Endothelial NMMHC IIA dissociation from PAR1 activates the CREB3/ARF4 signaling in thrombin-mediated intracerebral hemorrhage

Yujie Dai; Liangying Bao; Juan Huang; Miling Zhang; Junhe Yu; Yuanyuan Zhang; Fang Li; Boyang Yu; Shuaishuai Gong; Junping Kou

doi:10.1016/j.jare.2024.11.008

Endothelial NMMHC IIA dissociation from PAR1 activates the CREB3/ARF4 signaling in thrombin-mediated intracerebral hemorrhage

J Adv Res. 2024 Nov 7:S2090-1232(24)00500-9. doi: 10.1016/j.jare.2024.11.008. Online ahead of print.

Authors

Affiliations

¹ State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Pharmacology of Chinese Materia Medica, School of Traditional Pharmacy, China Pharmaceutical University, 639 Longmian Road, Nanjing 211198, PR China.
² State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Pharmacology of Chinese Materia Medica, School of Traditional Pharmacy, China Pharmaceutical University, 639 Longmian Road, Nanjing 211198, PR China. Electronic address: [email protected].
³ State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Pharmacology of Chinese Materia Medica, School of Traditional Pharmacy, China Pharmaceutical University, 639 Longmian Road, Nanjing 211198, PR China. Electronic address: [email protected].

PMID: 39521432
DOI: 10.1016/j.jare.2024.11.008

Abstract

Introduction: There is an urgent need for cerebroprotective interventions to improve the suboptimal outcomes with intracerebral hemorrhage (ICH). Despite the important role of nonmuscle myosin heavy chain IIA (NMMHC IIA) in the blood-brain barrier (BBB), its function in ICH remains unclear.

Objectives: The objective of this study is to explore how NMMHC IIA functions in ICH and to evaluate the effectiveness of targeting NMMHC IIA as a treatment for ICH.

Methods: We firstly examined the protein expression of NMMHC IIA in clinical patients and animal models with ICH. The function of NNMMHC IIA was then corroborated by using overexpress or knockdown NMMHC IIA specifically in ECs mice and pBMECs. In addition, we explored protein interacts with NMMHC IIA and signaling pathways after ICH by LC-MS/MS and transcriptomics analysis with an emphasis on the function of PAR1 and the CREB3/ARF4 signaling pathway, and validated them in three kind of animal models. To support the clinical translation of our results, we targeted NMMHC IIA to bicalutamide selected from a library of marketed drugs and examined to validate its ameliorative effect on ICH.

Results: We observed an upregulation of endothelial NMMHC IIA in the brain following the onset of ICH in both patients and mice, while inhibited NMMHC ⅡA improved ICH induced by thrombin, warfarin or tissue plasminogen activator (tPA) after ischemic stroke. Mechanistically, the head domain of NMMHC IIA interacted with protease-activated receptor 1 (PAR1) at the 380-430 aa region and subsequently dissociated and activated the CREB3/ARF4 signaling pathway. We found that bicalutamide and blebbistatin could bind to NMMHC IIA and effectively protect mice from thrombin-mediated ICH.

Conclusion: The findings indicated that NMMHC IIA dissociated from PAR1 and activated CREB3/ARF4 pathway, which aggravated BBB damage induced by thrombin. This suggested that NMMHC IIA was a novel potential therapeutic target for BBB-related diseases.

Keywords: Bicalutamide; Blood-brain barrier; CREB3/ARF4 signaling pathway; Intracerebral hemorrhage; Nonmuscle myosin heavy chain IIA; Protease-activated receptor 1.