Amyloid beta peptide-induced inhibition of endothelial nitric oxide production involves oxidative stress-mediated constitutive eNOS/HSP90 interaction and disruption of agonist-mediated Akt activation

Background: Amyloid beta (A beta)-induced vascular dysfunction significantly contributes to the pathogenesis of Alzheimer\u27s disease (AD). A beta is known to impair endothelial nitric oxide synthase (eNOS) activity, thus inhibiting endothelial nitric oxide production (NO). Method: In this study, we investigated A beta-effects on heat shock protein 90 (HSP90) interaction with eNOS and Akt in cultured vascular endothelial cells and also explored the role of oxidative stress in this process. Results: Treatments of endothelial cells (EC) with A beta promoted the constitutive association of HSP90 with eNOS but abrogated agonist (vascular endothelial growth factor (VEGF))-mediated HSP90 interaction with Akt. This effect resulted in blockade of agonist-mediated phosphorylation of Akt and eNOS at serine 1179. Furthermore, A beta stimulated the production of reactive oxygen species in endothelial cells and concomitant treatments of the cells with the antioxidant N-acetyl-cysteine (NAC) prevented A beta effects in promoting HSP90/eNOS interaction and rescued agonist-mediated Akt and eNOS phosphorylation. Conclusions: The obtained data support the hypothesis that oxidative damage caused by A beta results in altered interaction of HSP90 with Akt and eNOS, therefore promoting vascular dysfunction. This mechanism, by contributing to A beta-mediated blockade of nitric oxide production, may significantly contribute to the cognitive impairment seen in AD patients

Dual Anti-Inflammatory and Anti-Angiogenic Action of miR-15a in Diabetic Retinopathy

AbstractActivation of pro-inflammatory and pro-angiogenic pathways in the retina and the bone marrow contributes to pathogenesis of diabetic retinopathy. We identified miR-15a as key regulator of both pro-inflammatory and pro-angiogenic pathways through direct binding and inhibition of the central enzyme in the sphingolipid metabolism, ASM, and the pro-angiogenic growth factor, VEGF-A. miR-15a was downregulated in diabetic retina and bone marrow cells. Over-expression of miR-15a downregulated, and inhibition of miR-15a upregulated ASM and VEGF-A expression in retinal cells. In addition to retinal effects, migration and retinal vascular repair function was impaired in miR-15a inhibitor-treated circulating angiogenic cells (CAC). Diabetic mice overexpressing miR-15a under Tie-2 promoter had normalized retinal permeability compared to wild type littermates. Importantly, miR-15a overexpression led to modulation toward nondiabetic levels, rather than complete inhibition of ASM and VEGF-A providing therapeutic effect without detrimental consequences of ASM and VEGF-A deficiencies

Increased Oxidative and Nitrative Stress Accelerates Aging of the Retinal Vasculature in the Diabetic Retina.

Hyperglycemia-induced retinal oxidative and nitrative stress can accelerate vascular cell aging, which may lead to vascular dysfunction as seen in diabetes. There is no information on whether this may contribute to the progression of diabetic retinopathy (DR). In this study, we have assessed the occurrence of senescence-associated markers in retinas of streptozotocin-induced diabetic rats at 8 and 12 weeks of hyperglycemia as compared to normoglycemic aging (12 and 14 months) and adult (4.5 months) rat retinas. We have found that in the diabetic retinas there was an up-regulation of senescence-associated markers SA-β-Gal, p16INK4a and miR34a, which correlated with decreased expression of SIRT1, a target of miR34a. Expression of senescence-associated factors primarily found in retinal microvasculature of diabetic rats exceeded levels measured in adult and aging rat retinas. In aging rats, retinal expression of senescence associated-factors was mainly localized at the level of the retinal pigmented epithelium and only minimally in the retinal microvasculature. The expression of oxidative/nitrative stress markers such as 4-hydroxynonenal and nitrotyrosine was more pronounced in the retinal vasculature of diabetic rats as compared to normoglycemic aging and adult rat retinas. Treatments of STZ-rats with the anti-nitrating drug FeTPPS (10mg/Kg/day) significantly reduced the appearance of senescence markers in the retinal microvasculature. Our results demonstrate that hyperglycemia accelerates retinal microvascular cell aging whereas physiological aging affects primarily cells of the retinal pigmented epithelium. In conclusion, hyperglycemia-induced retinal vessel dysfunction and DR progression involve vascular cell senescence due to increased oxidative/nitrative stress

The role of indoleamine 2,3 dioxygenase in beneficial effects of stem cells in hind limb ischemia reperfusion injury.

Ischemia-Reperfusion (IR) injury of limb remains a significant clinical problem causing secondary complications and restricting clinical recovery, despite rapid restoration of blood flow and successful surgery. In an attempt to further improve post ischemic tissue repair, we investigated the effect of a local administration of bone marrow derived stem cells (BMDSCs) in the presence or absence of immune-regulatory enzyme, IDO, in a murine model. A whole limb warm ischemia-reperfusion model was developed using IDO sufficient (WT) and deficient (KO) mice with C57/BL6 background. Twenty-four hours after injury, 5 × 105 cells (5×105 cells/200 µL of PBS solution) BMDSCs (Sca1 + cells) were injected intramuscularly while the control group received just the vehicle buffer (PBS). Forty-eight to seventy-two hours after limb BMDSC injection, recovery status including the ratio of intrinsic paw function between affected and normal paws, general mobility, and inflammatory responses were measured using video micrometery, flow cytometry, and immunohistochemistry techniques. Additionally, MRI/MRA studies were performed to further study the inflammatory response between groups and to confirm reconstitution of blood flow after ischemia. For the first time, our data, showed that IDO may potentially represent a partial role in triggering the beneficial effects of BMDSCs in faster recovery and protection against structural changes and cellular damage in a hind limb IR injury setting (P = 0.00058)

SA-β-gal in RPE flat mounts.

<p>Bright-field images show SA-<b>β</b>-gal positive areas found in RPE flat mounts from age-matched (4.5 months old) normoglycemic control rats (<b>A</b>),STZ-rats, at 8 (<b>B</b>) and 12 weeks (<b>C</b>) of hyperglycemia, or in normoglycemic aging rats at12 (<b>D</b>) and 14 (<b>E</b>) months of age. Scale bar equal to <i>20</i><b>μ</b><i>m</i>.</p

Transgenic hepatitis B virus large envelope polypeptide (HBsAg) expression in hepatocytes with nuclear inclusions.

<p>(A, B) A high level of HBsAg expression in the cytoplasm of hepatocytes in nine-month-old transgenic mice. Antibody specificity was confirmed by lack of immuno-reactivity in wild-type control mice shown in the inset of (<b>B</b>). (<b>C</b>) Quantification of the relative number of hepatocytes from HBsAg transgenic mice that express the HBsAg in inclusion-bearing and normal hepatocytes (n = 5). p<0.01; ***,p<0.001; ****, p<0.0001, scale bar 50 µm.</p

Senescence-associated retinal changes with FeTPPS treatment.

<p><b>A,B</b>) Retinal frozen sections from diabetic and diabetic + FeTPPS groups were probed at pH6 for detection of SA-<b>β</b>-gal positivity (<i>blue</i>, <i>black arrows</i>). Representative images from frozen retinal sections probed with anti-SIRT1 (<b>C, D</b>) and anti-p16^INK4a (<b>E, F</b>) (<i>green</i>) to detect immunoreactivities in diabetic and retinas of FeTPPS- treated diabetic rats. Sections were co-labeled with anti-isolectin B4 (vascular structures, <i>red</i>). Hoescht staining was used to detect nuclei (<i>blue</i>). Scale bar equal to 50<b>μ</b>m.</p

Measurements of nitrative modifications.

<p>Representative images of immunohistochemical analysis using anti-nitrotyrosine in retinal flat mounts to demonstrate changes in retinal NY formation in the different treatment groups. Double labeling with isolectin B4 was used to specifically assess NY immunoreactivity in the retinal microvasculature. Areas of merging labeling (yellow) are indicated by the white arrows. Non-vascular areas immunoreactive to NY are indicated by white arrowheads. Scale bar equal to <i>20</i><b>μ</b><i>m</i>.</p