P2Y12-dependent regulation of emergency hematopoiesis after myocardial infarction

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
H Seung ◽  
C Wadle ◽  
T Hopp ◽  
D Duerschmied ◽  
I Hilgendorf ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Wound Healing ◽  
Left Ventricular ◽  
P2y12 Receptor ◽  
Myocardial Inflammation ◽  
Funding Source ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Adp Receptor

Abstract Introduction Inflammation is essential for wound healing after myocardial infarction (MI). Leukocytes, especially neutrophils and monocytes, orchestrate removal of necrosis and regulation of tissue remodeling. Beside local recruitment from the blood, leukocyte supply via increased hematopoiesis is of major relevance for post-ischemic myocardial inflammation. Little is known about the pathways that carry the signals for increased demand for leukocytes from the site of injury to upstream hematopoietic stem cells (LSK) in the bone marrow (BM). In this study, we investigate the role of the P2Y12-ADP-receptor mediated regulation for emergency hematopoiesis after MI. Methods For standardized MI, a model of permanent coronary ligation was used in C57/Bl6 and P2Y12−/− mice. Changes in plasmatic ADP levels after MI were screened using ELISA, whereas the expression of the P2Y12-ADP-receptor in cell populations isolated from the BM was investigated by qPCR. CFU assays added further functional insight on hematopoietic proliferation in vitro. The effect of P2Y12 on the hematopoietic system after MI was investigated by inhibiting the P2Y12-receptor via prasugrel and compared to inhibition of the thromboxane pathway via acetylsalicylic acid (ASA). Proliferation of LSK in BM and leukocyte composition in blood, BM and infarct tissue after MI were assessed via FACS. Leukocyte composition in the infarcted myocardium was validated by immunohistochemistry. Finally, left ventricular function (LV-EF) and remodeling were investigated by echocardiography. Results 24 h after MI, we found a peak of plasmatic ADP levels. LSK as upstream hematopoietic progenitors in the BM express a P2Y12-receptor, which was validated on transcriptional and protein level. Whereas ADP stimulation of LSK led to significantly larger colony growth in vitro on the one hand, percentage of cycling LSK were significantly reduced 48 h after MI in P2Y12−/− mice compared to WT mice, assessed by Ki67/DAPI cell cycle analysis. Prasugrel treatment showed similar effects, translating into reduced numbers of downstream hematopoietic progenitors GMP and MDP 72 h after MI. Treatment with ASA however had no significant effect neither on cycling LSK nor progenitor populations. Consequently, decreased medullary hematopoiesis under P2Y12-inhibition led to reduced infiltration of inflammatory cells in the infarct tissue 7 days after MI, finally resulting in significantly improved outcome in terms of LV-EF 3 weeks after MI. Conclusion In this study, we demonstrate that P2Y12-mediated signaling is involved in emergency hematopoiesis after MI and advocates post-MI inflammation. In turn, inhibition of P2Y12-mediated signaling contributes to improved wound healing and prevention of adverse cardiac remodeling after MI, which adds a yet unknown mechanism to the success story of modern P2Y12-receptor blockers. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Randomized, double blind, placebo-controlled, safety and efficacy study of dutogliptin in combination with filgrastim in early recovery post-MI: rationale, design and first interim analysis

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
D Von Lewinski ◽  
B Merkely ◽  
I Buysschaert ◽  
R.A Schatz ◽  
G.G Nagy ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Stem Cells ◽  
Adverse Events ◽  
Left Ventricular ◽  
Left Ventricular Ejection ◽  
Funding Source ◽  
Early Recovery ◽  
Ventricular Ejection Fraction ◽  
Combined Application

Abstract Background Regenerative therapies offer new approaches to improve cardiac function after acute ST-elevation myocardial infarction (STEMI). Mobilization of stem cells and homing within the infarcted area have been identified as the key mechanisms for successful treatment. Application of granulocyte-colony stimulating factor (G-CSF) is the least invasive way to mobilize stem cells while DDP4-inhibitor facilitates homing via stromal cell-derived factor 1 alpha (SDF-1α). Dutogliptin, a novel DPP4 inhibitor, combined with stem cell mobilization using G-CSF significantly improved survival and reduced infarct size in a murine model. Purpose We initiated a phase II, multicenter, randomized, placebo-controlled efficacy and safety study (N=140) analyzing the effect of combined application of G-CSF and dutogliptin, a small molecule DPP-IV-inhibitor for subcutaneous use after acute myocardial infarction. Methods The primary objective of the study is to evaluate the safety and tolerability of dutogliptin (14 days) in combination with filgrastim (5 days) in patients with STEMI (EF <45%) following percutaneous coronary intervention (PCI). Preliminary efficacy will be analyzed using cardiac magnetic resonance imaging (cMRI) to detect >3.8% improvement in left ventricular ejection fraction (LV-EF). 140 subjects will be randomized to filgrastim plus dutogliptin or matching placebos. Results Baseline characteristics of the first 26 patients randomized (24 treated) in this trial reveal a majority of male patients (70.8%) and a medium age of 58.4 years (37 to 84). During the 2-week active treatment period, 35 adverse events occurred in 13 patients, with 4 rated as serious (hospitalization due to pneumonia N=3, hospitalization due to acute myocardial infarction N=1), and 1 adverse event was rated as severe (fatal pneumonia), 9 moderate, and 25 as mild. 6 adverse events were considered possibly related to the study medication, including cases of increased hepatic enzymes (N=3), nausea (N=1), subcutaneous node/suffusion (N=1) and syncope (N=1). Conclusions Our data demonstrate that the combined application of dutogliptin and G-CSF appears to be safe on the short term and feasible after acute myocardial infarction and may represent a new therapeutic option in future. Funding Acknowledgement Type of funding source: Other. Main funding source(s): This research is funded by the sponsor RECARDIO, Inc., 1 Market Street San Francisco, CA 94150, USA. RECARDIO Inc. is funding the complete study. The Scientific Board of RECARDIO designed the study. Data Collection is at the participating sites. Interpretation of the data by the Scientific Board and Manuscript written by the authors and approved by the Sponsor

Prevalence and prognostic impact of left ventricular systolic dysfunction after acute myocardial infarction

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
T.J Jernberg ◽  
E.O Omerovic ◽  
E.H Hamilton ◽  
K.L Lindmark ◽  
L.D Desta ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Risk Factors ◽  
Myocardial Infarction ◽  
Atrial Fibrillation ◽  
Risk Factor ◽  
Reperfusion Therapy ◽  
Left Ventricular ◽  
Funding Source ◽  
Additional Risk Factor ◽  
Additional Risk

Abstract Background Left ventricular dysfunction after an acute myocardial infarction (MI) is associated with poor outcome. The PARADISE-MI trial is examining whether an angiotensin receptor-neprilysin inhibitor reduces the risk of cardiovascular death or worsening heart failure (HF) in this population. The aim of this study was to examine the prevalence and prognosis of different subsets of post-MI patients in a real-world setting. Additionally, the prognostic importance of some common risk factors used as risk enrichment criteria in the PARADISE-MI trial were specifically examined. Methods In a nationwide myocardial infarction registry (SWEDEHEART), including 87 177 patients with type 1 MI between 2011–2018, 3 subsets of patients were identified in the overall MI cohort (where patients with previous HF were excluded); population 1 (n=27 568 (32%)) with signs of acute HF or an ejection fraction (EF) <50%, population 2 (n=13 038 (15%)) with signs of acute HF or an EF <40%, and population 3 (PARADISE-MI like) (n=11 175 (13%)) with signs of acute HF or an EF <40% and at least one risk factor (Age ≥70, eGFR <60, diabetes mellitus, prior MI, atrial fibrillation, EF <30%, Killip III-IV and STEMI without reperfusion therapy). Results When all MIs, population 1 (HF or EF <50%), 2 (HF or EF <40%) and 3 (HF or EF <40% + additional risk factor (PARADISE-MI like)) were compared, the median (IQR) age increased from 70 (61–79) to 77 (70–84). Also, the proportion of diabetes (22% to 33%), STEMI (38% to 50%), atrial fibrillation (10% to 24%) and Killip-class >2 (1% to 7%) increased. After 3 years of follow-up, the cumulative probability of death or readmission because of heart failure in the overall MI population and in population 1 to 3 was 17.4%, 26.9%, 37.6% and 41.8%, respectively. In population 2, all risk factors were independently associated with death or readmission because of HF (Age ≥70 (HR (95% CI): 1.80 (1.66–1.95)), eGFR <60 (1.62 (1.52–1.74)), diabetes mellitus (1.35 (1.26–1.44)), prior MI (1.16 (1.07–1.25)), atrial fibrillation (1.35 (1.26–1.45)), EF <30% (1.69 (1.58–1.81)), Killip III-IV (1.34 (1.19–1.51)) and STEMI without reperfusion therapy (1.34 (1.21–1.48))) in a multivariable Cox regression analysis. The risk increased with increasing number of risk factors (Figure 1). Conclusion Depending on definition, post MI HF is present in 13–32% of all MI patients and is associated with a high risk of subsequent death or readmission because of HF. The risk increases significantly with every additional risk factor. There is a need to optimize management and improve outcomes for this high risk population. Figure 1 Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Novartis

scholarly journals Neutrophil extracellular traps induce MCP-1 release from endothelial cells at the plaque rupture site in acute myocardial infarction

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Ondracek ◽  
T.M Hofbauer ◽  
A Mangold ◽  
T Scherz ◽  
V Seidl ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Endothelial Cells ◽  
Plaque Rupture ◽  
Ex Vivo ◽  
Coronary Intervention ◽  
Neutrophil Extracellular Traps ◽  
Funding Source ◽  
Extracellular Traps

Abstract Introduction Leukocyte-mediated inflammation is crucial in acute myocardial infarction (AMI). We recently observed that neutrophil extracellular traps (NETs) are increased at the culprit site, promoting activation and differentiation of fibrocytes, cells with mesenchymal and leukocytic properties. Fibrocyte migration is mediated by monocyte chemoattractant protein (MCP)-1 and C-C chemokine receptor type 2 (CCR2). We investigated the interplay between NETs, fibrocyte function, and MCP-1 in AMI. Methods Culprit site and femoral blood of AMI patients was drawn during percutaneous coronary intervention. We characterized CCR2 expression of fibrocytes by flow cytometry. MCP-1 and the NET marker citrullinated histone H3 (citH3) were measured by ELISA. Fibrocytes were treated in vitro with MCP-1. Human coronary arterial endothelial cells (hCAECs) were stimulated with isolated NETs, and MCP-1 was measured by ELISA and qPCR. The influence of MCP-1 on NET formation in vitro was assessed using isolated neutrophils. Results We have included 50 consecutive AMI patients into the study. NETs and concentrations of MCP-1 were increased at the CLS. NET stimulation of hCAECs induced MCP-1 on mRNA and protein level. Increasing MCP-1 gradient was associated with fibrocyte accumulation at the site of occlusion. In the presence of higher MCP-1 these fibrocytes expressed proportionally less CCR2 than peripheral fibrocytes. In vitro, MCP-1 dose-dependently decreased fibrocyte CCR2 and reduced ex vivo NET release of healthy donor neutrophils. Conclusions NETs induce endothelial MCP-1 release, presumably promoting a chemotactic gradient for leukocyte and fibrocyte migration. MCP-1 mediated inhibition of NET formation could point to a negative feedback loop. These data will shed light on vascular healing. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund

scholarly journals Adropin-based dual treatment enhances the therapeutic potential of mesenchymal stem cells in rat myocardial infarction

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
HuiYa Li ◽  
DanQing Hu ◽  
Guilin Chen ◽  
DeDong Zheng ◽  
ShuMei Li ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Therapeutic Potential ◽  
Left Ventricular ◽  
Left Ventricular Ejection ◽  
Paracrine Factors ◽  
Dual Treatment

AbstractBoth weak survival ability of stem cells and hostile microenvironment are dual dilemma for cell therapy. Adropin, a bioactive substance, has been demonstrated to be cytoprotective. We therefore hypothesized that adropin may produce dual protective effects on the therapeutic potential of stem cells in myocardial infarction by employing an adropin-based dual treatment of promoting stem cell survival in vitro and modifying microenvironment in vivo. In the current study, adropin (25 ng/ml) in vitro reduced hydrogen peroxide-induced apoptosis in rat bone marrow mesenchymal stem cells (MSCs) and improved MSCs survival with increased phosphorylation of Akt and extracellular regulated protein kinases (ERK) l/2. Adropin-induced cytoprotection was blocked by the inhibitors of Akt and ERK1/2. The left main coronary artery of rats was ligated for 3 or 28 days to induce myocardial infarction. Bromodeoxyuridine (BrdU)-labeled MSCs, which were in vitro pretreated with adropin, were in vivo intramyocardially injected after ischemia, following an intravenous injection of 0.2 mg/kg adropin (dual treatment). Compared with MSCs transplantation alone, the dual treatment with adropin reported a higher level of interleukin-10, a lower level of tumor necrosis factor-α and interleukin-1β in plasma at day 3, and higher left ventricular ejection fraction and expression of paracrine factors at day 28, with less myocardial fibrosis and higher capillary density, and produced more surviving BrdU-positive cells at day 3 and 28. In conclusion, our data evidence that adropin-based dual treatment may enhance the therapeutic potential of MSCs to repair myocardium through paracrine mechanism via the pro-survival pathways.

Implication of anti-angiogenic VEGF-A165b in angiogenesis and systolic function after reperfused myocardial infarction

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
V Marcos Garces ◽  
C Rios-Navarro ◽  
L Hueso ◽  
A Diaz ◽  
C Bonanad ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Adjuvant Therapy ◽  
Ejection Fraction ◽  
Infarct Size ◽  
Systolic Function ◽  
Left Ventricular ◽  
Left Ventricular Ejection ◽  
Funding Source ◽  
Coronary Reperfusion ◽  
Ventricular Ejection Fraction

Abstract Background Angiogenesis participates in re-establishing microcirculation after myocardial infarction (MI). Purpose In this study, we aim to further understand the role of the anti-angiogenic isoform vascular endothelial growth factor (VEGF)-A165b after MI and explore its potential as a co-adjuvant therapy to coronary reperfusion. Methods Two mice MI models were formed: 1) permanent coronary ligation (non-reperfused MI), 2) transient 45-min coronary occlusion followed by reperfusion (reperfused MI); in both models, animals underwent echocardiography before euthanasia at day 21 after MI induction. Serum and myocardial VEGF-A165b levels were determined. In both experimental MI models, functional and structural implication of VEGF-A165b blockade was assessed. In a cohort of 104 ST-segment elevation MI patients, circulating VEGF-A165b levels were correlated with cardiovascular magnetic resonance-derived left ventricular ejection fraction at 6-months and with the occurrence of adverse events (death, heart failure and/or re-infarction). Results In both models, circulating and myocardial VEGF-A165b presence was increased 21 days after MI induction. Serum VEGF-A165b levels inversely correlated with systolic function evaluated by echocardiography. VEGF-A165b blockage increased capillary density, reduced infarct size, and enhanced left ventricular function in reperfused, but not in non-reperfused MI experiments. In patients, higher VEGF-A165b levels correlated with depressed ejection fraction and worse outcomes. Conclusions In experimental and clinical studies, higher serum VEGF-A165b levels associates with a worse systolic function. Its blockage enhances neoangiogenesis, reduces infarct size, and increases ejection fraction in reperfused, but not in non-reperfused MI experiments. Therefore, VEGF-A165b neutralization represents a potential co-adjuvant therapy to coronary reperfusion. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): This study was funded by “Instituto de Salud Carlos III” and “Fondos Europeos de Desarrollo Regional FEDER” (Exp. PIE15/00013, PI17/01836, PI18/00209 and CIBERCV16/11/00486).

Cardiac magnetic resonance T1 mapping allows for predicting adverse left ventricular remodeling post-reperfused st-elevation myocardial infarction

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
L Zhang ◽  
Y.K Guo ◽  
Z.G Yang ◽  
M.X Yang ◽  
K.Y Diao ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
T1 Mapping ◽  
Ventricular Remodeling ◽  
Tissue Injury ◽  
Left Ventricular ◽  
Funding Source ◽  
Native T1 ◽  
End Diastolic Volume ◽  
Significant Difference ◽  
Lv Remodeling

Abstract Background Cardiac magnet resonance (CMR) T1 mapping allows the quantitative characterization of the severity of tissue injury and predict functional recovery in acute myocardial infarction (AMI). Purpose The study aimed to investigate whether native T1 and ECV of infarct myocardium are influenced by microvascular obstruction (MVO) and have predictive value for adverse left ventricular (LV) remodeling post-infarction. Method A cohort of 54 patients with successfully reperfused STEMI underwent CMR imaging at a 3T scanner in AMI and 3 months post-infarction. Native T1 data was acquired using a modified Look-Locker inversion recovery (MOLLI) sequence, and ECV maps were calculated using blood sampled hematocrit. Manual regions-of-interest were drawn within the infarct myocardium to measure native T1 and ECV (native T1infarct and ECVinfarct, respectively). MVO identified as a low-intensity area within the infarct zone on LGE was eliminated. Results MVO was present in 36 patients (66.67%) in AMI. ECVinfarct in patients with MVO was different from those without (58.66±8.71% vs. 49.64±8.82%, P=0.001), while no significant difference in T1infarct was observed between patients with and without MVO (1474.7±63.5ms vs. 1495.4±98.0ms, P=0.352). ECV correlated well with the change in end-diastolic volume (all patients: r=0.564, P<0.001) and predicted LV remodeling in patients with and without MVO (rMVO absent = 0.626, P=0.005; rMVO present = 0.686, P<0.001; all patients: r=0.622, P<0.001); Native T1 was only associated with a 3-month change in LV end-diastolic volume (rMVO absent= 0.483, P=0.042) and predicted LV remodeling in patients without MVO (rMVO absent = 0.659, P=0.003). Furthermore, ECV had an association with LV remodeling (β=0.312, P=0.007) in multivariable logistic analysis. Conclusion Absolute native T1 in infarct myocardium might be affected by MVO but ECV isn't. ECV could predict LV remodeling in MI patients with and without MVO, while native T1 predict it in MI with MVO absent. Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): 1·3·5 project for disciplines of excellence, West China Hospital, Sichuan University

scholarly journals IL-6 blocks a discrete early step in lymphopoiesis

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 879-885 ◽  
Author(s):  
Kazuhiko Maeda ◽  
Yoshihiro Baba ◽  
Yoshinori Nagai ◽  
Kozo Miyazaki ◽  
Alexander Malykhin ◽  
...  
Keyword(s):  
Target Cells ◽  
Hematopoietic Progenitors ◽  
Early Step ◽  
Hematopoietic Stem ◽  
Lymphoid Lineage ◽  
Src Homology 2 ◽  
Multipotent Progenitors ◽  
Src Homology ◽  
Src Homology 2 Domain

Abstract Animals lacking Src homology 2 domain-containing inositol 5-phosphatase (SHIP) display a reduction in lymphopoiesis and a corresponding enhancement of myelopoiesis. These effects are mediated at least in part by elevated levels of interleukin 6 (IL-6). Here, we show the lymphopoiesis block in SHIP–/– mice is due to suppression of the lymphoid lineage choice by uncommitted progenitors. The suppression can be reproduced in vitro with recombinant IL-6, and IL-6 acts directly on hematopoietic progenitors. The block is partially overcome in SHIP–/– IL-6–/– double-deficient animals. IL-6 does not suppress but actually enhances proliferation of lymphoid-committed progenitors, indicating the IL-6 target cells are hematopoietic stem cells or multipotent progenitors. The findings suggest a mechanism for the lymphopenia that accompanies proinflammatory diseases.

Role of angiotensin-signalling in anthracycline-induced cardiotoxicity

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S Findlay ◽  
J.H Gill ◽  
R Plummer ◽  
C.J Plummer
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Angiotensin Ii ◽  
Late Onset ◽  
Left Ventricular Systolic Dysfunction ◽  
Left Ventricular ◽  
Cardiomyocyte Hypertrophy ◽  
Funding Source ◽  
Study Results

Abstract   Anthracycline chemotherapy remains a key component of cancer treatment regimens in both paediatric and adult patients. A significant issue with their use is the development of anthracycline-induced cardiotoxicity (AIC), with subclinical AIC and clinical heart failure observed in 13.8% and 3.1% of patients, respectively. The major clinical complication of AIC is the development of late-onset cardiotoxicity, occurring several years after drug administration, presenting as life-threatening heart failure (HF). Determining the relationship between subclinical AIC and late-onset HF, strategies for mitigation of AIC, and impacts upon the cancer survivor population remains a complex challenge. Administration of drugs targeting the angiotensin system, specifically angiotensin converting enzyme inhibitors (ACEi), have been reported to reduce AIC in the clinic. Whilst the therapeutic effect of ACEi in management of left ventricular systolic dysfunction and consequent HF is principally through optimisation of cardiac haemodynamics, the mechanism involved with mitigation of late-onset AIC several years after anthracycline exposure are currently unknown. Using a variety of human cardiomyocyte in vitro models we have previously demonstrated induction of cardiomyocyte hypertrophy by angiotensin II and anthracyclines. Importantly, selective blockade of the angiotensin II receptor 1 (ATR1) on cardiomyocytes mitigated the anthracycline-induced hypertrophic response, implicating synergism between AIC and angiotensin signalling in cardiomyocytes. Adult human ventricular cardiac myocyte AC10 cell-line were treated in vitro with a range of clinically relevant doxorubicin doses for clinically appropriate durations, with AT1 receptor gene expression evaluated using semi-quantitative PCR. Our results confirm a positive correlation between clinically-relevant concentration of doxorubicin and induction of genetic expression of ATR1 in AC10 cells, with up to 200% increases in ATR1 expression observed. Maximal doxorubicin-induced gene expression being observed at 8 and 24-hours, respectively. These preliminary results agreeing with clinical exposure parameters for this drug with protein expression studies being optimised to support these gene expression study results. Our preliminary studies also imply patients developing AIC carry a deleted polymorphism within intron 16 of the ACE gene and increased systemic levels of the ACE product angiotensin II, both with a known association to hypertrophic cardiomyopathy. Taken together, these data support our mechanistic hypothesis that a relationship exists between AIC and modulation of the angiotensin signalling pathway in cardiomyocytes, involving structural cellular changes and asymptomatic cardiac hypertrophy. An elevation in angiotensin II levels, potentially through polymorphisms in ACE, could thereby exacerbate anthracycline-induced hypertrophy and promote the development of late-onset anthracycline-induced HF. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Cancer Research UK funded PhD

Abstract 1770: Proteasome Switching is Associated with Adverse Cardiac Remodeling in a Transgenic Mouse Model of Sustained Inflammatory Signaling

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Wei Wang ◽  
Gabor Szalai ◽  
Natarajan Sivasubramanian ◽  
Douglas L Mann
Keyword(s):  
Proteolytic Activity ◽  
Cardiac Remodeling ◽  
Fold Increase ◽  
Left Ventricular ◽  
26S Proteasome ◽  
Myocardial Inflammation ◽  
Fluorescent Substrate ◽  
Lv Remodeling ◽  
Apoptotic Proteins

The 26S proteasome possess proteolytic activity and deubiquitinating (DUB) activity of ubiquitin tagged proteins. Whereas the proteolytic activity of the 26S proteasome facilitates protein degradation, proteasome DUB activity spares proteins from degradation by shortening the length of the ubiquitin chains, thereby preventing proteins from being degraded by the 26S proteasome. In yeast, increased DUB activity is beneficial by preventing depletion of ubiquitin pools critical for cell signaling. However, the role of DUB activity in mammalian systems is not known. We have shown that mice with cardiac restricted overexpression of tumor necrosis factor (sTNF mice) develop a heart failure phenotype characterized by progressive left ventricular (LV) remodeling and accumulation of pro-apoptotic proteins, including Smac/Diablo. To determine whether the adverse LV remodeling in sTNF mice was related to alterations in DUB activity we measured the cleavage of ubiquitin-AMC, an in vitro fluorescent substrate for DUBs, in purified preparations of the 26S proteasome obtained from hearts of 4 week old sTNF and littermate (LM) control mice. Compared to LM controls we observed a significant (p < 0.001) 60.8% decrease in activity of the 26S proteasome and a significant (p < 0.01) 24.2% increase in DUB activity in sTNF mouse hearts. There was also a significant (p < 0.01) 11-fold increase myocardial protein levels of USP14, a critical DUB associated with the 26S proteasome in sTNF mouse hearts. The decrease in 26S proteasome activity and increased DUB activity in sTNF mouse hearts was accompanied by an increase in myocardial levels of ubiquitinated SMAC/Diablo. Taken together these results show for the first time that sustained myocardial inflammation leads to switch in the function of the proteasome from a proteolytic function to a protein sparing function. Although this “proteasome switching” may provide a short-term adaptive benefit by preventing depletion of critical ubiquitin pools, it may lead to long-term maladaptive consequences by allowing the progressive accumulation of potentially harmful pro-apoptotic proteins in the cytosol, which may in turn promote programmed cell death and adverse cardiac remodeling.

Comparison of VASP-phosphorylation assay to light-transmission aggregometry in assessing inhibition of the platelet ADP P2Y12 receptor

2006 ◽  
Vol 96 (12) ◽  
pp. 767-773 ◽  
Author(s):  
Agnieszka Pampuch ◽  
Giovanni de Gaetano ◽  
Chiara Cerletti
Keyword(s):  
Light Transmission ◽  
Platelet Reactivity ◽  
Flow Cytometric Analysis ◽  
Individual Variability ◽  
P2y12 Receptor ◽  
Reactivity Index ◽  
Drug Induced ◽  
Light Transmission Aggregometry ◽  
Adp Receptor

SummaryThere is need to improve platelet function testing to monitor the response to antiplatelet drugs. We compared flow-cytometric analysis of intraplatelet vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) to light-transmission aggregometry for the detection of drug-induced in-vitro inhibition of the platelet P2Y12 ADP receptor on 22 healthy subjects (10 males, 12 females, 28.5 ± 6.6 years). The platelet reactivity index (PRI) of VASP was calculated both from mean fluorescence intensity (MFI) and percent of fluorescence-positive platelets in the presence of PGE1 with or without ADP (10 µM). Platelet aggregation was induced by ADP (1.25, 2.5 and 5 µM). Cangrelor, a competitive inhibitor of the P2Y12 receptor, preincubated 5 minutes, induced a concentration-dependent inhibition of platelet ADP-receptor function in both tests. Indeed PRI (%) based on either MFI or percent platelets gated were highly correlated with each other (r = 0.97, p<0.0001) and with aggregation in- duced by ADP. The IC50 of cangrelor against each of the three ADP concentrations used in aggregometry increased from 5.8 ± 3.4 nM to 23.1 ± 4.0 nM and to 98 ± 25 nM, respectively. The IC50 of cangrelor based on VASP-P was within the same range (25.5 ± 7.7 nM). No correlation was observed between IC50 values of cangrelor and ADP concentrations giving 50% effect (EC50) in the absence of the drug. However, at 10 nM cangrelor seven subjects could be identified by the VASP-P assay as “low responders” to the drug (PRI> 50%), and six of them also had an aggregation response to 5 µM ADP > 50%. These six subjects showed the lowest ADP EC50 values in the absence of the drug, possibly reflecting high sensitivity of their platelet P2Y12 receptors to ADP. In conclusion, both the VASP-P assay and light-transmission aggregometry detect in a comparable way in-vitro pharmacological inhibition of the platelet P2Y12 ADP receptor and its individual variability.

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