angiotensin ii/angiotensin ii receptor blockade affects...

E-Mail [email protected]

Original Paper

Cells Tissues Organs DOI: 10.1159/000464461

Angiotensin II/Angiotensin II Receptor Blockade Affects Osteoporosis via the AT1/AT2-Mediated cAMP-Dependent PKA Pathway

Yi Zhou Xiaoxu Guan Xiaoyi Chen Mengfei Yu Chaowei Wang

Xuepeng Chen Jiejun Shi Tie Liu Huiming Wang

Affiliated Hospital of Stomatology, Medical College, Zhejiang University, Hangzhou , China

ARB1 exhibits a greater capacity to increase bone mass than ARB2. The cAMP-dependent PKA pathway plays an impor-tant role in AngII/ARB on changing bone mass.

© 2017 S. Karger AG, Basel

Introduction

Osteoporosis and hypertension are 2 common age-re-lated diseases. Factors such as genetics and lifestyle habits, contribute to the pathogenesis and progression of both these disorders [Coffman, 2011; Hsu and Kiel, 2012]. These prevalent diseases contribute to fragility fractures,

Keywords

Osteoporosis · Hypertension · Angiotensin II · Olmesartan · PD123319 · cAMP

Abstract

Animal studies have reported on the benefits of ARB on bone mass. However, the underlying mechanism for angio-tensin II (AngII)/AngII receptor blockade (ARB) in regulating bone mass remains elusive. Since high levels of plasma and urine cAMP are observed in osteoporotic and hypertensive patients, we hypothesized that cAMP may be an important molecule for the downstream events of the activation of AT receptors, members of the G-protein-coupled receptor fam-ily, in regulating bone turnover. In this study, micro-CT and X-ray analyses indicated that AngII decreased bone mass via biasing bone resorption over bone formation in osteopo-rotic mice. However, these adverse effects were blocked by olmesartan and PD123319. In vitro, AngII was shown to downregulate osteogenic differentiation and matrix miner-alization, but to upregulate osteoclastic activity by mainly affecting osteoblasts producing osteoclastogenesis-associ-ated key soluble factors, including M-CSF and RANKL. Simi-larly, ARB treatment exhibited antagonistic effects on AngII. In conclusion, osteoblasts are the directly targeted cells.

Accepted after revision: February 15, 2017 Published online: May 6, 2017

Huiming Wang Affiliated Hospital of Stomatology, Medical College, Zhejiang University Yan’an Road 395 Hangzhou 310000 (China) E-Mail huimingwang1960 @ 163.com

© 2017 S. Karger AG, Basel

www.karger.com/cto

Abbreviations used in this paper

Ang II angiotensin IIARB angiotensin II receptor blockadeBMD bone mineral density BV/TV ratio of bone volume/total volumeELISA enzyme-linked immunosorbent assayMNCs multinucleate cellsOVX ovariectomyRT-qPCR real-time quantitative RT-PCRTRAP tartrate-resistant acid phosphatase

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM

Zhou/Guan/Chen/Yu/Wang/Chen/Shi/Liu/Wang

Cells Tissues OrgansDOI: 10.1159/000464461

2

cardiovascular diseases, and secondary complications, constituting a considerable global heath burden as theelderly population steadily increases. Epidemiological studies have established a strong association between high blood pressure and increased bone loss [Cappuccio et al., 2000]. Conversely, the significantly decreased low bone mineral density (BMD) is positively associated with death from stroke, a serious cardiovascular disease [Browner et al., 1991]. Although there is some evidence supporting the relationship between osteoporosis andhypertension, the underlying molecular mechanism re-mains unclear.

In the local milieu, bone remolding and homeostasis relies on the activities of 2 major bone cells, osteoblasts and osteoclasts. These 2 cell types usually reside close to each other, exchanging signals and regulating bone for-mation and resorption. Therefore, the molecules present in the bone microenvironment are critical for controlling the balance of bone turnover. Recently, animal studies have demonstrated the benefits of antihypertensive treat-ment such as angiotensin II (AngII) receptor blockade (ARB), in improving BMD and reducing the risk offractures, with the inactivation of the active component, AngII, proven as the reason for the beneficial effects of ARB on the skeletal system [Shimizu et al., 2008; Asaba et al., 2009; Izu et al., 2009]. However, the downstream events after AT receptor activation need to be fully clari-fied and this warrants further investigation.

AngII, an important molecule in Ras, exhibits its phys-iological and pathological roles via the transactivation of 2 mainly targeted receptors, AT1 and AT2 [Putnam et al., 2012]. AT1 and AT2 are members of the G-protein-cou-pled receptor family, and their activation leads to the re-lease of many important signaling molecules including cAMP, cGMP, IP3, DAG, and Rho [Brinks and Eckhart, 2010]. Among these factors, the most interesting mole-cule is cAMP, since elevated levels of plasma and urine cAMP have been observed both in osteoporotic and hy-pertensive patients [Neelon et al., 1973; Winther et al., 1986; Guan et al., 2011]. We speculated that the levels of cAMP in osteoporotic patients simultaneously suffering from hypertension are much higher than in patients with either osteoporosis or hypertension only. We thus postu-lated that the cAMP signaling pathway may be involved in AngII-related osteoporosis. Indeed, the hypertension-induced increased urinary calcium excretion and low lev-el of ionized calcium in serum can selectively activate ad-enylate cyclase subtypes [Resnick et al., 1983; Cappuccio et al., 2000; Willoughby, 2012]. In patients with low Ca 2+ , the activated adenylate cyclase 5/6 will catalyze ATP to

produce an abundance of second-messenger cAMP, an essential intracellular signaling molecule in controlling bone homeostasis [Yang et al., 2008; Guan et al., 2011; Willoughby, 2012].

Here, we hypothesized that AngII affects the osteopo-rotic process via the AT1/AT2-mediated cAMP/PKA pathway and that the potential benefits of ARB on bone may be via relieving the AngII-induced abnormal cAMP signals by reversing the level of this second messenger to a normal status.

Materials and Methods

Reagents All reagents and drugs such as AngII, olmesartan, PD123319,

and H-89, unless otherwise stated, were obtained from Sigma, St. Louis, MO, USA.

Animals, Ovariectomy, and Drug Treatments Animal care and experimental protocols were conducted in ac-

cordance with the guidelines of the Institutional Animal Care and Use Committee, Zhejiang University, Hangzhou, China.

Forty-eight female C57BL/65 mice underwent bilateral ovari-ectomy (OVX). In brief, after the mice were anesthetized with 10% chloral hydrate (350 mg/kg) by intraperitoneal injection, a midline ventral incision was performed at the level of the iliac crest and the ovaries were excised. Prophylactic antibiotics were then adminis-tered. Twelve weeks after surgery, osteoporotic mice models were established [Gao et al., 2009]. Osmotic minipumps containingAngII (Alzet model 2004; Alza, Palo Alto, CA, USA; 200 ng/kg/min) or saline were implanted [Nakagami et al., 2003]. The ARB1 and ARB2 used in the experiment were olmesartan and PD123319 by gavage, respectively [Shimizu et al., 2008; Asaba et al., 2009; Izu et al., 2009]. The oral administration of 2 AT1 and AT2 receptor blockades was set at 10 mg/kg/day [Shimizu et al., 2008; Asaba et al., 2009; Izu et al., 2009]. Mice were then randomly divided into4 groups: OVX ( n = 12), AngII ( n = 12), AngII + ARB1 ( n = 12) and AngII + ARB2 ( n = 12). The mice received drug treatment for 4 weeks. They were then sacrificed to collect the femurs, tibiae, and blood samples for radiographic evaluation, micro-CT measure-ment, real-time quantitative RT-PCR (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA).

X-Ray Measurement Radiographs of the collected tibiae ( n = 10) were taken using

RADspeed (HIMADZU Corp., Kyoto, Japan) at settings of 40 kV and 1.2 mA. BMD values (mg/cm 2 ) were assessed using Lunar iDXA (GE Healthcare Lunar, USA). The region of interest was de-fined as the same size as a longitudinal rectangle that was adjusted to cover the same sites for each tibia, including cancellous and cor-tical bone [Jones et al., 2008].

Micro-CT Evaluation Micro-CT evaluation was performed as described previously

[Hanyu et al., 2012]. Briefly, after drug treatment, the collected fe-murs ( n = 10) from ovariectomized mice were placed in physiolog-

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM

ARB Affects Osteoporosis via theAT1/AT2-Mediated cAMP/PKA Pathway


3

ical saline and a 3-dimensional CT measurement was conducted by using Scan-Xmate-E090 (Comscan Techno Co., Ltd., Sagami-hara, Japan). Microarchitecture parameters, such as the ratio of bone volume/total volume (BV/TV), connectivity density, trabec-ular thickness, trabecular separation, and trabecular number were assessed in the secondary trabecular regions at 2–4 mm away from the growth plate. The data were then analyzed and recorded using TRI/3D-Bon computer software (Ratoc System Engineering Co., Ltd., Japan).

RT-qPCR Total RNA was isolated from bone tissue ( n = 10) by using

TRIzol reagent as previously described [Carter et al., 2012]. cDNA was synthesized from 1 μg RNA using the PrimeScript TM RT re-agent kit (TAKARA, Dalian, China), according to the manufac-turer’s instructions. An RT-qPCR assay was then conducted on an ABI 7500 sequence detection system (Applied Biosystems, USA). The oligonucleotide primer sequences are summarized in Table 1 . GAPDH served as the control. Relative expression of detected genes was calculated using the comparative 2-ΔΔCt method.

Cell Isolation, Culture, Coculture, and Drug Treatments Preosteoblasts were harvested from the calvariae of ovariecto-

mized mice as previously described [Ryu et al., 2006; Shen et al., 2011; Kim et al., 2015; Li et al., 2015]. Cells were cultured in α-MEM supplemented with 10% FBS, both from Gibco BRL, Gaithersburg, MD, USA, and maintained in a 5% CO 2 humidified atmosphere at 37 ° C. Osteoclastic precursors were harvested from the femurs and tiabie of the ovariectomized mice and cultured as previously de-scribed, and the adherent bone marrow macrophages were used as osteoclast precursors [Ryu et al., 2006; Shen et al., 2011; Kim et al., 2015; Li et al., 2015]. Cells were also maintained in basic medium (10% FBS + α-MEM) and incubated in 5% CO 2 at 37 ° C. As for the coculture, 2 × 10 4 osteoclastic cells were cultured in 24-well plates in basic medium [Ryu et al., 2006; Kim et al., 2015]. Transwell chambers (pore diameter: 3 μm; Corning, NY, USA) cultured with 1 × 10 4 preosteoblasts were placed into 24-well plates [Shen et al., 2011; Li et al., 2015]. The osteogenic medium for conducting the assay on the osteoblastic cells contained 90% high-glucose Dul-becco’s modified Eagle’s medium (Gibco BRL, Gaithersburg, MD, USA), 10% FBS, 10 –6 M dexamethasone, 10 –2 M β-glycerol phos-

phate, and 50 μg/mL ascorbic acid. The osteoclastogenic medium for performing the assay on the osteoclastic cells contained 90% phenol red free α-MEM, 10% FBS, and 50 ng/mL RANKL and25 ng/mL M-CSF (both from R&D Systems Inc., Minneapolis, MN, USA). The 2 cell types were randomly divided into 4 groups: control ( n > 3), AngII ( n > 3), AngII + ARB1 ( n > 3), and AngII + ARB2 ( n > 3). The concentrations for AngII, ARB1 (olmesartan) and ARB2 (PD123319) in the following experiments were 10 –6 , 10 –5 , and 10 –5 M , respectively. The culture media were changed every 2 days. All media were supplemented with 100 IU/mL peni-cillin and 100 μg/mL streptomycin. After osteogenic differentia-tion for 14 days or osteoclastic differentiation for 4 days, cells were harvested for further biochemical assays.

MTT Assay Osteoblasts and osteoclast precursors were cultured in ba-

sic medium containing various concentrations of AngII(10 –3 to 10 –10 M ) for 2 days. MTT was performed to measure cell proliferation. Aliquots of MTT solution (0.5 mg/mL) were added and incubated at 37 ° C to form formazan crystals. The formed formazan was then solubilized with dimethyl sulfoxide. At 495 nm, OD values were recorded and final results were calculated as the absorbance reading from each well minus the OD value of blank wells. Because 10 –6 M AngII exhibited no effects on the prolifera-tion of osteoblasts and osteoclast precursors (Fig. 2a), 10 –6 M AngII was chosen for the following in vitro experiments.

Flow Cytometry Apoptosis and necrosis in osteoblasts and osteoclast precursors

following AngII, ARB1, or ARB2 treatment was measured using an annexin V-FITC/propidium iodide detection kit (BD Pharmin-gen, San José, CA, USA). Cells (5 × 10 5 cells/well) were collected using trypsin on day 2. Once the cells were stained with annexin V-FITC and propidium iodide, samples were immediately ana-lyzed on FACScan flow cytometry (Becton Dickinson, San José, CA, USA).

PKA Inhibition Study To assess the effects of PKA inhibition on in vitro osteoclasto-

genesis and osteogenic differentiation, 10 μ M H-89, an inhibitor of PKA, was added to osteoblasts and osteoclast precursors cultured

Table 1. Nucleotide sequences for real-time quantitative RT-PCR primers

Gene Sequences of primers (5′-3′) Amplicon, bp Temperature, °C

Runx2 Forward: AGGGACTATGGCGTCAAACA Reverse: CATAACAGCGGAGGCATTTC

171 60.5260.61

OC Forward: AAGCAGGAGGGCAATAAGGTReverse: TTTGTAGGCGGTCTTCAAGC

160 60.1060.39

RANKL Forward: AGAAGACAGCACTCACTGCTTTTAReverse: CCCACAATGTGTTGCAGTTC

166 59.7760.01

OPG Forward: TGTTCCGGAAACAGAGAAGCReverse: ACTCTCGGCATTCACTTTGG

160 60.3860.25

GAPDH Forward: GTATGAAGTGCCCCTCCTTGReverse: CCCCAGGCTGTACAAGACAT

190 59.5559.99

OC, osteocalcin; RANKL, receptor activator of NF-ĸB ligand; OPG, osteoprotegerin.

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM



4

alone or together for 4 or 14 days. The culture media were changed every 2 days. Osteoblastic and osteoclastic activity was assessed by the biochemical assays described below.

Von Kossa Staining Osteoblasts were fixed for 10 min in 4% formaldehyde, washed

in distilled water and stained in 5% silver nitrate solution (v/v) un-der UV light for 60 min, so that the deposited calcium in wells could be seen clearly.

Alkaline Phosphatase Activity The lysates of osteoblasts were collected and alkaline phospha-

tase activity was measured using the SensoLyte TM pNPP alkaline phosphatase assay kit (AnaSpec, CA, USA), according to the man-ufacturer’s instruction. The absorbance of p -nitrophenol was monitored at 405 nm with a spectrophotometer. Alkaline phos-phatase activities were then normalized to the total protein con-centrations of each sample.

Alizarin Red Staining Briefly, osteoblasts were fixed with 95% ethanol for 2 min,

rinsed 3 times and then stained with Alizarin red solution for 1 h.

Calcium Deposition Assay To measure the insoluble calcium content, the deposited cal-

cium in the extracellular matrix was decalcified with 0.5 N hydro-chloric acid. The concentrations of calcium in the supernatant were determined using a calcium assay kit (Sekisui Diagnostics, Stamford, CT, USA). Calcium content in each sample was then normalized to the total protein concentration.

Tartrate-Resistant Acid Phosphatase Assay Enzymatic Activity The p -nitrophenyl phosphate was used as a substrate to deter-

mine tartrate-resistant acid phosphatase (TRAP) activity as previ-ously described [Wittrant et al., 2008; Yoon et al., 2013]. Protein samples were incubated in the assay buffer containing 125 m M sodium acetate buffer (pH 5.2), 100 m M p -nitrophenyl phosphate and 1 m M L -sodium tartrate. The product of p -nitrophenol was measured at 405 nm at 37 ° C. Data were expressed as the mean OD/min/mg of total proteins.

Cell Staining A leukocyte acid phosphatase kit (Sigma) was used for TRAP

staining. Cells were fixed for 30 s with 4% formaldehyde and then incubated in TRAP staining solution in a dark room for 1 h at 37 ° C. After being rinsed in deionized water 3 times, samples were air-dried and evaluated microscopically. The number of TRAP-positive multinucleate cells (MNCs; at least 3 nuclei) was counted randomly in 8 fields under a ×4 objective and the number of os-teoclasts was analyzed statistically.

Western Blot The cytosolic and nuclear components in osteoblasts and os-

teoclast precursors were collected, respectively, as previously de-scribed [Wittrant et al., 2008; Di Paola et al., 2011; Zhou et al., 2015]; 20 μg of total protein were loaded on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Runx2, NF-ĸB p65, IKKα/β, phospho-IKKα/β (Ser180/181), IĸBα, phospho-IĸBα (Ser32), pPKAα/β/γ (Thr198), and β-actin were detected using

specific primary antibodies from Santa Cruz, CA, USA, and Ab-cam, Cambridge, UK. After incubation in secondary antibodies for 1 h, bands were visualized with enhanced chemiluminescence re-agents by using an ECL kit.

ELISA The cAMP level in the serum from ovariectomzied mice, and

RANKL and M-CSF in the supernatants from the cell culture me-dium following drug treatment were measured using ELISA kits (R&D Systems) according to the manufacturer’s instruction. Re-sults were expressed as the amount of cAMP, RANKL, or M-CSF in serum or supernatant. For intracellular cAMP, protein samples were collected from each well and cAMP levels were assessed by immunoassay using a commercial kit (R&D Systems). The parallel wells were utilized for cell number count by using a hemacytom-eter. cAMP contents were then expressed as pmol/10 6 cells.

Statistical Analysis All assays were conducted at least in triplicate with 3 indepen-

dent experiments. Data were presented as the mean ± standard deviation. One-way ANOVA was performed with the Dunnett post hoc test for statistical analysis. p < 0.05 was defined as statisti-cally significant.

Results

AngII Decreases Bone Mass and ARB1/2 Increases Bone Mass via Regulation of Osteoblastic and Osteoclastic Activity Forty-eight C57BL/65 ovariectomzied mice were treat-

ed daily with PBS (control), AngII, AngII + olmesartan or AngII + PD123319 for 4 weeks. To determine the effects of AngII, ARB1, and ARB2 on bone, micro-CT and X-ray evaluation were conducted after the drug treatments. Analysis of morphological parameters based on 3-dimen-sional micro-CT revealed that AngII treatment signifi-cantly reduced BV/TV by 27.58%, connectivity density by 42.83%, trabecular thickness by 39%, and trabecular

Fig. 1. Effects of AngII, ARB1, and ARB2 on bone mass, bone-specific mRNA expressions, and cAMP levels in ovariectomzied rats. a , b Representative radiographic films of mouse tibiae and quantitative data of X-ray measurement. c–h Representative mi-cro-CT images of mouse femurs and quantitative data of micro-CT examination. i–k Expressions of Runx2 ( i) , osteocalcin (j ), and RANKL/OPG mRNAs (k ) in bone tissue from ovariectomized mice following Ang II, ARB1, and ARB2 treatment. l cAMP levels in the serum of ovariectomzied mice following AngII, ARB1, and ARB2 treatments. Bars represent the mean ± standard deviation( n = 10). Comparisons between groups were performed using ANOVA. * p < 0.05 versus OVX; # p < 0.05 versus AngII-treated group; * * p < 0.05 versus ARB1-treated group. AngII, angiotensin II; ARB1/2, angiotensin II receptor blockade 1/2.

(For figure see next page.)

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM



5

AngII + ARB1AngIIOVX

AngII –

AngII + ARB2

40

32

24

16

8

0

BV/T

V, %

+ + +ARB1 – – + –ARB2c d– – – +

*

AngII + ARB1AngIIOVX AngII + ARB2

90

20100

Trab

ecul

arth

ickn

ess, μm

4030

6050

8070

f

*


1.0

0.8

0.6

0.4

0.2

0

Trab

ecul

arse

para

tion,

mm

h

*


25

20

15

10

5

0

Ost

eoca

lcin

j*


6

4

2

0

Seru

m c

AMP

(% o

f OVX

), pm

ol/m

L

l

*


12

9

6

3

0

Trab

ecul

arnu

mbe

r, m

m–1

e

*


907560

3045

150

Conn

ectiv

ityde

nsity

, mm

–3

g

*


20

16

12

8

4

0

Runx

2

i

*

AngII + ARB1AngIIOVX

*

*

AngII + ARB2

10 RANKL8

6

4

2

0

RAN

KL/O

PG

k

OPG

AngII + ARB1AngIIOVXAngII –

AngII + ARB2

0.20

0.15

0.10

0.05

0

BMD,

g/c

m2

+ + +ARB1 – – + –ARB2a

b

– – – +

*

*, #

* **,

* **,

*, #

*, # **,

*, # **,

# **,

# **,

*, #

*, #

*, #*, #

*, #

*, #*, #

*, #

*, # *, #

****

*#*

*##

1

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM



6

number by 33.11%, but increased trabecular separation by 72.64%, indicating that Ras activation may accelerate osteoporosis ( Fig. 1 c–h). These microarchitecture pa-rameters, however, were improved after ARB1 treatment as demonstrated by marked increases in BV/TV by 57%, connectivity density by 274%, trabecular number by 52%, trabecular thickness by 131% and a significant reduction in trabecular separation by 50% ( Fig. 1 c–h). PD123319 exhibited a similar pattern in improving bone parame-ters, but this beneficial effect was not so apparent as ARB1 ( Fig. 1 c–h). The effects of AngII, ARB1, and ARB2 on BMD as estimated by radiographic analysis demonstrat-ed a trend similar to that observed on micro-CT ( Fig. 1 a, b). RT-qPCR was then conducted to detect gene expres-sion in association with osteoblastic and osteoclastic ac-tivity. Interestingly, the AngII-inhibited Runx2 and os-teocalcin that are related to bone formation were signifi-cantly highly expressed after the ARB1 and ARB2 treatment, but the expression of genes such as RANKL / OPG (ratio), an important axis in controlling osteoclastic function, was dramatically decreased, implying that olmesartan and PD123319 have a dual effect, i.e. enhanc-ing bone formation but suppressing bone resorption ( Fig. 1 i–k). To test our hypothesis that cAMP levels are

much higher in osteoporotic mice with AngII treatment [Neelon et al., 1973; Winther et al., 1986; Guan et al., 2011], serum cAMP was assessed by ELISA. Dramatically elevated levels of cAMP were seen in the AngII-treated group, and ARB1/2 treatment was able to reverse cAMP back to a normal level ( Fig. 1 l), suggesting that the cAMP signaling pathway is involved in osteoporosis after AT receptor activation.

AngII Affects Osteoblast and Osteoclast Precursor Viability in a Concentration-Dependent Manner To further study the mechanism by which activating

or deactivating AT receptors results in a high bone-turn-over state with osteoblastic and osteoclastic activity, os-teoblasts and osteoclast precursors expressing AT recep-tors were used for in vitro cell viability and the osteo-genic and osteoclastogenic assay [Nishiya and Sugimoto, 2001; Ryu et al., 2013]. As shown in Fig. 2 a, various con-centrations of AngII (10 –3 to 10 –10 M ) affected cell growth in a concentration-dependent manner. AngII concentra-tions <10 –6 M exhibited beneficial effects on cell prolif-eration, but cell numbers were significantly decreased when AngII concentrations were >10 –6 M ( p < 0.05). Since 10 –6 M AngII had no impact on the growth of these bone

AngII + ARB1AngIIControl AngII + ARB2

302520151050

Cell

apop

tosi

s, %

c

OBOCP

OBOCP

AngII (log concentration), M–10

3 OB

–9 –8 –7 –6 –4–5 –3

2

1

0

MTT

, % o

f con

trol

a b

OCP


4

3

2

1

0

Cell

necr

osis

, %

d

Prop

idiu

m io

dide

Annexin-V-FITC

OB

OCP

AngllARB1ARB2

–––

++

+–

–

–

––

–

Fig. 2. Effects of AngII, ARB1, and ARB2 on the proliferation, ne-crosis, and apoptosis of preosteoblasts and osteoclast precursors. a The osteoblastic and osteoclastic proliferation following differ-ent concentrations of AngII was assessed by MTT assay. b–d Cell

apoptosis and necrosis rates were analyzed by flow cytometry. Bars represent the mean ± SD ( n ≥ 3). Comparisons between groups were performed using ANOVA. OB, osteoblasts; OCP, osteoclast precursors.

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM



7

cells, 10 –6 M AngII was selected as the concentration for in vitro osteogenic and osteoclastogenic assay, which was consistent with a previous study [Shimizu et al., 2008]. To examine whether 10 –6 M AngII, 10 –5 M olmesartan, and 10 –5 M PD123319 affect cell necrosis and apoptosis, flow cytometry was conducted. As shown in Fig. 2 b–d, there were no apparent differences in the percentage of apop-totic and necrotic cell populations for both cell types after the AngII, ARB1, and ARB2 treatments ( p > 0.05).

AngII, ARB1, and ARB2 Affect Bone Formation and Resorption via Directly Regulating Osteogenic Differentiation and Indirectly Regulating Osteoclastic Differentiation Osteoblast differentiation can be generally divided

into 3 stages: (1) osteogenic lineage commitment, (2) ex-tracellular matrix synthesis, and (3) matrix mineraliza-tion. In the AngII-treated group, markedly decreased os-teogenic lineage commitment, significantly reduced cal-cium deposition and matrix mineralization, as reflected by Runx2 levels, von Kossa staining, and calcium deposi-tion assays demonstrated the unfavorable role of AngII on osteogengesis ( Fig. 3 a–d). Although the PD123319

treatment did not appear to relieve the negative effect of AngII on bone like olmesartan did, both ARB1 and ARB2 showed an ability to recover and promote osteoblastic ac-tivity ( Fig. 3 a–d). However, in contrast to the in vivo find-ings, the in vitro study found that there was little impact of AngII, ARB1, and ARB2 on osteoclastic activity when osteoclast precursors were cultured in osteoclastogenic medium ( Fig. 4 ). To fully illuminate this contradictory phenomenon, preosteoblast and osteoclast precursors were cocultured without osteoclastogenic medium in or-der to mimic in vivo microenvironment. Interestingly and importantly, AngII is capable of significantly sup-pressing osteoclastogenesis as testified by TRAP staining ( Fig. 5 c, d). The RANKL-RANK system is essential for osteoclastic differentiation [Teitelbaum, 2000]. The ele-vated expression of NF-ĸB p65, phospho-IKKα/β, and phospho-IĸBα, 3 key factors in NF-ĸB activation, may be the reason for AngII-induced enhanced osteoclastic ac-tivity ( Fig. 5 a, b, e–g). Since RANKL, OPG, and M-CSF, the 3 key regulators of osteoclastic function, are secreted by osteoblasts and stromal cells, we observed their levels in coculture medium [Teitelbaum, 2000]. Although ELI-SA cannot detect OPG, AngII-treated osteoblasts secret-

AngII – + + +ARB1 – – + –ARB2b – – – +

AngII – + + +ARB1 – – + –ARB2

Runx2

a

c

– – – +

AngII – + + +ARB1 – – + –ARB2 – – – +

2.0

1.6

1.2

0.8

0.4

0

Runx

2,

* * **,

*, #

AngII – + + +ARB1 – – + –ARB2d – – – +

0.180.150.12

0.060.09

0.030

M * **

*, #

5 mm 5 mm 5 mm 5 mm

Fig. 3. Effects of AngII, ARB1, and ARB2 on the osteogenic differ-entiation of preosteoblasts. a , b Western blot analysis of Runx2 expression. c Von Kossa staining. d Calcium deposition measure-ment. Bars represent the mean ± SD ( n ≥ 3). Comparisons between

groups were performed using ANOVA. * p < 0.05 versus control; # p < 0.05 versus AngII-treated group; * * p < 0.05 versus ARB1-treated group.

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM



8

ed significantly greater levels of M-CSF and RANKL than the untreated control ( Fig. 5 h, i). However, by downregu-lating IKKα/β and IĸBα phosphorylation, NF-ĸB p65 ac-tivity, and M-CSF and RANKL secretion, both ARB1 and ARB2 were able to inhibit osteoblastic activity, but ARB1 showed more obvious effects on suppressing bone re-sorption ( Fig. 5 a–i). The in vitro data thus suggest that osteoblasts are the main target cells, and that AngII, ARB1, and ARB2 affect bone metabolism by directly reg-ulating osteogenesis but indirectly regulating osteoclasto-genesis.

Involvement of the cAMP/PKA Signaling Pathway in AngII, ARB1, and ARB-Regulated Osteoclastogenesis and Osteogenesis Having demonstrated that the cAMP signaling path-

way plays an important role in AngII, ARB1, and ARB2–regulated bone formation and resorption in vivo ( Fig. 1 l), we proceeded to further elucidate the role of cAMP in vitro. As shown in Fig. 6 a–f, PKA inhibition dramatically increased the cell osteogenic lineage commitment, extra-cellular matrix synthesis, and mineralization, as testified by Runx2 levels, alkaline phosphatase activity, and Aliza-rin red staining. However, PKA inhibition dramatically reduced osteoclastogenic activity, as reflected in NF-ĸB p65 levels and TRAP activity ( Fig. 7 ). This effect was more

apparent when H-89 was added to the ARB1- or ARB2-treated groups, pointing to the synergetic role of H-89 and ARB1/2 in relieving the negative effect of AnII on bone ( Fig. 6 , 7 ). It is therefore suggested that the cAMP-dependent PKA signaling pathway is essential in AngII-, ARB1-, and ARB2-conducted adaptive bone remolding.

Discussion

This work shows that AngII affects bone mass and mi-crostructure via the regulation of osteoblastic and osteo-clastic activity. Olmesartan and PD123319, 2 AT receptor blockades, are helpful in relieving the AngII-induced os-teoporotic process.

Animal and epidemiological data have confirmed that AT receptor activation contributes to calcium leakage, in-cluding elevated urinary calcium excretion and a low ion-ized calcium level in the serum. This, in turn, leads to the increased movement of calcium from the bone and re-sults in accelerated osteoporosis [Resnick et al., 1983; Cappuccio et al., 2000; Willoughby, 2012]. Although the action of AT receptors on the skeleton is clearly stated, the exact role of blocking AT1 and AT2 receptors in bone remains controversial and undetermined [Shimizu et al., 2008; Asaba et al., 2009; Izu et al., 2009]. It is important


AngII – + + +ARB1 – – + –ARB2b – – – +

AngII – + + +ARB1 – – + –ARB2

a

c

– – – +

AngII – + + +ARB1 – – + –ARB2 – – – +

1.2

0.9

0.3

0

,

d

180

12090

300

100 μm 100 μm 100 μm 100 μm

Fig. 4. Effects of AngII, ARB1, and ARB2 on the osteoclastic activ-ity. a , b Western blot analysis of NF-ĸB p65 expression. c , d Os-teoclastic activity was estimated by TRAP staining, and the num-

ber of TRAP-positive multinucleate cells (MNCs) was randomly counted in 8 fields. Bars represent the mean ± SD ( n ≥ 3). Com-parisons between groups were performed using ANOVA.

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM



9


180

306090

120150

0TRAP

-pos

itive

MN

Cs

d

AngII – + + +ARB1 – – + –ARB2

c

– – – + *

5

2

3

4

1

0

*

*, # *, #

AngII – + + +ARB1 – – + –ARB2b – – – +

NF-

,fo

ld in

crea

se

3.5

0.50

1.51.0

2.52.0

3.0*

*, #***, #,

AngII – + + +ARB1 – – + –ARB2f – – – +

Phos

pho-

I,

fold

incr

ease

3

2

1

0

*

#*, #

AngII – + + +ARB1 – – + –ARB2h – – – +

RAN

KL

65

234

10

*

# #

##

AngII – + + +ARB1 – – + –ARB2g – – – +

Phos

pho-

IKK

,fo

ld in

crea

se

2.0

0.81.21.6

0.40

*

AngII – + + +ARB1 – – + –ARB2i – – – +

*, # *, #

AngII – + + +ARB1 – – + –ARB2

NF-

a

– – – +

AngII – + + +ARB1 – – + –ARB2

Phospho-I

Phospho-IKK

IKK

I

Tubulin-e

– – – +

100 μm 100 μm 100 μm 100 μm

Fig. 5. Effects of AngII, ARB1, and ARB2 on the osteoclastic activ-ity when osteoclast precursors were cocultured with preosteoblas-tic cells. a , b Western blot analysis of NF-ĸB p65 expression. c , d Osteoclastic activity was estimated by TRAP staining, and the number of TRAP-positive multinucleate cells (MNCs) with >3 nu-clei was randomly counted in 8 fields. e–g Western blot analysis of

IKKα/β (e), IĸBα, and phospho-IĸBα (Ser32) (f), phospho-IKKα/β (Ser180/181) (g), expressions. h , i RANKL and M-CSF levels that secreted by osteoblasts were detected by ELISA. Bars represent the mean ± SD ( n ≥ 3). Comparisons between groups were performed using ANOVA. * p < 0.05 versus control; # p < 0.05 versus AngII-treated group; * * p < 0.05 versus ARB1-treated group.

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM



10

to note that AT1 deficiency and AT1 receptor blockade losartan has little impact on bone mass in male C57BL/65 mice, but knockout AT2 and AT2 receptor blockade PD123319 exhibits beneficial effects on bone [Izu et al., 2009]. However, in female Wistar rats, spontaneously hy-pertensive rats, or Wistar-Kyoto rats that undergo bilat-eral OVX, it has been shown that PD123329 (ARB2) treatment has no influence on the skeletal system [Shi-mizu et al., 2008]. Interestingly, the administration of olmesartan (ARB1) improves AngII-induced decreased

bone mass [Shimizu et al., 2008]. Partially consistent with these studies, both AT1 (olmesartan) and AT2 (PD123319) receptor blockades in this study increased BMD in C57BL/65 mice following OVX. The reason for such a discrepancy may be due to the various kinds of AT1/AT2 receptor blockades, personalized administration of drugs including drug dosage and different delivery methods, sex of mice or rats, and the variance in pathological status and severity.

**#,

56

234

10

*

**, #*, ##

*, ##

AngIIARB1ARB2

b

AngII – + + +ARB1 – – + –ARB2

d

– – – +

Runx

2,fo

ld in

crea

se

AngIIARB1ARB2

Runx2

pPKA

a

+ + +– + –– – +

–––– – – –

+––+

++–+

+–++

+ + +– + –– – +

–––– – – –

+––+

++–+

+–++

***,

10

2

4

6

0

*

**, #

##

AngIIARB1ARB2

f

pPKA fo

ld in

crea

se

+ + +– + –– – +

–––– – – –

+––+

++–+

+–++

1012

46

20

*

* **, ###

##

AngIIARB1ARB2

e

6 ce

lls

+ + +– + –– – +

–––– – – –

+––+

++–+

+–++

56

234

10

*

*, # *, #*, ##*, ##

AngIIARB1ARB2

c

n M

+ + +– + –– – +

–––– – – –

+––+

++–+

+–++

5 mm

5 mm 5 mm 5 mm

5 mm 5 mm 5 mm

Fig. 6. Involvement of cAMP/PKA pathway in AngII, ARB1, and ARB2-mediated osteogenesis. a , b , f Western blot analysis of Runx2 and pPKAα/β/γ (Thr198) expressions. c Alkaline phospha-tase activity was measured by spectrophotometry. d Osteoblastic activity was estimated by Alizarin red staining. e cAMP levels fol-

lowing AngII, ARB1, and ARB2 treatment were determined by immunoassay. Bars represent the mean ± SD ( n ≥ 3). Comparisons between groups were performed using ANOVA. * p < 0.05 versus control; # p < 0.05 versus AngII-treated group; * * p < 0.05 versus ARB1-treated group; ## p < 0.05 versus H-89-treated group.

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM



11

As members of the G-protein-coupled receptor fam-ily, the AT1 and AT2 receptors share a limited sequence homology (34% amino acid sequence identity) and thus respond differently to AngII at various concentrations in the context of the concrete biological environment [Sen-bonmatsu et al., 2003; Wang et al., 2012]. We thus hy-pothesized that exogenous AngII or different kinds and concentrations of ARB1/2 may affect the functions of these 2 receptors differently and subsequently induce dif-ferent biological responses in bone. It is important to note that, in the elderly population, osteoporosis is usually more severe and effectively curative in postmenopausal

AngIIARB1ARB2

a

+ + +– + –– – +

–––– – – –

+––+

++–+

+–++

1.2

0.30

*

* **

AngIIARB1ARB2

c

),

+ + +– + –– – +

–––– – – –

+––+

++–+

+–++

4

2

0

*

* ***

AngIIARB1ARB2

b

,

+ + +– + –– – +

–––– – – –

+––+

++–+

+–++

women [Tsai et al., 2013]. As such, we also suspected that, with a certain pathological status (such as comes with OVX or menopause), the interplay between estrogen and the AT1/AT2 receptors may alter the sensitivity of these receptors to AngII treatments, and thereby display differ-ent biological responses in bone [Nouet and Nahmias, 2000; Wassmann and Nickenig, 2006; Nakai et al., 2013].

Increased bone mass typically relies on enhanced bone formation by osteoblasts and decreased bone resorption by osteoclasts [Teitelbaum, 2000; Sobacchi et al., 2013]. Both ARB1 and ARB2 have a dual role in not only pos-itively regulating bone formation, but also negativelymediating bone resorption. The increased osteogenic commitment, highly synthesized extracellular matrix, and enhanced calcium deposition are attributable to the beneficial effects of ARB1/2 on bone. However, olmesar-tan and PD123319 have no direct impact on osteoclastic activity. The RANKL-RANK (NF-ĸB) system is a pivotal axis in regulating osteoclastogenesis [Teitelbaum, 2000]. By negatively affecting osteoblasts to secrete RANKL and M-CSF, by downregulating NF-ĸB p65 activity, and by reducing the phosphorylation of IKKα/β and IĸBα, ARB1/2 inhibited the osteoclast-conducted bone resorp-tion. This suggests that osteoblasts are the directly tar-geted cells but also that cell-to-cell (osteoblast/osteoclast) contact is essential for the dual action of ARB1/2 on bone.

In contrast to the harmful effects of AngII on bone mass, AngII exhibits different effects on cell proliferation; 10 –6 M of AngII is the critical concentration, since con-centrations between 10 –6 and 10 –3 M lessen cell viability and those between 10 –10 and 10 –6 M promote cell prolif-eration. The concentration-dependent effect of AngIIon cell growth was also observed by Rudic et al. [2013], who proved that AngII concentrations between 10 –8 and 10 –7 M have a deleterious effect on human osteosarcoma cells but that 10 –9 M AngII stimulates the growth of these cells.

cAMP, an important second messenger in bone ho-meostasis, plays a prominent but ambiguous role in de-termining the fate of cells, which is dependent on the physiological conditions such as the specific molecular and developmental context in which the cAMP signal is presented. It is known that intermittent administration of parathyroid hormone, an inducer of cAMP, increases bone formation, but that the continuous administration of parathyroid hormone results in bone loss [Kondo et al., 2002; Jilka, 2007; Kanis et al., 2008]. However, another cAMP inducer, forskolin, increases bone formation at low concentrations but inhibits it at higher concentra-tions [Turksen et al., 1990]. In addtion, cAMP displays

Fig. 7. Osteoclastogenesis was affected by PKA inhibition when preosteoclastic cells were cocultured with preosteoblasts. a , b Western blot analysis of NF-ĸB p65 expression. c TRAP activity was measured by spectrophotometry. Bars represent the mean ± SD ( n ≥ 3).Comparisons between groups were performed using ANOVA. * p < 0.05 versus control; # p < 0.05 versus AngII-treated group; * * p < 0.05 versus ARB1-treated group; ## p < 0.05 versusH-89-treated group.

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM



12

varying effects on different cell types. For example, the activation of PKA with the cAMP inducer db-cAMP pro-motes vascular calcification by increasing the calcifica-tion of vascular cells [Tintut et al., 1998]. On the other hand, cAMP inducers such as IBMX, forskolin, and Sp-cAMP inhibit the osteogenic differentiation and forma-tion of bone marrow-derived mesenchymal stromal cells [Yang et al., 2008]. In contrast, db-cAMP increases extra-cellular matrix synthesis and the mineralization of bone marrow-derived mesenchymal stromal cells [Siddappa et al., 2008]. Therefore, the role of cAMP in osteogenesis is dependent on different cAMP inducers, varying drug concentrations, and different targeted cells. It is difficult to elucidate the exact role of cAMP on bone. However, in the context of the AngII-treated ovariectomized mice, we postulated that the high levels of cAMP are associated with the decreased bone mass, and that the reduction of cAMP levels by ARB1/2 and H-89 can reverse the unde-sirable action of AngII on bone.

In conclusion, AngII accelerated the osteoporotic pro-cess via the AT-mediated cAMP/PKA pathway. ARB could relieve the AngII-induced abnormal cAMP signals by reversing the level of cAMP to a normal status. ARB1 showed more capacity than ARB2 in relieving AngII-in-duced osteoporosis.

Acknowledgements

We are grateful to Dr. Eric C. Chen and Prof. Chih-Ko Yeh for their assistance in English editing. This study was supported by the National Science Foundation of China (grant Nos.: 81400480, 81378820, and 81500891).

Disclosure Statement

The authors declare no conflicts of interest.

References

Asaba, Y., M. Ito, T. Fumoto, K. Watanabe, R. Fu-kuhara, S. Takeshita, Y. Nimura, J. Ishida, A. Fukamizu, K. Ikeda (2009) Activation of re-nin-angiotensin system induces osteoporosis independently of hypertension. J Bone Miner Res 24: 241–250.

Brinks, H.L., A.D. Eckhart (2010) Regulation of GPCR signaling in hypertension. Biochim Biophys Acta 1802: 1268–1275.

Browner, W.S., D.G. Seeley, T.M. Vogt, S.R. Cum-mings (1991) Non-trauma mortality in elder-ly women with low bone mineral density. Study of Osteoporotic Fractures Research Group. Lancet 338: 355–358.

Cappuccio, F.P., R. Kalaitzidis, S. Duneclift, J.B. Eastwood (2000) Unravelling the links be-tween calcium excretion, salt intake, hyper-tension, kidney stones and bone metabolism. J Nephrol 13: 169–177.

Carter, L.E., G. Kilroy, J.M. Gimble, Z.E. Floyd (2012) An improved method for isolation of RNA from bone. BMC Biotechnol 12: 5.

Coffman, T.M. (2011) Under pressure: the search for the essential mechanisms of hypertension. Nat Med 17: 1402–1409.

Di Paola, R., F. Briguglio, I. Paterniti, E. Mazzon, G. Oteri, D. Militi, G. Cordasco, S. Cuzzocrea (2011) Emerging role of PPAR-beta/delta in inflammatory process associated to experi-mental periodontitis. Mediators Inflamm 2011: 787159.

Gao, Y., E. Luo, J. Hu, J. Xue, S. Zhu, J. Li (2009) Effect of combined local treatment with zole-dronic acid and basic fibroblast growth factor on implant fixation in ovariectomized rats. Bone 44: 225–232.

Guan, X.X., Y. Zhou, J.Y. Li (2011) Reciprocal roles of angiotensin II and angiotensin II re-ceptors blockade (ARB) in regulating Cbfa1/RANKL via cAMP signaling pathway: possi-ble mechanism for hypertension-related os-teoporosis and antagonistic effect of ARB on hypertension-related osteoporosis. Int J Mol Sci 12: 4206–4213.

Hanyu, R., V.L. Wehbi, T. Hayata, S. Moriya, T.N. Feinstein, Y. Ezura, M. Nagao, Y. Saita, H. Hemmi, T. Notomi, T. Nakamoto, E. Schi-pani, S. Takeda, K. Kaneko, H. Kurosawa, G. Karsenty, H.M. Kronenberg, J.P. Vilardaga, M. Noda (2012) Anabolic action of parathy-roid hormone regulated by the beta2-adren-ergic receptor. Proc Natl Acad Sci USA 109: 7433–7438.

Hsu, Y.H., D.P. Kiel (2012) Clinical review: ge-nome-wide association studies of skeletal phenotypes: what we have learned and where we are headed. J Clin Endocrinol Metab 97: E1958–E1977.

Izu, Y., F. Mizoguchi, A. Kawamata, T. Hayata, T. Nakamoto, K. Nakashima, T. Inagami, Y. Ezura, M. Noda (2009) Angiotensin II type 2 receptor blockade increases bone mass. J Biol Chem 284: 4857–4864.

Jilka, R.L. (2007) Molecular and cellular mecha-nisms of the anabolic effect of intermittent PTH. Bone 40: 1434–1446.

Jones, C.B., C.T. Sabatino, J.M. Badura, D.L. Siet-sema, J.S. Marotta (2008) Improved healing efficacy in canine ulnar segmental defects with increasing recombinant human bone morphogenetic protein-2/allograft ratios. J Orthop Trauma 22: 550–559.

Kanis, J.A., J. Adams, F. Borgstrom, C. Cooper, B. Jonsson, D. Preedy, P. Selby, J. Compston (2008) The cost-effectiveness of alendronate in the management of osteoporosis. Bone 42: 4–15.

Kim, J.Y., J.Y. Min, J.M. Baek, S.J. Ahn, H.Y. Jun, K.H. Yoon, M.K. Choi, M.S. Lee, J. Oh (2015) CTRP3 acts as a negative regulator of osteo-clastogenesis through AMPK-c-Fos-NFATc1 signaling in vitro and RANKL-induced cal-varial bone destruction in vivo. Bone 79: 242–251.

Kondo, H., J. Guo, F.R. Bringhurst (2002) Cyclic adenosine monophosphate/protein kinase A mediates parathyroid hormone/parathyroid hormone-related protein receptor regulation of osteoclastogenesis and expression of RANKL and osteoprotegerin mRNAs by marrow stromal cells. J Bone Miner Res 17: 1667–1679.

Li, C.J., P. Cheng, M.K. Liang, Y.S. Chen, Q. Lu, J.Y. Wang, Z.Y. Xia, H.D. Zhou, X. Cao, H. Xie, E.Y. Liao, X.H. Luo (2015) Micro-RNA-188 regulates age-related switch be-tween osteoblast and adipocyte differentia-tion. J Clin Invest 125: 1509–1522.

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM



13

Nakagami, H., M. Takemoto, J.K. Liao (2003) NADPH oxidase-derived superoxide anion mediates angiotensin II-induced cardiac hy-pertrophy. J Mol cell Cardiol 35: 851–859.

Nakai, K., T. Kawato, T. Morita, T. Iinuma, N. Kamio, N. Zhao, M. Maeno (2013) Angioten-sin II induces the production of MMP-3 and MMP-13 through the MAPK signaling path-ways via the AT(1) receptor in osteoblasts. Biochimie 95: 922–933.

Neelon, F.A., B.M. Birch, M. Drezner, H.E. Lebo-vitz (1973) Urinary cyclic adenosine mono-phosphate as an aid in the diagnosis of hyper-parathyroidism. Lancet 1: 631–633.

Nishiya, Y., S. Sugimoto (2001) Effects of various antihypertensive drugs on the function of os-teoblast. Biol Pharm Bull 24: 628–633.

Nouet, S., C. Nahmias (2000) Signal transduction from the angiotensin II AT2 receptor. Trends Endocrinol Metab 11: 1–6.

Putnam, K., F. Batifoulier-Yiannikouris, K.G. Bharadwaj, E. Lewis, M. Karounos, A. Daugh-erty, L.A. Cassis (2012) Deficiency of angio-tensin type 1a receptors in adipocytes reduces differentiation and promotes hypertrophy of adipocytes in lean mice. Endocrinology 153: 4677–4686.

Resnick, L.M., J.H. Laragh, J.E. Sealey, M.H. Al-derman (1983) Divalent cations in essential hypertension. Relations between serum ion-ized calcium, magnesium, and plasma renin activity. N Engl J Med 309: 888–891.

Rudic, M., L. Milkovic, K. Zarkovic, S. Borovic-Sunjic, O. Sterkers, G. Waeg, E. Ferrary, G.A. Bozorg, N. Zarkovic (2013) The effects of an-giotensin II and the oxidative stress mediator 4-hydroxynonenal on human osteoblast-like cell growth: possible relevance to otosclerosis. Free Radic Biol Med 57: 22–28.

Ryu, J., H.J. Kim, E.J. Chang, H. Huang, Y. Banno, H.H. Kim (2006) Sphingosine 1-phosphate as a regulator of osteoclast differentiation and osteoclast-osteoblast coupling. EMBO J 25: 5840–5851.

Ryu, S., J.S. Shin, Y.W. Cho, H.K. Kim, S.H. Paik, J.H. Lee, Y.H. Chi, J.H. Kim, J.H. Kim, K.T. Lee (2013) Fimasartan, anti-hypertension drug, suppressed inducible nitric oxide syn-thase expressions via nuclear factor-kappa B and activator protein-1 inactivation. Biol Pharm Bull 36: 467–474.

Senbonmatsu, T., T. Saito, E.J. Landon, O. Wata-nabe, E.J. Price, R.L. Roberts, H. Imboden, T.G. Fitzgerald, F.A. Gaffney, T. Inagami (2003) A novel angiotensin II type 2 receptor signaling pathway: possible role in cardiac hy-pertrophy. EMBO J 22: 6471–6482.

Shen, W.J., L.F. Liu, S. Patel, F.B. Kraemer (2011) Hormone-sensitive lipase-knockout mice maintain high bone density during aging. FASEB J 25: 2722–2730.

Shimizu, H., H. Nakagami, M.K. Osako, R. Hanayama, Y. Kunugiza, T. Kizawa, T. To-mita, H. Yoshikawa, T. Ogihara, R. Morishita (2008) Angiotensin II accelerates osteopo-rosis by activating osteoclasts. FASEB J 22: 2465–2475.

Siddappa, R., A. Martens, J. Doorn, A. Leusink, C. Olivo, R. Licht, L. van Rijn, C. Gaspar, R. Fodde, F. Janssen, C. van Blitterswijk, J. de Boer (2008) cAMP/PKA pathway activation in human mesenchymal stem cells in vitro re-sults in robust bone formation in vivo. Proc Natl Acad Sci USA 105: 7281–7286.

Sobacchi, C., A. Schulz, F.P. Coxon, A. Villa, M.H. Helfrich (2013) Osteopetrosis: genetics, treat-ment and new insights into osteoclast func-tion. Nat Rev Endocrinol 9: 522–536.

Teitelbaum, S.L. (2000) Bone resorption by osteo-clasts. Science 289: 1504–1508.

Tintut, Y., F. Parhami, K. Bostrom, S.M. Jackson, L.L. Demer (1998) cAMP stimulates osteo-blast-like differentiation of calcifying vascular cells. Potential signaling pathway for vascular calcification. J Biol Chem 273: 7547–7553.

Tsai, J.N., A.V. Uihlein, H. Lee, R. Kumbhani, E. Siwila-Sackman, E.A. McKay, S.A. Burnett-Bowie, R.M. Neer, B.Z. Leder (2013) Teripa-ratide and denosumab, alone or combined, in women with postmenopausal osteoporosis: the DATA study randomised trial. Lancet 382: 50–56.

Turksen, K., A.E. Grigoriadis, J.N. Heersche, J.E. Aubin (1990) Forskolin has biphasic effects on osteoprogenitor cell differentiation in vi-tro. J Cell Physiol 142: 61–69.

Wang, N., G.D. Frank, R. Ding, Z. Tan, A. Racha-konda, P.P. Pandolfi, T. Senbonmatsu, E.J. Landon, T. Inagami (2012) Promyelocytic leukemia zinc finger protein activates GATA4 transcription and mediates cardiac hypertro-phic signaling from angiotensin II receptor 2. PLoS One 7: e35632.

Wassmann, S., G. Nickenig (2006) Pathophysio-logical regulation of the AT1-receptor and implications for vascular disease. J Hypertens Suppl 24: S15–S21.

Willoughby, D. (2012) Organization of cAMP signalling microdomains for optimal regula-tion by Ca2+ entry. Biochem Soc Trans 40: 246–250.

Winther, K., J.B. Knudsen, J. Gormsen, J. Jensen (1986) Effect of metoprolol and propranolol on platelet aggregation and cAMP level in hy-pertensive patients. Eur J Clin Pharmacol 29: 561–564.

Wittrant, Y., Y. Gorin, K. Woodruff, D. Horn, H.E. Abboud, S. Mohan, S.L. Abboud-Wer-ner (2008) High d(+)glucose concentration inhibits RANKL-induced osteoclastogenesis. Bone 42: 1122–1130.

Yang, D.C., H.J. Tsay, S.Y. Lin, S.H. Chiou, M.J. Li, T.J. Chang, S.C. Hung (2008) cAMP/PKA regulates osteogenesis, adipogenesis and ratio of RANKL/OPG mRNA expression in mes-enchymal stem cells by suppressing leptin. PLoS One 3: e1540.

Yoon, W.J., K.N. Kim, S.J. Heo, S.C. Han, J. Kim, Y.J. Ko, H.K. Kang, E.S. Yoo (2013) Sarga-chromanol G inhibits osteoclastogenesis by suppressing the activation NF-kappaB and MAPKs in RANKL-induced RAW 264.7 cells. Biochem Biophys Res Commun 434: 892–897.

Zhou, Y., X. Guan, T. Liu, X. Wang, M. Yu, G. Yang, H. Wang (2015) Whole body vibration improves osseointegration by up-regulating osteoblastic activity but down-regulatingosteoblast-mediated osteoclastogenesis via ERK1/2 pathway. Bone 71: 17–24.

Dow

nloa

ded

by:

Cor

nell

Uni

v.W

eill

Med

.Col

l.

207.

162.

240.

147

- 5/

11/2

017

9:27

:39

AM

本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP

图书馆。

图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页文献云下载图书馆入口外文数据库大全疑难文献辅助工具

http://www.xuebalib.com/cloud/

http://www.xuebalib.com/

http://www.xuebalib.com/cloud/


http://www.xuebalib.com/vip.html

http://www.xuebalib.com/db.php

http://www.xuebalib.com/zixun/2014-08-15/44.html


angiotensin ii/angiotensin ii receptor blockade affects...

Documents