Effect of TGF-β1 on the Migration and Recruitment of Mesenchymal Stem Cells after Vascular Balloon Injury: Involvement of Matrix Metalloproteinase-14

Abstract

Restenosis or occlusion after vascular procedures is ascribed to intimal hyperplasia. Transforming growth factor (TGF)-β1 is involved in recruitment of mesenchymal stem cells (MSCs) following arterial injury, and its release from latent TGF-binding protein by matrix metalloproteinase (MMP)-14-induced proteolysis contributes to neointima formation. However, the relationship between MMP-14 and TGF-β1 activation in restenosis is unknown. This study investigated the relationship using a rat model of balloon-induced injury. Rats were assigned to vehicle-, SB431542 (SB)-, or recombinant human (rh)TGF-β1-treated groups and examined at various time points after balloon-induced injury for expression of TGF-β1/Smad signalling pathway components, MMP-14 and MSCs markers including Nestin, CD29, and Sca1(+)CD29(+)CD11b/c(-)CD45(-). Intimal hyperplasia was reduced in SB- and rhTGF-β1-treated rats. The expression of TGF-β1, TGF-β1RI, and Smad2/3 was decreased, but the levels of phosphorylated Smad2/3 were higher in SB-treated rats than vehicle-treated after 7 days to 14 days. rhTGF-β1 administration decreased the expression of TGF-β1/Smad pathway proteins, except for TGF-β1RI. Nestin and CD29 expression and the number of Sca1(+)CD29(+)CD11b(-)CD45(-) cells were reduced, whereas MMP-14 expression was increased after SB431542 and rhTGF-β1 administration. These results suggest that TGF-β1/Smad signalling and MMP-14 act to recruit MSCs which differentiate to vascular smooth muscle cells and mesenchymal-like cells that participate in arterial repair/remodelling.

Figure 1. Neointimal thickness and degree of hyperplasia reduced by TβRI/Smad inhibitor and rhTGF-β1 in response to arterial remodelling.

(A) Haematoxylin and eosin staining of injured carotid arteries from rats treated with vehicle, SB431542 (SB)or rhTGF-β1. Scale bar, 200 μm. (B) I/M ratios. Results represent mean ± SD (n = 5 per group). *P < 0.05, SB-vs vehicle-treated group; ^#P < 0.05,rhTGF-β1- vs vehicle-treated group.

Figure 2. Expression levels of TGF-β1/Smad signalling proteins after arterial injury.

(A–C) Expression levels of TGF-β1 (12.5–25 kDa), TβRI (53 kDa), Smad2/3 (48 kDa), and P-Smad2/3 (52 kDa) after balloon angioplasty in rats treated with vehicle (A), SB431542 (B), or rhTGF-β1 (C), as detected by western blotting. The blots were cropped and the gels were run under the same experimental conditions. (D–G) Densitometry analysis of TGF-β1 (D), TβRI(E), Smad2/3 (F), and P-Smad2/3 (n = 5 per group per experiment). *P < 0.05, SB-vs vehicle-treated group; ^#P < 0.05, rhTGF-β1 vs vehicle-treated group.

Figure 3. Immunohistochemical analysis of TGF-β1/Smad signalling protein expression in injured arteries.

(A–C) Representative images of TGF-β1 (A), TβRI (B), and P-Smad2/3 (C) expression. Scale bar, 50 μm.

Figure 4. Injury-activated TGF-β1 induces the mobilisation of MSCs for tissue repair.

(A) Percentages of Sca1⁺CD29⁺CD11b⁻CD45⁻ cells in peripheral blood and bone marrow at indicated time points in injured rats treated with vehicle, SB431542 (SB), and rhTGF-β1. Results represent mean ± SD (n = 5 per group per time point). *P < 0.05, SB- vs. vehicle-treated rats. (B,C) Immunofluorescence analysis of Nestin (B) and CD29 (C) expression in vehicle-, SB-, and rhTGF-β1-treated injured rats. Scale bar, 50 μm.

Figure 5. Nestin expression after arterial injury.

Western blot analysis of Nestin(177 kDa) expression in vehicle-, SB431542 (SB)-, and rhTGF-β1-treated rats with common carotid arterial injury. *P < 0.05, SB- vs. vehicle-treated rats; ^#P < 0.05, rhTGF-β1- vs. vehicle-treated rats. The blots were cropped and the gels were run under the same experimental conditions.

Figure 6. Calponin, SMA, and Vimentin expression after arterial injury.

(A–C) Expression of Calponin (34 kDa), Vimentin (57 kDa), and MMP-14 (66 kDa) after balloon angioplasty in rats treated with vehicle (A), SB431542 (SB) (B), and rhTGF-β1, as detected by western blotting. The blots were cropped and the gels were run under the same experimental conditions. (D–F) Densitometry analysis of Calponin (D), Vimentin (E), and MMP-14 (F) expression (n = 5 per group per experiment). *P < 0.05, SB- vs. vehicle-treated group; ^#P < 0.05, rhTGF-β1- vs. vehicle-treated group.