Dissecting molecular mechanisms underlying ferroptosis in human umbilical cord mesenchymal stem cells: Role of cystathionine γ-lyase/hydrogen sulfide pathway

BACKGROUND

Ferroptosis can induce low retention and engraftment after mesenchymal stem cell (MSC) delivery, which is considered a major challenge to the effectiveness of MSC-based pulmonary arterial hypertension (PAH) therapy. Interestingly, the cystathionine γ-lyase (CSE)/hydrogen sulfide (H₂S) pathway may contribute to mediating ferroptosis. However, the influence of the CSE/H₂S pathway on ferroptosis in human umbilical cord MSCs (HUCMSCs) remains unclear.

AIM

To clarify whether the effect of HUCMSCs on vascular remodelling in PAH mice is affected by CSE/H₂S pathway-mediated ferroptosis, and to investigate the functions of the CSE/H₂S pathway in ferroptosis in HUCMSCs and the underlying mechanisms.

METHODS

Erastin and ferrostatin-1 (Fer-1) were used to induce and inhibit ferroptosis, respectively. HUCMSCs were transfected with a vector to overexpress or inhibit expression of CSE. A PAH mouse model was established using 4-wk-old male BALB/c nude mice under hypoxic conditions, and pulmonary pressure and vascular remodelling were measured. The survival of HUCMSCs after delivery was observed by in vivo bioluminescence imaging. Cell viability, iron accumulation, reactive oxygen species production, cystine uptake, and lipid peroxidation in HUCMSCs were tested. Ferroptosis-related proteins and S-sulfhydrated Kelch-like ECH-associating protein 1 (Keap1) were detected by western blot analysis.

RESULTS

In vivo, CSE overexpression improved cell survival after erastin-treated HUCMSC delivery in mice with hypoxia-induced PAH. In vitro, CSE overexpression improved H₂S production and ferroptosis-related indexes, such as cell viability, iron level, reactive oxygen species production, cystine uptake, lipid peroxidation, mitochondrial membrane density, and ferroptosis-related protein expression, in erastin-treated HUCMSCs. In contrast, in vivo, CSE inhibition decreased cell survival after Fer-1-treated HUCMSC delivery and aggravated vascular remodelling in PAH mice. In vitro, CSE inhibition decreased H₂S levels and restored ferroptosis in Fer-1-treated HUCMSCs. Interestingly, upregulation of the CSE/H₂S pathway induced Keap1 S-sulfhydration, which contributed to the inhibition of ferroptosis.

CONCLUSION

Regulation of the CSE/H₂S pathway in HUCMSCs contributes to the inhibition of ferroptosis and improves the suppressive effect on vascular remodelling in mice with hypoxia-induced PAH. Moreover, the protective effect of the CSE/H₂S pathway against ferroptosis in HUCMSCs is mediated via S-sulfhydrated Keap1/nuclear factor erythroid 2-related factor 2 signalling. The present study may provide a novel therapeutic avenue for improving the protective capacity of transplanted MSCs in PAH.

Key Words: Human umbilical cord mesenchymal stem cells, Cystathionine γ-lyase/hydrogen sulfide pathway, Ferroptosis, Pulmonary arterial hypertension, S-sulfhydration

Core Tip: Regulation of the cystathionine γ-lyase (CSE)/hydrogen sulfide (H₂S) pathway in human umbilical cord mesenchymal stem cells (HUCMSCs) contributes to the inhibition of ferroptosis and improves the suppressive effect of HUCMSCs on vascular remodelling in hypoxia-induced pulmonary arterial hypertension (PAH) mice. Moreover, the protective effect of the CSE/H₂S pathway against ferroptosis in HUCMSCs was mediated via S-sulfhydrated Kelch-like ECH-associating protein 1/nuclear factor erythroid 2-related factor 2 signalling. The present study may provide a novel therapeutic avenue for improving the protective capacity of transplanted MSCs in PAH.