Impairment of retinal vascular homeostasis is associated with the development and progression of diabetic retinopathy involving gap junction intercellular communication (GJIC) activity. The principal gap junction protein of intercellular communication, connexin, was investigated to determine the effects of high glucose concentrations on the expression of endothelial-specific connexins (Cx37, Cx40, and Cx43), connexin phosphorylation pattern, and GJIC activity. Rat microvascular endothelial (RME) cells grown in high (30 mmol/l)-glucose medium for 9 days had reduced Cx43 expression: Cx43 mRNA (68 ؎ 13% of control; P ؍ 0.019, n ؍ 5) and protein (55.6 ؎ 16% of control; P ؍ 0.003, n ؍ 5) levels were reduced; however, Cx37 and Cx40 expression was not affected. Using alkaline phosphatase and Western blot analyses, we identified three forms of Cx43: a nonphosphorylated form (P0) and two phosphorylated forms (P1 and P2). Expression of all three forms was decreased in cells grown in high-glucose medium: PO, 73 ؎ 15% of control (P ؍ 0.04); P1, 57 ؎ 16% of control (P ؍ 0.01); and P2, 42 ؎ 22% of control (P ؍ 0.006). Using immunofluorescence microscopy, we observed Cx43 localization at specific sites of contact (plaques) between adjacent cells. In cells grown in highglucose medium, we observed reduced plaque counts (63 ؎ 6% of control; P ؍ 0.009) and decreased intensity of Cx43 immunofluorescence compared with cells grown in normal medium. Furthermore, using scrape load dye transfer (SLDT) technique, we found that these cells exhibited reduced GJIC activity (60% of control; P ؍ 0.01, n ؍ 5). The reduction in GJIC activity correlated with the decreased Cx43 protein levels (r ؍ 0.9). These results indicate that high glucose concentrations inhibited GJIC activity by reducing Cx43 synthesis in RME cells. Impaired intercellular communication may contribute to breakdown of homeostatic balance in diabetic microangiopathy. Diabetes 51:1565-1571, 2002
Overexpression of extracellular matrix (ECM) components is closely associated with the development of vascular basement membrane (BM) thickening, a histological hallmark of diabetic microangiopathy. To determine whether BM thickening of retinal capillaries could be prevented by down regulating synthesis of fibronectin, an ECM component, we used antisense oligos targeted against translation initiation site of the fibronectin transcript in galactose-fed rat, an animal model of diabetic retinopathy. After 2 months of galactose-feeding, intravitreal administration of 3 mol/l antisense fibronectin oligos was initiated at monthly intervals for 3 months. The antisense strategy significantly reduced fibronectin mRNA and protein level in the retinas of treated eyes compared with untreated eyes of galactose-fed rats (130 ؎ 16 vs. 179 ؎ 18% of control, P < 0.01, and 144 ؎ 28 vs. 204 ؎ 22% of control, respectively, r ؍ 0.9) and resulted in partial reduction of retinal capillary BM width (123 ؎ 16 vs. 201 ؎ 12 nm, P < 0.03). In eyes treated with antisense fibronectin oligos, ϳ35% reduction in both pericyte loss and acellular retinal capillaries was observed (P < 0.04 and P < 0.03, respectively). Glycohemoglobin level was consistently elevated in the treated (6.9 ؎ 0.6%) and untreated (6.5 ؎ 0.7%) galactose-fed rats compared with control rats (4.5 ؎ 0.8%). Overall, these results indicate that downregulation of fibronectin synthesis reduces BM thickening in retinal capillaries with beneficial effect to retinal lesions. The antisense fibronectin oligos may provide a useful approach for reducing vascular lesions in diabetic retinopathy. The thickened vascular BM may be a potential therapeutic target for preventing retinal lesions in diabetic retinopathy. Diabetes 52: 1229 -1234, 2003
alpha-Sarcin, mitogillin, and restrictocin are small (approximately 17 kDa) basic robosome-inactivating proteins (RIPs) produced by the Aspergilli that catalytically inactivate the large ribosomal subunits of all organisms tested to date. These three fungal ribotoxins act as specific ribonucleases by hydrolyzing one single phosphodiester bond in the universally conserved alpha-sarcin domain of 23-28S rRNAs and are among the most potent inhibitors of protein synthesis known. Previous molecular studies of ribotoxins indicated that they belong to the superfamily of ribonucleases and analysis of the mitogillin gene employing PCR-mediated site-specific mutagenesis suggests that certain domains in ribotoxins, which share homologies with motifs in ribosome-related proteins, may be responsible for the targeting of ribotoxins to the ribosome. The applications of the ribotoxins as tools in research and their uses as therapeutic and diagnostic agents are also reviewed in this paper.
Mitogillin and the related Aspergillus fungal ribotoxins restrictocin and ␣-sarcin, are small basic proteins of ϳ17 kDa (kilodaltons). Mitogillin differs from restrictocin by only 1 amino acid and has 86% amino acid sequence identity with ␣-sarcin (1-4). The fungal ribotoxins are a family of highly specific ribonucleases which inactivate the ribosome by cleavage of the 23-28 S RNA of the large ribosomal subunit (5) at a single phosphodiester bond. The site of cleavage occurs between G 4325 and A 4326 (rat ribosome numbering) in a 14-base sequence (the ␣-sarcin loop) found in the large subunit ribosomal RNAs (rRNAs)
Ribotoxins are a family of potent cytotoxic proteins from Aspergillus whose members display a high sequence identity (85% for about 150 amino acid residues). The three-dimensional structures of two of these proteins, ␣-sarcin and restrictocin, are known. They interact with phospholipid bilayers, according to their ability to enter cells, and cleave a specific phosphodiester bond in the large subunit of ribosome thus inhibiting protein biosynthesis. Two nonconservative sequence changes between these proteins are located at the amino-terminal -hairpin of ␣-sarcin, a characteristic structure that is absent in other nontoxic structurally related microbial RNases. These two residues of ␣-sarcin, Lys 11 and Thr 20, have been substituted with the equivalent amino acids in restrictocin. The single mutants (K11L and T20D) and the corresponding K11L/T20D double mutant have been produced in Escherichia coli and purified to homogeneity. The spectroscopic characterization of the purified proteins reveals that the overall native structure is preserved. The ribonuclease and lipid-perturbing activities of the three mutants and restrictocin have been evaluated and compared with those of ␣-sarcin. These proteins exhibit the same ability to specifically inactivate ribosomes, although they show different activity against nonspecific substrate analogs such as poly(A). The mutant variant K11L and restrictocin display a lower phospholipid-interacting ability correlated with a decreased cytotoxicity. The results obtained are interpreted in terms of the involvement of the amino-terminal -hairpin in the interaction with both membranes and polyadenylic acid.
We have investigated whether antisense oligonucleotides delivered intravitreally could reduce gene expression specifically in the retina. In this study, phosphorothioate antisense oligonucleotides targeted to fibronectin transcripts were coupled to a novel carrier and used to specifically reduce fibronectin (FN) expression in retinal vascular cells. Using confocal microscopy, fluorescence from fluorescein isothio-cyanate-labeled FN-oligonucleotides was detected in retinal vascular cells at 24 h postinjection and persisted until day 6 (the end point of this study). The fibronectin mRNA level was consistently decreased to 86.7% +/- 7.9% of control (p<0.05) at day 2, and 46.7% +/- 4.9% of control (p<0.01) at day 6. In contrast, the beta-actin mRNA level, an internal control, was unaltered in rat retinas that received FN-oligonucleotides. Fibronectin protein level at day 6 was also significantly reduced to 61.4% +/- 16% of control (p<0.01). No toxic effect resulting from the carrier was detected histologically. Thus, intravitreal delivery of antisense oligonucleotides to modulate abnormal gene expression in retinal diseases may be an effective approach for ocular gene therapy.
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