To explore the changes of wall shear stress(WSS) effect on arterial endothelial cell(EC) apoptosis after reducing arterial blood flow. The reducing flow model was established in 60 rabbits. Endothelial stretched preparations were made at 8 different time intervals from 0 to 30 days. The apoptosis rate of arterial endothelial cells (AEC) was measured with TdT-mediated dUTP-biotin nick end labeling(TUNEL) method. The results showed that the apoptosis rate of AEC was significantly higher from 1 day to 7 days after decreasing WSS than that of control, which peaked on day 3. While with progressively increasing in WSS, the apoptosis rate restored to the level of control from 14 days to 30 days. These suggest that the apoptosis state of AEC might be markedly influenced by the changes of WSS. The persist decreasing of WSS may be the important factor which induces the cell apoptosis.
Animals ; Apoptosis ; Arteries ; cytology ; physiology ; Endothelium, Vascular ; cytology ; physiology ; Male ; Rabbits ; Regional Blood Flow ; Shear Strength

OBJECTIVETo investigate the variety of proliferating ability of umbilical endothelia (UE) transfected by plasmid pBABE-HYGR-hTERT.

METHODSUE was identified from two aspects: morphology and CD34 labeling technique. The plasmid was obtained and identified by alkali splitting and gel electrophoresis. Liposomes were used to transfect UE. RT-PCR based telomeric repeat amplification protocol (TRAP) assay was used to measure the telomerase activity of endothelia.

RESULTSUE arranged as "cobblestone" and were positive of CD34 labeling. Endothelia transfected by pBABE-HYGR-hTERT(HC) had an raised absorbance of 0.889. The shape of growth curve of HC was similar to UE. But the absorbance of MTT test and the amount of HC were prior to UE at every measuring time and the amount of HC increased four times within 8 days (P < 0.05).

CONCLUSIONThe transfection of pBABE-HYGRO-hTERT had greatly improved the proliferating abilities and activated the telomerase of UE.

Catalytic Domain ; genetics ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Humans ; Telomerase ; genetics ; Transfection ; Umbilical Veins ; cytology

Endothelium, Vascular ; cytology ; Humans ; Mucocutaneous Lymph Node Syndrome ; complications ; pathology ; Neovascularization, Pathologic ; Stem Cells ; cytology

The coupling between the endothelium and blood flow is an important biomedical problem and has drawn extensive research. Endothelial cells are known to adapt their shapes and functions in response to applied shear flow. Shear Stress being regarded as a primary triggering signal for cellular remodeling, it is important to understand the interaction mechanism between applied shear flow and endothelial cells. In present study we have established a theoretical model to simulate the coupling between the deformation of an endothelial cell and applied shear flow. A two dimensional computational fluid dynamic (CFD) is conducted to determine the local distributions of mechanical stress and pressure on cell surface. Our results show that: (1) the deformation of endothelial cell changes with alpha (corresponding to the shear stress imposed on cell surface by flow fluid). When alpha is greater than 0.021, the cell deformability increases greatly; (2) the distributions of stress and pressure on cell surface are not uniform, but the maximal shear stress and displacement are always at the top point of the cell. Meanwhile, we have measured the deformation of cultured human aortic endothelial cells (HAECs) exposed to shear flow by using a flow chamber. We found that the numerical results are well consistent with those of experiment. These results suggest that the non-uniformity distributions of mechanical stress and pressure on cell surface may play a particular role in the mechanism of cell activation and in the regulation of endothelial cells functions (modification of cytoskeleton, distributions of adhesion molecules, etc.). The present study offers a framework to facilitate the development of a comprehensive dynamic model for endothelial cells.
Aorta ; cytology ; Cells, Cultured ; Endothelium, Vascular ; cytology ; physiology ; Humans ; Models, Cardiovascular ; Stress, Mechanical

OBJECTIVE: To determine the biological traits and optimal condition for the induction and differentiation of endothelial progenitor cells from peripheral blood in healthy adults. METHODS: Mononuclear cells isolated from peripheral blood of healthy adults were cultured in M199 medium supplemented with VEGF, bFGF, IGF-1, and EGF. The appearing time of cell clusters or spindle-shaped cells was recorded respectively. Attached spindle-shaped cells were detached and labeled with a series of antibodies against blood vessel endothelial-specific markers. RESULTS: Attached spindle-like cells appeared 4 days after the culture, cell clusters were observed at 5 to 8 days, and cord-like structure was formed by 10th day. These cells expressed endothelial-specific markers. CONCLUSION Endothelial progenitors cells were derived from mononuclear cells of peripheral blood, which can be induced into endothelial cells at specific conditions.
Cell Differentiation ; Cell Separation ; Endothelial Cells ; cytology ; Endothelium, Vascular ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Stem Cells ; cytology

AIMIn order to establish a coculture system of ECs and SMCs and by which further study can be done.

METHODSECs in primary culture were grown on a side of Transwell membrane, and SMCs were grown on an other side of it or the bottom of culture well, so that two kinds of coculture systems were established, and detail observation on the coculture systems was carried out by transmission and scanning electron microscope.

RESULTSECs in primary culture were positive of VI factor by immunocytochemistry staining. ECs and SMCs were grown well on both sides of Transwell membrane, relative to ECs monolayer of "cobblestone appearance", SMCs were multilayer of "hills and valleys appearance". ECs and SMCs on both sides of Transwell membrane could form the gap junctions by micropores.

CONCLUSIONThe coculture systems of ECs and SMCs were established successfully by modeling the structural relationship of vascular wall.

Animals ; Aorta ; cytology ; Cell Communication ; Coculture Techniques ; Endothelial Cells ; cytology ; Endothelium, Vascular ; cytology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; Rabbits

In order to further investigate the effect of annexin II (Ann-II) on tissue plasminogen activator (t-PA)-dependent plasminogen (PLG) activation and its interactive mechanism, recombinant native Ann-II bound t-PA, PLG and plasmin with high affinity was examined. The flow cytometric assay showed that the ann-II expression rate was higher in the human umbilical vein endothelial cell (HUVEC) (87.65%) than in the HL-60 cells as controls (35.79%). Two irrelevant proteins, bovine serum albumin (BSA) and equine IgG (EIG) had no effect on the production of plasmin. Ann-II-mediated enhancement of t-PA-dependent PLG activation was inhibited by epsilon-aminocaproic acid or by pretreatment of Ann-II with carboxypeptidase B with the inhibitive rate being 77.8% and 77.0%, respectively. It was revealed that the effect of Ann-II on PLG activation was specific for t-PA. Urokinase didn't bind to Ann-II, demonstrating the role of receptor-related lysine residues on activation of PLG, showing that the Ann-II-PLG interaction was dependent upon carboxyl-terminal lysine residues. These findings suggest that annexin II-mediated co-assembly of t-PA and PLG may promote plasmin generation and play a key role in modulating fibrinolysis on the endothelial surface.
Annexin A2/*pharmacology ; Cells, Cultured ; Endothelium, Vascular/cytology ; Endothelium, Vascular/*metabolism ; Fibrinolysis ; Plasminogen/*metabolism ; Recombinant Proteins/pharmacology ; Tissue Plasminogen Activator/*metabolism ; Umbilical Veins/cytology

The effects of 3, 4-Dihydroxyacetophenone (3, 4-DHAP) on cytosolic free calcium [Ca2+]i in pulmonary artery endothelia (PAECs) and smooth muscle cells (PASMCs) during acute hypoxia were studied. Porcine pulmonary artery endothelial and smooth muscle cells (PASMCs) were cultured primarily, and they were divided into 4 groups: groups incubated under normoxia or hypoxia and those with or without treatment with 3,4-DHAP. The [Ca2+]i of both PAECs and PASMCs was measured by determining the fluorescence of fura 2 AM on spetrofluorometer. Our results showed that hypoxia caused significant elevation of [Ca2+]i, in both PAECs and PASMCs, 3,4-DHAP could attenuate the hypoxic elevation of [Ca2+]i only in PASMCs but not in PAECs. It is concluded that 3,4-DHAP decreases the hypoxic elevation of [Ca2+]i in PASMCs. This might contribute to its inhibitory effect on hypoxic pulmonary vasoconstriction.
Acetophenones/*pharmacology ; Calcium/*metabolism ; Cell Hypoxia ; Cells, Cultured ; Endothelium, Vascular/cytology ; Endothelium, Vascular/*metabolism ; Muscle, Smooth, Vascular/cytology ; Muscle, Smooth, Vascular/*metabolism ; Pulmonary Artery/cytology ; Pulmonary Artery/metabolism ; Swine

AIMTo analyze gene expression difference between human mesenchymal stem cells and umbilical vein endothelial cells, to discuss the feasibility of inducing hMSCs to differentiate into endothelial cells through in vitro gene transfection as well as the use and prospective as a seeding cell source of vascular tissue engineering.

METHODShMSCs and hUVECs were isolated, expanded in culture, and characterized by flow-cytometry, immunocytochemistry, immunofluorescence and transmission electron microscopy (TEM). Differential analysis of gene expression profiles between them was performed by Biostar H-40 cDNA microarrays. The properties of VEGF 165 transfected were also detected with RT-PCR, ELISA et al.

RESULTSNearly 86% genes were coexpressed in both cells and hMSCs expressed typical endothelial antigen marker of EC. After VEGF165 transfection, hMSCs (or committed hMSCs) were positive for CD31. To different extent, the expression of CD44 was down regulated and CD34, FVIIIAg, Flt-1 up regulated.

CONCLUSIONGeneration of functional EC or tissue engineered blood vessels from human MSCs is feasible utilizing an in vitro environment gene transfection.

Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Endothelial Cells ; cytology ; Endothelium, Vascular ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Transfection ; Vascular Endothelial Growth Factor A ; administration & dosage

Endothelial injury, smooth muscle cells (SMCs) proliferation and migration are the same common pathophysiological processes of many cardiovascular diseases, such as atherosclerosis, hypertension, diabetes and restenosis. It is important to determine the functional interactions between endothelial cells (ECs) and SMCs under pathologic conditions. This work was to study the effects of ECs growth states on the proliferation and migration of vascular SMCs in cell coculture system. (3)H-TdR incorporation and flow cytometry were used to determine the effects of ECs growth states on the proliferation of SMCs. The number of migrating SMCs was counted. RT-PCR was used to analyze the expression of alpha-SM-actin mRNA. The results showed that (3)H-TdR incorporation decreased significantly from 14,900+/-1035 cpm/well in the control group to 8,575+/-749 cpm/well in the confluent ECs group (n=6, P<0.01), and increased to 27,268+/-2310 cpm/well in the proliferative ECs group ( n=6, P<0.01). The transition of SMCs from G(0)/G(1) phase to G(2)/M and S phases was blocked in the confluent ECs group but promoted in the proliferative ECs group. Compared with the control group, the number of migrating cells was about 4 times higher in the proliferative ECs group (n=6, P<0.01), while it in the confluent ECs group was only the half of the number of the control(n=6, P<0.05). The expression of alpha-SM-actin mRNA was increased significantly in the confluent ECs group(2.3+/-0.11 vs 1.4+/-0.12, P<0.05), but decreased significantly in the proliferative ECs group(0.92+/-0.08 vs 1.4+/-0.12, P<0.05). The results suggest that the biologic features of SMCs are influenced by ECs growth states. The proliferative ECs promote SMCs proliferation, migration and downregulate alpha-SM-actin mRNA expression significantly.
Actins ; metabolism ; Aorta ; cytology ; Cell Differentiation ; Cell Division ; Cell Movement ; Cells, Cultured ; Coculture Techniques ; Endothelium, Vascular ; cytology ; Epithelial Cells ; cytology ; Muscle, Smooth, Vascular ; cytology