Griffin, C. T. | Search bioRxiv

bioRxiv2023

Epigenetic Regulation of Endothelial Extracellular Matrix Components is Critical for Murine Lung Development

BackgroundThe chromatin remodeling enzymes BRG1 (brahma-related gene 1) and CHD4 (chromodomain helicase DNA binding protein 4) independently regulate transcription of genes critical for vascular development, but their coordinated impact on vessels in late- stage embryos has not been explored. MethodsIn this study we genetically deleted endothelial Brg1 and Chd4 in mixed background mice (Brg1fl/fl;Chd4fl/fl;VE-Cadherin-Cre+), and littermates that were negative for Cre recombinase were used as controls. Perinatal lung tissue was analyzed by immunostaining, immunoblots, and flow cytometry. Quantitative reverse transcription PCR was used to determine gene expression, and chromatin immunoprecipitation revealed gene targets of BRG1 and CHD4 in cultured endothelial cells (ECs). ResultsWe found that Brg1/Chd4 double mutants died soon after birth with small and compact lungs. Despite having normal cellular composition, distal air sacs of the mutant lungs displayed diminished ECM (extracellular matrix) components and TGF{beta} (transforming growth factor beta) signaling, which typically promotes matrix synthesis. Transcripts for collagen- and elastin-related genes and the TGF{beta} ligand Tgfb1 were decreased in mutant lung ECs, but genetic deletion of endothelial Tgfb1 failed to recapitulate the small lungs and ECM defects seen in Brg1/Chd4 mutants. We instead found several ECM genes to be direct targets of BRG1 and CHD4 in cultured ECs. ConclusionsCollectively, our data highlight essential roles for ECs in promoting ECM deposition at late stages of embryonic lung development. Moreover, this endothelial ECM production is epigenetically regulated. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/552718v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@210be1org.highwire.dtl.DTLVardef@192ee2dorg.highwire.dtl.DTLVardef@1530595org.highwire.dtl.DTLVardef@1938529_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIGenetic deletion of the chromatin remodeling enzymes BRG1 and CHD4 in endothelial cells of late-stage mouse embryos (Brg1/Chd4-ECdko) results in small and compact lungs at birth. C_LIO_LIMutant embryos display reduced collagen IV deposition, dysregulated elastin fibers, and diminished TGF{beta}1 in the distal air sacs. C_LIO_LIOur combined in vitro and in vivo analyses indicate that BRG1 and CHD4 epigenetically regulate collagen IV- and elastin-related gene expression in embryonic ECs to promote proper lung development. C_LI

developmental biology↗

bioRxiv2023

Cytokine-Mediated Degradation of the Transcription Factor ERG Impacts the Pulmonary Vascular Response to Systemic Inflammatory Challenge

BackgroundDuring infectious diseases, pro-inflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury. MethodsCytokine-dependent ubiquitination and proteasomal degradation of ERG was analyzed in cultured Human Umbilical Vein ECs (HUVECs). Systemic administration of TNF or the bacterial cell wall component lipopolysaccharide (LPS) was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs (Ergfl/fl;Cdh5(PAC)CreERT2), and multiple organs were analyzed by histology, immunostaining, and electron microscopy. ResultsIn vitro, TNF promoted the ubiquitination and degradation of ERG in HUVECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNF or LPS resulted in a rapid and substantial degradation of ERG within lung ECs, but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Ergfl/fl;Cdh5(PAC)-CreERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek, a gene target of ERG previously implicated in maintaining pulmonary vascular stability during inflammation. ConclusionsCollectively, our data highlight a unique role for ERG in pulmonary vascular function. We propose that cytokine-induced ERG degradation and subsequent transcriptional changes in lung ECs play critical roles in the destabilization of pulmonary blood vessels during infectious diseases.

cell biology↗