Endothelial JMJD1C drives pathological ocular neovascularization by activating SREBF2-dependent cholesterol biosynthesis.

Multiple members of the caveolae-associated protein (Cavin) family are implicated in angiogenesis. However, the specific role of CAVIN3 in pathological angiogenesis within the eye remains unclear. The present study demonstrated that CAVIN3 knockdown in endothelial cells (ECs) promoted vascular normalization in ocular pathological neovascularization. Elevated CAVIN3 expression was observed in the ECs of retinal pigment epithelium/choroid complexes from patients with neovascular age-related macular degeneration and fibrovascular membranes from patients with proliferative diabetic retinopathy. Additionally, upregulated Cavin3 expression was detected in laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR) mouse models. In both OIR and CNV mice, Cavin3 knockdown inhibited pathological neovascularization. Cavin3 deficiency further disrupted EC proliferation and vascular sprouting, thereby promoting vascular normalization by partially restoring microenvironmental hypoxia and reestablishing pericyte-EC interactions. Mechanistically, we demonstrated that zinc finger E-box–binding homeobox 1 (ZEB1) regulated CAVIN3 transcription in ECs under hypoxic conditions. CAVIN3 deficiency modulated pathological vascularization by inhibiting ERK phosphorylation, which downregulated jagged 1 (JAG1) expression. Conclusively, this study elucidated the protective role of endothelial CAVIN3 deficiency in pathological neovascularization models, addressing a gap in understanding the regulatory role of Cavins in angiogenesis. These findings suggested a therapeutic direction for ocular neovascular diseases.

Pathological ocular neovascularization, caused by diabetic retinopathy, age-related macular degeneration, or retinopathy of prematurity, is a leading cause of blindness, yet much remains to be learned about its underlying causes. Here we used oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) to assess the contribution of the metalloprotease-disintegrin ADAM9 to ocular neovascularization in mice. Pathological neovascularization in both the OIR and CNV models was significantly reduced in Adam9(-/-) mice compared to wild-type controls. In addition, the level of ADAM9 expression was strongly increased in endothelial cells in pathological vascular tufts in the OIR model. Moreover, tumor growth from heterotopically injected B16F0 melanoma cells was reduced in Adam9(-/-) mice compared to controls. In cell-based assays, the overexpression of ADAM9 enhanced the ectodomain shedding of EphB4, Tie-2, Flk-1, CD40, VCAM, and VE-cadherin, so the enhanced expression of ADAM9 could potentially affect pathological neovascularization by increasing the shedding of these and other membrane proteins from endothelial cells. Finally, we provide the first evidence for the upregulation of ADAM9-dependent shedding by reactive oxygen species, which in turn are known to play a critical role in OIR. Collectively, these results suggest that ADAM9 could be an attractive target for the prevention of proliferative retinopathies, CNV, and cancer.

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.

PurposeThe present study aimed to evaluate the effect of acrizanib, a small molecule inhibitor targeting vascular endothelial growth factor receptor 2 (VEGFR2), on physiological angiogenesis and pathological neovascularization in the eye and to explore the underlying molecular mechanisms.MethodsWe investigated the potential role of acrizanib in physiological angiogenesis using C57BL/6J newborn mice, and pathological angiogenesis using the mouse oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) models. Moreover, vascular endothelial growth factor (VEGF)–treated human umbilical vein endothelial cells (HUVECs) were used as an in vitro model for studying the molecular mechanism underlying acrizanib's antiangiogenic effects.ResultsThe intravitreal injection of acrizanib did not show a considerable impact on physiological angiogenesis and retinal thickness, indicating a potentially favorable safety profile. In the mouse models of OIR and CNV, acrizanib showed promising results in reducing pathological neovascularization, inflammation, and vascular leakage, indicating its potential efficacy against pathological angiogenesis. Consistent with in vivo results, acrizanib blunted angiogenic events in VEGF-treated HUVECs such as proliferation, migration, and tube formation. Furthermore, acrizanib inhibited the multisite phosphorylation of VEGFR2 to varying degrees and the activation of its downstream signal pathways in VEGF-treated HUVECs.ConclusionsThis study suggested the potential efficacy and safety of acrizanib in suppressing fundus neovascularization. Acrizanib functioned through inhibiting multiple phosphorylation sites of VEGFR2 in endothelial cells to different degrees.Translational RelevanceThese results indicated that acrizanib might hold promise as a potential candidate for the treatment of ocular vascular diseases.

Ocular Neovascularization: Clarifying Complex Interactions

Background and ObjectivePathological ocular neovascularization is implicated in several blinding eye diseases such as retinopathy of prematurity, diabetic retinopathy, and wet or exudative age related macular degeneration (AMD). Wet AMD is characterized by choroidal neovascularization, where friable vessels grow under the retina causing distortion of the normally flat macula and loss of central vision. Apurinic/apyrimidinic endonuclease 1/Redox effector factor‐1 (APE1/Ref‐1 or Ref‐1) serves dual functions as a DNA repair protein and a redox‐sensitive transcriptional regulator. Key proangiogenic transcription factors such as HIF1α, STAT3 and NFκB are under the regulation of Ref‐1, hence its inhibition is a potential tool in the fight against pathological neovascularization. We investigated whether novel, second generation small molecule Ref‐1 inhibitors, APX2009 and APX2014, can ameliorate pathological ocular neovascularization.MethodsWe tested the inhibitors for antiproliferative effect on Human Retinal Microvascular Endothelial primary cells (HRECs) and the Rhesus choroidal endothelial cell line (Rf/6a). We then assessed their ability to affect endothelial cell properties of tubule formation in vitro. Cells treated with compound or DMSO were seeded on solidified Matrigel to assay their ability to form vascular tubes, and the scratch‐wound assay was also used to test migration, another important endothelial property. We then examined both compounds ex vivo in a choroidal sprouting assay, which models microvascular angiogenesis from tissue. Finally, we tested APX2009 in vivo in the laser‐induced choroidal neovascularization (L‐CNV) mouse model, which recapitulates features of wet AMD. Mice were treated with APX2009 twice daily via intraperitoneal injection after laser until 14 days post treatment, when neovascularization was quantified by isolectin staining.ResultsAPX2014 was more antiproliferative in both HRECs (GI50: 110 nM) and Rf/6a (GI50: 5 μM) than APX2009 (HREC GI50: 1.13 μM; Rf/6a GI50: 25.7 μM). Both compounds significantly reduced the ability of both HRECs and Rf/6a cells to form tubes at mid nanomolar concentrations compared to DMSO. Both APX2009 and APX2014 significantly inhibited HREC and Rf/6a cell migration into the scratched area at the lowest concentrations tested. Ex vivo, both APX2009 and APX2014 inhibited angiogenic sprouting at low micromolar and high nanomolar concentrations respectively. In vivo, intraperitoneal APX2009 treatment significantly decreased lesion volume by 4‐fold compared to vehicle (p<0.0001, ANOVA with Dunnett's post hoc tests), effectively treating L‐CNV, without obvious intraocular or systemic toxicity.ConclusionRef‐1 inhibition with APX2009 and APX2014 blocks ocular angiogenesis in vitro and ex vivo. In addition, APX2009 is an effective systemic therapy for CNV in vivo, thus establishing Ref‐1 inhibition as a promising therapeutic route to treat ocular neovascularization.Support or Funding InformationNIH/NEI R01EY025641, Research to Prevent Blindness, Apexian PharmaceuticalsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pathologic ocular neovascularization commonly causes blindness. It is critical to identify the factors altered in pathologically proliferating versus normally quiescent vessels to develop effective targeted therapeutics. MicroRNAs regulate both physiological and pathological angiogenesis through modulating expression of gene targets at the posttranscriptional level. However, it is not completely understood if specific microRNAs are altered in pathologic ocular blood vessels, influencing vascular eye diseases. Here we investigated the potential role of a specific microRNA, miR-150, in regulating ocular neovascularization. We found that miR-150 was highly expressed in normal quiescent retinal blood vessels and significantly suppressed in pathologic neovessels in a mouse model of oxygen-induced proliferative retinopathy. MiR-150 substantially decreased endothelial cell function including cell proliferation, migration, and tubular formation and specifically suppressed the expression of multiple angiogenic regulators, CXCR4, DLL4, and FZD4, in endothelial cells. Intravitreal injection of miR-150 mimic significantly decreased pathologic retinal neovascularization in vivo in both wild-type and miR-150 knockout mice. Loss of miR-150 significantly promoted angiogenesis in aortic rings and choroidal explants ex vivo and laser-induced choroidal neovascularization in vivo. In conclusion, miR-150 is specifically enriched in quiescent normal vessels and functions as an endothelium-specific endogenous inhibitor of pathologic ocular neovascularization.

A small molecular multi-targeting tyrosine kinase inhibitor, anlotinib, inhibits pathological ocular neovascularization

Vascular endothelial growth factor (VEGF) signaling via VEGF receptor 2 (VEGFR2) plays a crucial role in pathologic ocular neovascularization. In this study, we investigated the antiangiogenic effect of apatinib, a pharmacologic inhibitor of VEGFR2 tyrosine kinase, against oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) in mice. Western blotting and in vitro angiogenesis assays were performed using human retinal microvascular endothelial cells (HRMECs). OIR was induced in neonatal mice by exposure to 75% oxygen from postnatal day (P) 7 to P12 and to room air from P12 to P17. Experimental CNV was induced in mice using laser photocoagulation. Apatinib was intravitreally and orally administered to mice. Neovascularization and phosphorylation of VEGFR2 were evaluated by immunofluorescence staining. Apatinib inhibited VEGF-mediated activation of VEGFR2 signaling and substantially reduced VEGF-induced proliferation, migration, and cord formation in HRMECs. A single intravitreal injection of apatinib significantly attenuated retinal or choroidal neovascularization in mice with OIR or laser injury-induced CNV, respectively. Retinal or choroidal tissues of the eyes treated with apatinib exhibited substantially lower phosphorylation of VEGFR2 than those of controls injected with vehicle. Intravitreal injection of apatinib did not cause noticeable ocular toxicity. Moreover, oral administration of apatinib significantly reduced laser-induced CNV in mice. Our study demonstrates that apatinib inhibits pathologic ocular neovascularization in mice with OIR or laser-induced CNV. Apatinib may, therefore, be a promising drug for the prevention and treatment of ischemia-induced proliferative retinopathy and neovascular age-related macular degeneration.

Roundabout (ROBO) 1 and 2 are transmembrane receptors that bind secreted SLIT ligands through their extracellular domains (ECDs) and signal through their cytoplasmic domains to modulate the cytoskeleton and regulate cell migration, adhesion, and proliferation. SLIT-ROBO signaling regulates pathological ocular neovascularization, which is a major cause of vision loss worldwide, but pharmacological tools to prevent SLIT-ROBO signaling are lacking. Here, we developed human monoclonal antibodies (mAbs) against the ROBO1 and ROBO2 ECDs. One antibody that inhibited in vitro SLIT2 signaling through ROBO1 and ROBO2 (anti-ROBO1/2) also reduced ocular neovascularization in oxygen-induced retinopathy (OIR) and laser-induced corneal neovascularization (CNV) mouse models in vivo. Single-cell RNA sequencing of OIR retinas revealed that antibody treatment affected several cell types relevant to physiological and pathological angiogenesis, including endothelial cells, pericytes, and a heterogeneous population of myeloid cells. mAb treatment improved blood-retina barrier integrity and prevented pathological pericyte activation in OIR. SLIT-ROBO signaling inhibition prevented pathological activation of myeloid cells and increased neuroprotective myeloid populations normally seen in the developing retina. Microglia/infiltrating macrophage-specific ablation of Robo1 and Robo2 or knockout of the downstream effector phosphatidylinositol 3-kinase (Pik3cg) encoding PI3Kγ in both OIR and CNV models phenocopied anti-ROBO1/2 treatment, further demonstrating the key role of myeloid cells as drivers of ocular neovascular diseases. ROBO1/2 blocking antibodies may thus provide a promising strategy to combat inflammation in blinding eye diseases.

Background: Pathological ocular neovascularization, a characteristic feature of proliferative ocular diseases, is a primary contributor to global vision impairment. The dynamics of tubulin are crucial in maintaining ocular homeostasis, closely linked to cellular proliferation and angiogenesis. Elucidating the molecular mechanisms driving this process is vital for formulating effective therapeutic strategies. Methods: Multiple transcriptome analyses revealed upregulation of endothelial tubulin beta-6 chain (Tubb6) in oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) mice models. Transwell migration assay, wound healing assay, tube formation assay, flow cytometry, and immunofluorescent staining were employed to identify the role of TUBB6 knockout (KO) in vitro. The effects of Tubb6 silencing on retinal angiogenesis and choroidal neovascularization were subsequently evaluated. Results: We identified upregulated Tubb6 expression in retinas from OIR mice through combination analyses of single-cell RNA sequencing (scRNA-Seq) and bulk RNA-Seq. The RNA expression profiles of endothelial cells (ECs) from proliferative diabetic retinopathy (PDR) patients and neovascular age-related macular degeneration (nAMD) patients also exhibited an elevation in TUBB6. Notably, Tubb6 was abundantly expressed in ECs and pericytes, and was predominantly localized to proliferative ECs and vascular tip cells. Functional studies demonstrated that TUBB6 knockdown reduced the expression of proliferative and tip cell markers in vitro. Tubb6 deficiency decreased vascular sprouting and tip cell formation of OIR mice retina and retarded CNV progression in vivo. Mechanistically, YBX1, an RNA-binding protein, was identified as an upstream regulator of TUBB6 via binding to its 3' untranslated region (3'UTR) and maintaining mRNA stability. Transcriptome analysis further linked TUBB6 to the activity of WNT pathway. TUBB6 silencing suppressed the WNT signaling pathway, with WNT3A and FZD8 identified as downstream targets. Conclusions: Collectively, our research shed light on the pivotal function of TUBB6 in maintaining ocular homeostasis and uncovered the YBX1-TUBB6-WNT3A/FZD8 pathway's involvement in sprouting angiogenesis. Targeting TUBB6 and developing its specific inhibitor could pioneer new approaches for treating ocular microvascular diseases.

MicroRNA-145 Regulates Pathological Retinal Angiogenesis by Suppression of TMOD3

Topical Nanoemulsion of a Runt-related Transcription Factor 1 Inhibitor for the Treatment of Pathologic Ocular Angiogenesis.

Pathological ocular neovascularization is a major cause of blindness. Increased dietary intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) reduces retinal neovascularization and choroidal neovascularization (CNV), but ω-3 LCPUFA metabolites of a major metabolizing pathway, cytochrome P450 oxidase (CYP) 2C, promote ocular pathological angiogenesis. We hypothesized that inhibition of CYP2C activity will add to the protective effects of ω-3 LCPUFA on neovascular eye diseases. The mouse models of oxygen-induced retinopathy and laser-induced CNV were used to investigate pathological angiogenesis in the retina and choroid, respectively. The plasma levels of ω-3 LCPUFA metabolites of CYP2C were determined by mass spectroscopy. Aortic ring and choroidal explant sprouting assays were used to investigate the effects of CYP2C inhibition and ω-3 LCPUFA-derived CYP2C metabolic products on angiogenesis ex vivo. We found that inhibition of CYP2C activity by montelukast added to the protective effects of ω-3 LCPUFA on retinal neovascularization and CNV by 30% and 20%, respectively. In CYP2C8-overexpressing mice fed a ω-3 LCPUFA diet, montelukast suppressed retinal neovascularization and CNV by 36% and 39% and reduced the plasma levels of CYP2C8 products. Soluble epoxide hydrolase inhibition, which blocks breakdown and inactivation of CYP2C ω-3 LCPUFA-derived active metabolites, increased oxygen-induced retinopathy and CNV in vivo. Exposure to selected ω-3 LCPUFA metabolites of CYP2C significantly reversed the suppression of both angiogenesis ex vivo and endothelial cell functions in vitro by the CYP2C inhibitor montelukast. Inhibition of CYP2C activity adds to the protective effects of ω-3 LCPUFA on pathological retinal neovascularization and CNV.

Pathophysiological Function of Endogenous Calcitonin Gene–Related Peptide in Ocular Vascular Diseases