Smooth Muscle α Actin (Acta2) and Myofibroblast Function during Hepatic Wound Healing
Figure 5
Acta2 antisense oligodeoxynucleotides inhibit Acta2 expression, stellate cell contractility, and stellate cell motility.
Stellate cells were isolated from normal rat livers; after 24 hours, oligonucleotides were transfected as in Methods (the transfection mix containing oligonucleotides was replaced every 48 hours). Five days later, cells were harvested and lysates were subjected to immunoblotting to detect Acta2. In (A), different oligonucleotides (10 µM) were tested; specific Acta2 bands were scanned, quantitated and expressed graphically (n = 3, * p<0.01). In (B), the effect of different concentrations of sense and antisense oligonucleotides (the Acta2 3′UT #1 sequence) was examined. The upper portion of the figure depicts a representative immunoblot, and the graph below depicts scanned and quantitated data (n = 3, * p<0.01). Immunoblots with anti-cytoplasmic β-actin revealed no change in Acta2 expression (not shown). In (C), cells as above were placed on collagen lattices; oligonucleotides were added 24 hours later (all at 10 µM) and replaced at day 3 and 5 in culture. Serum free conditions were introduced and medium containing serum (10% horse/10% calf) was added to induce contraction. Lattices were dislodged from their plastic substrata and gel contraction was measured (contraction after 4 hours is shown, n = 4, *p<0.01 compared to lattices exposed to sense oligonucleotides). Cells exposed to only serum free or serum containing medium served as negative and positive controls, respectively. In (D–H), stellate cells from normal livers were isolated and allowed to undergo culture induced activation. Twenty-four hours after isolation, cells were transfected with oligodeoxynucleotides as in Methods. Seventy-two hours later, a linear scratch was applied to the cell monolayer. In (D), cells exposed to 3′UT #1 sense oligonucleotides (10 µM) are shown; in (E) cells exposed to 3′UT #1 antisense oligonucleotides (10 µM) are shown (representative images 24 hours after scratch wounding are shown) (bar = 50 microns). In (F), the number of cells per high-powered field entering the wounded area of the monolayer were counted and quantitated as in Methods (n = 6, *p<0.01 vs. cells exposed to sense oligonucleotides). In (G), the area in the wound remaining unoccupied by cells was quantitated by image analysis as in Methods (n = 6, *p<0.01 vs. cells exposed to sense oligonucleotides). In (H), the effect of Acta2 antisense oligodeoxynucleotides on stellate cell motility was assessed by measuring migration of stellate cells through polyethylene terphthalate membranes containing 8 µm pores as in Figure 2 (n = 3, *p<0.01 vs. to sense). Abbreviations: Init - initiation; UT – untranslated.