oa Biomechanical Stimuli Effects on Valve Endothelial Cell Anti-thrombotic Mechanisms

Because of the degeneration and thrombosis in artificial heart valve implants, it is important to understand the anti-thrombotic mechanisms of cardiac valve endothelial cells (VECs). These anti-thrombotic mechanisms can be integrated into poly(ethylene glycol) diacrylate (PEGDA) tissue-engineered heart valve (TEHV) design. This work will study the effects of (1) PEGDA hydrogel stiffness and (2) specific extracellular matrix (ECM) adhesive peptides on VEC phenotype and anti-thrombotic mechanisms. PEGDA 10% (w/v) hydrogels of MWs 3.4 and 20kDa were polymerized to apply different substrate rigidities in VEC culture. Thiol-ene reactions were used to covalently bind laminin- and fibronectin- derived peptides to the acrylate groups on PEGDA hydrogel surfaces. Laminin-derived peptide motif RKRLQVQLSIRT (RKR) and fibronectin adhesive peptide RGD were modified to include an additional cysteine at the end of each sequence, introducing a free thiol to undergo the thiol-ene reaction. Thiol-PEG-fluorescein (SH-PEG-FITC) served as a negative adhesive substrate control. Porcine aortic VECs were seeded onto each of the ECM-hydrogel combinations and cultured for 2, 6, and 10 days. Cell phenotype, adhesion, and proliferation were then assessed. Analysis of specific VEC anti-thrombotic protein regulation is in progress. At each time point, samples will be analyzed for maintenance of VEC phenotype and expression of thrombotic (von Willebrand Factor [VWF]) and anti-thrombotic (VWF cleaving enzyme [ADAMTS-13], eNOS, PGI2, tPA) proteins using histochemistry and qRT-PCR. Addition of histamine has been shown to stimulate rapid release of thrombogenic ultra-large VWF (ULVWF) strings by vascular endothelial cells. This method will be used to study VEC ULVWF string production and the associated cleavage activity of ADAMTS-13. Control of the hemostatic process will be quantified via western blot and ELISAs. The 3.4 and 20kDa MW PEGDA hydrogels had compressive moduli of 131±5 and 7.5±2kPa, respectively. Binding different concentrations of SH-PEG-FITC onto the gel surfaces showed that acrylate saturation was achieved for both MW compositions using ~5mM of peptide solution. After 2 days, the VECs on the stiffer 3.4kDa RKR gels appeared spread and elongated, whereas the 3.4kDa RGD seeded VECs had cobblestone morphology. VEC adhesion on the RKR and RGD 20kDa gels was observed, but with limited spreading. The cultured VECs may prefer the stiffer 3.4kDa gels over the softer 20kDa gels. After 10 days, VECs on the 3.4kDa RGD gels had minimal proliferation, while VECs on RKR grew confluent, were cobblestone shaped, and expressed VWF. Results suggest that both substrate rigidity and adhesive substrate greatly influence VEC survival, and likely affects anti-thrombotic regulation. Future studies include the use of basal lamina components collagen IV and perlecan peptides to evaluate changes in VEC behavior.