Hypoxic Preconditioning Research Articles

Hypoxic Preconditioning Alters Micro-RNAs Expression in Mesenchymal Stem Cells from Patients with Hypertensive Kidney Disease

Hypoxic Preconditioning Alters Micro-RNAs Expression in Mesenchymal Stem Cells from Patients with Hypertensive Kidney Disease

Positive effects of hypoxic preconditioning of the extracellular matrix and stromal vascular fraction from adipose tissue

BackgroundNumerous approaches have been developed to decelerate the aging process of facial skin. Synthetic fillers and cell-enriched fat grafts are the main procedures employed to fill wrinkles. ObjectiveThe aim of this study was to evaluate the in vitro and in vivo safety and efficiency of a new process developed by SYMBIOKEN: the AmeaCell, which facilitates the extraction of the stromal vascular fraction (SVF) and the associated hypoxia pre-conditioned matrix to promote fat graft survival. MethodsThe AmeaCell device allows the extraction from adipose tissue of SVF and pre-conditioned MatriCS and promotes a hypoxic environment. Experiments were carried out on human cells and then in mice. ResultsCharacterization of cells and MatriCS showed that after their extraction using the new process developed by SYMBIOKEN, the extracted cells expressed stem-cell markers. The presence of characteristic proteins and lipid fractions found in the adipose matrix were confirmed in MatriCS. Cobalt chloride treatment of the matrix using the AmeaCell device induced modifications in the matrix composition with a decrease in laminin and without collagen modification, both of which promote adhesion and differentiation of SVF or adipose-derived stromal cells. The combination of MatriCS and SVF (1 × 106 and 5 × 106, respectively) is safe and efficient to fill winkles induced by UVB irradiation. The cross-talk between MatriCS and SVF can act a durable filler compared to the filling performed using cells or matrix or fat alone, which need to be replaced frequently. ConclusionThese results indicate that the combination of MatriCS and SVF is safe and effective as a biological filler for achieving skin rejuvenation and wrinkle filling.

Chemical Hypoxic Preconditioning Improves Survival and Proliferation of Mesenchymal Stem Cells.

Increasing evidence has demonstrated that mesenchymal stem cells (MSCs) have been linked to tissue regeneration both in vitro and in vivo. However, poor engraftment and low survival rate of transplanted MSCs are still a major concern. It has been found that the proliferation, survival, and migration of MSCs are all increased by hypoxic preconditioning. However, the molecular mechanism through which hypoxic preconditioning enhances these beneficial properties of MSCs remains to be fully investigated. Therefore, the present study is aimed to investigate the mechanism by which hypoxic preconditioning enhances the survival of MSCs. We used proteomic analysis to explore the molecules that may contribute to the survival and proliferation of hypoxic preconditioned (HP) MSCs. The analysis revealed a higher expression of prelamin A/C (Lmna), glutamate dehydrogenase 1(Glud1), Actin, cytoplasmic 1(Actb), Alpha-enolase (Eno1), Glucose-6-phosphate 1-dehydrogenase (G6pd), Protein disulfide-isomerase A3 (Pdia3), Malate dehydrogenase (Mdh1), Peroxiredoxin-6 (Prdx6), Superoxide dismutase (Sod1), and Annexin A2 (Anxa2) in HP-MSCs. These proteins are possibly involved in cellular survival and proliferation through various cellular pathways. This research could aid in understanding the processes involved in hypoxic preconditioning of MSCs and designing of cell-based therapeutic strategies for tissue regeneration.

Hypoxic mesenchymal stem cell-derived exosomes promote the survival of skin flaps after ischaemia–reperfusion injury via mTOR/ULK1/FUNDC1 pathways

Flap necrosis, the most prevalent postoperative complication of reconstructive surgery, is significantly associated with ischaemia–reperfusion injury. Recent research indicates that exosomes derived from bone marrow mesenchymal stem cells (BMSCs) hold potential therapeutic applications in several diseases. Traditionally, BMSCs are cultured under normoxic conditions, a setting that diverges from their physiological hypoxic environment in vivo. Consequently, we propose a method involving the hypoxic preconditioning of BMSCs, aimed at exploring the function and the specific mechanisms of their exosomes in ischaemia–reperfusion skin flaps. This study constructed a 3 × 6 cm2 caudal superficial epigastric skin flap model and subjected it to ischaemic conditions for 6 h. Our findings reveal that exosomes from hypoxia-pretreated BMSCs significantly promoted flap survival, decrease MCP-1, IL-1β, and IL-6 levels in ischaemia–reperfusion injured flap, and reduce oxidative stress injury and apoptosis. Moreover, results indicated that Hypo-Exo provides protection to vascular endothelial cells from ischaemia–reperfusion injury both in vivo and in vitro. Through high-throughput sequencing and bioinformatics analysis, we further compared the differential miRNA expression profiles between Hypo-Exo and normoxic exosomes. Results display the enrichment of several pathways, including autophagy and mTOR. We have also elucidated a mechanism wherein Hypo-Exo promotes the survival of ischaemia–reperfusion injured flaps. This mechanism involves carrying large amounts of miR-421-3p, which target and regulate mTOR, thereby upregulating the expression of phosphorylated ULK1 and FUNDC1, and subsequently further activating autophagy. In summary, hypoxic preconditioning constitutes an effective and promising method for optimizing the therapeutic effects of BMSC-derived exosomes in the treatment of flap ischaemia–reperfusion injury.

Hypoxia improves self-renew and migration of urine-derived stem cells by upregulating autophagy and mitochondrial function through ERK signal pathway

Urine-derived stem cells (USCs) are autologous stem cells with self-renewal ability and multi-lineage differentiation potential. Our previous studies have shown that hypoxia preconditioning can improve self-renewal and migration abilities of USCs by up-regulating autophagy. The purpose of this study was to investigate the specific mechanism by which hypoxia treatment promotes the biological function of USCs. We found that hypoxia treatment upregulated the expression of phosphralated ERK protein without affecting the expression of total ERK protein. Inhibiting ERK signaling with the PD98059 inhibitor decreased cell proliferation, migration and colony formation during hypoxia treatment. Hypoxia increased ATP production, mitochondrial membrane potential and mt-DNA copy number, which were reversed by inhibiting the ERK signal. Additionally, the number of autophagosomes and autophagic lysosomes was significantly lower in PD98059 group than in the hypoxia group. PD98059 treatment inhibited the up-regulation of autophagy related proteins induced by hypoxia. Therefore, this study suggests that hypoxia improves the self-renewal and migration abilities of USCs by upregulating autophagy and mitochondrial function through ERK signaling pathway. This finding may provide a new therapeutic mechanism for hypoxia pretreated USCs as a source of stem cell transplantation.

Hypoxic Preconditioning Attenuates Neuroinflammation via Inhibiting NF-κB/NLRP3 Axis Mediated by p-MLKL after Transient Global Cerebral Ischemia.

Hypoxic preconditioning (HPC) has been reported to alleviate neuronal damage and microglial activation in hippocampal CA1 after transient global cerebral ischemia (tGCI). However, the molecular mechanism is unclear. Recent studies identified that nuclear factor-kappa-B (NF-κB)/oligomerization domain-like receptors protein (NLRP) 3 inflammasome pathway is mainly involved in the activation of microglia and that phosphorylated (p)-mixed lineage kinase domain-like (MLKL) is related to the regulation of NF-κB/NLRP3 axis. Hence, in this study, we set out to investigate whether HPC attenuates neuronal damage and microglial activation through inhibiting NF-κB/NLRP3 axis mediated by p-MLKL after tGCI in CA1 of male rats. We found that HPC decreased NLRP3 inflammasome in microglia and inhibited M1 polarization of microglia in CA1 after tGCI. Mechanistically, HPC inhibited the activation of NF-κB signaling pathway and reduced the mRNA and protein levels of NLRP3 inflammasome after tGCI. Additionally, the knockdown of p-MLKL by short hairpin RNA (shRNA) administration inhibited the activation of the NF-κB signaling pathway and reduced the formation of NLRP3 inflammasome, thus attenuating M1 polarization of microglia and decreasing the release of interleukin 1 beta (IL-1β) and necrosis factor alpha (TNF-α) in CA1 post ischemia. We consider that p-MLKL in microglia may be derived from necroptotic neurons after tGCI. In conclusion, the new finding in this study is that HPC-induced neuroprotection against tGCI through inhibiting NF-κB/NLRP3 pathway mediated by p-MLKL.

Deletion of the Neuronal Transcription Factor Satb1 Induced Disturbance of the Kinome and Mechanisms of Hypoxic Preconditioning.

Genetic disorders affecting the functioning of the brain lead not only to the development of numerous hereditary diseases but also to the development of neurodegenerative and cognitive disorders. The result of this may be the disability of part of the able-bodied population. Almost all pathological states of the brain are characterized by serious defects in the intracellular and intercellular signaling of neurons and glial cells. At the same time, the mechanisms of disruption of these signaling cascades are not well understood due to the large number of molecules, including transcription factors that, when mutated, cause brain malformations. The transcription factor Satb1 is one of the least studied factors in the cerebral cortex, and the effects of its deletion in the postnatal brain are practically not studied. Hyperexcitability of neurons is observed in many genetic diseases of the nervous system (Hirschsprung's disease, Martin-Bell syndrome, Huntington's disease, Alzheimer's, etc.), as well as in ischemic brain phenomena and convulsive and epileptic conditions of the brain. In turn, all these disorders of brain physiology are associated with defects in intracellular and intercellular signaling and are often the result of genetic disorders. Using Satb1 mutant mice and calcium neuroimaging, we show that Satb1 deletion in projection neurons of the neocortex causes downregulation of protein kinases PKC, CaMKII, and AKT/PKB, while a partial deletion does not cause a dramatic disruption of kinome and Ca2+ signaling. As a result, Satb1-null neurons are characterized by increased spontaneous Ca2+ activity and hyperexcitability when modeling epileptiform activity. As a result of the deletion of Satb1, preconditioning mechanisms are disrupted in neurons during episodes of hypoxia. This occurs against the background of increased sensitivity of neurons to a decrease in the partial pressure of oxygen, which may indicate the vulnerability of neuronal networks and be accompanied by impaired expression of the Satb1 transcription factor. Here, we show that Satb1 deletion impaired the expression of a number of key kinases and neuronal hyperexcitation in models of epileptiform activity and hypoxia.

Secretive derived from hypoxia preconditioned mesenchymal stem cells promote cartilage regeneration and mitigate joint inflammation via extracellular vesicles

Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great potential to enhance MSCs paracrine therapeutic effect. In our study, the paracrine effects of secretome derived from MSCs preconditioned in normoxia and hypoxia was compared through both in vitro functional assays and an in vivo rat osteochondral defect model. Specifically, the paracrine effect of total EVs were compared to that of soluble factors to characterize the predominant active components in the hypoxic secretome. We demonstrated that hypoxia conditioned medium, as well as the corresponding EVs, at a relatively low dosage, were efficient in promoting the repair of critical-sized osteochondral defects and mitigated the joint inflammation in a rat osteochondral defect model, relative to their normoxia counterpart. In vitro functional test shows enhancement through chondrocyte proliferation, migration, and matrix deposition, while inhibit IL-1β-induced chondrocytes senescence, inflammation, matrix degradation, and pro-inflammatory macrophage activity. Multiple functional proteins, as well as a change in EVs' size profile, with enrichment of specific EV-miRNAs were detected with hypoxia preconditioning, implicating complex molecular pathways involved in hypoxia pre-conditioned MSCs secretome generated cartilage regeneration.

Novel neutrophil extracellular trap-related mechanisms in diabetic wounds inspire a promising treatment strategy with hypoxia-challenged small extracellular vesicles.

Neutrophil extracellular traps (NETs) have been considered a significant unfavorable factor for wound healing in diabetes, but the mechanisms remain unclear. The therapeutic application of small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) has received considerable attention for their properties. Hypoxic preconditioning is reported to enhance the therapeutic potential of MSC-derived sEVs in regenerative medicine. Therefore, the aim of this study is to illustrate the detailed mechanism of NETs in impairment of diabetic wound healing and develop a promising NET-targeting treatment based on hypoxic pretreated MSC-derived sEVs (Hypo-sEVs). Excessive NETs were found in diabetic wounds and in high glucose (HG)-induced neutrophils. Further research showed that high concentration of NETs impaired the function of fibroblasts through activating endoplasmic reticulum (ER) stress. Hypo-sEVs efficiently promoted diabetic wound healing and reduced the excessive NET formation by transferring miR-17-5p. Bioinformatic analysis and RNA interference experiment revealed that miR-17-5p in Hypo-sEVs obstructed the NET formation by targeting TLR4/ROS/MAPK pathway. Additionally, miR-17-5p overexpression decreased NET formation and overcame NET-induced impairment in fibroblasts, similar to the effects of Hypo-sEVs. Overall, we identify a previously unrecognized NET-related mechanism in diabetic wounds and provide a promising NET-targeting strategy for wound treatment.

Obesity paradox or hypoxia preconditioning: How obstructive sleep apnea modifies the Obesity-MI relationship

ObjectivesThe objective of this study was to evaluate the interaction between obesity and obstructive sleep apnea on acute MI in hospital mortality. MethodsThis retrospective cohort study utilized Veterans Health Administration data from years 1999–2020. Participants were categorized according to their body mass index (BMI) to non-obese (BMI <30) and obese (BMI ≥30) groups. Clinical obstructive sleep apnea (SA) diagnosis was confirmed using ICD9/10 codes and the study subgroups included non-obese with no obstructive sleep apnea (nOB-nSA), non-Obese with obstructive sleep apnea (nOB-SA), obese with no obstructive sleep apnea (OB-nSA), and obese with obstructive sleep apnea (OB-SA). The primary outcome was odds ratio of in-hospital mortality during the hospitalization with acute MI as the principal diagnosis adjusted for age, gender, race, ethnicity, and Charlson comorbidity index (CCI) with the nOB-nSA group as the comparison group. ResultsAmong 72,036 veterans with acute-MI hospitalization, individuals with obesity and obstructive sleep apnea (OB-SA) had the lowest in-hospital mortality rate (1.0%) compared to those without obesity and obstructive sleep apnea (nOB-nSA, 2.8%), with obesity but without obstructive sleep apnea (OB-nSA, 2.4%), and with obesity and obstructive sleep apnea (nOB-SA, 1.4%). The adjusted odds ratio for mortality, compared to nOB-nSA, was 9% higher but not significant in OB-nSA (aOR, 1.09, 95%CI: 0.95, 1.25), 46% lower in OB-nSA (aOR, 0.54, 95%CI: 0.45, 0.66), and 52% lower in OB-SA (aOR, 0.48: 95%CI: 0.41, 0.57). ConclusionOur data suggest that the association between obesity and improved survival in acute MI is largely driven by the presence of sleep apnea.

Adipose-Derived Stem Cells (ADSCs) in preconditioning of hypoxic cultures compared to normoxic cultures to prevent the formation of fibrous tissue: a review article

Background: Stem cells are a key element in regenerative medicine for their ability to differentiate into various cell phenotypes. According to the literature review, one process that might improve the functionality of ADSCs is hypoxic culture conditions. Hypoxic oxygen culture conditions at 5% concentration are said to increase stem cell proliferation and viability after transplantation. Methods: A comprehensive literature search was conducted by the author to obtain relevant studies from the PubMed, MEDLINE, Embase, PreMEDLINE, Embase, PsycINFO, Scopus, and Cochrane databases for the last twenty years (January 2002 – July 2022). The author used a search strategy with the following keywords: Adipose-Derived Stem Cells, hypoxic culture, normoxic culture, or pre-conditioned stem cells. Discussion: Hypoxic culture preconditioning of ADSCs with an oxygen concentration of 5% can improve proliferation capabilities and stem cell viability post-transplantation. Hypoxic preconditioning will enhance the expression of HIF-1

COVID-19: Two years in the history and still a mystery! A case series of three interesting cases

COVID-19 was first identified as a new disease in December 2019. It’s now more than 2 years since its discovery and much research has been done. Still, there are some aspects in the pathogenesis and management that have not been completely understood. The cornerstone of therapy lies in the supportive treatment to maintain oxygenation. To the best of our knowledge, there are no earlier reports of severe COVID-19 with respiratory failure that have recovered without supplemental oxygen therapy. We present three unique cases of severe COVID-19 who were managed at home despite hypoxemia. The scarcity of oxygen during the second wave in Delhi deprived them of essential oxygen therapy. The patients survived and recovered well. Happy hypoxemia and hypoxic preconditioning played their role in symptoms tolerance. Therapeutically, the role of awake proning and steroid in the management cannot be undermined. Lastly, the technology in terms of video consultation definitely was useful in allaying the anxiety and guiding the right treatment.

Hypoxic Preconditioning Enhances Cellular Viability and Migratory Ability: Role of DANCR/miR-656-3p/HIF-1α Axis in Placental Mesenchymal Stem Cells.

Preeclampsia (PE) is a common complication of pregnancy characterized by new-onset hypertension, albuminuria, or end-stage organ dysfunction, which is seriously harmful to maternal and infant health. Mesenchymal stem cells (MSCs) are pluripotent stem cells derived from extraembryonic mesoderm. They have the potential for self-renewal, multidirectional differentiation, immunomodulation, and tissue regeneration. Several in vivo and in vitro experiments have confirmed that MSCs can delay the pathological progression of PE and improve maternal and fetal outcomes. However, the major limitations in the application of MSCs are their low-survival rates in ischemic and hypoxic disease areas after transplantation and their low rate of successful migration to the diseased regions. Therefore, enhancing cell viability and migration ability of MSCs in both ischemic and anoxic environments is important. This study aimed to investigate the effects of hypoxic preconditioning on the viability and migration ability of placental mesenchymal stem cells (PMSCs) and their underlying mechanisms. In this study, we found that hypoxic preconditioning enhanced the viability and migration ability of PMSCs, increased the expression of DANCR and hypoxia-inducible factor-1α (HIF-1α), and decreased the expression of miR-656-3p in PMSCs. Inhibiting the expression of HIF-1α and DACNR in PMSCs under hypoxia can inhibit the promotive effect of hypoxic preconditioning on viability and migration ability. In addition, RNA pull down and double luciferase assays confirmed that miR-656-3p could directly bind to DANCR and HIF-1α. In conclusion, our study showed that hypoxia could promote the viability and migration ability of PMSCs through the DANCR/miR-656-3p/HIF-1α axis.

#4067 HYPOXIC PRECONDITIONING FOR THE IMPROVEMENT OF THE REGENERATIVE CAPACITY OF MESENCHYMAL STEM CELLS AND ITS APPLICATION IN HYPERTENSIVE KIDNEY DISEASE

Abstract Background and Aims Autologous mesenchymal stem/stromal cells (MSCs) have emerged as a therapeutic option for many chronic diseases. Hypertensive kidney disease (HKD) might impair MSCs’ reparative ability by altering important biomolecular properties of the cell, but the characteristics of this impairment are unclear. In our previous pre-clinical studies, hypoxic preconditioning (HPC) enhanced MSC angiogenesis and reduced senescence through global gene and protein expression. Thus, we hypothesize that HPC would improve human MSCs by enhancing their functionality and angiogenesis, creating an anti-inflammatory and anti-senescence environment in MSCs from healthy control (HC), hypertensive patients (HTN), and hypertensive kidney disease patients (HKD). Method Human MSCs were collected from abdominal subcutaneous fat tissue biopsy of HC, HTN, and HKD patients (n=12 each). Samples were collected from healthy volunteers with no history of hypertension or kidney disease (HC), patients diagnosed with hypertension controlled with antihypertensive drugs (HTN), and hypertensive patients with eGFR&amp;lt;60 mL/min/1.73 m2 (HKD). MSCs were harvested and cultured in Normoxic (20% O2) or Hypoxic (1% O2) conditions for 24-48 hours. MSC functionality was measured by migration and proliferation. Angiogenic and inflammatory secretome in conditioned media was assessed along with MSC senescence by senescence-associated beta-galactosidase (SA-beta-gal) activity. In addition, transcriptome analysis using RNA-sequencing (n=3/group) and quantitative PCR (qPCR) were performed to evaluate gene expression. Results At baseline, Normoxic HTN-MSCs interestingly had higher proliferation compared to HC. HC-MSC subjected to HPC showed increased proliferation (Figure 1A). SA-β-gal activity tended to decrease after HPC, particularly in the HC group (P = .06). HPC did not affect the release of the pro-angiogenic protein VEGF from MSCs, but increased EGF in HC-MSC, and decreased HGF in HC and HKD MSCs. HPC upregulated the pro-angiogenetic genes, inflammatory gene, and a few senescence genes (Figure 1F). Conclusion HPC has a more favorable functional effect on HC- than on HKD-MSC, reflected in increased proliferation and EGF release, and tends to decrease senescence, whereas it has little effect on HTN or HKD MSCs. These observations may assist in developing novel therapeutic strategies to improve the regenerative capacity of MSCs in patients with hypertensive kidney disease.

Hypoxic Preconditioning Modulates BDNF and Its Signaling through DNA Methylation to Promote Learning and Memory in Mice.

Hypoxic preconditioning (HPC) as an endogenous mechanism can resist hypoxia/ischemia injury and exhibit protective effects on neurological function including learning and memory. Although underlying molecular mechanisms remain unclear, HPC probably regulates the expression of protective molecules by modulating DNA methylation. Brain-derived neurotrophic factor (BDNF) activates its signaling upon binding to the tropomyosin-related kinase B (TrkB) receptor, which is involved in neuronal growth, differentiation, and synaptic plasticity. Therefore, this study focused on the mechanism by which HPC regulates BDNF and BDNF/TrkB signaling through DNA methylation to influence learning and memory. Initially, the HPC model was established by hypoxia stimulations on ICR mice. We found that HPC downregulated the expression of DNA methyltransferase (DNMT) 3A and DNMT3B. Then, the upregulation of BDNF expression in HPC mice was generated from a decrease in DNA methylation of the BDNF gene promoter detected by pyrophosphate sequencing. Subsequently, upregulation of BDNF activated BDNF/TrkB signaling and ultimately improved learning and spatial memory in HPC mice. Moreover, after mice were intracerebroventricularly injected with the DNMT inhibitor, the restraint of DNA methylation accompanied by an increase of BDNF and BDNF/TrkB signaling was also discovered. Finally, we observed that the inhibitor of BDNF/TrkB signaling prevented HPC from ameliorating learning and memory in mice. However, the DNMT inhibitor promoted spatial cognition in mice. Thus, we suggest that HPC may upregulate BDNF by inhibiting DNMTs and decreasing DNA methylation of the BDNF gene and then activate BDNF/TrkB signaling to improve learning and memory in mice. This may provide theoretical guidance for the clinical treatment of cognitive dysfunction caused by ischemia/hypoxia disease.

The optimum oxygen level in hypoxic culture conditions of ligament derived stem cells: experimental research.

This study aimed to investigate the optimum oxygen level for rabbit cruciate ligament fibroblast cells culture and Ligament Derived Conditioned Medium/LD-CM (Secretome) preparation in vitro. Fibroblasts were isolated from the cruciate ligament of the rabbit's knee. Cultured of rabbit cruciate ligament Fibroblast Cells (fifth passage) were assigned to the slight (5% O2), middle (3% O2), and severe hypoxia (1% O2) groups and the normoxia (21% O2) group. Measurement of growth factors: TGF-β1, PDGF, FGF, and VEGF in LD-CM (Secretome) used an enzyme-linked immunosorbent assay. The highest number of cultured cells were in the 5% O2 group compared to the normoxia, 1 and 3% groups. The hypoxia 5% group also had increased productions of PDGF, FGF, and VEGF proteins in LD-CM (secretome) compared to the 1, 3%, and normoxia groups. TGF-β1 production was slightly higher in the 3 group than the 5% group. The hypoxic precondition of 5% oxygen was the optimum condition for ligament culture and ligament derived conditioned medium (secretome) preparation in vitro.

Hypoxic preconditioned mesenchymal stem cells ameliorate rat brain injury after cardiopulmonary resuscitation by suppressing neuronal pyroptosis.

Cardiac arrest (CA) can result in cerebral ischaemia-reperfusion injury and poor neurological outcomes. While bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to have protective effects in brain ischaemic disease, their efficacy can be reduced by the poor oxygen environment. In this study, we investigated the neuroprotective effects of hypoxic preconditioned BMSCs (HP-BMSCs) and normoxic BMSCs (N-BMSCs) in a cardiac arrest rat model by examining their ability to ameliorate cell pyroptosis. The mechanism underlying the process was also explored. Cardiac arrest was induced in rats for 8 min and surviving rats received 1 × 106 normoxic/hypoxic BMSCs or PBS via intracerebroventricular (ICV) transplantation. Neurological function of rats was evaluated using neurological deficit scores (NDSs) and examined for brain pathology. Serum S100B and neuron-specific enolase (NSE) levels and cortical proinflammatory cytokines were measured to evaluate brain injury. Pyroptosis-related proteins in the cortex after cardiopulmonary resuscitation (CPR) were measured using western blotting and immunofluorescent staining. Transplanted BMSCs were tracked using bioluminescence imaging. Results showed significantly better neurological function and neuropathological damage after transplantation with HP-BMSCs. In addition, HP-BMSCs reduced levels of pyroptosis-related proteins in the rat cortex after CPR and significantly reduced levels of biomarkers for brain injury. Mechanistically, HP-BMSCs alleviated brain injury by reducing the expressions of HMGB1, TLR4, NF-κB p65, p38 MAPK and JNK in the cortex. Our study demonstrated that hypoxic preconditioning could enhance the efficacy of BMSCs in alleviating post-resuscitation cortical pyroptosis. This effect may be related to the regulation of the HMGB1/TLR4/NF-κB, MAPK signalling pathways.

Hypoxia-Preconditioned Bone Marrow Mesenchymal Stem Cells Improved Cerebral Collateral Circulation and Stroke Outcome in Mice.

Adequate collateral circulation can remarkably improve patient prognoses for patients experiencing ischemic stroke. Hypoxic preconditioning enhances the regenerative properties of bone marrow mesenchymal stem cells (BMSCs). Rabep2 (RAB GTPase binding effector protein 2) is a key protein in collateral remodeling. We investigated whether BMSCs and hypoxia-preconditioned BMSCs (H-BMSCs) augment collateral circulation poststroke, particularly through Rabep2 regulation. BMSCs or H-BMSCs (1×106) were delivered intranasally in ischemic mice with distal middle cerebral artery occlusion at 6 hours poststroke. Two-photon microscopic imaging and vessel painting methods were used to analyze collateral remodeling. Blood flow, vascular density, infarct volume, and gait analysis were assessed to evaluate poststroke outcomes. Expressions of proangiogenic marker VEGF (vascular endothelial growth factor) and Rabep2 were determined by Western blotting. Western blot, EdU (5-ethynyl-2'-deoxyuridine) incorporation, and tube formation assays were conducted on cultured endothelial cells treated with BMSCs. BMSCs were more effectively transplanted in the ischemic brain after hypoxic preconditioning. The ipsilateral collateral diameter was increased by BMSCs and strengthened by H-BMSCs (P<0.05). BMSCs increased peri-infarct blood flow and vascular density and reduced infarct volume, gait deficits (P<0.05), and furthermore by H-BMSCs (P<0.05). VEGF and Rabep2 protein expression was increased by BMSCs (P<0.05), which was enhanced by preconditioning (P<0.01). Additionally, BMSCs increased Rabep2 expression, proliferation, and tube formation of endothelial cells in vitro (P<0.05). H-BMSCs enhanced these effects (P<0.05), which were annulled by Rabep2 knockdown. BMSCs increased collateral circulation and improved poststroke outcomes, through the upregulation of Rabep2. These effects were enhanced by hypoxic preconditioning.

Intermittent Hypoxic Preconditioning Plays a Cardioprotective Role in Doxorubicin-Induced Cardiomyopathy.

Intermittent hypoxic preconditioning (IHP) is a well-established cardioprotective intervention in models of ischemia/reperfusion injury. Nevertheless, the significance of IHP in different cardiac pathologies remains elusive. In order to investigate the role of IHP and its effects on calcium-dependent signalization in HF, we employed a model of cardiomyopathy induced by doxorubicin (Dox), a widely used drug from the class of cardiotoxic antineoplastics, which was i.p. injected to Wistar rats (4 applications of 4mg/kg/week). IHP-treated group was exposed to IHP for 2weeks prior to Dox administration. IHP ameliorated Dox-induced reduction in cardiac output. Western blot analysis revealed increased expression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) while the expression of hypoxia inducible factor (HIF)-1-α, which is a crucial regulator of hypoxia-inducible genes, was not changed. Animals administered with Dox had further decreased expression of TRPV1 and TRPV4 (transient receptor potential, vanilloid subtype) ion channels along with suppressed Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation. In summary, IHP-mediated improvement in cardiac output in the model of Dox-induced cardiomyopathy is likely a result of increased SERCA2a expression which could implicate IHP as a potential protective intervention in Dox cardiomyopathy, however, further analysis of observed effects is still required.

Metabolomic and Proteomic Identification of Serum Exosome for Hypoxic Preconditioning Participants.

In high-altitude areas, hypoxic stress can elicit a series of physiological responses in humans. Exosomes play important roles in both local and distal cellular communications. We used ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) studies to analyze the differentially expressed metabolomics and proteomics in serum exosome of hypoxic preconditioning participants and control subjects in the hypoxic conditions. Fifty-seven military personnel were divided into hypoxic preconditioning group (n = 27) and control group (n = 30). One hundred thirty-six differentially expressed serum exosomal metabolites were found between the hypoxic preconditioning and control groups in the hypoxic conditions, and these differentially expressed metabolites were enriched in pathways related to lysine degradation, butanoate metabolism, GABAergic synapse, histidine metabolism, and linoleic acid metabolism. In addition, hypoxic preconditioning participants showed 102 excellent differential expressions of proteomics compared to controls, which involved actin cytoskeleton organization, hemostasis, complement and coagulation cascades, vesicle-medicated transport, wound healing, etc. We revealed that the expression of exosomal metabolites and proteomics in hypoxic preconditioning participants was significantly different compared to controls in hypoxic conditions.