Angiotensin II receptor blockade with valsartan decreases ... - Nature

Journal of Human Hypertension (2011) 25, 334–339 & 2011 Macmillan Publishers Limited All rights reserved 0950-9240/11 www.nature.com/jhh

ORIGINAL ARTICLE

Angiotensin II receptor blockade with valsartan decreases plasma osteopontin levels in patients with essential hypertension M Kurata1,2, T Okura1, J Irita1, D Enomoto1, T Nagao1, M Jotoku1, K Miyoshi1, VR Desilva1 and J Higaki1 1

Department of Integrated Medicine and Informatics, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, Japan and 2Division of Pathogenomics, Department of Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, Japan

Osteopontin (OPN) has recently emerged as a key factor in both vascular remodelling and development of atherosclerosis. It has been reported that OPN is regulated by the renin–angiotensin–aldosterone system (RAAS). The aim of this study was to clarify the effect of angiotensin II receptor blockade with valsartan on plasma OPN levels in patients with essential hypertension (EHT). Forty-six patients (mean age, 64±11 years) with EHT were randomly assigned to treatment with amlodipine or valsartan. There were no significant differences in baseline clinical characteristics between the two groups. Blood sampling and blood pressure evaluation were performed before and after 24 weeks of treatment. After 24 weeks, both systolic blood pressure (SBP) and diastolic blood pressure (DBP) were decreased significantly and by the same degree in each

treatment group. However, valsartan but not amlodipine decreased plasma OPN levels (baseline and 24-week data—valsartan: 614±224 ng ml1, 472±268 ng ml1, P ¼ 0.006; amlodipine: 680±151 ng ml1, 687±234 ng ml1, P40.999). A positive correlation between the reduction in OPN and the log natural (ln) C-reactive protein (CRP) was seen in the valsartan-treated group. Stepwise regression analysis showed that treatment with valsartan and the reduction of ln CRP were associated with the reduction in OPN levels, and this association was independent of the reduction in SBP or aldosterone levels (valsartan: b ¼ 0.332, P ¼ 0.026; ln CRP reduction: b ¼ 0.366, P ¼ 0.015). These results suggest that suppression of the RAAS and inflammation may decrease plasma OPN levels. Journal of Human Hypertension (2011) 25, 334–339; doi:10.1038/jhh.2010.73; published online 22 July 2010

Keywords: osteopontin; essential hypertension; angiotensin II receptor blocker; C-reactive protein; inflammation

Introduction Osteopontin (OPN) has been characterized as a highly acidic, phosphoprotein component of bone matrix that is also present in organs such as kidney, brain, pancreas and vasculature.1 OPN has multiple functions including stimulation of specific cellular events, such as survival, proliferation, and migration in smooth muscle cells, endothelial cells and monocytes/macrophages.2–5 OPN is upregulated in a variety of acute and chronic inflammatory conditions, such as wound healing, fibrosis, autoimmune disease and atherosclerosis.6

Correspondence: Dr T Okura, Department of Integrated Medicine and Informatics, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan. E-mail: [email protected] Received 1 February 2010; revised 12 May 2010; accepted 30 May 2010; published online 22 July 2010

The renin–angiotensin–aldosterone system has a significant role in the advent and perpetuation of inflammation both directly and indirectly. OPN is an important cytokine that is regulated by the rennin–angiotensin–aldosterone system. In an in vitro study, Campos et al.7 reported that OPN mRNA was upregulated by angiotensin II (AII) in rat aortic smooth muscle cells using a DNA microarray method. We have reported that aldosterone also induces OPN mRNA and protein expression in rat renal fibroblasts.8 In an in vivo study, Bruemmer et al.9 showed that abdominal aortic aneurysms induced by AII were attenuated in OPN knockout mice. Clinically, plasma OPN levels have been shown to correlate with future cardiovascular events in patients with chronic stable angina.10 Even in patients without symptomatic cardiovascular disease, plasma OPN levels correlated with carotid atherosclerosis in patients with essential hypertension.11

Angiotensin II blockade reduces plasma osteopontin M Kurata et al 335

Many experimental studies showed that AII receptor blocker reduced inflammation as well as lowered blood pressure.12–15 Clinically, we have reported that valsartan reduced inflammatory cytokines16 and improved carotid arterial stiffness and haemodynamics, as evaluated by carotid ultrasound.16,17 In this study, we determined the effect of two different classes of antihypertensive agents, valsartan and amlodipine, on OPN concentrations in patients with essential hypertension. Furthermore, we attempted to determine the factors that affect changes in OPN levels in patients treated with these antihypertensive agents.

tion. Plasma aldosterone levels were measured by a radioimmunoassay kit (Bio Medical Laboratories, Tokyo, Japan). Plasma OPN levels were measured by an enzyme-linked immunosorbent assay kit (ImmunoBiological Laboratory, Gunma, Japan). All measurements were performed in duplicate. The detection limit and the intra-assay coefficient of variation for the OPN enzyme-linked immunosorbent assay measurements were o5 ng ml1. Blood chemistry, OPN, CRP, aldosterone and blood pressure were determined at baseline and after 24 weeks of treatment with each drug.

Statistical analysis

Methods Study subjects

The ethics committee of the Ehime University Graduate School of Medicine provided approval for the study. Informed consent was obtained from all participating patients. The patients were recruited from the outpatient clinic at Ehime University Hospital. Forty-six patients with essential hypertension without the use of antihypertensive medications were enrolled in this study. Hypertension was defined as a systolic blood pressure (SBP) X140 mm Hg or diastolic blood pressure (DBP) X90 mm Hg. The SBP and DBP were the average of three measurements taken with a brachial sphygmomanometer with the patient in the seated position. Patients with diabetes mellitus, congestive heart failure, previous myocardial infarction, angina pectoris, atrial fibrillation, chronic renal failure (serum creatinine 41.5 mg per 100 ml) or a history of stroke, malignant tumour or autoimmune disease were excluded. Patients were randomized into a group treated with 5 mg kg1 per day amlodipine (n ¼ 23) or a group treated with 80 mg kg1 per day valsartan (n ¼ 23). If blood pressure was not controlled after 4 weeks of treatment (SBP4140 mm Hg and/or DBP490 mm Hg), the doses of amlodipine or valsartan were titrated to 10 or 160 mg, respectively. Patients were followed-up at every outpatient clinic visit.

Blood sampling

Blood samples were drawn between 0800 and 1000 hours from the forearm in the supine position after a 20 min rest. Biochemical measurements were performed within 30 min of blood sampling, and blood samples for OPN were centrifuged and stored at 80 1C until assayed by commercially available enzyme-linked immunosorbent assay kits. The serum levels of creatinine, fasting glucose, total cholesterol, triglycerides, high-density lipoprotein cholesterol and high-sensitivity C-reactive protein (CRP) were measured on a 200FR analyzer (Toshiba, Tokyo, Japan). Low-density lipoprotein cholesterol levels were calculated using the Friedewald equa-

Normally distributed values were expressed as means±standard deviations. If residuals were considered not to be normally distributed, the data were log-transformed. In this study, only CRP was logtransformed. Comparisons between the groups were performed by a Student’s t-test or analysis of variance followed by post hoc tests adjusted with a Bonferroni correction factor. Pearson’s correlation coefficients were calculated to assess the association between continuous variables. Stepwise regression analysis was performed to clarify the independent determinant of change in OPN. A P-value of o0.05 was considered to be statistically significant.

Results The clinical characteristics of the study subjects are summarized in Table 1. None of the participants were taking any medication before this study. Fortysix patients (26 men; mean age, 64±11 years) were included in this study. There were 23 patients in each group, and there were no significant differences in the baseline clinical characteristics between the two groups (Table 1). No patients were withdrawn from the study for any reason, and there were no serious cardiovascular events during the study. Patients treated with amlodipine and valsartan had significant decreases of SBP (153±19 to 138±13 mm Hg, P ¼ 0.028; 156±16 to 143±14 mm Hg, P ¼ 0.040, respectively) (Table 2). There were no significant differences in the SBP or DBP reduction between the two treatment groups (reduction in SBP, P ¼ 0.586; reduction in DBP, P ¼ 0.531). Plasma OPN levels were decreased after 24 weeks in patients treated with valsartan (614±224 to 472±268 ng ml1, P ¼ 0.006); however, there was no significant change in amlodipine-treated patients (680±151 to 687±234 ng ml1, P40.999) (Figure 1). In the valsartan group, the reduction in OPN levels (baseline OPN minus 24 weeks OPN) was correlated with the reduction of ln CRP (baseline ln CRP minus 24 weeks ln CRP). In contrast, no correlation was found in the amlodipine group (Table 3, Figure 2). Stepwise regression analysis showed that treatment with valsartan and Journal of Human Hypertension


Table 1 Patients’ characteristic in amlodipine and valsartan group

Gender (male/female) Age (years) Current smoking (yes, %) BMI (kg m2) SBP (mm Hg) DBP (mm Hg) Creatinine (mmol l1) Blood glucose (mg per 100 ml) HbA1c (%) TC (mmol l1) HDL-C (mmol l1) LDL-C (mmol l1) Triglyceride (mmol l1) OPN (ng ml1) ln CRP Aldosterone (ng per 100 ml)

Amlodipine (n ¼ 23)

Valsartan (n ¼ 23)

P-value

15/8 63±10 4 (17) 24.7±2.2 153±19 92±14 69.9±23.0 100±15 5.29±0.35 5.25±0.72 1.47±0.36 3.27±0.53 2.64±1.03 680±151 2.35±1.19 0.86±0.39

11/12 66±12 6 (26) 24.5±2.3 155±16 86±12 70.8±36.2 110±22 5.43±0.63 5.02±0.75 1.4±0.39 3.00±0.76 3.18±0.91 614±224 2.07±1.26 0.88±0.41

0.157 0.554 0.324 0.713 0.705 0.138 0.969 0.060 0.346 0.267 0.521 0.249 0.063 0.229 0.447 0.655

Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; ln CRP, log natural C-reactive protein; OPN, osteopontin; SBP, systolic blood pressure; TC, total cholesterol. Statistical analysis was performed by Student’s t-test.

Table 2 Comparison of baseline and 24-week blood pressure and neurohumoral data Amlodipine

Valsartan

P-value

SBP (mm Hg) Baseline 6 months

153±19 138±13*

156±16 143±14*

o0.0001

DBP (mm Hg) Baseline 6 months

92±14 84±10

87±12 81±10

0.037

OPN (ng ml1) Baseline 6 months

680±151 687±234

614±224 472±268*

o0.0001

2.35±1.19 2.54±1.03

2.22±1.34 2.69±1.13*

0.030

8.34±4.14 7.01±3.73

0.682

ln CRP Baseline 6 months

Aldosterone (ng per 100 ml) Baseline 8.56±3.85 6 months 8.05±3.72

Abbreviations: DBP, diastolic blood pressure; ln CRP, log natural C-reactive protein; OPN, osteopontin; SBP, systolic blood pressure. P-value, comparison of groups using ANOVA. *Po0.05 vs each baseline using Bonferroni adjustment.

the reduction of ln CRP levels were associated with the reduction of OPN levels (reduction of valsartan: b ¼ 0.332, P ¼ 0.026; reduction of ln CRP: b ¼ 0.366, P ¼ 0.015), and these associations were independent of the reduction of SBP or aldosterone levels (reduction of SBP: b ¼ 0.055, P ¼ 0.700; reduction of aldosterone: b ¼ 0.031, P ¼ 0.824).

Discussion In this study, we showed that 24 weeks of treatment with valsartan but not amlodipine decreased Journal of Human Hypertension

plasma OPN levels. The degree of OPN reduction was dependent on the degree of ln CRP reduction. It is well known that the rennin–angiotensin– aldosterone system is a strong stimulator of OPN synthesis. In an in vitro study, Abe et al.18 reported that OPN expression induced by AII was blocked by valsartan in rat vascular smooth muscle cells. We also reported that aldosterone increased OPN and that the increase was abolished by the mineralocorticoid receptor antagonist, spironolactone, in renal fibroblast. In an in vivo study, Kramer et al.19 reported that the adriamycin nephropathy induced renal OPN expression and treatment with angiotensin-converting enzyme inhibitor and the combination of angiotensin-converting enzyme inhibitor/ spironolactone reduced renal OPN expression. These previous and present studies indicate that reduction of AII and aldosterone by valsartan may inhibit OPN expression. In transcriptional studies, both AII and aldosterone induced OPN expression through transcriptional factor activator protein 1 that is activated by the pro-inflammatory stimuli.8 AII receptor blockers decrease OPN both directly and indirectly, through suppression of inflammation, inhibition of macrophage accumulation and reduction of oxidative stress.20 In this study, the reduction of OPN levels was significantly correlated with the reduction of ln CRP levels only in the valsartan group. In addition, stepwise regression analysis showed that valsartan treatment and the reduction of ln CRP were independent determinants of the reduction of OPN levels. We previously reported that plasma OPN levels were associated with serum CRP levels in hypertensive patients without any prior treatment.21 Tanaka et al.22 reported a significant relationship between CRP and OPN in hyper-


p=0.006

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1000

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800

OPN (ng ml–1)

OPN (ng ml –1)

P >0.999

600 400

600 400 200

200 0

baseline

24 weeks

amlodipine

0

baseline

24 weeks

valsartan

Figure 1 Plasma OPN levels in the amlodipine and valsartan groups before and after 24 weeks of the treatment. Data are expressed as the mean±s.d.

Table 3 Correlation coefficients of changing of OPN with clinical variables Amlodipine

DSBP DDBP Dln CRP DAldosterone

Valsartan

r

P

r

P-value

0.192 0.164 0.157 0.328

0.393 0.479 0.474 0.117

0.150 0.027 0.620 0.298

0.498 0.906 0.002 0.190

Abbreviations: DBP, diastolic blood pressure; ln CRP, log natural C-reactive protein; SBP, systolic blood pressure. DSBP, 6 months SBP minus baseline SBP; DDBP, 6 months DBP minus baseline DBP; Dln CRP, 6 months ln CRP minus baseline ln CRP; Daldosterone, 6 months aldosterone minus baseline aldosterone.

cholesterolaemic patients. They showed that the reduction of OPN was correlated with the reduction of CRP, but not with the reduction of low-density lipoprotein cholesterol. These reports indicated a close relationship between the two molecules. We previously reported that valsartan reduced pro-inflammatory cytokines, such as interleukin-6 and tumour necrosis factor-a in hypertensive patients.16 Blocking AII with valsartan may suppress inflammation and reduce OPN levels. Furthermore, CRP is not only a marker of inflammation, but also a pro-inflammatory molecule itself.23 This raises the possibility of a direct interaction between CRP and OPN expression. Although we found that valsartan treatment and a reduction of ln CRP levels were independent determinants of a reduction in OPN levels, it is difficult to conclude that an antiinflammatory effect of valsartan is necessary to reduce OPN levels. Kurakawa et al.24 reported that in a murine model of allergic asthma, increased OPN

protein in the broncho-alveolar lavage was inhibited by the corticosteroids, anti-inflammatory drugs. Although their model is completely different in our study subject, anti-inflammatory drugs certainly reduce OPN expression. In this study, valsartan was effective in reducing plasma OPN levels in hypertensive patients. Although there was no difference in the reduction of blood pressure between the amlodipine and valsartan groups, plasma OPN levels were not changed in the amlodipine group. This result suggests that the reduction of OPN levels by valsartan was independent of its blood pressure lowering effect. There are some clinical trials that showed valsartan was effective in preventing cardiovascular events in hypertensive patients. JIKEI and the Kyoto Heart study showed that the addition of valsartan to conventional treatment prevented more cardiovascular events than supplementary conventional treatment.25,26 These beneficial effects cannot be entirely explained by blood pressure control. A relationship between OPN levels and atherosclerosis has been reported. Many factors that facilitate atherosclerosis, such as hypoxia, high plasma glucose,27 platelet-derived growth factor,28 AII,18,29 aldosterone8 and mechanical stress,30 also induce OPN expression. OPN has several effects that may facilitate the development and progression of cardiovascular lesions, including cell adhesion, migration, proliferation, angiogenesis and ectopic mineralization.31 Reduction of both CRP and OPN levels by AII receptor blockade with valsartan might have beneficial effects on atherosclerosis. We have no data on whether the reduction of OPN levels would prevent future cardiovascular events. However, the JUPITER trial32 clearly showed that lowering both low-density lipoprotein cholesterol and Journal of Human Hypertension


Δ OPN (ng ml-1)

600

Δ OPN (ng ml-1)

600

400

400

200

200

0

0

-200

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0

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Δ ln CRP

-2 0 2 y = 149.85x - 36.76

4

Δ ln CRP valsartan

amlodipine

Figure 2 Relationship between changes of OPN and ln CRP. D, delta (24 weeks value minus baseline value).

CRP levels is important in the prevention of cardiovascular events. That study concluded that the suppression of inflammation is important for the prevention of future events. It is clear that OPN levels reflect chronic inflammation, and that the reduction of OPN levels is associated with a reduction of inflammation. However, a prospective clinical trial with a large number of subjects is required to show a beneficial effect of OPN reduction on outcomes. The source of plasma OPN may be endothelial cells, vascular smooth muscle cells and macrophages that secrete OPN. They have been reported to express the AII receptor type I.2–5 However, we were not able to determine the precise source of OPN in this study. There were some limitations of our study. First, there were differences in SBP of 5 mm Hg (138 vs 143 mm Hg) and in DBP by 3 mm Hg (84 vs 81 mm Hg) between valsartan and amlodipine treatment, but they did not reach statistical significance. Second, we previously reported that plasma OPN levels were correlated with aldosterone concentrations,11 but in this study, we were not able to show a relationship between a reduction of aldosterone levels and a reduction of OPN levels. The reason for this result may be the small number of study subjects. Third, we have no data on impaired glucose tolerance in the study participants, because we did not perform a glucose tolerance test. Fourth, we did not include an agematched control group of normotensive subjects. Fifth, the precise mechanisms that control the relationship between CRP and OPN levels remain unknown. In conclusion, valsartan reduces plasma OPN levels and this effect is independent of its blood pressure lowering effect. Furthermore, lowering CRP is also important in reducing plasma OPN levels. Blocking AII and suppressing inflammation appear to be essential for a reduction in plasma OPN concentrations. Journal of Human Hypertension

What is known about the topic K Osteopontin (OPN) has been shown to be regulated by the rennin–angiotensin–aldosterone system in experimental studies, and plasma OPN levels reflected the severity of atherosclerosis in clinical studies. K Less is known about how OPN is affected by blocking angiotensin II using angiotensin II receptor blockers (ARBs). What this study adds K The plasma OPN level was reduced using the ARB, valsartan, but there was no change using the calcium channel blocker, amlodipine, in patients with essential hypertension. K The reduction of OPN levels was correlated with the reduction of C-reactive protein (CRP) levels, and the use of valsartan and a reduction of CRP were independent determinants of the reduction of OPN. K The results suggest that both suppression of angiotensin II and inflammation are needed to reduce plasma OPN levels and further study is needed to determine if a reduction of OPN levels will decrease future cardiovascular events.

Conflict of interest The authors declare no conflict of interest.

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