An ex vivo study of the biological properties of porcine aortic valves in response to circumferential cyclic stretch

Abstract

Normal physiological mechanical forces cause constant tissue renewal in aortic valve leaflets (AVL) while altered mechanical forces incite changes in their structural and biological properties. The current study aims at characterizing the remodeling properties of AVL subjected to cyclic circumferential stretch in a sterile ex vivo bioreactor. The leaflets cultured were stretched at a maximum rate of 300%s(-1) corresponding to a 15% strain for 48 h. Collagen, sulfated glycosaminoglycan (sGAG), and elastin contents of the stretched, fresh, and statically incubated leaflets were measured. Cusp morphology and cell phenotype were also examined. AVLs exposed to cyclic stretch showed a significant increase in collagen content (p < 0.05) when compared to fresh and statically incubated AVLs. sGAG content was significantly reduced in the stretched AVLs (p < 0.05) when compared to the fresh leaflets and was comparable between stretched and statically incubated AVLs. There was no statistically significant change in elastin content in all the three groups of AVLs (p > 0.05). Native aortic valve morphology was well preserved in stretched leaflets. Immunohistochemistry and immunoblotting studies showed an increased expression of alpha-smooth muscle actin (alpha-SMA) in stretched leaflets while alpha-SMA expression was reduced in statically incubated AVLs when compared to the fresh leaflets. To conclude, circumferential cyclic stretch altered the extracellular matrix remodeling activity of valvular cells, and consequently the extracellular matrix composition of the AVLs. Most interestingly, the contractile and fibrotic phenotypic expression of valve interstitial cells was enhanced. These results show that circumferential cyclic stretch is a possible mediator for AVL remodeling activity.

Figure 1.

Preparation of aortic valve leaflets for the experiment. Three sections were cut out in the circumferential direction from the base, belly and tip regions of the aortic valve leaflet. These sections were randomly chosen as stretched, fresh and static leaflet groups. Fresh sections were processed immediately. Static control sections were incubated for 48 h in DMEM. Stretched sections were stretched for 48 h.

Figure 2.

(a) Loading curve used in this study. A near physiologic loading curve was input to the actuator to stretch the leaflets. (b) Ex vivo tensile stretch bioreactor used in this study. A magnified image of the tissue chamber is shown on the right showing eight tissue wells, with aortic valve leaflets in four of the wells.

Figure 3.

Cyclic stretch increased collagen, decreased sGAG, while not affecting elastin contents in aortic valve leaflets. The leaflets were exposed to physiologic circumferential cyclic stretch (15% at heart rate of 70 bpm) or static control conditions for 48 h. The amounts of collagen, sGAG and elastin in the leaflets exposed to static or stretching were compared to those of fresh leaflets. The data were normalized by tissue dry weight and expressed as a mean value plus one standard error of the mean (* p < 0.05, n = 27).

Figure 4.

(a) Cyclic stretch did not damage tissue structure and morphology. H & E stained images of fresh, static and stretched aortic valve leaflets are depicted here. Cytoplasm was stained pink and cell nuclei were stained blue. The representative images show the three-layered morphology of the leaflets was intact in all three groups (F – fibrosa, S – spongiosa, V – ventricularis). (b) Cyclic stretch maintained native collagen architecture. Picrosirius red images of fresh, static and stretched aortic valve leaflets are shown. Mature collagen fibers were stained orange-red. Layered collagen morphology of leaflets was observed and crimp was preserved in stretched leaflets (F – fibrosa). (c) Cyclic stretch increased α-smooth muscle actin (α-SMA)-positive cells in the ventricularis side of the aortic valve leaflets. Fresh, static and stretched leaflets were examined by α-SMA IHC. Actin was stained red and cell nuclei were counterstained blue. Increased expression of α-SMA was observed in stretched leaflets, and α-SMA was reduced in static leaflets (V – ventricularis).

Figure 5.

(a) Fibrosa-to-spongiosa ratio and fibrosa-to-ventricularis ratio remained unchanged. There was no significant difference (p > 0.05, n = 8) in the relative thicknesses of the fibrosa, spongiosa and ventricularis layers between fresh, static and stretched leaflets. (b) Static leaflets exhibited reduced levels of newly synthesized collagen fibers. Newly synthesized fiber proportion was reduced in static leaflets signifying reduced levels of collagen synthesis. Newly synthesized fiber proportion was comparable between fresh and stretched leaflets, but the proportion of mature fibers was greater in stretched leaflets. (c) Cyclic stretch increases α-smooth muscle actin in the aortic valve leaflets. α-SMA immunopositive staining coverage was increased in stretched leaflets and reduced in static leaflets when compared with fresh leaflets.

Figure 6.

Cyclic stretch increased α-smooth muscle actin in the aortic valve leaflets. Fresh, static and stretched aortic valve leaflets were analyzed by Western blot with α-SMA antibody and analyzed by densitometry. When compared to fresh leaflets, α-SMA expression decreased in static leaflets, but increased in stretched leaflets (*p < 0.05, n = 10). Anti-GAPDH antibody was used as a loading control.