Pretreatment of Bioprosthetic Heart Valve Leaflets with Pyridoxamine (Vitamin B6) Enhances Endothelial Cell Adhesion and Viability

Introduction: Heart valve disease is one of the most common types of cardiovascular disorders, with millions of people affected globally. Currently there is no effective medical treatment; thus when disease progression is severe the only option is valve repair or replacement, with bioprosthetic heart valves (BHV) being most common type of device used. However, BHV, typically composed of glutaraldehyde fixed bovine pericardium (BP), over time undergo structural valve degeneration (SVD) due to the accumulation of advanced glycation end products (AGE) with associated serum proteins, and calcification. Our previous work has shown that modification of BP with the AGE-inhibitor Pyridoxamine (PYR), a B6 vitamer, was effective in mitigating AGE accumulation and serum protein uptake in vitro and in vivo. However, another significant issue with BHV is that they do not endothelialize, which contrasts with native valves, and further accelerates SVD. Thus, the goal of these studies was to examine the effect of PYR-modified BP on endothelial cell-BP interactions under both static and high shear flow conditions. Moreover, the effectiveness of PYR pretreatment under accelerated glycation conditions on endothelial cell retention under high shear flow conditions was also assessed. The hypothesis is that modification of BP with PYR will enhance BHV biocompatibility by promoting endothelialization.

Materials and

Methods: In vitro studies were performed using human bone marrow origin blood outgrowth endothelial cells (BOEC) derived from peripheral blood of healthy volunteers under an IRB approved protocol. PYR-BP stability was assessed over a 28-day period by quantifying PYR uptake in the tissue using a fluorescent based assay. To examine PYR pretreatment of BP vs unmodified BP on BOEC adhesion, BOEC were seeded on BP and after 24 hours cell adhesion was quantified using an Alamar Blue assay. Cytotoxicity of modifications was evaluated by incubating of BP samples in cell culture media. This conditioned media was used to assess cell viability of BOECs after 24 hours via Alamar Blue. Cell viability was visualized via calcein staining of PYR-BP or unmodified BP seeded with BOEC for 48 hours. For high shear flow studies, BOEC were seeded on PYR modified glutaraldehyde fixed collagen I coated slides or on respective non-PYR controls. For accelerated glycation conditions, after modification slides were incubated in 50mM glyoxal with 5% bovine serum albumin for 24 hours at 37℃. Non-glycated or glycated slides were seeded with BOEC, allowed to reach confluency for 48 hours, and placed under arterial level laminar shear flow (25 dynes/cm2) for 4 hours and then fixed in 4% paraformaldehyde. Slides only exposed to static conditions were used as a time zero. Slides were stained with Phalloidin and DAPI and nine random fields were imaged per slide; BOEC retention was determined by quantifying DAPI nuclei in 100x fields. Data are means±standard deviations, with t-tests.

Results, Conclusions, and Discussions: PYR stability studies showed that after three hours of pretreatment, there was a maximal uptake of 14.8± 0.3 ug PYR per mg of tissue; however, after 24 hours of rinsing, the PYR uptake reduced to 5.8± 1.1 ug PYR/ mg tissue but remained stable up to 28 days. In vitro adhesion studies showed that PYR-BP had a significantly higher number of cells adhered after 24 hours compared to controls(24,931±1663 cells vs 14,573±960; p< 0.001); a similar trend was seen with the calcein staining of BOEC seeded on PYR-BP versus unmodified BP, with a larger amount of live cells spread amongst the PYR-BP per field compared to unmodified BP. Cytotoxicity studies showed that incubation of BOECs in PYR-BP conditioned media showed a significant increase in cell viability compared to unmodified BP (98±3% vs 74±6.2%; p< 0.001). These results indicate that PYR modification of BP provides a more biocompatible and favorable surface for endothelial adhesion and viability compared to standard unmodified glut-fixed BP. Laminar flow studies demonstrated that modification with PYR maintained BOEC retention post flow while unmodified controls had a significant reduction in cell retention compared to their respective static controls, with a 18% loss in cell retention (p < 0.001). When challenging the system with an accelerated glycation condition, there was an overall reduction in BOEC adhesion on slides compared to non-glycated conditions. PYR pretreatment prior to glycation was effective in maintaining cell retention pre and post flow, while unmodified glycated slides had a significant reduction in cells post flow, with a 27% loss in cell retention compared post high shear flow (p < 0.001). These results further indicate that PYR pretreatment is effective in mitigating glycation, which has been shown to reduce endothelial adhesion, and can lead to endothelial dysfunction.

Conclusions: Modification of BP with PYR demonstrated both significantly increased BOEC adhesion and viability. In addition, PYR-modified collagen films showed increased BOEC adhesion under arterial level shear flow, providing evidence for PYR mediated enhanced endothelialization of BP. Further studies will be done to examine the impact of PYR pretreatment on mitigation of endothelial dysfunction under glycation conditions.

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