Extracellular Thioredoxin, not its Intracellular Counterpart, Mediates Homocysteine Thiolactone- Induced Vascular Endothelial Cell Dysfunction

Author(s): Ping Liu, Liangwei Zhong

Background: Our previous work demonstrated an inverse correlation between serum total homocysteine (Hcy) levels and thioredoxin (Trx) activity in patients with coronary artery stenosis. However, whether homocysteine thiolactone (HCTL), a highly reactive Hcy derivative, affects Trx remains unknown.

Methods: Using human cytosolic thioredoxin (hTrx1) as a model, we employed a multidisciplinary approach—integrating biochemical assays, mass spectrometry, electron microscopy, and vascular endothelial cell models—to investigate HCTL-induced regulation. We systematically examined the effects of HCTL exposure (0–250 μM) on hTrx1 structure and function, both intracellularly and extracellularly.

Key Findings: Hcy-hTrx1 complexes were present in human serum. While increasing extracellular HCTL elevated cellular levels of Hcy and Hcy-hTrx1 complexes, these changes did not compromise vascular endothelial cell viability. However, HCTL covalently modified extracellular hTrx1 at Lys³? and Lys³?, forming N-homocysteinylated hTrx1 (N-Hcy-hTrx1). This modification severely impaired hTrx1’s activity and properties, triggered amyloid-like fibril formation, and exhibited marked cytotoxicity—667-fold higher than HCTL alone. Additionally, we detected serum auto-antibodies specific to N-Hcy-hTrx1.

Conclusion: Extracellular N-Hcy-hTrx1, not free HCTL or intracellular hTrx1, serves as the primary cytotoxic mediator. The presence of serum auto-antibodies specifically targeting N-Hcy- hTrx1 further supports its pathogenic role in vivo. This study reveals a novel mechanism through which extracellular hTrx1 connects hyperhomocysteinemia to vascular endothelial cell dysfunction.