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Gene transfer into rat heart-derived endothelial cells
1Department of Cardiovascular Surgery, University of Kiel, Germany. MarcHein@compuserve.com
Objective:
Progressive graft arteriosclerosis is responsible for the majority of late deaths in cardiac transplant recipients. Despite many investigations, the pathogenesis of this disease remains undetermined and its control inadequate. A somatic gene transfer during the cold ischemic time and thus before transplantation might be a new therapeutic tool. This approach allows a long incubation time of the DNA and a safe transfer with liposomes and transferrin with less adverse effects for the organ recipient.
Methods:
The target cells (microvascular endothelial cells (MVECs)) for this gene transfer were isolated from rat hearts by perfusion with collagenase via an aortic cannulae. The cells were purified by changing the medium 30 min after subcultivation in order to remove fibroblasts and smooth muscle cells. The endothelial cells (ECs) were identified by typical morphology and the uptake of Dil-Ac-LDL. The gene transfer was carried out with a beta-galactosidase reporter plasmid (pCMVbeta), cationic liposomes (Lipofectin), and transferrin. Different transfection solutions were prepared with or without serum, and with different plasmid-liposome ratios and transferrin concentrations. The transfer rate was monitored with a semiquantitative orthonitrophenyl-beta/-D-galactoside (ONPG) assay and histologically by X-Gal staining. The cytotoxicity of this procedure was determined with a colorimetric ELISA with Alamar blue. The cardioplegic property of the transfection solution was tested in a Langendorff perfusion system monitoring the coronary blood flow over time after a cold ischemic time of 4 h.
Results:
The maximal gene expression could be detected after transfection with 4 microl Lipofectin, 2 microg pCMVbeta, and 16 microg transferrin/200 microl transfection solution. Under these conditions 60% of the cells showed a blue staining with X-Gal. Only 20% of the cells died during transfection. The lowest cytotoxicity during cold ischemic time for ECs was assessed with normal cell culture medium and the Buckberg solution. The best coronary flow rates after 4 h cold ischemia of the heart were measured for cardioplegia with St. Thomas and Buckberg solutions. In summary, the best transfection solution with a good cardioplegic property was the Buckberg solution.
Conclusions:
Finally, the results of this study show that an effective DNA delivery with a low toxicity into ECs is possible with a combination of liposomes and transferrin. This method might be useful for a safe and effective gene transfer into solid organs during the cold ischemic time and thus a therapeutic tool for chronic rejection.
Insights
Gene transfer using liposomes and transferrin effectively delivers DNA to endothelial cells with low toxicity. This method shows promise for preventing chronic rejection in cardiac transplant recipients.
Area of Science:
- Cardiovascular Research
- Gene Therapy
- Transplantation Immunology
Background:
- Progressive graft arteriosclerosis is a major cause of late mortality in cardiac transplant recipients.
- Current understanding of graft arteriosclerosis pathogenesis is limited, and effective treatments are lacking.
- Somatic gene transfer before transplantation offers a potential therapeutic strategy.
Purpose of the Study:
- To investigate somatic gene transfer into cardiac microvascular endothelial cells (MVECs) during cold ischemic time.
- To optimize a gene transfer method using liposomes and transferrin for efficacy and safety.
- To evaluate the cardioplegic properties of the optimized transfection solution.
Main Methods:
- Isolation and purification of rat cardiac MVECs.
- Gene transfer using a beta-galactosidase reporter plasmid with lipofectin and transferrin.
- Optimization of transfection solutions and assessment of gene transfer efficiency via ONPG assay and X-Gal staining.
- Cytotoxicity evaluation using Alamar blue assay and assessment of cardioplegic effects on coronary blood flow.
Main Results:
- Maximal gene expression achieved with specific concentrations of lipofectin, pCMVbeta plasmid, and transferrin.
- Transfection resulted in 60% gene-positive cells with only 20% cell death.
- Buckberg solution demonstrated good cardioplegic properties and was identified as the optimal transfection solution.
Conclusions:
- Effective DNA delivery with low toxicity into endothelial cells is achievable using a liposome-transferrin combination.
- This gene transfer approach during cold ischemic time may serve as a therapeutic tool for chronic rejection in solid organ transplantation.