Choroidal Neovascularization Model Research Articles

A novel ferrochelatase inhibitor as a therapy for ocular neovascularization

Background and Hypothesis: Ocular neovascularization, characterized by the abnormal growth of new blood vessels, underlies blindness-inducing diseases such as wet age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). This neovascularization can be either choroidal (in the case of wet AMD) or retinal (in the case of PDR). Inhibition of vascular endothelial growth factor is the target of current FDA approved therapies, however the need for intravitreal injections poses a drawback to such treatments. Furthermore, many patients are non-responders or refractory. Given this, the discovery of novel therapeutic targets is warranted. Ferrochelatase (FECH) is an enzyme involved in heme synthesis. The Corson lab has previously shown FECH to be necessary for angiogenesis both in vitro and in vivo. The Corson lab has been successful in developing novel compounds that inhibit FECH in vitro, based on high-throughput screening hits. The objective of this project was to test one such novel FECH inhibitor in a choroidal neovascularization mouse model. Experimental Design: FECH inhibition was investigated in the laser-induced choroidal neovascularization model. This model involved creating a laser-induced lesion into the retinal pigment epithelium/choroid of mice aged 6-8 weeks; this promotes neovascularization. The FECH inhibitor compound was delivered once intravitreally at the time of the laser. Neovascularization was analyzed in vivo using optical coherence tomography and fluorescein angiography one and two weeks after laser, then quantified ex vivo by isolectin staining. Results: There was a trend toward decreased ocular neovascularization with the administration of a FECH inhibitor, as measured by optical coherence tomography seven days after laser treatment. However, further analyses are ongoing to validate this finding. Conclusion and Potential Impact: The administration of a FECH inhibitor compound intravitreally may result in a decrease in CNV. This may be useful as it could lead to FECH serving as a target for future therapies.

Anti-VEGFR2 Antibody-modified Micelle for Triggered Drug Delivery and Effective Therapy of Choroidal Neovascularization.

This study aimed to examine whether DC101 (anti-VEGFR2 antibody)- modified micelles have applications as novel drug delivery devices, which allow small molecule antiangiogenic agents to deliver to angiogenic sites on a murine laser-induced choroidal neovascularization (CNV) model. CNV was induced by photocoagulation on the unilateral eye of each mouse under anesthesia. Immediately after laser coagulation, E7974-loaded DC101-modified micelles and motesanib-loaded DC101-modified micelles were intravitreally administrated. Two weeks after photocoagulation, CNV was visualized using fluorescein-conjugated dextran (MW=2,000 kDa), and the CNV area was measured in retinal pigment epithelium (RPE)-choroidal flat mounts. Intravitreal administration of both DC101-modified micelles loaded with E7974 at 2 µM and motesanib at 2 µM significantly reduced CNV area in the murine laser-induced CNV model at a clearly lower concentration than the effective dose of each agent. These results suggest that DC101-modified micelle might be effective drug carrier system for treating CNV and other ocular angiogenic diseases.

Novel anti-angiogenic PEDF-derived small peptides mitigate choroidal neovascularization

Abnormal migration and proliferation of endothelial cells (EC) drive neovascular retinopathies. While anti-VEGF treatment slows progression, pathology is often supported by decrease in intraocular pigment epithelium-derived factor (PEDF), an endogenous inhibitor of angiogenesis. A surface helical 34-mer peptide of PEDF, comprising this activity, is efficacious in animal models of neovascular retina disease but remains impractically large for therapeutic use. We sought smaller fragments within this sequence that mitigate choroidal neovascularization (CNV). Expecting rapid intravitreal (IVT) clearance, we also developed a method to reversibly attach peptides to nano-carriers for extended delivery. Synthetic fragments of 34-mer yielded smaller anti-angiogenic peptides, and N-terminal capping with dicarboxylic acids did not diminish activity. Charge restoration via substitution of an internal aspartate by asparagine improved potency, achieving low nM apoptotic response in VEGF-activated EC. Two optimized peptides (PEDF 335, 8-mer and PEDF 336, 9-mer) were tested in a mouse model of laser-induced CNV. IVT injection of either peptide, 2–5 days before laser treatment, gave significant CNV decrease at day +14 post laser treatment. The 8-mer also decreased CNV, when administered as eye drops. Also examined was a nanoparticle-conjugate (NPC) prodrug of the 9-mer, having positive zeta potential, expected to display longer intraocular residence. This NPC showed extended efficacy, even when injected 14 days before laser treatment. Neither inflammatory cells nor other histopathologic abnormalities were seen in rabbit eyes harvested 14 days following IVT injection of PEDF 336 (>200 μg). No rabbit or mouse eye irritation was observed over 12–17 days of PEDF 335 eye drops (10 mM). Viability was unaffected in 3 retinal and 2 choroidal cell types by PEDF 335 up to 100 μM, PEDF 336 (100 μM) gave slight growth inhibition only in choroidal EC. A small anti-angiogenic PEDF epitope (G-Y-D-L-Y-R-V) was identified, variants (adipic-Sar-Y-N-L-Y-R-V) mitigate CNV, with clinical potential in treating neovascular retinopathy. Their shared active motif, Y - - - R, is found in laminin (Ln) peptide YIGSR, which binds Ln receptor 67LR, a known high-affinity ligand of PEDF 34-mer.

FGF2-induced STAT3 activation regulates pathologic neovascularization

Cell-autonomous endothelial cell (EC) fibroblast growth factor receptor (FGFR) signaling through FGFR1/2 is essential for injury-induced wound vascularization and pathologic neovascularization as in blinding eye diseases such as age-related macular degeneration. Which FGF ligand(s) is critical in regulating angiogenesis is unknown. Utilizing ex vivo models of choroidal endothelial sprouting and in vivo models of choroidal neovascularization (CNV), we demonstrate here that only FGF2 is the essential ligand. Though FGF-FGFR signaling can activate multiple intracellular signaling pathways, we show that FGF2 regulates pathogenic angiogenesis via STAT3 activation. The identification of FGF2 as a critical mediator in aberrant neovascularization provides a new opportunity for developing multi-target therapies in blinding eye diseases especially given the limitations of anti-VEGF monotherapy.

SCF (Stem Cell Factor) and cKIT Modulate Pathological Ocular Neovascularization.

Aberrant neovascularization is a leading cause of blindness in several eye diseases, including age-related macular degeneration and proliferative diabetic retinopathy. The identification of key regulators of pathological ocular neovascularization has been a subject of extensive research and great therapeutic interest. Here, we explored the previously unrecognized role of cKIT and its ligand, SCF (stem cell factor), in the pathological ocular neovascularization process. Approach and Results: Compared with normoxia, hypoxia, a crucial driver of neovascularization, caused cKIT to be highly upregulated in endothelial cells, which significantly enhanced the angiogenic response of endothelial cells to SCF. In murine models of pathological ocular neovascularization, such as oxygen-induced retinopathy and laser-induced choroidal neovascularization models, cKIT and SCF expression was significantly increased in ocular tissues, and blockade of cKIT and SCF using cKit mutant mice and anti-SCF neutralizing IgG substantially suppressed pathological ocular neovascularization. Mechanistically, SCF/cKIT signaling induced neovascularization through phosphorylation of glycogen synthase kinase-3β and enhancement of the nuclear translocation of β-catenin and the transcription of β-catenin target genes related to angiogenesis. Inhibition of β-catenin-mediated transcription using chemical inhibitors blocked SCF-induced in vitro angiogenesis in hypoxia, and injection of a β-catenin agonist into cKit mutant mice with oxygen-induced retinopathy significantly enhanced pathological neovascularization in the retina. Conclusions; Our data reveal that SCF and cKIT are promising novel therapeutic targets for treating vision-threatening ocular neovascular diseases.

The role of sphingosine 1-phosphate receptors on retinal pigment epithelial cells barrier function and angiogenic effects

Sphingosine-1-phosphate (S1P) is a lysophospholipid mediator, promoting angiogenesis and inflammation via interactions with its receptors (S1P1–5), but the receptors and signaling pathways responsible for the progression of choroidal neovascularization (CNV) remain unknown. We investigated the roles of S1P/S1P receptors in RPE cells. ARPE-19 cells were treated with S1P dissolved in carrier proteins of albumin or apolipoprotein M (ApoM). The mRNA expression levels of interleukin-8 (IL-8), C-C motif chemokine ligand 2 (CCL2), and vascular endothelial growth factor (VEGF) were evaluated using quantitative real-time polymerase chain reaction. The protein level of hypoxia-inducible factor (HIF)-1α was assessed via enzyme-linked immunosorbent assay. HIF transcriptional activity was evaluated with a dual-reporter luciferase assay. Cellular barrier integrity was evaluated using transepithelial electrical resistance and the FITC-dextran permeability assay. The suppressive effect of an S1P antagonist on CNV progression was investigated with a laser-induced CNV model in mice. The increase in expression of IL-8, CCL2, and VEGF due to albumin-bound S1P was significantly mitigated by an S1P2 antagonist. The expression of HIF-1α significantly decreased with inhibition of S1P2 and S1P3. In addition, albumin-bound S1P disrupted the barrier integrity of retinal pigment epithelial cells via S1P2, whereas integrity was strengthened by ApoM-bound S1P. CNV lesions were significantly reduced in the mouse model with intravitreal injection of S1P2 antagonist.This study demonstrated that S1P significantly promotes angiogenesis, inflammation, and barrier integrity, which was attenuated by inhibition of S1P2 or S1P3, suggesting that regulation of S1P2 and S1P3 is a novel therapeutic target for CNV.

Enantioselective Synthesis of Homoisoflavanones by Asymmetric Transfer Hydrogenation and Their Biological Evaluation for Antiangiogenic Activity.

Neovascular eye diseases are a major cause of blindness. Excessive angiogenesis is a feature of several conditions, including wet age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity. Development of novel antiangiogenic small molecules for the treatment of neovascular eye disease is essential to provide new therapeutic leads for these diseases. We have previously reported the therapeutic potential of anti-angiogenic homoisoflavanone derivatives with efficacy in retinal and choroidal neovascularization models, although these are racemic compounds due to the C3-stereogenic center in the molecules. This work presents asymmetric synthesis and structural determination of anti-angiogenic homoisoflavanones and pharmacological characterization of the stereoisomers. We describe an enantioselective synthesis of homoisoflavanones by virtue of ruthenium-catalyzed asymmetric transfer hydrogenation accompanying dynamic kinetic resolution, providing a basis for the further development of these compounds into novel experimental therapeutics for neovascular eye diseases.

Promiscuous Chemokine Antagonist (BKT130) Suppresses Laser-Induced Choroidal Neovascularization by Inhibition of Monocyte Recruitment.

Background Age-related macular degeneration (AMD), the most common cause of blindness in the developed world, usually affects individuals older than 60 years of age. The majority of visual loss in this disease is attributable to the development of choroidal neovascularization (CNV). Mononuclear phagocytes, including monocytes and their tissue descendants, macrophages, have long been implicated in the pathogenesis of neovascular AMD (nvAMD). Current therapies for nvAMD are based on targeting vascular endothelial growth factor (VEGF). This study is aimed at assessing if perturbation of chemokine signaling and mononuclear cell recruitment may serve as novel complementary therapeutic targets for nvAMD. Methods A promiscuous chemokine antagonist (BKT130), aflibercept treatment, or combined BKT130+aflibercept treatment was tested in an in vivo laser-induced model of choroidal neovascularization (LI-CNV) and in an ex vivo choroidal sprouting assay (CSA). Quantification of CD11b+ cell in the CNV area was performed, and mRNA levels of genes implicated in CNV growth were measured in the retina and RPE-choroid. Results BKT130 reduced the CNV area and recruitment of CD11b+ cells by 30-35%. No effect of BKT130 on macrophages' proangiogenic phenotype was demonstrated ex vivo, but a lower VEGFA and CCR2 expression was found in the RPE-choroid and a lower expression of TNFα and NOS1 was found in both RPE-choroid and retinal tissues in the LI-CNV model under treatment with BKT130. Conclusions Targeting monocyte recruitment via perturbation of chemokine signaling can reduce the size of experimental CNV and should be evaluated as a potential novel therapeutic modality for nvAMD.

Apratoxin S4 Inspired by a Marine Natural Product, a New Treatment Option for Ocular Angiogenic Diseases.

Abnormal blood vessel formation is a defining feature of many blinding eye diseases. Targeting abnormal angiogenesis by inhibiting VEGF has revolutionized the treatment of many ocular angiogenic diseases over the last decade. However, a substantial number of patients are refractory to anti-VEGF treatment or may develop resistance over time. The objective of this study was to determine the efficacy and the mechanism of action of Apratoxin S4 in ocular angiogenesis. Retinal vascular cell proliferation, migration, and the ability to form tube-like structure were studied in vitro. Ex vivo aortic ring, choroid, and metatarsal assays were used to study Apratoxin S4's impact on vessel outgrowth in a multicellular environment. Apratoxin S4 was also tested in mouse models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), and in a rabbit model of persistent retinal neovascularization (PRNV). Western blot and ELISA were used to determine the expression of key angiogenic regulators after Apratoxin S4 treatment. Apratoxin S4 strongly inhibits retinal vascular cell activation by suppressing multiple angiogenic pathways. VEGF-activated vascular cells and angiogenic vessels are more susceptible to Apratoxin S4 treatment than quiescent vascular cells and vessels. Both intraperitoneal and intravitreal delivery of Apratoxin S4 are able to impede ocular neovascularization in vivo. Apratoxin S4 specifically attenuates pathological ocular angiogenesis and exhibits a combinatorial inhibitory effect with standard-of-care VEGF inhibitor drug (aflibercept). Apratoxin S4 is a potent antiangiogenic drug that inhibits the activation of retinal endothelial cells and pericytes through mediating multiple angiogenic pathways.

Development of an in vitro 3D choroidal neovascularization model using chemically induced hypoxia through an ultra-thin, free-standing nanofiber membrane.

Choroidal neovascularization (CNV) is the pathological growth of new blood vessels in the sub-retinal pigment epithelial (RPE) space from the choroid through a break in the Bruch's membrane (BM). Despite its importance in studying biological processes and drug discovery, the development of an in vitro CNV model that achieves the physiological structures of native RPE-BM-choroidal capillaries (CC) is still challenging. Here, we develop a novel 3D RPE-BM-CC complex biomimetic system on an ultra-thin, free-standing nanofiber membrane. The thickness of the pristine nanofiber membrane is 2.17 ± 0.81 μm, and the Matrigel-coated nanofiber membrane attains a permeability coefficient of 2.95 ± 0.25 × 10-6 cm/s by 40 kDa FITC-dextran, which is similar to the physiological value of the native BM. On the in vitro 3D RPE-BM-CC complex system, we demonstrate endothelial cell invasion across the 3D RPE-BM-CC complex and the mechanism of the invasion by imposing a hypoxic condition, which is thought to be the major pathological cause of CNV. Furthermore, alleviation of the invasion is achieved by treating with chrysin and anti-VEGF antibody. Thus, the in vitro 3D RPE-BM-CC complex biomimetic system can recapitulate essential features of the pathophysiological environment and be employed for the screening of drug candidates to reduce the number of costly and time-consuming in vivo tests or clinical trials.

Real-time OCT guidance and multimodal imaging monitoring of subretinal injection induced choroidal neovascularization in rabbit eyes

Choroidal neovascularization (CNV) is a major cause of vision loss that consists of abnormal growth of new blood vessels from the choroidal vasculature. High resolution in vivo imaging of animal models is essential to better elucidate and conduct research on CNV. This study evaluates a novel multimodal imaging platform combining optical coherence tomography (OCT) and photoacoustic microscopy (PAM). Using real-time OCT guidance subretinal injection to induce and multimodality imaging system to monitor CNV over time in rabbit eyes. The significance of our work lies in providing the optimal setting and conditions to make use of the OCT image guided system to improve the consistency and reproducibility of experimental results in subretinal injection induced CNV model in rabbits. For the first time, this study successfully demonstrated the dual-modality PAM-OCT system, without using exogenous contrast agents, can detect and visualize CNV in the rabbit eye with high resolution. This is promising system for diagnosing and monitoring CNV.

Brivanib, a multitargeted small-molecule tyrosine kinase inhibitor, suppresses laser-induced CNV in a mouse model of neovascular AMD.

In age-related macular degeneration (AMD), choroidal neovascularization (CNV), a major pathologic feature of neovascular AMD (nAMD), affects 10% of patients, potentially causing serious complications, including vision loss. Vascular endothelial growth factor receptor 2 (VEGFR2) and fibroblast growth factor receptor 1 (FGFR1) contribute to the pathogenesis of CNV. Brivanib is an oral selective dual receptor tyrosine kinase (RTK) inhibitor of FGFRs and VEGFRs, especially VEGFR2 and FGFR1. In this study, brivanib inhibited zebrafish embryonic angiogenesis without impairing neurodevelopment. In a mouse CNV model, brivanib intravitreal injection blocked phosphorylation of FGFR1 and VEGFR2 and reduced CNV leakage, area, and formation without causing intraocular toxicity. Moreover, brivanib oral gavage reduced CNV leakage and area. Accordingly, brivanib remained at high concentrations (above 14,000 ng/ml) in retinal/choroidal/scleral tissues following intravitreal injection. Similarly, brivanib remained at high concentrations (over 10,000 ng/ml) in retinal/choroidal/scleral tissues following oral gavage. Finally, in vitro cell experiments demonstrated that brivanib inhibited the proliferation, migration and tube formation of microvascular endothelial cells. In conclusion, our study suggested that brivanib treatment could be a novel therapeutic strategy for nAMD.

Inhibition of Experimental Age-Related Macular Degeneration by ZQMT in Mice

Age-related macular degeneration (AMD) is a progressive and irreversible eye disease. The anti-vascular endothelial growth factor (VEGF) therapy has revolutionized the treatment of neovascular AMD. However, the expense for such treatment is quite high. We used a traditional Chinese medicine ZQMT as an alternative therapeutic regimen for AMD. We employed two in vivo animal models mimicking dry and wet AMD respectively to assess the therapeutic efficacy of ZQMT on treating AMD-related retinopathy. AMD-related retinopathy in Crb1rd8 mice was evaluated from week 1 to 8 by fundus photography. Laser-induced choroidal neovascularization (CNV) was evaluated by fluorescein angiography and histopathology. ZQMT increased CX3CR1 expression in murine CD4+ T cells either cultured in vitro or directly isolated from animals treated with ZQMT. We also performed both in vitro and in vivo studies to confirm that ZQMT has no apparent toxic effects. ZQMT alleviated AMD-related retinopathy in both Crb1rd8 and CNV models. Depletion of CCL2 and CX3CR1 in Crb1rd8 mice abolished the efficacy of ZQMT, suggesting that CCL2 and/or CX3CR1 may underlie the mechanisms of ZQMT in treating AMD-related retinopathy in mice. In summary, our study supports the protective roles of a traditional Chinese medicine ZQMT in AMD.

Type I pig collagen enhances the efficacy of PEDF 34-mer peptide in a mouse model of laser-induced choroidal neovascularization.

Pigment epithelium-derived factor (PEDF)-derived 34-mer peptide (PEDF34, Asp44-Asn77) has anti-angiogenic activity but has limitations in clinical application because of an inverted bell-shaped dose-effect relationship and a short half-life. In this study, we attempted to mitigate these problems by mixing PEDF34 with type I collagen. The anti-angiogenic activity of the PEDF34/atelocollagen mixture was evaluated by HUVEC tube formation assay and in a laser-induced choroidal neovascular (CNV) mouse model. PEDF34 and/or collagen were administrated using intravitreal injections or eye drops. CNV lesion size was quantified using FITC-dextran-perfused retinal whole mounts. Western blot analysis and inhibitor assays were used to define the action mechanisms of PEDF34 and the mixture. Collagen broadened the effective dose range of PEDF34 in the tube formation assay by > 250 times (from 0.2 to 50nM). In the CNV model, five intravitreal injections of PEDF34 were required for therapeutic effect, whereas the mixture had a significant therapeutic effect following a single injection. Eye drops of the mixture showed significantly stronger CNV-suppressive effects than drops of PEDF34 alone. The anti-angiogenic activity of PEDF34 might be mediated by inhibition of ERK and JNK activation by VEGF, and collagen potentiated these effects. Collagen can serve as a carrier and reservoir of PEDF34. PEDF peptide/collagen mixture is easy to prepare than conventional methods for maintaining the therapeutic effect of PEDF peptide.

Differential Expressions of microRNAs and Transfer RNA-derived Small RNAs: Potential Targets of Choroidal Neovascularization

ABSTRACTPurpose: Choroidal neovascularization (CNV) is one of the major clinical characteristics of neovascular age-related macular degeneration (AMD). Small non-coding RNAs, such as microRNAs (miRNAs) and transfer RNA-derived small RNAs (tsRNAs) play key roles in diverse biological functions. The purpose of the study was to investigate the roles and possible functions of the miRNAs and tsRNAs in CNV.Methods: The mouse model of laser-induced CNV was conducted by laser photocoagulation. The expression profiles of miRNAs and tsRNAs were accessed by small RNA sequencing (RNA-Seq) in RPE-choroid-sclera complexes of mice in CNV group and control group. Selected altered miRNAs and tsRNAs were validated by qRT-PCR. Target genes were predicted by informatics analysis and intersected with the previous microarray study of altered mRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to reveal the biological functions and signaling pathways with which these target genes are most enriched.Results: The results revealed that 79 miRNAs and 72 tsRNAs in total were significantly altered in the RPE-choroid-sclera complexes of CNV mice. GO analysis revealed that the altered target genes of the selected miRNAs most enriched in immune response, integral component of membrane and peptide binding, while the altered target genes of tsRNAs most enriched in regulation of immune system process, extracellular region, and core promoter binding. Moreover, KEGG pathway analysis demonstrated that altered target genes of miRNAs and tsRNAs most enriched in hematopoietic cell lineage and nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway, respectively.Conclusions: Our study identified differential expressions of miRNAs and tsRNAs in CNV model, and these altered miRNAs and tsRNAs might be novel potential targets in treating CNVs in patients with neovascular AMD.

Human Umbilical Tissue-Derived Cells Secrete Soluble VEGFR1 and Inhibit Choroidal Neovascularization

Exudative age-related macular degeneration (AMD), characterized by choroidal neovascularization (CNV), is the leading cause of irreversible blindness in developed countries. Anti-vascular endothelial growth factor (VEGF) drugs are the standard treatment for AMD, but they have limitations. Cell therapy is a promising approach for ocular diseases, and it is being developed in the clinic for the treatment of retinal degeneration, including AMD. We previously showed that subretinal injection of human umbilical tissue-derived cells (hUTCs) in a rodent model of retinal degeneration preserved photoreceptors and visual function through rescue of retinal pigment epithelial (RPE) cell phagocytosis. Here we investigated the effect of hUTCs on a rat model of laser-induced CNV and on a human RPE cell line, ARPE-19, for VEGF production. We demonstrate that subretinal injection of hUTCs significantly inhibited CNV and lowered choroidal VEGF in vivo. VEGF release from ARPE-19 decreased when co-cultured with hUTCs. Soluble VEGF receptor 1 (sVEGFR1) is identified as the only factor in hUTC conditioned medium (CM) that binds to VEGF. The level of exogenous recombinant VEGF in hUTC CM was dramatically reduced and could be recovered with sVEGFR1-neutralizing antibody. This suggests that hUTC inhibits angiogenesis through the secretion of sVEGFR1 and could serve as a novel treatment for angiogenic ocular diseases, including AMD.

Novel multi-targeted inhibitors suppress ocular neovascularization by regulating unique gene sets

Neovascular diseases, such as many cancers and ocular disorders, are life threatening and devastating. Although anti-vascular endothelial growth factor A (VEGF-A) therapy is available, many patients are not responsive and drug resistance can develop. To try to overcome these problems, combination therapy targeting VEGF-A and platelet-derived growth factor B (PDGF-B) was tested. However, one obvious drawback was that the other VEGF and PDGF family members were not inhibited and therefore could compensate. Indeed, this was, at least to some extent, demonstrated by the disappointing outcomes. To this end, we designed novel multi-targeted inhibitors that can block most of the VEGF and PDGF family members simultaneously by making a fusion protein containing the ligand-binding domains of vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and platelet-derived growth factor receptor beta (PDGFRβ), which can therefore act as a decoy blocker for most of the VEGF and PDGF family members. Indeed, in cultured cells, the novel inhibitors suppressed the migration and proliferation of both vascular endothelial cells and smooth muscle cells, and abolished VEGFR2 and PDGFRβ activation. Importantly, in a choroidal neovascularization model in vivo, the novel inhibitor inhibited ocular neovascularization more efficiently than the mono-inhibitors against VEGFR or PDGFR alone respectively. Mechanistically, a genome-wide microarray analysis unveiled that the novel inhibitor regulated unique sets of genes that were not regulated by the mono-inhibitors, further demonstrating the functional uniqueness and superiority of the novel inhibitor. Together, we show that the multi-targeted inhibitors that can block VEGFR1, VEGFR2 and PDGFRβ simultaneously suppress pathological angiogenesis more efficiently than monotherapy, and may therefore have promising therapeutic value for the treatment of neovascular diseases.

HTRA1 synergizes with oxidized phospholipids in promoting inflammation and macrophage infiltration essential for ocular VEGF expression.

Understanding oxidative stress and HTRA1 locus in abnormal angiogenesis resulting in wet AMD pathology is an important step in developing a novel therapeutic approach. Using subretinal injection of oxLDL into C57BL/6 mice, we observed a lesion resembling the features of choroidal neovascularization (CNV), including macrophage infiltration, increased VEGF expression, and neovascularization. However, incubating ARPE-19 cells with oxLDL–a carrier of oxidized phospholipids–resulted in increased expression of inflammatory cytokines and chemoattractant proteins that recruited monocytes, but no substantial increase in expression of VEGF. Furthermore, incubation of ARPE-19 with oxLDL induced higher expression of HTRA1, which we showed to synergize with oxLDL in elevating the expression of inflammatory cytokines and chemoattractant factors. To investigate the role of macrophage infiltration on these expression changes, we treated cultured J774 macrophages with oxLDL and applied the conditioned medium onto ARPE-19 cells. This treatment was found to greatly enhance the expression of VEGF in ARPE-19, indicating the necessity of macrophage secretory products to induce increased expression of VEGF in retinal pigment epithelium. Gene expression analysis revealed that oxLDL induced the expression of Wnt3A in macrophages, a key activator of canonical Wnt signaling pathways. In addition, western blot analysis showed that the macrophage conditioned media further enhanced the reduction of phosphorylated β-catenin induced by oxLDL. Lastly, we investigated HTRA1 as a potential target for AMD therapeutics. We demonstrated the ability of anti-HTRA1 antibody in vitro to neutralize the protease activity of HTRA1 and reduce the inflammatory and angiogenic response to oxidative stress. Finally, we validated the neutralizing effect of anti-HTRA1 antibody in vivo by evaluating lesion size and protein expression in a laser-photocoagulation murine model of CNV. We found that the combination of oxLDL and HTRA1 enhanced CNV size, which was reversed by the addition of anti-HTRA1 antibody. This study not only provides preliminary evidence that HTRA1 may be a viable target for AMD therapeutics but also elucidates the biochemical mechanisms by which this therapeutic effect may be mediated.

Photothermal Optical Coherence Tomography of Anti-Angiogenic Treatment in the Mouse Retina Using Gold Nanorods as Contrast Agents

PurposeOptical coherence tomography (OCT) is widely used for ocular imaging in clinical and research settings. OCT natively provides structural information based on the reflectivity of the tissues it images. We demonstrate the utility of photothermal OCT (PTOCT) imaging of gold nanorods (GNR) in the mouse retina in vivo in the laser-induced choroidal neovascularization (LCNV) model to provide additional image contrast within the lesion.MethodsWild-type C57BL/6 mice were imaged following the intravenous injection of ICAM2-targeted or untargeted GNR. Mice were also imaged following the injection of ICAM2-targeted GNR with or without the additional ocular delivery of a neutralizing monoclonal anti-vascular endothelial growth factor (anti-VEGF) antibody.ResultsMice cohorts injected with untargeted or ICAM2-targeted GNR demonstrated increased lesion-associated photothermal signal during subsequent imaging relative to phosphate-buffered saline (PBS)-injected controls. Additionally, intravitreal injection of anti-VEGF antibody caused a detectable reduction in the extent of anatomic laser damage and lesion-associated photothermal signal density in mice treated in the LCNV model and injected with ICAM2-targeted GNR.ConclusionsThese experiments demonstrate the ability of PTOCT imaging of GNR to detect anti-VEGF-induced changes in the mouse retina using the LCNV model.Translational RelevanceThis study shows that PTOCT imaging of GNR in the LCNV model can be used to detect clinically relevant, anti-VEGF-induced changes that are not visible using standard OCT systems. In the future this technology could be used to aid in early detection of disease, monitoring disease progress, and assessing its response to therapies.

Silencing fibroblast growth factor 7 inhibits krypton laser-induced choroidal neovascularization in a rat model.

Choroidal neovascularization (CNV), a characteristic of age-related macular degeneration, is an underlying cause of severe vision loss among elderly patients. Fibroblast growth factor (FGF) is suggested to exert an important role in the pathogenesis of CNV. However, the molecular mechanisms governing this event are not fully elucidated. Herein, we identified the potential role of FGF7 in CNV. To examine the roles of FGF7 in the progression of CNV, rat CNV models were established and treated with small interfering RNA (siRNA) against FGF7 or FGF7 overexpression, followed by identification of expression of FGF7 in the CNV modeled rats. Next, proliferation and migration, and in vitro tube formation of human umbilical vein endothelial cells, as well as expression of vascular endothelial growth factor (VEGF) and transforming growth factor-beta 2 (TGF-β2) were evaluated. CNV led to upregulated FGF7 expression. Cells in the presence of FGF7 siRNA showed suppressed proliferation, migration, and tube formation, along with downregulated VEGF and TGF-β2 expression. Taken together, functional suppression of FGF7 inhibited the onset of CNV, ultimately highlighting a novel therapeutic target for suppressing CNV progression.