Hypoxic conditions promote a proliferative, poorly differentiated phenotype in COPD lung tissue progenitor cells in vitro.

PURPOSE: Chronic obstructive pulmonary disease (COPD) patients experience hypoxemia and lung tissue hypoxia, causing vasoconstriction, and at its most severe Cor pulmonale. However, minimal attention has been given to the effects of hypoxia at the cellular level. We hypothesize that a persistent progenitor cell population undergoes an aberrant differentiation process, influenced by changes in oxygen. METHODS: Distal lung progenitor cells from two emphysematous donors were cultured in 21% and 2% oxygen. Proliferation was determined on collagen-coated plastic and in 3T3-J2 co-culture. Epithelial (E-cadherin, pan-cytokeratin) and progenitor (TP63, cytokeratin 5) marker expressions were examined. Cells were differentiated at air-liquid interface, and ciliated, mucus-producing, and club cell populations identified by immunofluorescence. MUC5AC, MUC5B, CC10, and TP63 expression were determined using qRT-PCR, mucin5AC, and mucin5B protein levels by ELISA, and secreted mucin by periodic acid biotin hydrazide assay. RESULTS: Cells were positive for epithelial and progenitor markers at isolation and passage 5. Passage 5 cells in hypoxia increased the proportion of TP63 by 10% from 51.6 ± 1.2% to 62.6 ± 2.3% (p ≤ 0.01). Proliferative capacity was greater on 3T3J2 cells and in 2% oxygen, supporting the emergence of a proliferation unrestricted population with limited differentiation capacity. Differentiation resulted in ßIV tubulin positive-ciliated cells, mucin5AC, mucin5B, and CC10 positive secretory cells. Epithelial barrier formation was reduced (p ≤ 0.0001) in hypoxia-expanded cells. qRT-PCR showed higher mucin expression in 2% cells, significantly so with MUC5B (p ≤ 0.05). Although overall mucin5AC and mucin5B content was greater in 21% cells, normalization of secreted mucin to DNA showed a trend for increased mucin by low oxygen cells. CONCLUSIONS: These results demonstrate that hypoxia promotes a proliferative phenotype while affecting subsequent progenitor cell differentiation capacity. Furthermore, the retained differentiation potential becomes skewed to a more secretory phenotype, demonstrating that hypoxia may be contributing to disease symptoms and severity in COPD patients.

Endotenon-Derived Type II Tendon Stem Cells Have Enhanced Proliferative and Tenogenic Potential.

Tendon injuries caused by overuse or age-related deterioration are frequent. Incomplete knowledge of somatic tendon cell biology and their progenitors has hindered interventions for the effective repair of injured tendons. Here, we sought to compare and contrast distinct tendon-derived cell populations: type I and II tendon stem cells (TSCs) and tenocytes (TNCs). Porcine type I and II TSCs were isolated via the enzymatic digestion of distinct membranes (paratenon and endotenon, respectively), while tenocytes were isolated through an explant method. Resultant cell populations were characterized by morphology, differentiation, molecular, flow cytometry, and immunofluorescence analysis. Cells were isolated, cultured, and evaluated in two alternate oxygen concentrations (physiological (2%) and air (21%)) to determine the role of oxygen in cell biology determination within this relatively avascular tissue. The different cell populations demonstrated distinct proliferative potential, morphology, and transcript levels (both for tenogenic and stem cell markers). In contrast, all tendon-derived cell populations displayed multipotent differentiation potential and immunophenotypes (positive for CD90 and CD44). Type II TSCs emerged as the most promising tendon-derived cell population for expansion, given their enhanced proliferative potential, multipotency, and maintenance of a tenogenic profile at early and late passage. Moreover, in all cases, physoxia promoted the enhanced proliferation and maintenance of a tenogenic profile. These observations help shed light on the biological mechanisms of tendon cells, with the potential to aid in the development of novel therapeutic approaches for tendon disorders.

Human Mesenchymal Stem Cell Secretome Driven T Cell Immunomodulation Is IL-10 Dependent.

The Human Mesenchymal Stem Cell (hMSC) secretome has pleiotropic effects underpinning its therapeutic potential. hMSC serum-free conditioned media (SFCM) contains a variety of cytokines, with previous studies linking a changed secretome composition to physoxia. The Jurkat T cell model allowed the efficacy of SFCM vs. serum-free media (SFM) in the suppression of immunological aspects, including proliferation and polarisation, to be explored. Cell growth in SFM was higher [(21% O2 = 5.3 × 105 ± 1.8 × 104 cells/mL) and (2% O2 = 5.1 × 105 ± 3.0 × 104 cells/mL)], compared to SFCM [(21% O2 = 2.4 × 105 ± 2.5 × 104 cells/mL) and (2% O2 = 2.2 × 105 ± 5.8 × 103 cells/mL)]. SFM supported IL-2 release following activation [(21% O2 = 5305 ± 211 pg/mL) and (2% O2 = 5347 ± 327 pg/mL)] whereas SFCM suppressed IL-2 secretion [(21% O2 = 2461 ± 178 pg/mL) and (2% O2 = 1625 ± 159 pg/mL)]. Anti-inflammatory cytokines, namely IL-4, IL-10, and IL-13, which we previously confirmed as components of hMSC SFCM, were tested. IL-10 neutralisation in SFCM restored proliferation in both oxygen environments (SFM/SFCM+antiIL-10 ~1-fold increase). Conversely, IL-4/IL-13 neutralisation showed no proliferation restoration [(SFM/SFM+antiIL-4 ~2-fold decrease), and (SFM/SFCM+antiIL-13 ~2-fold decrease)]. Present findings indicate IL-10 played an immunosuppressive role by reducing IL-2 secretion. Identification of immunosuppressive components of the hMSC secretome and a mechanistic understanding of their action allow for the advancement and refinement of potential future cell-free therapies.

Physoxia Influences Global and Gene-Specific Methylation in Pluripotent Stem Cells.

Pluripotent stem cells (PSC) possess unlimited proliferation, self-renewal, and a differentiation capacity spanning all germ layers. Appropriate culture conditions are important for the maintenance of self-renewal, pluripotency, proliferation, differentiation, and epigenetic states. Oxygen concentrations vary across different human tissues depending on precise cell location and proximity to vascularisation. The bulk of PSC culture-based research is performed in a physiologically hyperoxic, air oxygen (21% O2) environment, with numerous reports now detailing the impact of a physiologic normoxia (physoxia), low oxygen culture in the maintenance of stemness, survival, morphology, proliferation, differentiation potential, and epigenetic profiles. Epigenetic mechanisms affect multiple cellular characteristics including gene expression during development and cell-fate determination in differentiated cells. We hypothesized that epigenetic marks are responsive to a reduced oxygen microenvironment in PSCs and their differentiation progeny. Here, we evaluated the role of physoxia in PSC culture, the regulation of DNA methylation (5mC (5-methylcytosine) and 5hmC (5-hydroxymethylcytosine)), and the expression of regulatory enzyme DNMTs and TETs. Physoxia enhanced the functional profile of PSC including proliferation, metabolic activity, and stemness attributes. PSCs cultured in physoxia revealed the significant downregulation of DNMT3B, DNMT3L, TET1, and TET3 vs. air oxygen, accompanied by significantly reduced 5mC and 5hmC levels. The downregulation of DNMT3B was associated with an increase in its promoter methylation. Coupled with the above, we also noted decreased HIF1A but increased HIF2A expression in physoxia-cultured PSCs versus air oxygen. In conclusion, PSCs display oxygen-sensitive methylation patterns that correlate with the transcriptional and translational regulation of the de novo methylase DNMT3B.

Therapeutic Effects of Hypoxic and Pro-Inflammatory Priming of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Inflammatory Arthritis.

Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling, including via secretion of extracellular vesicles (EVs) in the cell secretome. We evaluated the therapeutic potential of MSCs-derived small EVs in an antigen-induced model of arthritis (AIA). EVs isolated from MSCs cultured normoxically (21% O2, 5% CO2), hypoxically (2% O2, 5% CO2) or with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed post-arthritis induction through swelling and histopathological analysis of synovial joint structure. Activated CD4+ T cells from healthy mice were cultured with EVs or MSCs to assess deactivation capabilities prior to application of standard EVs in vivo to assess T cell polarisation within the immune response to AIA. All EVs treatments reduced knee-joint swelling whilst only normoxic and pro-inflammatory primed EVs improved histopathological outcomes. In vitro culture with EVs did not achieve T cell deactivation. Polarisation towards CD4+ helper cells expressing IL17a (Th17) was reduced when normoxic and hypoxic EV treatments were applied in vitro. Normoxic EVs applied into the AIA model reduced Th17 polarisation and improved Regulatory T cell (Treg):Th17 homeostatic balance. Normoxic EVs present the optimal strategy for broad therapeutic benefit. EVs present an effective novel technology with the potential for cell-free therapeutic translation.

Clerodane Diterpenoids from an Edible Plant Justicia insularis: Discovery, Cytotoxicity, and Apoptosis Induction in Human Ovarian Cancer Cells.

OBJECTIVES: The toxicity of chemotherapeutic anticancer drugs is a serious issue in clinics. Drug discovery from edible and medicinal plants represents a promising approach towards finding safer anticancer therapeutics. Justicia insularis T. Anderson (Acanthaceae) is an edible and medicinal plant in Nigeria. This study aims to discover cytotoxic compounds from this rarely explored J. insularis and investigate their underlying mechanism of action. METHODS: The cytotoxicity of the plant extract was evaluated in human ovarian cancer cell lines and normal human ovarian surface epithelia (HOE) cells using a sulforhodamine B assay. Bioassay-guided isolation was carried out using column chromatography including HPLC, and the isolated natural products were characterized using GC-MS, LC-HRMS, and 1D/2D NMR techniques. Induction of apoptosis was evaluated using Caspase 3/7, 8, and 9, and Annexin V and PI based flow cytometry assays. SwissADME and SwissTargetPrediction web tools were used to predict the molecular properties and possible protein targets of identified active compounds. Key finding: The two cytotoxic compounds were identified as clerodane diterpenoids: 16(α/ß)-hydroxy-cleroda-3,13(14)Z-dien-15,16-olide (1) and 16-oxo-cleroda-3,13(14)E-dien-15-oic acid (2) from the Acanthaceous plant for the first time. Compound 1 was a very abundant compound (0.7% per dry weight of plant material) and was shown to be more potent than compound 2 with IC50 values in the micromolar range against OVCAR-4 and OVCAR-8 cancer cells. Compounds 1 and 2 were less cytotoxic to HOE cell line. Both compounds induced apoptosis by increasing caspase 3/7 activities in a concentration dependent manner. Compound 1 further increased caspase 8 and 9 activities and apoptosis cell populations. Compounds 1 and 2 are both drug like, and compound 1 may target various proteins including a kinase. CONCLUSIONS: Clerodane diterpenoids (1 and 2) in J. insularis were identified as cytotoxic to ovarian cancer cells via the induction of apoptosis, providing an abundant and valuable source of hit compounds for the treatment of ovarian cancer.

Dose-Response Tendon-Specific Markers Induction by Growth Differentiation Factor-5 in Human Bone Marrow and Umbilical Cord Mesenchymal Stem Cells.

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) are utilized in tendon tissue-engineering protocols while extra-embryonic cord-derived, including from Wharton's Jelly (hWJ-MSCs), are emerging as useful alternatives. To explore the tenogenic responsiveness of hBM-MSCs and hWJ-MSCs to human Growth Differentiation Factor 5 (hGDF-5) we supplemented each at doses of 1, 10, and 100 ng/mL of hGDF-5 and determined proliferation, morphology and time-dependent expression of tenogenic markers. We evaluated the expression of collagen types 1 (COL1A1) and 3 (COL3A1), Decorin (DCN), Scleraxis-A (SCX-A), Tenascin-C (TNC) and Tenomodulin (TNMD) noting the earliest and largest increase with 100 ng/mL. With 100 ng/mL, hBM-MSCs showed up-regulation of SCX-A (1.7-fold) at Day 1, TNC (1.3-fold) and TNMD (12-fold) at Day 8. hWJ-MSCs, at the same dose, showed up-regulation of COL1A1 (3-fold), DCN (2.7-fold), SCX-A (3.8-fold) and TNC (2.3-fold) after three days of culture. hWJ-MSCs also showed larger proliferation rate and marked aggregation into a tubular-shaped system at Day 7 (with 100 ng/mL of hGDF-5). Simultaneous to this, we explored the expression of pro-inflammatory (IL-6, TNF, IL-12A, IL-1ß) and anti-inflammatory (IL-10, TGF-ß1) cytokines across for both cell types. hBM-MSCs exhibited a better balance of pro-inflammatory and anti-inflammatory cytokines up-regulating IL-1ß (11-fold) and IL-10 (10-fold) at Day 8; hWJ-MSCs, had a slight expression of IL-12A (1.5-fold), but a greater up-regulation of IL-10 (2.5-fold). Type 1 collagen and tenomodulin proteins, detected by immunofluorescence, confirming the greater protein expression when 100 ng/mL were supplemented. In the same conditions, both cell types showed specific alignment and shape modification with a length/width ratio increase, suggesting their response in activating tenogenic commitment events, and they both potential use in 3D in vitro tissue-engineering protocols.

In Vitro Innovation of Tendon Tissue Engineering Strategies.

Tendinopathy is the term used to refer to tendon disorders. Spontaneous adult tendon healing results in scar tissue formation and fibrosis with suboptimal biomechanical properties, often resulting in poor and painful mobility. The biomechanical properties of the tissue are negatively affected. Adult tendons have a limited natural healing capacity, and often respond poorly to current treatments that frequently are focused on exercise, drug delivery, and surgical procedures. Therefore, it is of great importance to identify key molecular and cellular processes involved in the progression of tendinopathies to develop effective therapeutic strategies and drive the tissue toward regeneration. To treat tendon diseases and support tendon regeneration, cell-based therapy as well as tissue engineering approaches are considered options, though none can yet be considered conclusive in their reproduction of a safe and successful long-term solution for full microarchitecture and biomechanical tissue recovery. In vitro differentiation techniques are not yet fully validated. This review aims to compare different available tendon in vitro differentiation strategies to clarify the state of art regarding the differentiation process.

Lung Regeneration: Endogenous and Exogenous Stem Cell Mediated Therapeutic Approaches.

The tissue turnover of unperturbed adult lung is remarkably slow. However, after injury or insult, a specialised group of facultative lung progenitors become activated to replenish damaged tissue through a reparative process called regeneration. Disruption in this process results in healing by fibrosis causing aberrant lung remodelling and organ dysfunction. Post-insult failure of regeneration leads to various incurable lung diseases including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis. Therefore, identification of true endogenous lung progenitors/stem cells, and their regenerative pathway are crucial for next-generation therapeutic development. Recent studies provide exciting and novel insights into postnatal lung development and post-injury lung regeneration by native lung progenitors. Furthermore, exogenous application of bone marrow stem cells, embryonic stem cells and inducible pluripotent stem cells (iPSC) show evidences of their regenerative capacity in the repair of injured and diseased lungs. With the advent of modern tissue engineering techniques, whole lung regeneration in the lab using de-cellularised tissue scaffold and stem cells is now becoming reality. In this review, we will highlight the advancement of our understanding in lung regeneration and development of stem cell mediated therapeutic strategies in combating incurable lung diseases.

Measurement of C-reactive protein, procalcitonin and neutrophil elastase in saliva of COPD patients and healthy controls: correlation to self-reported wellbeing parameters.

BACKGROUND: Saliva is increasingly promoted as an alternative diagnostic bio-sample to blood; however its role in respiratory disease requires elucidation. Our aim was to investigate whether C-reactive protein (CRP), procalcitonin (PCT) and neutrophil elastase (NE) could be measured in unstimulated whole saliva, and to explore differences between COPD patients and controls with normal lung function. We also determined the relationship between these salivary biomarkers and self-reported COPD-relevant metrics. METHODS: Salivary CRP, PCT and NE levels were measured at each of 3 visits over a 14-day period alongside spirometry and a daily self-assessment dairy in 143 subjects: 20 never-smokers and 25 smokers with normal spirometry; 98 COPD patients [GOLD Stage I, 16; Stage II, 32; Stage III, 39; Stage IV, 11]. Twenty-two randomly selected subjects provided simultaneous blood samples. RESULTS: Levels of each salivary biomarker could distinguish between the above cohorts. Significant differences remained for salivary CRP and NE (p < 0.05) following adjustment for age, gender, sampling time, gum disease and total co-morbidities; but not for BMI except for salivary NE, which remained higher in smokers compared to non-smokers and stable COPD subjects (p < 0.001). Patients with acute COPD exacerbations had a median increase in all 3 salivary biomarkers (p < 0.001); CRP: median 5.74 ng/ml, [interquartile range (IQR) 2.86-12.25], PCT 0.38 ng/ml, [IQR 0.22-0.94], and NE 539 ng/ml, [IQR 112.25-1264]. In COPD patients, only salivary CRP and PCT levels correlated with breathing scores (r = 0.14, p < 0.02; r = 0.13, p < 0.03 respectively) and sputum features but not with activities of daily living. Salivary CRP and PCT concentrations strongly correlated with serum counterparts [r = 0.82, (95% CI: 0.72-0.87), p < 0.001 by Spearman's; and r = 0.53, (95% CI: 0.33-0.69), p < 0.006 respectively]; salivary NE did not. CONCLUSIONS: CRP, PCT and NE were reliably and reproducibly measured in saliva, providing clinically-relevant information on health status in COPD; additionally NE distinguished smoking status. All 3 salivary biomarkers increased during COPD exacerbations, with CRP and PCT correlating well with patient-derived clinical metrics. These results provide the conceptual basis for further development of saliva as a viable bio-sample in COPD monitoring and exacerbation management.

TERT Promoter Methylation Is Oxygen-Sensitive and Regulates Telomerase Activity.

Telomere repeats protect linear chromosomes from degradation, and telomerase has a prominent role in their maintenance. Telomerase has telomere-independent effects on cell proliferation, DNA replication, differentiation, and tumorigenesis. TERT (telomerase reverse transcriptase enzyme), the catalytic subunit of telomerase, is required for enzyme activity. TERT promoter mutation and methylation are strongly associated with increased telomerase activation in cancer cells. TERT levels and telomerase activity are downregulated in stem cells during differentiation. The link between differentiation and telomerase can provide a valuable tool for the study of the epigenetic regulation of TERT. Oxygen levels can affect cellular behaviors including proliferation, metabolic activity, stemness, and differentiation. The role of oxygen in driving TERT promoter modifications in embryonic stem cells (ESCs) is poorly understood. We adopted a monolayer ESC differentiation model to explore the role of physiological oxygen (physoxia) in the epigenetic regulation of telomerase and TERT. We further hypothesized that DNMTs played a role in physoxia-driven epigenetic modification. ESCs were cultured in either air or a 2% O2 environment. Physoxia culture increased the proliferation rate and stemness of the ESCs and induced a slower onset of differentiation than in ambient air. As anticipated, downregulated TERT expression correlated with reduced telomerase activity during differentiation. Consistent with the slower onset of differentiation in physoxia, the TERT expression and telomerase activity were elevated in comparison to the air-oxygen-cultured ESCs. The TERT promoter methylation levels increased during differentiation in ambient air to a greater extent than in physoxia. The chemical inhibition of DNMT3B reduced TERT promoter methylation and was associated with increased TERT gene and telomerase activity during differentiation. DNMT3B ChIP (Chromatin immunoprecipitation) demonstrated that downregulated TERT expression and increased proximal promoter methylation were associated with DNMT3B promoter binding. In conclusion, we have demonstrated that DNMT3B directly associates with TERT promoter, is associated with differentiation-linked TERT downregulation, and displays oxygen sensitivity. Taken together, these findings help identify novel aspects of telomerase regulation that may play a role in better understanding developmental regulation and potential targets for therapeutic intervention.

A phosphate glass reinforced composite acrylamide gradient scaffold for osteochondral interface regeneration.

The bone-cartilage interface is defined by a unique arrangement of cells and tissue matrix. Injury to the interface can contribute to the development of arthritic joint disease. Attempts to repair osteochondral damage through clinical trials have generated mixed outcomes. Tissue engineering offers the potential of integrated scaffold design with multiregional architecture to assist in tissue regeneration, such as the bone-cartilage interface. Challenges remain in joining distinct materials in a single scaffold mass while maintaining integrity and avoiding delamination. The aim of the current work is to examine the possibility of joining two closely related acrylamide derivatives such as, poly n-isopropyl acrylamide (pNIPAM) and poly n­tert­butyl acrylamide (pNTBAM). The target is to produce a single scaffold unit with distinct architectural regions in the favour of regenerating the osteochondral interface. Longitudinal phosphate glass fibres (PGFs) with the formula 50P2O5.30CaO.20Na2O were incorporated to provide additional bioactivity by degradation to release ions such as calcium and phosphate which are considered valuable to assist the mineralization process. Polymers were prepared via atom transfer radical polymerization (ATRP) and solutions cast to ensure the integration of polymers chains. Scaffold was characterized using scanning electron microscope (SEM) and Fourier transform infra-red (FTIR) techniques. The PGF mass degradation pattern was inspected using micro computed tomography (µCT). Biological assessment of primary human osteoblasts (hOBs) and primary human chondrocytes (hCHs) upon scaffolds was performed using alizarin red and colorimetric calcium assay for mineralization assessment; alcian blue staining and dimethyl-methylene blue (DMMB) assay for glycosaminoglycans (GAGs); immunostaining and enzyme-linked immunosorbent assay (ELISA) to detect functional proteins expression by cells such as collagen I, II, and annexin A2. FTIR analysis revealed an intact unit with gradual transformation from pNIPAM to pNTBAM. SEM images showed three distinct architectural regions with mean pore diameter of 54.5 µm (pNIPAM), 16.5 µm (pNTBAM) and 118 µm at the mixed interface. Osteogenic and mineralization potential by cells was observed upon the entire scaffold's regions. Chondrogenic activity was relevant on the pNTBAM side of the scaffold only with minimal evidence in the pNIPAM region. PGFs increased mineralization potential of both hOBs and hCHs, evidenced by elevated collagens I, X, and annexin A2 with reduction of collagen II in PGFs scaffolds. In conclusion, pNIPAM and pNTBAM integration created a multiregional scaffold with distinct architectural regions. Differential chondrogenic, osteogenic, and mineralized cell performance, in addition to the impact of PGF, suggests a potential role for phosphate glass-incorporated, acrylamide-derivative scaffolds in osteochondral interface regeneration.

MSC-extracellular vesicle microRNAs target host cell-entry receptors in COVID-19: in silico modeling for in vivo validation.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has created a global pandemic with significant morbidity and mortality. SARS-CoV-2 primarily infects the lungs and is associated with various organ complications. Therapeutic approaches to combat COVID-19, including convalescent plasma and vaccination, have been developed. However, the high mutation rate of SARS-CoV-2 and its ability to inhibit host T-cell activity pose challenges for effective treatment. Mesenchymal stem cells (MSCs) and their extracellular vesicles (MSCs-EVs) have shown promise in COVID-19 therapy because of their immunomodulatory and regenerative properties. MicroRNAs (miRNAs) play crucial regulatory roles in various biological processes and can be manipulated for therapeutic purposes. OBJECTIVE: We aimed to investigate the role of lyophilized MSC-EVs and their microRNAs in targeting the receptors involved in SARS-CoV-2 entry into host cells as a strategy to limit infection. In silico microRNA prediction, structural predictions of the microRNA-mRNA duplex, and molecular docking with the Argonaut protein were performed. METHODS: Male Syrian hamsters infected with SARS-CoV-2 were treated with human Wharton's jelly-derived Mesenchymal Stem cell-derived lyophilized exosomes (Bioluga Company)via intraperitoneal injection, and viral shedding was assessed. The potential therapeutic effects of MSCs-EVs were measured via histopathology of lung tissues and PCR for microRNAs. RESULTS: The results revealed strong binding potential between miRNA‒mRNA duplexes and the AGO protein via molecular docking. MSCs-EVs reduced inflammation markers and normalized blood indices via the suppression of viral entry by regulating ACE2 and TMPRSS2 expression. MSCs-EVs alleviated histopathological aberrations. They improved lung histology and reduced collagen fiber deposition in infected lungs. CONCLUSION: We demonstrated that MSCs-EVs are a potential therapeutic option for treating COVID-19 by preventing viral entry into host cells.

Exploring the molecular mechanisms of MSC-derived exosomes in Alzheimer's disease: Autophagy, insulin and the PI3K/Akt/mTOR signaling pathway.

Alzheimer's disease (AD) is a devastating neurological condition characterized by cognitive decline, motor coordination impairment, and amyloid plaque accumulation. The underlying molecular mechanisms involve oxidative stress, inflammation, and neuronal degeneration. This study aimed to investigate the therapeutic effects of mesenchymal stem cell-derived exosomes (MSC-exos) on AD and explore the molecular pathways involved, including the PI3K/Akt/mTOR axis, autophagy, and neuroinflammation. To assess the potential of MSC-exos for the treatment of AD, rats were treated with AlCl3 (17 mg/kg/once/day) for 8 weeks, followed by the administration of an autophagy activator (rapamycin), or MSC-exos with or without an autophagy inhibitor (3-methyladenin; 3-MA+ chloroquine) for 4 weeks. Memory impairment was tested, and brain tissues were collected for gene expression analyses, western blotting, histological studies, immunohistochemistry, and transmission electron microscopy. Remarkably, the administration of MSC-exos improved memory performance in AD rats and reduced the accumulation of amyloid-beta (Aß) plaques and tau phosphorylation. Furthermore, MSC-exos promoted neurogenesis, enhanced synaptic function, and mitigated astrogliosis in AD brain tissues. These beneficial effects were associated with the modulation of autophagy and the PI3K/Akt/mTOR signalling pathway, as well as the inhibition of neuroinflammation. Additionally, MSC-exos were found to regulate specific microRNAs, including miRNA-21, miRNA-155, miRNA-17-5p, and miRNA-126-3p, further supporting their therapeutic potential. Histopathological and bioinformatic analyses confirmed these findings. This study provides compelling evidence that MSC-exos hold promise as a potential therapeutic approach for AD. By modulating the PI3K/Akt/mTOR axis, autophagy, and neuroinflammation, MSC-exos have the potential to improve memory, reduce Aß accumulation, enhance neurogenesis, and mitigate astrogliosis. These findings shed light on the therapeutic potential of MSC-exos and highlight their role in combating AD.

Club cells inhibit alveolar epithelial wound repair via TRAIL-dependent apoptosis.

Club cells (Clara cells) participate in bronchiolar wound repair and regeneration. Located in the bronchioles, they become activated during alveolar injury in idiopathic pulmonary fibrosis (IPF) and migrate into the affected alveoli, a process called alveolar bronchiolisation. The purpose of this migration and the role of club cells in alveolar wound repair is controversial. This study was undertaken to investigate the role of club cells in alveolar epithelial wound repair and pulmonary fibrosis. A direct-contact co-culture in vitro model was used to evaluate the role of club cells (H441 cell line) on alveolar epithelial cell (A549 cell line) and small airway epithelial cell (SAEC) wound repair. Immunohistochemistry was conducted on lung tissue samples from patients with IPF to replicate the in vitro findings ex vivo. Our study demonstrated that club cells induce apoptosis in alveolar epithelial cells and SAECs through a tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent mechanism resulting in significant inhibition of wound repair. Furthermore, in IPF lungs, TRAIL-expressing club cells were detected within the affected alveolar epithelia in areas of established fibrosis, together with widespread alveolar epithelial cell apoptosis. From these findings, we hypothesise that the extensive pro-fibrotic remodelling associated with IPF could be driven by TRAIL-expressing club cells inducing apoptosis in alveolar epithelial cells through a TRAIL-dependent mechanism.

Mesenchymal stem cells promote alveolar epithelial cell wound repair in vitro through distinct migratory and paracrine mechanisms.

BACKGROUND: Mesenchymal stem cells (MSC) are in clinical trials for widespread indications including musculoskeletal, neurological, cardiac and haematological disorders. Furthermore, MSC can ameliorate pulmonary fibrosis in animal models although mechanisms of action remain unclear. One emerging concept is that MSCs may have paracrine, rather than a functional, roles in lung injury repair and regeneration. METHODS: To investigate the paracrine role of human MSC (hMSC) on pulmonary epithelial repair, hMSC-conditioned media (CM) and a selected cohort of hMSC-secretory proteins (identified by LC-MS/MS mass spectrometry) were tested on human type II alveolar epithelial cell line A549 cells (AEC) and primary human small airway epithelial cells (SAEC) using an in vitro scratch wound repair model. A 3D direct-contact wound repair model was further developed to assess the migratory properties of hMSC. RESULTS: We demonstrate that MSC-CM facilitates AEC and SAEC wound repair in serum-dependent and -independent manners respectively via stimulation of cell migration. We also show that the hMSC secretome contains an array of proteins including Fibronectin, Lumican, Periostin, and IGFBP-7; each capable of influencing AEC and SAEC migration and wound repair stimulation. In addition, hMSC also show a strong migratory response to AEC injury as, supported by the observation of rapid and effective AEC wound gap closure by hMSC in the 3D model. CONCLUSION: These findings support the notion for clinical application of hMSCs and/or their secretory factors as a pharmacoregenerative modality for the treatment of idiopathic pulmonary fibrosis (IPF) and other fibrotic lung disorders.

Identification of different subsets of lung cells using Raman microspectroscopy and whole cell nucleus isolation.

Raman spectroscopy has been widely used to study its possible clinical application in cancer diagnosis. However, in order to make it into clinical practice, it is important that this technique is able not only to identify cancer cells from their normal counterparts, but also from the array of cells present in human tissues. To this purpose, we used Raman spectroscopy to assess whether this technique was able to differentiate not only between lung cancer cells and lung epithelial cells but also from lung fibroblasts. Furthermore, we studied whether the differences were due to cell lineage (epithelial versus fibroblast) or to different proliferative characteristics of cells, and where in the cell compartment these differences might reside. To answer these questions we studied cell cytoplasm, cell nucleus and isolated whole cell nuclei. Our data suggests that Raman spectroscopy can differentiate between lung cancer, lung epithelial cells and lung fibroblasts. More important, it can also differentiate between 2 cells from the same lineage (fibroblast) but with one of them rendered immortal and with an increased proliferative activity. Finally, it seems that the main spectral differences reside in the cell nucleus and that the study of isolated nuclei strengthens the differences between cells.

Aligned Ice Templated Biomaterial Strategies for the Musculoskeletal System.

Aligned pore structures present many advantages when conceiving biomaterial strategies for treatment of musculoskeletal disorders. Aligned ice templating (AIT) is one of the many different techniques capable of producing anisotropic porous scaffolds; its high versatility allows for the formation of structures with tunable pore sizes, as well as the use of many different materials. AIT has been found to yield improved compressive properties for bone tissue engineering (BTE), as well as higher tensile strength and optimized cellular alignment and proliferation in tendon and muscle repair applications. This review evaluates the work that has been done in the last decade toward the production of aligned pore structures by AIT with an outlook on the musculoskeletal system. This work describes the fundamentals of the AIT technique and focuses on the research carried out to optimize the biomechanical properties of scaffolds by modifying the pore structure, categorizing by material type and application. Related topics including growth factor incorporation into AIT scaffolds, drug delivery applications, and studies about immune system response will be discussed.

Injectable hydrogels: An emerging therapeutic strategy for cartilage regeneration.

The impairment of articular cartilage due to traumatic incidents or osteoarthritis has posed significant challenges for healthcare practitioners, researchers, and individuals suffering from these conditions. Due to the absence of an approved treatment strategy for the complete restoration of cartilage defects to their native state, the tissue condition often deteriorates over time, leading to osteoarthritic (OA). However, recent advancements in the field of regenerative medicine have unveiled promising prospects through the utilization of injectable hydrogels. This versatile class of biomaterials, characterized by their ability to emulate the characteristics of native articular cartilage, offers the distinct advantage of minimally invasive administration directly to the site of damage. These hydrogels can also serve as ideal delivery vehicles for a diverse range of bioactive agents, including growth factors, anti-inflammatory drugs, steroids, and cells. The controlled release of such biologically active molecules from hydrogel scaffolds can accelerate cartilage healing, stimulate chondrogenesis, and modulate the inflammatory microenvironment to halt osteoarthritic progression. The present review aims to describe the methods used to design injectable hydrogels, expound upon their applications as delivery vehicles of biologically active molecules, and provide an update on recent advances in leveraging these delivery systems to foster articular cartilage regeneration.

Tendon tissue engineering: An overview of biologics to promote tendon healing and repair.

Tendons are dense connective tissues with a hierarchical polarized structure that respond to and adapt to the transmission of muscle contraction forces to the skeleton, enabling motion and maintaining posture. Tendon injuries, also known as tendinopathies, are becoming more common as populations age and participation in sports/leisure activities increases. The tendon has a poor ability to self-heal and regenerate given its intrinsic, constrained vascular supply and exposure to frequent, severe loading. There is a lack of understanding of the underlying pathophysiology, and it is not surprising that disorder-targeted medicines have only been partially effective at best. Recent tissue engineering approaches have emerged as a potential tool to drive tendon regeneration and healing. In this review, we investigated the physiochemical factors involved in tendon ontogeny and discussed their potential application in vitro to reproduce functional and self-renewing tendon tissue. We sought to understand whether stem cells are capable of forming tendons, how they can be directed towards the tenogenic lineage, and how their growth is regulated and monitored during the entire differentiation path. Finally, we showed recent developments in tendon tissue engineering, specifically the use of mesenchymal stem cells (MSCs), which can differentiate into tendon cells, as well as the potential role of extracellular vesicles (EVs) in tendon regeneration and their potential for use in accelerating the healing response after injury.