Endothelial Gene Expression Research Articles

Laser speckle contrast imaging and Oxygen to See for assessing microcirculatory liver blood flow changes following different volumes of hepatectomy

ObjectivePortal hyperperfusion after extended hepatectomy or small-for-size liver transplantation may induce organ dysfunction and failure. This study was designed to monitor and characterize the hepatic microcirculatory perfusion following different volumes of hepatectomy in rats by using laser speckle contrast image (LSCI) and Oxygen to See (O2C), a spectrometric device. MethodsThe microcirculatory liver blood flow of the rats that underwent 68%, 85% and 90% hepatectomy (68PH, 85PH and 90PH) was monitored with LSCI and O2C before and following the hepatectomy. The portal venous flow (PVF) and hepatic arterial flow (HAF) were measured with an ultrasonic flowmeter. Liver regeneration, liver injury, histologic evaluation and gene expression were also assessed at 12h, 24h, 3d and 7d post hepatectomy. ResultsAll the 68PH and 85PH rats survived, and 57% of the 90PH rats survived. After hepatectomy, both PVF and HAF decreased transiently, with the PVF of the 85PH and 90PH rats significantly lower than that of the 68PH rats. In contrast, the PVF and HAF per gram of liver weight were greatly increased after liver resection and were proportional to the volume of resected liver. Correspondingly, the microcirculatory liver blood flow of the 68PH, 85PH and 90PH rats, as assessed by both LSCI and O2C, were increased after hepatectomy, and the 90PH group was significantly higher than the 68PH and 85PH groups. The hyperperfusion continued for approximately 3days and returned to baseline following the completion of liver regeneration. The liver venous oxygen saturation of the three groups decreased immediately after hepatectomy and returned to baseline from 24h after hepatectomy. The 90PH rats also showed delayed liver regeneration and the most severe liver injury, as reflected by increased serum ALT, AST and TBIL levels, hepatocellular vacuolization, and inflammatory and endothelial constriction gene expressions (TNF-α, IL-1β, MIP-1α, ET-1 and TM-1). ConclusionHepatic microcirculation hyperperfusion resulting from major and extended liver resection could be assessed by LSCI and O2C methods. The 90PH in rats led to extraordinary sinusoidal hyperperfusion, severe endothelial injury and liver failure. Monitoring the changes of hepatic microcirculation perfusion following extended hepatectomy or small-for-size liver transplantation may help to analyze the extent of hyperperfusion.

Interaction between the Stress Phase Angle (SPA) and the Oscillatory Shear Index (OSI) Affects Endothelial Cell Gene Expression.

Hemodynamic forces play an important role in the non-uniform distribution of atherosclerotic lesions. Endothelial cells are exposed simultaneously to fluid wall shear stress (WSS) and solid circumferential stress (CS). Due to variations in impedance (global factors) and geometric complexities (local factors) in the arterial circulation a time lag arises between these two forces that can be characterized by the temporal phase angle between CS and WSS (stress phase angle–SPA). Asynchronous flows (SPA close to -180°) that are most prominent in coronary arteries have been associated with localization of atherosclerosis. Reversing oscillatory flows characterized by an oscillatory shear index (OSI) that is great than zero are also associated with atherosclerosis localization. In this study we examined the relationship between asynchronous flows and reversing flows in altering the expression of 37 genes relevant to atherosclerosis development. In the case of reversing oscillatory flow, we observed that the asynchronous condition upregulated 8 genes compared to synchronous hemodynamics, most of them proatherogenic. Upregulation of the pro-inflammatory transcription factor NFκB p65 was confirmed by western blot, and nuclear translocation of NFκB p65 was confirmed by immunofluorescence staining. A comparative study between non-reversing flow and reversing flow found that in the case of synchronous hemodynamics, reversing flow altered the expression of 11 genes, while in the case of asynchronous hemodynamics, reversing flow altered the expression of 17 genes. Reversing flow significantly upregulated protein expression of NFκB p65 for both synchronous and asynchronous conditions. Nuclear translocation of NFκB p65 was confirmed for synchronous and asynchronous conditions in the presence of flow reversal. These data suggest that asynchronous hemodynamics and reversing flow can elicit proatherogenic responses in endothelial cells compared to synchronous hemodynamics without shear stress reversal, indicating that SPA as well as reversal flow (OSI) are important parameters characterizing arterial susceptibility to disease.

Abstract 13863: PGC-1α Mediates Differentiation Arrest of Murine Bone Marrow-derived Endothelial Progenitor Cells in Diabetes

Introduction: Endothelial progenitor cells (EPCs) in murine bone marrow lineage-negative c-kit+Sca-1+ (BM-KSL) fraction are mobilized in circulation upon tissue injury to mediate a neovascularization process called vasculogenesis, which requires proper differentiative capacity of EPCs. Diabetics suffer defective vasculogenesis, a substantial mechanism for diabetic angiopathy in ischemic limb and heart. How it occurs, however, remains largely unknown. We recently demonstrated that type 1 (T1) and T2 diabetes (DM) induces a transcriptional coactivator PGC-1α in endothelial cells (ECs), and that EC PGC-1α mediates impaired cell migration and angiogenesis in DM. PGC-1α was also found to increase in EPCs cultivated from T2DM patients PB-MNCs. Building on these, we hypothesized roles for PGC-1α in EPC dysfunction. Methods and Results: We used Tie2-Cre-driven PGC-1α conditional null mice (KO), with or without T1DM induction by streptozocine. DM increased PGC-1α mRNA in WT BM-KSL cells whereas this induction was blocked in KO cells. DM decreased the differentiation capacity of WT BM-KSL cells and PB-MNCs, as determined by the formation of primitive (pEPC) and definitive (dEPC) colonies. PGC-1α ablation in KO rescued the defective EPCs differentiation in DM, most notably PB-MNCs formation of dEPC colonies, the known vasculogenic population. Consistent with this, endothelial marker gene expression (KDR, CD31, vWF), EPCs abundance, and EPCs vasculogenic functions of PB-MNCs was blunted by DM but rescued by PGC-1α ablation in KO. Importantly, T2DM induction by high-fat diet feeding also caused reduction in the EPCs abundance and EC gene expression of PB-MNCs, which was rescued by PGC-1α ablation in KO. Conclusions: Our data demonstrate critical new roles for PGC-1α in vasculogenesis. PGC-1α mediates the vasculogenic differentiation defect of circulating EPCs in T1 and T2 DM, in sharp contrast to the anti-migratory effect of EC PGC-1α in mediating diabetic angiostasis. Our data broadens the relevance of PGC-1α in the etiology of diabetic angiopathy, now with a potential to therapeutically restore EPCs vasculogenicity, and may provide an opportunity to improve the efficacy of autologous adoptive EPC therapy for limb and myocardial ischemia in DM.

Role of MicroRNA in Endothelial Dysfunction and Hypertension.

Purpose of ReviewHypertension is either a cause or a consequence of the endothelial dysfunction and a major risk factor for cardiovascular disease (CVD). In vitro and in vivo studies established that microRNAs (miRNAs) are decisive for endothelial cell gene expression and function in various pathological conditions associated with CVD.This review provides an overview of the miRNA role in controlling the key connections between endothelial dysfunction and hypertension.Recent FindingsHerein we summarize the present understanding of mechanisms underlying hypertension and its associated endothelial dysfunction as well as the miRNA role in endothelial cells with accent on the modulation of renin-angiotensin-aldosterone-system, nitric oxide, oxidative stress and on the control of vascular inflammation and angiogenesis in relation to endothelial dysfunction in hypertension. In particular, latest insights in the identification of endothelial-specific microRNAs and their targets are added to the understanding of miRNA significance in hypertension.SummaryThis comprehensive knowledge of the role of miRNAs in endothelial dysfunction and hypertension and of molecular mechanisms proposed for miRNA actions may offer novel diagnostic biomarkers and therapeutic targets for controlling hypertension-associated endothelial dysfunction and other cardiovascular complications.

Biofluids, cell mechanics and epigenetics: Flow-induced epigenetic mechanisms of endothelial gene expression

Epigenetics is the regulation of gene expression (transcription) in response to changes in the cell environment through genomic modifications that largely involve the non-coding fraction of the human genome and that cannot be attributed to modification of the primary DNA sequence. Epigenetics is dominant in establishing cell fate and positioning during programmed embryonic development. However the same pathways are used by mature postnatal and adult mammalian cells during normal physiology and are implicated in disease mechanisms. Recent research demonstrates that blood flow and pressure are cell environments that can influence transcription via epigenetic pathways. The principal epigenetic pathways are chemical modification of cytosine residues of DNA (DNA methylation) and of the amino tails of histone proteins associated with DNA in nucleosomes. They also encompass the post-transcriptional degradation of mRNA transcripts by non-coding RNAs (ncRNA). In vascular endothelium, epigenetic pathways respond to temporal and spatial variations of flow and pressure, particularly hemodynamic disturbed blood flow, with important consequences for gene expression. The biofluid environment is linked by mechanotransduction and solute transport to cardiovascular cell phenotypes via signaling pathways and epigenetic regulation for which there is an adequate interdisciplinary infrastructure with robust tools and methods available. Epigenetic mechanisms may be less familiar than acute genomic signaling to Investigators at the interface of biofluids, biomechanics and cardiovascular biology. Here we introduce a biofluids / cellular biomechanics readership to the principal epigenetic pathways and provide a contextual overview of endothelial epigenetic plasticity in the regulation of flow-responsive transcription.

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Dietary glutamine supplementation enhances endothelial progenitor cell mobilization in streptozotocin-induced diabetic mice subjected to limb ischemia

Diabetes is a metabolic disorder with increased risk of vascular diseases. Tissue ischemia may occur with diabetic vascular complications. Bone marrow-derived endothelial progenitor cells (EPCs) constitute a reparative response to ischemic injury. This study investigated the effects of oral glutamine (GLN) supplementation on circulating EPC mobilization and expression of tissue EPC-releasing markers in diabetic mice subjected to limb ischemia. Diabetes was induced by a daily intraperitoneal injection of streptozotocin for 5 days. Diabetic mice were divided into 2 nonischemic groups and 6 ischemic groups. One of the nonischemic and 3 ischemic groups were fed the control diet, while the remaining 4 groups received diets with identical components except that part of the casein was replaced by GLN. The respective diets were fed to the mice for 3 weeks, and then the nonischemic mice were sacrificed. Unilateral hindlimb ischemia was created in the ischemic groups, and mice were sacrificed at 1, 7 or 21 days after ischemia. Their blood and ischemic muscle tissues were collected for further analyses. Results showed that plasma matrix metallopeptidase (MMP)-9 and the circulating EPC percentage increased after limb ischemia in a diabetic condition. Compared to groups without GLN, GLN supplementation up-regulated plasma stromal cell-derived factor (SDF)-1 and muscle MMP-9, SDF-1, hypoxia-inducible factor-1 and vascular endothelial growth factor gene expression. The CD31-immunoreactive intensities were also higher in the ischemic limb. These findings suggest that GLN supplementation enhanced circulating EPC mobilization that may promote endothelium repair at ischemic tissue in diabetic mice subjected to limb ischemia.

Gene expression profile of endothelial cells during perturbation of the gut vascular barrier

ABSTRACTIt has been widely demonstrated that tolerance against gut microbiota is compartmentalized to mucosal sites where microbes mostly reside. How the commensal bacteria are excluded from the entrance into the blood stream via intestinal capillaries that are located beneath the gut epithelium was not clear. We recently described the existence of a new anatomical structure, the ‘gut vascular barrier’ (GVB), both in murine and human intestines that plays a fundamental role in avoiding indiscriminate trafficking of bacteria from the gut into the blood circulation. The vascular barrier integrity could be altered by Salmonella typhimurium, a pathogen capable of systemic dissemination, through the modulation of the Wnt/β-catenin signaling pathway. Here we have analyzed the differences in gut endothelial gene expression profiles during Salmonella infection and have identified some interesting characteristics of endothelial to mesenchymal transition. These findings add new insights in the gut-liver axis.

Inhibition of microRNA-495 Enhances Therapeutic Angiogenesis of Human Induced Pluripotent Stem Cells.

Therapeutic angiogenesis has emerged as a promising strategy to regenerate the damaged blood vessels resulting from ischemic diseases such as myocardial infarction (MI). However, the functional integration of implanted endothelial cells (ECs) in infarcted heart remains challenging. We herein develop an EC generation approach by inhibiting microRNA-495 (miR-495) in human induced pluripotent stem cells (hiPSCs) and assess the angiogenic potential for MI treatment. The anti-angiogenic miR-495 belonging to Dlk1-Dio3 miR cluster was identified through expression profiling and computational analysis. Loss-of-function experiments for miR-495 were performed using a lentiviral transfer of antisense sequence in hiPSCs. The pluripotency of hiPSCs was not impacted by the genetic modification. Induced with differentiation medium, miR-495 inhibition enhanced the expression of EC genes of hiPSCs, as well as the yield of ECs. Newly derived ECs displayed prominent angiogenic characteristics including tube formation, cell migration, and proliferation. Mechanistically, miR-495 mediated the expression of endothelial or angiogenic genes by directly targeting vascular endothelial zinc finger 1. After transplantation in immunodeficient MI mice, the derived ECs significantly increased neovascularization in the infarcted heart, prevented functional worsening, and attenuated expansion of infarct size. The functional integration of the implanted ECs into coronary networks was also enhanced by inhibiting miR-495. miR-495 represents a new target not only for promoting EC generation from hiPSCs but also for enhancing angiogenesis and engraftment of hiPSC-derived ECs in ischemic heart. Stem Cells 2017;35:337-350.

High levels of HtrA4 observed in preeclamptic circulation drastically alter endothelial gene expression and induce inflammation in human umbilical vein endothelial cells

IntroductionPreeclampsia (PE) is a life-threatening pregnancy disorder characterized by wide-spread endothelial dysfunction. Placental factors circulating in the maternal blood are believed to cause endothelial dysfunction. Our previous study identified HtrA4 as a placenta-specific serine protease that is released into the maternal circulation and significantly increased in early-onset PE. In this study, we examined the impact of HtrA4 on expression of endothelial genes related to vessel biology, using human umbilical vein endothelial cells (HUVECs) as a model. MethodsHUVECs were treated with 0 or 3 μg/ml HtrA4 (highest concentration seen in PE circulation) for 24 h and analysed by an endothelial cell biology PCR array containing 84 genes. HtrA4-induced changes were then validated by real-time RT-PCR and ELISA for time and dose dependency. ResultsHigh levels of HtrA4 significantly altered the expression of a range of genes related to inflammation, vaso-activity, angiogenesis, cell adhesion, platelet activation and coagulation. In particular, pro-inflammatory genes IL6, PTGS2 (COX2) and IL1B were significantly increased by HtrA4. IL6 protein in HUVEC media was also drastically increased. THBD, an anticoagulant factor reported to be increased in PE, was significantly up-regulated by HtrA4. In contrast, THBS1, which is involved in many regulatory processes of endothelial cell biology, was severely down-regulated by HtrA4. DiscussionHtrA4 significantly increased the inflammatory responses of HUVECs, and altered their expression of a number of genes important for vessel biology. These data suggest that placenta-derived HtrA4 that is increased in PE circulation is a potential causal factor of endothelial dysfunction.

SY 15-2 ENDOTHELIAL EPIGENETICS AND ITS ROLE IN MEDIATING BIOMECHANICAL STRESS OF HYPERTENSION

Abstract Hemodynamics creates a constantly changing physical and chemical environment to which the arterial endothelium is exquisitely sensitive. Biomechanical stresses are intrinsic to blood flow characteristics and blood pressure and therefore are important considerations in hypertension. Near branching anatomical sites in arteries, blood flow separates from the main flow to undergo complex multi-directional characteristics for a part of each cardiac cycle (collectively referred to as disturbed flow). Atherosclerosis and aneurysmal pathology develop preferentially at disturbed flow locations, particularly when an additional cardiovascular risk factor such as hypercholesterolemia or high blood pressure are present. In the pre-disease state, a pre-inflammatory endothelial phenotype is characteristically present in such regions in contrast to nearby endothelium outside of the complex disturbed flow fields. A regulatory hierarchy of mechanisms controls endothelial gene and protein expression (and function) in response to the flow environment. Gene expression regulation, the transcription of the DNA (genomic) code to mRNA, is well described by transcription factors, enhancers and repressors, many of which are flow responsive. However, emerging studies describe the additional role of localized disturbed blood flow upon epigenetic mechanisms of endothelial responses to biomechanical stress. Epigenetics and epigenomics (global analyses of epigenetic changes across the entire genome) encompass heritable and non-heritable changes in nuclear chromatin leading to gene expression changes that cannot be attributed to changes in the primary DNA sequence. Hemodynamic stimuli have recently been shown to induce epigenetic responses including transcriptional regulation by proximal promoter DNA methylation, and regulation of gene and protein expression by histone/chromatin remodeling and by noncoding RNA-based mechanisms.Dynamic epigenomic responses to flow that result in regulation of endothelial gene expression in vivo and in vitro include: (i) regions of stable differentially methylated DNA in swine and mouse endothelial genomes that are arterial site-specific and map to atherosusceptibility, (ii) disturbed flow (but not undisturbed flow) applied to endothelial cells in vitro that induces DNA methylation through DNA methyltransferase enrichment of gene promoter regions; the resulting hypermethylation suppresses gene expression, (iii) histone acetylation / deacetylation and methylation that create histone marks that enable or suppress gene expression by controlling access of transcription factors to chromatin DNA, and (iv) short microRNAs and long noncoding RNAs that interact with highly specific binding sites of newly synthesized mRNA to promote its degradation and thus suppress transcription. Flow-mediated epigenomic responses intersect with cis and trans factor regulation to maintain endothelial function in a shear-stressed or pressure-stressed environment and may contribute to localized endothelial dysfunctions that promote localized vascular pathology.The Encyclopedia of DNA Elements (ENCODE) and the International Human Epigenome Consortia (IHEC) have uncovered thousands of putative epigenetic regulatory sites in non-coding regions of the genome. Methylation of cytosine nucleosides in DNA both at/near gene promoter regions as well as at some distance upstream is a potent epigenetic suppressor of transcription. Recently, complete methylated DNA immunoprecipitation sequencing (MeDIP-seq) of regions of arterial endothelium associated with disturbed vs undisturbed flow in vivo in pigs has identified many differentially methylated DNA profiles enriched in exons and 5'UTR sequences of annotated genes, 60 of which are linked to cardiovascular disease. Furthermore, in human arterial endothelial cells in culture we have demonstrated DNA methylation plasticity to be regulated by disturbed flow resulting in suppression (hypermethylation) or stimulation (hypomethylation) of transcription. We propose dynamic DNA methylation to be a genome-wide epigenetic mechanism of transcriptional control that regulates adaptive endothelial phenotype plasticity in response to the local environment, including biomechanical stresses associated with disturbed flow characteristics and hypertension.The epigenomic code therefore has the potential to figure prominently in disease susceptibility and pathogenesis arising from environmental influences independent of, or in concert with, mutations and single nucleotide polymorphisms (SNP) linked to many complex diseases, including hypertension and cardiovascular disease. The role of disturbed flow as a hemodynamic regulator of epigenomic DNA methylation-mediated and histone-mediated gene expression may play a prominent role in endothelial vascular function and dysfunction in hypertension, atherogenesis and aneurysm development. The cellular mechanisms that link hemodynamics and biomechanical stress to the epigenomic code remain unexplored.

Evaluation of vascular endothelial growth factor gene and protein expression in canine metastatic mammary carcinomas.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that could be associated with the induction of endothelial cell proliferation and metastasis. In this study, we evaluated VEGF gene and protein expression in canine mammary tumors (CMT), including metastatic carcinomas, to determine if there is an influence of this marker in the malignant processes and aggressiveness of CMT. We also compared VEGF protein levels with clinicopathological features. The VEGF gene and protein expression levels were evaluated by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively, in normal mammary gland samples, benign mammary tumors, nonmetastatic mammary carcinomas, and metastatic mammary carcinomas. High VEGF gene and protein levels were associated with malignant tumors compared with normal mammary glands (p = 0.0089 and p < 0.0001, respectively). Benign tumors showed an increased VEGF protein expression compared with normal samples (p = 0.0467). No significant differences in VEGF gene or protein levels were detected between benign and malignant tumors or between nonmetastatic and metastatic carcinomas, suggesting an absence in the correlation of VEGF with malignant processes and aggressiveness of CMT. No correlation of VEGF expression with clinical and histopathological parameters was observed, suggesting that VEGF could be important in the beginning of the mammary gland carcinogenic process and could be related to survival time.

Soluble adhesion molecules in patients with acute coronary syndrome after percutaneous coronary intervention with drug-coated balloon, drug-eluting stent or bare metal stent

Adhesion molecules play an important role in inflammation, atherosclerosis and coronary artery disease (CAD). These molecules are expressed on the surface of dysfunctional endothelial cells, causing inflammatory cells from the circulation to adhere and migrate through the endothelium. Their expression is upregulated in acute coronary syndrome (ACS) and after percutaneous coronary intervention (PCI). The contact between stent struts and endothelium upregulates endothelial cell gene expression, endothelial cell activation and inflammation. The paclitaxel or sirolimus eluting stents inhibited expression of adhesion molecules in several studies and reduced the incidence of major adverse cardiac events (MACE) after drug-eluting stent (DES) over bare metal stent (BMS) implantation. Therefore, we propose that elevated serum levels of the soluble adhesion molecules after primary PCI in patients treated with BMS or DES implantation versus drug-coated balloon (DCB) application to the vulnerable coronary plaque might be a predictor of MACE and further adverse outcomes. Consequently, DCB-only strategy in patients with ACS might be a superior approach in comparison to BMS implantation and non-inferior approach when compared to DES implantation.

Effect of Wnt/β-catenin signal pathway on of matrix metalloproteinase-7 and vascular endothelial growth factor gene expressions in endometriosis

Purpose: To explore the function of Wnt/&#x03B2;-catenin signal pathway on promoting the adhesion, invasion, and metastasis of endometriosis tissues by analyzing its effects on the expressions of matrix metalloproteinase-7 (MMP-7) and vascular endothelial growth factor in endometriosis. Materials and Methods: Endometriosis nude mice models were included. Small RNA interference technology was used to block Wnt/&#x03B2;-catenin signal pathway. HE staining technique was adopted to observe the difference of pathological morphology among groups. The immunohistochemistry and real-time quantitative PCR were performed to analyze the expressions of &#x03B2;-catenin, MMP-7 and VEGF from protein and mRNA levels. Results: Whether the Wnt/&#x03B2;-catenin signal pathway was blocked or not had little effect on the pathological morphology of lesions. The expressions of &#x03B2;-catenin, MMP-7 and VEGF in siRNA group were much lower than those in negative control group and control group (p p > 0.05). Conclusion: Blocking of Wnt/&#x03B2;-catenin signal pathway caused the decrease of MMP-7 and VEGF expressions, indicating that Wnt/&#x03B2;-catenin signal pathway plays an important role in the adhesion, invasion, and metastasis of endometriosis tissues.

Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.

Antihypertensive Therapy in Pre-Eclampsia: Effects of Plasma from Nonresponsive Patients on Endothelial Gene Expression

Over 40% of patients with pre-eclampsia are nonresponsive to antihypertensive therapy, but the underlying mechanisms are unknown. We examined the effects of plasma from nonresponsive and responsive patients on endothelial gene expression. PCR array was performed in human umbilical vein endothelial cells (HUVEC) incubated with plasma from nonresponsive (n = 4) and responsive (n = 4) patients. Gene networks and interactions with antihypertensive drugs used in pre-eclampsia were identified by Ingenuity Pathway Analysis. Nifedipine and hydralazine act by upregulate or downregulate genes found to be downregulated or upregulated in HUVEC incubated with plasma from nonresponsive patients. Our novel findings suggest that plasma from nonresponsive and responsive patients evoke different responses in HUVEC, and might advance the pharmacogenomics research in pre-eclampsia.

Inhibition of ALK1 signaling with dalantercept combined with VEGFR TKI leads to tumor stasis in renal cell carcinoma.

Treatment of metastatic renal cell carcinoma (mRCC) with agents that block signaling through vascular endothelial growth factor receptor 2 (VEGFR2) induces disease regression or stabilization in some patients; however, these responses tend to be short-lived. Therefore, development of combination therapies that can extend the efficacy of VEGFR antagonists in mRCC remains a priority.We studied murine xenograft models of RCC that become refractory to treatment with the VEGFR tyrosine kinase inhibitor (TKI) sunitinib. Dalantercept is a novel antagonist of Activin receptor-like kinase 1 (ALK1)/Bone morphogenetic protein (BMP) 9 signaling. Dalantercept inhibited growth in the murine A498 xenograft model which correlated with hyperdilation of the tumor vasculature and an increase in tumor hypoxia. When combined with sunitinib, dalantercept induced tumor necrosis and prevented tumor regrowth and revascularization typically seen with sunitinib monotherapy in two RCC models. Combination therapy led to significant downregulation of angiogenic genes as well as downregulation of endothelial specific gene expression particularly of the Notch signaling pathway.We demonstrate that simultaneous targeting of molecules that control distinct phases of angiogenesis, such as ALK1 and VEGFR, is a valid strategy for treatment of mRCC. At the molecular level, combination therapy leads to downregulation of Notch signaling.

Exercise training improves obesity-related lymphatic dysfunction.

Key points Obesity results in perilymphatic inflammation and lymphatic dysfunction.Lymphatic dysfunction in obesity is characterized by decreased lymphatic vessel density, decreased collecting lymphatic vessel pumping frequency, decreased lymphatic trafficking of immune cells, increased lymphatic vessel leakiness and changes in the gene expression patterns of lymphatic endothelial cells.Aerobic exercise, independent of weight loss, decreases perilymphatic inflammatory cell accumulation, improves lymphatic function and reverses pathological changes in gene expression in lymphatic endothelial cells. Although previous studies have shown that obesity markedly decreases lymphatic function, the cellular mechanisms that regulate this response remain unknown. In addition, it is unclear whether the pathological effects of obesity on the lymphatic system are reversible with behavioural modifications. The purpose of this study, therefore, was to analyse lymphatic vascular changes in obese mice and to determine whether these pathological effects are reversible with aerobic exercise. We randomized obese mice to either aerobic exercise (treadmill running for 30 min per day, 5 days a week, for 6 weeks) or a sedentary group that was not exercised and analysed lymphatic function using a variety of outcomes. We found that sedentary obese mice had markedly decreased collecting lymphatic vessel pumping capacity, decreased lymphatic vessel density, decreased lymphatic migration of immune cells, increased lymphatic vessel leakiness and decreased expression of lymphatic specific markers compared with lean mice (all P < 0.01). Aerobic exercise did not cause weight loss but markedly improved lymphatic function compared with sedentary obese mice. Exercise had a significant anti‐inflammatory effect, resulting in decreased perilymphatic accumulation of inflammatory cells and inducible nitric oxide synthase expression. In addition, exercise normalized isolated lymphatic endothelial cell gene expression of lymphatic specific genes, including VEGFR‐3 and Prox1. Taken together, our findings suggest that obesity impairs lymphatic function via multiple mechanisms and that these pathological changes can be reversed, in part, with aerobic exercise, independent of weight loss. In addition, our study shows that obesity‐induced lymphatic endothelial cell gene expression changes are reversible with behavioural modifications.

Integrated Regional Cardiac Hemodynamic Imaging and RNA Sequencing Reveal Corresponding Heterogeneity of Ventricular Wall Shear Stress and Endocardial Transcriptome

BackgroundUnlike arteries, in which regionally distinct hemodynamics are associated with phenotypic heterogeneity, the relationships between endocardial endothelial cell phenotype and intraventricular flow remain largely unexplored. We investigated regional differences in left ventricular wall shear stress and their association with endocardial endothelial cell gene expression.Methods and ResultsLocal wall shear stress was calculated from 4‐dimensional flow magnetic resonance imaging in 3 distinct regions of human (n=8) and pig (n=5) left ventricle: base, adjacent to the outflow tract; midventricle; and apex. In both species, wall shear stress values were significantly lower in the apex and midventricle relative to the base; oscillatory shear index was elevated in the apex. RNA sequencing of the endocardial endothelial cell transcriptome in pig left ventricle (n=8) at a false discovery rate ≤10% identified 1051 genes differentially expressed between the base and the apex and 327 between the base and the midventricle; no differentially expressed genes were detected at this false discovery rate between the apex and the midventricle. Enrichment analyses identified apical upregulation of genes associated with translation initiation including mammalian target of rapamycin, and eukaryotic initiation factor 2 signaling. Genes of mitochondrial dysfunction and oxidative phosphorylation were also consistently upregulated in the left ventricular apex, as were tissue factor pathway inhibitor (mean 50‐fold) and prostacyclin synthase (5‐fold)—genes prominently associated with antithrombotic protection.ConclusionsWe report the first spatiotemporal measurements of wall shear stress within the left ventricle and linked regional hemodynamics to heterogeneity in ventricular endothelial gene expression, most notably to translation initiation and anticoagulation properties in the left ventricular apex, in which oscillatory shear index is increased and wall shear stress is decreased.

The Epigenomic Code in Hemodynamic Regulation of Cell Phenotypes in Atherosusceptible Endothelium

Hemodynamics creates a constantly changing physical and chemical environment to which the arterial endothelium is exquisitely sensitive. Near branching anatomical sites in large and distributing arteries, blood flow separates from the main flow undergoing complex multi‐directional characteristics for a part of each cardiac cycle (collectively referred to as disturbed flow). Atherosclerosis develops preferentially at disturbed flow locations if an additional cardiovascular risk factor such as hypercholesterolemia is present. In the pre‐disease state, an atherosusceptible endothelial phenotype is characteristically present in such regions in contrast to the adjacent endothelium.A regulatory hierarchy of mechanisms control endothelial gene and protein expression (and function) in response to the flow environment. Gene expression regulation, the transcription of the DNA (genomic) code to mRNA, is well described by transcription factors, enhancers and repressors, many of which are flow responsive. However, emerging studies describe the additional role of localized disturbed blood flow upon epigenomic mechanisms of endothelial responses to biomechanical stress. Epigenetics and epigenomics encompass heritable and non‐heritable changes in nuclear chromatin leading to gene expression changes that cannot be attributed to changes in the primary DNA sequence. Hemodynamic stimuli have recently been shown to induce epigenomic responses including transcriptional regulation by proximal promoter DNA methylation, and regulation of gene and protein expression by histone/chromatin remodeling and by noncoding RNA‐based mechanisms.Dynamic epigenomic responses to flow that result in regulation of endothelial gene expression in vivo and in vitro include: (i) regions of stable differentially methylated DNA in swine and mouse endothelial genomes that arearterial site‐specific and map to atherosusceptibility, (ii) disturbed flow(but not undisturbed flow) applied to endothelial cells in vitro that induces DNA methylation through DNA methyltransferase enrichment of gene promoter regions; the resulting methylation suppresses gene expression, (iii) histone acetylation/deacetylation and methylation that create histone marks that enable or suppress gene expression by controlling access of transcription factors to chromatin DNA, and (iv) short microRNAs and long noncoding RNAs that interact with highly specific binding sites of promoter DNA. Flow‐mediated epigenomic responses intersect with cis and trans factor regulation to maintain endothelial function in a shear‐stressed environment and may contribute to localized endothelial dysfunctions that promoteatherosusceptibility.The epigenomic code therefore has the potential to figure prominently in disease susceptibility and pathogenesis arising from environmental influences independent of, or in concert with, mutations and single nucleotide polymorphisms (SNP) linked to many complex diseases, including cardiovascular disease. The role of disturbed flow as a hemodynamic regulator of epigenomic DNA methylation‐mediated and histone‐mediated gene expression may play a prominent role in endothelial vascular function and dysfunction. The mechanisms that link hemodynamics to the epigenomic code have not been identified.Support or Funding InformationSupported by AHA Postdoctoral Fellowship 13POST14070010, NIH (NHLBI) grants T32 HL07954 and P01 HL06220.