(261d) Monocyte Recruitment for Vascular Tissue Regeneration

Cardiovascular disease is the leading cause of death in the United States, claiming over 600,000 lives annually. Coronary artery disease is the most common form, with over 350,000 bypass grafting procedures performed every year, estimated at a total of $26 billion annually in healthcare costs, according to the American Heart Association. Tissue engineering approaches using native or synthetic scaffolds, or even scaffold-free strategies, have developed functional and implantable tissue engineered vessels (TEVs) that have been tested in small and large animal models. However, development of such TEVs typically requires the use of autologous cells, and weeks to months of cell expansion, tissue growth, and mechanical preconditioning before implantation. As a result, several laboratories have turned their attention to engineering cell-free vascular grafts. Due to lack of endothelial cells (EC), acellular (A)-TEV are more prone to acute thrombosis post implantation, necessitating strategies that promote anti-occlusive and self-endothelialization capacity. Several studies promoted endothelialization of the grafts by functionalizing the A-TEV lumen to attract circulating endothelial progenitor cells (EPCs) from blood or migration of adjacent EC from anastomotic sites to endothelialize the graft lumen, however, EPCs are rare and present <0.01% in circulating blood and trans-anastomotic endothelialization is very limited in large animals or humans.

In recent work from our laboratory, it was demonstrated that adherent MC differentiate into a mixed endothelial (EC) and macrophage (Mφ) phenotype and further develop into mature EC that align in the direction of flow and produce nitric oxide under high shear stress. Given the abundance of circulating MCs in blood as compared to circulating EPCs these results demonstrated the significance of MCs recruitment in ATEV endothelialiation and regeneration.

MCs are known to hover over and bind EC activated by injury or inflammation using integrin α4β1, which binds to the CS-1/CS-5 domain of the IIICS region of fibronectin as well as the immunoglobulin superfamily molecule VCAM1, which is expressed by activated endothelium. We hypothesized that engineering a lumen mimicking the activated endothelium might attract blood MCs that would turn into EC on the surface of the graft resulted in improving vascular graft regeneration. To this end, we generated novel fusion protein, H2R5, containing two domains: (i) the second heparin binding domain (H2) of fibronectin facilitates the H2R5 immobilization to heparin-coated ATEVs; and (ii) five tandem repeats of the flexible linker motif, GGGS followed by a peptide from CS-5 region of fibronectin, which is known for binding specifically to the α4β1 integrin (GGGS-HIPREDVDYH) denoted as R5. When immobilized on the surface of vascular grafts, the fusion protein, H2R5 could capture blood derived MC under static or flow conditions in a shear stress dependent manner. The bound MC turned into macrophages (Mφ) expressing both M1 and M2 phenotype specific genes. When H2R5 functionalized A-TEV were implanted into the mouse aorta, they remained patent and formed a continuous endothelium expressing both EC and MC specific proteins. Underneath the EC layer, multiple cells layers were formed co-expressing both smooth muscle cell (SMC) and MC specific markers. Lineage tracing analysis using a novel CX3CR1-confetti mouse model demonstrated that fluorescently labeled MC populated the graft lumen by two and four weeks post-implantation, providing direct evidence in support of MC/Mφ recruitment to the graft lumen. Given MC prevalence in the blood, circulating MCs may be a great source of cells that contribute directly to the endothelialization and vascular wall formation of acellular vascular grafts under the right chemical and biomechanical cues.