Project description:Formation and maturation of a functional blood vascular system is required for the development and maintenance of all tissues in the body. During the process of blood vessel development, primordial endothelial cells are formed and become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Specification of arterial and venous endothelial cells occurs in conjunction with suppression of endothelial cell cycle progression, and endothelial cell hyperproliferation is associated with potentially lethal arterial-venous malformations. However, the mechanistic role that cell cycle state plays in arterial-venous specification is unknown. Herein, studying vascular development in Cdh5-CreERT2;R26FUCCI2aR reporter mice, we find that venous and arterial endothelial cells exhibit a propensity for different cell cycle states during development and in adulthood. That is, venous endothelial cells are predominantly FUCCI-Negative, while arterial endothelial cells are enriched for the FUCCI-Red reporter. Single cell RNA sequencing analysis of developing retinal endothelial cells reveals that venous endothelial cells are enriched for the FUCCI-Negative state and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state and TGF-b signaling. Further transcriptional analyses and live imaging of cultured endothelial cells expressing the FUCCI reporter show that reporter-negative corresponds to an early G1 state and reporter-red corresponds to late G1 state. We find the early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-b1-induced arterial gene expression. In a mouse model of endothelial cell hyperproliferation and disrupted arterial-venous specification, pharmacological inhibition of endothelial cell cycle prevents the vascular defects. Collectively, our results show that endothelial cell cycle control plays a key role in arterial-venous network formation, and distinct cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous specification.
Project description:Formation and maturation of a functional blood vascular system is required for the development and maintenance of all tissues in the body. During the process of blood vessel development, primordial endothelial cells are formed and become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Specification of arterial and venous endothelial cells occurs in conjunction with suppression of endothelial cell cycle progression, and endothelial cell hyperproliferation is associated with potentially lethal arterial-venous malformations. However, the mechanistic role that cell cycle state plays in arterial-venous specification is unknown. Herein, studying vascular development in Cdh5-CreERT2;R26FUCCI2aR reporter mice, we find that venous and arterial endothelial cells exhibit a propensity for different cell cycle states during development and in adulthood. That is, venous endothelial cells are predominantly FUCCI-Negative, while arterial endothelial cells are enriched for the FUCCI-Red reporter. Single cell RNA sequencing analysis of developing retinal endothelial cells reveals that venous endothelial cells are enriched for the FUCCI-Negative state and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state and TGF-b signaling. Further transcriptional analyses and live imaging of cultured endothelial cells expressing the FUCCI reporter show that reporter-negative corresponds to an early G1 state and reporter-red corresponds to late G1 state. We find the early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-b1-induced arterial gene expression. In a mouse model of endothelial cell hyperproliferation and disrupted arterial-venous specification, pharmacological inhibition of endothelial cell cycle prevents the vascular defects. Collectively, our results show that endothelial cell cycle control plays a key role in arterial-venous network formation, and distinct cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous specification.
Project description:The vascular tree has considerable diversity, with discrete regions having different physiologic characteristics and permeability. Of note are venules that are significantly more sensitive to pro-inflammatory cytokines than arterioles. We used microarrays to identify molecular signatures that distinguish primary human venous endothelial cells from arterial endothelial cells. We used microarrays to identify genes differentially expressed by venous vs arterial human endothelial cells.
Project description:Circulating microRNAs (miRNAs) presented in venous plasma have recently been demonstrated as powerful biomarkers for the diagnosis and prognostic prediction of complex diseases like cancer. Nevertheless, those presented in arterial plasma have been ignored based on the assumption that the miRNA profiles in arterial and venous plasma would be identical. Here, we disputed this intuitive assumption by comparing arterial and venous plasma miRNA expression profiles from male rats using microarray technique. Though the microRNA profiles were largely similar, a considerable number of miRNAs showed significant differential expression, including 10 arterial highly expressed miRNAs and 14 venous highly expressed miRNAs. The differentially expressed miRNAs were validated by qRT-PCR. We performed computational analysis of the function enrichment and disease association of these miRNAs and their targets. Our analysis also suggested significant correlations between plasma miRNA expression and tissue miRNA expression. Four arterial highly expressed miRNAs showed enriched expression in specific tissues and thus could serve as novel biomarker candidates.
Project description:HUVEC-FUCCI cells were used to demonstrate that different endothelial cell cycle states provide distict windows of opportunity for gene expression in response to extrinsic signals. HUVEC-FUCCI were FACS-isolated into three different cell cycle states. Peptide digests from the resulting lysates showed differentially expressed proteins among the three cell cycles. These studies show that endothelial cell cycle state determines the propensity for arterial vs. venous fate specification.
Project description:Background: miRNAs derived from peripheral venous blood gained extensive attention as clinical biomarkers, while arterial miRNAs exhibited slightly different expression profiles. We compared the expression profiles of venous- and arterial-derived plasma miRNA between young and aged male SD rats by next-generation sequencing, in order to explore whether peripheral venous miRNAs can represent entire circulating vessels in abnormal conditions like aging. Results: MSigDB Hallmark Gene Set reference and TAM 2.0 server were used to investigate the enriched functions and associated diseases. The aging-related de-regulated miRNAs in artery and vein shown similar enriched functional terms. Of note, refer to the arterial-versus-venous differential-expressed miRNA profiles, only a few miRNAs shared between young and aged rats. Among them, miR-450a/b and miR-223 shown the similar tendency between young and aged rat, while miR-136 and miR-503 etc displayed the opposite direction under the same scenario. Since miRNAs are under the control of their specific transcriptional factors, we further analyzed upstream regulators which influence miRNAs level for vascular vessel location. TransmiR v2.0 tool was used and found enriched upstream transcription factors like NFκB and SIRT1. These transcriptional factors could be organ-specific expression and/or regulated in physiological and aging states as parts of plausible causal factors. Conclusion: This study screened and analyzed the differential differential-expressed miRNA profiles in arterial and venous plasma under aging conditions, suggesting the importance of origin of candidate circulating miRNA biomarkers upon the certain scenario and its potential regulatory rule.
Project description:Similar to bacterial proteins that are targeted to distinct macrophages organelles via extracellular vesicles, we propose that these vesicles also traffic small RNAs to modulate specific host factors. To test this, we aim to sequence extracellular vesicle derived sRNA, and whole bacterial small RNAs to determine selectivity, and to identify their bacterial and mammalian targets (Experimental Plan in Table-1). For this we will collect highly purified vesicles from N. gonorrhoeae (strain MS11A). We will also treat mouse derived primary macrophages with extracellular vesicles and compare their RNA response to untreated macrophages (Table-2). This will provide novel insights into how macrophages respond to N. gonorrhoeae infections. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:L1CAM-captured extracellular vesicles (LCEVs) were isolated and characterized meticulously. Whole-transcriptome of LCEVs was analyzed by lncRNA microarray and RNA-Sequencing. RNAs expressed differently in LCEVs from ASD sera vs. TD sera were screened, analyzed, and further validated.
Project description:Arterial and venous (A/V) thrombosis constitutes the greatest source of morbidity and mortality worldwide. Long considered as distinct entities, accumulating evidence indicates that A/V thrombosis can occur in the same populations suggesting that common mechanisms are likely operative. Although hyperactivation of the immune system is a common forerunner to the genesis of thrombotic events in both vascular systems, the key molecular control points remain poorly understood. Consequently, anti-thrombotic therapies targeting the immune system for therapeutic gain are lacking. Here we show that neutrophils are key effectors of both A/V thrombosis and can be targeted via novel immunoregulatory nanoparticles. Using antiphopholipid antibody syndrome (APS) as a model for devastating A/V thrombosis, we identified the transcription factor Krüppel-like factor 2 (KLF2) as a key regulator of neutrophil activation. Upon activation via genetic loss of KLF2 or administration of antiphospholipid antibodies, neutrophils cluster P-selectin glycoprotein ligand 1 (PSGL-1) via cortical actin remodeling, thereby increasing adhesion potential at thrombosis sites. Targeting clustered PSGL-1 using designer nanoparticles attenuates neutrophil-mediated A/V thrombosis in APS and KLF2 knockout models, illustrating the importance and feasibility of targeting activated neutrophils to prevent pathological thrombosis. Together, our results demosntrate a role for activated neutrophils to prevent pathological thrombosis. Together, our results demonstrate a role for activated neutorphils in both arterial and venous thrombosis and identify key molecular events that serve as potential targets for therapeutics against diverse causes of immunothrombosis.