Exosomal miR-17-5p from human embryonic stem cells prevents pulmonary fibrosis by targeting thrombospondin-2.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible lung disease characterized by pulmonary fibrosis and lung dysfunction, ultimately leading to respiratory failure. Many preclinical studies have investigated the therapeutic potential of stem cell-derived exosomes in this disease, particularly mesenchymal stem cell-derived exosomes. However, the effects of embryonic stem cell-derived exosomes in IPF remain unclear. METHODS: We established a bleomycin (BLM)-induced pulmonary fibrosis mice model and administered human embryonic stem cell exosomes (hESC-exo) from the first day after BLM treatment. The effects of hESC-exo were assessed by pulmonary function tests, biochemical analysis, histochemistry, quantitative real-time polymerase chain reaction (qPCR), and western blot (WB). RNA-seq was used to screen for the potential therapeutic targets of hESC-exo in fibrotic lungs; the identified signaling axis was characterized using a luciferase assay, qPCR, and WB. RESULTS: Results indicated hESC-exo administration notably alleviated inflammation, removed deposited collagen, and rescued alveolar architecture in the lungs of BLM-induced mice. In vivo and in vitro tests revealed that hESC-exo-derived miR-17-5p directly bound thrombospondin-2 (Thbs2) to regulate inflammation and fibrosis; thus, hESC-exo protected against BLM toxicity in the lungs via the miR-17-5p/Thbs2 axis. CONCLUSION: These results suggest a promising new treatment for fibrosis-associated diseases.

Circular RNA and intervertebral disc degeneration: unravelling mechanisms and implications.

Low back pain (LBP) is a major public health problem worldwide and a significant health and economic burden. Intervertebral disc degeneration (IDD) is the reason for LBP. However, we have not identified effective therapeutic strategies to address this challenge. With accumulating knowledge on the role of circular RNAs in the pathogenesis of IDD, we realised that circular RNAs (circRNAs) may have tremendous therapeutic potential and clinical application prospects in this field. This review presents an overview of the current understanding of characteristics, classification, biogenesis, and function of circRNAs and summarises the protective and detrimental circRNAs involved in the intervertebral disc that have been studied thus far. This review is aimed to help researchers better understand the regulatory role of circRNAs in the progression of IDD, reveal their clinical therapeutic potential, and provide a theoretical basis for the prevention and targeted treatment of IDD.

Mesenchymal stem cells alleviate aging in vitro and in vivo.

Background: Aging is a natural and multi-factorial phenomenon associated with multiple human pathologies. Mesenchymal stem cells (MSCs) hold great promise in clinical fields of medicine including tissue repair, cardiovascular disease, and brain ischemic injury. The purpose of this study was to explore the roles of MSCs in improving the condition of aging cells, repairing aging tissues and organs, and extending the life span of elderly mice. Methods: This study was carried out both in vitro and in vivo. We used MSCs to intervene with IMR-90 senescent cells induced by D-galactose and aged C57BL/6 mice. Results: After 48 hours of co-culturing the aged cells with MSCs, the up-regulated expression of inflammatory factor, interleukin 6 (IL6), and the down-regulated expression of several growth factors, such as transforming growth factor (TGFß1) and growth differentiation factor (GDF11), in D-galactose induced senescent cells were reversed. Moreover, compared with aged cells, the number of mitochondria and the telomere length were increased with MSC treatment. Similarly, in aged mice, the symptoms related to aging were improved after MSC treatment: the mouse hair became shiny and dense, and the symptoms of bladder overactivity were relieved. Hematoxylin and eosin (H&E) and Masson's trichrome staining showed that the histopathological changes in skin, bladder, liver, and lung were apparently improved. Conclusions: Treatment with MSCs effectively improves aging-related phenotypes and plays a beneficial role in improving aging and aging-related diseases.

Noncoding RNA PVT1 in osteosarcoma: The roles of lncRNA PVT1 and circPVT1.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and teenagers and is characterized by high malignant potential, rapid disease progression and high disability and mortality rates. Recently, noncoding RNAs (ncRNAs) have attracted the attention of many scholars due to their major regulatory roles in gene expression. Among them, lncRNA PVT1 and circPVT1 encoded by the PVT1 gene have been the focus of many studies; they are upregulated in OS, and abundant evidence indicates that lncRNA PVT1 and circPVT1 play key roles in the occurrence and development of OS. This review summarizes the mechanisms of action of lncRNA PVT1 and circPVT1 in regulating apoptosis, proliferation, glycolysis, invasion, migration and epithelial-mesenchymal transition (EMT) in OS and discusses their clinical applications in diagnosis, prognosis determination and drug resistance treatment, with the aim of helping researchers better understand the regulatory roles of lncRNA PVT1 and circPVT1 in OS progression and providing a theoretical basis for the development of early screening and accurate targeted treatment strategies and prognostic biomarkers for OS based on lncRNA PVT1 and circPVT1.

Systemic proteomics and miRNA profile analysis of exosomes derived from human pluripotent stem cells.

BACKGROUND: Increasing studies have reported the therapeutic effect of mesenchymal stem cell (MSC)-derived exosomes by which protein and miRNA are clearly characterized. However, the proteomics and miRNA profiles of exosomes derived from human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) remain unclear. METHODS: In this study, we isolated exosomes from hESCs, hiPSCs, and human umbilical cord mesenchymal stem cells (hUC-MSCs) via classic ultracentrifugation and a 0.22-µm filter, followed by the conservative identification. Tandem mass tag labeling and label-free relative peptide quantification together defined their proteomics. High-throughput sequencing was performed to determine miRNA profiles. Then, we conducted a bioinformatics analysis to identify the dominant biological processes and pathways modulated by exosome cargos. Finally, the western blot and RT-qPCR were performed to detect the actual loads of proteins and miRNAs in three types of exosomes. RESULTS: Based on our study, the cargos from three types of exosomes contribute to sophisticated biological processes. In comparison, hESC exosomes (hESC-Exos) were superior in regulating development, metabolism, and anti-aging, and hiPSC exosomes (hiPSC-Exos) had similar biological functions as hESC-Exos, whereas hUC-MSCs exosomes (hUC-MSC-Exos) contributed more to immune regulation. CONCLUSIONS: The data presented in our study help define the protein and miRNA landscapes of three exosomes, predict their biological functions via systematic and comprehensive network analysis at the system level, and reveal their respective potential applications in different fields so as to optimize exosome selection in preclinical and clinical trials.

CUGBP1, a crucial factor for heart regeneration in mice.

The mammalian heart is capable of achieving perfect regeneration following cardiac injury through sustained cardiomyocyte proliferation during the early period after birth. However, this regenerative capacity is lost by postnatal day 7 and throughout adulthood. CUGBP1 is critical for normal cardiac development but its role in heart regeneration remains unclear. Cardiac CUGBP1 levels are high in the early postnatal period and soon downregulate to adult levels within 1 week following birth in mice. The simultaneously diminished regenerative capacity and CUGBP1 levels by postnatal day lead us to hypothesize that CUGBP1 may be beneficial in heart regeneration. In this study, the function of CUGBP1 in heart regeneration was tested by a heart apex resection mouse model. We demonstrate that cardiac inactivation of CUGBP1 impairs neonatal heart regeneration at P1, in turn, replenishment of CUGBP1 levels prolong regenerative potential at P8 and P14. Furthermore, our results imply that the Wnt/ß-catenin signaling and GATA4 involve in the CUGBP1 modulated neonatal heart regeneration. Altogether, our findings support CUGBP1 as a key factor promoting post-injury heart regeneration and provide a potential therapeutic method for heart disease.

ERp44 is required for endocardial cushion development by regulating VEGFA secretion in myocardium.

OBJECTIVES: Endocardial cushions are precursors of the valve septum complex that separates the four heart chambers. Several genes have been implicated in the development of endocardial cushions. Specifically, ERp44 has been found to play a role in the early secretory pathway, but its function in heart development has not been well studied. MATERIALS AND METHODS: In this study, we established conditional and tissue-specific knockout mouse models. The morphology, survival rate, the development of heart and endocardial cushion were under evaluation. The relationship between ERp44 and VEGFA was investigated by transcriptome, qPCR, WB, immunofluorescence and immunohistochemistry. RESULTS: ERp44 knockout (KO) mice were smaller in size, and most mice died during early postnatal life. KO hearts exhibited the typical phenotypes of congenital heart diseases, such as abnormal heart shapes and severe septal and valvular defects. Similar phenotypes were found in cTNT-Cre+/- ; ERp44fl / fl mice, which indicated that myocardial ERp44 principally controls endocardial cushion formation. Further studies demonstrated that the deletion of ERp44 significantly decreased the proliferation of cushion cells and impaired the endocardial-mesenchymal transition (EndMT), which was followed by endocardial cushion dysplasia. Finally, we found that ERp44 was directly bound to VEGFA and controlled its release, further regulating EndMT. CONCLUSION: We demonstrated that ERp44 plays a specific role in heart development. ERp44 contributes to the development of the endocardial cushion by affecting VEGFA-mediated EndMT.

Bone marrow derived-mesenchymal stem cell improves diabetes-associated fatty liver via mitochondria transformation in mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has become a global epidemic disease. Its incidence is associated with type 2 diabetes mellitus (T2DM). Presently, there is no approved pharmacological agents specially developed for NAFLD. One promising disease-modifying strategy is the transplantation of stem cells to promote metabolic regulation and repair of injury. METHOD: In this study, a T2DM model was established through 28-week high-fat diet (HFD) feeding resulting in T2DM-associated NAFLD, followed by the injection of bone marrow mesenchymal stem cells (BMSCs). The morphology, function, and transfer of hepatocyte mitochondria were evaluated in both vivo and in vitro. RESULTS: BMSC implantation resulted in the considerable recovery of increasing weight, HFD-induced steatosis, liver function, and disordered glucose and lipid metabolism. The treatment with BMSC transplantation was accompanied by reduced fat accumulation. Moreover, mitochondrial transfer was observed in both vivo and vitro studies. And the mitochondria-recipient steatotic cells exhibited significantly enhanced OXPHOS activity, ATP production, and mitochondrial membrane potential, and reduced reactive oxygen species levels, which were not achieved by the blocking of mitochondrial transfer. CONCLUSION: Mitochondrial transfer from BMSCs is a feasible process to combat NAFLD via rescuing dysfunction mitochondria, and has a promising therapeutic effect on metabolism-related diseases.

Adipose-derived stem cells therapy effectively attenuates PM2.5-induced lung injury.

BACKGROUND: The adverse health effects of fine particulate matter (PM2.5) exposure are associated with marked inflammatory responses. Adipose-derived stem cells (ADSCs) have immunosuppressive effects, and ADSC transplantation could attenuate pulmonary fibrosis in different animal disease models. However, whether ADSCs affect PM2.5-induced lung injury has not been investigated. METHOD: C57BL/6 mice were exposed to PM2.5 every other day via intratracheal instillation for 4 weeks. After that, the mice received tail vein injections of ADSCs every 2 weeks. RESULTS: ADSC transplantation significantly attenuated systemic and pulmonary inflammation, cardiac dysfunction, fibrosis, and cell death in PM2.5-exposed mice. RNA-sequencing results and bioinformatic analysis suggested that the downregulated differentially expressed genes (DEGs) were mainly enriched in inflammatory and immune pathways. Moreover, ADSC transplantation attenuated PM2.5-induced cell apoptosis and pyroptosis in the lungs and hearts. CONCLUSION: ADSCs protect against PM2.5-induced adverse health effects through attenuating pulmonary inflammation and cell death. Our findings suggest that ADSC transplantation may be a potential therapeutic approach for severe air pollution-associated diseases.

A Novel System for Simple Rapid Adenoviral Vector Construction to Facilitate CRISPR/Cas9-Mediated Genome Editing.

Recombinant adenoviruses have broad applications for gene delivery and expression. Furthermore, the adenovirus packaging system facilitates the expression of RNA-guided CRISPR/Cas9 nuclease complexes. In this study, we developed a novel system, named AdBlue, for the construction of recombinant adenoviruses using an enzymatic assembly strategy. This system could significantly reduce the time and labor required to generate adenoviral vectors. When applied to CRISPR/Cas9 design, it simplifies the preparation of recombinant adenoviruses carrying nuclease complexes and can induce high levels of site-specific mutagenesis. Our system has outstanding advantages for adenovirus preparation and could be a useful molecular engineering tool for gene delivery and editing.

ERp44/CG9911 promotes fat storage in Drosophila adipocytes by regulating ER Ca2+ homeostasis.

Fat storage is one of the important strategies employed in regulating energy homeostasis. Impaired lipid storage causes metabolic disorders in both mammals and Drosophila. In this study, we report CG9911, the Drosophila homolog of ERp44 (endoplasmic reticulum protein 44) plays a role in regulating adipose tissue fat storage. Using the CRISPR/Cas9 system, we generated a CG9911 mutant line deleting 5 bp of the coding sequence. The mutant flies exhibit phenotypes of lower bodyweight, fewer lipid droplets, reduced TAG level and increased expression of lipolysis related genes. The increased lipolysis phenotype is enhanced in the presence of ER stresses and suppressed by a reduction of the ER Ca2+. Moreover, loss of CG9911 per se results in a decrease of ER Ca2+ in the fat body. Together, our results reveal a novel function of CG9911 in promoting fat storage via regulating ER Ca2+ signal in Drosophila.

Reduced Ca2+ spark activity contributes to detrusor overactivity of rats with partial bladder outlet obstruction.

We tested whether or not altered Ca2+ spark activity accounted for detrusor overactivity (DO) of Wistar rats after partial bladder outlet obstruction (PBOO). We constructed a DO model through PBOO and studied the Ca2+ spark activity of detrusor. By way of using confocal microscopy and the patch-clamp technique, Ca2+ sparks and spontaneous transient outward currents (STOCs) in detrusor myocytes were measured respectively. Our results indicated that Ca2+ spark activity and STOCs were significantly reduced in the DO detrusor myocytes compared to unafflicted control cells, and both of these had levels that were remarkably increased by applications of caffeine (10 µM), a RyR agonist, in DO myocytes. In addition, measures of detrusor contractions were also recorded by using freshly isolated detrusor strips. These results indicated that the spontaneous contraction of DO detrusor was significantly enhanced, and that the effect of caffeine (10 µM) upon detrusor contractions was reversed by applications of iberiotoxin (100 nM) which is a BK channel blocker. Western blotting (WB) analyses indicated that the levels of expression of ryanodine receptor type 2 (RyR2) and FK506 binding protein 12.6 (FKBP12.6) in bladder muscle were respectively decreased and increased in the samples from DO rats. Thus, we considered in the rat DO model wherein PBOO, the reduced Ca2+ spark activity in detrusor myocytes partly contributed to overactive detrusor contractions. The impaired Ca2+ spark activity may have resulted from decreased RyR2 expression and increased FKBP12.6 expression. Such novel findings in our research might help to provide means for better treatment outcomes for patients afflicted by bladder dysfunction.

The role of ERp44 in glucose and lipid metabolism.

Endoplasmic Reticulum Protein 44 (ERp44) is a member of the PDI family, named for a molecular weight of 44 kD. White adipose tissue has metabolic and endocrine functions that are important to metabolism. The role of ERp44 in glucose and lipid metabolism is not known yet. The current study was undertaken to investigate the implication of ERp44 in glucose and lipid metabolism. In this study, we generated and characterized ERp44-/- mice. We used type 2 diabetes models and ERp44 knockout mice to show the implication of ERp44 in glucose and lipid metabolism. Knockout newborns had lower blood glucose compared to wild-type. Adult knockouts had abnormal intraperitoneal, glucose, insulin and pyruvic acid tolerance. Lipocytes were smaller and fewer in knockout mice compared to wild-type. Knockouts resisted to high-fat diet-induced obesity. ERp44 expression in white adipose tissue decreased significantly in type 2 diabetes models. Results suggest that ERp44 is closely associated with glucose and lipid metabolism.

A chemical approach for global protein knockdown from mice to non-human primates.

Although conventional genetic modification approaches for protein knockdown work very successfully due to the increasing use of CRISPR/Cas9, effective techniques for achieving protein depletion in adult animals, especially in large animals such as non-human primates, are lacking. Here, we report a chemical approach based on PROTACs technology that efficiently and quickly knocks down FKBP12 (12-kDa FK506-binding) protein globally in vivo. Both intraperitoneal and oral administration led to rapid, robust, and reversible FKBP12 degradation in mice. The efficiency and practicality of this method were successfully demonstrated in both large and small animals (mice, rats, Bama pigs, and rhesus monkeys). Furthermore, we showed this approach can also be applied to effectively knockdown other target proteins such as Bruton's tyrosine kinase (BTK). This chemical protein knockdown strategy provides a powerful research tool for gene function studies in animals, particularly in large animals, for which gene-targeted knockout strategies may remain unfeasible.

A positive feedback regulation of Heme oxygenase 1 by CELF1 in cardiac myoblast cells.

As an RNA binding protein, CUG-BP Elav-like family (CELF) has been shown to be critical for heart biological functions. However, no reports have revealed the function of CELF1 in hypertrophic cardiomyopathy (HCM). Hinted by RNA immunoprecipitation-sequencing (RIP-seq) data, the influence of the CELF protein on heme oxygenase-1 (HO-1) expression was tested by modulating CELF1 levels. Cardiac hypertrophy is related to oxidative stress-induced damage. Hence, the cardiovascular system may be protected against further injury by upregulating the expression of antioxidant enzymes, such as HO-1. During the past two decades, research has demonstrated the central role of HO-1 in the protection against diseases. Thus, understanding the molecular mechanisms underlying the modulation of HO-1 expression is profoundly important for developing new strategies to prevent cardiac hypertrophy. To elucidate the molecular mechanisms underlying HO-1 regulation by the CELF protein, we performed RNA immunoprecipitation (RIP), biotin pull-down analysis, luciferase reporter and mRNA stability assays. We found that the expression of HO-1 was downregulated by CELF1 through the conserved GU-rich elements (GREs) in HO-1 3'UTR transcripts. Correspondingly, CELF1 expression was regulated by controlling the release of carbon monoxide (CO) in H9C2 cells. The CELF1-HO-1-CO regulation axis constituted a novel positive feedback circuit. In addition, we detected the potential involvement of CELF1 and HO-1 in samples from HCM patients. We found that CELF1 and CELF2, but not HO-1, were highly expressed in HCM heart samples. Thus, a manipulation targeting CELF1 could be developed as a potential therapeutic option for cardiac hypertrophy.

Polygonum aviculare L. extract and quercetin attenuate contraction in airway smooth muscle.

Because of the serious side effects of the currently used bronchodilators, new compounds with similar functions must be developed. We screened several herbs and found that Polygonum aviculare L. contains ingredients that inhibit the precontraction of mouse and human airway smooth muscle (ASM). High K+-induced precontraction in ASM was completely inhibited by nifedipine, a selective blocker of L-type voltage-dependent Ca2+ channels (LVDCCs). However, nifedipine only partially reduced the precontraction induced by acetylcholine chloride (ACH). Additionally, the ACH-induced precontraction was partly reduced by pyrazole-3 (Pyr3), a selective blocker of TRPC3 and stromal interaction molecule (STIM)/Orai channels. These channel-mediated currents were inhibited by the compounds present in P. aviculare extracts, suggesting that this inhibition was mediated by LVDCCs, TRPC3 and/or STIM/Orai channels. Moreover, these channel-mediated currents were inhibited by quercetin, which is present in P. aviculare extracts. Furthermore, quercetin inhibited ACH-induced precontraction in ASM. Overall, our data indicate that the ethyl acetate fraction of P. aviculare and quercetin can inhibit Ca2+-permeant LVDCCs, TRPC3 and STIM/Orai channels, which inhibits the precontraction of ASM. These findings suggest that P. aviculare could be used to develop new bronchodilators to treat obstructive lung diseases such as asthma and chronic obstructive pulmonary disease.

CD38 deficiency suppresses adipogenesis and lipogenesis in adipose tissues through activating Sirt1/PPARγ signaling pathway.

It has been recently reported that CD38 was highly expressed in adipose tissues from obese people and CD38-deficient mice were resistant to high-fat diet (HFD)-induced obesity. However, the role of CD38 in the regulation of adipogenesis and lipogenesis is unknown. In this study, to explore the roles of CD38 in adipogenesis and lipogenesis in vivo and in vitro, obesity models were generated with male CD38-/- and WT mice fed with HFD. The adipocyte differentiations were induced with MEFs from WT and CD38-/- mice, 3T3-L1 and C3H10T1/2 cells in vitro. The lipid accumulations and the alternations of CD38 and the genes involved in adipogenesis and lipogenesis were determined with the adipose tissues from the HFD-fed mice or the MEFs, 3T3-L1 and C3H10T1/2 cells during induction of adipocyte differentiation. The results showed that CD38-/- male mice were significantly resistant to HFD-induced obesity. CD38 expressions in adipocytes were significantly increased in WT mice fed with HFD, and the similar results were obtained from WT MEFs, 3T3-L1 and C3H10T1/2 during induction of adipocyte differentiation. The expressions of PPARγ, AP2 and C/EBPα were markedly attenuated in adipocytes from HFD-fed CD38-/- mice and CD38-/- MEFs at late stage of adipocyte differentiation. Moreover, the expressions of SREBP1 and FASN were also significantly decreased in CD38-/- MEFs. Finally, the CD38 deficiency-mediated activations of Sirt1 signalling were up-regulated or down-regulated by resveratrol and nicotinamide, respectively. These results suggest that CD38 deficiency impairs adipogenesis and lipogenesis through activating Sirt1/PPARγ-FASN signalling pathway during the development of obesity.

Distinct Effects of Ca2+ Sparks on Cerebral Artery and Airway Smooth Muscle Cell Tone in Mice and Humans.

The effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone, as well as the underlying mechanisms, are not clear. In this investigation, we elucidated the underlying mechanisms of the distinct effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone. In CASMCs, owing to the functional loss of Ca2+-activated Cl- (Clca) channels, Ca2+ sparks activated large-conductance Ca2+-activated K+ channels (BKs), resulting in a decreases in tone against a spontaneous depolarization-caused high tone in the resting state. In ASMCs, Ca2+ sparks induced relaxation through BKs and contraction via Clca channels. However, the integrated result was contraction because Ca2+ sparks activated BKs prior to Clca channels and Clca channels-induced depolarization was larger than BKs-caused hyperpolarization. However, the effects of Ca2+ sparks on both cell types were determined by L-type voltage-dependent Ca2+ channels (LVDCCs). In addition, compared with ASMCs, CASMCs had great and higher amplitude Ca2+ sparks, a higher density of BKs, and higher Ca2+ and voltage sensitivity of BKs. These differences enhanced the ability of Ca2+ sparks to decrease CASMC and to increase ASMC tone. The higher Ca2+ and voltage sensitivity of BKs in CASMCs than ASMCs were determined by the ß1 subunits. Moreover, Ca2+ sparks showed the similar effects on human CASMC and ASMC tone. In conclusions, Ca2+ sparks decrease CASMC tone and increase ASMC tone, mediated by BKs and Clca channels, respectively, and finally determined by LVDCCs.