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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 159-169, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4764

Insulin-Like Growth Factor-I Receptor–Mediated Vasculogenesis/Angiogenesis in Human Lung Development

Robin N. N. Han, Martin Post, A. Keith Tanswell and Stephen J. Lye

CIHR Group in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital; CIHR Group in Lung Development, Lung Biology Program, The Hospital for Sick Children; and Departments of Obstetrics and Gynaecology, Paediatrics and Physiology, University of Toronto, Toronto, Ontario, Canada

Address correspondence to: Robin N.N. Han, Rm. 870, Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, 600 University Avenue, Toronto, Canada, M5G 1X5. E-mail: hanrobin{at}aol.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The structural and functional development of the pulmonary system is dependent upon appropriate early vascularization of the embryonic lung. Our previous in vitro studies in a rat model indicated that insulin-like growth factor-I (IGF-I) is a potent angiogenic agent for fetal lung endothelial cells. To assess its role on human vascular lung development, we first examined the expression of IGF-I/II and IGF receptor type I (IGF-IR) in human embryonic and fetal lung tissues at 4–12 wk of gestation. Immunohistochemical and in situ hybridization studies revealed the presence of IGF-I/II–IGF-IR ligands and mRNA transcripts in embryonic lungs as early as 4 wk gestation. Immunotargeting using an anti–IGF-IR neutralizing antibody on human fetal lung explants demonstrated a significant blockade of IGF-IR signaling. Inactivation of IGF-IR resulted in a loss of endothelial cells, accompanied by dramatic changes in fetal lung explant morphology. Terminal transferase dUTP end-labeling assay and TEM studies of anti–IGF-IR–treated lungs demonstrated numerous apoptotic mesenchymal cells. Rat embryonic lung explant studies further validated the importance of the IGF–IGF-IR system for lung vascular development. These data provide the first demonstration of IGF-I/II expression in the human lung in early gestation and indicate that the IGF family of growth factors, acting through the IGF-IR, is required as a survival factor during normal human lung vascularization.

Abbreviations: angiopoietin 1, Ang-1 • endothelial cell, EC • electron microscopy, EM • insulin-like growth factors I and II, IGF-I/II • insulin-like growth factor receptor type I, IGF-IR • phosphate-buffered saline, PBS • smooth muscle {alpha}-actin, SM {alpha}-actin • smooth muscle cell, SMC • terminal transferase dUTP end-labeling, TUNEL • vascular endothelial growth factor, VEGF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human lung develops as an outpouch from the ventral wall of the foregut, at ~ 4 wk of gestation, which penetrates into the surrounding mesoderm. The lung bud subsequently divides through several generations of developing airways to reach an adult format of conducting airways at 16 wk of gestation. The surrounding mesoderm differentiates into several supporting cells and, most importantly, blood and lymph vessels. Both airway and vascular systems further develop in parallel with advancing gestation to become an efficient gas exchange organ at birth. Substantial growth occurs postnatally, with an adult complement of alveoli not being attained until the age of 8 yr (1). Several growth factors, their receptors, extracellular matrix, and associated molecules play a critical role in proliferation, migration, and differentiation during this well-coordinated growth process.

The importance of several growth factors and their cognate receptors in vasculogenesis and angiogenesis has been demonstrated in avian and murine species. However, studies of vascular development during early organogenesis in humans are limited. Gene targeting studies have suggested a pivotal role for specific growth factors, such as vascular endothelial growth factor (VEGF) (2) and angiopoietin 1 (Ang-1) (3), in vascular development.

Insulin-like growth factors (IGFs) are small peptides, which are closely related to proinsulin (4). IGF-I and IGF-II modulate cell proliferation and differentiation (5) during embryogenesis through paracrine (6) or autocrine (7) pathways. IGF receptor type I (IGF-IR) belongs to a receptor tyrosine kinase family, and has two extracellular {alpha}-subunits and two transmembrane ß-subunits (8). In vitro studies, using 3T3 cells, have demonstrated that IGF-IR is responsible for the determination of whether cells progress along a mitogenic pathway or undergo apoptosis (9). Previous studies have demonstrated that both IGF-I and IGF-II act via IGF-IR for mitogenic signaling in murine embryonic development (10). Our previous in vitro studies, using isolated fetal rat pulmonary microvascular endothelial cells (EC), had demonstrated that EC respond to exogenous IGF-I by a 2-fold increase in cell number (11). IGF-I/II are known potent angiogenic factors in a murine retinal model (12). The specific role of IGFs in human lung vascular development has not been reported.

Our objective was to examine human lung vascularization at its early stages of development. In particular, we focused on the spatial and temporal relationships of IGFI/II and IGF-IR during early lung vessel formation. Little is known about the temporal and spatial expressions of IGF-I/II and IGF-IR in early human embryonic lung development. Previously reported studies were mostly conducted on mid-gestation human fetal lung samples (13, 14). We first determined the temporal and spatial expressions of IGF-I, IGF-II, and IGF-IR in first-trimester human embryonic and fetal tissues. Both IGFs and IGF-IR were highly expressed in the airway epithelium and vascular endothelium. To elaborate the function of the IGF system in human lung vascular development, we proceeded with IGF-IR immunotargeting studies on human fetal lung explants using neutralizing antibodies. The resulting explants contained significantly less CD34-positive endothelial cells (EC). The importance of the IGF/IGF-IR system in lung vascular development was further validated by rat embryonic lung explant culture using an IGF-IR immunotargeting approach. These studies suggest that IGFs, acting through the IGF-IR, function as survival factors during vascular development of the human lung.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Preparation and Preparation of Sections
Human embryos and fetuses were collected from elective first-trimester abortuses of 4–12 wk gestation with prior approval of the Institutional Ethics Committee and following the patient's informed consent. Gestational ages of human tissue samples were assessed by morphometric analysis of embryos and fetuses using the Carnegie classification (15). Tissues were fixed in freshly prepared 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS; pH 7.2) at 4°C, and processed for paraffin embedding.

Immunohistochemistry
Mouse anti-human CD34 and CD31 (PECAM-1) were from Novocastra (Newcastle, UK). Rabbit anti-human vWF (von Willebrand Factor), mouse anti-human cytokeratin 18, a streptavidin-biotin alkaline phosphatase kit and Fast Red tablets were from DAKO (Carpinteria, CA). Monoclonal smooth muscle (SM) {alpha}-actin was from Zymed (San Francisco, CA). Rabbit anti-IGF-IR antibodies were purchased from Upstate Biotechnology Inc. (Lake Placid, NY) and from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-human IGF-I and II antibodies were provided by Dr. Victor Han (University of Western Ontario, London, ON, Canada). Immunohistochemical studies were conducted as described by Hsu and coworkers (16). For double immunostaining, the streptavidin-biotin alkaline phosphatase method was used, with Fast Red as color substrate. Slides were lightly counterstained with Carazzi's haematoxylin.

EC and Perivascular Markers
Three EC markers (vWF, CD31, and CD34) were used for our initial studies. There was a 100% correlation between CD31 and CD34 immunostaining in the mesenchymal cells of the developing lung. Both demonstrated essentially similar patterns of immunostaining for EC cells. However, vWF lacked positive staining in cells which appeared to be progenitors of EC and some microvascular EC. We subsequently used CD34 and SM {alpha}-actin (a marker for differentiated smooth muscle cells and pericytes) to study the ontogeny of lung vascular development. The specificities of IGF-I/II and IGF-IR antibodies (UBI) had been validated in a previous study (17). The specificity of IGF-IR (Santa Cruz) antibody was validated by immunoabsorption with a specific IGF-IR ligand followed by immunostaining.

Double Immunoflorescence Staining
CD34 and IGF-I colocalization studies were conducted on human embryonic and fetal lung sections using a double immunofluorescence method. FITC- and Texas Red–tagged secondary antibodies were used to visualize CD34 and IGF-I, respectively. Slides were mounted with Vectashield mounting media (Vector, Burlingame, CA) and observed under a MicroRadiance confocal microscope (Bio-Rad Laboratories, Hercules, CA).

Nonradioactive In Situ Hybridization
Preparation of cRNA probes. Human cDNA for both IGF-I and -II were generous gift of Dr. Victor Han (University of Western Ontario). Sense and antisense cRNA probes were transcribed from full-length cDNA fragments subcloned into vectors (phIGF-I-3Z and phIGF-II-4Z) using either SP6 or T7 polymerase; sense probes were used as controls. Riboprobe digoxigenin-labeling of RNAs was performed according to procedures provided by the manufacturer (Roche, Montreal, PQ, Canada). The efficiency and intensity of labeling was monitored by dot blots. Digoxigenin labeling was detected with alkaline phosphatase-conjugated anti-digoxigenin (Roche) (18) using Fast Red as color chromagen.

Microdissection and Organ Culture
DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) culture medium was from Gibco BRL (Burlington, ON, Canada) and Millicell-CM culture dish inserts were purchased from Millipore Corp. (Bedford, MA). Rabbit anti-human IGF-IR neutralizing antibody was purchased from R&D Systems (Minneapolis, MN). IGF-I ligand was purchased from Amgen Biologicals (Thousand Oaks, CA). Mouse monoclonal IgG2b antibody to phosphotyrosine (PY99) and goat anti-mouse IgG conjugated with peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ECL Western blotting detection reagents were from Amersham Life Science (Buckinghamshire, UK).

Human lungs from 12 wk gestation fetuses were carefully separated from other tissue under a dissecting microscope, and 1 x 1 x 1 mm cubes of lungs were teased out. Lung explants (5–6 pieces) were placed on Millicell-CM culture dish inserts (pore size 0.4 µm; Millipore), and incubated as a submersion culture in a serum-free medium consisting of DMEM/F-12 supplemented with 100 µg/ml streptomycin, 100 U/ml penicillin, and 0.25 mg/ml ascorbic acid, pH 7.4. The lung rudiments were then incubated over night at 37°C in 95% air and 5% CO2 (19). For IGF-IR immunotargeting experiments, each group was incubated with either 1 µg/ml or 10 µg/ml of rabbit anti–IGF-IR–neutralizing antibody (20). Control groups of lung explants were incubated with 1 µg/ml and 10 µg/ml of nonimmune rabbit IgG. Lung explants were monitored daily and photographs were recorded. Addition of antibodies and control IgG was repeated on the third day of culture. The whole experiment lasted for 5 d. Lung explants were washed in cold PBS before fixation with freshly prepared 2% (wt/vol) paraformaldehyde in PBS (pH 7.2) and embedded in paraffin. A piece of lung explant from each group was fixed and processed for transmission electron microscopic studies. In addition to above studies, a set of lung explants (both control and neutralizing anti–IGF-IR groups) was included for receptor phosphorylation studies. Lung explants from both control and anti–IGF-IR–treated groups were challenged with exogenous IGF-I (100–200 ng/ml) for the final 30 min of the experiment. Some groups of lung explants were not challenged with exogenous IGF-I. The purpose of this study was to demonstrate the neutralizing activity of the anti–IGF-IR antibody on IGFR signaling. After 30 min of exposure, lung explants were carefully washed with cold PBS and frozen until further processing for Western analysis. All of the above experiments were conducted on at least three separate human fetal lungs of 12 wk gestation.

Wistar rat embryonic lungs (E13 gestation) were carefully dissected out from embryos. We used the same submerged culture system as mentioned for human fetal lung explants above (similar concentrations of IGF-IR and isotypic IgG controls), except for the addition of 10% fetal bovine serum in the medium throughout the experiment. In addition to the IgG control, an additional set of controls was included by immunoabsorbing IGF-IR with IGF-IR peptide overnight at 4°C before the experiment. The whole experiment lasted 5 d, with treatments given at Day 1 and at 1.5 d with daily monitoring of explants and photographic recordings.

Terminal Transferase dUTP End-Labeling Assay
Tissue sections were dewaxed and treated with proteinase K (Gibco) 20 µg/ml for 15 min at 37°C. After washing tissue sections in PBS, the terminal transferase dUTP end-labeling (TUNEL) assay was conducted according to the manufacturer's instructions (in-situ cell death detection [fluorescein] kit; Roche). Images were digitally captured using a Leica imaging system.

Quantitative Studies for CD34-Positive and Apoptotic Cells
Quantitative studies of CD34-positive cells were performed at the light microscopy level in 10 random fields of each lung explant from three separate experiments. Quantitative analyses for apoptosis were performed on six random fields at EM level of each lung explant from three separate experiments. The criteria used to characterize an apoptotic cell were as described by Scavo and colleagues (21). The percentages of CD34-positive cells and apoptotic cells were calculated from the total number of mesenchymal cells in each lung explant.

Demonstration of IGF-IR Neutralization by Western Analysis for Tyrosine Phosphorylation
To determine whether IGF-IR neutralizing antibodies interfered with the IGF signaling pathway, we attempted to examine receptor phosphorylation following immunoprecipitation of IGF-IR in human lung explants. Unfortunately, with such small samples, we were unable to demonstrate phosphorylation of immunoprecipitated IGF-IR in human lung explants. We therefore took an indirect approach to stimulate lung explants with exogenous IGF-I (200 ng/ml) for 30 min at the end of experiment. Five lung explants from each control (IgG-treated) and anti–IGF-IR–treated groups were pooled and transferred to Eppendorf tubes containing 20 µl of lysis buffer. Western analysis for tyrosine phosphorylation was conducted as described by Liu and coworkers (22). Densitometric scanning was conducted using NIH image software.

Data Presentation
Numerical values for percentages of CD34-positive EC at the light microscopic level and percentages of apoptotic cells at the EM level (control versus anti–IGF-IR–treated lungs) are shown as mean ± SEM of three separate experiments. Statistical significance (P < 0.05) was determined by using Student's t test and comparison between treatment groups was determined by Kruskal-Wallis One Way Analysis of Variance on Ranks.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ontogeny of Human Embryonic and Fetal Lung Vascularization
Our initial studies, using EC markers, either alone or by a double immunolabeling technique (EC marker and smooth muscle/pericyte marker), allowed us to determine the pattern of vascular formation and the spatial relationship of the two cell types during vascular development. CD34-immunoreactive EC cells were detected in all human embryos and fetal lung tissues examined. CD34-positive EC lined the innermost layer of the primitive heart, the aorta, and all the primitive vessels in the mesenchyme of lung buds in sections of human embryos at 4 wk gestation (Figure 1A). This time point is from the embryonic stage of lung development (4–7 wk gestation). Under increased magnification of the human embryonic lung at 4 wk gestation (Figure 1B), CD34-immunoreactive EC were observed either as solitary cells or as groups forming a single layer of cells within the mesenchyme. Some of the CD34-positive cells demonstrated lumens adjacent to them. These cells were not in direct contact with the developing primitive airways. The number of CD34-immunoreactive EC increased, and appeared to form primary vascular plexuses surrounding the primitive airway, by 6 wk of gestation (Figure 1C). Vascular lumens were more apparent at this stage. By 8 wk of gestation (Figure 1D), CD34-immunoreactive EC formed loops or rings in the parenchyma. EC cells were also seen as groups forming linear cords, or as solitary cells, between the primitive airways. Vessel lumens were larger and more obvious than at earlier gestations. Similar findings were detected at 12 wk of gestation (Figure 1E). As can be noted, the number of CD34-immunoreactive EC increased in fetal lungs at 9–12 wk gestation (Figure 1E). The EC were juxtaposed to the developing airways at this stage of development. The pattern of lung vascular plexus formation became more complex with advancing gestation. By the canalicular stage of development (17–24 wk), the appearance of acini was accompanied by an explosion of microvessel formation. At this stage, CD34-immunoreactive EC lined the innermost layer of mature vessels and microvessels which were in close contact with canalicules (Figure 2G and 2I). The ontogeny of CD34 and SM {alpha}-actin colocalization studies were conducted in 4–12 wk gestation embryonic and fetal lungs. All embryonic and fetal lungs demonstrated two distinct patterns of fetal lung vascular development. One population of vascular plexuses were independent of SM {alpha}-actin–positive cells whereas another population of vascular plexuses were juxtaposed to SM {alpha}-actin–positive cells, as demonstrated in 4-wk gestation embryonic lung (Figure 1F) and in 12-wk fetal lung (Figure 1G).



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  		Figure 1. The progression of vascular development in human embryonic and fetal lungs at different gestational ages as assessed by CD34 (EC marker) immunostaining (A–E) and double immunostaining using CD34 and SM {alpha}–actin (perivascular cell marker) (F and G). Horizontal section of a 4-wk embryo (A) demonstrated EC (black) as inner linings of the heart and great vessels and in and around several developing organs. (B) Higher magnification of a lung bud from A demonstrated a single layer of EC organized around the developing airway (a). The dramatic increase of EC number and the organization of EC became more intricate as gestation advanced, as demonstrated in (C) 6-wk embryonic lung, (D) 8-wk fetal lung, and (E) 12-wk fetal lung. (F) 4-wk embryonic lung demonstrated two sets of EC (black), one independent set of EC in mesenchyme with lumens and one set of EC juxtaposed to perivascular cells (red). (G) 12-wk fetal lung with mature vessels surrounded by perivascular SM cells (red) in close proximity of developing airways, which were also surrounded by SM cells (red). Numerous EC without perivascular cells were also present in the mesenchyme. Developing airways (a) were present in all photomicrographs. Magnifications: A, x10; B–F, x40; G, x25.

 


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  		Figure 2. Confocal images of CD34 and IGF-I colocalization in human embryonic and fetal lungs by double immunofluorescence (A–I) and double immunolabeling of cytokeratin 18 and IGF-IR (J and K). CD34 immunofluorescence (A, D, G) and IGF-I immunofluorescence (B, E, H). CD34 and IGF-I colocalization (C, F, I). (A) CD34(+) cells (green; arrows) in mesenchyme of human embryonic lungs at 4 wk gestation. (a: airway epithelium). (B) Strong IGF-I immunoreactivity (red) in airway epithelium (a) and in mesenchymal cells (arrows) of 4-wk gestation lung. (C) Colocalization of CD34 and IGF-I in mesenchyme (yellow; arrows) of 4-wk human embryonic lung. Some cells in the mesenchyme demonstrated either CD34 (green) or IGF-I (red) immunoreactivity. (D) CD34(+) cells (green) in mesenchyme of 8-wk human embryonic lungs (a: airway epithelium). (E) Strong IGF-I (red) immunoreactivity in airway epithelium (a) and in mesenchymal cells of 8-wk gestation lung. (F) Most mesenchymal cells demonstrated colocalization of CD34 and IGF-I (yellow) of 8-wk human embryonic lung. Some mesenchymal cells and airway epithelial cells (a) demonstrated IGF-I (red) immunoreactivity. (G) Numerous CD34(+) cells (green) in the mesenchyme of human lung at 19 wk gestation (a: developing distal airways). (H) Strong IGF-I immunoreactivity in airway epithelial cells lining the distal airways (a). Very few mesenchymal cells demonstrated weak IGF-I immunoreactivity. (I) No CD34 and IGF-I colocalized cells (yellow) were detected in 19-wk human fetal lung. CD34 (+) cells (green) were in the mesenchyme and distal airway epithelial cells (a) showed IGF-I (+) immunoreactivity (red). (J and K) Double immunostaining of cytokeratin 18 (red) and IGF-IR (black) in 12-wk fetal lungs. Strong IGF-IR reactivity was detected in EC (arrows) and on airway (a) epithelial cells as punctate staining. Relatively weaker IGF-IR immunoreactivity was detected in smooth muscle cells (asterisks) adjacent to developing airways (a). Magnifications: A, B, C, G, H, and I, x40 (oil); D, E, and F, x20; J, x40; K, x100 (oil).

 
Ontogeny of IGF-I/II Ligand and IGF-IR Expression in Human Embryonic and Fetal Lungs
IGF-I/II immunolocalization studies were initially conducted using an immnoperoxidase method. Strong IGF-I immunoreactivity was detected in all whole human embryonic/fetal and lung sections examined. Immunoreactivity was particularly intense at early gestational ages. In a 4-wk gestation human embryo, IGF-I immunoreactivity was detected not only in lung buds, but in other developing organs, including liver, heart, limbs, myoblasts, spinal cord and spinal ganglia, and in EC lining developing large vessels (data not shown). At 4 wk of gestation, embryonic lung buds demonstrated strong IGF-I immunoreactivity in airway epithelial cells and EC lining the primary vascular plexuses (data shown as double immunoflorescence). IGF-II immunostaining demonstrated a similar pattern of immunoreactivity to that observed for IGF-I (data not shown).

Double immunoflorescence method was subsequently used to further elucidate the colocalization of IGF-I in CD34-positive cells. The number of CD34-positive cells increased dramatically from 4–19 wk gestation (Figures 2A, 2D, and 2G). Strong IGF-I immunoreactivity was observed in epithelial cells of the developing airways from 4–19 wk gestation (Figures 2B, 2E, and 2H). IGF-I colocalized with most CD34-positive cells in embryonic lung mesenchyme as early as 4 wk gestation (Figure 2C). It was more evident in the mesenchyme of 8 wk gestation fetal lung, whereas some cells demonstrated reactivity to IGF-I only (Figure 2F). However, no colocalization of CD34 and IGF-I was demonstrated in 19 wk gestation fetal lung (Figure 2I).

IGF-IR immunoreactivity (polyclonal antibody anti–IGF-IR, UBI) was detected in EC of 4 wk gestation embryonic lungs (data not shown). Double immunostaining (cytokeratin 18 and IGF-IR) demonstrated strong IGF-IR immunoreactivity in fetal lungs of 8–12 wk gestation in airway epithelial cells and in EC lining vessels (Figure 2J). Under increased magnification, IGF-IR immunoreactivity was detected in smooth muscle cells (SMCs) adjacent to airway epithelial cells and in some mesenchymal cells, in addition to airway epithelial and EC cells (Figure 2K). Immunohistolocalization of IGF-IR using an alternative antibody (polyclonal antibody anti–IGF-IR; Santa Cruz) on adjacent lung sections gave similar results (data not shown).

Ontogeny of IGF-I/II mRNAs in Human Embryonic and Fetal Lungs
IGF-I mRNA was detected in human embryonic and fetal lungs as early as 4 wk gestation (Figure 3A). Abundant IGF-I mRNA transcripts were detected in developing airway epithelial cells (embryonic/fetal), SMCs adjacent to developing airways, and in progenitor EC (Figure 3A). Abundant IGF-I transcripts were detected in developing liver (Figure 3C) and heart (Figure 3A). A similar IGF-I mRNA pattern was detected in 5-wk gestation embryonic lung (Figure 3D) and in 8 wk fetal lung (Figure 3E). Similarly, IGF-I mRNA was detected in airway ECs, SMCs adjacent to airway ECs, and in ECs of vessels in 12 wk gestation fetal lung (Figure 3F). Fetal lung sections, which were hybridized with sense IGF-I cRNA probe, demonstrated negative color reaction (Figure 3B). Abundant IGF-II transcripts were detected in human embryonic and fetal lungs in a fashion similar to that observed with IGF-I mRNA (Figures 3G and 3H).



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  		Figure 3. The spatial and temporal expression of IGF-I mRNA (A–F) and IGF-II mRNA (G and H) as demonstrated by in situ hybridization in human embryonic and fetal lungs. Strong IGF-I mRNA (red) was detected as early as 4-wk gestation embryonic lung (A) in developing airway (a) epithelial cells, smooth muscle cells adjacent to airway, and in EC (arrows) of the mesenchyme. Strong IGF-I mRNA expression was detected in the developing heart (ht). (C) Sagittal section of a 5-wk embryo demonstrated stronger IGF-I mRNA level in the developing liver (lv) than in the lung (lg). (D) Increased magnification of C demonstrated IGF-I mRNA in airway (a) epithelial cells, smooth muscle cells adjacent to airways, and in EC (arrows). Similar IGF-I mRNA distribution was evident in 8-wk fetal lung (E). Fetal lung of 12-wk gestation (F) demonstrated strong IGF-I mRNA transcripts in airway (a) epithelial cells, smooth muscle cells (asterisks) adjacent to airway, EC (arrows) that lined the developing vessels, and in solitary mesenchymal cells. (G) 5-wk gestation lung and (H) 12-wk gestation fetal lung demonstrated strong IGF-II mRNA transcripts (red) in airway (a) epithelial cells and EC (arrows). (B) Control (hybridized with sense cRNA probe for IGF-I) 7-wk fetal lung section demonstrated a negative color reaction. (A–F) counterstained with Carazzi's haematoxylin. G and H were without counterstain. Magnifications: C, x10; D and E, x20; A, B, G, and H, x40; and F, x100 (oil).

 
IGF-IR Immunotargeting Studies on Human Fetal Lung Explants
Control lung explants exposed to a nonspecific rabbit IgG exhibited a progressive budding of distal airways between Day 1 (Figure 4A) and Day 5 (Figure 4B) in culture. Lung explants that were not exposed to rabbit IgG (i.e., lung explants left under serum-free conditions) behaved similarly. Treatment with anti–IGF-IR neutralizing antibody attenuated this time-dependent increase in budding of distal airways (Figures 4F and 4N). Lung explants that were treated with anti–IGF-IR (1 µg/ml) (Figure 4F) did not increase in size to the same extent as explants in the control group, possibly due to a reduced amount of budding. Lung explants that were exposed to an increased concentration of anti–IGF-IR (10 µg/ml) demonstrated a significant perturbation of growth (Figure 4N). Gross histologic examination demonstrated that human fetal lung explants from the control group after 5 d in culture (Figure 4C) appeared essentially similar to freshly fixed lungs before culture. In contrast, lungs explants treated with the anti–IGF-IR antibody (1 µg/ml; Figure 4G) and anti–IGF-IR (10 µg/ml; Figure 4O) demonstrated airway collapse. In addition, the mesenchymal compartment was disrupted and demonstrated large gaps between the developing airways (Figures 4G and 4P).



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  		Figure 4. Morphologic changes of control and anti-IGF-IR treated human fetal lung explants. Top horizontal panels (A–D): IgG-1 µg/ml exposed (control) group; second horizontal panels (E–H): 1 µg/ml anti–IGF-IR neutralizing antibody group; third horizontal panels (I–L): IgG-10 µg/ml exposed (control) group; lowermost panels (M–P): 10 µg/ml anti–IGF-IR neutralizing antibody group. Day 1 (A, E, I, M) and Day 5 (B, F, J, N) of culture. Day 5 lung explants (B, F, and J) did not demonstrate dramatic differences in size compared with Day 1 explants. However, a significant growth reduction was demonstrated in explants exposed to 10 µg/ml anti-IGF-IR (N). Hematoxylin and eosin staining of lung explant from IgG-exposed (control) group (C and K) demonstrated essentially normal histology with patent airways. Gross macroscopic changes, as assessed by hematoxylin and eosin staining, were detected in lung explants that were exposed to anti–IGF-IR neutralizing antibody (G and O) . Airways (a) were collapsed with disrupted mesenchyme demonstrated as large gaps (asterisks). The number of EC as assessed by CD34 immunostaining in lung explants (D, H, L, and P). Numerous CD34-positive EC (black) and patent airways (a) were notable in lung explants of the control group (D and L). Very few CD34-positive EC (black) with collapsed airways (a) were found in lung explants exposed to anti–IGF-IR (H and P). Asterisks indicate large gaps in mesenchyme. Magnifications: A, B, E, F, I, J, M, and N, x5; K, x10; C, D, H, and L, x20; G, O, and P, x40.

 
The vascular developmental pattern, and the number of EC in lung explants, was assessed by CD34 immunostaining (Figures 4D, 4H, 4L, and 4P). The percentages of CD34-immunoreactive EC were calculated relative to the total mesenchymal cell population. There was a significant (5- to 10-fold) reduction (P < 0.001) in CD34-immunopositive ECs in anti–IGF-IR–treated lung explants compared with control IgG–exposed explants (Figure 5A).



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  		Figure 5. (A) Quantitative analyses of CD34 positive mesenchymal cells in control and IGF-IR–treated lung explants (see Figures 4D, 4H, 4L, and 4P). Comparison between lung explants exposed to either nonimmune IgG (control) or anti–IGF-IR antibodies (solid bars, 1 µg/ml; shaded bars, 10 µg/ml). Values (% of total mesenchymal cells) are mean ± SEM. *P < 0.001 determined by Student's t test and one-way ANOVA. n = 3 separate experiments. (B) IGF-IR–immunotargeted studies on rat embryonic (E13) lung explants. E13 rat embryonic lung at the start of experiment (I). Rat embryonic lung explants 36 h after treatment (II, III, IV). The number of blood islands (arrow) in IgG treated (control) explant (II) is comparable to an explant at Day 1 (I). A reduction of blood islands (arrow) is noted in lung explant treated with IGF-IR 10 µg/ml (III). The number of blood islands (arrow) in lung explant treated with IGF-IR, immunoabsorbed with IGF-IR peptide (IV) is comparable to IgG-treated explant (II). Magnification: x3.

 
IGF-IR Immunotargeting Studies on Rat Embryonic Lung Explants
To further validate the importance of the IGF system in the embryonic/fetal lung vascular development, we elected to conduct IGF-IR immunotargeting studies on readily available rat embryonic lungs. After 36 h in culture, IGF-IR–treated rat embryonic lungs (Figure 5B, III) demonstrated a loss of blood islands compared with embryonic lung explants that were treated with either control IgG (Figure 5B, II) or IGF-IR immunoabsorbed with IGF-IR peptide (Figure 5B, IV). The number of blood islands in both control rat embryonic lung explants (Figure 5B, II and IV) was comparable. IGF-IR–immunotargeted explant lungs demonstrated a complete absence of blood islands by the end of experiment at Day 5 (data not shown).

IGF-IR Immunotargeting and Apoptosis
An in-situ cell death detection (TUNEL) assay demonstrated a significant increase of apoptotic cells in IGF-IR–treated human lung explants compared with control IgG–exposed lung explants. Most TUNEL-positive apototic cells were detected in the mesenchyme of lung explants (Figures 6D and 6F). Very few TUNEL-positive cells were detected in freshly fixed human lung tissue of 12 wk gestation (Figure 6B). Transmission electron microscopy was used to assess the ultrastructural changes. A 9-fold increase in the number of cells demonstrating the characteristic changes associated with apoptosis was evident in lung explants exposed to anti–IGF-IR (1 µg/ml) (Figure 7A, III and IV) when compared with explants exposed to IgG (Figure 7A, I and II). Ultrastructural changes in lung explants that were treated with 10 µg/ml anti–IGF-IR (data not shown) were similar to explants exposed to 1 µg/ml anti–IGF-IR. However, lungs that were exposed to the higher concentration of anti–IGF-IR showed a further increase in the number of cells undergoing apoptosis (Figure 7B). In accordance with the TUNEL analysis (Figure 6), EM revealed that most apoptotic cells were mesenchymal cells. Few apoptotic airway epithelial cells were observed.



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  		Figure 6. TUNEL analysis of control and anti–IGF-IR–treated human fetal lung explants. 4',6'-diamidino-2-phenylindole staining (left panels) and TUNEL assay (right panels) of 12 wk gestation human fetal lung (A and B) and lung explants exposed to either control IgG or anti–IGF-IR antibodies (C–F). Freshly fixed 12-wk fetal lung (A and B), nonimmune IgG-treated lung explants (C and D), and anti–IGF-IR (1 µg/ml)-treated lung explants (E and F). Majority of TUNEL-positive cells were present in the mesenchyme (D and F). Very few TUNEL-positive cells were noted in airway (a) epithelium. Magnification: x25.

 


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  		Figure 7. (A) Ultrastructural changes of control and anti–IGF-IR–treated human fetal lung explants. (B) Quantitative analyses of apoptotic cells percentage in human lung explants. (C) Indirect evidence of IGF-IR signaling blockade in human fetal lung explants. Nonimmune (control) IgG-treated explants (A, panels I and II) and anti–IGF-IR (1 µg/ml)-treated lung explants (A, panels III and IV). (I) airway epithelium (a denotes lumen) and (II) mesenchymal cells of control explants demonstrated normal histology with very few apoptotic (arrow) cells in the mesenchyme. (III) Airway epithelium (a denotes lumen) of lung explants exposed to anti–IGF-IR demonstrated normal histology. However, numerous apoptotic cells (arrows) were detected in the mesenchymal compartment (IV) (asterisks denote disrupted mesenchyme with large gaps). Magnifications: I, II, and IV, x2,500; III, x1,850. (B) Comparison of the percentage of apoptotic cells between lung explants exposed to either nonimmune IgG (control) or anti–IGF-IR antibodies (bar graph). Values (% of total mesenchymal cells) are mean ± SEM. *P < 0.001 determined by Student's t test and one-way ANOVA (n = 3 separate experiments). (C) No phoshorylation activity in IGF-IR (1 µg/ml)-treated explants when not stimulated by exogenous IGF-I (lane 1). Tyrosine phosphorylation in control IgG (lane 2) and anti–IGF-IR (10 µg/ml)-treated human fetal lung explants (lane 3) after stimulated by IGF-I. A significant reduction of tyrosine phosphorylation in anti–IGF-IR–treated lung explants (lane 3) when compared with IgG-treated lung explants (lane 2).

 
Indirect Demonstration of IGF-IR Neutralization by Tyrosine Phosphorylation
Western analysis demonstrated phosphorylation activity at 90 kD, consistent with the molecular weight of IGF-IR (8) in control IgG–treated (Figure 7C, lane 2) and anti–IGF-IR–treated explants (Figure 7C, lane 3) that were challenged with exogenous IGF-I. An absence of phosphorylation activity was noted in anti–IGF-IR (1 µg/ml)-treated lung explants (Figure 7C, lane 1) that were not exposed to exogenous IGF-I. Western analysis indicated a significant reduction of tyrosine phosphorylation in lung explants that were exposed to anti–IGF-IR (10 µg/ml) (Figure 7C, lane 3) in comparison to lung explants treated with IgG (Figure 7C, lane 2).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Vascularization of the embryonic lung is critical for appropriate development of the lung in utero and for the capability of establishing optimal gas exchange at birth. Developmental studies on mice and other animal models have begun to document the relative contribution of vasculogenesis and angiogenesis to embryonic lung vascularization. However, there is very limited data on the ontogeny of lung vascularization in human embryos, and thus the extent to which animal studies are relevant to the human remains to be determined. In this study, we report for the first time the ontogeny of human lung vascularization during a critical period of embryonic development (4–7 wk gestation). Our data indicate that, as in mouse, at this early stage both vasculogenesis and angiogenesis contribute to the vascular development of the human embryonic lung. Our data further document that IGF-I contributes to vascularization through maintenance of the endothelial cell population, most likely through its action as a survival factor.

We identified solitary EC cells within the mesenchyme as early as 4 wk of gestation and observed lumen formation within some of these EC cells. Our preliminary studies demonstrated the correlation of both CD31+ and CD34+ immunostaining in those solitary cells. However, they were negative for vWF immunoreactivity. These findings suggested that those cells were progenitor ECs (angioblasts). The presence of both CD31 and CD34 immunoreactivity suggest that these cells are not hemangioblasts (CD31- and CD34+). Around 5 wk of gestation, EC cells began to form primary vascular plexuses indicative of vasculogenesis. Formation of EC loops or rings, an indication of intussusceptive microvascular growth or nonsprouting angiogenesis, as suggested by Wilting and Christ (23), was apparent at 8–12 wk of gestation. Based on CD34 and SM {alpha}-actin co-localization studies in embryonic and fetal lungs from 4–12 wk gestation, we were able to demonstrate that there were two patterns of fetal lung vascular development. The first pattern was the formation of primary vascular plexuses that were independent of perivascular cells, suggestive of vasculogenesis, and the second pattern was the formation of vascular plexuses that were juxtaposed to perivascular cells, suggestive of angiogenesis (24). Our data demonstrated the evidence of perivascular cells juxtaposed to EC cells as early as 4 wk of gestation. It is well documented that perivascular cells modulate angiogenesis, which is the formation of new vessels from primary vascular plexuses by either sprouting or nonsprouting (intussusception) mechanisms. This suggests a much earlier involvement of angiogenesis. The presence of both patterns of vascular development was evident as early as 4 wk gestation in embryonic lung and also in fetal lungs at later gestation. These data suggest that vasculogenesis and angiogenesis occur concomitantly in human lung vascular development. Our histologic analyses revealed marked similarities between vascular development in human and murine embryonic/fetal lungs as shown by de Mello and coworkers (25) using vascular casting and scanning EM techniques. Maeda and colleagues (26) recently demonstrated the two patterns of vascular development in human fetal lungs from the pseudoglandular stage onwards. Our data, from much earlier in gestation, is in agreement with their findings. Both studies used the same technique of double immunolabeling of CD34 and SM {alpha}-actin.

Insulin-like growth factors (IGF-I/II) play a crucial role in cell proliferation and differentiation (5) during embryogenesis. Our IGF-I/II–IGF-IR immunolocalization studies are consistent with IGFs acting as either autocrine and/or paracrine mediators of human fetal lung development. The colocalization of CD34-positive cells and IGF-I in embryonic and fetal lungs of 4–12 wk gestation, but not in the 19-wk fetal lung (canalicular stage), is intriguing. We speculate that IGF-I plays a role in both proliferation and maintaining the population of ECs in early stages of lung vascular development, but not in the midgestation of lung development.

The observation of intense expression (mRNA and protein) of both IGF-I and IGF-II, and of the IGF-I receptor, as early as 4 wk in ECs lining the primary vascular plexuses of human embryonic lungs is significant and highly suggestive of a role for this growth factor in early vascular development. This possibility was supported by studies in which we were able to demonstrate a dramatic reduction in the number of endothelial cells when 12-wk human fetal lung explants were treated with a neutralizing antibody to the IGF-IR. Both TUNEL and transmission electron microscopy analyses of these lung explants revealed significantly increased levels of apoptosis in mesenchymal cells. The apoptosis of mesenchymal cells was not limited to EC only. However, the quantitative analyses demonstrated a significant reduction of CD34-immunoreactive cells in the mesenchyme of anti–IGF-IR–exposed lung explants. This indirectly suggests that a significant number of apoptotic cells were EC. This is consistent with the transmission electron microscopy findings that EC lining the developing vessels and the mesenchymal cells both showed evidence of apoptosis. Furthermore, the presence of large gaps in the mesenchyme of lung explants that were exposed to anti–IGF-IR is consistent with a significant number of mesenchymal cells undergoing apoptosis. We conducted an experiment to prove that the neutralizing antibody we used for human fetal lung explants did have an effect on IGF-IR signaling. Our initial approach to immunoprecipitate IGF-IR followed by receptor phosphorylation on lung explants failed to demonstrate discernible bands. This could be due to the fact that only minute numbers of IGF-IR were present in lung explants that were treated in a serum-free medium for 5 d. We then used an indirect approach using exogenous IGF-I to stimulate the explants from both control and IGF-IR–treated lungs at the end of the experiment. Western analysis demonstrated two bands for phosphorylation approximately at 90 kD, consistent with the molecular weight of IGF-IR (8). The response to exogenous IGF-I stimulation demonstrated that the non-neutralized IGF-IRs in lung explants could still respond after 5 d in serum-free culture medium, and also demonstrated the viability of those explants. However, there was no tyrosine phosphorylation in lung explants that were not stimulated by exogenous IGF-I. Lung explants exposed to anti–IGF-IR neutralizing antibody showed a significant decrease in tyrosine phosphorylation, consistent with IGF-IR signaling being perturbed. However, even with the highest anti–IGF-IR concentration, an incomplete blockage of the IGF-IR signaling pathway was observed. This is in accordance with the observations of Karey and coworkers (27), who used the same antibody to inhibit MCF-7 cell proliferation. The authors reported a maximal inhibition of IGF-mediated cell proliferation of 40–60%.

Using double immunofluorescence for IGF-I and CD34 on embryonic and fetal lungs, we observed the colocalization of IGF-I and CD34 only in lungs at the embryonic and pseudoglanadular stages of lung development, but not in the canalicular stage (19 wk). We used a readily available rat embryonic (E13 gestation) lung explant system to confirm the importance of the IGF/IGF-IR system in lung vascular development. IGF-IR–immunotargeted lungs demonstrated a loss of blood islands compared with control lungs. These data suggest that the IGF system is crucial for the early phases of fetal lung vascular development.

IGF-IR expression has not previously been reported in embryonic/fetal EC. Studies indicate that IGF-I and IGF-IR are not expressed in adult EC (28), but are present in adult EC during periods of rapid EC proliferation (29). Thus the IGF/IGF-IR system appears to be a marker of proliferating EC, which in turn is a requirement of angiogenesis in both adult and fetal vessels.

IGF-I has been reported to upregulate the expression of VEGF (30, 31). Moreover, VEGF-induced retinal neovascularization can be blocked by suppressing IGF-IR activity (32). Therefore, it is possible that a decreased VEGF production in anti–IGF-IR–treated explants may also contribute to the reduction in EC population. Although the expression of IGFs/IGF-IR has not been previously reported in embryonic lung ECs, it has been reported to be present in other cell types of the developing human lung. Lellemand and colleagues (33) demonstrated the expression of IGF-I/IGF-II mRNA in epithelial and mesenchymal cells from 10 wk gestation onwards, whereas Birnbacher and coworkers (13) found IGF-II mRNA in all cell types of midgestation fetal lungs. We found expression of IGF-I/II and IGF-IR as early as 4 wk gestation in both ECs of developing airways and mesenchymal cells. These data suggest that IGFs may play an important role in lung branching morphogenesis through autocrine and/or paracrine pathways. This possibility is strengthened by our data showing that neutralization of IGF-IR in human embryonic lung explants led to a collapse of the developing airways. Blockade of chloride channels in rat fetal lung explants induces collapse of developing airways (34). However, there is no evidence to date that IGFs influence ion or water channel activities. The coincidental finding of airway collapse in immunotargeting IGF-IR in human lung explants needs further elucidation. The airway epithelial cells were somehow protected from undergoing apoptosis despite IGF-IR neutralization. This could be due to production of a survival factor or factors from genes upstream of IGF-IR. Alternatively, airway epithelial cells, unlike mesenchymal cells, may have IGF-I independent programmed cell death pathways.

The presence of IGF-IR on both epithelial and mesenchymal cells of human lung, as shown by immunohistochemistry, is in accordance with studies of IGF receptor mRNA expression in murine lungs (28). Interestingly, targeted disruption of the IGF-IR gene in mice leads to severe growth restriction and neonatal death (35). These authors suggested that the neonatal mice succumbed to respiratory failure as a consequence of poor diaphragm development. However, because no detailed analysis of lung vascular development was conducted, it is possible that defective pulmonary vascular development attenuated lung branching morphogenesis. Improper branching of developing airways might have contributed to the failure of efficient gas exchange at birth.

In conclusion, our data suggest that IGFs play a critical role in human fetal lung vascular development, most likely by acting as an EC survival factor.


   Acknowledgments
 
The authors would like to thank the research nurses, Christine Bedford and Ljiljana Petkovic, for their dedicated effort in the collection of tissue samples. This work was supported by grants from the Canadian Institutes of Health Research.

Received in original form November 1, 2001

Received in final form September 11, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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