Fibroblast growth factor-2 promotes in vitro mitral valve interstitial cell repair through transforming growth factor-β/Smad signaling

Abstract

Transforming growth factor (TGF)-β and fibroblast growth factor (FGF)-2 both promote repair in valve interstitial cell (VIC) injury models; however, the relationship between TGF-β and FGF-2 in wound repair are not well understood. VIC confluent monolayers were wounded by mechanical injury and incubated separately or in combination with FGF-2, neutralizing antibody to FGF-2, neutralizing antibody to TGF-β, and betaglycan antibody for 24 hours after wounding. Phosphorylated Smad2/3 (pSmad2/3) was localized at the wound edge (WE) and at the monolayer away from the WE. Down-regulation of pSmad2/3 protein expression via small-interfering RNA transfection was performed. The extent of wound closure was monitored for up to 96 hours. FGF-2 incubation resulted in a significant increase in nuclear pSmad2/3 staining at the WE. Neutralizing antibody to TGF-β alone or with FGF-2 present resulted in a similar significant decrease in pSmad2/3. Neutralizing antibody to FGF-2 alone or with FGF-2 present showed a similar significant decrease in pSmad2/3; however, significantly more staining was observed than treatment with neutralizing antibody to TGF-β. Incubation with betaglycan antibody inhibited FGF-2-mediated pSmad2/3 signaling. Wound closure corresponded with pSmad2/3 staining at the WE. Down-regulation of pSmad2/3 via small-interfering RNA transfection significantly reduced the extent to which FGF-2 promoted wound closure. Fibroblast growth factor-2 promotes in vitro VIC wound repair, at least in part, through the TGF-β/Smad2/3 signaling pathway.

Figure 1

Representative immunofluorescent confocal photomicrographs of a wounded VIC monolayer 24 hours after wounding at the WE stained for pSmad2/3 (green) (A and D) and nuclear localization with propidium iodide (blue) (B and E), and AWM strip parallel to WE stained for pSmad2/3 (C and F) in nontreated cultures (A–C), and FGF-2 (10 ng/ml) treated cultures (D–F). The VICs treated with exogenous FGF-2 show increased nuclear pSmad2/3 staining at WE compared with nontreated WE. No difference was observed in pSmad2/3 staining at the AWM between treated and nontreated VICs. Arrows indicate direction of wound closure. G: Percentage of VICs showing nuclear pSmad2/3 staining at the WE with no treatment and with FGF-2 treatment. The VICs at the FGF-2–treated WE exhibit a statistically significant increase in the percentage of VICs showing nuclear pSmad2/3 staining compared with nontreated WE. Statistical significance between indicated groups at *P < 0.05, (n = 9). Original magnifications, ×400.

Figure 2

Representative immunofluorescent confocal photomicrographs of a wounded VIC monolayer 24 hours after wounding at the WE stained for pSmad2/3 (green) (A, D, G, and J) and nuclear localization with propidium iodide (blue) (B, E, H, and K), and AWM strip parallel to WE stained for pSmad2/3 (C, F, I, and L) in wounded cultures treated with neutralizing antibody to TGF-β (15 μg/ml) (A–C), with neutralizing antibody to TGF-β (15 μg/ml) and exogenous FGF-2 (10 ng/ml) (D–F), with neutralizing antibody to FGF-2 (10 μg/ml) (G–I) and with neutralizing antibody to FGF-2 (10 μg/ml) and exogenous FGF-2 (10 ng/ml) (J–L). Same nuclear pSmad2/3 staining at the WE is observed for VICs treated with neutralizing antibody to TGF-β only and VICs treated with neutralizing antibody and exogenous FGF-2. The same nuclear pSmad2/3 staining at the WE is observed for VICs treated with neutralizing antibody to FGF-2 only and VICs treated with neutralizing antibody to FGF-2 and exogenous FGF-2. Treatment with neutralizing antibody to FGF-2 shows greater nuclear pSmad2/3 staining at the WE than VICs treated with neutralizing antibody to TGF-β at the WE. No difference was observed in pSmad2/3 staining at the AWM among the treatments. Arrows indicate direction of wound closure. M: Percentage of VICs showing nuclear pSmad2/3 staining at the WE with treatments of neutralizing antibody to TGF-β, exogenous FGF-2, and neutralizing antibody to TGF-β, neutralizing antibody to FGF-2, and exogenous FGF-2 and neutralizing antibody to FGF-2. Treatment of exogenous FGF-2 in combination with neutralizing antibody to TGF-β does not yield a statistically significant difference of pSmad2/3 staining at the WE from when treated with neutralizing antibody to TGF-β alone. Treatment of exogenous FGF-2 in combination with neutralizing antibody to FGF-2 does not yield a statistically significant difference of pSmad2/3 staining at the WE from when treated with neutralizing antibody to FGF-2 alone. A statistically significant increase is observed in percentage of VICs showing nuclear pSmad2/3 staining at the WE treated with neutralizing antibody to FGF-2 compared with WE treated with neutralizing antibody to TGF-β. Statistical significance between indicated groups at *P < 0.05, (n = 9). Original magnifications, ×400.

Figure 3

Representative immunofluorescent confocal photomicrographs of the WE of wounded VIC monolayers 24 hours after wounding stained for pSmad2/3 (green) (A, C, E, and G) and for nuclear localization with propidium iodide (blue) (B, D, F, and H) in wounded cultures with no treatment (A and B), with exogenous FGF-2 (10 ng/ml) (C and D), with betaglycan antibody (2 μg/ml) (E and F), and with betaglycan antibody (2 μg/ml) and exogenous FGF-2 (10 ng/ml) (G and H). Increased nuclear pSmad2/3 staining at the WE is observed for VICs treated with exogenous FGF-2 only compared with nontreated cultures. Same nuclear pSmad2/3 staining at the WE is observed for VICs treated with betaglycan antibody and VICs treated with betaglycan antibody and exogenous FGF-2. Decreased pSmad2/3 staining at the WE is observed in cultures treated with betaglycan antibody compared with cultures treated with exogenous FGF-2 only. Arrows indicate direction of wound closure. I: Percentage of VICs showing nuclear pSmad2/3 staining at the WE with no treatment, exogenous FGF-2, betaglycan antibody, and betaglycan antibody and FGF-2. A statistically significant increase is observed in percentage of VICs showing nuclear pSmad2/3 staining at the WE treated with exogenous FGF-2 compared with nontreated WE. Treatment of exogenous FGF-2 in conjunction with betaglycan antibody does not yield a statistically significant difference in nuclear pSmad2/3 staining at the WE from VICs treated with betaglycan antibody alone. Statistical significance between indicated groups at *P < 0.05 (n = 3 for nontreated and only exogenous FGF-2–treated cultures, and n = 9 for betaglycan antibody–treated cultures). Original magnifications, ×400.

Figure 4

Representative immunofluorescent confocal photomicrographs of subconfluent and confluent VIC monolayers, stained for α-SMA (red) and nuclear localization with propidium iodide (blue), 24 hours after no treatment (A and B), treatment with exogenous FGF-2 (C and D), and treatment with neutralizing antibody to FGF-2 (E and F), in subconfluent monolayers of 5000 cells/well inoculation (A, C, and E) and confluent monolayer of 100,000 cells/well inoculation (B, D, and F). α-SMA staining for all treatments is greater in subconfluent monolayer than their respective treatments in confluent monolayers. G: Percentage of VICs showing α-SMA staining in the monolayers with treatments of exogenous FGF-2 and neutralizing antibody to FGF-2. A statistically significant increase in α-SMA staining is observed in the subconfluent monolayer for nontreated and treated monolayers compared with their respective treatments in the confluent monolayer. Treatment with neutralizing antibody to FGF-2 resulted in a statistically significant decrease in α-SMA staining compared with the nontreated monolayers and monolayers treated with exogenous FGF-2. Nontreated and exogenous FGF-2–treated VIC subconfluent monolayers did not exhibit a statistically significant difference in α-SMA staining. Confluent monolayers did not yield statistically significant differences in α-SMA staining for different treatments. Significance between indicated groups at *P < 0.05, (n = 4). Original magnification, ×400.

Figure 5

Wound closure of a wounded VIC monolayer, up to 96 hours. Representative phase-contrast micrographs of wounded VICs at 0 (A), 24 (B), 48 (C), 72 (D), and 96 (E) hours. F: Measure of width of the wound, between the two WEs at points of 0, 24, 48, 72, and 96 hours, for various treatments of VICs. Treatment with exogenous FGF-2 resulted in a significantly smaller wound width; therefore, increased wound closure, compared with nontreated VICs, for all points up to complete closure of wounds. Treatment with neutralizing antibody to TGF-β resulted in significantly greater wound width and decreased wound closure compared with nontreated VICs for all points; further addition of FGF-2 in the presence of neutralizing antibody to TGF-β did not yield significantly different results from neutralizing antibody to TGF-β alone for all points. Treatment with neutralizing antibody to FGF-2 resulted in significantly greater wound width and decreased wound closure compared with nontreated VICs for all points up to wound closure, except for 24 hours after wounding; further addition of FGF-2 in the presence of neutralizing antibody to FGF-2 did not yield significantly different results from neutralizing antibody to FGF-2 alone for all points. Treatment with neutralizing antibody to FGF-2 yielded significantly greater wound closure than treatment with neutralizing antibody to TGF-β for all points. Treatment with betaglycan antibody did not result in significantly different wound width compared with nontreated VICs for all points; further addition of FGF-2 in the presence of betaglycan antibody did not yield significantly different results from betaglycan antibody alone for all points. Treatment of FGF-2 in the presence of, individually, neutralizing antibody to TGF-β, neutralizing antibody to FGF-2, or betaglycan antibody resulted in significantly greater wound width and decreased wound closure compared with treatment with FGF-2 alone for all points up to complete closure of wounds. Significance between indicated groups at *P < 0.05, (n = 3).

Figure 6

Down-regulation of pSmad2/3 in wounded VIC monolayers by Smad2/3 siRNA transfection. A: Representative pSmad2/3 protein expression of wounded VIC monolayer 72 hours after siRNA transfection detected by Western blot analysis with anti-pSmad2/3 antibody. The membrane was also probed with anti-GAPDH antibody as an internal control for equal loading and transfer. Down-regulated pSmad2/3 protein expression for Smad2/3 siRNA transfected VICs is observed compared with VICs with no siRNA transfection and control siRNA transfection. B: pSmad2/3 protein expression as detected by Western blot analysis and quantified by densitometry (ImageJ). Protein expression of pSmad2/3 in Smad2/3 siRNA–transfected VIC monolayers is significantly less than nontransfected and control siRNA-transfected VIC monolayer. Protein expression of pSmad2/3 in nontransfected and control siRNA-transfected VIC monolayers do not differ significantly. Protein expression of GAPDH for all transfections does not differ significantly from each another. Significance between indicated groups at *P < 0.05, (n = 3). C: Wounded VICs transfected with Smad2/3 siRNA showed significantly greater wound width and less wound closure at 72 hours with no treatment compared with nontransfected and control siRNA-treated VIC monolayers. For all transfections, FGF-2 treatment resulted in a statistically significant decrease of wound width and increased wound closure. Nontransfected and control siRNA-transfected VICs did not differ significantly in wound closure. Significance between indicated groups at *P < 0.05, (n = 3).

Figure 7

Summary of a putative pathway that regulates FGF-2–mediated wound repair through the TGF-β/Smad signaling pathway. The TGF-β promotes the phosphorylation of Smad2/3, resulting in VIC wound repair. Betaglycan binds endogenous TGF-β, resulting in less available TGF-β to bind to its signaling receptors. The FGF-2 also binds betaglycan, which makes more TGF-β available for receptor signaling.