The role of valvular endothelial cell paracrine signaling and matrix elasticity on valvular interstitial cell activation

Abstract

The effects of valvular endothelial cell (VlvEC) paracrine signaling on VIC phenotype and nodule formation were tested using a co-culture platform with physiologically relevant matrix elasticities and diffusion distance. 100 μm thin poly(ethylene glycol) (PEG) hydrogels of 3-27 kPa Young's moduli were fabricated in transwell inserts. VICs were cultured on the gels, as VIC phenotype is known to change significantly within this range, while VlvECs lined the underside of the membrane. Co-culture with VlvECs significantly reduced VIC activation to the myofibroblast phenotype on all gels with the largest percent decrease on the 3 kPa gels (~70%), while stiffer gels resulted in approximately 20-30% decrease. Additionally, VlvECs significantly reduced αSMA protein expression (~2 fold lower) on both 3 and 27 kPa gels, as well as the number (~2 fold lower) of nodules formed on the 27 kPa gels. Effects of VlvECs were prevented when nitric oxide (NO) release was inhibited with l-NAME, suggesting that VlvEC produced NO inhibits VIC activation. Withdrawal of l-NAME after 3, 5, and 7 days with restoration of VlvEC NO production for 2 additional days led to a partial reversal of VIC activation (~25% decrease). A potential mechanism by which VlvEC produced NO reduced VIC activation was studied by inhibiting initial and mid-stage cGMP pathway molecules. Inhibition of soluble guanylyl cyclase (sGC) with ODQ or protein kinase G (PKG) with RBrcGMP or stimulation of Rho kinase (ROCK) with LPA, abolished VlvEC effects on VIC activation. This work contributes substantially to the understanding of the valve endothelium's role in preventing VIC functions associated with aortic valve stenosis initiation and progression.

Keywords: Nitric oxide; PEG hydrogels; Valvular endothelial cells; Valvular interstitial cells; α-Smooth muscle actin.

Figure 1

(a) Example immunostaining images for VICs cultured alone or in direct or indirect contact with VlvECs on TCPS. Green is αSMA, red is cell tracker transfected into VlvECs, blue is the cell nuclei, and the scale bar is 100 μm. (b) Quantification of the percentage of VICs activated to the myofibroblast phenotype from immunostaining images for VICs cultured alone or co-cultured in director indirect contact with VlvECs on TCPS. VICs and VlvECs were co-cultured for 3 days in 1% FBS low glucose DMEM media at 10,000 cells/cm² and confluence, respectively, before fixation and immunostaining. * p<0.05 vs. VICs.

Figure 2

(a) Schematic of transwell insert PEG hydrogel fabrication material platform used to coculture VICs and VlvECs. (b) Representative fluorescent gel tagging confocal microscopy z-stack image and (c) quantification. (d) Young’s modulus as a function of crosslinking density for each of the four hydrogel formulations used for cell culture.

Figure 3

(a) Representative immunostaining images of VICs cultured on gels of increasing modulus, and in the absence of VlvECs, where green and blue are αSMA and nuclei, respectively. Scale bar = 100 μm. (b) Quantification of the percentage of VICs activated to the myofibroblast phenotype from immunostaining images for VICs cultured alone (diamond) or VICs co-cultured with VlvECs (square) as a function of gel modulus. VICs and VlvECs were co-cultured for 3 days in 1% FBS low glucose DMEM media at 10,000 cells/cm² and confluence, respectively, before fixation and immunostaining. (c) Percent decrease in myofibroblast activation with the addition of VlvECs.

Figure 4

(a) Percentage of myofibroblasts on low (E~3 kPa) and high (E~27 kPa) activating gel formulations and (b) Western blot bands and quantification of αSMA expression normalized to GAPDH ratio for VICs alone (white), coculture (grey), and coculture with 100 μM L-NAME (black) after 3 days in culture. VICs and VlvECs were co-cultured in 1% FBS low glucose DMEM media.

Figure 5

(a) Representative bright field images of VIC nodule formation on 27 kPa gels and (b) quantification of the number of the nodules on 3 and 27 kPa gels for VICs alone without (white) or with (light grey) 100 μM L-NAME and co-culture without (dark grey) or with 100 μM L-NAME (black) after 6 days in culture. VICs and VlvECs were co-cultured in 1% FBS low glucose DMEM media at confluence. Scale bar = 100μm.

Figure 6

The percentage of VICs activated to the myofibroblast phenotype as a function of culture time with 100 μM L-NAME exposure for 3, 5, or 7 days (black) and two additional days (i.e., 5, 7, or 9 total) without L-NAME (white) for VICs cultured alone (a) or in the presence of VlvECs (b). Lines were added to show trends. VICs and VlvECs were co-cultured in 1% FBS low glucose DMEM media at 10,000 cells/cm² and confluence, respectively, before immunostaining. * p<0.05.

Figure 7

Percentage of VICs activated to the myofibroblast phenotype as a function of culture conditions after 3 days (white) and after exposure to small molecule inhibitors of sGC (3 μM ODQ (a)) or PKG (0.5 mM RBrcGMP (b)), or the ROCK activator (20 μM LPA (c)) (black). VICs and VlvECs were co-cultured in 1% FBS low glucose DMEM media at 10,000 cells/cm² and confluence, respectively for 3 days before immunostaining. (d) Model for the intersection of stiffness and NO signaling. The increased prevalence of stabilized focal adhesions of VICs attached to stiff substrates increases myofibrotic pathways (red). NO secreted by local VECs acts to increase anti-fibrotic pathways (blue).