Endothelium : journal of endothelial cell research最新文献

Observations have been made linking the presence of psychosocial factors associated with elevated beta-endorphin concentrations with atherosclerosis. In this study, the authors assume an important role of the stress hormone beta-endorphin in several mechanisms that contribute to a dysbalance of human endothelial and monocytic endothelin (ET)-1 and nitric oxide (NO) release, mediated by mu1-opioid receptors. ET-1 and NO release were quantified via enzyme-linked immunosorbent assay (ELISA) or fluorometrically. mu1-Opioid receptors were identified by polymerase chain reaction (PCR) after stimulation with beta-endorphin. beta-Endorphin significantly increased endothelial and monocytic ET-1 release. The effect was mediated by mu1-opioid receptors and abolished by naloxonazine, a selective mu1-opioid receptor antagonist. In contrast, NO release was decreased under the influence of beta-endorphin. mu1-Opioid receptors on human monocytes and endothelial cells mediated a beta-endorphin-induced stimulation of ET-1 release, whereas NO release was decreased. Thus, the authors hypothesize a role of beta-Endorphin in the pathogenesis of stress-induced endothelial dysfunction through peripherally circulating beta-endorphin, which may offset the balance of vasoactive mediators, leading to an unopposed vasoconstriction. The data may also provide a new concept of mu1-opioid receptor antagonists, preventing beta-endorphin-induced disorders of vascular biology.

Vasospasm is a complication of cardiological procedures such as balloon angioplasty and may be related to vascular oxidant stress. Although nitric oxide donor drugs are often administered to prevent vasospasm, the response to these drugs in balloon-injured arteries has not been studied. Pig coronary arteries were balloon-injured in vitro and relaxations to nitric oxide (NO)-donating and NO-independent vasodilators studied. Generation of superoxide in response to injury was assayed using dihydroethidium. NO formation on addition of the NO donor drugs was studied using an amperometric sensor. Expression of nitrotyrosine, a peroxynitrite marker, was probed using immunocytochemistry. In vitro injury enhanced sensitivity to the NO donors SNAP and SpermineNONOate but blunted the response to isoprenaline or chromakalim. With both donors, NO formation was significantly enhanced in the presence of an injured vessel. Vascular superoxide generation was also increased throughout the vessel wall and a small increase in nitrotyrosine was detected in the injured vessel media following addition of SNAP. In conclusion, injured vessels were more sensitive to NO donors and this appears to be due to enhanced NO generation by the donor molecule. Increased formation of peroxynitrite within the injured vessel may contribute to the enhanced relaxation in injured vessels.

Historically, the etiology of local bone pathologies, such as avascular necrosis, has been related to intravascular occlusion. Recent reports have highlighted the occlusion of arteries, venules, and/or capillaries in bone tissue. Endothelium of bone presumably participates locally in the formation of the microvascular thrombosis. It is also known that endothelial cells (ECs) play a central role in angiogenesis, a process seen in osteosarcoma, amongst other bone diseases. Given the well-recognized heterogeneity of ECs throughout the body, investigations of local bone disease related to endothelium processes may be more appropriately targeted on bone ECs rather than other primary ECs or an immortalized EC line. In the current study, mechanical and enzymatic methods are described to isolate ECs from cancellous human bone tissue followed by immunomagnetic bead separation to purify the cell populations. The human bone-derived endothelial cells (hBDECs) were characterized based on endothelial cell antigen expression and functional assays. This study is the first report of isolation and expansion of ECs from human bone tissue. Isolation of hBDECs in human vascular bone diseases may facilitate the study of the molecular and/or genetic abnormalities in the vasculature system that contributes to the initiation and/or progression of the disease.

Mesenchymal stem cells (MSCs) are bone marrow-derived, pluripotent cells that possess the ability to transdifferentiate into various mesenchymal tissues such as bone, endothelium, and (heart) muscle. Therefore, these cells may provide a therapeutic tool, especially for the treatment of myocardial infarction. The interaction of the MSCs with the endothelial barrier and their ability to ultimately leave blood vessels after application are crucial in this context. In this study, the authors focused on the soluble factors produced by MSCs and their effect on the intracellular signal transduction of endothelial cells. The authors performed immunohistochemical measurements on human umbilical vein endothelial cells (HUVECs) treated with conditioned stem cell medium and took measurements of the intracellular nitric oxide (NO) levels and calcium changes. After application of conditioned stem cell medium, the authors detected an increase in endothelial NO synthase (eNOS) activity by translocation (Ca(2+)) and by phosphorylation (increase of pAKT and peNOS1177). Additionally, the authors observed an upregulation of pERK within the same time. The phosphorylated eNOS forms are linked to these findings and the increase of intracellular NO in the DAF measurements. Moreover, conditioned medium also increased intracellular calcium levels in endothelial cells. Concluding, the authors postulate that MSCs emit soluble factors that alter the NO and calcium levels of endothelial cells and may be important for facilitate crossing the endothelial barrier.

The relationships between cerebral and systemic endothelial (dys)function and between cerebral (dys)function and intima-media thickness (IMT) of carotid arteries in patients and healthy volunteers have not yet been clarified. In order to explore these issues, the authors performed a post hoc correlation analysis of cerebrovascular reactivity to L-arginine, a marker of cerebral endothelial function; flow-mediated dilatation (FMD), a marker of systemic endothelial function; and IMT of the carotid arteries, a marker of the extent of atherosclerosis. Correlations were analyzed in a heterogeneous group consisting of 20 patients with lacunar infarctions (LIs) and extensively impaired endothelial function, 21 patients with similar risk factors (SRs), but without LIs, and 21 healthy controls. Cerebrovascular reactivity to L-arginine was determined by the transcranial Doppler method (TCD), FMD by ultrasound measurements of the brachial artery after hyperemia, and IMT by measurement of the common carotid arteries. Analysis of correlations in the group of 62 subjects revealed that L-arginine reactivity, which was diminished in LI and SR patients, did not correlate with FMD, which was also diminished in both LI and SR patients (Rho = 0.10 with p = 0.458). On the contrary, a significant negative correlation was found between L-arginine reactivity and IMT (Rho = -0.30 with p = 0.015). In conclusion, our study investigating relations between cerebral and systemic endothelial dysfunction showed that cerebral endothelial function, determined by L-arginine reactivity, correlates well with the degree of atherosclerosis determined by IMT but does not correlate with FMD, suggesting that cerebral and systemic endothelial function may not be closely associated.

The therapeutic use of autologous cells with the capacity for extensive in vitro expansion and manipulation prior to host administration has been an area of significant investigation over the last decade. Blood outgrowth endothelial cells (BOECs) are derived from the circulation and exhibit proliferative growth, in vivo engraftment, and survival characteristics for long-term expression of endogenously secreted proteins, such as factor VIII (FVIII). The authors describe a modified method for the isolation, culture, and expansion of these cells that is readily accomplished using standard laboratory methods. Using a commercially available transfection reagent, approximately 30% of these primary cells can be routinely transfected with the nonviral Sleeping Beauty transposon for long-term, stable transgene expression. Moreover, the results indicate that these cells have the ability to secrete functionally active proteins that are synthesized endogenously by hepatocytes and require post-translational modification including alpha1-antitrypsin and clotting factors VII and IX. This, coupled with their notably long half-life of years, suggests that these cells may provide an appropriate vehicle for secretion of a variety of proteins produced by different cell types in vivo. Thus, BOECs have the potential to provide clinically relevant secreted proteins for diseases other than those of endothelial origin.

Vascular apoptosis-inducing proteins (VAPs) from hemorrhagic snake venom are apoptosis-inducing toxins targeting vascular endothelial cells. Well-characterized VAPs consist of disulfide-bridged double chains (ddVAPs). The authors previously described a single-chain VAP (scVAP), VAP2 from Crotalus atrox, which also induces apoptosis in endothelial cells (Masuda et al., 1998, European Journal of Biochemistry, 253, 36-41). The authors report here the whole cDNA sequences and some additional peptide characteristics of VAP2. In addition to the apoptosis-inducing activity of VAP2, the toxin displays a cell-detaching activity after incubation in high-salt conditions. These observations indicate that the apoptosis and cell-detaching functions can be discriminated. Analysis of the cell-detaching activity also revealed that VAP2 consists of two similar peptides, VAP2A and VAP2B, which are members of the PIII-type snake venom metalloproteases (SVMPs). The VAP2A cDNA encodes a 609-amino acid protein. In contrast, the peptide sequences of VAP2B were identical to that of catrocollastatin, an inhibitor of platelet aggregation. VAP2A and VAP2B interact with each other to form a noncovalent dimer similar to the ddVAPs, which was detected by native polyacrylamide gel electrophoresis. These data show some new characteristics of VAPs, which are important to clarify the apoptotic pathways in vascular endothelial cells.

Zoledronate exhibits antiangiogenic properties in vitro and in vivo. Integrins alphavbeta3 and alphavbeta5 are involved in angiogenesis. Because zoledronate inhibits endothelial cell adhesion, the authors explored the hypothesis that it could alter these integrins recruitment to focal adhesion sites. Human umbilical vein endothelial cells (HUVECs) were treated with zoledronate or with mevalonate pathway intermediates geranylgeraniol (GGOH) and farnesol (FOH). Zoledronate generated a significant decrease in alphavbeta3 and alphavbeta5 expression at HUVEC cell surface using flow cytometry and immunofluorescence. This inhibition was reversed by GGOH but not by FOH. Cells cotreated with zoledronate and GGOH were able to attach to vitronectin through alphavbeta3 and alphavbeta5, as confirmed by the use of specific function-blocking antibodies. The authors showed that zoledronate alters endothelial cell integrin-mediated adhesion. This effect is likely to contribute to the previously demonstrated antiangiogenic effect of zoledronate. Whether this mechanism of action also applies to metastatic tumor cells is under investigation.

Cells with an endothelial phenotype can be cultured from peripheral blood. These cells include cells of a monocytic origin with endothelial features (culture-modified mononuclear cells, CMMCs) and, at later time points, blood outgrowth endothelial cells (BOECs). Both are promising candidates for systemic cell-based cardiovascular therapies and each may have unique capabilities. Indeed, the combined use of both cell types has been shown to have synergistic therapeutic features requiring simultaneous delivery. However, the majority of preclinical studies of cell delivery have used splenectomized animals to increase systemic distribution. The goal of this study was to directly compare the distribution of these two cell types following systemic delivery in an intact animal model. A similar pattern of delivery was seen following delivery of both cell types with detection in the lung, liver, bone marrow, and spleen. Taken together, the data suggest that strategies using systemic delivery of circulation-derived cells must consider the distribution and efficiency of delivery in intact animals.