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Experimental Models of Pulmonary Arterial Hypertension

Experimental Models of Pulmonary Arterial Hypertension. Dr Figen Deveci F U , Department of Chest Disease s. PRESENTATION PLAN. 1. Definition of animal models of PAH 2. In generally, validity of animal models and differences from humans PAH.

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Experimental Models of Pulmonary Arterial Hypertension

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  1. Experimental Models of Pulmonary Arterial Hypertension Dr Figen Deveci FU, Department of Chest Diseases

  2. PRESENTATION PLAN 1. Definition of animal models of PAH 2. In generally, validity of animal models and differences from humans PAH

  3. In the last 100 years of PAH research (Zaiman A et al. Am J Respir Cell Mol Biol 2005;33:425-31)

  4. Animal models in PAH Aims; 1. Characterizing the pathophysiology of PAH 2. Researching its sequela such as RVH and failure 3. Testing novel therapeutic strategies Ideal animal model of PAH Creating 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. 1. Hypoxia induced PAH model Hypobaric hypoxia Normobaric hypoxia Normal air at hypobaric pressure Oxygen poor-air at normal pressure By lowering atmospheric pressure (one-half), (380 mmHg), 10-15 day Increased PAP50% (Ozaki M et al. Hypertension 2001;37:322-7) 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)

  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. 1A. The model of chronic hypoxia Acute pulmonary VC Acute hypoxic exposure PAP Remodeling of small pulmonary arteries Chronic hypoxic exposure (Voelkel NF, Tuder RM. J Clin Invest 2000;106:733-8)

  9. HPV in animal models • In animal experiments: PAO2< 70 mmHg elicits strong pulmonary arterial VC • HPV is common in mammals, differences are exist according to species • 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. Respiration1984;45:169-174) In animals, • Rapid increase in PVR with HPV • Gradually plateaus • Similar to in humans

  11. HPV in animal models Endothel isimportantin modulation of HPV Biphasic HPV Phase 1:immediate, end- independent constriction, peaks in 10 min Phase 2:slowly, end- dependent, sustained constriction, peaks in 40 min (Ward JP. Exp Physiol 1995; 80: 793-801) Monophasic HPV Denuded of endothelium, isolated PA: monophasic constriction (Archer SL et al. FASEB J 2001;15: 1801-3)

  12. Transition time of remodeling from HPV? Rat hiler PA • In humans, progress VC to remodeling in first 24 hour (Naeije R,Dewatchter L. Rev Mal Respir 2007; 24: 481-96) Adv Media In rats; • Endothelial changes at day 3th(Meyrick B, Reid L. Lab Invest. 1978;38:188-200) • RVH, at day 5th • 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)

  13. (Chen SJ et al. J Appl Physiol 1995;79 : 2122-31) 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) (Stenmark K et al. J Appl Physiol 1987;62:821-30) Endothelial cell proliferation not compose significantly (Chen SJ et al. J Appl Physiol 1995; 79 : 2122-31)

  14. In hypoxic neonatal calve; • Extreme elevation of PAP • Prominent intimal thickening In fast-growing broiler chicken • Remodeling in all layers (adventitia, media, intima) (Peacock AJ et al. Am Rev Respir Dis 1989;139:1524-30) “In a model ofhypoxia+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)

  15. Vascular remodeling in PA • Large proximal PA • Distal muscular PA • Non-muscular alveolar vessels • Adventitia • Endothel

  16. Remodeling in large proximal PA Large animals (calf, pig) • Early and dramatic medial thickening • Less thickening of adventitia 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)

  17. Adventitial remodeling • Early and dramatic in all animal species • 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) • 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)

  18. 1B. Intermittent hypoxia in animal models 1. Model (rat) 2. Model (mice) 3. Model (rat) 2min 10% O2 , followed by 2min normoxia, 8h/d, 4 week Hypobaric hypoxia,4-8 h/d, 5-7d/w,FiO2: 561ve 70mmHg2 Similar to OSA consecutive 30sec hypoxia30sec normoxicperiods, 8h/d, 5 week • Increase inmeanPAP • Increase in right ventricle weight (McGuire M, Bradford A. Eur Respir 2001;18: 279-85) • 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)

  19. 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. Differences between animals and humans in IH • 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)

  21. Differences between rats and humans in hypoxia-induced remodeling (Zielinski J. Eur Respir J 2005;25:173-80)

  22. “The rat model is the bad model for Hypoxic PH” (Heath D. Cardioscience 1992;3:1-6) 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)

  23. Animals age in hypoxia-induced PAH Newborn rat • Normal pulmonary arterial development is very similar to that seen in the human • Hypoxic/MNC induced PAH is more prominent than adult animals (Belik J et al. J appl Physiol 2003;94:2303-12) • The model of PAH in newborn rat is a useful model (Meyrick B, Reid L. Am Rev Respir Dis 1982;125:468–73)

  24. Hemodynamic measurements Closed-chest pressure recordings • Minimally invasive • Serial measurements • Technically demanding • Quality of signal was poor Direct pressure measurement -left lateral thoracotomy • More easily applicable • Without substantial differences in pressure values (Rabinovitch M et al. Am J Physiol 1979;236:818-27) (Kolettis T et al. Hellenic J Cardiol 2007;48:206-10)

  25. Right Ventricular Hypertrophy • Right ventricular free wall (RV) • Left ventricle together with septum (LV+S) • Weighed andexpressed as a ratio LV+S RV (Fulton RM et al. Br Heart J 1952;14:413-20)

  26. Quantitative morphological study Lung volumes • By water displacement • Angiograms (Ba-jelatin) Muscularization AWA% The thickness of medial muscular coat Calculating as the percentage of external diameter Reduction in the number of small arteries Increased ratio of the number of alveols/arteries (Rabinovitch M et al. Am J Physiol 1979;236:818-27)

  27. 1. Remodeling • Lumen occlusion due to medial and adventitial thickness • Not occurring maximal VD %MT=2xMT/external diameterx100 • Maximum VD • Transmuraldistending pressure • Medial cross-sectional area • Lumen area and MA/LA Increase in PAP,RVH (+) Medialthickness (+) Narrowing of lumen area(-) Outward remodeling (VanSuylen RJ et al. Am J Respir Crit Care Med 1998;157:1423-8) (Stenmark KR, McMurtry IF. Circ Res 2005;97:95-8)

  28. Quantitative stereology+confocalmicroscopy (Hyvelin JM et al. Circ Res 2005;97:185-91) (Howell K et al. J Physiol 2003;547:133-45)

  29. 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 Synd Hypoxia/hypoxemia COPD, ILD Hypoxia/hypoxemia + inflammation Eisenmenger Syndrome High pulmonary blood flow

  30. 2. Monocrotaline induced PAH model • 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 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 • MCT sensitivity different between rat strains Age: younger rat (2 w) more susceptible to theeffects of MCT Gender: female rats suffer more non-pulmonary organ damage (Schoental R, Head MA. Br J Cancer 1955;9:229-37)

  31. 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)

  32. Human PAH MCT induced PAH

  33. MCT induced PAH model Not develop plexiform lesions Standard modelfor human PAH 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)

  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 Shunt 1 week 1 week Left PA anastomosed directly end-to- end left subclavian artery MCT neointimal formation 4 week PAH (neointimal formation) (Tanaka Y et al. J Clin Invest 1996;98:434-42)

  36. Differences between MCT and CH models

  37. 3. Over-circulation induced PAH model • An aorta-to-pulmonary shunts with increased pulmonary blood flow in dogs • Between abdominal aorta and VCI in rats (Garcia R, Diebold S. Cardiovasc Res 1990; 24: 430-2) • 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)

  38. 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) As young as possible growing pigs “Blalock-Taussing operation” Left subclavian artery and pulmonary truncus were shunted because of naturally growing left-to-right shunt with the animal growth After the 3-4 mounth growing • Increases of PVR • Marked small pulmonary arteriolar medial hypertrophy • Severe PAH (PAP 30-40 mmHg)

  39. In-utero aorto-pulmonary shunts in the lamb -mPAP:40 mmHg, PVR increased -Dilatation and background haze increase -%WT increase (<200 µm) -Muscularization -The number of intra-acinar PA increase -Endothelial dysfunction • Late-gestation • Ascending aorta with main PA (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. 4. The models of newborn persistent PAH 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

  42. 1. Ductal ligation lamb model of PPHN • 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) • Increase in intra-uterin PAP, RVH • Medial hypertrophy • Muscularization • Adventitial remodeling • Severe PAH

  43. 2. Partial compression of DA in fetal lamb • Absence of fetal hypoxemia and high blood flow Intrauterine; • Sustained PAH, RVH • Altering fetal pulmonary vasoreactivity • PA; increases of medial thickness, luminal occlusion • The 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

  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. 3. The models of CDH 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)

  46. In-utero compression/ligation of the DA • CDH • 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 expressionwith different from human PAH (Ivy DD et al. Pediatr Res 1996;39:435-42)

  47. 4. Neonatal models of hypoxic PAH 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)

  48. 5. Hyperoxic PAH Model 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)

  49. 5. Hyperoxic PAH Model • The effect of 60% O2 exposure for 14 daysin 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) • 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)

  50. 5. Genetically modified models of PAH Composed to gen modification which have a role inPAHpathobiology

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