Haicheng She

Tumor Necrosis Factor-α Mediates Oligodendrocyte Death and Delayed Retinal Ganglion Cell Loss in a Mouse Model of Glaucoma

Glaucoma is a widespread ocular disease characterized by a progressive loss of retinal ganglion cells (RGCs). Previous studies suggest that the cytokine tumor necrosis factor-α (TNF-α) may contribute to the disease process, although its role in vivo and its mechanism of action are unclear. To investigate pathophysiological mechanisms in glaucoma, we induced ocular hypertension (OH) in mice by angle closure via laser irradiation. This treatment resulted in a rapid upregulation of TNF-α, followed sequentially by microglial activation, loss of optic nerve oligodendrocytes, and delayed loss of RGCs. Intravitreal TNF-α injections in normal mice mimicked these effects. Conversely, an anti-TNF-α-neutralizing antibody or deleting the genes encoding TNF-α or its receptor, TNFR2, blocked the deleterious effects of OH. Deleting the CD11b/CD18 gene prevented microglial activation and also blocked the pathophysiological effects of OH. Thus TNF-α provides an essential, although indirect, link between OH and RGC loss in vivo. Blocking TNF-α signaling or inflammation, therefore, may be helpful in treating glaucoma.

Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.

Photoreceptor apoptosis is a major cause of visual loss in retinal detachment (RD) and several other visual disorders, but the underlying mechanisms remain elusive. Recently, increased expression of monocyte chemoattractant protein 1 (MCP-1) was reported in vitreous humor samples of patients with RD and diabetic retinopathy as well as in the brain tissues of patients with neurodegenerative diseases, including Alzheimers disease and multiple sclerosis. Here we report that MCP-1 plays a critical role in mediating photoreceptor apoptosis in an experimental model of RD. RD led to increased MCP-1 expression in the Müller glia and increased CD11b+ macrophage/microglia in the detached retina. An MCP-1 blocking antibody greatly reduced macrophage/microglia infiltration and RD-induced photoreceptor apoptosis. Confirming these results, MCP-1 gene-deficient mice showed significantly reduced macrophage/microglia infiltration after RD and very little photoreceptor apoptosis. In primary retinal mixed cultures, MCP-1 was cytotoxic for recoverin+ photoreceptors, and this toxicity was eliminated through immunodepleting macrophage/microglia from the culture. In vivo, deletion of the gene encoding CD11b/CD18 nearly eliminated macrophage/microglia infiltration to the retina after RD and the loss of photoreceptors. Thus, MCP-1 expression and subsequent macrophage/microglia infiltration and activation are critical for RD-induced photoreceptor apoptosis. This pathway may be an important therapeutic target for preventing photoreceptor apoptosis in RD and other CNS diseases that share a common etiology.

HIV protease inhibitors provide neuroprotection through inhibition of mitochondrial apoptosis in mice.

Neuroprotection can be achieved by preventing apoptotic death of postmitotic cells. Apoptotic death can occur by either a caspase-dependent mechanism, involving cytochrome c, apoptosis protease-activating factor-1 (Apaf-1), and caspase-9, or a caspase-independent mechanism, involving apoptosis-inducing factor (AIF). HIV protease inhibitors (PIs) avert apoptosis in part by preventing mitochondrial outer membrane permeabilization (MOMP), but the precise mechanism by which they work is not known. Here, we evaluated the impact of the PIs in a mouse model of retinal detachment (RD) in vivo and in murine primary retinal cell cultures in vitro. Oral administration of the PIs nelfinavir and ritonavir significantly inhibited photoreceptor apoptosis, while preventing the translocation of AIF from mitochondria to the nucleus as well as the activation of caspase-9. RD-induced photoreceptor apoptosis was similarly inhibited in mice carrying hypomorphic mutations of the genes encoding AIF or Apaf-1. Nelfinavir attenuated apoptosis as well as mitochondrial release of AIF and cytochrome c, and subsequent activation of caspase-9 in vitro, in photoreceptor cultures exposed to starvation or monocyte chemoattractant protein-1-stimulated (MCP-1-stimulated) macrophages. Our results suggest that the MOMP inhibition by PIs involved interruption of both caspase-dependent and caspase-independent apoptosis pathways and that PIs may be clinically useful for the treatment of diseases caused by excessive apoptosis.

Endogenous endostatin inhibits choroidal neovascularization

Endostatin, a fragment of the basement membrane component collagen XVIII, exhibits antian‐ giogenic properties in vitro and in vivo when high doses are administered. It is not known whether endogenous endostatin at physiological levels has a protective role as an inhibitor of pathological angiogenesis, such as choroidal neovascularization (CNV) in age‐related macular degeneration. Using a laser injury model, we induced CNV in mice lacking collagen XVIII/endosta‐ tin and in control mice. CNV lesions in mutant mice were ~ 3‐fold larger than in control mice and showed increased vascular leakage. These differences were independent of age‐related changes at the choroid‐ retina interface. Ultrastructural analysis of the choroidal vasculature in mutant mice excluded morphological vascular abnormalities as a cause for the larger CNV lesions. When recombinant endostatin was administered to collagen XVIII/endostatin‐deficient mice, CNV lesions were similar to those seen in control mice. In control mice treated with recombinant endostatin, CNV lesions were almost undetectable. These findings demonstrate that endogenous endostatin is an inhibitor of induced angiogenesis and that administration of endostatin potently inhibits CNV growth and vascular leakage. Endostatin may have a regulatory role in the pathogenesis of CNV and could be used therapeutically to inhibit growth and leakage of CNV lesions.— Marneros, A. G., She, H., Zambarakji, H., Hashizume, H., Connolly, E. J., Kim, I., Gragoudas, E. S., Miller, J. W., Olsen, B. R. Endogenous endostatin inhibits choroidal neovascularization. FASEB J. 21, 3809–3818 (2007)

Vascular adhesion protein-1 blockade suppresses choroidal neovascularization

Vascular adhesion protein‐1 (VAP‐1) is an endothelial cell adhesion molecule involved in leukocyte recruitment. Leukocytes and, in particular, macrophages play an important role in the development of choroidal neovascularization (CNV), an integral component of age‐related macular degeneration (AMD). Previously, we showed a role for VAP‐1 in ocular inflammation. Here, we investigate the expression of VAP‐1 in the choroid and its role in CNV development. VAP‐1 was expressed in the choroid, exclusively in the vessels, and colocalized in the vessels of the CNV lesions. VAP‐1 blockade with a novel and specific inhibitor significantly decreased CNV size, fluorescent angiographic leakage, and the accumulation of macrophages in the CNV lesions. Furthermore, VAP‐1 blockade significantly reduced the expression of inflammation‐associated molecules such as tumor necrosis factor (TNF) ‐α, monocyte chemoattractant protein (MCP) ‐1, and intercellular adhesion molecule (ICAM) ‐1. This work provides evidence for an important role of VAP‐1 in the recruitment of macrophages to CNV lesions, establishing a novel link between VAP‐1 and angiogenesis. Inhibition of VAP‐1 may become a new therapeutic strategy in the treatment of AMD.—Noda, K., She, H., Nakazawa, T., Hisatomi, T., Nakao, S., Almulki, L., Zandi, S., Miyahara, S., Ito, Y., Thomas, K. L., Garland, R. C., Miller, J. W., Gragoudas, E. S., Mashima, Y., Hafezi‐Moghadam, A. Vascular adhesion protein‐1 blockade suppresses choroidal neovascularization. FASEB J. 22, 2928‐2935 (2008)

Inhibition of vascular adhesion protein-1 suppresses endotoxin-induced uveitis

Inflammatory leukocyte accumulation is a common feature of major ocular diseases, such as uveitis, diabetic retinopathy, and age‐related macular degeneration. Vascular adhesion protein‐1 (VAP‐1), a cell surface and soluble molecule that possesses semi‐carbazide‐sensitive amine oxidase (SSAO) activity, is involved in leukocyte recruitment. However, the expression of VAP‐1 in the eye and its contribution to ocular inflammation are unknown. Here, we investigated the role of VAP‐1 in an established model of ocular inflammation, the endotoxin‐induced uveitis (EIU), using a novel and specific inhibitor. Our inhibitor has a half‐maximal inhibitory concentration (IC50) of 0.007 μM against human and 0.008 μM against rat SSAO, while its IC50 against the functionally related monoamine oxidase (MAO) ‐A and MAO‐B is > 10 μM. In the retina, VAP‐1 was exclusively expressed in the vasculature, and its expression level was elevated during EIU. VAP‐1 inhibition in EIU animals significantly suppressed leukocyte recruitment to the anterior chamber, vitreous, and retina, as well as retinal endothelial P‐selectin expression. Our data suggest an important role for VAP‐1 in the recruitment of leukocytes to the immune‐privileged ocular tissues during acute inflammation. VAP‐1 inhibition may become a novel strategy in the treatment of ocular inflammatory diseases. Noda, K., Miyahara, S., Nakazawa, T., Almulki, L., Nakao, S., Hisatomi, T., She, H., Thomas, K. L., Garland, R. C., Miller, J. W., Gragoudas, E. S., Kawai, Y., Mashima, Y., Hafezi‐Moghadam, A. Inhibition of vascular adhesion protein‐1 suppresses endotoxin‐in‐duced uveitis. FASEB J. 22, 1094–1103 (2008)

Atrial Natriuretic Peptide Reduces Vascular Leakage and Choroidal Neovascularization

Atrial natriuretic peptide (ANP) is a hormone with diuretic, natriuretic, and vasodilatory properties. ANP blocks vascular endothelial growth factor (VEGF) production and signaling in vitro; however, its role in vascular leakage and angiogenesis is unknown. In vitro, retinal barrier permeability (transepithelial electrical resistance (TEER)) was measured in cultured retinal endothelial (HuREC) and retinal epithelial (ARPE-19) cells with VEGF (10 ng/ml), ANP (1 pM to 1 micromol/L), and/or isatin, an ANP receptor antagonist. In vivo, blood-retinal barrier (BRB) leakage was studied using the Evans Blue dye technique in rats treated with intravitreal injections of ANP, VEGF, or vehicle. Choroidal neovascularization was generated by laser injury, and 7 days later, lesion size and leakage was quantitated. ANP significantly reversed VEGF-induced BRB TEER reduction in both HuREC and ARPE-19 cells, modeling the inner and the outer BRB, respectively. Isatin, a specific ANP receptor antagonist, reversed ANPs effect. ANP reduced the response of ARPE-19 cells to VEGF apically but not basolaterally, suggesting polarized expression of the ANP receptors in these cells. ANPs TEER response was concentration but not time dependent. In vivo, ANP significantly reduced VEGF-induced BRB leakage and the size of laser-induced choroidal neovascularization lesions. In sum, ANP is an effective inhibitor of VEGF-induced vascular leakage and angiogenesis in vivo. These results may lead to new treatments for ocular diseases where VEGF plays a central role, such as age-related macular degeneration or diabetic retinopathy.

The Regulatory Roles of Apoptosis-Inducing Factor in the Formation and Regression Processes of Ocular Neovascularization

The role of apoptosis in the formation and regression of neovascularization is largely hypothesized, although the detailed mechanism remains unclear. Inflammatory cells and endothelial cells both participate and interact during neovascularization. During the early stage, these cells may migrate into an angiogenic site and form a pro-angiogenic microenvironment. Some angiogenic vessels appear to regress, whereas some vessels mature and remain. The control mechanisms of these processes, however, remain unknown. Previously, we reported that the prevention of mitochondrial apoptosis contributed to cellular survival via the prevention of the release of proapoptotic factors, such as apoptosis-inducing factor (AIF) and cytochrome c. In this study, we investigated the regulatory role of cellular apoptosis in angiogenesis using two models of ocular neovascularization: laser injury choroidal neovascularization and VEGF-induced corneal neovascularization in AIF-deficient mice. Averting apoptosis in AIF-deficient mice decreased apoptosis of leukocytes and endothelial cells compared to wild-type mice and resulted in the persistence of these cells at angiogenic sites in vitro and in vivo. Consequently, AIF deficiency expanded neovascularization and diminished vessel regression in these two models. We also observed that peritoneal macrophages from AIF-deficient mice showed anti-apoptotic survival compared to wild-type mice under conditions of starvation. Our data suggest that AIF-related apoptosis plays an important role in neovascularization and that mitochondria-regulated apoptosis could offer a new target for the treatment of pathological angiogenesis.

Photoreceptor Protection after Photodynamic Therapy Using Dexamethasone in a Rat Model of Choroidal Neovascularization

PURPOSE To study whether corticosteroids protect photoreceptors when combined with photodynamic therapy (PDT) in a laser-induced model of choroidal neovascularization (CNV). METHODS PDT was performed in 36 Brown-Norway rats 2 weeks after laser induction of CNV. The expressional change of several cytokines and chemokines in the CNV lesions after PDT was measured by real-time PCR in combination with laser-capture microdissection. Immunostaining for monocyte chemoattractant protein (MCP)-1, C-C chemokine receptor 2(CCR2), interleukin (IL)-1beta, and myeloperoxidase(MPO) were performed. To study the effect of corticosteroids in combination with PDT, either dexamethasone (100 mg/kg) or control was injected intraperitoneally 1 hour before PDT. Animals were killed 24 hours or 1 week after PDT. CNV was examined by fluorescein angiography and choroidal flatmount. Photoreceptor degeneration was evaluated by TUNEL assay. RESULTS MCP-1 and IL-1beta was increased in CNV lesions 24 hours after PDT. CCR2 was also expressed in laser-induced CNV but did not increase after PDT. Twenty-four hours after PDT, MPO-positive cells were noted in the CNV lesions. Dexamethasone-treated animals had significantly fewer TUNEL-positive cells in the photoreceptor layer than did the control animals (P < 0.05) after PDT. Fluorescein angiographic grading of CNV closure 6 days after PDT showed a closure rate in the dexamethasone-treated group of 31% (15/48 lesions) compared to 10% (4/42 lesions) in the control group (P < 0.05). CNV size was significantly smaller in the dexamethasone-treated group 1 week after PDT compared with the control (P < 0.05). CONCLUSIONS Systemic administration of dexamethasone combined with PDT reduces photoreceptor apoptosis, increases angiographic closure, and reduces CNV size compared with PDT alone in a rat model.