Conditional over-expression of RAGE by embryonic alveolar epithelium compromises the respiratory membrane and impairs endothelial cell differentiation

Abstract

BackgroundReceptors for advanced glycation end-products (RAGE) are cell surface receptors prominently expressed by lung epithelium. Previous research demonstrated that over-expression of RAGE by murine alveolar epithelial cells during embryogenesis caused severe lung hypoplasia and neonatal lethality. However, the effects of RAGE over-expression on adjacent matrix and endothelial cells remained unknown.MethodsRAGE transgenic (TG) mice were generated that conditionally over-expressed RAGE in alveolar type II cells when fed doxycycline (dox) from conception to E18.5. To evaluate effects on the basement membrane, immunostaining and immunoblotting were performed for collagen IV and MMP-9, a matrix metalloprotease capable of degrading basement membranes. To assess changes in vasculature, immunostaining, immunoblotting and qRT-PCR were performed for Pecam-1, a platelet endothelial cell adhesion marker also known as CD31. Lastly, to characterize potential regulatory mechanisms of endothelial cell differentiation, immunoblotting and qRT-PCR for FoxM1, a key endothelium-specific transcription factor of the Forkhead Box (Fox) family, were completed.ResultsQualitative immunostaining for collagen IV was less in RAGE TG mice compared to controls and immunoblotting revealed decreased collagen IV in the RAGE TG mouse lung. Additionally, elevated MMP-9 detected via immunostaining and immunoblotting implicated MMP-9 as a possible down stream effector in matrix destabilization mediated by RAGE signaling. Lastly, Pecam-1 assessment revealed a decrease in the prevalence of microvascular endothelial cells coincident with FoxM1 abrogation in RAGE TG mice compared to controls.ConclusionsRAGE over-expression by alveolar epithelium weakened the basement membrane and associated matrix via increased MMP-9 activity. Furthermore, over-expression of RAGE inhibited FoxM1, suggesting that anomalous transcriptional control contributes to decreased endothelial cell prevalence in the TG mouse lung.

The Receptor for Advanced Glycation End Products (RAGE) Contributes to the Progression of Emphysema in Mice

“…RAGE is expressed highly on alveolar type-I (AT-I) cells under normal conditions [ 19 , 40 ] serving as a marker of terminal differentiation on these cells [ 15 ]; hence the absence of RAGE may contribute to defective differentiation of the alveolar epithelial cells lining the lung parenchyma. In fact, transgenic mice that over-express RAGE in SP-C expressing alveolar type—II epithelial cells displayed significant alveolar hypoplasia, alterations in alveolar differentiation and weakened basement membrane [ 41 , 42 ]. In addition mouse models that over-express RAGE have poor alveolar septation during post-natal development [ 33 , 43 ].…”

The receptor for advanced glycation end products is a critical mediator of type 2 cytokine signaling in the lungs

“…17 The receptor for advanced glycation end products (RAGE) is a pattern recognition and danger signal receptor that is most abundantly expressed in the lungs 18,19 and contributes to lung physiology. 20,21 RAGE is a driving force of inflammation in both patients with nonpulmonary [22][23][24][25][26][27] and those with pulmonary [28][29][30][31] diseases. Human genome-wide association studies have identified sequence variants in the gene for RAGE (AGER), which are associated with decreased FEV 1 , a key respiratory parameter impaired in asthmatic patients.…”

Acute secondhand smoke-induced pulmonary inflammation is diminished in RAGE knockout mice

“…RAGE is a cell-surface membrane protein of the immunoglobulin superfamily expressed by many cell types including endothelial and vascular smooth muscle cells, fibroblasts, macrophages/monocytes, and epithelium (3,40). RAGE is purported to play an important role in intracellular signaling observed during the coordination of developmental tasks such as cell spreading and adherence during organ formation (46). Following organogenesis, however, RAGE expression persists at the basolateral membrane of differentiated alveolar epithelial cells, where it influences interactions with the extracellular matrix and may contribute to structural and apoptotic changes associated with alveolar epithelial cell maintenance (8,38).…”