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Published ahead of print on November 25, 2003, doi:10.1164/rccm.200306-774OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 505-511, (2004)
© 2004 American Thoracic Society

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

Po-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 Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China

Correspondence and requests for reprints should be addressed to Fon-Jou Hsieh, M.D., Department of Obstetrics and Gynecology, College of Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan. E-mail: fjhsieh{at}ha.mc.ntu.edu.tw


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To examine the role of placenta growth factor (PlGF) in the pathogenesis of pulmonary emphysema, we generated PlGF-transgenic (TG) mice using a phosphoglycerate kinase promoter. This resulted in constitutive overexpression of PlGF. In these TG mice, pulmonary emphysema, with enlarged air spaces and enhanced pulmonary compliance, first appeared at 6 months of age and became prominent at 12 months. Increased alveolar septal cell apoptosis was noted in their lungs. Fluorescence-activated cell sorter analysis suggests that these apoptotic septal cells are type II pneumocytes. At the same time, the messenger RNA of vascular endothelial growth factor and platelet-endothelial cell adhesion molecule-1, an endothelial cell marker, were downregulated indicating a reduced number of endothelial cells and its survival factor VEGF. In vitro, exogenous PlGF can inhibit the proliferation and promote the cell death of mouse type II pneumocytes. In normal newborn mice, abundant expression of PlGF messenger RNA was detected in the lungs during saccular division but was rapidly downregulated after alveolarization was complete. Thus, a persistently elevated PlGF was detrimental to the developed lung and causes the emphysematous change seen in our TG mice. Our study suggests that PlGF plays an important role in the pathogenesis of pulmonary emphysema via its action on type II pneumocytes.

Key Words: placenta growth factor • pulmonary emphysema • transgenic mice

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

Exogenous PlGF stimulates angiogenesis and induces vascular permeability when coinjected with VEGF (3, 4). The angiogenic activity of PlGF is probably caused by displacement of VEGF from the Flt-1 sink, thereby increasing the fraction of VEGF available for activation of Flk-1 (3, 5). Absence of PlGF had a negligible effect on vascular development and normal embryogenesis as demonstrated in PlGF knockout mice, but such a deficiency can reduce collateral vascular growth under pathologic conditions, such as in ischemia or inflammation (5).

In most normal tissues, PlGF messenger RNA (mRNA) is present 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 alveolar septal cell death due to the decreased expression of VEGF and Flk-1 may be a part of the pathogenesis of emphysema. VEGF-related signaling is required to maintain the alveolar structures of the lungs (7–9). Recently, Autiero and coworkers reported that PlGF can modulate the function of VEGF by regulating the intermolecular and intramolecular cross talk between Flt-1 and Flk-1. Moreover, PlGF alone can trigger its own intracellular signals, independent of VEGF/Flk-1 signaling, and can exert Flt-1–dependent biological effects, involving proliferation, apoptosis, or angiogenesis (10). Therefore, we hypothesize that 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 constitutively overexpressed PlGF. In these mice, pulmonary emphysema, mimicking human chronic obstructive pulmonary disease with enlarged air spaces and enhanced pulmonary compliance, was noted but without an inflammatory response. In vitro, we demonstrated that exogenous PlGF could inhibit the proliferation and promote the cell death of mouse pulmonary type II epithelial cells. Our study suggests the possibility of an important role for PlGF via its action on type II pneumocytes in the pathogenesis of pulmonary emphysema. Some of the results from this study have previously been reported in the form of an abstract (11).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
(More detailed information about METHODS is provided in the online supplement.).

Generation of PlGF TG Mice
The animal protocol was approved by the Animal Care and Use Committee of our hospital. The full coding sequence of mouse PlGF complementary DNA (nucleotides 318–794, GenBank NM 008827) was ligated into a phosphoglycerate kinase expression cassette. The linearized transgene was purified and injected into fertilized oocytes. The mice were screened for the presence of the transgene using a polymerase chain reaction (PCR).

Reverse Transcription–PCR for mRNA Expression
Total RNA was isolated from lung homogenates or cell lysates with Trizol. Reverse-transcribed complementary DNA products were amplified by PCR with primers specific for PlGF, VEGF, platelet-endothelial cell adhesion molecule-1, Flt-1, and the reduced form of glyceraldehyde-3-phosphate dehydrogenase. The amplification products were separated by agarose gel electrophoresis and visualized after staining with ethidium bromide.

Western Blotting
Proteins (50 µg) were subjected to electrophoresis on 10% gradient Bio-Tris gels (Novex, San Diego, CA) and transferred to PolyScreen polyvinylidine difluoride transfer membranes (Millipore Corp., Bedford, MA). The antibodies (1:1,000 dilution) used were anti-mouse PlGF, {alpha}-tubulin (R&D Systems, Minneapolis, MN), and anti-goat horseradish peroxidase–conjugated antibody (BioSource International, Camarillo, CA).

Bronchoalveolar Lavage and Quantification of PlGF Levels
Bronchoalveolar lavage fluid sample and blood were centrifuged and the supernatants were stored at -70°C until analysis. The amount of PlGF, in the bronchoalveolar lavage and in the serum, was measured using 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 tracheal instillation of 10% buffered formalin at constant pressure (25 cm H2O). Five-millimeter sections were stained with hematoxylin and eosin or terminal 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 Using a Digital Volumetric Imaging System
The formalin-fixed lung tissues were processed, and three-dimensional images were constructed with the help of a digital volumetric imaging system 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 respiratory properties of the mouse were measured (15).

Hemodynamic Study
Mice (three each of both wildtype [WT] and TG mice) were anesthetized with intraperitoneal urethane (120 mg/100 g body weight). The arterial (left carotid) and venous (portal) blood flows were measured by placement of a blood flow probe (0.5VB334; Transonic System Inc., Ithaca, NY) and the blood flow parameters were displayed using Ultransonic Systems (T206; Transonic Systems Inc.). The blood flow parameters were recorded simultaneously on an ADI system (Oxford Science Park, Oxford, UK).

TUNEL Staining for Apoptotic Cells in Emphysematous Lung Tissues
TUNEL was performed with a TdT-FragEL kit (Oncogene Research Products, San Diego, CA).

Effects of PlGF on Murine Type II Pneumocytes
Mouse pulmonary type II epithelial cells (MLE-15) were grown in Dulbecco's modified Eagle medium. The expression of VEGF, PlGF, and Flt-1 mRNA was measured, and the effects of PlGF on cell proliferation and cell death were examined by propidium iodide staining and BrdU incorporation study (16).

Fluorescence-activated Cell Sorter Scan for Pulmonary Primary Cells
The primary cells of mouse lung were harvested as previously reported with some modifications (17). Data acquisition and analysis was performed on a FACScalibur flow cytometer (18) to evaluate the percentages of apoptotic primary pulmonary type II cells in both the WT and TG groups.

Statistical Analysis
All the data are expressed as mean ± SEM. Statistical analysis was performed using SPSS 9.0 for Windows (Statistical Package for Social Sciences, Inc., Chicago, IL) and analyzed using analysis of variance, the Mann–Whitney test, and Pearson's correlation; p values less than 0.05 (*) were deemed statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pulmonary Emphysema in PlGF TG Mice
We generated PlGF TG mice using a construct containing mouse PlGF complementary DNA driven by a phosphoglycerate kinase promoter, which resulted in constitutive overexpression of PlGF. This was confirmed by reverse transcription–PCR and Western blot (Figures 1A–1D) . Both the TG and WT mice were killed at the age of 1 and 2 weeks and at 1, 2, 4, 6, and 12 months to carry out lung histology analysis (n = 5 in each age group). We found that lung development was normal from 1 week up to 4 months of age in PlGF TG mice. Enlarged airspaces in the lungs were noted, however, in TG mice from the age of 6 months, becoming prominent at 12 months of age (Figures 2A–2D) ; this phenotype simulates the pathologic findings of pulmonary emphysema. Apart from this phenotype, we observed no other organ or developmental abnormalities in the PlGF TG mice. The mean radial alveolar counts in the lungs of the PlGF TG mice were significantly lower than in the WT mice at 12 months of age (n = 5 in each group) (Figure 2E). The decrease in radial alveolar count and the degree of emphysematous change were more prominent in the TG mouse line T22, whose PlGF level was higher than that of line T29 (Figures 1D and 2E). Furthermore, the volume density of airspaces (Vv(air, lung)) was significantly higher 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 mRNA of PlGF in the lungs of the TG mice was persistently high from the newborn period to adulthood (Figure 7C). At 12 months of age, the PlGF serum levels (216.7 ± 19.6 vs. 114 ± 26.4 mg/dl, p < 0.05) and bronchoalveolar lavage (32.6 ± 9.9 vs. 10.5 ± 1.0, p < 0.01) in the PlGF T22 mice were significantly higher than in the WT mice (n = 5 in each group).



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  		Figure 1. Generation of placenta growth factor (PlGF)–transgenic (TG) mice. (A) The construct used in the generation of PlGF TG mice. (B) Reverse transcription–polymerase chain reaction (RT-PCR) of PlGF demonstrating increased PlGF messenger RNA (mRNA) expression in various organs in TG mice, when compared with wild-type (WT) mice. (C) and (D) Western blots of lung and brain revealing increased PlGF protein expression in two lines (T29 and T22) of TG mice, when compared with WT mice. Densitometry revealed that PlGF proteins in the lungs were increased approximately 2.3-fold in the T29 line and 2.8-fold in the T22 line when compared with the WT mice (n = 3 in each group).

 


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  		Figure 2. PlGF overexpression caused a phenotype similar to that in human emphysema. (A)–(D) Hematoxylin and eosin (H&E) staining of sections of the right lung fixed by tracheal instillation of 10% buffered formalin at constant pressure (25 cm H2O). The lungs did not reveal any vascular enlargement, edema, or inflammatory cell infiltration. Alveolar enlargement (emphysematous change) was seen in three different TG lines T29 (B), T22 (C), and T14 (D). Note the normal appearance of alveolus structure in WT mice (A). No inflammatory response was noted in the lung tissues of all TG mice. (E) Radial alveolar count (RAC), an assessment of the alveolarization of an acinus, was used to measure the alveolar development. RACs were significantly lower in PlGF TG mice, especially in lines T22 and T14, when compared with the WT mice. The decrease in RAC and the degree of emphysematous change were correlated well with the amounts of PlGF in TG mice when compared with Figure 1D (n = 5 in each group). (F) Pulmonary function tests revealed that the T22 mice had increased functional residual capacity (FRC), total lung capacity (TLC), and dynamic compliance (Crs) when compared with WT mice (n = 2 each). These patterns of change in RAC, FRC, TLC, and Crs are similar to those seen in patients with chronic obstructive pulmonary disease (COPD).

 
Pulmonary function tests in the 12-month-old TG mice revealed a picture similar to that of human patients with pulmonary emphysema, 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 WT mice, 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 PlGF TG mice were better illustrated using a Digital Volumetric Imaging system (Figure 3) .



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  		Figure 3. An emphysematous change in PlGF TG mice is illustrated with the use of a Digital Volumetric Imaging (DVI) system. For DVI, the fixed sample was stained and then embedded in a block polymer. After the block was mounted on a microtome, an image was captured directly off the 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 1,000 times on average. Because no slides were made and the sectioning was automated, thousands of images could be captured from a sample, thereby allowing 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.

 
Normal Hemodynamics in PlGF TG Mice
Although overexpression of PlGF may cause edema and vascular enlargement (19), we did not observe such findings in the histologic examination of their lungs. At 12 months of age, there was no significant change in portal venous blood flow (0.141 ± 0.010 and 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 pressure was also similar in both TG and WT mice (n = 3 in each group).

Platelet-Endothelial Cell Adhesion Molecule-1 and VEGF mRNA Was Decreased in Lungs of PlGF TG Mice
To assess whether microcirculation was affected in the PlGF TG mice, we analyzed the expression of platelet-endothelial cell adhesion molecule-1 (CD31), a specific endothelial cell marker, in 12-month-old TG mice by using semiquantitative reverse transcription–PCR. We also examined the expression of VEGF mRNA, which is known as a survival factor for endothelial cells. We found that the expression of both platelet-endothelial cell adhesion molecule-1 and VEGF mRNA was lower in the lungs of the PlGF TG mice than in the WT mice (n = 3 in each group) (Figures 4A and 4B) , suggesting a decrease and not an increase in vasculature.



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

 
Increased Pulmonary Septal Cell Death in Lungs of PlGF TG Mice
Histologic and histochemical examinations of lung sections obtained from the PlGF TG mice at 12 months of age revealed no evidence of fibrosis, edema, or inflammation. Focal and mild inflammations were occasionally seen in some of the TG mice older than 16 weeks. Blood vessels and bronchioles were unremarkable. We used the TUNEL technique to identify apoptotic cells in the lungs of the PlGF TG mice. TUNEL labeling of DNA-strand breaks in situ demonstrated increased apoptotic nuclear stains located at the periphery of alveolar spaces or at septa in the lungs of the PlGF TG mice (Figures 5A and 5B) . Virtually no apoptotic cells were seen in the lungs of the WT mice (Figure 5C). Many of these apoptotic cells appeared to be pneumocytes, judging by their oval and/or slightly elongated shape. In addition, by using fluorescence-activated cell sorter analysis, we demonstrated that the incidence of PI(+)/surfactant protein C(+) cells was significantly greater in the primary pulmonary cells of the PlGF TG mice (1.15 ± 0.14%) than in the cells of the WT mice (0.43 ± 0.14%) (p = 0.04) (Figure 5D). Because surfactant protein C is a specific marker for type II pneumocytes, we postulated that the apoptotic septal cells were mainly type II pneumocytes.



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  		Figure 5. (A) Increased apoptotic cells in the alveolar septa of PlGF TG mice. (A)–(C), terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) staining revealed increased apoptotic nuclei in TG alveolar septa (A and B), whereas no apoptotic cells were detected in the lungs of WT mice (C). Fluorescence-activated cell sorter (FACS) analysis for primary lung cells revealing that that the number of PI(+)/surfactant protein 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).

 
PlGF Promotes Pulmonary Epithelial Cells Death and Inhibits Cell Proliferation
Because we postulated that the apoptosis of type II pneumocytes in TG mice is related to the overexpression of PlGF, we examined the effects of recombinant PlGF protein on murine type II pneumocytes (MLE-15 cells) in vitro. The mRNAs of PlGF, VEGF, and Flt-1, (but not Flk-1), were demonstrated using reverse transcription–PCR in MLE-15 cells (Figure 6A) and were found to resemble the expression pattern in pneumocytes during "early" pulmonary development (see below). Nevertheless, we found that exogenous PlGF could inhibit the proliferation of MLE-15 cells in a dose-dependent manner and significantly promote the death of these cells (Figures 6B and 6C). Recombinant VEGF did not have the same effects. These data suggest that PlGF may play a significant regulatory role in the growth and survival of pneumocytes. In addition, elevated PlGF may result in a reduced proliferation and an increased death of pneumocytes.



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  		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-dependent fashion. Note that VEGF had no effects on MEL-15 cells.

 
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 in developing lungs, we collected total mRNA from murine lung tissues at different postnatal stages (from Days 3 to 14) and from 12-week-old adults. We examined the expression of PlGF mRNA by semiquantitative reverse transcription–PCR. Before alveolarization, the expression of PlGF mRNA in the lungs was relatively high but was shown to be downregulated during and after alveolarization (Figures 7A and 7B) . The PlGF mRNA expression was persistently high, however, in TG mice (Figure 7C).



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

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Pulmonary emphysema, defined as abnormal airspace enlargement distal to the terminal bronchioles, is a major component of chronic obstructive pulmonary disease, which is estimated to affect 16 million people in the United States. Currently it is the fourth leading cause of death worldwide (21). Although chronic obstructive pulmonary disease occurs predominantly in smokers, the fact that only 15 to 20% of cigarette smokers develop pulmonary emphysema suggests that other genetic and environmental 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 described as the mechanism causing pulmonary emphysema, including that caused by smoking (22, 26–31). Extracellular matrix proteases such as collagenase are also implicated as etiologic agents in the emphysematous process (29, 30, 32).

Inflammation may not be the sole mechanism responsible for the pathogenesis of emphysema. Adult mice expressing the platelet-derived growth factor-B with the lung-specific surfactant protein C promoter exhibited lung pathology characterized by enlarged airspaces, fibrosis, and inflammation (33). In these mice, emphysematous changes frequently occurred throughout the lung, but inflammatory lesions were usually confined to focal areas (33). Recently, Kasahara and coworkers demonstrated that chronic treatment of rats with a VEGF receptor-2 inhibitor led to endothelial and epithelial apoptosis followed by enlargement of the air spaces but without clear signs of acute or chronic inflammation (8). In contrast, inflammation such as that seen in lung tissues from patients with primary pulmonary hypertension and other conditions may not always lead to an emphysematous change (7). Taken together, these findings suggest that inflammation and/or protease–antiprotease imbalance may not be solely responsible for pulmonary emphysema.

In this study, we have demonstrated that the overexpression of PlGF in mice causes a phenotype and pulmonary dysfunction similar to human pulmonary emphysema with enlarged air spaces and enhanced pulmonary compliance but without an inflammatory response (Figure 2). We have shown that the overexpression of PlGF is associated with increased apoptosis events in the alveolar septa. These apoptotic cells are presumed to be type II pneumocytes as revealed by fluorescence-activated cell sorter analysis. We have further demonstrated that exogenous PlGF inhibited the proliferation of and promoted the cell death of cultured type II pneumocytes in a dose-dependent fashion in vitro. Simultaneously, mRNA of VEGF and platelet-endothelial cell adhesion molecule-1 in the lungs of TG mice were both downregulated, indicating a reduced number of endothelial cells and their survival factor, VEGF. The apoptotic effect of PlGF on pneumocytes was intriguing. Because Carmeliet and coworkers have reported that a high concentration of PlGF alone did not affect the survival of endothelial cells in vitro (5), it is reasonable to speculate that the reduced number of endothelial cells in the lungs of PlGF TG mice is secondary to the compromise of pneumocytes affected by elevated PlGF.

By examining resected human lung tissue, Kasahara and coworkers reported that in human emphysematous lungs, the number of TUNEL(+) septal cells is significantly higher than in normal lungs (7). Using double labeling with epithelial and endothelial markers, they suggested that both epithelial and endothelial cells were undergoing apoptosis. Moreover, they noted that in human emphysematous lung 25.7% of TUNEL(+) cells were epithelial cells, whereas only 5.5% of these TUNEL(+) cells were endothelial cells. Although they stressed the importance of endothelial cell death and decreased expression of VEGF and Flk-1 in human emphysema, they also suggested that the altered function of type I and type II cells (the main sites of VEGF production) may account for the decreased VEGF expression and increased epithelial cell apoptosis in emphysema. This is in line with our observation in the emphysematous lung of our PlGF TG mice where the apoptotic septal cells were mainly epithelial cells, which in turn affected the endothelial cells via reduced production of their survival factor,VEGF.

Increasing evidence points to a direct role for paracrine signaling between endothelial cells and surrounding target organ cells (34). It is overwhelmingly evident that tissues regulate vascular architecture by signaling to endothelial cells through potent local angiogenic agents such as the family of VEGF proteins. On the other hand, endothelial cells produce a variety of humoral factors, growth factors, cytokines, and cell surface molecules to communicate with surrounding cells. For example, bone morphogen protein-2 and brain-derived neurotrophic factor are endothelial signals to neurons (35, 36), transforming growth factor-ß and neuregulin are endothelial signals directed toward cardiomyocytes (37–39), nitric oxide is an endothelial signal to renal tubular cells (40, 41), and hepatocyte growth factor and interleukin-6 are endothelial signals to hepatocytes (42). In the pulmonary alveoli, the pneumocytes and endothelial cells may also use this kind of tissue–endothelial cell cross talk. Pneumocytes are the major sites of VEGF production, which supports the survival of endothelial cells. It is still not clear, however, how the PlGF, normally present in the pneumocyte, signals to the alveolar septal endothelial cells. Currently, endothelial signals directed to pneumocytes in the pulmonary alveoli are unknown.

The following questions remain to be answered: (1) do the endothelial cells signal to pneumocytes in the pulmonary alveoli and if so, how? (2) what is the physiologic role of PlGF in normal lungs? (3) what is the mechanism behind the observed harmful effects of overexpressed PlGF on pneumocytes? and (4) are apoptosis genes triggered by the autocrine regulation via the Flt-1 receptor on the pneumocytes?

We first hypothesized that overexpressed PlGF induced pneumocyte cell death leading to decreased VEGF production. This depletion resulted in endothelial cell compromise due to a lower availability of their survival factor, VEGF. The compromised microcirculation resulting from endothelial cell damage may further promote pneumocyte cell death. Finally, this vicious circle continues to promote alveolar septal cell death and eventually leads to pulmonary emphysema. We have also shown that abundant PlGF is present in the lungs of neonatal mice and is rapidly downregulated soon after alveolarization. This suggests that PlGF in the lung is developmentally regulated and that persistently elevated PlGF may be detrimental to developed lungs as seen in our PlGF TG mice (Figure 8)



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  		Figure 8. Intercellular signaling between pneumocytes and endothelial cells in normal and PlGF TG mice. (A) In the physiologic state, pneumocytes secrete VEGF, which exerts a trophic effect on endothelial cells (EC) via Flk-1. Pneumocytes also secrete certain amounts of PlGF, which may enhance VEGF-dependent angiogenesis through intramolecular and intermolecular cross talk between Flk-1 and Flt-1. PlGF may have some unknown autocrine effects on pneumocytes through Flt-1. Signals from endothelial cells to pneumocytes have not yet been discovered. (B) Increased PlGF production from pneumocytes is detrimental to PlGF TG mice through activation of Flt-1 on the pneumocytes, resulting in a decreased number of existing pneumocytes. Alternatively, with less VEGF available to endothelial cells, their survival is jeopardized.

 
Recently, Autiero and coworkers reported that PlGF regulates both intermolecular and intramolecular cross talk between Flt1 and Flk1. They state that activation of Flt1 by PlGF results in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1 (10). This finding is contrary to the negative effect of overexpressed PlGF on endothelial cells observed in our TG mice. Another significant finding is that although VEGF and PlGF both bind Flt1, each induces a distinct pattern of tyrosine phosphorylation and gene expression, suggesting that each activates Flt1 in a unique manner (10). In addition, PlGF alone can trigger its own intracellular signals, independent of VEGF/Flk1 signaling. These PlGF-regulated genes have presumed roles in proliferation and apoptosis (10). Such a mechanism is also likely to exist in pneumocytes because they express Flt1 but not Flk1 as demonstrated by reverse transcription–PCR.

In conclusion, our PlGF TG mice revealed a novel noninflammatory pathway in pulmonary emphysema. Elevated PlGF promotes pulmonary epithelial cell death leading to decreased VEGF production, which in turn affects the endothelial cells. The compromised microcirculation further jeopardizes the survival of pneumocytes and culminates in emphysema. Our observation of pulmonary emphysema in PlGF-overexpressing mice may shed new light on the pathogenesis of human pulmonary emphysema—a major component of chronic obstructive pulmonary disease.


   Acknowledgments
 
The authors thank Dr. T. J. Ley for kindly providing the PGF expression cassette and Dr. J. Whitsett for kindly providing the MLE-15 cell lines. They also thank Dr. Joel Moss and Dr. Martha Vaughan of the Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, and also Dr. James Mulshine of the Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, M.D. for critical reading of the manuscript. In addition, they thank M. J. Wu and C. Y. Su for excellent technical assistance and C.-L. Chien of the National Taiwan University, Taipei, Taiwan for guidance on light microscopy.


   FOOTNOTES
 
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 equal contributions to this study.

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

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 interest; C-A.C. has no declared conflict of interest; W-F.C. has no declared conflict of interest; S-C.W. has no declared conflict of interest; C-J.Y. has no declared conflict of interest; F-J.H. has no declared conflict of interest; S-M.H. has no declared conflict of interest.

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


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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