Advances in understanding of pulmonary arterial hypertension and the evolution of Experimental pulmonary hypertension models

PVRI Member Authors: Michiel Alexander de Raaf

“Primary Pulmonary Hypertension has been called the cardiologist’s cancer”

Greg Elliott in personal conversation with Norbert Voelkel, early 80’s:

Pulmonary Arterial Hypertension (PAH) can be a rapidly progressive and devastating disease characterized by dysfunction and remodeling of the pulmonary vasculature, leading to increased pulmonary vascular resistance. The increased vascular resistance pushes the right ventricle (RV) into adaptive compensatory remodeling by hypertrophy, but eventually RV dilatation, heart failure and death of the patient become inevitable [1, 2]. Today, 3 pathways are targeted in PAH treatment: the nitric oxide-cyclic guanosine monophosphate pathway, the endothelin pathway and the prostacyclin pathway [3]. Although treatments affecting these pathways delay disease progression and increase survival rates [3, 4], they do not cure PAH [3]. The pathogenic paradigm of PAH has shifted from pulmonary vasoconstriction driven by smooth muscle cells to vascular remodeling affecting all vessel wall layers. In this chapter, a brief overview will be given of how PAH research has converged on the role of the endothelial cell (EC). It will be argued that the development of new animal models has been critical to the development of a new PAH paradigm.

In 1970, the first morphologic characterization of the pulmonary hypertensive lung was published by Wagenvoort et al. [5, 6], followed by the first WHO classification of Pulmonary Hypertension in 1972. In this classification, the pathological focus in PAH, then called Primary Pulmonary Hypertension, was on the vascular media of the pulmonary arterioles, featuring hypertrophied and hyperplastic pulmonary artery smooth muscle cells (PASMC) [1, 2, 7]. The “vascular media paradigm” of PAH ascribed a major pathogenic role to sustained pulmonary vasoconstriction and was fueled by two PAH outbreaks related to the use of the appetite suppressants Aminorex (Menocil®) and fenfluramine (Ponderal®) in the late 60’s and 80’s. Both drugs are serotonin transporter substrates acting on the PASMC and promote vasoconstriction and PASMC hypertrophy and proliferation. At that time, the signature plexiform lesion was regarded as an epiphenomenon.

The description of idiopathic PAH as ‘the cardiologist’s cancer’ in the early 80’s meant at that time PAH and cancer shared a common clinical and scientific context. Both conditions were untreatable due to a profound lack of understanding and absence of technologies to study pathogenesis and pathobiology. As dedicated research did not lead to a medical cure for PAH, the idea that other vascular wall cells than smooth muscle cells were also involved in the disease was gradually accepted. For a better understanding of PAH, it was necessary to study the cellular and molecular aspects of the disease. An expanding research effort revealed the mechanisms by which PAH is characterized today: endothelial dysfunction, PASMC hypertrophy and hyperplasia, persistent inflammation and dysimmunity and dysregulated intra- and extracellular cell signaling in the cells of all layers of the pulmonary vessel bed, leading to pulmonary vessel remodeling [8–10]. Endothelial dysfunction is reflected in increased activity of contractive vasoactive agents as endothelin-1, serotonin, angiotensin II and decreased activity of dilative vasoactive agents as nitric oxide and prostacyclin, felt to lead to persistent vasoconstriction. The hyperproliferative endothelium in PAH disobeys the “law-of-the-monolayer” and has a monoclonal origin in plexiform lesions [11–13]. This hyperproliferation is mediated by increased activity of growth factors as fibroblast growth factor-2, platelet derived growth factor and epidermal growth factor and is accompanied by apoptopic resistance [14–17]. Also in the PASMC the increased activity of growth factors and apoptosis resistance are observed. As well in the EC, hyperproliferation of PASMC is mediated by migration and dysregulated BMPRII signaling, for example due to the germline BMPRII-mutation, which has a penetrance of approximately 75% in hereditary familial PAH [10, 14, 15]. Another germline mutation in PAH is found in the KNCK3 gene, which decreases the activity of TWIK-related acid-sensitive K+ channel-1 [15]. Together with alterations in the voltage-gated K+ channel, transient receptor potential 1 and 6 and calcium sensing receptor, the PASMC is hyperpolarized [9, 18, 19]. Moreover, the PASMC in PAH goes through an energetic shift to glycolysis [20], also found in fibroblast of the pulmonary adventitia [21], which is similar to the Warburg effect. These dysregulated mechanisms also affect the extracellular matrix [22], and contribute to the abnormal cell signaling leading to disordered angiogenesis [16, 17, 23], and recruit inflammatory cells in the pulmonary vasculature which can release cytokines and chemokines as for example interleukin 1 and 6 [11, 15, 24–26]. It is now recognized that all layers of the vessel wall contribute to the pulmonary vascular remodeling; including the hyperproliferative endothelium, and the plexiform lesion is no longer neglected as a hallmark of PAH [27, 28]. Many of the observations made during the study of pulmonary vascular remodeling in PAH, echoed the description of the hallmarks of cancer as described by Hanahan and Weinberg [28–31]. This expansion in knowledge gave rise to the exploration of potential treatment targets, which were already studied for their efficacy in cancer, such as Histone DeACetylase inhibitors (HDACis) and Tyrosine Kinase Inhibitors (TKIs) [32].

Experimental models to resemble pulmonary arterial hypertension

Translational research on the development of pulmonary hypertension and potential treatments of the disease has relied on animal models that replicate one or more important aspects of the disease [33–36]. Reeves and Grover were the first to use hypoxia induced pulmonary hypertension as a model of the human condition when they studied Brisket disease in cattle kept at high altitude [37, 38]. Exposure to hypoxia was soon also used to study pulmonary hypertension in rats. In addition, an animal model based on the administration of monocrotaline was first used in 1967 [39, 40]. The monocrotaline and chronic hypoxia animal models of pulmonary hypertension both exhibit profound remodeling of the vascular media and hypertrophy of PASMC. Because these phenomena were considered singularly important in human pulmonary hypertension, it was felt that there was little need to develop alternative animal models.

The expansion in pathobiological knowledge of PAH has coincided with the establishment of new animal models of the disease. When in 2001 the Sugen Hypoxia model (SuHx) of PAH was discovered [41], the striking resemblance was appreciated between the pathological changes in the SuHx rat and the human PAH lung. More specifically, it was noted that the intima remodeling and obliterative vascular remodeling that is typical of human PAH, was mimicked by the lung vascular changes found in the SuHx rat [35, 41, 42]. The SuHx model also brought the understanding that the emergence of a proliferative endothelium could require an initial phase of endothelial apoptosis, as it could be prevented by a broad spectrum caspase inhibitor [41, 43]. Also in other new animal models intimal proliferation were found in the combination of monocrotaline plus aortic caval shunt or monocrotaline plus pneumonectomy [44–46].

Despite increasing popularity, the SuHx model has not (yet) become a ‘gold standard’ for animal research in PAH. In comparison to the classic animal models, the SuHx model is more complex, has not been thoroughly characterized and is more difficult to implement and harmonize among laboratories. In addition, a vast amount of translational studies has been performed with the chronic hypoxia and monocrotaline models and this body of work is a heritage of historical literature to which new data is conveniently compared. Therefore, many translational studies are still performed using the traditional animal models. Remarkably, many potential treatments adopted from the cancer field, are still explored in the classic animal models that do not represent the pathobiology of angio-obliterative PAH nor offer a hyperproliferative endothelium as a treatment target. This might explain why therapeutic interventions seem curative in animal studies whereas human PAH remains refractory to treatment [47, 48].

Conclusion

Due to the ineffectiveness of vasodilating drugs and aided by many cellular and molecular findings, the pathogenic paradigm of PAH has shifted from vasoconstriction towards vessel remodeling with resemblances to malignancy. Concurrently, novel animal models have evolved, which feature the human hallmark of PAH, a hyperproliferative endothelium, that is not found in more traditional animal models. Therefore, when these traditional animal models are used to assess anti-proliferative drug targets of the endothelium, they become unpredictable in their translational value. Of course, due to the unknown pathogenesis of PAH, it is paradoxically true that no animal model can resemble PAH completely. However, with the increasing realization and evaluation of the different pathobiologies resembled by animal models [33–35], the selection of the used animal model should be explicitly motivated.

 

 

References

1. Simonneau G, Robbins IM, Beghetti M, Channick RN, Delcroix M, Denton CP, Elliott CG, Gaine SP, Gladwin MT, Jing Z-C, Krowka MJ, Langleben D, Nakanishi N, Souza R. Updated clinical classification of pulmonary hypertension. J. Am. Coll. Cardiol. 2009; 54: S43–S54.

2. Simonneau G, Gatzoulis MA, Adatia I, Celermajer D, Denton C, Ghofrani A, Gomez Sanchez MA, Krishna Kumar R, Landzberg M, Machado RF, Olschewski H, Robbins IM, Souza R. Updated clinical classification of pulmonary hypertension. J. Am. Coll. Cardiol. 2013; 62: D34–D41.

3. Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. N. Engl. J. Med. 2004; 351: 1425–1436.

4. D’Alonzo GE, Barst RJ, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, Fishman AP, Goldring RM, Groves BM, Kernis JT, others. Survival in patients with primary pulmonary hypertension. Ann. Intern. Med. 1991; 115: 343– 349.

5. Wagenvoort CA, Wagenvoort N. Primary Pulmonary Hypertension A Pathologic Study of the Lung Vessels in 156 Clinically Diagnosed Cases. Circulation 1970; 42: 1163–1184.

6. Wagenvoort CA. Vasoconstrictive primary pulmonary hypertension and pulmonary venoocclusive disease. Cardiovasc. Clin. 1972; 4: 97– 113.

7. Simonneau G, Galiè N, Rubin LJ, Langleben D, Seeger W, Domenighetti G, Gibbs S, Lebrec D, Speich R, Beghetti M, Rich S, Fishman A. Clinical classification of pulmonary hypertension. J. Am. Coll. Cardiol. 2004; 43: 5S – 12S.

8. Guignabert C, Tu L, Girerd B, Ricard N, Huertas A, Montani D, Humbert M. New molecular targets of pulmonary vascular remodeling in pulmonary arterial hypertension: importance of endothelial communication. Chest 2015; 147: 529–537.

9. Rabinovitch M. Molecular pathogenesis of pulmonary arterial hypertension. J. Clin. Invest. 2012; 122: 4306–4313.

10. Voelkel NF, Gomez-Arroyo J, Abbate A, Bogaard HJ, Nicolls MR. Pathobiology of pulmonary arterial hypertension and right ventricular failure. Eur. Respir. J. 2012; 40: 1555– 1565.

11. Tuder RM, Groves B, Badesch DB, Voelkel NF. Exuberant endothelial cell growth and elements of inflammation are present in plexiform lesions of pulmonary hypertension. Am. J. Pathol. 1994; 144: 275.

12. Voelkel NF, Cool C, Lee SD, Wright L, Geraci MW, Tuder RM. Primary pulmonary hypertension between inflammation and cancer. Chest 1998; 114: 225S – 230S.

13. Lee SD, Shroyer KR, Markham NE, Cool CD, Voelkel NF, Tuder RM. Monoclonal endothelial cell proliferation is present in primary but not secondary pulmonary hypertension. J. Clin. Invest. 1998; 101: 927–934.

14. Deng Z, Morse JH, Slager SL, Cuervo N, Moore KJ, Venetos G, Kalachikov S, Cayanis E, Fischer SG, Barst RJ, Hodge SE, Knowles JA. Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am. J. Hum. Genet. 2000; 67: 737–744.

15. Guignabert C, Tu L, Girerd B, Ricard N, Huertas A, Montani D, Humbert M. New molecular targets of pulmonary vascular remodeling in pulmonary arterial hypertension: importance of endothelial communication. Chest 2015; 147: 529–537. Learners’ Corner 59 Tu L, Dewachter L, Gore B, Fadel E, Dartevelle P, Simonneau G, Humbert M, Eddahibi S, Guignabert C. Autocrine fibroblast growth factor-2 signaling contributes to altered endothelial phenotype in pulmonary hypertension. Am. J. Respir. Cell Mol. Biol. 2011; 45: 311–322.

17. Schermuly RT. Reversal of experimental pulmonary hypertension by PDGF inhibition. J. Clin. Invest. 2005; 115: 2811–2821.

18. Bonnet S, Michelakis ED, Porter CJ, Andrade-Navarro MA, Thébaud B, Bonnet S, Haromy A, Harry G, Moudgil R, McMurtry MS, Weir EK, Archer SL. An abnormal mitochondrialhypoxia inducible factor-1alpha-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: similarities to human pulmonary arterial hypertension. Circulation 2006; 113: 2630–2641.

19. Michelakis ED, McMurtry MS, Wu X-C, Dyck JRB, Moudgil R, Hopkins TA, Lopaschuk GD, Puttagunta L, Waite R, Archer SL. Dichloroacetate, a metabolic modulator, prevents and reverses chronic hypoxic pulmonary hypertension in rats: role of increased expression and activity of voltage-gated potassium channels. Circulation 2002; 105: 244–250.

20. Xu W, Koeck T, Lara AR, Neumann D, DiFilippo FP, Koo M, Janocha AJ, Masri FA, Arroliga AC, Jennings C, Dweik RA, Tuder RM, Stuehr DJ, Erzurum SC. Alterations of cellular bioenergetics in pulmonary artery endothelial cells. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1342–1347.

21. Stenmark KR, Tuder RM, El Kasmi KC. Metabolic Reprogramming and Inflammation Act in Concert to Control Vascular Remodeling in Hypoxic Pulmonary Hypertension. J. Appl. Physiol. Bethesda Md 1985 2015; : jap.00283.2015.

22. Jones PL, Cowan KN, Rabinovitch M. Tenascin-C, proliferation and subendothelial fibronectin in progressive pulmonary vascular disease. Am. J. Pathol. 1997; 150: 1349–1360.

23. Tuder RM, Chacon M, Alger L, Wang J, Taraseviciene-Stewart L, Kasahara Y, Cool CD, Bishop AE, Geraci M, Semenza GL, Yacoub M, Polak JM, Voelkel NF. Expression of angiogenesisrelated molecules in plexiform lesions in severe pulmonary hypertension: evidence for a process of disordered angiogenesis. J. Pathol. 2001; 195: 367–374.

24. Huertas A, Perros F, Tu L, CohenKaminsky S, Montani D, Dorfmüller P, Guignabert C, Humbert M. Immune dysregulation and endothelial dysfunction in pulmonary arterial hypertension: a complex interplay. Circulation 2014; 129: 1332–1340.

25. Molossi S, Clausell N, Rabinovitch M. Reciprocal induction of tumor necrosis factoralpha and interleukin-1 beta activity mediates fibronectin synthesis in coronary artery smooth muscle cells. J. Cell. Physiol. 1995; 163: 19–29.

26. Steiner MK, Syrkina OL, Kolliputi N, Mark EJ, Hales CA, Waxman AB. Interleukin-6 overexpression induces pulmonary hypertension. Circ. Res. 2009; 104: 236–244, 28p following 244.

27. Adnot S, Eddahibi S. Lessons from oncology to understand and treat pulmonary hypertension. Int. J. Clin. Pract. Suppl. 2007; : 19–25. Molossi S, Clausell N, Rabinovitch M. Reciprocal induction of tumor necrosis factoralpha and interleukin-1 beta activity mediates fibronectin synthesis in coronary artery smooth muscle cells. J. Cell. Physiol. 1995; 163: 19–29.

28. Guignabert C, Tu L, Le Hiress M, Ricard N, Sattler C, Seferian A, Huertas A, Humbert M, Montani D. Pathogenesis of pulmonary arterial hypertension: lessons from cancer. Eur. Respir. Rev. Off. J. Eur. Respir. Soc. 2013; 22: 543–551. Lear

29. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: 57–70.

30. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144: 646– 674.

31. Rai PR, Cool CD, King JAC, Stevens T, Burns N, Winn RA, Kasper M, Voelkel NF. The cancer paradigm of severe pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 2008; 178: 558–564.

32. Stenmark KR, Rabinovitch M. Emerging therapies for the treatment of pulmonary hypertension. Pediatr. Crit. Care Med. J. Soc. Crit. Care Med. World Fed. Pediatr. Intensive Crit. Care Soc. 2010; 11: S85–S90.

33. Ryan J, Bloch K, Archer SL. Rodent models of pulmonary hypertension: harmonisation with the world health organisation’s categorisation of human PH. Int. J. Clin. Pract. Suppl. 2011; : 15– 34.

34. Stenmark KR, Meyrick B, Galie N, Mooi WJ, McMurtry IF. Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure. Am. J. Physiol. Lung Cell. Mol. Physiol. 2009; 297: L1013–L1032.

35. Nicolls MR, Mizuno S, TarasevicieneStewart L, Farkas L, Drake JI, Al Husseini A, Gomez-Arroyo JG, Voelkel NF, Bogaard HJ. New models of pulmonary hypertension based on VEGF receptor blockade-induced endothelial cell apoptosis. Pulm. Circ. 2012; 2: 434–442.

36. Voelkel NF, Schranz D, editors. The Right Ventricle in Health and Disease [Internet]. New York, NY: Springer New York; 2015 [cited 2015 May 18].Available from: http://link.springer. com/10.1007/978-1-4939-1065-6.

37. Reeves JT, Leathers JE. Hypoxic pulmonary hypertension of the calf with denervation of the lungs. J. Appl. Physiol. 1964; 19: 976–980.

38. Will DH, Alexander AF, Reeves JT, Grover RF. High Altitude-Induced Pulmonary Hypertension in Normal Cattle. Circ. Res. 1962; 10: 172–177.

39. Kay JM, Harris P, Heath D. Pulmonary hypertension produced in rats by ingestion of Crotalaria spectabilis seeds. Thorax 1967; 22: 176–179.

40. Rosenberg HC, Rabinovitch M. Endothelial injury and vascular reactivity in monocrotaline pulmonary hypertension. Am. J. Physiol. - Heart Circ. Physiol. 1988; 255: H1484–H1491.

41. Taraseviciene-Stewart L, Kasahara Y, Alger L, Hirth P, Mc Mahon G, Waltenberger J, Voelkel NF, Tuder RM. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 2001; 15: 427–438.

42. De Raaf MA, Schalij I, Gomez-Arroyo J, Rol N, Happé C, de Man FS, Vonk-Noordegraaf A, Westerhof N, Voelkel NF, Bogaard HJ. SuHx rat model: partly reversible pulmonary hypertension and progressive intima obstruction. Eur. Respir. J. 2014; 44: 160–168.

43. Sakao S, Taraseviciene-Stewart L, Lee JD, Wood K, Cool CD, Voelkel NF. Initial apoptosis is followed by increased proliferation of apoptosisresistant endothelial cells. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 2005; 19: 1178–1180.

44. Okada K, Tanaka Y, Bernstein M, Zhang W, Patterson GA, Botney MD. Pulmonary hemodynamics modify the rat pulmonary artery response to injury. A neointimal model of pulmonary hypertension. Am. J. Pathol. 1997; 151: 1019–1025.

45. White RJ, Meoli DF, Swarthout RF, Kallop DY, Galaria II, Harvey JL, Miller CM, Blaxall BC, Hall CM, Pierce RA, Cool CD, Taubman MB. Plexiform-like lesions and increased tissue factor expression in a rat model of severe pulmonary arterial hypertension. Am. J. Physiol. Lung Cell. Mol. Physiol. 2007; 293: L583–L590.

46. Van Albada ME, Bartelds B, Wijnberg H, Mohaupt S, Dickinson MG, Schoemaker RG, Kooi K, Gerbens F, Berger RMF. Gene expression profile in flow-associated pulmonary arterial hypertension with neointimal lesions. Am. J. Physiol. Lung Cell. Mol. Physiol. 2010; 298: L483– L491.

47. Voelkel NF, Mizuno S, Bogaard HJ. The role of hypoxia in pulmonary vascular diseases: a perspective. Am. J. Physiol. Lung Cell. Mol. Physiol. 2013; 304: L457–L465.

48. Gomez-Arroyo JG, Farkas L, Alhussaini AA, Farkas D, Kraskauskas D, Voelkel NF, Bogaard HJ. The monocrotaline model of pulmonary hypertension in perspective. Am. J. Physiol. Lung Cell. Mol. Physiol. 2012; 302: L363–L369.

 

Topics

Endothelin and Endothelium & Epithelium and Epithelial Transport
Nitric Oxide and Nitric Oxide Synthase
Pulmonary Hypertension

Published in:

PVRI Chronicle Vol 2: Issue 2 cover image

July 2015

PVRI Chronicle Vol 2: Issue 2

More from this Journal

Explore the PC Journal

Pulmonary Circulation allows diverse knowledge of research, techniques, and case studies to reach a wide readership of specialists in order to improve patient care and treatment outcomes.

shutterstock_136564397.jpg