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W4387191978 abstract "Full text Figures and data Side by side Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Nitric oxide (NO), as a gaseous therapeutic agent, shows great potential for the treatment of many kinds of diseases. Although various NO delivery systems have emerged, the immunogenicity and long-term toxicity of artificial carriers hinder the potential clinical translation of these gas therapeutics. Mesenchymal stem cells (MSCs), with the capacities of self-renewal, differentiation, and low immunogenicity, have been used as living carriers. However, MSCs as gaseous signaling molecule (GSM) carriers have not been reported. In this study, human MSCs were genetically modified to produce mutant β-galactosidase (β-GALH363A). Furthermore, a new NO prodrug, 6-methyl-galactose-benzyl-oxy NONOate (MGP), was designed. MGP can enter cells and selectively trigger NO release from genetically engineered MSCs (eMSCs) in the presence of β-GALH363A. Moreover, our results revealed that eMSCs can release NO when MGP is systemically administered in a mouse model of acute kidney injury (AKI), which can achieve NO release in a precise spatiotemporal manner and augment the therapeutic efficiency of MSCs. This eMSC and NO prodrug system provides a unique and tunable platform for GSM delivery and holds promise for regenerative therapy by enhancing the therapeutic efficiency of stem cells. Editor's evaluation The study provides compelling evidence that treatment with the newly designed NO prodrug, MGP, selectively triggers NO release from your genetically engineered MSCs. The significance of the study is that it provides in vivo demonstration that MSCs can release NO in a spatiotemporal manner in a mouse model of acute kidney injury thus contributing to regeneration. This constitutes a landmark finding with profound implications that are expected to have widespread influence. The work not only shows the therapeutic efficiency of MSCs, but also holds promises for regenerative therapy by enhancing the therapeutic efficiency of stem cells. Thus, it is felt that the newly generated tools will be used by many investigators thus making the findings interesting to a broad audience. https://doi.org/10.7554/eLife.84820.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Animals are made up of cells of different types, with each type of cell specializing on a specific role. But for the body to work properly, the different types of cells must be able to coordinate with each other to respond to internal and external stimuli. This can be achieved through signaling molecules, that is, molecules released by a cell that can elicit a specific response in other cells. There are many types of different molecules, including hormones and signaling proteins. Gases can also be potent signaling molecules, participating in various biological processes. Nitric oxide (NO) is a gas signaling molecule that can freely diffuse through the membranes of cells and has roles in blood vessel constriction and other disease processes, making it a promising therapeutic agent. Unfortunately, using artificial carriers to deliver nitric oxide to the organs and tissues where it is needed can lead to issues, including immune reactions to the carrier and long-term toxicity. One way to avoid these effects is by using cells to deliver nitric oxide to the right place. Huang, Qian, Liu et al. have used mesenchymal stem cells – which usually develop to form connective tissues such as bone and muscle – to develop a cell-based NO-delivery system. The researchers genetically modified the mesenchymal stem cells to produce a compound called β-GALH363A. On its own β-GALH363A does not do much, but in its presence, a non-toxic, non-reactive compound developed by Huang, Qian, Liu et al., called MGP, can drive the release of NO from cells. To confirm the usefulness of their cells as a delivery system, Huang, Qian, Liu et al. transplanted some of the genetically modified mesenchymal stem cells into the kidneys of mice, and then showed that when these mice were given MGP, the levels of NO increased in the kidneys but not in other organs. This result confirms that the cell-based delivery system provides spatial and temporal control of the production of NO. These findings demonstrate a new delivery system for therapies using gas molecules, which can be controlled spatiotemporally in mice. In the future, these types of systems could be used in the clinic for long-term treatment of conditions where artificial carriers could lead to complications. Introduction Nitric oxide (NO), as a gaseous signaling molecule (GSM), plays vital roles in various physiological processes, including tissue regeneration (Midgley et al., 2020). Due to the extremely short half-life of NO, efforts to extensively enhance the therapeutic efficacy of NO by various types of artificial carriers, such as polymers, peptides, and nanoparticles, have achieved many successes, and there are remaining challenges that need to be addressed, including off-targeting, toxicity, immunogenicity, and clinical translation (Wilhelm et al., 2016; Park, 2013). Successful NO delivery requires an appropriate carrier for delivering these therapeutic molecules to the target sites in an on-demand and controlled manner, as well as in a protected, pharmacologically active form (Weaver et al., 2014). Bioinspired delivery vehicles, including biological cells, exosomes, and isolated membrane ghosts, are highly attractive because they exhibit excellent biodistribution, immune compatibility, innate disease-targeting abilities, and reduced toxicity (Bush et al., 2021; Yoo et al., 2011; Samanta et al., 2018; Fang et al., 2018). Among these systems, mesenchymal stem cells (MSCs) have gained considerable attention as drug-delivery vehicles owing to their genetic tractability, payload diversity, intrinsic tropism for disease sites, and differentiation potential (Tran and Damaser, 2015; Thanuja et al., 2018). MSCs are adult stem cells capable of self-renewal and multilineage differentiation that have been reported to promote tissue regeneration mediated by immunomodulatory and proangiogenic properties via paracrine effects, and MSC-based cell therapeutics have entered multiple clinical trials (Baraniak and McDevitt, 2010). Previous studies revealed that MSCs do not express blood-group antigens or MHC class II antigens and have long been reported to be hypoimmunogenic or immune privileged, which possess the clinical potential of MSC-based cell therapy (Zhou and Shi, 2023). MSCs as delivery vehicles have been widely studied for the delivery of various drugs and bioactive molecules into target disease sites, including peptides, proteins, DNA, and RNA (Peng et al., 2012; Zhang et al., 2021). However, the concept of MSCs as vehicles to deliver GSMs has not been studied. In this regard, we propose an MSC-based gas-generating platform as a unique and tunable platform for extensively broad GSM therapies. To achieve a gas-generating platform based on living cells, MSCs are programmed to sustain the generation and release NO by taking advantage of enzyme prodrug therapy, a versatile and exploitable technique to convert inactive, nontoxic prodrugs to active drugs at the desired sites (Zhang et al., 2015). In the present study, we developed an engineered MSC (eMSC)-based NO delivery platform for controlled NO delivery. In this advanced delivery platform, the expression of mutant β-galactosidase (β-GALH363A) by eMSCs enabled the production of NO when the NO prodrug was administered. We hypothesized that eMSCs could successfully generate and release NO in a precise spatiotemporal manner, as well as augment the therapeutic effects of MSCs and result in superior efficacy of stem cell therapy. Results MSC-mediated NO release platform Other than mentioned in context, MSCs represent human placenta-derived MSCs. To establish a living cell-mediated gas generation platform, MSCs were genetically modified to produce mutant β-galactosidase (β-GALH363A), which triggers NO release from MSCs when the corresponding NO prodrug is applied and avoids the interference of endogenous glycosidase. We constructed a lentiviral vector to stably express mutant β-GALH363A, Renilla luciferase (Rluc), and red fluorescence protein (RFP), in which Rluc and RFP were used for molecular imaging and immunohistology, respectively (Figure 1A). The expression of β-GALH363A in eMSCs was confirmed (Figure 1B and C and Figure 1—figure supplement 1A). Moreover, Rluc expression was confirmed by in vitro molecular imaging (Figure 1—figure supplement 1B). Furthermore, compared with wild-type MSCs, eMSCs did not exhibit significant differences in morphology or MSC surface markers (Figure 1—figure supplement 2). Figure 1 with 5 supplements see all Download asset Open asset Evaluation of the nitric oxide (NO)-engineered mesenchymal stem cell (eMSC) system in NO generation and release. (A) Genetically modifying placenta-derived MSCs to express mutant β-galactosidase (β-GALH363A) through a lentiviral transduction system, thus enabling continuous production of β-GALH363A for NO delivery. Moreover, the MSCs also stably expressed Renilla luciferase (Rluc) and red fluorescence protein (RFP) for in vivo bioluminescence imaging and immunofluorescence analysis, respectively. (B) The protein expression of β-GALH363A in eMSCs was evaluated by 6×His tag protein detection due to β-GALH363A carrying a C-terminal 6×His tag. (C) Immunofluorescence staining of β-GALH363A (green) in eMSCs. Scale bars, 50 μm. (D) Structure of the NO prodrug MGP, 6-methyl-galactose-benzyl-oxy NONOate. (E) Construction of the NO-eMSC system and the mechanism of NO release from this system. (F) The NO release profile from eMSCs administered at different concentrations of NO-prodrug MGP was determined using the Griess assay. n=3. (G) NO release from eMSCs with MGP administration in vitro was assessed by electron paramagnetic resonance (EPR), and the characteristic triplet EPR signal was observed. eMSCs served as controls. (H) The amount of NO-Fe (DETC)2 was calibrated using TEMPO as a standard. A total of 1×106 eMSCs approximately released 1 nmol NO in 1 hr. n=3. (I) The intracellular NO release in eMSCs treated with MGP was measured by diaminofluorescein (DAF) staining, in which DAF-FM DA is a cell-permeable fluorescent probe for the detection of intracellular NO. Scale bar, 25 μm. (J) Quantification of DAF fluorescence intensity in eMSCs in the presence of 5 µg/mL MGP for 6 hr. eMSC+MGP, eMSCs with MGP administration. All data are presented as the mean ± SD, **p<0.01, ***p<0.001, ****p<0.0001. Figure 1—source data 1 Synthesis route of the NO-prodrug MGP. https://cdn.elifesciences.org/articles/84820/elife-84820-fig1-data1-v2.docx Download elife-84820-fig1-data1-v2.docx Based on our previous report (Hou et al., 2019), we designed a new NO prodrug, 6-methyl-galactose-benzyl-oxy NONOate, which was named MGP. MGP contains a lipid-soluble self-decomposition chain oil that enhances its water distribution coefficient and enables it to cross the cell membrane of eMSCs and then be catalyzed by β-GALH363A to release NO from eMSCs. The structure and synthesis routes of MGP are shown in Figure 1D and Figure 1—source data 1. To verify whether the redesigned NO prodrug MGP could release NO specifically by β-GALH363A catalysis, we measured the NO release profile of MGP in response to β-GALH363A in vitro. As shown in Figure 1—figure supplement 3, MGP can be specifically catalyzed by β-GALH363A and has sustained NO release behavior. In this novel NO release eMSC (NO-eMSC) system, eMSCs, as a manufacturing factory for producing β-GALH363A, could further release NO by catalyzing the NO prodrug MGP (Figure 1E). We detected NO production in the medium of eMSCs with MGP administration using the Griess method, confirming that MGP can diffuse into eMSCs and release NO (Figure 1F). We next directly measured the NO levels of eMSCs in vitro by an electron paramagnetic resonance (EPR) technique using the spin trap Fe•(DETC)2 colloid solution. The characteristic triplet EPR signal was observed in eMSCs with MGP administration (Figure 1G), and we further confirmed that 1×106 eMSCs could release 1 nmol NO (Figure 1H). Moreover, NO production of eMSCs with MGP administration was visualized by using diaminofluorescein (DAF)-FM diacetate, a cell-permeable fluorescent probe for the detection of intracellular NO, which also supported that eMSCs could specifically release NO (Figure 1I and J). Considering the cell-protective effects of a low NO concentration, our results revealed that the optimum concentration of MGP was 2 µg/mL by cell viability, immunostaining for the proliferation marker Ki67, and immunostaining for the expression of proliferation-related genes (Figure 1—figure supplement 4). We further verified the protective effects of NO in response to oxidative stress-induced eMSC apoptosis. The bioluminescence imaging (BLI) assay showed that eMSC proliferation was markedly ameliorated by H2O2-induced oxidative stress (Figure 1—figure supplement 5). Enhanced antioxidation properties of eMSCs To further investigate the transcriptomic profile of eMSCs with MGP administration, mRNA sequencing analysis was performed. RNA sequencing (RNA-seq) profiles from eMSCs and eMSCs with MGP administration passed quality control (Figure 2—figure supplement 1). We identified a total of 228 differentially expressed genes (DEGs), of which 116 were upregulated genes and 112 were downregulated genes (Figure 2—figure supplement 2A). Analysis of Gene Ontology (GO) categories was mainly enriched in biological processes, including protein transport, cell differentiation, morphogenesis, and oxidation-reduction processes (Figure 2A). Among them, the oxidation-reduction process attracted our attention, suggesting that NO may improve intracellular antioxidant capacity, which was confirmed by gene set enrichment analysis (GSEA) (Figure 2B and Figure 2—figure supplement 2B). Meanwhile, we noted that a significant increase in some of the DEGs associated with antioxidation was observed in eMSCs with MGP administration, namely, GSR, SRXN1, RRBP1, BMI1, RPL24P4, and MAFG, indicating that NO may indeed play a cytoprotective role in eMSCs by elevating antioxidation capacity (Figure 2C). We also found that NO led to robust increases in survival- or proliferation-related genes, including FER, NRSN2, UCK2, CAB39, SNHG4, and SYDE1 (Figure 2D), as well as angiogenesis-related genes, such as ARRB2, RHOQ, MYADM, and CFHR1 (Figure 2—figure supplement 2C). Consistent with the aforementioned findings, GSEA of the whole transcriptome also revealed negative regulation of cell apoptosis and positive regulation of angiogenesis by NO (Figure 2E, Figure 2—figure supplement 2D). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis highlighted pathways that may contribute to enhanced antioxidant capacity, including the Jak-STAT signaling pathway and MAPK signaling pathway (Figure 2—figure supplement 2E). Together, these results demonstrated that NO might be able to ameliorate the oxidative stress of eMSCs and inhibit cell apoptosis (Figure 2F). Figure 2 with 2 supplements see all Download asset Open asset RNA sequencing (RNA-seq) analysis revealed that nitric oxide (NO) improves the antioxidant capacity of engineered mesenchymal stem cells (eMSCs). (A) Gene Ontology (GO) category analysis of differentially expressed genes (DEGs) for biological processes. Only significantly enriched terms with corrected p< 0.05 are indicated. The top 20 enriched biological processes are ranked by the number of DEGs. (B) Gene set expression analysis (GSEA) revealed enrichment for the cell redox homeostasis pathway. The normalized enrichment score (NES), false discovery rate (FDR), and p value are indicated in the insert. (C, D) Heatmap of representative antioxidation (C) and proliferation/survival (D)-related genes. Fold change > 1.5, p < 0.05. Red indicates upregulation, and blue indicates downregulation. (E) GSEA revealed enrichment for apoptosis pathways. (F) Schematic representation of the prosurvival potential of the NO-eMSC system. eMSC+MGP, eMSCs with MGP administration. Enhanced renoprotection of eMSCs To further validate whether NO could promote eMSC survival in ischemia/reperfusion (I/R)-injured kidneys, BLI analysis was performed for the real-time longitudinal monitoring of eMSC survival. A robust BLI signal of eMSCs was observed, indicating successful eMSC transplantation (Figure 3A). Although all groups gradually experienced donor cell death in the following days, eMSCs with MGP administration strikingly exhibited increased cell retention and prolonged cell survival in comparison with the eMSC group (Figure 3B). Furthermore, immunohistology also confirmed that the eMSCs with MGP administration displayed higher cell proliferation and cell retention on day 3 (Figure 3C). Moreover, we found that the eMSCs treated with MGP showed significantly lower levels of SCr and BUN (Figure 3D and E). Further kidney histology examination showed that tubular dilation, cast formation, and massive loss of brush borders at the initial stage after injury (3 days) were obviously reduced after NO-eMSC system treatment (Figure 3F to H). Kidney fibrosis analysis demonstrated that the development of interstitial fibrosis was consistent with the mRNA expression of ECM synthesis- and fibrosis pathway-related genes (Figure 4). Figure 3 with 1 supplement see all Download asset Open asset The nitric oxide (NO)-engineered mesenchymal stem cell (eMSC) system confers renoprotection by increasing eMSC survival in vivo. (A) The survival ratio of eMSCs and eMSCs with MGP administration after transplantation in ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) model mice was tracked by bioluminescence imaging (BLI). Images are from representative animals receiving 1×106 eMSCs alone or eMSCs with MGPs. (B) Quantitative analysis of BLI signals demonstrates that eMSCs with MGP administration could prolong cell survival. Data are presented as the mean ± SD, *p<0.05, **p<0.01. (C) Representative photomicrographs display the engraftment of eMSCs and eMSCs with MGP administration (RFP, red) within kidneys on day 3. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. (D and E) Renal function indexes, including serum creatine (D) and blood urea nitrogen (E) levels, were measured on day 3 after AKI. n=9. (F and G) Semi-quantitative histological assessment of cast formation (F) and loss of brush border (G) of H&E staining on day 3 after AKI. n=10. (H) Representative images of H&E staining on day 3 post AKI. eMSC+MGP, eMSCs with MGP administration. Scale bar, 100 μm. All data are presented as the mean ± SD, eMSC+MGP, eMSCs with MGP administration. *p<0.05 versus eMSC; #p<0.05 versus PBS. Figure 4 Download asset Open asset Engineered mesenchymal stem cells (eMSCs) with MGP administration attenuate renal fibrosis. (A) Masson trichrome staining was performed to examine kidney fibrosis on day 28 post acute kidney injury (AKI). Scale bar, 100 μm. (B) COL-I immunofluorescence staining was performed to examine kidney fibrosis on day 28 post AKI. Scale bar, 100 μm. (C and D) Real-time polymerase chain reaction (PCR) analysis of fibrosis-related gene expression in the kidney on day 28 after AKI. n=5. All data are presented as the mean ± SD, *p<0.05 versus eMSC; #p<0.05 versus PBS. Furthermore, the expression of KIM-1, a kidney injury marker, was significantly decreased in the proximal tubules of the kidneys in the eMSC with MGP administration group compared to the eMSC group (Figure 3—figure supplement 1A and B). Simultaneously, we found that the level of caspase-3/cleaved caspase-3 exhibited an abrupt increase in the injured kidney of the PBS group but was attenuated by eMSCs with MGP administration (Figure 3—figure supplement 1C and D), suggesting that the NO-eMSC system has protective effects on apoptosis in tubular epithelial cells. Overall, these results indicated that the NO-eMSC system could increase eMSC survival in vivo and further improve kidney function. Evaluation of NO levels in the kidney To further evaluate in vivo NO release from the eMSCs, eMSCs were transplanted into the left kidney by renal parenchymal injection followed by intravenous injection of the NO prodrug MGP. The results from Griess (Figure 5A) and chemiluminescence assays (Figure 5B and C) showed that the NO level from eMSC-treated kidneys was significantly increased when the prodrug MGP was applied. We next directly measured the NO levels of eMSC-treated kidneys in vivo by an EPR technique using the spin trap ferrous N-methyl-d-glucamine dithiocarbamate complex ((MGD)2Fe2+). The EPR signal of NO was observed in all groups, while an obvious characteristic triplet EPR signal (NO signal) was observed in the eMSCs treated with MGP (Figure 5D and E), which further confirmed that eMSCs could release NO in vivo. Moreover, targeted NO delivery was evaluated by comparison of NO levels in different tissues of mice after the administration of the eMSC in the kidney. The quantitative data showed that NO production from the kidney was significantly higher than that from the heart and liver (Figure 5F to H), suggesting that eMSCs could release NO specifically in the kidney. Figure 5 with 3 supplements see all Download asset Open asset Detection of NO release from the NO-engineered mesenchymal stem cell (eMSC) system in vivo. (A) The Griess kit assay was used to measure NO levels in kidneys with MGP alone, eMSC alone, or eMSC treated with MGP. n=6. (B) Detection and quantification of NO levels in kidneys in vivo using chemiluminescence. The area of each peak represents the corresponding amount of NO. (C) Calculation of the NO concentration in each group from the standard curve. n=6. (D) NO release from eMSCs with MGP administration in kidneys was assessed by electron paramagnetic resonance (EPR). MGP and eMSC served as controls. (E) Quantitative analysis of NO production in each group. The amount of NO-Fe(DETC)2 was calibrated using TEMPO as a standard. n=6. (F) A Griess kit assay was used to measure NO levels in various tissues of renal eMSC-transplanted mice. n=3. (G) Detection and quantification of NO levels in different tissues from renal eMSC-transplanted mice in vivo using chemiluminescence. The area of each peak represents the corresponding amount of NO. (H) Calculation of the NO concentration in each group from the standard curve. n=3. eMSC+MGP, eMSCs with MGP administration. All data are presented as the mean ± SD, ***p<0.001, ****p<0.0001. Extended applications of this cell-based NO delivery platform To investigate the universality of our NO delivery approach for applications in AKI, we conducted additional experiments to confirm whether other MSCs will perform equally well. Human adipose-derived MSCs (AD-MSCs) and human umbilical cord MSCs (hUC-MSCs) were genetically modified to produce mutant β-galactosidase (β-GALH363A). NO production by engineered AD-MSCs (eAD-MSCs) and engineered hUC-MSCs (ehUC-MSCs) with MGP administration in vitro was detected by the EPR technique. The characteristic triplet EPR signal was observed in eAD-MSCs and ehUC-MSCs with MGP administration (Figure 5—figure supplement 1A and B, Figure 5—figure supplement 2A and B), which was further confirmed by the Griess method (Figure 5—figure supplement 1C and D, Figure 5—figure supplement 2C and D). Next, the therapeutic effects of eAD-MSCs or ehUC-MSCs with MGP administration in AKI mice were estimated via histological analysis and renal function analysis. We found that the eAD-MSCs and ehUC-MSCs treated with MGP showed significantly lower levels of SCr and BUN (Figure 5—figure supplements 1E and 2E). Further kidney histology examination showed tubular dilation, cast formation, and massive loss of brush borders at the initial stage after injury (3 days) (Figure 5—figure supplement 3A). Furthermore, the expression of KIM-1 (kidney injury marker) was significantly decreased in the proximal tubules of the kidneys in the eAD-MSC and ehUC-MSC with MGP administration groups (Figure 5—figure supplement 3B). Thus, our MSC and NO prodrug system provides a tunable platform for NO delivery and holds promise for regenerative therapy. Enhanced proangiogenic effects of eMSCs VEGFR2-Fluc transgenic mice were used to monitor renal angiogenesis in real time, which appeared as BLI signals. The schematic diagram is shown in Figure 6—figure supplement 1. According to the results of angiogenesis imaging, BLI signals were emitted in all groups, and the strongest signal was detected in the eMSCs with MGP administration group, which suggests that the NO-eMSC system could stimulate renal angiogenesis by activating the VEGF/VEGFR2 pathway (Figure 6A–C). We next verified neovascularization in damaged kidneys on day 7 post-surgery by histological examination. The CD31 immunostaining results revealed significantly enhanced CD31+ microvascular density in the group of eMSCs with MGP administration, which is consistent with the BLI results (Figure 6D). Moreover, the expression of the angiogenesis-related genes VEGFR2, bFGF, PLGF, Ang-1, and Ang-2 detected by real-time polymerase chain reaction (RT-PCR) also confirmed that the eMSCs with MGP administration facilitated renal angiogenesis by upregulating angiogenic factor expression (Figure 6—figure supplement 2). Recovery from renal I/R injury requires tubular cell proliferation to promote kidney regeneration. As shown in Figure 6E and F, the numbers of proliferating Ki-67+ tubular cells were largely stimulated in the group of eMSCs treated with MGP, suggesting that the NO-eMSC system promoted kidney regeneration by accelerating tubular cell proliferation. The promotion of renal angiogenesis contributes to the restoration and repair of damaged kidneys, which paves the way for successful kidney regeneration. Figure 6 with 3 supplements see all Download asset Open asset The nitric oxide (NO)-engineered mesenchymal stem cell (eMSC) system promoted kidney regeneration after acute kidney injury (AKI). (A) In vivo bioluminescence imaging (BLI) was used to monitor the spatiotemporal dynamics of VEGFR2 expression following eMSC administration in a mouse AKI model using Vegfr2-Fluc transgenic mice. (B) Quantification of BLI signals revealed that eMSCs with MGP could enhance angiogenesis in injured kidneys. The average radiance of Fluc was expressed as photons/s/cm2/sr. n=3. (C) Quantification of CD31-positive capillaries in injured kidneys on day 7 after AKI. CD31 is a marker of endothelial cells and can be used for angiogenesis evaluation. n=5. (D) Immunofluorescence staining of CD31 was performed on day 7 after AKI. Scale bar, 100 μm. LTL (green) was used to reveal proximal tubules. (E) Representative images of immunofluorescence staining of Ki67. (F) Quantification of the percentage of Ki67-positive cells in kidneys on day 3 after AKI. n=5. Scale bar, 50 μm. eMSC+MGP, eMSCs with MGP administration. All data are presented as the mean ± SD, *p<0.05 versus eMSCs; #p<0.05 versus PBS. Subsequently, we explored the mechanism of eMSCs with MGP administration-induced angiogenesis, and our data revealed that eMSCs boosted the migration ability of human umbilical vein endothelial cells (HUVECs) in a coculture system with Transwells, as evidenced by a scratch wound-healing assay (Figure 6—figure supplement 3A and B). Additionally, the angiogenic ability of HUVECs, as manifested by the number of nodes and branches, was visibly increased in the group of eMSCs treated with MGP, as assessed by a tube formation assay (Figure 6—figure supplement 3C, D, and E). Additionally, the protein level of cleaved caspase-3, a key apoptosis molecule, was detected by immunofluorescence staining, suggesting that eMSCs could inhibit the apoptosis of HUVECs (Figure 6—figure supplement 3F). In general, we proposed that eMSCs with MGP administration exert superior promotive effects on kidney angiogenesis after injury by enhancing the proangiogenic activities of vascular endothelial cells. Diminished modulated inflammatory responses We next explored the modulatory effect of eMSCs on the inflammatory response in I/R-injured kidneys, and our results showed that eMSCs with MGP administration increased the number of F4/80- and CD206-positive macrophages while decreasing the number of F4/80- and iNOS-positive macrophages, indicating that the NO-eMSC system could promote the polarization of M1 macrophages to M2 macrophages (Figure 7A and B). Meanwhile, mRNA expression profiling further confirmed this finding (Figure 7C and D). Oxidative stress-induced reactive oxygen species (ROS) are well known as the main cause of renal I/R injury, leading to subsequent augmented inflammation and extended tissue damage (Collard and Gelman, 2001). Damaged renal tubular cells were overloaded with excessive lipids caused by abnormal lipid metabolism, while elevated ROS levels can lead to irreversible oxidative damage to lipids and further result in cell damage and death. Excessive intracellular accumulation of lipids was observed in the renal tubules of the PBS group after I/R injury, while the eMSCs with MGP administration significantly decreased lipid deposition (Figure 7—figure supplement 1A and B). Meanwhile, the renal level of MDA, an index of ROS-mediated lipid peroxidation, was notably increased in the PBS group and significantly decreased in the eMSCs w" @default.
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W4387191978 title "Author response: Genetically engineered mesenchymal stem cells as a nitric oxide reservoir for acute kidney injury therapy" @default.
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