Pregnancy Outcomes in Women with Arrhythmias following Surgical Repair of Cardiac Defects.

OBJECTIVE: The goal of this study was to investigate whether preexisting cardiac arrhythmias are associated with adverse obstetrical outcomes in women with a history of open cardiac surgery. STUDY DESIGN: This was a retrospective cohort study of women with a history of open cardiac surgery who delivered at MedStar Washington Hospital Center (Washington, DC) from January 2007 through December 2018. Women with the isolated percutaneous cardiac surgical repair were excluded. Maternal and neonatal outcomes were compared between patients with preexisting cardiac arrhythmias and patients without preexisting cardiac arrhythmias. Maternal outcomes studied were intensive care unit admission, postpartum blood loss greater than 1,000 mL, congestive heart failure development, preeclampsia with severe features, postpartum readmission, postpartum cardiac events, and postpartum length of stay >5 days. Neonatal outcomes investigated were low birth weight <2,500 g, Apgar's scores <7 at 5 minutes, and neonatal intensive care unit admission. Multivariate logistic regression model was used to calculate the adjusted odds ratio (aOR) and 95% confidence intervals. RESULTS: The outcomes for 69 deliveries from 56 women with a history of open cardiac surgery were examined. Thirty-three women (48%) had arrhythmias after cardiac surgery with fourteen (20%) requiring implantable cardioverted defibrillators. Two women (6%) with preexisting arrhythmias after cardiac surgery developed postpartum volume overload requiring readmission (p = 0.06). After controlling for age, gestational age at delivery, and BMI, preeclampsia with severe features (p = 0.02) and low birth weight neonates (p = 0.02, aOR = 2.26 [0.56-9.03]) remained statistically more like to occur in patients with preexisting cardiac arrhythmias than in patients without preexisting arrhythmias. CONCLUSION: Women with a history of open cardiac surgery and preexisting cardiac arrhythmias prior to pregnancy are more likely to develop preeclampsia with severe features and have low birth weight neonates compared with women with a history of open cardiac surgery without preexisting cardiac arrhythmias. KEY POINTS: · Preexisting arrhythmias after cardiac surgery was associated with a risk of preeclampsia.. · Neonates of women with preexisting cardiac arrhythmias are more likely to be low birth weight.. · Forty-seven percent of women with open cardiac surgery developed subsequent arrhythmias..

Aquaporins 8 and 9 as Possible Markers for Adult Murine Lacrimal Gland Cells.

Aquaporins (AQPs) are proteins that selectively transport water across the cell membrane. Although AQPs play important roles in secretion in the lacrimal gland, the expression and localization of AQPs have not been clarified yet. In the current study, we investigated the expression pattern of AQP family members in the murine lacrimal gland during development. Lacrimal gland tissues were harvested from E13.5 and E17.5 murine embryos and from mice 8 weeks of age (adults). Corneal and conjunctival tissues from the latter served as controls. Total RNA was isolated and analyzed for the expression of AQP family members using qPCR. The localization of AQPs in the adult lacrimal gland in adult murine lacrimal glands was also analyzed. Expression of Aqp8 and Aqp9 mRNAs was detected in the adult lacrimal gland but not in the cornea, conjunctiva, or fetal lacrimal gland. AQP8 and AQP9 and α-SMA partially colocalized around the basal regions of the acinar unit. The levels of Aqp3 mRNAs and protein were much lower in the adult lacrimal gland but were readily detected in the adult cornea and conjunctiva. Our study suggests that AQP8 and AQP9 may serve as markers for adult murine lacrimal gland, ductal, and myoepithelial cells.

Two case reports of continued progression of chronic ocular graft-versus-host disease without concurrent systemic comorbidities treated by amniotic membrane transplantation.

BACKGROUND: Chronic ocular graft-versus-host disease (oGVHD) is an ocular comorbidity of graft-versus-host disease (GVHD) that usually occurs concurrently with systemic manifestations. Failure to detect and treat oGVHD in its early stages may lead to progression of ocular signs and symptoms leading to oGVHD that is refractory to conventional treatment. CASE PRESENTATION: We report the clinical course of a 19-year-old male and a 59-year-old female with severe and progressive chronic oGVHD without concurrent systemic signs of chronic graft-versus-host disease (cGVHD). Although their systemic conditions had been stable, both suffered from severe oGVHD and were referred to our clinic. Both cases exhibited marked improvement in conjunctival inflammation and fibrotic changes after amniotic membrane transplantation (AMT). Both cases underwent keratoplasty eventually to stabilize ocular surface conditions and to improve visual function. CONCLUSIONS: We reported the clinical outcomes of 2 cases of chronic oGVHD without concurrent systemic comorbidities that were treated with AMT. The clinician should be aware that cGVHD may persist in target organs even in the absence of concurrent systemic comorbidities following seemingly successful systemic treatment. A multidisciplinary team approach is essential in the early detection and therapeutic intervention for chronic oGVHD.

The Effect of Rebamipide Ophthalmic Solution on Cytokine and Mucin Secretion in Culture of Conjunctival Epithelial Cells From the Cu, Zn-Superoxide Dismutase-1 (SOD-1) Knock-Down Mouse.

OBJECTIVES: To evaluate the in vitro effects of 1-mM rebamipide ophthalmic solution on the expression of inflammatory cytokines and MUC5AC in Cu, Zn-superoxide dismutase-1 (SOD-1) knock-down conjunctival epithelium. METHODS: Conjunctival epithelium from C57BL/6 wild-type mice was cultured and treated with rebamipide ophthalmic solution. Using cytometric bead array, we examined the levels of interleukin-(IL)-6, IL-10, IL-17, monocyte chemoattractant protein-1, interferon-γ (INF-γ), tumor necrosis factor, and IL-12p70 in the culture supernatants. The culture supernatants were obtained from the culture medium of nontreated or SOD-1 knock-down conjunctival epithelium using small interfering RNA (siRNA). In addition, ELISA was performed to ascertain the MUC5AC concentration in the culture medium. RESULTS: After rebamipide ophthalmic solution was applied, IL-6 concentration in the supernatants of conjunctival epithelial cells treated with and without siRNA showed a significant timewise decrease from 0 to 24 hr (963±42 to 0.07±0.05 pg/mL and 932±168 to 2.2±0.05 pg/mL, respectively) (P<0.001). Compared with baseline values, MUC5AC concentrations significantly increased 24 hr after rebamipide application to the conjunctival cultures-both with and without SOD-1 siRNA treatment (P<0.05 in both cases). CONCLUSIONS: Rebamipide seems to increase MUC5AC levels and suppress inflammation by decreasing IL-6 levels in mouse conjunctival epithelial cell cultures. SOD-1 siRNA-treated mouse conjunctival epithelial cell culture is a practical method for investigating changes in mucosa-associated mucins and proinflammatory cytokines in response to therapeutic interventions.

Surgical site infections after cesarean delivery: epidemiology, prevention and treatment.

Cesarean delivery (CD) is one of the most common procedures performed in the United States, accounting for 32% of all deliveries. Postpartum surgical site infection (SSI), wound infection and endometritis is a major cause of prolonged hospital stay and poses a burden to the health care system. SSIs complicate a significant number of patients who undergo CD - 2-7% will experience sound infections and 2-16% will develop endometritis. Many risk factors for SSI have been described. These include maternal factors (such as tobacco use; limited prenatal care; obesity; corticosteroid use; nulliparity; twin gestations; and previous CD), intrapartum and operative factors (such as chorioamnionitis; premature rupture of membranes; prolonged rupture of membranes; prolonged labor, particularly prolonged second stage; large incision length; subcutaneous tissue thickness > 3 cm; subcutaneous hematoma; lack of antibiotic prophylaxis; emergency delivery; and excessive blood loss), and obstetrical care on the teaching service of an academic institution. Effective interventions to decrease surgical site infection include prophylactic antibiotic use (preoperative first generation cephalosporin and intravenous azithromycin), chlorhexidine skin preparation instead of iodine, hair removal using clippers instead of razors, vaginal cleansing by povidone-iodine, placental removal by traction of the umbilical cord instead of by manual removal, suture closure of subcutaneous tissue if the wound thickness is >2 cm, and skin closure with sutures instead of with staples. Implementation of surgical bundles in non-obstetric patients has been promising., Creating a similar patient care bundle comprised evidence-based elements in patients who undergo CD may decrease the incidence of this major complication. Each hospital has the opportunity to create its own CD surgical bundle to decrease surgical site infection.

Identification of transcription factors that promote the differentiation of human pluripotent stem cells into lacrimal gland epithelium-like cells.

Dry eye disease is the most prevalent pathological condition in aging eyes. One potential therapeutic strategy is the transplantation of lacrimal glands, generated in vitro from pluripotent stem cells such as human embryonic stem cells, into patients. One of the preceding requirements is a method to differentiate human embryonic stem cells into lacrimal gland epithelium cells. As the first step for this approach, this study aims to identify a set of transcription factors whose overexpression can promote the differentiation of human embryonic stem cells into lacrimal gland epithelium-like cells. We performed microarray analyses of lacrimal glands and lacrimal glands-related organs obtained from mouse embryos and adults, and identified transcription factors enriched in lacrimal gland epithelium cells. We then transfected synthetic messenger RNAs encoding human orthologues of these transcription factors into human embryonic stem cells and examined whether the human embryonic stem cells differentiate into lacrimal gland epithelium-like cells by assessing cell morphology and marker gene expression. The microarray analysis of lacrimal glands tissues identified 16 transcription factors that were enriched in lacrimal gland epithelium cells. We focused on three of the transcription factors, because they are expressed in other glands such as salivary glands and are also known to be involved in the development of lacrimal glands. We tested the overexpression of various combinations of the three transcription factors and PAX6, which is an indispensable gene for lacrimal glands development, in human embryonic stem cells. Combining PAX6, SIX1, and FOXC1 caused significant changes in morphology, i.e., elongated cell shape and increased expression (both RNAs and proteins) of epithelial markers such as cytokeratin15, branching morphogenesis markers such as BARX2, and lacrimal glands markers such as aquaporin5 and lactoferrin. We identified a set of transcription factors enriched in lacrimal gland epithelium cells and demonstrated that the simultaneous overexpression of these transcription factors can differentiate human embryonic stem cells into lacrimal gland epithelium-like cells. This study suggests the possibility of lacrimal glands regeneration from human pluripotent stem cells.

Evaluation of treatment for dry eye with 2-hydroxyestradiol using a dry eye rat model.

PURPOSE: 2-hydroxy estradiol (2-OHE2) is a catechol derivative of 17ß -Estradiol (E2) and it is synthesized from E2 catalyzed by cytochrome P4501A1. Previous studies reported that 2-OHE2 is a physiologic antioxidant in lipoproteins, liver microsomes, and the brain. Catechol derivatives show an anti-inflammatory effect through the inhibition of prostaglandin endoperoxide synthase (PGS) activity. Corneal erosion caused by dry eye is related to an increase in oxidative stress and inflammation in ocular surface cells. We investigated the therapeutic effects of 2-OHE2 on corneal damage caused by dry eye. METHODS: Steroidal radical scavenging activity was confirmed through the electron spin resonance (ESR) method. PGS activity was measured using the COX Fluorescent Activity Assay Kit. To evaluate the effect of 2-OHE2 on the treatment for dry eye, 2-OHE2 was applied as an eye drop experiment using dry eye model rats. RESULTS: 2-OHE2 scavenged tyrosyl radical and possibly suppressed oxidative stress in corneal epithelial cells. In addition, 2-OHE2 inhibited PGS activity, and 2-OHE2 is probably a competitive inhibitor of PGS. Corneal PGS activity was upregulated in the dry eye group. Therefore, 2-OHE2 eye drops improved corneal erosion in dry eye model rats. CONCLUSIONS: 2-OHE2 is a candidate for the treatment of dry eye through the suppression of inflammation and oxidative stress in the cornea.

Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells.

PURPOSE: Human and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs). METHODS: SGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis. RESULTS: SG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype. CONCLUSIONS: Epithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium.

Tissue Renin-Angiotensin System in Lacrimal Gland Fibrosis in a Murine Model of Chronic Graft-Versus-Host Disease.

Chronic graft-versus-host disease (cGVHD) is a serious complication known to occur after allogeneic hematopoietic stem cell transplantation. Clinical manifestation includes inflammation and fibrosis. Many peripheral tissues are capable of generating the renin-angiotensin system (RAS) components, called the tissue RAS, and have various roles in tissue-specific physiological and pathological functions of inflammation and fibrosis. This article reviews evidence for the presence of the tissue RAS in the normal mouse lacrimal gland, the role of the tissue RAS in the fibrotic pathogenesis of the lacrimal gland in cGVHD model mice, and the effect of angiotensin II receptor blockers on preventing lacrimal gland fibrosis. B10.D2→BALB/c (H-2d) major histocompatibility complex-compatible, minor histocompatibility antigen-mismatched mice were used as a model of cGVHD, which reflects the clinical and pathological symptoms of human cGVHD. We also describe the localization of RAS components in the normal mouse lacrimal gland. In addition, we characterize the inflammatory and fibrotic changes of the lacrimal gland in cGVHD model mice, demonstrate that fibroblasts strongly express angiotensin II, angiotensin II type 1 receptor (AT1R), and angiotensin II type 2 receptor, and show that mRNA expression of angiotensinogen increased in the lacrimal gland of cGVHD model mice. Inhibitory experiments revealed that lacrimal gland fibrosis was suppressed in mice treated with an AT1R blocker, but not in mice treated with an angiotensin II type 2 receptor blocker. Hence, we conclude that the tissue RAS is involved in the fibrotic pathogenesis of the lacrimal gland and that AT1R blockers have a therapeutic effect on lacrimal gland fibrosis in cGVHD model mice.

Long-term rebamipide and diquafosol in two cases of immune-mediated dry eye.

PURPOSE: Two new drugs with mucin-inducing and secretion-promotive effects, rebamipide and diquafosol, were recently approved as topical dry-eye treatments. We report two cases in which the long-term use of mucin-inducing eye drops improved chronic ocular graft-versus-host disease (cGVHD)-related dry eye and ocular cicatricial pemphigoid (OCP)-like disease. CASE REPORTS: Case 1. A 61-year-old woman had cGVHD-related dry eye that resisted traditional medications. Next, we use topical diquafosol in addition to conventional treatments. The patient used diquafosol for 6 months without experiencing any side effects. The symptoms, including dry-eye sensation, ocular pain, foreign body sensation, and photophobia, as well as ocular surface findings including fluorescein and rose bengal scores and tear break-up time (TBUT), partly improved. To further improve the clinical signs and symptoms and decrease chronic inflammation, rebamipide was added to diquafosol. The symptoms, TBUT, and fluorescein and rose bengal scores markedly improved after long-term dual treatment without any side effects for 6 months. Case 2. A 77-year-old woman had OCP-like disease with dry eye. The patient did not improve using the currently available conventional treatments. Next, we use topical rebamipide in addition to conventional treatments. Symptoms including asthenopia, dry-eye sensation, ocular pain, and dull sensation, as well as fluorescein and rose bengal scores and TBUT, partly improved. Specifically, functional visual acuity was markedly improved after commencement of rebamipide. To further improve the clinical signs and symptoms and increase tear film stability and tear film volume, diquafosol was added to rebamipide. The combination of diquafosol and rebamipide worked for the patient. Improvements were seen in several symptoms, fluorescein and rose bengal scores, Schirmer test value, and TBUT without any side effects for 12 months. CONCLUSIONS: Long-term treatment with topical rebamipide and diquafosol can improve dry eye in patients with cGVHD or OCP-like disease.

Clinical backgrounds of postoperative keratoplasty patients with spontaneous wound dehiscence or gaps after suture removal.

PURPOSE: To report that spontaneous postkeratoplasty wound dehiscence or gaps caused by suture removal may occur years after penetrating and deep lamellar keratoplasty. METHODS: We retrospectively reviewed the medical records of 667 keratoplasty patients (890 eyes). This study included 8 eyes of 8 patients (3 men and 5 women) aged 64 to 86 years who suffered from spontaneous wound dehiscence or gaps caused by suture removal. We examined their clinical backgrounds such as surgical procedure, reason for suture removal, and time interval between keratoplasty and suture removal. RESULTS: The surgical procedures included 6 penetrating keratoplasties and 2 deep anterior lamellar keratoplasties. The most common reason for suture removal was high corneal astigmatism. Time interval between keratoplasty and suture removal was 15.9 ± 7.7 months (7-26 months). Three patients underwent suture removal from 6 months to 1 year after keratoplasty, 3 patients from 1 to 2 years, and 2 patients after more than 2 years. CONCLUSIONS: Whenever postoperative keratoplasty suture is removed, it is important to consider that wound dehiscence or gaps may occur without incidence of trauma.

Ocular surface reconstruction by cultivated epithelial sheet transplantation.

Recent advances in ocular surface reconstruction for patients with severe ocular surface diseases have significantly improved the prognosis of patients with vision-impairing corneal abnormalities. The history of cultivated epithelial sheet transplantation is short, and debate on the current approaches for these procedures is continuing. Limbal stem cell transplantation, including conjunctivolimbal autograft and keratolimbal allograft, has brought opportunities for vision improvement. In addition, the use of cultivated limbal epithelial transplantation from both allogeneic and autologous sources has provided further options for immediate postoperative epithelialization of the corneal surface. Finally, cultivated oral mucosal epithelial transplantation, which allows autologous transplantation for patients with bilateral limbal stem cell deficiency, has provided the best overall midterm and long-term results. Its biggest advantages are the absence of rejection reactions and the reduction of postoperative complications associated with steroid therapy. However, a solitary surgical approach is not sufficient for obtaining a good clinical outcome. To maximize the possibility of success using these procedures, it is important to preoperatively enhance aggressive treatment of the ocular surface, especially with factors that facilitate moisture retention. In this review article, we also discuss our clinical experience in relation to these surgical procedures.

Effects of dietary supplementation with fish oil on dry eye syndrome subjects: randomized controlled trial.

The purpose of this study was to evaluate the efficacy of fish oil supplementation added to usual dry eye treatment in dry eye subjects in a randomized controlled trial. Twenty-seven typical dry eye subjects were selected from 43 candidates by the diagnostic criterion for dry eye in this study. They were assigned to the randomized fish oil group (n = 15) or the placebo group (n = 12). Fish oil group ingested fish oil capsules containing eicosapentaenoic acid (EPA, 1245 mg/day) and docosahexaenoic acid (DHA, 540 mg/day) for 12 weeks. Placebo group ingested placebo capsules without EPA or DHA. A visual analog scale test estimating subjective symptoms, the Schirmer I test, tear film break-up time (BUT) measurement, fluorescein staining, and rose bengal staining were performed every 4 weeks during the 12-week supplementation period and 4-week washout period. The subjective symptom of "eye pain", BUT, and changes in rose bengal staining score of the fish oil group were significantly improved after 8-12 weeks of supplementation and/or 4 weeks of washout, compared to those of the placebo group. These results suggest that fish oil supplementation added to usual care may be effective in the treatment of dry eye.

Functional lacrimal gland regeneration by transplantation of a bioengineered organ germ.

The lacrimal gland has a multifaceted role in maintaining a homeostatic microenvironment for a healthy ocular surface via tear secretion. Dry-eye disease, which is caused by lacrimal gland dysfunction, is one of the most prevalent eye diseases that cause corneal epithelial damage and results in significant loss of vision and a reduction in the quality of life. Here we demonstrate orthotopic transplantation of bioengineered lacrimal gland germs into adult mice with an extra-orbital lacrimal gland defect, a mouse model that mimics the corneal epithelial damage caused by lacrimal gland dysfunction. The bioengineered lacrimal gland germs and harderian gland germs both develop in vivo and achieve sufficient physiological functionality, including tear production in response to nervous stimulation and ocular surface protection. This study demonstrates the potential for bioengineered organ replacement to functionally restore the lacrimal gland.

Long-term maintenance of limbal epithelial progenitor cells using rho kinase inhibitor and keratinocyte growth factor.

Corneal epithelial stem cells are located in the limbus, the junction between the cornea and the conjunctiva. A limbal epithelium model in vitro would be useful for the study of epithelial stem cells, as well as improving the quality of cultivated epithelial sheets for the treatment of limbal stem cell deficiency. In this study, we succeeded in constructing a limbal epithelium-like structure that could be maintained for at least 5 months in vitro. We modified conventional medium by replacing epidermal growth factor with keratinocyte growth factor (KGF) and adding Y-27632, a rho kinase inhibitor. Using this medium, epithelial cells freshly isolated from human limbus were cocultured with human mesenchymal stem cell-derived feeder cells. Cells formed a stratified layer without air exposure, and both basal and suprabasal layers maintained their unique morphologies for up to 5 months. Basal layers expressed the progenitor marker p63 uniformly and K15 heterogeneously. Expressions of PAX6, K3, and K12 indicated that cell sheets underwent normal differentiation in the corneal epithelium lineage. Although medium was changed daily after day 7, cell debris was observed every day, suggesting that cell sheets underwent turnover. Furthermore, secondary colonies were observed from cells dissociated from 1-month and 3-month cultured sheets. In conclusion, human limbal epithelial cell sheet cultures with KGF and Y-27632 maintained stratification, high expression of both stem/progenitor markers and differentiation markers, and colony-forming cells long-term. This protocol may be useful as an in vitro limbal epithelial model for basic studies.

Angiotensin II type 1 receptor antagonist attenuates lacrimal gland, lung, and liver fibrosis in a murine model of chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2(d)) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.

Calorie restriction (CR) and CR mimetics for the prevention and treatment of age-related eye disorders.

The morbidity of ocular diseases, including macular degeneration, diabetic retinopathy, and dry eye disease, has been gradually increasing worldwide. Because these diseases develop from age-associated ocular dysfunctions, interventions against the aging process itself may be a promising strategy for their management. Among the several approaches to interrupt aging processes, calorie restriction (CR) has been shown to recover and/or slow age-related functional declines in various organs, including the eye. Here, we review interventions against the aging process as potential therapeutic approaches to age-related ocular diseases. The effects of CR and CR mimetics in animal models of age-related eye diseases are explored. Furthermore, we discuss the possibilities of expanding this research to prospective studies to elucidate the molecular mechanisms by which CR and/or CR mimetics preserve ocular functions.

Identification of keratocyte-like cells differentiated from circulating bone marrow-derived cells in the mouse cornea.

Bone marrow (BM)-derived stem cells have the potential to differentiate into multiple lineages of tissue resident cells. BM-derived cells have been detected in the mouse cornea, but most of these cells were found to be CD45(+) or CD11b(+) immunocompetent cells. Although some BM-derived cells in the cornea were negative for these cell surface markers, it has remained unclear whether cells of BM origin can differentiate into corneal resident cells. To address this issue, we subjected wild-type mice that had been exposed to a lethal dose of radiation to intravenous injection with BM cells from green fluorescent protein (GFP) transgenic mice. Two months after cell transplantation, fluorescence microscopy revealed the presence of numerous GFP(+) cells throughout the cornea, with intense GFP fluorescence being apparent around the limbal region. Immunohistofluorescence analysis of corneal cross-sections revealed that most of the BM-derived GFP(+) cells expressed CD45 or CD11b, although a few GFP(+) cells were negative for these markers. Immunostaining of individual cells isolated from the corneal stroma, however, showed that a small proportion (~1 %) of GFP(+) BM-derived cells expressed the keratocyte-specific proteoglycan keratocan. Our results suggest that BM-derived cells introduced intravenously are able to differentiate into resident cells of the corneal stroma.

Activation of Smad-mediated TGF-ß signaling triggers epithelial-mesenchymal transitions in murine cloned corneal progenitor cells.

Epithelial-mesenchymal transition (EMT), via activation of Wnt signaling, is prevailing in embryogenesis, but postnatally it only occurs in pathological processes, such as in tissue fibrosis and tumor metastasis. Our prior studies led us to speculate that EMT might be involved in the loss of limbal epithelial stem cells in explant cultures. To examine this hypothesis, we successfully grew murine corneal/limbal epithelial progenitors by prolonging the culture time and by seeding at a low density in a serum-free medium. Single cell-derived clonal growth was accompanied by a gradient of Wnt signaling activity, from the center to the periphery, marked by a centrifugal loss of E-cadherin and ß-catenin from intercellular junctions, coupled with nuclear translocation of ß-catenin and LEF-1. Large-colony-forming efficiency at central location of colony was higher than peripheral location. Importantly, there was also progressive centrifugal differentiation, with positive K14 keratin expression and the loss of p63 and PCNA nuclear staining, and irreversible EMT, evidenced by cytoplasmic expression of α-SMA and nuclear localization of S100A4; and by nuclear translocation of Smad4. Furthermore, cytoplasmic expression of α-SMA was promoted by high-density cultures and their conditioned media, which contained cell density-dependent levels of TGF-ß1, TGF-ß2, GM-CSF, and IL-1α. Exogenous TGF-ß1 induced α-SMA positive cells in a low-density culture, while TGF-ß1 neutralizing antibody partially inhibited α-SMA expression in a high-density culture. Collectively, these results indicate that irreversible EMT emerges in the periphery of clonal expansion where differentiation and senescence of murine corneal/limbal epithelial progenitors occurs as a result of Smad-mediated TGF-ß-signaling.

The semaphorin 3A inhibitor SM-345431 accelerates peripheral nerve regeneration and sensitivity in a murine corneal transplantation model.

BACKGROUND: Peripheral nerve damage of the cornea is a complication following surgery or infection which may lead to decreased visual function. We examined the efficacy of the semaphorin 3A inhibitor, SM-345431, in promoting regeneration of peripheral nerves in a mouse corneal transplantation model. METHODOLOGY/PRINCIPAL FINDINGS: P0-Cre/Floxed-EGFP mice which express EGFP in peripheral nerves cells were used as recipients of corneal transplantation with syngeneic wild-type mouse cornea donors. SM-345431 was administered subconjunctivally every 2 days while control mice received vehicle only. Mice were followed for 3 weeks and the length of regenerating nerves was measured by EGFP fluorescence and immunohistochemistry against ßIII tubulin. Cornea sensitivity was also measured by the Cochet-Bonnet esthesiometer. CD31 staining was used to determine corneal neovascularization as a possible side effect of SM-345431. Regeneration of ßIII tubulin positive peripheral nerves was significantly higher in SM-345431 treated mice compared to control. Furthermore, corneal sensitivity significantly improved in the SM-345431 group by 3 weeks after transplantation. Neovascularization was limited to the peripheral cornea with no difference between SM-345431 group and control. CONCLUSIONS/SIGNIFICANCE: Subconjunctival injections of SM-345431 promoted a robust network of regenerating nerves as well as functional recovery of corneal sensation in a mouse keratoplasty model, suggesting a novel therapeutic strategy for treating neurotrophic corneal disease.