Victor M. Elner

Mutations in TCF8 Cause Posterior Polymorphous Corneal Dystrophy and Ectopic Expression of COL4A3 by Corneal Endothelial Cells

Posterior polymorphous corneal dystrophy (PPCD, also known as PPMD) is a rare disease involving metaplasia and overgrowth of corneal endothelial cells. In patients with PPCD, these cells manifest in an epithelial morphology and gene expression pattern, produce an aberrant basement membrane, and, sometimes, spread over the iris and nearby structures in a way that increases the risk for glaucoma. We previously mapped PPCD to a region (PPCD3) on chromosome 10 containing the gene that encodes the two-handed zinc-finger homeodomain transcription factor TCF8. Here, we report a heterozygous frameshift mutation in TCF8 that segregates with PPCD in the family used to map PPCD3 and four different heterozygous nonsense and frameshift mutations in TCF8 in four other PPCD probands. Family reports of inguinal hernia, hydrocele, and possible bone anomalies in affected individuals suggest that individuals with TCF8 mutations should be examined for nonocular anomalies. We detect transcripts of all three identified PPCD genes (VSX1, COL8A2, and TCF8) in the cornea. We show presence of a complex (core plus secondary) binding site for TCF8 in the promoter of Alport syndrome gene COL4A3, which encodes collagen type IV alpha 3, and we present immunohistochemical evidence of ectopic expression of COL4A3 in corneal endothelium of the proband of the original PPCD3 family. Identification of TCF8 as the PPCD3 gene provides a valuable tool for the study of critical gene regulation events in PPCD pathology and suggests a possible role for TCF8 mutations in altered structure and function of cells lining body cavities other than the anterior chamber of the eye. Thus, this study has identified TCF8 as the gene responsible for approximately half of the cases of PPCD, has implicated TCF8 mutations in developmental abnormalities outside the eye, and has presented the TCF8 regulatory target, COL4A3, as a key, shared molecular component of two different diseases, PPCD and Alport syndrome.

Role of MCP‐1 and MIP‐1α in retinal neovascularization during postischemic inflammation in a mouse model of retinal neovascularization

Macrophages are important participants in neovascularization. This study was designed to examine the role of the monocyte/macrophage chemotactic proteins, monocyte chemotactic protein‐1 (MCP‐1), and macrophage inflammatory protein‐1α (MIP‐1α) in a mouse model of oxygen‐induced ischemic retinopathy and to determine whether the morphology and distribution of macrophages/microglia are concomitantly altered. The MCP‐1, MIP‐1α mRNA levels increased at 3 h after ischemia. MCP‐1, MIP‐1α, and vascular endothelial growth factor protein levels were also increased markedly and were maximal on days 1,0.5, and 1, respectively, after ischemia. In situ hybridization showed that MCP‐1 and MIP‐1α were localized in the hypoxic inner retina. Immunostaining demonstrated that the macrophages/microglia in the retina had morphological changes with enlarged processes, and some were closely associated with neovascular tufts at postnatal day 17. Coadministration of the neutralizing antibodies against MCP‐1 and MIP‐1α inhibited retinal neovascularization by 30%. Our data suggest that MCP‐1 and MIP‐1α are involved in the induction of retinal neovascularization and play a role in the inflammation induced by the ischemic retinopathy, possibly by modulating or attracting macrophages/microglia.

Interleukin-6 (IL-6) gene expression and secretion by cytokine-stimulated human retinal pigment epithelial cells

Retinal and choroidal inflammatory lesions are important causes of visual loss, but the mechanisms regulating intraocular inflammation remain poorly understood. By virtue of its position at the blood-retina barrier, the retinal pigment epithelium (RPE) cells may be critical to the initiation and propagation of ocular inflammation. Previously we showed that cytokine-stimulated RPE cells produce interleukin-8, a well-defined chemotactic factor for neutrophils and lymphocytes. In this study, we found that human RPE cells stimulated by human recombinant interleukin-1-beta (rIL-1 beta) or tumor necrosis factor-alpha (rTNF-alpha) produce interleukin-6 (IL-6). Using a plasmacytoma proliferation assay, significant levels of IL-6 were found in media of RPE cells stimulated with either rIL-1 beta or rTNF-alpha for 4 hr. Progressive accumulation of IL-6 in media overlying stimulated RPE cells occurred over the subsequent 20 hr. IL-1 beta was a significantly more potent stimulator of RPE IL-6 production than TNF-alpha, RPE IL-6 production in response to each of these cytokines was also dose-dependent over a range of 20 pg to 20 ng ml-1. Specific anti IL-6 antibody, but not control immunoglobulin, neutralized RPE-derived IL-6 activity in the plasmacytoma proliferation assays. RPE IL-6 mRNA levels were detectable 1 hr after cytokine stimulation, plateaued within 8 hr in 24-hr assays, and demonstrated dose-dependent kinetics in 6 hr assays. Lipopolysaccharide failed to induce RPE IL-6 mRNA expression or RPE IL-6 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Preliminary visual outcomes after three-dimensional conformal radiation therapy for optic nerve sheath meningioma

PURPOSE We assessed visual outcomes, local control, and toxicity associated with three-dimensional conformal radiation therapy (3D-CRT) for primary optic nerve sheath meningiomas (ONSM). METHODS Twenty-three patients diagnosed with ONSM were evaluated at the University of Michigan between 1986 and 2001. Fourteen patients were treated with 3D-CRT. Detailed pre- and postradiation treatment ophthalmologic examinations and MRIs were performed on all patients. Clinically significant visual acuity change was defined as a >or=three line change on the Snellen chart. Mean deviation change of >or=three decibels was defined as a clinically significant visual field change. Radiographic progression was defined as any increase in size on MRI. Acute and late toxicity was scored according to RTOG criteria. RESULTS Median follow-up was 51.3 months. Five patients had a clinically significant improvement in visual acuity. Seven had stable acuity, and only 2 worsened. Nine patients had clinically significant visual field improvement. One patient developed early radiation retinopathy, 1 experienced orbital pain, 1 developed dry eye, and 2 developed iritis. No patient has required additional treatment, and none have demonstrated radiographic progression. CONCLUSION 3D-CRT is effective in controlling tumor growth while improving or preserving vision in most patients with optic nerve sheath meningiomas.

Photodisruption in the human cornea as a function of laser pulse width

BACKGROUND We investigated the role of laser pulse width in determining fluence thresholds and efficiency for corneal photodisruption. METHODS A laser system that delivers a wide range of pulse energies and pulse widths was used to produce ablations at pulse widths from 100 femtoseconds (fs) to 7 nanoseconds (ns). The laser-induced breakdown fluence threshold at each pulse width was determined by monitoring individual plasma emissions. Using multiple shots, the photodisruption threshold and cutting depth at each pulse width were determined histologically. RESULTS Corneal breakdown thresholds decreased at a faster rate from 7 ns to approximately 10 picoseconds (ps), compared to further reductions in pulse width below 10 ps, where little variation was seen. Breakdown for pulse widths below 10 ps showed little intershot variability, resulting in highly reproducible fluence thresholds. Corneal tissue examined histologically showed similar fluence dependency. CONCLUSIONS Corneal tissue photodisruption thresholds demonstrate pulse width dependence. At pulse widths less than 10 ps and with fluences near the breakdown threshold, ablations are maximally precise and efficient. These findings suggest optimal laser parameters for corneal surgery.

Toll-like receptor 4 (TLR4) of retinal pigment epithelial cells participates in transmembrane signaling in response to photoreceptor outer segments.

Retinal pigment epithelial (RPE) cells mediate the recognition and clearance of effete photoreceptor outer segments (POS), a process central to the maintenance of normal vision. Given the emerging importance of Toll-like receptors (TLRs) in transmembrane signaling in response to invading pathogens as well as endogenous substances, we hypothesized that TLRs are associated with RPE cell management of POS. TLR4 clusters on human RPE cells in response to human, but not bovine, POS. However, TLR4 clustering could be inhibited by saturating concentrations of an inhibitory anti-TLR4 mAb. Furthermore, human POS binding to human RPE cells elicited transmembrane metabolic and calcium signals within RPE cells, which could be blocked by saturating doses of an inhibitory anti-TLR4 mAb. However, the heterologous combination of bovine POS and human RPE did not trigger these signals. The pattern recognition receptor CD36 collected at the POS–RPE cell interface for both homologous and heterologous samples, but human TLR4 only collected at the human POS–human RPE cell interface. Kinetic experiments of human POS binding to human RPE cells revealed that CD36 arrives at the POS–RPE interface followed by TLR4 accumulation within 2 min. Metabolic and calcium signals immediately follow. Similarly, the production of reactive oxygen metabolites (ROMs) was observed for the homologous human system, but not the heterologous bovine POS–human RPE cell system. As (a) the bovine POS/human RPE combination did not elicit TLR4 accumulation, RPE signaling, or ROM release, (b) TLR4 arrives at the POS–RPE cell interface just before signaling, (c) TLR4 blockade with an inhibitory anti-TLR4 mAb inhibited TLR4 clustering, signaling, and ROM release in the human POS–human RPE system, and (d) TLR4 demonstrates similar clustering and signaling responses to POS in confluent RPE monolayers, we suggest that TLR4 of RPE cells participates in transmembrane signaling events that contribute to the management of human POS.

Cytogenetic analysis of posterior uveal melanoma

Cytogenetic analysis was performed on short-term cultures of primary tumor samples from seven patients with posterior uveal melanoma. Informative data were obtained from four patients, all of whom had a near-diploid chromosomal number and clonal chromosomal alterations. Analysis of one patients tumor revealed monosomy 3 as the only cytogenetically distinguishable aberration. Trisomies of chromosome 8 and i(8)(q10) were detected in two other patients in combination with monosomy of chromosome 3. The fourth patients karyotype displayed two different translocations. One translocation, der(6)t(6;8)(q12;q13.1), resulted in the over-representation of 8q13.1-->qter and a partial monosomy of 6q12-->qter; the other translocation, der(9)t(6;9)(p12;p23), produced a partial trisomy of 6p12-->pter and a partial monosomy of 9p23-->pter. These results support the view that the recurring pattern of chromosomal rearrangements in ocular melanoma is unique from that associated with cutaneous malignant melanoma. Furthermore, these results help confirm that chromosomes 3, 6, and 8 are nonrandomly altered in ocular melanoma.

Human RPE-monocyte co-culture induces chemokine gene expression through activation of MAPK and NIK cascade

Cell-cell contact between human retinal pigment epithelium (hRPE) cells and monocytes occurs in many retinal diseases involving blood-retinal barrier breakdown. This study investigates chemokine secretion induced by co-culture of hRPE cells and monocytes and illustrates the roles of p38 kinase, ERK, JNK/SAPK and NF-kappaB-inducing kinase signaling pathways for hRPE IL-8 and MCP-1 secretion induced in hRPE by co-culture with monocytes. Co-culture of hRPE cells with monocytes increased steady-state IL-8 and MCP-1 mRNA and protein secretion. Stimulation of hRPE cells by monocytes resulted in prominent increases in p38, ERK1/2 and JNK/SAPK phosphorolation, IkappaBalpha degradation, and NF-kappaB nuclear translocation. The induced IL-8 and MCP-1 proteins were almost completely supporessed by U0126, a specific mitogen-activated protein kinase kinase (MEK) inhibitor, or by SB203580, a selective p38 inhibitor. Chemokine secretion was completely blocked by simultaneous administration of U0126 and SB203580. Induction of IL-8 and MCP-1 was abrogated by Ro318220, an inhibitor of PKC, as well as by genistein or herbimycin A, inhibitors of PTK. In addition, anti-inflammatory drugs dexamethasone (DEX) and cyclosporin A (CSA) both blocked activation of JNKS/SAPK and the cell-cell contact induced production of hRPE IL-8 and MCP-1, while activation of p38 and ERK was only inhibited by DEX, but not by CSA. These results suggest that activation of DEX-sensitive, CSA-resistant MEK/ERK and p38 pathways, and activation of NF-kappaB, PKC, and PTK are essential for IL-8 and MCP-1 expression by hRPE cells.

Dual Involvement of Caspase-4 in Inflammatory and ER Stress-Induced Apoptotic Responses in Human Retinal Pigment Epithelial Cells

PURPOSE To investigate the functional involvement of caspase-4 in human retinal pigment epithelial (hRPE) cells. METHODS Expression and activation of caspase-4 in hRPE cells were measured after stimulation with proinflammatory agents IL-1beta (2 ng/mL), TNF-alpha (20 ng/mL), lipopolysaccharide (1000 ng/mL), interferon-gamma (500 U/mL), or monocyte coculture in the absence or presence of immunomodulating agent cyclosporine (3 or 30 ng/mL), dexamethasone (10 microM), or IL-10 (100 U/mL) and endoplasmic reticulum (ER) stress inducer thapsigargin (25 nM) or tunicamycin (3 or 10 microM). The onset of ER stress was determined by expression of GRP78. The involvement of caspase-4 in inflammation and apoptosis was further examined by treating the cells with caspase-4 inhibitor Z-LEVD-fmk, caspase-1 and -4 inhibitor Z-YVAD-fmk, and pan-caspase inhibitor Z-VAD-fmk. RESULTS Caspase-4 mRNA expression and protein activation were induced by all the proinflammatory agents and ER stress inducers tested in this study. Caspase-4 activation was blocked or reduced by dexamethasone and IL-10. Elevated ER stress by proinflammatory agents and ER stress inducers was shown by increased expression of the ER stress marker GRP78. The induced caspase-4 and caspase-3 activities by tunicamycin and the stimulated IL-8 protein expression by IL-1beta were markedly reduced by caspase-4 inhibitor Z-LEVD-fmk. Although caspase-4 inhibitor Z-LEVD-fmk and caspase-1 and -4 inhibitor Z-YVAD-fmk reduced tunicamycin-induced hRPE apoptotic cell death by 59% and 86%, respectively, pan-caspase inhibitor Z-VAD-fmk completely abolished the induced apoptosis. CONCLUSIONS Caspase-4 is dually involved in hRPE proinflammatory and proapoptotic responses. Various proinflammatory stimuli and ER stress induce hRPE caspase-4 mRNA synthesis and protein activation. ER stress-induced hRPE cell death is caspase and, in part, caspase-4 dependent.

Fat adherence syndrome treated with intraoperative mitomycin-C: a rabbit model.

We used an animal model of restrictive strabismus analogous to the fat adherence syndrome in humans to test the efficacy of topical intraoperative mitomycin-C (MMC) in preventing the development of restrictive scar tissue. A cicatricial adhesion was created between the inferior rectus muscle and the inferior orbital rim of each eye in eight rabbits, and passive forced ductions were quantitatively measured with a spring scale. Eight eyes were treated intraoperatively with topical MMC 0.5 mg/mL, the other eight with sterile water. Passive forced ductions were again measured 4 weeks postoperatively and representative orbits were exenterated for histopathologic examination. Significant restriction of motility was produced in six of the eight control eyes. Though prophylactic treatment with MMC may have been beneficial in some cases, on average, the restriction developing in these eyes did not significantly differ from that in the control eyes. In addition, longer exposure times to MMC led to marked orbital inflammation and severe restriction of ocular motility. Finally, histopathologic evaluation of the orbits of the MMC-treated eyes revealed marked fibrosis of perimuscular connective tissues. Although MMC may have a role in the management of fat adherence syndrome, further study is needed to establish safe and efficacious methods of delivery.