Ideally, biomaterials designed to play specific physical and physiological roles in vivo should comprise components and microarchitectures analogous to those of the native tissues they intend to replace. For that, implantable biomaterials need to be carefully designed to have the correct structural and compositional properties, which consequently impart their bio-function. In this study, we showed that the control of such properties can be defined from the bottom-up, using smart surface templates to modulate the structure, composition, and bio-mechanics of human transplantable tissues. Using multi-functional peptide amphiphile-coated surfaces with different anisotropies, we were able to control the phenotype of corneal stromal cells and instruct them to fabricate self-lifting tissues that closely emulated the native stromal lamellae of the human cornea. The type and arrangement of the extracellular matrix comprising these corneal stromal Self-Lifting Analogous Tissue Equivalents (SLATEs) were then evaluated in detail, and was shown to correlate with tissue function. Specifically, SLATEs comprising aligned collagen fibrils were shown to be significantly thicker, denser, and more resistant to proteolytic degradation compared to SLATEs formed with randomly-oriented constituents. In addition, SLATEs were highly transparent while providing increased absorption to near-UV radiation. Importantly, corneal stromal SLATEs were capable of constituting tissues with a higher-order complexity, either by creating thicker tissues through stacking or by serving as substrate to support a fully-differentiated, stratified corneal epithelium. SLATEs were also deemed safe as implants in a rabbit corneal model, being capable of integrating with the surrounding host tissue without provoking inflammation, neo-vascularization, or any other signs of rejection after a 9-months follow-up. This work thus paves the way for the de novo bio-fabrication of easy-retrievable, scaffold-free human tissues with controlled structural, compositional, and functional properties to replace corneal, as well as other, tissues.

Purpose: To screen for and characterize compounds that protect corneal endothelial cells against unfolded protein response (UPR) and oxidative stress. Methods: Bovine corneal endothelial cells (BCECs) were treated for 48 hours with 640 compounds from a Food and Drug Administration (FDA)-approved drug library and then challenged with thapsigargin or H2O2 to induce UPR or oxidative stress, respectively. Cell viability was measured using the CellTiter-Glo survival assay. Selected "hits" were subjected to further dose-response testing, and their ability to modulate expression of UPR and oxidative stress markers was assessed by RT-PCR, Western blot, and measurement of protein carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) adducts in immortalized human corneal endothelial cells (iHCECs). Results: Forty-one drugs at 20 μM and 55 drugs at 100 μM increased survival of H2O2-challenged cells, and 8 drugs at 20 μM and 2 drugs at 100 μM increased survival of thapsigargin-challenged cells, compared with untreated control cells. Nicergoline, ergothioneine, nimesulide, oxotremorine, and mefenamic acid increased survival of both H2O2- and thapsigargin-challenged cells. Oxotremorine altered DNA damage inducible 3 (CHOP) gene expression, glucose-regulated protein 78 kDa (GRP78) and activating transcription factor 4 (ATF4) protein expression, and protein carbonyl and 8-OHdG levels. Mefenamic acid altered GRP78 protein expression and protein carbonyl and 8-OHdG levels. Conclusions: Oxotremorine and mefenamic acid are potential survival factors for corneal endothelial cells under UPR and oxidative stress. The described assay can be further expanded to screen additional drugs for potential therapeutic effect in corneal endothelial diseases such as Fuchs' endothelial corneal dystrophy.

Th17 cells are principal mediators of many autoimmune conditions. Recently, memory Th17 cells have been revealed as crucial in mediating the chronicity of various refractory autoimmune disorders; however, the underlying mechanisms maintaining memory Th17 cells have remained elusive. Here, using a preclinical model of ocular autoimmune disease we show that both IL-7 and IL-15 are critical for maintaining pathogenic memory Th17 cells. Neutralization of these cytokines leads to substantial reduction of memory Th17 cells; both IL-7 and IL-15 provide survival signals via activating STAT5, and IL-15 provides additional proliferation signals via activating both STAT5 and Akt. Topical neutralization of ocular IL-7 or IL-15 effectively reduces memory Th17 cells at the inflammatory site and draining lymphoid tissues, while topical neutralization of IL-17 alone, the major pathogenic cytokine secreted by Th17 cells, does not diminish memory Th17 cells at the draining lymphoid tissues. Our results suggest that the effective removal of pathogenic memory Th17 cells via abolishing environmental IL-7 or IL-15 is likely to be a novel strategy in the treatment of autoimmune diseases.

Specialized pro-resolving mediators (SPM), e.g. Resolvin D1, Protectin D1, Lipoxin A₄, and Resolvin E1 have each shown to be active in ocular models reducing inflammation. In general, SPMs have specific agonist functions that stimulate resolution of infection and inflammation in animal disease models. The presence and quantity of SPM in human emotional tears is of interest. Here, utilizing a targeted LC-MS-MS metabololipidomics based approach we document the identification of pro-inflammatory (Prostaglandins and Leukotriene B₄) and pro-resolving lipid mediators (D-series Resolvins, Protectin D1, and Lipoxin A₄) in human emotional tears from 12 healthy individuals. SPMs from the Maresin family (Maresin 1 and Maresin 2) were not present in these samples. Principal Component Analysis (PCA) revealed gender differences in the production of specific mediators within these tear samples as the SPMs were essentially absent in these female donors. These results indicate that specific SPM signatures are present in human emotional tears at concentrations known to be bioactive. Moreover, they will help to further appreciate the mechanisms of production and action of SPMs in the eye, as well as their physiologic roles in human ocular disease resolution.

Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions.

Purpose: Tumor necrosis factor (TNF)-α is upregulated in eyes following corneal alkali injury and contributes to corneal and also retinal damage. Prompt TNF-α inhibition by systemic infliximab ameliorates retinal damage and improves corneal wound healing. However, systemic administration of TNF-α inhibitors carries risk of significant complications, whereas topical eye-drop delivery is hindered by poor ocular bioavailability and the need for patient adherence. This study investigates the efficacy of subconjunctival delivery of TNF-α antibodies using a polymer-based drug delivery system (DDS). Methods: The drug delivery system was prepared using porous polydimethylsiloxane/polyvinyl alcohol composite fabrication and loaded with 85 μg of infliximab. Six Dutch-belted pigmented rabbits received ocular alkali burn with NaOH. Immediately after the burn, subconjunctival implantation of anti-TNF-α DDS was performed in three rabbits while another three received sham DDS (without antibody). Rabbits were followed with photography for 3 months. Results: After 3 months, the device was found to be well tolerated by the host and the eyes exhibited less corneal damage as compared to eyes implanted with a sham DDS without drug. The low dose treatment suppressed CD45 and TNF-α expression in the burned cornea and inhibited retinal ganglion cell apoptosis and optic nerve degeneration, as compared to the sham DDS treated eyes. Immunolocalization revealed drug penetration in the conjunctiva, cornea, iris, and choroid, with residual infliximab in the DDS 3 months after implantation. Conclusions: This reduced-risk biologic DDS improves corneal wound healing and provides retinal neuroprotection, and may be applicable not only to alkali burns but also to other inflammatory surgical procedures such as penetrating keratoplasty and keratoprosthesis implantation.

PURPOSE: To delineate and compare the kinetics of corneal angiogenesis after high-risk (HR) versus low-risk (LR) corneal transplantation. METHODS: In mice, intrastromal sutures were placed in the recipient graft bed 2 weeks before allogeneic transplantation to induce angiogenesis and amplify the risk of graft rejection. Control (LR) graft recipients did not undergo suture placement, and thus the host bed remained avascular at the time of transplantation. Graft hemangiogenesis and opacity scores were evaluated for 8 weeks by slit-lamp biomicroscopy. Immunohistochemistry was used to measure CD31 (blood vessels) and LYVE-1 (lymphatic vessels) cells. RESULTS: Biphasic kinetics were observed for hemangiogenesis in both HR and LR transplant recipients using clinical and immunohistochemical assessments. The biphasic kinetics were composed of a rise-fall (phase 1) followed by a second rise (phase 2) in the degree of vessels. Compared with LR recipients, HR recipients showed higher hemangiogenesis (whole cornea and graft) throughout 8 weeks. Analyzing grafts revealed sustained presence of lymphatic vessels in HR recipients; however, lymphatic neovessels regressed in LR recipients 2 weeks posttransplantation. In contrast to HR host beds, the LR host bed microenvironment cannot sustain the growth of lymphatic neovessels in allografts, whereas it can sustain continued hemangiogenesis. CONCLUSIONS: The sustained presence of lymphatic vessels in HR host beds can facilitate host immunity against allografts and is likely associated with ongoing higher risk of rejection of these grafts in the long term, suggesting that therapeutic interventions targeting inflammation and lymphatic vessels need to be sustained long term in the HR corneal transplant setting.

PURPOSE: The objective of this clinical trial (NCT02507934) was to assess the efficacy and safety of recombinant human lubricin as compared to a 0.18% sodium hyaluronate (HA) eye drop in subjects with moderate dry eye disease (DED). METHODS: DEWS Grade 2-3 subjects were randomized to use lubricin (N=19, 51.9 ± 11.8 years) or HA (N=20, 61.8 ± 13.3 years). After a saline washout period, subjects administered BID therapy for 7 days, followed by instillation as needed (2-6 drops per eye) for 7 days. Visual analog scale (VAS) including foreign body sensation, burning/stinging, itching, pain, sticky feeling, blurred vision and photophobia were primary outcomes, with secondary endpoints of corneal fluorescein staining, Schirmer test, tear film breakup time (TFBUT), eyelid and conjunctival erythema and number of instillations compared at day 14. RESULTS: The primary endpoint was met. Lubricin supplementation achieved greater than a 72% reduction from baseline in foreign body sensation (P<.013), burning/stinging, pain, sticky feeling (P<.0432), blurred vision (P<.0013), and photophobia (P<.011) in at least one eye. Lubricin also showed significant improvement in fluorescein staining (OD/OS: 43.8%/50.0%, vs. 26.5%/23.3%, P<.0398, P<.0232), TFBUT (P<.010), SANDE frequency (P<.0435), eyelid erythema (P<.004), conjunctival erythema (P<.0013), and instillations (P<.04) as compared to HA. No treatment-related adverse events occurred during the investigation. CONCLUSIONS: Recombinant human lubricin was shown to produce significant improvement in both signs and symptoms of dry eye disease as compared to HA.

PURPOSE: To investigate the levels of neutrophil elastase (NE), matrix metalloproteinases (MMPs), and myeloperoxidase (MPO) in tear washes of patients with ocular graft-vs-host disease (oGVHD). DESIGN: Case-control study. METHODS: Based on established criteria, oGVHD patients (n = 14; 28 eyes) and age-/sex-matched healthy controls (n = 14; 28 eyes) were enrolled. Tear washes were collected and analyzed for NE using a single-analyte enzyme-linked immunosorbent assay (ELISA). MMPs (1, 2, 3, 7, 8, 9, 12), MPO, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were analyzed using multianalyte bead-based ELISA assays. Total MMP activity was measured using a fluorimetric assay. Correlation studies were performed between NE, MMP-8, MMP-9, and MPO within study groups. RESULTS: NE, MMP-8, MMP-9, and MPO levels were elevated in oGVHD tears when compared with controls (P < .0001). NE was the most elevated analyte. MMP activity was higher and TIMP-1 levels were lower in oGVHD than in control (P < .0001). In oGVHD, NE significantly correlated with MMP-8 (r = 0.92), MMP-9 (r = 0.90), and MPO (r = 0.79) (P < .0001). MMP-8 correlated with MMP-9 (r = 0.96, P < .0001), and MPO (r = 0.60, P = .001). MMP-9 correlated with MPO (r = 0.55, P = .002). In controls, NE, MMP-9, and MPO significantly correlated with each other (P < .0001). CONCLUSIONS: The marked increase in NE in oGVHD tears that correlated strongly with elevated MMP-8, MMP-9, and MPO suggests a common neutrophilic source and provides evidence of neutrophil activity on the ocular surface of oGVHD patients.

In vivo confocal microscopy (IVCM) is becoming an indispensable tool for studying corneal physiology and disease. Enabling the dissection of corneal architecture at a cellular level, this technique offers fast and noninvasive in vivo imaging of the cornea with images comparable to those of ex vivo histochemical techniques. Corneal nerves bear substantial relevance to clinicians and scientists alike, given their pivotal roles in regulation of corneal sensation, maintenance of epithelial integrity, as well as proliferation and promotion of wound healing. Thus, IVCM offers a unique method to study corneal nerve alterations in a myriad of conditions, such as ocular and systemic diseases and following corneal surgery, without altering the tissue microenvironment. Of particular interest has been the correlation of corneal subbasal nerves to their function, which has been studied in normal eyes, contact lens wearers, and patients with keratoconus, infectious keratitis, corneal dystrophies, and neurotrophic keratopathy. Longitudinal studies have applied IVCM to investigate the effects of corneal surgery on nerves, demonstrating their regenerative capacity. IVCM is increasingly important in the diagnosis and management of systemic conditions such as peripheral diabetic neuropathy and, more recently, in ocular diseases. In this review, we outline the principles and applications of IVCM in the study of corneal nerves in various ocular and systemic diseases.

The cornea is the most commonly transplanted tissue in medicine. The main cause of corneal graft failure is allograft rejection. The incidence of graft rejection depends on the presence of high-risk characteristics, most notably corneal neovascularization. Although corneal grafting has a high success rates in the absence of these risk factors, high-risk keratoplasty is associated with low success rates because of a high incidence of immune-mediated graft rejection. To improve the survival of high-risk corneal transplantation, various preoperative, intraoperative, and postoperative measures can be considered.; however, the key step in the management of these grafts is the long-term use of local and/or systemic immunosuppressive agents. Although a number of immunosuppressive agents have been employed for this purpose, the results vary significantly across different studies. This is partly due to the lack of an optimized method for their use, as well as the lack of a precise stratification of the degree of risk in each individual patient. New targeted biologic treatments, as well as tolerance-inducing methods, show promising horizons in the management of high-risk corneal transplantation in near future.

Purpose: The purpose of this study was to evaluate the resistance to degradation by collagenase A of corneas that have been crosslinked with Rose Bengal and green light (RGX). Methods: The ex vivo crosslinking procedure was performed on enucleated rabbit corneas. Corneas were deepithelialized after applying 30% alcohol. Corneas were stained with Rose Bengal (RB, 0.1%) for 2 minutes and then exposed to green light (532 nm) at 0.25 W/cm2 for times to deliver doses of 50, 100, 150, or 200 J/cm2 (n = 5 per group). Five corneas were pretreated with riboflavin solution (0.1% riboflavin) for 15 minutes and irradiated with ultraviolet A (UVA) light (370 nm, 3 mW/cm2) for 30 minutes. Five corneas underwent only de-epithelialization and were otherwise untreated. Five corneas were stained with RB without light exposure. The central corneas of each group was removed with a 8.5-mm trephine and incubated at 37°C in 0.3% collagenase A solution. Time to dissolution of each cornea was compared across treatments. Results: Corneas treated with RGX were treated with light fluences of 50, 100, 150, and 200 J/cm2; these corneas dissolved completely at 8.3 ± 1.2, 11.1 ± 1.4, 12.4 ± 1.7, and 15.7 ± 1.8 hours, respectively. Corneas treated by riboflavin and UVA light dissolved at 15.7 ± 1.7 hours, and nontreated corneas dissolved at 6.1 ± 1.3 hours. Corneas treated with only RB (no green light) dissolved at 9.3 ± 1.7 hours. Compared with the untreated corneas, all of the RB groups and the riboflavin-UVA-treated group of corneas degraded statistically significantly slower than untreated corneas (P < 0.05). Conclusions: Crosslinking with RGX increased corneal resistance to digestion by collagenase comparable to that produced by riboflavin and UVA treatment.

Regulatory T cells (Tregs) are crucial for allograft survival. Tregs can be divided into thymus-derived natural Tregs (tTregs) and peripherally-derived induced Tregs (pTregs). Here, we determine whether the suppressive function of Treg subsets is hampered in hosts who are at high risk for rejecting their graft. To induce graft beds that promote high risk of transplant rejection, intrastromal corneal sutures were placed two weeks prior to the transplant procedure in mice. We demonstrate that in high-risk recipients the frequencies and function of pTregs (but not tTregs) are suppressed. Reduced function of pTregs correlated with decreased expression of CTLA-4, interleukin-10, and transforming growth factor-β. Adoptive transfer of pTregs from mice at low risk of subsequent graft rejection is able to rescue graft survival in recipients that are at high risk of rejecting their grafts. Our data suggest that impaired function of pTregs, but not tTregs, mediates the loss of immune tolerance and promotes allograft rejection.

PURPOSE: To compare the ocular surfaces of patients treated with rebamipide (REB) ophthalmic suspension or diquafosol (DQS) ophthalmic solution for dry eye syndrome after penetrating keratoplasty (PK). METHODS: A total of 40 eyes of 40 patients who had dry eyes after undergoing PK were enrolled and randomly divided into an REB group and a DQS group. Both REB and DQS groups used each eye drop four times. The tear breakup time (TBUT), corneal fluorescein staining scores, and dry eye-related quality-of-life score (DEQS) were evaluated before treatment, 2 weeks after start of treatment and 4 weeks after start of treatment. RESULTS: We found a significant improvement in TBUT (P < 0.001, Dunnett's test) and fluorescein scores (P < 0.001) 4 weeks after treatment in the REB group. Similar results were obtained in the DQS group (P < 0.001 and P = 0.01, respectively). No significant improvements in DEQS were found 4 weeks after treatment in each group (P = 0.15 and P = 0.63, analysis of variance, respectively). No significant differences were seen in these variables and in the changes between the groups after treatment. CONCLUSIONS: REB and DQS may be effective for the management of dry eye syndrome after PK in terms of ocular surface findings. In our study, effects of REB appear to be equivalent to those of DQS in the patients.

PURPOSE: To investigate morphological changes of the corneal epithelium and subbasal nerves in patients with corneal allodynia using in vivo confocal microscopy (IVCM). DESIGN: Case-control study of patients with corneal allodynia and healthy controls. METHODS: Ten eyes of six patients were diagnosed with corneal allodynia at a single center and compared to fifteen healthy eyes. IVCM of the central cornea was performed on all subjects and controls. Images were retrospectively analyzed numbers of total corneal subbasal nerves, main trunks and branches, total nerve length and density, nerve branching, and tortuosity, superficial and basal epithelial cell densities, and superficial epithelial cell size. RESULTS: Corneal allodynia was seen in patients with dry eye disease, recurrent corneal erosion syndrome, exposure to ultraviolet radiation, and Accutane use. Compared to controls, patients with corneal allodynia had a significant decrease in the total numbers of subbasal nerves (P=.014), nerve branches (P=.006), total nerve length (P=.0029), total nerve density (P=.0029) and superficial and basal epithelial cell densities (P=.0004, P=.0036) with an increase in superficial epithelial cell size (P=.016). There were no statistically significant differences in the number of subbasal nerve main trunks (P=.09), nerve branching (P=.21), and nerve tortuosity (P=.05). CONCLUSIONS: Corneal IVCM enables near-histological visualization and quantification of the cellular and neural changes in corneal allodynia. Regardless of etiology, corneal allodynia is associated with decreased corneal epithelial cell densities, increased epithelial cell size, and decreased numbers and lengths of subbasal nerves despite an unremarkable slit-lamp examination. Therefore, IVCM may be useful in the management of patients with corneal allodynia.

Purpose: We fabricated and investigated polymeric scaffolds that can substitute for the conjunctival extracellular matrix to provide a substrate for autologous expansion of human conjunctival goblet cells in culture. Methods: We fabricated two hydrogels and two silk films: (1) recombinant human collagen (RHC) hydrogel, (2) recombinant human collagen 2-methacryloylxyethyl phosphorylcholine (RHC-MPC) hydrogel, (3) arginine-glycine-aspartic acid (RGD) modified silk, and (4) poly-D-lysine (PDL) coated silk, and four electrospun scaffolds: (1) collagen, (2) poly(acrylic acid) (PAA), (3) poly(caprolactone) (PCL), and (4) poly(vinyl alcohol) (PVA). Coverslips and polyethylene terephthalate (PET) were used for comparison. Human conjunctival explants were cultured on scaffolds for 9 to 15 days. Cell viability, outgrowth area, and the percentage of cells expressing markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were determined. Results: Most of cells grown on all scaffolds were viable except for PCL in which only 3.6 ± 2.2% of the cells were viable. No cells attached to PVA scaffold. The outgrowth was greatest on PDL-silk and PET. Outgrowth was smallest on PCL. All cells were CK7-positive on RHC-MPC while 84.7 ± 6.9% of cells expressed CK7 on PDL-silk. For PCL, 87.10 ± 3.17% of cells were CK7-positive compared to PET where 67.10 ± 12.08% of cells were CK7-positive cells. Conclusions: Biopolymer substrates in the form of hydrogels and silk films provided for better adherence, proliferation, and differentiation than the electrospun scaffolds and could be used for conjunctival goblet cell expansion for eventual transplantation once undifferentiated and stratified squamous cells are included. Useful polymer scaffold design characteristics have emerged from this study.

Conjunctival goblet cells synthesize and secrete mucins which play an important role in protecting the ocular surface. Pro-resolution mediators, such as lipoxin A4 (LXA4), are produced during inflammation returning the tissue to homeostasis and are also produced in non-inflamed tissues. The purpose of this study was to determine the actions of LXA4 on cultured human conjunctival goblet cell mucin secretion and increase in intracellular [Ca(2+)] ([Ca(2+)]i) and on histamine-stimulated responses. LXA4 increased mucin secretion and [Ca(2+)]i, and activated ERK1/2 in human goblet cells. Addition of LXA4 before resolvin D1 (RvD1) decreased RvD1 responses though RvD1 did not block LXA4 responses. LXA4 inhibited histamine-stimulated increases in mucin secretion, [Ca(2+)]i, and ERK1/2 activation through activation of β-adrenergic receptor kinase 1. We conclude that conjunctival goblet cells respond to LXA4 through the ALX/FPR2 receptor to maintain homeostasis of the ocular surface and regulate histamine responses and could provide a new therapeutic approach for allergic conjunctivitis and dry eye diseases.

PURPOSE: To evaluate changes in corneal endothelial cell density over time in patients with dry eye disease (DED) and to correlate endothelial cell loss with corneal subbasal nerve density. METHODS: This retrospective study included 40 eyes of 20 patients with DED. Laser in vivo confocal microscopy had been performed in the central cornea of both eyes at an initial visit and repeated after a mean follow-up of 33.2 ± 10.2 months. The densities of corneal endothelial cells and subbasal nerves were measured in both visits and compared with 13 eyes of 13 normal age-matched controls. RESULTS: At the initial visit, the DED group had lower densities of corneal endothelial cells (2620 ± 386 cells/mm) and subbasal nerves (17.8 ± 7.5 mm/mm) compared with the control group (2861 ± 292 cells/mm and 22.8 ± 3.0 mm/mm, with P = 0.08 and P = 0.01, respectively). At the end of follow-up, although there was no significant change in subbasal nerve density (16.7 ± 7.2 mm/mm, P = 0.43), the mean corneal endothelial cell density significantly decreased to 2465 ± 391 cells/mm (P = 0.01), with a mean corneal endothelial cell loss of 2.1 ± 3.6% per year. The endothelial cell loss showed a statistically significant negative correlation with the initial subbasal nerve density (Rs = -0.55, P = 0.02). CONCLUSIONS: Patients with DED have an accelerated corneal endothelial cell loss compared with that reported in the literature for normal aging. Those with lower subbasal nerve density, in particular, are at a higher risk for endothelial cell loss over time.

The purpose of this study was to investigate the changes that occur in the lacrimal glands (LGs) in female thrombospondin 1 knockout (TSP1(-/-)) mice, a mouse model of the autoimmune disease Sjogren's syndrome. The LGs of 4, 12, and 24 week-old female TSP1(-/-) and C57BL/6J (wild type, WT) mice were used. qPCR was performed to measure cytokine expression. To study the architecture, LG sections were stained with hematoxylin and eosin. Cell proliferation was measured using bromo-deoxyuridine and immunohistochemistry. Amount of CD47 and stem cell markers was analyzed by western blot analysis and location by immunofluorescence microscopy. Expression of stem cell transcription factors was performed using Mouse Stem Cell Transcription Factors RT(2) Profiler PCR Array. Cytokine levels significantly increased in LGs of 24 week-old TSP1(-/-) mice while morphological changes were detected at 12 weeks. Proliferation was decreased in 12 week-old TSP1(-/-) mice. Three transcription factors were overexpressed and eleven underexpressed in TSP1(-/-) compared to WT LGs. The amount of CD47, Musashi1, and Sox2 was decreased while the amount of ABCG2 was increased in 12 week-old TSP1(-/-) mice. We conclude that TSP1 is necessary for maintaining normal LG homeostasis. Absence of TSP1 alters cytokine levels and stem cell transcription factors, LG cellular architecture, decreases cell proliferation, and alters amount of stem cell markers.