Endothelial Progenitor Cells Markers Research Articles

TRIM27 Promotes Endothelial Progenitor Cell Apoptosis in Patients with In-Stent Restenosis by Ubiquitinating TBK1.

Approximately 2-10% in-stent restenosis (ISR) may occur following percutaneous coronary intervention (PCI) despite the use of modern drug-eluting stents (DES); thus, our study aimed to explore the effects of tripartite motif-containing (TRIM) 27 on ISR and the underlying mechanism. For this purpose, a total of 42 patients undergoing coronary angiography who had prior coronary angiography with DES implantation were recruited. Endothelial progenitor cells (EPCs) markers (defined as CD34 and vascular endothelial growth factoreceptor-2 (VEGFR-2)) in peripheral blood were measured to asses the circulating EPC level. The TRIM family-related gene expressions were detected by reverse transcription-quantitative polymerase chain reaction. Results suggested that ISR patients had reduced CD34+VEGFR-2+ and increased apoptosis rate of EPCs, along with upregulated TRIM27 and TRIM37 and downregulated TRIM28. TRIM27 promoted and TBK1 inhibited the apoptosis rate of EPCs. Mechanically, TRIM27 interacted with TBK1 to ubiquitinate TBK1 in in vitro study. In summary, TRIM27 promoted the progression of ISR in patients after PCI by ubiquitinating TBK1, which might provide novel ideas for the clinical treatment of ISR.

Effect of M-CSF on the expression of endothelial progenitor cell markers in blood mononuclear cell culture in coronary heart disease

Aim. To evaluate the nature of changes in the expression of markers of endothelial progenitor cells (VEGFR2, CD34, CD14) and endothelial cells (CD146) in association with the expression of the leukocyte common antigen CD45 in the culture of blood mononuclear cells in the presence of M-CSF in patients with coronary heart disease (CHD) and healthy donors.Materials and methods. The study included 12 patients with CHD with class III–V angina pectoris and 10 healthy donors, from whom 30 ml of venous blood was taken on an empty stomach in the morning and stabilized with heparin. Blood mononuclear cells were isolated by Ficoll density gradient centrifugation (1.077 g / cm3) and subject to immunomagnetic separation using CD14-MicroBeads and CD34-MicroBead Kit (Miltenyi Biotec B.V. & Co. KG, Germany). The resulting CD14+ and CD34+ culture of mononuclear cells was incubated for 6 days in a complete nutrient medium with and without M-CSF 50 ng / ml (Cloud-Clone Corp., USA) with complete replacement of the medium and repeated application of M-CSF on day 3. After 6 days, the proportions of CD45+, CD14+, CD34+, VEGFR2+, and CD146+ cells in the culture were assessed by flow cytometry using CD14-FITC, CD34-PE, VEGFR2-Alexa Fluor 647; CD45-FITC and CD146-PerCP antibodies (BD Biosciences, USA).Results. It was shown that in healthy donors, the proportion of CD146+ cells in the co-culture of blood mononuclear cells with M-CSF exceeded their number in the sample without it, with comparable expression rates of CD45, CD14, and VEGFR2 markers between the control and stimulated cultures. In CHD patients, the number of CD146+ and VEGFR2+ cells did not change when M-CSF was added to the mononuclear cell culture; however, the proportion of CD14+ cells increased and the proportion of CD45+ cells decreased compared to the control sample. The number of CD34+ cells was comparable both between control and stimulated samples, and between the groups of examined individuals. At the same time, in patients with CHD, an increased proportion of VEGFR2+ cells was found in the control and stimulated samples compared to healthy individuals, while an increased proportion of CD14+ cells was detected only in the stimulated culture.Conclusion. The development of CHD disrupts the response of blood mononuclear cells to the effect of M-CSF, increasing the number of CD14+ and reducing the proportion of CD45+ cells in the culture in the absence of stimulating effects on the expression of endothelial cell marker CD146. At the same time, M-CSF does not affect the expression of CD34 and VEGFR2 in endothelial progenitor cells both in patients with CHD and in healthy individuals.

Development of a potency assay for CD34+ cell-based therapy

We have previously shown that intracardiac delivery of autologous CD34+ cells after acute myocardial infarction (AMI) is safe and leads to long term improvement. We are now conducting a multicenter, randomized, controlled Phase I/IIb study in post-AMI to investigate the safety and efficacy of intramyocardial injection of expanded autologous CD34+ cells (ProtheraCytes) (NCT02669810). Here, we conducted a series of in vitro studies characterizing the growth factor secretion, exosome secretion, gene expression, cell surface markers, differentiation potential, and angiogenic potential of ProtheraCytes clinical batches to develop a potency assay. We show that ProtheraCytes secrete vascular endothelial growth factor (VEGF) and its concentration is significantly correlated with the number of CD34+ cells obtained after expansion. ProtheraCytes also secrete exosomes containing proangiogenic miRNAs (126, 130a, 378, 26a), antiapoptotic miRNAs (21 and 146a), antifibrotic miRNAs (133a, 24, 29b, 132), and miRNAs promoting myocardial regeneration (199a and 590). We also show that ProtheraCytes have in vitro angiogenic activity, express surface markers of endothelial progenitor cells, and can differentiate in vitro into endothelial cells. After the in vitro characterization of multiple ProtheraCytes clinical batches, we established that measuring the concentration of VEGF provided the most practical, reliable, and consistent potency assay.

Abstract P1070: In Vivo Autotransplantation Of Spleen-derived Endothelial Progenitor Cells To Promote Angiogenesis In Ischemic Sites

Since mononuclear cells were first illuminated as potential endothelial progenitor cells (EPCs), bone marrow (BM) was extracted and processed as BM-derived EPCs for autotransplantation to the ischemic sites in previous studies. Still, numerous clinical trials based on this strategy showed only marginal effects, and the small number of BM-derived EPCs was suggested as a possible limitation. Meanwhile, spleen contains abundant mononuclear cells which can inherently target the ischemic sites, and these cells can be primed by hypoxia. If only they can be primed in vivo, spleen-derived EPCs can be inherently autotransplanted to ischemic sites with various advantages such as plentiful cell number, non-invasive extraction and delivery of these cells. In this study, splenic mononuclear cells were utilized to promote angiogenesis in ischemic sites. In vivo priming of splenic mononuclear cells, their inherent targeting and therapeutic effect on ischemic sites were sequentially explored towards the 'in vivo autotransplantation'. For hypoxic priming, the effect of hypoxia and hypoxic-mimetic agent (cobalt chloride) on isolated splenic mononuclear cells were comparable in both priming methods, regarding the morphology changes and the increase in expression of EPC markers. In align with these in vitro results, both hypoxic-mimetic agent-loaded nanoparticle delivery to spleen and splenic artery ligation resulted in increased expression of EPC markers in mice spleen. Moreover, the proportion and actual numbers of the cells presenting EPC marker were significantly higher in primed splenic cells than in BM cells. After the priming, these primed cells not only maintained the inherent targeting ability but also gained the ability to promote angiogenesis. Consequently, these primed cells presented therapeutic effects as evidenced by higher expression of angiogenic markers with enhanced blood flow recovery in mouse hindlimb ischemia model, compared with untreated or BM-derived EPCs' autotransplantation for conventional strategy. For EPC-based treatment of ischemia, 'In vivo autotransplantation' is simple and non-invasive strategy which can overcome the previous limitations of insufficient cell numbers.

Hyperbaric oxygen therapy efficacy on mandibular defect regeneration in rats with diabetes mellitus: an animal study

BackgroundThis study aimed to investigate the influence of hyperbaric oxygen therapy on mandibular critical-sized defect regeneration in rats with experimentally induced type I diabetes mellitus. Restoration of large osseous defects in an impaired osteogenic condition such as diabetes mellitus is a challenging task in clinical practice. Therefore, investigating adjunctive therapies to accelerate the regeneration of such defects is crucial.Materials and methodsSixteen albino rats were divided into two groups (n = 8/group). To induce diabetes mellitus, a single streptozotocin dosage was injected. Critical-sized defects were created in the right posterior mandibles and filled with beta-tricalcium phosphate graft. The study group was subjected to 90-min sessions of hyperbaric oxygen at 2.4 ATA, for 5 consecutive days per week. Euthanasia was carried out after 3 weeks of therapy. Bone regeneration was examined histologically and histomorphometrically. Angiogenesis was assessed by immunohistochemistry against vascular endothelial progenitor cell marker (CD34) and the microvessel density was calculated.ResultsExposure of diabetic animals to hyperbaric oxygen resulted in superior bone regeneration and increased endothelial cell proliferation, which were revealed histologically and immunohistochemically, respectively. These results were confirmed by histomorphometric analysis which disclosed a higher percentage of new bone surface area and microvessel density in the study group.ConclusionsHyperbaric oxygen has a beneficial effect on bone regenerative capacity, qualitatively and quantitively, as well as the ability to stimulate angiogenesis.

Granulocyte-colony stimulating factor mediates neovascularization in acellular dermal matrix-transplanted areas by promoting endothelial progenitor cell homing.

Acellular dermal matrix (ADM) is an ideal material for tissue engineering skin construction. Accelerating the vascularization of ADM is of great significance for improving the survival of skin transplantation. The purpose of this study is to investigate the function of granulocyte-colony stimulating factor (G-CSF) in endothelial progenitor cells (EPCs)-mediated neovascularization in ADM-transplanted skin area. Male Kunming mice were subcutaneous injected with 10 μg/kg GCSF at 5 days before skin in situ replantation or porcine ADM transplantation. The surrounding tissues of implanted skin or venous blood was collected from the mice before the operation, and after the operation for 48 h, 72 h, 1 week, and 2 weeks, respectively. Cells co-expressing EPC markers, CD133, CD34, and Flk-1 were detected by flow cytometry. Immunohistochemistry of BrdU was performed to evaluate neovascularization in ADM-transplanted skin area. The results showed that G-CSF treatment increased the number of CD133+-CD34+ cells and CD133+-Flk-1+ cells in ADMimplanted area as well as the number of CD34+-Flk-1+ cells in peripheral blood. Likewise, G-CSF also increased the number of capillaries in ADM-transplanted areas. To sum up, G-CSF mobilizes EPC migration from bone marrow to peripheral blood and homing to wound sites, thus inducing neovascularization in ADM-transplanted areas.

Astragaloside IV - mediated endothelial progenitor cell exosomes promote autophagy and inhibit apoptosis in hyperglycemic damaged endothelial cells via miR-21/PTEN axis.

As one of the basic components of Astragalus, Astragaloside IV (AS-IV) has a protective effect on endothelial injury caused by diabetes. AS-IV stimulated endothelial progenitor cells (EPCs) to secrete exosomes loaded with miR-21. This study aimed to investigate the effects of AS-IV-mediated EPCs exosomal miR-21 (EPC-exos-miR-21) on high glucose (HG) damaged endothelial cells. After the isolation of EPCs derived from fetal umbilical cord blood, exosomes of EPCs were obtained by differential centrifugation. The morphology of exosomes was observed by electron microscopy. The particle size distribution of exosomes was detected by Nanoparticle Tracking Analysis. Human umbilical vein endothelial cells (HUVECs) were treated with 33 mM glucose to establish an HG injury model. Flow cytometry and TUNEL assay were used to characterize the surface markers of primary EPCs and the apoptosis of HUVECs. The gene and protein expression were detected by qPCR, immunofluorescence, and Western blotting. A dual luciferase assay was used to verify the targeting relationship of miR-21 with PTEN. HG environment led to time- and dose-dependent inhibition and enhancement of autophagy and apoptosis in HUVECs. AS-IV stimulated EPCs to secrete exosomes loaded with miR-21. Exosomes secreted by EPCs pretreated with AS-IV [EPC-exo(ASIV)] promoted autophagy and inhibited apoptosis in HG-impaired HUVECs. PTEN is a target of miR-21. MiR-21 carried by EPC-exo(ASIV) repressed PTEN expression in HG-impaired HUVECs. In contrast, p-AKT, p-mTOR, p-PI3K, cleaved PARP and PARP levels were upregulated. Compared to the HG group, the expression of autophagy regulatory genes (ATG5, beclin1 and LC3) was enhanced in the EPC-exo(ASIV) group and EPC-exo(ASIV)-miR-21 mimic group. In contrast, apoptosis-positive regulatory genes (Bax, caspase-3 and caspase-9) were attenuated. Further overexpression of PTEN reversed the expression of these genes. AS-IV-mediated EPC-exos-miR-21 could enhance autophagy and depress apoptosis in HG-damaged endothelial cells via the miR-21/PTEN axis.

Bone Marrow-Derived Vasculogenic Mesenchymal Stem Cells Enhance In Vitro Angiogenic Sprouting of Human Umbilical Vein Endothelial Cells

Vasculogenic properties of bone marrow-derived mesenchymal stem cells (MSCs) have been reported, but it is still unclear whether the vasculogenic properties are restricted to some populations of MSCs or whether the entire population of MSCs has these properties. We cultured two different populations of MSCs in different culture media and their vasculogenic properties were evaluated using In vitro spheroid sprouting assay. Neither population of MSCs expressed markers of endothelial progenitor cells (EPCs), but they were different in the profiling of angiogenic factor expression as well as vasculogenic properties. One population of MSCs expressed basic fibroblast growth factor (bFGF) and another expressed hepatocyte growth factor (HGF). MSCs expressing HGF exhibited In vitro angiogenic sprouting capacity in response to bFGF derived from other MSCs as well as to their autocrine HGF. The vasculogenic mesenchymal stem cells (vMSCs) derived from the bone marrow also enhanced In vitro angiogenic sprouting capacity of human umbilical vein endothelial cells (HUVECs) in an HGF-dependent manner. These results suggest that MSCs exhibit different vasculogenic properties, and vMSCs that are different from EPCs may contribute to neovascularization and could be a promising cellular therapy for cardiovascular diseases.

Targeted Nanoparticles for the Binding of Injured Vascular Endothelium after Percutaneous Coronary Intervention

Percutaneous coronary intervention (PCI) is a common procedure for the management of coronary artery obstruction. However, it usually causes vascular wall injury leading to restenosis that limits the long-term success of the PCI endeavor. The ultimate objective of this study was to develop the targeting nanoparticles (NPs) that were destined for the injured subendothelium and attract endothelial progenitor cells (EPCs) to the damaged location for endothelium regeneration. Biodegradable poly(lactic-co-glycolic acid) (PLGA) NPs were conjugated with double targeting moieties, which are glycoprotein Ib alpha chain (GPIbα) and human single-chain antibody variable fragment (HuscFv) specific to the cluster of differentiation 34 (CD34). GPIb is a platelet receptor that interacts with the von Willebrand factor (vWF), highly deposited on the damaged subendothelial surface, while CD34 is a surface marker of EPCs. A candidate anti-CD34 HuscFv was successfully constructed using a phage display biopanning technique. The HuscFv could be purified and showed binding affinity to the CD34-positive cells. The GPIb-conjugated NPs (GPIb-NPs) could target vWF and prevent platelet adherence to vWF in vitro. Furthermore, the HuscFv-conjugated NPs (HuscFv-NPs) could capture CD34-positive cells. The bispecific NPs have high potential to locate at the damaged subendothelial surface and capture EPCs for accelerating the vessel repair.

Reticulated Platelets and Their Relationship with Endothelial Progenitor Cells during the Acute Phase of ST-Elevation Myocardial Infarction.

Endothelial progenitor cells (EPC) and reticulated platelets (RP) have central roles in the thrombotic and angiogenetic interactions during ST-elevation myocardial infarction (STEMI). The EPC and RP response in patients with STEMI treated by primary percutaneous intervention (PPCI) has not yet been investigated. We assessed EPC quantification by the expression of CD133+ and CD34+, and EPC function by the capacity of the cells to form colony-forming units (CFU) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) during the acute phase of STEMI. These measurements were correlated with RP at baseline and after 24 h following PPCI. Our cohort included 89 consecutive STEMI-diagnosed patients enrolled between December 2018 and July 2021. At baseline, there was a strong positive correlation between reticulated platelet quantity and MTT levels (R = 0.766 and R2 = 0.586, p < 0.001), CD34+ levels (R = 0.602, and R2 = 0.362, p < 0.001); CD133+ levels (R = 0.666 and R2 = 0.443, p < 0.001) and CFU levels (R = 0.437, R2 = 0.191, p < 0.001). The multiple linear regression showed that levels of MTT (adjusted R2 = 0.793; p < 0.001), CD34+ and CD133+ (adjusted R2 = 0.654; p < 0.001 and adjusted R2 = 0.627; p < 0.001, respectively) had strong independent correlations with RP response. At 24 h after PPCI, the correlation between RP quantity and EPC markers was not significant, except for MTT levels (R = 0.465, R2 = 0.216, p < 0.001). In patients with STEMI, higher levels of RP at baseline are significantly correlated with a more potent EPC response. The translational significance of these findings needs further investigation.

Characterization of Endothelial Progenitor Cell: Past, Present, and Future.

Endothelial progenitor cells (EPCs) are currently being studied as candidate cell sources for revascularization strategies. Despite these promising results, widespread clinical acceptance of EPCs for clinical therapies remains hampered by several challenges. The challenges and issues surrounding the use of EPCs and the current paradigm being developed to improve the harvest efficiency and functionality of EPCs for application in regenerative medicine are discussed. It has been observed that controversies have emerged regarding the isolation techniques and classification and origin of EPCs. This manuscript attempts to highlight the concept of EPCs in a sequential manner, from the initial discovery to the present (origin, sources of EPCs, isolation, and identification techniques). Human and murine EPC marker diversity is also discussed. Additionally, this manuscript is aimed at summarizing our current and future prospects regarding the crosstalk of EPCs with the biology of hematopoietic cells and culture techniques in the context of regeneration-associated cells (RACs).

CRISPR/Cas9-engineered human ES cells harboring heterozygous and homozygous c-KIT knockout

The receptor tyrosine kinase c-KIT (CD117) has a key role in hematopoiesis and is a marker for endothelial and cardiac progenitor cells. In vivo, deficiency of c-KIT is lethal and therefore using CRISPR/Cas9 editing we generated heterozygous and homozygous c-KIT knockout human embryonic stem cell (ES cell) lines. The c-KIT knockout left ES cell pluripotency unaffected as shown by immunofluorescence and trilineage differentiation potential. Heterozygous and homozygous c-KIT knockouts showed complete loss of exon 17, resulting in ablation of c-KIT protein from the cell surface. c-KIT knockout ES cells provide a valuable tool for further investigating c-KIT biology.

Endothelial Progenitor Cells and NADPH Oxidase Enzyme Activity in the Development of an Aortic Aneurysm.

IntroductionEndothelial progenitor cells (EPCs) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme activity may affect the vessel wall and have a role in development of aortic aneurysms. EPCs originate from hematopoietic stem cells and can be enumerated from peripheral blood samples by flow cytometry. In this study, we aimed to evaluate the relation of EPC number and NADPH oxidase enzyme activity in the development of thoracic aortic aneurysm (TAA).MethodsPatients with TAA (n=30) and healthy individuals without TAA (control, n=10) were included in our study. Characterization and enumeration of EPC from peripheral blood samples were performed by flow cytometry with panels including markers of EPCs (CD34/CD133/CD309/CD146/CD144). Additionally, NADPH oxidase enzyme activity (capacity) was also measured by the dihydrorhodamine 123 (DHR 123) test.ResultsThe enumeration of EPC with CD34+/CD146+ marker showed that the number of mean EPC/106 cells was increased in the patient group (41.5/106 cells), but not in the control group (20.50/105 cells) (P<0.01). Additionally, patients with TAA presented significantly lower NADPH oxidase activity by DHR assay than healthy controls (mean stimulation index: 60.40± 7.86 and 75.10±5.21, respectively) (P<0.01).ConclusionOur results showed that the number of EPCs is significantly higher in aortic aneurysm patients and may have a role in disease progression. The crosstalk between NADPH oxidase enzyme capacity and EPC number may be useful as a parameter to explain the clinical progression of TAA.

Endothelial progenitor cells and coronary artery disease: Current concepts and future research directions.

Vascular injury is a frequent pathology in coronary artery disease. To repair the vasculature, scientists have found that endothelial progenitor cells (EPCs) have excellent properties associated with angiogenesis. Over time, research on EPCs has made encouraging progress regardless of pathology or clinical technology. This review focuses on the origins and cell markers of EPCs, and the connection between EPCs and coronary artery disease. In addition, we summarized various studies of EPC-capturing stents and EPC infusion therapy, and aim to learn from past technology to predict the future.

BMP-7 Attenuates Inflammation-Induced Pyroptosis and Improves Cardiac Repair in Diabetic Cardiomyopathy.

In the present study, we investigated a novel signaling target in diabetic cardiomyopathy where inflammation induces caspase-1-dependent cell death, pyroptosis, involving Nek7-GBP5 activators to activate the NLRP3 inflammasome, destabilizes cardiac structure and neovascularization. Furthermore, we explored the therapeutic ability of bone morphogenetic protein-7 (BMP-7) to attenuate these adverse effects. C57BL/6J mice (n = 16 mice/group) were divided into: control (200 mg/kg, 0.9% saline intraperitoneal injection, i.p.); Streptozotocin (STZ) and STZ-BMP-7 groups (STZ, 200 mg/kg, i.p. injection). After 6 weeks, heart function was examined with echocardiography, and mice were sacrificed. Immunostaining, Western blotting, H&E, and Masson’s trichrome staining was performed on heart tissues. STZ-induced diabetic cardiomyopathy significantly increased inflammasome formation (TLR4, NLRP3, Nek7, and GBP5), pyroptosis markers (caspase-1, IL-1β, and IL-18), inflammatory cytokines (IL-6 and TNF-α), MMP9, and infiltration of monocytes (CD14), macrophage (iNOS), and dendritic cells (CD11b and CD11c) (p < 0.05). Moreover, a significant endothelial progenitor cells (EPCs) dysfunction (c-Kit/FLk-1, CD31), adverse cardiac remodeling, and reduction in left ventricular (LV) heart function were observed in STZ versus control (p < 0.05). Treatment with BMP-7 significantly reduced inflammasome formation, pyroptosis, and inflammatory cytokines and infiltrated inflammatory cells. In addition, BMP-7 treatment enhanced EPC markers and neovascularization and subsequently improved cardiac remodeling in a diabetic heart. Moreover, a significant improvement in LV heart function was achieved after BMP-7 administration relative to diabetic mice (p < 0.05). In conclusion, BMP-7 attenuated inflammation-induced pyroptosis, adverse cardiac remodeling, and improved heart function via the TLR4-NLRP3 inflammasome complex activated by novel signaling Nek7/GBP5. Our BMP-7 pre-clinical studies of mice could have significant potential as a future therapy for diabetic patients.

Endothelial GABBR2 Regulates Post-ischemic Angiogenesis by Inhibiting the Glycolysis Pathway.

Purpose: Angiogenesis post-ischemia plays an essential role in preventing ischemic damage to tissue by improving the blood recovery. Determining the regulatory mechanism of ischemic angiogenesis, therefore, could provide effective therapeutics for ischemic injury.Materials and Methods: The RNA sequencing (RNA-seq) database was used to predict the association of gamma-aminobutyric acid type B receptor subunit 2 (GABBR2) with endothelial-specific expression. The role of GABBR2 in angiogenesis was verified in vitro by downregulating GABBR2 in human umbilical vein endothelial cells (HUVECs) with lentiviral vectors. Besides, the in vivo effect of GABBR2 on the blood recovery of an ischemic hindlimb was demonstrated by establishing a hindlimb ischemia model in normal and GABBR2 adenoviral vector-infected mice. Then, the mobilization of endothelial progenitor cells (EPCs) in peripheral blood post-ischemia was determined by flow cytometry. Finally, the XF analyzer and Western blot were used to determine the effect of GABBR2 on endothelial metabolism.Results: The RNA-seq results indicated a strong association between GABBR2 and endothelial revascularization, and the upregulation of GABBR2 was detected in both hypoxia-treated HUVECs and ischemic mouse hindlimb. Hypoxia treatment for 6 h increased the proliferation, migration, and tube formation of HUVECs, which were inhibited by GABBR2 knockdown. Additionally, GABBR2 downregulation significantly decreased the blood flow recovery of mouse ischemic hindlimb. The expressions of the EPC markers CD34+ and CD133+ significantly decreased in the peripheral blood in hindlimb post-ischemia. Mechanically, glycolysis-dominated metabolism of HUVECs was compromised by GABBR2 knockdown. Evidences of the decreased expressions of HKII, PFKFB3, and PKM1 also supported the compromised glycolysis induced by GABBR2 downregulation.Conclusion: Our study demonstrated that GABBR2 regulated angiogenesis post-ischemia by inhibiting the glycolysis pathway.

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.

New endothelial markers: implications for coronary artery disease and chronic periodontitis

Abstract Funding Acknowledgements Type of funding sources: None. Introduction There is an association between chronic periodontitis (CP) and coronary artery disease (CAD). Endothelial function is increasingly used as an important outcome measure in cardiovascular diseases. But the data on early markers endothelial dysfunction in patients with CP is limited. Aim. To determine whether there are differences in parameters of tissue endothelial markers in gingiva between patients with CP, CAD or CAD and concomitant CP. Methods We examined 30 patients with CAD, 75 patients with CP and 35 patients with CAD and concomitant CP and 25 healthy control. There were no differences between the groups of patients with CP, CAD and co-morbidity in age, gender, smoking and obesity. In marginal gingiva NO-synthase-like cells (NOs-cells) and marker of endothelial progenitor cells (CD34+ cells) by immunohistochemical and morphometric analyses were assessed. Endothelium-dependent vasodilation (EDV) of the brachial artery was evaluated. Results Immunoreactivity to NOs and the CD34+ cells was found mainly in the vascular. In patients with average and severe CP significant decrease of expression of NOs–cells (6.2 ± 2.4% vs 7.9 ± 1.9%, p &amp;lt; 0.05) and of CD34+ -cells (6.3%±1.8 vs 8.9 ± 2.6%, p &amp;lt; 0.05) in gingiva was detected. There were no differences of endothelial immunohistochemical markers between the groups of patients with mild CP and healthy person. In patients with CAD also significant decrease of expression of NOs–cells (5.0 ± 2.1%) and of CD34+ -cells (4.9%±2.0%) in gingiva was found. The most significance changes of gingival expression of NOs–cells (4.3 ± 1.4%) and of CD34+ -cells (3.8%±1.7%) in patients with CAD and concomitant CP were observed. In the control group EDV 15.4 ± 6.5%, in patients with mild CP 13.9 ± 6.5, in patients with average and severe CP 10,9 ± 5,1% (p = 0.02 between healthy person). There were associations between the decrease of EDV of the brachial artery with gingival expression of NOs–cells and of CD34+ -cells (Spearman"s R = 0.575 and R = 0.578). Conclusion Average and severe periodontitis is associated with vascular effects outside the oral cavity. Periodontitis also as CAD are associated with changes in the cell expression of NOs and endothelial progenitor cells in the periodontal tissue. Immunohistochemical assessment of endothelial markers in periodontium is a promising method of early assessment of endothelial dysfunction in cardiovascular disease.

Circulating endothelial progenitor cells from septic patients are associated with different infectious organisms.

In sepsis, endothelial progenitor cells (EPCs) play a central role in the repair of endothelial injury by enhancing the processes of re-endothelialization and angiogenesis. However, the surface markers of EPCs have yet to be standardized, and Changes of EPCs in quantities and functions with different infectious organisms are still unclear. This study explored the relationship between the percentages of EPCs and various infectious organisms in patients with sepsis. Thirty-nine septic patients and 20 healthy controls were enrolled in this study. The percentages of CD34+/KDR+, CD133+/KDR+, CD34+/CD133+/KDR+, CD34+, CD133+, and KDR+ cells in different groups of septic patients and the healthy controls were analyzed by flow cytometry. The peripheral blood of septic patients had higher percentages of EPCs than that of the healthy controls. There were no significant differences in the percentages of EPCs between the sepsis and septic shock groups, nor between the survival group and the non-survival group. Additionally, the percentages of CD34+/CD133+/KDR+ cells in the gram-positive bacteremia group were significantly higher than those in the gram-negative bacteremia group and the negative blood culture group. The percentage of KDR+ cells in both the gram-positive bacteremia group and the gram-negative bacteremia group was significantly higher than that in the negative blood culture group. The percentages of circulating EPCs in patients with sepsis are associated with different infectious organisms.

Vagal \u03b17nAChR signaling regulates \u03b17nAChR+Sca1+ cells during lung injury repair

BackgroundThe distal airways of the lung and bone marrow are innervated by the vagus nerve. Vagal α7nAChR signaling plays a key role in regulating lung infection and inflammation; however, whether this pathway regulates α7nAChR+Sca1+ cells during lung injury repair remains unknown. We hypothesized that vagal α7nAChR signaling controls α7nAChR+Sca1+ cells, which contribute to the resolution of lung injury.MethodsPneumonia was induced by intratracheal challenge with E. coli. The bone marrow mononuclear cells (BM-MNCs) were isolated from the bone marrow of pneumonia mice for immunofluorescence. The bone marrow, blood, BAL, and lung cells were isolated for flow cytometric analysis by labeling with anti-Sca1, VE-cadherin, p-Akt1, or Flk1 antibodies. Immunofluorescence was also used to examine the coexpression of α7nAChR, VE-cadherin, and p-Akt1. Sham, vagotomized, α7nAChR knockout, and Akt1 knockout mice were infected with E. coli to study the regulatory role of vagal α7nAChR signaling and Akt1 in Sca1+ cells.ResultsDuring pneumonia, BM-MNCs were enriched with α7nAChR+Sca1+ cells, and this cell population proliferated. Transplantation of pneumonia BM-MNCs could mitigate lung injury and increase engraftment in recipient pneumonia lungs. Activation of α7nAChR by its agonist could boost α7nAChR+Sca1+ cells in the bone marrow, peripheral blood, and bronchoalveolar lavage (BAL) in pneumonia. Immunofluorescence revealed that α7nAChR, VE-cadherin, and p-Akt1 were coexpressed in the bone marrow cells. Vagotomy could reduce α7nAChR+VE-cadherin+ and VE-cadherin+p-Akt1+ cells in the bone marrow in pneumonia. Knockout of α7nAChR reduced VE-cadherin+ cells and p-Akt1+ cells in the bone marrow. Deletion of Akt1 reduced Sca1+ cells in the bone marrow and BAL. More importantly, 91.3 ± 4.9% bone marrow and 77.8 ± 4.9% lung α7nAChR+Sca1+VE-cadherin+ cells expressed Flk1, which is a key marker of endothelial progenitor cells (EPCs). Vagotomy reduced α7nAChR+Sca1+VE-cadherin+p-Akt1+ cells in the bone marrow and lung from pneumonia mice. Treatment with cultured EPCs reduced ELW compared to PBS treatment in E. coli pneumonia mice at 48 h. The ELW was further reduced by treatment with EPCs combining with α7nAChR agonist-PHA568487 compared to EPC treatments only.ConclusionsVagal α7nAChR signaling regulates α7nAChR+Sca1+VE-cadherin+ EPCs via phosphorylation of Akt1 during lung injury repair in pneumonia.