Rajala Rahul, Cleuren Audrey C A, Griffin Courtney T
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK.
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
bioRxiv. 2024 Jun 8:2024.06.07.598028. doi: 10.1101/2024.06.07.598028.
BACKGROUND & AIMS: Hepatic endothelial cell (EC) dysfunction and centrilobular hepatocyte necrosis occur with acetaminophen (APAP) overdose. The protease thrombin, which is acutely generated during APAP overdose, can signal through protease-activated receptors 1 and 4 (PAR1/PAR4). PAR1 is a high-affinity thrombin receptor that is known to signal on ECs, whereas PAR4 is a low-affinity thrombin receptor, and evidence for its expression and function on ECs is mixed. This study aims to exploit the high levels of thrombin generated during APAP overdose to determine (1) if hepatic endothelial PAR4 is a functional receptor, and (2) endothelial-specific functions for PAR1 and PAR4 in a high thrombin setting.
We generated mice with conditional deletion(s) of in ECs and overdosed them with APAP. Hepatic vascular permeability, erythrocyte congestion/bleeding, and liver function were assessed following overdose. Additionally, we investigated the expression levels of endothelial PARs and how they influence transcription in APAP-overdosed liver ECs using endothelial Translating Ribosome Affinity Purification followed by next-generation sequencing (TRAPseq).
We found that mice deficient in high-expressing endothelial or low-expressing had equivalent reductions in APAP-induced hepatic vascular instability but no effect on hepatocyte necrosis. Additionally, mice with loss of endothelial and had reduced permeability at an earlier time point after APAP overdose when compared to mice singly deficient in either receptor in ECs. We also found that endothelial PAR1-but not PAR4-can regulate transcription in hepatic ECs.
Low-expressing PAR4 can react similarly to high-expressing PAR1 in APAP-overdosed hepatic ECs, demonstrating that PAR4 is a potent thrombin receptor. Additionally, these receptors are functionally redundant but act divergently in their expression and ability to influence transcription in hepatic ECs.
对乙酰氨基酚（APAP）过量服用会导致肝内皮细胞（EC）功能障碍和小叶中心肝细胞坏死。在APAP过量服用期间会急性产生的蛋白酶凝血酶，可通过蛋白酶激活受体1和4（PAR1/PAR4）发出信号。PAR1是一种高亲和力凝血酶受体，已知其在EC上发出信号，而PAR4是一种低亲和力凝血酶受体，关于其在EC上的表达和功能的证据并不一致。本研究旨在利用APAP过量服用期间产生的高水平凝血酶来确定：（1）肝内皮PAR4是否为功能性受体；（2）在高凝血酶环境中PAR1和PAR4在内皮细胞中的特异性功能。
我们构建了内皮细胞中条件性缺失 的小鼠，并给它们过量服用APAP。过量服用后评估肝血管通透性、红细胞充血/出血情况以及肝功能。此外，我们使用内皮翻译核糖体亲和纯化技术结合下一代测序（TRAPseq），研究了内皮PARs的表达水平以及它们如何影响APAP过量服用的肝脏EC中的转录。
我们发现，高表达内皮 或低表达 的缺陷小鼠在APAP诱导的肝血管不稳定性方面有同等程度的降低，但对肝细胞坏死没有影响。此外，与内皮细胞中单一缺乏任一受体的小鼠相比，同时缺乏内皮 和 的小鼠在APAP过量服用后的更早时间点通透性降低。我们还发现，内皮PAR1而非PAR4可调节肝脏EC中的转录。
在APAP过量服用的肝脏EC中，低表达的PAR4与高表达的PAR1反应相似，表明PAR4是一种有效的凝血酶受体。此外，这些受体在功能上是冗余的，但在它们的表达以及影响肝脏EC转录的能力方面表现不同。
