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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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TGFβ3 treated cushions compact less than controls, but are under more residual tension.A) Bar graph of area ratios calculated from before and after images of 24 hour TGFβ3 treated cushions. Representative cushion images shown, scale bar = 100 µm. mean ± SEM, n≥12, *p<0.0001, t-test B) Opening angle of 24 hour TGFβ3 treated cushions is greater than control, indicating tissue is under greater residual tension. Inset shows representative images with opening angle, θ. mean ± SEM, n = 10–11, *p<0.001 t-test.
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pone-0042527-g002: TGFβ3 treated cushions compact less than controls, but are under more residual tension.A) Bar graph of area ratios calculated from before and after images of 24 hour TGFβ3 treated cushions. Representative cushion images shown, scale bar = 100 µm. mean ± SEM, n≥12, *p<0.0001, t-test B) Opening angle of 24 hour TGFβ3 treated cushions is greater than control, indicating tissue is under greater residual tension. Inset shows representative images with opening angle, θ. mean ± SEM, n = 10–11, *p<0.001 t-test.

Mentions: Ex vivo cultured AV cushions exhibited nonlinear mechanical behavior that was well described by the exponential constitutive model (Figure 1A). Administration of exogenous TGFβ3 (1 ng/ml) increased cushion stiffness 2.5 fold over controls (WTGFβ3 = 0.965±0.051 vs. WContr = 0.378±0.021, p<0.0001 Figure 1B). Inhibition of canonical TGFβ signaling via the TGFβ type 1 receptor Alk 5 (2.6 µM SB431542 [54]) blocked the increase in cushion stiffness (WT+TI = 0.245±0.043 Figure 1B). The Alk5 inhibitor alone had no effect on cushion biomechanics. TGFβ3-treated cushions compacted less than controls, with compaction quantified as the ratio of cross-sectional area before and after treatment (A/A0 = 0.925±0.028 vs. A/A0 = 0.508±0.017, p<0.0001 Figure 2A). This was unexpected because the Cytochalasin D (CytD, 1 µM) results suggested that compaction and stiffness are directly related. CytD inhibited cytoskeletal actin polymerization which resulted in a 5.3 fold decrease in strain energy density of the AV cushions relative to control (WCytD = 0.072±0.016, Figure S2A). Without actin polymerization the AV cushion cells did not compact the matrix, and the cushion did not remodel into the spherical configuration observed in all other treatments. Instead, the post-treatment cushion area was significantly larger than initial area, suggesting a relaxation of pre-treatment actin forces (A/A0 = 1.60±0.03, Figure S2B). The TGFβ3 results of stiffness increase with compaction decrease did not align with this trend. Alk5 inhibition did return compaction behavior to control levels (A/A0 = 0.570±0.035 Figure S3), indicating that the stiffness and compaction results are both dependent on activation of canonical TGFβ3 signaling. To better understand the relationship between stiffness and compaction, cushion opening angles were quantified to approximate differences in cell traction forces. The opening angle of TGFβ3 cushions was 1.29 fold larger than controls (74.6°±2.0° vs. 57.7°±1.4°, p<0.001 Figure 2B), indicating that TGFβ3 treated cushions did indeed have higher cell traction forces. Together, these results demonstrate that TGFβ3 induces cushion stiffening through Alk5, but with a concurrent reduction in tissue compaction that suggests other processes are also affected.


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)

TGFβ3 treated cushions compact less than controls, but are under more residual tension.A) Bar graph of area ratios calculated from before and after images of 24 hour TGFβ3 treated cushions. Representative cushion images shown, scale bar = 100 µm. mean ± SEM, n≥12, *p<0.0001, t-test B) Opening angle of 24 hour TGFβ3 treated cushions is greater than control, indicating tissue is under greater residual tension. Inset shows representative images with opening angle, θ. mean ± SEM, n = 10–11, *p<0.001 t-test.
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Related In: Results  -  Collection

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pone-0042527-g002: TGFβ3 treated cushions compact less than controls, but are under more residual tension.A) Bar graph of area ratios calculated from before and after images of 24 hour TGFβ3 treated cushions. Representative cushion images shown, scale bar = 100 µm. mean ± SEM, n≥12, *p<0.0001, t-test B) Opening angle of 24 hour TGFβ3 treated cushions is greater than control, indicating tissue is under greater residual tension. Inset shows representative images with opening angle, θ. mean ± SEM, n = 10–11, *p<0.001 t-test.
Mentions: Ex vivo cultured AV cushions exhibited nonlinear mechanical behavior that was well described by the exponential constitutive model (Figure 1A). Administration of exogenous TGFβ3 (1 ng/ml) increased cushion stiffness 2.5 fold over controls (WTGFβ3 = 0.965±0.051 vs. WContr = 0.378±0.021, p<0.0001 Figure 1B). Inhibition of canonical TGFβ signaling via the TGFβ type 1 receptor Alk 5 (2.6 µM SB431542 [54]) blocked the increase in cushion stiffness (WT+TI = 0.245±0.043 Figure 1B). The Alk5 inhibitor alone had no effect on cushion biomechanics. TGFβ3-treated cushions compacted less than controls, with compaction quantified as the ratio of cross-sectional area before and after treatment (A/A0 = 0.925±0.028 vs. A/A0 = 0.508±0.017, p<0.0001 Figure 2A). This was unexpected because the Cytochalasin D (CytD, 1 µM) results suggested that compaction and stiffness are directly related. CytD inhibited cytoskeletal actin polymerization which resulted in a 5.3 fold decrease in strain energy density of the AV cushions relative to control (WCytD = 0.072±0.016, Figure S2A). Without actin polymerization the AV cushion cells did not compact the matrix, and the cushion did not remodel into the spherical configuration observed in all other treatments. Instead, the post-treatment cushion area was significantly larger than initial area, suggesting a relaxation of pre-treatment actin forces (A/A0 = 1.60±0.03, Figure S2B). The TGFβ3 results of stiffness increase with compaction decrease did not align with this trend. Alk5 inhibition did return compaction behavior to control levels (A/A0 = 0.570±0.035 Figure S3), indicating that the stiffness and compaction results are both dependent on activation of canonical TGFβ3 signaling. To better understand the relationship between stiffness and compaction, cushion opening angles were quantified to approximate differences in cell traction forces. The opening angle of TGFβ3 cushions was 1.29 fold larger than controls (74.6°±2.0° vs. 57.7°±1.4°, p<0.001 Figure 2B), indicating that TGFβ3 treated cushions did indeed have higher cell traction forces. Together, these results demonstrate that TGFβ3 induces cushion stiffening through Alk5, but with a concurrent reduction in tissue compaction that suggests other processes are also affected.

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