Matched Paired Primary and Recurrent Meningiomas Points to Cell-Death Program Contributions to Genomic and Epigenomic Instability along Tumor Progression.

Meningioma (MN) is an important cause of disability, and predictive tools for estimating the risk of recurrence are still scarce. The need for objective and cost-effective techniques addressed to this purpose is well known. In this study, we present methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) as a friendly method for deepening the understanding of the mechanisms underlying meningioma progression. A large follow-up allowed us to obtain 50 samples, which included the primary tumor of 20 patients in which half of them are suffering one recurrence and the other half are suffering more than one. We histologically characterized the samples and performed MS-MLPA assays validated by FISH to assess their copy number alterations (CNA) and epigenetic status. Interestingly, we determined the increase in tumor instability with higher values of CNA during the progression accompanied by an increase in epigenetic damage. We also found a loss of HIC1 and the hypermethylation of CDKN2B and PTEN as independent prognostic markers. Comparison between grade 1 and higher primary MN's self-evolution pointed to a central role of GSTP1 in the first stages of the disease. Finally, a high rate of alterations in genes that are related to apoptosis and autophagy, such as DAPK1, PARK2, BCL2, FHIT, or VHL, underlines an important influence on cell-death programs through different pathways.

Desmoplastic infantile astrocytoma with atypical phenotype, PTEN homozygous deletion and BRAF V600E mutation.

Desmoplastic infantile astrocytoma (DIA) is rare, cystic and solid tumor of infants usually found in superficial cerebral hemispheres. Although DIA is usually benign, uncommon cases bearing malignant histological and aggressive clinical features have been described in the literature. We report a newborn patient who was diagnosed with a DIA and died postresection. Pathologic examination revealed that the main part of the tumor had benign features, but the internal region showed areas with a more aggressive appearance, with higher-proliferative cells, anaplastic GFAP positive cells with cellular polymorphism, necrosis foci, vascular hyperplasia with endothelial proliferation and microtrombosis. Genetic study, performed in both regions of the tumor, showed a BRAF V600E mutation and a homozygous deletion in PTEN, without changes in other relevant genes like EGFR, CDKN2A, TP53, NFKBIA, CDK4, MDM2 and PDGFRA. Although PTEN homozygous deletions are described in gliomas, the present case constitutes the first report of a PTEN mutation in a DIA, and this genetic feature may be related to the malignant behavior of a usually benign tumor. These genetic findings may point at the need of further and deeper genetic characterization of DIAs, in order to better understand the biology of this tumor and to obtain new prognostic approaches, a better clinical management and targeted therapies, especially in malignant cases of DIA.

Whole-exome sequencing, EGFR amplification and infiltration patterns in human glioblastoma.

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. This cancer shows rapid, highly infiltrative growth, that invades individually or in small groups the surrounding tissue. The aggressive tumor biology of GBM has devastating consequences with a median survival of 15 months. GBM often has Epidermal Growth Factor Receptor (EGFR) abnormalities. Despite recent advances in the study of GBM tumor biology, it is unclear whether mutations in GBM are related to EGFR amplification and relevant phenotypes like tumor infiltration. This study aimed to perform whole-exome sequencing analysis in 30 human GBM samples for identifying mutational portraits associated with EGFR amplification and infiltrative patterns. Our results show that EGFR-amplified tumors have overall higher mutation rates than EGFR-no-amplified. Six genes out of 2029 candidate genes show mutations associated with EGFR amplification status. Mutations in these genes for GBM are novel, not previously reported in GBM, and with little presence in the TCGA database. GPR179, USP48, and BLK show mutation only in EGFR-amplified cases, and all the affected cases exhibit diffuse infiltrative patterns. On the other hand, mutations in ADGB, EHHADH, and PTPN13, were present only in the EGFR-no-amplified group with a more diverse infiltrative phenotype. Overall, our work identified different mutational portraits of GBM related to well-established features like EGFR amplification and tumor infiltration.

Dental stem cell signaling pathway activation in response to hydraulic calcium silicate-based endodontic cements: A systematic review of in vitro studies.

OBJECTIVE: To present a qualitative synthesis of in vitro studies which analyzed human dental stem cell (DSC) molecular signaling pathway activation in response to hydraulic calcium silicate-based cements (HCSCs). METHODS: A systematic electronic search was performed in Medline, Scopus, Embase, Web of Science and SciELO databases on January 20 and last updated on March 20, 2020. In vitro studies assessing the implication of signaling pathways in activity related marker (gene/protein) expression and mineralization induced by HCSCs in contact with human DSCs were included. RESULTS: The search identified 277 preliminary results. After discarding duplicates, and screening of titles, abstracts, and full texts, 13 articles were considered eligible. All of the materials assessed by the included studies showed positive results in cytocompatibility and/or bioactivity assays. ProRoot MTA and Biodentine were the modal HCSCs studied, hDPSCs were the modal cell variant used, and the most studied signaling pathway was MAPK. In vitro assays measuring the expression of activity-related markers and mineralized nodule formation evidenced the involvement of MAPK (and its subfamilies ERK, JNK and P38), NF-κB, Wnt/ß-catenin, BMP/Smad and CAMKII pathways in the biological response of DSCs to HCSCs. SIGNIFICANCE: HCSCs considered in the present review elicited a favorable biological response from a variety of DSCs in vitro, thus supporting their use in biologically-based endodontic procedures. MAPK, NF-κß, Wnt/ß-catenin, BMP/Smad and CAMKII signaling pathways have been proposed as potential mediators in the biological interaction between DSCs and HCSCs. Understanding the signaling processes involved in tissue repair could lead to the development of new biomaterial compositions targeted at enhancing these mechanisms through biologically-based procedures.

Identification of New Genetic Clusters in Glioblastoma Multiforme: EGFR Status and ADD3 Losses Influence Prognosis.

Glioblastoma multiforme (GB) is one of the most aggressive tumors. Despite continuous efforts to improve its clinical management, there is still no strategy to avoid a rapid and fatal outcome. EGFR amplification is the most characteristic alteration of these tumors. Although effective therapy against it has not yet been found in GB, it may be central to classifying patients. We investigated somatic-copy number alterations (SCNA) by multiplex ligation-dependent probe amplification in a series of 137 GB, together with the detection of EGFRvIII and FISH analysis for EGFR amplification. Publicly available data from 604 patients were used as a validation cohort. We found statistical associations between EGFR amplification and/or EGFRvIII, and SCNA in CDKN2A, MSH6, MTAP and ADD3. Interestingly, we found that both EGFRvIII and losses on ADD3 were independent markers of bad prognosis (p = 0.028 and 0.014, respectively). Finally, we got an unsupervised hierarchical classification that differentiated three clusters of patients based on their genetic alterations. It offered a landscape of EGFR co-alterations that may improve the comprehension of the mechanisms underlying GB aggressiveness. Our findings can help in defining different genetic profiles, which is necessary to develop new and different approaches in the management of our patients.

Identification of a Novel BRCA1 Alteration in Recurrent Melanocytoma Resulting in Increased Proliferation.

Primary meningeal melanocytomas are rare tumors of the central nervous system. Although they are considered benign neoplasms, some reports describe recurrent rates up to 45%. Little is known about their genetic and epigenetic landscape because of their infrequency. Even less has been described about markers with prognostic value. Here we describe a patient who developed a primary meningeal melanocytoma, suffered 3 recurrences in a period of 6 years and died of the tumor. The genetic and epigenetic changes explored confirmed GNAQ mutation as an initiating event. We found an epigenetic alteration of GSTP1, a feature that has recently been described in meningiomas, from the beginning of the disease. In addition, there was loss of heterozygosity in BRCA1 beginning in the second recurrence that was linked to an increase in the proliferation index; this suggested a progression pathway similar to the one described in uveal melanomas. These findings underscore the necessity of further research focused on these tumors.

The Status of EGFR Modulates the Effect of miRNA-200c on ZEB1 Expression and Cell Migration in Glioblastoma Cells.

Migration of glioblastoma cells into surrounding tissue is one of the main features that makes this tumor incurable. We evaluated whole-genome miRNA expression profiling associated with different EGFR amplification patterns in 30 cases of primary glioblastoma. From the 64 miRNAs that showed differential expression between tumors with a high level of EGFR amplification and tumors without EGFR amplification, 40% were related with cell migration, being miR-200c the most differentially expressed between these two groups. We investigated the effect of miR-200c on ZEB1 expression and cell migration in an in vitro transfection model with a miR-200c mimic, a miR-200c inhibitor and siRNA targeting EGFR in three short-term cultures with different levels of EGFR amplification obtained from resected glioblastomas. The cell culture with the highest EGFR amplification level presented the lowest miR-200c expression and the status of EGFR modulated the effect of miR-200c on ZEB1 expression. Silencing EGFR led to miR-200c upregulation and ZEB1 downregulation in transfected cultures, except in the presence of high levels of EGFR. Likewise, miR-200c upregulation decreased ZEB1 expression and inhibited cell migration, especially when EGFR was not amplified. Our results suggest that modulating miR-200c may serve as a novel therapeutic approach for glioblastoma depending on EGFR status.

Pam3CSK4, a TLR2 ligand, induces differentiation of glioblastoma stem cells and confers susceptibility to temozolomide.

Glioblastoma multiforme (GBM) is the most aggressive human brain tumor, and GBM stem cells (GSC) may be responsible for its recurrence and therapeutic resistance. Toll-like receptors (TLRs), which recognize multiple ligands (endogenous and pathogen-associated) and trigger the immune response of mature immune cells, are also expressed by hematopoietic stem and progenitor cells, where their activation results in the differentiation of these cells into myeloid cells. Since TLR expression has been recently described in neural cells, including neural stem cells, we studied TLR expression by GSCs and the effect of stimulation by TLR ligands on promoting GSC differentiation into mature GBM cells. First, our results showed heterogeneous TLR expression by GBM cells from human tumors and, for the first time, by human GSCs defined by their CD133+ and CD44+ phenotypes. Next, the effect of TLR ligands was studied in in vitro cell cultures of neurospheres and CD44+ cells obtained from two GBM cell lines (U-87 and U-118). The expression of GSC markers diminished in the presence of Pam3CSK4 or LPS (TLR2 and TLR4 ligands, respectively), thus indicating TLR-dependent differentiation. Interestingly, simultaneous treatment with Pam3CSK4 plus temozolomide (TMZ), the reference drug in GBM treatment, significantly increased cell death compared to the effect of the ligand alone, which showed no toxicity, or TMZ alone. These results suggest a synergistic effect between Pam3CSK4 and TMZ based on the induction of TLR-dependent GSC differentiation towards mature GBM cells, which exhibited increased sensitivity to chemotherapy, and provide new perspectives in GBM therapy.

Somatic copy number alterations are associated with EGFR amplification and shortened survival in patients with primary glioblastoma.

Glioblastoma (GBM) is the most common malignant primary tumor of the central nervous system. With no effective therapy, the prognosis for patients is terrible poor. It is highly heterogeneous and EGFR amplification is its most frequent molecular alteration. In this light, we aimed to examine the genetic heterogeneity of GBM and to correlate it with the clinical characteristics of the patients. For that purpose, we analyzed the status of EGFR and the somatic copy number alterations (CNAs) of a set of tumor suppressor genes and oncogenes. Thus, we found GBMs with high level of EGFR amplification, low level and with no EGFR amplification. Highly amplified tumors showed histological features of aggressiveness. Interestingly, accumulation of CNAs, as a measure of tumor mutational burden, was frequent and significantly associated to shortened survival. EGFR-amplified GBMs displayed both a higher number of concrete CNAs and a higher global tumor mutational burden than their no EGFR-amplified counterparts. In addition to genetic changes previously described in GBM, we found PARK2 and LARGE1 CNAs associated to EGFR amplification. The set of genes analyzed allowed us to explore relevant signaling pathways on GBM. Both PARK2 and LARGE1 are related to receptor tyrosine kinase/PI3K/PTEN/AKT/mTOR-signaling pathway. Finally, we found an association between the molecular pathways altered, EGFR amplification and a poor outcome. Our results underline the potential interest of categorizing GBM according to their EGFR amplification level and the usefulness of assessing the tumor mutational burden. These approaches would open new knowledge possibilities related to GBM biology and therapy.

Characterization of a new glioblastoma cell line, GB-val4, with unusual TP53 mutation.

A novel cell line derived from a human glioblastoma (GB), named GB-val4, has been established and characterized. GB-val4 cells were hyperdiploid, with many numerical and structural chromosomal rearrangements. The cell line did not show mutations in IDH1/IDH2 genes or EGFR amplification, but it presented two missense mutations in TP53, which imply a very low p53 protein activity within the cell line. Cells also had gain of TP73 copies, hypermethylation of APC, CASP8 and RASSF1, increased expression of ARF1, CDH1 and NF-κB and decreased expression of CDKN2A. Tumorigenity was demonstrated by transplant of GB-val4 cells into athymic nude mice, where solid tumors were grown. Interestingly, a high percentage of GB-val4 cells presented expression of GSC markers CD133 or CD44. These GSC markers were increased in neurosphere cultures, which better mimic solid tumor conditions and maintain the genetic features of the tumor cells. In this study, we aimed to define the characteristics of this novel cell line and its applications in human cancer research. With its genetic features and a poor p53 activity, GB-val4 cells resemble GB tumors. Moreover, the important presence of GSCs in adherent cultures and especially in neurosphere cultures makes GB-val4 an attractive tool to study cancer stem cells, deepen in the knowledge the molecular pathways of GB and develop new therapeutic strategies for patients with these tumors.

Epigenetic changes underlie the aggressiveness of histologically benign meningiomas that recur.

Meningiomas are the most frequent primary brain tumor. Usually, they are curable by surgery, but even after seemingly complete resection, some low-grade lesions recur. Despite recent improvements, signatures having prognostic value in grade I tumors remain poorly characterized. The frequency and delicate location of these tumors suggest that the risk of recurrence might be more accurately predicted. Herein, we show an easy way to evaluate the methylation status of meningiomas and its correlation with the prognosis of the disease. A series of 120 meningiomas, including primary tumors and recurrences, were analyzed histopathologically, and 24 tumor suppressor genes (TSGs) were studied by methylation-specific multiple ligation probe amplification. Long-term follow-up was conducted to classify patients with grade I primary tumors according to their outcomes. We found that hypermethylation in at least one TSG is frequent. The number of hypermethylated TSG per case was significantly higher in recurrences than in primary tumors and in primary benign meningiomas that recurred than in tumors from patients who showed no evidence of disease during follow-up. Finally, hypermethylation in RASSF1A, MLH1, and CDKN2B was an independent prognostic factor associated with the time to recurrence of these benign tumors that were biologically aggressive. To our knowledge, this is one of the widest studies of primary grade I tumors of patients who developed a tumor recurrence. The frequency of epigenetic changes suggests that hypermethylation is an early event in meningiomas, whereas the accumulation of epigenetic changes is related to greater biological aggressiveness and may be a signature of potential clinical relevance.

Characterization of the new human pleomorphic undifferentiated sarcoma TP53-null cell line mfh-val2.

Pleomorphic undifferentiated sarcoma (PUS), also called malignant fibrous histiocytoma, is a soft tissue sarcoma which occurs predominantly in the extremities. Its origin is a poorly defined mesenchymal cell, which derives to histiocytic and fibroblastic cells. The patient, a 58 year-old man, presented a lesion located in the forearm composed by spindle cells and multinucleated giant cells, which expressed vimentin and adopted a histological pattern formed by irregular-swirling fascicles. Cells were cultured in vitro and a new cell line was established. We characterized this new cell line by histological analyses, cytogenetics (using G-bands and spectral karyotype technique) and cytometric analyses. Cells were grown in culture for more than 100 passages. They had elongated or polygonal morphology. The cells presented a saturation rate of 70,980 cells/cm2, a plating efficiency of 21.5% and a mitotic index of 21 mitoses per field. The cell line was tumorigenic in nude mice. The ploidy study using flow cytometry revealed an aneuploid peak with a DNA index of 1.43. A side population was detected, demonstrating the presence of stem and progenitor cells. Cytogenetics showed a hypotriploid range with many clonal unbalanced rearrangements. Loss of p53 gene was evidenced by MLPA. We describe, for the first time, the characterization of a new human PUS TP53-null cell line called mfh-val2. Mfh-val2 presents a wide number of applications as a TP53-null cell line and a great interest in order to characterize genetic alterations influencing the oncogenesis or progression of PUS and to advance in the biological investigation of this tumor.

Association between epidermal growth factor receptor amplification and ADP-ribosylation factor 1 methylation in human glioblastoma.

PURPOSE: Glioblastoma (GB) is the most frequent and most malignant primary brain tumor in adults. Previously, it has been found that both genetic and epigenetic factors may play critical roles in its etiology and prognosis. In addition, it has been found that the epidermal growth factor receptor gene (EGFR) is frequently over-expressed and amplified in primary GBs. Here, we assessed the promoter methylation status of 10 genes relevant to GB and explored associations between these findings and the EGFR gene amplification status. METHODS: Tumor samples were obtained from 36 patients with primary GBs. In addition, 6 control specimens were included from patients who were operated for diseases other than brain tumors. The amplification status of the EGFR gene, and its deletion mutant EGFRvIII, were evaluated using FISH and MLPA, respectively. The IDH1/2 gene mutation status was verified using Sanger sequencing. A commercial DNA methylation kit was used to assess the promoter methylation status of 10 pre-selected genes. Metabolic profiles were measured using HR-MAS NMR spectroscopy. The EGFR and ARF1 mRNA expression levels were quantified using qRT-PCR. RESULTS: Of the 10 genes analyzed, we found that only ARF1 promoter hypermethylation was significantly associated with EGFR gene amplification. ARF1 is a GTPase that is involved in vesicle trafficking and the Golgi apparatus. Subsequent tumor metabolism measurements revealed a positive association between EGFR amplification and different membrane precursors and methyl-donor metabolites. Finally, we found that EGFR gene amplifications were associated with distinct tumor infiltration patterns, thus representing a putative novel functional association between EGFR gene amplification and ARF1 gene promoter methylation in GB. CONCLUSIONS: The results reported here provide a basis for a new hypotheses connecting EGFR gene amplification in GB cells with ARF1 gene promoter methylation, vesicle trafficking, membrane turnover and tumor metabolism. The mechanism(s) underlying these connections and their functional consequences remain to be established.

Expression of the Chemokine Receptors CXCR3, CXCR4, CXCR7 and Their Ligands in Rhabdomyosarcoma.

Rhabdomyosarcomas (RMS) are soft tissue malignant tumors of childhood and adolescents. The mechanisms underlying their aggressiveness are still poorly understood. Chemokines are chemotactic proteins involved in pathological processes that have been intensely studied in several types of cancers because of their influence in migration, angiogenesis, or metastases. We analyzed the expression of the chemokine receptors CXCR3, CXCR4 and CXCR7 and their ligands CXCL9, CXCL10, CXCL11 and CXCL12, in 15 RMS samples derived from nine patients. Expression was measured in tumors and primary cultures of RMS by Real-Time Polymerase Chain Reaction, immunostaining and flow cytometry. Our results show that these receptors are widely expressed in RMS. A significant difference between CXCL12/CXCR4, CXCL12/CXCR7, CXCL11/CXCR7 expression ratios was found in alveolar versus embryonal RMS and similarly between CXCL12/CXCR4 and CXCL11/CXCR3 ratios in primary versus recurrent tumors. These findings suggest a possible association between the interrelation of chemokine/chemokine-receptor and an aggressive biological behavior in RMS.

Correlation between EGFR amplification and the expression of microRNA-200c in primary glioblastoma multiforme.

Extensive infiltration of the surrounding healthy brain tissue is a critical feature in glioblastoma. Several miRNAs have been related to gliomagenesis, some of them related with the EGFR pathway. We have evaluated whole-genome miRNA expression profiling associated with different EGFR amplification patterns, studied by fluorescence in situ hybridization in tissue microarrays, of 30 cases of primary glioblastoma multiforme, whose clinicopathological and immunohistochemical features have also been analyzed. MicroRNA-200c showed a very significant difference between tumors having or not EGFR amplification. This microRNA plays an important role in epithelial-mesenchymal transition, but its implication in the behavior of glioblastoma is largely unknown. With respect to EGFR status our cases were categorized into three groups: high level EGFR amplification, low level EGFR amplification, and no EGFR amplification. Our results showed that microRNA-200c and E-cadherin expression are down-regulated, while ZEB1 is up-regulated, when tumors showed a high level of EGFR amplification. Conversely, ZEB1 mRNA expression levels were significantly lower in the group of tumors without EGFR amplification. Tumors with a low level of EGFR amplification showed ZEB1 expression levels comparable to those detected in the group with a high level of amplification. In this study we provide what is to our knowledge the first report of association between microRNA-200c and EGFR amplification in glioblastomas.

Gene expression profiles of metabolic aggressiveness and tumor recurrence in benign meningioma.

Around 20% of meningiomas histologically benign may be clinically aggressive and recur. This strongly affects management of meningioma patients. There is a need to evaluate the potential aggressiveness of an individual meningioma. Additional criteria for better classification of meningiomas will improve clinical decisions as well as patient follow up strategy after surgery. The aim of this study was to determine the relationship between gene expression profiles and new metabolic subgroups of benign meningioma with potential clinical relevance. Forty benign and fourteen atypical meningioma tissue samples were included in the study. We obtained metabolic profiles by NMR and recurrence after surgery information for all of them. We measured gene expression by oligonucleotide microarray measurements on 19 of them. To our knowledge, this is the first time that distinct gene expression profiles are reported for benign meningioma molecular subgroups with clinical correlation. Our results show that metabolic aggressiveness in otherwise histological benign meningioma proceeds mostly through alterations in the expression of genes involved in the regulation of transcription, mainly the LMO3 gene. Genes involved in tumor metabolism, like IGF1R, are also differentially expressed in those meningioma subgroups with higher rates of membrane turnover, higher energy demand and increased resistance to apoptosis. These new subgroups of benign meningiomas exhibit different rates of recurrence. This work shows that benign meningioma with metabolic aggressiveness constitute a subgroup of potentially recurrent tumors in which alterations in genes regulating critical features of aggressiveness, like increased angiogenesis or cell invasion, are still no predominant. The determination of these gene expression biosignatures may allow the early detection of clinically aggressive tumors.

Genetic changes with prognostic value in histologically benign meningiomas.

Meningiomas add up to 25% of intracranial tumors. Although the majority is considered histologically benign, the prediction of their potential aggressiveness is still unclear. We studied the histopathology and aberrations of chromosomes 1p, 14, and 22 by FISH (fluorescence in situ hybridization) in histologically benign meningiomas of 70 patients for the purpose of defining the prognostic value of these alterations in tumoral progression and the risk of recurrence. According to the WHO histopathological criteria, the study set comprised 53 benign, 11 atypical, and 6 anaplastic meningiomas. In benign meningiomas, 25% of the cases displayed a normal karyotype, isolated monosomy 22 (36%), monosomy 22 + 1p deletion (14%), 1p deletion (10%), monosomy 22 + 14q deletion (5%), monosomy 22 + 1p deletion + 14q deletion (5%), or other alterations (5%). Grade II meningiomas presented losses in chromosome 14 in most of the cases (67%), and Grade III meningiomas showed alterations in chromosome 14 in all patients. We observed an overall relapse rate of 31%: recurrence was observed in 19% of Grade I meningiomas, 64% of Grade II, and 83% of Grade III. 9 out of 10 recurrent cases revealed abnormalities in chromosomes 1 and 14, which was a notably higher incidence compared to the series of tumors without relapse. Thus, benign meningiomas with cytogenetic alterations in chromosomes 1p and 14 may be more closely related to atypical meningiomas than benign meningiomas without these alterations, especially in terms of recurrence risk.

Arterial and venous endothelia display differential functional fractalkine (CX3CL1) expression by angiotensin-II.

OBJECTIVE: Angiotensin-II (Ang-II) promotes the interaction of mononuclear cells with arterioles and neutrophils with postcapillary venules. To investigate the mechanisms underlying this dissimilar response, the involvement of fractalkine (CX(3)CL1) was explored. METHODS AND RESULTS: Enhanced CX(3)CL1 expression was detected in both cremasteric arterioles and postcapillary venules 24 hours after Ang-II intrascrotal injection. Arteriolar leukocyte adhesion was the unique parameter significantly reduced (83%) in animals lacking CX(3)CL1 receptor (CX(3)CR1). Human umbilical arterial and venous endothelial cell stimulation with 1 µmol/L Ang-II increased CX(3)CL1 expression, yet neutralization of CX(3)CL1 activity only significantly inhibited Ang-II-induced mononuclear cell-human umbilical arterial endothelial cell interactions (73%) but not with human umbilical venous endothelial cells. The use of small interfering RNA revealed the involvement of tumor necrosis factor-α in Ang-II-induced CX(3)CL1 upregulation and mononuclear cell arrest. Nox5 knockdown with small interfering RNA or pharmacological inhibition of extracellular signal-regulated kinases1/2, p38 mitogen-activated protein kinase, and nuclear factor-κB also abolished these responses. Finally, when human umbilical arterial endothelial cells were costimulated with Ang-II, tumor necrosis factor-α, and interferon-γ, CX(3)CL1 expression and mononuclear cell adhesiveness were more pronounced than when each stimulus was provided alone. CONCLUSIONS: These results suggest that Ang-II induces functional CX(3)CL1 expression in arterial but not in venous endothelia. Thus, targeting endothelial CX(3)CL1-mononuclear leukocyte CX(3)CR1 interactions may constitute a new therapeutic strategy in the treatment of Ang-II-associated cardiovascular diseases.