NAD+ attenuates cardiac injury after myocardial infarction in diabetic mice through regulating alternative splicing of VEGF in macrophages.

Diabetic mellitus (DM) complicated with myocardial infarction (MI) is a serious clinical issue that remained poorly comprehended. The aim of the present study was to investigate the role of NAD+ in attenuating cardiac damage following MI in diabetic mice. The cardiac dysfunction in DM mice with MI was more severe compared with the non-diabetic mice and NAD+ administration could significantly improve the cardiac function in both non-diabetic and diabetic mice after MI for both 7 days and 28 days. Moreover, application of NAD+ could markedly reduce the cardiac injury area of DM complicated MI mice. Notably, the level of NAD+ was robustly decreased in the cardiac tissue of MI mice, which was further reduced in the DM complicated mice and NAD+ administration could significantly restore the NAD+ level. Furthermore, NAD+ was verified to facilitate the angiogenesis in the MI area of both diabetic mice and non-diabetic mice by microfil perfusion assay and immunofluorescence. Additionally, we demonstrated that NAD+ promoted cardiac angiogenesis after myocardial infarction in diabetic mice by promoting the M2 polarization of macrophages. At the molecular level, NAD+ promoted the secretion of VEGF in macrophages and therefore facilitating migration and tube formation of endothelial cells. Mechanistically, NAD+ was found to promote the generation of pro-angionesis VEGF165 and inhibit the generation of anti-angionesis VEGF165b via regulating the alternative splicing factors of VEGF (SRSF1 and SRSF6) in macrophages. The effects of NAD+ were readily reversible on deficiency of it. Collectively, our data showed that NAD+ could attenuate myocardial injury via regulating the alternative splicing of VEGF and promoting angiogenesis in diabetic mice after myocardial infarction. NAD+ administration may therefore be considered a potential new approach for the treatment of diabetic patients with myocardial infarction.

Comprehensive analysis of karyopherin alpha family expression in lung adenocarcinoma: Association with prognostic value and immune homeostasis.

Background: Karyopherin alpha (KPNA), a nuclear transporter, has been implicated in the development as well as the progression of many types of malignancies. Immune homeostasis is a multilevel system which regulated by multiple factors. However, the functional significance of the KPNA family in the pathogenesis of lung adenocarcinoma (LUAD) and the impact of immune homeostasis are not well characterized. Methods: In this study, by integrating the TCGA-LUAD database and Masked Somatic Mutation, we first conducted an investigation on the expression levels and mutation status of the KPNA family in patients with LUAD. Then, we constructed a prognostic model based on clinical features and the expression of the KPNA family. We performed functional enrichment analysis and constructed a regulatory network utilizing the differential genes in high-and low-risk groups. Lastly, we performed immune infiltration analysis using CIBERSORT. Results: Analysis of TCGA datasets revealed differential expression of the KPNA family in LUAD. Kaplan-Meier survival analyses indicated that the high expression of KPNA2 and KPNA4 were predictive of inferior overall survival (OS). In addition, we constructed a prognostic model incorporating clinical factors and the expression level of KPNA4 and KPNA5, which accurately predicted 1-year, 3-years, and 5-years survival outcomes. Patients in the high-risk group showed a poor prognosis. Functional enrichment analysis exhibited remarkable enrichment of transcriptional dysregulation in the high-risk group. On the other hand, gene set enrichment analysis (GSEA) displayed enrichment of cell cycle checkpoints as well as cell cycle mitotic in the high-risk group. Finally, analysis of immune infiltration revealed significant differences between the high-and low-risk groups. Further, the high-risk group was more prone to immune evasion while the inflammatory response was strongly associated with the low-risk group. Conclusions: the KPNA family-based prognostic model reflects many biological aspects of LUAD and provides potential targets for precision therapy in LUAD.

Cytokine levels in sputum, not serum, may be more helpful for indicating the damage in the lung and the prognosis of severe COVID-19 - A case series.

PURPOSE: To describe the relationship between the severity of lung damage and cytokine levels in sputum, bronchoalveolar lavage fluid (BALF), serum. METHOD: Eight severe patients infected with coronavirus disease 2019 (COVID-19) were admitted and their cytokines and chest computed tomography (CT) were analyzed. RESULTS: Compared with in serum, IL-6 and TNF-α in sputum and in BALF show more directly reflect the severity of COVID-19 critical patients. The gradient ratio of IL-6 levels may predict the prognosis of severe patients. CONCLUSION: Cytokine levels in the sputum may be more helpful for indicating lung damage. Local intervention through the respiratory tract is expected to benefit patients with severe COVID-19.

A feedback circuit comprising EHD1 and 14-3-3ζ sustains ß-catenin/c-Myc-mediated aerobic glycolysis and proliferation in non-small cell lung cancer.

Mammalian Eps15 homology domain 1 (EHD1) participates in the development of non-small cell lung cancer (NSCLC). However, its role in mediating aerobic glycolysis remains unclear. Herein, microarray analysis revealed that EHD1 expression was significantly correlated with the glycolysis/gluconeogenesis pathway. Clinically, EHD1 expression was positively correlated with the maximum standard uptake value (SUVmax) in 18F-FDG PET/CT scans. Additionally, EHD1 knockdown inhibited aerobic glycolysis and proliferation in vitro and in vivo. Furthermore, Wnt/ß-catenin signaling was identified as a critical EHD1-regulated pathway. Co-IP, native gel electrophoresis, and immunoblotting showed that EHD1 contributed to 14-3-3 dimerization via 14-3-3ζ and subsequent activation of ß-catenin/c-Myc signaling. Analysis of the EHD1 regulatory region via ENCODE revealed the potential for c-Myc recruitment, leading to transcriptional activation of EHD1 and formation of an EHD1/14-3-3ζ/ß-catenin/c-Myc positive feedback circuit. Notably, blocking this circuit with a Wnt/ß-catenin inhibitor dramatically inhibited tumor growth in vivo. The positive correlations among EHD1, 14-3-3ζ, c-Myc, and LDHA were further confirmed in NSCLC tissues. Collectively, our study demonstrated that EHD1 activates a 14-3-3ζ/ß-catenin/c-Myc regulatory circuit that synergistically promotes aerobic glycolysis and may constitute a promising therapeutic target for NSCLC.

Targeting the IL-1ß/EHD1/TUBB3 axis overcomes resistance to EGFR-TKI in NSCLC.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) significantly prolong the survival time of non-small-cell lung cancer (NSCLC) patients with EGFR-activating mutations, but resistance develops universally. Activation of the phosphatidyl inositol-3 kinase (PI3K)/AKT signaling pathway and phenotypic alterations in epithelial-mesenchymal transition (EMT) are both mechanisms of acquired resistance to EGFR-TKIs. However, the mechanisms underlying this resistance remain unclear. In this study, EHD1 depletion significantly increased NSCLC cell sensitivity to EGFR-TKI, which was accompanied by EMT reversal. Microarray analysis showed that the PTEN/PI3K/AKT signaling pathway is a crucial pathway regulated by EHD1. Moreover, a PTEN inhibitor abolished EHD1 shRNA regulation of EGFR-TKI sensitivity, EMT, and cancer progression. Mass spectrometry showed that TUBB3 is a novel EHD1-interacting protein. EHD1 modulated microtubule stability by interacting with TUBB3. Furthermore, TUBB3 depletion significantly attenuated EHD1-induced EGFR-TKI resistance and EMT. Bioinformatics analysis revealed that EHD1 is significantly associated with the gene set, "Cellular Response to Interleukin-1ß (IL-1ß)". As expected, treatment with IL-1ß led to increased expression of EHD1, activation of PTEN/PI3K/AKT signaling, and induction of EMT in NSCLC cells. In patient specimens, EHD1 was highly expressed in EGFR-TKI-refractory specimens. EHD1 was positively associated with TUBB3 and IL-1R1 but negatively associated with PTEN. In addition, targeting the IL-1ß/EHD1/TUBB3 axis mitigated cancer progression by inhibiting cell proliferation and metastasis and promoting apoptosis. Our study demonstrates the involvement of the IL-1ß/EHD1/TUBB3 axis in EGFR-TKI resistance and provides a potential therapeutic approach for treating patients with NSCLC that has acquired EGFR-TKI resistance.

Correction to: Tumor-associated neutrophils induce EMT by IL-17a to promote migration and invasion in gastric cancer cells.

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Tumor-associated neutrophils induce EMT by IL-17a to promote migration and invasion in gastric cancer cells.

PURPOSE: Epithelial to mesenchymal transition (EMT) can contribute to gastric cancer (GC) progression and recurrence following therapy. Tumor-associated neutrophils (TANs) are associated with poor outcomes in a variety of cancers. However, it is not clear whether TANs interact with the EMT process during GC development. METHODS: Immunohistochemistry was performed to examine the distribution and levels of CD66 + neutrophils in samples from 327 patients with GC. CD66b + TANs were isolated either directly from GC cell suspensions or were conditioned from healthy donor peripheral blood polymorphonuclear neutrophils (PMNs) stimulated with tumor tissue culture supernatants (TTCS) and placed into co-culture with MKN45 or MKN74 cells, after which migration, invasion and EMT were measured. Interleukin-17a (IL-17a) was blocked with a polyclonal antibody, and the STAT3 pathway was blocked with the specific inhibitor AG490. RESULTS: Neutrophils were widely distributed in gastric tissues of patients with GC and were enriched predominantly at the invasion margin. Neutrophil levels at the invasion margin were an independent predictor of poor disease-free survival (DFS) and disease-specific survival (DSS). IL-17a + neutrophils constituted a large portion of IL-17a-producing cells in GC, and IL-17a was produced at the highest levels in co-culture compared with that in TANs not undergoing co-culture. TANs enhanced the migration, invasion and EMT of GC cells through the secretion of IL-17a, which activated the Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT3) pathway in GC cells, while deprivation of IL-17a using a neutralizing antibody or inhibition of the JAK2/STAT3 pathway with AG490 markedly reversed these TAN-induced phenotypes in GC cells induced by TANs. CONCLUSIONS: Neutrophils correlate with tumor stage and predict poor prognosis in GC. TANs produce IL-17a, which promotes EMT of GC cells through JAK2/STAT3 signalling. Blockade of IL-17a signalling with a neutralizing antibody inhibits TAN-stimulated activity in GC cells. Therefore, IL-17a-targeted therapy might be used to treat patients with GC.

BTLA marks a less cytotoxic T-cell subset in diffuse large B-cell lymphoma with high expression of checkpoints.

Immunotherapy results in lymphoma have been encouraging. Preclinical and clinical trials have proven checkpoint blockade, such as PD-1 antibody, as an effective treatment for lymphoma, including diffuse large B-cell lymphoma (DLBCL). Combination of checkpoint blockades has emerged as a new way to treat lymphoma; however, the status of checkpoint expression and their function in DLBCL have not been fully elucidated yet. In this study, we examined the expression of BTLA, PD-1, TIM-3, LIGHT, and LAG-3 in tumor microenvironmental T cells of DLBCL using flow cytometry and compared the cytotoxicity and differentiation status of BTLA+ and BTLA- T-cells. We further characterized the relationship of STAT3 phosphorylation (p-STAT3) with BTLA expression. Our results suggest that BTLA+ T cells highly express other checkpoint molecules, including PD-1, TIM-3, LIGHT, and LAG-3. Moreover, high expression of BTLA is correlated with advanced stage of DLBCL. BTLA+ T cells have a less-differentiated phenotype, lower cytolytic function, and higher potential to proliferate compared with BTLA- T cells. Taken together, our data provide the first evidence that increased BTLA predicts poor prognosis in patients with DLBCL, and blockade of BTLA with other checkpoints may potentially represent a new strategy for immunotherapy of DLBCL.