The loss of normal alveolar capillary and deregulated angiogenesis occurs simultaneously in idiopathic pulmonary fibrosis (IPF); however the contributions of specific endothelial subpopulations in the development of pulmonary fibrosis are poorly understood. Herein, we perform single-cell RNA sequencing to characterize the heterogeneity of endothelial cells (ECs) in bleomycin (BLM)-induced lung fibrosis in rats. One subpopulation, characterized by the expression of Nos3 and Cav1, is mostly distributed in non-fibrotic lungs and also highly expresses genes related to the “response to mechanical stimulus” and “lung/heart morphogenesis” processes. Another subpopulation of ECs expanded in BLM-treated lungs, characterized by Cxcl12, is observed to be closely related to the pro-fibrotic process in the transcriptome data, such as “regulation of angiogenesis,” “collagen binding,” and “chemokine activity,” and spatially localized to BLM-induced neovascularization. Using CellPhoneDB software, we generated a complex cell–cell interaction network, which predicts the potential roles of EC subpopulations in recruiting monocytes, inducing the proliferation of fibroblasts and promoting the production and remolding of the extracellular matrix (ECM). Taken together, our data demonstrate the high degree of heterogeneity of ECs in fibrotic lung and it is proposed that the interaction between ECs, macrophages, and stromal cells contributes to pathologic IPF.

The occurrence and development of acute lung injury (ALI) involve a variety of pathological factors and complex mechanisms. How pulmonary cells communicate with each other and subsequently trigger an inflammatory cascade remains elusive. Extracellular vesicles (EVs) are a critical class of membrane-bound structures that have been widely investigated for their roles in pathophysiological processes, especially in immune responses and tumor progression. Most of the current knowledge of the functions of EVs is related to functions derived from viable cells (e.g., microvesicles and exosomes) or apoptotic cells (e.g., apoptotic bodies); however, there is limited understanding of the rapidly progressing inflammatory response in ALI. Herein, a comprehensive analysis of micron-sized EVs revealed a mass production of 1-5 μm pyroptotic bodies (PyrBDs) release in the early phase of ALI induced by lipopolysaccharide (LPS). Alveolar macrophages were the main source of PyrBDs in the early phase of ALI, and the formation and release of PyrBDs were dependent on caspase-1. Furthermore, PyrBDs promoted the activation of epithelial cells, induced vascular leakage and recruited neutrophils through delivery of damage-associated molecular patterns (DAMPs). Collectively, these findings suggest that PyrBDs are mainly released by macrophages in a caspase-1-dependent manner and serve as mediators of LPS-induced ALI.

Background:The presence of no-reflow can increase the risk of major adverse cardiac events and is widely regarded as an important sign of serious prognosis. Previous studies show that laminin receptor (LR) is closely related to the morphology and function of microvessels. However, whether LR is involved in the occurrence and development of no-reflow is still unknown. Methods: In vivo, positron emission tomography (PET) perfusion imaging was performed to detect the effects of intramyocardial gene (LR-AAV and LR-siRNA-AAV) delivery treatment on the degree of no-reflow. In vitro, LC-MS/MS analysis was conducted to identify the LR phosphorylation sites of human cardiac microvascular endothelial cells (HCMECs) treated with oxygen-glucose deprivation (OGD) for 4 h. Western blot analyses were used to evaluate the phosphorylation levels of LR at residues Tyr47 (phospho-Tyr47-LR/pY47-LR) and Thr125 (phospho-Thr125-LR/pT125-LR) and their effects on the phosphorylation of VE-cadherin residue Ser665 (phospho-Ser665-VE-cad). Findings: LR over-expression, LR T125A (phosphonull) and LR Y47A (phosphonull) treatments were found to reduce the level of phospho-Ser665-VE-cad, and subsequently maintain adherent junctions and endothelial barrier integrity in hypoxic environments. Mechanistically, TIMAP/PP1c can combine with LR on the cell membrane to form a novel LR-TIMAP/PP1c complex. The level of pY47-LR determined the stability of LR-TIMAP/PP1c complex. The binding of TIMAP/PP1c on LR activated the protein phosphatase activity of PP1c and regulated the level of pT125-LR. Interpretation: This study demonstrates that low level of phospho-LR reduces no-reflow area through stabilizing the LR-TIMAP/PP1c complex and promoting the stability of adherens junctions, and may help identify new therapeutic targets for the treatment of no-reflow.

Background Idiopathic pulmonary fibrosis (IPF) is a highly heterogeneous and fatal lung disease. In addition to dense fibrous tissue, abnormal angiogenesis is also an important feature of IPF. Pigment epithelium-derived factor (PEDF) is an angiogenesis inhibitor and a potential anti-fibrous factor. The purpose of this experiment is to observe the effect of PEDF on bleomycin (BLM)-induced pulmonary fibrosis in rats. Methods In vivo, pathological examination and detection of related factors were performed on pulmonary fibrosis induced by BLM in rats, and the temporal and spatial distribution of PEDF was investigated. Furthermore, lung gene delivery (PEDF-adeno-associated virus) was performed to investigate the effect of PEDF on pulmonary fibrosis. In vitro, lentiviral vectors were used to construct PEDF over-expression or knock out primary rat lung (PRL) fibroblasts. The effect of PEDF on fibroblast activation under TGF-β1 stimulation was evaluated, and the activation of TGF-β1/smad pathway and PPAR-γ expression (in the presence or absence of PPAR-γ inhibitors) were analyzed. Results In vivo results showed that PEDF expression decreased during the inflammatory phase and increased during the fibrotic phase. PEDF could inhibit the progression of pulmonary fibrosis in rats. In vitro results showed that PEDF could effectively inhibit TGF-β1-stimulated fibroblast activation and reduce the production of α-SMA and collagen-I. PEDF could inhibit the TGF-β1/smad pathway by up-regulating the activity of PPAR-γ. Conclusions PEDF can act as an anti-fibrotic factor, inhibit fibroblast activation by upregulating PPAR-γ activity and reduce BLM-induced pulmonary fibrosis in rats.