Corneal Neovascularization In Mice Research Articles

ALKBH5 Regulates Corneal Neovascularization by Mediating FOXM1 M6A Demethylation.

This study aims to explore the regulatory role and potential mechanisms of ALKBH5-mediated N6-methyladenosine (m6A) demethylation modification in corneal neovascularization (CNV). A mouse CNV model was established through corneal alkali burns. Total m6A levels were measured using an m6A RNA methylation quantification kit. The mRNA expression of candidate m6A-related enzymes was quantified by quantitative RT-PCR. Small interfering RNA targeting ALKBH5 was injected subconjunctivally into alkali-burned mice. The CNV area, corneal epithelial thickness, and pathological changes were evaluated. Protein expression was detected by western blot and immunofluorescence. Human umbilical vein endothelial cells (HUVECs) were treated with IL-6. Plasmid transfection knocked down ALKBH5 or overexpressed FOXM1 in IL-6-induced HUVECs. The assays of CCK8, wound healing, and tube formation evaluated the cell proliferation, migration, and tube formation abilities, respectively. The dual-luciferase assay examined the binding between ALKBH5 and FOXM1. Methylated RNA immunoprecipitation-qPCR detected the m6A levels of FOXM1. Significant CNV was observed on the seventh day. Total m6A levels were reduced, and ALKBH5 expression was increased in CNV corneas and IL-6-induced HUVECs. ALKBH5 knockdown alleviated corneal neovascularization and inflammation and countered IL-6-induced promotion of cell proliferation, migration, and tube formation in HUVECs. ALKBH5 depletion increased m6A levels and decreased VEGFA and CD31 expression both in vivo and in vitro. This knockdown in HUVECs elevated m6A levels on FOXM1 mRNA while reducing its mRNA and protein expression. Notably, FOXM1 overexpression can reverse ALKBH5 depletion effects. ALKBH5 modulates FOXM1 m6A demethylation, influencing CNV progression and highlighting its potential as a therapeutic target.

Genome Editing VEGFA Prevents Corneal Neovascularization In Vivo.

Corneal neovascularization (CNV) is a common clinical finding seen in a range of eye diseases. Current therapeutic approaches to treat corneal angiogenesis, in which vascular endothelial growth factor (VEGF) A plays a central role, can cause a variety of adverse side effects. The technology of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 can edit VEGFA gene to suppress its expression. CRISPR offers a novel opportunity to treat CNV. This study shows that depletion of VEGFA with a novel CRISPR/Cas9 system inhibits proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Importantly, subconjunctival injection of this dual AAV-SpCas9/sgRNA-VEGFA system is demonstrated which blocks suture-induced expression of VEGFA, CD31, and α-smooth muscle actin as well as corneal neovascularization in mice. This study has established a strong foundation for the treatment of corneal neovascularization via a gene editing approach for the first time.

Modulation of steroid pathway to preserve corneal transparency

Purpose: Corneal neovascularization (CN) is a major cause of blindness worldwide. We previously showed that endothelial mineralocorticoid receptor (MR) contributed to CN in mice. In this study, we investigated the mechanisms of action of MRA in a rat model of limbal deficiency.Methods: CN was induced in Lewis rats by total corneal de‐epithelialization and limbal scratching. Spironolactone (SPL, 25 mg/kg) or eplerenone was administered daily. Control eyes received vehicle. Rats were euthanized at day 3 and 7, and corneas were dissected for transcriptomic analysis. Proteins extracted from corneas at day 7 were used for western blot. Corneal innervation was evaluated by immunostaining of TUBB3 on corneal flat mounts at day 16. Corneal morphology and CN were assessed in vivo by Micron III optical coherence tomography and fluorescein angiography at day 14.Results: MR antagonists (MRA) favoured epithelial regeneration and nerve growth, reduced corneal angiogenesis, edema and inflammation in a rat model of CN. Transcriptomic analysis showed 75 differentially expressed genes in SPL groups. Gene set enrichment analysis highlighted pathways including wound healing and differentiation, inflammatory and immune responses, hypoxia, angiogenesis and neuroprotection. SPL upregulated Dcn, Col1a1, Timp2 and downregulated Lrg1 at day 3, and induced Pedf at day 7. Increase in DCN protein by SPL was confirmed by western blot.Conclusions: MRA regulates genes/proteins involved in anti‐inflammatory and anti‐angiogenic processes, and improves corneal re‐innervation in CN model. Dcn is one of the downstream regulated targets.

Preparation of a Sunitinib loaded microemulsion for ocular delivery and evaluation for the treatment of corneal neovascularization in vitro and in vivo.

Background: Corneal neovascularization (CNV) is a pathological condition that can disrupt corneal transparency, thus harming visual acuity. However, there is no effective drug to treat CNV. Sunitinib (STB), a small-molecule multiple receptor tyrosine kinase inhibitor, was shown to have an effect on CNV. The purpose of this study was to develop an STB microemulsion (STB-ME) eye drop to inhibit CNV by topical application. Methods: We successfully prepared an STB-ME by the phase inversion emulsification method, and the physicochemical properties of STB-MEs were investigated. The short-term storage stability, cytotoxicity to human corneal epithelial cells, drug release, ocular irritation, ocular pharmacokinetics and the inhibitory effect on CNV were evaluated in vitro and in vivo. Results: The optimal formulation of STB-ME is composed of oleic acid, CRH 40, Transcutol P, water and sodium hyaluronate (SH). It is a uniform spherical particle with a mean droplet size of 18.74 ± 0.09nm and a polydispersity index of 0.196 ± 0.004. In the in vitro drug release results, STB-ME showed sustained release and was best fitted by a Korsmeyer-Peppas model (R 2 = 0.9960). The results of the ocular pharmacokinetics in rabbits showed that the formulation containing SH increased the bioavailability in the cornea (2.47-fold) and conjunctiva (2.14-fold). STB-ME (0.05% and 0.1%), administered topically, suppressed alkali burn-induced CNV in mice more effectively than saline, and high-dose (0.1%) STB-ME had similar efficacy to dexamethasone (0.025%). Conclusion: This study provides a promising formulation of STB-ME for the inhibition of CNV by topical administration, which has the excellent characteristics of effectiveness, sustained release and high ocular bioavailability.

Gap26 inhibited angiogenesis through the β-catenin-VE-cadherin-VEGFR2-Erk signaling pathway

PurposeTo investigate the effect of connexin 43 (Cx43) on corneal neovascularization and its regulation of VEGFR2 on vascular endothelial cells. MethodsIn vivo, we used mouse corneal suture model to induce corneal neovascularization and discovered the function of gap26 in corneal neovascularization. In vitro, the effect of gap26 on HUVEC was observed by cell proliferation, tube formation and scratch experiments. WB and PCR detected the changes in angiogenic protein and mRNA expression. Knockdown of key mRNA in neovascularization using siRNA confirmed that Cx43 regulates neovascularization through the β-catenin-VE-cadherin-VEGFR2-Erk signaling pathway. ResultsIn vivo, gap26 can reduce mouse corneal neovascularization. In vitro, we show that Cx43 expression is increased in the presence of VEGFA stimulation, and when we use gap26 to inhibit Cx43 can reduce vascular endothelial cell proliferation, tube formation and migration. We found that the expression of pVEGFR2 and pErk increased in response to VEGFA, while they decreased after using gap26. And the expression of β-catenin and VE-cadherin decreased in response to VEGFA, while they increased after using gap26. Furthermore, we found that Cx43 regulates angiogenesis through the β-catenin-VE-cadherin-VEGFR2-Erk pathway. ConclusionsGap26 can downregulate VEGFR2 phosphorylation by stabilizing the expression of β-catenin and VE-cadherin on the cell membrane, thereby inhibiting VEGFA-induced HUVECs proliferation, migration and tube formation and inhibiting corneal neovascularization.

Targeted dexamethasone nano-prodrug for corneal neovascularization management

BackgroundTo overcome the drawbacks of traditional therapy for corneal neovascularization (CNV), we evaluated the efficacy of polyethylene glycol (PEG)-conjugated Ala-Pro-Arg-Pro-Gly (APRPG) peptide modified dexamethasone (Dex), a novel nano-prodrug (Dex-PEG-APRPG, DPA). MethodsCharacterization of DPA nano-prodrug were measured with transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses. Cytotoxicity and effects on cell migration and tube formation of DPA were evaluated in vitro. A murine CNV model was established by cornea alkali burn. The injured corneas were given eye drops of DPA (0.2 mM), Dex solution (0.2 mM), Dexp (2 mM), or normal saline three times a day. After two weeks, eyes were obtained for the analysis of histopathology, immunostaining, and mRNA expression. ResultsDPA with an average diameter of 30 nm, presented little cytotoxicity and had good ocular biocompatibility. More importantly, DPA showed specific targeting to vascular endothelial cells with efficient inhibition on cell migration and tube formation. In a mouse CNV model, clinical, histological, and immunohistochemical examination results revealed DPA had a much stronger angiogenesis suppression than Dex, resembling a clinical drug with an order of magnitude higher concentration. This was ascribed to the significant downregulations in the expression of pro-angiogenic and pro-inflammatory factors in the corneas. In vivo imaging results also demonstrated that APRPG could prolong ocular retention time. ConclusionsThis study suggests that DPA nano-prodrug occupies advantages of specific targeting ability and improved bioavailability over conventional therapy, and holds great potential for safe and efficient CNV therapy.

WNT16b promotes the proliferation and self-renewal of human limbal epithelial stem/progenitor cells via activating the calcium/calcineurin A/NFATC2 pathway.

Our previous finding revealed that WNT16b promoted the proliferation of human limbal epithelial stem cells (hLESCs) through a β-catenin independent pathway. Here, we aimed to explore its underlying molecular mechanism and evaluate its potential in the treatment of limbal stem cell deficiency (LSCD). Based on the findings of mRNA-sequencing, the expression of key molecules in WNT/calcineurin A/NFATC2 signalling pathway was investigated in WNT16b-co-incubated hLESCs and control hLESCs. An epithelial wound healing model was established on Wnt16b-KO mice to confirm the regulatory effect of WNT16b in vivo. The therapeutic potential of WNT16b-co-incubated hLESCs was also evaluated in mice with LSCD. Our findings showed that WNT16b bound with Frizzled7, promoted the release of Ca2+ and activated calcineurin A and NFATC2. With the translocation of NFATC2 into cell nucleus and the activation of HDAC3, WDR5 and GCN5L2, the expression of H3K4me3, H3K14ac and H3K27ac in the promoter regions of FoxM1 and c-MYC increased, which led to hLESC proliferation. The effect of the WNT16b/calcium/calcineurin A/NFATC2 pathway on LESC homeostasis maintenance and corneal epithelial repair was confirmed in Wnt16b-KO mice. Moreover, WNT16b-coincubated hLESCs could reconstruct a stable ocular surface and inhibit corneal neovascularization in mice with LSCD. In conclusion, WNT16b enhances the proliferation and maintains the stemness of hLESCs by activating the non-canonical calcium/calcineurin A/NFATC2 pathway in vitro and in vivo, and accelerates corneal epithelial wound healing.

Sorafenib-loaded nanostructured lipid carriers for topical ocular therapy of corneal neovascularization: development, in-vitro and in vivo study

Sorafenib (SRB), a multikinase inhibitor, is effective in reducing experimental corneal neovascularization (CNV) after oral administration; however, its therapeutic use in ocular surface disorders is restricted due to poor solubility and limited bioavailability. This study aimed to develop and optimize SRB-loaded nanostructured lipid carriers (SRB-NLCs) for topical ocular delivery by a central composite design response surface methodology (CCD-RSM). It was spherical and uniform in morphology with an average particle size of 111.87 ± 0.93 nm and a narrow size distribution. The in vitro drug release from the released SRB-NLC formulation was well fitted to Korsmeyer Peppas release kinetics. The cell counting kit-8 (CCK-8) cell viability assay demonstrated that SRB-NLC was not obviously cytotoxic to human corneal epithelial cells (HCECs). An in vivo ocular irritation test showed that SRB-NLC was well tolerated by rabbit eyes. Ocular pharmacokinetics revealed 6.79-fold and 1.24-fold increase in the area under concentration-time curves (AUC0-12h) over 12 h in rabbit cornea and conjunctiva, respectively, treated with one dose of SRB-NLC compared with those treated with SRB suspension. Moreover, SRB-NLC (0.05% SRB) and dexamethasone (0.025%) similarly suppressed corneal neovascularization in mice. In conclusion, the optimized SRB-NLC formulation demonstrated excellent physicochemical properties and good tolerance, sustained release, and enhanced ocular bioavailability. It is safe and potentially effective for the treatment of corneal neovascularization.

Monolith/Hydrogel composites as triamcinolone acetonide carriers for curing corneal neovascularization in mice by inhibiting the fibrinolytic system

Corneal neovascularization is a serious corneal pathological change caused by various factors. The drug delivery system is of great significance for the effective treatment of corneal neovascularization. Herein, we developed and characterized a monolith/hydrogel composite as the triamcinolone acetonide (TA) carrier for curing corneal neovascularization. The composite was prepared by photo-initiated free radical polymerization of multi-methacrylate substituted dodecamine organic molecular cage and post-modified by the sequential photo-initiated free radical polymerization of acrylated gelatin. The globular morphology and structural property of as-prepared composites were evaluated by scanning electron microscopy, Fourier-transform infrared spectroscopy and solid-state cross polarization magic angle spinning carbon-13 nuclear magnetic resonance. Then swelling ratio and the TA loading capacity were investigated then. Compared with gelatin hydrogel, the composites exhibited a decreased swelling ratio and an improved loading capacity. With good biocompatibility, the composite can sustainedly release TA for up to 28 days, and effectively inhibit corneal neovascularization with an alkali burn-induced corneal neovascularization model. Additionally, tandem mass tags-labeled quantitative proteomics were performed to identify differentially expressed proteins between vascularized and devascularized corneas. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the inhibition process could be primarily linked to the fibrinolytic system. These results demonstrated the potential of monolith/hydrogel composites as delivery systems in the therapy for biomedical diseases.

Inhibition of Neovascularization and Inflammation in a Mouse Model of Corneal Alkali Burns Using Cationic Liposomal Tacrolimus.

Eye drops account for more than 90% of commercialized ophthalmic drugs. However, eye drops have certain shortcomings, such as short precorneal retention time and weak corneal penetration. The requirement of frequent instillation of eye drops also causes poor patient compliance, which may lead to further aggravation of the disease. We aimed to develop a cationic liposome formulation to increase the bioavailability of the therapeutic agent and solve the aforementioned problems. In the present study, we prepared cationic liposomal tacrolimus (FK506) with a surface potential of approximately +30 mV, which could bind to the negatively charged mucin layer of the ocular surface. Our results showed that the content of FK506 in the cornea was increased by 93.77, 120.30, 14.24, and 20.36 times at 5, 30, 60, and 90 min, respectively, in the FK506 liposome group (0.2 mg/ml) compared with the free drug group (0.2 mg/ml). Moreover, FITC-labeled FK506 liposomes significantly prolonged the ocular surface retention time to 50 min after a single dose. In addition, the results of the Cell Counting Kit-8 assay, live and dead cell assay, sodium fluorescein staining, and hematoxylin and eosin staining all indicated that FK506 liposomes had good biological compatibility in both human corneal epithelial cells and mouse eyeballs. Compared with the free drug at the same concentration, FK506 liposomes effectively inhibited vascular endothelial growth factor-induced green fluorescent protein-transduced human umbilical vein endothelial cell migration and tube formation in vitro. In a mouse corneal neovascularization model induced by alkali burns, FK506 liposomes (0.2 mg/ml) enhanced corneal epithelial recovery, inhibited corneal neovascularization, and reduced corneal inflammation, and its therapeutic effect was better than those of the commercial FK506 eye drops (1 mg/ml) and the free drug (0.2 mg/ml). Collectively, these results indicate that cationic FK506 liposomes could increase the efficacy of FK506 in the corneal neovascularization model. Therefore, cationic FK506 liposomes can be considered as a promising ocular drug delivery system.

The functional role of decorin in corneal neovascularization in vivo

Our earlier decorin (Dcn) gene overexpression studies found that the targeted Dcn gene transfer into the cornea inhibited corneal angiogenesis in vivo using a rabbit model. In this study, we tested the hypothesis that anti-angiogenic effects of decorin in the cornea are mediated by alterations in a normal physiologic balance of pro- and anti-angiogenic factors using decorin deficient (Dcn−/−) and wild type (Dcn+/+) mice. Corneal neovascularization (CNV) in Dcn−/− and Dcn+/+ mice was produced with a standard chemical injury technique. The clinical progression of CNV in mice was monitored with stereo- and slit-lamp microscopes, and histopathological hematoxylin and eosin (H&E) staining. Protein and mRNA expression of pro- and anti-angiogenic factors in the cornea were evaluated using immunofluorescence and quantitative real-time PCR, respectively. Slit-lamp clinical eye examinations revealed significantly more CNV in Dcn−/- mice than the Dcn+/+ mice post-injury (p < 0.05) and AAV5-Dcn gene therapy significantly reduced CNV in Dcn−/- mice compered to no AAV5-Dcn gene therapy controls (p < 0.001). H&E-stained corneal sections exhibited morphology with several neovessels in injured corneas of the Dcn−/− mice than the Dcn+/+ mice. Immunofluorescence of corneal sections displayed significantly higher expression of α-smooth muscle actin (α-SMA) and endoglin proteins in Dcn−/- mice than Dcn+/+ mice (p < 0.05). Quantitative real-time PCR found significantly increased mRNA levels of pro-angiogenic factors endoglin (2.53-fold; p < 0.05), Vegf (2.47-fold; p < 0.05), and Pecam (2.14-fold; p < 0.05) and anti-angiogenic factor Vegfr2 (1.56-fold; p < 0.05) in the normal cornea of the Dcn−/− mice than the Dcn+/+ mice. Furthermore, neovascularized Dcn−/− mice corneas showed greater increase in mRNA expression of pro-angiogenic factors endoglin (4.58-fold; p < 0.0001), Vegf (4.16-fold; p < 0.0001), and Pdgf (2.15-fold; p < 0.0001) and reduced expression of anti-angiogenic factors Ang2 (0.12-fold; p < 0.05), Timp1 (0.22-fold; p < 0.05), and Vegfr2 (0.67-fold; p > 0.05) compared to neovascularized Dcn+/+ mice corneas. These gene deficience studies carried with transgenic Dcn−/− mice revealed decorin's role in influencing a physiologic balance between pro-and anti-angiogenic factors in the normal and injured cornea. We infer that the functional deletion of Dcn promotes irregular corneal repair and aggravates CNV.

Inhibition of PDGF-BB reduces alkali-induced corneal neovascularization in mice.

The aim of the present study was to investigate the role of platelet-derived growth factor (PDGF)-BB/PDGF receptor (R)-β signaling in an experimental murine corneal neovascularization (CrNV) model. Experimental CrNV was induced by alkali injury. The intra-corneal expression of PDGF-BB was examined using immunohistochemistry. The effect of PDGF-BB on CrNV was evaluated using immunofluorescence staining. The expression levels of PDGFR-β in human retinal endothelial cells (HRECs) under normal conditions or following cobalt chloride treatment, which induced hypoxic conditions, was assessed using reverse transcription-quantitative PCR. The effect of exogenous treatment of PDGF-BB on the proliferation, migration and tube formation of HRECs under normoxic or hypoxic conditions was evaluated in vitro using Cell Counting Kit-8, wound healing and 3D Matrigel capillary tube formation assays, respectively. The results indicated that the intra-corneal expression levels of the proteins of PDGF-BB and PDGFR-β were detectable on days 2 and 7 following alkali injury. The treatment with neutralizing anti-PDGF-BB antibody resulted in significant inhibition of CrNV. The intra-corneal expression levels of vascular endothelial growth factor A, matrix metallopeptidase (MMP)-2 and MMP-9 proteins were downregulated, while the expression levels of thrombospondin (TSP)-1, TSP-2, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 and ADAMTS-2 were upregulated significantly in mice treated with anti-PDGF-BB antibody. The expression levels of PDGFR-β were upregulated in HRECs under hypoxic conditions compared with those noted under normoxic conditions. Recombinant human PDGF-BB promoted the proliferation, migration and tube formation of HRECs under hypoxic conditions. The data indicated that PDGF-BB/PDGFR-β signaling was involved in CrNV and that it promoted endothelial cell proliferation, migration and tube formation. The pro-angiogenic effects of this pathway may be mediated via the induction of pro-angiogenic cytokine secretion and the suppression of anti-angiogenic cytokine secretion.

VEGF TrapR1R2 Suspended in the Semifluorinated Alkane F6H8 Inhibits Inflammatory Corneal Hem- and Lymphangiogenesis.

PurposeSemifluorinated alkanes (SFAs) are used at the ocular surface as lubricants or vehicles for drugs. The purpose of this study was to test the effect of vascular endothelial growth factor (VEGF) TrapR1R2 suspended in the SFA perfluorohexyloctane (Trap/F6H8) on corneal neovascularization.MethodsSuture placement was used to induce inflammatory corneal neovascularization in mice. Treatment groups were: Trap/F6H8, VEGF TrapR1R2 as aqueous formulation dissolved in phosphate buffer (Trap), F6H8, and phosphate buffer (controls). Eye drops were applied 3×/daily for 2 weeks. Afterward, corneas were stained with CD31 and LYVE-1 to analyze corneal hem- and lymphangiogenesis. To investigate the effect of on inflammatory cell recruitment, corneal CD45+ cells were quantified. In addition, epithelial wound closure after debridement was assessed by corneal fluorescein staining.ResultsTrap/F6H8 was as effective as Trap in inhibiting corneal hemangiogenesis and lymphangiogenesis after 2 weeks of treatment. After 3 days of treatment, Trap/F6H8 was even more effective than Trap in inhibiting corneal hemangiogenesis. Both treatment groups (Trap/F6H8 and Trap) significantly reduced corneal CD45+ cell recruitment. Epithelial closure after debridement was unaffected by Trap/F6H8 or Trap.ConclusionsIn this study, we demonstrate that F6H8 is a potential carrier for VEGF TrapR1R2 to topically treat corneal neovascularization. Our findings might open new treatment avenues for local anti-angiogenic therapy at the cornea, as F6H8 is already approved for the usage at the ocular surface.Translational RelevanceWith this study we show for the first time that SFAs can serve as carriers for anti-angiogenic drugs at the ocular surface.

Endothelial Desert Hedgehog and ectopic Sonic Hedgehog act antagonistically to regulate vessel integrity and angiogenesis

The therapeutic potential of Hedgehog (Hh) signaling agonists for vascular diseases is of growing interest. However, the molecular mechanisms underlying Hh signaling effects on the vasculature remains unclear and conflicting results have been published. Our goal is to clarify and compare the vascular effects of endothelial-derived Desert Hedgehog (eDhh) and soluble N-terminal Sonic Hedgehog (N-Shh). We first confirmed that in accordance with the literature, recombinant N-Shh is pro-angiogenic in the mouse corneal neovascularization assay. However, by using mice lacking Dhh expression in endothelial cells (ECs), we showed that eDhh limits VEGFA-dependent angiogenesis in the same model. Similarly, while we found that eDhh is necessary for blood-brain barrier (BBB) integrity, ectopic administration of soluble N-Shh induces BBB leakage. Consistent results were obtained in vitro in cultured ECs. Indeed, both Dhh knock down and Shh treatment promoted EC migration and increased ECs permeability. This first set of data demonstrates for the first time that Hedgehog ligands may induced opposite effects in ECs. Next, we found that mice lacking Smoothened (a positive regulator of Hh pathway) in ECs display a similar phenotype to those lacking eDhh, suggesting that eDhh stimulates Hh signaling in ECs while soluble N-Shh inhibits it. At a molecular level, we found that soluble Shh prevent Dhh binding with its receptor Ptch1 both in IP assays and proximity ligations assays. Altogether these results suggest that soluble Shh acts as an inhibitor of Hh signaling in endothelial cells by competing with Dhh. In conclusion the present data highlights for the first time that eDhh and soluble Shh have antagonistic properties especially in ECs. Such demonstration implies that Hh ligands cannot be used instead of another for rescue/therapeutic purposes.

Keratocytes promote corneal neovascularization through VEGFr3 induced by PPARα-inhibition

As the peroxisome proliferator – activated receptor alpha (PPARα) agonist, fenofibrate has been widely used to be a good lipid-regulating drug in the clinical application. In this study, we investigated the mechanism by which keratocytes inhibit the corneal neovascularization (CNV) through PPARα - activation. To do this, the CNV model was established by alkali burn, followed by being divided into three groups including control, fenofibrate and vehicle group. The expression of VEGFr3, MMP13 and PPARα in corneas of normal mouse and alkali-burned mouse was determined via quantitative RT- PCR (qRT-PCR) and Western blot analysis (WB). The CNV area was observed under a slit lamp microscope. The location of PPARα expression in the corneas was determined via immunohistochemistry. In cultured primary keratocytes, the effect of fenofibrate on PPARα, VEGFr3 and MMP13 expression was determined by qRT-PCR and WB. Besides, PPARα knockout (PPARα−/−) mouse CNV and keratocytes model were established to further confirm the effect of PPARα on VEGFr3 and MMP13 expression. We found that PPARα was expressed in epithelium, stroma and endothelium of the normal cornea, however, with relatively low level in the corneal stroma. Meanwhile, its expression was decreased markedly in the cornea during the stage of CNV formation. After treatment of fenofibrate, PPARα expression was promoted and the expression of VEGFr3 and MMP13 was inhibited in both CNV mice model and primary keratocytes, and CNV areas were decreased in CNV mice model. However, the results in PPARα−/− CNV and keratocytes model were opposite. Our results suggest that keratocytes could promote the expression of VEGFr3 and MMP13, and CNV formation through PPARα downregulation.

Development of Kaempferol-Loaded Gelatin Nanoparticles for the Treatment of Corneal Neovascularization in Mice.

Cornea is the transparent layer in front of the eye that does not contain blood vessels. Among eye diseases, corneal neovascularization (NV) is one of the major causes of vision loss, since it can also lead to blindness. An herbal extraction containing flavonoid, kaempferol (KA), with antiangiogenic effect was chosen as a candidate drug for inhibited vessel formation. The use of nanomedicine has led to higher drug bioavailability and slow release of the drug as an effective therapeutic formulation in ocular drug delivery. In this study, we prepared gelatin nanoparticles (GNP) with kaempferol encapsulation (GNP-KA) for corneal NV treatment by topical delivery, i.e., eye drops. We found that GNP with/without KA loading was in the size of 85−150 nm, and its zeta potential was around 22−26 mV. The KA entrapment rate of GNP-KA was around 90−98%, and the loading rate was about 4.6%. The TEM results clearly indicated the GNP-KA NPs to be round spheres. The in vitro test involved the adoption of human umbilical vein endothelial cells (HUVECs) for coculture with these nanoparticles. From WST-8 assay, and cell migration examinations, it was evident that GNP-KA had the capacity to inhibit the cell viability and function of HUVECs. The results from in vivo tests such as ocular vessels observation, hematoxylin & eosin (H&E) stain, and metalloproteinases (MMP)/vascular endothelial growth factor (VEGF) quantification revealed the mice’s eyes with corneal NV treated by eye drops containing GNP-KA once daily for 7 days had better therapeutic effects with less vessels in-growths in the cornea, compared to the KA solution group by reducing the production of MMP and VEGF in the cornea. Therefore, we expected to achieve a comfortable treatment with a simple method using nanomedicine (GNP-KA) as ophthalmological agent delivered as eye drops.

RIP1 kinase mediates angiogenesis by modulating macrophages in experimental neovascularization

Inflammation plays an important role in pathological angiogenesis. Receptor-interacting protein 1 (RIP1) is highly expressed in inflammatory cells and is known to play an important role in the regulation of apoptosis, necroptosis, and inflammation; however, a comprehensive description of its role in angiogenesis remains elusive. Here, we show that RIP1 is abundantly expressed in infiltrating macrophages during angiogenesis, and genetic or pharmacological inhibition of RIP1 kinase activity using kinase-inactive RIP1K45A/K45A mice or necrostatin-1 attenuates angiogenesis in laser-induced choroidal neovascularization, Matrigel plug angiogenesis, and alkali injury-induced corneal neovascularization in mice. The inhibitory effect on angiogenesis is mediated by caspase activation through a kinase-independent function of RIP1 and RIP3. Mechanistically, infiltrating macrophages are the key target of RIP1 kinase inhibition to attenuate pathological angiogenesis. Inhibition of RIP1 kinase activity is associated with caspase activation in infiltrating macrophages and decreased expression of proangiogenic M2-like markers but not M1-like markers. Similarly, in vitro, catalytic inhibition of RIP1 down-regulates the expression of M2-like markers in interleukin-4-activated bone marrow-derived macrophages, and this effect is blocked by simultaneous caspase inhibition. Collectively, these results demonstrate a nonnecrotic function of RIP1 kinase activity and suggest that RIP1-mediated modulation of macrophage activation may be a therapeutic target of pathological angiogenesis.

Epithelium-derived miR-204 inhibits corneal neovascularization

MicroRNA-204 (miR-204) is highly expressed in cornea, here we explored the role and mechanism of miR-204 in corneal neovascularization (CNV). Mouse CNV was induced by intrastromal placement of suture in BALB/c mice with the subconjunctival injection of miR-204 agomir or negative control. Human primary limbal epithelial cells (LECs) and immortalized microvascular endothelial cells (HMECs) were used to evaluate the expression changes and anti-angiogenic effects of miR-204 under biomechanical stress (BS). The expression and localization of miR-204, vascular endothelial growth factor (VEGF) and their receptors were detected by quantitative real-time PCR, in situ hybridization, immunohistochemistry and Western blot. The results showed that miR-204 expression was mainly localized in epithelium and down-expressed in vascularized cornea. Subconjunctival injection of miR-204 agomir inhibited CNV and reduced the expression of VEGF and VEGF receptor 2. Similarly, miR-204 overexpression attenuated the increased expression of VEGF by biomechanical stress in LECs, and suppressed the proliferation, migration, and tube formation of HMECs. These novel findings indicate that epithelium-derived miR-204 inhibits suture-induced CNV through regulating VEGF and VEGF receptor 2.

MicroRNA-296 mediated corneal neovascularization in an animal model of corneal burns after alkali exposures.

Alkali burns of the cornea may lead to permanent visual impairment or complete blindness. In the current study, the role of microRNA 296 (miR-296) was explored in mouse corneal neovascularization induced by alkali burns. An alkali burn model in Balb/c mice was developed to study chemical corneal injuries. The expression of the miR-296 gene was measured by reverse-transcription-quantitative polymerase chain reaction. Fibroblast growth factor 23 (FGF23) protein expression was measured by western blot analysis. Possible impacted pathways were analyzed by Kyoto Encyclopedia of Genes and Genomes pathway analysis. miR-296 gene expression was examined following chemical corneal injury and it was demonstrated that different topical eye medications decreased miR-296 gene expression. miR-296 may participate in cytokine-cytokine receptor interaction pathways to influence corneal inflammatory responses. It was also revealed that FGF23 was expressed following chemical corneal injury and that different treatments with topical eye drops decreased its expression. miR-296 is a novel molecular modulator for alkali burns in the mouse cornea.

Targeting VEGFR-3/-2 signaling pathways with AD0157: a potential strategy against tumor-associated lymphangiogenesis and lymphatic metastases

BackgroundLymphatic metastasis is one of the leading causes of death in patients with different types of cancer and is the main prognostic factor for the disease survival. The formation of new lymphatic vessels (lymphangiogenesis) in primary tumors facilitates tumor cell dissemination to regional lymph nodes and correlates with distant metastases. Lymphangiogenesis has thus emerged as a suitable therapeutic target to block metastases, but no anti-lymphangiogenic compounds have been approved for clinical use to date. Therefore, new or improved therapies blocking lymphatic metastases are urgently required.MethodsWe established murine breast tumors to assess the effect of AD0157 on tumor growth, lymphangiogenesis, and lymphatic dissemination. Then, a battery of in vivo (mouse corneal neovascularization and ear sponges), ex vivo (mouse lymphatic rings and rat mesentery explants), and in vitro (proliferation, tubulogenesis, wound-healing, Boyden chambers, and spheroids) assays was used to give insight into the lymphangiogenic steps affected by AD0157. Finally, we investigated the molecular pathways controlled by this drug.ResultsAD0157 was found to inhibit the growth of human breast cancer xenografts in mice, to strongly reduce tumor-associated lymphangiogenesis and to block metastatic dissemination to both lymph nodes and distant organs. The high anti-lymphangiogenic potency of AD0157 was further supported by its inhibitory activity at low micromolar range in two in vivo pathological models and in two ex vivo assays. In addition, AD0157 inhibited lymphatic endothelial cell proliferation, migration and invasion, cellular sprouting, and tube formation. Mechanistically, this compound induced apoptosis in lymphatic endothelial cells and decreased VEGFR-3/-2, ERK1/2, and Akt phosphorylations.ConclusionsThese findings demonstrate the suitability of AD0157 to suppress tumor-associated lymphangiogenesis. Beyond discovering a new potent anti-lymphangiogenic drug that is worth considering in future clinical settings, our study supports the interest of designing anti-lymphangiogenic therapies to avoid distant metastatic processes.