The analysis of human conjunctival epithelium proteome in ocular surface diseases using impression cytology and 2D-DIGE.
Conjunctival impression cytology samples from patients with meibomian gland dysfunction (MGD), dry eye (DE), and healthy subjects (CT) were collected for determination of the degree of squamous metaplasia (SM) by PAS-hematoxylin staining and for comparative proteomic analyses by 2D-DIGE. The protein spots with discriminant expression were identified by MALDI-TOF/TOF mass spectrometry. Three independent statistical studies were conducted: i). Analysis of differential protein expression between study groups: We observed increased expression of proteins S100A4, S100A8, retinal dehydrogenase-1, peroxiredoxin-1, annexin-A1, annexin-A2, α-enolase, and glutathione S-transferase-P in DE, whereas the highest expression of peroxiredoxin-6, actin cytoplasmic-1, peroxiredoxin-2, and heat shock protein HSP-90-α was observed in MGD; ii). Correlation between changes in the proteome profile and the grade of SM: The expression of 5 different cytokeratins (KRT1, KRT4, KRT8, KRT10, and KRT13) correlated with the degree of SM; iii). Proteome profile differences between pathological and CT groups: An overall proteome analysis revealed upregulation of 9 proteins in the pathological groups (Annexin-A1, α-enolase, Annexin-A2, S100A8, cytokeratin-1, Peroxiredoxin-2 and Leukocyte elastase inhibitor) and downregulation of 2 proteins (Galectin-3 and Lipocalin-1). In conclusion, a sensitive proteomic approach to study conjunctival tissue collected from minimally invasive impression cytology was implemented. Differential proteomics analyses showed that in comparison with the MGD, the DE patients presented higher overexpression of proteins related to antimicrobial defense, tissue-damage response, and regulation of body fluid secretions. Changes in MGD proteome were associated with oxidative stress and anti-apoptotic processes. We found a correlation between the grade of SM and expression of proteins associated with cytoskeleton and keratinization. The studied pathological groups shared elements related to the defense and inflammatory responses. Dot blot assays of proteins ANXA1, S100A8, and S100A4 validated the proteomic results obtained from 2D-DIGE experiments and confirmed the correlation between the expression of these proteins and the clinical parameters.
Bioftalmik Applied Research, Bizkaia Science and Technology Park, Building 612, E-48160 Derio, Bizkaia, Spain.
Javier
J
Soria
Bioftalmik Applied Research, Bizkaia Science and Technology Park, Building 612, E-48160 Derio, Bizkaia, Spain.
Arantxa
A
Acera
Instituto Clínico Quirúrgico de Oftalmología (ICQO), Virgen de Begoña Nº 34, E-48006 Bilbao, Bizkaia, Spain; Department of Ophthalmology, School of Medicine, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain.
Juan A
JA
Durán
Hospital la Paz, Pº de la Castellana, 261, E-28046 Madrid, Spain.
Ana
A
Boto-de-Los-Bueis
Hospital la Paz, Pº de la Castellana, 261, E-28046 Madrid, Spain.
Almudena
A
Del-Hierro-Zarzuelo
Bioftalmik Applied Research, Bizkaia Science and Technology Park, Building 612, E-48160 Derio, Bizkaia, Spain.
Nerea
N
González
Bioftalmik Applied Research, Bizkaia Science and Technology Park, Building 612, E-48160 Derio, Bizkaia, Spain.
Rebeca
R
Reigada
Bioftalmik Applied Research, Bizkaia Science and Technology Park, Building 612, E-48160 Derio, Bizkaia, Spain. Electronic address: tatiana.suarez@bioftalmik.com.
Tatiana
T
Suárez
D000328:Adult; D016022:Case-Control Studies; D003585:Cell Biology; D003228:Conjunctiva / Q000378:metabolism* / Q000473:pathology; D015352:Dry Eye Syndromes / Q000378:metabolism* / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D004848:Epithelium / Q000378:metabolism / Q000473:pathology; D005136:Eye Proteins / Q000378:metabolism*; D005141:Eyelid Diseases / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D015151:Immunoblotting; D008297:Male; D008537:Meibomian Glands / Q000378:metabolism* / Q000473:pathology; D008679:Metaplasia; D008875:Middle Aged; D011446:Prospective Studies; D020543:Proteome / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D057825:Two-Dimensional Difference Gel Electrophoresis
D005136:Eye Proteins; D020543:Proteome
2D-DIGE; Conjunctiva; Dry eye; Impression cytology; Meibomian gland dysfunction; Proteomics; Scarce samples; Squamous metaplasia