI score (19.0 ± 10.0) than those without xerostomia (12.9 ± 8.0; P<0.001). Moreover, xerostomia patients had more pathological meibum expressibility (0.9 ± 0.7) than those without xerostomia (0.7 ± 0.8; P = 0.046). Comparisons of OSDI and ocular signs were performed after controlling for the effects of sex, age and the number of systemic prescription drugs used. In conclusion, xerostomia patients demonstrated a higher DED symptom load and had poorer meibum expressibility than non-xerostomia patients.

Differential expression of vitamin D associated genes in the aorta of coronary artery disease patients with and without rheumatoid arthritis

Background Vitamin D has an important role in the immune system, and has been linked to rheumatoid arthritis (RA) and coronary artery disease (CAD). The exact mechanisms by which vitamin D is involved in these processes are still unclear. Therefore, we wanted to search for differences in expression of genes involved in the vitamin D receptor (VDR) activation pathway and genes that are known to alter upon vitamin D stimulation, in the aortic adventitia of CAD patients with and without RA. Methods Affymetrix microarray was used to determine gene expression profile in surgical specimens from the adventitia of the ascending aorta of CAD patients with RA (n = 8) and without RA (n = 8) from the Feiring Heart Biopsy Study. Results We identified three vitamin D associated genes that were differentially expressed between RA and non-RA patients: Growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A) (FC = 1.47; p = 0.006), Nuclear Receptor Co-repressor 1 (NCOR1) (FC = 1,21; p = 0.005) and paraoxonases 2 (PON2) (FC = -1.37; p = 0.01). High expression of GADD45A in RA tissues was confirmed by real-time qRT-PCR. GADD45A expression correlated with plasma levels of 1,25(OH)2D3 (rs = 0.69; p = 0.003). Conclusions Microarray analyses revealed higher expression of GADD45A and NCOR1; and lower expression of PON2 in the aortic adventitia of RA than non-RA patients. Further studies are needed to elucidate if and how GADD45A, NCOR1 and PON2 are involved in the development of accelerated atherosclerosis in RA. In theory, some of these factors might have proatherogenic effects whereas others might reflect an underlying vascular pathology promoting atherogenesis (such as vascular stress).

Identification of Objective Morphometric Markers of Xerostomia in the Oral Mucosa Epithelium with In Vivo Confocal Microscopy

The purpose of this work was to determine whether the morphology of the oral mucosa epithelium (OME) of patients with xerostomia differ from patients without xerostomia. In total, 34 patients with dry eye disease (DED) with or without xerostomia were examined at The Norwegian Dry Eye Disease Clinic with in vivo confocal microscopy of the lower lip. In addition, age- and gender-matched healthy controls (HC) were included. DED patients with xerostomia had a higher superficial to deep backscatter ratio compared with DED patients without xerostomia (p=0.002) and HC (p=0.001). Regression analysis demonstrated that this ratio was related to xerostomia independently of gender and age (p<0.001). Sensitivity and specificity of detecting xerostomia were 0.78 and 0.85, respectively, when using a superficial to deep backscatter ratio cut-off value of 0.995 (p=0.004). The mean nucleus to cytosol backscatter ratio in the superficial OME was lower in patients with xerostomia than in those without xerostomia (p=0.034). In vivo confocal microscopy is a potential tool for evaluating the oral cavity and to assess changes in the OME associated with xerostomia, objectively and quantitatively. The cause of the increased backscatter in the superficial OME in xerostomia, however, remains to be elucidated.

OP0282 Expression of Vitamin D Receptor Associated Genes in The Aorta of Coronary Artery Disease Patients with and without Rheumatoid Arthritis

Background Vitamin D has an important role in the immune system, and has been linked to inflammation, rheumatoid arthritis (RA) and coronary artery disease (CAD)[1, 2]. However, the exact mechanisms how vitamin D is involved in these processes are still unclear. Objectives To compare expression of vitamin D receptor (VDR) associated genes in the aortic adventitia of CAD patients with and without RA. Methods RNA was isolated, and Affymetrix microarray was used to determine the gene expression profile in specimens from the ascending aorta in 8 patients with CAD and 8 patients with CAD and RA from the Feiring Heart Biopsy Study. Partek Genomics Suite software was used to identify differentially expressed genes by one-way ANOVA (p<0.05; FC>1.1), and differences in expression of VDR associated genes were determined by Ingenuity Pathway Analysis. Results Among the 15586 transcripts that were identified, pathway analysis determined two genes within the VDR signaling pathway, Growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A) (p=0.006; FC=1.474) and Nuclear Receptor Corepressor 1 (NCOR1) (p=0.005; FC=1,210), that where both up-regulated in RA patients. Conclusions We found that GADD45A and NCOR1 were upregulated in the aorta of RA patients. GADD45A induces cell cycle arrest, DNA repair and apoptosis in response to various environmental stresses [3], while NCOR1 has an important role as a gene-specific integrator of positive and negative signals that control inflammation [4]. Based on this finding, we hypothesize that the accelerated atherosclerosis in RA might be related to the up-regulation of GADD45A and NCOR1 through the VDR signaling pathway. References Urruticoechea-Arana A, Martin-Martinez MA, Castaneda S, Piedra CA, Gonzalez-Juanatey C, Llorca J et al. Vitamin D deficiency in chronic inflammatory rheumatic diseases: results of the cardiovascular in rheumatology [CARMA] study. Arthritis Res Ther 2015;17: 211. Norman PE, Powell JT. Vitamin D and cardiovascular disease. Circ Res 2014;114 2: 379–93. Rosemary Siafakas A, Richardson DR. Growth arrest and DNA damage-45 alpha (GADD45alpha). The international journal of biochemistry & cell biology 2009;41 5: 986–9. Glass CK, Saijo K. Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells. Nat Rev Immunol 2010;10 5: 365–76. Disclosure of Interest None declared

SAT0101 The Increased Risk of Cardiovascular Disease in Rheumatoid Arthritis May be Related to NUPR1 Activation

Background The cause of accelerated atherosclerosis in rheumatoid arthritis (RA) is still unclear and appears to be multifactorial [1]. Besides the traditional risk factors, also RA-specific risk factors may play a role. Notably, vascular inflammation in the vascular adventitia may be a crucial factor. Elucidating the pathomechanism of cardiovascular (CV) disease in RA is essential in order to provide optimal CV prevention and treatment. Examination of vascular specimens may provide important clues for such an endeavor. Objectives To compare the gene expression profile in the aortic adventitia in coronary artery disease (CAD) patients with and without RA. Methods Total RNA was isolated from biopsies of the adventitia of the ascending aorta removed during coronary artery bypass grafting in patients with (n=8) and without (n=8) RA. The gene expression profile was determined using Affymetrix microarray. The CEL files were imported into the Partek Genomics Suite software, and differentially expressed genes were identified by one-way ANOVA (p<0.05; FC>1.1). Results Non-supervised hierarchical clustering analyses showed that the gene expression profiles clustered into two groups. A total of 15586 transcripts were identified, of which 201 were differentially expressed between the groups (p<0.05). Upstream analysis demonstrated activation of the stress-induced transcriptional regulator NUPR1 in RA patients (z-score: 3.0). Nine target molecules of NUPR1 were identified, including the endothelial dysfunction-related GADD45A [2], which was up-regulated in RA patients (p=0.006; FC=1.474) (Figure 1). Conclusions NUPR1 is a known key player in the cellular stress response [3]. Our results indicate that the increased CV risk in RA might be related to activation of NUPR1, with downstream activation of GADD45A, which in turn promotes endothelial dysfunction. Interestingly, NUPR1 has also been linked to heart failure [4]. In theory, NUPR1 might be a target for novel therapy. References Hollan I, Meroni PL, Ahearn JM, Cohen Tervaert JW, Curran S, Goodyear CS, Hestad KA, Kahaleh B, Riggio M, Shields K, Wasko MC: Cardiovascular disease in autoimmune rheumatic diseases. Autoimmunity reviews 2013, 12:1004-1015. Thum T, Borlak J: LOX-1 receptor blockade abrogates oxLDL-induced oxidative DNA damage and prevents activation of the transcriptional repressor Oct-1 in human coronary arterial endothelium. The Journal of biological chemistry 2008, 283:19456-19464. Goruppi S, Iovanna JL: Stress-inducible protein p8 is involved in several physiological and pathological processes. The Journal of biological chemistry 2010, 285:1577-1581. Goruppi S, Patten RD, Force T, Kyriakis JM: Helix-loop-helix protein p8, a transcriptional regulator required for cardiomyocyte hypertrophy and cardiac fibroblast matrix metalloprotease induction. Molecular and cellular biology 2007, 27:993-1006. Disclosure of Interest None declared