Objective
The objective of the current proposal is to produce stable, immortalized lines of human endothelial cells, safe for use, which retain their functional properties and have been fully evaluated for pharmacological and toxicological testing. This objective will require innovative strategies for "minimal immortalization" where metabolic disruption induced by the immortalizing gene product is engineered down to a minimum.
The endothelium constitutes the internal lining of the circulatory system and plays a pivotal role in the maintenance of the vessel wall integrity. The alteration of the functional properties of endothelial cells is the first step in the development of cardiovascular diseases such as astherosclerosis and myocardinal infarction. The possibility to isolate and study these cells invitro is limited by their short life span. This work concerns the possibility to immortalize cultured human endothelial cells by inserting specific oncogenes.
The first objectives of this work include immortalization of endothelial cells with retroviral vectors; construction of new viral vectors; and establishment of functional assay system for endothelial cells.
Results achieved to date are:
two human endothelial cell lines transfected with retroviral constructs and two distinct oncogenes have been developed and have been characterized for specific markers and for morphological parameters; plasmid vectors have been prepared; functional assays for evaluating a series of functional activities of endothelial cells have been set up; assays for the screening of substances with potential toxicology effects have been established.
Human endothelial cells (HEC) in culture are a vital cellular model in the study of vascular diseases. The development of HEC lines retaining their differentiated characteristics would prove an invaluable tool for pharmacological and toxicological studies. Previous attempts have resulted in HEC lines either with substantially altered properties or, when amphotropic pseudotypes of murine sarcoma virus were used, not fully safe in terms of infection of the workers. The current proposal describes innovative molecular biology strategies that will be used to test the effect of a series of genes with the potential to immortalize/transform cells. Three different approaches will be followed. The first two will use either retroviral or EBV vectors which will allow efficient introduction of the gene of interest into HEC and provide a screening method for a variety of oncogenes, growth factor genes and receptor genes. The third approach will use integrative
plasmids which will allow the manipulation of gene expression using heterologous promoters to achieve 'minimal immortalization' i.e. where metabolic disruption induced by the immortalizing gene product is engineered down to a mimimum. The HEC lines will be then characterized for their growth characteristics and specific endothelial markers. In addition a full pharmacological and toxicological assessment of the HEC lines' responses will be performed.
Fields of science
- medical and health sciencesclinical medicineangiologyvascular diseases
- natural sciencesbiological sciencesmicrobiologyvirology
- medical and health sciencesclinical medicinecardiologycardiovascular diseases
- medical and health sciencesbasic medicinetoxicology
- natural sciencesbiological sciencesmolecular biology
Topic(s)
Data not availableCall for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
20157 MILANO
Italy