Modulating the Nature of Ionizable Lipids and Number of Layers in Hyaluronan-Decorated Lipid Nanoparticles for In Vitro Delivery of RNAi.

Lipid nanoparticles (LNPs) have established their position as nonviral vectors for gene therapy. Tremendous efforts have been made to modulate the properties of LNPs to unleash their full clinical potential. Among the strategies being pursued, the layer-by-layer (LbL) technique has gained considerable attention in the biomedical field. Illuminated by our previous work, here we investigate if the LbL approach could be used to modify the LNP cores formulated with three different ionizable lipids: DODMA, MC3, and DODAP. Additionally, we wondered if more than three layers could be loaded onto LNPs without disrupting their gene transfection ability. Taking advantage of physicochemical analysis, as well as uptake and gene silencing studies, we demonstrate the feasibility of modifying the surface of LNPs with the LbL assembly. Precisely, we successfully modified three different LNPs using the layer-by-layer strategy which abrogated luciferase activity in vitro. Additionally, we constructed a 5×-layered HA-LNP containing the MC3 ionizable lipid which outperformed the 3×-layered counterpart in transfecting miRNA-181-5p to the pediatric GBM cell line, as a proof-of-concept in vitro experiment. The method used herein has been proven reproducible, of easy modification to adapt to different ionizable lipid-containing LNPs, and holds great potential for the translation of RNA-based therapeutic strategies.

Hyaluronan decorated layer-by-layer assembled lipid nanoparticles for miR-181a delivery in glioblastoma treatment.

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer. Current pharmacological interventions marginally increase the 12-month overall survival of patients with GBM. Among the novel therapeutic strategies being pursued, micro-RNAs, a class of non-coding RNAs, are receiving considerable attention for their regulation of several pathways implicated in tumorigenesis and survival. Notably, microRNA-181a-5p (miR-181a) has consistently been reported to be downregulated in GBM clinical samples, and its overexpression negatively affects tumor growth both in vitro and in vivo. To improve the delivery of miR-181a to GBM cells, we sought to develop a modified lipid-based nanocarrier capable of encapsulating and delivering miR-181a to GBM cells in vitro and in vivo. Optimized ionizable-lipid containing lipid nanoparticles (LNP) were constructed by covering the miR-181a-loaded LNP with alternating layers of miR-181a, poly-l-arginine and hyaluronic acid through the layer-by-layer technique. The resulting hyaluronan-decorated lipid nanoparticles (HA-LNP) targeted GBM cells more efficiently than non-modified LNP and mediated siRNA and miRNA transfection in vitro. Finally, delivery of miR-181a by HA-LNP induced significant cellular death of U87 GBM cells in vitro and delayed tumor growth in an in vivo subcutaneous tumor model.

MiR-181a-5p inhibits uveal melanoma development by targeting GNAQ and AKT3.

Uveal melanoma (UM) is the most common primary intraocular malignant tumor type in adults. Even after the treatment of the ocular tumor, the prognosis of patients with metastasis remains poor. Hence, an urgent unmet need exists to identify novel approaches to treat advanced UM. Previous studies have revealed G subunit alpha Q and alpha 11 (GNAQ/11) mutations in more than 85% of patients with UM, thus indicating the importance of GNAQ and downstream signaling pathways in UM occurrence. Here, we demonstrate that microRNA (miR)-181a-5p, a small non-coding RNA, effectively inhibited the viability, proliferation, and colony formation but induced apoptosis of UM cells. Furthermore, silencing GNAQ or AKT3 mimicked the anti-UM effects of miR-181a-5p, whereas overexpression of GNAQ or AKT3 rescued the anti-UM effects induced by miR-181a-5p. In addition, miR-181a-5p had a stronger effect in decreasing the viability of GNAQ mutant than GNAQ wild-type cells. Moreover, miR-181a-5p suppressed the total expression and phosphorylation of members of the ERK and PI3K/AKT/mTOR signaling pathways. Importantly, miR-181a-5p potently inhibited the growth of UM xenografts in nude mice. MiR-181a-5p also decreased the expression of Ki67, GNAQ, and AKT3, and induced the expression of cleaved-caspase3 in UM tumors. These results suggest that miR-181a-5p inhibits UM development by targeting GNAQ and AKT3.

Lymphocytic microparticles suppress retinal angiogenesis via targeting Müller cells in the ischemic retinopathy mouse model.

Retinopathy of prematurity (ROP) is the primary cause of visual impairment and vision loss in premature infants, which results from the formation of aberrant retinal neovascularization (NV). An emerging body of evidence has shown that Müller cells are the predominant source of vascular endothelial growth factor (VEGF), which also serves as a chemoattractant for monocyte/macrophage lineage. The recruitment of macrophages is increased during retinal NV, and they exert a pro-angiogenic role in ROP. We have shown that lymphocytic microparticles (microvesicles; LMPs) derived from apoptotic human T lymphocytes possess strong angiogenesis-inhibiting properties. Here, we investigated the effect of LMPs on the chemotactic capacity of Müller cells in vitro using rat Müller cell rMC-1 and mouse macrophage RAW 264.7. In addition, the impact of LMPs was determined in vivo using a mouse model of oxygen-induced ischemic retinopathy (OIR). The results revealed that LMPs were internalized by rMC-1 and reduced their cell proliferation dose-dependently without inducing cell apoptosis. LMPs inhibited the chemotactic capacity of rMC-1 on RAW 264.7 via reducing the expression of VEGF. Moreover, LMPs attenuated pathological retinal NV and the infiltration of macrophages in vivo. LMPs downregulated ERK1/2 and HIF-1α both in vitro and in vivo. These findings expand our understanding of the effects of LMPs, providing evidence of LMPs as a promising therapeutic approach for the treatment of retinal NV diseases.

Survivin silencing improved the cytotoxicity of carboplatin and melphalan in Y79 and primary retinoblastoma cells.

Survivin stands out as one of the most specific cancer targets discovered to date. Although single inhibition, e.g. through small interfering RNA (siRNA), has shown modest results in clinical trials, its combination with drugs holds promise to sensitize cancer cells to chemotherapeutics. In this study, we propose a sequential treatment of siRNA survivin followed by chemotherapy. Firstly, we demonstrated that siRNA-loaded switchable lipid nanoparticles (siLNP) silence survivin in a panel of cancer cell lines. Subsequently, we selected retinoblastoma (RB) as our model to screen four chemotherapeutic agents: carboplatin, topotecan, melphalan or teniposide. The effect of drugs on survivin expression and caspase-3 was investigated by RT-qPCR. The best drug combination was selected measuring the viability, survivin expression and the selectivity of the treatment. Our stepwise method revealed that siRNA delivery by switchable LNP sensitized Y79, but not the healthy APRE-19 cell line, to carboplatin and melphalan cytotoxicity. This ability was validated on primary human RB cells. Finally, the distinct behavior of the drugs demonstrated that a diligent screening of drugs should be envisioned when looking for synergy with survivin. Our sequential approach highlighted carboplatin and melphalan as agents to be investigated in future survivin-associated in vivo testing to tackle RB.

Extracellular microparticles exacerbate oxidative damage to retinal pigment epithelial cells.

Oxidative stress-induced retinal pigment epithelial cell (RPE) dysfunction is a primary contributing factor to early dry age-related macular degeneration (AMD). Oxidative injury to the retina may promote extracellular vesicles (EVs) released from RPE. In this study, we investigated the effects of oxidative-induced RPE cell-derived microparticles (RMPs) on RPE cell functions. The oxidative stress induced more RMPs released from RPE cells in vitro and in vivo, and significant more RMPs were released from aged RPE cells than that from younger RPE cells. RMPs were taken up by RPE cells in a time-dependent manner; however, blockage of CD36 attenuated the uptake process. Furthermore, the decrease of RPE cell viability by RMPs treatment was associated with an increased expression of cyclin-dependent kinase inhibitors p15 and p21. RMPs enhanced senescence and interrupted phagocytic activity of RPE cells as well. The present study demonstrated that RMPs produce a strong effect of inducing RPE cell degeneration. This finding further supports the postulate that RMPs exacerbate oxidative stress damage to RPE cells, which may uncover a potentially relevant process in the genesis of dry AMD.

Propranolol Attenuates Proangiogenic Activity of Mononuclear Phagocytes: Implication in Choroidal Neovascularization.

Purpose: Targeting ß-adrenergic receptor signaling with propranolol has emerged as a potential candidate to counteract choroidal neovascularization (CNV). Little is known of its effect on macrophages, which play a critical role in CNV. We investigated the effect of propranolol on angiogenic response of mononuclear phagocytes (MPs). Methods: The angiogenic effect of propranolol was evaluated in laser-induced CNV model. Mice received intraperitoneal injections of propranolol (6 mg/kg/d) or vehicle. CNV area and inflammatory cells were determined respectively by using lectin staining and an anti-IBA-1 antibody on RPE/choroid flat mounts. Inflammatory gene expression was evaluated by quantitative (q) PCR analysis. Mechanisms of propranolol was studied in MP cell lines J774 and RAW264.7 and in primary peritoneal macrophages. Expression of pro- and antiangiogenic mediators was studied. In addition, effects of propranolol treatment of MPs was assessed on choroidal explant. Results: CNV was attenuated by propranolol and concomitantly associated with decreased inflammatory mediators IL-6 and TNFα, albeit with accumulation of (ß-adrenoceptor harboring) MPs in the CNV area. Conditioned media from MPs preincubated with propranolol exerted antiangiogenic effects. Treatment of J774 confirmed the attenuation of inflammatory response to propranolol and increased cleaved caspase-3 on choroidal explant. We found that propranolol increased pigment epithelium-derived factor (PEDF) expression in MPs. Trapping of PEDF with an antibody abrogated antiangiogenic effects of propranolol. PEDF was also detected in CNV-associated MPs. Conclusions: We hereby show that propranolol confers on MPs antiangiogenic properties by increasing PEDF expression, which complements its effects on vascular tissue resulting in inhibition of choroidal vasoproliferation in inflammatory conditions. The study supports possible use of propranolol as a therapeutic modality for CNV.

Immunometabolic modulation of retinal inflammation by CD36 ligand.

In subretinal inflammation, activated mononuclear phagocytes (MP) play a key role in the progression of retinopathies. Little is known about the mechanism involved in the loss of photoreceptors leading to vision impairment. Studying retinal damage induced by photo-oxidative stress, we observed that cluster of differentiation 36 (CD36)-deficient mice featured less subretinal MP accumulation and attenuated photoreceptor degeneration. Moreover, treatment with a CD36-selective azapeptide ligand (MPE-001) reduced subretinal activated MP accumulation in wild type mice and preserved photoreceptor layers and function as assessed by electroretinography in a CD36-dependent manner. The azapeptide modulated the transcriptome of subretinal activated MP by reducing pro-inflammatory markers. In isolated MP, MPE-001 induced dissociation of the CD36-Toll-like receptor 2 (TLR2) oligomeric complex, decreasing nuclear factor-kappa B (NF-κB) and NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. In addition, MPE-001 caused an aerobic metabolic shift in activated MP, involving peroxisome proliferator-activated receptor-γ (PPAR-γ) activation, which in turn mitigated inflammation. Accordingly, PPAR-γ inhibition blocked the cytoprotective effect of MPE-001 on photoreceptor apoptosis elicited by activated MP. By altering activated MP metabolism, MPE-001 decreased immune responses to alleviate subsequent inflammation-dependent neuronal injury characteristic of various vision-threatening retinal disorders.

The Inability of the Choroid to Revascularize in Oxygen-Induced Retinopathy Results from Increased p53/miR-Let-7b Activity.

Retinopathy of prematurity (ROP) is characterized by an initial retinal avascularization, followed by pathologic neovascularization. Recently, choroidal thinning has also been detected in children formerly diagnosed with ROP; a similar sustained choroidal thinning is observed in ROP models. But the mechanism underlying the lack of choroidal revascularization remains unclear and was investigated in an oxygen-induced retinopathy (OIR) model. In OIR, evidence of senescence was detected, preceded by oxidative stress in the choroid and the retinal pigment epithelium. This was associated with a global reduction of proangiogenic factors, including insulin-like growth factor 1 receptor (Igf1R). Coincidentally, tumor suppressor p53 was highly expressed in the OIR retinae. Curtailing p53 activity resulted in reversal of senescence, normalization of Igf1r expression, and preservation of choroidal integrity. OIR-induced down-regulation of Igf1r was mediated at least partly by miR-let-7b as i) let-7b expression was augmented throughout and beyond the period of oxygen exposure, ii) let-7b directly targeted Igf1r mRNA, and iii) p53 knock-down blunted let-7b expression, restored Igf1r expression, and elicited choroidal revascularization. Finally, restoration of Igf1r expression rescued choroid thickness. Altogether, this study uncovers a significant mechanism for defective choroidal revascularization in OIR, revealing a new role for p53/let-7b/IGF-1R axis in the retina. Future investigations on this (and connected) pathway could further our understanding of other degenerative choroidopathies, such as geographic atrophy.

microRNA-181a inhibits ocular neovascularization by interfering with vascular endothelial growth factor expression.

AIM: Excess angiogenesis or neovascularization plays a key role in the pathophysiology of several ocular diseases such as retinopathy of prematurity, diabetic retinopathy, and exudative age-related macular degeneration. microRNA-181a (miR-181a) was found highly expressed in retina and choroidal tissues. This study intends to investigate the role of miR-181a in the regulation of ocular neovascularization in different pathophysiological conditions. METHOD: We performed the RNA sequence to identify the microRNAs components of anti-angiogenic lymphocyte-derived microparticles (LMPs). The effect of miR-181a on human retinal endothelial cells proliferation was assessed in vitro. The impact of miR-181a on angiogenesis was confirmed using in vitro angiogenesis assay, ex vivo choroidal explant, and in vivo retinal neovascularization. The expression of major angiogenic factors was assessed by real-time qPCR. RESULTS: RNA sequence revealed that miR-181a is selectively enriched in LMPs. Importantly, the inhibition of miR-181a significantly abrogated the effect of LMPs on endothelial viability, but overexpression of miR-181a reduced endothelial cell viability in a dose-dependent manner. miR-181a strongly inhibited in vitro angiogenesis and ex vivo choroidal neovascularization. The strong anti-angiogenic effect of miR-181a was also displayed on the retinal neovascularization of the in vivo mouse model of oxygen-induced retinopathy. In keeping with its effect, several angiogenesis-related genes were dysregulated in the miR-181a overexpressed endothelial cells. CONCLUSION: These data may open unexpected avenues for the development of miR-181a as a novel therapeutic strategy that would be particularly useful and relevant for the treatment of neovascular diseases.

Influences of Histidine-1 and Azaphenylalanine-4 on the Affinity, Anti-inflammatory, and Antiangiogenic Activities of Azapeptide Cluster of Differentiation 36 Receptor Modulators.

Azapeptide analogues of growth hormone releasing peptide-6 (GHRP-6) exhibit promising affinity, selectivity, and modulator activity on the cluster of differentiation 36 receptor (CD36). For example, [A1, azaF4]- and [azaY4]-GHRP-6 (1a and 2b) were previously shown to bind selectively to CD36 and exhibited respectively significant antiangiogenic and slight angiogenic activities in a microvascular sprouting assay using choroid explants. The influences of the 1- and 4-position residues on the affinity, anti-inflammatory, and antiangiogenic activity of these azapeptides have now been studied in detail by the synthesis and analysis of a set of 25 analogues featuring Ala1 or His1 and a variety of aromatic side chains at the aza-amino acid residue in the 4-position. Although their binding affinities differed only by a factor of 17, the analogues exhibited significant differences in ability to modulate production of nitric oxide (NO) in macrophages and choroidal neovascularization.

Ischemic Retinopathies: Oxidative Stress and Inflammation.

Ischemic retinopathies (IRs), such as retinopathy of prematurity (ROP), diabetic retinopathy (DR), and (in many cases) age-related macular degeneration (AMD), are ocular disorders characterized by an initial phase of microvascular changes that results in ischemia, followed by a second phase of abnormal neovascularization that may culminate into retinal detachment and blindness. IRs are complex retinal conditions in which several factors play a key role during the development of the different pathological stages of the disease. Increasing evidence reveals that oxidative stress and inflammatory processes are important contributors to the pathogenesis of IRs. Despite the beneficial effects of the photocoagulation and anti-VEGF therapy during neovascularization phase, the need to identify novel targets to prevent initial phases of these ocular pathologies is still needed. In this review, we provide an update on the involvement of oxidative stress and inflammation in the progression of IRs and address some therapeutic interventions by using antioxidants and anti-inflammatory agents.

Lymphocytic Microparticles Modulate Angiogenic Properties of Macrophages in Laser-induced Choroidal Neovascularization.

Pathological choroidal neovascularization (CNV) is the common cause of vision loss in patients with age-related macular degeneration (AMD). Macrophages possess potential angiogenic function in CNV. We have demonstrated that human T lymphocyte-derived microparticles (LMPs) exert a potent antiangiogenic effect in several pathological neovascularization models. In this study, we investigated the alteration of proangiogenic properties of macrophages by LMPs treatment in vitro and in vivo models. LMPs regulated the expression of several angiogenesis-related factors in macrophages and consequently stimulated their antiangiogenic effects evidenced by the suppression of the proliferation of human retinal endothelial cells in co-culture experiments. The involvement of CD36 receptor in LMPs uptake by macrophages was demonstrated by in vitro assays and by immunostaining of choroidal flat mounts. In addition, ex vivo experiments showed that CD36 mediates the antiangiogenic effect of LMPs in murine and human choroidal explants. Furthermore, intravitreal injection of LMPs in the mouse model of laser-induced CNV significantly suppressed CNV in CD36 dependent manner. The results of this study suggested an ability of LMPs to alter the gene expression pattern of angiogenesis-related factors in macrophages, which provide important information for a new therapeutic approach for efficiently interfering with both vascular and extravascular components of CNV.

Choroidal Involution Is Associated with a Progressive Degeneration of the Outer Retinal Function in a Model of Retinopathy of Prematurity: Early Role for IL-1ß.

Retinopathy of prematurity (ROP), the most common cause of blindness in premature infants, has long been associated with inner retinal alterations. However, recent studies reveal outer retinal dysfunctions in patients formerly afflicted with ROP. We have recently demonstrated that choroidal involution occurs early in retinopathy. Herein, we investigated the mechanisms underlying the choroidal involution and its long-term impact on retinal function. An oxygen-induced retinopathy (OIR) model was used. In vitro and ex vivo assays were applied to evaluate cytotoxic effects of IL-1ß on choroidal endothelium. Electroretinogram was used to evaluate visual function. We found that proinflammatory IL-1ß was markedly increased in retinal pigment epithelium (RPE)/choroid and positively correlated with choroidal degeneration in the early stages of retinopathy. IL-1ß was found to be cytotoxic to choroid in vitro, ex vivo, and in vivo. Long-term effects on choroidal involution included a hypoxic outer neuroretina, associated with a progressive loss of RPE and photoreceptors, and visual deterioration. Early inhibition of IL-1ß receptor preserved choroid, decreased subretinal hypoxia, and prevented RPE/photoreceptor death, resulting in life-long improved visual function in IL-1 receptor antagonist-treated OIR animals. Together, these findings suggest a critical role for IL-1ß-induced choroidal degeneration in outer retinal dysfunction. Neonatal therapy using IL-1 receptor antagonist preserves choroid and prevents protracted outer neuroretinal anomalies in OIR, suggesting IL-1ß as a potential therapeutic target in ROP.

SYK is a target of lymphocyte-derived microparticles in the induction of apoptosis of human retinoblastoma cells.

Retinoblastoma (Rb) is an aggressive childhood cancer of the developing retina. This disease is associated with epigenetic deregulation of several cancer pathways including upregulation of the proto-oncogene spleen tyrosine kinase (SYK). We have previously demonstrated that lymphocyte-derived microparticles (LMPs) possess strong cytotoxic effect on cancer cells. This report demonstrated that LMPs have potent pro-apoptotic properties on human Rb cells, which was associated with a strong reduction of SYK expression. Perturbing SYK activity in Rb cells induced cell apoptosis and upregulated expression of p53 and p21. Interestingly, inhibition of p53 or knockdown of p21, abolished LMP-induced caspase-3 activity and cell death. Blocking oxidized phospholipid-rich LMPs with a specific antibody significantly prevented LMP-induced Rb apoptosis and reversed the expression patterns of SYK, p53, p21. In summary, our results suggest that LMPs are important pro-apoptotic regulators for Rb cells through reduction of SYK expression and upregulation of the p53-p21 pathway which ultimately activates caspase-3. These data may open unexpected avenues for the development of LMPs as a novel therapeutic strategy that would be particularly useful and relevant for the treatment of Rb.

Generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells.

Interest in the biological roles of cell membrane-derived vesicles in cell-cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 µm to 1 µm, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte-derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.

Anti-proliferative and anti-tumour effects of lymphocyte-derived microparticles are neither species- nor tumour-type specific.

BACKGROUND: Unregulated cell proliferation or growth is a prominent characteristic of cancer. We have previously demonstrated that LMPs (cell membrane microparticles derived from apoptotic human CEM T lymphoma cells stimulated with actinomycin D) strongly suppress the proliferation of not only human endothelial cells but also mouse Lewis lung carcinoma cells. METHODS: LMPs were generated either from CEM T cells using different stimuli or from 3 different types of lymphocytes. The effects of LMPs on cancer cell proliferation were examined using cell lines from different species and tissues. The cell cycle kinetics was evaluated by FACS and the expression of cell cycle-related genes was determined using quantitative RT-PCR. The in vivo anti-tumor effect of LMPs was investigated using xenografts and allografts. RESULTS: LMPs at doses far above physiological levels dramatically suppressed the proliferation of cancer cells in a non species-specific manner. LMPs selectively target high proliferating cells and their anti-proliferative effect is not dependent on parental cell origin or stimuli. The anti-proliferative effect of LMPs was due to induction of cell-cycle arrest in G0/G1, with associated increases in expression of the cyclin-dependent kinase inhibitors p15(INK4b), p16(INK4a), and p21(Cip1). In vivo, LMPs significantly suppressed tumor growth in animal tumor models. CONCLUSION: These results highlight the potential role of LMPs in modulating the growth of high proliferating cells. Given that cell-based therapies are considered less toxic than pharmacologic approaches and have the potential to target multiple pathways in a synergistic manner, LMPs may serve as a veritable option for cancer treatment.

Choroid sprouting assay: an ex vivo model of microvascular angiogenesis.

Angiogenesis of the microvasculature is central to the etiology of many diseases including proliferative retinopathy, age-related macular degeneration and cancer. A mouse model of microvascular angiogenesis would be very valuable and enable access to a wide range of genetically manipulated tissues that closely approximate small blood vessel growth in vivo. Vascular endothelial cells cultured in vitro are widely used, however, isolating pure vascular murine endothelial cells is technically challenging. A microvascular mouse explant model that is robust, quantitative and can be reproduced without difficulty would overcome these limitations. Here we characterized and optimized for reproducibility an organotypic microvascular angiogenesis mouse and rat model from the choroid, a microvascular bed in the posterior of eye. The choroidal tissues from C57BL/6J and 129S6/SvEvTac mice and Sprague Dawley rats were isolated and incubated in Matrigel. Vascular sprouting was comparable between choroid samples obtained from different animals of the same genetic background. The sprouting area, normalized to controls, was highly reproducible between independent experiments. We developed a semi-automated macro in ImageJ software to allow for more efficient quantification of sprouting area. Isolated choroid explants responded to manipulation of the external environment while maintaining the local interactions of endothelial cells with neighboring cells, including pericytes and macrophages as evidenced by immunohistochemistry and fluorescence-activated cell sorting (FACS) analysis. This reproducible ex vivo angiogenesis assay can be used to evaluate angiogenic potential of pharmacologic compounds on microvessels and can take advantage of genetically manipulated mouse tissue for microvascular disease research.

p75 neurotrophin receptor participates in the choroidal antiangiogenic and apoptotic effects of T-lymphocyte-derived microparticles.

PURPOSE: Choroidal neovascularization (CNV) is a major cause of vision loss in which choroidal vessels penetrate the RPE-an important source of growth factors, including nerve growth factor (NGF), whose activation via the p75NTR receptor promotes apoptosis and inhibits angiogenesis. We demonstrated previously that human T-lymphocyte-derived microparticles (LMPs) significantly inhibit angiogenesis in several models of ocular neovascularization. We investigated how LMPs modulate pro- and antiangiogenic microenvironments during choroidal angiogenesis. METHODS: Antiangiogenic effects of LMPs were investigated using a rat model of choroidal angiogenesis. The impact of LMPs on expression of major angiogenic factors was assessed by real-time quantitative PCR (qPCR). To determine whether p75NTR signalling was implicated in LMPs-induced activities, we used a specific antibody and short hairpin RNA (shRNA) targeting p75NTR. Cellular apoptosis was determined via evaluation of activated caspase-3 and annexin V binding. RESULTS: The LMPs time-dependently inhibited choroidal angiogenesis by more than 64% after 48 hours of treatment. Removal of the RPE from choroidal explants abolished the antiangiogenic effects of LMPs. The mRNA levels of pigment epithelium-derived factor (PEDF) and NGF were increased significantly following LMPs treatment of intact, but not RPE-removed choroids. Downregulation of PEDF and p75NTR significantly blocked the antiangiogenic effects of LMPs. Finally, induction of choroidal endothelial cell apoptosis by LMPs was dependent on p75NTR. CONCLUSIONS: We demonstrate for the first time to our knowledge that LMPs markedly inhibit choroidal angiogenesis via mechanisms that are dependent on the integrity of the RPE, and that are mediated largely by the PEDF and proapoptotic activities of p75NTR.

Role of receptor-mediated endocytosis in the antiangiogenic effects of human T lymphoblastic cell-derived microparticles.

Microparticles possess therapeutic potential regarding angiogenesis. We have demonstrated the contribution of apoptotic human CEM T lymphocyte-derived microparticles (LMPs) as inhibitors of angiogenic responses in animal models of inflammation and tumor growth. In the present study, we characterized the antivascular endothelial growth factor (VEGF) effects of LMPs on pathological angiogenesis in an animal model of oxygen-induced retinopathy and explored the role of receptor-mediated endocytosis in the effects of LMPs on human retinal endothelial cells (HRECs). LMPs dramatically inhibited cell growth of HRECs, suppressed VEGF-induced cell migration in vitro experiments, and attenuated VEGF-induced retinal vascular leakage in vivo. Intravitreal injections of fluorescently labeled LMPs revealed accumulation of LMPs in retinal tissue, with more than 60% reductions of the vascular density in retinas of rats with oxygen-induced neovascularization. LMP uptake experiments demonstrated that the interaction between LMPs and HRECs is dependent on temperature. In addition, endocytosis is partially dependent on extracellular calcium. RNAi-mediated knockdown of low-density lipoprotein receptor (LDLR) reduced the uptake of LMPs and attenuated the inhibitory effects of LMPs on VEGF-A protein expression and HRECs cell growth. Intravitreal injection of lentivirus-mediated RNA interference reduced LDLR protein expression in retina by 53% and significantly blocked the antiangiogenic effects of LMPs on pathological vascularization. In summary, the potent antiangiogenic LMPs lead to a significant reduction of pathological retinal angiogenesis through modulation of VEGF signaling, whereas LDLR-mediated endocytosis plays a partial, but pivotal, role in the uptake of LMPs in HRECs.