Endothelial Gene Expression Research Articles

RF/6A Chorioretinal Cells Do Not Display Key Endothelial Phenotypes.

PurposeThe misuse of inauthentic cell lines is widely recognized as a major threat to the integrity of biomedical science. Whereas the majority of efforts to address this have focused on DNA profiling, we sought to anatomically, transcriptionally, and functionally authenticate the RF/6A chorioretinal cell line, which is widely used as an endothelial cell line to model retinal and choroidal angiogenesis.MethodsMultiple vials of RF/6A cells obtained from different commercial distributors were studied to validate their genetic, transcriptomic, anatomic, and functional fidelity to bona fide endothelial cells.ResultsTranscriptomic profiles of RF/6A cells obtained either de novo or from a public data repository did not correspond to endothelial gene expression signatures. Expression of established endothelial markers were very low or undetectable in RF/6A compared to primary human endothelial cells. Importantly, RF/6A cells also did not display functional characteristics of endothelial cells such as uptake of acetylated LDL, expression of E-selectin in response to TNF-α exposure, alignment in the direction of shear stress, and AKT and ERK1/2 phosphorylation following VEGFA stimulation.ConclusionsMultiple independent sources of RF/6A do not exhibit key endothelial cell phenotypes. Therefore, these cells appear unsuitable as surrogates for choroidal or retinal endothelial cells. Further, cell line authentication methods should extend beyond genomic profiling to include anatomic, transcriptional, and functional assessments.

A Comparative Study of Single and Dual Perfusion During End-ischemic Subnormothermic Liver Machine Preservation

BackgroundIt remains controversial if arterial perfusion in addition to portal vein perfusion during machine preservation improves liver graft quality. Comparative studies using both techniques are lacking. We studied the impact of using single or dual machine perfusion of donation after circulatory death rat livers. In addition, we analyzed the effect of pulsatile versus continuous arterial flow.MethodsDonation after circulatory death rat livers (n = 18) were preserved by 6 hours cold storage, followed by 1 hour subnormothermic machine perfusion (20°C, pressure of 40/5 mm Hg) and 2 hours ex vivo warm reperfusion (37°C, pressure of 80/11 mm Hg, 9% whole blood). Machine preservation was either through single portal vein perfusion (SP), dual pulsatile (DPP), or dual continuous perfusion (DCP) of the portal vein and hepatic artery. Hydrodynamics, liver function tests, histopathology, and expression of endothelial specific genes were assessed during 2 hours warm reperfusion.ResultsAt the end of reperfusion, arterial flow in DPP livers tended to be higher compared to DCP and SP grafts. However, this difference was not significant nor was better flow associated with better outcome. No differences in bile production or alanine aminotransferase levels were observed. SP livers had significantly lower lactate compared to DCP, but not DPP livers. Levels of Caspase-3 and tumor necrosis factor-α were similar between the groups. Expression of endothelial genes Krüppel-like-factor 2 and endothelial nitric oxide synthase tended to be higher in dual perfused livers, but no histological evidence of better preservation of the biliary endothelium or vasculature of the hepatic artery was observed.ConclusionsThis study shows comparable outcomes after using a dual or single perfusion approach during end-ischemic subnormothermic liver machine preservation.

Parahippocampal gyrus expression of endothelial and insulin receptor signaling pathway genes is modulated by Alzheimer's disease and normalized by treatment with anti-diabetic agents.

A large body of literature links risk of cognitive decline, mild cognitive impairment (MCI) and dementia with Type 2 Diabetes (T2D) or pre-diabetes. Accumulating evidence implicates a close relationship between the brain insulin receptor signaling pathway (IRSP) and the accumulation of amyloid beta and hyperphosphorylated and conformationally abnormal tau. We showed previously that the neuropathological features of Alzheimer’s disease (AD were reduced in patients with diabetes who were treated with insulin and oral antidiabetic medications. To understand better the neurobiological substrates of T2D and T2D medications in AD, we examined IRSP and endothelial cell markers in the parahippocampal gyrus of controls (N = 30), of persons with AD (N = 19), and of persons with AD and T2D, who, in turn, had been treated with anti-diabetic drugs (insulin and or oral agents; N = 34). We studied the gene expression of selected members of the IRSP and selective endothelial cell markers in bulk postmortem tissue from the parahippocampal gyrus and in endothelial cell enriched isolates from the same brain region. The results indicated that there are considerable abnormalities and reductions in gene expression (bulk tissue homogenates and endothelial cell isolates) in the parahippocampal gyri of persons with AD that map directly to genes associated with the microvasculature and the IRSP. Our results also showed that the numbers of abnormally expressed microvasculature and IRSP associated genes in diabetic AD donors who had been treated with anti-diabetic agents were reduced significantly. These findings suggest that anti-diabetic treatments may reduce or normalize compromised microvascular and IRSP functions in AD.

Endothelial FN (Fibronectin) Deposition by α5β1 Integrins Drives Atherogenic Inflammation.

Objective- Alterations in extracellular matrix quantity and composition contribute to atherosclerosis, with remodeling of the subendothelial basement membrane to an FN (fibronectin)-rich matrix preceding lesion development. Endothelial cell interactions with FN prime inflammatory responses to a variety of atherogenic stimuli; however, the mechanisms regulating early atherogenic FN accumulation remain unknown. We previously demonstrated that oxLDL (oxidized low-density lipoprotein) promotes endothelial proinflammatory gene expression by activating the integrin α5β1, a classic mediator of FN fibrillogenesis. Approach and Results- We now show that oxLDL drives robust endothelial FN deposition and inhibiting α5β1 (blocking antibodies, α5 knockout cells) completely inhibits oxLDL-induced FN deposition. Consistent with this, inducible endothelial-specific α5 integrin deletion in ApoE knockout mice significantly reduces atherosclerotic plaque formation, associated with reduced early atherogenic inflammation. Unlike TGFβ (transforming growth factor β)-induced FN deposition, oxLDL does not induce FN expression (mRNA, protein) or the endothelial-to-mesenchymal transition phenotype. In addition, we show that cell-derived and plasma-derived FN differentially affect endothelial function, with only cell-derived FN capable of supporting oxLDL-induced VCAM-1 (vascular cell adhesion molecule 1) expression, despite plasma FN deposition by oxLDL. The inclusion of alternative exon EIIIA (EDA) of FN (EIIIA) and alternative exon EIIIB (EDB) of FN (EIIIB) domains in cell-derived FN mediates this effect, as EIIIA/EIIIB knockout endothelial cells show diminished oxLDL-induced inflammation. Furthermore, our data suggest that EIIIA/EIIIB-positive cellular FN is required for maximal α5β1 recruitment to focal adhesions and FN fibrillogenesis. Conclusions- Taken together, our data demonstrate that endothelial α5 integrins drive oxLDL-induced FN deposition and early atherogenic inflammation. Additionally, we show that α5β1-dependent endothelial FN deposition mediates oxLDL-dependent endothelial inflammation and FN fibrillogenesis.

Maternal undernutrition during pregnancy alters the epigenetic landscape and the expression of endothelial function genes in male progeny

Recent studies point to the important role of in utero malnutrition in gene programming and in the development of vascular diseases. We hypothesize that maternal undernutrition affects vascular function in the offspring by promoting epigenetic changes that drive the differential expression of genes involved in endothelial function. To test this, we exposed mice to nutrient deprivation in utero and analyzed its effect on global DNA methylation and expression of endothelium-specific genes in the pulmonary endothelium of the adult progeny. Mice were kept either on ad libitum (AL) or energy-restricted (ER) diet during the second and third trimesters of gestation. Mice in the ER group received 65% of energy compared to mice in the AL diet group. Pulmonary endothelial cells were isolated from 6-week-old male offspring mice (AL-F1 and ER-F1). The expression of genes in the pulmonary endothelium was analyzed using quantitative reverse-transcription polymerase chain reaction array and confirmed by qRT-PCR. Several genes including fibronectin 1 and plasminogen activator inhibitor 1 were upregulated in the endothelium of male ER-F1 mice, whereas the expression of genes involved in regulation of histone acetylation was significantly attenuated. At the same time, the global DNA methylation did not change in pulmonary endothelial cells of ER-F1 mice compared to AL-F1 mice. Overall, we found that maternal undernutrition during pregnancy affects the expression of genes involved in regulation of endothelial cell function in the pulmonary vasculature of male progeny, which could potentially promote pulmonary vascular remodeling.

Angiogenic and Osteogenic Synergy of Human Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells Cocultured on a Nanomatrix

To date, bone tissue regeneration strategies lack an approach that effectively provides an osteogenic and angiogenic environment conducive to bone growth. In the current study, we evaluated the osteogenic and angiogenic response of human mesenchymal stem cells (hMSCs) and green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) cocultured on a self-assembled, peptide amphiphile nanomatrix functionalized with the cell adhesive ligand RGDS (PA-RGDS). Analysis of alkaline phosphatase activity, von Kossa staining, Alizarin Red quantification, and osteogenic gene expression, indicates a significant synergistic effect between the PA-RGDS nanomatrix and coculture that promoted hMSC osteogenesis. In addition, coculturing on PA-RGDS resulted in enhanced HUVEC network formation and upregulated vascular endothelial growth factor gene and protein expression. Though PA-RGDS and coculturing hMSCs with HUVECs were each previously reported to individually enhance hMSC osteogenesis, this study is the first to demonstrate a synergistic promotion of HUVEC angiogenesis and hMSC osteogenesis by integrating coculturing with the PA-RGDS nanomatrix. We believe that using the combination of hMSC/HUVEC coculture and PA-RGDS substrate is an efficient method for promoting osteogenesis and angiogenesis, which has immense potential as an efficacious, engineered platform for bone tissue regeneration.

Evaluation of Endothelial Nitric Oxide Synthase Gene Expression in Mice Genetically Heterozygous in CX43 (CX43 + / - Mice)

Gap junctions are proteins made of connexins which are involved in the regulation of vascular function. Deletion of connexins 43 (Cx43) modifies expression of genes known to be involved in the regulation of the vasculature, differentiation and function of vascular cells. Interestingly, mutant mice lacking endothelial nitric oxide synthase (eNOS) gene have been shown to be hypertensive, suggesting that nitric oxide (NO) plays a role in the physiological control of blood pressure. It was therefore hypothesised that the endothelial deletion of Cx43 in the pulmonary vasculature induces endothelial dysfunction and causes eNOS impairment thereby reducing NO biosynthesis, thus leading to vasoconstriction and vascular remodelling which subsequently leads to the development of pulmonary arterial hypertension (PAH). This project was aimed at evaluating eNOS gene expression in mice genetically heterozygous (HET) in Cx43. This was achieved by using lung tissues from four groups of wild type (W/T) and het Cx43 (male and female) mice. Ribonucleic acid (RNA) was isolated from the lung tissues using RNA II isolation system and was reverse transcribed to complementary deoxyribonucleic acid (cDNA). End point polymerase chain reaction (PCR) and real time PCR were used to measure the expression of eNOS gene. eNOS gene expression levels were found to be the same in all four groups of mice tested, with no significant difference. The result therefore suggests that eNOS gene is expressed in mice genetically heterozygous in Cx43.

Temporal clustering analysis of endothelial cell gene expression following exposure to a conventional radiotherapy dose fraction using Gaussian process clustering.

The vascular endothelium is considered as a key cell compartment for the response to ionizing radiation of normal tissues and tumors, and as a promising target to improve the differential effect of radiotherapy in the future. Following radiation exposure, the global endothelial cell response covers a wide range of gene, miRNA, protein and metabolite expression modifications. Changes occur at the transcriptional, translational and post-translational levels and impact cell phenotype as well as the microenvironment by the production and secretion of soluble factors such as reactive oxygen species, chemokines, cytokines and growth factors. These radiation-induced dynamic modifications of molecular networks may control the endothelial cell phenotype and govern recruitment of immune cells, stressing the importance of clearly understanding the mechanisms which underlie these temporal processes. A wide variety of time series data is commonly used in bioinformatics studies, including gene expression, protein concentrations and metabolomics data. The use of clustering of these data is still an unclear problem. Here, we introduce kernels between Gaussian processes modeling time series, and subsequently introduce a spectral clustering algorithm. We apply the methods to the study of human primary endothelial cells (HUVECs) exposed to a radiotherapy dose fraction (2 Gy). Time windows of differential expressions of 301 genes involved in key cellular processes such as angiogenesis, inflammation, apoptosis, immune response and protein kinase were determined from 12 hours to 3 weeks post-irradiation. Then, 43 temporal clusters corresponding to profiles of similar expressions, including 49 genes out of 301 initially measured, were generated according to the proposed method. Forty-seven transcription factors (TFs) responsible for the expression of clusters of genes were predicted from sequence regulatory elements using the MotifMap system. Their temporal profiles of occurrences were established and clustered. Dynamic network interactions and molecular pathways of TFs and differential genes were finally explored, revealing key node genes and putative important cellular processes involved in tissue infiltration by immune cells following exposure to a radiotherapy dose fraction.

Limited Endothelial Plasticity of Mesenchymal Stem Cells Revealed by Quantitative Phenotypic Comparisons to Representative Endothelial Cell Controls.

Considerable effort has been directed toward deriving endothelial cells (ECs) from adipose‐derived mesenchymal stem cells (ASCs) since 2004, when it was first suggested that ECs and adipocytes share a common progenitor. While the capacity of ASCs to express endothelial markers has been repeatedly demonstrated, none constitute conclusive evidence of an endothelial phenotype as all reported markers have been detected in other, non‐endothelial cell types. In this study, quantitative phenotypic comparisons to representative EC controls were used to determine the extent of endothelial differentiation being achieved with ASCs. ASCs were harvested from human subcutaneous abdominal white adipose tissue, and their endothelial differentiation was induced using well‐established biochemical stimuli. Reverse transcription quantitative real‐time polymerase chain reaction and parallel reaction monitoring mass spectrometry were used to quantify their expression of endothelial genes and corresponding proteins, respectively. Flow cytometry was used to quantitatively assess their uptake of acetylated low‐density lipoprotein (AcLDL). Human umbilical vein, coronary artery, and dermal microvascular ECs were used as positive controls to reflect the phenotypic heterogeneity between ECs derived from different vascular beds. Biochemically conditioned ASCs were found to upregulate their expression of endothelial genes and proteins, as well as AcLDL uptake, but their abundance remained orders of magnitude lower than that observed in the EC controls despite their global proteomic heterogeneity. The findings of this investigation demonstrate the strikingly limited extent of endothelial differentiation being achieved with ASCs using well‐established biochemical stimuli, and underscore the importance of quantitative phenotypic comparisons to representative primary cell controls in studies of differentiation. Stem Cells Translational Medicine 2019;8:35–45

Computing of Low Shear Stress-Driven Endothelial Gene Network Involved in Early Stages of Atherosclerotic Process.

Background In the pathogenesis of atherosclerosis, a central role is represented by endothelial inflammation with influx of chemokine-mediated leukocytes in the vascular wall. Aim of this study was to analyze the effect of different shear stresses on endothelial gene expression and compute gene network involved in atherosclerotic disease, in particular to homeostasis, inflammatory cell migration, and apoptotic processes. Methods HUVECs were subjected to shear stress of 1, 5, and 10 dyne/cm2 in a Flow Bioreactor for 24 hours to compare gene expression modulation. Total RNA was analyzed by Affymetrix technology and the expression of two specific genes (CXCR4 and ICAM-1) was validated by RT-PCR. To highlight possible regulations between genes and as further validation, a bioinformatics analysis was performed. Results At low shear stress (1 dyne/cm2) we observed the following: (a) strong upregulation of CXCR4; (b) mild upregulation of Caspase-8; (c) mild downregulation of ICAM-1; (d) marked downexpression of TNFAIP3. Bioinformatics analysis showed the presence of network composed by 59 new interactors (14 transcription factors and 45 microRNAs) appearing strongly related to shear stress. Conclusions The significant modulation of these genes at low shear stress and their close relationships through transcription factors and microRNAs suggest that all may promote an initial inflamed endothelial cell phenotype, favoring the atherosclerotic disease.

Increased macrophages and\xa0changed brain endothelial cell gene expression in the frontal cortex of people with schizophrenia displaying inflammation

Elevated pro-inflammatory cytokines exist in both blood and brain of people with schizophrenia but how this affects molecular indices of the blood–brain barrier (BBB) is unclear. Eight mRNAs relating to BBB function, a microglia and three immune cell markers were measured by qPCR in the prefrontal cortex from 37 people with schizophrenia/schizoaffective disorder and 37 matched controls. This cohort was previously grouped into “high inflammation” and “low inflammation” subgroups based on cortical inflammatory-related transcripts. Soluble intercellular adhesion molecule-1 (sICAM1) was measured in the plasma of 78 patients with schizophrenia/schizoaffective disorder and 73 healthy controls. We found that sICAM1 was significantly elevated in schizophrenia. An efflux transporter, ABCG2, was lower, while mRNAs encoding VE-cadherin and ICAM1 were higher in schizophrenia brain. The “high inflammation” schizophrenia subgroup had lower ABCG2 and higher ICAM1, VE-cadherin, occludin and interferon-induced transmembrane protein mRNAs compared to both “low inflammation” schizophrenia and “low inflammation” control subgroups. ICAM1 immunohistochemistry showed enrichment in brain endothelium regardless of diagnosis and was localised to astrocytes in some brains. Microglia mRNA was not altered in schizophrenia nor did it correlate with ICAM1 expression. Immune cell mRNAs were elevated in “high inflammation” schizophrenia compared to both “low inflammation” schizophrenia and controls. CD163+ perivascular macrophages were identified by immunohistochemistry in brain parenchyma in over 40% of “high inflammation” schizophrenia brains. People with high levels of cytokine expression and schizophrenia display changes consistent with greater immune cell transmigration into brain via increased ICAM1, which could contribute to other neuropathological changes found in this subgroup of people.

Protective effects of Danzhi jiangtang capsule on vascular endothelial damages induced by high-fat diet and palmitic acid

Danzhi jiangtang capsule (DJC) is mainly used to treat type 2 diabetes mellitus and vascular complication. Our aim was to investigate whether the protective effects of DJC by oral administration on high-fat diet (HFD) and palmitic acid-induced damages of endothelial cells are related to oxidative stress and endoplasmic reticulum (ER) stress. Male Sprague-Dawley rats were randomly divided into standard chow diet (SCD), HFD, HFD plus DJC-low dose (HFD + DJCL) and HFD plus DJC-high dose treatment groups (HFD + DJCH). The latter three groups were given HFD feeding for three months, then the HFD + DJCL and HFD + DJCH rats were treated with DJC (500 and 1000 mg/kg/day) via gavage for another two months respectively. Endothelium-dependent relaxations induced by acetylcholine were observed in isolated rat thoracic aortic ring preparation. Malondialdehyde (MDA), total-antioxidant capacity (T-AOC), superoxide dismutase (SOD), interleukin 1β (IL-1β), tumour necrosis factor α (TNFα), free fatty acids (FFA), endothelin-1 (ET-1) and nitric oxide (NO) levels in serum were assayed. The pathological changes, protein expression of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS) and ER stress-related genes in the thoracic aorta were measured. Human umbilical vein endothelial cells (HUVEC) were treated with serum-medicated DJC and then with palmitic acid to detect the reactive oxygen species (ROS) levels and C/EBP homologous protein (CHOP) distribution, expression of p-eNOS and ER stress-related genes. DJC therapy exhibited a potent antioxidant activity and effectively prevented the endothelial dysfunction (ED) manifested by promoting p-eNOS expression and enhancing NO release, decreasing lipid deposition (Oil-red O staining, CPT1b and ACC) and inflammation (IL-1β, TNFα, CD68 and p-JNK), alleviating oxidative and ER stress, and decreasing the apoptosis of endothelial cells (TUNEL, BCL-2 and BAX) induced by HFD and palmitic acid respectively. These findings suggest that the administration of DJC had endothelial protective effects against HFD-induced ED, inflammation and apoptosis by alleviating lipid deposition, oxidative and ER stress.

Atheroprone flow enhances the endothelial-to-mesenchymal transition.

The endothelial-to-mesenchymal transition (EndoMT) is a cellular process featuring decreased expression of endothelial marker genes but increased expression of mesenchymal marker genes. The EndoMT is involved in endothelial dysfunction and the pathogenesis of atherosclerosis. To investigate the dynamic expression of EndoMT genes in vascular endothelial cells under atheroprotective pulsatile shear stress (PS) and atheroprone oscillatory shear stress (OS), we analyzed RNA sequencing data from multitimepoint shear-stress experiments. This unbiased analysis involving next-generation sequencing confirmed that PS and OS had an opposite effect in regulating EndoMT genes. Further experimental validations with H2O2 and gain- and loss-of-function approaches indicated that reactive oxygen species are involved in OS-induced EndoMT, whereas AMP-activated protein kinase and sirtuin-1 could inhibit OS-induced EndoMT. Furthermore, compared with PS, OS increased the DNA methylation of the promoter regions of von Willebrand factor, CD31, and cadherin 5 genes but decreased that of cadherin 2, fibroblast-specific protein 1, and vimentin. The translational implication of the present study builds on the ability of the antidiabetic drug metformin and cholesterol-lowering drug atorvastatin to suppress the EndoMT in cultured endothelial cells and in mouse aortas. NEW & NOTEWORTHY Our RNA sequencing data provided a genome-wide and unbiased view of the shear stress regulation of the endothelial-to-mesenchymal transition (EndoMT) in the endothelium. Furthermore, epigenetic regulation (e.g., DNA methylation) is a key mechanism involved in shear stress-regulated EndoMT. The translational implication of this study is that cardiovascular medications such as statins and metformin have similar beneficial effects as that of atheroprotective flow by mitigating EndoMT.

Inference of cell type content from human brain transcriptomic datasets illuminates the effects of age, manner of death, dissection, and psychiatric diagnosis.

Psychiatric illness is unlikely to arise from pathology occurring uniformly across all cell types in affected brain regions. Despite this, transcriptomic analyses of the human brain have typically been conducted using macro-dissected tissue due to the difficulty of performing single-cell type analyses with donated post-mortem brains. To address this issue statistically, we compiled a database of several thousand transcripts that were specifically-enriched in one of 10 primary cortical cell types in previous publications. Using this database, we predicted the relative cell type content for 833 human cortical samples using microarray or RNA-Seq data from the Pritzker Consortium (GSE92538) or publicly-available databases (GSE53987, GSE21935, GSE21138, CommonMind Consortium). These predictions were generated by averaging normalized expression levels across transcripts specific to each cell type using our R-package BrainInABlender (validated and publicly-released on github). Using this method, we found that the principal components of variation in the datasets strongly correlated with the predicted neuronal/glial content of the samples. This variability was not simply due to dissection–the relative balance of brain cell types appeared to be influenced by a variety of demographic, pre- and post-mortem variables. Prolonged hypoxia around the time of death predicted increased astrocytic and endothelial gene expression, illustrating vascular upregulation. Aging was associated with decreased neuronal gene expression. Red blood cell gene expression was reduced in individuals who died following systemic blood loss. Subjects with Major Depressive Disorder had decreased astrocytic gene expression, mirroring previous morphometric observations. Subjects with Schizophrenia had reduced red blood cell gene expression, resembling the hypofrontality detected in fMRI experiments. Finally, in datasets containing samples with especially variable cell content, we found that controlling for predicted sample cell content while evaluating differential expression improved the detection of previously-identified psychiatric effects. We conclude that accounting for cell type can greatly improve the interpretability of transcriptomic data.

Is internal thoracic artery resistant to reperfusion injury? Evaluation of the storage of free internal thoracic artery grafts

ObjectivesThe in situ internal thoracic artery (ITA) is recognized as the best conduit for coronary artery bypass surgery. The ITA—if it is used as an in situ graft—has a much higher late patency rate than any other arterial graft, including a free ITA graft. We sought to determine if the use of the ITA as an in situ/free graft and its storage in preservation solutions, have an effect on endothelial function. MethodsThe ITA was harvested as either a free or in situ graft in a porcine model. Free grafts were stored in different preservation solutions (saline, Custodiol and Tiprotec [both Köhler Chemie GmbH, Bensheim, Germany]). The ITA was anastomosed off pump to the left anterior descending artery (as in situ/free graft). Freshly harvested ITA served as a control. After 2 hours of reperfusion, the implanted grafts were harvested. The assessment of endothelial function, histopathological analysis, and gene expression were performed. ResultsEndothelial function and integrity were severely impaired after reperfusion in the free ITA groups, however, it was partially preserved in the Tiprotec group. Reperfusion injury resulted in increased nitro-oxidative stress, DNA breakage, vascular cell adhesion protein 1, intercellular adhesion molecule-1, and caspase-3 scores, and a decreased endothelial nitric oxide synthase score in the free ITA groups. The in situ ITA graft showed no signs of injury. mRNA levels were significantly altered among the groups. ConclusionsAn early, severe endothelial dysfunction of the stored, free ITA as described, could be completely prevented by the use of an in situ ITA graft. Tiprotec might be a feasible option for storage of free arterial grafts during coronary artery bypass grafting.

Endothelial siRNA delivery in nonhuman primates using ionizable low-molecular weight polymeric nanoparticles.

Dysfunctional endothelial cells contribute to the pathophysiology of many diseases, including vascular disease, stroke, hypertension, atherosclerosis, organ failure, diabetes, retinopathy, and cancer. Toward the goal of creating a new RNA-based therapy to correct aberrant endothelial cell gene expression in humans, efficient gene silencing in the endothelium of nonhuman primates was achieved by delivering small interfering RNA (siRNA) with 7C1, a low–molecular weight, ionizable polymer that forms nanoparticles. After a single intravenous administration of 1 mg of siRNA per kilogram of animal, 7C1 nanoparticles delivering Tie2 siRNA caused Tie2 mRNA levels to decrease by approximately 80% in the endothelium of the lung. Significant decreases in Tie2 mRNA were also found in the heart, retina, kidney, pancreas, and bone. Blood chemistry and liver function analysis before and after treatment all showed protein and enzyme concentrations within the normal reference ranges. Furthermore, after controlling for siRNA-specific effects, no significant increases in inflammatory cytokine concentrations were found in the serum. Similarly, no gross lesions or significant underlying pathologies were observed after histological examination of nonhuman primate tissues. This study is the first demonstration of endothelial gene silencing in multiple nonhuman primate organs using systemically administered siRNA nanoparticles and highlights the potential of this approach for the treatment of disease in humans.

Abstract 250: Endothelial functional Regulation by Enhancer-Associated Long Non-Coding RNAs

Vascular endothelium constitutes the critical interface between circulating blood and vessel wall. Long non-coding RNAs (lncRNAs) are important regulators in gene expression and chromotin remodeling. We recently identified a l ncRNA that e nhances e ndothelial n itric oxide synthase (eNOS) e xpression (LEENE), that is encoded in a distal enhancer region and mediates long-range DNA interaction to promote the eNOS expression. We now extend from this prototype using a systematic approach to identify enhancer-associated lncRNAs and their molecular and functional regulation of endothelial gene expression. We integrate a variety of publicly available high-throughput sequencing datasets (including various chromatin immunoprecipitation-sequencing) that provide information on histone modifications and transcriptional activity and high-resolution chromatin conformation capture combined with sequencing (4C-seq) profiles generated in our own lab. From these analyses, we identified over 200 enhancer-associated lncRNA candidates with high potential to regulate endothelial gene expression. Quantitative PCR analysis verified that 7 out of the top 10 ranked candidates are differentially regulated by stimuli that alter endothelial gene expression and functions, including high-glucose, TNFα, atorvastatin, and metformin. Using CRISPR-cas9 gene editing to delete these lncRNA-associated enhancer regions and locked nucleic acid-mediated lncRNA inhibition, we demonstrated that the loss-of-function of these lncRNAs lead to significant alteration of endothelial gene expression, including those involved in angiogenesis and eNOS signaling. Collectively, these lncRNAs play an essential role in epigenetic regulation of endothelial functions; the identification of these lncRNAs can have significant impacts on understanding of endothelial homeostasis and dysfunction.

Abstract 606: The Differential Roles of the Adaptor Proteins Nck1 and Nck2 in Shear Stress-Induced Endothelial Activation in vitro and in vivo

Hemodynamic shear stress critically regulates endothelial activation and atherogenesis by affecting cytoskeletal dynamics and endothelial gene expression. The Nck adaptor proteins (Nck1 and Nck2) couple tyrosine kinase signaling to induction of cytoskeletal remodeling pathways, and we previously demonstrated that a peptide corresponding to a Nck-binding sequence in p21 activate kinase (PAK) blunts shear stress-induced proinflammatory signaling (PAK, NF-κB) and permeability in vitro and in vivo . However, the specific roles of Nck1 and Nck2 in flow-induced endothelial activation remain to be elucidated. Here, we demonstrate that Nck1, but not Nck2, critically regulates shear stress-induced endothelial activation. We show that Nck1 deficiency (siRNA knockdown, genetic knockout) decreases basal and shear stress-induced proinflammatory signaling (PAK2, NF-κB) and proinflammatory gene expression (ICAM-1, VCAM-1). In addition, only Nck1, but not Nck2, knockdown/knockout limited shear-induced endothelial permeability. However, other shear stress-dependent pathways, such as Akt activation, proved Nck1-independent. By contrast, selective Nck2 depletion paradoxically increased basal PAK2 activation without significant effects on NF-κB signaling or permeability. Using the partial carotid ligation model of disturbed flow, we found that Nck1 knockout mice showed significantly reduced proinflammatory gene expression that was not further diminished upon Nck2 deletion. However, Nck2 was not inconsequential to the flow response, as Nck2 knockdown or knockout reduced actin cytoskeletal alignment with laminar flow. Taken together, our data suggests that Nck1 plays a dominant role in flow-induced endothelial activation in response to shear stress, whereas Nck2 may critically regulate flow-induced cytoskeletal remodeling and endothelial alignment.

Abstract 690: Endothelial-Specific Krüppel-Like Factor 2 Inactivation Promotes Endothelial Dysfunction in the Setting of Uremia

Background: Uremic solutes that accumulate in end-stage renal disease (ESRD) contribute to endothelial dysfunction and subsequent cardiovascular disease in ESRD patients. However, the specific mechanisms which mediate uremia-induced endothelial dysfunction in ESRD are not understood. Kruppel-like factors (KLFs) are important regulators of endothelial homeostasis which may be affected by the uremic milieu. In this study, we examined the role and regulation of endothelial KLF2 in mediating endothelial dysfunction in the setting of uremia. Methods and Results: First, we assessed the impact of the uremic milieu on endothelial gene expression utilizing serum from uremic and non-uremic pigs with chronic renal insufficiency. We demonstrate that KLF2 expression was dose-dependently decreased with uremic serum in human umbilical vein endothelial cells (HUVECS) versus normal serum which was reversed by prolonged serum dialysis or the application of laminar shear stress. Carboxymethyl-lysine (CML) modified albumin, a uremic advanced glycation end-product (AGE), also inhibited KLF2 expression. This effect was completely abrogated by receptor for AGE (RAGE) siRNA, as well as by a constitutively active form of IκBα, implicating a role for RAGE activation and the NF-κB signaling pathway. KLF2 suppression also promoted endothelial dysfunction in vitro, as adenoviral overexpression of KLF2 inhibited reactive oxygen species production and leukocyte adhesion in HUVECs treated with uremic serum or CML-AGE. For in vivo translation, we utilized high-frequency ultrasound and quantified flow-mediated dilation of the femoral artery in endothelial-specific KLF2 conditional knockout (cKO) mice after 5 minutes of hindlimb ischemia and demonstrated a 50% reduction in vasodilation in KLF2 cKO mice compared to controls. The lack of femoral artery vasodilation in KLF2 cKO mice was also accompanied by attenuated return of wall shear stress to baseline independent of blood velocity. Conclusions: Collectively, these observations implicate loss of endothelial KLF2 as mediator of endothelial dysfunction in the setting of uremia and suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of cardiovascular disease in ESRD.

CCL17 blockade as a therapy for osteoarthritis pain and disease

BackgroundGranulocyte macrophage-colony stimulating factor (GM-CSF) has been implicated in the pathogenesis of a number of inflammatory diseases and in osteoarthritis (OA). We identified previously a new GM-CSF→Jmjd3→interferon regulatory factor 4 (IRF4)→chemokine (c-c motif) ligand 17 (CCL17) pathway, which is important for the development of inflammatory arthritis pain and disease. Tumour necrosis factor (TNF) can also be linked with this pathway. Here we investigated the involvement of the pathway in OA pain and disease development using the GM-CSF-dependent collagenase-induced OA (CiOA) model.MethodsCiOA was induced in C57BL/6 wild-type (WT), Irf4−/−, Ccl17E/E, Ccr4−/−, Tnf−/− and GM-CSF−/− mice. Additionally, therapeutic targeting of CCL17, Jmjd3 and cyclooxygenase 2 (COX-2) was evaluated. Development of pain (assessment of weight distribution) and OA disease (histologic scoring of synovitis, cartilage destruction and osteophyte size) were assessed. Synovial joint cells, including neutrophils, macrophages, fibroblasts and endothelial cells, were isolated (cell sorting) and gene expression analyzed (quantitative PCR).ResultsStudies in the gene-deficient mice indicated that IRF4, CCL17 and the CCL17 receptor, CCR4, but not TNF, were required for CiOA pain and optimal cartilage destruction and osteophyte size. Therapeutic neutralization of CCL17 and Jmjd3 ameliorated both pain and disease, whereas the COX-2 inhibitor only ameliorated pain. In the synovium Ccl17 mRNA was expressed only in the macrophages in a GM-CSF-dependent and IRF4-dependent manner.ConclusionsThe GM-CSF→Jmjd3→IRF4→CCL17 pathway is important for the development of CiOA, with CCL17 thus being a potential therapeutic target for the treatment of both OA pain and disease.