Alexander A. Veenstra

Photoreceptor cells are major contributors to diabetes-induced oxidative stress and local inflammation in the retina

Significance Studies of diabetic retinopathy have focused especially on the retinal vasculature, but recent studies suggest that the neural retina also is involved. Oxidative stress and local inflammatory changes have been shown to play important roles in the pathogenesis of this retinopathy, but the source of reactive oxygen species has been less clear. We now show that most of the oxidative stress in retinas of diabetic mice emanates from neural photoreceptor cells, and elimination of these cells in diabetes inhibits both the oxidative stress and inflammatory changes shown to cause the vascular lesions of diabetic retinopathy. These studies suggest a mechanism by which neural cells can initiate the vascular injury characteristic of diabetic retinopathy. Accumulating evidence suggests that photoreceptor cells play a previously unappreciated role in the development of early stages of diabetic retinopathy, but the mechanism by which this occurs is not clear. Inhibition of oxidative stress is known to inhibit the vascular lesions of early diabetic retinopathy, and we investigated whether the diabetes-induced oxidative stress in the retina emanates from photoreceptors. Superoxide generation was assessed in retinas of male C57BL/6J mice made diabetic for 2 mo (4 mo of age when killed) using histochemical (dichlorofluorescein and dihydroethidine) and bioluminescence (lucigenin) methods. Photoreceptors were eliminated in vivo by genetic (opsin−/−) and chemical (iodoacetic acid) techniques. Immunoblots were used to measure expression of intercellular adhesion molecule 1 and the inducible form of nitric oxide synthase. Diabetes increased the generation of superoxide by diabetic mouse retina more at night than during the day. Photoreceptors were the major source of reactive oxygen species in the retina, and their deletion (either genetically in opsin−/− mice or acutely with iodoacetic acid) inhibited the expected diabetes-induced increase in superoxide and inflammatory proteins in the remaining retina. Both mitochondria and NADPH oxidase contributed to the observed retinal superoxide generation, which could be inhibited in vivo with either methylene blue or apocynin. Photoreceptors are the major source of superoxide generated by retinas of diabetic mice. Pharmaceuticals targeting photoreceptor oxidative stress could offer a unique therapy for diabetic retinopathy.

Human endothelial cell growth and coagulant function varies with respect to interfacial properties of polymeric substrates

The in vitro coagulant function of human aortic endothelial cells (HAECs) was investigated when grown on a series of polymer surfaces that ranged from hydrophobic to hydrophilic. The polymer interface materials were prepared by radiofrequency plasma polymerization from hexamethyl-disilazane, gamma-butyrolactone, and N-vinyl-2-pyrrolidone and deposited onto tissue culture Permanox. The three plasma polymers were noncytotoxic. When precoated with fibronectin (FN), HAECs on all four polymer surfaces were similar with respect to cell proliferation and coagulant function. Without FN precoating, cell proliferation and spreading increased with increasing surface hydrophilicity. Normalized production of tissue-type plasminogen activator increased with increasing hydrophilicity of the polymers during early incubation times, as did tissue plasminogen activator/plasminogen activator inhibitor-1 ratios. In comparison, normalized von Willebrand factor release decreased on the more hydrophilic surfaces. Thus, both endothelial cell growth and some coagulant/fibrinolytic functions are improved with increasing substrate hydrophilicity.

Marrow-Derived Cells Regulate the Development of Early Diabetic Retinopathy and Tactile Allodynia in Mice

The hypothesis that marrow-derived cells, and specifically proinflammatory proteins in those cells, play a critical role in the development of diabetes-induced retinopathy and tactile allodynia was investigated. Abnormalities characteristic of the early stages of retinopathy and allodynia were measured in chimeric mice lacking inducible nitric oxide synthase (iNOS) or poly(ADP-ribosyl) polymerase (PARP1) in only their marrow-derived cells. Diabetes-induced capillary degeneration, proinflammatory changes, and superoxide production in the retina and allodynia were inhibited in diabetic animals in which iNOS or PARP1 was deleted from bone marrow cells only. Of the various marrow cells, neutrophils (and monocytes) play a major role in retinopathy development, because retinal capillary degeneration likewise was significantly inhibited in diabetic mice lacking the receptor for granulocyte colony-stimulating factor in their marrow-derived cells. Immunodepletion of neutrophils or monocytes inhibited the endothelial death otherwise observed when coculturing leukocytes from wild-type diabetic animals with retinal endothelium. iNOS and PARP1 are known to play a role in inflammatory processes, and we conclude that proinflammatory processes within marrow-derived cells play a central role in the development of diabetes complications in the retina and nerve.

The CorA Mg2+ transporter is a homotetramer

CorA is a primary Mg2+ transporter for Bacteria and Archaea. The C-terminal domain of approximately 80 amino acids forms three transmembrane (TM) segments, which suggests that CorA is a homo-oligomer. A Cys residue was added to the cytoplasmic C terminus (C317) of Salmonella enterica serovar Typhimurium CorA with or without mutation of the single periplasmic Cys191 to Ser; each mutant retained function. Oxidation of the Cys191Ser Cys317 CorA gave a dimer. Oxidation of Cys317 CorA showed a dimer plus an additional band, apparently cross-linked via both Cys317 and C191. To determine oligomer order, intact cells or purified membranes were treated with formaldehyde or carbon disulfide. Higher-molecular-mass bands formed, consistent with the presence of a tetramer. Cross-linking of the Bacillus subtilis CorA expressed in Salmonella serovar Typhimurium similarly indicated a tetramer. CorA periplasmic soluble domains from both Salmonella serovar Typhimurium and the archaeon Methanococcus jannaschii were purified and shown to retain structure. Formaldehyde treatment showed formation of a tetramer. Finally, previous mutagenesis of the CorA membrane domain identified six intramembrane residues forming an apparent pore that interacts with Mg2+ during transport. Each was mutated to Cys. In mutants carrying a single intramembrane Cys residue, spontaneous disulfide bond formation that was enhanced by oxidation with Cu(II)-1,10-phenanthroline was observed between monomers, indicating that these Mg2+-interacting residues within the membrane are very close to their cognate residue on another monomer. Thus, CorA appears to be a homotetramer with a TM segment of one monomer physically close to the same TM segment of another monomer.

Functional Characterization of Escherichia coli GlpG and Additional Rhomboid Proteins Using an aarA Mutant of Providencia stuartii

The Providencia stuartii AarA protein is a member of the rhomboid family of intramembrane serine proteases and required for the production of an extracellular signaling molecule that regulates cellular functions including peptidoglycan acetylation, methionine transport, and cysteine biosynthesis. Additional aarA-dependent phenotypes include (i) loss of an extracellular yellow pigment, (ii) inability to grow on MacConkey agar, and (iii) abnormal cell division. Since these phenotypes are easily assayed, the P. stuartii aarA mutant serves as a useful host system to investigate rhomboid function. The Escherichia coli GlpG protein was shown to be functionally similar to AarA and rescued the above aarA-dependent phenotypes in P. stuartii. GlpG proteins containing single alanine substitutions at the highly conserved catalytic triad of asparagine (N154A), serine (S201A), or histidine (H254A) residues were nonfunctional. The P. stuartii aarA mutant was also used as a biosensor to demonstrate that proteins from a variety of diverse sources exhibited rhomboid activity. In an effort to further investigate the role of a rhomboid protein in cell physiology, a glpG mutant of E. coli was constructed. In phenotype microarray experiments, the glpG mutant exhibited a slight increase in resistance to the beta-lactam antibiotic cefotaxime.

Antagonism of CD11b with Neutrophil Inhibitory Factor (NIF) Inhibits Vascular Lesions in Diabetic Retinopathy

Leukocytes and proteins that govern leukocyte adhesion to endothelial cells play a causal role in retinal abnormalities characteristic of the early stages of diabetic retinopathy, including diabetes-induced degeneration of retinal capillaries. Leukocyte integrin αmβ2 (CD11b/CD18, MAC1), a protein mediating adhesion, has been shown to mediate damage to endothelial cells by activated leukocytes in vitro. We hypothesized that Neutrophil Inhibitory Factor (NIF), a selective antagonist of integrin αmβ2, would inhibit the diabetes-induced degeneration of retinal capillaries by inhibiting the excessive interaction between leukocytes and retinal endothelial cells in diabetes. Wild type animals and transgenic animals expressing NIF were made diabetic with streptozotocin and assessed for diabetes-induced retinal vascular abnormalities and leukocyte activation. To assess if the leukocyte blocking therapy compromised the immune system, animals were challenged with bacteria. Retinal superoxide production, leukostasis and leukocyte superoxide production were increased in wild type mice diabetic for 10 weeks, as was the ability of leukocytes isolated from diabetic animals to kill retinal endothelial cells in vitro. Retinal capillary degeneration was significantly increased in wild type mice diabetic 40 weeks. In contrast, mice expressing NIF did not develop any of these abnormalities, with the exception that non-diabetic and diabetic mice expressing NIF generated greater amounts of superoxide than did similar mice not expressing NIF. Importantly, NIF did not significantly impair the ability of mice to clear an opportunistic bacterial challenge, suggesting that NIF did not compromise immune surveillance. We conclude that antagonism of CD11b (integrin αmβ2) by NIF is sufficient to inhibit early stages of diabetic retinopathy, while not compromising the basic immune response.

Retinylamine Benefits Early Diabetic Retinopathy in Mice.

Background: The development of diabetic retinopathy (DR) is incompletely understood. Administered retinylamine is stored in the retinal pigmented epithelium (RPE) where it affects the ocular visual cycle. Results: Retinylamine inhibited vascular and neural lesions of early DR. Conclusion: Both the RPE and visual cycle are novel targets for the inhibition of DR. Significance: Vision-related processes can contribute to DR. Recent evidence suggests an important role for outer retinal cells in the pathogenesis of diabetic retinopathy (DR). Here we investigated the effect of the visual cycle inhibitor retinylamine (Ret-NH2) on the development of early DR lesions. Wild-type (WT) C57BL/6J mice (male, 2 months old when diabetes was induced) were made diabetic with streptozotocin, and some were given Ret-NH2 once per week. Lecithin-retinol acyltransferase (LRAT)-deficient mice and P23H mutant mice were similarly studied. Mice were euthanized after 2 (WT and Lrat−/−) and 8 months (WT) of study to assess vascular histopathology, accumulation of albumin, visual function, and biochemical and physiological abnormalities in the retina. Non-retinal effects of Ret-NH2 were examined in leukocytes treated in vivo. Superoxide generation and expression of inflammatory proteins were significantly increased in retinas of mice diabetic for 2 or 8 months, and the number of degenerate retinal capillaries and accumulation of albumin in neural retina were significantly increased in mice diabetic for 8 months compared with nondiabetic controls. Administration of Ret-NH2 once per week inhibited capillary degeneration and accumulation of albumin in the neural retina, significantly reducing diabetes-induced retinal superoxide and expression of inflammatory proteins. Superoxide generation also was suppressed in Lrat−/− diabetic mice. Leukocytes isolated from diabetic mice treated with Ret-NH2 caused significantly less cytotoxicity to retinal endothelial cells ex vivo than did leukocytes from control diabetics. Administration of Ret-NH2 once per week significantly inhibited the pathogenesis of lesions characteristic of early DR in diabetic mice. The visual cycle constitutes a novel target for inhibition of DR.

A combination of G protein-coupled receptor modulators protects photoreceptors from degeneration

Degeneration of retinal photoreceptor cells can arise from environmental and/or genetic causes. Since photoreceptor cells, the retinal pigment epithelium (RPE), neurons, and glial cells of the retina are intimately associated, all cell types eventually are affected by retinal degenerative diseases. Such diseases often originate either in rod and/or cone photoreceptor cells or the RPE. Of these, cone cells located in the central retina are especially important for daily human activity. Here we describe the protection of cone cells by a combination therapy consisting of the G protein–coupled receptor modulators metoprolol, tamsulosin, and bromocriptine. These drugs were tested in Abca4−/−Rdh8−/− mice, a preclinical model for retinal degeneration. The specificity of these drugs was determined with an essentially complete panel of human G protein–coupled receptors. Significantly, the combination of metoprolol, tamsulosin, and bromocriptine had no deleterious effects on electroretinographic responses of wild-type mice. Moreover, putative G protein–coupled receptor targets of these drugs were shown to be expressed in human and mouse eyes by RNA sequencing and quantitative polymerase chain reaction. Liquid chromatography together with mass spectrometry using validated internal standards confirmed that metoprolol, tamsulosin, and bromocriptine individually or together penetrate the eye after either intraperitoneal delivery or oral gavage. Collectively, these findings support human trials with combined therapy composed of lower doses of metoprolol, tamsulosin, and bromocriptine designed to safely impede retinal degeneration associated with certain genetic diseases (e.g., Stargardt disease). The same low-dose combination also could protect the retina against diseases with complex or unknown etiologies such as age-related macular degeneration.