Tissue Factor as a Link Between Wounding and Tissue Repair

Efficacy of liposomal-mediated gene transfer. A: To determine transfection efficacy and stability of the transfected plasmids, somatic gene transfer of human TF (hTF) plasmid DNA was performed to allow differentiation between endogenous and transfected exogenous TF. The wounds were harvested 7 days after transfection, and hTF mRNA was detected by RT-PCR. Wounds transfected with hTF-plasmid DNA showed significant hTF transcription. B: Plasmid DNA carrying a luciferase reporter gene was delivered to cutaneous wounds of NOD mice. At the time points indicated in the figure, wounds were harvested. Total protein extracts were prepared before luciferase expression was detected by Western blot analysis. The bar graph summarizes the signal intensities determined in densitometric evaluation. C: Plasmid DNA carrying LacZ was delivered to cutaneous wounds of NOD mice, and the expression was detected after 7 days by immunostaining for X-gal. The picture shows a thin sheet of stratified squamous epithelium covered by a layer of fibrin, which contains inflammatory cells. Some inflammatory cells, such as macrophages, are visible in the dermis. Solid arrows mark the blue LacZ stains and open dotted arrows indicate possible staining of melanin in melanocytes (magnification 200×).

Effect of somatic gene transfer of vector (V) alone or TF on the transcription of TF, VEGF, and α-SMA. Wounds were prepared in diabetic NOD mice, and somatic gene transfer was performed using either vector or tissue factor on the same animal. A: Wounds were harvested after 1 day and analyzed by RT-PCR for TF, VEGF, α-SMA, and GAPDH mRNA. A representative result is shown on the top left side. The relative values of five mice determined by densitometry are shown below. The experiment was repeated three times with similar results. B: After harvesting wounds on days 1 or 7, expression of TF, VEGF, and α-SMA was determined using real-time PCR with a relative quantitative approach. After normalization with mGAPDH and a PCR efficiency correction, the relative amount of the respective gene in the vector-transfected wounds was compared with that of the TF-transfected wounds. For this purpose, the relative amount of gene from the vector-transfected wound was considered as one and the results are shown as standarized units. Statistics: Student’s t test was used. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01.

Effect of somatic gene transfer of vector (V) alone or TF on the expression of TF, VEGF, and α-SMA. Wounds were harvested after somatic gene transfer with vector (top) or TF (middle) transfection and stained with the respective antibodies as described in reasearch design and methods. A summary of the results analyzing sections of five independently transfected wounds is given in the bar graphs (bottom). A: TF expression at the wound edge 2 days after wounding (magnification 150×). The arrows indicate the migrating tip of the epidermis (where a stronger staining is seen in the TF-transfected wounds) and the large number of TF-positive inflammatory cells underlying the epidermis (middle). Two wound edges from two sections of each of the five independently transfected mice were analyzed, and the summary of the results is given at the bottom. B: VEGF expression at the wound edge 7 days after wounding (magnification 150×). In TF-transfected wounds (middle), a sharp increase of VEGF staining is seen in the wound edge and in sharp contrast to VEGF staining at the wound edge of vector control (top). The analysis of two sections from five wounds of five independently transfected mice is shown (bottom). C: α-SMA antigen expression in the wound bed 7 days after wounding (magnification 300×). In TF-transfected wounds (middle), the positive cells are surrounded by blood vessels, whereas less α-SMA–positive cells are seen in vector-transfected wounds (top). The number of α-SMA–positive vessels is significantly higher in TF- than in vector-transfected wounds (bottom). Sixteen wounds were analyzed from three independent experiments. Statistics: Student’s t test was used. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01.

Effect of somatic gene transfer of vector alone (V) or TF on fibrin/fibrinogen deposition. To study effects of somatic gene transfer with vector (left) or TF (right) on fibrin/fibrinogen deposition, wounds were harvested and stained with an anti-murine fibrin/fibrinogen antibody as previously described (41). Increased fibrin/fibrinogen deposition was more prominent in TF-transfected wounds (right). Four wounds for each condition from three independent experiments were analyzed (magnification 150×). Fibrin/fibrinogen positivity is indicated by dark brown color as indicated by arrows.

Effect of somatic gene transfer of vector alone (V) or TF on the formation of vessels. Wounds were prepared in diabetic NOD mice, and somatic gene transfer was performed using either vector or tissue factor on the same mouse. A: Mice were killed after 7 days, and vessels in vector (top)- or TF (bottom)-transfected wounds were visualized by ink injection as described in research design and methods. B: Wounds were harvested at day 7 and hematoxylin/eosin-stained as described in research design and methods to determine the number of vessels formed. Representative wounds transfected with vector (top) or TF (bottom) are shown. C: Statistical evaluation of blood vessel density in wound sections from eight independently transfected mice. The experiment was repeated three times with similar results. D: Mice were anesthetized and microbeads were injected as described in research design and methods. The number of beads recovered from 12 wounds is shown. Statistics: Student’s t test was used. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01.