Matthew Yan

Hypoxic Repression of Endothelial Nitric-oxide Synthase Transcription Is Coupled with Eviction of Promoter Histones

Hypoxia elicits endothelial dysfunction, in part, through reduced expression of endothelial nitric-oxide synthase (eNOS). Here we present evidence that hypoxia causes a rapid decrease in the transcription of the eNOS/NOS3 gene, accompanied by decreased acetylation and lysine 4 (histone H3) methylation of eNOS proximal promoter histones. Surprisingly, we demonstrate that histones are rapidly evicted from the eNOS proximal promoter during hypoxia. We also demonstrate endothelium-specific H2A.Z incorporation at the eNOS promoter and find that H2A.Z is also evicted by hypoxic stimulation. After longer durations of hypoxia, histones are reincorporated at the eNOS promoter, but these histones lack substantial histone acetylation. Additionally, we identify a key role for the chromatin remodeler, BRG1, in re-establishing eNOS expression following reoxygenation of hypoxic cells. We posit that post-translational histone modifications are required to maintain constitutive eNOS transcriptional activity and that histone eviction rapidly resets histone marks and is a proximal event in the hypoxic repression of eNOS. Although nucleosome eviction has been reported in models of transcriptional activation, the observation that eviction can also accompany transcriptional repression in hypoxic mammalian cells argues that eviction may be broadly relevant to both positive and negative changes in transcription.

Functional Importance of Dicer Protein in the Adaptive Cellular Response to Hypoxia

Background: Functional relationships between the microRNA and cellular hypoxia response pathways are unknown. Results: Dicer is down-regulated in chronic hypoxia; this mechanism maintains the induction of hypoxia-inducible factor-α subunits and hypoxia-responsive genes. Conclusion: Loss of Dicer-dependent microRNA regulation is important for maintaining the concerted cellular response to hypoxia. Significance: Altogether, we provide a newer perspective into the post-transcriptional pathways that regulate the cellular hypoxic response. The processes by which cells sense and respond to ambient oxygen concentration are fundamental to cell survival and function, and they commonly target gene regulatory events. To date, however, little is known about the link between the microRNA pathway and hypoxia signaling. Here, we show in vitro and in vivo that chronic hypoxia impairs Dicer (DICER1) expression and activity, resulting in global consequences on microRNA biogenesis. We show that von Hippel-Lindau-dependent down-regulation of Dicer is key to the expression and function of hypoxia-inducible factor α (HIF-α) subunits. Specifically, we show that EPAS1/HIF-2α is regulated by the Dicer-dependent microRNA miR-185, which is down-regulated by hypoxia. Full expression of hypoxia-responsive/HIF target genes in chronic hypoxia (e.g. VEGFA, FLT1/VEGFR1, KDR/VEGFR2, BNIP3L, and SLC2A1/GLUT1), the function of which is to regulate various adaptive responses to compromised oxygen availability, is also dependent on hypoxia-mediated down-regulation of Dicer function and changes in post-transcriptional gene regulation. Therefore, functional deficiency of Dicer in chronic hypoxia is relevant to both HIF-α isoforms and hypoxia-responsive/HIF target genes, especially in the vascular endothelium. These findings have relevance to emerging therapies given that we show that the efficacy of RNA interference under chronic hypoxia, but not normal oxygen availability, is Dicer-dependent. Collectively, these findings show that the down-regulation of Dicer under chronic hypoxia is an adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, thereby providing an essential mechanistic insight into the oxygen-dependent microRNA regulatory pathway.

Epigenetics and Cardiovascular Disease

A commonly-assumed paradigm holds that the primary genetic determinant of cardiovascular disease resides within the DNA sequence of our genes. This paradigm can be challenged. For example, how do sequence changes in the non-coding region of the genome influence phenotype? Why are all diseases not shared between identical twins? Part of the answer lies in the fact that the environment or exogenous stimuli clearly influence disease susceptibility, but it was unclear in the past how these effects were signalled to the static DNA code. Epigenetics is providing a newer perspective on these issues. Epigenetics refers to chromatin-based mechanisms important in the regulation of gene expression that do not involve changes to the DNA sequence per se. The field can be broadly categorized into three areas: DNA base modifications (including cytosine methylation and cytosine hydroxymethylation), post-translational modifications of histone proteins, and RNA-based mechanisms that operate in the nucleus. Cardiovascular disease pathways are now being approached from the epigenetic perspective, including those associated with atherosclerosis, angiogenesis, ischemia-reperfusion damage, and the cardiovascular response to hypoxia and shear stress, among many others. With increasing interest and expanding partnerships in the field, we can expect new insights to emerge from epigenetic perspectives of cardiovascular health. This paper reviews the principles governing epigenetic regulation, discusses their presently-understood importance in cardiovascular disease, and considers the growing significance we are likely to attribute to epigenetic contributions in the future, as they provide new mechanistic insights and a host of novel clinical applications.

Epigenetics of the vascular endothelium

Classical models of transcription in vascular endothelial cells, specifically the cis/trans paradigm, have limitations. For instance, how does the environment have chronic effects on gene expression in endothelial cells after weeks or years? When an endothelial cell divides, how is this information transmitted to daughter cells? Epigenetics refers to chromatin-based pathways important in the regulation of gene expression and includes three distinct, but highly interrelated, mechanisms: DNA methylation, histone density and posttranslational modifications, and RNA-based mechanisms. Together they offer a newer perspective on transcriptional control paradigms in vascular endothelial cells and provide a molecular basis for understanding how the environment impacts the genome to modify disease susceptibility. This alternative viewpoint for transcriptional regulation allows a reassessment of the cis/trans model and even helps explain some of its limitations. This review provides an introduction to epigenetic concepts for vascular biologists and uses topical examples in cell biology to provide insight into how cell types or even whole organisms, such as monozygotic human twins with the same DNA sequence, can exhibit heterogeneous patterns of gene expression, phenotype, or diseases prevalence. Using endothelial nitric oxide synthase (NOS3) as an example, we examine the growing body of evidence implicating epigenetic pathways in the control of vascular endothelial gene expression in health and disease.

A mechanistic role for DNA methylation in endothelial cell (EC)-enriched gene expression: relationship with DNA replication timing

Proximal promoter DNA methylation has been shown to be important for regulating gene expression. However, its relative contribution to the cell-specific expression of endothelial cell (EC)-enriched genes has not been defined. We used methyl-DNA immunoprecipitation and bisulfite conversion to analyze the DNA methylation profile of EC-enriched genes in ECs vs nonexpressing cell types, both in vitro and in vivo. We show that prototypic EC-enriched genes exhibit functional differential patterns of DNA methylation in proximal promoter regions of most (eg, CD31, von Willebrand factor [vWF], VE-cadherin, and intercellular adhesion molecule-2), but not all (eg, VEGFR-1 and VEGFR-2), EC-enriched genes. Comparable findings were evident in cultured ECs, human blood origin ECs, and murine aortic ECs. Promoter-reporter episomal transfection assays for endothelial nitric oxide synthase, VE-cadherin, and vWF indicated functional promoter activity in cell types where the native gene was not active. Inhibition of DNA methyltransferase activity indicated important functional relevance. Importantly, profiling DNA replication timing patterns indicated that EC-enriched gene promoters with differentially methylated regions replicate early in S-phase in both expressing and nonexpressing cell types. Collectively, these studies highlight the functional importance of promoter DNA methylation in controlling vascular EC gene expression.

Epigenetics in the Vascular Endothelium: Looking From a Different Perspective in the Epigenomics Era

Cardiovascular diseases are commonly thought to be complex, non-Mendelian diseases that are influenced by genetic and environmental factors. A growing body of evidence suggests that epigenetic pathways play a key role in vascular biology and might be involved in defining and transducing cardiovascular disease inheritability. In this review, we argue the importance of epigenetics in vascular biology, especially from the perspective of endothelial cell phenotype. We highlight and discuss the role of epigenetic modifications across the transcriptional unit of protein-coding genes, especially the role of intragenic chromatin modifications, which are underappreciated and not well characterized in the current era of genome-wide studies. Importantly, we describe the practical application of epigenetics in cardiovascular disease therapeutics. # Significance {#article-title-76}Cardiovascular diseases are commonly thought to be complex, non-Mendelian diseases that are influenced by genetic and environmental factors. A growing body of evidence suggests that epigenetic pathways play a key role in vascular biology and might be involved in defining and transducing cardiovascular disease inheritability. In this review, we argue the importance of epigenetics in vascular biology, especially from the perspective of endothelial cell phenotype. We highlight and discuss the role of epigenetic modifications across the transcriptional unit of protein-coding genes, especially the role of intragenic chromatin modifications, which are underappreciated and not well characterized in the current era of genome-wide studies. Importantly, we describe the practical application of epigenetics in cardiovascular disease therapeutics.

Epigenetics in the Vascular Endothelium

Cardiovascular diseases are commonly thought to be complex, non-Mendelian diseases that are influenced by genetic and environmental factors. A growing body of evidence suggests that epigenetic pathways play a key role in vascular biology and might be involved in defining and transducing cardiovascular disease inheritability. In this review, we argue the importance of epigenetics in vascular biology, especially from the perspective of endothelial cell phenotype. We highlight and discuss the role of epigenetic modifications across the transcriptional unit of protein-coding genes, especially the role of intragenic chromatin modifications, which are underappreciated and not well characterized in the current era of genome-wide studies. Importantly, we describe the practical application of epigenetics in cardiovascular disease therapeutics. # Significance {#article-title-76}Cardiovascular diseases are commonly thought to be complex, non-Mendelian diseases that are influenced by genetic and environmental factors. A growing body of evidence suggests that epigenetic pathways play a key role in vascular biology and might be involved in defining and transducing cardiovascular disease inheritability. In this review, we argue the importance of epigenetics in vascular biology, especially from the perspective of endothelial cell phenotype. We highlight and discuss the role of epigenetic modifications across the transcriptional unit of protein-coding genes, especially the role of intragenic chromatin modifications, which are underappreciated and not well characterized in the current era of genome-wide studies. Importantly, we describe the practical application of epigenetics in cardiovascular disease therapeutics.

Clinical Features of Patients With Philadelphia-Negative Myeloproliferative Neoplasms Complicated by Portal Hypertension

BACKGROUND Portal hypertension has been reported to afflict 7% to 18% of patients with Philadelphia-negative MPNs, with complications of variceal bleeding and ascites. The clinical features and outcomes of these patients are unclear. PATIENTS AND METHODS In this multicenter retrospective study, we evaluated the clinical features of 51 patients with MPNs complicated by PHTN. RESULTS The diagnosis of underlying MPN was most frequently PV (39%) and primary MF (35%), followed by post-PV MF (18%), ET (4%), and post-ET MF (4%). Frequency of Janus Kinase 2 V617F mutation appears as expected in the underlying MPN. Thrombosis within the splanchnic circulation was prevalent in patients with polycythemia compared with other MPNs (76% vs. 26%; P = .0007). CONCLUSION PV and MF patients have a greater incidence of PHTN in our population, with thrombosis contributing to PHTN development in PV patients. Patients with splanchnic circulation thrombosis are potential candidates for screening for PHTN. These data might be useful for developing screening strategies for early detection of PHTN in patients with MPN.

Thrombocytopenic syndromes in pregnancy.

The physiological changes in pregnancy result in platelet counts that are lower than in nonpregnant women. Consequently, thrombocytopenia is a common finding occurring in 7–12% of pregnant women. Gestational thrombocytopenia, the most common cause of low platelet counts, tends to be mild in most women and does not affect maternal, fetal or neonatal outcomes. Gestational thrombocytopenia needs to be distinguished from other less common causes of isolated thrombocytopenia, such as immune thrombocytopenia, which affects approximately 3% of thrombocytopenic pregnant women and can lead to neonatal thrombocytopenia. Hypertensive disorders of pregnancy and thrombotic microangiopathies are both associated with thrombocytopenia. They share a considerable number of similar characteristics and are associated with significant maternal and neonatal morbidity and rarely mortality. Accurate identification of the aetiology of thrombocytopenia and appropriate management are integral to optimizing the pregnancy, delivery and neonatal outcomes of this population. Clinical cases are described to illustrate the various aetiologies of thrombocytopenia in pregnancy and their treatment.

Sustained and significant increase in reporting of transfusion reactions with the implementation of an electronic reporting system

Sustained and significant increase in reporting of transfusion reactions with the implementation of an electronic reporting system We report that the implementation of an electronic reporting system (ERS) for transfusion reactions resulted in a sustained and significant increase in reporting at our center, a four-site, 767-bed, tertiary care hospital with high transfusion activity (e.g., 2014, 60,000 component and 30,000 derivative dispensations). In November 2009, we transitioned from a paper-based to a digital reporting process for suspected transfusion reactions (STRs). Six months before the transition to an ERS, hardcopy mount sheets for transfusion labels were revised to provide space for recording corresponding administration vital signs, with adjacent instructions on STR reporting. This first intervention in clinician support was followed by acute transfusion reaction policy refinements, which allowed for verbal reporting of minor reactions, without reflexively mandating recipient testing or termination of the transfusion. This second intervention aimed to remove STR reporting disincentives chiefly related to concerns of additional workload and/or material wastage. Technical tutorials for healthcare worker reporting spanned several months before implementation, without emphasizing the theoryor evidence-based value of hemovigilance. The process of electronically logging any STR was thus open to all bedside clinicians involved in the transfusion process or to the medical laboratory technologists verbally notified of a STR. Clinicians enter STR as they would other laboratory test or drug orders. Through this transition period, medical director reviews of STRs were documented in a transfusion laboratory database (recording imputability, reaction type, severity, and associated product(s)). The electronic logging of a STR was then further incentivized by the assurance of a feedback process in the ERS. Medical director conclusions are uploaded into the original event query/request for investigation after bench results on whether or not a serologic incompatibility was detected (or did not require examination). The ERS went live in the existing electronic health record (QuadraMed CPR, Citrix Systems Inc., 1990-2010, Version 12.1.44.1) with the revised policy in November 2009. Before this (July 9, 2009-October 31, 2009), the reported STR rate was 0.30/day (37 STR; 95% confidence interval [CI], 0.23-0.39). Subsequent to launch in the remaining quarter (November 1, 2009-December 12, 2009), the reported STR rate was 0.66/day (43 STR), representing a 220% increase (p< 0.05; Fig. 1). This daily reporting rate has been sustained through subsequent years with further increases (2010, 0.88; 2011, 0.87; 2012, 0.87; 2013, 0.88; 2014, 0.93), In aggregate, over the 5 years since the introduction of the ERS, 1668 STRs have been reported for an overall and stable rate of 0.88 (95% CI, 0.87-0.90; p< 0.05). The distribution of STR conclusions in the pre-ERS period was as follows: febrile nonhemolytic transfusion reaction (FNHTR), 40%; unrelated to transfusion reaction (UTR), 34%; allergic transfusion reaction (ATR), 7.3%; query bacterial contamination (BaCON), 4.9%; transfusion-related acute lung injury (TRALI), 3.6%; and transfusion-related circulatory overload (TACO), 2.4%. The distribution of STR conclusions after ERS (November 2009-December 2014) was as follows: ATR, 28.8%; UTR, 28.7%; FNHTR, 19.9%; TACO, 8.6%; transfusionassociated dyspnea, 4.7%; pain, 3.2%; query BaCON, 2.3%; and TRALI, 1.7%. In addition to uniquely demonstrating a sustained and significant increase in transfusion reaction reporting with an ERS, without an attributable increase in denominator transfusion activity, we did not see an increase in UTRs (34% vs. 25%) to account for the difference, arguing against “junk inflations” with the userfriendly process. Reporter suspicions have generally concurred with specialist conclusions on transfusion imputability. Two other studies investigated the implementation of an ERS for transfusion reaction reporting. Fujihara and colleagues showed an increase in reporting with an ERS, although findings were confounded by a marked increase in overall transfusion rates in the post-ERS period due to rising surgical volumes. In contrast, our denominator of blood utilization has been stable or decreasing across our sites over the past 5 years, with 3% of product recipients experiencing a STR in any quarter. Yeh and coworkers also reported an increase in postERS reporting rates, although this was only documented in the year following the implementation, without extended assessments for proof of a sustained effect. Large variations between passive/retrospective or active/prospective systems imply underreporting, which may be driven by unawareness of, nihilism on, or obstacles to, this responsibility. After electronic reporting, our STR reporting rate has consistently surpassed that of other sentinel sites in our province. Our findings suggest that despite a dearth of strategies to address this challenge, the solution may lie in removing disincentives while facilitating activities in familiar practice platforms. In robust hospital transfusion services, transfusion reaction reporting launches a doi:10.1111/trf.13549