Alexander Kennedy

Dendrimer-based targeted intravitreal therapy for sustained attenuation of neuroinflammation in retinal degeneration.

Retinal neuroinflammation, mediated by activated microglia, plays a key role in the pathogenesis of photoreceptor and retinal pigment epithelial cell loss in age-related macular degeneration and retinitis pigmentosa. Targeted drug therapy for attenuation of neuroinflammation in the retina was explored using hydroxyl-terminated polyamidoamine (PAMAM) dendrimer-drug conjugate nanodevices. We show that, upon intravitreal administration, PAMAM dendrimers selectively localize within activated outer retinal microglia in two rat models of retinal degeneration, but not in the retina of healthy controls. This pathology-dependent biodistribution was exploited for drug delivery, by covalently conjugating fluocinolone acetonide to the dendrimer. The conjugate released the drug in a sustained manner over 90 days. In vivo efficacy was assessed using the Royal College of Surgeons (RCS) rat retinal degeneration model over a four-week period when peak retinal degeneration occurs. One intravitreal injection of 1 μg of FA conjugated to 7 μg of the dendrimer was able to arrest retinal degeneration, preserve photoreceptor outer nuclear cell counts, and attenuate activated microglia, for an entire month. These studies suggest that PAMAM dendrimers (with no targeting ligands) have an intrinsic ability to selectively localize in activated microglia, and can deliver drugs inside these cells for a sustained period for the treatment of retinal neuroinflammation.

ATP causes retinal pericytes to contract in vitro

We evaluated the contractility of bovine retinal microvascular pericytes in culture by permeabilizing the cells with 0.1% Triton X-100 and measuring their response to MgATP. Sequential photographs of the cells were taken over 20 min and their surface areas were measured. Our study directly demonstrates that pericytes are contractile cells, which respond to MgATP in a dose-dependent fashion over a relatively short time course (minutes). Pericytes did not contract in response to GTP, pyrophosphate or beta, gamma-methylene ATP. Immunofluorescence study showed the presence of muscle actin in Triton X-100-treated cells before and after contraction, indicating preservation of this cytoskeletal protein even after treatment with the detergent. Similar experiments on human umbilical vein endothelial cells, bovine lens epithelial cells and human retinal pigment epithelial cells showed that these cells were significantly less contractile than retinal pericytes. That pericytes show substantial contraction over a short time course indicates that these cells may play a major role in regulating blood flow in the microcirculation.

Photoreceptor Neuroprotection in RCS Rats via Low-Dose Intravitreal Sustained-Delivery of Fluocinolone Acetonide

PURPOSE To study the neuroprotective effects of intravitreal fluocinolone acetonide (FA) in Royal College of Surgeons (RCS) rats. METHODS Five-week-old RCS rats were divided into four groups: 0.5 microg/d FA-loaded intravitreal drug-delivery implant (IDDI); 0.2 microg/d FA-loaded IDDI; inactive IDDI; and nonsurgical control. Electroretinography (ERG) and intraocular pressure (IOP) measurements were performed before surgery and weekly thereafter. Thicknesses of the retinal outer (ONL) and inner (INL) nuclear layers were evaluated at 9 weeks of age. ED-1-labeled activated microglia were counted. Total microglial cell counts were made by using Iba-1 antibody labeling. RESULTS At 9 weeks, control groups demonstrated an 80% reduction in ERG amplitudes (P < 0.001 for both groups). FA-treated groups demonstrated no statistically significant attenuation of ERG amplitudes at the end of the study, compared with the initial ERGs. Intraocular pressure (IOP) remained normal in all groups. ONL thickness in FA 0.2 microg/d-treated eyes was 2.1 +/- 0.5 times greater than in nonsurgical eyes (P < 0.001) and 3.4 +/- 0.7 times greater than in inactive IDDI-treated eyes (P < 0.0001). In FA 0.5 microg/d-treated eyes, ONL thickness was 1.5 +/- 0.1 times higher than in nonsurgical controls (P < 0.05) and 2.4 +/- 0.4 times higher than in inactive IDDI-treated eyes (P < 0.01). INL thickness was not different among groups. FA-treated eyes demonstrated significantly fewer activated microglia (P < 0.001) and overall number of microglia in the photoreceptor and outer debris zone layers (P < 0.001), compared with control groups. CONCLUSIONS Chronic intravitreal infusion of FA is neuroprotective in RCS rats, preserves ONL morphology and ERG amplitudes and reduces retinal neuroinflammation. These findings may have a therapeutic role in human photoreceptor cell degenerations.

Collagens of the retinal microvascular basement membrane and of retinal microvascular cells in vitro

We have analysed the collagens present in vascular basement membranes isolated from bovine retinal and cerebral microvessels and bovine renal glomeruli, and from the non-vascular basement membrane of bovine lens capsule. These are compared with the collagens produced by cultured bovine retinal microvascular pericytes and lens epithelial cells, and by canine retinal microvascular endothelial cells, in vitro. Biochemical and immunocytochemical analyses indicate that all of the vascular basement membrane preparations have an identical collagenous composition, consisting of the same polypeptides present in lens capsule (primarily type IV collagen), together with other polypeptides that are identified as type I, and a small amount of type III collagen. Identification of the latter is based on two-dimensional gel electrophoresis in the presence and absence of a reducing agent. Immunocytochemical studies, however, demonstrate type I, type IV and some type V collagen in the basement membranes of the isolated microvessels. The cultured microvascular cells produce predominantly type I collagen molecules, but they also produce other collagen peptides that appear to be type IV, and, at least in some experiments, small amounts of type III collagen. The biochemical identification of collagens type I and IV is confirmed by immunocytochemistry. However, results with anti-type I collagen and procollagen antibodies in cultured pericytes vary with antibodies from different sources. The quantities of the type IV peptides produced by the cultured cells also vary in different experiments.

The Influence of Glucose Concentration and Hypoxia on VEGF Secretion by Cultured Retinal Cells

Purpose: To study the relationship of oxygen level and glucose concentration on the secretion of vascular endothelial growth factor (VEGF) mRNA and protein in several types of cultured retinal cells. Materials and Methods: Several types of human and bovine retinal cells were cultured in medium without glucose, or containing 5 mM or 25 mM D-glucose or 5 mM D-glucose and 20 mM D-galactose. Cells were cultured in 20% O2 (“normoxia”) or in 1% O2 (“hypoxia”). After being cultured for 8–96 hr, we measured VEGF protein in the medium and VEGF mRNA in the cell layer, as well as the concentrations of glucose, lactate, and pyruvate in the medium. Results: Hypoxia increased VEGF mRNA and protein in these cells. In normoxia, culture in high glucose medium had no significant effect on basal VEGF production in normal glucose. However, culture in hypoxia and high glucose significantly blunted hypoxic VEGF up-regulation. Culture in normoxia, with no glucose in the medium, significantly increased VEGF. Culture in high galactose medium did not significantly affect VEGF production. Despite considerable lactate production, especially in the presence of 25 mM glucose, addition of strong buffers to the medium had little effect on VEGF production. Conclusions: Cultured retinal cells up-regulate their VEGF production when their energy supply, including glucose and/or O2, is inadequate. Supplying glucose to the cells in the presence of low O2 reduces their VEGF production. We suggest that “early worsening” of retinopathy results when diabetic patients with minimal to moderate retinopathy, whose retinal circulation and, hence, retinal oxygen supply is compromised, are placed on a “tight” glucose control regimen and their major remaining retinal energy source is reduced, with VEGF up-regulation as a compensatory mechanism.

Galactitol accumulation by glucose-6-phosphate deficient fibroblasts: A cellular model for resistance to the complications of diabetes mellitus

When incubated in high galactose media, fibroblasts from individuals with the severe (Mediterranean) variety of glucose-6-phosphate dehydrogenase (G6PD) deficiency accumulate significantly less galactitol than do fibroblasts from matched control subjects. The effect is not observed in fibroblasts from black subjects with the more common, and milder, A- variant of G6PD deficiency. Since aldose reductase and sorbitol dehydrogenase activities in experimental and control fibroblasts are identical, the effect is most likely due to the substantial reduction in NADPH levels in severely G6PD-deficient cells. Sorbitol does not accumulate either in control or in G6PD deficient fibroblasts incubated in high glucose medium, most likely because of the action of sorbitol dehydrogenase, and the presence of a carrier-mediated glucose transport system in the cell membrane which limits the concentration of glucose that can accumulate in these cells.