Ferrostatin-1 Reversed Chronic Intermittent Hypoxia-Induced Ferroptosis in Aortic Endothelial Cells via Reprogramming Mitochondrial Function.

Purpose: Chronic intermittent hypoxia (CIH) related arterial endothelium injury is a common cause of cardiovascular system injury. However, the mechanism still needs to be clarified. In this study, we aimed to clarify the role and mechanism of ferrostatin-1 (Fer-1) in CIH-related rat arterial endothelial cells (ROAEC) ferroptosis. Methods: ROAEC was divided into control group, CIH group, and CIH+ Fer-1 group. Cell viability was detected by cell counting kit 8 kits (CCK8). The apoptotic rate, reactive oxygen species (ROS) levels, Fe2+ levels, and lipid ROS levels were detected by flow cytometry. Malondialdehyde (MDA) levels and nicotinamide adenine dinucleotide (NAD+)/NADH ratio were detected via Elisa kits. The mRNA and protein levels of cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were detected by qRT-PCR and Western blot. Mitochondrial structure and function were observed by transmission electron microscope (TEM) and mitochondrial membrane potential (MMP). Central carbon metabolism was measured to compare metabolites among each group. Results: After the CIH exposure, ROAEC cell viability decreased; The levels of cell apoptosis, ROS, Fe2+, MDA, and lip ROS increased; The levels of NAD+/NADP ratio decreased; The mRNA and protein levels of GPX4 and SLC7A11 decreased (all p<0.05). Co-cultured with Fer-1 reversed the levels of apoptosis rate, cell viability, ROS, Fe2+, MAD, lipid ROS, NAD+/NADH ratio and the mRNA and protein expression of GPX4 and SLC7A11 (all p<0.05). The TEM results showed that damaged mitochondrial membrane and the matrix spillover in the CIH group. The results of the JC-1 assay showed decreased MMP in the CIH group. Fer-1 treatment ameliorated the mitochondrial injury. The results of central carbon metabolism found that CIH altered the metabolites in the TCA cycle, which were reversed by Fer-1 treatment. Conclusion: CIH-induced ferroptosis in ROAEC, which were reversed by Fer-1 via reprogramming mitochondrial function.

Increased oxidative stress response in circulating blood of systemic sclerosis patients - relation to disease characteristics and inflammatory blood biomarkers.

BACKGROUND: Systemic sclerosis (SSc) is a rare connective tissue disorder of unknown etiology characterized by organ fibrosis and microcirculation dysfunction. Emerging evidence suggests that SSc is related to increased oxidative stress, which contributes to further tissue and vascular damage. METHODS: Oxidative stress response in the peripheral blood was assessed in patients with SSc (n = 55) and well-matched controls (n = 44) using real-time monitoring of protein hydroperoxide (HP) formation by the coumarin boronic acid (CBA) assay. We also analyzed the relationship between HP generation and SSc clinics, systemic inflammation, and cellular fibronectin, an emerging biomarker of endothelial damage. RESULTS: SSc was characterized by a significantly faster (2-fold) fluorescent product generation in the CBA assay and higher cumulative HP formation (3-fold) compared to controls (p<0.001, both). The dynamics of HP generation were not associated with the form of the disease (diffuse vs. limited SSc), current immunosuppressive therapy use, presence of abnormal nailfold capillaries, and autoantibody profile. Still, it was enhanced in patients with more severe illness and certain clinical manifestations (i.e., pulmonary hypertension, digital ulcers, and cyclophosphamide treatment) and in smokers (current or past). Higher serum CRP, blood eosinophil count, and cellular fibronectin with lower hemoglobin levels were independent determinants of increased HP formation. CONCLUSIONS: Our data indicate a pro-oxidant imbalance in SSc, likely related to systemic inflammation and endothelial injury. However, extensive prospective studies are needed to verify whether it is also associated with clinical disease progression.

Evaluation of ex vivo produced endothelial progenitor cells for autologous transplantation in primates.

BACKGROUND: Autologous transplantation of endothelial progenitor cells (EPCs) is a promising therapeutic approach in the treatment of various vascular diseases. We previously reported a two-step culture system for scalable generation of human EPCs derived from cord blood CD34+ cells ex vivo. Here, we now apply this culture system to expand and differentiate human and nonhuman primate EPCs from mobilized peripheral blood (PB) CD34+ cells for the therapeutic potential of autologous transplantation. METHODS: The human and nonhuman primate EPCs from mobilized PB CD34+ cells were cultured according to our previously reported system. The generated adherent cells were then characterized by the morphology, surface markers, nitric oxide (NO)/endothelial NO synthase (eNOS) levels and Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake/fluorescein isothiocyanate (FITC)-lectin binding actives. Furthermore, the efficacy and safety studies were performed by autologous transplantation via hepatic portal vein injection in a nonhuman primate model with acute liver sinusoidal endothelial cell injury. RESULTS: The mobilized PB CD34+ cells from both human and nonhuman primate were efficiently expanded and differentiated. Over 2 × 108 adherent cells were generated from 20 mL mobilized primate PB (1.51 × 106 ± 3.39 × 105 CD34+ cells) by 36-day culture and more than 80% of the produced cells were identified as EPCs/endothelial cells (ECs). In the autologous transplant model, the injected EPC/ECs from nonhuman primate PB were scattered in the intercellular spaces of hepatocytes at the hepatic tissues 14 days post-transplantation, indicating successful migration and reconstitution in the liver structure as the functional EPCs/ECs. CONCLUSIONS: We successfully applied our previous two-step culture system for the generation of primate EPCs from mobilized PB CD34+ cells, evaluated the phenotypes ex vivo, and transplanted autologous EPCs/ECs in a nonhuman primate model. Our study indicates that it may be possible for these ex-vivo high-efficient expanded EPCs to be used in clinical cell therapy.

Trophic effect of PACAP on human corneal endothelium.

Cornea's posterior surface includes endothelium maintaining stromal hydration and clarity. Due to their limited proliferative capability, the loss of endothelial cells can outcome in permanent opacity. In the last years, different studies have demonstrated the protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in different ocular diseases. However, its role on human corneal endothelial cells (HCECs) has not been investigated, yet. Here, we have developed a culture protocol to differentiate HCECs from donor's cornea. PACAP treatment prevented damage induced by growth factors deprivation of cells grown on transwell supports as revealed by TERR measurements. Moreover, this peptide significantly increased tight junction proteins expression by conferring resistance to endothelial barrier. This effect is also related to promotion of cell viability as demonstrated by MTT assay. Furthermore, PACAP stimulated repairing of corneal endothelium lesion as shown by wound healing analysis. In conclusion, our data suggest that this peptide could represent an important trophic factor in maintaining functionality of human corneal endothelium.

Circulating platelet aggregates damage endothelial cells in culture.

BACKGROUND: Presence of circulating endothelial cells (CECs) in systemic circulation may be an indicator of endothelial damage and/or denudation, and the body's response to repair and revascularization. Thus, we hypothesized that aggregated platelets (AgPlts) can disrupt/denude the endothelium and contribute to the presence of CEC and EC-derived particles (ECDP). METHODS: Endothelial cells were grown in glass tubes and tagged with/without 0.5 µm fluorescent beads. These glass tubes were connected to a mini-pump variable-flow system to study the effect of circulating AgPlts on the endothelium. ECs in glass tube were exposed to medium alone, nonaggregated platelets (NAgPlts), AgPlts, and 90 micron polystyrene beads at a flow rate of 20 mL/min for various intervals. Collected effluents were cultured for 72 h to analyze the growth potential of dislodged but intact ECs. Endothelial damage was assessed by real time polymerase chain reaction (RT-PCR) for inflammatory genes and Western blot analysis for von Willebrand factor. RESULTS AND CONCLUSION: No ECs and ECDP were observed in effluents collected after injecting medium alone and NAgPlts, whereas AgPlts and Polybeads drastically dislodged ECs, releasing ECs and ECDP in effluents as the time increased. Effluents collected when endothelial cell damage was seen showed increased presence of von Willebrand factor as compared to control effluents. Furthermore, we analyzed the presence of ECs and ECDPs in heart failure subjects, as well as animal plasma samples. Our study demonstrates that circulating AgPlts denude the endothelium and release ECs and ECDP. Direct mechanical disruption and shear stress caused by circulating AgPlts could be the underlying mechanism of the observed endothelium damage.

An effective ex-vivo approach for inducing endothelial progenitor cells from umbilical cord blood CD34+ cells.

BACKGROUND: Transplantation of endothelial progenitor cells (EPCs)/endothelial cells (ECs) has been used for the treatment of ischemic diseases and hemophilia A, due to their great capacity for producing factor VIII and for repairing vascular damage. We established an effective approach to stimulate the expansion and differentiation of EPCs for potential therapeutic applications. METHODS: CD34+ cells isolated from human cord blood were cultured in a two-step system for 21 days. The generated adherent cells were characterized via flow cytometry and immunofluorescent staining. Moreover, single-cell clonogenic and tube-forming assays were carried out to evaluate their potential to proliferate and form vessel networks. Furthermore, these cells were transplanted into a mouse model of hepatic sinusoidal endothelium injury by hepatic portal vein injection to investigate their in-vivo behavior. RESULTS: The two-step culture protocol promoted the expansion and differentiation of human cord blood CD34+ cells efficiently, resulting in a large number of adherent cells within 3 weeks. The generated adherent cells were identified as EPCs/ECs based on the expression of CD31, CD144, vWF, and FVIII, and cell numbers showed a 1400-fold increase compared with the initial number. Moreover, these EPCs/ECs were capable of proliferating and establishing colonies as individual cells, and forming tube-like structures. More significantly, tissue examination of mice after transplantation revealed that the injected EPCs/ECs migrated and integrated into the liver, reconstituting the sinusoidal endothelial compartment. CONCLUSIONS: We developed an approach for the generation of cord blood-derived EPCs/ECs on a large scale, characterized them phenotypically, and demonstrated their in-vivo functional capacity. Our approach provides an excellent source of healthy EPCs/ECs for use in cell therapy in a clinical setting.

Transplantation of VEGFl65-overexpressing vascular endothelial progenitor cells relieves endothelial injury after deep vein thrombectomy.

AIM: The purpose of this study was to explore the therapeutic efficacy of VEGF165-overexpressing vascular endothelial progenitor cells (EPCs) in post-thrombotic syndrome. MATERIALS AND METHODS: A thrombus model was developed to mimic the in-vivo setting, and adenovirus transfection was used to overexpress VEGF165 in EPCs. These cells were transplanted into the animal model, and their ability to relieve endothelial injury was evaluated using haematoxylin and eosin staining, immunohistochemistry and scanning electron microscopy. RESULTS: Ferric chloride was used to build rat models of the inferior vena cava thrombosis, and HEK 293A cells were used to amplify adenovirus that overexpresses VEGF165. EPCs were infected with adenovirus, and this was confirmed by fluorescence microscopy. Transplantation of VEGH165-overexpressing EPCs into injured endothelial sites led to faster repair of the post-thrombotic tunica intima than wild-type EPCs. CONCLUSION: Transplantation of VEGF165-overexpressing EPCs was found to promote repair of the tunica intima, thus improving rehabilitation after surgery.

Relationship between graft-versus-host disease and endothelium injury following hematopoietic stem cells transplantation in mice / 中华器官移植杂志

Objective To study the relationship between graft-versus-host disease (GVHD) and endothelium injury following hematopoietic stem cells transplantation in mice. Methods C57BL/6 mice as donors and Balb/c mice as recipients were randomly divided into 4 groups: control group, bone marrow transplantation group, GVHD group, GVHD mitigation group. The clinical manifestations,circulating endothelial cells and tissue pathological changes were observed at different time points after transplantation. Results No manifestations of GVHD were found in each group at the day 5, while those were found in GVHD group at the day 9 and all died within 15 days. The counts of endothelial cells in peripheral blood showed no significant difference at the day 5 between GVHD group (7. 34 ±1.26 cells/μl) and bone marrow transplantation group (11.51 ± 7. 40 cells/μl) or GVHD mitigation group (7. 36 ± 0. 16 cells/μl), while among three groups there was statistically significant difference at the day 9 (GVHD group: 153. 64 ± 35. 35 cells/μl vs bone marrow transplantation group: 10. 49 ±5. 61 cells/μl and GVHD mitigation group: 47. 82 ± 4. 69 cells/μl). The scores of pathological aGVHD had no significant difference at the day 5 between GVHD group (4. 33± 1. 53) and bone marrow transplantation group (3. 33 ± 0. 58) or GVHD mitigation group (4. 00 ± 1.73), while among three groups there was statistically significant difference at the day 9 (GVHD group: 10. 0 vs bone marrow transplantation group: 3. 33 ± 1.15 or GVHD mitigation group: 4. 33 ± 0. 58) and at the day 14 (GVHD group: 10. 33 ± 2. 58 vs bone marrow transplantation group: 2. 33 ± 1.25 or GVHD mitigation group 3. 33 ± 1.15). Conclusion Occurrence of GVHD causes endothelial damage again and injured endothelium worsens the GVHD.

Changes of bonemarrow and circulating endothelial progenitor cells in mice with acute pancreatitis / 国际外科学杂志

Objective To investigate changes in number of endothelial progenitor cells(EPCs)from bone marrow and circulation in mice with acute pancreatitis.Methods BALB/c mice were assigned randomly to saline group and cerulein group.Animals were sacrificed at 12, 24 and 48 hours after injection.Bone marrow and circulating EPCs were detected by flow cyzometric analysis.Plasma VEGF, TNF-α and ET-1 were determined by enzyme-linked immunosorbent assay.The expression of VEGF in the pancreas was assessed by Western blotting.Apoptosis in situ was detected by TUNEL.Results The amounts of EPCs in bone marrow and circulation increased remarkably after cerulein injection(P < 0.05), also the levels of plasma VEGF TNF-α and ET-1(P < 0.05), the EPCs levels in bone marrow and circulation seen in the study closely mirrors the levels of VEGF detected in the circulation(r = 0.77, 0.67 individually).VEGF expression in pancreas was up-regulated after 12 h of cerulein injection compared with that of control group.Apoptosis of endothelial cells also increased in the cerulein group.Conclusion EPCs were mobilized by acute pancreatitis, which may be due to the mobilizing effect of increased levels of VEGF, EPCs may participate in the repair process of injured endothelium induced by acute pancreatitis.

Effect of Tongxinluo on the interaction between COX-2 and iNOS in vascular injury of rats with deficiency of vital energy or qi stagnation / 中成药

AIM:To investigate COX-2 and iNOS protein contents and their interaction in vascular endothelium injury of rats with deficiency of vital energy or qi stagnation,and the prevetion and treatment of Tongxinluo.METHODS:The model of vascular endothelium injury of rats with deficiency of vital energy or qi stagnation was established by using high L-Methionine,with load-carrying swimming or being fastened respectively.Western blotting was used to analyze protein contents of COX-2 and iNOS,co-immunoprecipitation and laser confocal microscopy were used to analyze the interaction between COX-2 and iNOS.Optical microscope and electronic microscope were used to evaluate pathological changes in vascular endothelium.RESULTS:The protein contents of COX-2 and iNOS,and their interaction increased significantly in deficiency of vital energy group and qi stagnation group,in accord with injury of vascular endothelium.Compared with deficiency of vital energy group and qi stagnation group respectively,their protein contents decreased and their interaction was weakened in Tongxinluo groups.CONCLUSION:When protein contents of COX-2 and iNOS increase and their interaction enhance after vascular endothelium injury of rats with deficiency of vital energy or qi stagnation,initiate exacerbations,Tongxinluo could attenuate the alterations and protect vascular endothelium from injury.

Changes of angiotensin1at different stages of atherosclerosis in rats / 第二军医大学学报

Objective:To investigate the relation between the angiotensinⅡ(AngⅡ)levels in thoracic aorta and plasma and atherosclerosis(AS).Methods:Totally40healthy male Wistar rats were randomly divided into4groups(n=10).Four different feeding methods,including normal diet,high lipid,high lipid+vitamin D overload,and high lipid+vitamin D over-load+endothelium injury,were used for inducing AS in rats.The thickness of intima was taken as the index of AS severity, radio-immunity analysis was used to assay AngⅡin each group in thoracic aorta and plasma.Results:Only rats in high lipid diet group did not develop AS plaque;high lipid+vitamin D only resulted in AS fibrous plaque with VSMC proliferation;high lipid+vitamin D+endothelium injury formed ripe AS plaque.The thickness of intima and AngⅡof thoracic aorta in-creased gradually compared with control group(P