Hypoxic Pretreatment Research Articles

Exosomes from Hypoxic Pretreatment ADSCs Ameliorate Cardiac Damage Post-MI via Activated circ-Stt3b/miR-15a-5p/GPX4 Signaling and Decreased Ferroptosis.

Accumulation studies confirmed that oxidative stress caused by ischemia after myocardial infarction (MI) is an important cause of ventricular remodeling. Exosome secretion through hypoxic pretreatment adipose-derived mesenchymal stem cells (ADSCs) ameliorates myocardial damaging post-MI. However, if ADSCs exosome can improve the microenvironment and ameliorate cardiac damage post-MI still unknown. Next-generation sequencing (NGS) was used to study abnormally expressed circRNAs in hypoxic pretreatment ADSC exosomes (HExos) and untreated ADSC exosomes (Exos). Bioinformatics and luciferase reporting were used to elucidate interaction correlation related to circRNA, mRNA, and miRNA. HL-1 cells were used to analyze the reactive oxygen species (ROS) and apoptosis under hypoxic conditions using immunofluorescence and flow cytometry. An MI mouse model was constructed and the therapeutic effect of Exos was determined using immunohistochemistry, immunofluorescence, and ELISA. The results showed that HExos had a more pronounced treatment effect than ADSC Exos on cardiac damage amelioration after MI. NGS showed that circ-Stt3b plays a role in HExo-mediated cardiac damage repair after MI. Overexpression of circ-Stt3b decreased apoptosis, ROS level, and inflammatory factor expression in HL-1 cells under hypoxic conditions. Bioinformatics and luciferase reporting data validated miR-15a-5p and GPX4 as downstream circ-Stt3b targets. GPX4 downregulation or miR-15a-5p overexpression reversed protective effect regarding circ-Stt3b upon HL-1 cells after exposure to a hypoxic microenvironment. Overexpression of circ-Stt3b increased the treatment effect of ASDSC Exos on cardiac damage amelioration after MI. Taken together, the study results demonstrated that Exos from hypoxic pretreatment ADSCs ameliorate cardiac damage post-MI through circ-Stt3b/miR-15a-5p/GPX4 signaling activation and decreased ferroptosis.

Circ-Erbb2ip from adipose-derived mesenchymal stem cell-derived exosomes promotes wound healing in diabetic mice by inducing the miR-670-5p/Nrf1 axis

BackgroundTo investigate the mechanism of exosomes (Exo) secretion by hypoxic pretreated adipose-derived mesenchymal stem cells (ADSCs) promoting skin wound healing in diabetic (DM) mice. MethodsHigh-throughput sequencing was used to investigate abnormal expression of circRNA in hypoxic pretreatment ADSCs exosome (HExo) and ADSCs exosome (Exo). Bioinformatics analysis and luciferase reporting analysis were used to clarify the interacted relationship among circRNA, miRNA and mRNA. EPCs cells were employ to analysis the ROS, inflammatory cytokines expression, angiogenic differentiation function under hypoxic condition by using immunofluorescence, ELISA detection and tube forming experiment. DM ulceration mice model were constructed and the therapeutic effect of Exo were detected using immunohistochemistry, immunofluorescence. ResultsThe result show that HExo have more treatment effect than Exo in promotes cutaneous wound healing of DM mice. High-throughput sequencing found that circ-Erbb2ip play a role in HExo mediated tissues repair. Downregulation circ-Erbb2ip decreased the therapeutic effect of HExo to wound healing in diabetic mice. Bioinformatics analysis and luciferase reporting analysis confirmed that both miR-670-5p and Nrf1 were downstream targets of circ-Erbb2ip. Downregulation of Nrf1 or overexpression of miR-670-5p reversed the protective effect of circ-Erbb2ip to EPCs after exposure to high glucose microenvironment. Upregulation circ-Erbb2ip increased the therapeutic effect of Exo to wound healing in diabetic mice by increased angiogenesis and decreased ROS, inflammatory cytokines expression. ConclusionIn conclusion, ADSC-Exos containing circ-Erbb2ip promotes wound healing by targeting miR-670-5p/Nrf1 pathway, and their effects in promoting soft tissue wound healing warrant further study.

Intermittent hypoxic pretreatment exacerbates house dust mite-induced asthma airway inflammation.

Asthma is widely recognized as an inflammatory disorder. In the context of this inflammatory microenvironment, the involvement of hypoxia and its impact on related pathways have drawn considerable attention. However, the exact role of hypoxia, a prevalent environmental factor, in the development and progression of asthma remains poorly understood. Mice were treated with house dust mite (HDM) extracts for 23 days to induce asthma. Mice were divided into room air (RA) group and intermittent hypoxic (IH) group by exposing to different conditions and IH preconditioning (IHP) were underwent to the above groups before the hypoxic regimen. Airway inflammation in mice was evaluated by airway hyperresponsiveness, excessive mucus secretion, and recruitment of inflammatory cells. Immunohistochemistry was employed to quantify the expression levels of NF-κB. Subsequently, the dose of allergen was modified to investigate whether the impact of hypoxia on asthma is affected by different doses of allergens. Compared to the RA and IH groups, HDM-treated mice in the IHP group exhibited aggravated inflammatory cell infiltration and airway hyperresponsiveness (p＜.05). Moreover, there was an increased release of inflammatory mediators and higher expression levels of NF-κB (p＜.05). Importantly, the impact ia on asthma was found to be influenced by high dose of allergen (p＜.05). IHP treatment potentially exacerbates HDM-induced airway inflammation in asthma, with the involvement of NF-κB, particularly under high-dose allergen stimulation.

Hypoxia preconditioning of human amniotic mesenchymal stem cells enhances proliferation and migration and promotes their homing via the HGF/C-MET signaling axis to augment the repair of acute liver failure

BackgroundTransplantation of mesenchymal stem cells (MSCs) is a newly developed strategy for treating acute liver failure (ALF). Nonetheless, the low survival rate of MSCs after transplantation and their poor homing to damaged tissues limit the clinical application of MSCs. The research assessed whether hypoxic preconditioning (HPC) can improve the biological activity of human amniotic mesenchymal stem cells (hA-MSCs), promote their homing ability to the liver of mice with ALF, and influence liver tissue repair. MethodsFlow cytometry, CCK8, Transwell, and Western blotting assays were conducted to assess the effects of hypoxic preconditioning on the phenotype, proliferation, and migration of hA-MSCs and the changes in the c-Met and CXCR4 gene expression levels were studied. To evaluate the effects of the transplantation of hypoxic preconditioning of hA-MSCs on the homing and repair of D-galactosamine (D-GalN)/LPS-induced ALF, the mechanism was elucidated by adding c-Met, CXCR4-specific blockers (SU11274 and AMD3100). ResultsAfter hypoxia pretreatment (1% oxygen volume fraction), hA-MSCs maintained the morphological characteristics of adherence and vortex colony growth and showed high CD44, CD90, and CD105 and low CD31, CD34, and CD45 expression levels. Hypoxic preconditioning of hA-MSCs significantly increased their proliferation and migration and highly expressed the c-Met and CXCR4 genes. In vivo and in vitro, this migration-promoting effect was suppressed by the c-Met specific blocker SU11274. In the acute liver failure mouse model, the HGF expression level was considerably elevated in the liver than that in the serum, lungs and kidneys. The transplantation of hypoxic preconditioned hA-MSCs introduced a remarkable improvement in the liver function and survival rate of mice with ALF and enhanced the anti-apoptosis ability of liver cells. The anti-apoptotic enhancing effect of hypoxic preconditioning was suppressed by the c-Met specific blocker SU11274. Hypoxic hA-MSCs administration was observed to have considerably increased the fluorescent cells in the liver than that recorded after administering normal oxygen-hA-MSCs. The number of hepatic fluorescent cells decreased remarkably after adding the c-Met inhibitor SU11274, compared to that recorded after hypoxic pretreatment, whereas the effect of c-Met inhibitor SU11274 on normal oxygen-hA-MSCs was not significant. ConclusionsHypoxic preconditioning depicted no impact on the morphology and phenotype features of the human amniotic mesenchymal stem cells, but it can promote their proliferation, migration, anti-apoptotic effect, and homing rate and improve the repair of acute liver failure, which might be mediated by the HGF/c-Met signaling axis.

Hypoxic bone marrow mesenchymal stem cell-derived exosomal lncRNA XIST attenuates lipopolysaccharide-induced acute lung injury via the miR-455-3p/Claudin-4 axis

Mesenchymal stem cell-derived exosomes and long non-coding RNAs (lncRNAs) have been identified to play a role in acute lung injury (ALI). In this study, we investigated whether exosomal lncRNAs could regulate ALI and the underlying mechanisms. Bone marrow mesenchymal stem cells (BM-MSCs) were pretreated with hypoxia or normoxia, and exosomes were subsequently extracted from normoxic BM-MSCs (Nor-exos) and hypoxic BM-MSCs (Hypo-exos). A rat model of ALI was established via an airway perfusion of lipopolysaccharide (LPS). Exosomes were administered via the tail vein to evaluate the in vivo effect of exosomes in ALI. LPS-exposed RLE-6TN cells were incubated with exosomes to explore their in vitro effect in ALI. A luciferase reporter assay was used to evaluate the interaction between lncRNA XIST and miR-455-3p, as well as miR-455-3p and Claudin-4. We found that the exosomes attenuated LPS-induced ALI and Hypo-Exos exerted a greater therapeutic effect compared with Nor-exos both in vitro and in vivo. Moreover, an abundance of lncRNA XIST was observed in Hypo-exos compared with Nor-exos. Mechanistically, LncRNA XIST functioned as a miR-455-3p sponge and targeted Claudin-4 in ALI. Our results provide novel insight into the role of exosomal lncRNA XIST for the treatment of ALI. Thus, hypoxic pretreatment may represent an effective method for improving the therapeutic effects of exosomes.

Exosomes from ADSCs ameliorate nerve damage in the hippocampus caused by post traumatic brain injury via the delivery of circ-Scmh1 promoting microglial M2 polarization

BackgroundTraumatic brain injury (TBI) is an urgent global health issue. Neuroinflammation, due partially to microglia, can worsen or even cause neuropsychiatric disorders after a TBI. An increasing number of studies have found that adipose-derived stem cell (ADSC) derived exosomes can alleviate many diseases by delivering non-coding RNAs including circRNA and miRNAs, but the mechanism of action remains unclear. MethodsIn the present investigation, we produced a TBI mouse model and isolated exosomes from their ADSCs before and after an hypoxic pretreatment. We then used next generation sequencing (NGS) to identify differentially expressed circRNAs and luciferase report assays to determine the relationship between the different noncoding RNAs (miRNA, circRNA and mRNA). ResultsThe results show that we successfully isolated ADSCs which possessed a multidirectional differentiation potential. We then isolated exosomes from untreated ADSCs (Exos) and from hypoxia pretreated ADSCs (HExos). The HExos significantly decreased hippocampal nerve injury after TBI by decreasing M1 microglia mediated inflammatory cytokine expression and caused recovery of cognitive function. NGS data revealed that abnormal circ-Scmh1 expression plays a role in HExo mediated brain tissue preservation after TBI. Furthermore, luciferase report analysis found that miR-154–5p and STAT6 were the targets for circ-Scmh1. Interestingly, miR-154–5p overexpression or STAT6 inhibition reversed the circ-Scmh1 induced M2 microglial polarization. Overexpression of circ-Scmh1 increased the therapeutic effect of Exo on hippocampal nerve injury after TBI by promotion of M2 microglial polarization and decreased inflammatory induced hippocampal nerve injury. ConclusionTaken together, we found that exosomes from ADSCs ameliorate nerve damage in the hippocampus post TBI through the delivery of circ-Scmh1 and the promotion of microglial M2 polarization.

Extracellular vesicles from hypoxia-pretreated adipose-derived stem cells regulate hypoxia/reoxygenation-induced human dermal microvascular endothelial apoptosis and autophagy in vitro

Recent studies suggest hypoxia can promote adipose-derived stem cells (ADSCs) to attenuate hypoxia/reoxygenation (H/R)-induced damage to human dermal microvascular endothelial cells (HDMECs). Extracellular vesicles (EVs), isolated from ADSCs, play an-important role in the fields of regenerative medicine. Here, we aimed to investigate the effect of EVs isolated from hypoxia-pretreated ADSCs (ADSC-EVs[H]) on HDMECs to attenuate ischemia/reperfusion injury of free skin flaps. First, we characterized EVs isolated from normoxia-cultured ADSCs (ADSC-EVs[N]) and ADSC-EVs(H). Experimental data indicated that EVs isolated from ADSCs consisted of lipid-bilayer vesicles that exhibited positive expression of vascular endothelial growth factor (VEGF) and marker proteins CD9, CD63 and CD81, and the mean particle size of EVs in the hypoxia-pretreated ADSCs (ADSC[H]) group was smaller (74.17 nm) than in the normoxic-cultured ADSCs (ADSC[N]) group (93.87 nm). Hypoxic pretreatment increased the number of EVs. Later, we favorably constructed the co-culture model of EVs isolated from ADSCs (ADSC-EVs) and H/R-induced HDMECs. Cell counting kit-8, Ethynyldeoxyuridine assay, western blotting and immunofluorescence staining showed that ADSC-EVs(H) promoted the survival of HDMECs and increased LC3 level. Apoptosis, reactive oxygen species (ROS) and JC-1 mitochondrial membrane potential (MMP) assays revealed that ADSC-EVs(H) reduced the apoptosis rate and ROS accumulation and increased MMP level in HDMECs, indicating that ADSC-EVs(H) effectively attenuated H/R-induced damage in HDMECs through autophagy activation and the-inhibition of apoptosis and oxidative stress. This study confirmed that ADSC-EVs(H) could effectively regulate the proliferation, apoptosis, oxidative stress, and autophagy expression of H/R-induced HDMECs in vitro, and therefore the transplantation of ADSC-EVs(H) may provide novel insights for the transplantation of free skin flaps.

Effects of Hypoxia on RNA Cargo in Extracellular Vesicles from Human Adipose-Derived Stromal/Stem Cells.

Mesenchymal stromal/stem cells and their derivates are the most promising cell source for cell therapies in regenerative medicine. The application of extracellular vesicles (EVs) as cell-free therapeuticals requires particles with a maximum regenerative capability to enhance tissue and organ regeneration. The cargo of mRNA and microRNA (miR) in EVs after hypoxic preconditioning has not been extensively investigated. Therefore, the aim of our study was the characterization of mRNA and the miR loading of EVs. We further investigated the effects of the isolated EVs on renal tubular epithelial cells in vitro. We found 3131 transcripts to be significantly regulated upon hypoxia. Only 15 of these were downregulated, but 3116 were up-regulated. In addition, we found 190 small RNAs, 169 of these were miRs and 21 were piwi-interacting RNAs (piR). However, only 18 of the small RNAs were significantly altered, seven were miRs and 11 were piRs. Interestingly, all seven miRs were down-regulated after hypoxic pretreatment, whereas all 11 piRs were up-regulated. Gene ontology term enrichment and miR-target enrichment analysis of the mRNAs and miR were also performed in order to study the biological background. Finally, the therapeutic effect of EVs on human renal tubular epithelial cells was shown by the increased expression of three anti-inflammatory molecules after incubation with EVs from hypoxic pretreatment. In summary, our study demonstrates the altered mRNA and miR load in EVs after hypoxic preconditioning, and their anti-inflammatory effect on epithelial cells.

Hypoxic mesenchymal stem cell-derived extracellular vesicles ameliorate renal fibrosis after ischemia–reperfusion injure by restoring CPT1A mediated fatty acid oxidation

BackgroundRenal fibrosis is a common pathological process of chronic kidney diseases induced by multiple factors. Hypoxic pretreatment of mesenchymal stem cells can enhance the efficacy of secreted extracellular vesicles (MSC-EVs) on various diseases, but it is not clear whether they can better improve renal fibrosis. The latest research showed that recovery of fatty acid oxidation (FAO) can reduce renal fibrosis. In this study, we aimed to examine whether hypoxic pretreatment with MSC extracellular vesicles (Hypo-EVs) can improve FAO to restore renal fibrosis and to investigate the underlying mechanism.MethodsHypo-EVs were isolated from hypoxia-pretreated human placenta-derived MSC (hP-MSC), and Norm-EVs were isolated from hP-MSC cultured under normal conditions. We used ischemia–reperfusion (I/R)-induced renal fibrosis model in vivo. The mice were injected with PBS, Hypo-EVs, or Norm-EVs immediately after the surgery and day 1 postsurgery. Renal function, kidney pathology, and renal fibrosis were assessed for kidney damage evaluation. For mechanistic exploration, fatty acid oxidation (FAO), mitochondrial morphological alterations, ATP production and mitochondrial mass proteins were detected in vivo. Mitochondrial membrane potential and reactive oxygen species (ROS) production were investigated in vitro.ResultsWe found that Hypo-EVs confer a superior therapeutic effect on recovery of renal structure damage, restoration of renal function and reduction in renal fibrosis. Meanwhile, Hypo-EVs enhanced mitochondrial FAO in kidney by restoring the expression of a FAO key rate-limiting enzyme carnitine palmitoyl-transferase 1A (CPT1A). Mechanistically, the improvement of mitochondrial homeostasis, characterized by repaired mitochondrial structure, restoration of mitochondrial mass and ATP production, inhibition of oxidative stress, and increased mitochondrial membrane potential, partially explains the effect of Hypo-EVs on improving mitochondrial FAO and thus attenuating I/R damage.ConclusionsHypo-EVs suppress the renal fibrosis by restoring CPT1A-mediated mitochondrial FAO, which effects may be achieved through regulation of mitochondrial homeostasis. Our findings provide further mechanism support for development cell-free therapy of renal fibrosis.

Metabolic adaptation of ‘Conference’ pear to postharvest hypoxia: The impact of harvest time and hypoxic pre-treatments

The choice of harvest time and low oxygen storage conditions are crucial parameters in the commercialization of ‘Conference’ pear and the prevention of market losses. To elucidate the effect of harvesting time and hypoxia on the metabolic adaptational responses in pear fruit, a 13C-feeding experiment was performed. Pear tissue disks obtained from fruit harvested at optimum harvest time, or 1 week earlier or later (commercial, early, and late maturity stages, respectively) were exposed for 24 h to 21 kPa O2 followed by 24 h incubation at 0.2 kPa O2 at a constant temperature of 18 °C in a liquid medium containing [U13C] sodium pyruvate. Results showed high fractional enrichment of organic acids, succinate, α-ketoglutarate and lactate, and amino acids, glutamate, and alanine, during hypoxia for all harvesting times suggesting a downregulated TCA cycle stopping at succinate, as suggested by its accumulation, and a possible upregulation of the γ-aminobutyric acid (GABA) shunt. Increased concentration of alanine under hypoxia only in late pears was connected to increased endogenous levels of ethanol and acetaldehyde prior the experiment and lower lactate enrichment. Concentration and fractional enrichment of the optimum harvest time under 0.2 kPa O2 were compared to previously published data from a similar experimental setup where a 4-day gradually declining hypoxic prolife from 21 kPa to 0.2 kPa was implemented. Results showed similar enrichment for all TCA cycle organic acids. Faster label propagation of lactate in the pre-treated tissue suggested faster adaptational mechanisms and an already initiated hypoxic response while higher concentration and enrichment of alanine in the non-acclimated fruit could be evidence of a tissue specific alanine response to sudden oxygen shortage or aneffect of the age of the acclimated sample.

Hypoxic pretreatment of adipose-derived stem cell exosomes improved cognition by delivery of circ-Epc1 and shifting microglial M1/M2 polarization in an Alzheimer's disease mice model.

Alzheimer’s disease (AD) is the most common dementia in the world. Increasing evidence has shown that exosomes from hypoxic pretreated adipose-derived stem cells (ADSCs) could be an effective cognitive function therapeutic in AD-associated pathophysiology. However, their role and regulatory mechanism remain largely unknown. High-throughput sequencing was used to identify differentially expressed circRNAs from ADSCs or hypoxia pretreated ADSC exosomes. Luciferase reporter assays and RT-qPCR were used to investigate the relationships between circ-Epc1, miR-770-3p, and TREM2. APP/PS1 double transgenic AD model mice were then used to study therapeutic effects regarding circ-Epc1 in ADSC exosomes. BV2 cells were used to show the regulatory relationships between circ-Epc1, miR-770-3p, and TREM2 and to show how these interactions modulated phenotypic transformations and inflammatory cytokine expressions in microglia. The results showed that exosomes from hypoxia pretreated ADSCs had a good therapeutic effect on improving cognitive functions by decreasing neuronal damage in the hippocampus. High-throughput sequencing showed that circ-Epc1 played an important role in hypoxia-pretreated ADSC exosomes regarding their ability to improve cognitive functions. Luciferase reporter assays showed that TREM2 and miR-770-3p were downstream targets of circ-Epc1. Overexpressing miR-770-3p or downregulating TREM2 reversed the effects of circ-Epc1 on M2 microglia during lipopolysaccharide treatment. In vivo experiments showed that circ-Epc1-containing ADSC exosomes increased the therapeutic effect of exosomes by improving cognitive function, decreasing neuronal damage, and shifting hippocampal microglia from the M1 polarization to the M2 polarization stages. Taken together, the data show that hypoxic pretreatment of ADSC exosomes improved cognition by delivery of circ-Epc1 and by shifting microglial M1/M2 polarization in an AD mouse model.

Circular RNA mmu_circ_0001295 from hypoxia pretreated adipose-derived mesenchymal stem cells (ADSCs) exosomes improves outcomes and inhibits sepsis-induced renal injury in a mouse model of sepsis

ABSTRACT Microvascular dysfunction causes mortality in the presence of sepsis and multi-organ failure. Previous studies have demonstrated that exogenous administration of exosomes from adipose-derived mesenchymal stem cells (ADSCs) protects against sepsis, improves organ function, decreases vascular leakage and increases survival. However, the underlying regulatory mechanism was largely unknown. Therefore, in this study, a mouse sepsis model based on cecal ligation and puncture (CLP) was constructed. Exosomes from various ADSCs were intravenously administered at 4 h post CLP. Treatment with ADSC exosomes (Exo), particularly those with hypoxic pretreatment (HExo), enhanced survival, suppressed renal vascular leakage and decreased kidney dysfunction in septic mice. HExo ameliorated sepsis-induced increases in chemokine and cytokine plasma levels. Furthermore, the HExo circRNA content, determined through next-generation sequencing, revealed abundant mmu_circ_0001295. Further studies demonstrated that downregulation of exosomal mmu_circ_0001295 suppressed the exosomes’ protective effects against sepsis. HExo prevented microvascular dysfunction, thus potentially improving sepsis outcomes via mmu_circ_0001295 delivery. In summary, the data indicated that HExo elongate sepsis-induced renal injury through delivering mmu_circ_0001295.

Hypoxia-Preconditioned Extracellular Vesicles from Mesenchymal Stem Cells Improve Cartilage Repair in Osteoarthritis.

In the past decade, mesenchymal stem cells (MSCs) have been widely used for the treatment of osteoarthritis (OA), and extracellular vesicles (EVs) may play a major role in the efficacy of this treatment. Hypoxia can change the cargo and biological functions of MSC-derived EVs (MSC-EVs). The aim of the present study was to determine whether the effects of hypoxia-preconditioned MSC-EVs on OA cartilage repair are superior to normoxia-preconditioned MSC-EVs. By using in vitro and in vivo OA models, we verified that hypoxia-preconditioned MSC-EVs improved chondrocyte proliferation and migration and suppressed chondrocyte apoptosis to a greater extent than normoxia-preconditioned MSC-EVs. Furthermore, we found that hypoxia altered the microRNA expression in MSC-EVs and identified four differentially expressed microRNAs: hsa-miR-181c-5p, hsa-miR-18a-3p, hsa-miR-376a-5p, and hsa-miR-337-5p. Bioinformatics analysis revealed that hypoxic pretreatment may promote cartilage repair by stimulating chondrocyte proliferation and migration and suppressing chondrocyte apoptosis through the miRNA-18-3P/JAK/STAT or miRNA-181c-5p/MAPK signaling pathway. Therefore, hypoxia-preconditioned EVs may be a novel treatment for OA.

Small extracellular vesicles from hypoxic mesenchymal stem cells alleviate intervertebral disc degeneration by delivering miR-17-5p

Minimally invasive repair strategies are a very promising approach for the treatment of intervertebral disc degeneration (IDD). In recent years, small extracellular vesicles (sEVs) secreted from mesenchymal stem cells (MSCs) have been shown great potential in alleviating IDD. However, in vitro experiments, MSCs are usually exposed to a normoxic micro-environment, which differs greatly from the hypoxic micro-environment in vivo. The primary purpose of our research was to determine whether sEVs isolated from MSCs under hypoxic status (H-sEVs) exhibit a more beneficial effect on protecting IDD compared with sEVs derived from MSCs under normoxic status (N-sEVs). A tail IDD rat model and a series of experiments in vitro were conducted to compare the beneficial effects of PBS, N-sEVs, and H-sEVs treatment. Then, to validate the role of sEVs miRNAs in IDD, a miRNA microarray sequencing analysis and a series of rescue experiments were conducted. Luciferase activity, RNA-ChIP and western blot were performed to explore the potential mechanisms. The results indicate that sEVs alleviate IDD by ameliorating the homeostatic imbalance between anabolism and catabolism in vivo and in vitro. Microarray sequencing result shows that miR-17-5p is maximally enriched in H-sEVs. Toll-like receptor 4 (TLR4) was determined to be a target downstream gene of miR-17-5p. Finally, it was found that H-sEVs miR-17-5p may modulate proliferation and synthesis of human nucleus pulposus cells (HNPCs) matrix via TLR4 pathway. In conclusion, H-sEVs miR-17-5p alleviate IDD via promoting HNPCs matrix proliferation and synthesis, providing new therapeutic targets for IDD. Statement of significanceIntervertebral disc degeneration (IDD) is the primary cause of low back pain (LBP), which is a huge burden to society. Our research demonstrates for the first time that hypoxic pretreatment of small extracellular vesicles (H-sEVs) effectively alleviated the progress of IDD. In short, in the present research, we found that H-sEVs miR-17-5p could modulate proliferation and synthesis of nucleus pulposus cells (NPCs) matrix via TLR4/PI3K/AKT pathway. Therefore, hypoxic pre-treatment is a prospective and efficient method to optimize the therapeutic effect of MSCs-derived sEVs. miRNA and MSCs-derived sEVs combination may be a promising therapeutic approach for IDD.

Circ-Klhl8 overexpression increased the therapeutic effect of EPCs in diabetic wound healing via the miR-212-3p/SIRT5 axis.

Previous studies found that hypoxic pretreatment of endothelial progenitor cells (EPCs) prior to transplantation had a greater therapeutic effect than untreated EPCs in promoting diabetic wound healing. However, the exact mechanism is uncertain. Here, circRNA expression in EPCs after hypoxic treatment was investigated. High-throughput sequencing was used to assess abnormal expression by EPCs of circular RNAs (circRNAs) following hypoxic pretreatment. Additionally, an in vivo full-thickness skin defect mouse model was used to assess the effects of transplanted EPCs on diabetic wound closure. Subsequently, the regulatory mechanism and targets were studied. The results showed that circ-Klhl8 overexpression suppressed hyper glucose-induced endothelial cell damage by activating autophagy. MiR-212-3p and SIRT5 were identified as the downstream targets of circ-Klhl8. Circ-Klhl8 overexpression promoted skin wound healing by regulating SIRT5-mediated autophagy. In conclusion, the study found that circ-Klhl8 overexpression increased the EPC therapeutic effect in promoting diabetic wound healing by targeting the miR-212-3p/SIRT5 axis.

Hypoxic Pretreatment of Adipose-Derived Stem Cells Accelerates Diabetic Wound Healing via circ-Gcap14 and HIF-1α/VEGF Mediated Angiopoiesis.

Background and ObjectivesAdipose-derived stem cell (ADSC) transplantation improves stem cell paracrine function and can enhance wound healing. However, in diabetic patients, glucose-associated effects on this function and cell survival lead to impaired wound closure, thereby limiting ADSC transplantation efficiency. The hypoxia-inducible factor HIF-1α has an important protective function during wound healing. Here, we aim to clarify the regulatory mechanism of ADSCs.Methods and ResultsADSCs were isolated from BALB/C mice adipose samples. We then used high-throughput sequencing to assess abnormal expression of circular RNAs (circRNAs). We also used an in vivo full-thickness skin defect mouse model to assess the effects of transplanted ADSC on diabetic wound closure. Hypoxic pretreatment of ADSCs accelerated diabetic wound closure, which enhanced angiogenic growth factor expression in our mouse model. High-throughput sequencing and RT-qPCR indicated that circ-Gcap14 was upregulated in hypoxic pretreated ADSCs. Similarly, circ-Gcap14 downregulation also decreased the therapeutic effects of ADSCs; however, circ-Gcap14 overexpression increased the effects of ADSC by promoting angiopoiesis. We also used a luciferase reporter assay to confirm that miR-18a-5p and HIF-1α were downstream targets of circ-Gcap14. HIF-1α expression plays an important role in increased VEGF level.ConclusionsBased on our data, we suggest that circ-Gcap14 plays an important role in accelerating hypoxic ADSC-mediated diabetic wound closure, by enhancing mouse angiogenic growth factor expression and regulating downstream miR-18a-5p/HIF-1α expression.

Hypoxia preconditioning improves structure and function of astrocytes mitochondria via PGC-1α/HIF signal

The protective effect of astrocytes on nerves was demonstrated by mitochondrial transfer. The neuroprotective effect of hypoxic pretreatment is widely accepted. The aim of this research is to investigate the role of hypoxic preconditioning on astrocytes mitochondria. Rat neuronal cells and astrocytes were isolated and cultured. A hypoxic preconditioned astrocyte and oxygen glucose deprivation (OGD) neuronal cell co-culture experiment was used to detect the effect of hypoxic preconditioning (HP) on nerve damage. The silencing of proliferatoractivated receptor γ coactivator-1α (PGC-1α) with siRNA was used to explore the role of HP in the repair of nerve damage and biogenesis of mitochondria. HP increased astrocyte viability and promoted neuroprotective factor secretion. The expression levels of antioxidant enzymes, PGC-1α and uncoupling protein2 (UCP2) were up-regulated by HP. In addition, HP improved mitochondrial function and reduced oxidative stress induced by OGD. It was found that HP astrocytes had a greater neuroprotective effect than normal astrocytes cells. Neuronal apoptosis and reactive oxygen species levels were down-regulated by cell co-culture. The PGC-1α siRNA experiment showed that hypoxia treatment promotes mitochondrial biogenesis and plays a neuroprotective role. HP significantly enhanced the efficacy of astrocytes in the treatment of neuronal injury.

Hypoxia enhances the cytotoxic effect of As4S4 on rat ventricular H9c2 cells through activation of ubiquitin–proteasome system

BackgroundAs4S4 is widely used in Chinese traditional medicine compound. However, based on some recent studies, we found that the cardiotoxicity risk of using As4S4 in ischemic heart disease patients may be increased. To study this potential risk, we compared the effects of As4S4 on rat ventricular H9c2 cell line with or without hypoxic pretreatment, and to elucidate mechanisms of c-Cbl mediated ubiquitination/degradation of integrin β1. MethodsThe present study was conducted on rat ventricular H9c2 cell line in the absence or presence of hypoxic pretreatment for 6 h followed by As4S4 treatment for 24 h. Following As4S4 treatment, cell viability assay, flow cytometric quantification of apoptotic cells, caspase-3 activity assay and DAPI staining were conducted. Western blotting was carried out to detect expressions of ubiquitination related proteins. In addition, the ubiquitination/degradation of integrin β1 and the role of c-Cbl in it was evaluated by immunoprecipitation and immunoblot assay. ResultsThe viability of cells with hypoxic pretreatment followed by As4S4 treatment was decreased significantly, apoptosis rate and the activity of caspase-3 were increased than As4S4 treatment alone. The ubiquitin-proteasome degradation pathway induced by As4S4 was further enhanced by hypoxic pretreatment. The results of IP and immunoblot assay showed hypoxic enhanced down-regulation effect of As4S4 on integrin β1 probably through c-Cbl activation. ConclusionsThis study demonstrated that the hypoxia enhanced cytotoxicity of As4S4 on H9c2 cells may through increasing the ubiquitin-proteasome degradation of integrin β1 mediated by the E3 ligase c-Cbl. The results provide an important clue that, in patients with ischemic heart disease, use of As4S4 may be associated with increased cardiotoxicity. We believe that the results worth to be further illuminated by in vivo and clinical research.

Hypoxic pretreatment of small extracellular vesicles mediates cartilage repair in osteoarthritis by delivering miR-216a-5p

Osteoarthritis (OA) is a regressive joint disease that mainly affects the cartilage and surrounding tissues. Mounting studies have confirmed that the paracrine effect is related to the potential mechanism of mesenchymal stem cell (MSC) transplantation and that small extracellular vesicles (sEVs) play an imperative role in this paracrine signaling. In fact, hypoxia can significantly improve the effectiveness of MSC transplantation in various disease models. However, it remains unknown whether MSCs in the state of a hypoxic environment can enhance OA cartilage repair and whether this enhancement is mediated by sEV signaling. The primary aim of the present study was to determine whether sEVs from MSCs in the state of hypoxia (Hypo-sEVs) have a superior effect on OA cartilage repair relative to sEVs from MSCs in the normoxia (Nor-sEVs) state. By using an OA model and performing in vitro studies, we verified that Hypo-sEV treatment facilitated the proliferation, migration, and apoptosis suppression of chondrocytes to a greater extent than Nor-sEV treatment. Furthermore, we verified the functional role of sEV miR-216a-5p in the OA cartilage repair process. We also identified JAK2 as the target gene of sEV miR-216a-5p through a series of experiments. Our findings indicated that HIF-1α induces hypoxic BMSCs to release sEVs, which promote the proliferation, migration, and apoptosis inhibition of chondrocytes through the miR-216a-5p/JAK2/STAT3 signaling pathway. Therefore, hypoxic pretreatment is a prospective and effective method to maximize the therapeutic effect of MSC-derived sEVs on OA.

Extracellular vesicles derived from human adipose-derived stem cells promote the exogenous angiogenesis of fat grafts via the let-7/AGO1/VEGF signalling pathway

Extracellular vesicles (EVs) derived from human adipose-derived stem cells (hADSCs) possess the proangiogenic potential for ischaemic diseases. Thus, our study aimed to evaluate the therapeutic effects of hADSC-EVs on fat grafting and explore the mechanism of hADSC-EVs promoting angiogenesis. The EVs released by hADSCs incubated under normal or hypoxic conditions were employed to supplement fat grafting in a nude mouse model. The proliferation, migration, tube formation and vascular endothelial growth factor (VEGF) secretion of vascular endothelial cells co-cultured with two kinds of hADSC-EVs were analysed. MicroRNA sequencing was performed to reveal the species and content of microRNAs in hADSC-EVs, the key microRNAs were blocked, and their effect in promoting angiogenesis was detected via above protocols as a reverse proof. The results demonstrate that hADSC-EVs could improve the survival of fat grafts by promoting exogenous angiogenesis and enhance the proliferation, migration, tube formation and VEGF secretion of vascular endothelial cells. In addition, the pro-angiogenic effect of hADSC-EVs in vivo and vitro could be enhanced by hypoxic pre-treatment. We found that the let-7 family, a kind of hypoxic-related microRNA, is enriched in hypoxic hADSC-EVs that contribute to angiogenesis via the let-7/argonaute 1 (AGO1)/VEGF signalling pathway.