Effect of angiotensin II receptor blockers,candesartan,on osteoprotegerin level in hypertensive patients: Link between bone and RAAS

Abstract

Introduction:

The renin-angiotensin-aldosterone system (RAAS) has recently been considered as a possible link between bone and vascular disease. The present study was designed to determine the effect of the angiotensin II receptor blocker candesartan on circulating osteoprotegerin (OPG) in hypertensive patients with multiple cardiovascular risk factors.

Material and methods:

A total of 69 hypertensive patients were randomized to two groups: Group 1 included patients treated with oral candesartan in doses of 16 mg to 32 mg per day in addition to routine standard of care (routine care + ARB), and Group 2 included patients who received routine standard of care other than ARBs or ACEIs, with no change to their treatment (routine care). Patients were evaluated for lipid profile, HbA1C, insulin, C-peptide, CRP, aldosterone, renin, homeostasis model assessment-insulin resistance (HOMA-IR) and OPG.

Results:

Baseline OPG levels did not differ significantly by treatment group. Post-treatment serum OPG levels were marginally lower in Group1 compared with Group 2; however, this decrease did not reach statistical significance (p = 0.077).

Conclusions:

In the present study, treatment with the ARB candesartan had no significant effect on circulating OPG levels in hypertensive patients with multiple cardiovascular risk factors. To the best of our knowledge, the present study is the first to estimate an effect of candesartan on bone remodeling marker such as OPG.

Keywords

Angiotensin II receptor blocker osteoprotegerin renin-angiotensin-aldosterone system

Introduction

Osteoprotegerin (OPG), a member of the tumor necrosis factor superfamily, is involved in pathological processes including bone remodeling, vascular inflammation and arterial calcification. OPG acts as a decoy receptor for the receptor activator of nuclear factor-κb ligand (RANKL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), regulates bone remodeling and applies antiapoptotic effects on endothelial cells, protecting them against TRAIL-induced cell death.^1–3 OPG, one of the major players in the balance between bone formation and bone resorption, exerts osteoprotective effects by inhibiting osteoclast differentiation and activation and promoting osteoclast apoptosis. However, in the clinical setting, the association between OPG and bone mineral density (BMD) as well as fragility fractures remains controversial. Increased OPG levels have been associated with higher BMD of the lumbar spine in men and total body BMD in women,^4,5 whereas in other studies a negative correlation or no correlation was found.^6,7 Recently, OPG has appeared to represent the molecular link between bone resorption and vascular calcification. Elevated serum OPG levels have been reported in patients with coronary artery disease and carotid atherosclerosis; further, increased OPG levels were positively associated with severity of vascular damage, atherosclerosis progression as well as with incident cardiovascular disease and mortality.^8–10

The renin-angiotensin-aldosterone system (RAAS), playing an integral role in cardiovascular homeostasis, has recently been considered as a possible link between bone and vascular disease. One of the final mediators of the RAAS cascade, angiotensin II (Ang II), indirectly promotes the differentiation and activation of osteoclasts through the upregulation of the activator RANKL in osteoblasts as well as stimulation of RANKL expression in osteoblasts and thereby osteoclastogenesis.^11,12 Ang II may also influence calcium metabolism by decreasing ionized calcium and increasing the parathyroid hormone level.¹³ Although previous studies have shown that interruption of the RAAS with angiotensin-converting enzyme inhibitors (ACEIs) is effective in increasing BMD and reduces the risk of fractures,^14,15 the results of trials evaluating effect of Ang II antagonist on bone metabolism are limited and equivocal. It has been shown that administration of an Ang II antagonist, losartan, had no effect on bone metabolism in normal rats,¹⁶ whereas another Ang II type 1 receptor blocker, olmesartan, ameliorates osteoclastogenesis through down-regulation of RANKL expression and improves bone density in ovariectomized spontaneously hypertensive rats.¹⁷ Differences in the affinity to the Ang II receptor as well as different immune and inflammatory effect may explain this variability.

The present study was designed to determine the effect of the angiotensin II receptor blocker (ARB) candesartan on circulating OPG levels in hypertensive patients with multiple cardiovascular risk factors.

Materials and methods

Participants

In the present study, 69 hypertensive patients were recruited from the outpatient clinic and evaluated for the study. Patients included in the study were stabilized on their previous medical treatment in the outpatient clinic for up to three months before entrance to the study, with an effort to minimize treatment change during the study. To optimize control of hypertension, individual medical advice was given to every patient at the beginning and offered throughout the duration of the study. Oral antihypertensive therapy, other than ARBs or ACEIs, was permitted in both groups. Following a three-month stabilization period, which included a washout period, all patients were consecutively randomized to two groups: Group 1 included patients treated with oral candesartan in doses of 16–32 mg per day in addition to routine standard of care (routine care + ARB), and Group 2 included patients receiving routine standard of care other than ARBs or ACEIs, with no change to their treatment (routine care).

Of the 69 patients recruited to the study, 67 completed the six-month treatment period (45 patients from Group 1 and 22 from Group 2).

Patients suspected of secondary hypertension, with history of major disease or surgery within the six months preceding entrance to the study, were excluded. Patients with hemodynamic instability, unbalanced endocrine disease, and any disease that might affect absorption of medications were excluded, as were patients with plasma creatinine > 2 mg/dl, elevation of liver enzymes to more than twice the upper normal limit and electrolyte abnormalities.

Biochemical parameters

Blood sampling for full chemistry and metabolic parameters, including total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, triglycerides, fasting glucose, HbA1C, c-peptide, insulin, and C-reactive protein (CRP), aldosterone and plasma renin activity were performed at baseline and at the end of the study. Samples for serum aldosterone and plasma renin activity were collected in a sitting position and measured using commercially available radioimmunoassay (DiaSorin, Italy, catalog number REF CA 1533). The lower limits of the serum aldosterone and plasma renin activity were 0.6 ng/dl and 0.1 ng/ml/h, respectively. The samples were measured in duplicate.

Serum OPG levels were determined by enzyme-linked immunosorbent assay (BioVendor). The intra- and interassay coefficients of variation for OPG were 2.4%–7.0 % and 3.4%–7.4%, respectively.

Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated by the following formula: fasting plasma insulin (mU/ml) × fasting plasma glucose (mg/dl)/405.

Statistical analysis

Analysis of data was carried out using SPSS 9.0 statistical analysis software (SPSS Inc, Chicago, IL, USA, 1999). For continuous variables, such as hemodynamic, biochemistry and arterial elasticity parameters, descriptive statistics were calculated and reported as mean ± standard deviation. Normalcy of distribution of continuous variables was assessed using the Kolmogorov-Smirnov test (cutoff at p = 0.01). Categorical variables such as sex and comorbidities were described using frequency distributions and are presented as frequency (%). Continuous variables were simultaneously across visits by group using GLM repeated-measures analysis. Significant differences were compared using the t-test for independent samples. Within a given treatment group, the Friedman test or GLM repeated-measures analysis was used to compare differences across visits, followed post hoc by the t-test for paired samples of the Wilcoxon signed ranks test as appropriate. Categorical variables were compared between groups using the chi-square test. All tests are two sided and considered significant at p < 0.05.

Results

Clinical characteristics of the study groups are present in Table 1. As can be seen the two groups were similar with respect to age, gender distribution, presence of cardiovascular risk factors, concomitant medications, baseline blood pressure level and baseline OPG levels.

Table 1.

Baseline characteristics of the study groups.

	Routine care+ candesartan group n = 47	Routine care group n = 22	p value
Sex (F/M)	27/20	14/8	ns
Age (years)	62.9 ± 7.9	62.0 ± 8.9	ns
BMI (kg/m²)	31.6 ± 5.3	30.6 ± 4.5	ns
Cardiovascular risk factors:
Diabetes mellitus (%)	30 (63.8%)	11 (50%)	ns
Dislipidemia (%)	35 (74.5%)	11 (50%)	ns
Smokers (%)	7 (14.9%)	3 (13/6%)	ns
Personal history of CVD (%)	12 (25.5%)	5 (22.7%)	ns
Concomitant_medications:
CCBs (%)	24 (51%)	10 (45%)	ns
B-blockers (%)	16 (34%)	10 (45%)	ns
Diuretics (%)	14 (29%)	4 (18%)	ns
Antidiabetic medications (%)	22 (59.6%)	10 (45.5%)	ns
Baseline SBP (mm/Hg)	152.9 ± 20.5	146.0 ± 15.9	ns
Baseline DBP (mm/Hg)	78.7 ± 8.8	78.6 ± 6.7	ns
Baseline CRP(mg/dl)	0.5 ± 0.4	0.3 ± 0.5	ns
Baseline HbA1C (%)	7.7 ± 1.6	6.7 ± 1.0	0.024
Baseline total cholesterol (mg/dl)	184.1 ± 35.9	178.5 ± 40.6	ns
Baseline LDL cholesterol (mg/dl)	106.2 ± 32.3	101.1 ± 33.1	ns
Baseline HDL cholesterol (mg/dl)	47.6 ± 9.0	47.5 ± 14.3	ns
Baseline insulin (IU/ml)	17.2 ± 16.9	11.9 ± 11.1	ns
Baseline C-peptide (IU/ml)	1.3 ± 0.9	1.3 ± 1.0	ns
Baseline HOMA-IR	7.3 ± 9.1	3.9 ± 4.6	0.048
Baseline aldosterone (ng/ml)	6.8 ± 3.7	5.9 ± 3.4	ns
Baseline OPG (pmol/l)	95.8 ± 57.1	98.6 ± 58.6	ns

F: female; M: male; BMI: body mass index; CVD: cardiovascular disease; CCBs: calcium channel blockers; SBP: systolic blood pressure; DBP: diastolic blood pressure; CRP: C-reactive protein; HbA1C: glycated hemoglobin; LDL: low-density lipoprotein; HDL: high-density lipoprotein; HOMA-IR: homeostasis model assessment-estimated insulin resistance; OPG: osteoprotegerin.

Between-group comparisons

Table 1 shows that both groups of patients were similar at baseline in terms of hemodynamic, metabolic and inflammatory parameters. There were no significant differences between the two groups in terms of systolic (SBP) as well as diastolic blood pressure (DBP) at baseline and after six months of treatment. Baseline OPG levels did not differ significantly by treatment group. However, post-treatment serum OPG levels were marginally lower in patients treated with candesartan compared with the routine care group (96.9±47.8 vs 129.2±34.5 mmol/l, p = 0.077) (Figure 1).

Figure 1.

Circulating osteoprotegerin (OPG) by groups during the six-month follow-up.

Baseline metabolic and inflammatory parameters including lipids, fasting insulin, C-peptide, and CRP did not differ significantly between the groups. After the six-month treatment period, no significant difference in these parameters was detected by group. Serum creatinine and electrolytes levels did not differ significantly between the groups at baseline and at the end of the study.

Changes in hemodynamic and metabolic parameters in hypertensive patients treated with candesartan

Table 2 shows hemodynamic and metabolic parameters in hypertensive patients treated with candesartan for six months. SBP decreased significantly from 151.5±18.3 at baseline to 129.5±12.8 mmHg after six months of treatment (p < 0.0001). DBP decreased significantly during the treatment period from 78.6±8.8 to 72.2±9.4 mmHg (p < 0.0001). Heart rate did not change during the study.

Table 2.

Change from baseline in homodynamic and metabolic variables in each group.

Variable	Routine care +candesartan group			Routine care group
	Baseline	6 months	p value	Baseline	6 months	p value
Systolic BP (mm/Hg)	151.5 ± 18.3	129.5 ± 12.8	0.000	146.0 ± 15.9	131.7 ± 14.1	0.001
Diastolic BP (mm/Hg)	78.6 ± 8.8	72.2 ± 9.4	0.000	78.6 ± 6.7	73.7 ± 9.2	0.019
HR (bpm)	69.6 ± 12.0	67.0 ± 10.7	0.067	65.6 ± 8.3	66.3 ± 10.1	0.699
Total cholesterol (mg/dl)	184.1 ± 35.9	188.6 ± 38.3	0.430	176.5 ± 40.6	186.8 ± 44.6	0.227
Triglycerides (mg/dl)	181.2 ± 178.6	172.5 ± 80.2	0.702	146.7 ± 65.9	154.6 ± 80.4	0.483
HDL cholesterol (mg/dl)	46.8 ± 8.5	43.9 ± 8.4	0.008	46.9 ± 14.5	44.9 ± 13.2	0.188
LDL cholesterol (mg/dl)	105.8 ± 33.8	108.9 ± 31.6	0.557	99.5 ± 33.3	110.1 ± 37.1	0.226
Fasting glucose (mg/dl)	150.4 ± 57.8	143.7 ± 59.1	0.364	123.0 ± 42.4	132.5 ± 52.8	0.156
HbA1c (%)	7.4 ± 1.6	7.4 ± 1.3	0.778	6.7 ± 1.1	6.9 ± 1.4	0.075
C-peptide (IU/ml)	1.3 ± 0.9	1.2 ± 0.7	0.596	1.3 ± 0.9	1.4 ± 1.0	0.756
Fasting insulin (IU/ml)	17.2 ± 17.0	13.1 ± 14.4	0.114	10.2 ± 6.1	11.3 ± 5.6	0.427
HOMA-IR	7.3 ± 9.1	5.3 ± 8.9	0.142	3.9 ± 4.6	3.2 ± 2.2	0.489
Urea (mg/dl)	40.3 ± 15.5	37.3 ± 11.8	0.072	32.4 ± 6.9	32.4 ± 9.3	0.981
Creatinine (mg/dl)	1.0 ± 0.3	1.0 ± 0.2	0.068	1.0 ± 0.1	1.0 ± 0.2	0.329
Potassium (mg/dl)	4.5 ± 0.4	4.7 ± 0.4	0.041	4.4 ± 0.4	4.5 ± 0.4	0.182
CRP (mg/dl)	0.5 ± 0.5	0.6 ± 0.2	0.563	0.4 ± 0.5	0.7 ± 0.6	0.026
Aldosterone (ng/ml)	6.6 ± 3.7	7.0 ± 9.7	0.802	5.9 ± 3.4	8.1 ± 5.9	0.085
OPG (pmol/l)	80.5 ± 55.9	99.6 ± 49.2	0.247	98.6 ± 58.7	119.4 ± 55.5	0.423

BP: blood pressure; HR: heart rate; HDL: high-density lipoprotein; LDL: low-density lipoprotein; HbA1C: glycated hemoglobin; HOMA-IR: homeostasis model assessment-estimated insulin resistance; CRP: C-reactive protein; OPG: osteoprotegerin.

Potassium levels increased from 4.5±0.4 to 4.7±0.4 mg/dl after six months of treatment (p = 0.041). Nevertheless, end-of-follow-up potassium values were within normal limits. OPG levels did not change during the study (p = 0.247). Parameters of glucose metabolism including lipids, HbA1C, fasting insulin, C-peptide and HOMA-IR did not change significantly during the study

Changes in hemodynamic and metabolic parameters in the routine care group

As shown in Table 2, SBP decreased significantly from 146.0±15.9 to 131.7±14.1 mm/Hg during the study (p = 0.001). DBP decreased significantly from 78.6±6.7 to 73.7±9.2 mm/Hg (p = 0.019). Pulse rate did not change during the follow-up.

Circulating OPG levels did not change during the study (p = 0.423). Neither glucose homeostasis parameters nor potassium levels changed significantly during the treatment period. CRP levels increased significantly during six months of follow-up (p = 0.026).

Discussion

Experimental, clinical and epidemiological evidence has shown that the RAAS plays an integral role in bone remodeling.

Using a chimeric model of transgenic hypertensive mouse expressing both the human renin as well as angiotensinogen genes, it has been shown that activation of the RAAS induces high turnover osteoporosis with accelerated bone resorption.¹¹ Ex vivo cultures showed that Ang II increased osteoclastogenesis-supporting cytokines, RANKL and vascular endothelial growth factor (VEGF), thereby stimulating the formation of osteoclasts. There have been several reports on the effects of Ang II on bone cell function in vitro, including inhibition of osteoblastic differentiation and mineralization, stimulation of proliferation and collagen synthesis in osteoblasts, and stimulation of osteoclastic bone resorption.^12,18,19 Furthermore, it was shown that ACEIs were effective in increasing BMD in women with the angiotensin-converting enzyme (ACE) DD polymorphism, which is associated with increased serum Ang II.²⁰ However, ACEIs also regulate the bradykinin-nitric oxide (NO) pathway as well as block Ang II production, different from ARBs. It is well known that the NO pathway regulates local blood flow in bone marrow capillaries, which might enhance bone marrow formation. Thus, the contribution of the NO pathway might have a role in the prevention of osteoporosis by ACE inhibition.

The results of trials evaluating the effect of Ang II antagonist on bone metabolism are equivocal. It has been shown that administration of an Ang II antagonist, losartan, had no effect on bone metabolism in normal rats,¹⁶ whereas other Ang II type 1 receptor blocker, olmesartan, ameliorates osteoclastogenesis through down-regulation of RANKL expression and improves bone density in ovariectomized spontaneously hypertensive rats.¹⁷ Alterations in the affinity to the Ang II receptor of the different representative ARBs may explain this variability. Among ARBs, olmesartan has been reported to show the highest level of binding with the Ang II type 1 receptor and, thus, has a stronger and more sustained hypotensive effect than that of the other ARBs. Moreover, the inhibitory effect of olmesartan on albuminuria was prominent and not affected by the dosing time, because olmesartan exhibits a strong antihypertensive effect that is sustained for 24 hours. Furthermore, additional properties such as reduction in oxidative stress, stimulation of adipogenesis with subsequent improvement in glucose transport and reduction in expression of proinflammatory vascular cell adhesion molecules, CRP and cytokines are different despite similar blood-lowering effects of various ARBs.^17,21,22

Another important observation in our study is the increase of CRP levels observed in the placebo group but not in the candesartan group. The pre-and post-treatment comparison of serum CRP levels was a secondary analysis and follows the statement that the between-group difference was not significant. Nevertheless, we think that the increase in CRP levels observed in the placebo group but not in the active treatment group is of interest. The findings of the present study concur with previous experimental and clinical trials that have demonstrated anti-inflammatory properties of ARBs. Recently published data evaluated the effect of two different classes of antihypertensive agents, valsartan and amlodipine, on CRP as well as osteopontin (OPN), a pleiotropic cytokine that has recently emerged as a key factor in vascular remodeling and is regulated by the RAAS. The significant reduction of OPN levels was correlated with the reduction of CRP levels, and the use of valsartan as well as reduction of CRP were independent determinants of the reduction of OPN.²³ Moreover, it has been shown that treatment with olmesartan as a mono-therapy as well as co-therapy with pravastatin resulted in a highly significant decrease of circulating levels of OPN in a large cohort of patients with essential hypertension, whereas OPN levels were correlated with a panel of adhesion molecules and inflammation markers such as interleukin-6 (IL-6), vascular cell adhesion protein 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and high-sensitivity C-reactive protein (hsCRP).²⁴

The present study has several limitations. We included a relatively small number of participants, and larger studies are required to establish the impact of the RAAS blockade on bone metabolism as well as bone density and fractures rates to demonstrate the overall clinical impact of RAAS blockade in different populations, especially patients at increased risk for osteoporosis.

Additionally, since the present study focused on OPG assessment, the effect of candesartan treatment on different bone remodeling markers such as OPN as well as RANKL and TRAIL expression will need to be evaluated by long-term studies in a hypertensive population.

In conclusion, significant changes in circulating OPG levels in hypertensive patients treated with the ARB candesartan were not observed. The findings of the present study are consistent with those of studies reporting a variability of the different representative ARBs in terms of RANKL expression. However, additional studies are required to establish the clinical impact of RAAS blockade by different ARBs on bone remodeling.

Footnotes

Author’s note

This manuscript represents original, unpublished material that is not under consideration for publication elsewhere; no portion of the manuscript has been published or posted on the Internet.

The corresponding author and all of the authors have read and approved the final submitted manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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