SMURF2 predisposes cancer cell toward ferroptosis in GPX4-independent manners by promoting GSTP1 degradation.

Ferroptosis is a non-apoptotic form of regulated cell death. Glutathione (GSH) peroxidase 4 (GPX4) and GSH-independent ferroptosis suppressor protein 1 (FSP1) have been identified as major defenses. Here, we uncover a protective mechanism mediated by GSH S-transferase P1 (GSTP1) by monitoring proteinomic dynamics during ferroptosis. Dramatic downregulation of GSTP1 is caused by SMURF2-mediated GSTP1 ubiquitination and degradation at early stages of ferroptosis. Intriguingly, GSTP1 acts in GPX4- and FSP1-independent manners by catalyzing GSH conjugation of 4-hydroxynonenal and detoxifying lipid hydroperoxides via selenium-independent GSH peroxidase activity. Genetic modulation of the SMURF2/GSTP1 axis or the pharmacological inhibition of GSTP1's catalytic activity sensitized tumor responses to Food and Drug Administration (FDA)-approved ferroptosis-inducing drugs both in vitro and in vivo. GSTP1 expression also confers resistance to immune checkpoint inhibitors by blunting ferroptosis. Collectively, these findings demonstrate a GPX4/FSP1-independent cellular defense mechanism against ferroptosis and suggest that targeting SMURF2/GSTP1 to sensitize cancer cells to ferroptosis has potential as an anticancer therapy.

Macrophage Ferroptosis Promotes MMP2/9 Overexpression Induced by Hemin in Hemorrhagic Plaque.

BACKGROUND: Intra-plaque hemorrhage (IPH) leads to rapid plaque progression and instability through upregulation of matrix metalloproteinases (MMPs) and collagen degradation. Hemoglobin-derived hemin during IPH promotes plaque instability. We investigated whether hemin affects MMP overexpression in macrophages and explored the underlying mechanisms. MATERIAL AND METHODS: In vivo, hemorrhagic plaque models were established in rabbits and ApoE-/- mice. Ferrostatin-1 was used to inhibit ferroptosis. Plaque size, collagen, and MMP2/9 levels were evaluated using immunohistochemistry, H&E, Sirius Red, and Masson staining. In vitro, mouse peritoneal macrophages were extracted. Western blot and ELISA were used to measure MMP2/9 levels. Bioinformatics analysis investigated the association between MMPs and ferroptosis pathway genes. Macrophage ferroptosis was assessed by evaluating cell viability, lipid reactive oxygen species, mitochondrial ultrastructure, iron content, and COX2 levels after pretreatment with cell death inhibitors. Hemin's impact on ferroptosis and MMP expression was studied using Ferrostatin-1 and SB202190. RESULTS: In the rabbit hemorrhagic plaques, hemin deposition and overexpression of MMP2/9 were observed, particularly in macrophage-enriched regions. In vitro, hemin induced ferroptosis and MMP2/9 expression in macrophages. Ferrostatin-1 and SB202190 inhibited hemin-induced MMP2/9 overexpression. Ferrostatin-1 inhibited p38 phosphorylation in macrophages. Ferostatin-1 inhibits macrophage ferroptosis, reduces MMP2/9 levels in plaques, and stabilizes the hemorrhagic plaques. CONCLUSION: Our results suggested that hemin-induced macrophage ferroptosis promotes p38 pathway activation and MMP2/9 overexpression, which may play a crucial role in increasing hemorrhagic plaque vulnerability. These findings provide insights into the pathogenesis of hemorrhagic plaques and suggest that targeting macrophage ferroptosis may be a promising strategy for stabilizing vulnerable plaque.

In-vivo programmable acoustic manipulation of genetically engineered bacteria.

Acoustic tweezers can control target movement through the momentum interaction between an acoustic wave and an object. This technology has advantages over optical tweezers for in-vivo cell manipulation due to its high tissue penetrability and strong acoustic radiation force. However, normal cells are difficult to acoustically manipulate because of their small size and the similarity between their acoustic impedance and that of the medium. In this study, we use the heterologous expression of gene clusters to generate genetically engineered bacteria that can produce numerous sub-micron gas vesicles in the bacterial cytoplasm. We show that the presence of the gas vesicles significantly enhances the acoustic sensitivity of the engineering bacteria, which can be manipulated by ultrasound. We find that by employing phased-array-based acoustic tweezers, the engineering bacteria can be trapped into clusters and manipulated in vitro and in vivo via electronically steered acoustic beams, enabling the counter flow or on-demand flow of these bacteria in the vasculature of live mice. Furthermore, we demonstrate that the aggregation efficiency of engineering bacteria in a tumour is improved by utilizing this technology. This study provides a platform for the in-vivo manipulation of live cells, which will promote the progress of cell-based biomedical applications.

Long noncoding RNA HITT coordinates with RGS2 to inhibit PD-L1 translation in T cell immunity.

Programmed cell death ligand 1 (PD-L1) is an immune checkpoint protein frequently expressed in human cancers that contributes to immune evasion through its binding to PD-1 on activated T cells. Unveiling the mechanisms underlying PD-L1 expression is essential for understanding the impact of the immunosuppressive microenvironment and is also crucial for the purpose of reboosting antitumor immunity. However, how PD-L1 is regulated, particularly at translational levels, remains largely unknown. Here, we discovered that a long noncoding RNA (lncRNA), HIF-1α inhibitor at translation level (HITT), was transactivated by E2F transcription factor 1 (E2F1) under IFN-γ stimulation. It coordinated with regulator of G protein signaling 2 (RGS2) in binding to the 5' UTR of PD-L1, resulting in reduced PD-L1 translation. HITT expression enhanced T cell-mediated cytotoxicity both in vitro and in vivo in a PD-L1-dependent manner. The clinical correlation between HITT/PD-L1 and RGS2/PD-L1 expression was also detected in breast cancer tissues. Together, these findings demonstrate the role of HITT in antitumor T cell immunity, highlighting activation of HITT as a potential therapeutic strategy for enhancing cancer immunotherapy.

Sonodynamic therapy reduces iron retention of hemorrhagic plaque.

Intraplaque hemorrhage (IPH) plays a major role in the aggressive progression of vulnerable plaque, leading to acute cardiovascular events. We previously demonstrated that sonodynamic therapy (SDT) inhibits atherosclerotic plaque progression. In this study, we investigated whether SDT could also be applied to treat more advanced hemorrhagic plaque and addressed the underlying mechanism. SDT decreased atherosclerotic burden, positively altered atherosclerotic lesion composition, and alleviated iron retention in rabbit hemorrhagic plaques. Furthermore, SDT reduced iron retention by stimulating ferroportin 1 (Fpn1) expression in apolipoprotein E (ApoE)-/- mouse plaques with high susceptibility to IPH. Subsequently, SDT inhibited iron-overload-induced foam-cell formation and pro-inflammatory cytokines secretion in vitro. Moreover, SDT reduced levels of the labile iron pool and ferritin expression via the reactive oxygen species (ROS)-nuclear factor erythroid 2-related factor 2 (Nrf2)-FPN1 pathway. SDT exerted therapeutic effects on hemorrhagic plaques and reduced iron retention via the ROS-Nrf2-FPN1 pathway in macrophages, thereby suggesting that it is a potential translational strategy for patients with advanced atherosclerosis in clinical practice.

Early modulation of macrophage ROS-PPARγ-NF-κB signalling by sonodynamic therapy attenuates neointimal hyperplasia in rabbits.

Disruption of re-endothelialization and haemodynamic balance remains a critical side effect of drug-eluting stents (DES) for preventing intimal hyperplasia. Previously, we found that 5-aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) suppressed macrophage-mediated inflammation in atherosclerotic plaques. However, the effects on intimal hyperplasia and re-endothelialization remain unknown. In this study, 56 rabbits were randomly assigned to control, ultrasound, ALA and ALA-SDT groups, and each group was divided into two subgroups (n = 7) on day 3 after right femoral artery balloon denudation combined with a hypercholesterolemic diet. Histopathological analysis revealed that ALA-SDT enhanced macrophage apoptosis and ameliorated inflammation from day 1. ALA-SDT inhibited neointima formation without affecting re-endothelialization, increased blood perfusion, decreased the content of macrophages, proliferating smooth muscle cells (SMCs) and collagen but increased elastin by day 28. In vitro, ALA-SDT induced macrophage apoptosis and reduced TNF-α, IL-6 and IL-1ß via the ROS-PPARγ-NF-κB signalling pathway, which indirectly inhibited human umbilical artery smooth muscle cell (HUASMC) proliferation, migration and IL-6 production. ALA-SDT effectively inhibits intimal hyperplasia without affecting re-endothelialization. Hence, its clinical application combined with bare-metal stent (BMS) implantation presents a potential strategy to decrease bleeding risk caused by prolonged dual-antiplatelet regimen after DES deployment.

Increased hepcidin in hemorrhagic plaques correlates with iron-stimulated IL-6/STAT3 pathway activation in macrophages.

Intraplaque hemorrhage (IPH) promotes the rapid progression of atherosclerotic plaques, resulting in cardiovascular events in a short time. Hepcidin increases iron retention and exerts proinflammatory effects in plaques. However, hepcidin expression levels in hemorrhagic plaques remain unknown. In the present study, we evaluated hepcidin expression in hemorrhagic plaques and the underlying mechanism. To investigate hepcidin expression in hemorrhagic plaques, carotid artery plaques were collected from patients undergoing carotid endarterectomy (CEA) and apolipoprotein E-deficient mice. The hepcidin expression level was increased in the area of IPH and positively correlated with the amount of hemorrhage as shown by immunohistochemistry. Hepcidin expression in macrophages within human plaques was confirmed by immunofluorescence. Furthermore, ferric ammonium citrate (FAC) was found to induce hepcidin and interleukin-6 (IL-6) expression in THP-1 macrophages and mouse peritoneal macrophages. Subsequently, activation of the IL-6/signal transducer and activator of transcription (STAT) 3 pathway was observed in rabbit hemorrhagic plaques. Macrophages were pretreated with antibodies that block IL-6/IL-6R interactions or STAT3 activation and dimerization inhibitor (STATTIC), and the results indicated that FAC induced hepcidin expression through the IL-6/STAT3 pathway. In conclusion, our data indicate that hepcidin levels are increased in hemorrhagic plaques, which correlates with iron-stimulated IL-6/STAT3 pathway activation in macrophages. Therefore, inhibition of the IL-6/STAT3 pathway may be a potential strategy to reduce hepcidin expression and further stabilize hemorrhagic plaques.