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Meibomian gland dysfunction (MGD) may be the leading cause of dry eye syndrome throughout the world. However, the precise mechanism(s) underlying the pathogenesis of this disease is unclear. This study was conducted to identify meibomian gland genes that may promote the development and/or progression of human MGD.
Lid tissues were obtained from male and female MGD patients and age-matched controls after eyelid surgeries (e.g., to correct entropion or ectropion). Meibomian glands were isolated and processed for RNA extraction and the analysis of gene expression.
The results show that MGD is associated with significant alterations in the expression of almost 400 genes in the human meibomian gland. The levels of 197 transcripts, including those encoding various small proline-rich proteins and S100 calcium-binding proteins, are significantly increased, whereas the expression of 194 genes, such as claudin 3 and cell adhesion molecule 1, is significantly decreased. These changes, which cannot be accounted for by sex differences, are accompanied by alterations in many gene ontologies (e.g., keratinization, cell cycle, and DNA repair). The findings also show that the human meibomian gland contains several highly expressed genes that are distinct from those in an adjacent tissue (i.e., conjunctival epithelium).
The results demonstrate that MGD is accompanied by multiple changes in gene expression in the meibomian gland. The nature of these alterations, including the upregulation of genes encoding small proline-rich proteins and S100 calcium-binding proteins, suggest that keratinization plays an important role in the pathogenesis of MGD.
Meibomian glands play a critical role in the health and well-being of the ocular surface.1–6 These glands secrete a lipid and protein mixture that provides a clear optical surface for the cornea, interferes with bacterial colonization, and retards tear overflow.1–8 The glandular secretions also enhance the stability and reduce the evaporation of the tear film.1–7 Conversely, meibomian gland dysfunction (MGD) destabilizes the tear film, increases its evaporation and osmolarity and is believed to be the key trigger for the induction of evaporative dry eye syndrome.1–6,8–14
The major cause of MGD appears to be excretory duct obstruction, due to hyperkeratinization of the ductal epithelium and an increased viscosity of meibum.15–22 This obstruction may lead to cystic dilatation of glandular ducts, acinar cell atrophy, and a loss of secretory meibocytes.15,23 The MGD may also facilitate bacterial growth on the lid margin24–26 and promote inflammation in the adjacent conjunctiva.27,28 The development of MGD has been linked to several risk factors, including aging,29–34 androgen deficiency,35–42 isotretinoin treatment,43–45 and possibly postmenopausal estrogen therapy.46–49 However, the molecular mechanisms that underlie the pathogenesis of MGD are unknown. This lack of information, in turn, has hampered the generation of safe and effective therapies for the treatment of MGD.
We hypothesize that alterations in gene expression promote the development and/or progression of human MGD. We also hypothesize that identification of such genetic changes will provide unique insight into the pathogenesis of MGD and will reveal novel glandular targets for possible therapeutic intervention. The purpose of this investigation was to begin to test these hypotheses by taking advantage of new advances in cDNA microarray technology, computational biology, and bioinformatics.
Human eyelid tissues were obtained from adult individuals with MGD (mean age, 73 ± 4 years) and from age-matched controls (mean age, 65 ± 9 years), after lid resection surgery at the Massachusetts Eye and Ear Infirmary (Table 1). Before surgery, normal or dysfunctional meibomian glands were diagnosed in the participants by two ophthalmologists (i.e., one evaluated two controls and two patients; the other four controls and four patients), according to a published classification system.44 In brief, the grading scheme was 0 for clear excreta with or without small particles, 1 for opaque cloudy excreta with normal viscosity, 2 for opaque excreta with increased viscosity, and 3 for secreta that retained shape after digital expression. Subjects were excluded from the study if they had active infection, used topical antiglaucoma or anti-inflammatory medications, or wore contact lenses. Lid segments were placed in RNA stabilizer (RNAlater; Ambion, Austin, TX) and stored at −80°C. Meibomian glands (2–5 glands/tarsal plate) were then isolated under a dissecting microscope (Bausch & Lomb, Rochester, NY), by the removal of skin, subcutaneous tissue, muscle, and palpebral conjunctiva, and were processed in entirety for molecular biological procedures. The use of human eyelid samples that would otherwise have been discarded was approved by the Institutional Review Boards of Massachusetts Eye and Ear Infirmary and Schepens Eye Research Institute and adhered to the tenets of the Declaration of Helsinki.
Total RNA was extracted (RNAqueous Kit; Ambion) and analyzed (RNA Nano 6000 Series II Chip with a 2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA) to verify RNA integrity. The RNA concentrations and 260/280-nm ratios were determined with spectrophotometer (a NanoDrop 1000; Thermo Scientific, Waltham MA).
The RNA (100 ng) samples were processed by Asuragen (Austin, TX) for the quantitation of mRNA levels by using microarray expression analysis (HumanHT-12 ver. 3 Expression BeadChips; Illumina, San Diego, CA). The chips target greater than 25,000 annotated genes with more than 48,000 probes derived from NCBI (National Center for Biotechnology Information, Bethesda, MD) reference sequences and the UniGene databases (http://www.ncbi.nlm.nih.gov/UniGene; NCBI). Briefly, biotin-labeled cRNA samples were prepared (MessageAmp II; Ambion, Inc.), quantitated by UV spectrophotometry and examined with a capillary electrophoresis system (2100 Bioanalyzer; Agilent). The labeled cRNAs were used to probe the chips. Hybridization, washing, and scanning of the arrays were performed according to the manufacturer's instructions. Data were processed with appropriate software (BeadStudio software ver. 3; Illumina), by using background subtraction and cubic spline normalization. Standardized hybridization intensity values were adjusted by adding a constant, such that the lowest intensity value for any sample was 16.51 Hybridization intensity measurements are generally relied on in microarray experiments to infer transcript abundance levels for specific genes.51
Normalized data were evaluated with commercial software (GeneSifter.Net; Geospiza, Seattle, WA). This comprehensive program also produced gene ontology and z-score reports. Ontologies encompassed biological processes, molecular functions, and cellular components and were organized according to the guidelines of the Gene Ontology Consortium (http://www.geneontology.org/).52 Gene expression data were analyzed, with and without log transformation, and statistical evaluation of these data was performed with Student's t-test (two-tailed, unpaired). Genes that were expressed in similar directions in different experiments were identified by using the intersector program (GeneSifter.Net; Geospiza; www.public.genesifter.net). All data from the microarray chips (BeadChips; Illumina) are accessible for download through the National Center for Biotechnology Information's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) via series accession number (GSE17822).
The differential expression of selected genes was confirmed by using quantitative real-time PCR (qPCR) procedures. The cDNAs were transcribed by using a transcriptase (SuperScript III Reverse Transcriptase; Invitrogen, Grand Island, NY) and random hexamer primers (Invitrogen). Real-time PCR reactions (n = 3 control and 5 MGD separate samples) were performed in triplicate (TaqMan Gene Assays; Applied Biosystems, Inc. [ABI], Foster City, CA) and specific primers and probes for small proline-rich protein 2A (Hs03046643_s1), keratin 10 (Hs00166289_m1), S100 calcium-binding protein A9 (S100A9; Hs00610058_m1), S100 calcium-binding protein A8 (Hs00374264_g1), and β-actin endogenous control (4326315E). Differential gene expression was calculated according to the comparative CT method (outlined in ABI User Bulletin 2; updated 2001).
Analysis of gene expression in human meibomian glands (n = 12 samples) led to the identification of 11,173 genes that exceeded the manufacturer's quality value of 0.95 (Illumina). Of these genes, 423 were associated with lipid metabolic processes, 185 with lipid biosynthesis, 116 with fatty acid metabolic processes, 89 with phospholipid metabolic processes, and 66 genes with lipid transport.
The intensity range for gene expression spanned from 29 to 29,219 units, with the mean values of 7 genes averaging above 20,000 units, 47 genes over 10,000 units, 204 genes above 3,000 units, and 689 genes between 1,000 to 10,000 units. Examples of high-intensity genes are shown in Table 2. These include transcripts such as stearoyl-CoA desaturase, mucin-like 1, keratinocyte differentiation-associated protein, fatty acid synthase, and S100 calcium-binding proteins A8 and A9 (S100A8, S100A9). Several of these glandular transcripts have recently been discovered as proteins in human meibomian gland secretions,8 including adenylate cyclase 7; dystrophin, Ellis-van Creveld syndrome protein; keratin-1, -5, -6, -7, -9, -10, -13, -14, and -16; the lacrimal proline-rich protein proline-rich 4 (similar to 1); lipocalin 1; seven transmembrane helix receptor mucin-like 1 (also termed small breast epithelial mucin); synaptonemal complex protein 3; transcription elongation factor A (SII)-like 1; UDP-N-acetylglucosamine pyrophosphorylase 1; and zinc metalloproteinase.
Our data also demonstrate that human meibomian gland gene expression is not the same as that of another lid tissue, human conjunctival epithelium. We compared 300 sequences with the highest average intensity in our samples' raw data to those from human conjunctival epithelia (Gene Expression Omnibus Series accession number GSE5543).53 By this process we found that 42.3% of the sequences matched and most were for ribosomal genes. If ribosomal genes were excluded, then only 24.1% of the remaining sequences were similar between the meibomian gland and conjunctiva. In effect, the most highly expressed genes in the human meibomian gland are unlike those in human conjunctival epithelia.
Evaluation of non- and log-transformed data from controls subjects (n = 6) and MGD patients (n = 6) showed that significant differences exist in meibomian gland gene expression between these groups. As demonstrated in Table 3, MGD was associated with a significant increase in the expression of 197 genes and a significant decrease in the activity of 194 genes, compared with that of the control population. Particularly notable was the MGD-related upregulation of genes for small proline-rich (SPRR) proteins 1A, 2A, 2E, 2F, and 3; cornulin; cystatin A; S100A8; and S100A9 (Table 4).
Many of the genes upregulated in MGD, as shown in Table 4, are located on chromosome 1. Significantly increased gene expression in MGD was also found on chromosomes 12 and 14, whereas higher gene activity was observed on chromosomes 13 and 17 in control tissues (Table 5).
To confirm in part the microarray results, we analyzed selected genes by qPCR. This experimental approach verified the significant (P < 0.05) upregulation of MGD on all tested genes, including small proline-rich protein 2A (2.6-fold ↑), S100A8 (12.8-fold ↑), S100A9 (5.9-fold ↑), and keratin 10 (4.6-fold ↑).
The influence of MGD was quite striking in its stimulation of various gene ontologies (Table 6), such as keratinization (Table 7); cell cycle (Table 8); DNA repair (Table 9); and energy generation, translation, structural molecule activity, cytoplasmic vesicles, and cornified envelope components (Table 10). In contrast, meibomian glands of control subjects presented with a significant increase in ontologies associated with cellular nitrogen compound metabolic processes, transport, transferase activity, iron and manganese binding, and intracellular membrane-bound organelles (Table 6).
Examination of the data also showed that certain gene ontologies were overrepresented in both control and dysfunctional meibomian glands, including nucleotide metabolic processes, proteasomal ubiquitin-dependent protein catabolic processes, DNase activity and cytoplasm (Table 6). Other gene ontologies were underrepresented in both groups, such as cell communication and signal transducer activity (Table 6).
Given that the control and patient groups were not sex matched (Table 1), it is possible that sex differences in meibomian gland gene expression may have contributed to the MGD-related results. More specifically, because more females were present in the MGD group, it may be that our data reflect female- rather than disease-associated genes. To explore this possibility, we compared meibomian gland gene expression between men (n = 5) and women (n = 7) and then compared these data to those between controls and patients.
Our analyses of non– and log-transformed data demonstrate that the sex of the individual has a significant impact on gene expression in human meibomian glands. We identified 257 genes that show sex-related differences, with 137 genes more highly expressed in men and 120 in women. Examples of these genes, which include prolactin-induced protein (male > female) and S100 calcium-binding protein A7 (female > male), are depicted in the Table 11. Sex also has a significant effect on the expression of gene ontologies in the human meibomian gland, such as processes involving Golgi vesicle transport, translation, amino acid ligase activity, protein binding, endoplasmic reticulum, and ribosomes (Table 12).
However, these sex-related differences do not account for the MGD-associated changes in gene expression. Comparison of these data sets showed that only four genes (e.g., zinc finger protein 658 and S100 calcium-binding protein A7) were upregulated, and only four genes were downregulated (e.g., der1-like domain family, member 2, and esterase D/formylglutathione hydrolase), in meibomian glands of both women (versus men) and patients (versus controls). Moreover, comparison of the ontologies listed in Tables 6 and and1212 demonstrate that, of the 40 biological process (36 different, 4 the same), 19 molecular function (16 different, 3 the same), and 31 cellular component (25 different, 6 the same) ontologies, the majority (n = 77) were not the same (n = 13) in both women and patients.
Our study shows that MGD exerts MGD exerted a significant effect on gene expression in the human meibomian gland. Thus, MGD was found to be associated with significant alterations in the expression of almost 400 genes, including those encoding SPRR, S100A8,and S100A9. These changes, which cannot be accounted for by sex-associated differences between control and patient populations, are accompanied by the up- and downregulation of many ontologies (e.g., keratinization, cell cycle, DNA repair, translation, cytoplasmic vesicles, and cornified envelope components). Our study also demonstrates that the human meibomian gland contains several highly expressed genes that are distinct from those in an adjacent tissue (i.e., conjunctival epithelium). Overall, the findings support our hypothesis that alterations in gene expression promote the development and/or progression of human MGD. Our data also reveal novel glandular targets for possible therapeutic intervention in MGD.
We identified several SPRR, S100A8, and S100A9 genes that are significantly upregulated in human MGD. The SPRRs are precursor proteins of the cornified cell envelope of terminally differentiating stratified squamous epithelia and influence the flexibility, rigidity, or toughness of this envelope.55–59 The increased expression of these SPRR genes reflect the stimulation of meibomian gland ontologies associated with keratinization; keratinocyte and epidermal cell differentiation; ectoderm, epidermis, and tissue development; and cornified envelope. Of particular interest, the SPRRs, whether alone55 or in combination with S100A8 and -A9,60 are known to promote keratinization, such as in the conjunctiva61,62 and elsewhere in the body.63,64 Further, increased S100A8 and S100A9 expression induces proliferation of human keratinocytes.65 If translated, these SPRR, S100A8, and S100A9 proteins may be responsible, at least in part, for the hyperkeratinization of meibomian gland ductal epithelium. This process is believed to be a primary cause of MGD.1–6,8–14
It is possible that SPRRs and S100A8 and S100A9 represent novel and unique targets for the potential topical treatment of MGD. First, downregulation of SPRRs may represent a mechanism to inhibit the keratinization process. Second, attenuation of S100A8 and S100A9 may permit suppression of both the keratinization and the possible conjunctival inflammation associated with MGD. The proteins S100A8 and S100A9 form a heterodimeric protein complex called calprotectin, which is a Toll-like receptor 4 agonist and a damage associated molecular pattern molecule (DAMP).66 A DAMP can initiate and perpetuate an immune response in noninfectious inflammation.66 Typically, calprotectin may play an important role in regulating cellular homeostasis (e.g., cytoskeleton), but after secretion may become a danger signal during inflammatory processes.66 The secreted S100A8 and -A9 amplifies inflammatory responses66,67 and could induce extravasation of leukocytes into the adjacent conjunctiva. Such conjunctival inflammation may occur during MGD.27,28 For comparison, the S100A8 and -A9 proteins may be involved in the pathophysiology of pterygium,68,69 as well as in Sjögren's syndrome, rheumatoid arthritis, inflammatory bowel disease, vasculitis, lichen planus, and lupus erythematosus.70–73
However, although calprotectin is commonly identified as a promoter and/or marker of inflammation,67,74–76 it may also act, depending on its concentration (e.g., high), in an anti-inflammatory manner.70,77 In fact, calprotectin may exert a variety of protective antioxidant, anti-invasive, and antimicrobial functions.70,77,78 An anti-inflammatory action of calprotectin would be especially intriguing, given that the most highly expressed gene in the human meibomian gland is leukocyte-associated immunoglobulin-like receptor (LAIR)-1. The LAIR-1 protein is an inhibitory receptor that attenuates immune cell activation and suppresses proinflammatory cytokine production.79,80 Working in tandem, calprotectin and LAIR-1 could theoretically be responsible for the typical absence of inflammation20,23,81 within the meibomian gland in MGD. In addition, the ability of calprotectin to make epithelial cells more resistant to bacterial invasion82 could be one reason why such bacterial infection is minimal or absent in obstructive MGD.20 Overall, calprotectin could possibly protect the meibomian gland against inflammation and infection in MGD. Whether it exerts either or both of these activities or is instead proinflammatory remains to be determined.
Additional meibomian gland genes that are modulated in MGD may play a role in these keratinization, antimicrobial, and structural processes. For example, the upregulated genes cornulin and cystatin A may promote epidermal differentiation,83,84 and the increased peptidase inhibitor 3, skin-derived, transcripts, if translated, may inhibit bacterial infection.84 In addition, the genes encoding claudin 3, cell adhesion molecule 1, and CD151 molecule are downregulated in MGD, a response that may disrupt cell adhesion.84 Our data also show that many of the most highly upregulated genes in MGD are from chromosome 1. In fact, the SPRR, S100A8, and S100A9 genes colocalize on chromosome 1 at q21, the region called the epidermal differentiation complex.60 There are more than 850 diseases related to this chromosome, including deafness, Alzheimer's, glaucoma, and breast cancer.85
The influence of MGD is striking in its stimulation of various gene ontologies, such as cell cycle, DNA repair, generation of precursor metabolites, and translation. These responses may be analogous to those in other sebaceous glands, wherein stem cell niches are mobilized to repair injured or diseased tissue.86,87 Of interest, a deficiency in the capacity to repair DNA may lead to neoplasia.88,89
A notable finding was our discovery that significant sex-related differences exist in gene expression in the human meibomian gland. Genes encoding such proteins as lysozyme and neurocalcin δ were upregulated in males (M), whereas leptin gene expression was increased in females (F). Analogous sex-associated differences have been observed for lysozyme in stimulated submandibular/sublingual saliva (M > F),90 neurocalcin δ in the olfactory bulb (M > F),91 and leptin in the plasma (F > M).92 More importantly, sex-associated variations are also known to occur in the morphologic appearance, neutral and polar lipid profiles, and secretory output of the meibomian gland, as well as in the gene expression of murine meibomian tissues.31,33–37,39,40,42,93–98 Many of these differences appear to be due to the effects of sex steroid hormones and sex-specific aromatase activities (Liu S, et al. IOVS 2007;43:ARVO E-Abstract 5657).49,98 It is possible that these sex-related differences in the meibomian gland contribute to the increased prevalence of dry eye disease in females.11
Our data demonstrate that the human meibomian gland contains several highly expressed genes that are associated with lipid dynamics, glandular structure, immunologic factors, and hormone action. For instance, these genes encode proteins involved in cholesterol transport (Niemann-Pick disease, type C2); cholesterol (farnesyl-diphosphate farnesyltransferase 1; sterol-C4-methyl oxidase-like; sterol-C5-desaturase-like), fatty acid (stearoyl-CoA desaturase; thyroid hormone responsive SPOT14; fatty acid synthase), and lipid (acyl-CoA synthetase short-chain family member 2; transaldolase 1) biosynthesis; fatty acyl-CoA reduction (fatty acyl CoA reductase 2) and elongation (hydroxysteroid (17β) dehydrogenase 12); and glycosphingolipid catabolism (prosaposin).84,99 The primary product of stearoyl-CoA desaturase is oleic acid, which has been identified in meibomian gland secretions100,101 and shown to be decreased in meibomianitis.102 Of interest, androgens upregulate stearoyl-CoA desaturase mRNA levels in mouse male and female meibomian glands,49,103 and the targeted disruption or this rate-limiting enzyme causes meibomian gland atrophy.104 Additional highly expressed genes are those related to extracellular matrix stabilization (inter-α (globulin) inhibitor H5), cell proliferation, migration and/or differentiation (clusterin associated protein 1; thymosin β4, X-linked; keratinocyte differentiation-associated protein; annexin A2), and proteasomal degradation (ubiquitin B and C).84,105,106 One of these structure-related genes encodes biglycan, which, if overexpressed, leads to failure of meibomian gland formation in mice.107 Immune-associated genes that present with high activities in the human meibomian gland are associated with antigen presentation (β2-microglobulin), dendritic and T-cell migration (chemokine (C-C motif) receptor 6), bacteriolytic action (lysozyme), and the inflammatory response (CCAAT/enhancer binding protein β, S100A8, S100A9, and LAIR-1).84 Highly expressed genes related to the endocrine system include those that may bind androgens (secretoglobin, family 2A, member 1) and catalyze the transformation of estrone into estradiol (hydroxysteroid [17β] dehydrogenase 12).84,108,109 Androgens are known to upregulate the prostatic levels of secretoglobin 2A1, a lipophilin that is also translated and strongly expressed in human meibomian gland epithelial cells.110
Several proteins encoded by meibomian gland genes have been identified in meibomian gland secretions.8 In this regard, it is noteworthy that S100A8, S100A9, prolactin-inducible protein, lipocalin-1, lysozyme, β2-microglobulin, and the lacrimal proline-rich protein proline-rich 4 have all been termed tear film biomarkers in dry eye,111,112 and some of these are proposed to be indicative of lacrimal gland aqueous deficiency.112 However, given that the meibomian gland contains transcripts for all these proteins, it may be that alterations in their translation and secretion influence the tear film levels.
In this study, a number of the subjects were taking medications, including lisinopril (antihypertensive), atorvastatin (lowers blood cholesterol levels), atenolol (β1 receptor selective antagonist for cardiovascular disease), levothyroxine (hypothyroid treatment), alendronate (osteoporosis therapy), escitalopram (antidepressant), hydrochlorothiazide (diuretic), and clonazepam (antiepileptic). It is possible that some of these pharmaceutical agents influenced the secretion quality and/or gene expression in the meibomian gland. For example, atorvastatin inhibits a rate-limiting enzyme involved in the cholesterol synthetic pathway113 and could theoretically alter the melting point and viscosity of meibomian gland secretions.5 Similarly, thyroxine could possibly influence meibomian gland activity, given that this hormone has been reported to regulate the function of other sebaceous glands,114 and antidepressant use is a risk factor for the development of dry eye syndrome.42 However, none of these pharmaceutical compounds has been shown to have any effect on the meibomian gland, and neither statins nor antihypertensive medications promote dry eye.42 Consequently, it remains to be determined whether any of these medications represented a confounding factor in this investigation.
Last, some limitations in this study should be considered. First, it is possible that very small pieces of adjacent lid tissue adhered to the meibomian glands during isolation under the dissecting microscope. However, such possible contamination would be similar for all tissues, given that the same individual removed glands from every lid segment in the same manner. Second, the number of tissue samples (i.e., five male and seven female) is relatively low for the analysis of sex-related differences in gene expression. Increasing the sample number is difficult, however, unless tissues are available from multiple lid surgeries. The tissue collection process for this study took more than 2 years to complete. Third, our data reflect mRNA levels. We have yet to determine whether these transcripts are translated into functional proteins.
Overall, our findings demonstrate that MGD is accompanied by multiple changes in gene expression in the meibomian gland. The nature of these alterations, including the upregulation of genes encoding SPRR and S100 calcium-binding proteins, suggest that keratinization plays an important role in the pathogenesis and/or progression of MGD.
Supported by Grant EY05612 from the National Institutes of Health and by Alcon Research, Ltd.
Disclosure: S. Liu, P; S.M. Richards, None; K. Lo, None; M. Hatton, None; A.M. Fay, None; D.A. Sullivan, Alcon (F), P