RESUMEN
Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.
Asunto(s)
Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Plaquetas/metabolismo , Encéfalo/irrigación sanguínea , Regulación de la Expresión Génica/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Sensibilidad y Especificidad , Análisis de la Célula Individual , Transgenes , Vísceras/irrigación sanguíneaRESUMEN
Full-length tissue factor (flTF), the coagulation initiator, is overexpressed in breast cancer (BrCa), but associations between flTF expression and clinical outcome remain controversial. It is currently not known whether the soluble alternatively spliced TF form (asTF) is expressed in BrCa or impacts BrCa progression. We are unique in reporting that asTF, but not flTF, strongly associates with both tumor size and grade, and induces BrCa cell proliferation by binding to ß1 integrins. asTF promotes oncogenic gene expression, anchorage-independent growth, and strongly up-regulates tumor expansion in a luminal BrCa model. In basal BrCa cells that constitutively express both TF isoforms, asTF blockade reduces tumor growth and proliferation in vivo. We propose that asTF plays a major role in BrCa progression acting as an autocrine factor that promotes tumor progression. Targeting asTF may comprise a previously unexplored therapeutic strategy in BrCa that stems tumor growth, yet does not impair normal hemostasis.
Asunto(s)
Empalme Alternativo , Neoplasias de la Mama/patología , Integrina beta1/fisiología , Tromboplastina/fisiología , Adulto , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Ratones , Persona de Mediana Edad , Tromboplastina/genéticaRESUMEN
Ephrin receptors constitute a large family of receptor tyrosine kinases in mammals that through interaction with cell surface-anchored ephrin ligands regulate multiple different cellular responses in numerous cell types and tissues. In the cardiovascular system, studies performed in vitro and in vivo have pointed to a critical role for Ephrin receptor B4 (EPHB4) as a regulator of blood and lymphatic vascular development and function. However, in this role, EPHB4 appears to act not as a classical growth factor receptor but instead functions to dampen the activation of the Ras-mitogen activated protein signaling (MAPK) pathway induced by other growth factor receptors in endothelial cells (EC). To inhibit the Ras-MAPK pathway, EPHB4 interacts functionally with Ras p21 protein activator 1 (RASA1) also known as p120 Ras GTPase-activating protein. Here, we review the evidence for an inhibitory role for an EPHB4-RASA1 interface in EC. We further discuss the mechanisms by which loss of EPHB4-RASA1 signaling in EC leads to blood and lymphatic vascular abnormalities in mice and the implications of these findings for an understanding of the pathogenesis of vascular anomalies in humans caused by mutations in EPHB4 and RASA1 genes. Last, we provide insights into possible means of drug therapy for EPHB4- and RASA1-related vascular anomalies.
RESUMEN
To elucidate the pathogenesis of vein of Galen malformations (VOGMs), the most common and most severe of congenital brain arteriovenous malformations, we performed an integrated analysis of 310 VOGM proband-family exomes and 336,326 human cerebrovasculature single-cell transcriptomes. We found the Ras suppressor p120 RasGAP (RASA1) harbored a genome-wide significant burden of loss-of-function de novo variants (2042.5-fold, p = 4.79 x 10-7). Rare, damaging transmitted variants were enriched in Ephrin receptor-B4 (EPHB4) (17.5-fold, p = 1.22 x 10-5), which cooperates with p120 RasGAP to regulate vascular development. Additional probands had damaging variants in ACVRL1, NOTCH1, ITGB1, and PTPN11. ACVRL1 variants were also identified in a multi-generational VOGM pedigree. Integrative genomic analysis defined developing endothelial cells as a likely spatio-temporal locus of VOGM pathophysiology. Mice expressing a VOGM-specific EPHB4 kinase-domain missense variant (Phe867Leu) exhibited disrupted developmental angiogenesis and impaired hierarchical development of arterial-capillary-venous networks, but only in the presence of a "second-hit" allele. These results illuminate human arterio-venous development and VOGM pathobiology and have implications for patients and their families.
Asunto(s)
Enfermedades Vasculares , Malformaciones de la Vena de Galeno , Humanos , Animales , Ratones , Malformaciones de la Vena de Galeno/genética , Malformaciones de la Vena de Galeno/patología , Células Endoteliales/patología , Mutación , Transducción de Señal/genética , Mutación Missense , Proteínas Activadoras de GTPasa/genética , Receptores de Activinas Tipo II/genética , Proteína Activadora de GTPasa p120/genéticaRESUMEN
To elucidate the pathogenesis of vein of Galen malformations (VOGMs), the most common and severe congenital brain arteriovenous malformation, we performed an integrated analysis of 310 VOGM proband-family exomes and 336,326 human cerebrovasculature single-cell transcriptomes. We found the Ras suppressor p120 RasGAP ( RASA1 ) harbored a genome-wide significant burden of loss-of-function de novo variants (p=4.79×10 -7 ). Rare, damaging transmitted variants were enriched in Ephrin receptor-B4 ( EPHB4 ) (p=1.22×10 -5 ), which cooperates with p120 RasGAP to limit Ras activation. Other probands had pathogenic variants in ACVRL1 , NOTCH1 , ITGB1 , and PTPN11 . ACVRL1 variants were also identified in a multi-generational VOGM pedigree. Integrative genomics defined developing endothelial cells as a key spatio-temporal locus of VOGM pathophysiology. Mice expressing a VOGM-specific EPHB4 kinase-domain missense variant exhibited constitutive endothelial Ras/ERK/MAPK activation and impaired hierarchical development of angiogenesis-regulated arterial-capillary-venous networks, but only when carrying a "second-hit" allele. These results illuminate human arterio-venous development and VOGM pathobiology and have clinical implications.
RESUMEN
We and others have identified FGFR4 as a direct transcriptional target of the alveolar rhabdomyosarcoma (ARMS) specific fusion protein, PAX3-FOXO1. We hypothesized fibroblast growth factor receptor 4 (FGFR4) may act as an effector of PAX3-FOXO1, contributing to PAX3-FOXO1 tumorigenic phenotypes. However, we demonstrate that enhanced expression of FGFR4 does not contribute to inhibited differentiation, enhanced proliferation, or transformation downstream of PAX3-FOXO1 in primary mouse myoblasts. Therefore we were unable to identify any contribution of up regulation of wild type FGFR4 to PAX3-FOXO1 driven tumorigenesis. Conversely, a constitutively active mutant of FGFR4 can enhance primary myoblast proliferation and transformation, indicating activating mutations of FGFR4 could contribute to the development and progression of ARMS. We sequenced the FGFR4 mRNA from five ARMS cell lines and identified no somatic mutations, nor any association with any human single nucleotide polymorphism within the FGFR4 coding region.
Asunto(s)
Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción Paired Box/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Rabdomiosarcoma Alveolar/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Datos de Secuencia Molecular , Mutación , Mioblastos/metabolismo , Mioblastos/patología , Proteínas de Fusión Oncogénica/genética , Factores de Transcripción Paired Box/genética , Polimorfismo de Nucleótido Simple , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Rabdomiosarcoma Alveolar/genética , Rabdomiosarcoma Alveolar/patología , Regulación hacia ArribaRESUMEN
A State of the Art lecture entitled "Molecular Analysis of Vascular Gene Expression" was presented at the ISTH Congress in 2021. Endothelial cells (ECs) form a critical interface between the blood and underlying tissue environment, serving as a reactive barrier to maintain tissue homeostasis. ECs play an important role in not only coagulation, but also in the response to inflammation by connecting these two processes in the host defense against pathogens. Furthermore, ECs tailor their behavior to the needs of the microenvironment in which they reside, resulting in a broad display of EC phenotypes. While this heterogeneity has been acknowledged for decades, the contributing molecular mechanisms have only recently started to emerge due to technological advances. These include high-throughput sequencing combined with methods to isolate ECs directly from their native tissue environment, as well as sequencing samples at a high cellular resolution. In addition, the newest technologies simultaneously quantitate and visualize a multitude of RNA transcripts directly in tissue sections, thus providing spatial information. Understanding how ECs function in (patho)physiological conditions is crucial to develop new therapeutics as many diseases can directly affect the endothelium. Of particular relevance for thrombotic disorders, EC dysfunction can lead to a procoagulant, proinflammatory phenotype with increased vascular permeability that can result in coagulopathy and tissue damage, as seen in a number of infectious diseases, including sepsis and coronavirus disease 2019. In light of the current pandemic, we will summarize relevant new data on the latter topic presented during the 2021 ISTH Congress.
RESUMEN
The COPII component SEC24 mediates the recruitment of transmembrane cargos or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E7.5) observed in Sec24c null mice. To test the potential overlap in function between SEC24C/D, we employed dual recombinase mediated cassette exchange to generate a Sec24cc-d allele, in which the C-terminal 90% of SEC24C has been replaced by SEC24D coding sequence. In contrast to the embryonic lethality at E7.5 of SEC24C-deficiency, Sec24cc-d/c-d pups survive to term, though dying shortly after birth. Sec24cc-d/c-d pups are smaller in size, but exhibit no other obvious developmental abnormality by pathologic evaluation. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/d genes rather than differences in cargo export function explain the early embryonic requirements for SEC24C and SEC24D.
Asunto(s)
Desarrollo Embrionario , Prueba de Complementación Genética , Proteínas de Transporte Vesicular , Animales , Ratones , Ratones Transgénicos , Proteínas de Transporte Vesicular/biosíntesis , Proteínas de Transporte Vesicular/genéticaRESUMEN
Monocyte differentiation into macrophages represents a cornerstone process for host defense. Concomitantly, immunological imprinting of either tolerance or trained immunity determines the functional fate of macrophages and susceptibility to secondary infections. We characterized the transcriptomes and epigenomes in four primary cell types: monocytes and in vitro-differentiated naïve, tolerized, and trained macrophages. Inflammatory and metabolic pathways were modulated in macrophages, including decreased inflammasome activation, and we identified pathways functionally implicated in trained immunity. ß-glucan training elicits an exclusive epigenetic signature, revealing a complex network of enhancers and promoters. Analysis of transcription factor motifs in deoxyribonuclease I hypersensitive sites at cell-type-specific epigenetic loci unveiled differentiation and treatment-specific repertoires. Altogether, we provide a resource to understand the epigenetic changes that underlie innate immunity in humans.