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Serotonin potentiates transforming growth factor-beta3 induced biomechanical remodeling in avian embryonic atrioventricular valves.

Buskohl PR, Sun MJ, Sun ML, Thompson RP, Butcher JT - PLoS ONE (2012)

Bottom Line: Blockade of TGFβ type I receptors via SB431542 inhibited the TGFβ3 effects.Elevated 5-HT in ovo resulted in elevated remodeling gene expression and increased TGFβ signaling activity, supporting our ex-vivo findings.Collectively, these results highlight TGFβ/5-HT signaling as a potent mechanism for control of biomechanical remodeling of AV cushions during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Embryonic heart valve primordia (cushions) maintain unidirectional blood flow during development despite an increasingly demanding mechanical environment. Recent studies demonstrate that atrioventricular (AV) cushions stiffen over gestation, but the molecular mechanisms of this process are unknown. Transforming growth factor-beta (TGFβ) and serotonin (5-HT) signaling modulate tissue biomechanics of postnatal valves, but less is known of their role in the biomechanical remodeling of embryonic valves. In this study, we demonstrate that exogenous TGFβ3 increases AV cushion biomechanical stiffness and residual stress, but paradoxically reduces matrix compaction. We then show that TGFβ3 induces contractile gene expression (RhoA, aSMA) and extracellular matrix expression (col1α2) in cushion mesenchyme, while simultaneously stimulating a two-fold increase in proliferation. Local compaction increased due to an elevated contractile phenotype, but global compaction appeared reduced due to proliferation and ECM synthesis. Blockade of TGFβ type I receptors via SB431542 inhibited the TGFβ3 effects. We next showed that exogenous 5-HT does not influence cushion stiffness by itself, but synergistically increases cushion stiffness with TGFβ3 co-treatment. 5-HT increased TGFβ3 gene expression and also potentiated TGFβ3 induced gene expression in a dose-dependent manner. Blockade of the 5HT2b receptor, but not 5-HT2a receptor or serotonin transporter (SERT), resulted in complete cessation of TGFβ3 induced mechanical strengthening. Finally, systemic 5-HT administration in ovo induced cushion remodeling related defects, including thinned/atretic AV valves, ventricular septal defects, and outflow rotation defects. Elevated 5-HT in ovo resulted in elevated remodeling gene expression and increased TGFβ signaling activity, supporting our ex-vivo findings. Collectively, these results highlight TGFβ/5-HT signaling as a potent mechanism for control of biomechanical remodeling of AV cushions during development.

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5-HT signaling modulates TGFβ3 induced AV cushion stiffness.Physiological dosages of 5-HT (470 nM, 5-HT) exacerbated TGFβ3 stiffening, while elevated dosages (47 µM, 5-HT+) eliminated it. Molecular inhibition of the 5-HT2a receptor (MDL100907 10 nM, anti-5-HT2a) and the serotonin transporter (Fluoxetine 10 µM, anti-SERT) did not affect TGFβ3 mediated biomechanical stiffening. Inhibition of the 5-HT2b receptor (SB204741 35 µM, anti-5-HT2b) however eliminated the stiffening effect of TGFβ3. mean ± SEM, n≥6, *p<0.0001 t-test relative to control, #p<0.05 2-way ANOVA with Tukey post-hoc test.
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pone-0042527-g004: 5-HT signaling modulates TGFβ3 induced AV cushion stiffness.Physiological dosages of 5-HT (470 nM, 5-HT) exacerbated TGFβ3 stiffening, while elevated dosages (47 µM, 5-HT+) eliminated it. Molecular inhibition of the 5-HT2a receptor (MDL100907 10 nM, anti-5-HT2a) and the serotonin transporter (Fluoxetine 10 µM, anti-SERT) did not affect TGFβ3 mediated biomechanical stiffening. Inhibition of the 5-HT2b receptor (SB204741 35 µM, anti-5-HT2b) however eliminated the stiffening effect of TGFβ3. mean ± SEM, n≥6, *p<0.0001 t-test relative to control, #p<0.05 2-way ANOVA with Tukey post-hoc test.

Mentions: The effect of 5-HT dose on biomechanical remodeling, independently and in combination with TGFβ3, was systematically evaluated through the stiffness and compaction metrics of the AV cushion organ culture system. 5-HT administration by itself had no statistically significant effect on cushion stiffness. Combined treatment of TGFβ3 with physiological 5-HT (470 nM) increased AV cushion stiffness (WT+5-HT = 1.136±0.035), but high 5-HT dose (5-HT+ = 47 µM) eliminated any TGFβ3 induced stiffening effect (WT+5-HT+ = 0.457±0.025, Figure 4). Neither selective inhibition of the 5-HT2a (MDL100907 10 nM), 5-HT2b (SB204741 2.6 µM) receptors, nor the serotonin transporter SERT (Flouxetine 10 µM) alone affected cushion stiffness (Figure 4). Yet in combination with TGFβ3, the anti-5-HT2b treatment completely blocked TGFβ3 dependent stiffness and compaction behavior (Figure 4 & Figure S3). Inhibition of the 5-HT2a receptor or SERT had no measurable effect on TGFβ3 induced cushion biomechanics. The compaction and stiffness changes induced by 5-HT potentiated TGFβ3 followed the same trend of TGFβ3 treatment alone, with compaction decreasing as stiffness increased and vice versa (Figure 4 & Figure S3). The additional stiffening effect of 5-HT with TGFβ3 was also eliminated with Alk5 inhibition, as shown through the combined treatment of TGFβ3+5-HT+anti-Alk5 in Figure S4. This combined treatment generated a strain energy density similar to the TGFβ3+anti-Alk5 treatment (0.209±0.023 Pa vs 0.245±0.16 Pa, respectively), and further supported that the effects of 5-HT signaling on AV valve remodeling is dependent on canonical TGFβ signaling. Together, these findings suggest that exogenous 5-HT acts through the 5-HT2b receptor to augment or impair TGFβ3 induced cushion stiffening and compaction in a dose-dependent manner.


Serotonin potentiates transforming growth factor-beta3 induced biomechanical remodeling in avian embryonic atrioventricular valves.

Buskohl PR, Sun MJ, Sun ML, Thompson RP, Butcher JT - PLoS ONE (2012)

5-HT signaling modulates TGFβ3 induced AV cushion stiffness.Physiological dosages of 5-HT (470 nM, 5-HT) exacerbated TGFβ3 stiffening, while elevated dosages (47 µM, 5-HT+) eliminated it. Molecular inhibition of the 5-HT2a receptor (MDL100907 10 nM, anti-5-HT2a) and the serotonin transporter (Fluoxetine 10 µM, anti-SERT) did not affect TGFβ3 mediated biomechanical stiffening. Inhibition of the 5-HT2b receptor (SB204741 35 µM, anti-5-HT2b) however eliminated the stiffening effect of TGFβ3. mean ± SEM, n≥6, *p<0.0001 t-test relative to control, #p<0.05 2-way ANOVA with Tukey post-hoc test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412853&req=5

pone-0042527-g004: 5-HT signaling modulates TGFβ3 induced AV cushion stiffness.Physiological dosages of 5-HT (470 nM, 5-HT) exacerbated TGFβ3 stiffening, while elevated dosages (47 µM, 5-HT+) eliminated it. Molecular inhibition of the 5-HT2a receptor (MDL100907 10 nM, anti-5-HT2a) and the serotonin transporter (Fluoxetine 10 µM, anti-SERT) did not affect TGFβ3 mediated biomechanical stiffening. Inhibition of the 5-HT2b receptor (SB204741 35 µM, anti-5-HT2b) however eliminated the stiffening effect of TGFβ3. mean ± SEM, n≥6, *p<0.0001 t-test relative to control, #p<0.05 2-way ANOVA with Tukey post-hoc test.
Mentions: The effect of 5-HT dose on biomechanical remodeling, independently and in combination with TGFβ3, was systematically evaluated through the stiffness and compaction metrics of the AV cushion organ culture system. 5-HT administration by itself had no statistically significant effect on cushion stiffness. Combined treatment of TGFβ3 with physiological 5-HT (470 nM) increased AV cushion stiffness (WT+5-HT = 1.136±0.035), but high 5-HT dose (5-HT+ = 47 µM) eliminated any TGFβ3 induced stiffening effect (WT+5-HT+ = 0.457±0.025, Figure 4). Neither selective inhibition of the 5-HT2a (MDL100907 10 nM), 5-HT2b (SB204741 2.6 µM) receptors, nor the serotonin transporter SERT (Flouxetine 10 µM) alone affected cushion stiffness (Figure 4). Yet in combination with TGFβ3, the anti-5-HT2b treatment completely blocked TGFβ3 dependent stiffness and compaction behavior (Figure 4 & Figure S3). Inhibition of the 5-HT2a receptor or SERT had no measurable effect on TGFβ3 induced cushion biomechanics. The compaction and stiffness changes induced by 5-HT potentiated TGFβ3 followed the same trend of TGFβ3 treatment alone, with compaction decreasing as stiffness increased and vice versa (Figure 4 & Figure S3). The additional stiffening effect of 5-HT with TGFβ3 was also eliminated with Alk5 inhibition, as shown through the combined treatment of TGFβ3+5-HT+anti-Alk5 in Figure S4. This combined treatment generated a strain energy density similar to the TGFβ3+anti-Alk5 treatment (0.209±0.023 Pa vs 0.245±0.16 Pa, respectively), and further supported that the effects of 5-HT signaling on AV valve remodeling is dependent on canonical TGFβ signaling. Together, these findings suggest that exogenous 5-HT acts through the 5-HT2b receptor to augment or impair TGFβ3 induced cushion stiffening and compaction in a dose-dependent manner.

Bottom Line: Blockade of TGFβ type I receptors via SB431542 inhibited the TGFβ3 effects.Elevated 5-HT in ovo resulted in elevated remodeling gene expression and increased TGFβ signaling activity, supporting our ex-vivo findings.Collectively, these results highlight TGFβ/5-HT signaling as a potent mechanism for control of biomechanical remodeling of AV cushions during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Embryonic heart valve primordia (cushions) maintain unidirectional blood flow during development despite an increasingly demanding mechanical environment. Recent studies demonstrate that atrioventricular (AV) cushions stiffen over gestation, but the molecular mechanisms of this process are unknown. Transforming growth factor-beta (TGFβ) and serotonin (5-HT) signaling modulate tissue biomechanics of postnatal valves, but less is known of their role in the biomechanical remodeling of embryonic valves. In this study, we demonstrate that exogenous TGFβ3 increases AV cushion biomechanical stiffness and residual stress, but paradoxically reduces matrix compaction. We then show that TGFβ3 induces contractile gene expression (RhoA, aSMA) and extracellular matrix expression (col1α2) in cushion mesenchyme, while simultaneously stimulating a two-fold increase in proliferation. Local compaction increased due to an elevated contractile phenotype, but global compaction appeared reduced due to proliferation and ECM synthesis. Blockade of TGFβ type I receptors via SB431542 inhibited the TGFβ3 effects. We next showed that exogenous 5-HT does not influence cushion stiffness by itself, but synergistically increases cushion stiffness with TGFβ3 co-treatment. 5-HT increased TGFβ3 gene expression and also potentiated TGFβ3 induced gene expression in a dose-dependent manner. Blockade of the 5HT2b receptor, but not 5-HT2a receptor or serotonin transporter (SERT), resulted in complete cessation of TGFβ3 induced mechanical strengthening. Finally, systemic 5-HT administration in ovo induced cushion remodeling related defects, including thinned/atretic AV valves, ventricular septal defects, and outflow rotation defects. Elevated 5-HT in ovo resulted in elevated remodeling gene expression and increased TGFβ signaling activity, supporting our ex-vivo findings. Collectively, these results highlight TGFβ/5-HT signaling as a potent mechanism for control of biomechanical remodeling of AV cushions during development.

Show MeSH
Related in: MedlinePlus