Updated SARS-CoV-2 single nucleotide variants and mortality association.

By analyzing newly collected SARS-CoV-2 genomes and comparing them with our previous study about SARS-CoV-2 single nucleotide variants (SNVs) before June 2020, we found that the SNV clustering had changed remarkably since June 2020. Apart from that the group of SNVs became dominant, which is represented by two nonsynonymous mutations A23403G (S:D614G) and C14408T (ORF1ab:P4715L), a few emerging groups of SNVs were recognized with sharply increased monthly incidence ratios of up to 70% in November 2020. Further investigation revealed sets of SNVs specific to patients' ages and/or gender, or strongly associated with mortality. Our logistic regression model explored features contributing to mortality status, including three critical SNVs, G25088T(S:V1176F), T27484C (ORF7a:L31L), and T25A (upstream of ORF1ab), ages above 40 years old, and the male gender. The protein structure analysis indicated that the emerging subgroups of nonsynonymous SNVs and the mortality-related ones were located on the protein surface area. The clashes in protein structure introduced by these mutations might in turn affect the viral pathogenesis through the alteration of protein conformation, leading to a difference in transmission and virulence. Particularly, we explored the fact that nonsynonymous SNVs tended to occur in intrinsic disordered regions of Spike and ORF1ab to significantly increase hydrophobicity, suggesting a potential role in the change of protein folding related to immune evasion.

Proteosomal degradation impairs transcytosis of AAV vectors from suprachoroidal space to retina.

Suprachoroidal injection provides a new route of delivery for AAV vectors to retinal pigmented epithelial cells and photoreceptors that can be done in an outpatient setting and is less invasive and potentially safer than subretinal injection, the most common route of delivery for ocular gene therapy. After suprachoroidal injection of AAV8 or AAV9 vectors, there is strong transduction of photoreceptors, but it is unclear how vector traverses the retinal pigmented epithelium. In this study, we found that transduction of photoreceptors was significantly increased after suprachoroidal injection of AAV2tYF-CBA-GFP versus AAV2-CBA-GFP vector. Compared with AAV2, AAV2tYF is more resistant to proteosomal degradation. Treatment with protease inhibitors significantly increased photoreceptor transduction after suprachoroidal injection of AAV5-GRK1-GFP. These data suggest that after suprachoroidal injection, AAV vectors access photoreceptors by transcytosis through retinal pigmented epithelial cells during which they are subject to proteosomal degradation, which if suppressed can enhance transduction of photoreceptors.

Inhibiting the NLRP3 inflammasome with MCC950 ameliorates retinal neovascularization and leakage by reversing the IL-1ß/IL-18 activation pattern in an oxygen-induced ischemic retinopathy mouse model.

Activation of the nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome plays an important role in ocular neovascularization. In our study, we found that the expression and activation levels of NLRP3 inflammasome components, including NLRP3, an apoptosis-associated speck-like protein (ASC) containing caspase activation and recruitment domain (CARD) and caspase-1 (CAS1), were significantly upregulated. In addition, we found interleukin (IL)-1ß activity increased while IL-18 activity decreased in the retinas of oxygen-induced ischemic retinopathy (OIR) mice. MCC950, an inhibitor of NLRP3, reversed the IL-1ß/IL-18 activation pattern, inhibited the formation of retinal neovascularization (RNV), decreased the number of acellular capillaries and reduced leakage of retinal vessels. Moreover, MCC950 could regulate the expression of endothelial cell- and pericyte function-associated molecules, such as vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)1, VEGFR2, matrix metalloproteinase (MMP)2, MMP9, tissue inhibitor of metalloproteinases (TIMP)1, TIMP2, platelet-derived growth factor receptor-ß (PDGFR-ß), platelet-derived growth factor-B (PDGF-B), and angiopoietin2 (Ang2). In vitro, recombinant human (r)IL-18 and rIL-1ß regulated the expression of endothelial cell- and pericyte function-associated molecules and the proliferation and migration of endothelial cells and pericytes. We therefore determined that inhibiting the NLRP3 inflammasome with MCC950 can regulate the function of endothelial cells and pericytes by reversing the IL-1ß/IL-18 activation pattern to ameliorate RNV and leakage; thereby opening new avenues to treat RNV-associated ocular diseases.

Inhibiting NF-κB Signaling Activation Reduces Retinal Neovascularization by Promoting a Polarization Shift in Macrophages.

Purpose: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling is involved in regulating tumor angiogenesis and metastasis; however, the exact mechanism of action in retinal neovascularization (RNV) remains unclear. The purpose of this study was to determine the role and underlying mechanism of NF-κB in regulating RNV in retinal neovascularization mice. Methods: Expression levels of NF-κB signaling were detected by immunofluorescence staining and western blotting in retinas of oxygen-induced retinopathy (OIR) mice. OIR mice were treated with either pyrrolidinedithiocarbamate (PDTC), a NF-κB signaling inhibitor, or PBS, and retinal flat-mounts were performed to quantify the area of RNV and the recruitment of retinal macrophages by immunofluorescence staining. Macrophage polarization detected by flow cytometric analysis and the expression of macrophage polarization-associated genes were evaluated by immunofluorescence staining, quantitative RT-PCR, and western blotting. Results: Expression levels of phosphorylated IκBα (p-IκBα) and p-p65 increased in OIR mice. Inhibiting NF-κB signaling activation by PDTC significantly reduced RNV. After treatment with PDTC, a reduction in the quantity of macrophages was observed: M1 polarized macrophages decreased, and M2 polarized macrophages increased; the expression of M1 macrophage-associated cytokines decreased and M2 macrophage-associated cytokines increased in the retinas of OIR mice. Conclusions: Blocking activation of NF-κB signaling reduces RNV by promoting polarization of M1 macrophages to M2 macrophages in OIR mice.

Sustained treatment of retinal vascular diseases with self-aggregating sunitinib microparticles.

Neovascular age-related macular degeneration and diabetic retinopathy are prevalent causes of vision loss requiring frequent intravitreous injections of VEGF-neutralizing proteins, and under-treatment is common and problematic. Here we report incorporation of sunitinib, a tyrosine kinase inhibitor that blocks VEGF receptors, into a non-inflammatory biodegradable polymer to generate sunitinib microparticles specially formulated to self-aggregate into a depot. A single intravitreous injection of sunitinib microparticles potently suppresses choroidal neovascularization in mice for six months and in another model, blocks VEGF-induced leukostasis and retinal nonperfusion, which are associated with diabetic retinopathy progression. After intravitreous injection in rabbits, sunitinib microparticles self-aggregate into a depot that remains localized and maintains therapeutic levels of sunitinib in retinal pigmented epithelium/choroid and retina for more than six months. There is no intraocular inflammation or retinal toxicity. Intravitreous injection of sunitinib microparticles provides a promising approach to achieve sustained suppression of VEGF signaling and improve outcomes in patients with retinal vascular diseases.

AAV8-vectored suprachoroidal gene transfer produces widespread ocular transgene expression.

There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8.GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGF-induced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.

A collagen IV-derived peptide disrupts α5ß1 integrin and potentiates Ang2/Tie2 signaling.

The angiopoietin (Ang)/Tie2 signaling pathway is essential for maintaining vascular homeostasis, and its dysregulation is associated with several diseases. Interactions between Tie2 and α5ß1 integrin have emerged as part of this control; however, the mechanism is incompletely understood. AXT107, a collagen IV-derived peptide, has strong antipermeability activity and has enabled the elucidation of this previously undetermined mechanism. Previously, AXT107 was shown to inhibit VEGFR2 and other growth factor signaling via receptor tyrosine kinase association with specific integrins. AXT107 disrupts α5ß1 and stimulates the relocation of Tie2 and α5 to cell junctions. In the presence of Ang2 and AXT107, junctional Tie2 is activated, downstream survival signals are upregulated, F-actin is rearranged to strengthen junctions, and, as a result, endothelial junctional permeability is reduced. These data suggest that α5ß1 sequesters Tie2 in nonjunctional locations in endothelial cell membranes and that AXT107-induced disruption of α5ß1 promotes clustering of Tie2 at junctions and converts Ang2 into a strong agonist, similar to responses observed when Ang1 levels greatly exceed those of Ang2. The potentiation of Tie2 activation by Ang2 even extended to mouse models in which AXT107 induced Tie2 phosphorylation in a model of hypoxia and inhibited vascular leakage in an Ang2-overexpression transgenic model and an LPS-induced inflammation model. Because Ang2 levels are very high in ischemic diseases, such as diabetic macular edema, neovascular age-related macular degeneration, uveitis, and cancer, targeting α5ß1 with AXT107 provides a potentially more effective approach to treat these diseases.

ATN-161 as an Integrin α5ß1 Antagonist Depresses Ocular Neovascularization by Promoting New Vascular Endothelial Cell Apoptosis.

BACKGROUND ATN-161 (Ac-PHSCN-NH2), an antagonist of integrin α5ß1, has shown an important influence in inhibiting tumor angiogenesis and metastasis of other tumor types. However, the mechanism of action of ATN-161 and whether it can inhibit ocular neovascularization (NV) are unclear. This study investigated the role of ATN-161 in regulating ocular angiogenesis in mouse models and explored the underlying signaling pathway. MATERIAL AND METHODS An oxygen-induced retinopathy (OIR) mouse model and a laser-induced choroidal neovascularization (CNV) mouse model were used to test integrin a5b1 expression and the effect of ATN-161 on ocular NV by immunofluorescence staining, Western blot analysis, and flat-mount analysis. The activation of nuclear factor-κB (NF-κB), matrix metalloproteinase-2/9 (MMP-2/9), and cell apoptosis were detected by immunofluorescence staining, Western blot, real-time RT-PCR, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). The cell proliferation was detected by BrdU labeling. RESULTS In OIR and CNV mice, the protein expression level of integrin α5ß1 increased compared with that in age-matched controls. The mice given ATN-161 had significantly reduced retinal neovascularization (RNV) and CNV. Blocking integrin a5b1 by ATN-161 strongly inhibited nuclear factor-κB (NF-κB) activation and matrix metalloproteinase-2/9 (MMP-2/9) expression and promoted cell apoptosis, but the effect of ATN-161 on proliferation in CNV mice was indirect and required the inhibition of neovascularization. Inhibiting NF-κB activation by ammonium pyrrolidinedithiocarbamate (PDTC) reduced RNV and promoted cell apoptosis in ocular NV. CONCLUSIONS Blocking integrin α5ß1 by ATN-161 reduced ocular NV by inhibiting MMP-2/MMP-9 expression and promoting the cell apoptosis of ocular NV.

Flt3 Regulation in the Mononuclear Phagocyte System Promotes Ocular Neovascularization.

Fms-like tyrosine kinase 3 (Flt3), a tyrosine kinase receptor expressed in CD34+ hematopoietic stem/progenitor cells, is important for both normal myeloid and lymphoid differentiation. It has been implicated in mice and humans for potential multilineage differentiation. We found that mice deficient in Flt3 or mice that received an Flt3 inhibitor (AC220) showed significantly reduced areas of ischemia-induced retinal neovascularization (RNV) and laser-induced choroidal NV (CNV) (P < 0.05). Increased Flt3 expression at the protein level was detected in retinas of oxygen-induced retinopathy (OIR) mice at P15 and P18 during retinal NV (RNV) progression. We subsequently found that macrophages (Mphi) polarization was regulated at the site of CNV in Flt3-deficient mice. Flow cytometry analysis demonstrated that Flt3 deficiency shifted Mphi polarization towards an M2 phenotype during RNV with significant reduction in M1 cytokine expression when compared to the wild-type controls (P < 0.05). Based on the above findings, we concluded that Flt3 inhibition alleviated ocular NV by promoting a Mphi polarization shift towards the M2 phenotype. Therapies targeting Flt3 may provide a new approach for the treatment of ocular NV.

VEGF/VEGFR2 blockade does not cause retinal atrophy in AMD-relevant models.

Intraocular injections of VEGF-neutralizing proteins provide tremendous benefits in patients with choroidal neovascularization (NV) due to age-related macular degeneration (AMD), but during treatment some patients develop retinal atrophy. Suggesting that VEGF is a survival factor for retinal neurons, a clinical trial group attributed retinal atrophy to VEGF suppression and cautioned against frequent anti-VEGF injections. This recommendation may contribute to poor outcomes in clinical practice from insufficient treatment. Patients with type 3 choroidal NV have particularly high risk of retinal atrophy, an unexplained observation. Herein we show in mouse models that VEGF signaling does not contribute to photoreceptor survival and functioning: (a) neutralization of VEGFR2 strongly suppresses choroidal NV without compromising photoreceptor function or survival; (b) VEGF does not slow loss of photoreceptor function or death in mice with inherited retinal degeneration, and there is no exacerbation by VEGF suppression; and (c) mice with type 3 choroidal NV develop retinal atrophy due to oxidative damage with no contribution from VEGF suppression. Intraocular injections of VEGF-neutralizing proteins, a highly effective treatment in patients with neovascular AMD, should not be withheld or reduced due to concern that they may contribute to long-term visual loss from retinal atrophy.

Endocan Blockade Suppresses Experimental Ocular Neovascularization in Mice.

Purpose: Ocular neovascularization (NV) is a pathologic process characterized by the proliferation and infiltration of various types of cells such as RPE, glial, and endothelial cells, which interact with proangiogenic factors and inflammatory cytokines. Endocan is known to be enriched in retinal endothelial tip cells under hypoxia, but the effect of endocan on ocular NV progression is largely unknown. In this study, we investigated the role of endocan in the ocular NV pathologic process and the possible mechanisms involved. Methods: In the eyes of mice with oxygen-induced retinopathy (OIR); choroidal NV (CNV); and rhodopsin promoter (rho)/VEGF transgenic mice, endocan expression was assessed by quantitative real-time PCR (RT-PCR) and Western blot. In vivo, a specific functional antibody was used to neutralize endocan and ocular NV levels were evaluated by RT-PCR, Western blot and immunostaining of flat-mounts. In vitro, the effect of endocan on human retinal microvascular endothelial cell (HREC) tube formation was observed using a routine method. Results: Endocan was significantly elevated in these three experimental mice models. Endocan blockade with the neutralizer intravitreal injection not only suppressed the area of retinal, choroidal and subretinal NV, but also resulted in a decrease in several angiogenesis-associated molecules. Recombinant endocan protein (rhEndocan) was found to induce tube formation on HRECs directly. Conclusions: The current data suggest that endocan is a potential therapeutic or an additional target for retinal and subretinal NV diseases.

AAV8-antiVEGFfab Ocular Gene Transfer for Neovascular Age-Related Macular Degeneration.

Sustained suppression of VEGF is needed in many patients with neovascular age-related macular degeneration (NVAMD), and gene transfer of a VEGF-neutralizing protein is a promising approach to achieve it. Initial clinical trials testing this approach have shown encouraging signals, but evidence of robust transgene expression and consistent antiangiogenic and antipermeability activity has been lacking. In this study, we demonstrate expression of an anti-human VEGF antibody fragment (antiVEGFfab) after subretinal injection of AAV8-antiVEGFfab. In transgenic mice expressing human VEGF in retina (rho/VEGF mice), a model of type 3 choroidal neovascularization (NV), eyes injected with ≥1 × 107 gene copies (GC) of AAV8-antiVEGFfab had significantly less mean area of NV than null vector-injected eyes. A dose-dependent response was observed with modest reduction of NV with ≤3 × 107, >50% reduction with ≥1 × 108 GC and almost complete elimination of NV with 3 × 109 or 1 × 1010 GC. In Tet/opsin/VEGF mice, in which doxycycline-induced high expression of VEGF leads to severe vascular leakage and exudative retinal detachment (RD), reduction of total RD by 70%-80% occurred with 3 × 109 or 1 × 1010 GC of AAV8-antiVEGFfab, an effect that was sustained for at least a month. These data strongly support initiating clinical trials testing subretinal injection of AAV8-antiVEGFfab in patients with NVAMD.

Reversible retinal vessel closure from VEGF-induced leukocyte plugging.

Clinical trials in patients with macular edema due to diabetic retinopathy or retinal vein occlusion (RVO) have shown that suppression of VEGF not only improves macular edema, but also reopens closed retinal vessels, prevents progression of vessel closure, and improves retinopathy. In this study, we show the molecular basis for those clinical observations. Increased retinal levels of VEGF in mice cause plugging of retinal vessels with leukocytes, vessel closure, and hypoxia. Suppression of VEGF reduces leukocyte plugging, causing reperfusion of closed vessels. Activation of VEGFR1 contributes to leukocyte recruitment, because it is significantly reduced by an anti-VEGFR1-neutralizing antibody. High VEGF increases transcriptional activity of NF-κB and expression of NF-κB target genes, particularly Vcam1. Injection of an anti-VCAM-1-neutralizing antibody reduces VEGF-induced leukocyte plugging. These data explain the broad range of benefits obtained by VEGF suppression in patients with ischemic retinopathies, provide an important insight into the pathogenesis of RVO and diabetic retinopathy, and suggest that sustained suppression of VEGF early in the course of these diseases may prevent vessel closure, worsening ischemia, and disease progression. This study also identifies VEGFR1 and VCAM-1 as molecular targets whose suppression could supplement VEGF neutralization for treatment of RVO and diabetic retinopathy.

Generation and characterization of ABBV642, a dual variable domain immunoglobulin molecule (DVD-Ig) that potently neutralizes VEGF and PDGF-BB and is designed for the treatment of exudative age-related macular degeneration.

Exudative age-related macular degeneration (AMD) is the most common cause of moderate and severe vision loss in developed countries. Intraocular injections of vascular endothelial growth factor (VEGF or VEGF-A)-neutralizing proteins provide substantial benefit, but frequent, long-term injections are needed. In addition, many patients experience initial visual gains that are ultimately lost due to subretinal fibrosis. Preclinical studies and early phase clinical trials suggest that combined suppression of VEGF and platelet-derived growth factor-BB (PDGF-BB) provides better outcomes than suppression of VEGF alone, due to more frequent regression of neovascularization (NV) and suppression of subretinal fibrosis. We generated a dual variable domain immunoglobulin molecule, ABBV642 that specifically and potently binds and neutralizes VEGF and PDGF-BB. ABBV642 has been optimized for treatment of exudative AMD based on the following design characteristics: 1) high affinity binding to all VEGF-A isoforms and both soluble and extracellular matrix (ECM)-associated PDGF-BB; 2) potential for extended residence time in the vitreous cavity to decrease the frequency of intraocular injections; 3) rapid clearance from systemic circulation compared with molecules with wild type Fc region for normal FcRn binding, which may reduce the risk of systemic complications; and 4) low risk of potential effector function. The bispecificity of ABBV642 allows for a single injection of a single therapeutic agent, and thus a more streamlined development and regulatory path compared with combination products. In a mouse model of exudative AMD, ABBV642 was observed to be more effective than aflibercept. ABBV642 has potential to improve efficacy with reduced injection frequency in patients with exudative AMD, thereby reducing the enormous disease burden for patients and society.

Targeting VE-PTP activates TIE2 and stabilizes the ocular vasculature.

Retinal and choroidal neovascularization (NV) and vascular leakage contribute to visual impairment in several common ocular diseases. The angiopoietin/TIE2 (ANG/TIE2) pathway maintains vascular integrity, and negative regulators of this pathway are potential therapeutic targets for these diseases. Here, we demonstrated that vascular endothelial-protein tyrosine phosphatase (VE-PTP), which negatively regulates TIE2 activation, is upregulated in hypoxic vascular endothelial cells, particularly in retinal NV. Intraocular injection of an anti-VE-PTP antibody previously shown to activate TIE2 suppressed ocular NV. Furthermore, a small-molecule inhibitor of VE-PTP catalytic activity (AKB-9778) activated TIE2, enhanced ANG1-induced TIE2 activation, and stimulated phosphorylation of signaling molecules in the TIE2 pathway, including AKT, eNOS, and ERK. In mouse models of neovascular age-related macular degeneration, AKB-9778 induced phosphorylation of TIE2 and strongly suppressed NV. Ischemia-induced retinal NV, which is relevant to diabetic retinopathy, was accentuated by the induction of ANG2 but inhibited by AKB-9778, even in the presence of high levels of ANG2. AKB-9778 also blocked VEGF-induced leakage from dermal and retinal vessels and prevented exudative retinal detachments in double-transgenic mice with high expression of VEGF in photoreceptors. These data support targeting VE-PTP to stabilize retinal and choroidal blood vessels and suggest that this strategy has potential for patients with a wide variety of retinal and choroidal vascular diseases.

Interleukin-18 has antipermeablity and antiangiogenic activities in the eye: reciprocal suppression with VEGF.

Interleukin-18 (IL-18) is increased along with IL-1ß by activation of the inflammasome and has been implicated in inflammatory and autoimmune diseases, but its role in the eye is uncertain. In patients with macular edema due to retinal vein occlusion, intraocular IL-18 levels increased significantly (P < 0.001) after treatment with ranibizumab particularly in patients with high baseline IL-18 which correlated with good visual outcome (P < 0.05). In mice with ischemic retinopathy, suppression of VEGF caused an increase in IL18 mRNA due to an increase in IL-18-positive myeloid cells. VEGF significantly and specifically inhibited IL-18 production by myeloid cells stimulated with lipopolysaccharide (P < 0.001). Intraocular injection of IL-18 reduced VEGF-induced leakage and neovascularization, and reversed VEGF-induced suppression of Claudin5 expression and Claudin 5 labeling of vascular tight junctions. Injection of IL-18 also increased expression of Thrombospondin 1 and reduced ischemia-induced retinal neovascularization relevant to diabetic retinopathy and subretinal neovascularization relevant to neovascular age-related macular degeneration. Thus, VEGF and IL-18 suppress each other's production and effects on the vasculature suggesting that IL-18 may provide benefit in multiple retinal/choroidal vascular diseases.

Antagonism of PDGF-BB suppresses subretinal neovascularization and enhances the effects of blocking VEGF-A.

Hypoxia-inducible factor-1 (HIF-1) plays an important role in retinal and subretinal neovascularization (NV). Increased levels of HIF-1 cause increased expression of vascular endothelial growth factor (VEGF-A) and current therapies for ocular NV focus on neutralizing VEGF-A, but there is mounting evidence that other HIF-1-responsive gene products may also participate. In this study, we tested the effect of a designed ankyrin repeat protein (DARPin) that selectively binds and antagonizes the hypoxia-regulated gene product PDGF-BB in three models of subretinal NV (relevant to neovascular age-related macular degeneration) and compared its effects to a DARPin that selectively antagonizes VEGF-A. Daily intraperitoneal injections of 10 mg/kg of the anti-PDGF-BB DARPin or 1 mg/kg of the anti-VEGF DARPin significantly suppressed subretinal NV from laser-induced rupture of Bruch's membrane. Injections of 1 mg/kg/day of the anti-PDGF-BB DARPin had no significant effect, but when combined with 1 mg/kg/day of the anti-VEGF-A DARPin there was greater suppression than injection of the anti-VEGF-A DARPin alone. In Vldlr (-/-) mice which spontaneously develop subretinal NV, intraocular injection of 1.85 µg of anti-PDGF-BB or anti-VEGF-A DARPin caused significant suppression of NV and when combined there was greater suppression than with either alone. The two DARPins also showed an additive effect in Tet/opsin/VEGF double transgenic mice, a particularly severe model of subretinal NV and exudative retinal detachment. In addition, intraocular injection of 1.85 µg of anti-PDGF-BB DARPin strongly suppressed ischemia-induced retinal NV, which is relevant to proliferative diabetic retinopathy and retinopathy of prematurity. These data demonstrate that PDGF-BB is another hypoxia-regulated gene product that along with VEGF-A contributes to ocular NV and suppression of both provides an additive effect.

Sustained delivery of a HIF-1 antagonist for ocular neovascularization.

Doxorubicin (DXR) and daunorubicin (DNR) inhibit hypoxia-inducible factor-1 (HIF-1) transcriptional activity by blocking its binding to DNA. Intraocular injections of DXR or DNR suppressed choroidal and retinal neovascularization (NV), but also perturbed retinal function as demonstrated by electroretinograms (ERGs). DXR was conjugated to novel copolymers of branched polyethylene glycol and poly(sebacic acid) (DXR-PSA-PEG3) and formulated into nanoparticles that when placed in aqueous buffer, slowly released small DXR-conjugates. Intraocular injection of DXR-PSA-PEG3 nanoparticles (1 or 10 µg DXR content) reduced HIF-1-responsive gene products, strongly suppressed choroidal and retinal NV, and did not cause retinal toxicity. In transgenic mice that express VEGF in photoreceptors, intraocular injection of DXR-PSA-PEG3 nanoparticles (10 µg DXR content) suppressed NV for at least 35 days. Intraocular injection of DXR-PSA-PEG3 nanoparticles (2.7 mg DXR content) in rabbits resulted in sustained DXR-conjugate release with detectable levels in aqueous humor and vitreous for at least 105 days. This study demonstrates a novel HIF-1-inhibitor-polymer conjugate formulated into controlled-release particles that maximizes efficacy and duration of activity, minimizes toxicity, and provides a promising new chemical entity for treatment of ocular NV.

VEGF receptor blockade markedly reduces retinal microglia/macrophage infiltration into laser-induced CNV.

Although blocking VEGF has a positive effect in wet age-related macular degeneration (AMD), the effect of blocking its receptors remains unclear. This was an investigation of the effect of VEGF receptor (VEGFR) 1 and/or 2 blockade on retinal microglia/macrophage infiltration in laser-induced choroidal neovascularization (CNV), a model of wet AMD. CNV lesions were isolated by laser capture microdissection at 3, 7, and 14 days after laser and analyzed by RT-PCR and immunofluorescence staining for mRNA and protein expression, respectively. Neutralizing antibodies for VEGFR1 or R2 and the microglia inhibitor minocycline were injected intraperitoneally (IP). Anti-CD11b, CD45 and Iba1 antibodies were used to confirm the cell identity of retinal microglia/macrophage, in the RPE/choroidal flat mounts or retinal cross sections. CD11b(+), CD45(+) or Iba1(+) cells were counted. mRNA of VEGFR1 and its three ligands, PlGF, VEGF-A (VEGF) and VEGF-B, were expressed at all stages, but VEGFR2 were detected only in the late stage. PlGF and VEGF proteins were expressed at 3 and 7 days after laser. Anti-VEGFR1 (MF1) delivered IP 3 days after laser inhibited infiltration of leukocyte populations, largely retinal microglia/macrophage to CNV, while anti-VEGFR2 (DC101) had no effect. At 14 days after laser, both MF1 and DC101 antibodies markedly inhibited retinal microglia/macrophage infiltration into CNV. Therefore, VEGFR1 and R2 play differential roles in the pathogenesis of CNV: VEGFR1 plays a dominant role at 3 days after laser; but both receptors play pivotal roles at 14 days after laser. In vivo imaging demonstrated accumulation of GFP-expressing microglia into CNV in both CX3CR1(gfp/gfp) and CX3CR1(gfp/+) mice. Minocycline treatment caused a significant increase in lectin(+) cells in the sub-retinal space anterior to CNV and a decrease in dextran-perfused neovessels compared to controls. Targeting the chemoattractant molecules that regulate trafficking of retinal microglia/macrophage appears to be a compelling therapeutic strategy to control CNV and treat wet AMD.

Topical pazopanib blocks VEGF-induced vascular leakage and neovascularization in the mouse retina but is ineffective in the rabbit.

PURPOSE: To test the effect of pazopanib, a tyrosine kinase inhibitor that blocks VEGF and platelet-derived growth factor (PDGF) receptors and c-Kit, on vascular leakage and neovascularization (NV) in the retina. METHODS: Pazopanib was tested to determine its effect on VEGF-induced vascular permeability via measurement of [(3)H]mannitol retina to lung (RLLR) and retina to renal leakage ratios (RRLR) and in rho/VEGF mice with subretinal NV. In rabbits, the effect of intravitreal, topical, and systemic pazopanib on VEGF-induced leakage was tested by vitreous fluorophotometry. RESULTS: In mice, oral pazopanib (40 mg/kg twice a day [bid]) reduced RLLR (0.84 to 0.58, P = 0.0014) and RRLR (0.55 to 0.30, P = 0.0018) in VEGF-injected eyes. After intraocular injection of VEGF into both eyes, topical pazopanib (10 mg/mL three times a day [tid] for 14 days) reduced RLLR (0.85 vs. 0.56, P = 0.001), RRLR (0.44 vs. 0.28, P = 0.0075), and immunoreactive albumin in the retina compared to values in fellow eye controls. Treatment of one eye of rho/VEGF mice with 10 mg/mL, but not 5 mg/mL, pazopanib tid reduced the mean area of subretinal NV compared to that in fellow eyes (0.0055 vs. 0.0025 mm(2), P = 0.020). In rabbits, intravitreal pazopanib suppressed VEGF-induced fluorescein leakage, but topical (10 mg/mL four times a day [qid] or 12 mg/mL bid) had no significant effect. Systemic administration of pazopanib by osmotic pump with or without 10 mg/mL drops tid also failed to suppress VEGF-induced leakage. CONCLUSIONS: Administration of pazopanib topically or systemically suppressed retinal vascular leakage in mice, but not rabbits. These data suggest differences in the blood-retinal barrier (BRB) of mice and rabbits and indicate that penetration through the outer BRB may be needed for topically administered drugs to exert effects in the retina.