Experimental Models of Pulmonary Arterial

Hypertension

Dr Figen DeveciDr Figen DeveciFFUU, Department, Department of of Chest Chest DiseaseDiseasess

2

PRESENTATION PLAN

1. Definition of animal

models of PAH

2. In generally, validity

of animal models

and differences

from humans PAH

3(Zaiman A et al. Am J Respir Cell Mol Biol 2005;33:425-31)

In the last 100 years of PAH research

4

Animal models in PAH

Aims;1. Characterizing the pathophysiology of

PAH2. Researching its sequela such as RVH and failure3. Testing novel therapeutic strategiesIdeal animal model of PAHCreating all these findings;

- Clinic, - Hemodynamic, - Histopathologic and - Biological characteristics

5

Animal models in PAH

1. Hypoxic exposure in rodents 2. Monocrotaline (MCT) injection in rodents

3. Chronic overcirculation-induced PAH in lambs and pigs

4. Models of newborn persistent PAH

5. Genetically modified rodents

6. Models of chronic embolic PH

7. Cell cultures(Naeije R,Dewatchter L. Rev Mal Respir 2007; 24: 481-96)

(Campian ME et al. Naunyn-Schmiedeberg’s Arch Pharmacol 2006;373:391-400)

6

By lowering atmospheric pressure (one-half), (380 mmHg), 10-15 day

Increased PAP 50%

(Ozaki M et al. Hypertension 2001;37:322-7)

Normal air at hypobaric

pressure Delivering system

with compressed air and N2. O2 content: 11%-12% (60-70 torr) (Fike CD. J Appl Physiol 1996;81:2078-87)

(Voelkel NF, Tuder RM. J Clin Invest 2000;106;733-8)(Voelkel NF, Tuder RM. J Clin Invest 2000;106;733-8)

Hypobaric hypoxia Normobaric hypoxia

Oxygen poor-air at normal pressure

1. Hypoxia induced PAH model

7

PAH was composed in mammals with chronic hypoxia

Vascular remodeling is not observed with chronic hypoxia in:

Pika, yak, snow pig, lama

Differences; - The animal species studied - The developmental stage - The sex

Lama

Snow pig

8(Voelkel NF, Tuder RM. J Clin Invest 2000;106:733-8)

1A. The model of chronic hypoxia

Acute hypoxic

exposure

Chronic hypoxicexposure

Acute pulmonary VC

Remodeling of small pulmonary arteries

PAP

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In animal experiments: PAO2< 70 mmHg elicits strong pulmonary arterial VC

HPV is common in mammals, differences are exist according to species

HPV in animal models

Rabbit; no reaction Dog, guinea-pig, lama; low levels Cat, pig, hoarse, cattle; strongest VC (+) Human < rat (moderate levels) (Reeve JT et al. Int Rev Physiol 1979; 20:289-310)

There is great variability among humans (Naeije R et al. Chest 1982; 82: 404-10)

10

HPV in animal models

The small resistance pulmonary arteries (<200µm)

The persistence of hypoxia results in downregulation of acute HPV, despite the occurrence of PAH

(Thompson BT et al. J Appl Physiol 1989; 66:920-28)(Greenlees KJ. Respiration 1984;45:169-174)

In animals,

•Rapid increase in PVR with HPV

•Gradually plateaus

•Similar to in humans

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Biphasic HPVPhase 1: immediate, end-independent constriction, peaks in 10 min Phase 2: slowly, end- dependent, sustainedconstriction, peaks in 40 min (Ward JP. Exp Physiol 1995; 80: 793-801)(Ward JP. Exp Physiol 1995; 80: 793-801)

Monophasic HPV

Denuded of endothelium,isolated PA: monophasicconstriction

(Archer SL et al. FASEB J 2001;15: 1801-3)(Archer SL et al. FASEB J 2001;15: 1801-3)

HPV in animal models

Endothel is important in

modulation of HPV

12

Medial thickness, at day 3th

Adventitial thickness, at day 3th

Newly muscularized arteries apparent from day 3,

increase to 7th day(Meyrick B, Reid J. Am J Pathol 1979;96:51-70)

Media

Adv

Rat hiler PA In humans, progress VC to remodeling in first 24 hour

(Naeije R,Dewatchter L. Rev Mal Respir 2007; 24: 481-96)

Transition time of remodeling from Transition time of remodeling from HPV?HPV?

In rats; Endothelial changes at day

3th (Meyrick B, Reid L. Lab Invest.

1978;38:188-200)

RVH, at day 5th

13

In many but not all animal species

Precapillary resistance arteriolar muscularization

Vascular SMC and adventitial fibroblast proliferation

(Rabinovitch M et al. Am J Physiol 1979;236:818-27)(Rabinovitch M et al. Am J Physiol 1979;236:818-27)

(Stenmark K et al. J Appl Physiol 1987;62:821-30)(Stenmark K et al. J Appl Physiol 1987;62:821-30)

(Chen SJ et al. J Appl Physiol 1995;

79 : 2122-31)

Endothelial cell proliferation not compose significantly

(Chen SJ et al. J Appl Physiol

1995;79 : 2122-31)

(Chen SJ et al. J Appl Physiol

1995;79 : 2122-31)

14

In hypoxic neonatal calve; Extreme elevation of PAP Prominent intimal

thickening

“In a model of hypoxia+VEGFR

blocker” Endothelial cell

proliferation Luminal obliteration Severe PAH

(Taraseciviene-Stewart L et al. FASEB J 2001;15:427-38)

(Voelkel NF, Tuder RM J Clin Invest 2000;106:733-8)

In fast-growing broiler chicken

Remodeling in all layers (adventitia, media, intima)

(Peacock AJ et al. Am Rev Respir Dis 1989;139:1524-30)

15

Vascular remodeling in PA

1. Large proximal PA 2. Distal muscular PA3. Non-muscular alveolar vessels4. Adventitia5. Endothel

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Remodeling in large proximal PA

Rat and mouse Early and dramatic adventitial thickness

(fibroblast) Thickening of the media lags behind (collagen

and elastin) (Stenmark KR et al. Circ Res 2006;99:675-91)

Large animals (calf, pig) Early and dramatic medial thickening Less thickening of adventitia

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Early and dramatic in all animal species

Hypoxic calf model; marked PA adventitial thickness (resembles the pathological picture in PPHN)

(Jeffery T. Prog Cardiovasc Dis 2002;45:173-202)

(Meyrick B. Clin Chest Med 2001:22:393-404)

More prominent in rats in contrast to

mice (Sobin SS. High Alt Med Biol

2000;1:311-22)

(Frid MG et al. Am J Pathol 2006;168:659-69)

Adventitial remodeling

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Hypobaric hypoxia,4-8 h/d,5-7 d/w, FiO2:561 ve 70 mmHg2

1. Model (rat)

1B. Intermittent hypoxia in animal models

3. Model (rat)

Similar to OSAconsecutive 30

sechypoxia 30 secnormoxic

periods,8h/d, 5 week

Increase in mean PAP

Increase in right ventricle weight

(McGuire M, Bradford A. Eur Respir2001;18: 279-85)

2. Model (mice)

Increase in RVSP RVH Pulmonary muscular arteriolar remodeling

(1 Sizemore DA et al. J Appl Physiol 1973;35:518-21)(2 Nattie EE et al. Am Rev Respir Dis 1978;118:653-8)

(Fagan AK. J Appl Physiol 2001;90:2502-7)

2 min 10% O2 ,

followed by 2 min normoxia, 8h/d, 4 week

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The duration of hypoxemia in experiment >

the duration of hypoxemia in human sleep

apnea syndrome (Fagan AK. J Appl Physiol

2001;90:2502-7)

“More shorter hypoxia-reoxygenation cycles”

(30-90 sec/min, 8 h/day, for several weeks)

increased1 / unchanged2 right ventricle mass

1 (McGuire M, Bradford A.Respir Physiol 1999;117::53-8)1 (Kraiczi H et al. J Appl Physiol 1999;87:2025-31)

2 (Bao G et al. J Appl Physiol 1997;83:95-101)2 (Fletcher EC et al. J Appl Phsiol 1992;72:1978-84)

“No defined presently rodent model of intermittent hypoxia-induced PAH”

(Fagan AK. J Appl Physiol 2001;90:2502-7)

20

In animal models; leads to development of PAH, regardless of the duration of the hypoxia/normoxia intervals

In humans, only a small, probably clinically unimportant, effects on pulmonary hemodynamics

(Campian ME et al. Naunyn-Schmiedeberg’s Arch Pharmacol 2006;373:391-400)

Differences between animals and humans in IH

21

Differences between rats and humans in hypoxia-induced

remodelingHypoxia Human RatProminent feature of remodeling

Intimal proliferation Muscularization

Changes with hypoxia

More less More severe

Alveolar hypoxia Uneven distribution

of inspired air (COPD, OSA)

Fairly uniform

In biological field eNOS NO PGI2

eNOS NO PGI2

(Zielinski J. Eur Respir J 2005;25:173-80)

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Disperancy between animal and human may be related to

an individual susceptibility to the hypoxic stimulus

(Weitzenblum E, Chauat A. Eur Respir J 2001; 18:251-39)

“The rat model is the

bad model for Hypoxic

PH” (Heath D. Cardioscience 1992;3:1-6)

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Animals age in hypoxia-induced PAH

Newborn rat Normal pulmonary arterial development is very similar to that seen in the human

The model of PAH in newborn rat is a useful model

(Meyrick B, Reid L. Am Rev Respir Dis 1982;125:468–73)

Hypoxic/MNC induced PAH is more prominent than

adult animals(Belik J et al. J appl Physiol 2003;94:2303-12)

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Hemodynamic measurementsClosed-chest pressure

recordings Minimally invasive Serial measurements Technically demanding Quality of signal was

poorDirect pressure measurement

-left lateral thoracotomy More easily applicable Without substantial

differences in pressure values

(Kolettis T et al. Hellenic J Cardiol 2007;48:206-10)

(Rabinovitch M et al. Am J Physiol 1979;236:818-27)

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Right Ventricular Hypertrophy

Right ventricular free wall (RV) Left ventricle together with septum

(LV+S) Weighed and expressed as a ratio

LV+SLV+S

RVRV

(Fulton RM et al. Br Heart J 1952;14:413-20)

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Quantitative morphological study

(Rabinovitch M et al. Am J Physiol 1979;236:818-27)

Lung volumes By water

displacement

Angiograms (Ba-jelatin)Muscularizatio

n AWA% The thickness of medial muscular coat Calculating as the percentage of external

diameterReduction in the number of small arteriesIncreased ratio of the number of alveols/arteries

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1. Remodeling Lumen occlusion due to

medial and adventitial thickness Not occurring maximal VD%MT=2xMT/external

diameterx100 Maximum VD Transmural distending pressure

Medial cross-sectional area Lumen area and MA/LA

Outward remodeling

Increase in PAP,RVH (+) Medial thickness (+)Narrowing of lumen area (-)

(VanSuylen RJ et al. Am J Respir Crit Care Med 1998;157:1423-8)

(Stenmark KR, McMurtry IF. Circ Res 2005;97:95-8)

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Quantitative stereology + confocal microscopy

Traditional method 2.Pruning

(rarefaction)

The number of barium-filled blood vessels/the number of alveoli

New method (Quantitative stereology + confocal microscopy)

Angiogenesis

Allows inferences about the 3D structural parameters of objects based on 2D information by histologic images

Totally capillary surface area and total length of intraacinar resistance vessels

(Hyvelin JM et al. Circ Res 2005;97:185-91)

(Howell K et al. J Physiol 2003;547:133-45)

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Which human PAH ways are showed by chronic hypoxic PAH model ? Similar to human PAH develops secondarily to

disorders of respiratory system (Bonnet S et al. Proc Natl Acad Sci U S A. 2003;100:9488-93)

“Pure hypoxic PAH”

-Chronic Mountain

Disease-Sleep Apnea

SyndHypoxia/

hypoxemia

COPD, ILD

Hypoxia/hypoxemia+ inflammation

Eisenmenger Syndrome

High pulmonary blood flow

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MCT is an alcaloids which takes places in “Crotalaria Spectabilis” plants

Dehidrogenation product “reactive MCT pyrrole” by hepatic cytochrome P450 3A is a toxic

(Reid et al. J Biochem Tox 1998;12:157-66)

Single dose (60 mg/kg ip/sc) MCT rapidly leads to severe pulmonary vascular disease similar to IPAH

Endothelial injury + medial hypertrophy Massive mononuclear infiltration into the

perivascular region (Nishimura T et al. Am J Respir Crit Care Med 2001;163:498-502)

MCT sensitivity different between rat strains

Age: younger rat (2 w) more susceptible to the effects

of MCTGender: female rats suffer more non-pulmonary organ

damage (Schoental R, Head MA. Br J Cancer 1955;9:229-37)

2. Monocrotaline induced PAHmodel

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MCT induced PAH is severe

mPAP 32 mmHg and prominent RVH

Firstly endothelial necrosis

Pulmonary edema which starting 24th h, continuously 1 week

Remodeling and cardiac injury when edema disappearing

(Sugita T et al.J Appl Physiol 1983; 54: 371-6)(Plestina R et al. J Pathol 1972; 106: 235-49)

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MCT induced PAH

Human PAH

Similarity The side of hemodynamic and histopathologic are severe The mortality rate is high

Differences

Endothelial barrier disappear early Prominent inflammatory adventitial proliferation Blocked with VD and/or anti-inflammatory agent

The biology of MCT induced PAH similar to the biology of PAH except the differences of ET-1 and eNOS levels (Naejie R, Dewachter L. Rev Mal Respir 2007;24:481-96)

33

MCT induced PAH model

MCT + one sided PNEUMONECTOMY (Rat)

Severe hemodynamic alterations (mPAP; 45mmHg)

Medial hypertrophy Prominent neointimal formation (shear stresses) Plexiform-like lesions Vascular obliteration

(Nishimura T et al. Circulation 2003;108:1640-5)

(White RJ et al Am J Physiol Lung Cell Mol Physiol 2007;293:583-90)

Not develop plexiform lesions

Standard modelfor human PAH

34

ETB-R deficient homozygous adult rat + MCT

Increased hemodynamic response Prominent medial hypertrophy, occlusion in

the vessel lumen (+) Plexiform neointimal proliferation (-)

(Kolettis T et al. Hellenic J Cardiol 2007;48:206-10)

Younger rat (4-6 w)+MCT More severe PAP Prominent neointimal lesion Medial hypertrophy Decreased arterial-to- alveolar ratio

(Ivy DD et al. Circulation 2005;111:2988-96)

35

MCT+Shunt

MCT

Left PA anastomosed directly end-to-end left subclavian artery

1 week

PAH(neointimal formation)

4 week

Shunt

1 week

MCT

neointimal formation

(Tanaka Y et al. J Clin Invest 1996;98:434-42)

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Differences between MCT and CH models

Hypothesis of proteomics approach; similar phenotype but may be different signaling pathways (Laudi S et al. Proteomics 2007;7:2469-78)

VC Remodeling

CH NO, CO VEGF

MCT Seretonin, catecholamine

ERp57, ERp29 from ERKC MCT

Medial hypertrophy + +++

Development time of increased PAP and induced RVH

3th day 21th day

Reduction of the lumen area

30-100µm (-)101-200 µm (-)

30-100µm (-)101-200 µm (+)

37

An aorta-to-pulmonary shunts with increased pulmonary blood flow in dogs

Growing piglets

Shunting between the thoracic aorta to the pulmonary trunk

The severity of PAH in this method limited with volume and radius of shunt

(D Canniere D et al. J Appl Physiol 1994;77:1591-6)

3. Over-circulation induced PAH model

Between abdominal aorta and VCI in rats(Garcia R, Diebold S. Cardiovasc Res 1990; 24: 430-2)

38

Left subclavian artery and pulmonary

truncus were shunted because of naturally

growing left-to-right shunt with theanimal growth

After the 3-4 mounth growing

As young as possible growing pigs

“Blalock-Taussing operation”

Increases of PVR Marked small pulmonary arteriolar medial

hypertrophy Severe PAH (PAP 30-40 mmHg)

Major morphological appearance: medial hypertrophy

Demonstrates to early stage of disease

Intimal and adventitial remodeling not compose, plexiform lesions not develop

Is it an accurate model of PAH in left-

to-right shunted accompanied to CHD?

(Rondelet B et al. Circulation 2003;107:1329-1335)

39

In-utero aorto-pulmonary shunts in the lamb

Late-gestation Ascending aorta

with main PA

-mPAP:40 mmHg, PVR mPAP:40 mmHg, PVR increasedincreased

-Dilatation and background -Dilatation and background hazehaze

increaseincrease

-%WT increase (<200 µm)-%WT increase (<200 µm)

-Muscularization-Muscularization

-The number of intra-acinar -The number of intra-acinar PAPA

increaseincrease

-Endothelial dysfunction-Endothelial dysfunction

(Reddy VM et al. Circulation 1995;92:606-13)

40

SHUNT Time Expansiveness Progressively increases in PAP

(Heath D et al. Br Heart J 1959; 21: 187-96)

Shunt models It is also, this model good mimics to PAH Minimally intimal and adventitial remodeling Usually it is limited with only prominent medial hypertrophy (Naeije R,Dewatchter L. Rev Mal Respir 2007; 24: 481-96)

41

1. Ductal ligation lamb model of PPHN

2. Partial compression of DA in fetal lamb

3. The models of congenital diaphragmatic hernia

4. Neonatal models of hypoxic PAH

5. Hyperoxic PAH model

4. The models of newborn persistent PAH

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Prenatal ligation of ductus arteriosus in pregnant ewes (127th day of gestation, term: 146 days)

After 9 day, cesarean section (Black SM et al. Pediatr Res 1998;44:821-30)

1. Ductal ligation lamb model of PPHN

Increase in intra-uterin PAP, RVH

Medial hypertrophy Muscularization Adventitial remodeling Severe PAH

43

Absence of fetal hypoxemia and high blood flow Intrauterine;

Sustained PAH, RVHAltering fetal pulmonary vasoreactivity PA; increases of medial thickness, luminal

occlusionThe failure of adaptation of the postnatal

pulmonary circulation (Abman SH et al. Clin Invest 1989;83:1849-58)

Postnatal periods; PAP is high, PVR is high Right-to-left shunt across to the ductus

2. Partial compression of DA in fetal lamb

44

3. The models of CDH

Immature lung Pulmonary vascular bed structural anomalies

causing PPHN

Excessive muscularization of the preaciner arteries

A reduce external diameter Increase in medial wall thickness of

prealveolar and intraalveolar arteries Reduce in luminal area Altering vasoreactivity

(Geggel RL et al. J Pediatr Surg 1985;107:457-64)

45

Three model:1. Surgical (lamb,rabbit)2. Teratogenic (rat,mouse)3. Genetic

Surgery 80-85th days of gestation Short incision of the left hemidiaphragm of fetus The stomach is pulled into the thorax (Thebaut B et al. An J Respir Cell Mol Biol 2002;27:42-7)(Beurskens N et al. Birth Defects Res A Clin Mol Teratol

2007;79:565-72)

3. The models of CDH

46

Histopathologic and biological features is more similar to human fetal and PPHN

No intimal proliferation

Absence of the prostacyclin, seretonine, ang 1, and BMPR changes

No change on Kv canal gen expression with different from human PAH

(Ivy DD et al. Pediatr Res 1996;39:435-42)

In-utero compression/ligation of the DA

CDH

47

I. Acute models; acute hypoxia and infusion of vasoconstrictors

II. Chronic models;

•Before and immediately after birth

•Generally, weanling rat and calf model

•Calf: prominent thickening of PA adventitia, and resembles the pathological picture in human neonatal PAH

(Meyrick B. Clin Chest Med. 2001;22:393–404)

•Rat: develops marked PA adventitial thickening

(Wistar-Kyoto rats)

4. Neonatal models of hypoxic PAH

48

Newborn rats Bronchopulmonary dysplasia1 PAH2 constructed

1 (Han RN et al. Pediatr Res 1996;39:921-9)2 (Koppel R et al. Pediatr Res1994;36:763-70)

Method: Sprague-Dawley pregnant rat, FiO2:60%,

control:21% O2

Dams and their litters 4,7,9,10,14. days 60% O2

(Jankov RP et al. Pediatr Res 2001;50:172-183)

(Buch S et al. Pediatr Res 2000; 48:423-33)

(Jankov RP et al. Am J Physiol Lung Cell Mol Physiol 2005;288:1162-70)

5. Hyperoxic PAH Model

49

Pre/postnatal rats exposed to 60% O2

for 14 days (a model for human BPD-newborn PAH model) develops

- RVH-Thickening of the medial layer -Expression of ET-1 increase

(Jankov RP et al. Am J Physiol Lung Cell Mol Physiol 2005;288:1162-70)

(Jankov RP. Pediatr Res 2000;48: 289–98)

The effect of 60% O2

exposure for 14 days in the adult rat ?

Chronic lung epithelial injury (Crapo

JD et al. Am J Physiol Lung Cell Mol Physiol 1994;267:797-806)

7 day, 60% O2 adult rat: minimal histological changes, limited vascular endothelial changes

(Hayatdavoudi G et al. J Appl Physiol 1981;51:1220-31)

5. Hyperoxic PAH Model

50

Composed to gen modification which have a

role in PAH pathobiology

5. Genetically modified models of PAH

51

gp91phox knockout mice + chronic hypoxia

More less superoxide production

VC response to ET-1 decrease

(Liu JQ et al. Chest 2005;128:594-6)

(Fresquet F et al. Br J Pharm 2006;148: 714-23)

Enhanced VC response to ET-1, the superoxide

overproduction via NADPH oxidase gp91phox plays a

central role

52

cav-1 knockout mice model

An useful model of PAH in lung fibrosis Cav-1 have a negative regulatory on eNOS

activity Cav-1 KO mice: - eNOS activity increase, NO increase,

occurs hyperactivity of the eNOS-NO pathway

Dysfunctional NO signaling have a pivotal role in

pathogenesis (Wunderlich C et al. Pulm Pharmacol Ther 2007; Doi:10.1016/j.pupt.2007.11.005)

53

Increase in in-situ thrombosis and procoagulant

activity

egr-1,TF expression and vascular fibrin deposition decrease due to hypoxia

PCK -egr-1-TF pathway induced by hypoxia may be

important to play a part in remodeling process (Yan SF et al. Biol Chem 2000;275:11921-8)

PKC null mice

Sgk-1 deficient mice

Thrombin increase ROS generation via activate NADPH oxidases

TF expression and activation stimulates by sgk-1

(BelAiba RS et al. Circ Res 2006;98:828-36)

54

HIF-1α +/- heterozygous null alleles

(Yu AY et al. J Clin Invest 1999;103:691-6)

In HIF-1α +/- mice with chronic hypoxia:

Development of PAH and RVH decrease Muscularization and medial thickness

decrease Development of polisitemia decrease

55

5-HT

Mice overexpressing 5-HTT (5-HTT+) develops spontaneous

PAH 5-HTT deficient

mice are less susceptible to hypoxia

(Guignabert C et

al. Cir Res 2006;98:1323-30)

(Edhabibi S et al. J Clin Invest 2000;105:1555–62)

tph1-/- mice

Expression of the tph1 gene increase in endothelial cells in IPAH

Tph deficiency mice must be protected to hypoxia induced PAH

(Morecroft I et al. Hypertension 2007;49:232-6)

56

PGIS overexpressing transgenic mice (tg+)

Activity of PGIS increased 2-fold No vascular remodeling was occured PGIS tg+ mice had not develope PAH after

exposure to chronic hypoxia (Geraci MW et al. J Clin Invest 1999;103:1509-15)

In rats, PGIS expressing plasmid+ HVC liposome

complex intratracheal transfection Intratracheal transfer of the PGIS gene

augmentes prostacyclin synthesis, protects MCT-induced

PAH (Nagaya N et al. Circulation 2000;102:2005-10)

57

VEGF-B -/- deficiency mice

Less sensible to hypoxia induced PAH (Wanstall JC et al. Cardiovasc Res 2002)

PAH with chronic hypoxia similar to wild type (Louzier V et al. Am J

Physiol Lung Cell Mol Physiol 2003;284:926-37)

Ad.VEGF A and ad.VEGF B overexpression mice

Protective effect against to hypoxic PAH (Louzier V et al. Am J Physiol Lung Cell Mol Physiol 2003;284:926-37)

58

VIP knockout mice (VIP-/-)

Development of moderate PAH in normoxia (Intimal proliferation is absent) A useful model for studying molecular

mechanisms of PAH and evaluating therapeutic approach

(Said SI et al. Circulation 2007;115:1260-8)

AM deficient heterozygous mice (AM+/-) Severe pulmonary vascular remodeling in

AM (+/-) mice with hypoxia (Matsui H et al. Circulation 2004;109:2246-51)

59

eNOS (-/-) deletion in mice

Mild PAH in normoxia Moderate PAH in mild hypoxemia No aggravated response with severe

hypoxemia (Fagan KA et al. J Clin Invest 1999;103:291–9)

Deletion of eNOS exaggerate pulmonary Hypertensive response to chronic hypoxia (Miller AA et al. Am J Physiol Lung Cell Mol Physiol 2005;289:299–306)

60

No increase in PAP under normoxia Similar to wild type PAH develops under

hypoxia Greater elevation of PAP with IL-1ß and ad.5-

LO overexpression and chronically serotonin infusion

(Long L. Circ Res 2006;98:818-27)

(Song Y. Circulation 2005;112:553-62)

Heterozygous BMPR-2 +/- mice

Homozygous BMPR-2 (-/-)

mice

in-utero exitus

Heterozygous BMPR-2 (+/-)

mice

Morphologicaly normal, PAH not

developed spontaneously

SMC spesific tg mouse expressing dominant (-) BMPR2

Spontaneously,moderate PAH and remodeling

(West J Circ Res 2004;94:1109-14)

61

AAV-Ang-1 injected rats

Advanced events of PAH similar to human disease without any additional stimulus

Rare plexiform lesion(Chu D. Ann Thorac Surg 2004;77:449-56)

AAV-sTIE2 rats

Molecular blocking of the interaction between ang-1 and TIE2 prevents MCT and ang-1 overexpression induced PAH

(Kido M. J Thorac Cardiovasc Surg 2005;129:268-76)

62

Fawn-Hooded Rat (FHR)

Develops severe PAH with mild hipoxia Marked medial thickness in PA Pruning Sustained pulmonary vasoconstriction

FHR is a appropriately genetic model that may be linked with the development of PAH

(Sato K. Am Rev Respir Dis 1992;145:793-7)

63

Repeated microembolizations with sephadex microspheres in dogs (Shelub I et al. J appl Physiol

1984;56:810-5)

Repeated microembolizations with polydextran microspheres in piglets

(Weimann J et al. J Crit Care 1999;14:133-40)

Continuous air embolization in sheeps (Johnson JE et al. Exp Lung Res 1997;23:459-73)

Formed repeated venous thrombi in-vivo + tranexamic acid in dogs

(Mosser KM et al. Circulation 1991;83:1371-9)

(Cantor JP et al. Am Rev Respir Dis 1988;137:185A)

Postobstructive pulmonary vasculopathy(Michel RP et al. J appl Physiol 1990;69:1022-32)

(Giaid A et al. J vasc Res 1993;30:333-338)

6. Chronic embolic PH

64

Mechanical obstruction and vasoconstriction

(Perkett et al. Am J Pathol 1988;132:444-54) (Kim et al. Eur Respir J 2000;15.640-8)

(Rectenwald JE et al. J vasc Surg

2006;43:800-8)

(Perkett et al. Am J Pathol 1988;132:444-54)

65

In vitro cultures of PA-SMC may be use investigation of

the effect of potantial antiproliferative agents and

mitogens

Growth of human distal PA-SMCs derived from the middle layer of the media

More rapid proliferation

Proximal PA-SMCs displayed more slow growth rate

Isolation of cells in resistive PA is difficult Heterogeneity of vascular SMC

(Wharton J et al. Circulation 2000;102:3130-6)

7. Cell cultures

66

The differences of growth stimulus (PDGF / serum) The evaluation index of proliferative activity (cell number / DNA synthesis) The concentration of agent

The evaluation of effectiveness to therapeutic

agents

(Clap LH et al. Am J Respir Cell Mol Biol 2002;26:194-201)

67

The disease was not composed on cell

bases or studied models of adopted to

likely hypothesis

Results

No animal model reproduces the full

spectrum of changes seen in human PAH

Signalization anomaly that begin and

continue to disease just don’t know(Naeije R,Dewatchter L. Rev Mal Respir 2007; 24: 481-96)

68

New animal models of PAH which neointimal lesiondeveloped should be preferred for the research ofnew therapies of PAH

Rodent models of distal PA neointimallesion formation

Left pneumonectomy + MCT

VEGF receptor blocker + chronic hypoxia

ETB receptor deficient rat + MCT

S100A4/Mts1 overexpressing mice model

Results

69

Animal models, even with their limitations:

A variety of combination therapies and more novel treatments will have been evaluated

Perhaps the use of several unnecessary or harmful therapies will be decreased

THANKS