Endothelial Progenitor Cells Group Research Articles

Role of Bushen Huoxue Formula and transplanted endothelial progenitor cells play in promoting endplate microcirculation and attenuating intervertebral disc degeneration

ObjectiveTo explore whether Bushen Huoxue Formula (BSHXF) improves the angiogenesis ability of transplanted endothelial progenitor cells (EPCs) in endplate and its potential mechanism in delaying intervertebral disc degeneration (IDD). MethodsBSHXF was analyzed via Ultra-High Performance Liquid Chromatography (UPLC). Rabbit axial compression lumbar IDD models were constructed and the effects of BSHXF, EPCs, and their combination in IDD were determined by MRI, histological evaluation, TUNEL, and immunofluorescence assays. Additionally, CCK-8 assay, flow cytometry, and tube formation assay were used to evaluate EPCs viability, proliferation, cell cycle and the angiogenesis ability of EPCs between groups. ResultsBSHXF and transplanted EPCs both attenuate the process of IDD in the rabbit model assessed by MRI, HE staining and Masson staining. TUNEL-positive NP cells were significantly reduced in the BSHXF group, EPCs group, and EPC + BSHXF group compared to the model group (P < 0.05), with the EPC + BSHXF group showing the most significant therapeutic effect. Immunofluorescence detection showed that VEGF, CD34 expression and quantity of microvessels in the endplate significantly increased in the EPC + BSHXF group compared to all the other groups (P < 0.05). Besides, the CCK-8 assay showed an upregulation of EPC viability and the tube formation assay demonstrated a significant increase in tube length and branching in EPCs cultured with BSHXF-containing serum (P < 0.05). Furthermore, BSHXF-containing serum increased VEGF expression in EPCs cultured in vitro (P < 0.05). ConclusionsBoth BSHXF and EPCs transplantation play an important role in increasing endplate angiogenesis and attenuating IDD. BSHXF can enhance the viability and tube-forming ability of EPCs and endplate microcirculation.

Abstract 17537: Myocardial Dysfunction is Associated With Impaired Angiogenesis in Patients With Heart Failure With Preserved Ejection Fraction

Background: Recent evidence suggests that myocardial microvascular damage may represent a key parameter in the pathogenesis of heart failure with preserved ejection fraction (HFpEF). Aim: We sought to investigate a potential correlation between parameters of angiogenesis and myocardial dysfunction in HFpEF patients. Methods: We enrolled 30 consecutive HFpEF patients with NYHA class III, left ventricular ejection fraction (LVEF) &gt;50%, E/e' &gt;15, and NT-proBNP &gt;300 pg/ml. In all patients endothelial progenitor cells (EPCs) were mobilized by granulocyte-colony stimulating factor (G-CSF; 10 mcg/kg, 5 days) and collected via apheresis. Before EPC mobilisation we performed echocardiography and measured plasma levels of biomarkers of angiogenesis. All patients underwent electroanatomical mapping for the measurement of myocardial viability; preserved viability was defined as mean unipolar voltage&gt; 8.3 mV. Results: Myocardial viability was preserved in 11 patients (37%; Group A) and decreased in 19 patients (63%; Group B). The groups did not differ in age (63±10 years in Group A vs. 61±8 years in Group B, P=0.78), gender (male: 73% vs. 79%, P=0.70), serum creatinine (1.07±0.34 mg/dL vs. 1.04±0.40 mg/dL, P=0.43), or LVEF (60±7% vs. 57±4%, P=0.32). In Group B we found higher levels of NT-proBNP (2370±1020 pg/mL vs. 920±455 pg/mL in Group A, P=0.04) and E/e’ (23.2±3.1 vs. 17.4±1.8 pg/ in Group A, P=0.02). After G-CSF stimulation, we found significantly lower numbers of EPCs in Group B (53±42 x10 6 cells/L) than in Group A (102±52 x10 6 cells/L, P=0.001). Similarly, apheresis yielded lower numbers of EPCs in Group B (85±82 x10 6 cells vs. 200±93 x10 6 cells in Group A, P=0.003). When compared to Group A, patients in Group B displayed lower levels of pro-angiogenic biomarkers (agiopoietin-1: 7.4±2.8 ng/mL vs. 12.4±5.1 ng/mL, P=0.02; vascular endothelial growth factor: 27.5±20.1 pg/mL vs. 51.6±24.2 pg/mL, P=0.01), and higher levels of anti-angiogenic biomarkers (endostatin: 56.5±19.1 ng/mL vs. 33.2±14.3 ng/mL, P=0.01; thrombospondin-2: 16.4±4.3 ng/mL vs. 9.2±5.0 ng/mL, P=0.03). Conclusion: In patients with HFpEF, myocardial dysfunction appears to correlate with reduced mobilisation of endothelial progenitor cells and impaired angiogenesis.

Transplantation of endothelial progenitor cells overexpressing mir-126-3p improves vascular repair in a diabetic rat model.

Transplantation of endothelial progenitor cells overexpressing mir-126-3p improves vascular repair in a diabetic rat model.

Infusion of Cultured Endothelial Progenitor Cells Boosts Hematopoietic Recovery and Increases the Expression of LepR+ Cells after Irradiation

The hemopoietic system's function of producing blood cells and immune cells during thorough life relies on the Maintainance and differentiation of hematopoietic stem cells (HSCs). HSCs activities were tightly and finely tuned by a specialized microenvironment called "niche" in the bone marrow. However, during the routine treatment of hematopoietic diseases, such as chemotherapy and radiotherapy, the niche was inevitably impaired which is one of the essential causes of hematopoietic suppression. Our previous research results showed that infusion of cultured endothelial progenitor cells (EPCs) could boost hematopoietic recovery after HSCT. And the latent mechanisms could be that EPCs can differentiate into endothelial cells (ECs) in vivo. However, the roles of EPCs play in the bone marrow niche and the specific mechanism remain unknown. Here we designed further to investigate the effect and mechanism of EPCs in boosting bone marrow microenvironment recovery after total body irradiation (TBI). This may offer a new cue for therapy of hematopoietic suppression after the treatment of malignant hematological diseases. Bone marrow-derived Primary EPCs isolation and culture were described as before. Briefly, Bone marrow cells from Balb/c or C57BL/6J mice aged 6-8 weeks were cultured with EGM-2 medium which was changed in 36 h, the adherent cells formed colonies identified as elongated sprouting cells radiating from a central core of round cells and identified as VEGFR2+; CD133+; CD144+; SCA1+ by FACS on days 4-6. To assess the effects of the EPCs in boosting hematopoiesis, Balb/c mice underwent TBI with a lethal dose and were injected withEPCs or the same volume of PBS via tail vein in 4-6 hours. The survival of the EPC group was elevated ( survival: 8/9 vs 0/13; p＜0.001). C57BL/6J mice underwent TBI with a sublethal dose and were injected with the cultured EPCs isolated from C57BL/6-Tg(CAG-EGFP) mice or the same volume of PBS via tail vein in 4-6 hours. Results showed that EPCs boost the recovery of hematopoietic cells in the bone marrow and peripheral blood cells; Immunofluence (IF) staining showed that Infused EPCs with GFP fluorescence were home to bone marrow and differentiated into ECs which were marked with endomucin (EMCN); Moreover, assessments of the alteration of bone marrow niche using H&E staining revealed that infusion of EPCs reduced the accumulation of fat and using multi-analyte flow assay showed that mice infused with EPCs express a higher level of M-CSF and TGF-β in plasma while the level of SCF and IL-6 both in plasma and bone marrow supernatant were lower compared to TBI. Furthermore, to assess the change of sinusoidal niche, we used IF staining and found that the sinusoids were dilated and perisinusoidal stromal cells identified as LepR+ were massively lost on the 7th day after TBI while the dilated sinusoidal lumens were narrowed (11.05±1.789 vs 20.86±4.573，p=0.0258) and LepR+ cells were upregulated on the 21st day after the infusion of EPCs which indicated that the sinusoidal niche has a better recovery compared to TBI. In Conclusion, EPCs could differentiate into endothelial cells in vivo boosting the recovery of the hematopoietic system. And the potential mechanism is that EPCs repair the bone marrow sinusoidal niche damaged by irradiation.

Establishment and biological effect evaluation of prevascularized porous β-tricalcium phosphate tissue engineered bone

To evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group ( n=6) and porous β-TCP scaffold as the control group ( n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. The isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel ( P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group ( P<0.05), except for area fraction at 4 weeks after implantation ( P>0.05). The prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair.

Impact of Endothelial Progenitor Cells in the Vascularization of Osteogenic Scaffolds.

The microvascular endothelial network plays an important role in osteogenesis, bone regeneration and bone tissue engineering. Endothelial progenitor cells (EPCs) display a high angiogenic and vasculogenic potential. The endothelialization of scaffolds with endothelial progenitor cells supports vascularization and tissue formation. In addition, EPCs enhance the osteogenic differentiation and bone formation of mesenchymal stem cells (MSCs). This study aimed to investigate the impact of EPCs on vascularization and bone formation of a hydroxyapatite (HA) and beta-tricalcium phosphate (ß-TCP)–fibrin scaffold. Three groups were designed: a scaffold-only group (A), a scaffold and EPC group (B), and a scaffold and EPC/MSC group (C). The HA/ß–TCP–fibrin scaffolds were placed in a porous titanium chamber permitting extrinsic vascularization from the surrounding tissue. Additionally, intrinsic vascularization was achieved by means of an arteriovenous loop (AV loop). After 12 weeks, the specimens were explanted and investigated by histology and CT. We were able to prove a strong scaffold vascularization in all groups. No differences regarding the vessel number and density were detected between the groups. Moreover, we were able to prove bone formation in the coimplantation group. Taken together, the AV loop is a powerful tool for vascularization which is independent from scaffold cellularization with endothelial progenitor cells’ prior implantation.

Human Endothelial Progenitor Cells Protect the Kidney against Ischemia-Reperfusion Injury via the NLRP3 Inflammasome in Mice.

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and progression to chronic kidney disease (CKD). However, no effective therapeutic intervention has been established for ischemic AKI. Endothelial progenitor cells (EPCs) have major roles in the maintenance of vascular integrity and the repair of endothelial damage; they also serve as therapeutic agents in various kidney diseases. Thus, we examined whether EPCs have a renoprotective effect in an IRI mouse model. Mice were assigned to sham, EPC, IRI-only, and EPC-treated IRI groups. EPCs originating from human peripheral blood were cultured. The EPCs were administered 5 min before reperfusion, and all mice were killed 72 h after IRI. Blood urea nitrogen, serum creatinine, and tissue injury were significantly increased in IRI mice; EPCs significantly improved the manifestations of IRI. Apoptotic cell death and oxidative stress were significantly reduced in EPC-treated IRI mice. Administration of EPCs decreased the expression levels of NLRP3, cleaved caspase-1, p-NF-κB, and p-p38. Furthermore, the expression levels of F4/80, ICAM-1, RORγt, and IL-17RA were significantly reduced in EPC-treated IRI mice. Finally, the levels of EMT-associated factors (TGF-β, α-SMA, Snail, and Twist) were significantly reduced in EPC-treated IRI mice. This study shows that inflammasome-mediated inflammation accompanied by immune modulation and fibrosis is a potential target of EPCs as a treatment for IRI-induced AKI and the prevention of progression to CKD.

LncRNA IGF2-AS Regulates Nucleotide Metabolism by Mediating HMGA1 to Promote Pyroptosis of Endothelial Progenitor Cells in Sepsis Patients.

Background Sepsis is one of the major causes of death worldwide, and its high mortality and pathological complexity hinder early accurate diagnosis. We aimed to investigate lncRNA IGF2-AS and HMGA1 effects on pyroptosis of endothelial progenitor cells (EPCs) in sepsis patients and the mechanisms involved. Methods Blood samples from sepsis patients and healthy subjects were collected, and EPCs were isolated and identified. We constructed cell lines that knocked down lncRNA IGF2-AS, HMGA1, and TYMS. Furthermore, lncRNA IGF2-AS was overexpressed. Subsequently, dNTP treatment with different concentrations was performed to investigate lncRNA IGF2-AS and HMGA1 effects on pyroptosis of EPCs in sepsis patients. Finally, exosomes were isolated from bone marrow mesenchymal stem cells (MSCs) to detect lncRNA IGF2-AS expression, and the influence of MSC-derived exosomal lncRNA IGF2-AS on sepsis was preliminarily discussed. Results Compared with Healthy group, lncRNA IGF2-AS, HMGA1, and TYMS were highly expressed in Sepsis group. Compared with si-NC group, si-lncRNA IGF2-AS group had increased proliferation ability, decreased pyroptosis, decreased HMGA1, RRM2, TK1, and TYMS expressions. lncRNA IGF2-AS played a regulatory role by binding HMGA1. Compared with si-NC group, the proliferation ability of si-HMGA1 group increased, pyroptosis decreased, and RRM2, TK1, and TYMS expressions also decreased. Compared with si-NC group, pyroptosis in si-TYMS group was reduced. In addition, HMGA1 was related and bound to TYMS. After overexpressing lncRNA IGF2-AS, dNTP level decreased, while the proliferation increased and pyroptosis decreased with higher concentration of dNTP. In addition, we found that EPCs took up MSC-exosomes. Compared with supernatant group, lncRNA IGF2-AS was expressed in exosomes group. Compared with EPCs group, EPCs+exosomes group had increased lncRNA IGF2-AS expression and increased pyroptosis. Conclusions lncRNA IGF2-AS regulated nucleotide metabolism by mediating HMGA1 to promote pyroptosis of EPCs in sepsis patients. This study provided important clues for finding new therapeutic targets for sepsis.

Effects of Shen-Yuan-Dan on Periprocedural Myocardial Injury and the Number of Peripheral Blood Endothelial Progenitor Cells in Patients with Unstable Angina Pectoris Undergoing Elective Percutaneous Coronary Intervention.

Objectives We aimed to investigate the effects of Shen-Yuan-Dan (SYD), a Chinese medicine preparation, on periprocedural myocardial injury (PMI) and the number of peripheral blood endothelial progenitor cells (EPCs) in patients with unstable angina pectoris (UA) who underwent elective percutaneous coronary intervention (PCI). Methods Patients were randomly divided into the experimental (group A) and control (group B) groups through the random number table method. In group A, patients concurrently received the conventional western treatment and SYD orally (4 capsules/time, 3 times/d, from 3 d before surgery to 7 d after surgery). In group B, patients received conventional Western medicine treatment. Both groups underwent coronary angiography, and patients undergoing PCI were eventually included in the study. The following patient data were collected: incidence of PMI, serum CK-MB content before PCI, 4 h, 24 h, and 7 d after PCI, number of CD45dim/-CD34+CD309+ peripheral venous EPCs, and number of CD184 coexpressed EPCs. The incidence of adverse reactions and 30-day major adverse cardiovascular events (MACEs) were also recorded. Results Sixty-two patients were finally included in this study, with 32 and 30 in groups A and B, respectively. In group A, the number of peripheral blood EPCs and the number of CD184 coexpressed EPCs at 1 h before surgery were higher than those at 3 d before surgery (37.24 ± 25.20 vs. 22.78 ± 9.60/ml; P < 0.001 and 23.38 ± 15.30 vs. 13.54 ± 8.08/ml; P < 0.001, resp.). The number of peripheral blood EPCs and number of CD184 coexpressed EPCs at 4 h after surgery were lower than those at 1 h before surgery (25.30 ± 11.90 vs. 37.24 ± 25.20/ml; P=0.019 and 15.38 ± 8.78 vs. 23.38 ± 15.30/ml; P=0.013, resp.), but there was no difference at 24 h and at 7 d after surgery in comparison with that at 1 h before surgery (P > 0.05). In group B, compared with that at 1 h before surgery, there existed a decline in the number of EPCs in peripheral blood and the number of CD184 coexpressed EPCs at 4 h after surgery, but without a statistical difference (P > 0.05). Comparing both groups, it was found that the incidence of PMI in group A was lower (6.25% vs. 26.67%; P=0.04), and the serum CK-MB content at 4 and 24 h after surgery was also lower than that in group B (17.33 ± 5.83 vs. 20.38 ± 4.32 U/l; P=0.048 and 15.79 ± 5.32 vs. 19.10 ± 4.93 U/l; P=0.030, resp.). The number of EPCs in peripheral blood and the number of CD184 coexpressed EPCs in group A were higher than those in group B at 1 h before surgery (37.24 ± 25.20 vs. 22.36 ± 12.26/ml; P=0.034 and 23.38 ± 15.30 vs. 13.12 ± 14.62/ml; P=0.013, resp.). In addition, there were no obvious adverse reactions and no 30-day MACEs in both groups during the trial. Conclusion SYD can reduce PMI and promote the mobilization of EPCs in the perioperative period of elective PCI in patients with UA.

Mechanism of isosorbide dinitrate combined with exercise training rehabilitation to mobilize endothelial progenitor cells in patients with coronary heart disease

ABSTRACT it was to explore effect of isosorbide dinitrate combined with exercise training and rehabilitation on endothelial progenitor cells (EPCs) in coronary heart disease. EPCs were isolated and cultured from peripheral blood of coronary heart disease patients, and morphology and surface markers were detected. Then, 116 patients were rolled into treatment group (isosorbide dinitrate + exercise rehabilitation training) and control group (isosorbide dinitrate). Characteristics of EPCs cells after treatment were compared. The mononuclear cells were round and small in size and were not evenly distributed in the culture plate. EPCs cells grew as colonies after 8d-culture, and the surrounding cells grew outward in a germinating manner with colonies as the center, forming multiple cell populations. Positive rates of EPCs surface markers CD133, CD34, and vascular endothelial growth factor receptor (KDR) were 11.25 ± 3.07%, 48.18 ± 9.13%, and 76.36 ± 8.27%, respectively. Proliferation activity of EPCs in the treatment group was dramatically higher versus controls at day three, five, and seven (P < 0.05). Adhesion ability of EPCs in treatment group was dramatically higher than controls at day three, five, and seven (P < 0.05). Migration ability of EPCs in treatment group was dramatically higher versus control group at day three, five, and seven (P < 0.05). In short, isosorbide dinitrate plus exercise rehabilitation greatly enhanced the proliferation activity, adhesion ability, and migration ability of EPCs cells, which also played a beneficial role in the repair of endothelial injury, with notable effects. Abbreviation listAbbreviation: Full name; EPCs: endothelial progenitor cells; RPMI: roswell Park Memorial Institute;VEGF: vascular endothelial growth factor; BFGF: basic fibroblast growth factor; DiL-acLDL: DiL-labeled acetyl low density lipoprotein; PBS: phosphate buffer; FITC-UEA-I: FITC-labeled Vitex agglutinin I; EDTA: ethylenediamine tetra-acetic acid; MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium.

Juxtaposition of Mesenchymal Stem Cells with Endothelial Progenitor Cells Promoted Angiogenic Potential Inside Alginate-Gelatin Microspheres.

Purpose: Here, we investigated the angiogenic potential of endothelial progenitor cells juxtaposed with mesenchymal stem cells (MSCs) inside alginate-gelatin microspheres with stromal derived factor-1α (SDF-1 α) for 7 days. Methods: Six encapsulated groups were allocated including endothelial progenitor cells (EPCs), EPCs/SDF-1α, MSCs, MSCs/SDF-1α, EPCs+MSCs and EPCs+MSCs/SDF-1α. Cells were encapsulated with a mixture of 1% alginate and 2% gelatin hydrogel. Cell survival was examined by MTT assay. Endothelial differentiation was determined by flow cytometry and ELISA. Tubulogenesis assay and Ac-Dil-LDL uptake were used to detect functional activity. Cell migration was analyzed by Transwell insert and gelatin zymography analyses. By using real-time polymerase chain reaction (PCR), we measured the transcription of Akt and PK1. Results: We found an increase in cell viability in MSCs/SDF-1α microspheres compared to EPCs group (P <0.05). EPC/MSCs co-culture contributed to the increase of CD133+ cells while we found high CD31 levels in MSCs group (P <0.05). Juxtaposition of EPC with MSCs increased tubulogenesis compared to SDF-1a-free condition (P <0.001). SDF-1α had the potential to increase in AC-LDL uptake in MSCs and EPCs/MSCs groups. Cells migration and MMP-9 activities increased after treatment with SDF-1α. SDF-1α upregulated PK1 and Akt in encapsulated cells, especially in a co-culture system. Conclusion: Alginate-gelatin microspheres could alter the angiogenic potential of progenitor cells in the presence of SDF-1α

Combined Transplantation of Adipose Tissue-Derived Stem Cells and Endothelial Progenitor Cells Improve Diabetic Erectile Dysfunction in a Rat Model

Erectile dysfunction (ED) is a common complication in men suffered with diabetic mellitus. Stem cell transplantation is a promising strategy for the treatment of diabetic ED (DED). In this study, we evaluated whether combined transplantation of adipose tissue-derived stem cells (ADSCs) and endothelial progenitor cells (EPCs) could improve the erectile function of the DED rat model. DED rats were induced via intraperitoneal injection of streptozotocin (50 mg/kg), and ED was screened by apomorphine (100 mg/kg). DED rats were divided into 4 groups (n = 14 each): DED, ADSC, EPC, and ADSC/EPC group. Another 14 age-matched male SD rats with normal erectile function were served as the normal group. The normal group and the DED group were received intracavernous injection with phosphate-buffered saline (PBS). And the other groups were received intracavernous injection with ADSCs (1 × 106), EPCs (1 × 106), and ADSCs/EPCs (0.5 × 106/0.5 × 106), respectively. The total intracavernous pressure (ICP) and mean arterial pressure (MAP) were recorded at day 28 after injection. The endothelium, smooth muscle, and penile dorsal nerves were assessed within cavernoursal tissue. On day 28 after injection, the ADSC/EPC group displayed more significantly enhanced ICP and ICP/MAP than the DED or ADSC or EPC group (p < 0.05). Immunofluorescent analysis and western blot demonstrated that the improvement of erectile function in the ADSC/EPC5 group was associated with increased expression of endothelial marker (CD31) and the correction of eNOS-cGMP-NO signaling. More 5-ethynyl-2′-deoxyuridine- (EdU-) positive EPCs could be found lining in the cavernous endothelial layer in the ADSC/EPC group than the EPC group, which was attributed to the paracrine of vascular endothelial growth factor (VEGF) and stromal-derived factor-1 (SDF-1) by ADSCs. Combined transplantation of ADSCs and EPCs has a synergic effect in repairing the endothelial function of DED rats, and the underlying mechanism might be the paracrine of VEGF and SDF-1 by ADSCs, which improves the recruitment and proliferation of EPCs in the cavernosum.

Endothelial progenitor cell transplantation restores vascular injury in mice after whole-brain irradiation

Vascular damage plays an important role in the pathogenesis of radiation-induced brain injury (RBI). Endothelial progenitor cells (EPCs) are responsible for maintaining and repairing endothelial function, and have become a promising method for the treatment of cerebrovascular diseases. However, whether EPC transplantation plays a protective role in RBI has not been fully elucidated. Therefore, the present study investigated the effects of bone marrow-derived EPC transplantation in a whole-brain irradiation (WBI) mouse model. Mice were divided into the three groups: control group, irradiation group and EPCs group. Phosphate buffered saline or EPCs were intravenously injected into mice one week after irradiation, and brains were analyzed eight weeks after injection. Flow cytometry demonstrated that irradiation led to a significant reduction in the peripheral blood EPC count; however, EPC transplantation led to a significant increase in the circulating EPCs. Intravital two-photon imaging and western blotting demonstrated that EPC transplantation reversed the effects of irradiation by decreasing blood–brain barrier permeability and increasing the expression of tight junction proteins in the brain. Additionally, immunofluorescence staining revealed that the brain microvascular density was higher in the EPCs group than the irradiation group. Therefore, EPC transplantation may restore damage caused by WBI to the blood–brain barrier, tight junctions, and cerebral capillary density. These results highlight the potential beneficial effects of EPC transplantation on vascular damage induced by RBI.

471-P: Transplantation of Apoptosis Resistant Endothelial Progenitor Cells (EPC) May Improve Renal Function in Both Type 1 and 2 Diabetic Kidney Disease (DKD)

Introduction: DKD is associated with glomerulosclerosis and poor perfusion. Transplanting apoptosis resistant p53 silenced (p53sh) EPCs may help to increased vascularization and renal perfusion. EPCs are known to improve perfusion and could be more beneficial than mouse mesenchymal stromal cell (mMSC) transplantation. Method: Hyperglycemia and proteinuria were confirmed at 10 weeks in STZ induced type 1 diabetic C57BL mice and db/db mice for type 2 diabetes mellitus (DM). We transplanted 0.3 million p53sh-EPCs, Null EPCs (control), MSC or saline under each kidney capsule. For type 2 DM model, we also transplanted supernatant of p53sh EPC. Urine was collected weekly for creatinine and protein estimation. Perfusion was measured by renal ultrasound at week 4 post transplantation. Kidneys were harvested for qRT PCR. Results: In type 1 DM model, reduction of protein/creatinine was observed in p53sh EPC transplantation group compared to null and mMSC (1.8 and 1.6 folds, p=0.03. 0.04 respectively) significantly. KDR expression was increased (1.2 fold, p=0.04) on p53sh EPC transplantation compared to null. We observed increased mean and peak renal blood velocity (1.3 folds, p= 0.01, 1.3 fold p=0.001 respectively) in p53sh EPC transplant group compared to null. In type 2 model, 2 fold reduction of protein concentration was noted in p53sh transplanted group compared to null and mMSC groups. Reduction of plasma TNFa, a marker for systemic inflammation was decreased by 2.7 and 1.6 folds in p53sh EPC group in comparison to null-EPC and mMSC respectively. We also noticed that blood glucose level decreased post treatment in all cell therapy groups including the supernatant group, compared to saline, indicating a possible paracrine systemic effect of the cell and supernatant therapy. Conclusion: Transplantation of p53sh EPC improves renal perfusion and renal function in both type 1 and type 2 DM and it is more beneficial than transplantation of mMSC. Disclosure N. Kundu: None. L.D. Asico: None. P.A. Jose: None. S. Sen: None.

The Paracrine Role of Endothelial Cells in Bone Formation via CXCR4/SDF-1 Pathway.

Vascularization is a prerequisite for bone formation. Endothelial progenitor cells (EPCs) stimulate bone formation by creating a vascular network. Moreover, EPCs secrete various bioactive molecules that may regulate bone formation. The aim of this research was to shed light on the pathways of EPCs in bone formation. In a subcutaneous nude mouse ectopic bone model, the transplantation of human EPCs onto β-TCP scaffold increased angiogenesis (p < 0.001) and mineralization (p < 0.01), compared to human neonatal dermal fibroblasts (HNDF group) and a-cellular scaffold transplantation (β-TCP group). Human EPCs were lining blood vessels lumen; however, the majority of the vessels originated from endogenous mouse endothelial cells at a higher level in the EPC group (p < 01). Ectopic mineralization was mostly found in the EPCs group, and can be attributed to the recruitment of endogenous mesenchymal cells ten days after transplantation (p < 0.0001). Stromal derived factor-1 gene was expressed at high levels in EPCs and controlled the migration of mesenchymal and endothelial cells towards EPC conditioned medium in vitro. Blocking SDF-1 receptors on both cells abolished cell migration. In conclusion, EPCs contribute to osteogenesis mainly by the secretion of SDF-1, that stimulates homing of endothelial and mesenchymal cells. This data may be used to accelerate bone formation in the future.

Proventive and Therapentic Effects of Endothelial Progenitor Cells on Monocrotaline-Induced Hepatic Vein Occlusion Disease

To investigate the preventive and therapeutic effects of endothelial progenitor cells on monocrotaline-induced hepatic vein occlusion disease in mice. C57BL/6 mice were randomly divided into 3 groups: saline group (n=15), monocrotaline group (n=15), and endothelial progenitor cell infusion group (n=15). Liver function (TBIL, ALT, AST), liver index, and serum levels of TNF-α and IL-6 were measured on the 8th day after intragastric administration. Hepatic sinusoidal endothelial cells, hepatic central venous endothelial cells and hepatocytes were observed by both HE and immunohistochemical staining. Hepatic fibrosis was observed by Masson's trichrome staining. By the light microscopy, the liver of the monocrotaline group showed moderate to the severe injuries of hepatic sinusoidal and central venous endothelial cells, and hepatic venous congestion. Masson staining showed moderate to severe hepatic fibrosis of central vein and hepatic sinus. In the endothelial progenitor cell group, hepatic sinusoidal and central venous endothelial cell injuries, and the fibrosis of central hepatic vein and hepatic sinus were mild to moderate. Hepatic venous congestion was reduced in comparison with that in the mice of the monocrotaline group. Compared with the endothelial progenitor cell group, the liver index was higher, the liver function was more abnormal, and the serum expression levels of TNF-α and IL-6 were higher in the monocrotaline group. The monocrotaline-induced damage of hepatic sinusoidal and central venous endothelial cells is an linitiating factor for hepatic vein occlusive disease. Infusion of endothelial progenitor cells can play a role in preventing and treating hepatic vein occlusion.

Proventive and Therapentic Effects of Endothelial Progenitor Cells on Monocrotaline-Induced Hepatic Vein Occlusion Disease

To investigate the preventive and therapeutic effects of endothelial progenitor cells on monocrotaline-induced hepatic vein occlusion disease in mice. C57BL/6 mice were randomly divided into 3 groups: saline group (n=15), monocrotaline group (n=15), and endothelial progenitor cell infusion group (n=15). Liver function (TBIL, ALT, AST), liver index, and serum levels of TNF-α and IL-6 were measured on the 8th day after intragastric administration. Hepatic sinusoidal endothelial cells, hepatic central venous endothelial cells and hepatocytes were observed by both HE and immunohistochemical staining. Hepatic fibrosis was observed by Masson's trichrome staining. By the light microscopy, the liver of the monocrotaline group showed moderate to the severe injuries of hepatic sinusoidal and central venous endothelial cells, and hepatic venous congestion. Masson staining showed moderate to severe hepatic fibrosis of central vein and hepatic sinus. In the endothelial progenitor cell group, hepatic sinusoidal and central venous endothelial cell injuries, and the fibrosis of central hepatic vein and hepatic sinus were mild to moderate. Hepatic venous congestion was reduced in comparison with that in the mice of the monocrotaline group. Compared with the endothelial progenitor cell group, the liver index was higher, the liver function was more abnormal, and the serum expression levels of TNF-α and IL-6 were higher in the monocrotaline group. The monocrotaline-induced damage of hepatic sinusoidal and central venous endothelial cells is an linitiating factor for hepatic vein occlusive disease. Infusion of endothelial progenitor cells can play a role in preventing and treating hepatic vein occlusion.

Effects of endothelial progenitor cells on acute lung rejection after lung transplantation

Objective To estimate the effects of endothelial progenitor cell(EPC) on acute rejection after lung transplantation. Methods Twenty-four Wistar rats were randomly divided into three groups (n=8): a sham operation group (opening chest alone, group S), a control group[intravenous injection of phosphate buffer saline (PBS) after orthopedic left lung transplantation, group C] and an endothelial progenitor cell group(intravenous injection of EPC+PBS after orthopedic left lung transplantation, group EPC). The donor was SD rat. Total sixteen SD rats were used. The blood of three groups of recipient rats was collected from femoral artery on Day 7 after operation. Then the oxygenation index (PaO2/FiO2) was measured by blood gas analysis while the concentrations of serum inflammatory factor intercellular cell adhesion molecule-1 (ICAM-1) and lymphocytefunctionassociatedantigen-1 (LFA-1) were measured by ELISA. The lungs of sacrificed rats were taken. Then, partial lung tissues were used to measure the dry and wet tissues specific gravity. other part of lung tissues were used to measure concentrations of inflammatory factors interferon-γ(IFN-γ) and interleukin(IL)-2, IL-10 by enzyme linked immunosorbent assay (ELISA) and to evaluate reject reaction by hematoxylin-eosin (H-E) staining. Results Compared with the group C, PaO2/FiO2 was increased whereas the wet/dry weight ratio and protein concentrations were decreased in group EPC(P<0.05). The levels of serum ICAM-1, LFA, IFN-γ and IL-2 in transplanted lung tissues were down-regulated, but the level of IL-10 was up-regulated in group EPC(P<0.05). The reject reaction score of group C (4.3±1.1) and group EPC(2.4±0.8) were higher than the score in group S(0). Conclusions EPC is able to relieve lung acute reject reaction after lung transplantation. Key words: Lung transplantation; Acute rejection; Endothelial progenitor cell

Infusion of Mesenchymal Stem Cells Combined with Epithelial Progenitor Cells Promotes Injured Intestinal Repairing after Hematopoietic Cell Transplantation

Pre-transplant chemoradiotherapy can impair intestinal barrier function and disrupt immune homeostasis, and thus increase the incidence of infection and other related complications. Mesenchymal stem cells (MSCs) have the potential to rescue Inflammatory Bowel Disease and intestinal graft versus host disease owing to their immunosuppressive capabilities. However, limited studies regarding MSC administration has been reported to aggravate T cell-mediated tissue injury. Plus, endothelial progenitor cells (EPCs) could improve endothelium repair, facilitate hematopoietic reconstitution and alleviate complications associated with hematopoietic cell transplantation (HCT). The key goals of this study were to i) identify the role of MSC derived soluble and contact dependent factors and how these affect intestinal injury; ii) investigate the effect of MSC combined EPC therapy for repairing the injured intestine. In this study, BALB/c mice were randomly divided into five groups, namely, total body irradiation only, bone marrow transplantation (BMT), MSC(BMT with 1 × 106MSC infusion), EPC(BMT with 5 × 105 EPCs infusion), and MSC+EPC (BMT with 1 × 106 MSC and 5 × 105 EPCs infusion). Results showed that the best performance of intestine was found in the MSC+EPC treated group, which showed more epithelial and goblet cells, and less apoptosis cells and adjacent crypts fusion in intestine morphology. EPC or MSC only group improved the intestinal injuries slightly compared with BMT group. The higher MECA-32 expression level was found in the intestinal tissue of MSC+EPC and MSC groups compared with other treated groups. The tight junction molecules occludin had highest expression level in the intestinal epithelial cells of MSC+EPC group, followed by MSC group and then EPC groups, but the expression level in BMT group was extremely low. Further study demonstrated that in MSC+EPC group the phosphorylated P38 enhanced heat shock protein HSP27 activation, which promoted cytoskeleton reconstruction and intestinal epithelial cells proliferation; and phosphorylated P38 also down-regulated the expression of apoptosis-related molecule caspase3. Moreover, the multiple effects of MSCs on cellular immunity may reflect their diverse influences on the different T-cell subpopulations. MSC+EPC infusion increased the number of IL-17A secreting cells (Th17 and Tc17) in mesenteric lymph nodes early after HCT, and decreased Th1 cells at day 10 and delayed expanding of Tc1 from day 10 to day 15. The soluble IL-17A in intestinal tissue was significantly increased after MSC+EPC infusion in according with IL-17A secreting cells in mesenteric lymph nodes. Furthermore, the gut bacterial information analyzed by high throughput sequencing uncovered that MSC and MSC+EPC groups had higher bacterial diversity and richness compared with other groups, and the bacterial community structures exhibited big changes regarding different treatment. Co-occurrence analysis demonstrated that the genus Akkermansia inducing PD-1 expression of T cells had significant correlation with phosphorylation of P38 and HSP27 in MSC+EPC and EPC treated groups, which indicated Akkermansia may be a key bacteria participanting the intestinal repairing in MSC and MSC+EPC groups. In conclusion, this study confirmed that the MSC+EPC infusion can obviously repair injured intestine, and gut bacteria Akkermansia correlated with phosphorylation of P38 and HSP27 expression and participated in intestinal repairing. Disclosures No relevant conflicts of interest to declare.

Experimental Study of Anti-Interventional Endothelial Progenitor Cells Combined with Mesenchymal Stem Cells in Large Femoral Defects

Mesenchymal stem cells (MSCs) and vascular endothelial progenitor cells (EPCs) have the potential for self-renewal and multi-directional differentiation. Their combination therapy is beneficial for treating orthopedic diseases. However, the role of antibiotic-interventional EPCs combined with MSCs in large femoral defects remains unclear. Rabbit MSCs and EPCs were isolated and EPCs were cultured in the presence of 1% green chain double antibody. The femoral segmental bone defect model was prepared and randomly divided into control group, MSCs group, EPCs group, EPCs + MSCs group, and autologous oxygen release nano-bionic scaffolds were combined with MSCs, EPCs, EPCs + MSCs followed by analysis of osteophytes by HE staining, bone density, ALP activity, TGF-β1 secretion by ELISA, and level of VEGF, Runx2 and OC by Real time PCR. MSCs, EPCs and combined treatment for large femoral bone defects can significantly improve osteophyte growth, promote bone density, increase ALP activity and TGF-β1 secretion, as well as increase VEGF, Runx2 and OC expression. Compared with control group, there was a significant difference (P &lt; 0.05) with combined treatment group having more significant effect on bone repair. Antibioticintervention of EPCs combined with MSCs promotes osteogenic activity and bone repair ability in large femoral defects.