Targeted delivery of nitric oxide via a 'bump-and-hole'-based enzyme-prodrug pair.

The spatiotemporal generation of nitric oxide (NO), a versatile endogenous messenger, is precisely controlled. Despite its therapeutic potential for a wide range of diseases, NO-based therapies are limited clinically due to a lack of effective strategies for precisely delivering NO to a specific site. In the present study, we developed a novel NO delivery system via modification of an enzyme-prodrug pair of galactosidase-galactosyl-NONOate using a 'bump-and-hole' strategy. Precise delivery to targeted tissues was clearly demonstrated by an in vivo near-infrared imaging assay. The therapeutic potential was evaluated in both rat hindlimb ischemia and mouse acute kidney injury models. Targeted delivery of NO clearly enhanced its therapeutic efficacy in tissue repair and function recovery and abolished side effects due to the systemic release of NO. The developed protocol holds broad applicability in the targeted delivery of important gaseous signaling molecules and offers a potent tool for the investigation of relevant molecular mechanisms.

Histone Deacetylase 7-Derived Peptides Play a Vital Role in Vascular Repair and Regeneration.

Cardiovascular disease is a leading cause of morbidity and mortality globally. Accumulating evidence indicates that local resident stem/progenitor cells play an important role in vascular regeneration. Recently, it is demonstrated that a histone deacetylase 7-derived 7-amino acid peptide (7A, MHSPGAD) is critical in modulating the mobilization and orientated differentiation of these stem/progenitor cells. Here, its therapeutic efficacy in vascular repair and regeneration is evaluated. In vitro functional analyses reveal that the 7A peptide, in particular phosphorylated 7A (7Ap, MH[pSer]PGAD), could increase stem cell antigen-1 positive (Sca1+) vascular progenitor cell (VPC) migration and differentiation toward an endothelial cell lineage. Furthermore, local delivery of 7A as well as 7Ap could enhance angiogenesis and ameliorate vascular injury in ischaemic tissues; these findings are confirmed in a femoral artery injury model and a hindlimb ischaemia model, respectively. Importantly, sustained delivery of 7A, especially 7Ap, from tissue-engineered vascular grafts could attract Sca1+-VPC cells into the grafts, contributing to endothelialization and intima/media formation in the vascular graft. These results suggest that this novel type of peptides has great translational potential in vascular regenerative medicine.

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model.

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 µm) and larger pores (~30 µm) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

Small-diameter hybrid vascular grafts composed of polycaprolactone and polydioxanone fibers.

Electrospun polycaprolactone (PCL) vascular grafts showed good mechanical properties and patency. However, the slow degradation of PCL limited vascular regeneration in the graft. Polydioxanone (PDS) is a biodegradable polymer with high mechanical strength and moderate degradation rate in vivo. In this study, a small-diameter hybrid vascular graft was prepared by co-electrospinning PCL and PDS fibers. The incorporation of PDS improves mechanical properties, hydrophilicity of the hybrid grafts compared to PCL grafts. The in vitro/vivo degradation assay showed that PDS fibers completely degraded within 12 weeks, which resulted in the increased pore size of PCL/PDS grafts. The healing characteristics of the hybrid grafts were evaluated by implantation in rat abdominal aorta replacement model for 1 and 3 months. Color Doppler ultrasound demonstrated PCL/PDS grafts had good patency, and did not show aneurysmal dilatation. Immunofluorescence staining showed the coverage of endothelial cells (ECs) was significantly enhanced in PCL/PDS grafts due to the improved surface hydrophilicity. The degradation of PDS fibers provided extra space, which facilitated vascular smooth muscle regeneration within PCL/PDS grafts. These results suggest that the hybrid PCL/PDS graft may be a promising candidate for the small-diameter vascular grafts.

Functional Modification of Electrospun Poly(ε-caprolactone) Vascular Grafts with the Fusion Protein VEGF-HGFI Enhanced Vascular Regeneration.

Synthetic artificial vascular grafts have exhibited low patency rate and severe neointimal hyperplasia in replacing small-caliber arteries (<6 mm) because of their failure to generate a functional endothelium. In this study, small-caliber (2.0 mm) electrospun poly(ε-caprolactone) (PCL) vascular grafts were modified with a fusion protein VEGF-HGFI which consists of the class I hydrophobin (HGFI) and vascular endothelial growth factor (VEGF), via hydrophobic interactions. Immunofluorescence staining with the anti-VEGF antibody showed that VEGF-HGFI formed a protein layer on the surface of fibers in the grafts. Scanning electron microscopy (SEM) and mechanical measurements showed that VEGF-HGFI modification had no effect on the structure and mechanical properties of PCL grafts. Blood compatibility tests demonstrated a lower level of fibrinogen (FGN) absorption, platelet activation, and aggregation on the VEGF-HGFI-modified PCL mats than that on the bare PCL mats. The hemolysis rate was comparable in both the modified and bare PCL mats. In vitro culture of human umbilical vein endothelial cells (HUVECs) demonstrated that VEGF-HGFI modification could remarkably enhance nitric oxide (NO) production, prostacyclin2 (PGI2) release, and the uptake of acetylated low-density lipoprotein (Ac-LDL) by HUVECs. The healing characteristics of the modified grafts were examined in the replacement of rat abdominal aorta for up to 1 month. Immunofluorescence staining revealed that endothelialization, vascularization, and smooth muscle cell (SMC) regeneration were markedly improved in the VEGF-HGFI-modified PCL grafts. These results suggest that modification with fusion protein VEGF-HGFI is an effective method to improve the regeneration capacity of synthetic vascular grafts.

Functional Modification of Fibrous PCL Scaffolds with Fusion Protein VEGF-HGFI Enhanced Cellularization and Vascularization.

The lack of efficient vascularization within frequently used poly(ε-caprolactone) (PCL) scaffolds has hindered their application in tissue engineering. Hydrophobin HGFI, an amphiphilic protein, can form a self-assembly layer on the surface of PCL scaffolds and convert their wettability. In this study, a fusion protein consisting of HGFI and vascular endothelial growth factor (VEGF) is prepared by Pichia pastoris expression system. Sodium dodecyl sulface-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting confirm that the VEGF-HGFI is successfully isolated and purified. Transmission electron microscope and water contact angle measurement demonstrate that VEGF-HGFI can form a self-assembly layer with about 25 nm in thickness on electrospun PCL fibers and increase their hydrophilicity. VEGF-HGFI modification can effectively enhance the adhesion, migration, and proliferation of human umbilical vein endothelial cells. Near-infrared fluorescence imaging shows that the VEGF-HGFI modification on PCL scaffolds can exist at least 21 d in vitro and at least 14 d in vivo. Bioluminescence imaging shows that VEGF-HGFI can effectively activate vascular endothelial growth factor receptor 2 receptors. Subcutaneous implantation in mice and rats reveal that cellularization and vascularization are significantly improved in VEGF-HGFI modified PCL scaffolds. These results suggest that VEGF-HGFI is a useful molecule for functional modification of scaffolds to enhance cellularization and vascularization in tissue engineering.

Three-Layered PCL Grafts Promoted Vascular Regeneration in a Rabbit Carotid Artery Model.

In this study, a three layered poly (ε-caprolactone) (PCL) graft (tPCL) was fabricated by electrospinning PCL and electrospraying poly (ethylene oxide) (PEO), which has a thin dense inner layer, a loose middle layer, and a dense outer layer. Regular PCL grafts (rPCL) with only a dense layer were used as control. In vivo evaluation was performed in rabbit carotid artery. Enhanced cell infiltration, rapid regeneration of endothelium and smooth muscle layers, and increased elastin deposition were observed within the tPCL graft wall. After 3 months, tPCL grafts showed faster PCL degradation than the rPCL grafts. Infiltrated macrophages in the tPCL grafts secreted higher level of monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) which enhanced vascular regeneration. In conclusion, the tPCL graft may be a useful vascular prosthesis and worth for further investigation.

Enhanced vascularization in hybrid PCL/gelatin fibrous scaffolds with sustained release of VEGF.

Creating a long-lasting and functional vasculature represents one of the most fundamental challenges in tissue engineering. VEGF has been widely accepted as a potent angiogenic factor involved in the early stages of blood vessel formation. In this study, fibrous scaffolds that consist of PCL and gelatin fibers were fabricated. The gelatin fibers were further functionalized by heparin immobilization, which provides binding sites for VEGF and thus enables the sustained release of VEGF. In vitro release test confirms the sustained releasing profile of VEGF, and stable release was observed over a time period of 25 days. In vitro cell assay indicates that VEGF release significantly promoted the proliferation of endothelial cells. More importantly, in vivo subcutaneous implantation reflects that vascularization has been effectively enhanced in the PCL/gelatin scaffolds compared with the PCL counterpart due to the sustained release of VEGF. Therefore, the heparinized PCL/gelatin scaffolds developed in this study may be a promising candidate for regeneration of complex tissues with sufficient vascularization.

Rapid in situ endothelialization of a small diameter vascular graft with catalytic nitric oxide generation and promoted endothelial cell adhesion.

Thrombosis and neointimal hyperplasia are the main causes for the failure of small diameter vascular grafts, and a complete and functional endothelium is essential in preventing these problems. Therefore, grafts that could be endothelialized rapidly are highly desirable. This study constructed a vascular graft with catalytic nitric oxide (NO) generation and promoted endothelial cell (EC) adhesion for rapid in situ endothelialization, and examined the in vivo performance of an NO-generating vascular graft for the first time. A macroporous electrospun polycaprolactone (PCL) graft was prepared and modified via layer-by-layer self-assembly. Organoselenium immobilized polyethyleneimine was loaded onto the graft for in situ catalytic NO generation, while hyaluronic acid was grafted with an EC specific peptide Arg-Glu-Asp-Val and deposited to promote EC adhesion. This dual-modified material generated a strong and sustained flow of NO from S-nitrosoglutathione and significantly enhanced EC adhesion in vitro. In a co-culture experiment of ECs and smooth muscle cells (SMCs), this material promoted the adhesion of ECs and increased the EC/SMC ratio. After implantation in rats, the modified grafts showed a remarkably promoted endothelialization compared to PCL ones with an endothelium coverage of 89% versus 55% after 4 weeks, and the ECs on modified grafts were better organized in a pattern similar to that of the native vessel. The results indicated that the combination of catalytic NO generation and promoted EC adhesion proposed in this work may be a promising method for rapid endothelialization of small diameter vascular grafts.