A nitric oxide releasing, self assembled peptide amphiphile matrix that mimics native endothelium for coating implantable cardiovascular devices
Cardiovascular disease is the number one cause of death in the United States. Deployment of stents and vascular grafts has been a major therapeutic method for treatment. However, restenosis, incomplete endothelialization, and thrombosis hamper the long term clinical success. As a solution to meet these current challenges, we have developed a native endothelial ECM mimicking self-assembled nanofibrous matrix to serve as a new treatment model. The nanofibrous matrix is formed by self-assembly of peptide amphiphiles (PAs), which contain nitric oxide (NO) donating residues, endothelial cell adhesive ligands composed of YIGSR peptide sequence, and enzyme-mediated degradable sites. NO was successfully released from the nanofibrous matrix rapidly within 48 h, followed by sustained release over period of 30 days. The NO releasing nanofibrous matrix demonstrated a significantly enhanced proliferation of endothelial cells (51 3% to 67 2%) but reduced proliferation of smooth muscle cells (35 2% to 16 3%) after 48 h of incubation. There was also a 150-fold decrease in platelet attachment on the NO releasing nanofibrous matrix (470 220 platelets/cm2) compared to the collagen-I (73 22 103 platelets/cm2) coated surface. The nanofibrous matrix has the potential to be applied to various cardiovascular implants as a selfassembled coating, thereby providing a native endothelial extracellular matrix (ECM) mimicking environment.
Cardiovascular disease is the leading cause of death in the United States .
Currently, stents and vascular grafts are the primary therapeutic methods for treatment of cardiovascular diseases. However, restenosis, incomplete endothelialization, and thrombosis hamper their long term clinical success [2–4]. Native endothelium consists of a monolayer of endothelial cells that adhere to the underlying nanofibrillar basement membrane and modulate vascular tone by release of soluble factors, such as nitric oxide (NO). The local release of NO plays a critical role in controlling the function of the human cardiovascular system by regulating vascular cell homeostasis [5–7]. Thus, the inevitable loss of this multi-functional endothelium associated with vascular stretch and injury at the implant sites of stents and vascular grafts triggers a cascade of restenosis by smooth muscle cells proliferation with accompanying extracellular matrix production. The risk of late thrombosis by platelet adhesion also compromises long term patency. Altogether, currently used stents and vascular grafts remain limited by incomplete re-endothelialization, restenosis, and late-thrombosis (Fig. 1a) [2–4,8].
Numerous therapeutic approaches have been investigated to overcome these problems with limited success. It is believed that the incorporation of endothelium specific factors will provide an enhanced clinical treatment, specifically tailoring biomaterials for cardiovascular implant coatings. To this effect, several NO releasing materials have been studied in the form of films or hydrogels and found to reduce platelet adhesion and intimal hyperplasia, both in vitro and in vivo [9–12]. However, none of the above materials are presently able to completely tackle all current clinical challenges, as they are limited by their inability to mimic the properties of native endothelium. Instead, a more multifunctional approach is required, which would provide a native endothelial extracellular matrix (ECM) mimicking environment on the surface of stents or vascular grafts to prevent restenosis and thrombosis by inhibiting smooth muscle proliferation and platelet adhesion, while enhancing re-endothelialization by promoting endothelial cell proliferation. Therefore, the goal of this study is to develop a native endothelial ECM mimicking nanofibrous matrix that consists of NO releasing peptide amphiphiles, and to study the behavior of endothelial cells, smooth muscle cells and platelets in vitro on this nanofibrous matrix.
Peptide amphiphiles (PAs) that consist of hydrophobic tails coupled to hydrophilic functional peptide sequences are attractive templates for biomimetic scaffolds because cell adhesion ligands and enzyme-mediated degradable sites can be incorporated into the hydrophilic domains of the PAs to mimic biochemical properties of the extracellular matrix (ECM) [13,14]. In order to mimic properties of a native endothelium, the designed hydrophilic functional peptide sequences consist of a matrix metalloprotease-2 (MMP2) mediated cleavage site, Gly-Thr-Ala-Gly-Leu-Ile-Gly-Gln (GTAGLIGQ), coupled to an endothelial cell-adhesive ligand, Tyr-Ile-Gly-Ser-Arg (YIGSR), or a polylysine (KKKKK) group to form NO (or nitrogen oxide) donating residues [17–19]. This study utilizes a bottom-up approach to achieve a unique synergistic effect by combining multiple components, including cell-adhesive ligand (YIGSR), cytokine molecule (NO), enzyme-mediated degradation (MMP-2), and self-assembly into a nano-fibrillar structure. NO is a natural mediator of vascular homeostasis and is produced by endothelial cells. It has been known to reduce platelet adhesion and smooth muscle cell proliferation, while concurrently stimulating endothelial cell proliferation [18,19]. Therefore, this nanofibrous matrix comprised two different PAs, PA-YIGSR (C16-GTAGLIGQYIGSR) and PA-KKKKK (C16- GTAGLIGQKKKKK). The nanofibrous matrix is designed to act as a surrogate reservoir of NO by replenishing the NO supply to the native artery during renewal of the injured endothelium. The incorporation of NO donating residues into the PA will allow controlled release of NO from the nanofibrous matrix coated on stents or vascular grafts into the local blood stream. NO will limit smooth muscle cell proliferation and platelet adhesion, while enhancing re-endothelialization onto stents or vascular grafts. Read More