The role of valvular endothelial cell paracrine signaling and matrix elasticity on valvular interstitial cell activation.
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.
Department of Chemical and Biological Engineering, The BioFrontiers Institute, Boulder, CO 80303, USA.
Sarah T
ST
Gould
Department of Chemical and Biological Engineering, The BioFrontiers Institute, Boulder, CO 80303, USA.
Emily E
EE
Matherly
Department of Chemical and Biological Engineering, The BioFrontiers Institute, Boulder, CO 80303, USA.
Jennifer N
JN
Smith
Departments of Internal Medicine and Pharmacology, University of Iowa Health Care, Iowa City, IA 52242, USA.
Donald D
DD
Heistad
Department of Chemical and Biological Engineering, The BioFrontiers Institute, Boulder, CO 80303, USA; Howard Hughes Medical Institute University of Colorado, Boulder, CO 80303, USA. Electronic address: Kristi.Anseth@Colorado.edu.
Kristi S
KS
Anseth
D000818:Animals; D001021:Aortic Valve / Q000166:cytology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D055119:Elastic Modulus / Q000187:drug effects; D004548:Elasticity / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D008970:Molecular Weight; D058628:Myofibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019899:Paracrine Communication / Q000187:drug effects; D010641:Phenotype; D011092:Polyethylene Glycols / Q000494:pharmacology; D011137:Polystyrenes / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D034421:Sus scrofa
D011137:Polystyrenes; D011092:Polyethylene Glycols
Nitric oxide; PEG hydrogels; Valvular endothelial cells; Valvular interstitial cells; α-Smooth muscle actin