Retinal pigment epithelium is protected against apoptosis by

PURPOSE. The degeneration of retinal pigment epithelial (RPE) cells is considered to be a crucial event in the pathophysiology of age-related macular degeneration (AMD). Cumulative oxidative damage has been implicated in the development of the changes seen in AMD. The present study was undertaken to evaluate the expression of the small heat shock protein ␣B-crystallin in the RPE in response to oxidative stress and to explore whether ␣B-crystallin expression confers an antiapoptotic cytoprotective effect on RPE cells. METHODS. Native human RPE cells from the macula and retinal periphery were analyzed by RT-PCR and Western blot analysis for expression of ␣B-crystallin. Monolayer cultures of human RPE cells were stressed by heat shock (42°C for 20 minutes) or oxidant-mediated injury (50 -300 M H 2 O 2 for 1 hour). Induction of ␣B-crystallin and the corresponding mRNA was assessed by Western and Northern blot analyses. To study the cytoprotective effect of ␣B-crystallin, human RPE cells were transfected with either a neomycin-selectable expression vector containing ␣B-crystallin cDNA or a control vector without ␣B-crystallin cDNA. Caspase-3 activity was determined by observing the cleavage of a colorimetric peptide substrate. Cell viability was quantified by combined propidium iodide and Hoechst 33342 staining. RESULTS. ␣B-crystallin is constitutively expressed in RPE under in vivo and in vitro conditions. Western blot analysis of freshly isolated RPE showed greater baseline expression levels in RPE derived from the macular area than in that from the more peripheral regions. Heat shock treatment and oxidative stress caused a significant increase in ␣B-crystallin mRNA and protein. Oxidant-mediated injury in RPE cells with baseline expression levels of ␣B-crystallin resulted in apoptotic cell death, as measured by caspase-3 activity, whereas RPE cells that had been stably transfected with ␣B-crystallin were more resistant to H 2 O 2 -induced cellular injury. CONCLUSIONS. ␣B-crystallin may function as a stress-inducible antiapoptotic protein in human RPE and is inducible by oxidative stress, a condition implicated in the pathogenesis of AMD. Overexpression of ␣B-crystallin may be an important mechanism for the RPE to prevent apoptotic cell death in response to cellular stress. (Invest Ophthalmol Vis Sci. 2002;43:3575-3582) A ge-related macular degeneration (AMD) is the leading cause of severe visual impairment in elderly individuals

Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies

Pulsed electron avalanche knife: new technology for cataract surgery

Background: The pulsed electron avalanche knife (PEAK-fc) is a new pulsed electrosurgical device that allows for precise, ``cold'' and traction-free tissue dissection.Aim: To evaluate the surgical applicability, safety and potential complications of PEAK-fc in complicated cataract surgery.Methods: The study included five children with congenital cataracts, two patients with advanced senile cataracts, six adults with mature cataracts, three of them with posterior iris synechia, three patients with post-traumatic cataracts with zonulolysis, one patient with intumescent traumatic cataract and three patients with massive anterior capsule opacification. Anterior and posterior capsulotomies, iris synechiolysis, dissection of anterior capsule opacification and fibrotic scar tissue were performed. PEAK-fc was set at voltages of 500--700 V, pulse duration of 0.1 m and repetition rate of 40--100 Hz.Results: Anterior and posterior capsulotomies were successfully and safely performed in all eyes. The edges of capsulotomies appeared sharp, showing only limited collateral damage. PEAK-fc worked best by just gently touching the capsule, thereby avoiding tractional forces or pressure on the lens capsule. Posterior iris synechiae could be released and anterior capsule opacification was dissected without complications.Conclusions: PEAK-fc is a very helpful cutting device for complicated cases of cataract surgery, especially for mature and congenital cataracts, traumatic zonulolysis or anterior segment complications after intraocular inflammation