Human Endothelial Progenitor Cells Research Articles

Impaired Bone Marrow Endothelial Progenitor Cells May Contribute to Acute Graft-Versus-Host Disease after Allotransplant

▪Background: Graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). While traditionally known as an immune cells-mediated immune injury, GVHD is also associated with endothelial injury, which determines the severity of GVHD. Moreover, emerging clinical and physiopathologic evidences suggest that vascular endothelium can be a target of aGVHD in the early phase and the increased circulating endothelial cells (ECs) represents a surrogate marker of endothelial damage. Bone marrow (BM) endothelial progenitor cells (EPCs), although the origin is not completely understood, are believed to be the precursors of ECs. Protective roles of EPCs in cardiovascular disorders have been extensively testified while their potential benefits in HSCT have rarely addressed. Moreover, BM suppression without infection is often observed in patients with acute GVHD (aGVHD) after allo-HSCT and the exact mechanism remains unclear. Therefore, it is imperative to evaluate whether the number and function of BM EPCs and hematopoietic stem cells (HSCs) in allo-HSCT patients with aGVHD differed from those without aGVHD. Furthermore, given that reactive oxygen species(ROS) exhibits the important role in maintaining endothelial function and vascular integrity, it will also be necessary to investigate whether aberrant levels of ROS in BM EPCs and HSCs play a role in the pathogenesis of aGVHD after allo-HSCT.Aims: To evaluate whether the number and function of BM EPCs and HSCs in allo-HSCT patients with aGVHD differed from those without aGVHD. Furthermore, to investigate whether aberrant levels of ROS in BM EPCs and HSCs play a role in the pathogenesis of aGVHD after allo-HSCT.Methods: In the current prospective case-pair study, 59 patients with aGVHD, 118 matched patients without aGVHD after allo-HSCT and 15 health donors as normal controls were enrolled. BM EPCs were cultured as previous reported. The number and function of BM EPCs were evaluated by cell counting, DiI-Ac-LDL and FITC-lectin-UEA-1 double staining, migration, tube formation, levels of ROS and apoptosis at 30 days, 60 days and 90 days after allo-HSCT. The number and function of BM HSCs were evaluated by cell counting, levels of ROS and apoptosis. Meanwhile, the quantity and function of synchronous peripheral blood (PB) ECs and HSCs were measured in patients with aGVHD at 15 days, 30 days, 60 days and 90 days after allo-HSCT.Results: Human BM EPCs were demonstrated as the spindle shape and the similar expression of CD34, VEGFR2 and CD133 at day 7 of cultivation among the enrolled subjects. Reduced and dysfunctional BM EPCs, characterized by decreased capacities of migration and angiogenesis, as well as higher levels of ROS and apoptosis, were observed in patients with the onset of aGVHD compared with those without aGVHD. Moreover, reduced and dysfunctional BM HSCs, characterized by higher levels of ROS and apoptosis, were identified in patients with aGVHD compared with those without aGVHD. In PB, increased frenquency of ECs and decreased HSCs with higher levels of ROS and apoptosis were detected in patients with aGVHD compared to those without aGVHD. In addition, the frequencies of BM EPCs and HSCs in patients with the onset of aGVHD ≥grade 2 were significantly lower than those with aGVHD <grade 2, while the levels of ROS and apoptosis in BM EPCs and HSCs were remarkably higher. At 30 days, 60 days and 90 days after allo-HSCT, the percentages of BM EPCs and HSCs were significant lower, whereas ROS levels in BM EPCs and HSCs were significant higher in patients with aGVHD than those without aGVHD. At 15 days, 30 days, 60 days and 90 days after allo-HSCT, the percentages of PB ECs and their ROS levels in patients with aGVHD were higher than those without aGVHD, while the percentages PB HSCs were lower than those without aGVHD at the different time points.Summary / Conclusion: These findings suggest that reduced and dysfunctional BM EPCs may be involved in the pathogenesis of aGVHD. Meanwhile, reduced and dysfunctional BM HSCs were also found in patients with aGVHD. Moreover, the impairment of EPCs and HSCs in BM and PB was associated with the elevated levels of ROS in patients with aGVHD. Therefore, our data indicate that it would be of value to investigate whether the improvement of BM EPCs would possibly ameliorate aGVHD, thereby providing a novel clinical intervention for aGVHD in the future. DisclosuresNo relevant conflicts of interest to declare.

The Inhibitory Effect of Lysophosphatidylcholine on Proangiogenesis of Human CD34+ Cells Derived Endothelial Progenitor Cells.

Increasing evidence reveals that lysophosphatidylcholine (LPC) is closely related to endothelial dysfunction. The present study aimed to investigate the mechanism of LPC in inhibiting the proangiogenesis and vascular inflammation of human endothelial progenitor cells (EPCs) derived from CD34+ cells. The early EPCs were derived from CD34+ hematopoietic stem cells whose purity was identified using flow cytometry analysis. The surface markers (CD34, KDR, CD31; VE-cadherin, vWF, eNOS) of EPCs were examined by flow cytometry analysis and immunofluorescence. RT-qPCR was used to detect the mRNA expression of inflammatory cytokines (CCL2, IL-8, CCL4) and genes associated with angiogenesis (VEGF, ANG-1, ANG-2) in early EPCs after treatment of LPC (10 μg/ml) or phosphatidylcholine (PC, 10 μg/ml, control). The angiogenesis of human umbilical vein endothelial cells (HUVECs) incubated with the supernatants of early EPCs was detected by a tube formation assay. The mRNA and protein levels of key factors on the PKC pathway (phosphorylated PKC, TGF-β1) were measured by RT-qPCR and western blot. The localization of PKC-β1 in EPCs was determined by immunofluorescence staining. We found that LPC suppressed the expression of CCL2, CCL4, ANG-1, ANG-2, promoted IL-8 expression and had no significant effects on VEGF expression in EPCs. EPCs promoted the angiogenesis of HUVECs, which was significantly inhibited by LPC treatment. Moreover, LPC was demonstrated to promote the activation of the PKC signaling pathway in EPCs. In conclusion, LPC inhibits proangiogenesis of human endothelial progenitor cells derived from CD34+ hematopoietic stem cells.

1230-P: Isolation of Functional Endothelial Progenitor Cells from Human Adipose Tissue

Endothelial progenitor cells (EPCs) are key for angiogenic responses in multiple tissues and mediate a coordinate augmentation of the capillary network as adipose tissue (AT) expands in response to positive energy balance. However, isolation and culture of EPCs from human AT has been difficult. Here we describe a new method for obtaining well-characterized EPCs from human AT. Subcutaneous and visceral AT specimens (approximately 1-2 g) were obtained from 10-20 obese subjects by needle biopsy of abdominal subcutaneous AT and removal of omental AT fragments during abdominal surgery, respectively. Human EPCs were isolated from both the digested (SVCI) and undigested (SVCII) stromal vascular fractions of AT by anti-CD31 beads. After 5 days of in vitro culture, EPCs from SVCII exhibited an endothelial-like cobblestone morphology as compared to EPCs from SVCI. Flow cytometry analysis showed that the percentage of CD31+ and CD144+ cells was much higher in the SVCII compared to SVCI fraction (SVCII: CD31 97.9±2.9%, CD144 93.3±4.7%; SVCI: CD31 4.0±1.2%, CD144 3.8±0.7%). Immunofluorescence staining demonstrated that CD31+ EPCs from SVCII expressed Von Willebrand Factor (vWF) and VE-cadherin. Additionally, significantly higher mRNA and protein levels of CD31, E-selectin, e-NOS, VEGFR, and CD34 were found in CD31+ EPCs from SVCII than in control cells (CD31- cells from SVCI). EPCs from SVCII maintained the expression of endothelial markers (CD144 mRNA and protein levels) up to the fourth cell passage. Lastly, CD31+ EPCs from SVCII, but not control cells, were able to form tubes in 3D scaffolds (Matrigel) resembling capillary structures, as well as to incorporate acetylated LDL in vitro. Thus, we have successfully isolated EPCs with high proliferative potential and specific functional properties from the human AT. This will potentially increase our understanding of AT biology particularly in relation to altered expansion of distinct AT depots in human obesity. Disclosure C. Caccioppoli: None. S. Perrini: None. V. Genchi: None. R. Doria: None. G. Palma: None. A. Cignarelli: None. A. Natalicchio: Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Lilly Diabetes, Sanofi. L. Laviola: Advisory Panel; Self; A. Menarini Diagnostics, Abbott, AstraZeneca, Lilly Diabetes, Novo Nordisk Inc., Roche Diabetes Care, Sanofi-Aventis, Speaker’s Bureau; Self; Boehringer Ingelheim International GmbH, Medtronic. F. Giorgino: Consultant; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Lilly Diabetes, Novo Nordisk, Roche Diabetes Care, Sanofi, Research Support; Self; Lilly Diabetes, Roche Diabetes Care.

MO334ERYTHROPOIETIN-STIMULATED HUMAN ENDOTHELIAL PROGENITOR CELLS AMELIORATE INFLAMMATION AND FIBROSIS VIA INFLAMMASOME IN ISCHEMIA/REPERFUSION-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Ischemia/reperfusion-induced AKI (IR-AKI) is a major cause of AKI and progress to chronic kidney disease. But an effective therapeutic intervention for IR-AKI is not established yet. Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells and is significantly upregulated during hypoxia. Endothelial progenitor cells (EPCs) are derived from the bone marrow or tissue-resident cells and play major roles in the maintenance of vascular integrity and the repair of endothelial damage. So, we investigated if EPO-stimulated human EPCs could have the renoprotective effects in an IR-AKI mouse model. Method EPCs originated from human peripheral blood were cultured with EPO (10 IU/mL). Mice were assigned to sham, IR only groups, IR with EPC, and IR with EPO-treated EPC. EPCs (5x105 cells, tail vein) were administered twice at 30 min prior to bilateral renal artery occlusion, and 5 min before reperfusion, with all mice sacrificed 24 h after IR-AKI. Results Both EPCs and EPO-treated EPCs significantly attenuated the renal dysfunction associated with IR-AKI, as well as tissue injury. Apoptotic cell death and oxidative stress were significantly reduced in EPC and EPO-treated EPC mice. Expression of PCNA, ICAM-1, MCP-1 and α-SMA were also significantly reduced in EPC and EPO-treated EPC mice. Furthermore, the expression of NLRP3 and caspase-1 via the activation of NF-κB signaling pathways were significantly reduced in EPC and EPO-treated EPC mice. These results show more effective in EPO-treated EPC than EPC alone and suggest EPO might be involved in the development of EPC. Conclusion This study provides that inflammasome-mediated inflammation and fibrosis might be a potential target of EPC as a treatment for IR-AKI.

The Combination of Tissue-Engineered Blood Vessel Constructs and Parallel Flow Chamber Provides a Potential Alternative to In Vivo Drug Testing Models.

Cardiovascular disease is a major cause of death globally. This has led to significant efforts to develop new anti-thrombotic therapies or re-purpose existing drugs to treat cardiovascular diseases. Due to difficulties of obtaining healthy human blood vessel tissues to recreate in vivo conditions, pre-clinical testing of these drugs currently requires significant use of animal experimentation, however, the successful translation of drugs from animal tests to use in humans is poor. Developing humanised drug test models that better replicate the human vasculature will help to develop anti-thrombotic therapies more rapidly. Tissue-engineered human blood vessel (TEBV) models were fabricated with biomimetic matrix and cellular components. The pro- and anti-aggregatory properties of both intact and FeCl3-injured TEBVs were assessed under physiological flow conditions using a modified parallel-plate flow chamber. These were perfused with fluorescently labelled human platelets and endothelial progenitor cells (EPCs), and their responses were monitored in real-time using fluorescent imaging. An endothelium-free TEBV exhibited the capacity to trigger platelet activation and aggregation in a shear stress-dependent manner, similar to the responses observed in vivo. Ketamine is commonly used as an anaesthetic in current in vivo models, but this drug significantly inhibited platelet aggregation on the injured TEBV. Atorvastatin was also shown to enhance EPC attachment on the injured TEBV. The TEBV, when perfused with human blood or blood components under physiological conditions, provides a powerful alternative to current in vivo drug testing models to assess their effects on thrombus formation and EPC recruitment.

Fuxinfang improves hypoxia-induced injury of human aortic endothelial cells by regulating c-Fos-NR4A1-p38 pathway

To explore the molecular mechanism of Fuxinfang for improving injury of human aortic endothelial progenitor cells (HAECs). Serum samples were collected from male SD rats treated with Fuxinfang (n=8) or saline (n= 5). HAECs cultured in normoxia or hypoxic condition (2% O2) were treated with serum from normal rats or with diluted serum (1% and 10%) from rats treated with Fuxinfang. The differentially expressed genes (DEGs) between Fuxinfang-treated and control cells were detected using high-throughput sequencing to screen the target DEGs that participated in arterial endothelial cell injury and underwent changes in response to both hypoxia and Fuxinfang treatment. AmiGo and String databases were used to infer the interactions among the target genes, and the expressions of the genes were analyzed in HAECs with different treatments using enzyme-linked immunosorbent assay (ELISA) and Western blotting. HAECs cultured in hypoxia did not show obvious changes in cell morphology or expressions of hypoxia-related factors in response to treatment with 1% or 10% serum from Fuxinfang-treated rats. The results of high-throughput sequencing showed a total of 7134 DEGs (4205 up-regulated and 2929 down-regulated genes) in HAECs in hypoxia model group and 762 DEGs (305 upregulated and 457 down-regulated genes) in Fuxinfang-treated HAECs. Analysis of AmiGo and String databases and the constructed protein-protein interaction network identified c-Fos, NR4A1, and p38MAPK as the target genes. The results of ELISA and Western blotting showed that the expressions of c-Fos, NR4A1, p38MAPK and pp38MAPK increased significantly in cells with hypoxic exposure (P < 0.05); treatment with the serum containing Fuxinfang significantly reduced the expression levels of c-Fos, NR4A1 and p-p38MAPK in hypoxic HAECs in a concentration-dependent manner (P < 0.05). The serum from Fuxinfang-treated rats can concentration-dependently inhibit the expressions of the DEGs occurring in hypoxia. Fuxinfang improves hypoxic injuries of HAECs possibly by down-regulating the expression of c-Fos to inhibit NR4A1 expression and suppressing hypoxia-induced p38 phosphorylation.

Additional alkaloids from Zoanthus vietnamensis with neuroprotective and anti-angiogenic effects

A chemical investigation of the zoantharian Zoanthus vietnamensis, collected off Taiwan, yielded eleven new alkaloids, 7α-hydroxykuroshine J (1), 18β-hydroxykuroshine J (2), 5α-hydroxyzoanthenamine (3), 5β-hydroxyzoanthenamine (4), 14α-hydroxyzoanthenamine (5), 30-hydroxyzoanthenamine (6), 11-dehydroxy-18-epi-kuroshine A (7), 5α-hydroxykuroshine A (8), 7β-hydroxykuroshine A (9), 11-keto-oxyzoanthamine (10), and 30-hydroxyzoanthamine (11), along with eight known compounds (12–19). The structures of these compounds were identified by detailed spectroscopic data, including HRESIMS, IR, NMR, and UV spectra. All secondary metabolites isolated from Z. vietnamensis were investigated for the anti-angiogenic effect in human endothelial progenitor cells (EPCs). Compounds 6, 7, 11, and 13 exhibited mild anti-angiogenic effect by blocking cell growth and tube formation of EPCs. The neuroprotective potential of four major compounds 12, 14, 15, and 19 against paclitaxel-induced neurotoxicity was evaluated. Pretreatment of 14 and 15 protected paclitaxel-damaged neurite outgrowth of dorsal root ganglion (DRG) neurons, without interfering the cytotoxic activity of paclitaxel on cervical cancer SiHa cells.

Activation of toll-like receptor signaling in endothelial progenitor cells dictates angiogenic potential: from hypothesis to actual state.

Human endothelial progenitor cells (EPCs) were isolated from cord blood samples and enriched by magnetic activated cell sorting method based on the CD133 marker. Cells were incubated with different doses of bacterial lipopolysaccharide, ranging from 2, 5, 10, 50, 100, 200, 250, 500, to 1000µg/ml, for 48h. The cell survival rate was determined by using MTT assay. To confirm activation of the toll-like receptor signaling pathway, PCR array analysis was performed. Protein levels of ERK1/2, p-ERK1/2, NF-ƙB and TRIF proteins were measured using western blotting. The content of TNF-α and lipoprotein lipase activity were analyzed by immunofluorescence imaging. Flow cytometric analysis of CD31 was performed to assess the maturation rate. Cell migration was studied by the Transwell migration assay. The expression of genes related to exosome biogenesis was measured using real-time PCR analysis. In vivo gel plug angiogenesis assay was done in nude mice. Lipopolysaccharide changed endothelial progenitor cells' survival in a dose-dependent manner with maximum viable cells in groups treated with 2µg/ml. PCR array analysis showed the activation of toll-like signaling pathways after exposure to LPS (p<0.05). Western blotting analysis indicated an induction of p-ERK1/2 and Erk1/2, NF-kB and TRIF in LPS-treated EPCs compared with the control (p<0.05). Immunofluorescence staining showed an elevation of TNF-α and lipoprotein lipase activity after lipopolysaccharide treatment (p<0.05). Lipopolysaccharide increased EPC migration and expression of exosome biogenesis-related genes (p<0.05). In vivo gel plug analysis revealed enhanced angiogenesis in cells exposed to bacterial lipopolysaccharide. Data highlighted the close relationship between the toll-like receptor signaling pathway and functional activity in EPCs.

TNF\u03b1 priming through its interaction with TNFR2 enhances endothelial progenitor cell immunosuppressive effect: new hope for their widespread clinical application

BackgroundBone marrow derived endothelial progenitor cells (EPCs) are immature endothelial cells (ECs) involved in neo-angiogenesis and endothelial homeostasis and are considered as a circulating reservoir for endothelial repair. Many studies showed that EPCs from patients with cardiovascular pathologies are impaired and insufficient; hence, allogenic sources of EPCs from adult or cord blood are considered as good choices for cell therapy applications. However, allogenic condition increases the chance of immune rejection, especially by T cells, before exerting the desired regenerative functions. TNFα is one of the main mediators of EPC activation that recognizes two distinct receptors, TNFR1 and TNFR2. We have recently reported that human EPCs are immunosuppressive and this effect was TNFα-TNFR2 dependent. Here, we aimed to investigate if an adequate TNFα pre-conditioning could increase TNFR2 expression and prime EPCs towards more immunoregulatory functions.MethodsEPCs were pre-treated with several doses of TNFα to find the proper dose to up-regulate TNFR2 while keeping the TNFR1 expression stable. Then, co-cultures of human EPCs and human T cells were performed to assess whether TNFα priming would increase EPC immunosuppressive and immunomodulatory effect.ResultsTreating EPCs with 1 ng/ml TNFα significantly up-regulated TNFR2 expression without unrestrained increase of TNFR1 and other endothelial injury markers. Moreover, TNFα priming through its interaction with TNFR2 remarkably enhanced EPC immunosuppressive and anti-inflammatory effects. Conversely, blocking TNFR2 using anti-TNFR2 mAb followed by 1 ng/ml of TNFα treatment led to the TNFα-TNFR1 interaction and polarized EPCs towards pro-inflammatory and immunogenic functions.ConclusionsWe report for the first time the crucial impact of inflammation notably the TNFα-TNFR signaling pathway on EPC immunological function. Our work unveils the pro-inflammatory role of the TNFα-TNFR1 axis and, inversely the anti-inflammatory implication of the TNFα-TNFR2 axis in EPC immunoregulatory functions. Priming EPCs with 1 ng/ml of TNFα prior to their administration could boost them toward a more immunosuppressive phenotype. This could potentially lead to EPCs’ longer presence in vivo after their allogenic administration resulting in their better contribution to angiogenesis and vascular regeneration.6Hzxhpu_E57htd_nED83pdVideo

Extracellular vesicle-based therapy for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) stands as a neurodegenerative disorder characterized by the rapid progression of motor neuron loss in the brain and spinal cord. Unfortunately, treatment options for ALS are limited, and therefore, novel therapies that prevent further motor neuron degeneration are of dire need. In ALS, the infiltration of pathological elements from the blood to the central nervous system (CNS) compartment that spur motor neuron damage may be prevented via restoration of the impaired blood-CNS-barrier. Transplantation of human bone marrow endothelial progenitor cells (hBM-EPCs) demonstrated therapeutic promise in a mouse model of ALS due to their capacity to mitigate the altered blood-CNS-barrier by restoring endothelial cell (EC) integrity. Remarkably, the hBM-EPCs can release angiogenic factors that endogenously ameliorate impaired ECs. In addition, these cells may produce extracellular vesicles (EVs) that carry a wide range of vesicular factors, which aid in alleviating EC damage. In an in vitro study, hBM-EPC-derived EVs were effectively uptaken by the mouse brain endothelial cells (mBECs) and cell damage was significantly attenuated. Interestingly, the incorporation of EVs into mBECs was inhibited via β1 integrin hindrance. This review explores preclinical studies of the therapeutic potential of hBM-EPCs, specifically via hBM-EPC-derived EVs, for the repair of the damaged blood-CNS-barrier in ALS as a novel treatment approach.

Effects of Astragaloside Ⅳ on exosome secretion and its microRNA-126 expression in human endothelial progenitor cells

Objective: To investigate the effects of Astragaloside Ⅳ on the secretion of exosomes in human endothelial progenitor cells (EPCs) and the expression of microRNA (miRNA)-126 in exosomes. Methods: The umbilical cord blood from one healthy full-term newborn from the Department of Obstetrics and Gynecology of the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine in 2019 was harvested for isolating mononuclear cells by density gradient centrifugation and cultured for 7 days. Morphological observation was performed during this period. Cells of the third passage were collected for identification by CD31 immunomagnetic bead sorting and double fluorescence staining. According to the random number table, the identified EPCs were divided into Astragaloside Ⅳ group and phosphate buffer solution (PBS) group. The cells in Astragaloside Ⅳ group were cultured with Astragaloside Ⅳ in final mass concentration of 100 mg/L for 24 hours, and the cells in PBS group were cultured with the same volume of PBS for 24 hours. After culture, the exosomes from the cell culture supernatant of the two groups were collected, and the expressions of characteristic markers of exosomes CD9, CD63, and CD81 were detected by Western blotting, the morphology of EPC exosomes (EPC-Exos) was observed under transmission electron microscope, and the particle size of EPC-Exos was detected by nanoparticle tracking analysis technique. The concentration of EPC-Exos was determined by dioctyl butyric acid method (the sample number was 3), and the expressions of miRNA-126-3p and miRNA-126-5p related to angiogenesis in EPC-Exos were determined by reverse transcription polymerase chain reaction (the sample number was 3). Data were statistically analyzed with independent sample t test. Results: (1) On the 4th day of culture, the cells began to adhere to the wall, and the multi-forms such as circle, fusiform, and strip appeared at the same time. On the 7th day of culture, the edge of the cells was clear and arranged like a paving stone, the central cells were round, and the surrounding cells were fusiform. (2) CD31 immunomagnetic beads sorting method identification showed that the membrane was stained with green fluorescence and the nucleus was stained with blue fluorescence. Double fluorescence staining method showed that the cells were orange-yellow. The cells were identified as EPCs. (3) After 24 hours of culture, the expressions of CD9, CD63, and CD81 in EPC-Exos were all positive, confirming that EPC-Exos were extracted successfully in this experiment. (4) After 24 hours of culture, the EPC-Exos of the two groups showed round membrane vesicles, and there was no significant difference in morphology. (5) After 24 hours of culture, the particle size of 98.7% EPC-Exos in Astragaloside Ⅳ group was 84.7 to 143.1 nm, and that of 98.0% EPC-Exos in PBS group was 88.7 to 123.5 nm. (6) After 24 hours of culture, the mass concentration of EPC-Exos in Astragaloside Ⅳ group was (310±5) μg/mL, which was significantly higher than (257±5) μg/mL in PBS group, t=13.369, P<0.01. (7) After 24 hours of culture, there were more miRNA-126-3p (t=16.062, P<0.01) and miRNA-126-5p (t=3.252, P<0.05) in EPC-Exos of Astragaloside Ⅳ group than in PBS group. Conclusions: Astragaloside Ⅳ can improve the function of human EPC secretory exosomes, and the secreted exosomes are loaded with miRNA-126.

Short term in vitro culture of human endothelial progenitor cells; so called early EPC: Does not belong to true endothelial lineage

Background and Aims: Isolation/characterization of Endothelial Progenitor Cells (EPC) is controversial. Two subtypes have been reported in culture: early and late EPCs. We examined whether the early EPCs are true endothelial cells and have a role in angiogenesis.

Effects of Human Endothelial Progenitor Cell and Its Conditioned Medium on Oocyte Development and Subsequent Embryo Development.

Human endothelial progenitor cells (EPCs) secrete numerous growth factors, and they have been applied to regenerative medicine for their roles in angiogenesis as well as neovascularization. Angiogenesis is one of the essential factors for the maturation of ovarian follicles; however, the physiological function of EPCs or their derivatives on in vitro culture systems has not been fully understood. The aim of this study was to evaluate the effectiveness of EPCs and their conditioned medium (EPC-CM) on oocyte development and subsequent embryo development. In the results, the oocyte development and subsequent embryo development were significantly improved in EPCs and the EPC-CM group. In addition, markedly increased levels of growth factors/cytokines, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), insulin growth factor-1 (IGF-1), interleukin-10 (IL-10), and epidermal growth factor (EGF), were observed in medium from the EPC-CM group. Additionally, EPC-CM after in vitro maturation (IVM) had significantly decreased reactive oxygen species (ROS) levels compared to those of other groups. Transcriptional levels of growth factor receptor-related genes (FGFR2, IGF1R) and anti-apoptotic-related gene (BCL2) were significantly upregulated in cumulus cells/oocytes from the EPC-CM group compared with those from the control. Furthermore, the expression levels of cumulus expansion-related genes (PTGS2, TNFAIP6, HAS2) and oocyte-maturation-related factors (GDF9, BMP15) were significantly enhanced in the EPC-CM group. Consequently, the present study provides the first evidence that EPC-CM contains several essential growth factors for oocyte development by regulating genes involved in oocyte maturation.

Predicting in vivo therapeutic efficacy of bioorthogonally labeled endothelial progenitor cells in hind limb ischemia models via non-invasive fluorescence molecular tomography

Human embryonic stem cells-derived endothelial progenitor cells (hEPCs) were utilized as cell therapeutics for the treatment of ischemic diseases. However, in vivo tracking of hEPCs for predicting their therapeutic efficacy is very difficult. Herein, we developed bioorthogonal labeling strategy of hEPCs that could non-invasively track them after transplantation in hind limb ischemia models. First, hEPCs were treated with tetraacylated N-azidomannosamine (Ac4ManNAz) for generating unnatural azide groups on the hEPCs surface. Second, near-infrared fluorescence (NIRF) dye, Cy5, conjugated dibenzocylooctyne (DBCO-Cy5) was chemically conjugated to the azide groups on the hEPC surface via copper-free click chemistry, resulting Cy5-hEPCs. The bioorthogonally labeled Cy5-hEPCs showed strong NIRF signal without cytotoxicity and functional perturbation in tubular formation, oxygen consumption and paracrine effect of hEPCs in vitro. In hind limb ischemia models, the distribution and migration of transplanted Cy5-hEPCs were successfully monitored via fluorescence molecular tomography (FMT) for 28 days. Notably, blood reperfusion and therapeutic neovascularization effects were significantly correlated with the initial transplantation forms of Cy5-hEPCs such as ‘condensed round shape’ and ‘spread shape’ in the ischemic lesion. The condensed transplanted Cy5-hEPCs substantially increased the therapeutic efficacy of hind limb ischemia, compared to that of spread Cy5-hEPCs. Therefore, our new stem cell labeling strategy can be used to predict therapeutic efficacy in hind limb ischemia and it can be applied a potential application in developing cell therapeutics for regenerative medicine.

The effect of fluid shear stress in hydrogen sulphide production and cystathionine γ-lyase expression in human early endothelial progenitor cells.

BackgroundPhysiological fluid shear stress has been shown to have a beneficial impact on vascular homeostasis. Endothelial progenitor cells (EPCs) make a significant contribution to maintaining endothelial integrity. Therefore, we hypothesised that shear stress-induced endothelium protection plays a role in hydrogen sulphide (H2S) production and up-regulation of cystathionine γ-lyase (CSE) expression in EPCs.MethodsHuman EPC-derived CSE activity was detected by colorimetric assay, and H2S production was evaluated by membrane adsorption method. Cell proliferation, migration, and adhesion were assessed by MTT, Transwell, and endothelial cell-mediated adhesion assays, respectively. Real-time polymerase chain reaction (RT-PCR) was carried out to analyse gene expression. Protein expression was analysed by western blot.ResultsHuman EPCs were treated with shear stress levels of 5–25 dyn/cm2 for up to 3 h, and 25 dyn/cm2 for up to 24 h. H2S production and CSE mRNA expression in the EPCs were increased by shear stress in a dose-dependent manner in vitro. Likewise, time-dependent shear stress also significantly enhanced CSE protein expression. Compared to static condition, shear stress improved EPCs proliferation, migration and adhesion capacity. Knockdown of CSE expression by small interfering RNA substantially eliminated the shear stress-induced above functions of human EPCs in vitro.ConclusionsThis study gives new insight into the regulatory effect of physiological shear stress on the CSE/H2S system in human EPCs. Our findings may contribute to the development of vascular protective research, although the relevant evidence is admittedly indirect.

Chemerin enhances the adhesion and migration of human endothelial progenitor cells and increases lipid accumulation in mice with atherosclerosis

BackgroundThe role of adipokines in the development of atherosclerosis (AS) has received increasing attention in recent years. This study aimed to explore the effects of chemerin on the functions of human endothelial progenitor cells (EPCs) and to investigate its role in lipid accumulation in ApoE-knockout (ApoE−/−) mice.MethodsEPCs were cultured and treated with chemerin together with the specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 in a time- and dose-dependent manner. Changes in migration, adhesion, proliferation and the apoptosis rate of EPCs were detected. ApoE−/− mice with high-fat diet-induced AS were treated with chemerin with or without SB 203580. Weights were recorded, lipid indicators were detected, and tissues sections were stained.ResultsThe data showed that chemerin enhanced the adhesion and migration abilities of EPCs, and reduced the apoptosis ratio and that this effect might be mediated through the p38 MAPK pathway. Additionally, chemerin increased the instability of plaques. Compared with the control group and the inhibitor group, ApoE−/− mice treated with chemerin protein had more serious arterial stenosis, higher lipid contents in plaques and decreased collagen. Lipid accumulation in the liver and kidney and inflammation in the hepatic portal area were enhanced by treatment with chemerin, and the size of adipocytes also increased after chemerin treatment. In conclusion, chemerin can enhance the adhesion and migration abilities of human EPCs and reduce the apoptosis ratio. In animals, chemerin can increase lipid accumulation in atherosclerotic plaques and exacerbate plaques instability. At the same time, chemerin can cause abnormal lipid accumulation in the livers and kidneys of model animals. After specifically blocking the p38 MAPK pathway, the effect of chemerin was reduced.ConclusionsIn conclusion, this study showed that chemerin enhances the adhesion and migration abilities of EPCs and increases the instability of plaques and abnormal lipid accumulation in ApoE−/− mice. Furthermore, these effects might be mediated through the p38 MAPK pathway.

Shear stress improves the endothelial progenitor cell function via the CXCR7/ERK pathway axis in the coronary artery disease cases

BackgroundDysfunction in the late Endothelial Progenitor Cells (EPCs) is responsible for endothelial repair in patients with Coronary Artery Disease (CAD), and the shear stress is beneficial for EPCs function. However, the impact of shear stress on the capacity of EPCs in CAD patients has not been elucidated yet. The C-X-C chemokine receptor 7/extracellular signal-regulated kinase (CXCR7)/(ERK) pathways are identified to regulate EPCs function in CAD patients. Here, we hypothesize that shear stress upregulates the CXCR7/ERK pathways, which restore the EPCs function in CAD patients.MethodsThe human Peripheral Blood Mononuclear Cells (PBMCs) were collected from healthy adults and CAD patients and then used for EPCs cultivation. The Lv-siRNA for human CXCR7 was transfected into induced EPCs isolated from the CAD patients. Meanwhile, the EPCs from CAD patients were subjected to shear stress generated by a biomimetic device. Next, the cell viability, migration, tube formation, and apoptosis were detected by CCK-8, Transwell assay, Matrigel, and flow cytometry, respectively. Also, the CXCR7/ERK pathways in human EPCs were analyzed by Western blotting and qRT-PCR.ResultCompared to the EPCs collected from normal adults, the CAD patient-derived EPCs showed reduced in vitro vasculogenic capacity. Also, the level of CXCR7 in CAD patient-derived EPCs was significantly reduced compared to the EPCs of healthy subjects. Meanwhile, the extracellular signal-regulated kinase (ERK), which represents a CXCR7 downstream signaling pathway, had decreased phosphorylation level. The shear stress treatment augmented the CXCR7 expression and also elevated ERK phosphorylation, which is comparable to the up-regulation of CAD patient-derived EPCs function. Further, the small interfering RNA (siRNA)-mediated CXCR7 knockdown diminished the enhanced migration, adhesion, and tube formation capacity of shear stress treated CAD patient-derived EPCs.ConclusionUp-regulation of the CXCR7/ERK pathways by shear stress can be a promising new target in enhancing the vasculogenic ability of CAD patient-derived EPCs.

Immobilization of BMP-2 and VEGF within Multilayered Polydopamine-Coated Scaffolds and the Resulting Osteogenic and Angiogenic Synergy of Co-Cultured Human Mesenchymal Stem Cells and Human Endothelial Progenitor Cells.

We have previously reported the fabrication of a polycaprolactone and hydroxyapatite composite scaffold incorporating growth factors to be used for bone regeneration. Two growth factors were incorporated employing a multilayered coating based on polydopamine (PDA). In particular, Bone morphogenetic protein-2 (BMP-2) was bound onto the inner PDA layer while vascular endothelial growth factor (VEGF) was immobilized onto the outer one. Herein, the in vitro release of both growth factors is evaluated. A fastest VEGF delivery followed by a slow and more sustained release of BMP-2 was demonstrated, thus fitting the needs for bone tissue engineering applications. Due to the relevance of the crosstalk between bone-promoting and vessel-forming cells during bone healing, the functionalized scaffolds are further assessed on a co-culture setup of human mesenchymal stem cells and human endothelial progenitor cells. Osteogenic and angiogenic gene expression analysis indicates a synergistic effect between the growth factor-loaded scaffolds and the co-culture conditions. Taken together, these results indicate that the developed scaffolds hold great potential as an efficient platform for bone-tissue applications.

Cell-Free Extracellular Vesicles Derived from Human Bone Marrow Endothelial Progenitor Cells as Potential Therapeutics for Microvascular Endothelium Restoration in ALS.

Repairing the damaged blood-CNS-barrier in amyotrophic lateral sclerosis (ALS) is necessary to prevent entry of detrimental blood-borne factors contributing to motor neuron dysfunction. Recently, we showed benefits of human bone marrow endothelial progenitor cell (hBM-EPC) transplantation into symptomatic ALS mice on barrier restoration by replacing damaged endothelial cells (ECs). Additionally, transplanted cells may endogenously repair ECs by secreting angiogenic factors as our subsequent in vitro study demonstrated. Based on these study results, hBM-EPCs may secrete extracellular vesicles, which may contain and transfer diverse vesicular biomolecules towards maintenance of EC functionality. The study aimed to characterize extracellular vesicles (EVs) derived from hBM-EPCs as potential cell-free therapeutics for endothelium repair in ALS. EVs were isolated from hBM-EPC media at different culture times and vesicle properties were evaluated. The protective effects of EVs on mouse brain endothelial cells (mBECs) exposed to ALS mouse plasma were investigated. Uptake and blockage of EVs from GFP-transfected hBM-EPCs in ECs were determined in vitro. Results showed that EVs isolated from hBM-EPCs as nanosized vesicles significantly reduced mBEC damage from the pathological environment and these EVs were taken up by cells. Blockage of β1 integrin on EVs prevented internalization of vesicles in mBECs. Together, these results provide evidence for potential of hBM-EPC-derived EVs as novel cell-free therapeutics for repair of endothelium in ALS. Although determining translational potential of hBM-EPC-derived EVs will require evaluation in vivo, this in vitro study represents a step towards an extracellular vesicle-based approach for repair of the damaged microvascular endothelium in ALS.

Identification of Potential Hub Genes and Signal Pathways Promoting the Distinct Biological Features of Cord Blood-Derived Endothelial Progenitor Cells Via Bioinformatics.

Background: Numerous studies, ranging from the alleviation of tissue ischemia to the assessment of cancer prognosis, have demonstrated the fundamental biological differences between human umbilical cord blood-derived endothelial progenitor cells (CB-EPCs) and adult peripheral blood-derived endothelial progenitor cells (PB-EPCs). However, the underlying molecular mechanisms that produce these differences are not clear.The purpose of this study was to identify potential hub genes, key protein interactive networks, and correlated signal pathways unique to CB-EPC biology via bioinformatic methods. Materials and Methods: We selected the microarray dataset GSE39763 and identified the differentially expressed genes (DEGs) using the "limma" package in the RStudio software. These DEGs were annotated by gene ontology enrichment analyses and signal pathway analyses. A protein-protein interaction (PPI) analysis was then performed to construct PPI networks and identify a hub protein module. We further validated candidate DEGs from the selected module in the gene expression profiling interactive analysis (GEPIA) database because the DEGs were enriched in cancer pathways. Results: Setting an adjusted p-value <0.01 and |Log2 fold change (FC)| ≥ 2 as cutoff criteria, a total of 346 DEGs, including 314 upregulated genes and 32 downregulated genes in CB-EPCs, were identified. Expression of the genes encoding the AT-Hook Containing Transcription Factor 1 (AHCTF1), the Cancer Susceptibility Candidate 5 (CASC5), the Centromere Protein C (CENPC), the Centromere Protein E (CENPE), the Centromere Protein F (CENPF), the NUF2 Component of NDC80 Kinetochore Complex (NUF2), the RAN-Binding Protein 2 (RANBP2), the Shugoshin-like 2 (SGOL2), the Structural Maintenance of Chromosomes 3 (SMC3), and the Spindle Apparatus Coiled-Coil Protein 1 (SPDL1) proteins were specifically associated with CB-EPCs. Except for CENPC, the other nine genes' expression are all associated with a poorer overall survival rate in cancers. The expression levels of the CENPF and NUF2 genes in tumor patients were significantly higher than those in the controls. Conclusion: The CB-EPCs express genes with greater potential for proliferation and increased migration compared to PB-EPCs; in this regard they are similar to cancer cells.