Journal of Undergraduate Research
Volume 1, Issue 6 - March 2000

Gene Transfer in Ischemic Rat Skin Flaps

Juan C. Varela

ABSTRACT

Ischemia, or inadequate blood supply, is a major cause of failure in skin flaps. Using a rat model, we demonstrated the feasibility of using plasmids (circular pieces of DNA) complexed with lipids to transfer genes into ischemic skin flaps even several days after the skin flap has been created. These results suggest that injection of plasmids expressing angiogenic growth factors might be able to reduce ischemic death of random skin flaps in patients.

BACKGROUND

Current estimates indicate that over 2,000,000 US citizens have chronic wounds each year, and the problem is increasing as the population ages. The cost of caring for chronic wounds reaches into the billions of dollars a year. Clearly, there is a need for better therapies to promote healing of chronic wounds. Ischemia is a major factor contributing to the failure of most chronic wounds to heal. Wound healing involves soluble factors that control a series of processes including inflammation, cellular proliferation and maturation (Robson. 1997). Pro-inflammatory cytokines such as tumor necrosis factor (TNF_) and interleukin-1_ (1L-1_), proteases, protease inhibitors, and growth factors play important roles in normal wound healing, and excessive production of these proteins can impede wound healing (Mast & Schultz. 1996). These factors only function normally when oxygen perfusion of wound tissue is adequate. Ischemia of wound tissues occurs frequently in patients with vascular disease such as venous hypertension, arterial insufficiency, or diabetes (Tarnuzzer & Schultz. 1996). Also, extended periods of pressure can cause ischemia in tissue pressure points in persons without nerve function who have lost nerve functions but are otherwise healthy such as quadriplegics or paraplegics. Thus, methods to restore adequate blood perfusion and reverse local tissue ischemia would promote healing of many chronic wounds. Our specific aim is to establish the foundation for the clinical use of gene therapy in chronic wounds using a rat model of ischemic-induced, impaired wound healing that recapitulates many of the characteristics of non-healing, ischemic, human wounds. We propose that gene therapy can be adapted for treatment of ischemic, non-healing wounds by delivering genes of angiogenic proteins to wound cells, specifically, vascular endothelial cell growth factor (VEGF). To demonstrate this concept, we will first transfect cells in normal and ischemic skin wounds in rats and measure the expression of reporter genes such as the chloramphenicol acetyl transferase (CAT) gene and the ß-Galactosidase gene in order to find out how much transfection we are obtaining and what cells are being transfected. Once this data is collected, we can utilize it to develop efficient experiments for gene transfer with the therapeutic gene VEGF.

METHODS

The animal model we will use is based on our modification of the single pedicle dorsal skin flap originally developed by Mcfarlane and colleagues (Mcfarlane, DeYoung, & Henry. 1965). A single pedicle skin flap measuring 11.5 cm x 2.5 cm will be raised at the inferior angle of the scapula extending distally to the level of the ischial tuberosity. After treatment of the flap, the flap will be repositioned and stapled in place (Figure 1). For analysis, the flap will be divided into three portions: Proximal, medial and distal. The proximal portion is the area of the flap closer to the scapula and is expected to behave similar to normal tissue as opposed to the distal portion where there will be severe ischemia and necrosis (Figure 1).

Figure 1. Single pedicle flap on a rat model.

We will utilize three different plasmids for the gene transfer: PMP6-CAT expressing the CAT gene, pTR-CLZ expressing the ß-Galactosidase gene and pTR-VEGF expressing the VEGF gene. The amount of CAT expressed will be measured through an Enzyme-Linked Immunosorbent Assay (ELISA), the area transfected with the ß-Galactosidase gene will be located through histochemical staining and the effectiveness of the VEGF transfection will be measured by evaluating flap viability and flap vascularization along with RT-PCR analysis.

RESULTS

Experiment #1: Transfection with PMP6-CAT on Ischemic vs. Non-Ischemic Rat Skin

The ischemic arm of the experiment consisted of 3 rats subjected to the single pedicle flap surgery. For the non-ischemic arm, 3 rats were also used but instead of lifting a single pedicle flap, a flap with the same dimension was drawn on their backs. A complex consisting of50ug of PMP6-CAT and 25nmoles of liposomes was injected at the distal and proximal portions of both the singe pedicle flaps and the drawn flaps. The tissue was collected after 48 hours, processed and analyzed through an ELISA test (Figure 2).

Figure 2. Elisa Test. a. Loaded wells before ELISA reaction. b. Wells after ELISA reaction.

Graph 1. Ischemic versus Non-ischemic.

The data shows that even at 48 hours after ischemia has been induced on rat skin, it has effect on gene transfer when compared with non-ischemic skin.

Experiment #2 Effect of Ischemia on Transfection of PMP6-CAT

This experiment consisted of injecting a complex consisting of50ug of PMP6-CAT and 25nmoles of liposomes at the proximal and distal portions of a single pedicle rat flap at 0, 2, 4 and 6 days after ischemia was induced through the flap surgery. The tissue was collected 48hours after injection, processed and analyzed through an ELISA test.

Graph 2. Time of Injection after Ischemia (days).

The results suggest an increase in transfection efficiency at the distal portion of the flap 3-4 days after ischemia has been induced.

CONCLUSIONS

Gene transfer is possible in this rat model of skin injury.

During the first 24-40 hrs after ischemia had been induced, the transfection efficiency on both the distal and proximal portions of the flap was essentially the same.

After 40-48hrs, the transfection efficiency of the distal portion increased notably when compared with transfection in the proximal portion. These results are in agreement with previous experiments, which showed the same pattern of increased distal transfection. From the data, it can be concluded that the period of this increased transfection is approximately 96 hrs. This is an encouraging observation which indicates that transfection with a therapeutic gene is likely to be successful.

FUTURE WORK

We have partially answered the question of how much transfection we obtain in an ischemic flap rat model using PMP6-CAT; more experiments on the effect of ischemia on transfection will be done in the very near future in order to obtain more data and confirm the results we have obtained so far. The next step is to localize the cells that are being transfected. Experiments with the ß-Galactosidase reporter gene are already on their way and the preliminary results are promising. Once we have obtained the necessary data from the reporter genes, we can begin the gene therapy with VEGF.

ACKNOWLEDGEMENTS

Thanks to all the staff at the Institute for Wound Research. Much thanks to Dr. Barry Byrne for providing the plasmids used in this experiments and to Dr. Greg Schultz for all the help and guidance provided for this project

REFERENCES

Ashcroft, G.S., Dodsworth, J., van Boxtel, E., Tarnuzzer, R.W., Horan, M.A., Schultz, G.S., & Ferguson, M.W.J. Estrogen Accelerates Cutaneous Wound Healing Associated with an Increase in TGF-b1 Levels. Nature Medicine, 3:1209-1215, 1997.
Mast, B.A., & Schultz, G.S. Interactions of cytokines, growth factors, and proteases in acute and chronic wounds. Wound Repair and Regeneration, 4:411-420, 1996.
McFarlane, R.M., DeYoung, G., & Henry, R.A. The Design of a Pedicle Flap in the Rat to Study Necrosis and Its Prevention. Plastic and Reconstructive Surgery, 35:177-182, 1965.
Nall, A.V., Brownlee, R.E., Colvin, C.P., Schultz, G., Fein, D., Cassisi, N.J., Nguyen, T., & Kalara, A. Transforming Growth Factor _1 Improves Wound Healing and Random Flap Survival in Normal and Irradiated Rats. Archives of Otolaryngology Head and Neck Surgery, 122:171-177, 1996.
Robson, M.C. The Role of Growth Factors in the Healing of Chronic Wounds. Wound Repair and Regeneration, 5:12-17, 1997.
Tarnuzzer, R.W., & Schultz, G.S. Biochemical Analysis of Acute and Chronic Wound Environments. Wound Rep Reg, 4:in press-in press, 1996.
Taub, P.J., Marmur, J.D., Zhang, W.X., Senderoff, D., Urken, M.L., Silver, L., & Weinberg, H. Effect of Time on the Viability of Ischemic Skin Flaps Treated With Vascular Endothelial Cell Growth Factor (VEGF) cDNA. Journal of Reconstructive Microsurgery, 14:387-390, 1998.

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