Live en face imaging of aortic valve leaflets under mechanical stress

Abstract

Soft tissues, such as tendons, skin, arteries, or lung, are constantly subject to mechanical stresses in vivo. None more so than the aortic heart valve that experiences an array of forces including shear stress, cyclic pressure, strain, and flexion. Anisotropic biaxial cyclic stretch maintains valve homeostasis; however, abnormal forces are implicated in disease progression. The response of the valve endothelium to deviations from physiological levels has not been fully characterized. Here, we show the design and validation of a novel stretch apparatus capable of applying biaxial stretch to viable heart valve tissue, while simultaneously allowing for live en face endothelial cell imaging via confocal laser scanning microscopy (CLSM). Real-time imaging of tissue is possible while undergoing highly characterized mechanical conditions and maintaining the native extracellular matrix. Thus, it provides significant advantages over traditional cell culture or in vivo animal models. Planar biaxial tissue stretching with simultaneous live cell imaging could prove useful in studying the mechanobiology of any soft tissue.

Inflammatory Cytokines Promote Mesenchymal Transformation in Embryonic and Adult Valve Endothelial Cells

“…While no gross calcification may be present, the valve endothelium exhibits non-negligible inflammatory activation 55-58 Paruchuri et al identified clonally isolated sub-populations of human pulmonary VEC that were capable of EndMT in response to TGFβ, which supports our findings in pure whole VEC populations 59 . Holliday et al recently showed that in vitro cultured human aortic VEC isolated from heart transplant surgeries express endothelial phenotype markers and elevated α-SMA, which is in contrast to in vivo expression patterns in non-diseased human and porcine aortic valves 16,60-62 . These co-expression findings suggest that human aortic valve endothelium from these patients may have an increased propensity or capacity for EndMT 60 .…”

Aortic Valve: Mechanical Environment and Mechanobiology

“…Although the above studies have investigated the mechanobiological effects of pressure in isolation, 66,67,95,117 it must be noted that pressure causes increased stretch on the leaflets through tensile-compressive and bending forces. Hence, future studies must investigate these two forces in combination, rather than in isolation.…”

“…32 Cell level effects are investigated by isolating the cells 36 and subjecting them to strain or by using imaging techniques to investigate the cell behavior while the entire AV leaflet tissue is stretched. 66 Even though different strain levels have been used in various in vitro and ex vivo studies to elucidate cell level effects, it has been established that non-physiological strain leads to pathological conditions in AV leaflets in terms of inflammation, remodeling, and calcification.…”

“…Expression of the various proteins normalized to β -actin and then the 10% stretch case (* p <0.05, # p <0.10). 66 …”

Prediction of matrix-to-cell stress transfer in heart valve tissues

“…Ex vivo bioreactor cultivation of native valvular tissues [19][20][21][22] and the in vitro application of stretch or defined substrate stiffness to VICs [23][24][25] and VIC-based engineered tissues [26,27] have collectively demonstrated that the VIC phenotype can be modulated by the mechanical environment [28,29]. Nevertheless, how heart valve leaflet tissue-level stresses and strains manifest locally at the cellular level remains unclear.…”