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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

ALDH1 expression predicts progression of premalignant lesions to cancer in Type I endometrial carcinomas.

In type 1 endometrial cancer, unopposed estrogen stimulation is thought to lead to endometrial hyperplasia which precedes malignant progression. Recent data from our group and others suggest that ALDH activity mediates stemness in endometrial cancer, but while aldehyde dehydrogenase 1 (ALDH1) has been suggested as a putative cancer stem cell marker in several cancer types, its clinical and prognostic value in endometrial cancer remains debated. The aim of this study was to investigate the clinical value of ALDH1 expression in endometrial hyperplasia and to determine its ability to predict progression to endometrial cancer. Interrogation of the TCGA database revealed upregulation of several isoforms in endometrial cancer, of which the ALDH1 isoforms collectively constituted the largest group. To translate its expression, a tissue microarray was previously constructed which contained a wide sampling of benign and malignant endometrial samples. The array contained a metachronous cohort of samples from individuals who either developed or did not develop endometrial cancer. Immunohistochemical staining was used to determine the intensity and frequency of ALDH1 expression. While benign proliferative and secretory endometrium showed very low levels of ALDH1, slightly higher expression was observed within the stratum basalis. In disease progression, cytoplasmic ALDH1 expression showed a step-wise increase between endometrial hyperplasia, atypical hyperplasia, and endometrial cancer. ALDH1 was also shown to be an early predictor of EC development, suggesting that it can serve as an independent prognostic indicator of patients with endometrial hyperplasia with or without atypia who would progress to cancer (p = 0.012).

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.

Increased epithelial membrane protein 2 expression in glioblastoma after treatment with bevacizumab.

BACKGROUND: Antiangiogenic therapy with bevacizumab has failed to provide substantial gains in overall survival. Epithelial membrane protein 2 (EMP2) is a cell surface protein that has been previously shown to be expressed in glioblastoma, correlate with poor survival, and regulate neoangiogenesis in cell lines. Thus, the relationship between bevacizumab and EMP2 was investigated. METHODS: Tumor samples were obtained from 12 patients with newly diagnosed glioblastoma at 2 time points: (1) during the initial surgery and (2) during a subsequent surgery following disease recurrence post-bevacizumab treatment. Clinical characteristics and survival data from these patients were collected, and tumor samples were stained for EMP2 expression. The IVY Glioblastoma Atlas Project database was used to evaluate EMP2 expression levels in 270 samples by differing histological areas of the tumor. RESULTS: Patients with high EMP2 staining at initial diagnosis had decreased progression-free and overall survival after bevacizumab (median progression-free survival 4.6 months vs 5.9 months; log-rank P = .076 and overall survival 7.7 months vs 14.4 months; log-rank P = .011). There was increased EMP2 staining in samples obtained after bevacizumab treatment in both unpaired (mean H-score 2.31 vs 1.76; P = .006) and paired analyses (mean difference 0.571; P = .019). This expression increase correlated with length of bevacizumab therapy (R 2  = 0.449; Pearson P = .024). CONCLUSIONS: Bevacizumab treatment increased EMP2 protein expression. This increase in EMP2 correlated with reduced mean survival time post-bevacizumab therapy. We hypothesize a role of EMP2 in clinical bevacizumab resistance and as a potential antiangiogenic therapeutic target in glioblastoma.

EMP2 Is a Novel Regulator of Stemness in Breast Cancer Cells.

Little is known about the role of epithelial membrane protein-2 (EMP2) in breast cancer development or progression. In this study, we tested the hypothesis that EMP2 may regulate the formation or self-renewal of breast cancer stem cells (BCSC) in the tumor microenvironment. In silico analysis of gene expression data demonstrated a correlation of EMP2 expression with known metastasis-related genes and markers of cancer stem cells (CSC) including aldehyde dehydrogenase (ALDH). In breast cancer cell lines, EMP2 overexpression increased and EMP2 knockdown decreased the proportion of stem-like cells as assessed by the expression of the CSC markers CD44+/CD24-, ALDH activity, or by tumor sphere formation. In vivo, upregulation of EMP2 promoted tumor growth, whereas knockdown reduced the ALDHhigh CSC population as well as retarded tumor growth. Mechanistically, EMP2 functionally regulated the response to hypoxia through the upregulation of HIF-1α, a transcription factor previously shown to regulate the self-renewal of ALDHhigh CSCs. Furthermore, in syngeneic mouse models and primary human tumor xenografts, mAbs directed against EMP2 effectively targeted CSCs, reducing the ALDH+ population and blocking their tumor-initiating capacity when implanted into secondary untreated mice. Collectively, our results show that EMP2 increases the proportion of tumor-initiating cells, providing a rationale for the continued development of EMP2-targeting agents.

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

Purpose: Retinopathy of prematurity (ROP) is a leading cause of childhood blindness. ROP occurs as a consequence of postnatal hyperoxia exposure in premature infants, resulting in vasoproliferation in the retina. The tetraspan protein epithelial membrane protein-2 (EMP2) is highly expressed in the retinal pigment epithelium (RPE) in adults, and it controls vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line. We, therefore, hypothesized that Emp2 knockout (Emp2 KO) protects against neovascularization in murine oxygen-induced retinopathy (OIR). Methods: Eyes were obtained from wildtype (WT) and Emp2 KO mouse pups at P7, P12, P17, and P21 after normoxia or hyperoxia (P7-P12) exposure. Following hyperoxia exposure, RNA sequencing was performed using the retina/choroid layers obtained from WT and Emp2 KO at P17. Retinal sections from P7, P12, P17, and P21 were evaluated for Emp2, hypoxia-inducible factor 1α (Hif1α), and VEGF expression. Whole mount images were generated to assess vaso-obliteration at P12 and neovascularization at P17. Results: Emp2 KO OIR mice demonstrated a decrease in pathologic neovascularization at P17 compared with WT OIR mice through evaluation of retinal vascular whole mount images. This protection was accompanied by a decrease in Hif1α at P12 and VEGFA expression at P17 in Emp2 KO animals compared with the WT animals in OIR conditions. Collectively, our results suggest that EMP2 enhances the effects of neovascularization through modulation of angiogenic signaling. Conclusions: The protection of Emp2 KO mice against pathologic neovascularization through attenuation of HIF and VEGF upregulation in OIR suggests that hypoxia-induced upregulation of EMP2 expression in the neuroretina modulates HIF-mediated neuroretinal VEGF expression.

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.

Gene-environment regulatory circuits of right ventricular pathology in tetralogy of fallot.

The phenotypic spectrum of congenital heart defects (CHDs) is contributed by both genetic and environmental factors. Their interactions are profoundly heterogeneous but may operate on common pathways as in the case of hypoxia signaling during postnatal heart development in the context of CHDs. Tetralogy of Fallot (TOF) is the most common cyanotic (hypoxemic) CHD. However, how the hypoxic environment contributes to TOF pathogenesis after birth is poorly understood. We performed Genome-wide transcriptome analysis on right ventricle outflow tract (RVOT) specimens from cyanotic and noncyanotic TOF. Co-expression network analysis identified gene modules specifically associated with clinical diagnosis and hypoxemia status in the TOF hearts. In particular, hypoxia-dependent induction of myocyte proliferation is associated with E2F1-mediated cell cycle regulation and repression of the WNT11-RB1 axis. Genes enriched in epithelial mesenchymal transition (EMT), fibrosis, and sarcomere were also repressed in cyanotic TOF patients. Importantly, transcription factor analysis of the hypoxia-regulated modules suggested CREB1 as a putative regulator of hypoxia/WNT11-RB1 circuit. The study provides a high-resolution landscape of transcriptome programming associated with TOF phenotypes and unveiled hypoxia-induced regulatory circuit in cyanotic TOF. Hypoxia-induced cardiomyocyte proliferation involves negative modulation of CREB1 activity upstream of the WNT11-RB1 axis. KEY MESSAGES: Genetic and environmental factors contribute to congenital heart defects (CHDs). How hypoxia contributes to Tetralogy of Fallot (TOF) pathogenesis after birth is unclear. Systems biology-based analysis revealed distinct molecular signature in CHDs. Gene expression modules specifically associated with cyanotic TOF were uncovered. Key regulatory circuits induced by hypoxia in TOF pathogenesis after birth were unveiled.

Aldehyde dehydrogenase isoforms and inflammatory cell populations are differentially expressed in term human placentas affected by intrauterine growth restriction.

OBJECTIVE: Intrauterine growth restriction (IUGR) is a complication of pregnancy that has both short- and long-term sequelae for affected mothers and offspring. The pathophysiology of disease stems from poor nutrient and oxygen provision to the fetus, resulting in increased oxidative stress within the placenta. As the milieu within the local microenvironment alters macrophage differentiation, we hypothesized that macrophage plasticity may be altered in placentas associated with IUGR, and that macrophages would show hallmarks of lipid peroxidation including altered aldehyde metabolism. METHODS: In human placentas taken from normal pregnancies resulting in appropriate-for-gestational-age (AGA) newborns and placentas associated with IUGR, placental macrophages were evaluated by immunohistochemistry and shown in IUGR to resemble pro-inflammatory activated M1-type macrophages. To link oxidative stress to macrophages, the expression of aldehyde dehydrogenase (ALDHs) isozymes ALDH1, ALDH2, and ALDH3 was assessed. RESULTS: All three isozymes displayed preferential staining for distinct cellular populations within the term human placenta. ALDH1 and ALDH2 were strongly expressed in placental Hofbauer and decidual stromal cells. ALDH3, in contrast, was present in extravillous trophoblasts. Comparing AGA and IUGR-associated placentas, ALDH1 and ALDH2 trended to have greater expression in macrophage populations but lower expression in decidual cell populations in IUGR-associated placentas. ALDH3 had higher expression in IUGR-associated placentas but localized specifically to extravillous trophoblast populations. CONCLUSION: Therefore, we speculate that specific ALDH isozymes have cell-specific functions related to differentiation, inflammation, or oxidative stress responses that are altered in IUGR-associated term human placentas. This family of isozymes may be a novel method to identify human placentas affected by placental insufficiency/IUGR.

The Placental Transcriptome in Late Gestational Hypoxia Resulting in Murine Intrauterine Growth Restriction Parallels Increased Risk of Adult Cardiometabolic Disease.

Intrauterine growth restriction (IUGR) enhances risk for adult onset cardiovascular disease (CVD). The mechanisms underlying IUGR are poorly understood, though inadequate blood flow and oxygen/nutrient provision are considered common endpoints. Based on evidence in humans linking IUGR to adult CVD, we hypothesized that in murine pregnancy, maternal late gestational hypoxia (LG-H) exposure resulting in IUGR would result in (1) placental transcriptome changes linked to risk for later CVD, and 2) adult phenotypes of CVD in the IUGR offspring. After subjecting pregnant mice to hypoxia (10.5% oxygen) from gestational day (GD) 14.5 to 18.5, we undertook RNA sequencing from GD19 placentas. Functional analysis suggested multiple changes in structural and functional genes important for placental health and function, with maximal dysregulation involving vascular and nutrient transport pathways. Concordantly, a ~10% decrease in birthweights and ~30% decrease in litter size was observed, supportive of placental insufficiency. We also found that the LG-H IUGR offspring exhibit increased risk for CVD at 4 months of age, manifesting as hypertension, increased abdominal fat, elevated leptin and total cholesterol concentrations. In summary, this animal model of IUGR links the placental transcriptional response to the stressor of gestational hypoxia to increased risk of developing cardiometabolic disease.

Epithelial Membrane Protein-2 (EMP2) Antibody Blockade Reduces Corneal Neovascularization in an In Vivo Model.

Purpose: Pathologic corneal neovascularization is a major cause of blindness worldwide, and treatment options are currently limited. VEGF is one of the critical mediators of corneal neovascularization but current anti-VEGF therapies have produced limited results in the cornea. Thus, additional therapeutic agents are needed to enhance the antiangiogenic arsenal. Our group previously demonstrated epithelial membrane protein-2 (EMP2) involvement in pathologic angiogenesis in multiple cancer models including breast cancer and glioblastoma. In this paper, we investigate the efficacy of anti-EMP2 immunotherapy in the prevention of corneal neovascularization. Methods: An in vivo murine cornea alkali burn model was used to study pathologic neovascularization. A unilateral corneal burn was induced using NaOH, and subconjunctival injection of either anti-EMP2 antibody, control antibody, or sterile saline was performed after corneal burn. Neovascularization was clinically scored at 7 days postalkali burn, and eyes were enucleated for histologic analysis and immunostaining including VEGF, CD31, and CD34 expression. Results: Anti-EMP2 antibody, compared to control antibody or vehicle, significantly reduced neovascularization as measured by clinical score and central cornea thickness, as well as by histologic reduction of neovascularization, decreased CD34 staining, and decreased CD31 staining. Incubation of corneal limbal cells in vitro with anti-EMP2 blocking antibody significantly decreased EMP2 expression, VEGF expression and secretion, and cell migration. Conclusions: This work demonstrates the effectiveness of EMP2 as a novel target in pathologic corneal neovascularization in an animal model and supports additional investigation into EMP2 antibody blockade as a potential new therapeutic option.