GENE EXPRESSION AND COLLAGEN FIBER MICROMECHANICAL INTERACTIONS OF THE SEMILUNAR HEART VALVE INTERSTITIAL CELL

Abstract

The semilunar (aortic and pulmonary) heart valves function under dramatically different hemodynamic environments, and have been shown to exhibit differences in mechanical properties, extracellular matrix (ECM) structure, and valve interstitial cell (VIC) biosynthetic activity. However, the relationship between VIC function and the unique micromechanical environment in each semilunar heart valve remains unclear. In the present study, we quantitatively compared porcine semilunar mRNA expression of primary ECM constituents, and layer- and valve-specific VIC-collagen mechanical interactions under increasing transvalvular pressure (TVP). Results indicated that the aortic valve (AV) had a higher fibrillar collagen mRNA expression level compared to the pulmonary valve (PV). We further noted that VICs exhibited larger deformations with increasing TVP in the collagen rich fibrosa layer, with substantially smaller changes in the spongiosa and ventricularis layers. While the VIC-collagen micro-mechanical coupling varied considerably between the semilunar valves, we observed that the VIC deformations in the fibrosa layer were similar at each valve's respective peak TVP. This result suggests that each semilunar heart valve's collagen fiber microstructure is organized to induce a consistent VIC deformation under its respective diastolic TVP. Collectively, our results are consistent with higher collagen biosynthetic demands for the AV compared to the PV, and that the valvular collagen microenvironment may play a significant role in regulating VIC function.

Figure 1

Histological assessment of porcine AV and PV leaflet stratification (inset: partially polarized image of an AV leaflet where C and R represent circumferential and radial directions, respectively). AV and PV leaflets were sectioned along the radial-transverse plane (line labelled as 1) and examined using Movat's pentachrome stain. Elastin fibers predominant in the ventricularis (V) layer are black, proteoglycans predominant in the spongiosa layer (S) are blue, and collagen fibers predominant in the fibrosa (F) are yellow. Relative thickness of the valve ECM layers is indicated by brackets. The AV had a thicker fibrosa layer than the PV. This is consistent with the higher fibrillar collagen gene expression in the AV compared to the PV.

Figure 2

(A) Differential ECM constituent gene expression in porcine AV and PV as determined by qRT-PCR showing that the AV has a significantly higher baseline expression of fibrillar collagen (Col1a1, Col3a1, and Hsp47) and the proteoglycan Biglycan than the PV. (B) Differential gene expression of VIC phenotypic markers was detected by qRT-PCR showing that AV has a significantly higher baseline expression of MGP but expression levels of periostin are not significantly different (*p<0.05).

Figure 3

Histological assessment of circumferential-transverse sections (see Fig. 1 line label 2) using Movat's pentachrome stain of porcine AV and PV leaflet tissue stratification at 0 and 90 mmHg TVP. At 90 mmHg the PV decreases in thickness proportionally more than the AV, and the VICs deformed more in the fibrosa layer compared to the spongiosa and ventricularis layer in both valve tissues.

Figure 4

(A) Cross-section of an AV leaflet in the circumferential-transverse (C-T) plane (see Fig. 1 line label 2) showing the fibrosa (F), spongiosa (S), and ventricularis (V) layers as visualized with second harmonic generation microscopy. The white boxes indicate the regions used for the 3D reconstruction in (B), and C, R, and T represent circumferential, radial and transverse directions, respectively. (B) Multi-photon microscopy images used to simultaneously image AV collagen (red) and cell nuclei (green) for a 60x80x20 μm localized volume, via second harmonic generation and two-photon excited fluorescence, respectively. The VIC nuclei (green) can been seen to undergo large deformations only in the fibrosa layer only under 90 mmHg, whereas in the spongiosa and ventricularis layers deformations remain modest.

Figure 5

(A) A schematic showing the coordinate system used to define the 3D VIC nuclei geometry, with principal axial directions e₁-e₂-e₃, ordered by decreasing magnitude. e₁ was thus used to define the nuclei 3D orientation using the spherical angles θ and Φ. The resulting nuclei angle probability distributions for θ (B) and Ф (C) as a function of TVP in the F-fibrosa layer and S+V - spongiosa and ventricularis layers. Results clearly demonstrated that the majority of the nuclei were oriented along the circumferential axis in the circumferential-radial plane. In addition, VIC nuclei aligned increased with TVP for the fibrosa layer for AV and PV; whereas the PV VIC orientations did not measurable change above 20 mmHg.

Figure 6

(A) Combined results for the AV and PV NAR as a function of TVP and layer, with F = fibrosa, and S+V = the combined spongiosa and ventricularis layers. Consistent with Fig. 5, VICs deformed significantly more in F compared to S+V. The vertical arrows denote approximate diastolic TVP of the AV and PV, while the horizontal dashed line highlights their respective NAR, and highlights the finding that the nuclei in both AV and PV experience very similar deformations at their respective TVPs.

Figure 7

(A) Observable straight collagen fiber area expressed as percent total measured area as a function of TVP for both the AV and PV (values for the AV taken from {Joyce, 2009 #22468}). The AV experienced both initially more rapid and also larger total changes compared to the PV. In order to highlight the relation between collagen crimp and VIC deformation, the NAR of the AV and PV fibrosa layer was assessed as a function of both observable straight collagen fiber area from percent total area and TVP in (B). From 0 to 70% observable straight collagen fiber area there was minimal VIC deformation, whereas above 70% observable straight collagen fiber area there was a rapid increase in NAR, with the PV having a higher rate of increase in NAR compared to the AV.

Figure 8

NAR of the AV and PV fibrosa (F) layer as a function of Cauchy stress, both the total (A) and fibrosa (B) thickness values. At diastolic TVP the AV was under approximately three times greater stress than the PV; yet their respective NAR values are similar. This suggests that the VICs in the AV may be more stress-shielded as a result of the AV fibrosa layer more robust collagen structure.