Changes In Endothelial Phenotype Research Articles

Signal Transduction and Gene Regulation in the Endothelium.

Extracellular signals act on G-protein-coupled receptors (GPCRs) to regulate homeostasis and adapt to stress. This involves rapid intracellular post-translational responses and long-lasting gene-expression changes that ultimately determine cellular phenotype and fate changes. The lipid mediator sphingosine 1-phosphate (S1P) and its receptors (S1PRs) are examples of well-studied GPCR signaling axis essential for vascular development, homeostasis, and diseases. The biochemical cascades involved in rapid S1P signaling are well understood. However, gene-expression regulation by S1PRs are less understood. In this review, we focus our attention to how S1PRs regulate nuclear chromatin changes and gene transcription to modulate vascular and lymphatic endothelial phenotypic changes during embryonic development and adult homeostasis. Because S1PR-targeted drugs approved for use in the treatment of autoimmune diseases cause substantial vascular-related adverse events, these findings are critical not only for general understanding of stimulus-evoked gene regulation in the vascular endothelium, but also for therapeutic development of drugs for autoimmune and perhaps vascular diseases.

Abstract 11024: Interferon-Gamma Impairs Human Coronary Artery Endothelial Glucose Metabolism via Tryptophan Catabolism and Activates Fatty Acid Oxidization

Introduction: Endothelial cells depend on glycolysis for much of energy production. Deranged endothelial glycolysis has been associated with various vascular pathobiologies. Interferon-gamma (IFN-γ) producing T-cells have been identified as the predominant pathologic cell subset in human atherosclerotic plaque. While the immunological consequences of these cells have been evaluated, their metabolic effects on endothelial cells remain unknown. The objective of this study was to determine the metabolic consequences of IFN-γ on human coronary artery endothelial cells (HCAEC). Hypothesis: We hypothesized that IFN-γ inhibits endothelial glucose metabolism and results in pathologic phenotypic changes in HCAEC. Methods: The metabolic effects of IFN-γ on primary HCAEC were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux and molecular mechanistic studies. Relevant cellular phenotype was assessed by measuring endothelial intracellular cGMP content and wound healing capacity. Results: IFN-γ inhibited glycolysis of HCAEC by 20 % ( p = 0.006) via global transcriptional suppression of glycolytic enzymes resulting from decreased basal nuclear hypoxia inducible factor 1α (HIF1α) availability. HIF1α destabilization (FC 0.72; p = 0.0039) was mediated by the sequestration of its binding partner HIF1β by aryl hydrocarbon receptor via a kynurenine surge (FC 6.24; p < 0.0001) resulting from IFN-γ-induced tryptophan degradation. Endothelial fatty acid oxidation (FAO) increased by 19% ( p = 0.014) with its inhibition leading to a 23% ATP deficit ( p < 0.0001). These metabolic derangements were associated with pathologic endothelial phenotypic changes, including decreased intracellular cGMP by 29 % ( p = 0.025) and impaired wound healing capacity by 38 % ( p = 0.0098). Conclusions: IFN-γ impairs endothelial glucose metabolism via tryptophan catabolism destabilizing HIF1α and results in a metabolic shift toward increased FAO. These findings suggest a novel mechanistic basis for pathologic T-lymphocyte-endothelial interaction in coronary artery disease mediated by IFN-γ, linking endothelial glucose, amino acid, and fatty acid metabolism and their adverse endothelial functional consequences.

Interferon-γ Impairs Human Coronary Artery Endothelial Glucose Metabolism by Tryptophan Catabolism and Activates Fatty Acid Oxidation.

Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. IFN-γ (interferon-γ)-producing CD4+ and CD8+ T lymphocytes have been identified as the predominant pathological cell subsets in human atherosclerotic plaques. Although the immunologic consequences of these cells have been extensively evaluated, their IFN-γ-mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN-γ, on human coronary artery endothelial cells. The metabolic effects of IFN-γ on primary human coronary artery endothelial cells were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cGMP content, wound-healing capacity, and adhesion molecule expression. IFN-γ exposure inhibited basal glycolysis of quiescent primary human coronary artery endothelial cells by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal HIF1α (hypoxia-inducible factor 1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN-γ-induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1β sequestration by the kynurenine-activated AHR (aryl hydrocarbon receptor). In addition, IFN-γ resulted in a 23% depletion of intracellular nicotinamide adenine dinucleotide in human coronary artery endothelial cells. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by >20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a proinflammatory state. IFN-γ impairs endothelial glucose metabolism by altered tryptophan catabolism destabilizing HIF1, depletes nicotinamide adenine dinucleotide, and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathological T lymphocyte-endothelial interactions in atherosclerosis mediated by IFN-γ, linking endothelial glucose, tryptophan, and fatty acid metabolism with the nicotinamide adenine dinucleotide balance and ATP generation and their adverse endothelial functional consequences.

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly.

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of the intact endothelium will provide valuable information for understanding the cellular and molecular events in the dysfunctional endothelial cells. Here, we describe a modified en face immunofluorescence staining technique which enables scientists to obtain clear images of the intact endothelial surface and analyze the molecule expression patterns in situ. The method is simple and reliable for observing the entire endothelial monolayer at different sites of the aorta. This technique may be a promising tool for understanding the pathophysiology of atherosclerosis, especially at an early stage.

Endothelial Cell Mechano-Metabolomic Coupling to Disease States in the Lung Microvasculature.

Lungs are the most vascular part of humans, accepting the totality of cardiac output in a volume much smaller than the body itself. Due to this cardiac output, the lung microvasculature is subject to mechanical forces including shear stress and cyclic stretch that vary with the cardiac and breathing cycle. Vessels are surrounded by extracellular matrix which dictates the stiffness which endothelial cells also sense and respond to. Shear stress, stiffness, and cyclic stretch are known to influence endothelial cell state. At high shear stress, endothelial cells exhibit cell quiescence marked by low inflammatory markers and high nitric oxide synthesis, whereas at low shear stress, endothelial cells are thought to “activate” into a pro-inflammatory state and have low nitric oxide. Shear stress' profound effect on vascular phenotype is most apparent in the arterial vasculature and in the pathophysiology of vascular inflammation. To conduct the flow of blood from the right heart, the lung microvasculature must be rigid yet compliant. It turns out that excessive substrate rigidity or stiffness is important in the development of pulmonary hypertension and chronic fibrosing lung diseases via excessive cell proliferation or the endothelial-mesenchymal transition. Recently, a new body of literature has evolved that couples mechanical sensing to endothelial phenotypic changes through metabolic signaling in clinically relevant contexts such as pulmonary hypertension, lung injury syndromes, as well as fibrosis, which is the focus of this review. Stretch, like flow, has profound effect on endothelial phenotype; metabolism studies due to stretch are in their infancy.

Vascular brain-derived neurotrophic factor pathway in rats with adjuvant-induced arthritis: Effect of anti-rheumatic drugs

Background and aimsIn rheumatoid arthritis, the control of both disease activity and standard cardiovascular (CV) risk factors is expected to attenuate the increased CV risk. Evidence that brain-derived neurotrophic factor (BDNF) plays a role in vascular biology led us to investigate the vascular BDNF pathway in arthritis rats as well as the interaction between endothelial nitric oxide (NO) and BDNF production. MethodsThe aortic BDNF pathway was studied in rats with adjuvant-induced arthritis, (AIA) using Western blot and immunohistochemical analysis. Control of arthritis score was achieved by administration (for 3 weeks) of an equipotent dosage of etanercept, prednisolone, methotrexate, celecoxib or diclofenac. Aortas were exposed to an NO donor or an NO synthase inhibitor and vasoreactivity experiments were performed using LM22A-4 as a TrkB agonist. ResultsVascular BDNF and full length tropomyosin-related kinase B receptor (TrkB-FL) were higher in AIA than in control rats. These changes coincided with decreased endothelial immunoreactivity in BDNF and pTrkBtyr816 and were disconnected from arthritis score. Among anti-rheumatic drugs, only prednisolone and methotrexate prevented AIA-induced vascular BDNF loss. The effect of AIA on aortic BDNF levels was reversed by an NO donor and reproduced by an NOS inhibitor. Finally, LM22A-4 induced both NO-dependent vasodilation and phosphorylation of endothelial NO synthase at serine 1177. ConclusionsOur study identified changes in the BDNF/TrkB pathway as a disease activity-independent component of AIA-associated changes in endothelial phenotype. It provides new perspectives in the understanding and management of the high CV risk reported in rheumatoid arthritis.

P-338 - NADPH oxidase regulates the expression of angiogenic growth factors in Cerebral Cavernous Malformation cellular models

Cerebral Cavernous Malformation (CCM) is a neurovascular disease characterized by abnormal vascular structures associated with defects on vascular lumen formation and vascular fragility, mostly localized in the brain. Lack of KRIT1 protein associated to CCM drives the stable and quiescent endothelium toward an active state characterized by loss of barrier function, increased vascular permeability, loss of adherens junction integrity, increased β-catenin nuclear localization and vascular endothelial growth factor (VEGF) expression and angiogenesis. Multiple pathways are responsible for changes in endothelial phenotype and we contributed to demonstrate that the dysregulation of reactive oxygen species (ROS) production following loss of KRIT1 expression play a key role in increasing vascular permeability and is linked to NADPH oxidase (NOX) up-regulation. By using wt and KRIT1-/- mouse embryonic fibroblasts (MEFs) we showed that loss of KRIT1 induced NOX4 overexpression that is associated with alteration of angiogenic growth factors expression and with the angiogenic switch of endothelial cells. Our data contribute to demonstrate the pivotal role of oxidative stress in the progression of CCM disease, indicating NOX4 and ROS as potential targets for pharmaceutical approaches.

Rapamycin activates TGF receptor independently of its ligand: implications for endothelial dysfunction.

Rapamycin, the macrolide immunosuppressant and active pharmaceutic in drug-eluting stents (DES), has a well-recognized antiproliferative action that involves inhibition of the mTOR pathway after binding to the cytosolic protein FKBP12. TGF receptor-type I (TGFRI) spontaneous activation is inhibited by the association with FKBP12. We hypothesized that rapamycin, in addition to inhibition of mTOR signaling, activates TGFRI independent of TGFβ. Human umbilical vein endothelial cells (HUVECs) were treated with rapamycin (10 nmol/l) and/or TGFβ RI kinase inhibitor (TGFRIi, 100 nmol/l) for 24 h. Rapamycin induced SMAD phosphorylation (SMAD1, SMAD2, and SMAD5) and PAI-1 up-regulation, which was specifically abrogated by SMAD2 knockdown. TGFRIi efficiently blocked phosphorylation of SMAD2, but not SMAD1/5. Interestingly, the inhibitor did not alter cell proliferation arrest induced by rapamycin. Active TGFβsecretion was not affected by the treatment. Neutralizing TGFβ experiments did not influence SMAD2 phosphorylation or PAI-1 expression indicating that activation of this pathway is independent of the ligand.In addition, rapamycin induction of endothelial-to-mesenchymal transition (EndMT) was potentiated by IL-1β and efficiently blocked by TGFRIi. In vivo, the prothrombogenic effects of rapamycin and up-regulation of PAI-1 in murine carotid arteries were reduced by TGFRIi treatment. In conclusion, we provide evidence that rapamycin activates TGF receptor independent of its ligand TGFβ, in concert with promotion of PAI-1 expression and changes in endothelial phenotype. These undesirable effects, the prothrombogenic state, and activation of EndMT are SMAD2-dependent and independent of the therapeutic rapamycin-induced cell proliferation arrest.

Angiotensin-(1-7) Inhibits Thrombin-Induced Endothelial Phenotypic Changes and Reactive Oxygen Species Production via NADPH Oxidase 5 Downregulation.

Background and Aims: The angiotensin-(1-7)/angiotensin-converting enzyme 2/Mas receptor axis counter-regulates the detrimental effects of angiotensin II. Beneficial effects of angiotensin-(1-7), including anti-inflammation, oxidative stress reduction, and anti-thrombosis, have been reported. Previous studies documented that ramipril decreased thrombin generation in human hypertension and that the anti-thrombotic effects of captopril and losartan were angiotensin-(1-7)-dependent, suggesting an interaction between thrombin and angiotensin-(1-7). However, it is not clear whether angiotensin-(1-7) can alleviate the endothelial phenotypic changes induced by thrombin. We have previously documented cytoskeleton remodeling, cell adhesion, and cell migration as dominant altered phenotypes in thrombin-stimulated human aortic endothelial cells (HAECs). In this study, we investigated whether angiotensin-(1-7) can modulate thrombin-induced phenotypic changes. Furthermore, we investigated whether NAPDH oxidase 5 (Nox5)-produced reactive oxygen species (ROS) play a significant role in angiotensin-(1-7)-mediated phenotypic changes.Methods: HAECs were pretreated with 100 nM angiotensin-(1-7) for 1 h, followed by stimulation with 2 units/mL thrombin for different times. Immunofluorescent assay, monocyte adhesion assay, wound-healing assay, ROS assay, real-time PCR, Western blotting, and Nox5 siRNA transfection were conducted. HAECs were pretreated with the ROS scavenger N-acetylcysteine (NAC) to determine whether thrombin-induced phenotypic changes depended on ROS production.Results: Angiotensin-(1-7) prevented thrombin-induced actin cytoskeleton derangements, monocyte adhesion, and migratory impairment. Nox5 siRNA transfection confirmed that thrombin-induced Nox5 expression stimulated ROS production and increased HO-1/NQO-1/ICAM-1/VCAM-1 gene expression, all of which were decreased by angiotensin-(1-7). Phenotypic changes induced by thrombin were prevented by NAC pretreatment.Conclusion: Angiotensin-(1-7) prevents actin cytoskeleton derangement, monocyte adhesion, and migration impairment induced by thrombin via downregulation of ROS production. In addition, thrombin-induced Nox5 expression is involved in the production of ROS, and angiotensin-(1-7) decreases ROS through its inhibitory effect on Nox5 expression.

Endothelial to mesenchymal transformation is induced by altered extracellular matrix in aortic valve endothelial cells

Alterations in shear stress, mechanical deformation, extracellular matrix (ECM) composition and exposure to inflammatory conditions are known to cause endothelial to mesenchymal transformation (EndMT). This change in endothelial phenotype has only recently been linked to adult pathologies such as cancer progression, organ fibrosis, and calcific aortic valve disease; and its function in adult physiology, especially in response to tissue mechanics, has not been rigorously investigated. EndMT is a response to mechanical and biochemical signals that results in the remodeling of underlying tissues. In diseased aortic valves, glycosaminoglycans (GAGs) are present in the collagen-rich valve fibrosa, and are deposited near calcified nodules. In this study, in vitro models of early and late-stage valve disease were developed by incorporating the GAGs chondroitin sulfate (CS), hyaluronic acid, and dermatan sulfate into 3D collagen hydrogels with or without exposure to TGF-β1 to simulate EndMT in response to microenvironmental changes. High levels of CS induced the highest rate of EndMT and led to the most collagen I and GAG production by mesenchymally transformed cells, which indicates a cell phenotype most likely to promote fibrotic disease. Mesenchymal transformation due to altered ECM was found to depend on cell-ECM bond strength and extracellular signal-regulated protein kinases 1/2 signaling. Determining the environmental conditions that induce and promote EndMT, and the subsequent behavior of mesenchymally transformed cells, will advance understanding on the role of endothelial cells in tissue regeneration or disease progression. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 105A: 2729-2741, 2017.

Abstract 80: A Three-Dimensional Tissue Engineered Platform to Analyze the Effect of Various Microvascular Shear Stress Conditions

PURPOSE: Open deep tissue wounds commonly result from severe burns, trauma, chronic diseases, irradiation, and infection. Autologous tissue transfer remains the gold standard for surgical reconstruction, but unfortunately can come with significant complications. The development of an on-demand, geometrically tunable tissue engineered substitute with an inherent vascular supply would transform reconstructive surgical practice, but regeneration of thicker or larger tissues of clinically relevant size remains a challenge due to poor oxygen diffusion into cells within these constructs. Hemodynamic shear stress alters cellular morphology and biological activity, especially luminal endothelial cells within blood vessels. However, these mechanisms have only been studied in isolation in vitro, and work within complex vascular networks has remained elusive. We have thus fabricated a unique three-dimensional vascular anatomy regime with various geometric angles to study the effects of differential shear stress on endothelial cell behavior. METHODS: A multi-hairpin channel geometry with a diameter of 1.5 mm was designed in SolidWorks and created using a 3D-printer. Flow parameters were calculated using fluid simulations performed by Ansys Fluent to determine expected shear stresses throughout the channel. A Pluronic-F127 multi-hairpin channel was sacrificed in type-I collagen creating a central microchannel with encapsulated human placental pericytes in the collagen bulk. Subsequently, human aortic smooth muscle cells were intraluminally seeded into the macrochannel, followed by human umbilical vein endothelial cells 24 hours later. Constructs were statically cultured for 72 hours followed by dynamic perfusion at 10 dynes/cm2 for 10 days. After 14 days of culture, constructs were analyzed for changes in endothelial phenotype. Images were analyzed in ImageJ (NIH). RESULTS: Fluid simulation verified the presence of different shear stresses at various regions in the channel. Acute angles provided regions of higher shear stress, and the plenum facilitated higher vorticity. The geometry allowed for multiple shear regimes within the construct for high throughput analysis. MicroCT analysis revealed the development of appropriately shaped microchannels that recapitulated the 3D printed geometry. Hemotoxylin and Eosin staining revealed the adherence of vascular cells along the microchannel. Regions of interest were stained with DAPI and confocal microscopy was used to take scans of the whole section. Images were converted to binary and the distribution of bulk cells around the vascularized microchannel was determined. Distribution of bulk cells was found to primarily localize around the microchannel, with 39% of cells being within 500μm of the microchannel. Although bulk cells were originally randomly mixed into a homogenous distribution, the localization of bulk cells suggested that the presence of shear stress promoted bulk cellular migration. CONCLUSION: Using our novel platform, we have fabricated an anatomically appropriate vascularized model with different geometric angles to study flow dynamics and the effects of differential shear stress on recruitment of bulk cells to stabilize the neovessel. This physiological model overcomes the limitations of previous 2D and 3D flow studies with the potential to recapitulate in-vivo cell organization. We have integrated the capabilities of 3D printing and tissue engineering to develop printable biologically derived, cell-friendly hydrogels.

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

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

Pathogenesis of Retinal Vein Occlusion

Retinal vein occlusion (RVO) has multiple pathogenetic mechanism that including local anatomical susceptibility, degenerative changes of the vessel wall and abnormal hematological abnormalities. All of these mechanisms could contribute to develop vascular obstruction. Anatomic features of arteriovenous crossings and common adventitial sheath shared by artery and vein could cause turbulent flow. Turbulence in vessel flow promotes endothelial phenotypic changes that cause endothelial f-actin expression increase. Intercellular adhesions and endothelial functions are affected by high f-actin levels. Damaged arteries and hypoxic tissues express endothelin-1 (ET-1). Endothelin-1 can diffuse from the neighboring veins and stimulate venous vasoconstriction. Vasoconstiriction of veins lead to an increase in pressure. High blood viscosity is another important factor in pathogenesis of RVO. Pathogenesis of RVO is very complex and these factors can lead to a vicious cycle of worsening the other factor.

Extracellular Vesicles Derived from HT-29 Colorectal Cancer Cells Induce Endothelial Cell Activation and Procoagulant Activity

Introduction: Hypercoagulability leading to thromboembolic complications is associated with significant morbidity and mortality in patients with colorectal cancer, however the underlying mechanisms are not well understood. Elevated levels of extracellular vesicles (EV), both microparticles (> 100 nm) and exosomes (< 100 nm), circulate in patients with colorectal cancer. While some studies suggest that EV-expressed tissue factor is associated with thrombosis in patients with cancer, overall results are inconsistent. However, EV contain many other mediators such as inflammatory cytokines and microRNAs (miRNA) that may be transferred to other cells and induce phenotypic changes. We hypothesized that EV derived from colorectal cancer cells in vitro would induce endothelial cell activation and promote endothelial cell procoagulant activity.Methods: Human colorectal adenocarcinoma cells (HT-29) were cultured at a density of 1.0×107 cells per T75 flask in EV-free media containing 2% FBS, and after removal of floating cells and cell debris, microparticles were collected by centrifugation at 20,000 x G for 15 minutes; each flask contained approximately 4.0 x 106 annexin V+ microparticles. Exosomes were collected by additional centrifugation of the supernatant at 100,000 x G for 90 minutes. Microparticles and exosomes, at different dilutions, were separately co-cultured with confluent endothelial cell monolayers in 6 or 96-well plates, for 6 hours, using TNF-α (4 ng/ml) as a positive control. Endothelial cell activation was assessed by measuring expression of E-selectin, vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), Il-1β and tissue factor by quantitative RT-PCR (qPCR) and immunoblotting. We also analyzed the effects of microparticles and exosomes on endothelial cell procoagulant activity by layering plasma over confluent endothelial cell monolayers. Briefly, after incubating endothelial cell monolayers with microparticles or exosomes (or 4 ng/ml TNFα as a positive control) for 6 hours, monolayers were washed three times with 20mM HEPES buffer (pH 7.4) containing 5mM calcium chloride and then overlaid with 100 µl per well of pooled normal human plasma. Plasma was re-calcified by the addition of 11 ul 200 mM calcium chloride, and plates were then placed in a kinetic microtiter-plate reader (Synergy HT) maintained at 37°C, and fibrin formation measured as an increase in the optical density at 405 nm.Results: Microparticles and exosomes constitutively released by HT-29 cells induced activation of endothelial cells in a concentration-dependent manner. Significant increases in the expression of E-selectin, ICAM-1, VCAM-1, tissue factor and IL-1β mRNA and protein were observed (Figure 1). In general, microparticles were more potent in inducing endothelial cell activation than exosomes when the EV were derived from the same number of HT-29 cells, though quantification of small exosomes is imprecise. Interestingly, while the response of endothelial cells to microparticles and exosomes was generally similar to that of 4 ng/ml TNFα, both types of EV appeared more potent in the induction of endothelial cell tissue factor, particularly at the mRNA level. HT-29 cell-derived EV, both microparticles and exosomes, also significantly shortened the clotting time of recalcified plasma added to endothelial cell monolayers (Figure 2).Conclusions: EV constitutively released from HT-29 cells, both microparticles and exosomes, directly activate cultured endothelial cells leading to increased expression of cell adhesion molecules, elaboration of IL-1β, and expression of tissue factor. The change in endothelial phenotype is not due to simple transfer of biomolecules since EV stimulated changes at the mRNA and protein level. While responses to microparticles and exosomes were similar, some differences were observed, suggesting that different underlying mechanisms may contribute. Endothelial cell activation in response to EV leads to accelerated clotting of plasma on the endothelial cell monolayer. These findings suggest a novel effect of cancer cell-derived EV that may contribute to the hypercoagulability present in patients with malignancy. [Display omitted] [Display omitted] DisclosuresKhorana:Daiichi Sankyo: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Leo Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; sanofi: Consultancy, Honoraria. McCrae:Momenta: Consultancy; Halozyme: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Syntimmune: Consultancy.

Blast Wave Exposure to the Extremities Causes Endothelial Activation and Damage.

ABSTRACTExtremity injury is a significant burden to those injured in explosive incidents and local ischaemia can result in poor functionality in salvaged limbs. This study examined whether blast injury to a limb resulted in a change in endothelial phenotype leading to changes to the surrounding tissue.The hind limbs of terminally anaesthetized rabbits were subjected to one of four blast exposures (high, medium, low, or no blast). Blood samples were analyzed for circulating endothelial cells pre-injury and at 1, 6, and 11 h postinjury as well as analysis for endothelial activation pre-injury and at 1, 6, and 12 h postinjury. Post-mortem tissue (12 h post-injury) was analysed for both protein and mRNA expression and also for histopathology. The high blast group had significantly elevated levels of circulating endothelial cells 6 h postinjury. This group also had significantly elevated tissue mRNA expression of IL-6, E-selectin, TNF-α, HIF-1, thrombomodulin, and PDGF. There was a significant correlation between blast dose and the degree of tissue pathology (hemorrhage, neutrophil infiltrate, and oedema) with the worst scores in the high blast group. This study has demonstrated that blast injury can activate the endothelium and in some cases cause damage that in turn leads to pathological changes in the surrounding tissue. For the casualty injured by an explosion the damaging effects of hemorrhage and shock could be exacerbated by blast injury and vice versa so that even low levels of blast become damaging, all of which could affect tissue functionality and long-term outcomes.

MRI-based assessment of endothelial function in mice in vivo.

While a healthy endothelium serves to maintain vascular haemostasis, a malfunctioning endothelium leads to various cardiovascular diseases, including atherothrombosis. Endothelial dysfunction is characterized by increased vascular permeability, impaired endothelium-dependent responses and various pro-inflammatory and pro-thrombotic changes in endothelial phenotype, all of which could provide the basis for an in vivo diagnosis of endothelial dysfunction. In the present review, we briefly summarize the magnetic resonance imaging (MRI)-based methods available for assessing endothelial function in animal models, especially in mice. These methods are aimed to assess biochemical phenotype using molecular imaging, endothelium-dependent responses or changes in endothelial permeability. All these approaches provide a complementary insight into the endothelial dysfunction in vivo and may offer a unique opportunity to study endothelium-based mechanisms of diseases and endothelial response to treatment.

Rapamycin Activates TGF Receptor Independently of Its Ligands: Implications for Endothelial Dysfunction

Rapamycin, the active drug in drug‐eluting stents, has a well‐recognized antiproliferative action: it binds to FKBP12 and the rapamycin‐FKBP complex inhibits the mTOR pathway inducing cell cycle arrest. FKBP12 is bounded to TGF receptor‐type I protecting its spontaneous activation. We hypothesize that rapamycin can displace FKBP12 from TGFRI promoting its activation. Human umbilical vein endothelial cells (HUVEC) were treated with rapamycin (10nmoL/L) and/or TGF‐β RI kinase inhibitor (TGFRIi, 100nmoL/L) for 24 hours. Rapamycin induced Smads phosphorylation (Smad1, Smad2 and Smad5) and PAI‐1 up‐regulation, which was specifically abrogated by Smad2 knockdown. TGFRIi efficiently blocked phosphorylation of Smad2, but not Smad1/5. TGF‐β secretion was not influenced by rapamycin indicating that Smad activation is independent of the ligand. Interestingly, rapamycin induced endothelial‐to‐mesenchymal transition was potentiated by IL‐1β, and efficiently blocked by TGFRIi. In vivo, the pro‐thrombogenic effects of rapamycin decreased by TGFRIi treatment and rapamycin‐induced PAI‐1 expression in carotid arteries were decreased by TGFRIi. Altogether, we provide evidence that rapamycin activates TGF receptor independent of its ligand TGF‐β, promotes PAI‐1 expression and induces changes in endothelial phenotype. These undesirable effects, the prothrombogenic state and activation of EndMT, are Smad 2‐dependent and independent of the therapeutic rapamycin‐induced cell proliferation arrest.

Molecular Imaging Reveals Rapid Reduction of Endothelial Activation in Early Atherosclerosis With Apocynin Independent of Antioxidative Properties

Antioxidative drugs continue to be developed for the treatment of atherosclerosis. Apocynin is an nicotinamide adenine dinucleotide phosphate oxidase inhibitor with anti-inflammatory properties. We used contrast-enhanced ultrasound molecular imaging to assess whether short-term apocynin therapy in atherosclerosis reduces vascular oxidative stress and endothelial activation Genetically modified mice with early atherosclerosis were studied at baseline and after 7 days of therapy with apocynin (4 mg/kg per day IP) or saline. Contrast-enhanced ultrasound molecular imaging of the aorta was performed with microbubbles targeted to vascular cell adhesion molecule 1 (VCAM-1; MB(V)), to platelet glycoprotein Ibα (MB(Pl)), and control microbubbles (MB(Ctr)). Aortic vascular cell adhesion molecule 1 was measured using Western blot. Aortic reactive oxygen species generation was measured using a lucigenin assay. Hydroethidine oxidation was used to assess aortic superoxide generation. Baseline signal for MBV (1.3 ± 0.3 AU) and MB(Pl )(1.5 ± 0.5 AU) was higher than for MBCtr (0.5 ± 0.2 AU; P<0.01). In saline-treated animals, signal did not significantly change for any microbubble agent, whereas short-term apocynin significantly (P<0.05) reduced vascular cell adhesion molecule 1 and platelet signal (MBV: 0.3 ± 0.1; MBPl: 0.4 ± 0.1; MBCtr: 0.3 ± 0.2 AU; P=0.6 between agents). Apocynin reduced aortic vascular cell adhesion molecule 1 expression by 50% (P<0.05). However, apocynin therapy did not reduce reactive oxygen species content, superoxide generation, or macrophage content. Short-term treatment with apocynin in atherosclerosis reduces endothelial cell adhesion molecule expression. This change in endothelial phenotype can be detected by molecular imaging before any measurable decrease in macrophage content and is not associated with a detectable change in oxidative burden.

In vivo knockdown of intersectin-1s alters endothelial cell phenotype and causes microvascular remodeling in the mouse lungs

Intersectin-1s (ITSN-1s) is a general endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, via MEK/Erk1/2MAPK signaling. To investigate the in vivo effects of ITSN-1s deficiency and the resulting ECs apoptosis on pulmonary vasculature and lung homeostasis, we used an ITSN-1s knocked-down (KDITSN) mouse generated by repeated delivery of a specific siRNA targeting ITSN-1 gene (siRNAITSN). Biochemical and histological analyses as well as electron microscopy (EM) revealed that acute KDITSN [3-days (3d) post-siRNAITSN treatment] inhibited Erk1/2MAPK pro-survival signaling, causing significant ECs apoptosis and lung injury; at 10d of KDITSN, caspase-3 activation was at peak, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive ECs showed 3.4-fold increase, the mean linear intercept (MLI) showed 48 % augment and pulmonary microvessel density as revealed by aquaporin-1 staining (AQP-1) decreased by 30 %, all compared to controls; pulmonary function was altered. Concomitantly, expression of several growth factors known to activate Erk1/2MAPK and suppress Bad pro-apoptotic activity increased. KDITSN altered Smads activity, downstream of the transforming growth factor beta-receptor-1 (TβR1), as shown by subcellular fractionation and immunoblot analyses. Moreover, 24d post-siRNAITSN, surviving ECs became hyper-proliferative and apoptotic-resistant against ITSN-1s deficiency, as demonstrated by EM imaging, 5-bromo-deoxyuridine (BrdU) incorporation and Bad-Ser112/155 phosphorylation, respectively, leading to increased microvessel density and repair of the injured lungs, as well as matrix deposition. In sum, ECs endocytic dysfunction and apoptotic death caused by KDITSN contribute to the initial lung injury and microvascular loss, followed by endothelial phenotypic changes and microvascular remodeling in the remaining murine pulmonary microvascular bed.Electronic supplementary materialThe online version of this article (doi:10.1007/s10495-012-0762-x) contains supplementary material, which is available to authorized users.

The CXCR4/CXCR7/SDF-1 pathway contributes to the pathogenesis of Shiga toxin–associated hemolytic uremic syndrome in humans and mice

Hemolytic uremic syndrome (HUS) is a potentially life-threatening condition. It often occurs after gastrointestinal infection with E. coli O157:H7, which produces Shiga toxins (Stx) that cause hemolytic anemia, thrombocytopenia, and renal injury. Stx-mediated changes in endothelial phenotype have been linked to the pathogenesis of HUS. Here we report our studies investigating Stx-induced changes in gene expression and their contribution to the pathogenesis of HUS. Stx function by inactivating host ribosomes but can also alter gene expression at concentrations that minimally affect global protein synthesis. Gene expression profiling of human microvascular endothelium treated with Stx implicated a role for activation of CXCR4 and CXCR7 by their shared cognate chemokine ligand (stromal cell-derived factor-1 [SDF-1]) in Stx-mediated pathophysiology. The changes in gene expression required a catalytically active Stx A subunit and were mediated by enhanced transcription and mRNA stability. Stx also enhanced the association of CXCR4, CXCR7, and SDF1 mRNAs with ribosomes. In a mouse model of Stx-mediated pathology, we noted changes in plasma and tissue content of CXCR4, CXCR7, and SDF-1 after Stx exposure. Furthermore, inhibition of the CXCR4/SDF-1 interaction decreased endothelial activation and organ injury and improved animal survival. Finally, in children infected with E. coli O157:H7, plasma SDF-1 levels were elevated in individuals who progressed to HUS. Collectively, these data implicate the CXCR4/CXCR7/SDF-1 pathway in Stx-mediated pathogenesis and suggest novel therapeutic strategies for prevention and/or treatment of complications associated with E. coli O157:H7 infection.