Department Name

We will be investigating several hypotheses related to vascular remodeling using genetic approaches in vitro and in the intact animal in the upcoming years. Some examples include: 

Hypothesis 1: Recombinant adeno-associated virus serotype 5 transduces vascular endothelial cells with greater efficiency than rAAV-2 or rAAV-1.

Project 1: Confluent monolayers of human umbilical vein endothelial cells, human iliac vein endothelial cells, and human saphenous vein endothelial cells will each be infected with rAAV-5-GFP, rAAV-2-GFP, or rAAV-1-GFP. The experiment will first be performed in the presence of helper wt Ad5, with identification of transduced cells as those cells that fluoresce green after 48hrs. Next, the experiment will be repeated using only the three rAAV serotypes, without adenovirus. FACS analysis will be used to quantify percent cells transduced by each vector.

Hypothesis 2: Transduction with rAAV results in long-term transgene expression in segments of arterialized vein in a rabbit model of vein graft stenosis without inciting a significant host inflammatory response. Project 2: A rabbit model of vein graft stenosis will be used to study the location, efficiency, and duration of transgene expression following intra-luminal application of rAAV-GFP to segments of arterialized vein. Jugular vein segments will be gently distended with vector solution prior to end-to-end placement of the vein segment into the carotid circulation. Animals will be sacrificed at various time points from 2 weeks to 1 year and the vein grafts will be harvested and examined microscopically. Cells that express the transgene will fluoresce green. Presence of inflammation (leukocyte invasion) and development of intimal hyperplasia will also be assessed histologically.

Gene therapy of a murine carotid artery

Hypothesis 3: rAAV-IL-10 will reduce the development of intimal hyperplasia in segments of arterialized vein in a rabbit model of vein graft stenosis.

Project 3: Repeat project 2, using rAAV-IL-10 instead of rAAV-GFP. RT-PCR will be used to confirm the presence of IL-10 mRNA in the treated vein graft segments. Development of inflammation and intimal hyperplasia will be assessed histologically.

Rabbit carotid interposition vein graft using cuff technique

Hypothesis 4: rAAV-elafin will reduce the development of intimal hyperplasia in segments of arterialized vein in a rabbit model of vein graft stenosis. Project 4: Repeat project 3, using rAAV-elafin instead of rAAV-IL-10. RT-PCR will be used to confirm the presence of elafin mRNA in the treated vein graft segments. Development of inflammation and intimal hyperplasia will be assessed histologically.

Hypothesis 5: Transgenic mice which over-express the serine elastase inhibitor, elafin, will show suppressed development of neointimal hyperplasia after carotid ligation compared to wild type mice.

Project 5: Unilateral carotid ligation will be performed on elafin over-expressing and control, wild type, mice. After various time intervals, vessels will be harvested to compare the inflammation and intimal thickness that has developed in each group.