Pulmonary hypertension: pathophysiology as a basis for clinical decision making

LUNG TRANSPLANT FORUM

Pulmonary Hypertension: Pathophysiology as a Basis for Clinical Decision Making Marlene Rabinovitch, MD

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tudies by our group of children with congenital heart disease first showed how examination of lung biopsy material in concert with hemodynamic assessment of pulmonary vascular resistance and angiographic evaluation of the pulmonary arteries could predict the success of a surgical repair and the potential for reversibility of pulmonary hypertension. With increasing evidence of the ability of continuous intravenous prostacylin to arrest progression, and even induce regression, of structurally advanced pulmonary vascular disease, the use of pathologic material for clinical decision making has undergone rethinking. While the presence of plexiform lesions was thought to represent irreversible disease and hypertrophy of the muscular media, the potential for irreversibility, this was often not borne out by the response to prostacyclin therapy. Moreover, the observation that ongoing cellular proliferation and connective tissue synthesis occurs even in advanced lesions (thought to represent end-stage “burnt-out” diseases) led to re-evaluation of the potential of reversing the disease process. Now there is an increasing need to carefully assess and document structural abnormalities in an effort to learn more about the disease process. Our laboratory has used clinical material, cultured cells, and studies in experimental animals to gain new insights From the Division of Cardiovascular Research, Research Institute, The Hospital for Sick Children, and the Departments of Pediatrics, Laboratory Medicine and Pathobiology and Medicine, University of Toronto, Toronto, Ontario, Canada. Submitted Jan. 21, 1999; accepted Feb. 15, 1999. Corresponding author and reprints: Marlene Rabinovitch, MD, Director, Division of Cardiovascular Research, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Telephone: 416-813-5918. Fax: 416-8137480. E-mail: [email protected] J Heart Lung Transplant 1999;11:1041–1053. Copyright © 1999 by the International Society for Heart and Lung Transplantation. 1053-2498/99/$–see front matter PII S1053-2498(99)00015-7

into some of the mechanisms that lead to the progression of vascular changes, and has used this information in strategies aimed at arresting progression and, more recently, inducing regression of pulmonary hypertension and associated vascular disease. Specifically, we have focused on the increased activity of an endogenous vascular elastase (EVE) and expression of the glycoproteins tenascin and fibronectin in the pathobiology of pulmonary hypertension. This article will first review our studies in children with congenital heart defects and the assessment of reversibility of pulmonary hypertension, and then discuss more recent work aimed at developing newer therapeutic strategies based upon cellular and molecular mechanisms.

PULMONARY VASCULAR DISEASE IN PATIENTS WITH CONGENITAL HEART DEFECTS Congenital heart defects with left-to-right shunts characterized by high pulmonary blood flow and high pressure induce increasing pulmonary hypertension related to progressive structural abnormalities1 associated with impaired growth of the pulmonary arteries. First observed is extension of muscle into peripheral, normally non-muscular, arteries (morphometric grade A) (Figure 1). Ultrastructural studies of lung biopsy tissue2 showed that this change is due to precocious differentiation of precursor cells (i.e., the pericyte in the non-muscular region of the artery and the intermediate cell in the partially muscular region) to mature smooth muscle cells. Since arteries become more muscular as they increase in size, we have speculated that, in the altered hemodynamic setting of chronic high flow and high pressure, “stretch” is the stimulus for smooth muscle cell differentiation from precursor cells. With grade B, there is, in addition to increased extension of muscle, severe medial hypertrophy of normally muscular arteries. When medial wall thick1041

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FIGURE 1 Schema showing peripheral pulmonary

arterial development through morphometric changes: extension of muscle into peripheral arteries, percent wall thickness, and artery number (alveolar-arterial ratio), as they relate to age. Upper panel, normal development. Bottom panel, abnormalities in all 3 features in a 2-year-old child with a hypertensive VSD. T.B., artery accompanying a terminal bronchiolus; R.B., artery accompanying a respiratory bronchiolus; A.D., artery accompanying an alveolar duct; A.W., artery accompanying an alveolar wall; ALV/Art, alveolararterial ratio. (Reproduced with permission from Rabinovitch M, et al.)1

ness is greater than 1.5 but less than 2 times normal (mild grade B), elevated mean pulmonary artery pressure is present; when medial wall thickness is more than twice normal (severe grade B), values are usually more than half the systemic level. The increase in medial thickness is due to hypertrophy as well as hyperplasia of pre-existing smooth muscle cells and also to an increase in the intercellular connective tissue proteins. With grade C, in addition to the findings of medial hypertrophy, arterial concentration is reduced. Patients with these changes usually have a pulmonary vascular resistance of greater than 3.5 ␮䡠m2. When the artery number is less than half of normal (severe grade C), pulmonary vascular resistance values are often in excess of 6 ␮䡠m2. The basis for grade C is likely the failure of new vessels to grow normally, although some loss of arteries may also occur. Morphometric grades A and B are refinements of Heath Edwards grade I (medial

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FIGURE 2 Heath-Edwards classification of pulmonary

vascular changes. A, grade I: medial hypertrophy. EVG, 150⫻. B, grade II: cellular intimal proliferation in an abnormally muscular artery. EVG, 250⫻. C, grade III: occlusive changes. Medium is thickened due to fasciculi of longitudinal muscle, and vessel is all but occluded by fibroelastic tissue. EVG, 150⫻. D, grade IV: dilatation. Vessel is dilated and medium is abnormally thin (arrow). Lumen is occluded by fibrous tissue. EVG, 150⫻. E, grade V: plexiform lesion. There is cellular intimal proliferation (arrow); clustered around are numerous thin-walled vessels that terminate as capillaries in the alveolar wall. EVG, 95⫻. F, grade VI: acute necrotizing arteritis. A severe reactive inflammatory exudate is seen through all layers of the vessel. HE, 250⫻. EVG, elastin-van Gieson stain; HE, hematoxylin eosin stain. (Reproduced with permission from Wagenvoort CA, Health D, Edwards JE. The pathology of the pulmonary vasculature. Springfield, Illinois: Charles C Thomas, 1964).

hypertrophy) (Figure 2). Grade C may be found with Heath Edwards grade I, is common with grade II (cellular neointimal formation), and is invariable with grade III (occlusive neointimal formation with fibrosis). In fact, when grade III is seen, arterial concentration is generally half normal or less. In general, features of grade III C or more severe abnormalities such as Heath Edwards grade IV (dilatation complexes), grade V angiomata formation grade VI (fibrinoid necrosis) correlate with severe elevation in pulmonary vascular resistance of ⬎8 ␮m2, which is refractory to vasodilators such as oxygen, prostacyclin, or nitric oxide.

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We subsequently determined whether the severity of these vascular abnormalities would predict the presence of pulmonary hypertension in the early postoperative period and at later follow-up. Patients with grade A or mild grade B changes have normal pulmonary artery pressures in the early postoperative period or only a minimal degree of elevation. The majority of patients with more severe medial hypertrophy, i.e., severe grade B and Heath Edwards I, have elevated values, but the pulmonary hypertension is frequently labile and almost always can be controlled by vasodilators and appropriate ventilation.3 Both the presence and the severity of early postoperative pulmonary hypertension are increasingly predictable when there are more advanced changes on lung biopsy, i.e., reduced artery number (grade C) and intimal hyperplasia (Heath Edwards II and III) (Figure 3A). Various methods of managing a postoperative increase in pulmonary vascular reactivity have been proposed, including prolonged anesthesia with fentanyl4 and vasodilators such as prostacyclin5 or, more recently, atrial natiuretic-peptide, or inhaled nitric oxide, which has proven to be particularly useful.6,7 There are, however, a few patients who maintain a high level of pulmonary vascular resistance and are refractory to vasodilator therapy despite what appears to be mild vascular changes on light microscopy (medial hypertrophy), and others who develop rapidly progressive pulmonary vascular disease despite early diagnosis and timely intervention. For these patients, the prognosis may be not much better than those with unexplained pulmonary hypertension.8 One year after repair, however, the presence and severity of residual pulmonary hypertension is based upon both the severity of the vascular changes and the age at the time of surgical repair. That is, patients operated on within the first 8 months of life tend to have normal pulmonary hemodynamics regardless of the severity of vascular changes on lung biopsy, as do all patients with severe grade B (Heath Edwards I) abnormalities, regardless of their age at repair. Patients surgically corrected between 9 months and 2 years of life with grade C and Heath Edwards II or III structural changes may have persistent elevation in pulmonary vascular resistance; this appears inevitable in those operated on after 2 years of life (Figure 3B).3 A variety of physiologic factors contribute to the severity and rate at which vascular disease develops in patients with congenital heart defects. Certain lesions, such as transposition of the great arteries with intact septum,9 but especially with a large ventricular septal defect10 or a patent ductus arteriosus,11 are at high risk. Curiously, there have been

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FIGURE 3 (A) Lung biopsy grade is correlated with mean

pulmonary arterial pressure recorded the day after surgical repair. The dashed vertical lines separate the normal from the abnormally elevated pressure values, and the dotted horizontal lines separate the biopsy grades. Note that with the more severe Health-Edwards changes on lung biopsy tissue, there is a trend toward a greater proportion of patients with elevated pulmonary arterial pressures and higher values. (B) Graph correlating lung biopsy grade with pulmonary vascular resistance 1 year after cardiac repair. Patients who underwent repair within the first 8 months of life, but not those operated on later, had normal pulmonary vascular resistance regardless of the severity of their structural changes. A, B, C, morphometric grades; m, mild, s, severe; N, I, II, III, HeathEdwards grades; n, normal; *, no patients in this group; VSD, ventricular septal defect; DTGA, d-transposition of the great arteries; CAVC, complete atrioventricular canal; complex, associated abnormality. (Reproduced with permission from Rabinovitch M, et al.)3

reports of progression of pulmonary vascular disease in infants with transposition of the great arteries even after successful surgical correction.12 Infants with a large ventricular septal defect13 and

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especially an atrioventricular septal defect14,15 also develop pulmonary vascular disease in early infancy, and, in the latter, there are advanced changes observed even within the first 6 months of life. Techniques of wedge angiography were developed to assess the structural state of the pulmonary vascular bed and obviate the need for a lung biopsy. We described a technique whereby the rate of tapering of the arteries is predictive of the severity of vascular disease assessed both morphometrically and by the Heath Edwards classification (Figure 4).16 When pulmonary vascular resistance calculations (based upon measured oxygen consumption) are elevated (ⱖ8 ␮m2 and non-reactive) and wedge angiography supports extensive structural abnormalities, then lung biopsy evaluation has not, in our experience, provided other than confirmatory evidence of severe pulmonary vascular disease. If, however, there are discrepancies, or if the patient is within the first 2 years of life, then the analysis of lung biopsy tissue, even prepared by frozen section, will help decide on a corrective procedure.17 The ability to predict from biopsy tissue whether even mild elevation in pulmonary vascular resistance will be present postoperatively is of increasing importance, particularly in the consideration of patients who require a Fontan procedure.18 Patients with tricuspid atresia who have had previous systemic to pulmonary artery shunts, and those with a single ventricle and previous pulmonary artery bands, are a particular problem. We currently consider a biopsy showing severe grade C (less than one-half the normal number of arteries) and grade III or greater changes in 20% of vessels to indicate vascular disease that is unlikely to regress postoperatively and, hence, precludes a successful result from closure of a ventricular septal defect. Severe grade B medial wall thickness greater than twice normal, and/or grade II changes in any vessels may preclude a favorable result from a Fontan procedure (direct right atrial to pulmonary artery communication). Recent studies have shown that even minor vascular changes on lung biopsy tissue (mild grade B) are associated with increased morbidity after Fontan’s procedure, as gauged by prolonged hospitalization due to the need for increased ventilator support and drainage protracted from chest tubes.

MECHANISMS UNDERLYING THE REACTIVE PULMONARY CIRCULATION There are many factors that could contribute to the hyper-reactive pulmonary circulation. Endothelial changes on scanning and transmission electron microscopy suggest a potential for altered function as well as

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FIGURE 4 Left, a wedge angiogram shows slow

tapering of the axial artery in a child with TGA and normal pulmonary artery pressure (PPA) and resistance (RP). Approximate segment length between 2.5 and 1.5 mm internal diameter is marked off (arrows). Right, a wedge angiogram in a child with a VSD shows rapid tapering of the artery when there is increased pulmonary artery pressure and resistance. An approximate segment length between 2.5 and 1.5 mm internal diameter is marked off (arrows). Large arrow denotes takeoff to the right pulmonary artery. (Reproduced with permission from Rabinovitch M. Quantitative structural analysis of the pulmonary vascular bed in congenital heart defects. In: Engle MA, ed. Pediatric cardiovascular disease. Philadelphia: FA Davis Co., 1981, pp. 149 –169.)

increased interaction with circulating blood elements, such as platelets and leukocytes.19 –23 This could result in a release of thromboxanes21 and other mediators causing pulmonary vasoconstriction. In addition, alterations in the subendothelium reflected in fragmentation of elastin suggested that an elastolytic enzyme

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FIGURE 5 Scanning electron photomicrographs of

pulmonary artery endothelial surfaces. (A) Normal pulmonary artery shows “corduroy” pattern, closely aligned ridges. (B) Hypertensive pulmonary artery shows “cable” pattern, deep knotted ridges, and numerous microvilli (mv). Magnification 810⫻. (Reproduced with permission from Rabinovitch M, et al.)19

might be stimulating the remodeling process, as well as altering the functional response of the vessel. On scanning electron microscopy, the endothelial surface of normal thin-walled pulmonary arteries has a corduroy-like appearance, in that the cells form narrow, even ridges (Figure 5). In contrast, the endothelial surface of hypertensive thick-walled pulmonary arteries has a cable-like texture, in that the cells form deep, twisted ridges. In patients with advanced pulmonary vascular changes, the endothelial surface exhibits a chenille pattern in which high ridges alternate with narrow, twisted misshapen ones. This abnormally contoured endothelium may be more likely to interact with marginating blood cells (platelets and leukocytes), resulting in the release of pulmonary vasoconstrictor substances such as thromboxanes and smooth muscle mitogens. On examination using transmission electron microscopy, endothelial cells from hypertensive compared to normotensive pulmonary arteries show an increased density of microfilament bundles and rough endoplasmic reticulum (Figure 6). The former suggests an altered cytoskeleton which may affect endothelial permeability and the latter indicates increased protein synthesis. We showed, by immunohistochemistry, that hypertensive endothelial cells produce increased von Willebrand factor (factor VIII). VIII:Ag levels were significantly higher in patients with congenital heart defects and elevated pulmonary artery pressure than in those with normal pressure, but the molecule being secreted was lacking in biological activity. Under con-

FIGURE 6 (A) A section of a pulmonary artery 92 ␮m in

diameter in a patient with normal pulmonary arterial pressure shows an intact elastic lamina. (B) In a section from a pulmonary artery 108 ␮m in diameter in a patient with increased pulmonary blood flow and pressure, microfibrillar material is present in the subendothelium but no true internal elastic lamina. The endothelial and smooth muscle cells are separated by only a thick basement membrane (bm). A myoendothelial contact is seen (e). Bar ⫽ 1 ␮m in both. (Reproduced with permission from Rabinovitch M, et al., 1986.)

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ditions that aggravate endothelial dysfunction, e.g., cardiopulmonary bypass, vWF biological activity was, however, markedly increased.22 This could account for the development of platelet fibrin microthrombi in the postoperative period and for abnormal release of vasoactive compounds causing increased vascular reactivity. An increased density of neuroepithelial bodies has been observed in the airways of patients at risk of this complication24 as well as an increase in vasoconstrictor neuropeptide-containing nerves.25 The neuroendocrine cells which are also oxygen sensors26 contain bombesin and serotonin, agents known to be potent vasoconstrictors. Since most of the pulmonary hypertensive crises occur upon weaning from the ventilator, it is tempting to speculate that swings in airway pressure might lead to degranulation of the neuroepithelial cells and release of the vasoconstrictor substances. Moreover, we have observed a striking decrease in lung compliance that accompanies the pulmonary hypertensive crisis.27 There is now very convincing evidence that production of endothelial dependent relaxing factor (EDRF) or nitric oxide is impaired at an early timepoint in patients with congenital heart defects and is lacking in those with advanced pulmonary vascular disease.28 –30 The further injury to the endothelium following cardiopulmonary bypass likely explains the successful use of nitric oxide as a pulmonary vasodilator in the postoperative patient. There is also evidence that production of the vasoconstrictor, endothelin, might also be increased in patients with pulmonary hypertension and congenital heart defects.31,32

NATURE OF THE VASCULAR LESIONS Our recent immunohistochemical studies carried out in lung biopsy tissue from patients with congenital heart defects have shown that there is a progressive increase in the deposition of the glycoproteins tenascin and fibronectin in the media and neointima (Figure 7).33 We have previously related increased expression of TN-C to vascular smooth muscle cell proliferation34 –35 and, in fact, there is co-localization of tenascin with proliferating smooth muscle cells and with expression of growth factors such as epidermal growth factor. We have also related fibronectin to increased smooth muscle cell migration in the context of neointimal formation.36,37 There is evidence from the studies of others that the neointimal lesions in primary pulmonary hypertension are associated with increased expression of transforming growth factor beta38 and procollagen39 (Figure 8) in addition to fibronectin.40 It is also proposed

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FIGURE 7 Representative photomicrographs showing

immunoperoxidase staining for tenascin-C (TN) (A, D, and G), proliferating cell nuclear antigen (PCNA) (B, E, and H), and epidermal growth factor (EGF) (C, F, and I) in graded lung biopsy tissue sections. A, B, and C, vessel showing a typical grade IA lesion; D, E, and F, vessel showing a typical grade IC lesion; G, H, and I, vessel showing a typical grade IIIC lesion. In low grade lesions (A), modest TN immunostaining was evident in the adventitia. With medial hypertrophy, TN immunoreactivity became more prominent in the periendothelium (D), with the most intense immunostaining being apparent within the neointima of high grade lesions showing occlusive neointimal formation (G). In the lowest grade of lesion, PCNA was negative (B), despite foci of EGF in the media. With medial hypertrophy, PCNA was expressed in the media (E), together with foci of EGF (F). With the development of higher grade occlusive lesions, TN (G), PCNA (H), and EGF (I) co-localized to the neointimal cell layers. Note that TN and PCNA staining was performed on serial sections, whereas EGF detection was carried out on similar vessels within the same biopsy. Original magnification 40⫻ (Reproduced with permission from Jones PL, Cowan KN, Rabinovitch M. Progressive pulmonary vascular disease is characterized by a proliferative response related to deposition of tenascin-C and is preceded by subendothelial accumulation of fibronectin. Am J Pathol 1997;150 in press.)

that endothelial cell proliferation and a form of angiogenesis is observed with plexiform lesions.41

PATHOPHYSIOLOGY OF PULMONARY VASCULAR DISEASE In experimental studies, aortopulmonary shunts surgically created in growing piglets were associated with a progressive increase in pulmonary artery pressure and with the development of structural changes; specifically, extension of muscle into peripheral arteries, medial hypertrophy of muscular arteries, and reduced arterial number.42 Several different experimental mod-

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FIGURE 9 Mean pulmonary artery pressures. S, saline;

V, vehicle; SC-I, SC-37698 (2 mg/day in the 2-week study and 3 mg/day in the 3-week study); M, monocrotaline. 2week study: S/V (n ⫽ 6); S/SC-I (n ⫽ 6); M/V (n ⫽ 7); M/SC-I (n ⫽ 6). 3-week study: S/V (n ⫽ 3); S/SC-I (n ⫽ 3); M/V (n ⫽ 6); M/SC-I (n ⫽ 6). Values were mean ⫾ standard error (SE). ***p ⬍ 0.001 and **p ⬍ 0.01, *p ⬍ 0.05. (Reproduced with permission from Ye C et al.)50

FIGURE 8 Immunohistochemistry was performed on

formalin-fixed, paraffin-embedded parenchyma obtained from patients with severe primary pulmonary hypertension at the time of single-lung transplantation. Tissue sections were then stained with antibodies to the amino-terminal end of the procollagen type 1 propeptide (B). This antibody identifies newly synthesized ␣-I(I) procollagen prior to cleavage of the amino-terminal propeptide following secretion and, therefore, can identify sites of active collagen deposition. An elastin-van Gieson stain demonstrates vascular structures (A). Procollagen immunoreactivity (B) is present only within the neointima of this occluded artery. Magnification 100⫻ (Reproduced with permission from Botney MD, et al.)39

els of high-flow congenital heart defects have been studied, e.g., lambs with a ventricular septal defect,43 aortopulmonary shunts created in utero,44 or sheep45 and calves46 with systemic to lobar pulmonary artery anastomoses. There are many common features linking the cellular and molecular pathophysiology of pulmonary vascular disease regardless of etiology. Ultrastructural study of lung biopsy tissue from patients with congenital heart defects provided the first clue that increased activity of an elastolytic enzyme might be important in the pathophysiology of pulmonary vascular disease. Fragmentation of elastin was also apparent in the pulmonary arteries of rats in which injection of the toxin monocrotaline was used to experimentally induce pulmonary hypertension.47 Biochemical studies

supported the structural observations by revealing high elastin turnover in pulmonary arteries from rats with monocrotaline-induced pulmonary hypertension, suggesting neosynthesis as well as degradation, and direct enzymatic assay confirmed increased activity of a serine elastase.48 –50 There was increased activity of this enzyme only 2 days after subjecting the rats to the inducing stimulus in both monocrotaline and hypoxicinduced pulmonary hypertension in rats.48,49 Hypoxicinduced pulmonary hypertension is potentially largely reversible, and the increase in enzyme activity was transient,49 whereas in the more malignant pulmonary hypertension induced by monocrotaline, increased elastase activity was also observed with development and progression of the structural changes in the pulmonary arteries.47 Infant rats injected with monocrotaline show spontaneous regression of pulmonary hypertension and lack the biochemical and structural evidence of the more sustained increase in elastase activity observed in the adult counterparts. A cause and effect relationship between elastase and pulmonary vascular disease was further established when serine elastase inhibitors49,50 including 5C-37698 (Searle) largely prevented the development and the progression of pulmonary hypertension in these experimental animals (Figure 9). An endogenous vascular elastase was isolated that appeared structurally related to the serine proteinase, adipsin;51 it was 20 kD in molecular weight, localized to smooth muscle cells, and therefore similar to the elastase enzyme reported in aortic smooth muscle cells and in atherosclerotic

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FIGURE 10 We have speculated as to how a stimulus could induce activity of an

elastolytic enzyme and how this might stimulate the remodeling process. The process of progressive pulmonary hypertension involves a series of switches in the smooth-muscle cell phenotype (i.e., differentiation of muscle from non-muscle precursor cells, smooth muscle cell hypertrophy, and proliferation accounting for medial hypertrophy, and smooth muscle cell migration resulting in neointimal formation). In response to a stimulus, such as high flow and pressure, the first “casuality” is the endothelial cell. As a result of structural and functional alterations in endothelial cells, some of the barrier function would be lost, allowing a leak into the subendothelium of a serum factor normally excluded from this region. The serum factor could induce activity of an endogenous vascular elastase (EVE). This enzyme released from precursor or mature smooth muscle cells would activate growth factors normally stored in the extracellular matrix in an inactive form, such as basic fibroblast growth factor and transforming growth factor-␤, which are known to induce smooth muscle hypertrophy and proliferation and increases in connective tissue protein (e.g., collagen and elastin) synthesis. bFGF (FGF-2) also induces tenascin, a matrix glycoprotein that amplifies the proliferative response as described in the text. This results in the differentiation of precursor cells to mature smooth muscle related to the muscularization of normally non-muscular small peripheral arteries. In the muscular arteries, the release of growth factors would result in hypertrophy of the vessel wall. Continued elastase activity would cause migration of smooth muscle cells in several ways. The elastin peptides or degradation products of elastin can stimulate fibronectin, a glycoprotein that is pivotal in altering smooth muscle cell shape and switches them from the contractile to motile phenotype. (Reproduced with permission from Rabinovitch M. It all begins with EVE (endogenous vascular elastase). Isr J Med Sci 1996;32:803– 8.)

tissues.52 The molecular nature of this enzyme is currently under investigation in our laboratory.

MECHANISM OF INDUCTION OF ELASTASE ACTIVITY To investigate how an increase in elastase might occur, and how it might cause the proliferative and obliterative changes that accompany progressive pulmonary hypertension cell culture, studies were carried out (Figure 10). We hypothesized that in response to a

perturbing stimulus, structural and functional alterations in endothelium would lead to the loss of barrier properties, and as a consequence, a serum factor would accumulate in the subendothelium, inducing activity of this EVE in vascular smooth muscle cells. Our studies confirmed that serum and endothelial factors can induce smooth muscle cell elastase activity. The factor(s) adhered to elastin and to the cell surface and induced binding of the elastin to the elastin binding protein (Figure 11).53 An elastin-binding se-

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oligonucleotides effectively repressed serum-treated elastin induction of elastase activity.

ELASTASE ACTIVITY LINKED TO SMOOTH MUSCLE CELL PROLIFERATION

FIGURE 11 A proposed schema indicating how serum

factor(s) or endothelial factor, i.e., Apo A1, tethers elastin to the smooth muscle cell surface, permitting engagement of the elastin binding protein (EBP) and co-operative interaction with integrin-receptors resulting in tyrosine kinase activity. The subsequent promotion of DNA transcription and mRNA translation culminate in the induction of vascular elastase. (Reproduced with permission from Thompson K, et al.)54

rum factor was eluted and shown to have elastaseinducing properties. N-terminal sequence analysis of the purified protein and molecular weight suggested that it was apolipoprotein A1.54 We showed that binding of elastin to cell surfaces via serum components initiated a signal transduction mechanism whereby tyrosine kinase activity was expressed. There was phosphorylation of focal adhesion kinase (FAK) and members of the MAP kinase family (ERK or extracellular regulated kinase-1).55 We identified the transcription factor AML1 as a target of ERK1 phosphorylation. AML1 is the transcription factor for neutrophil elastase and is a candidate transcription factor for vascular elastase.56 Differential display PCR suggested that a transcript that was differentially upregulated when cells are stimulated with serum-treated elastin to produce elastase was homologous to AML1, and we confirmed increased expression of AML1 protein.57 Moreover, inhibition of phosphorylation of ERK with MAP kinase inhibitor PD08059 resulted in reduced expression and DNA binding of the transcription factor AML1. We have documented by immunohistochemistry that AML1 is present in smooth muscle cells and expression is prominent in the nucleus with serum stimulation but not with MAP kinase inhibition. On gel mobility shift assay, AML1 is evident in nuclear extracts following serum-treated elastin induced elastase activity and forms a binding complex with radiolabelled oligonucleotides containing the AML1 concensus sequence. Moreover, AML1 antisense

Expression of EVE activity leads to the release of smooth muscle cell mitogens58 as has been shown for other serine proteinases.59 – 60 Both human leukocyte elastase and EVE induced by serum-treated elastin liberate fibroblast growth factor 2 (FGF-2) from the extracellular matrices of smooth muscle cells. In order for cells to respond optimally to growth factors, receptors must be available and in some way “primed.” Attachment of cells to the specific glycoprotein tenascin enhances their response to growth factors. The glycoprotein tenascin (TN)-C is induced by FGF-261 as well as elastase. It co-distributes with proliferating smooth muscle cells and its increased expression correlates with severity of the vascular lesion (Figure 7). When rat pulmonary artery smooth muscle cells were cultured on collagen gels supplemented with TN-C, there was increased cell proliferation upon addition of FGF-2. Supplementation with TN-C appeared to be a prerequisite for EGF-dependent pulmonary artery smooth muscle cell proliferation (Figure 12).35 Increased TN-C clusters ␤3 integrins, leading to formation of actin cytoskeleton focal adhesion contacts and tyrosine phosphorylation of a protein weighing 125 kd, which is likely focal adhesion kinase. Upon ligation with EGF there is brisk phosphorylation of the EGF receptor and transmission of a nuclear phosphorylation signal, which is reflected in cell growth (Figure 13). Proteolysis of collagen unmasks RGD sites which bind to ␤3 integrins, and signal TN-C gene transcription. When, however, proteinases are suppressed and TN-C is downregulated, apoptosis of smooth muscle cells is induced. We subsequently used organ culture to determine whether repression of TN-C induces smooth muscle cell apoptosis in the intact vessel and stimulates regression of vascular disease.62 Hypertrophied rat pulmonary arteries in organ cultures incubated with either a matrix metalloproteinase or a serine elastase inhibitor show markedly reduced tenascin C expression and regression of medial hypertrophy associated with SMC apoptosis.

FIBRONECTIN IN PULMONARY VASCULAR PATHOBIOLOGY Increased elastase activity also appears to underlie the mechanism of smooth muscle cell migration associated with neointimal formation and occlusive pulmonary vascular disease. We had previously shown increased

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FIGURE 12 Effect of exogenous tenascin (15 ␮g/mL)

on pulmonary artery smooth muscle and growth factordependent proliferation. Smooth muscle cell growth in serum-free medium (SFM) was unaffected by addition of exogenous tenascin. In contrast, addition of basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) to tenascin-treated cultures resulted in a significant increase in cell number. Values represent mean ⫾ SEM. p ⬍ 0.05. *, denotes difference from SFM level; †, denotes difference related to tenascin.

expression of the matrix glyocoprotein fibronectin with neointimal formation in the lamb ductus arteriosus in late gestation and in coronary arteries as a result of experimental heterotopic heart transplantation. In lung biopsy tissue from patients with congenital heart defects and pulmonary hypertension, fibronectin accumulates largely in the peri-endothelial region of the hypertrophied artery and is also seen in the neointima with advanced disease.33 This supported a fibronectin gradient in the pulmonary arteries as being of potential importance in stimulating SMC migration. In the ductus there is impaired elastin fiber assembly63 and in post-transplant coronary arteries and pulmonary arteries, we had shown that there is increased activity of a serine elastase.64 Cell culture studies had shown that elastin peptides, which can result from either impaired assembly or degradation of elastin, can upregulate fibronectin production.65– 67 Elafin, a selective serine elastase inhibitor, can prevent the development of neointimal formation68 related to fibronectin-dependent smooth muscle cell migration into the subendothelium and leukocyte transendothelial migration

FIGURE 13 Tenascin-C modifies the patterns of

distribution for filamentous actin, epidermal growth factor receptors, tyrosine phosphorylated proteins and vinculin. Representative immunofluorescence photomicrographs (from 2 different experiments) showing distribution patterns for F-actin, EGF-Rs and tyrosine phosphorylated (P-Tyr) proteins in SMC cultured in SFM (⫾50 ng/mL EGF for 30 minutes) on collagen alone, or on TN-C (15 ␮g/mL) supplemented collagen substrates. Rhodamine phalloidin staining of SMC on collagen revealed a longitudinal F-actin stress fiber pattern of distribution which was more cortical following treatment with EGF. Immunofluorescent staining for EGF-Rs and tyrosine phosphorylated proteins was diffuse in SMC cultured on collagen alone, and increased modestly following addition of EGF. In contrast, SMC cultured on TN-C supplemented collagen gels showed high intensity F-actin staining in regions that often overlapped with clusters of EGF-Rs and tyrosine phosphorylated proteins. Following addition of EGF to TN-C treated cultures, the levels and distribution of EGF-Rs remained pronounced, and high levels of tyrosine phosphorylated proteins were evident throughout the cell. Bar ⫽ 20 ␮M. (Reproduced with permission from Jones PL, et al.)34

(Figure 12).69 –71 The molecular mechanism that seems to modulate the increased fibronectin production in the smooth muscle cells is related to enhanced efficiency of translation of fibronectin messenger RNA (mRNA).72 We have shown that there is a cytoplasmic factor in the migratory cells which binds to the 3⬘ untranslated region of the fibronectin mRNA which

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can increase the efficiency of translation. This factor, light chain 3 (LC3) of microtubule-associated proteins 1A and 1B, is upregulated. Sequestration of LC3 by decoy RNA has been used by our group as a strategy in experimental animals to prevent migration of smooth muscle cells and the development of a neointima.73

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VASOACTIVE AGENTS ARE IMPORTANT MODULATORS OF STRUCTURAL REMODELING

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Nitric oxide donors reduce the serum stimulation of elastase in pulmonary artery smooth muscle cells, suggesting how nitric oxide might also suppress the vascular remodeling associated with pulmonary hypertension.74 On the other hand, nitric oxide donors, perhaps via peroxynitite production, increase fibronectin production and therefore can be expected to stimulate smooth muscle cell migration.75 Previous reports have also shown that increased expression of endothelin is associated with hypoxia-induced pulmonary hypertension,76 –78 and that endothelin receptor blockade is associated with the prevention of pulmonary vascular disease. Also, angiotensin II production in hypoxia also appears to be related to structural remodeling per se.79 The reduction in the pulmonary artery pressure and resistance observed following chronic infusion of prostacyclin, especially in patients with unexplained pulmonary hypertension who show no acute vasodilator response, suggests that there may be a direct effect on vascular remodeling.80 How these mediators interact with, and are influenced by, heightened EVE activity and the growth factors released by EVE will be of great interest in future studies, as will interactions between EVE and other growth factors such as vascular endothelial growth factor (VEGF),81 which stimulate new growth of blood vessels and proliferation of endothelial cells. A locus for familial primary pulmonary hypertension has been established on chromosome 282– 83 and so studies directed at the pathobiology of pulmonary hypertension might identify genes that segregate in that locus and that would be markers of patients at risk and targets of future therapy. Rather than using clinical and experimental tissues to indicate the severity of disease, it might be expected that our studies, and those of others, might identify novel therapeutic targets to prevent development, retard progression, and even induce regression of pulmonary vascular disease.

5.

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