Intact Blood Vessels Research Articles

Abstract P3008: Beta Adrenergic Receptor Activation Causes Endothelial Glycocalyx Degradation

The endothelial glycocalyx is a proteoglycan and glycoprotein matrix that coats the luminal surface of endothelial cells. It is an important component of the permeability barrier function of endothelial cells, including in the glomerular capillaries. Furthermore, it is a sensor of shear stress, and in vessels treated with enzymes to remove the glycocalyx do not exhibit flow-mediated vasodilation. Sparse previous studies have implicated a reduced glycocalyx thickness in hypertensive patients, but the causative signaling mechanisms have not been investigated. We and others have found that the endothelial glycocalyx can be studied in cultured endothelial cells if the medium contains significant soluble protein. We hypothesized that elevated catecholamines cause shedding of the endothelial glycocalyx in vitro . We cultured human umbilical vein endothelial cells (HUVECs) in M200 medium supplemented with 1% bovine serum albumin to permit development of a glycocalyx. We exposed these cells to concentrations of epinephrine, norepinephrine, phenylephrine, or isoproterenol from 0.01 to 10 μM for 30 minutes. We then imaged the glycocalyx using fluorescently labelled wheat germ agglutinin. We found that norepinephrine, epinephrine, and isoproterenol all caused a significant decrease in the endothelial glycocalyx staining intensity (5.82 ± 0.28 vs. 4.82 ± 0.21, 6.82 ± 0.28 vs. 5.09 ± 0.25, 7.46 ± 0.33 vs. 6.51 ± 0.28, at 1 μM, 1 μM, and 0.1 μM, respectively). However, phenylephrine caused an increase in glycocalyx staining intensity (6.27 ± 0.25 vs. 7.72 ± 0.32 at 1 μM). These results suggest that catecholamines acutely cause degradation of the glycocalyx in endothelial cells, but only when β adrenergic receptors are involved, since phenylephrine actually acted to increase glycocalyx. Future work will be needed to investigate the pathophysiological role of the reduced glycocalyx in hypertension. Furthermore, in vivo experiments are needed to determine whether these results are applicable to endothelial cells in the complex environment of intact blood vessels. Future work will also investigate the signaling mechanisms leading from adrenergic receptor activation to enzymatic degradation of the glycocalyx.

Transfusion of Resting Platelets Reduces Brain Hemorrhage After Intracerebral Hemorrhage and tPA-Induced Hemorrhage After Cerebral Ischemia.

BackgroundExacerbated blood-brain barrier (BBB) damage is related with tissue plasminogen activator (tPA)-induced brain hemorrhage after stroke. Platelets have long been recognized as the cellular orchestrators of primary haemostasis. Recent studies have demonstrated further that platelets are required for supporting intact mature blood vessels and play a crucial role in maintaining vascular integrity during inflammation. Therefore, we sought to investigate whether platelets could reduce tPA-induced deterioration of cerebrovascular integrity and lead to less hemorrhagic transformation.MethodsMice were subjected to models of collagenase-induced intracerebral hemorrhage (ICH) and transient middle cerebral artery (MCA) occlusion. After 2 h of MCA occlusion, tPA (10 mg/kg) was administered as an intravenous bolus injection of 1 mg/kg followed by a 9 mg/kg infusion for 30 min. Immediately after tPA treatment, mice were transfused with platelets. Hemorrhagic volume, infarct size, neurological deficit, tight junction and basal membrane damages, endothelial cell apoptosis, and extravascular accumulation of circulating dextran and IgG, and Evans blue were quantified at 24 h.ResultsPlatelet transfusion resulted in a significant decrease in hematoma volume after ICH. In mice after ischemia, tPA administration increased brain hemorrhage transformation and this was reversed by resting but not activated platelets. Consistent with this, we observed that tPA-induced brain hemorrhage was dramatically exacerbated in thrombocytopenic mice. Transfusion of resting platelets ameliorated tPA-induced loss of cerebrovascular integrity and reduced extravascular accumulation of circulating serum proteins and Evans blue, associated with improved neurological functions after ischemia. No changes were found for infarct volume. Inhibition of platelet receptor glycoprotein VI (GPVI) blunted the ability of platelets to attenuate tPA-induced BBB disruption and hemorrhage after ischemia.ConclusionOur findings demonstrate the importance of platelets in safeguarding BBB integrity and suggest that transfusion of resting platelets may be useful to improve the safety of tPA thrombolysis in ischemic stroke.

Depolarization of mitochondrial membrane by H2O2 regulates Ca2+ signalling in Endothelial Cells in Intact Blood Vessels

Hydrogen peroxide (H2O2) is a key mitochondrial derived reactive oxygen species (ROS) that acts as a signalling hub in regulating signalling transduction, vasocontraction and vasodilation. Although the physiological roles of ROS in mechano‐stress signal transduction, vascular relaxation and permeability of endothelial cells is acknowledged, there is uncertainty on the mechanisms by which how H2O2 regulates endothelial signalling. In the present study, the effects of H2O2 on Ca2+ signalling and on vasocontraction and vasodilation were investigated in the endothelium of intact rat mesenteric arteries. Endothelium‐dependent vasodilation of intact mesenteric arteries to acetylcholine (ACh) was suppressed by H2O2 (100 μM) and a greater vasoconstriction to phenylephrine occurred. To investigate the underlying endothelium‐dependent mechanisms of H2O2 on vascular response, intracellular Ca2+ signalling in endothelial cells of intact arteries was visualized and recorded with high‐speed and spatial‐resolution. H2O2 (100 nM – 1 mM) alone did not significantly alter basal Ca2+ activity in endothelial cells. However, H2O2 inhibited acetylcholine‐evoked Ca2+ signalling as revealed by decrease in the amplitude of the Ca2+ signal in each activated cell and a reduced number of cells activated. H2O2‐evoked inhibition of Ca2+ signalling was reversed by catalase (1000 U/ml). To determine if the effects of H2O2 were explained by decreased IP3 production or decreased IP3‐evoked Ca2+ release, photoactivatable caged‐inositol 1,4,5‐trisphosphate (cIP3) was used to bypass the synthesis of IP3. Ca2+ release evoked by photolysis of cIP3 supressed by 100 μM H2O2. These results suggest that H2O2 may alter the activity of IP3 receptors. One mechanism by which this may occur is by H2O2 altering mitochondrial activity. Mitochondria exert a profound control of IP3‐evoked Ca2+ release. Acetylchonline‐evoked Ca2+ release was inhibited by proton uncoupler carbonyl cyanide 3‐chlorophenylhydrazone (CCCP) and rotenone, a mitochondrial complex inhibitor. H2O2 (100 μM) depolarized mitochondria membrane potential as measured by the mitochondrial membrane potential sensitive dye, TMRE. The depolarization of mitochondria decreased the organelle's ability to produce ATP and sequester Ca2+ from the local microdomain near IP3 receptors. A higher local concentration of Ca2+ around IP3 receptors inhibited Ca2+ release from endoplasmic reticulum. In summary, H2O2 inhibit Ca2+ release from the internal store in endothelial cells by depolarizing mitochondrial membrane potential to suppress vasodilation in intact mesenteric arteries.Support or Funding InformationWellcome Trust and British Heart FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extracellular histones induce calcium signals in the endothelium of resistance-sized mesenteric arteries and cause loss of endothelium-dependent dilation.

Histone proteins are elevated in the circulation after traumatic injury owing to cellular lysis and release from neutrophils. Elevated circulating histones in trauma contribute to coagulopathy and mortality through a mechanism suspected to involve endothelial cell (EC) dysfunction. However, the functional consequences of histone exposure on intact blood vessels are unknown. Here, we sought to understand the effects of clinically relevant concentrations of histones on the endothelium in intact, resistance-sized, mesenteric arteries (MAs). EC Ca2+ was measured with high spatial and temporal resolution in MAs from mice selectively expressing the EC-specific, genetically encoded ratiometric Ca2+ indicator, Cx40-GCaMP-GR, and vessel diameter was measured by edge detection. Application of purified histone protein directly to the endothelium of en face mouse and human MA preparations produced large Ca2+ signals that spread within and between ECs. Surprisingly, luminal application of histones had no effect on the diameter of pressurized arteries. Instead, after prolonged exposure (30 min), it reduced dilations to endothelium-dependent vasodilators and ultimately caused death of ~25% of ECs, as evidenced by markedly elevated cytosolic Ca2+ levels (793 ± 75 nM) and uptake of propidium iodide. Removal of extracellular Ca2+ but not depletion of intracellular Ca2+ stores prevented histone-induced Ca2+ signals. Histone-induced signals were not suppressed by transient receptor potential vanilloid 4 (TRPV4) channel inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels or Toll-like receptor receptors. These data demonstrate that histones are robust activators of noncanonical EC Ca2+ signaling, which cause vascular dysfunction through loss of endothelium-dependent dilation in resistance-sized MAs. NEW & NOTEWORTHY We describe the first use of the endothelial cell (EC)-specific, ratiometric, genetically encoded Ca2+ indicator, Cx40-GCaMP-GR, to study the effect of histone proteins on EC Ca2+ signaling. We found that histones induce an influx of Ca2+ in ECs that does not cause vasodilation but instead causes Ca2+ overload, EC death, and vascular dysfunction in the form of lost endothelium-dependent dilation.

Vrozené a získané krvácivé stavy

Hemostasis can be characterized as an array of physiological mechanisms providing both blood fluidity in the intact blood vessels and hemostasis in the event of impaired continuity of the blood vessel wall. The impaired hemo-static balance may lead on the one hand to an increased tendency to bleed, either spontaneously or only in response to an external stimulus. At the opposite end of bleeding are thrombophilic conditions characterized by an increased tendency to blood coagulation and thereby to the development of venous or arterial thrombosis. The hemostatic balance is the result of normal functioning of the blood vessel wall, platelets and plasma agents which include the coagulation and fibrinolytic systems and their inhibitors. By coagulation we understand the process leading to the formation of fibrin networks including the controlled interaction of coagulation factors. It is a physiological process as opposed to thrombosis which can be defined as increased coagulation under pathological conditions. Key words: coagulation - coagulation factors - coagulopathy - hemophilia - inhibitors of coagulation factors deficiencies - purpura - thrombocytopathy - thrombocytopenia.

Diagnostika poruch hemostázy

Hemostasis is a complicated biological system, where the balance between procoagulation and anticoagulation processes maintains fluidity of blood through intact blood vessels and creates thrombi when it is needed to prevent bleeding from the impaired vessels. The modern model of hemostasis is divided into 2 principal phases, the first being defined as primary hemostasis which involves the platelet-vessel interplay, while the second, defined as secondary hemostasis, mainly involves coagulation factors and surfaces of activated cells. The activation and amplification of the coagulation cascade is regulated by natural inhibitors of coagulation. The blood clots which arise to prevent loss of blood must subsequently be broken down and the compact blood vessel wall must be restored. This process is called fibrinolysis. Bleeding and thromboses are manifestations of the impaired hemostatic balance. Detection of its cause is important for efficient treatment and prevention of the condition. This requires a combined evaluation of the family and personal history, a clinical anamnesis along with the evaluation of laboratory results. It is not reasonable or economical to perform all the available tests of hemostasis at the same time. It is recommendable to proceed from the global to the screening and then special tests and, through the process of elimination, obtain an explanation of the bleeding or prothrombotic phenotypes. The purpose of this report is to provide a brief overview of the principal causes of the hemostatic disorders and the practices which facilitate their diagnosis. Key words: diagnosis of hemorrhagic manifestations - diagnostics of thrombophilias - DIC diagnostics - hemophilia A - thrombopathy - von Willebrand disease.

AMP‐Activated Protein Kinase (AMPK) Modulates Myoendothelial Junctions (MEJ) in Murine Microvessels

Myoendothelial junctions (MEJ) in microvessels are not only the site of direct cell communication via gap junctions but also represent a signaling microdomain critical for regulating vascular processes such as endothelium dependent hyperpolarization (EDH). The 5′‐adenosine monophosphate‐activated protein kinase (AMPK) has been shown to influence vascular tone. It has not yet been studied, however, whether AMPK could also affect MEJ. Thus, the aim of this project was to elucidate the impact of AMPK on MEJ dynamics and its potential mechanism of action.We studied isolated small mesenteric and skeletal muscle resistance arteries of mice using a pressure myograph system developed in our laboratory. Using confocal and two photon excitation fluorescence microscopy we identified the internal elastic lamina, which in these vessels exhibits small holes that allow direct contact between endothelial and smooth muscle cells. On average, we found that in about 35% of the holes, structures with a bright actin core as assessed by using F‐actin fluorescence in LifeAct mice or by staining with phalloidin which we could define as sites of MEJ. These structures showed expression of Cx37 and Cx43 suggesting that gap junctions (MEGJs) were also localized in these structures. Over observation times of up to 2 hours, we found that the number and localization of these MEJ varied, while they always positioned in the area of holes of IEL only. Thus, for the first time we could investigate MEJ dynamics. AMPK negatively regulates MEJ formation, since in AMPK α1, but not α2 knockout (KO) mice, the number of MEJ was significantly increased. In accordance, in wild type mice, incubation with the AMPK inhibitory compound C (CC) significantly augmented the number of MEJ. Furthermore, KO of AMPK α1, in intact mice as well as in cultured human and porcine smooth muscle cells, went along with an increase in PAI‐1 expression. Accordingly, incubation of arteries with exogenous PAI‐1 also increased the number of MEJ. The negative effect of AMPK on PAI‐1 could be explained by enhanced expression of the silencer of the PAI‐1 gene, small heterodimer partner (SHP). Since MEJ are also involved in EDH, we studied whether an enhanced formation of MEJ correlated with the magnitude of EDH induced vasodilation. Indeed, a higher amount of MEJs in α1 KO mice went along with a left shift of acetylcholine (ACh) ‐induced dilation dose effect curve.This work describes for the first time a role of the α1 subunit of AMPK as a potent modulator of MEJ dynamics which is probably mediated by controlling PAI‐1 expression. Our data also for the first time demonstrate a dynamic regulation of MEJ expression in intact blood vessels.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Contractile Smooth Muscle and Active Stress Generation in Porcine Common Carotids.

The mechanical response of intact blood vessels to applied loads can be delineated into passive and active components using an isometric decomposition approach. Whereas the passive response is due predominantly to the extracellular matrix (ECM) proteins and amorphous ground substance, the active response depends on the presence of smooth muscle cells (SMCs) and the contractile machinery activated within those cells. To better understand determinants of active stress generation within the vascular wall, we subjected porcine common carotid arteries (CCAs) to biaxial inflation-extension testing under maximally contracted or passive SMC conditions and semiquantitatively measured two known markers of the contractile SMC phenotype: smoothelin and smooth muscle-myosin heavy chain (SM-MHC). Using isometric decomposition and established constitutive models, an intuitive but novel correlation between the magnitude of active stress generation and the relative abundance of smoothelin and SM-MHC emerged. Our results reiterate the importance of stretch-dependent active stress generation to the total mechanical response. Overall these findings can be used to decouple the mechanical contribution of SMCs from the ECM and is therefore a powerful tool in the analysis of disease states and potential therapies where both constituent are altered.

Calcium influx through TRPV4 channels modulates the adherens contacts between retinal microvascular endothelial cells.

Endothelial cells employ transient receptor potential isoform 4 (TRPV4) channels to sense ambient mechanical and chemical stimuli. In retinal microvascular endothelial cells, TRPV4 channels regulate calcium homeostasis, cytoskeletal signalling and the organization of adherens junctional contacts. Intracellular calcium increases induced by TRPV4 agonists include a significant contribution from calcium release from internal stores. Activation of TRPV4 channels regulates retinal endothelial barriers in vitro and in vivo. TRPV4 sensing may provide a feedback mechanism between sensing shear flow and eicosanoid modulators, vascular permeability and contractility at the inner retinal endothelial barrier. The identity of microvascular endothelial (MVE) mechanosensors that sense blood flow in response to mechanical and chemical stimuli and regulate vascular permeability in the retina is unknown. Using immunohistochemistry, calcium imaging, electrophysiology, impedance measurements and vascular permeability assays, we show that the transient receptor potential isoform 4 (TRPV4) plays a major role in Ca2+ /cation signalling, cytoskeletal remodelling and barrier function in retinal microvasculature in vitro and in vivo. Human retinal MVE cells (HrMVECs) predominantly expressed Trpv1 and Trpv4 transcripts, and TRPV4 was broadly localized to the plasma membrane of cultured cells and intact blood vessels in the inner retina. Treatment with the selective TRPV4 agonist GSK1016790A (GSK101) activated a nonselective cation current, robustly elevated [Ca2+ ]i and reversibly increased the permeability of MVEC monolayers. This was associated with disrupted organization of endothelial F-actin, downregulated expression of occludin and remodelling of adherens contacts consisting of vascular endothelial cadherin (VE-cadherin) and β-catenin. In vivo, GSK101 increased the permeability of retinal blood vessels in wild type but not in TRPV4 knockout mice. Agonist-evoked effects on barrier permeability and cytoskeletal reorganization were antagonized by the selective TRPV4 blocker HC 067047. Human choroidal endothelial cells expressed lower TRPV4 mRNA/protein levels and showed less pronounced agonist-evoked calcium signals compared to MVECs. These findings indicate a major role for TRPV4 in Ca2+ homeostasis and barrier function in human retinal capillaries and suggest that TRPV4 may differentially contribute to the inner vs. outer blood-retinal barrier function.

Bio-Inspired Microdevices that Mimic the Human Vasculature.

Blood vessels may be found throughout the entire body and their importance to human life is undeniable. This is evident in the fact that a malfunctioning blood vessel can result in mild symptoms such as shortness of breath or chest pain to more severe symptoms such as a heart attack or stroke, to even death in the severest of cases. Furthermore, there are a host of pathologies that have been linked to the human vasculature. As a result many researchers have attempted to unlock the mysteries of the vasculature by performing studies that duplicate the physiological structural, chemical, and mechanical properties known to exist. While the ideal study would consist of utilizing living, blood vessels derived from human tissue, such studies are not always possible since intact human blood vessels are not readily accessible and there are immense technical difficulties associated with such studies. These limitations have opened the door for the development of microdevices modeled after the human vasculature as it is believed by many researchers in the field that such devices can one day replace tissue models. In this review we present an overview of microdevices developed to mimic various types of vasculature found throughout the human body. Although the human body contains a diverse array of vascular systems for this review we limit our discussion to the cardiovascular system and cerebrovascular system and discuss such systems that have been fabricated in both 2D and 3D configurations.

Exposure of Induced Pluripotent Stem Cell-Derived Vascular Endothelial and Smooth Muscle Cells in Coculture to Hemodynamics Induces Primary Vascular Cell-Like Phenotypes

Human induced pluripotent stem cells (iPSCs) can be differentiated into vascular endothelial (iEC) and smooth muscle (iSMC) cells. However, because iECs and iSMCs are not derived from an intact blood vessel, they represent an immature phenotype. Hemodynamics and heterotypic cell:cell communication play important roles in vascular cell phenotypic modulation. Here we tested the hypothesis that hemodynamic exposure of iECs in coculture with iSMCs induces an in vivo‐like phenotype. iECs and iSMCs were cocultured under vascular region‐specific blood flow hemodynamics, and compared to hemodynamic cocultures of blood vessel‐derived endothelial (pEC) and smooth muscle (pSMC) cells. Hemodynamic flow‐induced gene expression positively correlated between pECs and iECs as well as pSMCs and iSMCs. While endothelial nitric oxide synthase 3 protein was lower in iECs than pECs, iECs were functionally mature as seen by acetylated‐low‐density lipoprotein (LDL) uptake. SMC contractile protein markers were also positively correlated between pSMCs and iSMCs. Exposure of iECs and pECs to atheroprone hemodynamics with oxidized‐LDL induced an inflammatory response in both. Dysfunction of the transforming growth factor β (TGFβ) pathway is seen in several vascular diseases, and iECs and iSMCs exhibited a transcriptomic prolife similar to pECs and pSMCs, respectively, in their responses to LY2109761‐mediated transforming growth factor β receptor I/II (TGFβRI/II) inhibition. Although there are differences between ECs and SMCs derived from iPSCs versus blood vessels, hemodynamic coculture restores a high degree of similarity in their responses to pathological stimuli associated with vascular diseases. Thus, iPSC‐derived vascular cells exposed to hemodynamics may provide a viable system for modeling rare vascular diseases and testing new therapeutic approaches. Stem Cells Translational Medicine 2017;6:1673–1683

Variability of Methylation Profiles of CpG Sites in microrNA Genes in Leukocytes and Vascular Tissues of Patients with Atherosclerosis.

In this study, we for the first time described the variability of methylation levels of 71 CpG sites in microRNA genes in leukocytes and blood vessels (coronary artery atherosclerotic plaques, intact internal thoracic arteries, and great saphenous veins) in patients with atherosclerosis using the Infinium HumanMethylation27 BeadChip microarray. Most of the analyzed CpG sites were characterized by the low variability, and most of these low-variable sites were hypomethylated in all tissue samples. CpG sites in coronary artery atherosclerotic plaques and leukocytes were similar in their methylation status. The highest variability of CpG methylation levels between different tissues was found for the CpG sites of the MIR10B gene; the methylation levels of these sites in leukocytes and atherosclerotic arteries were lower than in intact blood vessels. We also found that several cardiovascular disease risk factors, as well as medications, might affect methylation levels of CpG sites in microRNAs.

Pulsed ultrasound expands the extracellular and perivascular spaces of the brain

Diffusion within the extracellular and perivascular spaces of the brain plays an important role in biological processes, therapeutic delivery, and clearance mechanisms within the central nervous system. Recently, ultrasound has been used to enhance the dispersion of locally administered molecules and particles within the brain, but ultrasound-mediated effects on the brain parenchyma remain poorly understood. We combined an electron microscopy-based ultrastructural analysis with high-resolution tracking of non-adhesive nanoparticles in order to probe changes in the extracellular and perivascular spaces of the brain following a non-destructive pulsed ultrasound regimen known to alter diffusivity in other tissues. Freshly obtained rat brain neocortical slices underwent sham treatment or pulsed, low intensity ultrasound for 5min at 1MHz. Transmission electron microscopy revealed intact cells and blood vessels and evidence of enlarged spaces, particularly adjacent to blood vessels, in ultrasound-treated brain slices. Additionally, ultrasound significantly increased the diffusion rate of 100nm, 200nm, and 500nm nanoparticles that were injected into the brain slices, while 2000nm particles were unaffected. In ultrasound-treated slices, 91.6% of the 100nm particles, 20.7% of the 200nm particles, 13.8% of the 500nm particles, and 0% of the 2000nm particles exhibited diffusive motion. Thus, pulsed ultrasound can have meaningful structural effects on the brain extracellular and perivascular spaces without evidence of tissue disruption.

Effect of simvastatin on vascular tone in porcine coronary artery: Potential role of the mitochondria

Statins induce acute vasorelaxation which may contribute to the overall benefits of statins in the treatment of cardiovascular disease. The mechanism underlying this relaxation is unknown. As statins have been shown to alter mitochondrial function, in this study we investigated the role of mitochondria in the relaxation to simvastatin.Relaxation of porcine coronary artery segments by statins was measured using isolated tissue baths. Mitochondrial activity was determined by measuring changes in rhodamine 123 fluorescence. Changes in intracellular calcium levels were determined in freshly isolated smooth muscle cells with Fluo-4 using standard epifluorescent imaging techniques.Simvastatin, but not pravastatin, produced a slow relaxation of the coronary artery, which was independent of the endothelium. The relaxation was attenuated by the mitochondrial complex I inhibitor rotenone (10μM) and the complex III inhibitor myxothiazol (10μM), or a combination of the two. The complex III inhibitor antimycin A (10μM) produced a similar time-dependent relaxation of the porcine coronary artery, which was attenuated by rotenone. Changes in rhodamine 123 fluorescence showed that simvastatin (10μM) depolarized the membrane potential of mitochondria in both isolated mitochondria and intact blood vessels. Simvastatin and antimycin A both inhibited calcium-induced contractions in isolated blood vessels and calcium influx in smooth muscle cells and this inhibition was prevented by rotenone.In conclusion, simvastatin produces an endothelium-independent relaxation of the porcine coronary artery which is dependent, in part, upon effects on the mitochondria. The effects on the mitochondria may lead to a reduction in calcium influx and hence relaxation of the blood vessel.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries.

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2(.-)) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2(.-) levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2(.-), it is difficult to measure NO and O2(.-) directly in a blood vessel. Numerous methods have been developed to measure NO and O2(.-) production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2(.-) using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2(.-) levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2(.-) in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

Adventitia and perivascular cells.

The past few years have seen significant advances in our understanding of the multiple and dynamic roles of the adventitia and its companion perivascular tissues for vessel wall homeostasis and disease. It is now becoming clear that signals originating from within the adventitia and from perivascular cells play essential roles in regulation of vascular development, physiology, artery wall remodeling, immune surveillance, and vascular disease.1–4 The adventitia is in contact with and completely surrounds the media and is the interface between the vessel wall and its neighboring tissues. It contains many different interacting cell types including fibroblasts, microvascular endothelium, nerves, resident macrophages, T cells, B cells, mast cells, and dendritic cells.5–9 The adventitia is also home to resident vascular progenitor cells whose formation and maintenance depend, in part, on sonic hedgehog signaling.1,10–12 Perivascular cells are in close contact with the adventitia, particularly for the aorta and coronary arteries. Perivascular tissue includes adipocytes, lymphatic vessels, perivascular nerves, and stromal cells exhibiting mesenchymal stem cell–like properties.3,13 The adventitia and periadventitial cells function in concert. They are linked by microvessels, nerves, and migratory cells to regulate vascular physiology, homeostasis, structural remodeling, and exert major influences on the progression or regression of vascular disease. Crosstalk between intima, media, and adventitia further links the adventitia-periadventitial unit to the rest of the vessel wall. Much of the work advancing our concepts of the adventitia and periadventitial tissues has been published in ATVB , and some of those key studies are discussed in this highlights article. In the following summary, areas that have attracted the most interest over the past 2 years in ATVB are reviewed including the multiple roles of perivascular adipose tissue (PVAT) on control of vascular physiology and remodeling, adventitial progenitor …

HIF-1α and VEGF: Immunohistochemical Profile and Possible Function in Human Aortic Valve Stenosis

Calcific aortic stenosis (CAS) is the most common valvular disease in Western countries. Histological findings in patients with CAS extremely resemble those of atherosclerosis and include accumulation and modification of lipoproteins, inflammation, extracellular matrix remodeling, and calcification. Angiogenesis is another prominent feature of CAS; however, there is only a limited amount of data available regarding the mechanisms behind the pathological neovascularization of a structure that is originally avascular. The present study aims to identify the molecular basis that regulates blood vessel growth in stenotic aortic valves, focusing on the role of HIF-1α and VEGF pathway. A total of 19 native degenerating aortic valves obtained at valve replacement surgery have been processed for Western blot, immunohistochemical, morphometric, and ultrastructural analyses. First, we have demonstrated the adverse ECM remodeling and the significant thickening of the leaflet also showing that HIF-1α and VEGF are significantly upregulated in the stenotic valves, are locally produced and colocalize with angiogenesis and areas of calcification. Next, we have characterized, for the first time to the best of our knowledge, the morphological features of the neovasculature evidencing the presence of intact blood vessels in close proximity to the mineralized zones. These results suggest that the complex structural remodeling of the matrix might reduce oxygen availability in the valve cusp contributing to the stabilization of HIF-1α that in turn induces a metabolic adaptation through the upregulation of VEGF and the formation of new blood vessels not only to overcome the hypoxic state but also to sustain the calcification process.

Innate Immunity, Coagulation, and Thrombosis

In evolutionarily ancient animals such as insects and crustaceans, the host responses to physical injury and to invading pathogens can be mediated by the same mechanism of coagulum formation of the hemolymph. During vertebrate evolution hemostasis has emerged as an independent process primarily involved in the rapid repair of blood vessel injuries. The core processes of hemostasis are blood coagulation (resulting in fibrin formation) and platelet activation. Both processes can independently interact with inflammatory responses as apparent in a pathological context such as during development of disseminated intravascular coagulation (DIC). Moreover, extravascular fibrin formation can promote the trapping of pathogens and thereby help to contain infections. Nonetheless, the connections between fibrin formation, platelet activation and innate immunity are incompletely understood. We have recently shown that during early systemic infection with E. coli microvascular thrombi are formed which capture bacteria together with innate leukocytes. These thrombi are fibrin-rich and are in general observed in less than 10% of vessels with diameters < 25 µm. Their formation is not accompanied by marked activation of inflammation since the levels of pro-inflammatory markers are unchanged. Microvascular thrombosis is almost completely suppressed in mice deficient for the neutrophil serine proteases elastase and cathepsin G (NE/CG-/-) which are major microbicidal effectors of neutrophils. In the microcirculation of NE/CG-/- mice, microbes are mostly tissue-associated. In contrast, they are mostly present inside blood vessels in wild type mice. The results of experimental changes in microvascular fibrin formation show that intravascular blood coagulation is causally involved in the capturing of bacteria and of myeloid cells and, additionally, promotes the bacterial killing. Overall this suggests that microvascular thrombosis supports recognition, containment and elimination of bacteria without inducing noticeable damage to the host. It thus fulfills the criteria for a comprehensive intravascular process of innate immunity. This mechanism of intravascular immunity, which was termed "immunothrombosis," is supported by tissue factor (TF), the overall initiator of blood coagulation, and by factor XII, the starter protein of the contact pathway. In particular, extracellular nucleosomes (eNUC)/neutrophil extracellular traps (NETs) are indispensable for immunothrombosis. eNUC/NETs promote thrombosis by critically enhancing degradation of TFPI, the major antagonist of the coagulation start, via neutrophil elastase and by factor XII activation. Release of eNUC/NETs by neutrophils and induction of intravascular coagulation essentially require interaction of activated platelets with neutrophils. Interestingly, intravascular TF, factor XII, eNUC/NETs and innate leukocytes are almost completely dispensable for hemostasis. Furthermore, immunothrombosis in contrast to hemostasis develops in largely intact blood vessels. Together this indicates that thrombosis can be a physiological mechanism of innate immunity that is distinct from hemostasis. We have recently developed a new model for deep vein thrombosis (DVT) which closely reproduces the pathological changes in the vessel wall observed in most patients with DVT. Using this model, we show that intravascular TF, factor XII, eNUC/NETs, innate leukocytes and their interactions with platelets all critically promote DVT. Thus, DVT shares similar triggers (especially pathogens) and identical molecular and cellular mediators with immunothrombosis. In case of DIC, the connections to immunothrombosis are most likely similarly strong or even stronger. Finally, our results also show that mediators of immunothrombosis such as eNUC/NETs and neutrophil serine proteases are main triggers of arterial thrombosis. Hence, together with hemostasis, immunothrombosis likely constitutes the major biological template process for both (pathological) microvascular thrombosis and large vessel thrombosis. DisclosuresNo relevant conflicts of interest to declare.

The G protein‐coupled receptor (GPER/GPR30) activates endothelial nitric oxide synthase (1075.5)

Endogenous estrogens mediate protective effects in the cardiovascular system in part due to rapid activation of endothelial nitric oxide synthase (eNOS), which involves the classical estrogen receptor (ER) α. In intact blood vessels, nitric oxide (NO) bioactivity is also regulated by the G protein‐coupled estrogen receptor (GPER/GPR30), suggesting an alternative pathway of estrogen‐dependent eNOS activation. We have further investigated this mechanism by analyzing GPER signaling in telomerase‐immortalized human umbilical vein endothelial (TIVE) cells. Using an antibody recognizing the second extracellular loop of GPER, immunofluorescence microscopy revealed that GPER is expressed predominantly in intracellular membranes as determined by permeabilizing vs. non‐permeabilizing conditions. Similar to the non‐selective ER agonist 17β‐estradiol, the GPER‐selective agonist G‐1 stimulated phosphorylation of Akt (ser437) and eNOS (ser1171) in a dose‐dependent manner. Additional studies have examined the roles of EGFR and ERK in G‐1‐mediated eNOS activation. Pharmacological inhibition of GPER was used to assess its contribution to estrogen‐mediated NO production. Our results further characterize the signaling pathway of GPER‐mediated eNOS activation, suggesting that the beneficial NO‐dependent effects of endogenous estrogens in the cardiovascular system may partly be mediated by GPER.

Lipopolysaccharide induces a fibrotic‐like phenotype in endothelial cells

Endothelial dysfunction is crucial in endotoxaemia-derived sepsis syndrome pathogenesis. It is well accepted that lipopolysaccharide (LPS) induces endothelial dysfunction through immune system activation. However, LPS can also directly generate actions in endothelial cells (ECs) in the absence of participation by immune cells. Although interactions between LPS and ECs evoke endothelial death, a significant portion of ECs are resistant to LPS challenge. However, the mechanism that confers endothelial resistance to LPS is not known. LPS-resistant ECs exhibit a fibroblast-like morphology, suggesting that these ECs enter a fibrotic programme in response to LPS. Thus, our aim was to investigate whether LPS is able to induce endothelial fibrosis in the absence of immune cells and explore the underlying mechanism. Using primary cultures of ECs and culturing intact blood vessels, we demonstrated that LPS is a crucial factor to induce endothelial fibrosis. We demonstrated that LPS was able and sufficient to promote endothelial fibrosis, in the absence of immune cells through an activin receptor–like kinase 5 (ALK5) activity–dependent mechanism. LPS-challenged ECs showed an up-regulation of both fibroblast-specific protein expression and extracellular matrix proteins secretion, as well as a down-regulation of endothelial markers. These results demonstrate that LPS is a crucial factor in inducing endothelial fibrosis in the absence of immune cells through an ALK5-dependent mechanism. It is noteworthy that LPS-induced endothelial fibrosis perpetuates endothelial dysfunction as a maladaptive process rather than a survival mechanism for protection against LPS. These findings are useful in improving current treatment against endotoxaemia-derived sepsis syndrome and other inflammatory diseases.