Given that BOECs represent an easily acquired source of autologous endothelial cells and their potential for clinical applications, BOECs are an important cell type for investigating cellular attachment mechanisms to biomaterial surfaces. Others and we have shown that biomaterials can be modified to increase BOEC attachment and retention [3
]. In spite of their potential clinical importance, little is known of the molecular mechanisms that regulate BOEC physiology. In this paper we identified a novel role for CD47, a ubiquitously expressed cell surface receptor, in the attachment mechanism of BOECs to PU modified with cholesterol moieties. In addition these data begin to identify the molecular mechanism responsible for the increased BOEC binding to our novel cholesterol modified polyurethane.
We have previously reported that covalently linking cholesterol to polyurethane hard segments can increase the attachment rate and cell retention, under physiological shear forces, of BOECs and vascular endothelial cells [4
]. This observation was not completely unexpected, as hydrophobic surfaces, such as those resulting from the cholesterol modification, have demonstrated enhanced cell retention compared to more hydrophillic surfaces [24
]. To begin to understand the underlying molecular mechanisms influencing BOEC binding to synthetic surfaces, we focused on discerning the molecular mechanisms involved in BOEC attachment to PU-Chol. In our previous studies we observed that the maximal endothelial cell attachment to PU-Chol, compared to unmodified PU was achieved at thirty minutes post-seeding [4
]. Hence, in these current investigations, we examined the roles of CD47 and cholesterol related mechanisms upon BOEC attachment at thirty-minutes.
Appending cholesterol to synthetic surfaces by our group has been shown to enhance cell attachment [4
]. Of course cholesterol is a vital component of the cell membrane where it functions to maintain membrane integrity and to sequester membrane proteins. Work by others has shown that CD47 can form a cholesterol dependent multimeric protein complex with integrin αv
and the Gαi
]. Given the influence of cholesterol upon enhanced BOEC binding to PU that we had reported previously [4
], CD47 was identified as a molecule of interest in identifying the attachment mechanisms responsible for enhanced binding to PU-Chol. However, CD47 also has broadly defined functions independent of the previously mentioned integrin-signaling complex. These additional roles of CD47 do not require cholesterol and are responsible for immune recognition as well as fibroblast migration and aggregation [14
The results observed with the use of MβCD clearly demonstrate the significance of cholesterol in the previously observed enhanced oBOEC attachment on PU-Chol. The presence of MβCD significantly altered the attachment of oBOECs to PU-Chol, but not to unmodified PU. It probably disrupted the cholesterol-dependent CD47 multi-molecular complex as well as hydrophobic interactions and other pathways. We further assessed the contribution of appended cholesterol in the bulk modified PU-Chol by a pre-treatment of PU-Chol with MβCD prior to oBOEC seeding. Very interestingly, oBOEC attachment to PU-Chol was diminished with the pre-treatment, indicating that the surface oriented cholesterol in the bulk modified PU-Chol contributes to the attachment of BOECs to the PU surface. It is plausible that MβCD molecules sequester cholesterol molecules, becoming inaccessible for BOEC interactions even after washing with PBS, leading to the reduced cell attachment.
The sheep was our in vivo model in which we showed persistent BOEC seeding of PU-Chol pulmonary heart valve leaflets beyond 90 days implantation [13
]. As such, we used oBOECs to identify CD47 dependent attachment mechanisms. However, the extracellular region of CD47 has a high level of sequence variation between species and compatible reagents for ovine CD47 have not been completely characterized. Therefore, we used human BOECs to confirm our oBOEC observations. The use of blocking antibodies directed against CD47 and integrin αv
showed that these protein components were important in BOEC attachment to the PU-Chol film, but not to the unmodified PU. It was surprising that hBOEC attachment to unmodified PU was almost as robust as the attachment observed with hBOEC attachment to PU-Chol. Whether this is a result of variability between individual BOEC samples or if there actually is species variability in BOEC attachment to unmodified PU remains to be determined. As shown above, CD47 had a significant role in the attachment of both oBOEC and hBOEC, strongly suggesting a conserved CD47 attachment mechanism in both species.
We used an shRNA strategy to further confirm the role of CD47 in attachment to PU-Chol. We chose to use ovine BOECs for these investigations since sequence comparisons showed an 85% homology between the ovine and human genome in this region, and reduced gene expression was confirmed using Western blot analysis. Our BOECCD47− experiments showed that attachment of these cells to the PU-Chol surface was reduced by half compared to control cells. These numbers corresponded well with the reduction in CD47 expression. It was also clear that targeting CD47 or integrin αvβ3, via blocking antibodies as well as reducing CD47 expression via shRNA was not sufficient to completely block BOEC attachment to the PU-Chol surface. These results strongly suggest the existence of additional molecular pathways that contribute to BOEC attachment to PU-Chol.
CD47 has been demonstrated to have roles in both cell adhesion and immune evasion [14
]. Our current application of BOEC has been to seed autologous cells onto cholesterol-modified polyurethane [13
]. Thus, this study did not examine the role of CD47 in down regulating the immune response to implanted BOEC seeded materials. However, we have identified CD47 as an important molecular component in BOEC attachment to cholesterol-modified polyurethane. Further investigations into CD47 mediated signaling events in BOEC may assist in the development of future biomaterials that can both enhance BOEC attachment as well as reducing inflammatory responses to implanted biomaterials.