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Taher M, Nakao S, Zandi S, Melhorn MI, Hayes KC, Hafezi-Moghadam A.
Phenotypic transformation of intimal and adventitial lymphatics in atherosclerosis: a regulatory role for soluble VEGF receptor 2. FASEB J 2016;30(7):2490-9.
AbstractThe role of lymphatics in atherosclerosis is not yet understood. Here, we investigate lymphatic growth dynamics and marker expression in atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. The prolymphangiogenic growth factor, VEGF-C, was elevated in atherosclerotic aortic walls. Despite increased VEGF-C, we found that adventitial lymphatics regress during the course of formation of atherosclerosis (P < 0.01). Similar to lymphatic regression, the number of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1(+)) macrophages decreased in the aortic adventitia of apoE(-/-) mice with atherosclerosis (P < 0.01). Intimal lymphatics in the atherosclerotic lesions exhibited an atypical phenotype, with the expression of podoplanin and VEGF receptor 3 (VEGFR-3) but not of LYVE-1 and prospero homeobox protein 1. In the aortas of atherosclerotic animals, we found markedly increased soluble VEGFR-2. We hypothesized that the elevated soluble VEGFR-2 that was found in the aortas of apoE(-/-) mice with atherosclerosis binds to and diminishes the activity of VEGF-C. This trapping mechanism explains, despite increased VEGF-C in the atherosclerotic aortas, how adventitial lymphatics regress. Lymphatic regression impedes the drainage of lipids, growth factors, inflammatory cytokines, and immune cells. Insufficient lymphatic drainage could thus exacerbate atherosclerosis formation. Our study contributes new insights to previously unknown dynamic changes of adventitial lymphatics. Targeting soluble VEGFR-2 in atherosclerosis may provide a new strategy for the liberation of endogenous VEGF-C and the prevention of lymphatic regression.-Taher, M., Nakao, S., Zandi, S., Melhorn, M. I., Hayes, K. C., Hafezi-Moghadam, A. Phenotypic transformation of intimal and adventitial lymphatics in atherosclerosis: a regulatory role for soluble VEGF receptor 2.
Takeuchi K, Yanai R, Kumase F, Morizane Y, Suzuki J, Kayama M, Brodowska K, Nakazawa M, Miller JW, Connor KM, Vavvas DG.
EGF-like-domain-7 is required for VEGF-induced Akt/ERK activation and vascular tube formation in an ex vivo angiogenesis assay. PLoS One 2014;9(3):e91849.
AbstractEGFL7 is a secreted angiogenic factor, which in contrast to the well-known secreted angiogenic molecules VEGF and FGF-2, is almost exclusively expressed by endothelial cells and may act in an autocrine fashion. Prior studies have shown EGFL7 to mediate its angiogenic effects by interfering with the Notch pathway and/or via the intronic miR126. Less is known about its effects on VEGF signaling. We wanted to investigate the role of epidermal growth factor-like domain 7 (EGFL7) in VEGF-driven angiogenesis using an ex vivo Matrigel-embedded mouse eye cup assay and siRNA mediated knockdown of EGFL7 by siRNA. Our results suggested that VEGF-induced vascular tube formation was significantly impaired after siRNA downregulation of EGFL7. In addition, knockdown of EGFL7 suppressed VEGF upregulation of phospho-Akt and phospho-Erk(1/2) in endothelial cells, but did not alter VEGFR phosphorylation and neuropilin-1 protein expression or miR126 expression. Thus, in conclusion, EGFL7 is required for VEGF upregulation of the Akt/Erk (1/2) pathway during angiogenesis, and may represent a new therapeutic target in diseases of pathological neovascularization.
Tomita Y, Cakir B, Liu C-H, Fu Z, Huang S, Cho SS, Britton WR, Sun Y, Puder M, Hellström A, Talukdar S, Smith LEH.
Free fatty acid receptor 4 activation protects against choroidal neovascularization in mice. Angiogenesis 2020;23(3):385-394.
AbstractTo examine whether free fatty acid receptor 4 (FFAR4) activation can protect against choroidal neovascularization (CNV), which is a common cause of blindness, and to elucidate the mechanism underlying the inhibition, we used the mouse model of laser-induced CNV to mimic angiogenic aspects of age-related macular degeneration (AMD). Laser-induced CNV was compared between groups treated with an FFAR4 agonist or vehicle, and between FFAR4 wild-type (Ffar4) and knock out (Ffar4) mice on a C57BL/6J/6N background. The ex vivo choroid-sprouting assay, including primary retinal pigment epithelium (RPE) and choroid, without retina was used to investigate whether FFAR4 affects choroidal angiogenesis. Western blotting for pNF-ĸB/NF-ĸB and qRT-PCR for Il-6, Il-1β, Tnf-α, Vegf, and Nf-ĸb were used to examine the influence of FFAR4 on inflammation, known to influence CNV. RPE isolated from Ffar4 and Ffar4 mice were used to assess RPE contribution to inflammation. The FFAR4 agonist suppressed laser-induced CNV in C57BL/6J mice, and CNV increased in Ffar4 compared to Ffar4 mice. We showed that the FFAR4 agonist acted through the FFAR4 receptor. The FFAR4 agonist suppressed mRNA expression of inflammation markers (Il-6, Il-1β) via the NF-ĸB pathway in the retina, choroid, RPE complex. The FFAR4 agonist suppressed neovascularization in the choroid-sprouting ex vivo assay and FFAR4 deficiency exacerbated sprouting. Inflammation markers were increased in primary RPE cells of Ffar4 mice compared with Ffar4 RPE. In this mouse model, the FFAR4 agonist suppressed CNV, suggesting FFAR4 to be a new molecular target to reduce pathological angiogenesis in CNV.
Tomita Y, Shao Z, Cakir B, Kotoda Y, Fu Z, Smith LEH.
An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis. J Vis Exp 2020;(162)
AbstractPathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.
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Uemura A, Fruttiger M, D'Amore PA, De Falco S, Joussen AM, Sennlaub F, Brunck LR, Johnson KT, Lambrou GN, Rittenhouse KD, Langmann T.
VEGFR1 signaling in retinal angiogenesis and microinflammation. Prog Retin Eye Res 2021;84:100954.
AbstractFive vascular endothelial growth factor receptor (VEGFR) ligands (VEGF-A, -B, -C, -D, and placental growth factor [PlGF]) constitute the VEGF family. VEGF-A binds VEGF receptors 1 and 2 (VEGFR1/2), whereas VEGF-B and PlGF only bind VEGFR1. Although much research has been conducted on VEGFR2 to elucidate its key role in retinal diseases, recent efforts have shown the importance and involvement of VEGFR1 and its family of ligands in angiogenesis, vascular permeability, and microinflammatory cascades within the retina. Expression of VEGFR1 depends on the microenvironment, is differentially regulated under hypoxic and inflammatory conditions, and it has been detected in retinal and choroidal endothelial cells, pericytes, retinal and choroidal mononuclear phagocytes (including microglia), Müller cells, photoreceptor cells, and the retinal pigment epithelium. Whilst the VEGF-A decoy function of VEGFR1 is well established, consequences of its direct signaling are less clear. VEGFR1 activation can affect vascular permeability and induce macrophage and microglia production of proinflammatory and proangiogenic mediators. However the ability of the VEGFR1 ligands (VEGF-A, PlGF, and VEGF-B) to compete against each other for receptor binding and to heterodimerize complicates our understanding of the relative contribution of VEGFR1 signaling alone toward the pathologic processes seen in diabetic retinopathy, retinal vascular occlusions, retinopathy of prematurity, and age-related macular degeneration. Clinically, anti-VEGF drugs have proven transformational in these pathologies and their impact on modulation of VEGFR1 signaling is still an opportunity-rich field for further research.
Ueta T, Ishihara K, Notomi S, Lee J-J, Maidana DE, Efstathiou NE, Murakami Y, Hasegawa E, Azuma K, Toyono T, Paschalis EI, Aihara M, Miller JW, Vavvas DG.
RIP1 kinase mediates angiogenesis by modulating macrophages in experimental neovascularization. Proc Natl Acad Sci U S A 2019;116(47):23705-23713.
AbstractInflammation 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 RIP1 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.
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Wang JC, Miller JB.
For Mass Eye and Ear Special Issue: Optical Coherence Tomography Angiography: Review of Current Technical Aspects and Applications in Chorioretinal Disease. Semin Ophthalmol 2019;:1-7.
AbstractOptical coherence tomography angiography (OCT-A) has enabled fast, non-invasive, high-resolution visualization of vasculature within the eye. In the past few years, it has become increasingly utilized for a range of disorders including age-related macular degeneration, diabetic retinopathy, retinal vein occlusions, and uveitis among others. This article reviews technical aspects of OCT-A, its applications in chorioretinal disease, and known limitations of the technology.
Wang Z, Yemanyi F, Blomfield AK, Bora K, Huang S, Liu C-H, Britton WR, Cho SS, Tomita Y, Fu Z, Ma J-X, Li W-H, Chen J.
Amino acid transporter SLC38A5 regulates developmental and pathological retinal angiogenesis. Elife 2022;11
AbstractAmino acid (AA) metabolism in vascular endothelium is important for sprouting angiogenesis. SLC38A5 (solute carrier family 38 member 5), an AA transporter, shuttles neutral AAs across cell membrane, including glutamine, which may serve as metabolic fuel for proliferating endothelial cells (ECs) to promote angiogenesis. Here, we found that Slc38a5 is highly enriched in normal retinal vascular endothelium, and more specifically, in pathological sprouting neovessels. Slc38a5 is suppressed in retinal blood vessels from Lrp5-/- and Ndpy/- mice, both genetic models of defective retinal vascular development with Wnt signaling mutations. Additionally, Slc38a5 transcription is regulated by Wnt/β-catenin signaling. Genetic deficiency of Slc38a5 in mice substantially delays retinal vascular development and suppresses pathological neovascularization in oxygen-induced retinopathy modeling ischemic proliferative retinopathies. Inhibition of SLC38A5 in human retinal vascular ECs impairs EC proliferation and angiogenic function, suppresses glutamine uptake, and dampens vascular endothelial growth factor receptor 2. Together these findings suggest that SLC38A5 is a new metabolic regulator of retinal angiogenesis by controlling AA nutrient uptake and homeostasis in ECs.
Wang Y, Tadjuidje E, Pandey RN, Stefater JA, Smith LEH, Lang RA, Hegde RS.
The Eyes Absent Proteins in Developmental and Pathological Angiogenesis. Am J Pathol 2016;186(3):568-78.
AbstractManagement of neoangiogenesis remains a high-value therapeutic goal. A recently uncovered association between the DNA damage repair pathway and pathological angiogenesis could open previously unexplored possibilities for intervention. An attractive and novel target is the Eyes absent (EYA) tyrosine phosphatase, which plays a critical role in the repair versus apoptosis decision after DNA damage. This study examines the role of EYA in the postnatal development of the retinal vasculature and under conditions of ischemia-reperfusion encountered in proliferative retinopathies. We find that the ability of the EYA proteins to promote endothelial cell (EC) migration contributes to a delay in postnatal development of the retinal vasculature when Eya3 is deleted specifically in ECs. By using genetic and chemical biology tools, we show that EYA contributes to pathological angiogenesis in a model of oxygen-induced retinopathy. Both in vivo and in vitro, loss of EYA tyrosine phosphatase activity leads to defective assembly of γ-H2AX foci and thus to DNA damage repair in ECs under oxidative stress. These data reveal the potential utility of EYA tyrosine phosphatase inhibitors as therapeutic agents in inhibiting pathological neovascularization with a range of clinical applications.
Wang J, Wang X, Gao Y, Lin Z, Chen J, Gigantelli J, Shapiro JI, Xie Z, Pierre SV.
Stress Signal Regulation by Na/K-ATPase As a New Approach to Promote Physiological Revascularization in a Mouse Model of Ischemic Retinopathy. Invest Ophthalmol Vis Sci 2020;61(14):9.
AbstractPurpose: The identification of target pathways to block excessive angiogenesis while simultaneously restoring physiological vasculature is an unmet goal in the therapeutic management of ischemic retinopathies. pNaKtide, a cell-permeable peptide that we have designed by mapping the site of α1 Na/K-ATPase (NKA)/Src binding, blocks the formation of α1 NKA/Src/reactive oxygen species (ROS) amplification loops and restores physiological ROS signaling in a number of oxidative disease models. The aim of this study was to evaluate the importance of the NKA/Src/ROS amplification loop and the effect of pNaKtide in experimental ischemic retinopathy. Methods: Human retinal microvascular endothelial cells (HRMECs) and retinal pigment epithelium (ARPE-19) cells were used to evaluate the effect of pNaKtide on viability, proliferation, and angiogenesis. Retinal toxicity and distribution were assessed in those cells and in the mouse. Subsequently, the role and molecular mechanism of NKA/Src in ROS stress signaling were evaluated biochemically in the retinas of mice exposed to the well-established protocol of oxygen-induced retinopathy (OIR). Finally, pNaKtide efficacy was assessed in this model. Results: The results suggest a key role of α1 NKA in the regulation of ROS stress and the Nrf2 pathway in mouse OIR retinas. Inhibition of α1 NKA/Src by pNaKtide reduced pathologic ROS signaling and restored normal expression of hypoxia-inducible factor 1-α/vascular endothelial growth factor (VEGF). Unlike anti-VEGF agents, pNaKtide did promote retinal revascularization while inhibiting neovascularization and inflammation. Conclusions: Targeting α1 NKA represents a novel strategy to develop therapeutics that not only inhibit neovascularization but also promote physiological revascularization in ischemic eye diseases.
Whitmore HAB, Amarnani D, O'Hare M, Delgado-Tirado S, Gonzalez-Buendia L, An M, Pedron J, Bushweller JH, Arboleda-Velasquez JF, Kim LA.
TNF-α signaling regulates RUNX1 function in endothelial cells. FASEB J 2021;35(2):e21155.
AbstractRunt-related transcription factor 1 (RUNX1) acts as a mediator of aberrant retinal angiogenesis and has been implicated in the progression of proliferative diabetic retinopathy (PDR). Patients with PDR, retinopathy of prematurity (ROP), and wet age-related macular degeneration (wet AMD) have been found to have elevated levels of Tumor Necrosis Factor-alpha (TNF-α) in the eye. In fibrovascular membranes (FVMs) taken from patients with PDR RUNX1 expression was increased in the vasculature, while in human retinal microvascular endothelial cells (HRMECs), TNF-α stimulation causes increased RUNX1 expression, which can be modulated by RUNX1 inhibitors. Using TNF-α pathway inhibitors, we determined that in HRMECs, TNF-α-induced RUNX1 expression occurs via JNK activation, while NF-κB and p38/MAPK inhibition did not affect RUNX1 expression. JNK inhibitors were also effective at stopping high D-glucose-stimulated RUNX1 expression. We further linked JNK to RUNX1 through Activator Protein 1 (AP-1) and investigated the JNK-AP-1-RUNX1 regulatory feedback loop, which can be modulated by VEGF. Additionally, stimulation with TNF-α and D-glucose had an additive effect on RUNX1 expression, which was downregulated by VEGF modulation. These data suggest that the downregulation of RUNX1 in conjunction with anti-VEGF agents may be important in future treatments for the management of diseases of pathologic ocular angiogenesis.
Wu M, Matar DY, Yu Z, Chen Z, Knoedler S, Ng B, Darwish O, Haug V, Friedman L, Orgill DP, Panayi AC.
Modulation of Lymphangiogenesis in Incisional Murine Diabetic Wound Healing Using Negative Pressure Wound Therapy. Adv Wound Care (New Rochelle) 2023;12(9):483-497.
AbstractObjective: Despite the significant function of lymphatics in wound healing, and frequent clinical use of Negative Pressure Wound Therapy (NPWT), the effect of mechanical force application on lymphangiogenesis remains to be elucidated. We utilize a murine incisional wound healing model to assess the mechanisms of lymphangiogenesis following NPWT. Approach: Dorsal incisional skin wounds were created on diabetic mice (genetically obese leptin receptor-deficient mice [db/db]; n = 30) and covered with an occlusive dressing (Control, n = 15) or NPWT (-125 mmHg, continuous, 24 h for 7 days; NPWT, n = 15). The wounds were macroscopically assessed for 28 days. Tissue was harvested on day 10 for analysis. Qualitative functional analysis of lymphatic drainage was performed on day 28 using Evans Blue staining (n = 2). Results: NPWT increased lymphatic vessel density (40 ± 20 vs. 12 ± 6 podoplanin [PDPN]+ and 25 ± 9 vs. 14 ± 8 lymphatic vessel endothelial receptor 1 [LYVE-1]+) and vessel diameter (28 ± 9 vs. 12 ± 2 μm). Western blotting verified the upregulation of LYVE-1 with NPWT. Leukocyte presence was higher with NPWT (22% ± 3.7% vs. 9.1% ± 4.1% lymphocyte common antigen [CD45]+) and the leukocytes were predominately B cells clustered within vessels (8.8% ± 2.5% vs. 18% ± 3.6% B-lymphocyte antigen CD20 [CD20]+). Macrophage presence was lower in the NPWT group. Lymphatic drainage was increased in the NPWT group, which exhibited greater Evans Blue positivity. Innovation: The lymphangiogenic effects take place independent of macrophage infiltration, appearing to correlate with B cell presence. Conclusion: NPWT promotes lymphangiogenesis in incisional wounds, significantly increasing the lymph vessel density and diameter. This study highlights the potential of NPWT to stimulate lymphatic drainage and wound healing of surgical incisions.
Wu W, Duan Y, Ma G, Zhou G, Windhol C, D'Amore PA, Lei H.
AAV-CRISPR/Cas9-Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro. Invest Ophthalmol Vis Sci 2017;58(14):6082-6090.
AbstractPurpose: Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)-mediated CRISPR (clustered regularly interspaced short palindromic repeats)-associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events. Methods: The dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined. Results: AAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs. Conclusions: AAV-CRISRP/Cas9-mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis.
Wu W, Xia X, Tang L, Yao F, Xu H, Lei H.
Normal vitreous promotes angiogenesis via activation of Axl. FASEB J 2021;35(1):e21152.
AbstractVitreous has been reported to prevent tumor angiogenesis, but our previous findings indicate that vitreous activate the signaling pathway of phosphoinositide 3-kinase (PI3K)/Akt, which plays a critical role in angiogenesis. The goal of this research is to determine which role of vitreous plays in angiogenesis-related cellular responses in vitro. We found that in human retinal microvascular endothelial cells (HRECs) vitreous activates a number of receptor tyrosine kinases including Anexelekto (Axl), which plays an important role in angiogenesis. Subsequently, we discovered that depletion of Axl using CRISPR/Cas9 and an Axl-specific inhibitor R428 suppress vitreous-induced Akt activation and cell proliferation, migration, and tuber formation of HRECs. Therefore, this line of research not only demonstrate that vitreous promotes angiogenesis in vitro, but also reveal that Axl is one of receptor tyrosine kinases to mediate vitreous-induced angiogenesis in vitro, thereby providing a molecular basis for removal of vitreous as cleanly as possible when vitrectomy is performed in treating patients with proliferative diabetic retinopathy.
Wu W, Zhou G, Han H, Huang X, Jiang H, Mukai S, Kazlauskas A, Cui J, Matsubara JA, Vanhaesebroeck B, Xia X, Wang J, Lei H.
PI3Kδ as a Novel Therapeutic Target in Pathological Angiogenesis. Diabetes 2020;69(4):736-748.
AbstractDiabetic retinopathy is the most common microvascular complication of diabetes, and in the advanced diabetic retinopathy appear vitreal fibrovascular membranes that consist of a variety of cells, including vascular endothelial cells (ECs). New therapeutic approaches for this diabetic complication are urgently needed. Here, we report that in cultured human retinal microvascular ECs, high glucose induced expression of p110δ, which was also expressed in ECs of fibrovascular membranes from patients with diabetes. This catalytic subunit of a receptor-regulated PI3K isoform δ is known to be highly enriched in leukocytes. Using genetic and pharmacological approaches, we show that p110δ activity in cultured ECs controls Akt activation, cell proliferation, migration, and tube formation induced by vascular endothelial growth factor, basic fibroblast growth factor, and epidermal growth factor. Using a mouse model of oxygen-induced retinopathy, p110δ inactivation was found to attenuate pathological retinal angiogenesis. p110δ inhibitors have been approved for use in human B-cell malignancies. Our data suggest that antagonizing p110δ constitutes a previously unappreciated therapeutic opportunity for diabetic retinopathy.
Wu W, Ma G, Qi H, Dong L, Chen F, Wang Y, Mao X, Guo X, Cui J, Matsubara JA, Vanhaesebroeck B, Yan X, Zhao G, Zhang S, Lei H.
Genome Editing of Pik3cd Impedes Abnormal Retinal Angiogenesis. Hum Gene Ther 2023;34(1-2):30-41.
AbstractAbnormal angiogenesis is associated with myriad human diseases, including proliferative diabetic retinopathy (PDR). Signaling transduction through phosphoinositide 3-kinases (PI3Ks) plays a critical role in angiogenesis. Herein, we showed that p110δ, the catalytic subunit of PI3Kδ, was highly expressed in pathological retinal vascular endothelial cells (ECs) in a mouse model of oxygen-induced retinopathy (OIR) and in fibrovascular membranes from patients with PDR. To explore novel intervention with PI3Kδ expression, we developed a recombinant dual adeno-associated viral (rAAV) system for delivering CRISPR/Cas9 in which Streptococcus pyogenes (Sp) Cas9 expression was driven by an endothelial specific promoter of the intercellular adhesion molecule 2 (pICAM2) to edit genomic Pik3cd, the gene encoding p110δ. We then demonstrated that infection of cultured mouse vascular ECs with the dual rAAV1s of rAAV1-pICAM2-SpCas9 and rAAV1-SpGuide targeting genomic Pik3cd resulted in 80% DNA insertion/deletion in the locus of genomic Pik3cd and 70% depletion of p110δ expression. Furthermore, we showed that in the mouse model of OIR editing retinal Pik3cd with the dual rAAV1s resulted in not only a significant decrease in p110δ expression, and Akt activation, but also a dramatic reduction in pathological retinal angiogenesis. These findings reveal that Pik3cd editing is a novel approach to treating abnormal retinal angiogenesis.
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Yanagida K, Engelbrecht E, Niaudet C, Jung B, Gaengel K, Holton K, Swendeman S, Liu CH, Levesque MV, Kuo A, Fu Z, Smith LEH, Betsholtz C, Hla T.
Sphingosine 1-Phosphate Receptor Signaling Establishes AP-1 Gradients to Allow for Retinal Endothelial Cell Specialization. Dev Cell 2020;52(6):779-793.e7.
AbstractTranscriptional mechanisms that drive angiogenesis and organotypic vascular endothelial cell specialization are poorly understood. Here, we show that retinal endothelial sphingosine 1-phosphate receptors (S1PRs), which restrain vascular endothelial growth factor (VEGF)-induced angiogenesis, spatially restrict expression of JunB, a member of the activator protein 1 (AP-1) family of transcription factors (TFs). Mechanistically, VEGF induces JunB expression at the sprouting vascular front while S1PR-dependent vascular endothelial (VE)-cadherin assembly suppresses JunB expression in the nascent vascular network, thus creating a gradient of this TF. Endothelial-specific JunB knockout mice showed diminished expression of neurovascular guidance genes and attenuated retinal vascular network progression. In addition, endothelial S1PR signaling is required for normal expression of β-catenin-dependent genes such as TCF/LEF1 and ZIC3 TFs, transporters, and junctional proteins. These results show that S1PR signaling restricts JunB function to the expanding vascular front, thus creating an AP-1 gradient and enabling organotypic endothelial cell specialization of the vascular network.
Ying Y, Ueta T, Jiang S, Lin H, Wang Y, Vavvas D, Wen R, Chen Y-G, Luo Z.
Metformin inhibits ALK1-mediated angiogenesis via activation of AMPK. Oncotarget 2017;8(20):32794-32806.
AbstractAnti-VEGF therapy has been proven to be effective in the treatment of pathological angiogenesis. However, therapy resistance often occurs, leading to development of alternative approaches. The present study examines if AMPK negatively regulates ALK1-mediated signaling events and associated angiogenesis. Thus, we treated human umbilical vein endothelial cells with metformin as well as other pharmacological AMPK activators and showed that activation of AMPK inhibited Smad1/5 phosphorylation and tube formation induced by BMP9. This event was mimicked by expression of the active mutant of AMPKα1 and prevented by the dominant negative AMPKα1. Metformin inhibition of BMP9 signaling is possibly mediated by upregulation of Smurf1, leading to degradation of ALK1. Furthermore, metformin suppressed BMP9-induced angiogenesis in mouse matrigel plug. In addition, laser photocoagulation was employed to evaluate the effect of metformin. The data revealed that metformin significantly reduced choroidal neovascularization to a level comparable to LDN212854, an ALK1 specific inhibitor. In conjunction, metformin diminished expression of ALK1 in endothelium of the lesion area. Collectively, our study for the first time demonstrates that AMPK inhibits ALK1 and associated angiogenesis/neovascularization. This may offer us a new avenue for the treatment of related diseases using clinically used pharmacological AMPK activators like metformin in combination with other strategies to enhance the treatment efficacy or in the case of anti-VEGF resistance.
