Relationship between hepatic angiotensinogen mRNA expression and plasma angiotensinogen in patients with chronic hepatitis

Life Sciences, Vol. 60, No. 18, pp. 16211633, 1997 copyright 0 1997 Elfcvier science Inc. Printed in the USA. Ail tights reserved m2A-32a5/97 f17.00 + .cxl

ELSEVIER

PII SOO24-3205(97)00129-X

RELATIONSHTP BETWEEN HEPATIC ANCIOTENSlNOGEN AND PLASMA ANGIOTENSINOGEN

mRNA EXPRESSION

IN PATIENTS WITH CHRONIC HEPATITIS

Daisuke Takahashi, Kouichi Tamura, Toshiaki Ushikubo, Akihiko Moriya, Nobuyuki

Yokoyama, Nobuo Nyui, Eiko Chiba, Kiyoshi Hibi, Tomoaki Ishigami,

Machiko Yabana, Masakazu Tomiyama, Second Department

Satoshi Umcmura and Masao Ishii

of Internal Medicine, Yokohama City University Yokohama 236, Japan. (Received

School of Mcdicinc,

in final form January 31, 1997)

Summary Recent association and linkage studies suggested that angiotensinogen may play an important role in the pathogcnasis of csscntial hypertension. However, thcrc is little information in human conccming a relationship between plasma angiotensinogcn levels and the angiotcnsinogen mRNA cxprcssion in the liver, which is the main production site of angiotensinogen. Therefore, the aim of this study was to cxaminc whether hepatic angiotensinogen gene expression determines the level of circulating angiotensinogen and the activity of the rcnin-angiotensin system in humans. The subjects were 36 patients with chronic hepatitis. Blood was collected from each patients for estimation of plasma renin activity, plasma angiotcnsinogcn and angiotensin II concentrations and several parameters of liver function. In addition, totai RNA was isolated from liver biopsy specimens, which wcrc then used to measure angiotensinogcn mRNA with Northern blot analysis. Levels of angiotensinogen mRNA were detected easily in the liver biopsy specimens in all of the patients. Hepatic angiotcnsinogen mRNA levels were positively correlated with plasma angiotensinogen levels (t=O.41, P-0.013). In contrast, hepatic angiotensinogen mRNA levels did not show any significant relationship with plasma renin activity, plasma angiotcnsin II concentration, histological subgroup of hepatitis, histological activity index and parameters of liver function tests. The present study demonstrated, for the fast time, that hcpatic angiotensinogcn mRNA lcvcls correlated with plasma angiotensinogcn concentration in humans. Key Word:

angiotensinogen,

gene expression, liver biopsy, renin-angiotensin

system

The renin-angiotensin system (RAS) plays a major role in the regulation of blood prcssurc and in the maintenance of water and clcctrolytc balance. Angiotcnsinogen is the substrate of renin in the RAS and is the precursor of angiotensin I (Ang I), which is clcavcd by angiotcnsincorrespondence to: S. Umcmura, Second Department of Internal Mcdicinc, Yokohama City University School of Medicine, 3-9, Fukuura, Kanazawa-Ku, Yokohama 236, Japan.

1624

converting

enzyme to generate angiotensin

important

question

extracellular

is whether

reservoir

angiotensinogen

angiotensinogen positively

angiotcnsinogcn

angiotensin

concentration

angiotensinogen

In

gene showed associations

angiotensinogen

conditions

addition,

(5,6) and several cardiovascular genes have

of the RAS. One

of the RAS or is solely an

studies

have

suggcstcd

(1). In epidemiological and blood

recent

genetic

diseases

sumsted

stem cells. These mice do not product

were

of hypertension

by homologous

gene expression (SHR),

thereby

hypertension

factors to the proximal

hypertension

oligonucleotides

suggesting

structural

thcrcby

of blood prcssurc and the

functional

mRNA in cultured

that combined action of several

in spontaneously

changes

it would be interesting

gene is

expression of the gene (16-19).

inhibited the hepatic angiotensinogen

played

in human tissues.

is the liver, and

region of the angiotensinogen

significantly

anglotensinogen or

gene expression

carried out in cultured

promoter

which selectively

pressure

that

(20,21). Therefore,

angiotensinogen

in mouse

and arc hypotensivc,

and differentiation-dependent

decreased blood

and/or

of the

(9-11). Furthermore,

recombination

in the maintenance

In addition, we have reported

for the cell type-specific antisense

to be human

( 12,13).

cells, mice, and rats (1,14,15).

Furthermore,

found of the

mechanism

there have been many studies of the expression of hepatic angiotensinogen

novel transcription

plasma

studies using the rat and

the transcriptional

It is generally accepted that the primary source of plasma angiotensinogen

important

that

of this gene with high blood

angiotensinogen

indicating the critical importance of angiotensinogen

studies,

analysts

(7,8). Transgenic that

mice have been developed

of hypertension

pressure

linkage

of the polymorphism

gene may be involved in the pathogenesis

angiotensinogen-deficient

development

Several

(p-angiotensinogen)

(2-4).

angiotensinogen

embryonic

regulates activity

pcptidcs.

and pathophysiological

correlated

pressure

of

11 (Ang II), the bioactivc product

has an active regulatory function in both circulating blood and local tissues under

both physiological

human

Vol. 60, No. la,1997

Angiotensinogen mRNA in Human Liver

a role known

hypertensive

rats

in the

pathogcnesis

of

occur

in response

to

to

to investigate

the regulation

of the

However, most of these studies have been

cells and animals and little is known

concerning

the expression

and

regulation of the angiotensinogen

gene in humans. This can in part be explained by the difficulties

in obtaining sufficient quantities

of viable tissue samples for RNA extraction and low abundance

of specific mRNAs investigate

in the tissues. These factors have made it very difficult in the past to

the mRNA expression

of the components

biological methods such as Northern and dot blotting chain reaction

(PCR) method

of the RAS using established Recently, the development

as a new and extremely sensitive

molecular

of polymerase

method to investigate

small

amounts of DNA and mRNA has made it possible to study the expression of RAS component genes in very small tissue samples such as human biopsies (22). However, several difficulties existed in the quantification modification

of the mRNAs of the RAS irrespective

of PCR such as competitive

anglotensinogen

of the new application

PCR. On the other hand, fortunately,

gene has been reported to be expressed in the liver abundantly

of

the human enott& to be

Vol. 60, No. lf3,1!W7

Angiotensinogen mRNA in Human Liver

analyzed by Northern blot analysis of human angiotensinogen

1625

(23). Thercforc, as a first step, we cxamincd the cxprcssion

mRNA in liver biopsy

specimens

and compared

the mRNA lcvcls

with the values of plasma RAS activity and several parameters of liver function.

Methods

Participants:

Liver biopsy

specimens

(eleven female and twenty-five Twenty-eight

patients

were obtained

from 36 patients

with chronic hepatitis

male, ranging in age from 22 to 65 years old; mean age, 46).

had a positive

serum test for anti-hepatitis

positive

serum test for hepatitis

hepatitis

B surface antigen, and the remaining one patient

C antibodies,

scvcn had a

B surface antigen and negative test for antibodies

and B virus markers. Specimens were obtained by needle biopsy of each specimen was used for histological analysis,

against

tested ncgativc for both hepatitis usingultrasonography.

C

A part

and the remainder was frozen immediately

in liquid nitrogen and stored at -80°C until it was used for RNA extraction. lnfonncd

consent

was obtained from each subject before inclusion in the study.

Biochemical assavs: angiotensinogen anticoagulant,

Blood samples for measurement

and plasma Ang II concentration centrifuged immediately

of the plasma renin activity

(p-Ang II) were withdrawn

incubated with 5 pl 8-hydroxyquinoline,

Tokyo,

5 pl dimercaprol

buffer (0.1 M, pH 7.4) containing

generated angiotensin

For measurement

hydroxyquinoline,

(24). Briefly, 50 pi plasma was

, 25 pl NazEDTA (4%) and 165 1.11

0.1% lysozyme,

for 1 h at 37°C

coefficients

of p-angiotensinogen,

of variation

were 4.8 and 5.9%,

100 pl plasma was incubated with 5 yl 8-

5 pl dimercaprol , 25 pl Na*EDTA, 50 pl human kidney renin, and 65 pl Tris-

acetate buffer containing radioimmunoassay

lysozyme

for 12 h at 37’C, and the generated Ang I was mcasurcd by

(24). The mean intra- and interassay

7.9%, respectively.

coefficients

of variation wcrc 6.8 and

p-Ang II was determined by a specific direct radioimmunoassay,

anti-Ang II antibody as described previously,

by

a high-performance

using an

without extraction procedure (25). The mean intra-

and interassay coefficients of variation were 5.3 and 6.5%, respectively. were measured

and the

I (Ang I) was measured with a RENIN RIABEAD Ang I kit (Dainabot Ltd,

Japan). The mean intra- and interassay

respectively.

p-

and the plasma separated and kept frozen at -80°C until

the time of assay. PRA was measured by radioimmunoassay

Tris-acetate

(PRA),

with EDTA as

liquid

chromatographic

Plasma cathecholamincs

(HPLC)

method

using

an

automated HPLC analyzer (Tosoh Co, Tokyo, Japan). Details on this HPLC analyzer have been previously 7.2%,

reported (26). The mean intra- and intcrassay

respectively.

arninotransferase concentrations

Serum total

protein,

albumin,

(AST), alanine aminotransferase

School of Medicine.

y-globulin,

of variation wcrc 4.9 and

total

cholesterol,

(ALT) and y-glutamyl transpeptidasc

were measured by routine methods

Yokohama City University

coefficients

in the Department

aspartatc (y-GTP)

of Clinical Chemistry,

Angiotensinogen mRNA in Human Liver

1626

Isolation of RNA and Northern blot analvsis: by the acid guanidinium

Total RNA was cxtractcd from frozen liver tissue

thiocyanatc-phenol-chloroform

Tokyo, Japan). RNA concentration

Vol. 60, No. l&l997

method using ISOGEN (Nippon

was mcasurcd by spcctrophotomctry

Gcnc,

(absorbance

at 260

nm). RNA samples (4 pg per lane) wcrc separated by electrophorcsis

on 1.2% agarosc gels after

denaturation

on to nylon

with

glyoxal and dimethylsulfoxide,

(GeneScreen Plus, DuPont-New

and transferred

England Nuclear, Wilmington

mcmbrancs

DE) in 10 x SSC (1 x SSC = 0.15

M NaCl and 0.015 M sodium citrate). After 1 h of prehybridization

in 1% sodium dodccyl

sulfate (SDS), 1 M NaCl , 10% Dextran and 200 pg/ml salmon sperm DNA, the blots were then hybridized

at 65°C for 18 h with ‘*P-labelled probes for human angiotcnsinogcn

gcnc (23). The

18s ribosomal RNA (18s rRNA) probe was used as the intcmal control for each spccimcn (27). After

washing,

angiotensinogen

the

tiltcrs

were

subjected

mRNA was quantified

Photo Film, Japan), and normalized

to

autoradiography

using a FUJIX

BIO-Imaging

at -70°C. Analyzer

Expression BAS2000

of (Fuji

to the signal generated by probing for the constitutivcly

expressed 18s rRNA.

w:

Liver biopsy

and lobular inflammation

specimens

were graded for their degree of pcriportal,

and fibrosis according to the scoring system

Coded specimens were examined by a pathologist

Statistical

analvsis:

Univariate

correlation

quantitative

For statistical

analysis

portal

of Knodell et al. (28).

who had no knowlcdgc of their source.

of differences

analysis was used for examination

among groups, ANOVA

was used.

of relations bctwccn paramctcrs.

data are expressed as mean + SEM. PcO.05 was considered statistically

All

significant.

Results
The patients consist of 6 chronic persistent

hepatitis

(CPH), 19 chronic active hepatitis 2a (CAH2A) patients and 11 chronic active hepatitis

2b (CAH2B)

patients.

sig@ficant differences

The characteristics were observed

systolic blood pressure,

of the 36 patients

among the histological

diastolic blood pressure,

concentrations

other hand, the histological

activity

in Table 1. No

in terms of age, sex,

platelet count, serum total protein, albumin, y-

globulin, total cholesterol, AST, ALT and ?(-GTP concentrations. plasma cathecholamine

arc summarized subgroups

In addition, PRA, p-Ang II and

were similar among the histological index (HAI) scores were significantly

subgroups.

On the

different among the

histological subgroups (P
mRNA cxnression in each histoloeical erouv: Expression of hcpatic

mRNA was easily detected in all of 36 patients by Northcm blot hybridization

The band

length was 2 kb which

corresponded

to the rcportcd size (23). The HA1

Vol. 60,No. 18,1997

1627

Angiotensinogen mRNA in Human Liver

TABLE Patient Demographic Characteristics

Age (years) Gender (percent male) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Platelet count (x 104/mm3) Serum total protein (g/dl) Serum albumin (g/dl) Serum g-globulin (%) Serum total cholesterol (mg/dl) Serum AST (IU/I) Serum ALT (lU/l) Serum y-GTP (WI) Plasma renin activity (ng/ml/h) Plasma angiotensin II (pg/ml) Plasma noradrenaline (ng/ml) Plasma adrenaline (ng/ml) Histological activity index score

1

by Pathological Classification

CPH (n=6)

CAHPA

49.2k7.7 83.3 126.7*5.7 76.3*3.7

45.6k3.1

CAHPB (kll)

(n=l9)

57.9 127.3k4.4 74.2k3.1

16.3k2.6 7.8kO.3

15.5fl

.l

7.2fO. 1

4.4fO. 1

4.1kO.l

21522.1

21.1kl.l 159.5+10.1

165.3f8.0

92.1Ik15.0 70.7k30.5 150.5k28.4 38.7k5.9 66.8&l 1.9 1.82kO.68 0.94kO.27 3.7k1.5 2.9f0.8 0.31f0.06 0.18+0.02 0.028+0.015 0.017kO.020 4.2kO.4 8.2kO.4 42.8k9.8

Entries are the mean + SEM or percent

as indicated.

P

43.5k3.5 0.73 81.8 0.30 139.3k7.2 0.26 83.5f5.7 0.27 14.3&l .6 0.73 0.11 7.61kO.2 4.3k0.2 0.16 0.97 21.6f2.0 173.3zk7.0 0.60 0.11 120.4k28.1 181.8zk38.9 0.20 111.8k26.3 0.06 0.14 0.69kO.19 2.1f0.6 0.59 0.2WO.04 0.06 0.22 0.029+0.006 12.0&l .l <0.0001

Significance of pathological

effect in a one- way analysis of variance is given by P.

described

by Knodell et al. can be used as a semiquantitative

method to assess the degree of

histological injury in chronic liver disease (28). For each patient, and some subindexes biopsy

sample.

expression

were compared

However,

the scores for the total index

with the levels of angiotensinogcn

the hepatic

angiotcnsinogen

of 18s rRNA did not show significant

mRNA

relationship

mRNA in the same

levels standardized with histological

to the

subgroup

hepatitis (CPH, CAH2A and CAH2B) (Fig. 2) and HA1 (total index and any subindexes) not shown).

In addition, p-angiotensinogen

did not show statistically

significant

with each histological subgroup (Fig 2), although p-angiotensinogen

significantly

serum

Furthermore,

albumin

angiotensinogen liver function

concentration

(~0.45,

+0.0059)

(Fig.

mRNA levels did not have any statistical such as platelet

count

and serum

3).

of

(data

relationships correlated with the

hcpatic

relation with several parameters

total protein,

cholesterol, AST, ALT and y-GTP levels (Fig. 3 and data not shown).

albumin,

y -globulin,

of

total

Angioteasinogen mRNA in Human Liver

1628

Vol. 60, No. l&1997

-28s

ATNG

-18s

18s rRNA

-18s

Fig. 1 Representative liver biopsy

Northern blot of total RNA (4 pgper specimens.

to the human performed

The upper panel shows a single 2.0 kb band corresponding

angiotensinogen

using a specific

angiotensinogen.

(ATNG)

[“PI-1abelled

mRNA

transcript.

complementary

The

analysis

was

DNA probe for human

The lower panel shows the band of the 18 S ribosomal RNA on the

same blots, obtained

by hybridizing

DNA probe for 18 S ribosomal angiotensinogen

lane) extracted from the human

with a specitic r’P]-labelled

DNA and used to normalize

complementary the abundance

of

mRNA. Relative ATNG mRNA levels were dctermincd as indicated

in the methods section. Henatic

anaiotensinoaen

mRNA

exnression

and nlasma

studies indicated that control of angiotensinogen activity of the RAS and that conditions had influences

on the intravascular

mRNA levels with parameters

standardization

to the expression

si~ificantly

hepatic angiotensinogen Ang II (Fig. 4).

nrofiles:

was particularly

which altered circulatingangiotensinogen

angiotensinogen correlated

renin-antiotensin

biosynthesis

RAS activity of 18s rRNA,

with p-angiotensinogen

(14,29).

Thus,

the hepatic

concentrations

we compared

of the plasma renin-angiotensin angiotensinogcn

Previous

relevant to the

the hepatic

system.

After

mRNA

levels

(i-=0.41, P=O.O13) (Fig 4). In contrast,

mRNA levels did not show any significant relationship

the

with PRA or p-

Vol. 60, No. 18, l!N7

1629

Angiotensinogen mRNA in Human Liver

A

B

2ooo

NS

0.7 NS -0

0.6

P e

1500 =

0.6

I

f

Q

$

0.4

g

0.3

‘Ooo

1E

g

600

0.2

g B

0.1

0

0

CAU2A

CPU

CPU

CAU2B

CAH2A

CAH2B

Fig. 2 (A) Effects

of the histological

CAH2A,chronic plasma

active hepatitis

angiotensinogen

difference

subgoups

in p-ATNG

histological subgroups the expression

2a; CAH2B,

concentration among

difference

subgroups.

p-ATNG

chronic

There

(relative

ATNG

ATNG

is not

subgroups.

on the hepatic angiotensinogen

in relative

persistent

hepatitis;

chronic active hepatitis

(p-ATNG).

the histological

of 185 rRNA

significant

(CPH,

2b) on the a significant

(B) Effects

of the

mRNA levels standardized

mRNA

mRNA

levels).

levels

There

among

to

is not

a

the histological

and relative ATNG mRNA levels were dctcrmincd as indicated

in the methods section.

B

A

2400-

r=o.l. kO.0059

i%

0 0

2999.

3

loo_

2

0.8’

E (1 5

0.6 .tsO

L

0 0

0.4 . .o

0

00

3.8

4.2

4.6

5.0

‘0 O

0

~0.21,. 3.4

0

,

5.4

3.4

Serum Albumln Concantmtion (gldl)

3.8

a00 O”

0

00 0 00

,

,

4.2

0 e

,

,

4.6

,

,

5.0

, 5.4

Serum Albumin Concontmtlon (gldl)

Fig. 3 (A)

Correlation

angiotensinogen the

two

variables

concentration expression

between concentration

the

(P;O.O059).

and the hepatic

serum

(p-ATNG). (B)

albumin

concentration

and

plasma

There is a significant correlation between Correlation

angiotensinogen

between

mRNA

the

serum

albumin

levels standardized

to the

of 18s rRNA (relative ATNG mRNA levels). There is not a significant

correlation between the two variables.

Strum albumin concentration,

p-ATNG

relative ATNG mRNA levels were determined as indicated in the methods section.

and

1630

Angiotensinogen mRNA in Human Liver

0.2

0.4 mktb

0.6 nffi

0.6 mRNA

1.0

0.2

0.6

0.6

~16th

bfd6

ATNO

0.6 mm

Vol. 60, No. lS,l!W7

1.0

02

0.4 -

t.wd6

0.6 ATNG

0.6 mRNA

1.0

Lrma

Fig. 4 (A) Correlation the expression

between

the hepatic angiotensinogen

of 18s rRNA (rclativc ATNG

mRNA lcvcls standardized

to

mRNA levels) and the plasma rcnin

activity (PRA). There is not a significant correlation between the two variables. (B) Correlation between

the hepatic angiotensinogen

mRNA levels standardized

to the

expression of 18s rRNA (relative ATNG mRNA levels) and the plasma angiotcnsin II concentration variables.

(p-Ang II). There is not a significant

(C) Correlation

standardized

between

to the expression

plasma angiotensinogen

the hepatic

correlation between the two

angiotensinogen

mRNA

levels

of 18s rRNA (relative ATNG mRNA levels) and the

concentration

(p-ATNG).

between the two variables (FO.013).

There is a significant

PRA, p-Ang II, p-ATNG

correlation

and relative ATNG

mRNA levels were determined as indicated in the methods section.

Discussion

In this study, we found, for the first time, that the steady-state angiotensinogen angiotensinogen healthy

subjects

correlated

with

(14). This would be consistent concentration

of angiotensinogen

showed

p-angiotensinogen

in humans.

level as well as serum albumin concentration

with serum albumin

biosynthesis mRNA

correlated

also partly

relationship

which may directly

with our results that p-angiotensinogen

mRNA and p-angiotensinogen

p-

was

the major site for

with

the parameters

of any liver function

or

is not clear. Hcpatic angiotensinogen

affect p-angiotensinogen,

may be associated

not only with

mRNA expression but also with total liver size and function.

explain the relatively

angiotensinogen

patients,

were reported to be lower than in

since the liver represented

histological findings. The actual reason for this dissociation production,

In cirrhotic

and albumin. In contrast, the levels of hepatic angiotensinogen

no significant

hepatic angiotensinogen

mRNA levels of hepatic

small degree of correlation

This may

between hepatic angiotensinogen

observed in this study. Since the correlation between the hepatic

mRNA levels and p-angiotensinogen

was not so strong, several factors other

Vol. 60, No. 18,1997

Angiotensinogen mRNA in Human Liver

than hcpatic angiotensinogen

cxprcssion may participate

Although a variety of factors arc supposed the exact contributions synthesized

angiotensinogen

involved

in the regulation of p-angiotensinogen.

affected on hypertension

was significantly

production

tissue and level of angiotcnsinogen Although

angiotensinogen

the

level might result from an incrcasc in angiotcnsinogcn

gene expression

physiological

and

are not fully understood,

in adipocytes

fasting and refeeding

in a manner

irrespective of the lack of apparent

pathological

which parallels

adipocytc

adipogenic

angiotensinogen

in plasma

angiotensinogen

mRNA

angiotensinogen

was that tissue angiotensinogen expressions

gcnc

mRNA

by lcvcl

mRNA lcvcl (3 1). In

Rcccntly,

is regulated differently of hypertension concentration

WC have

in SHR and

is accompanied,

at least

as well as cardiac and

one possible

expression and p-angiotcnsinogen

such as adipogenic and/or cardiovascular

reasons why

the

was not so strong

angiotensinogcn

gcnc

might affect p-angiotensinogen.

In conclusion, liver biopsy

adipogenic

was modulated

(data not shown).

in SHR (32). Therefore,

correlation between hepatic angiotensinogen

the

between plasma lcvcls of angiotensinogcn

rats and indicated that the development with increases

differentiation

angiotensinogen

changes in the hepatic angiotensinogen

of tissue

of

regulated and blood pressure

mass index of the patients

that the expression

significance

in adipose

study showed that angiotensinogcn

this study, we could not find significant relationships

Wistar-Kyoto

was expressed abundantly

increased during adipogenic

a previous

was nutritionally

and body weight or body

For example, several

(30) and Bloem ct al. showed that p-

derived from body fat since angiotcnsinogen

temporally,

which is

related to body mass index in human children (4). They

suggested that a higher p-angiotensinogen

reported

Tissue angiotcnsinogcn,

in several tissues besides the liver such as the brain, heart, aortae, adrenals, kidneys

reports indicated that obesity

expression

in the regulation of p-angiotcnsinogcn.

to bc related to the regulation of p-angiotcnsinogcn,

of each factor remain to bc determined.

and fat, is also possibly

(17,lg).

1631

we have successfully

specimens

had statistically

detected angiotensinogen

by Northern blot analysis.

significant

relationship

mRNA expression in human

The hepatic angiotcnsinogen

with p-angiotensinogen

irrespective

mRNA levels of the lack of

association with PRA and p-Ang II. Although definitive results awaits further testing in a larger population,

these findings provide important

hepatic angiotensinogen

gene may contribute

insights into potential

relationships

to the regulation of p-angiotensinogen

whereby

the

in humans.

AcknowledPements This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan, the Uehara Memorial Foundation, and Yokohama Foundation for Advancement of Medical Science. Dr. Kouichi Tamura is supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. We would like to express our thanks to N. Inaba for excellent secretarial help.

1632

Angiotensbgen

mRNA in Human Liver

Vol. 60, No. l&l997

Rcfercnces

4. 5.

6. 7.

8.

9.

10.

11. 12. 13.

14. 15. 16. 17. 18.

19.

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