Isolation and characterization of endothelial progenitor cells from canine bone marrow

Chen, Chen; Dai, Pengxiu; Nan, Liangliang; Lu, Ruiqing; Wang, Xiuyuan; Tian, Yuanyuan; Zhang, Xinke; Gao, Yongping; Shi, Zheng; Zhang, Yihua

doi:10.1080/10520295.2020.1762001

Cited by 11 publications

(7 citation statements)

References 48 publications

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“…Phenotypic characterization of EPCs remains controversial 33 . However, CD31, CD34, VEGF-R2, and Scal-1 are widely accepted markers of EPCs 34 . CD117 was previously thought to be a marker of EPCs 35 .…”

Section: Discussionmentioning

confidence: 99%

Endothelial Progenitor Cell-Mediated Angiogenesis is Promoted by the Transforming Growth Factor Beta 1/CXC Chemokine Receptor 7 Signaling Axis in Diabetes Mellitus

Huang

Chen

et al. 2022

Preprint

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Background The role of the transforming growth factor (TGF)-β1/CXC chemokine receptor (CXCR) 7 signaling axis in angiogenesis mediated by endothelial progenitor cells (EPCs) in diabetes mellitus (DM) remains unclear. Methods An in vivo wound healing model was established in mice, which were treated with phosphate-buffered saline (PBS) or TGF-β1. Wound closure rate was measured to evaluate wound healing. Histological analysis and immunofluorescent staining were performed to investigate angiogenesis and the number of EPCs in vivo. EPCs were isolated from bone marrow via gradient centrifugation. An angiogenic medium was used to induce endothelial cells (ECs) from EPCs. Flow cytometry was used to identify EPCs and ECs. Lv-shCXCR7 was used to determine the role of CXCR7 in EPCs in vitro. EPC proliferation was measured via 5-ethynyl-2′-deoxyuridine staining. Scratch and transwell assays were performed to assess EPC migration. Results Remaining wound area from day 7 to 14 in the DM + TGF-β1 group was significantly lower than that in the DM + PBS group but higher than that in the PBS group. On day 14, the number of microvessels in wounds was significantly higher for TGF-β1-treated mice than for mice not treated with TGF-β1. The number of EPCs in the DM + TGF-β1 group was significantly higher than that in the DM + PBS group. The high-glucose environment significantly impaired the proliferation, migration, and tube-forming ability of EPCs; however, TGF-β1 partially restored the proliferation and tube-forming ability of EPCs (p < 0.05). Additionally, shCXCR7 lentiviral transfection resulted in a significant reduction in the proliferation and tube-forming ability of EPCs; however, it had no significant effect on EPC migration. Furthermore, the high-glucose environment caused a decrease in the mRNA and protein expression of CXCR7 in EPCs; however, this was partially reversed by TGF-β1 (p < 0.05). Conclusions Our results show for the first time that TGF-β1 partially restores the proliferation and tube-forming ability of EPCs in a high-glucose environment by upregulating CXCR7 expression, increasing the number of blood vessels, and accelerating wound healing. This novel TGF-β1 pathway may play a potentially therapeutic role from bench to bedside.

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Section: Discussionmentioning

confidence: 99%

Endothelial Progenitor Cell-Mediated Angiogenesis is Promoted by the Transforming Growth Factor Beta 1/CXC Chemokine Receptor 7 Signaling Axis in Diabetes Mellitus

Huang

Chen

et al. 2022

Preprint

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“…Notably, the expression levels of CD31 and CD133 in EPC's cellular markers varied at different stages. In this study, the majority of the EPCs used in our experiment were early-stage cells, allowing us to fully take use of their stem cell features [56,57].…”

Section: Discussionmentioning

confidence: 99%

Endothelial Progenitor-Derived exosomes Induce Macrophage M2 Polarization via SOCS3/JAK2/STAT3 axis and Improve the Outcome of Spinal Cord Injury

Yuan

Peng

Yang

et al. 2022

Preprint

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Background Macrophage in the spinal cord injury (SCI) area imparts a chronic pro-inflammation effect that challenges the recovery of SCI. Previously, endothelial progenitor cell-produced exosomes (EPC-EXOs) have been noticed to facilitate revascularization and inflammation control after SCI. However, their effects on macrophage polarization remained unclear. This study aimed to investigate the EPC-EXOs' role in macrophage polarization and reveal its underlying mechanism. Methods We extracted the macrophages and EPC from the bone marrow suspension of C57BL/L mice by centrifugation. After cell identification, the EPC-EXOs were collected by ultra-high-speed centrifugation and exosome extraction kits and identified by transmission electron microscopy and nanoparticle tracking analysis. Then, macrophages were cultured with EPC-EXOs in different concentrations. We labeled the exosome to confirm its internalization by macrophage and detected the macrophage polarization marker level both in vitro and in vivo. We further estimated EPC-EXOs' protective effects on SCI by mice spinal cord tissue H&E staining and motor behavior evaluation. Finally, we performed RT-qPCR to identify the upregulated miRNA in EPC-EXOs and manipulate its expression to estimate its role in macrophage polarization, SOCS3/JAK2/STAT3 pathway activation, and motor behavior improvement. Results We found that EPC-EXOs decreased the macrophages’ M1 polarization marker expression and increased their M2 polarization marker expression on the 7 and 14 days after SCI. The spinal cord H&E staining results showed that EPC-EXOs raised the tissue-sparing area rate significantly after 28 days of SCI and the motor behavior evaluation indicated an increased BMS score and motor evoked potential by EPC-EXOs treatment after SCI. The RT-qPCR assay identified that miR-222-3P was specifically upregulated in EPC-EXOs and its miRNA-mimic also decreased the M1 polarization and increased the M2 polarization of macrophages. Additionally, miR-222-3P mimic activated the SOCS3/JAK2/STAT3 pathway, and SOCS3/JAK2/STAT3 pathway inhibition blocked miR-2223P’s effects on macrophage polarization and mouse motor behavior. Conclusion Comprehensively, we discovered that EPC-EXOs-derived miR-222-3P affected macrophage polarization via SOCS3/JAK2/STAT3 pathway and promoted mouse functional repair after SCI. This reveals EPC-EXOs’ role in macrophage polarization and will provide a novel interventional strategy to induce the poste-SCI recovery.

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“…The images of fluorescently labeled cells were captured using a laser scanning confocal microscope (Leica, Germany). Cells positive for both markers are considered EPCs [ 30 ].…”

Section: Methodsmentioning

confidence: 99%

Experimental Study on the Effect of Allogeneic Endothelial Progenitor Cells on Wound Healing in Diabetic Mice

Leng

Peng

Pan

et al. 2021

Journal of Diabetes Research

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Endothelial progenitor cells (EPCs) are involved in the neovascularization in traumatic and ischemic sites, but EPCs are “detained” in bone marrow under diabetic conditions, which results in reduction of the number of EPCs and their biological activity in peripheral blood. Based on our previous study to mobilize autologous bone marrow EPCs by administering AMD3100+G-CSF to realize the optimal effect, our present study is aimed at exploring the effects of transplanting EPCs locally in a wound model of diabetic mice. First, we prepared and identified EPCs, and the biological functions and molecular characteristics were compared between EPCs from DB/+ and DB/DB mice. Then, we performed full-thickness skin resection in DB/DB mice and tested the effect of local transplantation of EPCs on skin wound healing. The wound healing process was recorded using digital photographs. The animals were sacrificed on postoperative days 7, 14, and 17 for histological and molecular analysis. Our results showed that DB/+ EPCs were biologically more active than those of DB/DB EPCs. When compared with the control group, local transplantation of EPCs accelerated wound healing in DB/DB mice by promoting wound granulation tissue formation, angiogenesis, and collagen fiber deposition, but there was no significant difference in wound healing between DB/+ EPCs and DB/DB EPCs transplanted into the wound. Furthermore, local transplantation of EPCs promoted the expression of SDF-1, CXCR4, and VEGF. We speculated that EPC transplantation may promote wound healing through the SDF-1/CXCR4 axis. This point is worth exploring further. Present data are of considerable significance because they raise the possibility of promoting wound healing by isolating autologous EPCs from the patient, which provides a new approach for the clinical treatment of diabetic wounds in the future.

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Isolation and characterization of endothelial progenitor cells from canine bone marrow

Cited by 11 publications

References 48 publications

Endothelial Progenitor Cell-Mediated Angiogenesis is Promoted by the Transforming Growth Factor Beta 1/CXC Chemokine Receptor 7 Signaling Axis in Diabetes Mellitus

Endothelial Progenitor Cell-Mediated Angiogenesis is Promoted by the Transforming Growth Factor Beta 1/CXC Chemokine Receptor 7 Signaling Axis in Diabetes Mellitus

Endothelial Progenitor-Derived exosomes Induce Macrophage M2 Polarization via SOCS3/JAK2/STAT3 axis and Improve the Outcome of Spinal Cord Injury

Experimental Study on the Effect of Allogeneic Endothelial Progenitor Cells on Wound Healing in Diabetic Mice

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