Kidney transplant injury occurs with ischemia and alloimmunity. Members of the receptor interacting protein kinase family (RIPK1,3) are key regulators of “necroptosis,” a newly recognized, regulated form of necrosis. Necroptosis and apoptosis death appear to be counterbalanced as caspase-8 inhibition can divert death from apoptosis to necrosis. Inhibition of necroptosis in donor organs to limit injury has not been studied in transplant models. In this study, necroptosis was triggered in caspase inhibited tubular epithelial cells (TEC) exposed to tumor necrosis factor alpha in vitro, while RIPK1 inhibition with necrostatin-1 or use of RIPK3^−/− TEC, prevented necroptosis. In vivo, short hairpin RNA silencing of caspase-8 in donor B6 mouse kidneys increased necroptosis, enhanced high-mobility group box 1 release, reduced renal function and accelerated rejection when transplanted into BALB/c recipients. Using ethidium homodimer perfusion to assess necrosis in vivo, necrosis was abrogated in RIPK3^−/− kidneys postischemia. Following transplantation, recipients receiving RIPK3^−/− kidneys had longer survival (p = 0.002) and improved renal function (p = 0.03) when compared to controls. In summary, we show for the first time that RIPK3-mediated necroptosis in donor kidneys can promote inflammatory injury, and has a major impact on renal ischemia–reperfusion injury and transplant survival. We suggest inhibition of necroptosis in donor organs may similarly provide a major clinical benefit.

Transient potential receptor vanilloid 1 (TRPV1) channels, which are expressed on sensory neurons, elicit cardioprotective effects during ischemia reperfusion injury by stimulating the release of neuropeptides, namely calcitonin gene-related peptide (CGRP) and substance P (SP). Recent studies show that TRPV1 channels are also expressed on cardiomyocytes and can exacerbate air pollutant-induced apoptosis. However, whether these channels present on cardiomyocytes directly modulate cell death and survival pathways during hypoxia/reoxygenation (H/R) injury remains unclear. In the present study, we investigated the role of TRPV1 in H/R induced apoptosis of H9C2 cardiomyocytes. We demonstrated that TRPV1 was indeed expressed in H9C2 cells, and activated by H/R injury. Although neuropeptide release caused by TRPV1 activation on sensory neurons elicits a cardioprotective effect, we found that capsaicin (CAP; a TRPV1 agonist) treatment of H9C2 cells paradoxically enhanced the level of apoptosis by increasing intracellular calcium and mitochondrial superoxide levels, attenuating mitochondrial membrane potential, and inhibiting mitochondrial biogenesis (measured by the expression of ATP synthase β). In contrast, treatment of cells with capsazepine (CPZ; a TRPV1 antagonist) or TRPV1 siRNA attenuated H/R induced-apoptosis. Furthermore, CAP and CPZ treatment revealed a similar effect on cell viability and mitochondrial superoxide production in primary cardiomyocytes. Finally, using both CGRP_8–37 (a CGRP receptor antagonist) and RP67580 (a SP receptor antagonist) to exclude the confounding effects of neuropeptides, we confirmed aforementioned detrimental effects as TRPV1^−/− mouse hearts exhibited improved cardiac function during ischemia/reperfusion. In summary, direct activation of TRPV1 in myocytes exacerbates H/R-induced apoptosis, likely through calcium overload and associated mitochondrial dysfunction. Our study provides a novel understanding of the role of myocyte TRPV1 channels in ischemia/reperfusion injury that sharply contrasts with its known extracardiac neuronal effects.

Background and Aims: HCC is a malignant disease. Compared with tyrosine kinase inhibitors (the classical therapy), immune checkpoint inhibitors are more effective in the treatment of HCC, despite their limited efficacy. Among these restricted factors, exhaustion of tumor-infiltrated lymphocytes, especially CD8⁺ T cells, is a core event. We aimed to determine the key factors contributing to CD8⁺ T-cell infiltration in HCC and investigate the underlying mechanisms. Approach and Results: Using machine learning and multiplex immunohistochemistry analysis, we showed that dedicator of cytokinesis protein 2 (DOCK2) was a potential indicator of infiltrated CD8⁺ T cells in HCC. Using RNA sequencing, flow cytometry analysis, and mouse HCC models, we demonstrated that DOCK2 inactivation accounted for infiltrated CD8⁺ T-cell exhaustion in tumors. Using quasi-targeted metabolomics, mass spectrum, and mass cytometry by time of flight analysis, we found that cholesterol sulfate synthesized by sulfotransferase 2B1 in tumor cells suppressed DOCK2 enzymatic activity of T cells. Through virtual screening, molecular docking simulation, and experiments validation, we demonstrated that tolazamide reversed DOCK2 inactivation-mediated CD8⁺ T-cell exhaustion and enhanced anti–programmed death-ligand 1 antibody+apatinib immunotherapeutic effects on HCC. Conclusions: This study indicates that DOCK2 controls CD8⁺ T-cell infiltration in HCC, and cholesterol sulfate synthesized by sulfotransferase 2B1 in tumor cells promotes effector T-cell exhaustion. The findings suggest that the usage of conventional drugs affects immunotherapy efficacy in HCC patients.

Recent studies suggest that circular RNA (circRNA)‐mediated post‐translational modification of RNA‐binding proteins (RBP) plays a pivotal role in metastasis of hepatocellular carcinoma (HCC). However, the specific mechanism and potential clinical therapeutic significance remain vague. This study attempts to profile the regulatory networks of circRNA and RBP using a multi‐omics approach. Has_circ_0006646 (circ0006646) is an unreported circRNA in HCC and is associated with a poor prognosis. Silencing of circ0006646 significantly hinders metastasis in vivo. Mechanistically, circ0006646 prevents the interaction between nucleolin (NCL) and the E3 ligase tripartite motif‐containing 21 to reduce the proteasome‐mediated degradation of NCL via K48‐linked polyubiquitylation. Furthermore, the change of NCL expression is proven to affect the phosphorylation levels of multiple proteins and inhibit p53 translation. Moreover, patient‐derived tumor xenograft and lentivirus injection, which is conducted to simulate clinical treatment confirmed the potential therapeutic value. Overall, this study describes the integrated multi‐omics landscape of circRNA‐mediated NCL ubiquitination degradation in HCC metastasis and provides a novel therapeutic target.

The efficacy of immune checkpoint inhibitors in the treatment of hepatocellular carcinoma (HCC) remains limited, highlighting the need for further investigation into the mechanisms underlying treatment resistance. Accumulating evidence indicates that tumor-associated macrophages (TAM) within the tumor microenvironment demonstrate a key role in immune evasion and treatment resistance. This study explored the role of TAMs in the HCC tumor microenvironment. Our findings reveal that TAMs expressing CX3C motif chemokine receptor 1 (CX3CR1) induced T-cell exhaustion through IL27 secretion in orthotopic models of HCC following treatment with anti-PD1. Moreover, we identified prostaglandin E2 (PGE2), released by immune-attacked tumor cells, as a key regulator of TAM transition to a CX3CR1+ phenotype. To augment the therapeutic response to anti-PD1 therapy, we propose targeting CX3CR1+ TAMs in addition to anti-PD1 therapy. Our study contributes to the understanding of the role of TAMs in cancer immunotherapy and highlights potential clinical implications for HCC treatment. The combination of targeting CX3CR1+ TAMs with anti-PD1 therapy holds promise for enhancing the efficacy of immunotherapeutic interventions in patients with HCC.