胎盤生長因子與肺臟相關疾病的探討及臨床意義

The Role of Placenta Growth Factor in Pulmonary Diseases and Clinical Implication

曹伯年(Po-Nien Tsao)

指導教授 ： 謝豐舟

共同指導教授 ： 李鴻(Hung Li)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.01115

摘要

在經過常規使用產前類固醇，和利用外加肺臟表面張力素(surfactant)來治療早產兒呼吸窘迫症候群，以及新生兒照顧上的進步，極低體重早產兒的存活率已經能夠大大的提昇，但早產兒慢性肺疾病仍然是這些早產兒最常見的併發症之一。在出生週數低於等於34週的早產兒身上，早產兒慢性肺疾病的發生率約在7.5到20%之間。目前這種早產兒慢性肺疾病的病理變化，主要是極度不成熟的肺臟，併隨因肺臟發育遲滯，導致的肺泡及肺部微血管生長發育的停滯或不完全和一些不正常的修復及纖維化。到底是什麼樣的機轉，導致在早產兒慢性肺疾病的嬰兒身上抑制肺臟的發育，仍不十分清楚。近年來，有些研究報告顯示，破壞血管內皮生長因子的訊息，會影響肺血管的生長，而可能在早產兒慢性肺疾病的成因上拌演一個角色。
胎盤生長因子是血管內皮生長因子(血管內皮生長因子)家族的成員之一，是一個含132個胺基酸，大小是50KDa的一個雙分子醣蛋白。它存在於釵h正常的組織中，特別是在胎盤、甲狀腺和肺臟中表現量較多，它是血管新生和血球新生過程中很重要的一個介質。不過，胎盤生長因子在這些組織中所扮演的角色仍不清楚。
之前有人研究顯示，若血管內皮生長因子的表現量減少，會讓肺臟上皮及內皮細胞死亡。另外，他們也認為第二型血管內皮生長因子接受器，在肺氣腫的病理成因上扮演一個角色，而且和血管內皮生長因子相關的訊息，對維持肺臟肺泡的構造是很重要的。最近，Autiero等人報告，認為胎盤生長因子可透過第一型接受器和第二型接受器，分子間或分子內的交流來調整血管內皮生長因子的訊息傳導。不僅如此，單獨的胎盤生長因子 也能引發它自己獨特的訊息，而此訊息和血管內皮生長因子/Flk-1的訊息無關；同時，此訊息可能和第一型接受器依賴性的生物弁遄虳M細胞增生、凋零或血管新生有關。為了探討胎盤生長因子在肺臟發育過程中所扮演的角色，以及和肺臟相關疾病之間的關聯，我們進行了以下的研究。

第一部份，這個研究的目的，是想瞭解在小鼠肺泡發育過程中，即出生後頭二週內，當肺泡微血管大量增生時，血管內皮生長因子、胎盤生長因子及其接受器，這個和血管新生最相關的系統，其mRNA的表現情形為何。我們利用FVB這種strain的老鼠，在出生後第3、7、10、14天及成鼠時，使用深度麻醉去取出它們的肺臟，並去除掉氣管及主要支氣管來抽取RNA，每個時間點使用3-4隻的小鼠。另外，在每個時間點，同時也使用2隻小鼠在犧牲後，由氣管灌入10%的福馬林，維持25公分水柱的壓力5分鐘，使其肺泡擴張，之後再整個放入4OC福馬林中固定24小時，最後包埋在臘塊中，供切片染色使用。結果我們證實在肺泡發育過程中，肺微血管數目的確有明顯的增加。同時我們也利用半定量RT-PCR的方式發現在肺泡發育過程中，血管內皮生長因子/Flk-1是被往上調控，而胎盤生長因子/Flt-1是維持一個相對較穩定的濃度去促進肺部微血管的發育。但是在成鼠的肺臟，當大部份的血管網路都長完全後，胎盤生長因子是被往下調控而Flt-1是被往上調控去阻止血管新生。同時，血管內皮生長因子/Flk-1是維持在相對較高的濃度，去維持成熟肺微血管網路構造。
第二部份為了瞭解胎盤生長因子對肺臟的影響，我們利用會在所有器官上皆能表現之PGK啟動子來製造全身細胞都會過度表達胎盤生長因子的胎盤生長因子基因轉殖鼠。我們發現胎盤生長因子之持續過度的表現會從六個月大開始造成肺部肺泡明顯地擴大，同時肺的順從性也增加，十分類似人類肺氣腫之變化，但是並沒有伴隨明顯的發炎反應。這種情形在12個月大以後更明顯。我們並發現這些基因轉殖鼠的肺泡壁細胞有的肺泡壁細胞有發現細胞凋零的現象。利用細胞流式分析儀分析發現，這些死亡的細胞主要是第二型肺泡上皮細胞。同時，肺臟血管內皮生長因子及血管內皮細胞的標記PECAM-1也都明顯被往下調控，表示血管內皮細胞及它的存活因子血管內皮生長因子都減少。於in vitro之實驗中，外加之胎盤生長因子會促進小鼠肺臟第二型上皮細胞之死亡並抑制其生長。而在正常小鼠的肺臟中，而在正常小鼠的肺臟中於肺囊泡期時胎盤生長因子會大量表現，但一旦完成所謂肺泡化的過程後 胎盤生長因子就要立刻被調控下來。而在我們的 胎盤生長因子基因轉殖鼠的 胎盤生長因子是持續過度表現的，這可能是導致其肺氣腫發生的原因。更進一步，我們也證實了在人類長期吸煙導致肺氣腫之患者身上，血清中之胎盤生長因子濃度明顯地比長期吸煙但沒有肺氣種及正常健康對照組來得高，並且胎盤生長因子的濃度之升高與肺氣腫之嚴重度有顯著相關。我們的研究證實了胎盤生長因子在肺氣腫之病理過程中扮演了一個重要的角色。且其作用乃是透過調節第二型肺細胞之生長所造成。
第三部份是想瞭解是否臍帶血中胎盤生長因子濃度較高的早產兒，日後比較容易會發生早產兒慢性肺疾病。本研究共收集了六十三位出生週數在34週以下含34週的早產兒，其中有2位因為在出生28天內死亡，因無法界定是否發生早產兒慢性肺疾病，而被排除。胎盤生長因子的測定是利用quantikine ELISA kit (R&D systems)來進行。本研究採用Mann-Whitney ran sum 測驗，Spearman correlation coefficients 以及多因子線性和回歸性分析，來做統計上的分析。結果我們發現將來發生早產兒慢性肺疾病的那組病人，臍帶血中有較高的胎盤生長因子值，他們的懷孕週數較低、出生體重也較低、有較高比率使用氣管插管、同時他們使用呼吸器的天數也比較久。雖然臍帶血中胎盤生長因子的濃度和出生週數及體重成反比；但是，利用多因子迴歸分析來調整所有相關因子後，只有臍帶血中胎盤生長因子的濃度及出生體重這二項和日後會不會發生早產兒慢性肺疾病有關；而且，臍帶血中胎盤生長因子的濃度和他使用呼吸器的天數成正相關，代表和早產兒慢性肺疾病的嚴重性是呈正相關。利用ROC curve我們發現，若將臍帶血中胎盤生長因子 的濃度定在17mg/dl ，超過此標準去預測日後會發生早產兒慢性肺疾病的專一性高達95%，敏感性有53%，陽性預測值有83%，而陰性預測值也有82%。因此，我們下一個結論，即在出生時檢測臍帶血中胎盤生長因子的濃度，可當做預測日後是否會發生早產兒慢性肺疾病的生物指標；如此，可提供早期治療的一個依據。
經過我們這一系列的研究，我們已經發現胎盤生長因子除了像別人報告過的在如缺血性病變、癌症的病理性血管新生及與單核球和巨嗜細胞引起的發炎反應相關外，它過度表現時會對肺臟造成傷害，類似成人的肺氣腫及早產兒的慢性肺疾病。因此，除了胎盤生長因子基因轉殖鼠可以提供成為日後研究肺氣腫及早產兒的慢性肺疾病的一個動物模式外，還可以利用此動物模式來研究肺泡的再生與重建，這在肺臟的基礎研究及臨床應用上將有很大的幫助。最後，胎盤生長因子可能可以當做一種新的治療標的去預防或治療成人的肺氣腫及早產兒慢性肺疾病。

關鍵字

胎盤生長因子 ； 肺臟

並列摘要

1. Angiogenic factors in alveolarization and bronchopulmonary dysplasia

During alveolarization, the lung undergoes marked vascular growth, as reflected by a 20-fold increase in alveolar and capillary surface areas from birth to adulthood.(Zeltner et al., 1987) Recently, Jakkula et al. demonstrated that angiogenesis is necessary for normal alveolarization during a critical period of lung development in the rat.(Jakkula et al., 2000) Although it is known that the correct temporal and spatial development of alveolar capillaries is critical to lung development, little is known about the factors that regulate alveolar capillary formation.

Blood vessels are constructed by two processes: vasculogenesis, whereby a primitive vascular network is established during embryogenesis from multipotential mesenchymal progenitors; and angiogenesis, in which pre-existing blood vessels send out capillary sprouts to produce new vessels.(Hanahan, 1997) Vascular endothelial growth factor (VEGF), the first characterized vascular-specific growth factor, has been identified as the most important driver of vascular formation.(Yancopoulos et al., 2000) Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2) are the endothelium-specific tyrosine kinase receptors of VEGF.(Yancopoulos et al., 2000) Placenta growth factor (PlGF) belongs to the VEGF family of proteins. It binds to the VEGF-1 receptor but not to the VEGF-2 receptor; the latter is thought to mediate most of the angiogenic and proliferative effects of VEGF. PlGF mRNA is present in most normal tissues, especially in the placenta, thyroid, and lungs.(DiPalma et al., 1996) Recently, Carmeliet et al. demonstrated synergy between VEGF and PlGF in angiogenesis and plasma extravasations in pathological conditions.(Carmeliet et al., 2001) In addition, two other angiogenic factors, angiopoietin 1 and 2 (Ang-1 and Ang-2), have been found to regulate the maturation of new vessels from proliferating endothelial cells. Tie-1 and Tie-2 comprise another family of endothelium-specific receptor tyrosine kinases; Ang-1 and Ang-2 are the specific ligands for Tie-2.(Maisonpierre et al., 1997; Suri et al., 1996)

Bhatt et al. demonstrated in 2000 that VEGF mRNA increases parallel to Flk-1 mRNA abundance during postnatal mouse lung development suggestive of a role for the VEGF/Flk-1 transduction system in lung microvascular development.(Bhatt et al., 2000) Furthermore, using a premature baboon model of bronchopulmonary dysplasia (BPD), it was demonstrated that expression of VEGF, Flt-1, Flk-1, Ang-1 and Tie-2 increases during gestation, but in BPD cases, only VEGF and Flt-1 significantly decreased.(Maniscalco et al., 2002) Similar findings were found in studies of human infants dying of BPD.(Bhatt et al., 2001) However, the roles of Ang-2 and PlGF during alveolarization are still unknown. Although the fetal baboon model of BPD mimics the human condition, it is problematic due to its high cost and long experimental duration. The most popular alternative BPD models are postnatal mice and rats undergoing hyperoxia or ventilation. Murine alveolar development begins on postnatal day 3 (P3), and sacular division is completed by the postnatal day 14 (P14).(Amy et al., 1977) This sequence and timing of alveolarization resembles human lung development. In addition, transgenic mice have become an increasingly popular model for investigating the role of specific factors in lung injury and prevention. Therefore, understanding the roles of these angiogenic factors and their receptors during postnatal lung development in mice is crucial to developing insights on human disease.
2. Angiogenic factors and their receptors in postnatal mouse developing lung

The aim of this study was to determine VEGF-VEGFR and Ang-Tie mRNA expression patterns in mouse lungs during the first two weeks after birth, when alveolarization occurs and pulmonary microvasculature increases. Using RT-PCR, we analyzed changes in VEGF, PlGF, Flk-1, Flt-1, Ang-1, Ang-2, and Tie-2 mRNA in infant mouse lungs at postnatal days 3, 7, 10, 14 and in adult mouse lungs in order to gain information about the VEGF-VEGFR and Ang-Tie systems regulate alveolar capillary formation.

2.1 The number of pulmonary capillaries markedly increases during alveolarization.

Murine alveolar development begins on postnatal day 3 (P3), and alveolarization is completed by the postnatal day 14 (P14).(Amy et al., 1977) The development of the postnatal lung is shown in Figure 1. At postnatal day 3 (P3) and day 7 (P7), the mouse lungs are in the sacular stage of lung development. The saccules are uncomplicated in appearance with thick walls. Some secondary crests (bulges) appear in the alveoli at P7 but not at P3; pulmonary capillaries are not abundant before 7 days old. At postnatal day 14 (P14), the number of pulmonary microsaccules has markedly increased, with numerous thin-walled alveoli (Fig. 1a-c). Using light microscopy, the alveolar structures at P14 are seen to be very similar to those of adult mice (AD) (Fig. 1 d). We analyzed the expression of a specific endothelial cell marker, PECAM-1 (CD31) by semiquantitative RT-PCR. We found that PECAM-1 mRNA were increased substantially to relatively high levels in the adult lung (Fig. 2).

2.2 The angiopoietin-Tie system is up regulated during alveolarization.

The expression patterns of Ang-1, Ang-2 and Tie-2 mRNA during postnatal mouse lung development were assessed by semi-quantitative RT-PCR. Using photoimaging analysis, we compared the above mRNA abundance (relative to GAPDH) in developing lung. The ratio of Ang-1, Ang-2 or Tie-2 to GAPDH at postnatal age of three days (P3) was assigned as value of 1. Before alveolarization (P3), levels of Ang-1, Ang-2 and Tie-2 mRNA were relatively low. During the first two weeks of postnatal development, when alveolarization occurred and pulmonary microvasculature increased, Ang-1 mRNA abundance increased 2.5-fold, Ang-2 2.2-fold and Tie-2 1.9-fold as compared with the levels at P3. These messages increased substantially to relatively high levels in the adult lung (Fig. 3).

2.3 The VEGF family and VEGFR system

Expression of VEGF isoforms
The pattern of VEGF isoform expression is shown in Fig. 4a. The relative amount of total VEGF mRNA increased substantially during postnatal mouse lung development, and reached a 1.6-fold increase in the adult lung as compared with the levels at P3 (Fig. 5a). The relative levels of the three VEGF isoform mRNAs did not change significantly during alveolarization: about 50% of the total VEGF message in VEGF 188, 30% in VEGF 164, and 20% in VEGF 120 (Fig. 5b).

PlGF is down-regulated during postnatal lung development
In contrast to angiopoietin and VEGF, PlGF mRNA expression was maintained at relatively high levels during alveolarization and decreased to 0.2-fold in the adult lung as compared with the levels at P3 (Fig. 4a & 5a).

Different expression patterns of Flk-1 and Flt-1
Similar to the Tie-2 mRNA expression profile, the amount of Flk-1 mRNA increased gradually during alveolarization and reached a 2.6-fold increase in the adult lung as compared with the levels at P3 (Fig. 4b & 5b). However, the expression pattern of Flt-1, another VEGF receptor, differed from that of Flk-1; the amount of Flt-1 mRNA was relatively high before alveolarization (P3) and slightly down regulated during alveolarization. When alveolarization was complete, Flt-1 mRNA levels were to the same as they were at the onset of alveolarization; levels increased to 2.0-fold in the adult lung as compared with the levels at P3 (Fig. 4b & 5b).

The formation of the microvascular network in mouse lungs during the postnatal period is crucial for alveolarization. During that time, lung development is characterized by endothelial cell proliferation and capillary remodeling.(Burri, 1974) The Ang/Tie-2 system plays a critical role in blood vessel formation. Tie (a tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) is predominantly expressed by vascular endothelial cells;(Schnurch and Risau, 1993) Ang-1 and Ang-2 are its ligands.(Yancopoulos et al., 2000) Ang-1 specifically binds the Tie-2 receptor, inducing Tie-2 tyrosine phosphorylation and transducing survival and sprouting signals.(Suri et al., 1996) Ang-2 also binds Tie-2; however, it does not induce phosphorylation of Tie-2, instead blocking Ang-1-mediated autophosphorylation of Tie-2.(Maisonpierre et al., 1997) Both Tie-2 and Ang-1 knockout mice die between 9.5-10.5 days after conception.(Dumont et al., 1994; Suri et al., 1996) Such embryos exhibit extensive malformation of the vascular network. This phenotype suggests a role for the Ang-1/Tie-2 signal in vascular network formation. Overexpression of Ang-2 has a similar phenotype to that of Ang-1 and Tie-2 knockout mice, indicating that Ang-2 is a natural antagonist for the Ang-1/Tie-2 pathway.(Maisonpierre et al., 1997)

In this study, we demonstrated that Ang-1 and Tie-2 mRNA expression increases in parallel during postnatal mouse lung development. Furthermore, the adult lung, which has a mature vascular network, retained relatively high levels of Ang-1 and Tie-2 mRNA expression. Together, these results suggest that Ang-1/Tie-2 signaling may play a role in alveolarization. Maniscalco et al. report similar findings.(Maniscalco et al., 2002) Recently, Jones et al. demonstrated that loss of Tie-2 induced rapid endothelial cell apoptosis in vivo and suggested that Tie-2 signals may function to maintain vasculature.(Jones et al., 2001) Our data support this hypothesis. As a survival factor, the Ang-1/Tie-2 signal may function to maintain the large pulmonary vasculature in the adult lung; this may explain the higher abundance of Ang-1 and Tie-2 mRNA observed in the adult lung.

An unexpected observation was that the abundance of Ang-2, the natural antagonist for Ang-1, increased in parallel to Ang-1 during postnatal mouse lung development. In a previous study, activation of Tie-2 by Ang-1 was antagonized by Ang-2 in endothelial cells.(Maisonpierre et al., 1997) However, recent work indicates that Ang-2 has an angiogenic activity in adult tissues and endothelial cells.(Kim et al., 2000; Mochizuki et al., 2002) Kim et al. found that high concentrations of Ang-2 can enhance endothelial cell survival.(Kim et al., 2000) Furthermore, Mochizuki et al. demonstrated that Ang-2 can induce Tie-2 tyrosine phosphorylation and lead to migration of and tube formation by murine capillary endothelial cells.(Mochizuki et al., 2002) This could indicate that Ang-2 also may play a role in alveolarization, which involves active angiogenesis and that it retains a high level of expression in the adult lung for the purpose of maintaining mature pulmonary microvasculature. Alternatively, it is also possible that Ang-2/Ang-1 may be kept at a constant level to maintain the lungs’ vascular network.

VEGF has been shown to play a pivotal role in the normal development of blood vessels during embryogenesis.(Ferrara et al., 1996) Our data show that VEGF and Flk-1 messages increase in parallel during postnatal mouse lung development. These data suggest that the VEGF/Flk-1 signal may play a role in regulating lung capillary development. In addition, the relatively high levels of VEGF and Flk-1 mRNA in adult lungs imply a role for pulmonary VEGF in endothelial cell maintenance and/or capillary permeability, a conclusion also supported by the work of Bhatt et al.(Bhatt et al., 2001; Maniscalco et al., 2002)

Variable mRNA splicing of the single mouse VEGF gene gives rise to at least three isoforms, VEGF 188, VEGF 164, VEGF 120.(Shima et al., 1996) In this study, we found that VEGF 188 was the predominant form of VEGF during mouse lung development with its relative level increasing slightly in the adult lung. This may indicate that VEGF 188, in contrast to VEGF 164 and VEGF 120, plays a more important role in the pulmonary microvascular development; Ng et al. had similar findings.(Ng et al., 2001)

PlGF belongs to the VEGF family. It binds to the Flt-1 VEGF receptor but not the KDR/Flk-1 receptor. PlGF mRNA is present in most normal tissues, especially in the placenta, thyroid and lung.(DiPalma et al., 1996) Carmeliet et al. reported that by up-regulating PlGF and the signaling subtype of Flt-1, endothelial cells amplify their responsiveness to VEGF during an angiogenic switch in many pathological disorders.(Carmeliet et al., 2001) However, the nature of PlGF regulation during postnatal mouse lung development is still unclear. Here, we have demonstrated that the abundance of PlGF mRNA is relatively high during alveolarization, but PlGF is down regulated in the adult lung, where little endothelial cell proliferation occurs. This may imply that PlGF also plays a synergistic role of VEGF in pulmonary microvascular development and is down regulated once active angiogenesis is complete.

Flt-1 may be involved in capillary formation. Flt-1 knockout mice die at mid-gestation, displaying vascular overgrowth and disorganization in autopsies,(Fong et al., 1995) however Hiratsuka et al. found that mice lacking the Flt-1 tyrosine kinase domain developed almost normal blood vessels.(Hiratsuka et al., 1998) These results indicated that the major role of Flt-1 is to negatively regulate the levels of endogenous VEGF by absorbing the ligand in the extracellular domain.(Shibuya, 2001) Although Bhatt et al. demonstrated that mRNA levels for Flt-1 and Tie-2 decreased in infants dying of BPD, who have abnormal alveolar microvessels, little was known about the role of Flt-1 in normal postnatal lung development. In this study, we found that expression of Flt-1 mRNA is slightly down regulated during alveolarization, but that of Flt-1 mRNA increased markedly in the adult lung. One explanation is that the Flt-1 signal negatively regulates developmental blood vessel formation,(Fong et al., 1995) therefore, it is down-regulated during pulmonary microvascular development and up-regulated when this process is finished. However, Maniscalco et al. have reported that Flt-1 mRNA peaks in late gestation and declines postnatally during baboon lung development.(Maniscalco et al., 2002) It is unclear whether these results are due to species differences or differences in methodology.

In summary, we demonstrate that, during alveolarization, VEGF/Flk-1, Ang-1, Ang-2, and Tie-2 are up regulated and PlGF/Flt-1 is kept at relatively constant level to promote pulmonary microvascular development. In the adult lung, when most of the vascular network is complete, PlGF is down regulated and Flt-1 is up-regulated to stop angiogenesis and VEGF/Flk-1, Ang-1, Ang-2 and Tie-2 are kept at relatively high levels to maintain mature pulmonary microvasculature. In our knowledge, this is the first study to demonstrate the expression of angiopoietin/Tie-2 system and vascular endothelial growth factor, PlGF, Flk-1 and Flt-1 during postnatal mouse lung development. A more comprehensive view of the roles of these angiogenic factors in pulmonary vascular development is currently underway by studies with transgenic mice.

3. Overexpression of placenta growth factor contributes to the pathogenesis of pulmonary emphysema
Vascular endothelial growth factor (VEGF), the vascular-specific growth factor first characterized, has been termed the most critical driver of vascular formation (Folkman and D'Amore, 1996). It binds to Flt-1 and 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 (Cao et al., 1996).
Exogenous PlGF stimulates angiogenesis and induces vascular permeability when co-injected with VEGF (Monsky et al., 1999; Park et al., 1994). 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 (Carmeliet et al., 2001; Park et al., 1994). 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 (Carmeliet et al., 2001).
In most normal tissues PlGF mRNA is present, most abundantly in the placenta, thyroid, and lungs (DiPalma et al., 1996), 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 (Kasahara et al., 2001; Kasahara et al., 2000; Santos et al., 2003). Recently, Autiero et al. 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 (Autiero et al., 2003b). Therefore, we hypothesize that PlGF may play some role in the pathogenesis of emphysema. Since no phenotype was obvious in the PlGF knockout mice (Carmeliet et al., 2001), we generated transgenic (TG) mice, which constitutively overexpressed PlGF. In these mice, pulmonary emphysema, mimicking human chronic obstructive pulmonary disease (COPD) 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 (Tsao et al., 2003a).

Pulmonary emphysema in PlGF transgenic mice

We generated PlGF TG mice using a construct containing mouse PlGF cDNA driven by a PGK promoter (Fig. 6a), which reslulted in constitutive over-expression of PlGF. This was confirmed by reverse transcription-PCR (RT-PCR) and Western blot (Fig. 6b & 7). Both the TG and WT mice were sacrificed at the age of 1 week, 2 weeks, 1 months, 2 months, 4 months, 6 months and 12 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 (Fig. 8); 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 RACs in the lungs of the PlGF TG mice were significantly lower than in the WT mice at 12 months old (n = 5 in each group) (Fig. 9). The decrease in RAC and the degree of emphysematous change were more prominent in the TG mouse line T22, whose PlGF level was higher than line T29. Furthermore, the volume density of airspaces (VV(air, lung)) was significantly higher in the PlGF TG mice than in the WT mice (79.62

並列關鍵字

lung ； Placenta growth factor

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