Genetic modification of smooth muscle cells to enhance the performance of tissue engineered vascular grafts.

摘要

Development of small-diameter blood vessels using tissue engineering has been promising as a novel therapy for patients who require coronary artery bypass grafting but lack suitable donor tissue. However, the performance of tissue engineered vascular grafts (TEVGs) has been challenged by the lack of a complete endothelium and by poor mechanical properties which contribute to blood incompatibility and burst failure in vivo respectively. In this work, these challenges have been addressed by genetically modifying vascular smooth muscle cells (VSMCs) to produce factors that will promote endothelialization and improve mechanical properties. Genetic engineering of VSMCs was explored using different methods to obtain the optimal transfection system. Viral transduction of cells resulted in the highest efficiency and stability of expression. Neomycin selection of virally transduced SMCs resulted in a homogenous population with high expression levels. To promote endothelialization of TEVGs, VSMCs were virally-transduced to produce vascular endothelial growth factor (VEGF) which acts as a chemoattractant and mitogen of endothelial cells (ECs). The proliferation of ECs significantly increased after exposure to VEGF-transfected SMCs or their conditioned media. The chemotactic response of ECs to the VEGF-producing cells was explored by different in vitro models which demonstrated increased migration of ECs in response to VEGF-transfected cells on both tissue culture treated surfaces as well as collagen-coated surfaces. To improve mechanical properties, lysyl oxidase (LO) was utilized to enzymatically crosslink extracellular matrix (ECM) proteins, particularly collagen and elastin, to enhance the mechanical integrity of the ECM and thereby impart mechanical strength to the engineered tissue. Viral transduction of VSMCs resulted in increased LO expression using Northern and Western analysis. Increased LO activity was demonstrated using a fluorescent enzyme substrate assay, and as increased levels of desmosine, a product of LO crosslinking, in the ECM. The mechanical effects of altered crosslink densities within tissue engineered constructs were demonstrated in a VSMC-populated collagen gel model. VSMCs transfected with lysyl oxidase were seeded in collagen gels; the tensile strength and elastic modulus in these constructs increased by approximately three-fold compared to constructs seeded with mock transfected VSMCs. Compositional analysis of the ECM deposited by the transformed cells showed similar collagen and elastin levels, and cell proliferation rates were similar as well, thus attributing increased mechanical properties to ECM crosslinking.