Inhibition of angiogenesis decreases alveolarization in the developing rat lung

Malathi Jakkula1, Timothy D. Le Cras1, Sarah Gebb1, K. Peter Hirth2, Rubin M. Tuder3, Norbert F. Voelkel4, and Steven H. Abman1

1 Pediatric Pulmonary Medicine, Pediatric Heart Lung Center, Department of Pediatrics, 3 Department of Pathology, and 4 Pulmonary Hypertension Center, University of Colorado School of Medicine, Denver, Colorado 80218; and 2 Sugen Incorporated, South San Francisco, California 94080


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To determine whether angiogenesis is necessary for normal alveolarization, we studied the effects of two antiangiogenic agents, thalidomide and fumagillin, on alveolarization during a critical period of lung growth in infant rats. Newborn rats were treated with daily injections of fumagillin, thalidomide, or vehicle during the first 2 wk of life. Compared with control treatment, fumagillin and thalidomide treatment reduced lung weight-to-body weight ratio and pulmonary arterial density by 20 and 36%, respectively, and reduced alveolarization by 22%. Because these drugs potentially have nonspecific effects on lung growth, we also studied the effects of Su-5416, an inhibitor of the vascular endothelial growth factor receptor known as kinase insert domain-containing receptor/fetal liver kinase (KDR/flk)-1. As observed with the other antiangiogenic agents, Su-5416 treatment decreased alveolarization and arterial density. We conclude that treatment with three different antiangiogenic agents attenuated lung vascular growth and reduced alveolarization in the infant rat. We speculate that angiogenesis is necessary for alveolarization during normal lung development and that injury to the developing pulmonary circulation during a critical period of lung growth can contribute to lung hypoplasia.

bronchopulmonary dysplasia; congenital diaphragmatic hernia; fumagillin; lung growth; lung hypoplasia; pulmonary circulation; pulmonary hypertension; thalidomide; vascular endothelial growth factor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NORMAL DEVELOPMENT of the human lung can be divided into five stages: embryonic (3-7 wk gestation), pseudoglandular (5-17 wk), canalicular (16-26 wk), saccular (24-38 wk), and alveolar (up to 2-3 yr of age) (7, 13, 16, 24, 29, 41, 42). Although these periods of lung development are similar across mammalian species, the relative timing and length of each stage varies between species. In the rat, alveolarization begins during late gestation but primarily occurs during the first 2 wk after birth (29). Alveoli are formed by the septation of large saccules that constitute the gas-exchange region of the immature lung during this critical period of lung growth. Secondary septae form as ridges that grow into distal air spaces, thereby increasing lung surface area and enhancing the capacity for gas exchange (7). Mechanisms that regulate alveolarization are poorly understood, but multiple stimuli modulate distal lung growth, including genetic factors, oxygen tension, nutrition, hormones, and autocrine and paracrine growth factors (5-7, 25, 29-31, 41, 42).

During the period of alveolarization, the lung also undergoes marked vascular growth as reflected by the 20-fold increase in alveolar and capillary surface areas from birth to adulthood (49). Lung vascular growth involves two basic processes: vasculogenesis, the formation of new blood vessels from endothelial cells within the mesenchyme, and angiogenesis, the formation of new blood vessels from sprouts of preexisting vessels (7, 14, 40, 41). Mechanisms that increase vascular surface area during late gestation and the early postnatal period are poorly understood, but it is clear that coordination of distal air space and vascular growth is essential for normal lung development. For example, the production and timing of the release from respiratory epithelial cells of angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor are likely to play key roles in normal lung vascular growth (2-4, 17, 33, 36, 38, 41, 42, 48, 51).

Experimental studies (5, 7, 29-31, 39) have shown that adverse stimuli such as hypoxia, hyperoxia, or glucocorticoid treatment during the critical period of postnatal lung growth in the rat can disrupt alveolarization and cause lung hypoplasia. These models of lung hypoplasia are also associated with abnormalities of the pulmonary circulation (25, 26, 39, 44a). In the clinical setting, lung hypoplasia is frequently associated with abnormalities of the pulmonary circulation in diverse diseases, including bronchopulmonary dysplasia (BPD), congenital diaphragmatic hernia, primary lung hypoplasia, and Down's syndrome (1, 8, 11, 28, 45, 47). It is generally presumed that disruption of alveolarization is likely to cause failure of lung vascular growth; whether injury to the developing pulmonary circulation or disruption of normal angiogenesis can also contribute to impaired alveolarization is unknown.

Antiangiogenic therapy with agents such as thalidomide and fumagillin has been studied in diverse experimental settings in order to examine basic mechanisms of angiogenesis and their potential role in pathological settings such as tumor angiogenesis (17, 50). Because fumagillin and thalidomide block angiogenesis by inhibiting endothelial cell proliferation (12, 17, 21, 22, 34, 37), which is critical for angiogenesis, these drugs provide useful pharmacological tools for studying the physiological roles of angiogenesis in different experimental settings. Therefore, to study the role of angiogenesis in postnatal lung growth, we proposed the hypothesis that disruption of angiogenesis during a critical period of lung growth would decrease alveolarization. To test this hypothesis, we studied the effects of fumagillin and thalidomide treatment during a critical period of postnatal lung growth in neonatal rats and report that these agents decrease lung weight, pulmonary arterial density, and alveolarization. Because these agents may have nonspecific effects on lung growth independent of their effects on endothelial cell proliferation, we further tested this hypothesis by treating neonatal rats with an antiangiogenic agent, Su-5416, an inhibitor of the VEGF receptor kinase insert domain-containing/fetal liver kinase (KDR/flk)-1 (18, 43). As observed with fumagillin and thalidomide, we report that Su-5416 treatment also decreased alveolarization and arterial density at 2 wk of age. These findings suggest that angiogenesis is necessary for normal alveolarization during a critical period of lung development in the rat.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Study Animals

All procedures and protocols used in this study were approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center. Timed-pregnant, pathogen-free Sprague-Dawley rats were purchased from Harlan Laboratories (Indianapolis, IN). The pregnant rats were maintained at Denver's altitude for at least 1 wk before giving birth. Animals were fed ad libitum and exposed to 12:12-h light-dark cycles.

Experimental Design

Daily injections of fumagillin, thalidomide, or vehicle (DMSO) were started 1 day after birth and continued until 13 days of age. Study group assignment was randomized for each litter. All drugs were freshly prepared on the day of study and stored at -20°C for the remainder of the study period. Fumagillin and thalidomide (Biomol Technologies, Plymouth Meeting, PA) were dissolved in DMSO before treatment. In the fumagillin group (n = 22 animals), fumagillin was administered by subcutaneous injection at a dose of 2 mg/kg body wt. Thalidomide (n = 14 animals) was administered by intraperitoneal injection at a dose of 10 mg/kg. An equivalent volume of 100% DMSO (Sigma, St. Louis, MO) was administered to the vehicle control study group (n = 18 animals) by intraperitoneal injection. Doses and routes of administration were based on published studies (12, 21, 22) on tumor angiogenesis and preliminary data from our laboratory. Study rats were killed on day 14 by intraperitoneal injection of pentobarbital sodium (0.3 mg/g body weight). Body, lung, and cardiac weights were measured, and the lungs were prepared for histology and morphometric analysis. At death, the hearts were removed and dissected to isolate the free wall of the right ventricle from the left ventricle and septum. The ratio of right ventricle weight to left ventricle plus septum weight (RV/LV+S) was used as an index of right ventricular hypertrophy.

The same protocol was used to study the effects of Su-5416 (Sugen, South San Francisco, CA), a selective inhibitor of the VEGF receptor KDR/flk-1 (18, 43). Newborn rats (n = 30 animals) from each litter were randomly assigned to treatment with Su-5416 (20 mg/kg) or vehicle control (carboxymethylcellulose), administered by subcutaneous injections on alternate days, beginning on day 1 and continuing through day 13. The dose of Su-5416 was based on a previous study (18) of tumor antiangiogenesis in mice. Study animals were killed, and the lungs were studied as outlined above.

Arterial Density Measurements After Barium Sulfate-Gelatin Infusions

Pulmonary arterial density was measured from lungs that were infused with barium sulfate according to previously established methods (13, 14). Immediately after death, PBS was infused through a main pulmonary artery catheter to flush the pulmonary circulation free of blood. A barium sulfate-gelatin mixture was heated to 70°C and infused into the main pulmonary artery at 74 mmHg. Pressure was maintained for at least 5 min to ensure penetration of the barium mixture. Lung tissue was subsequently fixed for histology as described in Lung Histology and Radial Alveolar Counts or used for arteriograms. Pulmonary arterial density was measured by counting (by two blinded observers) barium-filled arteries per high-power field (×100 magnification) in 8-10 randomly selected fields taken from at least two blocks of tissue.

Lung Histology and Radial Alveolar Counts

Lungs were fixed for histology by tracheal instillation of 10% buffered Formalin under constant pressure (20 cmH2O). The tracheae were ligated after sustained inflation, and the lungs were excised and immersed in Formalin overnight. Formalin-fixed lung tissue was cut into 4- to 5-mm-thick sections, placed in 10% buffered Formalin, and embedded in paraffin. Paraffin sections (5 µm thick) were serially mounted onto Superfrost Plus slides (Fisher Scientific, Fair Lawn, NJ) and stained with hematoxylin and eosin. At least three lung sections from each animal were assessed for morphometric analysis. Alveolarization was measured by the radial alveolar counts (RAC) methods of Cooney and Thurlbeck (9-11) and Emory and Mithal (16). Briefly, radial counts were performed by determining the number of septae that intersected a perpendicular line drawn from the center of a respiratory bronchiole to the distal acinus (connective tissue septum or pleura). At least 10 counts were performed on each lung section.

Statistical Analysis

Data are presented as means ± SE. Statistical analysis was performed with the Statview software package (Abacus Concepts, Berkeley, CA). Statistical comparisons were made with the use of ANOVA and Fisher's protected least significant difference test. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Thalidomide and Fumagillin Treatment

Lung, body, and cardiac weights. Body weights were not reduced by fumagillin or thalidomide treatment compared with weights of control rats (25.3 ± 2.0 g with fumagillin; 30.7 ± 0.6 g with thalidomide; 26.3 ± 1.8 g with vehicle). Lung weights were 0.44 ± 0.02 g for the fumagillin-treated, 0.41 ± 0.03 g for the thalidomide-treated, and 0.55 ± 0.06 g for control rats. Compared with the control group, lung-to-body weight ratios were reduced by 30 and 38% in the fumagillin and thalidomide treatment groups, respectively (0.017 ± 0.001 with fumagillin; 0.015 ± 0.001 with thalidomide; 0.023 ± 0.002 with vehicle control; P < 0.05 for control vs. other groups; Fig. 1). Right ventricular mass or hypertrophy as determined by the RV/LV+S was not different between the drug treatment and vehicle control groups (0.39 ± 0.01 with fumagillin; 0.38 ± 0.02 with thalidomide; 0.38 ± 0.02 with vehicle).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of fumagillin and thalidomide treatment on lung-to-body weight ratios in 14-day-old Sprague-Dawley rats. Compared with control rats, the ratio of lung-to-body weight was reduced in the treated rats. * P < 0.05 for treated vs. nontreated groups.

Pulmonary arterial density after barium sulfate-gelatin infusion. To determine whether treatment with antiangiogenic agents during a critical period of lung development could decrease arterial density, we infused the main pulmonary artery with a barium sulfate-gelatin suspension. Arteriograms from the left lungs of thalidomide- and fumagillin-treated rats showed a decrease in the filling of small pulmonary vessels (a decrease in background haze) compared with arteriograms of control animals (Fig. 2). The major conduit pulmonary arteries and their branches also appeared narrow compared with those in control rats, and lung histology revealed a reduction in the number of barium-filled arteries in the fumagillin- and thalidomide-treated rats (Fig. 3A). Pulmonary arterial density, as determined by barium-filled arteries per high-power field, was reduced by 20 and 36% in the fumagillin- and thalidomide-treated rats, respectively, compared with that in control animals (32.0 ± 0.2 with fumagillin; 29.0 ± 0.8 with thalidomide; and 44 ± 0.3 with vehicle; P < 0.05 vs. control group; Fig. 3B).


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 2.   Barium arteriograms of the left lungs of fumagillin- and thalidomide-treated 14-day-old rats. Lumen diameters of large pulmonary arteries and barium filling of small pulmonary arteries ("background haze") were reduced in treated (middle and right) compared with vehicle control rats (left).




View larger version (82K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of fumagillin and thalidomide treatment on the density of small pulmonary arteries in 14-day-old rats. A: barium-filled arteries appear sparse after antiangiogenic treatment of the developing rat (magnification, ×100). B: vessel density counts were reduced after antiangiogenic treatment. Counts were performed per high-power field (HPF; original magnification, ×100). * P < 0.05 for treated vs. control rats.

Lung histology and RAC. Lung histology revealed striking differences in lung structure between the fumagillin and thalidomide groups and the control group. Compared with vehicle control rat lungs, fumagillin and thalidomide treatment caused a histological pattern of alveolar simplification characterized by the presence of larger and fewer distal air spaces (Fig. 4A). To quantify the apparent decreases in alveolar number, we measured RAC in the fumagillin-, thalidomide-, and vehicle-treated groups. Compared with lungs from control rats, RAC were reduced by 22% in both the fumagillin- and thalidomide-treated rats (Fig. 4B).



View larger version (67K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of fumagillin and thalidomide treatment on lung histology and alveolarization in 14-day-old rats. A: in contrast with the distal lung of the vehicle control rats, lung histology demonstrates a pattern of alveolar simplification in both of the treatment groups (magnification, ×200). B: compared with that in control animals, alveolar number as determined by measurements of radial alveolar counts (RAC) was reduced after antiangiogenic treatment. * P < 0.05 for treated vs. control rats.

Effects of Su-5416 Treatment

Compared with vehicle-treated control group, treatment of neonatal rats with Su-5416, a specific inhibitor of the VEGF receptor KDR/flk-1, reduced both lung and body weight (see Table 1). Lung weight was decreased in the Su-5416-treated rats versus control animals (0.49 ± 0.02 vs. 0.33 ± 0.01 g; P < 0.05) as was body weight (33.74 ± 1.36 vs. 21.42 ± 0.77 g; P < 0.05). As demonstrated by light microscopy, Su-5416 treatment altered lung architecture, causing a histological pattern of enlarged distal air spaces ("alveolar simplification") with decreased arterial density (Fig. 5). Su-5416 treatment reduced RAC by 30% (9.8 ± 0.8 in control lungs vs. 6.8 ± 0.7 in treated lungs; P < 0.02; Fig. 6). Barium arteriograms from the left lungs of Su-5416-treated rats showed a decrease in the filling of small pulmonary vessels (Fig. 7). The major conduit pulmonary arteries and their branches were more narrowed compared with those in control animals, and pulmonary arterial density was reduced by Su-5416 treatment (Fig. 8). At the doses used in this study, Su-5416 had a more striking effect on the reduction of arterial density than fumagillin or thalidomide.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effects of Su-5416 on lung, cardiac, and body weights



View larger version (59K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of Su-5416 treatment on lung histology in 14-day-old rats. Compared with lungs from vehicle-treated control rats, Su-5416 treatment decreased alveolarization and vessel density.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of Su-5416 treatment on alveolarization in the developing rat lung as assessed by RAC. RAC were markedly reduced after Su-5416 treatment.



View larger version (92K):
[in this window]
[in a new window]
 
Fig. 7.   Barium arteriograms of the left lungs of Su-5416-treated 14-day-old rats. Compared with lungs from vehicle-treated control rats, the lumen diameters of large pulmonary arteries and barium filling of small pulmonary arteries ("background haze") were reduced in Su-5416-treated rats.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 8.   Effects of Su-5416 treatment on arterial density measured as vessels/HPF in the 14-day-old rat lung.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Extensive vascular growth accompanies the increase in alveolarization during the critical period of postnatal lung development in young rats, but mechanisms that coordinate alveolarization with growth of the pulmonary circulation are uncertain. We hypothesized that if vascular growth were necessary for the increase in alveolarization during this time period, then disruption of angiogenesis should cause lung hypoplasia. To test this hypothesis, we treated neonatal rats during the critical period of lung growth with three different antiangiogenic agents: thalidomide, fumagillin, and Su-5416. We report that treatment with thalidomide or fumagillin caused a histological pattern of alveolar simplification, reduced RAC and pulmonary arterial density, and decreased lung-to-body weight ratios. These findings support the hypothesis that angiogenesis may be necessary for alveolarization during normal lung development and that adverse stimuli that disrupt vascular growth may contribute to lung hypoplasia. Because thalidomide and fumagillin may have additional effects on lung growth that are independent of their well-established antiangiogenic activities and to determine the potential role of the VEGF receptor KDR/flk-1 during this critical period of postnatal lung growth, we performed a similar protocol with a KDR/flk-1 inhibitor, Su-5416. As observed with the other antiangiogenic agents, Su-5416 treatment reduced alveolarization as determined by RAC and decreased arterial density. These findings suggest that the KDR/flk-1 receptor contributes significantly to vascular growth in the postnatal rat lung and provide additional support for the hypothesis that angiogenesis is necessary for normal alveolarization during the postnatal period of rapid lung growth.

Previous studies (7, 29, 30) have demonstrated that alveoli are formed by the septation of large saccules that constitute the gas-exchange region of the immature lung. Secondary septae are formed by alternate upfolding of one of the two capillary layers on either side of the primary septum (7). The double-capillary network persists until later in maturation when focal fusions lead to the development of a single-capillary layer and thinning of the interstitium (7). This mechanism implies that the failure of capillary growth and maturation or disruption of the upfolding of the double-capillary network could limit alveolarization. Thus we speculate that failure of growth or maturation of the pulmonary circulation during the critical period of alveolarization could potentially decrease septation and cause lung hypoplasia. However, few studies have directly addressed the effects of disrupted angiogenesis on lung development during the neonatal period. Because alveolarization primarily occurs during late gestation and the early postnatal period (30), it is likely that disruption of alveolarization by abnormal intrauterine stimuli, premature birth, postnatal injury, or other mechanisms contributes to this problem. Past studies (5, 6, 29-31, 39) have shown that exposure to dexamethasone, hyperoxia, or hypoxia decreases alveolar number in neonatal rats. Premature infants with BPD and children with lung hypoplasia have distal air spaces that fail to septate, resulting in fewer alveoli and a reduced surface area for gas exchange (28, 32, 47). In BPD, lung injury occurs in premature newborns before the alveolar period of lung development. Several morphometric studies of older infants and children dying with BPD have demonstrated impaired alveolarization and abnormal lung vascular development (28, 47). Because hyperoxia, ventilator-induced stretch injury, inflammation, and hypoxia can directly injure the pulmonary circulation (15, 31, 39), we speculate that disruption of vascular growth early in development may contribute to the subsequent failure of alveolarization during infancy and childhood.

Possible limitations of this study include the potential for nonspecific effects of thalidomide and fumagillin on the developing lung. Fumagillin, an antibiotic secreted by Aspergillus fumigatus, and thalidomide, an immunomodulatory drug with sedative and teratogenic effects, have been shown to inhibit angiogenesis (12, 17, 21, 22, 34, 37). Thalidomide, known as a teratogen for its disruption of fetal limb formation (phocomelia), has been reintroduced as a therapeutic agent for use in macular degeneration, leprosy, Crohn's disease, Behcet's disease, arthritis, acquired immunodeficiency syndrome (AIDS), graft versus host lung disease after bone marrow transplantation, and metastatic cancer (17). Although thalidomide may inhibit the effects of tumor necrosis factor-alpha , recent studies (12, 22) have also shown that it can inhibit VEGF- and basic fibroblast growth factor-induced neovascularization in a mouse corneal model. In addition to thalidomide, fumagillin and its synthetic analog TNP-470 are also antiangiogenic, and effects on vascular growth have been extensively studied in diverse experimental settings. Fumagillin may act by binding endothelial methionine aminopeptidase II (21). Whether both of these drugs can also reduce alveolarization by direct inhibition of epithelial growth, elastin production in developing septae, or related nonvascular mechanisms is unknown.

In contrast, Su-5416 is a potent antagonist of the KDR/flk-1 receptor, has potent inhibitory effects on tumor angiogenesis, and is currently undergoing clinical trials in patients with cancer (18, 43). When used in similar treatment protocols as the other antiangiogenic agents, Su-5416 treatment also inhibits alveolarization. The exact mechanism by which inhibition of endothelial cell proliferation leads to decreased alveolar septation is uncertain; we speculate that these effects may be due to a trophic effect on vascular endothelium, altered production of endothelium-derived products such as nitric oxide or platelet-derived growth factor (PDGF), or other mechanisms (19, 23, 33, 35, 38, 46, 51). In addition to its effects on the KDR/flk-1 receptor, Su-5416 can also block the platelet-derived growth factor receptor at higher concentrations (IC50: 1.5 µm for KDR; 20 µM for PDGF receptor) (44). Whether part of the response to Su-5416 in this study is also in part due to inhibition of PDGF receptors is uncertain. However, a recent study (20) demonstrated that VEGF inhibition with the use of an inducible Cre-loxP-mediated gene-targeting approach or by treatment with a soluble VEGF receptor protein caused growth failure and high mortality in neonatal mice. In addition to reduced body and organ weights, VEGF inhibition also decreased lung weight and altered lung histology. A further study (20) revealed a marked alteration in endothelial cell appearance as viewed by electron microscopy and increased apoptosis, suggesting that VEGF is necessary for endothelial survival as well as for proliferation.

Recently, our laboratory has observed abnormal lung development in the Fawn-Hooded rat (FHR), a genetic strain that develops severe pulmonary hypertension with modest decreases in alveolar PO2 (25). Although the FHR strain has been generally considered as a genetic model of "primary" pulmonary hypertension, our laboratory (25) previously reported that FHR lungs showed a pattern of alveolar simplification and reduced pulmonary arterial density early during maturation. Similarly, in other rat models of lung hypoplasia caused by neonatal dexamethasone treatment (29) or hypoxia (5, 6, 31), pulmonary arterial density is also reduced, and there is an increased risk for late development of pulmonary hypertension (26, 44a). Whether decreased angiogenesis plays a significant role in the development of lung hypoplasia in these models is unknown, but data from this current report support the concept that inhibition of angiogenesis during a critical period of lung growth may contribute to impaired alveolarization.

In summary, we report that treatment of neonatal rats with two different inhibitors of angiogenesis during a critical period of postnatal lung growth caused lung hypoplasia as demonstrated by reduced lung-to-body weight ratios, alveolarization, and pulmonary arterial density. We also report that an inhibitor of the VEGF receptor KDR/flk-1 also reduced alveolarization, further suggesting that disruption of angiogenesis attenuates normal lung development. Based on these findings, we speculate that disruption of angiogenesis impairs alveolarization, suggesting that primary or acquired abnormalities of lung vascular growth can cause or contribute to lung hypoplasia. We further speculate that new strategies that stimulate vascular growth may provide an alternate approach to the treatment of abnormal lung growth in clinical disorders such as BPD and congenital diaphragmatic hernia.


    FOOTNOTES

Address for reprint requests and other correspondence: S. H. Abman, Dept. of Pediatrics, B-395, The Children's Hospital, 1056 E. Nineteenth Ave., Denver, CO 80218-1088 (E-mail: steven.abman{at}uchsc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 7 January 2000; accepted in final form 10 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abman, SH, and Sondheimer HM. Pulmonary circulation and cardiovascular sequelae of BPD. In: Diagnosis and Treatment of Pulmonary Hypertension, edited by Weir EK, Archer SL, and Reeves JT.. Mount Kisco, NY: Futura, 1992, p. 155-180.

2.   Acarregui, MJ, Penisten ST, Goss KL, Ramirez K, and Snyder JM. Vascular endothelial growth factor gene expression in human fetal lung in vitro. Am J Respir Cell Mol Biol 20: 14-23, 1999[Abstract/Free Full Text].

3.   Babaei, S, Teighert-Kuliszewska K, Monge JC, Mohamed F, Bendeck MP, and Stewart DJ. Role of NO in the angiogenic response in vitro to basic fibroblast growth factor. Circ Res 82: 1007-1015, 1998[Abstract/Free Full Text].

4.   Beck, L, and D'Amore PA. Vascular development: cellular and molecular regulation. FASEB J 11: 365-373, 1997[Abstract/Free Full Text].

5.   Blanco, LN, Massaro D, and Massaro GD. Alveolar size, number and surface area: developmentally dependent response to 13% O2. Am J Physiol Lung Cell Mol Physiol 261: L370-L377, 1991[Abstract/Free Full Text].

6.   Blanco, LN, Massaro GD, and Massaro D. Alveolar dimensions and number: developmental and hormonal regulation. Am J Physiol Lung Cell Mol Physiol 257: L240-L247, 1989[Abstract/Free Full Text].

7.   Burri, PH. Structural aspects of prenatal and postnatal development and growth of the lung. In: Lung Growth and Development, edited by McDonald JA.. New York: Dekker, 1997, p. 1-35.

8.   Chi, TPL, and Krovetz LJ. The pulmonary vascular bed in children with Down syndrome. J Pediatr 86: 533-538, 1975[ISI][Medline].

9.   Cooney, TP, and Thurlbeck WM. The radial alveolar count method of Emery and Mithal: a reappraisal 1---postnatal lung growth. Thorax 37: 580-583, 1982[Abstract].

10.   Cooney, TP, and Thurlbeck WM. The radial alveolar count method of Emery and Mithal: a reappraisal 2---intrauterine and early postnatal lung growth. Thorax 37: 572-579, 1982[Abstract].

11.   Cooney, TP, and Thurlbeck WM. Pulmonary hypoplasia in Down's syndrome. N Engl J Med 307: 1170-1173, 1982[Abstract].

12.   D'Amato, RJ, Loughman MS, Flynn E, and Folkman J. Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci USA 91: 4082-4085, 1994[Abstract].

13.   DeMello, DE, and Reid LM. Pre- and postnatal development of the pulmonary circulation. In: Basic Mechanisms of Pediatric Respiratory Disease: Cellular and Integrative, edited by Chernick V, and Mellins RB.. Philadelphia, PA: Decker, 1991, p. 6-54.

14.   DeMello, DE, Sawyer D, and Reid LM. Early fetal development of lung vasculature. Am J Respir Cell Mol Biol 16: 568-581, 1997[Abstract].

15.   Dreyfuss, D, Basset G, Soler P, and Saumon G. Intermittent positive pressure hyperventilation with high inflation pressure produces microvascular injury in rats. Am Rev Respir Dis 132: 880-884, 1985[ISI][Medline].

16.   Emory, JL, and Mithal A. The number of alveoli in the terminal respiratory unit of man during intrauterine life and childhood. Arch Dis Child 35: 483-485, 1960.

17.   Folkman, J. Clinical applications of research on angiogenesis. N Engl J Med 333: 1757-1763, 1995[Free Full Text].

18.   Fong, TAT, Shawver JK, Sun L, Tang C, App H, Powell TJ, Kim YH, Schreck R, Wang X, Risau W, Ullrich A, Hirth KP, and McMahon G. Su-5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res 59: 99-106, 1999[Abstract/Free Full Text].

19.   Garg, UC, and Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 83: 1774-1777, 1989[ISI][Medline].

20.   Gerber, HP, Hillan KJ, Ryan AM, Kowalski J, Keller GA, Rangell L, Wright BD, Radtke F, Aguet M, and Ferrara N. VEGF is required for growth and survival in neonatal mice. Development 126: 1149-1159, 1999[Abstract/Free Full Text].

21.   Griffith, EC, Niwayama S, Ramsay CA, Chang YH, and Liu JO. Molecular recognition of angiogenesis inhibitors fumagillin and ovalicin by methionine aminopeptidase 2. Proc Natl Acad Sci USA 95: 15183-15188, 1998[Abstract/Free Full Text].

22.   Kenyon, BM, Browne F, and D'Amato RJ. Effects of thalidomide and related metabolites in a mouse corneal model of neovascularization. Exp Eye Res 64: 971-978, 1997[ISI][Medline].

23.   Kroll, J, and Waltenberger J. VEGF-A induces expression of eNOS and iNOS in endothelial cells via VEGF receptor-2 (KDR). Biochem Biophys Res Commun 252: 743-746, 1998[ISI][Medline].

24.   Langston, C, Kida K, Reed M, and Thurlbeck WM. Human lung growth in late gestation and in the neonate. Am Rev Respir Dis 129: 607-613, 1984[ISI][Medline].

25.   Le Cras, TD, Kim DH, Gebb S, Markham NE, Shannon JM, Tuder RM, and Abman SH. Abnormal lung growth and the development of pulmonary hypertension in the Fawn-Hooded rat. Am J Physiol Lung Cell Mol Physiol 277: L709-L718, 1999[Abstract/Free Full Text].

26.   Le Cras, TD, Markham NE, Morris KG, Ahrens CR, McMurtry IF, and Abman SH. Neonatal dexamethasone treatment increases the risk for pulmonary hypertension in adult rats. Am J Physiol Lung Cell Mol Physiol 278: L822-L829, 2000[Abstract/Free Full Text].

28.   Margraf, LR, Tomashefski JF, Bruce MC, and Dahms BB. Morphometric analysis of the lung in bronchopulmonary dysplasia. Am Rev Respir Dis 143: 391-400, 1991[ISI][Medline].

29.   Massaro, D, and Massaro GD. Dexamethasone accelerates postnatal alveolar wall thinning and alters wall composition. Am J Physiol Regulatory Integrative Comp Physiol 251: R218-R224, 1986[ISI][Medline].

30.   Massaro, GD, and Massaro D. Formation of pulmonary alveoli and gas-exchange surface area: quantitation and regulation. Annu Rev Physiol 58: 73-92, 1996[ISI][Medline].

31.   Massaro, GD, Olivier J, and Massaro D. Short-term perinatal 10% oxygen alters postnatal development of lung alveoli. Am J Physiol Lung Cell Mol Physiol 257: L221-L225, 1989[Abstract/Free Full Text].

32.   Mitchell, SH, and Teague WG. Reduced gas transfer at rest and during exercise in school age survivors of bronchopulmonary dysplasia. Am J Respir Crit Care Med 157: 1406-1412, 1998[Abstract/Free Full Text].

33.   Morbidelli, L, Chang CH, Douglas JG, Granger HJ, Ledda F, and Ziche M. NO mediates mitogenic effect of VEGF on coronary venular endothelium. Am J Physiol Heart Circ Physiol 270: H411-H415, 1996[Abstract/Free Full Text].

34.   Mori, S, Ueda T, Kuratsu S, Hosono N, Izawa K, and Uchida A. Suppression of pulmonary metastasis by angiogenesis inhibitor TNP-470 in murine osteosarcoma. Int J Cancer 61: 148-152, 1995[ISI][Medline].

35.   Nakaki, T, Nakayama M, and Kato R. Inhibition by NO and NO-producing vasodilators of DNA synthesis in vascular smooth muscle cells. Eur J Pharmacol 189: 347-353, 1990[Medline].

36.   Neufeld, G, Coehn T, Gengrinovitch S, and Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 13: 9-22, 1999[Abstract/Free Full Text].

37.   Niwano, M, Arii S, Mori A, Ishigami S, Harada T, Mise M, Furutani M, Fujioka M, and Imamura M. Inhibition of tumor growth and microvascular angiogenesis by the potent angiogenesis inhibitor, TNP-470, in rats. Surg Today 28: 915-922, 1998[Medline].

38.   Papapetropoulos, A, Garcia-Cardena G, Madri JA, and Sessa WC. NO production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 100: 3131-3139, 1997[Abstract/Free Full Text].

39.   Randell, SH, Mercer RR, and Young SL. Neonatal hyperoxia alters the pulmonary alveolar and capillary structure of 40-day-old rats. Am J Pathol 136: 1259-1266, 1990[Abstract].

40.   Risau, W. Mechanisms of angiogenesis. Nature 386: 671-674, 1997[ISI][Medline].

41.   Roman, J. Cell-cell and cell-matrix interactions in development of the lung vasculature. In: Lung Growth and Development, edited by McDonald JA.. New York: Dekker, 1997, p. 365-400.

42.   Shannon, JM, and Deterding RR. Epithelial-mesenchymal interactions in lung development. In: Lung Growth and Development, edited by McDonald JA.. New York: Dekker, 1997, p. 81-118.

43.   Strawn, LM, McMahon G, App H, Schreck R, Kuchler WR, Longhi MP, Hui TH, Tang C, Levitzki A, Gazit A, Chen I, Keri G, Orfi L, Risau W, Flamme I, Ullrich A, Hirth KP, and Shawver LK. Flk-1 as a target for tumor growth inhibition. Cancer Res 56: 3540-3545, 1999[Abstract].

44.   Sun, L, Tran N, Tang F, App H, Hirth P, McMahon G, and Tang C. Synthesis and biological evaluations of 3-substituted indolin-2-ones: a novel class of tyrosine kinase inhibitors that exhibit selectivity toward particular tyrosine kinases. J Med Chem 41: 2588-2603, 1998[ISI][Medline].

44a.   Tang, JR, Le Cras TD, Morris KG, and Abman SH. Brief perinatal hypoxia increases the severity of pulmonary hypertension after reexposure to hypoxia in infant rats. Am J Physiol Lung Cell Mol Physiol 278: L356-L364, 2000[Abstract/Free Full Text].

45.   Thibeault, DW, and Haney B. Lung volume, pulmonary vasculature, and factors affecting survival in congenital diaphragmatic hernia. Pediatrics 101: 289-295, 1998[Abstract/Free Full Text].

46.   Thomae, KR, Nakayama DK, Billiar TR, Simmons RL, Pitt BR, and Davies P. Effect of NO on fetal pulmonary artery smooth muscle growth. J Surg Res 59: 337-343, 1995[ISI][Medline].

47.   Tomashefski, JF, Opperman HC, and Vaweighter GF. Bronchopulmonary dysplasia: a morphometric study with emphasis on the pulmonary vasculature. Pediatr Pathol 2: 469-487, 1984[Medline].

48.   Van der Zee, R, Murohara T, Luo Z, Zollmann F, Passeri J, Lekutat C, and Isner JM. Vascular endothelial growth factor/vascular permeability factor augments NO release from quiescent rabbit and human vascular endothelium. Circulation 95: 1030-1037, 1995[Abstract/Free Full Text].

49.   Zeltner, TB, Caaduff JH, Gehr P, Pfenninger J, and Burri PH. The postnatal development and growth of the human lung. I. Morphometry. Respir Physiol 67: 247-267, 1987[ISI][Medline].

50.   Zetter, BR. Angiogenesis and tumor metastasis. Annu Rev Physiol 49: 407-424, 1998.

51.   Ziche, M, Morbidelli L, Choudhuri R, Zhang HT, Donnini S, Granger HJ, and Bicknell R. NO synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest 99: 2625-2634, 1997[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 279(3):L600-L607
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society