Dioleoylphosphatidylglycerol Inhibits Heat Shock Protein B4 (HSPB4)-Induced Inflammatory Pathways In Vitro.

Our previous work shows that dioleoylphosphatidylglycerol (DOPG) accelerates corneal epithelial healing in vitro and in vivo by unknown mechanisms. Prior data demonstrate that DOPG inhibits toll-like receptor (TLR) activation and inflammation induced by microbial components (pathogen-associated molecular patterns, PAMPs) and by endogenous molecules upregulated in psoriatic skin, which act as danger-associated molecular patterns (DAMPs) to activate TLRs and promote inflammation. In the injured cornea, sterile inflammation can result from the release of the DAMP molecule, heat shock protein B4 (HSPB4), to contribute to delayed wound healing. Here, we show in vitro that DOPG inhibits TLR2 activation induced in response to HSPB4, as well as DAMPs that are elevated in diabetes, a disease that also slows corneal wound healing. Further, we show that the co-receptor, cluster of differentiation-14 (CD14), is necessary for PAMP/DAMP-induced activation of TLR2, as well as of TLR4. Finally, we simulated the high-glucose environment of diabetes to show that elevated glucose levels enhance TLR4 activation by a DAMP known to be upregulated in diabetes. Together, our results demonstrate the anti-inflammatory actions of DOPG and support further investigation into its development as a possible therapy for corneal injury, especially in diabetic patients at high risk of vision-threatening complications.

Innate Immune System Activation, Inflammation and Corneal Wound Healing.

Corneal wounds resulting from injury, surgeries, or other intrusions not only cause pain, but also can predispose an individual to infection. While some inflammation may be beneficial to protect against microbial infection of wounds, the inflammatory process, if excessive, may delay corneal wound healing. An examination of the literature on the effect of inflammation on corneal wound healing suggests that manipulations that result in reductions in severe or chronic inflammation lead to better outcomes in terms of corneal clarity, thickness, and healing. However, some acute inflammation is necessary to allow efficient bacterial and fungal clearance and prevent corneal infection. This inflammation can be triggered by microbial components that activate the innate immune system through toll-like receptor (TLR) pathways. In particular, TLR2 and TLR4 activation leads to pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) activation. Similarly, endogenous molecules released from disrupted cells, known as damage-associated molecular patterns (DAMPs), can also activate TLR2, TLR4 and NFκB, with the resultant inflammation worsening the outcome of corneal wound healing. In sterile keratitis without infection, inflammation can occur though TLRs to impact corneal wound healing and reduce corneal transparency. This review demonstrates the need for acute inflammation to prevent pathogenic infiltration, while supporting the idea that a reduction in chronic and/or excessive inflammation will allow for improved wound healing.

Vitamin D receptor and metabolite effects on corneal epithelial cell gap junction proteins.

Vitamin D is a fat-soluble prohormone that can be activated both systemically and within individual tissues. Our lab has previously demonstrated that the corneal epithelium can activate vitamin D and that the vitamin D metabolites 1,25(OH)2D3 and 24R,25(OH)2D3 can affect corneal epithelial migration, proliferation, and tight and gap junction function. These vitamin D-derived metabolites signal through the vitamin D receptor (VDR). The purpose of this study was to specifically determine the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell gap junction proteins. Connexin (Cx) 26, 30 and 43 protein expression was detected in a human corneal epithelial cell line (HCEC), wild type and vitamin D receptor knockout (VDR-/-) mouse corneas, and cultured mouse primary epithelial cells (MPCEC). In vitro gap junction function was assessed using the scrape loading/dye transfer assay. HCEC and MPCEC were treated with 1,25(OH)2D3 or 24R,25(OH)2D3. Western blotting was used to detect gap junction proteins. Vitamin D3 effects on epithelial intracellular Ca++ (Ca++i) were determined using the dye Cal-520. Cx26 and Cx43 protein levels were significantly increased in HCEC and MPCEC treated with both 1,25(OH)2D3 and 24R,25(OH)2D3. Cx30 and Cx43 protein levels were also significantly increased in VDR-/- MPCEC. In vitro gap junction connectivity was significanlty enhanced in HCEC and MPCEC cultured with 24R,25(OH)2D3 and 1,25(OH)2D3. Ca++i was not affected by 1,25(OH)2D3 or 24R,25(OH)2D3 in HCEC or MPCEC. We conclude that both 1,25(OH)2D3 and 24R,25(OH)2D3 are positive regulators of connexin proteins and gap junction communication in the corneal epithelium. These vitamin D metabolites appear to signal through both VDR-dependent and -independent pathways. The effects of vitamin D on corneal epithelial gap junctions do not seem to be dependent on Ca++i.

A new sensitive LC/MS/MS analysis of vitamin D metabolites using a click derivatization reagent, 2-nitrosopyridine.

There is an increased demand for comprehensive analysis of vitamin D metabolites. This is a major challenge, especially for 1α,25-dihydroxyvitamin D [1α,25(OH)2VitD], because it is biologically active at picomolar concentrations. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) was a revolutionary reagent in dramatically increasing sensitivity of all diene metabolites and allowing the routine analysis of the bioactive, but minor, vitamin D metabolites. A second generation of reagents used large fixed charge groups that increased sensitivity at the cost of a deterioration in chromatographic separation of the vitamin D derivatives. This precludes a survey of numerous vitamin D metabolites without redesigning the chromatographic system used. 2-Nitrosopyridine (PyrNO) demonstrates that one can improve ionization and gain higher sensitivity over PTAD. The resulting vitamin D derivatives facilitate high-resolution chromatographic separation of the major metabolites. Additionally, a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE) was developed to selectively extract 1α,25(OH)2VitD, while reducing 2- to 4-fold ion suppression compared with SPE alone. LLE-SPE followed by PyrNO derivatization and LC/MS/MS analysis is a promising new method for quantifying vitamin D metabolites in a smaller sample volume (100 µL of serum) than previously reported methods. The PyrNO derivatization method is based on the Diels-Alder reaction and thus is generally applicable to a variety diene analytes.

Bone turnover in long-term survivors of childhood acute lymphoblastic leukemia.

BACKGROUND: We investigated the effects of demographic, lifestyle (self-reported smoking status and physical activity levels), cancer-related treatment factors (radiation and chemotherapy), and diet (calcium and vitamin D intake) on bone turnover and the relationship of bone turnover to lumbar spine bone mineral density (BMD) Z-scores (LS-BMD Z-scores) determined by quantitative computed tomography (QCT) in 418 ≥5-year survivors of childhood acute lymphoblastic leukemia (ALL). PROCEDURE: Bone turnover was assessed by biomarkers including serum bone-specific alkaline phosphatase (BALP), osteocalcin (OC), and urinary N-telopeptide of type I collagen indexed to creatinine (NTX/Cr). The 215 males ranged in age from 9 to 36 years (median age 17 years). RESULTS: Age and tanner score were inversely associated with all biomarkers (BALP, OC, NTX/Cr) (P < 0.001). Males had higher BALP and OC than females (P < 0.001). Body mass index (BMI) was inversely associated with OC and NTX/Cr (P < 0.001). There was no significant association of biomarkers with lifestyle related factors, ALL treatment-related factors, dietary calcium, vitamin D, or LS-BMD Z-score. CONCLUSIONS: In this population of long-term survivors of ALL, bone turnover was significantly associated with age, gender, tanner stage, and BMI. ALL-related treatments did not influence bone turnover and bone turnover was not predictive of volumetric LS-BMD Z-score.

Transient plasma membrane disruption induced calcium waves in mouse and human corneal epithelial cells.

The purpose of this study was to examine transient plasma membrane disruptions (TPMDs) and TPMD-induced Ca++ waves (TPMD Ca++ Wvs) in human and mouse corneal epithelium (HCEC and MCEC). A multi-photon microscope was used to create laser-induced TPMDs in single cultured cells and in intact ex vivo and in vivo MCECs and ex vivo human cornea rim HCECs. Eye rubbing-induced TPMDs were studied by gentle rubbing with a cotton tipped applicator over a closed eyelid in ex vivo and in vivo MCECs. Ca++ sources for TPMD-induced Ca++ waves were explored using Ca++ channel inhibitors and Ca++-free media. TPMDs and TPMD Ca++ Wvs were observed in all cornea epithelial models examined, often times showing oscillating Ca++ levels. The sarcoplasmic reticulum Ca++ ATPase inhibitors thapsigargin and CPA reduced TPMD Ca++ Wvs. TRP V1 antagonists reduced TPMD Ca++ Wvs in MCECs but not HCECs. Ca++-free medium, 18α-GA (gap junction inhibitor), apyrase (hydrolyzes ATP), and AMTB (TRPM8 inhibitor) did not affect TPMD Ca++ Wvs. These results provide a direct demonstration of corneal epithelial cell TPMDs and TPMDs in in vivo cells from a live animal. TPMDs were observed following gentle eye rubbing, a routine corneal epithelial cell mechanical stress, indicating TPMDs and TPMD Ca++ Wvs are common features in corneal epithelial cells that likely play a role in corneal homeostasis and possibly pathophysiological conditions. Intracellular Ca++ stores are the primary Ca++ source for corneal epithelial cell TPMD Ca++ Wvs, with TRPV1 Ca++ channels providing Ca++ in MCECs but not HCECs. Corneal epithelial cell TPMD Ca++ Wv propagation is not influenced by gap junctions or ATP.

Effects of 1,25-Vitamin D3 and 24,25-Vitamin D3 on Corneal Nerve Regeneration in Diabetic Mice.

Corneal nerve homeostasis is essential for the functional integrity of the ocular surface. Vitamin D deficiency (VDD) and vitamin D receptor knockout (VDR KO) have been found to reduce corneal nerve density in diabetic mice. This is the first study to comprehensively examine the influence of vitamin D on nerve regeneration following corneal epithelial injury in diabetic mice. Corneal nerve regeneration was significantly retarded by diabetes, VDR KO, and VDD, and it was accelerated following topical 1,25 Vit D and 24,25 Vit D administration. Furthermore, topical 1,25 Vit D and 24,25 Vit D increased nerve growth factor, glial cell line-derived neurotropic factor, and neurotropin-3 protein expression, and it increased secretion of GDNF protein from human corneal epithelial cells. CD45+ cells and macrophage numbers were significantly decreased, and vitamin D increased CD45+ cell and macrophage recruitment in these wounded diabetic mouse corneas. The accelerated nerve regeneration observed in these corneas following topical 1,25 Vit D and 24,25 Vit D administration may be related to the vitamin D-stimulated expression, secretion of neurotrophic factors, and recruitment of immune cells.

A Method for Eliminating Fibroblast Contamination in Mouse and Human Primary Corneal Epithelial Cell Cultures.

PURPOSE: This study was designed to determine if previous approaches to eliminate fibroblast contamination in different cells types would be successful in eliminating fibroblast contamination from human and mouse primary corneal epithelial cell cultures, with the primary goal being to describe a simple, easy, and effective method to culture fibroblast-free primary mouse and human corneal epithelial cell cultures. METHODS: Primary human and mouse corneal stromal cells and epithelial cells were isolated and cultured from human corneal rims and mouse corneas, respectively. Several approaches previously used in other tissue types were evaluated using corneal epithelial cells and mixtures of fibroblasts and epithelial cells to determine the most effective purification method. Methods evaluated included 0.25% trypsin-EDTA, low temperature, mitomycin-C, and dispase. Degree of fibroblast contamination was examined using light microscopy evaluation of cell phenotype, immunofluorescence and western blotting using cell type-specific markers. Anti-pancytokeratin (PanCK) was used as the epithelial immunofluorescence label, and anti-α smooth muscle actin (αSMA) as the fibroblast immunofluorescence label. Epithelial western blot antibodies included PanCK, keratin 12, and E-cadherin, while αSMA, collagen 1A1 and collagen 3A1 were used to identify fibroblasts. RESULTS: Fibroblast contamination of human and mouse primary cornea epithelial cell cultures was best controlled using the 0.25% trypsin-EDTA method. The other methods examined were not effective at eliminating cornea fibroblast contamination. CONCLUSIONS: Trypsin-EDTA digestion is a simple and effective method for controlling fibroblast contamination of cultured primary human and mouse corneal epithelial cells.

Topical Spironolactone in the Treatment of Ocular Graft-Versus-Host Disease.

INTRODUCTION: This two-part study aimed to investigate the therapeutic potential of topical spironolactone in ocular graft-versus-host disease (oGVHD). While off-label use of topical spironolactone has been described in dry eye, its efficacy in managing signs and symptoms of oGVHD remains unstudied. Preclinically, we tested the hypothesis that spironolactone induces corneal lipid synthesis in a mouse model. Clinically, we assessed patient response to spironolactone with a retrospective observational design. METHODS: Both immortalized and primary human corneal epithelial cells were stained with oil red O after 9 days of treatment with spironolactone. C57BL/6 mice were dosed thrice daily with one drop in each eye for 18 days. Corneal tissue was stained with oil red O and BODIPY™. Twenty eyes with oGVHD, as defined by the International Chronic oGVHD Consensus Group, were studied. Corneal fluorescein staining, lid margin vascularity, meibomian gland obstruction, meibum turbidity, zone A posterior lid margin vascularity, and oGVHD diagnostic criteria severity grading were compared in a pre-post study. Follow-up times ranged from 7 to 21 weeks, with a median time of 12 weeks. Statistical analysis was done with STATA 17 by fitting data to a non-parametric model. RESULTS:  In vitro results showed an increased number and density of oil red O staining granules in the treatment group versus control in both primary and immortalized human corneal epithelium. In vivo, results showed translation to the mouse model with increased corneal epithelial BODIPY™ signal compared to untreated control. oGVHD patients had improved lid margin vascularity (p = 0.046), corneal fluorescein staining (p = 0.021), and International oGVHD Consensus Group severity scores (p = 0.011) after treatment with topical spironolactone. Minimal adverse effects were noted, the most common being mild stinging lasting less than a minute after instillation. CONCLUSION: The improved severity scores, lid margin inflammation, and corneal fluorescein staining after weeks of treatment support the rationale that topical spironolactone may benefit oGVHD. The observed lipid production by the corneal epithelium is thought to contribute to this protective effect against ocular surface erosive disease in oGVHD. A mineralocorticoid receptor antagonist, spironolactone may offer therapeutic benefits in oGVHD while avoiding undesirable side effects of topical or systemic glucocorticoids.

Lysophospholipids and lysophospholipase D in rabbit aqueous humor following corneal injury.

We previously found that lysophosphatidic acid (LPA)-like activity eliciting Cl(-) currents in Xenopus oocytes is increased in rabbit aqueous humor (AH) following corneal freeze wounds. The purpose of this study was to examine whether actual levels of LPA in AH from wounded eyes are higher than those from control eyes, and to determine the sources and enzymatic pathways of AH LPA in control and wounded conditions. Lysophospholipase D (lysoPLD) activity was measured by the enzymatic determination of choline following incubation of AH samples with exogenous lysophosphatidylcholines (LPCs). The molecular species compositions of LPA and LPC in fresh and incubated AH were determined by liquid chromatography-tandem mass spectrometry. A high, but similar activity of lysoPLD in the samples from both control and freeze-wounded eyes was detected. Its enzymatic properties resemble those of plasma lysoPLD, identified as autotaxin. Levels of LPCs, predominant substrates of lysoPLD in AH, were several times higher in the AH samples from injured eyes than those from the control eyes. Our results suggest that lysoPLD is constitutively released from corneal tissues and/or ciliary body into the AH, with no injury-induced increase in release following freeze-wounding. They also suggest that wound-induced increases in LPA-like biological activity are due to linoleoyl species-rich molecular composition in AH from wounded eyes. A possible mechanism of the altered molecular composition is an increase in the AH concentrations of LPCs, linoleoyl species of which are preferentially converted to corresponding unsaturated LPA by the constitutively active lysoPLD.

Effects of 1,25 and 24,25 Vitamin D on Corneal Fibroblast VDR and Vitamin D Metabolizing and Catabolizing Enzymes.

Purpose: To investigate the effects of 1,25-Vit D3 and 24,25-Vit D3 on corneal fibroblast expression of the vitamin D-associated enzymes CYP27B1 and CYP24A1 and the roles of the vitamin D receptor (VDR) and protein disulfide isomerase, family A, member 3 (Pdia3) in these cells.Methods: CYP24A1, CYP27B1, VDR, and Pdia3 expression in corneas was detected using immunohistochemistry. Western blotting was used to measure protein expression in human and mouse fibroblasts, including VDR KO mouse cells, treated with 1,25-Vit D3 (20 nM) and 24,25-Vit D3 (100 nM). The Pdia3 inhibitor LOC14 was used to explore the role of Pdia3 as a Vit D3 receptor in these cells.Results: CYP24A1, CYP27B1, VDR, and Pdia3 were all expressed in mouse and human corneal fibroblasts. 1,25-Vit D3 significantly increased VDR expression in human and mouse fibroblasts. 1,25-Vit D3 and 24,25-VitD3 significantly increased CYP24A1 and CYP27B1 expression level in human, VDR WT mouse, and VDR KO mouse corneal fibroblasts. CYP24A1 and CYP27B1 expression was unchanged in VDR KO mouse fibroblasts treated with 1,25-Vit D3 or 24,25-Vit D3 plus LOC14. Human fibroblast VDR, CYP24A1, and CYP27B1 expression were unaffected by LOC14.Conclusions: Vitamin D metabolic enzymes, VDR, and Pdia3 are all expressed in mouse and human corneal fibroblasts. 1,25-Vit D3 modulates fibroblast vitamin D enzymes through both the VDR and Pdia3 pathways in a species-dependent manner. 24,25-Vit D3 can increase expression of fibroblast CYP24A1 and CYP27B1 in the absence of VDR and is likely involved in fibroblast regulation independent of 1,25-Vit D3 or VDR.

Lysophosphatidic acid-activated Cl- current activity in human systemic sclerosis skin fibroblasts.

OBJECTIVES: SSc (scleroderma) is an often fatal disease characterized by widespread tissue fibrosis. Fibroblasts play a key role in SSc-associated fibrosis. This study was designed to determine: (i) whether fibroblasts isolated from skin of patients with SSc have increased lysophosphatidic acid-activated Cl- current (IClLPA) activity vs healthy controls; (ii) whether myofibroblast differentiation is involved in SSc skin fibrosis; and (iii) whether SSc fibroblasts have different proliferation rates vs controls. METHODS: Skin biopsies were taken from involved and uninvolved skin of SSc patients and controls. Whole-cell perforated patch-clamping was used to measure IClLPA activity in fibroblasts isolated and cultured from these biopsies. Western blotting was used to measure α-smooth muscle actin (α-SMA). Proliferation was measured using a colorimetric assay. RESULTS: Fibroblasts cultured from SSc skin show significantly increased IClLPA activity following LPA exposure compared with control skin fibroblasts. α-SMA protein was significantly increased in cultured SSc skin fibroblasts vs controls. No significant differences in proliferation rates were found. CONCLUSIONS: Elevated IClLPA activity is a hallmark of SSc skin fibroblasts. Blocking IClLPA activation may be a new therapeutic approach for treating SSc-associated fibrosis.

Teriparatide is safe and effectively increases bone biomarkers in institutionalized individuals with osteoporosis.

Institutionalized adults with severe developmental disabilities have a high rate of minimal trauma and appendicular fracture. There is little information about osteoporosis treatment in this population. In this efficacy and safety study, men and women with severe developmental disabilities and osteoporosis received 20 mcg teriparatide subcutaneously daily for 18-24 months. Markers of bone formation [procollagen type 1 intact N-terminal propeptide (P1NP)] and resorption [C-telopeptide (CTx)] were measured at three-month intervals. Serum calcium was measured at two-week intervals for 12 weeks and thereafter at three-month intervals. Twenty-seven individuals received at least one injection. The incidence of hypercalcemia was 11.1% but was persistent and led to medication discontinuation in only one participant. Biomarkers of bone formation increased rapidly, doubling by three months. At 12 months, P1NP and CTx remained elevated from baseline; P1NP had risen from 66.95 +/- 83.71 microg/l (mean +/- SD) to 142.42 +/- 113.85 microg/l (P = 0.05), and CTx had increased from 0.377 +/- 0.253 to 1.016 +/- 1.048 ng/ml (P = 0.01). The majority of participants had an increase in P1NP of over 10 microg/l. In conclusion, teriparatide is safe and effective in developmentally disabled institutionalized adults. Serial calcium measurements are warranted, particularly during the first three months of therapy.

Effects of Vitamin D Receptor Knockout and Vitamin D Deficiency on Corneal Epithelial Wound Healing and Nerve Density in Diabetic Mice.

Diabetic keratopathy occurs in ∼70% of all people with diabetes. This study was designed to examine the effects of vitamin D receptor knockout (VDR-/-) and vitamin D deficiency (VDD) on corneal epithelial wound healing and nerve density in diabetic mice. Diabetes was induced using the low-dose streptozotocin method. Corneal epithelial wounds were created using an Algerbrush, and wound healing was monitored over time. Corneal nerve density was measured in unwounded mice. VDR-/- and VDD diabetic mice (diabetic for 8 and 20 weeks, respectively) had slower healing ratios than wild-type diabetic mice. VDR-/- and VDD diabetic mice also showed significantly decreased nerve density. Reduced wound healing ratios and nerve densities were not fully rescued by a supplemental diet rich in calcium, lactose, and phosphate. We conclude that VDR-/- and VDD significantly reduce both corneal epithelial wound healing and nerve density in diabetic mice. Because the supplemental diet did not rescue wound healing or nerve density, these effects are likely not specifically related to hypocalcemia. This work supports the hypothesis that low vitamin D levels can exacerbate preexisting ophthalmic conditions, such as diabetes.

Transient Cell Membrane Disruptions induce Calcium Waves in Corneal Keratocytes.

The purpose of this study was to determine if transient cell membrane disruptions (TPMDs) in single keratocytes can trigger signaling events in neighboring keratocytes. Stromal cells were cultured from human corneas (HCSC) and mouse corneas (MCSC). TPMDs were produced using a multiphoton microscope in Cal-520-AM loaded cells. TPMD-induced calcium increases (Ca++i) were measured in Ca++-containing and Ca++-free solutions containing thapsigargin, ryanodine, BAPTA-AM, 18-α-glycyrrhetinic acid (18α-GA), apyrase, BCTC, AMG 9810, or AMTB. Fluorescence intensity was recorded as the number of cells responding and the area under the fluorescence versus time curve. The maximum distance of responding neighboring cells in ex vivo human corneas was measured. Connexin 43 protein in HCSC and MCSC was examined using immunofluorescence staining, and corneal rubbing was applied to confirm whether TPMDs occur following mechanical manipulation. Our results demonstrate that single cell TPMDs result in Ca++ waves in neighboring keratocytes both in culture and within ex vivo corneas. The source of Ca++ is both intra-and extra-cellular, and the signal can be mediated by ATP and/or gap junctions, and is species dependent. Stromal rubbing confirmed that TPMDs do occur following mechanical manipulation. Keratocyte TPMDs and their associated signaling events are likely common occurrences following minor or major corneal trauma.

Elevated serum levels of arachidonoyl-lysophosphatidic acid and sphingosine 1-phosphate in systemic sclerosis.

Systemic sclerosis (SSc) is an often fatal disease characterized by autoimmunity and inflammation, leading to widespread vasculopathy and fibrosis. Lysophosphatidic acid (LPA), a bioactive phospholipid in serum, is generated from lysophospholipids secreted from activated platelets in part by the action of lysophospholipase D (lysoPLD). Sphingosine 1-phosphate (S1P), a member of the bioactive lysophospholipid family, is also released from activated platelets. Because activated platelets are a hallmark of SSc, we wanted to determine whether subjects with SSc have altered serum lysophospholipid levels or lysoPLD activity. Lysophospholipid levels were measured using mass spectrometric analysis. LysoPLD activity was determined by quantifying choline released from exogenous lysophosphatidylcholine (LPC). The major results were that serum levels of arachidonoyl (20:4)-LPA and S1P were significantly higher in SSc subjects versus controls. Furthermore, serum LPA:LPC ratios of two different polyunsaturated phospholipid molecular species, and also the ratio of all species combined, were significantly higher in SSc subjects versus controls. No significant differences were found between other lysophospholipid levels or lysoPLD activities. Elevated 20:4 LPA, S1P levels and polyunsaturated LPA:LPC ratios may be markers for and/or play a significant role in the etiology of SSc and may be future pharmacological targets for SSc treatment.

Influence of Vitamin D on Corneal Epithelial Cell Desmosomes and Hemidesmosomes.

Purpose: We have observed noticably weak epithelial attachment in vitamin D receptor knockout mice (VDR KO) undergoing epithelial debridement. We hypothesized that VDR KO negatively affects corneal epithelial cell desmosomes and/or hemidesmosomes. Methods: Transcript levels of desmosome and hemidesmosome proteins in VDR KO corneas were assessed by qPCR. Western blotting and immunochemistry were used to detect proteins in cultured cells exposed to 1,25(OH)2D3 and 24R,25(OH)2D3. Results: VDR KO resulted in decreased corneal desmosomal desmoglein 1 (DSG1) and desmocollin 2 (DSC2) mRNA, and hemidesmosomal plectin mRNA. DSG1 and plectin protein expression were reduced in VDR KO corneas. DSG1 protein expression increased in VDR wild types (VDR WT) and VDR KO mouse primary epithelial cells (MPCEC) treated with 1,25(OH)2D3 and 24R,25(OH)2D3. 24R,25(OH)2D3 treatment resulted in increased plectin and integrin ß4 levels in VDR WT MPCEC, and decreased levels in VDR KO MPCEC. Treatment of human corneal epithelial cells (HCEC) with 1,25(OH)2D3 and 24R,25(OH)2D3 resulted in increased DSC2 and DSG1 protein expression. Plectin and integrin ß4 were only increased in 24R,25(OH)2D3 treated HCEC. Conclusions: VDR KO results in reduced desmosomal and hemidesmosomal mRNA and protein levels. 1,25(OH)2D3 and 24R,25(OH)2D3 increased DSG1 protein in all cells tested. For hemidesmosome proteins, 24R,25(OH)2D3 increased plectin and integrin ß4 protein expression in VDR WT and HCEC, with decreased expression in VDR KO MPCEC. Thus, vitamin D3 is involved in desmosome and hemidesmosome junction formation/regulation, and their decreased expression likely contributes to the loosely adherent corneal epithelium in VDR KO mice. Our data indicate the presence of a VDR-independent pathway.

PPIP5K2 and PCSK1 are Candidate Genetic Contributors to Familial Keratoconus.

Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future.

PEG-stabilized carbodiimide crosslinked collagen-chitosan hydrogels for corneal tissue engineering.

Implantable biomaterials that mimic the extracellular matrix (ECM) in key physical and physiological functions require components and microarchitectures that are carefully designed to maintain the correct balance between biofunctional and physical properties. Our goal was to develop hybrid polymer networks (HPN) that combine the bioactive features of natural materials and physical characteristics of synthetic ones to achieve synergy between the desirable mechanical properties of some components with the biological compatibility and physiological relevance of others. In this study, we developed collagen-chitosan composite hydrogels as corneal implants stabilized by either a simple carbodiimide cross-linker or a hybrid cross-linking system comprised of a long-range bi-functional cross-linker (e.g. poly(ethylene glycol) dibutyraldehyde (PEG-DBA)), and short-range amide-type cross-linkers (e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS)). Optimum hybrid hydrogel demonstrated significantly enhanced mechanical strength and elasticity by 100 and 20%, respectively, compared to its non-hybrid counterpart. It demonstrated excellent optical properties, optimum mechanical properties and suturability, and good permeability to glucose and albumin. It had excellent biocompatibility and when implanted into pig corneas for 12 months, allowed seamless host-graft integration with successful regeneration of host corneal epithelium, stroma, and nerves.

Recombinant human collagen for tissue engineered corneal substitutes.

We successfully fabricated transparent, robust hydrogels as corneal substitutes from concentrated recombinant human type I and type III collagen solutions crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). White light transmission through these gels is comparable or superior to that of human corneas. Hydrogels from both type I and type III collagens supported in vitro epithelium and nerve over-growth. While both these biocompatible hydrogels have adequate tensile strength and elasticity for surgical manipulation, type III collagen hydrogels tended to be mechanically superior. Twelve-month post-implantation results of type I recombinant collagen-based corneal substitutes into mini-pigs showed retention of optical clarity, along with regeneration of corneal cells, nerves and tear film. For clinical use, implants based on fully characterized, recombinant human collagen eliminate the risk of pathogen transfer or xenogeneic immuno-responses posed by animal collagens.