Jaclyn Bermudez

Photophysical characterization of anticancer drug valrubicin in rHDL nanoparticles and its use as an imaging agent.

Nanoparticles are target-specific drug delivery agents that are increasingly used in cancer therapy to enhance bioavailability and to reduce off target toxicity of anti-cancer agents. Valrubicin is an anti-cancer drug, currently approved only for vesicular bladder cancer treatment because of its poor water solubility. On the other hand, valrubicin carrying reconstituted high density lipoprotein (rHDL) nanoparticles appear ideally suited for extended applications, including systemic cancer chemotherapy. We determined selected fluorescence properties of the free (unencapsulated) drug vs. valrubicin incorporated into rHDL nanoparticles. We have found that upon encapsulation into rHDL nanoparticles the quantum yield of valrubicin fluorescence increased six fold while its fluorescence lifetime increased about 2 fold. Accordingly, these and potassium iodide (KI) quenching data suggest that upon incorporation, valrubicin is localized deep in the interior of the nanoparticle, inside the lipid matrix. Fluorescence anisotropy of the rHDL valrubicin nanoparticles was also found to be high along with extended rotational correlation time. The fluorescence of valrubicin could also be utilized to assess its distribution upon delivery to prostate cancer (PC3) cells. Overall the fluorescence properties of the rHDL: valrubicin complex reveal valuable novel characteristics of this drug delivery vehicle that may be particularly applicable when used in systemic (intravenous) therapy.

A Comparison of Gene Expression Profiles between Glucocorticoid Responder and Non-Responder Bovine Trabecular Meshwork Cells Using RNA Sequencing.

The most common ocular side effect of glucocorticoid (GC) therapy is GC-induced ocular hypertension (OHT) and GC-induced glaucoma (GIG). GC-induced OHT occurs in about 40% of the general population, while the other 60% are resistant. This study aims to determine the genes and pathways involved in differential GC responsiveness in the trabecular meshwork (TM). Using paired bovine eyes, one eye was perfusion-cultured with 100nM dexamethasone (DEX), while the fellow eye was used to establish a bovine TM (BTM) cell strain. Based on maximum IOP change in the perfused eye, the BTM cell strain was identified as a DEX-responder or non-responder strain. Three responder and three non-responder BTM cell strains were cultured, treated with 0.1% ethanol or 100nM DEX for 7 days. RNA and proteins were extracted for RNA sequencing (RNAseq), qPCR, and Western immunoblotting (WB), respectively. Data were analyzed using the human and bovine genome databases as well as Tophat2 software. Genes were grouped and compared using Student’s t-test. We found that DEX induced fibronectin expression in responder BTM cells but not in non-responder cells using WB. RNAseq showed between 93 and 606 differentially expressed genes in different expression groups between responder and non-responder BTM cells. The data generated by RNAseq were validated using qPCR. Pathway analyses showed 35 pathways associated with differentially expressed genes. These genes and pathways may play important roles in GC-induced OHT and will help us to better understand differential ocular responsiveness to GCs.

TGFβ2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways

Purpose Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor β2 (TGFβ2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFβ2 signaling pathways in CLAN formation. Methods Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFβ receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4′,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnetts post hoc test. Results TGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFβ receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFβ receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. Conclusions TGFβ2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.

Two-Dimensional Differential In-Gel Electrophoresis (2D-DIGE) Reveals Proteins Associated with Cross-Linked Actin Networks in Human Trabecular Meshwork Cells

Background: The primary risk factor for primary-open angle glaucoma (POAG) is increased intraocular pressure (IOP). In POAG patients, the outflow resistance through the trabecular meshwork (TM) is abnormally elevated. One of the important glaucoma-associated pathological changes in the TM is formation of excessive cross-linked actin networks (CLANs). CLANs are web-like polygonal structures found in confluent glaucoma TM cells and tissues. Glaucoma-associated factors transforming forming growth factor beta 2 (TGFβ2) and glucocorticoids induce CLAN formation in non-glaucoma TM cells (NTM). CLANs may increase cell stiffness and alter homeostasis, and thereby contribute to elevated IOP. Methods: We used a proteomic approach to identify CLAN-associated proteins. We treated confluent primary NTM cells with 0.1% ethanol (EtOH; vehicle), 100 nM dexamethasone (DEX), or 5 ng/ml TGFβ2 plus 0.1% EtOH for 7 days to induce CLANs. The Triton insoluble fraction containing cytoskeleton was extracted for two-dimensional differential in-gel electrophoresis (2D-DIGE). Mass spectrometry (MS) was used to identify the differential expressed proteins in the 2D-gels. Co-localization of identified proteins with CLANs was confirmed using immunocytofluorescence (ICF) microscopy. Results: 2D-DIGE revealed 103 differentially expressed proteins in both treatment groups. MSidentified 23 of the most enriched proteins. ICF showed that caldesmon, calponin, myosin light chain, and tropomyosin were colocalized with CLANs. Conclusions: We identified a subset of proteins differentially expressed in NTM cells with DEX or TGFβ2- induced CLAN formation. The potential involvement of these proteins in CLAN formation and/or maintenance requires further investigation. These proteins may provide new insights into the pathogenesis of glaucoma.

Cross-linked actin networks (CLANs) in glaucoma

One of the major causes of decreased vision, irreversible vision loss and blindness worldwide is glaucoma. Increased intraocular pressure (IOP) is a major risk factor associated with glaucoma and its molecular mechanisms are not fully understood. The trabecular meshwork (TM) is the primary site of injury in glaucoma, and its dysfunction results in elevated IOP. The glaucomatous TM has increased extracellular matrix deposition as well as cytoskeletal rearrangements referred to as cross-linked actin networks (CLANs) that consist of dome like structures consisting of hubs and spokes. CLANs are thought to play a role in increased aqueous humor outflow resistance and increased IOP by creating stiffer TM cells and tissue. CLANs are inducible by glucocorticoids (GCs) and TGFβ2 in confluent TM cells and TM tissues. The signaling pathways of these induction agents give insight into the possible mechanisms of CLAN formation, but to date, the mechanism of CLANs regulation by these pathways has yet to be determined. Understanding the role CLANs play in IOP elevation and their mechanisms of induction and regulation may lead to novel treatment options to help prevent or intervene in glaucomatous damage to the trabecular meshwork.

HDAC Inhibitor-Mediated Epigenetic Regulation of Glaucoma-Associated TGFβ2 in the Trabecular Meshwork.

Purpose Elevated intraocular pressure (IOP) in primary open-angle glaucoma (POAG) results from glaucomatous damage to the trabecular meshwork (TM). The glaucoma-associated factor TGFβ2 is increased in aqueous humor and TM of POAG patients. We hypothesize that histone acetylation has a role in dysregulated TGFβ2 expression. Methods Protein acetylation was compared between nonglaucomatous TM (NTM) and glaucomatous TM (GTM) cells using Western immunoblotting (WB). Nonglaucomatous TM cells were treated with 10 nM thailandepsin-A (TDP-A), a potent histone deacetylase inhibitor for 4 days. Total and nuclear proteins, RNA, and nuclear protein-DNA complexes were harvested for WB, quantitative PCR (qPCR), and chromatin immunoprecipitation (ChIP) assays, respectively. Paired bovine eyes were perfused with TDP-A versus DMSO, or TDP-A versus TDP-A plus the TGFβ pathway inhibitor LY364947 for 5 to 9 days. Intraocular pressure, TM, and perfusate proteins were compared. Results We found increased acetylated histone 3 and total protein acetylation in the GTM cells and TDP-A treated NTM cells. Chromatin immunoprecipitation assays showed that TDP-A induced histone hyperacetylation associated with the TGFβ2 promoter. This change of acetylation significantly increased TGFβ2 mRNA and protein expression in NTM cells. In perfusion-cultured bovine eyes, TDP-A increased TGFβ2 in the perfusate as well as elevated IOP. Histologic and immunofluorescent analyses showed increased extracellular matrix and cytoskeletal proteins in the TM of TDP-A treated bovine eyes. Cotreatment with the TGFβ pathway inhibitor LY364947 blocked TDP-A–induced ocular hypertension. Conclusions Our results suggest that histone acetylation has an important role in increased expression of the glaucoma-associated factor TGFβ2. Histone hyperacetylation may be the initiator of glaucomatous damage to the TM.

The Role of Wnt/β-Catenin Signaling and K-Cadherin in the Regulation of Intraocular Pressure

Purpose Wnt/β-catenin signaling in the trabecular meshwork (TM) is required for maintaining normal intraocular pressure (IOP), although the mechanism(s) behind this are unknown. We hypothesize that Wnt/β-catenin signaling regulates IOP via β-catenins effects on cadherin junctions. Methods Nonglaucomatous primary human TM (NTM) cells were treated with or without 100 ng/ml Wnt3a, 1 μg/ml sFRP1, or both for 4 to 48 hours. Cells were immunostained for β-catenin, total cadherins, or cadherin isoforms. Membrane proteins or whole-cell lysates were isolated for Western immunoblotting and probed for cadherin isoforms. RNA was extracted for cDNA synthesis and qPCR analysis of cadherin expression. Some NTM cells were cultured on electric plates for cell impedance assays. Ad5.CMV recombinant adenoviruses encoding K-cadherin, and/or sFRP1 were injected into eyes of 4- to 6-month-old female BALB/cJ mice (n = 8–10). Conscious IOPs were assessed for 35 days. Results Upon Wnt3a treatment, total cadherin expression increased and β-catenin accumulated at the TM cell membrane and on processes formed between TM cells. qPCR showed that Wnt3a significantly increased K-cadherin expression in NTM cells (P < 0.01, n = 3), and Western immunoblotting showed that Wnt3a increased K-cadherin in NTM cells, which was inhibited by the addition of sFRP1. Cell impedance assays showed that Wnt3a treatment increased transcellular resistance and anti-K-cadherin siRNA decreased transcellular resistance (P < 0.001, n = 4–6). Our in vivo study showed that K-cadherin significantly decreased sFRP1-induced ocular hypertension (P < 0.05, n = 6). Western immunoblotting also showed that K-cadherin alleviated sFRP1-induced β-catenin decrease in mouse anterior segments. Conclusions Our results suggest that cadherins play important roles in the regulation of TM homeostasis and IOP via the Wnt/β-catenin pathway.