HCC EV ECG score: An extracellular vesicle-based protein assay for detection of early-stage hepatocellular carcinoma.

BACKGROUND AND AIMS: The sensitivity of current surveillance methods for detecting early-stage hepatocellular carcinoma (HCC) is suboptimal. Extracellular vesicles (EVs) are promising circulating biomarkers for early cancer detection. In this study, we aim to develop an HCC EV-based surface protein assay for early detection of HCC. APPROACH AND RESULTS: Tissue microarray was used to evaluate four potential HCC-associated protein markers. An HCC EV surface protein assay, composed of covalent chemistry-mediated HCC EV purification and real-time immuno-polymerase chain reaction readouts, was developed and optimized for quantifying subpopulations of EVs. An HCC EV ECG score, calculated from the readouts of three HCC EV subpopulations ( E pCAM + CD63 + , C D147 + CD63 + , and G PC3 + CD63 + HCC EVs), was established for detecting early-stage HCC. A phase 2 biomarker study was conducted to evaluate the performance of ECG score in a training cohort ( n  = 106) and an independent validation cohort ( n  = 72).Overall, 99.7% of tissue microarray stained positive for at least one of the four HCC-associated protein markers (EpCAM, CD147, GPC3, and ASGPR1) that were subsequently validated in HCC EVs. In the training cohort, HCC EV ECG score demonstrated an area under the receiver operating curve (AUROC) of 0.95 (95% confidence interval [CI], 0.90-0.99) for distinguishing early-stage HCC from cirrhosis with a sensitivity of 91% and a specificity of 90%. The AUROCs of the HCC EV ECG score remained excellent in the validation cohort (0.93; 95% CI, 0.87-0.99) and in the subgroups by etiology (viral: 0.95; 95% CI, 0.90-1.00; nonviral: 0.94; 95% CI, 0.88-0.99). CONCLUSION: HCC EV ECG score demonstrated great potential for detecting early-stage HCC. It could augment current surveillance methods and improve patients' outcomes.

Understanding the Role of Endothelial Cells in Glioblastoma: Mechanisms and Novel Treatments.

Glioblastoma is a highly aggressive neoplasm and the most common primary malignant brain tumor. Endothelial tissue plays a critical role in glioblastoma growth and progression, facilitating angiogenesis, cellular communication, and tumorigenesis. In this review, we present an up-to-date and comprehensive summary of the role of endothelial cells in glioblastomas, along with an overview of recent developments in glioblastoma therapies and tumor endothelial marker identification.

Identification of epithelial membrane protein 2 (EMP2) as a molecular marker and correlate for angiogenesis in meningioma.

PURPOSE: Although intracranial meningiomas are the most common primary brain tumor in adults, treatment options are few and have traditionally been limited to surgical resection and radiotherapy. Additional targeted therapies and biomarkers are needed, especially as complete surgical resection is frequently not feasible in many patients. METHODS: Non-pathologic brain tissue from 3 patients undergoing routine autopsies and tumor specimens from 16 patients requiring surgical resection for meningioma were collected. EMP2 protein expression was evaluated by immunohistochemistry and western blot analysis. EMP2 mRNA expression was also investigated using surgical specimens and validated by analysis of several independent NCBI GEO databases. RESULTS: EMP2 mRNA expression levels were found to be higher in meningioma relative to non-pathologic meninges (P = 0.0013) and brain (P = 0.0011). Concordantly, strong EMP2 protein expression was demonstrated in 100% of meningioma specimens from all 16 patients, with no observable protein expression in normal brain tissue samples from 3 subjects (P < 0.001). EMP2 expression was confirmed by western blot analysis in five samples, with EMP2 protein intensity positively correlating with histologic staining score (R2 = 0.780; P = 0.047). No association was found between EMP2 mRNA or protein levels and WHO grade or markers of proliferation. However, EMP2 expression was positively associated with an angiomatous pattern on histologic evaluation (P = 0.0597), VEGF-A mRNA expression (P < 0.001), and clinical markers of tumor vascularity such as operative blood loss (P = 0.037). CONCLUSIONS: EMP2 is not found in normal brain tissue, yet has shown consistently high mRNA and protein expression in meningiomas, and may serve as a useful molecular marker for these tumors.

Epithelial membrane protein 2 (EMP2) deficiency alters placental angiogenesis, mimicking features of human placental insufficiency.

Epithelial membrane protein-2 (EMP2) is a tetraspan protein predicted to regulate placental development. Highly expressed in secretory endometrium and trophectoderm cells, previous studies suggest that it may regulate implantation by orchestrating the surface expression of integrins and other membrane proteins. In order to test the role of EMP2 in pregnancy, mice lacking EMP2 (Emp2-/- ) were generated. Emp2-/- females are fertile but have reduced litter sizes when carrying Emp2-/- but not Emp2+/- fetuses. Placentas of Emp2-/- fetuses exhibit dysregulation in pathways related to neoangiogenesis, coagulation, and oxidative stress, and have increased fibrin deposition and altered vasculature. Given that these findings often occur due to placental insufficiency resulting in an oxygen-poor environment, the expression of hypoxia-inducible factor-1 alpha (HIF-1α) was examined. Placentas from Emp2-/- fetuses had increased total HIF-1α expression in large part through an increase in uterine NK (uNK) cells, demonstrating a unique interplay between uNK cells and trophoblasts modulated through EMP2. To determine if these results translated to human pregnancy, placentas from normal, term deliveries or those complicated by placental insufficiency resulting in intrauterine growth restriction (IUGR) were stained for EMP2. EMP2 was significantly reduced in both villous and extravillous trophoblast populations in IUGR placentas. Experiments in vitro using human trophoblast cells lines indicate that EMP2 modulates angiogenesis by altering HIF-1α expression. Our results reveal a novel role for EMP2 in regulating trophoblast function and vascular development in mice and humans, and suggest that it may be a new biomarker for placental insufficiency. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Epithelial membrane protein-2 (EMP2) promotes angiogenesis in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor and is associated with an extremely poor clinical prognosis. One pathologic hallmark of GBM is excessive vascularization with abnormal blood vessels. Extensive investigation of anti-angiogenic therapy as a treatment for recurrent GBM has been performed. Bevacizumab, a monoclonal anti-vascular endothelial growth factor A (VEGF-A), suggests a progression-free survival benefit but no overall survival benefit. Developing novel anti-angiogenic therapies are urgently needed in controlling GBM growth. In this study, we demonstrate tumor expression of epithelial membrane protein-2 (EMP2) promotes angiogenesis both in vitro and in vivo using cell lines from human GBM. Mechanistically, this pro-angiogenic effect of EMP2 was partially through upregulating tumor VEGF-A levels. A potential therapeutic effect of a systemic administration of anti-EMP2 IgG1 on intracranial xenografts was observed resulting in both significant reduction of tumor load and decreased tumor vasculature. These results suggest the potential for anti-EMP2 IgG1 as a promising novel anti-angiogenic therapy for GBM. Further investigation is needed to fully understand the molecular mechanisms how EMP2 modulates GBM pathogenesis and progression and to further characterize anti-EMP2 therapy in GBM.

Epithelial membrane protein-2 (EMP2) activates Src protein and is a novel therapeutic target for glioblastoma.

Despite recent advances in molecular classification, surgery, radiotherapy, and targeted therapies, the clinical outcome of patients with malignant brain tumors remains extremely poor. In this study, we have identified the tetraspan protein epithelial membrane protein-2 (EMP2) as a potential target for glioblastoma (GBM) killing. EMP2 had low or undetectable expression in normal brain but was highly expressed in GBM as 95% of patients showed some expression of the protein. In GBM cells, EMP2 enhanced tumor growth in vivo in part by up-regulating αvß3 integrin surface expression, activating focal adhesion kinase and Src kinases, and promoting cell migration and invasion. Consistent with these findings, EMP2 expression significantly correlated with activated Src kinase in patient samples and promoted tumor cell invasion using intracranial mouse models. As a proof of principle to determine whether EMP2 could serve as a target for therapy, cells were treated using specific anti-EMP2 antibody reagents. These reagents were effective in killing GBM cells in vitro and in reducing tumor load in subcutaneous mouse models. These results support the role of EMP2 in the pathogenesis of GBM and suggest that anti-EMP2 treatment may be a novel therapeutic treatment.

Omega-3 reverses the metabolic and epigenetically regulated placental phenotype acquired from preconceptional and peri-conceptional exposure to air pollutants.

Air pollution is detrimental to pregnancy adversely affecting maternal and child health. Our objective was to unravel epigenetic mechanisms mediating the effect of preconception, periconception, and gestational exposure to inhaled air pollutants (AP) upon the maternal and placental-fetal phenotype and explore the benefit of an omega-3 rich dietary intervention. To this end, we investigated intranasal instilled AP during 8 weeks of preconception, periconception, and gestation (G; D0 to 18) upon GD16-19 maternal mouse metabolic status, placental nutrient transporters, placental-fetal size, and placental morphology. Prepregnant mice were glucose intolerant and insulin resistant, while pregnant mice were glucose intolerant but displayed no major placental macro-nutrient transporter changes, except for an increase in CD36. Placentas revealed inflammatory cellular infiltration with cellular edema, necrosis, hemorrhage, and an increase in fetal body weight. Upon examination of placental genome-wide epigenetic processes of DNA sequence specific 5'-hydroxymethylation (5'-hmC) and 5'-methylation (5'-mC) upon RNA sequenced gene expression profiles, revealed changes in key metabolic, inflammatory, transcriptional, and cellular processing genes and pathways. An omega-3 rich anti-inflammatory diet from preconception (8 weeks) through periconception and gestation (GD0-18), ameliorated all these maternal and placental-fetal adverse effects. We conclude that preconceptional, periconceptional and gestational exposures to AP incite a maternal inflammatory response resulting in features of pre-existing maternal diabetes mellitus with injury to the placental-fetal unit. DNA 5'-mC more than 5'-hmC mediated AP induced maternal inflammatory and metabolic dysregulation which together alter placental gene expression and phenotype. A dietary intervention partially reversing these adversities provides possibilities for a novel nutrigenomic therapeutic strategy.

Gestational exposure to air pollutants perturbs metabolic and placenta-fetal phenotype.

Air pollution (AP) is detrimental to pregnancies including increasing risk factors of gestational diabetes mellitus. We hypothesized that exposure to AP causes cardiovascular and metabolic disruption thereby altering placental gene expression, which in turn affects the placental phenotype and thereby embryonic/fetal development. To test this hypothesis, we investigated the impact of intra-nasal instilled AP upon gestational day 16-19 maternal mouse cardiovascular and metabolic status, placental nutrient transporters, and placental-fetal size and morphology. To further unravel mechanisms, we also examined placental total DNA 5'-hydroxymethylation and bulk RNA sequenced gene expression profiles. AP exposed pregnant mice and fetuses were tachycardic with a reduction in maternal left ventricular fractional shortening and increased uterine artery with decreased umbilical artery systolic peak velocities. In addition, they were hyperglycemic, glucose intolerant and insulin resistant, with changes in placental glucose (Glut3) and fatty acid (Fatp1 & Cd36) transporters, and a spatial disruption of cells expressing Glut10 that imports L-dehydroascorbic acid in protecting against oxidative stress. Placentas revealed inflammatory cellular infiltration with associated cellular edema and necrosis, with dilated vascular spaces and hemorrhage. Placental and fetal body weights decreased in mid-gestation with a reduction in brain cortical thickness emerging in late gestation. Placental total DNA 5'-hydroxymethylation was 2.5-fold higher, with perturbed gene expression profiles involving key metabolic, inflammatory, transcriptional, cellular polarizing and processing genes and pathways. We conclude that gestational exposure to AP incites a maternal inflammatory response resulting in features mimicking maternal gestational diabetes mellitus with altered placental DNA 5'-hydroxymethylation, gene expression, and associated injury.

Manuka Honey Inhibits Human Breast Cancer Progression in Preclinical Models.

Manuka honey (MH) exhibits potential antitumor activity in preclinical models of a number of human cancers. Treatment in vitro with MH at concentrations ranging from 0.3 to 5.0% (w/v) led to significant dose-dependent inhibition of proliferation of human breast cancer MCF-7 cells, but anti-proliferative effects of MH were less pronounced in MDA-MB-231 breast cancer cells. Effects of MH were also tested on non-malignant human mammary epithelial cells (HMECs) at 2.5% w/v, and it was found that MH reduced the proliferation of MCF-7 cells but not that of HMECs. Notably, the antitumor activity of MH was in the range of that exerted by treatment of MCF-7 cells with the antiestrogen tamoxifen. Further, MH treatment stimulated apoptosis of MCF-7 cells in vitro, with most cells exhibiting acute and significant levels of apoptosis that correlated with PARP activation. Additionally, the effects of MH induced the activation of AMPK and inhibition of AKT/mTOR downstream signaling. Treatment of MCF7 cells with increased concentrations of MH induced AMPK phosphorylation in a dose-dependent manner that was accompanied by inhibition of phosphorylation of AKT and mTOR downstream effector protein S6. In addition, MH reduced phosphorylated STAT3 levels in vitro, which may correlate with MH and AMPK-mediated anti-inflammatory properties. Further, in vivo, MH administered alone significantly inhibited the growth of established MCF-7 tumors in nude mice by 84%, resulting in an observable reduction in tumor volume. Our findings highlight the need for further research into the use of natural compounds, such as MH, for antitumor efficacy and potential chemoprevention and investigation of molecular pathways underlying these actions.

Safety and Efficacy of Laser Interstitial Thermal Therapy as Upfront Therapy in Primary Glioblastoma and IDH-Mutant Astrocytoma: A Meta-Analysis.

Although primary studies have reported the safety and efficacy of LITT as a primary treatment in glioma, they are limited by sample sizes and institutional variation in stereotactic parameters such as temperature and laser power. The current literature has yet to provide pooled statistics on outcomes solely for primary brain tumors according to the 2021 WHO Classification of Tumors of the Central Nervous System (WHO CNS5). In the present study, we identify recent articles on primary CNS neoplasms treated with LITT without prior intervention, focusing on relationships with molecular profile, PFS, and OS. This meta-analysis includes the extraction of data from primary sources across four databases using the Covidence systematic review manager. The pooled data suggest LITT may be a safe primary management option with tumor ablation rates of 94.8% and 84.6% in IDH-wildtype glioblastoma multiforme (GBM) and IDH-mutant astrocytoma, respectively. For IDH-wildtype GBM, the pooled PFS and OS were 5.0 and 9.0 months, respectively. Similar to rates reported in the prior literature, the neurologic and non-neurologic complication rates for IDH-wildtype GBM were 10.3% and 4.8%, respectively. The neurologic and non-neurologic complication rates were somewhat higher in the IDH-mutant astrocytoma cohort at 33% and 8.3%, likely due to a smaller cohort size.

Epithelial Membrane Protein 2 (EMP2) Blockade Attenuates Pathological Neovascularization in Murine Oxygen-Induced Retinopathy.

Purpose: Retinopathy of prematurity (ROP) results from postnatal hyperoxia exposure in premature infants and is characterized by aberrant neovascularization of retinal blood vessels. Epithelial membrane protein-2 (EMP2) regulates hypoxia-inducible factor (HIF)-induced vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line and genetic knock-out of Emp2 in a murine oxygen-induced retinopathy (OIR) model attenuates neovascularization. We hypothesize that EMP2 blockade via intravitreal injection protects against neovascularization. Methods: Ex vivo choroid sprouting assay was performed, comparing media and human IgG controls versus anti-EMP2 antibody (Ab) treatment. In vivo, eyes from wild-type (WT) mice exposed to hyperoxia from postnatal (P) days 7 to 12 were treated with P12 intravitreal injections of control IgG or anti-EMP2 Abs. Neovascularization was assessed at P17 by flat mount imaging. Local and systemic effects of anti-EMP2 Ab treatment were assessed. Results: Choroid sprouts treated with 30 µg/mL of anti-EMP2 Ab demonstrated a 48% reduction in vessel growth compared to control IgG-treated sprouts. Compared to IgG-treated controls, WT OIR mice treated with 4 µg/g of intravitreal anti-EMP2 Ab demonstrated a 42% reduction in neovascularization. They demonstrated down-regulation of retinal gene expression in pathways related to vasculature development and up-regulation in genes related to fatty acid oxidation and tricarboxylic acid cycle respiratory electron transport, compared to controls. Anti-EMP2 Ab-treated OIR mice did not exhibit gross retinal histologic abnormalities, vision transduction abnormalities, or weight loss. Conclusions: Our results suggest that EMP2 blockade could be a local and specific treatment modality for retinal neovascularization in oxygen-induced retinopathies, without systemic adverse effects.

89Zr-ImmunoPET for the Specific Detection of EMP2-Positive Tumors.

Epithelial membrane protein-2 (EMP2) is upregulated in a number of tumors and therefore remains a promising target for mAb-based therapy. In the current study, image-guided therapy for an anti-EMP2 mAb was evaluated by PET in both syngeneic and immunodeficient cancer models expressing different levels of EMP2 to enable a better understanding of its tumor uptake and off target accumulation and clearance. The therapeutic efficacy of the anti-EMP2 mAb was initially evaluated in high- and low-expressing tumors, and the mAb reduced tumor load for the high EMP2-expressing 4T1 and HEC-1-A tumors. To create an imaging agent, the anti-EMP2 mAb was conjugated to p-SCN-Bn-deferoxamine (DFO) and radiolabeled with 89Zr. Tumor targeting and tissue biodistribution were evaluated in syngeneic tumor models (4T1, CT26, and Panc02) and human tumor xenograft models (Ramos, HEC-1-A, and U87MG/EMP2). PET imaging revealed radioactive accumulation in EMP2-positive tumors within 24 hours after injection, and the signal was retained for 5 days. High specific uptake was observed in tumors with high EMP2 expression (4T1, CT26, HEC-1-A, and U87MG/EMP2), with less accumulation in tumors with low EMP2 expression (Panc02 and Ramos). Biodistribution at 5 days after injection revealed that the tumor uptake ranged from 2 to approximately 16%ID/cc. The results show that anti-EMP2 mAbs exhibit EMP2-dependent tumor uptake with low off-target accumulation in preclinical cancer models. The development of improved anti-EMP2 Ab fragments may be useful to track EMP2-positive tumors for subsequent therapeutic interventions.

EMP2 Serves as a Functional Biomarker for Chemotherapy-Resistant Triple-Negative Breast Cancer.

Breast cancer (BC) remains among the most commonly diagnosed cancers in women worldwide. Triple-negative BC (TNBC) is a subset of BC characterized by aggressive behavior, a high risk of distant recurrence, and poor overall survival rates. Chemotherapy is the backbone for treatment in patients with TNBC, but outcomes remain poor compared to other BC subtypes, in part due to the lack of recognized functional targets. In this study, the expression of the tetraspan protein epithelial membrane protein 2 (EMP2) was explored as a predictor of TNBC response to standard chemotherapy. We demonstrate that EMP2 functions as a prognostic biomarker for patients treated with taxane-based chemotherapy, with high expression at both transcriptomic and protein levels following treatment correlating with poor overall survival. Moreover, we show that targeting EMP2 in combination with docetaxel reduces tumor load in syngeneic and xenograft models of TNBC. These results provide support for the prognostic and therapeutic potential of this tetraspan protein, suggesting that anti-EMP2 therapy may be beneficial for the treatment of select chemotherapy-resistant TNBC tumors.

Epithelial membrane protein 2 (EMP2): A systematic review of its implications in pathogenesis.

OBJECTIVES: Epithelial membrane protein 2 (EMP2) is a cell surface protein composed of approximately 160 amino acids and encoded by the growth arrest-specific 3 (GAS3)/peripheral myelin protein 22 kDa (PMP22) gene family. Although EMP2 expression has been investigated in several diseases, much remains unknown regarding its mechanism of action and the extent of its role in pathogenesis. Our aim was to perform a systematic review on the involvement of EMP2 in disease processes and the current usage of anti-EMP2 therapies. METHODS: A Boolean search of the English-language medical literature was performed. PubMed, Scopus, Cochrane, and Web of Science were used to identify relevant citations. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: 52 studies met the inclusion criteria for qualitative analysis. Of those, 28 (53.8%) were human-only studies, 11 (21.2%) were animal-only studies, and 13 (25%) studies included both human and animal models. Furthermore, 34 (65.4%) studies focused on EMP2's role in neoplasms, while the remaining 18 (34.6%) articles evaluated its role in other pathologies. CONCLUSION: Overall, the evidence suggests the mechanisms of action of EMP2 are context dependent. Promising results have been produced by utilizing EMP2 as a biomarker and therapeutic target. More studies are warranted to better understand the mechanism and comprehend the role of EMP2 in the pathogenesis of diseases.

Exploring the Past, Present, and Future of Anti-Angiogenic Therapy in Glioblastoma.

Glioblastoma, a WHO grade IV astrocytoma, constitutes approximately half of malignant tumors of the central nervous system. Despite technological advancements and aggressive multimodal treatment, prognosis remains dismal. The highly vascularized nature of glioblastoma enables the tumor cells to grow and invade the surrounding tissue, and vascular endothelial growth factor-A (VEGF-A) is a critical mediator of this process. Therefore, over the past decade, angiogenesis, and more specifically, the VEGF signaling pathway, has emerged as a therapeutic target for glioblastoma therapy. This led to the FDA approval of bevacizumab, a monoclonal antibody designed against VEGF-A, for treatment of recurrent glioblastoma. Despite the promising preclinical data and its theoretical effectiveness, bevacizumab has failed to improve patients' overall survival. Furthermore, several other anti-angiogenic agents that target the VEGF signaling pathway have also not demonstrated survival improvement. This suggests the presence of other compensatory angiogenic signaling pathways that surpass the anti-angiogenic effects of these agents and facilitate vascularization despite ongoing VEGF signaling inhibition. Herein, we review the current state of anti-angiogenic agents, discuss potential mechanisms of anti-angiogenic resistance, and suggest potential avenues to increase the efficacy of this therapeutic approach.

Expression of thyroid transcription factor-1 in normal endometrium is associated with risk of endometrial cancer development.

Thyroid transcription factor-1 (TTF-1) is a DNA-binding protein that is mainly expressed in thyroid and lung tissue, but has also been found in gynecologic tissue. Recent studies have suggested that TTF-1 has tumor suppressor function in lung adenocarcinoma models. In the current study, we examined whether expression of TTF-1 in benign endometrium and endometrial hyperplasia might impact on the risk of developing endometrial cancer. Formalin-fixed paraffin-embedded endometrial tissues obtained from 535 cases were used to construct an endometrial tissue microarray. One hundred fifty of 207 patients had multiple serial endometrial specimens including 46 patients who progressed to endometrial cancer. The tissue microarray included a range of histopathologies including benign endometrium (n=231), simple hyperplasia (n=105), complex hyperplasia (n=36), simple atypical hyperplasia (n=10), complex atypical hyperplasia (n=44), and endometrial carcinoma (n=109). Expression of TTF-1 by immunohistochemistry in benign endometrium and endometrial hyperplasia was correlated with progression to cancer and clinical features known to be associated with increased risk of developing endometrial cancer. Carcinoma specimens showed a significantly greater expression of TTF-1 compared with benign endometrium and non-atypical hyperplasia (P=0.0007 and P=0.05). Presence of TTF-1 expression in benign endometrium was associated with a significantly decreased risk of cancer development (P=0.003, hazards ratio=0.104, 95% CI: 0.024-0.455). TTF-1 expression in hyperplasia did not significantly correlate with progression to cancer. The data from our study show that TTF-1 expression in normal endometrium is associated with a reduced risk of endometrial cancer development. This observation suggests that TTF-1 might function as a tumor suppressor in endometrial tissue. TTF-1 expression in normal endometrium could potentially provide clinically useful information as a biomarker for the risk of endometrial cancer.

Anti-EMP2 diabody blocks epithelial membrane protein 2 (EMP2) and FAK mediated collagen gel contraction in ARPE-19 cells.

Epithelial membrane protein 2 (EMP2) regulates collagen gel contraction by the retinal pigment epithelium cell line ARPE-19 by modulating FAK activation. Collagen gel contraction is one in vitro model for an aberrant wound healing response, proliferative vitreoretinopathy (PVR), which occurs as a complication of severe ocular trauma. The purpose of this study is to investigate whether EMP2 specific recombinant diabody decreases activation of FAK and collagen gel contraction in ARPE-19. Anti-EMP2 diabody was recombinantly constructed from a human phage library-derived clone selected for reactivity against an extracellular domain of human EMP2. ARPE-19 cells were exposed to an anti-EMP2 or control diabody, and toxicity, adhesion, and migration were assessed respectively through toluidine blue exclusion, binding to collagen type 1, and a migration assay. Collagen gel contraction was assessed using an in vitro assay. FAK activation was evaluated using Western blot. Exposure to anti-EMP2 diabody, resulted in a 75% reduction in EMP2 protein levels at 4 h. No significant toxicity was observed with anti-EMP2 diabody at levels that maximally reduced EMP2. Anti-EMP2 diabody, but not control diabody, significantly reduced collagen gel contraction (p < 0.001), without changes in adhesion or migration. Concordantly, anti-EMP2 diabody as compared to a control diabody reduced collagen stimulated FAK activation (p = 0.01). Anti-EMP2 diabody decreases EMP2 protein levels, FAK activation, and collagen gel contraction by ARPE-19 cells without an adverse effect on cell survival. Modulation of EMP2 using anti-EMP2 diabody could be a new approach for targeting EMP2 and pathologic consequences associated with EMP2.

Epithelial membrane protein 2 (EMP2) regulates hypoxia-induced angiogenesis in the adult retinal pigment epithelial cell lines.

Pathologic retinal neovascularization is a potentially blinding consequence seen in many common diseases including diabetic retinopathy, retinopathy of prematurity, and retinal vaso-occlusive diseases. This study investigates epithelial membrane protein 2 (EMP2) and its role as a possible modulator of angiogenesis in human retinal pigment epithelium (RPE) under hypoxic conditions. To study its effects, the RPE cell line ARPE-19 was genetically modified to either overexpress EMP2 or knock down its levels, and RNA sequencing and western blot analysis was performed to confirm the changes in expression at the RNA and protein level, respectively. Protein expression was evaluated under both normoxic conditions or hypoxic stress. Capillary tube formation assays with human umbilical vein endothelial cells (HUVEC) were used to evaluate functional responses. EMP2 expression was found to positively correlate with expression of pro-angiogenic factors HIF1α and VEGF at both mRNA and protein levels under hypoxic conditions. Mechanistically, EMP2 stabilized HIF1α expression through downregulation of von Hippel Lindau protein (pVHL). EMP2 mediated changes in ARPE-19 cells were also found to alter the secretion of a paracrine factor(s) in conditioned media that can regulate HUVEC migration and capillary tube formation in in vitro functional angiogenesis assays. This study identifies EMP2 as a potential mediator of angiogenesis in a human RPE cell line. EMP2 levels positively correlate with pro-angiogenic mediators HIF1α and VEGF, and mechanistically, EMP2 regulates HIF1α through downregulation of pVHL. This study supports further investigation of EMP2 as a promising novel target for therapeutic treatment of pathologic neovascularization in the retina.

Peripheral myelin protein-22 (PMP22) modulates alpha 6 integrin expression in the human endometrium.

BACKGROUND: PMP22, a member of the GAS3 family of tetraspan proteins, is associated with a variety of neurological diseases. Previous studies have shown that PMP22 is expressed in proliferative endometrium, but its function within this tissue is poorly understood. In this study, we first characterized the expression of PMP22 in the human menstrual cycle and began to characterize its function in the endometrium. METHODS: Using a combination of immunohistochemistry and quantitative PCR, we characterized the expression of PMP22 in both proliferative and secretory endometrium. Differences in PMP22 expression between proliferative and secretory endometrium were determined using a Mann-Whitney U test. In order to investigate the influence of PMP22 on α6 integrin expression, cells were created that ectopically overexpressed PMP22 or expressed a siRNA to inhibit its expression. These cells were analyzed for changes in integrins and binding to extracellular matrices. RESULTS: In this study, we show that PMP22 expression is higher in proliferative phase than secretory phase. Functionally, we have begun to characterize the functional significance of this expression. Previous studies have suggested a link between PMP22 and α6 integrin, and therefore we asked whether PMP22 could associate or potentially modulate the expression of α6 integrin. Expression of both PMP22 and α6 integrin were detectable in endometrial epithelial and stromal cells, and we show that both proteins can associate and colocalize with each other. To understand if PMP22 directly altered the expression of a6 integrin, we examined cell lines with modulated levels of the protein. Overexpression of PMP22 was sufficient to increase α6 integrin surface expression with a concominant increase in binding to the extracellular matrix laminin, while a reduction in PMP22 suppressed α6 integrin surface expression. CONCLUSION: These findings suggest a physiologic role for PMP22 on the expression of α6 integrin. We predict that this may be important for the maintainence of endometrial integrity and to the disease biology associated with altered levels of α6 integrin expression in the endometrium.

Epithelial membrane protein 2 (Emp2) modulates innate immune cell population recruitment at the maternal-fetal interface.

Epithelial membrane protein 2 (EMP2) is a tetraspan membrane protein that has been revealed in cancer and placental models to mediate a number of vascular responses. Recently, Emp2 modulation has been shown to have an immunologic effect on uterine NK cell recruitment in the mouse placenta. Given the importance of immune cell populations on both placental vascularization and maternal immune tolerance of the developing fetus, we wanted to better characterize the immunologic effects of Emp2 at the placental-fetal interface. We performed flow cytometry of WT and Emp2 KO C57Bl/6 mouse uterine horns at GD12.5 to characterize immune cell populations localized to the various components of the maternal-fetal interface. We found that Emp2 KO decidua and placenta showed an elevated overall percentage of CD45+ cells compared to WT. Characterization of CD45+ cells in the decidua of Emp2 KO dams revealed an increase in NK cells, whereas in the placenta, Emp2 KO dams showed an increased percentage of M1 macrophages (with an increased ratio of M1/M2 macrophages). Given the differences detected in uNK cell populations in the decidua, we further characterized the interaction between Emp2 genetic KO and NK cell deletion via anti-asialo GM1 antibody injections. While the double knock-out of Emp2 and NK cells did not alter individual pup birthweight, it significantly reduced total litter weight and size by ∼50 %. In conclusion, Emp2 appears to regulate uNK and macrophage cell populations in pregnancy.