Ki67 Immunofluorescence Research Articles

MiR-101-3p suppresses proliferation of orbital fibroblasts by targeting pentraxin-3 in thyroid eye disease.

Excessive proliferation of orbital fibroblasts (OFs) is an essential factor in the pathogenesis of thyroid eye disease (TED). While existing evidence indicates that various microRNAs (miRNAs) significantly contribute to TED development, the precise function and targets of miR-101-3p in TED pathogenesis remain unknown. This research aims to elucidate the effects of miR-101-3p on TED-OFs and identify its potential targets. Orbital adipose tissues were harvested from both TED patients and healthy controls to culture their fibroblasts. MiR-101-3p mimic or mimic negative control (mimic NC) was transfected into OFs from TED patients, with untreated OFs serving as an additional blank control group. Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay, Ki-67 immunofluorescence staining, and the EdU assay, while apoptosis was evaluated via flow cytometry. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure the expression levels of miR-101-3p and pentraxin-3 (PTX3), and PTX3 protein levels were quantified using western blot. A dual-luciferase assay was conducted to ascertain how miR-101-3p and PTX3 interacted. The results demonstrated a significant downregulation of miR-101-3p in fibroblasts and TED orbital adipose tissues. Transfection with the miR-101-3p mimic upregulated miR-101-3p levels, significantly reducing OFs proliferation without affecting apoptosis. Overexpression of miR-101-3p led to the downregulation of PTX3 in OFs. The dual-luciferase assay validated miR-101-3p binding to PTX3's 3'UTR, thereby repressing its expression. Moreover, overexpression of PTX3 partially rescued the miR-101-3p mimic's inhibitory effect on TED-OFs proliferation. Our findings illustrate miR-101-3p's role in targeting PTX3 to regulate TED-OFs proliferation, providing novel insights into the pathological mechanisms underlying TED development.

TMOD-24. DEVELOPMENT OF A 3D ORGANOTYPIC EXPLANT PLATFORM TO STUDY ONCOLYTIC VIRUS TREATMENT RESPONSE IN GLIOBLASTOMA

Abstract BACKGROUND Oncolytic viruses (OVs) are a promising strategy to induce an anti-tumor immune response in the lymphocyte-depleted environment of glioblastoma (GBM), however the mechanism of their oncolytic function is not well understood. Immune-intact experimental systems would be highly valuable in dissecting immune versus direct viral contribution to oncolysis. Here, we report on the development of an immune-intact organotypic explant (OE) model, amenable to perturbations and T-cell activity monitoring. METHODS OEs were made from fresh tumor samples and cultured in groups of 4-5. CAN-3310 OVs and T-cells were injected into each OE under a dissecting microscope. Cultured OEs were either fixed for immunofluorescence (IF) or dissociated into single-cell suspensions for flow cytometry and 5’ single-cell RNA (scRNA) and TCR sequencing. RESULTS Cultured OEs maintain the cellular density of the original tumor in both short- and long-term cultures. Ki67 IF shows that tumor cells retain their proliferative potential, allowing for renewal and repopulation. The TME, mainly microglia and macrophages, is maintained throughout culture. IF imaging of OV-treated OEs, 7 days post-treatment, shows significant GFP positivity in tumor cells demonstrating the feasibility of using OEs as an ex vivo model with OV. Additionally, autologous T-cells from the patient’s blood can survive in OEs, completing the TME with the equivalent of circulating T-cells. Finally, scRNA-seq of the original tumor and cultured OEs showed that the different cell types were represented at all time points. Tumor cell meta-module analysis showed an increase in hypoxic signatures recapitulating features of in vivo tumor cell growth. CONCLUSIONS OEs maintain tumor architecture and TME. OE co-culture with autologous T-cells allows us to better understand the oncolytic mechanisms of OVs. Future endeavors will include identifying expanded T-cell clones to create an OV-specific TCR repertoire. This OE system provides a new tool as an ex vivo platform to dissect glioma responses to therapy in a purely human system.

Kiss1 receptor knockout exacerbates airway hyperresponsiveness and remodeling in a mouse model of allergic asthma

BackgroundIn asthma, sex-steroids signaling is recognized as a critical regulator of disease pathophysiology. However, the paradoxical role of sex-steroids, especially estrogen, suggests that an upstream mechanism or even independent of estrogen plays an important role in regulating asthma pathophysiology. In this context, in our previous studies, we explored kisspeptin (Kp) and its receptor Kiss1R’s signaling in regulating human airway smooth muscle cell remodeling in vitro and airway hyperresponsiveness (AHR) in vivo in a mouse (wild-type, WT) model of asthma. In this study, we evaluated the effect of endogenous Kp in regulating AHR and remodeling using Kiss1R knockout (Kiss1R−/−) mice.MethodsC57BL/6J WT (Kiss1R+/+) and Kiss1R−/− mice, both male and female, were intranasally challenged with mixed-allergen (MA) and/or phosphate-buffered saline (PBS). We used flexiVent analysis to assess airway resistance (Rrs), elastance (Ers), and compliance (Crs). Following this, broncho-alveolar lavage (BAL) was performed for differential leukocyte count (DLC) and cytokine analysis. Histology staining was performed using hematoxylin and eosin (H&E) for morphological analysis and Masson’s Trichrome (MT) for collagen deposition. Additionally, lung sections were processed for immunofluorescence (IF) of Ki-67, α-smooth muscle actin (α-SMA), and tenascin-c.ResultsInterestingly, the loss of Kiss1R exacerbated lung function and airway contractility in mice challenged with MA, with more profound effects in Kiss1R−/− female mice. MA-challenged Kiss1R−/− mice showed a significant increase in immune cell infiltration and proinflammatory cytokine levels. Importantly, the loss of Kiss1R aggravated Th2/Th17 biased cytokines in MA-challenged mice. Furthermore, histology of lung sections from Kiss1R−/− mice showed increased collagen deposition on airway walls and mucin production in airway cells compared to Kiss1R+/+ mice. In addition, immunofluorescence analysis showed loss of Kiss1R significantly aggravated airway remodeling and subsequently AHR.ConclusionsThese findings demonstrate the importance of inherent Kiss1R signaling in regulating airway inflammation, AHR, and remodeling in the pathophysiology of asthma.

Sarcoplasmic reticulum calcium ATPase (SERCA) proteolysis by matrix metalloproteinase-2 contributes to vascular dysfunction in early hypertension

AimsHypertension is associated with an increased activity of matrix metalloproteinase (MMP)-2 in the vasculature, which, in turn, proteolyzes extra- and intracellular proteins that lead to vascular dysfunction. The activity of sarcoplasmic reticulum calcium ATPase (SERCA) is decreased in the aortas of hypertensive rats. Increased activity of MMP-2 proteolyzed SERCA in rat heart during ischemia and reperfusion injury, thus impairing cardiac function. Therefore, we examined whether increased activity of MMP-2 in early hypertension contributes to proteolyze SERCA in the aortas, thus leading to maladaptive vascular remodeling and dysfunction. Main methodsMale Sprague-Dawley rats were submitted to two kidney-one clip (2K-1C) or Sham surgery and treated with doxycycline. Systolic blood pressure (SBP) was assessed by tail-cuff plethysmography. After 7 days, aortas were collected for zymography assays, Western blot to SERCA, ATPase activity assay, vascular reactivity, Ki-67 immunofluorescence and hematoxylin/eosin stain. Key findingsSBP was increased in 2K-1C rats and doxycycline did not reduce it, but decreased MMP-2 activity and prevented SERCA proteolysis in aortas. Cross sectional area, media to lumen ratio and Ki-67 were all increased in the aortas of hypertensive rats and doxycycline decreased Ki-67. In 2K-1C rats, arterial relaxation to acetylcholine was impaired and doxycycline ameliorated it. Significancedoxycycline reduced MMP-2 activity in aortas of 2K-1C rats and prevented proteolysis of SERCA and its dysfunction, thus ameliorating hypertension-induced vascular dysfunction.

O65 SARCOPLASMIC CALCIUM ATPASE (SERCA) PROTEOLYSIS BY MATRIX METALLOPROTEINASE (MMP)-2 CONTRIBUTES TO VASCULAR DYSFUNCTION IN EARLY HYPERTENSION

Background and objective: Hypertension-induced increased matrix metalloproteinase (MMP)-2 activity proteolyzes extra- and intracellular proteins in aortas and contributes to vascular dysfunction. MMP-2 proteolyzes sarcoplasmic reticulum calcium ATPase (SERCA) in the heart during ischemia and reperfusion injury, thus impairing cardiac function. SERCA activity is also impaired in the arteries of hypertensive animals. We investigated whether hypertension-induced increased MMP-2 activity in aorta contributes to SERCA proteolysis, vascular remodeling and dysfunction. Methods: Aortas from male Sprague-Dawley (SD) rats, incubated or not with angiotensin II (AngII) and MMP inhibitors, ONO-4817 or doxycycline (Doxy), were used for zymography and co-immunoprecipitation of SERCA with MMP-2. Male SD rats were also submitted to two kidney-one clip (2K-1C) or Sham surgery and treated with Doxy from third to seventh day post-surgery. Systolic blood pressure (SBP) was assessed basally and daily by tail-cuff plethysmography. After 7 days, aortas were collected for gel and in situ zymography, western blot to SERCA, ATPase activity assay, vascular reactivity to phenylephrine and acetylcholine, Ki-67 immunofluorescence and hematoxylin/eosin stain. A7r5 cells were incubated or not with AngII, in the presence or absence of SERCA inhibitor, tapsigargin, to assess intracellular calcium concentrations. Results: AngII increased MMP-2 activity in aortas and MMP-2 co-immunoprecipitated with SERCA. SBP was increased in 2K-1C rats and Doxy did not reduce it, but decreased MMP-2 activity and prevented SERCA proteolysis in aortas. Intracellular calcium concentrations were increased in A7r5 cells incubated with AngII and/or SERCA inhibitor, tapsigargin. Cross sectional area, media to lumen ratio and Ki-67 were all increased in the aortas of hypertensive rats and Doxy decreased Ki-67 cell proliferation. In 2K-1C rats, arterial contraction to phenylephrine was accentuated but Doxy ameliorated only the impaired relaxation to acetylcholine. Conclusion: Doxy reduced MMP-2 activity in the aortas of 2K-1C rats and prevented the ATPase activity impairment and MMP-2-induced SERCA proteolysis and vascular dysfunction.

Testosterone-Induced H3K27 Deacetylation Participates in Granulosa Cell Proliferation Suppression and Pathogenesis of Polycystic Ovary Syndrome

Polycystic Ovary Syndrome (PCOS) is the leading cause of infertility in reproductive-age women. Hyperandrogenism, polycystic ovaries and chronic anovulation are its typical clinical features. However, the correlation between hyperandrogenism and ovarian follicle growth aberrations remains undisclosed. To advance our understanding of the molecular alterations in ovarian granulosa cells (GCs) with excessive androgen, epigenetic changes and affected gene expression in human granulosa-lutein cells (hGCs) and immortalized human GCs (KGN) were evaluated. A PCOS mouse model induced by dihydrotestosterone (DHT) was also established. This study found excessive testosterone significantly decreased the acetylation of lysine 27 on Histone H3 (H3K27ac). H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) data showed down-regulated expression of cell cycle-related genes (CCND1/CCND3/PCNA), which was confirmed by real-time quantitative PCR and western blot. Testosterone application impeding cell proliferation was also proved by Ki-67 immunofluorescence and flow-cytometric analysis. Moreover, testosterone influenced CK2α nuclear translocation, which increased the phosphorylation level of histone deacetylase 2 (HDAC2). Inhibition of CK2α nuclear translocation or silenced HDAC2 expression efficiently retarded H3K27 acetylation. Meanwhile, PCOS mouse model experiments also demonstrated decreased H3K27ac and enhanced HDAC2 phosphorylation in GCs. Cell proliferation-related genes were also downregulated in PCOS mouse GCs. In conclusion, hyperandrogenism in human and mouse GCs caused H3K27Ac aberrations, which are associated with CK2α nuclear translocation and HDAC2 phosphorylation, participating in abnormal follicle development in PCOS patients.

New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis

BackgroundBone marrow-derived mesenchymal stem cell (BMMSC)-based therapy has become a major focus for treating liver fibrosis/cirrhosis. However, although these cell therapies promote the treatment of this disease, the heterogeneity of BMMSCs, which causes insufficient efficacy during clinical trials, has not been addressed. In this study, we describe a novel Percoll–Plate–Wait procedure (PPWP) for the isolation of an active cell subset from BMMSC cultures that was characterized by the expression of neuroglial antigen 2 (NG2/BMMSCs).MethodsBy using the key method of PPWP and other classical biological techniques we compared NG2/BMMSCs with parental BMMSCs in biological and functional characteristics within a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis injury male C57BL/6 mouse model also in a culture system. Of note, the pathological alterations in the model is quite similar to humans’.ResultsThe NG2/BMMSCs revealed more advantages compared to parentalBMMSCs. They exhibited greater proliferation potential than parental BMMSCs, as indicated by Ki-67 immunofluorescence (IF) staining. Moreover, higher expression of SSEA-3 (a marker specific for embryonic stem cells) was detected in NG2/BMMSCs than in parental BMMSCs, which suggested that the “stemness” of NG2/BMMSCs was greater than that of parental BMMSCs. In vivo studies revealed that an injection of NG2/BMMSCs into mice with ongoing DEN-induced liver fibrotic/cirrhotic injury enhanced repair and functional recovery to a greater extent than in mice treated with parental BMMSCs. These effects were associated with the ability of NG2/BMMSCs to differentiate into bile duct cells (BDCs). In particular, we discovered for the first time that NG2/BMMSCs exhibit unique characteristics that differ from those of parental BMMSCs in terms of producing liver sinusoidal endothelial cells (LSECs) to reconstruct injured blood vessels and sinusoidal structures in the diseased livers, which are important for initiating hepatocyte regeneration. This unique potential may also suggest that NG2/BMMSCs could be an novel off-liver progenitor of LSECs. Ex vivo studies revealed that the NG2/BMMSCs exhibited a similar trend to that of their in vivo in terms of functional differentiation responding to the DEN-diseased injured liver cues. Additionally, the obvious core role of NG2/BMMSCs in supporting the functions of BMMSCs in bile duct repair and BDC-mediated hepatocyte regeneration might also be a novel finding.ConclusionsOverall, the PPWP-isolated NG2/BMMSCs could be a novel effective cell subset with increased purity to serve as a new therapeutic tool for enhancing treatment efficacy of BMMSCs and special seed cell source (BDCs, LSECs) also for bioliver engineering.

Inhibition of CIRBP represses the proliferation and migration of vascular smooth muscle cells via inhibiting Rheb/mTORC1 axis

The excessive migration and proliferation of vascular smooth muscle cells (VSMCs) plays a vital role in vascular intimal hyperplasia. CIRBP is involved in the proliferation of various cancer cells. This study was aimed to explore the role of CIRBP in the proliferation and migration of VSMCs. Adenovirus was used to interfere with cold-inducible RNA-binding protein (CIRBP) expression, while lentivirus was used to overexpress Ras homolog enriched in brain (Rheb). Western blotting and qRT-PCR were used to evaluate the expression of CIRBP, Rheb, and mechanistic target of rapamycin complex 1 (mTORC1) activity. The cell proliferation was determined by Ki67 immunofluorescence staining and CCK-8 assay. The wound healing assay was performed to assess cell migration. Additionally, immunohistochemistry was conducted to explore the role of CIRBP in intimal hyperplasia after vascular injury. We found that silencing CIRBP inhibited the proliferation and migration of VSMCs, decreased the expression of Rheb and mTORC1 activity. Restoration of mTORC1 activity via insulin or overexpression of Rheb via lentiviral transfection both attenuated the inhibitory effects of silencing CIRBP on the proliferation and migration of VSMCs. Moreover, Rheb overexpression abolished the inhibitory effect of silencing CIRBP on mTORC1 activity in VSMCs. CIRBP was upregulated in the injured carotid artery. Silencing CIRBP ameliorated intimal hyperplasia after vascular injury. In the summary, silencing CIRBP attenuates mTORC1 activity via reducing Rheb expression, thereby supressing the proliferation and migration of VSMCs and intimal hyperplasia after vascular injury.

Epigallocatechin gallate modulates ferroptosis through downregulation of tsRNA-13502 in non-small cell lung cancer

Ferroptosis, an iron-dependent regulated cell death mechanism, holds significant promise as a therapeutic strategy in oncology. In the current study, we explored the regulatory effects of epigallocatechin gallate (EGCG), a prominent polyphenol in green tea, on ferroptosis and its potential therapeutic implications for non-small cell lung cancer (NSCLC). Treatment of NSCLC cell lines with varying concentrations of EGCG resulted in a notable suppression of cell proliferation, as evidenced by a reduction in Ki67 immunofluorescence staining. Western blot analyses demonstrated that EGCG treatment led to a decrease in the expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) while increasing the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4). These molecular changes were accompanied by an increase in intracellular iron, malondialdehyde (MDA), and reactive oxygen species (ROS), alongside ultrastructural alterations characteristic of ferroptosis. Through small RNA sequencing and RT-qPCR, transfer RNA-derived small RNA 13502 (tsRNA-13502) was identified as a significant target of EGCG action, with its expression being upregulated in NSCLC tissues compared to adjacent non-tumorous tissues. EGCG was found to modulate the ferroptosis pathway by downregulating tsRNA-13502 and altering the expression of key ferroptosis regulators (GPX4/SLC7A11 and ACSL4), thereby promoting the accumulation of iron, MDA, and ROS, and ultimately inducing ferroptosis in NSCLC cells. This study elucidates EGCG’s multifaceted mechanisms of action, underscoring the modulation of ferroptosis as a viable therapeutic approach for enhancing NSCLC treatment outcomes.

P30 Exposure to particulate matter induces structural and pigmentary changes to human skin

Abstract Introduction and aims The majority of the world’s population reside in places with high levels of air pollution. The impact of air pollution on human health has been extensively studied and demonstrates a significant correlation between particulate matter (PM) and risk of cardiovascular and respiratory diseases. However, little is known about the impact of air pollution on human skin health. Here, we develop an ex vivo skin model to investigate cutaneous changes induced by the topical application of PM. Methods PM (0, 0.5, 3, 6, 10, 20 and 40 μg mL−1) was topically applied to Genoskin® NativeSkin Access® (Genoskin, Salem, MA, USA) human ex vivo skin samples of White European ancestry (n = 3) for 8 days, with reapplication on day 4. Resultant samples were assessed using haematoxylin and eosin staining (for morphometric analyses), immunofluorescence for Ki67 and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) (to determine cellular turnover), while pigmentary changes were analysed by colourimetry, Warthin–Starry (WS) staining and immunohistochemistry for the melanin markers tyrosinase (TYR), gp100 and c-kit. Results Morphological assessment revealed that epidermal thickness was significantly reduced following exposure to higher doses of PM (r = −0.9163, P < 0.01). In addition, there was reduced epidermal cell proliferation (Ki67) and increased apoptosis (TUNEL) at higher doses of PM. Exposure to PM resulted in a dose-dependent decrease in individual typology angle, which is indicative of skin colour becoming darker (r = −0.7895, P < 0.05). Furthermore, WS staining demonstrated significantly increased epidermal melanin (r = 0.9583, P < 0.01) and significantly increased expression of melanin markers TYR (P < 0.01), gp100 (P < 0.001) and c-kit (P < 0.01) in response to PM exposure. Conclusions This study confirms that PM induces pigmentary changes to ex vivo human skin by increasing epidermal melanin. Increased expression of c-kit in response to PM exposure suggests that increased pigmentation occurs owing to the upregulation of the microphthalmia-associated transcription factor signalling pathway. Further investigation of the mechanistic drivers of increased pigmentation is warranted.

GABPA-Mediated Expression of HPN-AS1 Facilitates Cell Apoptosis and Inhibits Cell Proliferation in Hepatocellular Carcinoma by Promoting eIF4A3 Degradation.

Hepatocellular carcinoma (HCC) represents a common neoplasm that presents a substantial worldwide health challenge. Nevertheless, the involvement of HPN-AS1 in HCC remains unknown. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was utilized to measure HPN-AS1 expression in HCC. The GABPA effects on the HPN-AS1 promoter were analyzed through chromatin immunoprecipitation and luciferase reporter assays. Cell proliferation potential was determined by deploying CCK-8 assay, Ki-67 immunofluorescence staining, and colony formation assay. Cell apoptosis was detected using acridine orange/ethidium bromide staining and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Western blotting was utilized to measure the protein levels of proliferation factors and apoptosis regulators. HPN-AS1 binding to eIF4A3 was accessed by RNA-binding protein immunoprecipitation assay. HPN-AS1 was significantly downregulated in both HCC cells and tissues. Lower HPN-AS1 levels indicate a poorer HCC prognosis. Moreover, we found that GABPA functions as a transcription factor for HPN-AS1. Functional studies revealed that HPN-AS1 displayed inhibitory effects on HCC cell proliferation and promoted apoptosis. Mechanically, HPN-AS1 bound to and facilitated translation initiation factor eIF4A3 degradation. Loss of HPN-AS1 augmented eIF4A3 protein levels rather than eIF4A3 mRNA levels. Exogenous expression of eIF4A3 could restore eIF4A3 protein levels and reverse HPN-AS1 overexpression-induced cell proliferation inhibition and cell apoptosis. Our study elucidated that HPN-AS1 downregulation was mediated by GABPA. HPN-AS acted as a tumor suppressor within HCC through binding and facilitating eIF4A3 degradation. The study provides a novel insight into the biological function of HPN-AS1 in HCC, suggesting that HPN-AS1 could be a promising biomarker and a potential target for HCC diagnosis and treatment.

Abstract 6774: Establishment and optimization of an air liquid interface co-culture system to evaluate phenotypic changes in primary human Barrett's esophagus cells upon different environmental exposures

Abstract Esophageal adenocarcinoma (EAC) is a deadly cancer with an increasing incidence which arises out of it's precancerous precursor, termed Barrett’s esophagus (BE). A better understanding of the factors that influence BE progression is essential for early detection and novel therapeutics. 3D Air Liquid Interface (ALI) cultures provide an edge over traditional 2D cultures as they better mimic the in vivo conditions of the esophagus. The aim of the study was to establish conditions for a BE ALI model and to evaluate the phenotypic and molecular changes in primary human BE cells upon interaction with different types of esophageal fibroblasts (EF) and exposure to bile acids(BA) (a major component of gastroesophageal reflux). ALI conditions that allowed growth of both primary BE and fibroblast for at least 30 days were optimized. Non dysplastic BE cells from two patients were utilized with one having EAC (P) and the other with no history of progression (NP). The BE cells were cultured in our ALI model with 3 types of EFs: normal esophageal fibroblasts(NFs), BE associated fibroblasts(BFs) and EAC associated fibroblasts(CFs). To determine the effect of BA exposure, ALI cultures underwent daily 1 hour exposure to bile salts. To determine if BE cells that lacked p53 responded differently to BA exposure, CRISPR Cas9 was utilized to knockout TP53. ALI cultures were conducted for 28 days. Phenotypic differences were determined by H&E staining and immunofluorescence (IF). Changes in cell proliferation was evaluated by Ki67 IF. Successful ALI cultures were validated by positive expression of laminin, pan-cytokeratin, Muc2 and Muc5ac by IF. Upon co-culture, we observed opposing histologic changes in the BE cells from the P and NP patients. The NP BE cells had a less complex structure upon culture with the BFs and CFs compared to NFs. The P BE cells showed the development of a complex morphology upon culture with the CFs compared to the BFs and the NFs. In both patients, we observed an increase in Ki67 positivity with fibroblast co culture; NFs (NP: 6.91%, p=0.070, P: 10.6%, p=0.001), BFs (NP: 12.49%, p=0.0001, P: 11.196%, p=0.003) and CFs (NP: 19.88%, p<0.0001, P:14.63 %, p=0.0001) compared to no fibroblasts (NP = 5.32%, P= 7.25% Ki67+). Co-culture with CFs increased Ki67 staining compared to either NFs or BFs (p=0.007,0.014). Exposure to BA showed a thinning but intact BE epithelial surface in both patients with and without TP53 knockout. Overall, primary BE cells and fibroblasts can be maintained long term in an ALI culture and co-culture with fibroblasts drive increased proliferation in BE cells. The histology changes observed in NP and P cell could be an indication of possible activation and interaction of differential signaling pathways. Ongoing studies aim to identify these pathways and determine transcriptional changes due to co-culture and BA exposure. Citation Format: Annesha Chatterjee, Jordana Maria Azevedo-Martins, Chira Chen Tanyolac, Qurat-Ul Ain, Hui Yu, Sophie Camilleri-Broet, Lorenzo Ferri, Philippe Gascard, Thea D. Tlsty, Matthew D. Stachler. Establishment and optimization of an air liquid interface co-culture system to evaluate phenotypic changes in primary human Barrett's esophagus cells upon different environmental exposures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6774.

Validation of hyperthermia as an enhancer of chemotherapeutic efficacy: insights from a bladder cancer organoid model

Objective This study aimed to evaluate the combined efficacy of hyperthermia and chemotherapy using a bladder cancer organoid model and to explore hyperthermia-related molecular pathways. Method Tumor organoids were generated by embedding RT4 bladder cancer cells into Matrigel. The resulting organoids were treated with pirarubicin or gemcitabine at 37 °C or 42 °C. Proliferation was determined by Ki67 immunofluorescence staining, and apoptosis was assessed using a TdT-mediated dUTP nick end labeling (TUNEL) assay. RNA sequencing was used to identify the differentially expressed genes. Results Bladder cancer organoids were successfully established and exhibited robust proliferative abilities. Treatment with gemcitabine or pirarubicin under hyperthermic conditions caused pronounced structural damage to the organoids and increased cell death compared to that in the normothermically treated group. Furthermore, Ki67 labeling and TUNEL assays showed that the hyperthermia chemotherapy group showed a significantly reduced proliferation rate and high level of apoptosis. Finally, RNA sequencing revealed the IFN-γ signaling pathway to be associated with hyperthermia. Conclusion Overall, hyperthermia combined with chemotherapy exerted better therapeutic effects than those of normothermic chemotherapy in grade 1-2 non-muscle-invasive bladder cancer, potentially through activation of the IFN-γ-JAK-STAT pathway.

S-propargyl-cysteine promotes the stability of atherosclerotic plaque via maintaining vascular muscle contractile phenotype.

Introduction: Plaque rupture in atherosclerosis contributes to various acute cardiovascular events. As a new sulfide-containing donor, S-propargyl-cysteine (SPRC) has been reported to play a beneficial role in cardioprotection, potentially through its anti-inflammatory, anti-oxidative and anti-atherogenic activities. Our previous study observed an increase in eNOS phosphorylation in endothelial cells. However, it remains unclear whether SPRC influences vascular smooth muscle cells (VSMCs) within the plaque and if this effect contributes to plaque stabilization. Methods: An atherosclerotic unstable plaque mouse model was established by subjecting ApoE-/- mice to tandem stenosis of the right carotid artery along with a Western diet. Daily SPRC administration was conducted for 13weeks. Plaque morphology and stability were assessed using MRI scanning and histopathological staining. In our in vitro studies, we stimulated human artery vascular smooth muscle cells (HAVSMCs) with platelet-derived growth factor-BB (PDGF-BB), both with and without 100μM SPRC treatment. Cell phenotype was assessed using both Western blot and Real-time PCR. Cell proliferation was assessed using the BrdU cell proliferation kit and immunofluorescence of Ki-67, while cell migration was measured using scratch wound healing and transwell assay. MiR-143-3p overexpression and knockdown experiments were used to investigate whether it mediates the effect of SPRC on VSMC phenotype. Results and Discussion: SPRC treatment reduced plasma lipid levels, increased collagen content and decreased cell apoptosis in atherosclerotic plaques, indicating improved plaque stability. Both in vivo and in vitro studies elucidated the role of SPRC in preserving the contractile phenotype of VSMCs through up-regulation of miR-143-3p expression. Furthermore, SPRC suppressed the pro-proliferation and pro-migration effects of PDGF-BB on HAVSMCs. Overall, these findings suggest that the inhibitory effect of SPRC on phenotype switch from contractile to synthetic VSMCs may contribute to its beneficial role in enhancing plaque stability.

Piezo1-ERK1/2-YAP Signaling Cascade Regulates the Proliferation of Urine-derived Stem Cells on Collagen Gels.

Urine-derived stem cells (USCs) were considered to be an ideal source of stem cells for repairing urological diseases. However, the proliferative ability of USCs significantly decreased when cultured on plastic dishes, which limited their clinical application. It was found that collagen gels could promote the proliferation of USCs, but the underlying molecular mechanisms were unclear. The study aims to investigate the role of the mechanically activated cation channel Piezo1 and the transcriptional coactivator YAP in the regulation of proliferation of USCs on collagen gels. USCs were cultured on collagen gels (group COL), or plastic dishes (group NON). MTT assay, Scratch assay, EDU staining, and immunofluorescence (IF) of Ki67 were performed to evaluate the proliferation of USCs; IF of YAP was conducted to observe its nuclear localization; calcium imaging experiment was executed to evaluate the function of Piezo1; western blot was used to compare changes in protein expression of YAP, LATS1, ERK1/2, and p-ERK1/2. In addition, the regulatory effect of YAP on the proliferative capacity of USCs was confirmed by intervening YAP with its inhibitor verteporfin (VP); and the inhibitor or activator of Piezo1, GsMTx4 or Yoda1 was used to explore the effect of Piezo1 on the nuclear localization of YAP, the proliferation of USCs and the regeneration of injured bladder. The results showed that cell proliferation was significantly enhanced in USCs in the COL group with the nuclear accumulation of YAP compared with the NON group and VP attenuated these effects. The expression and function of Piezo1 were higher in the COL group compared with the NON group. Blockage of Piezo1 by GsMTx4 decreased nuclear localization of YAP, the proliferation of USCs, and caused the failure of bladder reconstruction. Activation of Piezo1 by Yoda1 increased the nuclear expression of YAP, and the proliferation of USCs, which further improved the regeneration of the injured bladder. Finally, the ERK1/2 rather than LATS1 was revealed to participate in the Piezo1/YAP signal cascades of USCs proliferation. Taken together, Piezo1-ERK1/2-YAP signal cascades were involved in regulating the proliferation ability of USCs in collagen gels which would be beneficial for the regeneration of the bladder.

Effects of Photodynamic Therapy Using 5 -Aminolevulinic Acid (ALA) Loaded Acrylic Nanoparticles (ANPs) on HaCaT Cells.

ALA-PDT (5-aminolevulinic acid photodynamic therapy) is a central modality in the treatment of skin diseases. Increasing the bioavailability of ALA remains a critical issue. With this in mind, our study explores a novel route of ALA delivery by loading acrylic nanoparticles (ANPs). ALA-ANPs were synthesized by emulsion polymerisation and characterised by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The effects of ALA-ANPs on HaCaT cell line were evaluated, including characteristics, morphological changes, protoporphyrin IX (PpIX) fluorescence kinetics, reactive oxygen species (ROS) levels, mitochondrial membrane potential and ki67 expression in these cells. The ANPs had uniform sizes, smooth surfaces and excellent light transmittance, with diameters of 150-200 nm. In contrast, the ALA - ANPs had uneven surfaces and poor light transmittance, with diameters of 220-250 nm. During 12 hours of co-incubation of HaCaT cells with ALA, the intracellular accumulation of PpIX increased over time. Notably, after 6 hours of incubation, PpIX levels induced by 1.81 mg/mL ALA-ANPs exceeded those induced by 1.0 mM ALA (p < 0.01). CCK-8 results showed a positive correlation between PDT-induced inhibition of HaCaT cell proliferation and ALA concentration when ALA concentration remained below 2.0 mM. Compared to the 1.0 mM ALA group, the 1.81 mg/mL ALA-ANPs group showed decreased mitochondrial membrane potential, ki67 immunofluorescence intensity and cell proliferation. In contrast, ROS levels were significantly increased in the 1.81 mg/mL ALA-ANPs group (p < 0.01). Loading ANPs provide improved stability and potency for ALA. The ALA-ANPs-PDT approach has superior inhibitory effects on HaCaT proliferation in vitro.

Taurine Improved Autism-Like Behaviours and Defective Neurogenesis of the Hippocampus in BTBR Mice through the PTEN/mTOR/AKT Signalling Pathway.

Effective treatment of patients with autism spectrum disorder (ASD) is still absent so far. Taurine exhibits therapeutic effects towards the autism-like behaviour in ASD model animals. Here, we determined the mechanism of taurine effect on hippocampal neurogenesis in genetically inbred BTBR T+ tf/J (BTBR) mice, a proposed model of ASD. In this ASD mouse model, we explored the effect of oral taurine supplementation on ASD-like behaviours in an open field test, elevated plus maze, marble burying test, self-grooming test, and three-chamber test. The mice were divided into four groups of normal controls (WT) and models (BTBR), who did or did not receive 6-week taurine supplementation in water (WT, WT+ Taurine, BTBR, and BTBR+Taurine). Neurogenesis-related effects were determined by Ki67 immunofluorescence staining. Western blot analysis was performed to detect the expression of phosphatase and tensin homologue deleted from chromosome 10 (PTEN)/mTOR/AKT pathway-associated proteins. Our results showed that taurine improved the autism-like behaviour, increased the proliferation of hippocampal cells, promoted PTEN expression, and reduced phosphorylation of mTOR and AKT in hippocampal tissue of the BTBR mice. In conclusion, taurine reduced the autism-like behaviour in partially inherited autism model mice, which may be associa-ted with improving the defective neural precursor cell proliferation and enhancing the PTEN-associated pathway in hippocampal tissue.

Activation of PI3K/AKT/mTOR signaling axis by UBE2S inhibits autophagy leading to cisplatin resistance in ovarian cancer

BackgroundEpithelial ovarian cancer (OC) is the fourth leading cause of cancer-related deaths in women, with a 5-year survival rate of 30%-50%. Platinum resistance is the chief culprit for the high recurrence and mortality rates. Several studies confirm that the metabolic regulation of ubiquitinating enzymes plays a vital role in platinum resistance in OC.MethodsIn this study, we selected ubiquitin-conjugating enzyme E2S (UBE2S) as the candidate gene for validation. The levels of UBE2S expression were investigated using TCGA, GTEx, UALCAN, and HPA databases. In addition, the correlation between UBE2S and platinum resistance in OC was analyzed using data from TCGA. Cisplatin-resistant OC cell lines were generated and UBE2S was knocked down; the transfection efficiency was verified. Subsequently, the effects of knockdown of UBE2S on the proliferation and migration of cisplatin-resistant OC cells were examined through the CCK8, Ki-67 immunofluorescence, clone formation, wound healing, and transwell assays. In addition, the UBE2S gene was also validated in vivo by xenograft models in nude mice. Finally, the relationship between the UBE2S gene and autophagy and the possible underlying regulatory mechanism was preliminarily investigated through MDC and GFP-LC3-B autophagy detection and western blotting experiments. Most importantly, experimental validation of mTOR agonist reversion (the rescuse experiments) was also performed.ResultsUBE2S was highly expressed in OC at both nucleic acid and protein levels. The results of immunohistochemistry showed that the level of UBE2S expression in platinum-resistant samples was significantly higher relative to the platinum-sensitive samples. By cell transfection experiments, knocking down of the UBE2S gene was found to inhibit the proliferation and migration of cisplatin-resistant OC cells. Moreover, the UBE2S gene could inhibit autophagy by activating the PI3K/AKT/mTOR signaling pathway to induce cisplatin resistance in OC in vivo and in vitro.ConclusionIn conclusion, we discovered a novel oncogene, UBE2S, which was associated with platinum response in OC, and examined its key role through bioinformatics and preliminary experiments. The findings may open up a new avenue for the evaluation and treatment of OC patients at high risk of cisplatin resistance.

Platelet Lysate Promotes Proliferation and Angiogenic Activity of Dental Pulp Stem Cells via Store-operated Ca2+ Entry

AimsDental pulp stem cells (DPSCs) had been widely used in nerve tissue engineering. However, the low cell survival and invalid differentiation were urgent problems to be solved for cell-based therapy. Platelet lysate (PL) had attracted attentions for its good biocompatibility, low side effects and abundant growth factors. In this study, we aimed to evaluate the effect and mechanism of PL on the proliferation and angiogenesis of DPSCs. Materials and methodsIn our study, PL was prepared by repeated freeze-thaw methods. The effect of PL on the viability of DPSCs was screened by the Cell Counting Kit-8 (CCK-8) and Live/Dead assays. Then the store-operated Ca2+ entry (SOCE) agonist endothelin-1 (ET-1) and inhibitor 2-aminoethoxydiphenyl borate (2-APB) were used to clarify the biological functions of PL on DPSCs. We detected the mitosis of DPSCs by KI67 immunofluorescence staining. Moreover, Ca2+ influx in DPSCs was evaluated by fluorescence microscopy and a flow cytometry. The expression of p-SRC, VEGFA and CD31 as well as the number of formed tubules in each group were investigated, so as to further reveal the influence of PL on the DPSCs angiogenic activity. ResultsAs for CCK-8 and Live/Dead assays, the results indicated the 0.5% concentration of PL was better than that of 20ng/ml bFGF and 10% FBS in promoting cell survival within 24hours. KI67 staining showed that 0.5% PL promoted SOCE and cell proliferation. The expression levels of SRC phosphorylation (p-SRC) were increased in the 0.5% PL and ET-1 + 0.5% PL groups, as well as the expressions of endothelial markers CD31 and VEGFA were also increased in the ET-1, 0.5% PL and ET-1 + 0.5% PL groups. However, CD31 and VEGFA were not detected in 2-APB and 2-APB + 0.5% PL groups. After angiogenesis induction, capillary-like structures were observed in ET-1, 0.5% PL and ET-1 + 0.5% PL groups, whereas no vascular structures were observed in 2-APB and 2-APB + 0.5% PL groups. ConclusionOur results indicated that PL promoted the proliferation and vessel-formation of DPSCs via the activation of SOCE and the upregulation of p-SRC and VEGFA expressions, respectively. PL might act as a promising cell supplement in DPSCs survival and differentiation, furtherly applied for the repair and regeneration of peripheral nerve injury.

Distinctive Roles of Nitric Oxide Synthase Isoforms in Hydroxyurea-Mediated Cytostasis of Erythroid Progenitors

Chemotherapeutic hydroxyurea (HU) is used in the therapy of sickle cell anemia, thalassemia and myeloproliferative neoplasms according to its stimulation of fetal hemoglobin and myelosuppression. We reported that most nitric oxide (NO) in mammals is produced by the reduction of nitrate and nitrite. The NO properties of HU have been widely explored, while its enzymatic NO synthase (NOS) dependence is not completely defined. We have already demonstrated concomitant inhibition of erythroid cell proliferation by HU and NO donors. In this study, we investigated the NOS-mediation in HU regulation of proliferation, differentiation and cell cycle in HEL92.1.7 human erythroleukemic cell line and / or erythroid progenitors of Nos2- or Nos3-null mice. HU induced a dose-dependent increase in NOS2 and NOS3 protein levels and their enzymatic activity by increasing production of NO and citrulline in erythroleukemic cells. Inhibition of the NOS enzymes by pan-specific L-NAME, NOS2 inhibitor 1400W, or NOS2/NOS3 inhibitor DPI was sufficient to prevent the HU-mediated decrease in HEL92.1.7 proliferation assessed by Ki67 immunofluorescence. HU increased the frequency of cells in S-phase (66.58±2.44 vs. 55.18±1,06%) of the cycle at the expense of G0/G1 (17.07±1.67 vs. 28.55±1.01%) due to blocked DNA synthesis. The combined treatment of HU with 10μM DPI decreased the number of cells in G0/G1 (3.34±0.98 vs. 17.07±1.67%) and increased in S (72.3±0.96 vs. 66.58±%) and G2/M (24.36±0.19 vs. 16.35±2.28%) compared with HU alone. Moreover, NOS3 knock-down by shRNA decreased the percentage of cells in S phase from 69,19±2.68 to 34.23±1.07% (p&amp;lt;0.001) and increased in G2/M from 15.62±2.17 to 34.71±3.76 (p&amp;lt;0.001). HU-induced apoptosis was prevented by NOS2 (1400W) inhibition, determined by annexin V, decreasing the number of cells in early and late apoptosis in erythroleukemic cells. In accordance, NOS3 knock-down was not able to rescue HU-induced apoptosis. Furthermore, by performing in vitro enzymatic assay we showed that regulation of NOS2 activity seems to be achieved by direct interaction with HU leading to a dose- and time-dependent increase in NO and citrulline products. HU increased the expression of Nos1/2 isoforms by 20%, and Nos3 by 10% in mouse erythroid progenitors (mERP) of wild-type mice. Bone marrow cells isolated from Nos2- or Nos3-null mice treated orally with HU showed decreased protein nitrosylation in bone marrow compared to HU-treated wild-type mice. In ex vivo colony formation assay, both Nos2-/- and Nos3-/- bone marrow cells formed significantly more late erythroid colonies (CFU-E, 34- and 24-fold, respectively, p&amp;lt;0.001), and Nos2-/- also yielded more of the early erythroid (BFU-E, 9- and 7-fold, p&amp;lt;0.01) and granulocyte/macrophage (CFU-GM, 14- and 15-fold, p&amp;lt;0.01) colonies compared to wild-type mice untreated or HU-treated. HU treatment of mERP, of Nos2-/- and Nos3-/- origin, demonstrated decreased frequency of S-phase cells to levels comparable to untreated wild-type mice. Nos2 and Nos3 deficiency was able to rescue Caspase-3 expression in the presence of HU in mERP. Nevertheless, HU increased the quantity of late apoptotic cells in both Nos2 and Nos3 deficient mice. Our results demonstrated that HU induces the enzymatic activity of NOS proteins that mediate the cytostatic effect. NOS2/3 enzymes are involved in HU-inhibition of proliferation of human erythroleukemic cells and ex vivo differentiation of mERP. The observed NOS enzymes were fully and partially involved in HU-induced apoptosis of HEL92.1.7 cells and mERP, respectively. Uncovering all aspects of HU mechanism of action may help to increase the responsiveness or reduce the negative effects of cytotoxic chemotherapy.