Dorit Leshem-Lev

Response to Prasugrel and Levels of Circulating Reticulated Platelets in Patients With ST-Segment Elevation Myocardial Infarction

OBJECTIVES The aim of this study was to determine whether response to prasugrel is associated with the proportion of circulating reticulated platelets (RPs) in patients with ST-segment elevation myocardial infarction (STEMI). BACKGROUND Despite better pharmacodynamic properties and clinical efficacy of prasugrel compared with clopidogrel, antiplatelet responses to prasugrel are not uniform. The mechanism of this variability in response is not clear. RPs, young hyperactive forms, are increased during situations of enhanced platelet turnover. METHODS Patients with STEMI treated with primary percutaneous intervention (PCI) and prasugrel were tested for platelet reactivity using purinergic receptor P₂Y, G-protein coupled, 12 (P₂Y₁₂) assay and multiple electrode aggregometry (MEA). RP levels were determined using flow cytometry with thiazole orange staining. Tests were performed at 2 to 4 days and 30 days post-PCI. Platelet function was compared by varying levels of RPs, analyzed as continuous (regression analysis) and categorical (tertiles) variables. RESULTS Sixty-two patients were included (mean age: 57.5 ± 8 years; 21.2% women; 27.7% diabetes). At the early time point, RP levels were strongly correlated with platelet reactivity when evaluated by the P₂Y₁₂ assay (Spearmans correlation coefficient: 0.55 for P₂Y₁₂ reaction units, -0.49 for percent inhibition) and MEA (Spearmans: 0.50). The upper tertile of RPs displayed higher platelet reactivity compared with the middle and lower tertiles, according to P₂Y₁₂ assay and MEA. Similar results with strong correlations between RP and platelet reactivity were noted at 30 days post-PCI. CONCLUSIONS The proportion of circulating RPs strongly correlates with response to prasugrel in patients with STEMI treated with PCI. High levels of RPs are associated with increased platelet reactivity despite prasugrel treatment.

TGF-β1 signaling and Krüppel-like factor 10 regulate bone marrow–derived proangiogenic cell differentiation, function, and neovascularization

Emerging evidence demonstrates that proangiogenic cells (PACs) originate from the BM and are capable of being recruited to sites of ischemic injury where they contribute to neovascularization. We previously determined that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitor cells (GMPs) differentiate into PACs and possess robust angiogenic activity under ischemic conditions. Herein, we report that a TGF-β1-responsive Krüppel- like factor, KLF10, is strongly expressed in PACs derived from CMPs and GMPs, ∼ 60-fold higher than in progenitors lacking PAC markers. KLF10(-/-) mice present with marked defects in PAC differentiation, function, TGF-β responsiveness, and impaired blood flow recovery after hindlimb ischemia, an effect rescued by wild-type PACs, but not KLF10(-/-) PACs. Overexpression studies revealed that KLF10 could rescue PAC formation from TGF-β1(+/-) CMPs and GMPs. Mechanistically, KLF10 targets the VEGFR2 promoter in PACs which may underlie the observed effects. These findings may be clinically relevant because KLF10 expression was also found to be significantly reduced in PACs from patients with peripheral artery disease. Collectively, these observations identify TGF-β1 signaling and KLF10 as key regulators of functional PACs derived from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states.

Gold nanorods as absorption contrast agents for the noninvasive detection of arterial vascular disorders based on diffusion reflection measurements.

In this study we report the use of gold nanorods (GNRs) as absorption contrast agents in the diffusion reflection (DR) method for the in vivo detection of atherosclerotic injury. The early detection and characterization of atherosclerotic vascular disease is considered to be one of the greatest medical challenges today. We show that macrophage cells, which are major components of unstable active atherosclerotic plaques, uptake gold nanoparticles, resulting in a change in the optical properties of tissue-like phantoms and a unique DR profile. In vivo DR measurements of rats that underwent injury of the carotid artery showed a clear difference between the DR profiles of the injured compared with healthy arteries. The results suggest that DR measurements following GNRs administration represent a potential novel method for the early detection of atherosclerotic vascular disease.

Endothelial Progenitor Cell Function Inversely Correlates With Long-term Glucose Control in Diabetic Patients: Association With the Attenuation of the Heme Oxygenase-Adiponectin Axis

BACKGROUND Endothelial progenitor cells (EPCs) are attenuated, both in number and functionality, in animal models of chronic cardiovascular and metabolic disorders. This effect has subsequently been linked to the aggravation of long-term morbidity and mortality associated with such disorders. The objective was to examine EPC number and survival in chronic diabetic vs nondiabetic patients in conjunction with the examination of their redox, inflammatory, and antioxidant defense system (Nrf2 genes) status in serum and visceral fat. METHODS Visceral adipose tissue from diabetic and nondiabetic patients undergoing coronary artery bypass graft surgery was analyzed for Nrf2-dependent genes. Oxidative stress was evaluated using thiobarbituric acid-reactive substance assay (TBARS). Peripheral blood, collected 1 day prior to surgery, was evaluated for inflammatory cytokines and EPCs. RESULTS When compared with controls (P < 0.05), results of the thiobarbituric acid-reactive substance assay were higher in diabetic patients. Although Nrf2-dependent antioxidant proteins (thioredoxin-1 [Trx-1], nicotinamide adenine dinucleotide phosphate [NAD(P)H] quinone oxidoreductase [NQO1], glutathione S-transferase [GST]) were upregulated, heme oxygenase (HO-1) and adiponectin protein expression were lower in the diabetic group (P < 0.05). Serum levels of bilirubin were lower (P < 0.005) while the levels of inflammatory cytokines were higher in diabetic patients (P < 0.05). EPC levels and their colony forming units were significantly lower (P < 0.05) with reduced viability in diabetic patients as compared with nondiabetic patients. CONCLUSIONS These results demonstrate for the first time that in diabetic patients, there is an inadequate heme oxygenase-adiponectin axis response, which could compromise the compensatory antioxidant and anti-inflammatory effects consequently contributing toward EPC dysfunction in these patients.

Circulating endothelial progenitor cell levels and function in patients who experienced late coronary stent thrombosis

AIMS The pathogenesis of late coronary stent thrombosis may be related to impaired arterial healing. Endothelial progenitor cells (EPCs) have been shown to play an important role in repair and re-endothelialization following vascular injury. We hypothesized that patients who develop late stent thrombosis may have reduced or dysfunctional EPCs, and aimed to compare EPC level and function in patients who experienced stent thrombosis vs. matched controls. METHODS AND RESULTS Patients who developed late (> 30 days) stent thrombosis within the past 3 years were compared with matched patients who underwent stenting and did not develop stent thrombosis. All patients had blood samples taken ≥ 3 months from the stent thrombosis or index procedure. The proportion of peripheral mononuclear cells (PMNCs) expressing vascular endothelial growth factor receptor 2 (VEGFR-2), CD133, and CD34 was evaluated by flow cytometry. Endothelial progenitor cell colony forming units (CFUs) were grown from PMNCs, characterized and counted following 7 days of culture. The two groups (n = 30 each) were well-matched (93.3% men, mean age 60-62 years, 33.3% diabetes, 73-80% DESs). The proportion of cells co-expressing VEGFR-2, CD133, and CD34 was lower in the stent thrombosis group compared with the control [VEGFR-2(+)CD133(+): 0.18% (0.03-0.41%) vs. 0.47% (0.16-0.66%), P = 0.01; VEGFR-2(+)CD34(+): 0.32% (0.22-0.70%) vs. 0.66% (0.24-1.1%), P = 0.03]. The number of EPC CFUs was also lower in the stent thrombosis group [3.9% (3.2-5.5%) vs. 8.3% (6.5-13.4%) colonies/well, respectively, P < 0.0001]. CONCLUSION Patients who suffered late coronary stent thrombosis appear to have reduced levels of circulating EPCs and impaired functional properties of the cells. These findings require validation by further studies, but may contribute to understanding the pathogenesis of late stent thrombosis.

Elevated level of pro-inflammatory eicosanoids and EPC dysfunction in diabetic patients with cardiac ischemia

BACKGROUND Circulating endothelial progenitor cells (EPCs) are recruited from the blood system to sites of ischemia and endothelial damage, where they contribute to the repair and development of blood vessels. Since numerous eicosanoids including leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) have been shown to exert potent pro-inflammatory activities, we examined their levels in chronic diabetic patients with severe cardiac ischemia in conjunction with the level and function of EPCs. RESULTS Lipidomic analysis revealed a diabetes-specific increase (p<0.05) in inflammatory and angiogenic eicosanoids including the 5-lipoxygenase-derived LTB (4.11±1.17 vs. 0.96±0.27 ng/ml), the lipoxygenase/CYP-derived 12-HETE (117.08±35.05 vs. 24.34±10.03 ng/ml), 12-HETrE (17.56±4.43 vs. 4.15±2.07 ng/ml), and the CYP-derived 20-HETE (0.32±0.04 vs. 0.06±0.05 ng/ml) the level of which correlated with BMI (p=0.0027). In contrast, levels of the CYP-derived EETs were not significantly (p=0.36) different between these two groups. EPC levels and their colony-forming units were lower (p<0.05) with a reduced viability in diabetic patients compared with non-diabetics. EPC function (colony-forming units (CFUs) and MTT assay) also negatively correlated with the circulating levels of HgA1C. CONCLUSION This study demonstrates a close association between elevated levels of highly pro-inflammatory eicosonoids, diabetes and EPC dysfunction in patients with cardiac ischemia, indicating that chronic inflammation impact negatively on EPC function and angiogenic capacity in diabetes.

Circulating Endothelial Progenitor Cells in Patients With Dysfunctional Versus Normally Functioning Congenitally Bicuspid Aortic Valves

Patients with bicuspid aortic valve (BAV) may gradually develop significant valve dysfunction, whereas others remain free of dysfunction. Factors that determine the prognosis of BAV remain unclear. Because endothelial progenitor cells (EPCs) have a role in the repair of endothelial surfaces after injury, we hypothesized that EPCs may also be involved in preventing BAV degeneration. Accordingly, we compared EPC level and function in patients with BAV with versus without valve dysfunction. The study group included 22 patients with BAV and significant valve dysfunction (at least moderate aortic regurgitation and/or at least moderate aortic stenosis). The control group included 28 patients with BAV without valve dysfunction. All patients had 1 blood sample taken. Proportion of peripheral mononuclear cells expressing vascular endothelial growth factor receptor 2, CD133 and CD34 was evaluated by flow cytometry. EPC colony-forming units (CFUs) were grown from peripheral mononuclear cells, characterized, and counted after 7 days of culture. The 2 groups had similar clinical characteristics except for higher prevalence of hypertension in the dysfunctional valve group. Number of EPC CFUs was smaller in the dysfunctional valve group (32 CFUs/plate, 15 to 42.5, vs 48 CFUs/plate, 30 to 62.5, respectively, p = 0.01), and the migratory capacity of the cells in this group was decreased. In addition, the proportion of cells coexpressing vascular endothelial growth factor receptor 2, CD133, and CD34 tended to be smaller in the dysfunctional valve group. In conclusion, patients with BAV and significant valve dysfunction appear to have circulating EPCs with impaired functional properties. These findings require validation by further studies.

Effect of intensive glycaemic control on endothelial progenitor cells in patients with long-standing uncontrolled type 2 diabetes

Aims Endothelial progenitor cells (EPCs) have an important role in repair following vascular injury. However, in patients with diabetes, EPC number and function are markedly reduced. It is unclear whether intensive glycaemic control can modify EPC properties in diabetic patients. We aimed to examine whether glycaemic control can improve EPC number and function in patients with long-standing uncontrolled type 2 diabetes. Methods and Results Thirty-five patients with treated type 2 diabetes and HgA1c ≥8.5% were included. Patients were tested at baseline and after 3–4 months of an intensive glycaemic control programme, with the aim of achieving HgA1c of 7%. The diabetes group was compared to 20 patients without diabetes (control). Circulating EPC levels were assessed by flow cytometry for expression of VEGFR2, CD133, and CD34. The capacity of the cells to form colony-forming units (CFUs), and their migration and viability were quantified after 1 week of culture. Patients with diabetes (mean age 61.1 ± 7 years, 28.6% women, disease duration of 19.2 ± 8 years) had a baseline HgA1c of 9.4 ± 0.8%. After the glycaemic control period, HgA1c decreased to 8 ± 0.8%. Circulating EPC levels increased significantly after the intensive control period and reached a level similar to the control group. The number of EPC CFUs also increased significantly after glycaemic control but remained lower than the control group. All EPC functional assays improved following the glycaemic control. Conclusions In patients with uncontrolled long-standing type 2 diabetes, intensive glycaemic control was associated with an increase in the levels of circulating EPCs, and improvement in their functional properties.

Endothelial Progenitor Cells Are Suppressed in Anemic Patients with Acute Coronary Syndrome

OBJECTIVE Anemia is an independent predictor of poor prognosis in acute coronary syndrome. Endothelial progenitor cells are bone marrow-derived cells that are mobilized into the circulation in response to ischemia. The number of circulating endothelial progenitor cells increases within days of acute coronary syndrome. There is no confirmation regarding the correlation between the occurrence of anemia and the deficiency in endothelial progenitor cells in patients with acute coronary syndrome. The correlation between chronic anemia and endothelial progenitor cells in patients with acute coronary syndrome was investigated. METHODS Endothelial progenitor cells were examined in 26 patients with acute coronary syndrome. Fifteen patients had chronic nonprogressive anemia, and 11 patients had a normal blood count. Blood samples were drawn on the first day of admission and 4 to 7 days later. Mononuclear cells were separated and cultured on fibronectin-coated plates with EndoCult medium (StemCell Technologies, Vancouver, BC, Canada) for 5 days. Colony forming unit count and a migration assay were performed at each time point. RESULTS Baseline colony forming unit in the non-anemic group was higher than in the anemic group (P<.0001). There was a highly significant correlation between admission hemoglobin and colony forming unit count (R=0.83, P<.0001). Colony forming units increased in both groups on the second measurement but to a lower extent in the anemic group (P = .0004). The migration assay in the non-anemic group was higher than in the anemic group at baseline (P = .017) and 4 to 7 days later (P = .0054). CONCLUSION Patients with acute coronary syndrome with anemia demonstrate a reduced number of peripheral endothelial progenitor cells with impaired function, possibly representing a lower capacity for vascular healing. These phenomena may partly explain the poor prognosis observed in patients with acute coronary syndrome and anemia.

Effect of intensive glycemic control on platelet reactivity in patients with long-standing uncontrolled diabetes

BACKGROUND It has been previously shown that platelets of patients with diabetes are more reactive and less responsive to anti-platelet drugs compared with platelets from subjects without diabetes. Studies examining the effect of glycemic control on platelet reactivity have yielded conflicting data. Thus, in this study, we sought to explore the effect of tight glycemic control on platelet reactivity in patients with long standing uncontrolled diabetes. METHODS The study included 30 patients with long-standing treated diabetes and a baseline HbA1c level of ≥ 8.5%. All patients were treated with aspirin and statins. Patients were tested at baseline and after 3 months of intensive glycemic and metabolic control. The treatment goal was to achieve a HbA1c level of ≤ 7%. Platelet reactivity was assessed by light transmission aggregation in response to 5 and 10 μM ADP and to 0.5mg/ml arachidonic acid (AA). Additonally, platelet activation was assessed by plasma levels of soluble P-selectin using an enzyme-linked immunosorbent assay. RESULTS The mean duration of diabetes from the time of diagnosis was 20.46 ± 9.31 years. Baseline HbA1c was 9.4 ± 0.8%. Following the intensive glycemic control period, the HbA1C level decreased to 8.1 ± 0.8% (P < 0.0001). Other laboratory parameters did not change significantly except for triglyceride levels, which decreased. None of the platelet aggregation studies nor P-selectin levels differed between baseline and after 3 months of intensive glycemic control. CONCLUSIONS Intensive glycemic control in patients with longstanding uncontrolled diabetes does not seem to result in a reduction in platelet reactivity.