scholarly journals Extracorporeal Cardiac Shock Waves Therapy Improves the Function of Endothelial Progenitor Cells After Hypoxia Injury via Activating PI3K/Akt/eNOS Signal Pathway

2021 ◽  
Vol 8 ◽  
Author(s):  
Mingqiang Wang ◽  
Dan Yang ◽  
Zhao Hu ◽  
Yunke Shi ◽  
Yiming Ma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Shock Waves ◽  
Protein Expression ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Differentially Expressed Genes ◽  
Akt Signaling ◽  
Tube Formation ◽  
Cardiac Shock

Background: Extracorporeal cardiac shock waves (ECSW) have great potential in the treatment of coronary heart disease. Endothelial progenitor cells (EPCs) are a class of pluripotent progenitor cells derived from bone marrow or peripheral blood, which have the capacity to migrate to ischemic myocardium and differentiate into mature endothelial cells and play an important role in neovascularization and endothelial repair. In this study, we investigated whether ECSW therapy can improve EPCs dysfunction and apoptosis induced by hypoxia and explored the underlying mechanisms.Methods: EPCs were separated from ApoE gene knockout rat bone marrow and identified using flow cytometry and fluorescence staining. EPCs were used to produce in vitro hypoxia-injury models which were then divided into six groups: Control, Hypoxia, Hypoxia + ECSW, Hypoxia + LY294002 + ECSW, Hypoxia + MK-2206 + ECSW, and Hypoxia + L-NAME + ECSW. EPCs from the Control, Hypoxia, and Hypoxia + ECSW groups were used in mRNA sequencing reactions. mRNA and protein expression levels were analyzed using qRT-PCR and western blot analysis, respectively. Proliferation, apoptosis, adhesion, migration, and angiogenesis were measured using CCK-8, flow cytometry, gelatin, transwell, and tube formation, respectively. Nitric oxide (NO) levels were measured using an NO assay kit.Results: Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that differentially expressed genes were enriched in cancer signaling, PI3K-Akt signaling, and Rap1 signaling pathways. We selected differentially expressed genes in the PI3K-Akt signaling pathway and verified them using a series of experiments. The results showed that ECSW therapy (500 shots at 0.09 mJ/mm2) significantly improved proliferation, adhesion, migration, and tube formation abilities of EPCs following hypoxic injury, accompanied by upregulation of p-PI3K, p-Akt, p-eNOS, Bcl-2 protein and NO, PI3K, and Akt mRNA expression, and downregulation of Bax and Caspase3 protein expression. All these effects of ECSW were eliminated using inhibitors specific to PI3K (LY294002), Akt (MK-2206), and eNOS (L-NAME).Conclusion: ECSW exerted a strong repaired effect on EPCs suffering inhibited hypoxia injury by inhibiting cell apoptosis and promoting angiogenesis, mainly through activating the PI3K/Akt/eNOS signaling pathway, which provide new evidence for ECSW therapy in CHD.

scholarly journals Vascular endothelial growth factor-A signaling in bone marrow-derived endothelial progenitor cells exposed to hypoxic stress

2013 ◽  
Vol 45 (21) ◽  
pp. 1021-1034 ◽  
Author(s):  
Brian R. Hoffmann ◽  
Jordan R. Wagner ◽  
Anthony R. Prisco ◽  
Agnieszka Janiak ◽  
Andrew S. Greene
Keyword(s):  
Bone Marrow ◽  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Signaling Pathways ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Tube Formation ◽  
Vascular Endothelial ◽  
Endothelial Progenitor ◽  
Endothelial Growth Factor

Bone marrow-derived endothelial progenitor cells (BM-EPCs) are stimulated by vascular endothelial growth factor-A (VEGF-A) and other potent proangiogenic factors. During angiogenesis, an increase in VEGF-A expression stimulates BM-EPCs to enhance endothelial tube formation and contribute to an increase in microvessel density. Hypoxia is known to produce an enhanced angiogenic response and heightened levels of VEGF-A have been seen in oxygen deprived epithelial and endothelial cells, yet the pathways for VEGF-A signaling in BM-EPCs have not been described. This study explores the influence of hypoxia on VEGF-A signaling in rat BM-EPCs utilizing a novel proteomic strategy to directly identify interacting downstream components of the combined VEGF receptor(s) signaling pathways, gene expression analysis, and functional phenotyping. VEGF-A signaling network analysis following liquid chromatographic separation and tandem mass spectrometry revealed proteins related to inositol/calcium signaling, nitric oxide signaling, cell survival, cell migration, and inflammatory responses. Alterations in BM-EPC expression of common angiogenic genes and tube formation in response to VEGF-A during hypoxia were measured and combined with the proteomic analysis to enhance and support the signaling pathways detected. BM-EPC tube formation assays in response to VEGF-A exhibited little tube formation; however, a cell projection/migratory phenotype supported the signaling data. Additionally, a novel assay measuring BM-EPC incorporation into preformed endothelial cell tubes indicated a significant increase of incorporated BM-EPCs after pretreatment with VEGF-A during hypoxia. This study verifies known VEGF-A pathway components and reveals several unidentified mechanisms of VEGF-A signaling in BM-EPCs during hypoxia that may be important for migration to sites of vascular regeneration.

Transplantation of Ex Vivo Expanded Bone Marrow-Derived Endothelial Progenitor Cells Enhances Chronic Venous Thrombus Resolution and Recanalization

2011 ◽  
Vol 17 (6) ◽  
pp. E196-E201 ◽  
Author(s):  
Qingyou Meng ◽  
Xiaoqiang Li ◽  
Xiaobin Yu ◽  
Fengrui Lei ◽  
Kun Jiang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Protein Expression ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Vena Cava ◽  
Endothelial Progenitor ◽  
Venous Thrombus ◽  
Mrna And Protein Expression

Background: Preclinical and clinical studies indicated that endothelial progenitor cells (EPCs) enhanced blood vessel formation in many clinical situations. However, whether transplantation of EPCs would enhance chronic venous thrombus recanalization and resolution is unknown. Methods: Mononuclear cells were isolated from bone marrow of immature rats by density gradient centrifugation, cultured, and then transplanted into inferior vena cava of rats with experimentally induced thrombi. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), messenger RNA (mRNA), and protein expression levels were measured through real-time quantitative polymerase chain reaction and Western blotting of thrombi and adjacent caval walls 14 days following transplantation. Results: Transplantation of bone marrow-derived EPCs led to an increase in VEGF, bFGF, mRNA, and protein expression. In addition, transplantation of bone marrow-derived EPCs also resulted in reduced thrombus size and increased neovascularization in the specimen. Conclusions: Transplanted bone marrow-derived EPCs may be a therapeutic option for treating deep venous thrombosis.

scholarly journals Establishment of Tube Formation Assay of Bone Marrow-Derived Endothelial Progenitor Cells

2013 ◽  
Vol 19 (7) ◽  
pp. 533-535 ◽  
Author(s):  
Ji-Kuai Chen ◽  
Ya-Ping Deng ◽  
Guo-Jun Jiang ◽  
Yun-Zi Liu ◽  
Ting Zhao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Tube Formation ◽  
Endothelial Progenitor ◽  
Tube Formation Assay

scholarly journals Rosuvastatin for enhancement of aneurysm neck endothelialization after coil embolization: promotion of endothelial progenitor cells in a rodent model

2016 ◽  
Vol 124 (5) ◽  
pp. 1265-1274 ◽  
Author(s):  
Peixi Liu ◽  
Qingzhu An ◽  
Xi Chen ◽  
Jun Huang ◽  
Guo-Yuan Yang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Coil Embolization ◽  
Tube Formation ◽  
Invasive Technique ◽  
Sprague Dawley ◽  
Endothelial Progenitor ◽  
Risk Of Recurrence ◽  
Aneurysm Neck

OBJECT Coil embolization is a safe, efficient, and minimally invasive technique for the treatment of intracranial aneurysms. However, coil embolization is associated with a higher risk of recurrence than clip ligation. In this study, the authors explore a new approach through the promotion of endothelial progenitor cells (EPCs) to optimize endothelialization of the aneurysm neck and reduce the risk of recurrence. METHODS A coiled aneurysm model was created in 48 adult male Sprague-Dawley rats via microsurgery. Half of these animals were treated with rosuvastatin (20 mg/kg) in saline via gavage for 10, 20, or 30 days. The other half were administered saline without rosuvastatin. An additional 15 rats underwent “mock surgery” (identical anesthesia and saline gavage but no surgery). The endothelial repair process in the coiled aneurysms was evaluated via flow cytometry, im-munostaining, and electronic microscopy. The mock surgery group was used for comparison in flow cytometry studies. The effects of rosuvastatin on viability and functioning of Sprague-Dawley rat bone marrow-derived EPCs were also explored via MTT, migration, and tube formation assays. RESULTS The aneurysm neck repair score was significantly higher in the rosuvastatin-treated rats than in the untreated rats (p < 0.05). The circulating EPC count was increased and maintained at a higher level in rosuvastatin-treated rats compared with the aneurysm rats that did not receive rosuvastatin (p < 0.05). Immunostaining showed that the aneurysm neck endothelium was more integrated and the number of kinase insert domain receptor-positive cells was increased in the rosuvastatin-treated rats. Further study demonstrated that rosuvastatin promoted EPC proliferation, migration, and tube formation. CONCLUSIONS Rosuvastatin promoted endothelialization of the coiled aneurysm neck via induction of EPCs, suggesting that promoting endothelialization provides an additional therapeutic opportunity during vascular endothelium repair.

Gleevec and Rapamycin Synergistically Reduce Cell Viability and Inhibit Proliferation and Angiogenic Function of Mouse Bone Marrow-Derived Endothelial Progenitor Cells

2021 ◽  
pp. 1-12
Author(s):  
Ling Chen ◽  
Luping Dai ◽  
Dewen Yan ◽  
Boya Zhou ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Cell Apoptosis ◽  
Tube Formation ◽  
Endothelial Differentiation ◽  
Mouse Bone Marrow ◽  
Endothelial Progenitor

<b><i>Objective:</i></b> This study investigates the synergistic effects of Gleevec (imatinib) and rapamycin on the proliferative and angiogenic properties of mouse bone marrow-derived endothelial progenitor cells (EPCs). <b><i>Materials and Methods:</i></b> EPCs were isolated from mouse bone marrow and treated with different concentrations of Gleevec or rapamycin individually or in combination. The cell viability and proliferation were examined using the MTT assay. An analysis of cell cycle and apoptosis was performed using flow cytometry. Formation of capillary-like tubes was examined in vitro, and the protein expression of cell differentiation markers was determined using Western blot analysis. <b><i>Results:</i></b> Gleevec significantly reduced cell viability, cell proliferation, and induced cell apoptosis in EPCs. Rapamycin had similar effects on EPCs, but it did not induce cell apoptosis. The combination of Gleevec and rapamycin reduced the cell proliferation but increased cell apoptosis. Although rapamycin had no demonstratable effect on tube formation, the combined therapy of Gleevec and rapamycin significantly reduced tube formation when compared with Gleevec alone. Mechanistically, Gleevec, but not rapamycin, induced a significant elevation in caspase-3 activity in EPCs, and it attenuated the expression of the endothelial protein marker platelet-derived growth factor receptor α. Functionally, rapamycin, but not Gleevec, significantly enhanced the expression of endothelial differentiation marker proteins, while attenuating the expression of mammalian target of rapamycin signaling-related proteins. <b><i>Conclusions:</i></b> Gleevec and rapamycin synergistically suppress cell proliferation and tube formation of EPCs by inducing cell apoptosis and endothelial differentiation. Mechanistically, it is likely that rapamycin enhances the proapoptotic and antiangiogenic effects of Gleevec by promoting the endothelial differentiation of EPCs. Given that EPCs are involved in the pathogenesis of some cardiovascular diseases and critical to angiogenesis, pharmacological inhibition of EPC proliferation by combined Gleevec and rapamycin therapy may be a promising approach for suppressing cardiovascular disease pathologies associated with angiogenesis.

Lower Levels of Circulating Endothelial Progenitor Cells Are Associated with Increased Recurrence after Unprovoked Venous Thrombosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3805-3805
Author(s):  
Charlotte Ann Bradbury ◽  
Tracey Buckley ◽  
Kate Fletcher ◽  
Peter Rose ◽  
David Fitzmaurice
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Vascular Injury ◽  
Recurrence Risk ◽  
Endothelial Progenitor ◽  
Blood Samples ◽  
D Dimer

Abstract Introduction: Endothelial progenitor cells (EPCs) are multipotent progenitor cells mainly residing in human bone marrow. In response to stimuli such as arterial ischaemia or venous thrombosis, EPCs are mobilised from the bone marrow, released into the circulation and home to sites of vascular injury where they promote endothelial regeneration, revascularization, vasoactive and angiogenic factor secretion, proteinase generation and vein wall remodelling. There is significant research interest into the potential therapeutic role of EPCs in vascular injury. However, to our knowledge there is no published data on the measurement of EPCs in a larger population of patients with comparison to thrombotic outcomes and other biomarkers known to be predictive of recurrence risk for example, D Dimer. Methods: The EXACT study is a randomised controlled trial of extended warfarin treatment versus observation only for the prevention of recurrent venous thromboembolism (VTE) and post thrombotic syndrome (PTS) in patients with an unprovoked VTE. Patients recruited to this study had blood samples taken 3 months post VTE event whilst still on warfarin prior to stopping or continuing anticoagulation according to their randomisation group. All patients were clinically followed up for 2 years following randomisation and recurrent thrombotic events (confirmed by imaging) were recorded. The blood samples were processed for Full Blood Count, INR, D Dimers and quantitation of Endothelial Cells by flow cytometry (circulating mature and progenitor). Endothelial cells were identified by low side scatter, weak CD45 (unlike haematopoetic cells) and expression of endothelial markers (CD146, VEGFR, VEGFR-2). Endothelial progenitor cells also demonstrated at least one marker of immaturity (CD34 and CD133). Apoptotic or dead cells were detected and excluded using 7-amino-actinomycin D staining. The number of EPC and circulating endothelial cell (CEC) events was divided by the number of CD45 positive events and multiplied by the total white count, which will give a value x103/microlitre. Results: At 3 months of treatment with warfarin, in 193 patients, samples of sufficient quality were processed by flow cytometry for EPC quantitation. In 182 of these patients, a sufficiently full citrated sample was also available for D Dimer quantitation. High D Dimer levels were associated with low EPC levels and visa versa (fig 1). Out of the 193 patients, during 2 year follow up, 19 patients (10%) went on to have a recurrent VTE. D Dimers at 3 months treatment were not significantly different in patients who had a recurrence versus those who didn't (Figure 2). There was also no significant difference in circulating mature endothelial cells (CEC) and total WCC (table 2). However, EPC levels were significantly lower in patients who went on to develop a thrombosis vs those who didn't (p<0.05) figure 3. Discussion: According to current guidelines, patients with unprovoked VTE are considered for long term anticoagulation following a discussion with the patient regarding VTE recurrence risk off treatment vs bleeding risk on treatment. D Dimer results can be helpful to refine the estimated recurrence risk (e.g. using the DASH2 score) but testing is only validated off anticoagulation (for at least 7 days) which can be cumbersome to organise. This study was unable to support the predictive value of D Dimers taken from patients on warfarin. If EPC quantitation from blood samples of patients is confirmed to be predictive of VTE recurrence this could represent a new informative biomarker to aid decision making without the need to interrupt anticoagulation therapy. It also has the potential to provide complimentary information. For example, in defining a truly "low risk" group in those with negative D Dimers after completion of treatment. Further larger studies are needed to confirm the predictive value of EPC quantitation for VTE recurrence in patient groups continuing or discontinuing anticoagulation (this study included both groups) and to confirm whether the same results apply to patients on DOACs. Disclosures No relevant conflicts of interest to declare.

Abstract P375: Gender-based Cardio-protective Functional Dimorphism Of Bone Marrow Endothelial Progenitor Cells And Their Exosomes: Estrogen-independent Epigenetic Mechanisms

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Charan T Gurrala ◽  
Venkata Garikipati ◽  
Zhongjian Cheng ◽  
Vandana Mallaredy ◽  
Maria Cimini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Capillary Density ◽  
Tube Formation ◽  
Ischemic Myocardium ◽  
Epigenetic Mechanism ◽  
Epigenetic Mechanisms ◽  
Endothelial Progenitor

Introduction: Estrogen or estrogen receptor-dependent mechanisms in enhancing the cardioprotective efficacy of bone marrow endothelial progenitor cells (BM-EPC) is well-established in preclinical studies. However, the efficacy of estrogen does not reflect in the data from randomized cardiovascular clinical trials, suggesting an estrogen-independent role of female BM-EPC in eliciting enhanced cardiac protection compared to males. Hypothesis: Epigenetic mechanisms may contribute to the sex-specific dimorphism of Sca-1 + /CD31 + BM-EPC in regulating cell-homing, pro-angiogenic and anti-inflammatory functions in the ischemic myocardium leading to enhanced reparative function of female progenitor cells. Methods & Results: Transplantation of GFP-BM-mononuclear cells from male and female GFP transgenic mice into the BM of lethally irradiated recipient male C57BL/6 mice resulted in the enhanced mobilization of female Sca-1 + CD31 + /GFP + BM-EPC into circulation post-MI. A higher number of female BM-EPC homed to the ischemic myocardium and significantly improved LV functions and capillary density post-MI compared to male BM-EPC. Female BM-EPC showed increased expression of bFGF, VEGFR2, SDF-1α, and IL-10 genes, thereby efficiently promoted endothelial tube formation in vitro compared to male BM-EPC. Transplantation of female BM-EPC and their exosomes into post-MI male mice improved LV cardiac function, reduced scar size, and improved capillary density compared to male BM-EPC and exosomes. Male BM-EPC showed an increased expression of G9a/Ehmt2, an H3K9me3 methyltransferase, and Dnmt3a DNA methyltransferase compared to female BM-EPC. In contrast, Kdm6b/JMJD3, H3K27me3 demethylase was highly expressed in female BM-EPC compared to males. Treatment of BM-EPC of both sexes with 17-β-estradiol did not alter the expression of Kdm6b/JMJD3. Male BM-EPC highly expressed repressive gene marks, H3K9me3, and H3K27me3 compared to females. Compared to the male, BM-EPC from female and ovariectomized (OXV) female mice showed equally high expression of angiogenic genes ANGPT-1, MDK, PLAU, Tie-2, and VEGFR2 and lower levels of inflammatory cytokines, TNFα, IFNγ, IL-1β, and CCL3. Conditioned medium from female and OVX BM-EPC equally promoted enhanced migration and tube formation of HUVEC in vitro, compared to male BM-EPC. Conclusions: An estrogen-independent epigenetic mechanism likely governs the enhanced cardiac reparative properties of female BM-EPC.

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