Gene transfer into rat heart-derived endothelial cells

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.