Reconstitution of Kidney Side Population Cells after Ischemia-Reperfusion Injury by Self-Proliferation and Bone Marrow-Derived Cell Homing

The aim of this study was to examine the contribution of side population (SP) cells from kidney and bone marrow for reconstitution of kidney SP pools after ischemia-reperfusion injury (IRI). The SP and non-SP cells in kidneys following IRI were isolated and serially assessed by fluorescence-activated cell sorting. The apoptosis, proliferation, phenotype, and paracrine actions of SP cells were evaluated in vitro and in vivo. Results indicated that the SP cells from ischemic kidney were acutely depleted within one day following renal IRI and were progressively restored to baseline within 7 days after IRI, through both proliferation of remaining kidney SP cells and homing of bone marrow-derived cells to ischemic kidney. Either hypoxia or serum deprivation alone increased apoptosis of SP cells, and a combination of both further aggravated it. Furthermore, hypoxia in vivo and in vitro induced the increase in the secretion of vascular endothelial growth factor, insulin-like growth factor 1, hepatocyte growth factor, and stromal cell-derived factor-1α in kidney SP but not non-SP cells. In summary, these results suggest that following renal IRI, kidney SP cells are acutely depleted and then progressively restored to baseline levels by both self-proliferation and extrarenal source, that is, bone marrow-derived cell homing

Regulation of serotonin production by specific microbes from piglet gut

Abstract Background Serotonin is an important signaling molecule that regulates secretory and sensory functions in the gut. Gut microbiota has been demonstrated to affect serotonin synthesis in rodent models. However, how gut microbes regulate intestinal serotonin production in piglets remains vague. To investigate the relationship between microbiota and serotonin specifically in the colon, microbial composition and serotonin concentration were analyzed in ileum-cannulated piglets subjected to antibiotic infusion from the ileum when comparing with saline infusion. Microbes that correlated positively with serotonin production were isolated from piglet colon and were further used to investigate the regulation mechanisms on serotonin production in IPEC-J2 and a putative enterochromaffin cell line RIN-14B cells. Results Antibiotic infusion increased quantities of Lactobacillus amylovorus (LA) that positively correlated with increased serotonin concentrations in the colon, while no effects observed for Limosilactobacillus reuteri (LR). To understand how microbes regulate serotonin, representative strains of LA, LR, and Streptococcus alactolyticus (SA, enriched in feces from prior observation) were selected for cell culture studies. Compared to the control group, LA, LR and SA supernatants significantly up-regulated tryptophan hydroxylase 1 (TPH1) expression and promoted serotonin production in IPEC-J2 cells, while in RIN-14B cells only LA exerted similar action. To investigate potential mechanisms mediated by microbe-derived molecules, microbial metabolites including lactate, acetate, glutamine, and γ-aminobutyric acid were selected for cell treatment based on computational and metabolite profiling in bacterial supernatant. Among these metabolites, acetate upregulated the expression of free fatty acid receptor 3 and TPH1 while downregulated indoleamine 2,3-dioxygenase 1. Similar effects were also recapitulated when treating the cells with AR420626, an agonist targeting free fatty acid receptor 3. Conclusions Overall, these results suggest that Lactobacillus amylovorus showed a positive correlation with serotonin production in the pig gut and exhibited a remarkable ability to regulate serotonin production in cell cultures. These findings provide evidence that microbial metabolites mediate the dialogue between microbes and host, which reveals a potential approach using microbial manipulation to regulate intestinal serotonin biosynthesis

The Role of SDF-1-CXCR4/CXCR7 Axis in the Therapeutic Effects of Hypoxia-Preconditioned Mesenchymal Stem Cells for Renal Ischemia/Reperfusion Injury

In vitro hypoxic preconditioning (HP) of mesenchymal stem cells (MSCs) could ameliorate their viability and tissue repair capabilities after transplantation into the injured tissue through yet undefined mechanisms. There is also experimental evidence that HP enhances the expression of both stromal-derived factor-1 (SDF-1) receptors, CXCR4 and CXCR7, which are involved in migration and survival of MSCs in vitro, but little is known about their role in the in vivo therapeutic effectiveness of MSCs in renal ischemia/reperfusion (I/R) injury. Here, we evaluated the role of SDF-1-CXCR4/CXCR7 pathway in regulating chemotaxis, viability and paracrine actions of HP-MSCs in vitro and in vivo. Compared with normoxic preconditioning (NP), HP not only improved MSC chemotaxis and viability but also stimulated secretion of proangiogenic and mitogenic factors. Importantly, both CXCR4 and CXCR7 were required for the production of paracrine factors by HP-MSCs though the former was only responsible for chemotaxis while the latter was for viability. SDF-1α expression was upregulated in postischemic kidneys. After 24 h systemical administration following I/R, HP-MSCs but not NP-MSCs were selectively recruited to ischemic kidneys and this improved recruitment was abolished by neutralization of CXCR4, but not CXCR7. Furthermore, the increased recruitment of HP-MSCs was associated with enhanced functional recovery, accelerated mitogenic response, and reduced apoptotic cell death. In addition, neutralization of either CXCR4 or CXCR7 impaired the improved therapeutic potential of HP-MSCs. These results advance our knowledge about SDF-1-CXCR4/CXCR7 axis as an attractive target pathway for improving the beneficial effects of MSC-based therapies for renal I/R

Preparation and Characterization of Nanoliposomes Entrapping Medium-Chain Fatty Acids and Vitamin C by Lyophilization

The complex nanoliposomes encapsulating both a hydrophilic drug vitamin C (vit C) and hydrophobic drug medium-chain fatty acids (MCFAs) was prepared by combining double emulsion method with dynamic high pressure microfluidization. The complex nanoliposomes was further freeze-dried under −86 °C for 48 h with sucrose at the sucrose/lipids ratio of 2:1(w/w) in order to enhance its stability. The freeze-dried complex nanoliposomes under the suitable conditions exhibited high entrapment efficiency of MCFAs (44.26 ± 3.34)%, relatively high entrapment efficiency of vit C (62.25 ± 3.43)%, low average size diameter (110.4 ± 7.28) nm and good storage stability at 4 °C for 60 days with slight changes in mean particle diameter and drug entrapment efficiencies. The results of transmission electron microscopy of freeze-dried complex nanoliposomes also showed that the freeze-dried samples with sucrose were stable without great increase in their particle sizes and without destroying their spherical shape. The results indicated that sucrose presented well protection effects in MCFAs-vit C complex nanoliposomes, suggesting the possibility of further usage in commercial liposomes

Transcriptional and Proteolytic Regulation of the Toxin-Antitoxin Locus <i>vapBC10</i> (<i>ssr2962/slr1767</i>) on the Chromosome of <i>Synechocystis</i> sp. PCC 6803

<div><p>VapBC toxin-antitoxin (TA) systems are defined by the association of a PIN-domain toxin with a DNA-binding antitoxin, and are thought to play important physiological roles in bacteria and archaea. Recently, the PIN-associated gene pair PIN-COG2442 was proposed to encode VapBC-family TA system and found to be abundant in cyanobacteria. However, the features of these predicted TA loci remain under investigation. We here report characterization of the PIN-COG2442 locus <i>vapBC10</i> (<i>ssr2962/slr1767</i>) on the chromosome of <i>Synechocystis</i> sp. PCC 6803. RT-PCR analysis revealed that the <i>vapBC10</i> genes were co-transcribed under normal growth conditions. Ectopic expression of the PIN-domain protein VapC10 caused growth arrest of <i>Escherichia coli</i> that does not possess <i>vapBC</i> TA locus. Coincidentally, this growth-inhibition effect could be neutralized by either simultaneous or subsequent production of the COG2442-domain protein VapB10 through formation of the TA complex VapBC10 <i>in vivo</i>. In contrast to the transcription repression activity of the well-studied antitoxins, VapB10 positively auto-regulated the transcription of its own operon via specific binding to the promoter region. Furthermore, <i>in vivo</i> experiments in <i>E. coli</i> demonstrated that the <i>Synechocystis</i> protease ClpXP2s, rather than Lons, could cleave VapB10 and proteolytically activate the VapC10 toxicity. Our results show that the PIN-COG2442 locus <i>vapBC10</i> encodes a functional VapBC TA system with an alternative mechanism for the transcriptional auto-regulation of its own operon.</p></div

Effects of SDF-1-CXCR4/CXCR7 pathway on MSC paracrine actions.

<p>ELISA was performed to determine production of VEGF, β-FGF, IGF-1 and HGF from MSCs stimulated by hypoxia (3% O₂) or/and SDF-1α (50 ng/ml). The cells stimulated by neither hypoxia nor SDF-1α were used as control. (A) MSCs were stimulated with hypoxia or/and SDF-1α. *<i>P</i><0.05, vs control; ^†<i>P</i><0.05, vs SDF-1; ^‡<i>P</i><0.05, vs hypoxia. (B and C) The HP-MSCs stimulated with or whthout SDF-1α were treated with an anti-CXCR4 antibody (B), an anti-CXCR7 antibody (C), and the respective isotype-matched control antibodies. *<i>P</i><0.05, vs IgG2B (B) or IgG (C); ^†<i>P</i><0.05, vs αCXCR4 (B) or αCXCR7 (C); ^‡<i>P</i><0.05, vs SDF-1+IgG2B (B) or SDF-1+IgG (C). (D) NP-MSCs were transiently overexpressed with CXCR4 using pORF9-mCXCR4 vector or with CXCR7 using pORF9-mCXCR7 vector. *<i>P</i><0.05, vs empty vector; ^†<i>P</i><0.05, vs pORF9-mCXCR4 vector.</p

A model of regenerative potential of HP-MSCs in repair of I/R-AKI.

<p>Chemokine SDF-1 expression is upregulated in postischemic kidneys. HP enhances the expression of both SDF-1 receptors, CXCR4 and CXCR7, in MSCs. Intravenously injected HP-MSCs are recruited to the ischemic kidney and localized within the injured capillaries and in the interstitium through SDF-1α-CXCR4 interaction. The binding of SDF-1 to both CXCR4 and CXCR7 is responsible for the production of paracrine mediators, including VEGF, β-FGF, IGF-1 and HGF that exert mitogenic, anti-apoptotic, pro-angiogenic, and anti-inflammatory effects.</p