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.

Netrins are a family of matrix-binding proteins that function as guidance signals. Netrin-4 displays pathologic roles in tumorigenesis and neovascularization. To answer the question whether netrin-4 acts either pro- or anti-angiogenic, angiogenesis in the retina was assessed in Ntn-4(-/-) mice with oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), mimicking hypoxia-mediated neovascularization and inflammatory mediated angiogenesis. The basement membrane protein netrin-4 was found to be localised to mature retinal blood vessels. Netrin-4, but not netrin-1 mRNA expression, increased in response to relative hypoxia and recovered to normal levels at the end of blood vessel formation. No changes in the retina were found in normoxic Ntn-4(-/-) mice. In OIR, Ntn-4(-/-) mice initially displayed larger avascular areas which recovered faster to revascularization. Ganzfeld electroretinography showed faster recovery of retinal function in Ntn-4(-/-) mice. Expression of netrin receptors, Unc5H2 (Unc-5 homolog B, C. elegans) and DCC (deleted in colorectal carcinoma), was found in Müller cells and astrocytes. Laser-induced neovascularization in Nnt-4(-/-) mice did not differ to that in the controls. Our results indicate a role for netrin-4 as an angiogenesis modulating factor in O2-dependent vascular homeostasis while being less important during normal retinal developmental angiogenesis or during inflammatory neovascularization.

Id helix-loop-helix (HLH) proteins (Id1-4) bind E protein bHLH transcription factors, preventing them from forming active transcription complexes that drive changes in cell states. Id proteins are primarily expressed during development to inhibit differentiation, but they become re-expressed in adult tissues in diseases of the vasculature and cancer. We show that the genetic loss of Id1/Id3 reduces ocular neovascularization in mouse models of wet age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). An in silico screen identifies AGX51, a small-molecule Id antagonist. AGX51 inhibits the Id1-E47 interaction, leading to ubiquitin-mediated degradation of Ids, cell growth arrest, and reduced viability. AGX51 is well-tolerated in mice and phenocopies the genetic loss of Id expression in AMD and ROP models by inhibiting retinal neovascularization. Thus, AGX51 is a first-in-class compound that antagonizes an interaction formerly considered undruggable and that may have utility in the management of multiple diseases.

Choroidal neovascularization (CNV) is a major cause of vision loss in retinal diseases such as age-related macular degeneration (AMD). Previously, we demonstrated that semaphorin3A (Sema3A), which is a chemorepellent guidance molecule, inhibited the formation of retina neovascularization. In the present study, we investigated the antiangiogenic effects of Sema3A on transforming growth factor beta (TGF-β) in vitro and in vivo.

Purpose: To investigate the morphologic features and identify the risk factors of myopic choroidal neovascularization (CNV). Methods: Eighty-eight eyes of 69 consecutive patients with myopic CNV were included in this study. About 109 eyes of 78 pathologic myopia patients without myopic CNV were randomly selected as the control group. Morphologic features and parameters including scleral thickness (ST), choroidal thickness (CT), posterior staphyloma height and the presence of scleral perforating vessels were obtained and measured by swept-source optical coherence tomography (SS-OCT). Binary logistic regression analysis was performed to identify the risk factors for myopic CNV. Results: Patients with myopic CNV had relatively shorter axial length (P < 0.001) and thicker sclera (P < 0.001) compared to those without. After adjusting age, gender and axial length, thick sclera (OR = 1.333, P < 0.001 per 10-μm increase) and thin choroid (OR = 0.509, P < 0.001 per 10-μm increase) were associated with the presence of myopic CNV. Scleral perforating vessels were detected in the area of myopic CNV in 78.67% of the subjects. Conclusions: A relatively thicker sclera and a thinner choroid are the biological indicators for myopic CNV on SS-OCT. Scleral perforating vessels may also play a pivotal role in the formation of myopic CNV.

Diabetic retinopathy (DR) is a retinal microvascular disease characterized by inflammatory and angiogenic pathways. In this study, we evaluated NLRP3 inflammasome in a double transgenic mouse model, Akimba (Ins2 Akita xVEGF+/-), which demonstrates hyperglycemia, vascular hyperpermeability and neovascularization seen in the proliferative DR. Retinal structural integrity, vascular leakage and function were examined by fundus photography, fluorescein angiography, optical coherence tomography, retinal flat mounts, laser speckle flowgraphy (LSFG), and electroretinography in Akimba and its parental strains, Akita (Ins2 Akita ) and Kimba (trVEGF029) mice. Inflammatory mechanisms involving NLRP3 inflammasome were investigated using real time-PCR, immunohistochemistry, ELISA and western blots. We observed an increased vascular leakage, reduced retinal thickness, and function in Akimba retina. Also, Akimba retina depicts decreased relative flow volume measured by LSFG. Most importantly, high levels of IL-1β along with increased NLRP3, ASC, and Caspase-1 at mRNA and protein levels were observed in Akimba retina. However, the in vivo functional role remains undefined. In conclusion, increased activation of macroglia (GFAP), microglia (Iba-1 and OX-42) and perivascular macrophages (F4/80 and CD14) together with pro-inflammatory (IL-1β and IL-6) and pro-angiogenic markers (PECAM-1, ICAM-1, VEGF, Flt-1, and Flk-1), suggested a critical role for NLRP3 inflammasome in the Akimba mouse model depicting advanced stages of DR pathogenesis.

To investigate visual and morphological outcome in eyes with MRS and choroidal neovascularization (CNV) secondary to pathologic myopia treated with intravitreal (IVT) ranibizumab.

The META-Analysis of Pathologic Myopia Study group proposed a new classification system for myopic maculopathy (MM) with pathologic myopia (PM) defined as MM equal to/more serious than diffuse atrophy or the presence of plus lesions and myopic choroidal neovascularization (mCNV) defined as CNV in the eye with PM. However, CNV in elderly eyes with high myopia (HM) not meeting the PM definition (high-myopia CNV) are not classified as age-related macular degeneration (nAMD) or mCNV. This retrospective study included 39 eyes with high-myopia CNV, 20 eyes with mCNV, and 20 eyes with AMD. All patients were at least 40 years old. We compared the clinical characteristics and treatment outcomes among three groups. The high-myopia CNV group had significantly more CNV types, shorter axial length and fewer lacquer cracks (P < 0.0001, respectively); larger baseline greatest linear dimension (P = 0.0002), more fellow-eye drusen (P = 0.0106), more men (P = 0.0029), and more treatments (24 months, P = 0.0098) compared to the mCNV group. Compared with the nAMD group, the high-myopia CNV group was significantly younger (P = 0.0041), and had fewer CNV types (P = 0.0316), more lacquer cracks (P = 0.0079) and fewer drusen (affected-eye, P = 0.0006 and fellow-eye, P = 0.0222), and fewer treatments (24 months, P = 0.0030). Because the CNV in elderly eyes with HM not meeting the PM definition is classified as combined mCNV and nAMD, the clinical and angiographic findings are critical to determine the treatment strategy.

Ischemic retinopathies, including retinopathy of prematurity and diabetic retinopathy, are major causes of blindness. Both have two phases, vessel loss and consequent hypoxia-driven pathologic retinal neovascularization, yet relatively little is known about the transcription factors regulating these processes. Myocyte enhancer factor 2 (MEF2) C, a member of the MEF2 family of transcription factors that plays an important role in multiple developmental programs, including the cardiovascular system, seems to have a significant functional role in the vasculature. We, therefore, generated endothelial cell (EC)-specific MEF2C-deficient mice and explored the role of MEF2C in retinal vascularization during normal development and in a mouse model of oxygen-induced retinopathy. Ablation of MEF2C did not cause appreciable defects in normal retinal vascular development. However, MEF2C ablation in ECs suppressed vessel loss in oxygen-induced retinopathy and strongly promoted vascular regrowth, consequently reducing retinal avascularity. This finding was associated with suppression of pathologic retinal angiogenesis and blood-retinal barrier dysfunction. MEF2C knockdown in cultured retinal ECs using small-interfering RNAs rescued ECs from death and stimulated tube formation under stress conditions, confirming the endothelial-autonomous and antiangiogenic roles of MEF2C. HO-1 was induced by MEF2C knockdown in vitro and may play a role in the proangiogenic effect of MEF2C knockdown on retinal EC tube formation. Thus, MEF2C may play an antiangiogenic role in retinal ECs under stress conditions, and modulation of MEF2C may prevent pathologic retinal neovascularization.

Many neurodegenerative diseases are known to occur and progress because of oxidative stress, the presence of reactive oxygen species (ROS) in excess of the cellular defensive capabilities. Age related macular degeneration (AMD), diabetic retinopathy (DR) and inherited retinal degeneration share oxidative stress as a common node upstream of the blinding effects of these diseases. Knockout of the Vldlr gene results in a mouse that develops intraretinal and subretinal neovascular lesions within the first month of age and is an excellent model for a form of AMD called retinal angiomatous proliferation (RAP). Cerium oxide nanoparticles (nanoceria) catalytically scavenge ROS by mimicking the activities of superoxide dismutase and catalase. A single intravitreal injection of nanoceria into the Vldlr-/- eye was shown to inhibit: the rise in ROS in the Vldlr-/- retina, increases in vascular endothelial growth factor (VEGF) in the photoreceptor layer, and the formation of intraretinal and subretinal neovascular lesions. Of more therapeutic interest, injection of nanoceria into older mice (postnatal day 28) resulted in the regression of existing vascular lesions indicating that the pathologic neovessels require the continual production of excessive ROS. Our data demonstrate the unique ability of nanoceria to prevent downstream effects of oxidative stress in vivo and support their therapeutic potential for treatment of neurodegenerative diseases such as AMD and DR.

Crosstalk between pericytes and endothelial cells is critical for ocular neovascularization. Endothelial cells secrete platelet-derived growth factor (PDGF)-BB and recruit PDGF receptor β (PDGFRβ)-overexpressing pericytes, which in turn cover and stabilize neovessels, independent of vascular endothelial growth factor (VEGF). Therapeutic agents inhibiting PDGF-BB/PDGFRβ signaling were tested in clinical trials but failed to provide additional benefits over anti-VEGF agents. We tested whether an antibody-drug conjugate (ADC) - an engineered monoclonal antibody linked to a cytotoxic agent - could selectively ablate pericytes and suppress retinal and choroidal neovascularization.

To investigate the functional role of immunoproteasome subunit β5i in pathologic retinal neovascularization (RNV) and its ability to link the immunoproteasome and autophagy.

Pathological neovascularization of the outer retina is the hallmark of neovascular age-related macular degeneration (nAMD). Building on our previous observations that semaphorin 3F (Sema3f) is expressed in the outer retina and demonstrates anti-angiogenic potential, we have investigated whether Sema3f can be used to protect against subretinal neovascularization in two mouse models. Both in the very low-density lipid-receptor knockout (Vldlr-/-) model of spontaneous subretinal neovascularization as well as in the mouse model of laser-induced choroidal neovascularization (CNV), we found protective effects of Sema3f against the formation of pathologic neovascularization. In the Vldlr-/- model, AAV-induced overexpression of Sema3f reduced the size of pathologic neovascularization by 56%. In the laser-induced CNV model, intravitreally injected Sema3f reduced pathologic neovascularization by 30%. Combined, these results provide the first evidence from two distinct in vivo models for a use of Sema3f in protecting the outer retina against subretinal neovascularization.

Neurofibromatosis type 1 (NF1) is the result of inherited mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. Eye manifestations are common in NF1 with recent reports describing a vascular dysplasia in the retina and choroid. Common features of NF1 retinopathy include tortuous and dilated feeder vessels that terminate in capillary tufts, increased endothelial permeability, and neovascularization. Given the retinal vascular phenotype observed in persons with NF1, we hypothesize that preserving neurofibromin may be a novel strategy to control pathologic retinal neovascularization.

Pathologic angiogenesis in the retina leads to the catastrophic loss of vision. Retinopathy of prematurity (ROP), a vasoproliferative retinopathy, is a leading cause of blindness in children. We evaluated the inhibitory effect of decursin on retinal neovascularization.

Tight regulation of positive and negative regulators of angiogenesis is essential, particularly in the eye where their dysregulation can lead to vision loss. Thrombospondin-1 (TSP1) is a matricellular protein that negatively regulates angiogenesis and inflammation in the eye. It aids ocular vascular homeostasis such that its loss contributes to increased retinal vascular density and pathologic ocular neovascularization. Our previous studies demonstrated that mice globally lacking TSP1 expression had increased retinal vascular density, decreased hyperoxia-induced retinal vessel loss, and increased choroidal neovascularization. Here we determined the impact to the ocular vasculature of endothelial cell, pericyte, or astrocyte loss of TSP1 expression. Only lack of TSP1 expression in endothelial cells was sufficient to increase choroidal neovascularization with mice lacking expression in pericytes or astrocytes not demonstrating a significant impact. Although the global TSP1 knockout mice demonstrated increased retinal vascular density, individual cell type loss of TSP1 resulted in decreased retinal endothelial cell numbers before and/or after vascular maturation in a cell type specific fashion. Retinas from mice lacking TSP1 expression in endothelial cells, pericytes or astrocytes were not protected from retinal vessel regression in response to hyperoxia as we previously observed in the global knockout. Thus, modulation of TSP1 expression in individual cell types demonstrates a response that is unique to the role TSP1 plays in that cell type of interest, and their coordinated activity is critical for vision.

Pathologic ocular neovascularization commonly results in visual impairment or even blindness in numerous fundus diseases, including proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), and age-related macular degeneration (AMD). MicroRNAs regulate angiogenesis through modulating target genes and disease progression, making them a new class of targets for drug discovery. In this study, we investigated the potential role of miR-18a-5p in retinal neovascularization using a mouse model of oxygen-induced proliferative retinopathy (OIR). We found that miR-18a-5p was highly expressed in the retina of pups as well as retinal endothelial cells, and was consistently down-regulated during retinal development. On the other hand, miR-18a-5p was increased significantly during pathologic neovascularization in the retinas of OIR mice. Moreover, intravitreal administration of miRNA mimic, agomiR-18a-5p, significantly suppressed retinal neovascularization in OIR models. Accordingly, agomir-18a-5p markedly suppressed human retinal microvascular endothelial cell (HRMEC) function including proliferation, migration, and tube formation ability. Additionally, we demonstrated that miR-18a-5p directly down-regulated known vascular growth factors, fibroblast growth factor 1 (FGF1) and hypoxia-inducible factor 1-alpha (HIF1A), as the target genes. In conclusion, miR-18a-5p may be a useful drug target for pathologic ocular neovascularization.

Ciliary neurotrophic factor (CNTF) is a well-characterized neurotrophic factor currently in clinical trials for the treatment of macular telangiectasia type II. Our previous work showed that CNTF-induced STAT3 signaling is a potent inhibitor of pathologic preretinal neovascular tuft formation in the mouse model of oxygen-induced retinopathy. In this study, we investigated the effect of CNTF on outer retinal and choroidal angiogenesis and the mechanisms that underpin the observed decrease in outer retinal neovascularization following CNTF treatment.

Hypoxia-inducing pathologies as cancer develop pathologic and inefficient angiogenesis which rules tumor facilitating microenvironment, a key target for therapy. As such, the putative ability of endothelial precursor cells (EPCs) to specifically home to hypoxic sites of neovascularization prompted to design optimized, site-specific, cell-mediated, drug-/gene-targeting approach. Thus, EPC lines were established from aorta-gonad-mesonephros (AGM) of murine 10.5 dpc and 11.5 dpc embryo when endothelial repertoire is completed. Lines representing early endothelial differentiation steps were selected: MAgEC10.5 and MagEC11.5. Distinct in maturation, they differently express VEGF receptors, VE-cadherin and chemokine/receptors. MAgEC11.5, more differentiated than MAgEC 10.5, displayed faster angiogenesis in vitro, different response to hypoxia and chemokines. Both MAgEC lines cooperated to tube-like formation with mature endothelial cells and invaded tumor spheroids through a vasculogenesis-like process. In vivo, both MAgEC-formed vessels established blood flow. Intravenously injected, both MAgECs invaded Matrigel(TM)-plugs and targeted tumors. Here we show that EPCs (MAgEC11.5) target tumor angiogenesis and allow local overexpression of hypoxia-driven soluble VEGF-receptor2 enabling drastic tumor growth reduction. We propose that such EPCs, able to target tumor angiogenesis, could act as therapeutic gene vehicles to inhibit tumor growth by vessel normalization resulting from tumor hypoxia alleviation.

The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB) and its ligands brain derived neurotrophic factor (Bdnf), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) are known primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80(hi)CD11b(lo)CD11c(+) macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis.