Ferroptosis-related signature and immune infiltration characterization in acute lung injury/acute respiratory distress syndrome.

BACKGROUND: Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is one of the most life-threatening diseases in the intensive care unit with high mortality and morbidity. Ferroptosis is a newly discovered immune related cell death that is associated with various lung diseases. However, the role of immune-mediated ferroptosis in ALI/ARDS has not been elucidated. METHOD: We analyzed two Gene Expression Omnibus (GEO) datasets (GSE2411 and GSE109913) and extracted characteristic ferroptosis-related genes (FRGs) between the control and ALI groups through bioinformatic analysis. Then, we prospectively collected bronchoalveolar lavage fluid (BALF) from patients with ARDS and verified the expression of characteristic FRGs. Lastly, we constructed the ALI/ARDS model induced by LPS and isolated the primary neutrophils of mice. Erastin, an ferroptosis inducer, was used at the cellular level to verify the effect of neutrophils on ferroptosis in lung epithelium cells. RESULT: We identified three characteristic FRGs, Cp, Slc39a14 and Slc7a11, by analyzing two gene expression profiling datasets. Immune infiltration analysis showed that the three characteristic genes were significantly positively correlated with the infiltration levels of neutrophils. We collected BALF from 59 ARDS patients to verify the expression of Cp, Slc7a11 and Slc39a14 in humans. The results showed that Cp was elevated in patients with severe ARDS (p = 0.019), Slc7a11 was significantly elevated in patients with moderate ARDS (p = 0.021) relative to patients with mild ARDS. The levels of neutrophils in the peripheral blood of ARDS patients were positively correlated with the expression levels of Slc7a11 (Pearson's R2 = 0.086, p = 0.033). Three characteristic FRGs were significantly activated after the onset of ferroptosis (6 h) early in LPS induced ALI model, and that ferroptosis was alleviated after the organism compensated within 12 to 48 h. We extracted primary activated neutrophils from mice and co-cultured them with MLE-12 in transwell, Slc7a11, Cp and Slc39a14 in MLE-12 cells were significantly upregulated as the number of neutrophils increased. The results showed that neutrophil infiltration alleviated erastin-induced MDA accumulation, GSH depletion, and divalent iron accumulation, accompanied by upregulation of Slc7a11 and Gpx4, implying the existence of a compensatory effect of lipid oxidation in neutrophils after acute lung injury in the organism. CONCLUSION: We identified three immune-mediated ferroptosis genes, namely, Cp, Slc7a11 and Slc39a14, which possibly regulated by neutrophils during the development of ALI, and their pathways may be involved in anti-oxidative stress and anti-lipid metabolism. Thus, the present study contributes to the understanding of ALI/ARDS and provide novel targets for future immunotherapeutic.

Intermedin (adrenomedullin 2) promotes breast cancer metastasis via Src/c-Myc-mediated ribosome production and protein translation.

PURPOSE: Breast cancer is the most frequently diagnosed cancer and is the leading cause of cancer-associated mortality in women worldwide. Intermedin (IMD, also known as Adrenomedullin 2, ADM2) is an endogenous peptide that belongs to the calcitonin gene-related peptide family and has been reported to play important roles in several types of cancers, including breast cancer. In this study, we sought to investigate how IMD affects the behavior of breast cancer cells, the underlying mechanism of these effects, and whether blockade of IMD has a therapeutic effect against breast cancer. METHODS: Transcriptome sequencing (RNA-Seq), cell biological experiments, Western blotting, immunoprecipitation, and animal tumor models were used. RESULTS: IMD expression was significantly increased in breast cancer samples, and the IMD level was positively correlated with lymph node metastasis and Ki67 expression. Cell biological experiments showed that IMD promoted the anchorage-independent growth, migration, and invasive ability of breast cancer cells. Inhibiting IMD activity with an anti-IMD monoclonal antibody blocked these tumor-promoting effects. In addition, blockade of IMD reduced in situ tumor growth and significantly decreased lung metastasis of 4T1 breast cancer in vivo. IMD induced Src kinase phosphorylation, which triggered the transcription of c-Myc, a major oncoprotein controlling the expression of genes that encode ribosomal components. Our data suggest that IMD is involved in breast cancer cell invasion and metastasis, potentially through increasing ribosome biogenesis and protein translation via the Src/c-Myc signaling pathway. CONCLUSION: These results suggest that IMD may be a novel target for the treatment of breast cancer.

Sunitinib facilitates metastatic breast cancer spreading by inducing endothelial cell senescence.

BACKGROUND: Sunitinib, a receptor tyrosine kinase (RTK) inhibitor that targets multiple receptors such as vascular endothelial growth factor receptors (VEGFRs), was approved for cancer treatment in 2006. However, it was unsuccessful in treating certain cancers, particularly metastatic breast cancer (MBC), and the mechanism underlying this "sunitinib resistance" remains unclear. Herein, we investigated whether the sunitinib-associated inferior survival benefit in MBC was due to sunitinib-induced endothelial cell (EC) injury or EC senescence. METHODS: 4T1 murine breast cancer cells were used as the main breast tumor model for it produces a highly metastatic solid tumor that can spontaneously metastasize to the lung, which closely mimics highly metastatic human breast cancer. Senescence-associated ß-galactosidase (SA-ß-Gal, immunohistochemistry [IHC]-staining), P16, P53, and P57 (immunoblotting) were used as markers of cell senescence. A protein array containing 25 senescence-associated chemokines and the transwell chemotaxis assay were used to examine whether sunitinib increases inflammatory chemokine secretion which attracts tumor cells via chemokinesis. Flow cytometry and IHC were used to detect whether the sunitinib-induced senescent ECs recruit cancer-associated inflammatory myeloid cells. Finally, the spontaneous metastatic model was used to monitor whether sunitinib causes the formation of "pre-metastatic niche" which promotes MBC to metastasize to the lungs. RESULTS: We demonstrated that sunitinib induced a senescence-like endothelial cell (EC) phenotype. Inflammatory chemokine secretion and VCAM1 expression were significantly increased in senescent ECs, resulting in tumor cell (TC) chemotaxis and TC/EC interactions. Meanwhile, EC senescence caused loosening of EC junctions, facilitating TC transmigration through the endothelial barrier. Sunitinib-induced senescent ECs also recruited cancer-associated myeloid cells to form a "pre-metastatic niche"-like microenvironment. Alterations at the molecular level and in the tissue environment ultimately led to an increase in distant metastasis. CONCLUSION: Although sunitinib was designed to target the EC directly, the increase in tumor metastasis may ironically be due to sunitinib "correctly" playing its role. Our findings suggest that we should carefully weigh the pros and cons before using sunitinib and other antiangiogenic drugs that directly target the ECs.

High Serum Albumin Levels were Associated with Acute Kidney Injury in Pediatric Surgical Intensive Care Units.

INTRODUCTION: There are limited studies revealing the association between serum albumin concentrations and acute kidney injury (AKI) in critically ill children. METHODS: This was a multicenter retrospective study. Children consecutively admitted to four pediatric surgical intensive care units (PSICUs) between January 2016 and December 2020 were screened for analysis. Patients without recorded albumin values during the PSICU stay were excluded. Data were extracted from the electronic medical records systems of the hospitals. AKI was defined according to the Kidney Disease Improving Global Outcome (KDIGO) guidelines. The associations between serum albumin levels and AKI were assessed by using logistic regression models. RESULTS: A total of 7802 children were included in the analysis. The median age of the children was 1.0 (interquartile range (IQR), 0.0-4.0) years. There were 3214 (41.2 %) children who developed AKI. In the univariate logistic regression model, serum albumin levels were associated with AKI (odds ratio (OR) = 1.04, 95 % confidence interval (CI) 1.04-1.05). After adjusting for covariates, serum albumin showed an independent association with AKI (OR = 1.04, 95 % CI 1.03-1.05). Albumin levels above 39.43 g/L (OR = 1.036, 95 % CI 1.002-1.070) were associated with AKI in the unadjusted cubic spline. In the adjusted cubic spline, albumin levels above 40.41 g/L (OR = 1.061, 95 % CI 1.003-1.122) were associated with AKI. CONCLUSION: High serum albumin was associated with AKI in critically ill children in the PSICU. Further studies are needed to validate our findings. TYPE OF STUDY: Prognostic Study. LEVEL OF EVIDENCE: LEVEL II.

Heatstroke: a multicenter study in Southwestern China.

Background: An increase in Heatstroke cases occurred in southwest China in 2022 due to factors like global warming, abnormal temperature rise, insufficient power supply, and other contributing factors. This resulted in a notable rise in Heatstroke patients experiencing varying degrees of organ dysfunction. This descriptive study aims to analyze the epidemiology and clinical outcomes of Heatstroke patients in the ICU, providing support for standardized diagnosis and treatment, ultimately enhancing the prognosis of Heatstroke. Methods: A retrospective, multicenter, descriptive analysis was conducted on Heatstroke patients admitted to ICUs across 83 hospitals in southwest China. Electronic medical records were utilized for data collection, encompassing various aspects such as epidemiological factors, onset symptoms, complications, laboratory data, concurrent infections, treatments, and patient outcomes. Results: The dataset primarily comprised classic heatstroke, with 477 males (55% of total). The patient population had a median age of 72 years (range: 63-80 years). The most common initial symptoms were fever, mental or behavioral abnormalities, and fainting. ICU treatment involved respiratory support, antibiotics, sedatives, and other interventions. Among the 700 ICU admissions, 213 patients had no infection, while 487 were diagnosed with infection, predominantly lower respiratory tract infection. Patients presenting with neurological symptoms initially (n = 715) exhibited higher ICU mortality risk compared to those without neurological symptoms (n = 104), with an odds ratio of 2.382 (95% CI 1.665, 4.870) (p = 0.017). Conclusion: In 2022, the majority of Heatstroke patients in southwest China experienced classical Heatstroke, with many acquiring infections upon admission to the ICU. Moreover, Heatstroke can result in diverse complications.

Intermedin (adrenomedullin 2) plays a protective role in sepsis by regulating T- and B-cell proliferation and activity.

BACKGROUND: Sepsis is the major cause of death in intensive care units. We previously found that intermedin (IMD), a calcitonin family peptide, can protect against sepsis by dynamically repairing vascular endothelial junctions and can ameliorate the inflammatory response by inhibiting the infiltration of macrophages in peripheral tissues. The effects of IMD on inflammatory and immune responses indicate that IMD may play a role in immunity. However, whether IMD affects immune cell development, differentiation and response to infection remains unclear. METHODS: IMD-knockout (Adm2-/-) mice were generated in our previous work. Wild-type and IMD-KO mice were subjected to sham or cecal ligation and puncture (CLP) surgery, and bone marrow cells were obtained for RNA sequencing (RNA-Seq) analysis. The RNA-Seq results were verified by real-time RT-PCR. The effect of IMD KO or IMD rescue on the septic mice was explored using mild and severe infection models induced by CLP surgery at different levels of severity, and the survival outcomes were analyzed using Kaplan-Meier curves and the log-rank test. The mechanism underlying the effects of IMD in T/B cell proliferation and differentiation were investigated by PCR, Western blot (WB), and cell proliferation assays and flow cytometry analysis. RESULTS: RNA-Seq showed that IMD-KO mice exhibited a primary immunosuppression phenotype characterized by a marked decrease in the expression of T- and B-cell function-related genes. This immunosuppression made the IMD-KO mice vulnerable to pathogenic invasion, and even mild infection killed nearly half of the IMD-KO mice. Supplementation with the IMD peptide restored the expression of T/B-cell-related genes and significantly reduced the mortality rate of the IMD-KO mice. IMD is likely to directly promote T- and B-cell proliferation through ERK1/2 phosphorylation, stimulate T-cell differentiation via Ilr7/Rag1/2-controled T cell receptor (TCR) recombination, and activate B cells via Pax5, a transcription factor that activates at least 170 genes needed for B-cell functions. CONCLUSION: Together with previous findings, our results indicate that IMD may play a protective role in sepsis via three mechanisms: protecting the vascular endothelium, reducing the inflammatory response, and activating T/B-cell proliferation and differentiation. Our study may provide the first identification of IMD as a calcitonin peptide that plays an important role in the adaptive immune response by activating T/B cells and provides translational opportunities for the design of immunotherapies for sepsis and other diseases associated with primary immunodeficiency.

Risk Factor Analysis and Nomogram for Predicting In-Hospital Mortality in ICU Patients with Heat Stroke: A National Multicenter Study.

Objective: The aim of this nationwide multicenter study was to ascertain the risk factors associated with in-hospital mortality in patients with heat stroke admitted to intensive care units (ICUs) and to develop a nomogram for prognostic prediction. Methods: A retrospective analysis was conducted on clinical data collected from ICU patients diagnosed with heat stroke across multiple centers nationwide. Univariate and multivariate logistic regression analyses were performed to identify significant risk factors for in-hospital mortality. Based on the results of the multivariate analysis, a nomogram was constructed to estimate the individualized probability of mortality. Internal validation of the nomogram was performed, and its performance was assessed using receiver operating characteristic (ROC) curves, calibration plots, and decision curve analysis (DCA). Results: A total of 292 ICU patients with heat stroke were included in this study. Three risk factors, namely Cr (creatinine), AST (aspartate aminotransferase), and SBP (systolic blood pressure), were found to be significantly associated with in-hospital mortality. These risk factors were incorporated into the nomogram, which exhibited good discriminative ability (area under the ROC curve of the training and validation cohorts were 0.763 and 0.739, respectively) and calibration. Internal validation and decision curve analysis confirmed the stability and reliability of the nomogram. Conclusion: This nationwide multicenter study identified key risk factors for in-hospital mortality in ICU patients with heat stroke. The developed nomogram provides an individualized prediction of mortality risk and can serve as a valuable tool for clinicians in the assessment and management of ICU patients with heat stroke.

Intermedin facilitates hepatocellular carcinoma cell survival and invasion via ERK1/2-EGR1/DDIT3 signaling cascade.

As one of the most malignant cancer types, hepatocellular carcinoma (HCC) is highly invasive and capable of metastasizing to distant organs. Intermedin (IMD), an endogenous peptide belonging to the calcitonin family, has been suggested playing important roles in cancer cell survival and invasion, including in HCC. However, how IMD affects the behavior of HCC cells and the underlying mechanisms have not been fully elucidated. Here, we show that IMD maintains an important homeostatic state by activating the ERK1/2-EGR1 (early growth response 1) signaling cascade, through which HCC cells acquire a highly invasive ability via significantly enhanced filopodia formation. The inhibition of IMD blocks the phosphorylation of ERK1/2, resulting in EGR1 downregulation and endoplasmic reticulum stress (ER) stress, which is evidenced by the upregulation of ER stress marker DDIT3 (DNA damage-inducible transcript 3). The high level of DDIT3 induces HCC cells into an ER-stress related apoptotic pathway. Along with our previous finding that IMD plays critical roles in the vascular remodeling process that improves tumor blood perfusion, IMD may facilitate the acquisition of increased invasive abilities and a survival benefit by HCC cells, and it is easier for HCC cells to obtain blood supply via the vascular remodeling activities of IMD. According to these results, blockade of IMD activity may have therapeutic potential in the treatment of HCC.

Inhibition of Intermedin (Adrenomedullin 2) Suppresses the Growth of Glioblastoma and Increases the Antitumor Activity of Temozolomide.

Glioblastoma multiforme (GBM; grade IV glioma) is the most malignant type of primary brain tumor and is characterized by rapid proliferation and invasive growth. Intermedin (IMD) is an endogenous peptide belonging to the calcitonin gene-related peptide family and has been reported to play an important role in cell survival and invasiveness in several types of cancers. In this study, we found that the expression level of IMD was positively related to the malignancy grade of gliomas. The highest expression of IMD was found in GBM, indicating that IMD may play an important role in glioma malignancy. IMD increased the invasive ability of glioma cells by promoting filopodia formation, which is dependent on ERK1/2 activation. IMD-induced ERK1/2 phosphorylation also promoted GBM cell proliferation. In addition, IMD enhanced mitochondrial function and hypoxia-induced responses in GBM cells. Treatment with anti-IMD monoclonal antibodies not only inhibited tumor growth in both ectopic and orthotopic models of GBM but also significantly enhanced the antitumor activity of temozolomide. Our study may provide novel insights into the mechanism of GBM cell invasion and proliferation and provide an effective strategy to improve the therapeutic effect of GBM treatments.

Intermedin promotes vessel fusion by inducing VE-cadherin accumulation at potential fusion sites and to achieve a dynamic balance between VE-cadherin-complex dissociation/reconstitution.

To create a closed vascular system, angiogenic sprouts must meet and connect in a process called vessel fusion, which is a prerequisite for establishment of proper blood flow in nascent vessels. However, the molecular machinery underlying this process remains largely unknown. Herein, we report that intermedin (IMD), a calcitonin family member, promotes vessel fusion by inducing endothelial cells (ECs) to enter a "ready-to-anchor" state. IMD promotes vascular endothelial cadherin (VEC) accumulation at the potential fusion site to facilitate anchoring of approaching vessels to each other. Simultaneously, IMD fine-tunes VEC activity to achieve a dynamic balance between VEC complex dissociation and reconstitution in order to widen the anastomotic point. IMD induces persistent VEC phosphorylation. Internalized phospho-VEC preferentially binds to Rab4 and Rab11, which facilitate VEC vesicle recycling back to the cell-cell contact for reconstruction of the VEC complex. This novel mechanism may explain how neovessels contact and fuse to adjacent vessels to create a closed vascular system.

Intermedin protects against sepsis by concurrently re-establishing the endothelial barrier and alleviating inflammatory responses.

Sepsis is a life-threatening condition caused by dysregulated host responses to infection. Widespread vascular hyperpermeability and a "cytokine storm" are two pathophysiological hallmarks of sepsis. Here, we show that intermedin (IMD), a member of the calcitonin family, alleviates organ injury and decreases mortality in septic mice by concurrently alleviating vascular leakage and inflammatory responses. IMD promotes the relocation of vascular endothelial cadherin through a Rab11-dependent pathway to dynamically repair the disrupted endothelial junction. Additionally, IMD decreases inflammatory responses by reducing macrophage infiltration via downregulating CCR2 expression. IMD peptide administration ameliorates organ injuries and significantly improves the survival of septic mice, and the experimental results correlate with the clinical data. Patients with high IMD levels exhibit a lower risk of shock, lower severity scores, and greatly improved survival outcomes than those with low IMD levels. Based on our data, IMD may be an important self-protective factor in response to sepsis.