Expression Of Mesenchymal Markers Research Articles

Analysis of Fibroblast Growth Factor 2 Impact and Mechanism on Broncho-Pulmonary Dysplasia.

Epithelial-mesenchymal transition (EMT) of alveolar epithelial cells is an important mechanism for the onset and development of broncho-pulmonary dysplasia (BPD).The role of FGF-2 in BPD is currently unclear. The aim of our study is to investigate the expression of FGF-2 in lung tissue of BPD mice, to further clarify the effect of FGF-2 on EMT in alveolar epithelial cells and to actively search for possible signaling pathways. The BPD model was induced by exposure to hyperoxia. Lung tissue samples were collected and Hematoxylin and eosin (HE) staining was used to determine the modelling effect. Quantitative Real-time Polymerase Chain Reaction (QRT-PCR), immunohistochemistry were used to detect FGF-2 expression in BPD mice. To further investigate the effect of FGF-2 supplementation and deficiency on EMT in alveolar epithelial cells, A549 cells were cryopreserved, resuspended, cultured and passaged. Transforming growth factor-β1 (TGFβ1) was used to induce EMT. FGF-2 small interfering RNA fragments were synthesised and screened. Fbroblast growth factor receptor1 (FGFR1) expression was inhibited by BGJ398. (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium) (MTS) assay was used to detect the effect of FGF-2 and Infigratinib (BGJ398) on cell proliferation. We used qRT-PCR and Western blot to detect the expression of epithelial cell markers, mesenchymal cell markers and EMT-related signaling pathway proteins. Our results showed that the successful established hyperoxia mice model were characteristic by BPD. Hyperoxia decreased FGF-2 on day 4, upregulated FGF-2 on day 21, which resulted in EMT. In vitro, we found that FGF-2 alone increased the expression of mesenchymal markers, decreased the expression of epithelial markers and activated Phosphatidylinositol 3-kinase/Protein kinase B (PI3K/AKT), Small mother against decapentaplegic (Smad), mitogen-activated protein kinase (P38) and extracellular signal-regulated kinase (ERK) signaling pathways. FGF-2 could not reverse but synergistically promote TGF-β1-induced EMT of alveolar epithelial cells. Silencing FGF-2 increased the expression of epithelial marker E-cadherin, inhibited the PI3K/AKT, Smad, and P38 signaling pathways activated by TGF-β1, but activated ERK signaling. FGF-2 receptor inhibitor BGJ398 reversed TGF-β1-induced EMT, decreased the expression of FGFR1, and inhibited ERK signaling pathway activation. FGF2 was closely associated with EMT in BPD mice. Both high and low levels of FGF2 promoted EMT in A549. The FGF-2 receptor inhibitor BGJ398 reversed TGF-β1-induced EMT in A549 by inhibiting the FGFR1/P-ERK signaling pathway.

LGALS3 regulates endothelial-to-mesenchymal transition via PI3K/AKT signaling pathway in silica-induced pulmonary fibrosis

Silicosis is a progressive and chronic occupational lung disease characterized by lung inflammation, silicotic nodule formation, and diffuse pulmonary fibrosis(Jin et al. 2024). Emerging evidence indicates that endothelial-mesenchymal transition (EndoMT) plays a crucial role in the development of silicosis. Herein, we conducted a SiO2-induced EndoMT model and established a mouse model with pulmonary fibrosis by silica. We identified that SiO2 effectively increased the expression of mesenchymal markers while decreasing the levels of endothelial markers in endothelial cells. It’s further demonstrated that SiO2 induced the PI3K/Akt signaling pathway activation via LGALS3 synthesis. Next, interfering LGALS3 blocked the process of EndoMT by inhibiting the activity of PI3K/AKT signaling. In vivo, the administration of a specific PI3K inhibitor LY294002 significantly alleviated silica-induced pulmonary fibrosis. Collectively, these results identified that the LGALS3/PI3K/AKT pathway provided a rationale target for the clinical treatment and intervention of silicosis.

Prenatal glucocorticoid exposure and congenital abdominal wall defects: Involvement of CXCR4 – SDF-1 signaling

Developmental defects of the ventral abdominal wall, such as gastroschisis, have been associated with prenatal stress exposure. To investigate this further, dexamethasone (DEX), a synthetic glucocorticoid, was administered to fertilized chicken eggs on day 1 of incubation to simulate stress, and embryonic development was subsequently analyzed through in-situ hybridization, immunohistochemistry, and histological methods. Significant developmental abnormalities were displayed by DEX-treated embryos, including open abdomens, reduced MYOG expression in the abdominal wall, and disrupted muscle fiber formation, as indicated by altered Myosin heavy chain patterns. Additionally, early markers of muscle development, such as Pax3, and the CXCR4-SDF-1 signaling axis, crucial for the migration of myogenic precursors of the dermomyotome, were markedly affected. Significant alterations in the expression of mesenchymal markers, including Vimentin and Fibronectin in the lateral plate mesoderm, were observed, alongside alterations in Pitx2, BMP4 and TFAP2A expression. Importantly, a downregulation of Glucocorticoid Receptors was identified, emphasizing the chronic stress exposure. These results provide critical insights into how DEX interferes with key developmental pathways, particularly those involving chemokines like CXCR4 and SDF-1, and other markers of mesodermal differentiation. An advancement in the understanding of the mechanisms underlying ventral abdominal wall defects in the context of prenatal stress is provided by this research, with potential implications for preventing these congenital anomalies.

A microglia-containing cerebral organoid model to study early life immune challenges

Prenatal infections and activation of the maternal immune system have been proposed to contribute to causing neurodevelopmental disorders (NDDs), chronic conditions often linked to brain abnormalities. Microglia are the resident immune cells of the brain and play a key role in neurodevelopment. Disruption of microglial functions can lead to brain abnormalities and increase the risk of developing NDDs. How the maternal as well as the fetal immune system affect human neurodevelopment and contribute to NDDs remains unclear. An important reason for this knowledge gap is the fact that the impact of exposure to prenatal risk factors has been challenging to study in the human context. Here, we characterized a model of cerebral organoids (CO) with integrated microglia (COiMg). These organoids express typical microglial markers and respond to inflammatory stimuli. The presence of microglia influences cerebral organoid development, including cell density and neural differentiation, and regulates the expression of several ciliated and mesenchymal cell markers. Moreover, COiMg and organoids without microglia show similar but also distinct responses to inflammatory stimuli. Additionally, IFN-γ induced significant transcriptional and structural changes in the cerebral organoids, that appear to be regulated by the presence of microglia. Specifically, interferon-gamma (IFN-γ) was found to alter the expression of genes linked to autism. This model provides a valuable tool to study how inflammatory perturbations and microglial presence affect neurodevelopmental processes.

Alpha-lipoic acid-mediated inhibition of LTB4 synthesis suppresses epithelial-mesenchymal transition, modulating functional and tumorigenic capacities in non-small cell lung cancer A549 cells

BackgroundLeukotriene B4 (LTB4) plays a crucial role in carcinogenesis by inducing epithelial-mesenchymal transition (EMT), a process associated with tumor progression. The synthesis of LTB4 is mediated by leukotriene A4 hydrolase (LTA4H), and it binds to the receptors BLT1 and BLT2. Dysregulation in LTB4 production is linked to the development of various pathologies. Therefore, the identification or design of inhibitors of LTB4 synthesis or receptor antagonists represents an ongoing challenge. In this context, our laboratory previously demonstrated that alpha-lipoic acid (ALA) inhibits LTA4H. The objective of this study was to evaluate the effect of ALA on the expression of canonical EMT markers and the functional and tumorigenic capacities induced by LTB4 in A549 cells. MethodsThe expression of cPLA2, 5LOX, FLAP, LTA4H, BLT1, and LTB4 production in human adenocarcinomic alveolar basal epithelial A549 cells was assessed using Western blot, RT-qPCR, and ELISA, respectively. Subsequently, the expression of canonical EMT markers was evaluated by Western blot. Functional assays were performed to assess cell viability, proliferation, invasion, migration, and clonogenicity using MTT, Western blot, Transwell assays, and colony formation assays, respectively. Results were expressed as median with interquartile range (n≥3) and analyzed using the Kruskal-Wallis or Tukey multiple comparisons tests. ResultsA549 cells express key proteins involved in LTB4 synthesis and receptor binding, including LTA4H and BLT1, and ALA inhibits the production of LTB4. Additionally, LTA4H and BLT1 were detected in lung adenocarcinoma tissue samples. LTB4 was found to induce EMT, whereas ALA treatment enhanced the expression of epithelial markers and reduced the expression of mesenchymal markers. Furthermore, ALA treatment resulted in a decrease in LTB4 levels and attenuated the functional and tumorigenic capacities of A549 cells, including their viability, migration, invasion, and clonogenic potential. ConclusionsThese findings suggest that ALA may offer therapeutic potential in the context of lung cancer, as it could be integrated into conventional pharmacological therapies to enhance treatment efficacy and mitigate the adverse effects associated with chemotherapy. Further studies are warranted to confirm the clinical applicability of ALA as an adjunctive treatment in lung cancer.

Human epicardial adipose tissue secretome alters metabolomic profile and induces endothelial-to-mesenchymal transition in human lymphatic endothelial cells

Abstract Background Cardiac lymphatic systems play an important role in maintaining myocardial fluid homeostasis during inflammation. We have recently reported the epicardial adipose tissue (EAT)-derived factor induces inflammation and fibrosis in atrial myocardium, which lead to atrial fibrillation (AF). However, the direct effect of EAT on the cardiac lymphatic endothelial cells (LECs) remains unknown. Objective To elucidate the effect of EAT on cardiac LECs, as one of the potential causes of AF. Methods Human EAT samples were collected from 18 consecutive patients with or without AF patients during cardiovascular surgery. They were categorized into the sinus rhythm (SR) group (n=9, 72.7±8.6 years) and the AF group (n=9, 73.3±7.4 years). Preadipocytes were extracted from EAT by homogenization and collagenase treatment, and cultured to confluence. After differentiation to mature adipocytes, cell culture supernatant was collected, and cultured with human LECs. Cell permeability was measured by using FITC-dextran assay. Tube forming ability was analyzed by seeding LECs on the extracellular matrix gels. Metabolomic profiling of LECs was performed by GC-MS system. Results RT-PCR analysis revealed that cell culture supernatant from mature adipocytes of AF patients significantly increased mRNA expression of mesenchymal marker, TAGLN/SM22a (p<0.01) in human LECs, whereas mRNA expression of lymphatic endothelial markers, LYVE1 (p=0.0552) and PROX1 (p=0.0231) were decreased in human LECs, compared to those from mature adipocytes of SR patients. These data suggest cell culture supernatant from mature adipocytes of AF patients highly induce endothelial-to-mesenchymal transition (EndMT) of LECs. When assessing the endothelial barrier function by using FITC-dextran, cell culture supernatant from mature adipocytes of AF patients significantly decreased cell barrier function (p<0.01) compared to those from mature adipocytes of SR patients. Furthermore, cell culture supernatant from mature adipocytes of AF patients significantly decreased the tube forming ability of LECs (p<0.01) compared to those from mature adipocytes of SR patients. To assess the metabolomic changes in human LECs by EAT-secretome, we conducted the GC/MS analysis. Cell culture supernatant from mature adipocytes of AF patients significantly decreased several amino acids including branched-chain amino acid (BCAA; valine, leucine and isoleucine), compared to those from mature adipocytes of SR patients. Conclusion Our study with human EAT and LECs demonstrated that EAT-secretome from AF patients altered metabolomic profile in LECs, and highly caused EndMT in LECs, leading to impaired barrier function and the tube forming ability. Defects of lymphangiogenesis could be a therapeutic target for atrial cardiomyopathy.

Vitamin K2 and the Vitamin-K-Cycle-Enzyme VKORC1L1 improve endothelial regeneration by inhibiting endothelial-mesenchymal transition, inflammatory cell activation and ferroptosis

Abstract Introduction Vascular inflammation is a crucial contributor to atherosclerosis, in which oxidative stress, endoplasmic reticulum (ER) stress and transition of endothelial to mesenchymal cells (EndMT) are of critical importance. Vitamin-K-antagonists (VKA) promote vascular dysfunction, while dietary vitamin K intake was proposed to reduce incidence of coronary artery disease, but the mechanisms remain unresolved. Key enzyme for regeneration of vitamin K is the Vitamin K epoxide reductase complex subunit 1 (VKORC1), which also represents the pharmacological target of VKA. VKOR-like1 (VKORC1L1) is an isoenzyme of VKORC1, which resides at the ER-membrane, exerts antioxidative properties and is involved in vitamin K maintenance. Aim of this study was to investigate the role of Vitamin K2 and the VKOR-enzymes in endothelial cell inflammation. Methods and Results In human coronary artery endothelial cells (HCAEC), Vitamin K2 improves endothelial regeneration by increasing proliferation and cell viability and by reducing both apoptosis and ferroptosis (relative viability: RSL3 0.11 ± 0.01 vs 0.32 ± 0.05 RSL3+K2-1µM vs. 0.44 ± 0.11 RSL3+K2-10µM, p<0.001). Moreover, Vitamin K2-supplementation inhibits EndMT by reducing expression of mesenchymal markers (Calponin, SM22; Fig.1). In silico analyses of the proteome of human coronary atherosclerosis revealed increased expression of VKORC1L1, but not of VKORC1. In vitro induction of oxidative stress and ER-Stress promoted time- and dose-dependent enhanced expression of VKORC1L1, without altering VKORC1 expression (H2O2: 2.3-fold vs. 0.8-fold after 40 minutes, p=0.04; tunicamycin: 1 μg/ml: 1.43-fold vs. 0.8-fold, p<0.01). Likewise, siRNA-mediated VKORC1L1 knockdown induces an inflammatory gene signature with upregulation of NFkB-signalling and endoplasmic reticulum stress (NF-κB: 1.95-fold ± 0.13, GRP78: 1.32-fold ± 0.04 vs. control, p<0.01). Furthermore, VKORC1L1 downregulation increased formation of reactive oxygen species (ROS) and reduced proliferation (EdU signal: 0.56 ± 0.02 vs. control, p < 0.01). In contrast, VKORC1-knockdown does not impair endothelial regeneration and function (Fig.2). Vitamin K2 reduces vascular inflammation and endoplasmic reticulum stress in the case of VKORC1-, but not VKORC1L1-knockdown. In addition, inducing EndMT resulted in a reduction of VKORC1L1, but not of VKORC1, while VKORC1L1-knockdown aggravates EndMT. Conclusion VKORC1 and its isoenzyme VKORC1L1 exhibit divergent effects on endothelial cell inflammation. Further studies are warranted to shed light on the regulation of the enzymes to improve our understanding regarding vascular side effects of VKA treatment and beneficial effects of vitamin K supplementation.

Selective activation of PPARα by pemafibrate mitigates peritoneal inflammation and fibrosis through suppression of NLRP3 inflammasome and modulation of inflammation

Peritoneal inflammation and fibrosis remain major challenges to the long-term maintenance of peritoneal dialysis. Pemafibrate, a selective peroxisome proliferator-activated receptor α (PPARα) modulator, has been implicated in the management of fibrosis-related disorders. We investigated whether pemafibrate ameliorates peritoneal inflammation and fibrosis and explored the underlying mechanisms in mice with methylglyoxal (MGO)-induced peritoneal fibrosis (MGO mice). MGO mice exhibited peritoneal fibrosis with increased expression of mesenchymal markers, transforming growth factor-β1 (TGF-β1), and substantial deposition of extracellular matrix (ECM) proteins. Additionally, MGO mice exhibited peritoneal inflammation as indicated by elevated tumor necrosis factor-α expression and macrophage infiltration in peritoneal tissue. These effects were mitigated by pemafibrate treatment, which also restored peritoneal membrane function. Furthermore, pemafibrate promoted anti-inflammatory macrophage polarization in both mice and THP-1 cells. In human peritoneal mesothelial cells (HPMCs), pemafibrate effectively inhibited interferon-γ-induced production of TGF-β1 and ECM while suppressing the proinflammatory cytokines nuclear factor-κB (NF-κB) and activator protein 1. The NF-κB inhibitory effect of pemafibrate involved stabilization of the NF-κB inhibitory protein IkBα. Notably, pemafibrate hindered activation of the NLR family pyrin domain containing 3/caspase-1 axis in interferon-γ-stimulated THP-1 cells. These findings suggest that pemafibrate ameliorates peritoneal inflammation and fibrosis, making it a promising candidate for peritoneal fibrosis therapy.

6603 Impact of the Monocarbonyl Analogs of Curcumin, C66 and B2BrBC, on the Protein Expression of Epithelial and Mesenchymal Markers in MCF-7 Breast Cancer Cells

Abstract Disclosure: R. Stojchevski: None. A. Lavi: None. J. Bogdanov: None. N. Hadzi-Petrushev: None. M. Mladenov: None. L. Poretsky: None. D.B. Avtanski: None. The synthesis of monocarbonyl analogs of curcumin (MACs) presents a solution to the challenges posed by curcumin, a natural polyphenol extracted from the roots of the turmeric plant (Curcuma longa), which is known for its antioxidant and anti-inflammatory properties. The limitations of curcumin, including poor stability, low bioavailability, and rapid metabolism, hinder its application, and MACs, designed without a β-diketone moiety, address these drawbacks and exhibit enhanced stability and bioavailability. This study aimed to assess the effects of two monocarbonyl analogs of curcumin, (2E,6E)-2,6-bis[(2-trifluoromethyl)benzylidene]cyclohexanone (C66) and (2E,6E)-2,6-bis(2-bromobenzylidene)cyclohexanone (B2BrBC), on epithelial-to-mesenchymal transition (EMT) in MCF-7 breast cancer cells using western blot analysis. Our previous findings indicated that these analogs can potentially affect EMT by modulating the epithelial and mesenchymal gene expression. MCF-7 cells were cultured in complete medium supplemented with EMT-inducing factors for two days, followed by C66 and B2BrBC administration (100 µM) for three additional days. After the experiments, proteins were extracted, and western blot analyses were performed to measure the expression levels of various epithelial and mesenchymal markers (E-cadherin, N-cadherin, SNAI1, and Claudin-1). The results revealed that both C66 and B2BrBC increased the protein levels of the epithelial marker E-cadherin and suppressed those of the mesenchymal marker N-cadherin. Moreover, C66 and B2BrBC decreased the protein expression of the transcription factor SNAI1 and transmembrane protein Claudin-1. Between the two MACs, B2BrBC showed a more pronounced effect on N-cadherin expression, while C66 was more potent in suppressing SNAI1 protein expression. Our findings demonstrate that C66 and B2BrBC counteract EMT, contributing to the potential therapeutic applications of MACs in managing breast cancer progression. Presentation: 6/3/2024

VCAM-1 targeted nanocarriers of shRNA-Smad3 mitigate endothelial-to-mesenchymal transition triggered by high glucose concentrations and osteogenic factors in valvular endothelial cells

Endothelial to mesenchymal transition (EndMT) of valvular endothelial cells (VEC) is a key process in the development and progression of calcific aortic valve disease (CAVD). High expression of the Smad3 transcription factor is crucial in the transition process. We hypothesize that silencing Smad3 could hinder EndMT and provide a novel treatment for CAVD. We aimed at developing nanoparticles encapsulating short-hairpin (sh)RNA sequences specific for Smad3 targeted to the aortic valve. We synthesized VCAM-1-targeted lipopolyplexes encapsulating shRNA-Smad3 plasmid (V-LPP/shSmad3) and investigated their potential to reduce the EndMT of human VEC. VEC incubation in a medium containing high glucose concentrations and osteogenic factors (HGOM) triggers EndMT and increased expression of Smad3. Exposed to lipopolyplexes, VEC took up efficiently the V-LPP/shSmad3. The latter reduced the EndMT process in VEC exposed to HGOM by downregulating the expression of αSMA and S100A4 mesenchymal markers and increasing the expression of the CD31 endothelial marker. In vivo, V-LPP/shSmad3 accumulated in the aortic root and aorta of a murine model of atherosclerosis complicated with diabetes, without affecting the liver and kidney function. The results suggest that targeting activated VEC with lipopolyplexes to silence Smad3 could be an effective, novel treatment for CAVD mediated by the EndMT process.

PPARγ/ETV2 axis regulates endothelial-to-mesenchymal transition in pulmonary hypertension.

Endothelial-to-mesenchymal transition (EndoMT) plays an important role in pulmonary hypertension (PH) but the molecular mechanisms regulating EndoMT remain to be defined. We demonstrate that the axis of the transcription factors PPARγ (Peroxisome Proliferator-Activated Receptor gamma) and ETV2 (ETS variant 2) play important roles in the pathogenesis of PH. Decreased levels of the expression of PPARγ and ETV2 along with reduced endothelial and increased EndoMT markers are consistently observed in lungs and pulmonary artery endothelial cells (PAECs) of idiopathic pulmonary arterial hypertension patients, in hypoxia-exposed mouse lungs, human PAECs, and in induced-EndoMT cells. Etv2 +/- mice spontaneously developed PH and right ventricular hypertrophy (RVH), associated with increased EndoMT markers and decreased EC markers. Interestingly, chronic hypoxia exacerbated right ventricular systolic pressure and RVH in Etv2 +/- mice. PPARγ transcriptionally activates the ETV2 promoter. Consistently, while mice overexpressing endothelial PPARγ increases the expression of ETV2 and endothelial markers with reduced EndoMT markers, endothelial PPARγ KO mice show decreased ETV2 expression and enhanced EndoMT markers. Inducible overexpression of ETV2 under induced-EndoMT cell model reduces number of cells with mesenchymal morphology and decreases expression of mesenchymal markers with increased EC makers, compared to control. Therefore, our study suggests that PPARγ-ETV2 signaling regulates PH pathogenesis through EndoMT.

Cleistanthin A derivative disrupts autophagy and suppresses head and neck squamous cell carcinoma progression via targeted vacuolar ATPase

Head and neck squamous cell carcinoma (HNSCC) present a significant challenge due to its heterogeneity and limited treatment options, often resulting in severe side effects and poor survival rates with conventional chemoradiotherapy. Here, we investigated the anticancer activity of halogenated benzoate derivatives of cleistanthin A, ECDD-S16 and ECDD-S18, in HNSCC cells. Our findings revealed that ECDD-S18 exhibited remarkable cytotoxicity, surpassing that of cisplatin with minimal impact on normal and cisplatin-sensitive cells. Notably, ECDD-S18 induced apoptosis in a dose-dependent manner and effectively targeted vacuolar ATPase (V-ATPase), impairing lysosomal acidification. Intriguingly, ECDD-S18 inhibited autophagic flux, as evidenced by increased autophagosome but decreased autolysosome formation. Furthermore, proteomic analysis demonstrated downregulation of cathepsin D (CTSD), the lysosomal protease in ECDD-S18-treated HNSCC cells, concurrent with suppressed cell migration. ECDD-S18 also decreased expression of mesenchymal markers, suggesting inhibition of epithelial-mesenchymal transition (EMT). Importantly, cotreatment with ECDD-S18 and cisplatin enhanced the reduction in cell viability. Collectively, our results indicated that the anticancer activity of ECDD-S18 partly stems from its ability to disrupt lysosomal acidification and inhibit autophagy via targeted inhibition of V-ATPase. These findings underscore the therapeutic promise of ECDD-S18 in HNSCC treatment, either alone or in combination with existing drugs, while mitigating toxicity to normal cells.

Abstract A077: The role of RhoV in basal-like pancreatic cancer

Abstract Background Pancreatic ductal adenocarcinoma is a devastating disease lacking targeted therapy. Basal-like pancreatic cancer is a tumor subtype with a worse clinical outcome than other subtypes. These cancers are characterized by the expression of basal keratins and mesenchymal markers. However, the genes that drive the basal-like phenotype are poorly understood. Ras homolog family member V (RhoV) is one of the Rho GTPases, and it has been demonstrated to drive the progression of lung and breast cancers. The current study aims to reveal the role of RhoV in basal-like pancreatic cancer. Design RhoV, p63, and CK5/6 were stained in sequential sections by immunohistochemistry. Expression of RhoV in pancreatic cancer and cell lines was reanalyzed based on the data from ProteinAtlas and Broad Insitute DepMap Portal. Basal cell line, BxPC3, and non-basal cell line, Panc1 were selected for functional evaluation based on their differentiated RhoV expression. RhoV gene was overexpressed and knocked down using the CRISPR/gRNA-based Lentiviral system, followed by sphere formation, colony formation, migration, and invasion assays. Results RhoV, p63, and CK5/6 were expressed in the same histologic location of basal pancreatic cancer. The gene expression level of RhoV was co-expressed with p63 (a classic basal marker) in four pancreatic cancer cell lines (broadinstitute.org). Knockout of RhoV by three runs of gRNAs in BxPC3 cells all significantly attenuated tumor sphere formation with less colony formation as compared to mock knockout. Overexpression of RhoV in non-basal-like cells, Panc 1, significantly increased the size of sphere formation, numbers of colony formation, and p38 activation compared to EV control. Cell migration and invasion assays showed the same RhoV-dependent migration and invasion in corresponding cells. Panc1 cells with RhoV knockout significantly generated smaller tumor xenografts in immunodeficient mice and RhoV overexpression led to larger tumor sizes. Conclusion Finding the driver of the basal-like phenotype helps to reveal the pathophysiology of the basal-like subtype of pancreatic cancer and facilitates the identification of new targets for therapeutic purposes. Our preliminary results indicated that RhoV, a small G protein from the Rho family of GTPases, played essential roles in tumor proliferation and progression, by activating the p38 signaling pathway. Further study will be focused on revealing Rhov's downstream pathways and exploring the role of RhoV in apoptosis/cell cycle, cell metabolism, autophagy, etc. in basel-like pancreatic cancer. Citation Format: Shang Wu, Jia Feng, Selina Gao, Ting Wang, Zebang Li, Yifei Liu, Chiung-Kuei Huang, Shaolei Lu. The role of RhoV in basal-like pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A077.

Targeting Cross‐Talks of Notch and VEGF to Tweak the EMT and EPT Dynamics in Triple Negative Breast Cancer Cells

AbstractThe associations of the Notch pathway with the major oncogenic pathways (especially the receptor tyrosine kinases, RTKs) are primarily responsible for inducing EMT (epithelial to mesenchymal transition), angiogenesis, and chemoresistance. In this study, Axitinib is used in combination with LY411575 (γ‐secretase inhibitor) and it is observed that the co‐treatment synergistically induced apoptosis (by 37.36% in MDAMB231 and 27.9% in MDAMB468), arrests cells at the G2/M phase, decreases the stemness properties of the triple‐negative breast cancer (TNBC) cells. It also diminishes the spheroid forming ability, enhances the expression of epithelial markers, such as E‐cadherin (by 2.2 fold in MDAMB231 and 2.51 fold in MDAMB468), and downregulated the expression of mesenchymal markers. Additionally, the protein expression profile of the pro‐oncogenic and pro‐survival genes also reduces significantly after the administration of co‐therapy, which is highlighted by a reduction in the levels of pEGFR, pFAK, pMAPK, NF‐κB, etc. Moreover, the expression of pericyte markers (such as PDGFRs, α‐SMA, c‐kit, and NG2) reduces significantly in both TNBC cells upon co‐treatment, thereby hinting toward the inhibition of epithelial‐to‐pericyte transition (EPT). The current work endows with the effectiveness of the co‐therapy on the EMT and EPT dynamics of TNBC upon inhibition of the major crosstalk between the Vascular endothelial growth factor (VEGF)t and Notch pathway.

Prevention of Transition from Acute Kidney Injury to Chronic Kidney Disease Using Clinical-Grade Perinatal Stem Cells in Non-Clinical Study.

Acute kidney injury (AKI) is widely recognized as a precursor to the onset or rapid progression of chronic kidney disease (CKD). However, there is currently no effective treatment available for AKI, underscoring the urgent need for the development of new strategies to improve kidney function. Human placental mesenchymal stromal cells (hpMSCs) were isolated from donor placentas, cultured, and characterized with regard to yield, viability, flow cytometry, and potency. To mimic AKI and its progression to CKD in a rat model, a dedicated sensitive non-clinical bilateral kidney ischemia-reperfusion injury (IRI) model was utilized. The experimental group received 3 × 105 hpMSCs into each kidney, while the control group received IRI and saline and the untreated group received IRI only. Urine, serum, and kidney tissue samples were collected over a period of 28 days. The hpMSCs exhibited consistent yields, viability, and expression of mesenchymal lineage markers, and were also shown to suppress T cell proliferation in a dose-dependent manner. To ensure optimal donor selection, manufacturing optimization, and rigorous quality control, the rigorous Good Manufacturing Practice (GMP) conditions were utilized. The results indicated that hpMSCs increased rat survival rates and improved kidney function by decreasing serum creatinine, urea, potassium, and fractionated potassium levels. Furthermore, the study demonstrated that hpMSCs can prevent the initial stages of kidney structural fibrosis and improve kidney function in the early stages by mitigating late interstitial fibrosis and tubular atrophy. Additionally, a robust manufacturing process with consistent technical parameters was established.

Beauvericin Reverses Epithelial-to-Mesenchymal Transition in Triple-Negative Breast Cancer Cells through Regulation of Notch Signaling and Autophagy.

Metastasis stands as a prime contributor to triple-negative breast cancer (TNBC) associated mortality worldwide, presenting heightened severity and significant challenges due to limited treatment options. Addressing TNBC metastasis necessitates innovative approaches and novel therapeutics to specifically target its propensity for dissemination to distant organs. Targeted therapies capable of reversing epithelial-to-mesenchymal transition (EMT) play a crucial role in suppressing metastasis and enhancing the treatment response. Beauvericin, a promising fungal secondary metabolite, exhibits significant potential in diminishing the viability of EMT-induced TNBC cells by triggering intracellular oxidative stress, as evidenced by an enhanced reactive oxygen species level and reduced mitochondrial transmembrane potential. In monolayer cultures, it has exhibited an IC50 of 2.3 μM in both MDA-MB-468 and MDA-MB-231 cells, while in 3D spheroids, the IC50 values are 9.7 and 7.1 μM, respectively. Beauvericin has also reduced the migratory capability of MDA-MB-468 and MDA-MB-231 cells by 1.5- and 1.7-fold, respectively. Both qRT-PCR and Western blot analysis have shown significant upregulation in the expression of epithelial marker (E-cadherin) and downregulation in the expression of mesenchymal markers (N-cadherin, vimentin, Snail, Slug, and β-catenin), following treatment, indicating reversal of EMT. Furthermore, beauvericin has suppressed the Notch signaling pathway by substantially downregulating Notch-1, Notch-3, Hes-1, and cyclinD3 expression and induced autophagy as observed by elevated expression of autophagy markers LC3 and Beclin-1. In conclusion, beauvericin has successfully downregulated TNBC cell survival by inducing oxidative stress and suppressed their migratory potential by reversing EMT through the inhibition of Notch signaling and activation of autophagy.

Characterization of Central and Nasal Orbital Adipose Stem Cells and their Neural Differentiation Footprints.

The unique potential of stem cells to restore vision and regenerate damaged ocular cells has led to the increased attraction of researchers and ophthalmologists to ocular regenerative medicine in recent decades. In addition, advantages such as easy access to ocular tissues, non-invasive follow-up, and ocular immunologic privilege have enhanced the desire to develop ocular regenerative medicine. This study aimed to characterize central and nasal orbital adipose stem cells (OASCs) and their neural differentiation potential. The central and nasal orbital adipose tissues extracted during an upper blepharoplasty surgery were explant-cultured in Dulbecco's Modified Eagle Medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS). Cells from passage 3 were characterized morphologically by osteogenic and adipogenic differentiation potential and by flow cytometry for expression of mesenchymal (CD73, CD90, and CD105) and hematopoietic (CD34 and CD45) markers. The potential of OASCs for the expression of NGF, PI3K, and MAPK and to induce neurogenesis was assessed by real-time PCR. OASCs were spindle-shaped and positive for adipogenic and osteogenic induction. They were also positive for mesenchymal and negative for hematopoietic markers. They were positive for NGF expression in the absence of any significant alteration in the expression of PI3K and MAPK genes. Nasal OASCs had higher expression of CD90, higher potential for adipogenesis, a higher level of NGF expression under serum-free supplementation, and more potential for neuron-like morphology. We suggested the explant method of culture as an easy and suitable method for the expansion of OASCs. Our findings denote mesenchymal properties of both central and nasal OASCs, while mesenchymal and neural characteristics were expressed stronger in nasal OASCs when compared to central ones. These findings can be added to the literature when cell transplantation is targeted in the treatment of neuro-retinal degenerative disorders.

Unlocking the Secrets of Adipose Tissue: How an Obesity-Associated Secretome Promotes Osteoblast Dedifferentiation via TGF-β1 Signaling, Paving the Path to an Adipogenic Phenotype.

Obesity poses a significant global health challenge, given its association with the excessive accumulation of adipose tissue (AT) and various systemic disruptions. Within the adipose microenvironment, expansion and enrichment with immune cells trigger the release of inflammatory mediators and growth factors, which can disrupt tissues, including bones. While obesity's contribution to bone loss is well established, the direct impact of obese AT on osteoblast maturation remains uncertain. This study aimed to explore the influence of the secretomes from obese and lean AT on osteoblast differentiation and activity. SAOS-2 cells were exposed to the secretomes obtained by culturing human subcutaneous AT from individuals with obesity (OATS) or lean patients, and their effects on osteoblasts were evaluated. In the presence of the OATS, mature osteoblasts underwent dedifferentiation, showing an increased proliferation accompanied by a morphological shift towards a mesenchymal phenotype, with detrimental effects on osteogenic markers and the calcification capacity. Concurrently, the OATS promoted the expression of mesenchymal and adipogenic markers, inducing the formation of cytoplasmic lipid droplets in SAOS-2 cells exposed to an adipogenic differentiation medium. Additionally, TGF-β1 emerged as a key mediator of these effects, as the OATS was enriched with this growth factor. Our findings demonstrate that obese subcutaneous AT promotes the dedifferentiation of osteoblasts and increases the adipogenic profile in these cells.

Collagen VI deposition primes the glioblastoma microenvironment for invasion through mechanostimulation of β-catenin signaling.

While glioblastoma (GBM) progression is associated with extensive extracellular matrix (ECM) secretion, the causal contributions of ECM secretion to invasion remain unclear. Here we investigate these contributions by combining engineered materials, proteomics, analysis of patient data, and a model of bevacizumab-resistant GBM. We find that GBM cells cultured in engineered 3D hyaluronic acid hydrogels secrete ECM prior to invasion, particularly in the absence of exogenous ECM ligands. Proteomic measurements reveal extensive secretion of collagen VI, and collagen VI-associated transcripts are correspondingly enriched in microvascular proliferation regions of human GBMs. We further show that bevacizumab-resistant GBM cells deposit more collagen VI than their responsive counterparts, which is associated with marked cell-ECM stiffening. COL6A3 deletion in GBM cells reduces invasion, β-catenin signaling, and expression of mesenchymal markers, and these effects are amplified in hypoxia. Our studies strongly implicate GBM cell-derived collagen VI in microenvironmental remodeling to facilitate invasion.

Endothelial-to-mesenchymal transition enhances permissiveness to AAV vectors in cardiac endothelial cells

A major obstacle in inducing therapeutic angiogenesis in the heart is inefficient gene transfer to endothelial cells (ECs). Here, we identify compounds able to enhance the permissiveness of cardiac ECs to adeno-associated virus (AAV) vectors, which stand as ideal tools for invivo gene delivery. We screened a library of >1,500US Food and Drug Administration (FDA)-approved drugs, in combination with AAV vectors, in cardiac ECs. Among the top drugs increasing AAV-mediated transduction, we found vatalanib, an inhibitor of multiple tyrosine kinase receptors. The increased AAV transduction efficiency by vatalanib was paralleled by induction of the endothelial-to-mesenchymal transition, asdocumented by decreased endothelial and increased mesenchymal marker expression. Induction of the endothelial-to-mesenchymal transition by other strategies similarly increased EC permissiveness to AAV vectors. Invivo injection of AAV vectors in the heart after myocardial infarction resulted in the selective transduction of cells undergoing the endothelial-to-mesenchymal transition, which is known to happen transiently after cardiac ischemia. Collectively, these results point to the endothelial-to-mesenchymal transition as a mechanism for improving AAV transduction in cardiac ECs, with implications for both basic research and the induction of therapeutic angiogenesis in the heart.