Alter Cell Behavior Research Articles

4-aminopyridine attenuates inflammation and apoptosis and increases angiogenesis to promote skin regeneration following a burn injury in mice

Severe thermal skin burns are complicated by inflammation and apoptosis, which delays wound healing and contributes to significant morbidity. Diverse treatments demonstrate limited success in mitigating these processes to accelerate healing. Agents that alter cell behavior to improve healing would alter treatment paradigms. We repurposed 4-aminopyridine (4-AP), a drug approved by the US FDA for multiple sclerosis, to treat severe burns in mice (10-week-old C57BL/6 J male mice weighing 25 ± 3 g). We found that 4-AP, in the early stages of burn healing, significantly reduced the expression of pro-inflammatory cytokines IL1β and TNFα while increasing the expression of anti-inflammatory markers CD206, ARG-1, and IL10. We demonstrated increased intracellular calcium effects of 4-AP through Orai1-pSTAT6 signaling, where 4-AP significantly mitigated inflammatory effects by promoting M2 macrophage differentiation in in-vitro macrophages and post-skin burn tissues. 4-AP attenuated apoptosis, with decreases in apoptotic markers BAX, caspase-9, and caspase-3 and increases in anti-apoptotic markers BCL2 and BCL-XL. Furthermore, 4-AP promoted angiogenesis through increases in the expression of CD31, VEGF, and eNOS. Together, these likely contributed to accelerated burn wound closure, as demonstrated in increased keratinocyte proliferation (K14) and differentiation (K10) markers. In the later stages of burn healing, 4-AP increased TGFβ and FGF levels, which are known to mark the transformation of fibroblasts to myofibroblasts. This was further demonstrated by an increased expression of α-SMA and vimentin, as well as higher levels of collagen I and III, MMP 3, and 9 in mice treated with 4-AP. Our findings support the idea that 4-AP may have a novel, clinically relevant therapeutic use in promoting burn wound healing.

Enhancing Cell Aggregation and Migration via Double-Click Cross-Linking with Azide-Modified Hyaluronic Acid.

We present a novel approach to the formation of cell aggregates by employing click chemistry with water-soluble zwitterionic dibenzo cyclooctadiyne (WS-CODY) and azide-modified hyaluronic acid (HA-N3) as a linker to facilitate rapid and stable cell aggregation. By optimizing the concentrations of HA-N3 and WS-CODY, we achieved efficient cross-linking between azide-modified cell surfaces and HA-N3, generating cell aggregates within 10 min, and the resulting aggregates remained stable for up to 5 days, with cell viability maintained at approximately 80%. Systematic experiments revealed that a stoichiometric balance between HA-N3 and WS-CODY is important for effective cross-linking, highlighting the roles of both cell-surface azide modification and HA in the aggregate formation. We also investigated the genetic basis of altered cell behavior within these aggregates. Transcriptome analysis (RNA-seq) of aggregates postcultivation revealed a marked fluctuation of genes associated with 'cell migration' and 'cell adhesion', including notable changes in the expression of HYAL1, ICAM-1, CEACAM5 and RHOB. These findings suggest that HA-N3-mediated cell aggregation can induce intrinsic cellular responses that not only facilitate cell aggregate formation but also modulate cell-matrix interactions. We term this phenomenon 'chemo-resilience', The simplicity and efficacy of this click chemistry-based approach suggest it may have broad applicability for forming cell aggregates and modulating cell-matrix interactions in tissue engineering and regenerative medicine.

4-aminopyridine attenuates inflammation and apoptosis and increases angiogenesis to promote skin regeneration following a burn injury.

Severe thermal skin burns are complicated by inflammation and apoptosis, which delays wound healing and contributes to significant morbidity. Diverse treatments demonstrate limited success with mitigating these processes to accelerate healing. Agents that alter cell behavior to improve healing would alter treatment paradigms. We repurposed 4-aminopyridine (4-AP), a drug approved by the US FDA for multiple sclerosis, to treat severe burns. We found that 4-AP, in the early stages of burn healing, significantly reduced the expression of pro-inflammatory cytokines IL1β and TNFα while increasing the expression of anti-inflammatory markers CD206, ARG-1, and IL10. 4-AP attenuated apoptosis, with decreases in apoptotic markers BAX, caspase-9, and caspase-3 and increases in anti-apoptotic markers BCL2 and BCL-XL. Furthermore, 4-AP promoted angiogenesis through increases in the expression of CD31, VEGF, and eNOS. Together, these likely contributed to accelerated burn wound closure, as demonstrated in increased keratinocyte proliferation (K14) and differentiation (K10) markers. In the later stages of burn healing, 4-AP increased TGFβ and FGF levels, which are known to mark the transformation of fibroblasts to myofibroblasts. This was further demonstrated by an increased expression of α-SMA and vimentin, as well as higher levels of collagen I and III, MMP 3, and 9 in animals treated with 4-AP. Our findings support the idea that 4-AP may have a novel, clinically relevant therapeutic use in promoting burn wound healing.

Role of Emerin in regulating fibroblast differentiation and migration at the substrate of stiffness coupled topology.

In hypertrophic scars, the differentiation and migration of fibroblasts are influenced by the extracellular matrix microenvironment, which includes factors such as stiffness, restraint, and tensile force. These mechanical stresses incite alterations in cell behavior, accompanied by cytoskeletal protein reorganization. However, the role of nucleo-skeletal proteins in this context remains underexplored. In this study, we use a polyacrylamide hydrogel (PAA) to simulate the mechanical stress experienced by cells in scar tissue and investigate the impact of Emerin on cell behavior. We utilize atomic force microscopy (AFM) and RNA interference technology to analyze cell differentiation, migration, and stiffness. Our findings reveal that rigid substrates and cellular restriction elevate Emerin expression and diminish differentiation. Conversely, reducing Emerin expression leads to attenuated cell differentiation, where stiffness and constraining factors exert no notable influence. Furthermore, a softening of cells and an enhanced migration rate are also markedly observed. These observations indicate that variations in nuclear skeletal proteins, prompted by diverse matrix microenvironments, play a pivotal role in the pathogenesis of hypertrophic scars (HSs). This research offers novel insights and a reference point for understanding scar fibrosis formation mechanisms and preventing fibrosis.

The chondrocyte "mechanome": Activation of the mechanosensitive ion channels TRPV4 and PIEZO1 drives unique transcriptional signatures.

The mechanosensitive ion channels Transient Receptor Potential Vanilloid 4 (TRPV4) and PIEZO1 transduce physiologic and supraphysiologic magnitudes of mechanical signals in the chondrocyte, respectively. TRPV4 activation promotes chondrogenesis, while PIEZO1 activation by supraphysiologic deformations drives cell death. The mechanisms by which activation of these channels discretely drives changes in gene expression to alter cell behavior remain to be determined. To date, no studies have contrasted the transcriptomic response to activation of these channels nor has any published data attempted to correlate these transcriptomes to alterations in cellular function. This study used RNA sequencing to comprehensively investigate the transcriptomes associated with activation of TRPV4 or PIEZO1, revealing that TRPV4 and PIEZO drive distinct transcriptomes and also exhibit unique co-regulated clusters of genes. Notably, activation of PIEZO1 through supraphysiologic deformation induced a transient inflammatory profile that overlapped with the interleukin (IL)-1-responsive transcriptome and contained genes associated with cartilage degradation and osteoarthritis progression. However, both TRPV4 and PIEZO1 were also shown to elicit anabolic effects. PIEZO1 expression promoted a pro-chondrogenic transcriptome under unloaded conditions, and daily treatment with PIEZO1 agonist Yoda1 significantly increased sulfated glycosaminoglycan deposition invitro. These findings emphasize the presence of a broad "mechanome" with distinct effects of TRPV4 and PIEZO1 activation in chondrocytes, suggesting complex roles for PIEZO1 in both the physiologic and pathologic responses of chondrocytes. The identification of transcriptomic profiles unique to or shared by PIEZO1 and TRPV4 (distinct from IL-1-induced inflammation) could inform future therapeutic designs targeting these channels for the management and treatment of osteoarthritis.

Single Nuclei Transcriptomics in Diabetic Mice Reveals Altered Brain Hippocampal Endothelial Cell Function, Permeability, and Behavior

Single Nuclei Transcriptomics in Diabetic Mice Reveals Altered Brain Hippocampal Endothelial Cell Function, Permeability, and Behavior

Noncanonical functions of telomerase and telomeres in viruses-associated cancer.

The cause of cancer is attributed to the uncontrolled growth and proliferation of cells resulting from genetic changes and alterations in cell behavior, a phenomenon known as epigenetics. Telomeres, protective caps on the ends of chromosomes, regulate both cellular aging and cancer formation. In most cancers, telomerase is upregulated, with the telomerase reverse transcriptase(TERT) enzyme and telomerase RNA component(TERC) RNA element contributing to the maintenance of telomere length. Additionally, it is noteworthy that two viruses, human papillomavirus (HPV) and Epstein-Barr virus (EBV), utilize telomerase for their replication or persistence in infected cells. Also, TERT and TERC may play major roles in cancer not related to telomere biology. They are involved in the regulation of gene expression, signal transduction pathways, cellular metabolism, or even immune response modulation. Furthermore, the crosstalk between TERT, TERC, RNA-binding proteins, and microRNAs contributes to a greater extent to cancer biology. To understand the multifaceted roles played by TERT and TERC in cancer and viral life cycles, and then to develop effective therapeutic strategies against these diseases, are fundamental for this goal. By investigating deeply, the complicated mechanisms and relationships between TERT and TERC, scientists will open the doors to new therapies. In its analysis, the review emphasizes the significance of gaining insight into the multifaceted roles that TERT and TERC play in cancer pathogenesis, as well as their involvement in the viral life cycle for designing effective anticancer therapy approaches.

Effects of syndecan-4 silencing on the extracellular matrix remodeling in anoikis-resistant endothelial cells.

Anoikis is a process of programmed cell death induced by the loss of cell/matrix interactions. In previous work, we have shown that the acquisition of anoikis resistance upregulates syndecan-4 (SDC4) expression in endothelial cells. In addition, SDC4 gene silencing by microRNA interference reverses the transformed phenotype of anoikis-resistant endothelial cells. Due to this role of SDC4 in regulating the behavior of anoikis-resistant endothelial cells, we have evaluated that the functional consequences of SDC4 silencing in the extracellular matrix (ECM) remodeling in anoikis-resistant rabbit aortic endothelial cells submitted to SDC4 gene silencing (miR-Syn4-Adh-1-EC). For this, we evaluated the expression of adhesive proteins, ECM receptors, nonreceptor protein-tyrosine kinases, and ECM-degrading enzymes and their inhibitors. Altered cell behavior was monitored by adhesion, migration, and tube formation assays. We found that SDC4 silencing led to a decrease in migration and angiogenic capacity of anoikis-resistant endothelial cells; this was accompanied by an increase in adhesion to fibronectin. Furthermore, after SDC4 silencing, we observed an increase in the expression of fibronectin, collagen IV, and vitronectin, and a decrease in the expression of integrin α5β1 and αvβ3, besides that, silenced cells show an increase in Src and FAK expression. Quantitative polymerase chain reactionand Western blot analysis demonstrated that SDC4 silencing leads to altered gene and protein expression of MMP2, MMP9, and HSPE. Compared withparental cells, SDC4 silenced cells showed a decrease in nitric oxide production and eNOS expression. In conclusion, these data demonstrate that SDC4 plays an important role in ECM remodeling. In addition, our findings represent an important step towardunderstanding the mechanism by which SDC4 can reverse the transformed phenotype of anoikis-resistant endothelial cells.

Simulated microgravity improves maturation of cardiomyocytes derived from human induced pluripotent stem cells

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) possess tremendous potential for basic research and translational application. However, these cells structurally and functionally resemble fetal cardiomyocytes, which is a major limitation of these cells. Microgravity can significantly alter cell behavior and function. Here we investigated the effect of simulated microgravity on hiPSC-CM maturation. Following culture under simulated microgravity in a random positioning machine for 7 days, 3D hiPSC-CMs had increased mitochondrial content as detected by a mitochondrial protein and mitochondrial DNA to nuclear DNA ratio. The cells also had increased mitochondrial membrane potential. Consistently, simulated microgravity increased mitochondrial respiration in 3D hiPSC-CMs, as indicated by higher levels of maximal respiration and ATP content, suggesting improved metabolic maturation in simulated microgravity cultures compared with cultures under normal gravity. Cells from simulated microgravity cultures also had improved Ca2+ transient parameters, a functional characteristic of more mature cardiomyocytes. In addition, these cells had improved structural properties associated with more mature cardiomyocytes, including increased sarcomere length, z-disc length, nuclear diameter, and nuclear eccentricity. These findings indicate that microgravity enhances the maturation of hiPSC-CMs at the structural, metabolic, and functional levels.

Cell behaviors that pattern developing tissues: the case of the vertebrate nervous system.

Morphogenesis from cells to tissue gives rise to the complex architectures that make our organs. How cells and their dynamic behavior are translated into functional spatial patterns is only starting to be understood. Recent advances in quantitative imaging revealed that, although highly heterogeneous, cellular behaviors make reproducible tissue patterns. Emerging evidence suggests that mechanisms of cellular coordination, intrinsic variability and plasticity are critical for robust pattern formation. While pattern development shows a high level of fidelity, tissue organization has undergone drastic changes throughout the course of evolution. In addition, alterations in cell behavior, if unregulated, can cause developmental malformations that disrupt function. Therefore, comparative studies of different species and of disease models offer a powerful approach for understanding how novel spatial configurations arise from variations in cell behavior and the fundamentals of successful pattern formation. In this chapter, I dive into the development of the vertebrate nervous system to explore efforts to dissect pattern formation beyond molecules, the emerging core principles and open questions.

Silencing of lncRNA LOC105376794 promotes migration, invasion, and Gefitinib resistance of lung adenocarcinoma cells with EGFR 19Del mutation by ATF4/CHOP axis and ERK phosphorylation.

Epidermal growth factor receptor (EGFR) gene exon 19 in-frame deletion (19del) and exon 21 L858R point mutation (21L858R mutation) are prevalent mutations in lung adenocarcinoma. Lung adenocarcinoma patients with 19del presented with a better prognosis than the 21L858R mutation under the same epidermal growth factor receptor tyrosine kinase inhibitor treatment. Our study aimed to uncover the expression of long non-coding RNA LOC105376794 between 19del and 21L858R mutation, and explore the mechanism that regulates cells' biological behavior and gefitinib sensitivity in lung adenocarcinoma cells with 19del. Transcriptome sequencing was conducted to identify differentially expressed lncRNAs between EGFR 19del and 21L858R mutation in serum through the DNBSEQ Platform. Protein-protein interaction network and Kyoto Encyclopedia of Genes and Genomes pathway were conducted to analyze the relationship between lncRNAs and mRNAs through STRING and Dr. TOM. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression of lncRNA LOC105376794 in serum and cells. Loss-of-function experiments were used to validate the biological function and gefitinib sensitivity of LOC105376794 in lung adenocarcinoma cells. Protein levels were detected by western blotting. Through transcriptome resequencing and RT-qPCR, we found the expression levels of LOC105376794 in serum were increased in the 19del group compared with the 21L858R mutation group. Inhibition of LOC105376794 promoted proliferation, migration and invasion, and reduced apoptosis of HCC827 and PC-9 cells. The low expression of LOC105376794 reduced gefitinib sensitivity in PC-9 cells. Mechanistically, we found that the knockdown of LOC105376794 suppressed activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) signaling pathway and facilitated the expression of extracellular signal-regulated kinase 1/2 (ERK) phosphorylation. LOC105376794 altered cell biological behavior and gefitinib sensitivity of lung adenocarcinoma cells with 19del through the ATF4/CHOP signaling pathway and the expression of ERK phosphorylation. The results further illustrated the fact that lung adenocarcinoma patients with 19del presented with a more favorable clinical outcome and provided a theoretical basis for treatment strategy for lung adenocarcinoma patients with 19del.

Priming chondrocytes during expansion alters cell behavior and improves matrix production in 3D culture

ObjectiveCartilage tissue engineering strategies that use autologous chondrocytes require in vitro expansion of cells to obtain enough cells to produce functional engineered tissue. However, chondrocytes dedifferentiate during expansion culture, limiting their ability to produce chondrogenic tissue and their utility for cell-based cartilage repair strategies. The current study identified conditions that favor cartilage production and the mechanobiological mechanisms responsible for these benefits. DesignChondrocytes were isolated from juvenile bovine knee joints and cultured with (primed) or without (unprimed) a growth factor cocktail. Gene expression, cell morphology, cell adhesion, cytoskeletal protein distribution, and cell mechanics were assessed. Following passage 5, cells were embedded into agarose hydrogels to evaluate functional properties of engineered cartilage. ResultsPriming cells during expansion culture altered cell phenotype and chondrogenic tissue production. Unbiased RNA-sequencing analysis suggested, and experimental studies confirmed, that growth factor priming delays dedifferentiation associated changes in cell adhesion and cytoskeletal organization. Priming also overrode mechanobiological pathways to prevent chondrocytes from remodeling their cytoskeleton to accommodate the stiff, monolayer microenvironment. Passage 1 primed cells deformed less and had lower YAP1 activity than unprimed cells. Differences in cell adhesion, morphology, and cell mechanics between primed and unprimed cells were mitigated by passage 5. ConclusionsPriming suppresses mechanobiologic cytoskeletal remodeling to prevent chondrocyte dedifferentiation, resulting in more cartilage-like tissue-engineered constructs

STEM CELLS HOMEOSTASIS AND AGING

Abstract Adult (tissue) stem cells support tissue homeostasis and repair throughout the life of an individual. Numerous changes occur with age that result in altered stem cell behavior and reduced tissue maintenance and regeneration. Changes can be cell autonomous including changes in cell cycle progression, decreased bioenergetic efficiency, increased DNA damage, and epigenetic alterations. In addition, poorly understood changes to the local and systemic environments occur that result in decreased stem cell activity or alterations in commitment or differentiation potential. This symposium will cover the impact of aging on multiple adult stem cell systems, including hematopoietic stem cells (HSCs), intestinal stem cells (ISCs), and neural stem cells.

Combined Single-Cell and Spatial Transcriptomics Unveil the Complex Organization of the Non-Immune Human Bone Marrow Microenvironment during Aging

Hematopoietic stem cells (HSCs), reside in a specialized bone marrow (BM) microenvironment known as the hematopoietic stem niche. Despite the critical role of the niche in tightly controlling the processes of normal and malignant hematopoiesis, its cellular composition remains only partially understood. Recent studies, including our own, using single-cell RNA sequencing (scRNA-seq) approaches have provided valuable insights into the profiling of the human BM niche. However, a comprehensive effort to describe the cellular heterogeneity and regulatory circuitry of the aged BM microenvironment in elderly humans is still lacking. As individual age, multiple systems and organs experience a progressive loss of anatomical and physiological integrity. Aging of the HSC niche is accompanied by a reduction in the numbers and function of its constituents and a decrease in the levels of HSC-supporting factors. Whether niche aging can contribute to the defects observed in aged hematopoiesis, such as the clonal shift towards myelopoiesis, the decrease in immune surveillance, or age-associated metabolic diseases, remains unresolved. To address this, we utilized scRNA-seq and spatial transcriptomics to provide a detailed characterization of the molecular landscape and stromal interactions in the aged non-immune BM microenvironment. First, we performed scRNA-seq profiling on fluorescence-activated cell sorting-purified endothelial cells (ECs, TO-PRO-3 -/CD45 -/CD235 -/Lin -/CD31 +/CD9 +) and mesenchymal stromal cells (MSCs, TO-PRO-3 -/CD45 -/CD235 -/Lin -/CD271 +) from human BM samples of young (n=4) and elderly healthy donors (n=5). We analyzed a total of 1514 ECs and 3848 MSCs from older adults, grouped into 7 and 10 subclusters, respectively, defining distinct functional cell states. ECs and MSCs cells from young individuals were annotated using SingleR, utilizing the gene signature of each functional state in the elderly as a reference. Our results revealed significant shifts in the distribution of the functional states of BM niche cells during aging. Regarding ECs, we noticed reduced pathways associated with the cell cycle and RNA transcription pathways, indicating impaired cell cycle activity, coupled with decreased antioxidant defense (Figure 1A). In contrast, there was an increase in subgroups related to the response to foreign molecules, immune system activation, and vascular remodeling, suggesting an inflammatory response and increased vascular remodeling processes associated with aging. For MSCs, we observed a decline in osteogenesis and a reduction in the proportion of the early mesenchymal group related to immunity and the extracellular matrix (Figure 1B). These changes could contribute to the decreased ability of MSCs in elderly individuals to maintain HSCs function. On the contrary, we noticed increased adipogenic differentiation to the detriment of osteogenesis, which could be responsible for osteoporosis problems. Next, and to further explore the location and the direct interactions between niche cells, we are currently integrating spatially resolved transcriptomics with our scRNA-seq data to accurately comprehend the spatial distribution and interactions among the BM niche cells, using for the first time 10x Genomics Visium Spatial Gene Expression on bone BM tissue in humans. As a final validation step, we are using a mouse model to validate further the mechanisms of age-associated changes in the BM microenvironment. In summary, our results provide valuable insights into age-related transcriptional alterations in human BM ECs and MSCs, suggesting altered behavior of these niche cells during the aging of the hematopoietic system. This deeper understanding of the architecture of the aged hematopoietic system and its microenvironment offers the potential for developing novel therapeutic strategies preventing the detrimental effects of aging.

Single nuclei transcriptomics in diabetic mice reveals altered brain hippocampal endothelial cell function, permeability, and behavior

Type 2 diabetes mellitus (T2DM) is a metabolic disorder with cerebrovascular and cardiovascular sequelae. Yet, a clear pattern of gene dysregulation by T2DM in dementia has yet to be defined. We used single nuclei RNA sequencing technology to profile the transcriptome of endothelial cells (EC) from anatomically defined hippocampus of db/db mice to identify differentially expressed (DE) genes, gene pathways and networks, predicted regulating transcription factors, and targets of DE long noncoding RNAs. We also applied gadolinium (Gd) enhanced magnetic resonance imaging (MRI) to assess blood brain barrier (BBB) permeability, and functionally assessed cognitive behavior. The murine gene expression profiles were then integrated with those of persons with Alzheimer's disease (AD) and vascular dementia (VaD).We reveal that the transcriptome of the diabetic hippocampal murine brain endothelium differs substantially from control wild types with molecular changes characterized by differential RNA coding and noncoding pathways enriched for EC signaling and for endothelial functions for neuroinflammation, endothelial barrier disruption, and neurodegeneration. Gd enhanced structural brain MRI linked endothelial molecular alterations to BBB dysfunction by neuroimaging. Integrated multiomics of hippocampal endothelial gene dysregulation associated with impairments in cognitive adaptive capacity. In addition, the diabetic transcriptome significantly and positively correlated with that of persons with AD and VaD. Taken together, our results from comprehensive, multilevel, integrated, single nuclei transcriptomics support the hypothesis of T2DM-mediated neuroinflammation and endothelial cell and barrier disruption as key mechanisms in cognitive decline in T2DM, thereby suggesting potential endothelial-specific molecular therapeutic targets.

PTPRH promotes the progression of non-small cell lung cancer via glycolysis mediated by the PI3K/AKT/mTOR signaling pathway

BackgroundThe protein tyrosine phosphatase H receptor (PTPRH) is known to regulate the occurrence and development of pancreatic and colorectal cancer. However, its association with glycolysis in non-small cell lung cancer (NSCLC) is still unclear. In this study, we aimed to investigate the relationship between PTPRH expression and glucose metabolism and the underlying mechanism of action.MethodsThe expression of PTPRH in NSCLC cells was evaluated by IHC staining, qRT‒PCR and Western blotting. The effect of PTPRH on cell biological behavior was evaluated by colony assays, EdU experiments, Transwell assays, wound healing assays and flow cytometry. Changes in F-18-fluorodeoxyglucose (18F‐FDG) uptake and glucose metabolite levels after altering PTPRH expression were detected via a gamma counter and lactic acid tests. The expression of glycolysis-related proteins in NSCLC cells was detected by Western blotting after altering PTPRH expression.ResultsThe results showed that PTPRH was highly expressed in clinical patient tissue samples and closely related to tumor diameter and clinical stage. In addition, PTPRH expression was associated with glycometabolism indexes on 18F-FDG positron emission tomography/computed tomography (PET/CT) imaging, the expression level of Ki67 and the expression levels of glycolysis-related proteins. PTPRH altered cell behavior, inhibited apoptosis, and promoted 18F-FDG uptake, lactate production, and the expression of glycolysis-related proteins. In addition, PTPRH modulated the glycometabolism of NSCLC cells via the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, as assessed using LY294002 and 740Y-P (an inhibitor and agonist of PI3K, respectively). The same results were validated in vivo using a xenograft tumor model in nude mice. Protein expression levels of PTPRH, glycolysis-related proteins, p-PI3K/PI3K and p-AKT/AKT were measured by IHC staining using a subcutaneous xenograft model in nude mice.ConclusionsIn summary, we report that PTPRH promotes glycolysis, proliferation, migration, and invasion via the PI3K/AKT/mTOR signaling pathway in NSCLC and ultimately promotes tumor progression, which can be regulated by LY294002 and 740Y-P. These results suggest that PTPRH is a potential therapeutic target for NSCLC.Graphical

Cumulus expansion is impaired with advanced reproductive age due to loss of matrix integrity and reduced hyaluronan.

Reproductive aging is associated with ovulatory defects. Age-related ovarian fibrosis partially contributes to this phenotype as short-term treatment with anti-fibrotic compounds improves ovulation in reproductively old mice. However, age-dependent changes that are intrinsic to the follicle may also be relevant. In this study, we used a mouse model to demonstrate that reproductive aging is associated with impaired cumulus expansion which is accompanied by altered morphokinetic behavior of cumulus cells as assessed by time-lapse microscopy. The extracellular matrix integrity of expanded cumulus-oocyte complexes is compromised with advanced age as evidenced by increased penetration of fluorescent nanoparticles in a particle exclusion assay and larger open spaces on scanning electron microscopy. Reduced hyaluronan (HA) levels, decreased expression of genes encoding HA-associated proteins (e.g., Ptx3 and Tnfaip6), and increased expression of inflammatory genes and matrix metalloproteinases underlie this loss of matrix integrity. Importantly, HA levels are decreased with age in follicular fluid of women, indicative of conserved reproductive aging mechanisms. These findings provide novel mechanistic insights into how defects in cumulus expansion contribute to age-related infertility and may serve as a target to extend reproductive longevity.

The role of peroxidasin in solid cancer progression.

Peroxidasin is a heme-containing peroxidase enzyme that plays a vital role in the cross-linking of collagen IV molecules in basement membranes. Collagen IV cross-links are essential for providing structure and mechanical stability throughout tissue development, homeostasis, and wound healing. During cancer progression, the basement membrane is degraded, and proteins typically found in the basement membrane, including peroxidasin and collagen IV, can be found spread throughout the tumour microenvironment where they interact with cancer cells and alter cell behaviour. Whilst peroxidasin is reported to be up-regulated in a number of different cancers, the role that it plays in disease progression and metastasis has only recently begun to be studied. This review highlights the current literature exploring the known roles of peroxidasin in normal tissues and cancer progression, regulators of peroxidasin expression, and the reported relationships between peroxidasin expression and patient outcome in cancer.

Development and Characterization of an In Vitro Intestinal Model Including Extracellular Matrix and Macrovascular Endothelium.

In vitro intestinal models are used to study biological processes, drug and food absorption, or cytotoxicity, minimizing the use of animals in the laboratory. They usually consist of enterocytes and mucus-producing cells cultured for 3 weeks, e.g., on Transwells, to obtain a fully differentiated cell layer simulating the human epithelium. Other important components are the extracellular matrix (ECM) and strong vascularization. The former serves as structural support for cells and promotes cellular processes such as differentiation, migration, and growth. The latter includes endothelial cells, which coordinate vascularization and immune cell migration and facilitate the transport of ingested substances or drugs to the liver. In most cases, animal-derived hydrogels such as Matrigel or collagen are used as ECM in in vitro intestinal models, and endothelial cells are only partially considered, if at all. However, it is well-known that animal-derived products can lead to altered cell behavior and incorrect results. To circumvent these limitations, synthetic and modifiable hydrogels (Peptigel and Vitrogel) were studied here to mimic xenofree ECM, and the data were compared with Matrigel. Careful rheological characterization was performed, and the effect on cell proliferation was investigated. The results showed that Vitrogel exhibited shear-thinning behavior with an internal structure recovery of 78.9 ± 11.2%, providing the best properties among the gels investigated. Therefore, a coculture of Caco-2 and HT29-MTX cells (ratio 7:3) was grown on Vitrogel, while simultaneously endothelial cells were cultured on the basolateral side by inverse cultivation. The model was characterized in terms of cell proliferation, differentiation, and drug permeability. It was found that the cells cultured on Vitrogel induced a 1.7-fold increase in cell proliferation and facilitated the formation of microvilli and tight junctions after 2 weeks of cultivation. At the same time, the coculture showed full differentiation indicated by high alkaline phosphatase release of Caco-2 cells (95.0 ± 15.9%) and a mucus layer produced by HT29-MTX cells. Drug tests led to ex vivo comparable permeability coefficients (Papp) (i.e., Papp; antipyrine = (33.64 ± 5.13) × 10-6 cm/s, Papp; atenolol = (0.59 ± 0.16) × 10-6 cm/s). These results indicate that the newly developed intestinal model can be used for rapid and efficient assessment of drug permeability, excluding unexpected results due to animal-derived materials.

Frequent ZNF217 mutations lead to transcriptional deregulation of interferon signal transduction via altered chromatin accessibility in B cell lymphoma

Recent exome-wide studies discovered frequent somatic mutations in the epigenetic modifier ZNF217 in primary mediastinal B cell lymphoma (PMBCL) and related disorders. As functional consequences of ZNF217 alterations remain unknown, we comprehensively evaluated their impact in PMBCL. Targeted sequencing identified genetic lesions affecting ZNF217 in 33% of 157 PMBCL patients. Subsequent gene expression profiling (n = 120) revealed changes in cytokine and interferon signal transduction in ZNF217-aberrant PMBCL cases. In vitro, knockout of ZNF217 led to changes in chromatin accessibility interfering with binding motifs for crucial lymphoma-associated transcription factors. This led to disturbed expression of interferon-responsive and inflammation-associated genes, altered cell behavior, and aberrant differentiation. Mass spectrometry demonstrates that ZNF217 acts within a histone modifier complex containing LSD1, CoREST and HDAC and interferes with H3K4 methylation and H3K27 acetylation. Concluding, our data suggest non-catalytic activity of ZNF217, which directs histone modifier complex function and controls B cell differentiation-associated patterns of chromatin structure.