Extracellular Expression Research Articles

Neuroblastoma cell lines display heterogeneity in differentiation responses

Background Neuroblastoma is the most common extra-cranial childhood solid tumour, arising during development from stalled neural crest-derived precursor cells. In a subset of children younger than 18 months of age, neuroblastoma can undergo spontaneous regression driven by differentiation, leading to great interest in developing differentiation therapies to re-direct neuroblastoma cells down their correct developmental pathway. Recently, we have shown that combinatorial treatment with the CDK4/6 inhibitor palbociclib and differentiation-inducing agent retinoic acid inhibits proliferation and drives neuronal differentiation of adrenergic-type neuroblastoma cell lines. Methods Here, we explore the differentiation potential of neuroblastoma cell lines in vitro in response to palbociclib and retinoic acid treatment using microscopy, transcriptomic and qRT-PCR analyses. Results We present evidence suggesting that neuroblastoma cells can give rise to mixtures of neural crest-derived adrenal gland cell types, and that differentiation responses correspond to changes in patterns of extracellular matrix substrate expression. Conclusions This study builds a case to further investigate and consider heterogeneity in neuroblastoma cell differentiation and the role of the extracellular matrix in these cell fate decisions.

RIP1 inhibition reduces chondrocyte apoptosis through downregulating nuclear factor-kappa B signaling in a mouse osteoarthritis model.

Excessive chondrocyte death is a critical player in the process of osteoarthritis (OA). The present study was aimed to study the role of receptor-interacting serine/threonine kinase (RIP) 1-mediated signaling for programmed cell death in OA. In the present study, RIP1 protein expression was evaluated in mouse OA cartilage and cultured primary murine chondrocytes exposed to tumor necrosis factor-alpha (TNF-α). Protein expression involved in necroptosis and apoptosis and chondrocyte-derived extracellular matrix were examined. Inhibition of RIP1 was conducted using the RNAi technique and pharmacological inhibition. Western blot, immunohistochemistry, and immunofluorescence examination were applied. The protein presence of RIP1, but not RIP3, was increased in the mouse OA tissue and cultured chondrocytes exposed to TNF-α. Knockdown of RIP1 increased protein expression of collagen II and sex-determining region Y-box transcription factor 9, and reduced protein expression of matrix metallopeptidases 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5. Inhibition of RIP1 reduced the phosphorylated NF-κB signals, decreased cell apoptosis, and restored extracellular matrix expression in cultured chondrocytes. Both RNAi and pharmacological inhibition of RIP1 decelerated the progress of OA in mice. RIP1 regulates chondrocyte apoptosis through NF-κB signaling. Inhibition of RIP1 provides a novel therapeutic approach for OA therapy.

Intercellular junction-driven stromal cell stacking in a confined 3D microcavity

Understanding the detailed mechanisms driving fibroblast migration within native tissue settings during pathophysiological events presents a critical research challenge. In this study, we elucidate how stromal cells migrate and contribute to the development of three-dimensional (3D) cellular aggregates within confined microcavities. Integrin α5β1 and β-catenin (β-cat) are central in guiding this collective migration and achieving optimal filling of the microcavity. When β-cat is suppressed, cells tend to migrate more sporadically, leading to less efficient cellular organization. Furthermore, we also detail the pivotal roles of Cx43 and N-cadherin (N-cad) in orchestrating collective migration and in shaping efficient cellular stacking. Suppressing gap junctions, especially Cx43, significantly impacts the extracellular matrix expression, integrin α5 and β1, and other elements in the 3D construct, emphasizing the importance of physicochemical cell–cell interactions. The distribution patterns of N-cad and focal adhesion kinase (FAK) further corroborate the essential roles in forming cell–cell junctions and FAK in establishing the foundational layer that underpins the cell stacking within the microcavity. Interestingly, neither Rho-associated protein kinase (ROCK) nor RhoA significantly alter the cell migration pattern toward microcavity. These findings provide fresh perspectives on fibroblast activities in 3D space, enriching our understanding and offering implications for advancements in wound healing and tissue engineering.

Low, but Not High, Pulsating Fluid Shear Stress Affects Matrix Extracellular Phosphoglycoprotein Expression, Mainly via Integrin β Subunits in Pre-Osteoblasts

Matrix extracellular phosphoglycoprotein (Mepe), present in bone and dentin, plays important multifunctional roles in cell signaling, bone mineralization, and phosphate homeostasis. Mepe expression in bone cells changes in response to pulsating fluid shear stress (PFSS), which is transmitted into cells through integrin-based adhesion sites, i.e., α and β subunits. Whether and to what extent PFSS influences Mepe expression through the modulation of integrin α and/or β subunit expression in pre-osteoblasts is uncertain. Therefore, we aimed to test whether low and/or high PFSS affects Mepe expression via modulation of integrin α and/or β subunit expression. MC3T3-E1 pre-osteoblasts were treated with ± 1 h PFSS (magnitude: 0.3 Pa (low PFSS) or 0.7 Pa (high PFSS); frequency: 1 Hz). Single integrin fluorescence intensity in pre-osteoblasts was increased, but single integrin area was decreased by low and high PFSS. Expression of two integrin α subunit-related genes (Itga1 and Itga5 2) was increased by low PFSS, and one (Itga5 2) by high PFSS. Expression of five integrin β subunit genes (Itgb1, Itgb3, Itgb5, Itgb5 13, and Itgb5 123) was increased by low PFSS, and three (Itgb5, Itgb5 13, and Itgb5 123) by high PFSS. Interestingly, Mepe expression in pre-osteoblasts was only modulated by low, but not high, PFSS. In conclusion, both low and high PFSS affected integrin α and β subunit expression in pre-osteoblasts, while integrin β subunit expression was more altered by low PFSS. Importantly, Mepe gene expression was only affected by low PFSS. These results might explain the different ways that Mepe-induced changes in pre-osteoblast mechanosensitivity may drive signaling pathways of bone cell function at low or high impact loading. These findings might have physiological and biomedical implications and require future research specifically addressing the precise role of integrin α or β subunits and Mepe during dynamic loading in bone health and disease.

SynNotch-Programmed Macrophages for Cancerous Cell Detection and Sensing.

Synthetic Notch (synNotch) receptors have enabled mammalian cells to sense extracellular ligands and respond by activating user-prescribed transcriptional programs. Based on the synNotch system, we describe a cell-based in vivo sensor for cancerous cell detection. We attempted to engineer synNotch-programmed macrophages to sense cancer cells via urinary analysis of human chorionic gonadotropin (HCGB5). Principally, when the synNotch receptors of macrophages bind to the ligands of cancer cells, Notch is activated and undergoes intramembrane proteolysis to release the transcriptional activator into the nucleus. The transcriptional activator targets and activates downstream gene expression, such as human chorionic gonadotropin (HCGB5) in macrophages. When HCGB5 is secreted extracellularly into urine, it can be detected with commercial HCGB5 colloidal gold test strips. As a proof of principle, we demonstrated the feasibility of synNotch-programmed macrophages in detecting breast cancer cells engineered with artificial EGFP ligands. We demonstrated that HCGB5 expression was only induced when the cancer cell expressing EGFP ligands is present; thereby, extracellular HCGB5 expression is directly proportional to the number of cancer cells. Further optimizations of the synNotch system can realize the ultimate goal of establishing cell-based in vivo sensors as the paragon of cancer diagnostics for point-of-care testing and home self-test.

Immune cells mediate the causal relationship between uveitis and colorectal cancer via Mendelian randomization analysis

Colorectal cancer (CRC) is one of the most common and deadly malignancies worldwide, and immune regulation plays a critical role in its development. This study investigates the causal relationships between uveitis, specific immune cell traits, and CRC using Mendelian Randomization (MR) analyses. A total of 21 single nucleotide polymorphisms (SNPs) associated with uveitis were identified, and the analysis revealed that a 1 log-odds increase in uveitis was linked to a statistically significant 3.0% reduction in CRC odds (IVW OR = 0.970, 95% CI: 0.946–0.995, P = 0.021). This protective effect was also observed using the weighted median approach (OR = 0.963, 95% CI: 0.931–0.997, P = 0.034), reinforcing the robustness of the findings. Furthermore, both univariable and multivariable MR analyses highlighted the significant causal influence of specific immune cell traits on CRC odds. Notably, the levels of extracellular monocyte HLA-DR expression emerged as a critical factor, with an associated increase in CRC odds (IVW OR = 1.084, 95% CI: 1.008–1.165, P = 0.030). The proportion of CRC odds mediated by the levels of extracellular monocyte HLA-DR expression, calculated as the ratio of the indirect effect to the total effect using estimates from multivariable MR analyses, was approximately 34.1%(95% CI: 10.23−58.04%). These findings underscore the complex interplay between immune regulation and carcinogenesis, offering insights into potential mechanisms underlying CRC development and suggesting avenues for targeted prevention and therapeutic strategies.

RON receptor tyrosine kinase regulates glycolysis through MAPK/CREB signaling to affect ferroptosis and chemotherapy sensitivity of thyroid cancer cells.

Anaplastic thyroid cancer (ATC) is one of the deadliest and most aggressive human malignancies for which there is currently no effective treatment. Tyrosine kinase receptor RON is highly expressed in various cancer types, including colon, pancreatic and thyroid cancer. However, its underlying role in ATC is not fully understood. The present study investigated the therapeutic potential and molecular mechanism of RON in ATC. RON expression in thyroid cancer cells was detected by western blotting. Glycolysis was assessed by measuring the extracellular acidification rate, glucose uptake, lactate concentration, and expression levels of glucose transporter 1, hexokinase 2 and pyruvate kinase M1/2. In addition, ferroptosis was assessed by detecting the levels of total iron, lipid peroxide and reactive oxygen species, and the expression levels of ferroptosis‑related proteins. Furthermore, mitochondrial function were assessed by JC‑1 staining and detection kits, respectively. The results demonstrated that RON was highly expressed in thyroid cancer cell lines. Furthermore, RON interference inhibited glycolysis, promoted ferroptosis, elevated cell sensitivity to chemotherapy and affected mitochondrial function in thyroid cancer cells. Further experiments demonstrated that RON interference affected the ferroptosis levels in thyroid cancer cells by inhibiting the glycolysis process. Mechanistically, the present results indicated that RON may affect ferroptosis, glycolysis and chemotherapy sensitivity by regulating MAPK/cAMP‑response element binding protein (CREB) signaling in thyroid cancer cells. In conclusion, the present study demonstrated that RON affected ferroptosis, glycolysis and chemotherapy sensitivity in thyroid cancer cells by regulating MAPK/CREB signaling, demonstrating its potential as a therapeutic target in thyroid cancer cells.

The soluble factor milieu in idiopathic pulmonary fibrosis dysregulates epithelial differentiation.

In idiopathic pulmonary fibrosis (IPF), epithelial abnormalities are present including bronchiolization and alveolar cell dysfunction. We hypothesized that the IPF microenvironment disrupts normal epithelial growth and differentiation. We mimicked the soluble factors within an IPF microenvironment using an IPF cocktail (IPFc), composed of nine factors which are increased in IPF lungs (CCL2, IL-1β, IL-4, IL-8, IL-13, IL-33, TGF-β, TNFα, and TSLP). Using IPFc, we asked whether the soluble factor milieu in IPF affects epithelial growth and differentiation and how IPFc compares to TGF-β alone. Epithelial growth and differentiation were studied using mouse lung organoids (primary Epcam+ epithelial cells co-cultured with CCL206 fibroblasts). Organoids exposed to IPFc and TGF-β were re-sorted into epithelial and fibroblast fractions and subjected to RNA sequencing. IPFc did not affect the number of organoids formed. However, pro-surfactant protein C expression was decreased. On these parameters, TGF-β alone had similar effects. However, RNA sequencing of re-sorted organoids revealed that IPFc and TGF-β had distinct effects on both epithelial cells and fibroblasts. IPFc upregulated goblet cell markers, whereas these were inhibited by TGF-β. Although both IPFc and TGF-β increased extracellular matrix gene expression, only TGF-β increased myofibroblast markers. VEGF-C and Wnt signaling were among the most differentially regulated signaling pathways by IPFc versus TGF-β. Interestingly, Wnt pathway activation rescued Sftpc downregulation induced by IPFc. In conclusion, IPFc alters epithelial differentiation in a way that is distinct from TGF-β. Alterations in Wnt signaling contribute to these effects. IPFc may be a more comprehensive representation of the soluble factor microenvironment in IPF.

Tumor Necrosis Factor-α–Dependent Inflammation Upregulates High Mobility Group Box 1 To Induce Tumor Promotion and Anti–Programmed Cell Death Protein-1 Immunotherapy Resistance in Lung Adenocarcinoma

Tumor-associated chronic lung inflammation depends on tumor necrosis factor (TNF)-α to activate several cytokines as part of an inflammatory loop, which plays a critical role in tumor progression in lung adenocarcinoma. High mobility group box 1 (HMGB1) is a cytokine that mediates inflammation. Whether TNF-α–induced inflammation regulates HMGB1 to contribute to tumor progression and promotion in lung adenocarcinoma remains unclear. Thus, human samples and a urethane-induced inflammation-driven lung adenocarcinoma (IDLA) mouse model were used to explore the involvement of HMGB1 in tumorigenesis and tumor progression and efficacy of anti–programmed cell death protein (PD)-1 immunotherapy. High levels of HMGB1 were observed in human lung adenocarcinoma associated with poor overall survival in patients. HMGB1 upregulation was positively correlated with TNF-α–related inflammation and TIM-3+ infiltration. TNF-α upregulated intracellular and extracellular HMGB1 expression to contribute to tumor promotion in A549 cells in vitro. Using a urethane-induced IDLA mouse model, we found HMGB1 upregulation was associated with increased TIM-3+ T-cell infiltration. Blocking TNF-α–dependent inflammation downregulated HMGB1 expression and inhibited tumorigenesis in the IDLA model. Anti-PD-1 treatment alone did not inhibit tumor growth in the TNF-α–dependent IDLA, whereas anti-PD-1 combined with TNF-α blockade overcame anti-PD-1 immunotherapy resistance. Furthermore, anti-PD-1 combined with anti-HMGB1 also inhibited tumor growth in IDLA, suggesting that increased HMGB1 release by TNF-α contributes to the resistance of anti-PD-1 immunotherapy in IDLA. Thus, tumor-associated TNF-α–dependent inflammation upregulated intracellular and extracellular HMGB1 expression in an inflammatory loop, contributing to tumor promotion and anti-PD-1 immunotherapy resistance in lung adenocarcinoma.

ROCK Inhibitor Enhances Neurite Outgrowth In Vitro and Corneal Sensory Nerve Reinnervation In Vivo.

The intraepithelial corneal nerves are essential to corneal health. Rho kinase or ROCK inhibitors (RIs) have been reported to play a role in neuron survival after injury. Here we assess integrin and extracellular matrix expression in primary mouse neurons and determine whether treating cells with RI impacts neurite outgrowth in vitro and reinnervation after trephine and debridement injury in mice in vivo. Cocultures of human corneal limbal epithelial cells and E11.5 mouse trigeminal neurons and neurons alone were grown on glass coverslips. High-resolution imaging was performed to localize integrins and laminin on neurons and to determine whether RI impacts neurite outgrowth in vitro and in vivo after both 1.5-mm trephine and 1.5-mm debridement injuries. Several integrin α (α3, α6, αv) chains as well as β4 integrin are expressed on neuron axons and growth cones in cocultures. RI treatment of isolated neurons, cocultures, and in conditioned media increases neurite outgrowth. In vivo, RI positively impacts sensory nerve reinnervation after trephine and debridement injury. These studies are the first to demonstrate expression of β4 integrin on trigeminal sensory neurons and preferential adhesion of neurons to the laminin-enriched matrices found in footprints deposited by human corneal limbal epithelial cells. In addition, we also document for the first time the positive impact of RI on neurite outgrowth in vitro and reinnervation in vivo.

Recurrent CLTC::SYK fusions and CSF1R mutations in juvenile xanthogranuloma of soft tissue

AbstractJuvenile xanthogranuloma (JXG) is a histiocytic neoplasm that usually presents in the skin. Rarely, extracutaneous localizations occur; the genetic drivers of this clinical variant of JXG remain incompletely characterized. We present detailed clinicopathologic and molecular data of 16 children with extracutaneous JXG and 5 adults with xanthogranulomas confined to the central nervous system (CNS) or soft tissue. Tissue samples were obtained through the Dutch Nationwide Pathology Databank and analyzed with an innovative sequencing technique capable of detecting both small genomic variants and gene rearrangements. Targetable kinase alterations were detected in 16 of 16 children and 1 of 5 adults. Alterations included CLTC::SYK fusions in 6 children and CSF1R mutations in 7 others; all aged <2 years with soft tissue tumors. One child had a CSF1R mutation and MRC1::PDGFRB fusion. Most were treated surgically, although spontaneous regression occurred in 1 of 6 with CLTC::SYK and 2 of 7 with CSF1R mutations, underscoring that treatment is not always necessary. Tumors with CLTC::SYK fusions generally lacked Touton giant cells but exhibited many other histologic features of JXG and concordant methylation profiles. Using multispectral immunofluorescence, phosphorylated–spleen tyrosine kinase expression was localized to CD163+ histiocytes; tumors with CLTC::SYK fusions also demonstrated mTOR activation, cyclin D1 expression, and variable phosphorylated–extracellular signal-regulated kinase expression. BRAFV600E was detected in 1 child and 1 adult with CNS xanthogranulomas; both responded to BRAF inhibition. Finally, a TPM3::NTRK1 fusion or MAP2K1 deletion was detected in 2 children with systemic JXG who experienced spontaneous disease regression. This study advances the molecular understanding of histiocytic neoplasms and may guide diagnostics and clinical management.

Construction of a Kluyveromyces lactis strain with multi-copy integration for enhanced bovine chymosin production by CRISPR/Cas9 and UV mutagenesis

Bovine chymosin is an essential food enzyme widely used in cheese production in the dairy industry. This study used a codon-optimized prochymosin gene to construct an expression cassette for extracellular expression of bovine chymosin in Kluyveromyces lactis. After integration of the prochymosin gene into the host cell genome, the single-copy integration strain KLUcym showed the clotting activity of 40 U/mL in a shake flask. The CRISPR/Cas9 system was employed to delete amdS and construct the double-copy integration strain and triple-copy integration strain, which achieved the clotting activities of 70 U/mL and 78 U/mL in shake flasks, separately. Subsequently, multiple rounds of UV mutagenesis were performed on the double-copy strain KLUcymD, and a recombinant K. lactis strain with a high yield of bovine chymosin was obtained. This strain achieved the clotting activity of 270 U/mL in a shake flask and 600 U/mL in a 5 L bioreactor after 76 h. In summary, we construct a strain KLUcymD-M2 for high production of bovine chymosin, which lays a foundation of industrial fermentation.

Functional outcome of the anterior vaginal wall in a pelvic surgery injury rat model after treatment with stem cell-derived progenitors of smooth muscle cells

BackgroundStem-cell-derived therapy is a promising option for tissue regeneration. Human iPSC-derived progenitors of smooth muscle cells (pSMCs) exhibit limited proliferation and differentiation, which minimizes the risk of tumor formation while restoring smooth muscle cells (SMCs). Up to 29% of women suffer from recurrence of vaginal prolapse after prolapse surgery. Therefore, there is a need for therapies that can restore vaginal function. SMCs contribute to vaginal tone and contractility. We sought to examine whether human pSMCs can restore vaginal function in a rat model.MethodsFemale immunocompromised RNU rats were divided into 5 groups: intact controls (n = 12), VSHAM (surgery + saline injection, n = 35), and three cell-injection groups (surgery + cell injection using pSMCs from three patients, n = 14/cell line). The surgery to induce vaginal injury was analogous to prolapse surgery. Menopause was induced by surgical ovariectomy. The vagina, urethra, bladder were harvested 10 weeks after surgery (5 weeks after cell injection). Organ bath myography was performed to evaluate the contractile function of the vagina, and smooth muscle thickness was examined by tissue immunohistochemistry. Collagen I, collagen III, and elastin mRNA and protein expressions in tissues were assessed.ResultsVaginal smooth muscle contractions induced by carbachol and KCl in the cell-injection groups were significantly greater than those in the VSHAM group. Collagen I protein expression in the vagina of the cell-injections groups was significantly higher than in the VSHAM group. Vaginal elastin protein expression was similar between the cell-injection and VSHAM groups. In the urethra, gene expression levels of collagen I, III, and elastin were all significantly greater in the cell-injection groups than in the VSHAM group. Collagen I, III, and elastin protein expression of the urethra did not show a consistent trend between cell-injection groups and the VSHAM group.ConclusionsHuman iPSC-derived pSMCs transplantation appears to be associated with improved contractile function of the surgically injured vagina in a rat model. This is accompanied by changes in extracellular protein expression the vagina and urethra. These observations support further efforts in the translation of pSMCs into a treatment for regenerating the surgically injured vagina in women who suffer recurrent prolapse after surgery.

Effects of in utero exposure to Δ-9-tetrahydrocannabinol on cardiac extracellular matrix expression and vascular transcriptome in rhesus macaques.

Delta-9-tetrahydrocannabinol (THC), the psychoactive component of cannabis, remains a schedule I substance, thus safety data regarding the effects on the cardiovascular and prenatal health are limited. Importantly, there is evidence showing prenatal cannabis exposure can negatively impact fetal organ development, including the cardiovascular system. THC can cross the placenta and bind to cannabinoid receptors expressed in the developing fetus, including on endothelial cells. To understand the impact of prenatal THC exposure on the fetal cardiovascular system, we used our rhesus macaque model of prenatal daily edible THC consumption. Before conception, animals were acclimated to THC (2.5 mg/7 kg/day, equivalent to a heavy medical cannabis dose) and maintained on this dose daily throughout pregnancy. Fetal tissue samples were collected at gestational day 155 (full term is 168 days). Our model showed that in utero THC exposure was associated with a decreased heart weight-to-body weight ratio in offspring, warranting further mechanistic investigation. Histological examination of the fetal cardiac and vascular tissues did not reveal any significant effect of THC exposure on the maturity of collagen within the fetal heart or the aorta. Total collagen III expression and elastin production and organization were unchanged. However, bulk RNA-sequencing of vascular cells in the umbilical vein, umbilical artery, and fetal aorta demonstrated that THC alters the fetal vascular transcriptome and is associated with upregulated expression of genes involved in carbohydrate metabolism and inflammation. The long-term consequences of these findings are unknown but suggest that prenatal THC exposure may affect cardiovascular development in offspring.NEW & NOTEWORTHY Prenatal cannabis use is increasing and despite the public health relevance, there is limited safety data regarding its impact on offspring cardiovascular health outcomes. We used a translational, nonhuman primate model of daily edible Δ-9-tetrahydrocannabinol (THC) consumption during pregnancy to assess its effects on the fetal cardiovascular system. THC-exposed fetal vascular tissues displayed upregulation of genes involved in cellular metabolism and inflammation, suggesting that prenatal THC exposure may impact fetal vascular tissues.

Impact of metabolically healthy obesity during pregnancy on placental extracellular vesicle microRNA expression

Impact of metabolically healthy obesity during pregnancy on placental extracellular vesicle microRNA expression

The Effect of Tideglusib Application on Type 1 and Type 3 Collagen Expressions by Human Dental-Pulp Derived Stem Cells: A Preliminary Study.

Although Tideglusib cytotoxicity studies and its effects on human dental pulp-derived stem cells (DPSCs) have been examined in previous studies, there is no study investigating the expression of type 1 collagen and type 3 collagen by Tideglusib. The purpose of this study is to examine the effect of Wnt signaling activation using Tideglusib execution on human DPSC to determine its potential efficacy in collagen expression. Stem cell isolation was performed from five human third molar wisdom tooth pulps. DPSCs identified in only one sample were treated with 50 nM Tideglusib for 24 h and 1 week. Axin-2, type 1 and type 3 collagen expressions were evaluated by Western blot analysis. DPSCs without treatment served as a negative control. The Mann-Whitney U test was used for statistical analysis. The levels of type 1 collagen and Axin-2 in the test group were significantly higher than those in the control group at 24 h (P = 0.000, P = 0.001, respectively). Compared to the control group, a slight increase in type 3 collagen expression was observed in the test group at 24 h (P value = 0.063). Application of 50 nM Tideglusib for 1 week revealed marked decreases in type 1 and type 3 collagen expressions (P = 0.029, P = 0.038, respectively). In contrast, there was a significant increase in the level of Axin-2 (P = 0.000) compared to the control group. The fact that Wnt signaling pathway activation obtained by Tideglusib application on DPSCs confirmed by the finding in the increase of Axin-2 at short and long-term evaluation periods which is resulted in the increase in the type 1 collagen expression at 24 h and decrease at 1 week together with the decrease in type 3 collagen expression at 1 week warrants further studies to evaluate the effect of Tideglusib on extracellular matrix expression.

Digital Whole Slide Image Analysis of Elevated Stromal Content and Extracellular Matrix Protein Expression Predicts Adverse Prognosis in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis and limited treatment options. This study evaluates the prognostic value of stromal markers in TNBC, focusing on the tumor-stroma ratio (TSR) and overall stroma ratio (OSR) in whole slide images (WSI), as well as the expression of type-I collagen, type-III collagen, and fibrillin-1 on tissue microarrays (TMAs), using both visual assessment and digital image analysis (DIA). A total of 101 female TNBC patients, primarily treated with surgery between 2005 and 2016, were included. We found that high visual OSR correlates with worse overall survival (OS), advanced pN categories, lower stromal tumor-infiltrating lymphocyte count (sTIL), lower mitotic index, and patient age (p < 0.05). TSR showed significant connections to the pN category and mitotic index (p < 0.01). High expression levels of type-I collagen (>45%), type-III collagen (>30%), and fibrillin-1 (>20%) were linked to significantly worse OS (p = 0.004, p = 0.013, and p = 0.005, respectively) and progression-free survival (PFS) (p = 0.028, p = 0.025, and p = 0.002, respectively), validated at the mRNA level. Our results highlight the importance of stromal characteristics in promoting tumor progression and metastasis and that targeting extracellular matrix (ECM) components may offer novel therapeutic strategies. Furthermore, DIA can be more accurate and objective in evaluating TSR, OSR, and immunodetected stromal markers than traditional visual examination.

Anti-inflammatory effects of phytocannabinoids and terpenes on inflamed Tregs and Th17 cells in vitro

AimsPhytocannabinoids and terpenes from Cannabis sativa have demonstrated limited anti-inflammatory and analgesic effects in several inflammatory conditions. In the current study, we test the hypothesis that phytocannabinoids exert immunomodulatory effects in vitro by decreasing inflammatory cytokine expression and activation. Key methodsCD3/CD28 and lipopolysaccharide activated peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 6) were treated with phytocannabinoid compounds and terpenes in vitro. Flow cytometry was used to determine regulatory T cell (Treg) and T helper 17 (Th17) cell responses to treatments. Cell pellets were harvested for qRT-PCR gene expression analysis of cytokines, cell activation markers, and inflammation-related receptors. Cell culture supernatants were analysed by ELISA to quantify IL-6, TNF-α and IL-10 secretion. Main findingsIn an initial screen of 20 μM cannabinoids and terpenes which were coded to blind investigators, cannabigerol (GL4a), caryophyllene oxide (GL5a) and gamma-terpinene (GL6a) significantly reduced cytotoxicity and gene expression levels of IL6, IL10, TNF, TRPV1, CNR1, HTR1A, FOXP3, RORC and NFKΒ1. Tetrahydrocannabinol (GL7a) suppression of T cell activation was associated with downregulation of RORC and NFKΒ1 gene expression and reduced IL-6 (p < 0.0001) and IL10 (p < 0.01) secretion. Cannabidiol (GL1b) significantly suppressed activation of Tregs (p < 0.05) and Th17 cells (p < 0.05) in a follow-on in vitro dose-response study. IL-6 (p < 0.01) and IL-10 (p < 0.01) secretion was significantly reduced with 50 μM cannabidiol. SignificanceThe study provides the first evidence that cannabidiol and tetrahydrocannabinol suppress extracellular expression of both anti- and pro-inflammatory cytokines in an in vitro PBMC model of inflammation.

Role of pulmonary rehabilitation on extracellular matrix protein expression in vastus lateralis muscle in atrophic and non-atrophic patients with COPD

Role of pulmonary rehabilitation on extracellular matrix protein expression in vastus lateralis muscle in atrophic and non-atrophic patients with COPD

Au-modified ceria nanozyme prevents and treats hypoxia-induced pulmonary hypertension with greatly improved enzymatic activity and safety

BackgroundDespite recent advances the prognosis of pulmonary hypertension remains poor and warrants novel therapeutic options. Extensive studies, including ours, have revealed that hypoxia-induced pulmonary hypertension is associated with high oxidative stress. Cerium oxide nanozyme or nanoparticles (CeNPs) have displayed catalytic activity mimicking both catalase and superoxide dismutase functions and have been widely used as an anti-oxidative stress approach. However, whether CeNPs can attenuate hypoxia-induced pulmonary vascular oxidative stress and pulmonary hypertension is unknown.ResultsIn this study, we designed a new ceria nanozyme or nanoparticle (AuCeNPs) exhibiting enhanced enzyme activity. The AuCeNPs significantly blunted the increase of reactive oxygen species and intracellular calcium concentration while limiting proliferation of pulmonary artery smooth muscle cells and pulmonary vasoconstriction in a model of hypoxia-induced pulmonary hypertension. In addition, the inhalation of nebulized AuCeNPs, but not CeNPs, not only prevented but also blunted hypoxia-induced pulmonary hypertension in rats. The benefits of AuCeNPs were associated with limited increase of intracellular calcium concentration as well as enhancement of extracellular calcium-sensing receptor (CaSR) activity and expression in rat pulmonary artery smooth muscle cells. Nebulised AuCeNPs showed a favorable safety profile, systemic arterial pressure, liver and kidney function, plasma Ca2+ level, and blood biochemical parameters were not affected.ConclusionWe conclude that AuCeNPs is an improved reactive oxygen species scavenger that effectively prevents and treats hypoxia-induced pulmonary hypertension.Graphical