ALDH2 regulates mesenchymal stem cell senescence via modulation of mitochondrial homeostasis.

Although mitochondrial aldehyde dehydrogenase 2 (ALDH2) is involved in aging and aging-related diseases, its role in the regulation of human mesenchymal stem cell (MSC) senescence has not been investigated. This study aimed to determine the role of ALDH2 in regulating MSC senescence and illustrate the potential mechanisms. MSCs were isolated from young (YMSCs) and aged donors (AMSCs). Senescence-associated ß-galactosidase (SA-ß-gal) staining and Western blotting were used to assess MSC senescence. Reactive oxygen species (ROS) generation and mitochondrial membrane potential were determined to evaluate mitochondrial function. We showed that the expression of ALDH2 increased alongside cellular senescence of MSCs. Overexpression of ALDH2 accelerated YMSC senescence whereas down-regulation alleviated premature senescent phenotypes of AMSCs. Transcriptome and biochemical analyses revealed that an elevated ROS level and mitochondrial dysfunction contributed to ALDH2 function in MSC senescence. Using molecular docking, we identified interferon regulatory factor 7 (IRF7) as the potential target of ALDH2. Mechanistically, ectopic expression of ALDH2 led to mitochondrial dysfunction and accelerated senescence of MSCs by increasing the stability of IRF7 through a direct physical interaction. These effects were partially reversed by knockdown of IRF7. These findings highlight a crucial role of ALDH2 in driving MSC senescence by regulating mitochondrial homeostasis, providing a novel potential strategy against human aging-related diseases.

Unmasking Protein Phosphatase 2A Regulatory Subunit B as a Crucial Factor in the Progression of Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is one of the major causes of heart failure. Although significant progress has been made in elucidating the underlying mechanisms, further investigation is required for clarifying molecular diagnostic and therapeutic targets. In this study, we found that the mRNA level of protein phosphatase 2 regulatory subunit B' delta (Ppp2r5d) was altered in the peripheral blood plasma of DCM patients. Knockdown of Ppp2r5d in murine cardiomyocytes increased the intracellular levels of reactive oxygen species (ROS) and inhibited adenosine triphosphate (ATP) synthesis. In vivo knockdown of Ppp2r5d in an isoproterenol (ISO)-induced DCM mouse model aggravated the pathogenesis and ultimately led to heart failure. Mechanistically, Ppp2r5d-deficient cardiomyocytes showed an increase in phosphorylation of STAT3 at Y705 and a decrease in phosphorylation of STAT3 at S727. The elevated levels of phosphorylation at Y705 in STAT3 triggered the upregulation of interleukin 6 (IL6) expression. Moreover, the decreased phosphorylation at S727 in STAT3 disrupted mitochondrial electron transport chain function and dysregulated ATP synthesis and ROS levels. These results hereby reveal a novel role for Ppp2r5d in modulating STAT3 pathway in DCM, suggesting it as a potential target for the therapy of the disease.

Lentivirus-modified hematopoietic stem cell gene therapy for advanced symptomatic juvenile metachromatic leukodystrophy: A long-term follow-up pilot study.

Metachromatic leukodystrophy (MLD) is an inherited disease caused by a deficiency of the enzyme arylsulfatase A (ARSA). Lentivirus-modified autologous hematopoietic stem cell gene therapy (HSCGT) has recently been approved for clinical use in pre- and early-symptomatic children with MLD to increase ARSA activity. Unfortunately, this advanced therapy is not available for most patients with MLD who have progressed to more advanced symptomatic stages at diagnosis. Patients with late-onset juvenile MLD typically present with a slower neurological progression of symptoms and represent a significant burden to the economy and healthcare system, whereas those with early-onset infantile MLD die within a few years of symptom onset. We conducted a pilot study to determine the safety and benefit of HSCGT in patients with post-symptomatic juvenile MLD and report preliminary results. The safety profile of HSCGT was favorable in this long-term follow-up over nine years. The most common adverse events (AEs) within two months of HSCGT were related to busulfan conditioning, and all AEs resolved. No HSCGT-related AEs and no evidence of distorted hematopoietic differentiation during long-term follow-up for up to 9.6 years. Importantly, to date, patients have maintained remarkably improved ARSA activity with a stable disease state, including increased Functional Independence Measure (FIM) score and decreased magnetic resonance imaging (MRI) lesion score. This long-term follow-up pilot study suggests that HSCGT is safe and provides clinical benefit to patients with post-symptomatic juvenile MLD.

Exosomal miR-9-5p derived from iPSC-MSCs ameliorates doxorubicin-induced cardiomyopathy by inhibiting cardiomyocyte senescence.

Doxorubicin (DOX) is a chemotherapeutic agent widely used for tumor treatment. Nonetheless its clinical application is heavily limited by its cardiotoxicity. There is accumulated evidence that transplantation of mesenchymal stem cell-derived exosomes (MSC-EXOs) can protect against Dox-induced cardiomyopathy (DIC). This study aimed to examine the cardioprotective effects of EXOs isolated from human induced pluripotent stem cell-derived MSCs (iPSC-MSCs) against DIC and explore the potential mechanisms. EXOs were isolated from the cultural supernatant of human BM-MSCs (BM-MSC-EXOs) and iPSC-MSCs (iPSC-MSC-EXOs) by ultracentrifugation. A mouse model of DIC was induced by intraperitoneal injection of Dox followed by tail vein injection of PBS, BM-MSC-EXOs, or iPSC-MSC-EXOs. Cardiac function, cardiomyocyte senescence and mitochondrial dynamics in each group were assessed. In vitro, neonatal mouse cardiomyocytes (NMCMs) were subjected to Dox and treated with BM-MSC-EXOs or iPSC-MSC-EXOs. The mitochondrial morphology and cellular senescence of NMCMs were examined by Mitotracker staining and senescence-associated-ß-galactosidase assay, respectively. Compared with BM-MSC-EXOs, mice treated with iPSC-MSC-EXOs displayed improved cardiac function and decreased cardiomyocyte mitochondrial fragmentation and senescence. In vitro, iPSC-MSC-EXOs were superior to BM-MSC-EXOs in attenuation of cardiomyocyte mitochondrial fragmentation and senescence caused by DOX. MicroRNA sequencing revealed a higher level of miR-9-5p in iPSC-MSC-EXOs than BM-MSC-EXOs. Mechanistically, iPSC-MSC-EXOs transported miR-9-5p into DOX-treated cardiomyocytes, thereby suppressing cardiomyocyte mitochondrial fragmentation and senescence via regulation of the VPO1/ERK signal pathway. These protective effects and cardioprotection against DIC were largely reversed by knockdown of miR-9-5p in iPSC-MSC-EXOs. Our results showed that miR-9-5p transferred by iPSC-MSC-EXOs protected against DIC by alleviating cardiomyocyte senescence via inhibition of the VPO1/ERK pathway. This study offers new insight into the application of iPSC-MSC-EXOs as a novel therapeutic strategy for DIC treatment.

Downregulation of ALKBH5 rejuvenates aged human mesenchymal stem cells and enhances their therapeutic efficacy in myocardial infarction.

Despite promising results in myocardial infarction (MI), mesenchymal stem cell (MSC)-based therapy is limited by cell senescence. N6-methyladenosine (m6A) messenger RNA methylation has been reported to be closely associated with cell senescence. Nonetheless, its role in the regulation of MSC senescence remains unclear. We examined the role of ALKB homolog 5 (ALKBH5) in regulating MSC senescence and determined whether ALKBH5 downregulation could rejuvenate aged MSCs (AMSCs) to improve their therapeutic efficacy for MI. RNA methylation was determined by m6A dot blotting assay. MSC senescence was evaluated by senescence-associated ß-galactosidase (SA-ß-gal) staining. A mouse model of acute MI was established by ligation of the left anterior decedent coronary artery (LAD). Compared with young MSCs (YMSCs), m6A level was significantly reduced but ALKBH5 was greatly increased in AMSCs. Overexpression of ALKBH5 reduced m6A modification and accelerated YMSC senescence. Conversely, ALKBH5 knockdown increased m6A modifications and alleviated AMSC senescence. Mechanistically, ALKBH5 regulated the m6A modification and stability of CDKN1C mRNA, which further upregulated CDKN1C expression, leading to MSC senescence. CDKN1C overexpression ameliorated the inhibition of cellular senescence of ALKBH5 siRNA-treated AMSCs. More importantly, compared with AMSCs, shALKBH5-AMSCs transplantation provided a superior cardioprotective effect against MI in mice by improving MSC survival and angiogenesis. We determined that ALKBH5 accelerated MSC senescence through m6A modification-dependent stabilization of the CDKN1C transcript, providing a potential target for MSC rejuvenation. ALKBH5 knockdown rejuvenated AMSCs and enhanced cardiac function when transplanted into the mouse heart following infarction.

LINP1 represses unfolded protein response by directly inhibiting eIF2α phosphorylation to promote cutaneous squamous cell carcinoma.

BACKGROUND: Endoplasmic reticulum stress (ER stress) may destroy endoplasmic reticulum homeostasis (ER homeostasis) and leads to programmable cell death. Unfolded protein response (UPR) originally stimulated by ER stress is critical for the survival of tumor cells through trying to re-establish ER homeostasis as an adaption to harsh microenvironment. However, mechanisms involving key regulators in modulating UPR remain underexplored. METHODS: The expression of LINP1 in cutaneous squamous cell carcinoma (cSCC) tissues and cell lines was assessed. Subsequently, LINP1 was knocked out, knocked down or overexpressed in cSCC cells. CCK-8 assays, colony forming assays, transwell migration assays and invasiveness measurement by matrigel-coated transwell were performed to examine the role of LINP1 in cSCC development through gain-of-function and loss-of-function experiments. Transcriptomic sequencing (RNA-Seq) was conducted and indicated the key downstream signaling events regulated by LINP1 including UPR and apoptosis signaling. Furthermore, the direct interaction between LINP1 and eIF2α to modulate UPR and apoptosis was confirmed by RNA pulldown, RNA immunoprecipitation (RIP), ChIP-qPCR and in vitro phosphorylation assays. RESULTS: In this study, LncRNA in non-homologous end joining pathway 1 (LINP1) was identified to be one of the top ten highest-expressed LncRNAs in cSCC, the second most common cancer in the world. Functional studies using in vitro and in vivo models revealed that LINP1 functions as an oncogene to promote cell proliferation, colony formation, migration and invasiveness while inhibiting cell apoptosis in cSCC. Transcriptomic sequencing after knockdown of LINP1 indicated LINP1 negatively regulates UPR-related pathways involving key effectors for activating UPR and the apoptosis following the prolonged UPR. Mechanistic study showed LINP1 physically interacts with eIF2α to inhibit its phosphorylation for avoiding unmitigated UPR. Loss of LINP1 followed by enhanced eIF2α phosphorylation led to overactivated UPR and induced DDIT3 expression, contributing to ER stress-induced apoptosis and suppression of cSCC development. CONCLUSIONS: Our findings demonstrate a novel regulatory hierarchy of UPR by demonstrating LINP1 as a critical modulator for eIF2α phosphorylation and a suppressor of UPR-mediated apoptosis, which suggests a novel therapeutic target for cSCC treatment.

Direct administration of mesenchymal stem cell-derived mitochondria improves cardiac function after infarction via ameliorating endothelial senescence.

Mitochondrial dysfunction is considered to be a key contributor to the development of heart failure. Replacing injured mitochondria with healthy mitochondria to restore mitochondrial bioenergy in myocardium holds great promise for cardioprotection after infarction. This study aimed to investigate whether direct transplantation of exogenous mitochondria derived from mesenchymal stem cells (MSC-mt) is beneficial and superior in protecting cardiac function in a mouse model of myocardial infarction (MI) compared to mitochondria derived from skin fibroblast (FB-mt) and to explore the underlying mechanisms from their effects on the endothelial cells. The isolated MSC-mt presented intact mitochondrial morphology and activity, as determined by electron microscopy, JC-1 mitochondrial membrane potential assay, and seahorse assay. Direct injection of MSC-mt into the peri-infarct region in a mouse MI model enhanced blood vessel density, inhibited cardiac remodeling and apoptosis, thus improving heart function compared with FB-mt group. The injected MSC-mt can be tracked in the endothelial cells. In vitro, the fluorescence signal of MSC-mt can be detected in human umbilical vein endothelial cells (HUVECs) by confocal microscopy and flow cytometry after coculture. Compared to FB-mt, MSC-mt more effectively protected the HUVECs from oxidative stress-induced apoptosis and reduced mitochondrial production of reactive oxygen species. MSC-mt presented superior capacity in inducing tube formation, enhancing SCF secretion, ATP content and cell proliferation in HUVECs compared to FB-mt. Mechanistically, MSC-mt administration alleviated oxidative stress-induced endothelial senescence via activation of ERK pathway. These findings suggest that using MSCs as sources of mitochondria is feasible and that proangiogenesis could be the mechanism by which MSC-mt transplantation attenuates MI. MSC-mt transplantation might serve as a new therapeutic strategy for treating MI.

Resveratrol rescues cutaneous radiation-induced DNA damage via a novel AMPK/SIRT7/HMGB1 regulatory axis.

Cutaneous radiation injury (CRI) interrupts the scheduled process of radiotherapy and even compromises the life quality of patients. However, the current clinical options for alleviating CRI are relatively limited. Resveratrol (RSV) has been shown to be a promising protective agent against CRI; yet the mechanisms of RSV enhancing radioresistance were not fully elucidated and limited its clinical application. In this study, we demonstrate RSV promotes cutaneous radioresistance mainly through SIRT7. During ionizing radiation (IR) treatment, RSV indirectly phosphorylates and activates SIRT7 through AMPK, which is critical for maintaining the genome stability of keratinocytes. Immunoprecipitation and mass spectrometry identified HMGB1 to be the key interacting partner of SIRT7 to mediate the radioprotective function of RSV. Mechanistic study elucidated that SIRT7 interacts with and deacetylates HMGB1 to redistribute it into nucleus and "switch on" its function for DNA damage repair. Our findings establish a novel AMPK/SIRT7/HMGB1 regulatory axis that mediates the radioprotective function of RSV to alleviate IR-induced cutaneous DNA injury, providing an efficiently-curative option for patients with CRI during radiotherapy.

LILRB4, an immune checkpoint on myeloid cells.

Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor in the LILR family mainly expressed on normal and malignant human cells of myeloid origin. By binding to ligands, LILRB4 is activated and subsequently recruits adaptors to cytoplasmic immunoreceptor tyrosine inhibitory motifs to initiate different signaling cascades, thus playing an important role in physiological and pathological conditions, including autoimmune diseases, microbial infections, and cancers. In normal myeloid cells, LILRB4 regulates intrinsic cell activation and differentiation. In disease-associated or malignant myeloid cells, LILRB4 is significantly correlated with disease severity or patient survival and suppresses T cells, thereby participating in the pathogenesis of various diseases. In summary, LILRB4 functions as an immune checkpoint on myeloid cells and may be a promising therapeutic target for various human immune diseases, especially for cancer immunotherapy.

SALIS transcriptionally represses IGFBP3/Caspase-7-mediated apoptosis by associating with STAT5A to promote hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common subtype of liver cancer and the second most fatal cancer in the world despite the great therapeutic advances in the past two decades, which reminds us of the gap in fully understanding the oncogenic mechanism of HCC. To explore the key factors contributing to the progression of HCC, we identified a LncRNA, termed SALIS (Suppression of Apoptosis by LINC01186 Interacting with STAT5A), functions in promoting the proliferation, colony formation, migration and invasion while suppressing apoptosis in HCC cells. Mechanistic study indicated SALIS physically associates with transcription factor STAT5A and binds to the promoter regions of IGFBP3 and Caspase-7 to transcriptionally repress their expression and further inhibit apoptosis. Our findings identified SALIS as an oncogene to promote HCC by physically binding with STAT5A to inhibit the expression of pro-apoptotic IGFBP3 and Caspase-7, which suggests novel therapeutic targets for HCC treatments.

Intramyocardial injected human umbilical cord-derived mesenchymal stem cells (HucMSCs) contribute to the recovery of cardiac function and the migration of CD4+ T cells into the infarcted heart via CCL5/CCR5 signaling.

BACKGROUND: Human umbilical cord-derived mesenchymal stem cells (HucMSCs) have been recognized as a promising cell for treating myocardial infarction (MI). Inflammatory response post MI is critical in determining the cardiac function and subsequent adverse left ventricular remodeling. However, the local inflammatory effect of HucMSCs after intramyocardial injection in murine remains unclear. METHODS: HucMSCs were cultured and transplanted into the mice after MI surgery. Cardiac function of mice were analyzed among MI-N.S, MI-HucMSC and MI-HucMSC-C-C Motif Chemokine receptor 5 (CCR5) antagonist groups, and angiogenesis, fibrosis and hypertrophy, and immune cells infiltration of murine hearts were evaluated between MI-N.S and MI-HucMSC groups. We detected the expression of inflammatory cytokines and their effects on CD4+ T cells migration. RESULTS: HucMSCs treatment can significantly improve the cardiac function and some cells can survive at least 28 days after MI. Intramyocardial administration of HucMSCs also improved angiogenesis and alleviated cardiac fibrosis and hypertrophy. Moreover, we found the much higher numbers of CD4+ T cells and CD4+FoxP3+ regulatory T cells (Tregs) in the heart with HucMSCs than that with N.S treatment on day 7 post MI. In addition, the protein level of C-C Motif Chemokine Ligand 5 (CCL5) greatly increased in HucMSCs treated heart compared to MI-N.S group. In vitro, HucMSCs inhibited CD4+ T cells migration and addition of CCL5 antibody or CCR5 antagonist significantly reversed this effect. In vivo results further showed that addition of CCR5 antagonist can reduce the cardioprotective effect of HucMSCs administration on day 7 post MI injury. CONCLUSION: These findings indicated that HucMSCs contributed to cardiac functional recovery and attenuated cardiac remodeling post MI. Intramyocardial injection of HucMSCs upregulated the CD4+FoxP3+ Tregs and contributed to the migration of CD4+ T cells into the injured heart via CCL5/CCR5 pathway.

Apelin-13 Pretreatment Promotes the Cardioprotective Effect of Mesenchymal Stem Cells against Myocardial Infarction by Improving Their Survival.

Although mesenchymal stem cell- (MSC-) based therapy has shown promising results for myocardial infarction (MI), low cell survival heavily limits its beneficial effects. Apelin plays an essential regulatory role in cell proliferation. This study was aimed at determining whether Apelin-13 pretreatment could improve the survival of MSCs in the ischemic heart and enhance their cardioprotective efficacy against MI. MSCs were pretreated with or without Apelin-13 for 24 hours and then exposed to serum deprivation and hypoxia (SD/H) for 48 hours. The mitochondrial morphology of MSCs was assessed by MitoTracker staining. The apoptosis of MSCs was determined by TUNEL staining. The level of mitochondrial reactive oxygen species (ROS) of MSCs was detected by Mito-Sox staining. MSCs and Apelin-13-pretreated MSCs were transplanted into the peri-infarct region in a mouse MI model. Apelin-13 pretreatment protected MSCs against SD/H-induced mitochondrial fragmentation and apoptosis. Apelin-13 pretreatment reduced ROS generation induced by SD/H in MSCs. Furthermore, Apelin-13 pretreatment enhanced the angiogenesis of MSCs under SD/H conditions. Mechanistically, Apelin-13 pretreatment inhibited SD/H-induced MSC apoptosis by downregulating mitochondrial fission via activation of the ERK pathway, and these effects were partially abrogated by ERK inhibitor U0126. Apelin-13 pretreatment promoted the survival of MSCs in the ischemic heart. Moreover, transplantation with Apelin-13-pretreated MSCs improved heart function and increased angiogenesis accompanied by decreased fibrosis compared with MSC transplantation at 28 days following MI. These findings reveal that pretreatment with Apelin-13 improves MSCs survival and enhances their therapeutic efficacy for MI. Our study provides a novel approach to improve MSC-based therapy for cardiovascular disease.

PURPL represses autophagic cell death to promote cutaneous melanoma by modulating ULK1 phosphorylation.

Uncontrolled overactivation of autophagy may lead to autophagic cell death, suppression of which is a pro-survival strategy for tumors. However, mechanisms involving key regulators in modulating autophagic cell death remain poorly defined. Here, we report a novel long noncoding RNA, p53 upregulated regulator of p53 levels (PURPL), functions as an oncogene to promote cell proliferation, colony formation, migration, invasiveness, and inhibits cell death in melanoma cells. Mechanistic studies showed that PURPL promoted mTOR-mediated ULK1 phosphorylation at Ser757 by physical interacting with mTOR and ULK1 to constrain autophagic response to avoid cell death. Loss of PURPL led to AMPK-mediated phosphorylation of ULK1 at Ser555 and Ser317 to over-activate autophagy and induce autophagic cell death. Our results identify PURPL as a key regulator to modulate the activity of autophagy initiation factor ULK1 to repress autophagic cell death in melanoma and may represent a potential intervention target for melanoma therapy.

Hemin enhances the cardioprotective effects of mesenchymal stem cell-derived exosomes against infarction via amelioration of cardiomyocyte senescence.

BACKGROUND: Application of mesenchymal stem cell-derived exosomes (MSC-EXO) has emerged as a novel therapeutic strategy for myocardial infarction (MI). Our previous study showed that pretreatment with hemin, a potent heme oxygenase-1 (HO-1) inducer, enhanced the cardioprotective effects of MSCs in a mouse model of MI. This study aimed to investigate the therapeutic effects of EXO derived from hemin-pretreated MSCs (Hemin-MSC-EXO) in MI and explore the potential mechanisms. METHODS: MSC-EXO and Hemin-MSC-EXO were collected and characterized. MSC-EXO and Hemin-MSC-EXO were intramuscularly injected into the peri-infarct region in a mouse model of MI. Heart function of mice was assessed by echocardiography. The mitochondrial morphology of neonatal mice cardiomyocytes (NMCMs) under serum deprivation and hypoxic (SD/H) conditions was examined by Mitotracker staining. The cellular senescence of NMCMs was determined by senescence-associated-ß-galactosidase assay. A loss-of-function approach was adopted to determine the role of Hemin-MSC-exosomal-miR-183-5p in the regulation of cardiomyocyte senescence RESULTS: EXO were successfully isolated from the supernatant of MSCs and Hemin-pretreated MSCs. Compared with MSC-EXO, injection of Hemin-MSC-EXO significantly improved cardiac function and reduced fibrosis. Both MSC-EXO and Hemin-MSC-EXO ameliorated cardiomyocyte senescence and mitochondrial fission in vitro and in vivo, and the latter exhibited better protective effects. MicroRNA sequencing revealed a higher level of miR-183-5p in Hemin-MSC-EXO than in MSC-EXO. MiR-183-5p knockdown partially abrogated the protective effects of Hemin-MSC-EXO in attenuating mitochondrial fission and cellular senescence of cardiomyocytes induced by SD/H. High mobility group box-1 (HMGB1) abundance was lower in Hemin-MSC-EXO-treated than MSC-EXO-treated mouse hearts, and HMGB1 was identified as one of the potential target genes of miR-183-5p. Mechanistically, Hemin-MSC-EXO inhibited SD/H-induced cardiomyocyte senescence partially by delivering miR-183-5p into recipient cardiomyocytes via regulation of the HMGB1/ERK pathway. Furthermore, knockdown of miR-183-5p reduced the Hemin-MSC-EXO-mediated cardioprotective effects in a mouse model of MI. CONCLUSION: Our results reveal that Hemin-MSC-EXO are superior to MSC-EXO in treating MI. Exosomal miR-183-5p mediates, at least partially, the cardioprotective effects of Hemin-MSC-EXO by inhibiting cardiomyocyte senescence via regulation of the HMGB1/ERK pathway. This study highlights that MSC-EXO have high translational value in repairing cardiac dysfunction following infarction.

Conditioned medium from induced pluripotent stem cell-derived mesenchymal stem cells accelerates cutaneous wound healing through enhanced angiogenesis.

BACKGROUND: Mesenchymal stem cells (MSCs) can improve cutaneous wound healing via the secretion of growth factors. However, the therapeutic efficacy of MSCs varies depending upon their source. Induced pluripotent stem cells are emerging as a promising source of MSCs with the potential to overcome several limitations of adult MSCs. This study compared the effectiveness of conditioned medium of MSCs derived from induced pluripotent stem cells (iMSC-CdM) with that derived from umbilical cord MSCs (uMSC-CdM) in a mouse cutaneous wound healing model. We also investigated the mechanisms of protection. METHODS: The iMSC-CdM or uMSC-CdM were topically applied to mice cutaneous wound model. The recovery rate, scar formation, inflammation and angiogenesis were measured. We compared angiogenesis cytokine expression between iMSC-CdM and uMSC-CdM and their protective effects on human umbilical vein endothelial cells (HUVECs) under H2O2-induced injury. The effects of iMSC-CdM on energy metabolism, mitochondria fragmentation and apoptosis were measured. RESULTS: Topical application of iMSC-CdM was superior to the uMSC-CdM in accelerating wound closure and enhancing angiogenesis. Expression levels of angiogenetic cytokines were higher in iMSC-CdM than they were in uMSC-CdM. The iMSC-CdM protected HUVECs from H2O2 induced injury more effectively than uMSC-CdM did. Administration of iMSC-CdM stimulated HUVEC proliferation, tube formation and energy metabolism via the ERK pathway. Mechanistically, iMSC-CdM inhibited H2O2-induced mitochondrial fragmentation and apoptosis of HUVECs. CONCLUSION: Collectively, these findings indicate that iMSC-CdM is more effective than uMSC-CdM in treating cutaneous wounds, and in this way, iMSC-CdM may serve as a more constant and sustainable source for cell-free therapeutic approach.

Apelin Rejuvenates Aged Human Mesenchymal Stem Cells by Regulating Autophagy and Improves Cardiac Protection After Infarction.

The protective effects of mesenchymal stem cell (MSC)-based therapy for myocardial infarction (MI) are largely hampered as they age. Apelin is an endogenous ligand of its receptor APJ and plays an essential role in regulating multiple biological activities including MSC proliferation and survival. In this study, we investigated whether Apelin regulates MSC senescence and whether its overexpression could rejuvenate aged MSCs (AMSCs) to improve cardiac protection following infarction in mice. MSC senescence was evaluated by senescence-associated ß-galactosidase assays. Apelin level was examined by western blotting. Autophagy was determined by transmission electron microscopy. The cardioprotective effect of AMSCs with Apelin overexpression (Apelin-AMSCs) was assessed in a mouse MI model. Apelin expression was dramatically reduced in AMSCs. Interestingly, knockdown of Apelin induced young MSCs (YMSC) senescence, whereas overexpression rescued AMSC senescence. Apelin overexpression also increased AMSC angiogenic capacity. Mechanistically, Apelin overexpression upregulated the autophagy level of AMSCs by activating AMP-activated protein kinase (AMPK) signaling, thereby rejuvenating AMSCs. Compared with AMSCs, transplantation of Apelin-AMSCs achieved better therapeutic efficacy for MI by enhancing cell survival and angiogenesis. In conclusion, our results reveal that Apelin activates AMPK to rejuvenate AMSCs by increasing autophagy and promotes cardioprotection following infarction in mice. This study identified a novel target to rejuvenate AMSCs and enhance their therapeutic efficacy.

Myocardial ischemia-reperfusion induced cardiac extracellular vesicles harbour proinflammatory features and aggravate heart injury.

Extracellular vesicles (EVs) curb important biological functions. We previously disclosed that ischemia-reperfusion (IR) induces increased release of EVs (IR-EVs) in the heart. However, the role of IR-EVs in IR pathological process remains poorly understood. Here we found that adoptive transfer of IR-EVs aggravated IR induced heart injury, and EV inhibition by GW4869 reduced the IR injury. Our in vivo and in vitro investigations substantiated that IR-EVs facilitated M1-like polarization of macrophages with increased expression of proinflammatory cytokines. Further, we disclosed the miRNA profile in cardiac EVs and confirmed the enrichment of miRNAs, such as miR-155-5p in IR-EVs compared to EVs from the sham heart (S-EVs). In particular, IR-EVs transferred miR-155-5p to macrophages and enhanced the inflammatory response through activating JAK2/STAT1 pathway. Interestingly, IR-EVs not only boosted the local inflammation in the heart, but even triggered systemic inflammation in distant organs. Taken together, we newly identify an IR-EVs-miR-155-5p-M1 polarization axis in the heart post IR. The EVs derived from IR-injured heart contribute to both local and systemic inflammation. Importantly, EV inhibition by GW4869 is supposed to be a promising therapeutic strategy for IR injury.

Inhibition of miR-199a-5p rejuvenates aged mesenchymal stem cells derived from patients with idiopathic pulmonary fibrosis and improves their therapeutic efficacy in experimental pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an age-related disease with no cure. Mesenchymal stem cell (MSC)-based therapy has emerged as a novel strategy for IPF treatment. Nevertheless, MSCs derived from patients with IPF (IPF-MSCs) become senescent, thereby reducing their beneficial effects in IPF. MicroRNAs (miRNAs) mediate the senescence of MSCs, but the underlying mechanisms are not fully understood. We investigated the mechanisms by which miR-199a-5p regulates IPF-MSC senescence and whether its inhibition could rejuvenate IPF-MSCs and enhance their therapeutic efficacy. METHODS: Control-MSCs and IPF-MSCs were isolated from the adipose tissue of age-matched healthy and IPF donors, respectively. Cell senescence was examined by senescence-associated ß-galactosidase (SA-ß-gal) staining. The level of miR-199a-5p was measured by RT-PCR. Autophagy was determined using a transmission electron microscope (TEM). The therapeutic efficacy of anti-miR-199a-5p-IPF-MSCs was assessed using a mouse model of bleomycin-induced lung fibrosis. RESULTS: Despite similar surface makers, IPF-MSCs exhibited increased cellular senescence and decreased proliferative capacity compared with control-MSCs. The expression of miR-199a-5p was significantly enhanced in the serum of IPF patients and IPF-MSCs compared with that of healthy donors and control-MSCs. The upregulation of miR-199a-5p induced senescence of control-MSCs, whereas the downregulation rescued IPF-MSC senescence. Mechanistically, miR-155-5p suppressed autophagy of MSCs via the AMPK signaling pathway by downregulating the expression of Sirtuin 1(Sirt1), resulting in cellular senescence. Accordingly, miR-155-5p inhibition promoted autophagy and ameliorated IPF-MSC senescence by activating the Sirt1/AMPK signaling pathway. Compared with IPF-MSCs, the transplantation of anti-miR-199a-5p-IPF-MSCs increased the ability to prevent progression of pulmonary fibrosis in bleomycin-treated mice. CONCLUSIONS: Our study shows that miR-199a-5p regulates MSC senescence in patients with IPF by regulating the Sirt1/AMPK signaling pathway and miR-199a-5p is a novel target to rejuvenate IPF-MSCs and enhance their beneficial effects.

Adult mesenchymal stem cell ageing interplays with depressed mitochondrial Ndufs6.

Mesenchymal stem cell (MSC)-based therapy has emerged as a novel strategy to treat many degenerative diseases. Accumulating evidence shows that the function of MSCs declines with age, thus limiting their regenerative capacity. Nonetheless, the underlying mechanisms that control MSC ageing are not well understood. We show that compared with bone marrow-MSCs (BM-MSCs) isolated from young and aged samples, NADH dehydrogenase (ubiquinone) iron-sulfur protein 6 (Ndufs6) is depressed in aged MSCs. Similar to that of Ndufs6 knockout (Ndufs6-/-) mice, MSCs exhibited a reduced self-renewal and differentiation capacity with a tendency to senescence in the presence of an increased p53/p21 level. Downregulation of Ndufs6 by siRNA also accelerated progression of wild-type BM-MSCs to an aged state. In contrast, replenishment of Ndufs6 in Ndufs6-/--BM-MSCs significantly rejuvenated senescent cells and restored their proliferative ability. Compared with BM-MSCs, Ndufs6-/--BM-MSCs displayed increased intracellular and mitochondrial reactive oxygen species (ROS), and decreased mitochondrial membrane potential. Treatment of Ndufs6-/--BM-MSCs with mitochondrial ROS inhibitor Mito-TEMPO notably reversed the cellular senescence and reduced the increased p53/p21 level. We provide direct evidence that impairment of mitochondrial Ndufs6 is a putative accelerator of adult stem cell ageing that is associated with excessive ROS accumulation and upregulation of p53/p21. It also indicates that manipulation of mitochondrial function is critical and can effectively protect adult stem cells against senescence.

Acetylation dependent functions of Rab22a-NeoF1 Fusion Protein in Osteosarcoma.

Background: Rab22a-NeoF1 fusion gene containing the 1-38aa of Rab22a (Rab22a1-38) plays a decisive role in driving tumor metastasis by activating RhoA via binding to SmgGDS607. However, its intercellular regulation remains unknown. Methods: The Lys7 (K7) acetylation of Rab22a-NeoF1 was initially identified by mass spectrum. Co-transfection, immunoprecipitation and Western blotting were used to characterize the acetyltransferases and deacetylases responsible for the K7 acetylation of Rab22a-NeoF1, and to define the interaction of proteins. The specificity of K7 acetylation of Rab22a-NeoF1 was determined by its specific anti-K7ac-Rab22a-NeoF1 antibody and its K7R mutant. RhoA-GTP was measured by RhoA activation assay. The migration and invasion were assessed by Transwell assay without and with Matrigel matrix, respectively. The orthotopic osteosarcoma metastasis model in vivo was used to monitor the lung metastases of U2OS/MTX300-Luc stably expressing Vector, Rab22a-NeoF1 or its K7R mutant with or without C646, a relatively specific inhibitor of p300/CBP. The unpaired Student t test was used for the statistical significance. Results: The K7 of Rab22a-NeoF1 is acetylated by p300/CBP while is de-acetylated by both HDAC6 and SIRT1. The K7R mutant of Rab22a-NeoF1 lacks its binding to SmgGDS607 and subsequently lost its promoting functions, such as activation of RhoA, cell migration, invasion and lung metastasis in osteosarcoma in vitro and in vivo, which are also diminished by p300/CBP inhibitor C646. Conclusion:The promoting function of Rab22a-NeoF1 is dependent on its K7 acetylation in osteosarcoma, and targeting this acetylation (e.g., C646) may benefit cancer patients, in particular osteosarcoma patients, who are positive for the Rab22a1-38.