Choroidal Neovascularization Formation Research Articles

Role of Activating Transcription Factor 4 in Murine Choroidal Neovascularization Model.

Neovascular age-related macular degeneration (nAMD) featuring choroidal neovascularization (CNV) is the principal cause of irreversible blindness in elderly people in the world. Integrated stress response (ISR) is one of the intracellular signals to be adapted to various stress conditions including endoplasmic reticulum (ER) stress. ISR signaling results in the upregulation of activating transcription factor 4 (ATF4), which is a mediator of ISR. Although recent studies have suggested ISR contributes to the progression of some age-related disorders, the effects of ATF4 on the development of CNV remain unclear. Here, we performed a murine model of laser-induced CNV and found that ATF4 was highly expressed in endothelial cells of the blood vessels of the CNV lesion site. Exposure to integrated stress inhibitor (ISRIB) reduced CNV formation, vascular leakage, and the upregulation of vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE)-choroid-sclera complex. In human retinal microvascular endothelial cells (HRMECs), ISRIB reduced the level of ATF4 and VEGF induced by an ER stress inducer, thapsigargin, and recombinant human VEGF. Moreover, ISRIB decreased the VEGF-induced cell proliferation and migration of HRMECs. Collectively, our findings showed that pro-angiogenic effects of ATF4 in endothelial cells may be a potentially therapeutic target for patients with nAMD.

Subretinal Saline Protects the Neuroretina From Thermic Damage During Laser Induction of Experimental Choroidal Neovascularization in Pigs.

PurposeThe purpose of this study was to develop a porcine model for photocoagulation induced choroidal neovascularization (CNV) with high success rate and minimal thermic damage to the neuroretina.MethodsExperimental CNV was induced by laser photocoagulation in both eyes of 16 domestic pigs. In the left eyes, photocoagulation was preceded by subretinal injection of saline to protect the neuroretina from thermic damage, whereas the right eyes were treated with photocoagulation only. The development of the CNV after 3, 7, 14, 28, and 42 days was evaluated by optical coherence tomography (OCT) scanning, fluorescein angiography, and OCT angiography, and by histology after enucleation.ResultsFrom day 7 after the photocoagulation, OCT showed subretinal density in all lesions of 14 alive animals, and either fluorescein or OCT angiography confirmed CNV formation in 11 of 14 of the eyes that had received photocoagulation alone and those in which photocoagulation had been preceded by subretinal injection of saline. In all cases pretreated with subretinal saline, the neuroretina was protected from immediate thermic damage. The formation of CNVs were confirmed by histology. For both groups, the largest lesions were observed within 14 days after photocoagulation.ConclusionsInjection of subretinal saline can protect the retina from thermic damage induced by retinal photocoagulation without reducing the success rate in producing experimental CNV. The effect of interventional studies aimed at reducing photocoagulation induced experimental CNV in pigs can be evaluated within 2 weeks after photocoagulation.Translational RelevanceThis model provides a fundament to develop and evaluate novel treatment methods for neovascular retinal diseases.

Active Rap1-mediated inhibition of choroidal neovascularization requires interactions with IQGAP1 in choroidal endothelial cells.

Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness. The pathophysiology involves activation of choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelial (RPE) monolayer and form choroidal neovascularization (CNV) in the neural retina. The multidomain GTPase binding protein, IQGAP1, binds active Rac1 and sustains activation of CECs, thereby enabling migration associated with vision-threatening CNV. IQGAP1 also binds the GTPase, Rap1, which when activated reduces Rac1 activation in CECs and CNV. In this study, we tested the hypothesis that active Rap1 binding to IQGAP1 is necessary and sufficient to reduce Rac1 activation in CECs, and CNV. We found that pharmacologic activation of Rap1 or adenoviral transduction of constitutively active Rap1a reduced VEGF-mediated Rac1 activation, migration, and tube formation in CECs. Following pharmacologic activation of Rap1, VEGF-mediated Rac1 activation was reduced in CECs transfected with an IQGAP1 construct that increased active Rap1-IQGAP1 binding but not in CECs transfected with an IQGAP1 construct lacking the Rap1 binding domain. Specific knockout of IQGAP1 in endothelial cells reduced laser-induced CNV and Rac1 activation in CNV lesions, but pharmacologic activation of Rap1 did not further reduce CNV compared to littermate controls. Taken together, our findings provide evidence that active Rap1 binding to the IQ domain of IQGAP1 is sufficient to interfere with active Rac1-mediated CEC activation and CNV formation.

Expression of purinergic receptors on microglia in the animal model of choroidal neovascularisation

To investigate the effect of P2 receptor on microglia and its inhibitor PPADS on choroidal neovascularization. Forty CX3CR1GFP/+ mice were randomly divided into 8 groups. In addition to the normal group, the rest of groups were receiving laser treatment. The retina and choroid from the second, third, fourth and fifth group of mice were taken in the 1, 4, 7, 14 days after laser treatment. The mice in the sixth and seventh group received intravitreal injection of 2 µl PPADS or PBS respectively immediately after laser treatment. The mice in the eighth group received topical application of PPADS once per day of three days. The mice in sixth, seventh and eighth group received AF and FFA examination on the fourth day after laser treatment. Immunofluorescence histochemical staining and real-time quantitative PCR were used to evaluate P2 expression and its effect on choroidal neovascularization. After laser treatment, activated microglia can express P2 receptors (P2X4, P2X7, P2Y2 and P2Y12). The expression of P2 increased on the first day after laser damage, peaked on the fourth day (tP2X4 = 6.05, tP2X7 = 2.95, tP2Y2 = 3.67, tP2Y12 = 5.98, all P < 0.01), and then decreased. After PPADS inhibition, compared with the PBS injection group, the mRNA of P2X4, P2X7, P2Y2 and P2Y12 were decreased significantly in the PPADS injection group (tP2X4 = 5.54, tP2X7 = 9.82, tP2Y2 = 3.86, tP2Y12 = 7.91, all P < 0.01) and the PPADS topical application group (tP2X4 = 3.24, tP2X7 = 5.89, tP2Y2 = 6.75, tP2Y12 = 4.97, all P < 0.01). Compared with the PBS injection group, not only the activity of microglia cells but also the leakage of CNV decreased significantly (P < 0.01) in the PPADS injection group and the PPADS topical application group. But between two PPADS groups, the leakage of CNV had no difference (P = 0.864). After laser induced CNV, activated microglia can express P2 receptors. The P2 receptor inhibitor, PPADS, can significantly affect the function of microglia and inhibit the formation of choroidal neovascularization.

The P300/XBP1s/Herpud1 axis promotes macrophage M2 polarization and the development of choroidal neovascularization.

Neovascular age‐related macular degeneration (AMD), which is characterized by choroidal neovascularization (CNV), leads to vision loss. M2 macrophages produce vascular endothelial growth factor (VEGF), which aggravates CNV formation. The histone acetyltransferase p300 enhances the stability of spliced X‐box binding protein 1 (XBP1s) and promotes the transcriptional activity of the XBP1s target gene homocysteine inducible endoplasmic reticulum protein with ubiquitin‐like domain 1 (Herpud1). Herpud1 promotes the M2 polarization of macrophages. This study aimed to explore the roles of the p300/XBP1s/Herpud1 axis in the polarization of macrophages and the pathogenesis of CNV. Hypoxia‐induced p300 interacted with XBP1s to acetylate XBP1s in RAW264.7 cells. Additionally, hypoxia‐induced p300 enhanced the XBP‐1s‐mediated unfolded protein response (UPR), alleviated the proteasome‐dependent degradation of XBP1s and enhanced the transcriptional activity of XBP1s for Herpud1. The hypoxia‐induced p300/XBP1s/Herpud1 axis facilitated RAW264.7 cell M2 polarization. Knockdown of the p300/XBP1s/Herpud1 axis in RAW264.7 cells inhibited the proliferation, migration and tube formation of mouse choroidal endothelial cells (MCECs). The p300/XBP1s/Herpud1 axis increased in infiltrating M2‐type macrophages in mouse laser‐induced CNV lesions. Blockade of the p300/XBP1s/Herpud1 axis inhibited macrophage M2 polarization and alleviated CNV lesions. Our study demonstrated that the p300/XBP1s/Herpud1 axis in infiltrating macrophages increased the M2 polarization of macrophages and the development of CNV.

Guanabenz and Clonidine, α2-Adrenergic Receptor Agonists, Inhibit Choroidal Neovascularization.

Neovascular age-related macular degeneration (AMD) with choroidal neovascularization (CNV) is a leading cause of blindness in elderly people. Anti-vascular endothelial growth factor (anti-VEGF)-drugs are used to treat AMD patients; however, some patients are resistant to these therapies. The purpose of this study was to investigate the anti-angiogenic effects of α2-adrenergic agonists, including guanabenz and clonidine. We evaluated the anti-angiogenic effects of α2-adrenergic agonists in human retinal microvascular endothelial cells (HRMECs). A proliferation assay was conducted, and the migration ratio was evaluated. In a laser-induced CNV model, guanabenz and clonidine were delivered via intraperitoneal injection or implantation of an osmotic pump device. Fourteen days following CNV induction, CNV lesion size and fundus fluorescein angiography (FFA) were evaluated. Guanabenz and clonidine inhibited VEGF-induced retinal endothelial cell growth and migration. In the CNV model mice, CNV lesion sizes were reduced by intraperitoneal administration of guanabenz or clonidine. Data, including body weight, systolic blood pressure, and heart rate showed that guanabenz (0.5 and 2.0 mg/kg/day) had little effect on these parameters; conversely, a high dose of clonidine (1.0 mg/kg/day) did affect these parameters. Additionally, clonidine did not affect CNV size, but continuous administration of guanabenz attenuated both CNV size and leakage from neovessels. Our study suggests a key role for α2-adrenergic receptors during CNV formation. Therefore, we suggest that α2-adrenergic receptor agonists may represent novel therapeutic drugs for patients with neovascular AMD.

Intravitreal injection of anti-miRs against miR-142-3p reduces angiogenesis and microglia activation in a mouse model of laser-induced choroidal neovascularization.

Age-related macular degeneration (AMD) is a worldwide leading cause of blindness affecting individuals over 50 years old. The most aggressive form, wet AMD, is characterized by choroidal neovascularization (CNV) and inflammation involving microglia recruitment. By using a laser-induced CNV mouse model, we provide evidence for a key role played by miR-142-3p during CNV formation. MiR-142-3p was overexpressed in murine CNV lesions and its pharmacological inhibition decreased vascular and microglia densities by 46% and 30%, respectively. Consistently, miR-142-3p overexpression with mimics resulted in an increase of 136% and 126% of blood vessels and microglia recruitment. Interestingly, miR-142-3p expression was linked to the activation state of mouse microglia cells as determined by morphological analysis (cell solidity) through a computational method. In vitro, miR-142-3p overexpression in human microglia cells (HMC3) modulated microglia activation, as shown by CD68 levels. Interestingly, miR142-3p modulation also regulated the production of VEGF-A, the main pro-angiogenic factor. Together, these data strongly support the unprecedented importance of miR-142-3p-dependent vascular-inflammation axis during CNV progression, through microglia activation.

Suppression of choroidal neovascularization by silencing of long non-coding RNA IPW.

Long noncoding RNAs (lncRNAs) have emerged as the key regulators in the pathogenesis of human disorders. This study aimed to investigate the role of lncRNA-IPW in the progression of choroidal neovascularization (CNV) and the underlying molecular mechanism. IPW was significantly up-regulated in the choroidal tissues of laser-induced CNV mice and in the endothelial cells in response to hypoxic stress. IPW silencing led to reduced formation of CNV in laser-induced CNV model and ex vivo choroidal sprouting model, which could achieve similar therapeutic effects of anti-VEGF on CNV formation. Silencing or transgenic overexpression of IPW could alter endothelial cell viability, proliferation, migration, and tube formation ability in vitro. Mechanistically, IPW silencing led to increased expression of miR-370. Increased miR-370 could mimic the effects of IPW silencing on CNV formation and endothelial angiogenic phenotypes in vivo and in vitro. This study suggests that IPW silencing is a promising strategy for the treatment of neovascular ocular diseases.

CSF1/CSF1R-mediated Crosstalk Between Choroidal Vascular Endothelial Cells and Macrophages Promotes Choroidal Neovascularization.

PurposeThis study examined the role of the CSF1/CSF1Raxis in the crosstalk between choroidal vascular endothelial cells (CVECs) and macrophages during the formation of choroidal neovascularization (CNV).MethodsQuantitative reverse transcriptase (QRT)-PCR, Western blot and ELISA measured the production and release of CSF1 from human choroidal vascular endothelial cells (HCVECs) under hypoxic conditions. Western blot detected CSF1 released from HCVECs under hypoxic conditions that activated the PI3K/AKT/FOXO1 axis in human macrophages via binding to CSF1R. Transwell migration assay, qRT-PCR, and Western blot detected the effect of CSF1 released from HCVECs on macrophage migration and M2 polarization via the CSF1R/PI3K/AKT/FOXO1 pathway. Incorporation of 5-ethynyl-20-deoxyuridine, transwell migration, and tube formation assays detected the effects of CSF1/CSF1R on the behaviors of HCVECs. Fundus fluorescein angiography (FFA), indocyanine green angiography (ICGA), and immunofluorescence detected the effect of blockade of CSF1/CSF1R on mouse laser-induced CNV. Color fundus photograph, ICGA, and FFA detected CNV lesions in neovascular AMD (nAMD) patients. ELISA detected CSF1 and CSF1R in the aqueous humor of age-related cataract and nAMD patients.ResultsCSF1 released from HCVECs under hypoxic conditions activated the PI3K/AKT/FOXO1 axis in human macrophages via binding to CSF1R, promoting macrophage migration and M2 polarization via up-regulation of the CSF1R/PI3K/AKT/FOXO1 pathway. Human macrophages promoted the proliferation, migration, and tube formation of HCVECs in a CSF1/CSFR1-dependent manner under hypoxic conditions. CSF1/CSF1R blockade ameliorated the formation of mouse laser-induced CNV. CSF1 and CSF1R were increased in the aqueous humor of nAMD patients.ConclusionsOur results affirmed the crucial role of CSF1/CSF1R in boosting the formation of CNV and offered potential molecular targets for the treatment of nAMD.

The Antiangiogenic Effect of Sanguinarine Chloride on Experimental Choroidal Neovacularization in Mice via Inhibiting Vascular Endothelial Growth Factor.

Background: The purpose of this study is to investigate the antiangiogenic effect of Sanguinarine chloride (SC) on models of age-related macular degeneration (AMD) both in vivo and in vitro. Methods: Choroidal neovascularization (CNV) was conducted by laser photocoagulation in C57BL6/J mice. SC (2.5 μM, 2 μl/eye) was intravitreally injected immediately after laser injury. The control group received an equal amount of PBS. 7 days after laser injury, CNV severity was evaluated using fundus fluorescein angiography, hematoxylin and eosin (H&E) staining, and choroid flat-mount staining. Vascular endothelial growth factor (VEGF) expression in the retina/choroid complex was measured by western blot analysis and ELISA kit. In vitro, human retinal microvascular endothelial cells (HRMECs) were used to investigate the effects of SC on cell tube formation, migration, and cytotoxicity. The expression of VEGF-induced expression of extracellular signal-regulated kinase (ERK)1/2, protein kinase B (AKT), mitogen-activated protein kinases (p38-MAPK) in vitro and laser induced VEGF expression in vivo were also analyzed. Results: SC (≤2.5 μM) was safe both in vitro and in vivo. Intravitreal injection of SC restrained the formation of laser induced CNV in mice and decreased VEGF expression in the laser site of the retina/choroid complex. In vitro, SC inhibited VEGF-induced tube formation and endothelial cell migration by decreasing the phosphorylation of AKT, ERK1/2, and p38-MAPK in HRMECs. Conclusions: SC could inhibit laser-induced CNV formation via down-regulating VEGF expression and restrain the VEGF-induced tube formation and endothelial migration. Therefore, SC could be a potential candidate for the treatment of wet AMD.

Pirfenidone Alleviates Choroidal Neovascular Fibrosis through TGF-β/Smad Signaling Pathway.

Background. Transforming growth factor-β (TGF-β) plays a major role in CNV. However, the mechanism is unclear. This study investigates the effect of Pirfenidone (PFD) on TGF-β/Smad signaling pathway on the development of choroidal neovascular fibrosis in choroidal neovascularization (CNV) mouse model. C57BL/6J male mice (aged from 6 to 8 weeks) received intravitreal injections of phosphate-buffered saline (PBS)/PFD solution on 14 days after laser injury. Mice were anesthetized by intraperitoneal injection of 4% pentobarbital (0.05 mg/g body weight). Optical Coherence Tomography (OCT), Fundus Fluorescein angiography (FFA), and hematoxylin-eosin (HE) were used to assess CNV formation. The fibrosis area was monitored by staining the collagen type I (Col-I). Western blotting was used to analyze the expression of TGF-β2, Smad 2/3, phosphorylated Smad 2/3 (p-Smad 2/3), and α-smooth muscle actin (α-SMA). Terminal deoxynucleotidy1 transferase dUTP nick-end labelling (TUNEL) assay was performed on cryosections of mouse eyes to detect apoptosis. Our data showed PFD inhibited areas of fibrosis during day 21 to day 28. We also found that the levels of TGF-β2 protein expressions increasingly reached the peak till the 3rd week during the CNV development. The protein levels of Smad 2/3, p-Smad 2/3, and α-SMA also increased significantly in CNV mice, but this response was profoundly suppressed by the TGF-β inhibitor PFD. The results of this study suggest that TGF-β2 represents a target to prevent or treat choroidal neovascular fibrosis, and PFD may provide an alternative to traditional methods for Wet Age-related macular degeneration (wAMD) treatment.

The HIF-1α/p53/miRNA-34a/Klotho axis in retinal pigment epithelial cells promotes subretinal fibrosis and exacerbates choroidal neovascularization.

Wet age‐related macular degeneration (wAMD), characterized by choroidal neovascularization (CNV), is a leading cause of irreversible vision loss among elderly people in developed nations. Subretinal fibrosis, mediated by epithelial‐mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells, leads to unsuccessful anti‐vascular endothelial growth factor (VEGF) agent treatments in CNV patients. Under hypoxic conditions, hypoxia‐inducible factor‐1α (HIF‐1α) increases the stability and activation of p53, which activates microRNA‐34a (miRNA‐34a) transcription to promote fibrosis. Additionally, Klotho is a target gene of miRNA‐34a that inhibits fibrosis. This study aimed to explore the role of the HIF‐1α/p53/miRNA‐34a/Klotho axis in subretinal fibrosis and CNV. Hypoxia‐induced HIF‐1α promoted p53 stability, phosphorylation and nuclear translocation in ARPE‐19 cells (a human RPE cell line). HIF‐1α‐dependent p53 activation up‐regulated miRNA‐34a expression in ARPE‐19 cells following hypoxia. Moreover, hypoxia‐induced p53‐dependent miRNA‐34a inhibited the expression of Klotho in ARPE‐19 cells. Additionally, the HIF‐1α/p53/miRNA‐34a/Klotho axis facilitated hypoxia‐induced EMT in ARPE‐19 cells. In vivo, blockade of the HIF‐1α/p53/miRNA‐34a/Klotho axis alleviated the formation of mouse laser‐induced CNV and subretinal fibrosis. In short, the HIF‐1α/p53/miRNA‐34a/Klotho axis in RPE cells promoted subretinal fibrosis, thus aggravating the formation of CNV.

Vitelliform Distrofiye Sekonder Koroidal Neovaskülarizasyon: Optik Koherens Tomografi Anjiyografi ve Fluoresein Anjiyografi Bulguları

This case series aims to interpret the findings of optical coherence tomography angiography (OCTA) and fluorescein angiography (FA) in detecting choroidal neovascularization (CNV) in juvenile Best vitelliform macular dystrophy (BVMD) and adult-onset foveomacular vitelliform dystrophy (AOFVD). Two patients with BVMD and one patient with AOFVD were evaluated, and color fundus photography, spectral-domain optical coherence tomography, FA, and OCTA were performed. In 4 of 6 eyes, OCTA revealed neovascular formations in the outer retina and choriocapillaris. It is an effective, non-invasive method for showing CNV formation using OCTA in BVMD and AOFVD, and it has aspects that are superior to FA.

Laser-Induced Choroidal Neovascularization in Rats.

The laser-induced choroidal neovascularization (CNV) model has been widely used for research on wet age-related macular degeneration (wet-AMD) and other ocular neovascular diseases. In this model, the Bruch membrane is perforated by laser injury, resulting in neovascularization formed from the choroidal capillaries. It has become a standard method to evaluate the effect of different treatments on CNV progression in preclinical studies. This protocol can be used in various species, including rat, mouse, pig, and monkey. The rodent laser-induced CNV model is the most commonly used because of the advantages in both cost- and time-efficiency. It takes only 10-15min to complete the whole laser procedure after adequate training and practicing the technique. Peak CNV formation occurs at approximately 2weeks after laser application. The entire protocol may require up to 3weeks to complete the treatment, fundus image acquisition, and tissue collection for histologic analysis. This chapter describes the detailed procedures, protocols, and useful notes on how to induce CNV by laser.

Myeloid Lineage Contributes to Pathological Choroidal Neovascularization Formation Via SOCS3

Myeloid Lineage Contributes to Pathological Choroidal Neovascularization Formation Via SOCS3

Morphologic Features of Myopic Choroidal Neovascularization in Pathologic Myopia on Swept-Source Optical Coherence Tomography.

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.

Associations between the Complement System and Choroidal Neovascularization in Wet Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness affecting the elderly in the Western world. The most severe form of AMD, wet AMD (wAMD), is characterized by choroidal neovascularization (CNV) and acute vision loss. The current treatment for these patients comprises monthly intravitreal injections of anti-vascular endothelial growth factor (VEGF) antibodies, but this treatment is expensive, uncomfortable for the patient, and only effective in some individuals. AMD is a complex disease that has strong associations with the complement system. All three initiating complement pathways may be relevant in CNV formation, but most evidence indicates a major role for the alternative pathway (AP) and for the terminal complement complex, as well as certain complement peptides generated upon complement activation. Since the complement system is associated with AMD and CNV, a complement inhibitor may be a therapeutic option for patients with wAMD. The aim of this review is to (i) reflect on the possible complement targets in the context of wAMD pathology, (ii) investigate the results of prior clinical trials with complement inhibitors for wAMD patients, and (iii) outline important considerations when developing a future strategy for the treatment of wAMD.

Cytokine Profiles and the Effect of Intravitreal Aflibercept Treatment on Experimental Choroidal Neovascularization

Purpose: The purpose of our study was to investigate the profiles of inflammatory cytokines and the macrophage polarization gene in a choroidal neovascularization (CNV) mouse model before and after intravitreal aflibercept treatment. Methods: The CNV mouse model was conducted by laser photocoagulation. A total of 58 cytokines were measured by the multiplex mouse cytokine antibody array. The macrophage polarization genes were tested by reverse transcription polymerase chain reaction. The relationship between the cytokines and the CNV lesion area was analyzed by correlation. Results: MIP-1a on day 3 after laser photocoagulation, MCP-5 and Fas-L on day 7, and IL-15 and IL-7 on day 14 were significantly upregulated (p < 0.001, fold change >10.0). After the intravitreal aflibercept treatment, GM-CSF and MCP-1 on day 3 and TIMP-1 on days 7 and 14 were the most significantly upregulated cytokines (p < 0.001, fold change >10.0). MIP-1 on day 3, IL-13 and Fas-L on day 7, and Fas-L on day 14 were the most significantly downregulated cytokines after intravitreal aflibercept treatment (p < 0.001, fold change >5.0). M2 polarization and VEGFA genes were significantly increased in the CNV formation, whereas aflibercept suppressed M2 polarization and VEGFA genes. IL-7 was negatively related to the CNV lesion area on day 14 after intravitreal aflibercept treatment (r = −0.938, p = 0.006). Conclusion: The inflammatory cytokines and the M1/M2 macrophage genes significantly changed in the CNV mouse model. This result suggests that inflammatory cytokines and macrophages play a critical role in the physiopathology of CNV.

Fruquintinib inhibits VEGF/VEGFR2 axis of choroidal endothelial cells and M1-type macrophages to protect against mouse laser-induced choroidal neovascularization

Wet age-related macular degeneration, which is characterized by choroidal neovascularization (CNV) and induces obvious vision loss. Vascular endothelial growth factor (VEGF) family member VEGF-A (also named as VEGF) and its receptor VEGFR2 contribute to the pathogenesis of CNV. Choroidal endothelial cells (CECs) secret C–C motif chemokine ligand 2 (CCL2), which attracts macrophages to CNV lesion and promotes macrophage M1 polarization. Accordingly, infiltrating macrophages secret inflammatory cytokines to promote CNV. In vivo, intravitreal injection of fruquintinib (HMPL-013), an antitumor neovascularization drug, alleviated mouse CNV formation without obvious ocular toxicity. Meanwhile, HMPL-013 inhibited VEGF/VEGFR2 binding in CECs and macrophages, as well as macrophage M1 polarization. In vitro, noncontact coculture of human choroidal vascular endothelial cells (HCVECs) and macrophages under hypoxia conditions was established. HMPL-013 downregulated VEGF/VEGFR2/phosphoinositide-3-kinase/protein kinase B (AKT)/nuclear factor kappa B pathway and CCL2 secretion in HCVECs, as well as VEGF/VEGFR2-induced macrophage M1 polarization under hypoxia condition. In addition, HMPL-013 inhibited HCEVC derived CCL2-induced macrophage migration and M1 polarization, along with macrophage M1 polarization-induced HCVECs proliferation, migration, and tube formation. Altogether, HMPL-013 alleviated CNV formation might via breaking detrimental cross talk between CECs and macrophages.

Pyruvate dehydrogenase kinase/lactate axis: a therapeutic target for neovascular age-related macular degeneration identified by metabolomics.

Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in aging populations. Here, we applied metabolomics to human sera of patients with nAMD during an active (exudative) phase of the pathology and found higher lactate levels and a shift in the lipoprotein profile (increased VLDL-LDL/HDL ratio). Similar metabolomics changes were detected in the sera of mice subjected to laser-induced choroidal neovascularization (CNV). In this experimental model, we provide evidence for two sites of lactate production: first, a local one in the injured eye, and second a systemic site associated with the recruitment of bone marrow-derived inflammatory cells. Mechanistically, lactate promotes the angiogenic response and M2-like macrophage accumulation in the eyes. The therapeutic potential of our findings is demonstrated by the pharmacological control of lactate levels through pyruvate dehydrogenase kinase (PDK) inhibition by dichloroacetic acid (DCA). Mice treated with DCA exhibited normalized lactate levels and lipoprotein profiles, and inhibited CNV formation. Collectively, our findings implicate the key role of the PDK/lactate axis in AMD pathogenesis and reveal that the regulation of PDK activity has potential therapeutic value in this ocular disease. The results indicate that the lipoprotein profile is a traceable pattern that is worth considering for patient follow-up. KEY MESSAGES: Lactate and lipoprotein profile are associated with the active phase of AMD and CNV development. Lactate is a relevant and functional metabolite correlated with AMD progression. Modulating lactate through pyruvate dehydrogenase kinase led to a decrease of CNV progression. Pyruvate dehydrogenase kinase is a new therapeutic target for neovascular AMD.