American Journal of Respiratory and Critical Care Medicine

Pigment epithelium-derived factor (PEDF) is a 50-kD protein with angiostatic and neurotrophic activities that regulates vascular development within the eye. PEDF expression was increased in the lungs of patients with idiopathic pulmonary fibrosis (IPF) based on microarray analyses. Angiogenesis has been implicated in the pathogenesis of fibrotic lung diseases, we therefore hypothesized that regional abnormalities in vascularization occur in IPF as a result of an imbalance between PEDF and vascular endothelial growth factor. We demonstrated that vascular density is regionally decreased in IPF within the fibroblastic foci, and that within these areas PEDF was increased, whereas vascular endothelial growth factor was decreased. PEDF colocalized with the fibrogenic cytokine, transforming growth factor (TGF)-β1, particularly within the fibrotic interstitium and the fibroblastic focus, and prominently within the epithelium directly overlying the fibroblastic focus. This suggested that TGF-β1 might regulate PEDF expression. Using 3T3-L1 fibroblasts and human lung fibroblasts, we showed that PEDF was indeed a TGF-β1 target gene. Collectively, our findings implicate PEDF as a regulator of pulmonary angiogenesis and an important mediator in IPF.

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia, and is characterized histopathologically by the usual interstitial pneumonia (UIP) lesion (1). In contrast to other fibrosing interstitial lung diseases, such as cryptogenic organizing pneumonia (COP), which has an excellent prognosis (2), therapy for IPF is ineffective, and the 5-year survival is less than 50% (3). Thus, investigating the histopathologic features that are unique to UIP may permit a better understanding of the events that result in progressive fibrosis.

In UIP, discrete collections of fibroblasts and myofibroblasts, termed fibroblastic foci, are distributed in subepithelial areas throughout the interstitium, often in transition zones between normal and fibrotic lung. Fibroblastic foci are thought to represent areas of active collagen synthesis and are considered to be the “leading edge” of fibroproliferation in UIP (1, 4). Pathologically distinct areas of fibroproliferation, termed Masson bodies, are present in organizing pneumonia (OP), the histopathologic lesion of COP. However, these lesions localized to intact small airways and alveoli (5). These lesions, the fibroblastic foci of UIP, and the Masson bodies of OP may represent distinct wound repair responses to pulmonary injury; the progressive fibrosis of IPF may reflect abnormal wound repair, whereas that of COP may reflect normal wound repair in response to lung injury (68).

The activation and resolution of angiogenesis is fundamental to wound healing (911). Abnormal angiogenesis has been linked to the development of fibrosis (9, 12, 13), particularly within the lung (1421). The regulation of angiogenesis may therefore be important in both the normal response to injury and the development of fibrotic disorders.

In the eye, vascular development is tightly regulated by the coordinated expression of vascular endothelial growth factor (VEGF), a potent angiogenic cytokine, and pigment epithelium-derived factor (PEDF), a potent angiostatic cytokine (22, 23). These angiogenic regulators are expressed in a reciprocal fashion so that during angioproliferative conditions, such as macular degeneration or diabetic retinopathy, VEGF expression is enhanced and PEDF expression is suppressed (24). Whereas the importance of VEGF in lung development and maintenance has recently been demonstrated in several animal models (2528), data regarding inhibitors of angiogenesis in the lung, particularly PEDF, are lacking.

PEDF is a 50-kD protein first described in retinal pigmented epithelial cells (29), and then in young, proliferating fibroblasts (30). The angiostatic properties of PEDF are specific for new, developing vessels while sparing mature, existing ones (31). Recently, PEDF expression has been detected in the kidney, pancreas, prostate, testes, bone, pleura, and within peripheral blood (3237). In this context, we questioned whether PEDF might be expressed in the fibrotic lung, and if so, what might be its relationship to angiogenesis within the UIP lung and the fibroblastic focus.

To address these questions, we determined vascular density in the fibroproliferative areas of OP and UIP. Using oligonucleotide microarrays, we analyzed the expression of PEDF and VEGF in the IPF and normal lung. We show that vascular density is decreased in the fibroblastic foci in UIP but not in the Masson bodies of OP. Moreover, we find that there is increased PEDF expression, but decreased VEGF expression within the fibroblastic focus. Finally, we demonstrate that PEDF expression within the UIP lung is coincident with transforming growth factor (TGF)-β1 the prototypical fibrogenic cytokine, and that TGF-β1 treatment stimulates fibroblasts to express PEDF. Collectively, our findings provide a novel link between PEDF and TGF-β1, and suggest that the suppression of angiogenesis within the fibroblastic focus of UIP may be important in pulmonary fibrosis. Some of our results reported here have previously been reported in the form of abstracts (38, 39).

Patient Population and Biopsy Samples

Random bronchoalveolar lavage fluid (BALF) and/or surgical lung biopsy samples were obtained from the institutional review board–approved Interstitial Lung Disease Specialized Center of Research Tissue Bank at National Jewish Medical and Research Center. The histopathologic findings of usual interstitial pneumonitis (UIP) and OP were required for a diagnosis of IPF and COP, respectively (40) (see online supplement Table E1 for details and sample characteristics).


Immunohistochemistry was performed on 4% paraformaldehyde-fixed paraffin embedded tissue using standard techniques (41) (see online supplement for details).


Vascular density was analyzed using a modification of the protocol reported by King (42) (see online supplement for details). A minimum of five fibroproliferative areas were analyzed in five patients with IPF and five patients with COP.

Oligonucleotide Microarray Analyses

Oligonucleotide microarray analyses were performed using Hu6800 GeneChips (Affymetrix Inc., Santa Clara, CA). Total RNA was isolated using TRIzol reagent (InVitrogen, Carlsbad, CA), with subsequent preparation and analysis according to a standard protocol (43) (see online supplement for details).


VEGF and PEDF concentrations in BALF and lung homogenates were determined using ELISA kits: VEGF (R&D Systems, Minneapolis, MN) and PEDF (ELISATech, Aurora, CO).

Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and Western Blotting

Fifty μg of total lung protein from normal and IPF lungs were electrophoretically fractionated through a 10% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) and transferred electrophoretically to nitrocellulose membranes (44). PEDF was detected using one of two different PEDF antibodies (see online supplement for details).

Immunoflourescence Staining of α–Smooth Muscle Actin and PEDF

Immunofluorescence studies were performed on 4% paraformaldehyde–fixed paraffin–embedded tissue using a standard protocol and antigen retrieval (41) (see online supplement for details).

Northern Blotting

Quiescent 3T3-L1 cells were stimulated with TGF-β1 (10 ng/ml; R&D Systems) for 0–24 hours and subsequently harvested in RNAzol Reagent (tel-Test, Inc., Friendswood, TX). Northern blotting was performed, as described previously (45), using 32P-labeled human fibulin-5 cDNA probe (nucleotides 485–888) and a 32P-labeled human PEDF cDNA probe (nucleotides 150–558), which was excised from EST BG696757 by digestion with Kpn I and Bgl II (Promega, Madison, WI).

Primary Lung Fibroblast Isolation and Culture

Primary lung fibroblasts were isolated from surgical lung biopsy samples from normal and IPF/UIP lung and stimulated with TGF-β1 (10 ng/ml; R&D Systems) for 0–48 hours. PEDF expression was determined by reverse transcription and polymerase chain reaction (see online supplement for details).

In vitro Angiogenesis Assay

Human umbilical vein endothelial cells (HUVEC) were harvested at passage 3–6 for use in an in vitro endothelial migration assay (ECMatrix; Chemicon, Temecula, CA) according to the manufacturer's protocol. HUVEC-ECMatrix was supplemented with recombinant VEGF, recombinant PEDF, or homogenized lung extract from either IPF lung or normal lung samples. Assays were repeated in triplicate for each condition, and tubing morphogenesis was evaluated (see online supplement for details).

Statistical Analysis

Paired, 2-tailed t tests were used to identify differences for gene expression analyses and morphometric studies. Nonparametric Mann-Whitney analyses were used to identify differences between samples in ELISAs of VEGF and PEDF. Significance was accepted at p < 0.05.

Microvascular Density Is Decreased in the Fibroblastic Focus of IPF

Significant differences in vascular density were detected between the fibroblastic foci of UIP and the Masson bodies of OP. Whereas both lesions contain α–smooth muscle actin (α-SMA)–positive cells, consistent with a myofibroblast phenotype, vessels were prominent in the Masson bodies, whereas they were nearly undetectable within the fibroblastic focus (Figure 1)

. Considerable regional heterogeneity in vascularization was seen in UIP outside the fibroblastic focus, where numerous abnormal vessels were seen within regions of severe architectural distortion (honeycomb lung).

PEDF Is Upregulated in the IPF Lung

To identify mediators that may contribute to the abnormalities in the regional vascular density noted in the fibroblastic focus of UIP, we compared the global gene expression profiles of five normal human lungs and five IPF lungs using high-density oligonucleotide microarrays. Although not statistically significant, the expression of VEGF tended to be decreased in UIP (p = 0.09, 14 of 25 with a twofold decrease in VEGF in IPF lung). In contrast, the angiostatic mediator, PEDF, was increased more than 2.5-fold in the IPF lung in more than 80% of all possible comparisons (p < 0.05). Whereas we chose to focus on PEDF and VEGF, additional angiogenic and angiostatic cytokines were differentially expressed between normal and IPF lung and are listed in Table E2 in the online supplement).

PEDF Protein Is Expressed in BALF and Lung Homogenates from IPF

To confirm the gene expression data, PEDF was quantified in BALF (normal volunteers, n = 5 and subjects with IPF, n = 5) by ELISA analyses. PEDF was increased significantly in BALF of IPF patients compared with normal lung BALF (Figure 2A

, upper panel). PEDF expression was also analyzed by immunoblotting lung homogenates (normal volunteers, n = 3 and subjects with IPF, n = 3) with anti-PEDF antibodies. As shown in Figure 2B, PEDF was increased in each of the three IPF lung samples when compared with any of the normal lung samples. Quantitatively, PEDF was increased significantly in IPF lung homogenates compared with normal lung (Figure 2A, lower panel). Dual bands were present in both normal and IPF lung samples despite reducing conditions, suggesting that multiple forms of PEDF may exist within the lung, consistent with prior reports of extensive post-translational modification of PEDF (34).

VEGF Protein is Decreased in BALF from IPF

VEGF and PEDF are expressed in a reciprocal manner in the eye (24). We therefore wanted to determine whether a similar relationship exists within the lung. In doing so, we performed ELISA analyses to measure VEGF abundance in BALF and lung homogenates from a separate group of normal volunteers (n = 8) and patients with IPF (n = 15). As shown in Figure 3

, VEGF expression was decreased significantly in BALF subjects with IPF compared with that in normal control subjects, as previously suggested (46, 47). Surprisingly, VEGF was not significantly different in lung homogenates. Therefore, regional differences in VEGF expression may exist within the different compartments of the IPF lung.

PEDF Is Expressed in the Fibroblastic Focus of IPF

We performed immunohistochemical staining to assess the spatial distribution of PEDF in lung biopsy samples from a separate group of normal subjects and patients with COP and IPF. In normal lung, PEDF localized to the epithelium adjacent to mucous glands, vessels, alveolar structures, presumably type I pneumocytes, capillaries, and interstitial pericytes (Figures 4A–4C)

. In COP, PEDF was present in a similar distribution to that seen in the normal lung. PEDF was detected in 47 of 217 (17.8%) Masson bodies (Figures 4D–4F). In UIP, PEDF was present in a similar distribution to that seen in the normal lung but was prominently expressed within the fibrotic interstitium (Figures 4G–4I). In 81 of 89 (91.0%) fibroblastic foci from IPF/UIP samples, PEDF was present within fibroblastic foci. PEDF expression was present within the alveolar epithelium throughout the lung but most prominent in areas overlying the fibroblastic foci (Figures 4F and 4I). PEDF is, therefore, expressed to a greater extent within the fibroblastic foci in UIP than in the Masson bodies of COP.

VEGF Is Absent in the Fibroblastic Focus of IPF

In light of the spatial differences in PEDF expression, we wanted to assess whether there were regional differences in the expression of VEGF within the lung. In normal lung, VEGF was localized to the bronchial epithelium, alveolar type I cells, within the alveolar septae adjacent to alveolar capillaries, and within endothelium of vascular structures (Figures 5A–5C)

. VEGF was expressed in OP in a similar pattern to that seen in the normal lung but with additional staining within the alveolar mononuclear infilitrate (Figures 5G–5I). In the Masson bodies, VEGF localized to luminal structures as well as to the extracellular matrix. In contrast, VEGF was expressed in the IPF lung within the bronchial epithelium, the endothelium of vessels, and within areas of normal alveolar architecture, similar to that noted in the normal lung (Figures 5D–5F). Within the fibrotic interstitium and more specifically within the fibroblastic foci of IPF, VEGF expression was minimal.

PEDF is present in the epithelial cells directly overlying the fibroblastic focus in UIP and localizes with α-SMA in Masson bodies in OP and fibroblastic foci in UIP. To determine the cell types responsible for PEDF expression, colocalization of PEDF with α-SMA was performed. As shown in Figure 6

, PEDF expression in normal lung was detected in the epithelium and colocalizes with α-SMA in vessels. In OP, PEDF was detected in Masson bodies and colocalized with α-SMA–positive cells. In UIP, PEDF was expressed in the fibroblastic focus and localized to some extent with α-SMA. More prominent staining was detected in the epithelium directly overlying the fibroblastic foci in comparison with other areas of alveolar epithelium. Thus, both fibroblast/myofibroblasts and epithelial cells within the lung might express PEDF.

TGF-β1 Is Expressed in the Fibroblastic Focus of IPF

Given the colocalization of PEDF with myofibroblasts and fibroblasts, we determined the spatial distribution of TGF-β1 within normal lung, OP, and UIP (Figure 7)

. TGF-β1 immunolocalization revealed that in the normal lung, TGF-β1 was present in the alveolar macrophages, alveolar lining cells, and within the smooth muscle of arterial walls. In OP, TGF-β1 expression was visualized in arterial wall smooth muscle and the endothelium of vessels, but not within the Masson bodies. TGF-β1 was detected in many of the same locations in UIP as in normal lung. In contrast to OP, however, TGF-β1 staining was present within the fibroblastic focus, especially in its overlying epithelium, as well as in the regions of honeycomb change. Hence, the pattern of TGF-β1 staining in UIP overlaps that seen with PEDF.

Expression of PEDF by TGF-β1–stimulated Fibroblasts

Since our immunolocalization data suggested a similar distribution of TGF-β1 and PEDF within fibroblasts and the epithelium, we wished to determine whether PEDF was a TGF-β1-target gene. Following exposure to TGF-β1, PEDF expression was increased threefold after 8 hours in 3T3-L1 fibroblasts (Figure 8)

, comparable to fibulin-5, a known TGF-β1 inducible gene (45). A similar increase was present in fetal human lung fibroblasts, CCL171 (ATCC). In primary human lung fibroblasts from a normal human lung as well as those from a subject with IPF, PEDF was constitutively expressed but not appreciably increased in the presence of TGF-β1 (Figure 9) . Thus, TGF-β1 stimulated the expression of PEDF in murine fibroblasts and human lung fibroblasts.

IPF Lung Homogenate Decreased In Vitro Angiogenesis

To assess the angiogenic potential of lung homogenates from normal lung and IPF lung, we performed standard in vitro matrigel assays. Endothelial microtubular formation was suppressed in the presence of IPF lung protein relative to normal human lung protein (n = 3). Normal lung consistently demonstrated microtubule formation, whereas IPF lung protein supplementation impeded microtubule formation (n = 3), suggesting that the cytokine milieu within the IPF lung favors the suppression of angiogenesis rather than its induction (Figure 10)

. Control assays with VEGF and PEDF enhanced and suppressed microtubule formation, respectively.

The pathogenetic mechanisms responsible for the progressive fibrotic response in IPF have yet to be determined. The precise relationship between angiogenesis and fibrosis remains controversial, as considerable evidence supports both a positive and negative regulatory role for angiogenesis in progressive fibrosis. For instance, angiogenesis is associated with airways remodeling and fibrosis in chronic asthma and in synovial fibrosis in chronic, deforming arthritis (4850). An association between excessive angiogenesis and fibrosis in IPF has been suggested based on both human studies and bleomycin-induced murine pulmonary fibrosis (51). On the other hand, inadequate angiogenesis has also been implicated in a model of interstitial renal fibrosis. In this model, decreased renal microvascular density is accompanied with progressive renal impairment, excessive matrix deposition, and interstitial fibrosis that can be ameliorated with the infusion of VEGF, leading to a recovery of renal function with decreased interstitial renal fibrosis (52). Furthermore, decreased vascularization is also seen in hypertrophic scars and keloids (12). Hence, either excessive or inadequate angiogenesis may be associated with a progressive fibrotic response.

The regulation of angiogenesis is a complex process requiring transcriptional (53, 54) and post-transcriptional mechanisms (23, 55, 56), as well as matrix–matrix interactions, especially in the setting of wound repair (57). Although angiogenic mediators such as VEGF have been explored within the context of lung injury (58), the regional balance of angiogenic and angiostatic mediators within the lung is unknown and may be a more important regulator of angiogenesis. To our knowledge, the angiostatic mediator PEDF has not previously been detected in fibrotic human lung. In the current study, we have shown that PEDF is expressed in the normal human lung, that its expression is upregulated within the lungs of patients with IPF, and that it is localized to the fibroblastic focus, a site of active matrix synthesis where vascular density is low (5961). Our strategy was as follows: after confirming the decreased vascular density within the fibroblastic focus, we used oligonucleotide microarrays to detect potential anti-angiogenic mediators, which allowed us to identify PEDF as a potential modifier of angiogenesis within the IPF lung. We then chose to examine the regional expression of PEDF in relationship to the known landmarks in the lungs of patients with IPF and COP, and to examine the expression of VEGF, TGF-β1, and fibroblasts/myofibroblasts. Finally, using this information to guide a choice of cellular sources, we tested the ability of the fibrogenic cytokine TGF-β1 to induce the expression of PEDF in vitro.

In angioproliferative disorders, such as macular degeneration and diabetic retinopathy, PEDF expression is significantly decreased relative to that of VEGF (24, 62). Experimental models of macular degeneration also suggest that VEGF-induced retinopathy is reversed with the reexpression of PEDF (31). These studies have led to the notion that the relative abundance of VEGF and PEDF within the retina determines the resulting vascularity.

Within the repairing lung, differences in localization between PEDF and VEGF may predict the observed alterations in vascularity, as vascular remodeling appears to be regulated by the balanced expression of angiogenic and angiostatic mediators. In the fibroblastic focus of UIP, a structure whose density may predict a poor prognosis (6365), PEDF was present in the majority of lesions, whereas VEGF expression was nearly undetectable. In the Masson body of OP, a structure that resolves almost completely and is reminiscent of traditional granulation tissue given its degree of vascularity, VEGF was expressed to a much greater extent than within the fibroblastic focus. Although PEDF was present within a minority of Masson bodies, the relative abundance of VEGF compared with PEDF, and perhaps the resultant vascularity, was dramatically different within the fibroblastic focus of UIP. Spatial heterogeneity of vascularization was present in IPF; decreased vascular density was present within fibroblastic foci, but prominent interstitial vessels were noted. These observations are similar to those previously reported (59, 60, 66). The expression of VEGF in both IPF and COP we described was similar to that noted by Lappi-Blanco (67) and Ebina (66). In our study, we demonstrate for the first time the expression of the potent angiostatic mediator PEDF within the fibrotic lung, relative to that of VEGF, and suggest that the relative abundance of these mediators may regulate vascular development in the lung, as they do within the eye (68). Although we cannot directly implicate PEDF in the suppression of vascularization within the fibroblastic focus, our in vitro angiogenesis assays suggested a global angiostatic milieu within the IPF lung. This finding is contrary to prior studies using rat corneal micropocket assays, in which the angiogenesis was promoted rather than suppressed (69, 70). This observed difference may reflect differences between the angiogenesis assays or, perhaps, extraction techniques resulting in different sets of angiogenic and angiostatic mediators.

The cellular sources of PEDF are a subject of considerable interest. PEDF within Masson bodies and fibroblastic foci localized with α-SMA, suggesting myofibroblasts and fibroblasts as two sources. Although PEDF was present within fibroblastic foci, the predominant site within the IPF lung was in the fibrotic interstitium, presumably due to the binding of PEDF to type I collagen (71). PEDF was also preferentially expressed in epithelium directly overlying the fibroblastic focus. The significance of this regional, enhanced expression PEDF in the epithelium overlying the fibroblastic focus is unknown, but raises the question as to whether PEDF participates in abnormal reepithelialization and remodeling, as PEDF appears to regulate epithelial growth within the prostate and pancreas (72). The presence of PEDF within the lumen of vessels in normal lung, COP, and IPF might suggest the pulmonary endothelium as a potential source of PEDF. Transcriptional analyses of murine microvascular endothelial cells suggest that PEDF is not produced by the endothelium (73). Although presence of PEDF within vascular structure might appear counterintuitive, endothelial binding of PEDF does not uniformly result in endothelial apoptosis, as PEDF-induced Fas:Fas ligand apoptosis occurs in developing vessels but not mature or established vessels (74).

The mechanisms accounting for expression of PEDF are largely unknown, but studies suggest widespread expression in a variety of human tissues (32, 75). In retinal, pigmented epithelial cells, VEGF directly regulates PEDF expression (76). The PEDF promoter has been cloned and exhibits few conventional promoter elements, with the possible exception of a site for Ets transcription factors, but contains a dense cluster of Alu repeats, whose significance is unknown (75). In this context, the identification of PEDF as a TGF-β–responsive gene is novel and of considerable interest, as TGF-β is the cytokine most strongly linked to fibrosis. We do not suggest that PEDF is only regulated by TGF-β as the constitutive expression of PEDF in primary lung fibroblasts suggests additional regulators. In addition, however, there appears to be substantial post-transcriptional regulation of PEDF, suggesting other control mechanisms (77).

At present, there is no reliable animal model of IPF. Therefore, meticulously characterizing the human disease, at least until a more suitable animal model can be developed, will provide new insights into the pathogenetic mechanisms responsible for the progressive fibrosis of IPF. Our data suggest that within the IPF lung, regional abnormalities in vascularization exist, especially within the purported site of active wound healing, and that there is an unbalanced expression of important angiogenic and angiostatic mediators that favors the suppression of regional vascularization. As aberrant vascular development exists in other pathologic fibrosing conditions, we suggest that the progressive fibrotic response seen in IPF is a manifestation of disordered vascular development in the lung and possibly a failure of the normal repair process following injury.

Future studies are necessary to elucidate the mechanisms by which PEDF expression is coordinated with VEGF in the fibrotic lung, especially within the alveolar epithelium given the striking presence of PEDF in areas directly overlying fibroblastic foci in UIP. The manner in which extracellular matrix interactions regulate the bioavailability of PEDF will also need to be addressed in light of the abundance of PEDF within the fibrotic interstitium and the type I collagen binding site present in PEDF.

In summary, we describe for the first time the presence of the potent angiostatic mediator, PEDF, in the human lung. Its relative increased expression and location in the IPF lung as well as its stimulation by the profibrotic cytokine, TGF-β1, suggests a pathogenic role in both the abnormal regional angiogenesis and the progressive fibrosis that characterizes UIP.

The authors thank Dolly Kervistky and Marty Wallace for their assistance with tissue procurement, and Mark Moore, Aaron Carmody, and Tracy Gesell for their assistance with microarray analyses. Anonymous, nontransplanted normal lung samples were obtained from Tissue Transformation Technologies, Edison, NJ.

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Correspondence and requests for reprints should be addressed to G. Scott Worthen, M.D., National Jewish Medical and Research Center, D403, 1400 Jackson Street, Denver, CO 80206. E-mail:


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