Peter L. Gehlbach

Pigment Epithelium-Derived Factor Inhibits Retinal and Choroidal Neovascularization

In this study, we investigated whether overexpression of pigment epithelium‐derived factor (PEDF) by gene transfer can inhibit neovascularization by testing its effect in three different models of ocular neovascularization. Intravitreous injection of an adenoviral vector encoding PEDF resulted in expression of PEDF mRNA in the eye measured by RT‐PCR and increased immunohistochemical staining for PEDF protein throughout the retina. In mice with laser‐induced rupture of Bruchs membrane, choroidal neovascularization was significantly reduced after intravitreous injection of PEDF vector compared to injection of null vector or no injection. Subretinal injection of the PEDF vector resulted in prominent staining for PEDF in retinal pigmented epithelial cells and strong inhibition of choroidal neovascularization. In two models of retinal neovascularization (transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors and mice with oxygen‐induced ischemic retinopathy), intravitreous injection of null vector resulted in decreased neovascularization compared to no injection, but intravitreous injection of PEDF vector resulted in further inhibition of neovascularization that was statistically significant. These data suggest that sustained increased intraocular expression of PEDF by gene therapy might provide a promising approach for treatment of ocular neovascularization.

Inhibition of Choroidal Neovascularization by Intravenous Injection of Adenoviral Vectors Expressing Secretable Endostatin

Endostatin is a cleavage product of collagen XVIII that inhibits tumor angiogenesis and growth. Interferon alpha2a blocks tumor angiogenesis and causes regression of hemangiomas, but has no effect on choroidal neovascularization (CNV). Therefore, inhibitors of tumor angiogenesis do not necessarily inhibit ocular neovascularization. In this study, we used an intravenous injection of adenoviral vectors containing a sig-mEndo transgene consisting of murine immunoglobulin kappa-chain leader sequence coupled to sequence coding for murine endostatin to investigate the effect of high serum levels of endostatin on CNV in mice. Mice injected with a construct in which sig-mEndo expression was driven by the Rous sarcoma virus promoter had moderately high serum levels of endostatin and significantly smaller CNV lesions at sites of laser-induced rupture of Bruchs membrane than mice injected with null vector. Mice injected with a construct in which sig-mEndo was driven by the simian cytomegalovirus promoter had approximately 10-fold higher endostatin serum levels and had nearly complete prevention of CNV. There was a strong inverse correlation between endostatin serum level and area of CNV. This study provides proof of principle that gene therapy to increase levels of endostatin can prevent the development of CNV and may provide a new treatment for the leading cause of severe loss of vision in patients with age-related macular degeneration.

Inducible Expression of Vascular Endothelial Growth Factor in Adult Mice Causes Severe Proliferative Retinopathy and Retinal Detachment

Transgenic mice with vascular endothelial growth factor (VEGF) driven by the rhodopsin promoter (rho/VEGF mice) develop neovascularization that originates from the deep capillary bed of the retina and grows into the subretinal space. In rho/VEGF mice, VEGF expression in photoreceptors begins between postnatal days 5 and 7, the period when the deep capillary bed is developing. An important question is whether or not the developmental stage of the deep capillary bed is critical for occurrence of neovascularization. Also, although rho/VEGF mice are extremely useful for the study of ocular neovascularization, there are some applications for which the early onset of VEGF expression is a disadvantage. In this study, we used the reverse tetracycline transactivator (rtTA) inducible promoter system coupled to either the rhodopsin or interphotoreceptor retinoid-binding protein (IRBP) promoter to control the time of onset of VEGF transgene expression in photoreceptors. In the absence of doxycycline, adult double-transgenic rho/rtTA-TRE/VEGF or IRBP/rtTA-TRE/VEGF mice showed little VEGF transgene expression and no phenotype. The addition of doxycycline to the drinking water resulted in prominent transgene expression and evidence of neovascularization within 3 to 4 days. Like rho/VEGF mice, the neovascularization originated from the deep capillary bed of the retina, but it was more extensive and caused outer retinal folds followed by total retinal detachment. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay demonstrated that the mice with inducible expression of VEGF that developed retinal detachment had much higher ocular levels of VEGF mRNA and protein compared to rho/VEGF mice that manifest a much milder phenotype. These data demonstrate that regardless of developmental stage of the vascular bed, increased expression of VEGF in the retina is sufficient to cause neovascularization, and high levels of expression cause severe neovascularization and traction retinal detachment. Mice with inducible expression of VEGF in the retina provide a valuable new model of ocular neovascularization.

New steady-hand Eye Robot with micro-force sensing for vitreoretinal surgery

In retinal microsurgery, surgeons are required to perform micron scale maneuvers while safely applying forces to the retinal tissue that are below sensory perception. Real-time characterization and precise manipulation of this delicate tissue has thus far been hindered by human limits on tool control and the lack of a surgically compatible endpoint sensing instrument. Here we present the design of a new generation, cooperatively controlled microsurgery robot with a remote center-of-motion (RCM) mechanism and an integrated custom micro-force sensing surgical hook. Utilizing the forces measured by the end effector, we correct for tool deflections and implement a micro-force guided cooperative control algorithm to actively guide the operator. Preliminary experiments have been carried out to test our new control methods on raw chicken egg inner shell membranes and to capture useful dynamic characteristics associated with delicate tissue manipulations.

Phenotype and Genotype Characteristics of Age-related Macular Degeneration in a Japanese Population

PURPOSE To describe phenotype and genotype characteristics of age-related macular degeneration (AMD) in Japanese patients. DESIGN A case-control study. PARTICIPANTS A total of 550 case-control samples composed of 408 consecutive AMD cases and 142 controls. METHODS Clinical information assessing age, gender, affected eyes, fundus features, and fluorescein/indocyanine green angiograms were systematically evaluated. Four single nucleotide polymorphisms (SNPs; rs800292, rs1061170, rs1410996, rs2274700) in the complement factor H (CFH) gene, 1 SNP (rs11200638) in the high-temperature requirement factor A1 (HTRA1) gene, 3 SNPs (rs699947, rs1570360, rs2010963) in the vascular endothelial growth factor (VEGF) gene, and 4 SNPs (rs12150053, rs12948385, rs9913583, rs1136287) in the pigment epithelium-derived factor (PEDF) gene were assessed using TaqMan technology. MAIN OUTCOME MEASURES The clinical phenotype information and genotypes of CFH, HTRA1, VEGF, and PEDF polymorphisms. RESULTS Of Japanese patients with neovascular AMD (nAMD), 219 (58.7%) had typical nAMD and 154 (41.3%) had polypoidal choroidal vasculopathy (PCV). The frequency of bilateral exudative involvement was similar between typical nAMD (15.5%) and PCV (13.6%) (P = 0.613). Significant soft drusen were observed in the fellow eyes of 88 (47.6%) of 185 patients with unilateral typical nAMD and in 25 (18.8%) of 133 patients with unilateral PCV (P = 1.24x10(-7)). A serous pigment epithelium detachment was seen in 55 (25.1%) of 219 patients with typical nAMD and in 64 (41.6%) of 154 patients with PCV. A significant association was noted in CFH-rs800292, CFH-rs1410996, CFH-rs2274700, and HTRA1-rs11200638 with AMD development (P = 2.36x10(-5), 7.18x10(-5), 7.18x10(-5), 2.70x10(-7), respectively; population attributable risk = 57.3%, 57.8%, 57.8%, and 58.9%, respectively). We estimated the highest-risk group to have an approximately 70-fold greater risk of nAMD compared with the lowest-risk group when analyzing a combination of 4 SNPs in the CFH and HTRA1 genes. CONCLUSIONS The Japanese AMD phenotype is characterized by a higher frequency of PCV, male predominance, and lower frequency of bilateral presentation compared with Caucasian AMD. Genotype analyses demonstrate a significant population attributable risk for SNPs in the CFH and HTRA1 genes and demonstrate joint effects for both genes. Gene variants in both CFH and HTRA1 contribute significantly to the AMD phenotype in a Japanese population.

Comparison of epiretinal membranes of differing pathogenesis using optical coherence tomography.

Purpose To compare optical coherence tomography (OCT) images of idiopathic epiretinal membranes (ERMs) with those of secondary ERMs. Methods OCT was performed on 70 eyes of 63 consecutive patients with biomicroscopic evidence of ERMs and 23 eyes of 23 healthy volunteers without ERMs. OCT findings were correlated with the clinical pathogenesis of the ERM. Results Evaluation by OCT established that 48 of 70 ERMs were globally adherent to the retina and that 22 of 70 ERMs were focally adherent to the retina. When correlated to clinical pathogenesis, 20% of idiopathic membranes and 52% of secondary membranes were focally attached to the retina. There was a significant difference in the pattern of membrane attachment to the retina in the two pathogenic groups (P = 0.007). Eight of nine eyes with macular pseudoholes were associated with globally adherent membranes. Conclusion Secondary ERMs are more likely to be characterized by focal retinal adhesion than are primary ERMs. Primary ERMs tend to be globally adherent. This finding may contribute to understanding the underlying mechanisms of ERM formation in different clinical settings.

Periocular injection of an adenoviral vector encoding pigment epithelium-derived factor inhibits choroidal neovascularization

Gene transfer provides an exciting new approach for the treatment of retinal and choroidal diseases. Two areas of concern are the potential for vector-related toxicity and uncertainties associated with prolonged transgene expression. One way to address these concerns for transfer of genes encoding secreted proteins is to transduce cells on the outside of the eye, provided the gene product can gain access to the eye and have the desired effect. In this study, we investigated the feasibility of this approach. Periocular injection of an adenoviral vector encoding β-galactosidase (AdLacZ.10) resulted in LacZ-stained cells throughout the orbit and around the eye. Compared to periocular injection of 5 × 109 particles of control vector, periocular injection of 5 × 109 or 1 × 109 particles of an adenoviral vector expressing pigment epithelium-derived factor (PEDF) regulated by a CMV promoter (AdPEDF.11) resulted in significantly elevated intraocular levels of PEDF and suppression of choroidal neovascularization. Periocularly injected recombinant PEDF was also found to diffuse through the sclera into the eye. Although similar experiments are needed in an animal with a human-sized eye, these data suggest that periocular gene transfer deserves consideration for the treatment of choroidal diseases.

Periocular gene transfer of sFlt-1 suppresses ocular neovascularization and vascular endothelial growth factor-induced breakdown of the blood-retinal barrier

Vascular endothelial growth factor (VEGF) is a critical stimulus for both retinal and choroidal neovascularization, and for diabetic macular edema. We used mouse models for these diseases to explore the potential of gene transfer of soluble VEGF receptor-1 (sFlt-1) as a treatment. Intravitreous or periocular injection of an adenoviral vector encoding sFlt-1 (AdsFlt-1.10) markedly suppressed choroidal neovascularization at rupture sites in Bruchs membrane. Periocular injection of AdsFlt-1.10 also caused significant reduction in VEGF-induced breakdown of the blood-retinal barrier, but failed to significantly inhibit ischemia-induced retinal neovascularization. Periocular delivery of an adenoviral vector encoding pigment epithelium-derived factor (PEDF), another secreted protein, resulted in high levels of PEDF in the retinal pigmented epithelium and choroid, but not in the retina. This may explain why periocular injection of AdsFlt-1.10 inhibited choroidal, but not retinal neovascularization. Periocular delivery offers potential advantages over other routes of delivery and the demonstration that sFlt-1 enters the eye from the periocular space in sufficient levels to achieve efficacy in treating choroidal neovascularization and retinal vascular permeability is a novel finding that has important clinical implications. These data suggest that periocular gene transfer of sFlt-1 should be considered for treatment of choroidal neovascularization and diabetic macular edema.

Micro-force sensing in robot assisted membrane peeling for vitreoretinal surgery

Vitreoretinal surgeons use 0.5 mm diameter instruments to manipulate delicate tissue inside the eye while applying imperceptible forces that can cause damage to the retina. We present a system which robotically regulates user-applied forces to the tissue, to minimize the risk of retinal hemorrhage or tear during membrane peeling, a common task in vitreoretinal surgery. Our research platform is based on a cooperatively controlled microsurgery robot. It integrates a custom micro-force sensing surgical pick, which provides conventional surgical function and real time force information. We report the development of a new phantom, which is used to assess robot control, force feedback methods, and our newly implemented auditory sensory substitution to specifically assist membrane peeling. Our findings show that auditory sensory substitution decreased peeling forces in all tests, and that robotic force scaling with audio feedback is the most promising aid in reducing peeling forces and task completion time.

Single Fiber Optical Coherence Tomography Microsurgical Instruments for Computer and Robot-Assisted Retinal Surgery

We present initial prototype and preliminary experimental demonstration of a new class of microsurgical instruments that incorporate common path optical coherence tomography (CP-OCT) capabilities. These instruments may be used freehand or with robotic assistance. We describe a prototype 25 gauge microsurgical pick incorporating a single 125 microm diameter optical fiber interfaced to a Fourier Domain CP-OCT system developed in our laboratory. For initial experimentation, we have interfaced this instrument with an extremely precise, cooperatively controlled robot. We describe the tool, system design, and demonstration of three control methods on simple phantom models: 1) enforcement of safety constraints preventing unintentional collisions of the instrument with the retinal surface; 2) the ability to scan the probe across a surface while maintaining a constant distance offset; and 3) the ability to place the pick over a subsurface target identified in a scan and then penetrate the surface to hit the target.