Amniotic fluid stem cell-derived vesicles protect from VEGF-induced endothelial damage.
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ABSTRACT:
SUBMITTER: Sedrakyan S
PROVIDER: S-EPMC5715019 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background-Osteoporosis is characterized by defects in both quality and quantity of bone tissue, which imply high susceptibility to fractures with limitations of autonomy. Current therapies for osteoporosis are mostly concentrated on how to inhibit bone resorption but give serious adverse effects. Therefore, more effective and safer therapies are needed that even encourage bone formation. Here we examined the effect of extracellular vesicles secreted by human amniotic fluid stem cells (AFSC) (AFSC-EV) on a model of osteoporosis in vitro. Methods-human AFSC-EV were added to the culture medium of a human pre-osteoblast cell line (HOB) induced to differentiate, and then treated with dexamethasone as osteoporosis inducer. Aspects of differentiation and viability were assessed by immunofluorescence, Western blot, mass spectrometry, and histological assays. Since steroids induce oxidative stress, the levels of reactive oxygen species and of redox related proteins were evaluated. Results-AFSC-EV were able to ameliorate the differentiation ability of HOB both in the case of pre-osteoblasts and when the differentiation process was affected by dexamethasone. Moreover, the viability was increased and parallelly apoptotic markers were reduced. The presence of EV positively modulated the redox unbalance due to dexamethasone. Conclusion-these findings demonstrated that EV from hAFSC have the ability to recover precursor cell potential and delay local bone loss in steroid-related osteoporosis.
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Project description:The glyoxalase system is a ubiquitous cellular metabolic pathway whose main physiological role is the removal of methylglyoxal (MG). MG, a glycolysis byproduct formed by the spontaneous degradation of triosephosphates glyceraldehyde-3-phosphate (GA3P) and dihydroxyacetonephosphate (DHAP), is an arginine-directed glycating agent and precursor of the major advanced glycation end product arginine-derived, hydroimidazolone (MG-H1). Extracellular vesicles (EVs) are a heterogeneous family of lipid-bilayer-vesicular structures released by virtually all living cells, involved in cell-to-cell communication, specifically by transporting biomolecules to recipient cells, driving distinct biological responses. Emerging evidence suggests that included in the EVs cargo there are different metabolic enzymes. Specifically, recent research has pointed out that EVs derived from human amniotic fluid stem cell (HASC-EVs) contain glycolytic pay-off phase enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Since GAPDH catalyzes the sixth step of glycolysis using as a substrate GA3P, from which MG spontaneously origins, we wanted to investigate whether MG-derived MG-H1, as well as glyoxalases, could be novel molecule cargo in these EVs. By using immunoassays and spectrophotometric methods, we found, for the first time ever, that HASC-EVs contain functional glyoxalases and MG-H1, pioneering research to novel and exciting roles of these eclectic proteins, bringing them to the limelight once more.
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Project description:Myocardial ischemia is a condition with insufficient oxygen supporting the heart tissues, which may result from myocardial infarction or trauma-induced hemorrhagic shock. In order to develop better preventive and therapeutic strategies for myocardial ischemic damage, it is important that we understand the mechanisms underlying this type of injury. Mitochondrial-derived vesicles (MDVs) have been proposed as a novel player in maintaining mitochondrial quality control. This study aimed to investigate the role and possible mechanisms of MDVs in ischemia/hypoxia-induced myocardial apoptosis. H9C2 cardiomyocytes were used for the cellular experiments. A 40% fixed blood volume hemorrhagic shock rat model was used to construct the acute general ischemic models. MDVs were detected using immunofluorescence staining with PDH and TOM20. Exogenous MDVs were reconstituted in vitro from isolated mitochondria under different hypoxic conditions. The results demonstrate that MDV production was negatively correlated with cardiomyocyte apoptosis under hypoxic conditions; exogenous MDVs inhibited hypoxia-induced cardiomyocyte apoptosis; and MDV-mediated protection against hypoxia-induced cardiomyocyte apoptosis was accomplished via Bcl-2 interactions in the mitochondrial pathway. This study provides evidence that MDVs protect cardiomyocytes against hypoxic damage by inhibiting mitochondrial apoptosis. Our study used a novel approach that expands our understanding of MDVs and highlights that MDVs may be part of the endogenous response to hypoxia designed to mitigate damage. Strategies that stimulate cardiomyocytes to produce cargo-specific MDVs, including Bcl-2 containing MDVs, could theoretically be helpful in treating ischemic/hypoxic myocardial injury.
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Project description:Human amniotic fluid-derived stem (AFS) cells possess several advantages over embryonic and adult stem cells, as evidenced by expression of both types of stem cell markers and ability to differentiate into cells of all three germ layers. Herein, we examine endothelial differentiation of AFS cells in response to growth factors, shear force, and hypoxia. We isolated human AFS cells from amniotic fluid samples (1-4 cc/specimen) obtained from patients undergoing amniocentesis at 15-18 weeks of gestation (n = 10). Isolates maintained in nondifferentiating medium expressed the stem cell markers CD13, CD29, CD44, CD90, CD105, OCT-4, and SSEA-4 through passage 8. After 3 weeks of culture in endothelial growth media-2 (EGM-2), the stem cells exhibited an endothelial-like morphology, formed cord-like structures when plated on Matrigel, and uptook acetylated LDL/lectin. Additionally, mRNA and protein levels of CD31 and von Willebrand factor (vWF) significantly increased in response to culture in EGM-2, with further up-regulation when stimulated by physiological levels (12 dyne/cm(2)) of shear force. Culture in hypoxic conditions (5% O(2)) resulted in significant expression of vascular endothelial growth factor (VEGF) and placental growth factor (PGF) mRNA. This study suggests that AFS cells, isolated from minute amounts of amniotic fluid, acquire endothelial cell characteristics when stimulated by growth factors and shear force, and produce angiogenic factors (VEGF, PGF, and hepatocyte growth factor [HGF]) in response to hypoxia. Thus, amniotic fluid represents a rich source of mesenchymal stem cells potentially suitable for use in cardiovascular regenerative medicine.
| S-EPMC3139996 | biostudies-other
Project description:This review aims to encapsulate the current knowledge in extracellular vesicles extracted from amniotic fluid and amniotic fluid derived stem/stromal cells. Amniotic fluid (AF) bathes the developing fetus, providing nutrients and protection from biological and mechanical dangers. In addition to containing a myriad of proteins, immunoglobulins and growth factors, AF is a rich source of extracellular vesicles (EVs). These vesicles originate from cells in the fetoplacental unit. They are biological messengers carrying an active cargo enveloped within the lipid bilayer. EVs in reproduction are known to play key roles in all stages of pregnancy, starting from fertilisation through to parturition. The intriguing biology of AF-derived EVs (AF-EVs) in pregnancy and their untapped potential as biomarkers is currently gaining attention. EV studies in numerous animal and human disease models have raised expectations of their utility as therapeutics. Amniotic fluid stem cell and mesenchymal stromal cell-derived EVs (AFSC-EVs) provide an established supply of laboratory-made EVs. This cell-free mode of therapy is popular as an alternative to stem cell therapy, revealing similar, if not better therapeutic outcomes. Research has demonstrated the successful application of AF-EVs and AFSC-EVs in therapy, harnessing their anti-inflammatory, angiogenic and regenerative properties. This review provides an overview of such studies and discusses concerns in this emerging field of research.
| S-EPMC11010396 | biostudies-literature
Project description:BackgroundWith the widespread of Coronavirus Disease 2019 pandemic, in spite of the newly emerging vaccines, mutated strains remain a great obstacle to supportive and preventive measures. Coronavirus 19 survivors continue to face great danger of contacting the disease again. As long as no specific treatment has yet to be approved, a great percentage of patients experience real complications, including among others, lung fibrosis. High oxygen inhalation especially for prolonged periods is per se destructive to the lungs. Nevertheless, oxygen remains the first line support for such patients. In the present study we aimed at investigating the role of amniotic fluid-mesenchymal stem cells in preventing versus treating the hyperoxia-induced lung fibrosis in rats.MethodsThe study was conducted on adult albino rats; 5 pregnant female rats were used as amniotic fluid donors, and 64 male rats were randomly divided into two groups: Control group; where 10 rats were kept in normal atmospheric air then sacrificed after 2 months, and hyperoxia-induced lung fibrosis group, where 54 rats were exposed to hyperoxia (100% oxygen for 6 h/day) in air-tight glass chambers for 1 month, then randomly divided into the following 5 subgroups: Hyperoxia group, cell-free media-treated group, stem cells-prophylactic group, stem cells-treated group and untreated group. Isolation, culture and proliferation of stem cells were done till passage 3. Pulmonary function tests, histological examination of lung tissue under light and electron microscopes, biochemical assessment of oxidative stress, IL-6 and Rho-A levels, and statistical analysis of data were performed. F-test (ANOVA) was used for normally distributed quantitative variables, to compare between more than two groups, and Post Hoc test (Tukey) for pairwise comparisons.ResultsLabelled amniotic fluid-mesenchymal stem cells homed to lung tissue. Stem cells administration in the stem cells-prophylactic group succeeded to maintain pulmonary functions near the normal values with no significant difference between their values and those of the control group. Moreover, histological examination of lung tissues showed that stem cells-prophylactic group were completely protected while stem cells-treated group still showed various degrees of tissue injury, namely; thickened interalveolar septa, atelectasis and interstitial pneumonia. Biochemical studies after stem cells injection also showed decreased levels of RhoA and IL-6 in the prophylactic group and to a lesser extent in the treated group, in addition to increased total antioxidant capacity and decreased malondialdehyde in the stem cells-injected groups.ConclusionsAmniotic fluid-mesenchymal stem cells showed promising protective and therapeutic results against hyperoxia-induced lung fibrosis as evaluated physiologically, histologically and biochemically.
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Project description:Procoagulant activity in amniotic fluid (AF) is positively correlated with phosphatidylserine (PS) and tissue factor (TF)-expressing(+) extracellular vesicles (EVs). However, it is unknown if pathological fetal conditions may affect the composition, phenotype, and procoagulant potency of EVs in AF. We sought to evaluate EV-dependent procoagulant activity in AF from pregnant people with fetuses with or without diagnosed chromosomal mutations. AF samples were collected by transabdominal amniocentesis and assessed for common karyotype defects (total n = 11, 7 healthy and 4 abnormal karyotypes). The procoagulant activity of AF was tested using a fibrin generation assay with normal pooled plasma and plasmas deficient in factors XII, XI, IX, X, V, and VII. EV number and phenotype were determined by flow cytometry with anti-CD24 and anti-TF antibodies. We report that factor-VII-, X-, or V-deficient plasmas did not form fibrin clots in the presence of AF. Clotting time was significantly attenuated in AF samples with chromosomal mutations. In addition, CD24+, TF+, and CD24+ TF+ EV counts were significantly lower in this group. Finally, we found a significant correlation between EV counts and the clotting time induced by AF. In conclusion, we show that AF samples with chromosomal mutations had fewer fetal-derived CD24-bearing and TF-bearing EVs, which resulted in diminished procoagulant potency. This suggests that fetal-derived EVs are the predominant source of procoagulant activity in AF.
| S-EPMC9221817 | biostudies-literature
Project description:Embolization of amniotic fluid (AF) into the blood circulation leads to disseminated intravascular coagulation (DIC). Procoagulant phosphatidylserine (PS)- and tissue factor (TF)-exposing extracellular vesicles (EVs) might play an important role in AF embolism-induced DIC. It was the aim of the present study to perform analyses of the procoagulant properties of AF with a panel of functional coagulation assays and flow cytometry. We applied a prothrombinase assay (that quantifies PS exposure on EVs), an EV-associated TF activity assay, a fibrin generation assay, a thrombin generation assay, a whole blood clotting model, and flow cytometry in AF and control plasma. We found that PS exposure on EVs was 21-fold increased in AF compared with plasma. Also, EV-associated TF activity was highly increased in AF compared with plasma. AF-derived EVs activated the blood coagulation cascade via PS and TF in the fibrin and thrombin generation assays. In a whole blood clotting model, AF-derived EVs significantly shortened the clotting time from 734 ± 139 seconds in the presence to 232 ± 139 seconds in the absence of an anti-TF antibody. The contact activation pathway via factor XII (FXII) was not affected. Applying flow cytometry, a subpopulation of PS+ and TF+ EVs was identified in AF but not in control plasma. In conclusion, we investigated the effect of AF on blood coagulation and found that PS+ and TF+ EVs determine their procoagulant potential. Taken together, our data further delineate the pathomechanisms underlying AF-induced coagulopathy.
| S-EPMC6544525 | biostudies-literature
Project description:ObjectivesTo characterize mesenchymal stem cell-like cells isolated from human amniotic fluid for a new source of therapeutic cells.MaterialsFibroblastoid-type cells obtained from amniotic fluid at the time of birth.MethodsThe ability of ex vivo expansion was investigated until senescence, and stem cell-like characteristics were analyzed by examining differentiation potential, messenger RNA expression and immunophenotypes.Results and conclusionsA morphologically homogenous population of fibroblastoid-type (HAFFTs) cells, similar to mesenchymal stem cells from bone marrow (BM-MSCs), was obtained at the third passage. The cells became senescent after 27 passages over a period of 8 months while undergoing 66 population doublings. Under appropriate culture conditions, by the 8th passage they differentiated into adipocytes, osteocytes, chondrocytes and neuronal cells, as revealed by oil red O, von Kossa, Alcian blue and anti-NeuN antibody staining, respectively. Immunophenotype analyses at the 17th passage demonstrated the presence of TRA-1-60; SSEA-3 and-4; collagen types I, II, III, IV and XII; fibronectin; alpha-SMA; vimentin; desmin; CK18; CD44; CD54; CD106; FSP; vWF; CD31; and HLA ABC. Reverse transcriptase-polymerase chain reaction analysis of the HAFFTs from passages 6-20 showed consistent expression of Rex-1, SCF, GATA-4, vimentin, CK18, FGF-5 and HLA ABC genes. Oct-4 gene expression was observed up to the 19th passage but not at the 20th passage. HAFFTs showed telomerase activity at the 5th passage with a decreased level by the 21st passage. Interestingly, BMP-4, AFP, nestin and HNF-4alpha genes showed differential gene expression during ex vivo expansion. Taken together, these observations suggest that HAFFTs are pluripotent stem cells that are less differentiated than BM-MSCs, and that their gene expression profiles vary with passage number during ex vivo expansion.
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Project description:Necrotizing enterocolitis (NEC) is a devastating intestinal disease primarily affecting preterm neonates and causing high morbidity, high mortality, and huge costs for the family and society. The treatment and the outcome of the disease have not changed in recent decades. Emerging evidence has shown that stimulating the Wnt/β-catenin pathway and enhancing intestinal regeneration are beneficial in experimental NEC, and that they could potentially be used as a novel treatment. Amniotic fluid stem cells (AFSC) and AFSC-derived extracellular vesicles (EV) can be used to improve intestinal injury in experimental NEC. However, the mechanisms by which they affect the Wnt/β-catenin pathway and intestinal regeneration are unknown. In our current study, we demonstrated that AFSC and EV attenuate NEC intestinal injury by activating the Wnt signaling pathway. AFSC and EV stimulate intestinal recovery from NEC by increasing cellular proliferation, reducing inflammation and ultimately regenerating a normal intestinal epithelium. EV administration has a rescuing effect on intestinal injury when given during NEC induction; however, it failed to prevent injury when given prior to NEC induction. AFSC-derived EV administration is thus a potential emergent novel treatment strategy for NEC.
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