Extracellular matrix remodeling and cell phenotypic changes in dysplastic and hemodynamically altered semilunar human cardiac valves.

INTRODUCTION: Congenital cardiac valve disease is common, affecting ∼1% of the population, with substantial morbidity and mortality, but suboptimal treatment options. Characterization of the specific matrix and valve cell phenotypic abnormalities in these valves could lend insight into disease pathogenesis and potentially pave the way for novel therapies. METHODS: Thirty-five human aortic and pulmonic valves were categorized based on gross and microscopic assessment into control valves (n=21); dysplastic valves, all except one also displaying hemodynamic changes (HEMO/DYSP, n=6); and hemodynamically altered valves (HEMO, n=8). Immunohistochemistry was performed on valve sections and flow cytometry on valvular interstitial cells. RESULTS: While both hemodynamically altered aortic and pulmonic valves demonstrated increased collagen turnover and cell activation, prolyl 4-hydroxylase and hyaluronan increased in hemodynamically altered aortic valves but decreased in hemodynamically altered pulmonic valves relative to control valves (P<.001). HEMO/DYSP aortic valves demonstrated decreased collagen and elastic fiber synthesis and turnover compared to both hemodynamically altered aortic valves and control aortic valves (each P<.006). Valvular interstitial cells from both hemodynamically altered and HEMO/DYSP pulmonic valves showed altered cell phenotype compared to control valves (each P<.032), especially increased non-muscle myosin. Furthermore, valvular interstitial cells from hemodynamically altered pulmonic valves and HEMO/DYSP aortic and pulmonic valves each demonstrated greater size and complexity compared to control valves (each P<.05). CONCLUSIONS: Dysplastic semilunar valves displayed alterations in collagen and elastic fiber turnover that were distinct from valves similarly exposed to altered hemodynamics as well as to control valves. These results demonstrate that dysplastic valves are not simply valves with gross changes or loss of leaflet layers, but contain complex matrix and cell phenotype changes that, with future study, could potentially be targets for novel nonsurgical treatments.

Functional characterization of fibronectin-separated valve interstitial cell subpopulations in three-dimensional culture.

BACKGROUND AND AIM OF THE STUDY: Myxomatous mitral valves (MVs) contain elevated proportions of myofibroblasts, a valve interstitial cell (VIC) subpopulation that may be important in disease pathogenesis. A novel technique was recently developed for the isolation of VIC myofibroblasts using time-dependent adhesion to fibronectin (FN). Cells that adhere rapidly to FN ('FAST') demonstrate myofibroblast cell phenotype markers, in contrast to cells that fail to adhere after a longer time ('SLOW'). The study aim was to characterize the functionality of these subpopulations using three-dimensional (3D) collagen constructs. METHODS: The VICs were harvested from porcine mitral valve posterior leaflets. FAST and SLOW subpopulations, as well as unseparated VIC populations grown on FN and tissue culture plastic (TCP) (UNSEP FN, UNSEP TCP), were seeded within 3D collagen gels and cultured for three weeks. Collagen gel contraction was assessed throughout the culture duration; the mechanical properties of the resultant collagen constructs were assessed using uniaxial tensile testing. RESULTS: FAST cells demonstrated a greater contraction of collagen gels compared to SLOW cells, particularly after 10 days (p < 0.05). Interestingly, the collagen gel contraction by both FN-separated VIC subpopulations (FAST and SLOW) was greater than for gels seeded with UNSEP TCP VICs (p < 0.05). Further, the contraction of UNSEP FN gels was greater than UNSEP TCP throughout the culture duration (p < OR = 0.002), suggesting that the subculture of VICs on FN potentiated these phenotypic changes. Finally, the collagen constructs seeded with FAST cells were stiffer than those seeded with SLOW, followed by UNSEP TCP (p < 0.001). The same pattern was found for failure stress (p = 0.006). CONCLUSION: Time-dependent adhesion to FN produced a VIC subpopulation (FAST), the function of which in 3D culture was consistent with that of myofibroblasts; FN exposure alone also caused VICs to function similarly to myofibroblasts. This novel isolation method may prove valuable in future studies of myofibroblasts in valve disease.

The use of collagenase III for the isolation of porcine aortic valvular interstitial cells: rationale and optimization.

BACKGROUND AND AIM OF THE STUDY: Substantial heart valve research relies on the isolation of valvular interstitial cells (VICs). While a wide variety of conditions have been reported for VIC isolation, the effectiveness of these methods has rarely been compared. It is also likely that valve donor age will influence these valvular tissue dissociation conditions. The study aim was to increase the efficiency and cost-effectiveness of VIC isolation, while taking into account possible differences due to valve donor age. METHODS: Aortic valves were obtained from six-month-old (n = 24) and six-week-old (suckling) pigs (n = 45) within 24 h of death. After removal of endothelial cells, the tissues were minced and subjected to a variety of enzymatic digestions for variable lengths of time. RESULTS: The optimal concentration of collagenase III was determined as 1 mg/ml for six-week-old pigs, and 2 mg/ml for six-month-old pigs. The optimal duration of digestion was 4 h for both ages. The addition of neutral protease (2 mg/ml) further increased yield, while additional DNAse and hyaluronidase had no effect. Yield was not influenced by the volume of enzyme solution, nor the use of previously frozen enzyme solution. CONCLUSION: These findings provide age-specific conditions for improving the yield of VIC isolation, which should be of value in experimental studies of valvular cell biology and tissue engineering investigations.

Phenotypic characterization of isolated valvular interstitial cell subpopulations.

BACKGROUND AND AIM OF THE STUDY: Valvular interstitial cells (VICs) demonstrate a heterogeneous range of phenotypes such as variable expression of smooth muscle alpha-actin (SMalphaA). Myofibroblast-like VICs, expressing high levels of SMalphaA, are thought to be involved in myxomatous degeneration of mitral valves. The inability to isolate specific cell types has restricted potential investigations of valvular disease mechanisms. Thus, investigations were conducted into methods of isolating different cell subpopulations from primary VICs as a preparatory step for cell type-specific evaluations of heart valve disease. METHODS: VICs were isolated from porcine valves, cultured to 80% confluency, and subdivided using differential detachment or adhesion. The subdivided cells were further cultured and analyzed phenotypically by immunocytochemistry and flow cytometry to characterize SMalphaA expression. Roundness and growth rates were also analyzed. RESULTS: VICs that were relatively sensitive to trypsinization expressed low and heterogeneous levels of SMalphaA (15-35%), whereas more-adherent VICs expressed higher and homogeneous levels (>98%) suggestive of a myofibroblast-like phenotype. The more-adherent cells also had lower growth potential and were less round than less-adhesive VICs. Separated cell subtypes were found to maintain their phenotype through several cell passages. CONCLUSION: VICs are a mixed population of cells, many of which express high levels of SMalphaA. Differential detachment and adhesion can effectively separate cell subpopulations from primary cultures of VICs. The ability to study valve cell subpopulations has substantial implications for future analyses of valvular biology, disease, and tissue engineering.