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Adipose-derived mesenchymal stromal cells from aged patients with coronary artery disease keep mesenchymal stromal cell properties but exhibit characteristics of aging and have impaired angiogenic potential.
ABSTRACT: Tissue regeneration is impaired in aged individuals. Adipose-derived mesenchymal stromal cells (ADSCs), a promising source for cell therapy, were shown to secrete various angiogenic factors and improve vascularization of ischemic tissues. We analyzed how patient age affected the angiogenic properties of ADSCs. ADSCs were isolated from subcutaneous fat tissue of patients with coronary artery disease (CAD; n = 64, 43-77 years old) and without CAD (n = 31, 2-82 years old). ADSC phenotype characterized by flow cytometry was CD90(+)/CD73(+)/CD105(+)/CD45(-)/CD31(-) for all samples, and these cells were capable of adipogenic and osteogenic differentiation. ADSCs from aged patients had shorter telomeres (quantitative reverse transcription polymerase chain reaction) and a tendency to attenuated telomerase activity. ADSC-conditioned media (ADSC-CM) stimulated capillary-like tube formation by endothelial cells (EA.hy926), and this effect significantly decreased with the age of patients both with and without CAD. Angiogenic factors (vascular endothelial growth factor, placental growth factor, hepatocyte growth factor, angiopoetin-1, and angiogenin) in ADSC-CM measured by enzyme-linked immunosorbent assay significantly decreased with patient age, whereas levels of antiangiogenic factors thrombospondin-1 and endostatin did not. Expression of angiogenic factors in ADSCs did not change with patient age (real-time polymerase chain reaction); however, gene expression of factors related to extracellular proteolysis (urokinase and its receptor, plasminogen activator inhibitor-1) and urokinase-type plasminogen activator receptor surface expression increased in ADSCs from aged patients with CAD. ADSCs from aged patients both with and without CAD acquire aging characteristics, and their angiogenic potential declines because of decreasing proangiogenic factor secretion. This could restrict the effectiveness of autologous cell therapy with ADSCs in aged patients.
Project description:INTRODUCTION:Adipose-derived stem cells (ADSCs) have been isolated, expanded, and applied in the treatment of many diseases. ADSCs have also been used to treat injured articular cartilage. However, there is controversy regarding the treatment efficiency. We considered that ADSC transplantation with activated platelet-rich plasma (PRP) may improve injured articular cartilage compared with that of ADSC transplantation alone. In this study, we determined the role of PRP in ADSC transplantation to improve the treatment efficiency. METHODS:ADSCs were isolated and expanded from human adipose tissue. PRP was collected and activated from human peripheral blood. The effects of PRP were evaluated in vitro and in ADSC transplantation in vivo. In vitro, the effects of PRP on ADSC proliferation, differentiation into chondrogenic cells, and inhibition of angiogenic factors were investigated at three concentrations of PRP (10%, 15% and 20%). In vivo, ADSCs pretreated with or without PRP were transplanted into murine models of injured articular cartilage. RESULTS:PRP promoted ADSC proliferation and differentiation into chondrogenic cells that strongly expressed collagen II, Sox9 and aggrecan. Moreover, PRP inhibited expression of the angiogenic factor vascular endothelial growth factor. As a result, PRP-pretreated ADSCs improved healing of injured articular cartilage in murine models compared with that of untreated ADSCs. CONCLUSION:Pretreatment of ADSCs with PRP is a simple method to efficiently apply ADSCs in cartilage regeneration. This study provides an important step toward the use of autologous ADSCs in the treatment of injured articular cartilage.
Project description:Scleroderma is a group of skin-fibrosing diseases for which there are no effective treatments. A feature of the skin fibrosis typical of scleroderma is atrophy of the dermal white adipose tissue (DWAT). Adipose tissue contains adipose-derived mesenchymal stromal cells (ADSCs) that have regenerative and reparative functions; however, whether DWAT atrophy in fibrosis is accompanied by ADSC loss is poorly understood, as are the mechanisms that might maintain ADSC survival in fibrotic skin. Here, we have shown that DWAT ADSC numbers were reduced, likely because of cell death, in 2 murine models of scleroderma skin fibrosis. The remaining ADSCs showed a partial dependence on dendritic cells (DCs) for survival. Lymphotoxin ? (LT?) expression in DCs maintained ADSC survival in fibrotic skin by activating an LT? receptor/?1 integrin (LT?R/?1 integrin) pathway on ADSCs. Stimulation of LT?R augmented the engraftment of therapeutically injected ADSCs, which was associated with reductions in skin fibrosis and improved skin function. These findings provide insight into the effects of skin fibrosis on DWAT ADSCs, identify a DC-ADSC survival axis in fibrotic skin, and suggest an approach for improving mesenchymal stromal cell therapy in scleroderma and other diseases.
Project description:Adipose tissue-derived stromal cells (ADSCs) are of interest for regenerative medicine as they are isolated easily and can differentiate into multiple cell lineages. Studies of their in vitro proliferation, survival, and differentiation are common; however, genetic effects on these phenotypes remain unknown. To test if these phenotypes are genetically regulated, ADSCs were isolated from three genetically diverse inbred mouse strains--C57BL/6J (B6), BALB/cByJ (BALB), and DBA/2J (D2)--in which genetic regulation of hematopoietic stem function is well known. ADSCs from all three strains differentiated into osteogenic and chondrogenic lineages in vitro. ADSCs from BALB grew least well in vitro, probably due to apoptotic cell death after several days in culture. BALB ADSCs were also the most susceptible to the free radical inducers menadione and H2O2. ADSCs from the three possible F1 hybrids were employed to further define genetic regulation of ADSC phenotypes. D2, but not B6, alleles stimulated ADSC expansion in BALB cells. In contrast, B6, but not D2, alleles rescued BALB H2O2 resistance. We conclude that low oxidative stress resistance does not limit BALB ADSC growth in vitro, as these phenotypes are genetically regulated independently. In addition, ADSCs from these strains are an appropriate model system to investigate genetic regulation of ADSC apoptosis and stress resistance in future studies. Such investigations are essential to optimize cell expansion and differentiation and thus, potential for regenerative medicine.
Project description:Background:Adipose tissue-derived stromal cells (ADSCs) have great potential for cell-based therapies, including tissue engineering. However, various factors can influence the characteristics of isolated ADSCs. Methods:We studied the influence of the harvesting site, i.e., inner thigh (n = 3), outer thigh (n = 3), outer thigh (n = 3), outer thigh (. Results:We revealed higher initial cell yields from the outer thigh region than from the abdomen region. Negative pressure did not influence the cell yields from the outer thigh region, whereas the yields from the abdomen region were higher under high negative pressure than under low negative pressure. In the subsequent passage, in general, no significant relationship was identified between the different negative pressure and ADSC characteristics. No significant difference was observed in the characteristics of thigh ADSCs and abdomen ADSCs. Only on day 1, the diameter was significantly bigger in outer thigh ADSCs than in abdomen ADSCs. Moreover, we noted a tendency of thigh ADSCs (i.e., inner thigh+outer thigh) to reach a higher cell number on day 7. Discussion. The harvesting site and negative pressure can potentially influence initial cell yields from lipoaspirates. However, for subsequent in vitro culturing and for use in tissue engineering, it seems that the harvesting site and the level of negative pressure do not have a crucial or limiting effect on basic ADSC characteristics.in vitro culturing and for use in tissue engineering, it seems that the harvesting site and the level of negative pressure do not have a crucial or limiting effect on basic ADSC characteristics.
Project description:Background:Extracellular vesicles (EVs) secreted from adipose-derived mesenchymal stem cells (ADSCs) (ADSC-EVs) improve flap survival after ischemia–reperfusion injury. Exposure of parent ADSCs to oxidative stress has been shown to enhance this effect, but mechanisms are unclear. We aimed to determine whether angiogenesis-promoting protein and microRNA (miRNA) content is altered in EVs after preconditioning with hydrogen peroxide (H2O2 ADSC-EVs) and whether H2O2 ADSC-EVs can increase viability of random pattern skin flaps.Methods:EVs secreted by human ADSCs were isolated after culture in EV-depleted medium ± H2O2. Nanoparticle tracking analysis determined size and concentration of purified EVs. Mass spectrometry and small RNA next-generation sequencing were performed to compare proteomic and miRNA profiles. ADSC-EVs, H2O2 ADSC-EVs, or vehicle were injected into random pattern skin flaps of BALB/c mice (4–5 mice per group). Viable and necrotic areas were measured on day 7, and tissues underwent histologic analysis.Results:Angiogenic and antimicrobial protein content of EVs was altered with H2O2 preconditioning. Functional enrichment analysis identified constitutive photomorphogenesis 9 signalosome (known to direct vascular endothelial growth factor production) as the major enriched Gene Ontology term unique to H2O2 ADSC-EVs. Two miRNAs were increased, and 12 (including 10 antiangiogenic miRNAs) were reduced in H2O2 ADSC-EVs. Enhanced viability (P < 0.05) of flaps treated with H2O2 ADSC-EVs compared with vehicle corresponded to increased capillary density in the H2O2 group (P < 0.001).Conclusion:Altered protein and miRNA content in ADSC-EVs after H2O2 pretreatment likely contributes to enhanced therapeutic effects on flap survival observed in preclinical models.
Project description:Adipose-derived stem cell (ADSC)-based therapy is promising for critical limb ischemia (CLI) treatment, especially in patients with diabetes. However, the therapeutic effects of diabetic ADSCs (D-ADSCs) are impaired by the diabetes, possibly through intracellular reactive oxygen species (ROS) accumulation. The objective of the present study was to detect whether overexpression of methylglyoxal-metabolizing enzyme glyoxalase-1 (GLO1), which reduces ROS in D-ADSCs, can restore their proangiogenic function in a streptozotocin-induced diabetic mice model of CLI. GLO1 overexpression in D-ADSCs (G-D-ADSCs) was achieved using the lentivirus method. G-D-ADSCs showed a significant decrease in intracellular ROS accumulation, increase in cell viability, and resistance to apoptosis under high-glucose conditions compared with D-ADSCs. G-D-ADSCs also performed better in terms of migration, differentiation, and proangiogenic capacity than D-ADSCs in a high-glucose environment. Notably, these properties were restored to the same level as that of nondiabetic ADSCs under high-glucose conditions. G-D-ADSC transplantation induced improved reperfusion and an increased limb salvage rate compared D-ADSCs in a diabetic mice model of CLI. Histological analysis revealed higher microvessel densities and more G-D-ADSC-incorporated microvessels in the G-D-ADSC group than in the D-ADSC group, which was comparable to the nondiabetic ADSC group. Higher expression of vascular endothelial growth factor A and stromal cell-derived factor-1? and lower expression of hypoxia-induced factor-1? were also detected in the ischemic muscles from the G-D-ADSC group than that of the D-ADSC group. The results of the present study have demonstrated that protection from ROS accumulation by GLO1 overexpression is effective in reversing the impaired biological function of D-ADSCs in promoting neovascularization of diabetic CLI mice model and warrants the future clinical application of D-ADSC-based therapy in diabetic patients. Stem Cells Translational Medicine 2017;6:261-271.
Project description:Adipose-derived stem cells (ADSCs) have been shown to be beneficial in some pulmonary diseases, and the paracrine effect is the major mechanism underlying ADSC-based therapy. Autophagy plays a crucial role in maintaining stem cell homeostasis and survival. However, the role of autophagy in mediating ADSC paracrine effects has not been thoroughly elucidated. We examined whether ADSCs participate in lipopolysaccharide (LPS)-induced pulmonary microvascular endothelial cell (PMVEC) barrier damage in a paracrine manner and illuminated the role of autophagy in regulating ADSC paracrine effects. PMVECs and ADSCs with or without autophagy inhibition were cocultured without intercellular contact, and the microvascular barrier function was assessed after LPS treatment. ADSC paracrine function was evaluated by detecting essential growth factors for endothelial cells. For in vivo experiments, ADSCs with or without autophagy inhibition were transplanted into LPS-induced lung-injury mice, and lung injury was assessed. ADSCs significantly alleviated LPS-induced microvascular barrier injury. In addition, ADSC paracrine levels of VEGF, FGF, and EGF were induced by LPS treatment, especially in the coculture condition. Inhibiting autophagy weakened the paracrine function and the protective effects of ADSCs on microvascular barrier injury. Moreover, ADSC transplantation alleviated LPS-induced lung injury, and inhibiting autophagy markedly weakened the therapeutic effect of ADSCs on lung injury. Together, these findings show that ADSC paracrine effects play a vital protective role in LPS-induced pulmonary microvascular barrier injury. Autophagy is a positive mediating factor in the paracrine process. These results are helpful for illuminating the role and mechanism of ADSC paracrine effects and developing effective therapies in acute lung injury.
Project description:Adipose-derived stem cells (ADSCs) possess potent angiogenic properties and represent a source for cell-based approaches to delivery of bioactive factors to drive vascularization of tissues. Hypoxic signaling appears to be largely responsible for triggering release of these angiogenic cytokines, including vascular endothelial growth factor (VEGF). Three-dimensional (3D) culture may promote activation of hypoxia-induced pathways, and has furthermore been shown to enhance cell survival by promoting cell-cell interactions while increasing angiogenic potential. However, the development of hypoxia within ADSC spheroids is difficult to characterize. In the present study, we investigated the impact of spheroid size on hypoxia-inducible transcription factor (HIF)-1 activity in spheroid cultures under atmospheric and physiological oxygen conditions using a fluorescent marker. Hypoxia could be induced and modulated by controlling the size of the spheroid; HIF-1 activity increased with spheroid size and with decreasing external oxygen concentration. Furthermore, VEGF secretion was impacted by the hypoxic status of the culture, increasing with elevated HIF-1 activity, up to the point at which viability was compromised. Together, these results suggest the ability to use 3D culture geometry as a means to control output of angiogenic factors from ADSCs, and imply that at a particular environmental oxygen concentration an optimal culture size for cytokine production exists. Consideration of culture geometry and microenvironmental conditions at the implantation site will be important for successful realization of ADSCs as a pro-angiogenic therapy.
Project description:The aim of the present study was to compare the effects of adipose?derived mesenchymal stem cell (ADSC) and bone marrow mesenchymal stem cell (BMSC) transplantation into the corpora cavernosa of diabetic rats with erectile function. ADSCs and BMSCs were isolated and identified by flow cytometry. Rats with streptozocin?induced diabetes were screened using apomorphine to obtain a rat model of diabetic erectile dysfunction, followed by transplantation of ADSCs and BMSCs into the corpora cavernosa. Two weeks later, the rats were again injected with apomorphine, the intracavernous pressure (ICP) and mean arterial pressure (MAP) of the penile tissue were measured, and the corpus cavernosum tissues were harvested. Angiogenic endothelial nitric oxide synthase (eNOS) expression was detected by western blotting and immunofluorescence analysis. The blood vessels in the corpus cavernosum were observed following hematoxylin and eosin (H&E) staining, and the expression of collagen was detected by Sirius Red staining. The cellular ultrastructure was examined by transmission electron microscopy. Intracavernous injection of ADSCs significantly increased ICP and ICP/MAP. Western blotting and immunofluorescence results revealed that ADSC treatment improved the expression of eNOS in the penile tissue of diabetic rats. The H&E staining results demonstrated that ADSC treatment promoted revascularization of the corpus cavernosum, and the results of Sirius Red staining revealed that ADSC treatment reduced penile collagen in diabetic rats. Transmission electron microscopy examination revealed that the ultrastructure of the tissues in the ADSC?treated group was more complete compared with that in the untreated diabetic model group. In conclusion, ADSCs were found to be more effective compared with BMSCs in treating diabetes?related erectile dysfunction.
Project description:The abilities of intracavernous injection of autologous stromal vascular fraction (SVF) and adipose-derived stem cells (ADSCs) to facilitate recovery of erectile function in a rat model of cavernous nerve (CN) injury were compared. Forty male Sprague-Dawley rats were randomly divided into four groups: sham and control groups (intracavernous injection of phosphate-buffered saline), SVF group (intracavernous injection of SVF), and ADSC group (intracavernous injection of ADSCs). Rats in the latter three groups underwent bilateral CN injury prior to injection. The evaluation of erectile function and histomorphometric studies were performed 4 weeks after injection. The ratio of maximal intracavernous pressure to mean arterial pressure was significantly lower in the control group than in the sham group (0.18 vs. 0.56, p < .001). Intracavernous injection of SVF (0.36, p = .035) significantly improved erectile function compared with that in the control group, whereas the ADSC group (0.35, p = .052) showed marginally significant improvement. The smooth muscle/collagen ratio, smooth muscle content, number of neuronal nitric-oxide synthase-positive nerve fibers, and expression of von Willebrand factor were significantly higher in the SVF and ADSC groups than in the control group. Expression of endothelial nitric-oxide synthase was significantly increased in the SVF group. The increases in the smooth muscle/collagen ratio and von Willebrand factor expression were larger in the SVF group than in the ADSC group. Intracavernous injection of SVF or ADSCs was equally effective in recovering penile erection in a rat model of CN injury.