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

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

<i>In-vivo</i> PKCζ inhibition in 6-month-old-diabetic rats partially restores PKCζ staining pattern in cone outer segments.

<p>The phosphorylated active form PKCζ-P (green) was found in IS of both control (CTL) and diabetic (DIA) rats (a). Diabetic inhibitor (DIA+IZ) treated rats showed partial restoration of OS PKCζ (red) staining (a). We performed laser micro dissection of the photoreceptor layer on cryosections (b) to study layer-specific protein level by Western blotting analysis. The PKCζ-P immunoreactivity increase found in diabetic rats (DIA) was partially restored in treated rats (DIA+IZ).</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

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

Diabetes induces outer retinal edema and loss of cones photoreceptors.

<p>Diabetic retina (DIA) demonstrated increased extracellular spaces within the outer nuclear layer (ONL) and photoreceptor segment disorganization, consistent with an edematous aspect of the outer retina (Historesin (a) and semithin sections (b)). Peanut agglutinin labeling, a specific marker of cone extracellular matrix, evidenced a marked decreased in cell cone density (c). Whole retina quantification of cones evaluated the net loss to be around 20% (d) as compared to controls (p = 0.002, Mann–Whitney test).</p

PKCζ specifically regulates NF-κB signaling pathway which participates to diabetes-induced cone photoreceptor degeneration.

<p>In 6-month-old rat cryosections, the p65-P subunit of NF-κB (red) was only detected in diabetic (DIA) conditions in some nuclei of the outer nuclear layer (<i>arrows</i>) and not in controls (CTL) or treated rats (DIA+IZ). The triple staining with addition of PNA (green), a specific cone marker, confirmed that the nuclear translocation p65-P subunit of NF-κB exclusively occurred in cones (a). In age-matched rat cryosections, TUNEL assay confirmed apoptosis of photoreceptors in diabetic (DIA) conditions (b).</p

Influences of Histidine‑1 and Azaphenylalanine‑4 on the Affinity, Anti-inflammatory, and Antiangiogenic Activities of Azapeptide Cluster of Differentiation 36 Receptor Modulators

Azapeptide analogues of growth hormone releasing peptide-6 (GHRP-6) exhibit promising affinity, selectivity, and modulator activity on the cluster of differentiation 36 receptor (CD36). For example, [A¹, azaF⁴]- and [azaY⁴]-GHRP-6 (<b>1a</b> and <b>2b</b>) were previously shown to bind selectively to CD36 and exhibited respectively significant antiangiogenic and slight angiogenic activities in a microvascular sprouting assay using choroid explants. The influences of the 1- and 4-position residues on the affinity, anti-inflammatory, and antiangiogenic activity of these azapeptides have now been studied in detail by the synthesis and analysis of a set of 25 analogues featuring Ala¹ or His¹ and a variety of aromatic side chains at the aza-amino acid residue in the 4-position. Although their binding affinities differed only by a factor of 17, the analogues exhibited significant differences in ability to modulate production of nitric oxide (NO) in macrophages and choroidal neovascularization

PGF expression in diabetic retina.

<p><b>(a–b) Comparison of PGF staining in non diabetic (a) and diabetic (b) retinas.</b> (<b>a</b>) Sections of eyes from adult control non diabetic rat showed co-expression of PGF and GFAP in glial Muller cells from the gcl (arrowheads) to the inl, and PGF expression in glial Müller cells which are not immuno-reactive for GFAP (white star). Scale bar = 50 µm. (<b>b</b>) A similar pattern was observed on retinal sections of eyes from three-month-old diabetic rats, with a strong immuno-reactivity for PGF at the gcl level. <b>(c) PGF detection by Western-blot in diabetic and non-diabetic retinas, from 1, 2, 5 and 12 month-old rats</b>. For each lane in which 40 µg of proteins were deposited, the blood sugar level of the represented rats is indicated between parentheses. <b>(d–e) Immunostaining for PGF and GFAP in sections from pVAX2-rPGF-1 ET- treated diabetic rat eyes, one month after ET.</b> Sections show PGF-expressing infiltrating cells in the sub retinal space (<b>d</b>, arrows) and confirmed PGF expression by RMG cells (<b>d</b>, arrowheads). GFAP staining showed gliosis induced by RMG cells (<b>d, e</b>). <b>ch</b>, choroid; <b>gcl</b>, ganglion cell layer; <b>inl,</b> inner nuclear layer; <b>ipl</b>, inner plexiform layer; <b>onl</b>, outer nuclear layer; <b>rpe</b>, retinal pigmented epithelium; Scale bar = 100 µm.</p