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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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Remodeling behavior of TGFβ3 treated cushions is a balance of contractile differentiation, proliferation, and matrix synthesis.A) 24 hour TGFβ3 treated cushions upregulate contractile (αSMA, RhoA), proliferation (cyclin b), and extracellular matrix protein (col1α2) encoding genes. TGFβ3 administration also significantly stimulated its own production. mean ± SEM, n = 3–4 pooled samples of 8–10 cushions, *p<0.05, t-test. B) BrdU incorporation data (red) of TGFβ3 treated cushions normalized to DRAQ5 cell nuclei counter stain (blue). BrdU was administered 6 hours prior to completion of 24 hour treatment. Representative confocal images are shown above each bar, with a global view of cushion contained in the inset. mean ± SEM, n = 12, *p<0.0001, t-test.
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pone-0042527-g003: Remodeling behavior of TGFβ3 treated cushions is a balance of contractile differentiation, proliferation, and matrix synthesis.A) 24 hour TGFβ3 treated cushions upregulate contractile (αSMA, RhoA), proliferation (cyclin b), and extracellular matrix protein (col1α2) encoding genes. TGFβ3 administration also significantly stimulated its own production. mean ± SEM, n = 3–4 pooled samples of 8–10 cushions, *p<0.05, t-test. B) BrdU incorporation data (red) of TGFβ3 treated cushions normalized to DRAQ5 cell nuclei counter stain (blue). BrdU was administered 6 hours prior to completion of 24 hour treatment. Representative confocal images are shown above each bar, with a global view of cushion contained in the inset. mean ± SEM, n = 12, *p<0.0001, t-test.

Mentions: Contractile phenotype markers αSMA and RhoA were significantly upregulated with TGFβ3 treatment, 5.3±0.4 and 2.1±0.3 fold (± SEM) respectively (Figure 3A), suggesting that TGFβ3 induced residual tension is partially due to an increased migratory/contractile phenotype of resident cushion mesenchyme. TGFβ3 treatment also upregulated mRNA expression of col1α2 mRNA (3.8±0.9, p<0.05) and cyclin b2 (3.9±0.7 fold, p<0.05), indicative of increased collagen I synthesis and cell proliferation, respectively. BrdU incorporation confirmed that TGFβ3 increased cushion cell proliferation 2.26±0.36 fold over controls (p<0.0001, Figure 3B). Collectively, these results strongly suggest that while TGFβ3 treated AV cushion mesenchyme are more migratory/contractile, concomitant increases in cell proliferation and matrix synthesis work to counteract aggregate matrix compaction. This explains how the TGFβ3 treated cushions are biomechanically stiffer, but appear minimally compacted. Furthermore, TGFβ3 treatment increased TGFβ3 transcription (2.2±0.6 fold, p<0.05), indicating a potential positive feedback loop for TGFβ3 control of AV cushion 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)

Remodeling behavior of TGFβ3 treated cushions is a balance of contractile differentiation, proliferation, and matrix synthesis.A) 24 hour TGFβ3 treated cushions upregulate contractile (αSMA, RhoA), proliferation (cyclin b), and extracellular matrix protein (col1α2) encoding genes. TGFβ3 administration also significantly stimulated its own production. mean ± SEM, n = 3–4 pooled samples of 8–10 cushions, *p<0.05, t-test. B) BrdU incorporation data (red) of TGFβ3 treated cushions normalized to DRAQ5 cell nuclei counter stain (blue). BrdU was administered 6 hours prior to completion of 24 hour treatment. Representative confocal images are shown above each bar, with a global view of cushion contained in the inset. mean ± SEM, n = 12, *p<0.0001, t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042527-g003: Remodeling behavior of TGFβ3 treated cushions is a balance of contractile differentiation, proliferation, and matrix synthesis.A) 24 hour TGFβ3 treated cushions upregulate contractile (αSMA, RhoA), proliferation (cyclin b), and extracellular matrix protein (col1α2) encoding genes. TGFβ3 administration also significantly stimulated its own production. mean ± SEM, n = 3–4 pooled samples of 8–10 cushions, *p<0.05, t-test. B) BrdU incorporation data (red) of TGFβ3 treated cushions normalized to DRAQ5 cell nuclei counter stain (blue). BrdU was administered 6 hours prior to completion of 24 hour treatment. Representative confocal images are shown above each bar, with a global view of cushion contained in the inset. mean ± SEM, n = 12, *p<0.0001, t-test.
Mentions: Contractile phenotype markers αSMA and RhoA were significantly upregulated with TGFβ3 treatment, 5.3±0.4 and 2.1±0.3 fold (± SEM) respectively (Figure 3A), suggesting that TGFβ3 induced residual tension is partially due to an increased migratory/contractile phenotype of resident cushion mesenchyme. TGFβ3 treatment also upregulated mRNA expression of col1α2 mRNA (3.8±0.9, p<0.05) and cyclin b2 (3.9±0.7 fold, p<0.05), indicative of increased collagen I synthesis and cell proliferation, respectively. BrdU incorporation confirmed that TGFβ3 increased cushion cell proliferation 2.26±0.36 fold over controls (p<0.0001, Figure 3B). Collectively, these results strongly suggest that while TGFβ3 treated AV cushion mesenchyme are more migratory/contractile, concomitant increases in cell proliferation and matrix synthesis work to counteract aggregate matrix compaction. This explains how the TGFβ3 treated cushions are biomechanically stiffer, but appear minimally compacted. Furthermore, TGFβ3 treatment increased TGFβ3 transcription (2.2±0.6 fold, p<0.05), indicating a potential positive feedback loop for TGFβ3 control of AV cushion 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