Endothelial Gene Expression Research Articles

Shear stress in atherosclerotic plaque determination.

Atherosclerosis initiates at predictable focal sites near arterial branches and curves, where blood flow is disturbed and shear stress is complex. Endothelial shear stress is the tangential stress derived from the friction of the flowing blood on the endothelial surface of the arterial wall. It is a key factor in modulating endothelial cell gene expression and vascular development and remodeling. Increasing evidences suggest that shear stress patterns have a strong relationship with atherosclerotic features. Moreover, variations in the local artery geometry during atherogenesis further modify flow shear stress characteristics, which contribute to the rupture site at the plaque upstream. In this study, we summarize the mechanistic evidences that associate shear stress patterns with determined atherosclerotic plaque features. An enhanced understanding of the relationship and pathophysiological function of shear stress patterns in atherosclerotic plaque features is essential, which may provide early prediction of clinical risk and guide individualized treatment strategies. In the current review, we analyzed the function of shear stress on the determination of atherosclerotic lesion and provided an update on the mechanotransduction of shear stress, gene expression regulation, and atherosclerotic plaque development and rupture.

Chronic antibody-mediated rejection: new diagnostic tools - clinical significance of C4d deposition and improved detection and characterization of human leucocyte antigen antibodies.

Despite improvements in outcomes of solid organ transplantation (SOT), chronic deterioration of allografts remains an important problem and is associated with increased morbidity and mortality 1. With the implementation of sensitive and specific solid phase immunoassays (SPI) for identification of donor-specific anti-human leucocyte antigen (HLA) antibodies (DSA), and with the improved understanding of the pathogenic effect of alloantibodies, it is well accepted that chronic graft loss is part of the antibody-mediated rejection (AMR) spectrum 1,2. Circulating DSAs, either present prior to transplant or developed de novo, may bind to graft endothelial cells, promoting the classical pathway of complement activation, and the inactive product C4d bound covalently to tissue, provides the ‘footprint’ of antibody-mediated allograft damage 3. Correlation between the level of DSA in the serum, C4d positivity in the biopsy and severity of microvascular damage has been shown by several investigators 4,5. However, C4d-negative biopsies with circulating DSA also showed microcirculation damage and increased expression of endothelial genes 6. A recent publication summarizes the revised Banff criteria for AMR in renal and non-renal allografts, including C4d-negative AMR 7. DSAs have been shown to promote various types of AMR, from the acute to the chronic form of rejection also termed as transplant glomerulopathy in renal, cardiac allograft vasculopathy in heart and bronchiolitis obliterans in lung transplants. Although the HLA antibodies may appear before loss of function and are highly predictive of poor outcome, there is still controversy concerning: (i) whether antibodies detected solely by highly sensitive techniques are clinically relevant, (ii) how often to monitor post-transplant and (iii) when to implement antibody removal therapies in the absence of clinical dysfunction.

Cardiac extracellular matrix–fibrin hybrid scaffolds with tunable properties for cardiovascular tissue engineering

Solubilized cardiac extracellular matrix (ECM) is being developed as an injectable therapeutic that offers promise for promoting cardiac repair. However, the ECM alone forms a hydrogel that is very soft compared to the native myocardium. As both the stiffness and composition of the ECM are important in regulating cell behavior and can have complex synergistic effects, we sought to develop an ECM-based scaffold with tunable biochemical and mechanical properties. We used solubilized rat cardiac ECM from two developmental stages (neonatal, adult) combined with fibrin hydrogels that were cross-linked with transglutaminase. We show that ECM was retained within the gels and that the Young’s modulus could be tuned to span the range of the developing and mature heart. C-kit+ cardiovascular progenitor cells from pediatric patients with congenital heart defects were seeded into the hybrid gels. Both the elastic modulus and composition of the scaffolds impacted the expression of endothelial and smooth muscle cell genes. Furthermore, we demonstrate that the hybrid gels are injectable, and thus have potential for minimally invasive therapies. ECM–fibrin hybrid scaffolds offer new opportunities for exploiting the effects of both composition and mechanical properties in directing cell behavior for tissue engineering.

Effects of shear stress on endothelial progenitor cells.

Endothelial progenitor cells (EPCs) are adult stem cells that play a central role in neovascularization. EPCs are mobilized from bone marrow into peripheral blood, attach to existing endothelial cells, and then transmigrate across the endothelium into tissues, where they proliferate, differentiate, and form new blood vessels. In the process, EPCs are exposed to shear stress, a biomechanical force generated by flowing blood and tissue fluid flow. When cultured EPCs are exposed to controlled levels of shear stress in a flow-loading device, their bioactivities in terms of proliferation, anti-apoptosis, migration, production of bioactive substances, anti-thrombosis, and tube formation increase markedly. Expression of endothelial marker genes and proteins by EPCs also increases in response to shear stress, and they differentiate into mature endothelial cells. Great advances have been made in elucidating the mechanisms by which mature endothelial cells sense and respond to shear stress, but not in EPCs. Further study of EPC responses to shear stress will be necessary to better understand the physiological and pathophysiological roles of EPCs and to apply EPCs to new therapies in the field of regenerative medicine.

GENERATION AND UTILIZATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS-DERIVED-IPS FOR STUDYING ENDOTHELIAL GENE REGULATION

Damage to vascular endothelial cells (EC) is the core of various vascular diseases such as stroke and cardiovascular complications. Understanding molecular mechanisms that govern endothelial gene expression and function is crucial for developing novel therapeutic approaches for vascular endothelial dysfunction-related complications. Induced pluripotent stem cells (iPS) provide a valuable in vitro system to study the molecular processes, including EC-specific gene regulation, that contribute to establishment of EC phenotype. We aimed to study endothelial gene regulation by generating iPS from human umbilical vein EC (HUVECs) and differentiating the resulting iPS back into EC. This model provides a system with a homogenous genetic Background to explore how EC phenotype is revoked (HUVEC to iPS), and reestablished (iPS to EC). Colonies (iPS) were generated using originally reported transcription factors. Quantitative RT-PCR and immunofluorescence analysis were used to characterize iPS and cell lineages derived from iPS. Pluripotency of the iPS were demonstrated by generation of embryoid bodies (EB) and detection of the three germ layer markers. Additionally a novel method was used to directly differentiate iPS into neuronal cell lineages with high efficiency without the use of EB. Next EB generated from iPS were induced to differentiate into EC, thus establishing the EC to iPS back to EC system for analyses. In this system we explored the pattern of activation and repression of a highly EC-specific gene, von Willebrand factor (VWF), as well as transacting factors and cofactors that regulate the VWF promoter activity. Our analyses demonstrated that expression of transacting factors that function as activators of the VWF promoter mirrored that of VWF gene, while those functioning as repressors where either reversed or not significantly altered. Temporal expression of activators were observed during various stages of differentiation. Specifically during the progression from EB to EC we observed upregulation of GATA6 followed by Ets transactivators. Furthermore, the ability of iPS to incorporate into microvasculature was demonstrated in a mouse model of kidney vascular injury. Generation of iPS from EC and differentiation of iPS back to EC provides a unique opportunity to explore the molecular mechanisms that are involved in establishment of EC specific gene regulation and consequently the establishment of EC phenotype. This system will also provide an opportunity for exploring the potential of these EC derived iPS and their differentiated EC towards developing cell- therapy approaches for vascular diseases.

Spironolactone improves endothelial dysfunction in streptozotocin-induced diabetic rats.

Endothelial dysfunction is a critical initiator for developing diabetic vascular complications. Substantial clinical and experimental evidence suggests that aldosterone plays a crucial role in its pathogenesis. The present study aimed to investigate the effect of the mineralocorticoid receptor (MR) blocker, spironolactone, on diabetes-associated endothelial dysfunction and address the underlying mechanism(s) involved in this setting. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ) to rats and spironolactone was orally administered (50mg/kg/day). Our results showed a marked increase in aortic malondialdehyde (MDA) level and upregulation of the catalytic NADPH oxidase subunit, NOX2 gene expression alongside reducing catalase enzyme capacity, and the serum nitric oxide (NO) bioavailability in diabetic rats. This was associated with a significant reduction in endothelial nitric oxide synthase (eNOS) immunoreactivity and gene expression in diabetic aorta. The transforming growth factor-β (TGF-β) protein and the MR gene expression levels were significantly increased in the diabetic rat aorta. Moreover, the diabetic aorta showed a marked impairment in acetylcholine-mediated endothelium-dependent relaxation. Additionally, spironolactone significantly inhibited the elevated MDA, TGF-β, NOX2, and MR levels alongside correcting the dysregulated eNOS expression and the defective antioxidant function as well as NO bioavailability. Spironolactone markedly reversed the impaired endothelial function in the diabetic aorta. Collectively, our study demonstrates that spironolactone ameliorated the vascular dysfunction of diabetic aorta, at least partially via its anti-inflammatory and anti-oxidative effects alongside correcting the dysregulated eNOS and TGF-β expression. Thus, blockade of MR may represent a useful therapeutic approach against diabetic vasculopathy.

Isolation and characterization of a Sca-1+/CD31-progenitor cell lineage derived from mouse heart tissue

BackgroundMyocardial infarction remains the leading cause of mortality in developed countries despite recent advances in its prevention and treatment. Regenerative therapies based on resident cardiac progenitor cells (CPCs) are a promising alternative to conventional treatments. However, CPCs resident in the heart are quite rare. It is unclear how these CPCs can be isolated and cultured efficiently and what the effects of long-term culture in vitro are on their ‘stemness’ and differentiation potential, but this is critical knowledge for CPCs’ clinical application.ResultsHere, we isolated stem cell antigen-1 positive cells from postnatal mouse heart by magnetic active cell sorting using an iron-labeled anti-mouse Sca-1 antibody, and cultured them long-term in vitro. We tested stemness marker expression and the proliferation ability of long-term cultured Sca-1+ cells at early, middle and late passages. Furthermore, we determined the differentiation potential of these three passages into cardiac cell lineages (cardiomyocytes, smooth muscle and endothelial cells) after induction in vitro. The expression of myocardial, smooth muscle and endothelial cell-specific genes and surface markers were analyzed by RT-PCR and IF staining. We also investigated the oncogenicity of the three passages by subcutaneously injecting cells in nude mice. Overall, heart-derived Sca-1+ cells showed CPC characteristics: long-term propagation ability in vitro, non-tumorigenic in vivo, persistent expression of stemness and cardiac-specific markers, and multipotent differentiation into cardiac cell lineages.ConclusionsOur research may bring new insights to myocardium regeneration, for which even a small number of biopsy-derived CPCs could be enriched and propagated long term in vitro to obtain sufficient seed cells for cell injection or cardiac tissue engineering.

HDL inhibits the effects of oxidized phospholipids on endothelial cell gene expression via multiple mechanisms

Oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phospholcholine (OxPAPC) and its component phospholipids accumulate in atherosclerotic lesions and regulate the expression of >1,000 genes, many proatherogenic, in human aortic endothelial cells (HAECs). In contrast, there is evidence in the literature that HDL protects the vasculature from inflammatory insult. We have previously shown that in HAECs, HDL attenuates the expression of several proatherogenic genes regulated by OxPAPC and 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine. We now demonstrate that HDL reverses >50% of the OxPAPC transcriptional response. Genes reversed by HDL are enriched for inflammatory and vascular development pathways, while genes not affected by HDL are enriched for oxidative stress response pathways. The protective effect of HDL is partially mimicked by cholesterol repletion and treatment with apoA1 but does not require signaling through scavenger receptor class B type I. Furthermore, our data demonstrate that HDL protection requires direct interaction with OxPAPC. HDL-associated platelet-activating factor acetylhydrolase (PAF-AH) hydrolyzes short-chain bioactive phospholipids in OxPAPC; however, inhibiting PAF-AH activity does not prevent HDL protection. Our results are consistent with HDL sequestering specific bioactive lipids in OxPAPC, thereby preventing their regulation of select target genes. Overall, this work implicates HDL as a major regulator of OxPAPC action in endothelial cells via multiple mechanisms.

Rat Endothelial Cell Attachment, Behavior and Gene Expression on NaOH-treated Titanium Surfaces

189 INTRODUCTION Materials scientists and clinicians worldwide are working to improve the performance of dental implants, with the aim of accelerating and maintaining integration into hard and soft tissues, and extending their range of application. The surface characteristics of the implant material affect the rate and extent of osseointegration. Vandrovcova et al. 2 recently reviewed the growing evidence that surface-modified materials can increase adhesion, growth, and osteogenic differentiation of cells, thus promoting the integration of the implant into bone and maintaining its secondary stability. Recent advances in dental implant research allow for the modification of the surface of the implant material at the nanometer scale 3, . Surface modifications that increase surface area and Rat Endothelial Cell Attachment, Behavior and Gene Expression on NaOH-treated Titanium Surfaces

Role of flow-sensitive microRNAs in endothelial dysfunction and atherosclerosis: mechanosensitive athero-miRs.

Atherosclerosis preferentially occurs in arterial regions exposed to disturbed flow, in part, due to alterations in gene expression. MicroRNAs (miRNAs) are small, noncoding genes that post-transcriptionally regulate gene expression by targeting messenger RNA transcripts. Emerging evidence indicates that alteration of flow conditions regulate expression of miRNAs in endothelial cells both in vitro and in vivo. These flow-sensitive miRNAs, known as mechano-miRs, regulate endothelial gene expression and can regulate endothelial dysfunction and atherosclerosis. MiRNAs such as, miR-10a, miR-19a, miR-23b, miR-17-92, miR-21, miR-663, miR-92a, miR-143/145, miR-101, miR-126, miR-712, miR-205, and miR-155, have been identified as mechano-miRs. Many of these miRNAs were initially identified as flow sensitive in vitro and were later found to play a critical role in endothelial function and atherosclerosis in vivo through either gain-of-function or loss-of-function approaches. The key signaling pathways that are targeted by these mechano-miRs include the endothelial cell cycle, inflammation, apoptosis, and nitric oxide signaling. Furthermore, we have recently shown that the miR-712/205 family, which is upregulated by disturbed flow, contributes to endothelial inflammation and vascular hyperpermeability by targeting tissue inhibitor of metalloproteinase-3, which regulates metalloproteinases and a disintegrin and metalloproteinases. The mechano-miRs that are implicated in atherosclerosis are termed as mechanosensitive athero-miRs and are potential therapeutic targets to prevent or treat atherosclerosis. This review summarizes the current knowledge of mechanosensitive athero-miRs and their role in vascular biology and atherosclerosis.

Endothelial differentiation of diabetic adipose-derived stem cells

BackgroundDiabetic (DM) patients frequently lack autologous vascular tissue required for revascularization procedures and dialysis access creation. We have developed a tissue-engineered graft that uses adipose-derived stem cells (ASC) as endothelial cell substitutes. Here, we compare DM versus nondiabetic (NDM) ASC in terms of isolation efficiency, proliferation, commitment toward endothelial lineage, and seeding onto the luminal surface of a graft. MethodsASC were isolated from liposuction specimens of vascular surgery patients. Proliferation was assessed by constructing growth curves over 14 d. ASC were differentiated in endothelial growth medium (EGM2). Endothelial commitment was assessed by measuring endothelial cell-specific gene expression (CD31, von Willebrand factor) and by cord formation on Matrigel. Finally, ASC were seeded onto a vascular scaffold, flow conditioned, and imaged with confocal microscopy. ResultsDiabetes did not alter ASC isolation efficiency (224,028 ± 20,231 cells/g adipose for DM (n = 53) versus 259,345 ± 15,441 cells/g adipose for NDM (n = 145; P = 0.21). Growth curves for DM (n = 6) and NDM (n = 6) also appeared similar. After culture in EGM2, upregulation of CD31 and von Willebrand factor message was observed in NDM; these markers were found within the primary cultures of DM but no upregulation was observed after culture in EGM2. Both groups exhibited similar cord formation on Matrigel and retention to vascular scaffolds. ConclusionsIsolation and proliferation studies suggest that adipose is a promising source of stem cells for tissue engineering in the DM population. The angiogenic potential of DM ASC appears intact; however, differences in acquisition of endothelial cell markers suggest that differentiation may be inhibited or delayed by diabetes.

NUMERICAL STUDY OF BIDIRECTIONAL GLENN WITH UNILATERAL PULMONARY ARTERY STENOSIS

Purpose: Hypoplastic left heart syndrome (HLHS) is a congenital heart disease and is usually associated with pulmonary artery stenosis. The superior vena cava-to-pulmonary artery (bidirectional Glenn) shunt is used primarily as a staging procedure to the total cava-to-pulmonary connection for single-ventricle complex. When HLHS coexists with pulmonary artery stenosis, the surgeons then face a multiple problem. This leads to high demand of optimized structure of Glenn surgery. The objective of this article is to investigate the influence of various anastomotic structures and the direction of superior vena cava (SVC) in Glenn on hemodynamics under pulse inflow conditions and try to find an optimal structure of SVC in Glenn surgery with unilateral pulmonary artery stenosis.Method: First, 3D patient-specific models were constructed from medical images of a HLHS patient before any surgery by using the commercial software Mimics, and another software Free-form was used to deform the reconstructed models in the computer. Four 3D patient-specific Glenn models were constructed: model-1 (normal Glenn), model-2 (lean the SVC back to the stenotic pulmonary artery), model-3 (lean the SVC towards the stenotic pulmonary artery), model-4 (add patch at junction of the SVC toward stenosis at pulmonary artery). Second, a lumped parameter model (LPM) was established to predict boundary conditions for computational fluid dynamics (CFD). In addition, numerical simulations were conducted using CFD through the finite volume method. Finally, hemodynamic parameters were obtained and evaluated.Results: It was showed that model-4 have relatively balanced vena cava blood perfusion into the left pulmonary artery (LPA) and right pulmonary artery (RPA), this may be due to less helical flow and the patch at junction of the SVC. Near stenosis of pulmonary artery, model-4 performed with the higher wall shear stress (WSS), which would benefit endothelial cell function and gene expression. In addition, results showed that model-4 performed with the lower oscillatory shear index (OSI) and wall shear stress gradient (WSSG), which would decrease the opportunity of vascular intimal hyperplasia.Conclusion: It is benefited that surgeons adds patch at junction of the SVC towards stenosis at pulmonary artery. These results can impact the surgical design and planning of the Glenn surgery with unilateral pulmonary artery stenosis.

Regulation of endothelial cell-specific Robo4 gene expression by DNA methylation and non-lineage specific transcription factors

Studies of tissue-specific gene expression have suggested that tissue-specific transcription factors or tissue-specific combinations of non-cell-type-specific transcription factors regulate tissue-specific gene expression. Although the studies of endothelial cell (EC)-specific gene expression has identified several transcriptional activators such as SP1, ETS family proteins, and GATA proteins, their expression and combinations are not likely to be EC-specific. To investigate the mechanism of EC-specific gene expression, we analyzed the regulation mechanism of an EC-specific gene, Roundabout4 (Robo4). We identified the 3-kb Robo4 promoter and several transcription factors including SP1 and GABP that bind to the Robo4 promoter and promote Robo4 gene expression. However, we could not explain the mechanism for EC-specific Robo4 gene expression with only those factors because their expression and combination are not EC-specific. Therefore, we hypothesized the contribution of other mechanisms, especially epigenetic control, to Robo4 gene regulation and demonstrated the importance of DNA methylation for EC-specific Robo4 expression. In this review, we summarize our recent studies and discuss the novel regulation model of Robo4 gene expression by transcription factors and DNA methylation.

Turbulent Times for the Atherosclerosis Epigenome

Therapeutics targeting DNA methylation may reverse atherosclerosis.

VEGF-A isoforms differentially regulate ATF-2-dependent VCAM-1 gene expression and endothelial-leukocyte interactions.

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways.

CD47-dependent immunomodulatory and angiogenic activities of extracellular vesicles produced by T cells

Intercellular communication is critical for integrating complex signals in multicellular eukaryotes. Vascular endothelial cells and T lymphocytes closely interact during the recirculation and trans-endothelial migration of T cells. In addition to direct cell–cell contact, we show that T cell derived extracellular vesicles can interact with endothelial cells and modulate their cellular functions. Thrombospondin-1 and its receptor CD47 are expressed on exosomes/ectosomes derived from T cells, and these extracellular vesicles are internalized and modulate signaling in both T cells and endothelial cells. Extracellular vesicles released from cells expressing or lacking CD47 differentially regulate activation of T cells induced by engaging the T cell receptor. Similarly, T cell-derived extracellular vesicles modulate endothelial cell responses to vascular endothelial growth factor and tube formation in a CD47-dependent manner. Uptake of T cell derived extracellular vesicles by recipient endothelial cells globally alters gene expression in a CD47-dependent manner. CD47 also regulates the mRNA content of extracellular vesicles in a manner consistent with some of the resulting alterations in target endothelial cell gene expression. Therefore, the thrombospondin-1 receptor CD47 directly or indirectly regulates intercellular communication mediated by the transfer of extracellular vesicles between vascular cells.

Deep sequencing reveals novel Set7 networks.

Methyl-dependent regulation of transcription has expanded from a traditional focus on histones to encompass transcription factor modulation. While the Set7 lysine methyltransferase is associated with pro-inflammatory gene expression in vascular endothelial cells, genome-wide regulatory roles remain to be investigated. From initial characterization of Set7 as specific for methyl-lysine 4 of H3 histones (H3K4m1), biochemical activity toward non-histone substrates has revealed additional mechanisms of gene regulation. mRNA-Seq revealed transcriptional deregulation of over 8,000 genes in an endothelial model of Set7 knockdown. Gene ontology identified up-regulated pathways involved in developmental processes and extracellular matrix remodeling, whereas pathways regulating the inflammatory response as well as nitric oxide signaling were down-regulated. Chromatin maps derived from ChIP-Seq profiling of H3K4m1 identified several hundred loci with loss of H3K4m1 at gene regulatory elements associated with an unexpectedly subtle effect on gene expression. Transcription factor network analysis implicated six previously described Set7 substrates in mRNA-Seq changes, and we predict that Set7 post-translationally regulates other transcription factors associated with vascular endothelial gene expression through the presence of Set7 amino acid methylation motifs. We describe a role for Set7 in regulating developmental pathways and response to stimuli (inflammation/immune response) in human endothelial cells of vascular origin. Set7-dependent gene expression changes that occurred independent of H3K4m1 may involve transcription factor lysine methylation events. The method of mapping measured transcriptional changes to transcription factors to identify putative substrates with strong associations to functional changes is applicable to substrate prediction for other broad-substrate histone modifiers.

Flow-dependent epigenetic DNA methylation regulates endothelial gene expression and atherosclerosis.

In atherosclerosis, plaques preferentially develop in arterial regions of disturbed blood flow (d-flow), which alters endothelial gene expression and function. Here, we determined that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase-dependent (DNMT-dependent) manner. Induction of d-flow by partial carotid ligation surgery in a murine model induced DNMT1 in arterial endothelium. In cultured endothelial cells, DNMT1 was enhanced by oscillatory shear stress (OS), and reduction of DNMT with either the inhibitor 5-aza-2'-deoxycytidine (5Aza) or siRNA markedly reduced OS-induced endothelial inflammation. Moreover, administration of 5Aza reduced lesion formation in 2 mouse models of atherosclerosis. Using both reduced representation bisulfite sequencing (RRBS) and microarray, we determined that d-flow in the carotid artery resulted in hypermethylation within the promoters of 11 mechanosensitive genes and that 5Aza treatment restored normal methylation patterns. Of the identified genes, HoxA5 and Klf3 encode transcription factors that contain cAMP response elements, suggesting that the methylation status of these loci could serve as a mechanosensitive master switch in gene expression. Together, our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

Protective effect of Xin Mai Jia ultrafiltration extract on human umbilical vein endothelial cell injury induced by hydrogen peroxide and the effect on the NO-cGMP signaling pathway

The aim of the present study was to evaluate the protective effect of the ultrafiltration extract of Xin Mai Jia (XMJ) on a human umbilical vein endothelial cell (HUVEC) injury model induced by hydrogen peroxide (H2O2), by providing experimental data to investigate the mechanism and efficacy underlying the therapeutic effects on atherosclerosis. HUVECs were first injured by H2O2 and then varying final concentrations of the Chinese herb extract were added. Effects of the XMJ extract on morphology, activity, monolayer permeability, biochemical indicators, cytokines, endothelial nitric oxide synthase (eNOS) protein content and eNOS gene expression in the HUVECs were analyzed. H2O2 significantly promoted HUVEC injury. The XMJ ultrafiltration extract significantly improved the morphological changes in the injured HUVECs. In addition, XMJ treatment increased cell activity and decreased monolayer permeability. The expression levels of intracellular adhesion molecule-1, vascular adhesion molecule-1, interleukin-1 and -6 and nuclear factor-κB decreased, while the expression levels of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 increased with XMJ administration. Increased levels of nitric oxide (NO), eNOS protein and eNOS gene expression were also observed. Therefore, the XMJ ultrafiltration extract exhibits marked anti-inflammatory effects and antioxidant abilities. These properties significantly inhibited the H2O2-induced injury of HUVECs, which may be associated with the NO-cyclic guanosine monophosphate signaling pathway.

A systems-level integrative framework for genome-wide DNA methylation and gene expression data identifies differential gene expression modules under epigenetic control.

There is a growing number of studies generating matched Illumina Infinium HumanMethylation450 and gene expression data, yet there is a corresponding shortage of statistical tools aimed at their integrative analysis. Such integrative tools are important for the discovery of epigenetically regulated gene modules or molecular pathways, which play key roles in cellular differentiation and disease. Here, we present a novel functional supervised algorithm, called Functional Epigenetic Modules (FEM), for the integrative analysis of Infinium 450k DNA methylation and matched or unmatched gene expression data. The algorithm identifies gene modules of coordinated differential methylation and differential expression in the context of a human interactome. We validate the FEM algorithm on simulated and real data, demonstrating how it successfully retrieves an epigenetically deregulated gene, previously known to drive endometrial cancer development. Importantly, in the same cancer, FEM identified a novel epigenetically deregulated hotspot, directly upstream of the well-known progesterone receptor tumour suppressor pathway. In the context of cellular differentiation, FEM successfully identifies known endothelial cell subtype-specific gene expression markers, as well as a novel gene module whose overexpression in blood endothelial cells is mediated by DNA hypomethylation. The systems-level integrative framework presented here could be used to identify novel key genes or signalling pathways, which drive cellular differentiation or disease through an underlying epigenetic mechanism. FEM is freely available as an R-package from http://sourceforge.net/projects/funepimod.