Effect of Intrahippocampal Administration of α7 Subtype Nicotinic Receptor Agonist PNU-282987 and Its Solvent Dimethyl Sulfoxide on the Efficiency of Hypoxic Preconditioning in Rats

We have previously suggested a key role of the hippocampus in the preconditioning action of moderate hypobaric hypoxia (HBH). The preconditioning efficiency of HBH is associated with acoustic startle prepulse inhibition (PPI). In rats with PPI > 40%, HBH activates the cholinergic projections of hippocampus, and PNU-282987, a selective agonist of α7 nicotinic receptors (α7nAChRs), reduces the HBH efficiency and potentiating effect on HBH of its solvent dimethyl sulfoxide (DMSO, anticholinesterase agent) when administered intraperitoneally. In order to validate the hippocampus as a key structure in the mechanism of hypoxic preconditioning and research a significance of α7nAChR activation in the hypoxic preconditioning, we performed an in vivo pharmacological study of intrahippocampal injections of PNU-282987 into the CA1 area on HBH efficiency in rats with PPI ≥ 40%. We found that PNU-282987 (30 μM) reduced HBH efficiency as with intraperitoneal administration, while DMSO (0.05%) still potentiated this effect. Thus, direct evidence of the key role of the hippocampus in the preconditioning effect of HBH and some details of this mechanism were obtained in rats with PPI ≥ 40%. The activation of α7nAChRs is not involved in the cholinergic signaling initiated by HBH or DMSO via any route of administration. Possible ways of the potentiating action of DMSO on HBH efficiency and its dependence on α7nAChRs are discussed.

Hypoxic preconditioning in renal ischaemia–reperfusion injury: a review in pre-clinical models

Ischaemia–reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and chronic kidney disease, which consists of cellular damage and renal dysfunction. AKI is a major complication that is of particular concern after cardiac surgery and to a lesser degree following organ transplantation in the immediate post-transplantation period, leading to delayed graft function. Because effective therapies are still unavailable, several recent studies have explored the potential benefit of hypoxic preconditioning (HPC) on IRI. HPC refers to the acquisition of increased organ tolerance to subsequent ischaemic or severe hypoxic injury, and experimental evidences suggest a potential benefit of HPC. There are three experimental forms of HPC, and, for better clarity, we named them as follows: physical HPC, HPC via treated-cell administration and stabilised hypoxia-inducible factor (HIF)-1α HPC, or mimicked HPC. The purpose of this review is to present the latest developments in the literature on HPC in the context of renal IRI in pre-clinical models. The data we compiled suggest that preconditional activation of hypoxia pathways protects against renal IRI, suggesting that HPC could be used in the treatment of renal IRI in transplantation.

Serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of mesenchymal stem cells on experimental renal fibrosis

Background Mesenchymal stem cells (MSCs) repair injured tissue in a paracrine manner. To enhance their therapeutic properties, preconditioning with various factors has been researched. We have previously showed that MSCs cultured in serum-free medium (SF-MSCs) promote their immunosuppressive ability, thereby enhancing their anti-fibrotic effect. Here, we examined whether serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of MSCs on renal fibrosis in rats with ischemia–reperfusion injury (IRI). Methods SF-MSCs were incubated under 1% O2 conditions (hypo-SF-MSCs) or 21% O2 conditions (normo-SF-MSCs) for 24 h before collection. After IRI procedure, hypo-SF-MSCs or normo-SF-MSCs were injected through the abdominal aorta. At 7 or 21 days post-injection, the rats were killed and their kidneys were collected to evaluate inflammation and fibrosis. In in vitro experiments, we investigated whether hypo-SF-MSCs enhanced secretion of anti-fibrotic humoral factors using transforming growth factor (TGF)-β1-stimulated HK-2 cells incubated with conditioned medium from hypo-SF-MSCs or normo-SF-MSCs. Results Normo-SF-MSCs showed attenuation of senescence, which increased their proliferative capacity. Although no significant difference in cellular senescence was found between normo-SF-MSCs and hypo-SF-MSCs, hypo-SF-MSCs further increased their proliferative capacity compared with normo-SF-MSCs. Additionally, administration of hypo-SF-MSCs more strongly ameliorated renal fibrosis than that of normo-SF-MSCs. Moreover, although hypo-SF-MSCs strongly attenuated infiltration of inflammatory cells compared with the control rats, which were treated with PBS, this attenuation was almost equal between normo-SF-MSCs and hypo-SF-MSCs. In vitro experiments revealed that hypo-SF-MSCs more significantly inhibited transforming growth factor (TGF)-β/Smad signaling compared with normo-SF-MSCs. Moreover, hypoxic preconditioning increased hepatocyte growth factor (HGF) secretion even under serum-free conditions, whereas knockdown of HGF in hypo-SF-MSCs attenuated inhibition of TGF-β/Smad signaling. Conclusions These results indicate that administration of ex vivo-expanded, hypoxia-preconditioned SF-MSCs may be a useful cell therapy to prevent renal fibrosis.

Hypoxic preconditioning promotes survival of human adipocyte mesenchymal stem cell via expression of prosurvival and proangiogenic biomarkers

Background: Contributing factors for improved survival of human adipocytes mesenchymal stem cells (h-AMSCs) cultured through hypoxia preconditioning, in example apoptosis inhibition involving BCL2 and HSP27 expression, trigger signal expression (VEGF), SCF expression, OCT-4 expression, and CD44+ expression. The objective if this study was to explain the mechanism and role of hypoxic preconditioning and the optimal duration of hypoxic preconditioning exposure to improve survival of h-AMSCs. Methods: An experimental laboratory explorative study (in vitro) with hypoxic preconditioning in h-AMSCs cultures. This research was conducted through four stages. First, isolation of h-AMSCs culture from adipose tissue of patients. Second, the characterization of h-AMSCs from adipose tissue by phenotype (flowcytometry) through CD44+, CD90+ and CD45-expression before being pre-conditioned for hypoxic treatment. Third, the hypoxic preconditioning in h-AMSCs culture (in vitro) was performed with an oxygen concentration of 1% for 24, 48 and 72 hours. Fourth, observation of survival from h-AMSCs culture was tested on the role of CD44+, VEGF, SCF, OCT-4, BCL2, HSP27 with Flowcytometry and apoptotic inhibition by Tunnel Assay method. Results: The result of regression test showed that time difference had an effect on VEGF expression (p<0.001;β=-0.482) and hypoxia condition also influenced VEGF expression (p<0.001;β=0.774). The result of path analysis showed that SCF had effect on OCT-4 expression (p<0.001; β=0.985). The regression test results showed that time effects on HSP27 expression (p<0.001; β=0.398) and hypoxia precondition also affects HSP27 expression (p<0.001; β=0.847). Pathway analysis showed that BCL2 expression inhibited apoptosis (p=0.030; β=-0.442) and HSP27 expression also inhibited apoptosis (p<0,001;β=-0.487). Conclusion: Hypoxic preconditioning of h-AMSC culture has proven to increase the expression of VEGF, SCF, OCT-4, and BCL2 and HSP27. This study demonstrated and explained the existence of a new mechanism of increased h-AMSC survival in cultures with hypoxic preconditioning (O2 1%) via VEGF, SCF, OCT-4, BCL2, and HSP 27.

Hypoxic preconditioning rejuvenates mesenchymal stem cells and enhances neuroprotection following intracerebral hemorrhage via the miR-326-mediated autophagy

Background Intracerebral hemorrhage (ICH) is a major public health concern, and mesenchymal stem cells (MSCs) hold great potential for treating ICH. However, the quantity and quality of MSCs decline in the cerebral niche, limiting the potential efficacy of MSCs. Hypoxic preconditioning is suggested to enhance the survival of MSCs and augment the therapeutic efficacy of MSCs in ICH. MicroRNAs (miRNAs) are known to mediate cellular senescence. However, the precise mechanism by which miRNAs regulate the senescence of hypoxic MSCs remains to be further studied. In the present study, we evaluated whether hypoxic preconditioning enhances the survival and therapeutic effects of olfactory mucosa MSC (OM-MSC) survival and therapeutic effects in ICH and investigated the mechanisms by which miRNA ameliorates hypoxic OM-MSC senescence. Methods In the in vivo model, ICH was induced in mice by administration of collagenase IV. At 24 h post-ICH, 5 × 105 normoxia or hypoxia OM-MSCs or saline was administered intracerebrally. The behavioral outcome, neuronal apoptosis, and OM-MSC survival were evaluated. In the in vitro model, OM-MSCs were exposed to hemin. Cellular senescence was examined by evaluating the expressions of P16INK4A, P21, P53, and by β-galactosidase staining. Microarray and bioinformatic analyses were performed to investigate the differences in the miRNA expression profiles between the normoxia and hypoxia OM-MSCs. Autophagy was confirmed using the protein expression levels of LC3, P62, and Beclin-1. Results In the in vivo model, transplanted OM-MSCs with hypoxic preconditioning exhibited increased survival and tissue-protective capability. In the in vitro model, hypoxia preconditioning decreased the senescence of OM-MSCs exposed to hemin. Bioinformatic analysis identified that microRNA-326 (miR-326) expression was significantly increased in the hypoxia OM-MSCs compared with that of normoxia OM-MSCs. Upregulation of miR-326 alleviated normoxia OM-MSC senescence, whereas miR-326 downregulation increased hypoxia OM-MSC senescence. Furthermore, we showed that miR-326 alleviated cellular senescence by upregulating autophagy. Mechanistically, miR-326 promoted the autophagy of OM-MSCs via the PI3K signaling pathway by targeting polypyrimidine tract-binding protein 1 (PTBP1). Conclusions Our study shows that hypoxic preconditioning delays OM-MSC senescence and augments the therapeutic efficacy of OM-MSCs in ICH by upregulating the miR-326/PTBP1/PI3K-mediated autophagy.