Abstract Rationale Acute Respiratory Distress Syndrome (ARDS) represents the most severe manifestation of acute lung injury (ALI), characterized by alveolar cell death, increased permeability, and severe hypoxemia. Endothelial dysfunction is a hallmark of ARDS and leads to vascular leakage, leukocyte adhesion and loss of alveolar-capillary barrier integrity. We previously identified that endothelial microRNA-1 (miR-1) is downregulated during lung injury, and its restoration confers protection against endothelial death and permeability. We examined the miR-1 biogenesis pathway in endothelial cells (ECs) to find the upstream pathway controlling the miR-1 levels in the lung endothelium and found that it is regulated by a 3’ RNA modification. Methods Primary human lung endothelial cells were transfected with terminal nucleotide transferase-2 (TENT2) siRNA in in vitro knockdown experiments and treated with TNF-α for injury. In vivo, endothelial-targeted TENT2 knockdown was achieved via intranasal delivery of lentivirus expressing TENT2 shRNA under an endothelial-specific promoter in a murine lipopolysaccharide (LPS)-induced ALI model. Lung injury was assessed by measuring protein content, lactate dehydrogenase (LDH) levels, and neutrophil counts in bronchoalveolar lavage (BAL). Cell death in lung tissues and primary endothelial cells was analyzed by detecting DNA fragmentation using Terminal deoxynucleotidyl transferase dUTP nick-end labeling. RNA sequencing of TENT2 (or control) shRNA transduced primary lung endothelial cells identified TENT2-responsive endothelial-specific genes. Results Exposure to LPS or TNF-alpha decreased miR-1 levels in ECs without changing the levels of its precursors, suggesting that endothelial injury enhances mature miRNA decay. We measured the levels of adenylation enzymes known to regulate miRNA decay in stimulated ECs and found that TENT2 is induced by LPS. Knockdown of TENT2 increased miR-1 levels and reduced apoptosis in TNF-alpha-treated ECs. In murine models, LPS exposure increased TENT2 levels in the whole lung and specifically in the isolated ECs. Endothelial-specific knockdown of TENT2 decreased protein leakage, LDH release, and neutrophilia in the BAL of these mice, and TUNEL staining confirmed reduced cell death in the TENT2-deficient lungs. In a combinatorial screening study on HPMECs, we found that TENT2 knockdown downregulates pro-inflammatory genes (DDX58, CD36) and upregulates protective genes (VEGFA, NAGLU). Conclusion Acute lung injury induces TENT2 in the lung endothelial cells. TENT2 regulates a microRNA-driven network of genes controlling EC apoptosis and inflammation. Targeting TENT2 offers a novel RNA-based therapeutic approach to restore endothelial homeostasis and mitigate ALI/ARDS pathogenesis. This abstract is funded by: None
Korde et al. (Fri,) studied this question.