The Efficacy of Anti-PD-L1 Treatment in Melanoma Is Associated with the Expression of the ECM Molecule EMILIN2

The use of immune checkpoint inhibitors has revolutionized the treatment of melanoma patients, leading to remarkable improvements in the cure. However, to ensure a safe and effective treatment, there is the need to develop markers to identify the patients that would most likely respond to the therapies. The microenvironment is gaining attention in this context, since it can regulate both the immunotherapy efficacyand angiogenesis, which is known to be affected by treatment. Here, we investigated the putative role of the ECM molecule EMILIN-2, a tumor suppressive and pro-angiogenic molecule. We verified that the EMILIN2 expression is variable among melanoma patients and is associated with the response to PD-L1 inhibitors. Consistently, in preclinical settings, the absence of EMILIN-2 is associated with higher PD-L1 expression and increased immunotherapy efficacy. We verified that EMILIN-2 modulates PD-L1 expression in melanoma cells through indirect immune-dependent mechanisms. Notably, upon PD-L1 blockage, Emilin2^−/− mice displayed improved intra-tumoral vessel normalization and decreased tumor hypoxia. Finally, we provide evidence indicating that the inclusion of EMILIN2 in a number of gene expression signatures improves their predictive potential, a further indication that the analysis of this molecule may be key for the development of new markers to predict immunotherapy efficacy.     

N-Glycans in Immortalized Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for EV–Cell Interaction and Functional Activation of Endothelial Cells

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV–cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells’ interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton’s Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV–cell interactions and associated functions.     

Circulating YKL-40 Level,but not CHI3L1 Gene Variants,Is Associated with Atherosclerosis-Related Quantitative Traits and the Risk of Peripheral Artery Disease

YKL-40, a pleotropic cytokine, is emerging as a risk factor and a prognostic predictor of atherosclerotic cardiovascular disease. We attempted to elucidate the genetic, clinical and biochemical correlates of circulating YKL-40 level and, by combining it with CHI3L1 gene variants, with the risk and long-term mortality of peripheral artery disease (PAD). Plasma YKL-40 concentrations were measured in 612 Taiwanese individuals who had no clinically overt systemic disease. Clinical parameters, CHI3L1 gene promoter variants and 18 biomarker levels were analyzed. Eighty-six PAD patients were further enrolled for analysis. Significant associations were found between CHI3L1 genotypes/haplotypes and YKL-40 levels for the health examination subjects (smallest p = 8.36 × 10⁻⁷ for rs4950928 and smallest p = 1.72 × 10⁻¹⁰ for haplotype TGG) and also for PAD patients. For the health examination subjects, circulating YKL-40 level, but not CHI3L1 gene variants, were positively associated with age, smoking, and circulating levels of triglyceride, lipocalin 2 and multiple inflammatory biomarkers and negatively associated with low-density-lipoprotein cholesterol levels. Circulating YKL-40 level is also significantly associated with the risk of PAD (p = 3.3 × 10⁻²³). Circulating YKL40 level, but not CHI3L1 gene promoter variants, is associated with the risk of PAD in Taiwanese. The association of YKL-40 levels with multiple quantitative traits relating to the risk of PAD may provide a molecular basis linking YKL-40 to atherosclerotic cardiovascular disease.     

E3 Ubiquitin Ligase Midline 1 Regulates Endothelial Cell ICAM-1 Expression and Neutrophil Adhesion in Abdominal Sepsis

Septic lung damage is associated with endothelial cell and neutrophil activation. This study examines the role of the E3 ubiquitin ligase midline 1 (Mid1) in abdominal sepsis. Mid1 expression was increased in endothelial cells derived from post-capillary venules in septic mice and TNF-α challenge increased Mid1 levels in endothelial cells in vitro. The siRNA-mediated knockdown of Mid1 decreased TNF-α-induced upregulation of ICAM-1 and neutrophil adhesion to endothelial cells. Moreover, Mid1 silencing reduced leukocyte adhesion in post-capillary venules in septic lungs in vivo. The silencing of Mid1 not only decreased Mid1 expression but also attenuated expression of ICAM-1 in lungs from septic mice. Lastly, TNF-α stimulation decreased PP2Ac levels in endothelial cells in vitro, which was reversed in endothelial cells pretreated with siRNA directed against Mid1. Thus, our novel data show that Mid1 is an important regulator of ICAM-1 expression and neutrophil adhesion in vitro and septic lung injury in vivo. A possible target of Mid1 is PP2Ac in endothelial cells. Targeting the Mid1-PP2Ac axis may be a useful way to reduce pathological lung inflammation in abdominal sepsis.     

Impact of Cyanidin-3-Glucoside on Glycated LDL-Induced NADPH Oxidase Activation,Mitochondrial Dysfunction and Cell Viability in Cultured Vascular Endothelial Cells

Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC.     

Metalloproteinases 1 and 3 as Potential Biomarkers in Breast Cancer Development

Breast cancer continues to be one of the main causes of morbidity and mortality globally and was the leading cause of cancer death in women in Spain in 2020. Early diagnosis is one of the most effective methods to lower the incidence and mortality rates of breast cancer. The human metalloproteinases (MMP) mainly function as proteolytic enzymes degrading the extracellular matrix and plays important roles in most steps of breast tumorigenesis. This retrospective cohort study shows the immunohistochemical expression levels of MMP-1, MMP-2, MMP-3, and MMP-9 in 154 women with breast cancer and 42 women without tumor disease. The samples of breast tissue are assessed using several tissue matrices (TMA). The percentages of staining (≤50%–>50%) and intensity levels of staining (weak, moderate, or intense) are considered. The immunohistochemical expression of the MMP-1-intensity (p = 0.043) and MMP-3 percentage (p = 0.018) and intensity, (p = 0.025) present statistically significant associations with the variable group (control–case); therefore, expression in the tumor tissue samples of these MMPs may be related to the development of breast cancer. The relationships between these MMPs and some clinicopathological factors in breast cancer are also evaluated but no correlation is found. These results suggest the use of MMP-1 and MMP-3 as potential biomarkers of breast cancer diagnosis.     

Decreased Levels of Microfibril-Associated Glycoprotein (MAGP)-1 in Patients with Colon Cancer and Obesity Are Associated with Changes in Extracellular Matrix Remodelling

Objective: The protein microfibril-associated glycoprotein (MAGP)-1 constitutes a crucial extracellular matrix protein. We aimed to determine its impact on visceral adipose tissue (VAT) remodelling during obesity-associated colon cancer (CC). Methods: Samples obtained from 79 subjects (29 normoponderal (NP) (17 with CC) and 50 patients with obesity (OB) (19 with CC)) were used in the study. Circulating concentrations of MAGP-1 and its gene expression levels (MFAP2) in VAT were analysed. The impact of inflammation-related factors and adipocyte-conditioned media (ACM) on MFAP2 mRNA levels in colon adenocarcinoma HT-29 cells were further analysed. The effects of MAGP-1 in the expression of genes involved in the extracellular matrix (ECM) remodelling and tumorigenesis in HT-29 cells was also explored. Results: Obesity (p < 0.01) and CC (p < 0.001) significantly decreased MFAP2 gene expression levels in VAT whereas an opposite trend in TGFB1 mRNA levels was observed. Increased mRNA levels of MFAP2 after the stimulation of HT-29 cells with lipopolysaccharide (LPS) (p < 0.01) and interleukin (IL)-4 (p < 0.01) together with a downregulation (p < 0.05) after hypoxia mimicked by CoCl₂ treatment was observed. MAGP-1 treatment significantly enhanced the mRNA levels of the ECM-remodelling genes collagen type 6 α3 chain (COL6A3) (p < 0.05), decorin (DCN) (p < 0.01), osteopontin (SPP1) (p < 0.05) and TGFB1 (p < 0.05). Furthermore, MAGP-1 significantly reduced (p < 0.05) the gene expression levels of prostaglandin-endoperoxide synthase 2 (COX2/PTGS2), a key gene controlling cell proliferation, growth and adhesion in CC. Interestingly, a significant decrease (p < 0.01) in the mRNA levels of MFAP2 in HT-29 cells preincubated with ACM from volunteers with obesity compared with control media was observed. Conclusion: The decreased levels of MAGP-1 in patients with obesity and CC together with its capacity to modulate key genes involved in ECM remodelling and tumorigenesis suggest MAGP-1 as a link between AT excess and obesity-associated CC development.     

Farrerol Induces Cancer Cell Death via ERK Activation in SKOV3 Cells and Attenuates TNF-α-Mediated Lipolysis

Farrerol (FA) is a flavanone isolated from the Chinese herbal medicine “Man-shan-hong” (Rhododendron dauricum L.). In the present study, FA decreased the viability of SKOV3 cells in a dose- and time-dependent manner, and it induced G2/M cell cycle arrest and cell apoptosis. Cell cycle distribution analysis via flow cytometry showed that FA decreased G1 populations and increased G2/M populations in SKOV3 cells. Additionally, Western blotting confirmed an increase in the expression level of proteins involved in the cell cycle, e.g., CDK and cyclins. FA-induced apoptosis in SKOV3 cells was also investigated using a TUNEL assay, and increased expression levels of proapoptotic factors, including Caspase-3 and poly ADP ribose polymerase (PARP), through the Extracellular signal-regulated kinase (ERK)/MAPK pathway were investigated. Proinflammatory cytokines (e.g., IL-6, TNF-α, and IL-1) have been identified as a driver of the pathological mechanisms underlying involuntary weight loss and impaired physical function, i.e., cachexia, during cancer; in the present study, we showed that farrerol attenuates TNF-α-induced lipolysis and increases adipogenic differentiation in 3T3-L1 cells. Thus, farrerol could potentially be used as an anticancer agent or anticachetic drug.     

Suppression of Lipid Accumulation by Indole-3-Carbinol Is Associated with Increased Expression of the Aryl Hydrocarbon Receptor and CYP1B1 Proteins in Adipocytes and with Decreased Adipocyte-Stimulated Endothelial Tube Formation

This study investigated the effects of indole-3-carbinol (I3C) on adipogenesis- and angiogenesis-associated factors in mature adipocytes. The cross-talk between mature adipocytes and endothelial cells (ECs) was also explored by cultivating ECs in a conditioned medium (CM) by using I3C-treated adipocytes. The results revealed that I3C significantly inhibited triglyceride accumulation in mature adipocytes in association with significantly increased expression of AhR and CYP1B1 proteins as well as slightly decreased nuclear factor erythroid-derived factor 2–related factor 2, hormone-sensitive lipase, and glycerol-3-phosphate dehydrogenase expression by mature adipocytes. Furthermore, I3C inhibited CM-stimulated endothelial tube formation, which was accompanied by the modulated secretion of angiogenic factors in adipocytes, including vascular endothelial growth factor, interleukin-6, matrix metalloproteinases, and nitric oxide. In conclusion, I3C reduced lipid droplet accumulation in adipocytes and suppressed adipocyte-stimulated angiogenesis in ECs, suggesting that I3C is a potential therapeutic agent for treating obesity and obesity-associated disorders.     

HBx Protein Promotes Oval Cell Proliferation by Up-Regulation of Cyclin D1 via Activation of the MEK/ERK and PI3K/Akt Pathways

Growing evidence has shown that hepatic oval cells, also named liver progenitor cells, play an important role in the process of liver regeneration in various liver diseases. Oval cell proliferation has been reported in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and chronic liver disease. Studies have found expression of HBV surface and core antigens in oval cells in the livers of patients with HCC, suggesting that HBV infection of oval cells could be a mechanism of human hepatocarcinogenesis. In addition, there is evidence of multiplication of HBV in oval cell culture. However, little research has been performed to explore the role of HBV-encoded proteins in the proliferation of hepatic oval cells. Previously, we successfully transfected the HBV x (HBx) gene, one of the four genes in the HBV genome, into a rat LE/6 oval cell line. In this study, we tested whether or not the transfected HBx gene could affect oval cell proliferation in vitro. Our results show that overexpression of HBx promotes the proliferation of oval cells and increases cyclin D1 expression, assessed at both the mRNA and protein levels. We also found that HBx activated the PI-3K/Akt and MEK/ERK1/2 pathways in HBx-transfected oval cells. Furthermore, the HBx-induced increases in cyclin D1 expression and oval cell proliferation were completely abolished by treatment with either MEK inhibitor PD184352 or PI-3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. These results demonstrated that HBx has the ability to promote oval cell proliferation in vitro, and its stimulatory effects on cell proliferation and expression of cyclin D1 depend on the activation of the MEK/ERK and PI3K/Akt signaling pathways in cultured oval cells.