The Use of Bacteriophages and Immunological Monitoring for the Treatment of a Case of Chronic Septicemic Cutaneous Ulcerative Disease in a Loggerhead Sea Turtle Caretta caretta.

In this case study, phage therapy was applied to treat a multidrug-resistant case of septicemic cutaneous ulcerative disease (SCUD) caused by Citrobacter freundii in a loggerhead sea turtle Caretta caretta. Phages were applied topically, intravenously, into the carapace, and into the exhibit water using various phage cocktails specific to the causative agent over an 8-month period. This was performed in conjunction with antimicrobial therapy. The animal was monitored through weekly cultures, photographs, and complete blood cell counts, as well as immune assays (phagocytosis, plasma lysozyme and superoxide dismutase activity, and plasma electrophoresis profiles). The animal, in comparison to an untreated, unaffected control, had elevated antibody titers to the administered phages, which persisted for at least 35 weeks. Although cultures were clear of C. freundii after phage treatment, the infection did return over time and immune assays confirmed deficiencies when compared to a healthy loggerhead sea turtle. Immune parameters with statistically significant changes over the study period included the following: decreased phagocytosis, increased alpha- and gamma-globulin protein components, and an increased albumin : globulin ratio. When C. freundii appeared again, the multidrug-resistant status had reverted back to normal susceptibility patterns. Although not completely known whether it was another subspecies of bacteria, the therapy did resolve the multidrug-resistant challenge. Phage therapy in combination with antimicrobial agents may be an effective treatment for sea turtles with normally functioning immune systems or less-severe infections. Additional research is needed to better understand and quantify sea turtle immunology.

PRESENTATION AND MANAGEMENT OF DENTAL PAD FRACTURES IN TWO FLORIDA MANATEES (TRICHECHUS MANATUS LATIROSTRIS).

The Florida manatee (Trichechus manatus latirostris) has well-developed keratinized dental pads at the most rostral aspect of their mouth to assist with mastication. This unique development is thought to be an adaptive response to their highly abrasive diets that contain phytoliths and sediments that may accelerate dental wear. In May 2013, two Florida manatees presented with multiple fractures in their inferior dental pads. The fractures were successfully managed with nutritional modifications, dental pad trimming, and vigilant monitoring through behavioral husbandry training. Signs of spontaneous healing were observed as early as 60 days after initial presentation with subsequent full resolution. Although surgical intervention was planned, the spontaneous healing mitigated significant health risks associated with the procedure. To the authors' knowledge, these are the first reported cases of dental pad fractures and their spontaneous healing and resolution in manatees.

SURVEY OF GERIATRIC ELEPHANT MEDICAL CARE, NUTRITION, HUSBANDRY, AND WELFARE.

Improvements in husbandry, veterinary care, and nutrition have led to increased longevity of animals in human care, including elephants. The goal of this study was to collect and synthesize information pertaining to geriatric elephant medicine, management, husbandry, and nutrition. An electronic survey was created and distributed to American Association of Zoo Veterinarians members through an online link. A total of 61 responses were received from veterinarians, nutritionists, and elephant managers with data encompassing 314 elephants, of which 142 were geriatric (over 40 years old) and 51 were on their final set of molars. Following the initial survey, willing respondents were contacted for follow-up interviews. Osteoarthritis, foot disease, and colic were the most commonly reported diseases, and flunixin meglumine and phenylbutazone were the analgesics most often used. Respondents described diseases treated, husbandry changes specific for older animals, welfare assessments and quality of life monitoring, nutritional modifications for dental attrition, a variety of integrative medicine modalities, and unique cases. It is the hope that the information identified in this study can be used to improve treatment, management practices, and overall welfare for geriatric elephants.

Utility of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium for an In Vitro Model of Proliferative Vitreoretinopathy.

The advent of stem cell technology, including the technology to induce pluripotency in somatic cells, and direct differentiation of stem cells into specific somatic cell types, has created an exciting new field of scientific research. Much of the work with pluripotent stem (PS) cells has been focused on the exploration and exploitation of their potential as cells/tissue replacement therapies for personalized medicine. However, PS and stem cell-derived somatic cells are also proving to be valuable tools to study disease pathology and tissue-specific responses to injury. High-throughput drug screening assays using tissue-specific injury models have the potential to identify specific and effective treatments that will promote wound healing. Retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE) are well characterized cells that exhibit the phenotype and functions of in vivo RPE. In addition to their role as a source of cells to replace damaged or diseased RPE, iPS-RPE provide a robust platform for in vitro drug screening to identify novel therapeutics to promote healing and repair of ocular tissues after injury. Proliferative vitreoretinopathy (PVR) is an abnormal wound healing process that occurs after retinal tears or detachments. In this chapter, the role of iPS-RPE in the development of an in vitro model of PVR is described. Comprehensive analyses of the iPS-RPE response to injury suggests that these cells provide a physiologically relevant tool to investigate the cellular mechanisms of the three phases of PVR pathology: migration, proliferation, and contraction. This in vitro model will provide valuable information regarding cellular wound healing responses specific to RPE and enable the identification of effective therapeutics.

HEMATOLOGIC AND BIOCHEMICAL SUMMARY STATISTICS IN AQUARIUM-HOUSED SPOTTED EAGLE RAYS ( AETOBATUS NARINARI).

During this retrospective study, 18 plasma blood chemistry and 17 complete blood count (CBC) samples were analyzed from clinically healthy spotted eagle rays ( Aetobatus narinari) at Georgia Aquarium in order to generate hematological ranges for complete blood count (CBC) and biochemical profiles. Summary statistics were generated according to the American Society for Veterinary Clinical Pathology guidelines for the determination of reference intervals in veterinary species. 4 The mean packed cell volume (PCV) was 28.09% with a range of 23-35%. Mean total solids were 5.72 g/dl with a range of 5-7.0 g/dl. Lymphocytes were the dominant leukocyte observed on differential (67.35%), followed by fine eosinophilic granulocytes (FEGs) (15.41%), coarse eosinophilic granulocytes (CEGs) (10.24%), monocytes (1.88%), and basophils (1.24%). Chemistry samples were analyzed at two diagnostic laboratories, Michigan State University (MSU) and University of Miami (UMiami), and the results were compared. Both labs have the capacity to run blood chemistries on zoo and aquatic species, but utilize different methods to obtain chemistry analyte values. UMiami uses a thin-film dry-slide technology, whereas MSU uses an ion-selective electrode (ISE) and Beckman Coulter AU 640 analyzer. There is poor agreement between the analyzers used by the two laboratories for both alkaline phosphatase and BUN, because of proportional error. Establishing hematological ranges in spotted eagle rays and in elasmobranchs in general may enhance the understanding of the species and their health. This information may aid clinicians in deciding when and how to treat elasmobranchs.

Pathophysiology of blast-induced ocular trauma in rats after repeated exposure to low-level blast overpressure.

BACKGROUND: The incidence of blast-induced ocular injury has dramatically increased due to advances in weaponry and military tactics. A single exposure to blast overpressure (BOP) has been shown to cause damage to the eye in animal models; however, on the battlefield, military personnel are exposed to BOP multiple times. The effects of repeated exposures to BOP on ocular tissues have not been investigated. The purpose of this study is to characterize the effects of single or repeated exposure on ocular tissues. METHODS: A compressed air shock tube was used to deliver 70 ± 7 KPa BOP to rats, once (single blast overpressure [SBOP]) or once daily for 5 days (repeated blast overpressure [RBOP]). Immunohistochemistry was performed to characterize the pathophysiology of ocular injuries induced by SBOP and RBOP. Apoptosis was determined by quantification activated caspase 3. Gliosis was examined by detection of glial fibrillary acidic protein (GFAP). Inflammation was examined by detection of CD68. RESULTS: Activated caspase 3 was detected in ocular tissues from all animals subjected to BOP, while those exposed to RBOP had more activated caspase 3 in the optic nerve than those exposed to SBOP. GFAP was detected in the retinas from all animals subjected to BOP. CD68 was detected in optic nerves from all animals exposed to BOP. CONCLUSION: SBOP and RBOP induced retinal damage. RBOP caused more apoptosis in the optic nerve than SBOP, suggesting that RBOP causes more severe optic neuropathy than SBOP. SBOP and RBOP caused gliosis in the retina and increased inflammation in the optic nerve.

The ubiquitin/proteasome system mediates entry and endosomal trafficking of Kaposi's sarcoma-associated herpesvirus in endothelial cells.

Ubiquitination, a post-translational modification, mediates diverse cellular functions including endocytic transport of molecules. Kaposi's sarcoma-associated herpesvirus (KSHV), an enveloped herpesvirus, enters endothelial cells primarily through clathrin-mediated endocytosis. Whether ubiquitination and proteasome activity regulates KSHV entry and endocytosis remains unknown. We showed that inhibition of proteasome activity reduced KSHV entry into endothelial cells and intracellular trafficking to nuclei, thus preventing KSHV infection of the cells. Three-dimensional (3-D) analyses revealed accumulation of KSHV particles in a cytoplasmic compartment identified as EEA1+ endosomal vesicles upon proteasome inhibition. KSHV particles are colocalized with ubiquitin-binding proteins epsin and eps15. Furthermore, ubiquitination mediates internalization of both KSHV and one of its receptors integrin ß1. KSHV particles are colocalized with activated forms of the E3 ligase c-Cbl. Knock-down of c-Cbl or inhibition of its phosphorylation reduced viral entry and intracellular trafficking, resulting in decreased KSHV infectivity. These results demonstrate that ubiquitination mediates internalization of both KSHV and one of its cognate receptors integrin ß1, and identify c-Cbl as a potential E3 ligase that facilitates this process.

Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism.

The gene encoding IgH δ has been found in all species of teleosts studied to date. However, catfish (Ictalurus punctatus) is the only species of fish in which a secretory form of IgD has been characterized, and it occurs through the use of a dedicated δ-secretory exon, which is absent from all other species examined. Our studies have revealed that rainbow trout (Oncorhynchus mykiss) use a novel strategy for the generation of secreted IgD. The trout secretory δ transcript is produced via a run-on event in which the splice donor site at the end of the last constant domain exon (D7) is ignored and transcription continues until a stop codon is reached 33 nt downstream of the splice site, resulting in the production of an in-frame, 11-aa secretory tail at the end of the D7 domain. In silico analysis of several published IgD genes suggested that this unique splicing mechanism may also be used in other species of fish, reptiles, and amphibians. Alternative splicing of the secretory δ transcript resulted in two δ-H chains, which incorporated Cµ1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory δ mRNA and IgD(+) plasma cells were detected in all immune tissues at a lower frequency than secretory IgM. Our data demonstrate that secretory IgD is more prevalent and widespread across taxa than previously thought, and thus illustrate the potential that IgD may have a conserved role in immunity.

Microtubule- and dynein-dependent nuclear trafficking of rhesus rhadinovirus in rhesus fibroblasts.

We investigated the role of microtubules in rhesus rhadinovirus (RRV) nuclear trafficking in rhesus fibroblasts. Intact microtubules and microtubule dynamics are required for RRV trafficking to perinuclear regions. RRV trafficking was reduced by an inhibitor of the dynein motor and overexpression of dynamitin. Furthermore, RRV particles are colocalized with microtubules and dynein proteins. These results highlight the important roles of microtubules and dynein-dynactin complexes in the transport of RRV particles to nuclei during primary infection.

Outcomes from an anti-gonadotropin-releasing hormone immunotherapeutic trial in large flying foxes (Pteropus vampyrus).

OBJECTIVE: The anti-GnRH immunotherapeutic product Improvest was administered to intact male large flying foxes (Pteropus vampyrus) under managed care for androgen mitigation, leading to a decrease in agonistic behaviors, falls, and injuries from conspecific attention. ANIMALS: 12 males were included in this study. PROCEDURES: Eleven bats received subcutaneous (SC) Improvest interscapular, and 1 animal received Improvest SC in its leg. Assessments included clinical presentation, treatment, behavior, and urine and fecal glucocorticoid metabolites and testosterone (T5) concentrations. RESULTS: Eleven of the 12 bats developed reactions, which included facial edema, localized irritation, swelling of the head and neck, and pruritus with varying degrees of skin ulceration and subsequent necrosis. Three of the animals required extensive treatments, and the 1 animal who received the injection in its leg was unaffected. Posttreatment, fecal glucocorticoid metabolite and/or T5 values were at or below the nonbreeding season baseline for 3 successive breeding seasons, and there was a reduction in agonistic interactions, falls, and injuries. CLINICAL RELEVANCE: A behavioral characteristic of this species is to focus on areas of irritation that exacerbated the extent of the skin wounds. Some cases required medical, surgical, and behavioral intervention. Large flying foxes may be particularly sensitive to this immunotherapeutic when given subcutaneously in the interscapular region. Despite this reaction, the positive long-term effects on behavior and multiyear reduction of hormones suggest that the use of this immunotherapeutic warrants further investigation, although the results should be taken into consideration with other factors such as handling, treatments, chronicity of lesions.

Rhesus rhadinovirus infection of rhesus fibroblasts occurs through clathrin-mediated endocytosis.

Rhesus rhadinovirus (RRV) is a gammaherpesvirus closely related to Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus linked to the development of Kaposi's sarcoma and several other lymphoproliferative diseases, including primary effusion lymphoma and multicentric Castleman's disease. RRV naturally infects rhesus macaques and induces lymphoproliferative diseases under experimental conditions, making it an excellent model for the study of KSHV. Unlike KSHV, which grows poorly in cell culture, RRV replicates efficiently in rhesus fibroblasts (RFs). In this study, we have characterized the entry pathway of RRV in RFs. Using a luciferase-expressing recombinant RRV (RRV-luciferase), we show that the infectivity of RRV is reduced by inhibitors of endosomal acidification. RRV infectivity is also reduced by inhibitors of clathrin-mediated but not caveola-mediated endocytosis, indicating that RRV enters into RFs via clathrin-mediated endocytosis. Using a red fluorescent protein (RFP)-expressing recombinant RRV (RRV-RFP), we show that RRV particles are colocalized with markers of endocytosis (early endosome antigen 1) and clathrin-mediated endocytosis (clathrin heavy chain) during entry into RFs. RRV particles are also colocalized with transferrin, which enters cells by clathrin-mediated endocytosis, but not with cholera toxin B, which enters cells by caveola-mediated endocytosis. Inhibition of clathrin-mediated endocytosis with a dominant-negative construct of EPS15, an essential component of clathrin-coated pits, blocked the entry of RRV into RFs. Together, these results indicate that RRV entry into RFs is mediated by clathrin-mediated endocytosis.

Actin dynamics regulate multiple endosomal steps during Kaposi's sarcoma-associated herpesvirus entry and trafficking in endothelial cells.

The role of actin dynamics in clathrin-mediated endocytosis in mammalian cells is unclear. In this study, we define the role of actin cytoskeleton in Kaposi's sarcoma-associated herpesvirus (KSHV) entry and trafficking in endothelial cells using an immunofluorescence-based assay to visualize viral capsids and the associated cellular components. In contrast to infectivity or reporter assays, this method does not rely on the expression of any viral and reporter genes, but instead directly tracks the accumulation of individual viral particles at the nuclear membrane as an indicator of successful viral entry and trafficking in cells. Inhibitors of endosomal acidification reduced both the percentage of nuclei with viral particles and the total number of viral particles docking at the perinuclear region, indicating endocytosis, rather than plasma membrane fusion, as the primary route for KSHV entry into endothelial cells. Accordingly, a viral envelope protein was only detected on internalized KSHV particles at the early but not late stage of infection. Inhibitors of clathrin- but not caveolae/lipid raft-mediated endocytosis blocked KSHV entry, indicating that clathrin-mediated endocytosis is the major route of KSHV entry into endothelial cells. KSHV particles were colocalized not only with markers of early and recycling endosomes, and lysosomes, but also with actin filaments at the early time points of infection. Consistent with these observations, transferrin, which enters cells by clathrin-mediated endocytosis, was found to be associated with actin filaments together with early and recycling endosomes, and to a lesser degree, with late endosomes and lysosomes. KSHV infection induced dynamic actin cytoskeleton rearrangements. Disruption of the actin cytoskeleton and inhibition of regulators of actin nucleation such as Rho GTPases and Arp2/3 complex profoundly blocked KSHV entry and trafficking. Together, these results indicate an important role for actin dynamics in the internalization and endosomal sorting/trafficking of KSHV and clathrin-mediated endocytosis in endothelial cells.

Detection of Retinal Fibrosis in a Rabbit Model of Penetrating Eye Injury.

INTRODUCTION: To establish a rabbit model of posterior penetrating eye injury as a platform to test potential therapeutics. MATERIALS AND METHODS: Anesthetized rabbits received posterior penetrating eye injury in one eye, whereas contralateral eyes were maintained as uninjured controls. Rabbits were randomized into two experimental groups. Group A was euthanized on Day 14 postinjury to determine retinal fibrosis at an early phase of disease progression. Group B was euthanized on Day 28 postinjury to examine retinal fibrosis at a late phase of disease progression. We examined animals on postinjury Days 7, 14, 21, and 28 with indirect ophthalmoscope and fundus photography. After euthanasia, eyes were processed for histology and immunofluorescence labeling of fibrotic proteins α-smooth muscle actin and collagen I. RESULTS: Early fibrosis was detected by Day 14, as indicated by indirect ophthalmoscopy and fundus imaging. Fibrotic membranes were visible at sites of injury. Immunofluorescence analysis detected α-smooth muscle actin and collagen I within the fibrotic membranes. CONCLUSIONS: These data show that ocular fibrosis can be detected within 14 days after initial injury, with more severe fibrosis detected at 28 days postinjury. These results will be used to determine the optimal time points for later studies designed to test treatment strategies.

Pharmacokinetics of Ketoprofen in Nile Tilapia (Oreochromis niloticus) and Rainbow Trout (Oncorhynchus mykiss).

The objective of this study was to document the pharmacokinetics of ketoprofen following 3 mg/kg intramuscular (IM) and intravenous (IV) injections in rainbow trout (Oncorhynchus mykiss) and 8 mg/kg intramuscular (IM) injection in Nile tilapia (Oreochromis niloticus). Plasma was collected laterally from the tail vein for drug analysis at various time intervals up to 72 h following the injection of ketoprofen. In trout, area under the curve (AUC) levels were 115.24 µg hr/mL for IM and 135.69 µg hr/mL for IV groups with a half-life of 4.40 and 3.91 h, respectively. In both trout and tilapia, there were detectable ketoprofen concentrations in most fish for 24 h post-injection. In tilapia, there was a large difference between the R- and S-enantiomers, suggesting either chiral inversion from R- to S-enantiomer or more rapid clearance of the R-enantiomer. AUC values of the S- and R-enantiomers were 510 and 194 µg hr/Ml, respectively, corresponding to a faster clearance for the R-enantiomer. This study shows that there were very high plasma concentrations of ketoprofen in trout and tilapia with no adverse effects observed. Future studies on the efficacy, frequency of dosing, analgesia, adverse effects, and route of administration are warranted.

Topical delivery of a small molecule RUNX1 transcription factor inhibitor for the treatment of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment surgery failure. Despite significant advances in vitreoretinal surgery, it still remains without an effective prophylactic or therapeutic medical treatment. After ocular injury or retinal detachment, misplaced retinal cells undergo epithelial to mesenchymal transition (EMT) to form contractile membranes within the eye. We identified Runt-related transcription factor 1 (RUNX1) as a gene highly expressed in surgically-removed human PVR specimens. RUNX1 upregulation was a hallmark of EMT in primary cultures derived from human PVR membranes (C-PVR). The inhibition of RUNX1 reduced proliferation of human C-PVR cells in vitro, and curbed growth of freshly isolated human PVR membranes in an explant assay. We formulated Ro5-3335, a lipophilic small molecule RUNX1 inhibitor, into a nanoemulsion that when administered topically curbed the progression of disease in a novel rabbit model of mild PVR developed using C-PVR cells. Mass spectrometry analysis detected 2.67 ng/mL of Ro5-3335 within the vitreous cavity after treatment. This work shows a critical role for RUNX1 in PVR and supports the feasibility of targeting RUNX1 within the eye for the treatment of an EMT-mediated condition using a topical ophthalmic agent.

Kaposi's sarcoma-associated herpesvirus disrupts adherens junctions and increases endothelial permeability by inducing degradation of VE-cadherin.

Kaposi's sarcoma (KS) is a vascular tumor of proliferative endothelial cells caused by KS-associated herpesvirus (KSHV) infection. Aberrant vascular permeability is a hallmark of KS manifested as multifocal edematous skin and visceral lesions with dysregulated angiogenesis and vast inflammatory infiltrations. In this study, we showed that KSHV infection increased the permeability of confluent endothelial monolayers to serum albumin, blood-derived cells, KSHV-infected cells, and KSHV virions. KSHV-induced permeability was associated with the disruption of adherens junctions and the degradation of vascular endothelial cadherin (VE-cadherin) protein. Both the inactivation of KSHV virions by UV irradiation and the blockage of de novo protein synthesis with cycloheximide failed to reverse the KSHV-induced disruption of adherens junctions. However, soluble heparin that blocked KSHV entry into cells completely inhibited KSHV-induced permeability. Furthermore, the KSHV-induced degradation of VE-cadherin was dose dependent on the internalized virus particles. Together, these results indicate that KSHV infection induces vascular permeability by inducing VE-cadherin degradation during virus entry into cells. KSHV-induced aberrant vascular permeability could facilitate virus spread, promote inflammation and angiogenesis, and contribute to the pathogenesis of KSHV-induced malignancies.

Kaposi's sarcoma-associated herpesvirus latent gene vFLIP inhibits viral lytic replication through NF-kappaB-mediated suppression of the AP-1 pathway: a novel mechanism of virus control of latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency is central to the evasion of host immune surveillances and induction of KSHV-related malignancies. The mechanism of KSHV latency remains unclear. Here, we show that the KSHV latent gene vFLIP promotes viral latency by inhibiting viral lytic replication. vFLIP suppresses the AP-1 pathway, which is essential for KSHV lytic replication, by activating the NF-kappaB pathway. Thus, by manipulating two convergent cellular pathways, vFLIP regulates both cell survival and KSHV lytic replication to promote viral latency. These results also indicate that the effect of the NF-kappaB pathway on KSHV replication is determined by the status of the AP-1 pathway and hence provide a mechanistic explanation for the contradictory role of the NF-kappaB pathway in KSHV replication. Since the NF-kappaB pathway is commonly activated during infection of gammaherpesviruses, these findings might have general implications for the control of gammaherpesviral latency.

Molecular biology of KSHV in relation to AIDS-associated oncogenesis.

KSHV has been established as the causative agent of KS, PEL, and MCD, malignancies occurring more frequently in AIDS patients. The aggressive nature of KSHV in the context of HIV infection suggests that interactions between the two viruses enhance pathogenesis. KSHV latent infection and lytic reactivation are characterized by distinct gene expression profiles, and both latency and lytic reactivation seem to be required for malignant progression. As a sophisticated oncogenic virus, KSHV has evolved to possess a formidable repertoire of potent mechanisms that enable it to target and manipulate host cell pathways, leading to increased cell proliferation, increased cell survival, dysregulated angiogenesis, evasion of immunity, and malignant progression in the immunocompromised host. Worldwide, approximately 40.3 million people are currently living with HIV infection. Of these, a significant number are coinfected with KSHV. The complex interplay between the two viruses dramatically elevates the risk for development of KSHV-induced malignancies, KS, PEL, and MCD. Although HAART significantly reduces HIV viral load, the entire T-cell repertoire and immune function may not be completely restored. In fact, clinically significant immune deficiency is not necessary for the induction of KSHV-related malignancy. Because of variables such as lack of access to therapy noncompliance with prescribed treatment, failure to respond to treatment and the development of drug-resistant strains of HIV, KSHV-induced malignancies will continue to present as major health concerns.

Polarized Secretion of Matrix Metalloproteinases and Their Inhibitors by Retinal Pigment Epithelium Derived from Induced Pluripotent Stem Cells During Wound Healing.

PURPOSE: To characterize the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) by induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. We hypothesize that iPS-RPE secretes mediators of tissue remodeling such as MMPs and TIMPs to promote migration and proliferation of cells during wound healing. METHODS: iPS-RPE was grown on transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Conditioned media were collected from the apical and basolateral sides of the transwells every 72 h for 12 days. The media were analyzed by multiplex ELISA assays to detect secreted MMPs and TIMPs. Activity assays were performed to detect the active form of MMP-2 in conditioned media. RESULTS: MMP-2 and TIMP-1, -2, -3, and -4 were detected in conditioned media from iPS-RPE. The proteins were found to be secreted in a polarized manner. The apical secretion and activation of MMP-2 was elevated from days 3 to 12 after wounding. TIMP-1, -2, -3, and -4 were detected in conditioned media from both the apical and basolateral sides of wounded cells. Apical secretion of all 4 TIMPs increased within 3 days after wounding. CONCLUSIONS: These results indicate that iPS-RPE secretes MMP-2 and all 4 TIMPs in a polarized manner. After wounding, apical secretion of MMP-2 was higher compared to control. Apical secretion of all 4 TIMPs increased compared to control, while only TIMP-1 showed increased basolateral secretion compared to control.

Trichostatin A Inhibits Retinal Pigmented Epithelium Activation in an In Vitro Model of Proliferative Vitreoretinopathy.

PURPOSE: Proliferative vitreoretinopathy (PVR) is a blinding disorder that develops after a retinal tear or detachment. Activation of the retinal pigmented epithelium (RPE) is implicated in PVR; however, the mechanisms leading to enhanced RPE proliferation, migration, and contraction remain largely unknown. This study utilized an in vitro model of PVR to investigate the role of acetylation in RPE activation and its contribution to the progression of this disease. METHODS: ARPE-19 cells, primary cultures of porcine RPE, and induced pluripotent stem cell-derived RPE (iPS-RPE) were utilized for cellular and molecular analyses. Cells treated with transforming growth factor beta 2 (TGFß2; 10 ng/mL) alone or in the presence of the broad-spectrum histone deacetylase (HDAC) inhibitor, trichostatin A (TSA; 0.1 µM), were assessed for contraction and migration through collagen contraction and scratch assays, respectively. Western blotting and immunofluorescence analysis were performed to assess α-smooth muscle actin (α-SMA) and ß-catenin expression after TGFß2 treatment alone or in combination with TSA. RESULTS: TGFß2 significantly increased RPE cell contraction in collagen matrix and this effect was inhibited in the presence of TSA (0.1 µM). In agreement with these data, immunofluorescence analysis of TSA-treated iPS-RPE wounded monolayers revealed decreased α-SMA as compared with control. Scratch assays to assess wound healing revealed TSA inhibited TGFß2-mediated iPS-RPE cell migration. CONCLUSIONS: Our findings indicate a role of acetylation in RPE activation. Specifically, the HDAC inhibitor TSA decreased RPE cell proliferation and TGFß2-mediated cell contraction and migration. Further investigation of pharmacological compounds that modulate acetylation may hold promise as therapeutic agents for PVR.