Herein, we describe a protocol for the isolation of human embryonic stem cells (hESCs)-derived vascular cells at various stages of development. The cells are isolated from 10 to 15-d-old human embryoid bodies (EBs) cultured in suspension. After dissociation, cells are labeled with anti-CD34 or anti-CD31 (PECAM1) antibody and separated from the cell mixture by magnetic-activated cell separation (MACS) or fluorescent-activated cell sorting (FACS). Isolated vascular cells are then cultured in media conditions that support specific differentiation and expansion pathways. The resulting vascular cell populations contain > 80% endothelial-like or smooth muscle-like cells. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 1.5 months. Vascular cells isolated and differentiated under the described conditions may constitute a potential cell source for therapeutic application toward repair of ischemic tissues, preparation of tissue-engineered vascular grafts and design of cellular kits for drug screening applications.
The interaction between the vascular endothelium and hemodynamic forces (and more specifically, fluid shear stress), induced by the flow of blood, plays a major role in vascular remodeling and in new blood vessels formation via a process termed arteriogenesis. Tie1 is an orphan tyrosine kinase receptor expressed almost exclusively in endothelial cells and is required for normal vascular development and maintenance. The present study demonstrates that Tie1 expression is rapidly down-regulated in endothelial cells exposed to shear stress, and more so to shear stress changes. This down-regulation is accompanied by a rapid cleavage of Tie1 and binding of the cleaved Tie1 45 kDa endodomain to Tie2. The rapid cleavage of Tie1 is followed by a transcriptional down-regulation in response to shear stress. The activity of the Tie1 promoter is suppressed by shear stress and by tumor necrosis factor alpha. Shear stress-induced transcriptional suppression of Tie1 is mediated by a negative shear stress response element, localized in a region of 250 bp within the promoter. The rapid down-regulation of Tie1 by shear stress changes and its rapid binding to Tie2 may be required for destabilization of endothelial cells in order to initiate the process of vascular restructuring.
Ischemic heart diseases put a heavy economical burden on Western society. They remain one of the major causes of morbidity, and preventive or postoperative treatments are lengthy and expensive. In some patients of ischemic heart diseases, there is not a direct correlation between the degree of occlusion of major arteries and the development of medical symptoms or damage to the heart function. Interestingly, these patients develop well-formed collateral vessels that compensate for the decrease in blood supply to the heart wall. Clearly, the ability to understand why and how these patients develop collateral vessels may serve as a base for a new strategy to treat ischemic heart diseases by promoting collateral formation. The current article summarizes recent advances in the understanding of how collateral vessels develop and offers the authors' point of view on the central role of biomechanical forces in this process and the molecular mechanisms that underline it.
Heterophilic antibodies are well described, but poorly appreciated interferents and is often not a recognized problem affecting most immunoassays. We report a patient presented with ectopic Cushing's syndrome (CS), but repeated plasma adrenocorticotropic hormone (ACTH) concentrations conducted by immunoassay were inappropriately within the reference range and not elevated, most probably as a result of antibody interference. A 36-year-old woman, presented with large gastric neuroendocrine carcinoma and severe ectopic CS, while repeated plasma ACTH concentrations conducted by immunoassay were inappropriately within the reference range. As we expected ACTH concentration to be higher, we performed several tests to evaluate whether there was any assay interference causing falsely lower than expected ACTH results. We measured ACTH using a different immunoassay, assayed the sample in dilution, assayed the sample after being incubated in heterophilic antibody blocking agent tube and performed recovery studies. Tests indicated the presence of interfering compounds, most probably heterophilic antibodies. When clinicians find ACTH concentrations to be lower than expected, we recommend the laboratory investigate antibody interference.
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