RESUMEN
BACKGROUND: Randomized clinical trials are the gold standard for evaluating healthcare interventions and, more generally, add to the medical knowledge related to the treatment, diagnosis and prevention of diseases and conditions. Recent literature continues to identify health informatics methods that can help improve study efficiency throughout the life cycle of a clinical trial. Electronic medical record (EMR) data provides a mechanism to facilitate clinical trial research during the study planning and execution phases, and ultimately, can be utilized to enhance recruitment. The Department of Veterans Affairs (VA) has a strong history of clinical and epidemiological research with over four decades of data collected from Veterans it has served nationwide. The VA Informatics and Computing Infrastructure (VINCI) provides VA research investigators with a nationwide view of high-value VA patient data. Within VA, the Cooperative Studies Program (CSP) Network of Dedicated Enrollment Sites (NODES) is a consortium of nine sites that are part of an embedded clinical research infrastructure intended to provide systematic site-level solutions to issues that arise during the conduct of VA CSP clinical research. This paper describes the collaboration initiated by the Salt Lake City (SLC) node site to bring informatics and clinical trials together to enhance study planning and recruitment within the VA. METHODS: The SLC VA Medical Center physically houses both VINCI and a node site and the co-location of these two groups prompted a natural collaboration on both a local and national level. One of the functions of the SLC NODES is to enhance recruitment and promote the success of CSP projects. VINCI supports these efforts by providing VA researchers access to potential population pools. VINCI can provide 1) feasibility data during study planning, and 2) active patient lists during recruitment. The process for CSP study teams to utilize these services involves regulatory documentation, development of queries, revisions to the initial data request, and ongoing communications with several key study personnel including the requesting research team, study statisticians, and VINCI data managers. RESULTS: The early efforts of SLC NODES and VINCI aimed to provide patient lists exclusively to the SLC CSP study teams for the following purposes: 1) increasing recruitment for trials that were struggling to meet their respective enrollment goals, and 2) decreasing the time required by study coordinators to complete chart review activities. This effort was expanded to include multiple CSP sites and studies. To date, SLC NODES has facilitated the delivery of these VINCI services to nine active CSP studies. CONCLUSION: The ability of clinical trial study teams to successfully plan and execute their respective trials is contingent upon their proficiency in obtaining data that will help them efficiently and effectively recruit and enroll eligible participants. This collaboration demonstrates that the utilization of a model that partners two distinct entities, with similar objectives, was effective in the provision of feasibility and patient lists to clinical trial study teams and facilitation of clinical trial research within a large, integrated healthcare system.
RESUMEN
Kaposi sarcoma-associated herpesvirus (KSHV) infection is essential to the development of Kaposi sarcoma (KS). Notch signaling is also known to play a pivotal role in KS cell survival and lytic phase entrance of KSHV. In the current study, we sought to determine whether KSHV regulates Notch components. KSHV-infected lymphatic endothelial cells showed induction of receptors Notch3 and Notch4, Notch ligands Dll4 and Jagged1, and activated Notch receptors in contrast to uninfected lymphatic endothelial cells. In addition, KSHV induced the expression of endothelial precursor cell marker (CD133) and mural cell markers (calponin, desmin, and smooth muscle alpha actin), suggesting dedifferentiation and trans-differentiation. Overexpression of latency proteins (LANA, vFLIP) and lytic phase proteins (RTA, vGPCR, viral interleukin-6) further supported the direct regulatory capacity of KSHV viral proteins to induce Notch receptors (Notch2, Notch3), ligands (Dll1, Dll4, Jagged1), downstream targets (Hey, Hes), and endothelial precursor CD133. Targeting Notch pathway with gamma-secretase inhibitor and a decoy protein in the form of soluble Dll4 inhibited growth of KSHV-transformed endothelial cell line. Soluble Dll4 was also highly active in vivo against KS tumor xenograft. It inhibited tumor cell growth, induced tumor cell death, and reduced vessel perfusion. Soluble Dll4 is thus a candidate for clinical investigation.
Asunto(s)
Células Endoteliales , Infecciones por Herpesviridae/metabolismo , Herpesvirus Humano 8 , Receptores Notch/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al Calcio/metabolismo , Supervivencia Celular/fisiología , Transformación Celular Viral , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/fisiología , Células Endoteliales/virología , Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/fisiopatología , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptor Notch3 , Receptor Notch4 , Receptores Notch/genética , Proteínas Serrate-Jagged , Transfección , Trasplante Heterólogo , Arterias Umbilicales/citología , Venas Umbilicales/citología , Latencia del Virus/fisiologíaRESUMEN
The receptor tyrosine kinase EphB2 is expressed by colon progenitor cells; however, only 39% of colorectal tumors express EphB2 and expression levels decline with disease progression. Conversely, EphB4 is absent in normal colon but is expressed in all 102 colorectal cancer specimens analyzed, and its expression level correlates with higher tumor stage and grade. Both EphB4 and EphB2 are regulated by the Wnt pathway, the activation of which is critically required for the progression of colorectal cancer. Differential usage of transcriptional coactivator cyclic AMP-responsive element binding protein-binding protein (CBP) over p300 by the Wnt/beta-catenin pathway is known to suppress differentiation and increase proliferation. We show that the beta-catenin-CBP complex induces EphB4 and represses EphB2, in contrast to the beta-catenin-p300 complex. Gain of EphB4 provides survival advantage to tumor cells and resistance to innate tumor necrosis factor-related apoptosis-inducing ligand-mediated cell death. Knockdown of EphB4 inhibits tumor growth and metastases. Our work is the first to show that EphB4 is preferentially induced in colorectal cancer, in contrast to EphB2, whereby tumor cells acquire a survival advantage.
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Neoplasias Colorrectales/enzimología , Receptor EphB2/biosíntesis , Receptor EphB4/biosíntesis , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente , Células HT29 , Humanos , Immunoblotting , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Receptor EphB4/genética , Transfección , Trasplante Heterólogo , beta Catenina/metabolismoRESUMEN
Kaposi sarcoma (KS) is associated with human herpesvirus (HHV)-8 and is dependent on the induction of vascular endothelial growth factors (VEGFs). VEGF regulates genes that provide arterial or venous identity to endothelial cells, such as the induction of EphrinB2, which phenotypically defines arterial endothelial cells and pericytes, and represses EphB4, which defines venous endothelial cells. We conducted a comprehensive analysis of the Eph receptor tyrosine kinases to determine which members are expressed and therefore contribute to KS pathogenesis. We demonstrated limited Eph/Ephrin expression; notably, the only ligand highly expressed is EphrinB2. We next studied the biologic effects of blocking EphrinB2 using the extracellular domain of EphB4 fused with human serum albumin (sEphB4-HSA). sEphB4-HSA inhibited migration and invasion of the KS cells in vitro in response to various growth factors. Finally, we determined the biologic effects of combining sEphB4-HSA and an antibody to VEGF. sEphB4-HSA was more active than the VEGF antibody, and combination of the 2 had at least additive activity. sEphB4-HSA reduced blood vessel density, pericyte recruitment, vessel perfusion, and increased hypoxia, with an associated increase in VEGF and DLL4 expression. The combination of sEphB4-HSA and VEGF antibody is a rational treatment combination for further investigation.
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Efrina-B2/antagonistas & inhibidores , Efrina-B2/metabolismo , Receptor EphB4/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Sarcoma de Kaposi/fisiopatología , Neoplasias Vasculares/fisiopatología , Animales , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Células Endoteliales/citología , Efrinas/genética , Efrinas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Receptor EphB4/genética , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismo , Proteínas Recombinantes de Fusión/genética , Sarcoma de Kaposi/tratamiento farmacológico , Sarcoma de Kaposi/metabolismo , Albúmina Sérica/genética , Arterias Umbilicales/citología , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular/inmunología , Neoplasias Vasculares/tratamiento farmacológico , Neoplasias Vasculares/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The importance of Notch signaling pathway in the regulation of vascular development and angiogenesis is suggested by the expression of Notch receptors and ligands in vascular endothelial cells (ECs) and the observed vascular phenotypes in mutants of Notch receptors or ligands, especially Dll4. DLL4 is specifically expressed in arterial ECs during development, and haplo-insufficiency is embryonically lethal in mice. To address the role of Dll4 in vascular development, we produced mDll4 conditionally overexpressed transgenic mice that were crossed with constitutive recombinase cre lines. Double transgenic embryos displayed grossly enlarged dorsal aortae (DA) and died before embryonic day 10.5 (E10.5), showing a variable degree of premature arteriovenous fusion. Veins displayed ectopic expression of arterial markers. Other defects included reduced vascular sprouting, EC proliferation, and migration. mDll4 overexpression also inhibited VEGF signaling and increased fibronectin accumulation around the vessels. In vitro and in vivo studies of DLL4-FL (Dll4-full-length) in ECs recapitulate many of the mDll4 transgenics findings, including decreased tube formation, reduced vascular branching, fewer vessels, increased pericyte recruitment, and increased fibronectin expression. These results establish the role of Dll4 in arterial identity determination, and regulation of angiogenesis subject to dose and location.
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Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Arterias/anomalías , Arterias/embriología , Proteínas de Unión al Calcio , Movimiento Celular , Células Cultivadas , Pérdida del Embrión/genética , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Células Endoteliales/citología , Células Endoteliales/fisiología , Femenino , Corazón Fetal/anomalías , Corazón Fetal/embriología , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/fisiología , Masculino , Ratones , Ratones Transgénicos , Fenotipo , Embarazo , Receptores Notch/fisiología , Transfección , Venas/anomalías , Venas/embriologíaRESUMEN
Vascular development is dependent on various growth factors and certain modifiers critical for providing arterial or venous identity, interaction with the surrounding stroma and tissues, hierarchic network formation, and recruitment of pericytes. Notch receptors and ligands (Jagged and Delta-like) play a critical role in this process in addition to VEGF. Dll4 is one of the Notch ligands that regulates arterial specification and maturation events. In the current study, we have shown that loss of function by either targeted allele deletion or use of a soluble form of Dll4 extracellular domain leads to inhibition of Notch signaling, resulting in increased vascular proliferation but defective maturation. Newly forming vessels have thin caliber, a markedly reduced vessel lumen, markedly reduced pericyte recruitment, and deficient vascular perfusion. sDll4 similarly induced defective vascular response in tumor implants leading to reduced tumor growth. Interference with Dll4-Notch signaling may be particularly desirable in tumors that have highly induced Dll4-Notch pathway.