Charles S. Wallace

The Biocompatibility of Titanium Cardiovascular Devices Seeded With Autologous Blood-Derived Endothelial Progenitor Cells: EPC-Seeded Antithrombotic Ti Implants

Implantable and extracorporeal cardiovascular devices are commonly made from titanium (Ti) (e.g. Ti-coated Nitinol stents and mechanical circulatory assist devices). Endothelializing the blood-contacting Ti surfaces of these devices would provide them with an antithrombogenic coating that mimics the native lining of blood vessels and the heart. We evaluated the viability and adherence of peripheral blood-derived porcine endothelial progenitor cells (EPCs), seeded onto thin Ti layers on glass slides under static conditions and after exposure to fluid shear stresses. EPCs attached and grew to confluence on Ti in serum-free medium, without preadsorption of proteins. After attachment to Ti for 15 min, less than 5% of the cells detached at a shear stress of 100 dyne / cm(2). Confluent monolayers of EPCs on smooth Ti surfaces (Rq of 10 nm), exposed to 15 or 100 dyne/cm(2) for 48 h, aligned and elongated in the direction of flow and produced nitric oxide dependent on the level of shear stress. EPC-coated Ti surfaces had dramatically reduced platelet adhesion when compared to uncoated Ti surfaces. These results indicate that peripheral blood-derived EPCs adhere and function normally on Ti surfaces. Therefore EPCs may be used to seed cardiovascular devices prior to implantation to ameliorate platelet activation and thrombus formation.

Direct-contact co-culture between smooth muscle and endothelial cells inhibits TNF-α-mediated endothelial cell activation

We used a direct-contact endothelial cell-smooth muscle cell (EC-SMC) co-culture to examine whether quiescent SMCs regulate the EC inflammatory response to tumor necrosis factor (TNF)-alpha. ECs were cultured under static and physiological flow conditions. Compared with TNF-alpha-treated ECs in monoculture, TNF-alpha-treated ECs in co-culture had less NF-kappaB nuclear translocation; less intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin surface protein expression; no change in TNF receptor expression, but greater Kruppel-like factor 2 (KLF2) gene expression. After flow preconditioning for 24 h at 15 dyne/cm(2), and exposure of ECs to flow and TNF-alpha for 4.5 h, ECs in co-culture had less ICAM-1, VCAM-1, and E-selectin surface protein expression. Exposure to flow greatly increased KLF2 gene expression levels in both EC cultures; as a result, ECs in co-culture and monoculture had similar levels of post-flow KLF2 gene expression. The reduced levels of TNF-alpha-induced adhesion molecule expression in co-culture required the presence of quiescent SMCs; adhesion to decellularized extracellular matrix (ECM) or co-culture with fibroblasts produced only a modest reduction in EC adhesion molecule expression. Furthermore, co-culture of quiescent SMCs and ECs on the opposite side of a 10-microm-thick porous membrane did not alter the TNF-alpha-mediated ICAM-1 surface protein expression. Although the ECM produced by SMCs plays some role in reducing TNF-alpha-mediated inflammation, these results suggest that the direct contact between ECs and quiescent SMCs is required to inhibit TNF-alpha-mediated activation.

Late-outgrowth endothelial progenitors from patients with coronary artery disease: endothelialization of confluent stromal cell layers.

Patients with coronary artery disease (CAD) are the primary candidates to receive small-diameter tissue-engineered blood vessels (TEBVs). Peripheral blood derived endothelial progenitor cells (EPCs) from CAD patients (CAD EPCs) represent a minimally invasive source of autologous cells for TEBV endothelialization. We have previously shown that human CAD EPCs are highly proliferative and express many of the hallmarks of mature and healthy endothelial cells; however, their behavior on stromal cells that comprise the media of TEBVs has not yet been evaluated. Primary CAD EPCs or control human aortic endothelial cells (HAECs) were seeded over confluent, quiescent layers of human smooth muscle cells (SMCs) using a direct co-culture model. The percent coverage, adhesion strength, alignment under flow and generation of flow-induced nitric oxide of the seeded CAD EPCs were compared to that of HAECs. The integrin-binding profile of CAD EPCs was also evaluated over a layer of confluent, quiescent SMCs. Direct comparison of our CAD EPC results to analogous co-culture studies with cord blood EPCs show that both types of blood-derived EPCs are viable options for endothelialization of TEBVs.

Bi-ligand surfaces with oriented and patterned protein for real-time tracking of cell migration.

A bioactive platform for the quantitative observation of cell migration is presented by (1) presenting migration factors in a well-defined manner on 2-D substrates, and (2) enabling continuous cell tracking. Well-defined substrate presentation is achieved by correctly orienting immobilized proteins (chemokines and cell adhesion molecules), such that the active site is accessible to cell surface receptors. A thiol-terminated self-assembled monolayer on a silica slide was used as a base substrate for subsequent chemistry. The thiol-terminated surface was converted to an immobilized metal ion surface using a maleimido-nitrilotriacetic acid (NTA) cross-linker that bound Histidine-tagged recombinant proteins on the surface with uniform distribution and specific orientation. This platform was used to study the influence of surface-immobilized chemokine SDF-1α and cell adhesion molecule ICAM-1 on murine splenic B lymphocyte migration. While soluble SDF-1α induced trans-migration in a Boyden Chamber type chemotaxis assay, immobilized SDF-1α alone did not elicit significant surface-migration on our test-platform surface. Surface-immobilized cell adhesion protein, ICAM-1, in conjunction with activation enabled migration of this cell type on our surface. Controlled exposure to UV light was used to produce stable linear gradients of His-tagged recombinant SDF-1α co-immobilized with ICAM-1 following our surface chemistry approach. XPS and antibody staining showed defined gradients of outwardly oriented SDF-1α active sites. This test platform can be especially valuable for investigators interested in studying the influence of surface-immobilized factors on cell behavior and may also be used as a cell migration enabling platform for testing the effects of various diffusible agents.

Effect of Continuous Supression of Heparan Sulfate on Arterial Endothelial Alignment and Gene Expression Under Several Shear Stress Waveforms

The literature suggests that endothelial cells (ECs) possess many molecules that have mechanosensor capabilities, such as intracellular junction proteins, G proteins, ion channels, integrins, and the glycocalyx [1]. The endothelial glycocalyx is located on the luminal surface of ECs and interacts with the passing blood, thus making it a strong mechanosensor candidate. The glycocalyx is a negatively charged gel layer ranging from 0.5 μm thick in capillaries to 4.5 μm thick in carotid arteries [2]. The glycocalyx consists primarily of sialic acids, chondroitin sulfate, hyaluronic acid, and heparan sulfate (HS). HS is the most abundant glycosaminoglycan within the glycocalyx, and it is one of the most studied molecules in this layer [3].Copyright