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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 increases AV cushion pSmad2/3 expression in ovo.A) Representative images of pSmad2/3 staining. Arrows indicate pSmad2/3 positive cells. Cell nuclei – blue, pSmad2/3 – red. B) Embryos treated with systemic 5-HT at HH17 have increased pSmad2/3 expression at HH25 indicating elevated TGFβ signaling. n = 6, mean ± SEM *p<0.01, t-test.
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pone-0042527-g008: 5-HT increases AV cushion pSmad2/3 expression in ovo.A) Representative images of pSmad2/3 staining. Arrows indicate pSmad2/3 positive cells. Cell nuclei – blue, pSmad2/3 – red. B) Embryos treated with systemic 5-HT at HH17 have increased pSmad2/3 expression at HH25 indicating elevated TGFβ signaling. n = 6, mean ± SEM *p<0.01, t-test.

Mentions: Systemic 5-HT treatment at HH17 resulted in a statistically significant 1.4±0.2 fold increase in AV cushion stiffness over control at stage HH25 (strain energy density of 0.43±0.06 Pa vs. 0.31±0.03 Pa, *p<0.05, Figure 7A). We next analyzed the mesenchymal gene expression patterns in this in ovo system. 5-HT significantly upregulated TGFβ3 (1.7±0.1), αSMA (1.5±0.1), col1α2 (1.5±0.1), cyclin b (1.6±0.2), and RhoA (1.7±0.2) (*p<0.05, Figure 7B). Interestingly, the TGFβ3 mRNA expression was comparable to that observed in the ex ovo organ culture treatment of TGFβ3 alone (2.2±0.6), 5-HT alone (1.9±0.1), and TGFβ3+5-HT (2.0±0.3). αSMA and col1α2 mRNA were also upregulated in ovo with 5-HT, but less than with direct TGFβ3 administration in ex vivo culture (αSMA – 1.5 vs 5.7, RhoA – 1.7 vs 2.1). The similar mRNA profiles of the candidate genes in both models suggested that 5-HT also potentiates TGFβ signaling in AV cushions in ovo. To confirm that the 5-HT treatment was indeed modulating TGFβ signaling activity in ovo, we quantified nuclear pSmad2/3 expression in HH25 cushions with and without 5-HT treatment (Figure 8). 5-HT treatment increased the number of cell nuclei with localized pSmad2/3 expression 2.6±0.8 fold over control embryos (0.28±0.04 vs. 0.11±0.03, p<0.01). Together these results demonstrate that 5-HT potentiates TGFβ signaling in AV cushions to control contractile differentiation, proliferation, and biomechanical remodeling.


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 increases AV cushion pSmad2/3 expression in ovo.A) Representative images of pSmad2/3 staining. Arrows indicate pSmad2/3 positive cells. Cell nuclei – blue, pSmad2/3 – red. B) Embryos treated with systemic 5-HT at HH17 have increased pSmad2/3 expression at HH25 indicating elevated TGFβ signaling. n = 6, mean ± SEM *p<0.01, t-test.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412853&req=5

pone-0042527-g008: 5-HT increases AV cushion pSmad2/3 expression in ovo.A) Representative images of pSmad2/3 staining. Arrows indicate pSmad2/3 positive cells. Cell nuclei – blue, pSmad2/3 – red. B) Embryos treated with systemic 5-HT at HH17 have increased pSmad2/3 expression at HH25 indicating elevated TGFβ signaling. n = 6, mean ± SEM *p<0.01, t-test.
Mentions: Systemic 5-HT treatment at HH17 resulted in a statistically significant 1.4±0.2 fold increase in AV cushion stiffness over control at stage HH25 (strain energy density of 0.43±0.06 Pa vs. 0.31±0.03 Pa, *p<0.05, Figure 7A). We next analyzed the mesenchymal gene expression patterns in this in ovo system. 5-HT significantly upregulated TGFβ3 (1.7±0.1), αSMA (1.5±0.1), col1α2 (1.5±0.1), cyclin b (1.6±0.2), and RhoA (1.7±0.2) (*p<0.05, Figure 7B). Interestingly, the TGFβ3 mRNA expression was comparable to that observed in the ex ovo organ culture treatment of TGFβ3 alone (2.2±0.6), 5-HT alone (1.9±0.1), and TGFβ3+5-HT (2.0±0.3). αSMA and col1α2 mRNA were also upregulated in ovo with 5-HT, but less than with direct TGFβ3 administration in ex vivo culture (αSMA – 1.5 vs 5.7, RhoA – 1.7 vs 2.1). The similar mRNA profiles of the candidate genes in both models suggested that 5-HT also potentiates TGFβ signaling in AV cushions in ovo. To confirm that the 5-HT treatment was indeed modulating TGFβ signaling activity in ovo, we quantified nuclear pSmad2/3 expression in HH25 cushions with and without 5-HT treatment (Figure 8). 5-HT treatment increased the number of cell nuclei with localized pSmad2/3 expression 2.6±0.8 fold over control embryos (0.28±0.04 vs. 0.11±0.03, p<0.01). Together these results demonstrate that 5-HT potentiates TGFβ signaling in AV cushions to control contractile differentiation, proliferation, and biomechanical remodeling.

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