Endothelial Gene Expression Research Articles

Expression of Genes in Primo Vasculature Floating in Lymphatic Endothelium Under Lipopolysaccharide and Acupuncture Electric Stimulation

It is known that the primo vascular system (PVS) includes the primo nodes and vessels. However, the relevant genes in the PVS system for both pathologic and physiologic condition are poorly understood. Here, we first examined the gene expression in primo vessels (PVs) floating in lymphatic endothelium by isolation of PVS and lymphatic vessels (LVs) containing PVS. To investigate therapeutic effects, both PVs and LVs containing PVS were isolated after lipopolysaccharide injection and acupuncture electric stimulation at two acupoints Joksamni (ST36) and Hapgok (LI04) following lipopolysaccharide injection. We used reverse transcriptase–polymerase chain reaction to examine expression of lymphatic endothelial cell markers and inflammatory related genes. We found that lymphatic endothelial cell markers such as fms-related tyrosine kinase 4 (Flt4), lymphatic vessel endothelial receptor (Lyve-1), prospero homeobox protein 1 (Prox-1), and podoplanin (Pdpn) were highly expressed in PV compared to that of lymphatic endothelium, suggesting pivotal roles of PV in LV under inflammation. Furthermore, lymphatic-related genes including metal-response element-binding transcription factor 2 (Mtf2), hypoxia inducible factor (Hif1a), angiotensin II type 1 receptor (Agtr1), and angiotensin II type 2 receptor (Agtr2) were also overall increased in PV, and remarkably increased and these genes except peroxisome proliferator–activated receptor gamma (Pparg) after acupuncture electric stimulation in two acupoints implying central role of PV by gene activation.

Enhancer‐Associated Long Non‐Coding RNAs Regulate Vascular Endothelial Function

Serving as a bioactive interface between the blood flow and the vessel wall, the vascular endothelium responds dynamically to physiological and pathophysiological stimuli to develop distinct gene expression profiles. Among the extensive mechanistic studies on molecular and genetic levels, it is unclear whether and how chromatin remodeling plays a role in the regulation of endothelial gene expression. In this study, we explored the role of long non-coding RNAs (lncRNAs)-mediated chromatin remodeling in endothelial transcriptome. Combining RNA-sequencing, chromosome conformation capture technologies, and systems biology approaches, we identified in human endothelial cells (ECs) a set of long non-coding RNAs (lncRNAs) that are differentially regulated by endothelial protective vs. detrimental stimuli (e.g. shear stress, pro-inflammatory cytokine, and statins). As an example, we recently described the endothelial regulation by a “lncRNA that enhances endothelial nitric oxide synthase (eNOS) expression”, i.e., LEENE. LEENE is co-regulated with eNOS, the endothelial hallmark molecule, and that its enhancer resides in proximity to the eNOS promoter in ECs. Mechanistically, LEENE facilitates the recruitment of RNA polymerase II to the eNOS promoter to enhance eNOS nascent RNA transcription. By using a systematic and integrative approach to identify enhancer-associated lncRNAs and their regulation of endothelial gene expression, we identified >200 lncRNA candidates with high potentials to regulate endothelial function. Suppression of these lncRNAs with locked nucleic acids or CRISPR-Cas9-mediated genomic editing leads to altered endothelial gene expression and functional phenotypes. Furthermore, some of these lncRNAs can be detected in the circulating exosomes, suggesting their diagnostic and therapeutic potentials. Our findings unravel a new layer in endothelial regulation and provide novel insights into cardiovascular functions in health and disease. Support or Funding Information This work is supported by R00HL122368 and R01HL108735 from NIH, Ella Fitzgerald Foundation, and a Shared Resources Pilot Program and a Startup Fund from Beckman Research Institute, City of Hope. LEENE enhancer is in association proximity of eNOS promoter and is essential for eNOS expression This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Exercise‐Induced Shear Stress on Endothelial Gene Expression

PurposeEndothelial dysfunction is the first step in the development of atherosclerosis and one possible protective regulatory mechanism is exercise‐induced endothelial shear stress. It has been proposed that endurance exercise‐induced shear stress could guide treatment strategies in clinical settings; nevertheless, research on this topic is limited. The aims of this study are (1) to characterize exercise‐induced shear stress patterns and (2) to analyze the effect of simulated exercise‐induce shear stress on endothelial gene expression.MethodsFor the initial in vivo assessment, a total of 12 young healthy subjects (age 13–31) (10 males) were recruited to perform two exercise tests on a cycle ergometer. The first test was a maximal incremental test which established the workloads for the next session, according to blood lactate levels. The second one, performed at least 48 hours after the first exercise test, was a 3 workload steady‐state test at lactate levels of <2 mmol/L (low intensity) for 5 minutes, 2–4 mmol/L (moderate intensity) for 5 minutes, and >4 mmol/L (high intensity) for 3 minutes. Blood flow patterns were recorded during both tests through Doppler ultrasound of the brachial artery (GE Logic E) and shear stress was calculated by Womersley's approximation. In addition, measures of heart rate, oxygen uptake, blood lactate, and rate of perceived exertion (RPE) were obtained at the end of the third minute for each stage. After characterizing exercise‐induced shear stress patterns, in vitro experiments were performed. Cultured human vein endothelial cells (HUVEC) were exposed to anterograde pulsatile flow (OsciFlow Device) in a parallel flow plate (Flex Flow) during 13 hours to simulate resting and exercise conditions: (1) resting shear stress (13 hours at 15 dynes/cm2), (2) low‐to‐moderate shear stress (12 hours at 15 dynes/cm2 followed by 1 hour at 50 dynes/cm2), and (3) high shear stress (12 hours at 15 dynes/cm2 followed by 1 hour at 75 dynes/cm2). Finally, cells were collected and analyzed for pro‐atherogenic VCAM‐1 mRNA expression for each condition. Statistical analysis used one‐way repeated measures ANOVA with Fisher's LSD as post‐hoc analysis. Significance was set at p‐value ≤ 0.05.ResultsAnterograde shear stress showed an intensity‐dependent relationship (rest: 34.7 ± 5.8 dynes/cm2, low: 41.1 ± 13.7 dynes/cm2, moderate: 44.12 ± 17.2 dynes/cm2, and high: 56.96 ± 21.6 dynes/cm2), with significant difference only between low‐to‐moderate intensities vs. high intensity (p < 0.05). Meanwhile, retrograde shear stress exhibited the same intensity trend (rest: 6.9 ± 3.9 dynes/cm2, low: 11.5 ± 5.6 dynes/cm2, moderate: 17.37 ± 9.2 dynes/cm2, and high: 19.6 ± 8.4 dynes/cm2); however, significant differences were seen only between low intensity vs. high‐to‐moderate intensities (p < 0.01). An inverted U shaped curve for VCAM‐1 mRNA expression was observed for resting, low‐to‐moderate, and high simulated shear stress, respectively.ConclusionExercise‐induced shear stress increases in an intensity‐dependent manner. Shear stress observed during high‐intensity exercise might downregulate pro‐atherogenic gene expression on endothelial cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An efficient method to isolate Kupffer cells eliminating endothelial cell contamination and selective bias.

Multicolor flow cytometry and cell sorting are powerful immunologic tools for the study of hepatic mϕ, yet there is no consensus on the optimal method to prepare liver homogenates for these analyses. Using a combination of mϕ and endothelial cell reporter mice, flow cytometry, and confocal imaging, we have shown that conventional flow‐cytometric strategies for identification of Kupffer cells (KCs) leads to inclusion of a significant proportion of CD31hi endothelial cells. These cells were present regardless of the method used to prepare cells for flow cytometry and represented endothelium tightly adhered to remnants of KC membrane. Antibodies to endothelial markers, such as CD31, were vital for their exclusion. This result brings into focus recently published microarray datasets that identify high expression of endothelial cell‐associated genes by KCs compared with other tissue‐resident mϕ. Our studies also revealed significant and specific loss of KCs among leukocytes with commonly used isolation methods that led to enrichment of proliferating and monocyte‐derived mϕ. Hence, we present an optimal method to generate high yields of liver myeloid cells without bias for cell type or contamination with endothelial cells.

Effect of NRF2 Activation on Endothelial Function, Microvessel Density, and Gene Expression in Rats fed High Salt Diet

BACKGROUNDHigh salt (HS) diet promotes vascular oxidative stress and endothelial dysfunction in animal models and humans. Studies in animals indicate that salt‐induced suppression of antioxidant defense mechanisms plays a crucial role in endothelial dysfunction, microvascular rarefaction and vascular oxidative stress. We hypothesized that activation of the master antioxidant transcription factor nuclear factor (erythroid‐derived 2)‐like‐2 (NRF2) with the dietary supplement Protandim protects against salt‐induced vascular dysfunction and microvascular rarefaction by restoring redox homeostasis.OBJECTIVETo determine whether activation of Nrf2 via dietary supplementation with Protandim will prevent salt‐induced endothelial dysfunction, vascular oxidative stress, and microvascular rarefaction via effects on antioxidant enzyme expression.METHODSAge‐matched, male Sprague‐Dawley rats (12–15 weeks old) were fed a high salt (HS; 4.0% NaCl) diet ± ~60 mg/kg/day Protandim supplement for 2 weeks. mRNA from harvested liver samples was used for real time PCR evaluation of the effects of Nrf2 upregulation on a range of genes related to oxidative stress, as reported in the literature. Changes in protein expression were evaluated via Western blotting.RESULTSProtandim treatment restored endothelium‐dependent dilation to acetylcholine in middle cerebral arteries, reduced mitochondrial ROS levels (Mito‐SOX) in basilar arteries, and increased microvessel density in the cremaster muscle of HS‐fed rats. Protandim supplementation increased mRNA levels of thioredoxin reductase 1 (n=6, p=0.008), reduced mRNA expression of the pro‐oxidative transcription factor Kruppel‐like factor 9 (Klf9) (n=6, p < 0.001) and tended to increase the expression of mRNA for glutathione reductase (GSR) and catalase in liver samples compared to untreated rats fed HS diet alone. Other indicators were not significantly changed by Protandim treatment, either by qPCR or Western blot analysis.CONCLUSIONSTreatment of HS‐fed rats with the Nrf2 activator Protandim ameliorates salt‐induced endothelial dysfunction, vascular oxidative stress, and microvascular rarefaction via complex effects on enzymes and proteins affecting redox state.Support or Funding InformationNIH #R01‐HL128242 and # R21‐OD018309‐JHLThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Post‐septic Down‐regulation of Sulfatase‐1 Suppresses Pulmonary Endothelial ICAM‐1 Expression

RationalePost‐septic compensatory anti‐inflammatory syndrome (CARS) is a state of immunoparalysis that leaves patients susceptible to secondary infections. Interestingly, CARS coincides with post‐septic reconstitution of the pulmonary endothelial glycocalyx, a heparan sulfate (HS)‐enriched layer that regulates endothelial‐neutrophil adhesion and is degraded during early sepsis. CARS is generally described as a consequence of impaired leukocyte function, with little understanding of the role of the post‐septic endothelium. We hypothesized that the post‐septic pulmonary endothelial glycocalyx is characterized by changes in HS sulfation (via changes in sulfatase expression), leading to impaired endothelial inflammatory responses.MethodsWe induced sepsis in male C57BL/6J mice with cecal ligation and puncture (CLP). To recapitulate CARS, post‐septic mice (3 days after CLP) animals were treated with intratracheal lipopolysaccharide (LPS), and the degree of inflammation was assessed thereafter by bronchoalveolar lavage (BAL) cell count and protein concentration. To determine changes in post‐septic pulmonary endothelial gene expression during CARS, we flow‐sorted pulmonary endothelial cells and performed RNA microarray and confirmatory qRT‐PCR. To determine post‐septic changes in HS sulfation, we performed mass spectrometry of HS isolated from either the pulmonary endothelial glycocalyx or the plasma of post‐CLP or post‐sham mice. We determined the impact of altered endothelial HS sulfation on CARS by either (a) degrading HS from the endothelial glycocalyx by intravenous heparinase I or (b) administering exogenous sulfated HS intravenously prior to intratracheal LPS. We specifically measured the effect of sulfatase‐1 (sulf‐1, an extracellular enzyme that specifically cleaves 6‐O sulfate of HS) on endothelial cell ICAM‐1 expression using siRNA.ResultsIn comparison to sham mice, post‐septic mice showed decreased cellular and protein infiltration in BAL fluid 48 hrs after intratracheal LPS (n=5–6, P < 0.05). Microarray and qRT‐PCR analyses of flow‐sorted endothelial cells (harvested 48 hrs after CLP or sham) detected downregulation of sulf‐1 in CLP mice (n=3–4, P < 0.05). While these changes correlated with increased 6‐O sulfation in the endothelial glycocalyx and circulating HS, experimental modulation of these HS compartments did not influence CARS. However, in vitro, transfection of siRNA targeted to sulf‐1 resulted in 90% reduction of sulf‐1 mRNA and down‐regulation of ICAM‐1 by 57–70% (n=2). These in vitro changes surprisingly occurred independently of HS 6‐O sulfation.ConclusionsConcurrently with CARS, post‐septic pulmonary endothelial cells undergo downregulation of sulf‐1, causing loss of endothelial ICAM‐1 expression. Surprisingly, this impaired ICAM‐1 expression occurred in the absence of changes in HS 6‐O sulfation.Support or Funding InformationDepartment of Defense PR150655 (to EPS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Engineering a multicellular vascular niche to model hematopoietic cell trafficking

BackgroundThe marrow microenvironment and vasculature plays a critical role in regulating hematopoietic cell recruitment, residence, and maturation. Extensive in vitro and in vivo studies have aimed to understand the marrow cell types that contribute to hematopoiesis and the stem cell environment. Nonetheless, in vitro models are limited by a lack of complex multicellular interactions, and cellular interactions are not easily manipulated in vivo. Here, we develop an engineered human vascular marrow niche to examine the three-dimensional cell interactions that direct hematopoietic cell trafficking.MethodsUsing soft lithography and injection molding techniques, fully endothelialized vascular networks were fabricated in type I collagen matrix, and co-cultured under flow with embedded marrow fibroblast cells in the matrix. Marrow fibroblast (mesenchymal stem cells (MSCs), HS27a, or HS5) interactions with the endothelium were imaged via confocal microscopy and altered endothelial gene expression was analyzed with RT-PCR. Monocytes, hematopoietic progenitor cells, and leukemic cells were perfused through the network and their adhesion and migration was evaluated.ResultsHS27a cells and MSCs interact directly with the vessel wall more than HS5 cells, which are not seen to make contact with the endothelial cells. In both HS27a and HS5 co-cultures, endothelial expression of junctional markers was reduced. HS27a co-cultures promote perfused monocytes to adhere and migrate within the vessel network. Hematopoietic progenitors rely on monocyte-fibroblast crosstalk to facilitate preferential recruitment within HS27a co-cultured vessels. In contrast, leukemic cells sense fibroblast differences and are recruited preferentially to HS5 and HS27a co-cultures, but monocytes are able to block this sensitivity.ConclusionsWe demonstrate the use of a microvascular platform that incorporates a tunable, multicellular composition to examine differences in hematopoietic cell trafficking. Differential recruitment of hematopoietic cell types to distinct fibroblast microenvironments highlights the complexity of cell-cell interactions within the marrow. This system allows for step-wise incorporation of cellular components to reveal the dynamic spatial and temporal interactions between endothelial cells, marrow-derived fibroblasts, and hematopoietic cells that comprise the marrow vascular niche. Furthermore, this platform has potential for use in testing therapeutics and personalized medicine in both normal and disease contexts.

Addendum: Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification

Establishment of a functional vascular network is rate-limiting in embryonic development, tissue repair and engineering. During blood vessel formation, newly generated endothelial cells rapidly expand into primitive plexi that undergo vascular remodeling into circulatory networks, requiring coordinated growth inhibition and arterial-venous specification. Whether the mechanisms controlling endothelial cell cycle arrest and acquisition of specialized phenotypes are interdependent is unknown. Here we demonstrate that fluid shear stress, at arterial flow magnitudes, maximally activates NOTCH signaling, which upregulates GJA4 (commonly, Cx37) and downstream cell cycle inhibitor CDKN1B (p27). Blockade of any of these steps causes hyperproliferation and loss of arterial specification. Re-expression of GJA4 or CDKN1B, or chemical cell cycle inhibition, restores endothelial growth control and arterial gene expression. Thus, we elucidate a mechanochemical pathway in which arterial shear activates a NOTCH-GJA4-CDKN1B axis that promotes endothelial cell cycle arrest to enable arterial gene expression. These insights will guide vascular regeneration and engineering.

RNA expression profiling of renal allografts in a nonhuman primate identifies variation in NK and endothelial gene expression.

RNA transcript expression estimates are a promising method to study the mechanisms and classification of renal allograft rejections. Here we use the Nanostring platform to profile RNA expression in renal allografts in a nonhuman primate (NHP), the Cynomolgus monkey. We analyzed protocol and indication 278 archival renal allograft samples, both protocol and indication from 76 animals with diagnoses of chronic antibody-mediated rejection (CAMR), acute cellular rejection (TCMR), and MIXED (both CAMR and TCMR), plus normals and samples with no pathological rejection using a Cynomolgus-specific probe set of 67 genes. Analysis identified RNA expression heterogeneity of endothelial and NK genes within CAMR and TCMR, including the stages of CAMR. Three factors were partitioned into additional groups. One group with the longest allograft survival time is pure CAMR without NK or CD3. Three mixed groups show variation in NK and CD3. TCMR was split into 2 groups with variation in NK genes. Additional validation of the complete gene-set correlated many of the genes with diagnoses of CAMR, MIXED, and TCMR rejections and with Banff histologic criteria defined in human subjects. These NHP data demonstrate the utility of RNA expression profiling to identify additional heterogeneity of endothelial and NK RNA gene expressions.

Abstract 77: Circulating miRNome in Moyamoya Discordant MZ Twins - Implications for Endothelial Biofunctions

Objective: We aim to investigate circulating genome-wide microRNA (miRNome) profiles in Moyamoya disease-discordant monozygotic (MZ) twins with the RNF213 founder mutation (rs112735431), since the etiology remains unclear even after the identification of RNF213. Methods: Circulating miRNome was screened in a pair of Moyamoya-discordant MZ twins and a validation cohort (unrelated 9 cases and 8 normal controls) using microarray and validated by quantitative real-time PCR. Differential plasma microRNAs were quantified in endothelial cells differentiated from iPS cell line (iPSECs) derived from unrelated 3 cases and 3 controls. Gene expressions targeted by the significant microRNAs in iPSECs was studied using published iPSEC-transcriptome and bioinformatics tool. Results: A total of 537 circulating microRNAs were detected in the Moyamoya-discordant MZ twins and the validation cohort. Unsupervised cluster analysis demonstrated 2 discrete miRNome, predominantly by disease, but the MZ twins showed similar miRNome profiles, regardless of phenotypic discordance (Fig. 1A). Across the 143 and 98 differential microRNAs in the MZ twins and validation cohort, 23 microRNAs were overlapped (Fig. 1B). A cluster of plasma hsa-miR-6722-3p/-328-3p/150-5p was significantly up-regulated in patients in the validation cohort (p < 0.05) (Fig. 1C). In iPSECs, hsa-miR-6722-3p/-328-3p cluster was up-regulated with a greater than 3.0-fold change in patients. Bioinformatics analysis revealed that 94 target genes involving organismal injuries and cell survival/maintenance were significantly down-regulated in Moyamoya-iPSECs (p < 0.05, q < 0.1), suggesting the regulatory role of the miR-6722/-328 cluster in endothelial gene expression (Fig. 1D). Conclusions: Differential plasma-miRNAs in Moyamoya-discordant MZ twins may potentially serve as a diagnostic biomarker in Moyamoya disease and regulate endothelial gene expression.

The lncRNA GATA6-AS epigenetically regulates endothelial gene expression via interaction with LOXL2

Impaired or excessive growth of endothelial cells contributes to several diseases. However, the functional involvement of regulatory long non-coding RNAs in these processes is not well defined. Here, we show that the long non-coding antisense transcript of GATA6 (GATA6-AS) interacts with the epigenetic regulator LOXL2 to regulate endothelial gene expression via changes in histone methylation. Using RNA deep sequencing, we find that GATA6-AS is upregulated in endothelial cells during hypoxia. Silencing of GATA6-AS diminishes TGF-β2-induced endothelial–mesenchymal transition in vitro and promotes formation of blood vessels in mice. We identify LOXL2, known to remove activating H3K4me3 chromatin marks, as a GATA6-AS-associated protein, and reveal a set of angiogenesis-related genes that are inversely regulated by LOXL2 and GATA6-AS silencing. As GATA6-AS silencing reduces H3K4me3 methylation of two of these genes, periostin and cyclooxygenase-2, we conclude that GATA6-AS acts as negative regulator of nuclear LOXL2 function.

Flow-dependent epigenetic regulation of IGFBP5 expression by H3K27me3 contributes to endothelial anti-inflammatory effects.

Rationale: Atherosclerosis is a chronic inflammatory and epigenetic disease that is influenced by different patterns of blood flow. However, the epigenetic mechanism whereby atheroprotective flow controls endothelial gene programming remains elusive. Here, we investigated the possibility that flow alters endothelial gene expression through epigenetic mechanisms.Methods: En face staining and western blot were used to detect protein expression. Real-time PCR was used to determine relative gene expression. RNA-sequencing of human umbilical vein endothelial cells treated with siRNA of enhancer of zeste homolog 2 (EZH2) or laminar flow was used for transcriptional profiling.Results: We found that trimethylation of histone 3 lysine 27 (H3K27me3), a repressive epigenetic mark that orchestrates gene repression, was reduced in laminar flow areas of mouse aorta and flow-treated human endothelial cells. The decrease of H3K27me3 paralleled a reduction in the epigenetic “writer”-EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2). Moreover, laminar flow decreased expression of EZH2 via mechanosensitive miR101. Genome-wide transcriptome profiling studies in endothelial cells treated with EZH2 siRNA and flow revealed the upregulation of novel mechanosensitive gene IGFBP5 (insulin-like growth factor-binding protein 5), which is epigenetically silenced by H3K27me3. Functionally, inhibition of H3K27me3 by EZH2 siRNA or GSK126 (a specific EZH2 inhibitor) reduced H3K27me3 levels and monocyte adhesion to endothelial cells. Adenoviral overexpression of IGFBP5 also recapitulated the anti-inflammatory effects of H3K27me3 inhibition. More importantly, we observed EZH2 upregulation, and IGFBP5 downregulation, in advanced atherosclerotic plaques from human patients.Conclusion: Taken together, our findings reveal that atheroprotective flow reduces H3K27me3 as a chromatin-based mechanism to augment the expression of genes that confer an anti-inflammatory response in the endothelium. Our study exemplifies flow-dependent epigenetic regulation of endothelial gene expression, and also suggests that targeting the EZH2/H3K27me3/IGFBP5 pathway may offer novel therapeutics for inflammatory disorders such as atherosclerosis.

Tumoral Necrosis Factor (TNF) mRNA gene expression in relation to Vascular Endothelial Growth Factor (VEGF) and chemokines mRNA gene expression in tumoral microenvironment of endometrial cancer versus normal endometrium: Preliminary results

Tumoral Necrosis Factor (TNF) mRNA gene expression in relation to Vascular Endothelial Growth Factor (VEGF) and chemokines mRNA gene expression in tumoral microenvironment of endometrial cancer versus normal endometrium: Preliminary results

Evaluation of the effect of hesperidin on vascular endothelial growth factor gene expression in rat skin animal models following cobalt-60 gamma irradiation.

Skin is highly prone to radiation damage. Radiation burn is defined as damage to the skin or other biological tissues induced by radiofrequency or ionizing radiation. Vascular endothelial growth factor (VEGF) is a heparin-binded pro-angiogenic factor. Flavonoids belong to a family of polyphenol chemical compounds that are frequently present in fruits and vegetables. Hesperidin is an agent belonging to the flavonoid family. The aim of this study is to investigate whether hesperidin can affect the VEGF gene expression in rat skin following gamma irradiation or not. A total number of 36 male Sprague-Dawley rats were divided into three groups. First group: radiation group (n = 12), second group: radiation + hesperidin-treated group (n = 12), and third group: untreated control group (n = 12). The hesperidin administration dose was 100 mg/kg body weight. The rats received a 22 Gy single dose at a dose rate of about 0.3 Gy/min using a cobalt-60 external beam radiotherapy unit. The animals were euthanized 24 h postirradiation. VEGF gene expression data were analyzed using the equation 2-ΔΔCT, where ΔΔCT = (Threshold cycle [CT], of target gene - CT of housekeeping gene)treated group- (CT of target gene - CT of housekeeping gene)untreated control group. Glyceraldehyde-3-phosphate dehydrogenase gene was used as a housekeeping gene. VEGF gene in the radiation + hesperidin group overexpressed 25-fold relative to the control group. In addition, VEGF gene in the radiation group underexpressed 0.15-fold relative to the control group. When the three groups were compared relative to each other using the Kruskal-Wallis test, P < 0.001 was obtained. Based on the Mann-Whitney U-test, when all groups were compared to each in a binary model, P = 0.001 was achieved. These tests all showed statistically significant changes in VEGF gene expression. We can conclude that hesperidin is a potent angiogenic factor. Hesperidin as a radioprotector can initiate angiogenesis by VEGF gene induction. It may stimulate epithelialization, collagen deposition, and enhanced cellular proliferation. These changes can together accelerate wound healing, in particular, radiation-induced skin damage.

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.

Selection of reference genes for studies of human retinal endothelial cell gene expression by reverse transcription-quantitative real-time polymerase chain reaction

BackgroundHuman retinal endothelial cells are employed increasingly for investigations of retinal vascular diseases. Analysis of gene expression response to disease-associated stimuli by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is common. However, most reported work does not follow the minimum information for publication of qPCR experiments (MIQE) recommendation that multiple, stably expressed reference genes be used for normalization. MethodsTwo human retinal endothelial cell lines were treated with medium alone or containing stimuli that included: glucose at supraphysiological concentration, dimethyloxalyl-glycine, vascular endothelial growth factor, tumor necrosis factor-α, lipopolysaccharide and Toxoplasma gondii tachyzoites. Biological response of cells was confirmed by measuring significant increase in a stimulus-relevant transcript. Total RNA was reverse transcribed and analyzed by commercial PCR arrays designed to detect 28 reference genes. Stability of reference gene expression, for each and both cell lines, and for each and all conditions, was judged on gene-stability measure (M-value) <0.2 and coefficient of variation (CV-value) <0.1. ResultsReference gene expression varied substantially across stimulations and between cell lines. Of 27 detectable reference genes, 11–21 (41–78%) maintained expression stability across stimuli and cell lines. Ranking indicated substantial diversity in the most stable reference genes under different conditions, and no reference gene was expressed stably under all conditions of stimulation and for both cell lines. Four reference genes were expressed stably under 5 conditions: HSP90AB1, IPO8, PSMC4 and RPLPO. ConclusionsWe observed variation in stability of reference gene expression with different stimuli and between human retinal endothelial cell lines. Our findings support adherence to MIQE recommendations regarding normalization in RT-qPCR studies of human retinal endothelial cells.

Effect of Calcium-Infiltrated Hydroxyapatite Scaffolds on the Hematopoietic Fate of Human Umbilical Vein Endothelial Cells

Foamed hydroxyapatite offers a three-dimensional scaffold for the development of bone constructs, mimicking perfectly the in vivo bone structure. In vivo, calcium release at the surface is assumed to provide a locally increased gradient supporting the maintenance of the hematopoietic stem cells niche. We fabricated hydroxyapatite scaffolds with high surface calcium concentration by infiltration, and used human umbilical vein endothelial cells (HUVECs) as a model to study the effects on hematopoietic lineage direction. HUVECs are umbilical vein-derived and thus possess progenitor characteristics, with a prospective potential to give rise to hematopoietic lineages. HUVECs were cultured for long term on three-dimensional porous hydroxyapatite scaffolds, which were either infiltrated biphasic foams or untreated. Controls were cultured in two-dimensional dishes. The release of calcium into culture medium was determined, and cells were analyzed for typical hematopoietic and endothelial gene expressions, surface markers by flow cytometry, and hematopoietic potential using colony-forming unit assays. Our results indicate that the biphasic foams promoted a hematopoietic lineage direction of HUVECs, suggesting an improved in vivo-like scaffold for hematopoietic bone tissue engineering.

BRACHYURY directs histone acetylation to target loci during mesoderm development.

T-box transcription factors play essential roles in multiple aspects of vertebrate development. Here, we show that cooperative function of BRACHYURY (T) with histone-modifying enzymes is essential for mouse embryogenesis. A single point mutation (TY88A) results in decreased histone 3 lysine 27 acetylation (H3K27ac) at T target sites, including the T locus, suggesting that T autoregulates the maintenance of its expression and functions by recruiting permissive chromatin modifications to putative enhancers during mesoderm specification. Our data indicate that T mediates H3K27ac recruitment through a physical interaction with p300. In addition, we determine that T plays a prominent role in the specification of hematopoietic and endothelial cell types. Hematopoietic and endothelial gene expression programs are disrupted in TY88A mutant embryos, leading to a defect in the differentiation of hematopoietic progenitors. We show that this role of T is mediated, at least in part, through activation of a distal Lmo2 enhancer.

Abstract 21318: Long Non-Coding RNA Promotes Endothelial Homeostasis Through Chromatin Remodeling

Serving as a bioactive interface between the blood flow and the vessel wall, the vascular endothelium senses mechanical and chemical signals under various physiological and pathophysiological conditions, such as atherosclerosis and diabetes complications. Among the extensive mechanistic studies on multiple molecular levels, it is unclear whether and how chromatin remodeling plays a role in regulation of endothelial gene expression. In this study, we explored the role of long non-coding RNAs (lncRNAs)-mediated chromatin remodeling in endothelial transcriptome. Combining RNA-sequencing, chromosome conformation capture technologies, and systems biology approaches, we identified in human endothelial cells (ECs) a set of long non-coding RNAs (lncRNAs) that are differentially regulated by endothelial protective versus detrimental stimuli. Mechanistically, these lncRNAs can act as scaffolds to enhance transcription of key endothelial molecules through mediating the long-range DNA interaction and transcription factor binding. Suppression of these lncRNAs with locked nucleic acid-GapmerRs or CRISPR-Cas9-mediated genomic editing leads to altered endothelial transcriptome and phenotypes. Using mouse models, we demonstrate that some of these lncRNAs and their molecular functions may be conserved across-sepcies. Furthermore, these lncRNAs can be detected in the circulating exosomes, suggesting that they may bear diagnostics and therapeutics potentials. 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 diseases involving endothelial dysfunction.

Whey protein hydrolysate induced modulation of endothelial cell gene expression

Whey protein concentrate (WPC) hydrolysates generated using Neutrase®, Alcalase® and Flavourzyme® and their associated ultrafiltration fractions inhibited angiotensin-1-converting enzyme activity (71.14 ± 1.05, 73.22 ± 0.99 and 51.52 ± 5.80% inhibition when assayed at 14.3 µg/mL). Incubation of human umbilical vein endothelial cells with 5 kDa permeates of Neutrase®- and Alcalase®-hydrolysed WPC for 48 h resulted in the beneficial differential expression of genes relevant to blood pressure control, as measured by microarray. Furthermore, real-time reverse transcriptase polymerase chain reaction demonstrated an upregulation of endothelial nitric oxide synthase (+2.14 ± 0.45 and 2.36 ± 0.27-fold) and down-regulation of endothelin-1 (−0.58 ± 0.09 and −0.82 ± 0.11-fold) following incubation with the 5 kDa permeates of Alcalase®- and Neutrase®-hydrolysates, respectively. Peptide sequences within the 5 kDa permeate of the Alcalase®-hydrolysed WPC were identified, many of which have previously been demonstrated as having ACE-inhibitory and/or other bioactivities. These WPC hydrolysates potentially represent sources of bioactive peptides for the beneficial regulation of endothelial cell function.