Concentration-dependent inhibition of ADP-induced P-selectin expression using 2-MeSAMP and TGX-221.

<p>Heparinized human whole blood (n = 4) was treated for 5 minutes at 37°C with (A) PBS as control, 2-MeSAMP (10 and 100 µM; “2-MeS”) or a combination (100 µM each) of 2-MeSAMP and MRS2179 and (B) propylene glycol (“PG”) as control or TGX-221 (0.5 or 2.2 µM; “TGX”). Afterwards platelets were activated using ADP (final concentration: 20 µM) and the percentage of P-selectin expressing platelets under a pre-set histogram marker was analyzed in flow cytometry using an anti-P-selectin mAb. Data are given as means and SD.</p

P₂Y₁₂/P₂Y₁ blockade in combination with TGX-221 prevents upregulation of the Mac-1 receptor on granulocytes.

<p>Granulocyte activation was measured before (“before circ.”) and 30 minutes after hypothermic ECC in groups treated with PBS (control group), 2-MeSAMP and MRS2179 (“P₂Y block”; 100 µM each), propylene glycol (“PG”, control group), TGX-221 (“TGX”; 2.2 µM) and a combination of P₂Y block (100 µM each) and PI3K p110β inhibition with TGX-221 (2.2 µM; “P₂Y block + TGX”;). Mac-1 expression on granulocytes was evaluated using geometric mean values of antibody fluorescence in flow cytometry. Data are given as means (n = 6) and SD; groups were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; ***p<0.001.</p

Blockade of P₂Y₁₂ and P₂Y₁ as well as PI3K p110β inhibition profoundly inhibits hypothermic ECC-induced P-selectin expression on platelets and platelet microparticles.

<p>Prior to (“before circ.”) and after hypothermic ECC (28°C, 30 minutes) flow cytometric analysis of P-selectin expression on platelets and PMPs was performed in groups treated with either PBS as control, 2-MeSAMP and MRS2179 (100 µM each; “P₂Y block”), propylene glycol (“PG”) as control, TGX-221 to inhibit PI3K p110β (2.2 µM; “TGX”) or a combination of 2-MeSAMP and MRS2179 (100 µM each) as well as TGX-221 (2.2 µM; “P₂Y block + TGX”). Representative dot plot indicating the identification of PMPs, single platelets and aggregates according to their size and granularity (A). Representative histogram overlay including a marker to identify percentages of P-selectin expressing platelets before circulation without additional stimulation (grey filled), before circulation with addition of ADP (20 µM; grey solid line) as well as 30 minutes after hypothermic ECC (black solid line) (B). Percentages of platelets (C) and PMPs (D) expressing P-selectin are depicted. Data in (C) and (D) are given as means (n = 6) and SD; groups were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; **p<0.01, ***p<0.001.</p

Effects of hypothermic ECC and platelet inhibitor treatment on platelet adhesion to the ECC surface, platelet-granulocyte aggregate formation and platelet counts.

<p>Human blood samples were either left untreated or treated with PBS (control group), a combination of 2-MeSAMP and MRS2179 (100 µM each; “P₂Y block”), propylene glycol (“PG”, control group), TGX-221 (2.2 µM; “TGX”) or a combination of 2-MeSAMP, MRS2179 and TGX-221 (“P₂Y block + TGX”). Blood, which was treated accordingly, was circulated in an <i>ex vivo</i> ECC model at 28°C for 30 minutes. A specific ELISA method was employed to detect platelet adhesion to the ECC surface (A; n = 6). Platelet-granulocyte aggregate formation was measured in flow cytometry before circulation (“before circ.”) and after circulation in all treatment groups (B; n = 4) according to the fluorescence of an anti-CD15-PE antibody on aggregates (aggregate region in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038455#pone-0038455-g003" target="_blank">figure 3A</a>) under a pre-set histogram marker. Platelet counts were measured in all samples (C; n = 6). Data are given as means and SD; normally distributed data were compared using RM-ANOVA with Bonferroni’s multiple comparison test (A, B); not normally distributed data were analyzed using a non-parametrical test (Friedman test with Dunǹs multiple comparison test; C); *p<0.05; **p<0.01; ***p<0.001.</p

GPIIb/IIIa and GPIbα expression as well as vWF binding are not influenced by <i>ex vivo</i> hypothermic ECC and antiplatelet agents, while platelet PI3K p110β inhibition alone or in combination with P₂Y₁₂/P₂Y₁ receptor blockade reduce GPIIb/IIIa activation induced by hypothermic ECC.

<p>Human blood was left untreated (“before circ.”) or treated <i>ex vivo</i> with PBS (control), a combination of 2-MeSAMP and MRS2179 to block P₂Y receptors (100 µM each; “P₂Y block”), propylene glycol (“PG”, control), TGX-221 to inhibit PI3K p110β (2.2 µM; “TGX”) or a combination of 2-MeSAMP, MRS2179 (100 µM each) and TGX-221 (2.2 µM; “P₂Y block + TGX”). All treated samples were circulated in an ECC model for 30 minutes at 28°C. Expression of GPIIb (A; n = 4), activated GPIIb/IIIa (B; n = 6), GPIbα (C; n = 6) as well as vWF binding (D; n = 4) were evaluated in flow cytometry using specific antibodies. ADP stimulation (20 µM) before and after hypothermic ECC was performed as positive control for GPIbα expression (C) and vWF binding (D) in the PBS group. Geometric mean fluorescence values of fluorescently labeled antibodies are given in diagrams as means and SD; not normally distributed data were analyzed using a non-parametrical test (Friedman test with Dunǹs multiple comparison test; A, D); normally distributed data (B, C) were compared using RM-ANOVA with Bonferroni’s multiple comparison test; *p<0.05; **p<0.01; ***p<0.001.</p

Overview of a pharmacological strategy for platelet protection during hypothermic ECC employing P₂Y₁₂ and P₂Y₁ receptor blockers as well as a PI3K p110β inhibitor.

<p>Overview of a pharmacological strategy for platelet protection during hypothermic ECC employing P₂Y₁₂ and P₂Y₁ receptor blockers as well as a PI3K p110β inhibitor.</p

Image_2_Crosstalk between cytotoxic CD8+ T cells and stressed cardiomyocytes triggers development of interstitial cardiac fibrosis in hypertensive mouse hearts.tiff

Graphical AbstractCardiac fibrosis developed from fibrotic remodelling due to hypertension leads to heart failure. Immune cells are abundantly accumulated in fibrotic regions, but exact mechanisms how they contributed to cardiac fibrosis are not clear. Here we demonstrated that CD8 T cells are major contributor to cardiac fibrosis in hypertensive hearts. Stressed cardiomyocytes express STING-dependent RAE-1 that activates NKG2D + CD8 T cells to induce apoptosis of stressed cardiomyocytes. Preventing STING signalling in stressed cardiomyocytes attenuates cardiac fibrosis. Pharmacologically inhibiting cardiomyocayte-RAE-1 and CD8+ T cell-NKG2D axis may be a potential therapeutic strategy to prevent cardiac fibrosis in HFpEF patients.</p

Image_1_Crosstalk between cytotoxic CD8+ T cells and stressed cardiomyocytes triggers development of interstitial cardiac fibrosis in hypertensive mouse hearts.tiff

Graphical AbstractCardiac fibrosis developed from fibrotic remodelling due to hypertension leads to heart failure. Immune cells are abundantly accumulated in fibrotic regions, but exact mechanisms how they contributed to cardiac fibrosis are not clear. Here we demonstrated that CD8 T cells are major contributor to cardiac fibrosis in hypertensive hearts. Stressed cardiomyocytes express STING-dependent RAE-1 that activates NKG2D + CD8 T cells to induce apoptosis of stressed cardiomyocytes. Preventing STING signalling in stressed cardiomyocytes attenuates cardiac fibrosis. Pharmacologically inhibiting cardiomyocayte-RAE-1 and CD8+ T cell-NKG2D axis may be a potential therapeutic strategy to prevent cardiac fibrosis in HFpEF patients.</p

Equal blocking of monocyte binding to endothelial cells by anti-EPCR and APC under flow conditions.

<p>Monocyte binding to HUVECs in dynamic adhesion assay after 5 min (left) and 10 min venous flow (middle), and after 1 min arterial flow (right). Pre-treatment of HUVECs with anti-EPCR (dark grey bars), anti-Mac-1 (light grey bars), or with activated protein C (drotrecogin alfa; crosshatched bars) diminished monocyte adhesion to an equal extent compared to control. (*** <i>p</i><0.0001; ^###<i>p</i><0.0005; ^##<i>p</i><0.005; ^#<i>p</i><0.05 vs. no blocking; ns  =  statistically not signifiant).</p

<i>In vitro</i> Study of a Novel Stent Coating Using Modified CD39 Messenger RNA to Potentially Reduce Stent Angioplasty-Associated Complications

<div><p>Background</p><p>Stent angioplasty provides a minimally invasive treatment for atherosclerotic vessels. However, no treatment option for atherosclerosis-associated endothelial dysfunction, which is accompanied by a loss of CD39, is available, and hence, adverse effects like thromboembolism and restenosis may occur. Messenger RNA (mRNA)-based therapy represents a novel strategy, whereby <i>de novo</i> synthesis of a desired protein is achieved after delivery of a modified mRNA to the target cells.</p><p>Methods and Findings</p><p>Our study aimed to develop an innovative bioactive stent coating that induces overexpression of CD39 in the atherosclerotic vessel. Therefore, a modified CD39-encoding mRNA was produced by <i>in vitro</i> transcription. Different endothelial cells (ECs) were transfected with the mRNA, and CD39 expression and functionality were analyzed using various assays. Furthermore, CD39 mRNA was immobilized using poly(lactic-<i>co</i>-glycolic-acid) (PLGA), and the transfection efficiency in ECs was analyzed. Our data show that ECs successfully translate <i>in vitro</i>-generated CD39 mRNA after transfection. The overexpressed CD39 protein is highly functional in hydrolyzing ADP and in preventing platelet activation. Furthermore, PLGA-immobilized CD39 mRNA can be delivered to ECs without losing its functionality.</p><p>Summary</p><p>In summary, we present a novel and promising concept for a stent coating for the treatment of atherosclerotic blood vessels, whereby patients could be protected against angioplasty-associated complications.</p></div