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Contributes to Postnatal Pulmonary Development and Adult Lung Maintenance Program in Mice. Silvia Fineschi1, Giovanna De Cunto1, Fabrizio Facchinetti2, ...
Receptor for Advanced Glycation End Products Contributes to Postnatal Pulmonary Development and Adult Lung Maintenance Program in Mice Silvia Fineschi1, Giovanna De Cunto1, Fabrizio Facchinetti2, Maurizio Civelli2, Bruno P. Imbimbo2, Chiara Carnini2, Gino Villetti2, Benedetta Lunghi1, Stefania Stochino1, Deena L. Gibbons3, Adrian Hayday3, Giuseppe Lungarella1, and Eleonora Cavarra1 1

Departments of Physiopathology, Experimental Medicine, and Public Health, University of Siena, Siena, Italy; 2Preclinical Research and Development, Chiesi Farmaceutici S.p.A., Parma, Italy; and 3Peter Gorer Department of Immunobiology, King’s College London, Guy’s Hospital, London, United Kingdom

The role of the receptor for advanced glycation end products (RAGE) in promoting the inflammatory response through activation of NF-kB pathway is well established. Recent findings indicate that RAGE may also have a regulative function in apoptosis, as well as in cellular proliferation, differentiation, and adhesion. Unlike other organs, lung tissue in adulthood and during organ development shows relatively high levels of RAGE expression. Thus a role for the receptor in lung organogenesis and homeostasis may be proposed. To evaluate the role of RAGE in lung development and adult lung homeostasis, we generated hemizygous and homozygous transgenic mice overexpressing human RAGE, and analyzed their lungs from the fourth postnatal day to adulthood. Moderate RAGE hyperexpression during lung development influenced secondary septation, resulting in an impairment of alveolar morphogenesis and leading to significant changes in morphometric parameters such as airspace number and the size of alveolar ducts. An increase in alveolar cell apoptosis and a decrease in cell proliferation were demonstrated by the terminal deoxy-nucleotidyltransferase–mediated dUTP nick end labeling reaction, active caspase-3, and Ki-67 immunohistochemistry. Alterations in elastin organization and deposition and in TGF-b expression were observed. In homozygous mice, the hyperexpression of RAGE resulted in histological changes resembling those changes characterizing human bronchopulmonary dysplasia (BPD). RAGE hyperexpression in the adult lung is associated with an increase of the alveolar destructive index and persistent inflammatory status leading to “destructive” emphysema. These results suggest an important role for RAGE in both alveolar development and lung homeostasis, and open new doors to working hypotheses on the pathogenesis of BPD and chronic obstructive pulmonary disease. Keywords: RAGE; lung septation; developmental emphysema; TGF-b; destructive emphysema

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily (1) and a multiligand receptor for advanced glycation end products (AGEs) (2), S100/ calgranulins (3), amphoterin (4), and Mac-1 integrin (5). After being highly expressed during embryonic development, RAGE is down-regulated in most of healthy adult tissues, and becomes up-regulated wherever its ligands accumulate (6). The binding of these ligands to the receptor results in multiple cellular (Received in original form March 21, 2012 and in final form October 4, 2012) This work was supported by grant Fondo Investimenti Ricerca di Base RBIP06YM29 from the Italian Ministry for University and Research (Rome, Italy). Correspondence and requests for reprints should be addressed to Eleonora Cavarra, Ph.D., Department of Physiopathology, University of Siena, Via Aldo Moro 6, I-53100 Siena, Italy. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 48, Iss. 2, pp 164–171, Feb 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2012-0111OC on November 9, 2012 Internet address: www.atsjournals.org

CLINICAL RELEVANCE This research shows that the receptor for advanced glycation end products (RAGE) plays an important role in both alveolar development and adult lung homeostasis. The hyperexpression of RAGE during lung development influences secondary septation, resulting in an impairment of alveolar morphogenesis, with alteration in elastin organization. In adult lungs, RAGE hyperexpression is associated with an increase of the alveolar destructive index and a persistent inflammatory status leading to “destructive” emphysema. This study opens new doors to working hypotheses on the pathogenesis of bronchopulmonary dysplasia and chronic obstructive pulmonary disease.

effects, including the generation of reactive oxygen species (ROS) and the induction of the inflammatory response through the NF-kB pathway. Unlike other organs, adult pulmonary tissue shows a relatively high basal expression of RAGE, even under physiological conditions, but the exact localization of the receptor in the lung has remained a controversial issue. A selective localization of RAGE on the basolateral membrane of alveolar Type I (ATI) cells has been recently demonstrated (7), suggesting it plays a significant role in the induction of cell spreading and adherence (8). On the other hand, several studies indicated RAGE mRNA and protein expression in alveolar Type II cells (9, 10), where the receptor seemed to be involved in the epithelial–mesenchymal transition (11). The high expression of RAGE in normal adult pulmonary tissue suggests an important role for this receptor both in lung development and in maintaining lung homeostasis. However, much more work is necessary to demonstrate the involvement of RAGE in lung physiology. In the mouse, lung development is divided into four stages (12). During the pseudoglandular stage (Embryonic Days [E] 9.5–E16.5), the respiratory tree and pulmonary vasculature develop. At the canalicular stage (E16.5–E17.5), terminal bronchioles expand to form the respiratory ducts and sacs, and the saccular stage (E17.5 to Postnatal Day 5) is characterized by the differentiation of Type I and Type II pneumocytes. The septation of saccules that give rise to alveoli begins on Postnatal Day 5 (i.e., the alveolarization phase). The finely regulated temporal–spatial expression of RAGE during lung development has been observed in mice. This protein is poorly expressed in undifferentiated parenchymal cells during the canalicular stage, and is up-regulated during the postnatal period, when alveolar remodeling occurs (13). High levels of RAGE expression in ATI epithelial cells, vessels, and respiratory

Fineschi, De Cunto, Facchinetti, et al.: RAGE in Lung Development and Maintenance Program

epithelium are seen in this phase, characterized by cellular proliferation, migration, survival, and differentiation. This suggests that RAGE plays a role in later stages of lung morphogenesis (13). At the same time, several studies performed on various tissues stressed a physiologic role for this receptor in cellular survival (14), apoptosis (15, 16), and proliferation (17). To shed light on the role of RAGE in both lung development and lung homeostasis during adult life, we generated C57 transgenic mice overexpressing human full-length RAGE (C57Bl/ 6JTg(hRAGE)/1 mice) and analyzed their lungs from Postnatal Day 4 to adulthood. Some of the results of this study were previously reported in the form of abstract (18).

MATERIALS AND METHODS Detailed methods are available in the online supplement.

Generation of Human RAGE Transgenic Mice A 8.1-kb human bacterial artificial chromosome (BAC) clone (BAC RP11346J19) containing the entire genomic sequence of human RAGE, as accessed from public databases, was obtained from the Children’s Hospital Oakland Research Institute (Oakland, CA). The human RAGE (hRAGE) gene was under the control of the endogenous human promoter, and was flanked by a small amount of polylinker (z 35 bp). This BAC DNA (5 ng/ml) was microinjected into the pronuclei of C57 Bl/6J eggs. F0 (founder) pups were screened for their incorporation of the transgene, using the human-specific RAGE primer set 59-GTG GGG GAA AAG TAA CAT CAA CAC-39 and 39-CAT CAG TCC ATC AGG GCT GC-59. The expression of hRAGE in the lungs of transgenic mice was checked by RT-PCR and Western blotting. RAGE immunoreactive staining was also scored on lung sections from the different experimental groups at 20 days of age. The colony of hemizygous C57Bl/6JTg(hRAGE)/1 mice was maintained by breeding transgenic individuals to C57 Bl/6J mice. Tail biopsies were genotyped by PCR for hRAGE, using the already mentioned primers. Our laboratory has also bred C57Bl/6JTg(hRAGE)/1 male mice with C57Bl/6JTg(hRAGE)/1 female mice to generate homozygous C57Bl/6JTg (hRAGE)/(hRAGE) mice.

Morphology and Morphometry Eight mice in each group were killed at different ages (i.e., 4, 8, and 20 d, and 1, 6, 10, and 18 mo) under anesthesia with sodium pentobarbital. The lungs were removed for the assessment of mean linear intercepts (Lm), internal surface area (ISA), destructive index (DI) values, and secondary crest counts.

Real-Time PCR Real-time PCR was performed to determine the relative amounts of hRAGE and internal reference 18S ribosomal (r)RNA in the lungs of C57Bl/6JTg(hRAGE) /1 and C57Bl/6JTg (hRAGE)/(hRAGE) mice.

Immunohistochemistry and Weigert’s Resorcin–Fuchsin Elastin Staining The expression of RAGE, transforming growth factor (TGF)–b1, active caspase-3, phosphorylated SMAD2, and Ki-67 was assessed in 4-mm tissue sections from paraffin blocks, prepared as already described for lung morphometric analysis. Sections (5 mm) were stained for elastin with Weigert’s stain.

Terminal dUTP Nick-End Labeling Apoptosis Assay Apoptosis was evaluated by terminal deoxy-nucleotidyltransferase–mediated dUTP nick end labeling (TUNEL) assay.

Statistical Analysis For each parameter, both measured and calculated, the values for individual animals were averaged, and the SD was calculated. The significance

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of the differences between groups was calculated using one-way ANOVA (F-test). A P value of < 0.05 was considered significant.

RESULTS Characterization of C57Bl/6JTg (hRAGE)/1 and C57Bl/6JTg (hRAGE)/(hRAGE) Mice

Semiquantitative RT-PCR analysis was used to evaluate hRAGE gene expression in transgenic mice. As expected, hRAGE mRNA is undetectable in lung tissue from WT mice and is clearly detectable in the lung tissue of transgenic mice (see Figure E1A in the online supplement). To confirm RAGE overexpression in C57Bl/ 6JTg (hRAGE)/1 mice, lung tissue from these animals and from wild-type (WT) mice was analyzed by Western blotting and immunohistochemistry. The blots showed a single band of 50 kD, corresponding to human full-length RAGE in C57Bl/6JTg (hRAGE)/1 murine lungs, whereas that band was not detected in WT mice (Figure E1B). RAGE immunolocalization confirmed an overexpression of this protein in the epithelial cells of C57Bl/ 6JTg (hRAGE)/1 mice (Figure E2B). Generally, C57Bl/6JTg (hRAGE)/1 mice showed normal phenotypes at the ages studied. On the other hand, C57Bl/6JTg (hRAGE)/(hRAGE) mice showed delayed body development and, in some cases, respiratory failure with increased postnatal mortality (greater than 50%). Real-time PCR was performed to confirm the increased expression of the transgene in C57Bl/6JTg(hRAGE)/ (hRAGE) mice compared with C57Bl/6JTg(hRAGE)/1 mice. The hRAGE mRNA concentrations in C57Bl/6JTg(hRAGE)/1 mice were increased from fourfold to 6.2-fold compared with WT mice, and were further increased (up to 9.8-fold versus WT mice) in C57Bl/6JTg(hRAGE)/(hRAGE) mice, characterized by a more severe phenotype. The immunolocalization of RAGE in the lungs of the C57Bl/6JTg(hRAGE)/(hRAGE) animals confirmed a higher expression of the protein than that present in C57Bl/6JTg(hRAGE)/1 mice (Figure E2C). The overall RAGE immunoreactivity scores for the different experimental groups were 1.2 6 0.2 for WT, 2.0 6 0.4 for C57Bl/6JTg(hRAGE)/1, and 2.8 6 0.4 for C57Bl/6JTg(hRAGE)/(hRAGE) mice. Lung Morphology and Morphometry in Mice Overexpressing RAGE

To determine the role of RAGE in lung development and homeostasis, the lung morphology of C57Bl/6JTg(hRAGE)/1 mice was compared with that of littermates at various days of age. Throughout this period, upon gross examination, the lungs from control and transgenic mice revealed no differences in morphology (i.e., in terms of appearance, number of lobes, lung shape, and weight). Abnormalities in lung morphology and morphometry were observed early in C57Bl/6JTg(hRAGE)/1 mice, from Postnatal Day 4 (PN4). At this time in WT mice, secondary alveolar crests develop and extend to make new secondary septa. This results in an increase of alveolar units, accompanied by decreased mean alveolar size. Unlike WT animals, the lungs of transgenic mice showed diffuse airspace enlargement, short or absent secondary septa, and no inflammatory cell infiltration (Figures 1A–1F). These observations were confirmed by secondary crest counts and by the determination of Lm and ISA (Table 1). The average volume density ratio of secondary crests to lung parenchyma increased significantly both in control mice and C57Bl/6JTg(hRAGE)/1 mice between PN4 and PN20. However, the volume density ratio of secondary crests/lung parenchyma in the lungs of C57Bl/6JTg(hRAGE)/1 mice was significantly lower than that in control mice. On the fourth day after the birth, the lungs of transgenic mice already showed a significant increase of Lm values and a significant decrease of ISA values, compared with those of littermates. These

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Figure 1. Lung morphology in wild-type (WT) and C57Bl/6JTg(hRAGE)/1 mice. Histological sections from lungs of WT (A–C) and C57Bl/6JTg(hRAGE)/1 (D–I) mice at ages 4 days (A and D), 8 days (B and E), 20 days (C and F), 6 months (G), 10 months (H), and 18 months (I). (D) Defects of septation are already evident in the lung parenchyma of C57Bl/6JTg(hRAGE)/1 mice on Postnatal Day (PN) 4. On PN8 and PN20, alveolar areas are significantly increased in transgenic mice (E and F), compared with age-matched control mice (B and C). Ten-month-old (H) and 18month-old (I) C57Bl/6JTg(hRAGE)/1 mice show a progressive loss of alveolar structures, accompanied by significant inflammatory cell infiltration (insets) in respect to the lungs of 6-month-old C57Bl/6JTg(hRAGE)/1 mice (G). Hematoxylin and eosin staining. Scale bar ¼ 50 mm for A–F, and 90 mm for G–I. Tg, hRAGE, human receptor for advanced glycation end products; Tg, transgenic.

changes were more striking in C57Bl/6JTg(hRAGE)/1 murine lungs at 20 and 30 days of age. DI values in lungs of transgenic mice were not significantly different from those of control mice until 1 month of age (Table 1). Together, these results suggest that the airspace enlargement arising in C57Bl/6JTg(hRAGE)/1 mice is attributable to an altered alveolarization process. No significant changes in morphology and in morphometry (i.e., Lm, ISA, and DI) were observed between 1-month-old and 6-month-old WT and transgenic mice. This means that the postnatal growth of murine lungs is terminated at 1 month of age. From 10–18 months of age, C57Bl/6JTg(hRAGE)/1 adult mice showed a loss of alveolar structures, accompanied by significant inflammatory cell infiltration (Figures 1H and 1I) that resulted in a significant increase of Lm and a significant decrease of ISA values in respect to 6-month-old C57Bl/6JTg(hRAGE)/1 mice (Table 1 and Figure 1G). No significant changes in these parameters were observed in WT mice. The significant changes in DI we detected among transgenic mice from ages 6–18 months suggest that the variations in Lm and ISA values we observed in 18-month-old C57Bl/6JTg(hRAGE)/1 mice may be attributable to the destruction of alveolar walls (Table 1).

The highest expression of RAGE in homozygous C57Bl/ 6JTg(hRAGE)/(hRAGE) mice resulted in poor alveolar development (Figure 2A), with a significant increase of Lm (76.44 6 2.33 mm) and a significant decrease of ISA (319.96 6 72.79 cm2) and volume density ratio of secondary crests, compared with those of C57Bl/ 6JTg(hRAGE)/1 mice (Figure 3A). In these animals we also observed histological changes similar to those described in human bronchopulmonary dysplasia (BPD). These alterations included abnormal alveolar formation with thick interstitium and hypercellular septa, and abnormal vascular development. Vascular changes consisted of mild medial thickening and abnormalities in elastic fiber architecture. Homozygous C57Bl/6JTg(hRAGE)/(hRAGE) mice showed dilated airspaces, with thin alveolar walls (Figure 2C) interspersed between focal and widened septa (Figure 2B). This picture fits with the morphological pattern found in biopsies of very immature and low birth weight infants (19). Elastic Fiber Staining

The evaluation of alveolar elastic fibers was performed in lung tissue from WT, C57Bl/6JTg(hRAGE)/1, and C57Bl/6JTg(hRAGE)/(hRAGE) mice after Weigert’s resorcin–fuchsin staining. The fibers were

TABLE 1. LUNG MORPHOMETRY IN C57BL/6J AND C57BL/6J

WT 6 6 6 6

ISA (cm )

C57Bl/6J

4d 8d 20 d 1 mo

57.73 48.46 44.38 42.49

3.01 0.49* 1.88* 2.95*

6 mo 10 mo 18 mo

39.90 6 1.92 41.00 6 1.71 42.00 6 1.61

70.40 67.00 64.56 58.40

MICE

2

Lm (mm) Age

TG (hRAGE)/1

Tg(hRAGE)/1

6 6 6 6

5.07† 2.94† 4.09† 6.14†

60.00 6 2.01† 62.40 6 2.88† 65.00 6 1.90†,‡

WT

C57Bl/6J

DI (%) Tg(hRAGE)/1

6 20 142 6 27 6 105 522 6 83† 6 136 508 6 114† 6 195 641 6 144† Adult Life 856 6 38 617 6 42† 897 6 101 590 6 119† 885 6 102 535 6 113†,‡ 152 660 626 830

C57Bl/6JTg(hRAGE)/1

WT 10.56 9.40 11.60 12.30

6 6 6 6

1.04 2.35 2.67 2.07

13.00 6 0.50 12.4 6 3.50 14.40 6 2.23

11.20 10.00 12.60 13.20

6 6 6 6

1.50 2.60 2.21 6.30

13.70 6 2.31 15.60 6 2.90†,‡ 21.30 6 4.05†,‡

Definition of abbreviations: DI, destructive index; hRAGE, human receptor for advanced glycation end product; ISA, internal surface area of lungs; Lm, mean linear intercept; TG, transgenic; WT, wild-type. Data are presented as mean 6 SD. Eight mice were included in each group. * P , 0.05, versus 4-day-old WT mice. y P , 0.05, versus WT mice of the same age. z P , 0.05, versus 6-month-old mice of the same strain.

Fineschi, De Cunto, Facchinetti, et al.: RAGE in Lung Development and Maintenance Program

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Figure 2. Lung morphology in C57Bl/6JTg(hRAGE)/(hRAGE) mice. (A) The highest expression of RAGE in homozygous 20-day-old C57Bl/6JTg(hRAGE)/(hRAGE) mice results in poor alveolar development. Lung histology shows a severe histologic pattern characterized by dilated airspaces because of alveolar hypoplasia (C) and areas of alveolar interstitial thickening (B). Hematoxylin and eosin staining. Scale bar ¼ 70 mm for A, and 27 mm for B and C.

more densely packed and tight in control mice (Figure 3B) compared with fibers of transgenic mice, in which they were fragmented, thin, not well-organized, and distributed in the alveolar wall with an unraveled and loose appearance (Figure 3C). Elastin organization and deposition were even more impaired in the lungs of C57Bl/6JTg(hRAGE)/(hRAGE) mice (Figure 3D). TUNEL Test

As expected during postnatal lung development, in control mice 4 days after birth we observed a high number of cells with double DNA strand breaks (Figure E3A). Compared with WT mice (Figures E3A and E3D), C57Bl/6JTg(hRAGE)/1 mice showed a significant increase in the number of TUNEL-positive cells at both 4 days (Figure E3B) and 8 days (data not show) of age, as well as on Day 20 (Figure E3E) and Day 30 (data not shown). At the same time points, a further increase in TUNEL-positive cells was observed in the alveolar walls of C57Bl/6JTg(hRAGE)/(hRAGE) mice (Figures E3C and E3F). Active Caspase-3 and Ki-67 Immunohistochemistry

On Postnatal Days 4 (Figures 4A and 4B), 8 (data not shown), and 20 (Figures 4D and 4E), positive signals for active caspase-3 were predominantly detected in the alveolar epithelial cells of

lung tissue from both WT and C57Bl/6JTg(hRAGE)/1 mice. The immunoreaction for the active enzyme was significantly increased in lung epithelial cells of C57Bl/6JTg(hRAGE)/1 transgenic mice, compared with control mice. Of interest, positive signals for cleaved caspase-3 were also detected in 1-month-old transgenic murine lungs, when the alveolarization process was already completed (Figure E4). Immunostaining for active caspase-3 was much more evident in C57Bl/6JTg(hRAGE)/(hRAGE) lungs (Figures 4C and 4F). Cell proliferation was determined by Ki-67 immunostaining. As expected, a sharp rise in cell proliferation occurred just at the beginning of the alveolarization phase (Figure 5A), and slowly decreased from this point onward (Figure 5D). On the other hand, cell proliferation was drastically reduced in the epithelia of C57Bl/6JTg(hRAGE)/1 lungs at the same time point (Figures 5B and 5E). More evident was the reduction of epithelial cell proliferation in C57Bl/6JTg(hRAGE)/(hRAGE) lungs at the various time points (Figures 5C and 5F). The quantification of the immunohistochemical analysis for proliferation (Ki-67) and apoptosis (active caspase-3 and TUNEL) is presented in Figure E5A–E5C. These results suggest that the overexpression of RAGE may influence the apoptosis/proliferation balance by inducing impaired alveolar development with alveolar hypoplasia and a decrease in alveolar number and internal surface area.

Figure 3. hRAGE transgenic mice show reduced secondary crest formation and abnormal elastin structure in alveolar septa. (A) The volume density ratio of secondary crests/lung parenchyma increases significantly both in control mice and in C57Bl/6JTg(hRAGE)/1 mice from PN4 to PN20. However, the volume density of secondary crests in the lungs of C57Bl/6JTg(hRAGE)/1 mice is significantly lower than that of control mice. The decrease of secondary crest volume density is more pronounced in homozygous C57Bl/6JTg(hRAGE)/(hRAGE) mice. *P < 0.05, versus WT mice. yP < 0.05 versus C57Bl/6JTg(hRAGE)/1 mice. (B–D) Representative lungs from 20-day-old WT (B), C57Bl/6JTg(hRAGE)/1 (C), and C57Bl/6JTg(hRAGE)/(hRAGE) (D) mice after Weigert’s resorcin–fuchsin staining are shown. (C) Elastin fibers in the alveolar septa of C57Bl/ 6JTg(hRAGE)/1 are fragmented, tortuous, and irregularly distributed. (D) The elastin organization and deposition are even more impaired in lungs of C57Bl/ 6JTg(hRAGE)/(hRAGE) mice. Scale bar ¼ 20 mm.

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 48 2013 Figure 4. Effects of hRAGE expression on apoptosis. Lung sections from WT (A), C57Bl/6JTg(hRAGE)/1 (B), and C57Bl/6JTg(hRAGE)/(hRAGE) (C) mice at 4 days after birth were assayed for active caspase-3 by immunohistochemistry to detect apoptotic cells. Caspase-3 active signal was also tested in lungs from WT (D), C57Bl/6JTg(hRAGE)/1 (E), and C57Bl/ 6JTg(hRAGE)/(hRAGE) (F) mice at age 20 days. The active enzyme was detected primarily in alveolar epithelial cells of lung tissue from WT mice on PN4 (A) and PN20 (D). The immunoreaction was significantly increased in lung epithelial cells of C57Bl/ 6JTg(hRAGE)/1 both at PN4 (B) and at PN20 (E). An active caspase-3 signal is much more evident in C57Bl/6JTg(hRAGE)/(hRAGE) mice on PN4 (C) and on PN20 (F). Scale bar ¼ 20 mm.

TGF-b Immunohistochemistry

During lung development, apoptosis may be mediated by several proapoptotic factors such as TGF-b1 that are consistently expressed in the alveolar environment. As expected, this cytokine was highly expressed in the bronchiolar epithelium and alveolar cells of 4-day-old WT mice (Figure 6A). The reaction became more faint as the lungs developed, and was almost undetectable in adult murine lungs (Figure 6C). In the lungs of C57Bl/6JTg(hRAGE)/1 mice, the immunohistochemical staining for TGF-b1 was stronger than that observed in WT lungs. Moreover, the reaction was widespread in all alveolar structures, and was unchanged throughout all ages examined (Figures 6B and 6D). TGF-b1 expression was further increased in C57Bl/6JTg(hRAGE)/(hRAGE) lungs (Figure 6E). Activation of the TGF-b1 signaling pathway was confirmed by the phosphorylation of SMAD2, whose increase paralleled that of TGF-b1 expression (Figures E6A–E6F).

DISCUSSION In the present study, we used C57 transgenic mice overexpressing human full-length RAGE to investigate the role of this receptor both in lung development (specifically during the period of active alveolar formation) and in the maintenance of the adult lung. The results obtained demonstrate that hRAGE overexpression impairs alveolar morphogenesis during murine lung development, and induces further distal airspace enlargement in adult life.

During the postnatal period, the secondary alveolar crests of WT mice develop and extend to make new secondary septa. This process results in an increase of alveolar units and in significantly decreased Lm values (mean alveolar size) during the first weeks of age (20, 21). Unlike WT animals, the decrease in mean alveolar size was not observed among RAGE-overexpressing mice because of a defect in alveolarization. Notably, at 4 days after the birth, the lungs of RAGE transgenic mice already show significant airspace enlargement, in respect to WT mice of the same age. Thus, the impairment of alveologenesis during the postnatal period results in a reduction of the number of alveolar septa and an increase in size of the alveolar ducts (“developmental emphysema”) in transgenic mice. The absence of inflammatory infiltrate and the decrease of secondary crests in the lungs of these mice suggest that RAGE overexpression alters the alveolar phase of lung development by influencing secondary septation. Of interest, this “emphysematous-like” phenotype is observed in all transgenic animals, and seems directly related to levels of RAGE expression. Thus, homozygous C57Bl/6JTg (hRAGE)/(hRAGE) mice show a more severe histologic pattern, characterized by alveolar hypoplasia and by varying degrees of interstitial thickening, reminiscent of the pathological alterations described in BPD infants. This condition results in respiratory failure and high perinatal mortality in more than 50% of animals. The reduced number of airspaces in RAGE transgenic mice prompted us to investigate some alveologenic factors that may predispose these mice to altered terminal septation.

Figure 5. Effects of hRAGE expression on cell proliferation. Immunolocalization of Ki-67 was performed on lung sections from WT (A), C57Bl/6JTg(hRAGE)/1 (B), and C57Bl/6JTg(hRAGE)/(hRAGE) (C) mice at 4 days after birth. Ki-67 was also tested in lungs from WT (D), C57Bl/6JTg(hRAGE)/1 (E), and C57Bl/6JTg(hRAGE)/(hRAGE) (F) mice at age 20 days. Staining for Ki-67 was very intense in the epithelia of WT mice at the beginning of the alveolar phase, on PN4 (A), becoming less abundant on PN20 (D). C57Bl/6JTg(hRAGE)/1 (B and E) and C57Bl/6JTg(hRAGE)/(hRAGE) (C and F) lungs show a drastic reduction of cell proliferation with respect to age-matched WT control mice. Scale bar ¼ 10 mm.

Fineschi, De Cunto, Facchinetti, et al.: RAGE in Lung Development and Maintenance Program

169 Figure 6. Immunohistochemical staining for transforming growth factor (TGF)–b1. (A) TGF-b1 reaction in bronchiolar epithelium and alveolar cells of 4-day-old WT mice. (C) The reaction becomes most faint on PN20, when the lung develops. On PN4 (B) and at PN20 (D), the lung TGF-b1 signal in C57Bl/6JTg(hRAGE)/1 mice is stronger than that observed in WT lungs. (E) TGF-b1 expression is much more evident in C57Bl/6JTg(hRAGE)/(hRAGE) 20-dayold mice. Scale bar ¼ 40 mm.

The synthesis and deposition of elastin, a structural protein of the extracellular matrix, play an essential role during lung development, particularly in the formation of alveolar septa (22, 23). Mice overexpressing hRAGE show a fragmentation of their elastic fibers. TGF-b overexpression was recently reported to cause disorganized elastic fiber deposition in alveologenesis through the expression of connective tissue growth factor (24). This finding strongly suggests that under our experimental conditions, RAGE may interfere with elastin organization and deposition in alveolar septa through TGF-b expression. To determine more fully the cellular processes by which RAGE hyperexpression alters secondary crest formation, we evaluated the degree of cell apoptosis in the lungs of WT and transgenic mice. Apoptotic activity has been observed during all stages of lung development, suggesting its important role during lung organogenesis. Cell apoptosis occurs in a spatially and temporally ordered manner, involving specific cells during fetal and postnatal lung development. Normal lung development is associated with a progressive increase of apoptosis that constantly counteracts proliferation. Recent studies have shown that the engagement of RAGE promotes the apoptosis of osteoblastic cells (25), and that its inhibition suppresses LPS-induced apoptosis in alveolar Type I cells (26). In addition, the activation of RAGE, via high mobility group box 1 (HMGB1) or S100A14, was demonstrated to induce caspase-3–dependent apoptosis in peritoneal macrophages (27) and in esophageal squamous cell carcinoma cell lines (16), respectively. In agreement with these observations, we detected an increased number of apoptotic caspase-3–positive cells in the alveolar septa of C57Bl/6JTg(hRAGE)/1 mice, compared with those of WT mice. The molecular pathway by which RAGE mediates apoptosis is not yet clear, but it seems to be different according to the ligands and cell type involved in this process. A number of studies demonstrated that the interaction of AGEs or HMGB1 with RAGE can induce apoptosis in fibroblasts, endothelial progenitor cells, and macrophages, both directly (by the activation of caspases) and indirectly (by the formation of ROS) (27–29). Apoptosis may be regulated during lung development by a balance of proapoptotic factors such as TGF-b1 (30) and antiapoptotic factors such as insulin-like growth factor-1 (31), nitric oxide (32), and secreted apoptosis-related proteins (33) that are usually found in the alveolar environment. Because interactions between RAGE and its ligands can influence the expression of TGF-b1, we investigated the expression of the cytokine in the lungs of WT and transgenic mice (34, 35). In the present study, we observed increased TGF-b expression in the epithelial lung cells of RAGE-overexpressing mice (from Postnatal Day 4 to 1 month of age) and an activation of the TGF-b pathway (as revealed by phosphorylated SMAD2) that may result in the activation of the apoptosis cascade (36). TGF-b1 is well known to play a key role in lung morphogenesis. In fact, TGF-b inhibits the proliferation of Type II pneumocytes (37) and prevents, at high concentrations, the differentiation of Type II pneumocytes into Type I pneumocytes (38). Abnormal alveolarization was also observed to occur in mice overexpressing this cytokine immediately after birth (PN7–PN14) (39).

The data generated in our murine model overexpressing RAGE also suggest that high concentrations of TGF-b1 can induce not only the increased apoptosis of alveolar cells, but also a low proliferative rate of these cells, as revealed by Ki-67 immunostaining. The excessive amount of apoptotic cells and the significant decrease in proliferation rate that we observed during the last phases of transgenic lung development (PN4, PN8, and PN20) can induce an imbalance between cell proliferation and apoptosis, leading to impaired alveolar formation. The HMGB1–RAGE interaction was recently reported to be essential for the flattening and elongation of neurites (40), as well as for the mobility of neurons and epithelial cells during the development of the nervous system (41). HMGB1, the ligand with the greatest affinity for RAGE (4), is abundantly expressed in lung tissue under physiological conditions (42). Therefore, we suggest that HMGB1 may be responsible for the activation of RAGE during lung development, and consequentially for impaired alveolar formation when very high concentrations of the receptor are present. Of interest, in homozygous C57Bl/6JTg (hRAGE)/(hRAGE) mice we observed a number of morphological features characterizing human BPD, namely, abnormal elastin deposition, alveolar hypoplasia, and interstitial thickening (43), accompanied by TGF-b1 overexpression and increased apoptosis (44). Histologic alterations similar to those of BPD have been also reported in TGF-b1 transgenic mice (39), and increased concentrations of total TGF-b were found in the bronchoalveolar lavage fluid, tracheal aspirates, and serum of premature infants who subsequently developed BPD (45–47). Despite evidence accumulated over the last few years implicating a physiologic role of RAGE (13–17) in several processes involved in lung organogenesis, few studies have focused on the possible role of the receptor in lung development. As previously mentioned, Reynolds and colleagues (13) very recently demonstrated a fine-tuned regulation of RAGE expression during lung development. However, the real contribution of RAGE to lung morphogenesis has not yet been definitely established. During the preparation of this study, Reynolds and colleagues reported that RAGE overexpression during the early phases of embryonic development results in lung hypoplasia and airspace enlargement (48). Our results add some knowledge regarding the role of RAGE in lung development, demonstrating that this receptor may alter the apoptosis–proliferation balance by TGF-b1 expression, and signaling as revealed by SMAD2 phosphorylation. Although a great amount of data suggest that TGF-b1 activation may represent an important death signal (36), an unresolved issue involves the way in which caspase activation is regulated by TGF-b1. Much more work is necessary to generate complete and conclusive data. RAGE overexpression in mice provides a suitable model to study the molecular mechanisms involved in lung organogenesis. Understanding these mechanisms may be important in comprehending the pathogenic mechanisms implicated in the development of BPD.

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The persistence of RAGE expression in the adult lung suggests a role for this receptor in lung homeostasis. C57Bl/6JTg (hRAGE)/(1) mice showed a progressive loss of alveolar structures with age, accompanied by persistent inflammatory cell infiltration. This suggests that RAGE overexpression may also cause true (“destructive”) emphysema in adult mice. Aging is known to be associated with the accumulation of AGEs formed by the nonenzymatic glycation and oxidation of proteins (49). Because AGE–RAGE interactions activate the transcription of multiple genes encoding proinflammatory molecules, we propose that the AGE-mediated hyperactivation of RAGE determines, in the lungs of adult transgenic mice, a proinflammatory status leading to destructive emphysema (50). Several intracellular messengers and pathways involved in lung development are altered in chronic lung diseases such as chronic obstructive pulmonary disease (COPD) (51). An understanding of these mechanisms may be critical for an understanding of important pathogenic signaling. Further studies in this field may shed light on the mechanisms implicated in alveolar lung destruction, and may open the way for new working hypotheses on COPD. Author disclosures are available with the text of this article at www.atsjournals.org.

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