Ghrelin promotes cardiomyocyte differentiation of adipose tissue­derived mesenchymal stem cells by DDX17­mediated regulation of the SFRP4/Wnt/ß­catenin axis.

Adipose tissue­derived mesenchymal stem cells (ADMSCs) differentiate into cardiomyocytes and may be an ideal cell source for myocardial regenerative medicine. Ghrelin is a gastric­secreted peptide hormone involved in the multilineage differentiation of MSCs. To the best of our knowledge, however, the role and potential downstream regulatory mechanism of ghrelin in cardiomyocyte differentiation of ADMSCs is still unknown. The mRNA and protein levels were measured by reverse transcription­quantitative PCR and western blotting. Immunofluorescence staining was used to show the expression and cellular localization of cardiomyocyte markers and ß­catenin. RNA sequencing was used to explore the differentially expressed genes (DEGs) that regulated by ghrelin. The present study found that ghrelin promoted cardiomyocyte differentiation of ADMSCs in a concentration­dependent manner, as shown by increased levels of cardiomyocyte markers GATA binding protein 4, α­myosin heavy chain (α­MHC), ISL LIM homeobox 1, NK2 homeobox 5 and troponin T2, cardiac type. Ghrelin increased ß­catenin accumulation in nucleus and decreased the protein expression of secreted frizzled­related protein 4 (SFRP4), an inhibitor of Wnt signaling. RNA sequencing was used to determine the DEGs regulated by ghrelin. Functional enrichment showed that DEGs were more enriched in cardiomyocyte differentiation­associated terms and Wnt pathways. Dead­box helicase 17 (DDX17), an upregulated DEG, showed enhanced mRNA and protein expression levels following ghrelin addition. Overexpression of DDX17 promoted protein expression of cardiac­specific markers and ß­catenin and enhanced the fluorescence intensity of α­MHC and ß­catenin. DDX17 upregulation inhibited protein expression of SFRP4. Rescue assay confirmed that the addition of SFRP4 partially reversed ghrelin­enhanced protein levels of cardiac­specific markers and the fluorescence intensity of α­MHC. In conclusion, ghrelin promoted cardiomyocyte differentiation of ADMSCs by DDX17­mediated regulation of the SFRP4/Wnt/ß­catenin axis.

Structural measurements and vessel density of spectral-domain optic coherence tomography in early, moderate, and severe primary angle-closure glaucoma.

AIM: To compare the macular ganglion cell-inner plexiform layer (GCIPL) thickness, retinal nerve fiber layer (RNFL) thickness, optic nerve head (ONH) parameters, and retinal vessel density (VD) measured by spectral-domain optical coherence tomography (SD-OCT) and analyze the correlations between them in the early, moderate, severe primary angle-closure glaucoma (PACG) and normal eyes. METHODS: Totally 70 PACG eyes and 20 normal eyes were recruited for this retrospective analysis. PACG eyes were further separated into early, moderate, or severe PACG eyes using the Enhanced Glaucoma Staging System (GSS2). The GCIPL thickness, RNFL thickness, ONH parameters, and retinal VD were measured by SD-OCT, differences among the groups and correlations within the same group were calculated. RESULTS: The inferior and superotemporal sectors of the GCIPL thickness, rim area of ONH, average and inferior sector of the retinal VD were significantly reduced (all P<0.05) in the early PACG eyes compared to the normal and the optic disc area, cup to disc ratio (C/D), and cup volume were significantly higher (all P<0.05); but the RNFL was not significant changes in early and moderate PACG. In severe group, the GCIPL and RNFL thickness were obvious thinning with retinal VD were decreasing as well as C/D and cup volume increasing than other three groups (all P<0.01). In the early PACG subgroup, there were significant positive correlations between retinal VD and GCIPL thickness (except superonasal and inferonasal sectors, r=0.573 to 0.641, all P<0.05), superior sectors of RNFL thickness (r=0.055, P=0.049). More obvious significant positive correlations were existed in moderate PACG eyes between retinal VD and superior sectors of RNFL thickness (r=0.650, P=0.022), and temporal sectors of RNFL thickness (r=0.740, P=0.006). In the severe PACG eyes, neither GCIPL nor RNFL thickness was associated with retinal VD. CONCLUSION: The ONH damage and retinal VD loss appears earlier than RNFL thickness loss in PACG eyes. As the PACG disease progressed from the early to the moderate stage, the correlations between the retinal VD and RNFL thickness increases.

LNX2 involves in the role of ghrelin to promote the neuronal differentiation of adipose tissue-derived mesenchymal stem cells.

Adipose tissue-derived mesenchymal stem cells (ADSCs) have promising effects on nerve repair due to the differentiation ability to neural cells. Ghrelin has been shown to promote the neural differentiation of ADSCs. This work was designed to explore its underlying mechanism. Herein, we found high expression of LNX2 in ADSCs after neuronal differentiation. Knockdown of LNX2 might block neuronal differentiation of ADSCs, as evidenced by the decreased number of neural-like cells and dendrites per cell, and the reduced expressions of neural markers (including ß-Tubulin III, Nestin, and MAP2). We also demonstrated that LNX2 silencing suppressed the nuclear translocation of ß-catenin in differentiated ADSCs. Luciferase reporter assay indicated that LNX2 inhibited wnt/ß-catenin pathway by reducing its transcriptional activity. In addition, results showed that LNX2 expression was increased by ghrelin, and its inhibition diminished the effects of ghrelin on neuronal differentiation. Altogether, the results suggest that LNX2 is involved in the role of ghrelin to facilitate neuronal differentiation of ADSCs.

Krüppel-like factor 7 attenuates hippocampal neuronal injury after traumatic brain injury.

Our previous study has shown that the transcription factor Krüppel-like factor 7 (KLF7) promotes peripheral nerve regeneration and motor function recovery after spinal cord injury. KLF7 also participates in traumatic brain injury, but its regulatory mechanisms remain poorly understood. In the present study, an HT22 cell model of traumatic brain injury was established by stretch injury and oxygen-glucose deprivation. These cells were then transfected with an adeno-associated virus carrying KLF7 (AAV-KLF7). The results revealed that, after stretch injury and oxygen-glucose deprivation, KLF7 greatly reduced apoptosis, activated caspase-3 and lactate dehydrogenase, downregulated the expression of the apoptotic markers B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and cleaved caspase-3, and increased the expression of ßIII-tubulin and the antiapoptotic marker Bcl-2. Furthermore, KLF7 overexpression upregulated Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) phosphorylation in HT22 cells treated by stretch injury and oxygen-glucose deprivation. Immunoprecipitation assays revealed that KLF7 directly participated in the phosphorylation of STAT3. In addition, treatment with AG490, a selective inhibitor of JAK2/STAT3, weakened the protective effects of KLF7. A mouse controlled cortical impact model of traumatic brain injury was then established. At 30 minutes before modeling, AAV-KLF7 was injected into the ipsilateral lateral ventricle. The protein and mRNA levels of KLF7 in the hippocampus were increased at 1 day after injury and recovered to normal levels at 3 days after injury. KLF7 reduced ipsilateral hippocampal atrophy, decreased the injured cortex volume, downregulated Bax and cleaved caspase-3 expression, and increased the number of 5-bromo-2'-deoxyuridine-positive neurons and Bcl-2 protein expression. Moreover, KLF7 transfection greatly enhanced the phosphorylation of JAK2 and STAT3 in the ipsilateral hippocampus. These results suggest that KLF7 may protect hippocampal neurons after traumatic brain injury through activation of the JAK2/STAT3 signaling pathway. The study was approved by the Institutional Review Board of Mudanjiang Medical University, China (approval No. mdjyxy-2018-0012) on March 6, 2018.

Ghrelin promotes neural differentiation of adipose tissue-derived mesenchymal stem cell via AKT/mTOR and ß-catenin signaling pathways.

Adipose tissue-derived mesenchymal stem cells (ADSCs) are multipotent cells that can differentiate into various cell types. This study aimed to investigate the effect of ghrelin on the neural differentiation of rat ADSCs and underlying molecular mechanisms. Rat ADSCs were isolated and third-passage ADSCs were used in this study. The isolated ADSCs were characterized by flow cytometry analysis for MSCs' surface expression markers as evidenced by positive for CD90, CD44, and CD29 and negative for CD34, CD45, and CD11b/2f/c. The multilineage differentiation of ADSCs was confirmed by adipogenic, osteogenic, and neural differentiation. After induction of neurogenesis, the differentiated cells were identified by development of neuron-like morphology and expression of neural markers including glial fibrillary acidic protein, Nestin, MAP2, and ß-Tubulin III using immunofluorescence and western blot. Ghrelin concentration dependently elevated the proportion of neural-like cells and branching dendrites, as well as upregulated the expression of neural markers. Further, the expression of nuclear ß-catenin, p-GSK-3ß, p-AKT, and p-mTOR was increased by ghrelin, indicating an activation of ß-catenin and AKT/mTOR signaling after the ghrelin treatment. Importantly, inhibition of ß-catenin or AKT/mTOR signaling suppressed ghrelin-induced neurogenesis. Therefore, we demonstrate that ghrelin promotes neural differentiation of ADSCs through the activation of ß-catenin and AKT/mTOR signaling pathways.

Combinatory transplantation of mesenchymal stem cells with flavonoid small molecule in acellular nerve graft promotes sciatic nerve regeneration.

Previous animal studies have demonstrated that the flavonoid small-molecule TrkB agonist, 7, 8-dihydroxyflavone (DHF), promotes axon regeneration in transected peripheral nerves. In the present study, we investigated the combined effects of 7, 8-DHF treatment and bone marrow-derived stem/stromal cells (BMSCs) engraftment into acellular nerve allografts (ANAs) and explore relevant mechanisms that may be involved. Our results show that TrkB and downstream ERK1/2 phosphorylation are increased upon 7, 8-DHF treatment compared to the negative control group. Also, 7, 8-DHF promotes proliferation, survival, and Schwann-like cell differentiation of BMSCs in vitro. While selective ERK1/2 inhibitor U0126 suppressed the effect of upregulation of ERK1/2 phosphorylation and decreased cell proliferation, survival, and Schwann-like cell differentiation partially induced by 7, 8-DHF. In vivo, 7, 8-DHF promotes survival of transplanted BMSCs and upregulates axonal growth and myelination in regenerating ANAs. 7, 8-DHF+BMSCs also improved motor endplate density of target musculature. These benefits were associated with increased motor functional recovery. 7, 8-DHF+BMSCs significantly upregulated TrkB and ERK1/2 phosphorylation expression in regenerating ANA, and increased TrkB expression in the lumbar spinal cord. The mechanism of 7, 8-DHF action may be related to its ability to upregulate TrkB signaling, and downstream activation of survival signaling molecules ERK1/2 in the regenerating ANAs and spinal cord and improved survival of transplanted BMSCs. This study provides novel foundational data connecting the benefits of 7, 8-DHF treatment in neural injury and repair to BMSCs biology and function and demonstrates a potential combination approach for the treatment of injured peripheral nerve via nerve graft transplant.

Investigating multiple dysregulated pathways in rheumatoid arthritis based on pathway interaction network.

The traditional methods of identifying biomarkers in rheumatoid arthritis (RA) have focussed on the differentially expressed pathways or individual pathways, which however, neglect the interactions between pathways. To better understand the pathogenesis of RA, we aimed to identify dysregulated pathway sets using a pathway interaction network (PIN), which considered interactions among pathways. Firstly, RA-related gene expression profile data, protein-protein interactions (PPI) data and pathway data were taken up from the corresponding databases. Secondly, principal component analysis method was used to calculate the pathway activity of each of the pathway, and then a seed pathway was identified using data gleaned from the pathway activity. A PIN was then constructed based on the gene expression profile, pathway data, and PPI information. Finally, the dysregulated pathways were extracted from the PIN based on the seed pathway using the method of support vector machines and an area under the curve (AUC) index. The PIN comprised of a total of 854 pathways and 1064 pathway interactions. The greatest change in the activity score between RA and control samples was observed in the pathway of epigenetic regulation of gene expression, which was extracted and regarded as the seed pathway. Starting with this seed pathway, one maximum pathway set containing 10 dysregulated pathways was extracted from the PIN, having an AUC of 0.8249, and the result indicated that this pathway set could distinguish RA from the controls. These 10 dysregulated pathways might be potential biomarkers for RA diagnosis and treatment in the future.

Molecular examination of bone marrow stromal cells and chondroitinase ABC-assisted acellular nerve allograft for peripheral nerve regeneration.

The present study aimed to evaluate the molecular mechanisms underlying combinatorial bone marrow stromal cell (BMSC) transplantation and chondroitinase ABC (Ch-ABC) therapy in a model of acellular nerve allograft (ANA) repair of the sciatic nerve gap in rats. Sprague Dawley rats (n=24) were used as nerve donors and Wistar rats (n=48) were randomly divided into the following groups: Group I, Dulbecco's modified Eagle's medium (DMEM) control group (ANA treated with DMEM only); Group II, Ch-ABC group (ANA treated with Ch-ABC only); Group III, BMSC group (ANA seeded with BMSCs only); Group IV, Ch-ABC + BMSCs group (Ch-ABC treated ANA then seeded with BMSCs). After 8 weeks, the expression of nerve growth factor, brain-derived neurotrophic factor and vascular endothelial growth factor in the regenerated tissues were detected by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Axonal regeneration, motor neuron protection and functional recovery were examined by immunohistochemistry, horseradish peroxidase retrograde neural tracing and electrophysiological and tibialis anterior muscle recovery analyses. It was observed that combination therapy enhances the growth response of the donor nerve locally as well as distally, at the level of the spinal cord motoneuron and the target muscle organ. This phenomenon is likely due to the propagation of retrograde and anterograde transport of growth signals sourced from the graft site. Collectively, growth improvement on the donor nerve, target muscle and motoneuron ultimately contribute to efficacious axonal regeneration and functional recovery. Thorough investigation of molecular peripheral nerve injury combinatorial strategies are required for the optimization of efficacious therapy and full functional recovery following ANA.

Sciatic nerve regeneration in KLF7-transfected acellular nerve allografts.

OBJECTIVE: Krüppel-like Factor 7 (KLF7) is a transcription factor that promotes axon regeneration in the central nervous system. Here, we assessed whether KLF7 stimulates regeneration after peripheral nerve injury. METHODS: C57BL/6 mice received an acellular nerve allograft (ANA) injected with either adeno-associated virus 2 (AAV2) vector or AAV2-KLF7 for sciatic nerve gap repair. After 4 weeks, KLF7 was detected by RT-PCR, western blot and immunohistochemistry in regenerated nerves. Axonal regeneration and functional recovery were examined by immunohistochemistry, Fluorogold (FG) and cholera toxin B (CTB) retrograde neural tracing, sciatic function index (SFI), angle of ankle, Hargreaves test and electrophysiological analysis. RESULTS: With AAV2-KLF7 injection, KLF7 expression increased in regenerated nerves, and amplitude, score of SFI, angle of ankle and FG-labelled spinal cord neurons were increased. We observed elevated CTB-labelled neurons in dorsal root ganglia (DRG), neurofilaments, P0 (peripheral myelin) and S100 and decreased latency period and withdrawal latencies in the Hargreaves test. The SFI was significantly correlated with amplitude and regenerated axon number. Tyrosine kinase A (TrkA) and B (TrkB) receptors were also increased in the DRG. CONCLUSIONS: Our findings suggest that KLF7 promoted peripheral nerve axonal regeneration, further supporting a role for KLF7 as a growth-promoting transcription factor in the injured nervous system.

Expressions of type I collagen, α2 integrin and ß1 integrin in sclera of guinea pig with defocus myopia and inhibitory effects of bFGF on the formation of myopia.

AIM: To investigate the expressions of type I collagen, α2 integrin and ß1 integrin in the posterior sclera of guinea pigs with defocus myopia and whether basic fibroblast growth factor (bFGF) injection inhibits the formation and development of myopia by upregulating the expression of type I collagen, α2 integrin and ß1 integrin. METHODS: After 14 days of treatment, the refractive state and axial length were measured and the levels of type I collagen, α2 integrin and ß1 integrin were assayed in the posterior sclerae of groups of guinea pigs that wore a monocular -7D polymethylmethacrylate (PMMA) lens or had -7D lens wear followed by the peribulbar injection of Phosphate Buffer Solution (PBS) or bFGF. The untreated fellow eye served as a control. Guinea pigs with no treatment served as normal group. RESULTS: The results showed that 14 days of monocular defocus increased axial eye length and refraction, while bFGF delivery inhibited them markedly. Further, it was also found that the monocular -7D lens could decrease the levels of type I collagen, α2 integrin and ß1 integrin expressions, while, unlike PBS, bFGF increased them significantly in comparison to contralateral control eyes and normal eyes. CONCLUSION: bFGF can prevent the formation and development of defocus myopia by upregulating the expressions of type I collagen, α2 integrin and ß1 integrin. Taken together, our results demonstrate that bFGF promotes sclera remodeling to prevent myopia in guinea pigs.

Synergistic effects of bone mesenchymal stem cells and chondroitinase ABC on nerve regeneration after acellular nerve allograft in rats.

This study aimed to evaluate whether combination therapy of bone marrow stromal cells (BMSCs) transplantation and chondroitinase ABC (ChABC) treatment further enhances axonal regeneration and functional recovery after acellular nerve allograft repair of the sciatic nerve gap in rats. Eight Sprague-Dawley rats were used as nerve donors, and 32 Wistar rats were randomly divided into four groups: Group I: acellular rat sciatic nerve (ARSN) group; Group II: ChABC treatment; Group III: BMSCs transplantation; and Group IV: ChABC treatment and BMSCs transplantation. The results showed that compared with ARSN control group, BMSC transplantation promoted axonal regeneration, the secretion of neural trophic factors NGF, BDNF and axon angiogenesis in nerve graft. ChABC treatment degraded chondroitin sulfate proteoglycans in ARSN in vitro and in vivo and improved BMSCs survival in ARSN. The combination therapy caused much better beneficial effects evidenced by increasing sciatic function index, nerve conduction velocity, restoration rate of tibialis anterior wet muscle weight, and myelinated nerve number, but did not further boost the therapeutic effects on neurotrophic factor production, axon angiogenesis, and sensory functional recovery by BMSC transplantation. Taken together, for the first time, we demonstrate the synergistic effects of BMSC transplantation and BMSCs treatment on peripheral nerve regeneration, and our findings may help establish novel strategies for cell transplantation therapy for peripheral nerve injury.

Sciatic nerve repair by acellular nerve xenografts implanted with BMSCs in rats xenograft combined with BMSCs.

Acellular nerves possess the structural and biochemical features similar to those of naive endoneurial tubes, and have been proved bioactive for allogeneil graft in nerve tissue engineering. However, the source of allogenic donators is restricted in clinical treatment. To explore sufficient substitutes for acellular nerve allografts (ANA), we investigated the effectiveness of acellular nerve xenografts (ANX) combined with bone marrow stromal cells (BMSCs) on repairing peripheral nerve injuries. The acellular nerves derived from Sprague-Dawley rats and New Zealand rabbits were prepared, respectively, and BMSCs were implanted into the nerve scaffolds and cultured in vitro. All the grafts were employed to bridge 1 cm rat sciatic nerve gaps. Fifty Wistar rats were randomly divided into five groups (n = 10 per group): ANA group, ANX group, BMSCs-laden ANA group, BMSCs-laden ANX group, and autologous nerve graft group. At 8 weeks post-transplantation, electrophysiological study was performed and the regenerated nerves were assayed morphologically. Besides, growth-promoting factors in the regenerated tissues following the BMSCs integration were detected. The results indicated that compared with the acellular nerve control groups, nerve regeneration and functional rehabilitation for the xenogenic nerve transplantation integrated with BMSCs were advanced significantly, and the rehabilitation efficacy was comparable with that of the autografting. The expression of neurotrophic factors in the regenerated nerves, together with that of brain-derived neurotrophic factor (BDNF) in the spinal cord and muscles were elevated largely. In conclusion, ANX implanted with BMSCs could replace allografts to promote nerve regeneration effectively, which offers a reliable approach for repairing peripheral nerve defects.

Biocompatibility of acellular nerves of different mammalian species for nerve tissue engineering.

To explore the biocompatibility of acellular nerves of different mammalian species, for the acellular nerves derived from rats and rabbits, the morphology, immunocompatibility, and cytocompatibility with bone marrow stromal cells (BMSCs) were evaluated. The results indicated that the tridimensional architecture and main proteins of endoneurial tubes in both biomaterials were well retained. The nerve scaffolds did not show immunogenicity or cytotoxicity, but facilitated growth of BMSCs and secretion of neurotrophic factors in vitro. In conclusion, acellular nerves of different species possess favorable biocompatibility, and xenogenic acellular nerves combined with BMSCs have potential to replace allografts for peripheral nerve reconstruction.

Adipose-derived stem cells promote peripheral nerve repair.

INTRODUCTION: Recent evidence suggests that the implantation of bone marrow-derived mesenchymal stem cells improves peripheral nerve regeneration. In this study we aimed to investigate whether adipose-derived stem cells (ADSCs) can be used for peripheral nerve repair. MATERIAL AND METHODS: In a rat model, nerve regeneration was evaluated across a 15 mm lesion in the sciatic nerve by using an acellular nerve injected with allogenic ADSCs. The walking behaviour of rats was measured by footprint analysis, and electrophysiological analysis and histological examination were performed to evaluate the efficacy of nerve regeneration. RESULTS: Cultured ADSCs became morphologically homogeneous with a bipolar, spindle-like shape after ex vivo expansion. Implantation of ADSCs into the rat models led to (i) improved walking behaviour as measured by footprint analysis, (ii) increased conservation of muscle-mass ratio of gastrocnemius and soleus muscles, (iii) increased nerve conduction velocity, and (iv) increased number of myelinated fibres within the graft. CONCLUSIONS: Adipose-derived stem cells could promote peripheral nerve repair in a rat model. Although the detailed mechanism by which ADSCs promote peripheral nerve regeneration is being investigated in our lab, our results suggest that ADSCs transplantation represents a powerful therapeutic approach for peripheral nerve injury.