Research

Overall Goals of the Research Program of the Laboratory

The main area of my research is the elucidation of the molecular events that dictate the course of healing and especially inflammation and angiogenesis following the implantation of biomaterials and scaffolds for tissue engineering applications. Our primary research focus is on three molecules, MCP-1, MMP-9, and thrombospondin (TSP)-2 that we have shown to be critical to various aspects of these processes. In addition, through the process of molecular dissection of cell-matrix interactions, we aim to incorporate rational design in the development of bioengineering applications such as tissue-engineered vascular grafts.

My laboratory is also focused on elucidating the mechanism through which the endogeno us inhibitor of angiogenesis TSP-2 limits angiogenesis and art eriogene sis. We are utilizing an in vitro, TSP-2-sensitive, three-dimensional angiogenesis assay and we are currently investigating the effects of TSP-2 on endothelial cells. Our aim is to identify the signaling pathways that mediate the anti-angiogenic effect of TSP2. We have recently shown that TSP2 is critical for the recovery of blood flow in an experimental model of hindlimb ischemia, predominantly through enhanced arteriogenesis in the upper limb and increased angiogenesis in the lower limb. Finally, we are working on translating our basic findings regarding TSP2 function into a strategy for the generation of matrix-coated synthetic vascular grafts. Based on the assumption that a TSP2-null-derived matrix could confer anti-thrombotic and pro-angiogenic properties to the luminal surface of synthetic vessels, we have pursued several in vitro and in vivo studies in order to provide proof-of-principle for our hypothesis. Specifically, we have found that the TSP 2-null-derived matrix is more permissive for endothelial cell migration and compromised in its ability to induce platelet activation. Furthermore, transplantation of denuded aortic segments from TSP2-null to wild type mice indicates that they are resistant to thrombosis and, unlike transplanted wild type segments, remain patent in the long term.

Figures

Figure 1

Molecular events in the Foreign Body Response
When biomaterial and medical devices are surgically implanted into the body, a series of molecular and cellular events lead to its encapsulation and isolation from surrounding tissue. This series of events is known as the Foreign Body Reaction ( FBR) and can limit the device's overall biocompatibility and function. Every foreign surface first acquires a protein coat when within the body, composed of fibrinogen, albumin, fibronectin and other blood and interstitial-fluid proteins. The edema caused by the surgical procedure of implantation and several chemoattractants lead to leukocyte emigration from the blood and accumulation in the biomaterial site. Together with platelets, leukocytes bind on to the protein-coated surface . neutrophils have a short lifetime of about 2 days, while macrophages stay in the site for much longer. Around day 6/7 after implantation of the medical device, small multinucleated Foreign Body Giant Cells (FBGC) appear on the surface of the biomaterial. These cells can grow to include more than 100 nuclei since they are the result of robust macrophage fusion. Their phagocytic potential far exceeds that of all their component macrophages individually due to their ability to degrade targets extr acellularly (extroversion), and they are very similar to osteo clasts w hich arise from the same cellular precursor in the bone marrow. Both FBGC and macrophages secrete fibroblast attractants, and the incoming fibroblasts play a key role in creating a dense and organized collagenous matrix around the biomaterial, that has very low vasculature density. It is important to note that this matrix deposition is quite different from the regular process of healing, and the Foreign Body Response has a lot of characteristics that occur in the fibrotic process during a chronic inflammation. This ECM capsule isolates medical devices from the rest of the interstitial tissue and can also often contract, leading to adverse effects for the medical device function.

Figure 2

In vitro three-dimensional angiogenesis assay. Endothelial cells are placed in a collagen gel and form chords resembling blood vessels. Image shows cells stained with DAPI (blue nuclei), phalloidin (actin cytoskeleton). Green fluorescence is indicative of matrix metalloproteinase activity, which is required for angiogenesis.

Figure 3

Thrombospondin
Thrombospondin (TSP)-2 is an inhibitor of angiogenesis with pro-apoptotic and anti-proliferative effects on cultured endothelial cells. As a matricellular protein, TSP2 can modulate cell-matrix interactions and influence a number of processes including recovery from ischemia, wound healing, and the foreign body response. TSP2 has been shown to interact with a number of cell surface receptors, growth factors, extracellular matrix components, and enzymes. Thus, its participation in biological processes is thought to be complex.