American Journal of Respiratory Cell and Molecular Biology

Idiopathic pulmonary fibrosis (IPF) is a disabling disease of the lung parenchyma, characterized by progressive accumulation of scar tissue and myofibroblast activation after repetitive epithelial microinjury. The therapeutic options are limited, and patients usually die within a few years after diagnosis. Pulmonary hypertension (PH) in IPF has been increasingly recognized as a condition with relevance for the overall prognosis. Treatment trials are being designed, but to be effective, it is crucial to better understand the pathobiology of PH in IPF: the traditional concept, that hypoxic vasoconstriction and accumulation of scar tissue are mainly responsible for the development of PH in IPF, has been challenged. Recent studies, including our own in vivo research, suggest that the underlying pathobiology is much more complex, and includes a complicated interaction of epithelial cells, fibroblasts, and vascular cells. This interaction seems to be regulated by a large variety of angiogenesis promoters and inhibitors, as well as growth factors. Central components seem to be endothelial apoptosis and growth factor–induced remodeling of the pulmonary artery wall. The present review gives a conceptual overview about known and putative mechanisms that are involved in the development of PH in IPF. This report summarizes currently available therapeutic options, and also translates experimental research to discuss potential novel biomarkers and therapeutic strategies derived from new concepts in pathogenesis.

Idiopathic pulmonary fibrosis (IPF) is a crippling disease because of progressive scarring of the lung tissue and the lung function of patients with IPF is characterized by a restrictive volume pattern and reduced gas exchange capacity (13). The typical clinical course is determined by increasing shortness of breath, decreasing exercise capacity, and, ultimately, death. Usual interstitial pneumonia (UIP) represents the histopathology representative of IPF. Features of UIP include a heterogeneous, patchy distribution of fibrosis with subpleural and basal predominance, accumulation of activated myofibroblasts in subepithelial fibroblastic foci, and excessive deposition of extracellular matrix (ECM) components, such as collagen and fibronectin (2, 4, 5). Transforming growth factor (TGF)–β1 is a key factor which promotes fibrosis and is, therefore, a main focus for therapy. Inflammation is not crucial for the progression of fibrosis, but a perpetual activation of TGF-β signaling through the Smad3 pathway is (69). IPF is not sufficiently treatable, and the prognosis after diagnosis is limited to an average survival of 3–5 years (2, 4). The mortality is predicted by a progressive decrease of the forced vital capacity, reduced distance in the 6 minute walk test with elevated O2 desaturation, and presence of pulmonary hypertension (PH) (1018).

Pulmonary arterial hypertension (PAH) has been defined as an elevation of the mean pulmonary artery (PA) pressure (PAP) over 25 mm Hg at rest, and, at the same time, a normal pulmonary capillary wedge pressure (19). PH in IPF has been increasingly recognized, and treatment trials are being designed (3, 20).

The most recent clinical classification of PH categorizes PH in IPF into category 3.2 (PH associated with lung diseases and/or hypoxia; subclass, interstitial lung disease [ILD]) (21). The prevalence of PH in patients with IPF is between 32 and 85%, and PH seems to develop over time in most patients with IPF (17, 20, 2224). Although most of the PH is moderate, pressures of systemic levels can be found in patients with IPF. Increased levels of brain natriuretic peptide may predict PH in patients with IPF (25). Although early diagnosis of PH is important, the overlapping main symptoms—shortness of breath and exercise limitation—make it difficult to detect PH in these patients (3). Typical physical findings associated with PH are usually detected when the pulmonary vascular disease is advanced.

To be effective in the treatment of IPF-associated PH, we need to understand how PH develops and progresses in IPF. The pathobiology of PH in IPF is incompletely understood and research groups have only recently started to focus on the vascular aspects of chronic lung fibrosis. The results of recent clinical studies do not support the hypothesis that the predominant mechanisms for the development of PH in IPF are hypoxic vasoconstriction and pulmonary capillary loss after scar tissue accumulation: the presence of PH cannot be explained in all patients with IPF by hypoxemia or degree of lung function reduction (23, 2628). Considering the complex pathophysiology of IPF, it seems likely that the biological processes underlying fibrosis progression are also involved in the vascular remodeling and PH (20). Our group has recently shown, in a model of experimental pulmonary fibrosis (PF), that the development of fibrosis and PH are closely connected (29). Here, we review the reported data in this field and discuss findings in other lung diseases associated with interstitial fibrosis in the context of IPF to develop an overall concept that integrates the factors and the processes involved in the development of PH in PF.

Wound repair or fibrogenesis in the lung is associated with (or caused by) the reactivation of pathways and programs that have been involved in lung development (30). To understand the interactions between vascular and interstitial processes in PF, it is necessary to revisit how the pulmonary vasculature and the parenchyma relate to each other during lung embryology.

The pulmonary vascular system develops in close proximity to and synchronous with airway and alveolus formation (31). During the embryonic phase of lung development, lung buds appear and branch into the surrounding mesenchyme. These buds are supplied by capillaries that connect to the aortic sac via PAs, and to the prospective left atrium of the heart via pulmonary veins (3133). Cartilage, glands, and smooth muscle cells (SMCs) develop and epithelium differentiates in the airway walls during the pseudoglandular stage. In addition, this phase is also responsible for establishing preacinary airways, arteries, and veins (31). During the canalicular and saccular stages, further branching occurs into respiratory airways, which are accompanied by arteries and veins. Endothelial and epithelial basement membranes fuse, thereby facilitating gas exchange. A necessary requirement for the formation of an effective gas exchange is the close proximity of capillaries and epithelium after the differentiation of cuboid type 2 alveolar epithelial cells (AECs) into thin type 1 cells, and a decrease of the surrounding mesenchyme. AEC2 starts surfactant production around 24–25 weeks of gestation, alveoli appear, and cup-shaped alveoli with double capillary loops develop, thus increasing the area of the alveolar capillary bed. Airways, arteries, and veins increase in size (31).

Some studies suggest that blood vessels in the lung originate from existing vessels through angiogenesis (proliferation and migration of existing endothelial cells [ECs]). Other studies have produced evidence that angiogenesis may only play a role for proximal arteries, whereas vasculogenesis (new development and proliferation of EC) may be important for the development of peripheral arteries and capillaries, which later connect to the proximal arteries (3236). Several angiogenic growth factors have been implicated in lung vascular development. The most widely investigated is vascular endothelial growth factor (VEGF), which is involved in both angiogenesis and vasculogenesis. Epithelial cells seem to be a predominant source of VEGF during lung development, which suggests that airway and alveolar epithelium are crucial for the generation of the entire pulmonary vasculature (3740). Impaired VEGF expression and signaling do not only have detrimental effects on vascular development and maintenance, but also on the structure and integrity of the whole lung (4042). Other angiogenic factors, such as angiopoietin (Ang)-1, are also involved in blood vessel formation (43). In addition, tightly regulated levels and activity of growth factors, such as TGF-β, are important for coordinated vascular development, as illustrated by the findings that mice overexpressing TGF-β1 during lung development show impaired epithelial differentiation and decreased capillarization (44).

The three main sources of vascular SMCs (VSMCs) in the developing lung may explain some mechanisms of VSMC generation found later in PH pathophysiology: (1) bronchial SMCs form the inner layer of VSMCs in the arteries accompanying the penultimate airways (31, 33); (2) fibroblasts surrounding the arterial wall differentiate into VSMCs after the release of chemoattractant growth factors, such as platelet-derived growth factor (PDGF) or epidermal growth factor from the endothelium in response to Ang-1 secreted by mesenchymal cells (45); (3) VSMCs can also arise from the inside of the vessel through endothelial-to-mesenchymal transition (46). Similar transition mechanisms might be involved in the pulmonary arterial remodeling in the adult, fibrotic lung.

It is not surprising that vascular remodeling in a patchy, heterogeneous disease such as IPF is not homogeneous, and that the vascular density varies in IPF lungs. Fibrotic areas have less blood vessels, but adjacent, nonfibrotic tissue seems highly vascularized (Figures 1B and 1C) (4749). Fibrotic regions contain apoptotic, but also proliferating, ECs, eventually resulting in anastomoses between alveolar capillaries and pulmonary veins with an aberrant vascular architecture (Figures 1C–1H) (47, 50, 51). These changes result in a reduction in the cross-sectional vascular area throughout the lung. It appears that, in human IPF, vascular rarefaction develops not only after expansion of scar tissue, but also due to an imbalance between angiogenic and angiostatic factors: the reduced expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), is paralleled by elevation of angiostatic molecules, such as pigment epithelium–derived factor (PEDF) (47, 48). Although there is some controversy regarding up-regulated proangiogenic and down-regulated angiostatic chemokines in IPF, we were able to directly link, in our animal model of PF, decreased VEGF levels to EC apoptosis and PH (29, 5255). EC apoptosis could be also induced by elevated oxidative stress (3, 56). It is interesting in the context of EC apoptosis in IPF that reduced numbers of circulating endothelial progenitor cells (EPCs) were found in IPF. This suggests that either reduced availability or increased pulmonary homing to sites of injury, possibly associated with dysfunction of EPCs, could be involved in endothelial injury in IPF (57, 58). Figure 2 shows a synopsis of mechanisms that are putatively involved in EC apoptosis in IPF.

The histopathologic changes in PAs of UIP lungs show a broad range of structural alterations, from isolated thickening of the smooth muscle layer and proliferative intima lesions, to complete occlusion of the vessel by scar tissue and plexiform lesions (Figure 1D). The extent of these changes increases with advancing fibrosis of the surrounding tissue (59). We were able to correlate fibrosis, TGF-β activity, angiostatic environment, EC apoptosis, and PA muscularization in our animal model of PF. Based on these findings, we suggest that VSMC growth factors may be released from apoptotic EC in fibrotic regions, and that mediators from the surrounding fibrotic tissue also contribute to augmented PA muscularization (29, 60). Such molecules, likely involved in both processes, are TGF-β, angiotensin (AT) II, endothelin (ET)-1, and PDGF (20, 6062). Again, oxidative stress could also play a role for VSMC and fibroblast proliferation, and thereby contribute to pathological changes in all three vascular layers of the PA (3, 56). A number of other important mechanisms will probably contribute to vascular remodeling and PH in IPF, such as endothelial dysfunction, with reduced release of vasodilators and inhibitors of VSMC proliferation, notably nitric oxide (NO) and prostacyclin (29, 60, 61, 6365). Reduced vasodilator release and enhanced secretion of vasoconstrictors, such as ET-1, thromboxane A2, and AT II, points toward a possible component of vasoconstriction, thus contributing to increased PAP and remodeling (60, 61). Conceptually, these mechanisms are summarized in Figure 3. The same mediators may also promote reduction in cross-sectional vascular area through thrombotic obstruction of vessels (3). The complex biochemistry in the fibrotic tissue with release of growth factors, such as TGF-β, may also result in endothelial-to-mesenchymal transition, which might contribute to the myofibroblast accumulation in intima fibrosis or VSMC growth during media thickening (6669). Inhibition of bone-morphogenic protein–induced mesenchymal cell apoptosis through up-regulation of one of their antagonists, gremlin, might also add to vascular remodeling, similarly to findings in idiopathic PAH (7072). In addition, mast cells occur at sites of epithelial injury, and they might play a role in either repair or disease progression via tryptase, histamine, or ET (73). Mast cells have been found around PAs in both human and experimental PH, but experimental studies did not result in a definitive concept about mast cells contributing to vascular remodeling or mediating a protective mechanism (7476). Future investigation will need to uncover additional potential mechanisms, such as serotonin levels, inflammation, and alterations of Ca2+ and K+ currents in SMC dysfunction, as well as activity of Rho guanosin triphosphatases, due to their relevance in other PH classes (77). Figure 4 summarizes possible mechanisms that are likely to contribute to PA wall remodeling.

Systemic Sclerosis

Systemic sclerosis (SSc or scleroderma), an unusual autoimmune disease without known cause, is defined by skin fibrosis, Raynaud's phenomenon, and possible involvement of other organ systems, such as lung, kidney, and the musculoskeletal, cardiovascular, and gastrointestinal system (78). Pulmonary pathology is the predominant cause of death in SSc, and the main findings are ILD and PH (79). Nonspecific interstitial pneumonia is the preponderant histologic phenotype, but some patients with SSc also have UIP (80, 81). PH can be found with or without association to ILD, and has a prevalence of 45% in SSc-ILD (28). Lower estimates around 10% have been shown by other groups (82, 83).

Endothelial apoptosis has been identified as the initiating event of the pathological processes leading to SSc (84). Circulating autoantibodies, including anti-fibrillarin and anti-EC antibodies, represent the autoimmune component of SSc, and these autoantibodies are likely responsible for EC injury and apoptosis (8587). The damage leads to recruitment of inflammatory cells with predominantly CD8+ T cells and a T helper (Th) type 2 cytokine pattern (e.g., monocyte chemoattractant protein-1, CXC ligand (CXCL)8 and regulated upon activation, normal T cell expressed and secreted) (8891). Elevated levels of profibrotic growth factors, such as TGF-β, PDGF, CTGF (connective tissue growth factor), or ET-1, result in mesenchymal cell proliferation and ECM accumulation (9296). The initial stage of SSc is characterized by inflammation and an increased angiogenic response, with elevated levels of VEGF in the beginning. Later on, reversible vasospasm and reduction in capillary density are found, eventually culminating in obliterative vasculopathy with intima proliferation and adventitia fibrosis (77, 97). There is an overall antiangiogenic environment in advanced SSc, characterized by reduced capillary density, which could contribute to PH as in IPF (51, 97). Proliferative intima and media lesions might be a consequence of EC apoptosis, secondary mediators, and local inflammation (77). It is interesting that vascular and fibrotic processes can influence each other via production of profibrotic growth factors (98). Anti-fibroblast antibodies could promote vascular remodeling even without EC injury (99). There is a variety of additional potential mechanisms that might be involved in the development of PAH, which are more or less related to the autoimmune component of the disease. Potentially involved mechanisms include various other circulating autoantibodies, such as anti-PDGF receptor, anti-centromere, anti–topoisomerase 1, or anti–matrix metalloproteinase (MMP) 1–3 antibodies (96). The investigation of PH in SSc-associated ILD will generate new concepts regarding the close relationship and interaction between inflammation and autoimmunity, and interstitial and pulmonary vascular remodeling. In addition, we are inclined to believe that these processes argue in favor of similar mechanisms underlying PH in different forms of ILD.

Nevertheless, the complex pathobiology of SSc with potential involvement of multiple extrapulmonary organs will make it difficult to specifically treat the pulmonary vascular disease, and makes it understandable that therapy of PAH and ILD in SSc will have to be also a therapy of the systemic autoimmune disease. This is clearly illustrated by the potential involvement of the myocardium in SSc, which can be found in 15–35% of patients with SSc (96, 100). Inflammation, vascular lesions, and cardiac fibrosis may lead to impaired cardiac function, and cardiac dysfunction has been shown to occur in the right ventricle, but also in the left ventricle, in SSc (100102).


Sarcoidosis is a systemic inflammatory disease characterized by noncaseating granulomas. The pathology is almost always found in mediastinal lymph nodes and the lungs (103). An unknown antigenic stimulus activates a sequence of immune responses, followed by T cell and macrophage activation, and a Th1-type immune response (103). Typical findings are accumulation of inflammatory cells (T cells) in the lung due to locally increased cytokines and chemokines, such as monocyte chemoattractant protein–1, CXCL8, or regulated upon activation, normal T cell expressed and secreted (103, 104). The resulting granulomatous inflammation can improve without persistent damage to the lung, but can also progress to PF (Figure 5A). When PH is detected in patients with sarcoidosis, then PH is typically found together with already advanced PF. Similar to SSc, some patients may have PH without underlying ILD. Nevertheless, PH is a predictor of increased mortality in all of these patients (27). The prevalence of PH in sarcoidosis varies between 5.7 and 73.8% in advanced disease (28).

Several mechanisms seem to be involved in the development of PH in sarcoidosis: the granuloma tissue itself is rarely capillarized (Figure 5B). Due to their anatomical localization, PAs are frequently affected by the granulomatous inflammation with occlusion, perivascular fibrosis, or vasculitis (Figures 5C and 5D) (105107). Compression or invasion of pulmonary veins by inflammatory cells may also result in hemodynamic changes similar to pulmonary veno-occlusive disease (105109). Additional potential mechanisms are direct mechanical compression of PAs by enlarged lymphatic tissue, luminal obstruction by plexiform lesions, hypoxic vasoconstriction, and diastolic or systolic dysfunction due to myocardial sarcoidosis (105). Bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis shows enhanced levels of ET-1. Therefore, ET-1–induced mesenchymal cell proliferation is regarded as important for the development of vascular remodeling and PH in sarcoidosis (110). Although reduced BALF VEGF levels and changes in gene activity of apoptosis regulators have been shown, there is currently no clear evidence regarding a role of angiogenesis or EC injury in the development of sarcoidosis-associated PH (111113). Therapeutic considerations in sarcoidosis do not only include immunosuppression and specific PH therapy, but sarcoidosis treatment also has to target various extrapulmonary manifestations of sarcoidosis, including myocardial sarcoidosis, if existent (114).

This paragraph summarizes the potential contribution of selected factors to vascular remodeling in IPF, under consideration of a possible relevance for disease monitoring and therapy. Nevertheless, it has to be kept in mind that blood or BALF levels do not necessarily reproduce the expression or activity of the specified molecules within the pulmonary vascular system, especially in the heterogeneously altered lung of patients with IPF.


VEGF is a major angiogenic growth factor, and is essential for sprouting and migration of EC during angiogenesis, but it also has important functions for the maintenance of the vascular system: it is important for EC survival and proliferation, and inhibition of VEGF signaling in the adult healthy lung results in EC apoptosis and severe damage to the alveolar structure with the development of emphysema (115, 116). VEGF transcription is mainly induced by hypoxia-inducible factor–1α, which accumulates in the cells under hypoxia, but is also increased by TGF-β1 (117). Different receptors for VEGF have been characterized, but VEGF receptor 2 mediates vascular homeostasis and survival. Pulmonary VEGF expression can be detected in macrophages, epithelial cells, and ECs, but also in mesenchymal cells (118). In addition, a significant percentage of lung VEGF is matrix bound (118). Production of NO via endothelial NO synthase is required for many of the endothelial effects of VEGF, but NO and prostacyclin synthesis are also required for vasodilation (115).

In human IPF, VEGF has been shown to be reduced in BALF and fibrotic regions of the lung, especially within fibroblastic foci (47, 48, 119). In these areas, EC apoptosis is correlated with reduced vascular density and decreased capillary branching (47, 48, 51). We have shown that the local VEGF deficit in fibrotic tissue is directly involved in vascular rarefaction, PA remodeling, and PH through apoptosis of ECs (29). Interestingly, VEGF can also promote fibrosis through angiogenesis and collaboration with TGF-β, resulting in enhanced ECM production of fibroblasts (29). In addition, VEGF inhibition can reduce fibrosis in the bleomycin model of PF (120). The bleomycin model is widely used to investigate disease mechanisms in PF, and to evaluate new treatment options (121). Bleomycin induces epithelial injury in the rodent lung, with subsequent inflammation and transition to fibrosis after roughly 8–10 days (122). Studies investigating VEGF and angiogenesis almost exclusively used the bleomycin model, which differs from IPF, especially in terms of microvascular changes: as opposed to capillary loss found in IPF, bleomycin-induced fibrosis shows neovascularization (120). VEGF is significantly up-regulated during the inflammatory phase in this model, and inhibition of VEGF results in reduced fibrosis, most likely due to reduced leukocyte trafficking and protein leakage (120). In contrast to our recent work, most publications investigating angiogenesis inhibition as a therapeutic option for PF have not taken into consideration that this approach can also negatively affect vascular maintenance and promote the development of PH. The vasoprotective effects of VEGF have also been documented by several PH studies (123128). The current literature suggests two possible roles for VEGF in the clinic: (1) VEGF has potential as a therapeutic target due to its high angiogenic potential, but systemic therapy with VEGF also bears significant potential side effects—VEGF increases vascular permeability with the results of edema and hemorrhage, activates mesenchymal cells, and can promote the growth of occult tumors (29, 129). (2) VEGF inhibition can be useful to limit local angioproliferation, but it could also have detrimental effects on vascular integrity by promoting EC apoptosis. To find a satisfactory way out of this dilemma, compartment- or cell-specific treatment strategies need to be evaluated (e.g., by using genetically modified EPCs, as already investigated in other forms of PH [57, 58]). In addition to therapeutic considerations, VEGF levels have been frequently investigated in BALF and blood, but the results have so far been conflicting, and demonstrate that a factor might well be very important in pathogenesis, but not necessarily suitable as a potential biomarker (119, 130).


Decreased VEGF expression in the fibrotic tissue is accompanied by elevated levels of PEDF, predominantly in the epithelium overlying fibroblastic foci and the foci themselves. In the normal lung, PEDF is found in airway and AECs, ECs, and mesenchymal cells (48). PEDF shares structural homology with members of the serpin family of protease inhibitors, but does not seem to have inhibitory activity on its own (131). Among the various actions of PEDF, we find a very strong angiostatic component and inhibition of VEGF-induced angiogenesis, as well as induction of EC apoptosis and inhibition of fibroblast growth (132136). Recent studies have identified a putatitive 60-kD PEDF receptor on EC, the nonintegrin laminin receptor, and also an 80-kD receptor on neurons, PLA2/nutrin/patatin-like phospholipase domain–containing 2 (137142). The PEDF molecule contains two corresponding functional epitopes: a 34-mer peptide, and a 44-mer peptide (143). The 44-mer peptide binds to the 80-kD receptor on neurons and mediates neuroprotection (144). The 34-mer peptide is responsible for the anti-angiogenic effects of PEDF, and it seems to bind to the 60-kD receptor on ECs (141, 143, 144). The various functions of PEDF are highly regulated at the post-translational level, through phosphorylation at different amino acid residues and several binding sites for ECM components (e.g., collagen 1, heparin, or hyaluronan [145150]). These binding sites induce conformational changes (heparin), or are necessary for the antiangiogenic activity of PEDF (collagen 1) (148, 149). However, association with the ECM might be not only a mechanism to regulate the activity of PEDF, but could also explain the localization of PEDF to the ECM-rich fibrotic tissue (151). Other explanations for local PEDF accumulation in fibrosis are increased production, either from damaged epithelial cells or from activated myofibroblasts, as well as decreased degradation of PEDF, due to reduced MMP-2 and MMP-9 activity in fibrotic tissue paralleled with enhanced expression of tissue inhibitor of metalloproteinases (29, 48, 152, 153). Our data in the AdTGF-β1 model indicate that PEDF is early up-regulated in fibrotic areas (29). PEDF is likely involved in EC apoptosis through inhibition of VEGF, but the overall function of PEDF in fibrotic lung disease is unclear. Production in injured epithelial cells may be an attempt to reduce fibroblast activity and local inflammation (134, 154156). This makes sense when PEDF is regarded as a protective factor, as shown previously in kidney and brain (157160). However, the strong antiangiogenic effect of PEDF with subsequent EC apoptosis is likely detrimental in a highly vascularized organ such as the lung. PEDF inhibition (e.g., by antibodies or soluble receptor fragments), seems to be a promising therapeutic approach, once it has been clarified which role PEDF has in the development or progression of PH. Careful studies need to evaluate whether PEDF inhibition might also cause negative effects, such as increased fibrogenic activity, similar to increased VEGF activity (29). Further research is also needed to evaluate whether PEDF levels in blood or BALF can be used as a potential predictor of disease activity and, therefore, prognosis. Such studies have yielded interesting results in the field of oncology (with reduced PEDF levels as a negative outcome predictor), and recently also in patients with heart failure (161164).


The TGF-β family in mammals consists of three isoforms. All TGF-βs have a homologous sequence and similar functions during wound repair and tissue remodeling (165). They are major profibrotic growth factors involved in ECM turnover and mesenchymal cell activity. Most lung cells express TGF-β, and there is evidence of signaling. TGF-β also has important immune-suppressive functions during inflammation. TGF-β is mainly produced by macrophages, epithelial cells, and fibroblasts. Secreted TGF-β requires activation by cleavage of its latency-associated peptide. Two receptors, types I and II, bind TGF-β, and distinct intracellular pathways, such as the Smad2/3 pathway, mitogen-activated protein kinases, phosphoinositide 3-kinases, or Rho GTPases, mediate its intracellular actions. Mainly, the Smad pathway is relevant in tissue fibrosis (166). Importantly, signaling through the Smad2/3 pathway results in myofibroblast activation, characterized by elevated gene expression of ECM components and profibrotic mediators (166). TGF-β inhibits proliferation and/or function of various immune cells, such as T lymphocytes, natural killer cells, B lymphocytes, macrophages, dendritic cells, and granulocytes (167). In addition, TGF-β is also responsible for the induction of Forkhead box P3 (Foxp3) in CD4+ CD25 Foxp3 precursors of regulatory T cells outside of the thymus, but also for the maintenance of Foxp3 expression and the regulatory function of regulatory T cells (167171). However, TGF-β can also support inflammation (e.g., through differentiation of Th17 cells [172]). TGF-β1 is the most commonly investigated isoform, and has concentration-dependent effects on angiogenesis. At lower doses, TGF-β1 activates ECs through enhanced expression of angiogenic growth factors and proteinases (173). At high doses, it decreases EC growth or induces apoptosis, promotes basement membrane formation, and induces differentiation and recruitment of VSMCs (174, 175). Elevated TGF-β1 levels in fibrotic areas of IPF lungs may contribute to local EC apoptosis, and thereby vascular rarefaction, but also to locally increased muscularization of PAs (48, 176178). In summary, TGF-β has effects on a large number of different cell types, suggesting that a close coordination of TGF-β activation is of crucial importance to maintain tissue homeostasis (167). Therapeutic inhibition of TGF-β and its pathway appears to be very useful to target the fibrotic process, but also the pulmonary vascular disease, as demonstrated by in vivo studies (178, 179). The central importance of TGF-β would suggest that TGF-β could also be a valuable biomarker in IPF, but TGF-β measurements in BALF are highly variable, which can limit the value of this marker, and indicates the need for larger studies to establish more reliable fluid levels (180, 181).

Fibroblast Growth Factor–2

Fibroblast growth factor (FGF)–2 is a heparin-binding FGF with important functions in angiogenesis (182, 183). It signals through the high-affinity tyrosine kinase FGF receptors, FGFR1 and FGFR2, on ECs, and induces EC proliferation and migration (184). The main intracellular pathways that are activated are mitogen-activated protein kinase and protein kinase C (185). In addition to the effects on EC, FGF-2 also contributes to degradation of ECM through induction of MMPs and plasminogen activator expression as a preliminary step in angiogenesis (182). FGF-2 is reduced in fibroblastic foci of IPF lungs, but is expressed in the surrounding epithelium, similarly to VEGF (186). Although reduced levels of FGF-2 in fibrotic tissue are consistent with decreased capillary density, and suggest a potential contribution to EC apoptosis and vascular rarefaction, FGF-2 might also be involved in VSMC growth, similarly to human PH and experimental animal PH models, suggesting that FGF-2 might have a role in vascular remodeling in PF (187, 188). The relevance of FGF-2 in IPF is currently unclear: FGF-2 could help to reduce EC apoptosis, but the overall effects of FGF-2 on fibrotic process and PA remodeling warrant further investigation, as does the potential of FGF2 as a biomarker.

CXC Chemokines

Chemokines of the CXC family have four highly conserved cysteine residues, with the first two separated by a nonconserved amino acid, and a second, three-amino-acid motif determines the angiogenic activity. CXC chemokines with the three-amino-acid sequence, Glu-Leu-Arg (ELR), right ahead of the first cysteine amino acid, are proangiogenic ELR+ CXC chemokines, whereas ELR CXC chemokines have antiangiogenic properties (55, 189, 190). A representative sequence is shown in Figure 6.

Relevant ELR+ CXC chemokines are CXC ligand (CXCL) 1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8 (55, 189, 190). Interactions with various other factors are involved to promote angiogenesis (e.g., VEGF induces CXCL8 secretion from ECs, and thereby perpetuates angiogenesis [55, 191]). ELR+ CXC chemokines bind to CXC receptor (CXCR) 1 and CXCR2 (192). These receptors are expressed by ECs, although CXCR2 is involved in the major angiogenic effects in microvascular lung ECs in vitro (55, 192195). A CXC chemokine with profibrotic effects via stromal cell or mesenchymal progenitor recruitment is CXCL12, which also has angiogenic effects through its receptor, CXCR4 (55, 196).

The most frequently found ELR CXC chemokines are CXCL4, CXCL9, CXCL10, and CXCL11 (55). These chemokines represent a link between angiostasis and immune function, because they are induced by Th1-produced cytokines (55). Their receptor, CXCR3, is important for recruitment of leukocytes and inhibition of angiogenesis (55, 197).

In human IPF, the balance between ELR+ CXC chemokines and ELR CXC chemokines is shifted toward ELR+ CXC chemokines, which indicates a proangiogenic chemokine environment (53, 54, 198, 199). Depletion of ELR+ CXC chemokines or administration of ELR CXC chemokines in the bleomycin model of lung fibrosis results in a reduction of both angiogenesis and fibrosis (52, 200, 201). In apparent contrast, Ebina and colleagues (47) demonstrated decreased CXCL8 in fibrotic areas of IPF lungs, similar to VEGF. These contradictory results likely reflect the extensive heterogeneity of human UIP in terms of angiogenesis with evidence of EC proliferation and augmented capillary density in “interface” regions directly adjacent to the fibrotic tissue, where EC apoptosis and vascular rarefaction dominate (47, 51). The exact function of CXC chemokines in IPF and PH pathophysiology (counterregulatory process, contribution to PA remodeling, response to epithelial injury or inflammation) needs to be elucidated. This information is critical in deciding whether and which of the CXC chemokines might be useful as therapeutic targets or biomarkers in IPF-related PH.


PDGF-A and PDGF-B are best characterized and mainly investigated, whereas PDGF-C and PDGF-D have only been recently described (202). PDGF expression is found in many different cell types, including fibroblasts, epithelial cells, and platelets (182). PDGF is biologically active in the form of hetero- or homodimers of the A and B chains, and binds to complexes of α and β subtypes of PDGF receptors (202). PDGF is responsible for chemoattraction and proliferation of mesenchymal cells of the vessel wall, such as pericytes and VSMCs. Pericytes are α–smooth muscle actin+ mesenchymal cells surrounding capillary structures, which are required for angiogenesis and microvascular stability (182, 203, 204). In early IPF, PDGF is not only found in macrophages, fibroblasts, and AEC2, but also in EC and VSMC, suggesting a contribution of PDGF to vascular remodeling (205). In contrast, vessels in advanced IPF do not exhibit PDGF expression. In addition to fibroblast proliferation and migration, PDGF seems to be involved in PA wall remodeling through activation of VSMCs and fibroblasts, which results in enhanced muscularization, as well as intima and adventitia fibrosis (206). This hypothesis is supported by experiments in models of PH (207). Therefore, PDGF inhibition could evolve as a useful target to reduce both, fibrogenesis and vascular remodeling. In addition, serum or BALF levels of PDGF could be predictors of outcome, but future studies are needed to establish the relevance of PDGF.


The angiostatic endostatins are generated via cleavage of collagen XVIII at its protease-sensitive hinge region, and have molecular weights of 20–30 kD. Various pathways are involved in the angiostatic activity of endostatin, such as inhibition of VEGF receptor 2 and MMP-2 (208). The precursor molecule, collagen XVIII, is found in alveolar capillary and epithelial basement membranes (209, 210). IPF lungs contain elevated mRNA levels of collagen XVIII. Increased proteolysis of alveolar collagen XVIII by different enzymes, such as elastase, cathepsin, and different MMPs, seems to be responsible for the elevated levels of endostatin in IPF serum and lung (211, 212). It has been suggested that endostatin is produced by injured epithelial cells, which again, similarly to PEDF, may link epithelial injury and vascular rarefaction in IPF. Experimental data from hypoxic PH have implicated endostatin in PA vascular remodeling (213). Two main conclusions can be drawn: (1) inhibition of endostatin could reduce the consequences of epithelial injury on the pulmonary vasculature, and be, therefore, a useful target for future therapy; and (2) serum endostatin levels may represent a valuable parameter to evaluate the extent of pulmonary epithelial and vascular injury, but also disease progression.


The most important vascular functions of ET-1 are vasoconstriction and growth of VSMCs (61). This protein signals through interaction with ETA and ETB receptors, both G-protein coupled receptors (214). Expression of ET-1 is widely found throughout the lung, including in ECs, airway, and alveolar epithelium (215, 216). The levels of ET-1 seem to be increased in the lungs of patients with IPF, with the main expression localized to ECs and epithelial cells within fibrotic areas (216, 217). ET-1 has important profibrotic actions, and is involved in PA wall remodeling in different forms of human and experimental PH, as well as of lung fibrosis models (218220). The extent of PH correlates directly with ET-1 levels in the peripheral blood of patients with IPF (221). It is likely that ET-1 is involved in remodeling of all three artery layers in IPF, because ET-1 could be released by injured ECs and epithelial cells within the fibrotic areas, and thereby contribute to intima lesions and VSMC growth from the inside (release from ECs) and from the outside (secretion from epithelial cells) (29, 222). Although experimental research suggests that ET-1 would be a central target to treat IPF and associated PH, the current clinical data regarding ET receptor inhibition in IPF show only very limited success. There are only limited data available regarding the potential of ET-1 as a biomarker, but the results so far indicate that urine rather than plasma levels could be used (223).


The octapeptide, AT II, mediates the main effects of the renin–AT system. Renin and the AT-converting enzyme, which is prominently expressed in the lung capillary EC, cleaves angiotensinogen into its active form, AT II. AT II is not only a very potent vasoconstrictor, but is also involved in VSMC growth and proliferation (224). AT II is likely involved in the pathophysiology of PH, taking into account elevated expression of AT-converting enzyme in ECs of patients with PH; however, successful reduction of experimental PH through AT II inhibition has not been found in patients with PAH (224, 225). In addition, a putative, important role has been shown for AT II in epithelial cell apoptosis and fibrosis in both human IPF and experimental lung fibrosis (226, 227). Enhanced AT II in the fibrotic tissue may contribute to the development of PH through vasoconstriction and PA wall remodeling (60, 228). AT inhibition might be a valid therapeutic approach in IPF-associated PH, but the overall relevance needs to be established through ongoing research. AT II or AT-converting enzyme levels could be used as biomarkers, but the prognostic relevance needs to be established (229).


The Ang–Tie ligand–receptor system has important functions in regulating vascular integrity and maintenance of a quiescent EC state (230). The system consists of two tyrosine kinase receptors, Tie-1 and Tie-2, and four ligands, Ang-1, Ang-2, Ang-3, and Ang-4 (230, 231). Research has mainly focused on Ang-1 and Ang-2. The receptors have almost exclusively been detected on EC and hematopoietic stem cells (232235). Ang-1 is constitutively expressed by cells of mesenchymal lineage, such as pericytes, VSMC, fibroblasts, and some tumor cells (236238). The main role of Ang-1 is to keep ECs in a quiescent state, a function that requires signaling through the endothelial receptor, Tie-2 (230). Ang-1/Tie-2 interactions mainly activate the antiapoptotic Akt pathway (239). In contrast, Ang-2 expression is almost exclusively found in ECs, and is very low in quiescent ECs, but it dramatically rises once ECs are activated (230, 240, 241). The functions of EC-derived Ang-2 are highly context dependent: Ang-2 promotes angiogenesis in concert with VEGF, but results in EC apoptosis and subsequent vessel regression without VEGF (230, 242).

Current knowledge about the Ang–Tie system in IPF is very limited, but studies from kidney fibrosis show that Ang-1 therapy induces increased capillarization, most probably via stabilization of vessels (243, 244). There is some controversy about how Ang-1 effects experimental PH in rodents: although cell-based gene transfer of Ang-1 improved survival and pulmonary hemodynamics in monocrotaline-induced PH, Ang-1 overexpression induced PH in healthy rats (245, 246). Nevertheless, the Ang-1/Ang-2/Tie-2 system may have an important impact on understanding the complex processes leading to vascular rarefaction in lung fibrosis, and to discovering promising new treatment options. In IPF, activation of EC with induction of Ang-2 expression might contribute to EC apoptosis in the context of local angiogenesis inhibition in the fibrotic tissue. On the other side, reduced Ang-1 release from dysfunctional VSMC could also destabilize ECs locally, thereby contributing to EC damage. Therefore, supplementation of Ang-1, or inhibition of Ang-2 could help to restore endothelial integrity in IPF, possibly without the severe side effects of VEGF. Although Angs might be valuable biomarkers for the pulmonary vascular disease in IPF, there are currently no data available that would allow a statement.


Angiostatin is produced via proteolysis of plasminogen by a macrophage-derived metalloelastase and other MMPs, or by reduction of plasmin (247249). Angiostatin consists of the first four kringle domains of plasmin. The kringle is a triple loop structure linked by three pairs of disulfide bonds (250). Angiostatin inhibits proliferation, induces apoptosis, and prevents EC migration in vitro (251, 252). The molecular mechanisms include activation of focal adhesion kinase, ceramide generation, RhoA activation, and inhibition of an a/b ATP synthase on the surface of ECs (252256). Elevated levels of platelet angiostatin have been reported in some patients with idiopathic PAH (257). Pulmonary overexpression of angiostatin aggravated PH in a model of chronic hypoxia, believed to be because of decreased vascularization (258).

Angiostatin may be linked to the pathophysiology of IPF via altered plasminogen activation. In normal tissue, plasminogen is cleaved by tissue or urokinase plasminogen activator to form the active enzyme, plasmin, which contributes directly and via increased MMP activity to ECM degradation (259). In IPF, plasminogen activator inhibitor–1 is induced by TGF-β and inhibits plasminogen activation (259). This results in reduced decomposition of ECM and increased recruitment of inflammatory cells (259). Noncleaved plasminogen may then be cleaved increasingly to generate angiostatin, possibly by macrophage-derived metalloelastase, or by a direct effect of plasminogen activator inhibitor–1 on plasminogen. Locally elevated angiostatin levels in the fibrotic tissue may contribute to EC apoptosis and vascular rarefaction (260). However, the involvement of angiostatin in fibrosis and IPF-related PH needs to be established before angiostatin can be considered as a new therapeutic target, or even as a predictor of clinical outcome.

The current therapeutic options are quite limited for IPF, and even more so for IPF-associated PH. The major problem in treating both fibrosis and pulmonary vascular disease is the amount of organized scar tissue inside the fibrotic lung, which no longer takes part in active fibrogenesis. These areas are already fully organized, and active myofibroblasts are rarely found. This suggests that the IPF lung has areas with active fibrogenesis, which are typically characterized by the presence of fibroblastic foci, histologically normal appearing areas, and regions with the aforementioned organized scar tissue. The latter areas represent regions of final, nonreversible damage, not only to the interstitium, but very likely also to the pulmonary vasculature. This implies that these regions will likely not be accessible for therapy, and that therapeutic interventions will have to concentrate on the more active areas of the fibrotic lung.

Therapy of PH in IPF will have to be a multimodal therapeutic approach that has to be closely connected with IPF therapy itself. There are several aspects that will need attention when designing therapeutic interventions for IPF and associated PH: (1) the progression of the fibrotic process needs to be decelerated or inhibited, because this would also help to slow down PH development or progression due to the close relationship between fibrosis and pulmonary vascular pathology, as discussed previously; (2) the integrity of the lung vasculature needs to be stabilized, especially endothelial injury, and apoptosis warrants reduction, due to its importance for PA wall remodeling, vascular rarefaction, and angioproliferation; and (3) right ventricular function needs to be maintained.

Current IPF therapy has mainly focused on lung fibrosis itself, and has met rather limited success. Anti-inflammatory treatment using drugs, such as azathioprine, corticosteroids, cyclophosphamide, or methotrexate, has been the major strategy (3, 261). Overall, anti-inflammatory or immunosuppressive therapy has been useless in IPF, suggesting that inflammation might not be a central component of IPF (262). Recent clinical trials have shown small benefits (e.g., for the antifibrotic molecule, pirfenidone, or the antioxidant, N-acetylcysteine). Pirfenidone is a newly developed drug with combined anti-inflammatory, antioxidant, and antifibrotic effects, and clinical trial have shown small benefits (263, 264). Antioxidant strategies have been investigated before, after evidence that epithelial injury in IPF might be related to oxidative stress. A primarily antioxidant strategy using N-acetylcysteine seems to be beneficial for patients with IPF (265, 266). These therapeutic agents will be used to inhibit fibrosis progression. Other therapeutic agents targeting fibrogenesis are currently studied for IPF and IPF-PH, including IFNs, antagonists to cytokines, growth factors or their receptors, and antiangiogenic agents (details are available at (266). Especially antiangiogenic therapy will require careful evalulation to find out whether angiogenesis inhibition will result in antifibrotic and antiproliferative effects instead of a proapoptotic outcome with accelerated progression of PH.

There are currently no specific therapies for PH in IPF, and treatment guidelines for PH therapy have been primarily evaluated in other forms of PH. Although therapeutic trials for IPF-associated PH are currently underway, these studies mainly investigate drugs that are already used for other types of PH with more or less success (267). Prostacyclin-based therapies could reduce possible vasoconstriction and platelet aggregation (268, 269). Inhaled NO could be used as short-term therapy in advanced IPF or during acute deterioration (270). However, these vasodilators have to be tested carefully to rule out increased right–left shunting within the fibrotic lung. Sildenafil, a phosphodiesterase-5 inhibitor, is also a likely successful candidate in IPF, is frequently used in PAH, and has positive effects not only in the pulmonary vasculature (vasodilatation and inhibition of VSMC growth), but also selectively in the right ventricle (reduction of hypertrophy and elevated contractility) (270, 271). Whether or not Sildenafil can fulfill the promises from preliminary studies is currently being investigated in the STEP-IPF (Sildenafil Trial on Exercise Performance in IPF, and Pulmonary Arterial Hypertension Secondary to IPF and Treatment with Sildenafil Trial [details available at]) (271, 272). ET receptor antagonists could have some benefit by inhibiting vasoconstriction and VSMC growth, and bosentan, dual ET receptor antagonist, is being tested for IPF-associated PH, despite its limited previous success in IPF or other PAH forms (270, 273) in the Pulmonary Arterial Hypertension Secondary to Idiopathic Pulmonary Fibrosis and Treatment with Bosentan Trial ( Anticoagulation strategies with coumadin or warfarin, similar to what is recommended for PH, may also be helpful in IPF (274). Future research is needed to select specific angiogenic or angiostatic growth factors as targets for future therapeutic concepts (e.g., to reduce endothelial apoptosis or angioproliferation). An additional concept, which could gain relevance in IPF and is currently tested in PAH, is the use of genetically modified EPCs (e.g., endothelial NO synthase transfected) to support endothelial repair, where animal and preliminary human data are very promising (128, 275, 276).

There are limited therapeutic strategies currently aiming at right ventricular hypertrophy or failure. As mentioned, phoshodiesterase-5 inhibition can selectively improve right ventricular function. Further research is warranted to understand how the sick fibrotic lung affects the heart (e.g., whether there are systemic effects on angiogenesis in IPF), as could be suggested after Turner-Warwick's study (277), which might also affect capillarization, cardiomyocyte growth, or matrix production in the myocardium. Understanding these complex interactions will be important in designing the aforementioned multimodal therapeutic approach for IPF and assocated PH. Future therapeutic strategies could also include the use of mesenchymal stem cells for myocardial regeneration, as currently being investigated in left ventricular failure of various origins (278).

Lung transplantation (LTX) can be regarded as the ultimate of all therapeutic options, can be performed as single or double LTX, and results in decreased PAP after surgery (279). Due to the fast progression of IPF, integration with an LTX center should be considered early after diagnosis.

The pathobiology of IPF-associated pulmonary vascular disease is complicated, and the structural changes range from capillary loss and compression by fibrotic tissue to complex vascular lesions with intima and adventitia fibrosis and media thickening. Derived from studies in patient biopsies and our own experimental investigations, we believe that the main pathogenetic processes are a localized angiostatic shift, with reduction in angiogenic factors, such as VEGF, and an elevation of angiostatic factors, such as PEDF, EC injury, and apoptosis, vasoconstriction, and VSMC and fibroblast growth after growth factor release and activation. Several important translational aspects arise from our concept:

  1. The interaction of angiogenesis and growth factors in vascular pathology in IPF is a field that will require extensive attention to find successful therapies, and future studies need to include investigations of the pulmonary vascular integrity. This is particularly so when it comes to trials that interfere with angiogenesis and growth factors, as indicated by our own animal research demonstrating that a proangiogenic factor (VEGF) reduces EC apoptosis and thereby PH.

  2. Different pro- and anti-angiogenic and growth factors can be used as potential biomarkers to monitor disease progression by measuring their blood or BALF concentrations. Due to the variability in the measurements of these molecules, larger scale studies are warranted to find the best suitable marker or marker combination.

  3. After our own experimental data, indicating a close spatiotemporal relationship between fibrotic process, EC injury, and vascular remodeling (including angioproliferation, muscularization, adventitiafibrosis, and vascular rarefaction), therapy of PH in IPF will likely have to interfere with several of these changes to obtain best results. This could be accomplished by targeting various pathways at the same time. Future research should provide additional clarification of the complex interactions to help to design more successful therapies for IPF-related PH.

  4. Based on evidence in other forms of PH, it is likely that another important component of PH treatment in IPF will be to preserve right ventricular function.

  5. Depending on the results of future clinical trials, currently used PH therapies might or might not be a part of this concept, but the current clinical studies have shown a significant benefit of some PH treatments in IPF-associated PH.

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Correspondence and requests for reprints should be addressed to Martin Kolb, M.D., Ph.D., Departments of Medicine, Pathology and Molecular Medicine, McMaster University, Firestone Institute for Respiratory Health, 50 Charlton Avenue East, Room T2121, Hamilton, ON L8N 4A6, Canada. E-mail:


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