Cancer activity and bleeding events post-PCI

Purpose : Limited data exist about clinically relevant bleeding events related to antiplatelet therapy after percutaneous coronary intervention (PCI) in cancer patients. We investigated the risk factors for clinically relevant bleeding events in patients with cancer after PCI with stent implantation. Patients and Methods : Patients with solid cancer subjected to first PCI were divided into active (n = 45) and non-active cancer groups (n = 44). The active group included non-operable patients on treatment or with metastasis ; the non-active included those already subjected to or for whom radical surgery was planned within 3 months after the index PCI. Results : During a median follow-up of 2.2 years, 11 bleeding events occurred, with only one occurring in the non-active cancer group. Half of them occurred during the dual-antiplatelet therapy (DAPT) period, and the rest occurred during single-antiplatelet therapy (SAPT) period. Kaplan-Meier analysis showed significantly more bleeding events in the active cancer group (p = 0.010). Multivariate Cox regression hazard analysis revealed cancer activity as a significant independent risk factor for bleeding (p = 0.023) ; but not for three-point major adverse cardiovascular events. Conclusion : Clinically relevant bleeding risk after PCI was significantly lower in non-active cancer. Active cancer group had clinically relevant bleeding during both DAPT and SAPT periods

Molecular Characterization of Striated Muscle-Specific Gab1 Isoform as a Critical Signal Transducer for Neuregulin-1/ErbB Signaling in Cardiomyocytes

<div><p>Grb2-associated binder (Gab) docking proteins regulate signals downstream of a variety of growth factors and receptor tyrosine kinases. Neuregulin-1 (NRG-1), a member of epidermal growth factor family, plays a critical role for cardiomyocyte proliferation and prevention of heart failure via ErbB receptors. We previously reported that Gab1 and Gab2 in the myocardium are essential for maintenance of myocardial function in the postnatal heart via transmission of NRG-1/ErbB-signaling through analysis of Gab1/Gab2 cardiomyocyte-specific double knockout mice. In that study, we also found that there is an unknown high-molecular weight (high-MW) Gab1 isoform (120 kDa) expressed exclusively in the heart, in addition to the ubiquitously expressed low-MW (100 kDa) Gab1. However, the high-MW Gab1 has been molecularly ill-defined to date. Here, we identified the high-MW Gab1 as a striated muscle-specific isoform. The high-MW Gab1 has an extra exon encoding 27 amino acid residues between the already-known 3^rd and 4th exons of the ubiquitously expressed low-MW Gab1. Expression analysis by RT-PCR and immunostaining with the antibody specific for the high-MW Gab1 demonstrate that the high-MW Gab1 isoform is exclusively expressed in striated muscle including heart and skeletal muscle. The ratio of high-MW Gab1/ total Gab1 mRNAs increased along with heart development. The high-MW Gab1 isoform in heart underwent tyrosine-phosphorylation exclusively after intravenous administration of NRG-1, among several growth factors. Adenovirus-mediated overexpression of the high-MW Gab1 induces more sustained activation of AKT after stimulation with NRG-1 in cardiomyocytes compared with that of β-galactosidase. On the contrary, siRNA-mediated knockdown of the high-MW Gab1 significantly attenuated AKT activation after stimulation with NRG-1 in cardiomyocytes. Taken together, these findings suggest that the striated muscle-specific high-MW isoform of Gab1 has a crucial role for NRG-1/ErbB signaling in cardiomyocytes.</p></div

High-MW Gab1 is expressed in striated muscles of mouse embryo.

<p>A) and B) Immunohistochemical analyses of the high-MW Gab1 expression in mouse embryos. Sagittal sections of mouse embryos at E11.5 (A) and horizontal sections of mouse embryos at E17.5 were immunostained for the high-MW Gab1 (green) and α-Actinin (red). Representative images of 5 experiments (A) and 3 experiments (B) are shown.</p

High-MW mRNA is expressed in striated-muscle.

<p><b>A)</b> Expression pattern of Gab1 mRNA in mouse tissues and embryos. The primers for the high-MW Gab1 (top panel), Gab1 (middle panel), or GAPDH (control, bottom panel) were used to amplify cDNA from mouse tissues and embryos. B) and C) Quantitative expression analysis of <i>high-MW Gab1</i> (B) and <i>Gab1</i> (C) mRNAs in hearts at E11.5, E18.5, P7, and P28. (normalized to <i>GAPDH</i> mRNA; n = 3). Values are shown as mean±SEM for 3 separate experiments. One-way ANOVA followed by Tukey’s test was used to analyses differences. D) Quantitative expression analysis of <i>high-MW Gab1</i> to <i>Gab1</i> mRNA ratio in hearts at E11.5, E18.5, P7, and P28 (n = 3). Values are shown as mean±SEM for 3 separate experiments. One-way ANOVA followed by Tukey’s test was used to analyses differences. E) Expression patterns of high-MW Gab1 (arrowhead) and low-MW Gab1 (arrow) protein in mouse hearts at E18.5, P7, P28, and P56. Following immunoprecipitation with anti-Gab1 serum, expression of Gab1 was examined by immunoblotting analysis with anti-Gab1 antibody. SHP2 was examined as a loading control. Representative blots of 2 experiments are shown.</p

High-MW Gab1 is expressed in the heart.

<p>Expression patterns of A) Gab1 and B) high-MW Gab1 protein in mouse tissues. Following immunoprecipitation with A) anti-Gab1 or B) anti-high-MW Gab1 serum, expression of Gab1 was examined by immunoblotting analysis with anti-Gab1 antibody. The high-MW Gab1 is expressed in heart (arrowheads), and the low-MW Gab1 is commonly expressed (arrow). SHP2 was examined as a loading control. Results are representatives of three independent experiments.</p

Forced expression of high-MW Gab1 enhances activation of AKT in response to NRG-1 in NRCMs.

<p>A) Tyrosine phosphorylation of Gab1 and its association with p85 and SHP2 were analyzed by immunoprecipitation of the NRCMs lysates. NRCMs, infected with the indicated adenovirus vectors, were stimulated with NRG-1 (20 ng/ml) for indicated time periods and cell lysates were subjected with immunoprecipitation with anti-Gab1 serum, followed by immunoblotting analysis using the antibodies indicated at the left. Values are expressed relative to the control group. Representative blots of 2 experiments are shown. B) Phosphorylation of Gab1, ERK1/2, and AKT were assessed by phosphor-specific antibodies. Representative blots of 4 experiments are shown. C) Phosphorylation of ERK1/2 was quantified against total ERK1/2 (n = 4). D) Phosphorylation of AKT on Ser-473 was quantified against total AKT (n = 4). Values are shown as means±SEM for 4 separate experiments. One-way ANOVA followed by Tukey’s test was used to analyze statistical differences. **P<0.01, ***P<0.001 for the indicated groups. #P<0.01, ##P<0.001 vs β-gal expressing cells at the same time after stimulation.</p

SiRNA-mediated knockdown of high-MW Gab1 attenuates sustained activation of AKT after NRG-1 stimulation in NRCMs.

<p>A) Representative images of western blot analysis. NRCMs, transfected with the indicated siRNA, were stimulated with NRG-1 (20 ng/ml) for indicated time periods. Cell lysates were collected and subjected to immunoblotting analyses using the antibodies indicated at the left. Representative blots of 5 experiments are shown. B) Phosphorylation of ERK1/2 was quantified against total ERK1/2 (n = 5). C) Phosphorylation of AKT on Ser-473 was quantified against total AKT (n = 5). Values are shown as means±SEM for 5separate experiments. One-way ANOVA followed by Tukey’s test was used to analyze differences. *P<0.05, **P<0.01, and ***P<0.001 for the indicated groups. §P<0.05, #P<0.01, ##P<0.001 vs β-gal expressing cells at the same time after stimulation.</p

High-MW Gab1 is exclusively activated after stimulation with NRG-1 in heart.

<p>A) Tyrosine phosphorylation of the high-MW Gab1 was analyzed by immunoprecipitation of the heart lysates. Mouse heart lysates were prepared at 5 min after injection with the cytokines and growth factors listed at top. Heart lysates were subjected to immunoprecipitation with anti-high-MW Gab1 serum, followed by western blot analysis using the antibodies indicated at the left. Arrowheads denote the high-MW Gab1. B) Activation level of ErbB4 was assessed by phospho-specific antibody. C) Activation levels of ERK1/2 and AKT were assessed by phospho-specific antibodies. Heart lysates were subjected to western blot analysis with the indicated antibodies. Representative blots of 3 experiments are shown. D) Phosphorylation of ERK1/2 was quantified against total ERK1/2 (n = 3). E) Phosphorylation of AKT on Ser-473 was quantified against total AKT (n = 3). Values are shown as means±SEM for 3 separate experiments. One-way ANOVA followed by Tukey’s test was used to analyze differences. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 for the indicated groups.</p