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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 treatment increases stiffness of AV cushions through Alk5 mediated pathway.A) Representative pipette test data for TGFβ3 (1 ng/ml, TGFβ3+) and control media (TGFβ3−) treated cushions, n = 4. Strain energy density was calculated from the shaded regions beneath the ΔP vs λ curves. Inset: image of aspirated HH25 AV cushion after 24 hours of culture. The pipette radius, rp, and the aspirated length, L are indicated. Scale bar = 70 µm. B) AV cushion strain energy density increased with TGFβ3 treatment, but was blocked by Alk5 inhibition (SB431542, 2.6 µM). mean ± SEM, n≥7, *p<0.0001, 2-way ANOVA.
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pone-0042527-g001: TGFβ3 treatment increases stiffness of AV cushions through Alk5 mediated pathway.A) Representative pipette test data for TGFβ3 (1 ng/ml, TGFβ3+) and control media (TGFβ3−) treated cushions, n = 4. Strain energy density was calculated from the shaded regions beneath the ΔP vs λ curves. Inset: image of aspirated HH25 AV cushion after 24 hours of culture. The pipette radius, rp, and the aspirated length, L are indicated. Scale bar = 70 µm. B) AV cushion strain energy density increased with TGFβ3 treatment, but was blocked by Alk5 inhibition (SB431542, 2.6 µM). mean ± SEM, n≥7, *p<0.0001, 2-way ANOVA.

Mentions: Cushion mechanical properties were measured after 24 hour treatment in the ex vivo study and at HH25 in the in ovo study using the micromechanical pipette aspiration technique [22], [46], [47]. A glass micropipette (∼70–100 µm in diameter) was placed adjacent to the cushion surface, and a small vacuum pressure was incrementally applied. The pressure source was a 200 µL pipetter calibrated with a custom manometer. Previous strain history was mitigated by preconditioning with ∼20 cycles of low pressurization (<1 Pa). The tissue was then monotonically loaded with increasing static pressure loads, at which images were captured. Aspirated length L, measured as the length from the tip of the pipette to tip of the tissue furthest inside the pipette, was converted into an experimental “stretch ratio”, , by normalizing to the pipette radius, rp. The cushion was assumed to be an isotropic, incompressible, hyperelastic material with an exponential free energy law, , where IB is the first invariant of left Cauchy Green stretch tensor. AV cushion material isotropy at HH25 was supported by a lack of preferred matrix orientation as determined by ubiquitous protein stain 5-DTAF (50 µM Invitrogen; Figure S1). The ΔP vs. λ data was then fit to the axial stress equation of a uni-axially loaded bar of this exponential material, specifically, . From previous analysis [22], the ΔP vs. λ curve differs from the uniaxial load expression by a scale factor, γ. This scale factor was numerically determined to be a function of only the material parameter α. Due to the nonlinear nature of the data, the mechanical testing data is presented as strain energy density. This was calculated as the area under the ΔP vs. λ curve fit from λ = 1–2 (Figure 1A), which from our assumed material model is , where C* = γ C.


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 treatment increases stiffness of AV cushions through Alk5 mediated pathway.A) Representative pipette test data for TGFβ3 (1 ng/ml, TGFβ3+) and control media (TGFβ3−) treated cushions, n = 4. Strain energy density was calculated from the shaded regions beneath the ΔP vs λ curves. Inset: image of aspirated HH25 AV cushion after 24 hours of culture. The pipette radius, rp, and the aspirated length, L are indicated. Scale bar = 70 µm. B) AV cushion strain energy density increased with TGFβ3 treatment, but was blocked by Alk5 inhibition (SB431542, 2.6 µM). mean ± SEM, n≥7, *p<0.0001, 2-way ANOVA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042527-g001: TGFβ3 treatment increases stiffness of AV cushions through Alk5 mediated pathway.A) Representative pipette test data for TGFβ3 (1 ng/ml, TGFβ3+) and control media (TGFβ3−) treated cushions, n = 4. Strain energy density was calculated from the shaded regions beneath the ΔP vs λ curves. Inset: image of aspirated HH25 AV cushion after 24 hours of culture. The pipette radius, rp, and the aspirated length, L are indicated. Scale bar = 70 µm. B) AV cushion strain energy density increased with TGFβ3 treatment, but was blocked by Alk5 inhibition (SB431542, 2.6 µM). mean ± SEM, n≥7, *p<0.0001, 2-way ANOVA.
Mentions: Cushion mechanical properties were measured after 24 hour treatment in the ex vivo study and at HH25 in the in ovo study using the micromechanical pipette aspiration technique [22], [46], [47]. A glass micropipette (∼70–100 µm in diameter) was placed adjacent to the cushion surface, and a small vacuum pressure was incrementally applied. The pressure source was a 200 µL pipetter calibrated with a custom manometer. Previous strain history was mitigated by preconditioning with ∼20 cycles of low pressurization (<1 Pa). The tissue was then monotonically loaded with increasing static pressure loads, at which images were captured. Aspirated length L, measured as the length from the tip of the pipette to tip of the tissue furthest inside the pipette, was converted into an experimental “stretch ratio”, , by normalizing to the pipette radius, rp. The cushion was assumed to be an isotropic, incompressible, hyperelastic material with an exponential free energy law, , where IB is the first invariant of left Cauchy Green stretch tensor. AV cushion material isotropy at HH25 was supported by a lack of preferred matrix orientation as determined by ubiquitous protein stain 5-DTAF (50 µM Invitrogen; Figure S1). The ΔP vs. λ data was then fit to the axial stress equation of a uni-axially loaded bar of this exponential material, specifically, . From previous analysis [22], the ΔP vs. λ curve differs from the uniaxial load expression by a scale factor, γ. This scale factor was numerically determined to be a function of only the material parameter α. Due to the nonlinear nature of the data, the mechanical testing data is presented as strain energy density. This was calculated as the area under the ΔP vs. λ curve fit from λ = 1–2 (Figure 1A), which from our assumed material model is , where C* = γ C.

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