Transforming growth factor-β regulates the growth of valve interstitial cells in vitro

Abstract

Although valve interstitial cell (VIC) growth is an essential feature of injured and diseased valves, the regulation of VIC growth is poorly understood. Transforming growth factor (TGF)-β promotes VIC proliferation in early-stage wound repair; thus, herein, we tested the hypothesis that TGF-β regulates VIC proliferation under normal nonwound conditions using low-density porcine VIC monolayers. Cell numbers were counted during a 10-day period, whereas proliferation and apoptosis were quantified by bromodeoxyuridine staining and TUNEL, respectively. The extent of retinoblastoma protein phosphorylation and expression of cyclin D1, CDK 4, and p27 were compared using Western blot analysis. Adhesion was quantified using a trypsin adhesion assay, and morphological change was demonstrated by immunofluorescence localization of α-smooth muscle actin and vinculin. TGF-β-treated VICs were rhomboid; significantly decreased in number, proliferation, and retinoblastoma protein phosphorylation; and concomitantly had decreased expression of cyclin D1/CDK4 and increased expression of p27. TGF-β-treated VICs adhered better to substratum and had more vinculin plaques and α-smooth muscle actin stress fibers than did controls. Thus, the regulation of VIC growth by TGF-β is context dependent. TGF-β prevents excessive heart valve growth under normal physiological conditions while it promotes cell proliferation in the early stages of repair, when increased VICs are required.

Figure 1

p-Smad in nuclei. Immunofluorescent confocal micrographs of vehicle (A and C) and 5 ng/mL TGF-β–treated (B and D) subconfluent VIC monolayers 4 days (A and B) and 6 days (C and D) after first TGF-β addition are shown. VICs are stained for p-Smad2/3 (Ser423/425) (green; nuclei, red). There was more prominent p-Smad nuclear localization in treated VICs. Scale bar = 10 μm (D). Original magnification, ×600. E: Comparison of the percentages of p-Smad–incorporated nuclei in vehicle and TGF-β–treated monolayers after 4 and 6 days. *P < 0.0001 for statistical significance between groups. n = 9.

Figure 2

Phase-contrast micrographs of vehicle (A, C, E, G, and I) and 5 ng/mL TGF-β–treated (B, D, F, H, and J) VICs taken 1 day (A and B), 2 days (C and D), 4 days (E and F), 6 days (G and H), and 8 days (I and J) after initial treatment with TGF-β, showing the change over time in cell density and morphological features. Most treated cells appeared rhomboid, whereas the vehicle cells remained spindle shaped. Scale bar = 100 μm (J). Original magnification, ×100.

Figure 3

Growth curves of control and TGF-β–treated VICs. A: VIC growth in 0.1 or 0.5 ng/mL TGF-β and corresponding vehicle-treated subconfluent monolayers. TGF-β–treated VICs showed significantly delayed growth after 4 days of treatment. The growth-inhibitory effect is concentration dependent because 0.5 ng/mL treatment resulted in a significantly greater extent of inhibition than 0.1 ng/mL treatment. (Only the statistical significance between the two treatment groups is shown.) B: VIC growth in two independent groups. In the control group, DMSO was added to the culture 2 hours before the addition of TGF-β vehicle solution (4 mmol/L HCl and 0.1% bovine serum albumin). In the treatment group, 2 μmol/L SB431542 was added to the culture 2 hours before the addition of 5 ng/mL TGF-β. The TGF-β–mediated growth inhibition is completely abolished in the presence of SB431542, and the two growth curves overlap. C: VIC growth in DMSO and 1 μmol/L SB431542 treated subconfluent monolayers. The inhibitor alone inhibits VIC growth. D: VIC growth in vehicle- and TGF-β–neutralizing antibody (Ab)–treated monolayers. No significant change in cell number was observed. In all experiments, cell counts were performed in triplicate. Error bars denote SEM. Statistical significance between groups: *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4

BrdU incorporation in control and TGF-β–treated VICs. A comparison of the proportion of proliferating cells in vehicle (A, C, and E) and 5 ng/mL TGF-β–treated (B, D, and F) VICs 2 days (A and B), 3 days (C and D), and 4 days (E and F) after first TGF-β addition is shown in immunofluorescent micrographs of the nuclei of BrdU-incorporated VICs. Total nuclei are counterstained in red, with BrdU-stained nuclei shown in green. TGF-β significantly decreases the proportion of proliferating cells in the treated monolayers (B, D, and F). Scale bar = 10 μm (F). Original magnification, ×200. The percentages of proliferating cells in vehicle and 5 ng/mL or vehicle and 0.5 ng/mL TGF-β–treated subconfluent monolayers 2 days (G) and 4 days (H) after first TGF-β addition are compared. TGF-β at both concentrations tested significantly inhibits VIC proliferation at both time points. There is no significant difference in the percentages of proliferating cells treated with 5 and 0.5 ng/mL TGF-β. *P < 0.001 denotes significant differences between groups. n = 15.

Figure 5

A–D: Immuofluorescent micrographs of the nuclei of apoptotic VICs 6 days after first TGF-β treatment. Total nuclei are counterstained in red, with apoptotic nuclei shown in green. The positive control (A), with the addition of nuclease, shows a significant level of apoptosis, whereas the negative control (B), with the elimination of TdT enzyme, shows no sign of apoptosis; this is similar in the vehicle (C) and 5 ng/mL TGF-β–treated VIC monolayers. Scale bar = 10 μm. Original magnification, ×200.

Figure 6

The expression levels of phosphorylated pRb (Ser 807/811) in the cytoplasm and nucleus of vehicle and 0.5 ng/mL TGF-β–treated VICs (A) and vehicle and 5 μmol/L SB431542-treated VICs (B) are determined using Western blot analyses. Cell extracts were prepared 6 days after first TGF-β treatment. Extracts containing 20 μg of protein were separated on an 8% polyacrylamide gel. C: Expression of whole cell CDK4, cyclin D1, and nuclear p27 in vehicle and 0.5 ng/mL TGF-β–treated VICs. Cell extracts were prepared 6 days after first TGF-β treatment. Extracts containing 40 μg of protein were separated on a 12.5% polyacrylamide gel. Phosphorylated pRb, CDK4, cyclin D1, p27, and their corresponding loading controls (tubulin and histone) were detected by immunoblotting. D–F: The levels of expression are determined by densitometry and compared. Statistical significance between groups: *P < 0.01, **P < 0.001, and ***P < 0.0001.

Figure 7

α-SMA expression in control and TGF-β–treated VICs. Immunofluorescent confocal micrographs of vehicle and 5 ng/mL TGF-β–treated subconfluent VIC monolayers 4 days (A and B) and 6 days (C and D) after first TGF-β addition are shown. VICs are stained for α-SMA, which is expressed at a much higher level and organized into stress fibers in the treated group (green; nuclei, blue). Scale bar = 10 μm (D). Original magnification, ×600. E: Expression of whole cell α-SMA in vehicle and 0.5 ng/mL TGF-β–treated VICs. Cell extracts were prepared 6 days after first TGF-β treatment. Extracts containing 10 μg of protein were separated on an 8% polyacrylamide gel. α-SMA and tubulin loading control were detected by immunoblotting. F: The levels of expression in control and treated VICs are determined by densitometry and compared. *P < 0.001 denotes statistical significance between groups.

Figure 8

VIC adhesion in control and TGF-β–treated VICs. Immunofluorescent confocal micrographs of vehicle (A) and 5 ng/mL TGF-β–treated (B) subconfluent VIC monolayers 4 days after first TGF-β addition are shown. VICs are stained for vinculin (green; nuclei, blue), which is expressed at a higher level and organized into vinculin plaques in the treated group. Scale bar = 10 μm (B). Original magnification, ×600. C: Results from the trypsin adhesion assay showing proportions of cells detached during the given incubation period. TGF-β–treated VICs showed significantly greater attachment to the substratum. Three independent trials were performed. Error bars denote SEM. Statistical significance between groups: *P < 0.01, **P < 0.001.