Coronary Artery Disease as a Relevant Risk Factor in Screening of Abdominal Aortic Ectasia and Aneurysm.

BACKGROUND: The aim of this study was to investigate the prevalence of abdominal aortic aneurysm (AAA) and abdominal aortic ectasia (AAE) in coronary artery disease (CAD) patients in a multicenter setting to obtain significant data to establish an AAA screening program in our departments. METHODS: Between January and September 2016, 500 patients with suspected or diagnosed CAD planned for coronary angiography or coronary artery bypass graft (CABG) underwent a sonographic examination of the infrarenal abdominal aorta to diagnose AAA or AAE. We calculated the prevalence of AAA and AAE in patients diagnosed of CAD and investigated factors potentially associated with the occurrence of AAA. RESULTS: The overall prevalence in all grades of CAD for AAE was 35.1% and for AAA 5.4%. In patients with three-vessel CAD, the prevalence of AAE was 34% and of AAA 6.8%. Significant correlation was found between the three-vessel CAD and AAA (p = 0.039). The logistic regression analysis showed significant correlation between AAA and age > 65 years (p = 0.05). The multivariate analysis of risk factors and CAD revealed significant correlations between one-vessel CAD and arterial hypertension (AH) (p = 0.004) and age > 65 years (p = 0.001) as well as between three-vessel CAD and AH (p = 0.01), peripheral artery disease (p = 0.01), and age > 65 years (p = 0.03). CONCLUSION: Our results confirm, that in comparison to other data, the prevalence of AAA in patients with CAD is high. Thus, it is recommended to include patients with CAD, especially elderly patients with three-vessel CAD, in future AAA screening programs.

Purinergic Regulation of Endothelial Barrier Function.

Increased vascular permeability is a hallmark of several cardiovascular anomalies, including ischaemia/reperfusion injury and inflammation. During both ischaemia/reperfusion and inflammation, massive amounts of various nucleotides, particularly adenosine 5'-triphosphate (ATP) and adenosine, are released that can induce a plethora of signalling pathways via activation of several purinergic receptors and may affect endothelial barrier properties. The nature of the effects on endothelial barrier function may depend on the prevalence and type of purinergic receptors activated in a particular tissue. In this review, we discuss the influence of the activation of various purinergic receptors and downstream signalling pathways on vascular permeability during pathological conditions.

Venous access closure using a purse-string suture without heparin antagonism or additional compression after MitraClip implantation.

OBJECTIVES: This observational study was designed to analyze the safety and feasibility of percutaneous skin closure using a purse-string suture (PSS) after MitraClip procedures. METHODS: Forty-one consecutive patients with severe mitral regurgitation who underwent MitraClip implantation from February 2018 to January 2019 at our institution received a PSS after percutaneous mitral valve repair before withdrawal of the 24-French (Fr) sheath. Protamine was not administered after venous closure at procedure end. No compression therapy (e.g., compression bandage or pneumatic compression device) was used. Patients were on bed rest for 6 hrs prior to suture removal, which was accomplished 18-24 hrs after MitraClip implantation. We analyzed the occurrence of any vascular or thromboembolic complications during the hospital stay and until the 3-month follow-up. RESULTS: The primary endpoint-any access-related major complication-did not occur in any patients. None of the patients revealed a pseudoaneurysm or an arteriovenous fistula, a thromboembolic complication, or local stenosis related to the PSS closure. The secondary endpoint- minor access-site vascular complications (hematoma)- was documented in six (14.6%) patients. CONCLUSIONS: Venous access-site closure with a PSS without the need for protamine administration or compression therapy appears to be safe and feasible in patients undergoing MitraClip implantation with access via a 24-Fr sheath.

Inhibition of Protein Prenylation of GTPases Alters Endothelial Barrier Function.

The members of Rho family of GTPases, RhoA and Rac1 regulate endothelial cytoskeleton dynamics and hence barrier integrity. The spatial activities of these GTPases are regulated by post-translational prenylation. In the present study, we investigated the effect of prenylation inhibition on the endothelial cytoskeleton and barrier properties. The study was carried out in human umbilical vein endothelial cells (HUVEC) and protein prenylation is manipulated with various pharmacological inhibitors. Inhibition of either complete prenylation using statins or specifically geranylgeranylation but not farnesylation has a biphasic effect on HUVEC cytoskeleton and permeability. Short-term treatment inhibits the spatial activity of RhoA/Rho kinase (Rock) to actin cytoskeleton resulting in adherens junctions (AJ) stabilization and ameliorates thrombin-induced barrier disruption whereas long-term inhibition results in collapse of endothelial cytoskeleton leading to increased basal permeability. These effects are reversed by supplementing the cells with geranylgeranyl but not farnesyl pyrophosphate. Moreover, long-term inhibition of protein prenylation results in basal hyper activation of RhoA/Rock signaling that is antagonized by a specific Rock inhibitor or an activation of cAMP signaling. In conclusion, inhibition of geranylgeranylation in endothelial cells (ECs) exerts biphasic effect on endothelial barrier properties. Short-term inhibition stabilizes AJs and hence barrier function whereas long-term treatment results in disruption of barrier properties.

Uridine Triphosphate Thio Analogues Inhibit Platelet P2Y12 Receptor and Aggregation.

Platelet P2Y12 is an important adenosine diphosphate (ADP) receptor that is involved in agonist-induced platelet aggregation and is a valuable target for the development of anti-platelet drugs. Here we characterise the effects of thio analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation. Using human platelet-rich plasma, we demonstrate that UTP inhibits P2Y12 but not P2Y1 receptors and antagonises 10 µM ADP-induced platelet aggregation in a concentration-dependent manner with an IC50 value of ~250 °µM. An eight-fold higher platelet inhibitory activity was observed with a 2-thio analogue of UTP (2S-UTP), with an IC50 of 30 µM. The 4-thio analogue (4S-UTP) with an IC50 of 7.5 µM was 33-fold more effective. A three-fold decrease in inhibitory activity, however, was observed by introducing an isobutyl group at the 4S- position. A complete loss of inhibition was observed with thio-modification of the γ phosphate of the sugar moiety, which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y12 receptor was verified by P2Y12 receptor binding and cyclic AMP (cAMP) assays. These novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y12 receptor antagonists that may be useful for therapeutic intervention.

Diagnosing moyamoya syndrome using ultrasound - a case report.

BACKGROUND: Moyamoya syndrome is a vasculopathy characterised by progressive occlusion of the cerebral arteries resulting in the development of abnormal collateral circulation. To diagnose this syndrome, imaging of the cerebral arteries is required including CT- or MR-angiography and conventional angiography. We present a case of moyamoya disease with typical findings detected in the sonography. The diagnosis was suspected after reviewing the initial ultrasound images of the cerebral arteries with evidence for obliterated intracranial arteries and the detection of an existing collateral circulation network. CASE PRESENTATION: A 62 years old male patient presented in the hospital's emergency department with symptoms indicating a subacute cerebrovascular event. Immediate sonographic studies showed a right-sided pulsatile Doppler-signal in the common and internal carotid arteries, suggestive of distal stenoses. In addition, the transcranial examination indicated obliteration of both middle cerebral arteries. Numerous arterial vessels suggestive of leptomeningeal collateral arteries revealed a strong arterial leptomeningeal flow. At this stage of the diagnostic work-up, the collateral circulation network, characteristic of moyamoya disease, was indicated by sonography. Moyamoya syndrome was verified by conventional angiography. The aetiological work remained empty, so the diagnosis of moyamoya disease was established. CONCLUSION: Our case report indicates that sonography can be a useful tool for detecting the vaculopathy in moyamoya syndrome. In case routine procedures, such as the CT- or MR-angiography, with evidence for obliterated intracerebral arteries, ultrasound studies might provide important information regarding an existing collateral network in the scope of a moyamoya syndrome.

Hypoxia-reoxygenation-induced endothelial barrier failure: role of RhoA, Rac1 and myosin light chain kinase.

Hypoxia-reoxygenation induces loss of endothelial barrier function and oedema formation, which presents a major impediment for recovery of the organ. The integrity of the endothelial barrier is highly dependent on its contractile machinery and actin dynamics, which are precisely regulated by Rho GTPases. Perturbed activities of these Rho-GTPases under hypoxia-reoxygenation lead to derangement of the actin cytoskeleton and therefore may affect the integrity of the endothelial barrier. The aim of the present study was to analyse the role of these GTPases in regulating endothelial barrier function during hypoxia-reoxygenation in cultured porcine aortic endothelial cells and isolated perfused rat hearts. Hypoxia-reoxygenation induced an increase in albumin permeability of endothelial monolayers accompanied by an activation of the endothelial contractile machinery, derangement of the actin cytoskeleton and loss of VE-cadherin from cellular junctions. Inhibition of contractile activation with ML-7 partially protected against hypoxia-reoxygenation-induced hyperpermeability. Likewise, reoxygenation caused an increase in RhoA and a reduction in Rac1 activity accompanied by enhanced stress fibre formation and loss of peripheral actin. Inhibition of RhoA/rho kinase (Rock) signalling with RhoA or Rock inhibitors led to a complete depolymerisation and derangement of the actin cytoskeleton and worsened hypoxia-reoxygenation-induced hyperpermeability. Activation of Rac1 using a cAMP analogue, 8-CPT-O-Me-cAMP, which specifically activates Epac/Rap1 signalling, restored peripheral localisation of actin and VE-cadherin at cellular junctions and abrogated reoxygenation-induced hyperpermeability. Similar results were reproduced in isolated saline-perfused rat hearts. These data show that activation of Rac1 but not the inhibition of RhoA preserves endothelial integrity against reoxygenation-induced loss of barrier function.

Opposing effects of ATP and adenosine on barrier function of rat coronary microvasculature.

ATP can differentially affect the micro- and macrovascular endothelial barrier. It has been shown that it can both increase and/or decrease macromolecule permeability of microvascular endothelial cells and microvessels, in vivo. We hypothesised that the barrier stabilising effect is mediated by ATP itself via P2 receptors, while barrier-disrupting effect is mediated by its metabolite adenosine via adenosine receptors. The effects of ATP, ADP, AMP and adenosine on barrier function were studied in cultured rat coronary microvascular endothelial monolayers (RCEC) in vitro, as well as in rat mesentery vessels, and in rat hearts in vivo. ATP and ADP showed a biphasic effect on permeability of RCEC monolayers with a reduction followed by a later increase in albumin permeability. The permeability decreasing effect of ATP was enhanced by ecto-nucleotidase inhibitor ARL67156 while permeability increasing effect was enhanced by apyrase, an extracellular ecto-nucleotidase. Moreover, the permeability increasing effect was abrogated by adenosine receptor antagonists, 8-phenyltheophylline (8-PT) and DMPX. Adenosine and adenosine receptor agonists 5'-(N-ethylcarboxamido)-adenosine (NECA), CGS21680, and R-PIA enhanced albumin permeability which was antagonised by 8-PT, A(1), and A(2) but not by A(3) receptor antagonists. Likewise, immunofluorescence microscopy of VE-cadherin and actin showed that NECA induces a disturbance of intercellular junctions. Pre-incubation of ATP antagonised the effects of NECA on permeability, actin cytoskeleton and intercellular junctions. Similar effects of the applied substances were observed in rat mesentery artery by determining the vascular leakage using intravital microscopy as well as in rat hearts by assessing myocardial water contents in vivo. In conclusion, the study demonstrates that in RCEC, ATP, ADP, and its metabolite adenosine play opposing roles on endothelial barrier function.

Inhibition of STAT3 signaling prevents vascular smooth muscle cell proliferation and neointima formation.

Dedifferentiation, migration, and proliferation of resident vascular smooth muscle cells (SMCs) are key components of neointima formation after vascular injury. Activation of signal transducer and activator of transcription-3 (STAT3) is suggested to be critically involved in this process, but the complex regulation of STAT3-dependent genes and the functional significance of inhibiting this pathway during the development of vascular proliferative diseases remain elusive. In this study, we demonstrate that STAT3 was activated in neointimal lesions following wire-induced injury in mice. Phosphorylation of STAT3 induced trans-activation of cyclin D1 and survivin in SMCs in vitro and in neointimal cells in vivo, thus promoting proliferation and migration of SMCs as well as reducing apoptotic cell death. WP1066, a highly potent inhibitor of STAT3 signaling, abrogated phosphorylation of STAT3 and dose-dependently inhibited the functional effects of activated STAT3 in stimulated SMCs. The local application of WP1066 via a thermosensitive pluronic F-127 gel around the dilated arteries significantly inhibited proliferation of neointimal cells and decreased the neointimal lesion size at 3 weeks after injury. Even though WP1066 application attenuated the injury-induced up-regulation of the chemokine RANTES at 6 h after injury, there was no significant effect on the accumulation of circulating cells at 1 week after injury. In conclusion, these data identify STAT3 as a key molecule for the proliferative response of SMC and neointima formation. Moreover, inhibition of STAT3 by the potent and specific compound WP1066 might represent a novel and attractive approach for the local treatment of vascular proliferative diseases.

Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes.

Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

Insulin stabilizes microvascular endothelial barrier function via phosphatidylinositol 3-kinase/Akt-mediated Rac1 activation.

OBJECTIVE: Insulin is a key regulator of metabolism, but it also confers protective effects on the cardiovascular system. Here, we analyze the mechanism by which insulin stabilizes endothelial barrier function. METHODS AND RESULTS: Insulin reduced basal and antagonized tumor necrosis factor-alpha-induced macromolecule permeability of rat coronary microvascular endothelial monolayers. It also abolished reperfusion-induced vascular leakage in isolated-perfused rat hearts. Insulin induced dephosphorylation of the regulatory myosin light chains, as well as translocation of actin and vascular endothelial (VE)-cadherin to cell borders, indicating a reduction in contractile activation and stabilization of cell adhesion structures. These protective effects were blocked by genistein or Hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester (HNMPA-[AM](3)), a pan-tyrosine-kinase or specific insulin-receptor-kinase inhibitor, respectively. Insulin stimulated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and NO production, and it activated Rac1. Inhibition of PI3K/Akt abrogated Rac1 activation and insulin-induced barrier protection, whereas inhibition of the endothelial nitric oxide synthase/soluble guanylyl cyclase pathway partially inhibited them. Inhibition of Rac1 abrogated the assembly of actin at cell borders. Accordingly, it abolished the protective effect of insulin on barrier function of the cultured endothelial monolayer, as well as the intact coronary system of ischemic-reperfused hearts. CONCLUSIONS: Insulin stabilizes endothelial barrier via inactivation of the endothelial contractile machinery and enhancement of cell-cell adhesions. These effects are mediated via PI3K/Akt- and NO/cGMP-induced Rac1 activation.

Role of PI3K/Akt and MEK/ERK Signalling in cAMP/Epac-Mediated Endothelial Barrier Stabilisation.

BACKGROUND AND AIMS: Activation of the cAMP/Epac signalling stabilises endothelial barrier function. Moreover, its activation is accompanied by an activation of PI3K/Akt and MEK/ERK signalling in diverse cell types but their impact on endothelial barrier function is largely unknown. Here the role of PI3K/Akt and MEK/ERK signalling in cAMP/Epac-mediated endothelial barrier stabilisation was analysed. METHODS: Endothelial barrier function was analysed in cultured human umbilical vein endothelial cells (HUVECs) by measuring flux of albumin. A modified cAMP analogue 8-pCPT-2'-O-Me-cAMP (Epac agonist) was used to specifically activate cAMP/Epac signalling. RESULTS: Epac agonist reduces the basal and attenuates thrombin-induced endothelial hyperpermeability accompanied by an activation of PI3K/Akt and MEK/ERK signalling. The qPCR data demonstrate HUVECs express PI3Kα, PI3Kß, and PI3Kγ but not PI3Kδ isoforms. The western blot data demonstrate Epac agonist activates PI3Kα and PI3Kß isoforms. Inhibition of MEK/ERK but not PI3K/Akt pathway potentiates the endothelial barrier protective effects of cAMP/Epac signalling. Inhibition of MEK/ERK signalling in the presence of Epac agonist induces a reorganisation of actin cytoskeleton to the cell periphery, enhanced VE-cadherin localisation at cell-cell junctions, and dephosphorylation of myosin light chains (MLC) but not inhibition of RhoA/Rock signalling. Moreover, Epac agonist promotes endothelial cell (EC) survival via reduction in activities of pro-apoptotic caspases in a PI3K/Akt and MEK/ERK signalling-dependent manner. CONCLUSION: Our data demonstrate that the Epac agonist simultaneously activates diverse signalling pathways in ECs, which may have differential effects on endothelial barrier function. It activates PI3K/Akt and MEK/ERK signalling which mainly govern its pro-survival effects on ECs. Inhibition of MEK/ERK but not PI3K/Akt signalling enhances barrier stabilising and barrier protective effects of cAMP/Epac activation. CHEMICAL COMPOUNDS USED IN THIS STUDY: 8-pCPT-2'-O-Me-cAMP (PubChem CID: 9913268); Akt inhibitor VIII (PubChem CID: 10196499); AS-252424 (PubChem CID: 11630874); IC-87114 (PubChem CID: 9908783); PD 98059 (PubChem CID: 4713); PIK-75 (PubChem CID: 10275789); TGX-221 (PubChem CID: 9907093); Thrombin (PubChem CID: 90470996); U0126 (PubChem CID: 3006531); Wortmannin (PubChem CID: 312145).

Assessment of Cardiac Involvement in Fabry Disease (FD) with Native T1 Mapping.

PURPOSE: Fabry disease (FD) is an X-linked multi-organ disorder of lysosomal metabolism with cardiac disease being the leading cause of death. Identifying early FD-specific pathologies is important in the context of maximum therapeutic benefit in these stages. Therefore, the aim of this study was to investigate the value of quantitative cardiac T1 mapping as a potential disease-specific surrogate. METHODS: 16 consecutive FD patients (9 female, 7 male; median age: 54 years, IQR 17) and 16 control patients (9 female, 7 male; median age: 52 years, IQR 20) were investigated at 1.5 Tesla. Native T1 mapping was performed using a modified look locker inversion recovery sequence (MOLLI) and native T1 times were measured within the septal myocardium at the midventricular short-axis section. Also functional parameters, left ventricular morphology, presence of late-gadolinium enhancement, cTnI- and Lyso-Gb3-Levels were evaluated. RESULTS: The median native septal T1 time for FD was 889.0 ms and 950.6 ms for controls (p < 0.003). LGE and positive cTnI values (0.26 ±â€Š0.21) were present in 5 FD patients (31.25 %), and left ventricular hypertrophy (LVH) was present in 4 FD patients (25.00 %). The 4 cTnI and 8 Lyso-Gb3 positive FD patients had significantly lower native T1 values (p < 0.05, respectively p < 0.01). Assuming a T1 cut-off value of 900 ms for the identification of increased cardiac lipid deposit, 9 patients with FD (56.25 %) had pathologic values (4 patients cTnI and 8 patients Lyso-Gb3 positive). Moreover, native septal T1 showed a good negative correlation to Lyso-Gb3 (r = - 0.582; p = 0.018). CONCLUSION: A pathologic cardiac native T1 time obviously reflects cardiac involvement in the scope of FD at tissue level. In the future native T1 mapping as an imaging biomarker might allow identification of early stages of cardiac involvement in FD before morphological changes are obvious. KEY POINTS: · Native T1 values are significantly decreased in Fabry disease.. · Native T1 shows promising correlation to cardiac and Fabry-specific biomarkers.. · Native T1 mapping might have great potential for early disease detection and therapy monitoring.. CITATION FORMAT: · Roller FC, Fuest S, Meyer M et al. Assessment of Cardiac Involvement in Fabry Disease (FD) with Native T1 Mapping. Fortschr Röntgenstr 2019; 191: 932 - 939.

Inhibition of Cx43 attenuates ERK1/2 activation, enhances the expression of Cav­1 and suppresses cell proliferation.

In addition to being an important component of the gap junction, connexin 43 (Cx43) has been shown to regulate other cellular functions, including cell proliferation. This regulatory role of Cx43 may be important in therapeutic situations, including wound healing or ischemic injuries. Caveolin­1 (Cav­1) has been shown to regulate angiogenesis. The aim of the present study was to analyze whether Cx43 counter­regulates Cav­1 in controlling the proliferation and migration of endothelial cells. The inhibition of Cx43 with niflumic acid, flufenamic acid and 18­α­glycyrrhetinic acid in cultured human umbilical vein endothelial cells resulted in decreased phosphorylation of extracellular signal­regulated kinase (ERK)1/2 and increased expression of Cav­1, as shown by western blot analysis. Furthermore, the inhibition of Cx43 resulted in a 50±7% decrease in cell proliferation, determined using a crystal violet assay, a 48±5% decrease in migration, determined using a migration assay, and a 49±6% decrease in endothelial tube formation, determined using a Matrigel assay, compared with the control. Similar results were obtained following specific inhibition of Cx43 by mimetic peptides (Gap26 and Gap27). Inhibition of the mitogen­activated protein kinase kinase/ERK pathway with PD­98059 resulted in an increased expression of Cav­1 and a reduction in the expression of Cx43. Furthermore, cell proliferation, migration and tube formation in endothelial cells were impaired. By contrast, downregulation of the protein expression of Cav­1 by small interference RNA resulted in increased expression of Cx43 and phosphorylation of ERK1/2. Accordingly, the number of cells in the Cav­1 treated­group increased by 35±5% compared with the controls. The data of the present study showed that Cav­1 suppressed cell proliferation by inhibiting the activity of Cx43, which is upstream of ERK1/2. The downregulation of Cav­1 protein resulted in loss of the inhibitory activity of Cav­1 on cell proliferation and led to increased cell proliferation. This counter­regulatory effect of Cx43 may be of importance in therapeutic angiogenesis.

Accumulation of extracellular ATP protects against acute reperfusion injury in rat heart endothelial cells.

OBJECTIVE: Ischemia-reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial edema development that jeopardizes functional recovery of the heart during reperfusion. Here, we tested whether adenosine 5'-triphosphate (ATP), either exogenously applied or spontaneously released during reperfusion, protects the endothelial barrier against an imminent reperfusion injury and whether interventions preventing ATP breakdown augment this protective ATP effect. METHODS: Cultured microvascular coronary endothelial monolayers and isolated-perfused hearts of rat were used. RESULTS: After ischemic conditions were induced, reperfusion of endothelial monolayers activated the endothelial contractile machinery and caused intercellular gap formation. It also led to the release of ATP. When its breakdown was inhibited by 6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP (ARL 67156; 100 microM), a selective ectonucleotidase inhibitor, contractile activation and gap formation were significantly reduced. Reperfusion in the presence of exogenously added ATP (10 microM) plus ARL caused an additional reduction of both aforementioned effects. In contrast, elevation of ATP degradation by apyrase (1 U/ml), a soluble ectonucleotidase, or addition of adenosine (10 microM) provoked an increase in gap formation during reperfusion that could be completely inhibited by 8-phenyltheophylline (8-PT; 10 microM), an adenosine receptor antagonist. In Langendorff-perfused rat hearts, the reperfusion-induced increase in water content was significantly reduced by ARL plus ATP. Under conditions favouring ATP degradation, an increase in myocardial edema was observed that could be blocked by 8-PT. CONCLUSION: ATP, either released from cells or exogenously applied, protects against reperfusion-induced failure of the coronary endothelial barrier. Inhibition of ATP degradation enhances the stabilizing effect of ATP on barrier function.

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart.

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Effect of ticagrelor on endothelial calcium signalling and barrier function.

The P2Y12 receptor is a Gi-coupled receptor whose activation inhibits adenylyl cyclase and thereby reduces the concentration of intracellular cAMP. Here the hypothesis was tested whether AR-C 66096 or ticagrelor, two direct-acting and reversibly binding P2Y12 receptor antagonists, protect endothelial cell (EC) barrier function by raising intracellular cAMP in ECs. The study was carried out on primary human umbilical vein ECs (HUVECs) and human pulmonary microvascular ECs (hPMECs). AR-C66096 (10 µM) induced a 50 % increase in cAMP in ECs whereas ticagrelor (2-10 µM) had no effect. Likewise, AR-C666096 antagonised thrombin-induced hyperpermeability in both HUVECs and hPMECs, but ticagrelor had no effect on basal EC monolayer permeability. Ticagrelor, however, sensitised ECs for thrombin-induced hyperpermeability and potentiated the thrombin effect. Ticagrelor but not AR-C66096 caused an increase in cytosolic calcium ([Ca2+]i). This increase in [Ca2+]i was abrogated by LaCl3 (Ca2+ influx inhibitor) but not by xestospongin C (IP3 receptor antagonist) or by depletion of intracellular stores with thapsigargin, suggesting a Ca2+ influx from the extracellular space. Accordingly, ticagrelor caused an increase in myosin light chain (MLC) phosphorylation, an important regulator of EC contractile machinery and thus permeability, which was abrogated by LaCl3. The ability of ticagrelor to potentiate EC permeability was abrogated by a MLC kinase inhibitor (ML-7; 10 µM). Our data demonstrate that the P2Y12 receptor antagonist AR-C66096 exerts a protective effect on ECs in vitro, possibly by raising intracellular cAMP, whereas ticagrelor sensitises EC barrier function by inducing Ca2+ influx and activating downstream EC contractile machinery.

Targeting of Extracellular RNA Reduces Edema Formation and Infarct Size and Improves Survival After Myocardial Infarction in Mice.

BACKGROUND: Following myocardial infarction (MI), peri-infarct myocardial edema formation further impairs cardiac function. Extracellular RNA (eRNA) released from injured cells strongly increases vascular permeability. This study aimed to assess the role of eRNA in MI-induced cardiac edema formation, infarct size, cardiac function, and survival after acute MI and to evaluate the therapeutic potential of ribonuclease 1 (RNase-1) treatment as an eRNA-degrading intervention. METHODS AND RESULTS: C57BL/6J mice were subjected to MI by permanent ligation of the left anterior descending coronary artery. Plasma eRNA levels were significantly increased compared with those in controls starting from 30 minutes after ligation. Systemic application of RNase-1, but not DNase, significantly reduced myocardial edema formation 24 hours after ligation compared with controls. Consequently, eRNA degradation by RNase-1 significantly improved the perfusion of collateral arteries in the border zone of the infarcted myocardium 24 hours after ligation of the left anterior descending coronary artery, as detected by micro-computed tomography imaging. Although there was no significant difference in the area at risk, the area of vital myocardium was markedly larger in mice treated with RNase-1 compared with controls, as detected by Evans blue and 2,3,5-triphenyltetrazolium chloride staining. The increase in viable myocardium was associated with significantly preserved left ventricular function, as assessed by echocardiography. Moreover, RNase-1 significantly improved 8-week survival following MI. CONCLUSIONS: eRNA is an unrecognized permeability factor in vivo, associated with myocardial edema formation after acute MI. RNase-1 counteracts eRNA-induced edema formation and preserves perfusion of the infarction border zone, reducing infarct size and protecting cardiac function after MI.

ATP antagonism of thrombin-induced endothelial barrier permeability.

OBJECTIVES: Thrombin induces endothelial barrier failure by activating the contractile machinery of endothelial cells. Contractile activation is due to an increase in myosin light chain (MLC) phosphorylation. Here, it was investigated whether stimulation of endothelial cells with ATP can interrupt this thrombin-induced pathomechanism. METHODS: In cultured human umbilical vein endothelial cells, cytosolic calcium [Ca(2+)](i) (Fura 2 method), phosphorylation of MLC, isometric tension and permeability for albumin were studied. RESULTS: Thrombin (0.2 U/ml) increased [Ca(2+)](i) from a basal level of 78+/-8 to 570+/-63 nM (mean+/-S.D., n=5, P<0.05), MLC phosphorylation from 71+/-7 to 163+/-18%, isometric tension from 157+/-17 to 232+/-26 microN, and permeability from 2.8+/-0.4 to 11.6+/-1 x 10(-6) cm/s. Co-presence of ATP (10 microM) and thrombin did not alter the [Ca(2+)](i) rise, but reduced MLC phosphorylation to 59.8+/-10%, isometric tension to 174+/-14 microN, and permeability to 5.4+/-0.6 x 10(-6) cm/s. The thrombin-induced rise in MLC phosphorylation was sensitive to reduction of [Ca(2+)](i) It was accompanied by an increase in Rho activation, and was inhibited by Y-27632 (10 microM), a Rho-kinase blocker. The ATP-induced decrease in MLC phosphorylation was not sensitive to [Ca(2+)](i). It was not accompanied by changes in RhoA activation, and could not by suppressed by Y-27632. CONCLUSIONS: ATP antagonizes the Ca(2+)- and Rho-dependent effects of thrombin on MLC phosphorylation most likely by a Ca(2+)- and Rho-independent activation of MLC phosphatase. It thereby functionally antagonizes the thrombin-induced increase in monolayer tension and permeability.