Dataset Information

Human Lacrimal Gland Gene Expression.

ABSTRACT:

Background

The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development.

Methods

We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium.

Results

The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described.

Conclusions

Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas.

SUBMITTER: Aakalu VK

PROVIDER: S-EPMC5231359 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Human Lacrimal Gland Gene Expression.

Aakalu Vinay Kumar VK Parameswaran Sowmya S Maienschein-Cline Mark M Bahroos Neil N Shah Dhara D Ali Marwan M Krishnakumar Subramanian S

PloS one 20170112 1

<h4>Background</h4>The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our und ...[more]

PMID: 28081151

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Project description:Importance:Although a variety of well-characterized diseases, such as sarcoidosis and granulomatosis with polyangiitis, affect the lacrimal gland, many patients with dacryoadenitis are diagnosed as having nonspecific orbital inflammation (NSOI) on the basis of histology and systemic disease evaluation. The ability to further classify the disease in these patients should facilitate selection of effective therapies. Objective:To test the a priori hypothesis that gene expression profiles would complement clinical and histopathologic evaluations in identifying well-characterized diseases and in subdividing NSOI into clinically relevant groups. Design, Setting, and Participants:In this cohort study, gene expression levels in biopsy specimens of inflamed and control lacrimal glands were measured with microarrays. Stained sections of the same biopsy specimens were used for evaluation of histopathology. Tissue samples of patients were obtained from oculoplastic surgeons at 7 international centers representing 4 countries (United States, Saudi Arabia, Canada, and Taiwan). Gene expression analysis was done at Oregon Health & Science University. Participants were 48 patients, including 3 with granulomatosis with polyangiitis, 28 with NSOI, 7 with sarcoidosis, 4 with thyroid eye disease, and 6 healthy controls. The study dates were March 2012 to April 2017. Main Outcomes and Measures:The primary outcome was subdivision of biopsy specimens based on gene expression of a published list of approximately 40 differentially expressed transcripts in blood, lacrimal gland, and orbital adipose tissue from patients with sarcoidosis. Stained sections were evaluated for inflammation (none, mild, moderate, or marked), granulomas, nodules, or fibrosis by 2 independent ocular pathologists masked to the clinical diagnosis. Results:Among 48 patients (mean [SD] age, 41.6 [19.0] years; 32 [67%] female), the mclust algorithm segregated the biopsy specimens into 4 subsets, with the differences illustrated by a heat map and multidimensional scaling plots. Most of the sarcoidosis biopsy specimens were in subset 1, which had the highest granuloma score. Three NSOI biopsy specimens in subset 1 had no apparent granulomas. Thirty-two percent (9 of 28) of the NSOI biopsy specimens could not be distinguished from biopsy specimens of healthy controls in subset 4, while other examples of NSOI tended to group with gene expression resembling granulomatosis with polyangiitis or thyroid eye disease. The 4 subsets could also be partially differentiated by their fibrosis, granulomas, and inflammation pathology scores but not their lymphoid nodule scores. Conclusions and Relevance:Gene expression profiling discloses clear heterogeneity among patients with lacrimal inflammatory disease. Comparison of the expression profiles suggests that a subset of patients with nonspecific dacryoadenitis might have a limited form of sarcoidosis, while other patients with NSOI cannot be distinguished from healthy controls.

Project description:PurposeDry eye syndrome is a multifactorial chronic disabling disease mainly caused by the functional disruptions in the lacrimal gland. The treatment involves palliation like ocular surface lubrication and rehydration. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to patients. This study aimed to establish functionally competent lacrimal gland cultures in-vitro and explore the presence of stem cells in the native gland and the established in-vitro cultures.MethodsFresh human lacrimal gland from patients undergoing exenteration was harvested for cultures after IRB approval. The freshly isolated cells were evaluated by flow cytometry for expression of stem cell markers ABCG2, high ALDH1 levels and c-kit. Cultures were established on Matrigel, collagen and HAM and the cultured cells evaluated for the presence of stem cell markers and differentiating markers of epithelial (E-cadherin, EpCAM), mesenchymal (Vimentin, CD90) and myofibroblastic (?-SMA, S-100) origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme) post carbachol (100 µM) stimulation by ELISA.ResultsNative human lacrimal gland expressed ABCG2 (mean±SEM: 3.1±0.61%), high ALDH1 (3.8±1.26%) and c-kit (6.7±2.0%). Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15-20 days, containing a heterogeneous population of stem-like and differentiated cells. The epithelial cells formed 'spherules' with duct like connections, suggestive of ductal origin. The levels of scIgA (47.43 to 61.56 ng/ml), lysozyme (24.36 to 144.74 ng/ml) and lactoferrin (32.45 to 40.31 ng/ml) in the conditioned media were significantly higher than the negative controls (p<0.05 for all comparisons).ConclusionThe study reports the novel finding of establishing functionally competent human lacrimal gland cultures in-vitro. It also provides preliminary data on the presence of stem cells and duct-like cells in the fresh and in-vitro cultured human lacrimal gland. These significant findings could pave way for cell therapy in future.

Project description:ImportanceSarcoidosis is a major cause of ocular or periocular inflammation. The pathogenesis of sarcoidosis is incompletely understood and diagnosis often requires a biopsy.ObjectiveTo determine how gene expression in either orbital adipose tissue or the lacrimal gland affected by sarcoidosis compares with gene expression in other causes of orbital disease and how gene expression in tissue affected by sarcoidosis compares with gene expression in peripheral blood samples obtained from patients with sarcoidosis.Design, setting, and participantsIn a multicenter, international, observational study, gene expression profiling of formalin-fixed biopsy specimens, using GeneChipp U133 Plus 2 microarrays (Affymetrix), was conducted between October 2012 and January 2014 on tissues biopsied from January 2000 through June 2013. Participants included 12 patients with orbital sarcoidosis (7 in adipose tissue; 5 affecting the lacrimal gland) as well as comparable tissue from 6 healthy individuals serving as controls or patients with thyroid eye disease, nonspecific orbital inflammation, or granulomatosis with polyangiitis. In addition, results were compared with gene expression in peripheral blood samples obtained from 12 historical individuals with sarcoidosis.Main outcomes and measuresSignificantly differentially expressed transcripts defined as a minimum of a 1.5-fold increase or a comparable decrease and a false discovery rate of P < .05.ResultsSignals from 2449 probe sets (transcripts from approximately 1522 genes) were significantly increased in the orbital adipose tissue from patients with sarcoidosis. Signals from 4050 probe sets (approximately 2619 genes) were significantly decreased. Signals from 3069 probe sets (approximately 2001 genes) were significantly higher and 3320 (approximately 2283 genes) were significantly lower in the lacrimal gland for patients with sarcoidosis. Ninety-two probe sets (approximately 69 genes) had significantly elevated signals and 67 probe sets (approximately 56 genes) had significantly lower signals in both orbital tissues and in peripheral blood from patients with sarcoidosis. The transcription factors, interferon-response factor 1, interferon-response factor 2, and nuclear factor κB, were strongly implicated in the expression of messenger RNA upregulated in common in the 3 tissues.Conclusions and relevanceGene expression in sarcoidosis involving the orbit or lacrimal gland can be distinguished from gene expression patterns in control tissue and overlaps with many transcripts upregulated or downregulated in the peripheral blood of patients with sarcoidosis. These observations suggest that common pathogenic mechanisms contribute to sarcoidosis in different sites. The observations support the hypothesis that a pattern of gene expression profiles could provide diagnostic information in patients with sarcoidosis.

Project description:BackgroundAqueous-deficient dry eye disease (ADDED) resulting from dysfunction of the lacrimal gland (LG) is currently incurable. Although LG stem/progenitor cell-based therapy is considered to be a promising strategy for ADDED patients, the lack of a reliable serum-free culture method to obtain enough lacrimal gland stem cells (LGSCs) and the basic standard of LGSC transplantation are obstacles for further research.MethodsAdult mouse LGSCs were cultured in Matrigel-based 3D culture under serum-free culture condition, which contained EGF, FGF10, Wnt3A, and Y-27632. LGSCs were continuously passaged over 40 times every 7 days, and the morphology and cell numbers were recorded. LGSCs were induced to differentiate to ductal cells by reducing Matrigel rigidity, while fetal bovine serum was used for the induction of acinar cells. RT-PCR or qRT-PCR analysis, RNA-sequence analysis, H&E staining, and immunofluorescence were used for characterization and examining the differentiation of LGSCs. LGSCs were allotransplanted into diseased LGs to examine the ability of repairing the damage. The condition of eye orbits was recorded using a camera, the tear production was measured using phenol red-impregnated cotton threads, and the engraftments of LGSCs were examined by immunohistochemistry.ResultsWe established an efficient 3D serum-free culture for adult mouse LGSCs, in which LGSCs could be continuously passaged for long-term expansion. LGSCs cultured from both the healthy and ADDED mouse LGs expressed stem/progenitor cell markers Krt14, Krt5, P63, and nestin, had the potential to differentiate into acinar or ductal-like cells in vitro and could engraft into diseased LGs and relieve symptoms of ADDED after orthotopic injection of LGSCs.ConclusionWe successfully established an efficient serum-free culture for adult mouse LGSCs aiming at LG repair for the first time. Our approach provides an excellent theoretical and technical reference for future clinical research for ADDED stem cell therapy.

Dataset Information

Human Lacrimal Gland Gene Expression.

Background

Methods

Results

Conclusions

Publications

Human Lacrimal Gland Gene Expression.

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