GDF11 inhibits the malignant progression of hepatocellular carcinoma via regulation of the mTORC1­autophagy axis.

Hepatocellular carcinoma (HCC) is a common malignant tumor, which is associated with a poor prognosis and high mortality rate. It is well known that growth differentiation factor 11 (GDF11) acts as a tumor suppressor in various types of cancer, including HCC. The present study aimed to determine the tumor-suppressive properties of GDF11 in HCC and to assess the intrinsic mechanisms. In the present study, the human hepatoma cell line Huh-7 was transfected with the GDF11 overexpression plasmid (Oe-GDF11) for gain-of-function experiments to investigate the effects of GDF11 on the biological behaviors of HCC cells, including proliferation, colony formation, apoptosis, cell cycle arrest, migration, invasion, epithelial-mesenchymal transition (EMT) and angiogenesis. The proliferation, colony formation, apoptosis, cell cycle, migration, invasion and angiogenesis of HCC cells were assessed by CCK-8, EdU staining, colony formation, flow cytometry, wound healing, Transwell and tube formation assays, respectively. Apoptosis-, cell cycle-, EMT-related key factors were also determined by western blot assay. Furthermore, Oe-GDF11-transfected Huh-7 cells were treated with the mammalian target of rapamycin (mTOR) activator MHY1485 for rescue experiments to explore whether GDF11 could exert antitumor effects against HCC via mediating the mTOR complex 1 (mTORC1)-autophagy axis. In the present study, GDF11 was verified to be lowly expressed in HCC cells. Overexpression of GDF11 inhibited the proliferation, colony formation, migration, invasion, EMT and angiogenesis of HCC cells, and facilitated the apoptosis and cell cycle arrest of HCC cells. Additionally, it was verified that overexpression of GDF11 inactivated the mTORC1 signaling pathway to enhance autophagy in HCC cells. Treatment with the mTOR activator MHY1485 partially reversed the tumor-suppressive effects of GDF11 overexpression on HCC. In conclusion, GDF11 may exert tumor-suppressive properties in HCC cells through inactivating the mTORC1 signaling pathway to strengthen autophagy.

Ginsenoside Rg1 Alleviates Sepsis-Induced Acute Lung Injury by Reducing FBXO3 Stability in an m6A-Dependent Manner to Activate PGC-1α/Nrf2 Signaling Pathway.

BACKGROUND: Sepsis-induced acute lung injury (ALI) is one of the serious life-threatening complications of sepsis and is pathologically associated with mitochondrial dysfunction. Ginsenoside Rg1 has good therapeutic effects on ALI. Herein, the pharmacological effects of Rg1 in sepsis-induced ALI were investigated. METHODS: Sepsis-induced ALI models were established by CLP operation and LPS treatment. HE staining was adopted to analyze lung pathological changes. The expression and secretion of cytokines were measured by RT-qPCR and ELISA. Cell viability and apoptosis were assessed by MTT assay, flow cytometry and TUNEL staining. ROS level and mitochondrial membrane potential (MMP) were analyzed using DHE probe and JC-1 staining, respectively. FBXO3 m6A level was assessed using MeRIP assay. The interactions between FBXO3, YTHDF1, and PGC-1α were analyzed by Co-IP or RIP. RESULTS: Rg1 administration ameliorated LPS-induced epithelial cell inflammation, apoptosis, and mitochondrial dysfunction in a dose-dependent manner. Mechanically, Rg1 reduced PGC-1α ubiquitination modification level by inhibiting FBXO3 expression m6A-YTHDF1 dependently. As expected, Rg1's mitigative effect on LPS-induced inflammation, apoptosis and mitochondrial dysfunction in lung epithelial cells was abolished by FBXO3 overexpression. Moreover, FBXO3 upregulation eliminated the restoring effect of Rg1 on CLP-induced lung injury in rats. CONCLUSION: Rg1 activated PGC-1α/Nrf2 signaling pathway by reducing FBXO3 stability in an m6A-YTHDF1-dependent manner to improve mitochondrial function in lung epithelial cells during sepsis-induced ALI progression.

Effect of stratified dose of norepinephrine on cellular immune response in patients with septic shock and the construction of a prognostic risk model.

OBJECTIVE: To explore the effect of a stratified dose of norepinephrine (NE) on cellular immune response in patients with septic shock, and to construct a prognostic model of septic shock. METHODS: A total of 160 patients with septic shock (B group) and 58 patients with sepsis (A group) were given standard cluster therapy. Patients with septic shock were divided into four groups (B1-B4 groups: 0.01-0.2, 0.2-0.5, 0.5-1.0, and > 1 µg/kg/min) according to the quartile method of the early (72 h) time-weighted average dose of NE and clinical application. The cellular immune indexes at 24 h (T0) and 4-7 days (T1) after admission were collected. The difference method was used to explore the effect of NE stratified dose on cellular immune effect in patients with septic shock. A multivariate COX proportional risk regression model was used to analyze the independent prognostic risk factors, and a prognostic risk model was constructed. RESULTS: The differences of ΔIL-1ß, ΔIL-6, ΔIL-10, absolute value difference of T lymphocyte (ΔCD3+/CD45 + #) and Th helper T cell (ΔCD3+ CD4+/CD45 + #), CD64 infection index difference, ΔmHLA-DR, regulatory T lymphocyte ratio difference (ΔTregs%) between group A, B1, B2, B3 and B4 were statistically significant ( p < 0.05). There was a nonlinear relation between the stratified dose of NE and ΔIL-6, ΔIL-10, ΔCD3+/CD45 + #, ΔmHLA-DR%. The threshold periods of NE-induced pro-inflammatory and anti-inflammatory immune changes were 0.3-0.5 µg/kg/min. Multivariate COX model regression analysis showed that age, nutritional patterns, weighted average dose of norepinephrine, IL-6, absolute value of T lymphocytes, and mHLA-DR were independent risk factors affecting the prognosis of patients with septic shock ( p < 0.05). The prognostic risk model was constructed (AUC value = 0.813, 95%CI: 0.752-0.901). CONCLUSION: NE has a certain inhibitory effect on cellular immune function in patients with septic shock. A prognostic risk model was constructed with stronger prediction efficiency for the prognosis of patients with septic shock.

Characterization of tumor microenvironment and sensitive chemotherapy drugs based on cuproptosis-related signatures in renal cell carcinoma.

Cuproptosis is a novel type of copper-induced cell death and is considered as a new therapeutic target for many cancers. Distant metastases occur in about 40% of patients with advanced renal cell carcinoma (RCC), with a poor 5-year prognosis of about 10%. Through a series of comprehensive analyses, four differentially expressed cuproptosis-related lncRNAs (DECRLs) were identified as candidate biomarkers for RCC. The risk model constructed by using these four DECRLs can better predict the prognosis of patients with RCC, which is determined by the receiver operating characteristic (Time dependent area under curve value at 1-year, 3-year, 5-year, and 10-year were 0.82, 0.80, 0.76, and 0.73 respectively). There were significant differences in immune status between high-risk and low-risk RCC patients. The differentially expressed gene enrichment terms between high- and low-risk patients was also dominated by immune-related terms. The risk score was also correlated with immunotherapy as measured by the tumor immune dysfunction and exclusion (TIDE) score. In addition, we also found that the sensitivity of many chemotherapy drugs varies widely between high- and low-risk patients. The sensitivity of the three chemotherapy drugs (AZD4547, Vincristine, and WEHI-539) varied among high- and low-risk patients, and was significantly negatively correlated with risk values, suggesting that they could be used as clinical treatment drugs for RCC. Our study not only obtained four potential biomarkers, but also provided guidance for immunotherapy and chemotherapy treatment of RCC, as well as new research strategies for the screening of other cancer biomarkers and sensitive drugs.

Wnt signaling regulates MFSD2A-dependent drug delivery through endothelial transcytosis in glioma.

BACKGROUND: Systemic delivery of anti-tumor therapeutic agents to brain tumors is thwarted by the blood-brain barrier (BBB), an organotypic specialization of brain endothelial cells (ECs). A failure of pharmacological compounds to cross BBB is one culprit for the dismal prognosis of glioblastoma (GBM) patients. Identification of novel vascular targets to overcome the challenges posed by the BBB in tumors for GBM treatment is urgently needed. METHODS: Temozolomide (TMZ) delivery was investigated in CT2A and PDGFB-driven RCAS/tv-a orthotopic glioma models. Transcriptome analysis was performed on ECs from murine gliomas. Mfsd2a deficient, Cav1 deficient, and Mfsd2a EC-specific inducible mice were developed to study the underlying molecular mechanisms. RESULTS: We demonstrated that inhibiting Wnt signaling by LGK974 could increase TMZ delivery and sensitize glioma to chemotherapy in both murine glioma models. Transcriptome analysis of ECs from murine gliomas revealed that Wnt signaling inhibition enhanced vascular transcytosis as indicated by the upregulation of PLVAP and downregulation of MFSD2A. Mfsd2a deficiency in mice enhances TMZ delivery in tumors, whereas constitutive expression of Mfsd2a in ECs suppresses the enhanced TMZ delivery induced by Wnt pathway inhibition in murine glioma. In addition, Wnt signaling inhibition enhanced caveolin-1 (Cav1)-positive caveolae-mediated transcytosis in tumor ECs. Moreover, Wnt signaling inhibitor or Mfsd2a deficiency fails to enhance TMZ penetration in tumors from Cav1-deficient mice. CONCLUSIONS: These results demonstrated that Wnt signaling regulates MFSD2A-dependent TMZ delivery through a caveolae-mediated EC transcytosis pathway. Our findings identify Wnt signaling as a promising therapeutic target to improve drug delivery for GBM treatment.

ECO: An Integrated Gene Expression Omnibus for Mouse Endothelial Cells In Vivo.

Endothelial cell (EC) plays critical roles in vascular physiological and pathological processes. With the development of high-throughput technologies, transcriptomics analysis of EC has increased dramatically and a large amount of informative data have been generated. The dynamic patterns of gene expression in ECs under various conditions were revealed. Unfortunately, due to the lack of bioinformatics infrastructures, reuse of these large-scale datasets is challenging for many scientists. Here, by systematic re-analyzing, integrating, and standardizing of 203 RNA sequencing samples from freshly isolated mouse ECs under 71 conditions, we constructed an integrated mouse EC gene expression omnibus (ECO). The ECO database enables one-click retrieval of endothelial expression profiles from different organs under different conditions including disease models, genetic modifications, and clinically relevant treatments in vivo. The EC expression profiles are visualized with user-friendly bar-plots. It also provides a convenient search tool for co-expressed genes. ECO facilitates endothelial research with an integrated tool and resource for transcriptome analysis. The ECO database is freely available at https://heomics.shinyapps.io/ecodb/.

Uncovering a Key Role of ETS1 on Vascular Abnormality in Glioblastoma.

Glioblastoma (GBM) is the most aggressive type of brain tumor. Microvascular proliferation and abnormal vasculature are the hallmarks of the GBM, aggravating disease progression and increasing patient morbidity. Here, we uncovered a key role of ETS1 on vascular abnormality in glioblastoma. ETS1 was upregulated in endothelial cells from human tumors compared to endothelial cells from paired control brain tissue. Knockdown of Ets1 in mouse brain endothelial cells inhibited cell migration and proliferation, and suppressed expression of genes associated with vascular abnormality in GBM. ETS1 upregulation in tumor ECs was dependent on TGFß signaling, and targeting TGFß signaling by inhibitor decreased tumor angiogenesis and vascular abnormality in CT-2A glioma model. Our results identified ETS1 as a key factor regulating tumor angiogenesis, and suggested that TGFß inhibition may suppress the vascular abnormality driven by ETS1.

Uncovering a Distinct Gene Signature in Endothelial Cells Associated With Contrast Enhancement in Glioblastoma.

PURPOSE: Glioblastoma (GBM) is the most aggressive and lethal type of brain tumors. Magnetic resonance imaging (MRI) has been commonly used for GBM diagnosis. Contrast enhancement (CE) on T1-weighted sequences are presented in nearly all GBM as a result of high vascular permeability in glioblastomas. Although several radiomics studies indicated that CE is associated with distinct molecular signatures in tumors, the effects of vascular endothelial cells, the key component of blood brain barrier (BBB) controlling vascular permeability, on CE have not been thoroughly analyzed. METHODS: Endothelial cell enriched genes have been identified using transcriptome data from 128 patients by a systematic method based on correlation analysis. Distinct endothelial cell enriched genes associated with CE were identified by analyzing difference of correlation score between CE-high and CE-low GBM cases. Immunohistochemical staining was performed on in-house patient cohort to validate the selected genes associated with CE. Moreover, a survival analysis was conducted to uncover the relation between CE and patient survival. RESULTS: We illustrated that CE is associated with distinct vascular molecular imprints characterized by up-regulation of pro-inflammatory genes and deregulation of BBB related genes. Among them, PLVAP is up-regulated, whereas TJP1 and ABCG2 are down-regulated in the vasculature of GBM with high CE. In addition, we found that the high CE is associated with poor prognosis and GBM mesenchymal subtype. CONCLUSION: We provide an additional insight to reveal the molecular trait for CE in MRI images with special focus on vascular endothelial cells, linking CE with BBB disruption in the molecular level. This study provides a potential new direction that may be applied for the treatment optimization based on MRI features.

1p/19q co-deletion status is associated with distinct tumor-associated macrophage infiltration in IDH mutated lower-grade gliomas.

BACKGROUND: Tumor-associated macrophages (TAM)s are critical regulators of glioma progression. As yet, however, TAMs in isocitrate dehydrogenase (IDH) mutated lower-grade gliomas (LGGs) have not been thoroughly investigated. The aim of this study was to determine whether 1p/19q co-deletion status affects the TAM phenotype or its prevalence in IDH mutated LGGs. METHODS: TAMs in IDH mutated LGGs were analyzed using transcriptome data from 230 samples in the TCGA database in combination with transcriptome data from single-cell RNA sequencing of IDH-mutated LGGs. Proteins potentially involved in TAM regulation were examined by immuno-staining in primary LGG samples harboring IDH mutations. Essential signaling pathways regulating TAM phenotypes were investigated in a glioma mouse model using small molecule inhibitors. RESULTS: Most of the TAMs in IDH-mutated LGGs expressed the M1 activation markers CD86 and TNF, whereas a subset of individual TAMs co-expressed both M1 and M2-related markers. Bioinformatics analysis in combination with immuno-staining of IDH-mutated patient samples revealed higher amounts of TAMs expressing M2-related markers in 1p/19q non-codeletion IDH-mutated LGGs compared to 1p/19q codeletion LGGs. The levels of transforming growth factor beta 1 (TGFß1) and macrophage colony-stimulating factor (M-CSF) were significantly higher in 1p/19q non-codeletion LGGs than in 1p/19q codeletion LGGs. M-CSF and TGFß1 signal inhibition decreased tumor growth and modulated the TAM phenotype in a glioma mouse model. CONCLUSIONS: Our data indicate that 1p/19q co-deletion status relates to distinct TAM infiltration in gliomas, which is likely mediated by M-CSF and TGFß1 signaling. M-CSF and TGFß1 signaling may play a pivotal role in regulating the TAM phenotype in glioma.

An Integrated Transcriptome Analysis Reveals IGFBP7 Upregulation in Vasculature in Traumatic Brain Injury.

Vasculature plays critical roles in the pathogenesis and neurological repair of traumatic brain injury (TBI). However, how vascular endothelial cells respond to TBI at the molecular level has not been systematically reviewed. Here, by integrating three transcriptome datasets including whole cortex of mouse brain, FACS-sorted mouse brain endothelial cells, and single cell sequencing of mouse brain hippocampus, we revealed the key molecular alteration of endothelial cells characterized by increased Myc targets and Epithelial-Mesenchymal Transition signatures. In addition, immunofluorescence staining of patients' samples confirmed that IGFBP7 was up-regulated in vasculature in response to TBI. TGFß1, mainly derived from microglia and endothelial cells, sufficiently induces IGFBP7 expression in cultured endothelial cells, and is significantly upregulated in response to TBI. Our results identified IGFBP7 as a potential biomarker of vasculature in response to TBI, and indicate that TGFß signaling may contribute to the upregulation of IGFBP7 in the vasculature.

Risk of bleeding in patients with continued dual antiplatelet therapy during orthopedic surgery.

BACKGROUND: To prevent risk of life-threatening stent thrombosis, all patients need to undergo dual antiplatelet therapy (DAPT) for at least 6 weeks to 12 months after stent implantation. If DAPT is continued during noncardiac surgery, there is a risk of severe bleeding at the surgical site. Our study was to assess the risk of bleeding in patients with continued DAPT during orthopedic surgery. METHODS: The clinical data of 78 patients with coronary heart disease who underwent orthopedic surgery from February 2006 to July 2018 were retrospectively analyzed. Prior to orthopedic surgery, DAPT was continued in 16 patients (group I), 24 patients were treated with single antiplatelet therapy (group II), and 26 patients received low-molecular-weight heparin therapy for more than 5 days after the discontinuation of all antiplatelet therapies (group III). Twelve patients were excluded, as they had undergone minimally invasive surgery such as transforaminal endoscopy and vertebroplasty. The perioperative blood loss of each patient was calculated using Nadler's formula and Gross' formula. The intraoperative bleeding volume, total volume of intraoperative bleeding in addition to postoperative drainage, and total blood loss were compared between groups. The level of significance was set at P < 0.05. RESULTS: There were no significant differences between the three groups in age, intraoperative bleeding volume, total volume of intraoperative bleeding in addition to postoperative drainage, and total perioperative blood loss calculated by Nadler's formula and Gross' formula (all P > 0.05). Six patients experienced postoperative cardiovascular complications due to the delayed restart of antiplatelet therapy; one of these patients in group III died from myocardial infarction. CONCLUSIONS: Continued DAPT or single antiplatelet treatment during orthopedic surgery does not increase the total intraoperative and perioperative bleeding compared with switching from antiplatelet therapy to low-molecular-weight heparin. However, the discontinuation of antiplatelet therapy increases the risk of serious cardiac complications.