Allan R. Shepard

Adenoviral gene transfer of active human transforming growth factor-β2 elevates intraocular pressure and reduces outflow facility in rodent eyes.

Purpose. Glaucoma is a leading cause worldwide of blindness and visual impairment. Transforming growth factor-beta2 (TGFbeta2) has been implicated in the pathogenesis of primary open-angle glaucoma (POAG) based on elevated levels in glaucomatous aqueous humor and its ability to induce extracellular matrix (ECM) remodeling in the trabecular meshwork (TM). The goal of this study was to generate a rodent model of POAG using viral gene transfer of human TGFbeta2. Methods. Latent (hTGFbeta2(WT)) or active (C226S, C228S; hTGFbeta2(226/228)) TGFbeta2-encoding cDNA was cloned into the pac.Ad5.CMV.K-N.pA shuttle vector for generation of replication-deficient adenovirus. Empty adenovirus (Ad5.CMV.K-N.pA) was used as a control. Adenoviral expression of active and total TGFbeta2 was assayed in vitro by the transduction of Chinese hamster ovary and trabecular meshwork cells. BALB/cJ mice or Wistar rats were injected either intracamerally or intravitreally with the adenovectors and assessed for changes in intraocular pressure (IOP) using the rebound tonometer. At peak IOP, aqueous outflow facility and total TGFbeta2 levels in aqueous humor were measured. Mouse eye morphology was assessed by hematoxylin and eosin staining. Results. Adenoviral gene transfer of hTGFbeta2(226/228), but not hTGFbeta2(WT), to the rodent eye elevated IOP in rat (43%, P < 0.001) and mouse (110%, P < 0.001) and reduced aqueous humor outflow facility in the mouse. The TGFbeta2-induced ocular hypertension correlated with anterior segment TGFbeta2 expression levels (P < 0.0001). Conclusions. The adenoviral TGFbeta2 rodent model displays the glaucoma risk factors of elevated IOP and decreased aqueous outflow facility and may potentially serve as a model for studying glaucoma.

Exon-level expression profiling of ocular tissues.

The normal gene expression profiles of the tissues in the eye are a valuable resource for considering genes likely to be involved with disease processes. We profiled gene expression in ten ocular tissues from human donor eyes using Affymetrix Human Exon 1.0 ST arrays. Ten different tissues were obtained from six different individuals and RNA was pooled. The tissues included: retina, optic nerve head (ONH), optic nerve (ON), ciliary body (CB), trabecular meshwork (TM), sclera, lens, cornea, choroid/retinal pigment epithelium (RPE) and iris. Expression values were compared with publically available Expressed Sequence Tag (EST) and RNA-sequencing resources. Known tissue-specific genes were examined and they demonstrated correspondence of expression with the representative ocular tissues. The estimated gene and exon level abundances are available online at the Ocular Tissue Database.

Ion transporters and receptors in cDNA libraries from lens and cornea epithelia.

PURPOSE To construct tissue-specific cDNA libraries containing copies of genes of ion transporting and receptor proteins in lens and cornea epithelia. To screen the libraries for the cDNA of these molecules and to deduce the protein sequence from the cDNA sequence. METHOD Lens and corneal cDNA libraries are not commercially available and are difficult to prepare from microdissected single animal eyes or cadaver human eyes due to the small amount of tissue. To overcome these problems, we refined an approach for preparing high efficiency, non-PCR amplified plasmid cDNA libraries, where only nanogram quantities of poly (A) RNA are required. Plasmids were electrotransformed into DH10B bacteria and routinely yielded >10(6) independent colonies with typically >90% recombinants. Randomly selected colonies were subjected to colony PCR analysis to determine the libraries average insert size (typically approximately 1-kb). Primers to known genes were used in PCR amplification to check for representation of the genes in the cDNA libraries. RESULTS Using libraries from rabbit cornea epithelium and endothelium, cultured human lens epithelium, and alphaTN4 cells, we have found the libraries to contain the cDNA for 3 common housekeeping proteins expected to be at high copy numbers in all cells. In addition, we identified 22 rare proteins and for many we determined the complete coding regions. These include K+ channels, Cl- channels, Ca++ channels, Na+ channels, three exchangers, the Na+-HCO3- cotransporter, and three kinds of receptors. CONCLUSION Cornea and lens libraries have been constructed from single cornea or lens preparations. The libraries often contain the cDNA sequences for ion transporting and receptor proteins which are expected to be relatively rare in these cells. Many of these cDNAs have now been sequenced and the encoded proteins identified.

Characterization of rabbit myocilin: Implications for human myocilin glycosylation and signal peptide usage

BackgroundMutations in the gene encoding human myocilin (MYOC) have been shown to cause juvenile- and adult-onset glaucoma. In addition, myocilin has been associated with glucocorticoid-induced ocular hypertension and steroid-induced glaucoma. To better understand the role myocilin plays in steroid-induced glaucoma and open-angle glaucoma, we examined rabbit myocilin for use in the rabbit animal model of steroid-induced glaucoma.ResultsWe have cloned the rabbit ortholog of human MYOC. Rabbit MYOC consists of three exons and an open reading frame encoding a 490 amino acid, 54,882-Da protein, which is 14 amino acids shorter at the N-terminus than human myocilin but 84% identical overall. Rabbit myocilin migrates as a single electrophoretic band, vs. double-banded human myocilin, by SDS-PAGE/immunoblot analysis. We determined that the differential migration exhibited is due to an N-glycosylation site that is present in human (Asn57), monkey and mouse myocilin but absent in rabbit (Ser43), rat and bovine myocilin. Rabbit myocilin is secreted in vitro in trabecular meshwork cell culture and in vivo in aqueous humor. Secretion of human myocilin is shown to be dependent on the signal peptide and independent of the extra 14 amino acids not found in rabbit myocilin. Many of the amino acids in myocilin that are mutated in glaucoma patients are conserved across species.ConclusionWe have cloned the rabbit MYOC cDNA and determined that rabbit myocilin is secreted but not N-linked glycosylated. Knowledge of the rabbit MYOC cDNA sequence will facilitate future studies in the rabbit animal model examining the role of myocilin in steroid-induced glaucoma and the gain-of-function hypothesis in open-angle glaucoma.

TGF-β2-mediated ocular hypertension is attenuated in SPARC-null mice.

PURPOSE Transforming growth factor-β2 (TGF-β2) has been implicated in the pathogenesis of primary open-angle glaucoma through extracellular matrix (ECM) alteration among various mechanisms. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates ECM within the trabecular meshwork (TM), and is highly upregulated by TGF-β2. We hypothesized that, in vivo, SPARC is a critical regulatory node in TGF-β2-mediated ocular hypertension. METHODS Empty (Ad.empty) or TGF-β2-containing adenovirus (Ad.TGF-β2) was injected intravitreally into C57BL6-SV129 WT and SPARC-null mice. An initial study was performed to identify a stable period for IOP measurement under isoflurane. The IOP was measured before injection and every other day for two weeks using rebound tonometry. Additional mice were euthanized at peak IOP for immunohistochemistry. RESULTS The IOP was stable under isoflurane during minutes 5 to 8. The IOP was significantly elevated in Ad.TGF-β2-injected (n = 8) versus Ad.empty-injected WT (n = 8) mice and contralateral uninjected eyes during days 4 to 11 (P < 0.03). The IOPs were not significantly elevated in Ad.TGF-β2-injected versus Ad.empty-injected SPARC-null mice. However, on day 8, the IOP of Ad.TGF-β2-injected SPARC-null eyes was elevated compared to that of contralateral uninjected eyes (P = 0.0385). Immunohistochemistry demonstrated that TGF-β2 stimulated increases in collagen IV, fibronectin, plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and SPARC in WT mice, but only PAI-1 and CTGF in SPARC-null mice (P < 0.05). CONCLUSIONS SPARC is essential to the regulation of TGF-β2-mediated ocular hypertension. Deletion of SPARC significantly attenuates the effects of TGF-β2 by restricting collagen IV and fibronectin expression. These data provide further evidence that SPARC may have an important role in IOP regulation and possibly glaucoma pathogenesis.

Identification of PDE6D as a molecular target of anecortave acetate via a methotrexate-anchored yeast three-hybrid screen.

Glaucoma and age-related macular degeneration are ocular diseases targeted clinically by anecortave acetate (AA). AA and its deacetylated metabolite, anecortave desacetate (AdesA), are intraocular pressure (IOP)-lowering and angiostatic cortisenes devoid of glucocorticoid activity but with an unknown mechanism of action. We used a methotrexate-anchored yeast three-hybrid (Y3H) technology to search for binding targets for AA in human trabecular meshwork (TM) cells, the target cell type that controls IOP, a major risk factor in glaucoma. Y3H hits were filtered by competitive Y3H screens and coimmunoprecipitation experiments and verified by surface plasmon resonance analysis to yield a single target, phosphodiesterase 6-delta (PDE6D). PDE6D is a prenyl-binding protein with additional function outside the PDE6 phototransduction system. Overexpression of PDE6D in mouse eyes caused elevated IOP, and this elevation was reversed by topical ocular application of either AA or AdesA. The identification of PDE6D as the molecular binding partner of AA provides insight into the role of this drug candidate in treating glaucoma.

Chapter 4 Identification of Potassium Channels in Human Lens Epithelium

Publisher Summary Potassium channels are vital to the functioning of almost every cell type. Their general role is to set the resting membrane potential of the cell, although they are never the only transporters involved in this important function. If they were to dominate in resting membrane potential control, the transmembrane voltage would sit very near the potassium equilibrium potential, some –90 mV in most cells. This chapter discusses both electrophysiological characterization and some molecular biology characterizations of several potassium channels commonly found in the human lens epithelium. The channels are identified, and the kinds of approaches that can lead to their identification and characterization are also described. Because of the substantial sequence homology of human lens potassium channels with other published potassium-channel sequences, much more progress with the structure–function relationships of human lens channels has been made than with those from other ocular epithelia. Inward rectifier potassium currents are easily identified from either cultured or freshly dissociated human lens epithelial cells by measuring whole-cell currents.