Role of Excessive Autophagy Induced by Mechanical Overload in Vein Graft Neointima Formation: Prediction and Prevention

Little is known and able to predict the mechanical and molecular bases for vein graft restenosis. Stenosis initiation was elucidated by using a high‐frequency ultrasonic (HFU) echogenicity platform and estimating the yield stress from von‐Mises stress computation to predict the damage locations in living rats. The venous‐arterial transition induced the molecular cascades for autophagy and apoptosis in venous endothelial cells (ECs) to cause neointimal hyperplasia ( Fig. 1), which correlated with the high echogenicity in HFU images, and the large mechanical stress that exceeded the yield strength ( Fig. 2). The ex vivo perfusion of arterial laminar shear stress to isolated veins further confirmed the correlation ( Fig. 3). EC damage can be rescued by inhibiting autophagy formation using 3‐methyladenine (3MA) ( Fig. 4). Pretreatment of veins with 3MA prior to grafting reduced the pathological increases of echogenicity and neointima formation in rats ( Fig. 5). Therefore, this platform provides real‐time temporal spatial measurement and prediction of restenosis after venous‐arterial transition, as well as monitoring the progression of the treatments ( Fig. 6). Support or Funding Information This study was supported by grants from the Ministry of Science and Technology (NSC 102‐2320‐B‐006‐009‐MY3 to C.W.); (MOST 103‐2320‐B‐006‐011 to C.W.); and (MOST 103‐2911‐I‐006‐534 to C.W.) and the National Health Research Institutes of Taiwan (NHRI‐EX104‐10115EC to C.W.). 1 Vein graft stenosis was established by bridging the vein graft to carotid artery (CAV)Neointima hyperplasia significant increased in CAV rats (A). Loss of elastic fiber and fragments of the basal laminar were observed in the CAV graft (B). Cell death increased in intimal layer (C). Autophagy formation in CAV grafts was demonstrated by LC3 IHC staining (D). Scale bars in (A) and (B): 50 μm. Scale bars in (C) and (D): 20 μm. *significant difference from the artery graft rats, p<0.05.2 Spatial agreement of excessive autophagy and inflammation in areas with mechanical overloadLumen area were quantified from HFU (A). Venous endothelium is softer than arterial one (B). Von‐Mises stress was estimated during stenosis progression (C). Potential damages are predicted (red color) by threshold (D). Agreements were found among prediction, autophagy formation, COX‐2 expression, and echogenicity (arrowheads). Squares indicated high (red) and low stress region (blue) (E). *p<0.05.3 Detection of ALSS‐induced vein damage using HFU images in the ex vivo systemThe HFU images were acquired at different time points (A). ALSS increased the echogenicity of vein (B). ALSS induced venous EC autophagy formation, COX‐2 expression, and apoptosis (C). Echogenicity highly correlates with LC3 and COX‐2 expressions (D). The ALSS‐induced autophagy was confirmed by TEM images (E). Without arterial pressure, the ALSS still induced venous EC damage (F). Scale bar: 20 μm. *p<0.05.4 Transient inhibition of autophagy by 3MA prevented the loss of venous ECs under ALSSAfter 24 hrs of ALSS stimulation, venous ECs were peeled off with high expressions of autophagy formation (A). Autophagy inhibitor (3MA) preserved venous ECs after ALSS application (B). 3MA decreased ALSS‐induced LC3‐, COX‐2‐, and TUNEL‐positive staining (C), and echogenicity (D). Scale bar in (A): 50 μm. Scale bar in (C): 30 μm. *compare to static, p<0.05. # compare to non‐3MA treatment, p<0.05.5 Pre‐incubation of venous segment with 3MA prevented neointima formationReduction of neointimal hyperplasia by 3MA pretreatment in grafted veins (A). Quantification of neointima areas in grafted veins (B). The 3MA‐pretreated vein grafts inhibited of LC3‐ and TUNEL‐positive cells (C). Decreases of hyperechogenicity and neointimal thickness were determined by HFU in 3MA pretreated rats (D). *compare to non‐3MA treatment, p<0.05.6 Summary diagram on the monitoring of vein graft progressions by combining the HFU‐echogenecity and computational simulationThe prediction of mechanical overload from HFU and computational simulation with the consequent induction of EC damage is confirmed by the triggering of autophagic cell death detected by echogenicity. The autophagy induced by mechanical overload can be prevented by transient administration of 3MA prior to the venous‐arterial transition.