Interleukin-1 receptor antagonist overexpression in mesenchymal stem cells improves hemorrhagic cystitis outcomes via HtrA serine peptidase 3.

Background: Hemorrhagic cystitis (HC), a frequent complication of hematopoietic stem cell transplantation (HSCT), significantly affects quality of life and may worsen prognosis. Mesenchymal stem cells (MSCs) are known for their anti-inflammatory and tissue-regenerative properties. IL-1 receptor antagonist (IL-1Ra) blocks IL-1α and IL-1β by binding IL-1 receptors, offering potential therapeutic benefits. The aim of this study was to explore the therapeutic effect of MSCs overexpressing IL-1Ra on HC and investigate the underlying mechanisms.

Methods: MSCs were isolated from human umbilical cord tissues, and IL-1Ra-overexpressing MSCs (oeIL-1Ra-MSCs) were generated using lentiviral transfections. HC was induced in rats by cyclophosphamide administration. Rats received tail vein injections of either oeIL-1Ra-MSCs or control MSCs (Mock-MSCs). Hematuria and bladder tissue changes were assessed using test strips and hematoxylin & eosin (HE) staining. Immunohistochemistry detected molecular changes in bladder tissues. Gene expression differences between the two MSC groups were analyzed by mRNA sequencing and ChIP techniques.

Results: Treatment with oeIL-1Ra-MSCs significantly alleviated hematuria and reduced bladder edema and hemorrhage, and reduced mRNA expression levels of IL-1β, IL-6, and TNF-α in bladder tissues, compared with those in the Mock-MSC treatment group. Immunohistochemical staining showed a higher presence of CD105-positive cells (a marker for human MSCs) and CD31-positive vessels in bladder tissues treated with oeIL-1Ra-MSCs, indicating enhanced MSC migration and vascular stability. In vitro migration assay demonstrated a higher migration capacity of IL-1Ra overexpressing MSCs compared with that of control MSCs. Moreover, angiopoietin-1 (Ang-1) expression increased, while Angiopoietin-2 (Ang-2) expression decreased in bladder tissues treated with oeIL-1Ra-MSCs, suggesting enhanced blood vessel stabilization. Conditioned medium from oeIL-1Ra-MSC cultures stimulated human umbilical vein endothelial cell (hUVEC) migration, proliferation, and angiogenesis more effectively compared with that in control MSCs. mRNA sequencing revealed elevated HtrA3 expression in oeIL-1Ra-MSCs compared with that in control MSCs. Molecular analysis suggested that IL-1Ra overexpression in MSCs upregulated HtrA3 expression through inhibition of the JNK-c-Jun pathway and activation of the ERK-Egr-1 pathway.

Conclusion: Overexpression of IL-1Ra significantly enhances the therapeutic efficacy of MSCs in HC by promoting MSC migration to damaged bladder tissues, suppressing inflammation, stabilizing blood vessels, and upregulating angiogenesis via activation of HtrA3 signaling pathways.