Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2.

The endothelial glycocalyx, located at the luminal surface of the endothelium, plays an important role in the regulation of leukocyte adhesion, vascular permeability, and vascular homeostasis. Endomucin (EMCN), a component of the endothelial glycocalyx, is a mucin-like transmembrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that knockdown of EMCN impairs retinal vascular development in vivo and vascular endothelial growth factor 165 isoform (VEGF165)-induced cell migration, proliferation, and tube formation by human retinal endothelial cells in vitro and that EMCN is essential for VEGF165-stimulated clathrin-mediated endocytosis and signaling of VEGF receptor 2 (VEGFR2). Clathrin-mediated endocytosis is an essential step in receptor signaling and is of paramount importance for a number of receptors for growth factors involved in angiogenesis. In this study, we further investigated the molecular mechanism underlying EMCN's involvement in the regulation of VEGF-induced endocytosis. In addition, we examined the specificity of EMCN's role in angiogenesis-related cell surface receptor tyrosine kinase endocytosis and signaling. We identified that EMCN interacts with AP2 complex, which is essential for clathrin-mediated endocytosis. Lack of EMCN did not affect clathrin recruitment to the AP2 complex following VEGF stimulation, but it is necessary for the interaction between VEGFR2 and the AP2 complex during endocytosis. EMCN does not inhibit VEGFR1 and FGFR1 internalization or their downstream activities since EMCN interacts with VEGFR2 but not VEGFR1 or FGFR1. Additionally, EMCN also regulates VEGF121-induced VEGFR2 phosphorylation and internalization.

Macrophage efferocytosis with VEGFC and lymphangiogenesis: rescuing the broken heart.

Cardiac repair following ischemic injury is indispensable for survival and requires a coordinated cellular response involving the mobilization of immune cells from the secondary lymphoid organs to the site of damage. Efferocytosis, the engulfment of cell debris and dying cells by innate immune cells, along with lymphangiogenesis, the formation of new lymphatic vessels, are emerging as central to the cardiac healing response. In this issue of the JCI, Glinton et al. used state-of-the-art approaches to demonstrate that efferocytosis induced vascular endothelial growth factor C (VEGFC) in myeloid cells and stimulated lymphangiogenesis and cardiac repair. These findings provide impactful mechanistic information that can be leveraged to therapeutically target pathways in cardiac repair and ischemic heart failure.

Discovery of sterically-hindered phenol compounds with potent cytoprotective activities against ox-LDL-induced retinal pigment epithelial cell death as a potential pharmacotherapy.

Late-stage dry age-related macular degeneration (AMD) or geographic atrophy (GA) is an irreversible blinding condition characterized by degeneration of retinal pigment epithelium (RPE) and the associated photoreceptors. Clinical and genetic evidence supports a role for dysfunctional lipid processing and accumulation of harmful oxidized lipids in the pathogenesis of GA. Using an oxidized low-density lipoprotein (ox-LDL)-induced RPE death assay, we screened and identified sterically-hindered phenol compounds with potent protective activities for RPE. The phenol-containing PPARγ agonist, troglitazone, protected against ox-LDL-induced RPE cell death, whereas other more potent PPARγ agonists did not protect RPE cells. Knockdown of PPARγ did not affect the protective activity of troglitazone in RPE, confirming the protective function is not due to the thiazolidine (TZD) group of troglitazone. Prototypical hindered phenol trolox and its analogs potently protected against ox-LDL-induced RPE cell death whereas potent antioxidants without the phenol group failed to protect RPE. Hindered phenols preserved lysosomal integrity against ox-LDL-induced damage and FITC-labeled trolox was localized to the lysosomes in RPE cells. Analogs of trolox inhibited reactive oxygen species (ROS) formation induced by ox-LDL uptake in a dose-dependent fashion and were effective at sub-micromolar concentrations. Treatment with trolox analog 2,2,5,7,8-pentamethyl-6-chromanol (PMC) significantly induced the expression of the lysosomal protein NPC-1 and reduced intracellular cholesterol level upon ox-LDL uptake. Our data indicate that the lysosomal-localized hindered phenols are uniquely potent in protecting the RPE against the toxic effects of ox-LDL, and may represent a novel pharmacotherapy to preserve the vision in patients with GA.

VEGFR1 signaling in retinal angiogenesis and microinflammation.

Five 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.

Elements of the Endomucin Extracellular Domain Essential for VEGF-Induced VEGFR2 Activity.

Endomucin (EMCN) is the type I transmembrane glycoprotein, mucin-like component of the endothelial cell glycocalyx. We have previously shown that EMCN is necessary for vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2) internalization and downstream signaling. To explore the structural components of EMCN that are necessary for its function and the molecular mechanism of EMCN in VEGF-induced endothelial functions, we generated a series of mouse EMCN truncation mutants and examined their ability to rescue VEGF-induced endothelial functions in human primary endothelial cells (EC) in which endogenous EMCN had been knocked down using siRNA. Expression of the mouse full-length EMCN (FL EMCN) and the extracellular domain truncation mutants ∆21-81 EMCN and ∆21-121 EMCN, but not the shortest mutant ∆21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. ∆21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-type O-glycans were not required for its VEGFR2-related functions. Mutation of the two N-glycosylation sites on ∆21-121 EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal ∆21-121 EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated endothelial function and demonstrate an important role for N-glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.

ADAM10 and ADAM17 proteases mediate proinflammatory cytokine-induced and constitutive cleavage of endomucin from the endothelial surface.

Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte-EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.

Glycocalyx regulation of vascular endothelial growth factor receptor 2 activity.

We have previously shown that knockdown of endomucin (EMCN), an integral membrane glycocalyx glycoprotein, prevents VEGF-induced proliferation, migration, and tube formation in vitro and angiogenesis in vivo. In the endothelium, VEGF mediates most of its angiogenic effects through VEGF receptor 2 (VEGFR2). To understand the role of EMCN, we examined the effect of EMCN depletion on VEGFR2 endocytosis and activation. Results showed that although VEGF stimulation promoted VEGFR2 internalization in control endothelial cells (ECs), loss of EMCN prevented VEGFR2 endocytosis. Cell surface analysis revealed a decrease in VEGFR2 following VEGF stimulation in control but not siRNA directed against EMCN-transfected ECs. EMCN depletion resulted in heightened phosphorylation following VEGF stimulation with an increase in total VEGFR2 protein. These results indicate that EMCN modulates VEGFR2 endocytosis and activity and point to EMCN as a potential therapeutic target.-LeBlanc, M. E., Saez-Torres, K. L., Cano, I., Hu, Z., Saint-Geniez, M., Ng, Y.-S., D'Amore, P. A. Glycocalyx regulation of vascular endothelial growth factor receptor 2 activity.

AAV-CRISPR/Cas9-Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro.

Purpose: 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.

Endomucin inhibits VEGF-induced endothelial cell migration, growth, and morphogenesis by modulating VEGFR2 signaling.

Angiogenesis is central to both normal and pathologic processes. Endothelial cells (ECs) express O-glycoproteins that are believed to play important roles in vascular development and stability. Endomucin-1 (EMCN) is a type I O-glycosylated, sialic-rich glycoprotein, specifically expressed by venous and capillary endothelium. Evidence has pointed to a potential role for EMCN in angiogenesis but it had not been directly investigated. In this study, we examined the role of EMCN in angiogenesis by modulating EMCN levels both in vivo and in vitro. Reduction of EMCN in vivo led to the impairment of angiogenesis during normal retinal development in vivo. To determine the cellular basis of this inhibition, gain- and loss-of-function studies were performed in human retinal EC (HREC) in vitro by EMCN over-expression using adenovirus or EMCN gene knockdown by siRNA. We show that EMCN knockdown reduced migration, inhibited cell growth without compromising cell survival, and suppressed tube morphogenesis of ECs, whereas over-expression of EMCN led to increased migration, proliferation and tube formation. Furthermore, knockdown of EMCN suppressed VEGF-induced signaling as measured by decreased phospho-VEGFR2, phospho-ERK1/2 and phospho-p38-MAPK levels. These results suggest a novel role for EMCN as a potent regulator of angiogenesis and point to its potential as a new therapeutic target for angiogenesis-related diseases.

Introduction of the MDM2 T309G Mutation in Primary Human Retinal Epithelial Cells Enhances Experimental Proliferative Vitreoretinopathy.

Purpose: The murine double minute (MDM)2 is a critical negative regulator of the p53 tumor suppressor, and MDM2 SNP309G is associated with a higher risk of proliferative vitreoretinopathy (PVR); in addition, the MDM2 T309G created using clustered regularly interspaced short palindromic repeats (CRISPR)/associated endonuclease (Cas)9 enhances normal rabbit vitreous-induced expression of MDM2 and survival of primary human retinal pigment epithelial (hRPE) cells in vitro. The goal of this study was to determine whether this MDM2 T309G contributes to the development of experimental PVR. Methods: hRPE cells expressing MDM2 T309G or T309T only were treated with vitreous from human PVR donors (HV). The expression of MDM2 and p53 in the treated cells was examined by Western blot. The in vitro vitreous-induced cellular responses, such as contraction were assessed, and PVR was induced by intravitreal injection of the hRPE cells with MDM2 T309G or T309T only into rabbit eyes. Results: Western blot analyses indicated that treatment of hRPE cells with HV led to a significant increase (1.7 ± 0.2-fold) in the expression of MDM2 and a significant decrease in p53 in the cells expressing MDM2 T309G compared with those with MDM2 T309T. In addition, HV promoted contraction of the hRPE cells expressing MDM2 T309G significantly more than those with MDM2 T309T only. Furthermore, MDM2 T309G in the hRPE cells enhanced the development of PVR in a rabbit model. Conclusions: The MDM2 SNP309 in RPE cells enhances their potential of PVR pathogenesis.

Genome editing abrogates angiogenesis in vivo.

Angiogenesis, in which vascular endothelial growth factor receptor (VEGFR) 2 plays an essential role, is associated with a variety of human diseases including proliferative diabetic retinopathy and wet age-related macular degeneration. Here we report that a system of adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) is used to deplete VEGFR2 in vascular endothelial cells (ECs), whereby the expression of SpCas9 is driven by an endothelial-specific promoter of intercellular adhesion molecule 2. We further show that recombinant AAV serotype 1 (rAAV1) transduces ECs of pathologic vessels, and that editing of genomic VEGFR2 locus using rAAV1-mediated CRISPR/Cas9 abrogates angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization. This work establishes a strong foundation for genome editing as a strategy to treat angiogenesis-associated diseases.Abnormal angiogenesis causes many ocular diseases. Here the authors employ CRISPR/Cas9 gene editing technology to silence VEGFR2, a major regulator of angiogenesis, in retinal endothelium and abrogate angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization.

Identification of RUNX1 as a Mediator of Aberrant Retinal Angiogenesis.

Proliferative diabetic retinopathy (PDR) is a common cause of blindness in the developed world's working adult population and affects those with type 1 and type 2 diabetes. We identified Runt-related transcription factor 1 (RUNX1) as a gene upregulated in CD31+ vascular endothelial cells obtained from human PDR fibrovascular membranes (FVMs) via transcriptomic analysis. In vitro studies using human retinal microvascular endothelial cells (HRMECs) showed increased RUNX1 RNA and protein expression in response to high glucose, whereas RUNX1 inhibition reduced HRMEC migration, proliferation, and tube formation. Immunohistochemical staining for RUNX1 showed reactivity in vessels of patient-derived FVMs and angiogenic tufts in the retina of mice with oxygen-induced retinopathy, suggesting that RUNX1 upregulation is a hallmark of aberrant retinal angiogenesis. Inhibition of RUNX1 activity with the Ro5-3335 small molecule resulted in a significant reduction of neovascular tufts in oxygen-induced retinopathy, supporting the feasibility of targeting RUNX1 in aberrant retinal angiogenesis.

Editing VEGFR2 Blocks VEGF-Induced Activation of Akt and Tube Formation.

Purpose: Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in VEGF-induced angiogenesis. The goal of this project was to test the hypothesis that editing genomic VEGFR2 loci using the technology of clustered regularly interspaced palindromic repeats (CRISPR)-associated DNA endonuclease (Cas)9 in Streptococcus pyogenes (SpCas9) was able to block VEGF-induced activation of Akt and tube formation. Methods: Four 20 nucleotides for synthesizing single-guide RNAs based on human genomic VEGFR2 exon 3 loci were selected and cloned into a lentiCRISPR v2 vector, respectively. The DNA fragments from the genomic VEGFR2 exon 3 of transduced primary human retinal microvascular endothelial cells (HRECs) were analyzed by Sanger DNA sequencing, surveyor nuclease assay, and next-generation sequencing (NGS). In the transduced cells, expression of VEGFR2 and VEGF-stimulated signaling events (e.g., Akt phosphorylation) were determined by Western blot analyses; VEGF-induced cellular responses (proliferation, migration, and tube formation) were examined. Results: In the VEGFR2-sgRNA/SpCas9-transduced HRECs, Sanger DNA sequencing indicated that there were mutations, and NGS demonstrated that there were 83.57% insertion and deletions in the genomic VEGFR2 locus; expression of VEGFR2 was depleted in the VEGFR2-sgRNA/SpCas9-transduced HRECs. In addition, there were lower levels of Akt phosphorylation in HRECs with VEGFR2-sgRNA/SpCas9 than those with LacZ-sgRNA/SpCas9, and there was less VEGF-stimulated Akt activation, proliferation, migration, or tube formation in the VEGFR2-depleted HRECs than those treated with aflibercept or ranibizumab. Conclusions: The CRISPR-SpCas9 technology is a potential novel approach to prevention of pathologic angiogenesis.

Orbital Angiogenesis and Lymphangiogenesis in Thyroid Eye Disease: An Analysis of Vascular Growth Factors with Clinical Correlation.

PURPOSE: The human orbit is an environment that is vulnerable to inflammation and edema in the setting of autoimmune thyroid disease. Our study investigated the tenet that orbital adipose tissue lacks lymphatic vessels and analyzed the clinicopathologic differences between patients with acute and chronic thyroid eye disease (TED). The underlying molecular mediators of blood and lymphatic vessel formation within the orbital fat also were evaluated. DESIGN: Retrospective cohort study. PARTICIPANTS: The study included fat specimens from 26 orbits of 15 patients with TED undergoing orbital decompression. Orbital fat specimens from patients without TED as well as cadaveric orbital fat served as controls. METHODS: Tissue specimens were processed as formalin-fixed, paraffin-embedded sections or frozen cryosections for immunohistochemistry. Total RNA was extracted and analyzed via quantitative (real-time) reverse-transcription polymerase chain reaction. Clinicopathologic correlation was made by determining the clinical activity score (CAS) of each patient with TED. MAIN OUTCOME MEASURES: Samples were examined for vascular and lymphatic markers including podoplanin, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and cluster of differentiation 31 (CD31) by immunohistochemistry, as well as for mRNA levels of vascular endothelial growth factor (VEGF), VEGF receptors, semaphorin 3F, neuropilin 1, neuropilin 2, podoplanin, and LYVE-1 by quantitative (real-time) reverse-transcription polymerase chain reaction. RESULTS: Clinicopathologic correlation revealed increased staining of CD31-positive blood vessels in patients with acute TED with a CAS more than 4, as well as rare staining of podoplanin-positive lymphatic vessels within acutely inflamed orbital fat tissue. Additionally, quantitative (real-time) reverse-transcription polymerase chain reaction analysis demonstrated increased expression of VEGF receptor (VEGFR) 2 as well as VEGF signaling molecules VEGF-A, VEGF-C, and VEGF-D. CONCLUSIONS: In acute TED, compared with chronic TED and control orbital fat, there is increased blood vessel density, suggesting neovascularization and rare lymphatic vessels suggestive of limited lymphangiogenesis. This proangiogenic and prolymphangiogenic microenvironment is likely the result of the increased expression of VEGFR-2, VEGF-A, VEGF-C, and VEGF-D. These findings imply that orbital edema in acute TED may be mediated, in part, by both the formation of new, immature blood vessels and the formation of lymphatic capillaries that are functionally incapable of draining interstitial fluid.

Prevention of Proliferative Vitreoretinopathy by Suppression of Phosphatidylinositol 5-Phosphate 4-Kinases.

PURPOSE: Previous studies have shown that vitreous stimulates degradation of the tumor suppressor protein p53 and that knockdown of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Kα and -ß) abrogates proliferation of p53-deficient cells. The purpose of this study was to determine whether vitreous stimulated expression of PI5P4Kα and -ß and whether suppression of PI5P4Kα and -ß would inhibit vitreous-induced cellular responses and experimental proliferative vitreoretinopathy (PVR). METHODS: PI5P4Kα and -ß encoded by PIP4K2A and 2B, respectively, in human ARPE-19 cells were knocked down by stably expressing short hairpin (sh)RNA directed at human PIP4K2A and -2B. In addition, we rescued expression of PI5P4Kα and -ß by re-expressing mouse PIP4K2A and -2B in the PI5P4Kα and -ß knocked-down ARPE-19 cells. Expression of PI5P4Kα and -ß was determined by Western blot and immunofluorescence. The following cellular responses were monitored: cell proliferation, survival, migration, and contraction. Moreover, the cell potential of inducing PVR was examined in a rabbit model of PVR effected by intravitreal cell injection. RESULTS: We found that vitreous enhanced expression of PI5P4Kα and -ß in RPE cells and that knocking down PI5P4Kα and -ß abrogated vitreous-stimulated cell proliferation, survival, migration, and contraction. Re-expression of mouse PIP4Kα and -ß in the human PI5P4Kα and -ß knocked-down cells recovered the loss of vitreous-induced cell contraction. Importantly, suppression of PI5P4Kα and -ß abrogated the pathogenesis of PVR induced by intravitreal cell injection in rabbits. Moreover, we revealed that expression of PI5P4Kα and -ß was abundant in epiretinal membranes from PVR grade C patients. CONCLUSIONS: The findings from this study indicate that PI5P4Kα and -ß could be novel therapeutic targets for the treatment of PVR.

The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells.

The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR.

Endomucin prevents leukocyte-endothelial cell adhesion and has a critical role under resting and inflammatory conditions.

Endomucin is a membrane-bound glycoprotein expressed luminally by endothelial cells that line postcapillary venules, a primary site of leukocyte recruitment during inflammation. Here we show that endomucin abrogation on quiescent endothelial cells enables neutrophils to adhere firmly, via LFA-1-mediated binding to ICAM-1 constitutively expressed by endothelial cells. Moreover, TNF-α stimulation downregulates cell surface expression of endomucin concurrent with increased expression of adhesion molecules. Adenovirus-mediated expression of endomucin under inflammatory conditions prevents neutrophil adhesion in vitro and reduces the infiltration of CD45(+) and NIMP-R14(+) cells in vivo. These results indicate that endomucin prevents leukocyte contact with adhesion molecules in non-inflamed tissues and that downregulation of endomucin is critical to facilitate adhesion of leukocytes into inflamed tissues.

Neuropilin 1 Receptor Is Up-Regulated in Dysplastic Epithelium and Oral Squamous Cell Carcinoma.

Neuropilins are receptors for disparate ligands, including proangiogenic factors such as vascular endothelial growth factor and inhibitory class 3 semaphorin (SEMA3) family members. Differentiated cells in skin epithelium and cutaneous squamous cell carcinoma highly express the neuropilin-1 (NRP1) receptor. We examined the expression of NRP1 in human and mouse oral mucosa. NRP1 was significantly up-regulated in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). NRP1 receptor localized to the outer suprabasal epithelial layers in normal tongue, an expression pattern similar to the normal skin epidermis. However, dysplastic tongue epithelium and OSCC up-regulated NRP1 in basal and proliferating epithelial layers, a profile unseen in cutaneous squamous cell carcinoma. NRP1 up-regulation is observed in a mouse carcinogen-induced OSCC model and in human tongue OSCC biopsies. Human OSCC cell lines express NRP1 protein in vitro and in mouse tongue xenografts. Sites of capillary infiltration into orthotopic OSCC tumors correlate with high NRP1 expression. HSC3 xenografts, which express the highest NRP1 levels of the cell lines examined, showed massive intratumoral lymphangiogenesis. SEMA3A inhibited OSCC cell migration, suggesting that the NRP1 receptor was bioactive in OSCC. In conclusion, NRP1 is regulated in the oral epithelium and is selectively up-regulated during epithelial dysplasia. NRP1 may function as a reservoir to sequester proangiogenic ligands within the neoplastic compartment, thereby recruiting neovessels toward tumor cells.

Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy.

PURPOSE: Epiretinal fibrovascular membranes (FVMs) are a hallmark of proliferative diabetic retinopathy (PDR). Surgical removal of FVMs is often indicated to treat tractional retinal detachment. This potentially informative pathological tissue is usually disposed of after surgery without further examination. We developed a method for isolating and characterizing cells derived from FVMs and correlated their expression of specific markers in culture with that in tissue. METHODS: FVMs were obtained from 11 patients with PDR during diabetic vitrectomy surgery and were analyzed with electron microscopy (EM), comparative genomic hybridization (CGH), immunohistochemistry, and/or digested with collagenase II for cell isolation and culture. Antibody arrays and enzyme-linked immunosorbent assay (ELISA) were used to profile secreted angiogenesis-related proteins in cell culture supernatants. RESULTS: EM analysis of the FVMs showed abnormal vessels composed of endothelial cells with large nuclei and plasma membrane infoldings, loosely attached perivascular cells, and stromal cells. The cellular constituents of the FVMs lacked major chromosomal aberrations as shown with CGH. Cells derived from FVMs (C-FVMs) could be isolated and maintained in culture. The C-FVMs retained the expression of markers of cell identity in primary culture, which define specific cell populations including CD31-positive, alpha-smooth muscle actin-positive (SMA), and glial fibrillary acidic protein-positive (GFAP) cells. In primary culture, secretion of angiopoietin-1 and thrombospondin-1 was significantly decreased in culture conditions that resemble a diabetic environment in SMA-positive C-FVMs compared to human retinal pericytes derived from a non-diabetic donor. CONCLUSIONS: C-FVMs obtained from individuals with PDR can be isolated, cultured, and profiled in vitro and may constitute a unique resource for the discovery of cell signaling mechanisms underlying PDR that extends beyond current animal and cell culture models.