Proliferative Capacity Research Articles

Cardiomyocyte-Specific Overexpression of Activated Yes-Associated Protein Modified-RNA Promotes Cardiomyocyte Proliferation and Myocardial Regeneration.

The proliferative capacity of cardiomyocytes in adult mammalian hearts is far too low to replace the cells that are lost to myocardial infarction. Both cardiomyocyte proliferation and myocardial regeneration can be improved via the overexpression of a constitutively active variant of YAP5SA (Yes-associated protein, 5SA [active] mutant), but persistent overexpression of proliferation-inducing genes could lead to hypertrophy and arrhythmia, whereas off-target expression in fibroblasts and macrophages could increase fibrosis and inflammation. Transient overexpression of YAP5SA or GFP (green fluorescent protein; control) was targeted to cardiomyocytes via our cardiomyocyte-specific modified mRNA translation system (YAP5SACM-SMRTs or GFPCM-SMRTs, respectively). YAP5SA-cardiomyocyte specificity was confirmed via invitro experiments in cardiomyocytes and cardiac fibroblasts that had been differentiated from human induced- pluripotent stem cells and in human umbilical-vein endothelial cells, and the regenerative potency of YAP5SACM-SMRTs was evaluated in a mouse myocardial infarction model. In cultured human induced-pluripotent stem cells-cardiomyocytes, YAP was abundantly expressed for 3 days after YAP5SACM-SMRTs administration and was accompanied by increases in the expression of markers for proliferation, before declining to near-background levels after day 7, whereas GFP fluorescence remained high from days 1 to 3 after GFPCM-SMRTs treatment and then slowly declined. GFP fluorescence was also observed in human induced-pluripotent stem cells-cardiac fibroblasts and human umbilical-vein endothelial cells on day 1 after GFPCM-SMRTs administration but declined substantially by day 3. In the mouse myocardial infarction model, echocardiographic assessments of left-ventricular ejection fraction and fractional shortening were significantly greater, whereas infarct sizes were significantly smaller in YAP5SACM-SMRTs-treated mice than in vehicle-treated control animals, and YAP5SACM-SMRTs appeared to promote cardiomyocyte proliferation. The CM-SMRTs can be used to transiently and specifically overexpress YAP5SA in cardiomyocytes, and this treatment strategy significantly promoted cardiomyocyte proliferation and myocardial regeneration in a mouse myocardial infarction model.

Digoxin reduces senescence and restores dysfunctional endothelial-subcutaneous adipocyte cross-talk and dysregulated glucose homeostasis in patients with type 2 diabetes mellitus

Abstract Background Normal subcutaneous adipose tissue (SAT) function is dependent on SAT microvascular expansion. Heart failure with reduced ejection fraction (HF) and type 2 diabetes mellitus (DM) have a synergistic deleterious relationship and, of relevance to this, metabolic dysregulation may drive HF progression in DM. Chronic hyperinsulinemia is associated with adipocyte senescence, however any contributing role of SAT microvascular endothelial cell (SATMVEC) senescence on adverse microvascular expansion in type 2 diabetes mellitus (DM) remains unknown. Digoxin has been shown to have senolytic activity in murine cancer models. However, the potential therapeutic role of digoxin on SAT metabolic function is unexplored. Purpose We establish the presence of SATMVEC senescence and its effects on adipocyte function in people with HF and DM (HFDM). Moreover, we establish a therapeutic role for digoxin in targeting SATMVEC senescence, modulating SATMVEC-adipocyte crosstalk and restoring metabolic homeostasis. Methods We took pectoral SAT biopsies from 59 patients undergoing pacemaker implantation of whom 25 (42%) had HF and DM (HFDM). SATMVEC were isolated from SAT and were treated with 1ng/mL digoxin vs control for 24 hours before characterisation (cell function, expression of senescence-associated ß-galactosidase (ß-gal) and senescence associated secretory phenotype, SASP). Crosstalk between SATMVEC and subcutaneous white adipocytes were examined using co-culture techniques (Fig 1A). Paired digoxin vs control treated SATMVEC were seeded co-cultured with adipocytes for 24 hours, before adipocyte health was quantified, through 2-NBD-glucose uptake and expression of key adipogenic genes and proteins. Results Compared to HF alone, isolated SATMVEC from patients with HFDM had a senescent phenotype with increased SASP and ß-gal expression (p<0.05), reduced metabolic activity, ATP production, proliferative capacity, and impaired angiogenesis (p<0.05). In co-culture, SATMVEC from patients with HFDM had deleterious effects on adipocyte function: increasing expression of IL-6 (Fig 1C) and reducing 2-NBD-glucose uptake (p<0.05). Treatment of SATMVEC with digoxin reduced the SATMVEC senescent phenotype (Fig 1B) and increased 2-NBD-glucose adipocyte uptake (Figure 1D) (p<0.001). Conclusions SATMVEC from patients with HFDM have a senescent phenotype, and when in co-culture induce a proinflammatory adipocyte phenotype, resistant to glucose uptake. Digoxin reduces SATMVEC senescence, restores healthy adipocyte glucose homeostasis, and thus has the potential to repair dysfunctional SAT in patients with HFDM.

Characterisation of the novel SCN5A-P1891A variant, implicated in left ventricular noncompaction cardiomyopathy in a Finnish family, using human induced pluripotent stem cell-derived cardiomyocytes

Abstract Introduction Left ventricular noncompaction (LVNC) is a heterogenous cardiomyopathy characterised by an extensively trabeculated left ventricle (LV). Several genetic variants that perturb trabeculae compaction, and thus LV maturation, have been reported [1]. However, the precise mechanisms underpinning this have proven contentious with, for example, differentially aberrant cardiomyocyte proliferation implicated in the development of LVNC [2,3]. Purpose In this study, we investigate a novel SCN5A variant, P1891A, which is associated with LVNC in a Finnish family. We ascertain the effects of this variant through the characterisation of variant-carrying human induced pluripotent stem cell-derived cardiomyocytes (P1891A-hiPSC-CMs). Methods We isolated dermal fibroblasts from a symptomatic member of a Finnish family carrying the SCN5A-P1891A variant; derived P1891A-hiPSCs via nucleofection with reprogramming plasmids [4]; and differentiated these into hiPSC-CMs alongside healthy control-hiPSCs. We determined the action potential (AP) and sodium- (INa) and calcium-current (ICa) densities of hiPSC-CMs via patch-clamp. We investigated gene expression in hiPSC-CMs through qPCR and employed Multiple Approaches Combined (MCA) proteomics [5] in SCN5A-transfected HEK293-like cells to elucidate protein-protein interactions (PPI). We subjected hiPSC-CMs to mechanical stretch [6] to examine their stress response and assessed their proliferation capacity via high-content analysis for the percentage of BrdU positive hiPSC-CMs following mitogenic stimulation (5 µM CHIR99021 and 1 µM SB203580). Finally, we ascertained contractile kinetics and apparent contractile force using fibrin hydrogel-based engineered heart tissues (EHTs) [7]. Results The P1891A-hiPSC-CMs demonstrated elevated INa density alongside enhanced arrhythmogenicity relative to healthy control hiPSC-CMs although, AP kinetics and ICa density were unperturbed (Fig. 1). Under baseline conditions, P1891A-hiPSC-CMs exhibited higher SCN5A gene expression and proteomics revealed a reduction in PPI. Baseline proliferation was unchanged; however, aged P1891A-hiPSC-CMs had enhanced proliferative capacity following mitogenic stimulation. Further, the P1891A-hiPSC-CMs displayed impaired tolerance to mechanical stretch as evidenced by upregulated NPPB and ACTA1 expression. In the context of the EHT, P1891A-hiPSC-CMs yielded disparate phenotypes. Whilst a subset compacted fully and yielded weaker contractile parameters relative to healthy control EHTs, the majority of P1891A-hiPSC-CM EHTs failed to fully compact thereby recapitulating the LVNC phenotype (Fig. 2). Conclusions This study presents a unique aetiology of LVNC and links, for the first time, SCN5A mutations with enhanced human cardiomyocyte proliferation. The ability to recapture the noncompaction phenotype in EHTs presents a powerful model for future mechanistic research and drug development.

Growth plate closure and therapeutic interventions.

Height gains result from longitudinal bone growth, which is largely dependent on chondrocyte differentiation and proliferation within the growth plates of long bones. The growth plate, that is, the epiphyseal plate, is divided into resting, proliferative, and hypertrophic zones according to chondrocyte characteristics. The differentiation poten-tial of progenitor cells in the resting zone, continuous capacity for chondrocyte differentiation and proliferation within the proliferative zone, timely replacement by osteocytes, and calcification in the hypertrophic zone are the 3 main factors controlling longitudinal bone growth. Upon ade quate longitudinal bone growth, growth plate senescence limits human body height. During growth plate senescence, progenitor cells within the resting zone are deplet ed, proliferative chondrocyte numbers de crease, and hypertrophic chondrocyte number and size decrease. After senescence, hypertrophic chondrocytes are replaced by osteocytes, the extracellular matrix is calcified and va-scularized, the growth plate is closed, and longitudinal bone growth is complete. To date, go nadotropin-releasing hormone analogs, aromatase inhi bitors, C-type natriuretic peptide analogs, and fibroblast growth factor receptor 3 inhibitors have been studied or used as therapeutic interv-entions to delay growth plate closure. Complex networks of cellular, genetic, paracrine, and endocrine signals are involved in growth plate closure. However, the detailed mechanisms of this process remain unclear. Further eluci-dation of these mechanisms will enable the development of new thera peutic modalities for the treatment of short stature, precocious puberty, and skeletal dysplasia.

SAR1A Induces Cell Growth and Epithelial–Mesenchymal Transition Through the PI3K/AKT/mTOR Pathway in Head and Neck Squamous Cell Carcinoma: An In Vitro and In Vivo Study

Objectives: Head and neck squamous cell carcinoma (HNSCC) ranks sixth globally, with a 50% five-year survival rate. SAR1A exhibits high expression levels in various tumor types, yet its specific role in HNSCC remains to be clarified. Methods: In vitro assays, such as CCK8, EdU, colony formation, wound-healing, transwell, and Western blotting analyses, as well as in vivo assays, such as tumor xenografts and lung metastasis models, were conducted to evaluate the impacts of SAR1A on HNSCC proliferation, migration, and invasion. Transcriptome sequencing and KEGG enrichment pathway analysis revealed evident alterations in the PI3K/AKT/mTOR(PAM) pathways. LY294002 (a PI3K/AKT inhibitor) was used to investigate the role of the PAM pathway in proliferation, migration, and invasion in HNSCC. Results: Univariate and multivariate Cox regression were conducted to screen SAR1A as a gene prognostic biomarker in HNSCC, and it was validated in the Cancer Genome Atlas (TCGA) database. Functional assays demonstrated that the depletion of SAR1A leads to suppressed proliferation, migration, and invasion of HNSCC cells. This is accompanied by a decrease in the expression of epithelial–mesenchymal transition (EMT)-related markers in HNSCC cell lines. In addition, the diminished capacities of proliferation, migration, and invasion observed in SAR1A knockdown cells were reversed upon the overexpression of SAR1A. Furthermore, RNA-seq and KEGG enrichment analysis demonstrated a significant alteration in the PAM pathway following SAR1A knockdown. LY294002 effectively mitigated the increased proliferation, migration, and invasion induced by SAR1A overexpression. Conclusions: SAR1A facilitates HNSCC proliferation and EMT via the PI3K/AKT/mTOR pathway.

Stem cell seeding of decellularised porcine right ventricular outflow tracts to advance repair of congenital heart defects

Abstract Background Replacement of the right ventricular outflow tract (RVOT) is necessary in many critical congenital heart disease subtypes, often when the child is less than one year old. Poor availability of small-diameter cardiac homografts necessitates the use of xenografts in paediatric surgical reconstruction. Currently used xenografts are limited by immunoincompatibility and lack of growth, requiring repeat surgery to replace the defective graft. One approach to tackle acute immune rejection of animal tissue is aldehyde fixation of grafts prior to xenotransplantation. Though masking antigenicity, fixation is associated with multiple issues, including toxicity of fixative remnants and calcification. Decellularisation of biological grafts to remove foreign material is a promising alternative to reduce immunogenicity and dampen the risk of rejection. To combat lack of growth, in vitro cellularisation with stem cells is a promising solution. Purpose Current RVOT substitutes have not proven to be a lifelong solution for paediatric cardiac repair. This research has optimised a novel decellularisation technique for porcine RVOTs to produce a durable and cell-free scaffold with translational capability. Current work is looking to seed mesenchymal stem cells (MSCs) onto the acellular RVOT in vitro to enhance immune tolerance and endow the graft with growth capacity upon implant. Methods First, this decellularisation technique produces acellular RVOT scaffolds by simultaneously utilising three methods: enzymatic, chemical, and mechanical decellularisation. Novel to this method, the latter is realised using a 3D printed flow chamber to utilise the shear forces of continuous fluid flow for cell removal from the external and internal RVOTs structure. Coating the acellular graft with poly-lysine has then enabled recellularisation with MSCs isolated from the umbilical cord. Results Nuclei quantification demonstrates decellularisation of all structures of the RVOT, and elimination of residual nucleic acids is evidenced. Immunohistochemical analysis confirms the xenoantigen Galα1-3Galβ1-4GlcNAc-R is absent from the decellularised tissue. In parallel with removal of typical immunogens associated with the rejection response, the elastin and collagen fibres of the extracellular matrix are maintained, supported by histology and electron microscopy. Importantly, the tensile properties of the decellularised grafts are preserved. Finally, MSCs adhered onto the external pulmonary artery surface in vitro, achieving a uniform monolayer of cells. Conclusion RVOTs have been successfully decellularised and proven to possess seeding potential. Future work aims to characterise the seeded MSCs to assess parameters including proliferative capacity and expression of typical MSC markers. Overall, this work has the potential to produce a non-immunogenic graft possessing growth potential as a surgical alternative for paediatric RVOT replacement.

Transition from fetal to postnatal state in the heart: Crosstalk between metabolism and regeneration.

Cardiovascular disease is the leading cause of mortality worldwide. Myocardial injury resulting from ischemia can be fatal because of the limited regenerative capacity of adult myocardium. Mammalian cardiomyocytes rapidly lose their proliferative capacities, with only a small fraction of adult myocardium remaining proliferative, which is insufficient to support post-injury recovery. Recent investigations have revealed that this decline in myocardial proliferative capacity is closely linked to perinatal metabolic shifts. Predominantly glycolytic fetal myocardial metabolism transitions towards mitochondrial fatty acid oxidation postnatally, which not only enables efficient production of ATP but also causes a dramatic reduction in cardiomyocyte proliferative capacity. Extensive research has elucidated the mechanisms behind this metabolic shift, as well as methods to modulate these metabolic pathways. Some of these methods have been successfully applied to enhance metabolic reprogramming and myocardial regeneration. This review discusses recently acquired insights into the interplay between metabolism and myocardial proliferation, emphasizing postnatal metabolic transitions.

Human amniotic membrane scaffold enhances adipose mesenchymal stromal cell mitochondrial bioenergetics promoting their regenerative capacities.

The human amniotic membrane (hAM) has been applied as a scaffold in tissue engineering to sustain stem cells and enhance their regenerative capacities. We investigated the molecular and biochemical regulations of mesenchymal stromal cells (MSCs) cultured on hAM scaffold in a three-dimensional (3D) setting. Culture of adipose-MSCs (AMSCs) on decellularized hAM showed significant improvement in their viability, proliferative capacity, resistance to apoptosis, and enhanced MSC markers expression. These cultured MSCs displayed altered expression of markers associated with pro-angiogenesis and inflammation and demonstrated increased potential for differentiation into adipogenic and osteogenic lineages. The hAM scaffold modulated cellular respiration by upregulating glycolysis in MSCs as evidenced by increased glucose consumption, cellular pyruvate and lactate production, and upregulation of glycolysis markers. These metabolic changes modulated mitochondrial oxidative phosphorylation (OXPHOS) and altered the production of reactive oxygen species (ROS), expression of OXPHOS markers, and total antioxidant capacity. They also significantly boosted the urea cycle and altered the mitochondrial ultrastructure. Similar findings were observed in bone marrow-derived MSCs (BMSCs). Live cell imaging of BMSCs cultured in the same 3D environment revealed dynamic changes in cellular activity and interactions with its niche. These findings provide evidence for the favorable properties of hAM as a biomimetic scaffold for enhancing the in vitro functionality of MSCs and supporting their potential usefulness in clinical applications.

CD56 does not contribute to the anti-tumor, tissue homing, and glycolytic capacity of human NK cells.

Natural Killer (NK) cells are critical innate immune cells involved in the clearance of virally infected and malignant cells. Human NK cells are distinguished by their surface expression of CD56 and a lack of CD3. While CD56 expression and cell surface density has long been used as the prototypic marker to characterize primary human NK cell functional subsets, the exact functional role of CD56 in primary human NK cells is still not fully understood. Here we eliminated the expression of CD56 in human ex vivo expanded NK cells (CD56bright) using CRISPR/Cas9 in order to assess the function of CD56 in this highly activated and cytotoxic NK cell population. We show that the expression of CD56 has no effect on NK cell proliferative capacity or expression of various activation and inhibitory markers. Further, CD56 does not contribute to NK cell-mediated cytotoxicity, inflammatory cytokine production, or the ability of NK cells to control tumor engraftment in vivo. We also found that while deletion of CD56 did not impact NK cell glycolytic metabolism it did increase NK cell reliance on oxidative phosphorylation. Lastly, CD56 does not alter expanded NK cell in vivo tissue trafficking. Our results indicate that while CD56 expression could be used to indicate a hyper-functional state of NK cells, it does not directly influence the anti-tumor functions of expanded NK cells.

Impact of fetal umbilical cord blood CD34+ cells on breast cancer cell lines: a mechanism of fetal microchimerism?

Introduction Fetal microchimerism could be involved in the regulation of breast cancer oncogenesis. CD34+ cells could be of a particular interest as up to 12% of the CD34+ population in maternal blood are of fetal origin. The aim of this research was to analyze the impact of umbilical cord blood (UCB) CD34+ on MCF-7 and MDA-MB-231 breast cancer cell lines, in order to uncover novel biological mechanisms and suggest novel treatment options for breast cancer. Methods UCB CD34+ cells were obtained from healthy women at full-term delivery. Direct cultures were grown with MCF-7 and MDA-MB-231 cells. Proliferation, migration, invasion, and transcriptomic analysis of breast cancer cells were compared between cultures exposed and non-exposed to UCB CD34+ cells. Interactions between UCB CD34+ and breast cancer cells were analyzed under fluorescent microscopy. Functional analyses were generated with QIAGEN's Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA). Results Direct contact between UCB CD34+ and breast cancer cell lines induced a reduction in the proliferative capacities of MCF-7 and MDA-MB-231 and diminished the migration abilities of MDA-MB-231 cells. In 3D co-culture, UCB CD34+ cells were attracted by tumor spheroids and incorporated into tumor cells. These cell-to-cell interactions were responsible for transcriptome modifications coherent with observed functional modifications. Among the cytokines secreted by UCB CD34+, IFN was identified as a potential upstream regulator responsible for the molecular modifications observed in transcriptomic analysis of MCF-7 breast cancer cells exposed to UCB CD34+ cells, as was IL-17A in MDA-MB-231 cells. Conclusion Direct cell-to-cell contact induced functional modifications in breast cancer cells. Interactions between UCB CD34+ and breast cancer cells could induce cell fusion and signal transmission via cytokines. Further analysis of direct cell-to-cell interactions should be performed at a molecular level to further understand the potential role of fetal CD34+ cells in breast cancer.

One-carbon-mediated purine synthesis underlies temozolomide resistance in glioblastoma

Glioblastoma accounts for nearly half of all primary malignant brain tumors in adults, and despite an aggressive standard of care, including excisional surgery and adjuvant chemoradiation, recurrence remains universal, with an overall median survival of 14.6 months. Recent work has revealed the importance of passenger mutations as critical mediators of metabolic adaptation in cancer progression. In our previous work, we identified a role for the epigenetic modifier ID-1 in temozolomide resistance in glioblastoma. Here, we show that ID-1-mediated glioblastoma tumourigenesis is accompanied by upregulation of one-carbon (1-C) mediated de novo purine synthesis. ID-1 knockout results in a significant reduction in the expression of 1-C metabolism and purine synthesis enzymes. Analysis of glioblastoma surgical specimens at initial presentation and recurrence reveals that 1-C purine synthesis metabolic enzymes are enriched in recurrent glioblastoma and that their expression correlates with a shorter time to tumor recurrence. Further, we show that the 1-C metabolic phenotype underlies proliferative capacity and temozolomide resistance in glioblastoma cells. Supplementation with exogenous purines restores proliferation in ID-1-deficient cells, while inhibition of purine synthesis with AICAR sensitizes temozolomide-resistant glioblastoma cells to temozolomide chemotherapy. Our data suggest that the metabolic phenotype observed in treatment-resistant glioma cells is a potential therapeutic target in glioblastoma.

Identification of the proliferative activity of germline progenitor cells in the adult ovary of the bat Artibeus jamaicensis.

Until a few years ago, it was assumed that oocyte renewal did not take place in the ovary of adult organisms; however, the existence of germline progenitor cells (GPCs), which renew the ovarian follicular reserve, has now been documented in mammals. Specifically, in the adult ovary of bats, the presence of cells located in the cortical region with characteristics similar to GPCs, called adult cortical germ cells (ACGC), has been observed. One of the requirements that a GPC must fulfil is to be able to proliferate mitotically, so the evaluation of cell proliferation in ACGC is of utmost importance in order to be able to relate them to a parental lineage. Currently, there are several methods to determine cell proliferation, including BrdU labelling or the use of endogenous proliferation markers. Thus, the aim of this work was to evaluate the proliferative activity of ACGC in the adult ovary of the bat Artibeus jamaicensis, using different proliferation markers and correlating these with the protein expression of the transcription factor Oct4 and the germ line marker Ddx4. We found that the expression pattern of the proliferation markers BrdU, PCNA, Ki-67 and pH3 occurs at different times of the cell cycle, so co-localization of two or more of these markers allows us to identify proliferating cells. This allowed us to identify ACGC with proliferative capacity in the adult ovary of A. jamaicensis, suggesting that GPCs renew the follicle reserve during the adult life of the organism.

TRAF4 promotes lung cancer development by activating tyrosine kinase of EGFR

Objective: To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion. Methods: Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay. Results: The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin (r2: 0.438, 0.695, and 0.736, respectively, all P＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P=0.034). Traf4-/- cells had a weakened proliferative capacity than traf4+/+ cells and formed tumors with smaller size (P＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4-/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4+/+ cells [(62.3±10.3)%, P=0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4-/- cells were lower than those of the traf4+/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions: TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

The renoprotective efficacy and safety of genetically-engineered human bone marrow-derived mesenchymal stromal cells expressing anti-fibrotic cargo

BackgroundKidney fibrosis is a hallmark of chronic kidney disease (CKD) and compromises the viability of transplanted human bone marrow-derived mesenchymal stromal cells (BM-MSCs). Hence, BM-MSCs were genetically-engineered to express the anti-fibrotic and renoprotective hormone, human relaxin-2 (RLX) and green fluorescent protein (BM-MSCs-eRLX + GFP), which enabled BM-MSCs-eRLX + GFP delivery via a single intravenous injection.MethodsBM-MSCs were lentiviral-transduced with human relaxin-2 cDNA and GFP, under a eukaryotic translation elongation factor-1α promoter (BM-MSCs-eRLX + GFP) or GFP alone (BM-MSCs-eGFP). The ability of BM-MSCs-eRLX + GFP to differentiate, proliferate, migrate, produce RLX and cytokines was evaluated in vitro, whilst BM-MSC-eRLX + GFP vs BM-MSCs-eGFP homing to the injured kidney and renoprotective effects were evaluated in preclinical models of ischemia reperfusion injury (IRI) and high salt (HS)-induced hypertensive CKD in vivo. The long-term safety of BM-MSCs-RLX + GFP was also determined 9-months after treatment cessation in vivo.ResultsWhen cultured for 3- or 7-days in vitro, 1 × 106 BM-MSCs-eRLX + GFP produced therapeutic RLX levels, and secreted an enhanced but finely-tuned cytokine profile without compromising their proliferation or differentiation capacity compared to naïve BM-MSCs. BM-MSCs-eRLX + GFP were identified in the kidney 2-weeks post-administration and retained the therapeutic effects of RLX in vivo. 1–2 × 106 BM-MSCs-eRLX + GFP attenuated the IRI- or therapeutically abrogated the HS-induced tubular epithelial damage and interstitial fibrosis, and significantly reduced the HS-induced hypertension, glomerulosclerosis and proteinuria. This was to an equivalent extent as RLX and BM-MSCs administered separately but to a broader extent than BM-MSCs-eGFP or the angiotensin-converting enzyme inhibitor, perindopril. Additionally, these renoprotective effects of BM-MSCs-eRLX + GFP were maintained in the presence of perindopril co-treatment, highlighting their suitability as adjunct therapies to ACE inhibition. Importantly, no major long-term adverse effects of BM-MSCs-eRLX + GFP were observed.ConclusionsBM-MSCs-eRLX + GFP produced greater renoprotective and therapeutic efficacy over that of BM-MSCs-eGFP or ACE inhibition, and may represent a novel and safe treatment option for acute kidney injury and hypertensive CKD.

Chronic exposure to hexavalent chromium induces esophageal tumorigenesis via activating the Notch signaling pathway.

Hexavalent chromium Cr(VI), as a well-established carcinogen, contributes to tumorigenesis for many human cancers, especially respiratory and digestive tumors. However, the potential function and relevant mechanism of Cr(VI) on the initiation of esophageal carcinogenesis are largely unknown. Here, immortalized human esophageal epithelial cells (HEECs) were induced to be malignantly transformed cells, termed HEEC-Cr(VI) cells, via chronic exposure to Cr(VI), which simulates the progress of esophageal tumorigenesis. In vitro and in vivo experiments demonstrated that HEEC-Cr(VI) cells obtain the ability of anchorage-independent growth, greater proliferative capacity, cancer stem cell properties, and the capacity to form subcutaneous xenografts in BALB/c nude mice when compared to their parental cells, HEECs. Additionally, HEEC-Cr(VI) cells exhibited weakened cell motility and enhanced cell adhesion. Interestingly, HEECs with acute exposure to Cr(VI) failed to display those malignant phenotypes of HEEC-Cr(VI) cells, suggesting that Cr(VI)‍-induced malignant transformation, but not Cr(VI) itself, is the cause for the tumor characteristics of HEEC-Cr(VI) cells. Mechanistically, chronic exposure to Cr(VI) induced abnormal activation of Notch signaling, which is crucial to maintaining the capacity for malignant proliferation and stemness of HEEC-Cr(VI) cells. As expected, N-‍[N-‍(3,5-difluorophenacetyl)‍-L-alanyl]‍-S-phenylglycine t-butyl ester (DAPT), an inhibitor for the Notch pathway, drastically attenuated cancerous phenotypes of HEEC-Cr(VI) cells. In conclusion, our study clarified the molecular mechanism underlying Cr(VI)‍-induced esophageal tumorigenesis, which provides novel insights for further basic research and clinical therapeutic strategies about Cr(VI)‍-associated esophageal cancer.

Valproic acid alleviates total-body irradiation-induced small intestinal mucositis in mice

Purpose Gastrointestinal (GI) injury is one of the serious problems of total-body irradiation (TBI). However, no fundamental treatment for TBI and other radiation-induced GI injury has yet been established. Valproic acid (VPA) administration reduces mortality in mice subjected to total-body irradiation (TBI) with X-rays. This study aimed to evaluate the effects of VPA on GI injury induced by TBI in mice. Materials and methods Mice were subjected to TBI with X-rays to induce GI injury. Changes in survival and weight were observed after VPA administration. The small intestine was then sampled at 0, 1, 3, 7, and 10 d after irradiation for histological and immunohistological evaluation and measurement of myeloperoxidase (MPO) activity and inflammatory cytokine levels (IL-1β). Results VPA (200 and 600 mg/kg) increased survival rate and reduced weight loss in model mice. IL-1β expression 1 d after irradiation was significantly lower in the VPA group than that in the vehicle group. Furthermore, the increase in MPO activity at 3 and 7 d after irradiation was significantly suppressed by VPA administration. Histological examination (hematoxylin and eosin staining) revealed that 600 mg/kg VPA inhibited inflammatory cell infiltration. Immunostaining for the proliferating cell nuclear antigen involved in cell proliferation showed that VPA suppressed the irradiation-induced decrease in cell proliferative capacity. Conclusions Treatment with VPA in mice with GI injury caused by TBI suppressed inflammatory responses in small intestinal mucosal cells. These results suggest that VPA may be a useful therapeutic agent against TBI-induced small intestinal mucositis.

Lactobacillus rhamnosus GG-derived extracellular vesicles promote wound healing via miR-21-5p-mediated re-epithelization and angiogenesis

Extracellular vesicles (EVs), especially those derived from stem cells, have emerged as a novel treatment for promoting wound healing in regenerative medicine. However, the clinical application of mammalian cells-derived EVs is hindered by their high cost and low yields. Inspired by the ability of EVs to mediate interkingdom communication, we explored the therapeutic potential of EVs released by the probiotic strain Lactobacillus rhamnosus GG (LGG) in skin wound healing and elucidated the underlying mechanism involved. Using full-thickness skin wound-healing mouse models, we found that LGG-EVs accelerated wound healing procedures, including increased re-epithelialization and promoted angiogenesis. Using in vitro experiments, we further demonstrated that LGG-EVs boosted the proliferation and migration capacities of both epithelial and endothelial cells, as well as promoted endothelial tube formation. miRNA profiling analysis revealed that miR-21-5p was highly enriched in LGG-EVs and LGG-EV treatment significantly increased miR-21-5p level in recipient cells. Mechanically, LGG-EVs induced regulatory effects via miR-21-5p mediated metabolic signaling rewiring. Our results suggest that EVs derived from LGG could serve as a promising candidate for accelerating wound healing and possibly for treating chronic and impaired healing conditions.Graphical abstract

A transcriptomic analysis of dental pulp stem cell senescence in vitro

Background/purposeThe use of human dental pulp stem cells (hDPSCs) as autologous stem cells for tissue repair and regenerative techniques is a significant area of global research. The objective of this study was to investigate the effects of long-term in vitro culture on the multidifferentiation potential of hDPSCs and the potential molecular mechanisms involved.Materials and methodsThe tissue block method was used to extract hDPSCs from orthodontic-minus-extraction patients, which were then expanded and cultured in vitro for 12 generations. Stem cells from passages three, six, nine, and twelve were selected. Flow cytometry was used to detect the expression of stem cell surface markers, and CCK-8 was used to assess cell proliferation. β-Galactosidase staining was employed to detect cellular senescence, Alizarin Red S staining to assess osteogenic potential, and Oil Red O staining to evaluate lipogenic capacity. RNA sequencing (RNA-seq) was conducted to identify differentially expressed genes in DPSCs and investigate their potential mechanisms.ResultsWith increasing passage numbers, pulp stem cells showed an increase in senescence and a decrease in proliferative capacity and osteogenic–lipogenic multidifferentiation potential. The expression of stem cell surface markers CD34 and CD45 was stable, whereas the expression of CD73, CD90, and CD105 decreased with increasing passages. According to the RNA-seq analysis, the differentially expressed genes CFH, WNT16, HSD17B2, IDI1, and COL5A3 may be associated with stem cell senescence.ConclusionIncreased in vitro expansion induced cellular senescence in pulp stem cells, which resulted in a reduction in their proliferative capacity and osteogenic–lipogenic differentiation potential. The differential expression of genes such as CFH, WNT16, HSD17B2, IDI1, and COL5A3 may represent a potential mechanism for the induction of cellular senescence in pulp stem cells.

Inhibition of viability of human retinal microvascular endothelial cells by vialinin A under high glucose condition.

To investigate the effects of vialinin A on viability of human retinal endothelial cells (HRECs) under high glucose condition and its potential mechanism. The HRECs were divided into four groups: normal glucose control group (NG, 5 mmol/L D-glucose), high glucose group (HG, 30 mmol/L D-glucose), HG+1 µmol/L vialinin A group, and HG+5 µmol/L vialinin A group. The cell viabilities were measured with cell counting kit-8 (CCK-8) assay for proliferation, with scratch assay for migration, and tube formation, for evaluation of the impact of vialinin A on cellular behaviour. Real-time PCR and Western blotting were used to determine the expression level of vascular endothelial growth factor (VEGF). The proliferative capacity and migration of HRECs was reduced by 5 µmol/L vialinin A in high glucose environment (both P<0.05). Vialinin A also inhibited high-glucose-induced tube formation of HRECs. The expression level of VEGF and PI3K in HRECs was also significantly decreased by vialinin A (P<0.05). Vialinin A inhibits the cell viability of HRECs. It may serve as a potential target for anti-angiogenic therapy.

MerTK Signaling in Human Primary T cells Modulates Memory Potential and Improves Recall response.

Immune therapy using checkpoint inhibitors or adoptive cell transfer has revolutionized the treatment of several types of cancers. However, response to treatment is currently limited to a fraction of patients. Elucidation of immune modulatory mechanisms might optimize patient selection and present ways to modify anti-cancer immune responses. We recently discovered the expression and an important costimulatory role of TAM receptor MerTK signaling on activated human primary CD8+ T cells. Here we extend our study of the costimulatory role of MerTK expression in human CD8+ T cells. We uncover a clear link between MerTK expression and less differentiated Central Memory T cells based on an increased expression of CCR7, CD45RO, CD28, CD62L, and an altered metabolic profile. In addition, we observe an improved proliferative capacity and elevated expression of effector molecule IFNγ upon recall responses of MerTK-expressing cells in vitro. Finally, using gp100TCR-transduced T cells, we demonstrate how PROS1 treatment results in improved cytotoxicity and killing of tumors. Our findings describe a role of MerTK expression in T cells, which could be exploited in the search for improving immunotherapeutic approaches.