Telotristat ethyl reverses myxomatous changes in mice mitral valves.

Rationale: Myxomatous mitral valve degeneration is a common pathological manifestation of mitral valve regurgitation, with or without valvular prolapse. In addition to similarities between naturally occurring and serotonergic valve degeneration, an increasing body of evidence has recently suggested that serotonin signaling is a regulator of degenerative valvulopathies. Studies have found that serotonin can be synthesized locally by valvular cells and serotonin receptors in turn may be activated to promote signaling. Recently, telotristat ethyl (TE) has been introduced as a treatment for carcinoid disease, by selectively inhibiting tryptophan hydroxylase 1, the rate-limiting enzyme in peripheral serotonin synthesis. TE provides a unique tool to test inhibition of serotonin synthesis in vivo, without impacting brain serotonin, to further confirm the role of local serotonin synthesis on heart valves. Objective: To confirm the link between serotonin and myxomatous valvular disease in vivo. Methods and results: A hypertension-induced myxomatous mitral valve disease mouse model was employed to test the effect of TE on valvular degeneration. Circulating serotonin and local serotonin in valve tissues were tested by enzyme immunoassay and immunohistochemistry, respectively. TE was administrated in two modes: (1) parallel with angiotensin II (A2); (2) post A2 treatment. Myxomatous changes were successfully recapitulated in hypertensive mice, as determined by ECM remodeling, myofibroblast transformation, and serotonin signaling activation. These changes were at least partially reversed upon TE administration. Conclusion: This study provides the first evidence of TE as a potential therapeutic for myxomatous mitral disease, either used to prevent or reverse myxomatous degeneration.

Comparison of Serotonin-Regulated Calcific Processes in Aortic and Mitral Valvular Interstitial Cells.

Calcification is an important pathological process and a common complication of degenerative valvular heart diseases, with higher incidence in aortic versus mitral valves. Two phenotypes of valvular interstitial cells (VICs), activated VICs and osteoblastic VICs (obVICs), synergistically orchestrate this pathology. It has been demonstrated that serotonin is involved in early stages of myxomatous mitral degeneration, whereas the role of serotonin in calcific aortic valve disease is still unknown. To uncover the link between serotonin and osteogenesis in heart valves, osteogenesis of aortic and mitral VICs was induced in vitro. Actin polymerization and serotonin signaling were inhibited using cytochalasin D and serotonin inhibitors, respectively, to investigate the role of cell activation and serotonin signals in valvular cell osteogenesis. To evaluate calcification progress, calcium and collagen deposits along with the expression of protein markers, including the rate-limiting enzyme of serotonin synthesis [tryptophan hydroxylase 1 (TPH1)], were assessed. When exposed to osteogenic culture conditions and grown on soft surfaces, passage zero aortic VICs increased extracellular collagen deposits and obVIC phenotype markers. A more intense osteogenic process was observed in aortic VICs of higher passages, where cells were activated prior to osteogenic induction. For both, TPH1 expression was upregulated as osteogenesis advanced. However, these osteogenic changes were reversed upon serotonin inhibition. This discovery provides a better understanding of signaling pathways regulating VIC phenotype transformation and explains different manifestations of degenerative pathologies. In addition, the discovery of serotonin-based inhibition of valvular calcification will contribute to the development of potential novel therapies for calcific valvular diseases.

A survey of membrane receptor regulation in valvular interstitial cells cultured under mechanical stresses.

Degenerative valvular diseases have been linked to the action of abnormal forces on valve tissues during each cardiac cycle. It is now accepted that the degenerative behavior of valvular cells can be induced mechanically in vitro. This approach of in vitro modeling of valvular cells in culture constitutes a powerful tool to study, characterize, and develop predictors of heart valve degeneration in vivo. Using such in vitro systems, we expect to determine the exact signaling mechanisms that trigger and mediate propagation of degenerative signals. In this study, we aim to uncover the role of mechanosensing proteins on valvular cell membranes. These can be cell receptors and triggers of downstream pathways that are activated upon the action of cyclical tensile strains in pathophysiological conditions. In order to identify mechanosensors of tensile stresses on valvular interstitial cells, we employed biaxial cyclic strain of valvular cells in culture and quantitatively evaluated the expression of cell membrane proteins using a targeted protein array and interactome analyses. This approach yielded a high-throughput screening of all cell surface proteins involved in sensing mechanical stimuli. In this study, we were able to identify the cell membrane proteins which are activated during physiological cyclic tensile stresses of valvular cells. The proteins identified in this study were clustered into four interactomes, which included CC chemokine ligands, thrombospondin (adhesive glycoproteins), growth factors, and interleukins. The expression levels of these proteins generally indicated that cells tend to increase adhesive efforts to counteract the action of mechanical forces. This is the first study of this kind used to comprehensively identify the mechanosensitive proteins in valvular cells.

Local serotonin mediates cyclic strain-induced phenotype transformation, matrix degradation, and glycosaminoglycan synthesis in cultured sheep mitral valves.

This study addressed the following questions: 1) Does cyclic tensile strain induce protein expression patterns consistent with myxomatous degeneration in mitral valves? 2) Does cyclic strain induce local serotonin synthesis in mitral valves? 3) Are cyclic strain-induced myxomatous protein expression patterns in mitral valves dependent on local serotonin? Cultured sheep mitral valve leaflets were subjected to 0, 10, 20, and 30% cyclic strain for 24 and 72 h. Protein levels of activated myofibroblast phenotype markers, α-smooth muscle actin (α-SMA) and nonmuscle embryonic myosin (SMemb); matrix catabolic enzymes, matrix metalloprotease (MMP) 1 and 13, and cathepsin K; and sulfated glycosaminoglycan (GAG) content in mitral valves increased with increased cyclic strain. Serotonin was present in the serum-free media of cultured mitral valves and concentrations increased with cyclic strain. Expression of the serotonin synthetic enzyme tryptophan hydroxylase 1 (TPH1) increased in strained mitral valves. Pharmacologic inhibition of the serotonin 2B/2C receptor or TPH1 diminished expression of phenotype markers (α-SMA and SMemb) and matrix catabolic enzyme (MMP1, MMP13, and cathepsin K) expression in 10- and 30%-strained mitral valves. These results provide first evidence that mitral valves synthesize serotonin locally. The results further demonstrate that tensile loading modulates local serotonin synthesis, expression of effector proteins associated with mitral valve degeneration, and GAG synthesis. Inhibition of serotonin diminishes strain-mediated protein expression patterns. These findings implicate serotonin and tensile loading in mitral degeneration, functionally link the pathogeneses of serotoninergic (carcinoid, drug-induced) and degenerative mitral valve disease, and have therapeutic implications.

Signaling pathways in mitral valve degeneration.

Heart valves exhibit a highly-conserved stratified structure exquisitely designed to counter biomechanical forces delivered over a lifetime. Heart valve structure and competence is maintained by heart valve cells through a process of continuous turnover extracellular matrix (ECM). Degenerative (myxomatous) mitral valve disease (DMVD) is an important disease associated with aging in both dogs and humans. DMVD is increasingly regarded as a disease with identifiable signaling mechanisms that control key genes associated with regulation and dysregulation of ECM homeostasis. Initiating stimuli for these signaling pathways have not been fully elucidated but likely include both mechanical and chemical stimuli. Signaling pathways implicated in DMVD include serotonin, transforming growth factor ß (TGFß), and heart valve developmental pathways. High circulating serotonin (carcinoid syndrome) and serotoninergic drugs are known to cause valvulopathy that shares pathologic features with DMVD. Recent evidence supports a local serotonin signaling mechanism, possibly triggered by high tensile loading on heart valves. Serotonin initiates TGFß signaling, which in turn has been strongly implicated in canine DMVD. Recent evidence suggests that degenerative aortic and mitral valve disease may involve pathologic processes that mimic osteogenesis and chondrogenesis, respectively. These processes may be mediated by developmental pathways shared by heart valves, bone, and cartilage. These pathways include bone morphogenic protein (BMP) and Wnt signaling. Other signaling pathways implicated in heart valve disease include Notch, nitric oxide, and angiotensin II. Ultimately, increased understanding of signaling mechanisms could point to therapeutic strategies aimed at slowing or halting disease progression.

Static and cyclic tensile strain induce myxomatous effector proteins and serotonin in canine mitral valves.

OBJECTIVES: Degenerative (myxomatous) mitral valve disease is an important cardiac disease in dogs and humans. The mechanisms that initiate and propagate myxomatous pathology in mitral valves are poorly understood. We investigated the hypothesis that tensile strain initiates expression of proteins that mediate myxomatous pathology. We also explored whether tensile strain could induce the serotonin synthetic enzyme tryptophan hydroxylase 1 (TPH1), serotonin synthesis, and markers of chondrogenesis. ANIMALS: Mitral valves were obtained postmortem from dogs without apparent cardiovascular disease. METHODS: Mitral valves were placed in culture and subjected to 30% static or cyclic tensile strain and compared to cultured mitral valves subjected to 0% strain for 72 h. Abundance of target effector proteins, TPH1, and chondrogenic marker proteins was determined by immunoblotting. Serotonin was measured in the conditioned media by ELISA. RESULTS: Both static and cyclic strain increased (p < 0.05) expression of myxomatous effector proteins including markers of an activated myofibroblast phenotype, matrix catabolic and synthetic enzymes in canine mitral valves compared to unstrained control. Expression of TPH1 was increased in statically and cyclically strained mitral valves. Expression of chondrogenic markers was increased in statically strained mitral valves. Serotonin levels were higher (p < 0.05) in media of cyclically strained valves compared to unstrained valves after 72 h of culture. CONCLUSION: Static or cyclic tensile strain induces acute increases in the abundance of myxomatous effector proteins, TPH1, and markers of chondrogenesis in canine mitral valves. Canine mitral valves are capable of local serotonin synthesis, which may be influenced by strain.

Differential protein expression between normal, early-stage, and late-stage myxomatous mitral valves from dogs.

Valvular heart disease accounts for over 20 000 deaths and 90 000 hospitalizations yearly in the United States. Myxomatous valve disease (MVD) is the most common disease of the mitral valve in humans and dogs. MVD is pathologically identical in these species and its pathogenesis is poorly understood. The objectives of this study were to (i) develop proteomic methodology suitable for analysis of extracellular matrix-rich heart valve tissues and (ii) survey over- and under-expressed proteins that could provide mechanistic clues into the pathogenesis of MVD. Normal, early-stage, and late-stage myxomatous mitral valves from dogs were studied. A shotgun proteomic analysis was used to quantify differential protein expression. Proteins were classified by function and clustered according to differential expression patterns. More than 300 proteins, with 117 of those being differentially expressed, were identified. Hierarchical sample clustering of differential protein profiles showed that early- and late-stage valves were closely related. This finding suggests that proteome changes occur in early degeneration stages and these persist in late stages, characterizing a diseased proteome that is distinct from normal. Shotgun proteome analysis of matrix-rich canine heart valves is feasible, and should be applicable to human heart valves. This study provides a basis for future investigations into the pathogenesis of MVD.

Metaproteomic analysis of a bacterial community response to cadmium exposure.

Microbial communities are of great environmental, medical, and industrial significance. To date, biomolecular methods to study communities have focused on identifying species, with limited capabilities to reveal functions. Proteomics has the potential to yield functional information about these communities, but the application of proteomic methods to complex mixtures of unsequenced organisms is in its infancy. In this study, 2DE, MALDI-TOF/TOF MS, and de novo peptide sequencing were used for the separation and identification of proteins differentially expressed over time following exposure of a bacterial community to an inhibitory level of cadmium. Significant community proteome responses after 0.25, 1, 2, and 3 h of exposure to cadmium were observed, with more than 100 protein expression changes detected at each time point. Several temporal responses were observed, and the most common expression pattern was immediate up- or down-regulation within 15 min of shock followed by maintenance of that level. More than 100 unique differentially expressed proteins were identified through database searching and de novo sequencing. Proteins of importance in the cadmium shock included ATPases, oxidoreductases, and transport proteins. The ability of proteomics to detect the differential regulation of these proteins even during short cadmium exposures shows that it is a powerful tool in explaining cellular mechanisms for a mixed culture. This is the first report of the large-scale identification of proteins involved in the dynamic response of a community of unsequenced bacteria using de novo sequencing.