OBJECTIVETo investigate the effects of Salvianolic acid B preconditioned endothelial progenitor cells (EPCs) on the Nkx2.5 and GATA-4 gene expressions at the early stage of cell differentiation of bone mesenchymal stem cells (BMSc) transplanted into infarcted myocardium, in order to find out the best synergism for co-transplantation of the two kinds of cells.

METHODSBMSc and EPCs of rats were isolated and cultured, and rats were modeled into acute myocardial infarction (AMI) by left coronary artery ligation. Then the EPCs preconditioned with different concentrations of Salvianolic acid B and BMSc or DMEM medium were implanted into heart ischemia area. Expressions of Nkx2.5 and GATA-4 mRNA expressions in myocardium were detected by Real-time RT-PCR 4 weeks later.

RESULTSCompared with those in the non-implanted model rats' myocardium, the gene expression of Nkx2.5 and GATA-4 mRNA were significantly higher in all the transplantation receptive groups, comparisons between the implanted groups showed that the highest value of expressions (2. 654 +/- 0.606 of Nkx2.5 and 1.573 +/- 0.372 of GATA-4) displayed in the group contained more EPCs, for 8-fold to BMSc in volume.

CONCLUSIONBMSc can differentiate into cardiac muscle like cells, and condition of their differentiation is related with the degree of the internal environment improved.

Animals ; Benzofurans ; pharmacology ; Cells, Cultured ; Endothelial Cells ; cytology ; drug effects ; transplantation ; Gene Expression ; drug effects ; Male ; Mesenchymal Stem Cell Transplantation ; Myocardial Infarction ; metabolism ; therapy ; Myocardium ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Stem Cell Transplantation ; Stem Cells ; cytology ; drug effects

OBJECTIVETo observe the proliferation state of transplanted cells in acute myocardial infarction (AMI) rats, and the endothelial progenitor cells (EPCs) preconditioned by salvianolic acid B in different ratios with the bone mesenchymal stem cells (BMSCs).

METHODSThe cultivation and purification of EPCs were performed by density-gradient centrifugation and plastic adherence method. Two types of cells were identified by immunocytochemical method (CD34, CD133, and CD44). The rat model of AMI was prepared by ligation of left anterior descending artery. The EPCs were pre-treated with the optimal concentration of salvianolic acid B (8 microg/ mL). They were mixed with BMSCs in different proportions (EPCs/BMSCs in the ratio of 1:1, 2:1, 4:1, and 8:1, respectively). BMSCs and EPCs were injected into the myocardial infarction area. The infarcted area was determined by the N-BT staining and hematoxylin-eosin staining. The expression of Ki-67 was detected by immunohistochemical assay.

RESULTSCompared with the model group (19.60% +/- 3.23%), the myocardial infarction area of each implanted group obviously decreased (P < 0.05). Of them, the decrease was most obvious in the 4:1 group (11.37% +/- 2.18%) and the 8:1 group (9.23% +/- 2.35%, P < 0.05). Compared with the model group (cell/high magnification, 5.17 +/- 2.31), the Ki-67 positive cell number of each implanted groups significantly increased (P < 0.05). Of them, the Ki-67 positive cell number was obviously higher in the 8:1 group (15.00 +/- 3.16, P < 0.05).

CONCLUSIONSEPCs pretreated by salvianolic acid B combined with BMSCs could reduce the myocardial infarcted area, improve the proliferation of BMSCs in the peripheral infarction and local ischemia. Besides, along with the increase of the implant proportion of EPCs, the infarct area was gradually reduced, and the proliferative expression was gradually enhanced.

Animals ; Benzofurans ; pharmacology ; Bone Marrow Cells ; cytology ; drug effects ; Cell Proliferation ; Endothelial Cells ; cytology ; drug effects ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Myocardial Infarction ; metabolism ; pathology ; Rats ; Rats, Wistar ; Transplantation Conditioning

OBJECTIVETo compare the serum miR-663 levels in renal transplant patients with and without acute rejection (AR) and explore the role of miR-663 acute renal graft rejection.

METHODSReal time-PCR was used to determine serum miR-663 levels in renal transplant recipients with and without AR. MTT assay and Annexin V-FITC assay were employed to examine the viability and apoptosis of human renal glomerular endothelial cells (HRGEC) treated with a miR-663 mimic or a miR-663 inhibitor, and ELISA was performed to detect the expression of inflammation-related cytokines including IL-6, IFN-γ, CCL-2 and TNF-α in the cells. Transwell assay was used to examine the effect of miR-663 mimic and miR-663 inhibitor on the chemotactic capability of macrophages.

RESULTSSerum miR-663 level was significantly higher in renal transplant recipients with AR than in those without AR. The miR-663 mimic significantly inhibited the viability of HRGECs and increase the cell apoptosis rate, while miR-663 inhibitor suppressed the cell apoptosis. The miR-663 mimic increased the expression levels of inflammation-related cytokines and enhanced the chemotactic capability of macrophages.

CONCLUSIONmiR-663 might play important roles in acute renal graft rejection and may become a therapeutic target for treating AR.

Apoptosis ; Cells, Cultured ; Cytokines ; metabolism ; Endothelial Cells ; cytology ; Graft Rejection ; blood ; Humans ; Kidney Glomerulus ; cytology ; Kidney Transplantation ; Macrophages ; cytology ; drug effects ; MicroRNAs ; blood

Recent studies have shown that side population (SP) cells, isolated from adult myocardium, represent a distinct cardiac progenitor cell population that exhibits functional cardiomyogenic differentiation. However, information on the intrinsic characteristics and endothelial potential, of cardiac SP cells, is limited. The present study was designed to investigate whether cardiac SP cells exhibit endothelial differentiation potential. The cardiac SP cells more highly expressed the early cardiac transcription factors as well as endothelial cell markers compared to the bone marrow-SP cells. After treatment with VEGF, for 28 days, cardiac SP cells were able to differentiate into endothelial cells expressing von Willebrand factor as determined by immunocytochemistry. Furthermore, expression of endothelial cell markers increased several-fold in VEGF-treated cardiac SP cells compared to the control group when assessed by real-time PCR. We also confirmed that cardiac SP cells provided a significantly augmented ratio of ischemic/normal blood flow, in the cardiac SP cell-transplanted group compared with saline-treated controls on postoperative days 7, 14, 21 and 28, in a murine model. These results show that cardiac SP cells may contribute to regeneration of injured heart tissues partly by transdifferentiation into angiogenic lineages.
Animals ; Base Sequence ; Bone Marrow Cells/cytology/drug effects ; Cell Differentiation/drug effects ; Cell Separation ; Colony-Forming Units Assay ; DNA Primers/genetics ; Endothelial Cells/*cytology/drug effects/metabolism/transplantation ; Mice ; Mice, Inbred BALB C ; Myocardium/*cytology/metabolism ; Vascular Endothelial Growth Factor A/pharmacology

CD146 is a newly identified endothelial biomarker that has been implicated in angiogenesis. Though in vitro angiogenic function of CD146 has been extensively reported, in vivo evidence is still lacking. To address this issue, we generated endothelial-specific CD146 knockout (CD146(EC-KO)) mice using the Tg(Tek-cre) system. Surprisingly, these mice did not exhibit any apparent morphological defects in the development of normal retinal vasculature. To evaluate the role of CD146 in pathological angiogenesis, a xenograft tumor model was used. We found that both tumor volume and vascular density were significantly lower in CD146(EC-KO) mice when compared to WT littermates. Additionally, the ability for sprouting, migration and tube formation in response to VEGF treatment was impaired in endothelial cells (ECs) of CD146(EC-KO) mice. Mechanistic studies further confirmed that VEGF-induced VEGFR-2 phosphorylation and AKT/p38 MAPKs/NF-κB activation were inhibited in these CD146-null ECs, which might present the underlying cause for the observed inhibition of tumor angiogenesis in CD146(EC-KO) mice. These results suggest that CD146 plays a redundant role in physiological angiogenic processes, but becomes essential during pathological angiogenesis as observed in tumorigenesis.
Animals ; CD146 Antigen ; genetics ; metabolism ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Female ; Fibrosarcoma ; metabolism ; pathology ; Male ; Melanoma, Experimental ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; NF-kappa B ; metabolism ; Neovascularization, Physiologic ; drug effects ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Retinal Vein ; growth & development ; pathology ; Signal Transduction ; drug effects ; Transplantation, Homologous ; Vascular Endothelial Growth Factor A ; pharmacology ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

OBJECTIVETo study the anti-tumor angiogenesis effect of soluble VEGF receptor fragment by blocking the combination of VEGF and its receptor in vivo and in vitro.

METHODSRT-PCR technique was used to amplify Flk-1/KDR fragment from embryo mouse liver, which was recombinated to expression vector pET-28b(+) and retrovirus vector PLXSN, which was induced to be expressed, purified and identified with EcoR I and Hind III. Mouse endothelial cells were separated, cultured and identified by immunocytochemistrical staining using VIII factor-related antigen antibody. The expressed product was analyzed about its effect on endothelial cell's growth in vitro with MTT method. The retrovirus vector was transfected to tumor cell lines S180 and B16 by liposome method to observe the biological specificity in vitro after gene transfection.

RESULTS1000 bp size sVEGFR fragment was amplify from E9, E11 embryo mouse liver tissues, which was recombinated to TA clone vector and identified by sequence analysis. This fragment was cloned to expression vector pET-28b(+), the expressed product was purified and identified correctly. The in vitro study showed this expressed product can effectively inhibit endothelial cell(s), growth and proliferation. The fragment was then cloned to retrovirus vector PLXSN and transfected to tumor cell lines S180 and B16 successfully with RT-PCR and SDS-PAGE. The experiments in vivo showed that the weight of tumor smaller, the size decreased significantly, the microvessel density was fewer and Flk1 protein expression were higher in the group of gene transfection than that of control.

CONCLUSIONSoluble VEGFR fragment is a kind of effective gene engineer product for anti-tumor angiogenesis gene therapy and the development of anti-tumor drug.

Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cloning, Molecular ; Endothelial Cells ; cytology ; Genetic Vectors ; Melanoma, Experimental ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Neovascularization, Pathologic ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Retroviridae ; genetics ; Sarcoma 180 ; metabolism ; pathology ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics ; physiology

OBJECTIVEThe growth and metastasis of solid tumors are dependent on angiogenesis. Endostatin, the C-terminal proteolytic fragment of collagen XVIII, is a potent endogenous angiogenesis inhibitor. The authors designed a topical antiangiogenic gene therapy with recombinant human endostatin adenovirus (Ad-hEndo) and assessed its effects on the inhibition of angiogenesis in vitro, and tumor growth and metastasis in vivo.

METHODSMalignant cells (A549) were infected with Ad-hEndo. The expression of recombinant protein and the inhibition of cultured human umbilical vein endothelial were investigated. Immunodeficient A549 nude mice were treated with intratumoral injection of Ad-hEndo, the empty vector Ad-control or saline (NS). The dose-response, side effects, and serum concentration of endostatin were observed.

RESULTSRecombinant endostatin protein was detected in the infected tumor cells with different MOI Ad-hEndo and its inhibitory effect on endothelial cells growth was shown. In animal study, the volume of tumor and the number of pulmonary metastatic lesions in the Ad-hEndo treatment group were significantly smaller than those in the control groups (P<0.05).

CONCLUSIONThe present findings provide evidence of the anti-tumor effects of the endostatin and may be important for the further use of it in topical antiangiogenic gene therapy of cancer.

Adenoviridae ; genetics ; Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Disease Models, Animal ; Endostatins ; biosynthesis ; genetics ; therapeutic use ; Endothelial Cells ; Genetic Therapy ; Genetic Vectors ; Humans ; Kidney ; cytology ; metabolism ; Mice ; Mice, Nude ; Neoplasm Metastasis ; Neoplasm Transplantation ; Neoplasms, Experimental ; prevention & control ; Random Allocation ; Recombinant Proteins ; biosynthesis ; genetics ; therapeutic use ; Transfection ; Umbilical Veins ; cytology ; Xenograft Model Antitumor Assays

OBJECTIVETo observe and analyze the antitumor effect of endostar combined with docetaxel under different administration sequences.

METHODSNude mice with xenograft tumor (A549 cell line) were randomized into 3 groups, 8 mice/group: (1) Concurrent administration group (each mouse: endostar 400 µg/d, d1-d35, docetaxel 10 mg/kg, every 3 days, d1-d19); (2) Endo-first group (each mouse: endostar 400 µg/d, d1-d35, docetaxel 10 mg/kg, every 3 days, d16-d34); (3) Model group (positive control, tumor-bearing mice without treatment, each mouse: physiological saline, 100 µl/d, d1-d35, water for injection, 200 µl/d, d1-d35, every 3 days), and blank control group (negative control, normal mice without treatment, 8 mice), the administration method was the same to the model group. The volume of tumor and the weight of mouse were measured during treatment. Circulating endothelial cells (CECs) were detected by flowcytometry, and the expression of matrix metalloproteinase (MMP-2, MMP-9), the tissue inhibitor of MMP (TIMP-1, TIMP-2), the extracellular MMP inducer (EMMPRIN), CD34, α-smooth muscle actin (α-SMA) were determined by immunohistochemistry.

RESULTSThe tumor growth of concurrent administration group (39.94 mm(3)) was lower than that of the endo-first group [(99.57 ± 74.48) mm(3)] during treatment, both of them were smaller than that of the model group [(217.67 ± 95.44) mm(3), P < 0.05]. The amount of CECs in the endo-first group [(77.25 ± 24.02) cells/10(4) cells] was more than that of the concurrent administration group [(25.86 ± 11.77) cells/10(4) cells], the model group [(14.71 ± 11.07) cells/10(4) cells], and the blank control group [(12.90 ± 11.20) cells/10(4) cells, P < 0.01]. The expression of MMPs in the treatment groups was obviously downregulated. The expressions of TIMP-1 in the endo-first group and TIMP-2 in the concurrent administration group were upregulated (P < 0.05). The expression of EMMPRIN was significantly down-regulated in the concurrent administration group (P < 0.05). The MVD and α-SMA expressions of the treatment groups were less than that of the model group (P < 0.05).

CONCLUSIONIn comparison with the endo-first group, the anti-tumor effect and survival quality of the concurrent administration group are better. Both of the administration groups may have "vascular normalization effect" by down-regulating MMPs expression through different points, and inhibit the cancer-induced stromal reaction, restraining the cancer progress to a certain extent. The changes of CECs should be a dynamic process with an initial rise in the early-stage suggesting the decrease of vascular bed and subsequent decline ascribed to apoptosis of CECs and the tumor-regression after combined therapy. Investigation of its dynamic changes may be helpful to know the change of tumor burden and vascular bed and predict the antitumor effect.

Actins ; metabolism ; Angiogenesis Inhibitors ; administration & dosage ; pharmacology ; Animals ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Basigin ; metabolism ; Cell Line, Tumor ; Drug Administration Schedule ; Endostatins ; administration & dosage ; pharmacology ; Endothelial Cells ; cytology ; Female ; Lung Neoplasms ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microvessels ; drug effects ; Neoplasm Transplantation ; Taxoids ; administration & dosage ; pharmacology ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Tumor Burden ; drug effects

Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-β in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-β promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.
Acute Disease ; Adolescent ; Adult ; Angiogenesis Inducing Agents ; immunology ; metabolism ; pharmacology ; Angiopoietin-1 ; genetics ; immunology ; pharmacology ; Angiopoietin-2 ; genetics ; immunology ; pharmacology ; Antineoplastic Agents ; therapeutic use ; Female ; Gene Expression Regulation, Neoplastic ; Graft vs Host Disease ; genetics ; immunology ; pathology ; Hematopoietic Stem Cell Transplantation ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; immunology ; Humans ; Leukemia, Myeloid ; genetics ; immunology ; pathology ; therapy ; Lymphoma, Non-Hodgkin ; genetics ; immunology ; pathology ; therapy ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology ; pathology ; therapy ; Retrospective Studies ; Signal Transduction ; Transplantation, Homologous ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Endothelial Growth Factor A ; genetics ; immunology