Placental Growth Factor Contributes to Micro-Vascular Abnormalization and Blood-Retinal Barrier Breakdown in Diabetic Retinopathy

OBJECTIVE: There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1). MATERIALS AND METHODS: pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis. RESULTS: After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation. CONCLUSION: This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease

Etude du rôle de la PKC Zeta dans la rétinopathie diabétique

PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Ischemic Retinopathies: Oxidative Stress and Inflammation

Ischemic retinopathies (IRs), such as retinopathy of prematurity (ROP), diabetic retinopathy (DR), and (in many cases) age-related macular degeneration (AMD), are ocular disorders characterized by an initial phase of microvascular changes that results in ischemia, followed by a second phase of abnormal neovascularization that may culminate into retinal detachment and blindness. IRs are complex retinal conditions in which several factors play a key role during the development of the different pathological stages of the disease. Increasing evidence reveals that oxidative stress and inflammatory processes are important contributors to the pathogenesis of IRs. Despite the beneficial effects of the photocoagulation and anti-VEGF therapy during neovascularization phase, the need to identify novel targets to prevent initial phases of these ocular pathologies is still needed. In this review, we provide an update on the involvement of oxidative stress and inflammation in the progression of IRs and address some therapeutic interventions by using antioxidants and anti-inflammatory agents

PKCζ Mediates Breakdown of Outer Blood-Retinal Barriers in Diabetic Retinopathy.

AIMS/HYPOTHESIS: Diabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia. METHODS: Studies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor. RESULTS: PKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death. CONCLUSIONS: In the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology

Immunolocalization of PKCζ and occludin in retinal pigment epithelium (RPE) flatmounts.

<p>In 6-month-old controls, PKCζ staining (red) appeared as focal spots within the loops depicted by occludin (green) protrusions (a). In age-matched diabetic conditions, no PKCζ labeling (box and green arrow) was observed (b). After 12 month of diabetes marked disruption of intercellular junctions (arrows) was evidenced (c). At this stage the PKCζ at the junctions (TJ)/total amount of PKCζ ratio was significantly decreased for diabetic conditions (Image J software, National Institutes of Health, Bethesda, MD) (d) and occludin internalization from the cell membrane into the cytoplasm was found (e). The colocalization of PKCζ (red) and PKCζ-P (green) found in 12-month-old controls (f) was not present in age-matched diabetics (g). Furthermore the activated PKCζ-P form exhibited a discontinuous staining along the cell membrane (g).</p

Alteration of PKCζ distribution in the outer retina is associated with cone outer segment and OLM disruption in diabetic rats.

<p>PKCζ staining was located in inner segments (IS) of rod and cones and exclusively in cone OS as evidenced by PKCζ-PNA double labeling in 6-month-old control rats (a). In 6-month-old diabetic rats, no OS staining was detected (a). Furthermore PKCζ staining (red) was lost in S-cones, specifically marked by Blue opsin staining (green), as compared to controls. Some S-cone OS also showed marked structural alterations, suggesting early photoreceptor degeneration (b). In 12-month-old diabetic rats OLM discontinuity (arrows) was evidenced (c). OLM tight-junction disruption was further confirmed on retinal flatmounts by an occludin/PKCζ double staining (d, white arrows).</p

Diabetes destabilizes and down regulates PAR3/PAR6 PKCζ-associated protein complex.

<p>a) Upper panels: In 12-month-old diabetic rats (DIA) PAR6 distribution in flatmount RPE showed a clear cytoplasmic relocation of PAR6 compared to age-matched control rats (CTL) where PAR6 appeared as relative regular punctiform staining at the TJ levels. The same applied to PAR3 staining distribution (Lower panels). b) PAR6 and PAR3 immunoblotting on RPE cell extracts from 12-month-old rats showed a significant decrease of both protein levels in diabetic conditions (statistical analysis was performed on the PAR3 180 KDa isoform).</p

Characteristics of the study animals.

<p>a: diabetes versus controls, P value<0.05.</p

Diabetes stage-specific retinal pigment epithelium (RPE) level of PKCζ and its activated phosphorylated form PKCζ-P T410.

<p>No significant changes were observed for PKCζ level at any stage between control and diabetic conditions. To the contrary, PKCζ-P T410 immunoreactivity was significantly (*P<0.05, Mann–Whitney test) increased (by around 40%) at 6 months and then decreased (by around 60%) at 12 months of diabetes compared to controls.</p