Mild endoplasmic reticulum stress promotes retinal neovascularization via induction of BiP/GRP78.

Endoplasmic reticulum (ER) stress occurs as a result of accumulation of unfolded or misfolded proteins in the ER and is involved in the mechanisms of various diseases, such as cancer and neurodegeneration. The goal of the present study was to clarify the relationship between ER stress and pathological neovascularization in the retina. Proliferation and migration of human retinal microvascular endothelial cells (HRMEC) were assessed in the presence of ER stress inducers, such as tunicamycin and thapsigargin. The expression of ER chaperone immunoglobulin heavy-chain binding protein (BiP), known as Grp78, was evaluated by real time RT-PCR, immunostaining, and Western blotting. Tunicamycin or thapsigargin was injected into the intravitreal body of oxygen-induced retinopathy (OIR) model mice at postnatal day 14 (P14) and retinal neovascularization was quantified at P17. The expression and localization of BiP in the retina was also evaluated in the OIR model. Exposure to tunicamycin and thapsigargin increased the proliferation and migration of HRMEC. Tunicamycin enhanced the expression of BiP in HRMEC at both the mRNA level and at the protein level on the cell surface, and increased the formation of a BiP/T-cadherin immunocomplex. In OIR model mice, retinal neovascularization was accelerated by treatments with ER stress inducers. BiP was particularly observed in the pathological vasculature and retinal microvascular endothelial cells, and the increase of BiP expression was correlated with retinal neovascularization. In conclusion, ER stress may contribute to the formation of abnormal vasculature in the retina via BiP complexation with T-cadherin, which then promotes endothelial cell proliferation and migration

Severe ER stress-induced cell death of HRMEC <i>via</i> the apoptotis.

<p>(A) Representative images of migration test with tunicamycin at 10 µg/mL and thapsigargin at 10 µM are shown. (B) Both tunicamycin at 10 µg/mL and thapsigargin at 10 µM inhibited the cell migration in HRMEC. Fluorescence micrographs of Hoechst 33342 and PI staining are shown at 24 h after treatment for 2 h with (C) tunicamycin or (D) thapsigargin. Scale bar represents 100 µm. The dead cells were increased with (E) tunicamycin or (F) thapsigargin. Data are shown as mean ± S.E.M. (n = 6). *, p<0.05; **, p<0.01 vs. Control (Dunnett's multiple-comparison test). (G) Fluorescence photomicrographs of HRMEC triple-stained with propidium iodide (PI), annexin V-FITC, and Hoechst 33342, with tunicamycin. Red: Propidium Iodide, Green: Annexin V, blue: Hoechst33342. Scale bar represents 100 µm. (H) The apoptotic cells were significantly increased with tunicamycin at 3 and 10 µg/mL. Scale bar represents 100 µm. Data are shown as mean ± S.E.M. (n = 4). *, p<0.05; **, p<0.01 vs. Control (Dunnett's multiple-comparison test).</p

BiP protein levels regulates retinal neovascularization.

<p>(A) HRMEC were cultured in a 96-well plate (at a density of 2×10³ cells/well), and were then supplemented with the indicated concentrations of BiX for 1 h, and measurements were made by WST-8 assay. Data are shown as mean ± S.E.M. (n = 6). *, p<0.05 vs. Control (Dunnett's multiple-comparison test). Migration of HRMEC was assessed using a wound-healing assay. (B) Images of the wounded monolayer of HRMEC were taken at 24 h after treatment for 1 h with BiX. (C) The indicated concentrations of BiX increased migration compared to the control. Scale bars indicate 500 µm. Data are shown as mean ± S.E.M. (n = 4). **, p<0.01 vs. Control (Dunnett's multiple-comparison test). The original images (D, F and J, L), together with the analyzed images (E, G and K, M) obtained using the Angiogenesis Tube Formation module in Metamorph, are shown. Scale bars indicate 500 µm and 100 µm (in box). Quantitative analysis was performed on the entire retinal microvasculature in flat-mounted retinas obtained at P17. BiX at 1 µM and BiP small interfering (si) RNA at 1 µg/mL increased (vs. vehicle) both the number of nodes (E, K) and the node areas (F, L), which are indexes of pathological neovascularization as calculated using the Angiogenesis Tube Formation module. Data are shown as mean ± S.E.M. (n = 4 or 5). *, p<0.05 vs.Vehicle (Student's <i>t</i>-test).</p

BiP was expressed in the retinal vascular endothelial cells of a murine oxygen-induced retinopathy (OIR) model.

<p>(A) OIR model mice at P17, treated with 3 µg/ml of tunicamycin at P14, were perfused with FITC-dextran and the retinas were stained with anti-BiP antibody. The original images (green channel) and BiP stained images (red channel) are shown, along with the analyzed images. (B) BiP (green channel) and CD31 (red channel) staining in flat-mounted retinas of P17 mice, under normoxia or subjected to the OIR protocol, following tunicamycin injection. Arrowheads and arrows indicate normal vessels and newly formed vessels anterior to internal limiting membrane, respectively. Scale bars indicate 50 µm. The complexes of BiP and T-cadherin in the retina were immunoprecipitated with the anti-T-cadherin antibody and detected by using Western blotting with the anti-BiP antibody (C), or by the Coomassie-staining (D).</p

Tunicamycin at low concentration activated the transcription of <i>BiP</i> and <i>CHOP</i> mRNA with the mild dilation of ER in HRMEC.

<p>(A) Electron microscopic images are shown for 2 h with tunicamycin at 0.1 µg/mL. Tunicamycin induced the mild dilation of endoplasmic reticulum (ER). Scale bar represents 500 nm. Arrows indicate the ER in HRMEC. The mRNA levels of (B) <i>BiP</i> and (C) <i>CHOP</i> in HRMEC were determined by real-time RT-PCR and normalized against <i>β-actin</i>. Data are shown as mean ± S.E.M. (n = 4 to 6). *, p<0.05 vs. Control (Student's <i>t</i>-test). (D) <i>XBP-1</i> mRNA was not spliced by treatment with tunicamycin. HRMEC were harvested at 10 min, 30 min, 1 h, and 2 h after addition of tunicamycin, or at 10 and 22 h after incubation. Typical gel images of spliced or unspliced <i>XBP-1</i> bands at each sampling point were generated by MCE-202 MultiNA, a microchip based capillary electrophoresis system for DNA analysis (n = 3 or 4). (E) The mRNA level of <i>VEGF</i>, as a common angiogenic factor, was determined by real-time RT-PCR before and after the induction of <i>BiP</i> mRNA and <i>CHOP</i> mRNA, but no differences were apparent between the control and tunicamycin treated groups. Data are shown as mean ± S.E.M. (n = 4 or 5). For 1 h, p = 0.80; for 2 h, p = 0.91; for 12 h, p = 0.99 vs. Control (Student's <i>t</i>-test).</p

The expression of BiP was associated with retinal neovascularization of a murine oxygen-induced retinopathy (OIR) model.

<p>(A) The representative photographs are shown the retinal flat-mount (the upper images) and the higher magnification version of part of the corresponding upper images (the lower images). Arrows indicate newly formed vessels anterior to internal limiting membrane. Scale bars indicate 500 µm (Upper images) and 100 µm (Lower images). Quantitative analysis was performed on the entire retinal microvasculature time-dependently. In the OIR model, retinal neovascularization at P14 and P17 significantly increased both the number of nodes (B) and the nodes area (C), compared to that at P12. In the retina of the same individual, BiP induction was caused time-dependently (D). Relative intensity was described by the intensity of BiP divided by β-actin (E). Data are shown as mean ± S.E.M. (n = 6 to 9). *, p<0.05, **, p<0.01 vs. OIR mice at P12 (Dunnett's multiple-comparison test).</p

Tunicamycin and thapsigargin accelerated retinal neovascularization in a murine oxygen-induced retinopathy (OIR) model.

<p>The original images (A, C and G, I), together with the analyzed images (B, D and H, J) obtained using the Angiogenesis Tube Formation module in Metamorph, are shown. Scale bars indicate 500 µm and 100 µm (in box). Green labels in the analyzed images show the node regions. Quantitative analysis was performed on the entire retinal microvasculature in flat-mounted retinas obtained at P17. Tunicamycin at 3 µg/ml and thapsigargin at 1 µM increased (vs. vehicle) both the number of nodes (E, K) and the node areas (F, L), which are indexes of pathological neovascularization as calculated using the Angiogenesis Tube Formation module. Data are shown as mean ± S.E.M. (n = 5 to 9). *, p<0.05; **, p<0.01 vs.Vehicle (Student's <i>t</i>-test).</p

Diabetes Mellitus Aggravates Hemorrhagic Transformation after Ischemic Stroke via Mitochondrial Defects Leading to Endothelial Apoptosis

<div><p>Diabetes is a crucial risk factor for stroke and is associated with increased frequency and poor prognosis. Although endothelial dysfunction is a known contributor of stroke, the underlying mechanisms have not been elucidated. The aim of this study was to elucidate the mechanism by which chronic hyperglycemia may contribute to the worsened prognosis following stroke, especially focusing on mitochondrial alterations. We examined the effect of hyperglycemia on hemorrhagic transformation at 24 hours after middle cerebral artery occlusion (MCAO) in streptozotocin (STZ) -induced diabetic mice. We also examined the effects of high-glucose exposure for 6 days on cell death, mitochondrial functions and morphology in human brain microvascular endothelial cells (HBMVECs) or human endothelial cells derived from induced pluripotent stem cells (iCell endothelial cells). Hyperglycemia aggravated hemorrhagic transformation, but not infarction following stroke. High-glucose exposure increased apoptosis, capase-3 activity, and release of apoptosis inducing factor (AIF) and cytochrome c in HBMVECs as well as affected mitochondrial functions (decreased cell proliferation, ATP contents, mitochondrial membrane potential, and increased matrix metalloproteinase (MMP)-9 activity, but not reactive oxygen species production). Furthermore, morphological aberration of mitochondria was observed in diabetic cells (a great deal of fragmentation, vacuolation, and cristae disruption). A similar phenomena were seen also in iCell endothelial cells. In conclusion, chronic hyperglycemia aggravated hemorrhagic transformation after stroke through mitochondrial dysfunction and morphological alteration, partially via MMP-9 activation, leading to caspase-dependent apoptosis of endothelial cells of diabetic mice. Mitochondria-targeting therapy may be a clinically innovative therapeutic strategy for diabetic complications in the future.</p></div

Diagram illustrating the postulated mechanism through which hyperglycemia aggravates hemorrhagic transformation after ischemic stroke.

<p>Hyperglycemia increases the activity of MMP-9 in an ROS-independent manner, which promotes the opening of mitochondrial permeability transition pores (mitochondrial depolarization; Δ<b>ψ</b>_m ↓↓). The mitochondria that have lost normal function can no longer produce ATP, and emit various pro-apoptotic factors, such as AIF and cytochrome c into the cytosol. These factors subsequently activate caspase-3 and induce apoptotic cell death in HBMVECs. On the other hand, functional failure leads to morphological alteration of mitochondria, (fragmentation, vacuolation, cristae disruption). Eventually, both of these functional and morphological disturbances result in the aggravation of hemorrhagic transformation after ischemic stroke.</p