PERSPECTIVE
Prostacyclin Analogs as the Brakes for Pulmonary Artery Smooth Muscle Cell Proliferation
Is It Sufficient to Treat Severe Pulmonary Hypertension?

Rubin M. Tuder and Ari L. Zaiman

Division of Cardiopulmonary Pathology, Department of Pathology, and Division of Pulmonary Medicine, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland

The report by Clapp and coworkers in the current issue of the American Journal of Respiratory Cell and Molecular Biology advances our understanding of the antiproliferative effect of prostacyclin analogs on cultured pulmonary artery smooth muscle cells (1). The authors compared the antiproliferative effects and correlated these effects with the elevation of intracellular of cAMP of several prostacyclin analogs, which included Iloprost, Beraprost, Cicaprost, and the novel analog UT-15. The relevance of these studies lays on the importance of prostacyclin as one of the few effective drugs to treat severe pulmonary hypertension (SPH); in particular, the idiopathic form also known as primary pulmonary hypertension (PPH). SPH is characterized by extreme increases of pulmonary artery pressures (usually at levels above 45 mm Hg mean pulmonary pressures), ultimately resulting in patient's demise due to right heart failure. SPH can be sporadic, familial, or related to a variety of underlying conditions such as collagen vascular disease (the CREST variant of scleroderma or systemic lupus erythematosus), congenital heart malformations, HIV infection, cirrhosis, or anorectic drug use (2). SPH occurs infrequently as compared with pulmonary hypertension associated with chronic lung diseases such as chronic obstructive and interstitial pulmonary diseases.

The morphologic alterations of the pulmonary arteries in SPH differ from those seen in lungs with mild pulmonary hypertension. In SPH, the vascular lumen is obliterated because of proliferation of endothelial cells forming plexiform or concentric lesions, myofibroblasts embedded in a mucopolysaccharide matrix, and media vascular thickening (Figure 1) (2). In contrast, in mild pulmonary hypertension, the pulmonary vascular media appears thickened whereas the intima presents with the characteristic monolayer lining of endothelial cells.

View larger version (139K): [in this window] [in a new window]   Figure 1.   (A) Plexiform lesion in a lung of a patient with PPH. The proximal (A; feeder) segment shows marked intimal thickening. Distally, there is an irregular growth of endothelial cells (P), which are in different stages of differentiation toward the formation of capillaries (arrows) (Hematoxylin and Eosin, ×200). (B) Concentric lesion, which is characterized by an "onion-skinning" arrangement of endothelial cells. Note the presence of scattered lymphomononuclear infiltrating the outside boundaries of the lesion (Hematoxylin and Eosin, ×200). (C) Factor VIII-related antigen immunostaining of a segment of muscularized pulmonary arteries. Note the intense staining of endothelial cells in the concentric (C) and plexiform lesions (arrow, P) (Immunohistochemistry, ×100). (D) MIB-1 proliferation antigen immunostaining of a pulmonary artery with marked medial hypertrophy. Note the lack of nuclear immunoreactivity in the hypertrophic smooth muscle cells, whereas the basal epithelial cells in terminal bronchioles show expression of MIB-1 antigen, i.e., they are proliferating (inset) (Immunohistochemistry, ×100).

Excessive vasoconstriction has been the prevailing hypothesis of the pathobiology of SPH. Accordingly, several studies investigated whether a deficiency of vasodilators such as prostacyclin was present in patients with PPH. Patients with SPH were reported to have decreased serum levels of the vasodilator prostacyclin, as assessed by urinary excretion of the 2,3-dinor-6-keto PGF₁ metabolite, whereas the vasoconstrictor thromboxane was increased (3). This report supported the therapeutic supplementation of prostacyclin, as it suggested that lung synthesis of prostacyclin was decreased in patients with SPH. The direct histologic documentation of decreased expression of the enzyme responsible for the production of prostacyclin (prostacyclin synthase) in lungs of patients with SPH was accomplished seven years later (4). Initially introduced as a bridge to transplant, prostacyclin has been shown to improve hemodynamics and exercise tolerance, and to prolong survival in severe PPH (5). Furthermore, prostacyclin has been demonstrated to induce long term reductions in the pulmonary vascular resistance that exceed those of immediate vasodilation (6).

To better appreciate the overall impact of the study by Clapp and coworkers, we wish to bring into perspective the current knowledge of prostacyclin receptor signaling (in particular, the signaling in smooth muscle cells), the role of prostacyclin and prostacyclin receptor in experimental models of pulmonary hypertension, and the potential limitations of prostacyclin as an effective therapy for SPH in light of our recent understanding of the pathogenesis of SPH.

	Prostacyclin Signaling and Its Biologic Effects

Prostacyclin, produced predominantly by lung endothelial cells, is a vasodilatory prostanoid and the main cyclooxygenase product of arachidonic acid in vascular tissues. Initially, these molecules were thought to mediate their effects by traversing the lipid membrane; however, biochemical evidence suggested the presence of a membrane-bound receptor. The seven transmembrane G-protein-coupled receptor for thromboxane A₂ was the first prostanoid receptor cloned. Homology screening was used to clone all subsequent prostanoid receptors. The prostacyclin receptor (IP) is located on a variety of cell types, enabling prostacyclin to exert a range of biologic actions by means of raising intracellular levels of cAMP (7). Prostacyclin has broad vascular actions that span vasodilation, cellular proliferation, and thrombosis. In platelets, prostacyclin reduces dense granule release and therefore _II3-mediated platelet aggregation. Prostacyclin also acts at the endothelial cells to provide an anti-inflammatory, antiplatelet, and antithrombotic surface vital for the proper function of the pulmonary circulation. Of most significance in the pathogenesis of pulmonary hypertension is the effect of prostacyclin on contractility, growth, and matrix-producing properties of vascular smooth muscle cells. As SPH is a prothrombotic, proliferative, and inflammatory disease, SPH appeared to be the ideal setting for the broad protective actions of prostacyclin (8).

The studies of Clapp and coworkers highlight the ability of therapeutically useful prostacyclin analogs to inhibit the growth properties of smooth muscle cells in vitro. The novelty of the study lies in the new information provided with UT-15, as compared with previously characterized analogs. Although this growth suppression is hypothesized to be cAMP-dependent, the intracellular levels of cAMP could not predict uniform growth retardation effects of prostacyclin analogs. UT-15 led to the greatest suppression of smooth muscle cell proliferation and the highest cAMP elevation among the analogs tested. The experiments by Clapp and coworkers did not offer new information as to whether suppression of smooth muscle cell proliferation may occur by cAMP-independent pathways. Because the studies by Clapp and coworkers were performed in cultured smooth muscle cells obtained from the first two segments of normal pulmonary arteries, it is not possible at the present time to extend the information gathered with these cells with the efficacy and cAMP-dependency of prostacyclin analogs in peripheral artery smooth muscle cells in PPH, which are phenotypically distinct when compared with normal pulmonary artery smooth muscle cells. Indeed, PPH smooth muscle cells have decreased expression of the  subunit of the K⁺_v channels and increased growth stimulation by serotonin as compared with smooth muscle cells obtained from normal pulmonary arteries (9, 10).

As mentioned above, prostacyclin has broad cellular actions, which ultimately preserve the function of blood vessels. Because of its focus, the impact of the studies by Clapp and coworkers depends on the role of vascular smooth muscle cell proliferation (vis-à-vis other cellular properties of prostacyclin) in SPH. Experimental models of pulmonary hypertension triggered by chronic hypoxia or monocrotaline are characterized by smooth muscle cell proliferation, which is restricted to the first 3 wk of treatment (11, 12). It is noteworthy that, despite the continuous stimulation by the inciting agent, i.e., chronic hypoxia and monocrotaline, the proliferative capacity of vascular smooth muscle cell in pulmonary vascular hypertension is finite, and it is followed by cellular hypertrophy (which may be triggered by the cyclin kinase inhibitor p27 [13]) as a prototypic response of the pulmonary blood vessels in pulmonary hypertension. Both smooth muscle cell hypertrophy and an increase in extracellular matrix probably contribute to pulmonary vascular thickening in the chronic disease. Clapp and colleagues did not address the effects of prostacyclin analogs on smooth muscle cell hypertrophy or matrix production.

	Role of Prostacyclin and Prostacyclin Receptors in Models of Pulmonary Hypertension

Because of the limited knowledge of the pathobiology of SPH, most of the information concerning the alterations of vascular smooth muscle cells in pulmonary hypertension has originated from experimental models of severe pulmonary hypertension, in particular, the chronic hypoxia and the monocrotaline rodent models. The relevance of the chronic hypoxia model of pulmonary hypertension has been recently reviewed (14). This system models the mild pulmonary hypertension in humans, a condition that is not usually fatal and not a target of prostacyclin treatment. The monocrotaline model is based on a lung vascular cytotoxic effect of the liver metabolite of the alkaloid monocrotaline pyrrole. This model, as the chronic hypoxia model, lacks several of the features seen in human SPH, and therefore, the evidence accumulated in studies employing these models should be considered carefully as to the extent that applies to the human disease.

As based in transgenic mouse studies, prostacyclin and the prostacyclin receptor appear to have a distinct role in the regulation of pulmonary artery pressures under normoxic or hypoxic conditions when compared with nitric oxide, which has shown to modulate basal pulmonary vascular tone (15). Prostacyclin receptor knock-out mice have normal pulmonary artery pressures at mild hypoxia conditions (Denver altitude, 18% inspired oxygen) but higher pulmonary artery pressures and more pronounced pulmonary artery remodeling following chronic hypoxia as compared with wild-type controls (16). Lung-specific overexpression of prostacyclin synthase did not result in abnormal resting pulmonary artery pressures, but prevented the development of pulmonary hypertension and pulmonary vascular remodeling when the mice were exposed to chronic hypoxia (17). Recently, a similar observation with regards to the protective effects of prostacyclin was extended to the monocrotaline rodent model of pulmonary hypertension (18). These rodent models highlight the requirement of prostacyclin/prostacyclin receptor signaling in the control of the vascular smooth muscle cell remodeling and pulmonary artery pressures.

The mechanisms of smooth muscle cell growth (hypertrophy and hyperplasia) in experimental models of pulmonary hypertension involve serotonin, angiotensin II, the matrix protein tenascin, and v3 integrin signaling (19) (Figure 2). These stimulatory signals, despite triggering different pathways of intracellular signaling, all result in smooth muscle cell growth. Whether prostacyclin or prostacyclin analogs alter smooth muscle cell responses to these smooth muscle cell stimulatory signals is unknown. It is possible that prostacyclin inhibits cell growth by means of increasing the levels of cyclin kinase inhibitors such as p27 (22).

View larger version (17K): [in this window] [in a new window]   Figure 2.   Growth factor signaling pathways in smooth muscle cell.

	Severe Pulmonary Hypertension: Need for Novel Therapies

Marked smooth muscle cell thickening and proliferation of endothelial cells characterize human SPH. The latter finding is not present in pulmonary arteries of the chronic hypoxia or monocrotaline rodent models. Smooth muscle cell proliferation may not play a significant role in the established stages of SPH, as vascular smooth muscle cells in lungs of patients with SPH do not exhibit markers of cellular proliferation (Figure 1). Although Clapp and coworkers conclusively demonstrated the antiproliferative effects of prostacyclin-related compounds, there is no evidence that, in addition to its antiproliferative effects, prostacyclin and prostacyclin analogs can arrest the hypertrophy of smooth muscle cells or the growth of pulmonary endothelial cells with features of neoplasia such as seen in SPH (23). Because there is recent evidence that lungs of patients have decreased expression of the prostacyclin receptor (Bischoff and colleagues, submitted, 2001), the overall impact of prostacyclin in the clinical improvement of patients with SPH may be related to non-receptor-mediated effects in the pulmonary vessels or, alternatively, prostacyclin effects on the heart or in the recruitment of less remodeled pulmonary arteries, which have preserved prostacyclin receptor signaling.

Although prostacyclin treatment has represented a large step forward in the management of SPH, immense challenges in the areas of the pathobiology and therapy of SPH still remain. Importantly, chronic prostacyclin treatment does not induce regression of the medial vascular hypertrophy and the endothelial cell proliferative lesions in SPH (24). The proliferation of endothelial cells in SPH is the signature feature that distinguishes this group of disorders from those that are associated with potentially less severe forms of pulmonary hypertension, such as that present in interstitial lung diseases. Because we lack fundamental knowledge about the natural history of SPH, it is unknown whether a predominantly vasoconstrictive or a growth-prone medial smooth muscle cell or an abnormally proliferative endothelial cell plays a determinant role in the initiation of SPH; i.e., we do not have key pathogenetic information on how the disease starts. However, in the past two years, there have been significant developments in our understanding of the molecular pathogenesis of PPH. One of the genes that affords susceptibility to the hereditary form of primary or unexplained pulmonary hypertension has been identified as germline mutations in the bone morphogenetic protein receptor II (BMPR-II) (25, 26). The proliferating endothelial cells in sporadic PPH are monoclonal and harbor microsatellite (inactivating) mutations of the transforming growth factor- receptor II (TGF--RII) and/or the antiapoptotic molecule Bax (27), as described in bona fide neoplastic processes. How these genetic events translate into the initiation and progression in PPH is presently undetermined. These insights into the molecular pathogenesis of SPH point to cellular proliferative events, in particular in endothelial cells, as key to the development of SPH.

Prostacyclin and prostacyclin analogs have offered enormous relief to patients with SPH, yet several of the beneficial effects of prostacyclin may not be solely related to their effect on smooth muscle cell proliferation. Novel therapies that are aimed at the components of the pulmonary vascular pathology ought to be added to the current standards of therapy in SPH. The development of rodent models of SPH, either characterized by marked intima scarring (due to the combination of monocrotaline treatment and pulmonectomy to increase pulmonary blood flow to the other lung [28]) or by intravascular endothelial proliferation (29) may be employed to screen novel antiproliferative compounds, which may alter the disease course alone or in combination with prostacyclin.

	Footnotes

Address correspondence to: Rubin M. Tuder, M.D., Division of Cardiopulmonary Pathology, Department of Pathology, The Johns Hopkins University School of Medicine, 720 Rutland Ave., Ross Building, Room 519, Baltimore, MD 21205. E-mail: Rtuder{at}JHMI.EDU

(Received in original form December 21, 2001).

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