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

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

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

BACKGROUNDThough there have been various methods for harvesting and preserving descemet membrane (DM) and intact endothelium, there is no literature about the morphological evaluation of endothelium after graft preparation for descemet membrane endothelial keratoplasty (DMEK). The aim of this study was to establish and improve a simple method for preparing, preserving, and morphologically evaluating the donor graft for DMEK.

METHODSTo obtain a donor graft, an air bubble was formed by injecting a 29 G needle with 1 ml sterile air into a small edge created outside the Schwalbe line. Another needle was inserted into the bubble through the stroma to aspirate the air or replace half the air with organ culture medium. Trypan blue was used to mark the location for small incision to improve the success rate. Frozen sections were stained with hematoxylin and eosin (HE). Based on the air bubble, DM grafts were divided into four groups: group A (normal control), graft without any operative technique; group B, graft with zero-pressure air bubble; group C, graft with full-pressure air bubble; group D, graft with half-pressure air bubble. The four groups of grafts were preserved for 24 hours to observe the effect of bubbles on cells. The gross and ultrastructure morphologies were evaluated using alizarin red and scanning electron microscopy (SEM), respectively.

RESULTSDonor grafts were harvested via the air bubble technique, facilitated by prior trypan blue staining. HE-stained sections revealed a pure graft without stroma. There were no significant changes under light microscope. In group A, SEM revealed a confluent layer of polygonal endothelium with distributed microvilli exhibiting characteristics of interdigitating junctions. In group B, intercellular borders became thinner. In group C, interdigitations were almost flat and microvilli were observed less frequently. In group D, other than less microvilli, there were minimal changes.

CONCLUSIONSThe donor graft preparation method appears to be effective and convenient. Properly decreasing the air pressure could protect and preserve the endothelium.

Animals ; Descemet Membrane ; cytology ; Descemet Stripping Endothelial Keratoplasty ; methods ; Endothelium, Corneal ; cytology ; Rabbits ; Tissue Donors

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

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

Angioplasty, Balloon, Coronary ; Coronary Restenosis ; prevention & control ; Endothelial Cells ; cytology ; Endothelium ; cytology ; Humans ; Stem Cells ; cytology ; Stents

PURPOSE: The goal of this study was to characterize the morphology of the mucinous layer on rabbit, bovine, owl, and human corneal endothelial cells. METHODS: Corneoscleral buttons were fixed using cetylpyridinium chloride to stabilize "mucus" and the tissue was prepared for transmission electron microscopy. Photomicrographs were measured to determine the thickness of the endothelial and epithelial mucinous layer in the central cornea. RESULTS: The endothelial mucinous layer was seen as a nearly uniform electrodense region on the apical aspect of the endothelium. It was found to be 0.9 microm, 0.9 microm, 0.9 microm, and 0.5 microm thick in rabbit, bovine, owl, and human, respectively. The owl endothelium had an additional less electrodense layer with a granular appearance and a thickness of about 200 microm. The mucinous layer on the epithelium was similar in appearance to that on the endothelium and across species. CONCLUSIONS: The morphologic similarity of the endothelial and epithelial mucinous layers is a serendipitous finding that should prove valuable in experimental design. Ultimately, it is hoped that studies of the posterior corneal surface will deepen our knowledge of endothelial protection.
Adult ; Animal ; Cytokines/pharmacology ; Endothelium, Corneal/ultrastructure ; Endothelium, Corneal/metabolism* ; Endothelium, Corneal/cytology ; Human ; Microscopy, Electron ; Mucins/ultrastructure ; Mucins/metabolism* ; Owls ; Rabbits ; Staining and Labeling

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