Abstract
Background. The precise pathophysiological processes underlying the prothrombotic or hypercoagulable state in atrial fibrillation (AF) remain uncertain. We hypothesized a relationship between abnormal endothelial damage/dysfunction, coagulation, and angiogenic factors, thereby contributing to increased thrombogenicity.
Methods. Plasma levels of von Willebrand factor (vWF, an index of endothelial damage/dysfunction) and tissue factor (TF, an index of coagulation), as well as the angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin‐1 (Ang‐1) and angiopoietin‐2 (Ang‐2), were measured by enzyme‐linked immunosorbant assay (ELISA) in 59 chronic AF patients. Data were compared to 40 age‐ and sex‐matched healthy controls in sinus rhythm.
Results. Plasma vWF, VEGF and Ang‐2 were significantly higher in AF patients compared to healthy controls (P = 0.005, P = 0.0055 and P<0.0001 respectively) but there were no significant differences in plasma Ang‐1 or TF levels between the two groups (P = 0.925 and P = 0.121 respectively). Significant correlations were found between VEGF and vWF levels (Spearman, r = 0.262, P = 0.011) and between VEGF and Ang‐2 (r = 0.333, P = 0.001).
Conclusions. Raised VEGF in association with Ang‐2 and vWF may reflect a link between abnormal endothelial damage/dysfunction and angiogenic factors. These may act together to alter TF expression and endothelial integrity, thereby contributing to the prothrombotic state in AF.
Introduction
Atrial fibrillation (AF) is associated with thromboembolic tendency and is a leading cause of stroke and thromboembolism Citation1, probably due to abnormalities in haemostasis, platelets and indices of endothelial damage/dysfunction Citation2 that seem independent of associated structural heart disease or underlying aetiology of AF Citation3. However, the precise pathophysiological processes underlying the prothrombotic or hypercoagulable state in atrial fibrillation (AF) remain uncertain.
Endothelial damage/dysfunction may be central to the pathogenesis of the prothrombotic state in AF Citation4. Indeed, indices of endothelial damage/dysfunction, such as plasma von Willebrand factor (vWF) are consistently abnormal in AF Citation5–7 and vWF has even been associated with stroke risk stratification and adverse outcomes in AF patients Citation8, Citation9. As well as vWF, other endothelial measures such as forearm plethysmography, nitric oxide production, plasma E‐selectin and soluble thrombomodulin are also abnormal in AF Citation10–12, Citation6.
There are many possible indices of angiogenesis. For example, vascular endothelial growth factor (VEGF) is one such marker, and is a potent endothelium‐specific angiogenic factor, which acts to promote endothelial cell survival, proliferation and migration Citation13. The VEGF gene and protein are both strongly expressed in actively angiogenic areas, and it exerts its biological effects through binding its high‐affinity tyrosine kinase receptors Flt‐1 and Flk‐1/KDR. VEGF is thought to play an essential role in angiogenesis by increasing endothelial monolayer permeability Citation14, but in addition, this molecule also acts as a pro‐inflammatory cytokine by inducing the expression of adhesion molecules that bind leucocytes to endothelial cells Citation15, as well as increasing tissue factor expression and pro‐coagulant activity Citation16. The importance of tissue factor (TF) is its powerful procoagulant activity on forming a complex with factor VII(a), and it is widely regarded as the physiological trigger to blood clotting as it drives thrombin formation Citation17. Importantly, VEGF has been shown to be raised in AF Citation18, Citation19. Raised VEGF has been associated with increased TF levels in AF, linking angiogenesis (or VEGF) to thrombogenesis Citation19, in keeping with data from cancer biology Citation20.
More recently, oncology research has identified other growth factors, such as those of the angiopoietin family, that appear to have a major role in angiogenesis and metastasis Citation21–23. The roles of two members of this family, angiopoietin‐1 and angiopoietin‐2 (Ang‐1 and Ang‐2) are becoming understood, and may act alongside VEGF. VEGF is also known to increase Ang‐2 levels Citation24, and can also have an influence on TF activity. Indeed, the balance of Ang‐1 and Ang‐2 levels has been proposed to have an important homeostatic role via their respective agonist and antagonistic actions on Tie‐2 (an endothelial receptor tyrosine kinase). When the balance is in favour of Ang‐1, endothelial stability is promoted – whereas when the actions of Ang‐2 predominate, endothelial destabilization, new vessel sprouting and facilitation of the actions of other vascular cytokines such as VEGF are favoured.
We hypothesised a link between abnormal endothelial damage/dysfunction and angiogenic factors, which may act together to alter tissue factor expression and endothelial integrity, thereby contributing to increased thrombogenicity. We tested this hypothesis in a cross‐sectional study of 59 chronic AF patients and 40 controls in sinus rhythm, in whom levels of vWF and TF, as well as the angiogenic factors, VEGF and Ang‐1&2, were measured by ELISA.
Key messages
The precise pathophysiological processes underlying the prothrombotic or hypercoagulable state in atrial fibrillation (AF) remain uncertain. Abnormalities of angiogenesis and endothelial damage/dysfunction may have a role.
In this study we found abnormal vascular endothelial growth factor (VEGF, associated with angiogenesis) in association with angiopoietin‐2 (another index of angiogenesis) and vWF (an index of endothelial damage/dysfunction) in AF, which may reflect a link between an abnormal endothelium and angiogenic factors.
These abnormalities may act together to alter tissue factor expression and endothelial integrity, thereby contributing to the prothrombotic state in AF.
Methods
We studied 59 outpatients with chronic AF attending our specialist clinic. All patients had a history of AF of >6 weeks, and no evidence of valvular heart disease or left ventricular dysfunction (by clinical examination and echocardiography). Exclusion criteria also included previous myocardial infarction, documented coronary artery disease at angiography, diabetes mellitus, a history of an acute cerebrovascular event (within 1 month), malignancy, connective tissue or inflammatory disease, acute or chronic infection, hepatic impairment and renal impairment.
Results were compared to 40 age‐ and sex‐matched healthy control subjects, recruited from hospital staff and relatives or friends of patients who attended hospital. Healthy controls had no history of diabetes, hypertension, neoplastic, connective tissue or cardiovascular disease and all subjects underwent careful screening with baseline bloods, blood pressure measurement, electrocardiography and echocardiography. All healthy controls were ‘healthy’ by virtue of normal clinical history and examination, as well as basic blood screening tests, electrocardiogram (ECG) and echocardiography. The study was conducted in accordance with the Declaration of Helsinki and following the approval of the local research ethics committee. Written informed consent was obtained from all participants.
Laboratory
Venous blood was obtained from subjects in the fasted state and, where possible, >8 hours following any medication. Citrated plasma samples were collected and immediately placed on ice for a maximum of 20 minutes before processing. Platelet‐free citrated plasma was obtained from venous blood by centrifugation at 3000 rpm for 20 minutes at 4°C. Plasma was then aliquoted and frozen at −70°C for subsequent batch analysis. Plasma vWF was measured using an established enzyme‐linked immunosorbant assay (ELISA) (Dako, Glostrup, Denmark). Tissue factor (pg/mL) was measured by ELISA using reagents and standards from Axis‐Shield (Dundee). VEGF, Ang‐1 and Ang‐2 (ng/mL) were all measured by ELISA using reagents from R&D systems (Abingdon, Oxfordshire, UK). Intra‐assay coefficients of variation for all ELISAs were <5% and interassay variances <10%.
Power calculations and statistical analysis
We have previously reported plasma Ang‐2 to be significantly raised (by 0.5 SD) in 40 patients with chronic congestive heart failure compared to 17 healthy controls (P<0.001) Citation25. In the present study, we set our power calculation at a minimum of 32 subjects per group to enable us to detect a difference of one standard deviation with 1−β = 0.8 and P<0.05 between groups.
Continuous data were subjected to the Anderson‐Darling test to assess distribution, and are expressed as mean±standard deviation (SD) or median and inter‐quartile range (IQR) as appropriate. Differences between the patient group and controls were analysed by t‐testing or Mann‐Whitney tests, as appropriate. Categorical data were compared using chi‐squared test. Correlations were performed using Spearman's rank correlation method. Multivariate analysis was performed by stepwise multiple regression analysis using research indices as the dependent variable(s) and clinical variables (e.g. age, gender, hypertension, etc.) and the presence/absence of AF as predictors. A two‐tailed P value <0.05 was considered statistically significant.
Results
Demographic and clinical data for patients and controls are summarized in . The median duration of AF (defined from the time of first electrocardiographic confirmation) was 6.5 months (IQR 3.75–24) and of note, 40 of the AF group were already receiving warfarin, 13 were on aspirin and 6 had not yet started antithrombotic therapy at the time of blood sampling, although antithrombotic therapy has not been noted to alter our research indices Citation3.
Table I. Demographic and clinical data for AF cases and healthy controls.
Results are summarized in . In patients with AF there were, as expected, higher plasma levels of vWF compared to healthy controls (P = 0.005). VEGF and Ang‐2 were also significantly raised in AF (P = 0.0055 and P<0.0001 respectively). There was, however, no significant difference in Ang‐1 or TF levels in comparison with control subjects (P = 0.925 and P = 0.121 respectively).
Table II. Plasma levels of vWF, tissue factor, VEGF, angiopoietin‐1 and angiopoietin‐2 in AF patients in comparison to healthy controls.
Effects of age, co‐morbidity and concomitant warfarin or aspirin use in AF patients
There were no significant differences in vWF, TF, VEGF, Ang‐1 or Ang‐2 levels between AF patients when separated into those above and below the median age of 65 (IQR 64–68) years old (). There were also no differences in research indices in those patients with AF who also had a diagnosis of hypertension when compared to those with no past medical history of hypertension (), and AF patients with other co‐morbidities known to influence angiopoietin levels such as diabetes or known ischaemic heart disease (angina or prior myocardial infarction) were excluded from this study from the outset.
Table III. Difference in research indices in the AF group with age (group divided into those above and below the median age of 65).
Table IV. Difference in research indices in the AF group dependent on a pre‐existent diagnosis of hypertension.
Warfarin use in the AF group did not influence either vWF plasma levels or plasma levels of the other endothelium‐related indices, with no significant differences between those patients on and off warfarin treatment at the time of sampling (). Similarly the use of aspirin had no influence on plasma vWF, VEGF, Ang‐1, Ang‐2 or TF (data not shown).
Table V. Difference in research indices in the AF group dependent on whether on or not the patient was on warfarin at the time of blood sampling.
Correlations and multivariate analyses
In the entire cohort, there were weak but significant correlations found between vWF and VEGF levels (Spearman r = 0.262, P = 0.011), and VEGF and Ang‐2 (r = 0.333, P = 0.001). Correlations of the plasma levels of vWF, VEGF and Ang‐2 with clinical variables (AF, sex, age hypertension, current smoking and prior cerebrovascular accident) and with the use of concurrent medication (listed in ), showed a significant association between vWF and AF (r = 0.247, P = 0.007), but no significant positive correlations with the other clinical indices or treatment listed. VEGF levels showed significant correlations with presence of AF (r = 0.236, P = 0.005) and previous CVA (r = 0.239, P = 0.019). Ang‐2 levels correlated with AF (r = 0.425, P<0.0001) and previously diagnosed hypertension (r = 0.259, P = 0.02).
To determine those factors independently associated with plasma vWF, VEGF and Ang‐2, we used stepwise multiple regression analyses. The presence of AF was the only independent predictor of vWF levels (P = 0.008) and was also the only independent positive predictor of Ang‐2 (P<0.001) levels. The presence of AF just failed to reach significance for independently predicting VEGF levels (P = 0.053), and CVA was not associated with VEGF on multiple regression analysis (P = 0.784).
Discussion
In this study we have shown that Ang‐2 is increased in AF in association with VEGF and vWF, thus demonstrating alterations in the balance of angiogenic factor expression in these patients. Furthermore, correlations between vWF and VEGF and between VEGF and Ang‐2 would indicate a connection (by a common drive, origin or pathophysiological process) that links their expression.
Raised vWF in AF is consistent with numerous previous studies Citation2, Citation5–7 and is almost certainly a reflection of endothelial damage/dysfunction in association with this arrhythmia, especially within the left atrium Citation26. The increased expression of vWF in the endocardium can be associated with enlarged left atrial dimensions in mitral valvular disease or increased myocyte diameters in the underlying myocardium, as well as platelet adhesion/aggregation on the endocardium Citation27. Indeed, the increased vWF in the endocardium may be a local predisposing factor for thrombogenesis in overloaded human atrial appendage Citation28. Theories as to the cause of endothelial damage/dysfunction in AF vary, but we postulate these could be associated with altered shear stresses to the vascular endothelium, either localized to the atrium or systemically due to a lack of regular pulsatile laminar flow Citation29, Citation30. Changes in shear stress in AF may influence adhesion molecule expression and lead to a down‐regulation in endothelial nitric oxide synthase expression and therefore endothelial cell nitric oxide production. These could be the first steps in a cascade of events with resultant platelet aggregation and adhesion, leucocyte adhesion, inflammatory cytokine release, further endothelial damage and vWF release into the circulation, thus promoting thrombus formation. The presence of other cardiovascular risk factors would, of course, compound this by further causing endothelial dysfunction therefore escalating the drive for thrombus formation in AF. Alternatively, alterations in endothelial indices may simply reflect associated vascular disease, although we did try to minimize the influence of this by exclusion of patients with known coronary or peripheral artery disease from our study.
Although there is no obvious requirement for angiogenesis in AF, we found VEGF levels again to be raised in this cohort, consistent with previous studies Citation18, Citation19. In the current study, efforts were made to exclude other co‐morbidities that have been associated with angiogenesis (such as diabetes mellitus or documented coronary artery disease), which would have confounded our results. Although patients with treated cardiovascular risk factors (hypertension and hyperlipidaemia) were included in our study group, both mean blood pressure and serum total cholesterol levels were comparable in cases and controls and therefore should not have influenced our results.
In this study we have again shown the association of raised VEGF levels with AF. The drive for this is uncertain, but left atrial enlargement has been postulated to play a role, as mechanical stretch has been shown to induce rapid secretion of VEGF by cultured rat cardiac myocytes in vitroCitation31 and similarly smooth muscle cells when stretched exhibit up‐regulation of hypoxia‐inducible factor‐1 (HIF‐1)α, which in turn increases the expression of VEGF (as VEGF is the target gene for HIF‐1) Citation32. In the former, the increased expression of VEGF has been suggested to be a response to relative tissue hypoxia, with a role in the protection of cardiac myocytes from tachyarrhythmia‐induced stress and improvement of coronary microcirculation. However, generalized tissue hypoxia and angiogenesis are not features of AF, and an alternative explanation that we would put forward is that of a rise in VEGF being part of a maladaptive response to endothelial perturbation that acts to perpetuate rather than fix the problem. Another possible source of VEGF may be activated platelets Citation13, which may be implicated in the hypercoagulable state of AF Citation2.
We have previously suggested that both VEGF and Ang‐2, rather than being angiogenic in congestive heart failure patients, may be necessary for endothelial cell repair and cell turnover and their increased secretion may be stimulated by endothelial damage or dysfunction Citation25. Indeed, increased VEGF levels may have a similar role in AF, and could promote the hypercoagulable state by increasing endothelial cell expression of adhesion molecules such as E‐selectin, thereby encouraging leucocyte and platelet adhesion and activation, and could also act to increase endothelial tissue factor expression and procoagulant activity. In the present study, plasma VEGF levels also correlated with Ang‐2 levels, in keeping with data showing that VEGF may increase Ang‐2 expression Citation24. However, the relationship between vWF and Ang‐2 can also be explained by the recent finding that they are co‐secreted by endothelial cells as they are contained within a subset of Weibel Palade bodies Citation33.
Whatever the mechanism of Ang‐2 increase, we do know that Ang‐1 and Ang‐2 are natural co‐antagonists in that they compete for the same active binding site on Tie‐2 Citation34, Citation35 so although Ang‐1 levels are not depressed in our patient group, with the balance in favour of Ang‐2 we may assume that the actions of Ang‐1 are inhibited. If this were the case, the raised Ang‐2 levels in AF would be of relevance in that they would result in a loss of negative regulation of VEGF‐induced tissue factor activity by Ang‐1, and although TF levels were not significantly increased in this particular study group, we have previously demonstrated raised TF plasma levels in AF compared with healthy controls Citation19.
Therefore, rather than promoting angiogenesis per se, we suggest that an imbalance of Ang‐1 and ‐2 could act to disrupt endothelial integrity, and increases in Ang‐2 in association with increased VEGF could increase TF expression and procoagulant activity therefore playing an integral role in generation of the prothrombotic state. Indeed, the link between alterations in angiogenic factors and thrombogenicity proposed in atherosclerotic disease Citation36 may also be relevant in the setting of AF. The lack of influence of VEGF and Ang‐2, on promoting new vessel formation would have to be further explored, but we speculate could be explained by concurrent suppression of the signalling pathways to angiogenesis. In AF, this could be explained by down‐regulation of endothelial nitric oxide synthetase (eNOS) by reduced shear stress, rather than increased VEGF causing eNOS up‐regulation, thereby disrupting the usual angiogenic signalling pathway of VEGF Citation37. Atrial natriuretic peptide (ANP), which is known to be raised in AF Citation38, has been shown to interrupt VEGF signalling via natriuretic peptide clearance receptor specific ligand (NPRC) and guanylate cyclase (GC) receptors Citation39. Therefore alterations in angiopoietin expression associated with concurrent increases in VEGF levels could be acting to increase TF without promoting angiogenesis.
This study is, of course, limited by its cross‐sectional design and only allows us to explore novel associations therefore no causality is implied. The influence of co‐morbidities such as hypertension and hyperlipidaemia, although treated, cannot be discounted. Additionally as there is evidence for alterations in angiogenic factor expression in cerebral infarction and ischaemia Citation40, Citation41, the influence of previous cerebrovascular events also cannot be discounted, and although acute strokes (within 1 month) are excluded, it is acknowledged that this may be contributory to our findings.
We conclude that the raised VEGF levels in association with increases in Ang‐2 and vWF demonstrated by this study may reflect a link between abnormal endothelial damage/dysfunction and angiogenic factors, which may act together to alter TF expression and endothelial integrity, thereby contributing to the prothrombotic state in AF. Indeed in light of the recently published finding that Ang‐2 co‐localizes with vWF in Weibel Palade bodies and of their co‐export from stimulated endothelial cells Citation33, our study would support a functional role for Ang‐2 alongside vWF in vascular haemostasis in AF, or at the very least shows Ang‐2 as yet another marker of endothelial perturbation in AF.
Acknowledgements
We acknowledge the support of the City Hospital Research & Development Programme for the Haemostasis Thrombosis and Vascular Biology Unit.
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