Overexpression of Placenta Growth Factor Contributes to the Pathogenesis of Pulmonary Emphysema

Overexpression of Placenta Growth Factor Contributesto the Pathogenesis of Pulmonary EmphysemaPo-Nien Tsao, Yi-Ning Su, Hung Li, Pei-Hsin Huang, Chiang-Ting Chien, Yih-Loong Lai, Chien-Nan Lee,Chi-An Chen, Wen-Fang Cheng, Shu-Chen Wei, Chong-Jen Yu, Fon-Jou Hsieh, and Su-Ming Hsu

Departments of Medical Genetics, Pediatrics, Pathology, Medical Research Administration, Obstetrics and Gynecology, and Internal Medicine,National Taiwan University Hospital; Department of Physiology, College of Medicine, National Taiwan University; and Institute of MolecularBiology, Academia Sinica, Taipei, Taiwan, Republic of China

To examine the role of placenta growth factor (PlGF) in the patho-genesis of pulmonary emphysema, we generated PlGF-transgenic(TG) mice using a phosphoglycerate kinase promoter. This resultedin constitutive overexpression of PlGF. In these TG mice, pulmonaryemphysema, with enlarged air spaces and enhanced pulmonarycompliance, first appeared at 6 months of age and became promi-nent at 12 months. Increased alveolar septal cell apoptosis wasnoted in their lungs. Fluorescence-activated cell sorter analysis sug-gests that these apoptotic septal cells are type II pneumocytes. Atthe same time, the messenger RNA of vascular endothelial growthfactor and platelet-endothelial cell adhesion molecule-1, an endothe-lial cell marker, were downregulated indicating a reduced numberof endothelial cells and its survival factor VEGF. In vitro, exogenousPlGF can inhibit the proliferation and promote the cell death ofmouse type II pneumocytes. In normal newborn mice, abundantexpression of PlGF messenger RNA was detected in the lungs duringsaccular division but was rapidly downregulated after alveolarizationwas complete. Thus, a persistently elevated PlGF was detrimental tothe developed lung and causes the emphysematous change seen inour TG mice. Our study suggests that PlGF plays an important rolein the pathogenesis of pulmonary emphysema via its action on typeII pneumocytes.

Keywords: placenta growth factor; pulmonary emphysema; transgenicmice

Vascular endothelial growth factor (VEGF), the vascular-specificgrowth factor first characterized, has been termed the most criticaldriver of vascular formation (1). It binds to fms-like tyrosine kinase1 (Flt-1) and fetal liver kinase 1 (Flk-1). The latter is thought tomediate most of the angiogenic and proliferative effects of VEGF.However, little is known about a VEGF homolog called placentagrowth factor (PlGF). PlGF binds to Flt-1 but not to Flk-1, andit may function by modulating VEGF activity (2).

Exogenous PlGF stimulates angiogenesis and induces vascu-lar permeability when coinjected with VEGF (3, 4). The angio-genic activity of PlGF is probably caused by displacement ofVEGF from the Flt-1 sink, thereby increasing the fraction of

(Received in original form June 12, 2003; accepted in final form November 24, 2003)

Supported by grants from the National Taiwan University Hospital 91A17 (P-N.T.)and from the Institute of Biomedical Science, Academia Sinica IBMS-CRC90-T02(F-J.H.) and by grant 89-B-FA01-1-4 (for light microscopy).

P-N.T. and Y-N.S. share first authorship, and H.L., P-H.H., and C-T.C. made equalcontributions to this study.

Correspondence and requests for reprints should be addressed to Fon-Jou Hsieh,M.D., Department of Obstetrics and Gynecology, College of Medicine, NationalTaiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan. E-mail:[email protected]

This article has an online data supplement, which is accessible from this issue’stable of contents online at www.atsjournals.org

Am J Respir Crit Care Med Vol 169. pp 505–511, 2004Originally Published in Press as DOI: 10.1164/rccm.200306-774OC on November 25, 2003Internet address: www.atsjournals.org

VEGF available for activation of Flk-1 (3, 5). Absence of PlGFhad a negligible effect on vascular development and normalembryogenesis as demonstrated in PlGF knockout mice, butsuch a deficiency can reduce collateral vascular growth underpathologic conditions, such as in ischemia or inflammation (5).

In most normal tissues, PlGF messenger RNA (mRNA) ispresent most abundantly in the placenta, thyroid, and lungs(6), although the roles of PlGF in these tissues remain unclear.Previous evidence suggests that epithelial and endothelial alveo-lar septal cell death due to the decreased expression of VEGFand Flk-1 may be a part of the pathogenesis of emphysema.VEGF-related signaling is required to maintain the alveolarstructures of the lungs (7–9). Recently, Autiero and coworkersreported that PlGF can modulate the function of VEGF byregulating the intermolecular and intramolecular cross talk be-tween Flt-1 and Flk-1. Moreover, PlGF alone can trigger its ownintracellular signals, independent of VEGF/Flk-1 signaling, andcan exert Flt-1–dependent biological effects, involving prolifera-tion, apoptosis, or angiogenesis (10). Therefore, we hypothesizethat PlGF may play some role in the pathogenesis of emphysema.Because no phenotype was obvious in the PlGF knockout mice(5), we generated transgenic (TG) mice, which constitutivelyoverexpressed PlGF. In these mice, pulmonary emphysema,mimicking human chronic obstructive pulmonary disease withenlarged air spaces and enhanced pulmonary compliance, wasnoted but without an inflammatory response. In vitro, we demon-strated that exogenous PlGF could inhibit the proliferation andpromote the cell death of mouse pulmonary type II epithelialcells. Our study suggests the possibility of an important role forPlGF via its action on type II pneumocytes in the pathogenesisof pulmonary emphysema. Some of the results from this studyhave previously been reported in the form of an abstract (11).

METHODS

(More detailed information about Methods is provided in the onlinesupplement.).

Generation of PlGF TG Mice

The animal protocol was approved by the Animal Care and Use Com-mittee of our hospital. The full coding sequence of mouse PlGF comple-mentary DNA (nucleotides 318–794, GenBank NM 008827) was ligatedinto a phosphoglycerate kinase expression cassette. The linearizedtransgene was purified and injected into fertilized oocytes. The micewere screened for the presence of the transgene using a polymerasechain reaction (PCR).

Reverse Transcription–PCR for mRNA Expression

Total RNA was isolated from lung homogenates or cell lysates withTrizol. Reverse-transcribed complementary DNA products were ampli-fied by PCR with primers specific for PlGF, VEGF, platelet-endothelialcell adhesion molecule-1, Flt-1, and the reduced form of glyceraldehyde-3-phosphate dehydrogenase. The amplification products were separatedby agarose gel electrophoresis and visualized after staining with ethid-ium bromide.

506 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 169 2004

Western Blotting

Proteins (50 �g) were subjected to electrophoresis on 10% gradientBio-Tris gels (Novex, San Diego, CA) and transferred to PolyScreenpolyvinylidine difluoride transfer membranes (Millipore Corp., Bed-ford, MA). The antibodies (1:1,000 dilution) used were anti-mousePlGF, �-tubulin (R&D Systems, Minneapolis, MN), and anti-goat horse-radish peroxidase–conjugated antibody (BioSource International, Ca-marillo, CA).

Bronchoalveolar Lavage and Quantification of PlGF Levels

Bronchoalveolar lavage fluid sample and blood were centrifuged andthe supernatants were stored at �70�C until analysis. The amount ofPlGF, in the bronchoalveolar lavage and in the serum, was measuredusing an ELISA assay (R&D Systems).

Morphologic and Histopathologic Assessment

We performed light microscopic examination of the heart, brain, liver,kidney, and lungs of the PlGF TG mice. The lungs were fixed by trachealinstillation of 10% buffered formalin at constant pressure (25 cm H2O).Five-millimeter sections were stained with hematoxylin and eosin orterminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling(TUNEL). Alveolarization was assessed by the radial alveolar count(12, 13), and volume density of the airspaces (Vv(air, lung) %) was estimated(14).

Microanalysis of Three-dimensional Images Usinga Digital Volumetric Imaging System

The formalin-fixed lung tissues were processed, and three-dimensionalimages were constructed with the help of a digital volumetric imagingsystem from Resolution Sciences Corporation (Corte Madera, CA).

Pulmonary Function Testing of PlGF TG Mice

After anesthesia with pentobarbital sodium (70 mg/kg, intraperitoneal),each animal’s trachea, carotid artery, and jugular vein were cannulated.Each animal was artificially ventilated, and the mechanical respiratoryproperties of the mouse were measured (15).

Hemodynamic Study

Mice (three each of both wildtype [WT] and TG mice) were anesthe-tized with intraperitoneal urethane (120 mg/100 g body weight). Thearterial (left carotid) and venous (portal) blood flows were measuredby placement of a blood flow probe (0.5VB334; Transonic System Inc.,Ithaca, NY) and the blood flow parameters were displayed using Ul-transonic Systems (T206; Transonic Systems Inc.). The blood flow pa-rameters were recorded simultaneously on an ADI system (OxfordScience Park, Oxford, UK).

TUNEL Staining for Apoptotic Cells in EmphysematousLung Tissues

TUNEL was performed with a TdT-FragEL kit (Oncogene ResearchProducts, San Diego, CA).

Figure 1. Generation of placenta growth fac-tor (PlGF)–transgenic (TG) mice. (A ) The con-struct used in the generation of PlGF TG mice.(B ) Reverse transcription–polymerase chainreaction (RT-PCR) of PlGF demonstrating in-creased PlGF messenger RNA (mRNA) ex-pression in various organs in TG mice, whencompared with wild-type (WT) mice. (C ) and(D ) Western blots of lung and brain revealingincreased PlGF protein expression in two lines(T29 and T22) of TG mice, when comparedwith WT mice. Densitometry revealed thatPlGF proteins in the lungs were increasedapproximately 2.3-fold in the T29 line and2.8-fold in the T22 line when compared withthe WT mice (n � 3 in each group).

Effects of PlGF on Murine Type II Pneumocytes

Mouse pulmonary type II epithelial cells (MLE-15) were grown inDulbecco’s modified Eagle medium. The expression of VEGF, PlGF,and Flt-1 mRNA was measured, and the effects of PlGF on cell prolifer-ation and cell death were examined by propidium iodide staining andBrdU incorporation study (16).

Fluorescence-activated Cell Sorter Scan for PulmonaryPrimary Cells

The primary cells of mouse lung were harvested as previously reportedwith some modifications (17). Data acquisition and analysis was per-formed on a FACScalibur flow cytometer (18) to evaluate the percent-ages of apoptotic primary pulmonary type II cells in both the WT andTG groups.

Statistical Analysis

All the data are expressed as mean � SEM. Statistical analysis wasperformed using SPSS 9.0 for Windows (Statistical Package for SocialSciences, Inc., Chicago, IL) and analyzed using analysis of variance,the Mann–Whitney test, and Pearson’s correlation; p values less than0.05 (*) were deemed statistically significant.

RESULTS

Pulmonary Emphysema in PlGF TG Mice

We generated PlGF TG mice using a construct containing mousePlGF complementary DNA driven by a phosphoglycerate kinasepromoter, which resulted in constitutive overexpression of PlGF.This was confirmed by reverse transcription–PCR and Westernblot (Figures 1A–1D). Both the TG and WT mice were killedat the age of 1 and 2 weeks and at 1, 2, 4, 6, and 12 months tocarry out lung histology analysis (n � 5 in each age group). Wefound that lung development was normal from 1 week up to 4months of age in PlGF TG mice. Enlarged airspaces in the lungswere noted, however, in TG mice from the age of 6 months,becoming prominent at 12 months of age (Figures 2A–2D); thisphenotype simulates the pathologic findings of pulmonary em-physema. Apart from this phenotype, we observed no otherorgan or developmental abnormalities in the PlGF TG mice.The mean radial alveolar counts in the lungs of the PlGF TGmice were significantly lower than in the WT mice at 12 monthsof age (n � 5 in each group) (Figure 2E). The decrease in radialalveolar count and the degree of emphysematous change weremore prominent in the TG mouse line T22, whose PlGF levelwas higher than that of line T29 (Figures 1D and 2E). Further-more, the volume density of airspaces (Vv(air, lung)) was significantlyhigher in the PlGF TG mice than in the WT mice (79.62 � 0.6,86.3 � 0.5, 88.2 � 0.5, and 73.0 � 0.5% in lines T29, T22, T14,and WT mice, respectively; p � 0.01). It is noteworthy that the

Tsao, Su, Li, et al.: Pulmonary Emphysema in PlGF TG Mice 507

Figure 2. PlGF overexpression caused a phenotype similar tothat in human emphysema. (A )–(D) Hematoxylin and eosin (H&E) staining of sections of the right lung fixed by tracheal instilla-tion of 10% buffered formalin at constant pressure (25 cm H2O).The lungs did not reveal any vascular enlargement, edema, orinflammatory cell infiltration. Alveolar enlargement (emphyse-matous change) was seen in three different TG lines T29 (B ),T22 (C ), and T14 (D ). Note the normal appearance of alveolusstructure in WT mice (A ). No inflammatory response was notedin the lung tissues of all TG mice. (E ) Radial alveolar count (RAC),an assessment of the alveolarization of an acinus, was used tomeasure the alveolar development. RACs were significantly lowerin PlGF TG mice, especially in lines T22 and T14, when comparedwith the WT mice. The decrease in RAC and the degree of emphy-sematous change were correlated well with the amounts of PlGFin TG mice when compared with Figure 1D (n � 5 in eachgroup). (F ) Pulmonary function tests revealed that the T22 micehad increased functional residual capacity (FRC), total lung ca-pacity (TLC), and dynamic compliance (Crs) when comparedwith WT mice (n � 2 each). These patterns of change in RAC,FRC, TLC, and Crs are similar to those seen in patients withchronic obstructive pulmonary disease (COPD).

mRNA of PlGF in the lungs of the TG mice was persistentlyhigh from the newborn period to adulthood (Figure 7C). At 12months of age, the PlGF serum levels (216.7 � 19.6 vs. 114 �26.4 mg/dl, p � 0.05) and bronchoalveolar lavage (32.6 � 9.9vs. 10.5 � 1.0, p � 0.01) in the PlGF T22 mice were significantlyhigher than in the WT mice (n � 5 in each group).

Pulmonary function tests in the 12-month-old TG mice re-vealed a picture similar to that of human patients with pulmonaryemphysema, including a higher functional residual capacity(0.6 � 0 and 0.4 � 0.14 ml in TG and WT mice, respectively),total lung capacity (1.67 � 0.34 and 1.05 � 0.14 ml in TG and WTmice, respectively), and respiratory system compliance (0.023 �0.003 and 0.0125 � 0.004 in TG and WT mice, respectively) (n �2 in each group) (Figure 2F). Emphysematous changes in the PlGFTG mice were better illustrated using a Digital Volumetric Imagingsystem (Figure 3).

Normal Hemodynamics in PlGF TG Mice

Although overexpression of PlGF may cause edema and vascularenlargement (19), we did not observe such findings in the histo-

Figure 3. An emphysematous change in PlGF TG mice is illustrated with the use of a Digital Volumetric Imaging (DVI) system. For DVI, the fixedsample was stained and then embedded in a block polymer. After the block was mounted on a microtome, an image was captured directly offthe cut surface of the block using a CCD camera. A section was cut to the depth of the first image, and the process was repeated about 1,000times on average. Because no slides were made and the sectioning was automated, thousands of images could be captured from a sample, therebyallowing micron range–resolution three-dimensional images to be generated. The lung sections of WT mice (A and B ) and TG mice (T22) (C )were viewed by RESView software.

logic examination of their lungs. At 12 months of age, there wasno significant change in portal venous blood flow (0.141 � 0.010and 0.147 � 0.011 ml/minute in WT and TG mice, respectively)and in left carotid arterial blood flow (0.100 � 0.005 and 0.107 �0.006 ml/minute, in WT and TG mice, respectively). Blood pres-sure was also similar in both TG and WT mice (n � 3 in eachgroup).

Platelet-Endothelial Cell Adhesion Molecule-1 and VEGFmRNA Was Decreased in Lungs of PlGF TG Mice

To assess whether microcirculation was affected in the PlGF TGmice, we analyzed the expression of platelet-endothelial celladhesion molecule-1 (CD31), a specific endothelial cell marker,in 12-month-old TG mice by using semiquantitative reversetranscription–PCR. We also examined the expression of VEGFmRNA, which is known as a survival factor for endothelial cells.We found that the expression of both platelet-endothelial celladhesion molecule-1 and VEGF mRNA was lower in the lungsof the PlGF TG mice than in the WT mice (n � 3 in each group)(Figures 4A and 4B), suggesting a decrease and not an increasein vasculature.


Figure 4. RT-PCR of total lung mRNA, revealing that platelet-endothelial cell adhesion molecule-1 (PECAM-1) and vascular en-dothelial growth factor (VEGF) mRNA were downregulated inTG mice (A ). Densitometry showed that amounts of VEGF andPECAM-1 mRNA were about 60% of those found in WT mice (B )(n � 3 in each group).

Increased Pulmonary Septal Cell Deathin Lungs of PlGF TG Mice

Histologic and histochemical examinations of lung sections ob-tained from the PlGF TG mice at 12 months of age revealed noevidence of fibrosis, edema, or inflammation. Focal and mildinflammations were occasionally seen in some of the TG miceolder than 16 weeks. Blood vessels and bronchioles were unre-markable. We used the TUNEL technique to identify apoptoticcells in the lungs of the PlGF TG mice. TUNEL labeling ofDNA-strand breaks in situ demonstrated increased apoptoticnuclear stains located at the periphery of alveolar spaces or atsepta in the lungs of the PlGF TG mice (Figures 5A and 5B).Virtually no apoptotic cells were seen in the lungs of the WTmice (Figure 5C). Many of these apoptotic cells appeared to bepneumocytes, judging by their oval and/or slightly elongatedshape. In addition, by using fluorescence-activated cell sorteranalysis, we demonstrated that the incidence of PI(�)/surfactantprotein C(�) cells was significantly greater in the primary pulmo-nary cells of the PlGF TG mice (1.15 � 0.14%) than in the cellsof the WT mice (0.43 � 0.14%) (p � 0.04) (Figure 5D). Becausesurfactant protein C is a specific marker for type II pneumocytes,we postulated that the apoptotic septal cells were mainly typeII pneumocytes.

PlGF Promotes Pulmonary Epithelial Cells Deathand Inhibits Cell Proliferation

Because we postulated that the apoptosis of type II pneumocytesin TG mice is related to the overexpression of PlGF, we examinedthe effects of recombinant PlGF protein on murine type II pneu-mocytes (MLE-15 cells) in vitro. The mRNAs of PlGF, VEGF,and Flt-1, (but not Flk-1), were demonstrated using reverse tran-

Figure 5. (A ) Increased apoptotic cells in the alveolar septa of PlGF TG mice. (A )–(C ), terminal deoxynucleotidyl transferase–mediated dUTP nickend-labeling (TUNEL) staining revealed increased apoptotic nuclei in TG alveolar septa (A and B ), whereas no apoptotic cells were detected in thelungs of WT mice (C ). Fluorescence-activated cell sorter (FACS) analysis for primary lung cells revealing that that the number of PI(�)/surfactantprotein C (SP-C) (�) cells were significantly higher in the primary pulmonary cells of PlGF TG mice than in the WT mice (D ) (n � 3 in each group).

scription–PCR in MLE-15 cells (Figure 6A) and were found toresemble the expression pattern in pneumocytes during “early”pulmonary development (see below). Nevertheless, we found thatexogenous PlGF could inhibit the proliferation of MLE-15 cellsin a dose-dependent manner and significantly promote the deathof these cells (Figures 6B and 6C). Recombinant VEGF did nothave the same effects. These data suggest that PlGF may play asignificant regulatory role in the growth and survival of pneumo-cytes. In addition, elevated PlGF may result in a reduced prolifera-tion and an increased death of pneumocytes.

PlGF Shown to Be Developmentally Regulated in the Lung

Murine alveolar development begins on postnatal Day 3,whereas saccular division is completed by postnatal Day 14 (20).To determine whether PlGF is developmentally regulated indeveloping lungs, we collected total mRNA from murine lungtissues at different postnatal stages (from Days 3 to 14) andfrom 12-week-old adults. We examined the expression of PlGFmRNA by semiquantitative reverse transcription–PCR. Beforealveolarization, the expression of PlGF mRNA in the lungs wasrelatively high but was shown to be downregulated during andafter alveolarization (Figures 7A and 7B). The PlGF mRNAexpression was persistently high, however, in TG mice (Figure7C).

DISCUSSION

Pulmonary emphysema, defined as abnormal airspace enlarge-ment distal to the terminal bronchioles, is a major componentof chronic obstructive pulmonary disease, which is estimated toaffect 16 million people in the United States. Currently it is the


Figure 6. (A ) RT-PCR revealed expression of VEGF, Flt-1, and PlGF in murine pulmonary type II epithelial cells (MLE-15). (B ) Recombinant PlGF,but not recombinant VEGF, significantly enhanced cell death of MLE-15 cells. (C ) PlGF inhibited proliferation of MLE-15 cells in a dose-dependentfashion. Note that VEGF had no effects on MEL-15 cells.

fourth leading cause of death worldwide (21). Although chronicobstructive pulmonary disease occurs predominantly in smokers,the fact that only 15 to 20% of cigarette smokers develop pulmo-nary emphysema suggests that other genetic and environmen-tal factors interact with cigarette smoke to cause emphysema(22–25).

An increased protease or more precisely a protease–antiprotease imbalance induced by inflammation has been de-scribed as the mechanism causing pulmonary emphysema,including that caused by smoking (22, 26–31). Extracellular ma-trix proteases such as collagenase are also implicated as etiologicagents in the emphysematous process (29, 30, 32).

Inflammation may not be the sole mechanism responsiblefor the pathogenesis of emphysema. Adult mice expressing theplatelet-derived growth factor-B with the lung-specific surfactantprotein C promoter exhibited lung pathology characterized byenlarged airspaces, fibrosis, and inflammation (33). In these mice,emphysematous changes frequently occurred throughout thelung, but inflammatory lesions were usually confined to focalareas (33). Recently, Kasahara and coworkers demonstrated thatchronic treatment of rats with a VEGF receptor-2 inhibitor ledto endothelial and epithelial apoptosis followed by enlargementof the air spaces but without clear signs of acute or chronicinflammation (8). In contrast, inflammation such as that seen inlung tissues from patients with primary pulmonary hypertensionand other conditions may not always lead to an emphysematouschange (7). Taken together, these findings suggest that inflam-

Figure 7. (A ) and (B ) Downregulation of PlGF during lung develop-ment. PlGF mRNA expression was downregulated during alveolariza-tion, from P3 to P14, and decreased 0.2-fold in adult lungs (n � 3 ineach age group). (C ) Constitutional expression of PlGF mRNA duringlung development in PlGF TG mice (n � 3 in each age group).

mation and/or protease–antiprotease imbalance may not besolely responsible for pulmonary emphysema.

In this study, we have demonstrated that the overexpressionof PlGF in mice causes a phenotype and pulmonary dysfunctionsimilar to human pulmonary emphysema with enlarged airspaces and enhanced pulmonary compliance but without an in-flammatory response (Figure 2). We have shown that the overex-pression of PlGF is associated with increased apoptosis eventsin the alveolar septa. These apoptotic cells are presumed to betype II pneumocytes as revealed by fluorescence-activated cellsorter analysis. We have further demonstrated that exogenousPlGF inhibited the proliferation of and promoted the cell deathof cultured type II pneumocytes in a dose-dependent fashionin vitro. Simultaneously, mRNA of VEGF and platelet-endothelialcell adhesion molecule-1 in the lungs of TG mice were bothdownregulated, indicating a reduced number of endothelial cellsand their survival factor, VEGF. The apoptotic effect of PlGF onpneumocytes was intriguing. Because Carmeliet and coworkershave reported that a high concentration of PlGF alone did notaffect the survival of endothelial cells in vitro (5), it is reasonableto speculate that the reduced number of endothelial cells inthe lungs of PlGF TG mice is secondary to the compromise ofpneumocytes affected by elevated PlGF.

By examining resected human lung tissue, Kasahara and co-workers reported that in human emphysematous lungs, the num-ber of TUNEL(�) septal cells is significantly higher than innormal lungs (7). Using double labeling with epithelial and endo-thelial markers, they suggested that both epithelial and endothe-lial cells were undergoing apoptosis. Moreover, they noted thatin human emphysematous lung 25.7% of TUNEL(�) cells wereepithelial cells, whereas only 5.5% of these TUNEL(�) cellswere endothelial cells. Although they stressed the importanceof endothelial cell death and decreased expression of VEGFand Flk-1 in human emphysema, they also suggested that thealtered function of type I and type II cells (the main sites ofVEGF production) may account for the decreased VEGF ex-pression and increased epithelial cell apoptosis in emphysema.This is in line with our observation in the emphysematous lungof our PlGF TG mice where the apoptotic septal cells weremainly epithelial cells, which in turn affected the endothelialcells via reduced production of their survival factor,VEGF.

Increasing evidence points to a direct role for paracrine sig-naling between endothelial cells and surrounding target organcells (34). It is overwhelmingly evident that tissues regulate vas-cular architecture by signaling to endothelial cells through potentlocal angiogenic agents such as the family of VEGF proteins.On the other hand, endothelial cells produce a variety of humoralfactors, growth factors, cytokines, and cell surface molecules to


Figure 8. Intercellular signaling be-tween pneumocytes and endo-thelial cells in normal and PlGF TGmice. (A ) In the physiologic state,pneumocytes secrete VEGF, whichexerts a trophic effect on endothe-lial cells (EC) via Flk-1. Pneumo-cytes also secrete certain amountsof PlGF, which may enhance VEGF-dependent angiogenesis throughintramolecular and intermolecularcross talk between Flk-1 and Flt-1.PlGF may have some unknownautocrine effects on pneumocytesthrough Flt-1. Signals from endo-thelial cells to pneumocytes havenot yet been discovered. (B ) In-creased PlGF production frompneumocytes is detrimental toPlGF TG mice through activationof Flt-1 on the pneumocytes, re-sulting in a decreased number ofexisting pneumocytes. Alterna-tively, with less VEGF available toendothelial cells, their survival isjeopardized.

communicate with surrounding cells. For example, bone mor-phogen protein-2 and brain-derived neurotrophic factor are en-dothelial signals to neurons (35, 36), transforming growthfactor- and neuregulin are endothelial signals directed towardcardiomyocytes (37–39), nitric oxide is an endothelial signal torenal tubular cells (40, 41), and hepatocyte growth factor andinterleukin-6 are endothelial signals to hepatocytes (42). In thepulmonary alveoli, the pneumocytes and endothelial cells mayalso use this kind of tissue–endothelial cell cross talk. Pneumo-cytes are the major sites of VEGF production, which supportsthe survival of endothelial cells. It is still not clear, however,how the PlGF, normally present in the pneumocyte, signals to thealveolar septal endothelial cells. Currently, endothelial signalsdirected to pneumocytes in the pulmonary alveoli are unknown.

The following questions remain to be answered: (1) do theendothelial cells signal to pneumocytes in the pulmonary alveoliand if so, how? (2) what is the physiologic role of PlGF in normallungs? (3) what is the mechanism behind the observed harmfuleffects of overexpressed PlGF on pneumocytes? and (4) areapoptosis genes triggered by the autocrine regulation via theFlt-1 receptor on the pneumocytes?

We first hypothesized that overexpressed PlGF induced pneu-mocyte cell death leading to decreased VEGF production. Thisdepletion resulted in endothelial cell compromise due to a loweravailability of their survival factor, VEGF. The compromisedmicrocirculation resulting from endothelial cell damage may fur-ther promote pneumocyte cell death. Finally, this vicious circlecontinues to promote alveolar septal cell death and eventuallyleads to pulmonary emphysema. We have also shown that abun-dant PlGF is present in the lungs of neonatal mice and is rapidlydownregulated soon after alveolarization. This suggests thatPlGF in the lung is developmentally regulated and that persis-tently elevated PlGF may be detrimental to developed lungs asseen in our PlGF TG mice (Figure 8)

Recently, Autiero and coworkers reported that PlGF regulatesboth intermolecular and intramolecular cross talk between Flt1and Flk1. They state that activation of Flt1 by PlGF results inintermolecular transphosphorylation of Flk1, thereby amplifyingVEGF-driven angiogenesis through Flk1 (10). This finding is con-

trary to the negative effect of overexpressed PlGF on endothelialcells observed in our TG mice. Another significant finding is thatalthough VEGF and PlGF both bind Flt1, each induces a distinctpattern of tyrosine phosphorylation and gene expression, suggestingthat each activates Flt1 in a unique manner (10). In addition,PlGF alone can trigger its own intracellular signals, independent ofVEGF/Flk1 signaling. These PlGF-regulated genes have presumedroles in proliferation and apoptosis (10). Such a mechanism is alsolikely to exist in pneumocytes because they express Flt1 but notFlk1 as demonstrated by reverse transcription–PCR.

In conclusion, our PlGF TG mice revealed a novel nonin-flammatory pathway in pulmonary emphysema. Elevated PlGFpromotes pulmonary epithelial cell death leading to decreasedVEGF production, which in turn affects the endothelial cells.The compromised microcirculation further jeopardizes the sur-vival of pneumocytes and culminates in emphysema. Our obser-vation of pulmonary emphysema in PlGF-overexpressing micemay shed new light on the pathogenesis of human pulmonaryemphysema—a major component of chronic obstructive pulmo-nary disease.

Conflict of Interest Statement : P-N.T. has no declared conflict of interest; Y-N.S.has no declared conflict of interest; H.L. has no declared conflict of interest; P-H.H.has no declared conflict of interest; C-T.C. has no declared conflict of interest;Y-L.L. has no declared conflict of interest; C-N.L. has no declared conflict of inter-est; C-A.C. has no declared conflict of interest; W-F.C. has no declared conflictof interest; S-C.W. has no declared conflict of interest; C-J.Y. has no declaredconflict of interest; F-J.H. has no declared conflict of interest; S-M.H. has nodeclared conflict of interest.

Acknowledgment : The authors thank Dr. T. J. Ley for kindly providing thePGF expression cassette and Dr. J. Whitsett for kindly providing the MLE-15 celllines. They also thank Dr. Joel Moss and Dr. Martha Vaughan of the Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, NationalInstitutes of Health, and also Dr. James Mulshine of the Cell and Cancer Bio-logy Branch, Center for Cancer Research, National Cancer Institute, NationalInstitutes of Health, Bethesda, M.D. for critical reading of the manuscript. Inaddition, they thank M. J. Wu and C. Y. Su for excellent technical assistance andC.-L. Chien of the National Taiwan University, Taipei, Taiwan for guidance onlight microscopy.

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Overexpression of Placenta Growth Factor Contributes to the Pathogenesis of

Pulmonary Emphysema

Po-Nien Tsao1,2, Yi-Ning Su1, Hung Li3, Pei-Hsin Huang4, Chiang-Ting Chien5,

Yih-Loong Lai6, Chien-Nan Lee7, Chi-An Chen7, Wen-Fang Cheng7, Shu-Chen Wei8,

Chong-Jen Yu8, Fon-Jou Hsieh7*, and Su-Ming Hsu4

ONLINE DATA SUPPLEMENT

Methods

Generation of PlGF transgenic mice

The animal protocol was approved by the Animal Care and Use Committee of the

National Taiwan University Hospital. FVB strain mice were purchased from a

commercial vendor and housed in the Animal Care Facility of the National Taiwan

University Hospital. The full coding sequence of mouse PlGF cDNA (nucleotides

318-794, GenBank NM 008827) was ligated into a PGK expression cassette. The

linearized transgene was purified and injected into fertilized oocytes. Mice were

screened for the transgene by polymerase chain reaction (PCR) of genomic DNA and

the following primers: 1) forward 5'-TCCTTCGCTTTCTGGGCTCAGAG-3', reverse

5’-CGAGAGAGAAAGAAAGAAGCCAG -3' (amplification product, 793 bp).

Reverse transcription-PCR for mRNA expression

Total RNA was isolated from lung homogenates or cell lysates with Trizol according

to the manufacturer’s instructions. RNA (0.5 µg/ sample) was converted to

complementary DNA (cDNA) by the oligo dT-primed reverse transcriptase reaction

according to the manufacturer’s protocol. Reverse-transcribed cDNA products were

amplified by PCR with primers specific for PlGF, VEGF, PECAM-1, Flt-1, and

GAPDH. PCR reactions consisted of 10 pmol of each primer, 0.25 mM of each

deoxynucleotide triphosphate, 2.5 mM MgCl2, 60 mM Tris-HCl (pH 8.5), 12.5 mM

(NH4)2SO4, and 0.1 U Taq polymerase in a total reaction volume of 25 µl. The

amplification products were separated by agarose gel electrophoresis and visualized

by staining with ethidium bromide.

Western blotting

Proteins (50 µg) were electrophoresed on 10% gradient Bio-Tris gels (Novex, San

Diego, CA) and transferred to PolyScreen PVDF transfer membrane (Millipore Corp.,

Bedford, MA) in Tris-glycine buffer containing 10% methanol. The antibodies

(1:1000 dilution) used were anti-mouse PlGF, α-tubulin (R&D Systems, Minneapolis,

MN), and anti-goat HRP-conjugated antibody (BioSource International, Camarillo,

CA).

Bronchoalveolar lavage and quantification of PlGF Levels

After the mice were sacrificed, the tracheas were isolated by blunt dissection, and

small caliber tubing was inserted and secured in the airways. Two volumes of 0.6 ml

of PBS were then instilled, gently aspirated, and pooled. Each bronchoalveolar lavage

(BAL) fluid sample was centrifuged and the supernatants were stored at -70°C until

used. At the time of sacrifice, blood was also obtained and centrifuged within 15

minutes of collection, and the serum was kept at -700C until assayed. The levels of

PlGF in the BAL and serum samples were determined using a commercial ELISA

(R&D Systems) according to the manufacturer's instructions.

Morphologic and histopathologic assessment

We performed light microscopic examination of the heart, brain, liver, kidney, and

lung from PlGF TG mice. The lungs were fixed by tracheal instillation of 10%

buffered formalin at constant pressure (25 cm H2O). Afterwards, they were removed

and immersed in 10% buffered formalin for 24 hours at 4°C. The lungs were then

immersed in paraffin and stored at room temperature. Five-millimeter sections were

stained with H&E or terminal deoxynucleotidyl transferase–mediated dUTP nick

end-labeling (TUNEL). Alveolarization was assessed by the radial alveolar count

(RAC) method of Emery and Mithal, as described (E1, 2). Volume density of the

airspaces (Vv(air, lung) %) was estimated according to Heemskerk-Gerritsen et al

(E3).

Microanalysis of three-dimensional images using the Digital Volumetric Imaging

System

The formalin-fixed lung tissues were processed for three-dimensional image analysis

as instructed by Resolution Sciences Corporation (Resolution Sciences Corporation,

Corte Madera, CA). We sorted through the three-dimensional images section by

section (in the x, y, and z planes) and rotated the images to show the overall

architecture of the lungs.

Pulmonary function testing of PlGF transgenic mice

After anesthetizing the animals with pentobarbital sodium (70 mg/kg IP), each

animal’s trachea, carotid artery, and jugular vein were cannulated with an 18-gauge

needle and PE-10 tube. After being paralyzed with gallamine triethiodide (1 mg/kg),

the animal was artificially ventilated with a tidal volume of 8-10 ml/kg and a

frequency of 120 breaths/minute by a small-animal ventilator. Under such conditions,

the respiratory mechanical properties of the mice were measured as previously

described (E4).

Hemodynamic study

The study was intended to examine whether overexpression of PlGF leads to changes

in the microcirculation and hemodynamics of the TG mice. A group consisting of

three wild type and three TG mice was anesthetized with intraperitoneal urethane (120

mg/100 g body weight). Arterial (left carotid) and venous (portal) blood flows were

measured by placement of a blood flow probe (0.5VB334, Transonic System Inc.,

Ithaca, NY) around the respective blood vessel, and the blood flow parameter was

displayed in Ultransonic Systems (T206, Transonic Systems Inc., Ithaca, NY).

Parameters of blood flow were recorded simultaneously on an ADI system (Oxford

Science Park, Oxford, England).

TUNEL staining for apoptotic cells in emphysematous lung tissues

TUNELwas performed with a TdT-FragELTM kit (Oncogene Research Products, San

Diego, CA), following the manufacturer’s instructions with minor modifications.

Effects of PlGF on murine type II pneumocytes

Mouse pulmonary type II epithelial cells (MLE-15)) were grown in DMEM and used

to demonstrate the expression of VEGF, PlGF, and VEGFR-1 mRNA and the effects

of PlGF on cell proliferation and cell death. Recombinant mouse (rm) PlGF and

rmVEGF164 were obtained from R&D Systems. MLE-15 cells were seeded at 105/well

in a 6-well plate and incubated at 370C in a 5% CO2 atmosphere overnight. Different

concentrations of rmPlGF or rmVEGF (up to 100 ng/ml) were added. Cell apoptosis

was measured by PI staining as described previously (E5). For the cell proliferation

assay, an enzyme immunoassay by quantification of BrdU incorporation into DNA

was performed according to the manufacturer's instruction (Roche Diagnostics GmbH,

Mannheim, Germany).

FACScan for pulmonary primary cells

The primary cells of mouse lung were harvested as previously reported with

some modification (E6). Briefly, the lungs were dissected, trypsinized, and then

passed through a metal sieve with two rounded tweezers. After rinsing the sieve with

40 ml of PBS, the cell suspensions were centrifuged at 400 x g for 10 minutes.

Apoptosis of pulmonary primary cells was measured. The cells were first washed with

FACScan buffer. Then the cells were incubated with rabbit anti-mouse surfactant

protein C (SP-C) polyclonal antibody (Chemicon, Temecula, CA), the type II

pneumocyte specific marker, for 30 minutes on ice. Afterwards, The cells were

washed with FACScan buffer. PI (PharMingen, San Diego, CA) and FITC-conjugated

goat anti-rabbit IgG (Cappel, Durham, NC) were stained for 30 minutes on ice and

washed with FACScan buffer. Data acquisition and analysis were performed on a

FACScalibur flow cytometer as described earlier (E7) to evaluate the percentages of

apoptotic primary pulmonary type II cells in the WT and TG groups.

Statistical analysis

All the data are expressed as mean±SEM. Statistical analysis was performed with

SPSS 9.0 for Windows (Statistical Package for Social Sciences, Inc., Chicago, IL) and

analyzed by ANOVA, Mann-Whitney test, and Pearson’s correlation; P values <0.05

(*) were deemed statistically significant.

References

E1. Cooney, T. P., and W. M. Thurlbeck. The radial alveolar count method of Emery

and Mithal: a reappraisal 1--postnatal lung growth. Thorax 1982;37:572-9.

E2. Cooney, T. P., and W. M. Thurlbeck. The radial alveolar count method of Emery

and Mithal: a reappraisal 2--intrauterine and early postnatal lung growth. Thorax

1982;37:580-3.

E3. Heemskerk-Gerritsen, B. A., J. H. Dijkman, and A. A. Ten Have-Opbroek.

Stereological methods: a new approach in the assessment of pulmonary emphysema.

Microsc Res Tech 1996;34:556-62.

E4. Lai, Y. L., and H. Chou. Respiratory mechanics and maximal expiratory flow in

the anesthetized mouse. J Appl Physiol 2000;88:939-43.

E5. Nutku, E., Q. Zhuang, A. Soussi-Gounni, F. Aris, B. D. Mazer, and Q. Hamid.

Functional expression of IL-12 receptor by human eosinophils: IL-12 promotes

eosinophil apoptosis. J Immunol 2001;167:1039-46.

E6. Pabst, R., A. Luhrmann, I. Steinmetz, and T. Tschernig. A single intratracheal dose

of the growth factor Fms-like tyrosine kinase receptor-3 ligand induces a rapid

differential increase of dendritic cells and lymphocyte subsets in lung tissue and

bronchoalveolar lavage, resulting in an increased local antibody production. J

Immunol 2003;171:325-30.

E7. Cheng, W. F., C. F. Hung, C. Y. Chai, K. F. Hsu, L. He, M. Ling, and T. C. Wu.

Tumor-specific immunity and antiangiogenesis generated by a DNA vaccine encoding

calreticulin linked to a tumor antigen. J Clin Invest 2001;108(5):669-78.

Overexpression of Placenta Growth Factor Contributes to the Pathogenesis of Pulmonary Emphysema

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