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. 2002 Dec;161(6):2111-21.
doi: 10.1016/s0002-9440(10)64489-6.

Serotonin mechanisms in heart valve disease I: serotonin-induced up-regulation of transforming growth factor-beta1 via G-protein signal transduction in aortic valve interstitial cells

Bo Jian  1 , Jie Xu, Jeanne Connolly, Rashmin C Savani, Navneet Narula, Bruce Liang, Robert J Levy
Affiliations

Serotonin mechanisms in heart valve disease I: serotonin-induced up-regulation of transforming growth factor-beta1 via G-protein signal transduction in aortic valve interstitial cells

Bo Jian et al. Am J Pathol. 2002 Dec.

Abstract

Clinical disorders associated with increased serotonin [5-hydroxytryptamine (5-HT)] levels, such as carcinoid syndrome, and the use of serotonin agonists, such as fenfluoramine have been associated with a valvulopathy characterized by hyperplastic valvular and endocardial lesions with increased extracellular matrix. Furthermore, 5-HT has been demonstrated to up-regulate transforming growth factor (TGF)-beta in mesangial cells via G-protein signal transduction. We investigated the hypothesis that increased exposure of heart valve interstitial cells to 5-HT may result in increased TGF-beta1 expression and activity because of serotonin receptor-mediated signal transduction with activation of Galphaq, and subsequently up-regulation of phospholipase C. Thus, in the present study we performed a clinical-pathological investigation of retrieved carcinoid and normal valve cusps using immunohistochemical techniques to detect the presence of TGF-beta1 and other proteins associated with TGF-beta expression, including TGF-beta receptors I and II, latent TGF-beta-associated peptide (LAP), and alpha-smooth muscle actin. Carcinoid valve cusps demonstrated the unusual finding of widespread smooth muscle actin involving the interstitial cells in the periphery of carcinoid nodules; these same cells were also positive for LAP. Normal valve cusps were only focally positive for smooth muscle actin and LAP. In sheep aortic valve interstitial cell cultures 5-HT induced TGF-beta1 mRNA production and increased TGF-beta1 activity. 5-HT also increased collagen biosynthesis at the dosages studied. Furthermore, TGF-beta1 added to SAVIC cultures increased the production of sulfated glycan and hyaluronic acid. In addition, overexpression of Galphaq using an adenoviral expression vector for a constitutively active Galphaq mutant (Q209L-Galphaq) resulted in increased phospholipase C activity as well as up-regulation of TGF-beta expression and activity. These results strongly support the view that G-protein-related signal transduction is involved in 5-HT up-regulation of TGF-beta1. In conclusion, 5-HT-associated valve disease may be, in part, because of TGF-beta1 mechanisms.

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Figures

Figure 1.
Figure 1.
A: Representative micrographs of a carcinoid tricuspid valve cusp with prominent SMA staining by peroxidase immunochemistry (brown staining) with a hematoxylin counterstain. B: Normal control tricuspid valve cusp shows sparse subendothelial SMA staining (arrows indicate SMA-positive cells). C: A carcinoid valve cusp with strongly positive LAP-TGF-β1 immunostaining (arrows) localized in hyperplastic areas around carcinoid nodules. D: Focal subendothelial LAP-TGF-β1 immunostaining (arrows) in control valves. E: Negative IgG control shows an absence of peroxidase activity. Asterisk denotes carcinoid nodule. All micrographs were prepared with immunoperoxidase and hematoxylin staining techniques. Original magnifications, ×400.
Figure 2.
Figure 2.
Immunostaining results demonstrating that SAVICs are immunopositive (brown) for the 5-HT2A receptor (A), compared to B, which demonstrates an absence of immunopositive cells using a nonspecific antibody. TGF-β1 exposure results in increased α/τ-SMA-immunopositive cells (brown) with an elongated cytoskeleton (C), compared to D. Cells not exposed to TGF-β1 are predominant by immunonegative for α/τ-SMA. Nonspecific IgG results were negative for α/τ-SMA (E). Peroxidase immunohistochemistry. Original magnifications: ×200 (A, B); ×400 (C–E).
Figure 3.
Figure 3.
Serotonin up-regulates TGF-β1 expression and activity in AVICs. A: Time-dependent TGF-β1 mRNA induction by 10 μmol/L of 5-HT treatment evaluated by real-time RT-PCR. Results are expressed as mean ± SEM of four to six independent experiments (**, P < 0.01; *, P < 0.05; analysis of variance followed by Tukey-Kramer multiple comparisons post test). B: A standard calibration curve for the PAI/L construct with directly added TGF-β1 demonstrating a dose response. C: Active TGF-β stimulated by 10 μmol/L of 5-HT after 72 hours, per PAI/luciferase assays, and blocked by a TGF-β1 function-blocking antibody. D: Total TGF-β (active and latent), examined by PAI/luciferase assays, increased because of 72 hours exposure to 5-HT, and blocked by TGF-β1 function-blocking antibody. For B, C, and D, the results are expressed as mean ± SEM of triplicates, which are typical of four independent experiments (*, P < 0.05, significant from control; **, P < 0.01, significant from 5-HT; analysis of variance followed by Tukey-Kramer multiple comparisons post test).
Figure 4.
Figure 4.
Both 5-HT and TGF-β1 stimulate collagen synthesis by SAVICs. A: Stimulation of [3H]proline incorporation into cells by TGF-β1, significant stimulation was observed at 0.01 to 10 ng/ml of TGF-β1 (**, P < 0.01). B: The effects of TGF-β1 on total protein synthesis measured by [3H]leucine incorporation, demonstrating significant inhibition of protein synthesis at 10 ng/ml of TGF-β1 (*, P < 0.05). C: Stimulation of [3H]proline incorporation into cells by 5-HT, significant stimulation was observed from 1.0 to 100 μmol/L of 5-HT (*, P < 0.05; **, P < 0.01). D: Anti-TGF-β antibody blocked [3H]proline incorporation into cells stimulated by 5-HT. A significant blocking effect was observed at concentrations as low as 5 μg/ml of anti-TGF-β antibody (*, P < 0.05; **, P < 0.01). Results are expressed as mean ± SEM of four independent experiments (*, P < 0.05; **, P < 0.01, analysis of variance followed by Tukey-Kramer multiple comparisons post test).
Figure 5.
Figure 5.
TGF-β1 stimulates both total glycosaminoglycan and HA production by SAVICs in a dose- and time-dependent manner. A: Time- and dose-dependent stimulation of total sulfated glycan by TGF-β1. B: Dose-dependent stimulation of HA production by TGF-β1, with a significant increase in HA production observed at 10 ng/ml or greater of TGF-β1 for 24 hours treatment. 5-HT at 10 μmol/L showed no significant effect on HA production. C: Time-dependent stimulation of HA production by TGF-β1, with the maximum HA production observed after 48 hours of incubation with TGF-β1. Results are expressed as mean ± SEM of triplicates, which are typical of three or four independent experiments (*, P < 0.05; **, P < 0.01, followed by Tukey-Kramer multiple comparisons post test).
Figure 6.
Figure 6.
Overexpression of Gαq via transduction of SAVICs with a replication defective adenovirus stimulates PLC activity and TGF-β1 expression, where S1 is the M199 medium (control); S2 is the AdCMV-GFP, 107 PFU; S3 is the AdCMV-Gαq, 107 PFU. Overexpression of Gαq significantly increases the accumulation of total inositol phosphates (A), stimulates TGF-β1 mRNA expression (B); increases the production of active TGF-β1 evaluated by PAI/L assay (C); and increases the production of total (active and latent) TGF-β1 evaluated by PAI/L assay (D). Results are expressed as mean ± SEM of triplicates, which are typical of three independent experiments (*, P < 0.05; **, P < 0.01, analysis of variance followed by Tukey-Kramer multiple comparisons post test).
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