Shu Kachi

The SDF-1/CXCR4 ligand/receptor pair is an important contributor to several types of ocular neovascularization

Hypoxia causes increased expression of several proteins that have the potential to promote neovascularization. Vascular endothelial growth factor (VEGF) is up‐regulated by hypoxia in the retina and plays a central role in the development of several types of ocular neovascularization, but the effects of other hypoxia‐regulated proteins are less clear. Stromal‐de‐rived factor‐1 (SDF‐1) and its receptor, CXCR4, have hypoxia response elements in the promoter regions of their genes and are increased in hypoxic liver and heart. In this study, we found that SDF‐1 and CXCR4 are increased in hypoxic retina, with SDF‐1 localized in glial cells primarily near the surface of the retina and CXCR4 localized in bone marrow‐derived cells. Glial cells also expressed CXCR4, which suggested the possibility of autocrine stimulation, but influx of bone marrow‐derived cells is the major source of increased levels of CXCR4. High levels of VEGF in the retina in the absence of hypoxia also increased levels of Cxcr4 and Sdf1 mRNA. CXCR4 antagonists reduced influx of bone marrow‐derived cells into ischemic retina and strongly suppressed retinal neovascularization, VEGF‐induced subretinal neovascularization, and choroidal neovascularization. These data suggest that SDF‐1 and CXCR4 contribute to the involvement of bone marrow‐derived cells and collaborate with VEGF in the development of several types of ocular neovascularization. They provide new targets for therapeutic intervention that may help to bolster and supplement effects obtained with VEGF antagonists.—Lima e Silva, R., Shen, J., Hackett, S. F., Kachi, S., Akiyama, H., Kiuchi, K., Yokoi, K., Hatara, M. C., Lauer, T., Aslam, S., Gong, Y. Y., Xiao, W‐H., Khu, N. H., Thut, C., Campochiaro, P. A. The SDF‐1/CXCR4 ligand/receptor pair is an important contributor to several types of ocular neo‐vascularization. FASEB J. 21, 3219–3230 (2007)

Different effects of angiopoietin-2 in different vascular beds: new vessels are most sensitive

In this study, we used double transgenic mice with inducible expression of angiopoietin‐2 (Ang2) to investigate the role of Ang2 in the retinal and choroidal circulations and in three models of ocular neovascularization (NV). Mice with induced expression of Ang2 ubiquitously, or specifically in the retina, survived and appeared grossly normal. They also had normal‐appearing retinal and choroidal circulations, demonstrating that high levels of Ang2 did not induce regression of mature retinal or choroidal vessels. When Ang2 expression was induced soon after birth, there was increased density of the deep capillary bed on postnatal day (P) 11 that returned to normal by P18, the time that retinal vascular development is usually completed. In mice with ischemic retinopathy, induction of Ang2 during the ischemic period resulted in a significant increase in retinal NV, but induction of Ang2 at a later time point when ischemia (and vascular endothelial growth factor [VEGF]) was less, hastened regression of NV. In triple transgenic mice that coexpressed VEGF and Ang2, the increased expression of Ang2 inhibited VEGF‐induced NV in the retina. Increased expression of Ang2 also resulted in regression of choroidal neovascularization. These data suggest that ocular neovascularization, but not mature retinal or choroidal vessels, is sensitive to Ang2; a high Ang2/VEGF ratio promotes regression, while high Ang2 in the setting of hypoxia and/or concomitantly high Ang2 and VEGF stimulate neovascularization.

Characteristics of Bone Marrow–Derived Microglia in the Normal and Injured Retina

PURPOSE To compare the distribution and immunologic characteristics of bone marrow (BM)-derived and resident microglia in the retina. METHODS Mice were irradiated and injected with enhanced green fluorescent protein-positive (EGFP(+)) BM cells. One month to 12 months after BM transplantation, eyes were analyzed histologically for the expression of EGFP and various monocyte/microglia/macrophage markers (Iba-1, F4/80, GS-1, major histocompatibility complex [MHC] class II). N-methyl-N-nitrosourea (MNU) was injected or retinal detachment was created to induce retinal damage. RESULTS Many BM-derived EGFP(+) cells were found in the ciliary body and choroid and around the optic nerve in the uninjured eyes. Within the retina, few such cells existed at the retinal margin and juxtapapillary area at 3 to 12 months after BM transplantation. However, after MNU injection, many EGFP(+) cells were found in the retina adjacent to the retinal vessels, optic nerve, and ciliary body that rapidly spread throughout the retina. Most of them showed morphologic and immunohistochemical features of microglia. By 7 days after MNU injection, EGFP(+) BM-derived cells occupied approximately 15% of the total Iba-1(+) retinal microglia. Meanwhile, the proportion of MHC class II(+) cells was larger among BM-derived (EGFP(+)/Iba-1(+)) than resident (EGFP(-)/Iba-1(+)) microglia. In the eyes with retinal detachment, EGFP(+)/F4/80(+) cells engrafted exclusively around the detached retina. CONCLUSIONS In response to retinal damage, numerous BM-derived cells migrated to the retina from the ciliary body, optic nerve, and retinal vessels and differentiated into microglia. The higher rate of immunologic activation and the increased specificity to the damaged site appeared to be the characteristic features of BM-derived microglia.

Retinal Dysfunction in Basigin Deficiency

PURPOSE To examine the retina of basigin (Bsg) knockout mice by electrophysiological and histologic methods and thereby to determine the possible function of Bsg in phototransduction and retinal development. METHODS Scotopic and photopic electroretinograms (ERGs) were recorded from 11 wild-type, 12 heterozygous, and 8 homozygous Bsg gene knockout mice of different ages. The retinas were also examined by histologic and immunolabeling methods. RESULTS Bsg knockout mice of 5 to 41 weeks of age showed a decrease in the amplitude of all components of both the photopic and scotopic ERGs. In contrast, the fundus and the fluorescein fundus angiography and morphology of the retina at the light microscopic level appeared to be normal until 8 weeks of age in Bsg knockout mice. Thereafter, the length of outer segment and outer nuclear layers decreased with increasing age. Immunohistochemical analysis localized Bsg protein in a variety of cells in the retina, especially in the pigment epithelium, the upper outer plexiform layer and the inner segments of photoreceptor cells. CONCLUSIONS The results demonstrated that both rod and cone function were severely affected from an early age by the targeted disruption of the Bsg gene. In spite of abnormal ERGs, the photoreceptor cells maintained normal morphology up to 8 weeks. Thereafter, the photoreceptor cells degenerated gradually and were almost ablated by 41 weeks.

Intravitreal injection of bevacizumab for macular edema secondary to branch retinal vein occlusion:results after 12 months and multiple regression analysis.

Purpose: To evaluate the 12-month follow-up results of intravitreal bevacizumab therapy for macular edema secondary to branch retinal vein occlusion and to identify the pretreatment factors that were associated with an improvement of the final visual outcome. Methods: Fifty eyes of 50 patients with macular edema secondary to branch retinal vein occlusion received an injection of 1.25 mg/0.05 mL bevacizumab. Additional injections were done when recurrence of macular edema occurred or the treatment was not effective. The best-corrected visual acuity and foveal thickness were measured. Stepwise multiple regression analyses were also performed. Results: The mean logarithm of the minimum angle of resolution visual acuity improved significantly from 0.53 to 0.26, and the mean foveal thickness decreased significantly from 523 to 305 &mgr;m during the 12-month follow-up period. The mean number of injections was 2.0 (range, 1–4). Stepwise multiple regression analyses showed that younger patients had both better visual acuity at 12 months and greater improvement of visual acuity during 12 months. In addition, better pretreatment visual acuity was associated with better visual acuity at 12 months but with less improvement of the visual acuity. Conclusion: Intravitreal bevacizumab therapy can be a long-term effective treatment for macular edema secondary to branch retinal vein occlusion.

Increased expression of VEGF in retinal pigmented epithelial cells is not sufficient to cause choroidal neovascularization.

Increased expression of vascular endothelial cell growth factor (VEGF) in the retina is sufficient to stimulate sprouting of neovascularization from the deep capillary bed of the retina, but not the superficial retinal capillaries or the choriocapillaris. Coexpression of VEGF and angiopoietin 2 (Ang2) results in sprouting of neovascularization from superficial and deep retinal capillaries, but not the choriocapillaris. However, retina‐derived VEGF and Ang2 may not reach the choriocapillaris, because of tight junctions between retinal pigmented epithelial (RPE) cells. To eliminate this possible confounding factor, we used the human vitelliform macular dystrophy 2 (VMD2) promoter, an RPE‐specific promoter, combined with the tetracycline‐inducible promoter system, to generate double transgenic mice with inducible expression of VEGF in RPE cells. Adult mice with increased expression of VEGF in RPE cells had normal retinas and choroids with no choroidal neovascularization (CNV), but when increased expression of VEGF in RPE cells was combined with subretinal injection of a gutless adenoviral vector containing an expression construct for Ang2 (AGVAng2), CNV consistently occurred. In contrast, triple transgenic mice with induced expression of Ang2 and VEGF in RPE cells, did not develop CNV. These data suggest that increased expression of VEGF and/or Ang2 in RPE cells is not sufficient to cause CNV unless it is combined with a subretinal injection of a gutless adenoviral vector, which is likely to perturb RPE cells. These data also suggest that the effects of angiogenic proteins may vary among vascular beds, even those that are closely related, and, therefore, generalizations should be avoided.

Nonviral ocular gene transfer

In this study, we explored the use of electroporation or media that promote lipoplex formation for nonviral gene transfer in the eye. There was no detectable staining for LacZ after subretinal, intravitreous, or periocular injection of a plasmid containing a CMV promoter expression cassette for LacZ, but when plasmid injection in each of the three sites was combined with electroporation, there was efficient transduction. Specific staining for LacZ was seen in retinal pigmented epithelial (RPE) cells after subretinal injection of a plasmid containing a vitelliform macular dystrophy 2 (VMD2) promoter expression cassette, demonstrating that this approach can be used to evaluate purported tissue-specific promoters in vivo. Electroporation with 10 V/mm resulted in strong LacZ staining, but was damaging to photoreceptors; substantial transduction with no evidence of retinal damage was seen using 3.4 V/mm. Staining for LacZ was also seen after subretinal or periocular, but not intravitreous, injection of plasmid DNA in medium containing 40% Lipofectamine2000 (Lf). Injection of 40% Lf into the subretinal space caused damage to photoreceptors, but subretinal injection of plasmid DNA in medium containing 10% NeuroPorter resulted in transduction of RPE cells with no adverse effects on retinal morphology or function as assessed by electroretinograms (ERGs). After either electroporation or lipofection, LacZ staining was detectable for at least 14 days, and could be reinduced by a second procedure. These data suggest that electroporation or lipofection can be used as experimental tools for ocular gene transfer to evaluate tissue-specific promoter fragments or to evaluate the effects of transgene expression in the retina. Also, with additional optimization, nonviral gene transfer may prove to be a valuable approach for the treatment of retinal and choroidal diseases.

Suppression and Regression of Choroidal Neovascularization by Systemic Administration of an α5β1 Integrin Antagonist

Integrin α5β1 plays an important role in developmental angiogenesis, but its role in various types of pathologic neovascularization has not been completely defined. In this study, we found strong up-regulation of α5β1 in choroidal neovascularization. Implantation of an osmotic pump delivering 1.5 or 10 μg/h (∼1.8 or 12 mg/kg/day) of 3-(2-{1-alkyl-5-[(pyridin-2-ylamino)-methyl]-pyrrolidin-3-yloxy}-acetylamino)-2-(alkylamino)-propionic acid (JSM6427), a selective α5β1 antagonist, caused significant suppression of choroidal neovascularization; the area of neovascularization was reduced by 33 to 40%. When an osmotic pump delivering 10 μg/h of JSM6427 was implanted 7 days after rupture of Bruchs membrane, there was terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in vascular cells within the neovascularization and significant regression of the neovascularization over the next week. JSM6427 also induced apoptosis of cultured vascular endothelial cells. Fibronectin stimulates phosphorylation of extracellular signal-regulated kinase (ERK) in α5β1-expressing cells that is blocked by JSM6427. These data suggest that α5β1 plays a role in the development and maintenance of choroidal neovascularization and provides a target for therapeutic intervention.

Topical Administration of a Multi-Targeted Kinase Inhibitor Suppresses Choroidal Neovascularization and Retinal Edema

Age‐related macular degeneration, diabetic retinopathy, and retinal vein occlusions are complicated by neovascularization and macular edema. Multi‐targeted kinase inhibitors that inhibit select growth factor receptor tyrosine kinases and/or components of their down‐stream signaling cascades (such as Src kinases) are rationale treatment strategies for these disease processes. We describe the discovery and characterization of two such agents. TG100572, which inhibits Src kinases and selected receptor tyrosine kinases, induced apoptosis of proliferating endothelial cells in vitro. Systemic delivery of TG100572 in a murine model of laser‐induced choroidal neovascularization (CNV) caused significant suppression of CNV, but with an associated weight loss suggestive of systemic toxicity. To minimize systemic exposure, topical delivery of TG100572 to the cornea was explored, and while substantial levels of TG100572 were achieved in the retina and choroid, superior exposure levels were achieved using TG100801, an inactive prodrug that generates TG100572 by de‐esterification. Neither TG100801 nor TG100572 were detectable in plasma following topical delivery of TG100801, and adverse safety signals (such as weight loss) were not observed even with prolonged dosing schedules. Topical TG100801 significantly suppressed laser‐induced CNV in mice, and reduced fluorescein leakage from the vasculature and retinal thickening measured by optical coherence tomography in a rat model of retinal vein occlusion. These data suggest that TG100801 may provide a new topically applied treatment approach for ocular neovascularization and retinal edema. J. Cell. Physiol. 216: 29–37, 2008.

Optical Coherence Tomography for the Noninvasive Evaluation of the Cornea

Purpose. To determine the optical coherence tomographic (OCT) characteristics of normal corneas and to characterize the OCT images of abnormal corneal lesions. Methods. Eleven eyes from 10 patients were examined at the Cornea Service of the Nagoya University Hospital: 4 had corneal pathologies, 4 underwent keratoplasty, and 2 were normal controls; 1 enucleated eye was also examined. OCT (OCT 2000 Zeiss-Humphrey) was used to study the normal cornea and various corneal abnormalities. We compared the OCT images to the observations made by slit-lamp biomicroscopy. Results. Fluid spaces were detected as black images. A highly reflective reflex was observed at the interface of different tissues, and intensive backscattering (reflex) was seen when the incident ray hit the laminated layers vertically. Corneal opacities were not clearly imaged when they were diffuse and mild, or when they were arranged axially in a small area, as was the scar of the graft–host junction. It was possible to obtain images from the region of the cornea that was not clearly visible by slit-lamp examination because of a corneal opacity. Conclusion. OCT is a noncontact and noninvasive technique that can be performed safely on diseased corneas. OCT can provide objective documentation of corneal disorders that cannot be obtained by slit-lamp examination. The use of OCT in conjunction with other conventional instruments should provide a more complete image of the cornea.