Endothelial Gene Expression Profiles Research Articles

KLF4 recruits SWI/SNF to increase chromatin accessibility and reprogram the endothelial enhancer landscape under laminar shear stress

Physiologic laminar shear stress (LSS) induces an endothelial gene expression profile that is vasculo-protective. In this report, we delineate how LSS mediates changes in the epigenetic landscape to promote this beneficial response. We show that under LSS, KLF4 interacts with the SWI/SNF nucleosome remodeling complex to increase accessibility at enhancer sites that promote the expression of homeostatic endothelial genes. By combining molecular and computational approaches we discover enhancers that loop to promoters of KLF4- and LSS-responsive genes that stabilize endothelial cells and suppress inflammation, such as BMPR2, SMAD5, and DUSP5. By linking enhancers to genes that they regulate under physiologic LSS, our work establishes a foundation for interpreting how non-coding DNA variants in these regions might disrupt protective gene expression to influence vascular disease.

Generation of a nitric oxide signaling pathway in mesenchymal stem cells promotes endothelial lineage commitment.

Enhancing differentiation of mesenchymal stem cells (MSCs) to endothelial cells may improve their ability to vascularize tissue and promote wound healing. This study describes a novel role for nitric oxide (NO) in reprogramming MSCs towards an endothelial lineage and highlights the role of Wnt signaling and epigenetic modification by NO. Rat MSCs were transduced with lentiviral vectors expressing endothelial nitric oxide synthase (pLV-eNOS) and a mutated caveolin gene (pLV-CAV-1F92A ) to enhance NO generation resulting in increased in vitro capillary tubule formation and endothelial marker gene expression. An exogenous source of NO could also stimulate CD31 expression in MSCs. NO was associated with an arterial-specific endothelial gene expression profile of Notch1, Dll4, and Hey2 and significantly reduced expression of venous markers. Wnt signaling associated with NO was evident through increased gene expression of Wnt3a and β-catenin protein, and expression of the endothelial marker Pecam-1 could be significantly reduced by treatment with the Wnt signaling inhibitor Dkk-1. The role of NO as an epigenetic modifier was evident with reduced gene expression of the methyltransferase, DNMT1, and bisulfite sequencing of the endothelial Flt1 promoter region in NO-producing MSCs showed significant demethylation compared to control cells. Finally, subcutaneous implantation of NO-producing MSCs seeded in a biomaterial scaffold (NovoSorb®) resulted in survival of transplanted cells and the formation of blood vessels. In summary, this study describes, NO as a potent endothelial programming factor which acts as an epigenetic modifier in MSCs and may provide a novel platform for vascular regenerative therapy.

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.

Investigating the effect of hypoxic culture on the endothelial differentiation of human amniotic fluid-derived stem cells.

Amniotic fluid-derived stem cells (AFSCs) are a unique stem cell source that may have great potential for use in tissue engineering (TE) due to their pluripotentiality. AFSCs have previously shown angiogenic potential and may present an alternative cell source for endothelial-like cells that could be used in range of applications, including the pre-vascularisation of TE constructs and the treatment of ischaemic diseases. This study investigated the ability of these cells to differentiate down an endothelial lineage with the aim of producing an endothelial-like cell suitable for use in pre-vascularisation. As hypoxia and the associated HIF-1 pathway have been implicated in the induction of angiogenesis in a number of biological processes, it was hypothesised that culture in hypoxic conditions could enhance the endothelial differentiation of AFSCs. The cells were cultured in endothelial cell media supplemented with 50 ng mL(-1) of VEGF, maintained in normoxia, intermittent hypoxia or continuous hypoxia and assessed for markers of endothelial differentiation at day 7 and 14. The results demonstrated that AFSCs subjected to these culture conditions display an endothelial gene expression profile and adopted functional endothelial cell characteristics indicative of early endothelial differentiation. Culture in continuous hypoxia enhanced endothelial gene expression but did not enhance functional endothelial cell characteristics. Overall, AFSCs subjected to endothelial stimuli demonstrated a less mature endothelial gene expression profile and phenotype when compared with HUVECs, the endothelial cell control. However, this study is the first time that the positive effect of an extended period of continuous hypoxic culture on endothelial differentiation in AFSCs has been demonstrated.

Abstract A23: Metformin as a potent antiangiogenic factor: From diabetes to cancer angioprevention

Abstract Metformin, an anti-diabetic drug commonly used for type 2 diabetes therapy, is associated with a decreased risk for certain cancer. Due to extensive reports of its in vitro and in vivo anti-tumor activity, we and others have focused on the anti-angiogenic potential as a possible mechanism through which metformin could affect tumor progression, and to propose its use as an angiopreventive drug. Metformin was able to interfere with endothelial cell proliferation, apoptosis and invasion in vitro. Gene arrays analyses were performed to verify the effect of metformin on endothelial gene expression profiles and the matrigel sponge assay to test its effect in vivo. We show that metformin inhibited endothelial cell proliferation by effecting cell cycle regulation rather than inducing apoptosis. In vitro, metformin reduces endothelial cell invasion and represses their ability to organize into capillary-like networks. These effects are partially reverted by compound C, a specific inhibitor of AMPK, confirming that metformin could interfere with angiogenesis responses by activating AMPK signaling. Gene expression profiles of endothelial cells treated with metformin showed down-regulation of several angiogenesis-related genes but up-regulation of chemokine pathways. In vivo, metformin inhibited angiogenesis induced by VEGF. Considering the strict correlation between type II diabetes and obesity, we evaluated the ability of Metformin to modulate endothelial precursor cells in an obese murine model and we reported that Metformin decreases the number of adipose tissue progenitor endothelial cells in vivo only in obese mice. These data suggest that metformin has anti-angiogenic effect associated with down-regulation of specific molecule although it also induces some inflammatory mediators, and that it may be most effective in the context of obesity. Citation Format: Katiuscia Dallaglio, Antonino Bruno, Anna Rita Cantelmo, Ulrich Pfeffer, Francesco Bertolini, Douglas M. Noonan, Adriana Albini. Metformin as a potent antiangiogenic factor: From diabetes to cancer angioprevention. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A23.

Endothelial Cell Whole Genome Expression Analysis in a Mouse Model of Early-Onset Fuchs' Endothelial Corneal Dystrophy

To investigate the endothelial gene expression profile in a Col8a2 Q455K mutant knock-in mouse model of early-onset Fuchs' endothelial corneal dystrophy (FECD) and identify potential targets that can be correlated to human late-onset FECD. Diseased or normal endothelial phenotypes were verified in 12-month-old homozygous Col8a2(Q455K/Q455K) mutant and wild-type mice by clinical confocal microscopy. An endothelial whole genome expression profile was generated by microarray-based analysis. Result validation was performed by real-time PCR. Endothelial COX2 and JUN expression was further studied in human late-onset FECD compared to normal samples. Microarray analysis demonstrated endothelial expression of 24,538 genes (162 up-regulated and 172 down-regulated targets) and identified affected gene ontology terms including Response to Stress, Protein Metabolic Process, Protein Folding, Regulation of Apoptosis, and Transporter Activity. Real-time PCR assessment confirmed increased Cox2 (P = 0.001) and Jun mRNA (P = 0.03) levels in Col8a2(Q455K/Q455K) mutant compared to wild-type mice. In human FECD samples, real-time PCR demonstrated a statistically significant increase in COX2 mRNA (P < 0.0001) and JUN mRNA (P = 0.002) and tissue microarray analysis showed increased endothelial COX2 (P = 0.02) and JUN protein (P = 0.04). The present study provides the first endothelial whole genome expression analysis in an animal model of FECD and represents a useful resource for future studies of the disease. In particular endothelial COX2 up-regulation warrants further investigation of its role in FECD.

Endothelial differentiation of mesenchymal stromal cells.

Human mesenchymal stromal cells (hMSCs) are increasingly used in regenerative medicine for restoring worn-out or damaged tissue. Newly engineered tissues need to be properly vascularized and current candidates for in vitro tissue pre-vascularization are endothelial cells and endothelial progenitor cells. However, their use in therapy is hampered by their limited expansion capacity and lack of autologous sources. Our approach to engineering large grafts is to use hMSCs both as a source of cells for regeneration of targeted tissue and at the same time as the source of endothelial cells. Here we investigate how different stimuli influence endothelial differentiation of hMSCs. Although growth supplements together with shear force were not sufficient to differentiate hMSCs with respect to expression of endothelial markers such as CD31 and KDR, these conditions did prime the cells to differentiate into cells with an endothelial gene expression profile and morphology when seeded on Matrigel. In addition, we show that endothelial-like hMSCs are able to create a capillary network in 3D culture both in vitro and in vivo conditions. The expansion phase in the presence of growth supplements was crucial for the stability of the capillaries formed in vitro. To conclude, we established a robust protocol for endothelial differentiation of hMSCs, including an immortalized MSC line (iMSCs) which allows for reproducible in vitro analysis in further studies.

Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow

AbstractRecently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.

Prelesional arterial endothelial phenotypes in hypercholesterolemia: universal ABCA1 upregulation contrasts with region-specific gene expression in vivo

Atherosclerosis originates as focal arterial lesions having a predictable distribution to regions of bifurcations, branches, and inner curvatures where blood flow characteristics are complex. Distinct endothelial phenotypes correlate with regional hemodynamics. We propose that systemic risk factors modify regional endothelial phenotype to influence focal susceptibility to atherosclerosis. Transcript profiles of freshly isolated endothelial cells from three atherosusceptible and three atheroprotected arterial regions in adult swine were analyzed to determine the initial prelesional effects of hypercholesterolemia on endothelial phenotypes in vivo. Cholesterol efflux transporter ATP-binding cassette transporter A1 (ABCA1) was upregulated at all sites in response to short-term high-fat diet. Proinflammatory and antioxidative endothelial gene expression profiles were induced in atherosusceptible and atheroprotected regions, respectively. However, markers for endoplasmic reticulum stress, a signature of susceptible endothelial phenotype, were not further enhanced by brief hypercholesterolemia. Both region-specific and ubiquitous (ABCA1) phenotype changes were identified as early prelesional responses of the endothelium to hypercholesterolemia.

HOXA13 Is Essential for Placental Vascular Patterning and Labyrinth Endothelial Specification

In eutherian mammals, embryonic growth and survival is dependent on the formation of the placenta, an organ that facilitates the efficient exchange of oxygen, nutrients, and metabolic waste between the maternal and fetal blood supplies. Key to the placenta's function is the formation of its vascular labyrinth, a series of finely branched vessels whose molecular ontogeny remains largely undefined. In this report, we demonstrate that HOXA13 plays an essential role in labyrinth vessel formation. In the absence of HOXA13 function, placental endothelial cell morphology is altered, causing a loss in vessel wall integrity, edema of the embryonic blood vessels, and mid-gestational lethality. Microarray analysis of wild-type and mutant placentas revealed significant changes in endothelial gene expression profiles. Notably, pro-vascular genes, including Tie2 and Foxf1, exhibited reduced expression in the mutant endothelia, which also exhibited elevated expression of genes normally expressed in lymphatic or sinusoidal endothelia. ChIP analysis of HOXA13–DNA complexes in the placenta confirmed that HOXA13 binds the Tie2 and Foxf1 promoters in vivo. In vitro, HOXA13 binds sequences present in the Tie2 and Foxf1 promoters with high affinity (Kd = 27–42 nM) and HOXA13 can use these bound promoter regions to direct gene expression. Taken together, these findings demonstrate that HOXA13 directly regulates Tie2 and Foxf1 in the placental labyrinth endothelia, providing a functional explanation for the mid-gestational lethality exhibited by Hoxa13 mutant embryos as well as a novel transcriptional program necessary for the specification of the labyrinth vascular endothelia.

Vascular inflammation in aging: role of increased mitochondrial H2O2 production in endothelial NF‐kB activation

Aging elicits vascular oxidative stress and a pro‐inflammatory shift in endothelial gene expression profile. To elucidate the link between increased oxidative stress and vascular inflammation in aging the carotid arteries and aortas of young and aged (24 mo) F344 rats were compared. In aged vessels there was an increased NF‐kB activity (assessed by luciferase reporter gene assay and NF‐kB binding assay), which was attenuated by scavenging H2O2. Aging did not alter the vascular expression of p65 and p50 subunits of NF‐kB. In endothelial cells of aged vessels there was an increased production of H2O2, which was attenuated by the mitochondrial uncoupler FCCP. In young arteries and cultured endothelial cells antimycin A plus succinate significantly increased mitochondrial H2O2 generation, which was associated with activation of NF‐kB. In aged vessels inhibition of NF‐kB (by PDTC, resveratrol) significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness. Thus, increased mitochondrial oxidative stress contributes to endothelial NF‐kB activation, which contributes to the pro‐inflammatory phenotypic alterations in the aged vasculature.

Increased mitochondrial H2O2 production promotes endothelial NF-κB activation in aged rat arteries

Previous studies have shown that the aging vascular system undergoes pro-atherogenic phenotypic changes, including increased oxidative stress and a pro-inflammatory shift in endothelial gene expression profile. To elucidate the link between increased oxidative stress and vascular inflammation in aging, we compared the carotid arteries and aortas of young and aged (24 mo old) Fisher 344 rats. In aged vessels there was an increased NF-kappaB activity (assessed by luciferase reporter gene assay and NF-kappaB binding assay), which was attenuated by scavenging H(2)O(2). Aging did not alter the vascular mRNA and protein expression of p65 and p50 subunits of NF-kappaB. In endothelial cells of aged vessels there was an increased production of H(2)O(2) (assessed by 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester fluorescence), which was attenuated by the mitochondrial uncoupler FCCP. In young arteries and cultured endothelial cells, antimycin A plus succinate significantly increased FCCP-sensitive mitochondrial H(2)O(2) generation, which was associated with activation of NF-kappaB. In aged vessels inhibition of NF-kappaB (by pyrrolidenedithiocarbamate, resveratrol) significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness. Thus increased mitochondrial oxidative stress contributes to endothelial NF-kappaB activation, which contributes to the pro-inflammatory phenotypic alterations in the aged vaculature. Our model predicts that by reducing mitochondrial H(2)O(2) production and/or directly inhibiting NF-kappaB novel anti-aging pharmacological treatments (e.g., calorie restriction mimetics) will exert significant anti-inflammatory and vasoprotective effects.

Plasma Factors in Severe Early-Onset Preeclampsia Do Not Substantially Alter Endothelial Gene Expression In Vitro

Systemic endothelial dysfunction is a central feature in the pathophysiology of preeclampsia. Its cell biologic and molecular basis is poorly understood. One leading hypothesis argues that endothelial dysfunction is caused by (at present largely unknown) circulating factors released from the ischemic placenta. This study investigated the effects of plasma factors of severe, early-onset preeclamptic women versus healthy pregnant women on endothelial gene expression in vitro. Plasma samples were taken from eight severe early-onset preeclamptic women and eight matched pregnant control women. Primary human umbilical vein endothelial cell (HUVEC) and human glomerular microvascular endothelial cell (hGMEC) cultures were incubated with 20% (vol/vol) plasma for 4, 12, and 24 hours. Identical amounts of RNA isolated from HUVEC from three preeclamptic and three control samples were pooled for each time point, and subsequently hybridized on human 60-mer oligonucleotide microarrays containing 17,000 genes. Gene expression levels of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), and interleukin-6 (IL-6) in HUVEC and hGMEC were quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Microarray analyses of individual genes identified no genes that were up- or down-regulated more than 2.7-fold, and analyses of gene ontologies showed no gene ontology significantly up- or down-regulated in HUVEC by preeclamptic plasma. IL-8 gene expression was modestly induced by preeclamptic plasma after 4, 12, and 24 hours of HUVEC and hGMEC incubation, as identified by real-time RT-PCR. The other genes analyzed did not show altered regulation by preeclamptic plasma factors. In vitro, plasma from preeclamptic patients does not substantially alter endothelial gene expression profile. Only modest induction of IL-8 gene expression was observed. These results indicate that mechanisms other than soluble plasma constituents are likely involved in systemic endothelial cell activation in preeclampsia.

Transcriptional profiling of IKK2/NF-κB— and p38 MAP kinasedependent gene expression in TNF-α—stimulated primary human endothelial cells

AbstractInflammatory stimulation of endothelial cells by tumor necrosis factor α (TNF-α) involves activation of nuclear factor κB (NF-κB) and p38 mitogen-activated protein (MAP) kinase signaling pathways. A reliable analysis of the gene expression program elicited by TNF-α and its assignment to distinct signaling pathways is not available. A sophisticated analysis of oligonucleotide microarrays covering more than 13 000 genes allowed definition of the TNF-α-regulated endothelial gene expression profile and novel TNF-α-induced genes. Virtually all TNF-α-inducible genes were dependent on IκB kinase 2 (IKK2)/NF-κB activation, whereas a minor number was additionally modulated by p38. Furthermore, genes suppressed by IKK2/NF-κB were newly identified. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry confirmed reliability of data. Thus, these results define a list of primary candidates for targeted modulation of endothelial functions during inflammation. (Blood. 2004;103:3365-3373)

Adaptation of the endothelium to fluid flow: in vitro analyses of gene expression and in vivo implications.

Biomechanical forces generated by blood flow play an important role in the pathogenesis of vascular disease. For example, regions exposed to non-uniform shear stresses develop early atherosclerotic lesions while areas exposed to uniform shear stresses are protected. A variety of in vitro flow apparatuses have been created to apply well-characterized flow patterns to endothelial cells in an effort to dissect the cellular and molecular pathways involved in these distinct processes. Recent advances in biotechnology have permitted large-scale transcriptional profiling techniques to replace candidate gene screens and have allowed the genome-wide examination of biomechanical force-induced endothelial gene expression profiles. This review provides an overview of biomechanical force-induced modulation of endothelial phenotype. It examines the effect of sustained laminar shear stress (LSS), a type of uniform shear stress, on in vitro endothelial gene expression by synthesizing data from the early candidate gene and differential display polymerase chain reaction (PCR) approaches to the numerous, recent, high throughput functional genomic analyses. These studies demonstrate that prolonged LSS regulates the expression of only a small percentage (approximately 1-5%) of endothelial genes, and this transcriptional profile produces an endothelial phenotype that is quiescent, being protected from apoptosis, inflammation and oxidative stress. These observations provide a possible molecular mechanism for the strong correlation between patterns of blood flow and the occurrence of vascular pathologies, such as atherosclerosis, in vivo.

Gene expression in donor corneal endothelium.

To report gene expression profiles of normal human corneal endothelium with microarray analysis and serial analysis of gene expression (SAGE). Corneal endothelium was removed from normal human corneas obtained from eye banks. Total RNA was isolated and SAGE analysis was performed. The same RNA source was used to construct a complementary DNA library that was hybridized to microarrays containing 12 558 transcripts. A total of 9530 SAGE tags were sequenced, representing 4724 unique tags. Microarray analysis identified 542 distinct transcripts. A database of human corneal endothelial gene expression was compiled. Of the SAGE tags, 1720 matched known genes, 478 corresponded to expressed sequence tags, and 2526 had no known match to public databases. The 5 most abundantly expressed SAGE tags were cytochrome c oxidase subunit II, adenosine triphosphate synthase F(0) subunit 6, carbonic anhydrase XII, 12S ribosomal RNA, and ferritin, heavy polypeptide 1. Thirty-four percent of the transcripts (n = 1616) were specific to the corneal endothelium, when compared with other publicly available SAGE libraries. The 5 most abundant unique tags were keratin 12, angiopoietinlike factor, annexin A8, and 2 tags with no match to the database. Many endothelial pump function enzymes were confirmed, including several plasma membrane Na( +)/K(+) adenosine triphosphatases and a recently reported bicarbonate transporter. Corneal endothelial gene expression profiles by the current analysis provide an understanding of endothelial metabolism, structure, and function; enable comparisons to diseased endothelium; and provide baseline data that may lead to the discovery of novel endothelial genes.

Molecular Profiling of Angiogenic Markers: A Step Towards Interpretive Analysis of a Complex Biological Function

Gene expression profiling is now being used routinely to define complex biological events. The profiling of a large array of genes expressed in the progression of a biological response opens the door to our understanding the unique relationships between genes and their functions. Angiogenesis, the process of new blood vessel development, is a necessary component of both normal and pathological physiology. In this issue of The American Journal of Pathology, Shih and colleagues 1 have used quantitative molecular profiling of angiogenic-related factors to define some of the elements required for angiogenic profiling. Although presented as a technical advance, the basic concept of this work is that the use of quantitative molecular profiling of gene expression gives additional insight into functional interrelationships between the genes expressed during the angiogenic process. This approach can be applied in large array format as a diagnostic tool for experimental systems and pathological samples.