Marwan Ali

Histatin-1 Expression in Human Lacrimal Epithelium

Background Study of human lacrimal cell biology is limited by poor access to tissue samples, heterogeneous cell composition of tissue and a lack of established lacrimal epithelial markers. In order to further our understanding of lacrimal cell biology, we sought to find a better marker for human lacrimal epithelial cells, compared to what has been reported in the literature. Methods We utilized human Muller’s muscle conjunctival resection (MMCR) specimens containing accessory lacrimal gland (ALG) and cadaveric main lacrimal gland (MLG) as sources of lacrimal tissue. Candidate markers were sought using human ALG tissue from MMCR specimens, isolated by laser capture microdissection (LCM). Affymetrix® analysis was performed on total RNA isolated from FFPE samples to profile transcription in ALG. MMCR tissue sections were assessed by immunofluorescence using antibodies for histatin-1, lactoferrin, E-cadherin (E-cad) and alpha-smooth muscle actin (ASMA). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed to analyze the expression of histatin-1, E-cad and lactoferrin from cadaveric MLG. Results Histatin-1 is expressed in ALG and MLG, localizes to lacrimal epithelium, and to a greater degree than do other putative lacrimal epithelial markers. Conclusions Histatin-1 is a good marker for human lacrimal epithelium in ALG and MLG and can be used to identify lacrimal cells in future studies.

Human Lacrimal Gland Gene Expression

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.

Evaluation of Accessory Lacrimal Gland in Muller’s Muscle Conjunctival Resection Specimens for Precursor Cell Markers and Biological Markers of Dry Eye Disease

ABSTRACT Purpose: The accessory lacrimal glands (ALGs) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential, and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics. Materials and Methods: ALG tissues obtained from Muller’s muscle conjunctival resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers. Results: Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2, and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES. Conclusions: Thus, we report for the first time that human ALG tissues contain precursor marker-positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES, and isolate candidate precursor cells from ALG tissue.

Effects of histatin-1 peptide on human corneal epithelial cells

Purpose Ocular surface and corneal epithelial wounds are common and potentially debilitating problems. Ideal treatments for these injuries would promote epithelial healing without inflammation, infection and scarring. In addition the best treatments would be cost-efficient, effective, non-toxic and easily applied. Histatin-1 peptides have been shown to be safe and effective enhancers of epithelial wound healing in other model systems. We sought to determine whether histatin-1 peptides could enhance human corneal epithelial wound healing in vitro. Methods Histatin-1 peptides were applied to human corneal epithelial cells and compared over useful dose ranges in scratch assays using time-lapse microscopy. In addition, path finding analysis, cell spreading assays, toxicity and proliferation assays were performed to further characterize the effects of histatin-1 peptide on human corneal limbal epithelial (HCLE). Results Histatin-1 enhanced human corneal epithelial wound healing in typical wound healing models. There was minimal toxicity and no significant enhancement of proliferation of corneal epithelium in response to histatin-1 application. Corneal epithelial spreading and pathfinding appeared to be enhanced by the application of histatin-1 peptides. Conclusions Histatin -1 peptide may enhance migration of HCLE cells and wound healing in vitro. These peptides may have benefit in corneal epithelial wounds and need to be investigated further.

Strategies for Regenerating the Lacrimal Gland

Purpose of ReviewAqueous deficient dry eye disease, a significant cause of morbidity worldwide, is due to dysfunction of the main and accessory lacrimal glands. Recent advances in efforts to regenerate lacrimal gland are reviewed.Recent FindingsSeveral strategies are being explored: ex vivo culture models of human and non-human lacrimal gland epithelial and myoepithelial cells, isolation and characterization of adult precursor cells within lacrimal glands, directed differentiation of stem cells to lacrimal gland cells, and organogenesis and engraftment techniques.SummaryConditions for primary cell culture and expansion are being established and will help in the characterization of lacrimal cells. Presumed adult precursor cells have been isolated, laying down foundations for regeneration. Stem cells have been induced to express features of lacrimal gland cells. Engraftment of ex vivo cultured lacrimal tissue is proof of concept that lacrimal gland regeneration and repopulation is possible.