Bone morphogenetic receptor-II

Mutations in the transforming growth factor (TGF)-β receptor superfamily have been genetically linked to PAH and likely play a causative role in disease development. A rare group of patients with hereditary hemorrhagic telangiectasia (HHT) and idiopathic PAH harbor specific mutations in ALK1 or endoglin, genes encoding for two such members of the TGF-β receptor superfamily.^9,10 However, a more prevalent cohort of PAH patients carries mutations in the bone morphogenetic protein receptor type II (BMPR2 gene which encodes for BMPR2).^11,12 Over 140 mutations in BMPR2 have been reported in patients with familial PAH,¹³ mainly located in the extracellular ligand-binding domain, cytoplasmic serine/threonine kinase domain, or long carboxyterminal domain. These account for 70% of all familial pedigrees of PAH and 10% to 30% of idiopathic PAH cases.⁸ BMPR2 loss-of-function mutations have only been found in the heterozygous state. The absence of BMPR2 mutations in some familial cohorts and in most sporadic cases indicates that additional unidentified genetic mutations can also predispose to development of PAH. Furthermore, the presence of incomplete penetrance (approximately 25% of carriers in affected families develop clinical PAH) and genetic anticipation suggests that BMPR2 mutations are necessary but insufficient alone to result in clinically significant disease.

The mechanism of action of BMPR2 is complex, and its role in PAH progression is still unclear (Fig. 56-4A). It functions as a receptor with serine/threonine kinase activity, and it activates a broad and complex range of intracellular signaling pathways (as reviewed in¹⁴). Upon binding one of many possible bone morphogenetic protein (BMP) ligands, BMPR2 forms a heterodimer with one of three type-I receptors. BMPR2 phosphorylates the bound type-I receptor, which in turn phosphorylates one of the Smad family of proteins to allow for nuclear translocation, binding to deoxyribonucleic acid (DNA), and regulation of gene transcription. Alternatively, BMPR2 activation can also lead to signaling via the LIM kinase pathway, p38/ mitogen-activated protein kinase/extracellular signal regulated kinase/c-jun NH 2-terminal kinase (MAPK/ERK/JNK) pathways, or c-Src pathway, independent of Smad activation.

Figure 56-4 Molecular signaling mechanisms of pulmonary arterial hypertension (PAH).

A, Bone morphogenetic protein receptor type II (BMPR2) signaling in pulmonary vasculature. Heterozygous loss-of-function mutations in BMPR2 are found throughout the gene, leading to a complex cascade of dysregulated signaling that predisposes to PAH. When activated by a BMP ligand, BMPR2 heterodimerizes with BMPR1 to activate SMAD transcription factors. SMAD activity can control cellular differentiation, vascular tone, and proliferation, among other functions. BMPR1 can also activate signaling through XIAP (X-linked inhibitor of apoptosis), which controls activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK), both of which facilitate proinflammatory signaling. BMPR2 also carries a long cytoplasmic tail that binds to numerous signaling molecules including SRC, TCTEX, RACK1 (receptor for activated C-kinase 1), and LIMK1 (LIM domain kinase 1). LIMK1 can phosphorylate cofilin (Cfl1), which influences F-actin organization and glucocorticoid receptor (GR) nuclear translocation, important in inflammation. B, Serotonin signaling in pulmonary vasculature. Increased serotonin bioavailability is observed during PAH progression. This stems from increased release by platelets and increased production by endothelial cells (ECs). On vascular smooth muscle cells (VSMCs), serotonin receptors (5HT-2A, 5HT-2B, 5HT-1B/1D) are activated and induce vasoconstriction and remodeling. Overexpression of serotonin transporter (SERT) enhances the mitogenic effect of serotonin. Serotonin receptors on platelets potentiate aggregation. C, Nitric oxide (NO), endothelin-1 (ET-1), and prostacyclin are dysregulated vasoactive effectors in PAH. In pulmonary vasculature, NO is predominantly generated in ECs and transported to VSMCs; there, it stimulates production of cyclic guanosine monophosphate (cGMP) to induce vasorelaxation and decrease proliferation. Endothelin-1 is also predominantly synthesized and released from ECs. It activates endothelin receptor subtype A (ET-A) on smooth muscle cells (SMCs) to induce vasoconstriction and proliferation, while it stimulates NO and prostacyclin release via endothelin receptor subtype B (ET-B) on endothelial cells. Prostacyclin is produced from arachidonic acid and released from endothelial cells. In vascular smooth muscle cells, it activates production of cyclic adenosine monophosphate (cAMP) to promote vasorelaxation and inhibit proliferation. In PAH, NO and prostacyclin levels are significantly reduced while ET-1 levels are markedly elevated. This leads to a profound imbalance of these vasoactive effectors and exaggerated vasoconstriction and abnormal vascular smooth muscle proliferation. AM, adrenomedullin; AMP, adenosine monophosphate; ATP, adenosine triphosphate; BH4, tetrahydrobiopterin; eNOS, endothelial nitric oxide synthase; GMP, guanosine triphosphate; PDE, phosphodiesterase; TGFβ, transforming growth factor beta; TGFβR, transforming growth factor beta receptor; VIP, vasoactive intestinal peptide.

(Adapted from Archer SL, Weir EK, Wilkins MR: Basic science of pulmonary arterial hypertension for clinicians: new concepts and experimental therapies. Circulation 121:2045–2066, 2010.)

Cellular effects of BMPR2 activation are multiform. In the adult, BMPR2 is expressed predominantly in pulmonary endothelium, medial SMCs, and macrophages.¹⁵ Under normal conditions, BMP ligands bind BMPR2 to suppress growth of VSMCs. In contrast, binding of BMP2 and BMP7 to BMPR2 in pulmonary endothelium protects against apoptosis. A widely held hypothesis contends that failure of the suppressive effects of BMP ligands on vascular smooth muscle and failure of the protective effects of BMP ligands on endothelium may trigger vascular proliferation and remodeling. Accordingly, in VSMCs derived from patients with familial PAH harboring BMPR2 mutations, exposure of BMP ligands does not suppress proliferation. Unlike the response in wild-type endothelium, exposure of ECs cultured from patients with idiopathic PAH to BMP2 does not protect against apoptosis.¹⁶ These dysfunctional signaling pathways have been corroborated in some rodent models of PAH. In correlation, pulmonary levels of BMPR2 are reduced both in familial cases of PAH without any BMPR2 mutation and in cases of secondary PAH.¹⁵ Thus, dysregulation of the BMP signaling pathway may be a common pathogenic finding in multiple types of PAH due to genetic or exogenous stimuli, but the definitive in vivo effects of these mutations have been difficult to decipher. Specifically, mouse models harboring specific BMPR2 heterozygous mutations have failed to exhibit robust PAH under static conditions,^17,18 again suggesting that dysfunctional BMPR2 is likely insufficient alone to cause disease. As a result, a clear mechanistic explanation of the impact of BMPR2 mutations on pathogenesis remains elusive.

Additional genetic pathways

In addition to BMPR2 haploinsufficiency, alternative mechanisms involving complementary “modifier” genes may also contribute to a genetic predisposition to PAH. The most promising data identifying such modifier genes have analyzed the association of particular single nucleotide polymorphisms (SNPs) with the development of PAH. Thus far, SNP variants have suggested certain genes such as the serotonin transporter (SERT), Kv1.5, and the trp cation channel, subfamily C, member 6 (TRPC6).³ Such associations do not always suggest a causal relationship, so additional mechanistic data are necessary for proper interpretation.

In the case of SERT and serotonin signaling, supportive data are more prevalent. Serotonin is both a vasoconstrictor and mitogen that promotes smooth muscle hyperplasia and hypertrophy (see Fig. 56-4B). Primarily stored in platelet granules, secreted serotonin binds G protein–coupled serotonin receptors (GPCRs) present on pulmonary artery SMCs. Activation of these receptors leads to a decrease in adenylyl cyclase and cyclic adenosine monophosphate (cAMP), resulting in increased contraction. Furthermore, the cell-surface SERT allows for transport of extracellular serotonin into the cytoplasm of SMCs, thereby activating cellular proliferation directly through the action of serotonin or indirectly via potential pleiotropic mechanisms.

A number of observations in idiopathic and familial disease, congenital disease, and environmental exposure have implicated the proliferative effects of serotonin in PAH.¹⁹ In idiopathic PAH, pulmonary expression of serotonin receptors is increased, and plasma levels of serotonin are chronically elevated. A mouse model of hypoxic PH parallels these changes.²⁰ A positive association has been noted among patients with congenital platelet defects in serotonin uptake (i.e., delta storage pool disease) and development of PAH. Chronic exposure to anorexigens, such dexfenfluramine (an inhibitor of serotonin reuptake and stimulator of serotonin secretion), leads to increased levels of circulating free serotonin. In mice, these changes are accompanied by increased 5HT receptor type 2B response and inhibition of SERT responses. In humans, these changes correlate with an increased risk for development of PAH. In addition, the L-allelic variant of SERT is associated with increased expression of the transporter and enhanced smooth muscle proliferation. In some human studies,²¹ this variant has been associated with an increased risk of PAH in the homozygous population.

Animal models of PH have also implicated the activated serotonin pathway in disease progression. Treatment with serotonin and chronic hypoxia in a rat model led to worsened hemodynamics and increased vessel remodeling.²² Exposure to increased serotonin led to worsened PH in a BMPR2^+/− heterozygote murine model.²³ Similarly, overexpression of SERT in mice resulted in spontaneous development of PAH in the absence of hypoxia and exaggeration of PH after a hypoxic stimulus.²⁴ Conversely, vessel remodeling and hypoxic PH are reduced in a 5HT1B receptor–null mouse²⁵ and are abrogated in a 5HT2B receptor–null mouse.²⁰ As a result, serotonin signaling modulates pulmonary smooth muscle function in both normal and disease states and likely contributes to disease progression of PAH. However, attempts at using selective serotonin reuptake inhibitors (SSRIs) as a therapeutic approach have yielded mixed results³ to date. The exact contribution of this mechanism in PAH requires further clarification.

Chronic hypoxia

Pulmonary vascular response to hypoxia has been well studied in cell culture and animal models,²⁶ but its impact on PAH is unclear. In general, pulmonary vascular responses in acute and chronic hypoxia likely allow for the propagation of PAH, and therefore may contribute to later stages of the disease. Acute hypoxia induces vasodilation in systemic vessels but induces vasoconstriction in pulmonary arteries. This acute and reversible effect is mediated in part by up-regulation of endothelin-1 (ET-1) and serotonin, and in part by hypoxia- and redox-sensitive potassium channel activity in pulmonary VSMCs. Coordinately, these events lead to membrane depolarization in SMCs, increase in cytosolic calcium, and vasoconstriction.²⁷ In contrast, chronic hypoxia induces vascular remodeling and less reversible changes, including migration and proliferation of VSMCs and deposition of ECM. These cellular events in chronic hypoxia correlate with the remodeling events inend-stage PAH; however, because the histopathology of hypoxic PH does not recapitulate all aspects of PAH, some mechanistic differences in pathogenesis certainly exist but have not yet been fully identified. These are important considerations, especially in the context of interpreting studies of hypoxic PH and extrapolating those findings to the pathogenesis of PAH.

Hemoglobin disorders

Pulmonary arterial hypertension is associated with hemoglobinopathies, especially thalassemias and sickle cell anemia.²⁸ Hemolysis accompanying these disorders may lead to destruction of bioactive nitric oxide (NO) by free hemoglobin or reactive oxygen species (ROS). Furthermore, ROS may lead to increased levels of oxyhemoglobin, which further impairs delivery of NO to the vessel wall. As a result of the lack of available NO, an inflammatory and proliferative cascade may ensue, with culmination in PAH. Accordingly, decreased NO bioavailability with development of PH has been reported after hemolysis in a murine model of sickle cell disease (SCD),²⁹ and acutely in a murine model of intravascular hemolysis.³⁰ In vivo correlation to human disease is pending.

Portopulmonary hypertension

Portopulmonary hypertension is defined as PAH associated with portal hypertension (portal pressure >10 mmHg), with or without hepatic disease.³¹ Approximately 9% of patients with severe PAH are reported to have portal hypertension. Portopulmonary hypertension affects 4% to 6% of patients referred for liver transplantation. Liver transplantation perioperative mortality is significantly increased in patients with a mean PAP above 35 mmHg. Diagnosis of PH is usually made 4 to 7 years after the diagnosis of portal hypertension, but it has occasionally been reported to precede onset of portal hypertension. Risk of PH increases with duration of disease. The correlation between severity of portal hypertension and development of PH is debated. The female predominance of primary PAH is not seen in portopulmonary hypertension.

Survival is much worse than in PAH of other causes, with a median survival of 6 months. Because patients with nonhepatic causes of portal hypertension have been reported with this entity, it appears that portal hypertension, not cirrhosis, triggers development of PAH, yet the mechanism of portopulmonary hypertension is unknown. Hypotheses include inability of the liver to metabolize serotonin and other vasoactive substances. Alternatively, shear stress from increased pulmonary blood flow may result in endothelial injury, triggering a cascade of events that result in the characteristic adverse remodeling described earlier.

Collagen vascular disease

Pulmonary arteriopathy complicates autoimmune diseases, especially in the setting of the CREST (calcinosis, Raynaud phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia) variant of limited systemic sclerosis and, to a lesser degree, in mixed connective tissue disease, systemic lupus erythematosus (SLE), and rheumatoid arthritis.³² Pulmonary arterial hypertension has been reported in approximately 10% to 30% of patients with mixed connective tissue disease, 5% to 10% of patients with SLE, and more rarely in the settings of rheumatoid arthritis, dermatomyositis, and polymyositis.³³ Sjögren syndrome may rarely be complicated by rapidly progressive PAH. It is particularly important to distinguish between PAH and thromboembolic PH in patients with SLE and antiphospholipid syndrome. Occurrence of PAH in each disease has been associated with Raynaud phenomenon, suggesting at least some similarities in pathogenesis.³⁴ Presence of interstitial lung disease and pulmonary fibrosis, seen at varying frequency in these autoimmune syndromes, may represent a common pathogenic factor in development of PAH. Accordingly, in the setting of pulmonary fibrosis and hypoxia, significant perivascular inflammation and deposition of ECM have been observed, which may increase vasoconstriction, proliferation, and vessel remodeling. Murine models of interstitial lung disease may prove important in further elucidating pathogenic mechanisms.

Human immunodeficiency virus

An association between HIV infection and PH has been noted in approximately 0.5% of all patients with HIV infection, a rate 6 to 12 times higher than the general population.³⁵ Notably, HIV does not infect pulmonary arterial endothelium, but mechanisms of disease have been proposed that directly stem from effects of HIV infection.³⁶ These include infection of SMCs with subsequent dysregulation of proliferation, imbalance of vascular mitogens in response to systemic HIV infection, and endothelial injury precipitated by HIV-infected T cells. The direct actions of HIV-encoded proteins may also factor into PAH development.⁸ The HIV gp120 protein may induce pulmonary endothelial dysfunction and apoptosis. In a macaque model of simian immunodeficiency virus infection, a pathogenic interaction of the viral Nef protein with the pulmonary vessel wall has been reported, leading to pulmonary arteriopathy. Cell culture studies have also demonstrated a role for the HIV Tat protein in repression of BMPR2 transcription, potentially provoking a proliferative response in the vessel wall.

It had been proposed that human herpesvirus 8 (HHV-8), the causative agent of Kaposi sarcoma and an opportunistic pathogen highly associated with HIV infection, may play a role in PAH development with progression to plexiform lesions. Although it was initially reported that HHV-8 infection is associated with idiopathic PAH,³⁷ that link has not been consistently validated after study of additional populations. Nonetheless, PAH in the setting of HIV infection likely results from multifactorial effects, and the underlying pathogenesis may involve both direct results of viral infection and indirect consequences of associated pathogens.

Shunt physiology

Increased flow through the pulmonary circulation has long been associated with development of PAH. Certain types of congenital heart disease with functional systemic-to-pulmonary shunts, such as unrestricted ventricular septal defects (VSDs) and large patent ductus arteriosus (PDA), invariably lead to pulmonary vascular remodeling and the clinical syndrome of PAH during childhood (Eisenmenger’s syndrome).³⁸ Approximately a third of patients with uncorrected VSDs and PDAs die from PAH. The timing of surgical repair greatly influences the outcome. If the shunt is repaired within the first 8 months of life, patients tend to have normal pulmonary pressures regardless of pathological findings; by contrast, patients operated on after age 2 tend to have persistent PAH. Importantly, when PVR equals or exceeds systemic vascular resistance, surgical correction of a shunt will increase the load on an already overburdened right ventricle (RV), worsen the patient’s clinical condition, and not reverse PAH.

The presence of atrial septal defects (ASD) with systemic-to-pulmonary shunts may also lead to PAH over time.³⁹ Yet, in contrast to cases of unrestricted VSD and PDA, only 10% to 20% of all persons with ASDs progress to PAH. This observation may reflect differences in the response of the pulmonary vasculature to pressure overload (as seen in shunts with VSD and PDA) as compared to volume overload (as seen in shunts with ASD). Furthermore, patients with ASD may harbor a specific unidentified genetic predisposition to the development of PAH that may exacerbate the increased volume load to the pulmonary circulation.

At the molecular level, the physiological flow patterns of laminar shear stress, turbulent flow, and cyclic strain are all recognized by ECs, leading to transduction of intracellular signals and modulation of a wide variety of phenotypic changes.⁴⁰ Significant prior work has focused mainly on the endothelium of the peripheral vasculature, suggesting that laminar flow induces a vasoprotective quiescent vascular state, whereas turbulent flow leads to a proinflammatory and thrombogenic state. It is unclear whether these flow-dependent phenotypes are recapitulated in the pulmonary vasculature. In part, this stems from the difficulty of directly studying directly the in vivo flow patterns at the anatomical level of the pulmonary arteriole. Ex vivo modeling of pulsatile flow with high levels of shear stress and chronic vascular endothelial growth factor (VEGF) inhibition has demonstrated apoptosis of pulmonary artery ECs, followed by outgrowth and selection for proliferating apoptosis-resistant cells.⁴¹ Therefore, chronically elevated flow may allow for selection of cells with dysregulated EC growth and resulting clonal or polyclonal expansion to plexiform lesions.

Persistent pulmonary hypertension of the newborn

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by persistent elevation of PVR, right-to-left shunting, and severe hypoxemia. It can occur with pulmonary parenchymal diseases including sepsis, meconium aspiration, pneumonia, maladaptation of the pulmonary vascular bed, or without an apparent cause.⁴² Persistent PH in newborns may lead to death during the neonatal period, or it may be transient, leading to spontaneous and complete recovery. Inadequate production of NO may be an important contributor to persistent PH in infants, and inhaled NO is useful in treating this disorder.

Miscellaneous disorders

Pulmonary arterial hypertension is more rarely reported in patients suffering from other clinical syndromes such as pulmonary veno-occlusive disease, chronic myelodysplastic syndromes with thrombocytosis, and idiopathic thrombocythemia, as well as in persons exposed to stimulants of the central nervous system, such as methamphetamines and cocaine. Dysregulation of serotoninergic signaling may contribute⁸ but does not explain these associations entirely. Finally, idiopathic PAH demonstrates a gender predilection of unclear etiology, with a high predominance of affected females.⁴³

Nitric oxide

Gaseous vasoactive molecules regulate pulmonary vascular homeostasis, and alterations in their endogenous production have been linked to progression of PAH. Nitric oxide is the best described of these factors (see Fig. 56-4C).⁴⁴ It is a potent pulmonary arterial vasodilator as well as a direct inhibitor of platelet activation and VSMC proliferation. Nitric oxide diffuses into recipient cells (e.g., vascular muscle), where it activates soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). In turn, cGMP interacts with at least three different groups of effectors: cGMP-dependent protein kinases, cGMP-regulated phosphodiesterase (PDE), and cyclic nucleotide-gated ion channels. Synthesis of NO is mediated by a family of NO synthase (NOS) enzymes. In the pulmonary vasculature, the endothelial (eNOS) isoform figures most prominently and is regulated by a multitude of vasoactive factors and physiological stimuli including hypoxia, inflammation, and oxidative stress. Reduced levels and reduced activity of eNOS, reduced NO bioavailability, dysregulated intracellular NO transport via caveolae, and increased degradation of cGMP all aggravate PAH progression in human studies and/or animal models.

Dysregulation of NO may also depend upon still incompletely characterized processes of NO transport in blood.⁴⁵ Specific polymorphisms of NOS have also been associated with pulmonary hypertension. Together, these effects indicate a coordinated mechanism of dysregulated vasoconstriction. Correspondingly, murine models that genetically lack eNOS, GTP cyclohydrase-1 (the rate limiting enzyme for synthesis of an essential cofactor of NOS, tetrahydrobiopterin), or dimethylarginine dimethylaminohydrolase (DDAH, an enzyme important in degradation of NOS inhibitors) all display exaggerated susceptibility to developing PH in response to other endogenous stimuli.³ Finally, the impact of NO has been reflected in its therapeutic role in PAH, such as inhaled NO⁴⁶ and the NO-dependent phosphodiesterase type-5 (PDE5) inhibitors (discussed later in detail).

Prostacyclin/thromboxanes

The arachidonic acid metabolites prostacyclin and thromboxane A₂ (TxA₂) also play crucial roles in vasoconstriction, thrombosis, and to a certain degree, vessel wall proliferation (see Fig. 56-4C). Prostacyclin (prostaglandin I₂) activates cAMP-dependent pathways and serves as a vasodilator, antiproliferative agent for vascular smooth muscle, and inhibitor of platelet activation and aggregation. In contrast, TxA₂ increases vasoconstriction and activates platelets.⁴⁷ Protein levels of prostacyclin synthase are decreased in small and medium-sized pulmonary arteries in patients with PAH, particularly with the idiopathic form.⁴⁸ Biochemical analysis of urine in patients with PAH has shown decreased levels of a breakdown product of prostacyclin (6-ketoprostacyclin F₂α), accompanied by increased levels of a metabolite of TxA₂ (thromboxane B₂).⁴⁹ Therefore, it appears that production of these effectors is coordinately regulated, with the imbalance toward TxA₂ favored in the development of PAH. The mode of this regulation remains to be characterized. Nonetheless, recognition of this imbalance has led to the success of prostacyclin therapy and improvement of hemodynamics, clinical status, and survival in patients with severe PAH.⁵⁰

Endothelin-1

Endothelin-1 is expressed by pulmonary ECs and has been identified as a significant vascular mediator in PAH (see Fig. 56-4C).⁵¹ It acts as both a potent pulmonary arterial vasoconstrictor and mitogen of pulmonary smooth muscle cells. The vasoconstrictor response relies upon binding to the endothelin receptor A (ET-A) on vascular smooth muscle cells. This leads to an increase in intracellular calcium, along with activation of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and the early growth response genes c-fos and c-jun.⁵² The mitogenic action of ET-1 on pulmonary VSMCs can occur through either the ET-A and/or the ET-B receptor subtype, depending on the anatomical location of cells. Endothelin receptor A predominantly mediates mitogenesis in the main pulmonary artery, whereas mitogenesis in resistance arteries relies upon contributions from both subtypes. The resulting vasoconstriction, mitogenesis, and vascular remodeling are thought to lead to significant hemodynamic changes in the pulmonary vasculature and to PAH. Plasma levels of ET-1 are increased in animal and human subjects suffering from PAH due to a variety of etiologies⁵³ and correlate with disease severity.⁵⁴ Again, improvement in hemodynamics, clinical status, and survival of PAH patients treated with chronic ET receptor antagonists highlights the significance of these effects.⁵⁵

Vasoactive intestinal peptide

Down-regulation of vasoactive intestinal peptide (VIP) may also play a pathogenic role. Vasoactive intestinal peptide is a pulmonary vasodilator, an inhibitor of proliferation of VSMCs, an inhibitor of platelet aggregation, and free radical scavenger. Decreased concentrations of VIP and VIP receptors have been reported in serum and lung tissue of patients with PAH.⁵⁶ Vasoactive intestinal peptide-null mice suffer from moderate pulmonary hypertension.⁵⁷ Both pulmonary arterial pressure (PAP) and PVR in humans decrease after treatment with VIP.^58,59 Key questions regarding the mode(s) of regulation of VIP expression and its putative causative role in PAH remain unanswered.

Peroxisome proliferator-activated receptor γ

Peroxisome proliferator-activated receptor gamma (PPAR-γ)and apolipoprotein E (apoE) may function as integral factors in PAH.^60,61 PPARs are ligand-activated nuclear transcription factors that heterodimerize with the retinoid X receptor for subsequent binding to PPAR response elements in the promoters of target genes. Idiopathic PAH patients carry reduced pulmonary transcript levels of PPAR-γ and apoE. PPAR-γ in particular is a direct target of BMP2 in human PASMCs, leading to stimulation of apoE synthesis and downstream inhibition of vascular smooth muscle proliferation. PPAR-γ also regulates a host of protein kinases implicated in PASMC proliferation and migration. PPAR-γ agonists are antiinflammatory and induce pro-apoptotic phenotypes, both theoretically inhibitory to the pathogenesis of PAH. Mice deficient in smooth muscle–specific PPAR-γ are prone to PAH. Similarly, when fed a high-fat diet, male mice deficient in apoE develop PAH. This condition is reversed by rosiglitazone, a PPAR-γ agonist. Nonetheless, less robust results have been seen when using rosiglitazone hypoxic-PH rats, suggesting that these results offer a partial explanation to the complex molecular pathogenesis of BMPR2 signaling and PAH pathogenesis.

Natriuretic peptides

The atrial and brain natriuretic peptides (ANP and BNP) are produced by myocardium in response to stretch. They bind guanylyl cyclase receptors (NPR-A and NPR-B) that induce production of cGMP. Increased plasma concentrations of these peptides in PAH have been used as markers of the extent of RV dysfunction.⁶² Genetic inactivation of NPR-A in mice is associated with PH⁶³; in contrast, administration of atrial natriuretic peptide (ANP) ameliorates PAH in rodent models.⁶⁴ Additionally, inhibition of the metabolic breakdown of natriuretic peptides via neutral endopeptidase inhibitors has shown promise in animal studies of PAH. Human studies have yet to confirm efficacy.

Adrenomedullin

Adrenomedullin is an endogenous peptide that activates signaling pathways (i.e., cAMP, NO-cGMP, phosphatidylinositol-3-kinase/Akt) to induce vasodilation and inhibit proliferation. It decreases mean PAP and RV hypertrophy in hypoxic rats.⁶⁵ A related peptide, adrenomedullin-2, binds the same cellular receptors as adrenomedullin, and it is elevated in some rodent forms of PAH.

Miscellaneous effectors

Other potential contributing factors include angiopoietin-1, the vasodilatory gases carbon monoxide and hydrogen sulfide, phosphodiesterase I, survivin, the calcium binding protein S100A4/Mts, the transient receptor potential channels, and Notch 3.⁶⁶ These may represent important but as yet incompletely described pathogenic contributors.⁸ Other mitogenic and angiogenic growth factors, such as VEGF, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF)-2, insulin-like growth factor (IGF)-1, and epidermal growth factor (EGF) all may play downstream roles in later stages of PAH.

Mitochondrial metabolism

Mitochondrial and metabolic dysfunction may be common in PAH. Endothelial and VSMCs from human PAH tissue display dysmorphic and hyperpolarized mitochondria.⁶⁸ Metabolically, these cells preferentially exhibit a down-regulation of mitochondrial metabolism with an induction of glycolysis for energy production.⁶⁹ Under hypoxic conditions, this so-called Pasteur effect is a normal adaptive event that improves cellular survival by optimizing adenosine triphosphate (ATP) production while reducing oxygen radicals generated from the mitochondrial electron transport chain. Under normoxic conditions, however, such a shift to glycolysis (the Warburg effect) is thought to confer inappropriate resistance to apoptosis and is prominently seen in various cancer lineages. Such metabolic changes in the mitochondria are dependent upon a master transcription factor of hypoxia, hypoxia inducible factor 1 alpha (HIF-1α), and its critical downstream targets such as pyruvate dehydrogenase kinase (PDK1), among others.⁷⁰ In PAH, HIF-1α and PDK1 are dysregulated; conversely, treatment with dichloroacetate, a PDK inhibitor, ameliorates multiple forms of PAH in rodent models (chronic hypoxic PH, monocrotaline PAH, and fawn-hooded rat PAH).⁷¹ It is conceivable that therapies aimed at reversing this metabolic dysregulation may result in improvement and/or regression of the PAH phenotype.

Right ventricular dysfunction

After birth, PAPs typically decline, leading to involution of the RV to a thin-walled structure in the adult. During pathological conditions of increased PAPs in PAH, however, RV hypertrophy (RVH) and strain ensue, followed by RV failure if left untreated. Historically, the molecular pathways governing left ventricular (LV) failure, which have been better characterized, had been assumed to play primary roles in RV failure as well. Contemporary evidence suggests there may be distinct molecular and physiological differences between LV and RV failure that are just beginning to be explored.⁷² For instance, PDE5, which is expressed in the fetal RV, is selectively up-regulated in the hypertrophied RV. Interestingly, inhibition of PDE5 enhances RV contractility without affecting the LV, where PDE5 expression is absent.

Unique metabolic changes in the RV have also been reported. The normal RV can use either fatty acids or glucose as needed for energy production. In RVH, the RV nearly exclusively relies upon glycolysis. Adenosine monophosphate (AMP)-activated protein kinase is activated in RVH, which preserves ATP levels by increasing glucose transport and glycolysis while inhibiting the tricarboxylic acid (TCA) cycle via repression of acetyl-coenzyme A carboxylase.⁷³ Furthermore, during RVH, PDK is activated, inducing a metabolic switch away from oxidative phosphorylation to glycolysis. As a result, the hypertrophied RV has been hypothesized to behave as “hibernating myocardium,” which may suggest that the RV can be targeted therapeutically in PAH.³

Potassium channel biology

Modulation of voltage-gated potassium channels (Kv) may also represent an overarching pathogenic mechanism of PAH.⁷¹ Kv channels are tetrameric membrane-spanning channels that selectively conduct potassium ions; in PASMCs, Kv channels aid in regulation of the resting membrane potential. In response to Kv inhibition or down-regulation, depolarization leads to the opening of voltage-gated calcium channels, increase in intracellular calcium, and initiation of a number of intracellular signaling cascades promoting vasoconstriction and proliferation and inhibiting apoptosis. Expression array analysis has demonstrated a depletion of Kv1.5 channels in pulmonary tissue derived from PAH patients.⁷⁴ It is currently unknown whether these Kv channel abnormalities are congenital or acquired, but a number of polymorphisms in the Kv1.5 channel gene (KCNA5) have been described, which may suggest a genetic predisposition to channel depletion.⁷⁵ Appetite suppressants (e.g., dexfenfluramine, aminorex) that are risk factors for development of PAH can also directly inhibit Kv1.5 and Kv2.1. A variety of transcription factors (HIF-1α, NFAT, and c-Jun) are increased in PAH, with resulting down-regulation of Kv1.5 expression in PASMCs. Inhibition of these factors consequently increases Kv1.5 expression, with resulting improvements in Kv current and in some cases, improved pulmonary arterial remodeling in hypoxic rodent models of PH. Inhibition of Kv currents in pulmonary SMCs may be regulated by serotonin, TxA₂, and perhaps NO. Furthermore, BMP signaling can regulate Kv receptor expression. Taken together, the Kv pathway may represent a common point of regulation in pathogenesis. Accordingly, augmentation of Kv activation would be predicted to induce vasodilation and perhaps allow for regression of vessel remodeling. In vivo gene transfer of Kv channels in chronically hypoxic rats has led to improvement of PH and suggests its therapeutic potential.⁷⁶

Tyrosine kinases

End stages of PAH are marked by exaggerated expression or activity of multiple growth factors, including PDGF, fibroblast growth factor 1 (FGF-1), FGF-2, EGF, and VEGF. All appear to contribute substantially to the overall obstructive arterial remodeling characteristic of PAH, via synergistic and combinatorial pathways. Some of the receptors for these growth factors are transmembrane receptor tyrosine kinases that activate a diverse and overlapping set of intracellular signaling pathways. Inhibition of EGF⁷⁷ and PDGF⁷⁸ receptors demonstrates improved hemodynamics and survival in PAH. Case reports exist of a beneficial effect of adding imatinib (a nonselective tyrosine kinase inhibitor)⁷⁹ or sorafenib (a “multikinase inhibitor”)⁸⁰ to baseline PAH therapy; however, further mechanistic clarity of the specific role of tyrosine kinases in the pathogenesis of PAH is needed.

Rho-kinase signaling

Multiple vascular cell types rely upon the rho kinase signaling pathway for homeostatic function and response to injury.⁸¹ Rho is a guanosine triphosphate (GTP) binding protein that activates its downstream target, rho-kinase, in response to activation of a variety of GPCRs (including those related to BMP/SMAD signaling and serotonin signaling). In vitro activation of these signaling cascades results in modulation of multiple cellular processes, including enhanced vasoconstriction, proliferation, impaired endothelial response to vasodilators, chronic pulmonary remodeling, and up-regulation of vasoactive cytokines via the nuclear factor kappa beta (NFκB) transcription pathway. Rho-kinase activity has also been linked specifically to a number of known effectors of PAH, including ET-1, serotonin, and eNOS, among others. Elevated rho-kinase activity has been demonstrated in animal models of PAH,^82,83 and intravenous fasudil, a selective rho-kinase inhibitor, has induced pulmonary vasodilation and regression of PAH in various animal models (as reviewed in⁸), as well as in patients with severe PAH who were otherwise refractory to conventional therapies.^84,85 Taken together, these data suggest that rho-kinase may control a master molecular “switch” in the pulmonary artery, initiating an activated state in disease from a quiescent state in health. As a result, rho-kinase represents an attractive and novel upstream therapeutic target for treatment of PAH.

Inflammation

As reflected by a strong association of PAH with various autoimmune and infectious states as well as a significant presence of T cells, B cells, and macrophages in plexiform lesions, a severe inflammatory state predominates end-stage PAH. Athymic nude rats, which lack functioning T cells, display greater sensitivity to PAH.⁸⁶  T-cell dysregulation may figure prominently because regulatory T cells (T_reg) are increased, whereas CD8⁺ cytotoxic cells are decreased, in idiopathic PAH.⁸⁷ In addition, a number of soluble chemoattractants and proinflammatory cytokines from the pulmonary artery are up-regulated in human and animal models of severe PAH. These include interleukin (IL)-1β, TGF-β₁, bradykinin, monocyte chemotactic protein-1, fractalkine, RANTES, and leukotrienes, among others. 5-Lipoxygenase (5-LO) regulates synthesis of leukotrienes, which in turn can promote cytokine release. 5-Lipoxygenase may represent a possible upstream factor involved in inciting this proinflammatory state, and elevated levels of 5-LO have been detected in macrophages and pulmonary endothelium derived from patients suffering from idiopathic PAH.⁸⁸ In various murine models of PAH, overexpression of 5-LO has worsened PH and vascular remodeling,⁸⁹ while 5-LO inhibitors have attenuated PH. It is thus tempting to speculate that 5-LO itself may possess an upstream regulatory role in PAH progression.

Extracellular matrix biology

Vascular-specific serine elastase activity has been implicated in PAH pathogenesis via regulation of the remodeling response in the extracellular matrix.⁹⁰ In pulmonary arterioles, serine elastases are secreted into the extracellular space to activate matrix metalloproteinases (MMP) and inhibit tissue inhibitors of MMP (TIMP). Both MMP and elastases degrade most components of the ECM leading to an up-regulation of fibronectin (FN) and subsequent enhancement of cellular migration. Matrix degradation also increases integrin signaling, with resulting expression of the glycoprotein tenascin C. Tenascin C acts cooperatively with other factors to enhance smooth muscle proliferation. Increased degradation of elastin⁹¹ has been observed in pulmonary arteries from patients suffering from congenital heart disease and resultant PAH. In pulmonary tissue of PAH patients harboring BMPR2 mutations and rat models of PAH, increased production and activity of vascular elastases⁹² and tenascin C⁹³ have been reported. This up-regulation of elastase function may be induced by a number of vascular effectors implicated in PAH, including NO, serotonin, and theoretically, the BMP pathway. Elastase inhibitors can induce apoptosis of SMCs in cell culture and can improve PAH in animal models.^94,95 Thus, elastase function may also represent a novel therapeutic target.

Symptoms

By the time patients develop symptoms, PAH is usually advanced, and CO is often reduced. The nonspecificity of presenting symptoms can cause a long delay in diagnosis in most patients. The most common presenting symptom is dyspnea on exertion, which affects nearly all patients as disease progresses. Other presenting symptoms include fatigue, syncope or near syncope, chest pain, lower-extremity edema, or palpitations. Dyspnea may be due to impaired oxygen delivery during exercise, secondary to inability to increase CO to accommodate increased oxygen demand. Syncope occurs when CO is severely limited and inadequate cerebral blood flow with exertion ensues. Chest pain in PAH may be caused by subendocardial RV ischemia.

Physical findings

Clinical findings in PAH are initially subtle. The first signs of disease may be an RV heave, a loud pulmonic second heart sound, and a right-sided fourth heart sound. Eventually a right-sided third heart sound and a left parasternal systolic murmur of tricuspid regurgitation may be audible. Findings of jugular venous distension, ascites, and peripheral edema indicate overt right heart failure. Physical examination must include evaluation for signs associated with specific diseases associated with PAH, including collagen vascular disease, liver disease, HIV, HHT, thyroid disease, and all secondary causes of PH.

Laboratory studies

Secondary causes of PAH should be sought with serology for HIV and collagen vascular diseases, liver function tests (LFTs), and toxic exposures. Thyroid function should be evaluated. Thrombocytopenia may be present in severe PAH and has multiple contributing causes, including platelet activation and aggregation, pulmonary vascular sequestration, hepatosplenomegaly with splenic sequestration, and an autoimmune-mediated syndrome similar to idiopathic thrombocytopenic purpura.⁹⁷ Thrombocytopenia may accompany microangiopathic hemolysis when blood flows through fibrin deposits in plexiform lesions, with subsequent shearing of red blood cells and platelets. Prostacyclin may also induce thrombocytopenia. Thrombocytosis may be present in patients following splenectomy.

In patients with PH, high levels of ANP and BNP parallel decreased RV function. Levels of both peptides decrease with prostacyclin treatment and ensuing hemodynamic improvement. A subsequent increase in plasma BNP has been demonstrated to be an independent predictor of mortality.⁹⁸

Radiographic studies

Chest radiographs in PH usually show an enlarged pulmonary trunk and hilar pulmonary arteries, pruning of peripheral vessels, and obliteration of the retrosternal clear space by the enlarged RV (Fig. 56-6). Occasionally the chest radiograph may appear normal.⁵ High-resolution computed tomography (CT) is used to evaluate lung parenchyma for interstitial lung disease. Helical CT is used to evaluate the central pulmonary arteries for presence of thrombi. Ventilation/perfusion scans are used to rule out chronic pulmonary thromboemboli. In patients with PAH, these scans are normal or show only patchy defects. If inconclusive, a pulmonary angiogram, which will show pruning of peripheral vessels in PAH, must be performed to definitively exclude thromboembolic disease. Use of pulmonary magnetic resonance angiography (MRA) has not been widely reported, but has recently been shown to identify patients with PAH with high sensitivity and negative predictive values.⁹⁸

Figure 56-6 Chest radiograph in pulmonary arterial hypertension (PAH).

Pulmonary arteries are highly prominent bilaterally, with abrupt tapering (or “pruning”) of vessels due to increased peripheral vascular resistance (PVR) and diminished flow. There is right atrial and ventricular enlargement. Lung parenchyma is normal.

Electrocardiogram

The electrocardiogram (ECG) is not a sensitive or specific screening tool. In advanced disease, the ECG usually shows signs of right heart strain and enlargement, including right axis deviation and evidence of RV hypertrophy. Presence of a conduction abnormality is not typical of PAH. Electrocardiogram evidence of right heart strain has been associated with decreased survival.²

Pulmonary function tests

Pulmonary function tests (PFTs) are important in excluding secondary causes of PH, particularly chronic obstructive airways disease. Airway obstruction is not typical of PAH, although cases of bronchial obstruction due to enlarged pulmonary arteries have been reported. Pulmonary arterial hypertension patients typically demonstrate borderline restrictive physiology, a reduced diffusing capacity for carbon monoxide (DLCO), and hypoxemia with hypocapnea.⁵ Reduction in DLCO results from reduced blood volume in alveolar capillaries. In a study of pulmonary function in 79 patients presenting with PAH, significantly reduced DLCO (mean 68% of predicted values) was observed in 75% of patients, and mild restrictive physiology was seen in 50%.⁹⁹

Echocardiography

Transthoracic echocardiography is a crucial diagnostic tool in evaluating patients for PH.⁹⁶ It can determine the presence of left-sided heart disease, valvular disease, and intracardiac shunts, and it allows noninvasive measurement of PAP. A finding of elevated PAP must be further evaluated with pulmonary artery catheterization. Echocardiography of PAH patients frequently shows RV hypertrophy and dilation, right atrial enlargement, and decreased LV cavity size due to bowing of the interventricular septum in advanced disease. The inferior vena cava is typically distended and does not collapse during inspiration in advanced disease. Systolic PAP can be estimated using Doppler measurement of the tricuspid regurgitant flow velocity. The upper limit of normal systolic PAP is generally considered 40 to 50 mmHg at rest, corresponding to a tricuspid regurgitant velocity of 3 to 3.5 m/s (although this value varies with age, body mass index [BMI], and right atrial pressure).

Limitations to Doppler measurement of PAP do exist, however; false-negative studies are possible in patients with poor-quality views or moderate elevations in PAP. Doppler estimates of pressures are also operator dependent. Studies comparing Doppler-derived PAP values with pressures determined by catheterization yield varying results, with some reporting underestimation of systolic PAP by Doppler.⁹⁸ Echocardiography is probably best employed for its negative predictive value, although a number of echocardiographic features of PAH suggest worsened prognosis.¹⁰⁰ These mostly include echocardiographic markers of RV dysfunction (i.e., right atrial or ventricular size, septal shift toward the LV during diastole, tricuspid annular plane excursion to approximate RV ejection fraction, RV myocardial index). Notably, however, echocardiographic estimates of RV systolic pressure do not predict survival.¹⁰¹

Exercise echocardiography is a more sensitive test for the presence of early PAH, which is particularly valuable in pediatric cases where it may influence decisions regarding surgery. Exercise echocardiography has been studied as a screening tool to identify asymptomatic carriers of BMPR2 mutations in PAH families.¹⁰² A resting echocardiogram demonstrating normal PA pressure usually excludes a diagnosis of PH. When PAH is strongly suspected or a patient has unexplained dyspnea, however, exercise echocardiography performed during exercise may reveal exercise-induced PAH.

Cardiac catheterization

Cardiac catheterization remains the gold standard for establishing the diagnosis and type of PAH. This procedure can directly measure right heart and PA pressures, as well as pulmonary capillary wedge pressure and CO. It can also be used to assess vasodilation reserve (see explanation later in the section on vasodilator therapy) and is the major determinant in prognosis of PH. Cardiac catheterization can also be performed with exercise to assess the possibility of exercise-induced PH, in which the resting PAP is normal, but PAP during exercise is abnormally high.

Exercise testing

Exercise testing is not required for a diagnosis of PH, but it may provide valuable information regarding prognosis. The most widely used and reproducible exercise test is the 6-minute walk test. This test is usually done after the diagnosis is confirmed by cardiac catheterization, and at regular intervals to monitor functional status. The distance walked in 6 minutes has been shown to decrease in proportion to the NYHA functional class and is a strong independent predictor of mortality. Patients with PAH who walk 300 or fewer meters and display decreased arterial oxygen saturation by 10% at maximal distance suffer from a significantly increased rate of mortality.⁹⁸ Maximal exercise testing must be avoided because syncope and sudden death have been reported. Cardiopulmonary exercise testing with or without echocardiography has been increasingly used in research studies and can be helpful in distinguishing PAH from PH secondary to diastolic heart failure.

Screening

Screening asymptomatic patients at high risk for PAH is recommended.¹⁰³ The exact population recommended for screening is controversial, since the prevalence of disease is low even in categories of patients at increased risk. Patient groups who likely benefit from screening include those with already known genetic mutations predisposing to PAH, first-degree relatives in a familial PAH cluster, patients with scleroderma, portal hypertension prior to liver transplantation, and patients with congenital systemic-to-pulmonary shunts.¹⁰⁴ Some patients should be evaluated for PAH only if they present with symptoms suggestive of the disease, including those with collagen vascular disease other than scleroderma, HIV, intravenous drug users, patients exposed to appetite-suppressant drugs, and patients with portal hypertension who are not being considered for transplantation. Screening asymptomatic or minimally symptomatic patients should begin with a thorough history and physical examination to elicit symptoms or signs consistent with PH, followed by diagnostic testing if inconclusive. Transthoracic echocardiogram is the best noninvasive test used for screening patients.

Genetic testing

Genetic testing of asymptomatic individuals is not currently advocated as an effective method for diagnosis, considering the low penetrance of disease manifestation even if a predisposing mutation is present. The risk for disease in first-degree relatives of a patient with PAH is relatively low, which has led to uncertainty in genetic screening of their family members. Advocates suggest that screening asymptomatic patients may enhance knowledge of the prevalence of familial PAH and shed light on whether early treatment influences disease pathogenesis. Screening also allows at-risk individuals to be aware of known risks that theoretically may augment penetrance of the disease.¹⁰⁵ However, genetic testing results can be confusing; even if family members have inherited a predisposing mutation, about an 80% chance exists that no discernible disease phenotype will manifest. Notably, such circumstances may result in detrimental psychological, employment, and insurance effects and, if pursued, must be supported by appropriate genetic counseling.

Initial screening for vasodilator reserve

Acute vasoreactivity testing is generally used as an initial screen to assess for vasodilator reserve and potential response to vasodilator therapy.¹⁰⁹ A significant response to vasodilator testing is generally accepted as a decrease in PVR by at least 25%, with a decrease in mean PAP to less than 40 mmHg. Other parameters include a greater than 20% decrease in systolic PAP, a greater than 10 mmHg drop in mean PAP, or an increase in cardiac index by 30% or more. The most widely used drugs for acute vasoreactivity testing include inhaled NO and intravenous prostacyclin (epoprostenol), adenosine, and iloprost, a prostacyclin analog with less effect on the systemic vasculature. Testing more than one drug offers no advantage. Vasodilator challenge must be performed with care because drug-induced systemic hypotension (e.g., with prostacyclin) may reduce RV coronary blood flow and cause RV ischemia.

Most studies on PAH report the proportion of responders to vasodilators as between 12% and 25%.⁹⁶ There is no particular clinical or disease characteristic that reliably predicts vasodilator response. Lack of response to acute vasodilators predicts response to oral vasodilator therapy (i.e., nonresponders to inhaled NO do not respond to oral calcium channel blockers). Response to acute vasodilators, however, does not predict a response to prostacyclin. A trial of long-term calcium channel blocker therapy is usually recommended in PAH patients who respond to vasodilator challenge with a decrease in PVR of 50 or more.

Calcium channel blockers

Calcium channel blockers are the oral drugs of choice for treating patients who have a significant response to acute vasodilators.¹⁰⁹ These agents have been shown to improve hemodynamics, RV function, and survival in the subpopulation of PAH patients responding to acute vasoreactivity testing.¹¹⁰ Empirical therapy without vasodilator testing is not recommended because of the high rate of treatment failure. Both nifedipine and diltiazem are effective in appropriate patients, and the choice between these two drugs is guided by resting heart rate. Doses of these drugs required to lower PVR are much higher than those used for other indications, which makes systemic side effects a significant problem, particularly hypotension and lower-extremity edema. Verapamil is not used because of its greater negative inotropic effects. Acute administration of amlodipine causes pulmonary vasodilation, but its long-term efficacy has not been studied.

Endothelin-1 receptor antagonists

Presently, three endothelin receptor antagonists are approved for oral use in PAH: two in the United States (bosentan [Tracleer] and ambrisentan [Letairis]) and one in Europe and Canada (sitaxsentan [Thelin]).¹¹¹ Bosentan is a nonspecific antagonist recognizing both endothelin receptor subtypes A (ET-A) and B (ET-B), and ambrisentan and sitaxsentan are specific for ET-A. It is thought that specific inhibition of ET-A may provide more benefit by decreasing the vasoconstrictor effects of ET-A while allowing the vasodilator and ET-1 clearance functions of ET-B receptors. All three agents have been found to improve exercise capacity and hemodynamics in 12- to 16-week clinical trials.^112–114 Bosentan has also improved survival in open-label studies and comparison with historical control data.⁹⁶ Long-term survival data for the selective endothelin inhibitors appear favorable as well, with some preliminary data suggesting that time to clinical worsening favors sitaxsentan over bosentan. No clear survival data are yet available from properly designed clinical trials. Because of its efficacy and ease of use, bosentan is currently recommended for stable functional class III PAH patients as first- or second-line agents.

A major complication of bosentan includes a dose-dependent increase in liver transaminases, which necessitates discontinuation in 2% and dose adjustment in 8% to 12% of patients. Hepatic toxicity can occur. Teratogenicity is a class effect of these medications. Delayed hemodynamic benefit, compared to the immediate effect of prostacyclins, should also be expected. Bosentan can alter levels of concurrently dosed oral contraceptives, PDE inhibitors, or HIV antiretroviral drugs. Sitaxsentan use has been complicated by supertherapeutic anticoagulation.

Phosphodiesterase inhibitors

Originally marketed for erectile dysfunction (ED), oral PDE5 inhibitors such as sildenafil (Revatio) selectively inhibit the cGMP-specific enzyme PDE5, enabling endogenous NO to exert a more sustained effect.¹¹⁵ This PDE is highly abundant in pulmonary vasculature, making such medications relatively selective pulmonary vasodilators. A more recently approved PDE5 inhibitor for use in PAH includes tadalafil. In patients with lung fibrosis and PAH, acute administration of sildenafil surpasses the vasodilating effect of NO. The effect is similar to intravenous epoprostenol, except sildenafil is more selective for better ventilated areas of the lung, resulting in improved gas exchange. Clinical efficacy has been reported with use of sildenafil in patients with primary PAH and PAH associated with congenital shunts, SLE, and HIV. Benefits with treatment include improved symptoms and hemodynamics, and increased 6-minute walk distance. In a multicenter double-blinded, randomized, placebo-controlled study of sildenafil treatment for PAH, significant improvements in exercise ability and quality of life have been observed; in the open-label follow-up study, 95% survival was measured at 1 year.¹¹⁶

Major side effects of PDE5 inhibitors include systemic hypotension, especially in the setting of nitroglycerin use. Approved dosing of sildenafil for PAH is three times daily, whereas tadalafil is typically a once-daily medication. Currently, PDE inhibitors are approved for functional class II-III PAH patients as first-line agents, and for class IV patients as second-line agents.

Prostacyclin and prostacyclin analogs

Prostacyclin is an endogenous prostaglandin (prostaglandin I₂) that causes vasodilation and inhibits platelet aggregation. It also has antiproliferative and weak fibrinolytic activities. Intravenous prostacyclin (epoprostenol [Flolan]) was first used to treat PAH in the early 1980s. Since then, multiple trials of epoprostenol have demonstrated improved survival, exercise capacity, and hemodynamics in patients with PAH compared with conventional treatment.¹¹⁷ Epoprostenol was approved for use in PAH in 1995; it is now considered the treatment of choice in patients with functional class IV PAH and is an alternative to lung transplantation.¹⁰⁷ A chronic, perhaps remodeling, effect of epoprostenol has additionally been suggested by several findings. Patients who have no acute response may demonstrate a delayed hemodynamic improvement. Hemodynamic improvement increases in patients who show an initial response, and RV dysfunction improves after long-term therapy.

Epoprostenol has an extremely short half-life (approximately 3-6 minutes), must be kept cold, and must be administered through a central venous catheter. It is started in the hospital, employing a PA catheter with continuous monitoring. Some clinicians continue the dose escalation until limited by side effects or until there is a plateau in the hemodynamic response. Notably, initiation of epoprostenol can lead to increased CO with LV strain, or isolated pulmonary edema, with rapid clinical deterioration. Long-term dose requirements are highly variable among patients, and further dose increases are made on an outpatient setting on the basis of clinical symptoms, exercise testing, and hemodynamic measurements. Common side effects include jaw pain, headache, diarrhea, flushing, leg pain, nausea, and vomiting. Patients also tend to develop tachyphylaxis to epoprostenol over time.

Major limitations to use of epoprostenol include the need for permanent central venous access, with the associated small risk of catheter-related infection or air embolism, the capacity to handle the catheter and pump, and the high cost of the drug. Complexities of epoprostenol administration have led investigators to search for alternative agents.¹¹⁸

The prostacyclin analogs treprostinil (Remodulin), iloprost (Ventavis), and beraprost have been tested in 12-week placebo-controlled trials.¹¹⁷ All these agents have demonstrated significant improvements in mean exercise capacity. Treprostinil (Remodulin, UT-15) has a longer half-life (≈︀ 4 hours) than epoprostenol and can be delivered intravenously or subcutaneously with a pump system similar to that used with subcutaneous insulin. Treprostinil does not require reconstitution or cold temperature. Pain at the site of subcutaneous infusion is a frequent problem that requires cessation of the drug in 8% to 12% of patients. Iloprost is another chemically stable analog that can be given intravenously and by inhaled routes. Iloprost can be used in a nebulized form that must be inhaled 6 to 9 times daily for a continuous effect. Beraprost is an oral analog taken four times daily. Treprostinil and iloprost are approved for PAH therapy—treprostinil for functional class II-IV and iloprost for functional class II-IV.

Alternative nitric oxide–based therapy

In addition to PDE5 inhibitors, a number of therapeutic approaches designed to improve NO production, release, and activity in the pulmonary vasculature are under investigation. Inhaled NO gas has been successfully used to decrease PAP and improve oxygenation and hemodynamics in diverse forms of PAH. However, long-term NO therapy is cumbersome, necessitates continuous inhalation, and must be monitored carefully to avoid toxicity with nitrogen oxides and methemoglobin. Alternative strategies to deliver NO include the use of NONOates, compounds that release predictable levels of NO when exposed to physiological pH. Such compounds have been used by daily nebulization in rodent PAH models, with reduction of PAP without systemic hypotension.¹³⁴

Another avenue of increasing NO levels in PAH relies upon enhancing NOS activity. Tetrahydrobiopterin (BH4) is an important NOS cofactor that enzymatically links oxygenation of L-arginine to create NO and L-citrulline. BH4 levels may be decreased in PAH patients, so administration of BH4 in its synthetic form (sapropterin) may hold promise in PAH to increase NOS activity and NO levels in the pulmonary vasculature.³

Finally, NO-independent sGC activators have been developed for PAH patients.⁴⁴ Both heme-dependent (BAY 41-2272) and heme-independent (BAY 58-2667 and HMR-1766) sGC activators exist.¹³⁵ BAY 41-2272 stimulates sGC directly and sensitizes sGC to NO, resulting in synergism. It improves pulmonary hemodynamics in models of PH. NO-independent sGC activators appear to provide an additive rather than synergistic effect when combined with NO donors. Both BAY 41-2272 and BAY 58-2667 ameliorate PAH in rodent models, yet this benefit is partially dependent on endogenous NOS activity. BAY 63-2521 is an oral agent, has a favorable safety profile, and is currently under phase III clinical study in PAH. Interestingly, dehydroepiandrosterone (DHEA), an endogenous steroid, also increases sGC expression, activates vasodilatory calcium-sensitive potassium channels (BKCa) in vascular smooth muscle, and inhibits hypoxic PH in rats.¹³⁶ More data are necessary before testing in human trials.

Serotonin and serotonin transporter inhibitors

Terguride is a potent antagonist of 5-hydroxytryptamine receptors and is currently in a phase II study for PAH patients.³ It has already received orphan drug status in Europe. PRX-08066 is a selective 5-hydroxytryptamine 2B receptor antagonist that inhibits hypoxia-mediated increases in PAP in humans and is also under phase II trial study. Given the importance of SERT in the biology of serotonin signaling, synergistic inhibition of both SERT and serotonin receptors may represent a potent future strategy for therapeutic benefit.

BMPR2-targeted therapies

Recent advances linking BMPR2 haploinsufficiency to PAH suggests that enhancing BMPR2 expression or activity may benefit or reverse PAH progression. Gene replacement therapy has been proposed but has yet to show success in animal models. It has also been found that some mutant forms of BMPR2 in PAH result in impaired trafficking to the cell surface rather than decreased expression.¹³⁷ In that case, medications that improve protein trafficking (e.g., sodium 4-phenybutyrate as used in cystic fibrosis) may be useful; however, it remains uncertain how much mutant BMPR2 must reach the cell membrane to result in a relevant effect in vivo. Finally, dysfunction of BMPR2 can lead to increased signaling via other TGF receptor superfamily members such as the TGF-β/activin receptor-like kinase 5. Notably, SB525334, an inhibitor of TGF-β/activin receptor-like kinase 5, reverses PAH and RVH in a rodent model and may have therapeutic benefit.¹³⁸

Tyrosine kinase inhibitors

Initial experience with the tyrosine kinase inhibitor imatinib and the “multikinase inhibitor” sorafenib in PAH patients have been encouraging but results are still preliminary. In humans, there are case reports of beneficial effects in adding imatinib to conventional therapy.⁷⁹ Sorafenib reverses PAH and RV remodeling in monocrotaline-treated rats.⁸⁰ Phase I clinical trials studying both medications have been performed recently.

Rho kinase inhibitors

Rho kinase inhibitors (i.e., Y-27632, fasudil) have been developed for study in the PAH population.¹³⁹ These medications reduce PAP in multiple rodent models of PAH. In humans, fasudil induces modest but immediate reductions in PVR. However, a common adverse side effect includes systemic vasodilation and hypotension. Alternative delivery options such as inhaled or nebulized formulations may allow circumvention of this side effect.

Interestingly, one of the pleiotropic effects of HMG CoA reductase inhibitors (statins) includes inhibition of rho kinase activity. In some rodent studies, simvastatin reduces PAH and demonstrates a significant improvement in survival^140,141; however, these beneficial effects have not been replicated in all rodent models.¹⁴² Nonetheless, several statin trails in human PAH are underway.

Miscellaneous pathways

Therapeutic targets in several additional pathogenic pathways have been studied to varying degrees. Inhaled adrenomedullin modestly reduces PVR and increases peak oxygen consumption during exercise in humans with PAH.¹⁴³ Nebulized VIP improves pulmonary hemodynamics in PAH to a larger degree, and if continued for 3 months, reduces PVR and improves 6-minute walk distance⁵⁶; however, VIP is susceptible to rapid degradation. A sustained-release liposomal VIP preparation is currently under development. Heparins and inhibitors of angiopoietin 1, STAT3, polyamines, survivin, and the cell cycle have also been proposed for therapeutic benefit, but with varying degrees of success.³

Endothelial progenitor cells

Progenitor cell infusion represents a potentially powerful, but currently nebulous, therapeutic possibility. Specifically, endothelial progenitor cells (EPCs) arise from mesodermal stem cells or hemangioblasts in the bone marrow. From there, they are released and circulate in blood until they engraft into sites of ischemia or injury, leading to angiogenesis and revascularization. Circulating EPCs (defined by CD34⁺/KDR⁺ and CD34⁺/CD133⁺/KDR⁺ cells) are lower in patients with Eisenmenger’s syndrome.¹⁴⁴ Some reports have indicated reduced levels of EPCs in idiopathic PAH patients.¹⁴⁵ Furthermore, the functions of EPCs in cell culture (i.e., colony forming activity, adherence, migration, apoptotic potential) differ from those isolated from PAH patients as compared with normal controls.^144–146 Administration of EPCs in rats has resulted in improvement in pulmonary hemodynamics, vascular remodeling, and survival in models of PAH.¹⁴⁷ Additionally, two small studies in which adults¹⁴⁸ and children¹⁴⁹ with idiopathic PAH were given a single infusion of autologous mononuclear cells provides support for the therapeutic potential of such therapy. A therapeutic trial has begun to assess the safety of administering such cells in patients with PAH.

Much still remains unclear regarding regenerative cell-based therapy. Differences in markers used to identify EPCs have complicated interpretation of results. The complex biology of EPCs upon engraftment has not been delineated and may carry detrimental consequences in certain clinical contexts. Previously reported beneficial effects of progenitor cells may very likely result from paracrine effects of secreted factors, rather than true engraftment and transdifferentiation to healthy endothelium. In addition, some investigators argue that EPCs promote development of plexogenic lesions, suggesting that their use may worsen end-stage disease. Caution is certainly warranted before such a therapeutic avenue can be pursued on a large scale.