Cardiovascular Tissue Engineering: the role of immune cells in vascular graft regeneration.

(NHLBI, R01HL151196-01)

Stelios T. Andreadis Lab

Introduction & Significance

The Andreadis group aims to understand methods to engineer small-diameter, transplantable blood vessels using adult mesenchymal stem cells from bone marrow, hair follicles, and human iPSC.

Cardiovascular disease is the leading cause of death in the United States, claiming 610,000 lives in 2014 2. 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. The Andreadis laboratory developed methods to engineer small-diameter, transplantable blood vessels using adult mesenchymal stem cells from bone marrow, hair follicles, and human iPSC 3-6. While successful, production 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.

Zoom image: Fig. 1: Schematic indicating the use of VEGF to capture VEGFR-expressing blood cells and repopulate the graft lumen.  1.1.

Fig. 1: Schematic indicating the use of VEGF to capture VEGFR-expressing blood cells and repopulate the graft lumen. 

Research Findings

To address these limitations, the laboratory developed an acellular (A)-TEV using a native biodegradable material, small intestinal submucosa (SIS), which was coated with heparin-bound vascular endothelial growth factor (VEGF) to prevent thrombosis and promote endothelialization 7,8. This signifies a major advancement in the development of clinically relevant off-the-shelf vascular grafts. This A-TEV is made with tubular laminated SIS, with bound heparin by EDC-NHS chemistry, and subsequent addition of VEGF. VEGF binds to heparin via its heparin binding domain resulting in immobilization and cells expressing the VEGF-receptor to bind (Fig. 1). These A-TEVs have been tested in a preclinical ovine carotid model for up to 6 months demonstrating well over 90% patency. A functional confluent endothelium was formed in the lumen of these grafts as early as one month post implantation. Most notably, these VEGF based A-TEVs integrated seamlessly with the native vasculature and grew with the host, when implanted into neonatal lambs 9. These results suggest that they might be suitable for treatment of congenital heart disorders to alleviate the need for repeated surgeries, currently the standard practice for pediatric patients. 

Most notably, the lab discovered that instead of endothelial cells (EC), blood monocytes (MC) attached to the graft lumen and under the influence of biochemical factors in blood or immobilized VEGF, as well as biophysical factors such as shear stress, MC turned into endothelial-like cells, that kept the grafts patent and functional 10. Adherent MC differentiated into a mixed EC and macrophage (Mf) phenotype, and further developed into mature EC, which aligned in the direction of flow and produced nitric oxide under high shear stress. In-vivo, newly recruited cells after one week post implantation on the vascular lumen expressed only MC markers. By 1- and 3- months post-implantation, they co-expressed MC and EC-specific proteins and maintained graft patency. In addition, an in vitro protocol was developed to differentiate VEGF-captured MC into EC within 14 days. RT-PCR and single cell (sc)RNA-seq (Fig. 2) showed that MC differentiated to macrophages before expressing EC markers. When MC-derived EC were subjected to shear stress, they downregulated venous and upregulated arterial genes, demonstrating the effect of shear on generating arterial EC. 

Zoom image: Fig. 2: Monocytes differentiated on VEGF surfaces using our optimized protocol and further subjected to physiological shear differentiate to a mature endothelial phenotype. (Top Panels) Immunostaining shows expression of key monocyte/macrophage and endothelial markers on monocyte derived endothelial cells after exposure to physiological shear on an immobilized VEGF surface: (left) CD163 (red) and CD144 (green); (right) CD31 (red) and CD14 (green); white arrows indicate the direction of fluid flow. (Bottom Panel) Single cell RNA sequencing and bioinformatic analysis shows that monocyte derived endothelial cells (MCEC) are transcriptionally similar to human carotid artery endothelial cells (HCAEC) and are distinct from their monocytic (MC) origins. 1.2.

Fig. 2: Monocytes differentiated on VEGF surfaces using our optimized protocol and further subjected to physiological shear differentiate to a mature endothelial phenotype.

(Top Panels) Immunostaining shows expression of key monocyte/macrophage and endothelial markers on monocyte derived endothelial cells after exposure to physiological shear on an immobilized VEGF surface: (left) CD163 (red) and CD144 (green); (right) CD31 (red) and CD14 (green); white arrows indicate the direction of fluid flow.

(Bottom Panel) Single cell RNA sequencing and bioinformatic analysis shows that monocyte derived endothelial cells (MCEC) are transcriptionally similar to human carotid artery endothelial cells (HCAEC) and are distinct from their monocytic (MC) origins.

Future Work

Current and future work in this area includes lineage tracing analysis of monocytes populating vascular grafts using Confetti mice (see recent publication 11); understanding the epigenetic and metabolic requirements during monocyte/macrophage trans-differentiation to EC in the vascular microenvironment; as well as the effects of aging and genetic diseases on macrophage driven endothelialization. This work is a paradigm shift in vascular biology and bioengineering showing direct contribution of MC to endothelialization. As such, it may influence, not only on the clinical implementation of vascular grafts but also other tissues e.g., heart valves as well as understanding vascular repair and regeneration following injury or infections. 

Student Researchers

Post-docs: Mohamed Alaa Mohamed, Ph.D., Karthik Ramachandran, Ph.D.

Graduate Students: Ari Das, Bita Nasiri, Yulun Yu

Collaborators: Chris Breuer (Nationwide Childrens’ Hospital, Ohio State University).

References

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  2.  Mozaffarian, D., Benjamin, E.J., Go, A.S., Arnett, D.K., Blaha, M.J., Cushman, M., de Ferranti, S., Despres, J.P., Fullerton, H.J., Howard, V.J., Huffman, M.D., Judd, S.E., Kissela, B.M., Lackland, D.T., Lichtman, J.H., Lisabeth, L.D., Liu, S., Mackey, R.H., Matchar, D.B., McGuire, D.K., Mohler, E.R., 3rd, Moy, C.S., Muntner, P., Mussolino, M.E., Nasir, K., Neumar, R.W., Nichol, G., Palaniappan, L., Pandey, D.K., Reeves, M.J., Rodriguez, C.J., Sorlie, P.D., Stein, J., Towfighi, A., Turan, T.N., Virani, S.S., Willey, J.Z., Woo, D., Yeh, R.W. & Turner, M.B. Heart disease and stroke statistics--2015 update: a report from the American Heart Association. Circulation 131, e29-322 (2015).
  3. Row, S., Peng, H., Schlaich, E.M., Koenigsknecht, C., Andreadis, S.T. & Swartz, D.D. Arterial grafts exhibiting unprecedented cellular infiltration and remodeling in vivo: the role of cells in the vascular wall. Biomaterials 50, 115-126 (2015).
  4. Peng, H., Schlaich, E.M., Row, S., Andreadis, S.T. & Swartz, D.D. A novel ovine ex vivo arteriovenous shunt model to test vascular implantability. Cells Tissues Organs 195, 108-121 (2012).
  5. Liu, J.Y., Swartz, D.D., Peng, H.F., Gugino, S.F., Russell, J.A. & Andreadis, S.T. Functional tissue-engineered blood vessels from bone marrow progenitor cells. Cardiovasc Res 75, 618-628 (2007).
  6. Swartz, D.D., Russell, J.A. & Andreadis, S.T. Engineering of fibrin-based functional and implantable small-diameter blood vessels. Am J Physiol Heart Circ Physiol 288, H1451-1460 (2005).
  7. Koobatian, M.T., Row, S., Smith, R.J., Jr., Koenigsknecht, C., Andreadis, S.T. & Swartz, D.D. Successful endothelialization and remodeling of a cell-free small-diameter arterial graft in a large animal model. Biomaterials 76, 344-358 (2016).
  8. Smith, R.J., Jr., Koobatian, M.T., Shahini, A., Swartz, D.D. & Andreadis, S.T. Capture of endothelial cells under flow using immobilized vascular endothelial growth factor. Biomaterials 51, 303-312 (2015).
  9. Nasiri, B., Row, S., Smith, R.J., Jr., Swartz, D.D. & Andreadis, S.T. Cell-free vascular grafts that grow with the host. Adv Funct Mater 30(2020).
  10. Smith, R.J., Jr., Nasiri, B., Kann, J., Yergeau, D., Bard, J.E., Swartz, D.D. & Andreadis, S.T. Endothelialization of arterial vascular grafts by circulating monocytes. Nature communications 11, 1622 (2020).
  11. Nasiri, B., Yi, T., Wu, Y., Smith, R.J., Jr., Podder, A.K., Breuer, C.K. & Andreadis, S.T. Monocyte Recruitment for Vascular Tissue Regeneration. Advanced healthcare materials, e2200890 (2022).