Chronic treatment with angiotensin AT1 receptorantagonists reduced serum but not bone TGF-b1

levels in ovariectomized rats

Yong-Qi Li, Hui Ji, Yang Shen, Li-Ju Ding, Pei Zhuang, Yu-Lin Yang, andQiu-Ju Huang

Abstract: Approximately 50% of hypertensive patients are postmenopausal women; therefore, any antihypertensive ther-apy must not adversely affect bone loss in this population. Recently, however, concern has been raised that use of angio-tensin AT1 receptor antagonists may increase the tendency to develop postmenopausal osteoporosis by decreasingtransforming growth factor-b1 (TGF-b1), which has been implicated in bone mass maintenance. In the present study, we se-lected telmisartan and valsartan as representatives of angiotensin AT1 receptor antagonists and used ovariectomized (OVX)rats as a model of human postmenopausal osteoporosis. After 3 months treatment with telmisartan (5 mg/kg daily) or val-sartan (10 mg/kg daily), OVX rats showed no signs of adverse effects on bone mineral density of the lumbar vertebrae(L1–L5) or the total femur, nor did treatment affect serum levels of osteocalcin and osteoclast-derived tartrate-resistantacid phosphatase (TRACP-5b). Bone TGF-b1 content remained unchanged, although treatment with telmisartan and valsar-tan significantly reduced serum TGF-b1 levels (p < 0.05). In conclusion, chronic treatment with angiotensin AT1 receptorantagonists reduced serum but not bone TGF-b1 levels and did not accelerate ovariectomy-induced bone loss in rats.

Key words: angiotensin AT1 receptor antagonists, bone mineral density, osteoporosis, ovariectomized rats, transforminggrowth factor-b1, TGF-b1.

Resume : Environ 50 % des patients souffrant d’hypertension sont des femmes postmenopausees, de sorte que les traite-ments antihypertenseurs ne doivent pas contribuer a la perte osseuse chez cette population. Recemment, on s’est inquietedu fait que l’utilisation d’antagonistes des recepteurs AT1 de l’angiotensine puisse augmenter le developpement de l’osteo-porose postmenopausique en diminuant le facteur de croissance transformant-b1 (TGF-b1), qui a ete lie au maintien de lamasse osseuse. Dans la presente etude, nous avons choisi le telmisartan et le valsartan comme antagonistes representatifsdes recepteurs AT1 de l’angiotensine, et des rats ovariectomises (OVX) comme modele d’osteoporose postmenopausiquehumain. Le traitement des rats OVX pendant 3 mois par telmisartan (5 mg/kg par jour) ou valsartan (10 mg/kg par jour)n’a pas eu d’effets negatifs sur la TMO des vertebres lombaires (L1–L5) et du femur, et n’a pas affecte les taux seriquesd’osteocalcine et de TRACP-5b. La teneur en TGF-b1 osseux est demeuree stable, bien que le traitement par ces deux me-dicaments ait significativement diminue les taux de TGF-b1 seriques (p < 0,05). En conclusion, un traitement chronique al’aide des antagonistes des recepteurs AT1 de l’angiotensine a reduit les taux seriques, mais pas les taux osseux, de TGF-b1, et n’a pas accelere la perte osseuse induite par une ovariectomie chez les rats.

Mots-cles : antagonistes des recepteurs AT1 de l’angiotensine, teneur minerale osseuse, osteoporose, rats ovariectomises,facteur de croissance transformant-b1 (TGF-b1).

[Traduit par la Redaction]

IntroductionHypertension affects 1 billion people worldwide, approxi-

mately 65 million people in the United States alone, and it isthe leading cause of death in industrialized countries

(Kearney et al. 2005; Smith and Ashiya 2007). AngiotensinAT1 receptor antagonists are widely used in the treatment ofhypertension and its related diseases, and they account for15% of all antihypertensive prescriptions in the UnitedStates (Smith and Ashiya 2007). Global sales will continueto grow for their beneficial effects on insulin resistance, acentral feature of metabolic syndrome (Benndorf et al.2006; Deedwania and Schmieder 2006; Karagiannis et al.2007; Scheen 2004; Usui et al. 2007).

Both osteoporosis and hypertension are major causes ofmorbidity and mortality in elderly women. Because approx-imately 50% of human hypertensive patients are postmeno-pausal females (Safar et al. 1994), any antihypertensivetherapy must not adversely affect the bone loss in this popu-lation. Recently, however, concern has been raised that useof angiotensin AT1 receptor antagonists may increase the

Received 1 July 2008. Accepted 22 October 2008. Published onthe NRC Research Press Web site at cjpp.nrc.ca on 6 January2009.

Y.Q. Li, H. Ji,1 Y. Shen, L.J. Ding, P. Zhuang, andQ.J. Huang. Department of Pharmacology, ChinaPharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009,P.R. China.Y.L. Yang. School of Pharmacy, Wenzhou Medical College,Wenzhou 325035, P.R. China.

1Corresponding author (e-mail: huijicpu@163.com).

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tendency to develop postmenopausal osteoporosis by de-creasing transforming growth factor-b1 (TGF-b1) which hasbeen implicated in bone mass maintenance (Akinci et al.2007; Finkelman et al. 1991, 1992; Gulcan et al. 2008; Nic-olas et al. 1994). So it is urgent and important to test thishypothesis and clarify the effects of angiotensin AT1 recep-tor antagonists on postmenopausal bone loss.

In the present study, we selected telmisartan and valsartanas representatives of angiotensin AT1 receptor antagonistsand ovariectomized rats as a model of human postmeno-pausal osteoporosis. After 3 months of treatment, dual en-ergy X-ray absorptiometry (DEXA), biomechanical testing,and serum biochemical assay, reflecting the formation ofnew bone (bone turnover marker osteocalcin) or providingan index of osteoclast activity (tartrate-resistant acid pho-phatase 5b (TRACP-5b), were conducted. TGF-b1 levels inserum and bone were also determined.

Materials and methods

Animals and experimental proceduresForty 5-month-old nulliparous female Sprague–Dawley

rats weighting 230–260 g were obtained from QinglongMountain Farm (Nanjing, China). Rats were housed instandard cages (5 rats per cage) and they were maintainedat 25 ± 1 8C and constant humidity (60%) with a 12 hlight : 12 h dark cycle. The animals were allowed free accessto water and standard pellet diet. Animal care and treatmentwere conducted in accordance with the institutional guide-lines.

Animals were randomly divided into 4 groups (10 rats ineach group) and treated with a daily oral gavage for3 months. Group 1, sham-operated, received vehicle (0.5%carboxymethylcellulose sodium (CMC-Na)). Group 2, ovar-iectomized (OVX), also received vehicle. Group 3 andgroup 4 were OVX treated with 5 mg/kg telmisartan or10 mg/kg valsartan, respectively, dissolved in the vehicle.The dosages of telmisartan and valsartan (Center of DrugDiscovery, China Pharmaceutical University, Nanjing,China) were similar to those commonly used in clinicalpractice and in a previous report (Mori et al. 2007). Treat-ments were begun one week after the surgery. Body weightswere monitored weekly to adjust the dosage of each drug.At the end of the study, the rats were fasted overnight, andblood samples were collected from the abdominal aorta,centrifuged, and stored at –80 8C until analysis. The ratswere then euthanized, their right femoral diaphyses werequickly removed, cleaned, snap-frozen, and stored in liquidnitrogen for determination of TGF-b1.

Bone mineral density assessment by DEXABone mineral density (BMD) was measured at the end of

the study by using DEXA (GE Lunar, Wisconsin, USA) inrats anesthetized with ether. BMD and bone mineral contentof the lumbar vertebrae (L1–L5) and the total femur wererecorded. The investigator performing the measurementswas unaware of the treatments the rats had received.

Biomechanical testImmediately after the rats were euthanized, their left fe-

murs were collected for biomechanical test by using a 3-

point bending method as we described previously (Ren etal. 2007).

Serum levels of osteocalcin, TRACP-5b, and TGF-b1Serum levels of osteocalcin (IDS, Boldon, England),

TRACP-5b (IDS), and TGF-b1 (R&D Systems, Minnesota,USA) were determined with commercially available ratELISA kits according to the manufacture’s instructions. Allthe serum samples were assayed in duplicate.

Preparation of bone extracts and determination of boneTGF-b1

According to the method described in previous reports(Finkelman et al. 1991, 1992), bone extracts were preparedand analyzed by using a rat ELISA kit for the determinationof TGF-b1. The content of total protein in bone extracts wasquantitated by Bradford assay (BioRad, Hercules, USA).TGF-b1 content was expressed as nanograms per milligramof total protein (ng/mg).

Statistical analysisAll data were expressed as means ± SE. Statistical analy-

sis was performed using the Student’s t test or one-wayANOVA followed by the post hoc Tukey’s test. A valueof p < 0.05 was considered to be statistically significant.

Results

No adverse effects related to drug administration were ob-served. Figure 1 shows body weight gains of the rats in eachgroup. The sham-operated rats increased their body weightsby 14% during the 3-month experimental period. Greaterweight gains were observed in all the OVX groups, and nobetween-group differences were detected.

Figure 2 illustrates BMD of the lumbar vertebrae (L1–L5)and the total femur at the end of the study. The femur BMDwas significantly reduced (approximately15%, p < 0.001versus sham-operated) in the OVX rats. Treatment with tel-misartan (5 mg/kg daily) or valsartan (10 mg/kg daily) for3 months had no effect on BMD of the OVX rats. Similar

Fig. 1. Three months treatment with telmisartan (5 mg/kg) or val-sartan (10 mg/kg) daily by oral gavage did not affect body weightgains in the OVX rats. 0.5% CMC-Na served as vehicle control.Data are means ± SE of 10 animals. ***, Significant at p < 0.001versus sham-operated rats by Student’s t test. OVX, ovariecto-mized; CMC-Na,carboxymethylcellulose sodium.

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results were observed in the biomechanical test (data notshown).

Serum levels of osteocalcin and TRACP-5b were bothelevated in the OVX rats (Figs. 3a and 3b), indicating an ac-celerated bone turnover. Three months treatment with telmi-sartan or valsartan had no effect on osteocalcin and TRACP-5b. Although treatment with telmisartan and valsartan re-duced serum TGF-b1 levels significantly (Fig. 4a) comparedwith those of CMC-Na in OVX (group 2), bone TGF-b1content (Fig. 4b) remained unchanged.

DiscussionOvariectomized rats have been widely used as a model of

postmenopausal osteoporosis and have been validated as aclinically relevant model of human postmenopausal boneloss (Kalu 1991; Wronski et al. 1993). In a previous study,administration of losartan in a dose similar to that recom-mended for the treatment of hypertension did not cause sig-nificant changes in bone density, ash and mineral content, ormorphometric parameters of the femur in healthy intact fe-male rats (Broulik et al. 2001). In the current study, 3months treatment with telmisartan (5 mg/kg daily) or valsar-tan (10 mg/kg daily) in the OVX rats showed no adverse ef-fects on BMD of the lumbar vertebrae (L1–L5) or the total

femur and did not affect serum levels of osteocalcin andTRACP-5b. Similar results were observed in the biomechan-ical test (data not shown). Together, the results of thesestudies confirmed the safe use of angiotensin AT1 receptorantagonist for long-term management of blood pressure inpostmenopausal hypertensive patients.

TGF-b1 is a ubiquitous, multifunctional growth factor.Despite many positive reports supporting the therapeutic useof TGF-b1 as a bone-forming agent, conflicting data exist re-garding the role of TGF-b1 in the pathogenesis and treat-ment of bone loss or osteoporosis (Janssens et al. 2005).Specifically, direct associations between low TGF-b1 levelsin serum or bone and osteoporosis remain controversial(Akinci et al. 2007; Finkelman et al. 1992; Georgescu et al.2004; Grainger et al. 1999). Several studies demonstratedthat angiotensin AT1 receptor antagonists significantly de-creased TGF-b1 levels in human plasma and in rat liver andaortic grafts (Esmatjes et al. 2001; Jin et al. 2007; Yoshiji etal. 2001; Zezina et al. 2001; Zhao et al. 2007). In line withfindings from previous studies, our study showed treatmentof OVX rats with telmisartan and valsartan reduced the se-rum TGF-b1 levels, but did not affect bone TGF-b1 contentand showed no adverse effects on BMD of the lumbar verte-brae (L1–L5) or total femur, nor on bone turnover markers

Fig. 2. Three months treatment with telmisartan (5 mg/kg) or val-sartan (10 mg/kg) daily by oral gavage had no effect on BMD ofthe lumbar vertebrae (L1–L5) (a) or of the total femur (b) in theOVX rats. 0.5% CMC-Na served as vehicle control. Data aremeans ± SE of 10 animals. ***, Significant at p < 0.001 versussham-operated rats by Student’s t test. BMD, bone mineral density;OVX, ovariectomized; CMC-Na, carboxymethylcellulose sodium.

Fig. 3. Three months treatment with telmisartan (5 mg/kg) or val-sartan (10 mg/kg) daily by oral gavage did not alter serum levels ofosteocalcin (a) and TRACP-5b (b) in the OVX rats. 0.5% CMC-Naserved as vehicle control. Data are means ± SE of 10 animals.*, Significant at p < 0.05 and ***, p < 0.001 versus sham-operatedrats by Student’s t test. TRACP-5b, osteoclast-derived tartrate-re-sistant acid phophatase; OVX, ovariectomized; CMC-Na, carboxy-methylcellulose sodium.

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(osteocalcin and TRACP-5b). Simultaneous determination ofTGF-b1 levels in serum and bone appears to be more clini-cally relevant than determination of either alone. The con-clusions of studies (Akinci et al. 2007; Grainger et al. 1999)where only serum TGF-b1 levels were detected may need tobe reconsidered, given our finding that reduction of thisgrowth factor in serum may not be accompanied by its re-duction in bone. Unchanged TGF-b1 levels in the bones oftelmisartan- and valsartan-treated OVX rats may be ex-plained by (i) poor availability of these 2 drugs in bone andinsufficient effects on the local renin–angiotensin system(Haznedaroglu and Ozturk 2003), (ii) TGF-b1 interactionwith a wide range of other growth factors and hormones inbone (Janssens et al. 2005), which generates an intricate net-work of interpathway crosstalk and thus provokes a complexresponse upon slight changes that maintains a local homeo-stasis, or (iii) other confounding factors.

In the elderly, hypertension seldom occurs in isolationfrom other cardiovascular risk factors. Circulating TGF-b1levels have been found to be higher in hypertensive individ-uals with obesity, renal failure, African American ethnicity,and end-organ damage (Cottone et al. 2002; Derhaschnig etal. 2002; Parrinello et al. 2005; Porreca et al. 2002; Suthan-thiran et al. 2000). The results of our study indicate that itmay be helpful to use angiotensin AT1 receptor antagonists

to reduce circulating TGF-b1 levels and to control comorbid-ities and hypertension-related diseases in these patients.

In conclusion, chronic treatment with angiotensin AT1 re-ceptor antagonists does not accelerate ovariectomy-inducedbone loss in rats. These drugs can be helpful for the treat-ment of hypertension and hypertension-related comorbid-ities.

AcknowledgementsSpecial thanks to Shan Yu, Ying Chen, and Xing-Li Gu

for their excellent technical assistance (Department of Phar-macology, China Pharmaceutical University). This work wassupported in part by the China National Science Foundation(No. 30801424) and Research Initiation Fund(No. 01211101) from China Pharmaceutical University.

ReferencesAkinci, B., Bayraktar, F., Saklamaz, A., Demir, T., Yener, S.,

Comlekci, A., et al. 2007. Low transforming growth factor-beta1 serum levels in idiopathic male osteoporosis. J. Endocri-nol. Invest. 30(5): 350–355. PMID:17598964.

Benndorf, R.A., Rudolph, T., Appel, D., Schwedhelm, E., Maas,R., Schulze, F., et al. 2006. Telmisartan improves insulin sensi-tivity in nondiabetic patients with essential hypertension. Meta-bolism, 55(9): 1159–1164. doi:10.1016/j.metabol.2006.04.013.PMID:16919533.

Broulik, P.D., Tesar, V., Zima, T., and Jirsa, M. 2001. Impact ofantihypertensive therapy on the skeleton: effects of enalapriland AT1 receptor antagonist losartan in female rats. Physiol.Res. 50(4): 353–358. PMID:11551140.

Cottone, S., Mule, G., Amato, F., Riccobene, R., Vadala, A.,Lorito, M.C., et al. 2002. Amplified biochemical activation ofendothelial function in hypertension associated with moderate tosevere renal failure. J. Nephrol. 15(6): 643–648.PMID:12495277.

Deedwania, P.C., and Schmieder, R. 2006. Angiotensin receptorblockers: cardiovascular protection in the metabolic syndrome.J. Renin Angiotensin Aldosterone Syst. 7(Suppl 1): S12–S18.doi:10.3317/jraas.2006.018. PMID:16986230.

Derhaschnig, U., Shehata, M., Herkner, H., Bur, A.,Woisetschlager, C., Laggner, A.N., et al. 2002. Increased levelsof transforming growth factor-beta1 in essential hypertension.Am. J. Hypertens. 15(3): 207–211. doi:10.1016/S0895-7061(01)02327-5. PMID:11939608.

Esmatjes, E., Flores, L., Inigo, P., Lario, S., Ruilope, L.M., andCampistol, J.M. 2001. Effect of losartan on TGF-beta1 and urin-ary albumin excretion in patients with type 2 diabetes mellitusand microalbuminuria. Nephrol. Dial. Transplant. 16(Suppl 1):90–93. PMID:11369831.

Finkelman, R.D., Linkhart, T.A., Mohan, S., Lau, K.H., Baylink,D.J., and Bell, N.H. 1991. Vitamin D deficiency causes a selec-tive reduction in deposition of transforming growth factor betain rat bone: possible mechanism for impaired osteoinduction.Proc. Natl. Acad. Sci. U.S.A. 88(9): 3657–3660. doi:10.1073/pnas.88.9.3657. PMID:2023915.

Finkelman, R.D., Bell, N.H., Strong, D.D., Demers, L.M., andBaylink, D.J. 1992. Ovariectomy selectively reduces the concen-tration of transforming growth factor beta in rat bone: implica-tions for estrogen deficiency-associated bone loss. Proc. Natl.Acad. Sci. U.S.A. 89(24): 12190–12193. doi:10.1073/pnas.89.24.12190. PMID:1465458.

Georgescu, C., Seck, T., Diel, I., Minne, H., Duncea, I., and

Fig. 4. Three months treatment with telmisartan (5 mg/kg) or val-sartan (10 mg/kg) daily by oral gavage reduced serum (a) but notbone (b) TGF-b1 levels in the OVX rats. 0.5% CMC-Na served asvehicle control. Data are means ± SE of 10 animals. *, Significantat p < 0.05 and ***, p < 0.001 versus sham-operated rats by Stu-dent’s t test. #, p < 0.05 versus CMC-Na in OVX rats by post hocTukey’s test. TGF-b1, transforming growth factor-b1; OVX, ovar-iectomized; CMC-Na, carboxymethylcellulose sodium.

54 Can. J. Physiol. Pharmacol. Vol. 87, 2009

Published by NRC Research Press

Pfeilschifter, J. 2004. Bone matrix insulin-like growth factor(IGF)-I, IGF-II and transforming growth factor (TGF)-beta1 le-vels in men and postmenopausal women with osteoporosis: lackof association with circulating growth factors and bone mineraldensity. Rev. Med. Chir. Soc. Med. Nat. Iasi, 108(2): 281–286.PMID:15688799.

Grainger, D.J., Percival, J., Chiano, M., and Spector, T.D. 1999.The role of serum TGF-beta isoforms as potential markers of os-teoporosis. Osteoporos. Int. 9(5): 398–404. doi:10.1007/s001980050163. PMID:10550458.

Gulcan, E., Gulcan, A., Toker, S., and Cosar, E. 2008. Are thereany effects of angiotensin II receptor blockers on postmenopau-sal osteoporosis? Med. Hypotheses, 70(3): 701–702. doi:10.1016/j.mehy.2007.07.011. PMID:17714882.

Haznedaroglu, I.C., and Ozturk, M.A. 2003. Towards the under-standing of the local hematopoietic bone marrow renin-angiotensin system. Int. J. Biochem. Cell Biol. 35(6): 867–880.doi:10.1016/S1357-2725(02)00278-9. PMID:12676173.

Janssens, K., ten Dijke, P., Janssens, S., and Van Hul, W. 2005.Transforming growth factor-beta1 to the bone. Endocr. Rev.26(6): 743–774. doi:10.1210/er.2004-0001. PMID:15901668.

Jin, H., Yamamoto, N., Uchida, K., Terai, S., and Sakaida, I. 2007.Telmisartan prevents hepatic fibrosis and enzyme-altered lesionsin liver cirrhosis rat induced by a choline-deficient L-aminoacid-defined diet. Biochem. Biophys. Res. Commun. 364(4):801–807. doi:10.1016/j.bbrc.2007.10.083. PMID:17963695.

Kalu, D.N. 1991. The ovariectomized rat model of postmenopausalbone loss. Bone Miner. 15(3): 175–191. doi:10.1016/0169-6009(91)90124-I. PMID:1773131.

Karagiannis, A., Mikhailidis, D.P., Athyros, V.G., Kakafika, A.I.,Tziomalos, K., Liberopoulos, E.N., et al. 2007. The role of re-nin-angiotensin system inhibition in the treatment of hyperten-sion in metabolic syndrome: are all the angiotensin receptorblockers equal? Expert Opin. Ther. Targets, 11(2): 191–205.doi:10.1517/14728222.11.2.191. PMID:17227234.

Kearney, P.M., Whelton, M., Reynolds, K., Muntner, P., Whelton,P.K., and He, J. 2005. Global burden of hypertension: analysisof worldwide data. Lancet, 365(9455): 217–223. PMID:15652604.

Mori, Y., Itoh, Y., and Tajima, N. 2007. Angiotensin II receptorblockers downsize adipocytes in spontaneously type 2 diabeticrats with visceral fat obesity. Am. J. Hypertens. 20(4): 431–436.doi:10.1016/j.amjhyper.2006.09.016. PMID:17386352.

Nicolas, V., Prewett, A., Bettica, P., Mohan, S., Finkelman, R.D.,Baylink, D.J., et al. 1994. Age-related decreases in insulin-likegrowth factor-I and transforming growth factor-beta in femoralcortical bone from both men and women: implications for boneloss with aging. J. Clin. Endocrinol. Metab. 78(5): 1011–1016.doi:10.1210/jc.78.5.1011. PMID:8175953.

Parrinello, G., Licata, A., Colomba, D., Di Chiara, T., Argano, C.,Bologna, P., et al. 2005. Left ventricular filling abnormalities

and obesity-associated hypertension: relationship with overpro-duction of circulating transforming growth factor beta1. J. Hum.Hypertens. 19(7): 543–550. doi:10.1038/sj.jhh.1001864. PMID:15944724.

Porreca, E., Di Febbo, C., Vitacolonna, E., Baccante, G., DiCastelnuovo, A., Angelini, A., et al. 2002. Transforming growthfactor-beta1 levels in hypertensive patients: association withbody mass index and leptin. Am. J. Hypertens. 15(9): 759–765.doi:10.1016/S0895-7061(02)02978-3. PMID:12219869.

Ren, P., Ji, H., Shao, Q., Chen, X., Han, J., and Sun, Y. 2007. Pro-tective effects of sodium daidzein sulfonate on trabecular bonein ovariectomized rats. Pharmacology, 79(3): 129–136. doi:10.1159/000098115. PMID:17179740.

Safar, M., Stimpel, M., and Zanchetti, A. 1994. Hypertension inpostmenopausal women. Berlin: Springer-Verlag. PMID:17598964.

Scheen, A.J. 2004. Prevention of type 2 diabetes mellitus through inhi-bition of the renin-angiotensin system. Drugs, 64(22): 2537–2565.doi:10.2165/00003495-200464220-00004. PMID:15516153.

Smith, R.E.T., and Ashiya, M. 2007. Antihypertensive therapies.Nat. Rev. Drug Discov. 6(8): 597–598. doi:10.1038/nrd2354.PMID:17821828.

Suthanthiran, M., Li, B., Song, J.O., Ding, R., Sharma, V.K.,Schwartz, J.E., et al. 2000. Transforming growth factor-beta 1hyperexpression in African-American hypertensives: A novelmediator of hypertension and/or target organ damage. Proc.Natl. Acad. Sci. U.S.A. 97(7): 3479–3484. doi:10.1073/pnas.050420897. PMID:10725360.

Usui, I., Fujisaka, S., Yamazaki, K., Takano, A., Murakami, S.,Yamazaki, Y., et al. 2007. Telmisartan reduced blood pressureand HOMA-IR with increasing plasma leptin level in hypertensiveand type 2 diabetic patients. Diabetes Res. Clin. Pract. 77(2): 210–214. doi:10.1016/j.diabres.2006.11.014. PMID:17240472.

Wronski, T.J., Dann, L.M., Qi, H., and Yen, C.F. 1993. Skeletaleffects of withdrawal of estrogen and diphosphonate treatmentin ovariectomized rats. Calcif. Tissue Int. 53(3): 210–216.doi:10.1007/BF01321840. PMID:8242475.

Yoshiji, H., Kuriyama, S., Yoshii, J., Ikenaka, Y., Noguchi, R.,Nakatani, T., et al. 2001. Angiotensin-II type 1 receptor interac-tion is a major regulator for liver fibrosis development in rats.Hepatology, 34(4 Pt 1): 745–750. PMID:11584371.

Zezina, L., Larsson, E., and Fellstrom, B. 2001. Candesartan cilex-etil reduces graft arteriosclerosis in aortic transplantation modelin rat. Transpl. Immunol. 8(4): 245–251. doi:10.1016/S0966-3274(01)00028-4. PMID:11316067.

Zhao, C., Wang, Y.Y., Xiao, Z.X., Wang, Y.P., and Zhang, Q.Y.2007. The relationship between the gene polymorphism ofTGF-beta1 and early renal injury in patients with essential hy-pertension, and the effect of the gene polymorphism of TGF-beta1 on the individual treatment with valsartan. Zhonghua YiXue Yi Chuan Xue Za Zhi, 24(4): 428–431. PMID:17680535.

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