- 作者: Ting-Hein Lee, Sung-Tsang Hsieh and Hou-Yu Chiang
- 作者服務機構: 1. Department of Anatomy, College of Medicine, Chang Gung University, 259 Wenhua 1st Rd., Guishan Dist, Taoyuan City, 33302, Taiwan 2. Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan 3. Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital, Linkou, Taiwan 4. Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan 5. Graduate Institute of Brain and Mind Sciences, College of Medicine, National Taiwan University, Taipei, Taiwan 6. Department of Neurology, National Taiwan University Hospital, Taipei, Taiwan
- 中文摘要:
- 英文摘要:
Background
The blood–spinal cord barrier (BSCB) is composed of a monolayer of endothelium linked with tight junctions and extracellular matrix (ECM)-rich basement membranes and is surrounded by astrocyte foot processes. Endothelial permeability is regulated by interaction between endothelial cells and ECM proteins. Fibronectin (FN) is a principal ECM component of microvessels. Excessive FN deposition disrupts cell–cell adhesion in fibroblasts through β1 integrin ligation. To determine whether excessive FN deposition contributes to the disruption of endothelial integrity, we used an in vitro model of the endothelial monolayer to investigate whether the FN inhibitor pUR4 prevents FN deposition into the subendothelial matrix and attenuates endothelial leakage.
Methods
To correlate the effects of excessive FN accumulation in microvessels on BSCB disruption, spinal nerve ligation—which induces BSCB leakage—was applied, and FN expression in the spinal cord was evaluated through immunohistochemistry and immunoblotting. To elucidate the effects by which pUR4 modulates endothelial permeability, brain-derived endothelial (bEND.3) cells treated with tumor necrosis factor (TNF)-α were used to mimic a leaky BSCB. A bEND.3 monolayer was preincubated with pUR4 before TNF-α treatment. The transendothelial electrical resistance (TEER) measurement and transendothelial permeability assay were applied to assess the endothelial integrity of the bEND.3 monolayer. Immunofluorescence analysis and immunoblotting were performed to evaluate the inhibitory effects of pUR4 on TNF-α-induced FN deposition. To determine the mechanisms underlying pUR4-mediated endothelial permeability, cell morphology, stress fiber formation, myosin light chain (MLC) phosphorylation, and β1 integrin–mediated signaling were evaluated through immunofluorescence analysis and immunoblotting.
Results
Excessive FN was accumulated in the microvessels of the spinal cord after spinal nerve ligation; moreover, pUR4 inhibited TNF-α-induced FN deposition in the bEND.3 monolayer and maintained intact TEER and endothelial permeability. Furthermore, pUR4 reduced cell morphology alteration, actin stress fiber formation, and MLC phosphorylation, thereby attenuating paracellular gap formation. Moreover, pUR4 reduced β1 integrin activation and downstream signaling.
Conclusions
pUR4 reduces TNF-α-induced β1 integrin activation by depleting ECM FN, leading to a decrease in endothelial hyperpermeability and maintenance of monolayer integrity. These findings suggest therapeutic benefits of pUR4 in pathological vascular leakage treatment. - 中文關鍵字:
- 英文關鍵字: Endothelial permeability, Fibronectin, β1 integrin, Stress fiber