Elsevier

Cardiovascular Pathology

Volume 21, Issue 3, May–June 2012, Pages 206-213
Cardiovascular Pathology

Original Article
Elevated cyclic stretch and serotonin result in altered aortic valve remodeling via a mechanosensitive 5-HT2A receptor-dependent pathway

https://doi.org/10.1016/j.carpath.2011.07.005Get rights and content

Abstract

Introduction

Serotonin/5-hydroxytryptamine (5-HT) has been implicated in valve disease and in the modulation of valve mechanical properties. Several 5-HT receptor subtypes are also known to be mechanosensitive in other cell types, but this has not been studied in the context of the valve. In this study, we sought to understand the effects of elevated 5-HT levels and stretch overload on aortic valve remodeling and the dominant 5-HT receptor subtype that regulates these processes.

Methods and results

Collagen biosynthesis and tissue mechanical properties of porcine aortic valve cusps were evaluated after 10% (physiologic) and 15% (pathologic) dynamic stretch. These studies were performed in normal medium or medium supplemented with 5-HT (1, 10, 100 μM) in the absence and presence of 5-HT2A or 5-HT2B receptor antagonists. Fresh valves served as controls. Valve collagen content was maximal at the 10-μM 5-HT concentration for both 10% and 15% stretch. The 5-HT2A receptor antagonist reduced collagen synthesis, cell proliferation, and hsp47 expression under elevated and normal stretch, whereas the 5-HT2B receptor antagonist was effective only at normal stretch. The pretransition stiffness of the valve cusps was also increased in response to 5-HT via a stretch-sensitive 5-HT2A mechanism, with the post-transition stiffness unaltered.

Conclusions

Combined elevated stretch and 5-HT resulted in increased valve collagen biosynthesis, cell proliferation, and tissue stiffness. These responses were inhibited by a 5-HT2A antagonist. This strongly suggests that the 5-HT2A receptor subtype is sensitive to elevated stretch.

Introduction

Serotonin or 5-hydroxytryptamine (5HT) is a neurotransmitter that is targeted pharmacologically to regulate anger, aggression, mood, sleep, appetite, and metabolism, and forms the basis for several antidepressants, appetite suppressant drugs, and therapeutics for Parkinson's disease [1], [2]. Apart from their neuromodulatory effects, elevated circulating levels of 5-HT have been implicated in cardiovascular pathologies such as cardiac valve fibrosis, and systemic and pulmonary hypertension [3]. The in vivo hallmarks of 5-HT-related valvulopathy include excessive cellular proliferation and increased extracellular matrix synthesis [4], [5], which can result in abnormal thickening of the valve cusp and altered valve mechanical properties [3], [6].

Prior work has reported that 5-HT results in increased collagen biosynthesis [7], altered mechanical properties, and increased rate of leakage through the valve in a concentration-dependent manner [4], [8], [9]. Recent evidence indicates that these pathologies generally occur via 5-HT2-receptor-mediated mechanisms [10], [11], [12]. Static cell culture studies using aortic valve interstitial cells demonstrated that the 5-HT2A receptor regulates the downstream synthesis of TGF-β in valve cells and, to some extent, regulated the ERK-1/2 signaling via the MAP kinase pathway [13]. Data from this study indicated that other receptor subtypes may also be involved in serotonin signaling, and the role of 5-HT2B and 5-HT2C receptor subtypes was hypothesized. Additionally, Liang et al. [14] reported that the 5-HT2B receptor was mechanosensitive in cardiomyocytes, postulating a role for mechanics in regulating 5-HT-mediated cellular responses.

We therefore hypothesized that mechanical stretch magnitude in the aortic valve alters its response to 5-HT via 5-HT2A and 5HT2B receptor subtypes. These specific receptor subtypes are especially relevant in valve pathology [7], [11], [14], [15], [16], [17] and are also highly up-regulated by mechanical stretch [18]. We report that higher magnitudes of mechanical stretch enhance 5-HT-induced collagen synthesis, leading to altered valve mechanical properties and that these responses could be mediated by 5-HT2A inhibition.

Section snippets

Tissue harvest and cyclic stretch experimental setup

Fresh aortic valve specimens were explanted as in our previous studies [19], [20], [21] (Fig. 1A), randomized, and assigned to three mechanical treatments—fresh controls, 10% dynamic stretch (physiologic [22]), and 15% dynamic stretch (pathologic [20], [23]). Dynamic stretch experiments were conducted for 72 h, which has been demonstrated as sufficient for significant structural and compositional changes within the valve cusp [7], [13], [19], [20].

Serotonin and serotonin receptor antagonist treatment groups

The control treatment groups consisted of

Effect of cyclic stretch and serotonin concentration on valve collagen content

Collagen content (Fig. 2A) was comparable between fresh controls and 10% stretch in normal media, but increased at 15% stretch in the same media as expected [19]. At 10% stretch, 1 µM 5-HT did not alter collagen compared to the no-5-HT control, while collagen content increased significantly at 10 µM 5-HT. At 100 µM, a drop in collagen content was observed in comparison to the preceding concentration, but not fresh control. At 15% stretch, 1 µM 5-HT did not significantly increase collagen

Discussion

Results from this study demonstrate two key findings: (I) 5-HT2A and 5-HT2B receptor subtypes regulate 5-HT-induced collagen remodeling in aortic valve tissue; (II) mechanical stretch magnitude governs the dominant receptor subtype in 5-HT-induced remodeling in the aortic valve. Considering the active, dynamic nature of the aortic valve, understanding the mechanosensitive response of the valve to circulating 5-HT is crucial in defining a complete picture of serotonin-related valvulopathy.

Limitations

The ex vivo model used in this study limits the study of molecular mechanisms relating to 5-HT-mediated disease as compared with cell culture studies. However, it is a powerful, high-throughput, bench-top model which can provide assessments of whole valve tissue under dynamic loading conditions. We also acknowledge that our ex vivo model does not simulate all in vivo mechanical conditions such as shear stress; however, we expect the addition of shear stress to compound the observed results.

Acknowledgments

The authors would like to acknowledge Holifield Farms, Covington, GA, for donating the porcine hearts for this study.

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    K. Balachandran was supported by an American Heart Association Predoctoral Fellowship (09PRE2060605).

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