Organ culture as a tool to identify early mechanisms of serotonergic valve disease.

BACKGROUND AND AIM OF THE STUDY: Although the late effects of serotonergic valve disease are known, the early mechanisms of the characteristic plaque formation are poorly understood. METHODS: To model conditions leading to plaque formation on mitral valves, samples (n = 6-8 per treatment) cultured in a splashing bioreactor were exposed to serotonin (5HT) and norfenfluramine (NF). In order to assess the role of 5HT2B receptor activation, the effects of these drugs were also tested with a 5HT2B receptor antagonist. After two weeks, tissue samples were stained immunohistochemically to localize changes in multiple extracellular matrix (ECM) components and synthesis mediators. RESULTS: Decorin and versican expression tended to increase with 5HT treatment compared to NF or baseline controls, regardless of the presence of the receptor antagonist. Samples treated with 5HT or with the receptor antagonist tended to express less collagen (types I and III) and biglycan than NF or the baseline controls. Heat shock protein 47, prolyl-4-hydroxylase, matrix metalloproteinase 9 (MMP9) and MMP13 tended to be down-regulated with 5HT or NF exposure, although some samples treated with the antagonist displayed normal levels of these mediators. Superficial plaques grew on a subgroup of the NF-treated organ cultures, but on none of the 5HT and control valves. CONCLUSION: Although both serotonin agents lead to plaque formation in a clinical setting, the early effects of exposure to the different drugs were found to be quite different. Additionally, the different drug responses suggest that a mechanism other than 5HT2B receptor activation might contribute to plaque formation.

Design and validation of a novel splashing bioreactor system for use in mitral valve organ culture.

Previous research in our lab suggested that heart valve tissues cultured without mechanical stimulation do not retain their in vivo microstructure, i.e., cell density decreased within the deep tissue layers and increased at the periphery. In this study, a splashing rotating bioreactor was designed to apply mechanical stimulation to a mitral valve leaflet segment. Porcine valve segments (n = 9-10 per group) were cultured in the bioreactor for 2 weeks (dynamic culture), negative controls were cultured without mechanical stimulation (static culture), and baseline controls were fresh uncultured samples. Overall changes in cellularity and extracellular matrix (ECM) structure were assessed by H&E and Movat pentachrome stains. Tissues were also immunostained for multiple ECM components and turnover mediators. After 2 weeks of culture, proliferating cells were distributed throughout the tissue in segments cultured in the bioreactor, in contrast to segments cultured without mechanical stimulation. Most ECM components, especially collagen types I and III, better maintained normal expression patterns and magnitudes (as found in baseline controls) over 2 weeks of dynamic organ culture compared to static culture. Lack of mechanical stimulation changed several aspects of the tissue microstructure, including the cell distribution and ECM locations. In conclusion, mechanical stimulation by the bioreactor maintained tissue integrity, which will enable future in vitro investigation of mitral valve remodeling.

Abundance and location of proteoglycans and hyaluronan within normal and myxomatous mitral valves.

INTRODUCTION: Extracellular matrix changes occur in many heart valve pathologies. For example, myxomatous mitral valves are reported to contain excess proteoglycans and hyaluronan. However, it is unknown which specific proteoglycans are altered in myxomatous valves. Because proteoglycans perform varied functions in connective tissues, this study was designed to identify and localize three matrix-associated proteoglycans, as well as hyaluronan and the hyaluronan receptor for endocytosis, within myxomatous and normal mitral valves. METHODS: Human mitral posterior leaflets (control, n=6-9; myxomatous, n=14-21; mean age, 61 years for all groups) were histochemically stained for proteoglycan core proteins, hyaluronan, and the hyaluronan receptor for endocytosis. Stain intensity was semiquantitatively graded to determine differences in marker abundance between normal and myxomatous valves. The proteoglycans were localized to different regions of the leaflet by correspondence to parallel Movat-stained sections. RESULTS: The proteoglycans decorin, biglycan, and versican were more abundant in myxomatous valves than in normal controls (P<.03). There was a gender effect on proteoglycan presence, but no age-related trends were observed. Hyaluronan and the hyaluronan receptor for endocytosis were distributed throughout all valves. There was no significant difference in hyaluronan between groups, but expression of the hyaluronan receptor for endocytosis was reduced in myxomatous valves compared to normal controls (P<.002). CONCLUSION: Excess decorin, biglycan, and versican may be associated with the remodeling of other matrix components in myxomatous mitral valves. Decreased expression of the hyaluronan receptor for endocytosis in myxomatous valves suggests that hyaluronan metabolism could be altered in myxomatous mitral valve disease. These findings contribute towards elucidating the pathogenesis of myxomatous mitral valve disease and developing potential new therapies.

Age-related structural changes in cardiac valves: implications for tissue-engineered repairs.

Elderly patients would receive substantial benefits from tissue-engineered heart valves (TEHVs), but most TEHV research has not focused on applications for this growing patient population. There will be numerous technical challenges involved in developing TEHVs for the elderly, such as designing tissues to accommodate higher blood pressure and larger aortic roots that may be friable or calcified. Concomitant medications may also affect the biology of the TEHV. Due to the predominantly senescent behavior of cells from older persons, a nonautologous cell source may be required to develop the TEHV. Decellularized heart valve allografts from elderly donors may not be durable enough to use as a scaffold, but several polymer and natural biodegradable scaffolds may provide promising alternatives. The selection of cell sources, scaffolds, and mechanical/biologic conditioning will need to be precisely targeted to meet the diverse physiological, medical, and surgical requirements of elderly patients.