High-Yield Method for Isolation and Culture of Endothelial Cells from Rat Coronary Blood Vessels Suitable for Analysis of Intracellular Calcium and Nitric Oxide Biosynthetic Pathways

We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This methods makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed

Aspirin inhibits Chlamydia pneumoniae:Induced nuclear factor-kappa B activation, cytokine expression, and bacterial development in human endothelial cells

Objective-Chlamydia pneumoniae has been associated with atherosclerosis. Infection of vascular endothelial cells with C pneumoniae increases the expression of proatherogenic cytokines mediated by nuclear factor (NF)-kappaB, a transcription factor. The present study was designed to test the effect of aspirin on C pneumoniae-induced NF-kappaB activation, interleukin expression, and bacterial development in cultured human endothelial cells. Methods and Results-Aspirin, its metabolite salicylic acid, and 2 other unrelated NF-kappaB inhibitors showed a strong concentration-dependent inhibitory effect on chlamydial growth, indicated by the reduction of bacterial inclusions and the titer of infectious progeny. Involvement of the transcription factor NF-kappaB was confirmed by electrophoretic mobility shift assay and by transfection experiments with appropriate decoy oligodeoxynucleotides. Attenuation of the C pneumoniae-induced activation of NF-kappaB by aspirin also reduced the secretion of interleukin-6 and interleukin-8, indicating efficient inhibition of NF-kappaB gene expression. Reduction of chlamydial growth was not caused by apoptosis of the host cell, as determined by monitoring characteristic chromatin condensation. Conclusions-These data provide evidence that NF-kappaB-mediated gene activation represents a crucial step in the developmental cycle of C pneumoniae. Aspirin exerts an anti-chlamydial effect that is due to the inhibition of C pneumoniae-induced NF-kappaB activation, which might account for some of the cardioprotective activity of aspirin

Characterization of immortalized human umbilical and iliac vein endothelial cell lines after transfection with SV40 large T-antigen

Most in vitro studies of human endothelial cells have relied on cells derived from human umbilical veins (HUVEC); however, heterogeneity of primary cultured endothelial cells can make critical interpretation of results difficult. Several endothelial cell, lines have been produced to serve as a more constant source of endothelial cells. In this study, we characterized the endothelial cell lines EVLB3 and EVLC2 derived from HUVEC, and EVLK1 and EVLK2 derived from human iliac vein endothelial cells (HIVEC). These cell lines maintained the typical endothelial cell cobblestone morphology and appeared to be growth factor independent. They lost PECAM-1 and von Willebrand factor, GP96 was reduced to the level of vascular smooth muscle cells (SMC), but alpha SMC-actin was far less than in vascular SMC. Antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) were comparable with young endothelial cells, and mRNA was present for tPA, PAI-1, tissue factor (TF), tissue factor pathway inhibitor and thrombomodulin. This study revealed that mRNA and protein expression of coagulation and fibrinolytic factors was influenced by the stage of cell confluence. No differences could be detected between the endothelial cell lines derived from HUVEC and HIVEC. These cell lines may be a useful tool for studies on cellular interactions of fibrinolytic components or exploring the regulation of TF expression. (C) 2000 Lippincott Williams & Wilkins

Characterization of immortalized human umbilical and iliac vein endothelial cell lines after transfection with SV40 large T-antigen

Most in vitro studies of human endothelial cells have relied on cells derived from human umbilical veins (HUVEC); however, heterogeneity of primary cultured endothelial cells can make critical interpretation of results difficult. Several endothelial cell, lines have been produced to serve as a more constant source of endothelial cells. In this study, we characterized the endothelial cell lines EVLB3 and EVLC2 derived from HUVEC, and EVLK1 and EVLK2 derived from human iliac vein endothelial cells (HIVEC). These cell lines maintained the typical endothelial cell cobblestone morphology and appeared to be growth factor independent. They lost PECAM-1 and von Willebrand factor, GP96 was reduced to the level of vascular smooth muscle cells (SMC), but alpha SMC-actin was far less than in vascular SMC. Antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) were comparable with young endothelial cells, and mRNA was present for tPA, PAI-1, tissue factor (TF), tissue factor pathway inhibitor and thrombomodulin. This study revealed that mRNA and protein expression of coagulation and fibrinolytic factors was influenced by the stage of cell confluence. No differences could be detected between the endothelial cell lines derived from HUVEC and HIVEC. These cell lines may be a useful tool for studies on cellular interactions of fibrinolytic components or exploring the regulation of TF expression. (C) 2000 Lippincott Williams & Wilkins

Scanning electron microscopic analysis of endothelial cell coverage and quality in large vessels from multi-organ donors:effects of preservation on endothelial cell integrity

Endothelial cell integrity (coverage and quality) of large donor vessels is important because these vessels are used for vascular reconstructions in solid-organ transplantation. Disruption of the endothelial cell monolayer will initiate blood coagulation and may lead to thrombosis of large vessels, often resulting in the loss of the transplanted organ. Iliac arteries and veins, removed from 10 heart-beating multi-organ donors at the end of the donor procedure. were analyzed using scanning electron microscopy at three different rime points of preservation. Endothelial cell coverage and quality were determined immediately after removal from the donor, after 10 h (time of transplantation) and 7 d storage in 'University of Wisconsin' cold preservation solution (UW). Endothelial cell coverage decreased during the preservation of arteries, but was maintained in veins. Storage of the veins for 7 d in plastic bags showed a decreased endothelial cell coverage compared to storage in glass vials. Early removal of the blood vessels and proper storage, free floating and in clean UW, may improve maintenance of the endothelial cell integrity. These findings may be important in order to reduce the risk of thrombosis and, consequently, organ failure after transplantation. Furthermore, vessels with maintained endothelial cell integrity after 7 d may be used for in vitro research