Zhi-Ren Zhang

BACKGROUND: The use of anti–programmed cell death-1 (PD-1) antibody increases heart failure (HF) risk in patients with cancer with preexisting cardiovascular conditions. However, the underlying mechanism remains incompletely understood. METHODS: To evaluate the effects of anti–PD-1 antibody on transverse aortic constriction (TAC)–induced cardiac remodeling and HF, anti–PD-1 antibody–treated mice; T cell–, myeloid-, and CD8 ⁺ T cell–specific Pdcd1 knockout; C-X-C motif chemokine receptor 3 ( Cxcr3 ) knockout; and granzyme B ( Gzmb ) knockout mice combined with flow cytometry, Western blotting, immunofluorescence staining, pharmacological approaches, and bulk RNA-sequencing analyses were used. RESULTS: Administration of anti–PD-1 antibody, T cell–, or CD8 ⁺ T cell–specific Pdcd1 deletion, but not myeloid-specific Pdcd1 knockout, aggravated TAC-induced cardiomyopathy and HF in mice. Mechanistically, PD-1 blockade or deletion increased myocardial infiltration of CXCR3 ⁺ CD8 ⁺ T cells, leading to granzyme B/perforin–mediated impairment of cardiomyocyte mitochondrial complex I to exacerbate TAC-induced cardiac injury and HF. TAC-enhanced chemotaxis, between cardiac fibroblast–derived CXCL9/CXCL10 and CXCR3 ⁺ CD8 ⁺ T cells, was a driving force for recruiting CXCR3 ⁺ CD8 ⁺ T cells under the conditions of PD-1 blockade or deletion. The worsened TAC-induced cardiomyopathy caused by anti–PD-1 antibody or T cell–specific Pdcd1 deletion was rescued by genetic deletion or pharmacological blockade of granzyme B and CXCR3. CONCLUSIONS: Anti–PD-1 antibody enhances myocardial infiltration of CXCR3 ⁺ CD8 ⁺ T cells under TAC condition through CXCL9/CXCL10–mediated chemotaxis. The increased granzyme B and perforin likely derived from CD8 ⁺ T cells impair function of mitochondrial respiratory chain complexes to cause cardiomyocyte apoptosis, thereby exacerbating TAC-induced cardiomyopathy and HF. CXCL9/CXCL10-CXCR3 ⁺ CD8 ⁺ T cell axis may represent a promising target for combating anti–PD-1 antibody–associated cardiotoxicity.

Natural killer (NK) cell-based therapies are under assessment for the treatment of various cancers due to their intrinsic ability to distinguish between malignant and healthy cells in an allogeneic context, enabling off-the-shelf manufacturing possibilities. However, cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. In this study, we evaluated three cryoprotectants (CryoStor 10; ZKCELL FM-01; FBS + DMSO) for the cryopreservation of NK cells. Post-thaw viability, ATP levels, and cytotoxicity were assessed and found to have persistent differences between cryopreserved and fresh cells. Transmission electron microscopy, flow cytometry, and Western blot analysis revealed a complex mode of cell death in cryopreserved cells, which could be partially mitigated by adding some death inhibitors. We further investigated the effects of centrifugation on thawed cells, identifying lysosomal stability as a key determinant of cell death. Pretreatment with low-dose LLOMe prior to cryopreservation induced stress granule formation, stabilizing lysosomes and improving cell recovery rates without compromising effector functional capacity. These findings offer new insights for optimizing NK cell cryopreservation and facilitating their clinical application.

BACKGROUND: Cardiac fibrosis is a major determinant of adverse clinical outcomes of many heart diseases; currently, therapeutic strategy directly targeting fibroblasts is lacking. Nitric oxide–mediated nitrosative stress is associated with cardiac injury, and excessive nitric oxide can trigger S-nitrosylation (SNO) to specific cysteine thiol. This study aims to investigate the role of SNO in cardiac fibrosis and to identify potential therapeutic target. METHODS: SNO proteomic analysis was performed in cardiac tissue isolated from both mice subjected to transverse aortic constriction and spontaneous hypertensive rats. Elevated SNO of pyruvate kinase M2 (PKM2) was identified in cardiac fibroblasts, which was merely detected in cardiomyocytes. Cardiac fibroblast–specific PKM2 knockout mice and mice transfected with wild-type or SNO-resistant PKM2 mutant were used to determine the involvement of SNO of PKM2 (SNO-PKM2) in cardiac fibrosis. Unbiased proteomics and coimmunoprecipitation combined with mass spectrometry analysis were conducted to explore effectors mediating SNO-PKM2–induced activation of cardiac fibroblasts. A recently approved drug for rare blood disorder, mitapivat, was shown to dose-dependently relieve cardiac fibrosis. RESULTS: SNO of PKM2 at cysteine 49 and 326 increased in the heart tissue of patients with heart failure, heart tissue of murine cardiac fibrosis models, and cardiac fibroblasts stimulated with angiotensin II. SNO-PKM2 reduced pyruvate kinase activity and tetramerization of PKM2, and cardiac fibroblast–specific PKM2 knockout aggravated cardiac fibrosis, whereas cardiac fibroblast–specific PKM2 knockout mice transfected with SNO-resistant mutant rather than wild-type PKM2 had cardiac function. Mechanistically, SNO-PKM2 drove excessive mitochondrial fission and mitochondrial dysfunction through interfering with its interaction with actin regulatory protein gelsolin. TEPP-46, a pharmacological PKM2 activator, alleviated mitochondrial fission and cardiac fibrosis. Moreover, the US Food and Drug Administration–approved drug mitapivat showed preventive and therapeutical effects on cardiac fibrosis through activating PKM2. CONCLUSIONS: SNO-PKM2 specifically increases in cardiac fibroblasts and activated cardiac fibroblasts by inducing excessive mitochondrial fission through a gelsolin-dependent manner. Mitapivat is a potential therapeutic option for attenuating cardiac fibrosis.

Abdominal aortic aneurysm (AAA) and the attendant catastrophic event of rupture remain a leading cause of death. Germline mutation of RRAS2 cause Noonan Syndrome, yet little is known about its function in AAA pathogenesis. Integrated analysis of RNA-seq data revealed RRAS2 a potential regulator of AAA. Its mRNA and protein levels reduced in abdominal aorta of AAA patients and AAA mice. Mice with SMCs-specific knockout of Rras2 were more vulnerable to Ang II and porcine pancreatic elastase-induced AAA, while overexpression of Rras2 in SMCs inhibited AAA progression. Maternally expressed gene 3 (MEG3) prevented RRAS2 mRNA degradation through binding with ELAV-like protein 1 (ELAVL1/HuR). Mechanistically, loss of R-Ras2 reduces phosphorylation, nuclear translocation, and transcriptional activity of general transcription factor 2I (GTF2-I/TFII-I) to reduce contractile-related genes expression in a MET tyrosine kinase-dependent manner. Here, we show an essential role of R-Ras2 in preserving VSMCs homeostasis and provide potential therapeutic targets for AAA.

Endothelial dysfunction is one of the earliest processes in diabetes and a major contributor to diabetic vascular complications, which often exhibit limited response to glucose-lowering therapies. We identified up-regulated S -nitrosoglutathione reductase (GSNOR) as a critical factor associated with diabetic vascular complications by unbiased proteomics. Elevated GSNOR expression was observed in the endothelium of patients with type 2 diabetes and in streptozotocin (STZ)–induced type 1 diabetes mice as well as in db/db type 2 diabetes mouse models. Genetic ablation of endothelial Gsnor promoted angiogenesis, maintained vascular permeability, and improved vasodilation in type 1 diabetes mice induced by STZ. GSNOR deficiency protected against high glucose–induced endothelial dysfunction in vitro, as evidenced by rescued tube formation, enhanced spheroid sprouting, maintained barrier integrity, and reduced permeability. Mechanistically, GSNOR orchestrated endothelial dysfunction independently of its enzymatic activity by binding the transcription factor ETS-related gene (ERG) and triggered its nuclear export through chromosome region maintenance 1. We synthesized NYY-001, an oral agent, that selectively blocks the GSNOR-ERG interaction. The direct targeting of NYY-001 to GSNOR was determined by resolving the crystal structure of their complex using cryo–electron microscopy. NYY-001 treatment enhanced postischemic neovascularization and restored vascular permeability in the peripheral vasculature in STZ-induced type 1 diabetes and db/db type 2 diabetes mouse models. These findings reveal a mechanistic role for the GSNOR-ERG complex in diabetic vascular complications and highlight NYY-001 as a promising therapeutic candidate.