Increased Platelet Reactivity in Idiopathic Pulmonary Fibrosis Is Mediated by a Plasma Factor

Introduction

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, incurable fibrotic interstitial lung disease with a prognosis worse than many cancers. Its pathogenesis is poorly understood. Activated platelets can release pro-fibrotic mediators that have the potential to contribute to lung fibrosis. We determine platelet reactivity in subjects with IPF compared to age-matched controls.

Methods

Whole blood flow cytometry was used to measure platelet-monocyte aggregate formation, platelet P-selectin expression and platelet fibrinogen binding at basal levels and following stimulation with platelet agonists. A plasma swap approach was used to assess the effect of IPF plasma on control platelets.

Results

Subjects with IPF showed greater platelet reactivity than controls. Platelet P-selectin expression was significantly greater in IPF patients than controls following stimulation with 0.1 µM ADP (1.9% positive ±0.5 (mean ± SEM) versus 0.7%±0.1; p = 0.03), 1 µM ADP (9.8%±1.3 versus 3.3%±0.8; p<0.01) and 10 µM ADP (41.3%±4.2 versus 22.5%±2.6; p<0.01). Platelet fibrinogen binding was also increased, and platelet activation resulted in increased platelet-monocyte aggregate formation in IPF patients. Re-suspension of control platelets in plasma taken from subjects with IPF resulted in increased platelet activation compared to control plasma.

Conclusions

IPF patients exhibit increased platelet reactivity compared with controls. This hyperactivity may result from the plasma environment since control platelets exhibit increased activation when exposed to IPF plasma.     

The Role of Whole Blood Impedance Aggregometry and Its Utilisation in the Diagnosis and Prognosis of Patients with Systemic Inflammatory Response Syndrome and Sepsis in Acute Critical Illness

Objective

To assess the prognostic and diagnostic value of whole blood impedance aggregometry in patients with sepsis and SIRS and to compare with whole blood parameters (platelet count, haemoglobin, haematocrit and white cell count).

Methods

We performed an observational, prospective study in the acute setting. Platelet function was determined using whole blood impedance aggregometry (multiplate) on admission to the Emergency Department or Intensive Care Unit and at 6 and 24 hours post admission. Platelet count, haemoglobin, haematocrit and white cell count were also determined.

Results

106 adult patients that met SIRS and sepsis criteria were included. Platelet aggregation was significantly reduced in patients with severe sepsis/septic shock when compared to SIRS/uncomplicated sepsis (ADP: 90.7±37.6 vs 61.4±40.6; p<0.001, Arachadonic Acid 99.9±48.3 vs 66.3±50.2; p = 0.001, Collagen 102.6±33.0 vs 79.1±38.8; p = 0.001; SD ± mean)). Furthermore platelet aggregation was significantly reduced in the 28 day mortality group when compared with the survival group (Arachadonic Acid 58.8±47.7 vs 91.1±50.9; p<0.05, Collagen 36.6±36.6 vs 98.0±35.1; p = 0.001; SD ± mean)). However haemoglobin, haematocrit and platelet count were more effective at distinguishing between subgroups and were equally effective indicators of prognosis. Significant positive correlations were observed between whole blood impedance aggregometry and platelet count (ADP 0.588 p<0.0001, Arachadonic Acid 0.611 p<0.0001, Collagen 0.599 p<0.0001 (Pearson correlation)).

Conclusions

Reduced platelet aggregometry responses were not only significantly associated with morbidity and mortality in sepsis and SIRS patients, but also correlated with the different pathological groups. Whole blood aggregometry significantly correlated with platelet count, however, when we adjust for the different groups we investigated, the effect of platelet count appears to be non-significant.     

Platelet Expression of Stromal Cell-Derived Factor-1 Is Associated with the Degree of Valvular Aortic Stenosis

Background and Purpose

Platelet surface expression of stromal-cell-derived factor-1 (SDF-1) is increased during platelet activation and constitutes an important factor in hematopoetic progenitor cell trafficking at sites of vascular injury and ischemia. Enhanced platelet SDF-1 expression has been reported previously in patients suffering from acute coronary syndrome (ACS). We hypothesized that expression of platelet associated SDF-1 may also be influenced by calcified valvular aortic stenosis (AS).

Methods

We consecutively evaluated 941 patients, who were admitted to the emergency department with dyspnea and chest pain. Platelet surface expression of SDF-1 was determined by flow cytometry, AS was assessed using echocardiography and hemodynamic assessment by heart catheterization. A 1∶1 propensity score matching was implemented to match 218 cases with 109 pairs adjusting for age, sex, cardiovascular risk factors, and medication including ACE inhibitors, angiotensin receptor blockers, beta blockers, statins, aspirin, clopidogrel, GPIIb/IIIa antagonists, and vitamin K antagonists.

Results

Patients with valvular AS showed enhanced platelet SDF-1 expression compared to patients without AS (non-valvular disease, NV) independent of ACS and stable coronary artery disease (SAP) [mean fluorescence intensity (MFI) for ACS (AS vs. NV): 75±40.4 vs. 39.5±23.3; P = 0.002; for SAP (AS vs. NV): 54.9±44.6 vs. 24.3±11.2; P = 0.008]. Moreover, the degree of AS significantly correlated with SDF-1 platelet surface expression (r = 0.462; P = 0.002).

Conclusions

Valvular AS is associated with enhanced platelet-SDF-1 expression; moreover the degree of valvular AS correlates with SDF-1 platelet surface expression. These findings may have clinical implications in the future.     

Fiber-Optic System for Dual-Modality Imaging of Glucose Probes 18F-FDG and 6-NBDG in Atherosclerotic Plaques

Background

Atherosclerosis is a progressive inflammatory condition that underlies coronary artery disease (CAD)–the leading cause of death in the United States. Thus, the ultimate goal of this research is to advance our understanding of human CAD by improving the characterization of metabolically active vulnerable plaques within the coronary arteries using a novel catheter-based imaging system. The aims of this study include (1) developing a novel fiber-optic imaging system with a scintillator to detect both ¹⁸F and fluorescent glucose probes, and (2) validating the system on ex vivo murine plaques.

Methods

A novel design implements a flexible fiber-optic catheter consisting of both a radio-luminescence and a fluorescence imaging system to detect radionuclide ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) and the fluorescent analog 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-Deoxyglucose (6-NBDG), respectively. Murine macrophage-rich atherosclerotic carotid plaques were imaged ex vivo after intravenous delivery of ¹⁸F-FDG or 6-NBDG. Confirmatory optical imaging by IVIS-200 and autoradiography were also performed.

Results

Our fiber-optic imaging system successfully visualized both ¹⁸F-FDG and 6-NBDG probes in atherosclerotic plaques. For ¹⁸F-FDG, the ligated left carotid arteries (LCs) exhibited 4.9-fold higher radioluminescence signal intensity compared to the non-ligated right carotid arteries (RCs) (2.6×10⁴±1.4×10³ vs. 5.4×10³±1.3×10³ A.U., P = 0.008). Similarly, for 6-NBDG, the ligated LCs emitted 4.3-fold brighter fluorescent signals than the control RCs (1.6×10²±2.7×10¹ vs. 3.8×10¹±5.9 A.U., P = 0.002). The higher uptake of both ¹⁸F-FDG and 6-NBDG in ligated LCs were confirmed with the IVIS-200 system. Autoradiography further verified the higher uptake of ¹⁸F-FDG by the LCs.

Conclusions

This novel fiber-optic imaging system was sensitive to both radionuclide and fluorescent glucose probes taken up by murine atherosclerotic plaques. In addition, 6-NBDG is a promising novel fluorescent probe for detecting macrophage-rich atherosclerotic plaques.     

Fibrin Clot Structure and Platelet Aggregation in Patients with Aspirin Treatment Failure

Background

Aspirin is a cornerstone in prevention of cardiovascular events and modulates both platelet aggregation and fibrin clot formation. Some patients experience cardiovascular events whilst on aspirin, often termed aspirin treatment failure (ATF). This study evaluated both platelet aggregation and fibrin clot structure in patients with ATF.

Methods

We included 177 stable coronary artery disease patients on aspirin monotherapy. Among these, 116 (66%) had ATF defined as myocardial infarction (MI) whilst on aspirin. Platelet aggregation was assessed by Multiplate® aggregometry and VerifyNow®, whereas turbidimetric assays and scanning electron microscopy were employed to study fibrin clot characteristics.

Results

Enhanced platelet aggregation was observed in patients with ATF compared with non-MI patients following stimulation with arachidonic acid 1.0 mM (median 161 (IQR 95; 222) vs. 97 (60; 1776) AU*min, p = 0.005) and collagen 1.0 µg/mL (293 (198; 427) vs. 220 (165; 370) AU*min, p = 0.03). Similarly, clot maximum absorbance, a measure of fibrin network density, was increased in patients with ATF (0.48 (0.41; 0.52) vs. 0.42 (0.38; 0.50), p = 0.02), and this was associated with thinner fibres (mean ± SD: 119.7±27.5 vs. 127.8±31.1 nm, p = 0.003) and prolonged lysis time (552 (498; 756) vs. 519 (468; 633) seconds; p = 0.02). Patients with ATF also had increased levels of C-reactive protein (CRP) (1.34 (0.48; 2.94) and 0.88 (0.32; 1.77) mg/L, p = 0.01) compared with the non-MI group. Clot maximum absorbance correlated with platelet aggregation (r = 0.31–0.35, p-values<0.001) and CRP levels (r = 0.60, p<0.001).

Conclusions

Patients with aspirin treatment failure showed increased platelet aggregation and altered clot structure with impaired fibrinolysis compared with stable CAD patients without previous MI. These findings suggest that an increased risk of aspirin treatment failure may be identified by measuring both platelet function and fibrin clot structure.     

Homocysteine and Carotid Plaque Stability: A Cross-Sectional Study in Chinese Adults

Background and Purpose

This study aimed to explore the possible association of plasma total homocysteine with carotid plaque stability.

Methods

A cross-sectional study was conducted from 2010 to 2011. A stratified random sample of 2,919 Chinese participants aged 40 years or older was enrolled. Plasma total homocysteine levels were measured and carotid plaques were evaluated by ultrasonography. Logistic regression model was used to analyze the association of homocysteine levels to the progression of carotid plaque development, while adjusting for demographics and vascular risk factors.

Results

The mean level of plasma homocysteine in the subjects was 14.9 µmol/l. Along with increase in homocysteine level, the risk of advanced carotid plaque elevated (odds ratio = 1.28; 95% confidence interval = 1.09–1.51) after adjusting for age, sex, and other potential confounders. Stratified by sex, higher homocysteine level was strongly associated with advanced carotid plaque in men (OR = 1.41; 95% confidence interval = 1.17–1.70), but not in women.

Conclusion

The findings suggest that plasma level of homocysteine may be associated with advanced carotid plaque, which constitutes high risks of stroke, in male Chinese adults.     

Impact of Aspirin and Clopidogrel Interruption on Platelet Function in Patients Undergoing Major Vascular Surgery

Aims

To investigate functional platelet recovery after preoperative withdrawal of aspirin and clopidogrel and platelet function 5 days after treatment resumption.

Methods/Results

We conducted an observational study, which prospectively included consecutive patients taking aspirin, taking clopidogrel, and untreated controls (15 patients in each group). The antiplatelet drugs were withdrawn five days before surgery (baseline) and were reintroduced two days after surgery. Platelet function was evaluated by optical aggregation in the presence of collagen, arachidonic acid (aspirin) and ADP (clopidogrel) and by VASP assay (clopidogrel). Platelet-leukocyte complex (PLC) level was quantified at each time-point. At baseline, platelet function was efficiently inhibited by aspirin and had recovered fully in most patients 5 days after drug withdrawal. PLC levels five days after aspirin reintroduction were similar to baseline (+4±10%; p = 0.16), in line with an effective platelet inhibition. Chronic clopidogrel treatment was associated with variable platelet inhibition and its withdrawal led to variable functional recovery. PLC levels were significantly increased five days after clopidogrel reintroduction (+10±15%; p = 0.02), compared to baseline.

Conclusions

Aspirin withdrawal 5 days before high-bleeding-risk procedures was associated with functional platelet recovery, and its reintroduction two days after surgery restored antiplaletet efficacy five days later. This was not the case of clopidogrel, and further work is therefore needed to define its optimal perioperative management.     

Platelet Turnover in Stable Coronary Artery Disease – Influence of Thrombopoietin and Low-Grade Inflammation

Background

Newly formed platelets are associated with increased aggregation and adverse outcomes in patients with coronary artery disease (CAD). The mechanisms involved in the regulation of platelet turnover in patients with CAD are largely unknown.

Aim

To investigate associations between platelet turnover parameters, thrombopoietin and markers of low-grade inflammation in patients with stable CAD. Furthermore, to explore the relationship between platelet turnover parameters and type 2 diabetes, prior myocardial infarction, smoking, age, gender and renal insufficiency.

Methods

We studied 581 stable CAD patients. Platelet turnover parameters (immature platelet fraction, immature platelet count, mean platelet volume, platelet distribution width and platelet large cell-ratio) were determined using automated flow cytometry (Sysmex XE-2100). Furthermore, we measured thrombopoietin and evaluated low-grade inflammation by measurement of high-sensitive CRP and interleukin-6.

Results

We found strong associations between the immature platelet fraction, immature platelet count, mean platelet volume, platelet distribution width and platelet large cell ratio (r = 0.61–0.99, p<0.0001). Thrombopoietin levels were inversely related to all of the platelet turnover parameters (r = −0.17–−0.25, p<0.0001). Moreover, thrombopoietin levels were significantly increased in patients with diabetes (p = 0.03) and in smokers (p = 0.003). Low-grade inflammation evaluated by high-sensitive CRP correlated significantly, yet weakly, with immature platelet count (r = 0.10, p = 0.03) and thrombopoietin (r = 0.16, p<0.001). Also interleukin-6 correlated with thrombopoietin (r = 0.10, p = 0.02).

Conclusion

In stable CAD patients, thrombopoietin was inversely associated with platelet turnover parameters. Furthermore, thrombopoietin levels were increased in patients with diabetes and in smokers. However, low-grade inflammation did not seem to have a substantial impact on platelet turnover parameters.     

Decreased Platelet Count in Patients Receiving Continuous Veno-Venous Hemofiltration: A Single-Center Retrospective Study

Background

A decreased platelet count may occur and portend a worse outcome in patients receiving continuous renal replacement therapy (CRRT). We aim to investigate the incidence of decreased platelet count and related risk factors in patients receiving CRRT.

Methods

In this retrospective study, we screened all patients receiving continuous veno-venous hemofiltration (CVVH) at Jinling Hospital between November 2008 and October 2012. The patients were included who received uninterrupted CVVH for more than 72 h and had records of blood test for 4 consecutive days after ruling out pre-existing conditions that may affect the platelet count. Platelet counts before and during CVVH, illness severity, CVVH settings, and outcomes were analyzed.

Results

The study included 125 patients. During the 3-day CVVH, 44.8% and 16% patients had a mild decline (20–49.9%) and severe decline (≥50%) in the platelet count,respectively; 37.6% and 16.0% patients had mild thrombocytopenia (platelet count 50.1–100×10⁹/L) and severe thrombocytopenia (platelet count ≤50×10⁹/L), respectively. Patients with a severe decline in the platelet count had a significantly lower survival rate than patients without a severe decline in the platelet count (35.0% versus 59.0%, P = 0.012), while patients with severe thrombocytopenia had a survival rate similar to those without severe thrombocytopenia (45.0% versus 57.1%, P = 0.308). Female gender, older age, and longer course of the disease were independent risk factors for a severe decline in the platelet count.

Conclusions

A decline in the platelet count and thrombocytopenia are quite common in patients receiving CVVH. The severity of the decline in the platelet count rather than the absolute count during CVVH may be associated with hospital mortality. Knowing the risk factors for a severe decline in the platelet count may allow physicians to prevent such an outcome.     

A Non-Invasive Laboratory Panel as a Diagnostic and Prognostic Biomarker for Thrombotic Microangiopathy: Development and Application in a Chinese Cohort Study

Background

Thrombotic microangiopathy (TMA) in the kidney is a histopathologic lesion that occurs in a number of clinical settings and is often associated with poor renal prognosis. The standard test for the diagnosis of TMA is the renal biopsy; noninvasive parameters such as potential biomarkers have not been developed.

Methods

We analyzed routine parameters in a cohort of 220 patients with suspected TMA and developed a diagnostic laboratory panel by logistic regression. The levels of candidate markers were validated using an independent cohort (n = 46), a cohort of systemic lupus erythematosus (SLE) (n = 157) and an expanded cohort (n = 113), as well as 9 patients with repeat biopsies.

Results

Of the 220 patients in the derivation cohort, 51 patients with biopsy-proven TMA presented with a worse renal prognosis than those with no TMA (P = 0.002). Platelet and L-lactate dehydrogenase (LDH) levels showed an acceptable diagnostic value of TMA (AUC = 0.739 and 0.756, respectively). A panel of 4 variables - creatinine, platelets, ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats 13) activity and LDH - can effectively discriminate patients with TMA (AUC = 0.800). In the validation cohort, the platelet and LDH levels and the 4-variable panel signature robustly distinguished patients with TMA. The discrimination effects of these three markers were confirmed in patients with SLE. Moreover, LDH levels and the 4-variable panel signature also showed discrimination values in an expanded set. Among patients undergoing repeat biopsy, increased LDH levels and panel signatures were associated with TMA status when paired evaluations were performed. Importantly, only the 4-variable panel was an independent prognostic marker for renal outcome (hazard ratio = 3.549; P<0.001).

Conclusions

The noninvasive laboratory diagnostic panel is better for the early detection and prognosis of TMA compared with a single parameter, and may provide a promising biomarker for clinical application.     

LDL-Migration Index (LDL-MI), an Indicator of Small Dense Low-Density Lipoprotein (sdLDL), Is Higher in Non-Alcoholic Steatohepatitis than in Non-Alcoholic Fatty Liver: A Multicenter Cross-Sectional Study

Background

Non-alcoholic fatty liver disease (NAFLD) is associated with increased risks of atherosclerotic diseases, including cardiovascular disease. However, the difference in risk between patients with non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) has not yet been determined. Accumulating evidence has shown that high amounts of small dense low-density lipoprotein (sdLDL) are closely associated with atherosclerotic diseases. This study investigated differences in risk factors for atherosclerotic diseases, especially LDL-migration index (LDL-MI), an indicator of sdLDL, between patients with NAFL and NASH.

Methods

LDL-MI was analyzed in a primary cohort of 156 patients with NAFLD, including 53 with NAFL and 103 with NASH, and a validation cohort of 69 patients with NAFLD, including 25 with NAFL and 44 with NASH.

Results

In the primary cohort, NASH was associated with elevated LDL-MI (p = 0.039). Multiple regression analysis showed that NASH and the non-use of lipid lowering medications were independently correlated with higher LDL-MI in all patients with NAFLD. Among patients not on lipid lowering medications, those with NASH had significantly higher LDL-MI than those with NAFL (p = 0.001). These findings were confirmed in a validation cohort, in that LDL-MI was significantly higher in patients with NASH than with NAFL (p = 0.043).

Conclusion

This study is the first to show that LDL-MI, an indicator of sdLDL, was higher in patients with NASH than with NAFL, suggesting that the risk of atherosclerotic diseases may be higher in NASH than NAFL. Patients with NASH should be followed closely, especially for the progression of liver pathology and atherosclerotic diseases.

Trial Registration

UMIN000009614     

Atherosclerotic plaques occur in absence of intima-media thickening in both systemic sclerosis and systemic lupus erythematosus: a duplexsonography study of carotid and femoral arteries and follow-up for cardiovascular events

Introduction

The objective of this cross-sectional and retrospective cohort study was (1) to determine the usefulness of intima-media thickness (IMT) in contrast to plaque assessment, (2) to examine the value of additive femoral artery sonography and (3) to identify potential risk factors for atherosclerosis and incident cardiovascular events in systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) patients.

Methods

In this study, 90 SSc and 100 SLE patients were examined by duplexsonography. IMT was measured in common carotid and common femoral arteries, plaques were assessed in common, internal and external carotid and common, proximal superficial and deep femoral arteries. Different definitions of pathological IMT (pIMT) were compared with the presence of plaque. Results were evaluated in relation to traditional and non-traditional risk factors for baseline atherosclerosis (logistic regression) and their predictive value for cardiovascular events during follow-up (cox regression).

Results

Definite atherosclerosis occurred frequently without signs of subclinical atherosclerosis in both diseases: pIMT >0.9 mm was present in only 17/59 (28.9%) SSc and 13/49 (26.5%) SLE patients with already present atherosclerotic plaques. Using age-adjusted pIMT definitions, this rate was even lower (5.1-10.3% in SSc, 14.3-26.5% in SLE). Plaques were located only at the carotid or only at the femoral arteries in 26 (13.7%) and 24 (12.6%) patients, respectively. Age and nicotine pack-years were independently associated with atherosclerotic plaques in SLE and SSc patients, as well as the cumulative prednisolone dose in SSc subgroup, and ssDNA positive SLE patients had a lower risk for atherosclerotic plaque. During follow-up (available for 129/190 (67.9%) patients, 650 person-years), cardiovascular events occurred more often in patients with coronary heart disease (adjusted-hazards ratio (HR) 10.19, 95% confidence interval (CI) 3.04 to 34.17, P <0.001), male patients (adjusted-HR 8.78, 95% CI 2.73 to 28.19, P <0.001) and in patients with coexistent carotid and femoral plaques (adjusted-HR 5.92, 95% CI 1.55 to 22.67, P = 0.009). Patients with solely carotid or femoral plaque were not at higher risk.

Conclusion

Atherosclerotic plaque lesions can be found frequently in absence of intima-media thickening in both SSc and SLE patients. As well as routine sonography of carotid arteries, the sonography of femoral arteries is recommended to identify additional atherosclerotic lesions and to detect patients at a high risk for cardiovascular events.     

Clinical Presentations,Antiplatelet Strategies and Prognosis of Patients with Stent Thrombosis: An Observational Study of 140 Patients

Background

Until now there has been scarce evidence regarding an optimal antiplatelet strategy and clinical outcomes for patients who had suffered from stent thrombosis (ST).

Methods and Results

140 patients who suffered from stent thrombosis were prospectively registered. Patients received dual (aspirin and 150 mg clopidogrel, N = 66) or triple (additional cilostazol, N = 74) antiplatelet therapy at the physician’s discretion. Thereafter platelet reactivity and one year clinical outcomes were analyzed. The primary outcome included the composite of cardiac death, non-fatal myocardial infarction (MI) or stroke at one year,which developed in 41 (29.3%) patients, consisting of 31 (22.1%) cardiac death, 9 (6.4%) non-fatal MI and 1 (1.4%) stroke. Recurrent definite and probable ST according to ARC definition was observed in 8 (5.7%) and 14 (10.0%) patients, respectively. Triple therapy was associated with significantly lower platelet reactivities (50.2±17.8, % vs. 59.6±17.2, %, P = 0.002) compared to high dose dual antiplatelet therapy. However, the incidence of primary events (24.3% vs. 34.8%, P = 0.172) did not differ between triple and dual antiplatelet therapies. High on-treatment platelet reactivity (HR: 8.35, 95% CI: 2.234∼30.867, P = 0.002) and diabetes (HR: 3.732, 95% CI: 1.353∼10.298, P = 0.011) were independent predictors of primary events.

Conclusions

Patients who suffered from stent thrombosis have a poor prognosis even after revascularization with intensive antiplatelet therapy. Triple antiplatelet therapy was more effective in reducing on-treatment platelet reactivity, compared to high dose dual antiplatelet therapy.     

Effects of Neck Radiation Therapy on Extra-Cranial Carotid Arteries Atherosclerosis Disease Prevalence: Systematic Review and a Meta-Analysis

Introduction

Radiation arteritis following neck irradiation as a treatment for head and neck malignancy has been well documented. The long-term sequelae of radiation exposure of the carotid arteries may take years to manifest clinically, and extra-cranial carotid artery (ECCA) stenosis is a well-recognised vascular complication. These carotid lesions should not be regarded as benign and should be treated in the same manner as standard carotid stenosis. Previous studies have noted increased cerebrovascular events such as stroke in this cohort of patients because of high-grade symptomatic carotid stenosis resulting in emboli.

Aim

To evaluate the effect of radiation therapy on ECCA atherosclerosis progression.

Methods

Online search for case-control studies and randomised clinical trials that reported on stenosis in extra-cranial carotid arteries in patients with neck malignancies who received radiation therapy (RT) comparing them to patients with neck malignancies who did not receive RT.

Results

Eight studies were included in the final analysis with total of 1070 patients – 596 received RT compared to 474 in the control group. There was statistically significant difference in overall stenosis rate (Pooled risk ratio  =  4.38 [2.98, 6.45], P  =  0.00001) and severe stenosis (Pooled risk ratio  =  7.51 [2.78, 20.32], P <0.0001), both being higher in the RT group. Pooled analysis of the five studies that reported on mild stenosis also showed significant difference (Pooled risk ratio  =  2.74 [1.75, 4.30], 95% CI, P  =  0.0001).

Conclusion

The incidence of severe ECCA stenosis is higher among patients who received RT for neck malignancies. Those patients should be closely monitored and screening programs should be considered in all patients who receive neck RT.     

Increased Systemic and Local Interleukin 9 Levels in Patients with Carotid and Coronary Atherosclerosis

Objective

Atherosclerosis is a chronic inflammatory disorder that involves a range of inflammatory mediators. Although interleukin (IL)-9 has been related to inflammation, there are at present no data on its role in atherosclerosis. Here we have examined IL-9 and IL-9 receptor (IL-9R) systemically and locally in patients with coronary and carotid atherosclerosis.

Methods

Plasma IL-9 was quantified by enzyme immunoassay and multiplex technology. IL-9 and IL-9R mRNA were quantified by real-time RT-PCR, and their localization within the lesion was assessed by immunohistochemistry.

Results

The main findings were: (i) Patients with carotid atherosclerosis had significantly raised IL-9 plasma levels compared with healthy controls (n = 28), with no differences between asymptomatic (n = 56) and symptomatic (n = 88) patients. (ii) On admission, patients with acute ST-elevation myocardial infarction (STEMI) (n = 42) had markedly raised IL-9 plasma levels which gradually declined during the first week post-MI. (iii) T cells and monocytes from patients with unstable angina (n = 17) had increased mRNA levels of IL-9 as compared with controls (n = 11). (iv) Carotid plaques (n = 68) showed increased mRNA levels of IL-9 and IL-9R compared to non-atherosclerotic vessels (n = 10). Co-localization to T cells (IL-9 and IL-9R) and macrophages (IL-9) were shown by immunohistochemistry. (v) IL-9 increased IL-17 release in peripheral blood mononuclear cells from patients with unstable angina (n = 5) and healthy controls (n = 5) with a particularly enhancing effect in cells from the patient group.

Conclusion

Our findings show increased IL-9 levels in different atherosclerotic disorders both systemically and within the lesion, suggesting a role for the IL-9/IL-9R axis in the atherosclerotic process, potentially involving IL-17 mediated mechanisms. However, the functional consequences of these findings should be further investigated.     

Fish-Free Diet in Patients with Phenylketonuria Is Not Associated with Early Atherosclerotic Changes and Enhanced Platelet Activation

Background and Purpose

Since patients with phenylketonuria (PKU) have to follow a lifelong restriction of natural protein to lower phenylalanine-intake, they never eat fish. This diet may lead to a chronic deficit of omega-3 and omega-6 fatty acids with the risk of early atherosclerotic changes. The aim of the study was to analyse the fatty acid profile of PKU patients and to correlate the results with surrogate markers of early atherosclerotic changes [enhanced carotid intima media thickness (CIMT) and ß-stiffness index] and platelet activation.

Methods

In 43 PKU patients and in 58 healthy controls we prospectively examined the fatty acid profile, CIMT, ß-stiffness index and platelet activation (flow cytometric determination of markers of platelet activation). CIMT was measured bilaterally by ultrasound. CIMT _mean was defined as the mean value of the sum of CIMT _left and CIMT _right.

Results

Despite of lower HDL-cholesterol and higher triglyceride concentrations in the PKU group, there was no significant difference in the omega-6 or omega-3 fatty acid profile, CIMT, ß-stiffness index between both groups. Platelet activation was not enhanced in the PKU group.

Conclusions

Fish-free diet does not induce early atherosclerotic changes or enhanced platelet activation in PKU patients.     

Different Influences of Hematocrit on the Results of Two Point-Of-Care Platelet Function Tests,the VerifyNow Assay and Multiple Electrode Platelet Aggregometry

Objective

Previous studies have reported a considerable association between the VerifyNow (Accumetrics, San Diego, CA, USA) P2Y12 assay results and hematocrit. No reports, however, have described an association between the multiple electrode platelet aggregometry (MEA; Dynabyte, Munich, Germany) adenosine diphosphate (ADP) assay results and hematocrit. This study was conducted to evaluate the influence of hematocrit on the results of 2 different point-of-care platelet function tests.

Methods

A total of 462 consecutive patients who were undergoing percutaneous coronary intervention were enrolled. Platelet function was evaluated with both the VerifyNow P2Y12 and MEA ADP assays.

Results

Anemic patients (n = 152, 32.9%) demonstrated a significantly higher rate of cardiac death, myocardial infarction, and stroke (5.3% vs. 2.3%, p = 0.046) during the follow-up (median: 18.8 months). Although the VerifyNow P2Y12 assay results demonstrated a significant inverse correlation with hematocrit (r = −0.409, p<0.001), there was no such correlation between the MEA ADP assay results and hematocrit (r = 0.039, p = 0.401). In the multivariate analysis, anemia was an independent predictor of high on-treatment platelet reactivity, defined as a VerifyNow P2Y12 reaction unit level of ≥252.5 (odds ratio = 2.21, 95% confidence interval = 1.39–3.52; p = 0.001). Importantly, this association was independent of an intrinsic change in platelet reactivity as measured by the MEA ADP assay. Adjusting for the influence of hematocrit improved the strength of the correlation between the VerifyNow P2Y12 and MEA ADP assay results.

Conclusions

Hematocrit significantly influenced the VerifyNow P2Y12 assay results, a phenomenon that was presumably in-vitro. Hematocrit level should therefore be considered when interpreting results of the VerifyNow P2Y12 assay.     

Fatty Acid binding protein 4 is associated with carotid atherosclerosis and outcome in patients with acute ischemic stroke

Background and Purpose

Fatty acid binding protein 4 (FABP4) has been shown to play an important role in macrophage cholesterol trafficking and associated inflammation. To further elucidate the role of FABP4 in atherogenesis in humans, we examined the regulation of FABP4 in carotid atherosclerosis and ischemic stroke.

Methods

We examined plasma FABP4 levels in asymptomatic (n = 28) and symptomatic (n = 31) patients with carotid atherosclerosis, as well as in 202 subjects with acute ischemic stroke. In a subgroup of patients we also analysed the expression of FABP4 within the atherosclerotic lesion. In addition, we investigated the ability of different stimuli with relevance to atherosclerosis to regulate FABP4 expression in monocytes/macrophages.

Results

FABP4 levels were higher in patients with carotid atherosclerosis, both systemically and within the atherosclerotic lesion, with particular high mRNA levels in carotid plaques from patients with the most recent symptoms. Immunostaining of carotid plaques localized FABP4 to macrophages, while activated platelets and oxidized LDL were potent stimuli for FABP4 expression in monocytes/macrophages in vitro. When measured at the time of acute ischemic stroke, high plasma levels of FABP4 were significantly associated with total and cardiovascular mortality during follow-up, although we did not find that addition of FABP4 to the fully adjusted multivariate model had an effect on the prognostic discrimination for all-cause mortality as assessed by c-statistics.

Conclusions

FABP4 is linked to atherogenesis, plaque instability and adverse outcome in patients with carotid atherosclerosis and acute ischemic stroke.     

Association of the sirtuin and mitochondrial uncoupling protein genes with carotid plaque

Objective

Sirtuins (SIRTs) and mitochondrial uncoupling proteins (UCPs) have been implicated in cardiovascular diseases through the control of reactive oxygen species production. This study sought to investigate the association between genetic variants in the SIRT and UCP genes and carotid plaque.

Methods

In a group of 1018 stroke-free subjects from the Northern Manhattan Study with high-definition carotid ultrasonography and genotyping, we investigated the associations of 85 single nucleotide polymorphisms (SNPs) in the 11 SIRT and UCP genes with the presence and number of carotid plaques, and evaluated interactions of SNPs with sex, smoking, diabetes and hypertension as well as interactions between SNPs significantly associated with carotid plaque.

Results

Overall, 60% of subjects had carotid plaques. After adjustment for demographic and vascular risk factors, T-carriers of the SIRT6 SNP rs107251 had an increased risk for carotid plaque (odds ratio, OR = 1.71, 95% CI = 1.23–2.37, Bonferroni-corrected p = 0.03) and for a number of plaques (rate ratio, RR = 1.31, 1.18–1.45, Bonferroni-corrected p = 1.4×10⁻⁵), whereas T-carriers of the UCP5 SNP rs5977238 had an decreased risk for carotid plaque (OR = 0.49, 95% CI = 0.32–0.74, Bonferroni-corrected p = 0.02) and plaque number (RR = 0.64, 95% CI = 0.52–0.78, Bonferroni-corrected p = 4.9×10⁻⁴). Some interactions with a nominal p≤0.01 were found between sex and SNPs in the UCP1 and UCP3 gene; between smoking, diabetes, hypertension and SNPs in UCP5 and SIRT5; and between SNPs in the UCP5 gene and the UCP1, SIRT1, SIRT3, SIRT5, and SIRT6 genes in association with plaque phenotypes.

Conclusion

We observed significant associations between genetic variants in the SIRT6 and UCP5 genes and atherosclerotic plaque. We also found potential effect modifications by sex, smoking and vascular risk factors of the SIRT/UCP genes in the associations with atherosclerotic plaque. Further studies are needed to validate our observations.     

Association of Localized Hypersensitivity and In-Stent Neoatherosclerosis with the Very Late Drug-Eluting Stent Thrombosis

Background

Localized hypersensitivity reaction, delayed arterial healing, and neoatherosclerosis inside the stent have been suggested as the underlying pathologic mechanisms of very late stent thrombosis (VLST) of drug-eluting stent (DES). The present study sought to explore the prevalence of inflammatory cell infiltrates and evidence for fragments of atherosclerotic plaques in the aspirated thrombi in patients with DES VLST.

Methods and Results

From April 2004 to September 2012, 48 patients with stent thrombosis (ST) of DES underwent thrombus aspiration with retrieved material sufficient for the histopathologic analysis; early ST (EST, within 30 days): N = 17, late ST (LST, between 31 and 365 days): N = 7, and very late ST (VLST, >1 year): N = 24. Eosinophil fraction in the aspirated thrombi was significantly higher in patients with VLST (8.2±5.7%) as compared with those with EST (4.3±3.0%) and LST (5.5±3.8%) (P = 0.03). Eosinophil fraction in the aspirated thrombi was significantly higher in 12 VLST patients with angiographic peri-stent contrast staining (PSS) and/or incomplete stent apposition (ISA) by intravascular ultrasound than in 12 VLST patients without PSS or ISA (10.6±6.1% versus 5.8±4.1%, P = 0.03). Evidences for fragments of atherosclerotic plaques in the aspirated thrombi were observed only in 3 (13%) out of 24 patients with DES VLST.

Conclusions

Eosinophil fraction in the aspirated thrombi was significantly higher in patients with DES VLST as compared with those with EST and LST. Evidences for fragments of atherosclerotic plaques were relatively uncommon in patients with DES VLST.