Protein Expression In Endothelial Cells Research Articles

Suppression of Kv1.5 protects against endothelial apoptosis induced by palmitate and in type 2 diabetes mice

AimsPalmitate, a common saturated free fatty acid, induces endothelial apoptosis in vitro in culture endothelial cells and in vivo in type 2 diabetes mellitus (T2DM) patients. The present study aimed to investigate whether Kv1.5 regulates palmitate-induced endothelial apoptosis and endothelial dysfunction in T2DM. Main methodsIn vitro experiments were carried out in primary human HUVECs. Apoptosis was analyzed by flow cytometry. Cell viability was determined by Cell Counting Assay Kit-8. The siRNA transfection was employed to knockdown Kv1.5 protein expression. Intracellular and mitochondrial ROS, and mitochondrial membrane potential were detected using fluorescent probes. Male C57BL/6 mice fed with high-sucrose/fat diet were injected with streptozotocin (35mg/kg body weight) to establish T2DM animal model. Key findingsWe found that palmitate-induced endothelial apoptosis was parallel to a significant increase in endogenous Kv1.5 protein expression in endothelial cells. Silencing of Kv1.5 with siRNA reduced palmitate-induced endothelial apoptosis, intracellular ROS generation, mitochondrial ROS generation and membrane potential (Δψm) alteration and cleaved caspase-3 protein expression; while increased cell viability and ratio of Bcl-2/Bax. Furthermore, we observed that Kv1.5 protein expression increased in endothelial cells of thoracic aorta of T2DM mice. Silencing of Kv1.5 significantly improved the endothelium-dependent vasodilation in thoracic aortic rings of T2DM mice. SignificanceThese results demonstrate that suppression of Kv1.5 protects endothelial cells against palmitate-induced apoptosis via inhibiting mitochondria-mediated excessive ROS generation and apoptotic signaling pathway, suggesting that Kv1.5 may serve as a therapeutic target of treatment for endothelial dysfunction induced by palmitate and lipid metabolism in T2DM patients.

Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

Background. Both endothelin receptor type B ([ETBR], a G protein-coupled receptor that mediates the vascular effects of the potent vasoconstrictor endothelin-1) and human cytomegalovirus ([HCMV], a ubiquitous herpesvirus) have been implicated in the pathogenesis of cardiovascular disease (CVD). The effects of HCMV infection on ETBR expression are unknown. We hypothesized that HCMV may contribute to the pathogenesis of CVD via ETBR modulation.Methods. Human CMV effects on ETBR were studied in vitro in endothelial cells (ECs) and smooth muscle cells (SMCs) and ex vivo in human carotid plaque tissue specimens. Expression of ETBR and viral immediate-early were quantified using quantitative polymerase chain reaction. Functional consequences after ETBR blockade in ECs were examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation, wound healing, tube formation, and flow adhesion assays.Results. Human CMV is capable of upregulating both ETBR mRNA and protein expression in ECs and SMCs. The ETBR was also abundantly expressed in ECs, foam cells, and SMCs, and, more importantly, in HCMV-positive cells in human carotid plaques. Endothelin receptor type B blockade led to decreased proliferation and reduced tumor necrosis factor α-mediated leukocyte recruitment in both uninfected and HCMV-infected ECs. Direct HCMV infection was antimigratory and antiangiogenic in ECs.Conclusions. Human CMV may contribute to CVD via ETBR induction.

Mesenchymal stem cells stabilize the blood-brain barrier through regulation of astrocytes.

IntroductionThe blood–brain barrier (BBB) protects the brain against potentially neurotoxic molecules in the circulation, and loss of its integrity may contribute to disease progression in neurodegenerative conditions. Recently, the active role of reactive astrocytes in BBB disruption has become evident in the inflamed brain. In the present study, we investigated whether mesenchymal stem cell (MSC) treatment might modulate reactive astrocytes and thus stabilize BBB integrity through vascular endothelial growth factor A (VEGF-A) signaling in inflammatory conditions.MethodsFor the inflamed brain, we injected LPS using a stereotaxic apparatus and MSCs were injected into the tail vein. At 6 hours and 7 days after LPS injection, we analyzed modulatory effects of MSCs on the change of BBB permeability through VEGF-A signaling using immunochemistry and western blot. To determine the effects of MSCs on VEGF-A-related signaling in cellular system, we had used endothelial cells treated with VEGF-A and co-cultured astrocyte and BV 2 cells treated with lipopolysaccharide (LPS) and then these cells were co-cultured with MSCs.ResultsIn LPS-treated rats, MSCs restored Evans blue infiltration and the number of endothelial-barrier antigen (EBA) and P-glycoprotein (p-gp)-expressing cells, which were significantly altered in LPS-treated animals. Additionally, MSC administration following LPS treatment markedly increased the density of astrocytic filaments around vessels and reversed LPS-induced elevations in VEGF-A levels as well as endothelial nitric oxide synthase (eNOS)-dependent downregulation of tight junction proteins in the endothelium. Consequently, MSC treatment reduced neutrophil infiltration and enhanced survival of midbrain dopaminergic neurons in LPS-treated animals. In cellular system, MSC treatment led to a significant reversion of VEGF-A-induced eNOS and tight junction protein expression in endothelial cells, which led to increased EBA expressing cells. Additionally, MSC treatment significantly attenuated LPS-induced increased expressions of IL-1β in microglia and VEGF-A in astrocytes with an increase in IL-10 levels.ConclusionThe present study indicated that MSCs may stabilize BBB permeability by modulating astrocytic endfeet and VEGF-A signaling, which may be relevant to the treatment of Parkinsonian diseases as a candidate for disease modifying therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0180-4) contains supplementary material, which is available to authorized users.

Identifying novel mechanisms regulating expression of inflammatory chemokine CXCL2 (CCR3P.206)

Abstract Mammalian cells produce inflammatory mediators in response to the innate immune signal and their expression is tightly regulated. Chemokine (C-X-C motif) ligand 2 (CXCL2), also known as MIP2-alpha, belongs to the CXC chemokine family. CXCL2 is chemotactic for neutrophils. Elevated expression of CXCL2 is associated with many inflammatory and autoimmune diseases. Recently we have found that the Fli-1 transcription factor is a novel regulator in modulating the expression of many inflammatory mediators. including MCP-1,CCL5) and IL-6. Since we found that lupus-prone mice with reduced expression of Fli-1 had significantly decreased neutrophil infiltration into the kidneys, we investigated if Fli-1 regulates the expression of CXCL2. We found that Fli-1 protein expression in endothelial cells transfected with Fli-1 specific siRNA was significantly decreased compared to the expression of Fli-1 in cells transfected with control siRNA after stimulation by Toll-like receptor (TLR) 4 ligands, and tumor necrosis factor (TNF) alpha. The production of CXCL2 in endothelial cells with LPS stimulation is dose-dependent. We found that Fli-1 binds to the CXCL2 promoter by Chromatin immunoprecipitation‎ (ChIP) assay. Transient transfection assays show that Fli-1 drives transcription from the CXCL2 promoter in a dose-dependent manner. These results indicate that Fli-1 is a novel, critical transcription factor in regulating the expression of the inflammatory chemokine CXCL2.

Abstract 61: Deficiency of the RNA Editing Enzyme ADAR2 Impairs Inflammation, Neovascularization and Functional Recovery of Ischemic Tissues

Background: Adenosine deaminase acting on RNA-2 (ADAR2) enzyme catalyzes adenosine-to-inosine (A-to-I) RNA editing of mRNAs and microRNAs and controls brain development. However, the role of endothelial cell ADAR2 in vascular biology and inflammation has not been described so far. Methods and Results: ADAR2 is expressed in human and murine endothelial cells and is 2-fold induced by hypoxia or hind limb ischemia in mice (P<0.05 for all). ADAR2 deficiency resulted in 73±12% impairment of leukocyte infiltration, in 53±4% reduced neovascularization, and a 40±6% decreased blood-flow recovery of ischemic muscle tissues in a hindlimb ischemia mouse model (P<0.001 for all). Mechanistically, among the highly ADAR2-regulated transcripts was interleukin-6 signal transducer (IL6ST or gp130), the receptor of interleukin-6 (IL-6). Silencing of ADAR2 resulted in a downregulation of gp130 mRNA and protein expression in endothelial cells by 65±5% and 50±5%, respectively (P<0.001 for both). Similarly, the expression of gp130 mRNA was decreased by 50±25% (P<0.05) in murine lung endothelial cells, which derived from transgenic ADAR2-null mice. In order to investigate the physiological relevance we treated endothelial cells, which were transfected with a siRNA against ADAR2 or scrambled control, with IL-6 and sIL6Ra. Silencing of ADAR2 reduced by at least 2-fold the IL-6-mediated STAT3 phosphorylation, the mRNA expression of the IL-6-induced downstream genes MCP-1, VCAM-1 and E-selectin, and the IL-6-induced platelet and leukocyte adhesion on endothelial cells and endothelial cell network formation (P<0.05 for all). Of interest, ADAR2 regulated gp130 mRNA stability. Co-silencing of ADAR2 and Drosha completely restored the ADAR2-regulated gp130 mRNA expression indicating that ADAR2 regulates the maturation of specific miRNAs. Indeed, ADAR2 regulates by at least 2-fold the expression of 20 microRNAs, including of miR-579 and miR-320e, which are predicted to bind to the 3’-untranslated region of IL6ST mRNA. Conclusions: Taken together, ADAR2 constitutes a potential candidate for therapeutic modulation of angiogenesis and inflammation in ischemic tissues.

Hypoxic trophoblast HMGB1 induces endothelial cell hyperpermeability via the TRL-4/caveolin-1 pathway.

High mobility group box 1 (HMGB1) plays an important role in the pathologic processes of endothelial permeability under oxidative stress. Trophoblast oxidative stress has been implicated in the pathophysiology of preeclampsia (PE). HMGB1 serum levels are increased in PE. However, the potential roles of HMGB1 in endothelial permeability in PE remain unclear. We assessed the effects of the hypoxic trophoblast on the permeability of the endothelial monolayer. Our results showed that the hypoxic trophoblast displayed higher HMGB1 mRNA, intracellular HMGB1 protein, and HMGB1 in conditioned medium than those of the normoxic trophoblast did. The hypoxic trophoblast conditioned medium increased the endothelial monolayer permeability and increased TLR 4 and caveolin-1 (CAV-1) protein expression in endothelial cells, which was inhibited by glycyrrhizic acid and HMGB1 small interfering RNA transfection to trophoblasts before hypoxia. The increased endothelial permeability induced by hypoxic trophoblast conditioned medium could be inhibited with TLR4 or CAV-1 gene silencing in endothelial cells. Immunoprecipitation showed that CAV-1 and TLR4 are colocalized in HUVECs and C57BL/6 mouse kidney. TLR4 small interfering RNA suppressed CAV-1 protein expression in endothelial cells upon stimulation of hypoxic trophoblast conditioned medium or HMGB1. We conclude that hypoxic trophoblasts play an important role in the mechanism of general edema (including protein urine) in PE via increasing endothelial monolayer permeability through the HMGB1/TLR4/CAV-1 pathway.

SIRT3 interactions with FOXO3 acetylation, phosphorylation and ubiquitinylation mediate endothelial cell responses to hypoxia.

The endothelial cells (ECs) that line the vascular lumen are exposed to a wide variety of environmental stresses, such as hypoxia. Maladaptation to stress in ECs is a key event in the development of cardiovascular disease. Sirtuin 3 (SIRT3) is an NAD+-dependent protein deacetylase that modulates various proteins to control mitochondrial function and metabolism. We found that hypoxia elicits an increase in SIRT3 mRNA and protein expression in ECs. Under the same hypoxic conditions, the forkhead box class O transcription factor FOXO3 is deacetylated by SIRT3. The SIRT3-mediated deacetylation of FOXO3 further reduces FOXO3 phosphorylation, ubiquitination and degradation, thereby stabilizing FOXO3 proteins. As a result, the level of FOXO3 protein is increased during hypoxia. Moreover, a set of FOXO3-dependent mitochondrial antioxidant enzymes, including manganese superoxide dismutase (MnSOD), peroxiredoxin 3 (Prx3), Prx5 and thioredoxin 2 (Trx2), are up-regulated in ECs to facilitate ROS detoxification in response to hypoxia. The SIRT3-mediated deacetylation of FOXO3 preserves mitochondrial bioenergetic function and increases cell survival under hypoxic conditions. These results indicate that SIRT3 stabilizes FOXO3 via deacetylation, which enhances the mitochondrial antioxidant defence system to increase the adaptive capacity of ECs during hypoxia. This finding provides a direction for ameliorating the development of cardiovascular diseases.

Assessment of vascular function in patients with chronic kidney disease.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The RNA binding protein hnRNP-K mediates post-transcriptional regulation of uncoupling protein-2 by angiopoietin-1

Angiopoietin-1 (Ang1) is a ligand for the receptor tyrosine kinase Tie2 and has key roles in the development of the vascular system and vascular protection. In a screen to define signalling pathways regulated by Ang1 in endothelial cells we found the RNA-binding protein hnRNP-K to be phosphorylated in response to Ang1. The ligand stimulated both tyrosine phosphorylation of hnRNP-K and recruitment of the tyrosine kinase Src to the RNA-binding protein. In endothelial cells hnRNP-K was found bound to mRNA encoding the mitochondrial protein uncoupling protein-2 (UCP2). Ang1 stimulation of cells resulted in the release of UCP2 mRNA from hnRNP-K. Using in vitro assays we confirmed direct binding between hnRNP-K and UCP2 mRNA. Furthermore Src induced phosphorylation of purified hnRNP-K and prevented UCP2 mRNA binding. Tyrosine 458 in the RNA-binding protein was found to be required for suppression of UCP2 mRNA binding by Src phosphorylation. In addition to releasing UCP2 mRNA from hnRNP-K, Ang1 induced an increase in UCP2 protein expression in endothelial cells without affecting total UCP2 mRNA levels. Consistent with the known effects of UCP2 to suppress generation of reactive oxygen species, Ang1 limited ROS production in endothelium stimulated with tumour necrosis factor-α. Taken together these data suggest that UCP2 mRNA is present in endothelial cells bound to hnRNP-K, which holds it in a translationally inactive state, and that Ang1 stimulates Src interaction with hnRNP-K, phosphorylation of the RNA-binding protein, release of these transcripts and upregulation of UCP2 protein expression. This study demonstrates a new mechanism for post-transcriptional regulation of UCP2 by the vascular protective ligand Ang1. The ability to rapidly upregulate UCP2 protein expression may be important in protecting endothelial cells from excessive generation of potentially damaging reactive oxygen species.

Gas6‐induced tissue factor expression in endothelial cells is mediated through caveolin‐1–enriched microdomains

Gas6 has been shown to interact with Axl in endothelial cells and to induce several signaling pathways involved in cell survival and proliferation. However, the interaction of Gas6/Axl with lipid raft/caveolin-1 in endothelial cells and its role in thrombosis are unknown. We tested whether Axl and/or caveolin-1 is involved in Gas6-induced Akt, ERK1/2, and c-Src activation leading to altered tissue factor expression in endothelial cells. Gas6-treated endothelial cells were transfected with small interfering RNA (siRNA) for Axl, caveolin-1, c-Src, and Akt or treated with pharmacological inhibitors of c-Src and ERK1/2. Sucrose gradient centrifugation and confocal microscopy were used to study lipid raft/caveolin-1-enriched fractions. Akt, ERK1/2, p38, and c-Src activation was analyzed by Western blot analysis. Tissue factor expression was assessed by real-time quantitative polymerase chain reaction and immunofluorescence. Gas6 induced Axl and c-Src localization into lipid raft/caveolin-1-enriched fractions. Gas6 increased the phosphorylation of Akt, ERK1/2, and c-Src but not p38. Using siRNA, we demonstrated that Axl is required for Akt, ERK1/2, and c-Src activation after Gas6 stimulation. siRNA for caveolin-1 blocked Gas6-induced phosphorylation of Akt, ERK1/2, and c-Src. c-Src downregulation inhibited Gas6-induced Akt but not ERK1/2 phosphorylation. Finally, Gas6 increased tissue factor mRNA and protein expression in endothelial cells. Tissue factor expression was blocked by siRNA for Axl, caveolin-1, or Akt as well as c-Src inhibition. These data demonstrate that the signaling pathway Gas6/Axl/caveolin-1/c-Src/Akt is required for tissue factor expression in endothelial cells, providing mechanistic insight into how Gas6 exerts its prothrombotic role in the vasculature.

Antioxidant and drug metabolism

The production of physiological amount of free radical is essential to fight against an unfavorable environment. The free radicals like nitric oxide (NO), superoxide anion, and related reactive oxygen species (ROS) play an important role as regulatory mediators in signaling processes. In higher organisms, NO and ROS regulates vascular tone, oxygen tension in control of ventilation and erythropoietin production.1 Free radicals activate a signal transduction from membrane receptors, for example ROS is essential for tissue factor protein expression in endothelial cells, TNF-γ-induced vascular cell adhesion protein 1 (VCAM-1) in vascular smooth muscle cells (VSMCs), tyrosine phosphorylation in fibroblasts and lymphocytes, etc. Similarly reactive nitrogen species (RNS) is important in norepinephrine-induced prostacycline release in hypothalamus, formations of synaptic connections in olfactory receptor neurons, N-methyl-D-aspartic acid (NMDA) receptor activity in cortical neurons, etc.2

Exercise training and vascular cell phenotype in a swine model of familial hypercholesterolaemia: conduit arteries and veins

What is the central question of this study? Does endurance exercise training cause anti-atherogenic effects on the endothelium in a swine model of familial hypercholesterolaemia (FH), and how are these effects distributed across veins, arteries and multiple vascular territories within each system? What is the main finding and its importance? Coronary artery endothelium-dependent vasomotor function was depressed in sedentary FH pigs compared with sedentary control animals, and exercise training did not change vasomotor function within FH. In systemic conduit arteries and veins, few effects of FH on endothelial cell protein expression were noted, including both pro- and anti-atherogenic changes. These findings suggest that exercise training does not produce a consistently improved endothelial cell phenotype in either coronary or systemic conduit vessels in this swine model of FH. Exercise training has emerged as an intervention for the primary and secondary prevention of coronary artery disease, but the mechanisms through which training reduces relative risk are not completely understood. The goal of this study was to investigate the impact of endurance exercise training on vasomotor function and vascular cell phenotype in coronary arteries and systemic conduit arteries and veins against a background of advanced atherosclerosis. We tested the hypothesis that exercise training restores endothelial vasomotor function and produces an anti-atherogenic endothelial and smooth muscle cell phenotype in familial hypercholesterolaemic (FH) swine. The study included 30 FH (15 exercised and 15 sedentary) and 13 non-FH control male castrated swine. The exercise-training intervention consisted of treadmill running 5 days per week for 16-20 weeks. Tissues sampled at sacrifice included vascular rings from the coronary circulation for vasomotor function experiments (dose-dependent bradykinin-induced vasorelaxation) and endothelial cells (ECs) from isolated segments of the thoracic aorta, the carotid, brachial, femoral and renal arteries, as well as each corresponding regionally associated vein, and from the abdominal vena cava, the right coronary and internal mammary arteries. Smooth muscle cells were sampled from the right coronary artery only. Vascular cell phenotype was assessed by immunoblotting for a host of both pro- and anti-atherogenic markers [e.g. endothelial nitric oxide synthase, p67phox, superoxide dismutase 1 (SOD1)]. Coronary artery endothelium-dependent vasomotor function was depressed in sedentary FH pigs compared with sedentary control pigs, and exercise training did not change vasomotor function within FH. In contrast, only scattered effects of FH on EC phenotype were noted across the vasculature, which included both pro- and anti-atherogenic changes in EC protein expression (e.g. increased endothelial nitric oxide synthase in carotid artery ECs, decreased p67phox in brachial artery ECs, but decreased expression of the antioxidant protein SOD1 in thoracic vena cava; all P < 0.05). In thoracic vena cava ECs, this deficit was corrected by exercise training, while no other effects of exercise were observed in conduit vessel EC phenotype. Thus, while exercise training abrogated the adverse effect of hypercholesterolaemia on thoracic vena cava SOD1 expression, it appears that exercise training does not produce a consistently improved EC phenotype in either coronary or systemic conduit vessels in this FH swine model.

Suppression of protein kinase C-ζ attenuates vascular leakage via prevention of tight junction protein decrease in diabetic retinopathy

To investigate the effect of protein kinase C (PKC)-ζ inhibition on vascular leakage in diabetic retinopathy, streptozotocin-induced diabetic mice were intravitreously injected with siPKC-ζ. According to the fluorescein angiography of the retinal vessels, suppression of PKC-ζ effectively attenuated vascular leakage in diabetic retina. Further evaluation on the retina with western blot analysis and immunohistochemistry revealed accompanying restoration of tight junction proteins on retinal vessels. As two major contributors to vascular leakage in diabetic retinopathy, vascular endothelial growth factor (VEGF) and advanced glycation end products (AGEs) were investigated on the tight junction protein expression in endothelial cells. Inhibition of PKC-ζ attenuated VEGF-induced decrease of tight junction proteins and accompanying hyperpermeability in human retinal microvascular endothelial cells (HRMECs). PKC-ζ inhibition also attenuated AGE-induced decrease of tight junction proteins in HRMECs. Our findings suggest that inhibition of PKC-ζ could be an alternative treatment option for compromised blood-retinal barrier in diabetic retinopathy.

Mapping the Extracellular and Membrane Proteome Associated with the Vasculature and the Stroma in the Embryo

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments.

Mineralocorticoid receptors modulate vascular endothelial function in human obesity

Obesity increases linearly with age and is associated with impaired vascular endothelial function and increased risk of cardiovascular disease. MRs (mineralocorticoid receptors) contribute to impaired vascular endothelial function in cardiovascular disease; however, their role in uncomplicated human obesity is unknown. Because plasma aldosterone levels are elevated in obesity and adipocytes may be a source of aldosterone, we hypothesized that MRs modulate vascular endothelial function in older adults in an adiposity-dependent manner. To test this hypothesis, we administered MR blockade (eplerenone; 100 mg/day) for 1 month in a balanced randomized double-blind placebo-controlled cross-over study to 22 older adults (ten men, 55-79 years) varying widely in adiposity [BMI (body mass index): 20-45 kg/m²], but who were free from overt cardiovascular disease. We evaluated vascular endothelial function [brachial artery FMD (flow-mediated dilation)] via ultrasonography) and oxidative stress (plasma F2-isoprostanes and vascular endothelial cell protein expression of nitrotyrosine and NADPH oxidase p47phox) during placebo and MR blockade. In the whole group, oxidative stress (P>0.05) and FMD did not change with MR blockade (6.39 ± 0.67 compared with 6.23 ± 0.73%; P=0.7). However, individual improvements in FMD in response to eplerenone were associated with higher total body fat (BMI: r=0.45, P=0.02; and dual-energy X-ray absorptiometry-derived percentage body fat: r=0.50, P=0.009) and abdominal fat (total: r=0.61, P=0.005; visceral: r=0.67, P=0.002; and subcutaneous: r=0.48, P=0.03). In addition, greater improvements in FMD with eplerenone were related to higher baseline fasting glucose (r=0.53, P=0.01). MRs influence vascular endothelial function in an adiposity-dependent manner in healthy older adults.

Crosstalk between hydrogen sulfide and nitric oxide in endothelial cells

Hydrogen sulfide (H2S) and nitric oxide (NO) are major gasotransmitters produced in endothelial cells (ECs), contributing to the regulation of vascular contractility and structural integrity. Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H2S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs-926) with NaHS (a H2S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H2S had little effect on eNOS protein expression in ECs. L-cysteine, a precursor of H2S, stimulated NO production whereas blockage of the activity of H2S-generating enzyme, cystathionine gamma-lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3-K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H2S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS-specific siRNA or nitro-L-arginine methyl ester (L-NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H2S on ECs. Our results suggest that H2S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt-dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H2S-induced angiogenesis.

Abstract 5080: Inhibiting the ALK1/BMP9 signaling pathway with dalantercept as an antiangiogenic therapy.

Abstract Activin receptor-like kinase 1 (ALK1) is an endothelial cell-specific receptor for bone morphogenetic proteins (BMP) 9 and 10, and is a key regulator of angiogenesis and vascular morphogenesis. Preclinical data suggest that the BMP9/ALK1 pathway is operating in the resolution and maturation stage of angiogenesis, which is distinct from the VEGF/VEGFR pathway which drives the initiation stage of angiogenesis. Dalantercept is an ALK1 extracellular domain-Fc fusion protein that inhibits the BMP9/ALK1 pathway. Thus, dalantercept may prove useful either as a monotherapy to inhibit tumor angiogenesis or in combination with VEGFR inhibitors to target different phases of vascular network formation and potentially to overcome acquired resistance to anti-VEGF therapy. To test this hypothesis, the A498 renal cell carcinoma xenograft model was used to test dalantercept efficacy either alone or in combination with sunitinib. In this model, tumor progression was defined as a 2 mm increase in tumor length. In the placebo group, mean (± SD) tumor progression was 5.8 ±0.6 days. Treatment with dalantercept delayed tumor progression to 10.8 ± 1.7 days (p&amp;lt;0.01 vs placebo) and sunitinib delayed tumor progression to 13.8 ± 2.5 days (p&amp;lt;0.01 vs placebo). The combination treatment of dalantercept plus sunitinib had a greater beneficial effect with a delay in tumor progression to 35 ± 12.6 days (p=0.01 vs sunitinib). We used immunohistochemistry (IHC) analysis to evaluate effects of dalantercept on tumor vasculature and to look for potential biomarkers of response to treatment in A498 tumors. Id1 is a transcription factor expressed in endothelial cells and is known to be regulated via the BMP9/ALK1/SMAD pathway. We observed a decrease in Id1 protein expression in endothelial cells of A498 xenografts treated with dalantercept, consistent with our previous experiments. Because BMP9 is one of the main target ligands of dalantercept, IHC analysis of archived human tumor samples was performed to determine BMP9 expression levels in various tumor types. In squamous cell carcinoma of the head and neck it was shown that out of 28 tumor samples 79% had high or medium levels of BMP9 staining, with little to no staining in normal tissues. Data from BMP9 expression in renal cell carcinoma and hepatocellular carcinoma will also be presented. In a Phase 1 study in patients with advanced, refractory solid tumors, dalantercept was generally well tolerated and exhibited signs of clinical activity including patients with objective response and prolonged stable disease. Dalantercept is currently being tested in several Phase 2 oncology studies, including a study of dalantercept in combination with the anti-VEGFR TKI axitinib in second-line advanced renal cell carcinoma. Citation Format: Marat Alimzhanov, Nicolas Solban, Michael Lee, Phaethon Philbrook, Xiaoen Wang, Lin Wei, Jiaxi Song, Sabina Signoretti, R. Scott Pearsall, Matthew L. Sherman, Ravindra Kumar, Rupal S. Bhatt. Inhibiting the ALK1/BMP9 signaling pathway with dalantercept as an antiangiogenic therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5080. doi:10.1158/1538-7445.AM2013-5080

Targeted Proteomics: Endothelial Cell Membrane Response to TNF‐α

Endothelial cells play a key role in regulating vascular function. One key role of the endothelium is to control the inflammatory response, limiting it to acute periods of defense. Produced by macrophages, one of the first cytokines produced in the inflammatory response is tumor necrosis factor alpha (TNFα). TNFα signaling to endothelial cells is essential for inducing the expression of proteins that recruit additional cells of the inflammatory response. TNFα signaling is complex, having two receptors that each signal through multiple pathways. In this study, we determine the effect of low dose TNFα on differential membrane protein expression in endothelial cells, identifying cell surface proteins that are upregulated, downregulated or uniquely expressed.We developed a novel proteomics technique, cell membrane associated enrichment (CMAE) for analyzing proteins contained on the outer cell surface and those closely associated with the cell surface. CMAE, targeting plasma membrane proteins with a surface amine group, combined with a more well‐established surface N‐glycoproteome enrichment (CSC) allowed increased coverage of the cell surface proteome. After CSC and CMAE results were combined allowing us to specifically target differentially regulated plasma membrane proteins in that were then validated by flow cytometry analysis of endothelial cells.

Acute mineralocorticoid receptor blockade leads to decreased vascular endothelial function in healthy older adults

Mineralocorticoid receptors (MR) modulate vascular endothelial function (VEF) in chronic heart failure and in healthy young adults, but their influence in healthy older adults is unknown. To test the hypothesis that MR modulate VEF in healthy aging, we administered acute MR blockade (MRB: 100 mg Eplerenone for 2 days) in a randomized, double‐blind, placebo‐controlled cross‐over study in 22 adults (8 men, 61±1 y; mean±SE, 53–79 y) free from clinical disease. We measured VEF (brachial flow‐mediated dilation [FMD]; ultrasonography), plasma oxidized LDL and vascular endothelial cell protein expression (VECPE) of eNOS, phosphorylated Ser1177 eNOS (PeNOS), NADPH oxidase (p47‐ phox), nitrotyrosine (NT), and TNF‐α (biopsy coupled with quantitative immunofluorescence) after MRB and placebo. In the overall group, FMD decreased (6.9±0.5 vs. 5.6±0.6%, P=0.02; Eplerenone vs. placebo) with MRB. However, MRB had no effect on oxidized LDL and VECPE of eNOS, PeNOS, p47‐phox, NT and TNF‐α (P≥0.4). Greater decreases in FMD were associated with lower baseline white blood cell and neutrophil (r=0.49, P=0.02 and r=0.57, P=0.006, respectively) and greater lymphocyte (r=−0.53, P=0.01) levels. In conclusion, MR modulate VEF in healthy older adults in an immune cell‐specific manner. MR activation appears to be physiologically important for maintaining vascular function.Supported by NIH Grant AG032067 &amp; AHA Grant 0865117F.

Vascular endothelial cell protein expression of adiponectin is related with vascular endothelial function in healthy older adults

Adiponectin (APN) is associated with enhanced vascular endothelial function in humans. Recently, there is some evidence that human renal glomerular endothelial cells express APN mRNA and protein but this has not been established in human vascular endothelial cells. We sought to 1) measure vascular endothelial cell protein expression (VECPE) of APN and circulating plasma APN; and 2) examine their relation to vascular endothelial function in healthy older adults. We measured vascular endothelial function (brachial artery flow‐mediated dilation [FMD] using ultrasonography), APN VECPE (biopsy coupled with quantitative immunofluorescence) and plasma APN (Mercodia, ELISA) in 23 adults (64±2 years; mean±SE, 55 – 79 years; 11 men and 12 women) free from clinical disease. APN VECPE was not related with plasma APN (r=−0.14, P=0.6). Greater APN VECPE was related with greater FMD (r=0.43, P=0.047), lower baseline (r=−0.45, P=0.03) and post‐occlusion shear stress (r=−0.46, P=0.03) and greater FMD/shear stress (r=0.56, P=0.007). Both plasma and APN VECPE were related with impaired renal function (r=0.44 to 0.62, P≤0.04). In conclusion, APN VECPE is associated with enhanced vascular endothelial function and is elevated with impaired renal function in older adults free from clinical disease.Supported by NIH Grant AG032067 &amp; AHA Grant 0865117F.