αA-Crystallin inhibits optic nerve astrocyte activation induced by oxygen-glucose deprivation in vitro.

AIMS: A previous study reported that intravitreal injection of αA-crystallin inhibits glial scar formation after optic nerve traumatic injury. The purpose of this study was to investigate the effect of αA-crystallin on optic nerve astrocytes induced by oxygen glucose deprivation (OGD) in vitro. MATERIALS AND METHODS: Optic nerve astrocytes from newborn Long Evans rats were cultured with αA-crystallin (10-4 g/l) to detect the effects of αA-crystallin on astrocytes. Using a scratch assay, the effect of αA-crystallin treatment on astrocyte migration was assessed. Astrocytes were exposed to OGD and glucose reintroduction/reoxygenation culture for 24 h and 48 h. The expression of glial fibrillary acidic protein (GFAP) and neurocan were subsequently evaluated via immunocytochemistry and western blot. BMP2/4, BMPRIa/Ib and Smad1/5/8 mRNA expression levels were detected by RT-PCR. KEY FINDINGS: The results showed that αA-crystallin slowed the migration of astrocytes in filling the scratch gaps. GFAP and neurocan expression in astrocytes was increased after OGD. However, after treatment with αA-crystallin, GFAP and neurocan expression levels clearly decreased. Furthermore, RT-PCR showed that BMP2 and BMP4 mRNA expression levels decreased significantly. SIGNIFICANCE: These results suggest that αA-crystallin inhibits the activation of astrocytes after OGD injury in vitro. Inhibition of the BMP/Smad signaling pathway might be the mechanism underlying this effect.

Reduction of experimental diabetic vascular leakage and pericyte apoptosis in mice by delivery of αA-crystallin with a recombinant adenovirus.

AIMS/HYPOTHESIS: The study aimed to evaluate the efficacy of recombinant adenovirus expressing αA-crystallin (Ad-αAc-Gfp) in reducing pericyte loss within retinal vasculature in early diabetes. METHODS: Diabetes was induced by streptozotocin injection into C57BL/6 mice. Ad-αAc-Gfp was delivered by intravitreous injection to the right eyes of mice 2 weeks before induction of diabetes. Vascular leakage was determined by fluorescent angiography, Evans Blue leakage assay and leucocyte adhesion test. Production of αA-crystallin was analysed by immunoblotting and double immunostaining and pericyte loss was analysed by pericyte count. RESULTS: Vessel leakage and pericyte loss were observed in the streptozotocin-induced diabetic retina. Decreased abundance of αA-crystallin in retinas 2 and 6 months after the induction of diabetes was confirmed by two-dimensional electrophoretic analysis, immunoblotting and RT-PCR. Double immunofluorescence staining for αA-crystallin and NG2 chondroitin sulphate proteoglycan revealed that αA-crystallin was predominantly produced in the retinal pericyte and that the number of αA-crystallin-producing pericytes decreased in the diabetic retina. Retinal infection with Ad-αAc-Gfp led to decreased pericyte loss and vascular leakage compared with control. CONCLUSIONS/INTERPRETATION: Intravitreal delivery of Ad-αAc-Gfp protects against vascular leakage in the streptozotocin-induced model of diabetes. This effect is associated with the inhibition of diabetic retinal pericyte loss in early diabetes, suggesting that αA-crystallin has a role in preventing the pathogenesis of early diabetic retinopathy.

Small heat shock protein αA-crystallin prevents photoreceptor degeneration in experimental autoimmune uveitis.

The small heat shock protein, αA-crystallin null (αA-/-) mice are known to be more prone to retinal degeneration than the wild type mice in Experimental Autoimmune Uveoretinitis (EAU). In this report we demonstrate that intravenous administration of αA preserves retinal architecture and prevents photoreceptor damage in EAU. Interestingly, only αA and not αB-crystallin (αB), a closely related small heat shock protein works, pointing to molecular specificity in the observed retinal protection. The possible involvement of αA in retinal protection through immune modulation is corroborated by adaptive transfer experiments, (employing αA-/- and wild type mice with EAU as donors and Rag2-/- as the recipient mice), which indicate that αA protects against the autoimmune challenge by modulating the systemic B and T cell immunity. We show that αA administration causes marked reduction in Th1 cytokines (TNF-α, IL-12 and IFN-γ), both in the retina and in the spleen; notably, IL-17 was only reduced in the retina suggesting local intervention. Importantly, expression of Toll-like receptors and their associated adaptors is also inhibited suggesting that αA protection, against photoreceptor loss in EAU, is associated with systemic suppression of both the adaptive and innate immune responses.

αA-crystallin in the pathogenesis and intervention of experimental murine corneal neovascularization.

This study was to determine the potential roles of lens crystallins in the pathogenesis of corneal neovascularization (CorNV) and implications in therapy of CorNV-related diseases. Suture- or chemical burn-induced CorNV in different strains of mice were used. Changes of gene expression patterns were analyzed by microarray, and the results of interesting genes were confirmed by real-time quantitative PCR and Western blot. Mice deficient in αA-crystallin gene were used to evaluate the role of αA-crystallin in the development of CorNV. In some animals, exogenous αA-crystallin proteins were injected around time of CorNV induction. CorNV was assessed by slit-lamp, flat-mounts and histology. In BALB/C mice, the expression of α-, ß-, and γ-crystallins were up-regulated at day 5 and returned to baseline level at day 10 of suture-induced CorNV, but remained up-regulated from day 6 to day 14 of chemical burn-induced CorNV. In chemical burn-induced CorNV in C57BL/6J mice, however, they were down-regulated at day 6. Corneal crystallins were down-regulated in both CorNV models at all time points in both BALB/c and C57BL/6J mice. Comparison of CorNV development in αA-crystallin-deficient mice and that in wild-type mice revealed no significant difference. Subconjunctival injection of αA-crystallin significantly attenuated suture-induced CorNV, and the inhibitory activity might be implemented by the increased expression of soluble VEGFR-1. In conclusion, the expression patterns of lens crystallins were time- and strain-dependent but different from that of corneal crystallins in mouse CorNV models. Exogenous αA-crystallin protein attenuated CorNV, potentially by increasing the expression of soluble VEGFR-1.