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Effects of Aspirin on Intra-Platelet Vascular Endothelial Growth Factor, Angiopoietin-1, and P-Selectin Levels in Hypertensive Patients
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2006; 19:970 –977
Mechanisms
Effects of Aspirin on Intra-Platelet Vascular Endothelial Growth Factor, Angiopoietin-1, and P-Selectin Levels in Hypertensive Patients Sunil Nadar, Andrew D. Blann, and Gregory Y.H. Lip Background: Although aspirin is useful in reducing platelet activation and cardiovascular events, its effects on platelet levels of angiogenic factors, such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), and markers of platelet activation in hypertension are unknown. The aim of this study was to study the effects of aspirin on the platelet morphology, plasma and platelet levels of VEGF (sVEGF and pVEGF respectively), Ang-1 (sAng-1 and pAng-1 respectively), and P-selectin (sPsel and pPsel respectively) in patients with well controlled hypertension.
well as pVEGF (P ⫽ .008), pAng-1(P ⫽ .001), sPsel (P ⫽ .02), pPsel (P ⬍ .001), and mean platelet mass (P ⫽ .01) when compared with control subjects. After treatment with aspirin for 3 months, there were significant reductions in plasma VEGF (P ⫽ .01), pAng-1 (P ⫽ .04), sPsel (P ⫽ .001), and pPsel (P ⬍ .001) levels, but not levels of platelet VEGF and plasma Ang-1. Neither pVEGF nor pAng-1 correlated with blood pressure or with their respective plasma levels.
Methods: A total of 35 aspirin-naive, hypertensive patients (29 male and six female; mean age 64 years) were compared with 30 (23 male, seven female, mean age 59 years) normotensive control subjects. Blood was collected for plasma VEGF, P-selectin, and Ang-1 (enzyme-linked immunoassay), intra-platelet levels of VEGF, Ang-1, and P-selectin, and platelet volume and mass. Research indices in hypertensive patients were studied before and after 3 months treatment with aspirin 75 mg daily.
Conclusions: The use of aspirin in high-risk hypertensive patients leads to a reduction in intra-platelet angiogenic growth factors and platelet activation. This may have implications for the use of aspirin in conditions (such as vascular disease) that have been associated with an increase in angiogenesis and platelet activation. Am J Hypertens 2006;19:970 –977 © 2006 American Journal of Hypertension, Ltd.
Results: Hypertensive patients had significantly higher plasma levels of VEGF (P ⫽ .04), Ang-1 (P ⬍ .001), as
Key Words: Hypertension, aspirin, vascular endothelial growth factor, angiopoeitin-1, P-selectin, platelets.
ypertension is known to be associated with an increased risk of thrombotic complications (strokes, myocardial infarction), which are likely to result from the prothrombotic or hypercoagulable state associated with this condition.1,2 Aspirin is the most widely used antiplatelet agent for reducing the risk of cardiovascular and cerebrovascular events in both primary prevention and secondary prevention.3 Its mode of action is in inhibiting cyclooxygenases (COX), enzymes that catalyse the conversion of arachidonic acid to eichosanoids and thus may play an important role in platelet– vessel wall interactions. Indeed, thromboxane is the major COX product in platelets, and its biosynthesis is increased
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in conditions associated with platelet activation such as unstable angina and peripheral vascular disease. Therefore, its inhibition leads to a reduction in platelet activation.4 – 6 Platelet activation can be assessed by a number of methods, such as the expression of P-selectin at the platelet cell surface, or by increased plasma levels. Platelet size, as well as membrane and plasma P-selectin are also increased in hypertension and acute stroke,7–9 and we have also shown that the total mass of P-selectin within each platelet (as determined by measuring levels of P-selectin in the lysate formed by solubilizing a fixed number of these cells) are increased
Received November 21, 2005. First decision January 29, 2006. Accepted March 7, 2006. From the Haemostasis Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham, England. This work was supported by the Peel Medical Research Trust and the Sandwell and West Birmingham Hospitals NHS Trust Research and
Development programme for the Haemostasis Thrombosis and Vascular Biology Unit. Address correspondence and reprint requests to Prof. Gregory Y.H. Lip, Haemostasis Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham B18 7QH, England; e-mail: [email protected]
0895-7061/06/$32.00 doi:10.1016/j.amjhyper.2006.03.001
© 2006 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.
AJH–September 2006 –VOL. 19, NO. 9
in atrial fibrillation, a condition often associated with hypertension.10 Of further interest is the fact that platelets are carriers of angiogenic growth factors, such as vascular endothelial growth factor (VEGF)11,12 and angiopoietin-1 (Ang-1).13 Indeed, platelet-derived VEGF and Ang-1 have been shown previously to be important in the pathogenesis of complications and metastasis in cancers, and aspirin has also been demonstrated to inhibit the release of these factors from platelets in patients with malignancies.14,15 We have previously reported raised levels of plasma VEGF in hypertension, although neither the clinical nor pathophysiolgic implications of this finding are clear.16 However, abnormal angiogenesis has been demonstrated in hypertension, and there seems to be an impaired ability for vascular growth resulting from structural alteration of the microvascular network, which includes capillary rarefaction, increased arteriolar length and tortuosity.17–19 Hence, we do know the following: 1) there is ample evidence of the involvement of platelets in the pathogenesis of hypertension, especially its vascular consequences (such as myocardial infarction and stroke); 2) aspirin is an effective antiplatelet agent; and 3) platelets may contribute to the complications of hypertension by virtue of their carriage of angiogenic (and other) growth factors. We therefore hypothesized that aspirin would cause a reduction in markers of platelet activation (P-selectin and changes in morphology) and alter the levels of both plasma and platelet angiogenic growth factors (VEGF and Ang-1). We tested our hypothesis in a simple case-control study of hypertensive patients and healthy control subjects, and a longitudinal study of the effects of introducing aspirin 75 mg/day in aspirin-naive hypertensive patients.
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the hospital for routine cataract or hernia surgery. These subjects were normotensive (ⱕ140/90 mm Hg) and in sinus rhythm. Exclusion criteria for all subjects included women of child-bearing potential in whom pregnancy could not be ruled out, malignancy, inflammatory disease, secondary hypertension, coexisting medical conditions such as peripheral vascular disease, previous strokes, or myocardial infarctions, previous malignant hypertension, or congestive cardiac failure. Informed consent was obtained from all study participants. The study was approved by the local ethics committee of the hospital. Laboratory Procedures Blood was drawn from an antecubital vein with atraumatic venepuncture. Citrated plasma was collected after 30 min centrifugation at 1000 g and stored at ⫺70°C for batch testing for soluble P-selectin (ie, sPsel), VEGF (sVEGF), and Ang-1 (sAng-1) by enzyme-linked immunoassay (R&D Systems, Abingdon, England). Other aliquots of citrated plasma were centrifuged at 200 g for 10 min to produce a platelet-rich plasma. From this, 108 platelets were pelleted by centrifugation at 2500 rpm (1000 g) for 20 min, washed in phosphate-buffered saline, and lysed by a small volume (250 L) of 0.1% Triton X-100 (Sigma, Poole, England). The total amount of P-selectin, VEGF and Ang-1 in an aliquot of this lysate (ie, pPsel, pVEGF, and pAng-1) was determined by the same enzyme-linked immunoassays; after adjustment these are reported as picograms per number of platelets.10 Platelet parameters total platelet count, mean platelet volume, and mean platelet mass were measured on Advia Haematology 120 autoanalyser (Bayer, Newbury, Berks, England).
Materials and Methods Study Patients
Data Analysis and Power Calculations
Patients, recruited from the hypertension clinic of City Hospital, Birmingham, were 45 years of age or more, with essential hypertension, but had additional risk factors justifying starting on aspirin 75 mg daily, according to recommendations in the British Hypertension Society guidelines.20 Patients who were already on antiplatelet agents (eg, aspirin or clopidogrel) and nonsteroidal antiinflammatory drugs were excluded. After baseline blood samples were taken, all subjects (who were aspirin naive) were started on aspirin 75 mg daily; during this time, no patients had any other new medication prescribed to them, in terms of either antihypertensive or lipid-lowering therapy. Hypertensive patients who had stable and controlled blood pressure, and for whom the need for additional antihypertensive agents would not arise, were recruited into this study. Indeed, at the end of the follow-up, there were no statistical differences in the mean blood pressure from the time of recruitment. We also recruited 30 healthy normal subjects from hospital staff, relatives of the patients or those attending
We hypothesized an increase of one half of a standard deviation in a normally distributed index in the patients compared with the control subjects. To achieve this at P ⬍ .05 and 1-beta ⫽ 0.8, a total of 32 subjects per group are required. This number of subjects, in an intervention study, provides the power to detect a fall in a research index of half a standard deviation at P ⬍ .05 and 1- ⫽ 0.86. Results are expressed as mean with standard deviation (SD) or as median with interquartile range (IQR) for the normally distributed data and skewed data respectively. Data between patients and control subjects was analyzed by the unpaired t test or Mann-Whitney U test test, as appropriate. Data before and after use of aspirin was analyzed by the paired Student t test or by the Wilcoxon test. Correlations were performed by the Spearman correlation method (where necessary, on logged data). All statistical calculations were performed on a microcomputer using a commercially available statistical package (Minitab release 13, Minitab Inc, State College, PA). A P value ⬍ .05 was considered statistically significant.
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EFFECT OF ASPIRIN IN HYPERTENSIVE PATIENTS
Table 1. Clinical characteristics of hypertensive patients and control subjects
Characteristic Age (y) Sex (male:female) Ethnicity White Asian Afro-Caribbean SBP (mm Hg) DBP (mm Hg) Diabetes mellitus Smoker Serum glucose (mmol/L) Serum creatinine (mmol/L) Serum cholesterol (mmol/L) Antihypertensive medication ␣-Blocker Diuretic ARB Moxinidine ACE-I -Blocker Lipid-lowering agent
Control subjects (n ⴝ 30)
Hypertensive patients (n ⴝ 35)
59 ⫾ 12 23:7
64 ⫾ 7 29:6
21 7 2 128 ⫾ 9 76 ⫾ 9 0 0 5.0 ⫹ 0.7 80 (63–88) 5.2 ⫹ 0.9
26 3 5 140 ⫾ 15 79 ⫾ 9 10 (33%) 1 (3%) 6.2 ⫾ 1.2 102 (94–112) 5.0 ⫾ 1.0 22 21 4 11 9 22 8
P value .055 .53 .2 ⬍.001 .13 ⬍.001 ⬍.001 .3
(67%) (61%) (11%) (32%) (26%) (67%) (23%)
Values are mean ⫾ standard deviation, or median (interquartile range); analysis by Students t test, 2 test, or Mann-Whitney U test as appropriate. ACE-I ⫽ angiotensin-converting enzyme inhibitor; ARB ⫽ angiotensin receptor blocker; DBP ⫽ diastolic blood pressure; SBP ⫽ systolic blood pressure.
Results The demographic and clinical details of the control and hypertensive subjects are shown in Table 1, research indices in Table 2. As expected, the hypertensive patients had significantly higher baseline plasma levels of sPsel and sVEGF,10,16,21 In keeping with our previous work, the hypertensive patients also had increased pPsel, pVEGF, sAng-1, and pAng-1.22–24 Also, as would be expected, there was a strong correlation was between mean platelet volume and mean platelet mass (Spearman, r ⫽ 0.584, P ⬍ .001). There were weak correlations between sPsel and
mean platelet volume (r ⫽ 0.3, P ⫽ .01), and between pPsel and mean platelet mass (r ⫽ 0.295, P ⫽ .017). No other statistically significant correlations were noted. Of the 35 hypertensive patients who were recruited, 30 completed the follow-up phase, inasmuch as five were required to stop taking aspirin because of gastric side effects or were noncompliant; therefore the pre/post paired analyses are confined to the 30 patients with complete data. During this period, there were no new antihypertensive medications or lipid-lowering agents added. At 3 months, there were no significant changes in mean blood
Table 2. Plasma/platelet vascular endothelial growth factor (VEGF), angiopoietin-1 and other research indices in hypertensive patients and control subjects
Characteristic 6
3
Platelet count (⫻10 /mm ) MPM (pg) MPV (fl) sPsel (ng/mL) pPsel (⫻106/mm3) VEGF (pg/mL) pVEGF (pg/cell) Ang-1 (pg/mL) pAng-1 (pg/cell)
Control Subjects (n ⴝ 30)
Hypertensive Patients (n ⴝ 35)
P Value
224 ⫾ 43 1.78 ⫾ 0.19 6.4 ⫾ 0.63 70 (53–90) 115 (78–131) 275 (35–925) 30 (10–287) 10 (10–10) 10 (10–32)
250 ⫾ 62 1.89 ⫾ 0.19 6.7 ⫾ 1.3 100 (60–150) 250 (160–250) 600 (100–5000) 100 (50–2000) 20 (10–50) 50 (15–60)
.051 .01 .25 .02 ⬍.001 .04 .008 ⬍.001 .001
Values are mean ⫾ standard deviation, or median (interquartile range). Analysis by Mann-Whitney U test or Student t test as appropriate. Ang-1 ⫽ angiopoietin-1; MPM ⫽ mean platelet mass; MPV ⫽ mean platelet volume; pAng-1 ⫽ platelet Ang-1; pPsel ⫽ platelet P-selectin; SBP ⫽ systolic blood pressure; sPsel ⫽ soluble P-selectin.
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sPsel (ng/ml) 250
sPsel (ng/ml)
200
150
100
50
0
Pre-treatment
Post-treatment
sPsel- soluble P selectin FIG. 1 Effects of aspirin on individual levels of plasma soluble P-selectin (sPsel, ng/mL).
pressure levels or in serum glucose, cholesterol, or creatinine levels. However, there were significant reductions in plasma VEGF and sPsel (Fig. 1), and in platelet Ang-1 and pPsel (Table 3 and Fig. 2). There were no correlations between the reduction of these factors and changes in any other parameter. There also were no changes in the plasma Ang-1, or platelet VEGF. Similarly, there were no changes in the mean platelet mass or volume with aspirin therapy.
Discussion Our patient population was a group in which we expected to see chronic platelet activation, specifically hypertensive subjects at high cardiovascular risk, with modest (33%) proportion of patients with diabetes.2 Despite being on optimal therapy, with fairly well controlled blood pressure as well as controlled glucose and cholesterol levels, our cross-sectional data show that compared with healthy subjects, hypertensive patients had elevated levels of the growth factor angiopoieitin-1 in their plasma, along with increased P-selectin, VEGF, and angiopoietin-1 in their platelets (ie, pPsel, pVEGF, and pAng-1). This, in addition
to the elevated plasma P-selectin (sPsel) and VEGF, point to differences in platelet physiology that can be interpreted as indicating excess activity. These cross-sectional data confirm previous reports,22–24 in which patients with hypertension only have been shown to undergo these changes on their own. We acknowledge that the presence of diabetic patients, smokers, and patients with renal dysfunction among the hypertensive patient population could have contributed further to the increased platelet activation that was seen in this group; however, our cross-sectional analysis of baseline data for these patients compared with control subjects was not powered to examine the effects all of these confounders, as the primary objective of the study was to assess the effect of introducing aspirin. In the present study, platelets in hypertensive patients also had greater mass but equivalent volume; the latter are at variance with previous work9 but are in agreement with data from other groups.25 The lack of significant difference in the platelet volume could be caused by the smaller sample size in the present analysis.
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Table 3. Patient values after treatment with aspirin for 3 months Characteristic
Pre-treatment (n ⴝ 30)
Post-treatment (n ⴝ 30)
P value
SBP (mm Hg) DBP (mm Hg) Cholesterol (mmol/L) Creatinine (mol/L) Glucose (mmol/L) VEGF (pg/mL) pVEGF (⫻10⫺6 pg/cell) Ang-1 (ng/mL) pAng-1 (⫻10⫺6 ng/cell) MPM (pg) MPV (fl) sPsel (ng/mL) pPsel (⫻10⫺6 pg/cell)
141 ⫾ 16 80 ⫾ 9 4.3 ⫾ 0.6 107 ⫾ 14 7.2 ⫾ 2.1 800 (100–6,250) 100 (50–625) 20 (10–50) 50 (15–52) 1.8 ⫾ 0.1 6.6 ⫾ 1.3 85 (60–142) 250 (157–250)
137 ⫾ 11 78 ⫾ 8 5.1 ⫾ 1.4 102 ⫾ 12 6.8 ⫾ 1.5 200 (100–10,000) 100 (100–125) 10 (10–30) 25 (19–30) 1.84 ⫾ 0.19 6.47 ⫾ 0.99 50 (40–92) 150 (87–250)
.14 .258 .29 .06 .634 .01 .98 .06 .04 .338 .655 .001 ⬍.001
Values are mean ⫾ standard deviation, or median (interquartile range). Analysis is by Student t test or Wilcoxon test as appropriate. Abbreviations as in Tables 1 and 2.
pPsel (ng/platelet x 10-8) 300
pPsel (ng/platelet x10-8)
250
200
150
100
50
0
Pre-treatment
pPsel- Platelet P selectin FIG. 2 Effects of aspirin on individual levels of platelet P-selectin (pPsel; ng/platelet ⫻ 10⫺8).
Post-treatement
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The P-selectin data are to some extent understandable, given the existing knowledge, and support the general concept of platelet activation in patients at risk for cardiovascular disease.21 However, the angiopoietin and VEGF data break new ground. Our pilot data16 pertained simply to elevated VEGF levels in hypertension, but we now show increased levels of another growth factor as well as increased levels of both in platelets. Growth factors in platelets are established11,13,26 and may have pathophysiologic consequences in angiogenic aspects of metastatic cancer.27 However, although there are data regarding abnormal angiogenesis in hypertension,18,19 this is not as well established a concept as in oncology. There is, however, growing evidence of this in overt cardiovascular disease.28 –30 Although VEGF correlates with the Framingham risk score in subjects with hypertension and other disease,31 it is still not possible to determine whether this general concept of excess angiogenic potential is an epiphenomenon or a direct participant in the disease process. One possible speculative link may be that excess platelet activity, possibly driven by processes related to cardiovascular disease and thus thrombosis, may result in release of the growth factors at the site of thrombosis or elsewhere, which may in turn promote angiogenesis. Our cross-sectional data are supplemented by interventional data on the effects of aspirin. Broadly speaking, these are in line with expectations, with a reduction in plasma VEGF and P-selectin as well as in platelet Ang-1 and P-selectin levels. However, there was no fall in pVEGF, although the fall in plasma Ang-1 only just missed significance. A different response of the growth factors are interesting, but as nothing is known of their distribution within or metabolism by the platelet, further discussion would be speculative. Again, there may be a place for nonplatelet sources of the growth factors.32,33 Nevertheless our data support a mechanism by which aspirin is of benefit in certain cancers, specifically through reduction of angiogenic growth factors.15,27 Part of the change in platelet values with the use of aspirin could arguably be related to microscopic or macroscopic blood loss from the gastrointestinal tract or elsewhere as a result of the use of aspirin. However, in our study, there is no reason to suspect gastrointestinal blood loss, with no change in the platelet count or hemoglobin or serum urea levels (data not shown). Similarly, there was no change in either the mass or volume of platelets to suggest an increase in “new” platelets. Of note, COX and prostaglandins have recently been demonstrated to have an important role in angiogenesis,34 and both isoforms of COX (1 and 2) have been implicated.35,36 In malignancies, COX-2 inhibitors have been shown to reduce the levels of angiogenesis, both in vitro and in vivo.37–39 Aspirin, being an inhibitor of both the forms of COX, would probably cause a reduction in both the intraplatelet and plasma levels of VEGF and Ang-1 through this mechanism. Platelets, being a rich source of COX-1, would therefore be expected to play an
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important role in the modulation of angiogenesis.40 In our study, we were not able to demonstrate any correlation between the changes in platelet activation with aspirin and the changes in angiogenic factors. This may reflect the multiple other pathways that are responsible for angiogenesis besides the COX pathways. Of note, the use of aspirin in reducing markers of angiogenesis has been demonstrated in other conditions such as myocardial infarction,41 cancer15 and also in vitro.42,43 As far as we know, this is the first study on the effects of aspirin on angiogenesis in hypertensive patients. Similarly, we believe that this is the first published report on the effects of aspirin on Ang-1. A reduction in both plasma and platelet P-selectin (ie, sPsel and pPsel respectively) is consistent with platelet biology21 and adds to the general concept of the effects of aspirin on the platelet.3,44 Some studies have failed to demonstrate marked changes in plasma sPel levels by introducing aspirin.45– 47 However, these studies were short term and were conducted in normal subjects; furthermore, platelet activation was induced by artificial means (such as smoking or the injection of endotoxin) in healthy young volunteers and often as a single dose. This methodology differs from that used in our patient group, in whom platelet activation may be more chronic, and a result of multiple factors; aspirin was also given over a longer period of time. Given the increasing data on the pathophysiologic importance of P-selectin, it is therefore of significance that aspirin, by virtue of its antiplatelet effects, leads to a decrease both in the intraplatelet and soluble forms of P-selectin. Thus we speculate that part of the benefit achieved by aspirin therapy in patients at high cardiovascular risk3 could be caused by the reduction in the P-selectin levels within the platelets. The exact nature of the events leading to the reduction in both soluble and intraplatelet P-selectin by aspirin are not certain. Part of this effect could be a direct result of the prevention of platelet activation by aspirin via the cyclooxygenase pathway. There is no evidence to suggest that aspirin achieves this by a reduction, for example, in RNA levels. Nonetheless, platelet activation is the end result of a chain of events that lead to the platelets changing their shape and size and increasing their “stickiness,” causing them to exhibit more adhesion and aggregation; P-selectin levels are a reflection, as well as an end result, of this activation. Aspirin has been known to affect megakaryocytes48 and the endothelial cell reduction of P-selectin,43 and could also account in part for the reduction in Pselectin. Aspirin has also previously been shown to decrease plasma levels of VEGF in patients with ischemic heart disease who were undergoing coronary artery bypass grafting.49 More experimental data would be needed to ascertain the precise mechanisms but would also be limited by extrapolating any ex vivo behavior of platelets to the (clinical) in vivo situation. In summary, we studied patients with fairly well controlled hypertension who were at high risk for cardiovascular disease, and we found increased platelet activation markers
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but also high levels of angiogenic growth factors. The high cardiovascular risk justified the use of aspirin, which led to a significant decrease in the levels of plasma VEGF and pAng-1. This may have implications for the use of aspirin in conditions in which increased angiogenesis is implicated, such as vascular disease, diabetic retinopathy, and malignancy.
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19. Sullivan JM, Prewitt RL, Josephs JA: Attenuation of the microcirculation in young patients with high-output borderline hypertension. Hypertension 1983;5:844 – 851. 20. Ramsay L, Williams B, Johnston G, MacGregor G, Poston L, Potter J, et al: Guidelines for management of hypertension: report of the third working party of the British Hypertension Society. J Hum Hypertens 1999;13:569 –592. 21. Blann AD, Nadar SK, Lip GY: The adhesion molecule P-selectin and cardiovascular disease. Eur Heart J 2003;24:2166 –2179. 22. Nadar S, Blann AD, Lip GY: Platelet morphology and plasma indices of platelet activation in essential hypertension: effects of amlodipine-based antihypertensive therapy. Ann Med 2004;36:552– 557. 23. Nadar SK, Blann AD, Lip GY: Plasma and platelet-derived vascular endothelial growth factor and angiopoietin-1 in hypertension: effects of antihypertensive therapy. J Intern Med 2004;256:331–337. 24. Nadar SK, Blann AD, Kamath S, Beevers DG, Lip GY: Platelet indexes in relation to target organ damage in high-risk hypertensive patients: a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT). J Am Coll Cardiol 2004;44:415– 422. 25. Bath PM, Carney C, Markandu ND, MacGregor GA: Platelet volume is not increased in essential hypertension. J Hum Hypertens 1994;8:457– 459. 26. Maloney JP, Silliman CC, Ambruso DR, Wang J, Tuder RM, Voelkel NF: In vitro release of vascular endothelial growth factor during platelet aggregation. Am J Physiol 1998;275:H1054 –H1061. 27. Salgado R, Benoy I, van Marck E, Vermeulen P, Dirix L: The importance of the VEGF-load in platelets in cancer patients. Ann Oncol 2002;13:1689 –1690. 28. Chin BS, Chung NA, Gibbs CR, Blann AD, Lip GY: Vascular endothelial growth factor and soluble P-selectin in acute and chronic congestive heart failure. Am J Cardiol 2002;90:1258 –1260. 29. Chung NA, Lydakis C, Belgore F, Blann AD, Lip GY: Angiogenesis in myocardial infarction. An acute or chronic process? Eur Heart J 2002;23:1604 –1608. 30. Makin AJ, Chung NA, Silverman SH, Lip GY: Vascular endothelial growth factor and tissue factor in patients with established peripheral artery disease: a link between angiogenesis and thrombogenesis? Clin Sci (Lond) 2003;104:397– 404. 31. Felmeden DC, Spencer CG, Chung NA, Belgore FM, Blann AD, Beevers DG, et al: Relation of thrombogenesis in systemic hypertension to angiogenesis and endothelial damage/dysfunction (a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial [ASCOT]). Am J Cardiol 2003;92:400 – 405. 32. Dvorak HF, Brown LF, Detmar M, Dvorak AM: Vascular permeability factor/vascular endothelial growth factor, microvascular hyper-permeability, and angiogenesis. Am J Pathol 1995;146:1029 – 1039. 33. Ferrara N, Winer J, Burton T: Aortic smooth muscle cells express and secrete vascular endothelial growth factor. Growth Factors 1991;5:141–148. 34. Leahy KM, Koki AT, Masferrer JL: Role of cyclooxygenases in angiogenesis. Curr Med Chem 2000;7:1163–1170. 35. Sales KJ, Katz AA, Howard B, Soeters RP, Millar RP, Jabbour HN: Cyclooxygenase-1 is up-regulated in cervical carcinomas: autocrine/paracrine regulation of cyclooxygenase-2, prostaglandin e receptors, and angiogenic factors by cyclooxygenase-1. Cancer Res 2002;62:424 – 432. 36. Tamura M, Sebastian S, Gurates B, Yang S, Fang Z, Bulun SE: Vascular endothelial growth factor up-regulates cyclooxygenase-2 expression in human endothelial cells. J Clin Endocrinol Metab 2002;87:3504 –3507. 37. Sawaoka H, Tsuji S, Tsujii M, Gunawan ES, Sasaki Y, Kawano S, et al: Cyclooxygenase inhibitors suppress angiogenesis and reduce tumor growth in vivo. Lab Invest 1999;79:1469 –1477. 38. Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN: Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998;93:705–716.
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39. Dermond O, Ruegg C: Inhibition of tumor angiogenesis by non-steroidal anti-inflammatory drugs: emerging mechanisms and therapeutic perspectives. Drug Resist Update 2001;4: 314 –321. 40. Rhee JS, Black M, Schubert U, Fischer S, Morgenstern E, Hammes HP, et al: The functional role of blood platelet components in angiogenesis. Thromb Haemost 2004;92:394 – 402. 41. Gerrah R, Fogel M, Gilon D: Aspirin decreases vascular endothelial growth factor release during myocardial ischaemia. Int J Cardiol 2004; 94:25–29. 42. Shtivelband MI, Juneja HS, Lee S, Wu KK: Aspirin and salicylate inhibit colon cancer me. J Thromb Haemost 2003;1:2225–2233. 43. Pearce HR, Kalia N, Bardhan KD, Brown NJ: Effects of aspirin and indomethacin on endothelial cell proliferation in vitro. J Gastroenterol Hepatol 2003;18:1180 –1187. 44. Blann AD, Nadar S, Lip GY: Pharmacological modulation of platelet function in hypertension. Hypertension 2003;42:1–7.
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45. Pernerstorfer T, Eichler HG, Stohlawetz P, Speiser W, Jilma B: Effects of heparin and aspirin on circulating P-selectin, E-selectin and von Willebrand factor levels in healthy men. Atherosclerosis 2001;155:389 –393. 46. Jilma B, Blann A, Pernerstorfer T, Stohlawetz P, Eichler HG, Vondrovec B, et al: Regulation of adhesion molecules during human endotoxemia. No acute effects of aspirin. Am J Respir Crit Care Med 1999;159:857– 863. 47. Pernerstorfer T, Stohlawetz P, Stummvoll G, Kapiotis S, Szekeres T, Eichler HG, et al: Low-dose aspirin does not lower in vivo platelet activation in healthy smokers. Br J Haematol 1998;102:1229 –1231. 48. van Pampus EC, Huijgens PC, Zevenbergen A, Twaalfhoven H, van Kamp GJ, Langenhuijsen MM: Influence of aspirin on human megakaryocyte prostaglandin synthesis. Eur J Haematol 1993;50:264–268. 49. Gerrah R, Fogel M, Gilon D: Aspirin decreases vascular endothelial growth factor release during myocardial ischemia. Int J Cardiol 2004; 94:25–29.
2006; 19:970 –977
Mechanisms
Effects of Aspirin on Intra-Platelet Vascular Endothelial Growth Factor, Angiopoietin-1, and P-Selectin Levels in Hypertensive Patients Sunil Nadar, Andrew D. Blann, and Gregory Y.H. Lip Background: Although aspirin is useful in reducing platelet activation and cardiovascular events, its effects on platelet levels of angiogenic factors, such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), and markers of platelet activation in hypertension are unknown. The aim of this study was to study the effects of aspirin on the platelet morphology, plasma and platelet levels of VEGF (sVEGF and pVEGF respectively), Ang-1 (sAng-1 and pAng-1 respectively), and P-selectin (sPsel and pPsel respectively) in patients with well controlled hypertension.
well as pVEGF (P ⫽ .008), pAng-1(P ⫽ .001), sPsel (P ⫽ .02), pPsel (P ⬍ .001), and mean platelet mass (P ⫽ .01) when compared with control subjects. After treatment with aspirin for 3 months, there were significant reductions in plasma VEGF (P ⫽ .01), pAng-1 (P ⫽ .04), sPsel (P ⫽ .001), and pPsel (P ⬍ .001) levels, but not levels of platelet VEGF and plasma Ang-1. Neither pVEGF nor pAng-1 correlated with blood pressure or with their respective plasma levels.
Methods: A total of 35 aspirin-naive, hypertensive patients (29 male and six female; mean age 64 years) were compared with 30 (23 male, seven female, mean age 59 years) normotensive control subjects. Blood was collected for plasma VEGF, P-selectin, and Ang-1 (enzyme-linked immunoassay), intra-platelet levels of VEGF, Ang-1, and P-selectin, and platelet volume and mass. Research indices in hypertensive patients were studied before and after 3 months treatment with aspirin 75 mg daily.
Conclusions: The use of aspirin in high-risk hypertensive patients leads to a reduction in intra-platelet angiogenic growth factors and platelet activation. This may have implications for the use of aspirin in conditions (such as vascular disease) that have been associated with an increase in angiogenesis and platelet activation. Am J Hypertens 2006;19:970 –977 © 2006 American Journal of Hypertension, Ltd.
Results: Hypertensive patients had significantly higher plasma levels of VEGF (P ⫽ .04), Ang-1 (P ⬍ .001), as
Key Words: Hypertension, aspirin, vascular endothelial growth factor, angiopoeitin-1, P-selectin, platelets.
ypertension is known to be associated with an increased risk of thrombotic complications (strokes, myocardial infarction), which are likely to result from the prothrombotic or hypercoagulable state associated with this condition.1,2 Aspirin is the most widely used antiplatelet agent for reducing the risk of cardiovascular and cerebrovascular events in both primary prevention and secondary prevention.3 Its mode of action is in inhibiting cyclooxygenases (COX), enzymes that catalyse the conversion of arachidonic acid to eichosanoids and thus may play an important role in platelet– vessel wall interactions. Indeed, thromboxane is the major COX product in platelets, and its biosynthesis is increased
H
in conditions associated with platelet activation such as unstable angina and peripheral vascular disease. Therefore, its inhibition leads to a reduction in platelet activation.4 – 6 Platelet activation can be assessed by a number of methods, such as the expression of P-selectin at the platelet cell surface, or by increased plasma levels. Platelet size, as well as membrane and plasma P-selectin are also increased in hypertension and acute stroke,7–9 and we have also shown that the total mass of P-selectin within each platelet (as determined by measuring levels of P-selectin in the lysate formed by solubilizing a fixed number of these cells) are increased
Received November 21, 2005. First decision January 29, 2006. Accepted March 7, 2006. From the Haemostasis Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham, England. This work was supported by the Peel Medical Research Trust and the Sandwell and West Birmingham Hospitals NHS Trust Research and
Development programme for the Haemostasis Thrombosis and Vascular Biology Unit. Address correspondence and reprint requests to Prof. Gregory Y.H. Lip, Haemostasis Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham B18 7QH, England; e-mail: [email protected]
0895-7061/06/$32.00 doi:10.1016/j.amjhyper.2006.03.001
© 2006 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.
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in atrial fibrillation, a condition often associated with hypertension.10 Of further interest is the fact that platelets are carriers of angiogenic growth factors, such as vascular endothelial growth factor (VEGF)11,12 and angiopoietin-1 (Ang-1).13 Indeed, platelet-derived VEGF and Ang-1 have been shown previously to be important in the pathogenesis of complications and metastasis in cancers, and aspirin has also been demonstrated to inhibit the release of these factors from platelets in patients with malignancies.14,15 We have previously reported raised levels of plasma VEGF in hypertension, although neither the clinical nor pathophysiolgic implications of this finding are clear.16 However, abnormal angiogenesis has been demonstrated in hypertension, and there seems to be an impaired ability for vascular growth resulting from structural alteration of the microvascular network, which includes capillary rarefaction, increased arteriolar length and tortuosity.17–19 Hence, we do know the following: 1) there is ample evidence of the involvement of platelets in the pathogenesis of hypertension, especially its vascular consequences (such as myocardial infarction and stroke); 2) aspirin is an effective antiplatelet agent; and 3) platelets may contribute to the complications of hypertension by virtue of their carriage of angiogenic (and other) growth factors. We therefore hypothesized that aspirin would cause a reduction in markers of platelet activation (P-selectin and changes in morphology) and alter the levels of both plasma and platelet angiogenic growth factors (VEGF and Ang-1). We tested our hypothesis in a simple case-control study of hypertensive patients and healthy control subjects, and a longitudinal study of the effects of introducing aspirin 75 mg/day in aspirin-naive hypertensive patients.
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the hospital for routine cataract or hernia surgery. These subjects were normotensive (ⱕ140/90 mm Hg) and in sinus rhythm. Exclusion criteria for all subjects included women of child-bearing potential in whom pregnancy could not be ruled out, malignancy, inflammatory disease, secondary hypertension, coexisting medical conditions such as peripheral vascular disease, previous strokes, or myocardial infarctions, previous malignant hypertension, or congestive cardiac failure. Informed consent was obtained from all study participants. The study was approved by the local ethics committee of the hospital. Laboratory Procedures Blood was drawn from an antecubital vein with atraumatic venepuncture. Citrated plasma was collected after 30 min centrifugation at 1000 g and stored at ⫺70°C for batch testing for soluble P-selectin (ie, sPsel), VEGF (sVEGF), and Ang-1 (sAng-1) by enzyme-linked immunoassay (R&D Systems, Abingdon, England). Other aliquots of citrated plasma were centrifuged at 200 g for 10 min to produce a platelet-rich plasma. From this, 108 platelets were pelleted by centrifugation at 2500 rpm (1000 g) for 20 min, washed in phosphate-buffered saline, and lysed by a small volume (250 L) of 0.1% Triton X-100 (Sigma, Poole, England). The total amount of P-selectin, VEGF and Ang-1 in an aliquot of this lysate (ie, pPsel, pVEGF, and pAng-1) was determined by the same enzyme-linked immunoassays; after adjustment these are reported as picograms per number of platelets.10 Platelet parameters total platelet count, mean platelet volume, and mean platelet mass were measured on Advia Haematology 120 autoanalyser (Bayer, Newbury, Berks, England).
Materials and Methods Study Patients
Data Analysis and Power Calculations
Patients, recruited from the hypertension clinic of City Hospital, Birmingham, were 45 years of age or more, with essential hypertension, but had additional risk factors justifying starting on aspirin 75 mg daily, according to recommendations in the British Hypertension Society guidelines.20 Patients who were already on antiplatelet agents (eg, aspirin or clopidogrel) and nonsteroidal antiinflammatory drugs were excluded. After baseline blood samples were taken, all subjects (who were aspirin naive) were started on aspirin 75 mg daily; during this time, no patients had any other new medication prescribed to them, in terms of either antihypertensive or lipid-lowering therapy. Hypertensive patients who had stable and controlled blood pressure, and for whom the need for additional antihypertensive agents would not arise, were recruited into this study. Indeed, at the end of the follow-up, there were no statistical differences in the mean blood pressure from the time of recruitment. We also recruited 30 healthy normal subjects from hospital staff, relatives of the patients or those attending
We hypothesized an increase of one half of a standard deviation in a normally distributed index in the patients compared with the control subjects. To achieve this at P ⬍ .05 and 1-beta ⫽ 0.8, a total of 32 subjects per group are required. This number of subjects, in an intervention study, provides the power to detect a fall in a research index of half a standard deviation at P ⬍ .05 and 1- ⫽ 0.86. Results are expressed as mean with standard deviation (SD) or as median with interquartile range (IQR) for the normally distributed data and skewed data respectively. Data between patients and control subjects was analyzed by the unpaired t test or Mann-Whitney U test test, as appropriate. Data before and after use of aspirin was analyzed by the paired Student t test or by the Wilcoxon test. Correlations were performed by the Spearman correlation method (where necessary, on logged data). All statistical calculations were performed on a microcomputer using a commercially available statistical package (Minitab release 13, Minitab Inc, State College, PA). A P value ⬍ .05 was considered statistically significant.
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EFFECT OF ASPIRIN IN HYPERTENSIVE PATIENTS
Table 1. Clinical characteristics of hypertensive patients and control subjects
Characteristic Age (y) Sex (male:female) Ethnicity White Asian Afro-Caribbean SBP (mm Hg) DBP (mm Hg) Diabetes mellitus Smoker Serum glucose (mmol/L) Serum creatinine (mmol/L) Serum cholesterol (mmol/L) Antihypertensive medication ␣-Blocker Diuretic ARB Moxinidine ACE-I -Blocker Lipid-lowering agent
Control subjects (n ⴝ 30)
Hypertensive patients (n ⴝ 35)
59 ⫾ 12 23:7
64 ⫾ 7 29:6
21 7 2 128 ⫾ 9 76 ⫾ 9 0 0 5.0 ⫹ 0.7 80 (63–88) 5.2 ⫹ 0.9
26 3 5 140 ⫾ 15 79 ⫾ 9 10 (33%) 1 (3%) 6.2 ⫾ 1.2 102 (94–112) 5.0 ⫾ 1.0 22 21 4 11 9 22 8
P value .055 .53 .2 ⬍.001 .13 ⬍.001 ⬍.001 .3
(67%) (61%) (11%) (32%) (26%) (67%) (23%)
Values are mean ⫾ standard deviation, or median (interquartile range); analysis by Students t test, 2 test, or Mann-Whitney U test as appropriate. ACE-I ⫽ angiotensin-converting enzyme inhibitor; ARB ⫽ angiotensin receptor blocker; DBP ⫽ diastolic blood pressure; SBP ⫽ systolic blood pressure.
Results The demographic and clinical details of the control and hypertensive subjects are shown in Table 1, research indices in Table 2. As expected, the hypertensive patients had significantly higher baseline plasma levels of sPsel and sVEGF,10,16,21 In keeping with our previous work, the hypertensive patients also had increased pPsel, pVEGF, sAng-1, and pAng-1.22–24 Also, as would be expected, there was a strong correlation was between mean platelet volume and mean platelet mass (Spearman, r ⫽ 0.584, P ⬍ .001). There were weak correlations between sPsel and
mean platelet volume (r ⫽ 0.3, P ⫽ .01), and between pPsel and mean platelet mass (r ⫽ 0.295, P ⫽ .017). No other statistically significant correlations were noted. Of the 35 hypertensive patients who were recruited, 30 completed the follow-up phase, inasmuch as five were required to stop taking aspirin because of gastric side effects or were noncompliant; therefore the pre/post paired analyses are confined to the 30 patients with complete data. During this period, there were no new antihypertensive medications or lipid-lowering agents added. At 3 months, there were no significant changes in mean blood
Table 2. Plasma/platelet vascular endothelial growth factor (VEGF), angiopoietin-1 and other research indices in hypertensive patients and control subjects
Characteristic 6
3
Platelet count (⫻10 /mm ) MPM (pg) MPV (fl) sPsel (ng/mL) pPsel (⫻106/mm3) VEGF (pg/mL) pVEGF (pg/cell) Ang-1 (pg/mL) pAng-1 (pg/cell)
Control Subjects (n ⴝ 30)
Hypertensive Patients (n ⴝ 35)
P Value
224 ⫾ 43 1.78 ⫾ 0.19 6.4 ⫾ 0.63 70 (53–90) 115 (78–131) 275 (35–925) 30 (10–287) 10 (10–10) 10 (10–32)
250 ⫾ 62 1.89 ⫾ 0.19 6.7 ⫾ 1.3 100 (60–150) 250 (160–250) 600 (100–5000) 100 (50–2000) 20 (10–50) 50 (15–60)
.051 .01 .25 .02 ⬍.001 .04 .008 ⬍.001 .001
Values are mean ⫾ standard deviation, or median (interquartile range). Analysis by Mann-Whitney U test or Student t test as appropriate. Ang-1 ⫽ angiopoietin-1; MPM ⫽ mean platelet mass; MPV ⫽ mean platelet volume; pAng-1 ⫽ platelet Ang-1; pPsel ⫽ platelet P-selectin; SBP ⫽ systolic blood pressure; sPsel ⫽ soluble P-selectin.
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sPsel (ng/ml) 250
sPsel (ng/ml)
200
150
100
50
0
Pre-treatment
Post-treatment
sPsel- soluble P selectin FIG. 1 Effects of aspirin on individual levels of plasma soluble P-selectin (sPsel, ng/mL).
pressure levels or in serum glucose, cholesterol, or creatinine levels. However, there were significant reductions in plasma VEGF and sPsel (Fig. 1), and in platelet Ang-1 and pPsel (Table 3 and Fig. 2). There were no correlations between the reduction of these factors and changes in any other parameter. There also were no changes in the plasma Ang-1, or platelet VEGF. Similarly, there were no changes in the mean platelet mass or volume with aspirin therapy.
Discussion Our patient population was a group in which we expected to see chronic platelet activation, specifically hypertensive subjects at high cardiovascular risk, with modest (33%) proportion of patients with diabetes.2 Despite being on optimal therapy, with fairly well controlled blood pressure as well as controlled glucose and cholesterol levels, our cross-sectional data show that compared with healthy subjects, hypertensive patients had elevated levels of the growth factor angiopoieitin-1 in their plasma, along with increased P-selectin, VEGF, and angiopoietin-1 in their platelets (ie, pPsel, pVEGF, and pAng-1). This, in addition
to the elevated plasma P-selectin (sPsel) and VEGF, point to differences in platelet physiology that can be interpreted as indicating excess activity. These cross-sectional data confirm previous reports,22–24 in which patients with hypertension only have been shown to undergo these changes on their own. We acknowledge that the presence of diabetic patients, smokers, and patients with renal dysfunction among the hypertensive patient population could have contributed further to the increased platelet activation that was seen in this group; however, our cross-sectional analysis of baseline data for these patients compared with control subjects was not powered to examine the effects all of these confounders, as the primary objective of the study was to assess the effect of introducing aspirin. In the present study, platelets in hypertensive patients also had greater mass but equivalent volume; the latter are at variance with previous work9 but are in agreement with data from other groups.25 The lack of significant difference in the platelet volume could be caused by the smaller sample size in the present analysis.
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EFFECT OF ASPIRIN IN HYPERTENSIVE PATIENTS
Table 3. Patient values after treatment with aspirin for 3 months Characteristic
Pre-treatment (n ⴝ 30)
Post-treatment (n ⴝ 30)
P value
SBP (mm Hg) DBP (mm Hg) Cholesterol (mmol/L) Creatinine (mol/L) Glucose (mmol/L) VEGF (pg/mL) pVEGF (⫻10⫺6 pg/cell) Ang-1 (ng/mL) pAng-1 (⫻10⫺6 ng/cell) MPM (pg) MPV (fl) sPsel (ng/mL) pPsel (⫻10⫺6 pg/cell)
141 ⫾ 16 80 ⫾ 9 4.3 ⫾ 0.6 107 ⫾ 14 7.2 ⫾ 2.1 800 (100–6,250) 100 (50–625) 20 (10–50) 50 (15–52) 1.8 ⫾ 0.1 6.6 ⫾ 1.3 85 (60–142) 250 (157–250)
137 ⫾ 11 78 ⫾ 8 5.1 ⫾ 1.4 102 ⫾ 12 6.8 ⫾ 1.5 200 (100–10,000) 100 (100–125) 10 (10–30) 25 (19–30) 1.84 ⫾ 0.19 6.47 ⫾ 0.99 50 (40–92) 150 (87–250)
.14 .258 .29 .06 .634 .01 .98 .06 .04 .338 .655 .001 ⬍.001
Values are mean ⫾ standard deviation, or median (interquartile range). Analysis is by Student t test or Wilcoxon test as appropriate. Abbreviations as in Tables 1 and 2.
pPsel (ng/platelet x 10-8) 300
pPsel (ng/platelet x10-8)
250
200
150
100
50
0
Pre-treatment
pPsel- Platelet P selectin FIG. 2 Effects of aspirin on individual levels of platelet P-selectin (pPsel; ng/platelet ⫻ 10⫺8).
Post-treatement
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The P-selectin data are to some extent understandable, given the existing knowledge, and support the general concept of platelet activation in patients at risk for cardiovascular disease.21 However, the angiopoietin and VEGF data break new ground. Our pilot data16 pertained simply to elevated VEGF levels in hypertension, but we now show increased levels of another growth factor as well as increased levels of both in platelets. Growth factors in platelets are established11,13,26 and may have pathophysiologic consequences in angiogenic aspects of metastatic cancer.27 However, although there are data regarding abnormal angiogenesis in hypertension,18,19 this is not as well established a concept as in oncology. There is, however, growing evidence of this in overt cardiovascular disease.28 –30 Although VEGF correlates with the Framingham risk score in subjects with hypertension and other disease,31 it is still not possible to determine whether this general concept of excess angiogenic potential is an epiphenomenon or a direct participant in the disease process. One possible speculative link may be that excess platelet activity, possibly driven by processes related to cardiovascular disease and thus thrombosis, may result in release of the growth factors at the site of thrombosis or elsewhere, which may in turn promote angiogenesis. Our cross-sectional data are supplemented by interventional data on the effects of aspirin. Broadly speaking, these are in line with expectations, with a reduction in plasma VEGF and P-selectin as well as in platelet Ang-1 and P-selectin levels. However, there was no fall in pVEGF, although the fall in plasma Ang-1 only just missed significance. A different response of the growth factors are interesting, but as nothing is known of their distribution within or metabolism by the platelet, further discussion would be speculative. Again, there may be a place for nonplatelet sources of the growth factors.32,33 Nevertheless our data support a mechanism by which aspirin is of benefit in certain cancers, specifically through reduction of angiogenic growth factors.15,27 Part of the change in platelet values with the use of aspirin could arguably be related to microscopic or macroscopic blood loss from the gastrointestinal tract or elsewhere as a result of the use of aspirin. However, in our study, there is no reason to suspect gastrointestinal blood loss, with no change in the platelet count or hemoglobin or serum urea levels (data not shown). Similarly, there was no change in either the mass or volume of platelets to suggest an increase in “new” platelets. Of note, COX and prostaglandins have recently been demonstrated to have an important role in angiogenesis,34 and both isoforms of COX (1 and 2) have been implicated.35,36 In malignancies, COX-2 inhibitors have been shown to reduce the levels of angiogenesis, both in vitro and in vivo.37–39 Aspirin, being an inhibitor of both the forms of COX, would probably cause a reduction in both the intraplatelet and plasma levels of VEGF and Ang-1 through this mechanism. Platelets, being a rich source of COX-1, would therefore be expected to play an
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important role in the modulation of angiogenesis.40 In our study, we were not able to demonstrate any correlation between the changes in platelet activation with aspirin and the changes in angiogenic factors. This may reflect the multiple other pathways that are responsible for angiogenesis besides the COX pathways. Of note, the use of aspirin in reducing markers of angiogenesis has been demonstrated in other conditions such as myocardial infarction,41 cancer15 and also in vitro.42,43 As far as we know, this is the first study on the effects of aspirin on angiogenesis in hypertensive patients. Similarly, we believe that this is the first published report on the effects of aspirin on Ang-1. A reduction in both plasma and platelet P-selectin (ie, sPsel and pPsel respectively) is consistent with platelet biology21 and adds to the general concept of the effects of aspirin on the platelet.3,44 Some studies have failed to demonstrate marked changes in plasma sPel levels by introducing aspirin.45– 47 However, these studies were short term and were conducted in normal subjects; furthermore, platelet activation was induced by artificial means (such as smoking or the injection of endotoxin) in healthy young volunteers and often as a single dose. This methodology differs from that used in our patient group, in whom platelet activation may be more chronic, and a result of multiple factors; aspirin was also given over a longer period of time. Given the increasing data on the pathophysiologic importance of P-selectin, it is therefore of significance that aspirin, by virtue of its antiplatelet effects, leads to a decrease both in the intraplatelet and soluble forms of P-selectin. Thus we speculate that part of the benefit achieved by aspirin therapy in patients at high cardiovascular risk3 could be caused by the reduction in the P-selectin levels within the platelets. The exact nature of the events leading to the reduction in both soluble and intraplatelet P-selectin by aspirin are not certain. Part of this effect could be a direct result of the prevention of platelet activation by aspirin via the cyclooxygenase pathway. There is no evidence to suggest that aspirin achieves this by a reduction, for example, in RNA levels. Nonetheless, platelet activation is the end result of a chain of events that lead to the platelets changing their shape and size and increasing their “stickiness,” causing them to exhibit more adhesion and aggregation; P-selectin levels are a reflection, as well as an end result, of this activation. Aspirin has been known to affect megakaryocytes48 and the endothelial cell reduction of P-selectin,43 and could also account in part for the reduction in Pselectin. Aspirin has also previously been shown to decrease plasma levels of VEGF in patients with ischemic heart disease who were undergoing coronary artery bypass grafting.49 More experimental data would be needed to ascertain the precise mechanisms but would also be limited by extrapolating any ex vivo behavior of platelets to the (clinical) in vivo situation. In summary, we studied patients with fairly well controlled hypertension who were at high risk for cardiovascular disease, and we found increased platelet activation markers
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but also high levels of angiogenic growth factors. The high cardiovascular risk justified the use of aspirin, which led to a significant decrease in the levels of plasma VEGF and pAng-1. This may have implications for the use of aspirin in conditions in which increased angiogenesis is implicated, such as vascular disease, diabetic retinopathy, and malignancy.
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