A temporally-restricted pattern of endothelial cell collagen 4 alpha 1 expression during embryonic development determined with a novel knockin Col4a1-P2A-eGFP mouse line.

Classical collagen type IV comprising of a heterotrimer of two collagen IV alpha 1 chains and one collagen IV alpha 2 chain is the principal type of collagen synthesized by endothelial cells (EC) and is a major constituent of vascular basement membranes. In mouse and man, mutations in genes that encode collagen IV alpha 1 and alpha 2 result in vascular dysfunction. In addition, mutations in genes that encode the Ephrin receptor B4 (EPHB4) and the p120 Ras GTPase-activating protein (RASA1) that cause increased activation of the Ras mitogen-activated protein kinase (MAPK) signaling pathway in EC result in vascular dysfunction as a consequence of impaired export of collagen IV. To understand the pathogenesis of collagen IV-related vascular diseases and phenotypes it is necessary to identify at which times collagen IV is actively synthesized by EC. For this purpose, we used CRISPR/Cas9 targeting in mice to include immediately after the terminal Col4a1 codon a sequence that specifies a P2A peptide followed by enhanced green fluorescent protein (eGFP). Analysis of eGFP expression in Col4a1-P2A-eGFP mice revealed active embryonic EC synthesis of collagen IV alpha 1 through mid to late gestation followed by a sharp decline before birth. These results provide a contextual framework for understanding the basis for the varied vascular abnormalities resulting from perturbation of EC expression and export of functional collagen IV.

Mutation of key signaling regulators of cerebrovascular development in vein of Galen malformations.

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.

EPHB4-RASA1-Mediated Negative Regulation of Ras-MAPK Signaling in the Vasculature: Implications for the Treatment of EPHB4- and RASA1-Related Vascular Anomalies in Humans.

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.

Genetic dysregulation of an endothelial Ras signaling network in vein of Galen malformations.

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.

Molecular analysis of vascular gene expression.

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.

Murine SEC24D can substitute functionally for SEC24C during embryonic development.

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.

Clinical performance and sample freeze-thaw stability of the cobas®6800 SARS-CoV-2 assay for the detection of SARS-CoV-2 in oro-/nasopharyngeal swabs and lower respiratory specimens.

INTRODUCTION: Studies describing the performance characteristics of the cobas®6800 system for SARS-CoV-2 detection in deep respiratory specimens and freeze-thaw stability are limited. The current study compares the clinical performance of the automated SARS-CoV-2 assay on the cobas®6800 system to a lab-developed assay (LDA) and the cobas impact of freeze-thawing combined with lysis buffer. METHODS: Both retrospective and prospectively selected deep respiratory samples and oro- and nasopharyngeal samples in either E-swab® or GLY- were tested using the SARS-CoV-2 assay on the cobas®6800 System and compared to a lab developed assay. Additonally, SARS-CoV-2 RNA stability was assessed after one freeze-thaw cycle with or without lysis buffer. RESULTS: In total, 221 (58.3 %) oro- and nasopharyngeal swabs, 131 (34.6 %) deep respiratory specimens, and n = 25 (6.6 %) swabs of unknown origin were included to study clinical performance. Only 4 samples gave discrepant results, all being positive in the LDA and not the cobas®6800 system. For stability testing, 66 samples without and 110 with lysis buffer were included. No clinically significant difference was found in test results after one freeze-thaw cycle and addition of lysis buffer. CONCLUSION: Based on our findings, the cobas®6800 SARS-CoV-2 RNA assay yielded similar results as the LDA in oro-/nasopharyngeal swabs and deep respiratory specimens. Moreover, the cobas®6800 SARS-CoV-2 RNA assay yielded similar results before and after a freeze-thaw cycle, with better preservation of low viral loads in lysis buffer.

The in vivo endothelial cell translatome is highly heterogeneous across vascular beds.

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.

The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease.

Many common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (<5%) and rare (<1%) variants with a strong impact on blood cell phenotypes. Our data highlight general properties of the allelic architecture of complex traits, including the proportion of the heritable component of each blood trait explained by the polygenic signal across different genome regulatory domains. Finally, through Mendelian randomization, we provide evidence of shared genetic pathways linking blood cell indices with complex pathologies, including autoimmune diseases, schizophrenia, and coronary heart disease and evidence suggesting previously reported population associations between blood cell indices and cardiovascular disease may be non-causal.

Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity.

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.

Transcriptional diversity during lineage commitment of human blood progenitors.

Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine.

Alternatively spliced tissue factor promotes breast cancer growth in a ß1 integrin-dependent manner.

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.

PAX3-FOXO1 and FGFR4 in alveolar rhabdomyosarcoma.

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.