Loss of DPP4 activity is related to a prothrombogenic status of endothelial cells: implications for the coronary microvasculature of myocardial infarction patients

Pro-coagulant and pro-inflammatory intramyocardial (micro)vasculature plays an important role in acute myocardial infarction (AMI). Currently, inhibition of serine protease dipeptidyl peptidase 4 (DPP4) receives a lot of interest as an anti-hyperglycemic therapy in type 2 diabetes patients. However, DPP4 also possesses anti-thrombotic properties and may behave as an immobilized anti-coagulant on endothelial cells. Here, we studied the expression and activity of endothelial DPP4 in human myocardial infarction in relation to a prothrombogenic endothelial phenotype. Using (immuno)histochemistry, DPP4 expression and activity were found on the endothelium of intramyocardial blood vessels in autopsied control hearts (n = 9). Within the infarction area of AMI patients (n = 73), this DPP4 expression and activity were significantly decreased, coinciding with an increase in Tissue Factor expression. In primary human umbilical vein endothelial cells (HUVECs), Western blot analysis and digital imaging fluorescence microscopy revealed that DPP4 expression was strongly decreased after metabolic inhibition, also coinciding with Tissue Factor upregulation. Interestingly, inhibition of DPP4 activity with diprotin A also enhanced the amount of Tissue Factor encountered and induced the adherence of platelets under flow conditions. Ischemia induces loss of coronary microvascular endothelial DPP4 expression and increased Tissue Factor expression in AMI as well as in vitro in HUVECs. Our data suggest that the loss of DPP4 activity affects the anti-thrombogenic nature of the endothelium

Homocysteine Induces Phosphatidylserine Exposure in Cardiomyocytes through Inhibition of Rho Kinase and Flippase Activity.

AIMS: Increased levels of homocysteine (Hcy) form an independent risk factor for cardiovascular disease. In a previous study we have shown that Hcy induced phosphatidylserine (PS) exposure to the outer leaflet of the plasma membrane in cardiomyocytes, inducing a pro-inflammatory phenotype. In the present study the mechanism(s) involved in Hcy-induced PS exposure were analyzed. METHODS: H9c2 rat cardiomyoblasts were subjected to 2.5 mM D,L-Hcy and analyzed for RhoA translocation and activity, Rho Kinase (ROCK) activity and expression and flippase activity. In addition, the effect of ROCK inhibition with Y27632 on Hcy-induced PS exposure and flippase activity was analyzed. Furthermore, GTP and ATP levels were determined. RESULTS: Incubation of H9c2 cells with 2.5 mM D,L-Hcy did not inhibit RhoA translocation to the plasma membrane. Neither did it inhibit activation of RhoA, even though GTP levels were significantly decreased. Hcy did significantly inhibit ROCK activation, but not its expression, and did inhibit flippase activity, in advance of a significant decrease in ATP levels. ROCK inhibition via Y27632 did not have significant added effects on this. CONCLUSION: Hcy induced PS exposure in the outer leaflet of the plasma membrane in cardiomyocytes via inhibition of ROCK and flippase activity. As such Hcy may induce cardiomyocytes vulnerable to inflammation in vivo in hyperhomocysteinaemia patients

Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47(phox).

Item does not contain fulltextWe previously found that homocysteine (Hcy) induced plasma membrane flip-flop, apoptosis, and necrosis in cardiomyocytes. Inactivation of flippase by Hcy induced membrane flip-flop, while apoptosis was induced via a NOX2-dependent mechanism. It has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in hyperhomocysteinemia (HHC)-induced pathogenesis of cardiovascular disease. Therefore, we evaluated whether the observed cytotoxic effect of Hcy in cardiomyocytes is SAH dependent. Rat cardiomyoblasts (H9c2 cells) were treated under different conditions: (1) non-treated control (1.5 nM intracellular SAH with 2.8 muM extracellular L -Hcy), (2) incubation with 50 muM adenosine-2,3-dialdehyde (ADA resulting in 83.5 nM intracellular SAH, and 1.6 muM extracellular L -Hcy), (3) incubation with 2.5 mM D, L -Hcy (resulting in 68 nM intracellular SAH and 1513 muM extracellular L -Hcy) with or without 10 muM reactive oxygen species (ROS)-inhibitor apocynin, and (4) incubation with 100 nM, 10 muM, and 100 muM SAH. We then determined the effect on annexin V/propodium iodide positivity, flippase activity, caspase-3 activity, intracellular NOX2 and p47(phox) expression and localization, and nuclear ROS production. In contrast to Hcy, ADA did not induce apoptosis, necrosis, or membrane flip-flop. Remarkably, both ADA and Hcy induced a significant increase in nuclear NOX2 expression. However, in contrast to ADA, Hcy additionally induced nuclear p47(phox) expression, increased nuclear ROS production, and inactivated flippase. Incubation with SAH did not have an effect on cell viability, nor on flippase activity, nor on nuclear NOX2-, p47phox expression or nuclear ROS production. HHC-induced membrane flip-flop and apoptosis in cardiomyocytes is due to increased Hcy levels and not primarily related to increased intracellular SAH, which plays a crucial role in nuclear p47(phox) translocation and subsequent ROS production.1 december 201

PX-18 Protects Human Saphenous Vein Endothelial Cells under Arterial Blood Pressure

BACKGROUND: Arterial blood pressure-induced shear stress causes endothelial cell apoptosis and inflammation in vein grafts after coronary artery bypass grafting. As the inflammatory protein type IIA secretory phospholipase A2 (sPLA2-IIA) has been shown to progress atherosclerosis, we hypothesized a role for sPLA2-IIA herein. METHODS: The effects of PX-18, an inhibitor of both sPLA2-IIA and apoptosis, on residual endothelium and the presence of sPLA2-IIA were studied in human saphenous vein segments (n = 6) perfused at arterial blood pressure with autologous blood for 6 hrs. RESULTS: The presence of PX-18 in the perfusion blood induced a significant 20% reduction in endothelial cell loss compared to veins perfused without PX18, coinciding with significantly reduced sPLA2-IIA levels in the media of the vein graft wall. In addition, PX-18 significantly attenuated caspase-3 activation in human umbilical vein endothelial cells subjected to shear stress via mechanical stretch independent of sPLA2-IIA. CONCLUSIONS: In conclusion, PX-18 protects saphenous vein endothelial cells from arterial blood pressure-induced death, possibly also independent of sPLA2-IIA inhibition

A Sensitive, Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

<div><p>Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.</p></div

Antibody reagents for immunofluorescence detection of dystrophin and spectrin.

<p>Dilution of primary antibodies in PBS containing 0.05% Tween, 5% FBS: ^a1∶60,^b1∶300, ^c1∶200.</p><p>Dilution of secondary antibodies in PBS containing 0.05% Tween: ^d1∶250,^e1∶1000.</p><p>FBS, fetal bovine serum; Ig, immunoglobulin; PBS, phosphate-buffered saline.</p><p>Antibody reagents for immunofluorescence detection of dystrophin and spectrin.</p

Immunofluorescence analysis of dystrophin intensities per fiber in a muscle fiber population.

<p><b>A.</b> Cryosections of a healthy control muscle sample (control 3, quadriceps muscle) co-stained with anti-dystrophin antibody MANDYS106 and an anti-spectrin antibody. The sections were imaged using a confocal microscope, with the green channel (Alexa488) showing dystrophin and the red channel (Alexa594) showing spectrin. <b>B.</b> Images were analyzed using customized Definiens software, which automatically identifies individual muscle fiber membranes using their spectrin signal and measures the membrane dystrophin intensity per muscle fiber. <b>C.</b> Dystrophin intensity distribution in muscle fiber population analyzed, shown as a regular histogram (blue line) or a cumulative histogram (red line). (au: arbitrary units).</p

Dystrophin signal specificity using MANDYS106.

<p>Immunofluorescence intensities measured in pre-treatment biopsies of different patients with DMD at microscope settings suitable for imaging DMD samples (7% laser intensity). <b>A.</b> (Left): Dystrophin staining (AlexaFluor488). All fibers exhibit varying intensities of dystrophin (trace dystrophin) and certain fibers termed “revertants” express higher levels of dystrophin. (Middle): Spectrin staining (AlexaFluor594): Fibers with equivalent spectrin intensities exhibited varying levels of dystrophin staining. (Right): Histogram of the mean dystrophin intensity in the fiber population depicting the fibers expressing trace dystrophin and revertant fibers. The mean dystrophin intensity of the fiber population expressing trace dystrophin is indicated by the arrow. <b>B.</b> Specificity of MANDYS106 antibody compared with isotype control for measuring dystrophin expression. Immunofluorescence images of MANDYS106 (left) or negative control isotype staining (right) of patients DMD 4 (deletion exon 45) and DMD 6 (deletion exon 43), and the corresponding histograms of mean dystrophin intensity (cumulative graphs). <b>C.</b> Dystrophin intensities in patients with DMD with different exon deletions (DMD patient 1: deletion exon 48–50; DMD patient 3: deletion exon 50; DMD patient 5: deletion exon 45; DMD patient 6: deletion exon 45). These samples were analyzed in the same experiment. (au: arbitrary units; del: deletion; DMD: Duchenne muscular dystrophy).</p

Dystrophin intensity in control muscle samples, and pre-treatment DMD and BMD muscle biopsies.

<p><b>A.</b> Representative immunofluorescence images (1 of 10 images) from the tibialis anterior muscle samples (from one control subject (CTRL2), two patients with DMD and four patients with BMD. Analysis was performed in the same experiment using the MANDYS106 antibody and ‘control’ imaging settings (1% laser intensity). <b>B.</b> Corresponding dystrophin membrane intensity distribution in the fiber populations analyzed (cumulative graphs). (au: arbitrary units; BMD: Becker muscular dystrophy; CTRL: control; DMD: Duchenne muscular dystrophy).</p