Effects of atorvastatin on circulating CD34+/CD133+/ CD45− progenitor cells and indices of angiogenesis (vascular endothelial growth factor and the angiopoietins 1 and 2) in atherosclerotic vascular disease and diabetes mellitus

Abstract. Jaumdally RJ, Goon PKY, Varma C, Blann AD, Lip GYH (City Hospital, Birmingham, United Kingdom). Effects of atorvastatin on circulating CD34+/CD133+/CD45? progenitor cells and indices of angiogenesis (vascular endothelial growth factor and the angiopoietins 1 and 2) in atherosclerotic vascular disease and diabetes mellitus. J Intern Med 2010; 267 : 385–393. Background. Cardiovascular disease (CVD) remains a major cause of morbidity and mortality, especially in the presence of diabetes, possibly because of endothelial damage. Increased circulating progenitor cells (CPCs) and increased plasma markers of angiogenesis [vascular endothelial growth factor (VEGF) and the angiopoietins (Ang‐1 and ‐2)] may be evidence of this damage. Treatment with hydroxy‐methyl‐glutaryl (HMG‐CoA) reductase inhibitors (‘statins’) improves outcomes in patients with vascular disease, including diabetic patients. We hypothesized that 80 mg per day atorvastatin influences CPC counts of VEGF and the angiopoietins in patients with atherosclerotic CVD with or without diabetes mellitus. Methods. Cardiovascular disease patients with diabetes mellitus (Group A, n = 14) and nondiabetic patients with CVD only (Group B, n = 10) took atorvastatin 80 mg per day for a period of 8–10 weeks. CPCs (CD34+/CD133+/CD45?) were defined by flow cytometry, plasma levels VEGF and Ang‐1 and Ang‐2 by ELISA). Results. Circulating progenitor cell counts increased (P < 0.001) in Group A compared with a nonsignificant change in Group B (P = 0.37). VEGF levels fell significantly in Group A (P = 0.04) but no significant change was seen in Group B (P = 0.16). Whilst Ang‐1 remained unchanged (P = 0.41), Ang‐2 levels increased markedly in both groups (P < 0.05). These effects were independent of LDL and total cholesterol changes but were associated with HDL changes Conclusion. High‐dose atorvastatin increased circulating CPCs, reduced VEGF and increased Ang‐2 in patients with diabetes and CVD, providing another possible pathophysiological mechanism for the beneficial effects of statins in CVD.     

Soluble CD40 ligand and atrial fibrillation: relationship to platelet activation, and endothelial damage/dysfunction

Increased plasma soluble CD40L (sCD40L) is present in many cardiovascular diseases and predicts poor outcome, but levels in atrial fibrillation (AF) are unknown. Although the platelet is frequently cited as the source of sCD40L, this view is not universal. We hypothesised (a) raised sCD40L in non-rheumatic AF, and (b) that sCD40L correlates with platelet, but not endothelial, markers, thus suggesting a platelet origin. Plasma sCD40L, platelet marker soluble P-selectin and endothelial markers von Willebrand factor (vWf) and soluble E-selectin were measured by ELISA in 54 AF patients free of diabetes or major cardiovascular disease, and in 28 age/sex matched controls. Median (inter-quartile range) sCD40L in AF was 0.82 (0-4.8) ng/mL compared to 0.21 (0-5.5 ng/mL) in controls (p=0.0397). vWf and soluble P-selectin (p<0.005), but not soluble E-selectin, were raised in AF, but none of the indices inter-correlated significantly. We find that sCD40L is marginally raised in AF but the stimulus for this is unclear. The lack of clear correlation with relevant plasma markers suggests that the source is unlikely to be the endothelium or platelet alone.     

Stroke risk and suboptimal thromboprophylaxis in Chinese patients with atrial fibrillation: Would the novel oral anticoagulants have an impact?

Background

The risk of stroke associated with atrial fibrillation (AF) is higher in Far Eastern population than in Western population, and warfarin use suboptimal. There is uncertainty whether the novel oral anticoagulants (NOACs) would have a major impact on stroke prevention in Far Eastern populations with AF.

Objectives

We investigated current antithrombotic therapy use on stroke and bleeding risk, determinants of warfarin use and performed a modeling analysis of the net clinical benefit of the NOACs (apixaban, dabigatran) in a large cohort of Chinese patients with AF.

Methods

We studied 1034 Chinese patients (27.1% female, median age 75 years, interquartile range [IQR]: 63–83) with AF who were followed-up for an average of 1.9 years (IQR: 1.43–2.64). Stroke/thromboembolism (TE), and major bleeding associated to antithrombotic treatment were investigated. A modeling analysis was performed for the net clinical benefit balancing major bleeding against stroke/TE for dabigatran 110 mg bid, dabigatran 150 mg bid and apixaban, using their respective recent clinical trial outcome data.

Results

Using a Cox proportional hazard model, the Hazard Ratio [HR (95% confidence interval, CI)] for stroke/TE compared to no-antithrombotic therapy (no ATT) was 1.27 (0.65–2.50) on aspirin, 1.40 (0.35–3.57) on clopidogrel, 1.52 (0.72–3.23) on dual antiplatelets and 1.65 (0.76–3.57) on warfarin. The risk for major bleeding was 0.35 (0.14–0.85) on aspirin, 0.74 (0.24–2.29) on clopidogrel, 0.35 (0.11–1.10) on dual antiplatelets, and 0.88 (0.36–2.17) on warfarin. Binary logistic regression analysis showed persistent/permanent AF (Odds Ratio, OR, 2.03 [95%CI 1.05–3.92], p = 0.035) was associated with warfarin use, but age ≥ 75 years (0.26 [0.16–0.42], p < 0.001), aspirin (0.18 [0.12–0.27], p < 0.001) and clopidogrel (0.17 [0.08–0.33], p < 0.001) were independent determinants of non-use of warfarin.On modeling net clinical benefit (per 100 person-years [95% CI]), apixaban use compared to antiplatelet agents or no ATT was 3.29 (2.15–4.30) using Singer's method and 2.08 (1.18–3.21) with Connolly's method amongst high-risk patients. The use of dabigatran 110 mg bid and 150 mg bid compared to antiplatelet agents could reduce an additional 18.1 stroke/TE and 24.3 stroke/TE events, respectively. Compared to warfarin, dabigatran 150 mg bid had the best net clinical benefit.

Conclusions

There was suboptimal stroke prevention with no difference between antiplatelet and OAC treated patients, perhaps reflecting an inappropriate Target INR range. On modeling analyses, the use of the NOACs (apixaban and dabigatran) could provide better stroke prevention compared to antiplatelet (or warfarin) use in this Chinese AF population, with a positive net clinical benefit.     

von Willebrand factor and its relevance to cardiovascular disorders.

In the past decade the importance of the vascular endothelium in cardiovascular pathophysiology has become more apparent. One substance that is synthesised by and stored in endothelial cells is von Willebrand factor (vWF). When released, vWF seems to mediate platelet aggregation and adhesion to the vascular endothelium. Because the release of vWF is increased when endothelial cells are damaged, vWF has been proposed as an indicator of endothelial disturbance or dysfunction. The availability of such an index of endothelial dysfunction may have clinical value, because measurement of such a marker can be a non-invasive way of assisting in diagnosis or as an indicator of disease progression. The known association between vWF, thrombogenesis, and atherosclerotic vascular disease also suggests that high concentrations of vWF may be an indirect indicator of atherosclerosis and/or thrombosis. In addition, high vWF concentrations have prognostic implications in patients with ischaemic heart disease and peripheral vascular disease.     

Mechanism of antithrombin III inhibition of factor VIIa/tissue factor activity on cell surfaces. Comparison with tissue factor pathway inhibitor/factor Xa-induced inhibition of factor VIIa/tissue factor activity

Recent studies have shown that antithrombin III (AT III)/heparin is capable of inhibiting the catalytic activity of factor VIIa bound either to relipidated tissue factor (TF) in suspension or to TF expressed on cell surfaces. We report studies of the mechanism of which by AT III inhibits factor VIIa bound to cell surface TF and compare this inhibitory mechanism with that of tissue factor pathway inhibitor (TFPI)-induced inhibition of factor VIIa/TF. AT III alone and AT III/heparin to a greater extent reduced factor VIIa bound to cell surface TF. Our data show that the decrease in the amount of factor VIIa associated with cell surface TF in the presence of AT III was the result of (1) accelerated dissociation of factor VIIa from cell surface TF after the binding of AT III to factor VIIa/TF complexes and (2) the inability of the resultant free factor VIIa-AT III complexes to bind effectively to a new cell surface TF site. Binding of TFPI/factor Xa to cell surface factor VIIa/TF complexes markedly decreased the dissociation of factor VIIa from the resultant quaternary complex of factor VIIa/TF/TFPI/factor Xa. Addition of high concentrations of factor VIIa could reverse the AT III-induced inhibition of cell surface factor VIIa/TF activity but not TFPI/factor Xa-induced inhibition of factor VIIa/TF activity.     

Von Willebrand factor, P-selectin and fibrinogen levels in patients with acute ischaemic and haemorrhagic stroke, and their relationship with stroke sub-type and functional outcome

Although ischaemic stroke is associated with accentuated platelet function, it remains unclear whether this applies to all sub-types, especially lacunar infarcts, which differ from cortical infarction in their patho-aetiology. Similarly, conflicting evidence suggests that haemorrhagic stroke may, or may not, be associated with a hypothrombotic state. Levels of von Willebrand factor (vWF), fibrinogen, and P-selectin were measured within 48 h of ictus in 163 patients with acute ischaemic stroke and 40 patients with acute primary intracerebral haemorrhage, and 33 age, gender and race matched-controls. vW F (IU/dl) was significantly increased in both cortical and lacunar ischaemic stroke, and haemorrhagic stroke, as compared with controls, median (semiquartile range): 158 (25) vs 144 (19) vs 147 (24) vs 114 (16), respectively. Similarly, fibrinogen (g/litre) was increased: 4.80 (0.90) vs 4.65 (0.70) vs 4.35 (0.83) vs 3.70 (0.70). In contrast, soluble P-selectin (ng/ml) was increased in cortical stroke as compared with lacunar infraction patients or controls: 408 (101) vs 300 (108) vs 324 (121), respectively; P-selectin was not increased in haemorrhagic stroke, 360 (153). Both vW F and fibrinogen correlated with 3-month functional outcome (modified Rankin score): r = 0.371 (2 P = 0.0006), and r = 0.195 (2 P = 0.042), respectively; however, P-selectin was not associated with outcome: r = 0.188 (2 P = 0.084). The results suggest that increases in vW F and fibrinogen in all types of stroke reflect an acute phase response; in contrast, increased soluble P-selectin levels in cortical stroke, but not lacunar infarction, suggest that platelets contribute to the patho-aetiology of some subtypes of ischaemic stroke.     

Binding of factor VIIa to tissue factor permits rapid antithrombin III/heparin inhibition of factor VIIa

Because free factor VIIa is inactivated only very slowly by a plasma concentration of antithrombin III (AT III) even in the presence of heparin, it has been assumed that AT III plays no significant role in regulating the initiation of tissue factor-dependent blood coagulation. However, in the present study, we present evidence that factor VIIa bound to tissue factor, unlike free factor VIIa, is readily inactivated by AT III in the presence of heparin. In a reaction mixture containing calcium ions and approximately equimolar concentrations of relipidated tissue factor (8.9 nmol/L) and factor VIIa (10 nmol/L), AT III (100 micrograms/mL) plus heparin (1 U/mL) inhibited 50% of the factor VIIa coagulant activity of the reaction mixture within 5 minutes. AT III/heparin was also shown to inhibit the catalytic activity towards factor X of factor VIIa/tissue factor complexes formed on monolayers of an ovarian carcinoma cell line (OC-2008) that constitutively expresses surface membrane tissue factor. AT III, even in the absence of exogenously added heparin, substantially inhibited the functional activity of factor VIIa/cell surface tissue factor complexes on intact monolayers. AT III alone and AT III/heparin, to a greater extent, also inhibited factor VIIa on "nonfunctional" factor VIIa/tissue factor complexes on intact monolayers, with resultant inhibition of their expression of factor VIIa/tissue factor catalytic activity toward factor X after cell lysis. The potential physiologic significance of these findings is discussed.     

A cross-sectional and diurnal study of thrombogenesis among patients with chronic atrial fibrillation

OBJECTIVES

First, we sought to determine whether there is diurnal variation in hemostatic factors related to thrombogenesis and hypercoagulability among patients with chronic atrial fibrillation (AF). Second, we sought to determine whether levels of soluble thrombomodulin (sTM), a marker of endothelial function, or soluble P-selectin (sP-sel), an index of platelet activation, are altered in patients with AF as compared with subjects in sinus rhythm.

BACKGROUND

Atrial fibrillation is associated with an increased risk of stroke and thromboembolism and is known to confer a hypercoagulable state, with abnormalities of thrombosis, platelet activation and endothelial cell function. Many cardiovascular events, such as acute myocardial infarction, have thrombosis as an underlying process, and they undergo diurnal variation.

METHODS

Fifty-two patients (45 men, mean [±SD] age 66 ± 6 years) with chronic AF, none of whom received antithrombotic therapy, were studied. Baseline levels of fibrinogen, sP-sel, sTM and von Willebrand factor (vWF) were compared to those levels in matched healthy control subjects in sinus rhythm. In a subgroup of 20 patients, five venous blood samples were collected through an indwelling cannula at 6-h intervals from 12 to 12 the following day and were analyzed for the same markers.

RESULTS

Patients with chronic AF had higher plasma sP-sel, sTM, vWF and fibrinogen levels as compared with control subjects in sinus rhythm. Significant correlations were found between fibrinogen and sP-sel in patients with AF (r = 0.567 [Spearman], p < 0.001) and in control subjects (r = 0.334, p = 0.016). There was no significant diurnal variation in plasma levels of sP-sel, sTM, vWF or fibrinogen over the 24-h study period (repeated measures analysis of variance, p = NS).

CONCLUSIONS

There is no circadian or diurnal variation in the hypercoagulable state seen in AF, as assessed by plasma fibrinogen and markers of platelet (sP-sel) and endothelial function (vWF and sTM). The persistent hypercoagulable state, together with the loss of diurnal variation in various hemostatic markers, in chronic AF may contribute to the high risk of stroke and thromboembolic complications in these patients.     

Laboratory assessment of anti-thrombotic therapy in heart failure,atrial fibrillation and coronary artery disease: insights using thrombelastography and a micro-titre plate assay of thrombogenesis and fibrinolysis

As heart failure, coronary artery disease and atrial fibrillation all bring a risk of thrombosis, anti-thrombotic therapy is recommended. Despite such treatment, major cardiovascular events such as myocardial infarction and stroke still occur, implying inadequate suppression of thrombus formation. Accordingly, identification of patients whose haemostasis remains unimpaired by treatment is valuable. We compared indices for assessing thrombogenesis and fibrinolysis by two different techniques in patients on different anti-thrombotic agents, i.e. aspirin or warfarin. We determined fibrin clot formation and fibrinolysis by a microplate assay and thromboelastography, and platelet marker soluble P selectin in 181 patients with acute or chronic heart failure, coronary artery disease who were taking either aspirin or warfarin. Five thromboelastograph indices and four microplate assay indices were different on aspirin versus warfarin (p < 0.05). In multivariate regression analysis, only microplate assay indices rate of clot formation and rate of clot dissolution were independently related to aspirin or warfarin use (p ≤ 0.001). Five microplate assay indices, but no thrombelastograph index, were different (p < 0.001) in aspirin users. Three microplate assay indices were different (p ≤ 0.002) in warfarin users. The microplate assay indices of lag time and rate of clot formation were abnormal in chronic heart failure patients on aspirin, suggesting increased risk of thrombosis despite anti-platelet use. Soluble P selectin was lower in patients on aspirin (p = 0.0175) but failed to correlate with any other index of haemostasis. The microplate assay shows promise as a tool for dissecting thrombogenesis and fibrinolysis in cardiovascular disease, and the impact of antithrombotic therapy. Prospective studies are required to determine a role in predicting thrombotic risk.     

Plasma von Willebrand factor, fibrinogen and soluble P-selectin levels in paroxysmal, persistent and permanent atrial fibrillation. Effects of cardioversion and return of left atrial function.

BACKGROUND: Atrial fibrillation is associated with increased risk of stroke and thromboembolism, possibly by conferring a prothrombotic or hypercoagulable state. However, it is unclear whether or not this differs in the clinical subgroups of chronic atrial fibrillation patients, that is, in those with paroxysmal, persistent or permanent atrial fibrillation. We therefore hypothesized that: (i) there are differences in the prothrombotic state between these patients; and (ii) reduction in indices of hypercoagulability would follow elective electrical cardioversion of persistent atrial fibrillation and the return of left atrial function. PATIENTS AND METHODS: We studied 69 patients with chronic atrial fibrillation: 23 with paroxysmal atrial fibrillation (16 males; mean age 65 years+/-SD 13); 23 with persistent atrial fibrillation (16 males; 65 years+/-13), with a mean duration of atrial fibrillation of 3 months (range 2 to 6 months); and 23 with permanent atrial fibrillation (16 males; 67 years+/-10). Blood results were compared to 20 age- and sex-matched healthy controls. The patients with persistent atrial fibrillation then underwent elective DC cardioversion, with Doppler echocardiographic examinations and bloods tests performed prior to cardioversion, and at 3 and 12 weeks afterwards. The prothrombotic state was quantified by measurement of plasma levels of fibrinogen, soluble P-selectin (an index of platelet activation) and von Willebrand factor (a marker of endothelial dysfunction). RESULTS: Permanent atrial fibrillation was associated with significantly raised levels of von Willebrand factor, soluble P-selectin and fibrinogen (all P<0.001); paroxysmal atrial fibrillation with significantly elevated levels of plasma von Willebrand factor (P=0.0067) and fibrinogen (P=0.0001) but not soluble P-selectin (P=0.472); and persistent atrial fibrillation with normal levels of fibrinogen, von Willebrand factor and soluble P-selectin when compared to healthy controls (all P=ns). Stepwise multiple regression analyses demonstrated that the presence of atrial fibrillation was an independent predictor of abnormal von Willebrand factor, fibrinogen and soluble P-selectin levels. Electrical cardioversion of the patients with persistent atrial fibrillation did not significantly alter levels of von Willebrand factor (P=0.766), soluble P-selectin (P=0.726) or fibrinogen (P=0.50) despite maintenance of sinus rhythm and a significant return of left atrial systolic function (as quantified by the presence of A wave on Doppler echocardiography) at 3 months. CONCLUSION: There were significant differences in the prothrombotic state when patients with paroxysmal and permanent atrial fibrillation are compared to matched patients with persistent atrial fibrillation or controls in sinus rhythm. Cardioversion of persistent atrial fibrillation did not significantly alter indices of hypercoagulability even after 3 months maintenance of sinus rhythm, despite the return of atrial systole.