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ObjectiveTo investigate the mechanism of Jiedu Huoxue prescription in promoting the reendothelialization of injured vessels by regulating the nuclear factor （NF）-κB/NOD-like receptor protein 3 （NLRP3）/cysteine-aspartic acid protease （Caspase）-1-mediated pyroptosis. MethodA rat model of injured thoracic aorta was established by balloon injury， and 36 rats were assigned into shame surgery， model， low-， medium-， and high-dose Jiedu Huoxue prescription， and atorvastatin calcium tablet groups. The injured aortic segment was collected 28 days after surgery. Hematoxylin-eosin （HE） staining and Evans blue staining were conducted to reveal the changes of vascular structural morphology and the reendothelialization of blood vessels， respectively. The enzyme-linked immunosorbent assay （ELISA） was employed to determine the levels of tumor necrosis factor-α （TNF-α）， intercellular adhesion molecule-1 （ICAM-1）， interleukin （IL）-1β， and nitric oxide （NO） in the serum. Western blotting was employed to determine the expression of endothelial nitric oxide synthase （eNOS）， NF-κB p65， phospho-NF-κB p65 （p-NF-κB p65）， NLRP3， and Caspase-1 in the vascular tissue. ResultThe model group showed thickened endovascular membrane， proliferation and disarrangement of smooth muscle cells of the artery wall， obvious inflammatory cell infiltration， and narrowed luminal area. Jiedu Huoxue prescription and atorvastatin calcium tablets mitigated the pathological changes of the thoracic aorta in different degrees. After balloon injury， the endothelial coverage rate of the model group decreased significantly， while Jiedu Huoxue prescription and atorvastatin calcium tablets increased the reendothelialization rate （P<0.05）. Compared with the shame surgery group， the model group showed elevated levels of TNF-α， ICAM-1， and IL-1β （P<0.01） and lowered NO level （P<0.01） in the serum. In addition， the model group presented down-regulated protein level of eNOS （P<0.01） and up-regulated phosphorylation of pyroptosis-associated proteins NLPR3， Caspase-1， and NF-κB p65 in the vascular tissue （P<0.05， P<0.01）. Compared with the model group， Jiedu Huoxue prescription and atorvastatin calcium tablets lowered TNF-α， ICAM-1， and IL-1β levels （P<0.05， P<0.01） and elevated the NO level in the serum （P<0.05， P<0.01）. Moreover， the drugs up-regulated the expression of eNOS （P<0.01） and down-regulated the expression of NLRP3， Caspase-1， and NF-κB p65 （P<0.05， P<0.01） in the vascular tissue. ConclusionJiedu Huoxue prescription can promote the reendothelialization and inhibit the intimal hyperplasia of vessels after balloon injury by regulating the NF-κB/NLRP3/Caspase-1 pathway to inhibit pyroptosis and reduce endothelial inflammatory injury.
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<p><b>OBJECTIVE</b>To evaluate whether the berberine treatment can improve endothelial repair capacity of early endothelial progenitor cells (EPCs) from prehypertensive subjects through increasing CXC chemokine receptor 4 (CXCR4) signaling.</p><p><b>METHODS</b>EPCs were isolated from prehypertensive and healthy subjects and cultured. In vivo reendothelialization capacity of EPCs from prehypertensive patients with or without in vitro berberine treatment was examined in a nude mouse model of carotid artery injury. The protein expressions of CXCR4/Janus kinase-2 (JAK-2) signaling of in vitro EPCs were detected by Western blot analysis.</p><p><b>RESULTS</b>CXCR4 signaling and alteration in migration and adhesion functions of EPCs were evaluated. Basal CXCR4 expression was significantly reduced in EPCs from prehypertensive patients compared with normal subjects (P<0.01). Also, the phosphorylation of JAK-2 of EPCs, a CXCR4 downstream signaling, was significantly decreased (P<0.01). Berberine promoted CXCR4/JAK-2 signaling expression of in vitro EPCs (P<0.01). Transplantation of EPCs pretreated with berberine markedly accelerated in vivo reendothelialization (P<0.01). The increased in vitro function and in vivo reendothelialization capacity of EPCs were inhibited by CXCR4 neutralizing antibody or pretreatment with JAK-2 inhibitor AG490, respectively (P<0.01).</p><p><b>CONCLUSION</b>Berberinemodified EPCs via up-regulation of CXCR4 signaling contributes to enhanced endothelial repair capacity in prehypertension, indicating that berberine may be used as a novel potential primary prevention means against prehypertension-related atherosclerotic cardiovascular disease.</p>
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AIM:To study the effect of crocin on the mobilization of endothelial progenitor cells (EPCs) in the peripheral blood of the mice with carotid arterial injury and its mechanism.METHODS:The carotid artery injury model of the C57BL/6 mice was established by the method of wire injury.The animals were divided into sham operation group,saline-treated model group,and low dose,medium dose and high dose (10,50 and 100 μ mol· kg-1 · L-1,respectively) of crocin treatment groups.The mobilization of the EPCs in peripheral blood of the mice with carotid artery injury was detected by flow cytometry at 3 d.The changes of vascular endothelial growth factor (VEGF),stromal-derived factor-1 (SDF-1),basic fibroblast growth factor (bFGF),epidermal growth factor (EGF) and matrix metalloproteinase-9 (MMP-9) in the peripheral blood of the mice with carotid artery injury were detected by enzyme-linked immunosorbent assay at 7 d.The vascular re-endothelialization and intimal hyperplasia of the mice with carotid artery injury were detected by Evans blue and hematoxylin-eosin staining.At the same time,real-time PCR was used to detect the mRNA expression of vascular repair factor-related receptors,vascular endothelial growth factor receotor-2 (VEGFR-2),CXC chemokine receptor-4 (CXCR4),basic fibroblast growth factor receptor (bFGFR) and epidermal growth factor receptor (EGFR),in the injured segments of carotid arteries.RESULTS:Compared with sham group,the EPCs mobilization and the content of vascular repair factors VEGF,SDF-1,bFGF,EGF and MMP-9 in peripheral blood were increased in model group (P ＜0.05).The area of vascular endothelium was decreased,while the area of intimal hyperplasia and the ratio of intimal to medial membrane area were increased significantly (P ＜ 0.05).The expression levels of VEGFR-2,CXCR4,bFGFR and EGFR were also increased in the injured segments of carotid arteries (P ＜ 0.05).Compared with model group,the EPCs mobilization and the content of vascular repair factors VEGF,SDF-1,bFGF,EGF and MMP-9 in peripheral blood were significantly increased in different concentrations of crocin-treated mice with carotid artery injury (P ＜ 0.05).The area of vascular endothelium was gradually increased,while the area of intimal hyperplasia and the ratio of intimal to medial membrane area were gradually decreased (P ＜ 0.05).The expression levels of VEGFR-2,CXCR4,bFGFR and EGFR were also gradually increased in the injured segments of cartid arteries (P ＜ 0.05).CONCLUSION:Crocin promotes the mobilization of EPCs and the re-endothelialization of damaged blood vessels in the mice with carotid artery injury,thus repairing the injured vasculature.
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AIM:To study the effect of crocin on the mobilization of endothelial progenitor cells (EPCs) in the peripheral blood of the mice with carotid arterial injury and its mechanism.METHODS:The carotid artery injury model of the C57BL/6 mice was established by the method of wire injury.The animals were divided into sham operation group,saline-treated model group,and low dose,medium dose and high dose (10,50 and 100 μ mol· kg-1 · L-1,respectively) of crocin treatment groups.The mobilization of the EPCs in peripheral blood of the mice with carotid artery injury was detected by flow cytometry at 3 d.The changes of vascular endothelial growth factor (VEGF),stromal-derived factor-1 (SDF-1),basic fibroblast growth factor (bFGF),epidermal growth factor (EGF) and matrix metalloproteinase-9 (MMP-9) in the peripheral blood of the mice with carotid artery injury were detected by enzyme-linked immunosorbent assay at 7 d.The vascular re-endothelialization and intimal hyperplasia of the mice with carotid artery injury were detected by Evans blue and hematoxylin-eosin staining.At the same time,real-time PCR was used to detect the mRNA expression of vascular repair factor-related receptors,vascular endothelial growth factor receotor-2 (VEGFR-2),CXC chemokine receptor-4 (CXCR4),basic fibroblast growth factor receptor (bFGFR) and epidermal growth factor receptor (EGFR),in the injured segments of carotid arteries.RESULTS:Compared with sham group,the EPCs mobilization and the content of vascular repair factors VEGF,SDF-1,bFGF,EGF and MMP-9 in peripheral blood were increased in model group (P ＜0.05).The area of vascular endothelium was decreased,while the area of intimal hyperplasia and the ratio of intimal to medial membrane area were increased significantly (P ＜ 0.05).The expression levels of VEGFR-2,CXCR4,bFGFR and EGFR were also increased in the injured segments of carotid arteries (P ＜ 0.05).Compared with model group,the EPCs mobilization and the content of vascular repair factors VEGF,SDF-1,bFGF,EGF and MMP-9 in peripheral blood were significantly increased in different concentrations of crocin-treated mice with carotid artery injury (P ＜ 0.05).The area of vascular endothelium was gradually increased,while the area of intimal hyperplasia and the ratio of intimal to medial membrane area were gradually decreased (P ＜ 0.05).The expression levels of VEGFR-2,CXCR4,bFGFR and EGFR were also gradually increased in the injured segments of cartid arteries (P ＜ 0.05).CONCLUSION:Crocin promotes the mobilization of EPCs and the re-endothelialization of damaged blood vessels in the mice with carotid artery injury,thus repairing the injured vasculature.
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<p><b>OBJECTIVE</b>This study was aimed to investigate the effects of carbon monoxide releasing molecule (CORM-2), a novel carbon monoxide carrier, on the reendothelialization of carotid artery in rat endothelial denudation model.</p><p><b>METHODS</b>Male rats subjected to carotid artery balloon injury were treated with CORM-2, inactive CORM-2 (iCORM-2) or dimethyl sulfoxide (DMSO). The reendothelialization capacity was evaluated by Evans Blue dye and the immunostaining with anti-CD31 antibody. The number of circulating endothelial progenitor cells (EPCs) was detected by flow cytometry. The proliferation, migration, and adhesion of human umbilical vein endothelial cells (HUVECs) were assessed by using [3H]thymidine, Boyden chamber and human fibronectin respectively. The expressions of protein were detected by using western blot analysis.</p><p><b>RESULTS</b>CORM-2 remarkably accelerated the re-endothelialization 5 d later and inhibited neointima formation 28 d later. In addition, the number of peripheral EPCs significantly increased in CORM-2-treated rats than that in iCORM-2 or DMSO-treated rats after 5 d later. In vitro experiments, CORM-2 significantly enhanced the proliferation, migration and adhesion of HUVECs. The levels of Akt, eNOS phosphorylation, and NO generation in HUVECs were also much higher in CORM-2 treated group. Blocking of PI3K/Akt/eNOS signaling pathway markedly suppressed the enhanced migration and adhesion of HUVECs induced by CORM-2.</p><p><b>CONCLUSION</b>CORM-2 could promote endothelial repair, and inhibit neointima formation after carotid artery balloon injury, which might be associated with the function changes of HUVECs regulated by PI3K/Akt/eNOS pathway.</p>
Subject(s)
Animals , Humans , Male , Rats , Carbon Monoxide , Metabolism , Pharmacology , Carotid Artery Injuries , Drug Therapy , Allergy and Immunology , Metabolism , Pathology , Carotid Artery, Common , Allergy and Immunology , Metabolism , Pathology , Cell Adhesion , Disease Models, Animal , Endothelial Cells , Allergy and Immunology , Metabolism , Pathology , Endothelium, Vascular , Metabolism , Pathology , Rats, Sprague-DawleyABSTRACT
Objective To investigate the effects of shear stress on late endothelial progenitor cells (EPCs) functions in vitro and in vivo. Methods Density gradient centrifugation-isolated rat bone marrow mononuclear cells were cultured in EGM-2MV and induced into EPCs. The 3rd~4th generation of EPCs, namely late EPCs, were treated with shear stress (1.2 Pa). Then cell biological functions, such as proliferation, adhesion, migration and ability of tube formation, were assayed with EdU incorporation assay, adhesion testing, Boyden chamber assay and Matrigel, respectively. The gene expression of VEFG was analyzed by real time RT-PCR. The apoptosis and aging situation of late EPCs were assayed by FACS and senescence-associated β-galactosidase (SA-β-gal) staining. The reendothelialization capacity of late EPCs treated by shear stress was evaluated by establishing models of freshly balloon-injured carotid arteries of rats and cell transplantation in situ. Results Shear stress could increase proliferation, adhesion, migration and tube formation of late EPCs (P＜0.05), upregulate the gene expression of VEGF, inhibit EPC apoptosis and delayed EPC aging (P＜0.05). Transplantation of late EPCs treated by shear stress facilitated in vivo reendothelialization in the injured arterial segment and inhibited neointima formation. Conclusions Shear stress within the physiological range can improve the functions of late EPCs and enhance their therapeutic ability of repairing vascular endothelial injury, which provides experimental basis for the clinic application of EPCs and shear stress-mediated cell therapy.
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The purpose of our study was to create a novel rat aorta stent implantation model. Stainless steel bare metal stents (BMS) or paclitaxel-eluting stents (PES) were implanted in male Sprague-Dawley rats (BW 400 +/- 20 g). Two and four weeks after stent implantation, the aorta were collected, fixed with 2% glutaraldehyde, and cut into two segments. One segment was used for scanning electron microscopy analysis to evaluate re-endothelialization, and the other segment was used to calculate the neointimal area. At 2 weeks after stenting, the appearance of neointimal hyperplasia was less in the PES group than in the BMS group. At 4 weeks after stenting, no significant difference in neointimal hyperplasia was observed between two groups. On the other hand, the PES group showed more thrombus formation and less re-endothelialization compared to the BMS group. This study demonstrated the ability of a novel rat model of aorta stenting via a common carotid artery to measure the efficacy and safety of commercially available drug-eluting stents.
Subject(s)
Animals , Male , Rats , Angioplasty/methods , Aorta, Thoracic/surgery , Coronary Artery Disease/surgery , Drug-Eluting Stents , Histocytochemistry , Microscopy, Electron, Scanning , Models, Animal , Neointima/pathology , Paclitaxel/administration & dosage , Rats, Sprague-DawleyABSTRACT
Background Stromal cell-derived factor-1_?(SDF-1_?)has been demonstrated to be essential for stern cell mobilization/homing.Recent evidence indicates that SDF-1_? has been expressed in injured carotid arter- ies.Besides,high SDF-1_? plasma levels are clinically associated with stable coronary artery disease.Objective To investigate whether SDF 1 involves in mobilization of endothelial progenitor cells(EPC)and reendothelialization after vascular injury.Methods SDF-1_? was detected by RT-PCR and Western blot in carotid arteries of mice at different time points after wire-induced injury.SDF-1_? determination in peripheral blood samples and BM was per- formed by SDF-1_? enzyme-linked immunosorbent assay(ELISA)kit.EPC in peripheral blood collected at different time points after vascular injury were quantified by flow cytornetry.In subgroup,blocking SDF-1 rnonoclonal anti- body was injected,peripheral blood EPC were quantified after vascular injury and reendothelialization of injured ar- teries was determined 14 days later.Results Expression of SDF-1_? was evident at day 1,and peaked at day 3 after arterial injury.A rise in plasmatic concentration of SDF-1_? and a significant reduction of SDF-1_? in bone marrow concentration was noticed at all time points following injury.The amount of circulating EPC was increased shortly after induction of vascular injury and persisted up to 7 days(P
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AIM: To investigate the function and the mechanism of transplanting bone marrow derived peripheral blood mesenchymal stem cells(PBMSCs) on restenosis after carotid balloon angioplasty in the model of carotid atherosclerosis rabbits,and to determine if the functions of PBMSCs are enhanced after hypoxia preconditioning.METHODS: Bone marrow cells were mobilized by granulocyte colony-stimulating factor(G-CSF),and PBMSCs were collected through density gradient centrifugation and adherent culture,labeled with enhancement type green fluorescent protein(EGFP) genes.All animals with carotid atherosclerosis stenosis were randomly divided into three groups: hypoxia preconditioning group(n=24,received intravenous transplantation of PBMSCs with hypoxia preconditioning),non-hypoxia preconditioning group(n=24,received normal culture of PBMSCs) and control group(n=24,only received equal-volume of culture medium).Vascular endothelial growth factor(VEGF) was determined by enzyme linked immunosorbent assay(ELISA) at 7 d,14 d and 28 d post-angioplasty,respectively.The vessel morphology,the homing of MSCs and the reendothelialization were analyzed with Weigert staining and immunohistochemistry.RESULTS: Compared to control group,the level of VEGF significantly increased in both hypoxia preconditioning group and non-hypoxia preconditioning group at all time points(P