Yoko Fukushima

Sema3E-PlexinD1 signaling selectively suppresses disoriented angiogenesis in ischemic retinopathy in mice

During development, the retinal vasculature grows toward hypoxic areas in an organized fashion. By contrast, in ischemic retinopathies, new blood vessels grow out of the retinal surfaces without ameliorating retinal hypoxia. Restoration of proper angiogenic directionality would be of great benefit to reoxygenize the ischemic retina and resolve disease pathogenesis. Here, we show that binding of the semaphorin 3E (Sema3E) ligand to the transmembrane PlexinD1 receptor initiates a signaling pathway that normalizes angiogenic directionality in both developing retinas and ischemic retinopathy. In developing mouse retinas, inhibition of VEGF signaling resulted in downregulation of endothelial PlexinD1 expression, suggesting that astrocyte-derived VEGF normally promotes PlexinD1 expression in growing blood vessels. Neuron-derived Sema3E signaled to PlexinD1 and activated the small GTPase RhoJ in ECs, thereby counteracting VEGF-induced filopodia projections and defining the retinal vascular pathfinding. In a mouse model of ischemic retinopathy, enhanced expression of PlexinD1 and RhoJ in extraretinal vessels prevented VEGF-induced disoriented projections of the endothelial filopodia. Remarkably, intravitreal administration of Sema3E protein selectively suppressed extraretinal vascular outgrowth without affecting the desired regeneration of the retinal vasculature. Our study suggests a new paradigm for vascular regeneration therapy that guides angiogenesis precisely toward the ischemic retina.

Vascular RhoJ is an effective and selective target for tumor angiogenesis and vascular disruption.

Current antiangiogenic therapy is limited by its cytostatic nature and systemic side effects. To address these limitations, we have unveiled the role of RhoJ, an endothelial-enriched Rho GTPase, during tumor progression. RhoJ blockade provides a double assault on tumor vessels by both inhibiting tumor angiogenesis and disrupting the preformed tumor vessels through the activation of the RhoA-ROCK (Rho kinase) signaling pathway in tumor endothelial cells, consequently resulting in a functional failure of tumor vasculatures. Moreover, enhanced anticancer effects were observed when RhoJ blockade was employed in concert with a cytotoxic chemotherapeutic agent, angiogenesis-inhibiting agent, or vascular-disrupting agent. These results identify RhoJ blockade as a selective and effective therapeutic strategy for targeting tumor vasculature with minimal side effects.

Sustained inflammation after pericyte depletion induces irreversible blood-retina barrier breakdown.

In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRβ antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.

Arhgef15 Promotes Retinal Angiogenesis by Mediating VEGF-Induced Cdc42 Activation and Potentiating RhoJ Inactivation in Endothelial Cells

Background Drugs inhibiting vascular endothelial growth factor (VEGF) signaling are globally administered to suppress deregulated angiogenesis in a variety of eye diseases. However, anti-VEGF therapy potentially affects the normal functions of retinal neurons and glias which constitutively express VEGF receptor 2. Thus, it is desirable to identify novel drug targets which are exclusively expressed in endothelial cells (ECs). Here we attempted to identify an EC-specific Rho guanine nucleotide exchange factor (GEF) and evaluate its role in retinal angiogenesis. Methodology/Principal Findings By exploiting fluorescence-activated cell sorting and microarray analyses in conjunction with in silico bioinformatics analyses, we comprehensively identified endothelial genes in angiogenic retinal vessels of postnatal mice. Of 9 RhoGEFs which were highly expressed in retinal ECs, we show that Arhgef15 acted as an EC-specific GEF to mediate VEGF-induced Cdc42 activation and potentiated RhoJ inactivation, thereby promoting actin polymerization and cell motility. Disruption of the Arhgef15 gene led to delayed extension of vascular networks and subsequent reduction of total vessel areas in postnatal mouse retinas. Conclusions/Significance Our study provides information useful to the development of new means of selectively manipulating angiogenesis without affecting homeostasis in un-targeted tissues; not only in eyes but also in various disease settings such as cancer.

EphrinB2–EphB4 Signals Regulate Formation and Maintenance of Funnel-Shaped Valves in Corneal Lymphatic Capillaries

PURPOSE To elucidate the role of signals mediated by EphB4 receptor tyrosine kinase and its transmembrane ephrinB2 ligand in corneal lymphatic capillaries. METHODS To detect expression of ephrinB2 and EphB4 in mouse corneas, immunohistochemistry of flat-mount corneas from 6- to 10-week-old wild-type, Efnb2-lacZ, and Ephb4-lacZ mice on a C57BL/6 background was performed. To induce formation of new blood vessels and lymphatic vessels, mouse corneal epithelia were swabbed with 0.1 M sodium hydroxide. To antagonize endogenous receptor-ligand interactions in corneal lymphatic vessels, recombinant EphB4/Fc proteins were injected into the subconjunctival spaces. To visualize the corneal lymphatic flow, FITC-dextran was injected subconjunctivally. RESULTS In lymphatic capillaries of adult mouse corneas, EphB4 was intensively expressed in lymphatic endothelial cells (LECs) of funnel-shaped valves, which were segregated from ephrinB2-expressing LECs. The number of corneal lymphatic valves was significantly decreased by Efnb2 haploinsufficiency, and subconjunctival EphB4/Fc injections resulted in the deformation of preexisting valves of corneal lymphatic capillaries. In alkali-burn corneas, ephrinB2 and EphB4 were highly expressed in LECs of valve-forming areas. Subconjunctival EphB4/Fc injections perturbed the morphologic maturation of new lymphatic valves, leading to reflux of FITC-dextran to peripheral lymphatic branches. CONCLUSIONS The results demonstrate a pivotal role of ephrinB2-EphB4 signals in the formation and maintenance of funnel-shaped valves in corneal lymphatic capillaries, and further suggest the potential of ephrinB2-EphB4 signals as a target to therapeutically manipulate corneal lymphangiogenesis.

Retinal Microvasculature and Visual Acuity in Eyes With Branch Retinal Vein Occlusion: Imaging Analysis by Optical Coherence Tomography Angiography

Purpose To investigate microvascular changes in the superficial capillary plexus (SCP) and deep capillary plexus (DCP) in eyes with resolved branch retinal vein occlusion (BRVO) and their association with best-corrected visual acuity (BCVA). Methods Eighty-five eyes (82 consecutive patients) with BRVO after resolution of the macular edema were retrospectively evaluated. All patients underwent optical coherence tomography angiography (OCTA) for assessment of microvascular changes, including capillary telangiectasia, microaneurysm, and disruption of the foveal avascular zone (FAZ). The areas of vascular perfusion and FAZ in the SCP and DCP were quantitatively evaluated. Best-corrected visual acuity was measured on the same day as OCTA examination. The correlation between BCVA and OCTA findings was assessed. Results In eyes with resolved BRVO, the mean vascular perfusion areas in the SCP and DCP within a 3 × 3-mm area were 3.75 ± 0.49 and 3.80 ± 0.55 mm2, respectively. The mean FAZ areas of the SCP and DCP were 0.57 ± 0.36 and 0.76 ± 0.38 mm2, respectively. Better BCVA was significantly associated with a larger vascular perfusion area in the SCP (P < 0.001) and DCP (P < 0.001), and a smaller FAZ area in the SCP (P = 0.025) and DCP (P = 0.017). Stepwise multiple regression analysis revealed that the vascular perfusion area in the DCP was the most important parameter significantly correlated with BCVA (R2 = 0.33, P < 0.001). Conclusions Preservation of the deep retinal vasculature is crucial for better visual function in BRVO.

Massive Subretinal Hemorrhage After Photodynamic Therapy for Polypoidal Choroidal Vasculopathy After Macular Translocation Surgery

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Antioxidant role of autophagy in maintaining the integrity of glomerular capillaries

ABSTRACT Autophagy is a lysosomal degradation system by which cytosolic materials and damaged organelles are broken down into basic components. To explore the physiological role of autophagy in glomerular endothelial cells (GEnCs), we compared the autophagic flux among cells in the kidney under starvation. Inhibition of autophagy by chloroquine administration significantly increased the number of autophagosomes or autolysosomes in GEnCs and proximal tubular cells, but not in podocytes, suggesting that the GEnCs exhibit substantial autophagic activity. Next, we analyzed endothelial and hematopoietic cell-specific atg5-deficient mice (atg5-conditional KO [cKO] mice). Glomeruli of 4-wk-old atg5-cKO mice exhibited slightly distended capillary loops accompanied by an accumulation of reactive oxygen species (ROS). Glomeruli of 8-wk-old atg5-cKO mice showed a lobular pattern with thickening of the capillary loops and mesangial matrix expansion; however, the vasculature of other organs was preserved. The atg5-cKO mice died by 12 wk of age, presumably due to pancytopenia resulting from the defect in their hematopoietic lineages. Therefore, we subjected 4-wk atg5-cKO mice to irradiation followed by bone marrow transplantation from normal littermates. Transplanted mice recapitulated the glomerular phenotypes of the atg5-cKO mice with no obvious histological changes in other organs. Twelve-mo-old transplanted mice developed mesangiolysis and glomerulosclerosis with significant deterioration of kidney function. Administration of N-acetyl-l-cysteine, a ROS scavenger, to atg5-cKO mice rescued the glomerular phenotypes. These data suggest that endothelial autophagy protects glomeruli from oxidative stress and maintains the integrity of glomerular capillaries. Enhancing endothelial autophagy may provide a novel therapeutic approach to minimizing glomerular diseases.

Radial optic neurotomy for the management of hemicentral retinal vein occlusion: long-term follow-up study.

Purpose: To assess the efficacy of radial optic neurotomy (RON) for hemicentral retinal vein occlusion (hemi-CRVO) in patients with follow-up of >12 months. Methods: Five consecutive patients with hemi-CRVO were enrolled in the study and underwent vitrectomy with RON. The effects of this treatment were assessed by visual acuity, optical coherence tomography, indocyanine green angiography, and kinetic visual field testing. Results: The median and mean visual acuities improved from 20/250 to 20/50 and from 20/286 to 20/51, respectively. Four patients (80%) had visual acuity improvement of ≥3 lines; visual acuity in 1 patient (20%) remained the same. Visual acuity in all four eyes with preoperative visual acuity of <20/200 improved to >20/200 12 months after surgery. Foveal thickness was gradually reduced during the follow-up period. Chorioretinal anastomosis was detected by indocyanine green angiography in 3 eyes (60%). A temporal visual field defect was found in two eyes. No neovascularization on the iris, angle, optic disk, or retina was detected postoperatively. Conclusion: Visual acuity may improve more with RON compared with the natural history of the disease, and no neovascular formation may occur after RON, suggesting that this procedure may be an effective treatment for hemi-CRVO.

Simulation of panretinal laser photocoagulation using geometric methods for calculating the photocoagulation index

Purpose To establish geometrically based methods for simulating panretinal laser photocoagulation (PRP) for the photocoagulation index. Methods A formula for calculating the curved surface area of a spherical dome was used for the simulation. If the radius of the dome is c and the height of the dome is h, then the curved surface area (S) of the dome is S = π (c2 + h2). We calculated the area of the whole retina using this formula and the anatomical dimensions of the standard eyeball. To simulate PRP with a 400-μm spot on the retina with 1-spot spacing, we drew 400-μm-diameter circles, separated by 400 μm, on a retinal map. We calculated the ratio of the total retinal photocoagulated area to the whole retina, termed the photocoagulation index, in order to investigate the impact of the extent of the photocoagulated area and the pulse duration on PRP. Results The whole retinal area was 1,092 mm2. The numbers of spots in the scattered and full-scattered PRP were 1,222 and 1,814, respectively. The photocoagulation index was 14.1% and 20.9% for scattered and full-scattered PRP, respectively. These values changed to 14.3% (5.6%) and 21.3% (8.3%), respectively, for PRP with a 100-ms pulse or a 20-ms pulse. Conclusions This method will be useful for investigating the impact of various PRP parameters (duration, spacing, intensity of burns, extent of photocoagulated area, etc.) on the photocoagulation index.